Endoplasmic reticulum calcium pump expression and control of cell growth

Intracellular Ca²⁺ pump expression and Ca²⁺ pool function are shown to be closely associated with growth and proliferation of DDT₁MF-2 hamster smooth muscle cells. The Ca²⁺ pump blocker thapsigargin induces sustained Ca²⁺ pool emptying and entry of cells into a quiescent G₀-like state (Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4986-4990). Using DDT₁MF-2 cells growth-arrested by exposure to 3 μM thapsigargin for 24 h, treatment with 20% serum for 6 h without thapsigargin induced expression of functional Ca²⁺ pump protein detected as a 110-kDa thapsigargin-sensitive phosphorylated intermediate; 2.5% serum treatment resulted in no functional pump expression. Western analysis revealed only a slight serum-induced increase in total Ca²⁺ pump protein. New functional Ca²⁺ pump protein could be detected within 1 h of high serum treatment of thapsigargin- arrested cells, increasing over a 6-h period and correlating with the appearance of new Ca²⁺ pools. Induction of Ca²⁺ pools required serum at 10% or higher; no pools appeared with 5% serum or less. Significantly, high serum was required for only a brief but precise period of time. Exposure of thapsigargin-arrested cells to a 45-min pulse of 20% serum followed by continued culture in 2.5% serum was sufficient for full induction of new functional Ca²⁺ pump protein and Ca²⁺ pools; in contrast, no pumps or pools were detected after a 30-min serum pulse. A 40-min high serum pulse resulted in arrested cells reentering the cell cycle, synthesizing DNA, and resuming normal proliferation; in contrast, 35 min of serum treatment resulted in cells remaining totally quiescent. The results provide important evidence for the necessity of functional endoplasmic reticulum Ca²⁺ pumps in serum-induced cell growth and reflect a remarkably precise signaling period during which quiescent cells become committed to a progression of events including Ca²⁺ pump expression, Ca²⁺ pool function, reentry into the cell cycle, and cell division.