Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors

Hypoxia and induction of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) is a hallmark of many tumors. Under normal oxygen tension HIF-alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re-introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF-1 and HIF-2 and found that siRNA targeted degradation of HIF-1alpha and HIF-2alpha results in decreased hypoxia-induced PHD3 expression. Ectopic overexpression of HIF-2alpha in two different cell lines provided a much better induction of PHD3 gene than HIF-1alpha. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF-2alpha. However, induction of PHD2 by hypoxia has HIF-1-independent and -dependent components. Short-term hypoxia (4 h) results in induction of PHD2 independent of HIF-1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA-mediated degradation of HIF-1alpha subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation.     

Effects of point mutations in pVHL on the binding of HIF-1α

The von Hippel-Lindau tumor suppressor protein (pVHL) has an essential role in the regulation of the hypoxia response pathway in animal cells. Under normoxic conditions, the hypoxia-inducible factor (HIF) undergoes trans-4-prolyl hydroxylation and is subsequently recognised by the β-domain of pVHL, leading to the ubiquitination and degradation of HIF. Mutations of pVHL alter the binding of HIF. A subset of relevant clinically observed mutations to pVHL are thought to cause weaker binding of HIF-1α and are associated with cancer and cardiovascular diseases. Here, we present computational studies analyzing the interaction of HIF with mutant forms of pVHL, describing at atomic detail the local structural reorganization caused by substitution of certain residues of pVHL. The results reveal that the canonical configuration in the wild-type system is vital for the efficient functioning of the complex and that mutation of any of the residues implicated in the h-bond network in the binding site disrupts HIF binding. Although the experimentally observed ordering of binding energies for mutants of Tyr98 is reproduced, our examination of a broader range of mutations does not support the hypothesis of a correlation between the degree of disruption of the pVHL/HIF-1α interaction caused by a mutation and the phenotype with which the mutation is associated. We suggest that disruption of the binding interaction is one of many factors behind the manifestation of VHL disease.     

Loss of VHL confers hypoxia-inducible factor (HIF)-dependent resistance to vesicular stomatitis virus: role of HIF in antiviral response

Hypoxia-inducible factor (HIF) is a central regulator of cellular responses to hypoxia, and under normal oxygen tension the catalytic alpha subunit of HIF is targeted for ubiquitin-mediated destruction via the VHL-containing E3 ubiquitin ligase complex. Principally known for its association with oncogenesis, HIF has been documented to have a role in the antibacterial response. Interferons, cytokines with antiviral functions, have been shown to upregulate the expression of HIF-1alpha, but the significance of HIF in the antiviral response has not been established. Here, using renal carcinoma cells devoid of VHL or reconstituted with functional wild-type VHL or VHL mutants with various abilities to negatively regulate HIF as an ideal model system of HIF activity, we show that elevated HIF activity confers dramatically enhanced resistance to vesicular stomatitis virus (VSV)-mediated cytotoxicity. Inhibition of HIF activity using a small-molecule inhibitor, chetomin, enhanced cellular sensitivity to VSV, while treatment with hypoxia mimetic CoCl2 promoted resistance. Similarly, targeting HIF-2alpha by RNA interference also enhanced susceptibility to VSV. Expression profiling studies show that upon VSV infection, the induction of genes with known antiviral activity, such as that encoding beta interferon (IFN-beta), is significantly enhanced by HIF. These results reveal a previously unrecognized role of HIF in the antiviral response by promoting the expression of the IFN-beta gene and other genes with antiviral activity upon viral infection.     

The VHL tumor suppressor in development and disease: functional studies in mice by conditional gene targeting

The von Hippel-Lindau tumor suppressor pVHL plays a critical role in the pathogenesis of familial and sporadic clear cell carcinomas of the kidney and hemangioblastomas of the retina and central nervous system. pVHL targets the oxygen sensitive alpha subunit of hypoxia-inducible factor (HIF) for proteasomal degradation, thus providing a direct link between tumorigenesis and molecular pathways critical for cellular adaptation to hypoxia. Cell type specific gene targeting of VHL in mice has demonstrated that proper pVHL mediated HIF proteolysis is fundamentally important for survival, proliferation and differentiation of many cell types and furthermore, that inactivation of pVHL may, unexpectedly, inhibit tumor growth under certain conditions. Mouse knock out studies have provided novel mechanistic insights into VHL associated tumorigenesis and established a central role for HIF in the development of the VHL phenotype.     

pVHL modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is the cause of the familial VHL disease and most sporadic renal clear-cell carcinomas (RCC). pVHL has been shown to play a role in the destruction of hypoxia-inducible factor alpha (HIF-alpha) subunits via ubiquitin-mediated proteolysis and in the regulation of fibronectin matrix assembly. Although most disease-causing pVHL mutations hinder the regulation of the HIF pathway, every disease-causing pVHL mutant tested to date has failed to promote the assembly of the fibronectin matrix, underscoring its potential importance in VHL disease. Here, we report that a ubiquitin-like molecule called NEDD8 covalently modifies pVHL. A nonneddylateable pVHL mutant, while retaining its ability to ubiquitylate HIF, failed to bind to and promote the assembly of the fibronectin matrix. Expression of the neddylation-defective pVHL in RCC cells, while restoring the regulation of HIF, failed to promote the differentiated morphology in a three-dimensional growth assay and was insufficient to suppress the formation of tumors in SCID mice. These results suggest that NEDD8 modification of pVHL plays an important role in fibronectin matrix assembly and that in the absence of such regulation, an intact HIF pathway is insufficient to prevent VHL-associated tumorigenesis.     

HIF-2α downregulation in the absence of functional VHL is not sufficient for renal cell differentiation

Background  

Mutational inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene has been linked to hereditary as well as sporadic clear cell renal carcinomas. The product of the VHL gene, pVHL, acts to target hypoxia-inducible factor alpha (HIF-α) subunits for ubiquitination and subsequent degradation. Using an RNA interference approach to lower levels of HIF-2α in two different renal cell lines that lack functional pVHL, we have tested the contribution of HIF-2α toward cellular pVHL activities.     

VHL Type 2B Mutations Retain VBC Complex Form and Function

Background

von Hippel-Lindau disease is characterized by a spectrum of hypervascular tumors, including renal cell carcinoma, hemangioblastoma, and pheochromocytoma, which occur with VHL genotype-specific differences in penetrance. VHL loss causes a failure to regulate the hypoxia inducible factors (HIF-1α and HIF-2α), resulting in accumulation of both factors to high levels. Although HIF dysregulation is critical to VHL disease-associated renal tumorigenesis, increasing evidence points toward gradations of HIF dysregulation contributing to the degree of predisposition to renal cell carcinoma and other manifestations of the disease.

Methodology/Principal Findings

This investigation examined the ability of disease-specific VHL missense mutations to support the assembly of the VBC complex and to promote the ubiquitylation of HIF. Our interaction analysis supported previous observations that VHL Type 2B mutations disrupt the interaction between pVHL and Elongin C but maintain partial regulation of HIF. We additionally demonstrated that Type 2B mutant pVHL forms a remnant VBC complex containing the active members ROC1 and Cullin-2 which retains the ability to ubiquitylate HIF-1α.

Conclusions

Our results suggest that subtypes of VHL mutations support an intermediate level of HIF regulation via a remnant VBC complex. These findings provide a mechanism for the graded HIF dysregulation and genetic predisposition for cancer development in VHL disease.     

Role of iron (II)-2-oxoglutarate-dependent dioxygenases in the generation of hypoxia-induced phosphatidic acid through HIF-1/2 and von Hippel-Lindau-independent mechanisms

Hypoxia-inducible factors (HIF-1/HIF-2) govern the expression of critical genes for cellular adaptation to low oxygen tensions. We have previously reported that the intracellular level of phosphatidic acid (PA) rises in response to hypoxia (1% O(2)). In this report, we have explored whether components of the canonical HIF/von Hippel-Lindau (VHL) pathway are involved in the induction of PA. We found that hypoxia induces PA in a cell line constitutively expressing a stable version of HIF-1alpha. PA induction was also found in HIF-1alpha- and 2alpha-negative CHO Ka13 cells, as well as in HIF-beta-negative HepaC4 cells. These data indicate that HIF activity is neither sufficient nor necessary for oxygen-dependent PA accumulation. PA generation was also detected in cells deficient for the tumor suppressor VHL, indicating that the presence of VHL was not required for the induction of PA. Here we show that PA accumulation also occurs at moderate hypoxia (5% O(2)), although to a lesser extent to that seen at 1% O(2), revealing that PA is induced at the same hypoxia range required to activate HIF-1. Prolyl hydroxylases (PHD) and asparaginyl hydroxylase (FIH) belong to the iron (II) and 2-oxoglutarate-dependent dioxygenase family and have been proposed as oxygen sensors involved in the regulation of HIFs. Chemical inhibition of these activities by treatment with iron chelators or 2-oxoglutarate analogs also results in a marked PA accumulation similar to that observed in hypoxia. Together these data show that PA accumulation in response to hypoxia is both HIF-1/2- and VHL-independent and indicate a role of iron (II)-2-oxoglutarate-dependent dioxygenases in the oxygen-sensing mechanisms involved in hypoxia-driven phospholipid regulation.     

The Positive Regulation of p53 by the Tumor Suppressor VHL

The ubiquitin-mediated degradation of hypoxia-inducible factor-α (HIF-α) by a von Hippel-Lindau tumor suppressor protein (pVHL) is mechanistically responsible for controlling gene expression due to oxygen availability. Germline mutations in the VHL gene cause dysregulation of HIF and induce an autosomal dominant cancer syndrome referred to as VHL disease. However, it is unclear whether HIF accumulation caused by VHL mutations is sufficient for tumorigenesis. Recently, we found that pVHL directly associates and positively regulates the tumor suppressor p53 by inhibiting Mdm2-mediated ubiquitination, and by subsequently recruiting p53-modifying enzymes. Moreover, VHL-deleted RCC cells showed attenuated apoptosis or abnormal cell-cycle arrest upon DNA damage, but became normal when pVHL was restored. Thus, pVHL appears to play a pivotal role in tumor suppression by participating actively as a component of p53 transactivation complex during DNA damage response.