Prolyl Hydroxylase Domain Protein Research Articles

Abstract 370: BACH1 metastatic activity is regulated by oxygen-dependent proline hydroxylation in triple-negative breast cancer

Abstract Hypoxia, low oxygen availability at the tissue level, is a key driver of metastasis in solid tumors. Oxygen-sensitive activation of hypoxia-induced transcription factors is in part regulated by prolyl hydroxylase domain proteins (PHDs). We and others previously showed that the BTB and CNC homolog 1 (BACH1) transcription factor promotes metastasis in various cancer types. In accompanying work, we show that BACH1 is prolyl-hydroxylated and degraded in an oxygen-dependent manner in triple-negative breast cancer (TNBC). Here we show that BACH1 lacking its hydroxylated proline sites (i.e., mutant BACH1) expressed in TNBC cells exhibits increased DNA binding to a canonical BACH1 binding site as well as greater transcriptional output of metastasis and hypoxia-response-related genes relative to wild-type BACH1. Furthermore, in vivo assays showed that mutant BACH1 promotes greater tumor metastasis of TNBC cells than wild-type BACH1. Our findings suggest that the proline hydroxylation of BACH1 directly regulates BACH1’s transcriptional activity by modulating its DNA-binding activity. Together, these results reveal a mechanism underlying oxygen-sensitive regulation of BACH1 that could potentially contribute to BACH1-mediated pro-metastatic signaling and therapeutic resistance under hypoxia in TNBC. Citation Format: Thomas Li, Long Chi Nguyen, Christopher Dann, Madeline Henn, Dongbo Yang, Emily Shi, Lydia Robinson-Mailman, Kazuhiko Igarashi, Marsha R. Rosner. BACH1 metastatic activity is regulated by oxygen-dependent proline hydroxylation in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 370.

The Prolyl Hydroxylase Domain 2 (PHD2) Deficiency Enhances Maximal Exercise Capacity

PURPOSE: Hypoxic training is beneficial for improving endurance performance. Hypoxia-inducible factor (HIF) is a key transcription factor that regulates the hypoxic-responsive pathway. Stabilization of HIF by inactivating prolyl hydroxylase domain protein 2 (PHD2) under low oxygen tension, triggers angiogenesis, erythropoiesis, and glycolysis. Glycolytic capacity is essential to perform anaerobic exercise. This study aims to verify whether the upregulation of glycolytic metabolism under the hypoxic-responsive pathway enhances exercise capacity.METHODS: We employed tamoxifen-inducible conditional PHD2 knock-out (KO) mice that exhibited stabilized HIF-alpha. To evaluate anaerobic exercise capacity, a rodent treadmill exercise test was performed. RNA sequencing (RNA-seq) analysis was conducted using C2C12 myoblasts after PHD2 siRNA transfection.RESULTS: PHD2KO mice exhibited increased red blood cell counts, hemoglobin, and hematocrit values, along with enhanced maximal exercise performance compared to littermate wild-type (WT) mice. PHD2 deficiency promoted maximal exercise capacity and resulted in lower blood lactate levels. RNAseq analysis revealed that PHD2-deficient myoblasts increased the expression of genes related to glycolysis, gluconeogenesis, and muscle contraction.CONCLUSIONS: These findings suggest that PHD2 deficiency-induced activation of the hypoxic-responsive pathway enhances maximal exercise capacity.

Growing concerns about using hypoxia-inducible factor prolyl hydroxylase inhibitors for the treatment of renal anemia.

Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) have emerged as a novel therapeutic class for treating anemia in patients with chronic kidney disease. Small molecule analogs of α-ketoglutarate (AKG), an essential substrate for 2-oxoglutarate-dependent dioxygenases (2-OGDDs), including prolyl hydroxylase domain proteins (PHDs), inhibit PHDs pharmacologically and thereby prevent HIF degradation. HIF stabilization alleviates anemia through several stimulatory effects on erythropoiesis, but it also affects the expression of many anemia-unrelated genes whose protein products exert important functions in vivo. Therefore, the pleiotropic effects of HIF stabilization under normoxic conditions deserve to be examined in more detail. Specifically, we believe that particular attention should be given to epigenetic modifications among the various AKG-based metabolic systems that may be altered by HIF-PHIs. It is noteworthy that AKG has been reported to exert health-protective actions. AKG-based metabolic systems include enzymes associated with the tricarboxylic acid cycle and amino acid metabolism, as well as 2-OGDD-mediated processes, which play important roles in many biological reactions. In this review, we examine the multifaceted effects of HIF-PHIs, encompassing not only their on-target effect of HIF stabilization but also their off-target inhibitory effects on various AKG-based metabolic systems. Furthermore, we examine its potential relevance to cardiovascular complications, based on clinical and animal studies suggesting its involvement in vascular calcification, thrombogenesis and heart failure. In conclusion, although HIF-PHIs offer a promising avenue for anemia treatment in CKD patients, their broader impact on multiple biological systems raises substantial concerns. The intricate interplay between HIF stabilization, AKG competition and cardiovascular complications warrants extensive, long-term investigations to ensure the safety and usefulness of HIF-PHIs in clinical practice.

Low nuclear expression of HIF-hydroxylases PHD2/EGLN1 and PHD3/EGLN3 are associated with poor recurrence-free survival in clear cell renal cell carcinoma.

Hypoxia inducible factors, HIF-1α and HIF-2α, and their main regulators, the prolyl hydroxylase domain proteins (PHDs), mediate cellular response to hypoxia and contribute to tumor progression in clear cell renal cell carcinoma (ccRCC). These biomarkers may improve the value of traditional histopathological features in predicting disease progression after nephrectomy for localized ccRCC and guide patient selection for adjuvant treatments. In this study, we analyzed the associations of PHD2 and PHD3 with histopathological tumor features and recurrence-free survival (RFS) in a retrospective cohort of 173 patients who had undergone surgery for localized ccRCC at Helsinki University Hospital (HUH), Finland. An external validation cohort of 191 patients was obtained from Turku University Hospital (TUH), Finland. Tissue-microarrays (TMA) were constructed using the primary tumor samples. Clinical parameters and follow-up information from 2006 to 2019 were obtained from electronic medical records. The cytoplasmic and nuclear expression of PHD2, and PHD3 were scored based on immunohistochemical staining and their associations with histopathological features and RFS were evaluated. Nuclear PHD2 and PHD3 expression in cancer cells were associated with lower pT-stage and Fuhrman grade compared with negative nuclei. Patients with positive nuclear expression of PHD2 and PHD3 in cancer cells had favorable RFS compared with patients having negative tumors. The nuclear expression of PHD2 was independently associated with a decreased risk of disease recurrence or death from RCC in multivariable analysis. These results were observed in both cohorts. The absence of nuclear PHD2 and PHD3 expression in ccRCC was associated with poor RFS and the nuclear expression of PHD2 predicted RFS regardless of other known histopathological prognostic factors. Nuclear PHD2 and PHD3 are potential prognostic biomarkers in patients with localized ccRCC and should be further investigated and validated in prospective studies.

Prolyl hydroxylase 2 inhibits glycolytic activity in colorectal cancer via the NF‑κB signaling pathway.

A variety of malignancies preferentially meet energy demands through the glycolytic pathway. Hypoxia‑induced cancer cell adaptations are essential for tumor development. However, in cancerous glycolysis, the functional importance and underlying molecular mechanism of prolyl hydroxylase domain protein 2 (PHD2) have not been fully elucidated. Gain‑ and loss‑of‑function assays were conducted to evaluate PHD2 functions in colon cancer cells. Glucose uptake, lactate production and intracellular adenosine‑5'‑triphosphate/adenosine diphosphate ratio were measured to determine glycolytic activities. Protein and gene expression levels were measured by western blot analysis and reverse transcription‑quantitative PCR, respectively. The human colon cancer xenograft model was used to confirm the role of PHD2 in tumor progression invivo. Functionally, the data demonstrated that PHD2 knockdown leads to increased glycolysis, while PHD2 overexpression resulted in suppressed glycolysis in colorectal cancer cells. In addition, the glycolytic activity was enhanced without PHD2 and normalized after PHD2 reconstitution. PHD2 was shown to inhibit colorectal tumor growth, suppress cancer cell proliferation and improve tumor‑bearing mice survival invivo. Mechanically, it was found that PHD2 inhibits the expression of critical glycolytic enzymes (glucose transporter 1, hexokinase 2 and phosphoinositide‑dependent protein kinase 1). In addition, PHD2 inhibited Ikkβ‑mediated NF‑κB activation in a hypoxia‑inducible factor‑1α‑independent manner. In conclusion, the data demonstrated that PHD2/Ikkβ/NF‑κB signaling has critical roles in regulating glycolysis and suggests that PHD2 potentially suppresses colorectal cancer.

BCLAF1-induced HIF-1α accumulation under normoxia enhances PD-L1 treatment resistances via BCLAF1-CUL3 complex

Bcl-2-associated transcription factor-1 (BCLAF1), an apoptosis-regulating protein of paramount significance, orchestrates the progression of various malignancies. This study reveals increased BCLAF1 expression in hepatocellular carcinoma (HCC) patients, in whom elevated BCLAF1 levels are linked to escalated tumor grades and diminished survival rates. Moreover, novel BCLAF1 expression is particularly increased in HCC patients who were not sensitive to the combined treatment of atezolizumab and bevacizumab, but not in patients who had tumors that responded to the combined regimen. Notably, overexpression of BCLAF1 increases HCC cell proliferation in vitro and in vivo, while the conditioned medium derived from cells overexpressing BCLAF1 strikingly enhances the tube-formation capacity of human umbilical vein endothelial cells. Furthermore, compelling evidence demonstrates that BCLAF1 attenuates the expression of prolyl hydroxylase domain protein 2 (PHD2) and governs the stability of hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions without exerting any influence on transcription, as determined by Western blot and RT‒qPCR analyses. Subsequently, employing coimmunoprecipitation and immunofluorescence, we validated the reciprocal interaction between BCLAF1 and Cullin 3 (CUL3), through which BCLAF1 actively upregulates the ubiquitination and degradation of PHD2. The Western blot and RT‒qPCR results suggests that programmed death ligand-1 (PD-L1) is one of the downstream responders to HIF-1α in HCC. Thus, we reveal the pivotal role of BCLAF1 in promoting PD-L1 transcription and, through binding to CUL3, in promoting the accumulation of HIF-1α under normoxic conditions, thereby facilitating the ubiquitination and degradation of PHD2.

Hypoxia stabilizes SETDB1 to maintain genome stability.

Von Hippel-Lindau (VHL) is a tumor suppressor that functions as the substrate recognition subunit of the CRL2VHL E3 complex. While substrates of VHL have been identified, its tumor suppressive role remains to be fully understood. For further determination of VHL substrates, we analyzed the physical interactome of VHL and identified the histone H3K9 methyltransferase SETBD1 as a novel target. SETDB1 undergoes oxygen-dependent hydroxylation by prolyl hydroxylase domain proteins and the CRL2VHL complex recognizes hydroxylated SETDB1 for ubiquitin-mediated degradation. Under hypoxic conditions, SETDB1 accumulates by escaping CRL2VHL activity. Loss of SETDB1 in hypoxia compared with that in normoxia escalates the production of transposable element-derived double-stranded RNAs, thereby hyperactivating the immune-inflammatory response. In addition, strong derepression of TEs in hypoxic cells lacking SETDB1 triggers DNA damage-induced death. Our collective results support a molecular mechanism of oxygen-dependent SETDB1 degradation by the CRL2VHL E3 complex and reveal a role of SETDB1 in genome stability under hypoxia.

Oxygen-sensing pathways and the pulmonary circulation.

The unique property of the pulmonary circulation to constrict in response to hypoxia, rather than dilate, brings advantages in both health and disease. Hypoxic pulmonary vasoconstriction (HPV) acts to optimise ventilation-perfusion matching - this is important clinically both in focal disease (such as pneumonia) and in one-lung ventilation during anaesthesia for thoracic surgery. However, during global hypoxia such as that encountered at high altitude, generalised pulmonary vasoconstriction can lead to pulmonary hypertension. There is now a growing body of evidence that links the hypoxia-inducible factor (HIF) pathway and pulmonary vascular tone - in both acute and chronic settings. Genetic and pharmacological alterations to all key components of this pathway (VHL - von Hippel-Lindau ubiquitin E3 ligase; PHD2 - prolyl hydroxylase domain protein 2; HIF1 and HIF2) have clear effects on the pulmonary circulation, particularly in hypoxia. Furthermore, knowledge of the molecular biology of the prolyl hydroxylase enzymes has led to an extensive and ongoing body of research into the importance of iron in both HPV and pulmonary hypertension. This review will explore these relationships in more detail and discuss future avenues of research.

Molecular Characterization and Functional Analysis of Hypoxia-Responsive Factor Prolyl Hydroxylase Domain 2 in Mandarin Fish (Siniperca chuatsi).

With increased breeding density, the phenomenon of hypoxia gradually increases in aquaculture. Hypoxia is primarily mediated by the hypoxia-inducible factor 1 (HIF-1) signaling pathway. Prolyl hydroxylase domain proteins (PHD) are cellular oxygen-sensing molecules that regulate the stability of HIF-1α through hydroxylation. In this study, the characterization of the PHD2 from mandarin fish Siniperca chuatsi (scPHD2) and its roles in the HIF-1 signaling pathway were investigated. Bioinformation analysis showed that scPHD2 had the conserved prolyl 4-hydroxylase alpha subunit homolog domains at its C-terminal and was more closely related to other Perciformes PHD2 than other PHD2. Tissue-distribution results revealed that scphd2 gene was expressed in all tissues tested and more highly expressed in blood and liver than in other tested tissues. Dual-luciferase reporter gene and RT-qPCR assays showed that scPHD2 overexpression could significantly inhibit the HIF-1 signaling pathway. Co-immunoprecipitation analysis showed that scPHD2 could interact with scHIF-1α. Protein degradation experiment results suggested that scPHD2 could promote scHIF-1α degradation through the proteasome degradation pathway. This study advances our understanding of how the HIF-1 signaling pathway is regulated by scPHD2 and will help in understanding the molecular mechanisms underlying hypoxia adaptation in teleost fish.

Degradation-Resistant Hypoxia Inducible Factor-2α in Murine Osteocytes Promotes a High Bone Mass Phenotype.

Molecular oxygen levels vary during development and disease. Adaptations to decreased oxygen bioavailability (hypoxia) are mediated by hypoxia-inducible factor (HIF) transcription factors. HIFs are composed of an oxygen-dependent α subunit (HIF-α), of which there are two transcriptionally active isoforms (HIF-1α and HIF-2α), and a constitutively expressed β subunit (HIFβ). Under normoxic conditions, HIF-α is hydroxylated via prolyl hydroxylase domain (PHD) proteins and targeted for degradation via Von Hippel-Lindau (VHL). Under hypoxic conditions, hydroxylation via PHD is inhibited, allowing for HIF-α stabilization and induction of target transcriptional changes. Our previous studies showed that Vhl deletion in osteocytes (Dmp1-cre; Vhl f/f ) resulted in HIF-α stabilization and generation of a high bone mass (HBM) phenotype. The skeletal impact of HIF-1α accumulation has been well characterized; however, the unique skeletal impacts of HIF-2α remain understudied. Because osteocytes orchestrate skeletal development and homeostasis, we investigated the role of osteocytic HIF-α isoforms in driving HBM phenotypes via osteocyte-specific loss-of-function and gain-of-function HIF-1α and HIF-2α mutations in C57BL/6 female mice. Deletion of Hif1a or Hif2a in osteocytes showed no effect on skeletal microarchitecture. Constitutively stable, degradation-resistant HIF-2α (HIF-2α cDR), but not HIF-1α cDR, generated dramatic increases in bone mass, enhanced osteoclast activity, and expansion of metaphyseal marrow stromal tissue at the expense of hematopoietic tissue. Our studies reveal a novel influence of osteocytic HIF-2α in driving HBM phenotypes that can potentially be harnessed pharmacologically to improve bone mass and reduce fracture risk. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Hypoxia signaling pathway: A central mediator in endocrine tumors.

Adequate oxygen levels are essential for the functioning and maintenance of biological processes in virtually every cell, albeit based on specific need. Thus, any change in oxygen pressure leads to modulated activation of the hypoxia pathway, which affects numerous physiological and pathological processes, including hematopoiesis, inflammation, and tumor development. The Hypoxia Inducible Factors (HIFs) are essential transcription factors and the driving force of the hypoxia pathway; whereas, their inhibitors, HIF prolyl hydroxylase domain (PHDs) proteins are the true oxygen sensors that critically regulate this response. Recently, we and others have described the central role of the PHD/HIF axis in various compartments of the adrenal gland and its potential influence in associated tumors, including pheochromocytomas and paragangliomas. Here, we provide an overview of the most recent findings on the hypoxia signaling pathway in vivo, including its role in the endocrine system, especially in adrenal tumors.

CHD1 deletion stabilizes HIF1α to promote angiogenesis and glycolysis in prostate cancer.

Chromodomain-helicase-DNA-binding protein 1 (CHD1) deletion is among the most common mutations in prostate cancer (PCa), but its role remains unclear. In this study, RNA sequencing was conducted in PCa cells after clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-based CHD1 knockout. Gene set enrichment analysis (GSEA) indicated upregulation of hypoxia-related pathways. A subsequent study confirmed that CHD1 deletion significantly upregulated hypoxia-inducible factor 1α (HIF1α) expression. Mechanistic investigation revealed that CHD1 deletion upregulated HIF1α by transcriptionally downregulating prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase catalyzing the hydroxylation of HIF1α and thus promoting its degradation by the E3 ligase von Hippel-Lindau tumor suppressor (VHL). Functional analysis showed that CHD1 deletion promoted angiogenesis and glycolysis, possibly through HIF1α target genes. Taken together, these findings indicate that CHD1 deletion enhances HIF1α expression through PHD2 downregulation and therefore promotes angiogenesis and metabolic reprogramming in PCa.

Tailless and hypoxia inducible factor-2α cooperate to sustain proangiogenic states of retinal astrocytes in neonatal mice.

Tailless (TLX, an orphan nuclear receptor) and hypoxia inducible factor-2α (HIF2α) are both essential for retinal astrocyte and vascular development. Tlx-/- mutation and astrocyte specific Hif2α disruption in Hif2αf/f/GFAPCre mice are known to cause defective astrocyte development and block vascular development in neonatal retinas. Here we report that TLX and HIF2α support retinal angiogenesis by cooperatively maintaining retinal astrocytes in their proangiogenic states. While Tlx+/- and Hif2αf/+/GFAPCre mice are phenotypically normal, Tlx+/-/Hif2αf/+/GFAPCre mice display precocious retinal astrocyte differentiation towards non-angiogenic states, along with significantly reduced retinal angiogenesis. In wild-type mice, TLX and HIF2α coexist in the same protein complex, suggesting a cooperative function under physiological conditions. Furthermore, astrocyte specific disruption of Phd2 (prolyl hydroxylase domain protein 2), a manipulation previously shown to cause HIF2α accumulation, did not rescue retinal angiogenesis in Tlx-/- background, which suggests functional dependence of HIF2α on TLX. Finally, the expression of fibronectin and VEGF-A is significantly reduced in retinal astrocytes of neonatal Tlx+/-/Hif2αf/+/GFAPCre mice. Overall, these data indicate that TLX and HIF2α cooperatively support retinal angiogenesis by maintaining angiogenic potential of retinal astrocytes.

Effects of hypoxia and reoxygenation on mitochondrial functions and transcriptional profiles of isolated brain and muscle porcine cells

Oxygen fluctuations might occur in mammalian tissues under physiological (e.g. at high altitudes) or pathological (e.g. ischemia–reperfusion) conditions. Mitochondria are the key target and potential amplifiers of hypoxia-reoxygenation (H-R) stress. Understanding the mitochondrial responses to H-R stress is important for identifying adaptive mechanisms and potential therapeutic solutions for pathologies associated with oxygen fluctuations. We explored metabolic response to H-R stress in two tissue types (muscle and brain) with different degrees of hypoxia tolerance in a domestic pig Sus scrofa focusing on the cellular responses independent of the systemic regulatory mechanisms. Isolated cells from the skeletal muscle (masseter) and brain (thalamus) were exposed to acute short-term (15 min) hypoxia followed by reoxygenation. The mitochondrial oxygen consumption, reactive oxygen species (ROS) production rates and transcriptional profiles of hypoxia-responsive mRNA and miRNA were determined. Mitochondria of the porcine brain cells showed a decrease in the resting respiration and ATP synthesis capacity whereas the mitochondria from the muscle cells showed robust respiration and less susceptibility to H-R stress. ROS production was not affected by the short-term H-R stress in the brain or muscle cells. Transcriptionally, prolyl hydroxylase domain protein EGLN3 was upregulated during hypoxia and suppressed during reoxygenation in porcine muscle cells. The decline in EGLN3 mRNA during reoxygenation was accompanied by an upregulation of hypoxia-inducible factor subunit α (HIF1A) transcripts in the muscle cells. However, in the brain cells, HIF1A mRNA levels were suppressed during reoxygenation. Other functionally important transcripts and miRNAs involved in antioxidant response, apoptosis, inflammation, and substrate oxidation were also differentially expressed between the muscle and brain cells. Suppression of miRNA levels during acute intermittent hypoxia was stronger in the brain cells affecting ~ 55% of all studied miRNA transcripts than in the muscle cells (~ 25% of miRNA) signifying transcriptional derepression of the respective mRNA targets. Our study provides insights into the potential molecular and physiological mechanisms contributing to different hypoxia sensitivity of the studied tissues and can serve as a starting point to better understand the biological processes associated with hypoxia stress, e.g. during ischemia and reperfusion.

Role of prolyl hydroxylase/HIF-1 signaling in vascular calcification.

Morbidity and mortality of chronic kidney disease (CKD) patients are largely associated with vascular calcification, an actively regulated process in which vascular smooth muscle cells (VSMCs) change into cells similar to osteocytes/chondrocytes, known as trans-differentiation. Cellular and systemic response to low oxygen (hypoxia) is regulated by the prolyl hydroxylase/hypoxia-inducible factor (HIF)-1 pathway. Recent studies highlighted that hypoxia-mediated activation of HIF-1 induces trans-differentiation of VSMCs into bone-forming type through an increase in osteo-/chondrogenic genes. Inhibition of the HIF-1 pathway abolished osteochondrogenic differentiation of VSMCs. Hypoxia strongly enhanced elevated phosphate-induced VSMC osteogenic trans-differentiation and calcification. HIF-1 was shown to be essential for phosphate enhanced VSMC calcification. O2-dependent degradation HIF-1 is triggered by the prolyl hydroxylase domain proteins (PHD). Prolyl hydroxylase inhibitors, daprodustat and roxadustat, increase high phosphate-induced VC in VSMCs, stabilizing HIF-1α and activating the HIF-1 pathway in these cells. Whether the use of these PHD inhibitors to treat anemia in CKD patients will favor the development and progression of vascular calcification remains to be explored.

The ribosomal chaperone NACA recruits PHD2 to cotranslationally modify HIF-α.

Prolyl hydroxylase domain protein 2 (PHD2)-catalyzed modification of hypoxia-inducible factor (HIF)-α is a key event in oxygen sensing. We previously showed that the zinc finger of PHD2 binds to a Pro-Xaa-Leu-Glu (PXLE) motif. Here, we show that the zinc finger binds to this motif in the ribosomal chaperone nascent polypeptide complex-α (NACA). This recruits PHD2 to the translation machinery to cotranslationally modify HIF-α. Importantly, this cotranslational modification is enhanced by a translational pause sequence in HIF-α. Mice with a knock-in Naca gene mutation that abolishes the PXLE motif display erythrocytosis, a reflection of HIF pathway dysregulation. In addition, human erythrocytosis-associated mutations in the zinc finger of PHD2 ablate interaction with NACA. Tibetans, who have adapted to the hypoxia of high altitude, harbor a PHD2 variant that we previously showed displays a defect in zinc finger binding to p23, a PXLE-containing HSP90 cochaperone. We show here that Tibetan PHD2 maintains interaction with NACA, thereby showing differential interactions with PXLE-containing proteins and providing an explanation for why Tibetans are not predisposed to erythrocytosis.

Differential methylation in EGLN1 associates with blood oxygen saturation and plasma protein levels in high-altitude pulmonary edema

BackgroundHigh-altitude (HA, 2500 m) hypoxic exposure evokes a multitude of physiological processes. The hypoxia-sensing genes though influence transcriptional output in disease susceptibility; the exact regulatory mechanisms remain undetermined in high-altitude pulmonary edema (HAPE). Here, we investigated the differential DNA methylation distribution in the two genes encoding the oxygen-sensing HIF-prolyl hydroxylases, prolyl hydroxylase domain protein 2 (PHD2) and factor inhibiting HIF-1α and the consequent contributions to the HAPE pathophysiology.MethodsDeep sequencing of the sodium bisulfite converted DNA segments of the two genes, Egl nine homolog 1 (EGLN1) and Hypoxia Inducible Factor 1 Subunit Alpha Inhibitor (HIF1AN), was conducted to analyze the differential methylation distribution in three study groups, namely HAPE-patients (HAPE-p), HAPE-free sojourners (HAPE-f) and healthy HA natives (HLs). HAPE-p and HAPE-f were permanent residents of low altitude (< 200 m) of North India who traveled to Leh (3500 m), India, and were recruited through Sonam Norboo Memorial (SNM) hospital, Leh. HLs were permanent residents of altitudes at and above 3500 m. In addition to the high resolution, bisulfite converted DNA sequencing, gene expression of EGLN1 and HIF1AN and their plasma protein levels were estimated.ResultsA significantly lower methylation distribution of CpG sites was observed in EGLN1 and higher in HIF1AN (P < 0.01) in HAPE-p compared to the two control groups, HAPE-f and HLs. Of note, differential methylation distribution of a few CpG sites, 231,556,748, 231,556,804, 231,556,881, 231,557,317 and 231,557,329, in EGLN1 were significantly associated with the risk of HAPE (OR = 4.79–10.29; P = 0.048–004). Overall, the methylation percentage in EGLN1 correlated with upregulated plasma PHD2 levels (R = − 0.36, P = 0.002) and decreased peripheral blood oxygen saturation (SpO2) levels (R = 0.34, P = 0.004). We also identified a few regulatory SNPs in the DNA methylation region of EGLN1 covering chr1:231,556,683–231,558,443 suggestive of the functional role of differential methylation distribution of these CpG sites in the regulation of the genes and consequently in the HIF-1α signaling.ConclusionsSignificantly lower methylation distribution in EGLN1 and the consequent physiological influences annotated its functional epigenetic relevance in the HAPE pathophysiology.

Regulation of Erythropoiesis by the Hypoxia-Inducible Factor Pathway: Effects of Genetic and Pharmacological Perturbations.

Red blood cells transport O2 from the lungs to body tissues. Hypoxia stimulates kidney cells to secrete erythropoietin (EPO), which increases red cell mass. Hypoxia-inducible factors (HIFs) mediate EPO gene transcriptional activation. HIF-α subunits are subject to O2-dependent prolyl hydroxylation and then bound by the von Hippel-Lindau protein (VHL), which triggers their ubiquitination and proteasomal degradation. Mutations in the genes encoding EPO, EPO receptor, HIF-2α, prolyl hydroxylase domain protein 2 (PHD2), or VHL cause familial erythrocytosis. In addition to O2, α-ketoglutarate is a substrate for PHD2, and analogs of α-ketoglutarate inhibit hydroxylase activity. In phase III clinical trials evaluating the treatment of anemia in chronic kidney disease, HIF prolyl hydroxylase inhibitors were as efficacious as darbepoetin alfa in stimulating erythropoiesis. However, safety concerns have arisen that are focused on thromboembolism, which is also a phenotypic manifestation of VHL or HIF-2α mutation, suggesting that these events are on-target effects of HIF prolyl hydroxylase inhibitors.

Myeloid Angiotensin II Type 1 Receptor Mediates Macrophage Polarization and Promotes Vascular Injury in DOCA/Salt Hypertensive Mice.

Activation of the renin–angiotensin system has been implicated in hypertension. Angiotensin (Ang) II is a potent proinflammatory mediator. The present study investigated the role of myeloid angiotensin type 1 receptor (AT1R) in control of macrophage phenotype in vitro and vascular injury in deoxycorticosterone acetate (DOCA)/salt hypertension. In human THP-1/macrophages, Ang II increased mRNA expressions of M1 cytokines and decreased M2 cytokine expressions. Overexpression of AT1R further increased Ang II-induced expressions of M1 cytokines and decreased M2 cytokines. Silenced AT1R reversed Ang II-induced changes in M1 and M2 cytokines. Ang II upregulated hypoxia-inducible factor (HIF)1α, toll-like receptor (TLR)4, and the ratio of pIκB/IκB, which were prevented by silenced AT1R. Silenced HIF1α prevented Ang II activation of the TLR4/NFκB pathway. Furthermore, Ang II increased HIF1α via reactive oxygen species-dependent reduction in prolyl hydroxylase domain protein 2 (PHD2) expression. The expressions of AT1R and HIF1α and the ratio of pIκB/IκB were upregulated in the peritoneal macrophages of DOCA hypertensive mice, and the specific deletion of myeloid AT1R attenuated cardiac and vascular injury and vascular oxidative stress, reduced the recruitment of macrophages and M1 cytokine expressions, and improved endothelial function without significant reduction in blood pressure. Our results demonstrate that Ang II/AT1R controls the macrophage phenotype via stimulating the HIF1α/NFκB pathway, and specific myeloid AT1R KO improves endothelial function, vascular inflammation, and injury in salt-sensitive hypertension. The results support the notion that myeloid AT1R plays an important role in the regulation of the macrophage phenotype, and dysfunction of this receptor may promote vascular dysfunction and injury in salt-sensitive hypertension.

Prolyl Hydroxylase Domain Protein Inhibitor Not Harboring a 2-Oxoglutarate Scaffold Protects against Hypoxic Stress.

Hypoxia-inducible factor-α (HIF-α) activation has shown promising results in the treatment of ischemia, such as stroke, myocardial infarction, and chronic kidney disease. A number of HIF-α activators have been developed to improve the symptoms of these diseases. Many feature 2-oxoglutarate (2-OG) scaffolds that interact with the active centers of prolyl hydroxylase domain-containing proteins (PHDs), displacing the coenzyme 2-OG. This stabilizes HIF-α. Therefore, the specificity of the 2-OG analogs is not high. Here, we identified 5-(1-acetyl-5-phenylpyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid (PyrzA) among over 10 000 compounds as a novel HIF activator that does not contain a 2-OG scaffold. In cultured cells, PyrzA enhanced HIF-α stability and upregulated the expression of HIF target genes. Interestingly, PyrzA decreased HIF-1α prolyl hydroxylation, suggesting that PyrzA may activate HIF to prevent the degradation of HIF-α. These results indicate that PyrzA stabilizes HIF via a novel mechanism and could be a potential HIF activator candidate.