TAK1 is a pivotal therapeutic target for tumor progression and bone destruction in myeloma

Along with the tumor progression, the bone marrow microenvironment is skewed in multiple myeloma (MM), which underlies the unique pathophysiology of MM and confers aggressiveness and drug resistance in MM cells. TGF-β-activated kinase-1 (TAK1) mediates a wide range of intracellular signaling pathways. We demonstrate here that TAK1 is constitutively overexpressed and phosphorylated in MM cells, and that TAK1 inhibition suppresses the activation of NF-κB, p38MAPK, ERK and STAT3 to decrease the expression of critical mediators for MM growth and survival, including PIM2, MYC, Mcl-1, IRF4, and Sp1, along with a substantial reduction in the angiogenic factor VEGF in MM cells. Intriguingly, TAK1 phosphorylation was also induced along with upregulation of vascular cell adhesion molecule-1 (VCAM-1) in bone marrow stromal cells (BMSCs) in cocultures with MM cells, which facilitated MM cell-BMSC adhesion while inducing IL-6 production and receptor activator of nuclear factor κ-Β ligand (RANKL) expression by BMSCs. TAK1 inhibition effectively impaired MM cell adhesion to BMSCs to disrupt the support of MM cell growth and survival by BMSCs. Furthermore, TAK1 inhibition suppressed osteoclastogenesis enhanced by RANKL in cocultures of bone marrow cells with MM cells, and restored osteoblastic differentiation suppressed by MM cells or inhibitory factors for osteoblastogenesis overproduced in MM. Finally, treatment with the TAK1 inhibitor LLZ1640-2 markedly suppressed MM tumor growth and prevented bone destruction and loss in mouse MM models. Therefore, TAK1 inhibition may be a promising therapeutic option targeting not only MM cells but also the skewed bone marrow microenvironment in MM.


Pim-2 is a critical target for treatment of osteoclastogenesis enhanced in myeloma.


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.


Involvement of interleukin23 induced by Porphyromonas endodontalis lipopolysaccharide in osteoclastogenesis

Periapical lesions are characterized by the destruction of periapical bone, and occur as a result of local inflammatory responses to root canal infection by microorganisms including Porphyromonas endodontalis (P. endodontalis). P. endodontalis and its primary virulence factor, lipopolysaccharide (LPS), are associated with the development of periapical lesions and alveolar bone loss. Interleukin-23 (IL-23) is critical in the initiation and progression of periodontal disease via effects on peripheral bone metabolism. The present study investigated the expression of IL-23 in tissue where a periapical lesion was present, and the effect of P. endodontalis LPS on the expression of IL-23 in periodontal ligament (PDL) cells. Reverse transcription- quantitative polymerase chain reaction and immunohistochemistry revealed increased levels of IL-23 expression in tissue with periapical lesions compared with healthy PDL tissue. Treatment with P. endodontalis LPS increased the expression of IL-23 in the SH-9 human PDL cell line. BAY11-7082, a nuclear factor κB inhibitor, suppressed P. endodontalis LPS-induced IL-23 expression in SH-9 cells. Treatment of RAW264.7 cells with conditioned medium from P. endodontalis LPS-treated SH-9 cells promoted osteoclastogenesis. By contrast, RAW264.7 cells treated with conditioned medium from IL-23-knockdown SH-9 cells underwent reduced levels of osteoclastogenesis. The results of the present study indicated that the expression of IL-23 in PDL cells induced by P. endodontalis LPS treatment may be involved in the progression of periapical lesions via stimulation of the osteoclastogenesis process.


Protein phosphatase 2A Cα regulates proliferation, migration, and metastasis of osteosarcoma cells.

Osteosarcoma is the most frequent primary bone tumor. Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes, such as cell cycle, growth, apoptosis, and signal transduction. In this study, we examined the expression and function of PP2A Cα in osteosarcoma cells. PP2A Cα expression was expected to be higher in malignant osteosarcoma tissues. PP2A Cα expression level and PP2A activity was higher in malignant osteosarcoma LM8 cells compared with that in primary osteoblasts and in the osteoblast-like cell line MC3T3-E1. Okadaic acid, an inhibitor of PP2A, reduced cell viability and induced apoptosis in LM8 cells. PP2A Cα-knockdown LM8 cells (shPP2A) exhibited less striking filopodial and lamellipodial structures than that in original LM8 cells. Focal adhesion kinase phosphorylation and NF-κB activity decreased in shPP2A-treated cells. Sensitivity to serum deprivation-induced apoptosis increased in shPP2A-treated cells, accompanied by a lower expression level of anti-apoptotic BCL-2 in these cells. Reduction of PP2A Cα resulted in a decrease in the migration ability of LM8 cells in vitro. Reduction in PP2A Cα levels in vivo suppressed proliferation and metastasis in LM8 cells. PP2A Cα expression was also higher in human osteosarcoma MG63 and SaOS-2 cells than that in primary osteoblasts and MC3T3-E1 cells, and reduction in PP2A Cα levels suppressed the cell proliferation rate and migration ability of MG63 cells. These results indicate that PP2A Cα has a critical role in the proliferation and metastasis of osteosarcoma cells; therefore, its inhibition could potentially suppress the malignancy of osteosarcoma cells.

Laboratory Investigation; a Journal of Technical Methods and Pathology


Histone demethylase Jmjd3 regulates osteoblast apoptosis through targeting anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim.

Posttranslational modifications including histone methylation regulate gene transcription through directly affecting the structure of chromatin. Trimethylation of histone H3K27 (H3K27me3) contributes to gene silencing and the histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes the methylation of H3K27me3, followed by the activation of gene expression. In the present study, we explored the roles of Jmjd3 in regulating osteoblast apoptosis. Knockdown of Jmjd3 promoted osteoblast apoptosis induced by serum deprivation with decreased mitochondrial membrane potential and increased levels of caspase-3 activation, PARP cleavage, and DNA fragmentation. B cell lymphoma-2 (Bcl-2), an anti-apoptotic protein, was down-regulated by knockdown of Jmjd3 through retaining H3K27me3 on its promoter region. Knockdown of Jmjd3 increased the pro-apoptotic activity of Bim through inhibiting ERK-dependent phosphorylation of Bim. Protein kinase D1 (PKD1), which stimulates ERK phosphorylation, decreased in the Jmjd3-knockdown cells and introduction of PKD1 relieved osteoblast apoptosis in the Jmjd3-knockdown cells through increasing ERK-regulated Bim phosphorylation. These results suggest that Jmjd3 regulates osteoblast apoptosis through targeting Bcl-2 expression and Bim phosphorylation.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research


PKR regulates LPS-induced osteoclast formation and bone destruction in vitro and in vivo.

PKR plays a pivotal role in LPS-induced bone loss in PD, and, thus, has potential as a therapeutic target for PD. This article is protected by copyright. All rights reserved.


Histone demethylase Utx regulates differentiation and mineralization in osteoblasts.

Alteration of methylation status of lysine 27 on histone H3 (H3K27) associates with dramatic changes in gene expression in response to various differentiation signals. Demethylation of H3K27 is controlled by specific histone demethylases including ubiquitously transcribed tetratricopeptide repeat X chromosome (Utx). However, the role of Utx in osteoblast differentiation remains unknown. In this study, we examined whether Utx should be involved in osteoblast differentiation. Expression of Utx increased during osteoblast differentiation in MC3T3-E1 cells and primary osteoblasts. GSK-J1, a potent inhibitor of H3K27 demethylase, increased the levels of trimethylated H3K27 (H3K27me3) and decreased the expressions of Runx2 and Osterix and ALP activity in MC3T3-E1 cells. Stable knockdown of Utx by shRNA attenuated osteoblast differentiation and decreased ALP activity, calcium content, and bone-related gene expressions. Silencing of Utx increased the level of H3K27me3 on the promoter regions of Runx2 and Osterix and decreased the promoter activities of Runx2 and Osterix. Taken together, our present results propose that Utx plays important roles in osteoblast differentiation by controlling the expressions of Runx2 and Osterix.


Double stranded RNA-dependent protein kinase is necessary for TNF--induced osteoclast formation in vitro and in vivo.

Double-stranded RNA-dependent protein kinase (PKR) is involved in cell cycle progression, cell proliferation, cell differentiation, tumorgenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification in osteoblasts. TNF- plays a key role in osteoclast differentiation. However, it is unknown about the roles of PKR in the TNF--induced osteoclast differentiation. The expression of PKR in osteoclast precursor RAW264.7 cells increased during TNF--induced osteoclastogenesis. The TNF--induced osteoclast differentiation in bone marrow-derived macrophages and RAW264.7 cells was markedly suppressed by the pre-treatment of PKR inhibitor, 2-aminopurine (2AP), as well as gene silencing of PKR. The expression of gene markers in the differentiated osteoclasts including TRAP, Calcitonin receptor, cathepsin K and ATP6V0d2 was also suppressed by the 2AP treatment. Bone resorption activity of TNF--induced osteoclasts was also supressed by 2AP treatment. Inhibition of PKR supressed the TNF--induced activation of NF-B and MAPK in RAW264.7 cells. 2AP inhibited both the nuclear translocation of NF-B and its transcriptional activity in RAW264.7 cells. 2AP inhibited the TNF--induced expression of NFATc1 and c-fos, master transcription factors in osteoclastogenesis. TNF--induced nuclear translocation of NFATc1 in mature osteoclasts was clearly inhibited by the 2AP treatment. The PKR inhibitor C16 decreased the TNF--induced osteoclast formation and bone resorption in mouse calvaria. The present study indicates that PKR is necessary for the TNF--induced osteoclast differentiation in vitro and in vivo. This article is protected by copyright. All rights reserved.


Tumor necrosis factor-α induces interleukin-34 expression through nuclear factor-κB

Osteoblasts produce various types of cytokines under pathological conditions and control osteoclast differentiation. Tumor necrosis factor-α (TNF-α) has been demonstrated to exert complex effects in osteoblasts under local inflammatory conditions, including in periodontal and periapical diseases. Interleukin-34 (IL-34) has been recently identified as a novel regulatory factor for the differentiation and function of osteoclasts. The present study provides the first evidence, to the best of our knowledge, that the expression of IL-34 is induced by TNF-α through nuclear factor-κB (NF-κB) activation in MC3T3-E1 osteoblastic cells. TNF-α induced IL-34 expression in a dose- and time-dependent manner. Immunocytochemistry with an NF-κB antibody demonstrated that NF-κB was mainly localized in the cytoplasm of the untreated MC3T3-E1 cells. Rapid translocation of NF-κB from the cytoplasm to the nucleus was observed in the cells treated with TNF-α for 15 min. Translocation and transcriptional activity of NF-κB were also determined by western blotting and a luciferase reporter assay, respectively. Pretreatment with 100 µM CAPE, an inhibitor of NF-κB, significantly inhibited TNF-α-induced IL-34 expression. These results indicate that TNF-α induces IL-34 expression via NF-κB in osteoblasts.


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.


Calcium hydroxide suppresses the virulence of lipopolysaccharide from Porphyromonas endodontalis to bone cells

Porphyromonas endodontalis and its main virulence factor, lipopolysaccharide (LPS), are associated with the development of periapical diseases and alveolar bone loss. Calcium hydroxide is commonly used for endodontic therapy. However, the effects of calcium hydroxide on the virulence of P. endodontalis LPS and the mechanism of P. endodontalis LPS-induced bone destruction are not clear. Calcium hydroxide rescued the P. endodontalis LPS-suppressed viability of MC3T3-E1 cells and activity of nuclear factor-κB (NF-κB) in these cells, resulting in the reduced expression of interleukin-6 and tumor necrosis factor-α. In addition, calcium hydroxide inhibited P. endodontalis LPS-induced osteoclastogenesis by decreasing the activities of NF-κB, p38, and ERK1/2 and the expression of nuclear factor of activated T-cell cytoplasmic 1 in RAW264.7 cells. Calcium hydroxide also rescued the P. endodontalis LPS-induced osteoclastogenesis and bone destruction in mouse calvaria. Taken together, our present results indicate that calcium hydroxide suppressed bone destruction by attenuating the virulence of P. endodontalis LPS on bone cells.


Histone demethylase Jmjd3 regulates osteoblast differentiation via transcription factors Runx2 and Osterix

Post-translational modifications of histones including methylation play important roles in cell differentiation. Jumonji domain-containing 3 (Jmjd3) is a histone demethylase, which specifically catalyzes the removal of trimethylation of histone H3 at lysine 27 (H3K27me3). In this study, we examined the expression of Jmjd3 in osteoblasts and its roles in osteoblast differentiation. Jmjd3 expression in the nucleus was induced in response to the stimulation of osteoblast differentiation as well as treatment of bone morphogenetic protein-2 (BMP-2). Either treatment with Noggin, an inhibitor of BMP-2, or silencing of Smad1/5 suppressed Jmjd3 expression during osteoblast differentiation. Silencing of Jmjd3 expression suppressed osteoblast differentiation through the expression of bone-related genes including Runx2, osterix, osteopontin, bone sialoprotein (BSP), and osteocalcin (OCN). Silencing of Jmjd3 decreased the promoter activities of Runx2 and osterix and increased the level of H3K27me3 on the promoter regions of Runx2 and osterix. Introduction of the exogenous Runx2 and osterix partly rescued osteoblast differentiation in the shJmjd3 cells. The present results indicate that Jmjd3 plays important roles in osteoblast differentiation and regulates the expressions of BSP and OCN via transcription factors Runx2 and osterix.


Stimulation of osteoclast formation by RANKL requires interferon regulatory factor-4 and is inhibited by simvastatin in a mouse model of bone loss

Diseases of bone loss are a major public health problem. Here, we report the novel therapeutic action of simvastatin in osteoclastogenesis and osteoprotection, demonstrated by the ability of simvastatin to suppress osteoclast formation in vitro and in vivo. We found that in vitro, IRF4 expression is upregulated during osteoclast differentiation induced by RANKL (receptor activator of nuclear factor-κB ligand), while simvastatin blocks RANKL-induced osteoclastogenesis and decreases expression of NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1), IRF4 and osteoclast markers. We also show that IRF4 acts in cooperation with NFATc2 and NF-κB on the promoter region of NFATc1 to accelerate its initial transcription during the early stage of osteoclastogenesis. Moreover, our study using IRF4 siRNA knockdown directly demonstrates the requirement for IRF4 in NFATc1 mRNA transcription and its necessity in RANKL-induced osteoclast differentiation. Our results suggest that the reduction in osteoclastogenesis is partly due to the inhibition of IRF4 production in RANKL-induced osteoclast differentiation. To investigate the in vivo effects of simvastatin in RANKL-treated mice, we examined the bone mineral density (BMD) of a mouse model of bone loss, and found that simvastatin significantly reduced bone loss by suppressing osteoclast numbers in vivo, even in the presence of high concentrations of RANKL. These results suggest that the depletion of osteoclasts is not due to the reduction in RANKL produced by osteoblasts in vivo. The results are consistent with the hypothesis that simvastatin blocks RANKL-induced IRF4 expression in osteoclastogenesis. We propose that the expression of IRF4 by osteoclasts could be a promising new therapeutic target in bone-loss diseases.


Okadaic acid activates the PKR pathway and induces apoptosis through PKR stimulation in MG63 osteoblast-like cells.

Double-stranded RNA-dependent protein kinase (PKR) is one of the players in the cellular antiviral responses and is involved in transcriptional stimulation through activation of NF-κB. Treatment of the human osteosarcoma cell line MG63 with the protein phosphatase inhibitor okadaic acid stimulated the expression and phosphorylation of IκBα, as judged from the results of real-time PCR and western blot analysis. We investigated the functional relationship between PKR and signal transduction of NF-κB by establishing PKR-K/R cells that produced a catalytically inactive mutant of PKR. Phosphorylation of eIF-2α, a substrate of PKR, was not stimulated by okadaic acid in the PKR-K/R cells, whereas okadaic acid induced phosphorylation of eIF-2α in MG63 cells. Phosphorylation of NF-κB in MG63 cells was stimulated by okadaic acid; however, okadaic acid did not induce phosphorylation of NF-κB in the PKR-K/R cells. Finally, okadaic acid-induced apoptosis was inhibited in the PKR-K/R cells. Our results suggest that okadaic acid-induced phosphorylation of IκBα was mediated by PKR kinase activity, thus, indicating the involvement of this kinase in the control mechanism governing the activation of NF-κB and induction of apoptosis.


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.


Double-stranded RNA-dependent protein kinase regulates insulin-stimulated chondrogenesis in mouse clonal chondrogenic cells, ATDC-5.

Double-stranded RNA-dependent protein kinase (PKR) is an interferon-induced protein that has been identified and characterized as a translational inhibitor in an interferon-regulated antiviral pathway. PKR is also reported to play important roles in the regulation of cell growth and differentiation. We have previously demonstrated that PKR inactivation suppresses osteoblast calcification and osteoclast formation. However, reports concerning the roles of PKR in chondrogenesis are limited. In this study, we have demonstrated that PKR is required for the in vitro differentiation of the mouse clonal chondrogenic cell line ATDC-5. ATDC-5 cells treated with insulin differentiated into chondrocytes and produced an alcian-blue-positive cartilage matrix. The protein expression of signal transducers and activators of transcription (STAT) peaked at day 7 of differentiation, whereas the expression of SRY-box-containing gene 9 (Sox-9), which is a transcription factor for chondrocyte differentiation, increased gradually. When the cells were treated with a PKR inhibitor (2-aminopurine), the cartilage matrix formation decreased. The protein expression of STAT1 continued to increase up to day 21, whereas the expression of Sox-9 was low and did not increase. We also demonstrated that PKR was localized to a marginal region of the mandibular condyle cartilage in mouse embryos. Our findings suggest that PKR has important functions in the differentiation of chondrocytes through the modulation of STAT1 and Sox-9 expression.


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 of protein phosphatase 1δ with nucleophosmin in human osteoblastic cells

Protein phosphorylation and dephosphorylation has been recognized as an essential mechanism in the regulation of cellular metabolism and function in various tissues. Serine and threonine protein phosphatases (PP) are divided into four categories: PP1, PP2A, PP2B, and PP2C. At least four isoforms of PP1 catalytic subunit in rat, PP1α, PP1γ1, PP1γ2, and PP1δ, were isolated. In the present study, we examined the localization and expression of PP1δ in human osteoblastic Saos-2 cells. Anti-PP1δ antibody recognized a protein present in the nucleolar regions in Saos-2 cells. Cellular fractionation revealed that PP1δ is a 37 kDa protein localized in the nucleolus. Nucleophosmin is a nucleolar phosphoprotein and located mainly in the nucleolus. Staining pattern of nucleophosmin in Saos-2 cells was similar to that of PP1δ. PP1δ and nucleophosmin were specifically stained as dots in the nucleus. Dual fluorescence images revealed that PP1δ and nucleophosmin were localized in the same regions in the nucleolus. Similar distribution patterns of PP1δ and nucleophosmin were observed in osteoblastic MG63 cells. The interaction of PP1δ and nucleophosmin was also shown by immunoprecipitation and Western analysis. These results indicated that PP1δ associate with nucleophosmin directly in the nucleolus and suggested that nucleophosmin is one of the candidate substrate for PP1δ.


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


Double stranded RNA-dependent protein kinase is involved in osteoclast differentiation of RAW264.7 cells in vitro

Double-stranded RNA-dependent protein kinase (PKR) plays a critical role in antiviral defence of the host cells. PKR is also involved in cell cycle progression, cell proliferation, cell differentiation, tumorigenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification of osteoblasts. However, it is unknown about the role of PKR in osteoclast differentiation. A dominant-negative PKR mutant cDNA, in which the amino acid lysine at 296 was replaced with arginine, was transfected into RAW264.7 cells. We have established the cell line that stably expresses the PKR mutant gene (PKR-K/R). Phosphorylation of PKR and α-subunit of eukaryotic initiation factor 2 was not stimulated by polyinosic-polycytidylic acid in the PKR-K/R cells. RANKL stimulated the formation of TRAP-positive multinuclear cells in RAW264.7 cells. However, TRAP-positive multinuclear cells were not formed in the PKR-K/R cells even when the cells were stimulated with higher doses of RANKL. A specific inhibitor of PKR, 2-aminopurine, also suppressed the RANKL-induced osteoclast differentiation in RAW264.7 cells. The expression of macrophage fusion receptor and dendritic cell-specific transmembrane protein significantly decreased in the PKR-K/R cells by real time PCR analysis. The results of RT-PCR revealed that the mRNA expression of osteoclast markers (cathepsin K and calcitonin receptor) was suppressed in the PKR-K/R cells and RAW264.7 cells treated with 2-aminopurine. Expression of NF-κB protein was suppressed in the PKR-K/R cells and 2-aminopurine-treated RAW264.7 cells. The level of STAT1 protein expression was elevated in the PKR-K/R cells compared with that of the wild-type cells. Immunohistochemical study showed that PKR was localized in osteoclasts of metatarsal bone of newborn mouse. The finding that the PKR-positive multinuclear cells should be osteoclasts was confirmed by TRAP-staining. Our present study indicates that PKR plays important roles in the differentiation of osteoclasts.


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.


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.


Role of transcription factors in multidrug resistance and apoptosis


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.


Cleavage of nucleolin and AgNOR proteins during apoptosis induced by anticancer drugs in human salivary gland cells

To investigate the behavior of nuclear proteins in apoptosis induced by anticancer drugs in cultured human salivary gland (HSG) cells. Dynamic alternations of nucleolin and argyrophilic nucleolar organizer region (AgNOR) proteins in anticancer drug-induced apoptosis of HSG cells and in a cell-free apoptotic system were examined using Western blot analysis and immunocytochemical method. The 110-kDa form of nucleolin and AgNOR protein decreased and the 80- and 95-kDa forms appeared during apoptosis in HSG cells and in a cell-free apoptotic system. In addition, the induction of DNA ladder formation coincided with the appearance of alternation of nucleolin and AgNOR proteins in a cell-free apoptosis. Nucleolin diffusely spread out into the nuclear material in the apoptotic body of HSG cells. The present results indicate that alternations of nucleolin and AgNOR proteins are associated with the induction of DNA fragmentation and the final active phase of apoptosis induced by anticancer drugs in malignant salivary gland cells.


Association of protein phosphatase 1 delta with nucleolin in osteoblastic cells and cleavage of nucleolin in apoptosis-induced osteoblastic cells

Protein phosphorylation and dephosphorylation has been recognized as a key mechanism in cell function. Okadaic acid is a potent inhibitor of PP1 and PP 2A and induces apoptosis in human or mouse cells including osteoblasts. Nucleolin is an abundantly expressed nucleolar phosphoprotein and is located mainly in the nucleolus. The staining pattern of nucleolin in cultured osteoblastic cells is similar to that of PP1δ. Nucleolin was demonstrated to bind to PP1δ in nucleolus in human osteoblastic cells by using immunocytochemical and immunoprecipitation methods. AgNORs and nucleolin, visible as dots in the nuclei of the control osteoblastic cells, disappeared from the nuclei of the osteoblastic cells treated with okadaic acid. A major band, 110 kDa, was detected in the proteins obtained from osteoblastic cells. The level of the 110 kDa protein decreased in the apoptotic osteoblastic cells, whereas an additional band, 80 kDa, appeared and the level of this protein increased in the proteins prepared from okadaic acid-induced apoptotic osteoblastic cells. Our results indicate that PP1δ directly binds to nucleolin in the nucleolus of osteoblastic cells and that nucleolin is cleaved during apoptosis in osteoblastic cells.


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


Suppression of Egr-1 expression in human oral squamous carcinoma cells by okadaic acid.

We examined the expression of early growth response-1 (Egr-1) gene in human oral squamous carcinoma cell lines SCCKN and SCC-25 cells and human osteoblastic cell lines Saos-2 and MG63 cells treated with okadaic acid, a potent inhibitor of protein phosphatases type 1 and type 2A. Western blot analysis revealed that Egr-1 was strongly expressed in SCCKN cells and that okadaic acid decreased the expression of Egr-1 protein in these cells. However, Egr-1 was expressed at lower levels in SCC-25, Saos-2, and MG63 cells and transiently increased with the okadaic acid treatment. Suppression of Egr-1 protein expression in okadaic acid-treated SCCKN cells stemmed from the suppression of the Egr-1 mRNA level, as determined by the RT-RCR method. Formaldehyde-fixed and alcohol-permeabilized cultured SCCKN cells were reacted with the anti-Egr-1 antibody using immunohistochemical methods. Intense fluorescence was observed in the nuclei of the control SCCKN cells interacted with anti-Egr-1 antibody. However, only a weak reaction was observed in the nuclei in SCCKN cells treated with okadaic acid. A gel mobility shift assay showed that treatment of SCCKN cells with okadaic acid suppressed Egr-1 binding to the DIG-labeled Egr-1 consensus oligonucleotide probe. The present results indicate that the alteration of phosphorylation states in SCCKN cells regulates Egr-1 binding to its consensus sequence and its expression at the transcriptional level.


Differentioal histochemical localization patterns of reduced nicotinamide adenine dinucleotide phosphate-diaphorase and neuronal nitric oxide synthase during postnatal development of the rat vomeronasal organ

The present study was undertaken to examine the localization patterns of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) by enzyme histochemistry and neuronal nitric oxide synthase (NOS) by immunohistochemistry in the vomeronasal organ of rat from postnatal day 0 for 8 weeks (adult). Nicotinamide adenine dinucleotide phosphate-diaphorase activity was not observed in the sensory epithelium of the vomeronasal organ at postnatal day 0 (the day of birth) and at day 1. At postnatal day 2, NADPH-d activity was observed in several vomeronasal neurons and on the surface of the sensory epithelium. From 25 days through adulthood, the number of vomeronasal neurons having NADPH-d activity increased gradually. On the other hand, neuronal NOS immunoreactivity was not observed in the sensory epithelium of the vomeronasal organ in newborns or in the adult rat. In this study, it is suggested that the nitric oxide pathway in the sensory epithelium of the vomeronasal organ comes into play beyond postnatal day 3. Moreover, it was found that NADPH-d and neuronal NOS are not colocalized in the sensory epithelium of the developing rat vomeronasal organ.


Interaction of protein phosphatase 1 delta with nucleolin in human osteoblastic cells

We examined the expression and cytolocalization of the protein phosphatase type 1 delta (PP1delta) isoform and nucleolin in human osteoblastic MG63 and Saos-2 cells. Cellular fractionation of MG63 cells was done and protein was prepared from each fraction. Anti-nucleolin antibody interacted with the 100- and 95-kD proteins present in the whole-cell lysate. The 100-kD protein was detected in nuclear and nucleolar fractions. The 95-kD protein was detected in cytosolic and nucleoplasmic fractions. PP1delta and nucleolin were co-localized in the nucleolus in MG63 and Saos-2 cells revealed by an immunofluorescence method. PP1delta and nucleolin were also co-immunoprecipitated with anti-nucleolin and anti-PP1delta antibodies. In the actinomycin D-treated cells, the subcellular localization of PP1delta and nucleolin was changed. Expression of PP1delta was upregulated with actinomycin D treatment. The level of 100-kD protein did not change in the actinomycin D-treated cells. However, the level of the 95-kD band increased with actinomycin D treatment. These results indicate that PP1delta was associated with nucleolin in the nucleolus of MG63 and Saos-2 cells and that nucleolin is a possible candidate substrate for PP1delta.

The Journal of Histochemistry and Cytochemistry


Okadaic acid stimulates apoptosis through expression of Fas receptor and Fas ligand in human squamous cell carcinoma cells.

Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to Fas receptor leads to activation of the latter and the induction of intracellular signals that result in apoptotic cell death. In the present study, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis to examine the expression of mRNAs and proteins of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. The PCR product of Fas receptor mRNA was detected in the cells and a protein with an estimated molecular weight of 35,000 was also expressed in them. Expression of Fas receptor mRNA stimulated by okadaic acid was elevated in dose- and time-dependent manners as judged by semiquantitative RT-PCR analysis, with the maximum expression level at 50 nM and 8 h treatment. Fas ligand mRNA expression was also stimulated by okadaic acid in SCC-25 cells in dose- and time-dependent manners. Okadaic acid also stimulated the expression of Fas ligand protein in the cells. Okadaic acid in serum-free medium induced apoptosis in SCC-25 cells in a time-dependent manner up to 24 h as determined by nuclear condensation and fragmentation of chromatin and DNA ladder formation. The present results indicate that the expression of Fas receptor and Fas ligand is negatively regulated by a protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in SCC-25 cells. Our results also suggest that Fas receptor and Fas ligand system might regulate apoptosis in SCC-25 cells in an autocrine fashion.


Peplomycin-induced apoptosis in oral squamous carcinoma cells depend on bleomycin sensitivity.

Oral squamous carcinoma cell line SSCKN cells were shown to be highly sensitive to bleomycin, whereas SCCTF cells were minimally sensitive to this reagent. To determine whether the anticancer drug resistance to oral squamous carcinoma cells could be related to the degree of the drug-induced apoptosis, we examined the effects of peplomycin on induction of apoptosis in these cells. After reaching subconfluence, SCCKN and SCCTF cells were exposed to various concentrations of peplomycin. Peplomycin caused cytotoxicity in both SCCKN and SCCTF cells in a dose-dependent fashion with the maximal effect at concentrations of 1 and 10 microM, respectively, as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, we observed marked nuclear condensation and fragmentation of chromatin in SCCKN cells treated with 1 microM peplomycin. However, SCCTF cells treated with 1 microM peplomycin showed neither nuclear condensation nor fragmentation. DNA ladder formation was also detected in both cell lines by treatment with peplomycin. The induced DNA ladder formation in SCCKN and SCCTF cells was dose-dependent, with the maximal effect at concentrations of 5 and 50 microM, respectively. Bleomycin also induced DNA ladder formation in SCCKN and SCCTF cells with different sensitivities. Mitomycin C induced DNA laddering in both SCCKN and SCCTF cells; however, the intensity of DNA ladder formation was almost the same in both cell lines. The present results indicate that peplomycin-induced apoptosis in oral squamous carcinoma cell lines depends on the sensitivity of these cells to bleomycin.


Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human squamous carcinoma cells.

The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 kDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein. In the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis.


Fas antigen expression and outcome of oral squamous cell carcinoma.

Paraffin sections of biopsy specimens obtained from 46 patients with oral squamous cell carcinomas were stained with both anti-peptide antibody against human Fas antigen and monoclonal mouse antibody against human proliferating cell nuclear antigen (PCNA). The patients received chemotherapy with a combination of carboplatin and peplomycin sulfate or mitomycin C and peplomycin sulfate before surgery. The relation between the expression of Fas antigen and the clinical features of each case was examined. The correlation between PCNA and Fas antigen expression was also studied. The mean PCNA labeling index of the 22 Fas-negative cases was 46.9%, which was significantly higher than that of the 24 Fas-positive cases (39.5%). Strong correlations were found between the expression of Fas antigen and the response to chemotherapy, tumor recurrence, and survival. The Fas-negative group had only a minor response to chemotherapy and a poor outcome, whereas the Fas-positive group had a better response to chemotherapy and a good outcome. Although lymph node metastasis was significantly related to survival, there was no correlation between Fas antigen expression and lymph node metastasis. The Kaplan-Meier survival curve of patients positive for Fas antigen was significantly better than that of patients negative for Fas antigen. Our results suggest that Fas antigen expression is an independent predictor of outcome whose usefulness should be evaluated in prospective studies.

International Journal of Oral and Maxillofacial Surgery


Quantitative radiographic changes in the mandible and the tibia in systematically loaded rats fed a low-calcium diet.


Upregulation of the expression of Fas antigen and Fas ligand in a human submandibular gland ductal cell line by okadaic acid.

Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to the receptor leads to receptor activation and the induction of intracellular signals that result in apoptotic cell death. In the present study, the expression of mRNA and proteins of Fas receptor and Fas ligand were examined in human submandibular gland ductal (HSG) cells treated with okadaic acid by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Six hundred and eighty-two bp of the PCR product of Fas receptor mRNA was detected in HSG cells and a protein with an estimated molecular weight of 58,000 was expressed in HSG cells. Treatment of HSG cells with an agonistic anti-Fas monoclonal antibody resulted in death of HSG cells, indicating that the functional Fas receptor protein is expressed in HSG cells. Fas receptor protein expression stimulated by okadaic acid was elevated in a dose- and time-dependent manner, with maximal expression at 20 nM and 48 h treatment. Fas ligand mRNA was also detected constitutively in HSG cells by RT-PCR. Okadaic acid stimulated the expression of Fas ligand protein in HSG cells in a time-dependent manner, while the expression of the ligand was low in untreated HSG cells. The molecular weight of Fas ligand was estimated as 68,000. An antagonistic anti-Fas ligand monoclonal antibody prevented okadaic acid-induced death in HSG cells in a dose-dependent fashion as determined by WST-1 assay. The results indicate that the expression of Fas receptor and ligand is regulated by protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in HSG cells. The results also suggest that the Fas receptor-ligand system might regulate apoptosis in HSG cells.


Fas expression and Fas monoclonal antibody-induced apoptosis in human squamous cell carcinoma cell line SCC-25 cells.

Fas antigen is a cell surface protein that mediates apoptosis via signal transduction from the plasma membrane. Using reverse transcriptase-polymerase chain reaction (RT-PCR), messenger RNA for Fas antigen was detected in the human oral squamous cell carcinoma cell line, SCC-25. In serum-free medium, a monoclonal anti-Fas antibody (CH-11) induced Fas antigen expression in SCC-25 cells, as determined by immunocytochemistry and Western blotting, using an anti-Fas polyclonal antibody (Fas D) as primary antibody. Fas antigen was localized to the cytoplasm and the cell membrane. The molecular weight of the protein recognized by Western blot analysis was 35,000, consistent with the value reported for the Fas antigen. The CH-11 antibody did not induce Fas antigen expression in serum-containing medium. To determine whether CH-11 could induce apoptosis in oral squamous cell carcinoma, we examined its effects on the survival of cultured SCC-25 cells. Anti-Fas monoclonal antibody in serum-free medium induced cytotoxicity in SCC-25 cells in a time-dependent manner up to 8 h, as determined by phase-contrast microscopy and WST-1 assay. Marked nuclear condensation and fragmentation of chromatin were observed in the CH-11-treated cells using Hoechst 33342 staining. This anti-Fas monoclonal antibody also induced DNA ladder formation in SCC-25 cells in a time-dependent manner. The present results indicate that the anti-Fas monoclonal antibody (CH-11) may mediate apoptosis by binding to the Fas antigen expressed in SCC-25 cells.


Induction of apoptosis in human oral squamous carcinoma cell line SCC-25 cells by protein phosphatase inhibitors.


The significance of PCNA labeling index at tumour invasion front in oral squamous cell carcinoma

Asian Journal of Oral and Maxillofacial Surgery


Malignant peripheral nerve sheath tumour in the maxilla associated with von Recklinghausen's diseases

Malignant transformation of neurofibromatosis is one of the most serious complications of von Recklinghausen's disease (VRD). The most common associated malignancy is the malignant peripheral nerve sheath tumour (MPNST). Few cases of MPNST associated with VRD in the maxillary region have been reported. This report describes a rare case of MPNST in the maxilla and the aggressive nature of MPNST associated with VRD.


The protein phosphatase inhibitors, okadaic acid and calyculin A, induce apoptosis in human submandibular gland cell line HSG cells.

To investigate a possible relationship between protein phosphorylation or dephosphorylation status and apoptosis in salivary gland cells, we examined the effects of okadaic acid and calyculin A, the protein phosphatase inhibitors, on cultured human submandibular gland ductal cell line, HSG cells. HSG cells at subconfluent stages were exposed to varying concentrations of okadaic acid or calyculin A. Apoptoses were analysed in HSG cells by phase-contrast microscopy, WST-1 cytotoxicity assay, Hoechst 33342 staining, and DNA ladder formation. Both okadaic acid and calyculin A induced cell death in HSG cells in a dose-dependent fashion. Marked nuclear condensation and fragmentation of chromatin was observed in HSG cells. DNA ladder formation was also detected in HSG cells by treatment with okadaic acid or calyculin A. The induced DNA ladder formation was dose-dependent with maximal effect at concentrations of 50 nM okadaic acid and 2 nM calyculin A, respectively, and were time-dependent from 14 h to 48 h. To further determine if new gene transcription and protein synthesis regulate okadaic acid-induced apoptosis in HSG cells, the cells were treated with cycloheximide or actinomycin D in the presence of 20 nM okadaic acid. Neither inhibitor protected the cells against okadaic acid-induced apoptosis. Based on the known selectivity of okadaic acid and calyculin A, our results indicate that the pathway of the apoptosis in the cultured salivary gland cells is regulated by protein phosphatase type 1 or type 2A. Our results also suggest that new protein synthesis and/or mRNA expression are not involved in okadaic acid-induced apoptosis in HSG cells.


Molecular cloning and characterization of a novel dual specificity protein phosphatase possibly involved in spermatogenesis.

Dual-specificity protein phosphatases (DSPs) play roles in the regulation of mitogenic signal transduction for extracellular stimulation and the cell cycle. In the present study, we identified a novel DSP, termed TMDP (testis- and skeletal-muscle-specific DSP). Nucleotide sequence analysis of TMDP cDNA indicated that the open reading frame of 597 bp encodes a protein of 198 amino acid residues with a predicted molecular mass of 22.5 kDa. The deduced amino acid sequence contains a motif for a conserved catalytic domain of DSPs and shows highest similarity to human Vaccinia HI-related phosphatase (45.5% identity) but low homology to the mitogen-activated protein kinase phosphatase and CDC25 subfamilies of DSPs. Recombinant TMDP protein exhibited intrinsic phosphatase activity towards both phospho-seryl/threonyl and -tyrosyl residues of myelin basic protein, with similar specific activities in vitro. Northern-blot analysis revealed that TMDP is most abundantly expressed in the testis. The expression in the testis is characterized as follows: (i) TMDP mRNA first appeared 3 weeks after birth, corresponding to the time that meiosis begins; (ii) TMDP mRNA was abundant in fractionated spermatocytes and round spermatids; and (iii) hybridization in situ showed that the TMDP mRNA is localized in spermatocytes and/or spermatids in seminiferous tubules. These data demonstrate that TMDP is a novel DSP abundantly expressed in the testis and suggest that TMDP may be involved in the regulation of meiosis and/or differentiation of testicular germ cells during spermatogenesis.


Inhibitors of protein synthesis and RNA synthesis protect against okadaic acid induced apoptosis in human osteosarcoma cell line MG63 cells but not in Saos-2 cells.

In a previous study, we demonstrated that the protein phosphatase inhibitors, okadaic acid and calyculin A, induced apoptosis in human osteosarcoma cell lines, Saos-2 and MG63 cells. In the present study, to determine if new gene transcription and protein synthesis are required for okadaic acid-induced apoptosis in Saos-2 and MG63 cells, the cells were treated for 48h with varying concentrations of the inhibitors of protein or RNA synthesis, i.e., cycloheximide, actinomycin D, and puromycin, in the presence of a fixed dose of okadaic acid. All these reagents in different concentrations prevented the okadaic acid-induced apoptosis in MG63 cells in a dose-dependent fashion. The same concentrations of cycloheximide, actinomycin D, or puromycin alone did not induce any apoptotic features in MG63 cells. However, not all the aforementioned reagents affected okadaic acid-induced apoptosis in Saos-2 cells. Okadaic acid-induced and cycloheximide-prevented apoptosis was shown by phase-contrast microscopy, WST-1 assay, direct visualization of nuclear condensation and fragmentation of chromatin, and the characteristic DNA ladder formation on agarose gel electrophoresis. The present results indicate that the induction of new cell death genes and ongoing protein synthesis may have a role in okadaic acid-induced apoptosis in MG63 cells and that such proteins are not required in Saos-2 cells.


Prilocaine induces apoptosis in osteoblastic cells.

To determine whether prilocaine, a local anesthetic, induces apoptosis in osteoblastic cells. After reaching subconfluence, human osteoblastic Saos-2 and MG63 cells and mouse osteoblastic MC3T3-E1 cells were exposed for 48 hr to varying concentrations of prilocaine up to 10 mM and the cytotoxicity of the cells was analyzed by phase-contrast microscopy and WST-1 assay. Saos-2 cells treated for 48 hr with 5 mM prilocaine were stained with Hoechst 33342 and nuclear fragmentation was examined under a fluorescence microscope. DNA was extracted from the cells treated with 5 mM prilocaine and DNA ladder formation (a hallmark of apoptosis) was analyzed by agarose gel electrophoresis. Prilocaine induced cell death in Saos-2 cells in a dose- and time-dependent manner up to the concentration of 10 mM. Marked nuclear condensation and fragmentation of chromatin were observed in the prilocaine-treated cells. DNA ladder formation also was induced by prilocaine treatment. Prilocaine-induced DNA ladder formation was dose-dependent with maximal effect at a concentration of 5 mM and was time-dependent from 12 to 48 hr. DNA ladder formation was also induced by prilocaine treatment in human osteoblastic MG63 cells and mouse osteoblastic MC3T3-E1 cells. Cycloheximide prevented prilocaine-induced apoptosis in Saos-2 cells in a dose-dependent fashion up to 20 microM as determined by WST-1 assay and DNA ladder formation in agarose gel electrophoresis. Osteoblastic cells treated with prilocaine exhibit both morphological and biochemical features indicative of apoptosis. The apoptotic mechanisms involve transcriptional regulation of specific proteins or protein synthesis.


Extracts of Actinobacillus actinomycetemcomitans induce apoptotic cell death in human osteoblastic MG63 cells.

Whether an extracellular component of periodontal-disease-causing bacteria induces apoptotic cell death in bone-related cells is unknown. To study the effects on osteoblasts of extracts obtained from sonicated Actinobacillus actinomycetemcomitans and Prevotella intermedia, we cultured human osteoblastic cell lines MG63 and Saos-2 cells and mouse osteoblastic cell line MC3T3-E1 cells in the presence of such extracts. The addition of the extracts from Actinobacillus actinomycetemcomitans induced cell death in MG63 cells in a dose- and time-dependent fashion over the concentration range of 0.1 to 10 microg/mL. By contrast, the extracts from Prevotella intermedia did not induce cell death in these cells, even in the presence of 10 microg/mL protein. By using the Hoechst 33342 staining technique, we observed marked nuclear condensation and fragmentation of chromatin in MG63 cells treated with the extracts of Actinobacillus actinomycetemcomitans. DNA ladder formation, a hallmark of apoptosis, also was detected in MG63 cells treated with extracts from Actinobacillus actinomycetemcomitans. In MG63 cells, DNA ladder formation was dose-dependent, with a maximal effect at a concentration of 10 microg/mL, and time-dependent, from 12 to 48 hrs. However, the extracts from Prevotella intermedia did not induce DNA fragmentation in MG63, Saos-2, or MC3T3-E1 cells. The extracts from Actinobacillus actinomycetemcomitans did not induce cell death and DNA fragmentation in Saos-2 and MC3T3-E1 cells. Sonicated extracts of Actinobacillus actinomycetemcomitans that had been treated with heat and trypsin did not induce DNA ladder formation in MG63 cells, suggesting that the apoptosis-inducing factors are proteinaceous. Cycloheximide prevented the Actinobacillus actinomycetemcomitans-induced DNA ladder formation in MG63 cells in a dose-dependent fashion, suggesting that new gene transcription and protein synthesis are regulated for Actinobacillus actinomycetemcomitans-induced apoptosis in MG63 cells. Our results indicate that apoptosis in alveolar bone cells induced by Actinobacillus actinomycetemcomitans plays an important role in periodontal diseases.


Localization of Fas antigen in oral squamous cell carcinoma.

Fas antigen is a cell-surface protein that transduces an apoptotic signal from the cell surface into the cytoplasm. The localization of Fas antigen in human oral squamous cell carcinoma (SCC) was examined by immunohistochemistry using a monospecific polyclonal antibody with a high titre. This antibody, designated as Fas D, was raised against a synthetic polypeptide segment corresponding to a specific extracellular domain of human Fas antigen (aa 104-114). Thirty-eight specimens of oral SCCs were stained with Fas D antibody and 26 (68%) reacted intensely. The specimens were graded as 'well', 'moderately', or 'poorly differentiated', according to the histopathological criteria. Out of 24 cases of the well differentiated tumours examined, 22 had reacted to Fas staining. The tumour cells formed nests that encompassed keratin pearls; staining was confined to cytoplasmic granules of peripheral cells. Among the moderately differentiated tumours, 4 out of 11 cases had reacted to Fas staining. No Fas-positive cells were observed in the poorly differentiated tumours, but only three specimens were examined. The expression of Fas antigen seems to be related to the degree of tumour differentiation.

The British Journal of Oral & Maxillofacial Surgery


Subcellular localization of protein phosphatase type 1 isotypes in mouse osteoblastic cells.

The cytolocalization of protein phosphatase type 1 catalytic subunits in exponentially growing mouse osteoblastic MC3T3-E1 cells was determined. Formaldehyde-fixed and alcohol-permeabilized cultured cells were reacted with the PP1 alpha, PP1 delta, PP1 gamma 1, and PP1 gamma 2 antibodies using immunohistochemical methods. With PP1 alpha antibody intense staining occurred in the nuclei, while with PP1 delta antibody nucleolus-like bodies were intensely stained. PP1 gamma 1 localized in the perinuclear region and in the nucleus of the cultured cells, with the staining reaction of the former being much stronger than that in the latter. An immunoreaction did not occur in the cells interacted with PP1 gamma 2 antibody or with the normal rabbit serum. Proteins were prepared from the exponentially growing cells and subconfluent cells. Cellular fractionation was also done with the exponentially growing cells and proteins were prepared from each fraction. Each protein preparation was subjected to SDS-PAGE followed by Western blot analysis with the antibodies. PP1 alpha recognized the 38 kDa proteins mainly present in the nucleus, whereas PP1 delta interacted with the proteins in the nucleolar fraction whose molecular weight was estimated as 37 kDa. PP1 gamma 1 antibody recognized a band corresponding to an estimated molecular weight of 36 kDa mainly in the cytosolic fraction. PP1 gamma 2 antibody and the normal rabbit serum did not interact with any proteins prepared from the cultured cells. Our observations show that four different isozymes of protein phosphatases occupy distinct compartments in MC3T3-E1 cells. This differential distribution suggests that these isozymes may play different roles in cellular functions.

Biochemical and Biophysical Research Communications


Histopathologic and immunohistochemical examination of diffuse invasion type SCC transformed from leukoplakia of the tongue -a case report-

浸潤式 4C 型扁平上皮癌へと移行した舌白板症症例の病理学的，免疫組織化学的検討


Variability of argyrophilic nucleolar organizer regions in osteoblastic cells.

The changes in the number of AgNORs per nucleus in normal cells are obscure. In the present study, we examined the variability of AgNORs in osteoblastic MC3T3-E1 cells. We designed to enumerate the number of AgNORs during cell differentiation. The cells were grown for up to 20 days in the presence of fetal bovine serum. At the days 3, 6, 9, 13 and 20 after plating, the cells were stained by AgNOR method and also used for measurement of ALP activity. The number of AgNORs per nucleus increased from 6.9±2.15 (at the day 3) to 11.7±2.79 (at the day 6), decreased to 7.9±2.25 (at the day 9) and then leveled to 7.7±2.19 (at the day 20). The ALP activity was maximal at the day 9 after plating. In osteoblastic cells, the number of AgNORs was maximized in the earlier matrix maturation stage, not in the proliferating stage and was variable during cell differentiation.

鍍銀染色法の一つであるAgNORs法は細胞増殖能を知ることができることから腫瘍病理学の分野では, 病理学的診断と新生物の予後をはかるマーカーとして注目を集めている.本法は免疫組織学的手法に依らないことから, 短時間で検鏡できる点でも迅速な診断を求められる臨床の場において広く応用される可能性を秘めている.現在までに, AgNORs法を用いて悪性腫瘍の診断と予後をはかる報告は数多くみられるが, ある一つの細胞を対象に細胞分化とAgNORs数との関連を調べたものはほとんどない.今回我々は骨細胞に分化する系統の骨芽細胞様細胞MC3T3-E1を用いて細胞増殖期から細胞外基質成熟期まで核内のAgNORs数の変化を調べ, 以下の結論を得た.1. 骨芽細胞の核内AgNORs数は一定でない.2. 細胞が基質成熟期の初期のときAgNORs数が最大を示した.AgNORs法は細胞増殖能をよく反映することから新生物の病理組織診断や予後に応用されつつあるが, 本法を応用する際には細胞の成長と分化度を考慮する必要があることが示唆された.

The Journal of the Kyushu Dental Society


Extracts of Prevotella intermedia and Actinobacillus actinomycetemcomitans inhibit alkaline phosphatase activity in osteoblastic cells in vitro.

In order to study the effects of sonicated extracts from Prevotella intermedia, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and other oral-related bacteria, as well as Escherichia coli on bone formation, clone MC3T3-E1 cells, which have retained osteoblastic activity, were cultured with various bacterial extracts. The addition of the sonicated extracts from Prevotella intermedia and Actinobacillus actinomycetemcomitans decreased the alkaline phosphatase activity in a dose-dependent fashion over the concentration range of 1-1000 ng ml-1 compared with the control. By contrast, the sonicated extracts from other oral bacteria including Porphyromonas gingivalis, Capnocytophaga ochracea, Streptococcus milleri and Streptococcus sanguis, and Escherichia coli did not decrease the alkaline phosphatase activity even in the presence of 100 ng ml-1 protein. The addition of Prevotella intermedia and Actinobacillus actinomycetemcomitans extracts that had been treated with heat and trypsin to the cell cultures also inhibited alkaline phosphatase activity in the cells, suggesting that inhibitory factors are not proteinaceous. Polymyxin B did not change the alkaline phosphatase activity in the cells treated with the extracts from Prevotella intermedia and Actinobacillus actinomycetemcomitans, suggesting that the inhibitory activity of the extracts is not lipopolysaccharide. The inhibitory effect of both extracts was observed in the molecular mass over 290 kDa eluted from Sephadex G-200 column. The inhibitory substances of Prevotella intermedia were partially purified and showed broad band with estimated molecular weight of 170-190 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicate that Prevotella intermedia and Actinobacillus actinomycetemcomitans may play an important role in inhibiting bone formation as well as in stimulating bone resorption.