摘要： Leukocyte extravasation is a fundamental process of the inflammatory responses. The mechanisms that control remodelling of endothelial (EC) shape and adhesive contacts during leukocyte transendothelial migration (TEM) are not completely understood. We studied the role of EC phosphatidylinositol 3-kinase (PI3K) activity in lymphocyte TEM under shear stress conditions. Inhibition of EC PI3K activity by its pan inhibitors decreased lymphocyte diapedesis in a step after VE-cadherin opening. The importance of PI3K catalytic isoforms (p110α, p110β, p110δ and p110γ) were studied in TEM. Treatment of EC with isoform inhibitors of p110β, p110δ and p110γ did not affect lymphocyte TEM. Inhibition of p110α activity or expression reduced lymphocyte diapedesis. PI3K activity was measured in EC exposed to shear stress alone or shear stress on cells where ICAM-1 or VCAM-1 were cross-linked. The most significant effect was seen in cells cross-linked with ICAM-1 and exposed to shear stress. This suggests that cooperation of shear-induced mechanotransduction and ICAM-1 during leukocyte interaction with EC facilitates leukocyte diapedesis by inducing PI3K. We hypothesized that Rho GTP proteins downstream of PI3K activity are involved in leukocyte TEM. We studied the role of IQGAP1, a Rac1/Cdc42 effector, during lymphocyte TEM. EC IQGAP1 knockdown decreases both microtubule (MT) tethered to the adherens junction (AJ) and lymphocyte TEM. Similarly, loss of AJ-associated MT induced by brief nocodazole (ND) treatment decreases lymphocyte TEM. Neither intervention affected leukocyte migration to the interendothelial junctions. These data indicate that IQGAP1 contributes to MT stability at endothelial junctions and is involved in the junction remodelling required for efficient lymphocyte diapedesis. We studied a candidate Rho guanine nucleotide exchange factor named FGD5 upstream of IQGAP1. Inhibition of FGD5 expression resulted in more sensitivity to apoptotic stimuli and a higher rate of a

关键词： apoptosis   ECyd   focused proteomics   IQGAP1   RNase L  

摘要： Mammalian intracellular ribonuclease L ( RNase L) is a latent endoribonuclease that functions against viral infections as an apoptosis-inducing protein, and its activity requires intracellular 5'-end- triphosphorylated-2',5' oligoadenylates (2-5A) as an activator. Previously, we showed that RNase L can be activated in human cancer cell line HT1080 by an RNA polymerase I inhibitor, 1-(3-C-ethynyl-beta-d-ribo-pentofuranosyl) cytosine (3'-ethynylcytidine;ECyd). In ECyd-treated cells, knockdown of the RNase L resulted in a marked decrease in c-jun N-terminal kinase (JNK) phosphorylation, thereby inhibiting apoptosis. We investigate RNase L binding partners by focused proteomic approach using immunoprecipitation with anti-RNase L IgG and mass spectrometry. We found that the IQ motif-containing Ras GTPase-activating-like protein 1 (IQGAP1) can associate with RNase L, and that phosphorylation occurs on the IQGAP1. ECyd-induced JNK phosphorylation and apoptosis were inhibited when IQGAP1 was knocked down with a small interfering RNA. These results raise the interesting possibility that the RNase L-IQGAP1 association may regulate JNK phosphorylation in RNase L-madiated apoptosis. It is likely IQGAP1 works as a regulator in apoptosis.

关键词： Normal Liver   SNU387 Cell   Hepatic Adenoma   Liver Cancer Cell Line   Calponin Homology Domain  

摘要： Background: IQGAP1 and IQGAP2 are homologous members of the IQGAP family of scaffold proteins. Accumulating evidence implicates IQGAPs in tumorigenesis. We recently reported that IQGAP2 deficiency leads to the development of hepatocellular carcinoma (HCC) in mice. In the current study we extend these findings, and investigate IQGAP1 and IQGAP2 expression in human HCC. Methods: IQGAP1 and IQGAP2 protein expression was assessed by Western blotting and immunohistochemistry. IQGAP mRNA was measured by quantitative RT-PCR. The methylation status of the Iqgap2 promoter was determined by pyrosequencing of bisulfite-treated genomic DNA. Results: IQGAP1 and IQGAP2 expression was reciprocally altered in 6/6 liver cancer cell lines. Similarly, immunohistochemical staining of 82 HCC samples showed that IQGAP2 protein expression was reduced in 64/82 (78.0%), while IQGAP1 was present in 69/82 (84.1%). No IQGAP1 staining was detected in 23/28 (82.1%) normal livers, 4/4 (100.0%) hepatic adenomas and 23/23 (100.0%) cirrhosis cases, while IQGAP2 was increased in 22/28 (78.6%), 4/4 (100.0%) and 23/23 (100.0%), respectively. Although the Iqgap2 promoter was not hypermethylated in HCC at any of the 25 CpG sites studied (N = 17), IQGAP2 mRNA levels were significantly lower in HCC specimens (N = 23) than normal livers (N = 6). Conclusions: We conclude that increased IQGAP1 and/or decreased IQGAP2 contribute to the pathogenesis of human HCC. Furthermore, downregulation of IQGAP2 in HCC occurs independently of hypermethylation of the Iqgap2 promoter. Immunostaining of IQGAP1 and IQGAP2 may aid in the diagnosis of HCC, and their pharmacologic modulation may represent a novel therapeutic strategy for the treatment of liver cancer.

关键词： Animals   Macrophages/pathology   Mice   Mice, Knockout   Mice, Knockout   Neovascularization, Pathologic   ras GTPase-Activating Proteins/genetics   ras GTPase-Activating Proteins/physiology   Reverse Transcriptase Polymerase Chain Reaction  

摘要： Background: Neovascularization is an important repair mechanism in response to ischemic injury and is dependent on inflammation, angiogenesis and reactive oxygen species (ROS). IQGAP1, an actin-binding scaffold protein, is a key regulator for actin cytoskeleton and motility. We previously demonstrated that IQGAP1 mediates vascular endothelial growth factor (VEGF)-induced ROS production and migration of cultured endothelial cells (ECs);however, its role in post-ischemic neovascularization is unknown. Methodology/Principal Findings: Ischemia was induced by left femoral artery ligation, which resulted in increased IQGAP1 expression in Mac3(+) macrophages and CD31(+) capillary-like ECs in ischemic legs. Mice lacking IQGAP1 exhibited a significant reduction in the post-ischemic neovascularization as evaluated by laser Doppler blood flow, capillary density and a-actin positive arterioles. Furthermore, IQGAP1(-/-) mice showed a decrease in macrophage infiltration and ROS production in ischemic muscles, leading to impaired muscle regeneration and increased necrosis and fibrosis. The numbers of bone marrow (BM)-derived cells in the peripheral blood were not affected in these knockout mice. BM transplantation revealed that IQGAP1 expressed in both BM-derived cells and tissue resident cells, such as ECs, is required for post-ischemic neovascularization. Moreover, thioglycollate-induced peritoneal macrophage recruitment and ROS production were inhibited in IQGAP1(-/-) mice. In vitro, IQGAP1(-/-) BM-derived macrophages showed inhibition of migration and adhesion capacity, which may explain the defective macrophage recruitment into the ischemic tissue in IQGAP1(-/-) mice. Conclusions/Significance: IQGAP1 plays a key role in post-ischemic neovascularization by regulating, not only, ECs-mediated angiogenesis but also macrophage infiltration as well as ROS production. Thus, IQGAP1 is a potential therapeutic target for inflammation-and angiogenesis-dependent ischemic cardiovascular d

关键词： calponin   Actins   Domain   Homologous Sequences  

关键词： Human embryonic stemcells   microRNA   Cell migration   SLUG   IQGAP1  

摘要： Precise control of gene expression is of paramount importance for proper embryonic development. Although a number of microRNAs (miRNAs) has been implicated in fine-tuning mRNA translation during development, their exact roles for gastrulation, particularly in connection with functional targets, have yet to be clarified, with regard to stage-specific cell migration to form three embryonic germ layers. We found that miR-124a is expressed in human embryonic stem cells (hESC), but is gradually downregulated during embryoid body (EB) formation in vitro. We also provide evidence that SLUG and IQGAP1, which modulates rearrangement of the migratory cytoskeleton, are specific targets for miR-124a during EB forma-tion. Furthermore, we show that the beginning of cell migration, a hallmark event in gastrulation, is tightly coupled with downregulation of miR-124a during EB formation and induction of SLUG and IQGAP1. Overexpressed miR-124a in hESC reduced expression of SLUG and IQGAP1 and blocked migratory cell behavior in EB. An expression level of MIXL1, associated with gastulation process, was also inversely correlated with expression of miR-124a. Taken together, our results strongly suggest that miR-124a may play an active role in inhibiting hESCs from differentiation into EB by downregulating expression of SLUG and IQGAP1, thereby maintaining stemness. STEM CELLS 2010;28:1550-1559

关键词： carcinoma   hepatocellular   IQ motif containing GTPase activating protein   mTOR protein   oncogenes   proto-oncogene proteins c-akt  

摘要： The scaffold protein IQGAP1 shows elevated levels in several cancer types, but its expression in hepatocellular carcinoma is unknown. We found that 58% of human hepatocellular carcinoma tissue samples had increased IQGAP1 expression compared to adjacent normal tissue. Overexpressing IQGAP1 raised the in vivo tumorigenicity of hepatocellular carcinoma cells, and forced overexpression of IQGAP1 in vitro stimulated cell proliferation. Cell growth was reduced by knockdown or mutation of IQGAP1, or by treatment of cells with a phosphotidylinositol 3-kinase inhibitor. To determine the mechanism by which IQGAP1 overexpression affected hepatocellular carcinoma cells, we confirmed its interaction in these cells with mammalian target of rapamycin (mTOR), a serine/threonine kinase that integrates signals about nutrient and energy status with downstream effectors that influence cell division. In addition, we discovered a new interaction involving IQGAP1, mTOR and Akt, which is a downstream target of mTOR. Akt phosphorylation on Ser-473, which is catalyzed by mTOR and required for Akt activation, increased with increasing amounts of IQGAP1, and decreased with IQGAP1 mutation. We hypothesize that IQGAP1 is a scaffold that facilitates mTOR and Akt interaction.

关键词： 胃癌   IQGAP1   基因   蛋白质  

摘要： 目的检测含有IQ模体的Ras GTP酶活化蛋白1(IQ motif-containing GTPase activating protein 1,IQGAP1)在胃癌组织中的表达,分析其与临床病理参数的相关性。方法分别用RT-PCR和western blot,对29例胃癌患者的癌组织及其对应癌旁组织进行IQGAP1 mRNA和蛋白质表达的检测,分析胃癌与癌旁组织IQGAP1的表达差异及其与临床病理参数的关系。结果胃癌组织中IQGAP1的表达高于对应癌旁组织者的为75.9%(22/29);胃癌组织IQGAP1的表达在基因和蛋白质水平上均与肿瘤分化程度呈负相关(P<0.05)。结论胃癌组织中IQGAP1的表达可作为癌分化程度的参考指标。

关键词： IQGAP1   colorectal carcinoma   cell adhesion   tumor invasion   metastasis  

摘要： IQGAP1 is a multifunctional protein involved in actin cytoskeleton assembly and E-cadherin-mediated cell adhesion. We reported previously IQGAP1 overexpression in human colorectal carcinomas especially at the invasion front (IF) and that such overexpression tended to correlate with lymph node metastasis in advanced cases. Thus, in this study, we investigated the clinicopathological significance of IQGAP1 expression in 85 cases of pT2-3 colorectal carcinomas with special reference to its expression pattern and prognosis, followed by analysis of the role of IQGAP1 in cancer invasion in vitro. Quantitative reverse transcription-PCR showed significant upregulation of IQGAP1 in colorectal carcinomas compared with normal mucosa. Immunohistochemically, IQGAP1 expression pattern was classified into diffuse (20%), IF-associated (35.3%) and focal (44.7%). The diffuse pattern was associated with higher rates of distant metastasis. Patients with IQGAP1 overexpression and diffuse pattern had significantly shorter survival (p < 0.0001) than others, and the diffuse pattern was an independent predictor of poor survival by multivariate analysis. In vitro invasion assays using three human colon carcinoma cell lines showed that IQGAP1 siRNA significantly suppressed hepatocyte growth factor (HGF)-stimulated cell invasion. HGF reduced membranous localization of alpha-catenin, but did not alter localization of E-cadherin, beta-catenin and IQGAP1 in membranes. Suppression of IQGAP1 expression by siRNA did not alter membranous localization of alpha-catenin even in the presence of HGF. Our results indicate that IQGAP1 plays a critical role in colon cancer cell invasion, and therefore diffuse and high expression of IQGAP1 predicts poor prognosis in patients with colorectal carcinoma.

关键词： Cell biology   Cell migration   Honokiol   Quantitative proteomics   Ras GTPase-activating-like protein   Stable isotope labeling by amino acids in cell culture  

摘要： Honokiol (HNK), a natural small molecular product, inhibited proliferation of HepG2 cells and exhibited anti-tumor activity in nude mice. In this article, we applied a novel sensitive stable isotope labeling with amino acids in cell culture-based quantitative proteomic method and a model of nude mice to investigate the correlation between HNK and the hotspot migration molecule Ras GTPase-activating-like protein (IQGAP1). The quantitative proteomic analysis showed that IQGAP1 was 0.53-fold down-regulated under 10 mu g/mL HNK exposure for 24 h on HepG2 cells. Migration ability of HepG2 cells under HNK treatment was correlated with its expression level of IQGAP1. In addition, the biochemical validation on HepG2 cells and the tumor xenograft model further demonstrated that HNK decreased the expression level of IQGAP1 and its upstream proteins Cdc42/Rac1. These data supported that HNK can modulate cell adhesion and cell migration by acting on Cdc42/Rac1 signaling via IQGAP1 interactions with its upstream Cdc42/Rac1 proteins, which is a new molecular mechanism of HNK to exert its anti-tumor activity.