Intracellular calcium requirements for beta1 integrin activation.
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.
Rowin
M E
ME
Department of Pediatrics, University of Utah School of Medicine, Salt Lake City 84132, USA.
Whatley
R E
RE
Yednock
T
T
Bohnsack
J F
JF
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D000939:Epitopes / Q000737:chemistry; D005353:Fibronectins / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D004075:Diglycerides; D000939:Epitopes; D005353:Fibronectins; D019012:Integrin beta1; D007476:Ionophores; D010455:Peptides; C044295:1,2-dioctanoylglycerol; D015759:Ionomycin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium