Abstract 599: Regulated Alternative Splicing Of Plasma Membrane Calcium Atpase-4 During Cell Cycle Progression Of Vascular Smooth Muscle Cells
Background: We and others have shown the functional importance of the ubiquitously expressed plasma membrane calcium ATPase-4 (PMCA4) gene to cell cycle progression, differentiation, and contractile function in vascular smooth muscle cells (VSMC) and cardiomyocytes. Alternative splicing of exon-20 (near the C-terminus) of PMCA4 generates two distinct splice variants, namely -CI and -CII, in which the respective presence and absence of a PDZ-binding motif determines caveolin-based association and functional regulation of the PDZ-harboring isoform nNOS in VSMC and cardiomyocytes. However, the mechanism(s) regulating PMCA4 splicing are unknown.
Methods & Results: Full-length PMCA4-CI and -CII cDNA were cloned from total RNA extracted from mouse VSMC and brain respectively. When synthetic capped RNA synthesized in vitro from cDNA clones were injected, Xenopus oocytes over-expressing mouse PMCA4-CI or -CII exhibited efflux rates that were 6- or 8-fold higher than control-injected oocytes respectively. Two unique chicken polyclonal antibodies (Ab) against PMCA4-CI and -CII specific epitopes were developed and shown to recognize an ~130 kDa (PMCA4) protein in cardiac extracts from C57BL6 but not PMCA4 knockout mice. Quantitative RT-PCR with PMCA4-CI- and -CII-specific primers with comparable amplification efficiencies showed that splice variant-specific mRNA copy numbers were reduced 9-fold (-CI) and 6-fold (-CII) respectively as cell cycle-synchronized VSMC move from G0 to G1/S. Treatment with the adenylate-cyclase stimulant Forskolin abolished PMCA4 isoform repression, increasing PMCA4-CI mRNA levels 2- (G0) to 10-fold (G1/S) and PMCA4-CII mRNA levels 4- (G0) to 17-fold (G1/S) during cell cycle progression. Western blot with the novel anti-PMCA4-CI chicken polyclonal antibody also showed a decrease in PMCA-CI protein during G0 to G1/S progression in VSMC, and an increase in PMCA4-CI protein after Forskolin treatment.
Conclusion: These data suggest that a cAMP-driven PKA-dependent CREB phosphorylation regulates splicing proteins which modulate physiologically relevant alternative splicing at exon-20 of the PMCA4 pre-mRNA during cell cycle progression of VSMC.