Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia

Nitric oxide (NO) is known to have various biologic and pathophysiologic effects on organisms. The molecular mechanisms by which NO exerts harmful effects are unknown, although various O2 radicals and ions that result from reactivity of NO are presumed to be involved. Here we report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-l-glutathione and 3-morpholinosydnonimine, giving IC50 values of 7.8, 211, and 490 μM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC50 = 6.6 μM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC50 = 2.5 μM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1α or HIF-1α-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1α to a DNA-binding form.

The transcription factor EPAS-1/hypoxia-inducible factor 2α plays an important role in vascular remodeling

We have studied the role of the basic helix–loop–helix–PAS transcription factor EPAS-1/hypoxia-inducible factor 2α in vascular development by gene targeting. In ICR/129 Sv outbred background, more than half of the mutants displayed varying degrees of vascular disorganization, typically in the yolk sac, and died in utero between embryonic day (E)9.5 and E13.5. In mutant embryos directly derived from EPAS-1−/− embryonic stem cells (hence in 129 Sv background), all embryos developed severe vascular defects both in the yolk sac and embryo proper and died between E9.5 and E12.5. Normal blood vessels were formed by vasculogenesis but they either fused improperly or failed to assemble into larger vessels later during development. Our results suggest that EPAS-1 plays an important role at postvasculogenesis stages and is required for the remodeling of the primary vascular network into a mature hierarchy pattern.

Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.

Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2, consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia.

Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein.

Inactivation of the von Hippel-Lindau protein (pVHL) has been implicated in the pathogenesis of renal carcinomas and central nervous system hemangioblastomas. These are highly vascular tumors which overproduce angiogenic peptides such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Renal carcinoma cells lacking wild-type pVHL were found to produce mRNAs encoding VEGF/VPF, the glucose transporter GLUT1, and the platelet-derived growth factor B chain under both normoxic and hypoxic conditions. Reintroduction of wild-type, but not mutant, pVHL into these cells specifically inhibited the production of these mRNAs under normoxic conditions, thus restoring their previously described hypoxia-inducible profile. Thus, pVHL appears to play a critical role in the transduction of signals generated by changes in ambient oxygen tension.

Characterization of the α1B-adrenergic receptor gene promoter region and hypoxia regulatory elements in vascular smooth muscle

We previously demonstrated that α1B-adrenergic receptor (AR) gene transcription, mRNA, and functionally coupled receptors increase during 3% O2 exposure in aorta, but not in vena cava smooth muscle cells (SMC). We report here that α1BAR mRNA also increases during hypoxia in liver and lung, but not heart and kidney. A single 2.7-kb α1BAR mRNA was detected in aorta and vena cava during normoxia and hypoxia. The α1BAR 5′ flanking region was sequenced to −2,460 (relative to ATG +1). Transient transfection experiments identify the minimal promoter region between −270 and −143 and sequence between −270 and −248 that are required for transcription of the α1BAR gene in aorta and vena cava SMC during normoxia and hypoxia. An ATTAAA motif within this sequence specifically binds aorta, vena cava, and DDT1MF-2 nuclear proteins, and transcription primarily initiates downstream of this motif at approximately −160 in aorta SMC. Sequence between −837 and −273 conferred strong hypoxic induction of transcription in aorta, but not in vena cava SMC, whereas the cis-element for the transcription factor, hypoxia-inducible factor 1, conferred hypoxia-induced transcription in both aorta and vena cava SMC. These data identify sequence required for transcription of the α1BAR gene in vascular SMC and suggest the atypical TATA-box, ATTAAA, may mediate this transcription. Hypoxia-sensitive regions of the α1BAR gene also were identified that may confer the differential hypoxic increase in α1BAR gene transcription in aorta, but not in vena cava SMC.

Human interleukin-10-related T cell-derived inducible factor: Molecular cloning and functional characterization as an hepatocyte-stimulating factor

IL-10-related T cell-derived inducible factor (IL-TIF or IL-21) is a new cytokine structurally related to IL-10 and originally identified in the mouse as a gene induced by IL-9 in T cells and mast cells. Here, we report the cloning of the human IL-TIF cDNA, which shares 79% amino acid identity with mouse IL-TIF and 25% identity with human IL-10. Recombinant human IL-TIF was found to activate signal transducer and activator of transcription factors-1 and -3 in several hepatoma cell lines. IL-TIF stimulation of HepG2 human hepatoma cells up-regulated the production of acute phase reactants such as serum amyloid A, α1-antichymotrypsin, and haptoglobin. Although IL-10 and IL-TIF have distinct activities, antibodies directed against the β chain of the IL-10 receptor blocked the induction of acute phase reactants by IL-TIF, indicating that this chain is a common component of the IL-10 and IL-TIF receptors. Similar acute phase reactant induction was observed in mouse liver upon IL-TIF injection, and IL-TIF expression was found to be rapidly increased after lipopolysaccharide (LPS) injection, suggesting that this cytokine contributes to the inflammatory response in vivo.

The Caenorhabditis elegans hif-1 gene encodes a bHLH-PAS protein that is required for adaptation to hypoxia

Hypoxia-inducible factor, a heterodimeric transcription complex, regulates cellular and systemic responses to low oxygen levels (hypoxia) during normal mammalian development or tumor progression. Here, we present evidence that a similar complex mediates response to hypoxia in Caenorhabditis elegans. This complex consists of HIF-1 and AHA-1, which are encoded by C. elegans homologs of the hypoxia-inducible factor (HIF) α and β subunits, respectively. hif-1 mutants exhibit no severe defects under standard laboratory conditions, but they are unable to adapt to hypoxia. Although wild-type animals can survive and reproduce in 1% oxygen, the majority of hif-1-defective animals die in these conditions. We show that the expression of an HIF-1:green fluorescent protein fusion protein is induced by hypoxia and is subsequently reduced upon reoxygenation. Both hif-1 and aha-1 are expressed in most cell types, and the gene products can be coimmunoprecipitated. We conclude that the mechanisms of hypoxia signaling are likely conserved among metazoans. Additionally, we find that nuclear localization of AHA-1 is disrupted in an hif-1 mutant. This finding suggests that heterodimerization may be a prerequisite for efficient nuclear translocation of AHA-1.

Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays

The pleiotropic activities of interferons (IFNs) are mediated primarily through the transcriptional regulation of many downstream effector genes. The mRNA profiles from IFN-α, -β, or -γ treatments of the human fibrosarcoma cell line, HT1080, were determined by using oligonucleotide arrays with probe sets corresponding to more than 6,800 human genes. Among these were transcripts for known IFN-stimulated genes (ISGs), the expression of which were consistent with previous studies in which the particular ISG was characterized as responsive to either Type I (α, β) or Type II (γ) IFNs, or both. Importantly, many novel IFN-stimulated genes were identified that were diverse in their known biological functions. For instance, several novel ISGs were identified that are implicated in apoptosis (including RAP46/Bag-1, phospholipid scramblase, and hypoxia inducible factor-1α). Furthermore, several IFN-repressed genes also were identified. These results demonstrate the usefulness of oligonucleotide arrays in monitoring mammalian gene expression on a broad and unprecedented scale. In particular, these findings provide insights into the basic mechanisms of IFN actions and ultimately may contribute to better therapeutic uses for IFNs.

Targeted mutation of the murine arylhydrocarbon receptor nuclear translocator 2 (Arnt2) gene reveals partial redundancy with Arnt

The ubiquitously expressed basic helix–loop–helix (bHLH)-PAS protein ARNT (arylhydrocarbon receptor nuclear transporter) forms transcriptionally active heterodimers with a variety of other bHLH-PAS proteins, including HIF-1α (hypoxia-inducible factor-1α) and AHR (arylhydrocarbon receptor). These complexes regulate gene expression in response to hypoxia and xenobiotics, respectively, and mutation of the murine Arnt locus results in embryonic death by day 10.5 associated with placental, vascular, and hematopoietic defects. The closely related protein ARNT2 is highly expressed in the central nervous system and kidney and also forms complexes with HIF-1α and AHR. To assess unique roles for ARNT2 in development, and reveal potential functional overlap with ARNT, we generated a targeted null mutation of the murine Arnt2 locus. Arnt2−/− embryos die perinatally and exhibit impaired hypothalamic development, phenotypes previously observed for a targeted mutation in the murine bHLH-PAS gene Sim1 (Single-minded 1), and consistent with the recent proposal that ARNT2 and SIM1 form an essential heterodimer in vivo [Michaud, J. L., DeRossi, C., May, N. R., Holdener, B. C. & Fan, C. (2000) Mech. Dev. 90, 253–261]. In addition, cultured Arnt2−/− neurons display decreased hypoxic induction of HIF-1 target genes, demonstrating formally that ARNT2/HIF-1α complexes regulate oxygen-responsive genes. Finally, a strong genetic interaction between Arnt and Arnt2 mutations was observed, indicating that either gene can fulfill essential functions in a dose-dependent manner before embryonic day 8.5. These results demonstrate that Arnt and Arnt2 have both unique and overlapping essential functions in embryonic development.

Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability

Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3′ untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3′ untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3′ untranslated region and distinct mRNA-binding proteins in human tumor cells.

Vascular endothelial growth factor: Direct neuroprotective effect in in vitro ischemia

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic peptide with recently identified neurotrophic effects. Because some neurotrophic factors can protect neurons from hypoxic or ischemic injury, we investigated the possibility that VEGF has similar neuroprotective properties. In HN33, an immortalized hippocampal neuronal cell line, VEGF reduced cell death associated with an in vitro model of cerebral ischemia: at a maximally effective concentration of 50 ng/ml, VEGF approximately doubled the number of cells surviving after 24 h of hypoxia and glucose deprivation. To investigate the mechanism of neuroprotection by VEGF, the expression of known target receptors for VEGF was measured by Western blotting, which showed that HN33 cells expressed VEGFR-2 receptors and neuropilin-1, but not VEGFR-1 receptors. The neuropilin-1 ligand placenta growth factor-2 failed to reproduce the protective effect of VEGF, pointing to VEGFR-2 as the site of VEGF's neuroprotective action. Two phosphatidylinositol 3′-kinase inhibitors, wortmannin and LY294002, reversed the neuroprotective effect of VEGF, implicating the phosphatidylinositol 3′-kinase/Akt signal transduction system in VEGF-mediated neuroprotection. VEGF also protected primary cultures of rat cerebral cortical neurons from hypoxia and glucose deprivation. We conclude that in addition to its known role as an angiogenic factor, VEGF may exert a direct neuroprotective effect in hypoxic-ischemic injury.

Role of transforming growth factor-α in von Hippel– Lindau (VHL)−/− clear cell renal carcinoma cell proliferation: A possible mechanism coupling VHL tumor suppressor inactivation and tumorigenesis

Mutations of the VHL tumor suppressor gene occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL−/− RCC). Loss of VHL protein function is associated with constitutive expression of mRNAs encoding hypoxia-inducible proteins, such as vascular endothelial growth factor. Overproduction of angiogenic factors might explain why VHL−/− RCC tumors are so highly vascularized, but whether this overproduction is sufficient for oncogenesis still remains unknown. In this report, we examined the activity of transforming growth factor-α (TGF-α), another VHL-regulated growth factor. We show that TGF-α mRNA and protein are hypoxia-inducible in VHL−/− RCC cells expressing reintroduced VHL. In addition to its overexpression by VHL−/− RCC cells, TGF-α can also act as a specific growth-stimulatory factor for VHL−/− RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of RCC. This role is in contrast to those of other growth factors overexpressed by VHL−/− RCC cells, such as vascular endothelial growth factor and TGF-β1, which do not stimulate RCC cell proliferation. A TGF-α-specific antisense oligodeoxynucleotide blocked TGF-α production in VHL−/− RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture. Growth of VHL−/− RCC cells was also significantly reduced by a drug that specifically inhibits the epidermal growth factor receptor, the receptor through which TGF-α stimulates proliferation. These results suggest that the generation of a TGF-α autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells may provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.

Activation of HIF1α ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex

Mutations in the VHL tumor suppressor gene result in constitutive expression of many hypoxia-inducible genes, at least in part because of increases in the cellular level of hypoxia-inducible transcription factor HIF1α, which in normal cells is rapidly ubiquitinated and degraded by the proteasome under normoxic conditions. The recent observation that the VHL protein is a subunit of an Skp1-Cul1/Cdc53-F-box (SCF)-like E3 ubiquitin ligase raised the possibility that VHL may be directly responsible for regulating cellular levels of HIF1α by targeting it for ubiquitination and proteolysis. In this report, we test this hypothesis directly. We report development of methods for production of the purified recombinant VHL complex and present direct biochemical evidence that it can function with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme to activate HIF1α ubiquitination in vitro. Our findings provide new insight into the function of the VHL tumor suppressor protein, and they provide a foundation for future investigations of the mechanisms underlying VHL regulation of oxygen-dependent gene expression.

Vascular tumors in livers with targeted inactivation of the von Hippel–Lindau tumor suppressor

von Hippel–Lindau (VHL) disease is a pleomorphic familial tumor syndrome that is characterized by the development of highly vascularized tumors. Homozygous disruption of the VHL gene in mice results in embryonic lethality. To investigate VHL function in the adult we have generated a conditional VHL null allele (2-lox allele) and null allele (1-lox allele) by Cre-mediated recombination in embryonic stem cells. We show here that mice heterozygous for the 1-lox allele develop cavernous hemangiomas of the liver, a rare manifestation of the human disease. Histologically these tumors were associated with hepatocellular steatosis and focal proliferations of small vessels. To study the cellular origin of these lesions we inactivated VHL tissue-specifically in hepatocytes. Deletion of VHL in the liver resulted in severe steatosis, many blood-filled vascular cavities, and foci of increased vascularization within the hepatic parenchyma. These histopathological changes were similar to those seen in livers from mice heterozygous for the 1-lox allele. Hypoxia-inducible mRNAs encoding vascular endothelial growth factor, glucose transporter 1, and erythropoietin were up-regulated. We thus provide evidence that targeted inactivation of mouse VHL can model clinical features of the human disease and underline the importance of the VHL gene product in the regulation of hypoxia-responsive genes in vivo.

The human granulocyte-macrophage colony- stimulating factor gene is autonomously regulated in vivo by an inducible tissue-specific enhancer

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is part of a cytokine gene cluster and is directly linked to a conserved upstream inducible enhancer. Here we examined the in vitro and in vivo functions of the human GM-CSF enhancer and found that it was required for the correctly regulated expression of the GM-CSF gene. An inducible DNase I-hypersensitive site appeared within the enhancer in cell types such as T cells, myeloid cells, and endothelial cells that express GM-CSF, but not in nonexpressing cells. In a panel of transfected cells the human GM-CSF enhancer was activated in a tissue-specific manner in parallel with the endogenous gene. The in vivo function of the enhancer was examined in a transgenic mouse model that also addressed the issue of whether the GM-CSF locus was correctly regulated in isolation from other segments of the cytokine gene cluster. After correction for copy number the mean level of human GM-CSF expression in splenocytes from 11 lines of transgenic mice containing a 10.5-kb human GM-CSF transgene was indistinguishable from mouse GM-CSF expression (99% ± 56% SD). In contrast, a 9.8-kb transgene lacking just the enhancer had a significantly reduced (P = 0.004) and more variable level of activity (29% ± 89% SD). From these studies we conclude that the GM-CSF enhancer is required for the correct copy number-dependent expression of the human GM-CSF gene and that the GM-CSF gene is regulated independently from DNA elements associated with the closely linked IL-3 gene or other members of the cytokine gene cluster.