Regulation of tyrosine-hydroxylase (TH) gene expression by hypoxia and von Hippel-Lindau tumor suppressor complex

Abstract

Loss of von Hippel-Lindau (VHL) gene function leads to a familial cancer syndrome, VHL disease, characterized by CNS and retinal hemangioblastomas, renal clear cell carcinomas, and pheochromocytomas. Pheochromocytomas, tumors of the chromaffin cells of the adrenal medulla, secrete large amounts of catecholamines. This results from enhanced expression of TH, the rate-limiting enzyme in catecholamine biosynthesis. Overexpression of exogenous pVHL in PC12 cells lead to a substantial repression of TH protein and mRNA [1]. Here we examined the role of endogenous pVHL in the regulation of TH gene expression. We developed PC12 clonal cell lines that stably express VHL antisense RNA and have reduced levels of endogenous pVHL by 5–10 fold. In these cells, TH protein and mRNA were increased 2–3 fold as compared to control cells. Nuclear run-on analysis revealed an increase in the overall TH gene transcription. In addition, we measured augmented efficiency of the TH transcript elongation in the distal part of the gene. Transient co-transfections of -773 kb TH-CAT promoter-reporter constructs with the VHL antisense RNA resulted in an approximately 2–3 fold increase in reporter gene activity. TH is a hypoxia-inducible gene. The overall accumulation of THmRNA and the rate of TH gene transcription were substantially enhanced in cells with decreased levels of pVhl exposed to 1% O2 for 16 h. These results indicate that endogenous pVhl constitutively represses TH gene expression in PC12 cells. TH promoter contains a functional hypoxia-responsive element (HRE) - like sequence. Transient co-transfections of the HIF1α and HIF2α expression vectors with the wild type, but not mutated TH promoter revealed significant 3–4 fold induction of the reporter gene activity. Gel-shift analysis showed presence of the hypoxia-inducible complexes associated with the wild type but not mutated THHRE. Electroelution of protein complexes from the hypoxia-inducible complex showed presence of the HIF2α, and to lesser extent of the HIF1 subunit. pVHL is part of the E3 ubiquitin ligase complex that targets the HIF alphas for ubiquitination and degradation. Changes in the levels of the pVHL in PC12 cells by either overexpression of pVHL or expression of VHL antisense RNA resulted in respectively decreased and increased HIF-and hypoxia-mediated activation of the TH promoter. Stable changes in the pVHL levels in PC12 cells resulted in accumulation of HIF alphas during normoxia in cells with decreased amounts of pVHL, and in decreased expression of HIF alphas during hypoxia in cells overex-pressing pVHL. The changes in HIFα protein accumulation induced by pVHL resulted from ubiquitination of HIF alphas by pVHL.