AMP-activated protein kinase inhibitor decreases prostaglandin F2α-stimulated interleukin-6 synthesis through p38 MAP kinase in osteoblasts.

We previously showed that prostaglandin F(2α) (PGF(2α)) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in part via p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase but not stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of AMP-activated protein kinase (AMPK), an intracellular energy sensor, in PGF(2α)-stimulated IL-6 synthesis in MC3T3-E1 cells. PGF(2α) time-dependently induced the phosphorylation of the AMPK α-subunit. Compound C, an inhibitor of AMPK, dose-dependently suppressed PGF(2α)-stimulated IL-6 release. Compound C reduced the PGF(2α)-induced acetyl-CoA carboxylase phosphorylation. In addition, PGF(2α)-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The IL-6 mRNA expression induced by PGF(2α) was markedly reduced by compound C. Downregulation of the AMPK α1-subunit by short interfering RNA (siRNA) significantly suppressed the PGF(2α)-stimulated IL-6 release. PGF(2α)-induced phosphorylation of p38 MAP kinase was inhibited by compound C, which failed to affect the p44/p42 MAP kinase phosphorylation. These results strongly suggest that AMPK regulates PGF(2α)-stimulated IL-6 synthesis via p38 MAP kinase in osteoblasts.

AMPK limits IL-1-stimulated IL-6 synthesis in osteoblasts: involvement of IκB/NF-κB pathway.

AMP-activated protein kinase (AMPK) is currently known to act as a key regulator of metabolic homeostasis. Several biosynthetic enzymes for fatty acid or glycogen are recognized as the targets of AMPK. In the present study, we investigated the role of AMPK in the interleukin-1 (IL-1)-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. IL-1 induced phosphorylation of AMPK-α (Thr-172), which regulates AMPK activities, and acetyl-CoA carboxylase, a direct substrate of AMPK. Compound C, an inhibitor of AMPK, which suppressed the IL-1-induced phosphorylation of acetyl-CoA carboxylase, increased the release and the mRNA level of IL-6 stimulated by IL-1. Transfection of AMPK siRNA-α also amplified the IL-1-stimulated IL-6 release compared to the control cells. On the other hand, IL-1 elicited the phosphorylation of IκB, which caused subsequent decrease of total level of IκB. Wedelolactone, an inhibitor of IκB kinase, which reduced the phosphorylation both of IκB and NF-κB, significantly enhanced the IL-1-stimulated IL-6 synthesis. Compound C remarkably suppressed the IL-1-induced phosphorylation of IκB. These results strongly suggest that AMPK negatively regulates IL-1-stimulated IL-6 synthesis through the IκB/NF-κB pathway in osteoblasts.

Involvement of AMP-activated protein kinase in TGF-ß-stimulated VEGF synthesis in osteoblasts.

It is generally recognized that AMP-activated protein kinase (AMPK) acts as a key regulator of energy homeostasis. We have previously shown that transforming growth factor-ß (TGF-ß) stimulates synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether AMPK is involved in the TGF-ß-stimulated VEGF synthesis in osteoblast-like MC3T3-E1 cells. TGF-ß time-dependently induced the phosphorylation of the AMPK α-subunit (Thr172) and the AMPK ß-subunit (Ser108). Compound C, an AMPK inhibitor, significantly reduced the TGF-ß-stimulated VEGF release. The inhibitory effect of compound C was also observed in normal human osteoblasts (NHOst). Although compound C failed to affect the TGF-ß-induced phosphorylation of SAPK/JNK, p38 MAP kinase or Smad2, it markedly suppressed the TGF-ß-induced phosphorylation of both MEK1/2 and p44/p42 MAP kinase. In addition, compound C significantly suppressed the VEGF mRNA expression induced by TGF-ß. Taken together, our results strongly suggest that AMPK is involved in TGF-ß-stimulated VEGF synthesis, and that it functions at a point upstream of MEK1/2.

AMP-activated protein kinase regulates PDGF-BB-stimulated interleukin-6 synthesis in osteoblasts: involvement of mitogen-activated protein kinases.

AIM: We have previously reported that platelet-derived growth factor (PDGF)-BB stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is implicated in the IL-6 synthesis. In the present study,we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in the PDGF-BB-stimulated IL-6 synthesis in MC3T3-E1 cells. MAIN METHODS: The levels of IL-6 were measured by ELISA. The phosphorylation of each protein kinases was analyzed by Western blotting. The mRNA levels of IL-6 were determined by real-time RT-PCR. KEY FINDINGS: PDGF-BB time-dependently induced the phosphorylation of AMPK. Compound C, an inhibitor of AMPK, which reduced PDGF-BB-induced acetyl-CoA carboxylase phosphorylation, dose-dependently suppressed the PDGF-BB-stimulated IL-6 release. In addition, the PDGF-BB-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The mRNA expression of IL-6 induced by PDGF-BB was markedly reduced by compound C. The PDGF-BB-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK was inhibited by compound C. SIGNIFICANCE: These results strongly suggest that AMPK positively regulates PDGF-BB-stimulated IL-6 synthesis via the MAP kinases in osteoblasts.

Role of HSP27 in tumor necrosis factor-α-stimulated interleukin-6 synthesis in osteoblasts.

Heat shock protein 27 (HSP27) is known to act as a molecular chaperone. We have recently reported that HSP27 regulates osteocalcin synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the role of HSP27 in tumor necrosis factor-α (TNF-α)-stimulated interleukin-6 (IL-6) synthesis in MC3T3-E1 cells. The levels of IL-6 release and IL-6 mRNA stimulated by TNF-α in MC3T3-E1 cells transfected with HSP27 was significantly higher than those in the control cells. In addition, the levels of secreted IL-6 and IL-6 mRNA in the phospho-mimic HSP27-overexpressing cells were significantly higher than those in the non-phosphoryl-atable HSP27-overexpressing cells. Furthermore, we observed no significant differences in the phosphorylation levels of IκB/NFκB, Akt, and p44/p42 mitogen-activated protein kinase among the 4 types of transfected cells. Therefore, these results strongly suggest that HSP27 enhances TNF-α-stimulated IL-6 synthesis, and that the phosphorylation status of HSP27 is related to IL-6 synthesis in osteoblasts.

Rho-kinase regulates thrombin-stimulated interleukin-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts.

We have previously reported that thrombin stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of thrombin in the thrombin-stimulated IL-6 synthesis and the involvement of Rho-kinase in MC3T3-E1 cells. Thrombin time-dependently induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and myosin phosphatase targeting subunit-1 (MYPT-1), a Rho-kinase substrate. While SP600125, an inhibitor of SAPK/JNK, failed to reduce IL-6 synthesis, PD98059, a specific inhibitor of MEK, and SB203580 and BIRB0796, potent inhibitors of p38 MAP kinase, suppressed the IL-6 synthesis induced by thrombin. Y27632, a specific Rho-kinase inhibitor, significantly reduced thrombin-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed thrombin-stimulated IL-6 synthesis. Y27632 and fasudil failed to affect thrombin-induced phosphorylation of p44/p42 MAP kinase. Y27632 as well as fasudil attenuated thrombin-induced phosphorylation of p38 MAP kinase. These results strongly suggest that Rho-kinase regulates thrombin-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.

Wnt3a upregulates transforming growth factor-ß-stimulated VEGF synthesis in osteoblasts.

It is recognized that Wnt3a affects bone metabolism via the canonical Wnt/ß-catenin signalling pathway. We have previously shown that transforming growth factor-ß (TGF-ß) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TGF-ß-stimulated VEGF synthesis in these cells. Wnt3a, which alone had little effect on the VEGF levels, significantly enhanced the TGF-ß-stimulated VEGF release. Lithium chloride and SB216763, inhibitors of glycogen synthase kinase 3ß, markedly amplified the TGF-ß-stimulated VEGF release. Wnt3a failed to affect the TGF-ß-induced phosphorylation of Smad2, p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. Wnt3a and lithium chloride strengthened the VEGF mRNA expression induced by TGF-ß. These results strongly suggest that Wnt3a upregulates VEGF synthesis stimulated by TGF-ß via activation of the canonical pathway in osteoblasts.

(-)-Epigallocatechin gallate inhibits thyroid hormone-stimulated osteocalcin synthesis in osteoblasts.

It is recognized that catechin possesses beneficial properties for bone metabolism. We previously revealed that triiodothyronine (T3)-activated p38 mitogen-activated protein (MAP) kinase, but not p44/p42 MAP kinase, is involved in the synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of (-)-epigallocatechin gallate (EGCG), the predominant green tea polyphenol, on the synthesis of osteocalcin in MC3T3-E1 cells. EGCG significantly suppressed T3-stimulated osteocalcin synthesis. The inhibitory effect of EGCG was dose-dependent in the range of 3 to 30 µM. On the other hand, T3-induced phosphorylation of p38 MAP kinase was not affected by EGCG. EGCG profoundly inhibited T3-stimulated transcriptional activity. These results strongly suggest that EGCG suppresses T3-stimulated osteocalcin synthesis upstream of the transcriptional level in osteoblast-like MC3T3-E1 cells.

Enhancement of basic fibroblast growth factor-stimulated VEGF synthesis by Wnt3a in osteoblasts.

It is currently recognized that the Wnt signaling pathway regulates bone mass. We have previously reported that the basic fibroblast growth factor (FGF-2) stimulates the synthesis of the vascular endothelial growth factor (VEGF) at least in part via the p44/p42 mitogen-activated protein (MAP) kinase and the stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on FGF-2-stimulated VEGF synthesis in MC3T3-E1 cells. Wnt3a significantly augmented the FGF-2-stimulated VEGF release in a dose-dependent manner in the range between 1 and 30 ng/ml. Lithium chloride and SB216763, inhibitors of glycogen synthase kinase 3ß (GSK3ß), enhanced the FGF-2-stimulated VEGF release. Wnt3a did not affect the phosphorylation of the p44/p42 MAP kinase, SAPK/JNK, Akt, p38 MAP kinase or the p70 S6 kinase induced by FGF-2. Wnt3a and SB216763 increased the levels of VEGF mRNA expression induced by FGF-2. These results strongly suggest that Wnt3a enhances VEGF synthesis stimulated by FGF-2 via activation of the canonical Wnt/ß-catenin pathway in osteoblasts.

cAMP regulates ADP-induced HSP27 phosphorylation in human platelets.

Elevation of cAMP in platelets is recognized to play a suppressive role in platelet functions. We have previously shown that adenosine diphosphate (ADP)-induced phosphorylation of heat shock protein 27 (HSP27) via p38 mitogen-activated protein (MAP) kinase is correlated with platelet-derived growth factor (PDGF)-AB secretion and soluble CD40 ligand (sCD40L) release. In the present study, we investigated the relationship between cAMP and HSP27 phosphorylation in platelet function. 8-Bromoadenosine-3',5'-cyclic monophosphate (8-bromo-cAMP), a plasma membrane-permeable cAMP analogue, or cilostazol, an inhibitor of cAMP phosphodiesterase, markedly attenuated the ADP-induced phosphorylation levels of p38 MAP kinase. In addition, the ADP-induced HSP27 phosphorylation was suppressed by 8-bromo-cAMP or cilostazol. 8-Bromo-cAMP, forskolin and cilostazol remarkably reduced the ADP-stimulated PDGF-AB secretion and sCD40L release. These results strongly suggest that cAMP regulates ADP-stimulated platelet activation due to inhibition of HSP27 phosphorylation via p38 MAP kinase.

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.

AMP-activated protein kinase positively regulates FGF-2-stimulated VEGF synthesis in osteoblasts.

AMP-activated protein kinase (AMPK) is recognized as a regulator of energy homeostasis. We have previously reported that basic fibroblast growth factor (FGF-2) stimulates vascular endothelial growth factor (VEGF) release through the activation of 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. In the present study, we investigated the involvement of AMPK in FGF-2-stimulated VEGF release in these cells. FGF-2 time-dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an AMPK inhibitor, which suppressed the FGF-2-induced phosphorylation of AMPK, significantly inhibited the VEGF release stimulated by FGF-2. The AMPK inhibitor also reduced the mRNA expression of VEGF induced by FGF-2. The FGF-2-induced phosphorylation of both p44/p42 MAP kinase and SAPK/JNK was attenuated by compound C. These results strongly suggest that AMPK positively regulates the FGF-2-stimulated VEGF synthesis via p44/p42 MAP kinase and SAPK/JNK in osteoblasts.

Antithrombin III reduces collagen-stimulated granule secretion of PDGF-AB and the release of soluble CD40 ligand from human platelets.

Although antithrombin-III (AT-III), an anti-coagulant, has been shown to affect human platelet functions, the direct effect of AT-III on platelets is still unknown. We recently reported that the collagen-induced phosphorylation of the heat shock protein 27 (HSP27) via the p44/p42 mitogen-activated protein (MAP) kinase is sufficient for granule secretion and the release of soluble CD40 ligand (sCD40L) from platelets but not platelet aggregation. In the present study, we investigated whether AT-III affects the collagen-induced secretion of the platelet-derived growth factor (PDGF)-AB and sCD40L release. AT-III inhibited collagen-stimulated platelet aggregation. The collagen-induced secretion of PDGF-AB was significantly suppressed by AT-III. AT-III also reduced sCD40L release. AT-III markedly attenuated the collagen-induced phosphorylated levels of p44/p42 MAP kinase. In addition, AT-III suppressed collagen-induced HSP27 phosphorylation. These results strongly suggest that AT-III reduced collagen-stimulated platelet granule secretion due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

HSP90 inhibitors induce desensitization of EGF receptor via p38 MAPK-mediated phosphorylation at Ser1046/1047 in human pancreatic cancer cells.

Heat shock protein (HSP) 90 is known to be a molecular chaperone whose association is required for the stability and function of oncogenic protein including epidermal growth factor receptor (EGFR) that promotes cancer cell growth. Therefore, HSP90 is a promising target for therapy against cancer including in the pancreas, some of which are highly dependent on EGFR. We investigated the effects of HSP90 inhibitors on cytotoxicity and desensitization of EGFR in human pancreatic cancer cells (KP3, BxPc3 and AsPc1). 17-allylamino-17-demethoxy-geldanamycin (17-AAG), an inhibitor of HSP90, caused de-sensitization of EGFR in a time-dependent manner, concurrently inducing phosphorylation of EGFR at Ser1046/1047 (Ser1046/7), a site which plays an important role in EGFR desensitization in these pancreatic cancer cells. We also found similar effects in KP3 cells treated with other HSP90 inhibitors, geldanamycin and 17-dimethylamino-ethylamino-17-demethoxy-geldanamycin (17-DMAG). In KP3 cells, 17-AAG induced activation of either p44/p42 mitogen-activated protein kinase (MAPK) or p38 MAPK. Interestingly, whereas the inhibition of p44/p42 MAPK attenuated neither phosphorylation of EGFR at Ser1046/7 nor desensitization of EGFR, the phosphorylation at Ser1046/7 induced by 17-AAG was markedly attenuated by the inhibition of p38 MAPK, indicating that p38 MAPK induced this phosphorylation. Moreover, the inhibition of p38 MAPK significantly attenuated 17-AAG-induced EGFR desensitization. These results strongly suggest that EGFR phosphorylation at Ser1046/7 via activation of p38 MAPK induced by HSP90 inhibitors plays a pivotal role in EGFR desensitization in human pancreatic cancer cells.

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.