Abstract 270: Insulin-Like Growth Factor-1 Promotes Vascular Smooth Muscle Survival By 26S Proteasome-Mediated Degradation of Wilms’ Tumor 1-Associating Protein

Abstract

Apoptosis of vascular smooth muscle cells (SMC) is a culprit event in atherosclerotic plaque destabilization. We recently discovered that Wilms’ tumor 1-associating protein (WTAP) is a dynamically expressed transcriptional regulator that can be pro-apoptotic for human SMCs (Circ Res, 2006). To identify upstream regulators of this nuclear protein, we screened growth factors for their capacity to impact WTAP expression and found that insulin-like growth factor-1 (IGF-1), a potent survival factor for SMCs, stimulated a striking decline in WTAP protein abundance, to 10% at 12 h. We further determined that this decline in WTAP was due specifically to WTAP protein degradation, established by pulse-chase analysis of 35 S-labeled WTAP and the absence of an acute effect of IGF-1 on WTAP mRNA abundance. IGF-1-mediated WTAP degradation was blocked by two mechanistically distinct IGF-1 receptor inhibitors (picropodophyllin and PQ401) and by inhibition of phosphatidylinositol 3 (PI3)-kinase but not by MEK inhibition. In addition, IGF-1 induced the association of WTAP with ubiquitin, established by coimmunoprecipitation, and the downregulation of WTAP by IGF-1 was abrogated by inhibiting 26S proteasome activity with lactacystin or MG132. Interestingly, IGF-1 also stimulated phosphorylation of WTAP, that preceded the association of WTAP with ubiquitin, and hyperphosphorylation of WTAP through phosphatase-inhibition further accelerated WTAP degradation. Finally, to determine if WTAP downregulation was necessary for IGF-1-mediated SMC survival, surface expression of phosphatidylserine was quantified by flow cytometry of SMCs infected with retrovirus containing WTAP cDNA. Whereas IGF-1 enhanced the survival of vector-infected SMCs this was completely abrogated in WTAP-overexpressing SMCs. Conclusions: IGF-1-mediated SMC survival is dependent on the rapid depletion of WTAP from the nucleus, a degradation cascade that is heralded by WTAP phosphorylation. This WTAP phosphorylation and clearance response represents a novel consequence of PI3-kinase activation and highlights WTAP as a key negative regulator of SMC survival during vascular remodeling.