关键词： Animal models   Beta-catenin signaling   Biochemistry   Biology   Developmental biology   Developmental signaling   Embryology   Mechanisms of signal transduction   Model organisms   Molecular cell biology   Molecular development   Protein interactions   Proteins   Research Article   Signal transduction   Signaling   Signaling cascades   Signaling in cellular processes   Signaling in selected disciplines   Signaling pathways   Xenopus laevis  

摘要： Dishevelled (DVL) is a central factor in the Wnt signaling pathway, which is highly conserved among various organisms. DVL plays important roles in transcriptional activation in the nucleus, but the molecular mechanisms underlying their nuclear localization remain unclear. In the present study, we identified IQGAP1 as a regulator of DVL function. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of DVL, and expression of Wnt target genes during early embryogenesis. The domains in DVL and IQGAP1 that mediated their interaction are also required for their nuclear localization. Endogenous expression of Wnt target genes was reduced by depletion of IQGAP1 during early embryogenesis, but notably not by depletion of other IQGAP family genes. Moreover, expression of Wnt target genes caused by depletion of endogenous IQGAP1 could be rescued by expression of wild-type IQGAP1, but not IQGAP1 deleting DVL binding region. These results provide the first evidence that IQGAP1 functions as a modulator in the canonical Wnt signaling pathway.

关键词： 乳腺肿瘤   支架蛋白   IQGAP1   Transwell   Westenblot  

摘要： 目的:通过免疫组化方法探讨支架蛋白IQGAP1在乳腺癌中的表达及意义。方法:(1)选取乳腺癌、癌旁组织及良性病变组织的存档蜡块,制成组织芯片;(2)通过免疫组织化学EnVision二步法检测IQGAP1蛋白的表达情况,运用SPSS 13.0进行统计学分析。结果:(1)82例导管癌组织标本中69例为阳性表达,81例癌旁组织标本中63例为阳性表达,两者差异无统计学意义(P>0.05);28例良性病变中9例为胞浆阳性表达,与癌组织比较差异有显著统计学意义(P<0.001)。(2)导管癌82例阳性表达标本中组织学分级为3级者膜阳性表达为12例(12/14),差异有统计学意义(P<0.05),2级为29例(29/47),差异无统计学意义(P>0.05),1级为0例(0/8),差异有显著统计学意义(P<0.001)。(3)肿瘤组织呈条索样生长的标本阳性表达有27例,其中膜表达23例;非条索样生长的标本阳性表达42例,膜表达18例,两者差异有显著统计学意义(P<0.001)。结论:(1)IQGAP1蛋白表达部位与肿瘤分化程度有关,并可能参与肿瘤局部浸润的微环境变化;(2)IQGAP1蛋白与肿瘤细胞的迁徙运动密切相关。

关键词： MYOFIBROBLASTS   METASTASIS   UBIQUITIN   CELLULAR signal transduction   CELL membranes   PERICYTES  

摘要： In the tumor microenvironment, TGF-beta induces transdifferentiation of quiescent pericytes and related stromal cells into myofibroblasts that promote tumor growth and metastasis. The mechanisms governing myofibroblastic activation remain poorly understood, and its role in the tumor microenvironment has not been explored. Here, we demonstrate that IQ motif containing GTPase activating protein 1 (IQGAP1) binds to TGF-beta receptor II (T beta RII) and suppresses T beta RII-mediated signaling in pericytes to prevent myofibroblastic differentiation in the tumor microenvironment. We found that TGF-beta 1 recruited IQGAP1 to T beta RII in hepatic stellate cells (HSCs), the resident liver pericytes. Iqgap1 knockdown inhibited the targeting of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor 1 (SMURF1) to the plasma membrane and T beta RII ubiquitination and degradation. Thus, Iqgap1 knockdown stabilized T beta RII and potentiated TGF-beta 1 transdifferentiation of pericytes into myofibroblasts in vitro. Iqgap1 deficiency in HSCs promoted myofibroblast activation, tumor implantation, and metastatic growth in mice via upregulation of paracrine signaling molecules. Additionally, we found that IQGAP1 expression was downregulated in myofibroblasts associated with human colorectal liver metastases. Taken together, our studies demonstrate that IQGAP1 in the tumor microenvironment suppresses T beta RII and TGF-beta dependent myofibroblastic differentiation to constrain tumor growth.

关键词： Anatomy and physiology   Biochemistry   Biology   Cell differentiation   Cellular neuroscience   Developmental biology   Developmental neuroscience   Immunochemistry   Medicine   Molecular cell biology   Morphogenesis   Neurochemistry   Neurological system   Neuroscience   Physiology   Research Article   Synaptic plasticity  

摘要： IQGAP1 is a scaffolding protein that regulates spine number. We now show a differential role for IQGAP1 domains in spine morphogenesis, in which a region of the N-terminus that promotes Arp2/3-mediated actin polymerization and branching stimulates spine head formation while a region that binds to Cdc42 and Rac is required for stalk extension. Conversely, IQGAP1 rescues spine deficiency induced by expression of dominant negative Cdc42 by stimulating formation of stubby spines. Together, our observations place IQGAP1 as a crucial regulator of spine number and shape acting through the N-Wasp Arp2/3 complex, as well as upstream and downstream of Cdc42.

关键词： Breast cancer   Cancer cell migration   Invasive tumors   Guanosine triphosphatase   Breast tumors   Co-immunoprecipitation   Immunostaining   Cell motility  

摘要： beta-arrestins play critical roles in chemotaxis and cytoskeletal reorganization downstream of several receptor types, including G protein-coupled receptors (GPCRs), which are targets for greater than 50% of all pharmaceuticals. Among them, receptors for lysophosphatidic acid (LPA), namely LPA(1) are overexpressed in breast cancer and promote metastatic spread. We have recently reported that beta-arrestin2 regulates LPA(1)-mediated breast cancer cell migration and invasion, although the underlying molecular mechanisms are not clearly understood. We show here that LPA induces activity of the small G protein, Rap1 in breast cancer cells in a beta-arrestin2-dependent manner, but fails to activate Rap1 in non-malignant mammary epithelial cells. We found that Rap1A mRNA levels are higher in human breast tumors compared to healthy patient samples and Rap1A is robustly expressed in human ductal carcinoma in situ and invasive tumors, in contrast to the normal mammary ducts. Rap1A protein expression is also higher in aggressive breast cancer cells (MDA-MB-231 and Hs578t) relative to the weakly invasive MCF-7 cells or non-malignant MCF10A mammary cells. Depletion of Rap1A expression significantly impaired LPA-stimulated migration of breast cancer cells and invasiveness in three-dimensional Matrigel cultures. Furthermore, we found that beta-arrestin2 associates with the actin binding protein IQGAP1 in breast cancer cells, and is necessary for the recruitment of IQGAP1 to the leading edge of migratory cells. Depletion of IQGAP1 blocked LPA-stimulated breast cancer cell invasion. Finally, we have identified that LPA enhances the binding of endogenous Rap1A to beta-arrestin2, and also stimulates Rap1A and IQGAP1 to associate with LPA1. Thus our data establish novel roles for Rap1A and IQGAP1 as critical regulators of LPA-induced breast cancer cell migration and invasion.

关键词： IQGAP1蛋白   气道上皮细胞   损伤修复   肺癌   侵袭转移  

摘要： IQGAP1蛋白为小的真核细胞蛋白家族中的一员，在细胞增殖、分化、转移等过程中起着重要作用。最近研究发现其参与了气道炎性损伤、肺癌侵袭转移等呼吸系统疾病变化。本文就IQGAP1蛋白的结构和功能、在呼吸系统疾病中的调控作用作一综述。

关键词： 非小细胞肺癌   脑转移   IQGAP1  

摘要： 目的:探讨IQGAP1的表达对非小细胞肺癌脑转移的影响。方法:应用Western blotting技术检测肺癌低潜力脑转移细胞与高潜力脑转移细胞中IQGAP1表达的差异;构建质粒载体pcDNA3.1-IQGAP1,将其转染至非小细胞型肺癌低潜力脑转移细胞系,并用RT-PCR方法鉴定转染效果;进而应用Transwell的方法和免疫荧光检测上调IQGAP1表达后对细胞侵袭和迁移的影响。结果:通过Western blot检测,PC14/B细胞内IQGAP1表达明显高于PC14细胞;外源性高表达IQGAP1的质粒载体pcDNA3.1-IQGAP1成功转染至PC14细胞,同时细胞形态经过转染后也发生显著变化;外源性上调的IQGAP1表达显著增强了非小细胞肺癌细胞株PC14的细胞侵袭和迁移能力。结论:IQGAP1在肺癌脑转移的过程中可能发挥了作用,为进一步研究肺癌脑转移的具体机制提供了新的思路。

关键词： FaS   GTP酶激活蛋白质类   血管紧张素Ⅱ   肾小球   细胞凋亡   细胞外   调节蛋白激酶1   2  

摘要： 目的研究血管紧张素Ⅱ（AngⅡ）输注对大鼠肾小球rasGTP酶活化蛋白（IQGAP1）表达及细胞凋亡的影响，探讨IQGAP1在AngⅡ诱导肾小球细胞凋亡中的作用。方法36只雄性Wistar大鼠按随机数字法分为正常对照组、生理盐水输注组和AngⅡ输注组，每隔7d测量大鼠血压及尿蛋白量。分别于实验第14天、第28天处死动物取。肾，PAS染色观察肾组织病理学改变；免疫组化、免疫荧光法观察IQGAP1在肾小球的表达及分布；Western印迹法检测。肾组织IQGAP1、半胱氨酸蛋白酶3（caspase．3）及细胞外调节蛋白激酶1／2（ERK1／2）蛋白表达；TUNEL法检测肾小球细胞凋亡。结果AngⅡ输注组大鼠血压升高，实验第14天达峰值并维持在较高水平；第7天出现蛋白尿，且随时间的延长尿蛋白量持续增加，与对照组比较，差异有统计学意义（P〈0．05）。AngⅡ输注组出现轻度系膜细胞增殖和系膜基质增加，生理盐水输注组和正常对照组均未见明显病理改变。TUNEL检测结果显示，AngⅡ输注后肾小球细胞凋亡显著增加；Western印迹发现，AngⅡ输注组大鼠肾小球caspase．3活化明显增加，与对照组比较，差异有统计学意义（P〈0．05）。正常对照组IQGAP1呈低水平表达并沿毛细血管袢线性分布，AngⅡ输注后IQGAP1表达量显著增加（P〈0．05），且其蛋白表达量与caspase-3活化量呈正相关（r=0．689，P〈0．05）。与对照组比较，AngⅡ输注组磷酸化的ERK1／2（p-ERK1／2）表达量显著增加（P〈0．05），且与IQGAP1蛋白表达呈正相关（r=0．658，P〈0．05）。结论IQGAP1表达上调可能通过激活ERK1/2信号通路参与AngⅡ诱导肾小球细胞凋亡的病理过程。

关键词： Rac1   肺炎衣原体   血管平滑肌细胞   细胞迁移   PI3K   IQGAP1  

摘要： 目的 利用肺炎衣原体(Chlamydia pneumoniae, ***)体外感染大鼠原代血管平滑肌细胞(Vascular smooth muscle cell, VSMC)模型,观察Racl活化在***感染诱导VSMC迁移中的作用以及在此过程中Racl活化与PI3K、IQGAP1的联系,探讨其相关分子机制。 方法 ***增殖培养后体外感染大鼠原代VSMC; 2. GST pull-down实验检测***感染VSMC后不同时间点Racl的活性； 3.用Racl特异性抑制剂NSC23766抑制Rac1的活性,GST pull-down实验检测VSMC内GTP-Racl的表达变化,Wound-healing实验和Transwell实验观察VSMC迁移能力的改变； 4.用PI3K特异性抑制剂LY294002抑制PI3K的活性,GST pull-down实验检测VSMC内Rac1活性的变化,明确PI3K在***感染诱导VSMC内Rac1活化过程中的作用； 5.用Rac1特异性抑制剂NSC23766抑制Racl的活性,Western blot实验检测VSMC内IQGAP1表达的变化,明确***感染后VSMC内Racl活化对其下游效应蛋白IQGAP1的影响。 结果 1.目的质粒转染大肠杆菌,体外制备足量有效与GTP-Racl特异结合的GST-PBD融合蛋白；分别于***感染VSMC0h、0.5h、1h、2h、3h、6h、8h和12h后提取总蛋白,GST pull-down实验结果显示,***感染VSMC0.5h后GTP-Racl表达水平开始升高；随着感染时间延长其表达水平逐渐升高,3h左右达高峰,8h开始下降,并持续至12h；而各时间点VSMC中总Racl表达水平无显著差异。 2. GST pull-down实验结果显示,使用Rac1特异性抑制剂NSC23766预处理VSMC后,***感染诱导的VSMC内GTP-Racl的表达水平明显降低。用NSC23766预处理的***感染组的VSMC内GTP-Racl的表达水平较单纯***感染组明显降低(P<0.05),差异有统计学意义；各组VSMC中总Rac1表达无显著差异。 3. Wound-healing实验结果显示,NSC23766预处理的***感染组VSMC向划痕中央迁移的面积明显小于单纯***感染组(P<0.05)；Transwell实验结果显示,NSC23766预处理的***感染组VSMC的穿膜细胞数明显低于单纯***感染组(P<0.05),差异有统计学意义。 4.使用PI3K抑制剂LY294002预处理VSMC, GST pull-down实验结果显示,LY294002预处理的***感染组VSMC内GTP-Racl的表达水平较单纯***感染组明显降低(P<0.05),差异有统计学意义；各组VSMC中总Rac1表达无显著差异。 5.使用Rac1特异性抑制剂NSC23766预处理VSMC, Western blot实验结果显示,50μMNSC23766预处理的***感染组VSMC内IQGAP1的表达水平虽较单纯***感染组有所下降(P>0.05),但差异无统计学意义；100μMNSC23766预处理的***感染组VSMC内IQGAP1的表达水平较单纯***感染组则明显降低(P<0.05),差异有统计学意义。 结论 ***感染可能通过激活Rac1促进VSMC迁移,且在此过程中Rac1的活化依赖PI3K的酶活性,活化的Rac1可影响其下游效应蛋白IQGAP1的表达,即***感染可能通过PI3K活化Rac1进而激活IQGAP1促进VSMC迁移。

关键词： 乳腺导管癌   具有IQ基序的GTP激酶活化蛋白   Transwell迁移实验  

摘要： 目的探讨支架蛋白IQGAP1在乳腺浸润性导管癌中的表达及沉默IQGAP1表达对肿瘤细胞迁移能力的影响。方法利用免疫组织化学染色和Western blotting技术检测IQGAP1蛋白在乳腺浸润性导管癌组织、癌旁组织以及良性乳腺病变组织中的表达;分析IQGAP1蛋白在乳腺浸润性导管癌组织中的表达与临床病理参数的关系。构建载有shRNA-IQGAP1的慢病毒表达载体,将其稳定转染高转移乳腺癌MDA-MB-435细胞,获得稳定抑制IQGAP1蛋白表达的细胞株(IQGAP1-shRNA组)及相应的空载细胞株(Vector组)、空白对照细胞株(Blank组);运用基于Transwell系统的体外迁移实验观察沉默IQGAP1的表达对MDA-MB-435细胞迁移能力的影响。结果 IQGAP1蛋白在乳腺浸润性导管癌组织中的阳性表达率为83.44%(136/163),与良性乳腺病变组织(32.14%,9/28)比较,差异具有统计学意义(P<0.01);IQGAP1蛋白在癌旁组织中的阳性表达率(77.78%,63/81)与乳腺浸润性导管癌组织比较,差异无统计学意义(P>0.05)。163例乳腺浸润性导管癌中WHO分级为3级者IQGAP1蛋白表达为阴性、胞质阳性和胞膜阳性的例数分别为1、3和22,2级者分别为21、40和61;1级者分别为5、9和1,差异具有统计学意义(P<0.01);IQGAP1蛋白的表达与肿瘤的大小有关(P<0.01),与淋巴结是否转移无关(P>0.05)。Blank组、Vector组和IQGAP1-shRNA组中穿过Transwell小室的平均细胞数分别为(294.3±16.3)、(287.7±16.9)和(93.5±7.6)个,IQGAP1-shRNA组显著低于其他两组(P<0.01)。结论 IQGAP1蛋白在乳腺浸润性导管癌组织中的表达明显高于良性乳腺病变组织;IQGAP1蛋白表达部位与肿瘤分化程度有关;IQGAP1蛋白在乳腺癌转移过程中起促进作用。