Calcium Efflux Activity of Plasma Membrane Ca2+ ATPase-4 (PMCA4) Mediates Cell Cycle Progression in Vascular Smooth Muscle Cells

Background: PMCA4 actions in vascular smooth muscle are not understood. Results: Alternative splicing of PMCA4 changes during vessel injury. Cell cycle arrest and downstream effectors of PMCA4 deletion are rescued by PMCA4a, PMCA4b, and PMCA4b unable to bind PDZ- proteins but not by inactive PMCA4. Conclusion: Ca2+ efflux activity of PMCA4 regulates G(1) progression. Significance: PMCA4 Ca2+ efflux regulates cell cycle. We explored the role played by plasma membrane calcium ATPase-4 (PMCA4) and its alternative splice variants in the cell cycle of vascular smooth muscle cells (VSMC). A novel variant (PMCA4e) was discovered. Quantitative real-time-PCR-quantified PMCA4 splice variant proportions differed in specific organs. The PMCA4a:4b ratio in uninjured carotid arteries (approximate to 1:1) was significantly reduced by wire denudation injury (to approximate to 1:3) by modulation of alternative splicing, as confirmed by novel antibodies against PMCA4a/e and PMCA4b. Laser capture microdissection localized this shift to the media and adventitia. Primary carotid VSMC from PMCA4 knock-out (P4KO) mice showed impaired [H-3]thymidine incorporation and G(1) phase arrest as compared with wild type (P4WT). Electroporation of expression constructs encoding PMCA4a, PMCA4b, and a PMCA4b mutant lacking PDZ binding rescued this phenotype of P4KO cells, whereas a mutant with only 10% of normal Ca2+ efflux activity could not. Microarray of early G(1)-synchronized VSMC showed 39-fold higher Rgs16 (NFAT (nuclear factor of activated T-cells) target; MAPK inhibitor) and 69-fold higher Decorin (G(1) arrest marker) expression in P4KO versus P4WT. Validation by Western blot also revealed decreased levels of Cyclin D1 and NFATc3 in P4KO. Microarrays of P4KO VSMC rescued by PMCA4a or PMCA4b expression showed reversal of perturbed Rgs16, Decorin, and NFATc3 expression levels. However, PMCA4a rescue caused a 44-fold reduction in AP-2, a known anti-proliferative transcription factor, whereas PMCA4b rescue resulted in a 50-fold reduction in p15 (Cyclin D1/Cdk4 inhibitor). We conclude that Ca2+ efflux activity of PMCA4 underlies G(1) progression in VSMC and that PMCA4a and PMCA4b differentially regulate specific downstream mediators.