MEK/ERK信号通路与脑缺血再灌注损伤

在脑缺血病灶中,中心区神经元坏死为主,周围以缺血半暗带凋亡为主,抑制半暗带细胞的凋亡,可以减少细胞的死亡和脑梗死的面积,因此改善半暗带是治疗脑卒中的关键环节.目前发现MAPK分布于整个中枢神经系统中,MEK/ERK信号通路参与细胞生长、发育、细胞抗凋亡等过程,在脑缺血再灌注损伤过程中有MEK/ERK信号通路的参与,MEK/ERK通路通过影响Bcl-2家族成员的活化和表达调控内源性凋亡途径,ERK通过对细胞周期的调控,抑制胶质细胞大量活化和过度增殖,减少了有害因子并改善局部微循环,从而减少神经元的凋亡.可能为脑血管的防治开辟一条新的途径.本文就MEK/ERK信号通路的结构特点与脑缺血再灌注损伤相关作用机制作一综述.     

Targeting Cadherin-17 Inactivates Ras/Raf/MEK/ERK Signaling and Inhibits Cell Proliferation in Gastric Cancer

Cadherin-17 (CDH17), one member of 7D-cadherin superfamily, was overexpressed in gastric cancer (GC) and was associated with poor survival, tumor recurrence, metastasis, and advanced tumor stage. So far the cellular function and signaling mechanism of CDH17 in GC remains unclear. In this study, we showed that over 66% of GC cell lines (20/30) were CDH17 positive. Tissue microarray (TMA) assay showed that 73.6% Chinese GC tissues (159/216) were CDH17 positive, while 37% respective adjacent normal tissues were CDH17 positive. Knockdown of CDH17 inhibited cell proliferation, migration, adhesion and colony formation, and also induced a cell cycle arrest and apoptosis in AGS human GC cells. On the other side, overexpression of CDH17 facilitated MGC-803 GC tumor growth in nude mice. Antibody array and Western blotting assay demonstrated that knockdown of CDH17 in AGS cells down-regulated integrin β series proteins, further inactivated the Ras/Raf/MEK/ERK pathway and led to p53 and p21 accumulation, which resulted in proliferation inhibition, cell-cycle arrest and apoptosis induction. Collectively, our data firstly demonstrate the capacity of CDH17 to regulate the activity of Ras/Raf/MEK/ERK pathway for cell proliferation in GC, and suggest that CDH17 can serve as an attractive therapeutic target for future research.     

Interstitial Fluid Flow Increases Hepatocellular Carcinoma Cell Invasion through CXCR4/CXCL12 and MEK/ERK Signaling

Hepatocellular carcinoma (HCC) is the most common form of liver cancer (~80%), and it is one of the few cancer types with rising incidence in the United States. This highly invasive cancer is very difficult to detect until its later stages, resulting in limited treatment options and low survival rates. There is a dearth of knowledge regarding the mechanisms associated with the effects of biomechanical forces such as interstitial fluid flow (IFF) on hepatocellular carcinoma invasion. We hypothesized that interstitial fluid flow enhanced hepatocellular carcinoma cell invasion through chemokine-mediated autologous chemotaxis. Utilizing a 3D in vitro invasion assay, we demonstrated that interstitial fluid flow promoted invasion of hepatocellular carcinoma derived cell lines. Furthermore, we showed that autologous chemotaxis influences this interstitial fluid flow-induced invasion of hepatocellular carcinoma derived cell lines via the C-X-C chemokine receptor type 4 (CXCR4)/C-X-C motif chemokine 12 (CXCL12) signaling axis. We also demonstrated that mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling affects interstitial fluid flow-induced invasion; however, this pathway was separate from CXCR4/CXCL12 signaling. This study demonstrates, for the first time, the potential role of interstitial fluid flow in hepatocellular carcinoma invasion. Uncovering the mechanisms that control hepatocellular carcinoma invasion will aid in enhancing current liver cancer therapies and provide better treatment options for patients.     

Maspin Regulates Endothelial Cell Adhesion and Migration through an Integrin Signaling Pathway

Maspin has been identified as a potent angiogenesis inhibitor. However, the molecular mechanism responsible for its anti-angiogenic property is unclear. In this study, we examined the effect of maspin on endothelial cell (EC) adhesion and migration in a cell culture system. We found that maspin was expressed in blood vessels ECs and human umbilical vein endothelial cells (HUVECs). Maspin significantly enhanced HUVEC cell adhesion to various matrix proteins. This effect was dependent on the activation of integrin β₁, which subsequently led to distribution pattern changes of vinculin and F-actin. These results indicated that maspin affects cell adhesion and cytoskeleton reorganization through an integrin signal transduction pathway. Analysis of HUVECs following maspin treatment revealed increased integrin-linked kinase activities and phosphorylated FAK levels, consistent with increased cell adhesion. Interestingly, when HUVECs were induced to migrate by migration stimulatory factor bFGF, active Rac1 and cdc42 small GTPase levels were decreased dramatically at 30 min following maspin treatment. Using phosphorylated FAK at Tyr³⁹⁷ as an indicator of focal adhesion disassembly, maspin-treated HUVECs had elevated FAK phosphorylation compared with the mock treated control. The results were a reduction in focal adhesion disassembly and the retardation in EC migration. This study uncovers a mechanism by which maspin exerts its effect on EC adhesion and migration through an integrin signal transduction pathway.     

MEK5/ERK5 pathway: The first fifteen years

While conventional MAP kinase pathways are one of the most highly studied signal transduction molecules, less is known about the MEK5 signaling pathway. This pathway has been shown to play a role in normal cell growth cycles, survival and differentiation. The MEK5 pathway is also believed to mediate the effects of a number of oncogenes. MEK5 is the upstream activator of ERK5 in many epithelial cells. Activation of the MEK-MAPK pathway is a frequent event in malignant tumor formation and contributes to chemoresistance and anti-apoptotic signaling. This pathway may be involved in a number of more aggressive, metastatic varieties of cancer due to its role in cell survival, proliferation and EMT transitioning. Further study of this pathway may lead to new prognostic factors and new drug targets to combat more aggressive forms of cancer.     

A Non-canonical MEK/ERK Signaling Pathway Regulates Autophagy via Regulating Beclin 1

Autophagy-essential proteins are the molecular basis of protective or destructive autophagy machinery. However, little is known about the signaling mechanisms governing these proteins and the opposing consequences of autophagy in mammals. Here we report that a non-canonical MEK/ERK module, which is positioned downstream of AMP-activated protein kinase (AMPK) and upstream of tuberous sclerosis complex (TSC), regulates autophagy by regulating Beclin 1. Depletion of ERK partially inhibited autophagy, whereas specific inhibition on MEK completely inhibited autophagy. MEK could bypass ERK to promote autophagy. Basal MEK/ERK activity conferred basal Beclin 1 by preventing disassembly of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2. Activation of MEK/ERK by AMPK upon autophagy stimuli disassembled mTORC1 via binding to and activating TSC but disassembled mTORC2 independently of TSC. Inhibition of mTORC1 or mTORC2 by transiently or moderately activated MEK/ERK caused moderately enhanced Beclin 1 resulting in cytoprotective autophagy, whereas inhibition of both mTORC1 and mTORC2 by sustained MEK/ERK activation caused strongly pronounced Beclin 1 leading to cytodestructive autophagy. Our findings thus propose that the AMPK-MEK/ERK-TSC-mTOR pathway regulation of Beclin 1 represents different thresholds responsible for a protective or destructive autophagy.Autophagy is an evolutionally conserved machinery involving the degradation and turnover of cytoplasmic material in lysosomes. Autophagy plays a role in cellular homeostasis (1), antiaging (2–4), development (1, 5), protection of the genome (6), and regulation of cell size (7). Autophagy may act as a means of defense against bacterium and virus invasion and be linked to various diseases including cancer (8–10), cardiomyopathy (11), and neurodegenerative disorders (12).Autophagy starts with the formation of an autophagosome, enclosed within a double membrane that engulfs part of the cytoplasm. During periods of autophagy stimuli, cells respond to either maintain the metabolism essential for survival or execute cell death. Autophagy-essential proteins (Atg)² are the molecular basis of autophagy machinery. About 30 Atg proteins in yeast and 10 in mammals have been identified. In yeast, the protein kinase target of rapamycin (TOR) mediates autophagy via Atg1-Atg13 kinase complex. Atg1 interacts with multiple components of the autophagic machinery through direct association, phosphorylation, and/or intracellular localization (13, 14).In mammalian systems, autophagosomes fuse with lysosomes to generate autophagolysosomes, which undergo a maturation process by fusing with endocytic compartments and lysosomes (15). Because it is not known how the Atg1 homolog acts in mammals, a different mechanism may be involved in regulating autophagy. Beclin 1/Atg6, microtubule-associated protein 1 light chain 3 (LC3)/Atg8, Atg5, Atg12, and Atg13 are essential for autophagosome formation in mammalian species (5, 16–20). Atg7 and Atg3 are required in the conjugation reaction between Atg12 and Atg5 and in the lipidation of LC3. During the formation of autophagosomes in mammalian cells, LC3 is lipidated via a ubiquitylation-like system (17, 21), generating a soluble form, LC3-I. LC3-I is further modified to a membrane-bound form, LC3-II, which is subsequently localized to autophagosomes and autolysosomes until being degraded by the lysosome.Beclin 1 was initially isolated as a B-cell lymphoma-2 (Bcl2)-interacting tumor suppressor in mammalian cells (22). Overexpression of Bcl2 attenuates the formation of the kinase complex Beclin 1-class III phosphatidylinositol 3-kinase (PI3KC3) essential for the formation of autophagosomes (23). The UV radiation resistance-associated gene tumor suppressor and the activating molecule in Beclin 1-regulated autophagy protein 1 (Ambra 1) were identified as new Beclin 1-binding partners that also regulate autophagy by regulating the Beclin 1-PI3KC3 kinase complex. Association of Beclin 1 with PI3KC3 is negatively regulated by Bcl2 (22) and positively regulated by UV radiation resistance-associated gene tumor suppressor and Ambra 1 (24, 25). Beclin 1 is homoallelically deleted in many human tumors. A decreased Beclin 1 level causes defective autophagy and breast cancer, but restoration of Beclin 1 induces autophagy and inhibits tumorigenicity of human breast cancer cells (18). These reports evidence the dependence on Beclin 1 for a functional autophagy mechanism.Diverse signaling pathways have been reported in the regulation of autophagy in mammalian cells (26, 27). In contrast to yeast, mammalian cells regulate autophagy via both class I and class III PI3K. Class I PI3K plays an inhibitory role, whereas class III PI3K kinase complex, which includes Beclin 1, plays a stimulatory role in autophagy by promoting the nucleation of autophagic vesicles (28, 29). A recent study also indicates that hVps15 is required in regulation of class III PI3K in mammalian cells (30). However, the signaling mechanisms controlling autophagy-essential proteins, in particular Beclin 1, and the opposing consequences of autophagy remain to be resolved.Our present studies identified and positioned a non-canonical MEK/ERK pathway downstream of AMPK and upstream of TSC and mTOR. This MEK/ERK module regulated autophagy via regulating the Beclin 1 level through the AMPK-MEK/ERK-TSC-mTOR pathway. Moderately enhanced Beclin 1 by transient or moderate activation of MEK/ERK and subsequent inhibition on mTORC1 and mTORC2 individually caused protective autophagy. Strongly pronounced Beclin 1 by sustained or strong activation of MEK/ERK followed by dual inhibition on mTORC1 and mTORC2 caused destructive autophagy. Our results thus reveal interesting Beclin 1 thresholds in regulating autophagy.     

MEK/ERK信号通路对人结膜上皮细胞增殖的影响及其机制研究

目的:探讨MEK/ERK信号通路对人结膜上皮细胞增殖的影响及其可能的机制。方法:采用不同浓度(0、12.5、25、50、100μmol/L)的MEK抑制剂PD98059处理人结膜上皮细胞(HConEpiC),通过CCK-8法检测不同浓度PD98059作用不同时间(0、12、24、48 h)对人结膜上皮细胞增殖的影响,Western blot检测不同浓度PD98059对人结膜上皮细胞ERK1/2、P-ERK1/2表达的影响。结果:相比对照组(0μmol/L),不同浓度(12.5、25、50、100μmol/L)PD98059处理后的人结膜上皮细胞增殖率明显下降,呈剂量-效应关系,且随处理时间增加(12、24、48 h)其抑制作用也显著增强,差异均有统计学意义(P0.05)。不同浓度PD98059处理人结膜上皮细胞24 h后,其ERK及p-ERK1/2表达随处理浓度增加而降低,与对照组(0μmol/L)相比差异有统计学意义(P0.05),且二者表达量与细胞增值抑制率均呈显著负相关(r=-0.995、r=-0.968,P0.05)。结论:PD98059可抑制人结膜上皮细胞增殖,这可能与其下调ERK表达和减少其活化有关。     

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。     

黏着斑激酶与细胞迁移

细胞迁移过程始于细胞前端板状伪足的形成、外周黏附的建立、细胞体的收缩和尾部的解离.黏着斑激酶是一种非受体酪氨酸蛋白激酶,通过其激酶活性和"脚手架"的功能在细胞迁移的各个过程中发挥关键作用.现重点介绍黏着斑激酶介导的信号转导通路及其在调控细胞迁移方面的研究进展.     

EphB/ephrinB Signaling in Cell Adhesion and Migration

Eph receptors and their ligands, ephrins, represent the largest group of the receptor tyrosine kinase (RTK) family, and they mediate numerous developmental processes in a variety of organisms. Ephrins are membrane-bound proteins that are mainly divided into two classes: A class ephrins, which are linked to the membrane by a glycosylphosphatidylinositol (GPI) linkage, and B class ephrins, which are transmembrane ligands. Based on their domain structures and affinities for ligand binding, the Eph receptors are also divided into two groups. Trans-dimerization of Eph receptors with their membrane-tethered ligands regulates cell-cell interactions and initiates bidirectional signaling pathways. These pathways are intimately involved in regulating cytoskeleton dynamics, cell migration, and alterations in cellular dynamics and shapes. The EphBs and ephrinBs are specifically localized and modified to promote higher-order clustering and initiate of bidirectional signaling. In this review, we present an in-depth overview of the structure, mechanisms, cell signaling, and functions of EphB/ephrinB in cell adhesion and migration.     

Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL), a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF) signaling and angiogenesis in endothelial cells (ECs). We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day) recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis.     

Up-Regulation of Hepatitis C Virus Replication and Production by Inhibition of MEK/ERK Signaling

Background

Viruses interact with and exploit the host cellular machinery for their multiplication and propagation. The MEK/ERK signaling pathway positively regulates replication of many RNA viruses. However, whether and how this signaling pathway affects hepatitis C virus (HCV) replication and production is not well understood.

Methods and Results

In this study, we took advantage of two well-characterized MEK/ERK inhibitors and MEK/ERK dominant negative mutants and investigated the roles of the MEK/ERK signaling pathway in HCV gene expression and replication. We showed that inhibition of MEK/ERK signaling enhanced HCV gene expression, plus- and minus-strand RNA synthesis, and virus production. In addition, we showed that this enhancement was independent of interferon-α (IFN-α) antiviral activity and did not require prior activation of the MEK/ERK signaling pathway. Furthermore, we showed that only MEK and ERK-2 but not ERK-1 was involved in HCV replication, likely through regulation of HCV RNA translation.

Conclusions

Taken together, these results demonstrate a negative regulatory role of the MEK/ERK signaling pathway in HCV replication and suggest a potential risk in targeting this signaling pathway to treat and prevent neoplastic transformation of HCV-infected liver cells.     

微波辐射对PC12细胞Raf/MEK/ERK信号通路相关分子表达的影响

体外培养PC12细胞,将其诱导分化为神经元后,建立微波辐射细胞模型,采用免疫印迹技术和图像分析技术研究微波辐射后Raf/MEK/ERK信号通路相关分子的动态表达变化规律,进一步探讨微波辐射损伤的分子机制。结果发现,微波辐射后6h～3d,假辐射组和辐射组PC12细胞中Raf-1、ERK表达均呈先增加后减少趋势,两组差别不显著,但辐射组Raf-1、ERK和CREB的磷酸化水平均较假辐射组明显升高,表明Raf/MEK/ERK信号通路活化增强可能是微波辐射致神经细胞损伤的重要机制。     

Kinase Suppressor of Ras Forms a Multiprotein Signaling Complex and Modulates MEK Localization

Genetic screens for modifiers of activated Ras phenotypes have identified a novel protein, kinase suppressor of Ras (KSR), which shares significant sequence homology with Raf family protein kinases. Studies using Drosophila melanogaster and Caenorhabditis elegans predict that KSR positively regulates Ras signaling; however, the function of mammalian KSR is not well understood. We show here that two predicted kinase-dead mutants of KSR retain the ability to complement ksr-1 loss-of-function alleles in C. elegans, suggesting that KSR may have physiological, kinase-independent functions. Furthermore, we observe that murine KSR forms a multimolecular signaling complex in human embryonic kidney 293T cells composed of HSP90, HSP70, HSP68, p50(CDC37), MEK1, MEK2, 14-3-3, and several other, unidentified proteins. Treatment of cells with geldanamycin, an inhibitor of HSP90, decreases the half-life of KSR, suggesting that HSPs may serve to stabilize KSR. Both nematode and mammalian KSRs are capable of binding to MEKs, and three-point mutants of KSR, corresponding to C. elegans loss-of-function alleles, are specifically compromised in MEK binding. KSR did not alter MEK activity or activation. However, KSR-MEK binding shifts the apparent molecular mass of MEK from 44 to >700 kDa, and this results in the appearance of MEK in membrane-associated fractions. Together, these results suggest that KSR may act as a scaffolding protein for the Ras-mitogen-activated protein kinase pathway.     

Noncatalytic Function of ERK1/2 Can Promote Raf/MEK/ERK-mediated Growth Arrest Signaling

Kinase activity is known as the key biochemical property of MAPKs. Here, we report that ERK1/2 also utilizes its noncatalytic function to mediate certain signal transductions. Sustained activation of the Raf/MEK/ERK pathway induces growth arrest, accompanied by changes in cell cycle regulators (decreased retinoblastoma phosphorylation, E2F1 down-regulation, and/or p21^CIP1 up-regulation) and cell type-specific changes in morphology and expression of c-Myc or RET in the human tumor lines LNCaP, U251, and TT. Ablation of ERK1/2 by RNA interference abrogated all these effects. However, active site-disabled ERK mutants (ERK1-K71R, ERK2-K52R, and ERK2-D147A), which competitively inhibit activation of endogenous ERK1/2, could not block Raf/MEK-induced growth arrest as well as changes in the cell cycle regulators, although they effectively blocked phosphorylation of the ERK1/2 catalytic activity readouts, p90^RSK and ELK1, as well as the cell type-specific changes. Because this indicated a potential noncatalytic ERK1/2 function, we generated stable lines of the tumor cells in which both ERK1 and ERK2 were significantly knocked down, and we further investigated the possibility using rat-derived kinase-deficient ERK mutants (ERK2-K52R and ERK2-T183A/Y185F) that were not targeted by human small hairpin RNA. Indeed, ERK2-K52R selectively restored Raf-induced growth inhibitory signaling in ERK1/2-depleted cells, as manifested by regained cellular ability to undergo growth arrest and to control the cell cycle regulators without affecting c-Myc and morphology. However, ERK2-T183A/Y185F was less effective, indicating the requirement of TEY site phosphorylation. Our study suggests that functions of ERK1/2 other than its “canonical” kinase activity are also involved in the pathway-mediated growth arrest signaling.