Association of upregulated Ras/Raf/ERK1/2 signaling with autism

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. BTBR mouse is currently used as a model for understanding mechanisms that may be responsible for the pathogenesis of autism. Growing evidence suggests that Ras/Raf/ERK1/2 signaling plays death-promoting apoptotic roles in neural cells. Recent studies showed a possible association between neural cell death and autism. In addition, two studies reported that a deletion of a locus on chromosome 16, which includes the MAPK3 gene that encodes ERK1, is associated with autism. We thus hypothesized that Ras/Raf/ERK1/2 signaling could be abnormally regulated in the brain of BTBR mice that models autism. In this study, we show that expression of Ras protein was significantly elevated in frontal cortex and cerebellum of BTBR mice as compared with B6 mice. The phosphorylations of A-Raf, B-Raf and C-Raf were all significantly increased in frontal cortex of BTBR mice. However, only C-Raf phosphorylation was increased in the cerebellum of BTBR mice. In addition, we further detected that the activities of both MEK1/2 and ERK1/2, which are the downstream kinases of Ras/Raf signaling, were significantly enhanced in the frontal cortex. We also detected that ERK1/2 is significantly over-expressed in frontal cortex of autistic subjects. Our results indicate that Ras/Raf/ERK1/2 signaling is upregulated in the frontal cortex of BTBR mice that model autism. These findings, together with the enhanced ERK1/2 expression in autistic frontal cortex, imply that Ras/Raf/ERK1/2 signaling activities could be increased in autistic brain and involved in the pathogenesis of autism.     

NBS1 is required for IGF-1 induced cellular proliferation through the Ras/Raf/MEK/ERK cascade

NBS1 is a member of the Mre11–Rad50–NBS1 complex, which plays a role in cellular responses to DNA damage and the maintenance of genomic stability. Transgenic mice models and clinical symptoms of NBS patients have shown that NBS1 exerts pleiotropic actions on the growth and development of mammals. The present study showed that after repression of endogenous NBS1 levels using short interfering RNA, hTERT-RPE cells demonstrated impaired proliferation and a poor response to IGF-1. NBS1 down-regulated cells displayed disturbances in periodical oscillations of cyclin E and A and delayed cell cycle progression. Remarkably, lower phosphorylation levels of c-Raf and diminished activity of Erk1/2 in response to IGF-1 suggest a link among NBS1, IGF-1 signaling and the Ras/Raf/MEK/ERK cascade. The functional relevance of NBS1 in mitogenic signaling and initiation of cell cycle progression were demonstrated in NBS1 down-regulated cells where IGF-1 had a limited ability to induce the FOS and CCND1 expressions. In conclusion, our findings provide strong evidence that NBS1 has a functional role in IGF-1 signaling for the promotion of cell proliferation via the Ras/Raf/MEK/ERK cascade.     

Growth arrest signaling of the Raf/MEK/ERK pathway in cancer

The Raf/MEK/extraceUular signal-regulated kinase （ERK） pathway has a pivotal role in facilitating cell proliferation, and its deregulated activation is a central signature of many epithelial cancers. However paradoxically, sustained activity of Raf/MEK/ERK can also result in growth arrest in many different cell types. This anti-proliferative Raf/MEK/ERK signaling also has physiological significance, as exemplified by its potential as a tumor suppressive mechanism. Therefore, significant questions include in which cell types and by what mechanisms this pathway can mediate such an opposing context of signaling. Particularly, our understating of the role of ERK1 and ERK2, the focal points of pathway signaling, in growth arrest signaling is still limited. This review discusses these aspects of Raf/MEK/ ERK-mediated growth arrest signaling.     

Three-dimensional matrix induces sustained activation of ERK1/2 via Src/Ras/Raf signaling pathway

Research in cell signaling often depends on tissue culture, but the artificial substrates used to grow cells in vitro are likely to distort the conclusions, particularly when adhesion-mediated signaling events are investigated. Studies of signal transduction pathways operating in cells grown in three-dimensional (3D) matrices provide a better system, giving a closer insight of the cell signaling in vivo. We compared the steady-state levels of ERK1/2 activity in primary human fibroblasts, induced by cell-derived 3D fibronectin matrix or fibronectin, coated on flat surfaces. 3D environment caused ERK1/2 stimulation concomitant with a 2.5-fold increase in Ras GTP loading and Src activation. Under these conditions FAK autophosphorylation was suppressed. Treatment with Src inhibitor PP2 abolished these effects indicating that 3D fibronectin matrix activated ERK1/2 through Src/Ras/Raf pathway, bypassing FAK. These observations suggest that within in vivo-like conditions Src may have a leading role in the induction of sustained ERK1/2 activation.     

Involvement of ERK1/2 signaling pathway in DJ-1-induced neuroprotection against oxidative stress

Parkinson’s disease (PD) is a progressive neurodegenerative disorder. Although the precise mechanism remains unclear, mounting evidence suggests that oxidative stress plays an important role in the pathogenesis of PD. DJ-1 gene is associated with oxidative stress and mutations in DJ-1 are involved in an autosomal recessive, early onset familial form of PD. The ERK1/2 signaling pathway contributes to neuroprotection during oxidative stress. However, the correlation between DJ-1 and the ERK1/2 signaling pathway remains unknown. To test for an association of DJ-1 with the ERK1/2 signaling pathway, we transfected wild-type and L166P mutated DJ-1 into COS-7 and MN9D cells. The results showed that over-expression of WT-DJ-1 dramatically enhanced the phosphorylation of ERK1/2 and its upstream kinase MEK1/2. Meanwhile, WT-DJ-1, but not L166P-DJ-1 inhibited the expression of protein phosphatase 2A (PP2A), an inhibitor of the ERK1/2 signaling pathway. Moreover, over-expression of WT-DJ-1 increased cell viability and decreased cell sensitivity to H₂O₂-induced neurotoxicity. Inhibition of the ERK1/2 signaling pathway with a MEK1/2 inhibitor reversed these changes. We conclude that DJ-1 does affect the ERK1/2 signaling pathway and change the susceptibility of cells to oxidative stress.     

GRP75经由Raf/Mek/Erk1/2信号转导通路抑制缺糖诱导的细胞凋亡

本文旨在探讨促存活信号通路Raf/Mek/Erk1/2是否参与了葡萄糖调节蛋白75(glucose-regulated protein75,GRP75)对缺糖诱导的细胞凋亡的抑制作用。GRP75过表达的PC12细胞给予Raf/Mek/Erk1/2通路抑制剂U0126预处理之后,无糖培养6、12和24h,同时以DMSO预处理的GRP75过表达PC12细胞组为对照。Western blot检测Erk1/2的磷酸化和表达水平,MTT实验检测细胞存活率,Hoechst 33258染色观察凋亡细胞核的形态学改变,流式细胞仪检测细胞亚二倍体峰,免疫荧光检测细胞色素c(cytochrome c,Cytc)向胞浆的弥散情况。结果显示:U0126在没有影响Erk1/2表达水平的前提下,阻断了GRP75对Erk1/2磷酸化水平的维持;U0126处理组的凋亡率明显高于对照组;U0126处理组Cytc从线粒体向胞浆释放的时间明显早于对照组,同时Cytc向胞浆的弥散程度大于对照组。以上结果提示,U0126通过抑制Erk1/2磷酸化,阻断了缺糖状态下GRP75对Cytc释放和细胞凋亡的抑制作用,这表明GRP75是通过Raf/Mek/Er...     

Wentilactone A as a novel potential antitumor agent induces apoptosis and G2/M arrest of human lung carcinoma cells,and is mediated by HRas-GTP accumulation to excessively activate the Ras/Raf/ERK/p53-p21 pathway

Chemotherapy remains the common therapeutic for patients with lung cancer. Novel, selective antitumor agents are pressingly needed. This study is the first to investigate a different, however, effective antitumor drug candidate Wentilactone A (WA) for its development as a novel agent. In NCI-H460 and NCI-H446 cell lines, WA triggered G2/M phase arrest and mitochondrial-related apoptosis, accompanying the accumulation of reactive oxygen species (ROS). It also induced activation of mitogen-activated protein kinase and p53 and increased expression of p21. When we pre-treated cells with ERK, JNK, p38, p53 inhibitor or NAC followed by WA treatment, only ERK and p53 inhibitors blocked WA-induced apoptosis and G2/M arrest. We further observed Ras (HRas, KRas and NRas) and Raf activation, and found that WA treatment increased HRas–Raf activation. Knockdown of HRas by using small interfering RNA (siRNA) abolished WA-induced apoptosis and G2/M arrest. HRas siRNA also halted Raf, ERK, p53 activation and p21 accumulation. Molecular docking analysis suggested that WA could bind to HRas-GTP, causing accumulation of Ras-GTP and excessive activation of Raf/ERK/p53-p21. The direct binding affinity was confirmed by surface plasmon resonance (SPR). In vivo, WA suppressed tumor growth without adverse toxicity and presented the same mechanism as that in vitro. Taken together, these findings suggest WA as a promising novel, potent and selective antitumor drug candidate for lung cancer.     

km23‐1/DYNLRB1 regulation of MEK/ERK signaling and R‐Ras in invasive human colorectal cancer cells

We previously found that km23‐1/DYNLRB1 is required for transforming growth factor‐β (TGFβ) production through Ras/ERK pathways in TGFβ‐sensitive epithelial cells and in human colorectal cancer (CRC) cells. Here we demonstrate that km23‐1/DYNLRB1 is required for mitogen‐activated protein kinase kinase (MEK) activation in human CRC cells, detected by km23‐1/DYNLRB1‐siRNA inhibition of phospho‐(p)‐MEK immunostaining in RKO cells. Furthermore, we show that CRISPR‐Cas9 knock‐out (KO) of km23‐1/DYNLRB1 reduced cell migration in two additional CRC models, HCT116 and DLD‐1. Of interest, in contrast to our previous work showing that dynein motor activity was required for TGFβ‐mediated nuclear translocation of Smad2, in the current report, we demonstrate for the first time that disruption of dynein motor activity did not reduce TGFβ‐mediated activation of MEK1/2 or c‐Jun N‐terminal kinase (JNK). Moreover, size exclusion chromatography of RKO cell lysates revealed that B‐Raf, extracellular signal‐regulated kinase (ERK), and p‐ERK were not present in the large molecular weight fractions containing dynein holocomplex components. Furthermore, sucrose gradient fractionation of cell lysates from both HCT116 and CBS CRC cells demonstrated that km23‐1/DYNLRB1 co‐sedimented with Ras, p‐ERK, and ERK in fractions that did not contain components of holo‐dynein. Thus, km23‐1/DYNLRB1 may be associated with activated Ras/ERK signaling complexes in cell compartments that do not contain the dynein holoprotein complex, suggesting dynein‐independent km23‐1/DYNLRB1 functions in Ras/ERK signaling. Finally, of the Ras isoforms, R‐Ras is most often associated with cell migration, adhesion, and protrusive activity. Here, we show that a significant fraction of km23‐1/DYNLRB1 and RRas wase co‐localized at the protruding edges of migrating HCT116 cells, suggesting an important role for the km23‐1/DYNLRB1‐R‐Ras complex in CRC invasion.     

hSef co-localizes and interacts with Ras in the inhibition of Ras/MAPK signaling pathway

To study the mechanism of the inhibitory effects of Sef (similar expression to fgf genes) on Ras/mitogen-activated protein kinase (MAPK) signaling pathway, we observed cellular localization of this protein. Immunofluorescent staining results show that Sef locates in the vesicles of the cytoplasm without bFGF treatment but co-localizes with Ras on the plasma membrane (PM) in response to bFGF stimulation. The coimmunoprecipitation assay demonstrates that Sef interacts with Ras or RasG12V, respectively. We observed that Sef inhibited FGF induced, but not RasG12V mediated, signal transduction. We propose that Sef interacted with Ras in the inhibition of Ras/MAPK signaling pathway.     

Diverse and converging roles of ERK1/2 and ERK5 pathways on mesenchymal to epithelial transition in breast cancer

The epithelial to mesenchymal transition (EMT) is characterized by a loss of cell polarity, a decrease in the epithelial cell marker E-cadherin, and an increase in mesenchymal markers including the zinc-finger E-box binding homeobox (ZEB1). The EMT is also associated with an increase in cell migration and anchorage-independent growth. Induction of a reversal of the EMT, a mesenchymal to epithelial transition (MET), is an emerging strategy being explored to attenuate the metastatic potential of aggressive cancer types, such as triple-negative breast cancers (TNBCs) and tamoxifen-resistant (TAMR) ER-positive breast cancers, which have a mesenchymal phenotype. Patients with these aggressive cancers have poor prognoses, quick relapse, and resistance to most chemotherapeutic drugs. Overexpression of extracellular signal-regulated kinase (ERK) 1/2 and ERK5 is associated with poor patient survival in breast cancer. Moreover, TNBC and tamoxifen resistant cancers are unresponsive to most targeted clinical therapies and there is a dire need for alternative therapies.In the current study, we found that MAPK3, MAPK1, and MAPK7 gene expression correlated with EMT markers and poor overall survival in breast cancer patients using publicly available datasets. The effect of ERK1/2 and ERK5 pathway inhibition on MET was evaluated in MDA-MB-231, BT-549 TNBC cells, and tamoxifen-resistant MCF-7 breast cancer cells. Moreover, TU-BcX-4IC patient-derived primary TNBC cells were included to enhance the translational relevance of our study. We evaluated the effect of pharmacological inhibitors and lentivirus-induced activation or inhibition of the MEK1/2-ERK1/2 and MEK5-ERK5 pathways on cell morphology, E-cadherin, vimentin and ZEB1 expression. Additionally, the effects of pharmacological inhibition of trametinib and XMD8-92 on nuclear localization of ERK1/2 and ERK5, cell migration, proliferation, and spheroid formation were evaluated. Novel compounds that target the MEK1/2 and MEK5 pathways were used in combination with the AKT inhibitor ipatasertib to understand cell-specific responses to kinase inhibition. The results from this study will aid in the design of innovative therapeutic strategies that target cancer metastases.     

Role of ERK1/2 signaling during EGF-induced inhibition of palatal fusion

During mammalian palatal fusion, the medial edge epithelial (MEE) cells must stop DNA synthesis prior to the initial contact of opposing palatal shelves and thereafter selectively disappear from the midline. Exogenous EGF has been shown to inhibit the cessation of DNA synthesis and induce cleft palate; however, the precise intracellular mechanism has not been determined. We hypothesized that EGF signaling acting via ERK1/2 would maintain MEE DNA synthesis and cell proliferation and consequently inhibit the process of palatal fusion. Palatal shelves from E13 mouse embryos were maintained in organ cultures and stimulated with EGF. EGF-treated palates failed to fuse with intact MEE and had significant ERK1/2 phosphorylation. Both EGF-induced ERK1/2 phosphorylation and BrdU-incorporation were localized in the nucleus of MEE cells. Subsequent inhibition assays using U0126, a specific inhibitor of ERK1/2 phosphorylation, were conducted. U0126 inhibited EGF-induced ERK1/2 phosphorylation in a dose-dependent manner and consequently MEE cells stopped proliferation. The threshold of ERK1/2 inactivation to stop MEE DNA synthesis coincides with the level required to rescue the EGF-induced cleft palate phenotype. These results indicate that EGF-induced inhibition of palatal fusion is dependent on nuclear ERK1/2 activation and that this mechanism must be tightly regulated during normal palatal fusion.     

ERK1/2和p-ERK1/2在肺癌组织中的表达及意义

目的研究细胞外信号调节激酶1/2（extracellular regulated kinase 1/2,ERK1/2）及其磷酸化状态（p-ERK1/2）在不同分化程度肺癌中的表达情况,探讨二者与肺癌侵袭、转移的关系。方法采用免疫组化（Envision）法,检测79例肺癌组织及l2例癌旁正常肺组织中ERK1/2和p-ERK1/2的表达。结果ERK1/2在高、中、低分化组表达率分别为13.6%,39.4%,66.7%,p-ERK1/2在高、中、低分化组表达率分别14.3%,27.3%,79.2%（P〈0.05）;无淋巴结转移者阳性率为20%,有淋巴结转移者阳性率为50.1%（P〈0.05）。ERK1/2和p-ERK1/2的表达在不同年龄、性别、肿瘤大小、肿瘤病理类型无显著性差异,而与分化程度有关,其中p-ERK1/2的表达还与有无淋巴结转移有关。结论ERK1/2和p-ERK1/2在肺癌组织中高表达且与分化程度有关。