Von Hippel-Lindau Protein Induces Hypoxia-regulated Arrest of Tyrosine Hydroxylase Transcript Elongation in Pheochromocytoma Cells

S.L Kroll,W.R Paulding,P.O Schnell,M.C Barton,J.W Conaway,R.C Conaway,M.F Czyzyk-Krzeska

doi:10.1074/jbc.274.42.30109

Abstract

Rat pheochromocytoma (PC12) cells were stably transfected with either wild type or mutated human von Hippel-Lindau tumor suppressor protein (hpVHL). These proteins have opposing effects on regulating expression of the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Whereas wild type hpVHL represses levels of TH mRNA and protein 5-fold, a truncated pVHL mutant, pVHL(1-115), induces accumulation of TH mRNA and protein 3-fold. hpVHL-induced inhibition of TH gene expression does not involve either a decrease in TH mRNA stability or repression of TH promoter activity. However, repression results from inhibition of RNA elongation at a downstream region of the TH gene. This elongation pause is accompanied by hpVHL sequestration in the nuclear extracts of elongins B and C, regulatory components of the transcription elongation heterotrimer SIII (elongin A/B/C). Hypoxia, a physiological stimulus for TH gene expression, alleviates the elongation block. A truncated pVHL mutant, pVHL(1-115), stimulates TH gene expression by increasing the efficiency of TH transcript elongation. This is the first report showing pVHL-dependent regulation of specific transcript elongation in vivo, as well as dominant negative activity of pVHL mutants in pheochromocytoma cells.

Highlights

Pheochromocytoma tumors arise from chromaffin cells of the adrenal medulla
In cell lines derived from renal clear cell carcinoma tumors that have mutated form of pVHL, wild type pVHL has been shown to down-regulate the expression of a number of genes that may be involved in the pathogenesis of von Hippel-Lindau (VHL) disease-associated tumors, including vascular endothelial growth factor (VEGF) [12,13,14], glucose transporters [12, 14], and carbonic anhydrases [15]
Repression of tyrosine hydroxylase (TH) Gene Expression by hpVHL—A hemagglutinin (HA)-tagged human wild type pVHL, its truncated mutant, pVHL[1–115], and a pRC cytomegalovirus vector with no insert were stably expressed in PC12 cells

Summary

Introduction

Pheochromocytoma tumors arise from chromaffin cells of the adrenal medulla. The primary phenotype of these tumors is the ability to synthesize and release large amounts of catecholamines. This is the first report showing pVHL-dependent regulation of specific transcript elongation in vivo, as well as dominant negative activity of pVHL mutants in pheochromocytoma cells. We report that wild type pVHL represses the expression of TH in rat pheochromocytoma PC12 cells at the level of transcript elongation.

Results

Conclusion