【作者】 崔凌凌；

【导师】 曾宪录；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2009， 博士

【摘要】 白细胞沿着血管内皮滚动、稳定粘附和跨内皮迁移,最终到达炎症部位是一个复杂的多步骤的过程,它的精确调控与机体的宿主防御和炎症反应密切相关。有多种粘附分子参与这一事件,其中β2整合素主要介导白细胞稳定粘附于血管内皮上和跨内皮的迁移。β2整合素是一类仅表达于白细胞表面的粘附分子家族,它包括四个成员,由一个共同的β亚基分别与四种α亚基形成非共价结合的异源二聚体。在静息的白细胞中,β2整合素以一种低亲和力和非粘附的状态存在。在炎症部位产生的细胞因子和趋化因子的刺激下,β2整合素能够迅速活化与其配体结合,这个过程为整合素的“inside-out”信号。活化的整合素能产生“outside-in”信号来调节细胞的骨架变化以及信号转导。β2整合素的信号对于中性粒细胞的稳定粘附和伸展非常关键,大量证据表明中性粒细胞的呼吸爆发、脱颗粒、蛋白的酪氨酸磷酸化等生理事件都依赖于β2整合素介导的细胞粘附。但是,对于中性粒细胞中β2整合素是如何调控细胞粘附的机制还不十分清楚。在中性粒细胞中,粘附引起的β2整合素的交联能够活化多种非受体酪氨酸激酶,这些激酶与细胞的骨架重排、生长、存活、运动密切相关。c-Abl激酶是一种非受体酪氨酸激酶,它参与多种受体引起的信号转导,并且参与了F-肌动蛋白依赖的细胞骨架变化。我们推测,c-Abl激酶可能参与了β2整合素介导的中性粒细胞粘附。本文主要探讨了c-Abl激酶在中性粒细胞的β2整合素信号转导中的功能和作用机制。我们的研究发现,在中性粒细胞中,β2整合素的交联能够增强c-Abl激酶的活性。细胞因子TNF-α能够引起细胞表面整合素表达的增加和活性的增强,但是c-Abl激酶抑制剂STI571并不影响这一变化,表明c-Abl激酶不参与TNF-α引起的整合素inside-out活化。而c-Abl激酶的抑制能显著降低细胞的稳定粘附和伸展,当干涉胞内的c-Abl激酶表达后也有相似的作用,因此c-Abl激酶主要参与了β2整合素的outside-in信号通路。在静息的中性粒细胞中,c-Abl激酶均匀的分布在细胞质内,而β2整合素介导细胞粘附之后,c-Abl激酶就募集到β2整合素富集的部位。通过蛋白质免疫沉淀和GST pull-down等实验,我们发现在β2整合素交联的中性粒细胞中,β2整合素与c-Abl激酶存在于同一个蛋白质复合体中,但二者的结合不是直接的。talin是一类骨架蛋白,它的头部与中性粒细胞中的β2整合素的胞浆尾部直接并持续地结合。活化的c-Abl激酶通过其SH3结构域与talin的头部结合,进而募集到β2整合素的胞浆尾部。Vav是一类鸟苷酸交换因子,它参与了β2整合素对中性粒细胞粘附的调控。我们的实验发现,在中性粒细胞中c-Abl激酶与Vav持续结合,并且在β2整合素产生的信号通路中c-Abl激酶能够调节Vav的活性。二者的作用方式是通过c-Abl激酶的SH3结构域与Vav的SH2结构域的结合。通过点突变实验,我们发现c-Abl激酶SH3结构域中的134位酪氨酸对于二者的结合很关键。我们的结果证明了,在β2整合素介导的中性粒细胞粘附过程中,c-Abl激酶能够被β2整合素活化。talin作为持续与β2整合素胞浆尾部结合的骨架蛋白,对于c-Abl激酶募集到β2整合素非常关键。活化的c-Abl激酶能够通过调节Vav的活性进而参与了对中性粒细胞的稳定粘附和伸展的调控。本论文揭示了c-Abl激酶在天然免疫过程中的新作用。更多还原

【Abstract】 Recruitment of leukocytes from blood into tissues plays an important role in host defense and inflammation, including tethering, rolling, adhesion and transmigration. This process is controlled by a variety of adhesion molecules, and members ofβ2 integrin subfamily are critical for the regulation of cellular adhesive activity in this event.β2 integrins are noncovalently associated heterodimers composed of a commonβ-chain and one of four uniqueα-chains.β2 integrins are in low affinity and nonadhesive state in resting leukocytes, and are rapidly activated to bind ligands in response to local inflammatory stimuli, such as cytokines or chemoattractants, a process known as“inside-out”activation. Once activated, integrins can deliver“outside-in”signals to regulate the cytoskeleton and signaling complex. Integrin signaling is critical for neutrophil firm adhesion and spreading, and oxidative burst, degranulation and protein tyrosine phosphorylation are all dependent on the cell adhesion mediated byβ2 integrin. However, the molecular mechanisms by which integrins regulate adhesive events in neutrophils are largely undefined. In neutrophils, adhesion-induced ligation ofβ2 integrin activates various non-receptor tyrosine kinases, which have been demonstrated critical in integrin signaling and leads to cytoskeleton rearrangements, cell growth, survival, and motility. c-Abl kinase as a non-receptor protein tyrosine kinase plays an essential role in signaling transduction from various receptors and in F-actin reassembly. Thus, we speculate that c-Abl kinase plays a role in neutrophilβ2 integrin signaling. In this paper, we focus on the function and mechnism of c-Abl kinase in neutrophils induced byβ2 integrin engagement.c-Abl kinase is activated byβ2 integrin engagement. The expression ofβ2 integrin on neutrophils induced by TNF-αis not affected by c-Abl kinase inhibitor STI571, suggesting that c-Abl kinase is not involved in TNF-αinduced integrin inside-out activation. Inhibiton of c-Abl kinase impaires neutrophil sustained adhesion and spreading, which is similar with c-Abl kinase knock-out cells. Thus, we conclude that c-Abl kinase is mainly involved inβ2 integrin outside-in signaling.In resting neutrophils, c-Abl kinase is approximatively uniform in the cytosol. After neutrophil adhesion mediated byβ2 integrin, c-Abl kinase is redistributed and colocalized withβ2 integrin. We have shown that c-Abl kinase interacts with the cytoplasmic domain ofβ2 integrin afterβ2 integrin engagement by immunoprecipitation and GST pull-down assay, but this interaction is not direct. Talin is a major cytoskeletal protein and constitutively and directly binds to integrinβ2 cytoplasmic domains with its head domain. The recruitment of c-Abl kinase toβ2 integrin is dependent on the interaction between c-Abl kinase SH3 domain and talin head domain.The Vav family proteins are cytoplasmic guanine nucleotide exchange factors, and involved in the regulation of neutrophil adhesion mediated byβ2 integrin. Our results show that c-Abl kinase constitutively and directly associates with Vav, and the activity of Vav is regulated by c-Abl kinase inβ2 integrin signaling transduction. c-Abl kinase SH3 domain and Vav SH2 domain are involved in this association, and Tyr134 in c-Abl kinase SH3 domain is critical.Our results have defined that c-Abl kinase can be activated byβ2 integrin engagemnet in neutrophils. Talin head domain, as aβ2 integrin cytoplasmic binding protein, is critical for the recruitment of c-Abl kinase toβ2 integrin. After activated, c-Abl kinase can regulate neutrophil adhesion and spreading via Vav. The c-Abl kinase has a novel role in innate immunity.更多还原