Regulation of vascular smooth muscle cell proliferation by plasma membrane Ca(2+)-ATPase

Abstract

We have previously shown that reductions in c-Myb-dependent transcription inhibit cell cycle progression and decrease intracellular Ca2+ concentrations in vascular smooth muscle cells (VSMC). We now report that these effects are largely mediated by a 4- to 10-fold increased rate of La(3+)-sensitive 45Ca extrusion, which is associated with 2- to 4-fold increased levels of plasma membrane Ca(2+)-ATPase 1 (PMCA1) mRNA and protein. PMCA4 mRNA, present at much lower concentrations, undergoes similar changes during suppression of c-Myb activity. We also report that PMCA1 expression is regulated during VSMC cell cycle progression, such that levels of PMCA1 are 40% lower at the G1/S interface than at G0. Moreover, transient overexpression of PMCA1a in VSMC elevates the 45Ca efflux rate by approximately 2-fold, decreases resting and peak thapsigargin-releasable Ca2+ concentrations at G1/S by 43% (68 nM) and 52% (160 nM), respectively, and reduces the rate of cell proliferation by over 2.5-fold. These data define a mechanism for c-Myb-dependent Ca2+ homeostasis and support a critical role for PMCA in the regulation of VSMC growth.

c-Myb-binding Sites Mediate G1/S-associated Repression of the Plasma Membrane Ca2+-ATPase-1 Promoter

“…Cells were washed twice with phosphate-buffered saline and incubated in Dulbecco's modified Eagle's medium containing 0.25% fetal bovine serum for 48 h at which point the cells were either harvested (G 0 stage cells), or the medium was replaced with Dulbecco's modified Eagle's medium and 10% fetal bovine serum for 8 (G 1 stage cells), 16 (G 1 /S stage cells), or 24 h (G 2 /M stage cells) prior to harvest. The degree and extent of cell cycle synchronization, as assessed by DNA quantitative flow-cytometry, was identical to previously published reports (data not shown) (6,9,10). Primary VSMC were isolated by the method of Cornwell and Lincoln (27) using 10 -12 aortas from C57Bl6 mice (Charles River Laboratories, Inc., Wilmington, MA).…”

“…These manipulations resulted in a 30% reduction in mean resting [Ca 2ϩ ] i , a 42% decrease in mean S phase entry, and a 36% decrease in the mean cell proliferation rate of synchronized rat VSMC populations (10). We also demonstrated that a direct 2-fold overexpression of PMCA1, independent of any manipulation of c-Myb activity, resulted in similarly significant reduc- tions in [Ca 2ϩ ] i , G 1 to S transitions, and rate of cell proliferation (9). Having demonstrated the physiological importance of PMCA1 regulation in VSMC, we now examine more closely the molecular interaction between c-Myb and the PMCA1 gene.…”

“…We have shown that c-Myb activity regulates [Ca 2ϩ ] i in both VSMC and fibroblasts (9 -11). Experiments employing either wild-type or dominant negative forms of c-Myb, in which cell cycle-associated Ca 2ϩ homeostasis was monitored, implicated the plasma membrane Ca 2ϩ -ATPase (PMCA) family of Ca 2ϩ efflux pumps as critical mediators of c-Myb-dependent [Ca 2ϩ ] i (9,10).…”

“…Indeed, the resting [Ca 2ϩ ] i in many cell types is critically regulated by the level of PMCA activity (9,21,22). However, despite the obvious importance of PMCAs to cellular Ca 2ϩ handling, little is known of the mechanisms regulating their expression.…”

Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension

“…Removal of extracellular free Ca 2ϩ or blockade of Ca 2ϩ channels inhibits PASMC proliferation (22,26,27,49). Depletion of SR Ca 2ϩ attenuates smooth muscle cell proliferation (25) and promotes apoptosis (50). In contrast, elevated [Ca 2ϩ ] cyt may induce PASMC proliferation by initiating and promoting the cell cycle, and by triggering or promoting gene transcription via transcription factor phosphorylation (21,25,37).…”