HIF-1α Knockout Research Articles

Chronic Intermittent Hypoxia Induces Atherosclerosis via Activation of Adipose Angiopoietin-like 4

Obstructive sleep apnea is a risk factor for dyslipidemia and atherosclerosis, which have been attributed to chronic intermittent hypoxia (CIH). Intermittent hypoxia inhibits a key enzyme of lipoprotein clearance, lipoprotein lipase, and up-regulates a lipoprotein lipase inhibitor, angiopoietin-like 4 (Angptl4), in adipose tissue. The effects and mechanisms of Angptl4 up-regulation in sleep apnea are unknown. To examine whether CIH induces dyslipidemia and atherosclerosis by increasing adipose Angptl4 via hypoxia-inducible factor-1 (HIF-1). ApoE(-/-) mice were exposed to intermittent hypoxia or air for 4 weeks while being treated with Angptl4-neutralizing antibody or vehicle. In vehicle-treated mice, hypoxia increased adipose Angptl4 levels, inhibited adipose lipoprotein lipase, increased fasting levels of plasma triglycerides and very low density lipoprotein cholesterol, and increased the size of atherosclerotic plaques. The effects of CIH were abolished by the antibody. Hypoxia-induced increases in plasma fasting triglycerides and adipose Angptl4 were not observed in mice with germline heterozygosity for a HIF-1α knockout allele. Transgenic overexpression of HIF-1α in adipose tissue led to dyslipidemia and increased levels of adipose Angptl4. In cultured adipocytes, constitutive expression of HIF-1α increased Angptl4 levels, which was abolished by siRNA. Finally, in obese patients undergoing bariatric surgery, the severity of nocturnal hypoxemia predicted Angptl4 levels in subcutaneous adipose tissue. HIF-1-mediated increase in adipose Angptl4 and the ensuing lipoprotein lipase inactivation may contribute to atherosclerosis in patients with sleep apnea.

SHH-Dependent Knockout of HIF-1 Alpha Accelerates the Degenerative Process in Mouse Intervertebral Disc

Hypoxia-inducible factor-1alpha (HIF-1 alpha) has been reported to have an important role in the metabolism and synthesis of extracellular matrix of the nucleus pulposus cells (NPCs) and was assumed to be involved in the process of intervertebral disc degeneration. The objective of this study was to investigate the role of HIF-1alpha in disc degeneration in vivo using a conditional HIF-1alpha knockout (KO) mouse model. ShhCre transgenic mice were mated with HIF-1 alpha fl/fl mice to generate conditional HIF-1alpha KO mice (HIF-1alpha fl/fl-ShhCre+). Three mice of each genotype (Wide-type and HIF-1alpha KO) at the age of 3 days, 6, and 12 weeks were sacrificed after genotyping. Five lumbar disc samples were harvested from each mouse, with a total of 45 disc samples for each genotype. In situ hybridization and immunohistochemical analysis were used to check the efficacy of HIF-1alpha knockout. Histological grading of the disc degeneration was performed according to the classification system proposed by Boos et al. Picro-sirius red staining, Safranine O/fast green staining and immunohistochemical study were used to evaluate the expression of aggrecan, type-II collagen and vascular endothelial growth factor (VEGF). Histologic analysis revealed more NPC deaths and signs of degeneration in HIF-1alpha KO mice and the degeneration scores of HIF-1alpha KO mice were significantly higher than those of the Wide-type mice at the age of 6 weeks and 12 weeks. There were less expressions of aggrecan, type-II collagen and VEGF in the intervertebral discs of HIF1-alpha KO mice than in those of wild-type mice. Taken together, the results of our study indicated that HIF-1alpha is a pivotal contributor to NPC survival and the homeotasis of extracellular matrix through the HIF-1alpha/VEGF signaling pathway, and plays an important role in the development of disc degeneration.

Myeloid Hypoxia-Inducible Factor-1α Is Essential for Skeletal Muscle Regeneration in Mice

The outstanding regeneration ability of skeletal muscle is based on stem cells that become activated and develop to myoblasts after myotrauma. Proliferation and growth of myoblasts result in self-renewal of skeletal muscle. In this article, we show that myotrauma causes a hypoxic microenvironment leading to accumulation of the transcription factor hypoxia-inducible factor-1 (HIF-1) in skeletal muscle cells, as well as invading myeloid cells. To evaluate the impact of HIF-1 in skeletal muscle injury and repair, we examined mice with a conditional HIF-1α knockout targeted to skeletal muscle or myeloid cells in a model of soft tissue trauma. No differences in acute trauma size were detected between control and HIF-1α knockout mice. However, muscles of myeloid HIF-1α knockout mice showed a significant delay in myoblast proliferation and growth of regenerating myofibers, in association with decreased expression of cyclooxygenase-2 in HIF-1α-deficient myeloid cells. Moreover, the removal of necrotic cell debris and the regeneration of endothelial cell structure were impaired in myeloid HIF-1α knockout mice that showed delayed invasion of macrophages to the injury site. Our findings for the first time, to our knowledge, demonstrate that myeloid HIF-1α is required for adequate skeletal muscle regeneration.

Myeloid cell HIF-1α regulates asthma airway resistance and eosinophil function.

Hypoxia-inducible factor (HIF)-1α is a master regulator of inflammatory activities of myeloid cells, including neutrophils and macrophages. These studies examine the role of myeloid cell HIF-1α in regulating asthma induction and pathogenesis, and for the first time, evaluate the roles of HIF-1α and HIF-2α in the chemotactic properties of eosinophils, the myeloid cells most associated with asthma. Wild-type (WT) and myeloid cell-specific HIF-1α knockout (KO) C57BL/6 mice were studied in an ovalbumin (OVA) model of asthma. Administration of the pharmacological HIF-1α antagonist YC-1 was used to corroborate findings from the genetic model. WT, HIF-1α, and HIF-2α KO eosinophils underwent in vitro chemotaxis assays. We found that deletion of HIF-1α in myeloid cells and systemic treatment with YC-1 during asthma induction decreased airway hyperresponsiveness (AHR). Deletion of HIF-1α in myeloid cells in OVA-induced asthma also reduced eosinophil infiltration, goblet cell hyperplasia, and levels of cytokines IL-4, IL-5, and IL-13 in the lung. HIF-1α inhibition with YC-1 during asthma induction decreased eosinophilia in bronchoalveolar lavage, lung parenchyma, and blood, as well as decreased total lung inflammation, IL-5, and serum OVA-specific IgE levels. Deletion of HIF-1α in eosinophils decreased their chemotaxis, while deletion of the isoform HIF-2α led to increased chemotaxis. This work demonstrates that HIF-1α in myeloid cells plays a role in asthma pathogenesis, particularly in AHR development. Additionally, treatment with HIF-1α inhibitors during asthma induction decreases AHR and eosinophilia. Finally, we show that HIF-1α and HIF-2α regulate eosinophil migration in opposing ways.

Abstract P5-03-10: HIF-1alpha knockout radiosensitizes select Inflammatory Breast Cancer cells through reduction of stem-like cancer cells

Abstract Independent of the tumor stroma, HIF-1α regulates tumor cell metabolism, cell proliferation and DNA repair pathways, which may produce conflicting responses to radiation, promoting either radioresistance or radiosensitization. Recently, HIF-1α was shown to increase tumor initiating cells (TICs) activity in normoxic tumor cells derived from the MMTV-PyMT trasngenic mouse model of luminal-like breast cancer. While TICs have been reported to be relatively radio-resistant, the role of HIF-1α in mediating the radation response in breast cancer TICs remains unknown. Herein, we examined radiosenstitivity of TICs derived from either PyMT HIF-1α knock out (KO) tumor cells or human breast cancer cell lines transduced with a HIF-1α dominant negative (DN) construct. Consistent prior results that loss of HIF-1 activity reduces TIC frequency, down-regulation of HIF-1 activity through the HIF DN construct reduced mammosphere formation in SUM-159, SUM-149 and MCF-7 cells (by 5.0-, 2.0-, and 2.0-fold respectively). Moreover, β-catenin expression was down-regulated in SUM-149 and SUM-159 cells transfected with the HIF-1α DN construct. Moreover, SUM-149 cells expressing the HIF-1α DN construct exhibited delayed tumor growth in vivo (p = 0.05). Standard clonogenic assays demonstrated that PyMT HIF-1α KO cells and SUM-149 cells expressing the HIF-1α DN construct were more sensitive to radiation therapy (SF6 of PyMT, HIF-1α:Control, 0.016:0.087), but that the PyMT KO and SUM-149 mammospheres that persisted after radiation were completely radioresistant (SF6 of PyMT, HIF-1 α: Control, 0.48:0.44). In contrast, MCF-7 cells were not radiosensitized in either standard or mammosphere assays. Interestingly, in HIF-1α DN MCF-7 cells, molecular features of IBC were observed, such as the increased expression of E-Cadherin and loss of Wisp3. But, Notch1 protein expression was unchanged between HIF-1α DN MCF7 or SUM149 cells. Moreover, concurrent radiation in the presence of a gamma secretase inhibitor or with a p53-MDM2 inhibitor nutlin failed to radiosensitize HIF KO mammosphere clonogens. We conclude that downregulation of HIF-1 activity selectively radiosensitizes IBC clonogenic cells but fails to radiosensitize the residual mammospheres. These data suggest that the known HIF-1α mediated mechanisms that favor radiosensitivity, such as the promotion of glycolysis and proliferation under stress, may predominate in mammospheres, which ultimately leads to radioresistance in residual mammospheres after HIF inhibition. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-10.

Normal glucose uptake in the brain and heart requires an endothelial cell-specific HIF-1α–dependent function

Although intimately positioned between metabolic substrates in the bloodstream and the tissue parenchymal cells that require these substrates, a major role of the vascular endothelium in the regulation of tissue metabolism has not been widely appreciated. We hypothesized that via control of transendothelial glucose transport and contributing paracrine mechanisms the endothelium plays a major role in regulating organ and tissue glucose metabolism. We further hypothesized that the hypoxia-inducible factor -1α (HIF-1α) plays an important role in coordinating these endothelial functions. To test these hypotheses, we generated mice with endothelial cell-specific deletion of HIF-1α. Loss of HIF in the endothelium resulted in significantly increased fasting blood glucose levels, a blunted insulin response with delayed glucose clearance from the blood after i.v. loading, and significantly decreased glucose uptake into the brain and heart. Endothelial HIF-1α knockout mice also exhibited a reduced cerebrospinal fluid/blood glucose ratio, a finding consistent with reduced transendothelial glucose transport and a diagnostic criterion for the Glut1 deficiency genetic syndrome. Endothelial cells from these mice demonstrated decreased Glut1 levels and reduced glucose uptake that was reversed by forced expression of Glut1. These data strongly support an important role of the vascular endothelium in determining whole-organ glucose metabolism and indicate that HIF-1α is a critical mediator of this function.

Impacts of Hypoxia-Inducible Factor-1 Knockout in the Retinal Pigment Epithelium on Choroidal Neovascularization

Hypoxia-inducible factor (HIF)-1 is a key oxygen sensor and is believed to play an important role in neovascularization (NV). The purpose of this study is to determine the role of retinal pigment epithelium (RPE)-derived HIF-1α on ocular NV. Conditional HIF-1α knockout (KO) mice were generated by crossing transgenic mice expressing Cre in the RPE with HIF-1α floxed mice, confirmed by immunohistochemistry, Western blot analysis, and fundus fluorescein angiography. The mice were used for the oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) models. HIF-1α levels were significantly decreased in the RPE layer of ocular sections and in primary RPE cells from the HIF-1α KO mice. Under normal conditions, the HIF-1α KO mice exhibited no apparent abnormalities in retinal histology or visual function as shown by light microscopy and electroretinogram recording, respectively. The HIF-1α KO mice with OIR showed no significant difference from the wild-type (WT) mice in retinal levels of HIF-1α and VEGF as well as in the number of preretinal neovascular cells. In the laser-induced CNV model, however, the disruption of HIF-1α in the RPE attenuated the over expression of VEGF and the intercellular adhesion molecule 1 (ICAM-1), and reduced vascular leakage and CNV area. RPE-derived HIF-1α plays a key role in CNV, but not in ischemia-induced retinal NV.

HIF1A C1772T polymorphism leads to HIF-1α mRNA overexpression in prostate cancer patients

Hypoxia-inducible factor 1α (HIF-1α) gene polymorphisms have been investigated for a possible role in mediating genetic predisposition to cancer. Our previous data show that men homozygous to C1772T polymorphism had 4-fold risk to develop prostate cancer. Therefore, we studied the effects of C1772T polymorphism on HIF-1α expression. HIF-1α mRNA expression levels were significantly higher in peripheral blood leukocytes of prostate cancer patients with the TT genotype compared with the CC genotype. Expression of C1772T HIF-1α in HIF-1α knockout cancer cells showed higher expression levels and stabilization of HIF-1α mRNA compared with the wild-type. Mutated HIF-1α protein half-life was similar to that of the wild-type. Hence, our data provide evidence that C1772T polymorphism causes activation of HIF-1α as a gain-of-function mechanism driven by stabilization of HIF-1α mRNA. These findings may also explain the increased risk of men homozygous to this mutation to develop prostate cancer.

Transient Ureteral Obstruction Prevents against Kidney Ischemia/Reperfusion Injury via Hypoxia-Inducible Factor (HIF)-2α Activation

Although the protective effect of transient ureteral obstruction (UO) prior to ischemia on subsequent renal ischemia/reperfusion (I/R) injury has been documented, the underlying molecular mechanism remains to be understood. We showed in the current study that 24 h of UO led to renal tubular hypoxia in the ipsilateral kidney in mice, with the accumulation of hypoxia-inducible factor (HIF)-2α, which lasted for a week after the release of UO. To address the functions of HIF-2α in UO-mediated protection of renal IRI, we utilized the Mx-Cre/loxP recombination system to knock out target genes. Inactivation of HIF-2α, but not HIF-1α blunted the renal protective effects of UO, as demonstrated by much higher serum creatinine level and severer histological damage. UO failed to prevent postischemic neutrophil infiltration and apoptosis induction in HIF-2α knockout mice, which also diminished the postobstructive up-regulation of the protective molecule, heat shock protein (HSP)-27. The renal protective effects of UO were associated with the improvement of the postischemic recovery of intra-renal microvascular blood flow, which was also dependent on the activation of HIF-2α. Our results demonstrated that UO protected the kidney via activation of HIF-2α, which reduced tubular damages via preservation of adequate renal microvascular perfusion after ischemia. Thus, preconditional HIF-2α activation might serve as a novel therapeutic strategy for the treatment of ischemic acute renal failure.

Hypoxia-Inducible Factor Activation in Myeloid Cells Contributes to the Development of Liver Fibrosis in Cholestatic Mice

Macrophages play an integral role in the development of liver fibrosis by releasing mediators, such as platelet-derived growth factor-B (PDGF-B) and transforming growth factor-β1, which stimulate hepatic stellate cell proliferation, chemotaxis, and collagen production. However, the mechanism by which chronic liver injury stimulates macrophages to release these mediators is not completely understood. We tested the hypothesis that chronic liver injury activates hypoxia-inducible factor (HIF) transcription factors in macrophages that regulate the production of mediators that promote fibrosis. To test this hypothesis, Cre/lox technology was used to generate myeloid cell-specific HIF-1α or HIF-1β knockout mice. When these mice were subjected to bile duct ligation (BDL), levels of α-smooth muscle actin and type I collagen in the liver were reduced compared with those of mice with normal levels of HIFs. The deficiency of HIFs in macrophages did not affect liver injury or inflammation after BDL but reduced PDGF-B mRNA and protein, suggesting that HIF activation in macrophages may promote fibrosis by regulating the production of PDGF-B. Consistent with a role for HIFs in liver fibrosis in cholestatic liver disease, nuclear HIF-1α protein was present in macrophages, hepatocytes, and fibroblasts in the livers from patients with primary biliary cirrhosis and primary sclerosing cholangitis. These studies demonstrate that HIFs are important regulators of profibrotic mediator production by macrophages during the development of liver fibrosis and suggest that HIFs may be a novel therapeutic target for the treatment of chronic liver disease in patients.

Loss of epidermal hypoxia-inducible factor-1α accelerates epidermal aging and affects re-epithelialization in human and mouse

In mouse and human skin, HIF-1α is constitutively expressed in the epidermis, mainly in the basal layer. HIF-1α has been shown to have crucial systemic functions: regulation of kidney erythropoietin production in mice with constitutive HIF-1α epidermal deletion, and hypervascularity following epidermal HIF-1α overexpression. However, its local role in keratinocyte physiology has not been clearly defined. To address the function of HIF-1α in the epidermis, we used the mouse model of HIF-1α knockout targeted to keratinocytes (K14-Cre/Hif1a(flox/flox)). These mice had a delayed skin phenotype characterized by skin atrophy and pruritic inflammation, partly mediated by basement membrane disturbances involving laminin-332 (Ln-332) and integrins. We also investigated the relevance of results of studies in mice to human skin using reconstructed epidermis and showed that HIF-1α knockdown in human keratinocytes impairs the formation of a viable reconstructed epidermis. A diminution of keratinocyte growth potential, following HIF-1α silencing, was associated with a decreased expression of Ln-322 and α6 integrin and β1 integrin. Overall, these results indicate a role of HIF-1α in skin homeostasis especially during epidermal aging.

Abstract 8401: Switching of Hif-α Isoforms is Critical in the Resolution of Inflammation

Hypoxia is a pathological condition in which the tissue is deprived of adequate oxygen supply. It occurs in myocardial ischemia, cardiac hypertrophy and atherosclerosis, and accelerates the inflammatory processes in cardiac tissues. Each cell exerts its own responses to hypoxia, and most of them are mediated through a pair of transcription factors, hypoxia inducible factors- 1α (HIF- 1α) and HIF- 2α. Macrophages, key mediators of inflammation, are activated and accumulate in hypoxic area. Macrophages are currently classified into M1 (pro-inflammatory) and M2 (anti-inflammatory). However, the molecular mechanisms of M1 and M2-specific activations remain elusive. Here we revealed M1 and M2 specific functions of HIF-1α and HIF-2α, which play essential roles in the initiation and resolution of inflammation. HIF-1α was acutely induced in M1 activation, whereas HIF-2α was exclusively expressed in M2, but not in M1 macrophages. Th1 cytokines such as IFNγ induced HIF-1α, but strongly suppressed HIF-2α. In contrast, Th2 cytokine, including IL-4 increased HIF-2α level. We identified inducible NO synthase (iNOS, a M1 marker gene) as a HIF-1α target, and arginase1 (arg1, a M2 marker gene) as a HIF-2α target gene, respectively. These two enzymes compete for the common substrate, l-arginine, and inhibit each other's function. HIF-1α elicits NO production in M1, however, HIF-2α suppresses NO production through arginase1 induction in M2 macrophages. The balance between HIF-1α and HIF-2α determine that of iNOS/arg1, resulting in the regulation of NO production. To verify the roles of HIF-α in vivo, we generated macrophage specific HIF-1α knockout mice (mHIF-1α KO) and HIF-2α knockout (mHIF-2α KO). Intraperitoneal injection of lipopolysaccaride (LPS) induces plasma NO rises acutely (6hr), and it normalizes within 24 hours. In mHIF-1α KO mice, peak NO rise was attenuated. In contrast, mHIF-2α KO mice exhibited delayed recovery of NO level after LPS injection. Those results implied the essential role of HIF-1α in initiating NO production, whereas HIF-2α is critically important in the resolution. The switching of HIF-α isoforms could account for the functional diversity between M1 and M2, and contribute to the initiation and resolution of the inflammation.

Hypoxia-Inducible Factor (HIF)-1α Is a Therapeutic Target in Myeloma-Induced Angiogenesis

Abstract 3927Hypoxia-inducible factor (HIF)-1α is a critical trigger and regulator of tumor associated angiogenesis. It has been previously reported that bone marrow (BM) microenvironment is hypoxic in multiple myeloma (MM) patients and that HIF-1α is overexpressed by CD138+ MM cells and modulates the transcriptional and pro-angiogenic profiles of MM cells. The potential role of HIF-1α as a therapeutic target in MM is under investigation. To deepen this issue, in this study we explored the effect of a stable HIF-1α inhibition in MM cells on cell proliferation, survival and on MM-induced angiogenesis either in vitro or in vivo using a plasmocytoma model. Human myeloma cell lines (HMCL)s were screened for HIF-1α protein expression in normoxia condition finding that 2 out 5 HMCLs were positive (JJN3 and RPMI-8226). Anti-HIF-1α Lentivirus sh RNA pool was used for HIF-1α stable knock-out in human myeloma cell lines (HMCL)s whereas the pKLO.1 lentiviral vector was used as the empty control vector. HMCLs have been infected and selected with 0.4 ug/ml puromycin for 21 days and the selected clones screened for HIF-1α, HIF-1β, HIF-2α and HIF-3α. A selective inhibition on HIF-1α mRNA and protein expression was observed both in normoxic and hypoxic conditions. HMCLs proliferation and apoptosis were not affected by HIF-1α inhibition neither in hypoxia nor normoxia.Thereafter the transcriptional profiles on the HMCL JJN3 transduced with shRNA anti-HIF-1α (JJN3-anti-HIF-1α), as compared to those infected with the control vector pKLO.1 (JJN3-pKLO.1) have been analyzed by U133 Plus2.0 Arrays (Affymetrix ®) either in hypoxic or normoxic conditions. The raw intensity signals were extracted from CEL files and normalized using the RMA package for Bioconductor and custom GeneAnnot-based Chip Definition Files in R software. Absolute fold change higher than 1.5 was used to select differentially expressed genes. Among the genes significantly modulated (327 and 361 genes in hypoxic and normoxic condition, respectively) we found that the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, MMP9 were down-regulated in JJN3-anti-HIF-1α as compared to JJN3-pKLO.1 either in hypoxic or normoxic conditions. Microarray data were further validated by quantitative real time PCR. In line with this observation, we found that the pro-angiogenic properties of JJN3, evaluated by an in vitro angiogenesis assay (Angio-Kit, TCS), were significantly inhibited by the HIF-1α knock-out. Finally the potential involvement of HIF-1α in MM-induced angiogenesis was assessed in vivo in NOD/SCID mice in a subcutaneous model. We found that the mice which were injected subcutaneously with JJN3-anti-HIF-1α, showed a dramatic reduction of the weight and volume of the plasmocytoma mass than mice inoculated with the JJN3-pKLO.1 (P=0.0007 and P=0.012; respectively). Moreover we observed a significant reduction of the number of vessels X field stained by CD31 antigen (anti-HIF-1α vs. pKLO.1: −76%; P=0.003) and VEGF immunostaining, whereas the Ki67 index of the plasmocytoma mass only showed a slight reduction (anti-HIF-1α vs. pKLO.1: −14%, P=NS).Overall our data indicate that HIF-1α suppression in myeloma cells significantly blocks MM-induced angiogenic switch both in vitro and in vivo and consequently reduces the MM tumoral burden suggesting that HIF-1α is a potential therapeutic target in MM. Disclosures:Bolzoni:Celgene Italy: Research Funding. Giuliani:Celgene: Research Funding; Novartis: Research Funding.

Disruption of Hypoxia-Inducible Factor 1 in Adipocytes Improves Insulin Sensitivity and Decreases Adiposity in High-Fat Diet–Fed Mice

OBJECTIVEObesity, insulin resistance, and type 2 diabetes form a tightly correlated cluster of metabolic disorders in which adipose is one of the first affected tissues. The role of hypoxia and hypoxia-inducible factor 1 (HIF1) in the development of high-fat diet (HFD)–induced obesity and insulin resistance was investigated using animal models.RESEARCH DESIGN AND METHODSMice with adipocyte-specific targeted disruption of the genes encoding the HIF1 obligatory subunits Hif1α or Arnt (Hif1β) were generated using an aP2-Cre transgene with the Cre/LoxP system. The mice were fed an HFD for 12 weeks and their metabolic phenotypes were determined. Gene expression patterns in adipose tissues were also determined by microarray and quantitative PCR.RESULTSOn an HFD, adipocyte-specific ARNT knockout mice and adipocyte-specific HIF1α knockout mice exhibit similar metabolic phenotypes, including reduced fat formation, protection from HFD-induced obesity, and insulin resistance compared with similarly fed wild-type controls. The cumulative food intake remained similar; however, the metabolic efficiency was lower in adipocyte-specific HIF1α knockout mice. Moreover, indirect calorimetry revealed respiratory exchange ratios were reduced in adipocyte-specific HIF1α knockout mice. Hyperinsulinemic-euglycemic clamp studies demonstrated that targeted disruption of HIF1α in adipocytes enhanced whole-body insulin sensitivity. The improvement of insulin resistance is associated with decreased expression of Socs3 and induction of adiponectin.CONCLUSIONSInhibition of HIF1 in adipose tissue ameliorates obesity and insulin resistance. This study reveals that HIF1 could provide a novel potential therapeutic target for obesity and type 2 diabetes.

Ischaemia-induced retinal neovascularisation and diabetic retinopathy in mice with conditional knockout of hypoxia-inducible factor-1 in retinal Müller cells

Retinal Müller cells are known to produce inflammatory and angiogenic cytokines, which play important roles in diabetic retinopathy. Hypoxia-inducible factor (HIF)-1 has been shown to play a crucial role in retinal inflammation and neovascularisation. We sought to determine the role of Müller cell-derived HIF-1 in oxygen-induced retinopathy (OIR) and diabetic retinopathy using conditional Hif-1α (also known as Hif1a) knockout (KO) mice. Conditional Hif-1α KO mice were generated by crossing mice expressing cyclisation recombinase (cre, also known as P1_gp003) in Müller cells with floxed Hif-1α mice and used for OIR and streptozotocin-induced diabetes to induce retinal neovascularisation and inflammation, respectively. Abundance of HIF-1α and pro-angiogenic and pro-inflammatory factors was measured by immunoblotting and immunohistochemistry. Retinal neovascularisation was visualised by angiography and quantified by counting pre-retinal nuclei. Retinal inflammation was evaluated by leucostasis and vascular leakage. While the Hif-1α KO mice showed significantly decreased HIF-1α levels in the retina, they exhibited no apparent histological or visual functional abnormalities under normal conditions. Compared with wild-type counterparts, Hif-1α KO mice with OIR demonstrated attenuated overproduction of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1, reduced vascular leakage and alleviated neovascularisation in the retina. Under diabetes conditions, disruption of Hif-1α in Müller cells attenuated the increases of retinal vascular leakage and adherent leucocytes, as well as the overproduction of VEGF and ICAM-1. Müller cell-derived HIF-1α is a key mediator of retinal neovascularisation, vascular leakage and inflammation, the major pathological changes in diabetic retinopathy. Müller cell-derived HIF-1α is therefore a promising therapeutic target for diabetic retinopathy.

PGC-1α regulates a HIF2α-dependent switch in skeletal muscle fiber types

The coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) coordinates a broad set of transcriptional programs that regulate the response of skeletal muscle to exercise. However, the complete transcriptional network controlled by PGC-1α has not been described. In this study, we used a qPCR-based screen of all known transcriptional components (Quanttrx) to identify transcription factors that are quantitatively regulated by PGC-1α in cultured skeletal muscle cells. This analysis identified hypoxia-inducible factor 2 α (HIF2α) as a major PGC-1α target in skeletal muscle that is positively regulated by both exercise and β-adrenergic signaling. This transcriptional regulation of HIF2α is completely dependent on the PGC-1α/ERRα complex and is further modulated by the action of SIRT1. Transcriptional profiling of HIF2α target genes in primary myotubes suggested an unexpected role for HIF2α in the regulation of muscle fiber types, specifically enhancing the expression of a slow twitch gene program. The PGC-1α-mediated switch to slow, oxidative fibers in vitro is dependent on HIF2α, and mice with a muscle-specific knockout of HIF2α increase the expression of genes and proteins characteristic of a fast-twitch fiber-type switch. These data indicate that HIF2α acts downstream of PGC-1α as a key regulator of a muscle fiber-type program and the adaptive response to exercise.

Targeted Knockdown of SEPT9_v1 Inhibits Tumor Growth and Angiogenesis of Human Prostate Cancer Cells Concomitant with Disruption of Hypoxia-Inducible Factor-1 Pathway

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor in the hypoxic response pathway. We recently identified a novel interaction between HIF-1alpha and the mammalian septin family member, septin 9 protein, isoform 1 (SEPT9_i1), a protein product of septin 9 transcript variant 1 (SEPT9_v1). Septins are a highly conserved family of GTP-binding cytoskeletal proteins that are implicated in multiple cellular functions, including oncogenesis. SEPT9_i1 binds and stabilizes HIF-1alpha protein and stimulates HIF-1 transcriptional activity by preventing its RACK1-mediated ubiquitination and degradation. SEPT9_i1-HIF-1 activation promotes tumor growth and angiogenesis. The effect of SEPT9_v1 silencing in prostate cancer cells was studied. SEPT9_v1 stable knockdown was generated in PC-3 cells using a specific shRNA. SEPT9_v1 silencing reduced HIF-1alpha protein expression and inhibited HIF-1 transcriptional activity. SEPT9_v1 knockdown affected cell morphology, deregulated cell cycle, and decreased migration. The antiproliferative effect of shSEPT9_v1 was abolished in HIF-1alpha knockout colon cancer cells. In vivo, SEPT9_i1 depletion reduced HIF-1alpha protein expression, cellular proliferation, tumor growth, and angiogenesis. These results provide new insights and validation for applying SEPT9_v1 as a potential target for antitumor therapy by interrupting the HIF-1 pathway.

HIF-1α Promotes A Hypoxia-Independent Cell Migration.

Hypoxia-inducible factor-1α (HIF-1α) is known as a transactivator for VEGF gene promoter. It can be induced by hypoxia. However, no study has been done so far to dissect HIF-1α-mediated effects from hypoxia or VEGF-mediated effects. By using a HIF-1α knockout (HIF-1α KO) cell system in mouse embryonic fibroblast (MEF) cells, this study analyzes cell migration and HIF-1α, hypoxia and VEGF activation. A hypoxia-mediated HIF-1α induction and VEGF transactivation were observed: both HIF-1α WT lines had significantly increased VEGF transactivation, as an indicator for HIF-1α induction, in hypoxia compared to normoxia; in contrast, HIF-1α KO line had no increased VEGF transactivation under hypoxia. HIF-1α promotes cell migration: HIF-1α-KO cells had a significantly reduced migration compared to that of the HIF-1α WT cells under both normoxia and hypoxia. The significantly reduced cell migration in HIF-1α KO cells can be partially rescued by the restoration of WT HIF-1α expression mediated by adenoviral-mediated gene transfer. Interestingly, hypoxia has no effect on cell migration: the cells had a similar cell migration rate under hypoxic and normoxic conditions for both HIF-1α WT and HIF-1α KO lines, respectively. Collectively, these data suggest that HIF-1α plays a role in MEF cell migration that is independent from hypoxia-mediated effects.

Identification of the KAI1 metastasis suppressor gene as a hypoxia target gene

KAI1 is a metastasis suppressor gene known to inhibit cancer metastasis without affecting primary tumorigenicity. Although KAI1 expression has been reported to undergo transcriptional regulation, how its expression is up- or down-regulated by specific upstream signaling pathways has not been studied in detail. In this study, we characterized the regulatory elements within the 500bp upstream region of mouse KAI1 gene and identified a functional hypoxia-response element (HRE) within the promoter region. Hypoxia-dependent induction of KAI1 was directly mediated by hypoxia-inducible factor (HIF)-1α binding on the promoter, which subsequently caused increased recruitment of RNA polymerase II for transcriptional activation. The failure of HIF-1α recruitment to the KAI1 promoter was observed in Hif-1α knockout mouse embryonic fibroblasts. Furthermore, KAI1 protein synthesis was markedly increased in ischemic tissues, suggesting that KAI1 is a hypoxia target gene in vivo.

HIF-1α Promotes A Hypoxia-Independent Cell Migration

Hypoxia-inducible factor-1α (HIF-1α) is known as a transactivator for VEGF gene promoter. It can be induced by hypoxia. However, no study has been done so far to dissect HIF-1α-mediated effects from hypoxia or VEGF-mediated effects. By using a HIF-1α knockout (HIF-1α KO) cell system in mouse embryonic fibroblast (MEF) cells, this study analyzes cell migration and HIF-1α, hypoxia and VEGF activation. A hypoxia-mediated HIF-1α induction and VEGF transactivation were observed: both HIF-1α WT lines had significantly increased VEGF transactivation, as an indicator for HIF-1α induction, in hypoxia compared to normoxia; in contrast, HIF-1α KO line had no increased VEGF transactivation under hypoxia. HIF-1α promotes cell migration: HIF-1α-KO cells had a significantly reduced migration compared to that of the HIF-1α WT cells under both normoxia and hypoxia. The significantly reduced cell migration in HIF-1α KO cells can be partially rescued by the restoration of WT HIF-1α expression mediated by adenoviral-mediated gene transfer. Interestingly, hypoxia has no effect on cell migration: the cells had a similar cell migration rate under hypoxic and normoxic conditions for both HIF-1α WT and HIF-1α KO lines, respectively. Collectively, these data suggest that HIF-1α plays a role in MEF cell migration that is independent from hypoxia-mediated effects.