【作者】 汪永平；

【导师】 吴人亮；

【作者基本信息】 华中科技大学 ， 病理学， 2008， 博士

【摘要】 第一部分气道上皮细胞中机械划伤致IQGAP1磷酸化对损伤修复的影响实验背景:由于气道(气管和支气管)粘膜暴露于外界环境,易受到损伤。气道上皮受损后将立即启动修复过程,从而保持正常的肺功能。这个损伤修复过程需要气道上皮细胞伸展,迁移和增生最终覆盖受损区域。气道受损是气道疾病例如慢性支气管炎和COPD的重要的发病过程。因此气道上皮自身的修复能力是解决这些气道疾病的关键步骤。然而,参与气道上皮损伤修复的分子机制尚未完全明了。IQGAP1(IQ domain GTPase-activating protein)在许多细胞生命活动中是一个非常有意义的蛋白,但是尚未有人阐述其在气道上皮损伤修复中的作用。磷酸化作为翻译后的主要手段,参与调节蛋白质及细胞骨架的功能。有报导显示,IQGAP1丝氨酸残基能被高磷酸化,结果能促进神经元轴突外生性生长。PKC的一个新的亚型PKCε可能有助于IQGAP1的磷酸化。我们推测,在气道上皮损伤修复过程中IQGAP1磷酸化可能发挥重要作用。目的:使用目前广泛应用的刮伤气道上皮细胞的体外模型,初步探讨机械划伤后IQGAP1的磷酸化水平以及对损伤修复的影响。方法:建立机械划痕引起体外气道上皮损伤模型,采用损伤分析实验、免疫组化、western blot、免疫荧光共聚焦成像、免疫沉淀、瞬时转染等方法观测IQGAP1磷酸化、IQGAP1与PKCε在损伤前后的变化,并通过激活和抑制PKCε活性,观察对损伤愈合的影响。结果:形态学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。在机械性损伤后,气道上皮细胞可成片状的单向迁移,且其迁移方向垂直于划痕方向。损伤6 h细胞形态即发生显著改变,形成具有特征性的极化形态学改变,损伤12 h更为显著。损伤24 h后迁移的气道上皮细胞能掩盖划线部分。本实验发现,IQGAP1在多种动物气道上皮细胞中表达丰富。它与微管骨架共定位,但能被微管解聚剂nocodazole破坏。机械划伤后,IQGAP1磷酸化水平迅速增加,在损伤后1小时达到峰值,持续9小时。过表达PKCε或添加PKC活化剂PMA,能促进IQGAP1磷酸化。相反,PKC抑制剂GF109203X能消除划伤引起的IQGAP1磷酸化。通过免疫荧光共聚焦和免疫共沉淀方法,我们发现机械划伤能加强IQGAP1与PKCε结合。损伤分析实验结果显示,刮擦24h后,正常对照组细胞划痕已经闭合,而过表达PKCε或PMA处理组细胞划痕提前愈合。相反,GF109203X处理组不仅没有闭合,而且其划痕宽度仍占原始划痕的32%,提示IQGAP1磷酸化介导了气道上皮细胞的划痕愈合。结论本实验表明,IQGAP1在气道上皮细胞中有丰富的表达,且PKCε介导的IQGAP1磷酸化参与了机械划伤引起的气道上皮细胞损伤愈合过程。第二部分IQGAP1以不依赖于Rac1和Cdc42的方式参与气道上皮损伤修复的增殖过程实验背景:香烟烟雾、大气污染、感染等多种致病因素在机体抵抗力降低时,都能引起气道上皮损伤,导致肺疾病的发生。气道损伤后立即启动修复过程,这是一个复杂的多步骤过程,包括细胞延伸、迁移和增殖。Rho GTPases是小G蛋白超家族的主要成员,其中RhoA,Rac1和Cdc42研究最为广泛。Rho GTPases在GTP结合的活化状态和GDP结合的失活状态间转换。通过与许多效应子的作用,如IQGAP1、PAK(p21 activated kinase)和WASP(Wiskott-Aldrich syndrome protein),Rho GTPases在组织形成、细胞极化和迁移过程中发挥重要作用。作为Rac1和Cdc42的效应子,IQGAP1能与细胞骨架成分、细胞黏附复合体及微管相关蛋白结合,参与了细胞黏附、极化和定向迁移过程。目前尚未有人阐述Rho GTPases及IQGAP1在肺上皮细胞中的作用。本研究推断上述信号可能在气道上皮细胞损伤修复中发挥作用,并探讨其可能的机制。目的:使用了体外损伤模型来研究Rho GTPases及IQGAP1的功能。检测上述因子在气道上皮不同损伤修复时期的动态表达变化,初步研究其在气道上皮损伤修复过程中的作用,继而为深入探讨损伤修复机制提供理论和实验依据。方法:在机械划伤单层细胞建立的体外损伤模型中,通过pull-down方法检测Rac1及Cdc42的活性。采用免疫细胞荧光、RT-PCR、荧光定量PCR、western blot、分离核浆蛋白、质粒瞬时转染和RNA干扰方法观测IQGAP1及相关因子在气道上皮损伤模型中的表达和分布变化,并探讨上述信号影响损伤修复的可能机制。结果:机械划伤能降低GTP结合的Rac1和Cdc42,但能促进IQGAP1 mRNA及蛋白表达。我们证实IQGAP1能结合组成性激活型Rac1(Rac1V12)和Cdc42(Cdc42V12),但不能结合显性负突变型Rac1(Rac1N17)和Cdc42(Cdc42N17)。在实验中我们应用了IQGAP1的突变体T1050AX2,该突变体在1050-1052位氨基酸均被丙氨酸替代,缺乏与Rho GTPases结合的能力。研究发现,划伤和过表达外源性IQGAP1(wild type和T1050AX2)都能使β-catenin核转位从而活化Tcf/Lef信号。同时,过表达上述外源性IQGAP1也能促进Tcf靶基因c-myc和cyclinD1表达。此外,刮伤可影响IQGAP1与β-catenin、adenmatous polyposis coli(APC)及cytoplasmic linker protein- 170(CLIP-170)之间的相互作用。通过小干扰RNA(siRNA)消除IQGAP1的表达可明显延迟损伤愈合。我们总结认为,IQGAP1信号参与了气道上皮细胞的损伤修复,且以不依赖于Rac1和Cdc42的方式介导了细胞增殖过程。结论:上述结果表明:(1)IQGAP1可能通过表达量上调参与气道上皮受损后启动的修复。(2)IQGAP1以不依赖于Rac1和Cdc42的方式促进β-catenin核转位,活化Tcf信号并诱导其靶基因c-myc和cyclin-D1表达。(3)划伤能减弱β-catenin与α-catenin的结合,但加强β-catenin与IQGAP1的结合。(4)机械损伤能增强IQGAP1与微管相关蛋白APC、CLIP-170之间的作用。(5)敲低IQGAP1能明显阻滞划痕愈合。本实验提示:IQGAP1可通过表达上调而活化β-catenin/Tcf信号途径,参与机械刮擦致气道上皮细胞的损伤以及损伤后修复过程。Rac1和Cdc42并不参与IQGAP1介导的细胞增殖过程,而磷酸化的IQGAP1有助于损伤愈合。这些结论对阐明呼吸系统疾病的发病机制及其逆转病变的进展具有重要意义。更多还原

【Abstract】 Part I Effects of phosphorylation of IQGAP1 induced by mechanical scraching on wound and repair in airway epithelial cellsBackground:Airway (trachea and bronchi) epithelium is frequently injured because of its exposure to the external environment. After injury, the airway epithelium initiates a wound repair process to keep normal lung function, which requires spreading, migration, and eventually proliferation of airway epithelial cells into the injured area. Damage to airway epithelium is critical to the pathogenesis of airway disorders such as chronic bronchitis and COPD, so the ability of the airway epithelium to repair itself is an important step in the resolution of airway disease. However, the molecular mechanism involved in the injury and repair of airway epithelium has not been well understood.IQGAP1 (IQ domain GTPase-activating protein) is a key actor in a series of cell processes, but has not been identified in lung epithelial cells. Phosphorylation is a major post-translational method for regulating protein function, including that of the cytoskeleton. The serine residues of IQGAP1 are phosphorylated, which promote neurite outgrowth. PKCεmay be one of the candidates responsible for phosphorylation of IQGAP1. We suppose that phosphorylation of IQGAP1 may play roles in the process of injury and repair.Objective:In this study, we utilized a widely used model in vitro by scratching bronchial epithelial cells (BECs) and investigated the effects of phosphorylation of IQGAP1 in the process of wound closure. Method:We established an in vitro injury and repair model by scratching a monolayer of BECs. Then the following tests were undertaken: wound assays, immunohistochemistry, western blot, confocal laser sanning, immunoprecipitation, transient transfection and so on. We aimed to study phosphorylation of IQGAP1, the association between IQGAP1 and PKCεand the effects of PKCεfor wound closure after activation or inhibition its activity.Results:Cell morphological observation demonstrated cultured BECs showed a classic cobblestone epithelial morphology that was three-dimensional, slightly raised and closely adherent. After scratching, bronchial epithelial cells moved unidirectionally as sheets or groups, perpendicular to the direction of the wound. A polarized morphology developed 6 h following scratching and became pronounced after 12 h. The wound gap was approximately closed in 24 h after scratching.Our results showed that IQGAP1 was abundant in BECs of several species. It was colocalized with tubulin cytoskeleton, but was destroyed by microtubule disassembled reagent of nocodazole. Soon after scratching, phosphorylation of IQGAP1 increased and reached a maximum at 1 h, lasting for at least 9 h. Overexpression of PKCεor treatment with PMA (the PKC activator) increased the amounts of phosphorylation of IQGAP1. On the contrary, phosphorylation of IQGAP1 induced by scratching was blocked by GF109203X (the PKC inhibitor). We next found that scratching induced close association between IQGAP1 and PKCεby immunofluorescence and immunoprecipitation methods. Our data from wound assays suggested that the wound gap closed within 24 h in control, while overexpression of PKCεand treatment with PMA promoted wound closure. On the other hand, the group treated with GF109203X was shown to impair the wound closure and still had 32% of original width. These results suggested that phosphorylation of IQGAP1 mediated the process of wound closure in BECs. Conclusion:Our results showed that IQGAP1 was generously expressed in BECs and suggested that PKCεmediated phosphorylation of IQGAP1 was involved in the early stage of wound closure. Part II IQGAP1 is involved in the proliferation process of injury and repair in BECs independent of Rac1 and Cdc42Background:Airway epithelium is repeatedly subjected to injury by cigarette smoke, pathogens, toxicants and infection. Immediately after injury, the airway epithelium initiates a multiple wound healing process to keep normal lung function, which involves so much signaling pathways and factors including cell extension, migration and proliferation. Rho GTPases are main branches of the Ras small GTPase superfamily, in which RhoA, Rac1 and Cdc42 are widely studied. Most GTPases cycle between the GTP-bound active and GDP-bound inactive forms. Through the effectors such as IQGAP1, PAK (p21 activated kinase) and WASP (Wiskott-Aldrich syndrome protein), Rho GTPases play critical roles in many processes, such as organization, chemotaxis and injury and healing. As an effector of Rac1 and Cdc42, IQGAP1 is an integral protein of cytoskeletal organization and interacts with cytoskeleton, cell adhesion complex and microtubule associated proteins (MAPs), thus IQGAP1 is considered to play roles in cell adhesion, polarization and migration. The mechanisms of Rho GTPases and their effector IQGAP1 are poor known in the process of airway epithelium. We supposed that Rho GTPases-IQGAP1 signal regulated the process of injury and repair in BECs and discussed the possible mechanism.Objective:We aim to study whether and how Rac1, Cdc42 and IQGAP1 work in injury and repair of BECs in vitro. The dynamic expressions of these factors during different stages of injury and repair were detected and their roles involved in the process were discussed. This study may bring theoretical and experimental evidences for further exploring the mechanism of injury and repair.Methods:In the model of injury and repair by scratching a monolayer of BECs in vitro, we detected the activities of Rac1 and Cdc42 by pull down assay. Next, we observed and examined the localizations and expressions of IQGAP1 and related factors in the model of injury and repair by immunofluorescence, RT-PCR, fluorescence quantitative PCR, western blot, isolation of cytoplasmic and nuclear proteins, transient transfection and RNA interference methods.Results:We found that scratching decreased GTP-bound Rac1 and Cdc42, but increased the amounts of IQGAP1 in both mRNA and protein at different time points. Next, we confirmed that IQGAP1 interacted with constitutively active Rac1 (Rac1V12) and Cdc42 (Cdc42V12) rather than dominant negative Rac1 (Rac1N17) and Cdc42 (Cdc42N17). Overexpressions of wild type (WT) IQGAP1 and its mutant (T1050AX2), which was mutated at 1050-1052 amino acids instead for alanine and was defective to interact with Rho GTPase, induced translocation ofβ-catenin from the cytoplasm into the nucleus. These results activated Tcf/Lef and induced expressions of its target genes of c-myc and cyclin D1. As similar, scratching increased the amounts of c-myc and cyclin D1. On the other hand, scratching altered the associations of IQGAP1 withβ-catenin, adenmatous polyposis coli (APC) and cytoplasmic linker protein-170 (CLIP-170). Depletion of IQGAP1 by small interference RNA (siRNA) blocked the wound closure. We conclude that IQGAP1 signal is involved in injury and repair and promotes cell proliferation in a manner independent of Rac1 and Cdc42 in the process of BECs.Conclusion:Our results indicated that: (1) Increasing amounts of IQGAP1 should play role in the process of wound closure. (2) IQGAP1 promotedβ-catenin translocation, activated Tcf and induced expressions of its target genes of c-myc and cyclin-D1 in a manner independent of Rac1 and Cdc42. (3) Scratching induced weak associations betweenα- andβ-catenin, but strong associations between IQGAP1 andβ-catenin. (4) IQGAP1 was shown to have strong associations with APC and CLIP-170 after scratching. (5) Depletion of IQGAP1 blocked the scratched wound closure.Our data suggested that: The process of wound closure in airway epithelial cells involves several coordinated events that are dependent on up-regulating of IQGAP1. The participation of Rac1 or Cdc42 is not necessary for this effect, but phosphylation of IQGAP1 is important at the early stage of injury and repair. These results may help to elucidate the mechanisms of the diseases in respiratory system and arrest their progression.更多还原