Shakib, Kaveh; (2002) Transcriptional regulation of the collagen alpha1(1) gene by hypoxia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis investigates the molecular mechanisms of hypoxic regulation of the collagen α1(1) (COL1A1) gene. Collagen-1 is the most abundant extracellular protein in vertebrates. Transcription of this protein is normally under tight physiological control. However, under pathological conditions this fine control is lost leading to accumulation of collagen, scarring and fibrosis. Hypoxia, as a result of microvascular injury, has been implicated in the pathogenesis of fibrosis and studies in this laboratory had shown that hypoxia is a pro-fibrogenic stimulus increasing production and decreasing turnover of interstitial Collagen-1 in vitro. Hypoxia is a potent regulator of gene expression and hypoxia response elements (HRE) have been identified in a wide variety of genes. These HREs mostly, but not exclusively, contain consensus binding sites for the hypoxia-inducible factor-1 (HIF-1). Transfection experiments and deletion analysis of the COL1A1 promoter demonstrated that the HRE(s) for this gene lies between -220/+115. This evolutionary conserved region lacks any consensus HIF-1 binding sites suggesting HIF-1-independent transcriptional activation of COL1A1 in response to low O2. In addition TGF-β1, the preeminent fibrogenic stimulus, did not stimulate reporter gene activity in this region of COL1A1, indicating TGF-β1 independent hypoxic activation. Subsequent studies focussed on identification of c/s-acting DNA elements and transcription factors mediating the hypoxia-induced COL1A1 gene transcription: DNA elements; DNAse-l footprinting of the mouse COL1A1 -220/+115 with nuclear extracts from normoxic fibroblasts showed 2 protected regions. Hypoxic nuclear proteins demonstrated increased affinity to the promoter producing 11 protected areas representing higher transcriptional activity. Transcription factors: Nuclear extracts from normoxic and hypoxic fibroblasts were resolved on two dimensional gels and proteins identified by proteomics. Results of this study produced a broad spectrum of nuclear proteins therefore a more targeted approach was devised, where specific DNA binding proteins were identified by Southwestern blotting and characterised with proteomics. On Southwesterns, 7 normoxic nuclear proteins hybridised to the -220/+115 COL1A1 radiolabelled probe, while hypoxic nuclear extracts produced 12 strong bands, again illustrating higher transcriptional activity in hypoxia. Proteomic analysis of the bands identified several transcription factors with Sp1 displaying the highest increase in DNA binding in hypoxia. DNA-Protein interaction: Sequence scanning of COL1A1 -220/+115 revealed 3 putative Sp1 binding sites (-123/-114, -98/-82, -63/-53). Binding of Sp1 to the 2 more upstream sites has been previously reported. EMSAs and antibody supershifts showed Sp1 binding to the two upstream sites (-123/-114 and -98/-82) in normoxic and hypoxic nuclear extracts consistent with a role for this factor in basal and hypoxic transcriptional regulation of COL1A1. The binding of Sp1 to both sites increased in hypoxia with the most 5' site (-123/-114) showing the highest increase. The level of binding by normoxic and hypoxic nuclear proteins to the most proximal putative Sp1 binding site was weak. To assess the functional role of Sp1 the effect of inhibiting Sp1 binding to the COL1A1 promoter was examined. Mithramycin is a GC-specific DNA-binding drug, which selectively inhibits transcription of genes with GC-rich promoter. In transfection experiments mithramycin treatment completely inhibited the hypoxic induction of COL1A1 -220/+115 to a level blow the basal activity of the promoter. The increased binding of Sp1 to the promoter in hypoxia could be due to either upregulation of Sp1 with increased availability of Sp1 in the nucleus or to "activation" of Sp1 increasing the binding affinity of this factor for the promoter. By Western blotting there was no difference in nuclear Sp1 levels in hypoxia and normoxia suggesting that hypoxia increases Sp1 activation and DNA binding. In conclusion, these studies demonstrate Sp1 is an important mediator of hypoxia-induced COL1A1 gene transcription in fibroblasts acting via hypoxia response elements in the -220/+115 region of COL1A1.

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