Abstract 13260: Regulated Splicing of Plasma Membrane Ca2+ ATPase-4 Controls Cell Cycle Progression in Vascular Smooth Muscle Cells
Objective: To explore the functional relevance of plasma membrane calcium ATPase-4 (PMCA4) splice variants (PMCA4a/4b).
Methods & Results: A novel splice site C variant (PMCA4e) was discovered. qRT-PCR- quantified PMCA4 splice variant relative proportions differ in specific organs. PMCA4a:4b ratio in uninjured carotid arteries (~1:1) is shifted by wire denudation injury (to ~1:3) via modulation of alternative splicing which is confirmed at the protein level using new antibodies for PMCA4b and PMCA4a/e. Laser capture microdissection documented this shift in the injured media and remodeled adventitia. Next, primary vascular smooth muscle cells (VSMC) from PMCA4-/- (P4KO) mice showed impaired 3H-thymidine incorporation and G1-phase arrest as compared to wild-type (P4WT). Microarray of flow-sorted early G1 VSMC subpopulations showed a 39-fold increase in Rgs16 (NFAT target) and a 69-fold increase in Decorin (G1 arrest marker) in P4KO vs. P4WT. Validation of these results by Western blot also revealed decreased levels of Cyclin D1 and NFATc3. PMCA4b overexpression in P4KO cells rescued the G1 phase arrest and reversed the perturbed expression of Rgs16, Decorin and NFATc3.
Conclusions: PMCA4 alternative splicing is modulated to increase PMCA4b expression during proliferation in vivo. PMCA4 deletion produces a G1 arrest which is reversed by PMCA4b expression.
- © 2012 by American Heart Association, Inc.