Calcineurin-independent regulation of plasma membrane Ca2+ ATPase-4 in the vascular smooth muscle cell cycle

Calcineurin mediates repression of plasma membrane Ca2+-ATPase-4 (PMCA4) expression in neurons, whereas c-Myb is known to repress PMCA1 expression in vascular smooth muscle cells (VSMC). Here, we describe a novel mouse VSMC line (MOVAS) in which Ca-45 efflux rates decreased 50%, fura 2-AM-based intracellular Ca2+ concentrations ([Ca2+](i)) increased twofold, and real-time RT-PCR and Western blot revealed a similar to40% decrease in PMCA4 expression levels from G(0) to G(1)/S in the cell cycle, where PMCA4 constituted similar to20% of total PMCA protein. Although calcineurin activity increased fivefold as MOVAS progressed from G(0) to G(1)/S, inhibition of this increase with either BAPTA or retroviral transduction with peptide inhibitors of calcineurin (CAIN), or its downstream target nuclear factor of activated T cells (NFAT) (VIVIT), had no effect on the repression of PMCA4 mRNA expression at G(1)/S. By contrast, Ca2+-independent activity of the calmodulin-dependent protein kinase-II (CaMK-II) increased eightfold as MOVAS progressed from G(0) to G(1)/S, and treatment with an inhibitor of CaMK-II (KN-93) or transduction of a c-Myb-neutralizing antibody significantly alleviated the G(1)/S-associated repression of PMCA4. These data show that G(1)/S-specific PMCA4 repression in proliferating VSMC is brought about by c-Myb and CaMK-II and that calcineurin may regulate cell cycle-associated [Ca2+](i) through alternate targets.