Calcium efflux activity of plasma membrane Ca2+ ATPase-4 (PMCA4) mediates cell cycle progression in vascular smooth muscle cells.
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 (∼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 knock-out (P4KO) mice showed impaired [(3)H]thymidine incorporation and G1 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 Ca(2+) efflux activity could not. Microarray of early G1-synchronized VSMC showed 39-fold higher Rgs16 (NFAT (nuclear factor of activated T-cells) target; MAPK inhibitor) and 69-fold higher Decorin (G1 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-2β, a known anti-proliferative transcription factor, whereas PMCA4b rescue resulted in a 50-fold reduction in p15 (Cyclin D1/Cdk4 inhibitor). We conclude that Ca(2+) efflux activity of PMCA4 underlies G1 progression in VSMC and that PMCA4a and PMCA4b differentially regulate specific downstream mediators.
Division of Experimental Therapeutics, Toronto General Research Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada.
Talat
T
Afroze
Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Ge
G
Yang
Division of Experimental Therapeutics, Toronto General Research Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada.
Amir
A
Khoshbin
Division of Experimental Therapeutics, Toronto General Research Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada.
Mansoor
M
Tanwir
Division of Experimental Therapeutics, Toronto General Research Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada.
Taha
T
Tabish
Division of Experimental Therapeutics, Toronto General Research Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada.
Abdul
A
Momen
Division of Experimental Therapeutics, Toronto General Research Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada; Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Heart and Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, Toronto, Ontario M5G 1L7; Department of Medicine, University of Toronto, Toronto, Ontario M5G 2C4, Canada. Electronic address: mansoor.husain@uhn.ca.
Mansoor
M
Husain
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002339:Carotid Arteries / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D014018:Tissue Distribution
D002118:Calcium; D000252:Calcium-Transporting ATPases; D053499:Plasma Membrane Calcium-Transporting ATPases; D020033:Protein Isoforms; C000589038:ATP2B4 protein, mouse
AP-2β; Alternative Splicing; Calcium ATPase; Cell Cycle; Cell Proliferation; Decorin; Plasma Membrane Calcium Atpase-4; Rgs16; Vascular Smooth Muscle Cells; p15(Ink4b)