Nuclear Degradation of Wilms Tumor 1-associating Protein and Survivin Splice Variant Switching Underlie IGF-1-mediated Survival

Abstract

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...

Coiled-Coil Domain-Containing Protein 98 (CCDC98) Regulates Cyclin B1 Expression by Affecting WTAP Protein Stability

“…HeLa cells transfected with indicated siRNAs were fixed and stained with histone-specific anti-pH3 (a mitotic marker) and propidium iodide. [9]. These data provide additional evidence that WTAP expression may be regulated in the protein levels.…”

“…Figure 5B showed that the depletion of CCDC98 led to a dramatic decrease in the half-life of WTAP compared to that for control siRNA transfected HeLa cells. Previous data showed the ubiquitination of WTAP [9]. These data indicate that WTAP protein is degraded by CCDC98 via the ubiquitination/proteosome pathway.…”

“…These data indicate that WTAP protein is degraded by CCDC98 via the ubiquitination/proteosome pathway. It has been reported that WTAP knockdown led to a decrease in cyclin A2 at the mRNA and protein levels [9]. Therefore, we investigated whether or not CCDC98 knockdown also affects cyclins A2 and B1 at the mRNA levels.…”

IGF-1 deficiency in a critical period early in life influences the vascular aging phenotype in mice by altering miRNA-mediated post-transcriptional gene regulation: implications for the developmental origins of health and disease hypothesis

“…Recent studies show that WTAP inhibits the proliferation of vascular smooth muscle cells and endothelial cells and promotes apoptosis, regulating vascular remodeling (Small et al 2006(Small et al , 2007. During development of the vasculature IGF-1 was shown to downregulate WTAP, which is necessary for IGF-1 to confer its antiapoptotic effects, regulating smooth muscle cell fate (Small and Pickering 2009). Another factors affected by developmental IGF-1 deficiency are endothelin receptor A, versican and O-GlcNAc transferase (Ogt), and Wwtr1.…”

RNA sequencing: current and prospective uses in metabolic research

“…As there are data showing that different splice variants have different functions (Small & Pickering 2009, Kahkhaie et al 2014, Michel et al 2014, it is important to remember that a genes function is so much more than just gene expression levels. Therefore in the future it is likely that RNA sequencing can be used much in the same way as HDL/LDL measurements are being used today, but instead of looking at the lipid levels in the blood the biological response in the form of specific splice variants can be used.…”