Assessment of HIF-1alpha function and identification of a novel degradation mechanism

Abstract

HIF-1 is a heterodimeric complex of two bHLH/PAS transcription factors, HIF-1alpha and ARNT. 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 and binds hypoxia-responsive elements present in target genes. HIF-1 functions as a master regulator of adaptive responses to hypoxia by activating genes important for physiological processes. These processes range from oxygen supply, cellular metabolism, and cell growth and apoptosis.

The interaction between the von Hippel-Lindau tumor suppressor gene product (pVHL, 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 (PHD). They hydroxylate two conserved proline residues (Pro402 and/or Pro563 within mouse HIF-1alpha), targeting HIF-1alpha for degradation. Initially, it was suggested that mutagenesis of these prolines to alanine residues that cannot be hydroxylated generates a constitutively stabilized form of HIF-1alpha, allowing the protein to be expressed independently of oxygen levels.

We have investigated the effects on neoangiogenesis by mHIF-1alpha(P402A/P563A), using adeno-associated virus (AAV) gene delivery to skeletal muscle. Additionally, we have compared these effects to those produced by previously known vascular growth factors, such as the VEGF. mHIF-1alpha(P402A/P563A) was shown to be capable of inducing formation of functional neovasculature without increased leakiness (a well-characterized side-effect of VEGF). Our data further suggest that the use of the mHIF-1alpha mutant in pro-angiogenic gene therapy may have the ability to circumvent most of the present problems in cardiovascular gene transfer studies. This is due to the fact that HIF-1alpha is capable of inducing endogenous angiogenic cascades that lead to the activation of multiple and necessary vasculogenic growth factors.

We have also evaluated the expression of mHIF-1alpha and mHIF-1alpha(P402A/P563A) in cultured cells and observed normoxia-dependent degradation of these proteins. Our studies show that degradation of these proteins is mediated by the ubiquitin-proteasome system, through a pVHL-dependent mechanism. Furthermore, PHDs failed to induce normoxiadependent degradation of the mHIF-1alpha(P402A/P563A) mutant. pVHL-mediated degradation of both mHIF-1alpha and the double proline mutant proved to be intrinsically independent of the hydroxylation status of the HIF-1alpha proteins. Taken together, these data suggest the existence of yet unraveled mechanisms of degradation of HIF-1alpha.

Subsequently, we evaluated the contribution of the small ubiquitin-like modifier (SUMO) and the SUMO-specific protease 1 (SENP1) to HIF-1alpha degradation. Our results show that RNAi directed to SENP1 impairs protein accumulation of both mHIF-1alpha and mHIF- 1alpha(P402A/P563A) at hypoxia. At normoxia, silencing of the SUMO conjugating enzyme (Ubc9) resulted in stabilization of endogenous HIF-1alpha in cells where PHD2 was down regulated. Additionally, silencing of Ubc9 is sufficient to promote stability of the HIF-1alpha double proline mutant at normoxia.

In conclusion, our studies indicate that mHIF-1alpha(P402A/P563A) is a bona fide proangiogenic factor with potential therapeutic effects. Still, this protein is regulated in an oxygen-dependent manner by proteasome-mediated degradation. The degradation of mHIF-1alpha(P402A/P563A) proved to be pVHL-dependent yet PHD-independent. Further investigations indicated a role for SUMO modification in the HIF-1alpha degradation pathway. The presence of SUMO moieties on mHIF-1alpha(P402A/P563A) are sufficient for the recruitment of pVHL and thus promote degradation of the protein at normoxia, suggesting SUMOylation as the novel mechanism for degradation of HIF-1alpha.