关键词： 博莱霉素   Akt   GSK3β   NF-κB   气道上皮细胞   甲醛   IQGAP1   β-连环素/Tcf信号   APC   气道上皮细胞  

摘要： 第一部分 博莱霉素致气道上皮细胞毒性损伤中GSK3β的活性变化对NF-κB信号通路的影响 实验背景 博莱霉素是一种有效的化疗药物，但所引起的急性肺损伤与肺纤维化的副作用却不容忽视。气道上皮是呼吸系统对抗毒性刺激的第一道防线，因此气道上皮细胞通常被认为是博莱霉素肺毒性的主要靶点。暴露于博莱霉素的细胞可发生各种细胞反应，包括细胞凋亡、细胞坏死及前炎症因子分泌等，这些细胞反应共同促进了博莱霉素导致的肺损伤与肺纤维化的发展过程。大量研究显示，核因子-κB(nuclear factor-κB，NF-κB)信号参与调节细胞存活、细胞分化、增殖、凋亡及炎症过程的多种基因的转录，与博莱霉素引起的肺毒性密切相关。 正常情况下，NF-κB与抑制蛋白IκB相结合并隐匿于静息细胞的胞浆，保持无活性的状态。外界刺激可引起IκB进入泛素化降解途径从而使NF-κB被释放并转位入核，激活下游靶基因的转录。糖原合成酶激酶3(glycogen synthase kinase 3，GSK3)是一种多功能的丝／苏氨酸蛋白激酶，与NF-κB不同，GSK3β在静息细胞内呈组成性激活的状态，其N末端丝氨酸残基可发生PI3K(phosphatidylinositol3-kinase)／Akt(also called protein kinase B，PKB)依赖性的磷酸化并导致其活性抑制。近年来的研究显示，GSK3β可能参与调节与NF-κB信号相关的多种疾病的病理生理过程，但具体机制并不明确。因此，本研究针对GSK3β在博莱霉素肺毒性中的活性变化及其对NF-κB信号通路的影响进行了初步探讨。 实验目的 本实验使用博莱霉素刺激建立体外肺毒性损伤模型，初步探讨在博莱霉素作用下气道上皮细胞中GSK3β的活性变化对NF-κB信号通路的影响，从而进一步分析肺纤维化发生发展的机制。 实验方法 使用博莱霉素刺激建立体外肺毒性损伤模型，首先采用相差显微镜及细胞荧光染色观察气道上皮细胞在博莱霉素刺激下的形态变化，并进一步使用MTT方法检测不同浓度下细胞活力的变化。然后使用Western blot、免疫荧光共聚焦成像、核浆分离、免疫共沉淀、瞬时转染及报告基因分析等方法观测博莱霉素处理后NF-κB信号的活化状态、Akt／GSK3β通路的活性变化及其对NF-κB信号的影响。 实验结果 相差显微镜下观察显示培养的气道上皮细胞呈现上皮细胞典型的铺路石状形态，其特点是立体的，稍微凸起的，细胞间紧密连接。经博莱霉素毒性刺激后，细胞间隙增宽，伸展扁平，立体感消失，死细胞增多，在荧光显微镜下可见大量细胞核呈现皱缩变圆、亮蓝色的凋亡形态。MTT结果显示博莱霉素可引起浓度依赖性的细胞活力降低。 然后，我们使用瞬时转染及报告基因分析方法发现博莱霉素可引起气道上皮细胞中NF-κB信号的活化，而且在博莱霉素刺激2 h时，NF-κB的抑制蛋白IκBα迅速发生降解并几近消失，同时核浆分离实验及免疫荧光共聚焦结果均显示NF-κB亚单位p65发生了向核内的转位，与文献所报道的体内实验的结果相符。接着我们发现，博莱霉素刺激以后，Akt与GSK3β发生了明显的磷酸化，而使用抑制剂LY294002抑制二者的磷酸化以后，NF-κB信号的活性也随之降低。因此我们推测，博莱霉素所引起的NF-κB信号的活化必与GSK3β的活化状态有着密切的关系。于是我们进一步使用持续激活型GSK3βS9A质粒转染入细胞后再使用博莱霉素刺激，结果发现GSK3β的持续活化抑制了博莱霉素诱导的NF-κB信号活化，并保护内源性IκBα不受降解，继而阻止了NF-κB亚单位p65转位入核。为了更深入的探究这种现象的可能原因所在，我们使用免疫共沉淀的方法将GSK3β与IκBα互相沉淀，发现二者可以共同沉淀下来，并在博莱霉素刺激后共同沉淀的蛋白减少，而在沉淀的IκBα蛋白中并未检测到磷酸化的GSK3β，提示GSK3β与IκBα存在于同一个复合物中，并且很有可能对IκBα有着某种机制的保护作用，进一步说明GSK3β可能通过与IκBα的这种相互作用从而影响了博莱霉素诱导的NF-κB信号活化。 实验结论 本实验表明：(1)博莱霉素可导致气道上皮细胞发生形态学改变及细胞活力降低。(2)博莱霉素可诱导气道上皮细胞NF-κB信号的活化。(3)博莱霉素诱导的气道上皮细胞中Akt及GSK3β的磷酸化可能参与了NF-κB信号的活化。(4)GSK3β的活性改变可能通过与NF-κB抑制蛋白IκBα的相互作用从而负向调节博莱霉素诱导的气道上皮细胞中NF-κB信号的活化过程。 第二部分 甲醛暴露致气道上皮毒性损伤中IQGAP1的表达变化对APC蛋白及

关键词： ACTIN   ESCHERICHIA coli   CALCIUM   CALMODULIN   CYTOSKELETON  

摘要： During infection, enteropathogenic Escherichia coli (EPEC) injects effector proteins into the host cell to manipulate the actin cytoskeleton and promote formation of actin pedestals. IQGAP1 is a multidomain protein that participates in numerous cellular functions, including Rac1/Cdc42 and Ca2+/calmodulin signaling and actin polymerization. Here we report that IQGAP1, Ca2+, and calmodulin modulate actin pedestal formation by EPEC. Infection with EPEC promotes both the interaction of IQGAP1 with calmodulin and the localization of IQGAP1 and calmodulin to actin pedestals while reducing the interaction of IQGAP1 with Rac1 and Cdc42. IQGAP1-null fibroblasts display a reduced polymerization of actin in response to EPEC. In addition, antagonism of calmodulin or chelation of intracellular Ca2+ reduces EPEC-dependent actin polymerization. Furthermore, IQGAP1 specifically interacts with Tir in vitro and in cells. Together these data identify IQGAP1, Ca2+, and calmodulin as a novel signaling complex regulating actin pedestal formation by EPEC.

关键词： Phosphorylation   Endothelial cells   Immunoprecipitation   Small interfering RNA   Angiogenesis   Tyrosine   VEGF signaling   Protein kinases  

摘要： Background: Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood. Methodology/Principal Findings: In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an in vivo angiogenesis model of chicken chorioallantoic membrane (CAM). Conclusions/Significance: Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2;it phosphorylates VEGFR-2 at Y1173, facilitates association and activation of IQGAP1 and other signaling proteins to VEGFR-2. IQGAP1-dependent signaling, in part, is critically required for endothelial cell proliferation, a key step in angiogenesis. Thus, Y1057 of VEGFR-2 serves to regul

关键词： Ovarian Cancer   Ovarian Carcinoma   Ovarian Cancer Cell   Ovarian Cancer Cell Line   Cell Migration Assay  

摘要： Background: IQGAP1 is a scaffolding protein and overexpressed in many human tumors, including ovarian cancer. However, the contribution of IQGAP1 to invasive properties of ovarian cancer cells remains unknown. Here, we investigated the effect of IQGAP1-specific short hairpin RNA (shRNA) expressing plasmids on metastatic potential of ovarian cancer HO-8910PM cells. Methods: We used RT-PCR and Western blot analysis to characterize expression of IQGAP1 in three human ovarian cancer-derived cell lines SK-OV-3, HO-8910 and HO-8910PM. We then determined whether expression of endogenous IQGAP1 correlated with invasive and migratory ability by using an in vitro Matrigel assay and cell migration assay. We further knocked down IQGAP1 using shRNA expressing plasmids controlled by U1 promoter in HO-8910PM cells and examined the proliferation activity, invasive and migration potential of IQGAP1 shRNA transfectants using MTT assay, in vitro Matrigel-coated invasion assay and migration assay. Results: IQGAP1 expression level seemed to be closely associated with the enhanced invasion and migration in ovarian cancer cell lines. Levels of both IQGAP1 mRNA and protein were significantly reduced in HO-8910PM cells transfected with plasmid-based IQGAP1-specific shRNAs. RNAimediated knockdown of IQGAP1 expression in HO-8910PM cells resulted in a significant decrease in cell invasion and migration. Conclusion: Our findings support the hypothesis that IQGAP1 promotes tumor progression and identify IQGAP1 as a potential therapeutic strategy for ovarian cancer and some other tumors with over-expression of the IQGAP1 gene.

关键词： IQ序列三磷酸鸟苷酶激活蛋白   细胞分裂周期42蛋白   气态甲醛   气道上皮   损伤修复  

摘要： 目的探讨包含IQ序列的三磷酸鸟苷酶(GTP)激活蛋白1(IQGAP1)和细胞分裂周期42蛋白(Cdc42)在气道上皮细胞损伤修复不同阶段的作用。方法选用健康昆明种小鼠24只,5 mg/m3气态甲醛,染毒4 h/d,分别染毒3,7和60 d后处死,采用免疫组织化学染色、蛋白印迹(Western blot)检测和免疫荧光共聚焦成像方法,观察IQ-GAP1和Cdc42蛋白在染毒小鼠气道上皮细胞损伤修复过程中的表达和定位变化。结果IQGAP1在染毒3和7 d时表达明显增强(P<0.01),60 d时恢复至对照组水平;Cdc42蛋白在染毒3 d时表达明显减弱(P<0.01),7 d时表达有所恢复。对照组中IQGAP1主要定位于气道上皮细胞的胞膜处,而Cdc42蛋白弥散分布于气道上皮细胞(AECs)胞质中;3 d和7 d时共同定位于细胞极化前缘;60 d时其定位与对照组相似。结论Cdc42蛋白和IQGAP1在气道上皮细胞损伤修复过程中的表达及定位呈动态变化,提示其在这一过程中发挥着重要作用。

关键词： Calcium ions   Cytoskeleton   EF-hand   IQ motif   Mlc1sa   Native gel  

摘要： IQGAPs are cytoskeletal scaffolding proteins which link signalling pathways to the reorganisation of actin and microtubules. Human IQGAP1 has four IQ motifs each of which binds to calmodulin. The same region has been implicated in binding to two calmodulin-like proteins, the myosin essential light chain Mlc1sa and the calcium and zinc ion binding protein S100B. Using synthetic peptides corresponding to the four IQ motifs of human IQGAP1, we showed by native gel electrophoresis that only the first IQ motif interacts with Mlc1sa. This IQ motif, and also the fourth, interacts with the budding yeast myosin essential light chain Mlc1p. The first and second IQ motifs interact with S100B in the presence of calcium ions. This clearly establishes that S100B can interact with its targets through IQ motifs in addition to interacting via previously reported sequences. These results are discussed in terms of the function of IQGAP1 and IQ motif recognition.

关键词： actin cytoskeleton   cleaving embryo   meiotic spindle   oocyte   maturation   Cdc42   IQGAP1   toxin B   cytokinesis   oogenesis  

摘要： Cdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 Was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and Mitotic Cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte. (c) 2008 Elsevier Inc. All rights reserved.

关键词： scleroderma   lung fibrosis   connective tissue growth factor   cell migration  

摘要： Bogatkevich GS, Ludwicka-Bradley A, Singleton CB, Bethard JR, Silver RM. Proteomic analysis of CTGF- activated lung fibroblasts: identification of IQGAP1 as a key player in lung fibroblast migration. Am J Physiol Lung Cell Mol Physiol 295: L603-L611, 2008. First published August 1, 2008;doi: 10.1152/ajplung.00530.2007.-Connective tissue growth factor ( CTGF, CCN2) is overexpressed in lung fibroblasts isolated from patients with interstitial lung disease (ILD) and systemic sclerosis (SSc, scleroderma) and is considered to be a molecular marker of fibrosis. To understand the significance of elevated CTGF, we investigated the changes in lung fibroblast proteome in response to CTGF overexpression. Using 2-dimensional gel electrophoresis followed by in-gel proteolytic digestion and mass spectrometric analysis, we identified 13 proteins affected by CTGF. Several of the CTGF- induced proteins, such as pro-alpha ( I) collagen and cytoskeletal proteins vinculin, moesin, and ezrin, are known to be elevated in pulmonary fibrosis, whereas 9 of 13 proteins have not been studied in pulmonary fibrosis and are, therefore, novel CTGF- responsive molecules that may have important roles in ILD. Our study demonstrates that 1 of the novel CTGF- induced proteins, IQ motif containing GTPase activating protein ( IQGAP) 1, is elevated in lung fibroblasts isolated from scleroderma patients with ILD. IQGAP1 is a scaffold protein that plays a pivotal role in regulating migration of endothelial and epithelial cells. Scleroderma lung fibroblasts and normal lung fibroblasts treated with CTGF demonstrated increased rate of migration in a wound healing assay. Depletion of IQGAP1 expression by small interfering RNA inhibited CTGF- induced migration and MAPK ERK1/2 phosphorylation in lung fibroblasts. MAPK inhibitor U0126 decreased CTGF- induced cell migration and did not interfere with CTGF- induced IQGAP1 expression, suggesting that MAPK pathway is downstream of IQGAP1. These findings further impli

关键词： Rho GTPases   IQGAP1   Proliferation   Injury and repair   Bronchial epithelial cells (BECs)  

摘要： The process of injury and repair involves spreading, migration and cell proliferation. The functions of Rho GTPases and their effector IQGAP1 are poor known in this process of airway epithelium. In the present study, we employed a widely used in vitro model by scratching a monolayer of BECs. We found that scratching induced decreasing of the GTP-bound Rac1 and Cdc42, but increasing the amounts of IQGAP1 at different time points. Next, we confirmed that IQGAP1 interacted with the constitutively active Rac1 (Rac1(V12)) and Cdc42(Cdc42(V12)) rather than the dominant negative Rac1 (Rac1(N17)) and Cdc42 (Cdc42(N17)). Over-expressions of wild type (WT) IQGAP1 and its mutant (T1050AX2), which was defective to interact with Rho GTPases, induced translocation of beta-catenin from the cytoplasm into the nucleus. These results activated Tcf/Lef and increased the expression levels of its target genes of c-myc and cyclin D1. Likewise, the amounts of c-myc and cyclin D1 increased after scratching. Our results suggested that IQGAP1 mediated cell proliferation through activating Tcf in a manner independent of Racl and Cdc42 in wound repair of BECs. (C) 2008 Elsevier Inc. All rights reserved.

关键词： GROWTH-FACTOR RECEPTOR   PROTEIN-KINASE   ACTIN-BINDING   CELL-ADHESION   MAP KINASE   CALMODULIN   ACTIVATION   CALCIUM   CDC42   CA2+  

摘要： Ca2+ and calmodulin modulate numerous cellular functions, ranging from muscle contraction to the cell cycle. Accumulating evidence indicates that Ca2+ and calmodulin regulate the MAPK signaling pathway at multiple positions in the cascade, but the molecular mechanism underlying these observations is poorly defined. We previously documented that IQGAP1 is a scaffold in the MAPK cascade. IQGAP1 binds to and regulates the activities of ERK, MEK, and B-Raf. Here we demonstrate that IQGAP1 integrates Ca2+ and calmodulin with B-Raf signaling. In vitro analysis reveals that Ca2+ promotes the direct binding of IQGAP1 to B-Raf. This interaction is inhibited by calmodulin in a Ca2+-regulated manner. Epidermal growth factor (EGF) is unable to stimulate B-Raf activity in fibroblasts treated with the Ca2+ ionophore A23187. In contrast, chelation of intracellular free Ca2+ concentrations ([Ca2+](i)) significantly enhances EGF-stimulated B-Raf activity, an effect that is dependent on IQGAP1. Incubation of cells with EGF augments the association of B-Raf with IQGAP1. Moreover, Ca2+ regulates the association of B-Raf with IQGAP1 in cells. Increasing [Ca2+](i) with Ca2+ ionophores significantly reduces co-immunoprecipitation of B- Raf and IQGAP1, whereas chelation of Ca2+ enhances the interaction. Consistent with these findings, increasing and decreasing [Ca2+](i) increase and decrease, respectively, co-immunoprecipitation of calmodulin with IQGAP1. Collectively, our data identify a previously unrecognized mechanism in which the scaffold protein IQGAP1 couples Ca2+ and calmodulin signaling to B-Raf function.