摘要： Epidermal growth factor receptor (EGFR) and its downstream phosphoinositide 3-kinase (PI3K) pathway are commonly deregulated in cancer. Recently, we have shown that the IQ motif-containing GTPase-activating protein 1 (IQGAP1) provides a molecular platform to scaffold all the components of the PI3K-Akt pathway and results in the sequential generation of phosphatidylinositol-3,4,5-trisphosphate (PI3,4,5P(3)). In addition to the PI3K-Akt pathway, IQGAP1 also scaffolds the Ras-ERK pathway. To define the specificity of IQGAP1 for the control of PI3K signaling, we have focused on the IQ3 motif in IQGAP1 as PIPKI alpha and PI3K enzymes bind this region. An IQ3 deletion mutant loses interactions with the PI3K-Akt components but retains binding to ERK and EGFR. Consistently, blocking the IQ3 motif of IQGAP1 using an IQ3 motif-derived peptide mirrors the effect of IQ3 deletion mutant by reducing Akt activation but has no impact on ERK activation. Also, the peptide disrupts the binding of IQGAP1 with PI3K-Akt pathway components, while IQGAP1 interactions with ERK and EGFR are not affected. Functionally, deleting or blocking the IQ3 motif inhibits cell proliferation, invasion, and migration in a non-additive manner to a PIPKIa inhibitor, establishing the functional specificity of IQ3 motif towards the PI3K-Akt pathway. Taken together, the IQ3 motif is a specific target for suppressing activation of the PI3K-Akt but not the Ras-ERK pathway. Although EGFR stimulates the IQGAP1-PI3K and-ERK pathways, here we show that IQGAP1-PI3K controls migration, invasion, and proliferation independent of ERK. These data illustrate that the IQ3 region of IQGAP1 is a promising therapeutic target for PI3K-driven cancer.

关键词： phospholipase D   PA   VSMC   migration  

摘要： Very little is known about how lipid signaling regulates intima hyperplasia after vascular injury. Herein, we report that deletion and pharmacological inhibition of phospholipase D (PLD)2, which generates the signaling lipid phosphatidic acid (PA), reduced neointimal formation in the mouse carotid artery ligation model. PLD2 deficiency inhibits migration of vascular smooth muscle cells (VSMCs) into the intima in mice as well as migration and formation of membrane ruffles in primary VSMCs. PA specifically binds to the IQ motif-containing guanosine triphosphatase-activating protein 1 (IQGAP1) scaffold protein. The binding between PA and IQGAP is required for the plasma membrane recruitment of IQGAP1. Similar to PLD2 inhibition, knockdown of IQGAP1 blocks ruffle formation and migration in VSMCs, which are rescued by expression of the exogenous IQGAP1 but not the PA binding-deficient mutant. These data reveal that the PLD2-PA-IQGAP1 pathway plays an important role in VSMC migration and injury-induced vascular remodeling, and implicate PLD2 as a candidate target for therapeutic interventions.-Wang, Z., Cai, M., Tay, L. W. R., Zhang, F., Wu, P., Huynh, A., Cao, X., Di Paolo, G., Peng, J., Milewicz, D. M., Du, G. Phosphatidic acid generated by PLD2 promotes the plasma membrane recruitment of IQGAP1 and neointima formation.

摘要： IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that participates in several cellular functions, including cytoskeletal regulation, cell adhesion, gene transcription and cell polarization. IQGAP1 has been implicated in the tumorigenesis and progression of several human cancers. However, the role of IQGAP1 in pancreatic ductal adenocarcinoma (PDAC) is still unknown. We found that IQGAP1 expression was an independent prognostic factor for PDAC. IQGAP1 upregulation significantly promoted cell proliferation, migration, invasion and epithelial-mesenchyma I transition (EMT), whereas IQGAP1 downregulation impaired its oncogenic functions. Overexpression of IQGAP1 increased the protein level of Dishevelled2 (DVL2) and enhanced canonical Wnt signaling as evidenced by increased DVL2 level, beta-catenin transcriptional activity, beta-catenin nuclear translocation and expression of the direct target genes of beta-catenin (cyclin D1 and c-myc). In contrast, knockdown of IQGAP1 decreased the level of DVL2 and attenuated Wnt/beta-catenin signaling. In vivo results revealed that IQGAP1 promoted tumor growth and metastasis. Co-immunoprecipitation studies demonstrated that IQGAP1 interacted with both DVL2 and beta-catenin. Moreover, knockdown of DVL2 reversed IQGAP1-induced EMT. Our findings thus confirmed that IQGAP1 could be used as a potential target for PDAC treatment.

关键词： 胶质瘤   IQGAP1基因   细胞侵袭   细胞迁移   免疫抑制   STAT3信号通路  

摘要： 目的:探讨敲减IQGAP1基因表达对脑胶质瘤细胞侵袭、迁移及免疫抑制的影响及机制。方法:将人脑胶质瘤U251细胞随机分为空白组、阴性对照组和si-IQGAP1组,AG490作为STAT3信号通路抑制剂。转染48 h后,MTT法检测细胞活力;Western blot检测IQGAP1、血管内皮生长因子(VEGF)、转化生长因子β1(TGF-β1)、STAT3和p-STAT3的蛋白水平;Transwell小室检测细胞的侵袭和迁移能力。结果:si-IQGAP1-1组和si-IQGAP1-2组的IQGAP1蛋白表达均显著低于空白组(P<0.05)。与空白组比较,si-IQGAP1组和AG490组的细胞活力、侵袭能力和迁移能力均显著降低,VEGF、TGF-β1和p-STAT3的蛋白水平均显著降低(P<0.05)。与AG490组比较,AG490+si-IQGAP1组的细胞活力、侵袭能力和迁移能力均显著降低,VEGF和TGF-β1的蛋白表达均显著降低(P<0.05)。结论:敲减IQGAP1基因表达可降低脑胶质瘤细胞的侵袭和迁移能力,减弱细胞免疫抑制因子VEGF和TGF-β1的表达,机制与下调STAT3信号通路有关。

关键词： 翼状胬肉   睑裂斑   p-ERK   IQGAP1  

摘要： 目的探讨翼状胬肉与睑裂斑的病理改变差异和磷酸化细胞外信号调节激酶(p-ERK)及含IQ模序的GTP酶活化蛋白1(IQGAP1)的表达,比较其在翼状胬肉、睑裂斑中的差异。方法 8例翼状胬肉、睑裂斑病理及正常结膜组织,HE染色显示组织结构,免疫组化检测各组织中p-ERK和IQGAP1的表达。结果上皮层数比较:翼状胬肉>睑裂斑>结膜。血管数量比较:翼状胬肉>睑裂斑>结膜。翼状胬肉、睑裂斑组织中p-ERK、IQGAP1表达水平明显高于正常结膜。相关分析显示翼状胬肉中p-ERK与IQGAP1表达呈正相关。结论翼状胬肉和睑裂斑中的p-ERK和IQGAP1表达增加,共同参与翼状胬肉、睑裂斑的发病过程。翼状胬肉和睑裂斑的发病机制可能有一定相似性。

关键词： Cell Locomotion   Traffic   Cell Adhesion   Endosomes   Leading edges   GTPase-Activating Proteins   Plasma membrane   Alanine Aminopeptidase   Cell Migration   Guanosine Triphosphate Phosphohydrolases   Integrins  

摘要： Cell attachment to the extracellular matrix (ECM) requires a balance between integrin internalization and recycling to the surface that is mediated by numerous proteins, emphasizing the complexity of these processes. Upon ligand binding in various cells, the beta 1 integrin is internalized, traffics to early endosomes, and is returned to the plasma membrane through recycling endosomes. This trafficking process depends on the cyclical activation and inactivation of small guanosine triphosphatases (GTPases) by their specific guanine exchange factors (GEFs) and their GTPase-activating proteins (GAPs). In this study, we found that the cell surface antigen CD13, a multifunctional transmembrane molecule that regulates cell-cell adhesion and receptor-mediated endocytosis, also promoted cell migration and colocalized with beta 1 integrin at sites of cell adhesion and at the leading edge. A lack of CD13 resulted in aberrant trafficking of internalized beta 1 integrin to late endosomes and its ultimate degradation. Our data indicate that CD13 promoted ARF6 GTPase activity by positioning the ARF6-GEF EFA6 at the cell membrane. In migrating cells, a complex containing phosphorylated CD13, IQGAP1, GTP-bound (active) ARF6, and EFA6 at the leading edge promoted the ARF6 GTPase cycling and cell migration. Together, our findings uncover a role for CD13 in the fundamental cellular processes of receptor recycling, regulation of small GTPase activities, cell-ECM interactions, and cell migration.

关键词： 足细胞   细胞骨架重排   高糖   IQ结构域GTP酶活化蛋白1   细胞分裂周期蛋白42  

摘要： 目的观察高糖环境下IQ结构域GTP酶活化蛋白1(IQGAP1)在足细胞中的表达情况,并探讨IQGAP1在调节高糖诱导的足细胞骨架重排中的作用及其可能的机制。方法 (1)体外培养永生化的小鼠足细胞。取一部分足细胞加入不同浓度葡萄糖(5 mmol/L、10 mmol/L、15 mmol/L、20 mmol/L、25 mmol/L、30 mmol/L)刺激48 h,另取一部分细胞加入高浓度葡萄糖(30 mmol/L)刺激不同时间(0 h、3 h、6 h、12 h、24 h、48 h),检测各组足细胞IQGAP1蛋白的表达情况。(2)将足细胞分为正常糖浓度组(5 mmol/L葡萄糖,NG组)、高糖组(30 mmol/L葡萄糖,HG组)、高渗对照组(5 mmol/L葡萄糖+25 mmol/L甘露醇,HO组)、HG+IQGAP1质粒转染组、HG+空质粒转染组、NG+IQGAP1质粒转染组、NG+空质粒转染组。给予相应干预后,比较NG组、HG组、HO组足细胞的IQGAP1蛋白表达情况及纤维状肌动蛋白(F-actin)骨架分布,比较NG组、HG组、HG+IQGAP1质粒转染组、HG+空质粒转染组足细胞的F-actin骨架分布,比较6组足细胞的IQGAP1蛋白及细胞分裂周期蛋白42(Cdc42)表达情况、IQGAP1和Cdc42蛋白的结合情况。结果 (1) 30 mmol/L葡萄糖刺激后足细胞的IQGAP1蛋白表达水平低于5 mmol/L葡萄糖刺激后的水平(P<0.05),高糖刺激48 h后的足细胞IQGAP1蛋白表达水平低于高糖刺激0 h后的水平(P<0.05)。(2) HG组足细胞的F-actin排列紊乱,F-actin形成核外周环,F-actin外周环评分(CFS)高于NG组(P<0.05)。(3) HG+IQGAP1质粒转染组的足细胞骨架重排部分逆转,CSF低于HG组(P<0.05)。(4) HG+IQGAP1质粒转染组的IQGAP1蛋白及Cdc42表达水平、两者结合水平均高于HG组(均P<0.05)。结论高糖刺激可减少IQGAP1及Cdc42蛋白表达以及两者结合,并诱导足细胞骨架重排,而IQGAP1可能通过与Cdc42结合调节细胞骨架重排。

关键词： IQGAP1   Human glomeruli   Human renal tubules   F-actin  

摘要： IQGAP1 is a multifunctional, 190-kDa scaffolding protein that plays an important role in the regulation of cell adhesion, migration, proliferation, differentiation, polarization and cytoskeletal remodeling. IQGAP1 is ubiquitously expressed in human organs and is highly expressed in the kidney. Currently, the site-specific expression of IQGAP1 in the human nephrons is unclear. We performed Western blotting analysis, immunohistochemistry and double-immunolabeling confocal microscopic analysis of IQGAP1 with specific biomarkers of each nephron segment to study the expression and distribution of IQGAP1 in human nephrons. We found that IQGAP1 was strongly expressed in human podocytes and glomerular endothelial cells, but weakly expressed in glomerular mesangial cells. In human renal tubules, IQGAP1 was strongly expressed in the collecting duct, moderately expressed in the proximal tubule, medullary loop, distal convoluted tubule and connecting tubule. IQGAP1 staining was much stronger in the apical membrane in the proximal tubule, thick descending limb and thick ascending limb of medullary loop and collecting duct. However, the expression of IQGAP1 was mainly in the basolateral membrane of the connecting tubule, and diffusely in the thin limb of medullary loop and distal convoluted tubule. The interaction between IQGAP1 and F-actin suggested that cytoskeleton regulation may be the underlying mechanism mediating the effect of IQGAP1 in human nephrons. To the best of our knowledge, this is the first report of specific expression and differential subcellular location of IQGAP1 in human nephrons. The site-specific expression pattern of IQGAP1 suggests that IQGAP1 may play diverse roles in various human nephron segments.

关键词： 食管鳞状细胞癌   TE-2细胞   具有IQ结构域的GTP激酶活化蛋白1   小干扰RNA   增殖   侵袭  

摘要： 目的:探讨具有IQ结构域的GTP激酶活化蛋白1(Ras GTPase-activating-like protein, IQGAP1)在食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)组织与细胞中的表达及其对TE-2细胞增殖及侵袭能力的影响。方法:收集2015年1月至2016年12月郑州大学附属肿瘤医院125例ESCC手术切除的癌及癌旁组织标本,以及ESCC细胞系TE-2、TE-3、ECA109和正常食管上皮细胞株Het-1A。用免疫组化染色法检测癌组织中IQGAP1的表达,分析其表达水平与临床病理特征的关系;用qPCR和Western blotting检测ESCC细胞中IQGAP1 mRNA和蛋白的表达。用si-IQGAP1(阳性转染组)、si-CTRL(阴性对照组)质粒转染TE-2细胞,用MTT法、Transwell小室法和Western blotting分别检测沉默IQGAP1对TE-2细胞增殖、侵袭能力以及上皮钙黏蛋白和神经钙黏蛋白表达的影响。结果:IQGAP1在ESCC组织中的阳性表达率明显高于癌旁组织(P<0.05),其高表达与肿瘤分期和分级密切相关(均P<0.05);IQGAP1 m RNA和蛋白在TE-2、TE-3、ECA109细胞中表达水平显著高于Het-1A细胞(均P<0.05)。沉默IQGAP1后,与阴性对照组和空白组比较,阳性转染组TE-2细胞中IQGAP1 mRNA及蛋白的表达水平明显下降(均P<0.05);细胞的增殖和侵袭能力显著降低(均P<0.05),上皮钙黏蛋白表达水平升高(P<0.05),神经钙黏蛋白表达水平下降(P<0.05)。结论:IQGAP1在ESCC组织中高表达,敲减IQGAP1可抑制ESCC细胞的增殖和侵袭能力,其在ESCC的发生发展过程中起重要的作用。

关键词： IQGAP1   上皮间质转化   缺氧诱导因子1α   血管内皮生长因子A   胃癌  

摘要： 研究目的:通过肿瘤公共数据库分析IQGAP1（IQ motif containing GTPase activating protein 1）在胃癌组织中的表达,检测IQGAP1在胃癌细胞（SGC7901、HGC27、MGC803）和胃黏膜上皮细胞（GES1）蛋白质水平,研究下调蛋白IQGAP1对胃癌细胞迁移能力、上皮间质转化（epithelial mesenchymal transition,EMT）的生物学行为的影响,探讨下调IQGAP1在胃癌细胞EMT中可能作用机制。研究方法:1.通过肿瘤数据库分析IQGAP1在胃癌组织中mRNA、蛋白的表达情况。通过Western blot检测IQGAP1在SGC7901、HGC27、MGC803三种胃癌细胞株以及胃黏膜上皮细胞株（GES1）的表达水平。2.在IQGAP1表达相对较高的细胞株（HGC27、MGC803）中,利用IQGAP siRNA稳定下调细胞中IQGAP1表达,通过Transwell实验以及划痕实验检测下调IQGAP1对胃癌细胞迁移能力影响。通过Western blot法检测下调IQGAP1对胃癌细胞EMT相关蛋白指标的影响。3.通过Western blot法检测血管内皮生长因子A（vascular endothelial growth factor-A,VEGF-A）在胃癌细胞、胃粘膜上皮细胞、低表达IQGAP1胃癌细胞中表达。通过ELISA法检测VEGF-A胃癌细胞胞外分泌水平。***2溶液稳定诱导胃癌细胞缺氧后,通过Western blot法检测下调IQGAP1胃癌缺氧细胞中缺氧诱导因子1α（Hypoxia-inducible factors 1α,HIF1α）和VEGF-A蛋白表达,ELISA法检测细胞外VEGF-A分泌水平,核浆分离实验检测胃癌细胞中HIF1α的核质分布情况。研究结果:1.肿瘤数据库发现胃腺癌组织中的IQGAP1在mRNA水平高于正常胃组织,各个分期的胃腺癌组织IQGAP1在mRNA水平高于正常胃组织,免疫组化提示胃腺癌组织中的IQGAP1蛋白水平明显高于正常胃组织;Western blot结果表明IQGAP1蛋白表达水平在HGC27、MGC803细胞相对较高,SGC7901细胞次之,GES1细胞最低。2.在HGC27、MGC803细胞中下调IQGAP1后,细胞迁移能力下降;Western blot结果表明:与对照组相比,IQGAP1-siRNA组EMT相关指标N-cadherin、Vimentin、Slug、Snail、β-catenin蛋白表达量降低,而E-cadherin蛋白表达水平增高。***7901、HGC27、MGC803、GES1细胞中IQGAP1和VEGF-A蛋白呈相关性表达;下调胃癌细胞IQGAP1后发现细胞内VEGF-A蛋白表达及细胞外分泌水平同步下降。4.在缺氧刺激低表达IQGAP1胃癌细胞株中,HIF1α蛋白表达较对照组明显下降,缺氧细胞外VEGF-A分泌受到抑制;核浆分离实验表明:与对照组相比,低表达IQGAP1实验组中HIF1α在细胞核内表达降低。研究结论:IQGAP1在胃癌组织和胃癌细胞中呈相对高表达。下调IQGAP1具有抑制胃癌细胞迁移、EMT的生物学作用。下调IQGAP1可能通过HIF1α/VEGF-A信号通路抑制胃癌细胞EMT过程。