Abstract 15713: UAP56 is a Novel Interacting Partner of Bcr in Vascular Smooth Muscle Cell Proliferation
Background: We have previously reported that breakpoint cluster region (Bcr) is an important mediator of Angiotensin II (AngII)/platelet-derived growth factor (PDGF) mediated signaling in vascular smooth muscle cells (VSMC). We demonstrated that Bcr, a serine/threonine kinase, acts in part via phosphorylation and inhibition of peroxisome proliferator-activated receptor gamma (PPARγ). In addition, we identified a novel substrate for Bcr kinase, the DExD/H box protein UAP56 (also known as BAT1). UAP56 has ATPase activity and is also an ATP dependent RNA helicase, utilizing the energy from ATP hydrolysis to unwind double stranded RNA. UAP56 plays an important role in RNA metabolism including RNA splicing, mRNA export and protein synthesis. Given that other DExD/H box proteins play a role in cell growth, we hypothesized that UAP56 is an important mediator of Bcr induced VSMC proliferation.
Methods and Results: Co-immunoprecipitation studies confirmed that Bcr binds to UAP56. Assessment by in vitro kinase assay demonstrated that Bcr phosphorylates UAP56. Utilizing UAP56 siRNA, we demonstrated that knockdown of UAP56 inhibited AngII/PDGF mediated VSMC DNA synthesis (41% ± 5% inhibition, p<0.01) as measured by thymidine incorporation. Overexpression of Bcr increased DNA synthesis (2.1 ± 0.3 fold, p<0.01) compared with control, an effect that was inhibited by UAP56 siRNA. DN-UAP56 also inhibited Bcr induced DNA synthesis (51% ± 13% inhibition, p<0.01). Because DExD/H box proteins are thought to affect cell growth by regulating the transcription of cell cycle proteins, we examined the role of Bcr and UAP56 in the expression of cyclins and cyclin dependent kinase inhibitors and demonstrated that overexpression of Bcr increased the expression of cyclin E and decreased the expression of p21 and p27. Knockdown of UAP56 reversed the effect of Bcr on cyclin E, p21 and p27 expression. Knockdown of UAP56 also inhibited PDGF induced E2F transcriptional activation. Utilizing a partial ligation model of the left carotid artery, we demonstrated that UAP56 is present in the intima following injury.
Conclusions: Our data identify UAP56 as an important binding partner of Bcr in AngII/PDGF signaling and a novel target for improving vascular proliferative diseases.
- © 2010 by American Heart Association, Inc.