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。

关键词： IQGAP1   squamous cell carcinoma   inverted papilloma   tumor invasion   metastasis   Level of Evidence: NA  

摘要： Objectives/Hypothesis: The scaffold protein IQGAP1 is a ubiquitously expressed 190 kDa protein that participates in protein-protein interactions, integrating diverse signaling pathways. In this study, we examined the expression of IQGAP1 in sinonasal inverted papillomas (IP) and sinonasal squamous cell carcinomas (SCC), which include IP with SCC (IPcSCC) and SCC alone. We also tried to elucidate whether the amount of IQGAP1 expression is related with any clinicopathologic features of sinonasal SCCs. Study Design: This was a retrospective study. Methods: The expression of IQGAP1 was analyzed by means of immunohistochemistry. The degrees of IQGAP1 expression and subcellular localization (rim formation) were analyzed with respect to the following clinicopathologic variables: age, gender, T stage, histologic differentiation, local recurrence, and distant metastasis. Results: The expression of IQGAP1 was significantly higher in patients with IPcSCC (3.62 +/- 0.14), and SCC (2.93 +/- 0.34) compared with IP (1.43 +/- 0.26). The rim formation scale was significantly higher in IPcSCC (2.39 +/- 0.24) compared with IP (1.68 +/- 0.15). Distant metastasis in patients with SCC significantly correlated with an increased IQGAP1 rim formation (p = 0.011). Histologic differentiation was also significantly correlated with a positive staining scale for IQGAP1 in IPcSCCs (p = 0.038). Conclusion: These results indicated that IQGAP1 expression is associated with both the histologic differentiation of IPcSCCs and is present in SCC cases where distant metastasis has occurred.

关键词： CHD   Cytoskeletal scaffolding protein   Calcium-dependent interaction   Protein-protein crosslinking   ANS displacement assay  

摘要： IQGAPs are cytoskeletal scaffolding proteins which collect information from a variety of signalling pathways and pass it on to the microfilaments and microtubules. There is a well-characterised interaction between IQGAP and calmodulin through a series of IQ-motifs towards the middle of the primary sequence. However, it has been shown previously that the calponin homology domain (CHD), located at the N-terminus of the protein, can also interact weakly with calmodulin. Using a recombinant fragment of human IQGAP1 which encompasses the CHD, we have demonstrated that the CHD undergoes a calcium ion-dependent interaction with calmodulin. The CHD can also displace the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulphonate from calcium-calmodulin, suggesting that the interaction involves non-polar residues on the surface of calmodulin. Molecular modelling identified a possible site on the CHD for calmodulin interaction. The physiological significance of this interaction remains to be discovered.

关键词： Gastric cancer   Small interfering RNA   Cyclins   Cell proliferation   Transfection   MTT assay   Cell cycle and cell division   Co-immunoprecipitation  

摘要： Our previous research results showed that both Ras homolog family member C (RhoC) and IQ-domain GTPase-activating protein 1 (IQGAP1) were over-expressed in gastric cancer tissues and cells, but their role in tumorigenensis has not been addressed clearly. Herein we reported the proliferation stimulating effect of RhoC and IQGAP1 on gastric cancer cells and the interaction between two proteins in regulating the proliferation of gastric cancer cells. Plasmids and viral constructs encoding target siRNA and DNA were used to alter the expression of RhoC and IQGAP1. MTT method and BrdU incorporation assay were used for analyzing the effect of RhoC and different structures of IQGAP1 on proliferation. Protein levels of IQGAP1 and RhoC in cell lines were detected by Western blotting. Immunofluorescence and Co-Immunoprecipitation assays were applied to investigate the localization and binding between RhoC and IQGAP1. The results showed that RhoC, IQGAP1 and the C-terminal fragment of IQGAP1 significantly stimulated the proliferation of gastric cancer cells, and enhanced the expression of cyclin E and cyclin D1. By contrast, reduction of endogenous IQGAP1 or RhoC by siRNA attenuated cell proliferation. The depletion of IQGAP1 expression by siRNA significantly blocked the proliferative activity of constitutively active RhoC, while RhoC silencing by siRNA had no effect on IQGAP1-induced proliferation in gastric cancer cells. Co-immunoprecipitation and Immunofluorescence assays showed that RhoC and IQGAP1 bound each other. In conclusion, our results suggest that RhoC stimulates the proliferation of gastric cancer cells through recruiting IQGAP1 as an effector.

关键词： Breast Cancer   Cell Migration   Cell Proliferation   Protein-Protein Interactions   Rho GTPases   IQGAP1   Rho Effector   Rho Isoforms  

摘要： We performed a proteomics screen for Rho isoform-specific binding proteins to clarify the tumor-promoting effects of RhoA and C that contrast with the tumor-suppressive effects of RhoB. We found that the IQ-motif-containing GTPase-activating protein IQGAP1 interacts directly with GTP-bound, prenylated RhoA and RhoC, but not with RhoB. Co-immunoprecipitation of IQGAP1 with endogenous RhoA/C was enhanced when RhoA/C were activated by epidermal growth factor (EGF) or transfection of a constitutively active guanine nucleotide exchange factor (GEF). Overexpression of IQGAP1 increased GTP-loading of RhoA/C, while siRNA-mediated depletion of IQGAP1 prevented endogenous RhoA/C activation by growth factors. IQGAP1 knockdown also reduced the amount of GTP bound to GTPase-deficient RhoA/C mutants, suggesting that IQGAP enhances Rho activation by GEF(s) or stabilizes Rho-GTP. IQGAP1 depletion in MDA-MB-231 breast cancer cells blocked EGF- and RhoA-induced stimulation of DNA synthesis. Infecting cells with adenovirus encoding constitutively active RhoA(L63) and measuring absolute amounts of RhoA-GTP in infected cells demonstrated that the lack of RhoA(L63)-induced DNA synthesis in IQGAP1-depleted cells was not due to reduced GTP-bound RhoA. These data suggested that IQGAP1 functions downstream of RhoA. Overexpression of IQGAP1 in MDA-MB-231 cells increased DNA synthesis irrespective of siRNA-mediated RhoA knockdown. Breast cancer cell motility was increased by expressing a constitutively-active RhoC(V14) mutant or overexpressing IQGAP1. EGF- or RhoC-induced migration required IQGAP1, but IQGAP1-stimulated migration independently of RhoC, placing IQGAP1 downstream of RhoC. We conclude that IQGAP1 acts both upstream of RhoA/C, regulating their activation state, and downstream of RhoA/C, mediating their effects on breast cancer cell proliferation and migration, respectively.

关键词： Chlamydophila (Chlamydia) pneumoniae   VSMC   Cell adhesion   Cell migration   IQGAP1   ShRNA  

摘要： The mechanisms for Chlamydophila (Chlamydia) pneumoniae (C. pneumoniae) infection-induced atherosclerosis are still unclear. Cell adhesion has important roles in vascular smooth muscle cell (VSMC) migration required in the development of atherosclerosis. However, it is still unknown whether IQ domain GTPase-activating protein 1 (IQGAP1) plays pivotal roles in C. pneumoniae infection-induced the adhesion and migration of rat primary VSMCs. Accordingly, in this study, we demonstrated that rat primary VSMC adhesion (P < 0.001) and migration (P < 0.01) measured by cell adhesion assay and Transwell assay, respectively, were significantly enhanced after C. pneumoniae infection. Reverse transcription-polymerase chain reaction analysis revealed that the mRNA expression levels of IQGAP1 in the infected rat primary VSMCs were found to increase gradually to reach a peak and then decrease gradually to a level similar to the control. We further showed that the increases in rat primary VSMC adhesion to Matrigel (P < 0.001) and migration (P < 0.01) caused by C. pneumoniae infection were markedly inhibited after IQGAP1 knockdown by a pool of four short hairpin RNAs. Taken together, our results suggest that C pneumoniae infection may promote the adhesion and migration of VSMCs possibly by upregulating the IQGAP1 expression. (c) 2012 Elsevier Ltd. All rights reserved.