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

January 21st, 2014 by Afroze, T., Yang, G., Khoshbin, A., Tanwir, M., Tabish, T., Momen, A., Husain, M.

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. qRT-PCR-quantified PMCA4 splice variant proportions differed in specific organs. The PMCA4a:4b ratio in uninjured carotid arteries (~1:1) was significantly reduced by wire denudation injury (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 knockout (P4KO) mice showed impaired 3H-thymidine incorporation and G1-phase arrest as compared to wild-type (P4WT). Electroporation of expression constructs encoding PMCA4a, PMCA4b, and a PMCA4b mutant lacking PDZ binding rescued this phenotype of P4KO cells, while a mutant with only 10% of normal Ca2+ efflux activity could not. Microarray of early G1-synchronized VSMC showed 39-fold higher Rgs16 (NFAT target; MAPK inhibitor) and 69-fold higher Decorin (G1 arrest marker) expression in P4KO vs. 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 β, a known anti-proliferative transcription factor, while PMCA4b rescue resulted in a 50-fold reduction in p15 (Cyclin D1/Cdk4 inhibitor). We conclude that Ca2+-efflux activity of PMCA4 underlies G1 progression in VSMC, and that PMCA4a and PMCA4b differentially regulate specific downstream mediators.
  • Posted in Journal of Biological Chemistry, Publications
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