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

<p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><h3>List of scientific papers</h3><p>I. Pajusola K, Künnapuu J, Vuorikoski S, Soronen J, André H, Pereira T, Korpisalo P, Ylä-Herttuala S, Poellinger L, Alitalo K (2005). Stabilized HIF-1alpha is superior to VEGF for angiogenesis in skeletal muscle via adeno-associated virus gene transfer. FASEB J. 19(10): 1365-7. Epub 2005 Jun 15 <br><a href="https://pubmed.ncbi.nlm.nih.gov/15958522">https://pubmed.ncbi.nlm.nih.gov/15958522</a><br><br></p><p>II. André H, Pereira TS (2008). Identification of an alternative mechanism of degradation of the hypoxia-inducible factor-1alpha. J Biol Chem. 283(43): 29375-84. Epub 2008 Aug 11 <br><a href="https://pubmed.ncbi.nlm.nih.gov/18694926">https://pubmed.ncbi.nlm.nih.gov/18694926</a><br><br></p><p>III. André H, Pereira TS (2009). Modification of HIF-1alpha by the Small Ubiquitin-like Modifier (SUMO) targets the protein for degradation at normoxia. [Manuscript]</p>