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At least 235 records · Page 13Linked to original sources
PubMed2002

Role of nitric oxide in the regulation of HIF-1alpha expression during hypoxia.

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor consisting of HIF-1alpha and HIF-1beta subunits, controls the expression of a large number of genes involved in the regulation of cellular responses to reduced oxygen availability. The oxygen-regulated subunit, HIF-1alpha, is stabilized in cells exposed to hypoxia. The regulation of hypoxic responses by nitric oxide (NO) is believed to have wide pathophysiological relevance, thus we investigated whether NO affects HIF-1 activation in hypoxic cells. Here we show that NO generated from NO donors prevented HIF-1alpha hypoxic accumulation in Hep 3B and PC-12 cells. Addition of a glutathione analog or peroxynitrite scavengers prevented the NO-induced inhibition of HIF-1alpha accumulation in both cell lines. Exposure to NO was associated with inhibition of mitochondrial electron transport and compensatory glycolysis, which maintained normal cellular ATP content. Succinate, a Krebs cycle intermediate and respiratory chain substrate, restored HIF-1alpha hypoxic induction in the cells, suggesting involvement of mitochondria in regulation of HIF-1alpha accumulation during hypoxia. Regulation of HIF-1alpha by NO is an additional important mechanism by which NO might modulate cellular responses to hypoxia in mammalian cells.

Animals↗
PubMed2006

Sex-related dimorphic response of HIF-1 alpha expression in myocardial ischemia.

Hypoxia-inducible factor-1 alpha (HIF-1 alpha) plays a role in a number of cell-protective pathways after ischemia. There are clear sex-related differences in the remodeling process, and hearts from males tend to dilate in response to pathological loads and ischemia to a greater degree than do hearts from females. Thus we hypothesized that there would be a sex-related dimorphic response of HIF-1 alpha to an ischemic event. Male and female rats were euthanized 5 and 24 h after coronary ligation (M-MI and F-MI; MI, myocardial ischemia), and HIF-1 alpha expression was determined by immunohistochemistry, Western blot, and quantitative RT-PCR. Sham-operated male and female animals served as controls (M-SH and F-SH). In the ischemic area, histochemical analysis at 5 h showed that HIF was expressed in 33% of cell nuclei in M-MI and in 55% in F-MI. At 24 h, HIF expression increased to 49% in M-MI and to 82% in F-MI (P < 0.05 vs. SH and also M-MI vs. F-MI). This difference was not only statistically significant between the two sexes at 24 h but also within each sex at 5 and 24 h after ligation. Western blots confirmed that, at 24 h after ischemia, HIF protein increased significantly in both male and female hearts relative to sham-operated animals but that the increase in females was 60% greater than that seen in males. mRNA expression of HIF was significantly increased at 24 h in F-MI versus M-MI and sham-operated animals. Expression of downstream HIF target genes (heme oxygenase and brain natriuretic peptide) was increased in proportion to the levels of HIF expression. These data suggest a novel cellular mechanism to explain the sex-related dimorphic response to ischemia and also the possibility that exogenous modulation of HIF might represent a new therapeutic approach to preventing left ventricular remodeling.

Animals↗
PubMed2003

Redox regulation of HIF-1alpha levels and HO-1 expression in renal medullary interstitial cells.

The present study hypothesized that superoxide (O2(-)*) importantly contributes to the regulation of hypoxia-inducible factor (HIF)-1alpha expression at posttranscriptional levels in renal medullary interstitial cells (RMICs) of rats. By Western blot analysis, it was found that incubation of RMICs with O2(-)* generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or CoCl(2)-induced increase in HIF-1alpha levels and completely blocked the increase in HIF-1alpha levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under normoxic conditions, a cell-permeable O2(-)* dismutase (SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) and PEG-SOD, significantly increased HIF-1alpha levels in RMICs. Two mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1alpha levels in these renal cells. Moreover, introduction of an anti-sense oligodeoxynucleotide specific to NAD(P)H oxidase subunit, p22(phox), into RMICs markedly increased HIF-1alpha levels. In contrast, the OH* scavenger tetramethylthiourea had no effect on the accumulation of HIF-1alpha in these renal cells. By Northern blot analysis, scavenging or dismutation of O2(-)* by TEMPOL and PEG-SOD was found to increase the mRNA levels of an HIF-1alpha-targeted gene, heme oxygenase-1. These results indicate that increased intracellular O2(-)* levels induce HIF-1alpha degradation independently of H(2)O(2) and OH* radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1alpha in these cells under physiological conditions.

Animals↗
PubMed2006

Expression and actions of HIF prolyl-4-hydroxylase in the rat kidneys.

Hypoxia inducible factor (HIF) prolyl-4-hydroxylase domain-containing proteins (PHDs) promote the degradation of HIF-1alpha. Because HIF-1alpha is highly expressed in the renal medulla and HIF-1alpha-targeted genes such as nitric oxide synthase, cyclooxygenase, and heme oxygenase are important in the regulation of renal medullary function, we hypothesized that PHD regulates HIF-1alpha levels in the renal medulla and, thereby, participates in the control of renal Na(+) excretion. Using real-time RT-PCR, Western blot, and immunohistochemical analyses, we have demonstrated that all three isoforms of PHD, PHD1, PHD2, and PHD3, are expressed in the kidneys and that PHD2 is the most abundant isoform. Regionally, all PHDs exhibited much higher levels in renal medulla than cortex. A furosemide-induced increase in renal medullary tissue Po(2) significantly decreased PHD levels in renal medulla, whereas hypoxia significantly increased mRNA levels of PHDs in cultured renal medullary interstitial cells, indicating that O(2) regulates PHDs. Functionally, the PHD inhibitor l-mimosine (l-Mim, 50 mg x kg(-1) x day(-1) i.p. for 2 wk) substantially upregulated HIF-1alpha expression in the kidneys, especially in the renal medulla, and remarkably enhanced (by >80%) the natriuretic response to renal perfusion pressure in Sprague-Dawley rats. Inhibition of HIF transcriptional activity by renal medullary transfection of HIF-1alpha decoy oligodeoxynucleotides attenuated l-Mim-induced enhancement of pressure natriuresis, which confirmed that HIF-1alpha mediated the effect of l-Mim. These results indicate that highly expressed PHDs in the renal medulla make an important contribution to the control of renal Na(+) excretion through regulation of HIF-1alpha and its targeted genes.

Animals↗
PubMed2006

Oxygen treatment after experimental hypoxia-ischemia in neonatal rats alters the expression of HIF-1alpha and its downstream target genes.

Recently, mounting evidence has emerged to suggest that hyperbaric oxygenation (HBOT)-induced neuroprotection after experimental global ischemia and subarachnoid hemorrhage entails a decrease in the expression of hypoxia-inducible factor-1alpha (HIF-1alpha). Therefore, the purpose of this study was to test the hypothesis that oxygen-induced neuroprotection after neonatal hypoxia-ischemia involves alterations in the expression of HIF-1alpha. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2 h of hypoxia (8% O(2) at 37 degrees C). Pups were then treated with HBOT (2.5 ATA) or normobaric oxygenation treatment (NBOT) for 2 h. The expression and phosphorylation status of HIF-1alpha was evaluated at intervals up to 24 h after the insult, as was the expression of glucose transporter (GLUT)-1, GLUT-3, lactate dehydrogenase (LDH), aldolase (Ald), and p53. The protein-protein interaction of HIF-1alpha and p53 was also examined. An elevated expression of HIF-1alpha, GLUT-1, GLUT-3, Ald, and LDH was observed after the insult. An increase in the dephosphorylated form of HIF-1alpha was followed by an increase in the association of HIF-1alpha with p53 and an increase in p53 levels. Both HBOT and NBOT reduced the elevated expression of HIF-1alpha and decreased its dephosphorylated form. Furthermore, both treatments promoted a transient increase in the expression of GLUT-1, GLUT-3, LDH, and Ald, while decreasing the HIF-1alpha-p53 interaction and decreasing the expression of p53. Therefore, the alteration of the HIF-1alpha phenotype by a single oxygen treatment may be one of the underlying mechanisms for the observed oxygen-induced neuroprotection seen when oxygen is administered after a neonatal hypoxic-ischemic insult.

Adaptation, Physiological↗
PubMed2005

Hypoxic induction of an HIF-1alpha-dependent bFGF autocrine loop drives angiogenesis in human endothelial cells.

Hypoxia is a major pathophysiological condition for the induction of angiogenesis, which is a crucial aspect of growth in solid tumors. In mammalian cells, the transcriptional response to oxygen deprivation is largely mediated by hypoxia-inducible factor 1 (HIF-1), a heterodimer composed of HIF-1alpha and HIF-1beta subunits. However, the response of endothelial cells to hypoxia and the specific involvement of HIF-alpha subunits in this process are still poorly understood. We show that human umbilical vein endothelial cells (HUVECs) cultured in the absence of growth factors survive and form tubelike structures when cultured under hypoxic, but not normoxic, conditions. HUVECs expressed both HIF-1alpha and HIF-2alpha when cultured under hypoxic conditions. Transfection of HIF-1alpha, but not HIF-2alpha, siRNA to HUVECs completely abrogated hypoxic induction of cords. Neutralizing antibodies to bFGF, but not IGF-1, VEGF, or PDGF-BB, blocked survival and sprouting of HUVECs under hypoxic conditions, suggesting the existence of an autocrine loop induced by low oxygen levels. Notably, bFGF-dependent induction of cord formation under normoxic conditions required HIF-1alpha activity, which was also essential for hypoxic induction of bFGF mRNA and protein expression. These results uncover the existence of an HIF-1alpha-bFGF amplification pathway that mediates survival and sprouting of endothelial cells under hypoxic conditions.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed2006

Cloning and expression analysis of two distinct HIF-alpha isoforms--gcHIF-1alpha and gcHIF-4alpha--from the hypoxia-tolerant grass carp, Ctenopharyngodon idellus.

BACKGROUND: Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs. RESULTS: We have cloned and characterized two distinct HIF-alpha cDNAs--gcHIF-1alpha and gcHIF-4alpha--from the hypoxia-tolerant grass carp. The deduced gcHIF-1alpha protein is highly similar to the HIF-1alphas (57-68%) from various vertebrate species, while gcHIF-4alpha is a novel isoform, and shows an equivalent degree of amino acid identity (41-47%) to the HIF-1alpha, HIF-2alpha and HIF-3alpha proteins so far described. Parsimony analysis indicated that gcHIF-4alpha is most closely related to the HIF-3alpha proteins. Northern blot analysis showed that mRNA levels of gcHIF-1alpha and gcHIF-4alpha differ substantially under normoxic and hypoxic conditions, while Western blot studies demonstrated that the endogenous protein levels for both gcHIF-1alpha and gcHIF-4alpha are similarly responsive to hypoxia. Our findings suggest that both gcHIF-1alpha and gcHIF-4alpha are differentially regulated at the transcriptional and translational levels. HRE-luciferase reporter assays show that both proteins function as transcription activators and play distinct roles in modulating the hypoxic response in grass carp. CONCLUSION: There are at least two distinct HIF-alpha isoforms--gcHIF-1alpha and gcHIF-4alpha--in the hypoxia-tolerant grass carp, which are differentially expressed and regulated in different fish organs in response to hypoxic stress. Overall, the results suggest that unique molecular mechanisms operate through these two HIF-alpha isoforms, which underpin the hypoxic response in the hypoxia-tolerant grass carp.

Amino Acid Sequence↗
PubMed2003

HIF-1alpha controls extracellular matrix synthesis by epiphyseal chondrocytes.

The transcription factor HIF-1alpha plays a crucial role in modifying gene expression during low oxygen tension. In a previous study, we demonstrated that HIF-1alpha is essential for chondrocyte growth arrest and survival in vivo. To explore further the role of HIF-1alpha in cartilage biology, we undertook studies with primary epiphyseal chondrocytes with a targeted deletion of HIF-1alpha. In this study, we show that HIF-1alpha is necessary for regulating glycolysis under aerobic and anaerobic conditions. HIF-1alpha-null chondrocytes were unable to maintain ATP levels in hypoxic microenvironments, indicating a fundamental requirement for this factor for the regulation of chondrocyte metabolism. Synthesis of the angiogenic factor vascular endothelial growth factor was also significantly induced by hypoxia, and this increase is lost in HIF-1alpha-null mutant cells. Under hypoxic conditions, aggrecan mRNA and protein levels were significantly reduced in chondrocytes lacking the HIF-1alpha transcription factor. Interestingly, strongly increased type-II collagen protein levels were detected in wild-type cells after 44 hours of hypoxia. In addition, type-II collagen mRNA and protein levels were strongly decreased under low oxygen in chondrocytes lacking HIF-1alpha. In summary, our results clearly demonstrate the importance of HIF-1alpha in maintenance of anaerobic glycolysis, and thereby extracellular matrix synthesis, of epiphyseal chondrocytes.

Adenosine Triphosphate↗
PubMedDate not supplied

HIF hydroxylation and cellular oxygen sensing.

Hypoxia-inducible factor (HIF) is a transcriptional complex that mediates a broad range of cellular and systemic responses to hypoxia. Analysis of HIF-alpha subunits has demonstrated that its activity is regulated by a series of oxygen-dependent enzymatic hydroxylations at specific prolyl and asparaginyl residues. Combined structural/genetic approaches have identified the relevant enzymes as members of the 2-oxoglutarate-dependent dioxygenase superfamily, possessing a beta-barrel 'jelly-roll' conformation that aligns a 2-histidine/1-carboxylate iron co-ordination motif at the catalytic centre. HIF prolyl hydroxylation is performed by a closely related set of isoenzymes (PHD1-3) that differ in abundance and subcellular localisation. Hydroxylation of either human HIF-1alpha Pro402 or Pro564 promotes interaction with the von Hippel-Lindau tumour suppressor protein (pVHL). In oxygenated cells this process targets HIF-alpha for rapid proteasomal destruction. HIF asparaginyl hydroxylation is performed by a protein termed factor inhibiting HIF (FIH). In oxygenated cells hydroxylation of human HIF-1alpha Asn803 prevents interaction with the p300 transcriptional co-activator, providing a second mechanism by which HIF-mediated transcription is inactivated. Genetic studies demonstrate a critical function for both types of enzyme in regulating the HIF transcriptional cascade. Limitation of activity in hypoxia supports a central role of these hydroxylases in cellular oxygen sensing. Regulation of the amount of hydroxylase protein, and the supply of other co-substrates and co-factors, particularly the cellular availability of iron, also contribute to tuning the physiological response to hypoxia.

Animals↗
PubMed2006

Cytokines and hormones in the regulation of hypoxia inducible factor-1alpha (HIF-1alpha).

Hypoxia inducible factor-1 (HIF-1) is a central component of the oxygen sensing system that coordinates cellular responses to conditions of decreased oxygen availability. The hypoxia inducible transcription factor HIF-1 is a heterodimer composed of the helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) proteins HIF-1alpha and the aryl hydrocarbon nuclear translocator (ARNT) also known as HIF-1beta. Transactivation of HIF-1 transmits a hypoxic signal into patho-physiological responses such as angiogenesis, erythropoiesis, vasomotor control, an altered energy metabolism, as well as cell survival decisions by regulating a staidly growing number of target genes. Among recent advances are the discoveries that cytokines and growth factors make use of the 'hypoxic signaling system' under normoxia. Here we summarize current knowledge and existing concepts that help to understand how cytokines and hormones affect protein accumulation of HIF-1alpha and discuss potential implications of activating HIF-1 under normoxia. Considering the fundamental role of cytokines during inflammation may predict a role of HIF-1alpha in coordinating cellular responses to pathogens and point to the connection of cancer and inflammation. Moreover, we will address potential feed-back mechanisms showing an impact of HIF-1 on cytokine production. These considerations suggest an intimate signaling cross-talk between cytokines and the HIF-1 system.

Cytokines↗
PubMed2004

Expression of HIF-2alpha/EPAS1 in hepatocellular carcinoma.

AIM: To investigate the expression of hypoxia-inducible factor (HIF)-2alpha/endothelial PAS domain protein1 (EPAS1) in hepatocellular carcinoma (HCC). METHODS: Expression of HIF-2alpha/EPAS1 was investigated immunohistochemically on paraffin-embedded sections from 97 patients with HCC. To further confirm that HIF-2alpha/EPAS1 in HCC tissues also correlated with angiogenesis, a parallel immunohistchemistry study of vascular endothelial growth factor (VEGF) was performed on these 97 cases. RESULTS: HIF-2alpha/EPAS1 could be detected in 50 of 97 cases (51.6%), including 19 weakly positive (19.8%), and 31 strongly positive (31.1%), the other 47 cases were negative (48.4%). The expression of HIF-2alpha/EPAS1was significantly correlated with tumor size, capsule infiltration, portal vein invasion, and necrosis. A parallel immunohistochemical analysis of VEGF demonstrated its positive correlation with capsule infiltration, portal vein invasion, and HIF-2alpha/EPAS1 overexpression, which supported the correlation of HIF-2alpha/EPAS1up-regulation with tumor angiogenesis. No apparent correlation was observed between HIF-2alpha/EPAS1 and capsular formation, presence of cirrhosis, and histological grade. CONCLUSION: HIF-2alpha/EPAS1 is expressed in most of HCC with capsular infiltration and portal vein invasion, which indicates a possible role of HIF-2alpha/EPAS1 in HCC metastasis.

Adult↗
PubMed2005

Expression of HIF-1alpha and Glut-1 in human bladder cancer.

HIF-1 is a heterodimer consisting of the HIF-1alpha and HIF-1beta subunits, and HIF-1alpha is the unique oxygen regulated subunit that determines HIF-1 activity. HIF-1alpha upgrades many gene products which include the glucose transporter protein 1 (Glut-1). Immunohistochemical studies using a monoclonal antibody specific for HIF-1alpha indicate that the overexpression of HIF-1alpha occurs in the most common forms of human cancer, including bladder cancer. The expression of Glut-1 in human bladder cancer is associated with poor prognosis and a low survival rate. To our knowledge, this is the first study to compare the expression of both HIF-1alpha and Glut-1 with clinicopathological characteristics in superficial and invasive human bladder cancer (all invasive bladder cancer patients received radical radiotherapy). The Kaplan-Meier survival analysis curve shows a significant association of HIF-1alpha expression with recurrence and survival in superficial bladder cancer and shows a significant association of Glut-1 with survival in invasive bladder cancer [chi2 (4)=10.52; Pr >chi2 =0.0012].

Antibodies, Monoclonal↗
PubMed2005

[The relationship between the expression of HIF-1alpha and the angiogenesis in infancy hemangioma].

OBJECTIVE: To investigate the expression of HIF-1alpha and its roles on the vascular endothelial cell growth factor (VEGF) and microvessel density (MVD ) in hemangioma. METHODS: Immunohistochemical streptavidin/peroxidase(SP) stain was applied to examine the expression of HIF-1alpha, VEGF and CD34. The amount of microvessel was counted, which the endothelial cell expressed CD34. RESULTS: The nuclei and cytoplasm of the endothelial cell showed positive staining for HIF-1alpha in hemangioma. In 28 cases, positive rate for HIF-1alpha was 64%, for VEGF was 71.4%. In the proliferating phase, positive rate for HIF-1alpha was 87.5%, for VEGF was 93.7%. However, the positive rate for HIF-1alpha, in the involuting phase, was 33.3%, and for VEGF was 41.7%. There was a very significantly different between two phases (P < 0.01). In the proliferating phase, MVD was 73.4 +/- 14.63 . In the involuting phase, MVD was 30.2 +/- 9.1 (P < 0.01). The correlation between the HIF-1alpha and VEGF was positive (P < 0.01). There was a positive correlation between the HIF-1alpha and MVD (P < 0.01). CONCLUSIONS: There might be a special hypoxia microenvironment in endothelial cell of the hemangioma in proliferating phase , which could up-regulate the expression of the HIF-1alpha protein. The HIF-1alpha protein could also up-regulate the VEGF protein and then result in the angiogenesis.

Adolescent↗
PubMed2006

[Reversion the multidrug resistance of human breast carcinoma cells by RNA interference targeting HIF-1 alpha gene].

OBJECTIVE: To reverse the multidrug resistant (MDR) phenotype of human breast carcinoma cells by small hairpin RNA (shRNA) technique targeting hypoxia-inducible factor (HIF)-1alpha gene. METHODS: Small hairpin RNA (shRNA) eukaryotic expression vector targeting HIF-1alpha gene, named pSilencer-HIF, was constructed and transfected into MCF-7/ADR human breast cancer cells by liposome technique. Tumor cell livability (TCL) and Rhodamine 123 efflux assay were used to monitor the biological changes of the transfected cells. The mRNA and protein expression of HIF-1alpha and mdr-1 were investigated by RT-PCR and Western blot. RESULTS: The successful construction of pSilencer-HIF plasmid was confirmed by DNA sequencing. HIF-1alpha mRNA and protein levels were significantly decreased in MCF-7/ADR cells after the transfection and there was a direct correlation between HIF-1alpha and mdr-1 expression. By comparing the cells transfected with control vector and the MCF-7/ADR cells transfected with pSilencer-HIF, a reduced TCL from 76% to 43%, and an increased Rhodamine 123 fluorescence intensity from 22.0% to 86.6% were observed. CONCLUSIONS: pSilencer-HIF-1alpha has been successfully constructed. The inhibition of HIF-1alpha expression through shRNA technique can significantly reverse the multidrug resistance phenotype of MCF-7/ADR cells.

Breast Neoplasms↗
PubMed2004

Pretreatment evaluation of combined HIF-1alpha, p53 and p21 expression is a useful and sensitive indicator of response to radiation and chemotherapy in esophageal cancer.

Tumor hypoxia has been known to be associated with resistance to radiation and chemotherapy (CRT). Hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor induced by hypoxic condition, plays a major role in the pleiotropic response observed under hypoxic conditions. It encodes proteins that play key roles in critical development and physiologic processes, including angiogenesis, glucose transport and erythropoiesis. On the other hand, cell cycle- and apoptosis-control genes p53 and p21 may play major roles in the tumor response to cytotoxic agents such as radiation and chemotherapy. Previous reports have suggested that the regulation of p53 and p21 is HIF-1-dependent. Our aim was to evaluate the expression of the HIF-1alpha, p53 and p21 proteins by immunohistochemistry in biopsy specimens of esophageal squamous cell carcinoma, which were obtained endoscopically from 65 patients before CRT, and then determine whether the levels of expression of these proteins predicted the clinical effectiveness of CRT in individual cancers. Also, to assess the relationship between expression of these proteins and cell death and cellular proliferation activity, we evaluated Ki67 expression and the apoptosis index (TUNEL). HIF-1alpha expression in esophageal cancer was significantly and negatively related to the response to CRT, independently of p53 and p21 expression. Interestingly, 44.4% (12/27) of the HIF-1alpha-negative group showed a complete response to therapy. There was no significant correlation between the expression of HIF-1alpha, p53 and p21 and proliferation and apoptosis. HIF-1alpha overexpression may predict resistance to CRT and may be a helpful guide in choosing between therapeutic strategies, such as intensive combined modality therapy vs. palliative therapy. Combined immunohistochemical evaluation of HIF-1alpha, p53 and p21 protein expression at the pretreatment biopsy is a very useful and powerful indicator of sensitivity to CRT in human esophageal cancer. Our data also indicate the importance of having a clear grasp of the degree of hypoxia (HIF-1alpha) of a tumor, rather than its cellular character (proliferation and apoptosis), to indicate the likely impact of CRT.

Biomarkers, Tumor↗
PubMed2006

Induction of human endometrial cancer cell senescence through modulation of HIF-1alpha activity by EGLN1.

Previous observations indicate that transfer of human chromosome (chr.) 1 induces senescence of endometrial cancer cells. To identify the gene(s) responsible for the senescence, we first analyzed the structural integrity of the introduced chr. 1 in immortal revertant from chr.1-transferred HHUA cells. The data demonstrated a correlation between nonrandom deletions within the 1q31-qter region and reversion to immortality. Next, by using a panel of 12 microsatellite markers, we found high frequencies of loss of heterozygosity in the particular 1q region (1q41-42), in surgically removed samples. Then, we screened the genetic mutation of the genes involved in this region, with endometrial cancer panel. Among them, EGLN1, that is a member of prolyl hydroxylase and can facilitate HIF-1 degradation by ubiquitination through the hydroxylation of HIF-1, was mutated at significantly higher frequencies (12/20, 60%). Introduction of wild-type EGLN1 into endometrial cancer cell lines (HHUA, Ishikawa and HWCA), that carry EGLN1 gene mutations induced senescence. This was invoked through the negative regulation of HIF-1 expression. In addition, alternative way of negative regulation of HIF-1 by Factor inhibiting HIF-1(FIH), SiRNA against HIF-1, and HIF-1 inhibitor, YC-1, could also induce senescence. Thus, EGLN1 can be considered as a candidate tumor suppressor on chr. 1q, and our observation could open the new aspect in exploring the machinery of senescence induction associated with HIF-1 signal transduction. These results also suggested the availability of negative regulation of HIF-1 signals for uterine cancer treatment, especially for uterine sarcomas that have worse prognosis and show a high frequency of EGLN1 gene abnormality.

Animals↗
PubMed2006

HIF-1alpha has an anti-apoptotic effect in human airway epithelium that is mediated via Mcl-1 gene expression.

Hypoxia-inducible factor-1alpha (HIF-1alpha) and myeloid cell leukemia-1 (Mcl-1) proteins have been shown to regulate apoptosis in some cell systems but have not been studied in this context in airway epithelium. Using a model of anoxia/reoxygenation (A/R), the present study employed RNA interference (RNAi) targeting HIF-1alpha and Mcl-1 to evaluate their possible anti-apoptotic effects on HBE1 cells, an immortalized human bronchial epithelial cell line. The cells were either cultured under normoxic conditions or were transfected with small interfering RNA (siRNA) duplexes targeting HIF-1alpha or Mcl-1 mRNA and then immediately exposed to A/R. As controls, non-transfected HBE1 cells and cells transfected with scrambled RNA duplexes were subjected to A/R. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and RNAi was assessed by knockdown of HIF-1alpha and Mcl-1 mRNA and protein expression using real-time quantitative RT-PCR (Q-PCR), immunohistochemistry, and Western blots. HBE1 cells transfected with siRNA duplexes targeting either HIF-1alpha or Mcl-1 and subjected to A/R manifested considerable apoptosis, a finding not observed in either non-transfected cells or cells transfected with scrambled RNA duplexes. Specific knockdown of mRNA and protein expression by RNAi in HBE1 cells after A/R was shown for siRNA duplexes targeting either HIF-1alpha or Mcl-1. Unexpectedly, knockdown of HIF-1alpha induced parallel knockdown of Mcl-1 mRNA and protein expression, whereas Mcl-1 knockdown had no noticeable effect on HIF-1alpha expression. Thus, although both of these proteins were shown to be anti-apoptotic, the action of HIF-1alpha appeared to be mediated in part via Mcl-1.

Apoptosis↗
PubMed2003

Role of ERK and calcium in the hypoxia-induced activation of HIF-1.

Oxygen-dependent regulation of HIF-1 activity occurs at multiple levels in vivo. The mechanisms regulating HIF-1alpha protein expression have been most extensively analyzed but the ones modulating HIF-1 transcriptional activity remain unclear. Changes in the phosphorylation and/or redox status of HIF-1alpha certainly play a role. Here, we show that ionomycin could activate HIF-1 transcriptional activity in a way that was additive to the effect of hypoxia without affecting HIF-1alpha protein level. In addition, a calmodulin dominant negative mutant and W7, a calmodulin antagonist, as well as BAPTA, an intracellular calcium chelator, inhibited the hypoxia-induced HIF-1 activation. These results indicate that elevated calcium in hypoxia could participate in HIF-1 activation. Furthermore, ERK but not JNK phosphorylation was evidenced in both conditions, ionomycin and hypoxia. PD98059, an inhibitor of the ERK pathway as well as a ERK1 dominant negative mutant also blocked HIF-1 activation by hypoxia and by ionomycin. A MEKK1 (a kinase upstream of JNK) dominant negative mutant had no effect. In addition, BAPTA, calmidazolium, a calmodulin antagonist and PD98059 inhibited VEGF secretion by hypoxic HepG2. All together, these results suggest that calcium and calmodulin would act upstream of ERK in the hypoxia signal transduction pathway.

Animals↗