Expression Of PHD1 Research Articles

Effects of hypoxia on the gill morphological structure, apoptosis and hypoxia-related gene expression in blunt snout bream (Megalobrama amblycephala).

The blunt snout bream (Megalobrama amblycephala) is a typical hypoxia-sensitive fish, and hypoxia stress leads to reduced vitality and yield during aquaculture. To explore the specific adaptation mechanism under hypoxia, the blunt snout bream was treated with hypoxia (DO = 2.0 ± 0.1mg/L) for 24h, followed by 3h of recovery. Our results depicted that the gill filament structure of blunt snout bream changed after hypoxia. During hypoxia for 24h, the gill filament structure was altered, including a more than 80% expansion of the lamellar respiratory surface area and a proportionate apoptosis decrease in interlamellar cell mass (ILCM) volume. Thus, the water-blood diffusion distance was shortened to less than 46%. During hypoxia for 24h, the activity of ROS in gill tissue increased significantly (p < 0.05), while the mitochondrial membrane potential decreased significantly (p < 0.05). During hypoxia, mRNA expression level of anti-apoptotic gene Bcl-2 in the gills of blunt snout bream decreased significantly (p < 0.05), while the expression of pro-apoptotic gene Bax mRNA increased significantly (p < 0.05). Thus, the ratio of Bax/Bcl-2 mRNA increased in the gills of blunt snout bream to promote the activity of Caspase-3. Together, our results indicated hypoxia-induced apoptosis in the gills of blunt snout bream through the mitochondrial pathway. In addition, a decreased expression of Phd1 and an increased expression of Hif-1α in gills under hypoxia stress indicates that blunt snout bream may cope with hypoxia-induced apoptosis by enhancing the HIF pathway. These results provide new insights into fish's adaptation strategies and mechanisms of hypoxia.

Gene Expression Levels of the Prolyl Hydroxylase Domain Proteins PHD1 and PHD2 but Not PHD3 Are Decreased in Primary Tumours and Correlate with Poor Prognosis of Patients with Surgically Resected Non-Small-Cell Lung Cancer.

Simple SummaryHypoxia correlates with poor prognosis in several cancer types, including lung cancer. Prolyl hydroxylase domain proteins (PHDs) belong to an evolutionarily conserved superfamily of dioxygenases that play a role in cell oxygen sensing and homeostasis. In this study, we evaluated PHD1, PHD2 and PHD3 mRNA expression in 60 NSCLC tumours and compared it to that in normal lungs and evaluated the prognostic significance of these differences for distinguishing the survival of NSCLC patients treated with radical surgery. Our results showed that the mRNA expression PHD1 and PHD2 in NSCLC primary tumours was decreased, which correlated with larger tumour size and poor prognosis of patients. PHD1 also showed borderline independent prognostic value in multivariate analysis. In contrast, we found no associations between PHD3 expression and any of the observed parameters. Our results suggest that loss of PHD1 and PHD2 expression is associated with the development and progression of NSCLC, whereas PHD1 could be further assessed as a prognostic marker in NSCLC.Background: Hypoxia correlates with poor prognosis in several cancer types, including lung cancer. Prolyl hydroxylase domain proteins (PHDs) play a role in cell oxygen sensing, negatively regulating the hypoxia-inducible factor (HIF) pathway. Our study aim was to evaluate PHD1, PHD2 and PHD3 mRNA expression levels in primary tumours and normal lungs of non-small-cell lung cancer (NSCLC) patients and to correlate it with selected regulators of HIF signalling, with clinicopathological characteristics and overall survival (OS). Methods: Tumour tissue samples were obtained from 60 patients with surgically resected NSCLC who were treated with radical surgery. In 22 out of 60 cases, matching morphologically normal lung tissue was obtained. PHD1, PHD2 and PHD3 mRNA expressions were measured using RT-qPCR. Results: The PHD1 and PHD2 mRNA levels in primary tumours were significantly decreased compared to those in normal lungs (both p < 0.0001). PHD1 and PHD2 expression in tumours was positively correlated (rs = 0.82; p < 0.0001) and correlated well with HIF pathway downstream genes HIF1A, PKM2 and PDK1. Decreased PHD1 and PHD2 were associated with larger tumour size, higher tumour stage (PHD1 only) and squamous cell carcinoma. Patients with low PHD1 and patients with low PHD2 expression had shorter OS than patients with high PHD1 (p = 0.02) and PHD2 expression (p = 0.01). PHD1 showed borderline independent prognostic values in multivariate analysis (p = 0.06). In contrast, we found no associations between PHD3 expression and any of the observed parameters. Conclusions: Our results show that reduced expression of PHD1 and PHD2 is associated with the development and progression of NSCLC. PHD1 could be further assessed as a prognostic marker in NSCLC.

Baicalin promotes extracellular matrix synthesis in chondrocytes via the activation of hypoxia-inducible factor-1α.

Chinese herbal extracts are being used increasingly to treat osteoarthritis (OA) in recent years. Baicalin (BA) is an active component of Scutellaria baicalensis Georgi extracts and protects chondrocytes against damage. The aim of the present study was to examine the mechanism of action of BA on chondrocytes from mouse articular cartilage. In total, 44 µM BA and 10 µM hypoxia-inducible-factor-1α (HIF-1α) inhibitor BAY-87-2243 were screened by the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] method. Alcian blue and Safran O staining were used to investigate the synthesis of extracellular matrix (ECM) in chondrocytes treated with BA. The expression of HIF-1α and chondrogenic marker genes including SOX9, AGG and Col2α was detected by western blotting or reverse-transcription quantitative (RT-qPCR), the expression of PHD1,2,3 and catabolic genes including ADAMTS5, MMP9 and MMP13 were detected by RT-qPCR. To investigate the effect of BA on the ECM synthesis of chondrocytes, 44 µM BA and 10 µM BAY were chosen for further experimentation. It was confirmed that BA at a concentration of 44 µM could significantly promote the secretion of ECM. The expressions of genes including HIF-1α, SOX9, collagen type 2 (Col2α) and aggrecan (AGG) were elevated following BA pretreatment and decreased by subsequent BAY-87-2243 stimulation for 24 h. Compared with untreated chondrocytes, the expressions of genes including ADAMTS5, MMP9, MMP13, PHD1, PHD2 and PHD3 in chondrocytes treated by BA were downregulated, however, BAY-87-2243 reversed the effect of BA on the genes including ADAMTS5, MMP9, MMP13, PHD1, PHD2 and PHD3 in chondrocytes. The findings of the present study suggest that BA may promote ECM synthesis and marker gene expression in chondrocytes by activating HIF-1α. Therefore, BA may represent a novel clinical drug for OA.

Effects of Simultaneous Downregulation of PHD1 and Keap1 on Prevention and Reversal of Liver Fibrosis in Mice

Background and Aim: To investigate whether double-knockdown of PHD1 and Keap1 in mice could enhance the resolution of carbon tetrachloride (CCl4)-induced liver fibrosis.Methods: The liver fibrosis model of mice was established by intraperitoneal injection of 25% CCl4 in olive oil (4 ul/g) twice a week for 8 weeks. PHD1shRNA and Keap1shRNA eukaryotic expression plasmids were simultaneously administered from the beginning of the first to fourth week (preventive group) or from the fifth to eighth week of CCl4 injection (therapeutic group) via hydrodynamic-based tail vein injection. Successful transfection was confirmed with the expression of red fluorescent protein and green fluorescent protein in hepatocytes. Western blot was used for determining the expression of PHD1 and Keap1, HE, Sirius red, and Masson staining for evaluating the histopathological stages of fibrosis. Immunohistochemical techniques were applied to evaluate the expression of a-SMA.Results: The fluorescence of red and green were observed mainly in hepatocytes, and downregulation of PHD1 and Keap1 expression in liver was detected by western blot. Meanwhile, double-knockdown of PHD1 and Keap1 in mice alleviated liver fibrosis, and the effect was further enhanced especially in the preventive group. Immunocytochemical staining showed decreased expression of a-SMA when both PHD1 and Keap1 were knockdown.Conclusion: Downregulation of PHD1 and Keap1 expression in the liver could be achieved via hydrodynamic injection of PHD1shRNA and Keap1shRNA, thereby, preventing liver fibrosis.

Contribution of prolyl hydroxyl binding proteins (PHD) to MYD88/NFkB pathways in Nitric Oxide deficiency‐induced hypertensive renal injury

Oxidative stress and associated inflammation, features of hypertension and kidney disease, involve many mediators that include Nrf2, a transcription factor, the primary cellular defense against oxidative stress, MYD88, an adaptor protein implicated in NFkB‐mediated inflammation. PHD, oxygen sensor in hypoxia is intricately linked to NO. This study evaluated the role played by PHD and its association with Nrf2, MYD88 and NFKB in hypertension‐induced renal injury.Adult rats were placed on LNNA, inhibitor of NO synthase and/or DMOG, an inhibitor of PHD. Blood pressure (BP) was determined on Days 1, 4, 7, and 14 and 24‐hour urine collected for protein excretion. Kidneys were collected to determine expression of PHD1 and PHD2, Nrf2, p65, and MYD88 in nuclear fractions. KIM‐1 expression, index of tubular damage, was assessed in whole kidneys. Plasma TNFa and IL‐1b levels, markers of inflammation, were measured by ELISA. Renal generation of oxidant species was evaluated as xanthine (3,5,10 mg/kg)‐induced generation of H2O2. LNNA, inhibitor of NO synthase increased BP (35%, P&lt;0.05), kidney weight (15%, P&lt;0.05), protein excretion (3.5‐fold, P&lt;0.05) and KIM‐1 expression (3.8‐fold, P&lt;0.05). DMOG blunted the increases. LNNA also reduced PHD2 (20%) but not PHD1 expression or Nrf2 expression. There was increased nuclear expression of p65 (34%, P&lt;0.05) and MYD88 (32%, P&lt;0.05) and increased TNFa and IL‐1b (P&lt;0.05) in LNNA‐treated rats. DMOG blunted these effects. Renal production of H2O2 by xanthine was greater (P&lt;0.05) in LNNA‐treated rats. These data suggest a role for PHD2 in hypertensive renal injury and inflammation induced by NO deficiency. The accompanying changes in MYD88/p65 expression and cytokine production confirm the ongoing inflammatory process. However, despite the increase in production of reactive oxygen species, Nrf2 does not appear to play a part in NO deficiency‐induced cardiovascular effect.Support or Funding InformationNational Institutes of Health grant 5G12 MD007605

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Objective: Previously, we demonstrated that 7 days of high salt (HS) intake down-regulated hypoxia inducible factor 1 alpha (HIF-1α) and cyclooxygenase 1 and 2 (COX1 and COX2) genes in the brain blood vessels (BBV) compared to control Sprague-Dawley (SD) rats fed standard rat chow (Physiology 2014 (London, UK), PCB177). HS diet suppresses the angiotensin II levels thus down-regulating all antioxidative enzymes genes which impairs antioxidative defense systems and increases oxidative stress. The aim of this study was to assess the effects of TEMPOL on the expression of antioxidative enzymes (Cu/Zn SOD, MnSOD, EC-SOD), COX 1,2, HIF-1α, HIF prolyl hydroxylases (PHD1, PHD2 and PHD3), vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in BBV. Design and method: Three groups of male SD rats were included in the study (N = 5 per group, age 11weeks) - control on a normal chow, HS (4%NaCl) and HS+TEMPOL (4% NaCl + TEMPOL 100 μmol/L in drinking water ad libidum). Following diet protocol, rats were anesthetized with ketamine (75 mg/kg) and midazolam (2.5 mg/kg) and sacrificed by decapitation and their BBV harvested. mRNA levels were determined by quantitative real-time PCR. Data were analyzed using t-test and p < 0.05 was considered significant. Results: There was significantly higher expression of EC-SOD, HIF-1α and VEGF in HS+TEMPOL group compared to both, control and HS group. Expression of COX2 and iNOS was significantly decreased in HS+TEMPOL compared to control group of animals. Expression of PHD1 was increased in HS + TEMPOL group in compared to HS group. Cu/Zn SOD and PHD3 gene expression was increased in HS+TEMPOL compared to both group, and expression of COX 1 and PHD2 in compared to HS group, but did not reach statistical significance. eNOS and MnSOD expression did not changed in compared to control or HS group. Conclusions: Our results show that expression of HIF-1α, VEGF and EC-SOD depends on the level of superoxide. Oxidative stress also affects the expression of COX-2 which is important in mediating functional vascular responses and may be a potential target, or gene interrelated with HIF-1α.

Study on the Expressions of PHD and HIF in Placentas from Normal Pregnant Women and Patients with Preeclampsia

Objective: To investigate the relationship between oxygen sensitivity of trophoblast and hypoxia in preeclamptic placenta by the study on the expressions of hypoxia-inducible factor prolyl 4-hydroxylase (PHD) and hypoxia-inducible factor (HIF) in placentas from normal pregnant women and patients with pre-eclampsia.Methods: Subjects were chosen from the in-patients or the out-patients from May 2003 to May 2004. They were divided into 5 groups: early pregnancy group (EP), 13 cases; middle pregnancy group (MP), 9 cases; late pregnancy group (LP, or control group), 12 cases; preeclampsia (PE) group, 20 cases; gestational hypertension group (GH), 10 cases. The mRNA expressions of PHD-1 and -2 and -3 in placentas from all the subjects were assessed by in situ hybridization and Real-time PCR. The expressions of HIF-1α and -2α in placentas from different groups were assessed by immunohistochemistry and western blot.Results: PHD-1,-2 and -3 mRNA were mainly expressed in cytoplasm of trophoblast, especially strongly expressed in extravillous trophoblast. During the progress of pregnancy, the expression of PHD-1 increased significantly (R=0.616, P<0.001). The PHD-1mRNA expression in placentas from PE group decreased significantly compared with that from control group, P<0.05. A significant direct correlation between the PHD-1 mRNA expression in placentas from PE group and their placenta weight was found (R=0.457, P<0.05). The HIF-2α, not the HIF-1α expression, from PE group was significantly higher than that from control group, P<0.01; The HIF-2α expression in trophoblast from PE was inversely correlated to the date of the onset of the disease (R=-0.730, P<0.01).Conclusions: PHD-1 played an important role in hypoxic response pathway of trophoblast through modulating the level of HIF-2α. The overly activated hypoxic response pathway of trophoblast in preeclamptic placenta, which is manifested as the result of HIF-2α over-expression, is the key point to hypoxic dysfunction of trophoblast.

Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most common and comprehensively studied malignancies. Hypoxic conditions during formation of CRC may support the development of more aggressive cancers. Hypoxia inducible factor (HIF), a major player in cancerous tissue adaptation to hypoxia, is negatively regulated by the family of prolyl hydroxylase enzymes (PHD1, PHD2, PHD3) and asparaginyl hydroxylase, called factor inhibiting HIF (FIH).MethodsPHD1, PHD2, PHD3 and FIH gene expression was evaluated using quantitative RT-PCR and western blotting in primary colonic adenocarcinoma and adjacent histopathologically unchanged colonic mucosa from patients who underwent radical surgical resection of the colon (n = 90), and the same methods were used for assessment of PHD3 gene expression in HCT116 and DLD-1 CRC cell lines. DNA methylation levels of the CpG island in the promoter regulatory region of PHD1, PHD2, PHD3 and FIH were assessed using bisulfite DNA sequencing and high resolution melting analysis (HRM) for patients and HRM analysis for CRC cell lines.ResultsWe found significantly lower levels of PHD1, PHD2 and PHD3 transcripts (p = 0.00026; p < 0.00001; p < 0.00001) and proteins (p = 0.004164; p = 0.0071; p < 0.00001) in primary cancerous than in histopathologically unchanged tissues. Despite this, we did not observe statistically significant differences in FIH transcript levels between cancerous and histopathologically unchanged colorectal tissue, but we found a significantly increased level of FIH protein in CRC (p = 0.0169). The reduced PHD3 expression was correlated with significantly increased DNA methylation in the CpG island of the PHD3 promoter regulatory region (p < 0.0001). We did not observe DNA methylation in the CpG island of the PHD1, PHD2 or FIH promoter in cancerous and histopathologically unchanged colorectal tissue. We also showed that 5-Aza-2’-deoxycytidine induced DNA demethylation leading to increased PHD3 transcript and protein level in HCT116 cells.ConclusionWe demonstrated that reduced PHD3 expression in cancerous tissue was accompanied by methylation of the CpG rich region located within the first exon and intron of the PHD3 gene. The diminished expression of PHD1 and PHD2 and elevated level of FIH protein in cancerous tissue compared to histopathologically unchanged colonic mucosa was not associated with DNA methylation within the CpG islands of the PHD1, PHD2 and FIH genes.

Inhibition of prolyl hydroxylase domain-containing protein on hypertension/renal injury induced by high salt diet and nitric oxide withdrawal

Nitric oxide just as prolyl hydroxylase domain-containing protein (PHD) is a regulator of hypoxia inducible factor-1 α (HIF-1α), a transcription factor complex that controls the expression of most genes involved in hypoxia and cardiovascular diseases. In the absence of nitric oxide, it is not clear how HIF-1α and PHD are regulated and to what extent they contribute to the ensuing disorder. Using the nitric oxide withdrawal/high salt diet model of hypertensive renal injury, this study tested the hypothesis that removal of the inhibition by nitric oxide on PHD predisposes to increased PHD but reduced HIF-1α expression, hypertension and renal injury. In animals treated with N-nitro-L-arginine (L-NNA; 250 mg/l in drinking water for 14 days) and high salt diet (4% NaCl), there was hypertension (41±5%, P<0.05), proteinuria (three-fold, P<0.05), kidney (22±3%, P<0.05) and heart enlargement (24±3%, P<0.05), as well as increased renal osteopontin (21±3%, P<0.05) and collagen IV (24±4%, P<0.05) expression. Accompanying these effects were increased expression of PHD1 (24±4%, P<0.05) and PHD2 (36±4%, P<0.05) but reduced HIF-1α (35±6%, P < 0.05) expression. Dimethyloxallyl glycine (5mg/kg), a PHD inhibitor, paradoxically exacerbated hypertension (46±7%, P<0.05), proteinuria (two-fold, P <0.05), and increased osteopontin (15±2%, P<0.05) and HIF-1α (31±5%, P<0.05) expression with no change in PHD1/2 expression or kidney and heart enlargement. These data suggest that the protective effect of physiological levels of nitric oxide may be by virtue of inhibition of PHD or increased HIF-1α expression, hence, the pathological changes produced following its withdrawal was accompanied by increased PHD or decreased HIF-1α expression. Exacerbation of hypertension and renal injury following PHD inhibition suggests a deleterious effect in the chronic setting and challenges the dogma that inhibition of PHD is useful in cardiovascular diseases.

Abstract 309: Sirt3 Regulates Oxygen Sensors and Blood Vessel Formation in the Heart

New blood vessels formation is driven by deprivation of nutrients and oxygen during myocardial ischemia. The interaction between nutrients and oxygen sensors in the regulation of new blood vessel formation still remains incompletely understood. Sirtuins belong to a family of NAD+-dependent deacetylases which are involved in the regulation of angiogenesis and energy hemostasis. In the present study, we investigated the functional role of Sirtuin 3 (Sirt3), a metabolic sensor, in the regulation of blood vessel formation and its interaction with oxygen sensors-prolyl hydroxylases (PHDs)/hypoxia-inducible factors (HIFs) system. The levels of PHD1-3, HIFs, and angiogenic growth factors as well as blood vessel formation were examined in the hearts of Sirt3 knockout (Sirt3 KO) mice and wild type (WT) mice. Our data showed that the expression of angiopoietin-1 and angiopoietin-2 was significantly reduced, whereas the expression of Tie-2 and VEGF was unchanged in the hearts of Sirt3 KO mice compared to WT mice. Intriguingly, loss of Sirt3 had different effects on the PHDs/HIFs system. The expression of PHD1, PHD2, and HIF1α was significantly up-regulated, but PHD3 and HIF2α expression was down-regulated in Sirt3 KO mice. Ex vivo angiogenesis assay revealed that vessel outgrowth was significantly reduced in Sirt3 KO mice compared to WT mice. Our immunohistochemistry study further confirmed that the arteriole formation was significantly reduced in the hearts of Sirt3 KO mice compared to that in WT mice. This was accompanied by a reduction of cardiac ejection fraction (EF %) and fraction shortening (FS %). Moreover, the phosphorylation level of Akt was dramatically decreased together with increased Wnt7b, caspase-3 expression and apoptosis in the hearts of Sirt3 KO mice. Therefore, our data implicates that Sirt3 is important in controlling of blood vessel growth and cell survival by affecting oxygen sensors and angiopoietins expression in the heart.

Expression of Prolyl Hydroxylases (PHDs) Is Selectively Controlled by HIF-1 and HIF-2 Proteins in Nucleus Pulposus Cells of the Intervertebral Disc: DISTINCT ROLES OF PHD2 AND PHD3 PROTEINS IN CONTROLLING HIF-1α ACTIVITY IN HYPOXIA

Adaptive response to hypoxia in nucleus pulposus cells of the intervertebral disc is regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Moreover, oxygen-dependent turnover of HIF-1α in these cells is controlled by the prolyl-4-hydroxylase domain (PHD) family of proteins. Whether HIF homologues control expression of PHDs and whether PHDs control hypoxia-inducible factor (HIF) turnover and/or activity under hypoxia is not known. Here, we show that in nucleus pulposus cells, hypoxia robustly induces PHD3 expression and, to a lesser extent, of PHD2 and PHD1. Reporter analysis shows that the hypoxic induction of the PHD2 promoter is HIF-1α dependent, whereas PHD3 promoter/enhancer activity is dependent on both HIF-1α and HIF-2α. Lentiviral delivery of HIF-1α, ShHIF-1α, and ShHIF-1β confirmed these observations. Noteworthy, HIF-1α maintains basal expression of PHD1 in hypoxia at the posttranscriptional level. Finally, loss of function studies using lentiviral transduction of ShPHDs clearly shows that even at 1% O(2), PHD2 selectively degrades HIF-1α. In contrast, in hypoxia, PHD3 enhances HIF-1α transcriptional activity without affecting protein levels. To correlate these observations with disc disease, a condition characterized by tissue vascularization, we analyzed human tissues. Increased PHD1 mRNA expression but decreased PHD2 and PHD3 expression is observed in degenerate tissues. Interestingly, the hypoxic responsiveness of all the PHDs is maintained in isolated nucleus pulposus cells regardless of the disease state. We propose that PHD2 and PHD3 can be used as a biomarker of tissue oxygenation in the disc and that, as such, it may have important clinical implications.

Abstract 344: Oxygen sensors are poor prognosticators for survival in NSCLC

Abstract Background: Most solid tumors are at least partly hypoxic. To survive hypoxia, cancer cells must adapt by hypoxic gene activation. Hypoxia-inducible factors (HIFs) have been recognized as key regulators of this transcriptional control. However, oxygen tension is sensed by three HIF prolyl hydroxylases (PHD1, PHD2 and PHD3) and one asparaginyl hydroxylase, FIH (Foctor inhibiting HIF). This study evaluates the prognostic impact of these four oxygen sensors in the biggest killer in cancer, Non-Small Cell Lung Cancer (NSCLC). Methods: 335 unselected stage I-IIIA NSCLC-patients were included. Duplicate 0.6 mm diameter cores from both the viable neoplastic cell compartment (tumor) and stroma were collected in tissue microarrays (TMAs). Immunohistochemistry (IHC) was used to semiquantitatively evaluate the expression. Results: There was only scorable expression in the tumor compartment. In univariate analyses high expression of all the oxygen sensors in tumor were significantly associated with a poor prognosis (PHD1, P = 0.023; PHD2, P = 0.013; PHD3, P = 0.018; FIH, P = 0.033). There were only weak correlations between these markers (strongest between PHD2 and FIH; r = 0.18, P = 0.001). In the multivariate analysis, entering all the significant clinicopathological variables and the oxygen sensors, we found only high expression of PHD2 (HR 2.03; CI 95% 1.20-3.42; P = 0.008) and PHD1 (HR 1.45; CI 95% 1.01-2.10; P = 0.047) of the molecular variables to be independent poor prognosticators. Conclusions: Oxygen sensors were only scorable in tumor. High expression of all the oxygen sensors was associated with a poor prognosis. High expression of PHD1 and PHD2 were also significantly and independently associated with a dismal prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 344. doi:10.1158/1538-7445.AM2011-344

Oxygen Sensing in Drosophila: Multiple Isoforms of the Prolyl Hydroxylase Fatiga Have Different Capacity to Regulate HIFα/Sima

BackgroundThe Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia. Three HIF-prolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing.Methodology and Principal FindingsWe have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter.Conclusions and SignificanceDiversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain.

Expression of key hypoxia sensing prolyl‐hydroxylases PHD1, ‐2 and ‐3 in pancreaticobiliary cancer

Tumour hypoxia is associated with an aggressive phenotype and resistance to chemotherapy and radiotherapy. The aim was to investigate whether key hypoxia sensing prolyl hydroxylases PHD1, PHD2 and PHD3 are dysregulated in pancreaticobiliary cancers, and to evaluate their potential clinical significance. Formalin-fixed human pancreatic tissue from 120 consecutive patients undergoing pancreatic resections between June 2001 and June 2006 was constructed into tissue microarrays. Expression of PHD1, PHD2 and PHD3 was analysed using immunohistochemistry and correlated with clinicopathological variables and disease-specific overall survival. PHD1, PHD2 and PHD3 were significantly overexpressed in pancreaticobiliary tumours compared with normal pancreatic ductal tissues (P = 0.03, P < 0.0001 and P < 0.0001, respectively). PHD3 expression in tumour tissue was associated with a trend towards worse overall disease-specific survival in ampullary adenocarcinomas (P = 0.035) and pancreatic adenocarcinomas (P = 0.084). Absence of PHD1 expression was significantly associated with perineural invasion in pancreatic adenocarcinomas (P = 0.02) and a trend towards significance was also seen for absence of PHD2 expression in pancreatic adenocarcinomas (P = 0.04). Our results provide the first clinical evidence that PHD1, PHD2 and PHD3 may be involved in pancreaticobiliary tumorigenesis.

Enhancement of angiogenic effectors through hypoxia-inducible factor in preterm primate lung in vivo

Development of lung microvasculature is critical for distal airway formation. Both processes are arrested in the lungs of preterm newborns with bronchopulmonary dysplasia (BPD), a chronic form of lung disease. We hypothesized that activation of hypoxia-inducible factors (HIFs) augments lung vascular development. Pulmonary angiogenic factors were assessed by quantitative real-time PCR, Western blot, and immunohistochemistry in preterm baboons (125 days+14 days pro re nata O2 model) treated for 14 days with intravenous FG-4095, an inhibitor of prolyl hydroxylase domain-containing proteins (PHDs) that initiates HIF degradation. HIF-1alpha, but not HIF-2alpha, mRNA and protein were increased (8- and 3-fold, respectively) in FG-4095-treated baboons relative to untreated controls. Expression of PHD-1, -2, and -3 was unchanged. Of note, mRNA and/or protein for platelet-endothelial cell adhesion molecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) were increased by FG-4095. Moreover, PECAM-1-expressing capillary endothelial cells detected by immunohistochemistry were augmented in FG-4095-treated baboons to levels comparable to those in fetal age-matched controls. Alveolar septal cell expression of Ki67, a proliferative marker, and VEGF were similar in untreated controls and FG-4095-treated neonates. These results indicate that HIF stimulation by PHD inhibition enhances lung angiogenesis in the primate model of BPD.

Oxygen-sensitive reset of hypoxia-inducible factor transactivation response: Prolyl hydroxylases tune the biological normoxic set point

Cellular O 2 sensing enables physiological adjustments to variations in tissue pO 2. Under basal conditions, cells are adjusted to an O 2 environment biologically read as normoxia. Any sharp departure from that state of normoxia triggers O 2-sensitive biological responses. The stabilization of hypoxia-inducible factor (HIF) signifies a robust biological readout of hypoxia. In the presence of sufficient O 2, HIF is hydroxylated and degraded. HIF prolyl hydroxylation is catalyzed by prolyl hydroxylase isoenzymes PHD1, 2, and 3. Using HT22 neurons stably transfected with a HIF reporter construct, we tested a novel hypothesis postulating that biological cells are capable of resetting their normoxic set point by O 2-sensitive changes in PHD expression. Results of this study show that the pO 2 of the mouse brain cortex was 35 mm Hg or 5% O 2. Exposure of HT22, adjusted to growing in 20% O 2, to 5% O 2 resulted in HIF-driven transcription. However, cells adjusted to growing in 5% O 2 did not report hypoxia. Cells adjusted to growing in 30% O 2 reported hypoxia when acutely exposed to room air culture conditions. When grown under high O 2 conditions, cells reset their normoxic set point upward by down-regulating the expression of PHD1–3. When grown under low O 2 conditions, cells reset their normoxic set point downward by inducing the expression of PHD1–3. Exposure of mice in vivo to a hypoxic 10% O 2 environment lowered blood as well as brain pO 2. Such hypoxic exposure induced PHD1–3. Exposure of mice to a hyperoxic 50% O 2 ambience repressed the expression of PHD1–3, indicating that O 2-sensitive regulation of PHD expression is effective in the brain in vivo. siRNA dependent knockdown of PHD expression revealed that O 2-sensitive regulation of PHD may contribute to tuning the normoxic set point in biological cells.

Hypoxia-dependent regulation of PHD1: cloning and characterization of the human PHD1/EGLN2 gene promoter

The recent identification of hypoxia-inducible-factor (HIF) prolyl hydroxylases (PHD1, 2, and 3), which modify HIF-1α in an oxygen-dependent manner, provided an important link between oxygen availability and hypoxia-induced gene expression. However, little is known about the regulation of the PHDs. To investigate the transcriptional regulation of PHD1, we cloned the PHD1 gene promoter. Here, we report that the expression of PHD1 is reduced under hypoxic conditions. Furthermore, we identified binding sites for aryl hydrocarbon nuclear translocator (ARNT/HIF-1β) within the PHD1 promoter, and showed that ARNT is associated in vivo with the PHD1 promoter following hypoxia, which implies a role for ARNT in the hypoxia-dependent regulation of PHD1. Taken together, our findings suggest a hypoxia-induced regulatory loop of PHD1 expression, mediated by ARNT.