Hypoxia-inducible Factor Target Research Articles

PIAS3 enhances the transcriptional activity of HIF-1α by increasing its protein stability

The transcription factor hypoxia-inducible factor-1 (HIF-1) functions as a master regulator of hypoxic response by inducing the transcription of various genes responsible for cellular adaptation to hypoxia. In this study, we investigated the effects of protein inhibitor of activated STAT3 (PIAS3), a small ubiquitin-related modifier (SUMO) E3 ligase, on HIF-1-mediated transcriptional activation. We found that PIAS3 physically associated with HIF-1α. Moreover, PIAS3 overexpression enhanced the transcriptional activity of HIF-1α independently of its SUMO E3 ligase activity. Conversely, quantitative RT-PCR analysis showed that RNAi-mediated PIAS3 knockdown reduced the expression of HIF-1 target genes under hypoxia. In addition, PIAS3 knockdown induced the destabilization of HIF-1α protein, and the destabilization was reversed by the proteasome inhibitor MG132. Taken together, these results suggest that PIAS3 functions as a positive regulator of HIF-1α-mediated transcription by increasing its protein stability.

Endothelial HIF signaling regulates pulmonary fibrosis-associated pulmonary hypertension.

Pulmonary hypertension (PH) complicating chronic parenchymal lung disease, such as idiopathic pulmonary fibrosis, results in significant morbidity and mortality. Since the hypoxia-inducible factor (HIF) signaling pathway is important for development of pulmonary hypertension in chronic hypoxia, we investigated whether HIF signaling in vascular endothelium regulates development of PH related to pulmonary fibrosis. We generated a transgenic model in which HIF is deleted within vascular endothelial cells and then exposed these mice to chronic intraperitoneal bleomycin to induce PH associated with lung fibrosis. Although no differences in the degree of fibrotic remodeling were observed, we found that endothelial HIF-deficient mice were protected against development of PH, including right ventricle and pulmonary vessel remodeling. Similarly, endothelial HIF-deficient mice were protected from PH after a 4-wk exposure to normobaric hypoxia. In vitro studies of pulmonary vascular endothelial cells isolated from the HIF-targeted mice and controls revealed that endothelial HIF signaling increases endothelial cell expression of connective tissue growth factor, enhances vascular permeability, and promotes pulmonary artery smooth muscle cell proliferation and wound healing ability, all of which have the potential to impact the development of PH in vivo. Taken together, these studies demonstrate that vascular endothelial cell HIF signaling is necessary for development of hypoxia and pulmonary fibrosis associated PH. As such, HIF and HIF-regulated targets represent a therapeutic target in these conditions.

Lnc RNAs, hypoxia and metastasis.

In a variety of pathophysiological conditions oxygen demand exceeds its supply, leading to a compromising condition referred to as hypoxia, which triggers complex adaptive mechanisms mediated primarily through transcriptional reprogramming by hypoxia inducible factors (HIFs). Solid tumors are an example of this condition, as deregulated proliferation generates oxygen deficient microenvironments to which cancer cells need to adapt. Among the adaptive processes, the growth of new vessels (angiogenesis) and metabolic reprogramming are central to cope with low oxygen tension. However, in the context of solid tumors, hypoxic areas perpetuate and expand due to the aberrant activation of angiogenesis which gives rise to abnormal tumor vessels and a dysfunctional tumor vasculature. In fact, the prognostic significance of hypoxic regions is well recognized and tumor hypoxia is causally associated with resistance to therapy. Additionally, hypoxia induces metastasis through regulation of several critical processes implicated in the complex metastatic cascade, especially epithelial-mesenchymal transition and cancer stem cell renewal [1]. Recent studies have implicated HIF target genes in nearly every step of the metastatic process; however, the precise molecular and cellular mechanisms underlying hypoxia-induced metastatic dissemination are just beginning to be uncovered.

Propranolol reduces viability and induces apoptosis in hemangioblastoma cells from von Hippel-Lindau patients.

BackgroundVon Hippel-Lindau (VHL) disease is a rare oncological disease with an incidence of 1:36,000, and is characterized by the growth of different types of tumors: hemangioblastomas in the central nervous system (CNS) and retina, renal carcinoma, pheochromocytomas, pancreatic serous cystadenoma, and endolymphatic sac tumors. These tumors do not express VHL protein (pVHL). pVHL ubiquitinates hypoxia inducible factor (HIF) protein for degradation by the proteasome; in the absence of VHL, HIF translocates to the nucleus to activate the expression of its target genes. Targeting VHL-derived tumors with drugs that have reduced side effects is urgent to avoid repeat CNS surgeries. Recent reports have shown that propranolol, a β-blocker used for the treatment of hypertension and other cardiac and neurological diseases, is the best option for infantile hemangioma (IH). Propranolol could be an efficient treatment to control hemangioblastoma growth in VHL disease because of its antiangiogenic effects demonstrated in IH and the hypothetical impact on HIF levels.MethodsHeLa 9X (HRE) hypoxia responsive element cell line and primary hemangioblastoma-derived cells were subjected to propranolol treatment and cell viability and apoptosis were evaluated. HIF1-α and Hif-2α expression after propranolol treatment was analyzed by western blotting. Quantitative PCR was performed to study the mRNA expression of HIF target genes. Vascular endothelial growth factor (VEGF) was measured in culture supernatants by immunoassay.ResultsPropranolol downregulated HIF-dependent transcription in HeLa 9XHRE cells. Under hypoxic conditions, propranolol decreased the expression of HIF target genes in hemangioblastoma cells, which stopped proliferating and died following long-term treatment. These results suggests that propranolol treatment promoted reduced HIF protein expression and corresponding downregulation of HIF target genes, and inhibited cell proliferation in parallel with induction of cell death by apoptosis.ConclusionsOur results suggest that propranolol could reduce the growth of HIF-dependent tumors and may thus be a promising treatment to delay surgery in VHL patients.

Synthesis and Biological Evaluation of Manassantin Analogues for Hypoxia-Inducible Factor 1α Inhibition.

To cope with hypoxia, tumor cells have developed a number of adaptive mechanisms mediated by hypoxia-inducible factor 1 (HIF-1) to promote angiogenesis and cell survival. Due to significant roles of HIF-1 in the initiation, progression, metastasis, and resistance to treatment of most solid tumors, a considerable amount of effort has been made to identify HIF-1 inhibitors for treatment of cancer. Isolated from Saururus cernuus, manassantins A (1) and B (2) are potent inhibitors of HIF-1 activity. To define the structural requirements of manassantins for HIF-1 inhibition, we prepared and evaluated a series of manassantin analogues. Our SAR studies examined key regions of manassantin’s structure in order to understand the impact of these regions on biological activity and to define modifications that can lead to improved performance and drug-like properties. Our efforts identified several manassantin analogues with reduced structural complexity as potential lead compounds for further development. Analogues MA04, MA07, and MA11 down-regulated hypoxia-induced expression of the HIF-1α protein and reduced the levels of HIF-1 target genes, including cyclin-dependent kinase 6 (Cdk6) and vascular endothelial growth factor (VEGF). These findings provide an important framework to design potent and selective HIF-1α inhibitors, which is necessary to aid translation of manassantin-derived natural products to the clinic as novel therapeutics for cancers.

Dihydrotanshinone I inhibits the translational expression of hypoxia-inducible factor-1α

The hypoxia-inducible factor-1 (HIF-1) has been known to be correlated to the adaptation and proliferation of tumor cells; therefore HIF-1 has become an important target in the development of anticancer drugs. Dihydrotanshinone I (DHTS) is a phenanthrenequinone compound from Salvia miltiorrhiza Bunge, which has been used in oriental medicine for its antitumor activities. However, the mechanisms by which DHTS inhibits tumor growth are not fully understood. We here demonstrated the effect of DHTS on hypoxia-inducible factor-1 (HIF-1) activation. DHTS dose-dependently decreased the hypoxia-induced accumulation and activation of HIF-1α protein. Further analysis revealed that DHTS inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), extracellular signal-regulated kinase-1/2 (ERK1/2), ribosomal protein S6 kinase (p70S6K), eIF4E binding protein-1 (4E-BP1), and eukaryotic initiation factor 4E (eIF4E) were dose-dependently suppressed by DHTS, but no significant effect on total protein levels was observed. Furthermore, DHTS prevented hypoxia-induced expression of HIF-1 target genes and flow cytometric analysis indicated that DHTS induced G1 phase arrest in HeLa cell. In vivo studies further confirmed the inhibitory effect of DHTS on the expression of HIF-1α proteins, leading to a decrease in growth of HeLa cells in a xenograft tumor model. These results show that DHTS inhibited HIF-1α protein synthesis by downregulating the mTOR/p70S6K/4E-BP1 and MEK/ERK pathways. These conclusions suggest that DHTS is an effective inhibitor of HIF-1 and provide new perspectives into the mechanism of its anticancer activity.

Deferoxamine Preconditioning of Neural-Like Cells Derived from Human Wharton's Jelly Mesenchymal Stem Cells as a Strategy to Promote Their Tolerance and Therapeutic Potential: An In Vitro Study.

Transplantation of neural-like cells is considered as a promising therapeutic strategy developed for neurodegenerative disease in particular for ischemic stroke. Since cell survival is a major concern following cell implantation, a number of studies have underlined the protective effects of preconditioning with hypoxia or hypoxia mimetic pharmacological agents such as deferoxamine (DFO), induced by activation of hypoxia inducible factor-1 (HIF-1) and its target genes. The present study has investigated the effects of DFO preconditioning on some factors involved in cell survival, angiogenesis, and neurogenesis of neural-like cells derived from human Wharton's jelly mesenchymal stem cells (HWJ-MSCs) in presence of hydrogen peroxide (H2O2). HWJ-MSCs were differentiated toward neural-like cells for 14days and neural cell markers were identified using immunocytochemistry. HWJ-MSC-derived neural-like cells were then treated with 100µM DFO, as a known hypoxia mimetic agent for 48h. mRNA and protein expression of HIF-1 target genes including brain-derived neurotrophic factors (BDNF) and vascular endothelial growth factor (VEGF) significantly increased using RT-PCR and Western blotting which were reversed by HIF-1α inhibitor, while, gene expression of Akt-1, Bcl-2, and Bax did not change significantly but pAkt-1 was up-regulated as compared to poor DFO group. However, addition of H2O2 to DFO-treated cells resulted in higher resistance to H2O2-induced cell death. Western blotting analysis also showed significant up-regulation of HIF-1α, BDNF, VEGF, and pAkt-1, and decrease of Bax/Bcl-2 ratio as compared to poor DFO. These results may suggest that DFO preconditioning of HWJ-MSC-derived neural-like cells improves their tolerance and therapeutic potential and might be considered as a valuable strategy to improve cell therapy.

Abstract 533: S100A10 as a novel biomarker in hepatocellular carcinoma

Abstract Background and Objective: Hepatocellular carcinoma (HCC) is the dominant form of primary liver tumor and the second most lethal malignancy in worldwide. From transcriptome sequencing analysis, we observed that S100A10 was overexpressed in our cohort of 16 HCC patients. S100A10 is a calcium binding protein and accumulating studies have indicated that annexin A2- S100A10 heterotetramer on the cell surface is important for the conversion of plasminogen to plasmin, which promotes activation of matrix metalloproteinases and degradation of extracellular matrix. This enhances cancer invasion and migration. While annexin A2 overexpression has been documented in various malignancies and shows significant association with local invasion and metastasis, information regarding the expression of S100A10 in human cancers is scarce. Furthermore, how S100A10 was regulated remains unexplored. Hypoxia is a common phenomenon in HCC microenvironment and stabilizies transcription factors, hypoxia inducible factor (HIF)-1α and -2α, to initiate transcription of a wide repertoire of genes for cancer progression. The major aim of this study is to evaluate the possible clinical relevance and diagnostic value of S100A10 in HCC and study the molecular mechanism that drives S100A10 overexpression under the influence of hypoxia in HCC. Experimental Procedures: We evaluated S100A10 expression in human HCC and the corresponding non-tumorous liver (NT) tissues in a cohort of 67 HCC patients by RT-PCR. S100A10 expression was correlated with various HCC clinico-pathological features by Student's t-test and survival outcome of HCC patients by Kaplan-Meier plot followed by log rank test. Promoter of S100A10 was analyzed for potential hypoxia responsive elements -A/GCGTG- and confirmed by ChIP assay with HIF antibodies. To evaluate the impact of HIF on S100A10 expression, we knocked-down (shRNA) and knocked-out (TALEN) HIF-1/2α in a HCC cell line, MHCC97L and evaluated S100A10 mRNA and protein expression. Results: S100A10 was significantly upregulated (P = 0.001, Wilcoxon signed rank test) in HCC as compared to NT tissues. Overexpression of S100A10 (≥ 2 fold) was found in 50.746% (34/67) of HCC patients and was closely correlated with direct liver invasion (P = 0.038), tumor microsatellite formation (P = 0.050) and absence of tumor encapsulation (P = 0.048). More importantly, S100A10 overexpression was associated with poorer 5-year survival rate in HCC patients (P = 0.062). ROC analysis suggested that S100A10 could be a potential diagnostic marker for HCC detection (Area Under Curve = 0.685, P = 0.001). Hypoxia induced S100A10 mRNA and protein expression levels in multiple HCC cell lines and this was abolished in HIF-1/2α knockdown or knockout HCC cells. Conclusion: Our current data have presented evidence that S100A10 can be a potential diagnostic and prognostic marker in HCC and S100A10 is a transcriptional target of HIF. Citation Format: David Kung-Chun Chiu, Carmen Chak-Lui Wong, Irene Oi-Lin Ng, Aki Pui-Wah Tse. S100A10 as a novel biomarker in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 533. doi:10.1158/1538-7445.AM2015-533

Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis.

BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1α and Hif target genes, including those involved in glycolysis and angiogenesis—two processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunction—caused by defects in mitophagy—can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

Identification of a hypoxia-regulated miRNA signature in bladder cancer and a role for miR-145 in hypoxia-dependent apoptosis.

Background:Hypoxia leads to the stabilisation of the hypoxia-inducible factor (HIF) transcription factor that drives the expression of target genes including microRNAs (miRNAs). MicroRNAs are known to regulate many genes involved in tumourigenesis. The aim of this study was to identify hypoxia-regulated miRNAs (HRMs) in bladder cancer and investigate their functional significance.Methods:Bladder cancer cell lines were exposed to normoxic and hypoxic conditions and interrogated for the expression of 384 miRNAs by qPCR. Functional studies were carried out using siRNA-mediated gene knockdown and chromatin immunoprecipitations. Apoptosis was quantified by annexin V staining and flow cytometry.Results:The HRM signature for NMI bladder cancer lines includes miR-210, miR-193b, miR-145, miR-125-3p, miR-708 and miR-517a. The most hypoxia-upregulated miRNA was miR-145. The miR-145 was a direct target of HIF-1α and two hypoxia response elements were identified within the promoter region of the gene. Finally, the hypoxic upregulation of miR-145 contributed to increased apoptosis in RT4 cells.Conclusions:We have demonstrated the hypoxic regulation of a number of miRNAs in bladder cancer. We have shown that miR-145 is a novel, robust and direct HIF target gene that in turn leads to increased cell death in NMI bladder cancer cell lines.

A glycolytic phenotype is associated with prostate cancer progression and aggressiveness: a role for monocarboxylate transporters as metabolic targets for therapy.

Metabolic adaptation is considered an emerging hallmark of cancer, whereby cancer cells exhibit high rates of glucose consumption with consequent lactate production. To ensure rapid efflux of lactate, most cancer cells express high levels of monocarboxylate transporters (MCTs), which therefore may constitute suitable therapeutic targets. The impact of MCT inhibition, along with the clinical impact of altered cellular metabolism during prostate cancer (PCa) initiation and progression, has not been described. Using a large cohort of human prostate tissues of different grades, in silico data, in vitro and ex vivo studies, we demonstrate the metabolic heterogeneity of PCa and its clinical relevance. We show an increased glycolytic phenotype in advanced stages of PCa and its correlation with poor prognosis. Finally, we present evidence supporting MCTs as suitable targets in PCa, affecting not only cancer cell proliferation and survival but also the expression of a number of hypoxia‐inducible factor target genes associated with poor prognosis. Herein, we suggest that patients with highly glycolytic tumours have poorer outcome, supporting the notion of targeting glycolytic tumour cells in prostate cancer through the use of MCT inhibitors. © 2015 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF target gene expression

Abnormal accumulation of oncometabolite fumarate and succinate is associated with inhibition of mitochondrial function and carcinogenesis. By competing with α-ketoglutarate, oncometabolites also activate hypoxia inducible factors (HIFs), which makes oncometabolite mimetics broadly utilised in hypoxia research. We found that dimethyloxalylglycine (DMOG), a synthetic analogue of α-ketoglutarate, commonly used to induce HIF signalling, inhibits O2 consumption in cancer cell lines HCT116 and PC12, well before activation of HIF pathways. A rapid suppression of cellular respiration was accompanied by a decrease in histone H4 lysine 16 acetylation and not abolished by double knockdown of HIF-1α and HIF-2α. In agreement with this, production of NADH and state 3 respiration in isolated mitochondria were down-regulated by the de-esterified DMOG derivative, N-oxalylglycine. Exploring the roles of DMOG as a putative inhibitor of glutamine/α-ketoglutarate metabolic axis, we found that the observed suppression of OxPhos and compensatory activation of glycolytic ATP flux make cancer cells vulnerable to combined treatment with DMOG and inhibitors of glycolysis. On the other hand, DMOG treatment impairs deep cell deoxygenation in 3D tissue culture models, demonstrating a potential to relieve functional stress imposed by deep hypoxia on tumour, ischemic or inflamed tissues. Indeed, using a murine model of colitis, we found that O2 availability in the inflamed colon tissue rapidly increased after application of DMOG, which could contribute to the known therapeutic effect of this compound. Overall, our results provide new insights into the relationship between mitochondrial function, O2 availability, metabolic reprogramming and associated diseases.

Targeting Hypoxia-Inducible Factor 1α in a New Orthotopic Model of Glioblastoma Recapitulating the Hypoxic Tumor Microenvironment.

Tissue hypoxia and necrosis represent pathophysiologic and histologic hallmarks of glioblastoma (GBM). Although hypoxia inducible factor 1α (HIF-1α) plays crucial roles in the malignant phenotypes of GBM, developing HIF-1α-targeted agents has been hampered by the lack of a suitable preclinical model that recapitulates the complex biology of clinical GBM. We present a new GBM model, MGG123, which was established from a recurrent human GBM. Orthotopic xenografting of stem-like MGG123 cells reproducibly generated lethal tumors that were characterized by foci of palisading necrosis, hypervascularity, and robust stem cell marker expression. Perinecrotic neoplastic cells distinctively express HIF-1α and are proliferative in both xenografts and the patient tissue. The xenografts contain scattered hypoxic foci that were consistently greater than 50 μm distant from blood vessels, indicating intratumoral heterogeneity of oxygenation. Hypoxia enhanced HIF-1α expression in cultured MGG123 cells, which was abrogated by the HIF-1α inhibitors digoxin or ouabain. In vivo, treatment of orthotopic MGG123 xenografts with digoxin decreased HIF-1α expression, vascular endothelial growth factor mRNA levels, and CD34-positive vasculature within the tumors, and extended survival of mice bearing the aggressive MGG123 GBM. This preclinical tumor model faithfully recapitulates the GBM-relevant hypoxic microenvironment and stemness and is a suitable platform for studying disease biology and developing hypoxia-targeted agents.

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping.

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the −82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the −82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the −82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia.

HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity.

Intestinal epithelial cells (IECs) are exposed to profound fluctuations in oxygen tension and have evolved adaptive transcriptional responses to a low-oxygen environment. These adaptations are mediated primarily through the hypoxia-inducible factor (HIF) complex. Given the central role of the IEC in barrier function, we sought to determine whether HIF influenced epithelial tight junction (TJ) structure and function. Initial studies revealed that short hairpin RNA-mediated depletion of the HIF1β in T84 cells resulted in profound defects in barrier and nonuniform, undulating TJ morphology. Global HIF1α chromatin immunoprecipitation (ChIP) analysis identified claudin-1 (CLDN1) as a prominent HIF target gene. Analysis of HIF1β-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1β-deficient cells resulted in resolution of morphological abnormalities and restoration of barrier function. ChIP and site-directed mutagenesis revealed prominent hypoxia response elements in the CLDN1 promoter region. Subsequent in vivo analysis revealed the importance of HIF-mediated CLDN1 expression during experimental colitis. These results identify a critical link between HIF and specific tight junction function, providing important insight into mechanisms of HIF-regulated epithelial homeostasis.

Repression of hypoxia-inducible factor α signaling by Set7-mediated methylation.

Hypoxia-inducible factor (HIF)-1α and HIF-2α are the main regulators of cellular responses to hypoxia. Post-translational modifications of HIF-1α and 2α are necessary to modulate their functions. The methylation of non-histone proteins by Set7, an SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. In this study, we show that Set7 methylates HIF-1α at lysine 32 and HIF-2α at lysine K29; this methylation inhibits the expression of HIF-1α/2α targets by impairing the occupancy of HIF-α on hypoxia response element of HIF target gene promoter. Set7-null fibroblasts and the cells with shRNA-knocked down Set7 exhibit upregulated HIF target genes. Set7 inhibitor blocks HIF-1α/2α methylation to enhance HIF target gene expression. Set7-null fibroblasts and the cells with shRNA-knocked down Set7 or inhibition of Set7 by the inhibitor subjected to hypoxia display an increased glucose uptake and intracellular adenosine triphosphate levels. These findings define a novel modification of HIF-1α/2α and demonstrate that Set7-medited lysine methylation negatively regulates HIF-α transcriptional activity and HIF-1α-mediated glucose homeostasis.

The effect of lentiviral vector-mediated RNA interference targeting hypoxia-inducible factor 1α on the uptake of fluorodeoxyglucose ((18)f) in the human pancreatic cancer cell line, patu8988.

Hypoxia can stimulate (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in cultured tumor cells. This study has investigated the effect of lentiviral vector-mediated RNA interference (RNAi) targeting hypoxia-inducible factor 1α (HIF-1α) on the changes in HIF-1 and glucose transporter 1 (Glut-1) expression, the cell growth, and the uptake of (18)F-FDG in the human pancreatic cancer cell line, Patu8988. Lentiviral RNAi vector targeting the HIF-1α gene (LV-HIF-1αRNAi) was constructed and used to treat cells at various concentrations (25-200 nM). The expression changes of HIF-1α and Glut-1 in hypoxic Patu8988 cells after RNAi treatment were determined using real time reverse transcription-polymerase chain reaction (real-time PCR). The inhibition rate of cell proliferation 48 hours after the addition of 10 μL of different concentrations of LV-HIF-1αRNAi (25-200 nM) was assayed using the MTT method. Meanwhile, the cell uptake of (18)F-FDG was also assessed. After RNAi transfection, the relative expression levels of HIF-1α mRNA and Glut-1 under hypoxia were reduced and the relative expression levels of HIF-1α protein also decreased. Compared with the control group, the inhibition rates of cell proliferation under different viral dosages were 5.98%, 15.65%, 26.42%, and 40.81%, respectively, positively correlated with the viral doses (r=0.558, p<0.05). Under hypoxia, Glut-1 mRNA expression in Patu8988 cells treated with 200 nM of LV-HIF-1αRNAi for 24, 48, and 72 hours, respectively, was positively correlated with the inhibition rate of cell proliferation (r=0.618, p<0.05) as well as the inhibition rate of (18)F-FDG uptake (r=0.664, p<0.05), while the latter two displayed a positive correlation with each other too (r=0.582, p<0.05). Under hypoxia, RNAi targeting HIF-1α significantly inhibited the expression of Glut-1 mRNA in Patu8988 pancreatic cancer cells and their uptake of (18)F-FDG. These results suggest that LV-HIF-1αRNAi may form a new treatment for pancreatic cancer, and the effectiveness of the treatment can be readily assessed with (18)F-FDG imaging.

Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin.

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1-3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic-osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic-osteogenic coupling.

HIF Upregulates AQP1 Expression in Pulmonary Arterial Smooth Muscle Cells (PASMCs) in a Calcium‐Dependent Manner

Prolonged exposure to hypoxia can have adverse effects on the pulmonary vasculature, including structural remodeling and sustained contraction, resulting in the development of pulmonary hypertension. Both PASMC proliferation and migration contribute to the vascular remodeling. The expression of AQP1, a membrane water channel protein, is elevated during hypoxia. Studies in other cell types suggest that AQP1 is a direct transcriptional target of hypoxia inducible factor (HIF)‐1. Moreover, an increase in intracellular calcium ([Ca2+]i) concentration is a hallmark of hypoxic exposure in PASMCs. Thus, we wanted to determine whether HIF regulates AQP1 in PASMCs and, if so, whether the process was Ca2+‐dependent. PASMCs were incubated with DMOG, which inhibits HIF protein degradation. DMOG produced a time‐dependent increase in AQP1 protein, but not mRNA. Interestingly, incubation with DMOG increased [Ca2+]i in PASMCs, and this elevation was prevented by the voltage‐gated Ca2+ channel inhibitor, verapamil (VER). VER also blocked upregulation of AQP1 protein by DMOG, but had no effect on expression of GLUT1, a canonical HIF transcriptional target. Thus, our results show that contrary to reports in other cell types, in PASMCs, AQP1 does not appear to be a direct target for HIF transcriptional regulation. Instead, AQP1 protein may be upregulated by a mechanism involving HIF‐dependent increases in [Ca2+]i.

Microbe‐Host Crosstalk between Short‐Chain Fatty Acids and Intestinal Epithelial HIF Provides a New Mechanism to Augment Tissue Barrier Function

The interactions between the enteric microbiota and distal gut play important roles in regulating human health. Short chain fatty acids (i.e. butyrate) are produced by anaerobic microbes and are a major energy source for the colonic epithelium. Decreased concentrations of butyrate is strongly associated with colonic disease. Interestingly, the low oxygen conditions that enable butyrate production also place unusual metabolic demands on the colonic mucosa. However, the colonic epithelium is uniquely adapted to this austere oxygen environment through the stabilization of hypoxia‐inducible factor (HIF) and expression of HIF target genes. Because anaerobes are the major producers of butyrate, we hypothesized that microbe‐derived butyrate regulates host HIF. Studies with human intestinal epithelial cells (IECs) revealed that butyrate promotes mitochondrial‐dependent oxygen consumption and stabilizes HIF1α. Furthermore, butyrate activated HIF target genes in IECs and antibiotics decreased butyrate and HIF expression in the mouse colon. Germ free mice also showed decreased HIF1α expression in the colonic epithelium. Finally, we found that butyrate rescues physiologic hypoxia and protects the intestinal barrier via HIF. This indicates that butyrate participates in a diverse set of functions in the gut, including regulation of barrier function, and could be a potential therapeutic for mucosal protection.