Prolyl Hydroxylase Domain Research Articles

Post-ischemic inactivation of HIF Prolyl Hydroxylases in endothelium promotes maladaptive kidney repair by inducing glycolysis.

Ischemic acute kidney injury (AKI) is common in hospitalized patients and increases the risk for chronic kidney disease (CKD). Impaired endothelial cell (EC) functions are thought to contribute in AKI to CKD transition, but the underlying mechanisms remain unclear. Here, we identify a critical role for endothelial oxygen sensing prolyl hydroxylase domain (PHD) enzymes 1-3 in regulating post-ischemic kidney repair. In renal endothelium, we observed compartment-specific differences in the expression of the three PHD isoforms in both mice and humans. Post-ischemic concurrent inactivation of endothelial PHD1, PHD2, and PHD3 but not PHD2 alone promoted maladaptive kidney repair characterized by exacerbated tissue injury, fibrosis, and inflammation. Single-cell RNA-seq analysis of the post-ischemic endothelial PHD1, PHD2 and PHD3 deficient (PHDTiEC) kidney revealed an endothelial hypoxia and glycolysis related gene signature, also observed in human kidneys with severe AKI. This metabolic program was coupled to upregulation of the SLC16A3 gene encoding the lactate exporter monocarboxylate transporter 4 (MCT4). Strikingly, treatment with the MCT4 inhibitor syrosingopine restored adaptive kidney repair in PHDTiEC mice. Mechanistically, MCT4 inhibition suppressed pro-inflammatory EC activation reducing monocyte-endothelial cell interaction. Our findings suggest avenues for halting AKI to CKD transition based on selectively targeting the endothelial hypoxia-driven glycolysis/MCT4 axis.

Human prolyl hydroxylase domain 2 reacts with O2 and 2-oxoglutarate to enable formation of inactive Fe(III).2OG.hypoxia-inducible-factor α complexes

Hypoxia inducible transcription factors (HIFs) mediate the hypoxic response in metazoans. When sufficient O2 is present, Fe(II)/2-oxoglutarate (2OG)-dependent oxygenases (human PHD1-3) promote HIFα degradation via prolyl-hydroxylation. We report crystallographic, spectroscopic, and biochemical characterization of stable and inactive PHD2.Fe(III).2OG complexes. Aerobic incubation of PHD2 with Fe(II) and 2OG enables formation of PHD2.Fe(III).2OG complexes which bind HIF1-2α to give inactive PHD2.Fe(III).2OG.HIF1-2α complexes. The Fe(III) oxidation state in the inactive complexes was shown by EPR spectroscopy. L-Ascorbate hinders formation of the PHD2.Fe(III).2OG.(+/-HIFα) complexes and slowly regenerates them to give the catalytically active PHD2.Fe(II).2OG complex. Crystallographic comparison of the PHD2.Fe(III).2OG.HIF2α complex with the analogous anaerobic Fe(II) complex reveals near identical structures. Exposure of the anaerobic PHD2.Fe(II).2OG.HIF2α crystals to O2 enables in crystallo hydroxylation. The resulting PHD2.product structure, manifests conformational changes compared to the substrate structures. The results have implications for the role of the PHDs in hypoxia sensing and open new opportunities for inhibition of the PHDs and other 2OG dependent oxygenases by promoting formation of stable Fe(III) complexes.

Endothelial PHD2 deficiency induces apoptosis resistance and inflammation via AKT activation and AIP1 loss independent of HIF2α.

In hypoxic and pseudohypoxic rodent models of pulmonary hypertension (PH), hypoxia-inducible factor (HIF) inhibition attenuates disease initiation. However, HIF activation alone, due to genetic alterations or use of inhibitors of prolyl hydroxylase domain (PHD) enzymes, has not been definitively shown to cause PH in humans, indicating the involvement of other mechanisms. Given the association between endothelial cell dysfunction and PH, the effects of pseudohypoxia and its underlying pathways were investigated in primary human lung endothelial cells. PHD2 silencing or inhibition, while activating HIF2α, induced apoptosis-resistance and IFN/STAT activation in endothelial cells, independent of HIF signaling. Mechanistically, PHD2 deficiency activated AKT and ERK, inhibited JNK, and reduced AIP1 (ASK1-interacting protein 1), all independent of HIF2α. Like PHD2, AIP1 silencing affected these same kinase pathways and produced a similar dysfunctional endothelial cell phenotype, which was partially reversed by AKT inhibition. Consistent with these in vitro findings, AIP1 protein levels in lung endothelial cells were decreased in Tie2-Cre/Phd2 knockout mice compared with wild-type controls. Lung vascular endothelial cells from patients with pulmonary arterial hypertension (PAH) showed IFN/STAT activation. Lung tissue from both SU5416/hypoxia PAH rats and patients with PAH all showed AKT activation and dysregulated AIP1 expression. In conclusion, PHD2 deficiency in lung vascular endothelial cells drives an apoptosis-resistant and inflammatory phenotype, mediated by AKT activation and AIP1 loss independent of HIF signaling. Targeting these pathways, including PHD2, AKT, and AIP1, holds the potential for developing new treatments for endothelial dysfunction in PH.NEW & NOTEWORTHY HIF activation alone does not conclusively lead to human PH, suggesting that HIF-independent signaling may also contribute to hypoxia-induced PH. This study demonstrated that PHD2 silencing-induced pseudohypoxia in human lung endothelial cells suppresses apoptosis and activates STAT, effects that persist despite HIF2α inhibition or knockdown and are attributed to AKT and ERK activation, JNK inhibition, and AIP1 loss. These findings align with observations in lung endothelial cells and tissues from PAH rodent models and patients.

NADPH Oxidase 4: Crucial for Endothelial Function under Hypoxia-Complementing Prostacyclin.

Aim: The primary endothelial NADPH oxidase isoform 4 (NOX4) is notably induced during hypoxia, with emerging evidence suggesting its vasoprotective role through H2O2 production. Therefore, we aimed to elucidate NOX4's significance in endothelial function under hypoxia. Methods: Human vessels, in addition to murine vessels from Nox4-/- mice, were explored. On a functional level, Mulvany myograph experiments were performed. To obtain mechanistical insights, human endothelial cells were cultured under hypoxia with inhibitors of hypoxia-inducible factors. Additionally, endothelial cells were cultured under combined hypoxia and laminar shear stress conditions. Results: In human occluded vessels, NOX4 expression strongly correlated with prostaglandin I2 synthase (PTGIS). Hypoxia significantly elevated NOX4 and PTGIS expression and activity in human endothelial cells. Inhibition of prolyl hydroxylase domain (PHD) enzymes, which stabilize hypoxia-inducible factors (HIFs), increased NOX4 and PTGIS expression even under normoxic conditions. NOX4 mRNA expression was reduced by HIF1a inhibition, while PTGIS mRNA expression was only affected by the inhibition of HIF2a under hypoxia. Endothelial function assessments revealed hypoxia-induced endothelial dysfunction in mesenteric arteries from wild-type mice. Mesenteric arteries from Nox4-/- mice exhibited an altered endothelial function under hypoxia, most prominent in the presence of cyclooxygenase inhibitor diclofenac to exclude the impact of prostacyclin. Restored protective laminar shear stress, as it might occur after thrombolysis, angioplasty, or stenting, attenuated the hypoxic response in endothelial cells, reducing HIF1a expression and its target NOX4 while enhancing eNOS expression. Conclusions: Hypoxia strongly induces NOX4 and PTGIS, with a close correlation between both factors in occluded, hypoxic human vessels. This relationship ensured endothelium-dependent vasodilation under hypoxic conditions. Protective laminar blood flow restores eNOS expression and mitigates the hypoxic response on NOX4 and PTGIS.

Optimizing the dosing regimen of roxadustat in kidney transplant recipients with early post-transplant anemia

IntroductionRoxadustat, an oral inhibitor of hypoxia-inducible factor prolyl hydroxylase domain enzymes, has been approved for the treatment of renal anemia. However, there is a lack of study on its pharmacokinetics in kidney transplant recipients (KTRs) with early posttransplant anemia (PTA). Therefore, the aim of this study is to elucidate the pharmacokinetic characteristics of roxadustat in KTRs with early PTA and optimize the dosing regimen. MethodsA population pharmacokinetic (PopPK) analysis was performed based on 72-hour full concentration-time profiles collected from 52 Chinese KTRs. Covariates influencing exposure were assessed using stepwise covariate modelling. Monte Carlo simulations were conducted to recommend the dosing regimen for patients with different levels of covariates. ResultsPopPK analysis showed that the concentration-time data can be fully described by a two-compartment model. Body weight (BW) and direct bilirubin (DBIL) levels significant affected the apparent clearance of roxadustat. Based on the established model and the estimated exposures of roxadustat by Monte Carlo simulations, a recommended dosing regimen for KTRs with early PTA at varying BW and DBIL levels were developed. Roxadustat at 100 mg three times weekly were suitable for the majority of KTRs with a DBIL level around 3 μmol/L and BW between 50 and 75 kg. The required dose may need to be increased with higher BW and lower DBIL levels, while decreased with lower BW and higher DBIL levels. ConclusionsIt was the first PopPK analysis of roxadustat in KTRs with early PTA, which provide a research basis for optimizing the dosing regimen.

Genetic modifications of EGLN1 reactivate HbF production in β0-thalassemia/HbE

Reactivation of fetal hemoglobin (HbF, α2γ2) potentially alleviates clinical presentation in β-thalassemia. Prolyl hydroxylase domain enzymes (PHDs) play roles in the canonical oxygen-sensing pathway and maintain the stability of cellular hypoxia-inducible factor α (HIF-α) in response to low oxygen levels or hypoxia. Pharmacological inhibition of PHDs has been shown to increase HbF production in erythroid progenitors derived from healthy donors. Here, we demonstrated the relationship between PHD2, the main PHD isoform, and clinical phenotypes in β0-thalassemia/HbE disease. Although the targeted sequencing annotated several common variants within EGLN1, the gene encoding PHD2, none of these variants were located in the functional domains of PHD2 and were irrelevant to the clinical phenotypes. CRISPR-mediated EGLN1 modifications at the functional regions; however, led to significantly reduce PHD2 expression and increase HbF expression levels in severe β-thalassemia erythroblasts. Moreover, these beneficial phenotypes were independent to the two well-known HbF regulators including BCL11A and GATA1. Our findings introduce an additional mechanism for HbF regulation in β-thalassemia and propose that targeting the canonical oxygen-sensing pathway, particularly PHD2 functional domains, might offer a promising therapeutic strategy to β-thalassemia diseases.

Renoprotective Effects of Daprodustat in Patients with Chronic Kidney Disease and Renal Anemia.

Many large-scale studies revealed that exogenous erythropoietin, erythropoiesis-stimulating agents, have no renoprotective effects. We reported the renoprotective effects of endogenous erythropoietin production on renal function in ischemic reperfusion injury (IRI) of the kidney using the prolyl hydroxylase domain (PHD) inhibitor, Roxadustat. The purpose of this study was to investigate the effects of daprodustat on the progression of chronic renal failure. We retrospectively investigated the effects of daprodustat on the progression of chronic renal failure and renal anemia in patients with stages 3a-5 chronic kidney diseases (estimated glomerular filtration rate, eGFR < 60 mL/min/1.73 m2). The results show that daprodustat largely slowed the reduction in eGFR. The recovery of renal function was observed in some patients. Daprodustat is useful not only for renal anemia but also for the preservation of renal function. The renoprotective effect of daprodustat was small in patients with serum creatinine larger than 3-4 mg/dL because of low residual renal function. The appearance of renal anemia would be a sign of the time to start using daprodustat.

Evolution of Key Oxygen-Sensing Genes Is Associated with Hypoxia Tolerance in Fishes.

Low dissolved oxygen (hypoxia) is recognized as a major threat to aquatic ecosystems worldwide. Because oxygen is paramount for the energy metabolism of animals, understanding the functional and genetic drivers of whole-animal hypoxia tolerance is critical to predicting the impacts of aquatic hypoxia. In this study, we investigate the molecular evolution of key genes involved in the detection of and response to hypoxia in ray-finned fishes: the prolyl hydroxylase domain (PHD)-hypoxia-inducible factor (HIF) oxygen-sensing system, also known as the EGLN (egg-laying nine)-HIF oxygen-sensing system. We searched fish genomes for HIFA and EGLN genes, discovered new paralogs from both gene families, and analyzed protein-coding sites under positive selection. The physicochemical properties of these positively selected amino acid sites were summarized using linear discriminants for each gene. We employed phylogenetic generalized least squares to assess the relationship between these linear discriminants for each HIFA and EGLN and hypoxia tolerance as reflected by the critical oxygen tension (Pcrit) of the corresponding species. Our results demonstrate that Pcrit in ray-finned fishes correlates with the physicochemical variation of positively selected sites in specific HIFA and EGLN genes. For HIF2A, two linear discriminants captured more than 90% of the physicochemical variation of these sites and explained between 20% and 39% of the variation in Pcrit. Thus, variation in HIF2A among fishes may contribute to their capacity to cope with aquatic hypoxia, similar to its proposed role in conferring tolerance to high-altitude hypoxia in certain lineages of terrestrial vertebrates.

Zebrafish phd1 enhances mavs-mediated antiviral responses in a hydroxylation-independent manner.

PHD1 is a member of the prolyl hydroxylase domain protein (PHD1-4) family, which plays a prominent role in the post-translational modification of its target proteins by hydroxylating proline residues. The best-characterized targets of PHD1 are hypoxia-inducible factor α (HIF-1α and HIF-2α), two master regulators of the hypoxia signaling pathway. In this study, we show that zebrafish phd1 positively regulates mavs-mediated antiviral innate immunity. Overexpression of phd1 enhances the cellular antiviral response. Consistently, zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. Further assays indicate that phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, zebrafish phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. However, the enzymatic activity of phd1 is not required for phd1 to activate mavs. In conclusion, this study reveals a novel function of phd1 in the regulation of antiviral innate immunity.IMPORTANCEPHD1 is a key regulator of the hypoxia signaling pathway, but its role in antiviral innate immunity is largely unknown. In this study, we found that zebrafish phd1 enhances cellular antiviral responses in a hydroxylation-independent manner. Phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. Zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. These findings reveal a novel role for phd1 in the regulation of mavs-mediated antiviral innate immunity.

Caulerpa chemnitzia polysaccharide exerts immunomodulatory activity in macrophages by mediating the succinate/PHD2/HIF-1α/IL-1β pathway

Algal polysaccharide is an important food functional factor with diverse bioactive and low toxicity. Previous studies have confirmed Caulerpa chemnitzia polysaccharides (CRVP) have immunomodulatory activity, but the immunomodulatory mechanism of CRVP in macrophages has not been thoroughly explored yet. In our research, we found that CRVP has outstanding immunomodulatory activity in macrophages, which is reflected in promoting cell proliferation, upregulating cytokines (IL-1β, IL-6, and TNF-α) expression, and increasing NO and ROS levels. Additionally, the result of joint analysis of untargeted metabolomics showed metabolism played a major role in the immunomodulatory of CRVP and suggested succinic acid was a key metabolite. Further verification indicated that the accumulation of succinic acid in macrophages after administered with CRVP, induced the down-regulation of prolyl hydroxylase domain 2 (PHD2) and up-regulation of hypoxia-inducible factor-1α (HIF-1α), thereby enhancing IL-1β expression. Together, the immunomodulatory activity of CRVP in macrophages via succinate/PHD2/HIF-1α/IL-1β pathway.

Abstract We078: Endothelial-specific PHD2 Modulates Cardiac Inflammation, Hypertrophy, and Dysfunction in an Age-dependent Manner

Background: Prolyl hydroxylase domain proteins (PHD) are oxygen-sensing molecules that degrade hypoxia-inducible factor-α. Deletion of endothelial-specific PHD2 (PHD2ECKO) shows different degrees of pulmonary arterial hypertension and cardiac hypertrophy. Whether EC-specific PHD2 deletion plays a role in regulating cardiac inflammation and dysfunction is still unclear. We investigated the effect of endothelial-specific PHD2 deficiency on cardiac inflammation and dysfunction in mice. Methods: Briefly, PHD2ECKO mice were obtained by crossing PHD2-floxed mice with VE-Cadherin-Cre transgenic mice. The effect of PHD2ECKO on cardiac structure and function was determined in male young (6 months old) and old (12-16 months old) mice. Results: While PHD2ECKO caused significant left ventricular (LV) hypertrophy in mice at 6 months, PHD2ECKO had no significant impact on LV fibrosis and dysfunction in these mice. However, PHD2ECKO caused significant LV leukocyte infiltration, fibrosis, cardiomyocyte hypertrophy, and LV dysfunction in mice over 12 months. Most interestingly, we found that PHD2ECKO caused a significant increase of cardiac CD4 + T cells and CD8 + T cells, activation, and their proinflammatory cytokine productions in mice at the age of 12 months. Moreover, we found that PHD2ECKO resulted in a significant upregulation of several adhesion molecules important for CD4 + and CD8 + T cell homing, retention, and proliferation. However, the cytokine production by other immune cell subsets was largely unaffected. Conclusions: Our findings demonstrated that endothelial-PHD2 exerts an important role in proliferation, activation, and proinflammatory cytokine production by CD4 and CD8 T cells, and the aging-dependent LV hypertrophy and dysfunction.

Pre-activation of hypoxia-inducible factor 1-α using prolyl hydroxylase domain inhibitors reduces cisplatin-induced nephrotoxicity

Pre-activation of hypoxia-inducible factor 1-α using prolyl hydroxylase domain inhibitors reduces cisplatin-induced nephrotoxicity

Endothelial HIF2α suppresses retinal angiogenesis in neonatal mice by upregulating NOTCH signaling.

Prolyl hydroxylase domain (PHD) proteins are oxygen sensors that use intracellular oxygen as a substrate to hydroxylate hypoxia-inducible factor (HIF) α proteins, routing them for polyubiquitylation and proteasomal degradation. Typically, HIFα accumulation in hypoxic or PHD-deficient tissues leads to upregulated angiogenesis. Here, we report unexpected retinal phenotypes associated with endothelial cell (EC)-specific gene targeting of Phd2 (Egln1) and Hif2alpha (Epas1). EC-specific Phd2 disruption suppressed retinal angiogenesis, despite HIFα accumulation and VEGFA upregulation. Suppressed retinal angiogenesis was observed both in development and in the oxygen-induced retinopathy (OIR) model. On the other hand, EC-specific deletion of Hif1alpha (Hif1a), Hif2alpha, or both did not affect retinal vascular morphogenesis. Strikingly, retinal angiogenesis appeared normal in mice double-deficient for endothelial PHD2 and HIF2α. In PHD2-deficient retinal vasculature, delta-like 4 (DLL4, a NOTCH ligand) and HEY2 (a NOTCH target) were upregulated by HIF2α-dependent mechanisms. Inhibition of NOTCH signaling by a chemical inhibitor or DLL4 antibody partially rescued retinal angiogenesis. Taken together, our data demonstrate that HIF2α accumulation in retinal ECs inhibits rather than stimulates retinal angiogenesis, in part by upregulating DLL4 expression and NOTCH signaling.

CD8 Cell PHD2 Deficiency Amplifies Pressure Overload-Induced Cardiac Inflammation through Metabolic Reprogramming of CD8 Cells

Prolyl hydroxylase domain (PHD) proteins function as oxygen sensors and regulate various cellular metabolism. PHD2 is well-known for its role in oxygen sensing and has been implicated in cardiac hypertrophy and fibrosis through HIF-2α. However, the specific impact and the mechanism of CD8 cell PHD2 deficiency on cardiac inflammation and heart failure (HF) development remains unclear. Here, we explored the role of CD8 cell-specific PHD2 deficiency (CD8-PHD2 KO) in HF development by using transverse aortic constriction (TAC) mouse model. Our findings reveal that CD8 PHD2 knockout exacerbated TAC-induced left ventricular (LV) hypertrophy, as evidenced by increased LV weight relative to bodyweight or tibial length. CD8-PHD2 KO also leads to a significant increase in lung and right ventricular weight relative to bodyweight or tibial length following TAC. Furthermore, CD8-PHD2 KO worsens TAC-induced reductions in LV ejection fraction, LV fractional shortening, and LV dilatation. Additionally, CD8-PHD2 KO exacerbates TAC-induced LV cardiomyocyte hypertrophy, fibrosis, and immune cell infiltration. Moreover, our study demonstrated that CD8-PHD2 KO amplified the infiltration and activation of antigen-presenting cells, CD4 T cells, and CD8 T cells in the heart and lungs after TAC. At the molecular level, we characterized the PHD2-deficient CD8 cells showed typical features of anaerobic glycolysis, which were paralleled by augmented hypoxia-inducible factor 1α (HIF-1α) protein levels. Collectively, these results highlight the critical role of PHD2 in CD8 cells in regulating cardiac inflammation and HF development in response to systolic overload. National Institutes of Health Project Number 5R01HL161085-02. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Rhabdomyolysis and Neuroleptic Malignant Syndrome in a Postrenal Transplant Patient: Is Desidustat a Culprit?

We report a case of a 46-year-old female – a postrenal transplant patient, having developed rhabdomyolysis and neuroleptic malignant syndrome (NMS), after 2.5 months of desidustat initiation – for anemia. Post renal transplant anemia (PTA) is common among renal transplant recipients. Apart from treating it with iron supplements, folic acid, andrecombinant human erythropoietin (EPO), novel agents like hypoxia-inducible factor–prolyl hydroxylase domain (HIF–PHI) are also used, with a very few studies available on its efficacy and safety in PTA patients. Anemia occurring in the first 6 months is called early PTA – occurring most commonly due to iron deficiency, while anemia occurring after 6 months is called late PTA – associated with impaired graft function. The optimal target of hemoglobin in transplant recipients is 12.5–13 gm/dl, which is higher than targets in chronic kidney disease patients. NMS is a life-threatening emergency – mainly seen in patients on antipsychotic medications. It needs to be differentiated from other closely related conditions such as central nervous system infections (meningitis and encephalitis), tetanus, heatstroke, serotonin syndrome, and thyrotoxicosis. Desidustat is an oral HIF–PHI that stimulates erythropoiesis by preventing EPO degradation. Its adverse effects include high blood pressure, myocardial infarction, heart failure, high potassium, and dizziness; however, there is only one case report of rhabdomyolysis associated with desidustat, and none of NMS. It is, therefore, important to be aware of this potential severe side effect of the drug that was not known earlier so that practicing physicians can be vigilant of the same.

Abstract 373: Transcriptional and post-transcriptional positive regulatory loops between HIF-1a and the RNA-Binding protein hnRNPA18

Abstract Hypoxia, the scarcity of oxygen, is a hallmark of solid tumors to which they adapt by activating the hypoxia-inducible transcription factor 1 (HIF-1). HIF activates the transcription of genes bearing hypoxia responsive elements (RE) in their promoter regions including genes related to angiogenesis, glucose metabolism, cell proliferation, survival, invasion, and metastasis. The HIF-1 pathway is thus an attractive target to prevent cancer aggressiveness and improve the effectiveness of cancer therapy. HIF-1α activity is known to be mainly regulated through post-translational modification by prolyl hydroxylase domain (PHD) enzymes, but accumulating evidence indicate that it is also regulated by other mechanisms such as transcriptional initiation, translational initiation, protein-protein interaction, post-translational modifications and post-transcriptionally, primarily through the action of trans-acting factors (noncoding RNAs and RNA-binding proteins) that interact with the HIF-1α mRNA to regulate its decay and translational rates. We recently identified the RNA binding protein hnRNP A18 as a regulator of HIF-1α translation under hypoxic conditions. hnRNP A18 is a nuclear stress responsive protein that translocates to the cytosol in response to cellular stress including hypoxia to bind to a recognition motif in the 3’UTR of its targeted transcripts including HIF-1α to stabilize the transcripts and increase their translation. Using a bicistronic reporter plasmid containing a HIF-1α internal ribosome entry site (IRES) we now show that hnRNP A18 can also regulate HIF-1α through its IRES in the 5’UTR. In fact, cells expressing hnRNP A18 significantly increase translation of a reporter CAT protein under the control of HIF-1α IRES in the presence of the hypoxia mimetic agent CoCl2. On the other hand, deletion of hnRNP A18 prevents the translation of the CAT reporter protein even in the presence of CoCl2. Moreover, we also identified several HIF-1α REs in hnRNP A18 promoter. Two of the predicted HIF-1α REs, located at -57 and -226 upstream of the hnRNP A18 start codon were validated as bonafide HIF-1α binding sites by Electromobility Shift Assay with recombinant HIF-1α protein and by Chromatin Immunoprecipitation assay in human pancreatic cancer cells Panc 01. These data thus indicate that a positive regulatory loop between hnRNP A18 and HIF1-1α exist to amplify HIF-1α expression under cellular stress. hnRNP A18 can upregulate HIF1α protein expression by stabilizing its transcript at the 3’UTR and by binding to its IRES. On the other hand, HIF-1α can upregulate hnRNP A18 by binding to its proximal promoter. Targeting hnRNP A18 could thus provide a new mechanism to regulate HIF-1α expression and sensitize cancer cells to therapies. Citation Format: Eduardo Solano-Gonzalez, France Carrier. Transcriptional and post-transcriptional positive regulatory loops between HIF-1a and the RNA-Binding protein hnRNPA18 [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 373.

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.

Dimethyloxalylglycine Suppresses SREBP1c and Lipogenic Gene Expressions in Hepatocytes Independently of HIF1A.

Dimethyloxalylglycine (DMOG) is a representative inhibitor of the prolyl hydroxylase domain (PHD), which mediates the degradation of hypoxia-inducible factor-1-alpha (HIF1A). DMOG exerts its pharmacological effects via the canonical pathway that involves PHD inhibition; however, it remains unclear whether DMOG affects lipogenic gene expression in hepatocytes. We aimed to elucidate the effects of DMOG on sterol regulatory element-binding protein-1c (SREBP1c), a master regulator of fatty acid synthesis in hepatocytes. DMOG treatment inhibited SREBP1c mRNA and protein expression in HepG2 and AML12 hepatocytes and reduced the transcript levels of SREBP1c-regulated lipogenic genes. A luciferase reporter assay revealed that DMOG inhibited the transcriptional activity of SREBP1c. Moreover, DMOG suppressed SREBP1c expression in mice liver. Mechanistically, treatment with DMOG enhanced the expression of HIF1A and insulin-induced gene 2 (INSIG2), which inhibits the activation of SREBP1c. However, HIF1A or INSIG2 knockdown failed to reverse the inhibitory effect of DMOG on SREBP1c expression, suggesting a redundant role of HIF1A and INSIG2 in terms of repressing SREBP1c. DMOG did not function through the canonical pathway involving inhibition of SREBP1c by PHD, highlighting the presence of non-canonical pathways that mediate its anti-lipogenic effect.

Mechanism of ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix regulates HIF-1α signaling pathway mediated by renal hypoxia to ameliorate renal fibrosis in DKD rats

This study explored the mechanism of the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix in ameliorating renal fibrosis in the rat model of diabetic kidney disease(DKD) based on the expression of hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF) and HIF-1α/platelet-derived growth factor(PDGF)/platelet-derived growth factor receptor(PDGFR) signaling pathways in the DKD rats. After 1 week of adaptive feeding, 50 male SPF-grade Wistar rats were randomized into a blank group(n=7) and a modeling group. After 24 h of fasting, the rats in the modeling group were subjected to intraperitoneal injection of streptozocin and fed with a high-sugar and high-fat diet to establish a DKD model. After modeling, the rats were randomly assigned into model(n=7), low-dose ultrafiltration extract(n=7), medium-dose ultrafiltration extract(n=7), irbesartan(n=8), and high-dose ultrafiltration extract(n=8) groups. After intervention by corresponding drugs for 12 weeks, the general conditions of the rats were observed. The body weights and blood glucose levels of the rats were measured weekly, and the 24 h urinary protein(24hUP) was measured at the 6th and 12th weeks of drug administration. After the last drug administration, the renal function indicators were determined. Masson staining was employed to observe the pathological changes of the renal tissue. The expression of prolyl hydroxylase domain 2(PHD2) and HIF-1α in the renal tissue was detected by immunohistochemistry(IHC). Real-time qPCR was employed to determine the mRNA levels of PHD2, VEGF, PDGF, and PDGFR in the renal tissue. Western blot was employed to determine the protein levels of HIF-1α, VEGF, PDGF, and PDGFR in the renal tissue. The results showed that compared with the model group, drug administration lowered the levels of glycosylated serum protein(GSP), aerum creatinine(Scr), and blood urea nitrogen(BUN) in a dose-dependent manner(P&lt;0.05 or P&lt;0.01) and mitigated the pathological changes in the renal tissue. Furthermore, drug administration up-regulated mRNA level of PHD2(P&lt;0.05 or P&lt;0.01), down-regulated the mRNA levels of VEGF, PDGF, and PDGFR(P&lt;0.05 or P&lt;0.01) and the protein levels of HIF-1α, VEGF, PDGF, and PDGFR(P&lt;0.01) in the renal tissue, and increased the rate of PHD2-positive cells(P&lt;0.01). In conclusion, the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix effectively alleviated the renal fibrosis in DKD rats by inhibiting the expression of key proteins in the HIF-1α signaling pathway mediated by renal hypoxia and reducing extracellular matrix(ECM) deposition.

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.