Platelet-derived growth factor (PDGF) has been implicated in the pathogenesis of vascular occlusive disorders such as atherosclerosis and restenosis, in part due to its regulation of smooth muscle cell (SMC) phenotype. The molecular mechanisms regulating the expression of PDGF-receptor-?? (PDGF-R-??), which binds all known dimeric forms of PDGF except PDGF-DD, are poorly understood. Here it is demonstrated that PDGF-R-?? protein and transcriptional regulation in SMCs is under the positive regulatory influence of the zinc finger nuclear protein, Sp1. An atypical G-rich Sp1-binding element from -61 to -52 bp (-61G10 - 52) upstream of the transcriptional start site, was identified in the PDGF-R-?? promoter via electrophoretic mobility shift, competition and supershift analysis. Mutation of this sequence ablated endogenous Sp1 binding and activation of the PDGF-R-?? promoter. In addition to Sp1 regulation of PDGF-R-??, it was shown that fibroblast growth factor-2 (FGF-2) represses PDGF-R-?? transcription, mRNA and protein expression in SMCs. The FGF-2-induced inhibition of PDGF-R-?? was rescued by block of extracellular signal-regulated kinase-1/2 (ERK1/2) signaling. Interestingly, mutation of the aforementioned novel Sp1-response-element blocked FGF-2-induced repression of PDGF-R-?? transcription. FGF-2 was also shown to stimulate Sp1 phosphorylation in an ERK1/2-dependent manner, enhancing its interaction with the PDGF-R-?? promoter. Further analysis revealed that mutations of residues Thr453 and Thr739 in Sp1 resulted in loss of FGF-2-mediated repression of PDGF-R-?? transcription. These findings demonstrate that FGF-2 stimulates ERK1/2- dependent Sp1 phosphorylation, thereby repressing PDGF-R-?? transcription via Sp1 binding the -61/-52 element in the PDGF-R-?? promoter. Thus, phosphorylation triggered by FGF-2, switches Sp1 from an activator to a repressor of PDGF-R-?? transcription, a finding previously unreported in any Sp1-dependent gene. It is also shown in this thesis that the proto-oncogene Ets-1 controls PDGF-R-?? transcription and mRNA expression in SMCs. Mutational, electrophoretic mobility shift and chromatin immunoprecipitation assays revealed the existence of a reverse Ets binding motif (-45TTCC-42) in the proximal region of the PDGFR- ?? promoter which bound both recombinant and endogenous Ets-1. Ets-1- inducible PDGF-R-?? expression was dependent upon the integrity of both the - 45TTCC-42 motif and the previously identified -61G10 -52 element, which resides upstream of -45TTCC-42 and mediates Sp1 induction. Hydrogen peroxide (H2O2) at nanomolar concentrations, stimulated levels of Ets-1 and increased PDGF-R- ?? transcription and mRNA expression without affecting Sp1 expression. Disruption of the -45TTCC-42 motif or -61G10 -52 element blocked H2O2 activation of the PDGF-R-?? promoter. These studies identify a functional Ets motif in the PDGF-R-?? promoter which plays a pivotal role in agonist-inducible PDGF-R-?? transcription. The interplay between transcription factors such as Sp1 and Ets- 1 in the promoter of genes can exert profound influences on gene regulation. Modulating gene expression affects biological processes such as SMC proliferation and phenotype changes, which contributes to changes in vessel integrity, a hallmark of atherosclerosis. This study provides a greater insight in the functional consequences of Sp1 and Ets-1 interplay in PDGF-R-?? gene regulation and in general, provides a greater understanding of the functional regulation of cooperating transcription factors.
| Identifer | oai:union.ndltd.org:ADTP/235870 |
| Date | January 2005 |
| Creators | Bonello, Michelle Rita, Medical Sciences, Faculty of Medicine, UNSW |
| Publisher | Awarded by:University of New South Wales. School of Medical Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Michelle Rita Bonello, http://unsworks.unsw.edu.au/copyright |
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