关键词： 人肾脏足细胞   细胞凋亡   IQGAP1   ERK1/2蛋白   p-ERK1/2蛋白  

摘要： 研究背景糖尿病是临床上一种常见内分泌性疾病,它是由于多种病因引起的以慢性高血糖为特征的代谢性疾病,是由于胰岛素分泌和（或）作用缺陷所致。持续高血糖与长期代谢紊乱等可导致全身组织器官的多种并发症,尤其是眼、肾、心血管及神经系统的慢性进行性病变、功能减退或衰竭。糖尿病的全身血管并发症分为大血管并发症和微血管并发症,糖尿病肾病（diabetic nephropathy,DN）是糖尿病最常见的微血管并发症之一,其临床表现主要为高血压、水肿、严重蛋白尿、低白蛋白血症和进行性肾功能减退;肾脏病理主要表现为结节状或弥漫性系膜细胞增殖、基底膜(（GBM）增厚,系膜细胞外基质积聚、足细胞足突融合,肾小球硬化及肾小管间质纤维化。糖尿病肾病是目前世界范围内导致终末期肾病（end stage renal disease,ESRD）的首位位病因,增加各国卫生保健事业支出的同时也给予社会、家庭、个人带来沉重负担。在我国糖尿病肾病发病率以及在终末期肾衰竭透析患者中所占的比例也成逐年增加趋势,严重影响了患者的生存质量。因此,探讨糖尿病肾病的发病机制、及早发现糖尿病肾病并给予适当干预治疗措施对广大糖尿病肾病患者具有重要的社会和现实意义。DN的发病机制十分复杂,涉及到糖脂代谢紊乱、炎症介质释放、血流动力学异常、细胞因子、氧化应激以及细胞凋亡等多因素的作用。近年来的研究显示,肾小球滤过屏障结构和功能的改变,尤其是作为肾小球滤过屏障重要组成成分的足细胞损伤,在糖尿病肾病的进展中发挥了举足轻重的作用,被认为是引起DN蛋白尿和肾小球硬化的关键因素。因此,研究足细胞的损伤为DN的预防和治疗带来了新的契机。足细胞又称为脏层肾小球上皮细胞,附着于肾小球基底膜外侧,与肾小球基底膜（glomerular basement membrane,GBM）、内皮细胞共同构成肾小球滤过屏障。足细胞的主要作用是对蛋白质的滤过及GBM成分的更新起着举足轻重的作用。由于足细胞是高度分化细胞,增殖能力较差,当足细胞受到损伤时,尿蛋白漏出增加,从而加重肾衰竭的发生,而蛋白尿又进一步加重足细胞的损伤。在DN的发生发展中,足细胞的改变主要包括足细胞数目减少,细胞凋亡增加,流动性增强,直至足细胞从GBM上脱落随尿液排出,导致GBM裸露,形成局灶粘连和肾小球硬化。DN足细胞损伤机制复杂,其不是单由某个因素所诱发,而是多个因素的联合作用。主要包括肾蛋白（nephrin）、高血糖、整合素、机械应力、炎性反应、肾素-血管紧张素-醛固酮系统（RAAS）的激活、AGEs的蓄积、GH-胰岛素样生长因子（insulin-like growth factor,IGF）-I轴、血管内皮生长因子（VEGF）、类肝素硫酸蛋白聚糖（HSPG）、肿瘤抑制基因（wT1）、Podocalyxin蛋白、过氧化物酶体增殖物激活受体（perox-isome proliferatoractivated receptor,PPAR）、脂联素、微小 RNA（micro RNA,miRNA）、TGF-β1、氧化应激及细胞凋亡等。有研究显示,IQ结构域GTP酶活化蛋白1（IQdomainGTPase-activating protein 1,IQGAP1）是一种含有多个蛋白结合域的支架蛋白,近期研究发现IQGAP1在细胞黏附迁移、维持细胞形态以及调节细胞凋亡等众多生命过程中发挥了举足轻重的作用[1,2]。IQGAP1是否参与高糖诱导的足细胞凋亡尚未见报道,基于上述理论基础,我们设计并进行了本次研究。本课题分为两部分,第一步部分,采用体外培养人肾脏足细胞（HPC）,观察高糖刺激对IQGAP1表达和分布以及对足细胞凋亡的影响。第二部分,进一步观察IQGAP1与丝裂原活化蛋白激酶（MAPK）信号通路以及肾素□血管紧张素□醛固酮系统（RAAS）在足细胞凋亡中的作用,探讨高糖诱导肾脏足细胞凋亡的分子机制,从而为更好的治疗糖尿病肾病提供理论支持。第一部分高糖刺激对足细胞中IQGAP1表达和分布以及凋亡的影响研究目的:1、观察人肾脏足细胞在体外高糖刺激下IQGAP1表达情况;2、观察人肾脏足细胞在体外高糖刺激下IQGAP1分布情况;3、观察人肾脏足细胞在体外高糖刺激下细胞凋亡情况。研究方法:HPC在33℃、5%CO2培养箱中,以含有10%胎牛血清及γ-干扰素（10 U/ml）的RPMI 1640培养液培养细胞使其增殖,传代后更换为不含Y-干扰素的RPMI 1640培养液,置入37° C、5%C02培养箱中培养10～14天,促进细胞分化成熟,用于后续实验。HPC被随机分为3组:A组:正常对照组（Normal Control,NC,）、B组:甘露醇对照组（Manitol+Glucose Control,MG）和C组:高糖组（High Glucose,HG）。NC组细胞给

关键词： LEGIONELLALEGIONELLACEAELEGIONELLA pneumophilaQUORUM sensing (Microbiology)CELL interaction (Biology)  

摘要： Small molecule signaling promotes the communication between bacteria as well as between bacteria and eukaryotes. The opportunistic pathogenic bacterium Legionella pneumophila employs LAI-1 (3-hydroxypentadecane-4-one) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including natural competence for DNA uptake and pathogen-host cell interactions. In this study, we analyze the role of LAI-1 in inter-kingdom signaling. L. pneumophila lacking the autoinducer synthase LqsA no longer impeded the migration of infected cells, and the defect was complemented by plasmid-borne lqsA. Synthetic LAI-1 dose-dependently inhibited cell migration, without affecting bacterial uptake or cytotoxicity. The forward migration index but not the velocity of LAI-1-treated cells was reduced, and the cell cytoskeleton appeared destabilized. LAI-1-dependent inhibition of cell migration involved the scaffold protein IQGAP1, the small GTPase Cdc42 as well as the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or RhoA, Rac1 or Ran GTPase. Upon treatment with LAI-1, Cdc42 was inactivated and IQGAP1 redistributed to the cell cortex regardless of whether Cdc42 was present or not. Furthermore, LAI-1 reversed the inhibition of cell migration by L. pneumophila, suggesting that the compound and the bacteria antagonistically target host signaling pathway(s). Collectively, the results indicate that the L. pneumophila quorum sensing compound LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42 and ARHGEF9.

关键词： IQGAP1   口腔鳞状细胞癌(OSCC)   增殖   侵袭  

摘要： 目的:研究IQ结构域三磷酸鸟苷合酶-激活蛋白1(IQGAP1)在口腔鳞状细胞癌(OSCC)组织中的表达,探讨其对肿瘤细胞增殖和侵袭的影响及其作用机制。方法:免疫印迹和荧光定量PCR检测IQGAP1在癌组织和癌旁组织中的表达水平;利用脂质体转染法将重组质粒pc DNA3.1-IQGAP1转入人口腔鳞状细胞癌细胞系SCC-4,并用Akt抑制剂预处理,免疫印迹检测Akt磷酸化水平、MTT法和Transwell侵袭实验分别测定细胞增殖和侵袭能力的变化。结果:IQGAP1蛋白和mRNA表达量显著高于癌旁组织(P<0.05),重组质粒转染后,SCC-4细胞中IQGAP1表达量明显增加,细胞的增殖能力和侵袭能力增强(P<0.05)。IQGAP1过表达后细胞中p Akt水平明显升高;当用Akt抑制剂处理后,IQGAP1诱导的p Akt的水平明显降低,细胞增殖和侵袭能力降低(P<0.05)。结论:IQGAP1在OSCC组织中高表达,可能通过调节Akt通路的活化来介导癌细胞的增殖和侵袭能力。

关键词： 肝细胞肝癌   IQ模体的Ras   GTP酶活化蛋白1   肝切除术   预后  

摘要： 目的探讨含有IQ模体的Ras GTP酶活化蛋白1（IQGAP1）在肝细胞肝癌组织中的表达及其临床意义。方法收集2007~2009年期间在笔者所在医院行肝癌切除治疗的79例肝细胞肝癌患者的临床资料,采用免疫组化法检测肝癌组织中IQGAP1的表达,并分析其与肝癌患者临床病理特征的关系,比较IQGAP1表达阳性和阴性患者肝癌切除术后的累积无瘤生存率及总体生存率。结果本组79例肝癌患者中肝癌组织中IQGAP1表达阳性者43（54.4%）,IQGAP1表达阳性的患者多为低分化及存在微血管侵犯者。IQGAP1表达阳性与阴性患者术后1、3及5年的累积无瘤生存率分别为67.4%、39.5%及23.3%和100%、94.4%及83.3%,前者明显低于后者（P〈0.001）;IQGAP1表达阳性患者术后1、3及5年的累积总体生存率为97.7%、71.5%和53.3%,明显低于IQGAP1表达阴性者的100%、97.2%和88.9%（P〈0.001）。结论肝细胞肝癌患者中IQGAP1阳性表达者预后较差。IQGAP1可能是肝细胞肝癌预后的指标。

关键词： colorectal cancer   cell invasion   DNAJB6   IQGAP1  

摘要： DNAJB6 is a member of the heat shock protein 40 (Hsp40) family. We here investigated the clinical correlation and biological role of DNAJB6 overexpression in colorectal cancer (CRC). The expression of DNAJB6 protein was examined in 200 cases of colorectal adenocarcinomas by immunohistochemistry (IHC) technology. Gene transfection and RNA interference were performed to determine the effect of DNAJB6 expression on the invasion of CRC cells and to explore the underlying molecular mechanisms in vitro and in vivo. Overexpression of DNAJB6 was found in 39% (78/200) of the CRC tissues, especially in tumors at pT4 as compared with at pT1-3 (P = 0.02). A Kaplan-Meier survival analysis revealed a correlation between DNAJB6 expression and overall survival (OS) times (P = 0.003). Multivariate analysis confirmed that DNAJB6 overexpression was an independent prognostic factor for CRC (P = 0.002). RNA interference-mediated silencing of the DNAJB6 gene inhibited the invasion of CRC cells in vitro were accompanied by a significant reduction in the protein levels of IQ-domain GTPase-activating protein 1 (IQGAP1) and phosphorylated ERK (pERK). An in vivo assay showed that inhibition of DNAJB6 expression decreased the lung metastases of CRC cells. IHC analysis of serial sections showed that there was a positive correlation between DNAJB6 and IQGAP1 expression in primary CRC tissues (P = 0.013). The data suggest that DNAJB6 plays an important oncogenic role in CRC cell invasion by up-regulating IQGAP1 and activating the ERK signaling pathway and that DNAJB6 may be used as a prognostic marker for CRC. (C) 2014 Wiley Periodicals, Inc.

关键词： Research Article  

摘要： The intracellular bacterial pathogen Shigella infects and spreads through the human intestinal epithelium. Effector proteins delivered by Shigella into cells promote infection by modulating diverse host functions. We demonstrate that the effector protein OspB interacts directly with the scaffolding protein IQGAP1, and that the absence of either OspB or IQGAP1 during infection leads to larger areas of S. flexneri spread through cell monolayers. We show that the effect on the area of bacterial spread is due to OspB triggering increased cell proliferation at the periphery of infected foci, thereby replacing some of the cells that die within infected foci and restricting the area of bacterial spread. We demonstrate that OspB enhancement of cell proliferation results from activation of mTORC1, a master regulator of cell growth, and is blocked by the mTORC1-specific inhibitor rapamycin. OspB activation of mTORC1, and its effects on cell proliferation and bacterial spread, depends on IQGAP1. Our results identify OspB as a regulator of mTORC1 and mTORC1-dependent cell proliferation early during S. flexneri infection and establish a role for IQGAP1 in mTORC1 signaling. They also raise the possibility that IQGAP1 serves as a scaffold for the assembly of an OspB-mTORC1 signaling complex.

关键词： actin   cell migration   membrane   microtubule   scaffold protein   microtubule dynamics  

摘要： Cell migration is orchestrated by dynamic interaction of microtubules with the plasma membrane cortex. However, the regulatory mechanisms underlying the cortical actin cytoskeleton and microtubule dynamics are less characterized. Our earlier study showed that small GTPase-activating proteins, IQGAPs, regulate polarized secretion in epithelial cells (1). Here, we show that IQGAP1 links dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein, SKAP. Biochemical characterizations revealed that IQGAP1 and SKAP form a cognate complex and that their binding interfaces map to the WWIQ motif and the C-terminal of SKAP, respectively. The WWIQ peptide disrupts the biochemical interaction between IQGAP1 and SKAP in vitro, and perturbation of the IQGAP1-SKAP interaction in vivo using a membrane-permeable TAT-WWIQ peptide results in inhibition of directional cell migration elicited by EGF. Mechanistically, the N-terminal of SKAP binds to EB1, and its C terminus binds to IQGAP1 in migrating cells. Thus, we reason that a novel IQGAP1 complex orchestrates directional cell migration via coupling dynamic microtubule plus-ends to the cell cortex.

关键词： Actin   Calcium-binding protein   Cell cortex   EF hand   Membrane  

摘要： The cortical cytoskeleton constitutes an important subcellular structure that determines cell shape and regulates cell migration as well as membrane traffic to and from the plasma membrane. Many components of the cortical cytoskeleton have been identified including structural and scaffolding proteins, membrane-cytoskeleton linker proteins and signaling intermediates. We describe here an association of the membrane-F-actin linker protein ezrin with the scaffolding protein IQGAP1 that serves as a hub for concentrating different signaling complexes. Both, ezrin and IQGAP1 bind in a Ca2+-dependent manner to the EF hand protein S100P and complexes consisting of Ca2+-bound S100P, IQGAP1 and ezrin can be isolated by immunoprecipitation. Ezrin and IQGAP1 also interact in the absence of Ca2+, thus independent of S100P. Direct ezrin-IQGAP1 interaction can be shown with the purified proteins. It is mediated via the N-terminal FERM domain of ezrin and the IQ domain of IQGAP1, respectively. Ezrin and IQGAP1 colocalize in the submembraneous cytoskeleton and in cellular protrusions of human epithelial cells and knockdown of ezrin reduces the cortical localization of IQGAP1. Thus, ezrin appears to participate in recruiting IQGAP1 to the cell cortex thereby establishing a close connection between membrane-F-actin contacts and actin regulators that can be assembled by IQGAP1. This article is part of a Special Issue entitled: 13th European Symposium on Calcium. (C) 2014 Elsevier B.V. All rights reserved.

关键词： Actin   Calcium-binding protein   Cell cortex   EF hand   Membrane  

摘要： The cortical cytoskeleton constitutes an important subcellular structure that determines cell shape and regulates cell migration as well as membrane traffic to and from the plasma membrane. Many components of the cortical cytoskeleton have been identified including structural and scaffolding proteins, membrane–cytoskeleton linker proteins and signaling intermediates. We describe here an association of the membrane-F-actin linker protein ezrin with the scaffolding protein IQGAP1 that serves as a hub for concentrating different signaling complexes. Both, ezrin and IQGAP1 bind in a Ca 2 + -dependent manner to the EF hand protein S100P and complexes consisting of Ca 2 + -bound S100P, IQGAP1 and ezrin can be isolated by immunoprecipitation. Ezrin and IQGAP1 also interact in the absence of Ca 2 + , thus independent of S100P. Direct ezrin–IQGAP1 interaction can be shown with the purified proteins. It is mediated via the N-terminal FERM domain of ezrin and the IQ domain of IQGAP1, respectively. Ezrin and IQGAP1 colocalize in the submembraneous cytoskeleton and in cellular protrusions of human epithelial cells and knockdown of ezrin reduces the cortical localization of IQGAP1. Thus, ezrin appears to participate in recruiting IQGAP1 to the cell cortex thereby establishing a close connection between membrane-F-actin contacts and actin regulators that can be assembled by IQGAP1. This article is part of a Special Issue entitled: 13th European Symposium on Calcium. [ABSTRACT FROM AUTHOR]

关键词： GTPASE-activating protein   RESEARCH   GROWTH factors   CELL proliferation   EPIDERMAL growth factor   HOMEOSTASIS  

摘要： IQ motif-containing GTPase-activating protein (IQGAP) scaffolding proteins regulate many essential cellular processes including growth factor receptor signaling, cytoskeletal rearrangement, adhesion, and proliferation and are highly expressed in many cancers. Using genetically engineered human skin tissue in vivo, we demonstrate that diminished, sub-physiologic expression of IQGAP1 or IQGAP3 is sufficient to maintain normal epidermal homeostasis, whereas significantly higher levels are required to support tumorigenesis. To target this tumor-specific IQGAP requirement in vivo, we engineered epidermal keratinocytes to express individual IQGAP protein domains designed to compete with endogenous IQGAPs for effector protein binding. Expression of the IQGAP1-IQ motif decoy domain in epidermal tissue in vivo inhibits oncogenic Ras-driven mitogen-activated protein kinase signaling and antagonizes tumorigenesis, without disrupting normal epidermal proliferation or differentiation. These findings define essential non-redundant roles for IQGAP1 and IQGAP3 in the epidermis and demonstrate the potential of IQGAP antagonism for cancer therapy.