【作者】 王峰松；

【导师】 姚雪彪；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2008， 博士

【摘要】 细胞可塑性与动力学特征是维系组织及器官功能特异性及个体健康的基础,在分子水平上细胞可塑性与动力学特征则体现为蛋白质作用网络的时空调节。细胞极化是行使其功能区域化的基础,亦是细胞可塑性与动力学特征的综合表征之一。胃壁上皮细胞是一个典型的极化细胞,负责胃酸与内因子的分泌。为此,胃壁细胞酸分泌不仅为研究胞吐分子机制提供了一个极好的模式体系,同时对其机制的研究亦为阐明胃壁细胞分子生理学及校正胃酸分泌异常奠定基础。幽门螺旋杆菌长久地驻留于人类肠胃之中,是人类多种胃病的罪魁祸首,与萎缩性胃炎、胃癌等休戚相关。虽然很久之前人们已经知道幽门螺旋杆菌感染能够导致胃酸分泌的减少,但其分子机制至今仍然悬而未决。以往的研究多集中在免疫调节细胞,我们的研究结果表明,VacA毒素能够直接诱导胃壁细胞顶膜通透性的增加,导致胃酸分泌的减少。为此,我们建立了原代培养的胃腺、胃壁细胞等研究模型和良好的评估壁细胞分泌能力的技术平台,包括氨基比林吸收实验、激光共聚焦扫描技术等,对VacA感染所导致的壁细胞生理特征的改变进行了深入细致的研究。结果表明:VacA诱导的细胞膜通透性的增加导致了胞外钙离子的内流,进而引起胞内钙依赖性蛋白水解酶CalpainⅠ蛋白酶的活化,将Ezrin在469与470之间进行切开,从而使得Ezrin分子从壁细胞膜上脱离下来。Ezrin是一个膜蛋白与皮层骨架之间的连接器,它还参与了信号转导途径。在壁细胞中,它定位于顶膜,并受到PKA磷酸化修饰的调控,进而影响到HCl的分泌。VacA处理的细胞通过阻止H,K-ATPase管状囊泡到顶膜上的招募抑制了胃酸的分泌。电镜结果也显示,VacA处理导致了Ezrin在壁细胞中完整性的缺失,破坏了壁细胞顶膜微绒毛中肌动蛋白纤维的放射状排布。值得关注的是:在外源性表达Ezrin水解位点突变体的壁细胞中,虽然进行了VacA感染的处理,但其依然能够保持泌酸功能的正常进行。我们对VacA感染的细胞中Ezrin的水解现象进行研究,为全面地认识幽门螺旋杆菌诱导胃酸分泌减少的现象提供了一个全新的方位。我们的结果表明,VacA能够破坏胃壁细胞顶膜与骨架之间的精细连接,进而引发胃酸分泌的减少。在研究细胞膜与微丝骨架动力学调控机制过程中我们鉴定出一个新的蛋白复合体,其中包括Ezrin-ACAP4-Arf6。然而,ACAP4与Ezrin之间是否存在着直接的相互作用,它们之间又是如何协调控制着膜-骨架之间的重构还不是很清楚。利用壁细胞酸分泌为模式体系,我们发现:Ezrin能够依赖PKA介导的磷酸化修饰与ACAP4相互作用。我们的体外生化研究结果表明,ACAP4依赖其氨基端400个氨基酸区域与模拟磷酸化状态的Ezrin相互作用,这说明它们之间的相互作用依赖于蛋白自身的构象变化。更为重要的是,在分泌性的壁细胞中,Ezrin特异性地将ACAP4靶向到顶膜,不论是敲除掉Ezrin,还是表达缺失Ezrin结合区域的ACAP4的片断,都削弱了ACAP4在顶膜的定位。ACAP4是一个Arf6特异性的GTP酶激活蛋白,它能够特异性地识别并结合Arf6-GTP。过量表达GAP活性缺陷型的ACAP4突变体抑制了顶膜-骨架的重构和壁细胞酸的分泌。总之,我们的结果为Arf6-ACAP4-Ezrin之间的相互作用和极化上皮细胞的分泌之间提供了一个全新的分子机制的诠释。综上所述,本研究阐述了Arf6-ACAP4-ezrin蛋白质作用网络在壁细胞酸分泌过程中的重要作用及调控分子机制,同时我们研究揭示了幽门螺旋杆菌直接作用于壁细胞并干扰胃酸分泌的病理学分子机制。因此,本研究为系统阐明胃壁细胞分子生理学及防治壁细胞生理学异常所致的消化道疾病提供了新的思路。更多还原

【Abstract】 Cell plasticity and dynamics are the determinants underlying the formation and specification of tissues and organs,which is essential for animal development and health.Cellular polarity governs functional specificities of different domains of a given cell,which synthesizes the cellular plasticity and dynamics.Gastric parietal cells is a typical polarized epithelial cell responsible for HCl secretion and intrinsic factor release.Because of its great cytological features associated with secretion-coupled transformation stimulated by gastric hormones,gastric parietal cell becomes a unique model system to dissect molecular mechanisms underlying cAMP-regulated exocytosis.In turn,a better understanding of parietal cell physiology offers efficient strategies to minimize or eradicate gastrointestinal diseases associated with aberrant parietal cell function.Helicobacter pylori persistently colonize the human stomach and have been linked to atrophic gastritis and gastric carcinoma.Although it is well-known that H. pylori infection can result in hypochlorhydria,the molecular mechanisms underlying this phenomenon remain poorly understood.Most of formerly study focused on the immunoregulatory cells.Here we show that VacA permeabilizes the apical membrane of gastric parietal cells and induces hypochlorhydria.The functional consequences of VacA infection on parietal cell physiology were studied using freshly isolated rabbit gastric glands and cultured parietal cells.Secretory activity of parietal cells was judged by aminopyrine uptake assay and confocal microscopic examination.VacA permeabilization induces an influx of extracellular calcium followed by activation of the calcium dependent proteolytic enzyme calpainⅠand subsequent proteolysis of Ezrin at ⁴⁶⁹Mer-Thr⁴⁷⁰,which results in the liberation of ezrin from the apical membrane of the parietal cells.Ezrin provides a regulated linkage between membrane proteins aud the cortical cytoskeleton,and also participates in signal-transduction pathways.Ezrin is localized to the apical membrane of parietal cells and couples the PKA activation cascade to the regulated HCl secretion in gastric parietal cells.VacA treatment inhibits acid secretion by preventing the recruitment of H,K-ATPase-containing tubulovesicles to the apical membrane of gastric parietal cells.Electron microscopic examination revealed that VacA treatment disrupts the radial arrangement of actin filaments in apical microvilli due to the loss of Ezrin integrity in parietal cells.Significantly,expression of calpain-resistant Ezrin restored the functional activity of parietal cells in the presence of VacA.Proteolysis of Ezrin in VacA-infected parietal cell is a novel mechanism underlying H.pylori-induced inhibition of acid secretion.Our results indicate that VacA disrupts the apical membrane-cytoskeletal interactions in gastric parietal cells,and thereby causes hypochlorhydria.The second aspect of my research seeks to define the functional role of actin-based cytoskeleton dynamics underlying HCl secretion.Earlier experiments in our laboratory demonstrated that ezrin,a F-actin binding protein,provides a regulated linkage between cAMP-mediated signaling cascade and volatile membrane-cytoskeletal remodeling.To delineate the molecular mechanisms underlying parietal cell acid secretion,we have identified a new protein complex containing Ezrin-ACAP4-ARF6 responsible for volatile membrane-cytoskeletal reorganization.Using the gastric acid secretion as our model system,here we provide the first evidence that ezrin interacts with ACAP4 in PKA-mediated phosphorylation-dependent manner.ACAP4 resides on cytoplasmic membrane of resting parietal cells and relocates with H,K-ATPase tot he apical membrane upon histamine-stimulation.Our biochemical studies indicate that the N-terminal ACAP4 （1-400 aa）binds to phospho-mimicking mutant ezrin in vitro,suggesting a conformation-oriented interaction.Importantly,ezrin specifies the apical distribution of ACAP4 in secreting gastric parietal cells as either suppression of ezrin eliminates the apical localization of ACAP4.ACAP4 is an ARF6 GTPase-activating protein and binds directly to ARF6-GTP.Over-expressing GAP-deficient ACAP4 results in an inhibition of apical membrane-cytoskeletal remodeling and parietal cell secretion. Taken together,these results define a novel molecular mechanism linking ARF6-ACAP4-Ezrin interactions to polarized epithelial secretion.In sum,my research work has discovered a novel Ezrin-ACAP4-ARF6 protein network and elucidated its molecular function in gastric HCl section.My study on Helicobacter pylori has identified that VacA toxin disrupts the apical membrane-cytoskeleton via hydrolysis of ezrin.The information derived from this research will better our understanding of molecular physiology of parietal cell secretion which ultimately aid to minimize or perhaps eradicate the discomforts associated with gastrointestinal diseases of aberrant HCl secretion.更多还原