Analysis of HIF-1alpha degradation and function

Sammanfattning: HIF-1 is a heterodimeric complex of two bHLH/PAS transcription factors, HIF- 1alpha and ANRT. In contrast to the constitutively expressed ARNT, HIF-1alpha protein levels are regulated by oxygen tension. Under normoxic conditions, HIF-1alpha is rapidly degraded by the ubiquitinproteasome pathway. Upon exposure to hypoxia, HIF-1alpha is stabilized and translocated to the nucleus where it heterodimerizes with ARNT to bind hypoxia-responsive elements present in target genes. HIF-1 functions as a master regulator of adaptive responses to hypoxia by activating genes that regulate oxygen supply (erythropoietin, VEGF, NOS), cellular metabolism (GLUT-1, PGK-1, LDH-A), and cell growth and apoptosis (TGF-beta3, telomerase). The interaction between the von Hippel-Lindau tumor suppressor gene product (part of an E3 ubiquitin-ligase) and HIF-1alpha has been shown to be regulated by three soluble dioxygenases. These dioxygenases are termed prolyl hydroxylase domain proteins, since they hydroxylate two conserved proline residues (Pro" and/or Pro` within mHIF-1alpha), targeting HIF-1alpha for degradation. Subsequent studies have indicated that mutagenesis of these prolines to alanine residues generates a constitutively stabilized form of HIF-1alpha, allowing the protein to be expressed independently of oxygen levels. Here we have evaluated the expression of mHIF-1alpha and several mutants in cultured cells and examined normoxia-dependent degradation of these proteins. Under certain conditions we observed degradation of these mutants by the ubiquitin-proteasome system. Moreover, PHD1 or PHD3 failed to induce normoxia-dependent degradation of the mHIF-1alpha(P402A/P563A) mutant. pVHLmediated degradation of both mHIF-1alpha and the double Pro mutant proved to be intrinsically dependent on the hydroxylation status of the overexpressed HIF-1alpha proteins. Taken together, these data suggest the existence of yet unraveled mechanisms of degradation of HIF-1alpha. Furthermore, we have investigated the effects on neoangiogenesis by mHlF-1alpha(P402A/P563A), using adenoassociated virus gene delivery to skeletal muscle. Additionally, we have compared these effects to those produced by previously known vascular growth factors, such as the VEGF. mHIF1alpha(P402A/P563A) was shown to be capable of inducing formation of functional neovasculature without increased leakiness (a well-cheracterized side-effect of VEGF). Our data further suggest that the use of the mHIF-1alpha mutant in pro-angiogenic gene therapy may be capable to circumvent most of the present problems in cardiovascular gene transfer studies. This is due to the fact that HIF1alpha is capable of inducing endogenous angiogenic cascades, that lead to the activation of multiple, necessary vasculogenenic growth factors.