Rowin M E, Whatley R E, Yednock T, Bohnsack J F
Department of Pediatrics, University of Utah School of Medicine, Salt Lake City 84132, USA.
J Cell Physiol. 1998 May;175(2):193-202. doi: 10.1002/(SICI)1097-4652(199805)175:2<193::AID-JCP9>3.0.CO;2-J.
Human polymorphonuclear leukocytes (PMNs) express beta1 integrins that mediate adhesion to extracellular matrix proteins following stimulation with agonists that induce an increase in intracellular calcium. The purpose of these studies was to determine the contribution made by alterations in intracellular calcium ([Ca++]i) to inside-out activation of beta1 integrins using dimethyl sulfoxide (DMSO)-differentiated granulocytic HL60 cells as a model of human PMNs. Activation of beta1 integrins was determined by measuring the expression of an activation-dependent epitope on the beta1 subunit that is recognized by monoclonal antibody (mAb) 15/7. Exposure of granulocytic HL60 cells to calcium ionophore ionomycin (800 nM) alone did not increase the binding of mAb 15/7 to the cell surface, nor did it increase beta1 integrin-mediated adhesion of the cells to fibronectin. Similarly, exposure of the cells to the direct protein kinase C (PKC) activator, dioctanoylglycerol (di-C8) at 100 microM, neither increased binding of mAb 15/7 to these cells nor adhesion to fibronectin. Simultaneous addition of di-C8 and ionomycin, however, caused a significant increase in the expression of the 15/7 epitope and cell adhesion, suggesting synergy between elevating [Ca++]i and stimulating PKC in beta1 integrin activation. Chelation of [Ca++]i with Quin-2 and EGTA reduced both basal (unstimulated) expression of the 15/7 epitope and basal adhesion of granulocytic HL60 cells to fibronectin. In addition, chelation of [Ca++]i caused a significant decrease in 15/7 binding and adhesion stimulated by low (1 ng/ml) concentrations of phorbol myristate acetate (PMA). The inhibitory effect of [Ca++]i chelation on beta1 integrin activation was reversed by repleting [Ca++]i with ionomycin in a Ca++-containing buffer, or by the addition of higher concentrations of PMA (10 ng/ml). These data suggest a role for [Ca++]i in inside-out activation of beta1 integrins, probably through a synergistic effect with PKC activation.
人类多形核白细胞（PMNs）表达β1整合素，在受到诱导细胞内钙增加的激动剂刺激后，β1整合素介导与细胞外基质蛋白的黏附。这些研究的目的是使用二甲基亚砜（DMSO）分化的粒细胞性HL60细胞作为人类PMNs的模型，确定细胞内钙（[Ca++]i）变化对β1整合素外向内激活的作用。通过测量单克隆抗体（mAb）15/7识别的β1亚基上激活依赖性表位的表达来确定β1整合素的激活。单独将粒细胞性HL60细胞暴露于钙离子载体离子霉素（800 nM）不会增加mAb 15/7与细胞表面的结合，也不会增加细胞通过β1整合素介导的与纤连蛋白的黏附。同样，将细胞暴露于100 μM的直接蛋白激酶C（PKC）激活剂二辛酰甘油（di-C8），既不会增加mAb 15/7与这些细胞的结合，也不会增加与纤连蛋白的黏附。然而，同时添加di-C8和离子霉素会导致15/7表位的表达和细胞黏附显著增加，表明在β1整合素激活过程中，升高[Ca++]i和刺激PKC之间存在协同作用。用喹啉-2和乙二醇双乙醚四乙酸（EGTA）螯合[Ca++]i会降低15/7表位的基础（未刺激）表达以及粒细胞性HL60细胞与纤连蛋白的基础黏附。此外，螯合[Ca++]i会导致低浓度（1 ng/ml）佛波酯肉豆蔻酸酯（PMA）刺激的15/7结合和黏附显著降低。在含Ca++的缓冲液中用离子霉素补充[Ca++]i，或添加更高浓度的PMA（10 ng/ml），可逆转[Ca++]i螯合对β1整合素激活的抑制作用。这些数据表明[Ca++]i在β1整合素外向内激活中起作用，可能是通过与PKC激活的协同效应。
