Involvement of SAPK/JNK in basic fibroblast growth factor-induced vascular endothelial growth factor release in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) activates p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates VEGF release. In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in FGF-2-induced VEGF release in these cells. FGF-2 markedly induced the phosphorylation of SAPK/JNK. SP600125, an inhibitor of SAPK/JNK, markedly reduced the FGF-2-induced VEGF release. SP600125 suppressed the FGF-2-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase induced by FGF-2. PD98059, an inhibitor of upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase, failed to affect the FGF-2-induced phosphorylation of SAPK/JNK. A combination of SP600125 and SB203580 suppressed the FGF-2-stimulated VEGF release in an additive manner. These results strongly suggest that FGF-2 activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a part in FGF-2-induced VEGF release.     

Involvement of SAPK/JNK in prostaglandin E(1)-induced VEGF synthesis in osteoblast-like cells

We previously reported that prostaglandin E₁ (PGE₁) activates both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase via cAMP-dependent protein kinase in osteoblast-like MC3T3-E1 cells, and that p38 MAP kinase but not p42/p44 MAP kinase is involved in PGE₁-induced synthesis of vascular endothelial growth factor (VEGF). In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in the PGE₁-induced VEGF synthesis in MC3T3-E1 cells. PGE₁ induced the phosphorylation of SAPK/JNK. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced the PGE₁-induced VEGF synthesis. Forskolin, a direct activator of adenylyl cyclase, elicited the phosphorylation of SAPK/JNK, and 8bromo-cAMP, a plasma membrane-permeable cAMP analogue-stimulated VEGF synthesis was significantly reduced by SP600125. SP600125 suppressed the PGE₁-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p38 MAP kinase induced by PGE₁. The phosphorylation of c-Jun induced by PGE₁ was also inhibited by SP600125. SB203580, a p38 MAP kinase inhibitor, failed to reduce the PGE₁ induced phosphorylation of SAPK/JNK. A combination of SP600125 and SB203580 suppressed the PGE₁-stimulated VEGF synthesis in an additive manner. These results strongly suggest that PGE₁ activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a part in PGE₁-induced VEGF synthesis.     

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.     

p38 MAP kinase is required for vasopressin-stimulated HSP27 induction in aortic smooth muscle cells

We previously showed that arginine vasopressin (AVP) stimulates heat shock protein 27 (HSP27) induction through protein kinase C activation in aortic smooth muscle A10 cells. In the present study, we examined whether the mitogen-activated protein (MAP) kinase superfamily is involved in the AVP-stimulated HSP27 induction in A10 cells. AVP stimulated the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. On the contrary, AVP had little effect on SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) phosphorylation. The HSP27 accumulation by AVP was not affected by PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, suppressed the AVP-induced accumulation of HSP27. 12-O-tetradecanoylphorbol 13-acetate, an activator of protein kinase C, induced accumulation of HSP27 and was not inhibited by PD98059 but was inhibited by SB203580. Calphostin C and ET-18-OCH(3), inhibitors of protein kinase C, reduced the phosphorylation of p38 MAP kinase by AVP. SB203580 and PD169316 suppressed the AVP-increased levels in mRNA for HSP27. Dissociation of the aggregated HSP27 to the dissociated HSP27 was induced by AVP. These results strongly suggest that p38 MAP kinase takes part in the pathway of the AVP-stimulated induction of HSP27 in vascular smooth muscle cells.     

Involvement of AMP-activated protein kinase in thrombin-stimulated interleukin 6 synthesis in osteoblasts

We previously demonstrated that thrombin stimulates synthesis of interleukin 6 (IL6), a potent bone resorptive agent, in part via p44/p42 MAP kinase and p38 MAP kinase but not through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In this study, we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in thrombin-stimulated IL6 synthesis in MC3T3-E1 cells. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, or AMPK was determined by western blot analysis. The release of IL6 was determined by the measurement of IL6 concentration in the conditioned medium using an ELISA kit. The expression of IL6 mRNA was determined by RT-PCR. Thrombin time dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an inhibitor of AMPK, dose-dependently suppressed the thrombin-stimulated IL6 release in the range between 0.3 and 10 μM. Compound C reduced thrombin-induced acetyl-CoA carboxylase phosphorylation. The IL6 mRNA expression induced by thrombin was markedly reduced by compound C. Downregulation of AMPK by siRNA suppressed the thrombin-stimulated IL6 release. The thrombin-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was inhibited by compound C, which failed to affect SAPK/JNK phosphorylation. These results strongly suggest that AMPK regulates thrombin-stimulated IL6 synthesis via p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.     

Limitation by p70 S6 kinase of platelet-derived growth factor-BB-induced interleukin 6 synthesis in osteoblast-like MC3T3-E1 cells

It has been reported that platelet-derived growth factor-BB (PDGF-BB) stimulates interleukin 6 (IL-6) in osteoblasts. In the present study, we investigated the mechanism of IL-6 synthesis induced by PDGF-BB in osteoblast-like MC3T3-E1 cells. Platelet-derived growth factor-BB 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 p70 S6 kinase. PD98059 (an inhibitor of MAP kinase/extracellular signal-regulated kinase kinase [MEK]), SB203580 (an inhibitor of p38 MAP kinase), or SP600125 (an inhibitor of SAPK/JNK) suppressed the IL-6 synthesis induced by PDGF-BB. Rapamycin, an inhibitor of p70 S6 kinase, significantly enhanced the PDGF-BB-stimulated IL-6 synthesis. The PDGF-BB-induced phosphorylation of p70 S6 kinase was suppressed by rapamycin. Rapamycin failed to affect the PDGF-BB-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, or SAPK/JNK. These results strongly suggest that PDGF-BB stimulates IL-6 synthesis through activation of 3 MAP kinases in osteoblasts and that p70 S6 kinase negatively regulates the IL-6 synthesis.     

Mechanism of prostaglandin E2-stimulated heat shock protein 27 induction in osteoblast-like MC3T3-E1 cells

We investigated the effect of prostaglandin E2 (PGE2) on the induction of heat shock protein 27 (HSP27) and HSP70, and the mechanism behind the induction in osteoblast-like MC3T3-E1 cells. PGE2 time-dependently increased the level of HSP27 without affecting the level of HSP70. PGE2 stimulated the accumulation of HSP27 dose-dependently in the range between 10 nM and 10 microM. PGE2 stimulated the increase in the level of the mRNA for HSP27. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), suppressed the PGE2-induced HSP27 accumulation. The effect of PGE2 on HSP27 accumulation was reduced in the PKC down-regulated cells. BAPTA/AM, a chelator of intracellular Ca2+, or TMB-8, an inhibitor of intracellular Ca2+ mobilization, reduced the accumulation of HSP27 induced by PGE2. Dibutyryl cAMP had little effect on the basal level of HSP27. PGE2 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059 and U-0126, inhibitors of the upstream kinase of p44/p42 MAP kinase, reduced the accumulation of HSP27 induced by PGE2. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the HSP27 accumulation induced by PGE2. U-73122, an inhibitor of phospholipase C, and calphostin C reduced the PGE2-induced phosphorylation of both p44/p42 MAP kinase and p38 MAP kinase. These results indicate that PGE2 stimulates the induction of HSP27 through PKC-dependent activations of both p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.     

p38 mitogen-activated protein (MAP) kinase but not p44/p42 MAP kinase is involved in prostaglandin E1-induced vascular endothelial growth factor synthesis in osteoblasts

We investigated the mechanism underlying vascular endothelial growth factor (VEGF) synthesis stimulated by prostaglandin E1 (PGE1) in osteoblast-like MC3T3-E1 cells. PGE1 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. SB203580, a specific inhibitor of p38 MAP kinase, inhibited the PGE1-stimulated VEGF synthesis as well as PGE1-induced phosphorylation of p38 MAP kinase. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, which reduced the PGE1-induced phosphorylation of p44/p42 MAP kinase, had little effect on the VEGF synthesis stimulated by PGE1. AH-6809, an antagonist of the subtypes of the PGE receptor, EP1 and EP2, or SC-19220, an antagonist of EP1 receptor, did not inhibit the PGE1-induced VEGF synthesis. H-89, an inhibitor of cAMP-dependent protein kinase, and SQ22536, an inhibitor of adenylate cyclase, reduced the VEGF synthesis induced by PGE1. Cholera toxin, an activator of G(s), and forskolin, an activator of adenylate cyclase, induced VEGF synthesis. SB203580 and PD169316, another specific inhibitor of p38 MAP kinase, reduced the cholera toxin-, forskolin- or 8bromo-cAMP-stimulated VEGF synthesis. However, PD98059 failed to affect the VEGF synthesis stimulated by cholera toxin, forskolin or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP). SB203580 reduced the phosphorylation of p38 MAP kinase induced by forskolin or 8bromo-cAMP. These results strongly suggest that p44/p42 MAP kinase activation is not involved in the PGE1-stimulated VEGF synthesis in osteoblasts but that p38 MAP kinase activation is involved.     

Mitogen-activated protein (MAP) kinases are involved in interleukin-1 (IL-1)-induced IL-6 synthesis in osteoblasts: modulation not of p38 MAP kinase, but of p42/p44 MAP kinase by IL-1-activated protein kinase C.

We previously reported that interleukin-1alpha (IL-1alpha)-induced activation of protein kinase C (PKC) via phosphatidylcholine-specific phospholipase C (PC-PLC) limits IL-6 synthesis induced by IL-1alpha itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism behind IL-1alpha-induced IL-6 synthesis in MC3T3-E1 cells. IL-1alpha time-dependently stimulated the phosphorylation of both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059, a specific inhibitor of the upstream kinase that activates p42/p44 MAP kinase, inhibited the IL-1alpha-induced IL-6 synthesis as well as the phosphorylation of p42/p44 MAP kinase induced by IL-1alpha. SB203580, a specific inhibitor of p38 MAP kinase, also reduced both the phosphorylation of p38 MAP kinase and the IL-6 synthesis. 1-Oleoyl-2-acetylglycerol, an activator of PKC, suppressed the IL-1alpha-induced IL-6 synthesis. Calphostin C, a specific inhibitor of PKC, or D-609, a specific inhibitor of PC-PLC, significantly enhanced the IL-1alpha-induced phosphorylation of p42/p44 MAP kinase without affecting the phosphorylation of p38 MAP kinase. The phosphorylation of p42/p44 MAP kinase by IL-1alpha was markedly increased in PKC-down-regulated MC3T3-E1 cells. Neither 12-O-tetradecanoylphorbol-13-acetate, known to be an activator of PKC, nor 1-oleoyl-2-acetylglycerol affected the phosphorylation of p38 MAP kinase induced by IL-1alpha. These results strongly suggest that IL-1alpha-induced IL-6 synthesis is mediated via activations of both p42/p44 MAP kinase and p38 MAP kinase in osteoblasts, and that PKC activated by IL-1alpha itself negatively regulates IL-6 synthesis at a point upstream from p42/p44 MAP kinase.     

Endothelin-mediated vascular growth requires p42/p44 mitogen-activated protein kinase and p70 S6 kinase cascades via transactivation of epidermal growth factor receptor.

Endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor peptide, exerts a growth-promoting effect on vascular smooth muscle cells, implicating its pathogenic role in vascular remodeling. To gain insight into the cellular and molecular mechanism whereby ET-1 induces vascular growth, we studied whether transactivation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor, are required for activation of p42/p44 mitogen-activated protein (MAP) kinase and p70 S6 kinase (p70S6K), and subsequent growth-promotion by ET-1 in cultured rat vascular smooth muscle cells. Immunoblotting with antiphosphotyrosine antibody revealed that ET-1 rapidly (within 2 min) and transiently induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR. ET-1 rapidly increased association of EGFR and Shc with glutathione-S-transferase-Grb2 fusion protein. The ET-1-induced activation of MAP kinase was reduced by an EGFR kinase inhibitor (AG1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG1296). AG1478 dose-dependently decreased ET-1-stimulated MAP kinase activity as well as [3H]leucine and [3H]thymidine uptake. The ET-1-induced tyrosine phosphorylation of EGFR, as well as MAP kinase activation, was inhibited by an ETA receptor antagonist and intracellular Ca2+ antagonists but not by an ETB receptor antagonist, pertussis toxin, or protein kinase C inhibitors. In addition, dominant negative mutant of H-Ras and a MAP kinase kinase (MEK-1) inhibitor (PD98059) completely blocked ET-1-induced MAP kinase activation as well as [3H]leucine and [3H]thymidine uptake. Both AG1478 and PD98059 inhibited ET-1-induced phosphorylation and activation of p70S6K. Furthermore, rapamycin, a selective inhibitor of mammalian target of rapamycin, completely blocked ET-1-stimulated [3H]leucine and [3H]thymidine uptake. These results suggest that ETA receptor-mediated vascular growth by ET-1 requires both MAP kinase and p70S6K cascades mediated partly via Ca2+-dependent EGFR transactivation.     

Involvement of p44/p42 MAP kinase in insulin-like growth factor-I-induced alkaline phosphatase activity in osteoblast-like-MC3T3-E1 cells

It has been shown that insulin-like growth factor-I (IGF-I) stimulates the activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblasts. In the present study, we investigated the involvement of the mitogen-activated protein (MAP) kinase superfamily in the IGF-I-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. IGF-I-stimulated alkaline phosphatase activity dose dependently in the range between 1 nM and 0.1 microM. IGF-I induced the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase but not stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). PD98059 and U0126, specific inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly suppressed the IGF-I-induced alkaline phosphatase activity. On the contrary, SB203580 and PD169316, specific inhibitors of p38 MAP kinase, failed to affect the activity induced by IGF-I. Specific inhibitors for phosphatidylinositol 3-kinase (PI3K)/Akt pathway (LY294002 and wortmannin) also had no significant effect on IGF-I-induced p44/p42 MAP kinase phosphorylation. The phosphorylation of p44/p42 MAP kinase induced by IGF-I was reduced by U0126. These results strongly suggest that p44/p42 MAP kinase among the MAP kinase superfamily plays a role in the IGF-I-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells.     

Activation of p38 mitogen-activated protein kinase mediates thyroid hormone-stimulated osteocalcin synthesis in osteoblasts

It is well known that thyroid hormone modulates osteoblast cell function. We have previously shown that triiodothyronine (T(3)) activates p44/p42 mitogen-activated protein (MAP) kinase, which limits T(3)-induced alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether p44/p42 MAP kinase or p38 MAP kinase is involved in the thyroid hormone-stimulated osteocalcin synthesis in these cells. T(3) markedly induced the phosphorylation of p38 MAP kinase in addition to p44/p42 MAP kinase. PD98059 and U0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the T(3)-induced synthesis of osteocalcin. On the contrary, the T(3)-induced osteocalcin synthesis was significantly reduced by SB203580 and PD169316, inhibitors of p38 MAP kinase. SB203580, PD169316 or PD98059 suppressed the T(3)-phosphorylation of myelin basic protein. T(3)-induced osteocalcin synthesis was significantly reduced by SB203580 or PD169316 also in primary cultured mouse osteoblasts. These results strongly suggest that p38 MAP kinase but not p44/p42 MAP kinase takes part in the thyroid hormone-stimulated osteocalcin synthesis in osteoblasts.     

Inhibitors of mitogen-activated protein kinases differentially regulate eosinophil-activating cytokine release from human airway smooth muscle

Airway smooth muscle (ASM) is a potential source of multiple proinflammatory cytokines during airway inflammation. In the present study, we examined a requirement for mitogen-activated protein (MAP) kinase activation for interleukin (IL)-1beta-stimulated GM-CSF, RANTES, and eotaxin release. IL-1beta induced concentration-dependent phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs), p38 MAP kinase, and c-Jun amino-terminal kinase (SAPK/JNK). p42/p44 ERK and p38 MAP kinase phosphorylation peaked at 15 min and remained elevated up to 4 h. SAPK/JNK phosphorylation also peaked at 15 min but fell to baseline within 60 min. SB 203580 selectively inhibited IL-1beta-stimulated activation of p38 MAP kinase; U 0126 was selective against p42/p44 ERK activity. SB 202474, an inactive analog, had no effect on p42/p44 ERK, p38 MAP kinase, or SAPK/JNK activation, or on eotaxin or RANTES release. Eotaxin release was inhibited by SB 203580 and U 0126, whereas RANTES release was prevented by U 0126 only. GM-CSF release was inhibited by U 0126 but enhanced by SB 203580. These data indicate that RANTES release is dependent on p42/p44 ERK activation but occurs independently of p38 MAP kinase activity. Eotaxin release, however, is dependent on both p38 MAP kinase- and p42/p44 ERK-dependent mechanisms. GM-CSF release is p42/p44 ERK dependent and is tonically suppressed by a mechanism that is partially dependent on p38 MAP kinase, though direct inhibition of cyclooxygenase (COX) activity due to poor inhibitor selectivity may also contribute.     

Cytochrome P4502E1 sensitizes to tumor necrosis factor alpha-induced liver injury through activation of mitogen-activated protein kinases in mice

The goal of this study was to evaluate the role of mitogen-activated protein kinase (MAPK) in cytochrome P4502E1 (CYP2E1) potentiation of lipopolysaccharide or tumor necrosis factor alpha (TNF-alpha)-induced liver injury. Treatment of C57/BL/6 mice with pyrazole (PY) plus lipopolysaccharide (LPS) induced liver injury compared with mice treated with PY or LPS alone. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 or p38 MAPK inhibitor SB203580 prevented this liver injury. PY plus LPS treatment activated p38 MAPK and JNK but not extracellular signal-regulated kinase (ERK). PY plus LPS treatment triggered oxidative stress in the liver with increases in lipid peroxidation, decrease of glutathione (GSH) levels, and increased production of 3-nitrotyrosine adducts and protein carbonyl formation. This oxidative stress was blocked by SP600125 or SB203580. PY plus LPS treatment elevated TNF-alpha production, and this was blocked by SP600125 or SB203580. Neither SP600125 nor SB203580 affected CYP2E1 activity or protein levels. Treating C57/BL/6 mice with PY plus TNF-alpha also induced liver injury and increased lipid peroxidation and decreased GSH levels. Prolonged activation of JNK and p38 MAPK was observed. All of these effects were blocked by SP600125 or SB203580. In contrast to wild-type SV 129 mice, treating CYP2E1 knockout mice with PY plus TNF-alpha did not induce liver injury, thus validating the role of CYP21E1 in this potentiated liver injury. Liver mitochondria from PY plus LPS or PY plus TNF-alpha treated mice underwent calcium-dependent, cyclosporine A-sensitive swelling, which was prevented by SB203580 or SP600125. CONCLUSION: These results show that CYP2E1 sensitizes liver hepatocytes to LPS or TNF-alpha and that the CYP2E1-enhanced LPS or TNF-alpha injury, oxidant stress, and mitochondrial injury is JNK or p38 MAPK dependent.     

软脂酸通过丝裂原活化蛋白激酶通路促进血管内皮细胞凋亡

目的探讨软脂酸(PA)诱导的血管内皮细胞凋亡中丝裂原活化蛋白激酶(MAPK)通路的作用。方法将人脐静脉内皮细胞(HUVEC)分对照组、PA组、MAPK通路干预组[分别先用p38抑制剂SB203580、氨基末端激酶(JNK)抑制剂PD98059、细胞外信号调节激酶(ERK)抑制剂SP600125干预]再分为PA+SB组、PA+PD组、PA+SP组。流式细胞仪检测细胞凋亡率;Western blot法检测caspase-3、磷酸化p38、JNK和ERK1/2表达水平;分光光度法检测caspase-3的活性。结果与对照组比较,PA组、PA+SB组、PA+PD组、PA+SP组HUVEC凋亡及caspase-3表达和活性明显增加,PA组磷酸化p38MAPK表达明显增加(P<0.05)。与PA组比较,PA+SB组HUVEC细胞凋亡率、caspase-3表达和活性明显降低(P<0.05);而PA+PD组和PA+SP组HUVEC凋亡率、caspase-3表达和活性无明显变化(P>0.05)。结论 PA通过p38MAPK通路促进内皮细胞凋亡。     

PTH regulation of c-Jun terminal kinase and p38 MAPK cascades in intestinal cells from young and aged rats

In the present study, we examined the role of Parathyroid hormone (PTH) on the c-Jun N-terminal kinase (JNK) 1/2 and p38 mitogen-activated protein kinase (MAPK) members of the MAPK family as it relates to ageing by measuring hormone-induced changes in their activity in enterocytes isolated from young (3 month old) and aged (24 month old) rats. Our results show that PTH induces a transient activation of JNK 1/2, peaking at 1 min (+threefold). The hormone also stimulates JNK 1/2 tyrosine phosphorylation, in a dose-dependent fashion, this effect being maximal at 10 nM. PTH-induced JNK 1/2 phosphorylation was suppressed by its selective inhibitor SP600125. Moreover, hormone-dependent activation of JNK 1/2 was dependent on calcium, since pretreatment of cells with BAPTA-AM or EGTA blocked PTH effects. With ageing, the response to PTH was significantly reduced. JNK basal protein expression was not different in the enterocytes from young and aged rats, however, basal protein phosphorylation increased with ageing. PTH did not stimulate, within 1–10 min, the basal activity and phosphorylation of p38 MAPK in rat intestinal cells. The hormone increased enterocyte DNA synthesis; the response was dose-dependent and decreased (-40%) with ageing. In agreement with the mitogenic role of the MAPK cascades, this effect was blocked by specific inhibitors of extracellular signal-regulated protein kinase (ERK) 1/2 and JNK 1/2. The results obtained in this work expand our knowledge on the mechanism of action of PTH in duodenal cells.     

Opposing effect of p38 MAP kinase and JNK inhibitors on the development of heart failure in the cardiomyopathic hamster

OBJECTIVE: p38 MAP kinase (p38 MAPK) and c-Jun NH2-terminal kinase (JNK) have been implicated in the pathophysiology of heart failure. We investigated the effects of chronic treatment with p38 MAPK and JNK inhibitors on the development of heart failure in dilated cardiomyopathy (DCM) hamster heart. METHODS AND RESULTS: BIO14.6 hamster hearts showed markedly increased p38 MAPK and JNK activities at 6 weeks of age when there was no significant increase in the area of fibrosis, heart weight/body weight, left ventricular (LV) chamber dilation and LV dysfunction. p38 MAPK and JNK activities were attenuated at 26 weeks of age and abolished at 40 weeks of age in BIO14.6 hamster hearts. BIO14.6 hamsters and the control BIOF1B hamsters were chronically treated (i.p.) with the p38 MAPK inhibitors, SB203580 (1 mg/kg/day) and FR167653 (3 mg/kg/day), or the JNK inhibitor, SP600125 (1 mg/kg/day) or vehicle for 20 weeks starting from 6 weeks of age. Treatment of BIO14.6 hamster hearts with SB203580 and FR167653 reduced the number of TUNEL-positive myocytes, the area of fibrosis and heart weight/body weight associated with a significant decrease of LV dimension and an increase in LV ejection fraction and LV contractility compared to the vehicle-treated counterpart. In contrast, treatment with SP600125 increased the number of TUNEL-positive myocytes and the area of interstitial fibrosis associated with aggravation of LV chamber dilation and LV dysfunction. CONCLUSIONS: These results suggest that chronic treatment with p38 MAPK and JNK inhibitors produces opposing effects on the development of heart failure in the DCM hamster heart.     

P70 S6 kinase negatively regulates fibroblast growth factor 2-stimulated interleukin-6 synthesis in osteoblasts: function at a point downstream from protein kinase C

We have previously reported that protein kinase C negatively regulates basic fibroblast growth factor (FGF-2)-stimulated synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells. To further clarify the mechanism underlying the synthesis of IL-6 in osteoblasts, we investigated whether p70 S6 kinase is involved in the FGF-2-stimulated IL-6 synthesis in these cells. Rapamycin, an inhibitor of p70 S6 kinase, significantly enhanced the FGF-2-stimulated IL-6 synthesis in a dose-dependent manner. Downregulation of p70 S6 kinase by siRNA markedly amplified the FGF-2-stimulated IL-6 synthesis. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a direct activator of protein kinase C, induced the phosphorylation of p70 S6 kinase. Go6976 and bisindolylmaleimide I, inhibitors of protein kinase C, suppressed the TPA-stimulated phosphorylation of p70 S6 kinase. Additionally, protein kinase C inhibitors markedly reduced the phosphorylation of p70 S6 kinase induced by FGF-2. These results strongly suggest that p70 S6 kinase functions at a point downstream of protein kinase C and limits the FGF-2-stimulated IL-6 synthesis in osteoblasts.