Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that regulates cellular responses to low oxygen environments. HIF-1 is composed of two subunits: hypoxia-inducible HIF-1α and constitutively-expressed HIF-1β. During hypoxic conditions, HIF-1α heterodimerizes with HIF-1β and translocates to the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes implicated in cell growth and survival. HIF-1α protein expression is elevated in many solid tumors, including those of the cervix and brain, where cells that are the greatest distance from blood vessels, and therefore the most hypoxic, express the highest levels of HIF-1α. Therapeutic blockade of the HIF-1 signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer drugs. To identify small molecule inhibitors of the HIF-1 pathway, we have developed a cell-based reporter gene assay and screened a large compound library by using a quantitative high-throughput screening (qHTS) approach.
The assay is based upon a β-lactamase reporter under the control of a HRE. We have screened approximate 73,000 compounds by qHTS, with each compound tested over a range of seven to fifteen concentrations. After qHTS we have rapidly identified three novel structural series of HIF-1 pathway Inhibitors. Selected compounds in these series were also confirmed as inhibitors in a HRE β-lactamase reporter gene assay induced by low oxygen and in a VEGF secretion assay. Three of the four selected compounds tested showed significant inhibition of hypoxia-induced HIF-1α accumulation by western blot analysis.
The use of β-lactamase reporter gene assays, in combination with qHTS, enabled the rapid identification and prioritization of inhibitors specific to the hypoxia induced signaling pathway.
The importance of hypoxia-inducible factor (HIF) in promoting angiogenesis and vasculogenesis during wound healing has been demonstrated. It is widely accepted that HIF activity can be promoted by many factors, including hypoxia in the wound or cytokines from inflammatory cells infiltrating the wound. However, there has not been a systematic exploration of the relationship between HIF activity and hypoxia in the burn wound. The location of the hypoxic tissue has not been clearly delineated. The time course of the appearance of hypoxia and the increased activity of HIF and appearance of HIF’s downstream transcription products has not been described. The aim of this study was to utilize pimonidazole, a specific tissue hypoxia marker, to characterize the spatial and temporal course of hypoxia in a murine burn model and correlate this with the appearance of HIF-1α and its important angiogenic and vasculogenic transcription products VEGF and SDF-1. Hypoxia was found in the healing margin of burn wounds beginning at 48 hours after burn and peaking at day 3 after burn. On sequential sections of the same tissue block, positive staining of HIF-1α, SDF-1, and VEGF all occurred at the leading margin of the healing area and peaked at day 3, as did hypoxia. Immunohistochemical analysis was used to explore the characteristics of the hypoxic region of the wound. The localization of hypoxia was found to be related to cell growth and migration, but not to proliferation or inflammatory infiltration.
ypoxia; Hypoxia-inducible factor -10α; Burn; Wound Healing; Ki67 Cell Proliferation; Keratin17
Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a
critical mediator of the cellular response to hypoxia. Enhanced levels of
HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated
with increased tumour angiogenesis, metastasis, therapeutic resistance and
poor prognosis. It is in this context that we previously demonstrated that
under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth
Factor (VEGF)-mediated tumour angiogenesis.
By using human melanoma cell lines and their stable or transient derivative
bcl-2 overexpressing cells, the current study identified HIF-1α
protein stabilization as a key regulator for the induction of HIF-1 by bcl-2
under hypoxia. We also demonstrated that bcl-2-induced accumulation of
HIF-1α protein during hypoxia was not due to an increased gene
transcription or protein synthesis. In fact, it was related to a modulation
of HIF-1α protein expression at a post-translational level, indeed
its degradation rate was faster in the control lines than in bcl-2
transfectants. The bcl-2-induced HIF-1α stabilization in response to
low oxygen tension conditions was achieved through the impairment of
ubiquitin-dependent HIF-1α degradation involving the molecular
chaperone HSP90, but it was not dependent on the prolyl hydroxylation of
HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90
proteins form a tri-complex that may contribute to enhancing the stability
of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic
conditions. Finally, by using genetic and pharmacological approaches we
proved that HSP90 is involved in bcl-2-dependent stabilization of
HIF-1α protein during hypoxia, and in particular the isoform
HSP90β is the main player in this phenomenon.
We identified the stabilization of HIF-1α protein as a mechanism
through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells
involving the β isoform of molecular chaperone HSP90.
Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron- and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1α and HIF-2α. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1α and up-regulate HIF-dependent target genes (e.g. enolase, p21waf1/cip1, vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress.
The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important molecular target for anticancer drug discovery. In a T47D cell-based reporter assay, the Caulerpa spp. algal pigment caulerpin (1) inhibited hypoxia-induced as well as 1,10-phenanthroline-induced HIF-1 activation. The angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF-1. Caulerpin (10 μM) suppressed hypoxic induction of secreted VEGF protein and the ability of hypoxic T47D cell-conditioned media to promote tumor angiogenesis in vitro. Under hypoxic conditions, 1 (10 μM) blocked the induction of HIF-1α protein, the oxygen-regulated subunit that controls HIF-1 activity. Reactive oxygen species produced by mitochondrial complex III are believed to act as a signal of cellular hypoxia that leads to HIF-1α protein induction and activation. Further mechanistic studies revealed that 1 inhibits mitochondrial respiration at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Under hypoxic conditions, it is proposed that 1 may disrupt mitochondrial ROS-regulated HIF-1 activation and HIF-1 downstream target gene expression by inhibiting the transport or delivery of electrons to complex III.
Cellular metabolism depends on the availability of oxygen and the major regulator of oxygen homeostasis is hypoxia-inducible factor 1 (HIF-1), a highly conserved transcription factor that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimeric transcription factor composed of hypoxia-inducible HIF-1α and constitutively expressed HIF-1β. Under hypoxic conditions, the two subunits dimerize, allowing translocation of the HIF-1 complex to the nucleus where it binds to hypoxia-response elements (HREs) and activates expression of target genes implicated in angiogenesis, cell growth, and survival. The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia. Thus, there is considerable interest in identifying compounds that modulate the HIF-1 signaling pathway. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, we screened a National Toxicology Program collection of 1408 compounds using a cell-based β-lactamase HRE reporter gene assay in a quantitative high-throughput screening (qHTS) format. Twelve active compounds were identified. These compounds were tested in a confirmatory assay for induction of vascular endothelial growth factor, a known hypoxia target gene, and confirmed compounds were further tested for their ability to mimic the effect of a reduced-oxygen environment on hypoxia-regulated promoter activity. Based on this testing strategy, three compounds (o-phenanthroline, iodochlorohydroxyquinoline, cobalt sulfate heptahydrate) were confirmed as hypoxia mimetics, whereas two compounds (7-diethylamino-4-methylcoumarin and 7,12-dimethylbenz(a)anthracence) were found to interact with HIF-1 in a manner different from hypoxia. These results demonstrate the effectiveness of qHTS in combination with secondary assays for identification of HIF-1α inducers and for distinguishing among inducers based on their pattern of activated hypoxic target genes. Identification of environmental compounds having HIF-1α activation activity in cell-based assays may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.
cobalt sulfate heptahydrate; 7-diethylamino-4-methylcoumarin; 7,12-dimethylbenz(a)anthracence; HIF-1α; inducers; iodochlorohydroxyquinoline; NTP 1408 compound library; o-phenanthroline; qHTS
Angiogenesis and bone formation are intimately related processes. Hypoxia during early bone development stabilizes hypoxia-inducible factor-1α (HIF-1α) and increases angiogenic signals including vascular endothelial growth factor (VEGF). Furthermore, stabilization of HIF-1α by genetic or chemical means stimulates bone formation. On the other hand, deficiency of Runx2, a key osteogenic transcription factor, prevents vascular invasion of bone and VEGF expression. This study explores the possibility that HIF-1α and Runx2 interact to activate angiogenic signals. Runx2 over-expression in mesenchymal cells increased VEGF mRNA and protein under both normoxic and hypoxic conditions. In normoxia, Runx2 also dramatically increased HIF-1α protein. In all cases, the Runx2 response was inhibited by siRNA-mediated suppression of HIF-1α and completely blocked by the HIF-1α inhibitor, echinomycin. Similarly, treatment of preosteoblast cells with Runx2 siRNA reduced VEGF mRNA in normoxia or hypoxia. However, Runx2 is not essential for the HIF-1α response since VEGF is induced by hypoxia even in Runx2-null cells. Endogenous Runx2 and HIF-1α were colocalized to the nuclei of MC3T3-E1 preosteoblast cells. Moreover, HIF-1α and Runx2 physically interact using sites within the Runx2 RUNT domain. Chromatin immunoprecipitation also provided evidence for colocalization of Runx2 and HIF-1α on the VEGF promoter. In addition, Runx2 stimulated HIF-1α-dependent activation of an HRE-luciferase reporter gene without requiring a separate Runx2-binding enhancer. These studies indicate that Runx2 functions together with HIF-1α to stimulate angiogenic gene expression in bone cells and may in part explain the known requirement for Runx2 in bone vascularization.
Osteoblast; vascularization; angiogenesis; transcriptional factors; hypoxia
The mechanisms that regulate angiogenesis in hypoxia or hypoxic microenvironment are modulated by several pro- and antiangiogenic factors. Hypoxia-inducible factors (HIFs) have been established as the basic and major inducers of angiogenesis, but understanding the role of interacting proteins is becoming increasingly important to elucidate the angiogenic processes of a hypoxic response. In particular, with regard to wound healing and the novel therapies for vascular disorders such as ischemic brain and heart attack, it is essential to gain insights in the formation and regulation of HIF transcriptional machineries related to angiogenesis. Further, identification of alternative ways of inhibiting tumor growth by disrupting the growth-triggering mechanisms of increasing vascular supply via angiogenesis depends on the knowledge of how tumor cells develop their own vasculature. Here, we review our findings on the interactions of basic HIFs, HIF-1α and HIF-2α, with their regulatory binding proteins, histone deacetylase 7 (HDAC7) and translation initiation factor 6 (Int6), respectively. The present results and discussion revealed new regulatory interactions of HIF-related mechanisms.
angiogenic proteins; anoxia; eukaryotic initiation factors; histone deacetylases; hypoxia-ischemia, brain; neovascularization, pathologic
Angiogenesis is an essential process during follicular development and corpora lutea (CL) formation. Recent studies have shown that vascular endothelial growth factor (VEGF) is an essential regulator of ovarian angiogenesis. Several lines of evidence have indicated that the production of VEGF is regulated by hypoxia inducible factor-1α (HIF-1α), especially under hypoxic conditions, but the expression of HIF-1α has not been well characterized in the porcine ovary. The present study examined the expression of HIF-1α mRNA and its localization in porcine ovaries at different stages of the estrous cycle. Northern blot analyses of total CL RNA indicated hybridization of the porcine HIF-1α probe to transcripts of approximately 3.8 kb. The mRNA expression of HIF-1α was highest in CL during the early luteal phase, followed by a decrease during the mid- and late-luteal phases. Using in situ hybridization, abundant HIF-1α mRNA was evident in follicles and CL. Within non-atretic follicles, HIF-1α mRNA was highly expressed in the granulosa cell layer, while weaker labeling was evident in the theca interna. These results suggest that HIF-1α may play a role in the regulation of cellular metabolism and blood supply during follicular growth and CL formation.
Hypoxia, which occurs in the brain when oxygen availability drops below the normal level, is a major cause of perinatal hypoxic-ischemic injury (HII). The transcriptional factor hypoxia inducible factor-1 (HIF-1) is a key regulator in the pathophysiological response to the stress of hypoxia. Genes regulated by HIF-1 are involved in energy metabolism, erythropoiesis, angiogenesis, vasodilatation, cell survival and apoptosis. Compared with the adult brain, the neonatal brain is different in physiological structure, function, cellular composition and signaling pathways related gene activation and response after hypoxia. The purpose of this review is to determine if developmental susceptibility of the brain after hypoxic/ischemic injury is related to HIF-1a, which also plays a pivotal role in the normal brain development. HIF-1a regulates both prosurvival and prodeath responses in the neonatal brain and various mechanisms underlie the apparent contradictory effects, including duration of ischemic injury and severity, cell-types, and/or dependent on the nature of the stimulus after HII. Studies report an excessive induction of HIF-1 in the immature brain, which suggests that a cell death promoting role of HIF may prevail. Inhibition of HIF-1a and targeted activation of its prosurvival genes appear as a favorable therapeutic strategy. However, a better understanding of multifaceted HIF-1 function during brain development is required to explore potential targets for further therapeutic interventions in the neonate.
Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in driving tumor growth is receiving increased attention. Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular responses to hypoxia. It is a heterodimeric transcription factor composed of α and β subunits. The α subunit is stable in hypoxic conditions but is rapidly degraded in normoxia. The function of HIF-1 is also modulated by several molecular mechanisms that regulate its synthesis, degradation, and transcriptional activity. Upon stabilization or activation, HIF-1 translocates to the nucleus and induces transcription of its downstream target genes. Most important to gliomagenesis, HIF-1 is a potent activator of angiogenesis and invasion through its upregulation of target genes critical for these functions. Activation of the HIF-1 pathway is a common feature of gliomas and may explain the intense vascular hyperplasia often seen in glioblastoma multiforme. Activation of HIF results in the activation of vascular endothelial growth factors, vascular endothelial growth factor receptors, matrix metalloproteinases, plasminogen activator inhibitor, transforming growth factors α and β, angiopoietin and Tie receptors, endothelin-1, inducible nitric oxide synthase, adrenomedullin, and erythropoietin, which all affect glioma angiogenesis. In conclusion, HIF is a critical regulatory factor in the tumor microenvironment because of its central role in promoting proangiogenic and invasive properties. While HIF activation strongly promotes angiogenesis, the emerging vasculature is often abnormal, leading to a vicious cycle that causes further hypoxia and HIF upregulation.
Transcription factor hypoxia-inducible factor (HIF)-1 protein accumulates and activates the transcription of genes that are of fundamental importance for oxygen homeostasis – including genes involved in energy metabolism, angiogenesis, vasomotor control, apoptosis, proliferation, and matrix production – under hypoxic conditions. We speculated that HIF-1α may have an important role in chondrocyte viability as a cell survival factor during the progression of osteoarthritis (OA). The expression of HIF-1α mRNA in human OA cartilage samples was analyzed by real-time PCR. We analyzed whether or not the catabolic factors IL-1β and H2O2 induce the expression of HIF-1α in OA chondrocytes under normoxic and hypoxic conditions (O2 <6%). We investigated the levels of energy generation, cartilage matrix production, and apoptosis induction in HIF-1α-deficient chondrocytes under normoxic and hypoxic conditions. In articular cartilages from human OA patients, the expression of HIF-1α mRNA was higher in the degenerated regions than in the intact regions. Both IL-1β and H2O2 accelerated mRNA and protein levels of HIF-1α in cultured chondrocytes. Inhibitors for phosphatidylinositol 3-kinase and p38 kinase caused a significant decrease in catabolic-factor-induced HIF-1α expression. HIF-1α-deficient chondrocytes did not maintain energy generation and cartilage matrix production under both normoxic and hypoxic conditions. Also, HIF-1α-deficient chondrocytes showed an acceleration of catabolic stress-induced apoptosis in vitro. Our findings in human OA cartilage show that HIF-1α expression in OA cartilage is associated with the progression of articular cartilage degeneration. Catabolic-stresses, IL-1β, and oxidative stress induce the expression of HIF-1α in chondrocytes. Our results suggest an important role of stress-induced HIF-1α in the maintenance of chondrocyte viability in OA articular cartilage.
The von Hippel-Lindau tumor suppressor pVHL regulates the stability of Hypoxia-Inducible Factors (HIF) -1 and –2, oxygen-sensitive basic helix-loop-helix transcription factors, which mediate the hypoxic induction of angiogenic growth factors such as vascular endothelial growth factor (VEGF). Loss of VHL function results in constitutive activation of HIF-1 and HIF-2 and is associated with the development of highly vascularized tumors in multiple organs. We have used a conditional gene targeting approach to investigate the relative contributions of HIF-1 and HIF-2 to VHL-associated vascular tumorigenesis in a mouse model of liver hemangiomas. Here we demonstrate genetically that conditional inactivation of HIF-2α suppressed the development of VHL-associated liver hemangiomas and that angiogenic gene expression in hepatocytes is predominantly regulated by HIF-2 and not by HIF-1. These findings suggest that HIF-2 is the dominant HIF in the pathogenesis of VHL-associated vascular tumors and that pharmacologic targeting of HIF-2 may be an effective strategy for their treatment.
Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator composed of HIF-1α and HIF-1β subunits. Several dozen HIF-1 targets are known, including the gene encoding vascular endothelial growth factor (VEGF). Under hypoxic conditions, HIF-1α expression increases as a result of decreased ubiquitination and degradation. The tumor suppressors VHL (von Hippel-Lindau protein) and p53 target HIF-1α for ubiquitination such that their inactivation in tumor cells increases the half-life of HIF-1α. Increased phosphatidylinositol 3-kinase (PI3K) and AKT or decreased PTEN activity in prostate cancer cells also increases HIF-1α expression by an undefined mechanism. In breast cancer, increased activity of the HER2 (also known as neu) receptor tyrosine kinase is associated with increased tumor grade, chemotherapy resistance, and decreased patient survival. HER2 has also been implicated as an inducer of VEGF expression. Here we demonstrate that HER2 signaling induced by overexpression in mouse 3T3 cells or heregulin stimulation of human MCF-7 breast cancer cells results in increased HIF-1α protein and VEGF mRNA expression that is dependent upon activity of PI3K, AKT (also known as protein kinase B), and the downstream kinase FRAP (FKBP-rapamycin-associated protein). In contrast to other inducers of HIF-1 expression, heregulin stimulation does not affect the half-life of HIF-1α but instead stimulates HIF-1α synthesis in a rapamycin-dependent manner. The 5′-untranslated region of HIF-1α mRNA directs heregulin-inducible expression of a heterologous protein. These data provide a molecular basis for VEGF induction and tumor angiogenesis by heregulin-HER2 signaling and establish a novel mechanism for the regulation of HIF-1α expression.
During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1α (HIF-1α), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B, and plasminogen activator inhibitor-1 (PAI-1) in the liver during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1α in liver cell types. Accordingly, the hypothesis was tested that HIF-1α is activated in hypoxic hepatocytes and regulates production of profibrotic mediators by these cells.
In this study, hepatocytes were isolated from the livers of control and HIF-1α or HIF-1β-Deficient mice and exposed to hypoxia.
Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1α and upregulated PAI-1, vascular endothelial cell growth factor, and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1α-Deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2α, may also regulate these genes. In support of this, HIF-2α was activated in hypoxic hepatocytes, and exposure of HIF-1β-Deficient hepatocytes to 1% oxygen completely prevented upregulation PAI-1, VEGF, and ADM-1, suggesting that HIF-2α may also contribute to upregulation of these genes in hypoxic hepatocytes.
Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes.
Hypoxia-inducible factor; hepatocytes; liver fibrosis
Angiogenesis is essential for promoting growth and metastasis of solid tumors by ensuring blood supply to the tumor mass. Targeting angiogenesis is therefore an attractive approach to therapeutic intervention of cancer. Tumor angiogenesis is a process that is controlled by a complex network of molecular components including sensors, signaling transducers, and effectors, leading to cellular responses under hypoxic conditions. Positioned at the center of this network are the hypoxia-inducible factors (HIFs). HIF-1 is a major transcription factor that consists of two subunits, HIF-1α and HIF-1β. It mediates transcription of a spectrum of gene targets whose products are essential for mounting hypoxic responses. HIF-1α protein level is very low in the normoxic condition but is rapidly elevated under hypoxia. This dramatic change in the cellular HIF-1α level is primarily regulated through the proteosome-mediated degradation process. In the past few years, scientific progress has clearly demonstrated that HIF-1α phosphorylation is mediated by several families of protein kinases including GSK3β and ERKs both of which play crucial roles in the regulation of HIF-1α stability. Recent research progress has identified that Polo-like kinase 3 (Plk3) phosphorylates HIF-1α at two previously unidentified serine residues and that the Plk3-mediated phosphorylation of these residues results in destabilization of HIF-1α. Plk3 has also recently been found to phosphorylate and stabilize PTEN phosphatase, a known regulator of HIF-1α and tumor angiogenesis. Given the success of targeting protein kinases and tumor angiogenesis in anti-cancer therapies, Plk3 could be a potential molecular target for the development of novel and effective therapeutic agents for cancer treatment.
Plk3; Tumor angiogenesis; Tumor suppression; HIF-1α; PTEN
Hypoxia-inducible factor 1α (HIF-1α) and HIF-2α display unique and sometimes opposing activities in regulating cellular energy homeostasis, cell fate decisions, and oncogenesis. Macrophages exposed to hypoxia accumulate both HIF-1α and HIF-2α, and overexpression of HIF-2α in tumor-associated macrophages (TAMs) is specifically correlated with high-grade human tumors and poor prognosis. However, the precise role of HIF-2α during macrophage-mediated inflammatory responses remains unclear. To fully characterize cellular hypoxic adaptations, distinct functions of HIF-1α versus HIF-2α must be elucidated. We demonstrate here that mice lacking HIF-2α in myeloid cells (Hif2aΔ/Δ mice) are resistant to lipopolysaccharide-induced endotoxemia and display a marked inability to mount inflammatory responses to cutaneous and peritoneal irritants. Furthermore, HIF-2α directly regulated proinflammatory cytokine/chemokine expression in macrophages activated in vitro. Hif2aΔ/Δ mice displayed reduced TAM infiltration in independent murine hepatocellular and colitis-associated colon carcinoma models, and this was associated with reduced tumor cell proliferation and progression. Notably, HIF-2α modulated macrophage migration by regulating the expression of the cytokine receptor M-CSFR and the chemokine receptor CXCR4, without altering intracellular ATP levels. Collectively, our data identify HIF-2α as an important regulator of innate immunity, suggesting it may be a useful therapeutic target for treating inflammatory disorders and cancer.
Hypoxia-inducible factors (HIFs) are transcription factors that activate the transcription of target genes involved in crucial aspects of cancer development. This study investigated the expression of HIFs and their contribution to the regulation of target genes related to angiogenesis and glucose metabolism in gastric cancer. The data showed that HIFs were over-expressed in gastric cancer and that activation of the target genes was observed mainly in the early stages. Moreover, the results of the present study revealed that only HIF-1α, but not HIF-2α dimerizes with HIF-1β and then regulates expression of target genes in response to hypoxia. The results of the present study demonstrate that HIF-1α and HIF-1β enhances expression of VEGF and glucose metabolism-related genes in response to hypoxia in gastric cancer. These data offer important information regarding HIF pathways in the development of gastric cancer.
glucose metabolism disorders; hypoxia-inducible factor 1; neovascularization, pathologic; stomach neoplasms; vascular endothelial growth factor A
Evidence suggests that the activation of the transcription factor hypoxia-inducible factor 1α (HIF-1α) may promote cell survival in hypoxic or ischemic brain. To help understand the role of HIF-1α in neonatal hypoxic-ischemic brain injury, mice with conditional neuron-specific inactivation of HIF-1α underwent hypoxia-ischemia (HI). Mice heterozygous for Cre recombinase under the control of the calcium/calmodulin-dependent kinase II promoter were bred with homozygous ‘floxed’ HIF-1α transgenic mice. The resulting litters produced mice with a forebrain predominant neuronal deletion of HIF-1α (HIF-1αΔ/Δ), as well as littermates without the deletion. In order to verify reduction of HIF-1α at postnatal day 7, HIF-1αΔ/Δ and wild-type mice were exposed to a hypoxic stimulus (8% oxygen) or room air for 1 h, followed by immediate collection of brain cortices for determination of HIF-1α expression. Results of Western blotting of mouse cortices exposed to hypoxia stimulus or room air confirmed that HIF-1αΔ/Δ cortex expressed a minimal amount of HIF-1α protein compared to wild-type cortex with the same hypoxic stimulus. Subsequently, pups underwent the Vannucci procedure of HI at postnatal day 7: unilateral ligation of the right common carotid artery followed by 30 min of hypoxia (8% oxygen). Immunofluorescent staining of brains 24 h after HI confirmed a relative lack of HIF-1α in the HIF-1αΔ/Δ cortex compared to the wild type, and that HIF-1α in the wild type is located in neurons. HIF-1α expression was determined in mouse cortex 24 h after HI. Histological analysis for the degree of injury was performed 5 days after HI. HIF-1α protein expression 24 h after HI showed a large increase of HIF-1α in the hypoxic-ischemic cortex of the wild-type compared to the hypoxic only cortex. Histological analysis revealed that HI injury was increased in the neuronally deficient HIF-1αΔ/Δ mouse brain (p < 0.05) and was more severe in the cortex. Genetic reduction of neuronal HIF-1α results in a worsening of injury after neonatal HI, with a region-specific role for HIF-1α in the setting of neonatal brain injury.
Brain injury; Transcription factor; Hypoxia-inducible factor 1α; Neonatal hypoxia-ischemia
Localized tissue hypoxia is a consequence of vascular compromise or rapid cellular proliferation and is a potent inducer of compensatory angiogenesis. The oxygen-responsive transcriptional regulator hypoxia-inducible factor 2α (HIF-2α) is highly expressed in vascular ECs and, along with HIF-1α, activates expression of target genes whose products modulate vascular functions and angiogenesis. However, the mechanisms by which HIF-2α regulates EC function and tissue perfusion under physiological and pathological conditions are poorly understood. Using mice in which Hif2a was specifically deleted in ECs, we demonstrate here that HIF-2α expression is required for angiogenic responses during hindlimb ischemia and for the growth of autochthonous skin tumors. EC-specific Hif2a deletion resulted in increased vessel formation in both models; however, these vessels failed to undergo proper arteriogenesis, resulting in poor perfusion. Analysis of cultured HIF-2α–deficient ECs revealed cell-autonomous increases in migration, invasion, and morphogenetic activity, which correlated with HIF-2α–dependent expression of specific angiogenic factors, including delta-like ligand 4 (Dll4), a Notch ligand, and angiopoietin 2. By stimulating Dll4 signaling in cultured ECs or restoring Dll4 expression in ischemic muscle tissue, we rescued most of the HIF-2α–dependent EC phenotypes in vitro and in vivo, emphasizing the critical role of Dll4/Notch signaling as a downstream target of HIF-2α in ECs. These results indicate that HIF-1α and HIF-2α fulfill complementary, but largely nonoverlapping, essential functions in pathophysiological angiogenesis.
Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.
In rapidly growing tumors, hypoxia commonly develops due to the imbalance between O2 consumption and supply. Hypoxia Inducible Factor (HIF)-1α is a transcription factor responsible for tumor growth and angiogenesis in the hypoxic microenvironment; thus, its inhibition is regarded as a promising strategy for cancer therapy. Given that CamKII or PARP inhibitors are emerging anticancer agents, we investigated if they have the potential to be developed as new HIF-1α-targeting drugs. When treating various cancer cells with the inhibitors, we found that a CamKII inhibitor, KN-62, effectively suppressed HIF-1α specifically in hepatoma cells. To examine the effect of KN-62 on HIF-1α-driven gene expression, we analyzed the EPO-enhancer reporter activity and mRNA levels of HIF-1α downstream genes, such as EPO, LOX and CA9. Both the reporter activity and the mRNA expression were repressed by KN-62. We also found that KN-62 suppressed HIF-1α by impairing synthesis of HIF-1α protein. Based on these results, we propose that KN-62 is a candidate as a HIF-1α-targeting anticancer agent.
CaMKII; HIF1-α; Hepatocellular carcinoma; Hypoxia; KN-62
Hypoxia-inducible factor 1α (HIF-1α) controls the cellular responses to hypoxia, activating transcription of a range of genes involved in adaptive processes such as increasing glycolysis and promoting angiogenesis. However, paradoxically, HIF-1α also participates in hypoxic cell death. Several gene products, such as BNip3, RTP801, and Noxa, were identified as HIF-1α-responsive proapoptotic proteins, but the complicated hypoxic cell death pathways could not be completely explained by the few known genes. Moreover, molecules linking the proapoptotic signals of HIF-1α directly to mitochondrial permeability transition are missing. In this work, we report the identification of an HIF-1α-responsive proapoptotic molecule, HGTD-P. Its expression was directly regulated by HIF-1α through a hypoxia-responsive element on the HGTD-P promoter region. When overexpressed, HGTD-P was localized to mitochondria and facilitated apoptotic cell death via typical mitochondrial apoptotic cascades, including permeability transition, cytochrome c release, and caspase 9 activation. In the process of permeability transition induction, the death-inducing domain of HGTD-P physically interacted with the voltage-dependent anion channel. In addition, suppression of HGTD-P expression by small interfering RNA or antisense oligonucleotides protected against hypoxic cell death. Taken together, our data indicate that HGTD-P is a new HIF-1α-responsive proapoptotic molecule that activates mitochondrial apoptotic cascades.
The hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the α subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1α in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).
Patients with IE were screened for changes in the HIF-1α coding sequence, and a change in the ODD domain that converts Pro-582 to Ser was identified in several patients. This same change, however, was also detected at a significant frequency, 0.073, in unaffected controls compared to 0.109 in the IE patient group. In vitro hydroxylation assays examining this amino acid change failed to reveal a discernible effect on HIF hydroxylation at Pro-564.
The Pro582Ser change represents a common polymorphism of HIF-1α that does not impair HIF-1α prolyl hydroxylation. Although the Pro582Ser polymorphism is located in the ODD domain of HIF-1α it does not diminish the association of HIF-1α with VHL. Thus, it is unlikely that this polymorphism accounts for the erythrocytosis in the group of IE patients studied.
3,3′-Diindolylmethane (DIM) is a chemopreventive and chemotherapeutic phytochemical derived from the metabolism of indoles found at high concentrations in cruciferous vegetables. We have previously shown that DIM exhibits anti-angiogenic properties in cultured vascular endothelial cells and in Matrigel plug assays in rodents. In the present study, we demonstrate that DIM reduces the level of hypoxia-inducible factor (HIF)-1α in hypoxic tumor cell lines, as well as HIF-1 transcriptional activity as measured by a reporter assay. Moreover, DIM inhibited the expression of HIF-1-responsive endogenous genes, resulting in the reduced expression of key hypoxia responsive factors, VEGF, furin, enolase-1, glucose transporter-1 and phosphofructokinase. DIM reduced the level of HIF-1α in hypoxic cells by increasing the rate of the prolylhydroxylase- and proteosome-mediated degradation of HIF-1α, and by decreasing the rate of HIF-1α transcription. Using enzyme kinetics studies, we established that DIM interacts with the oligomycin-binding site on the F1 transmembrane component of mitochondrial F1F0-ATPase. The contributions of the resulting increases in levels of ROS and O2 in hypoxic cells to the inhibitory effects of DIM on HIF-1α expression are discussed. These studies are the first to show that DIM can decrease the accumulation and activity of the key angiogenesis regulatory factor, HIF-1α, in hypoxic tumor cells.
3,3′-Diindolylmethane; angiogenesis; hypoxia-inducible factor; cancer; ATPase; prolylhydroxylase