The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-α subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-α for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione S-transferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.
The central transcriptional response to hypoxia is mediated by the Prolyl Hydroxylase Domain protein (PHD):Hypoxia Inducible Factor (HIF) pathway. In this pathway, PHD prolyl hydroxylates and thereby negatively regulates the α-subunit of the transcription factor HIF (HIF-α). An important HIF target gene is that for Erythropoietin (EPO), which controls red cell mass. Recent studies have identified PHD2 as the critical PHD isoform regulating the EPO gene. Other studies have shown that the inducibility of the HIF pathway diminishes as a function of age. Thus, an important question is whether the PHD2:EPO pathway is altered in the aging. Here, we employed a mouse line with a globally-inducible Phd2 conditional knockout allele to examine the integrity of the Phd2:Epo axis in young (six to eight month old) and aging (sixteen to twenty month old) mice. We find that acute global deletion of Phd2 results in a robust erythrocytosis in both young and aging mice, with both age groups showing marked extramedullary hematopoiesis in the spleen. Epo mRNA is dramatically upregulated in the kidney, but not in the liver, in both age groups. Conversely, other Hif targets, including Vegf, Pgk1, and Phd3 are upregulated in the liver but not in the kidney in both age groups. These findings have implications for targeting this pathway in the aging.
Erythropoietin; Prolyl Hydroxylase Domain protein; Hypoxia Inducible Factor; Prolyl hydroxylation; Gene regulation
Originally identified as the enzymes responsible for catalysing the oxidation of specific, conserved proline residues within hypoxia-inducible factor-1α (HIF-1α), the additional roles for the prolyl hydroxylase domain (PHD) proteins have remained elusive. Of the four identified PHD enzymes, PHD2 is considered to be the key oxygen sensor, as knockdown of PHD2 results in elevated HIF protein. Several recent studies have highlighted the importance of PHD2 in tumourigenesis. However, there is conflicting evidence as to the exact role of PHD2 in tumour angiogenesis. The divergence seems to be because of the contribution of stromal-derived PHD2, and in particular the involvement of endothelial cells, vs tumour-derived PHD2. This review summarises our current understanding of PHD2 and tumour angiogenesis, focusing on the influences of PHD2 on vascular normalisation and neovascularisation.
PHD2; tumour angiogenesis; HIF
Activation of hypoxia-inducible factors (HIFs), responsible for tumor angiogenesis and glycolytic switch, is regulated by reduced oxygen availability. Normally, HIF-α proteins are maintained at low levels, controlled by site-specific hydroxylation carried out by HIF prolyl hydroxylases (PHDs), and subsequent proteasomal degradation via the von Hippel-Lindau (VHL) ubiquitin ligase. Using a yeast-two hybrid screen, we identified an interaction between MAGE-11 cancer-testis antigen and the major HIF-α hydroxylating enzyme PHD2. The interaction was confirmed by pull-down assay, co-immunoprecipitation and co-localization in both normoxic and hypoxic conditions. Furthermore, MAGE-9, the closest homolog of MAGE-11, was also found to interact with PHD2. MAGE-11 inhibited PHD activity without affecting protein levels. This inhibition was accompanied by stabilization of ectopic or endogenous HIF-1α protein. Knock-down of MAGE-11 by siRNA results in decreased hypoxic induction of HIF-1α and its target genes. Inhibition of PHD by MAGE-11 and following activation of hypoxia-inducible factors is a novel tumor associated HIF regulatory mechanism. This finding provides new insights into the significance of MAGE expression in tumors and may provide valuable tools for therapeutic intervention because of the restricted expression of the MAGE gene family in cancers but not in normal tissues.
HIF; hypoxia; MAGE-11; PHD2
The prolyl-hydroxylase domain family of enzymes (PHD1-3) plays an important role in the cellular response to hypoxia by negatively regulating HIF-α proteins. Disruption of this process can lead to up-regulation of factors that promote tumorigenesis. We observed decreased basal expression of PHD3 in prostate cancer tissue and tumor cell lines representing diverse tissues of origin. Furthermore, some cancer lines displayed a failure of PHD3 mRNA induction when introduced to a hypoxic environment. This study explores the mechanism by which malignancies neither basally express PHD3 nor induce PHD3 under hypoxic conditions.
Using bisulfite sequencing and methylated DNA enrichment procedures, we identified human PHD3 promoter hypermethylation in prostate, breast, melanoma and renal carcinoma cell lines. In contrast, non-transformed human prostate and breast epithelial cell lines contained PHD3 CpG islands that were unmethylated and responded normally to hypoxia by upregulating PHD3 mRNA. Only treatment of cells lines containing PHD3 promoter hypermethylation with the demethylating drug 5-aza-2′-deoxycytidine significantly increased the expression of PHD3.
We conclude that expression of PHD3 is silenced by aberrant CpG methylation of the PHD3 promoter in a subset of human carcinoma cell lines of diverse origin and that this aberrant cytosine methylation status is the mechanism by which these cancer cell lines fail to upregulate PHD3 mRNA. We further show that a loss of PHD3 expression does not correlate with an increase in HIF-1α protein levels or an increase in the transcriptional activity of HIF, suggesting that loss of PHD3 may convey a selective advantage in some cancers by affecting pathway(s) other than HIF.
The human prolyl-4-hydroxylase domain (PHD) proteins 1–3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathways.
PHD1, PHD2, and PHD3 are prolyl hydroxylase domain proteins that regulate the stability of hypoxia-inducible factor α subunits (HIF-α). To determine the roles of individual PHDs during mouse development, we disrupted all three Phd genes and found that Phd2−/− embryos died between embryonic days 12.5 and 14.5 whereas Phd1−/− or Phd3−/− mice were apparently normal. In Phd2−/− mice, severe placental and heart defects preceded embryonic death. Placental defects included significantly reduced labyrinthine branching morphogenesis, widespread penetration of the labyrinth by spongiotrophoblasts, and abnormal distribution of trophoblast giant cells. The expression of several trophoblast markers was also altered, including an increase in the spongiotrophoblast marker Mash2 and decreases in the labyrinthine markers Tfeb and Gcm1. In the heart, trabeculae were poorly developed, the myocardium was remarkably thinner, and interventricular septum was incompletely formed. Surprisingly, while there were significant global increases in HIF-α protein levels in the placenta and the embryo proper, there was no specific HIF-α increase in the heart. Taken together, these data indicate that among all three PHD proteins, PHD2 is uniquely essential during mouse embryogenesis.
The 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 Findings
We 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 Significance
Diversity 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.
We previously reported that interferons (IFNs) regulate transcription of HIF-1α in human endothelial cells (ECs), linking immunity and hypoxia. Prolyl hydroxylases (PHDs) regulate expression of HIF-1α in response to hypoxia. We examined whether IFNs affect PHD expression and whether PHDs regulate the EC response to IFNs.
Methods and Results
Human cell cultures were treated with various cytokines and PHD expression was examined using qRT-PCR and immunoblotting. IFNγ and, to a lesser extent, IFNα significantly induced PHD3, but not PHD1 or 2, mRNA and protein expression selectively in ECs directly via a JAK/STAT1 pathway as demonstrated by pharmacological inhibition, siRNA knockdown and chromatin immunoprecipitation. Inhibition of PHD activity with dimethyloxallyl glycine or desferroxamine reduced IFNg-dependent responses in these same cells.
IFNγ induces PHD3 through a JAK/STAT1-dependent mechanism in human ECs. Induction is independent of HIF-1α and may contribute to expression of IFNγ-dependent genes.
IFNγ signaling; endothelial cells; hypoxia; PHD3; HIF-1α
Hypoxia inducible factor (HIF) is a product of tumor cells that plays an important role in protecting tumor cells and adjusting to low oxygen tension through driving the progression and aggressiveness of tumors and changing the growth, angiogenesis, differentiation and metastasis of tumors. Prolyl hydroxylase 3 (PHD3) is a member of PHDs that are induced in hypoxia. Many studies have shown that PHD3 not only can hydroxylate HIF-1α, but also has various other biological functions. Thus PHD3 plays significant roles in suppressing the growth, angiogenesis, differentiation and metastasis of tumors and promoting apoptosis of tumors under hypoxic conditions. It may become a new tumor suppressor gene and also may become a new approach to investigate tumors.
prolyl hydroxylase 3; cancer; hypoxia inducible factors
The HIF prolyl hydroxylases (PHDs/EGLNs) are central regulators of the molecular responses to oxygen availability. One isoform, PHD3, is expressed in response to hypoxia and causes apoptosis in oxygenated conditions in neural cells. Here we show that PHD3 forms subcellular aggregates in an oxygen-dependent manner. The aggregation of PHD3 was seen under normoxia and was strongly reduced under hypoxia or by the inactivation of the PHD3 hydroxylase activity. The PHD3 aggregates were dependent on microtubular integrity and contained components of the 26S proteasome, chaperones, and ubiquitin, thus demonstrating features that are characteristic for aggresome-like structures. Forced expression of the active PHD3 induced the aggregation of proteasomal components and activated apoptosis under normoxia in HeLa cells. The apoptosis was seen in cells prone to PHD3 aggregation and the PHD3 aggregation preceded apoptosis. The data demonstrates the cellular oxygen sensor PHD3 as a regulator of protein aggregation in response to varying oxygen availability.
High salt induces the expression of transcription factor hypoxia-inducible factor (HIF)-1α and its target genes in the renal medulla, which is an important renal adaptive mechanism to high salt intake. HIF prolyl hydroxylase domain-containing proteins (PHDs) have been identified as major enzymes to promote the degradation of HIF-1α. PHD2 is the predominant isoform of PHDs in the kidney and primarily expressed in the renal medulla. The present study tested the hypothesis that PHD2 responds to high salt and mediates high salt-induced increase in HIF-1α levels in the renal medulla. In normotensive rats, high salt intake (4% NaCl, 10 days) significantly inhibited PHD2 expressions and enzyme activities in the renal medulla. Renal medullary overexpression of PHD2 transgene significantly decreased HIF-1α levels. PHD2 transgene also blocked high salt-induced activation of HIF-1α target genes heme oxygenase-1 and nitric oxide synthase-2 in the renal medulla. In Dahl salt-sensitive hypertensive rats, however, high salt intake did not inhibit the expression and activities of PHD2 in the renal medulla. Correspondingly, renal medullary HIF-1α levels were not up-regulated by high salt intake in these rats. After transfection of PHD2 shRNA, HIF-1α and its target genes were significantly up-regulated by high salt intake in Dahl S rats. Overexpression of PHD2 transgene in the renal medulla impaired renal sodium excretion after salt loading. These data suggest that high salt intake inhibits PHD2 in the renal medulla, thereby upregulating the HIF-1α expression. The lack of PHD-mediated response to high salt may represent a pathogenic mechanism producing salt sensitive hypertension.
hypertension; transcription factor; gene transfection; Dahl salt-sensitive rats; siRNA
The protein “amplified in osteosarcoma-9” (OS-9) has been shown previously to interact with the prolyl hydroxylases PHD2 and PHD3. These enzymes initiate oxygen-dependent degradation of the α-subunit of hypoxia-inducible factor (HIF), a transcription factor that adapts cells to insufficient oxygen supply (hypoxia). A new model has been proposed where OS-9 triggers PHD dependent degradation of HIF-α. It was the aim of our study to define the molecular mode of action of OS-9 in the regulation of PHD and HIF activity. Although initial co-immunoprecipitation experiments confirmed physical interaction between OS-9 and PHD2, neither overexpression nor lentiviral inhibition of OS-9 expression affected HIF regulation. Subcellular localization experiments revealed a distinct reticular staining pattern for OS-9 while PHD2 was mainly localized in the cytoplasm. Further cell fractionation experiments and glycosylation tests indicated that OS-9 is a luminal ER protein. In vivo protein interaction analysis by fluorescence resonance energy transfer (FRET) showed no significant physical interaction of overexpressed PHD2-CFP and OS-9-YFP. We conclude that OS-9 plays no direct functional role in HIF degradation since physical interaction of OS-9 with oxygen sensing HIF prolyl hydroxylases cannot occur in vivo due to their different subcellular localization.
The regulation of neutrophil lifespan by induction of apoptosis is critical for maintaining an effective host response and preventing excessive inflammation. The hypoxia-inducible factor (HIF) oxygen-sensing pathway has a major effect on the susceptibility of neutrophils to apoptosis, with a marked delay in cell death observed under hypoxic conditions. HIF expression and transcriptional activity are regulated by the oxygen-sensitive prolyl hydroxylases (PHD1–3), but the role of PHDs in neutrophil survival is unclear. We examined PHD expression in human neutrophils and found that PHD3 was strongly induced in response to hypoxia and inflammatory stimuli in vitro and in vivo. Using neutrophils from mice deficient in Phd3, we demonstrated a unique role for Phd3 in prolonging neutrophil survival during hypoxia, distinct from other hypoxia-associated changes in neutrophil function and metabolic activity. Moreover, this selective defect in neutrophil survival occurred in the presence of preserved HIF transcriptional activity but was associated with upregulation of the proapoptotic mediator Siva1 and loss of its binding target Bcl-xL. In vivo, using an acute lung injury model, we observed increased levels of neutrophil apoptosis and clearance in Phd3-deficient mice compared with WT controls. We also observed reduced neutrophilic inflammation in an acute mouse model of colitis. These data support what we believe to be a novel function for PHD3 in regulating neutrophil survival in hypoxia and may enable the development of new therapeutics for inflammatory disease.
Prolyl hydroxylase domain 2 (PHD2) has been implicated in several pathways of cell signaling, most notably in its regulation of hypoxia inducible factor (HIF)-1α stability. In normoxia, PHD2 hydroxylates proline residues on HIF-1α, rendering it inactive. However in hypoxia, PHD2 is inactive, HIF-1α is stabilized and downstream effectors such as VEGF and FGF-2 are produced to promote angiogenesis. In the present study we utilize RNAi to PHD2 to promote therapeutic angiogenesis in a diabetic wound model, presumably by the stabilization of HIF-1α.
Stented wounds were created on the dorsum of diabetic Lpr db/db mice. Mice were treated with PHD2 siRNA or nonsense siRNA. Wounds were measured photometrically on days 0–28. Wounds were harvested for histology, protein, and RNA analysis.
Diabetic wounds treated with siRNA closed within 21 +/−1.2 days; sham treated closed in 28 +/−1.5 days. By day 7, Western blot revealed near complete suppression of PHD protein and corresponding increased HIF-1α. Angiogenic mediators VEGF and FGF-2 were elevated, corresponding to increased CD31 staining in the treated groups.
siRNA-mediated silencing of PHD2 increases HIF-1α and several mediators of angiogenesis. This corresponded to improved time to closure in diabetic wounds compared to sham treated wounds. These findings suggest that impaired wound healing in diabetes can be ameliorated with therapeutic angiogenesis.
diabetic wound; PHD2; HIF-1
Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor induced by hypoxia. Under normoxic conditions, site-specific proline hydroxylation of the α subunits of HIF allows recognition by the von Hippel-Lindau tumor suppressor protein (VHL), a component of an E3 ubiquitin ligase complex that targets these subunits for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this hydroxylation is inhibited, allowing the α subunits of HIF to escape VHL-mediated degradation. Three enzymes, prolyl hydroxylase domain-containing proteins 1, 2, and 3 (PHD1, -2, and -3; also known as HIF prolyl hydroxylase 3, 2, and 1, respectively), have recently been identified that catalyze proline hydroxylation of HIF α subunits. These enzymes hydroxylate specific prolines in HIF α subunits in the context of a strongly conserved LXXLAP sequence motif (where X indicates any amino acid and P indicates the hydroxylacceptor proline). We report here that PHD2 has the highest specific activity toward the primary hydroxylation site of HIF-1α. Furthermore, and unexpectedly, mutations can be tolerated at the −5, −2, and −1 positions (relative to proline) of the LXXLAP motif. Thus, these results provide evidence that the only obligatory residue for proline hydroxylation in HIF-1α is the hydroxylacceptor proline itself.
Germline mutations in the von Hippel–Lindau disease (VHL) and succinate dehydrogenase subunit B (SDHB) genes can cause inherited phaeochromocytoma and/or renal cell carcinoma (RCC). Dysregulation of the hypoxia-inducible factor (HIF) transcription factors has been linked to VHL and SDHB-related RCC; both HIF dysregulation and disordered function of a prolyl hydroxylase domain isoform 3 (PHD3/EGLN3)-related pathway of neuronal apoptosis have been linked to the development of phaeochromocytoma. The 2-oxoglutarate-dependent prolyl hydroxylase enzymes PHD1 (EGLN2), PHD2 (EGLN1) and PHD3 (EGLN3) have a key role in regulating the stability of HIF-α subunits (and hence expression of the HIF-α transcription factors). A germline PHD2 mutation has been reported in association with congenital erythrocytosis and recurrent extra-adrenal phaeochromocytoma. We undertook mutation analysis of PHD1, PHD2 and PHD3 in two cohorts of patients with features of inherited phaeochromocytoma (n=82) and inherited RCC (n=64) and no evidence of germline mutations in known susceptibility genes. No confirmed pathogenic mutations were detected suggesting that mutations in these genes are not a frequent cause of inherited phaeochromocytoma or RCC.
Tumor hypoxia induces the up-regulation of Hif-1alpha which in turn induces the expression of genes including VEGF to recruit new blood vessel outgrowth, enabling tumor growth and metastasis. Interference with the Hif-1 pathway and neoangiogenesis is an attractive anti-tumor target. The hydroxylation of Hif-1alpha by PHD proteins during normoxia serves as a recognition motif for its proteasomal degradation. However, under hypoxic conditions, hydroxylation is inhibited and furthermore, PHD proteins are themselves poly-ubiquitylated and degraded by Siah ubiqiuitin ligases. Our data demonstrate for the first time that inhibition of the interaction between Siah and PHD proteins using a peptide derived from a Drosophila protein interferes with the PHD degradation. Furthermore, cells stably expressing the inhibitor display reduced up-regulation of Hif-1alpha protein levels and Hif-1 mediated gene expression under hypoxia. In a syngeneic mouse model of breast cancer, the inhibitor reduced tumor growth and neoangiogenesis and prolonged survival of the mice. In addition, levels of Hif-1alpha and its target Glut-1 are reduced in the inhibitor expressing tumors. These data demonstrate, in a proof-of-principle study, that Siah protein, the most upstream component of the hypoxia pathway yet identified, is a viable drug target for anti-tumor therapies.
Caloric restriction remains the most reproducible measure known to extend life span or diminish age-associated changes. Previously, we have described an elevated expression of the prolyl-4-hydroxylase domain (PHD) 3 with increasing age in mouse and human heart. PHDs modulate the cellular response towards hypoxia by regulating the stability of the α-subunit of the transcriptional activator hypoxia inducible factor (HIF). In the present study we demonstrate that elevated PHD3, but not PHD1 or PHD2, expression is not restricted to the heart but does also occur in rat skeletal muscle and liver. Elevated expression of PHD3 is counteracted by a decrease in caloric intake (40% caloric restriction applied for 6 months) in all three tissues. Age-associated changes in PHD3 expression inversely correlated with the expression of the HIF-target gene macrophage migration inhibitory factor (MIF), which has been previously described to be involved in cellular HIF-mediated anti-ageing effects. These data give insight into the molecular consequences of caloric restriction, which influences hypoxia-mediated gene expression via PHD3.
Ageing; HIF-1α; Hypoxia; Prolyl-4-hydroxylase domain
Colorectal 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).
PHD1, 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.
We 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.
We 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.
Hypoxia-inducible transcription factor (HIF)-prolyl hydroxylases domain (PHD-1–3) are oxygen sensors that regulate the stability of the HIFs in an oxygen-dependent manner. Suppression of PHD enzymes leads to stabilization of HIFs and offers a potential treatment option for many ischemic disorders, such as peripheral artery occlusive disease, myocardial infarction, and stroke. Here, we show that homozygous disruption of PHD-1 (PHD-1−/−) could facilitate HIF-1α-mediated cardioprotection in ischemia/reperfused (I/R) myocardium. Wild-type (WT) and PHD-1−/− mice were randomized into WT time-matched control (TMC), PHD-1−/− TMC (PHD1TMC), WT I/R, and PHD-1−/− I/R (PHD1IR). Isolated hearts from each group were subjected to 30 min of global ischemia followed by 2 h of reperfusion. TMC hearts were perfused for 2 h 30 min without ischemia. Decreased infarct size (35% ± 0.6% vs. 49% ± 0.4%) and apoptotic cardiomyocytes (106 ± 13 vs. 233 ± 21 counts/100 high-power field) were observed in PHD1IR compared to wild-type ischemia/reperfusion (WTIR). Protein expression of HIF-1α was significantly increased in PHD1IR compared to WTIR. mRNA expression of β-catenin (1.9-fold), endothelial nitric oxide synthase (1.9-fold), p65 (1.9-fold), and Bcl-2 (2.7-fold) were upregulated in the PHD1IR compared with WTIR, which was studied by real-time quantitative polymerase chain reaction. Further, gel-shift analysis showed increased DNA binding activity of HIF-1α and nuclear factor-kappaB in PHD1IR compared to WTIR. In addition, nuclear translocation of β-catenin was increased in PHD1IR compared with WTIR. These findings indicated that silencing of PHD-1 attenuates myocardial I/R injury probably by enhancing HIF-1α/β-catenin/endothelial nitric oxide synthase/nuclear factor-kappaB and Bcl-2 signaling pathway. Antioxid. Redox Signal. 15, 1789–1797.
The purpose of the present study was to investigate the relationship of expression of hypoxia inducible factor (HIF)-1α-modifying enzymes prolyl hydroxylase (PHD)1, PHD2 and PHD3 to response of tumours and survival in breast cancer patients enrolled in a phase II trial of neoadjuvant anthracycline and tamoxifen therapy.
The expression of PHD1, PHD2 and PHD3 together with HIF-1α and the HIF-inducible genes vascular endothelial cell growth factor (VEGF) and carbonic anhydrase IX were assessed by immunohistochemistry using a tissue microarray approach in 211 patients with T2-4 N0-1 breast cancer enrolled in a randomised trial comparing single-agent epirubicin versus epirubicin and tamoxifen as the primary systemic treatment.
PHD1, PHD2 and PHD3 were detected in 47/179 (26.7%), 85/163 (52.2%) and 69/177 (39%) of tumours at baseline. PHD2 and PHD3 expression was moderate/strong whereas PHD1 expression was generally weak. There was a significant positive correlation between HIF-1α and PHD1 (P = 0.002) and PHD3 (P < 0.05) but not PHD2 (P = 0.41). There was a significant positive relationship between VEGF and PHD1 (P < 0.008) and PHD3 (P = 0.001) but not PHD2 (P = 0.09). PHD1, PHD2 and PHD3 expression was significantly increased after epirubicin therapy (all P < 0.000) with no significant difference in PHD changes between the treatment arms. There was no significant difference in response in tumours that expressed PHDs and PHD expression was not associated with survival.
Although expression of the PHDs was not related to response or survival in patients receiving neoadjuvant epirubicin, our data provide the first evidence that these enzymes are upregulated on therapy in breast cancer and that the biological effects independent of HIF make them therapeutic targets.
Hypertension induces cardiovascular hypertrophy and fibrosis. Infiltrated macrophages are critically involved in this process. We recently reported that inhibition of prolyl hydroxylase domain protein 2 (PHD2), which hydroxylates the proline residues of hypoxia‐inducible factor‐α (HIF‐α) and thereby induces HIF‐α degradation, suppressed inflammatory responses in macrophages. We examined whether myeloid‐specific Phd2 deletion affects hypertension‐induced cardiovascular remodeling.
Methods and Results
Myeloid‐specific PHD2‐deficient mice (MyPHD2KO) were generated by crossing Phd2‐floxed mice with LysM‐Cre transgenic mice, resulting in the accumulation of HIF‐1α and HIF‐2α in macrophage. Eight‐ to ten‐week‐old mice were given NG‐nitro‐L‐arginine methyl ester (L‐NAME), a nitric oxide synthase inhibitor, and Angiotensin II (Ang II) infusion. L‐NAME/Ang II comparably increased systolic blood pressure in control and MyPHD2KO mice. However, MyPHD2KO mice showed less aortic medial and adventitial thickening, and macrophage infiltration. Cardiac interstitial fibrosis and myocyte hypertrophy were also significantly ameliorated in MyPHD2KO mice. Transforming growth factor‐β and collagen expression were decreased in the aorta and heart from MyPHD2KO mice. Echocardiographic analysis showed that left ventricular hypertrophy and reduced ejection fraction induced by L‐NAME/Ang II treatment in control mice were not observed in MyPHD2KO mice. Administration of digoxin that inhibits HIF‐α synthesis to L‐NAME/Ang II‐treated MyPHD2KO mice reversed these beneficial features.
Phd2 deletion in myeloid lineage attenuates hypertensive cardiovascular hypertrophy and fibrosis, which may be mediated by decreased inflammation‐ and fibrosis‐associated gene expression in macrophages. PHD2 in myeloid lineage plays a critical role in hypertensive cardiovascular remodeling.
fibrosis; hypertrophy; hypoxia; macrophages; migration
Cellular O2 sensing enables physiological adjustments to variations in tissue pO2. Under basal conditions, cells are adjusted to an O2 environment biologically read as normoxia. Any sharp departure from that state of normoxia triggers O2-sensitive biological responses. The stabilization of hypoxia-inducible factor (HIF) signifies a robust biological read-out of hypoxia. In the presence of sufficient O2, 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 O2-sensitive changes in PHD expression. Results of this study show that the pO2 of the mouse brain cortex was 35 mm Hg or 5% O2. Exposure of HT22, adjusted to growing in 20% O2, to 5% O2 resulted in HIF-driven transcription. However, cells adjusted to growing in 5% O2 did not report hypoxia. Cells adjusted to growing in 30% O2 reported hypoxia when acutely exposed to room air culture conditions. When grown under high O2 conditions, cells reset their normoxic set-point upwards by down-regulating the expression of PHD1–3. When grown under low O2 conditions, cells reset their normoxic set-point downwards by inducing the expression of PHD1–3. Exposure of mice in vivo to a hypoxic 10% O2 environment lowered blood as well as brain pO2. Such hypoxic exposure induced PHD1–3. Exposure of mice to a hyperoxic 50% O2 ambience repressed the expression of PHD1–3 indicating that O2-sensitive regulation of PHD expression is effective in the brain in vivo. siRNA dependent knock-down of PHD expression revealed that O2-sensitive regulation of PHD may contribute to tuning the normoxic set-point in biological cells.
Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1α. The effects of PHD3 in tumour growth are largely unknown.
PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model.
PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo, PHD3 inhibited tumour growth by abrogation of tumour angiogenesis.
Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.
angiogenesis; HIF-1; hypoxia; pancreatic cancer; PHD3