Haruka Okabayashi, Miki Yasuda, Chinatsu Nii, Ryo Sugishita, Keijo Fukushima, Kouki Yuasa, Satoshi Kotoura and Hiromichi Fujino : Phosphatidylcholine-Plasmalogen-Oleic Acid Reduces BACE1 Expression in Human SH-SY5Y Cells., Biological & Pharmaceutical Bulletin, Vol.47, No.1, 192-195, 2024.
(Summary)
Plasmalogens are a family of glycerophospholipids containing one vinyl-ether bond at the sn-1 position in the glycerol backbone, and play important roles in cellular homeostasis including neural transmission. Therefore, reductions of plasmalogens have been associated with neurodegenerative disorders, such as Alzheimer's disease (AD). To evaluate the potential protective effects of plasmalogens against the pathology of AD, protein expression levels of key factors in amyloid precursor protein (APP) metabolic processes were examined using human neuroblastoma SH-SY5Y cells. Here, phosphatidylcholine-plasmalogen-oleic acid (PC-PLS-18) was shown to reduce protein expression levels of β-site APP cleaving enzyme 1 (BACE1), clusterin, and Tau, factors involved in the amyloid β-associated pathogenesis of AD. Thus, PC-PLS-18 may have preventive effects against AD by delaying the onset risk for a certain period.
A strong hypoxic environment has been observed in pancreatic ductal adenocarcinoma (PDAC) cells, which contributes to drug resistance, tumor progression, and metastasis. Therefore, we performed bioinformatics analyses to investigate potential targets for the treatment of PDAC. To identify potential genes as effective PDAC treatment targets, we selected all genes whose expression level was related to worse overall survival (OS) in The Cancer Genome Atlas (TCGA) database and selected only the genes that matched with the genes upregulated due to hypoxia in pancreatic cancer cells in the dataset obtained from the Gene Expression Omnibus (GEO) database. Although the extracted 107 hypoxia-responsive genes included the genes that were slightly enriched in angiogenic factors, TCGA data analysis revealed that the expression level of endothelial cell (EC) markers did not affect OS. Finally, we selected CA9 and PRELID2 as potential targets for PDAC treatment and elucidated that a CA9 inhibitor, U-104, suppressed pancreatic cancer cell growth more effectively than 5-fluorouracil (5-FU) and PRELID2 siRNA treatment suppressed the cell growth stronger than CA9 siRNA treatment. Thus, we elucidated that specific inhibition of PRELID2 as well as CA9, extracted via exhaustive bioinformatic analyses of clinical datasets, could be a more effective strategy for PDAC treatment.
Ayaka Hamaguchi, Hayato Fukuda, Koichi Fujiwara, Tomofumi Harada, Keijo Fukushima, Satoshi Shuto and Hiromichi Fujino : Individual resolvin E family members work distinctly and in a coordinated manner in the resolution of inflammation., Prostaglandins & Other Lipid Mediators, Vol.168, 106759, 2023.
(Summary)
Three main E-type resolvins (RvEs): RvE1, RvE2, and RvE3, have roles in the resolution of inflammation as anti-inflammatory activities. To investigate the roles of each RvE in the resolution of inflammation, timing of interleukin (IL)- 10 release and IL-10 receptor expressions, and phagocytosis evoked by each RvE in differentiated human monocytes, macrophage-like U937 cells were examined. Here, we show that RvEs enhance the expression of IL-10, and IL-10 receptor-mediated signaling pathways and IL-10-mediated-signaling-independent resolution of inflammatory effects by activating the phagocytotic function. Thus, RvE2 mainly evoked an IL-10-mediated anti-inflammatory function, whereas RvE3 principally activated phagocytotic activity of macrophages, which may be involved in tissue repair. On the other hand, RvE1 showed both functions, although not prominent but rather acting as a relief mediator that takes over the RvE2 function and passes over to the RvE3 function. Therefore, each RvE may act as an important role/stage-specific mediator in a coordinated manner with other RvEs in the processes of the resolution of inflammation.
Takahiro Niimura, Yoshito Zamami, Koji Miyata, Takahisa Mikami, Mizuho Asada, Keijo Fukushima, Masaki Yoshino, Satoru Mitsuboshi, Naoto Okada, Hirofumi Hamano, Takumi Sakurada, Rie Matsuoka-Ando, Fuka Aizawa, Kenta Yagi, Mitsuhiro Goda, Masayuki Chuma, Toshihiro Koyama, Yuki Izawa-Ishizawa, Hiroaki Yanagawa, Hiromichi Fujino, Yoshihiro Yamanishi and Keisuke Ishizawa : Characterization of Immune Checkpoint Inhibitor-Induced Myasthenia Gravis Using the US Food and Drug Administration Adverse Event Reporting System., Journal of Clinical Pharmacology, Vol.63, No.4, 473-479, 2022.
(Summary)
Myasthenia gravis (MG) is a rare but fatal adverse event of immune checkpoint inhibitors (ICIs). We assessed whether patient characteristics differed between those with ICI-related myasthenia gravis and those with idiopathic myasthenia gravis. Reports from the US Food and Drug Administration Adverse Event Reporting System were analyzed. Multivariate analyses were conducted to evaluate the associations between age, sex, and ICI treatment and the reporting rate of myasthenia gravis. Among 5 464 099 cases between 2011 and 2019, 53 447 were treated with ICIs. Myasthenia gravis was reported more often in ICI users. Multiple logistic regression analyses showed that the reporting rate of ICI-related myasthenia gravis did not differ significantly between men and women; however, it was higher in older people than in younger people (adjusted odds ratio, 2.4 [95%CI, 1.84-3.13]). We also investigated useful signs for the early detection of myositis and myocarditis, which are fatal when overlapping with ICI-related myasthenia gravis. Patients with elevated serum creatine kinase or troponin levels were more likely to have concurrent myositis and myocarditis. Unlike idiopathic myasthenia gravis, there was no sex difference in the development of ICI-related myasthenia gravis, which may be more common in older people. Considering the physiological muscle weakness that occurs in the elderly, it may be necessary to monitor ICI-related myasthenia gravis more closely in older people.
(Keyword)
Male / United States / Humans / Female / Aged / Immune Checkpoint Inhibitors / United States Food and Drug Administration / Myocarditis / Myasthenia Gravis / Myositis
Natsuki Yamagiwa, Haruka Kobayashi, Haruka Okabayashi, Miki Yasuda, Keijo Fukushima, Jun Kawamura, Satoshi Kotoura and Hiromichi Fujino : Phosphatidylcholine-Plasmalogen-Oleic Acid Has Protective Effects against Arachidonic Acid-Induced Cytotoxicity., Biological & Pharmaceutical Bulletin, Vol.45, No.5, 643-648, 2022.
(Summary)
Plasmalogens are a group of glycerophospholipids containing a vinyl-ether bond at the sn-1 position in the glycerol backbone. Cellular membrane plasmalogens are considered to have important roles in homeostasis as endogenous antioxidants, differentiation, and intracellular signal transduction pathways including neural transmission. Therefore, reduced levels of plasmalogens have been suggested to be associated with neurodegenerative diseases such as Alzheimer's disease. Interestingly, although arachidonic acid is considered to be involved in learning and memory, it could be liberated and excessively activate neuronal activity to the excitotoxic levels seen in Alzheimer's disease patients. Here, we examined the protective effects of several kinds of plasmalogens against cellular toxicity caused by arachidonic acid in human neuroblastoma SH-SY5Y cells. As a result, only phosphatidylcholine-plasmalogen-oleic acid (PC-PLS-18) showed protective effects against arachidonic acid-induced cytotoxicity based on the results of lactate dehydrogenase release and ATP depletion assays, as well as cellular morphological changes in SH-SY5Y cells. These results indicate that PC-PLS-18 protects against arachidonic acid-induced cytotoxicity, possibly via improving the stability of the cellular membrane in SH-SY5Y cells.
Yoshito Zamami, Takahiro Niimura, Takehiro Kawashiri, Mitsuhiro Goda, Yutaro Naito, Keijo Fukushima, Soichiro Ushio, Fuka Aizawa, Hirofumi Hamano, Naoto Okada, Kenta Yagi, Koji Miyata, Kenshi Takechi, Masayuki Chuma, Toshihiro Koyama, Daisuke Kobayashi, Takao Shimazoe, Hiromichi Fujino, Yuki Izawa-Ishizawa and Keisuke Ishizawa : Identification of prophylactic drugs for oxaliplatin-induced peripheral neuropathy using big data., Biomedicine & Pharmacotherapy, Vol.148, 2022.
(Summary)
Thus, drug repositioning using data from large-scale basic and clinical databases enables the discovery of new indications for approved drugs with a high probability of success.
(Keyword)
Animals / Anticholesteremic Agents / Antineoplastic Agents / Big Data / Databases, Factual / Drug Repositioning / Humans / Hyperalgesia / Japan / Male / Mice / Mice, Inbred BALB C / Oxaliplatin / Peripheral Nervous System Diseases / Pre-Exposure Prophylaxis / Rats / Rats, Sprague-Dawley / Retrospective Studies / Simvastatin
Kana Kitagawa, Ayaka Hamaguchi, Keijo Fukushima, Yuki Nakano, W John Regan, Masato Mashimo and Hiromichi Fujino : Interleukin-4 may suppress expression of E-type prostanoid receptor4 in human colorectal cancer HCA-7 cells, European Journal of Pharmacology, Vol.920, 2022.
(Summary)
) and cyclooxygenase-2 (COX-2), the hallmarks of colorectal carcinogenesis. In the present study, using an in silico analysis and pharmacological experiments, we demonstrated that interleukin (IL)-4, a signature cytokine of Th2 cells, down-regulated the expression of COX-2 and PGE
(Keyword)
Colonic Neoplasms / Cyclooxygenase 2 / Dinoprostone / Down-Regulation / Humans / Interleukin-4 / Prostaglandins E / Receptors, Prostaglandin E, EP2 Subtype / Receptors, Prostaglandin E, EP4 Subtype
Keijo Fukushima, Kanaho Senoo, Naoki Kurata, W John Regan and Hiromichi Fujino : The Gαs-protein-mediated pathway may be steadily stimulated by prostanoid EP2 receptors, but not by EP4 receptors., FEBS Open Bio, Vol.12, No.4, 775-783, 2022.
(Summary)
causes accumulation of cAMP in EP2 receptors, whereas markedly low levels of cAMP accumulated in EP4 receptors. By applying the Black/Leff operational model calculation, we found that EP2 receptors have a biased ability to intrinsically activate the Gαs-protein-mediated pathway, whereas EP4 receptors have strong biased activity for the Gαi-protein-mediated pathway. Thus, EP2 and EP4 receptors may not be similar Gαs-coupled receptors but instead substantially different receptors with distinct roles.
(Keyword)
Prostaglandins / Receptors, Prostaglandin E, EP2 Subtype / Receptors, Prostaglandin E, EP4 Subtype / Signal Transduction
Keijo Fukushima and Hiromichi Fujino : Identification and Characterization of Human Colorectal Cancer Cluster Predominantly Expressing EP3 Prostanoid Receptor Subtype., Biological & Pharmaceutical Bulletin, Vol.45, No.6, 698-702, 2022.
(Summary)
) is a well-known mediator of colorectal cancer through stimulation of four E-type prostanoid (EP) receptor subtypes: EP1, EP2, EP3, and EP4 receptors. All subtypes of EP receptors are involved in CRC promotion or malignancy. However, the characteristics of CRC that highly expresses EP receptor subtypes have not been clarified. In the present study, we classified CRC from a cancer genomic database and identified CRC clusters which highly express EP receptor subtypes. Most of these clusters predominantly expressed one subtype of EP receptor and showed different gene expression patterns. Among them, we focused on the cluster highly expressing the EP3 receptor (CL-EP3). As the result of characterization of gene expression, CL-EP3 was characterized as: epithelial mesenchymal transition (EMT)-induced progressed cancer with activation of transforming growth factor-β pathway, activation of hypoxia-inducible factor-1α, and suppression of runt-related transcription factor 3. Since we previously reported that EP3 receptor is involved in and induce colon cancer cell migration, EP3 receptor-expressing CRC may induce metastasis through these signaling pathways. Thus, the findings suggest the effectiveness of cancer clustering by gene expression of the EP receptor subtype to elucidate the mechanism of human CRC.
(Keyword)
Colorectal Neoplasms / Dinoprostone / Humans / Receptors, Prostaglandin E / Signal Transduction
Hirofumi Hamano, Yasumasa Ikeda, Mitsuhiro Goda, Keijo Fukushima, Seiji Kishi, Masayuki Chuma, Michiko Yamashita, Takahiro Niimura, Kenshi Takechi, Masaki Imanishi, Yoshito Zamami, Yuya Horinouchi, Izawa-Ishizawa Yuki, Licht Miyamoto, Ishizawa Keisuke, Hiromichi Fujino, Toshiaki Tamaki, Ken-ichi Aihara and Koichiro Tsuchiya : Diphenhydramine may be a preventive medicine against cisplatin-induced kidney toxicity, Kidney International, Vol.99, No.4, 885-889, 2021.
(Summary)
Cisplatin is widely used as an anti-tumor drug for the treatment of solid tumors. Unfortunately, it causes kidney toxicity as a critical side effect, limiting its use, given that no preventive drug against cisplatin-induced kidney toxicity is currently available. Here, based on a repositioning analysis of the Food and Drug Administration Adverse Events Reporting System, we found that a previously developed drug, diphenhydramine, may provide a novel treatment for cisplatin-induced kidney toxicity. To confirm this, the actual efficacy of diphenhydramine was evaluated in in vitro and in vivo experiments. Diphenhydramine inhibited cisplatin-induced cell death in kidney proximal tubular cells. Mice administered cisplatin developed kidney injury with significant dysfunction (mean plasma creatinine: 0.43 vs 0.15 mg/dl) and showed augmented oxidative stress, increased apoptosis, elevated inflammatory cytokines, and MAPKs activation. However, most of these symptoms were suppressed by treatment with diphenhydramine. Furthermore, the concentration of cisplatin in the kidney was significantly attenuated in diphenhydramine-treated mice (mean platinum content: 70.0 vs 53.4 μg/g dry kidney weight). Importantly, diphenhydramine did not influence or interfere with the anti-tumor effect of cisplatin in any of the in vitro or in vivo experiments. In a selected cohort of 98 1:1 matched patients from a retrospective database of 1467 patients showed that patients with malignant cancer who had used diphenhydramine before cisplatin treatment exhibited significantly less acute kidney injury compared to ones who did not (6.1 % vs 22.4 %, respectively). Thus, diphenhydramine demonstrated efficacy as a novel preventive medicine against cisplatin-induced kidney toxicity.
Motofumi Suzuki, Atsumi Urabe, Sayaka Sasaki, Ryo Tsugawa, Satoshi Nishio, Haruka Mukaiyama, Yoshiko Murata, Hiroshi Masuda, M. Sann Aung, Akane Mera, Masaki Takeuchi, Keijo Fukushima, Michika Kanaki, Kaori Kobayashi, Yudai Chiba, Binod Babu Shrestha, Hiromi Nakanishi, T. Watanabe, Atsushi Nakayama, Hiromichi Fujino, Takanori Kobayashi, Keiji Tanino, Naoko Nishizawa and Kosuke Namba : Development of a mugineic acid family phytosiderophore analog as an iron fertilizer, Nature Communications, Vol.12, No.1, 1558, 2021.
(Summary)
Iron (Fe) is an essential nutrient, but is poorly bioavailable because of its low solubility in alkaline soils; this leads to reduced agricultural productivity. To overcome this problem, we first showed that the soil application of synthetic 2'-deoxymugineic acid, a natural phytosiderophore from the Poaceae, can recover Fe deficiency in rice grown in calcareous soil. However, the high cost and poor stability of synthetic 2'-deoxymugineic acid preclude its agricultural use. In this work, we develop a more stable and less expensive analog, proline-2'-deoxymugineic acid, and demonstrate its practical synthesis and transport of its Fe-chelated form across the plasma membrane by Fe(III)·2'-deoxymugineic acid transporters. Possibility of its use as an iron fertilizer on alkaline soils is supported by promotion of rice growth in a calcareous soil by soil application of metal free proline-2'-deoxymugineic acid.
(Keyword)
Azetidinecarboxylic Acid / Fertilizers / Iron / Siderophores / Soil
The roles of cancer-associated fibroblasts (CAFs) have been studied in the tumor progression, and CAFs are expected to become the new targets for cancer pharmacotherapies. CAFs contribute to tumor cell survival and proliferation, tumor angiogenesis, immune suppression, tumor inflammation, tumor cell invasion and metastasis, and extracellular matrix remodeling. However, detailed mechanisms of how CAFs function in the living system remain unclear. CAFs include α-smooth muscle actin, expressing activated fibroblasts similar to myofibroblasts, and are highly capable of producing collagen. Several reports have demonstrated the contributions of extracellular-signal-regulated kinase 5 (ERK5) in fibroblasts to the fibrotic processes; however, the roles of CAF-derived ERK5 remain unclear. To investigate the roles of CAF-derived ERK5 in the tumor progression, we created mice lacking the ERK5 gene specifically in fibroblasts. Colon-26 mouse colon cancer cells were implanted into the mice subcutaneously, and the histological analyses of the tumor tissue were performed after 2 weeks. Immunofluorescence analyses showed that recipient-derived fibroblasts existed within the tumor tissue. The present study demonstrated that fibroblast-specific ERK5 deficiency exacerbated tumor progression and it was accompanied with thicker tumor vessel formation and the increase in the number of activated fibroblasts. We combined the results of The Cancer Genome Atlas (TCGA) database analysis with our animal studies, and indicated that regulating ERK5 activity in CAFs or CAF invasion into the tumor tissue can be important strategies for the development of new targets in cancer pharmacotherapies.
Hirofumi Hamano, Takahiro Niimura, Yuya Horinouchi, Yoshito Zamami, Kenshi Takechi, Mitsuhiro Goda, Masaki Imanishi, Masayuki Chuma, Yuki Izawa-Ishizawa, Licht Miyamoto, Keijo Fukushima, Hiromichi Fujino, Koichiro Tsuchiya, Keisuke Ishizawa, Toshiaki Tamaki and Yasumasa Ikeda : Proton pump inhibitors block iron absorption through direct regulation of hepcidin via the aryl hydrocarbon receptor-mediated pathway, Toxicology Letters, Vol.318, 86-91, 2020.
(Summary)
Proton pump inhibitors (PPIs) have been used worldwide to treat gastrointestinal disorders. A recent study showed that long-term use of PPIs caused iron deficiency; however, it is unclear whether PPIs affect iron metabolism directly. We investigated the effect of PPIs on the peptide hepcidin, an important iron regulatory hormone. First, we used the FDA Adverse Event Reporting System database and analyzed the influence of PPIs. We found that PPIs, as well as H2 blockers, increased the odds ratio of iron-deficient anemia. Next, HepG2 cells were used to examine the action of PPIs and H2 blockers on hepcidin. PPIs augmented hepcidin expression, while H2 blockers did not. In fact, the PPI omeprazole increased hepcidin secretion, and omeprazole-induced hepcidin upregulation was inhibited by gene silencing or the pharmacological inhibition of the aryl hydrocarbon receptor. In mouse experiments, omeprazole also increased hepatic hepcidin mRNA expression and blood hepcidin levels. In mice treated with omeprazole, protein levels of duodenal and splenic ferroportin decreased. Taken together, PPIs directly affect iron metabolism by suppressing iron absorption through the inhibition of duodenal ferroportin via hepcidin upregulation. These findings provide a new insight into the molecular mechanism of PPI-induced iron deficiency.
Naoki Kurata, Natsumi Tokashiki, Keijo Fukushima, Takaya Misao, Nanae Hasuoka, Kana Kitagawa, Masato Mashimo, W John Regan, Toshihiko Murayama and Hiromichi Fujino : Short chain fatty acid butyrate uptake reduces expressions of prostanoid EP4 receptors and their mediation of cyclooxygenase-2 induction in HCA-7 human colon cancer cells., European Journal of Pharmacology, Vol.853, 308-315, 2019.
Hirofumi Hamano, Marin Mitsui, Yoshito Zamami, Kenshi Takechi, Takahiro Nimura, Naoto Okada, Keijo Fukushima, Masaki Imanishi, Masayuki Chuma, Yuya Horinouchi, Yuki Izawa-Ishizawa, Yasushi Kirino, Toshimi Nakamura, Kazuhiko Teraoka, Yasumasa Ikeda, Hiromichi Fujino, Hiroaki Yanagawa, Toshiaki Tamaki and Keisuke Ishizawa : Irinotecan-induced neutropenia is reduced by oral alkalization drugs: analysis using retrospective chart reviews and the spontaneous reporting database., Supportive Care in Cancer, Vol.27, No.3, 849-856, 2019.
(Summary)
These data indicate that oral alkalization drugs may reduce the frequency of neutropenia caused by irinotecan administration, making it possible to increase the dose safely.
Naofumi Seira, Kazuyuki Yamagata, Keijo Fukushima, Yumi Araki, Naoki Kurata, Naoki Yanagisawa, Masato Mashimo, Hiroyuki Nakamura, W John Regan, Toshihiko Murayama and Hiromichi Fujino : Cellular density-dependent increases in HIF-1α compete with c-Myc to down-regulate human EP4 receptor promoter activity through Sp-1-binding region., Pharmacology Research & Perspectives, Vol.6, No.6, 2018.
(Summary)
The up-regulated expression of E-type prostanoid (EP) 4 receptors has been implicated in carcinogenesis; however, the expression of EP4 receptors has also been reported to be weaker in tumor tissues than in normal tissues. Indeed, EP4 receptors have been suggested to play a role in the maintenance of colorectal homeostasis. This study aimed to examine the underlying mechanisms/reasons for why inconsistent findings have been reported regarding EP4 receptor expression levels in homeostasis and carcinogenesis by focusing on cellular densities. Thus, the human colon cancer HCA-7 cells, which retain some functional features of normal epithelia, and luciferase reporter genes containing wild-type or mutated EP4 receptor promoters were used for elucidating the cellular density-dependent mechanisms about the regulation of EP4 receptor expression. In silico analysis was also utilized for confirming the relevance of the findings with respect to colon cancer development. We here demonstrated that the expression of EP4 receptors was up-regulated by c-Myc by binding to Sp-1 under low cellular density conditions, but was down-regulated under high cellular density conditions via the increase in the expression levels of HIF-1α protein, which may pull out c-Myc and Sp-1 from DNA-binding. The tightly regulated EP4 receptor expression mechanism may be a critical system for maintaining homeostasis in normal colorectal epithelial cells. Therefore, once the system is altered, possibly due to the transient overexpression of EP4 receptors, it may result in aberrant cellular proliferation and transformation to cancerous phenotypes. However, at the point, EP4 receptors themselves and their mediated homeostasis would be no longer required.
Masaki Imanishi, Yuki Izawa-Ishizawa, T Sakurada, Y Kohara, Yuya Horinouchi, E Sairyo, Yoshito Zamami, Kenshi Takechi, Masayuki Chuma, Keijo Fukushima, Yasumasa Ikeda, Hiromichi Fujino, M Yoshizumi, Koichiro Tsuchiya, Toshiaki Tamaki and Keisuke Ishizawa : Nitrosonifedipine, a Photodegradation Product of Nifedipine, Suppresses Pharmacologically Induced Aortic Aneurysm Formation., Pharmacology, Vol.102, No.5-6, 281-286, 2018.
(Summary)
We have reported that nitrosonifedipine (NO-NIF), a photodegradation product of nifedipine, has strong antioxidant and endothelial protective effects, and can suppress several cardiovascular diseases in animal models. The objective of the present study was to investigate the effects of NO-NIF on aortic aneurysm formation. The mice were infused with β-aminopropionitrile for 2 weeks and angiotensin II for 6 weeks to induce aortic aneurysm formation. The oxidative stress was measured by dihydroethidium staining and nitrotyrosine staining. The expressions of inflammation-related genes were assessed by quantitative real-time PCR and immunohistochemical staining. To clarify the mechanisms of how NO-NIF suppresses vascular cell adhesion molecule (VCAM)-1, endothelial cells were used in in vitro system. NO-NIF suppressed pharmacologically induced the aortic aneurysm formation and aortic expansion without blood pressure changes. NO-NIF suppressed elastin degradation and matrix metalloproteinase-2 mRNA expression. NO-NIF suppressed the reactive oxygen species-cyclophilin A positive feedback loop. Upregulated mRNA expressions of inflammation-related genes and endothelial VCAM-1 were suppressed by NO-NIF co-treatment in aortae. NO-NIF has the potential to be a new, nifedipine-derived therapeutic drug for suppressing aortic aneurysm formation by directly improving aortic structure with its strong ability to reduce oxidative stress and inflammation.
Yuya Horinouchi, Yasumasa Ikeda, Keijo Fukushima, Masaki Imanishi, Hirofumi Hamano, Yuki Izawa-Ishizawa, Yoshito Zamami, Kenshi Takechi, Licht Miyamoto, Hiromichi Fujino, Keisuke Ishizawa, Koichiro Tsuchiya and Toshiaki Tamaki : Renoprotective effects of a factor Xa inhibitor: fusion of basic research and a database analysis., Scientific Reports, Vol.8, No.1, 2018.
(Summary)
Renal tubulointerstitial injury, an inflammation-associated condition, is a major cause of chronic kidney disease (CKD). Levels of activated factor X (FXa), a blood coagulation factor, are increased in various inflammatory diseases. Therefore, we investigated the protective effects of an FXa inhibitor against renal tubulointerstitial injury using unilateral ureteral obstruction (UUO) mice (a renal tubulointerstitial fibrosis model) and the Food and Drug Administration Adverse Events Reporting System (FAERS) database. The renal expression levels of FX and the FXa receptors protease-activated receptor (PAR)-1 and PAR-2 were significantly higher in UUO mice than in sham-operated mice. UUO-induced tubulointerstitial fibrosis and extracellular matrix expression were suppressed in UUO mice treated with the FXa inhibitor edoxaban. Additionally, edoxaban attenuated UUO-induced macrophage infiltration and inflammatory molecule upregulation. In an analysis of the FAERS database, there were significantly fewer reports of tubulointerstitial nephritis for patients treated with FXa inhibitors than for patients not treated with inhibitors. These results suggest that FXa inhibitors exert protective effects against CKD by inhibiting tubulointerstitial fibrosis.
Aurpita Shaha, Hiroyuki Mizuguchi, Yoshiaki Kitamura, Hiromichi Fujino, Masami Yabumoto, Noriaki Takeda and Hiroyuki Fukui : Receptor and Interleukin-9 Gene Expressions Responsible for the Pathogenesis of the Allergic Rhinitis., Biological & Pharmaceutical Bulletin, Vol.41, No.9, 1440-1447, 2018.
(Summary)
phosphorylation of PKCδ in HeLa cells. In RBL-2H3 cells, RJ and BGPP also suppressed NFAT-mediated IL-9 gene expression. These results suggest that RJ and BGPP improve allergic symptoms by suppressing PKCδ and NFAT signaling pathways, two important signal pathways for the AR pathogenesis, and suggest that RJ and BGPP could be good therapeutics against AR.
Rezwanul Islam, Hiroyuki Mizuguchi, Aurpita Shaha, Kohei Nishida, Masami Yabumoto, Hisashi Ikeda, Hiromichi Fujino, Yoshiaki Kitamura, Hiroyuki Fukui and Noriaki Takeda : Effect of wild grape on the signaling of histamine H1 receptor gene expression responsible for the pathogenesis of allergic rhinitis., The Journal of Medical Investigation : JMI, Vol.65, No.3.4, 242-250, 2018.
(Summary)
receptor (H1R) and IL-9 gene expressions, respectively, are responsible for the pathogenesis of allergic rhinitis. We explore anti-allergic compounds that suppress these signaling pathways and found that wild grape (WG) contains such compounds. Here, we investigated the effect of WG hot water extract (WGE) on the signaling pathways for PKCδ-mediated H1R and NFAT-mediated IL-9 gene expressions. WGE suppressed histamine/PMA-induced H1R gene up-regulation in HeLa cells. Toluene-2,4-diisocyanate (TDI)-induced H1R mRNA elevation in TDI-sensitized rats was also suppressed by WGE treatment. Treatment with WGE in combination with Awa-tea, suppresses NFAT signaling-mediated IL-9 gene, markedly alleviated nasal symptoms. Furthermore, WGE suppressed PMA-induced IL-33 gene up-regulation in Swiss 3T3 cells. Data suggest that combination of WGE, suppresses PKCδ signaling with Awa-tea, suppresses NFAT signaling would have distinct clinical and therapeutic advantages as a substitute for anti-allergic drugs. In addition, as the expression level of IL-33 mRNA was correlated with the blood eosinophils number in patients with pollinosis, WG could alleviate eosinophilic inflammation through the suppression of IL-33 gene expression. J. Med. Invest. 65:242-250, August, 2018.
Takahiro Niimura, Yoshito Zamami, Toshihiro Koyama, Yuki Izawa-Ishizawa, Masashi Miyake, Tadashi Koga, Keisaku Harada, Ayako Ohshima, Toru Imai, Yutaka Kondo, Masaki Imanishi, Kenshi Takechi, Keijo Fukushima, Yuya Horinouchi, Yasumasa Ikeda, Hiromichi Fujino, Koichiro Tsuchiya, Toshiaki Tamaki, Shiro Hinotsu, R Mitsunobu Kano and Keisuke Ishizawa : Hydrocortisone administration was associated with improved survival in Japanese patients with cardiac arrest., Scientific Reports, Vol.7, No.1, 2017.
(Summary)
There are few reports on hydrocortisone administration after cardiac arrest, and those that have been published included few subjects. This study aimed to evaluate the effect of hydrocortisone administration on the outcomes of patients who experienced cardiac arrest. We investigated the survival discharge rates and the length of hospital stay from cardiac arrest to discharge, stratified by use of hydrocortisone, using a Japanese health-insurance claims dataset that covers approximately 2% of the Japanese population. The study included the data of 2233 subjects who experienced either in-hospital or out-of-hospital cardiac arrest between January 2005 and May 2014. These patients were divided into two groups, based on the administration of hydrocortisone. We adjusted the baseline characteristics, medical treatment, and drug administration data of the two groups using propensity scores obtained via the inverse probability of treatment weighted method. The hydrocortisone group had a significantly higher survival discharge rate (13/61 [21.1%] vs. 240/2172 [11.0%], adjusted odds ratio: 4.2, 95% CI: 1.60-10.98, p = 0.004). In addition, the administration of hydrocortisone was independent predictor of survival to discharge (hazard ratio: 4.6, p < 0.001). The results demonstrate a correlation between hydrocortisone administration and the high rates of survival to discharge.
Araki Yumi, Suganami Akiko, Endo Suzu, Masuda Yuta, Keijo Fukushima, Regan W. John, Murayama Toshihiko, Tamura Yutaka and Hiromichi Fujino : PGE1 and E3 show lower efficacies than E2 to -catenin-mediated activity as biased ligands of EP4 prostanoid receptors., FEBS Letters, Vol.591, No.22, 3771-3780, 2017.
(Summary)
The 2-series of prostaglandin E (PGE ) is regarded as a pro-cancer prostanoid, whereas the 1-series (PGE ) and the 3-series (PGE ) are considered to act as anti-cancer prostanoids. In the present study, we provide possible reasons why PGE and PGE , but not PGE , exert anti-cancer effects by focusing on each diverged E-type prostanoid (EP)4 receptor-mediated signaling pathway. PGE , PGE and PGE function as full agonists in terms of G - and G -protein-mediated signaling. However, PGE and PGE function as partial agonists of T-cell factor (TCF)/β-catenin (β-cat)-mediated activity, the well-known cancer-related signaling pathway. Furthermore, pretreatment with PGE or PGE almost completely reduces PGE -induced TCF/β-cat activity. These results provide a plausible reason why PGE and PGE function as anti-cancer prostanoids as a result of novel biased activity for EP4 receptors.
Akiko Suganami, Hiromichi Fujino, Iori Okura, Naoki Yanagisawa, Hajime Sugiyama, W John Regan, Yutaka Tamura and Toshihiko Murayama : Human DP and EP2 prostanoid receptors take on distinct forms depending on the diverse binding of different ligands., The FEBS Journal, Vol.283, No.21, 3931-3940, 2016.
(Summary)
Human D-type prostanoid (DP) and E-type prostanoid 2 (EP2) receptors are G protein-coupled receptors and are regarded as the most closely related receptors among prostanoid receptors because they are generated by tandem duplication. The DP receptor-cognate ligand, prostaglandin D2 (PGD2 ) has the ability to activate not only DP receptors but also EP2 receptors. Likewise, the EP2 receptor-cognate ligand, prostaglandin E2 (PGE2 ) has the ability to activate DP receptors in addition to EP receptors in order to stimulate cAMP formation. However, since PGD2 and/or PGE2 activate DP and EP2 receptors to similar maximal levels, that is, their similar efficacies, differences between the ligands in each receptor have not yet been determined in detail except for their different affinities. Herein we demonstrated, using an in silico simulation to predict binding patterns among DP or EP2 receptors and PGD2 , PGE2 , or prostaglandin F2 as the reference prostanoid, that DP and EP2 receptors plausibly take on distinct forms depending on the diverse binding of different ligands. Since these ligands have the potential to make these receptors form distinct conformations with discrete signaling pathways, they are consequently regarded as endogenous biased ligands. Moreover, by using functional assays, the susceptibilities of the DP receptors to the noncognate ligands were approximately 10 times lower than those of EP2 receptors. Thus, EP2 receptors seem to be able to distinguish endogenous ligands better than DP receptors, thereby both receptors are plausibly gaining role-sharing functions with respect to one another as the copies of duplicated gene.
Hiromichi Fujino, Naofumi Seira, Naoki Kurata, Yumi Araki, Hiroyuki Nakamura, W John Regan and Toshihiko Murayama : Prostaglandin E2-stimulated prostanoid EP4 receptors induce prolonged de novo prostaglandin E2 synthesis through biphasic phosphorylation of extracellular signal-regulated kinases mediated by activation of protein kinase A in HCA-7 human colon cancer cells., European Journal of Pharmacology, Vol.768, 149-159, 2015.
(Summary)
Approximately two decades have passed since E-type prostanoid 4 (EP4) receptors were cloned, and the signaling pathways mediated by these receptors have since been implicated in cancer development through the alliance of Gi-protein/phosphatidylinositol 3-kinase (PI3K)/extracellular signal-regulated kinases (ERKs) activation. Although prostanoid EP4 receptors were initially identified as Gs-coupled receptors, the specific/distinctive role(s) of prostanoid EP4 receptor-induced cAMP/protein kinase A (PKA) pathways in cancer development have not yet been elucidated in detail. We previously reported using HCA-7 human colon cancer cells that prostaglandin E2 (PGE2)-stimulated prostanoid EP4 receptors induced cyclooxygenase-2 (COX-2) as an initiating event in development of colon cancer. Moreover, this induction of COX-2 was mediated by transactivation of epidermal growth factor (EGF) receptors. However, direct activation of EGF receptors by EGF also induced similar amounts of COX-2 in this cell line. Thus, the emergence of unique role(s) for prostanoid EP4 receptors is expected by clarifying the different signaling mechanisms between PGE2-stimulated prostanoid EP4 receptors and EGF-stimulated EGF receptors to induce COX-2 and produce PGE2. We here demonstrated that prostanoid EP4 receptor activation by PGE2 in HCA-7 cells led to PKA-dependent re-activation of ERKs, which resulted in prolonged de novo synthesis of PGE2. Although EGF-stimulated EGF receptors in cells also induced COX-2 and the de novo synthesis of PGE2, the activation of this pathway was transient and not mediated by PKA. Therefore, the novel mechanism underlying prolonged de novo synthesis of PGE2 has provided an insight into the importance of prostanoid EP4 receptor-mediated Gs-protein/cAMP/PKA pathway in development of colon cancer.
Satoshi Yamane, Ryouya Nomura, Madoka Yanagihara, Hiroyuki Nakamura, Hiromichi Fujino, Kenjiro Matsumoto, Syunji Horie and Toshihiko Murayama : L-Cysteine/D,L-homocysteine-regulated ileum motility via system L and B°(,+) transporter: Modification by inhibitors of hydrogen sulfide synthesis and dietary treatments., European Journal of Pharmacology, Vol.764, 471-479, 2015.
(Summary)
Previous studies including ours demonstrated that L-cysteine treatments decreased motility in gastrointestinal tissues including the ileum via hydrogen sulfide (H2S), which is formed from sulfur-containing amino acids such as L-cysteine and L-homocysteine. However, the amino acid transport systems involved in L-cysteine/L-homocysteine-induced responses have not yet been elucidated in detail; therefore, we investigated these systems pharmacologically by measuring electrical stimulation (ES)-induced contractions with amino acids in mouse ileum preparations. The treatments with L-cysteine and D,L-homocysteine inhibited ES-induced contractions in ileum preparations from fasted mice, and these responses were decreased by the treatment with 2-aminobicyclo[2.2.1]heptane-2-carboxylate (BCH), an inhibitor of systems L and B°(,+). The results obtained using ileum preparations and a model cell line (PC12 cells) with various amino acids and BCH showed that not only L-cysteine, but also aminooxyacetic acid and D,L-propargylglycine, which act as H2S synthesis inhibitors, appeared to be taken up by these preparations/cells in L and B°(,+) system-dependent manners. The L-cysteine and D,L-homocysteine responses were delayed and abolished, respectively, in ileum preparations from fed mice. Our results suggested that the regulation of ileum motility by L-cysteine and D,L-homocysteine was dependent on BCH-sensitive systems, and varied depending on feeding in mice. Therefore, the effects of aminooxyacetic acid and D,L-propargylglycine on transport systems need to be considered in pharmacological analyses.
(Keyword)
Amino Acid Transport System y+L / Amino Acid Transport Systems, Neutral / Amino Acids, Cyclic / Animals / Cysteine / Diet / Eating / Electric Stimulation / Fasting / Gastrointestinal Motility / Homocysteine / Hydrogen Sulfide / Ileum / In Vitro Techniques / Male / Mice / PC12 Cells / Postprandial Period / Rats / Time Factors
Sho Otake, Kenji Yoshida, Naofumi Seira, M Christopher Sanchez, W John Regan, Hiromichi Fujino and Toshihiko Murayama : Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1 in HCA-7 human colon cancer cells., Pharmacology Research & Perspectives, Vol.3, No.1, e00083, 2014.
(Summary)
Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1 (HIF-1). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1 and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1 levels increased in a cellular density-dependent manner. The knockdown of HIF-1 by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1 expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1 may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.
Satoshi Yamane, Toshio Kanno, Hiroyuki Nakamura, Hiromichi Fujino and Toshihiko Murayama : Hydrogen sulfide-mediated regulation of contractility in the mouse ileum with electrical stimulation: roles of L-cysteine, cystathionine -synthase, and K+ channels., European Journal of Pharmacology, Vol.740, 112-120, 2014.
(Summary)
Hydrogen sulfide (H2S) is considered to be a signaling molecule. The precise mechanisms underlying H2S-related events, including the producing enzymes and target molecules in gastrointestinal tissues, have not been elucidated in detail. We herein examined the involvement of H2S in contractions induced by repeated electrical stimulations (ES). ES-induced contractions were neurotoxin-sensitive and increased by aminooxyacetic acid, an inhibitor of cystathionine -synthase (CBS) and cystathionine -lyase, but not by D,L-propargylglycine, a selective inhibitor of cystathionine -lyase, in an ES trial-dependent manner. ES-induced contractions were markedly decreased in the presence of L-cysteine. This response was inhibited by aminooxyacetic acid and an antioxidant, and accelerated by L-methionine, an activator of CBS. The existence of CBS was confirmed. NaHS transiently inhibited ES- and acetylcholine-induced contractions, and sustainably decreased basal tone for at least 20 min after its addition. The treatment with glibenclamide, an ATP-sensitive K+ channel blocker, reduced both the L-cysteine response and NaHS-induced inhibition of contractions. The NaHS-induced decrease in basal tone was inhibited by apamin, a small conductance Ca2+-activated K+ channel blocker. These results suggest that H2S may be endogenously produced via CBS in ES-activated enteric neurons, and regulates contractility via multiple K+ channels in the ileum.
T Hosoya, K Matsumoto, K Tashima, H Nakamura, Hiromichi Fujino, T Murayama and S Horie : TRPM8 has a key role in experimental colitis-induced visceral hyperalgesia in mice., Neurogastroenterology and Motility, Vol.26, No.8, 1112-1121, 2014.
(Summary)
Increased expression of TRPM8 may contribute to the visceral hyperalgesia of experimental colitis.
Satomi Oyama, Hiromichi Fujino, Risa Yamazaki, Iori Okura, W John Regan, Atsuko Awata, Takayoshi Arai and Toshihiko Murayama : A novel indole compound, AWT-489, inhibits prostaglandin D2-induced CD55 expression by acting on DP prostanoid receptors as an antagonist in LS174T human colon cancer cells., Archives of Biochemistry and Biophysics, Vol.541, 21-29, 2013.
(Summary)
Indoles are composed of a common core structure, the indole ring, and are widely used as pharmaceuticals and their precursors. In this study, a newly composed relatively small indole compound, AWT-489 was examined to find a novel specific antagonist for DP receptors; the cognate receptors for prostaglandin D2 (PGD2), to prevent colon cancer malignancy. Here we showed that AWT-489 antagonized DP receptor-mediated cyclic AMP formation, and expression of CD55, an inhibitor of the complement system that correlates with poor survival in patients with colorectal cancer, in LS174T human colon cancer cells. Interestingly, unlike a popular indole compound, indomethacin, AWT-489 did not act on the cyclooxygenases as a non-steroidal anti-inflammatory drug. Moreover, AWT-489 exhibited a better inhibitory effect than that of the well-used DP receptor antagonist, BWA868C when a dose close to the physiological concentration of PGD2 was used. These results suggest that AWT-489 can act as a novel human DP receptor antagonist to reduce the expression of CD55 in LS174T human colon cancer cells. We believe that AWT-489 has potential as a lead compound for designing a new DP receptor antagonist that may help improve PGD2-related diseases, especially colon cancer in the near future.
Ryouya Nomura, Madoka Yanagihara, Hiromi Sato, Kenjiro Matsumoto, Kimihito Tashima, Shunji Horie, Shuonan Chen, Hiromichi Fujino, Kouichi Ueno and Toshihiko Murayama : Bee venom phospholipase A2-induced phasic contractions in mouse rectum: independent roles of eicosanoid and gap junction proteins and their loss in experimental colitis., European Journal of Pharmacology, Vol.718, No.1-3, 314-322, 2013.
(Summary)
Various events including digestion and inflammation are regulated by secreted phospholipase A2 (sPLA2) in gastrointestinal tissues, however, the role of sPLA2 on contractile activity has not been elucidated. We investigated the effect of bee venom PLA2 (bvPLA2), which is homologous to the central domain of group III sPLA2, on contractile activity in mouse rectum. The longitudinal preparations of rectum showed rhythmic phasic contractions (RPCs) with varied amplitude and high frequency. Treatment with bvPLA2 at 1 g/ml increased amplitudes of RPCs without marked changes in frequency and basal tone. RPCs by bvPLA2 were affected neither by atropine nor by inhibition of nitric oxide synthase, and partly inhibited by dual inhibition of the cyclooxygenase and lipoxygenase pathways. Pretreatment of bvPLA2 with dithiothreitol, which inhibits the enzyme activity, partly reduced bvPLA2-induced RPCs, and arachidonic acid-increased RPCs were completely abolished by cyclooxygenase/lipoxygenase inhibition. Phasic contractions have been shown to be regulated by gap junction and to be decreased in gastrointestinal tissues with experimental colitis. Treatment with inhibitors of gap junction proteins, 50 M 18-glycyrrhetinic acid and 100 M carbenoxolone, partly and almost completely reduced bvPLA2-induced RPCs without and with the cyclooxygenase/lipoxygenase inhibitors, respectively, but not arachidonic acid-induced RPCs. In rectum from mouse having colitis, where total levels and modified forms of connexin43 increased, bvPLA2-induced RPCs were markedly decreased. Our results suggest that both arachidonic acid metabolism and gap junction proteins independently regulated the sPLA2-induced RPCs in mouse rectum. An increased expression and/or modification of connexin43 may influence sPLA2-induced RPCs in rectum with colitis.
Kenji Yoshida, Hiromichi Fujino, Sho Otake, Naofumi Seira, W John Regan and Toshihiko Murayama : Induction of cyclooxygenase-2 expression by prostaglandin E2 stimulation of the prostanoid EP4 receptor via coupling to Gi and transactivation of the epidermal growth factor receptor in HCA-7 human colon cancer cells., European Journal of Pharmacology, Vol.718, No.1-3, 408-417, 2013.
(Summary)
Increased expressions of cyclooxygenase-2 (COX-2) and its downstream metabolite, prostaglandin E2 (PGE2), are well documented events in the development of colorectal cancer. Interestingly, PGE2 itself can induce the expression of COX-2 thereby creating the potential for positive feedback. Although evidence for such a positive feedback has been previously described, the specific E-type prostanoid (EP) receptor subtype that mediates this response, as well as the relevant signaling pathways, remain unclear. We now report that the PGE2 stimulated induction of COX-2 expression in human colon cancer HCA-7 cells is mediated by activation of the prostanoid EP4 receptor subtype and is followed by coupling of the receptor to Gi and the activation of phosphatidylinositol 3-kinase. Subsequent activation of metalloproteinases releases membrane bound heparin-binding epidermal growth factor-like growth factor resulting in the transactivation of epidermal growth factor receptors and the activation of the extracellular signal-regulated kinases and induction of COX-2 expression. This induction of COX-2 expression by PGE2 stimulation of the prostanoid EP4 receptor may underlie the upregulation of COX-2 during colorectal cancer and appears to be an early event in the process of tumorigenesis.
Tatsuo Kawashima, Risa Yamazaki, Yasuo Matsuzawa, Erika Yamaura, Mamoru Takabatake, Sho Otake, Yuta Ikawa, Hiroyuki Nakamura, Hiromichi Fujino and Toshihiko Murayama : Contrary effects of sphingosine-1-phosphate on expression of -smooth muscle actin in transforming growth factor 1-stimulated lung fibroblasts., European Journal of Pharmacology, Vol.696, No.1-3, 120-129, 2012.
(Summary)
Transforming growth factor-1 (TGF1) plays a pivotal role in fibrosis in various organs including the lung. Following pulmonary injury, TGF1 stimulates conversion of fibroblasts to myofibroblasts that are mainly characterized by up-regulation of -smooth muscle actin (SMA) expression, and the resulting excess production of extracellular matrix proteins causes fibrosis with loss of alveolar function. The present study was undertaken to define the role of the sphingosine-1-phosphate (S1P) pathway in TGF1-induced expression of SMA in human fetal lung fibroblasts, HFL1 cells. Analysis of mRNA revealed the existence of S1P(1), S1P(2), and S1P(3) receptor mRNAs. Treatment with TGF1 increased sphingosine kinase (SphK) activity and S1P(3) receptor mRNA at 24h after stimulation, and pharmacological data showed the involvement of sphingomyelinase, SphK, and S1P(3) receptor in the TGF1-induced up-regulation of SMA with and without serum. Treatment with pertussis toxin and S1P(1) receptor antagonist W146 enhanced SMA expression by TGF1/serum, and S1P decreased and increased SMA levels with and without serum, respectively. TGF1 increased cyclooxygenase-2 expression in a manner dependent on serum and the sphingomyelinase/SphK pathway, and the response was decreased by pertussis toxin. Prostaglandin E(2), formed by TGF1/serum stimulation, decreased the TGF1-induced expression of SMA via EP prostanoid receptor. These data suggest that S1P formed by TGF1 stimulation has diverse effects on the expression of SMA, inhibition via the S1P(1) receptor-mediated and serum-dependent expression of cyclooxygenase-2 and the resulting formation of prostaglandin E(2), and stimulation via the S1P(3) receptor in a serum-independent manner.
Hiroyuki Nakamura, Kana Yasufuku, Tomohiko Makiyama, Ikiru Matsumoto, Hiromichi Fujino and Toshihiko Murayama : Arachidonic acid metabolism via cytosolic phospholipase A2 induces cytotoxicity in niemann-pick disease type C cells., Journal of Cellular Physiology, Vol.227, No.7, 2847-2855, 2012.
(Summary)
Niemann-Pick disease type C (NPC) is a neurodegenerative lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids. Here, we report that NPC1-deficient Chinese hamster ovary cells exhibit increased release of arachidonic acid (AA) and synthesis of prostaglandin E(2) compared with wild-type cells. The enhanced release of AA was inhibited by both treatment with the selective inhibitor of cytosolic phospholipase A(2) (cPLA(2) ) and cultivation in lipoprotein-deficient medium. There was no difference in the expression of both cyclooxygenase-1 and -2 between NPC cells and wild-type cells. U18666A, a cholesterol transport-inhibiting agent commonly used to mimic NPC, also increased the release of AA in L929 mouse fibrosarcoma cells. Furthermore, U18666A-induced formation of reactive oxygen species (ROS) resulted in the induction of cell death and cell cycle delay/arrest in L929 cells. Interestingly, these responses induced by U18666A were much weaker in cPLA(2) knockdown L929 cells. These results suggest that cPLA(2) -AA pathway plays important roles in the cytotoxicity and the ROS formation in NPC cells.
(Keyword)
Androstenes / Animals / Arachidonic Acid / CHO Cells / Carrier Proteins / Cell Cycle Checkpoints / Cell Death / Cholesterol / Cricetinae / Cyclooxygenase 1 / Cyclooxygenase 2 / Cytosol / Dinoprostone / Fibrosarcoma / Group IV Phospholipases A2 / Membrane Glycoproteins / Mice / Niemann-Pick Disease, Type C / Reactive Oxygen Species / Tumor Cells, Cultured
Yuta Ikawa, Hiromichi Fujino, Sho Otake and Toshihiko Murayama : Indomethacin antagonizes EP(2) prostanoid receptor activation in LS174T human colon cancer cells., European Journal of Pharmacology, Vol.680, No.1-3, 16-21, 2012.
(Summary)
Increases in the level of cyclooxygenase (COX)-2 and prostanoids such as prostaglandin E(2) (PGE(2)) are considered biomarkers of colorectal cancer. Therefore, non-steroidal anti-inflammatory drugs (NSAID) have been used to reduce the risk of cancer development by reducing prostanoid biosynthesis as COX inhibitors. Along with their activity as COX inhibitors, NSAID have been reported to have other effects. One major NSAID, indomethacin, has been shown to have several effects independent of COX inhibition. To further examine the COX-inhibition-independent effects of indomethacin on colorectal cancer, we used human colon cancer LS174T cells, known to have express little COX-2 and have no detectable PGE(2) production. Here we show that indomethacin has a potential antagonizing effect on human EP(2) receptors. We believe this study raises the reasons to use indomethacin as a lead-compound for setting up another EP(2) receptor-specific antagonist as a relatively cost-efficient strategy for anti-cancer medication in the future.
Koichi Kawada, Masayuki Kaneko, Yasuyuki Nomura, Seisuke Mimori, Hiroshi Hamana, Kiyokazu Ogita, Toshihiko Murayama, Hiromichi Fujino and Yasunobu Okuma : Expression of the ubiquitin ligase HRD1 in neural stem/progenitor cells of the adult mouse brain., Journal of Pharmacological Sciences, Vol.117, No.3, 208-212, 2011.
(Summary)
Neural stem/progenitor cells (NSCs) reside in the subventricular zone (SVZ) and subgranular zone of the hippocampal dentate gyrus in adult mammals. The ubiquitin ligase HRD1 is associated with degradation of amyloid precursor protein and believed to be specifically expressed in neurons and not in astrocytes. We investigated expression of HRD1 using immunohistochemistry and found colocalization of HRD1 with the NSC marker protein nestin and glial fibrillary acidic protein in the NSCs of the SVZ (the SVZ astrocytes) but not in the hippocampus. In the hippocampal dentate gyrus, HRD1 is localized in the nucleus of nestin-positive cells.
Yuri Hamada, Erina Kato, Hiroyuki Nakamura, Hiromichi Fujino, Kenjiro Matsumoto, Kimihito Tashima, Shunji Horie and Toshihiko Murayama : Decrease of guanylyl cyclase 1 subunit and nitric oxide (NO)-induced relaxation in mouse rectum with colitis and its reproduction on long-term NO treatment., Naunyn-Schmiedeberg's Archives of Pharmacology, Vol.385, No.1, 81-94, 2011.
(Summary)
Nitric oxide (NO) influences motility in the colon in patients with ulcerative colitis, but the exact mechanism involved remains unknown. Colitis was induced in mice by the oral administration of 2.5% dextran sodium sulfate (DSS), and the motility in longitudinal preparations from rectum and distal colon and expression of β1 subunit of soluble guanylyl cyclase (sGCβ1) were analyzed. Electrical stimulation (ES) caused a transient relaxation via the NO pathway in both rectum and colon from control mice. Stimulation with sodium nitroprusside (SNP) caused relaxation in the two regions, and the half-time (T (1/2)) of the maximal relaxation induced by 100 μM SNP was 8.1 ± 1.0 s in rectum. DSS treatment (1) abolished the ES-induced relaxation, but not dibutyryl cyclic GMP-induced response, in both regions, (2) decreased the maximal response to SNP accompanied by a loss of immunoreactive sGCβ1 protein in rectum, but did not affect the amplitude of the relaxant response or the protein in distal colon, and (3) caused an increase in the T (1/2) value in response to SNP in both regions. Pretreatment of both preparations from control mice with 600 μM SNP for 30 min decreased both ES- and SNP-induced relaxation, SNP-induced cyclic GMP formation, and immunoreactive sGCβ1 levels. NO-mediated relaxation was impaired by a dysfunctional sGC with and without a loss of immunoreactivity to sGCβ1 in rectum and colon from DSS-treated mice, respectively. Long-term exposure of the tissues with an excess amount of NO changes the sGC-mediated relaxation.
Erina Kato, Satoshi Yamane, Ryoya Nomura, Kenjiro Matsumoto, Kimihito Tashima, Shunji Horie, Takeshi Saito, Hiromichi Fujino and Toshihiko Murayama : Dysfunction of neurogenic VIP-mediated relaxation in mouse distal colon with dextran sulfate sodium-induced colitis., Pharmacological Research, Vol.65, No.2, 204-212, 2011.
(Summary)
Vasoactive intestinal peptide (VIP) regulates various functions including motility and immune homeostasis in colon. The VIP system including its receptors has been established to control the development of ulcerative colitis, but the functional changes of the system-regulated motility in colon with ulcerative colitis are not well understood. In this study, we investigated VIP-related contractile responses in distal colon from mice with dextran sulfate sodium (DSS)-induced acute colitis. Electrical stimulation (ES) under our conditions caused relaxation during ES and contraction after withdrawal of ES in a tetrodotoxin-sensitive manner. Pharmacological analyses showed two phases of ES-induced relaxation: a transient neuronal nitric oxide (NO) synthase-dependent phase (I), and a continued VIP receptor-mediated phase (II). Inhibition of VIP receptors and protein kinase A decreased both phases. In colon from DSS-treated mice, ES-induced phase II (also phase I) and VIP-induced, but not cyclic AMP analog-induced, relaxation were decreased. Stimulation with VIP significantly increased cyclic AMP formation in colon preparations from control but not DSS-treated mice. In colon from DSS-treated mice, the basal cyclic AMP level was markedly greater without changes in the level of VIP receptor VPAC(2). Isoprenaline- and forskolin-induced relaxation and cyclic AMP formation were not changed by DSS treatment. These findings suggest that dysfunction of VIP receptors in muscles, in addition to loss of the neuronal VIP and NO pathways, are involved in abnormal motility in mouse colon with DSS-induced colitis.
Tomoko Kato, Hiromichi Fujino, Satomi Oyama, Tatsuo Kawashima and Toshihiko Murayama : Indomethacin induces cellular morphological change and migration via epithelial-mesenchymal transition in A549 human lung cancer cells: a novel cyclooxygenase-inhibition-independent effect., Biochemical Pharmacology, Vol.82, No.11, 1781-1791, 2011.
(Summary)
Levels of cyclooxygenase (COX)-2 and its metabolite prostaglandin E(2) (PGE(2)) are frequently increased in colon cancer and other cancers including lung cancer. Non-steroidal anti-inflammatory drugs are considered to have chemo-preventive effects on these diseases by reducing the biosynthesis of PGE(2) via their inhibition of COX-2. Although the COX-2/PGE(2) pathway may directly impact on lung carcinogenesis, some population-based cohort studies of NSAIDs showed no significant protective effects. In this study, using human non-small-cell lung cancer A549 cells, we examined the effects of indomethacin, a potent NSAID, on the growth and motility of lung cancer cells. Besides inhibiting PGE(2) production and cellular growth, indomethacin caused drastic morphological changes with a loss of stress fibers in a time- and dose-dependent manner. Interestingly, the change in cellular shape caused by indomethacin was not seen when the cells were treated with aspirin or diclofenac, two other NSAIDs, despite the concentrations used being sufficient to inhibit PGE(2) production. The indomethacin-induced morphological changes in A549 cells were accompanied by a reduction in levels of the adhesion molecule E-cadherin and a component of basal lamina, collagen IV, as well as an increase in the activity of a collagenase, matrix metalloprotease-9. Furthermore, indomethacin-induced shape changes resulted in enhanced motility via regulation of peroxisome proliferator-activated receptor . The dual effects of indomethacin, inhibition of cellular growth and enhancement of migration, would explain, to some extent, the difficulty in using this NSAID for lung cancer therapy.
Yasuo Matsuzawa, Tatsuo Kawashima, Risa Yamazaki, Erika Yamaura, Tomohiko Makiyama, Hiromichi Fujino and Toshihiko Murayama : Inhibitory effects of clinical reagents having anti-oxidative activity on transforming growth factor-1-induced expression of -smooth muscle actin in human fetal lung fibroblasts., The Journal of Toxicological Sciences, Vol.36, No.6, 733-740, 2011.
(Summary)
Excessive production of transforming growth factor-1 (TGF1) plays an important role in lung fibrosis, in which the differentiation of fibroblasts into myofibroblasts is a key process. Increased formation of reactive oxygen species (ROS) induced by TGF1 is a common pathological feature of fibrosis. In the present study, probucol and lovastatin, which are therapeutics used for hyperlipidemia and proposed to act as anti-oxidants, were examined in terms of their effect on TGF1-induced formation of ROS and expression of -smooth muscle actin (SMA), a myofibroblast marker, in human fetal lung fibroblasts (HFL1 cells). The effects of anti-oxidative enzymes and reagents including N-acetyl-L-cysteine, -tocopherol, and lecithinized-superoxide dismutase (SOD) on TGF1-induced expression of SMA were also examined. Treatment with probucol (30 µM) and lovastatin (1 µM and 3 µM), in addition to lecithinized-SOD, significantly inhibited the TGF1-induced formation of ROS and SMA. Other anti-oxidants including N-acetyl-L-cysteine had marginal effects on SMA expression under the conditions. Probucol and lovastatin, established therapeutics, may be worth trying in patients with both lung fibrosis and hypercholesterolemia.
Hiromichi Fujino, Kaori Toyomura, Xiao-bo Chen, W John Regan and Toshihiko Murayama : Prostaglandin E2 regulates cellular migration via induction of vascular endothelial growth factor receptor-1 in HCA-7 human colon cancer cells., Biochemical Pharmacology, Vol.81, No.3, 379-387, 2010.
(Summary)
An important event in the development of tumors is angiogenesis, or the formation of new blood vessels. Angiogenesis is also known to be involved in tumor cell metastasis and is dependent upon the activity of the vascular endothelial growth factor (VEGF) signaling pathway. Studies of mice in which the EP3 prostanoid receptors have been genetically deleted have shown a role for these receptors in cancer growth and angiogenesis. In the present study, human colon cancer HCA-7 cells were used as a model system to understand the potential role of EP3 receptors in tumor cell migration. We now show that stimulation of HCA-7 cells with PGE₂ enhanced the up-regulation of VEGF receptor-1 (VEGFR-1) expression by a mechanism involving EP3 receptor-mediated activation of phosphatidylinositol 3-kinase and the extracellular signal-regulated kinases. Moreover, the PGE₂ stimulated increase in VEGFR-1 expression was accompanied by an increase in the cellular migration of HCA-7 cells. Given the known involvement of VEGFR-1 in cellular migration, our results suggest that EP3 receptors may contribute to tumor cell metastasis by increasing cellular migration through the up-regulation of VEGFR-1 signaling.
Takashi Orido, Hiromichi Fujino, Tatsuo Kawashima and Toshihiko Murayama : Decrease in uptake of arachidonic acid by indomethacin in LS174T human colon cancer cells; a novel cyclooxygenase-2-inhibition-independent effect., Archives of Biochemistry and Biophysics, Vol.494, No.1, 78-85, 2009.
(Summary)
Nonsteroidal anti-inflammatory drugs (NSAIDs) have chemopreventive activity and may be suitable for treatment of colorectal cancer. A popular and potent NSAID, indomethacin, is known to cause serious side-effects, for this reason its therapeutic usefulness is limited. However, these side-effects are likely to be attributed to the additional effects of indomethacin besides its cyclooxygenase inhibition. In this study, we examined the effect of indomethacin on arachidonic acid uptake using LS174T human colon cancer cells. We here show that treatment of LS174T cells with indomethacin reduced arachidonic acid uptake as well as reduced expressions of fatty acid translocase/CD36 and peroxisome proliferators-activated receptor gamma. Since arachidonic acid is a major substrate of inflammatory mediators such as prostaglandins and leukotrienes, we believe this novel effect of indomethacin may apply to new treatment strategies that aim to suppress these mediators by decreasing the uptake of their substrates, which would eventually inhibit colorectal cancer malignancy.
Yasuo Matsuzawa, Yoshiaki Kiuchi, Kaori Toyomura, Ikiru Matsumoto, Hiroyuki Nakamura, Hiromichi Fujino, Toshihiko Murayama and Tatsuo Kawashima : Activation of cytosolic phospholipase A2alpha by epidermal growth factor (EGF) and phorbol ester in HeLa cells: different effects of inhibitors for EGF receptor, protein kinase C, Src, and C-Raf., Journal of Pharmacological Sciences, Vol.111, No.2, 182-192, 2009.
(Summary)
In several types of cancer cells, prostaglandins produced via the over-expression of epidermal growth factor receptor (EGFR) and cyclooxygenases regulate cell growth. We investigated the signaling mechanisms for the release of arachidonic acid (AA, a precursor for prostaglandins) in human cervical carcinoma HeLa cells. Treatment with EGF and 4beta-phorbol 12-myristate 13-acetate (PMA) with A23187 released AA accompanied by the phosphorylation of extracellular signal-regulated kinases (ERK1/2). Pharmacological experiments showed that the responses (ERK phosphorylation and AA release) induced by EGF and PMA were mediated by a mitogen-activated protein kinase/ERK kinase (MEK)-ERK-alpha-type cytosolic phospholipase A(2) (cPLA(2)alpha) pathway and that EGFR couples with the pathway in a manner insensitive to sorafenib, an inhibitor of B- and C-Raf, enzymes upstream of MEK. Activation of protein kinase C by PMA couples with the pathway partly in a sorafenib-sensitive and probably C-Raf-mediated manner and partly in a family of Src tyrosine kinases (Src)-dependent and sorafenib-insensitive manner. Co-treatment with sorafenib and an inhibitor of Src family members additionally inhibited the PMA-induced release of AA. Cross-talk between EGFR and protein kinase C was not observed. In human lung carcinoma A549 cells, the release of AA by EGF was insensitive to sorafenib. Possible mechanisms for the sorafenib-insensitive activation of the MEK-ERK-cPLA(2)alpha pathway are discussed.
(Keyword)
Benzenesulfonates / Cytosol / Enzyme Activation / Enzyme Inhibitors / Epidermal Growth Factor / Group IV Phospholipases A2 / HeLa Cells / Humans / Mitogen-Activated Protein Kinase 1 / Mitogen-Activated Protein Kinase 3 / Models, Biological / Niacinamide / Phenylurea Compounds / Phosphorylation / Protein Kinase C / Protein Kinase Inhibitors / Proto-Oncogene Proteins c-raf / Pyridines / Receptor, Epidermal Growth Factor / Signal Transduction / Temperature / Tetradecanoylphorbol Acetate / Time Factors / src-Family Kinases
Yuri Hamada, Ikuo Murakami, Erina Kato, Satoshi Yamane, Hiromichi Fujino, Kenjiro Matsumoto, Kimihito Tashima, Shunji Horie and Toshihiko Murayama : Neurogenic contraction of mouse rectum via the cyclooxygenase pathway: Changes of PGE2-induced contraction with dextran sulfate sodium-induced colitis., Pharmacological Research, Vol.61, No.1, 48-57, 2009.
(Summary)
Recent reports suggest that cyclooxygenases (COXs) including COX-2 are constitutively expressed, and prostaglandins (PGs) regulate motility and/or contraction in the colon and rectum. This study examines the role of COXs in the regulation of neuromuscular function in longitudinal preparations of isolated rectum and distal colon (Side A, close to the transverse colon; and Side B, close to the rectum) in normal mice and after the induction of colitis by dextran sulfate sodium (DSS). In control rectum, electrical stimulation (ES)-induced contractions were inhibited by atropine and by COX inhibitors, in an independent manner. PGE(2) at 3microM caused a marked contraction, but the secondary response at 20min after the first application was 60% desensitized. In rectum from DSS-treated mice, spontaneous and ES-induced contractions were significantly less intense than in the control preparations, and the response to PGE(2) was abolished but that to 3microM acetylcholine was not. In control distal colon, the responses to PGE(2) in neither side were desensitized by the repeated application. In DSS-treated distal colon, PGE(2) response was impaired in the two regions, and was desensitized on Side B more than Side A. DSS treatment impaired contractions by 40mM KCl in rectum and on Side B but not Side A. DSS treatment increased COX-2 expression in rectum, but not in distal colon. These findings suggest that the induction of colitis by DSS affects ES- and PGE(2)-regulated motility in the order rectum>distal colon close to the rectum>distal colon in mice.
Takeshi Kurosawa, Hiroyuki Nakamura, Erika Yamaura, Hiromichi Fujino, Yasuo Matsuzawa, Tatsuo Kawashima and Toshihiko Murayama : Cytotoxicity induced by inhibition of thioredoxin reductases via multiple signaling pathways: role of cytosolic phospholipase A(2)alpha-dependent and -independent release of arachidonic acid., Journal of Cellular Physiology, Vol.219, No.3, 606-616, 2009.
(Summary)
The thioredoxin (Trx) system, comprising Trx, the selenoprotein thioredoxin reductase (TrxR), and NADPH, functions as an antioxidant system. Trx has various biological activities including growth control and anti-apoptotic properties, and the Trx system offers a target for the development of drugs to treat and/or prevent cancer. We evaluated the role of TrxR inhibition in the release of arachidonic acid (AA), cell toxicity, and intracellular signaling pathways in L929 mouse fibrosarcoma cells. Treatment with 1-chloro-2,4-dinitrobenzene (DNCB, an inhibitor of TrxR) under conditions involving limited inhibition of TrxR activity in cells, released AA before causing cytotoxicity. Treatment with an inhibitor of p38 kinase, a downstream enzyme of the apoptosis signal-regulating kinase 1 pathway, and pyrrophenone (an inhibitor of alpha-type cytosolic phospholipase A(2), cPLA(2)alpha) partially but significantly decreased the DNCB-induced release of AA and cell death. The responses were much weaker in cPLA(2)alpha knockdown L929 cells. Exogenously added AA showed cytotoxicity. DNCB increased intracellular reactive oxygen species (ROS) levels, and butylated hydroxyanisole (an antioxidant) reduced DNCB-induced ROS formation and cell toxicity but not the phosphorylation of p38 kinase and release of AA. Auranofin, another inhibitor of TrxR having a different formula, released AA resulting in toxicity in L929 cells. DNCB caused the release of AA and cytotoxicity in A549 human lung carcinoma cells, and caused p38 kinase-dependent toxicity in PC12 rat pheochromocytoma cells. Our data suggest that a dysfunctional Trx system triggers multiple signaling pathways, and that the AA released by cPLA(2)alpha-dependent and -independent pathways is important to cytotoxicity. J. Cell. Physiol. 219: 606-616, 2009. (c) 2009 Wiley-Liss, Inc.
(Keyword)
Animals / Arachidonic Acid / Cell Death / Cell Line, Tumor / Cytosol / Dinitrochlorobenzene / Enzyme Inhibitors / Group IV Phospholipases A2 / Humans / Imidazoles / MAP Kinase Kinase Kinase 5 / MAP Kinase Signaling System / Mice / PC12 Cells / Pyridines / Pyrrolidines / Rats / Reactive Oxygen Species / Signal Transduction / Thioredoxin-Disulfide Reductase / p38 Mitogen-Activated Protein Kinases
Masaya Shimizu, Yuki Muramatsu, Eiko Tada, Takeshi Kurosawa, Erika Yamaura, Hiroyuki Nakamura, Hiromichi Fujino, Yuuya Houjyo, Yuri Miyasaka, Yuuki Koide, Atsushi Nishida and Toshihiko Murayama : Effects of synthetic sphingosine-1-phosphate analogs on cytosolic phospholipase A2alpha-independent release of arachidonic acid and cell toxicity in L929 fibrosarcoma cells: the structure-activity relationship., Journal of Pharmacological Sciences, Vol.109, No.3, 431-443, 2009.
(Summary)
Sphingolipid metabolites including ceramide, sphingosine, and their phosphorylated products [sphingosine-1-phosphate (S1P) and ceramide-1-phosphate] regulate cell functions including arachidonic acid (AA) metabolism and cell death. The development of analogs of S1P may be useful for regulating these mediator-induced cellular responses. We synthesized new analogs of S1P and examined their effects on the release of AA and cell death in L929 mouse fibrosarcoma cells. Among the analogs tested, several compounds including DMB-mC11S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMB-mC9S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-nonyl)phenylpropyl phosphate] released AA within 1 h and caused cell death 6 h after treatment. The release of AA was observed in C12 cells [a L929 variant lacking a type alpha cytosolic phospholipase A(2) (cPLA(2)alpha)] and L929-cPLAalpha-siRNA cells (L929 cells treated with small interference RNA for cPLA(2)alpha). Treatment with pharmacological inhibitors of secretory and Ca(2+)-independent PLA(2)s decreased the DMB-mC11S-induced release of AA. The effect of the S1P analogs tested on the release of AA was comparable to that on cell death in L929 cells, and a high correlation coefficient was observed. Two analogs lacking a butoxycarbonyl moiety [DMAc-mC11S (dimethyl (2S,3R)-2-acetamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMAm-mC11S [dimethyl (2S,3R)-2-amino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate)] had inhibitory effects on the release of AA and cell toxicity induced by DMB-mC11S. Synthetic phosphorylated lipid analogs may be useful for studying PLA(2) activity and its toxicity in cells. [Supplementary Fig. 1: available only at http://dx.doi.org/10.1254/jphs.08284FP].
(Keyword)
Animals / Arachidonic Acid / Cell Death / Cell Line, Tumor / Fibrosarcoma / Group IV Phospholipases A2 / Lysophospholipids / Mice / RNA, Small Interfering / Sphingosine / Structure-Activity Relationship / Time Factors
Ikuo Murakami, Yuri Hamada, Satoshi Yamane, Hiromichi Fujino, Shunji Horie and Toshihiko Murayama : Nicotine-induced neurogenic relaxation in the mouse colon: changes with dextran sodium sulfate-induced colitis., Journal of Pharmacological Sciences, Vol.109, No.1, 128-138, 2009.
(Summary)
Nicotine has been shown to reduce both tone and muscular activity in the human colon by releasing nitric oxide (NO) from nerves. To our knowledge, however, the effect of nicotine on mouse colon has not been elucidated, and the response in tissue from ulcerative colitis (UC) has not been investigated. We examined nicotine-induced responses in colon from control mice and mice with dextran sodium sulfate (DSS)-induced UC. In controls, bath application of nicotine caused a transient relaxation in longitudinal preparations from the transverse and distal colons but not from the rectum. The response was observed in the presence of bethanechol, abolished by treatment with tetrodotoxin and hexamethonium, and mediated partially (>50%) by the NO pathway. In longitudinal preparations of the distal colon from DSS-treated mice, spontaneous contractions decreased markedly, and nicotine caused contraction without relaxation in half of the preparations tested. Nicotine-induced relaxation in the presence of bethanechol was significantly decreased in the DSS-treated distal colon without changing bethanechol-induced contractions. These data suggest that 1) responses to nicotine differ dependent on colon regions, 2) DSS treatment predominantly caused nicotine-sensitive neurogenic changes in distal colon, and 3) DSS treatment may reverse the direction of nicotine-evoked responses in the colon, in mice.
Masaya Shimizu, Eiko Tada, Tomohiko Makiyama, Kana Yasufuku, Yuta Moriyama, Hiromichi Fujino, Hiroyuki Nakamura and Toshihiko Murayama : Effects of ceramide, ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2alpha; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling., Cellular Signalling, Vol.21, No.3, 440-447, 2008.
(Summary)
Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A(2) (PLA(2)) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca(2+) binding regions in the C2 domain of alpha type cytosolic PLA(2) (cPLA(2)alpha), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA(2)alpha has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4beta-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca(2+) ionophore (A23187) -induced release of AA via cPLA(2)alpha's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca(2+) nor the phosphorylation of cPLA(2)alpha in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA(2)alpha that is independent of the Ca(2+) signal and the PKC-ERK-mediated phosphorylation signal.
(Keyword)
Active Transport, Cell Nucleus / Animals / CHO Cells / Calcium Signaling / Ceramidases / Ceramides / Cricetinae / Cricetulus / Cytosol / Extracellular Signal-Regulated MAP Kinases / Gene Expression Regulation, Enzymologic / Group IV Phospholipases A2 / Humans / Phosphorylation / Phosphotransferases (Alcohol Group Acceptor) / Protein Kinase C / Protein Transport / Up-Regulation
Maiko Nabemoto, Masato Mashimo, Akiyoshi Someya, Hiroyuki Nakamura, Tetsuya Hirabayashi, Hiromichi Fujino, Masayuki Kaneko, Yasunobu Okuma, Takeshi Saito, Naoto Yamaguchi and Toshihiko Murayama : Release of arachidonic acid by 2-arachidonoyl glycerol and HU210 in PC12 cells; roles of Src, phospholipase C and cytosolic phospholipase A(2)alpha., European Journal of Pharmacology, Vol.590, No.1-3, 1-11, 2008.
(Summary)
The phospholipase A(2) (PLA(2))-prostanoid cascade is involved in cannabinoid receptor-mediated neuronal functions. We investigated the signaling mechanism for the release of arachidonic acid by cannabinoids, 2-arachidonoyl glycerol (2-AG) and HU210, in rat PC12 cells and in primary cultured cells from the mouse cerebellum. The effect of selective inhibitors for signaling pathways and/or enzymes (alpha type cytosolic PLA(2) (cPLA(2)alpha), G protein, Src kinases, phospholipase C, protein kinase C) was assessed. Methods included translocation of the chimeric protein GFP-cPLA(2)alpha, the activities of Src family kinases, Ca(2+)-dependent fluorescence and cyclic AMP accumulation. Treatment with 2-AG and HU210 at greater concentrations than 3 muM caused the release of arachidonic acid, and the response was inhibited by AM251 (an antagonist of cannabinoid CB(1) receptor) and by pyrrophenone (a selective inhibitor of cPLA(2)alpha) in PC12 cells. The cannabinoid treatment caused the intracellular translocation of cPLA(2)alpha and an increase in the intracellular Ca(2+) level. Treatment with HU210 caused tyrosine phosphorylation of Src and Fyn, and increased their kinase activities. Pretreatment with inhibitors of tyrosine kinases or phospholipase C abolished the cannabinoids-induced release of arachidonic acid and Ca(2+) response, and protein kinase C inhibitor reduced the release of arachidonic acid. 2-AG caused the release of arachidonic acid from cultured cells of the mouse cerebellum via similar mechanisms. These data reveal that cannabinoids activated cPLA(2)alpha in a Src-phospholipase C-protein kinase C-dependent manner probably via cannabinoid CB(1) receptor and/or CB(1)-like receptor in neuronal cells.
(Keyword)
Animals / Arachidonic Acid / Arachidonic Acids / Cyclic AMP / Cytosol / Dronabinol / Endocannabinoids / Glycerides / Group IV Phospholipases A2 / Mice / Mice, Inbred ICR / PC12 Cells / Phosphorylation / Piperidines / Protein Kinase C / Proto-Oncogene Proteins c-fyn / Pyrazoles / Rats / Receptor, Cannabinoid, CB1 / Signal Transduction / Type C Phospholipases / src-Family Kinases
Wei Xu, Chih-Ling Chou, Haipeng Sun, Hiromichi Fujino, M Qin Chen and W John Regan : FP prostanoid receptor-mediated induction of the expression of early growth response factor-1 by activation of a Ras/Raf/mitogen-activated protein kinase signaling cascade., Molecular Pharmacology, Vol.73, No.1, 111-118, 2007.
(Summary)
FP prostanoid receptors are G-protein-coupled receptors whose physiological activator is prostaglandin-F(2alpha) (PGF(2alpha)). PGF(2alpha) has been implicated in wound healing and cardiac hypertrophy, which are both known to involve the induction of the immediate-early response gene, early growth response factor-1 (EGR-1). We hypothesized that activation of the human FP receptor by PGF(2alpha) could induce the expression of EGR-1 and found that 1 muM PGF(2alpha) produced a time-dependent induction of both mRNA and protein expression for EGR-1. This FP receptor-mediated induction of EGR-1 expression involved activation of the small GTPase Ras followed by activation of C-Raf and the mitogen-activated protein (MAP) kinase kinases 1 and 2 (MEK1/2). Thus, induction of EGR-1 expression by PGF(2alpha) was blocked using dominant-negative constructs of Ras and C-Raf and the Raf kinase inhibitor 4-(4-(3-(4-chloro-3-trifluoromethylphenyl)ureido)phenoxy)-pyridine-2-carboxyllic acid methyamide-4-methylbenzenesulfonate (BAY43-9006). Likewise, the MEK1/2 inhibitor 2'-amino-3'-methoxyflavone (PD98059) blocked the induction of EGR-1 expression by PGF(2alpha). FP receptor stimulation by PGF(2alpha) induced the phosphorylation of C-Raf, MEK1/2, and extracellular signal-regulated kinases 1 and 2, consistent with the activation of a MAP kinase signaling cascade. PGF(2alpha) was also found to induce the expression of EGR-1 in rat cardiomyocytes through the activation of endogenous FP receptors. This induction of EGR-1 expression in cardiomyocytes also involved the activation of Raf and MAP kinase signaling and was dependent on the activation of protein kinase C. This is the first report to show the regulation of EGR-1 expression after PGF(2alpha) activation of FP receptors and suggests that this could be an early event involved in wound healing and cardiac hypertrophy.
(Keyword)
Early Growth Response Protein 1 / Enzyme Activation / Humans / MAP Kinase Signaling System / Receptors, Prostaglandin / Signal Transduction
Tomoko Taniguchi, Masaya Shimizu, Hiroyuki Nakamura, Tetsuya Hirabayashi, Hiromichi Fujino, Takeshi Saito and Toshihiko Murayama : Vanadate-induced activation of cytosolic phospholipase A2alpha in L929 cells: Roles of tyrosine kinase, protein kinase C, and extracellular signal-regulated kinase., Biochemical Pharmacology, Vol.73, No.6, 854-862, 2006.
(Summary)
Orthovanadate (Na3VO4), which acts as an inhibitor of protein tyrosine phosphatases, has a various pharmacological effects including the release of arachidonic acid (AA) from cells. We investigated roles of alpha-type cytosolic phospholipase A2 (cPLA2alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of AA induced by Na3VO4 from a murine fibroblast cell line, L929. C12 cells, a variant of L929 that lacks expression of cPLA2alpha, were used along with a clone of C12 cells that are stably expressing cPLA2alpha (C12-cPLA2alpha cells). In the presence of a Ca2+ ionophore (10 microM A23187), 5 and 10mM Na3VO4 synergistically stimulated AA release from L929 and C12-cPLA2alpha cells, and to a much lesser extent from control C12 cells. The release of AA by Na3VO4/A23187 was inhibited by a selective cPLA2alpha inhibitor (3 microM pyrrophenone). The release of AA by Na3VO4/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X), in PKC-depleted cells, by a Src inhibitor (2 microM PP2) and by an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (10 microM U0126). The phosphorylation of ERK1/2 was stimulated by Na3VO4, and the response was significantly decreased by inhibitors of Src, PKC and ERK1/2 kinase. Our data show that Na3VO4 stimulates AA release largely via cPLA2alpha activation in Ca2+-dependent manner, and the cross-talk between Src and PKC and the ERK-dependent pathways are involved in Na3VO4-induced AA release from L929 cells.
(Keyword)
Animals / Arachidonic Acid / Calcium / Cells, Cultured / Enzyme Activation / Extracellular Signal-Regulated MAP Kinases / Hydrogen Peroxide / Mice / Phospholipases A / Phospholipases A2 / Protein Kinase C / Protein Transport / Receptor, Epidermal Growth Factor / Signal Transduction / Vanadates / src-Family Kinases
Tomoko Taniguchi, Masaya Shimizu, Hiroyuki Nakamura, Tetsuya Hirabayashi, Hiromichi Fujino and Toshihiko Murayama : Hydrogen peroxide-induced arachidonic acid release in L929 cells; roles of Src, protein kinase C and cytosolic phospholipase A2alpha., European Journal of Pharmacology, Vol.546, No.1-3, 1-10, 2006.
(Summary)
Hydrogen peroxide (H(2)O(2)) stimulates the release of arachidonic acid from cells, but the signaling mechanism(s) involved remains to be elucidated. We investigated the roles of alpha-type cytosolic phospholipase A(2) (cPLA(2)alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of arachidonic acid from L929 cells (a murine fibroblast cell line), C12 cells (a variant of L929 that lacks cPLA(2)alpha) and a stable clone of C12 cells expressing cPLA(2)alpha (C12-cPLA(2)alpha cells). In the presence of 10 muM A23187, 100 nM phorbol myristate acetate (PMA) and 1 mM H(2)O(2) synergistically stimulated arachidonic acid release from L929 cells and C12-cPLA(2)alpha cells, and to a much lesser extent from C12 cells. The reagents alone and co-treatment with PMA and H(2)O(2) without A23187 had marginal effects. No arachidonic acid was released by PMA/A23187 or H(2)O(2)/A23187 in CaCl(2)-free buffer and the release was inhibited by a selective cPLA(2)alpha inhibitor (3 microM pyrrophenone). Addition of 10 microM H(2)O(2), which did not stimulate arachidonic acid release with A23187, enhanced the response to PMA/A23187. The release induced by PMA/A23187 and by H(2)O(2)/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X) and in PKC-depleted cells, and by a Src inhibitor (2 microM PP2). The phosphorylation of extracellular signal-regulated kinase 1/2 induced by PMA/A23187 and H(2)O(2)/A23187 was significantly decreased by inhibitors of PKC and Src. These findings suggest that H(2)O(2) with Ca(2+) stimulates arachidonic acid release via cPLA(2)alpha in a Src- and PKC-dependent manner in L929 cells. The role of cross-talk between Src and PKC in arachidonic acid release is discussed.
(Keyword)
Animals / Arachidonic Acid / Calcimycin / Calcium / Cell Line, Tumor / Cytosol / Dose-Response Relationship, Drug / Enzyme Inhibitors / Fibroblasts / Group IV Phospholipases A2 / Humans / Hydrogen Peroxide / Indoles / Ionophores / Maleimides / Mice / Phospholipases A / Protein Kinase C / Protein Kinase C-alpha / Protein Transport / Pyrimidines / Pyrrolidines / Signal Transduction / Tetradecanoylphorbol Acetate / Transfection / src-Family Kinases
Nobuteru Akiyama, Maiko Nabemoto, Yoshio Hatori, Hiroyuki Nakamura, Tetsuya Hirabayashi, Hiromichi Fujino, Takeshi Saito and Toshihiko Murayama : Up-regulation of cytosolic phospholipase A2alpha expression by N,N-diethyldithiocarbamate in PC12 cells; involvement of reactive oxygen species and nitric oxide., Toxicology and Applied Pharmacology, Vol.215, No.2, 218-227, 2006.
(Summary)
Disulfiram (an alcohol-aversive drug) and related compounds are known to provoke several side effects involving behavioral and neurological complications. N,N-diethyldithiocarbamate (DDC) is considered as one of the main toxic species of disulfiram and acts as an inhibitor of superoxide dismutase. Since arachidonic acid (AA) formation is regulated by reactive oxygen species (ROS) and related to toxicity in neuronal cells, we investigated the effects of DDC on AA release and expression of the alpha type of cytosolic phospholipase A(2) (cPLA(2)alpha) in PC12 cells. Treatment with 80-120 microM DDC that causes a moderate increase in ROS levels without cell toxicity stimulated cPLA(2)alpha mRNA and its protein expression. The expression was mediated by extracellular-signal-regulated kinase (ERK1/2), one of the mitogen-activated protein kinases. Treatment with N(G) nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase, 1 mM) and oxy-hemoglobin (a scavenger of nitric oxide, 2 mg/mL) abolished the DDC-induced responses (ERK1/2 phosphorylation and cPLA(2)alpha expression). We also showed DDC-induced up-regulation of the mRNA expression of lipocortin 1, an inhibitor of PLA(2). Furthermore, DDC treatment of the cells enhanced Ca(2+)-ionophore-induced AA release in 30 min, although the effect was limited. Changes in AA metabolism in DDC-treated cells may have a potential role in mediating neurotoxic actions of disulfiram. In this study, we show the first to demonstrate the up-regulation of cPLA(2)alpha expression by DDC treatment in neuronal cells.
Hiromichi Fujino and W John Regan : EP(4) prostanoid receptor coupling to a pertussis toxin-sensitive inhibitory G protein., Molecular Pharmacology, Vol.69, No.1, 5-10, 2005.
(Summary)
The EP(2) and EP(4) prostanoid receptor subtypes are G-protein-coupled receptors for prostaglandin E(2) (PGE(2)). Both receptor subtypes are known to couple to the stimulatory guanine nucleotide binding protein (Galpha(s)) and, after stimulation with PGE(2), can increase the formation of intracellular cAMP. In addition, PGE(2) stimulation of the EP(4) receptor can activate phosphatidylinositol 3-kinase (PI3K) leading to phosphorylation of the extracellular signal-regulated kinases (ERKs) and induction of early growth response factor-1 (EGR-1). We now report that the PGE(2)-mediated phosphorylation of the ERKs and induction of EGR-1 can be blocked by pretreatment of EP(4)-expressing cells with pertussis toxin (PTX). Furthermore, pretreatment with PTX increased the amount of PGE(2)-stimulated intracellular cAMP formation in EP(4)-expressing cells but not in EP(2)-expressing cells. These data indicate that the EP(4) prostanoid receptor subtype, but not the EP(2), couples to a PTX-sensitive inhibitory G-protein (Galpha(i)) that can inhibit cAMP-dependent signaling and activate PI3K/ERK-dependent signaling.
(Keyword)
Cell Line / Cyclic AMP / Cyclic AMP-Dependent Protein Kinases / Dinoprostone / Extracellular Signal-Regulated MAP Kinases / GTP-Binding Proteins / Humans / Pertussis Toxin / Phosphorylation / Protein Binding / Receptors, Prostaglandin E / Receptors, Prostaglandin E, EP4 Subtype / Signal Transduction
Hiromichi Fujino, Sambhitab Salvi and W John Regan : Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP2 and EP4 prostanoid receptors by prostaglandin E2., Molecular Pharmacology, Vol.68, No.1, 251-259, 2005.
(Summary)
The EP2 and EP4 prostanoid receptors are G-protein-coupled receptors whose activation by their endogenous ligand, prostaglandin (PG) E2, stimulates the formation of intracellular cAMP. We have previously reported that the stimulation of cAMP formation in EP4-expressing cells is significantly less than in EP2-expressing cells, despite nearly identical levels of receptor expression (J Biol Chem 277:2614-2619, 2002). In addition, a component of EP4 receptor signaling, but not of EP2 receptor signaling, was found to involve the activation of phosphatidylinositol 3-kinase (PI3K). In this study, we report that PGE2 stimulation of cells expressing either the EP2 or EP4 receptor results in the phosphorylation of the cAMP response element binding protein (CREB) at serine-133. Pretreatment of cells with N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), an inhibitor of protein kinase A (PKA), attenuated the PGE2-mediated phosphorylation of CREB in EP2-expressing cells, but not in EP4-expressing cells. Pretreatment of cells with wortmannin, an inhibitor of PI3K, had no effects on the PGE2-mediated phosphorylation of CREB in either EP2- or EP4-expressing cells, although it significantly increased the PGE2-mediated activation of PKA in EP4-expressing cells. However, combined pretreatment with H-89 and wortmannin blocked PGE2-mediated phosphorylation in EP2-expressing cells as well as in EP2-expressing cells. PGE2-mediated intracellular cAMP formation was not affected by pretreatment with wortmannin, or combined treatment with wortmannin and H-89, in either the EP2- or EP4-expressing cells. These findings suggest that PGE2 stimulation of EP4 receptors, but not EP2 receptors, results in the activation of a PI3K signaling pathway that inhibits the activity of PKA and that the PGE2-mediated phosphorylation of CREB by these receptors occurs through different signaling pathways
(Keyword)
Androstadienes / Cell Line / Cyclic AMP Response Element-Binding Protein / Dinoprostone / Humans / Phosphorylation / Receptors, Prostaglandin E / Receptors, Prostaglandin E, EP2 Subtype / Receptors, Prostaglandin E, EP4 Subtype
Hiromichi Fujino, A George Vielhauer and W John Regan : Prostaglandin E2 selectively antagonizes prostaglandin F2alpha-stimulated T-cell factor/beta-catenin signaling pathway by the FPB prostanoid receptor., The Journal of Biological Chemistry, Vol.279, No.42, 43386-43391, 2004.
(Summary)
FP prostanoid receptors are G-protein-coupled receptors that consist of two isoforms named FPA and FPB. Both isoforms activate inositol phosphate second messenger signaling pathways by their endogenous ligand prostaglandin F2alpha (PGF2alpha). Previously we have shown that both isoforms undergo Rho-mediated cell rounding following treatment with PGF2alpha. Following the removal of PGF2alpha, however, FPA-expressing cells return to their original morphology, whereas FPB-expressing cells do not. It was also found that PGF2alpha-could activate T-cell factor (Tcf)/beta-catenin signaling in cells expressing the FPB isoform but not in cells expressing the FPA isoform. We now show that prostaglandin E2 (PGE2) can induce cell rounding and stimulate the formation of inositol phosphates to the same extent as PGF2alpha in cells expressing either the FPA or FPB isoforms. However, PGE2 has much lower efficacy as compared with PGF2alpha for the activation of Tcf/beta-catenin signaling in FPB-expressing cells, and the cell rounding is reversible. Interestingly, pretreatment of FPB-expressing cells with PGE2-attenuated PGF2alpha-stimulated Tcf/beta-catenin signaling in a dose-dependent manner while having no effect on PGF2alpha-stimulated inositol phosphates formation. Thus, the ratio of endogenous PGE2 and PGF2alpha has the potential to selectively regulate one signaling pathway over another. This represents a novel mechanism for the regulation of cell signaling that is distinct from regulation occurring at the level of the receptor and its effector pathways.
A George Vielhauer, Hiromichi Fujino and W John Regan : Cloning and localization of hFP(S): a six-transmembrane mRNA splice variant of the human FP prostanoid receptor., Archives of Biochemistry and Biophysics, Vol.421, No.2, 175-185, 2004.
(Summary)
FP prostanoid receptors are G-protein coupled receptors that mediate the actions of prostaglandin F2alpha. Two isoforms, designated FP(A) and FP(B), have been previously described. We now report the cloning of a FP receptor mRNA alternative splice variant from human heart and placenta cDNA, named hFP(S). The cDNA encoding hFP(S) has a 71 bp insert that produces a frame shift resulting in a truncated receptor lacking transmembrane-7 and the intracellular carboxyl tail. This 71 bp sequence has been identified as a distinct exon localized in the human FP receptor gene on chromosome one. Northern blot analysis suggests that hFPs is expressed in skeletal muscle as well as human heart and placenta. Immunohistochemical microscopy showed positive immunoreactivity on vascular endothelial, trophoblast, and decidual cells from human placenta. hFPs represents the first confirmed alternative splice variant of the human FP prostanoid receptor gene, however, its function is presently unknown.
(Keyword)
Alternative Splicing / Amino Acid Sequence / Base Sequence / Computational Biology / Endothelium / Humans / Molecular Sequence Data / Organ Specificity / Placenta / RNA, Messenger / Receptors, Prostaglandin / Reverse Transcriptase Polymerase Chain Reaction / Sequence Analysis, DNA / Umbilical Veins
Hiromichi Fujino, Wei Xu and W John Regan : Prostaglandin E2 induced functional expression of early growth response factor-1 by EP4, but not EP2, prostanoid receptors via the phosphatidylinositol 3-kinase and extracellular signal-regulated kinases., The Journal of Biological Chemistry, Vol.278, No.14, 12151-12156, 2003.
(Summary)
Prostaglandin E(2) (PGE(2)) mediates its physiological effects by interactions with a subfamily of G-protein-coupled receptors known as EP receptors. These receptors consist of four primary subtypes named EP(1), EP(2), EP(3), and EP(4). The EP(2) and EP(4) subtypes are known to couple to Galpha(s) and stimulate intracellular cyclic 3,5- adenosine monophosphate formation, whereas the EP(1) and EP(3) receptors are known to couple to Galpha(q) and Galpha(i), respectively. Recently we found that EP(2) and EP(4) receptors can activate T-cell factor signaling; however, EP(2) receptors did this primarily through a cAMP-dependent protein kinase-dependent pathway, whereas EP(4) receptors primarily utilized a phosphatidylinositol 3-kinase (PI3K)-dependent pathway (Fujino, H., West, K. A., and Regan, J. W. (2002) J. Biol. Chem. 277, 2614-2619). We now report that PGE(2) stimulation of EP(4) receptors, but not EP(2) receptors, leads to phosphorylation of the extracellular signal-regulated kinases (ERKs) through a PI3K-dependent mechanism. Furthermore, this activation of PI3K/ERK signaling by the EP(4) receptors induces the functional expression of early growth response factor-1 (EGR-1). Under the same conditions induction of EGR-1 protein expression was not observed following PGE(2) stimulation of EP(2) receptors. These findings point to important differences in the signaling potential of the EP(2) and EP(4) receptors, which could be significant with respect to the potential involvement of EP(4) receptors in inflammation and cancer.
(Keyword)
Androstadienes / DNA-Binding Proteins / Dinoprostone / Early Growth Response Protein 1 / Enzyme Inhibitors / Gene Expression / Humans / Immediate-Early Proteins / Kidney / MAP Kinase Signaling System / Mitogen-Activated Protein Kinase 10 / Mitogen-Activated Protein Kinases / Phosphatidylinositol 3-Kinases / Phosphorylation / Promoter Regions, Genetic / Protein-Tyrosine Kinases / Receptors, Prostaglandin E / Receptors, Prostaglandin E, EP2 Subtype / Receptors, Prostaglandin E, EP4 Subtype / Transcription Factors / p38 Mitogen-Activated Protein Kinases
Hiromichi Fujino, Dinesh Srinivasan and W John Regan : Cellular conditioning and activation of beta-catenin signaling by the FPB prostanoid receptor., The Journal of Biological Chemistry, Vol.277, No.50, 48786-48795, 2002.
(Summary)
FP prostanoid receptors have been identified as two isoforms named FP(A) and FP(B). We have shown that the FP(B) isoform, but not the FP(A), activates beta-catenin-mediated transcription. We now report that the mechanism of this FP(B)-specific activation of beta-catenin signaling occurs in two steps. The first is a conditioning step that involves an agonist-independent association of the FP(B) receptor with phosphatidylinositol 3-kinase followed by constitutive internalization of a receptor complex containing E-cadherin and beta-catenin. This constitutive internalization conditions the cell for subsequent beta-catenin signaling by increasing the cellular content of cytosolic beta-catenin. The second step involves agonist-dependent activation of Rho followed by cell rounding. Because of the conditioning step, this agonist-dependent step results in a stabilization of beta-catenin and activation of transcription. Although stimulation of the FP(A) isoform activates Rho and induces cellular shape change, it does not activate beta-catenin signaling, because the FP(A) does not undergo constitutive internalization and does not condition the cell for beta-catenin signaling. The cellular conditioning described here for the FP(B) illustrates the potential of the receptor to alter the signaling environment of a cell even in the absence of agonist and has general significance for understanding G-protein-coupled receptor signaling.
Dinesh Srinivasan, Hiromichi Fujino and W John Regan : Differential internalization of the prostaglandin f(2alpha) receptor isoforms: role of protein kinase C and clathrin., The Journal of Pharmacology and Experimental Therapeutics, Vol.302, No.1, 219-224, 2002.
(Summary)
FP prostanoid receptors are G-protein-coupled receptors that mediate the actions of prostaglandin F(2alpha) (PGF(2alpha)). Alternative mRNA splicing gives rise to two isoforms, FP(A) and FP(B), which are identical except for their intracellular carboxyl termini. In this study, we examined the internalization of recombinant FLAG-epitope-tagged FP(A) and FP(B) receptors that were stably expressed in human embryonic kidney-293 cells. Cell surface receptors on live cells were labeled with anti-FLAG antibodies either in the presence or absence of PGF(2alpha) and were examined by immunofluorescence microscopy. In the absence of PGF(2alpha), FP(A)-expressing cells were labeled predominantly on the cell surface; however, FP(B)-expressing cells were labeled on both the cell surface and intracellularly, indicating constitutive internalization of the FP(B) isoform. After treatment with PGF(2alpha), FP(A)-expressing cells were labeled intracellularly, reflecting receptor internalization, which could be mimicked with phorbol 12-myristyl 13-acetate (PMA), an activator of protein kinase C (PKC). Pretreatment of FP(A)-expressing cells with Gö 6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbozole], an inhibitor of PKC, blocked both PGF(2alpha)- and PMA-induced receptor internalization. However, Gö 6976 did not block constitutive internalization of the FP(B) isoform, suggesting that the mechanisms of receptor internalization differ between the FP(A) and FP(B) isoforms. Furthermore, pretreatment with sucrose, an inhibitor of clathrin-dependent internalization, blocked PGF(2alpha)-induced internalization of the FP(A) isoform but did not block constitutive internalization of the FP(B) isoform. In conclusion, the FP(A) receptor isoform shows an agonist-induced internalization involving PKC and clathrin, whereas the FP(B) isoform undergoes agonist-independent internalization that does not involve PKC or clathrin.
L Todd Anthony, Hiromichi Fujino, L Kristen Pierce, J Andrea Yool and W John Regan : Differential regulation of Ca(2+)-dependent Cl- currents by FP prostanoid receptor isoforms in Xenopus oocytes., Biochemical Pharmacology, Vol.63, No.10, 1797-1806, 2002.
(Summary)
The FP(A) and FP(B) prostanoid receptor isoforms are G-protein-coupled receptors that are activated by prostaglandin F(2alpha) (PGF(2alpha)). Differences in their carboxyl termini prompted us to examine the intracellular calcium (Ca(2+)) signaling of these receptor isoforms using the Xenopus oocyte expression system. Protein expression was determined by immunofluorescence microscopy and whole cell binding with [3H]PGF(2alpha). Positive immunolabeling was observed on the outer membranes of oocytes expressing FLAG-tagged FP receptor isoforms, but not on control (water-injected) oocytes. Intracellular signaling was examined using a two-electrode voltage clamp. Specific whole-cell binding was also detected for both receptor isoforms. Bath application of 10 microM PGF(2alpha) to FP(A)-expressing oocytes produced a chloride (Cl-) current response similar to that of an injection of inositol 1,4,5-trisphosphate (InsP(3)) (5.76+/-0.6 microA, peak current; N=23) that returned to control levels within 25 min. In FP(B)-expressing oocytes the activation of the Cl- current was delayed or completely absent (1.38+/-0.2 microA, peak current; N=18). Control oocytes were not responsive to the application of PGF(2alpha) (0.87+/-0.1 microA, peak current; N=10). Activation of Cl- currents for both FP receptor isoforms was dependent upon intracellular Ca(2+) stores as a 30-min pretreatment with thapsigargin (1 microM; N=5) blocked the PGF(2alpha) induction of the Cl- current. These data indicate that the FP prostanoid receptor isoforms differ in their ability to activate Ca(2+)-dependent Cl- channels when expressed in Xenopus oocytes. The difference appears to be in the ability of the two FP prostanoid receptor isoforms to mobilize intracellular calcium.
Hiromichi Fujino, A Kimberly West and W John Regan : Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2., The Journal of Biological Chemistry, Vol.277, No.4, 2614-2619, 2001.
(Summary)
Recently we have shown that the FP(B) prostanoid receptor, a G-protein-coupled receptor that couples to Galpha(q), activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-mediated transcriptional activation (Fujino, H., and Regan, J. W. (2001) J. Biol. Chem. 276, 12489-12492). We now report that the EP(2) and EP(4) prostanoid receptors, which couple to Galpha(s), also activate Tcf/Lef signaling. By using a Tcf/Lef-responsive luciferase reporter gene, transcriptional activity was stimulated approximately 10-fold over basal by 1 h of treatment with prostaglandin E(2) (PGE(2)) in HEK cells that were stably transfected with the human EP(2) and EP(4) receptors. This stimulation of reporter gene activity was accompanied by a PGE(2)-dependent increase in the phosphorylation of both glycogen synthase kinase-3 (GSK-3) and Akt kinase. H-89, an inhibitor of protein kinase A (PKA), completely blocked the agonist-dependent phosphorylation of GSK-3 in both EP(2)- and EP(4)-expressing cells. However, H-89 pretreatment only blocked PGE(2)-stimulated Lef/Tcf reporter gene activity by 20% in EP(4)-expressing cells compared with 65% inhibition in EP(2)-expressing cells. On the other hand wortmannin, an inhibitor of phosphatidylinositol 3-kinase, had the opposite effect and inhibited PGE(2)-stimulated reporter gene activity to a much greater extent in EP(4)-expressing cells as compared with EP(2)-expressing cells. These findings indicate that the activation of Tcf/Lef signaling by EP(2) receptors occurs primarily through a PKA-dependent pathway, whereas EP(4) receptors activate Tcf/Lef signaling mainly through a phosphatidylinositol 3-kinase-dependent pathway. This is the first indication of a fundamental difference in the signaling potential of EP(2) and EP(4) prostanoid receptors.
(Keyword)
Androstadienes / Binding, Competitive / Blotting, Western / Calcium-Calmodulin-Dependent Protein Kinases / Cell Differentiation / Cell Division / Cell Line / Cyclic AMP-Dependent Protein Kinases / DNA-Binding Proteins / Dinoprostone / Dose-Response Relationship, Drug / Enzyme Inhibitors / Genes, Reporter / Glycogen Synthase Kinase 3 / Glycogen Synthase Kinases / Humans / Isoquinolines / Luciferases / Lymphoid Enhancer-Binding Factor 1 / Phosphatidylinositol 3-Kinases / Phosphorylation / Protein Binding / Radioligand Assay / Receptors, Prostaglandin E / Receptors, Prostaglandin E, EP2 Subtype / Receptors, Prostaglandin E, EP4 Subtype / Signal Transduction / Sulfonamides / Time Factors / Transcription Factors / Transcription, Genetic / Transfection
Hiromichi Fujino, T Uehara, T Murayama, Y Okuma, H Ariga and Y Nomura : Extracellular signal regulated protein kinase and c-jun N-terminal kinase are involved in ml muscarinic receptor-enhanced interleukin-2 production pathway in Jurkat cells., Biological & Pharmaceutical Bulletin, Vol.23, No.10, 1198-1205, 2000.
(Summary)
We have previously shown that m1 and m2 muscarinic receptors were expressed on human peripheral blood lymphocytes (hPBL) and that pre-stimulation of these receptors enhanced phytohemagglutinin (PHA)-induced interleukin-2 (IL-2) production. Possible intracellular signal pathways of muscarinic receptors to regulate IL-2 production were examined in human T cell line Jurkat cells. Pretreatment of the cells with muscarinic receptor agonist, oxotremorine M (Oxo-M), enhanced IL-2 production induced by phorbol 12-myristate 13-acetate (PMA)/A23187, while Oxo-M by itself did not affect IL-2 production. The enhancement of IL-2 production by Oxo-M was inhibited by 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) an ml/m3 receptor antagonist. When the cells were pretreated with AF-DX116, an m2 antagonist, the IL-2 production enhanced by Oxo-M was further stimulated. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that ml and m2 muscarinic receptors exist on Jurkat cells. The stimulation of ml receptors enhanced the PMA/A23187-induced binding activity to AP-1 consensus sequences in IL-2 promoter and production of c-Fos and c-Jun protein. The stimulation of ml receptors did not modify the DNA binding of NF-kappaB, NF-AT or Oct-1. When ml receptors were stimulated, activities of mitogen-activated protein kinase (MAPK)/extracellular signal regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) were increased, while p38 MAPK was not affected. Incubation with Oxo-M induced a transient increase in [Ca2+]i, which was abolished by pretreatment with 4-DAMP. Treatment with cyclosporin A markedly decreased the PMA/A23187-induced IL-2 promoter activity. This treatment, however, did not affect the enhancement of the promoter activity induced by ml receptor stimulation. The results suggest that transcription factor AP-1 is involved in the ml receptor-mediated enhancement of IL-2 transcript in Jurkat cells, and that pathways via MAPK/ERK and JNK, but not via p38 MAPK, are involved in the ml receptor-mediated enhancement of IL-2 promoter activity.
Hiromichi Fujino, L K Pierce, D Srinivasan, E C Protzman, H A Krauss, F D Woodward and W J Regan : Delayed reversal of shape change in cells expressing FP(B) prostanoid receptors. Possible role of receptor resensitization., The Journal of Biological Chemistry, Vol.275, No.38, 29907-29914, 2000.
(Summary)
Prostaglandin F(2 alpha) (PGF(2 alpha)) receptors are G-protein-coupled receptors consisting of two alternative mRNA splice variants, named FP(A) and FP(B). As compared with the FP(A) isoform, the FP(B) isoform lacks the last 46 amino acids of the carboxyl terminus and, therefore, represents a truncated version of the FP(A). We recently found (Pierce, K. L., Fujino, H., Srinivasan, D., and Regan, J. W. (1999) J. Biol. Chem. 274, 35944-35949) that stimulation of both isoforms with PGF(2 alpha) leads to activation of a Rho signaling pathway, resulting in tyrosine phosphorylation of p125 focal adhesion kinase, formation of actin stress fibers, and cell rounding. Although the activation of Rho and subsequent cell rounding occur at a similar rate for both isoforms, we now report that following the removal of PGF(2 alpha) the reversal of cell rounding is much slower for cells expressing the FP(B) isoform as compared with the FP(A) isoform. Thus, in HEK-293 cells that stably express the FP(A) isoform, the reversal of cell rounding appears to be complete after 1 h, whereas for FP(B)-expressing cells there is essentially no reversal even after 2 h. Similarly, the disappearance of stress fibers and dephosphorylation of p125 focal adhesion kinase following removal of agonist are much slower in FP(B)-expressing cells than in FP(A)-expressing cells. The mechanism of this differential reversal appears to involve a difference in receptor resensitization following the removal of agonist. Based upon whole cell radioligand binding, agonist-induced stimulation of inositol phosphate formation, and mobilization of intracellular Ca(2+), the FP(B) isoform resensitizes more slowly than the FP(A) isoform. These findings suggest that the carboxyl terminus of the FP(A) is critical for resensitization and that the slower resensitization of the FP(B) isoform leads to prolonged signaling. This differential signaling distinguishes the FP(A) and FP(B) receptor isoforms and could be important toward understanding the physiological actions of PGF(2 alpha).
(Keyword)
Calcium / Cell Line / Cell Size / Humans / Protein Isoforms / Receptors, Prostaglandin / Signal Transduction
Hiromichi Fujino, D Srinivasan, L K Pierce and W J Regan : Differential regulation of prostaglandin F(2alpha) receptor isoforms by protein kinase C., Molecular Pharmacology, Vol.57, No.2, 353-358, 2000.
(Summary)
Prostaglandin F(2alpha) receptors (FP) are G protein-coupled receptors that bind prostaglandin F(2alpha) (PGF(2alpha)), resulting in the activation of an inositol phosphate (IP) second messenger pathway. Alternative mRNA splicing generates two FP receptor isoforms. These isoforms, designated FP(A) and FP(B), are otherwise identical except for their carboxyl termini. FP(B) is essentially a truncated version of FP(A) that lacks the 46 carboxyl-terminal amino acids, including four putative protein kinase C (PKC) phosphorylation sites. Until now, functional differences between these FP receptor isoforms have not been identified. We now report that pretreatment with the PKC inhibitor bisindolylmaleimide I enhanced PGF(2alpha)-stimulated IP accumulation in transfected cells stably expressing the FP(A) isoform but not in cells stably expressing the FP(B) isoform. Whole-cell phosphorylation experiments showed a strong agonist-dependent phosphorylation of the FP(A) isoform but little or no phosphorylation of the FP(B). Pretreatment of cells with bisindolylmaleimide I decreased PGF(2alpha)-stimulated phosphorylation of the FP(A) isoform consistent with a PKC-dependent phosphorylation. In vitro phosphorylation of an FP(A) carboxyl-terminal fusion protein by recombinant PKCalpha showed that the carboxyl terminus of the FP(A) is a substrate for PKC. These results suggest that PKC-dependent phosphorylation is responsible for differential regulation of second messenger signaling by FP prostanoid receptor isoforms.
(Keyword)
Cell Line / Dinoprost / Glutathione Transferase / Humans / Inositol Phosphates / Isoenzymes / Phosphorylation / Protein Isoforms / Protein Kinase C / Protein Kinase C-alpha / Receptors, Prostaglandin / Recombinant Fusion Proteins
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 10648645
L K Pierce, Hiromichi Fujino, D Srinivasan and W J Regan : Activation of FP prostanoid receptor isoforms leads to Rho-mediated changes in cell morphology and in the cell cytoskeleton., The Journal of Biological Chemistry, Vol.274, No.50, 35944-35949, 1999.
(Summary)
Prostaglandin F(2alpha) (PGF(2alpha)) exerts its biological effects by binding to and activating FP prostanoid receptors. These receptors, which include two isoforms, the FP(A) and FP(B), have been cloned from a number of species and are members of the superfamily of G-protein-coupled receptors. Previous studies have shown that the activation of FP receptors leads to phosphatidylinositol hydrolysis, intracellular calcium release, and activation of protein kinase C. Here, we demonstrate that PGF(2alpha) treatment of 293-EBNA (Epstein-Barr nuclear antigen) cells that have been stably transfected with either the FP(A) or FP(B) receptor isoforms leads to changes in cell morphology and in the cell cytoskeleton. Specifically, cells treated with PGF(2alpha) show retraction of filopodia and become rounded, and actin stress fibers are formed. Pretreatment of the cells with bisindolylmaleimide I, a protein kinase C inhibitor, has no effect on the PGF(2alpha)-induced changes in cell morphology, although it does block the effects of phorbol myristate acetate on cell morphology. On the other hand, the PGF(2alpha)-induced changes in cell morphology and formation of actin stress fibers can be blocked by pretreatment of the cells with C3 exoenzyme, a specific inhibitor of the small G-protein, Rho. Consistent with FP receptor induced formation of actin stress fibers and focal adhesions, FP(A) receptor activation also leads to rapid (within two minutes) tyrosine phosphorylation of p125 focal adhesion kinase (FAK) which can be blocked by pretreating the cells with C3 exoenzyme. Taken together, these results suggest that the FP receptor isoforms are coupled to at least two second messenger pathways, one pathway associated with protein kinase C activation, and the other with activation of Rho.
C Hayashi, Hiromichi Fujino, M Ogata, Y Sato, M S Iguchi-Ariga and H Ariga : Determination of the functional domain of a mouse autonomous replicating sequence., Biological & Pharmaceutical Bulletin, Vol.20, No.6, 690-693, 1997.
(Summary)
We previously isolated from mouse cells an autonomous replicating sequence (ARS) ARS65 (Ariga, Itani and Iguchi-Ariga, Mol. Cell. Biol. 7, 1-6, 1987). Here we report the nucleotide sequence of ARS65. The sequence from BgIII to EcoRI sites cloned as ARS was 2658 bp long. There exist three interesting domains: a TA repeat, a myc like box (essential sequence for c-myc ARS), and a T rich region. Cloned DNAs containing various segments of pARS65 were transfected to rat 3Y1 cells together with the hygromycinB resistance expression vector, and hygromycinB resistant clones were isolated. Established cell lines transfected with plasmids carrying either a myc-like box or a T rich region harbored the replicated plasmids, indicating that these two elements are necessary for the ARS function of pARS65.
(Keyword)
Animals / Anti-Bacterial Agents / Base Sequence / Cell Line / Cloning, Molecular / DNA Replication / DNA, Recombinant / Drug Resistance / Hygromycin B / Mice / Molecular Sequence Data / Plasmids / Rats / Regulatory Sequences, Nucleic Acid / Transfection
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 9212992
Hiromichi Fujino, Y Kitamura, T Yada, T Uehara and Y Nomura : Stimulatory roles of muscarinic acetylcholine receptors on T cell antigen receptor/CD3 complex-mediated interleukin-2 production in human peripheral blood lymphocytes., Molecular Pharmacology, Vol.51, No.6, 1007-1014, 1997.
(Summary)
It is known that there are some bidirectional interactions between the nervous and the immune systems via neurotransmitters and cytokines. To clarify whether any neurotransmitters modulate lymphocyte functions, we examined the effects of oxotremorine-M (Oxo-M) on interleukin-2 (IL-2) production in human peripheral blood lymphocytes by using enzyme-linked immunosorbent assays, Northern blot analyses, reverse transcriptase-polymerase chain reaction, and fluorescence-activated cell sorter. Pretreatment of cells with Oxo-M (10 nM to 10 microM) for 4-24 hr enhanced phytohemagglutinin (PHA)-induced IL-2 mRNA expression and markedly increased IL-2 production compared with those induced by PHA alone. Oxo-M alone did not affect IL-2 mRNA expression and IL-2 production. In CD3-positive T cells, pretreatment with Oxo-M for 24 hr enhanced PHA-induced IL-2 production. Furthermore, pretreatment with Oxo-M enhanced PHA-induced mRNA expression of the alpha and beta subunits of IL-2 receptors and DNA synthesis. Cytometric analysis showed Oxo-M treatment did not up-regulate expression of cell surface molecules such as CD3, CD2, CD4, CD8, and IL-2 receptors. These results suggest that activation of muscarinic receptors enhances T cell antigen receptor/CD3-induced IL-2 production.
Masato Mashimo, Asuka Shimizu, Aimi Mori, Ayaka Hamaguchi, Keijo Fukushima, Naofumi Seira, Takeshi Fujii and Hiromichi Fujino : PARP14 regulates EP4 receptor expression in human colon cancer HCA-7 cells., Biochemical and Biophysical Research Communications, Vol.623, 133-139, 2022.
(Summary)
E type prostanoid 4 (EP4) receptors and their signaling pathways have been implicated in the development and malignant transformation of colorectal cancer. We herein demonstrated that the mono(ADP-ribosyl)ation of histone deacetylase (HDAC)1 and HDAC2 by poly(ADP-ribose) polymerase 14 (PARP14) may be required to induce the expression of EP4 receptors. The suppression of PARP14 activity by siRNA and/or its inhibitors reduced the mRNA expression of EP4 receptors. Thus, the expression of their proteins to approximately 50-80% in human colon cancer HCA-7 cells, however, which retained the activities of EP4 receptors to some extent. Since the expression levels of EP4 receptors are important factors for the maintenance of homeostasis, the adequate inhibition of PARP14 activity will be a good target for the prevention of colon cancer and/or as an alternative therapy for this disease. Since non-steroidal anti-inflammatory drugs (NSAIDs) are associated with a risk of heart attacks and stroke, novel PARP14 inhibitors will supersede NSAIDs without causing heart attacks and stroke, while maintaining appropriate EP4 receptor-mediated intestinal homeostasis.
Seira Naofumi, Yanagisawa Naoki, Suganami Akiko, Takuya Honda, Wasai Makiko, Regan W John, Keijo Fukushima, Yamaguchi Naoto, Tamura Yutaka, Arai Takayoshi, Murayama Toshihiko and Hiromichi Fujino : Anti-cancer effects of MW-03, a novel indole compound, by inducing 15-hydroxyprostaglandin dehydrogenase and cellular growth inhibition in the LS174T human colon cancer cell line, Biological & Pharmaceutical Bulletin, Vol.40, No.10, 1806-1812, 2017.
(Summary)
Increases in the expression of prostaglandin E (PGE) are widely known to be involved in aberrant growth in the early stage of colon cancer development. We herein demonstrated that the novel indole compound MW-03 reduced PGE-induced cAMP formation by catalization to an inactive metabolite by inducing 15-hydroxyprostaglandin dehydrogenase through the activation of peroxisome proliferator-activated receptor-γ. MW-03 also inhibited colon cancer cell growth by arresting the cell cycle at the S phase. Although the target of MW-03 for cell cycle inhibition has not yet been identified, these dual anti-cancer effects of MW-03 itself and/or its leading compound(s) on colon cancer cells may reduce colon cancer development and, thus, have potential as a novel treatment for the early stage of this disease.
Jun Tanimoto, Hiromichi Fujino, Hiroki Takahashi and Toshihiko Murayama : Human EP2 prostanoid receptors exhibit more constraints to mutations than human DP prostanoid receptors., FEBS Letters, Vol.589, No.6, 766-772, 2015.
(Summary)
Human D-type prostanoid (DP) and E-type prostanoid 2 (EP2) receptors are regarded as the most closely related receptors among prostanoid receptors. Although these receptors are generated by tandem duplication, their physiological outputs often oppose one another. In the present study, we extracted mutations occurring in the coding regions of both receptors using the 1000 genome project database and found that EP2 receptors have 8-fold fewer amino acid mutations. We speculate that EP2 receptors exhibit more constraints to mutations than DP receptors.
Takashi Orido, Hiromichi Fujino, Yuta Hasegawa, Kaori Toyomura, Tatsuo Kawashima and Toshihiko Murayama : Indomethacin decreases arachidonic acid uptake in HCA-7 human colon cancer cells., Journal of Pharmacological Sciences, Vol.108, No.3, 389-392, 2008.
(Summary)
Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the incidence of colorectal cancer. However, evidence is accumulating that NSAIDs have anti-cancer effects in addition to inhibiting cyclooxygenase (COX)-mediated prostanoid biosynthesis. We now show that indomethacin, a popular NSAID, significantly reduced the [3H]-arachidonic acid uptake in HCA-7 human colon cancer cells. Interestingly, no decrease in the uptake of [3H]-arachidonic acid occurred when the cells were treated with aspirin, diclofenac, and sulindac even though the concentrations of these NSAIDs were high enough to inhibit COX-2 activity. These findings suggest that indomethacin has a novel anti-cancer effect that may be independent of COX-2 inhibition.
Tomoko Taniguchi, Hiromichi Fujino, D Davelene Israel, W John Regan and Toshihiko Murayama : Human EP3(I) prostanoid receptor induces VEGF and VEGF receptor-1 mRNA expression., Biochemical and Biophysical Research Communications, Vol.377, No.4, 1173-1178, 2008.
(Summary)
A critical event in tumor development is the formation of new blood vessels to provide oxygen, nutrients and growth factors to the rapidly growing cancer cells. This process of angiogenesis is complex, however, it is well established that vascular endothelial growth factor (VEGF)-mediated signaling is an important early event. Knockout mice studies have implicated the EP3 receptor in tumor development and angiogenesis; however, the signaling mechanism involved with this effect is unclear. We now show that stimulation of the EP3(I) isoform of the human EP3 receptor with prostaglandin E(2) increases the mRNA expression of both VEGF and its cognate receptor VEGF receptor-1 (VEGFR-1). These inductions by the EP3(I) receptor involve the sequential activation of phosphatidylinositol 3-kinase and the extracellular signal-regulated kinases. Up-regulation of VEGF and VEGFR-1 mRNA by the human EP3(I) receptor has not been previously reported and further strengthen the role of this receptor in tumor-associated angiogenesis.
(Keyword)
Adipogenesis / Butadienes / Dinoprostone / Humans / Mitogen-Activated Protein Kinase 1 / Mitogen-Activated Protein Kinase 3 / Nitriles / Phosphorylation / RNA, Messenger / Receptors, Prostaglandin E / Receptors, Prostaglandin E, EP3 Subtype / Up-Regulation / Vascular Endothelial Growth Factor A / Vascular Endothelial Growth Factor Receptor-1
Hiromichi Fujino, Xiao-bo Chen, W John Regan and Toshihiko Murayama : Indomethacin decreases EP2 prostanoid receptor expression in colon cancer cells., Biochemical and Biophysical Research Communications, Vol.359, No.3, 568-573, 2007.
(Summary)
Nonsteroidal anti-inflammatory drugs (NSAIDs) can decrease the risk of colorectal cancer; however, it has not been established if this effect is solely through their ability to inhibit cyclooxygenase (COX). In this study the effects of indomethacin, a potent NSAID and nonselective COX inhibitor, was examined in LS174T human colon cancer cells. These cells were found to express EP2 prostanoid receptors, but not the EP1, EP3 or EP4 subtypes. Pretreatment of LS174T cells with indomethacin produced a complete inhibition of prostaglandin E(2) (PGE(2)) stimulated cyclic AMP (cAMP) formation in a dose dependent manner with an IC(50) of 21 microM. Interestingly, the inhibition of PGE(2)-stimulated cAMP formation by indomethacin was accompanied by a decrease in EP2 mRNA expression and by a decrease in the whole cell specific binding of [(3)H]PGE(2). Thus, treatment of LS174T cells with indomethacin causes a down regulation of EP2 prostanoid receptors expression that may be independent of COX inhibition.
Hiromichi Fujino and W John Regan : Prostaglandin F2alpha amplifies tumor necrosis factor-alpha promoter activity by the FPB prostanoid receptor., Biochemical and Biophysical Research Communications, Vol.317, No.4, 1114-1120, 2004.
(Summary)
This study examines the regulation of tumor necrosis factor-alpha (TNF-alpha) promoter activity by prostaglandin F2alpha ( PGF2alpha ) in HEK cells stably expressing either the FPA or FPB prostanoid receptors. Cells were transiently transfected with a luciferase reporter plasmid under the control of a TNF-alpha promoter and luciferase activity was measured. In the absence of PGF2alpha basal TNF-alpha reporter gene activity is elevated in FPB cells as compared with FPA cells. This elevated basal activity is blocked by pretreatment with a Rho inhibitor, but not by pretreatment with an inhibitor of protein kinase C (PKC). TNF-alpha reporter activity in FPB cells is stimulated by PGF2alpha and this is decreased by pretreatment with a chelator of intracellular calcium or by a gap junction inhibitor. In FPB cells pretreatment with a Rho inhibitor combined with either a calcium chelator or a gap junction inhibitor decreases both basal and PGF2alpha stimulated TNF-alpha reporter activity. Interestingly post-treatment of FPB cells with an inhibitor of PKC decreased PGF2alpha stimulated TNF-alpha reporter gene activity even though pretreatment did not. It, therefore, appears that PGF2alpha stimulated TNF-alpha reporter activity in FPB cells is amplified by a Rho-dependent mechanism involving calcium, gap junctions, and PKC. These findings may help in understanding the function of the FPB isoform in the corpus luteum.
Hiromichi Fujino and W John Regan : Prostaglandin F2α stimulation of cyclooxygenase-2 promoter activity by the FPB prostanoid receptor., European Journal of Pharmacology, Vol.465, No.1-2, 39-41, 2003.
(Summary)
We have recently shown that the FP(B) prostanoid receptor activates beta-catenin signaling through the activation of Rho in human embryo kidney (HEK)-293 cells stably expressing the FP(B) receptors. We now report that the FP(B) receptor can stimulate cyclooxygenase-2 promoter activity and may, therefore, regulate the expression of cyclooxygenase-2. This stimulation of cyclooxygenase-2 promoter activity is blocked by pretreatment with an inhibitor of Rho, but not with an inhibitor of protein kinase C (PKC). Potential up regulation of cyclooxygenase-2 expression by the FP(B) receptor would establish a positive feedback loop that would drive beta-catenin signaling and could be involved in cancer.
Hiromichi Fujino and W J Regan : FP prostanoid receptor activation of a T-cell factor/beta -catenin signaling pathway., The Journal of Biological Chemistry, Vol.276, No.16, 12489-12492, 2001.
(Summary)
FP prostanoid receptors are G-protein-coupled receptors (GPCR) that consist of two known isoforms, FP(A) and FP(B). These isoforms, which are generated by alternative mRNA splicing, are identical except for their carboxyl-terminal domains. Previously we have shown that stimulation of both isoforms with prostaglandin F(2alpha) (PGF(2alpha)) activates the small G-protein Rho, leading to morphological changes consisting of cell rounding and the formation of cell aggregates. Following the removal of PGF(2alpha), however, FP(A)-expressing cells show rapid reversal of cell rounding, whereas FP(B)-expressing cells do not. We now show that acute treatment of FP(B)-expressing cells with PGF(2alpha) leads to a subcellular reorganization of beta-catenin, a decrease in the phosphorylation of cytoplasmic beta-catenin, and persistent stimulation of Tcf/Lef-mediated transcriptional activation. This does not occur in FP(A)-expressing cells and may underlie the differences between these isoforms with respect to the reversal of cell rounding. The Tcf/beta-catenin signaling pathway is known to mediate the actions of Wnt acting through the heptahelical receptor, Frizzled, and has not been associated previously with GPCR activation. Our findings expand the signaling possibilities for GPCRs and suggest novel roles for FP receptors in normal tissue development and malignant transformation.
Hiromichi Fujino : The Biased Activities of Prostanoids and Their Receptors: Review and Beyond., Biological & Pharmaceutical Bulletin, Vol.45, No.6, 684-690, 2022.
(Summary)
Since the discovery of β-arrestin, a new concept/viewpoint has arisen in G-protein coupled receptor (GPCR)-mediated signaling. The Lock and Key concept of GPCR was previously recognized as basically a single- or mono-originated pathway activated from a single receptor. However, the new concept/viewpoint allows for many- or more-than-one-originated pathways activated from a single receptor; namely, biased activities. It is well-recognized that prostanoids exhibit preferences for their corresponding cognate receptors, while promiscuous cross-reactivities have also been reported among endogenous prostanoids and their receptor family. However, of particular interest, such cross-reactivities have led to reports of their physiologically significant roles. Thus, this review discusses and considers that the endogenous prostanoids are not showing random cross-reactivities but what are showing important physiological and pathological activities as biased ligands. Moreover, why and how the biased activities are evoked by endogenous structurally similar prostanoid ligands are discussed. Furthermore, when the biased activities of endogenous prostanoids first arose is also discussed and considered. These biased activities of endogenous prostanoids are also discussed from the perspective that they may provide many benefits and/or disadvantages for all living things, any-where on this planet, who/which are utilizing, had utilized, and will utilize the prostanoids and their receptor system, as a marked driving force for evolution.
Hiromichi Fujino : The Roles of EP4 Prostanoid Receptors in Cancer Malignancy Signaling., Biological & Pharmaceutical Bulletin, Vol.39, No.2, 149-155, 2016.
(Summary)
The lipid mediator prostanoids consist of prostaglandins and thromboxanes, and are synthesized from arachidonic acid by the action of cyclooxygenase. There are five major prostanoids, including prostaglandin E2 (PGE2), and they are involved in a variety of biological responses such as inflammation, allergy, parturition, and tumorigenesis. These prostanoids exert their effects via activation of their cognate G protein coupled receptors, e.g., E-type prostanoid (EP) receptors for PGE2. The EP receptors are composed of four subtypes, namely EP1 to EP4. Here, breakthroughs in the last dozen years of research are introduced, with a special focus on some important findings of EP4 receptor-mediated signaling and the signaling associated with cancer development, particularly in colon cancer.
Hiromichi Fujino, Toshihiko Murayama and W John Regan : Assessment of constitutive activity in E-type prostanoid receptors., Methods in Enzymology, Vol.484, 95-107, 2010.
(Summary)
The potential for G-protein-coupled receptors (GPCRs) to show constitutive activity is emerging as one of the fundamental properties of GPCRs signal transduction. Indeed, of the four subtypes of E-type prostanoid (EP) receptors, the EP3 and EP4 subtypes show constitutive activity in addition to their innate ligand-dependent activation of signaling pathways. The constitutive activity of the EP3 and EP4 receptor subtypes was discovered during the initial characterizations of these receptors and may be important for setting the basal level of cellular tone in the given signaling pathway. This chapter introduces some of the methods that can be used to study the constitutive activity of the EP receptors.
(Keyword)
Animals / Biological Assay / Cyclic AMP / Humans / Receptors, Prostaglandin E / Receptors, Prostaglandin E, EP3 Subtype / Receptors, Prostaglandin E, EP4 Subtype
Hiromichi Fujino and W John Regan : Prostanoid receptors and phosphatidylinositol 3-kinase: a pathway to cancer?, Trends in Pharmacological Sciences, Vol.24, No.7, 335-340, Jul. 2003.
(Keyword)
Animals / Humans / Neoplasms / Phosphatidylinositol 3-Kinases / Receptors, Prostaglandin / Second Messenger Systems
Niimura Takahiro, Yoshito Zamami, Yuki Izawa-Ishizawa, Mitsuhiro Goda, Kenshi Takechi, Masayuki Chuma, Keijo Fukushima, Yuya Horinouchi, Hiromichi Fujino, Koichiro Tsuchiya and Keisuke Ishizawa : Nicorandil improve prognosis of cardiac arrest patient: A large-scale medical information analysis, FIP2019, Abu Dhabi, Sep. 2019.
2.
Hiromichi Fujino : The functions of receptors for prostanoids are tossed up and down by endogenous cognate and/or non-cognate ligands, The 4th symposium of integrated research institute for drug development, College of Pharmacy, Dongguk University, Oct. 2018.
3.
Yuya Horinouchi, Yasumasa Ikeda, Keijo Fukushima, Masaki Imanishi, Yuki Izawa-Ishizawa, Yoshito Zamami, Hiromichi Fujino, Keisuke Ishizawa, Koichiro Tsuchiya and Toshiaki Tamaki : Utilizing Real-World Big Data in the Search for New Renoprotective Drugs, Joint Hypertension 2018 Scientific Sessions, Sep. 2018.
4.
Seira Naofumi, Yamagata Kazuyuki, Keijo Fukushima, Araki Yumi, Kurata Naoki, Yanagisawa Naoki, Mashimo Masato, Nakamura Hiroyuki, Regan W. John, Murayama Toshihiko and Hiromichi Fujino : Hypoxia inducible factor-1alpha regulates human EP4 receptor expression by binding to specificity protein-1, The 3rd Chiba University-Mahidol University Joint Symposium on Pharmaceutical Sciences, Aug. 2018.
Hiramatsu Miharu, Mizuguchi Hiroyuki, Wakugawa Tomoharu, Nagamine Kenichi, Tanabe Hideya, Shinohara Keiko, Sawada Eiji, Hiromichi Fujino, Kitamura Yoshiaki, Takeda Noriaki and Fukui Hiroyuki : Isolation of anti-allergic compound from lotus roots, World Histamine Symposium 2018, Jul. 2018.
7.
Islam Rezwanul, Shaha Aurpita, Okamoto Kentaro, Wakugawa Tomoharu, Nishida Kohei, Yabumoto Masami, Ikeda Hisashi, Hiromichi Fujino, Fukui Hiroyuki, Takeda Noriaki and Mizuguchi Hiroyuki : Nasal symptoms in allergic model rats markedly alleviated by the combination therapy with wild grapes and Awa-tea, World Histamine Symposium 2018, Jul. 2018.
8.
Shaha Aurpita, Mizuguchi Hiroyuki, Islam Rezwanul, Wakugawa Tomoharu, Hiromichi Fujino, Yabumoto Masami, Takeda Noriaki and Fukui Hiroyuki : Effect of Royal jerry and Brazilian green propolis on the signaling for histamine H1 receptor and interleukin-9 gene expressions responsible for the pathogenesis of the allergic rhinitis, World Histamine Symposium 2018, Jul. 2018.
9.
Konishi Yuki, Mizuguchi Hiroyuki, Kitamura Yoshiaki, Hiromichi Fujino, Takeda Noriaki and Fukui Hiroyuki : Effect of Sho-seiryu-to on histamine H1 receptor and interleukin-33 gene expression, World Histamine Symposium 2018, Jul. 2018.
10.
Kyuta Ayumi, Mizuguchi Hiroyuki, Kawai Makiko, Hiromichi Fujino, Kitamura Yoshiaki, Takeda Noriaki and Fukui Hiroyuki : Effect of anti-allergic compound (-)-maackiain isolated from kujin on steroid signaling, World Histamine Symposium 2018, Jul. 2018.
11.
Sanada Nanae, Mizuguchi Hiroyuki, Kamimura Seiichiro, Fujii Tatuya, Yamada Takuya, Kitamura Yoshiaki, Hiromichi Fujino, Takeda Noriaki and Fukui Hiroyuki : Effect of narrow-band UVB on up-regulation of histamine H1 receptor gene expression in HeLa cells, World Histamine Symposium 2018, Jul. 2018.
12.
Yuya Horinouchi, Yasumasa Ikeda, Keijo Fukushima, Masaki Imanishi, Yuki Izawa-Ishizawa, Yoshito Zamami, Hiromichi Fujino, Keisuke Ishizawa, Koichiro Tsuchiya and Toshiaki Tamaki : Renoprotective effects of edoxaban, a factor Xa inhibitor, 18th World Congress of Basic and Clinical Pharmacology, Jul. 2018.
13.
Yoshito Zamami, Yuki Izawa-Ishizawa, Kenshi Takechi, Masaki Imanishi, Keijo Fukushima, Yuya Horinouchi, Yasumasa Ikeda, Hiromichi Fujino, Toshiaki Tamaki and Keisuke Ishizawa : Search for drugs that attenuate the anti tumor effect of bevacizumab using adverse event database, 18th World Congress of Basic and Clinical Pharmacology, Jul. 2018.
14.
Wakugawa Tomoharu, Mizuguchi Hiroyuki, Hiramatsu Miharu, Nagamine Ken-ichi, Tanabe Hideya, Shinohara Keiko, Sawada Eiji, Hiromichi Fujino, Fukui Hiroyuki and Takeda Noriaki : Isolation of anti-allergic compound from Lotus Root, 18th World Congress of Basic and Clinical Pharmacology, Jul. 2018.
15.
Hiromichi Fujino, Araki Yumi, Suganami Akiko, Endo Suzu, Takano Harumi, Masuda Yuta, Keijo Fukushima, Regan W. John, Murayama Toshihiko and Tamura Yutaka : PGE1 and E3 show lower efficacies than E2 to beta-catenin-mediated activity as biased ligands of EP4 prostanoid receptors, 18th World Congress of Basic and Clinical Pharmacology, Jul. 2018.
16.
Islam Rezwanul, Shaha Aurpita, Okamoto Kentaro, Wakugawa Tomoharu, Hiramatsu Miharu, Ikeda Hisashi, Hiromichi Fujino, Fukui Hiroyuki and Mizuguchi Hiroyuki : Wild grapes in a combination with Awa tea markedly alleviated nasal symptoms in toluene-2,4-diisocyanate-sensitized allergy model rats., The 18th International Congress of International Society for Ethnopharmacology& the 5th International Congress of the Society for Ethnopharmacology, Jan. 2018.
17.
Seira Naofumi, Hiromichi Fujino, Otake Sho, Regan W. John, Takahashi Hiroki and Murayama Toshihiko : EP4 prostanoid receptor expression by HIF-1a in human colon cancer HCA-7 cell line., 2nd World Congress of Cancer Research & Therapy, Oct. 2017.
18.
Masaki Imanishi, Kyohei Tanaka, Raiki Ikutoh, Yoshito Zamami, Kenshi Takechi, Yuya Horinouchi, Yuki Izawa-Ishizawa, Yasumasa Ikeda, Hiromichi Fujino, Koichiro Tsuchiya, Toshiaki Tamaki and Keisuke Ishizawa : THE EFFECTS OF FEBUXOSTAT ON ANGIOTENSIN II-INDUCED VASCULAR REMODELING, 27th European Meeting on Hypertension and Cardiovascular Protection, Jun. 2017.
19.
Yamazaki Risa, Fujita Tesuo, Yanagihara Madoka, Nakamura Hiroyuki, Hiromichi Fujino, Kasuya Yoshitoshi and Murayama Toshihiko : Cyclosporin A has anti-fibrotic effects in human lung fibroblasts and in bleomycin-induced lung fibrosis model mice, Annual Meeting of American Society for Pharmacology and Experimental Therapeutics (ASPET)-2016 (San Diego, USA), Apr. 2016.
Takaya Misao, Keijo Fukushima and Hiromichi Fujino : 内因性カンナビノイドによる抗結腸がんメカニズムの解明, 第63回 日本生化学会 中国・四国支部例会, May 2022.
32.
Haruka Kobayashi, Haruka Okabayashi, Miki Yasuda, Keijo Fukushima, 川村 純, 琴浦 聡 and Hiromichi Fujino : アラキドン酸による神経毒生を緩和するプラズマローゲン作用の解明, 日本薬学会第142年会，名古屋, Mar. 2022.
33.
Ayaka Hamaguchi, Keijo Fukushima, 福田 隼人, 藤原 広一, 周東 智 and Hiromichi Fujino : Resolvin E-seriesの炎症収束作用の検討, 日本薬学会第142年会，名古屋, Mar. 2022.
34.
Kanaho Senoh, Hitomi Yamamoto, Suzu Endoh, W John Regan, Keijo Fukushima and Hiromichi Fujino : プロスタグランジンD2の代謝物はDPプロスタノイド受容体に対してバイアスアゴニストとして働く, 第95回日本薬理学会年会，福岡, Mar. 2022.
35.
Akito Ohnishi, Akiko Higashiyama, Shunya Yanagawa, 清良 尚史, W John Regan, Kento Ohkawachi, Masaya Denda, Keijo Fukushima, Akira Otaka and Hiromichi Fujino : プロスタノイドEP4受容体の1アミノ酸変異によるシグナル伝達プロファイル変化, 第95回日本薬理学会年会，福岡, Mar. 2022.
36.
Yasumasa Ikeda, Hirofumi Hamano, Mitsuhiro Goda, Keijo Fukushima, Seiji Kishi, Masayuki Chuma, Yoshito Zamami, Licht Miyamoto, 石澤 啓介, Hiromichi Fujino, Ken-ichi Aihara, Koichiro Tsuchiya and Toshiaki Tamaki : Identification of a candidate drug for the prevention of cisplatin-induced nephrotoxicity by a database analysis -basic research-clinical study, 第94回日本薬理学会年会, Mar. 2021.
37.
Nanae Hasuoka, Miho Agata, 間下 雅士, Keijo Fukushima and Hiromichi Fujino : PGD2およびその代謝物によるCRTH2受容体へのバイアス作用の解明, 日本薬学会第141年会, Mar. 2021.
38.
Kiyoka Matsumoto, Yuki Nakano, 高橋 弘喜, 楠屋 陽子, 村山 俊彦, Keijo Fukushima and Hiromichi Fujino : PGE2による結腸がん細胞内代謝変化の解析, 日本薬学会第141年会, Mar. 2021.
39.
Harumi Takano, Kanaho Senoh, Mayu Yamashita, Yumi Araki, Regan John W, Keijo Fukushima and Hiromichi Fujino : EP4プロスタノイド受容体を介したプロスタグランジンD2のバイアス性による抗癌作用, 第94回日本薬理学会年会, Mar. 2021.
40.
Rei Matsumoto, Kaori Ueki, Yuta Masuda, 間下 雅士, Keijo Fukushima and Hiromichi Fujino : 結腸癌転移に関与するヒスタミンH1受容体作用の解明, Mar. 2021.
森 愛実, 清水 未来華, Hiromichi Fujino and 間下 雅士 : PARP14による大腸癌の増殖に関与するEP4受容体の発現調節機構の解明, 日本薬学会第140年会, Mar. 2020.
43.
Manabu Sugiyama, Akito Ohnishi, Takuya Morisaki, Akira Shigenaga, Keijo Fukushima, Akira Otaka and Hiromichi Fujino : inteinシステムを用いたプロスタノイドEP4受容体の局在解析を目指して, 日本薬学会第140年会, Mar. 2020.
44.
Natsuki Yamagiwa, Haruka Kobayashi, Keijo Fukushima, Haruka Okabayashi, 川村 純, 琴浦 聡 and Hiromichi Fujino : アラキドン酸による神経毒性作用を緩和する有効な生理活性脂質としてのプラズマローゲン種の同定, 日本薬学会第140年会, Mar. 2020.
45.
Kana Kitagawa, Ayaka Hamaguchi, 間下 雅士, Regan John W., Keijo Fukushima and Hiromichi Fujino : ヒト結腸がんHCA-7細胞においてインターロイキン4はEP4プロスタノイド受容体発現を抑制する, 第93回日本薬理学会年会, Mar. 2020.
46.
Suzu Endoh, Kanaho Senoh, Harumi Takano, Yumi Araki, Regan John W., Keijo Fukushima and Hiromichi Fujino : PGE2代謝物 15-keto-PGE2はバイアスアゴニストとしてEP2およびEP4プロスタノイド受容体に作用する, 第93回日本薬理学会年会, Mar. 2020.
山形 一行, 清良 尚史, 福島 圭稔, Yumi Araki, 倉田 直希, 柳澤 直樹, 間下 雅士, 中村 浩之, W John Regan, 村山 俊彦 and Hiromichi Fujino : HIF-1α and c-Myc oppositely regulate human EP4 receptor promoter activity in human colon cancer HCA-7 cells, 第92回日本薬理学会年会, Mar. 2019.
Explore the irreversible check point from normal to malignant transformation of the cells by changing in the balance of the expression levels of the EP prostanoid receptor subtypes. (Project/Area Number: 20K07084 )
Exploring the mechanism of EP4 prostanoid receptor-evoked colorectal cancer development during the collapse of homeostasis and its ameliorating effects by substances of type 2 innate immune system (Project/Area Number: 17K08308 )
Mechanisms of colon cancer malignancy regulation by two endogenous prostanoid receptors (Project/Area Number: 25460091 )
Exploring the novel cycloocygenase-inhibition-independent effects of indomethacin (Project/Area Number: 22590079 )