Modulation of endothelial cell shape by SPARC does not involve chelation of extracellular Ca2+ and Mg2+.
SPARC (secreted protein, acidic and rich in cysteine) is an extracellular, Ca(2+)-binding protein that inhibits the spreading of newly plated cells and elicits a rounded morphology in spread cells. In this study, I investigated whether the rounding effect of SPARC depends on the ability of the protein to chelate Ca2+ at the cell surface. Bovine aortic endothelial cells were plated in the presence of different concentrations of SPARC and Ca2+; control experiments were performed with 1 mM EGTA and with Mg2+. Quantitative estimates of cell rounding were calculated according to a rounding index. SPARC, at concentrations between 0.15 and 0.58 microM, elicited rounding (or prevented spreading) of cells cultured for 16-38 h in 0.5-2.0 mM Ca2+. Addition of 0.5-2.0 mM Mg2+ to cells previously rounded in the presence of SPARC did not abrogate the effect of SPARC. When the levels of extracellular Ca2+ were adjusted with 1 mM EGTA to maximum values ranging from 7.1 to 320 microM, cells displayed a rounded morphology in the presence of exogenous SPARC. Although the rounding induced by 1 mM EGTA was essentially reversed by the inclusion of 2 mM Ca2+, cultures containing these reagents together with SPARC maintained the rounded phenotype. These results do not support a mechanism that involves the abstraction of Ca2+ from proteins at the cell surface or the provision of Ca2+ from native extracellular SPARC to cells. Therefore, SPARC does not appear to act as a local chelator of extracellular Ca2+ and Mg2+ and presumably exerts its function as a modulator of cell shape via a different pathway.
Sage
E H
EH
Department of Biological Structure, University of Washington, Seattle 98195.
D000818:Animals; D001011:Aorta / Q000166:cytology; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008274:Magnesium / Q000494:pharmacology; D009024:Morphogenesis / Q000187:drug effects / Q000502:physiology*; D015676:Osteonectin / Q000494:pharmacology*
D015676:Osteonectin; D004533:Egtazic Acid; D008274:Magnesium; D002118:Calcium