THE EXIT FROM G(0) INTO THE CELL-CYCLE REQUIRES AND IS CONTROLLED BY SARCO(ENDO)PLASMIC RETICULUM CA2+ PUMP

The intracellular calcium pump sarco(endo)plasmic reticulum Ca2+ (SERC A) is responsible for the formation of the Ca2+ gradient across the en doplasmic reticulum membrane, and this gradient is used to generate th e Ca2+ signal during agonist-stimulated cell growth. In the present st udy, the role of SERCA in both cell cycle and growth control was inves tigated using cultured rat aortic endothelial cells (RAEC). Using a no vel DNA transfection approach, cell lines were established that showed varying degree of SERCA activity through the down-regulation of the e ndogenous SERCA gene (B. F. Liu, X. Xu, R. Fridman, S. Muallem, and T. H. Kuo, J. Biol. Chem. 271, 1-9, 1996), Cell proliferation studies in dicated that the lower SERCA expressing cells exhibited a slower growt h pattern without altering the doubling time which was similar for bot h parental and transfected RAEC lines. G(1) to S phase transition was prolonged with a smaller proportion of cells entering DNA synthesis as indicated by thymidine incorporation assay. Comparison of transfected cell lines indicated a tight coupling of SERCA activity and the lengt h of the G(1) period. Down-regulation of SERCA gene expression was acc ompanied by increased mRNA levels of p21 (WAF1/CIP1), a universal cell cycle inhibitor. The delay in G(1) to S progression also coincided wi th the up-regulation of p53 mRNA and underphosphorylation of the retin oblastoma protein. This study suggests that Ca2+ metabolism in the ago nist mobilizable pool controls the cell cycle through the regulation o f genes operating in the critical G(1) to S checkpoint. (C) 1996 Acade mic Press, Inc.