Lorenz Poellinger

Mechanism of regulation of the hypoxia-inducible factor-1α by the von Hippel-Lindau tumor suppressor protein

In normoxic cells the hypoxia‐inducible factor‐1α (HIF‐1α) is rapidly degraded by the ubiquitin‐proteasome pathway, and activation of HIF‐1α to a functional form requires protein stabilization. Here we show that the product of the von Hippel‐Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF‐1α under normoxic conditions via interaction with the core of the oxygen‐dependent degradation domain of HIF‐1α. The region of VHL mediating interaction with HIF‐1α overlapped with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF‐1α and subsequent degradation. Interestingly, the VHL binding site within HIF‐1α overlapped with one of the minimal transactivation domains. Protection of HIF‐1α against degradation by VHL was a multistep mechanism, including hypoxia‐induced nuclear translocation of HIF‐1α and an intranuclear hypoxia‐dependent signal. VHL was not released from HIF‐1α during this process. Finally, stabilization of HIF‐1α protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.

REGULATION OF THE HYPOXIA-INDUCIBLE TRANSCRIPTION FACTOR 1ALPHA BY THE UBIQUITIN-PROTEASOME PATHWAY

HIF-1α (hypoxia-inducible factor 1α) is a basic-helix-loop-helix PAS (Per/Arnt/Sim) transcription factor that, under hypoxic conditions, dimerizes with a partner factor, the basic-helix-loop-helix/PAS protein Arnt, to recognize hypoxia-responsive elements of target genes. It has recently been demonstrated that HIF-1α protein but not mRNA levels are dramatically up-regulated in response to hypoxia. Here we show that inhibitors of 26 S proteasome activity produced a dramatic accumulation of endogenous as well as transfected HIF-1α protein under normoxic conditions, whereas the levels of Arnt protein were not affected. HIF-1α was polyubiquitinated in vivo under normoxic conditions, indicating rapid degradation via the ubiquitin-proteasome pathway. This degradation process appeared to target a region within the C terminus of HIF-1α. Importantly, HIF-1α ubiquitination was drastically decreased under hypoxic conditions. Up-regulation of HIF-1α protein by proteasome inhibitors did not result in transcriptional activation of reporter genes, indicating either the requirement of additional regulatory steps to induce functional activity of HIF-1α or the inability of polyubiquitinated forms of HIF-1α to mediate hypoxic signal transduction. In support of both these notions, we demonstrate that HIF-1α showed hypoxia-dependent translocation from the cytoplasm to the nucleus and that this regulatory mechanism was severely impaired in the presence of proteasome inhibitors. Taken together, these data demonstrate that the mechanism of hypoxia-dependent activation of HIF-1α is a complex multistep process and that stabilization of HIF-1α protein levels is not sufficient to generate a functional form.

Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha.

In response to decreased cellular oxygen concentrations the basic helix‐loop‐helix (bHLH)/PAS (Per, Arnt, Sim) hypoxia‐inducible transcription factor, HIF‐1α, mediates activation of networks of target genes involved in angiogenesis, erythropoiesis and glycolysis. Here we demonstrate that the mechanism of activation ofHIF‐1α is a multi‐step process which includes hypoxia‐dependent nuclear import and activation (derepression) of the transactivation domain, resulting in recruitment of the CREB‐binding protein (CBP)/p300 coactivator. Inducible nuclear accumulation was shown to be dependent on a nuclear localization signal (NLS) within the C‐terminal end of HIF‐1α which also harbors the hypoxia‐inducible transactivation domain. Nuclear import of HIF‐1α was inhibited by either deletion or a single amino acid substitution within the NLS sequence motif and, within the context of the full‐length protein, these mutations also resulted in inhibition of the transactivation activity of HIF‐1α and recruitment of CBP. However, nuclear localization per se was not sufficient for transcriptional activation, since fusion of HIF‐1α to the heterologous GAL4 DNA‐binding domain generated a protein which showed constitutive nuclear localization but required hypoxic stimuli for function as a CBP‐dependent transcription factor. Thus, hypoxia‐inducible nuclear import and transactivation by recruitment of CBP can be functionally separated from one another and play critical roles in signal transduction by HIF‐1α.

Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression.

Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300.

Hypoxia‐inducible factor 1 α (HIF1α) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C‐terminal half. Their sequences are conserved between HLF and HIF1α. One is designated NAD (N‐terminal activation domain), while the other is CAD (C‐terminal activation domain). Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two‐hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co‐transfection with CREB‐binding protein (CBP) prey, whereas CAD, but not NAD, enhanced β‐galactosidase expression in yeast by CBP co‐expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co‐transfection experiments revealed that expression of Ref‐1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia‐inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations.

Notch signaling mediates hypoxia-induced tumor cell migration and invasion

Tumor hypoxia is linked to increased metastatic potential, but the molecular mechanisms coupling hypoxia to metastasis are poorly understood. Here, we show that Notch signaling is required to convert the hypoxic stimulus into epithelial–mesenchymal transition (EMT), increased motility, and invasiveness. Inhibition of Notch signaling abrogated hypoxia-induced EMT and invasion, and, conversely, an activated form of Notch could substitute for hypoxia to induce these processes. Notch signaling deploys two distinct mechanisms that act in synergy to control the expression of Snail-1, a critical regulator of EMT. First, Notch directly up-regulated Snail-1 expression by recruitment of the Notch intracellular domain to the Snail-1 promoter, and second, Notch potentiated hypoxia-inducible factor 1α (HIF-1α) recruitment to the lysyl oxidase (LOX) promoter and elevated the hypoxia-induced up-regulation of LOX, which stabilizes the Snail-1 protein. In sum, these data demonstrate a complex integration of the hypoxia and Notch signaling pathways in regulation of EMT and open up perspectives for pharmacological intervention with hypoxiainduced EMT and cell invasiveness in tumors.

Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor.

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia and dioxin signalling, respectively. Both proteins are conditionally regulated basic helix-loop-helix (bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) region of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt. Here we demonstrate that HIF-1 alpha required Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and PAS motifs of Arnt were critical for dimerization with HIF-1 alpha. Strikingly, HIF-1 alpha exhibited very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha inhibited ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated signalling pathways were not mutually exclusive, since activation of dioxin receptor function did not impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs were expressed constitutively in a large number of human tissues and cell lines, and these steady-state expression levels were not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1 alpha function. Interestingly, we observed that HIF-1 alpha was associated with the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism, possibly by binding an as yet unknown class of ligands.

Redox-regulated recruitment of the transcriptional coactivators CREB-binding protein and SRC-1 to hypoxia-inducible factor 1alpha.

ABSTRACT Hypoxia-inducible factor 1α (HIF-1α) functions as a transcription factor that is activated by decreased cellular oxygen concentrations to induce expression of a network of genes involved in angiogenesis, erythropoiesis, and glucose homeostasis. Here we demonstrate that two members of the SRC-1/p160 family of transcriptional coactivators harboring histone acetyltransferase activity, SRC-1 and transcription intermediary factor 2 (TIF2), are able to interact with HIF-1α and enhance its transactivation potential in a hypoxia-dependent manner. HIF-1α contains within its C terminus two transactivation domains. The hypoxia-inducible activity of both these domains was enhanced by either SRC-1 or the CREB-binding protein (CBP)/p300 coactivator. Moreover, at limiting concentrations, SRC-1 produced this effect in synergy with CBP. Interestingly, this effect was strongly potentiated by the redox regulatory protein Ref-1, a dual-function protein harboring DNA repair endonuclease and cysteine reducing activities. These data indicate that all three proteins, CBP, SRC-1, and Ref-1, are important components of the hypoxia signaling pathway and have a common function in regulation of HIF-1α function in hypoxic cells. Given the absence of cysteine residues in one of the Ref-1-regulated transactivation domains of HIF-1α, it is thus possible that Ref-1 functions in hypoxic cells by targeting critical steps in the recruitment of the CBP–SRC-1 coactivator complex.

Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype.

Insufficient oxygen and nutrient supply often restrain solid tumor growth, and the hypoxia-inducible factors (HIF) 1α and HIF-2α are key transcription regulators of phenotypic adaptation to low oxygen levels. Moreover, mouse gene disruption studies have implicated HIF-2α in embryonic regulation of tyrosine hydroxylase, a hallmark gene of the sympathetic nervous system. Neuroblastoma tumors originate from immature sympathetic cells, and therefore we investigated the effect of hypoxia on the differentiation status of human neuroblastoma cells. Hypoxia stabilized HIF-1α and HIF-2α proteins and activated the expression of known hypoxia-induced genes, such as vascular endothelial growth factor and tyrosine hydroxylase. These changes in gene expression also occurred in hypoxic regions of experimental neuroblastoma xenografts grown in mice. In contrast, hypoxia decreased the expression of several neuronal/neuroendocrine marker genes but induced genes expressed in neural crest sympathetic progenitors, for instance c-kit and Notch-1. Thus, hypoxia apparently causes dedifferentiation both in vitro and in vivo. These findings suggest a novel mechanism for selection of highly malignant tumor cells with stem-cell characteristics.

Stabilization of HIF-1α is critical to improve wound healing in diabetic mice

Relative hypoxia is essential in wound healing since it normally plays a pivotal role in regulation of all the critical processes involved in tissue repair. Hypoxia-inducible factor (HIF) 1α is the critical transcription factor that regulates adaptive responses to hypoxia. HIF-1α stability and function is regulated by oxygen-dependent soluble hydroxylases targeting critical proline and asparaginyl residues. Here we show that hyperglycemia complexly affects both HIF-1α stability and activation, resulting in suppression of expression of HIF-1 target genes essential for wound healing both in vitro and in vivo. However, by blocking HIF-1α hydroxylation through chemical inhibition, it is possible to reverse this negative effect of hyperglycemia and to improve the wound healing process (i.e., granulation, vascularization, epidermal regeneration, and recruitment of endothelial precursors). Local adenovirus-mediated transfer of two stable HIF constructs demonstrated that stabilization of HIF-1α is necessary and sufficient for promoting wound healing in a diabetic environment. Our findings outline the necessity to develop specific hydroxylase inhibitors as therapeutic agents for chronic diabetes wounds. In conclusion, we demonstrate that impaired regulation of HIF-1α is essential for the development of diabetic wounds, and we provide evidence that stabilization of HIF-1α is critical to reverse the pathological process.