Transcription Of Hypoxia-inducible Genes Research Articles

USP51 facilitates colorectal cancer stemness and chemoresistance by forming a positive feed-forward loop with HIF1A.

In the current study, we have shown that USP51 promotes colorectal cancer stemness and chemoresistance, and high expression of USP51 predicts survival disadvantage in colorectal cancer patients. Mechanically, USP51 directly binds to Elongin C (ELOC) and forms a larger functional complex with VHL E3 ligase (USP51/VHL/CUL2/ELOB/ELOC/RBX1) to regulate the ubiquitin-dependent proteasomal degradation of HIF1A. USP51 efficiently deubiquitinates HIF1A and activates hypoxia-induced gene transcription. Conversely, the activation of HIF1A under hypoxia transcriptionally upregulates the expression of USP51. Thus, USP51 and HIF1A form a positive feedback loop. Further, we found that the SUMOylation of ELOC at K32 inhibits its binding to USP51. SUMO-specific protease 1 (SENP1) mediates the deSUMOylation of ELOC, promoting the binding of USP51 to ELOC and facilitating the deubiquitination and stabilization of HIF1A by USP51. Importantly, USP51 plays a crucial role in promoting the HIF1A and SENP1-dependent proliferation, migration, stemness, and chemoresistance under hypoxia in colorectal cancer. Together, our data revealed that USP51 is an oncogene stabilizing the pro-survival protein HIF1A, offering a potential therapeutic target for colorectal cancer.

Pyruvate dehydrogenase phosphatase 1 (PDP1) stimulates HIF activity by supporting histone acetylation under hypoxia.

Cancer cells, when exposed to the hypoxic tumour microenvironment, respond by activating hypoxia-inducible factors (HIFs). HIF-1 mediates extensive metabolic re-programming, and expression of HIF-1α, its oxygen-regulated subunit, is associated with poor prognosis in cancer. Here we analyse the role of pyruvate dehydrogenase phosphatase 1 (PDP1) in the regulation of HIF-1 activity. PDP1 is a key hormone-regulated metabolic enzyme that dephosphorylates and activates pyruvate dehydrogenase (PDH), thereby stimulating the conversion of pyruvate into acetyl-CoA. Silencing of PDP1 down-regulated HIF transcriptional activity and the expression of HIF-dependent genes, including that of PDK1, the kinase that phosphorylates and inactivates PDH, opposing the effects of PDP1. Inversely, PDP1 stimulation enhanced HIF activity under hypoxia. Alteration of PDP1 levels or activity did not have an effect on HIF-1α protein levels, nuclear accumulation or interaction with its partners ARNT and NPM1. However, depletion of PDP-1 decreased histone H3 acetylation of HIF-1 target gene promoters and inhibited binding of HIF-1 to the respective hypoxia-response elements (HREs) under hypoxia. Furthermore, the decrease of HIF transcriptional activity upon PDP1 depletion could be reversed by treating the cells with acetate, as an exogenous source of acetyl-CoA, or the histone deacetylase (HDAC) inhibitor trichostatin A. These data suggest that the PDP1/PDH/HIF-1/PDK1 axis is part of a homeostatic loop which, under hypoxia, preserves cellular acetyl-CoA production to a level sufficient to sustain chromatin acetylation and transcription of hypoxia-inducible genes.

PSUN375 What Blindness and Paraganglioma Have in Common? A Rare Case of Paraganglioma Syndrome

Abstract Background Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors with an annual incidence in the U.S. of 500-1600 cases per year. Approximately 30-35% of PPGLs harbor germline mutations, while 35-40% are due to somatic mutations. Recent studies have identified the association between somatic mutations of the hypoxia-inducible factor 2 alpha (HIF2A) gene and a new syndrome of PPGLs associated with congenital polycythemia. Patients present with recurrent PPGLs, congenital malformation of intracranial veins, malformation of macula and retina, and type I Chiari malformations. Case A 44-year-old man with history of in-utero stroke, congenital blindness, polycythemia and spinal stenosis had an incidental para-aortic lesion found during workup of back pain. He experienced episodic palpitations associated with diaphoresis, headache and uncontrolled hypertension for the past eight year. There was no family history of neuroendocrine tumors. Dedicated abdominal computer tomography with contrast showed a right adrenal enhancing lesion measuring 1.4×1.2 cm and a conglomerate of heterogenous enhancing periaortic lesions measuring up to 5 cm in the mid-abdomen. Biochemical work up revealed plasma free normetanephrine of 27.5 nmol/L (0.00-0.89) and plasma free metanephrines of 0.49 nmol/L (0.00-0.49). Gallium 68 DOTATATE scan showed multiple avid retroperitoneal lesions measuring up to 4.4 cm and an avid right adrenal nodule. He underwent right adrenalectomy and para-aortic lesion resection with perio-operative alpha blockade. Pathology confirmed synchronous pheochromocytoma (1.5 cm, Ki-67< 1%, with capsular and periadrenal fat invasion) and paraganglioma (9.3 cm, Ki-67 < 1%, with lymphovascular invasion and one out of nine lymph nodes positive for metastatic disease). Post-operatively, his metanephrines normalized and a six-months positron emission tomography scan showed no evidence of disease. Genetic testing has been delayed. Discussion This case illustrates a rare syndrome of PPGLs, congenital blindness, and polycythemia, likely due to somatic mutations of HIF2a. HIF is an alpha/beta heterodimer. HIF2a becomes a transcription factor after dimerization with HIF2b. Any mutations that stabilize HIF protein leads to transcription of hypoxia inducible genes resulting in angiogenesis, polycythemia, and tumorigenesis. In our patient, we suspect the stroke in-utero may have been related to abnormal intracranial angiogenesis and/or polycythemia. Recognition of this syndrome and lifelong surveillance is imperative due to risk of recurrence of PPGL. Targeted therapies such as a HIF2a inhibitor have been proposed and trials are ongoing. Our patient remains disease free on radiographic and biochemical surveillance. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Abstract IA-017: Chromatin and gene transcription in hypoxia

Abstract Oxygen is essential for the life of most multicellular organism and changes in oxygen availability have implications in physiological and pathophysiological conditions. In response to hypoxia (low oxygen availability) transcriptional changes are induced. The transcriptional response to hypoxia is mediated primarily by the Hypoxia Inducible Factor (HIF) family of transcription factors. HIFs are activated in hypoxia via inhibition of prolyl hydroxylases (PHDs), a group of molecular dioxygenases, which require oxygen for their activity. Whilst the role of HIFs in the transcriptional response to hypoxia is well characterized, little is known with regards to chromatin structure in low oxygen stress and how this coordinates the transcriptional response. It was recently shown that chromatin is sensitive to oxygen levels, with hypoxia altering histone methylation at hypoxia responsive genes via inhibition of specific JmjC histone demethylases. To further investigate chromatin structure and its role in hypoxia induced gene transcription, we have performed ATAC-seq and RNA-seq in human cancer cell lines in acute and prolonged hypoxia. We find that acute hypoxia leads to increased promoter accessibility at a subset of hypoxia inducible genes, and this mostly precedes changes in mRNA expression, with further changes in chromatin structure after prolonged hypoxia. In addition, ChIP-seq data on histone methylation marks (H3K4me and H3K36me3), revealed similar earlier patterns of genomic distribution. Furthermore, we dissected the impact of HIF in the observed changes using a chemical probe and specific HIF1beta depletion analysis of ATAC-seq. This work gives insights into the role of chromatin dynamics in regulating the transcriptional response to hypoxia and elucidating the mechanisms behind these changes may lead to targeted therapies for hypoxia driven diseases. Citation Format: Michael Batie, Julianty Frost, Sonia Rocha. Chromatin and gene transcription in hypoxia [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-017.

Dual targeting of the androgen receptor and hypoxia-inducible factor 1α pathways synergistically inhibits castration-resistant prostate cancer cells.

Enzalutamide is a potent second-generation androgen receptor (AR) antagonist with activity in metastatic castrate-resistant prostate cancer (CRPC). Although enzalutamide is initially effective, disease progression inevitably ensues with the emergence of resistance. Intratumoral hypoxia is also associated with CRPC progression and treatment resistance. Given that both AR and hypoxia inducible factor-1 α (HIF-1α) are key regulators of these processes, dual targeting of both signaling axes represents an attractive therapeutic approach. Crosstalk of the AR and HIF-1α signaling pathways were examined in prostate cancer cell lines (LNCaP, 22Rv1) with assays measuring the effect of androgen and hypoxia on AR-dependent and hypoxia-inducible gene transcription, protein expression, cell proliferation, and apoptosis. HIF-1α inhibition was achieved by siRNA silencing HIF-1α or via chetomin, a disruptor of HIF-1α-p300 interactions. In prostate cancer cells, the gene expression of AR targets (KLK3, FKBP5, TMPRSS2) was repressed by HIF-signaling; conversely, specific HIF-1α target expression was induced by dihydrotestosterone-mediated AR signaling. Treatment of CRPC cells with enzalutamide or HIF-1α inhibition attenuated AR-regulated and HIF-1α-mediated gene transcription. The combination of enzalutamide and HIF-1α inhibition was more effective than either treatment alone. Similarly, the combination also reduced vascular endothelial growth factor protein levels. HIF-1α siRNA synergistically enhanced the inhibitory effect of enzalutamide on cell growth in LNCaP and enzalutamide-resistant 22Rv1 cells via increased enzalutamide-induced apoptosis. In conclusion, the combination of enzalutamide with HIF-1α inhibition resulted in synergistic inhibition of AR-dependent and gene-specific HIF-dependent expression and prostate cancer cell growth.

Dual targeting of the androgen receptor and hypoxia-inducible factor-1α pathways to synergistically inhibit castrate-resistant prostate cancer.

270 Background: Enzalutamide is a potent second-generation androgen receptor (AR) antagonist with activity in metastatic castrate-resistant prostate cancer (CRPC). While enzalutamide is initially effective, disease progression inevitably ensues with the emergence of resistance. Intratumoral hypoxia is also associated with CRPC progression and treatment resistance. Given that both AR and HIF-1α are key regulators of these processes, dual targeting of both signaling axis represents an attractive therapeutic approach. Methods: Crosstalk of the AR and HIF-1α signaling pathways were examined in prostate cancer cell lines (LNCaP, 22Rv1) with assays measuring the effect of androgen and hypoxia on AR-dependent and hypoxia-inducible gene transcription, protein expression, cell proliferation, and apoptosis. Cells were stimulated with dihydrotestosterone (DHT) or the hypoxia mimetic cobalt chloride. HIF-1α inhibition was achieved by siRNA silencing HIF-1α or via chetomin, a disruptor of HIF-1α-p300 interactions. Results: In prostate cancer cells, gene expressions of AR targets (KLK3, FKBP5, TMPRSS2) were repressed by HIF-signaling; conversely, HIF-1α target expressions (VEGF, ENO1, LDHA) were induced by DHT-mediated AR signaling. Treatment of CRPC cells with enzalutamide and chetomin or HIF-1α siRNA attenuated AR-regulated and HIF-1α-mediated gene transcription. The combination of enzalutamide and HIF-1α inhibition was more effective than either treatment alone. Similarly, the combination also reduced VEGF protein levels. HIF-1α siRNA synergistically enhanced the inhibitory effect of enzalutamide on cell growth in LNCaP and enzalutamide-resistant 22Rv1 cells. Additionally, HIF-1α siRNA synergistically increased enzalutamide-induced apoptosis. Conclusions: Combination of enzalutamide with HIF-1α inhibition resulted in synergistic inhibition of AR-dependent and gene specific HIF-dependent expression, prostate cancer cell growth, and apoptosis.

Human cytomegalovirus inhibits erythropoietin production.

Anemia is a feature of CKD and a complication of renal transplantation, often caused by impaired production of erythropoietin. The kidney is a target organ for human cytomegalovirus (hCMV) in such patients, but it is not known whether hCMV effects erythropoietin production. We found that kidneys from patients with CKD were positive for hCMV protein and that blood levels of hCMV IgG inversely correlated with red blood cell count. In mice, systemic murine cytomegalovirus infection decreased serum erythropoietin levels. In human erythropoietin-producing cells, hCMV inhibited hypoxia-induced expression of erythropoietin mRNA and protein. hCMV early gene expression was responsible, as ultraviolet-inactivated virus had no effect and valganciclovir treatment showed that late gene expression was nonessential. Hypoxia-induced gene transcription is controlled by the transcription factors hypoxia-inducible transcription factor (HIF)-1α and HIF2α, which are constitutively produced but stable only under low oxygen conditions. We found that hCMV inhibited constitutive production of HIF2α mRNA. HIF2α is thought to be the master regulator of erythropoietin transcription. Single-cell analysis revealed that nuclear accumulation of HIF2α was inhibited in hCMV-infected cells, and the extent of inhibition correlated with hCMV protein expression. Our findings suggest that renal hCMV infection could induce or exacerbate anemia in patients.

Superoxide induced by a high-glucose concentration attenuates production of angiogenic growth factors in hypoxic mouse mesenchymal stem cells

Previous reports have shown that the paracrine system may be an important mediator in bone-marrow-derived mesenchymal stem cell (MSC) therapy for ischemic diseases. Hyperglycemia and hypoxia have been associated with increased levels of reactive oxygen species; oxidative stress may therefore influence the paracrine effects of MSCs under hypoxic conditions in diabetic patients, although the mechanism underlying this effect remains unknown. Hypoxia-inducible factor 1α (HIF-1α) regulates the transcription of hypoxia-inducible genes. We determined the effect of high-glucose concentrations on the production of angiogenic growth factors via HIF-1α induction in hypoxic MSCs. MSCs were cultured with different glucose concentration (5.6, 11, 20, or 30 mM) for 24 h. The cells were then incubated in a hypoxic chamber (5% O₂) or under normoxia (21% O₂) for 6 or 24 h. Protein levels of HIF-1α, vascular endothelial growth factor A₁₆₅ (VEGF-A₁₆₅), and platelet-derived growth factor B (PDGF-B) were attenuated by glucose in hypoxic MSCs in a dose-dependent manner. Treatment with MG132, a specific inhibitor of proteasome activity, significantly reversed the inhibitory effect of high-glucose concentrations in hypoxic MSCs. 4-Hydroxyl-tetramethylpiperidin-oxyl (a cell-permeable superoxide scavenger) or Apocynin (a NADPH oxidase inhibitor) significantly reversed glucose-induced attenuation of VEGF-A₁₆₅, PDGF-B, and HIF-1α protein levels. Stimulation with a high-glucose concentration (30 mM) significantly increased intracellular superoxide levels in hypoxic MSCs. Our results suggest that in hypoxic MSCs the increase in intracellular superoxide levels induced by high-glucose concentrations may attenuate hypoxia-induced HIF-1α expression, which in turn attenuates hypoxia-induced VEGF-A₁₆₅ and PDGF-B transcription.

Factor inhibiting HIF limits the expression of hypoxia-inducible genes in podocytes and distal tubular cells

The two hypoxia-inducible factors (HIF-1α and HIF-2α) are transcription factors that regulate the response to hypoxia. Recently, the factor inhibiting HIF (FIH1) was identified as a molecular oxygen-dependent dioxygenase that blunts the transcriptional activity of HIF and has also been implicated in HIF-dependent and -independent hypoxia responses. Interestingly, HIF accumulation in the kidney has been shown to confer renal protection and to also cause glomerular injury or enhance renal fibrosis. In order to better understand the regulation of hypoxia-inducible genes, we determined the expression of FIH1 in the kidney and its functional role in isolated renal cells. FIH1 was expressed only in distal tubules and in podocytes, thus showing a very distinct expression pattern, partially overlapping with sites of HIF-1α expression. In tubular cells, RNA silencing of FIH1 caused transcriptional activation of HIF target genes during hypoxia. In contrast, FIH1 silencing in podocytes enhanced transcription of hypoxia-inducible genes in an HIF-independent manner. Using the anti-Thy.1 rat model of glomerulonephritis, we found a gradual decrease of glomerular FIH1 expression during disease progression paralleled by an increase in hypoxia-inducible genes including CXCR4, a mediator of glomerular inflammation. Thus, FIH1 appears to be a suppressor of oxygen-dependent genes in the kidney, operating through HIF-dependent and -independent mechanisms.

Activating transcription factor 2 increases transactivation and protein stability of hypoxia-inducible factor 1α in hepatocytes

HIF-1 (hypoxia inducible factor 1) performs a crucial role in mediating the response to hypoxia. However, other transcription factors are also capable of regulating hypoxia-induced target-gene transcription. In a previous report, we demonstrated that the transcription factor ATF-2 (activating transcription factor 2) regulates hypoxia-induced gene transcription, along with HIF-1alpha. In the present study, we show that the protein stability of ATF-2 is induced by hypoxia and the hypoxia-mimic CoCl2 (cobalt chloride), and that ATF-2 induction enhances HIF-1alpha protein stability via direct protein interaction. The knockdown of ATF-2 using small interfering RNA and translation-inhibition experiments demonstrated that ATF-2 plays a key role in the maintenance of the expression level and transcriptional activity of HIF-1alpha. Furthermore, we determined that ATF-2 interacts directly with HIF-1alpha both in vivo and in vitro and competes with the tumour suppressor protein p53 for HIF-1alpha binding. Collectively, these results show that protein stabilization of ATF-2 under hypoxic conditions is required for the induction of the protein stability and transactivation activity of HIF-1alpha for efficient hypoxia-associated gene expression.

Autonomous Tracheal Sprouting

Hypoxia-inducible factor (HIF) signaling is important for regulating the oxidative stress response as well as for branching morphogenesis of tubular oxygen-delivering tissues such as vertebrate blood vessels. Centanin et al . investigated the role of Similar (Sima), a homolog of the transcription factor HIF-α, and its repressor Fatiga (Fga) in Drosophila tracheal development. Under normal oxygen conditions, Sima is marked for degradation by Fga-mediated hydroxylation; under hypoxic conditions, this oxygen-dependent reaction slows and Sima accumulates, stimulating transcription of hypoxia-inducible genes. Trachea of fga loss-of-function mutants sprouted more branches than wild-type trachea, and overexpression of sima in the trachea, but not in tissues surrounding the trachea, was sufficient to induce extra branching. sima loss-of-function mutants, however, had wild-type tracheal branching when reared in normoxic conditions. Under hypoxic conditions, sima induced expression of the fibroblast growth factor (FGF) receptor breathless ( btl ) in trachea and the FGF branchless ( bnl ) in tissue around the trachea. In normal development, Btl and Bnl are essential for tracheal branching: btl -expressing tracheal cells are attracted to target tissues that secrete Bnl. These data suggest that all aspects of Drosophila tracheal branching are genetically programmed but that under hypoxic conditions the HIF pathway stimulates extra tracheal branching via the well-characterized Btl/Bnl chemoattraction system. Thus, the Sima-Fga oxygen sensor is required in both trachea and surrounding tissues for hypoxia-induced branching, and the trachea may initiate branching independently of signaling from the target tissue. L. Centanin, A. Dekanty, N. Romero, M. Irisarri, T. A. Gorr, P. Wappner, Cell autonomy of HIF effects in Drosophila : Tracheal cells sense hypoxia and induce terminal branch sprouting. Dev. Cell 14 , 547-558 (2008). [PubMed]

Decreasing Intracellular Superoxide Corrects Defective Ischemia-induced New Vessel Formation in Diabetic Mice

Tissue ischemia promotes vasculogenesis through chemokine-induced recruitment of bone marrow-derived endothelial progenitor cells (EPCs). Diabetes significantly impairs this process. Because hyperglycemia increases reactive oxygen species in a number of cell types, and because many of the defects responsible for impaired vasculogenesis involve HIF1-regulated genes, we hypothesized that HIF1 function is impaired in diabetes because of reactive oxygen species-induced modification of HIF1alpha by the glyoxalase 1 (GLO1) substrate methylglyoxal. Decreasing superoxide in diabetic mice by either transgenic expression of manganese superoxide dismutase or by administration of an superoxide dismutase mimetic corrected post-ischemic defects in neovascularization, oxygen delivery, and chemokine expression, and normalized tissue survival. In hypoxic fibroblasts cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the EPC mobilizing chemokine stromal cell-derived factor-1 (SDF-1) and of vascular epidermal growth factor, which modulates growth and differentiation of recruited EPCs. In hypoxic EPCs cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the SDF-1 receptor CXCR4, and endothelial nitric-oxide synthase, an enzyme essential for EPC mobilization. HIF1alpha modification by methylglyoxal reduced heterodimer formation and HIF1alpha binding to all relevant promoters. These results provide a basis for the rational design of new therapeutics to normalize impaired ischemia-induced vasculogenesis in patients with diabetes.

Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan

Mesenchymal stem cells (MSC) are capable of differentiating into bone, fat, cartilage, tendon and other organ progenitor cells. Despite the abundance of MSC within the organism, little is known about their in vivo properties or about their corresponding in vivo niches. We therefore isolated MSC from spongy (cancellous) bone biopsies of healthy adults. When compared with the surrounding marrow, a fourfold higher number of colony-forming units was found within the tight meshwork of trabecular bone surface. At these sites, oxygen concentrations range from 1% to 7%. In MSC cultured at oxygen as low as 3%, rates for cell death and hypoxia-induced gene transcription remained unchanged, while in vitro proliferative lifespan was significantly increased, with about 10 additional population doublings before reaching terminal growth arrest. However, differentiation capacity into adipogenic progeny was diminished and no osteogenic differentiation was detectable at 3% oxygen. In turn, MSC that had previously been cultured at 3% oxygen could subsequently be stimulated to successfully differentiate at 20% oxygen. These data support our preliminary finding that primary MSC are enriched at the surface of spongy bone. Low oxygen levels in this location provide a milieu that extends cellular lifespan and furthermore is instructive for the stemness of MSC allowing proliferation upon stimulation while suppressing differentiation.

Two transactivation mechanisms cooperate for the bulk of HIF-1-responsive gene expression

The C-terminal activation domain (C-TAD) of the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha binds the CH1 domains of the related transcriptional coactivators CREB-binding protein (CBP) and p300, an oxygen-regulated interaction thought to be highly essential for hypoxia-responsive transcription. The role of the CH1 domain in vivo is unknown, however. We created mutant mice bearing deletions in the CH1 domains (DeltaCH1) of CBP and p300 that abrogate their interactions with the C-TAD, revealing that the CH1 domains of CBP and p300 are genetically non-redundant and indispensable for C-TAD transactivation function. Surprisingly, the CH1 domain was only required for an average of approximately 35-50% of global HIF-1-responsive gene expression, whereas another HIF transactivation mechanism that is sensitive to the histone deacetylase inhibitor trichostatin A (TSA(S)) accounts for approximately 70%. Both pathways are required for greater than 90% of the response for some target genes. Our findings suggest that a novel functional interaction between the protein acetylases CBP and p300, and deacetylases, is essential for nearly all HIF-responsive transcription.

Mutations of von Hippel-Lindau Tumor-Suppressor Gene and Congenital Polycythemia

The von Hippel-Lindau (pVHL) protein plays an important role in hypoxia sensing. It binds to the hydroxylated hypoxia-inducible factor 1 alpha (HIF-1 alpha) and serves as a recognition component of an E3-ubiquitin ligase complex. In hypoxia or secondary to a mutated VHL gene, the nondegraded HIF-1 alpha forms a heterodimer with HIF-beta and leads to increased transcription of hypoxia-inducible genes, including erythropoietin (EPO). The autosomal dominant cancer-predisposition von Hippel-Lindau (VHL) syndrome is due to inheritance of a single mutated allele of VHL. In contrast, we recently showed that homozygous germline 598C-->T VHL mutation leads to Chuvash polycythemia (CP). We subsequently found VHL mutations in three unrelated individuals unaffected with CP, one of whom was compound heterozygous for the 598C-->T mutation and another VHL mutation. We now report seven additional polycythemic patients with VHL mutations in both alleles. Two Danish siblings and another American boy were homozygous for the VHL 598C-->T mutation. Three unrelated white Americans were compound heterozygotes for 598C-->T and another VHL mutation, 562C-->G in two and 574C-->T in the third. Additionally, a Croatian boy was homozygous for a 571C-->G VHL mutation, the first example of homozygous VHL germline mutation causing polycythemia, other than the VHL 598C-->T mutation. We have not observed VHL syndrome-associated tumors in polycythemic subjects or their heterozygous relatives; however, this will need to be evaluated by longitudinal studies. Over all, we found that up to half of the consecutive patients with apparent congenital polycythemia and increased serum Epo we have examined have mutations of both VHL alleles. Those findings, along with reports of CP, underscore that VHL mutations are the most frequent cause of congenital polycythemia and define a new class of polycythemic disorder, polycythemias due to augmented hypoxia sensing.

Regulation of gene expression by the hypoxia-inducible factors.

Many molecular and physiological responses to hypoxia in mammals are controlled by the transcription factors Hypoxia-Inducible Factor-1alpha (HIF-1alpha) and HIF-2alpha. Their ability to promote the transcription of hypoxia-inducible genes is mediated by protein stability and regulation of a C-terminal transactivation domain. Oxygen-dependent hydroxylation of conserved proline and asparagine residues in HIF-alpha are required for targeting HIF-alpha to proteasomes for destruction, and for inhibiting its capacity for CBP/p300-dependent transactivation, respectively. In hypoxia, the O2 required for prolyl and asparaginyl hydroxylation is limiting, and HIF-alpha is thus stabilized and competent for transcription. Because these proteins participate in angiogenesis, glycolysis, programmed cell death, cancer, and ischemia, HIF-alpha and its mediators are attractive therapeutic targets.

A dominant-negative isoform lacking exons 11 and 12 of the human hypoxia-inducible factor-1α gene

Hypoxia-inducible factor-1α (HIF-1α), a member of the transcription family characterized by a basic helix-loop-helix (bHLH) domain and a PAS domain, regulates the transcription of hypoxia-inducible genes involved in erythropoiesis, vascular remodelling and glucose/energy metabolism. It contains bHLH/PAS domains in the N-terminal half, and a nuclear localization signal (NLS) and two transactivation domains (TADs) in the C-terminal half. It also has an oxygen-dependent degradation (ODD) domain, which is required to degrade HIF-1α protein by the ubiquitin—proteasome pathway. In this study, we identified a new alternatively spliced variant of human HIF-1α mRNA, which lacked both exons 11 and 12, producing a frame shift and giving a shorter form of HIF-1α. In the corresponding protein, a part of the ODD domain, both TADs and the C-terminal NLS motif were missing. Expression of endogenous HIF-1α variant protein was identified using immunoprecipitation and immunoblotting methods. The expressed HIF-1α variant exhibited neither the activity of transactivation nor hypoxia-induced nuclear translocation. In contrast with HIF-1α, the variant was strikingly stable in normoxic conditions and not up-regulated to such an extent by hypoxia, cobalt ions or desferrioxamine. It was also demonstrated that the HIF-1α variant competed with endogenous HIF-1α and suppressed HIF-1 activity, resulting in the down-regulation of mRNA expression of hypoxia-inducible genes. The association of the variant and arylhydrocarbon receptor nuclear translocator in the cytoplasm may be related to the inhibition of HIF-1 activity. It is assumed that this isoform preserves the balance between aerobic and anaerobic metabolism by counteracting the overaction of HIF-1α.

A dominant-negative isoform lacking exons 11 and 12 of the human hypoxia-inducible factor-1alpha gene.

Hypoxia-inducible factor-1alpha (HIF-1alpha), a member of the transcription family characterized by a basic helix-loop-helix (bHLH) domain and a PAS domain, regulates the transcription of hypoxia-inducible genes involved in erythropoiesis, vascular remodelling and glucose/energy metabolism. It contains bHLH/PAS domains in the N-terminal half, and a nuclear localization signal (NLS) and two transactivation domains (TADs) in the C-terminal half. It also has an oxygen-dependent degradation (ODD) domain, which is required to degrade HIF-1alpha protein by the ubiquitin-proteasome pathway. In this study, we identified a new alternatively spliced variant of human HIF-1alpha mRNA, which lacked both exons 11 and 12, producing a frame shift and giving a shorter form of HIF-1alpha. In the corresponding protein, a part of the ODD domain, both TADs and the C-terminal NLS motif were missing. Expression of endogenous HIF-1alpha variant protein was identified using immunoprecipitation and immunoblotting methods. The expressed HIF-1alpha variant exhibited neither the activity of transactivation nor hypoxia-induced nuclear translocation. In contrast with HIF-1alpha, the variant was strikingly stable in normoxic conditions and not up-regulated to such an extent by hypoxia, cobalt ions or desferrioxamine. It was also demonstrated that the HIF-1alpha variant competed with endogenous HIF-1alpha and suppressed HIF-1 activity, resulting in the down-regulation of mRNA expression of hypoxia-inducible genes. The association of the variant and arylhydrocarbon receptor nuclear translocator in the cytoplasm may be related to the inhibition of HIF-1 activity. It is assumed that this isoform preserves the balance between aerobic and anaerobic metabolism by counteracting the overaction of HIF-1alpha.

C-Jun and hypoxia-inducible factor 1 functionally cooperate in hypoxia-induced gene transcription.

Under low-oxygen conditions, cells develop an adaptive program that leads to the induction of several genes, which are transcriptionally regulated by hypoxia-inducible factor 1 (HIF-1). On the other hand, there are other factors which modulate the HIF-1-mediated induction of some genes by binding to cis-acting motifs present in their promoters. Here, we show that c-Jun functionally cooperates with HIF-1 transcriptional activity in different cell types. Interestingly, a dominant-negative mutant of c-Jun which lacks its transactivation domain partially inhibits HIF-1-mediated transcription. This cooperative effect is not due to an increase in the nuclear amount of the HIF-1alpha subunit, nor does it require direct binding of c-Jun to DNA. c-Jun and HIF-1alpha are able to associate in vivo but not in vitro, suggesting that this interaction involves the participation of additional proteins and/or a posttranslational modification of these factors. In this context, hypoxia induces phosphorylation of c-Jun at Ser(63) in endothelial cells. This process is involved in its cooperative effect, since specific blockade of the JNK pathway and mutation of c-Jun at Ser(63) and Ser(73) impair its functional cooperation with HIF-1. The functional interplay between c-Jun and HIF-1 provides a novel insight into the regulation of some genes, such as the one for VEGF, which is a key regulator of tumor angiogenesis.

The Von Hippel-Lindau Protein Interacts with Heteronuclear Ribonucleoprotein A2 and Regulates Its Expression

The product of the von Hippel-Lindau (VHL) tumor suppressor gene, pVHL, functions as a ubiquitin-protein isopeptide ligase in regulating HIF-1 protein turnover, thus accounting for the increased transcription of hypoxia-inducible genes that accompanies VHL mutations. The increased vascular endothelial growth factor mRNA stability in cells lacking pVHL has been hypothesized to be due to a similar regulation of an RNA-binding protein. We report the expression of the GLUT-1 3'-untranslated region RNA-binding protein, heteronuclear ribonucleoprotein (hnRNP) A2, is specifically increased in pVHL-deficient cell lines. Enhanced hnRNP A2 expression was apparent in all cell fractions, including polysomes, where a similar modest effect on hnRNP L (a GLUT-1 and VEGF 3'-untranslated region-binding protein), was seen. Steady state levels of hnRNP A2 mRNA were unaffected. Regulation of hnRNP A2 levels correlated with the ability of pVHL to bind elongin C. Proteasome inhibition of cells expressing wild type pVHL selectively increased cytoplasmic hnRNP A2 levels to that seen in pVHL-deficient cells. Finally, an in vivo interaction between pVHL and hnRNP A2 was demonstrated in both the nucleus and the cytoplasm. Collectively, these data indicate that hnRNP A2 expression is regulated by pVHL in a manner that is dependent on elongin C interactions as well as functioning proteasomes.