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 microM 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.
Waldron
R T
RT
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Short
A D
AD
Meadows
J J
JJ
Ghosh
T K
TK
Gill
D L
DL
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D003470:Culture Media; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D010766:Phosphorylation; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D013997:Time Factors
D003470:Culture Media; D013729:Terpenes; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium