A Specific Oligodeoxynucleotide Promotes the Differentiation of Osteoblasts via ERK and p38 MAPK Pathways

A specific oligodeoxynucleotide (ODN), ODN MT01, was found to have positive effects on the proliferation and activation of the osteoblast-like cell line MG 63. In this study, the detailed signaling pathways in which ODN MT01 promoted the differentiation of osteoblasts were systematically examined. ODN MT01 enhanced the expression of osteogenic marker genes, such as osteocalcin and type I collagen. Furthermore, ODN MT01 activated Runx2 phosphorylation via ERK1/2 mitogen-activated protein kinase (MAPK) and p38 MAPK. Consistently, ODN MT01 induced up-regulation of osteocalcin, alkaline phosphatase (ALP) and type I collagen, which was inhibited by pre-treatment with the ERK1/2 inhibitor U0126 and the p38 inhibitor SB203580. These results suggest that the ERK1/2 and p38 MAPK pathways, as well as Runx2 activation, are involved in ODN MT01-induced up-regulation of osteocalcin, type I collagen and the activity of ALP in MG 63 cells.     

Activation of ERK and p38 Reduces AZD8055-Mediated Inhibition of Protein Synthesis in Hepatocellular Carcinoma HepG2 Cell Line

Protein synthesis is important for maintaining cellular homeostasis under various stress responses. In this study, we screened an anticancer drug library to select compounds with translational repression functions. AZD8055, an ATP-competitive mechanistic target of rapamycin complex 1/2 (mTORC1/2) inhibitor, was selected as a translational suppressor. AZD8055 inhibited protein synthesis in mouse embryonic fibroblasts and hepatocellular carcinoma HepG2 cells. Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) were activated during the early phase of mTORC1/2 inhibition by AZD8055 treatment. Combined treatment of AZD8055 with the MAPK kinase1/2 (MEK1/2) inhibitor refametinib or the p38 inhibitor SB203580 markedly decreased translation in HepG2 cells. Thus, the inhibition of ERK1/2 or p38 may enhance the efficacy of AZD8055-mediated inhibition of protein synthesis. In addition, AZD8055 down-regulated the phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and AZD8055-induced phosphorylation of ERK1/2 and p38 had no effect on phosphorylation status of 4E-BP1. Interestingly, AZD8055 modulated the 4E-BP1 mRNA pool by up-regulating ERK1/2 and p38 pathways. Together, these results suggest that AZD8055-induced activation of MAPKs interferes with inhibition of protein synthesis at an early stage of mTORC1/2 inhibition, and that it may contribute to the development of resistance to mTORC1/2 inhibitors.     

ZnO nanoparticle-induced oxidative stress triggers apoptosis by activating JNK signaling pathway in cultured primary astrocytes

It has been documented in in vitro studies that zinc oxide nanoparticles (ZnO NPs) are capable of inducing oxidative stress, which plays a crucial role in ZnO NP-mediated apoptosis. However, the underlying molecular mechanism of apoptosis in neurocytes induced by ZnO NP exposure was not fully elucidated. In this study, we investigated the potential mechanisms of apoptosis provoked by ZnO NPs in cultured primary astrocytes by exploring the molecular signaling pathways triggered after ZnO NP exposure. ZnO NP exposure was found to reduce cell viability in MTT assays, increase lactate dehydrogenase (LDH) release, stimulate intracellular reactive oxygen species (ROS) generation, and elicit caspase-3 activation in a dose- and time-dependent manner. Apoptosis occurred after ZnO NP exposure as evidenced by nuclear condensation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. A decrease in mitochondrial membrane potential (MMP) with a concomitant increase in the expression of Bax/Bcl-2 ratio suggested that the mitochondria also mediated the pathway involved in ZnO NP-induced apoptosis. In addition, exposure of the cultured cells to ZnO NPs led to phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK). Moreover, JNK inhibitor (SP600125) significantly reduced ZnO NP-induced cleaved PARP and cleaved caspase-3 expression, but not ERK inhibitor (U0126) or p38 MAPK inhibitor (SB203580), indicating that JNK signaling pathway is involved in ZnO NP-induced apoptosis in primary astrocytes.     

Lysophosphatidic Acid Mediates Imiquimod-Induced Psoriasis-like Symptoms by Promoting Keratinocyte Proliferation through LPAR1/ROCK2/PI3K/AKT Signaling Pathway

Psoriasis is a chronic inflammatory skin disease. Recently, lysophosphatidic acid (LPA)/LPAR5 signaling has been reported to be involved in both NLRP3 inflammasome activation in macrophages and keratinocyte activation to produce inflammatory cytokines, contributing to psoriasis pathogenesis. However, the effect and molecular mechanisms of LPA/LPAR signaling in keratinocyte proliferation in psoriasis remain unclear. In this study, we investigated the effects of LPAR1/3 inhibition on imiquimod (IMQ)-induced psoriasis-like mice. Treatment with the LPAR1/3 antagonist, ki16425, alleviated skin symptoms in IMQ-induced psoriasis-like mouse models and decreased keratinocyte proliferation in the lesion. It also decreased LPA-induced cell proliferation and cell cycle progression via increased cyclin A2, cyclin D1, cyclin-dependent kinase (CDK)2, and CDK4 expression and decreased p27^Kip1 expression in HaCaT cells. LPAR1 knockdown in HaCaT cells reduced LPA-induced proliferation, suppressed cyclin A2 and CDK2 expression, and restored p27^Kip1 expression. LPA increased Rho-associated protein kinase 2 (ROCK2) expression and PI3K/AKT activation; moreover, the pharmacological inhibition of ROCK2 and PI3K/AKT signaling suppressed LPA-induced cell cycle progression. In conclusion, we demonstrated that LPAR1/3 antagonist alleviates IMQ-induced psoriasis-like symptoms in mice, and in particular, LPAR1 signaling is involved in cell cycle progression via ROCK2/PI3K/AKT pathways in keratinocytes.     

Apoptosis Induction of Human Prostate Carcinoma DU145 Cells by Diallyl Disulfide via Modulation of JNK and PI3K/AKT Signaling Pathways

Diallyl disulfide (DADS), a sulfur compound derived from garlic, has various biological properties, such as anticancer, antiangiogenic and anti-inflammatory effects. However, the mechanisms of action underlying the compound’s anticancer activity have not been fully elucidated. In this study, the apoptotic effects of DADS were investigated in DU145 human prostate carcinoma cells. Our results showed that DADS markedly inhibited the growth of the DU145 cells by induction of apoptosis. Apoptosis was accompanied by modulation of Bcl-2 and inhibitor of apoptosis protein (IAP) family proteins, depolarization of the mitochondrial membrane potential (MMP, ΔΨm) and proteolytic activation of caspases. We also found that the expression of death-receptor 4 (DR4) and Fas ligand (FasL) proteins was increased and that the level of intact Bid proteins was down-regulated by DADS. Moreover, treatment with DADS induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular-signal regulating kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK). A specific JNK inhibitor, SP600125, significantly blocked DADS-induced-apoptosis, whereas inhibitors of the ERK (PD98059) and p38 MAPK (SB203580) had no effect. The induction of apoptosis was also accompanied by inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt and the PI3K inhibitor LY29004 significantly increased DADS-induced cell death. These findings provide evidence demonstrating that the proapoptotic effect of DADS is mediated through the activation of JNK and the inhibition of the PI3K/Akt signaling pathway in DU145 cells.     

p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells

Saturated stearic acid (SA) induces apoptosis in the human pancreatic β-cells NES2Y. However, the molecular mechanisms involved are unclear. We showed that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway in these cells. Therefore, we tested the role of p38 MAPK signaling pathway activation in apoptosis induction by SA in NES2Y cells. Crosstalk between p38 MAPK pathway activation and accompanying ERK pathway inhibition after SA application was also tested. The inhibition of p38 MAPK expression by siRNA silencing resulted in a decrease in MAPKAPK-2 activation after SA application, but it had no significant effect on cell viability or the level of phosphorylated ERK pathway members. The inhibition of p38 MAPK activity by the specific inhibitor SB202190 resulted in inhibition of MAPKAPK-2 activation and noticeable activation of ERK pathway members after SA treatment but in no significant effect on cell viability. p38 MAPK overexpression by plasmid transfection produced an increase in MAPKAPK-2 activation after SA exposure but no significant influence on cell viability or ERK pathway activation. The activation of p38 MAPK by the specific activator anisomycin resulted in significant activation of MAPKAPK-2. Concerning the effect on cell viability, application of the activator led to apoptosis induction similar to application of SA (PARP cleavage and caspase-7, -8, and -9 activation) and in inhibition of ERK pathway members. We demonstrated that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway and that this activation could be involved in apoptosis induction by SA in the human pancreatic β-cells NES2Y. However, this involvement does not seem to play a key role. Crosstalk between p38 MAPK pathway activation and ERK pathway inhibition in NES2Y cells seems likely. Thus, the ERK pathway inhibition by p38 MAPK activation does not also seem to be essential for SA-induced apoptosis.     

TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function

Migration of vascular smooth muscle cells (VSMCs) into the intima is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of Toll-like receptor 4 (TLR4) in the progression of atherogenesis, its function in the regulation of VSMC migration remains unclear. The goal of the present study was to elucidate the mechanism by which TLR4 regulates VSMC migration. Inhibitor experiments revealed that TLR4-induced IL-6 secretion and VSMC migration were mediated via the concerted actions of MyD88 and TRIF on the activation of p38 MAPK and ERK1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited TLR4 agonist-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed TLR4-induced IL-6 production and VSMC migration. Rac-1 inhibitor suppressed TLR4-driven VSMC migration but not IL-6 production. Importantly, the serum level of IL-6 and TLR4 endogenous ligand HMGB1 was significantly higher in patients with coronary artery diseases (CAD) than in healthy subjects. Serum HMGB1 level was positively correlated with serum IL-6 level in CAD patients. The expression of both HMGB1 and IL-6 was clearly detected in the atherosclerotic tissue of the CAD patients. Additionally, there was a positive association between p-CREB and HMGB1 in mouse atherosclerotic tissue. Based on our findings, we concluded that, upon ligand binding, TLR4 activates p38 MAPK and ERK1/2 signaling through MyD88 and TRIF in VSMCs. These signaling pathways subsequently coordinate an additive augmentation of CREB-driven IL-6 production, which in turn triggers Rac-1-mediated actin cytoskeleton to promote VSMC migration.     

The Apoptotic Volume Decrease Is an Upstream Event of MAP Kinase Activation during Staurosporine-Induced Apoptosis in HeLa Cells

Persistent cell shrinkage, called apoptotic volume decrease (AVD), is a pivotal event of apoptosis. Activation of the volume-sensitive outwardly rectifying Cl(-) channel (VSOR) is involved in the AVD induction. On the other hand, activation of the MAP kinase (MAPK) cascade is also known to play a critical role in apoptosis. In the present study, we investigated the relationship between the AVD induction and the stress-responsive MAPK cascade activation during the apoptosis process induced by staurosporine (STS) in HeLa cells. STS was found to induce AVD within 2-5 min and phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK after over 20-30 min. VSOR blockers suppressed not only STS-induced AVD but also phosphorylation of JNK and p38 as well as activation of caspase-3/7. Moreover, a p38 inhibitor, SB203580, and a JNK inhibitor, SP600125, failed to affect STS-induced AVD, whereas these compounds reduced STS-induced activation of caspase-3/7. Also, treatment with ASK1-specific siRNA suppressed STS-induced caspase-3/7 activation without affecting the AVD induction. Furthermore, sustained osmotic cell shrinkage per se was found to trigger phosphorylation of JNK and p38, caspase activation, and cell death. Thus, it is suggested that activation of p38 and JNK is a downstream event of AVD for the STS-induced apoptosis of HeLa cells.     

p38 MAPK抑制剂SB203580对缺氧致人脐静脉内皮细胞损伤的保护作用

目的观察p38MAPK特异性抑制剂SB203580对缺氧致人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)损伤的保护作用。方法将HUVECs分为正常对照组、缺氧培养组、SB203580+正常对照组和SB203580+缺氧培养组,培养24h后,流式细胞术检测各组细胞的凋亡率;Western blot分析各组细胞p38MAPK蛋白及其磷酸化水平;Transwell小室模型检测各组细胞的迁移率;ELISA法检测各组细胞培养上清中可溶性血管内皮生长因子受体-1(sFlt-1)及可溶性Endoglin(sEng)的含量。结果与正常对照组相比,缺氧培养组细胞的凋亡率、p38MAPK的磷酸化水平、sFlt-1及sEng含量显著增加(P<0.01),细胞体外迁移能力下降;SB203580+缺氧培养组细胞的凋亡率、p38MAPK的磷酸化水平、sFlt-1和sEng的释放较缺氧培养组均下降,体外迁移能力增强(P<0.05);磷酸化p38MAPK的释放水平与sFlt-1及sEng含量呈正相关(r1=0.69,P<0.05;r2=0.71,P<0.05)。结论 SB203580通过特异性阻断p38MAPK信号转导通路,对缺氧培养的HUVECs产生保护作用。     

Possible Mechanisms of Di(2-ethylhexyl) Phthalate-Induced MMP-2 and MMP-9 Expression in A7r5 Rat Vascular Smooth Muscle Cells

Proliferation and migration of vascular smooth muscle cells (VSMC) are important in the development and/or progression of many cardiovascular diseases, including atherosclerosis. Evidence shows that matrix metalloproteinase (MMP)-2 and MMP-9 are related to the pathogenesis of atherosclerosis. The expressions of MMP-2 and MMP-9 in atherosclerosis are regulated via various pathways, such as p38 mitogen activated protein kinase (MAPK), extracellular signal regulated kinase 1 and 2 (ERK1/2), Akt, and nuclear factor kappa (NF-κB). Di(2-ethylhexyl) phthalate (DEHP) has been shown to induce atherosclerosis by increasing tumor necrosis factor (TNF)-α, interleukin (IL)-6, and intercellular adhesion molecule (ICAM) productions. However, whether DEHP poses any effects on MMP-2 or MMP-9 expression in VSMC has not yet been answered. In our studies, rat aorta VSMC was treated with DEHP (between 2 and 17.5 ppm) and p38 MAPK, ERK1/2, Akt, NF-κB, and MMP-2 and MMP-9 proteins and activities were measured. Results showed that the presence of DEHP can induce higher MMP-2 and MMP-9 expression than the controls. Similar results on MMP-regulating proteins, i.e., p38 MAPK, ERK1/2, Akt, and NF-κB, were also observed. In summary, our current results have showed that DEHP can be a potent inducer of atherosclerosis by increasing MMP-2 and MMP-9 expression at least through the regulations of p38 MAPK, ERK1/2, Akt, and NF-κB.     

MAPK/ERK1/2对乳酸杆菌介导的子宫内膜上皮细胞增殖的影响

目的研究乳酸杆菌对子宫内膜上皮细胞增殖的丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)通路的作用机制。方法将103个/mL乳酸杆菌与子宫内膜上皮细胞共培养20、40、60 min,各时间点收集细胞,采用Western blot法检测子宫内膜上皮细胞MAPK通路中ERK1/2、JNK、P38蛋白及其磷酸化水平。在子宫内膜上皮细胞中加入40 ng/mL U0126,再加入10^3个/mL乳酸杆菌,共培养20、40、60 min,各时间点收集细胞,采用Western blot及CCK-8法检测U0126对ERK1/2、JNK、P38、p90RSK蛋白磷酸化及细胞增殖的影响。结果乳酸杆菌能促进子宫内膜上皮细胞MAPK通路中ERK1/2蛋白发生磷酸化,共培养40和60 min组ERK1/2磷酸化水平显著增加,与对照(0 min)和20 min组相比,差异有统计学意义(P <0. 05),但与40和60 min组比较,差异无统计学意义(P> 0. 05);MAPK通路中JNK和P38总蛋白水平和磷酸化蛋白水平均无明显变化(P> 0. 05)。40 ng/mL U0126对JNK、P38蛋白磷酸化水平无影响,但能抑制ERK1/2信号通路下游蛋白p90RSK的磷酸化,同时能抑制乳酸杆菌促进子宫内膜上皮细胞增殖。结论乳酸杆菌介导的促进子宫内膜上皮细胞增殖的作用是通过MAPK信号通路中的ERK1/2磷酸化发挥作用的。     

Increased formation of phosphatidylinositol-4-phosphate in human platelets stimulated with lysophosphatidic acid

Lysophosphatidic acid (LPA, 1-acyl-sn-glycerol 3-phosphate), at a concentration of 1–40 μM, was found to induce the formation of [³H]inositol-labelled phosphatidylinositol-4-phosphate (PIP) without significantly altering the levels of either phosphatidylinositol (PI) or phosphatidylinositol bisphosphate (PIP₂) in washed human platelets. Preincubation of platelets with the cyclooxygenase/lipoxygenase inhibitor, BW755C at 100 μM, did not alter the LPA-induced formation of PIP. Activation of platelets with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), elicited a similar response (induction of PIP formation). The specific protein kinase C (PKC) inhibitor, GF109203X (10 μM), completely blocked the effect of PMA but not the LPA-induced generation of PIP. The present results indicate that LPA can induce PIP formationvia PI-4-kinase activation, through processes which are independent of the eicosanoid/TxA₂ pathway and are not PKC-dependent.     

MAPKs and Signal Transduction in the Control of Gastrointestinal Epithelial Cell Proliferation and Differentiation

Mitogen-activated protein kinase (MAPK) pathways are activated by several stimuli and transduce the signal inside cells, generating diverse responses including cell proliferation, differentiation, migration and apoptosis. Each MAPK cascade comprises a series of molecules, and regulation takes place at different levels. They communicate with each other and with additional pathways, creating a signaling network that is important for cell fate determination. In this review, we focus on ERK, JNK, p38 and ERK5, the major MAPKs, and their interactions with PI3K-Akt, TGFβ/Smad and Wnt/β-catenin pathways. More importantly, we describe how MAPKs regulate cell proliferation and differentiation in the rapidly renewing epithelia that lines the gastrointestinal tract and, finally, we highlight the recent findings on nutritional aspects that affect MAPK transduction cascades.     

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD) simulations of: (1) MK5 alone; (2) MK5 in complex with an inhibitor; and (3) MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS) calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.     

Induction of Apoptosis by PQ1, a Gap Junction Enhancer that Upregulates Connexin 43 and Activates the MAPK Signaling Pathway in Mammary Carcinoma Cells

The mechanism of gap junction enhancer (PQ1) induced cytotoxicity is thought to be attributed to the change in connexin 43 (Cx43) expression; therefore, the effects of Cx43 modulation in cell survival were investigated in mammary carcinoma cells (FMC2u) derived from a malignant neoplasm of a female FVB/N-Tg(MMTV-PyVT)634Mul/J (PyVT) transgenic mouse. PQ1 was determined to have an IC₅₀ of 6.5 µM in FMC2u cells, while inducing an upregulation in Cx43 expression. The effects of Cx43 modulation in FMC2u cell survival was determined through transfection experiments with Cx43 cDNA, which induced an elevated level of protein expression similar to that seen with PQ1 exposure, or siRNA to silence Cx43 protein expression. Overexpression or silencing of Cx43 led to a reduction or an increase in cell viability, respectively. The mitogen-activated protein kinase (MAPK) family has been implicated in the regulation of cell survival and cell death; therefore, the gap junctional intercellular communication (GJIC)-independent function of PQ1 and Cx43 in the Raf/Mitogen-activated protein kinase/ERK kinase/extracellular-signal-regulated kinase (Raf-MEK-ERK) cascade of cellular survival and p38 MAPK-dependent pathway of apoptosis were explored. PQ1 treatment activated p44/42 MAPK, while the overexpression of Cx43 resulted in a reduced expression. This suggests that PQ1 affects the Raf-MEK-ERK cascade independent of Cx43 upregulation. Both overexpression of Cx43 and PQ1 treatment stimulated an increase in the phosphorylated form of p38-MAPK, reduced levels of the anti-apoptotic protein Bcl-2, and increased the cleavage of pro-caspase-3. Silencing of Cx43 protein expression led to a reduction in the phosphorylation of p38-MAPK and an increase in Bcl-2 expression. The mechanism behind PQ1-induced cytotoxicity in FMC2u mammary carcinoma cells is thought to be attributed to the change in Cx43 expression. Furthermore, PQ1-induced apoptosis through the upregulation of Cx43 may depend on p38 MAPK, highlighting that the effect of PQ1 on gap junctions as well as cellular survival via a MAPK-dependent pathway.     

Wound Healing Effect of Gintonin Involves Lysophosphatidic Acid Receptor/Vascular Endothelial Growth Factor Signaling Pathway in Keratinocytes

Gintonin, a novel compound of ginseng, is a ligand of the lysophosphatidic acid (LPA) receptor. The in vitro and in vivo skin wound healing effects of gintonin remain unknown. Therefore, the objective of this study was to investigate the effects of gintonin on wound healing-linked responses, especially migration and proliferation, in skin keratinocytes HaCaT. In this study, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, Boyden chamber migration assay, scratch wound healing assay, and Western blot assay were performed. A tail wound mouse model was used for the in vivo test. Gintonin increased proliferation, migration, and scratch closure in HaCaT cells. It also increased the release of vascular endothelial growth factor (VEGF) in HaCaT cells. However, these increases, induced by gintonin, were markedly blocked by treatment with Ki16425, an LPA inhibitor, PD98059, an ERK inhibitor, 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester), a calcium chelator, and U73122, a PLC inhibitor. The VEGF receptor inhibitor axitinib also attenuated gintonin-enhanced HaCaT cell proliferation. Gintonin increased the phosphorylation of AKT and ERK1/2 in HaCaT cells. In addition, gintonin improved tail wound healing in mice. These results indicate that gintonin may promote wound healing through LPA receptor activation and/or VEGF release-mediated downstream signaling pathways. Thus, gintonin could be a beneficial substance to facilitate skin wound healing.