Regulation by heat shock protein 27 of osteocalcin synthesis in osteoblasts.

We have previously reported that various stimuli, including sphingosine 1-phosphate, are able to induce heat shock protein (HSP) 27 in osteoblast-like MC3T3-E1 cells. However, the precise role of HSP27 in bone metabolism has not been satisfactory clarified. In this study, we investigated the effect of HSP27 on osteocalcin synthesis induced by bone morphogenetic protein (BMP)-4 or T3 in these cells. In MC3T3-E1 cells, pretreatment with sphingosine 1-phosphate, sodium arsenite, or heat stress caused the attenuation of osteocalcin synthesis induced by BMP-4 or T3 with concurrent HSP27 induction. To further investigate the effect of HSP27, we established stable HSP27-transfected cells. The osteocalcin synthesis was significantly reduced in the stable HSP27-transfected MC3T3-E1 cells and normal human osteoblasts compared with empty-vector transfected cells. On the other hand, anisomycin, a p38 MAPK activator, caused the phosphorylation of HSP27 in both sphingosine 1-phosphate-stimulated untransfected MC3T3-E1 cells and HSP27-transfected MC3T3-E1 cells. An immunofluorescence microscopy study showed that the phosphorylated HSP27 induced by anisomycin concentrated perinuclearly in these cells, in which it colocalized with the endoplasmic reticulum. We also established stable mutant-HSP27-transfected cells. Osteocalcin synthesis induced by either BMP-4 or T3 was markedly suppressed in the nonphosphorylatable HSP27-overexpressing MC3T3-E1 cells compared with the phosphomimic HSP27-overexpressing cells. In contrast, the matrix mineralization was more obvious in nonphosphorylatable HSP27-overexpressing cells than that in phosphomimic HSP27-overexpressing cells. Taken together, these results strongly suggest that unphosphorylated HSP27 has an inhibitory effect on osteocalcin synthesis, but has a stimulatory effect on mineralization, in osteoblasts.

Role of heat shock protein 27 in transforming growth factor-ß-stimulated vascular endothelial growth factor release in osteoblasts.

We have previously reported that transforming growth factor-ß (TGF-ß) stimulates heat shock protein 27 (HSP27) induction via p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase in osteoblast-like MC3T3-E1 cells, and that the release of vascular endothelial growth factor (VEGF) is induced by TGF-ß in these cells. In the present study, we investigated the effect of HSP27 knockdown on the TGF-ß-stimulated VEGF release in these cells. Gene silencing using short interfering RNA against HSP27 (HSP27-siRNA) significantly suppressed the TGF-ß-induced VEGF release. Immunofluorescence microscopy also revealed that HSP27-siRNA suppressed the TGF-ß-stimulated VEGF induction as well as the reduction of HSP27 induction in these cells. However, the mRNA expression of VEGF stimulated by TGF-ß was not reduced even in cells transfected with HSP27-siRNA. These results strongly suggest that HSP27 induction is critical for TGF-ß-induced VEGF release in osteoblasts.

Wnt3a regulates tumor necrosis factor-α-stimulated interleukin-6 release in osteoblasts.

It is recognized that Wnt pathways regulate bone metabolism. We have previously shown that tumor necrosis factor-α (TNF-α) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3-kinase)/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TNF-α-stimulated IL-6 synthesis in these cells. Wnt3a, which alone did not affect the IL-6 levels, significantly suppressed the TNF-α-stimulated IL-6 release. Lithium Chloride (LiCl), which is an inhibitor of GSK3ß, markedly reduced the TNF-α-stimulated IL-6 release, similar to the results with Wnt3a. The suppression by Wnt3a or LiCl was also observed in the intracellular protein levels of IL-6 elicited by TNF-α. Wnt3a failed to affect the TNF-α-induced phosphorylation of p44/p42 MAP kinase, Akt, IκB or NFκB. Either Wnt3a or LiCl failed to reduce, rather increased the IL-6 mRNA expression stimulated by TNF-α. Lactacystin, a proteasome inhibitor, and bafilomycin A1, a lysosomal protease inhibitor, significantly restored the suppressive effect of Wnt3a on TNF-α-stimulated IL-6 release. Taken together, our results strongly suggest that Wnt3a regulates IL-6 release stimulated by TNF-α at post-transcriptional level in osteoblasts.

Mechanism of collagen-induced release of 5-HT, PDGF-AB and sCD40L from human platelets: role of HSP27 phosphorylation via p44/p42 MAPK.

Collagen plays a crucial role in hemostasis and thrombosis by activating platelets and reportedly induces the phosphorylation of heat shock protein (HSP) 27 in human platelets. However, the exact role of HSP27 phosphorylation in human platelets has not yet been clarified. In the present study, we investigated the mechanism of collagen-induced HSP27 phosphorylation and the role in human platelets. The collagen-effect on the phospholylation of HSP27 was dose-dependent in the range between 0.03 and 1.0 microg/ml. The phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK) was also stimulated by collagen. PD98059, a specific inhibitor of MAPK kinase (MEK1/2), reduced collagen-induced HSP27 phosphorylation as well as p44/p42 MAPK phosphorylation. PD98059 significantly suppressed collagen-induced releases of serotonin (5-HT), platelet-derived growth factor (PDGF)-AB and soluble CD40 ligand (sCD40L) while it had little effect on the platelet aggregation. These results strongly suggest that the collagen-induced phosphorylation of HSP27 via p44/p42 MAPK is sufficient for releases of 5-HT, PDGF-AB and sCD40L from human platelets.

Synergistic effect of vasoactive intestinal peptides on TNF-alpha-induced IL-6 synthesis in osteoblasts: amplification of p44/p42 MAP kinase activation.

We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of vasoactive intestinal peptide (VIP) on TNF-alpha-induced IL-6 synthesis in these cells. VIP, which by itself slightly stimulated IL-6 synthesis, synergistically enhanced the TNF-alpha-induced IL-6 synthesis in MC3T3-E1 cells. The synergistic effect of VIP on the TNF-alpha-induced IL-6 synthesis was concentration-dependent in the range between 1 and 70 nM. We previously reported that VIP stimulated cAMP production in MC3T3-E1 cells. Forskolin, a direct activator of adenylyl cyclase, or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP), a plasma membrane-permeable cAMP analogue, markedly enhanced the TNF-alpha-induced IL-6 synthesis as well as VIP. VIP markedly up-regulated the TNF-alpha-induced p44/p42 MAP kinase phosphorylation. The Akt phosphorylation stimulated by TNF-alpha was only slightly affected by VIP. PD98059, a specific inhibitor of MEK1/2, significantly suppressed the enhancement of TNF-alpha-induced IL-6 synthesis by VIP. The synergistic effect of a combination of VIP and TNF-alpha on the phosphorylation of p44/p42 MAP kinase was diminished by H-89, an inhibitor of cAMP-dependent protein kinase. These results strongly suggest that VIP synergistically enhances TNF-alpha-stimulated IL-6 synthesis via up-regulating p44/p42 MAP kinase through the adenylyl cyclase-cAMP system in osteoblasts.

Rho-kinase limits FGF-2-stimulated VEGF release in osteoblasts.

We previously reported that basic fibroblast growth factor (FGF-2) stimulates the release of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates the VEGF release in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is involved in FGF-2-stimulated VEGF release in MC3T3-E1 cells. FGF-2 induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632, a specific inhibitor of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly enhanced the FGF-2-stimulated VEGF release. Fasudil, another Rho-kinase inhibitor, also amplified the VEGF release. FGF-2 significantly stimulated VEGF accumulation and fasudil enhanced FGF-2-stimulated VEGF accumulation also in whole cell lysates. Neither Y27632 nor fasudil affected the phosphorylation levels of p44/p42 MAP kinase or p38 MAP kinase. Y27632 and fasudil markedly strengthened the FGF-2-induced phosphorylation of SAPK/JNK. Y27632 as well as fasudil enhanced FGF-2-stimulated VEGF release and Y27632 enhanced the FGF-2-induced phosphorylation levels of SAPK/JNK also in human osteoblasts. These results strongly suggest that Rho-kinase negatively regulates FGF-2-stimulated VEGF release in osteoblasts.

p70 S6 kinase limits tumor necrosis factor-alpha-induced interleukin-6 synthesis in osteoblast-like cells.

Our previous study demonstrated that tumor necrosis factor-alpha (TNF-alpha) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether p70 S6 kinase is involved in TNF-alpha-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-alpha time dependently induced the phosphorylation of p70 S6 kinase. Rapamycin, an inhibitor of p70 S6 kinase, which attenuated the phosphorylation of p70 S6 kinase induced by TNF-alpha, significantly amplified the TNF-alpha-stimulated IL-6 synthesis. TNF-alpha-induced phosphorylations of both p44/p42 MAP kinase and Akt were markedly enhanced by rapamycin. The amplification by rapamycin of TNF-alpha-induced IL-6 synthesis was reduced by PD98059, a specific inhibitor of MEK1/2, or Akt inhibitor. Rapamycin enhanced the IL-6 synthesis and the phosphorylation of Akt induced by TNF-alpha also in human osteoblasts. Taken together, these results strongly suggest that p70 S6 kinase limits the TNF-alpha-stimulated IL-6 synthesis at a point upstream from p44/p42 MAP kinase and Akt in osteoblast-like cells.

Rho-kinase regulates prostaglandin D(2)-stimulated heat shock protein 27 induction in osteoblasts.

We previously reported that prostaglandin D(2) (PGD(2)) stimulates heat shock protein 27 (HSP27) induction through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In addition, we recently showed that PGD(2) activates Rho-kinase, resulting in the regulation of interleukin-6 synthesis via activation of p38 MAP kinase but not p44/p42 MAP kinase in these cells. In the present study, in order to investigate whether Rho-kinase is involved in the PGD(2)-stimulated HSP27 induction in MC3T3-E1 cells, we examined the effects of Rho-kinase inhibitors on HSP27 induction. Y27632 and fasudil, Rho-kinase inhibitors, markedly suppressed the HSP27 induction stimulated by PGD(2) in a dose-dependent manner without affecting levels of HSP70 in the presence of PGD(2). Immunofluorescence microscopy studies also revealed that Y27632 and fasudil markedly suppressed the induction of HSP27. Y27632 and fasudil attenuated the PGD(2)-induced phosphorylation levels of SAPK/JNK. In conclusion, Rho-kinase inhibitors regulate PGD(2)-stimulated HSP27 induction via activation of both SAPK/JNK and p38 MAP kinase in osteoblasts.

Antithrombin III suppresses ADP-induced platelet granule secretion: inhibition of HSP27 phosphorylation.

Antithrombin III (AT-III), an anti-coagulant, has recently been reported to directly affect human platelet functions. However, the exact mechanism of AT-III in platelets remains to be clarified. We have previously shown that adenosine diphosphate (ADP)-induced phosphorylation of heat shock protein 27 (HSP27) via p44/p42 mitogen-activated protein kinase (MAPK) and p38 MAPK is correlated with platelet granule secretion. In the present study, we investigated the relationship between AT-III and the ADP-induced platelet granule secretion. The ADP-induced secretion of platelet-derived growth factor (PDGF)-AB and serotonin (5-HT) were significantly suppressed by AT-III. The ADP-induced soluble CD40 ligand (sCD40L) release was inhibited by either PD98059, a MEK inhibitor, or SB203580, a p38 MAPK inhibitor. AT-III also inhibited the sCD40L release. AT-III markedly attenuated the ADP-induced phosphorylation levels of p44/p42 MAPK and p38 MAPK. Furthermore, the ADP-induced HSP27 phosphorylation was suppressed by AT-III. These results strongly suggest that AT-III directly acts on platelets and suppresses ADP-induced platelet granule secretion due to inhibiting HSP27 phosphorylation via p44/p42 MAPK and p38 MAPK.

(-)-Epigallocatechin gallate attenuates the induction of HSP27 stimulated by sphingosine 1-phosphate via suppression of phosphatidylinositol 3-kinase/Akt pathway in osteoblasts.

We previously reported that sphingosine 1-phosphate stimulates the induction of heat shock protein 27 (HSP27) through p38 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the induction of HSP27 in these cells and its mechanism, since it was previously reported that catechin, including EGCG, suppresses bone resorption. EGCG significantly reduced the induction of HSP27 stimulated by sphingosine 1-phosphate in a dose-dependent manner between 10 and 30 microM. Immunofluorescence microscopy studies revealed that sphingosine 1-phosphate certainly stimulated the induction of HSP27 in the cytosol of these cells, and that EGCG clearly suppressed its induction. However, sphingosine 1-phosphate-stimulated phosphorylation of p38 MAP kinase or MAPKAP-2 was not affected by EGCG. By contrast, EGCG markedly suppressed the phosphorylations of both Akt and glycogen synthase kinase-3beta stimulated by sphingosine 1-phosphate. These results strongly suggest that EGCG suppresses the induction of HSP27 stimulated by sphingosine 1-phosphate via attenuation of, not the p38 MAP kinase pathway, but of the phosphatidylinositol 3-kinase/Akt pathway in osteoblasts.

Involvement of Rho-kinase in prostaglandin E(1)-stimulated VEGF synthesis through stress-activated protein kinase/c-Jun N-terminal kinase in osteoblast-like MC3T3-E1 cells.

We have previously shown that prostaglandin E(1) (PGE(1)) stimulates the synthesis of vascular endothelial growth factor (VEGF) through p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rho-kinase in the PGE(1)-stimulated VEGF synthesis in these cells. PGE(1) induced within 3min the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632 and fasudil, specific inhibitors of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly suppressed the PGE(1)-stimulated VEGF synthesis. Y27632 and fasudil markedly reduced the PGE(1)-induced phosphorylation of SAPK/JNK without affecting the phosphorylation levels of p38 MAP kinase or p44/p42 MAP kinase. These results strongly suggest that Rho-kinase functions at a point upstream of SAPK/JNK and regulates PGE(1)-stimulated VEGF synthesis in osteoblasts.

Involvement of Rho-kinase in sphingosine 1-phosphate-stimulated HSP27 induction in osteoblasts.

We previously reported that sphingosine 1-phosphate induces heat shock protein 27 (HSP 27) via activation of phosphatidylinositol 3-kinase (PI3K)/Akt and p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in sphingosine 1-phosphate-stimulated induction of HSP27 in MC3T3-E1 cells. Sphingosine 1-phosphate time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced sphingosine 1-phosphate-stimulated HSP27 induction, as well as MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, also suppressed sphingosine 1-phosphate-stimulated HSP27 induction. Y27632, as well as fasudil, attenuated sphingosine 1-phosphate-induced phosphorylation of p38 MAP kinase. However, Akt phosphorylation induced by sphingosine 1-phosphate was not affected by either Rho-kinase inhibitor. These results strongly suggest that Rho-kinase regulates sphingosine 1-phosphate-stimulated induction of HSP27 at a point upstream of p38 MAP kinase in osteoblasts.

Tacrolimus but not cyclosporine A enhances FGF-2-induced VEGF release in osteoblasts.

We previously reported that basic fibroblast growth factor (FGF-2) stimulates the release of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates VEGF release. In addition, p70 S6 kinase activated by FGF-2 negatively regulates VEGF release via SAPK/JNK. In the present study, we investigated the effects of tacrolimus (FK506) and cyclosporine A, well-known immunosuppressants, on the FGF-2-induced VEGF release in these cells. Tacrolimus, but not cyclosporine A which alone had no effect on VEGF basal levels, significantly enhanced FGF-2-stimulated VEGF release. Tacrolimus markedly enhanced FGF-2-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP or p38 MAP kinases. SP600125, a specific inhibitor of SAPK/JNK, reduced the amplification by tacrolimus of the FGF-2-induced VEGF release. The FGF-2-induced phosphorylation of p70 S6 kinase was suppressed by tacrolimus. These results strongly suggest that tacrolimus enhances FGF-2-stimulated VEGF release via up-regulation of SAPK/JNK through modulating p70 S6 kinase in osteoblasts.

Rho-kinase inhibitors decrease TGF-beta-stimulated VEGF synthesis through stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts.

We have previously reported that transforming growth factor-beta (TGF-beta) stimulates the synthesis of vascular endothelial growth factor (VEGF) through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In order to investigate whether Rho-kinase is involved in the TGF-beta-stimulated VEGF synthesis in these cells we examined the effects of Rho-kinase inhibitors on the VEGF synthesis. TGF-beta time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1) which is a well known substrate of Rho-kinase. Y27632 and fasudil, Rho-kinase inhibitors, significantly reduced the TGF-beta-stimulated VEGF synthesis as well as the MYPT-1 phosphorylation. Y27632 and fasudil failed to affect the TGF-beta-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or Smad2. On the contrary, Y27632 as well as fasudil markedly suppressed the TGF-beta-induced phosphorylation of SAPK/JNK. Taken together, our results strongly suggest that Rho-kinase regulates TGF-beta-stimulated VEGF synthesis via SAPK/JNK activation in osteoblasts.

Involvement of Rho-kinase in TGF-ß-stimulated heat shock protein 27 induction in osteoblasts.

We previously reported that transforming growth factor-ß (TGF-ß) stimulates heat shock protein 27 (HSP27) induction through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In addition, we recently reported that the Rho-kinase inhibitors Y27632 and fasudil suppressed the TGF-ß-induced phosphorylation of SAPK/JNK, but not p44/p42 MAP kinase, p38 MAP kinase or Smad2. In the present study, to investigate whether Rho-kinase is involved in TGF-ß-stimulated HSP27 induction in MC3T3-E1 cells, we examined the effects of Rho-kinase inhibitors on HSP27 induction. Y27632 and fasudil significantly suppressed the HSP27 induction stimulated by TGF-ß in a dose-dependent manner, without affecting the protein levels of HSP70 or HSP90. Immunofluorescence microscopy also revealed that TGF-ß clearly stimulated, while Y27632 and fasudil markedly suppressed, HSP27 induction in the cytosol of these cells. Taken together, these findings indicate that Rho-kinase regulates TGF-ß-stimulated HSP27 induction via SAPK/JNK activation in osteoblasts.

Raloxifene-induced acceleration of platelet aggregation.

A 59-year-old postmenopausal woman diagnosed to have primary osteoporosis began to take 60 mg daily of oral raloxifene. The platelet aggregation induced by 1 microM adenosine diphosphate (ADP) and the alpha2-antiplasmin activity were accelerated significantly after 8 weeks from the beginning of raloxifene-treatment, and gradually deteriorated up to 24 weeks. ADP markedly caused the phosphorylation of Akt in the platelets obtained at 24 weeks. Although there were no subjective complaints at 24 weeks, the medication was stopped with her consent to avoid any adverse effects due to thrombus formation. The platelet hyper-aggregability and Akt phosphorylation induced by ADP disappeared at 4 weeks after the cessation of medication. These results strongly suggest that raloxifene caused the acceleration of platelet aggregation and subclinical thrombus formation through the Akt signal pathway in this case.

Function of Rho-kinase in prostaglandin D2-induced interleukin-6 synthesis in osteoblasts.

We have previously reported that prostaglandin D2 (PGD2) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in the PGD2-stimulated IL-6 synthesis in MC3T3-E1 cells. PGD2 time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced the PGD2-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed the PGD2-stimulated IL-6 synthesis. The PGD2-stimulated IL-6 synthesis was reduced by PD98059, a MEK inhibitor, and SB203580, an inhibitor of p38 mitogen-activated protein (MAP) kinase, but not SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). However, Y27632 and fasudil failed to affect the PGD2-induced phosphorylation of p44/p42 MAP kinase. On the other hand, Y27632 as well as fasudil markedly attenuated the PGD2-induced phosphorylation of p38 MAP kinase. In addition, PGD2 additively induced IL-6 synthesis in combination with endothelin-1 which induces IL-6 synthesis through p38 MAP kinase regulated by Rho-kinase. These results strongly suggest that Rho-kinase regulates PGD2-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.

Involvement of Rho-kinase in prostaglandin F2alpha-stimulated interleukin-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts.

We have previously reported that prostaglandin F(2alpha) (PGF(2alpha)) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in the PGF(2alpha)-stimulated IL-6 synthesis in MC3T3-E1 cells. PGF(2alpha) time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced the PGF(2alpha)-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed the PGF(2alpha)-stimulated IL-6 synthesis. Y27632 and fasudil failed to affect the PGF(2alpha)-induced phosphorylation of p44/p42 MAP kinase. SB203580 and BIRB0796, potent inhibitors of p38 MAP kinase, suppressed the IL-6 synthesis induced by PGF(2alpha). While SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), failed to reduce the synthesis. Y27632 as well as fasudil attenuated the PGF(2alpha)-induced phosphorylation of p38 MAP kinase. These results strongly suggest that Rho-kinase regulates PGF(2alpha)-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.

Phosphorylated heat shock protein 27 represses growth of hepatocellular carcinoma via inhibition of extracellular signal-regulated kinase.

Heat shock protein 27, one of the low molecular weight stress proteins, is recognized as a molecular chaperone; however, other functions have not yet been well established. Phosphorylated heat shock protein 27 levels inversely correlate with the progression of human hepatocellular carcinoma. This study shows that phosphorylated heat shock protein 27 interferes with cell growth of the hepatocellular carcinoma-derived HuH7 cells in the presence of the proinflammatory cytokine, tumor necrosis factor-alpha, via inhibition of the sustained activation of the extracellular signal-regulated kinase signal pathway. The activities of Raf/extracellular signal-regulated kinase and subsequent activator protein-1 transactivation and the induction levels of cyclin D1 were lower in HuH7 cells transfected with phosphorylated heat shock protein 27 than those with unphosphorylated heat shock protein 27. Moreover, phosphorylated heat shock protein 27 up-regulated the levels of p38 mitogen-activated protein kinase and mitogen-activated protein kinase phosphatase-1, an inhibitory protein of extracellular signal-regulated kinase. These results indicate that phosphorylated heat shock protein 27 might suppress the extracellular signal-regulated kinase activity in the hepatocellular carcinoma cells via two separate pathways in an inflammatory state. The extracellular signal-regulated kinase activity is inversely correlated with phosphorylated heat shock protein 27 at serine 15 and also in human hepatocellular carcinoma tissues in vivo. Because the extracellular signal-regulated kinase signal pathway is a major proliferation signal of hepatocellular carcinoma, activator protein-1 activation is an early event in hepatocarcinogenesis. These findings strongly suggest that the control of the phosphorylated heat shock protein 27 levels could be a new therapeutic strategy especially to counter the recurrence of hepatocellular carcinoma.

HSP27 phosphorylation is correlated with ADP-induced platelet granule secretion.

Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis by activating platelets. ADP has been reported to induce heat-shock protein (HSP) 27 phosphorylation in human platelets. However, the exact role of HSP27 phosphorylation in human platelets has not yet been clarified. In the present study, we investigated the mechanisms and the roles of ADP-induced HSP27 phosphorylation in human platelets. We showed for the first time that both of decreased phosphorylation levels of HSP27 by PD98059, a MEK1/2 inhibitor and SB203580, a p38 MAPK inhibitor were correlated with the suppressed levels of platelet granule secretion but not with platelet aggregation. Furthermore, the inhibition of either the p44/p42 MAPK or p38 MAPK pathways had no effect on ADP-induced platelet aggregation. These results strongly suggest that the ADP-induced phosphorylation of HSP27 via p44/p42 MAPK and/or p38 MAPK is therefore sufficient for platelet granule secretion but not for platelet aggregation in humans.