Porphyromonas gingivalis outer membrane vesicles increase vascular permeability by inducing stress fiber formation and degrading vascular endothelial-cadherin in endothelial cells.

Porphyromonas gingivalis (Pg) is a keystone bacterium associated with systemic diseases, such as diabetes mellitus and Alzheimer's disease. Outer membrane vesicles (OMVs) released from Pg have been implicated in systemic diseases by delivering Pg virulence factors to host cells in distant organs and inducing cellular dysfunction. Pg OMVs also have the potential to enter distant organs via the bloodstream. However, the effects of Pg OMVs on the vascular function are poorly understood. Here, we showed that Pg OMVs increase vascular permeability by promoting stress fiber formation and lysosome/endosome-mediated vascular endothelial-cadherin (VEc) degradation in human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs). F-actin, visualized via fluorescein isothiocyanate-phalloidin, became thicker and longer, leading to the formation of radical stress fibers in response to Pg OMVs in HUVECs and HPMECs. Western blotting and quantitative real-time polymerase chain reaction analyses revealed that Pg OMVs decreased VEc protein levels in a gene-independent manner. Pg OMVs enhanced vesicular VEc accumulation in the cytoplasm around lysosome-associated membrane protein 1-positive structures during pretreatment with the lysosomal inhibitor chloroquine. This suggests that Pg OMVs decrease VEc protein levels by accelerating their internalization and degradation via lysosomes and endosomes. A27632 inhibition of Rho kinases impaired the Pg OMV-induced stress fiber formation and VEc degradation, resulting in the recovery of hyperpermeability. These findings provide new insights into the pathogenesis of systemic diseases that are associated with periodontal diseases.


Lipocalin 2 inhibits the expressions of interleukin-8 and macrophage inflammatory protein-1α in human neutrophil-like cells

Lipocalin 2 (LCN2) is a glycoprotein with multiple functions, including antimicrobial activity, inflammatory response modulation, and cell migration. LCN2 is expressed in some cells, such as epithelial cells and neutrophils, and its levels are increased in inflammatory diseases. This study investigated the presence of LCN2 receptor (24p3R) in cells around periodontal tissues and function of LCN2 in cells with a receptor to explore the role of LCN2 in periodontal diseases. The presence of 24p3R was examined in periodontal cells, including human gingival fibroblasts, periodontal ligament fibroblasts, human oral epithelial cells (HOECs), and neutrophil-like cells (HL-60), by Western blotting. Changes in periodontal disease-associated proteins in the presence of recombinant LCN2 (rLCN2) were examined using a protein array in differentiated HL-60 (dHL-60) cells. Interleukin-8 (IL-8) and macrophage inflammatory protein-1α (MIP-1α) mRNA expressions were analyzed by qRT-PCR, and IL-8 and MIP-1α levels in dHL-60 cells treated with rLCN2 or Porphyromonas gingivalis-lipopolysaccaharide (P.g-LPS) were determined by enzyme-linked immunosorbent assay. We detected 24p3R in dHL-60 cells. IL-8 was highly expressed and MIP-1α was weakly expressed in dHL-60 cells using a protein array. rLCN2 significantly decreased IL-8 mRNA and protein levels and suppressed P.g-LPS-induced IL-8 production in dHL-60 cells. As dHL-60 cells were co-cultured with HOECs in which LCN2 was knocked down, IL-8 mRNA expression increased in dHL-60 cells. Furthermore, rLCN2 inhibited MIP-1α production in dHL-60 cells. LCN2 suppresses inflammatory responses by regulating IL-8 and MIP-1α expression in periodontal diseases.


Porphyromonas gingivalis infection alters microRNA composition in extracellular vesicles.

Periodontitis, commonly associated with Porphyromonas gingivalis (Pg), involves intricate alterations of oral intercellular interactions, in which extracellular vesicles (EVs) play a pivotal role. The understanding of the miRNA profiles in the EVs derived from Pg-infected cells (Pg-EVs) remains incomplete despite acknowledging their importance in intercellular communication during periodontitis. Therefore, our objective was to identify and characterize the miRNAs enriched in Pg-EVs. Microarray analysis was conducted to examine the miRNA profiles in the EVs derived from Pg-infected THP-1 cells. We compared the identified miRNAs with those upregulated in the EVs after stimulation with LPS. Additionally, we explored how inhibiting TLR signaling during Pg infection affects the transcription of specific miRNAs. We investigated the unique sequence motifs specific to the miRNAs concentrated in Pg-EVs. The levels of eleven miRNAs, including miR-155, were increased in Pg-EVs compared with those elevated after LPS stimulation. The Pg-induced miR-155 upregulation via TLR2 but not TLR4 signaling suggests the influence of TLR signaling on the miRNA composition of EVs. Furthermore, the miRNAs upregulated in Pg-EVs contained AGAGGG and GRGGSGC sequence motifs. Our findings demonstrate that Pg-induced alterations in EV-containing miRNA composition occur in a TLR4-independent manner. Notably, the concentrated miRNAs in Pg-EVs harbor specific motifs with a high G + C content within their sequences. The upregulation of specific miRNAs in EVs under infectious conditions suggests the influence of both innate immune receptor signals and miRNA sequence characteristics.


Synthesis of secretory leukocyte protease inhibitor using cell-free protein synthesis system

Secretory leukocyte protease inhibitor (SLPI) functions as a protease inhibitor that modulates excessive proteolysis in the body, exhibits broad-spectrum antimicrobial activity, regulates inflammatory responses, and plays an important role in the innate immunity. The purpose of the study was to artificially synthesize a SLPI, an antimicrobial peptide, and investigate its effect on antimicrobial activity against Porphyromonas gingivalis and interleukin-6 (IL-6) production. SLPI protein with a molecular weight of approximately 13 kDa was artificially synthesized using a cell-free protein synthesis (CFPS) system and investigated by western blotting and enzyme-linked immunosorbent assay (ELISA). Disulfide bond isomerase in the protein synthesis mixture increased the amount of SLPI synthesized. The synthesized SLPI (sSLPI) protein was purified and its antimicrobial activity was investigated based on the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells. The effect of sSLPI on IL-6 production in human periodontal ligament fibroblasts (HPLFs) was examined by ELISA. Our results showed that sSLPI significantly inhibited the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells and further inhibited IL-6 production by HPLFs. These results suggested that SLPI artificially synthesized using the CFPS system may play a role in the prevention of periodontal diseases through its antimicrobial and anti-inflammatory effects.


Porphyromonas gingivalis outer membrane vesicles in cerebral ventricles activate microglia in mice

Objective: Porphyromonas gingivalis (Pg) is thought to be involved in the progression of Alzheimer's disease (AD). Whether Pg or its contents can reach the brain and directly affect neuropathology is, however, unknown. Here, we investigated whether outer membrane vesicles (OMVs) of Pg translocate to the brain and induce the pathogenic features of AD. Material and Methods: Pg OMVs were injected into the abdominal cavity of mice for 12 weeks. Pg OMV translocation to the brain was detected by immunohistochemistry using an anti-gingipain antibody. Tau protein and microglial activation in the mouse brain were examined by western blotting and immunohistochemistry. The effect of gingipains on inflammation was assessed by real-time polymerase chain reaction using human microglial HMC3 cells. Results: Gingipains were detected in the region around cerebral ventricles, choroid plexus, and ventricular ependymal cells in Pg OMV-administered mice. Tau and phosphorylated Tau protein increased and microglia were activated. Pg OMVs also increased the gene expression of proinflammatory cytokines in HMC3 cells in a gingipain-dependent manner. Conclusion: Pg OMVs, including gingipains, can reach the cerebral ventricle and induce neuroinflammation by activating microglia. Pg OMVs may provide a better understanding of the implications of periodontal diseases in neurodegenerative conditions such as AD.


Neuropilin 1 (NRP1) Positively Regulates Adipogenic Differentiation in C3H10T1/2 Cells

Neuropilin 1 (NRP1), a non-tyrosine kinase receptor for several ligands, is highly expressed in many kinds of mesenchymal stem cells (MSCs), but its function is poorly understood. In this study, we explored the roles of full-length NRP1 and glycosaminoglycan (GAG)-modifiable NRP1 in adipogenesis in C3H10T1/2 cells. The expression of full-length NRP1 and GAG-modifiable NRP1 increased during adipogenic differentiation in C3H10T1/2 cells. NRP1 knockdown repressed adipogenesis while decreasing the levels of Akt and ERK1/2 phosphorylation. Moreover, the scaffold protein JIP4 was involved in adipogenesis in C3H10T1/2 cells by interacting with NRP1. Furthermore, overexpression of non-GAG-modifiable NRP1 mutant (S612A) greatly promoted adipogenic differentiation, accompanied by upregulation of the phosphorylated Akt and ERK1/2. Taken together, these results indicate that NRP1 is a key regulator that promotes adipogenesis in C3H10T1/2 cells by interacting with JIP4 and activating the Akt and ERK1/2 pathway. Non-GAG-modifiable NRP1 mutant (S612A) accelerates the process of adipogenic differentiation, suggesting that GAG glycosylation is a negative post-translational modification of NRP1 in adipogenic differentiation.

International Journal of Molecular Sciences


Lipocalin 2, synthesized using a cell-free protein synthesis system and encapsulated into liposomes, inhibits the adhesion of Porphyromonas gingivalis to human oral epithelial cells.


β-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells.


Porphyromonas gingivalis Outer Membrane Vesicles Stimulate Gingival Epithelial Cells to Induce Pro-Inflammatory Cytokines via the MAPK and STING Pathways

() is a keystone pathogen associated with chronic periodontitis and produces outer membrane vesicles (OMVs) that contain lipopolysaccharide (LPS), gingipains, and pathogen-derived DNA and RNA. -OMVs are involved in the pathogenesis of periodontitis. -OMV-activated pathways that induce the production of the pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in the human gingival epithelial cell line, OBA-9, were investigated. The role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in levels of -OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. -OMVs induced IL-6 and IL-8 production via the extracellular signal-regulated kinase (Erk) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, and NF-κB signaling pathways in OBA-9 cells. In addition, the stimulator of interferon genes (STING), an essential innate immune signaling molecule, was triggered by a cytosolic pathogen DNA. -OMV-induced IL-6 and IL-8 mRNA expression and production were significantly suppressed by STING-specific small interfering RNA. Taken together, these results demonstrated that -OMV-activated Erk1/2, JNK, p38 MAPK, STING, and NF-κB signaling pathways resulting in increased IL-6 and IL-8 expression in human gingival epithelial cells. These results suggest that -OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.


O-GlcNAcylation drives calcium signaling toward osteoblast differentiation: A bioinformatics-oriented study


Extracellular vesicles of P. gingivalis-infected macrophages induce lung injury

Periodontal diseases are common inflammatory diseases that are induced by infection with periodontal bacteria such as Porphyromonas gingivalis (Pg). The association between periodontal diseases and many types of systemic diseases has been demonstrated; the term "periodontal medicine" is used to describe how periodontal infection/inflammation may impact extraoral health. However, the molecular mechanisms by which the factors produced in the oral cavity reach multiple distant organs and impact general health have not been elucidated. Extracellular vesicles (EVs) are nano-sized spherical structures secreted by various types of cells into the tissue microenvironment, and influence pathophysiological conditions by delivering their cargo. However, a detailed understanding of the effect of EVs on periodontal medicine is lacking. In this study, we investigated whether EVs derived from Pg-infected macrophages reach distant organs in mice and influence the pathophysiological status. EVs were isolated from human macrophages, THP-1 cells, infected with Pg. We observed that EVs from Pg-infected THP-1 cells (Pg-inf EVs) contained abundant core histone proteins such as histone H3 and translocated to the lungs, liver, and kidneys of mice. Pg-inf EVs also induced pulmonary injury, including edema, vascular congestion, inflammation, and collagen deposition causing alveoli destruction. The Pg-inf EVs or the recombinant histone H3 activated the NF-κB pathway, leading to increase in the levels of pro-inflammatory cytokines in human lung epithelial A549 cells. Our results suggest a possible mechanism by which EVs produced in periodontal diseases contribute to the progression of periodontal medicine.

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease


Conversion from epithelial to partial-EMT phenotype by Fusobacterium nucleatum infection promotes invasion of oral cancer cells.

The ability of cancer cells to undergo partial-epithelial mesenchymal transition (p-EMT), rather than complete EMT, poses a higher metastatic risk. Although Fusobacterium nucleatum mainly inhabits in oral cavity, attention has been focused on the F. nucleatum involvement in colorectal cancer development. Here we examined the p-EMT regulation by F. nucleatum in oral squamous cell carcinoma (OSCC) cells. We cultured OSCC cells with epithelial, p-EMT or EMT phenotype with live or heat-inactivated F. nucleatum. Expression of the genes involved in epithelial differentiation, p-EMT and EMT were examined in OSCC cells after co-culture with F. nucleatum by qPCR. Cell growth and invasion of OSCC cells were also examined. Both live and heat-inactivated F. nucleatum upregulated the expression of p-EMT-related genes in OSCC cells with epithelial phenotype, but not with p-EMT or EMT phenotype. Moreover, F. nucleatum promoted invasion of OSCC cells with epithelial phenotype. Co-culture with other strains of bacteria other than Porphyromonas gingivalis did not alter p-EMT-related genes in OSCC cells with epithelial phenotype. F. nucleatum infection may convert epithelial to p-EMT phenotype via altering gene expression in OSCC. Oral hygiene managements against F. nucleatum infection may contribute to reduce the risk for an increase in metastatic ability of OSCC.


Involvement of Fusobacterium Species in Oral Cancer Progression: A Literature Review Including Other Types of Cancer

Chronic inflammation caused by infections has been suggested to be one of the most important cause of cancers. It has recently been shown that there is correlation between intestinal bacteria and cancer development including metastasis. As over 700 bacterial species exist in an oral cavity, it has been concerning that bacterial infection may cause oral cancer. However, the role of bacteria regarding tumorigenesis of oral cancer remains unclear. Several papers have shown that species deriving the oral cavities, especially, play a crucial role for the development of colorectal and esophageal cancer. is a well-known oral bacterium involved in formation of typical dental plaque on human teeth and causing periodontal diseases. The greatest characteristic of is its ability to adhere to various bacteria and host cells. Interestingly, is frequently detected in oral cancer tissues. Moreover, detection of is correlated with the clinical stage of oral cancer. Although the detailed mechanism is still unclear, species have been suggested to be associated with cell adhesion, tumorigenesis, epithelial-to-mesenchymal transition, inflammasomes, cell cycle, etc. in oral cancer. In this review, we introduce the reports focused on the association of species with cancer development and progression including oral, esophageal, and colon cancers.


N-(3-oxododecanoyl)-homoserine lactone regulates osteoblast apoptosis and differentiation by mediating intracellular calcium

Pseudomonas aeruginosa (P. aeruginosa) is associated with periapical periodontitis. The lesions are characterized by a disorder in osteoblast metabolism. Quorum sensing molecular N-(3-oxododecanoyl)-homoserine lactone (AHL) is secreted by P. aeruginosa and governs the expression of numerous virulence factors. AHL can trigger intracellular calcium ([Ca]) fluctuations in many host cells. However, it is unclear whether AHL can regulate osteoblast metabolism by affecting [Ca] changes or its spatial correlation. We explored AHL-induced apoptosis and differentiation in pre-osteoblastic MC3T3-E1 cells and evaluated [Ca] mobilization using several extraction methods. The spatial distribution pattern of [Ca] among cells was investigated by Moran's I, an index of spatial autocorrelation. We found that 30 μM and 50 μM AHL triggered opposing osteoblast fates. At 50 μM, AHL inhibited osteoblast differentiation by promoting mitochondrial-dependent apoptosis and negatively regulating osteogenic marker genes, including Runx2, Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). In contrast, prolonged treatment with 30 μM AHL promoted osteoblast differentiation concomitantly with cell apoptosis. The elevation of [Ca] levels in osteoblasts treated with 50 μM AHL was spatially autocorrelated, while no such phenomenon was observed in 30 μM AHL-treated osteoblasts. The blocking of cell-to-cell spatial autocorrelation in the osteoblasts provoked by 50 μM AHL significantly inhibited apoptosis and partially restored differentiation. Our observations suggest that AHL affects the fate of osteoblasts (apoptosis and differentiation) by affecting the spatial correlation of [Ca] changes. Thus, AHL acts as a double-edged sword for osteoblast function.


Outer membrane vesicles derived from Porphyromonas gingivalis induced cell death with disruption of tight junctions in human lung epithelial cells.


Suppresive effects of 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer on the adherence of Candida species and MRSA to acrylic denture resin.


Extracellular Vesicles Enriched With Moonlighting Metalloproteinase Are Highly Transmissive, Pro-Tumorigenic, and Trans-Activates Cellular Communication Network Factor ( CCN2/CTGF): CRISPR Against Cancer

Matrix metalloproteinase 3 (MMP3) plays multiple roles in extracellular proteolysis as well as intracellular transcription, prompting a new definition of moonlighting metalloproteinase (MMP), according to a definition of protein moonlighting (or gene sharing), a phenomenon by which a protein can perform more than one function. Indeed, connective tissue growth factor (CTGF, aka cellular communication network factor 2 (CCN2)) is transcriptionally induced as well as cleaved by MMP3. Moreover, several members of the MMP family have been found within tumor-derived extracellular vesicles (EVs). We here investigated the roles of MMP3-rich EVs in tumor progression, molecular transmission, and gene regulation. EVs derived from a rapidly metastatic cancer cell line (LuM1) were enriched in MMP3 and a C-terminal half fragment of CCN2/CTGF. MMP3-rich, LuM1-derived EVs were disseminated to multiple organs through body fluid and were pro-tumorigenic in an allograft mouse model, which prompted us to define LuM1-EVs as oncosomes in the present study. Oncosome-derived MMP3 was transferred into recipient cell nuclei and thereby trans-activated the promoter, and induced CCN2/CTGF production in vitro. TRENDIC and other cis-elements in the promoter were essential for the oncosomal responsivity. The CRISPR/Cas9-mediated knockout of MMP3 showed significant anti-tumor effects such as the inhibition of migration and invasion of tumor cells, and a reduction in CCN2/CTGF promoter activity and fragmentations in vitro. A high expression level of MMP3 or CCN2/CTGF mRNA was prognostic and unfavorable in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers. These data newly demonstrate that oncogenic EVs-derived MMP is a transmissive trans-activator for the cellular communication network gene and promotes tumorigenesis at distant sites.


Inhibitory effect of retinoic acid receptor agonists on in vitro chondrogenic differentiation.

Retinoic acid (RA), an active metabolite of vitamin A, plays pivotal roles in a wide variety of biological processes, such as body patterning, organ development, and cell differentiation and proliferation. RA signaling is mediated by nuclear retinoic acid receptors, α, β, and γ (RARα, RARβ, and RARγ). RA is a well-known regulator of cartilage and skeleton formation and RARs are also essential for skeletal growth and hypertrophic chondrocyte-specific gene expression. These important roles of RA and RARs in chondrogenesis have been widely investigated using in vivo mouse models. However, few reports are available on the function of each subtype of RARs on in vitro chondrocyte differentiation. Here, we examined the effect of specific agonists of RARs on chondrogenic differentiation of ATDC5 and C3H10T1/2 cells. Subtype-specific RAR agonists as well as RA decreased the expressions of chondrogenic differentiation marker genes and inhibited chondrogenic differentiation, which was accompanied with morphological change to spindle-shaped cells. Among RAR agonists, RARα and RARγ agonists revealed a strong inhibitory effect on chondrogenic differentiation. RARα and RARγ agonists also hampered viability of ATDC5 cells. These observations suggested that RARα and RARγ are dominant receptors of RA signaling that negatively regulate chondrogenic differentiation.


Quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone: An all-rounder in mammalian cell modification.

BACKGROUND: Bacteria exhibit multi-cellular social behavior, such as biofilm formation, virulence generation, bioluminescence, or sporulation, through cell-to-cell communication involving a quorum sensing (QS) system capable of sensing species density. Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous gram-negative opportunistic pathogen that is frequently isolated from immunocompromised patients. It is particularly detected in patients with severe periodontitis and persistent endodontic infections, forcing a rethink of the role of this opportunistic pathogen in oral lesions.HIGHLIGHT: N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) is a pivotal QS molecule, which regulates numerous virulence genes in P. aeruginosa and exhibits broad biological modulation effects in mammalian cells. In this review, we highlight the diverse OdDHL-mediated apoptosis and immunomodulatory effects on host cells. The structural properties, signaling pathways, targeted genes and proteins, and intracellular metabolism of OdDHL are also discussed to clarify the interactions between P. aeruginosa and the host.CONCLUSION: The purpose of this review is to identify a valid target for quenching OdDHL, which could potentially eliminate the pathogenic effect of P. aeruginosa.


Outer membrane vesicles of Porphyromonas gingivalis attenuate insulin sensitivity by delivering gingipains to the liver

Outer membrane vesicles (OMVs) are nanosized particles derived from the outer membrane of gram-negative bacteria. Oral bacterium Porphyromonas gingivalis (Pg) is known to be a major pathogen of periodontitis that contributes to the progression of periodontal disease by releasing OMVs. The effect of Pg OMVs on systemic diseases is still unknown. To verify whether Pg OMVs affect the progress of diabetes mellitus, we analyzed the cargo proteins of vesicles and evaluated their effect on hepatic glucose metabolism. Here, we show that Pg OMVs were equipped with Pg-derived proteases gingipains and translocated to the liver in mice. In these mice, the hepatic glycogen synthesis in response to insulin was decreased, and thus high blood glucose levels were maintained. Pg OMVs also attenuated the insulin-induced Akt/ glycogen synthase kinase-3 (GSK-3 signaling in a gingipain-dependent fashion in hepatic HepG2 cells. These results suggest that the delivery of gingipains mediated by Pg OMV elicits changes in glucose metabolisms in the liver and contributes to the progression of diabetes mellitus.

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease


Advanced glycation end-products increase IL-6 and ICAM-1 expression via RAGE, MAPK and NF-kB pathways in human gingival fibroblasts

Diabetes mellitus (DM) is a risk factor for periodontal diseases and may exacerbate the progression of the pathogenesis of periodontitis. Advanced glycation end-products (AGEs) cause DM complications relative to levels of glycemic control and larger amounts accumulate in the periodontal tissues of patients with periodontitis and DM. In the present study, we investigated the effects of AGEs on the expression of inflammation-related factors in human gingival fibroblasts (HGFs) to elucidate the impact of AGEs on DM-associated periodontitis. HGFs were cultured with or without AGEs. Cell viability was examined, and RNA and protein fractions were isolated from AGE-treated cells. The expression of interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1), and the receptor for AGE (RAGE) was investigated using reverse transcription-polymerase chain reaction, quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and reactive oxygen species activity was measured using a kit with 2',7'-dichlorofluorescin diacetate. Human monocytic cells (THP-1) labeled with a fluorescent reagent were co-cultured with HGFs treated with AGEs and IL-6 siRNA, and the adhesive activity of THP-1 cells to HGFs was assessed. The expression of IL-6 and ICAM-1 was examined when HGFs were pretreated with recombinant human IL-6, the siRNAs of RAGE and IL-6, and inhibitors of MAPK and NF-κB, and then cultured with and without AGEs. The phosphorylation of MAPK and NF-κB was assessed using western blotting. AGEs increased the mRNA and protein expressions of RAGE, IL-6, ICAM-1 and reactive oxygen species activity in HGFs, and promoted the adhesion of THP-1 cells to HGFs, but had no effect on cell viability until 72 hours. Recombinant human IL-6 increased ICAM-1 expression in HGFs, while the siRNAs of RAGE and IL-6 inhibited AGE-induced IL6 and ICAM1 mRNA expression, and IL-6 siRNA depressed AGE-induced THP-1 cell adhesion. AGEs increased the phosphorylation of p38 and ERK MAPKs, p65 NF-κB and IκBα, while inhibitors of p38, ERK MAPKs and NF-κB significantly decreased AGE-induced IL-6 and ICAM-1 expression. AGEs increase IL-6 and ICAM-1 expression via the RAGE, MAPK and NF-κB pathways in HGFs and may exacerbate the progression of the pathogenesis of periodontal diseases.


Reduction of protein phosphatase 2A Ca promotes in vivo bone formation and adipocyte differentiation.

Serine/threonine protein phosphatase 2A (PP2A) regulates diverse physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Previously, we demonstrated that silencing of the α-isoform of PP2A catalytic subunit (PP2A Cα) in osteoblasts accelerated osteoblast differentiation, whereas its overexpression suppressed differentiation. In this study, we examined the role of PP2A Cα in in vivo bone formation by generating transgenic mice (PP2A-Tg), in which the dominant negative form of PP2A Cα was specifically expressed in osteoblasts. PP2A-Tg mice exhibited an increase in body weight, cortical bone mineral density, and cortical bone thickness. Interestingly, they also displayed higher amounts of adipose tissue in the bone marrow of tibiae. The co-culture study showed that PP2A Cα-knockdown osteoblasts stimulated adipocyte differentiation from undifferentiated mesenchymal cells via upregulation of the adipocyte marker genes, such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). These results indicated that the reduction of PP2A Cα levels in osteoblasts promoted bone formation in vivo. Additionally, PP2A Cα in osteoblasts was also potentially involved in controlling adipocyte differentiation through a paracrine mechanism.


Advanced glycation end-products and Porphyromonas gingivalis lipopolysaccharide increase calprotectin expression in human gingival epithelial cells.

Accumulation of advanced glycation end-products (AGEs) in periodontal tissues of patients with diabetes mellitus aggravates periodontitis, but the mechanisms are unknown. Calprotectin, a heterocomplex of S100A8 and S100A9 proteins, is a constitutive cytoplasmic component of healthy gingival epithelial cells. This study aimed at investigating the effects of AGE and Porphyromonas gingivalis lipopolysaccharide (PgLPS) on calprotectin expression in the human gingival epithelial cell line OBA-9. AGE and PgLPS increased the expression of S100A8 and S100A9 mRNAs, and AGE + PgLPS co-stimulation amplified their expression in OBA-9 cells. A higher concentration of calprotectin in cell lysates was also induced by stimulation with AGE and/or PgLPS. S100A8 was mainly translocated from the nucleus to the cytoplasm by AGE stimulation, while cytoplasmic localization of S100A9 was not altered following stimulation with AGE and/or PgLPS. Calprotectin was found in the cytoplasm of BSA-treated cells, but cytoplasmic and nuclear localization was observed following stimulation with AGE and/or PgLPS. AGE-induced S100A8, and S100A9 mRNA expression was partially suppressed by RAGE-specific siRNA. In contrast, PgLPS-induced S100A8 and S100A9 mRNA expression was strongly suppressed by TLR2-specific siRNA. Furthermore, the inhibition of p38, JNK MAPK, and NF-κB attenuated AGE- and PgLPS-induced S100A8 and S100A9 mRNA expression. Taken together, these results demonstrate that AGE acts in synergy with PgLPS to stimulate RAGE and TLR2 expression and activate p38, JNK MAPK, and NF-κB signaling pathways, resulting in increased activation of calprotectin (S100A8/S100A9) in human gingival epithelial cells. Our results suggest that calprotectin may be involved in the pathogenesis of diabetic periodontitis. This article is protected by copyright. All rights reserved.


PKR induces the expression of NLRP3 by regulating the NF-κB pathway in Porphyromonas gingivalis-infected osteoblasts

The double-stranded RNA-dependent kinase (PKR), which is activated by double stranded RNA, induces inflammation by regulating NF-κB signaling. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome also modulates inflammation in response to infection. Porphyromonas gingivalis (P.gingivalis) is an oral bacterium which is implicated in the pathogenesis of periodontal diseases. We previously reported that PKR is a key modulator of bone metabolism and inflammation in the periodontal tissue. PKR was also reported to induce inflammation in response to microbes by regulating the NLRP3 inflammasome, suggesting that PKR could affect inflammation along with NLRP3 in periodontal diseases. In this study, we investigated the effects of PKR on NLRP3 expression and NF-κB activity in P. gingivalis infected osteoblasts. We first constructed a SNAP26b-tagged P.gingivalis (SNAP-P. g.) and traced its internalization into the cell. SNAP-P. g. increased the activity of PKR and NF-κB and also induced NLRP3 expression in osteoblasts. Inhibition of NF-κB attenuated SNAP-P. g.-induced NLRP3 expression. The knockdown of PKR using shRNA decreased both the activity of NF-κB and the expression of NLRP3 induced by SNAP-P.g.. We therefore concluded that in osteoblasts, P. gingivalis activated PKR, which in turn increased NLRP3 expression by activating NF-κB. Our results suggest that PKR modulates inflammation by regulating the expression of the NLRP3 inflammasome through the NF-κB pathway in periodontal diseases.


Role of Protein Phosphatase 2A in Osteoblast Differentiation and Function.

The reversible phosphorylation of proteins plays hugely important roles in a variety of cellular processes, such as differentiation, proliferation, and apoptosis. These processes are strictly controlled by protein kinases (phosphorylation) and phosphatases (de-phosphorylation). Here we provide a brief history of the study of protein phosphorylation, including a summary of different types of protein kinases and phosphatases. One of the most physiologically important serine/threonine phosphatases is PP2A. This review provides a description of the phenotypes of various PP2A transgenic mice and further focuses on the known functions of PP2A in bone formation, including its role in osteoblast differentiation and function. A reduction in PP2A promotes bone formation and osteoblast differentiation through the regulation of bone-related transcription factors such as Osterix. Interestingly, downregulation of PP2A also stimulates adipocyte differentiation from undifferentiated mesenchymal cells under the appropriate adipogenic differentiation conditions. In osteoblasts, PP2A is also involved in the ability to control osteoclastogenesis as well as in the proliferation and metastasis of osteosarcoma cells. Thus, PP2A is considered to be a comprehensive factor in controlling the differentiation and function of cells derived from mesenchymal cells such as osteoblasts and adipocytes.


Porphyromonas gingivalis attenuates the insulin-induced phosphorylation and translocation of forkhead box protein O1 in human hepatocytes


Preventive effect of Daiokanzoto (TJ-84) on 5-fluorouracil-induced human gingival cell death through the inhibition of reactive oxygen species production

Daiokanzoto (TJ-84) is a traditional Japanese herbal medicine (Kampo formulation). While many Kampo formulations have been reported to regulate inflammation and immune responses in oral mucosa, there is no evidence to show that TJ-84 has beneficial effects on oral mucositis, a disease resulting from increased cell death induced by chemotherapeutic agents such as 5-fluorouracil (5-FU). In order to develop effective new therapeutic strategies for treating oral mucositis, we investigated (i) the mechanisms by which 5-FU induces the death of human gingival cells and (ii) the effects of TJ-84 on biological events induced by 5-FU. 5-FU-induced lactate dehydrogenase (LDH) release and pore formation in gingival cells (Sa3 cell line) resulted in cell death. Incubating the cells with 5-FU increased the expression of nucleotide-binding domain and leucine-rich repeat containing PYD-3 (NLRP3) and caspase-1. The cleavage of caspase-1 was observed in 5-FU-treated cells, which was followed by an increased secretion of interleukin (IL)-1β. The inhibition of the NLRP3 pathway slightly decreased the effects of 5-FU on cell viability and LDH release, suggesting that NLRP3 may be in part involved in 5-FU-induced cell death. TJ-84 decreased 5-FU-induced LDH release and cell death and also significantly inhibited the depolarization of mitochondria and the up-regulation of 5-FU-induced reactive oxygen species (ROS) and nitric oxide (NO) production. The transcriptional factor, nuclear factor-κB (NF-κB) was not involved in the 5-FU-induced cell death in Sa3 cells. In conclusion, we provide evidence suggesting that the increase of ROS production in mitochondria, rather than NLRP3 activation, was considered to be associated with the cell death induced by 5-FU. The results also suggested that TJ-84 may attenuate 5-FU-induced cell death through the inhibition of mitochondrial ROS production.


Reduction of PP2A C stimulates adipogenesis by regulating the Wnt/GSK-3/-catenin pathway and PPAR expression

Serine/threonine protein phosphatase 2A (PP2A) regulates several physiological processes such as the cell cycle, cell growth, apoptosis, and signal transduction. In this study, we examined the expression and role of PP2A Cα in adipocyte differentiation. PP2A Cα expression and PP2A activity decreased during adipocyte differentiation in C3H10T1/2 and 3T3-L1 cells and the expression of adipocyte marker genes such as PPARγ and adiponectin increased. To further clarify the role of PP2A Cα in adipocyte differentiation, we constructed PP2A knockdown cells by infecting C3H10T1/2 cells with a lentivirus expressing a shRNA specific for the PP2A Cα (shPP2A cells). Silencing of PP2A Cα in C3H10T1/2 cells dramatically stimulated adipocyte differentiation and lipid accumulation, which were accompanied by expression of adipocyte marker genes. Silencing of PP2A Cα suppressed Wnt10b expression and reduced the levels of the inactivated form of GSK-3β (phospho-GSK-3β), leading to the reduction of β-catenin levels in the nucleus and its transcriptional activity. Treatment with LiCl, a GSK-3β inhibitor, and inhibition of PPARγ expression suppressed the accelerated adipogenesis of shPP2A cells. Our data indicate that PP2A Cα plays an important role in the regulation of adipocyte differentiation by regulating the Wnt/GSK-3β/β-catenin pathway and PPARγ expression.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research


Oral porphyromonas gingivalis translocates to the liver and regulates hepatic glycogen synthesis through the Akt/GSK-3b signaling pathway

Periodontal diseases are common chronic inflammatory disorders that result in the destruction of tissues around teeth. Many clinical studies suggest that periodontal diseases are risk factors for insulin resistance and diabetic mellitus development. However, the molecular mechanisms by which periodontal diseases regulate the progress of diabetes mellitus remain unknown. In this study, we investigated whether Porphyromonas gingivalis (P.g.), a major pathogen of periodontal diseases, present in the oral cavity, moves to the liver and affects hepatic glycogen synthesis. SNAP26b-tagged P.g. (SNAP-P.g.) was introduced into the oral cavity to induce periodontal disease in 4-week old female Balb/c mice. SNAP-P.g. was detected in the liver extracted from SNAP-P.g.-treated mice using nested PCR analysis. High blood glucose levels tended to promote SNAP-P.g. translocation from the oral cavity to the liver in mice. Periodic acid-Schiff staining suggested that hepatic glycogen synthesis decreased in SNAP-P.g.-treated mice. SNAP-P.g. was also internalized into the human hepatoma cell line HepG2, and this attenuated the phosphorylation of insulin receptor substrate (IRS)-1, Akt and glycogen synthase kinase-3β induced by insulin. Insulin-induced glycogen synthesis was suppressed by SNAP-P.g. in HepG2 cells. Our results suggest that P.g. translocation from the oral cavity to the liver may contribute to the progress of diabetes mellitus by influencing hepatic glycogenesis.

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease


Protein phosphatase 2A Cα regulates osteoblast differentiation and the expressions of bone sialoprotein and osteocalcin via osterix transcription factor.

Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Osterix is a zinc-finger-containing transcription factor that is essential for osteoblast differentiation and regulation of many bone-related genes. We have recently reported that decrease in α-isoform of PP2A catalytic subunit (PP2A Cα) accelerates osteoblast differentiation through the expression of bone-related genes. In this study, we further examined the role of PP2A Cα in osteoblast differentiation by establishing the stable cell lines that overexpress PP2A Cα. Overexpression of PP2A Cα reduced alkaline phosphatase (ALP) activity. Osteoblast differentiation and mineralization were also decreased in PP2A Cα-overexpressing cells, with reduction of bone-related genes including osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). Luciferase assay showed that the transcriptional activity of the Osterix promoter region was decreased in PP2A Cα-overexpressing cells. Introduction of ectopic Osterix rescued the expression of Bsp and OCN in PP2A Cα-overexpressing cells. These results indicate that PP2A Cα and its activity play a negative role in osteoblast differentiation and Osterix is a key factor responsible for regulating the expressions of Bsp and OCN during PP2A Cα-mediated osteoblast differentiation.


Protein phosphatase 2A C is involved in osteoclastogenesis by regulating RANKL and OPG expression in osteoblasts.

We examined whether alteration of PP2A Cα expression in osteoblasts is involved in osteoclast differentiation. Reduction of PP2A Cα in MC3T3-E1 cells (shPP2A) decreased receptor activator of nuclear factor κB ligand (RANKL) expression and increased osteoprotegerin (OPG) expression. The conditioned medium from shPP2A cells failed to induce NFATc1 as well as the expression of osteoclast marker genes cathepsin K and osteoclast-associated receptor (OSCAR) in bone marrow macrophage cells. Treatment of bone marrow macrophage cells with the conditioned medium from shPP2A cells impaired osteoclastogenesis. These results suggest that alteration of PP2A Cα expression in osteoblasts modulates the expressions of RANKL and OPG, which are involved in osteoclastogenesis via the NFATc1 transcription factor.


PKR plays a positive role in osteoblast differentiation by regulating GSK-3b activity through a b-catenin-independent pathway

Double-stranded RNA-dependent protein kinase (PKR) is involved in various cellular functions. We previously reported that PKR regulates osteoblast differentiation, but the specific mechanisms by which this occurs remain unclear. In this study, we investigated the role of PKR in Glycogen synthase kinase 3 (GSK-3) regulation of osteoblast differentiation. Lithium chloride (LiCl), a GSK-3 inhibitor, increased GSK-3 phosphorylation in MC3T3-E1 and MG-63 cells. LiCl also inhibited Runx2 and expression of its regulated genes, causing inhibition of Alkaline phosphatase activity and mineralization. LiCl injection to the calvaria in mice suppressed bone formation. Further, GSK-3 phosphorylation was increased in osteoblasts, by Akt-independent mechanisms, in which PKR was constitutively inactivated. A PKR inhibitor, 2-aminopurine, also induced GSK-3 phosphorylation in MC3T3-E1 and MG-63 cells. Further, Runx2 and its regulated genes were inhibited in PKR-inactivated osteoblasts, and differentiation was suppressed through a -catenin-independent pathway. PKR positively regulates the differentiation of osteoblasts by mediating GSK-3 activity through a -catenin-independent pathway.


Interaction between PKR and PACT mediated by LPS-inducible NF-kB in human gingival cells

The double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase expressed constitutively in mammalian cells. PKR is activated upon virus infection by double-stranded RNA (dsRNA), and plays a critical role in host antiviral defense mechanisms. PKR is also known to regulate various biological responses, including cell differentiation and apoptosis. However, whether PKR is involved in the progress of periodontitis is not clear. The present study explained the phosphorylation of PKR by LPS in the human gingival cell line, Sa3. Expression of genes encoding LPS receptors was detected in Sa3 cells and treatment of cells with 1 µg/mL LPS for 6 h caused PKR phosphorylation. LPS elevated the expression of the protein activator of PKR (PACT) mRNA and protein, followed by the enhanced association between PACT and PKR within 3 h. In addition, LPS treatment induced the translocation of NF-κB to the nucleus after 30 min, and inhibition of NF-κB decreased the PACT-PKR interaction induced by LPS. The level of pro-inflammatory cytokine mRNA, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), appeared within 45 min and reached at the maximal levels by 90 min after the addition of LPS. This induction of pro-inflammatory cytokines was not affected by RNAi-mediated silencing of PKR and a pharmacological inhibitor of PKR, whereas the inhibition of NF-κB decreased it. These results indicated that LPS induces PKR phosphorylation and the PACT-PKR association in Sa3 cells. Our results also suggest that NF-κB is involved in the PACT-PKR interaction and the production of pro-inflammatory cytokines in periodontitis.


Reduction of protein phosphatase 2A Cα enhances bone formation and osteoblast differentiation through the expression of bone-specific transcription factor Osterix.

The serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as control of cell cycle, growth, and division. On the other hand, Osterix is a zinc-finger-containing transcription factor that is essential for the differentiation of osteoblasts and regulation of many bone-related genes. Here we examined the effect of okadaic acid (OA), a specific inhibitor of PP2A, on bone formation in vivo and the molecular mechanism regulated by PP2A Cα in osteoblast differentiation. Administration of 1nM OA to the calvarial region in mice increased bone mineral density, as shown by μCT, while histomorphological analysis showed an increase in mineral apposition and bone thickness in the same region. In addition, treatment with 1nM OA stimulated osteoblast differentiation and the expression of Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN) in mouse osteoblastic MC3T3-E1 cells. Moreover, the expression and phosphatase activity of PP2A Cα was decreased in the initial step of osteoblast differentiation, which was in parallel with an increase in Osterix expression. To further clarify the role of PP2A Cα in osteoblast differentiation, we constructed PP2A knock-down cells by infecting MC3T3-E1 cells with a lentivirus expressing shRNA specific for the PP2A Cα. Accordingly, the silencing of PP2A Cα in MC3T3-E1 cells dramatically increased osteoblast differentiation and mineralization, which were accompanied with expressions of Osterix, Bsp, and OCN. Our data indicate that PP2A Cα plays an important role in the regulation of bone formation and osteoblast differentiation through the bone-related genes.


Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells.

(Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-B translocation in human hepatic HepG2 cells, ILY did not affect NF-B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.)

Biochemical and Biophysical Research Communications


High glucose levels increase osteopontin production and pathologic calcification in rat dental pulp tissues.

INTRODUCTION: Pulp stones are frequently formed as a pathologic calcification product in dental pulp tissues, but the pathogenesis is poorly understood. We previously found that osteopontin (OPN) was produced by dental pulp cells, and its expression was associated with formation of the pulp stone matrix. It was reported that amorphous calcification appeared in the dental pulp of diabetic patients. The aim of this study was to determine the relationship between OPN expression and pathologic calcification in rat diabetic pulp. METHODS: The effect of glucose on OPN production and alkaline phosphatase activity in cultured rat dental pulp cells (RPC-C2A) was investigated, and then dental pulp calcification and OPN expression in diabetic rats were determined and compared with those in healthy rats by histologic and immunohistochemical analyses. RESULTS: In RPC-C2A cells, biochemical analysis showed that a high concentration of glucose (50 mmol/L) increased OPN protein production and alkaline phosphatase activity 1.3-fold and 1.5-fold, respectively. Histologic observations showed more calcified particles in dental pulp tissues in diabetic than in nondiabetic rats. Moreover, a thickened layer of predentin was formed in the radicular pulp of diabetic rats. OPN was more strongly stained around the calcified particles and in the odontoblast zone under the thickened predentin in diabetic rats. CONCLUSIONS: OPN might be a key molecule involved in the increase of pathologic pulp calcifications, which are frequently observed in diabetic patients.


Negative regulation of TIMP1 is mediated by transcription factor TWIST1

TWIST1 is involved in tumor invasion and metastasis by promoting epithelial-to-mesenchymal transition. However, the molecular target of TWIST1 involving this mechanism is poorly understood. To identify the TWIST1-regulated genes, we established the Saos-2 cells stably expressing TWIST1 gene by transfecting TWIST1 cDNA into the cells and performed a differential display approach by using annealing control primers. Here, we report that one of the genes that were down-regulated in TWIST1 expressing cells is predicted to be TIMP1. TIMP1 has been reported as the naturally occurring specific inhibitor of matrix metalloproteinases (MMPs). Overexpression of TWIST1 gene suppressed the expression of TIMP1 mRNA but had no effect on TIMP2 and MMP-2 expression, as determined by semi-quantitative RT-PCR. We showed that TWIST1 was up-regulated in SCCBHY cells, which have a strong capacity of invasion into mandibular bone compared with SCCHN cells. Our present results suggest that TWIST1 is involved in tumor invasion by regulating the expression of TIMP1.


PKR-mediated degradation of STAT1 regulates osteoblast differentiation

The double-stranded RNA-dependent protein kinase (PKR) plays a critical role in various biological responses including antiviral defense, cell differentiation, apoptosis, and tumorigenesis. In this study, we investigated whether PKR could affect the post-translational modifications of STAT1 protein and whether these modifications regulate osteoblast differentiation. We demonstrated that PKR was necessary for the ubiquitination of STAT1 protein. The expressions of bone-related genes such as type I collagen, integrin binding sialoprotein, osteopontin, and osterix were suppressed in osteoblasts lacking PKR activity. In contrast, the expressions of interleukin-6 and matrix metalloproteinases 8 and 13 increased in PKR-mutated osteoblasts. The expression and degradation of STAT1 protein were regulated by PKR in a SLIM-dependent pathway. Inhibition of SLIM by RNA interference resulted in the decreased activity of Runx2 in osteoblasts. Stimulation of interleukin-6 expression and suppression of alkaline phosphatase activity were regulated through by SLIM-dependent pathway. However, expressions of bone-related genes and MMPs were regulated by SLIM-independent pathway. Our present results suggest that the aberrant accumulation of STAT1 protein induced by loss of PKR regulate osteoblast differentiation through both SLIM/STAT1-dependent and -independent pathways.


徳島大学パイロット事業支援プログラム「歯科医療系学生の口腔保健・福祉体験実習による健康長寿支援」

Journal of University Education Research


Calcineurin regulates phosphorylation satus of transcriptionfactor osterix

Osterix is an osteoblast-specific transcriptional factor that is essential for osteoblast differentiation and bone formation. Calcineurin regulates bone formation through modulating osteoblast differentiation. However, post-translational modification of osterix such as phosphorylation and interactions between osterix and calcineurin remains unclear. In the present study, we demonstrated that calcineurin interacted with osterix determined by immunoprecipitation assay and Western analysis. Immunocytochemical study also revealed that osterix and calcineurin were co-localized in nucleus. Deletion of calcineurin binding motif on osterix molecule disrupted osterix-calcineurin interaction. Phosphorylation status of osterix was augmented by treatment with phosphatase inhibitors, FK506 and calyculin A. In contrast, treatment of recombinant calcineurin reduced phosphorylation status of osterix. Our present study suggests that calcineurin has an important role in the function of osterix through its modification of phosphorylation.


Histone H1.2 is translocated to mitochondria and associates with Bak in bleomycin-induced apoptotic cells

Bleomycin induces single- and double-stranded breaks in DNA, with consequent mitochondrial membrane aberrations that lead to the apoptotic cell death. It is poorly understood how DNA damage-inducing apoptotic signals are transmitted to mitochondria, from which apoptotic factors are released into the cytoplasm. Here, we investigated the localization of histone H1.2 in the bleomycin-treated human squamous carcinoma SCCTF cells. The presence of DNA double-strand breaks in the bleomycin-treated cells was examined by Western analysis using antibody against phosphorylated histone H2AX (gamma-H2AX). Incubation of SCCTF cells for 48 h with 10 microM bleomycin induced apoptosis, as determined by cleavage of lamin B1 to 28 kDa fragment and DNA ladder formation. The mitochondrial permeabilization causing apoptotic feature was also detected with MitoCapture in the bleomycin-treated cells. Histone H1.2 was translocated from the nucleus to the mitochondria after treatment with bleomycin and co-localized with Bak in mitochondria. Our present results suggest that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following double-stranded breaks of DNA by bleomycin.


Cobalt chloride induces apoptosis and zinc chloride suppresses cobalt-induced apoptosis by bcl-2 expression in human submandibular gland HSG cells

To determine the effects of cobalt chloride on human submandibular gland cells, HSG cells were exposed to various concentrations of cobalt chloride. Cobalt chloride induced cytotoxicity and cell death in HSG cells as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, marked nuclear condensation and fragmentation of chromatin were observed in cobalt chloride-treated cells. Cobalt chloride induced DNA ladder formation in HSG cells in both dose- and time-dependent manner with maximal effect at a concentration of 0.5 mM and 48 h, respectively. Cobalt chloride inhibited the expression of both Bcl-2 protein and mRNA in dose- and time-dependent manner. Zinc chloride recovered the cobalt-suppressed Bcl-2 expression and protected against cobalt-induced apoptosis in HSG cells. Our results show that the pathway of the apoptosis in HSG cells is regulated by cobalt chloride and zinc chloride. Our results also indicate that cobalt-induced apoptotic steps in HSG cells are related to the production of Bcl-2 protein.


Expression of PTEN and Akt phosphorylation in Lippopolysacccharide-treated NIH3T3 cells

PTEN is a tumor suppressor gene encoding a phosphatase, and it negatively regulates cell survival mediated by the phosphoinositol 3-kinase (PI3-Kinase)-Akt pathway. To elucidate PTEN expression and its effect on the PI3-kinase-Akt pathway in fibroblasts and macrophages, we investigated the expression of PTEN and the phosphorylation status of Akt in NIH3T3 and RAW264.7 cells treated with LPS. Phosphorylation of Akt was induced by LPS treatment in a dose-dependent manner in RAW264.7 cells, but not in NIH3T3 cells. LPS induced the expression of PTEN in a dose and time-dependent manner in NIH3T3 cells (0-1 microg/ml, 0-6h). However, LPS did not stimulate PTEN expression in RAW264.7 cells. These data indicate the existence of diverse mechanisms for PTEN expression and Akt activation in fibroblasts and macrophages. RNA interference using double-stranded RNA specific for the PTEN gene reduced both mRNA and protein levels of PTEN in NIH3T3 cells treated or not with LPS. The phosphorylation status of Akt in NIH3T3 cells stimulated with LPS did not change when the PTEN expression had been inhibited by RNA interference. The present results suggest that the up-regulation of PTEN expression by LPS is not involved in the activation of Akt in NIH3T3 cells. PTEN expression might be involved in the diverse inflammatory responses to LPS in fibroblasts and macrophages.


Calyculin A induces apoptosis and stimulates phosphorylation of p65NF-κB in human osteoblastic osteosarcoma MG63 cells

Previously, we reported that okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, induced apoptosis in human osteoblastic cells. However, it is not clear whether calyculin A, another inhibitor of protein phosphatases, would induce apoptosis in human osteoblastic cells and if so, which mechanisms are involved and whether the phosphorylation status of NF-kappaB could be affected by the treatment with calyculin A. In this report, we demonstrate that calyculin A induced apoptosis in MG63 cells, as judged by WST-8 assay, nuclear fragmentation, and DNA ladder formation. Expression of PTEN, FasL, and FasR mRNA was stimulated by calyculin A treatment in MG63 cells. Calyculin A also enhanced the phosphorylation level of NF-kappaB, as judged from the results of Western blot analysis and an in vitro dephosphorylation assay. Western blot analysis with anti-phospho-p65NF-kappaB antibody disclosed that the NF-kappaB was phosphorylated on serine 536 in cytosol and translocated into nucleus with calyculin A-treatment. The phosphorylation status of p65NF-kappaB was further confirmed by using the phosphorylation site-mutated p65NF-kappaB gene transfected into HEK293 cells. Unlike TNF-alpha, calyculin A treatment did not degraded IkappaBalpha within 10 min, while it degraded IkappaBalpha at 2-h treatment. Our findings indicate that calyculin A elicit phosphorylation of NF-kappaB on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-kappaB to the nucleus, thereby promoting transcriptional activity of NF-kappaB-related genes.


Differntial expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PP1) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PP1 isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PP1s and nucleolin was not changed. Anti-nucleolin antibody interacted with 110-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G(2)/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PP1 and nucleolin were differently expressed at G(1)/S and G(2)/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression.


Calyculin A stimulates the expression of TNF-a mRNA via phosphorylation of Akt in mouse osteoblastic MC3T3-E1 cells

Intracellular phosphatase activity has been recognized to play a central role in signal transduction. In the present study, we investigated the effects of calyculin A, an inhibitor of protein phosphatases, on the expression of TNF-alpha mRNA and the possible signaling pathways in mouse osteoblastic MC3T3-E1 cells. The result of semiquantitative RT-PCR showed that calyculin A increased the expression of TNF-alpha mRNA in MC3T3-E1 cells. Pre-treatment of LY294002 and Wortmannin, inhibitors of PI3K, inhibited the calyculin A-stimulated TNF-alpha mRNA expression. Western blot result disclosed that calyculin A increased the phosphorylation status of Akt at Ser473. However, U0126 and SB203580, specific inhibitor of MEK1/2 and p38MAPK, respectively, had no effect on calyculin A-stimulated expression of TNF-alpha mRNA. BAY11-7085 and CAPE, inhibitors of NF-kappaB activity, did not alter the calyculin A-stimulated TNF-alpha mRNA expression. Indirect immunofluorescent study confirmed that NF-kappaB was not translocated to the nucleus by calyculin A treatment. Our present results suggest that inhibition of phosphatase activity by calyculin A stimulate the phosphorylation of Akt at Ser473 by PI3K/Akt signaling pathway, resulting in the expression TNF-alpha mRNA.


The transcriptional factor Osterix directly interacts with RNA helicase A

Osterix is an osteoblast-specific transcriptional factor, required for bone formation and osteoblast differentiation. Here, we identified new Osterix interacting factors by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among the candidates, RNA helicase A was identified to interact with Osterix. To determine the interaction of Osterix with RNA helicase A, immunoprecipitation assay was performed. Western analysis confirmed the association between Osterix and RNA helicase A. Immunocytochemical analysis also showed that Osterix and RNA helicase A were co-localized in HEK 293 cells. Our data suggest that RNA helicase A might be a component of Osterix regulation.

Biochemical and Biophysical Research Communications


Arachidonic acid inhibits osteoblast differentiation through cytosolic phospholipase A2-dependent pathway.

Arachidonic acid, a precursor of prostaglandins (PGs), is released by phospholipase A2 (PLA2) and plays an important role in biological reactions. We examined the roles of arachidonic acid on the pathway of PG synthesis and osteoblast differentiation by using clone MC3T3-E1 cells. The effect of arachidonic acid was evaluated by the measurement of alkaline phosphatase activity, cells shape, production of arachidonic acid and the expression of cyclooxygenase (COX). Arachidonic acid dose dependently decreased alkaline phosphatase activity and increased PGE2 production in MC3T3-E1 cells. The cell shape changed from polygonal to fibroblastic following treatment with arachidonic acid. These effects were recovered by the treatment of NS-398 and indomethacin. Arachidonic acid increased the expression of COX-2 mRNA and the PGE2 production. The exogenous arachidonic acid induced the release of cellular arachidonic acid in MC3T3-E1 cells. Moreover, methylarachidonyl fluorophosphonate suppressed the arachidonic acid release and the expression of COX-2 mRNA. The present results indicate that exogenous arachidonic acid stimulated the activity of PLA2, leading to the new release of membranous arachidonic acid. The amplified arachidonic acid enhanced PGE2 production by COX-2, which inhibits the differentiation of MC3T3-E1 cells. Our results provide a new insight into the molecular mechanisms by which exogenous arachidonic acid plays a role as a paracrine/autocrine amplifier of PGE2 biosynthesis by coupling with PLA2 and COX-2.


Okadaic acid induces phosphorylation of p65NF-κB on serine 536 adn activates NF-κB transcriptional activity in human osteoblastic MG63 cells

Nuclear factor-kappa B (NF-kappaB) is an essential transcription factor in the control of expression of genes involved in cell growth, differentiation, inflammation, and neoplastic transformation. Previously, we reported that okadaic acid (OA), which is a specific inhibitor of serine/threonine protein phosphatases, induced apoptosis in cells of human osteosarcoma cell line MG63. However, to date, it is not clear whether the phosphorylation status of NF-kappaB could be affected by the treatment with OA. In this report, we demonstrate that treatment of MG63 cells with OA enhanced the phosphorylation level of NF-kappaB, as judged from the results of Western blot analysis and a lambda protein phosphatase dephosphorylation assay. The phosphorylation level of NF-kappaB was enhanced in both time- and dose-dependent manners. In the cells treated with 100 nM OA for 3 h, consequential translocation of NF-kappaB from the cytosol to the nucleus occurred. Western blotting experiments with an anti-phospho-p65NF-kappaB antibody disclosed that the NF-kappaB was phosphorylated on serine 536. Furthermore, OA stimulated the transcriptional activity of NF-kappaB in MG63 cells, as judged from the results of a luciferase assay. Our findings indicate that OA elicit phosphorylation of NF-kappaB on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-kappaB to the nucleus, thereby promoting transcriptional activity of genes.


Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro

In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1alpha (STAT1alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse osteoblastic MC3T3-E1 cells; thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1alpha protein in PKR mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1alpha and/or Runx2 expression.


Okadaic acid induces tyrosine phosphorylation of IkBa that mediated by PKR pathway in human osteoblastic MG63 cells

Treatment of human osteosarcoma cell line MG 63 cells with okadaic acid stimulated phosphorylation of IkappaBalpha, as judged from the results of Western blot analysis and a lambda protein phosphatase dephosphorylation assay. The stimulated phosphorylation of IkappaBalpha was both time- and dose-dependent. The phosphorylation sites of IkappaBalpha were taken to be tyrosine residues because the anti-phospho-tyrosine antibody bound to the samples immunoprecipitated with the anti-IkappaBalpha antibody. In the cells treated with 100 nM okadaic acid consequential translocation of NF-kappaB p65 from the cytosol to the nucleus occurred. Double-stranded RNA-dependent protein kinase (PKR) is a player in the cellular antiviral response and is involved in transcriptional stimulation through activation of NF-kappaB. We investigated the functional relationship between PKR and IkappaBalpha phosphorylation by constructing MG 63 PKR K/R cells that produced a catalytically inactive mutant PKR. NF-kappaB p65 was detected in the nucleus of these cells, even in the unstimulated cells. Although IkappaBalpha was degraded phosphorylation of eIF-2 alpha, a substrate of PKR, did not occur in the mutant cells treated with okadaic acid. Our results suggest that okadaic acid-induced tyrosine phosphorylation of IkappaBalpha was mediated by PKR kinase activity, thus indicating the involvement of this kinase in the control mechanism governing the activation of NF-kappaB.


PTEN expression by Egr-1 transcription factor in calyculin A-induced apoptotic cells

PTEN is a tumor suppressor gene encoding a phosphatase that negatively regulates cell survival mediated by the PI3-kinase-Akt pathway. The gene for transcription factor EGR-1 is an early response gene essential for cellular growth, proliferation, and differentiation. Protein phosphatase inhibitors including calyculin A and okadaic acid are potent inducers of apoptosis in several cell lines; however, the molecular mechanisms underlying their action are unknown. The purpose of this study was to examine the expression of PTEN and EGR-1 and the phosphorylation status of EGR-1 and Akt in calyculin A-treated human squamous carcinoma cells (SCCTF). Phosphorylation of EGR-1 and upregulation of PTEN expression were observed to occur in SCCTF cells treated with calyculin A in time- and dose-dependent fashions. The level of phosphorylated Akt decreased as the expression of PTEN protein increased in the calyculin A-treated SCCTF cells. Calyculin A-stimulated expression of EGR-1 and PTEN might be p53 independent, because the expression of them was also detected in p53-null Saos-2 cells. RNA interference using double-stranded RNA specific for the EGR-1 gene inhibited not only EGR-1 expression but also PTEN expression in SCCTF cells treated or not with calyculin A. Calyculin A induced nuclear fragmentation and chromatin condensation in SCCTF cells. The present results suggest that the level of PTEN expression and the phosphorylation status of Akt were associated with apoptosis induced by calyculin A. These observations also support the view that EGR-1 regulates PTEN expression in the initial steps of the apoptotic pathway.


Okadaic acid induces apoptosis through double-stranded RNA-dependent protein kinase/eukaryotic initiation factor-2a pathway in human osteoblastic MG63 cells

Double-stranded RNA-dependent protein kinase (PKR) is a participant in the cellular antiviral response and phosphorylates the alpha-subunit of eukaryotic translation initiation factor 2alpha (eIF-2alpha) to block protein synthesis. Treatment of human osteosarcoma cell line MG63 cells with a serine and threonine protein phosphatase inhibitor, okadaic acid, at the concentration of 100 nM, but not at 20 nM, induced apoptosis. To investigate the functional relationship between phosphatases and apoptosis, we examined the phosphorylation levels of PKR and eIF-2alpha by Western blot analysis. During treatment of cells with it at the higher concentration (100 nM), okadaic acid increased the level of phosphorylated PKR in MG63 cells, this kinase phosphorylating eIF-2alpha. However, at the lower concentration (20 nM), okadaic acid did not affect the level of phosphorylated PKR. In the cells treated with 100 nM okadaic acid, activation of NF-kappaB also occurred. Even though inhibition of translation occurred simultaneously in MG63 cells, the expression of pro-apoptotic proteins Fas and Bax was not affected by 100 nM okadaic acid in these cells. We concluded that the inhibition of translation decreased anti-apoptotic protein expression, thus resulting in apoptosis. Our results also suggest that the inhibition of the protein phosphatase activity by okadaic acid induced apoptosis in MG63 cells through PKR and eIF-2alpha.


Transcription factor NF-Y regulates mdr1 expression through binding to inverted CCAAT sequence in drug-resistant human squamous carcinoma cells

In this study, the expression and transcriptional regulation of the multidrug resistance-1 (MDR1) gene in multidrug-resistant SCCTF cells and -sensitive SCCKN cells derived from human squamous carcinoma were investigated. RT-PCR revealed that mdr1 mRNA was highly expressed in SCCTF cells while it was under the limit of detection in SCCKN cells. With an electrophoretic mobility shift assay using the mdr1 promoter region, a DNA-protein complex was detected strongly in SCCTF cells, but weakly in SCCKN cells. Incubation of the DNA-protein complex with an anti-NF-Y antibody caused a supershift in the migration to a position near the origin of the gel. Chromatin immunoprecipitation assay with an anti-NF-Y antibody showed that NF-Y binds to mdr1 promoter in SCCTF cells. The mdr1 promoter region including its NF-Y binding sequence was cloned into the luciferase reporter plasmid pGL3-basic vector, and this vector was used to transfect SCCTF and SCCKN cells. The luciferase assay showed that the inverted CCAAT sequence in the mdr1 promoter region is involved in the positive regulation of mdr1 promoter activity. NF-YA protein was expressed at higher levels in SCCTF cells than that in SCCKN cells. Hoechst dye staining also showed that MDR1 protein acts more effectively as an efflux pump in SCCTF cells than that in SCCKN cells.


Double-stranded RNA mediates selective gene silencing of protein phosphatase type 1 delta isoform in HEK-293 cells

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1delta) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP1delta. This RNA interference did not affect the expression of lphaand gamma1 isoforms of PP1. Transfection of antisense RNA specific for PP1delta also suppressed the expression of PP1delta. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.

Journal of Enzyme Inhibition and Medicinal Chemistry


Okadaic acid stimulates expression of Fas receptor and Fas ligand by activation of nuclear factor kappa-B in human oral squamous carcinoma cells

In the present study, we used western blot and RT-PCR analysis to examine the expression of proteins and mRNAs of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Treatment with okadaic acid enhanced the expression of proteins and mRNAs of both Fas receptor and Fas ligand in SCC-25 cells. The amount of IkappaB-alpha in whole cell lysates decreased, while the level of NF-kappaB in nucleus increased, in the okadaic acid-treated cells. Okadaic acid-treatment also alters the cellular localization of NF-kappaB, from cytoplasm to nuclei. To investigate the activation of NF-kappaB in okadaic acid-treated SCC-25 cells, we performed electrophoretic mobility gel shift assay using nuclear extracts and the consensus oligonucleotide for NF-kappaB DNA binding site. The binding of nuclear proteins to the oligonucleotide of NF-kappaB increased when the cells had been treated with 20 nM okadaic acid for 4 h. We transfected the cells with pFLF1, which has the promoter region of Fas receptor gene containing NF-kappaB binding site. A luciferase reporter gene assay demonstrated that the activity in the cells transfected with pFLF1 and treated with 20 nM okadaic acid increased in a time-dependent manner and that the activity was more than three-fold over that in the control cells. Our results suggest that NF-kappaB activated at early stages in the okadaic acid-treated SCC-25 cells stimulated the promoter activity of Fas receptor in the cells leading to the apoptotic death of these cells.


Transblot and cytochemical identification of avidin-interacting proteins in mitochondoria of cultured cells

Cell lysates prepared from 3T3-L1 cells were analyzed by western blotting using the avidin-biotin complex system and anti-Bax antibody. The antibody interacted with bands of proteins with estimated molecular weights of 120, 74, 72, and 25 kDa. However, only the 25-kDa band was detected with the anti-Bax antibody when the direct immunoblotting method was used. Peroxidase-conjugated avidin interacted with the 120-, 74-, and 72-kDa bands. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these three proteins in MC3T3-E1, YROS, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Avidin-peroxidase also interacted with these three proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. FITC-streptavidin was also localized in mitochondria in the cultured cells. The localization of avidin/streptavidin-interacting proteins in mitochondria was confirmed by using double staining with FITC-streptavidin and Mito-tracker.


Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in

To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2.

Biochemical and Biophysical Research Communications


Okadaic acid stimulates the expression of receptor activator of nuclear factor-kappa B ligand RANKL in mouse osteoblastic cells


アラキドン酸による骨芽細胞の分化抑制作用およびその機構解明に関する研究

アラキドン酸による骨芽細胞の分化抑制作用およびその機構解明に関する研究