Date of Award
2009
Degree Type
Thesis
Degree Name
Doctor of Philosophy
Program
Biochemistry
Supervisor
Dr. J. Geoffrey Pickering
Second Advisor
Dr. Frederick Dick
Third Advisor
Dr. Murray Huff
Abstract
The purpose of this thesis was to assess the role of Wilms' tumor 1-associating protein (WTAP), a nuclear constituent of uncertain function, in vascular smooth muscle cells (SMCs). The index finding for this thesis was our observation that WTAP was upregulated as SMCs shifted from a proliferative to non-proliferative state. Using laser capture microscopy, I determined that 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, together with reduced apoptosis. WTAP was found to associate with the Wilms' tumor-1 (WT1) 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. I also identified that insulin-like growth factor-1 (IGF-1) uniquely reduced WTAP abundance in SMCs. 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 receptor- phosphatidylinositol 3-kinase-Akt signaling axis that directed WTAP degradation via a nuclear 26S proteasome. Moreover, by promoting the degradation of WTAP, IGF-1 shifted the pre-mRNA splicing program survivin, to reduce expression of survivin-2B, which is pro-apoptotic, 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. Finally, using co-immunoprecipitation studies and site-directed mutagenesis, I identified that WTAP is phosphorylated by ATM kinase at serine residues 200 and 244. Furthermore, ATM-dependant phosphorylation at these residues released WTAP from the spliceosome and skewed the survivin splicing output toward survivin-2B. In summary, I have identified that WTAP is a critical regulator of both transcriptional and posttranscriptional events. These actions regulate the proliferation and survival of vascular SMCs and implicate WTAP as a multifaceted regulator of vascular remodeling.
Recommended Citation
Small, Theodore Whitfield, "THE ROLE OF WILMS TUMOR 1-ASSOCIATING PROTEIN IN VASCULAR SMOOTH MUSCLE CELLS" (2009). Digitized Theses. 4000.
https://ir.lib.uwo.ca/digitizedtheses/4000