Ethanol-induced oxidative stress is mediated by p38 MAPK pathway in mouse hippocampal cells

It has been known that ethanol causes neuronal cell death through oxidative stress. Ethanol itself and reactive oxygen species (ROS) produced by ethanol modulate intracellular signaling pathways including mitogen-activated protein kinase (MAPK) cascades. This study was conducted to examine the impact of ethanol on MAPK signaling in HT22 cells. Ethanol (100 and 400 mM) caused activation of ERK, p38 MAPK, and JNK. ERK activation occurred in early time and p38 MAPK activation was evident when ERK activation was diminished. Specific inhibitor of p38 MAPK (SB203580) protected HT22 cells against ethanol, which was accompanied by an inhibition of ROS accumulation. However, inhibitors of ERK (U0126) and JNK (SP600125) had no effects on ethanol-induced neuronal cell death when they are treated with ethanol for 24 h. These results suggest that p38 MAPK may have important roles in ROS accumulation during ethanol-induced oxidative stress in HT22 cells.     

Aldosterone upregulates connective tissue growth factor gene expression via p38 MAPK pathway and mineralocorticoid receptor in ventricular myocytes

The effect of aldosterone on connective tissue growth factor (CTGF) was examined in rat embryonic ventricular myocytes. Upon aldosterone treatment, CTGF expression was significantly increased in a dose and time-dependent manner. To explore the molecular mechanism for this upregulation, we examined the role of mineralocorticoid receptor. Pre-treatment of an antagonist (spironolactone) at 5-fold excess of aldosterone blocked the CTGF induction by aldosterone, suggesting that the upregulation was mediated by mineralocorticoid receptor. Aldosterone treatment resulted in activation of ERK1/2, p38 MAPK, and JNK pathways with a more transient pattern in p38 MAPK. Blocking studies using pretreatment of the inhibitor of each pathway revealed that p38 MAPK cascade may be important for aldosterone-mediated CTGF upregulation as evidenced by the blocking of CTGF induction by SB203580 (p38 MAPK inhibitor), but not by PD098059 (ERK1/2 inhibitor) and JNK inhibitor I. Interestingly, JNK inhibitor I and PD098059 decreased the basal level of CTGF expression. On the other hand, pretreatment of spironolactone abrogated the p38 MAPK activation, indicating that mineralocorticoid receptor mechanism is linked to p38 MAPK pathway. Taken together, our findings suggest that aldosterone induces CTGF expression via both p38 MAPK cascade and mineralocorticoid receptor and that cross-talk exists between the two pathways.     

p38 MAPK- Pim-3通路可能介导预处理对抗心肌细胞缺氧/复氧损伤

目的： 研究MAPK通路在原癌基因Pim-3抗心肌急性缺氧复氧损伤中的作用。方法：采用原代培养新生大鼠的心肌细胞,随机分为4组：正常对照组（control）、缺氧复氧组(A/R)、缺氧预适应组(APC+A/R)、阻断剂组。在缺氧预处理前分别用终浓度为10 μmol/L SB203850(p38 MAPK阻断剂)、U0126（ERK1/2阻断剂）、SP600125（SAPK/JNK阻断剂）与细胞孵育30 min。实验结束后测定MAPKs通路中ERK1/2、JNK、p38 MAPK 磷酸化蛋白表达水平及Pim-3蛋白的表达水平,同时检测培养液中乳酸脱氢酶(LDH) 活性、四唑盐（MTT）比色试验测定细胞存活率、TUNEL法检测细胞凋亡。结果： SB203850、U0126、SP600125能分别取消由APC或A/R所诱导ERK1/2、JNK、p38 MAPK的磷酸化水平的升高;由APC所诱导的Pim-3表达的升高在p38 MAPK通路被阻断后明显下调(P<0.01),并且心肌细胞LDH值升高,细胞存活率则下降,心肌细胞的凋亡指数升高。结论： p38 MAPK的激活可上调原癌基因Pim-3的表达,从而可能对心肌细胞起到保护作用。     

Cinobufagin exerts anti-proliferative and pro-apoptotic effects through the modulation ROS-mediated MAPKs signaling pathway

Abstract

Cinobufagin (CBG) is a cardiotoxic bufanolide steroid secreted by the skin and parotid venom glands of the Asiatic toad Bufo bufo gargarizans (called Chan-Su). Although CBG is known to exhibit anti-cancer activities, very little is known about its potential mechanism(s) of action. In this study, we investigated whether CBG mediates its effect through the modulation of the mitogen-activated protein kinases (MAPKs) signaling pathway in human multiple myeloma (MM) U266 cells. We found that CBG caused the significant activation of ERK, JNK and p38 MAPK in U266 cells. CBG showed much higher cytotoxicity against U266 cells as compared to peripheral blood mononuclear cells (PBMC). Induction of CBG increased reactive oxygen species (ROS) generation from mitochondria, which is associated with the induction of apoptosis as characterized by increased sub-G1 DNA contents of cell cycle, positive Annexin V binding, activation of caspase-3 and cleavage of PARP. Inhibition of ROS generation by N-acetyl-l-cysteine (NAC) significantly prevented CBG-induced ERK, JNK and p38 MAPK activation and apoptosis. CBG also down-regulated the expression of various downstream gene products that mediate cell proliferation, survival, angiogenesis and metastasis. Interestingly, ERK, JNK and p38MAPK pharmacological inhibitors blocked CBG-induced MAPKs activation and ERK inhibitor (PD98059) also prevented the CBG-induced caspase-3 activation and PARP cleavage in U266 cells. Taken together, these findings suggest that CBG can act as a potent anticancer agent against MM and possibly exerts its effects through the ROS-mediated activation of ERK, JNK and p38 MAPK leading to the activation of caspase-3 in U266 cells.     

c-Jun N-terminal kinase and p38 mitogen-activated protein kinase mediate double-strand RNA-induced inducible nitric oxide synthase expression in microglial cells

Double-stranded RNA (dsRNA) has been implicated as a potential immune stimulant in activating microglia, which can cause chronic neurodegeneration. In this study, we examined the involvement of different types of mitogen-activated protein kinases (MAPKs) in the induction of inducible nitric oxide synthase (iNOS) by dsRNA in microglial cells. Nitric oxide production was increased after exposure of microglia to 50 μg/mL dsRNA. Levels of dsRNA-induced nitrite production in a line of immortalized murine microglia (BV2) and in primary cultures of murine microglia were decreased by inhibition of JNK or p38 MAPK, but were increased by inhibition of extracellular signal-regulated kinase. Similar results were shown in the levels of dsRNA-induced iNOS gene expression in BV2 cells. Phosphorylation levels of p38 MAPK were increased, depending on p38 MAPK inhibitor concentrations, while activation levels of MAPKAPK2, a known p38 substrate, were inhibited. Thus, it is likely that SB203580 inhibited the kinase activity of p38 MAPK, resulting in the loss of a feedback inhibition regulatory loop of p38 MAPK in BV2 cells. These findings suggest that dsRNA stimulated iNOS expression via MAPK signaling pathways, including JNK and p38 MAPK.     

活性氧与丝裂原激活蛋白激酶通路的相互作用介导高糖引起的心肌细胞损伤

目的探讨活性氧(ROS)与丝裂原激活蛋白激酶(MAPK)通路的相互作用在高糖损伤H9c2心肌细胞中的作用。方法应用细胞计数盒(CCK-8)检测细胞存活率;Hoechst 33258核染色检测凋亡细胞形态及数量的改变;双氯荧光素(DCFH-DA)染色荧光显微镜照相检测细胞内ROS水平;Western blot测定蛋白质表达水平。结果高糖(35 mmol/L葡萄糖)处理H9c2心肌细胞24 h可引起明显的损伤,表现为细胞存活率下降,凋亡细胞数量及ROS水平明显升高。另方面,高糖可明显地上调磷酸化(p)p38MAPK、细胞外信号调节蛋白激酶1/2(ERK1/2)及c-Jun N端激酶(JNK)(为MAPK家族的3个成员)的表达水平。N-乙酰半胱氨酸(NAC,为ROS清除剂)能抑制高糖引起的心肌细胞毒性和细胞凋亡,也能阻断高糖对p-p38MAPK、p-ERK1/2及p-JNK表达的上调作用。此外,p38MAPK、ERK1/2和JNK的选择性抑制剂均能抑制高糖引起的心肌损伤,并能抑制ROS生成增多。结论在高糖损伤H9c2心肌细胞中,存在ROS与MAPK通路的正相互作用,这种相互作用可能在高糖引起的心肌细胞损伤中起着重要的作用。     

Different involvement of the MAPK family in inflammatory regulation in human pulmonary microvascular endothelial cells stimulated with LPS and IFN-γ

Pulmonary endothelial injury is central in the pathogenesis of acute lung injury (ALI). The MAPK signaling cascades are generally thought to be involved in the molecular mechanism underlying the ALI development, but their roles in pulmonary endothelial injury is poorly understood. We thus examined the involvement of the MAPK family member in inflammatory responses of human pulmonary microvascular endothelial cells (HPMVECs) stimulated with LPS and IFN-γ. HPMVECs were found to exhibit the upregulation of expression of Toll-like receptor 4 by IFN-γ, resulting in potentiation of inflammatory cytokine release by LPS stimulation. All MAPKs, ERK1/2, JNK, and p38, were activated by simultaneous stimulation with LPS/IFN-γ. JNK activation in cells stimulated with LPS/IFN-γ was significantly potentiated by the two different p38 inhibitors, SB203580 and RWJ67657, suggesting the negative regulation of JNK activation by p38 in HPMVECs. The mRNA and protein expression levels of ICAM-1 were eliminated by the JNK inhibitor, suggesting that ICAM-1 expression is positively regulated by JNK. The p38 inhibitor significantly enhanced ICAM-1 expression. ERK1/2 activation was not responsible for the LPS/IFN-γ-induced ICAM-1 upregulation in HPMVECs. THP-1 monocyte adhesion to HPMVECs under LPS/IFN-γ stimulation was inhibited by the JNK inhibitor and enhanced by the p38 inhibitor. We conclude that, in HPMVECs stimulated with LPS/IFN-γ, JNK mediates ICAM-1 expression that can facilitate leukocyte adherence and transmigration, while p38 MAPK negatively regulates the upregulation of ICAM-1 through inhibition of JNK activation.     

Role of mitogen-activated protein kinases in influenza virus induction of prostaglandin E2 from arachidonic acid in bronchial epithelial cells

BACKGROUND: Influenza virus (IV) infection causes airway inflammation; however, it has not been determined whether IV infection could catabolize arachidonic acid cascade in airway epithelial cells. In addition, the responsible intracellular signalling molecules that catabolize arachidonic acid cascade have not been determined. OBJECTIVE: In the present study, to clarify these issues, we examined the cyclooxygenase (COX) expression, cytosolic phospholipase A2 (cPLA2) phosphorylation and prostaglandin E2 (PGE2) release in human bronchial epithelial cells (BEC) upon IV infection, and the role of mitogen-activated protein kinase (MAPK) including extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun-NH2-terminal kinase (JNK) in catabolizing arachidonic acid cascade in BEC. METHODS: COX-2 expression, phosphorylation of cPLA2 and phosphorylation of ERK, JNK and p38 MAPK were determined by Western blot. The concentrations of PGE2 were determined by ELISA. PD 98059 as a specific inhibitor of MAPK kinase-1 (MEK-1), an up-stream kinase of ERK, SB 203580 as a specific inhibitor of p38 MAPK and CEP-11004 as a specific inhibitor of JNK cascade were used to investigate the role of ERK, p38 MAPK and JNK in catabolizing arachidonic acid cascade in BEC. RESULTS: The results showed that (1) IV infection increases COX-2 expression, cPLA2 phosphorylation and PGE2 release, (2) ERK, p38 MAPK and JNK were phosphorylated, (3) CEP-11004 and PD 98059 predominantly attenuated COX-2 expression and cPLA2 phosphorylation, respectively, (4) SB 203580 did not remarkably affect COX-2 expression and cPLA2 phosphorylation, and (5) each inhibitor dose-dependently attenuated PGE2 release by various extents. CONCLUSION: These results indicate that IV infection activates three distinct MAPKs, ERK, p38 MAPK and JNK, to participate to various extents in the induction of PGE2 synthesis from arachidonic acid in BEC.     

Close association of p38 and JNK with plasminogen-dependent upregulation of PAI-1 in rat astrocytes in vitro

As reported previously, stimulation of astrocytes with plasminogen (PLGn) remarkably enhances their production/release of plasminogen activator inhibitor-1 (PAI-1). In addition, both p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) are activated in these astrocytes. However, it remains to be determined whether the MAPK activation is associated with the PAI-1 induction in PLGn-stimulated astrocytes. In the present study, we investigated the relationship between MAPK activity and PAI-1 induction in PLGn-stimulated astrocytes. PLGn stimulation led to definitive phosphorylation of three MAPKs: external signal regulated kinase (ERK), JNK and p38. These results suggest that all of these MAPKs, either alone or in combination, are involved in PAI-1 induction. To verify this association, an inhibition experiment was carried out by using inhibitors specific for each MAPK. The results of the immunoblotting analysis indicated that 20 μM SB203580 (the p38 inhibitor) or SP600125 (the JNK inhibitor) suppressed approximately 85% or 40% of PLGn-inducible PAI-1, respectively. Only 20% inhibition was achieved by pretreatment of astrocytes with 20 μM PD98059 (the inhibitor of MEK1/2, an upstream kinase of ERK). In conclusion, p38 and JNK were shown to be the major MAPKs involved in the signaling cascade leading to PAI-1 induction in astrocytes stimulated with PLGn.     

Role of caspases in dexamethasone-induced apoptosis and activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase in human eosinophils

Eosinophils are the principal effector cells for the pathogenesis of allergic inflammation. Glucocorticoids such as dexamethasone have long been used therapeutically for eosinophilia in allergic inflammation by inducing eosinophil apoptosis, but little is known about the intracellular mechanisms mediating dexamethasone-induced apoptosis. In the present study, we investigated the effect of dexamethasone on three mitogen-activated protein kinases (MAPK) involved in the intracellular signalling pathway: c-Jun NH2-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK). We found that dexamethasone could activate JNK and p38 MAPK in a time-dependent manner but not ERK. Further, SB 203580, a specific p38 MAPK inhibitor, was additive with dexamethasone in inducing eosinophil apoptosis, while JNK1/2 antisense phosphorothioate oligodeoxynucleotides did not show any significant effect. These suggest that dexamethasone-induced JNK1/2 and p38 MAPK activation are not crucial to the induction of apoptosis. Pretreatment of eosinophils with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.FMK), a broad-spectrum caspase inhibitor, could inhibit dexamethasone-induced apoptosis in eosinophils dose-dependently. Moreover, Z-VAD.FMK partially inhibited dexamethasone-activated JNK and p38 MAPK activities. However, dexamethasone treatment did not activate specific caspase-3, -8 activity in eosinophils compared with spontaneous apoptosis. We therefore conclude that dexamethasone-induced apoptosis and activation of JNK and p38 MAPK activity in eosinophils are regulated by caspases but not through the common apoptosis-related caspase-3, -8 as in other cell types. Elucidation of the important role of caspases in eosinophil apoptosis may facilitate the development of more specific and effective treatment for allergic inflammation.     

Tumor necrosis factor-alpha up-regulates the expression of CCL2 and adhesion molecules of human proximal tubular epithelial cells through MAPK signaling pathways

Both circulating and urinary tumor necrosis factor (TNF)-alpha levels have been shown to increase in inflammatory chronic kidney diseases and TNF-alpha can induce secretion of other inflammatory mediators from many cell types. Chemokine, mononuclear chemoattractant protein-1 (CCL2/MCP-1), and cell surface adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), in renal proximal tubular epithelial cells (PTEC) are important for promoting recruitment and adhesion of infiltrating macrophages and lymphocytes to inflamed renal tissue. This study aimed to investigate the effect of TNF-alpha on the expression of these inflammation-related molecules of human PTEC and the underlying intracellular mitogen-activated protein kinase (MAPK) regulatory signaling mechanisms. Cytokine expression profile of TNF-alpha-activated PTEC was assayed by protein array. The concentration of CCL2 was analyzed by ELISA, while the expression of cell surface ICAM-1 and VCAM-1 and intracellular phosphorylated p38 MAPK, c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) was assessed using flow cytometry. TNF-alpha could significantly induce CCL2, ICAM-1 and VCAM-1 expression of PTEC. Selective inhibitors of p38 MAPK (SB203580), JNK (SP600125) and ERK (PD98059) could suppress TNF-alpha-induced CCL2 and ICAM-1 expression, while only p38 MAPK and ERK inhibitors could suppress TNF-alpha-induced VCAM-1 expression. JNK inhibitor was found to up-regulate VCAM-1 expression but did not elicit any additive effect with TNF-alpha on VCAM-1 expression. Moreover, p38 MAPK inhibitor was found to abrogate the TNF-alpha-induced ERK phosphorylation, suggesting that there was a one-way interaction between p38 MAPK and ERK pathways during the TNF-alpha activation. TNF-alpha can play a crucial role in the immunopathogenesis of nephritis by the induction of CCL2, ICAM-1 and VCAM-1 expression via the activation of the intracellular MAPK signaling pathway, which may contribute to macrophage and lymphocyte infiltration.     

C-Jun NH2 terminal kinase (JNK) is an essential mediator of Toll-like receptor 2-induced corneal inflammation

TLRs play an important role in the host inflammatory response to bacteria and bacterial products by activating a cascade of intracellular events leading to production of proinflammatory and chemotactic cytokines. To determine the role of MAPKs in TLR- induced corneal inflammation, we stimulated human corneal epithelial (HCE) cells with TLR2 ligands, tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3Cys) or inactivated Staphylococcus aureus, and examined the time course of expression of MAPKs and the effect of MAPK inhibition on IkBalpha degradation and CXC chemokine production. We found that S. aureus and Pam3Cys stimulate phosphorylation of JNK, p38 MAPK, and ERK within 4 h and that blockade of JNK, but not p38 or ERK phosphorylation, had an inhibitory effect on IkBalpha degradation and CXC chemokine production. To determine if JNK is also important in TLR2-induced corneal inflammation in vivo, we examined JNK1(-/-) mice and pharmacological inhibitors in a murine model of TLR2-induced corneal inflammation which is characterized by neutrophil recruitment to the corneal stroma and development of corneal haze. We found that corneal inflammation was significantly impaired in JNK1(-/-) mice compared with control mice, and in mice treated with the JNK inhibitor compared with vehicle control. Taken together with results from HCE cells, these findings demonstrate that JNK has an essential role in TLR2-induced corneal inflammation.     

Stress-activated protein kinases mediate cell migration in human airway epithelial cells

Airway epithelial cell (AEC) repair immediately after injury requires coordinated cell spreading and migration at the site of injury. Stress-activated protein kinases such as p38 MAPK and c-Jun N-terminal Protein Kinase (JNK) modulate several responses to cell stress and injury, but their role in AEC migration is not clear. We examined migration in confluent 16HBE14o(-) human AEC lines and in primary AEC grown on collagen-IV. Wounds were created by mechanical abrasion and followed to closure using digital microscopy. Inhibitors of either p38 extracellular signal-regulated kinase (ERK)1/2 (PD98059), mitogen-activated protein kinase (MAPK) (SB203580), or JNK (SP600125) could block cell migration substantially. Inhibiting JNK but not p38 MAPK or ERK1/2 blocked extension of cells into the wound region from the original line of injury. Initial migration was associated with phosphorylation of ERK, p38 MAPK, and JNK within 5-15 min. The downstream effector of p38, heat shock protein 27, also was phosphorylated rapidly after injury; phosphorylation could be blocked by prior treatment with SB203580 but not SP600125. The downstream effector of JNK, c-Jun, likewise was phosphorylated rapidly after injury and could be blocked by inhibiting JNK. Our data demonstrate that p38 MAPK, JNK, and ERK1/2 participate in the early stages of AEC migration.     

The action of nitric oxide to enhance cell survival in chick cardiomyocytes is mediated through a cGMP and ERK1/2 pathway while p38 mitogen-activated protein kinase-dependent pathways do not alter cell death

The objective of this study was to determine whether the dual action of nitric oxide (NO) on cardiomyocyte cell viability is mediated through p38 mitogen-activated protein kinase (MAPK)-induced cell death and extracellular signal-regulated kinase (ERK1/2)-mediated cell survival pathways, and whether either of these is mediated through a cGMP-protein kinase G (PKG) pathway. Cell viability of embryonic chick cardiomyocytes was assessed by the MTT assay, which is based on the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The NO donor sodium nitroprusside (SNP) produced a significant (P < 0.01) concentration-dependent reduction in cell viability or increase in cell death. Sodium nitroprusside induced ERK1/2 phosphorylation, and the mitogen-activated protein kinase (MEK1/2) inhibitor PD 98059 significantly increased cell death. In contrast, SB202190, a relatively selective inhibitor of p38 MAPK, did not affect SNP-induced cell death. The cardioprotective effect of NO was prbably mediated in part via cGMP because 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-sensitive guanylyl cyclase, produced a significant enhancement of SNP-induced cell death. In contrast, the PKG inhibitor KT5823 did not affect cell viability. In summary, these data suggest that NO, via stimulation of soluble guanylyl cyclase, activates MEK1/2 whose product, ERK1/2, protects against cell death. In contrast, SNP-induced p38 MAPK activation does not modulate NO-induced cardiomyocyte cell death. Not all cGMP targets affect NO-induced cell death, since the PKG pathway does not enhance or suppress NO-induced cardiomyocyte cell death. Enhancement of the ERK1/2 responses to NO may permit the beneficial effects of NO to predominate.     

Neurite outgrowth of PC12 mutant cells induced by orange oil and d-limonene via the p38 MAPK pathway

We studied the effects of natural essential oil on neurite outgrowth in PC12m3 neuronal cells to elucidate the mechanism underlying the action of the oils used in aromatherapy. Neurite outgrowth can be induced by nerve growth factor (NGF), where ERK and p38 MAPK among MAPK pathways play important roles in activating intracellular signal transduction. In this study, we investigated whether d-limonene, the major component of essential oils from oranges, can promote neurite outgrowth in PC12m3 cells, in which neurite outgrowth can be induced by various physical stimulations. We also examined by which pathways, the ERK, p38 MAPK or JNK pathway, d-limonene acts on PC12m3 cells. Our results showed that neurite outgrowth can be induced when the cells are treated with d-limonene. After treatment with d-limonene, we observed that p38 MAPK is strongly activated in PC12m3 cells, while ERK is weakly activated. In contrast, JNK shows little activity. A study using an inhibitor of p38 MAPK revealed that neurite outgrowth in PC12m3 cells is induced via the activation of p38 MAPK by d-limonene. The results thus indicate that d-limonene may promote neural cell differentiation mainly via activation of the p38 MAPK pathway.     

柚皮苷在促大鼠骨髓间充质干细胞骨向分化过程中对MAPK信号通路的影响

目的: 研究中药单体柚皮苷(NG)对体外培养的大鼠骨髓间充质干细胞(MSCs)向成骨细胞分化过程中MAPK信号通路的影响。方法: 观察在正常、加入p38、ERK和JNK通路抑制剂SB203580、PD98059、SP600125及3种抑制剂全部加入的情况下,各组碱性磷酸酶(ALP)、骨钙素(BGP)、I型胶原(Col I)等骨向分化指标的差异。用Western blotting技术检测各组p38、ERK1/2和JNK蛋白的磷酸化水平,用荧光定量PCR技术检测细胞因子转化生长因子β₁(TGF-β₁)、骨形成蛋白2(BMP-2)和核心结合因子α1(Cbfα1) mRNA的表达。结果: (1)10^-7mol/L为本实验中NG的最佳促骨向分化浓度。(2) NG最佳浓度组的ALP和BGP含量比其它各组都高(P<0.05),Col I含量无明显差异(P>0.05);与NG组相比,加入不同抑制剂组的ALP、BGP和ColⅠ表达量出现不同程度的降低。(3)与空白组相比,NG组JNK蛋白的磷酸化水平升高(P<0.05),p38蛋白的磷酸化水平降低(P<0.01),ERK1/2蛋白的磷酸化水平无明显差异(P>0.05)。与NG组相比,加入不同抑制剂组的p38、ERK1/2和JNK蛋白的磷酸化水平有升高也有降低。(4) NG组上调BMP-2的表达(P<0.05),下调Cbfα1的表达(P<0.05),而对TGF-β₁的表达无明显影响(P>0.05)。与NG组相比,加入不同抑制剂组的TGF-β₁、BMP-2和Cbfα1 mRNA表达量出现不同程度的降低。结论: NG主要通过激活MAPK信号通路中ERK通路、JNK通路以及上调BMP-2的表达,促进MSCs的骨向分化。NG上调BMP-2的表达受MAPK通路中p38通路的影响较大。     

MAPK signaling has stage-dependent osteogenic effects on human adipose-derived stem cells in vitro

Overview: The use of pro-osteogenic growth factors, such as BMP2, in human adipose-derived stem cell (ASC) osteogenesis is well described. Because these growth factors work via signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) cascade, a study of the relationship between MAPK signaling and ASC osteogenesis was conducted. Materials and Methods: ERK, JNK, and p38MAPK activation were measured in ASCs osteo-induced using either dexamethasone or vitamin D3 and correlated with mineralization. Activation and mineralization were also measured without dexamethasone or using the glucocorticoid, cortisone. The expression of the MAPK phosphatase, MKP1, and its relationship to mineralization was also assessed. The effect of decreasing MAPK activation on mineralization through the use of exogenous inhibitors was examined along with siRNA-knockdown and adenoviral overexpression of ERK1/2. Finally, the effect of ERK1/2 overexpression on ASCs induced on PLGA scaffolds was assessed. Results: ASC mineralization in dexamethasone or vitamin D3-induced ASCs correlated with both increased ERK1/2 and JNK1/2 activation. ASCs induced without dexamethasone also mineralized, with JNK1/2 signaling possibly mediating this event. No link between cortisone induction and MAPK signaling could be ascertained. ASCs treated with ERK, JNK, or p38MAPK inhibitors showed decreased osteogenic gene expression and diminished mineralization. Mineralization levels were also affected by viruses designed to inhibit or augment ERK1/2 expression and activity. Finally, ASC mineralization appeared to be a balance between the MAPK kinase activity and MKP1. Conclusions: It is likely that MAPK signaling plays a significant role in ASC osteogenesis, affecting differentiation in kinase- and stage-specific manners.     

Leptin Induces Terminal Differentiation of Rat Annulus Fibrosus Cells via Activation of MAPK Signaling

Both leptin and its receptor are expressed in rat annulus fibrosus (AF) cells. However, little is known about their role and mechanism during disc degeneration. The mitogen activating protein kinase (MAPK) pathway which mediates leptin‐induced terminal differentiation of rat AF cells was analyzed using PCR, Western‐blot and immunocytochemistry. It was found that leptin‐induced AF cells terminal differentiation, which may be attributed to upregulated p38 and ERK1/2 phosphorylation, however, JNK phosphorylation was not observed. Specific inhibitors of p38 or ERK1/2, but not JNK, could inhibit the stimulative activity of leptin on collagen X and MMP‐13 protein levels. This study, for the first time, shows that the MAPK pathway, especially p38 and ERK1/2 signaling, plays a distinct role in leptin‐induced AF cells terminal differentiation. Anat Rec, 296:1806–1812, 2013. © 2013 Wiley Periodicals, Inc.     

Sodium salicylate-induced apoptosis of human peripheral blood eosinophils is independent of the activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase

BACKGROUND: It has been shown that the inhibition of eosinophilic apoptosis is an important mechanism for the development of blood and tissue eosinophilia in allergic diseases. Considerable attention has recently been focused on the role played by different intracellular kinase cascades in the control of apoptosis. In the present study, we investigated the effect of sodium salicylate (NaSal), a nonsteroidal anti-inflammatory drug, on mitogen-activated protein kinases (MAPK) and apoptosis of human eosinophils. METHODS: Human blood eosinophils were purified from buffy coat. NaSal-induced apoptosis of eosinophils was assessed by morphological changes and Annexin-V binding assay. Changes of MAPK activity upon treatment with NaSal were measured by kinase activity assay and Western blot. RESULTS: NaSal could induce apoptosis of human blood eosinophils in a dose- and time-dependent manner. It could also activate c-Jun N-terminal kinase (JNK) and p38 MAPK but not extracellular signal-regulated protein kinase (ERK) activity within 1 h. Pretreatment of eosinophils with p38 MAPK and JNK anti-sense (AS) phosphorothioate oligodeoxynucleotides (ODN) or specific p38 MAPK inhibitor SB 203580 did not have any significant effect on NaSal-induced apoptosis. However, ERK AS ODNs could trigger the apoptosis of normal eosinophils. CONCLUSION: There is no direct relationship between the activation of JNK and p38 MAPK pathways and NaSal-induced apoptosis in human peripheral blood eosinophils.