Cyclosporin A通过MEK/ERK1/2信号通路调节滋养细胞titin表达

探讨MEK/ERK1/2信号通路在Cyclosporin A(CsA)诱导滋养细胞表达titin中的作用。应用RT-PCR、Western blot检测CsA诱导的滋养细胞titin的表达水平,Western blot检测CsA作用于滋养细胞后ERK1/2的活化程度,并观察MEK特异性抑制剂U0126对其mRNA转录的影响。发现CsA以时间和剂量依赖方式诱导titin表达,并刺激滋养细胞ERK1/2的活化,U0126以剂量依赖方式抑制CsA诱导的titin表达。结果表明CsA通过活化MEK/ERK1/2信号通路诱导滋养细胞titin 的表达,改变其生物学行为,从而有利于胚胎着床及早期发育。     

Ganglioside GD1a regulation of caveolin-1 and Stim1 expression in mouse FBJ cells:Augmented expression of caveolin-1 and Stim1 in cells with increased GD1a content

GD1a was previously shown responsible for regulating cell motility, cellular adhesiveness to vitronectin, phosphorylation of c-Met and metastatic ability of mouse FBJ osteosarcoma cells. To determine the particular molecules regulated by GD1a, FBJ cells were assessed for tumor-related gene expression by semi-quantitative RT-PCR. Caveolin-1 and stromal interaction molecule 1 (Stim1) expression in FBJ-S1 cells, rich in GD1a, were found to be 6 and 4 times as much, respectively, than in FBJ-LL cells devoid of GD1a. Enhanced production of caveolin-1 in protein was confirmed by Western blotting. A low-metastatic FBJ-LL cell variant, having high GD1a expression through β1-4GalNAcT-1 (GM2/GD2 synthase) cDNA transfection (Hyuga S, et al, Int J Cancer 83: 685-91, 1999), showed enhanced production of caveolin-1 and Stim1 in mRNA and protein, compared to mock-transfectant M5. Incubation of FBJ-M5 cells with exogenous GD1a augmented the expression of caveolin-1 in mRNA and protein and Stim1 in mRNA as well. Treatment of FBJ-S1 with fumonisin B1, an inhibitor of N-acylsphinganine synthesis, for 15 days caused the complete depletion of gangliosides and suppressed the expression of caveolin-1 and Stim1. St3gal5 siRNA transfected cells showed decreased expression of caveolin-1 and Stim1 mRNA, as well as St3gal5 mRNA. These findings clearly indicate ganglioside GD1a to be involved in the regulation of the transformation suppressor genes, caveolin-1 and Stim1. Moreover, treatment with GD1a of mouse melanoma B16 cells and human hepatoma HepG2 cells brought about elevated expression of caveolin-1 and Stim1. Li Wang and Shizuka Takaku are equal contributors to the present work     

Glycogen synthase kinase-3beta is tyrosine-phosphorylated by MEK1 in human skin fibroblasts

Glycogen synthase kinase-3beta (GSK-3beta) can be associated with several proteins in cell. We analyzed the immunoprecipitates by an anti-GSK-3beta antibody from cell lysate of human fibroblasts and found that this protein was co-precipitated with mitogen-activated protein kinase kinase (MEK1/2). U0126, a MEK1/2 inhibitor, inhibited tyrosine phosphorylation of GSK-3beta, suggesting that MEK1/2 was involved in the phosphorylation of Tyr(216) in GSK-3beta. In vitro kinase assay was carried out using a recombinant human active MEK1 and we found that GSK-3beta was phosphorylated on Tyr(216) by this kinase in a dose- and time-dependent manner. Further, the pretreatment of fibroblasts with U0126 inhibited serum-induced nuclear translocation of GSK-3beta. These results suggested that MEK1/2 induces tyrosine phosphorylation of GSK-3beta and this cellular event might induce nuclear translocation of GSK-3beta. This is the first report to suggest that MEK1/2 phosphorylates not only ERK1/2 but also GSK-3beta.     

细胞MEK1/2蛋白及其相关通路在单纯疱疹病毒Ⅱ型（HSV-2）复制中的作用

基于细胞Raf/MEK/ERK信号通路与病毒复制的关系,应用Western印迹检测 p-ERK1/2蛋白的表达、用终点滴定法测定病毒增殖量（TCID_５０）,以及观察感染细胞的细胞病变效应（CPE）等,揭示单纯疱疹病毒Ⅱ型(HSV-2)复制与 ERK通路的关系. 结果表明,HSV-2的复制可引起细胞ERK通路的活化;用U0126预先抑制ERK通路的活化,或用特异性siRNA敲减MEK1/2基因的表达可显著地抑制病毒复制.提示ERK信号通路以及MEK1/2蛋白对HSV-2的复制具有重要的作用.该研究对进一步阐明细胞ERK通路各激酶蛋白在病毒复制中的作用机制、寻找抗病毒作用靶标等奠定了良好的基础.     

Angiotensin II induces tyrosine nitration and activation of ERK1/2 in vascular smooth muscle cells

Angiotensin II (Ang II) induces a prominent and sustained nitration and activation of ERK1/2 in rat vascular smooth muscle cells, both mediated via AT1 receptor. Nitration and activation was also shown for recombinant non-activated extracellular signal-regulated kinase (ERK) and MEK. Nitration and phosphorylation of ERK1/2 by Ang II was significantly inhibited by NAD(P)H inhibitors and scavengers of oxygen and nitrogen reactive species and completely blocked by a selective inducible nitric-oxide synthase inhibitor. MEK inhibitor U0126 did not affect ERK nitration but completely blocked activation. These data indicate that Ang II nitrates and activates ERK1/2 via a reactive species-sensitive pathway.     

Cyclosporin A通过MEK／ERK1／2信号通路调节滋养细胞titin表达

探讨MEK／ERK1／2信号通路在CyclosporinA(CsA)诱导滋养细胞表达titin中的作用。应用RT—PCR、Western blot检测CsA诱导的滋养细胞titin的表达水平,Western blot检测CsA作用于滋养细胞后ERK1／2的活化程度,并观察MEK特异性抑制剂U0126对其mRNA转录的影响。发现CsA以时间和剂量依赖方式诱导titin表达,并刺激滋养细胞ERK1／2的活化,U0126以剂量依赖方式抑制CsA诱导的titin表达。结果表明CsA通过活化MEK／ERK1／2信号通路诱导滋养细胞titin的表达,改变其生物学行为,从而有利于胚胎着床及早期发育。     

Sphingosine 1-phosphate induces EGFR expression via Akt/NF-kappaB and ERK/AP-1 pathways in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) has been shown to regulate expression of several genes in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating epidermal growth factor receptor (EGFR) expression by S1P in aortic VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced EGFR mRNA and protein expression in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and phosphatidylinositide 3-kinase (PI3K; wortmannin), and transfection with dominant negative mutants of ERK and Akt, respectively. These results suggested that S1P-induced EGFR expression was mediated through p42/p44 MAPK and PI3K/Akt pathways in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt which was attenuated by U0126 and wortmannin, respectively. Furthermore, S1P-induced EGFR upregulation was blocked by a selective NF-kappaB inhibitor helenalin. Immunofluorescent staining and reporter gene assay revealed that S1P-induced activation of NF-kappaB was blocked by wortmannin, but not by U0126, suggesting that activation of NF-kappaB was mediated through PI3K/Akt. Moreover, S1P-induced EGFR expression was inhibited by an AP-1 inhibitor curcumin and tanshinone IIA. S1P-stimulated AP-1 subunits (c-Jun and c-Fos mRNA) expression was attenuated by U0126 and wortmannin, suggesting that MEK and PI3K/ERK cascade linking to AP-1 was involved in EGFR expression. Upregulation of EGFR by S1P may exert a phenotype modulation of VSMCs. This hypothesis was supported by pretreatment with AG1478 or transfection with shRNA of EGFR that attenuated EGF-stimulated proliferation of VSMCs pretreated with S1P, determined by XTT assay. These results demonstrated that in VSMCs, activation of Akt/NF-kappaB and ERK/AP-1 pathways independently regulated S1P-induced EGFR expression in VSMCs. Understanding the mechanisms involved in S1P-induced EGFR expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.     

Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor gamma (PPARgamma ) and C/EBPalpha gene expression during the differentiation of 3T3-L1 preadipocytes

We demonstrate that exposure of post-confluent 3T3-L1 preadipocytes to insulin, isobutylmethylxanthine (MIX), dexamethasone (DEX), and fetal bovine serum induces a rapid but transient activation of MEK1 as indicated by extensive phosphorylation of ERK1 and ERK2 during the initial 2 h of adipogenesis. Inhibition of this activity by treating the cells with a MEK1-specific inhibitor (U0126 or PD98059) prior to the induction of differentiation significantly attenuated the expression of peroxisome proliferator-activated receptor (PPAR) gamma, CCAAT/enhancer-binding protein (C/EBP) alpha, perilipin, and adipocyte-specific fatty acid-binding protein (aP2). Treating the preadipocytes with troglitazone, a potent PPARgamma ligand, could circumvent the inhibition of adipogenic gene expression by U0126. Fibroblast growth factor-2 (FGF-2), in the presence of dexamethasone, isobutylmethylxanthine, and insulin, induces a prolonged activation of the MEK/ERK signaling pathway, which lasts for at least 12 h post-induction, and this activity is less sensitive to the MEK inhibitors. Consequently, preadipocytes treated with U0126 in the presence of fibroblast growth factor-2 (FGF-2) express normal post-induction levels of MEK activity, and, in so doing, are capable of undergoing adipogenesis. We further show that activation of MEK1 significantly enhances the transactivation of the C/EBPalpha minimal promoter during the early phase of the differentiation process. Our results suggest that activation of the MEK/ERK signaling pathway during the initial 12 h of adipogenesis enhances the activity of factors that regulate both C/EBPalpha and PPARgamma expression.     

GD3 synthase gene expression in PC12 cells results in the continuous activation of TrkA and ERK1/2 and enhanced proliferation

A rat pheochromocytoma cell line (PC12) transfected with ganglioside GD3 synthase gene showed a marked change in the ganglioside profile and enhanced proliferation and no response of neurite extension to nerve growth factor (NGF) stimulation. In these transfectant cells, a continuous phosphorylation of TrkA and the activation of ERK1/2 without NGF treatment were observed. Proliferation inhibition experiments with kinase inhibitors such as herbimycin A, K-252a, and PD98059 revealed that the enhanced proliferation was actually due to the activation of the Ras/MEK/ERK pathway. A TrkA dimer was detected in the GD3 synthase transfectant cells regardless of NGF treatment by cross-linking and immunoblotting. The increased expression of GD1b and GT1b in these transfectant cells might induce the conformational change of TrkA to form a dimer and to be activated continuously. These results may indicate regulatory roles of gangliosides in cell proliferation under physiological and malignant processes.     

CCN2 expression and localization in melanoma cells

The matricellular protein connective tissue growth factor (CTGF, CCN2) is overexpressed in several forms of cancer and may represent a novel target in anti-cancer therapy. However, whether CCN2 is expressed in melanoma cells is unknown. The highly metastatic murine melanoma cell line B16(F10) was used for our studies. Real time polymerase chain reaction analysis was used to detect mRNA expression of CCN1, CCN2, CCN3 and CCN4 in Western blot and immunofluorescence analyses were used to detect CCN2 protein. Inhibitors of signal transduction cascades were used to probe the mechanism underlying CCN2 expression in B16(F10) cells. CCN2 was expressed in B16(F10) cells, and was reduced by the FAK/src inhibitor PP2 and the MEK/ERK inhibitor U0126 indicating that CCN2 acts downstream of these pathways in B16(F10) murine melanoma cells. Expression of CCN1, CCN3 and CCN4 was not reduced by PP2 or U0126; in fact, expression of CCN4 mRNA was elevated by PP2 or U0126 treatment. To our surprise, CCN2 protein was detected in the nuclei of B16(F10) cells, and was undetectable in the cytoplasm. CCN2 was expressed in B16(F10) melanoma cells, adding to the list of cancer cells in which CCN2 is expressed. Of the CCN family members tested, only CCN2 is downstream of the highly oncogenic MEK/ERK pathway. CCN2 should be further evaluated for a possible role in melanoma growth and progression.     

IL-10 synergistically enhances GM-CSF-induced CCR1 expression in myelomonocytic cells

CC chemokine receptor 1 (CCR1) has been implicated in inflammation. The present study examined the signaling mechanisms that mediate GM-CSF/IL-10-induced synergistic CCR1 protein expression in monocytic U937 cells. GM-CSF alone markedly increased both the mRNA and protein expression of CCR1. IL-10 augmented GM-CSF-induced CCR1 protein expression with no effect on mRNA expression. PD098059 and U0126 (two MEK inhibitors), and LY294002 (a PI3K inhibitor) inhibited GM-CSF/IL-10-induced CCR1 gene and protein expression. PD098059, U0126, and LY294002 also attenuated chemotaxis of GM-CSF/IL-10-primed U937 cells in response to MIP-1alpha. Immunoblotting studies show that GM-CSF alone induced ERK2 phosphorylation; whereas, IL-10 alone induced p70(S6k) phosphorylation in U937 cells. Neither cytokine when used alone induced PKB/Akt phosphorylation. Combined GM-CSF/IL-10 treatment of U937 cells induced phosphorylation of ERK2, p70(S6k), and PKB/Akt. PD098059 and U0126 completely abrogated ERK2 phosphorylation; whereas, LY294002 completely blocked PKB/Akt and p70(S6k) phosphorylation. Our findings indicate that IL-10 may potentiate GM-CSF-induced CCR1 protein expression in U937 cells via activation of PKB/Akt and p70(S6k).     

Analysis of CCN2 promoter activity in PANC-1 cells: regulation by ras/MEK/ERK

Connective tissue growth factor (CTGF, CCN2) is overexpressed in pancreatic cancer. We mapped the minimal CCN2 promoter active in PANC-1 cells, a human pancreatic cancer cell line. Within this region, Sp1, BCE-1 and Ets elements were important for the activity of the CCN2 promoter. Constitutive hyperactivated ras is a hallmark of cancers, including that of the pancreas. Treatment of PANC-1 cells with the MEK inhibitor U0126 or the Sp1 inhibitor mithramycin reduced CCN2 mRNA and promoter activity. Mutation of the BCE-1, but not Sp1 or Ets, site abolished the responsiveness of the CCN2 promoter to U0126. Overexpressing constitutively active MEK1 or ras activated CCN2 promoter activity. Thus CCN2 is likely to act downstream of ras in PANC-1 cells. CCN2 is overexpressed in cancer cells. Activated ras/MEK/ERK is a hallmark of cancer, and we have shown that the elevated CCN2 expression in pancreatic cancer cells is dependent on this pathway.     

SPARCL1通过MEK/ERK信号通路调节非小细胞肺癌细胞的增殖、凋亡和侵袭

摘要 目的：分析富含半胱氨酸的酸性分泌蛋白类似蛋白1（SPARCL1）对非小细胞肺癌（NSCLC）细胞增殖、凋亡、侵袭的影响,并探讨分裂原活化抑制剂（MEK）/细胞外调节蛋白激酶（ERK）通路在其中发挥的作用。方法：收集2019年9月~2021年6月期间本院接受手术治疗的84例NSCLC患者癌组织与相应癌旁组织,实时定量逆转录聚合酶链反应（qRT-PCR）法测定并比较各组织以及正常肺上皮细胞HBEpiC、NSCLC细胞A549、HCC827、H1299、H292中SPARCL1 信使RNA（mRNA）表达水平,选取A549、HCC827培养并分组,分为对照组、NC siRNA组、SPARCL1 siRNA组、U0126组（MEK/ERK特异性抑制剂）、SPARCL1 siRNA加U0126组,细胞计数法（CCK8）以及平板克隆法测定A549、HCC827细胞增殖,流式细胞仪测定A549、HCC827细胞凋亡,Transwell小室法测定A549、HCC827细胞侵袭能力,蛋白质印迹法（western blot）检测SPARCL1、p-MEK、MEK、p-ERK1/2、ERK1/2蛋白表达。结果：SPARCL1在NSCLC组织中mRNA表达水平低于癌旁组织（P＜0.05）;与HBEpiC细胞相比,NSCLC细胞A549、HCC827、H1299、H292细胞中SPARCL1 mRNA表达水平降低（P＜0.05）;与对照组相比,SPARCL1 siRNA组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率降低（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达升高（P＜0.05）,U0126组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率升高（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达降低（P＜0.05）;与SPARCL1 siRNA组相比,SPARCL1 siRNA加U0126组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率升高（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达降低（P＜0.05）。结论：SPARCL1可能通过调控MEK/ERK通路影响NSCLC A549、HCC827细胞增殖、侵袭与凋亡。     

p21(Cip-1/SDI-1/WAF-1) expression via the mitogen-activated protein kinase signaling pathway in insulin-induced chondrogenic differentiation of ATDC5 cells

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21(Cip-1/SDI-1/WAF-1), is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression.     

Involvement of integrin up‐regulation in RANKL/RANK pathway of chondrosarcomas migration

Invasion of tumor cells is the primary cause of therapeutic failure in malignant chondrosarcomas treatment. Receptor activator of nuclear factor‐κB ligand (RANKL) and its receptor, RANK, play a key roles in osteoclastogenesis and tumor metastasis. We found that the RANKL and RANK expression in human chondrosarcoma tissues was higher than that in normal cartilage. We also found that RANKL directed the migration and increased cell surface expression of β1 integrin in human chondrosarcoma cells (JJ012 cells). Pretreatment of JJ012 cells with MAPK kinase (MEK) inhibitors, PD98059 or U0126, inhibited the RANKL‐induced migration and integrin expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited RANKL‐induced cells migration and integrin up‐regulation. Taken together, these results suggest that the RANKL acts through MEK/ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activation of β1 integrin and contributing to the migration of human chondrosarcoma cells. J. Cell. Biochem. 111: 138–147, 2010. © 2010 Wiley‐Liss, Inc.     

Involvement of p42/p44 MAPK, JNK, and NF-kappaB in IL-1beta-induced ICAM-1 expression in human pulmonary epithelial cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and contributes to inflammatory responses. However, the mechanisms regulating ICAM-1 expression by IL-1beta in human A549 cells was not completely understood. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB pathways for IL-1beta-induced ICAM-1 expression were investigated in A549 cells. IL-1beta induced expression of ICAM-1 protein and mRNA in a time- and concentration-dependent manner. The IL-1beta induction of ICAM-1 mRNA and protein were partially inhibited by U0126 and PD98059 (specific inhibitors of MEK1/2) and SP600125 [a specific inhibitor of c-Jun-N-terminal kinase (JNK)]. U0126 was more potent than other inhibitors to attenuate IL-1beta-induced ICAM-1 expression. Consistently, IL-1beta stimulated phosphorylation of p42/p44 MAPK and JNK which was attenuated by pretreatment with U0126 or SP600125, respectively. Moreover, transfection with dominant negative mutants of MEK1/2 (MEK K97R) or ERK2 (ERK2 K52R) also attenuated IL-1beta-induced ICAM-1 expression. The combination of PD98059 and SP600125 displayed an additive effect on IL-1beta-induced ICAM-1 gene expression. IL-1beta-induced ICAM-1 expression was almost completely blocked by a specific NF-kappaB inhibitor helenalin. Consistently, IL-1beta stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha which was blocked by helenalin, U0126, or SP600125. Taken together, these results suggest that activation of p42/p44 MAPK and JNK cascades, at least in part, mediated through NF-kappaB pathway is essential for IL-1beta-induced ICAM-1 gene expression in A549 cells. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in the airway disease.     

MicroRNA-181a regulates the activation of the NLRP3 inflammatory pathway by targeting MEK1 in THP-1 macrophages stimulated by ox-LDL

Atherosclerosis (AS) is a chronic inflammatory disease that is characterized by the deposition of lipids in the vascular wall and the formation of foam cells. Macrophages play a critical role in the development of this chronic inflammation. An increasing amount of research shows that microRNAs affect many steps of inflammation. The goal of our study was to investigate the regulatory effect of miR-181a on the NLRP3 inflammasome pathway and explore its possible mechanism. Compared with the control group, the expression of miR-181a was downregulated in the carotid tissue of AS group mice, while the expression of MEK1 and NLRP3-related proteins was upregulated significantly. In vitro, when THP-1 macrophages were stimulated with oxidized low-density lipoprotein (ox-LDL), the expression of miR-181a was decreased, the MEK/ERK/NF-κB inflammatory pathways were activated and the expression of NLRP3 inflammasome-related proteins was upregulated. Exogenous overexpression of miR-181a downregulated the activation of the MEK/ERK/NF-κB pathway and decreased the expression of NLRP3 inflammasome-related proteins (such as NLRP3, caspase-1, interleukin-18 [IL-18], IL-1β, etc). Exogenous miR-181a knockdown showed the opposite results to those of overexpression group. A luciferase reporter assay proved that miR-181a inhibited the expression of MEK1 by binding to its 3′-untranslated region. When we knocked down miR-181a and then treated cells with U0126 before ox-LDL stimulation, we found that U0126 reversed the increased activation of the MEK/ERK/NF-κB pathway and upregulation of NLRP3 inflammasome-related proteins (NLRP3, caspase-1, IL-18, IL-1β) that resulted from miR-181a knockdown. Our study suggests that miR-181a regulates the activation of the NLRP3 inflammatory pathway by altering the activity of the MEK/ERK/NF-κB pathway via targeting of MEK1.     

Up‐regulation of TRPM6 transcriptional activity by AP‐1 in renal epithelial cells

Transient receptor potential melastatin 6 (TRPM6) channel is involved in the reabsorption of magnesium in the kidney. We recently found that TRPM6 expression is up‐regulated by EGF, but the regulatory mechanism has not been clear. TRPM6 mRNA was endogenously expressed in HEK293 cells. TRPM6 mRNA expression was increased by EGF, which was inhibited by U0126, an MEK inhibitor. Promoter activity of human TRPM6 was observed in the TRPM6 5′‐flanking region from ?1,214 to ?718. This promoter activity was enhanced by EGF and inhibited by U0126. Three putative AP‐1 binding sites were identified within the region of ?1,214/?718. The mutation of the putative AP‐1 binding site (?741/?736) completely inhibited the EGF‐induced promoter activity. EGF increased p‐ERK1/2, c‐Fos, c‐Jun, and p‐c‐Jun levels, which were inhibited by U0126. The introduction of c‐Fos or c‐Jun siRNA inhibited the EGF‐induced promoter activity. A chromatin immunoprecipitation assay revealed that c‐Fos and c‐Jun bind to the AP‐1 binding site within the region of ?1,214/?718. These results suggest that EGF up‐regulates TRPM6 mRNA expression mediate via the activation of ERK/AP‐1‐dependent pathway. J. Cell. Physiol. 222: 481–487, 2010. © 2009 Wiley‐Liss, Inc.