Spleen tyrosine kinase participates in src-mediated migration and proliferation by PDGF-BB in rat aortic smooth muscle cells

Tyrosine kinases, Src and spleen tyrosine kinase (Syk), play crucial roles in cell responses to platelet-derived growth factor (PDGF) and may have their functional interactions. In this study, we focused on investigating the roles of Syk in the regulation of Src signaling in PDGF-mediated vascular cell responses. Migration, proliferation, and activity of kinases were determined in rat aortic smooth muscle cells (RASMCs). PDGF-BB (10 ng/mL) induced the migration and proliferation of RASMCs, which were significantly inhibited by PP2 (10 microM) and piceatannol (30 microM), inhibitors of Src and Syk, respectively. The phosphorylation of Syk induced by PDGF-BB was abolished by PP2. PDGF-BB increased the co-association of the PDGFbeta-receptor and the kinases, Src or Syk, and its maximal binding to Src was achieved in a shorter time than that to Syk. PDGF-BB stimulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2, which was inhibited by PP2 and piceatannol. PDGF-BB-induced proliferation and migration were inhibited by SB203580 (30 microM) and PD98059 (30 microM), inhibitors of p38 MAPK and ERK1/2, respectively. These results imply that Syk is regulated by Src kinase, which participates in migration and proliferation in response to PDGF-BB in RASMCs.     

Endothelin-1 induces contraction via a Syk-mediated p38 mitogen-activated protein kinase pathway in rat aortic smooth muscle

Although spleen tyrosine kinase (Syk) has crucial roles in various cells, its function on vascular smooth muscle contraction has not been determined. In the present study, we performed experiments to determine if Syk contributes to the endothelin-1 (ET-1)-mediated contraction in rat aortic smooth muscle. ET-1-induced contraction of aortic strips was inhibited by piceatannol, PD98059, and SB203580, inhibitors of Syk, extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK), respectively. Piceatannol also attenuated high K(+)-induced contraction. ET-1 dose-dependently enhanced the activity of Syk and this was inhibited by piceatannol in both rat aortic strip and rat aortic smooth muscle cells. The phosphorylation of p38 MAPK and heat shock protein 27 (HSP27), but not that of ERK1/2, in response to ET-1 was inhibited by both piceatannol and SB203580. These results suggest that Syk may play an important role in the regulation of aortic smooth muscle contraction induced by ET-1, which may be mediated by the p38 MAPK/HSP27 signaling pathway.     

Src and Cas are essentially but differentially involved in angiotensin II-stimulated migration of vascular smooth muscle cells via extracellular signal-regulated kinase 1/2 and c-Jun NH2-terminal kinase activation

Angiotensin II (Ang II) plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth and migration. Src activity is known to be required for the migration of a number of cell types. p130Cas was reported to be essential for cell migration and actin filament reorganization. Mitogen-activated protein (MAP) kinases were also reported to be critical regulatory factors for growth and migration of VSMC. However, precise intracellular mechanisms involving c-Src, p130Cas, and MAP kinases in Ang II-stimulated migration of VSMC have not been well elucidated. Here we demonstrated that Ang II rapidly and significantly stimulated tyrosine phosphorylation of Src and Cas and their association in rat aortic smooth muscle cells (RASMC). Ang II-stimulated tyrosine phosphorylation of Src and Cas and activation of ERK1/2 and JNK, but not p38, were potently inhibited by Src family tyrosine kinase inhibitors, herbimycin A (HA) and PP2. Ang II-stimulated Src and Cas association, tyrosine phosphorylation of Cas, and activation of ERK1/2 and JNK were suppressed in kinase-inactive Src (KI Src)-overexpressed RASMC. Ang II-stimulated JNK activation but not ERK1/2 activation was blocked in substrate domain-deleted Cas (DeltaSD Cas)-overexpressed RASMC. In addition, HA, PP2, ERK1/2 inhibitor, 2'-amino-3'-methoxyflavone (PD98059) and JNK inhibitor, and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) significantly inhibited Ang II-stimulated migration of RASMC. Ang II-induced colocalization of Src and Cas and migration were inhibited in both KI Src- and DeltaSD Cas-overexpressed RASMC. These findings suggest that Src and Cas are essentially but differentially involved in Ang II-stimulated migration of VSMC through the activation of ERK1/2 and JNK.     

Olibanum Extract Inhibits Vascular Smooth Muscle Cell Migration and Proliferation in Response to Platelet-Derived Growth Factor

Olibanum (Boswellia serrata) has been shown to have anti-inflammatory, anti-arthritic and anti-cancer effects. This study determined the role of a water extract of olibanum in platelet-derived growth factor (PDGF)-stimulated proliferation and migration of rat aortic smooth muscle cells (RASMCs). PDGF-BB induced the migration and proliferation of RASMCs that were inhibited by olibanum extract in a dose-dependent manner. The PDGF-BB-increased phosphorylation of p38 mitogen-activated protein kinase (MAPK); the heat shock protein (Hsp) 27 was significantly inhibited by the olibanum extract. The effects of PDGF-BB-induced extracellular signal-regulated kinase1/2 was not altered by the olibanum extract. Treatment with olibanum extract inhibited PDGF-BB-stimulated sprout out growth of aortic rings. These results suggest that the water extract of olibanum inhibits PDGF-BB-stimulated migration and proliferation in RASMCs as well as sprout out growth, which may be mediated by the inhibition of the p38 MAPK and Hsp27 pathways.     

Fluorofenidone inhibits transforming growth factor-beta1-induced cardiac myofibroblast differentiation

Cardiac myofibroblast differentiation, characterized by expression of alpha-smooth muscle actin (alpha-SMA) and fibrillar collagens, plays a key role in the adverse myocardial remodeling. Fluorofenidone (1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD) is a novel pyridone antifibrotic agent, which exerts a strong antifibrotic effect. This study investigated the potential role of AKF-PD in suppressing cardiac myofibroblast conversion induced by transforming growth factor-beta1 (TGF-beta1) and the related mitogen-activated protein kinase (MAPK) signaling pathways in neonatal rat cardiac fibroblasts. The MAPK inhibitors used for pathway determination are c-Jun NH(2)-terminal kinase (JNK) inhibitor II (JNK inhibitor), PD98059 (extracellular signal-regulated kinase inhibitor (ERK) inhibitor) and SB203580 (p38 MAPK inhibitor). Cell proliferation was evaluated by multiply-table tournament (MTT) assay. The expressions of fibronectin (FN), alpha-SMA, phosphorylated ERK1/2 (pERK1/2) and ERK1/2 were investigated using Western blot analysis. AKF-PD remarkablely reduced the proliferative response of cardiac fibroblasts by 27.57% compared with TGF-beta1 stimulated group. AKF-PD, PD98059, and JNK inhibitor II completely prevented TGF-beta1-induced FN protein production. In addition, AKF-PD, PD98059 and SB203580 greatly attenuated alpha-SMA expression induced by TGF-beta1. Furthermore, AKF-PD significantly blocked TGF-beta1-induced phosphorylation of ERK. These results indicate that (1) AKF-PD inhibits TGF-beta1-induced myofibroblast differentiation; (2) the anti-fibrotic effects of AKF-PD are partially mediated by ERK phosphorylation.     

Contribution of extracellular signal-regulated kinases to the IL-1-induced growth inhibition of human melanoma cells A375

The role of ERK1/2 in the IL-1-induced growth inhibition was investigated using human melanoma A375-6 cells. A selective inhibitor of ERK1/2 pathway, PD98059 and a selective inhibitor of p38MAPK, SB203580 each alone significantly reversed the IL-1-induced growth inhibition of A375-6 cells. Co-treatment with PD98059 and SB203580 completely reversed the IL-1-induced growth inhibition. ERK1/2 was constitutively activated in A375-6 cells, and IL-1 further augmented ERK activation. Antiproliferative effect of IL-1 was attenuated by the expression of dominant negative form of ERK2. IL-1 induced cell cycle arrest in G(0)/G(1) phase, expression of p21 and p27 proteins, and down-regulation of cyclin D/cyclin-dependent kinase (CDK) 2 and CDK4 activities. These effects of IL-1 were reversed by PD98059. PD98059 also reversed the IL-1-induced hypophosphorylation of RB protein (pRB) and down-regulation of E2F activity. These findings demonstrate that ERK1/2 contribute to the IL-1-induced growth inhibition through induction of CDK inhibitors, down-regulation of CDK activity, pRB phosphorylation and E2F activity.     

Phorbol 12-myristate 13-acetate (PMA)-induced migration of glioblastoma cells is mediated via p38MAPK/Hsp27 pathway

We investigated the mechanism of phorbol 12-myristate 13-acetate (PMA)-induced migration of glioblastoma cells focusing on the p38 mitogen-activated protein kinase (MAPK)/heat shock protein 27 (Hsp27) pathway. PMA-induced cell migration and activation of p38MAPK in A172 glioblastoma cells. PMA-induced formation of lamellipodia and focal complexes was blocked by inhibiting p38MAPK with SB203580 or small interfering RNA (siRNA). Furthermore, activation of p38MAPK resulted in phosphorylation of an F-actin polymerization regulator, Hsp27. Immunohistochemical analysis showed that upon PMA stimulation, both unphosphorylated and phosphorylated Hsp27 were translocated to the lamellipodia. SB203580 or p38MAPK siRNA blocked these phenomena, indicating that Hsp27 phosphorylation and translocation from cytosol to membrane were mediated by p38MAPK. To address the question of whether endogenous Hsp27 participates in PMA-induced migration, we inhibited the expression of Hsp27 using Hsp27 siRNA. Although knockdown of Hsp27 by siRNA had little effect on p38MAPK activation, lamellipodia and focal complex formation was markedly inhibited. Migration was also abolished in Hsp27 siRNA-transfected cells. In conclusion, p38MAPK activation followed by Hsp27 phosphorylation was required for PMA-induced migration. Furthermore, Hsp27 itself played critical roles in PMA-induced migration. Our data provide substantial evidence for a model elucidating the molecular mechanisms of regulation of actin dynamics and migration by PMA-activated protein kinase C in glioblastoma cells.     

SPK1调控人脐静脉内皮细胞SIRT1表达及细胞迁移的机制研究

目的 探讨鞘氨醇激酶1(sphingosine kinase1,SPK1)调控沉默信息调节因子1(silent mating type infor-mation regulation 2 homolog 1,SIRT1)表达及对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)迁移能力的机制.方法 用病毒感染复数(MOI)=20的pLKO.1-shSPK1-GFP干涉慢病毒(shSPK1组)和pPIG-U6-Scramble-GFP对照慢病毒(GFP-SC组)感染HUVECs,48 h后提取细胞蛋白并用蛋白免疫印迹法检测ERK、P38 MAPK及AKT磷酸化水平.分别用PD98059、SB203580及Wortmannin处理HUVECs 1 h,检测ERK、P38 MAPK及AKT信号表达及SIRT1蛋白表达情况.取抑制剂干预后的细胞进行Transwell小室迁移实验,检测细胞迁移能力.结果 shSPK1组ERK、P38 MAPK及AKT的磷酸化水平均低于GFP-SC组(P ＜0.05,P＜0.01).PD98059、SB203580及Wortmannin组ERK、P38 MAPK、AKT磷酸化水平及SIRT1蛋白表达水平低于对照组,HUVECs的迁移能力较对照组弱(P＜0.01).结论 SPK1可以通过ERK、P38 MAPK及AKT通路调控HUVECs SIRT1的表达及迁移能力.     

Thrombin induces inducible nitric oxide synthase expression via the MAPK, MSK1, and NF-κB signaling pathways in alveolar macrophages

In this study, we investigated the roles of mitogen activated protein kinase (MAPK), mitogen stress-activated protein kinase 1 (MSK1), and nuclear factor-κB (NF-κB) signaling pathways in thrombin-induced inducible nitric oxide synthase (iNOS) expression in alveolar macrophages (NR8383). Treatment of NR8383 cells with thrombin caused an increase in iNOS expression in a concentration- and time-dependent manner. Treatment of NR8383 cells with SB203580 (4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1H-imidazole, a p38 MAPK inhibitor), PD98059 (2'-amino-3'-methoxyflavone, a MAPK kinase (MEK) inhibitor), and SP600125 (anthra[1-9-cd]pyrazol-6(2H)-one, a JNK inhibitor) all inhibited thrombin-induced iNOS expression. Stimulation of cells with thrombin caused an increase in p38 MAPK, ERK, and JNK phosphorylation. Treatment of cells with Ro 31-8220 (an MSK1 inhibitor) and MSK1 small interfering RNA (MSK1 siRNA) both inhibited thrombin-induced iNOS expression. Thrombin caused time-dependent activation of MSK1 Ser531 phosphorylation, which was inhibited by SB203580 and PD98059, but not by SP600125. Treatment of cells with pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) inhibited thrombin-induced iNOS expression in a concentration-dependent manner. Treatment of NR8383 cells with thrombin induced κB-luciferase activity and p65 Ser276 phosphorylation. Thrombin-induced increases in p65 Ser276 phosphorylation and κB-luciferase activity were inhibited by SB203580, PD98059, Ro 31-8220, and MSK1 siRNA. Taken together, these results suggest that the signaling pathways of MAPK, MSK1, and NF-κB play important roles in thrombin-induced iNOS expression in alveolar macrophages.     

Intracellular regulation of evodiamine-induced A375-S2 cell death

We have reported that in A375-S2 cells, evodiamine isolated from Evodia rutaecarpa induces cell death of human melanoma, A375-S2, through two distinct pathways: apoptosis and necrosis. In the present study, we further demonstrate two different mechanisms by which evodiamine induces apoptosis and necrosis. Although caspase-1 and -10 inhibitors failed to block cell death, pan-caspase inhibitor and caspase-3, -8, and -9 inhibitors had marked inhibitory effects on apoptosis induced by 15 microM evodiamine. Furthermore, evodiamine-induced activation of caspase-3 resulted in the down-regulation of anti-apoptotic Bcl-2 expression and up-regulation of proapoptotic Bax expression. After 24 h incubation with evodiamine, no caspase inhibitor had any influence on cell death, but p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) attenuated cell death; in contrast, extracellular signal-regulated protein kinase (ERK) MAPK inhibitor (PD98059) augmented cell death, as was further confirmed by cotreatment with SB203580 or PD98059 and pan-caspase inhibitor. Moreover, evodiamine increased the phosphorylation of p38 and decreased the expression and phosphorylation of ERK in caspase-independent necrosis. Consequently, evodiamine induced the caspase- and Bax-mediated apoptosis at an early stage, but, initiated MAPKs-dependent necrosis at a later stage.     

Attenuated expression and function of the RECK tumor suppressor under hypoxic conditions is mediated by the MAPK signaling pathways

Downregulation of the tumor suppressor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) has been reported under hypoxic conditions (Lee et al., 2010); however, the signaling pathways involved in this downregulation have not yet been identified. Hypoxia causes the silencing of RECK mRNA expression, but treatment with inhibitors of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated kinase (MAPK) (PD98059, SP600125, and SB203580 respectively) or their dominant negative mutants recovered RECK suppression induced by hypoxia as analyzed with semiquantitative RT-PCR analysis and a RECK promoter luciferase assay. Hypoxia increased phosphorylation of ERK1/2, JNK and p38 MAPKs. The activities of matrix metalloproteinase (MMP)-9 and MMP-2 were increased under hypoxic conditions but treatment with PD98059, SP600125 and SB203580 inhibited their activation in cancer cells, as seen by zymography. Moreover, treatment with the inhibitors blocked cancer cell migration induced by hypoxia in H-Ras transformed MCF10A mammary cells. RECK suppression under hypoxic conditions was inversely related to HIF-1α expression; however, treatment with PD98059, SP600125 and SB203580 did not influence binding of HIF-1α to the reverse hypoxia responsive element site of the RECK promoter in a DNA precipitation assay. These results suggest that the ERK, JNK and p38 MAPK signaling pathways are indirectly involved in RECK suppression but are not involved in the binding activity of HIF-1α to the reverse hypoxia responsive element site on the RECK promoter under hypoxic conditions.     

Src tyrosine kinases mediate crystalline silica-induced NF-kappaB activation through tyrosine phosphorylation of IkappaB-alpha and p65 NF-kappaB in RAW 264.7 macrophages.

Protein tyrosine kinases (PTKs) and mitogen-activated protein kinases (MAPKs) have been demonstrated to play a crucial role in the signaling pathways induced by silica. In the present study, we investigated whether Src family TKs play a role in crystalline silica-induced NF-kappaB activation and whether NF-kappaB activation requires Src TK-dependent MAPK activity in RAW 264.7 cells, a mouse peritoneal macrophage cell line. Selective Src TK inhibitors, damnacanthal or PP1, inhibited silica-induced NF-kappaB activation in a dose-dependent manner. Furthermore, these kinase inhibitors suppressed silica-induced tyrosine phosphorylation of IkappaB-alpha and p65 NF-kappaB. Within a similar time frame, c-Src and Lck were physically associated with IkappaB-alpha and with p65 NF-kappaB. Silica stimulated the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), but not p38 MAPK and c-Jun NH(2)-terminal kinase 1 and 2 (JNK1/2). Damnacanthal or PP1 substantially blocked the silica-induced activation of ERK1/2. Moreover, PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 MAPK, failed to inhibit silica-induced NF-kappaB activation. These results suggest that c-Src and Lck act for silica-induced NF-kappaB activation by mediating the tyrosine phosphorylations of IkappaB-alpha and p65 NF-kappaB. However, the Src TK-dependent activation of ERK1/2 may not be involved in the silica signaling pathway leading to NF-kappaB activation.     

5-Hydroxytryptamine induces cyclooxygenase-2 in rat vascular smooth muscle cells: Mechanisms involving Src, PKC and MAPK activation [corrected

Considering the importance of 5-hydroxytryptamine (5-HT) and cyclooxygenase (COX) products in vascular pathology, we investigated the effects of 5-HT on COX expression in rat vascular smooth muscle cells (VSMCs), and to provide mechanistic insights into these effects. VSMCs were enzymatically isolated from aortic media of Wistar rats. Incubation of VSMCs with 5-HT for 24h stimulated prostaglandin I(2) production, but this stimulation was completely suppressed by NS-398, a selective COX-2 inhibitor. 5-HT induced transient COX-2, but not COX-1, protein and mRNA expression in concentration- and time-dependent manners. This effect of 5-HT was completely inhibited by sarpogrelate, a 5-HT(2A) receptor antagonist. 5-HT-induced COX-2 expression was markedly blunted by Ca(2+) depletion; GF 109203X, a protein kinase C (PKC) inhibitor; PP2, an inhibitor of Src-family tyrosine kinase (Src); PD 98059, an inhibitor of extracellular signal-regulated kinase (ERK) activation; SB 203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK); and SP 600125, an inhibitor of c-Jun N-terminal kinase (JNK). 5-HT activated ERK and p38 MAPK, followed by JNK activation. PP2 inhibited these activations, while GF 109203X inhibited only JNK activation. Furthermore, PD 98059 inhibited JNK activation. These results suggest that 5-HT induces COX-2 expression in rat VSMCs, and that PKC, Src, and MAPK activation are each essential for the full expression of COX-2 pathways.     

Cordycepin Attenuates Neointimal Formation by Inhibiting Reactive Oxygen Species–Mediated Responses in Vascular Smooth Muscle Cells in Rats

We determined the action mechanism of cordycepin, a major bioactive component of Cordyceps militaris, on responses of rat aortic smooth muscle cells (RASMCs) and on vascular disorders, especially neointimal formation. Cordycepin inhibited platelet-derived growth factor-BB (PDGF-BB)-induced RASMCs migration and proliferation in a dose-dependent manner. However, pre-treatment with N^ω-nitro-L-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor, and 1,3-dipropyl-8-sulphophenylxanthine (DPSPX), an A₁/A₂ adenosine– receptor antagonist, abolished the inhibitory role of cordycepin. Cordycepin suppressed the phosphorylation of p38 mitogen–activated protein kinase (p38 MAPK) and heat shock protein 27 (Hsp27), but not that of extracellular signal-regulated kinase (ERK) 1/2 in RASMCs stimulated by PDGF-BB. The production of reactive oxygen species (ROS), O₂^? and H₂O₂, induced by PDGF-BB was abolished by the treatment of cordycepin. Moreover, the sprout outgrowth of aortic rings by PDGF-BB was inhibited by cordycepin. In vivo neointimal formation evoked by balloon-injury was significantly attenuated by the administration of cordycepin. These results demonstrate that cordycepin may exert inhibitory effects on PDGF-BB–induced migration and proliferation via interfering with adenosine receptor–mediated NOS pathways, thus resulting in the attenuation of neointima formation. In conclusion, cordycepin may be a potent, promising anti-atherosclerosis agent.     

Cryptotanshinone inhibits chemotactic migration in macrophages through negative regulation of the PI3K signaling pathway

BACKGROUND AND PURPOSE: Cryptotanshinone, the major tanshinone isolated from Salvia miltiorrhiza Bunge, exhibits anti-inflammatory activity. However, there is no report on the effect of cryptotanshinone on recruitment of leukocytes to inflammatory sites. We therefore assessed the effects of cryptotanshinone on macrophage chemotaxis. EXPERIMENTAL APPROACH: Macrophage migration induced by complement 5a (C5a) or macrophage inflammatory protein-1alpha (MIP-1alpha) was measured in vitro. Intracellular kinase translocation and phosphorylation was assessed by Western blotting. KEY RESULTS: RAW264.7 cell migration towards C5a (1 microg ml(-1)) was significantly inhibited by cryptotanshinone (1, 3, 10 and 30 microM) in a concentration-dependent manner. Primary human macrophages stimulated by C5a were similarly inhibited. C5a-evoked migration in RAW264.7 cells was significantly suppressed by wortmannin (phosphatidylinositol 3-kinase (PI3K) inhibitor), PD98059 (MEK1/2 inhibitor) and SB203580 (p38 mitogen-activated protein kinase (MAPK) inhibitor), but not by SP600125 (c-Jun N-terminal kinase (JNK) inhibitor), suggesting that activation of PI3K, ERK1/2 and p38 MAPK signal pathways was involved in responses to C5a. Western blotting revealed that cryptotanshinone significantly inhibited PI3K-p110gamma membrane translocation and phosphorylation of Akt (PI3K downstream effector protein) and ERK1/2 induced by C5a. However, neither p38 MAPK nor JNK phosphorylation was affected by cryptotanshinone. Wortmannin significantly attenuated C5a-induced PI3K-p110gamma translocation, Akt and ERK1/2 phosphorylation. PD98059 suppressed ERK1/2 phosphorylation but failed to modify PI3K-p110gamma translocation by C5a stimulation. Furthermore, MIP-1alpha-induced cell migration and PI3K-p110gamma translocation were also inhibited by cryptotanshinone in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: Inhibition of macrophage migration by cryptotanshinone involved inhibition of PI3K activation with consequent reduction of phosphorylation of Akt and ERK1/2.     

Involvement of reactive oxygen species and stress-activated MAPKs in satratoxin H-induced apoptosis

Satratoxins, members of the trichothecene mycotoxin family, have been known to be harmful to health. However, the mechanisms underlying the toxicity still remain unclear. The present study is undertaken to elucidate the mechanisms of the satratoxin H-induced cytotoxicity in PC12 cells. Satratoxin H caused cytotoxicity, which was reflected from apoptosis determined by chromatin staining and flow cytometry. Satratoxin H stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Pre-incubation with SB203580, a p38 MAPK inhibitor, or SP600125, a JNK inhibitor, but not PD98059, an ERK inhibitor, reduced satratoxin-induced cytotoxicity. Co-incubation of cells with glutathione, N-acetyl-L-cysteine or glutathione reductase inhibited cytotoxicity and the phosphorylation of p38 MAPK induced by satratoxin H. Our data suggest that satratoxin H-induced apoptosis in PC12 cells is dependent on the activation of p38 MAPK/JNK and the increase in reactive oxygen species.     

Activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) induces cell death through MAPK-dependent mechanism in osteoblastic cells

The present study was undertaken to determine the role of the mitogen-activated protein kinase (MAPK) subfamilies in cell death induced by PPARgamma agonists in osteoblastic cells. Ciglitazone and troglitazone, PPARgamma agonists, resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. But a PPARalpha agonist ciprofibrate did not affect the cell death. Ciglitazone caused reactive oxygen species (ROS) generation and ciglitazone-induced cell death was prevented by antioxidants, suggesting an important role of ROS generation in the ciglitazone-induced cell death. ROS generation and cell death induced by ciglitazone were inhibited by the PPARgamma antagonist GW9662. Ciglitazone treatment caused activation of extracellular signal-regulated kinase (ERK) and p38. Activation of ERK was dependent on epidermal growth factor receptor (EGFR) and that of p38 was independent. Ciglitazone-induced cell death was significantly prevented by PD98059, an inhibitor of ERK upstream kinase MEK1/2, and SB203580, a p38 inhibitor. Ciglitazone treatment increased Bax expression and caused a loss of mitochondrial membrane potential, and its effect was prevented by N-acetylcysteine, PD98059, and SB203580. Ciglitazone induced caspase activation, which was prevented by PD98059 and SB203580. The general caspase inhibitor z-DEVD-FMK and the specific inhibitor of caspases-3 DEVD-CHO exerted the protective effect against the ciglitazone-induced cell death. The EGFR inhibitors AG1478 and suramin protected against the ciglitazone-induced cell death. Taken together, these findings suggest that the MAPK signaling pathways play an active role in mediating the ciglitazone-induced cell death of osteoblasts and function upstream of a mitochondria-dependent mechanism. These data may provide a novel insight into potential therapeutic strategies for treatment of osteoporosis.