Piccardoni Paola, Manarini Stefano, Federico Lorenzo, Bagoly Zsuzsa, Pecce Romina, Martelli Nicola, Piccoli Antonio, Totani Licia, Cerletti Chiara, Evangelista Virgilio
Laboratory of Vascular Biology and Pharmacology, Consorzio Mario Negri Sud, Via Nazionale 1, 66030, Santa Maria Imbaro, Italy.
Biochem J. 2004 May 15;380(Pt 1):57-65. doi: 10.1042/BJ20040151.
In human PMN (polymorphonuclear cells), challenged by P-selectin, the beta2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108-116]. This suggested that an SRC-dependent outside-in signalling strengthens the beta2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock beta2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor beta2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common beta2 chain, by a combination of antibodies against alphaL and alphaM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by beta2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by beta2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize beta2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.
在受到P-选择素刺激的人多形核细胞（PMN）中，β2整合素Mac-1（巨噬细胞抗原-1）促进了SRC（劳斯肉瘤病毒致癌蛋白的细胞同源物）家族成员HCK（造血细胞激酶）和LYN（一种SRC家族蛋白酪氨酸激酶）的激活以及P-110（110 kDa蛋白）的磷酸化。反过来，SRC激酶活性对于巨噬细胞抗原-1介导的黏附是必需的[皮卡多尼、西代里、马纳里尼、皮科利、马尔泰利、德加埃塔诺、塞尔letti和埃万杰利斯塔（2001年）《血液》98卷，第108 - 116页]。这表明一种依赖SRC的由外向内信号传导增强了β2整合素与配体的相互作用。为了进一步支持这一假设，在本研究中，我们使用单克隆抗体KIM127或锰将β2整合素锁定在高亲和力状态，并分析同型PMN黏附以监测β2整合素的黏附功能。KIM127或锰诱导了PMN同型黏附和P-110磷酸化。这两个过程都被针对共同β2链的阻断抗体、针对αL和αM的抗体组合或SRC活性抑制剂所消除。共聚焦显微镜显示，激活表位由与黏附位点处F-肌动蛋白斑块共定位的β2整合素表达。SRC激酶或肌动蛋白聚合的阻断阻止了激活的整合素聚集以及F-肌动蛋白积累。流式细胞术分析表明，SRC抑制剂既不改变基础的也不改变锰诱导的KIM127结合。由β2整合素引发的依赖SRC的由外向内信号传导对于白细胞介素-8触发的黏附也是必需的。这些结果证实了这样的假设，即由高亲和力和配体结合引发的依赖SRC的由外向内信号传导对于稳定β2整合素介导的黏附是必需的。允许激活的整合素聚集，SRC可能将高亲和力与高亲合力状态联系起来。富含脯氨酸的酪氨酸激酶-2似乎参与了这一过程。
