Abstract-Smooth muscle cells (SMCs) are called on to proliferate during vascular restructuring but must return to a nonproliferative state if remodeling is to appropriately terminate. To identify mediators of the reacquisition of replicative quiescence, we undertook gene expression screening in a uniquely plastic human SMC line. As proliferating SMCs shifted to a contractile and nonproliferative state, expression of TIMP-3, Axl, and KIAA0098 decreased whereas expression of complement C1s, cathepsin B, cellular repressor of E1A-activated genes increased. Wilms' tumor 1-associating protein (WTAP), a nuclear constituent of unknown function, was also upregulated as SMCs became nonproliferative. Furthermore, WTAP in the intima of injured arteries was substantially upregulated in the late stages of repair. Introduction of WTAP complementary DNA into human SMCs inhibited their proliferation, with a corresponding decrease in DNA synthesis and an increase in apoptosis. Knocking down endogenous WTAP increased SMC proliferation, because of increased DNA synthesis and G 1 /S phase transition, together with reduced apoptosis. WTAP was found to associate with the Wilms' tumor-1 protein in human SMCs and WTAP overexpression inhibited the binding of WT1 to an oligonucleotide containing a consensus WT1 binding site, whereas WTAP knockdown accentuated this interaction. Expression of the WT1 target genes, amphiregulin and Bcl-2, was suppressed in WTAP-overexpressing SMCs and increased in WTAP-deficient SMCs. Moreover, exogenous amphiregulin rescued the antiproliferative effect of WTAP. These findings identify WTAP as a novel regulator of the cell cycle and cell survival and implicate a WTAP-WT1 axis as a novel pathway for controlling vascular SMC phenotype. (Circ Res. 2006;99:1338-1346.)Key Words: amphiregulin Ⅲ smooth muscle cells Ⅲ Wilms' tumor 1-associating protein Ⅲ vascular smooth muscle cell proliferation P henotype plasticity is a feature of adult vascular smooth muscle cells (SMCs). A widely studied example of this is the dedifferentiation of mature, nonproliferative SMCs into proliferative SMCs, a process central to vascular remodeling. 1,2 Although less well studied, an equally important manifestation of SMC plasticity is the reverse shift, whereby proliferative adult SMCs convert back to a nonproliferative state. This particular phenotype switch is essential for limiting SMC accumulation and for terminating vascular remodeling. As such, the regulatory factors that drive proliferative SMCs into a nonproliferative state, and hold them in that state, are critical for effective vascular remodeling and for limiting vascular disease.We have generated unique lines of nonimmortalized human SMCs that are capable of converting between proliferative and nonproliferative states. 2,3 In the presence of serum, these SMCs proliferate, migrate, and elaborate extracellular matrix similar to primary SMCs. On withdrawal of serum however they undergo a reproducible program of cellular maturation whereby they exit the cell cycle, migrate...

WTAP (Wilms tumor 1-associating protein) is a recently identified nuclear protein that is essential for mouse embryo development. The Drosophila homolog of WTAP, Fl(2)d, regulates pre-mRNA splicing; however, the role of WTAP in mammalian cells is uncertain. To elucidate a context for WTAP action, we screened growth and survival factors for their effects on WTAP expression in vascular smooth muscle cells (SMCs), a cell type previously found to express WTAP dynamically. This revealed that insulin-like growth factor-1 (IGF-1) uniquely reduced WTAP abundance. This decline in WTAP proved to be necessary for IGF-1 to confer its antiapoptotic properties, which were blocked by transducing the WTAP gene into SMCs. WTAP down-regulation by IGF-1 was mediated by an IGF-1 receptorphosphatidylinositol 3-kinase-Akt signaling axis that directed WTAP degradation via a nuclear 26 S proteasome. Moreover, by promoting the degradation of WTAP, IGF-1 shifted the pre-mRNA splicing program for the survival factor, survivin, to reduce expression of survivin-2B, which is proapoptotic, and increase expression of survivin, which is antiapoptotic. Knockdown of survivin-2B rescued the ability of IGF-1 to promote survival when WTAP was overexpressed. These data uncover a novel regulatory cascade for human SMC survival based on adjusting the nuclear abundance of WTAP to define the splice variant balance among survivin isoforms. Smooth muscle cell (SMC)3 survival is critical to vascular stability (1). This is especially important during atherosclerosis, a condition that depends on replicating SMCs to mechanically stabilize the artery wall. An abundance of proapoptotic stimuli within an atherosclerotic lesion can deplete the SMC population, resulting in plaque rupture and myocardial infarction (1, 2). Pathways that enable SMCs to replicate and perform reparative functions and simultaneously resist apoptotic signals are therefore vital to cardiovascular health.WTAP (Wilms tumor 1-associating protein) is a recently identified nuclear protein that, as its name implies, can interact with the WT1 suppressor protein (3, 4). The precise molecular actions of WTAP are not well understood, but the importance of this protein is highlighted by the early lethality of WTAP-null mouse embryos (5, 6). WTAP is widely expressed in adult tissues and might therefore play a constitutive role. However, expression of WTAP can also be dynamic. We found that WTAP expression in the adult artery wall varied, depending on whether the vessel was quiescent or remodeling. In particular, WTAP abundance decreased as vascular SMCs were induced to proliferate and increased as remodeling subsided and SMC accumulation terminated. Furthermore, overexpression of WTAP in cultured SMCs suppressed their accumulation, whereas WTAP knockdown stimulated population growth. These effects were mediated, at least in part, by the capacity for WTAP to activate apoptosis (4).The basis by which WTAP impacts cell function is uncertain, and several possibilities have been raised. One group has repor...

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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.