Abstract
Type IV collagen (Coll IV), a component of the extracellular matrix, stimulates motility in the A2058 human melanoma cell line, a response that is inhibited by pertussis toxin (PT). Fibronectin (FN)-induced chemotaxis in this cell line is not affected by PT. To understand the mechanism of cellular signaling, single cell intracellular Ca2+ responses to Coll IV and FN were studied using Fura-2 and digital imaging fluorescence microscopy. Coll IV, at a dose that stimulates motility (100 micrograms/ml, 185 nM), induces a significant rise in cytosolic free Ca2+ concentration ([Ca2+]i) within 100 s. This response is not inhibited by PT. Treatment of the cells with FN 30 micrograms/ml (70 nM), a dose that stimulates near-maximal chemotaxis, does not increase [Ca2+]i appreciably. Removal of extracellular Ca2+ fails to inhibit the Coll IV-stimulated rise in Ca2+ in all cells. Depletion of extracellular Ca2+ and pretreatment of cells with Ca2+ channel blockers only partially inhibits Coll IV-induced motility. Depletion of intracellular Ca2+ inhibits both chemotaxis and the Coll IV-induced increase in intracellular Ca2+. Coll IV does not stimulate membrane phosphoinositide hydrolysis. We conclude that Coll IV treatment induces an inositol 1,4,5-trisphosphate-independent release of intracellular Ca2+ stores which appears to play a necessary role in the chemotactic response of A2058 cells but is not mediated by a PT-sensitive G-protein. This response is not seen in cells exposed to FN, suggesting different intracellular signaling mechanisms for stimulated motility between these two extracellular matrix molecules.
Highlights
From the $Medicine Branch and Laboratoroyf Pathology, National Cancer Institute, National Instituteosf Health, Bethesda, Maryland 20892, the TLaboratory of Developmental Neurobiology, National Institutoef Child Health and Development, National Institutes of Health, Bethesda, Maryland 20892, the IIIstituto di Farmacologia, IZ Policlinico, Naples, Italy, and the **Laboratory of Pathology, National Cancer Institute, National Instituteosf Health, Bethesda, Maryland 20892
Specific receptors for ECM moleculeshave been shown to exist on the surface of cells [9], it has never been shown that any component of the ECM cantransmitan intracellular signal in tumor cells
Hibit the Coll IV-stimulated rise in Ca2+ in all cells. triggers chemotaxis in the A2058 melanoma cell line
Summary
We con- degrade collagenous components of the basement membrane; clude that Coll IV treatment induces an inos1i,t4o,l5- levels of type IV collagenase activity have been shown to trisphosphate-independent release oifntracellular Ca2+stores which appears to play a necessary role i n the chemotacticresponse of A 2 0 5 8 cells but is not mediated bya PT-sensitive G-protein. This responisse not seen in cells exposed to FN, suggesting different intracellular signaling mechanisms for stimulatmedocorrelate with invasiveness and thepropensity to metastasize [11, 12]. We have studied the etiology of the Ca" response, the contribution of G-proteins through the use of PT, and have correlated the changes in [Ca2+Iiwith motility in response to Coll IV
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