Thrombin-induced NF-κB activation and IL-8/CXCL8 release is mediated by c-Src-dependent Shc,Raf-1, and ERK pathways in lung epithelial cells

In addition to its functions in thrombosis and hemostasis, thrombin also plays an important role in lung inflammation. Our previous report showed that thrombin activates the protein kinase C (PKC)α/c-Src and Gβγ/Rac1/PI3K/Akt signaling pathways to induce IκB kinase α/β (IKKα/β) activation, NF-κB transactivation, and IL-8/CXCL8 expressions in human lung epithelial cells (ECs). In this study, we further investigated the mechanism of c-Src-dependent Shc, Raf-1, and extracellular signal-regulated kinase (ERK) signaling pathways involved in thrombin-induced NF-κB activation and IL-8/CXCL8 release. Thrombin-induced increases in IL-8/CXCL8 release and κB-luciferase activity were inhibited by the Shc small interfering RNA (siRNA), p66Shc siRNA, GW 5074 (a Raf-1 inhibitor), and PD98059 (a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor). Treatment of A549 cells with thrombin increased p66Shc and p46/p52Shc phosphorylation at Tyr239/240 and Tyr317, which was inhibited by cell transfection with the dominant negative mutant of c-Src (c-Src DN). Thrombin caused time-dependent phosphorylation of Raf-1 and ERK, which was attenuated by the c-Src DN. Thrombin-induced IKKα/β phosphorylation was inhibited by GW 5074 and PD98059. Treatment of cells with thrombin induced Gβγ, c-Src, and p66Shc complex formation in a time-dependent manner. Taken together, these results show for the first time that thrombin activates Shc, Raf-1, and ERK through Gβγ, c-Src, and Shc complex formation to induce IKKα/β phosphorylation, NF-κB activation, and IL-8/CXCL8 release in human lung ECs.     

Urotensin-2 promotes collagen synthesis via ERK1/2-dependent and ERK1/2-independent TGF-β1 in neonatal cardiac fibroblasts

U2 (urotensin-2) is the most potent vasoconstrictor in mammals which is involved in cardiac remodelling, including cardiac hypertrophy and cardiac fibrosis. Although the cellular mechanisms of the U2-induced vasoconstriction have been extensively studied, the signalling pathways involved in U2-induced TGF-β1 (transforming growth factor-β1) expression and collagen synthesis remain unclear. In this study, we show that U2 promoted collagen synthesis and ERK1/2 (extracellular signal-regulated kinase 1/2) activation in neonatal cardiac fibroblasts. The U2-induced collagen synthesis and TGF-β1 production were significantly but not completely inhibited by blocking ERK1/2. Both ERK1/2 inhibitor and TGF-β1 antibody could separately inhibit U2-induced collagen synthesis, and the synergistic inhibition effect was observed by blocking ERK1/2 and TGF-β1 simultaneously. These data suggest that U2 promotes collagen synthesis via ERK1/2-dependent and independent TGF-β1 pathway in neonatal cardiac fibroblasts.     

Integrin αvβ6 Promotes Lung Cancer Proliferation and Metastasis through Upregulation of IL-8–Mediated MAPK/ERK Signaling

Lung cancer is notorious for high morbidity and mortality around the world. Interleukin (IL)-8, a proinflammatory chemokine with tumorigenic and proangiogenic effects, promotes lung cancer cells growth and migration and contributes to cell aggressive phenotypes. Integrin αvβ6 is a receptor of transmembrane heterodimeric cell surface adhesion, and its overexpression correlates with poor survival from non–small cell lung cancer. However, the cross talk between αvβ6 and IL-8 in lung cancer has not been characterized so far. Herein, human lung cancer samples were analyzed, and it revealed that the immunohistochemical and mRNA expression of integrin αvβ6 was significantly correlated with the expression of IL-8. Furthermore, in vitro, integrin αvβ6 increased cell proliferation, migration, and invasion by impairing the expressions of MMP-2 and MMP-9 and inhibited cell apoptosis in human lung cancer cells A549 and H460. In addition, integrin αvβ6 upregulated IL-8 expression through activating MAPK/ERK signaling. The in vivo experiment showed that integrin αvβ6 promoted tumor growth in xenograft model mice by accelerating tumor volume and reducing apoptosis. Meanwhile, lung metastasis model experiment suggested that integrin αvβ6 stimulated tumor metastasis with the increase of lung/total weight and tumor nodules. Simultaneously, integrin αvβ6 upregulated IL-8 expression detected by both Western blots and immunohistochemistry, along with the activation of MAPK/ERK signaling. Overall, these data suggested that, in vitro and in vivo, integrin αvβ6 promoted lung cancer proliferation and metastasis, at least in part, through upregulation of IL-8–mediated MAPK/ERK signaling. Thus, the inhibition of integrin αvβ6 and IL-8 may be the key for the treatment of lung cancer.     

IL-8通过激活PKC/ERK信号通路促进肾癌细胞上皮细胞-间质细胞转化

上皮细胞-间质细胞转化(EMT)在肿瘤转移方面起着非常重要的作用．肾癌发生EMT的具体分子机制尚不清楚．IL-8是一个重要的炎症趋化因子,研究表明肾癌细胞可以分泌IL-8,但IL-8是否参与肾癌细胞EMT的调节目前尚无报道．我们研究发现,IL-8可以促进肾癌细胞形态发生间质化改变,IL-8刺激后E-钙黏蛋白表达水平下降, N-钙黏蛋白表达上调．另外,IL-8可以促进肾癌细胞侵袭,但对肾癌细胞增殖的影响并不明显．进一步研究显示,IL-8通过激活蛋白激酶C(PKC)引起细胞外调节性激酶(ERK)磷酸化．因此,我们认为IL-8可能通过PKC/ERK信号通路促进肾癌细胞发生EMT,这可能是肾癌转移的重要机制之一．     

Transforming growth factor-β1 suppresses serum deprivation-induced apop-tosis via activation of ERK1/ERK2 and the inhibition of p38 activity and ceramide production

In this report we studied the effects and mechanism of transforming growth factor-β1 (TGF-β1) on serum deprivation-induced cell apoptosis. Serum deprivation induces apoptosis, which is associated with an increase in intracellular ceramide level and with the activation of p38 mitogen-activated protein (MAP) kinase. Inhibition of p38 MAP kinase by SB203580 significantly reduced apoptosis induced by serum-deprivation. Treatment of cells with TGF-β1 stimulated cell proliferation and suppressed the serum deprivation-induced apoptotic response. The anti-apoptotic effect of TGF-β1 is correlated with its ability to inhibit the serum deprivation-induced activation of p38 MAP kinase and the increase in intracellular ceramide level. In     

AS-703026 Inhibits LPS-Induced TNFα Production through MEK/ERK Dependent and Independent Mechanisms

Chronic obstructive pulmonary disease (COPD) is characterized by intense lung infiltrations of immune cells (macrophages and monocytes). Lipopolysaccharide (LPS) activates macrophages/monocytes, leading to production of tumor necrosis factor α (TNFα) and other cytokines, which cause subsequent lung damages. In the current study, our results demonstrated that AS-703026, a novel MEK/ERK inhibitor, suppressed LPS-induced TNFα mRNA expression and protein secretion in RAW 264.7 murine macrophages, and in murine bone marrow-derived macrophages (BMDMs). Meanwhile, TNFα production in LPS-stimulated COPD patents’ peripheral blood mononuclear cells (PBMCs) was also repressed by AS-703026. At the molecular level, we showed that AS-703026 blocked LPS-induced MEK/ERK activation in above macrophages/monocytes. However, restoring ERK activation in AS-703026-treated RAW 264.7 cells by introducing a constitutive-actively (CA)-ERK1 only partially reinstated LPS-mediated TNFα production. Meanwhile, AS-703026 could still inhibit TNFα response in ERK1/2-depleted (by shRNA) RAW 264.7 cells. Significantly, we found that AS-703026 inhibited LPS-induced nuclear factor κB (NFκB) activation in above macrophages and COPD patients’ PBMCs. In vivo, oral administration of AS-703026 inhibited LPS-induced TNFα production and endotoxin shock in BALB/c mice. Together, we show that AS-703026 in vitro inhibits LPS-induced TNFα production in macrophages/monocytes, and in vivo protects mice from LPS-induced endotoxin shock. Thus, it could be further studied as a useful anti-inflammatory therapy for COPD patients.     

β-Arrestin prevents cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt pathways

Our previous studies have shown that β-arrestin 2 plays an anti-apoptotic effect. However, the mechanisms by which β-arrestin contribute to anti-apoptotic role remain unclear. In this study, we show that a deficiency of either β-arrestin 1 or β-arrestin 2 significantly increases serum deprivation (SD)-induced percentage of apoptotic cells. β-arrestin 2 deficient-induced apoptosis was inhibited by transfection with β-arrestin 2 full-length plasmid, revealing that SD-induced apoptosis is dependent on β-arrestin 2. Furthermore, in the absence of either β-arrestin 1 or β-arrestin 2 significantly enhances SD-induced the level of pro-apoptotic proteins, including cleaved caspase-3, extracellular-signal regulated kinase 1/2 (ERK1/2) and p38, members of mitogen-activated protein kinases (MAPKs). In addition, a deficiency of either β-arrestin 1 or β-arrestin 2 inhibits phosphorylation of Akt. The SD-induced changes in cleaved caspase-3, ERK1/2 and p38 MAPKs, Akt, and apoptotic cell numbers could be blocked by double knockout of β-arrestin 1/2. Our study thus demonstrates that β-arrestin inhibits cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt signaling pathways.     

Requirement for PLCγ2 in IL-3 and GM-CSF-stimulated MEK/ERK phosphorylation in murine and human hematopoietic stem/progenitor cells

Even though the involvement of intracellular Ca(2+) Ca(i)(2+) in hematopoiesis has been previously demonstrated, the relationship between Ca(i)(2+) signaling and cytokine-induced intracellular pathways remains poorly understood. Herein, the molecular mechanisms integrating Ca(2+) signaling with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in primary murine and human hematopoietic stem/progenitor cells stimulated by IL-3 and GM-CSF were studied. Our results demonstrated that IL-3 and GM-CSF stimulation induced increased inositol 1,4,5-trisphosphate (IP(3) ) levels and Ca(i)(2+) release in murine and human hematopoietic stem/progenitor cells. In addition, Ca(i)(2+) signaling inhibitors, such as inositol 1,4,5-trisphosphate receptor antagonist (2-APB), PKC inhibitor (GF109203), and CaMKII inhibitor (KN-62), blocked phosphorylation of MEK activated by IL-3 and GM-CSF, suggesting the participation of Ca(2+) -dependent kinases in MEK activation. In addition, we identify phospholipase Cγ2 (PLCγ2) as a PLCγ responsible for the induction of Ca(2+) release by IL-3 and GM-CSF in hematopoietic stem/progenitor cells. Furthermore, the PLCγ inhibitor U73122 significantly reduced the numbers of granulocyte-macrophage colony-forming units after cytokine stimulation. Similar results were obtained in both murine and human hematopoietic stem/progenitor cells. Taken together, these data indicate a role for PLCγ2 and Ca(2+) signaling through the modulation of MEK in both murine and human hematopoietic stem/progenitor cells.     

GPR54 regulates ERK1/2 activity and hypothalamic gene expression in a Gα(q/11) and β-arrestin-dependent manner

G protein-coupled receptor 54 (GPR54) is a G(q/11)-coupled 7 transmembrane-spanning receptor (7TMR). Activation of GPR54 by kisspeptin (Kp) stimulates PIP(2) hydrolysis, Ca(2+) mobilization and ERK1/2 MAPK phosphorylation. Kp and GPR54 are established regulators of the hypothalamic-pituitary-gonadal (HPG) axis and loss-of-function mutations in GPR54 are associated with an absence of puberty and hypogonadotropic hypogonadism, thus defining an important role of the Kp/GPR54 signaling system in reproductive function. Given the tremendous physiological and clinical importance of the Kp/GPR54 signaling system, we explored the contributions of the GPR54-coupled G(q/11) and β-arrestin pathways on the activation of a major downstream signaling molecule, ERK, using G(q/11) and β-arrestin knockout mouse embryonic fibroblasts. Our study revealed that GPR54 employs the G(q/11) and β-arrestin-2 pathways in a co-dependent and temporally overlapping manner to positively regulate ERK activity and pERK nuclear localization. We also show that while β-arrestin-2 potentiates GPR54 signaling to ERK, β-arrestin-1 inhibits it. Our data also revealed that diminished β-arrestin-1 and -2 expression in the GT1-7 GnRH hypothalamic neuronal cell line triggered distinct patterns of gene expression following Kp-10 treatment. Thus, β-arrestin-1 and -2 also regulate distinct downstream responses in gene expression. Finally, we showed that GPR54, when uncoupled from the G(q/11) pathway, as is the case for several naturally occurring GPR54 mutants associated with hypogonadotropic hypogonadism, continues to regulate gene expression in a G protein-independent manner. These new and exciting findings add significantly to our mechanistic understanding of how this important receptor signals intracellularly in response to kisspeptin stimulation.     

TNFα Signaling Regulates Cystic Epithelial Cell Proliferation through Akt/mTOR and ERK/MAPK/Cdk2 Mediated Id2 Signaling

Tumor necrosis factor alpha (TNFα) is present in cyst fluid and promotes cyst growth in autosomal dominant polycystic kidney disease (ADPKD). However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive. In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation. The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation. TNFα levels increase in cystic kidneys in response to macrophage infiltration and thus might contribute to cyst growth and enlargement during the progression of disease. As such, this study elucidates a novel mechanism for TNFα signaling in regulating cystic renal epithelial cell proliferation in ADPKD.     

Beta-tricalcium phosphate exerts osteoconductivity through α2β1 integrin and down-stream MAPK/ERK signaling pathway

Beta-tricalcium phosphate (β-TCP) has been clinically used as a bone graft substitute for decades because of its excellent osteoconductivity. However, the exact mechanism(s) by which β-TCP exerts osteoconductivity are not fully documented. This study was aimed to investigate the molecular mechanism(s) by which β-TCP modulates the biological response of primary human osteoblasts (HOBs). It was showed that HOBs seeded into the β-TCP scaffolds expressed significantly higher levels of osteogenic genes, compared to those cultured on tissue culture plastic; meanwhile these cells showed 7-fold increase in α2 integrin subunit gene expression and the activation of the mitogen-activated protein kinase (MAPK)/extracellular related kinase (ERK) signaling pathway. In addition, the osteogenic conduction by β-TCP scaffolds was attenuated directly by inhibiting MAPK/ERK or indirectly by blocking the α2β1 integrin signaling pathway. We concluded that β-TCP scaffold exerts osteoconductivity through α2β1 integrin and down-stream MAPK/ERK signaling pathway, suggesting a feasible approach to consider when designing or fabricating the scaffolds for bone tissue engineering.     

P53 modulates hepatic insulin sensitivity through NF-κB and p38/ERK MAPK pathways

Besides its well-established oncosuppressor activity, the role of p53 in regulating metabolic pathways has been recently identified. Nevertheless, the function of p53 with respect to insulin resistance appears highly controversial. To address this issue, we investigated the expression of p53 in experimental model of insulin resistance. Then we used activator (nutlin-3α) and inhibitor (pifithrin-α, PFT-α) of p53 in HepG2 cell. Here we showed that p53 protein level was decreased in the hepatic tissue of high-fat diet-induced insulin resistance mice, genetically diabetic ob/ob mice and palmitate (PA) treated HepG2 cells. And high expression of phosphor-p38, ERK1/2 and nuclear factor kappa B (NF-κB) p65 accompanied with low expression of p53. But activation of p53 with nutlin-3α prevented PA-induced reduction of glucose consumption and suppression of insulin signaling pathways. At the same time, nutlin-3α downregulated the activation of NF-κB, p38 and ERK1/2 pathways upon stimulation with PA. In contrast, inhibition of p53 with PFT-α decreased glucose consumption and suppressed insulin signaling pathway. Furthermore, PFT-α activated NF-κB, p38 and ERK1/2 pathways in HepG2 cells. Overall, these results suggest that p53 is involved in improving insulin sensitivity of hepatic cells via inhibition of mitogen-activated protein kinases (MAPKs) and NF-κB pathways.     

SDF-1α inhibits hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells through PI3K/Akt and ERK1/2 signaling pathways

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated to be a promising cell sources for cardiac regeneration. Poor survival rate of transplanted BMSCs in infarcted myocardium attenuated its clinical application. It’s reported that stromal-derived factor-1 (SDF-1) could protect progenitor cells including endothelial progenitor cells and embryonic stem cells from apoptosis. But little is known whether SDF-1α protein has the same protective effects on BMSCs under conditions of hypoxia and serum deprivation (hypoxia/SD). In present study, we verified that SDF-1α (0.50–2.0 μg/ml) inhibited hypoxia/SD induced apoptosis of BMSCs through mitochondrial pathway. After administration of SDF-1α, the loss of mitochondrial membrane potential and cytochrome c released from mitochondria to cytosol were significantly inhibited, and caspase 3 activity also declined. Furthermore, the effect of SDF-1α on mitochondrial pathway was neutralized by using PI3K inhibitor (Wortmannin) and ERK1/2 inhibitor (U0126). Our observations suggested that SDF-1α inhibits hypoxia/SD induced BMSCs apoptosis through PI3K/Akt and ERK1/2 signaling pathways. These data also imply that the anti-apoptotic effect mediated by SDF-1α may enhance cell survival after cell transplantation.     

MAPK/ERK and Wnt/β-Catenin pathways are synergistically involved in proliferation of Sca-1 positive hepatic progenitor cells

Hepatic progenitor cells (HPCs) persist in adulthood and have the potential to play a major role in regenerating diseased liver. However, the signaling pathways that both directly and indirectly regulate HPCs’ self-renewal and differentiation remain elusive. Previously, we identified a bipotent, stem cell antigen-1 (Sca-1) positive HPC population from naïve adult liver tissue. In the present study, we aimed to investigate the involvement of various signaling pathways in Sca-1⁺ HPC proliferation. Epidermal growth factor (EGF) supplementation shows a significant increase in Sca-1⁺ HPC proliferation and colony formation while stimulating phosphorylation of ERK1/2 and activating the induction of Cyclin D1. There were no demonstrable effects of EGF on Akt. The MEK inhibitor, PD0325901, inhibits proliferation and ERK1/2 phosphorylation while also suppressing the expression of Cyclin D1. In addition, activation of either IL-6/STAT3 or Wnt/β-Catenin pathway did not independently support cell proliferation and colony formation of HPCs. The Wnt/β-Catenin pathway can cooperate with EGF to significantly promote HPC colony formation ratio and maintain long-term HPC in vitro. The data indicates that the MAPK/ERK pathway is both essential and critical for HPC proliferation, and the Wnt signaling pathway is not sufficient, while it works synergistically with the MAPK/ERK signaling pathway to promote HPC proliferation.     

MiR-21 induced angiogenesis through AKT and ERK activation and HIF-1α expression

MicroRNAs (miRNAs) are endogenous, small noncoding RNAs that play important roles in various cellular functions and tumor development. Recent studies have indicated that miR-21 is one of the important miRNAs associated with tumor growth and metastasis, but the role and molecular mechanism of miR-21 in regulating tumor angiogenesis remain to be elucidated. In this study, miR-21 was overexpressed by transfecting pre-miR-21 into human prostate cancer cells and tumor angiogenesis was assayed using chicken chorioallantoic membrane (CAM). We found that overexpression of miR-21 in DU145 cells increased the expression of HIF-1α and VEGF, and induced tumor angiogenesis. AKT and extracellular regulated kinases (ERK) 1/2 are activated by miR-21. Inhibition of miR-21 by the antigomir blocked this process. Overexpression of the miR-21 target, PTEN, also inhibited tumor angiogenesis by partially inactivating AKT and ERK and decreasing the expression of HIF-1 and VEGF. The AKT and ERK inhibitors, LY294002 and U0126, suppressed HIF-1α and VEGF expression and angiogenesis. Moreover, inhibition of HIF-1α expression alone abolished miR-21-inducing tumor angiogenesis, indicating that HIF-1α is required for miR-21-upregulated angiogenesis. Therefore, we demonstrate that miR-21 induces tumor angiogenesis through targeting PTEN, leading to activate AKT and ERK1/2 signaling pathways, and thereby enhancing HIF-1α and VEGF expression; HIF-1α is a key downstream target of miR-21 in regulating tumor angiogenesis.