Prolyl Hydroxylase Research Articles

Developmental growth plate cartilage formation suppressed by artificial light at night via inhibiting BMAL1-driven collagen hydroxylation.

Exposure to artificial light at night (LAN) can induce obesity, depressive disorder and osteoporosis, but the pernicious effects of excessive LAN exposure on tissue structure are poorly understood. Here, we demonstrated that artificial LAN can impair developmental growth plate cartilage extracellular matrix (ECM) formation and cause endoplasmic reticulum (ER) dilation, which in turn compromises bone formation. Excessive LAN exposure induces downregulation of the core circadian clock protein BMAL1, which leads to collagen accumulation in the ER. Further investigations suggest that BMAL1 is the direct transcriptional activator of prolyl 4-hydroxylase subunit alpha 1 (P4ha1) in chondrocytes, which orchestrates collagen prolyl hydroxylation and secretion. BMAL1 downregulation induced by LAN markedly inhibits proline hydroxylation and transport of collagen from ER to golgi, thereby inducing ER stress in chondrocytes. Restoration of BMAL1/P4HA1 signaling can effectively rescue the dysregulation of cartilage formation within the developmental growth plate induced by artificial LAN exposure. In summary, our investigations suggested that LAN is a significant risk factor in bone growth and development, and a proposed novel strategy targeting enhancement of BMAL1-mediated collagen hydroxylation could be a potential therapeutic approach to facilitate bone growth.

Hypoxia-inducible factor stabilisation-related lncRNAs in retinopathy of prematurity

Long non-coding RNAs (lncRNAs) play an important role in the response to many diseases. The previous study reported the transcriptomes of mice that were cured of oxygen-induced retinopathy (OIR, retinopathy of prematurity (ROP) model) by hypoxia-inducible factor (HIF) stabilisation via HIF prolyl hydroxylase inhibition using the isoquinolone Roxadustat or the 2-oxoglutarateanalog dimethyloxalylglycine (DMOG). However, there is little understanding of how those genes are regulated. In the present study, 6918 known lncRNAs and 3654 novel lncRNAs were obtained, and a series of differentially expressed lncRNAs (DELncRNAs) were also identified. By cis- and trans-regulation analyses, the target genes of DELncRNAs were predicted. Functional analysis demonstrated that multiple genes were involved in the MAPK signalling pathway, adipocytokine signalling pathway was regulated by the DELncRNAs. By HIF-pathway analysis, two lncRNAs Gm12758 and Gm15283 were found that can regulate the HIF-pathway by targeting the Vegfa, Pgk1, Pfkl, Eno1, Eno1b and Aldoa genes. In conclusion, the present study provided a series of lncRNAs for further understanding and protecting the extremely premature infant from oxygen toxicity.

Exaptation of Inactivated Host Enzymes for Structural Roles in Orthopoxviruses and Novel Folds of Virus Proteins Revealed by Protein Structure Modeling.

Viruses with large, double-stranded DNA genomes captured the majority of their genes from their hosts at different stages of evolution. The origins of many virus genes are readily detected through significant sequence similarity with cellular homologs. In particular, this is the case for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs, meaning that their origins remain enigmatic. We explored the potential origins of such proteins that are encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus that includes major human pathogens. To this end, we used AlphaFold2 to predict the structures of all 214 proteins that are encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded clear indications of origin for 14 of them and validated several inferences that were previously made via sequence analysis. A notable emerging trend is the exaptation of enzymes from cellular organisms for nonenzymatic, structural roles in virus reproduction that is accompanied by the disruption of catalytic sites and by an overall drastic divergence that precludes homology detection at the sequence level. Among the 16 orthopoxvirus proteins that were found to be inactivated enzyme derivatives are the poxvirus replication processivity factor A20, which is an inactivated NAD-dependent DNA ligase; the major core protein A3, which is an inactivated deubiquitinase; F11, which is an inactivated prolyl hydroxylase; and more similar cases. For nearly one-third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement that yielded unique protein folds. IMPORTANCE Protein structures are more strongly conserved in evolution than are amino acid sequences. Comparative structural analysis is particularly important for inferring the origins of viral proteins that typically evolve at high rates. We used a powerful protein structure modeling method, namely, AlphaFold2, to model the structures of all orthopoxvirus proteins and compared them to all available protein structures. Multiple cases of recruitment of host enzymes for structural roles in viruses, accompanied by the disruption of catalytic sites, were discovered. However, many viral proteins appear to have evolved unique structural folds.

Abstract 1003: The prognostic significance of prolyl 4-hydroxylase subunit alpha 1 and the therapeutic efficacy of diethyl-pythiDC, its small molecule inhibitor, in pancreatic ductal adenocarcinoma

Abstract Background: Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) has an oncogenic role in pancreatic ductal adenocarcinoma (PDAC). Since high expression of P4HA1 in PDAC is suggested to correlate with poor prognosis, targeting P4HA1 could have therapeutic benefits. Herein, we assessed the prognostic significance of P4HA1 expression at the RNA and protein levels in PDAC using publicly available data. We also evaluated the efficacy of diethyl-pythiDC, a small molecule inhibitor of P4HA1, in PDAC cells. Methods: UALCAN (ualcan.path.uab.edu), a comprehensive proteogenomic cancer data analysis portal, was used to obtain gene expression P4HA1 as transcripts per million reads (TPM) (PDAC n=178) from The Cancer Genome Atlas (TCGA); levels of P4HA1 protein expression data were obtained from (normal pancreas, n=74, PDAC n=137) the Clinical Proteomic Tumor Analysis consortium (CPTAC). Immunohistochemical analysis obtained from Human Protein Atlas (HPA) confirmed increased P4HA1 protein expression. Overall survival (OS) between patients with high and low/medium TPM (TPM values above and below upper quartile, respectively) and high and low protein expression score (based on staining intensity and fraction of stained cells) was calculated using the log-rank Wilcoxon test. To determine the efficacy of diethyl-pythiDC in PDAC cells (PANC-1 and BxPC-3), we performed clonogenic assay for colony formation, MTT assay for cell proliferation, flow cytometry for cell cycle analysis, and western blotting for markers of the epithelial-mesenchymal transition (EMT). Results: In the TCGA data, high P4HA1 expression (TPMs) was associated with a poor prognosis (high expression vs. low/medium expression, log-rank p=0.009). Analysis of HPA data showed that the 5-year OS of patients with high P4HA1 expression was 12% vs. 58% for patients with low P4HA1 (log-rank p<0.001). CPTAC data showed higher P4HA1 protein expression in patients with receptor tyrosine kinase (RTK) pathway (p<0.001), mammalian target of rapamycin (mTOR) pathway (p=0.045) and SWItch/Sucrose Non-Fermentable (SWI-SNF) complex (p=0.022) alterations. Diethyl-pythiDC treatment of PDAC cells reduced proliferation, decreased colony formation, and increased G2/M cell cycle arrest. In addition, diethyl-pythiDC increased protein expression of E-cadherin, an epithelial marker, and decreased the expression of N-cadherin and vimentin, the mesenchymal protein markers. Conclusions: Patients with high P4HA1 expression had poor OS. CPTAC data showed higher P4HA1 protein expression in patients with RTK, mTOR and SWI-SNF alterations. Targeting P4HA1 with diethyl-pythiDC reduced proliferation by arresting cells in G2/M phase of the cell cycle and modulated EMT markers. Thus, targeting of P4HA1 can be a viable strategy and may be considered in future PDAC clinical trials. Citation Format: Moh'd Khushman, Farrukh Afaq, Prachi Bajpai, Sameer Al Diffalha, Darryl Outlaw, Grant R. Williams, Rojymon Jacob, Kondal Rao Kyanam Kabir, Ali Ahmed, Peter Shajan, Sooryanarayana Varambally, Upender Manne. The prognostic significance of prolyl 4-hydroxylase subunit alpha 1 and the therapeutic efficacy of diethyl-pythiDC, its small molecule inhibitor, in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1003.

Abstract 1107: Exploring the hypoxia pathway for gastrointestinal radioprotection in human minigut model

Abstract Introduction: When pancreatic cancer cannot be removed by surgery, radiation can be used to approximate a resection. Unfortunately, conventional radiation therapy cannot achieve ablative doses in the pancreas due to potential radiotoxicity to the nearby intestinal tissues. Thus, treatment-related gastrointestinal (GI) toxicity may be the most significant barrier to improving treatment responses for unresectable pancreatic cancer. There are no medications that can selectively protect the intestine from radiation damage, but we previously demonstrated that the genetic or pharmacologic inhibition of the EGLN prolyl hydroxylases protects murine intestines from radiation injury. To translate these findings to patients, we sought to test the EGLN inhibitor, FG-4592, and its mechanism of selective radioprotection in patient-derived intestinal organoids (also known as enteroids or mini-guts). Methods: The patient-derived enteroids (PDEs) were grown from duodenal tissue of non-oncologic patients, embedded in Matrigel, and cultured in media impregnated with essential growth factors. These PDEs have the capacity to recapitulate the full array of intestinal cells that are present in the crypt-villus axis. We first treated PDEs with FG-4592 then irradiation at 24 hours followed by RT-qPCR at 72 hrs post-exposure to gauge the effects of radiation on markers associated with intestinal stem cells (ISCs) and differentiated epithelial cells. Single cell RNA seq techniques were used to identify shifts in cellular populations FG-4592 and/or radiation treatment of human enteroids. Results: Irradiation produced a signature of enhanced Crypt Base Columnar (CBC) cells and +4 quiescent stem cells markers (Bmi1, Tert, Dclk1) suggesting these specialized cells awaken upon crypt damage and start to proliferate to restore the ISC pool. The +4 cell markers are further enhanced in FG-4592 treated irradiated group, suggesting EGLN inhibition leads to activation of these specialized cell population along with restoration of other intestinal cells like secretory progenitors and Paneth cells. Further, gene set enrichment analysis after FG-4592 treatment showed the activation of key pathways involved in proliferation, maintaining homeostasis, metabolism, and DNA repair that could lead to intestinal proliferation and recovery from radiation damage. Conclusion: Taken together, these preliminary data suggest that HIF stabilization by FG-4592 upregulates the normally quiescent +4 stem cells, a radioresistant stem population historically thought to become active upon radiation injury, along with activation of several other pathways required for normal proliferation and recovery from radiation injury. Our study justifies further exploration in clinical trials as a radioprotective or radio mitigative agent. Citation Format: Neeraj Kumari, Maya Ferrell, Carolina G. Garcia, Cullen M. Taniguchi. Exploring the hypoxia pathway for gastrointestinal radioprotection in human minigut model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1107.

Abstract 4807: BACH1 proline hydroxylation regulates the hypoxia response and metastasis in triple negative breast cancer

Abstract Hypoxia is an important hallmark of aggressive solid tumors. Here we show that the pro-metastatic BTB and CNC homology 1 (BACH1) transcription factor is prolyl-hydroxylated by the HIF Prolyl Hydroxylase 1 (PHD1) in an oxygen-dependent manner. Using mass spectrometry, we identified two major prolyl hydroxylation sites in BACH1 and demonstrate that prolyl hydroxylation increased protein turnover in normoxia. Notably, a clinically relevant BACH1 mutant (BACH1M) resistant to hydroxylation displays enhanced BACH1 DNA binding capacity. Triple-negative breast cancer (TNBC) cells expressing BACH1M showed higher invasion in vitro and increased metastasis in vivo. Loss of BACH1 reduced the cellular transcriptional hypoxic response. Under hypoxia, BACH1 increases chromatin accessibility and gene expression through chromatin remodeling involving direct BACH1 binding and indirect epigenetic regulation. These results indicate that hypoxia stabilizes and enhances the pro-metastatic transcriptional activity of BACH1 through loss of prolyl hydoxylation in TNBC. Our findings identify BACH1 as an oxygen sensitive effector of the hypoxia response and a clinically relevant target for attenuating hypoxia induced, pro-metastatic signaling and therapeutic resistance in cancers. Citation Format: Long Chi Nguyen, Christopher Dann, Dongbo Yang, Emily Shi, Thomas Li, Joseph Wynne, Letícia Stock, Madeline Henn, Zeyu Qiao, Andrea Valdespino, Raven Watson, Wenchao Liu, Lydia Robinson-Mailman, Galina Khramtsova, Mitsuyo Matsumoto, Raymond Moellering, Olufunmilayo I. Olopade, Kazuhiko Igarashi, Marsha R. Rosner. BACH1 proline hydroxylation regulates the hypoxia response and metastasis in triple negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4807.

Effects of hypoxia-inducible factor prolyl hydroxylase inhibitors versus erythropoiesis-stimulating agents on iron metabolism and inflammation in patients undergoing dialysis: A systematic review and meta-analysis

AimsThis study aimed to evaluate the effects of hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) on iron metabolism and inflammation in dialysis-dependent chronic kidney disease (DD-CKD) patients. MethodsPubMed, Embase, Web of Science, Cochrane Library, and ClinicalTrials.gov websites were searched for randomized controlled trials (RCTs) investigating HIF-PHIs versus ESAs for DD-CKD patients. Key findingsTwenty studies with 14,737 participants were included in the meta-analysis, which demonstrated no significant difference in the effect of transferrin saturation and ferritin between HIF-PHIs and the ESAs group (MD, 0.65; 95%CI, −0.45 to 1.75; very low certainty; SMD, −0.03; 95% CI, −0.13 to 0.07; low certainty). However, HIF-PHIs significantly increased the iron (MD, 2.30; 95% CI, 1.40 to 3.20; low certainty), total iron-binding capacity (SMD, 0.82; 95% CI, 0.66 to 0.98; low certainty), and transferrin (SMD, 0.90; 95%CI, 0.74 to 1.05; moderate certainty) levels when compared with the ESAs group. In contrast, the hepcidin level and dosage of intravenous iron were significantly decreased in the HIF-PHIs group compared with the ESAs group (MD, −15.06, 95%CI, −21.96 to −8.16; low certainty; MD, −18.07; 95% CI, −30.05 to −6.09; low certainty). The maintenance dose requirements of roxadustat were independent of baseline CRP or hsCRP levels with respect to the effect on inflammation. SignificanceHIF-PHIs promote iron utilization and reduce the use of intravenous iron therapy. Furthermore, HIF-PHIs, such as roxadustat, maintain the erythropoietic response independent of the inflammatory state. Thus, HIF-PHIs may be an alternative treatment strategy for anemia in DD-CKD patients, where ESA is hyporesponsive due to iron deficiency and inflammation.

Front Cover

FEBS LettersVolume 597, Issue 8 p. 1053-1054 Issue InformationFree Access Front Cover First published: 25 April 2023 https://doi.org/10.1002/1873-3468.14386AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Cover illustration HuR in lung cancer cells transfected with myc-tagged mutant OGFOD1 (H155A, D157A). HuR was stained in green, OGFOD1 in red, and the nuclei in blue. The image refers to Fujisaki et al. “The prolyl hydroxylase OGFOD1 promotes cancer cell proliferation by regulating the expression of cell cycle regulators”. Volume597, Issue8April 2023Pages 1053-1054 RelatedInformation

High Glucose Activates Prolyl Hydroxylases and Disrupts HIF-α Signaling via the P53/TIGAR Pathway in Cardiomyocyte.

The induction of hypoxia tolerance has emerged as a novel therapeutic strategy for the treatment of ischemic diseases. The disruption of hypoxic signaling by hyperglycemia has been shown to contribute to diabetic cardiomyopathy. In this study, we explored the potential molecular mechanisms by which high glucose (HG) impairs hypoxia-inducible factor-α (HIF-α) signaling in cardiomyocytes. The exposure of H9c2 cell lines to HG resulted in time- and concentration-dependent decreases in HIF-1α and HIF-2α expression together with an increase in prolyl hydroxylase-1,2 (PHD1 and PHD2) expression, the main regulators of HIF-α destabilization in the heart. The exposure of H9c2 cells to normal glucose (5.5 mM) and high glucose (15, 30, and 45 mM) led to dose-dependent increases in p53 and TIGAR and a decrease in SIRT3 expression. The pretreatment of H9c2 with p53 siRNA to knockdown p53 attenuated PHD1 and PHD2 expression, thus significantly enhancing HIF-1α and HIF-2α expression in H9c2 cells under HG conditions. Interestingly, pretreatment with p53 siRNA altered H9c2 cell metabolism by reducing oxygen consumption rate and increasing glycolysis. Similarly, pretreatment with TIGAR siRNA blunted HG-induced PHD1 and PHD2 expression. This was accompanied by an increase in HIF-1α and HIF-2α expression with a reduction in oxygen consumption rate in H9c2 cells. Furthermore, pretreatment with adenovirus-SIRT3 (Ad-SIRT3) significantly reduced the HG-induced expression of p53 and PHDs and increased HIF-1α levels in H9c2 cells. Ad-SIRT3 treatment also regulated PHDs-HIF-1α levels in the hearts of diabetic db/db mice. Our study revealed a novel role of the HG-induced disruption of PHDs-HIF-α signaling via upregulating p53 and TIGAR expression. Therefore, the p53/TIGAR signaling pathway may be a novel target for diabetic cardiomyopathy.

Decreased expression of prolyl hydroxylase 1 is associated with poor prognosis in colorectal cancers

BackgroundProlyl hydroxylase 1 (PHD1) is a prognostic marker in several cancers.Aims and scopesThis study was undertaken to elucidate the clinical relevance of PHD1 in colorectal cancer (CRC) prognosis.Materials and methodsWe compared PHD1 expression on a tissue microarray (TMA) containing samples from 1800 CRCs with corresponding clinicopathological tumor variables and patient survival.ResultsWhile PHD1 staining was always high in benign colorectal epithelium, high PHD1 staining was detectable in only 71.8% of CRCs. Low PHD1 staining was associated with advanced tumor stage (p = 0.0101) and shortened overall survival in CRC patients (p = 0.0011). In a multivariable analysis including tumor stage, histological type and PHD1 staining revealed tumor stage and histological type (p < 0.0001 each), but also PHD1 staining (p = 0.0202) to be independent prognostic markers for CRC.ConclusionsIn our cohort, loss of PHD1 expression independently identified a subset of CRC patients with poor overall survival and might, thus, be a promising prognostic marker. PHD1 targeting may even allow for specific therapeutic approaches for these patients.

Thiosulfinate-Enriched Allium sativum Extract Exhibits Differential Effects between Healthy and Sepsis Patients: The Implication of HIF-1α

Garlic (Allium sativum) has historically been associated with antioxidant, immunomodulatory, and microbiocidal properties, mainly due to its richness in thiosulfates and sulfur-containing phytoconstituents. Sepsis patients could benefit from these properties because it involves both inflammatory and refractory processes. We evaluated the effects of thiosulfinate-enriched Allium sativum extract (TASE) on the immune response to bacterial lipopolysaccharide (LPS) by monocytes from healthy volunteers (HVs) and patients with sepsis. We also explored the TASE effects in HIF-1α, described as the key transcription factor leading to endotoxin tolerance in sepsis monocytes through IRAK-M expression. Our results showed TASE reduced the LPS-triggered reactive oxygen species (ROS) production in monocytes from both patients with sepsis and HVs. Moreover, this extract significantly reduced tumor necrosis factor (TNF)-α, interleukin-1β, and interleukin-6 production in LPS-stimulated monocytes from HVs. However, TASE enhanced the inflammatory response in monocytes from patients with sepsis along with increased expression of human leukocyte antigen-DR. Curiously, these dual effects of TASE on immune response were also found when the HV cohort was divided into low- and high-LPS responders. Although TASE enhanced TNFα production in the LPS-low responders, it decreased the inflammatory response in the LPS-high responders. Furthermore, TASE decreased the HIF-1α pathway-associated genes IRAK-M, VEGFA and PD-L1 in sepsis cells, suggesting HIF-1α inhibition by TASE leads to higher cytokine production in these cells as a consequence of IRAK-M downregulation. The suppression of this pathway by TASE was confirmed in vitro with the prolyl hydroxylase inhibitor dimethyloxalylglycine. Our data revealed TASE's dual effect on monocyte response according to status/phenotype and suggested the HIF-1α suppression as the possible underlying mechanism.

Dimethyloxalylglycine Attenuates Steroid-Associated Endothelial Progenitor Cell Impairment and Osteonecrosis of the Femoral Head by Regulating the HIF-1α Signaling Pathway

Endothelial impairment and dysfunction are closely related to the pathogenesis of steroid-associated osteonecrosis of the femoral head (SONFH). Recent studies have showed that hypoxia inducible factor-1α (HIF-1α) plays a crucial role in endothelial homeostasis maintenance. Dimethyloxalylglycine (DMOG) could suppress HIF-1 degradation and result in nucleus stabilization by repressing prolyl hydroxylase domain (PHD) enzymatic activity. Our results showed that methylprednisolone (MPS) remarkably undermined biological function of endothelial progenitor cells (EPC) by inhibiting colony formation, migration, angiogenesis, and stimulating senescence of EPCs, while DMOG treatment alleviated these effects by promoting HIF-1α signaling pathway, as evidenced by senescence-associated β-galactosidase (SA-β-Gal) staining, colony-forming unit, matrigel tube formation, and transwell assays. The levels of proteins related to angiogenesis were determined by ELISA and Western blotting. In addition, active HIF-1α bolstered the targeting and homing of endogenous EPCs to the injured endothelium in the femoral head. Histopathologically, our in vivo study showed that DMOG not only alleviated glucocorticoid-induced osteonecrosis but also promoted angiogenesis and osteogenesis in the femoral head as detected by microcomputed tomography (Micro-CT) analysis and histological staining of OCN, TRAP, and Factor Ⅷ. However, all of these effects were impaired by an HIF-1α inhibitor. These findings demonstrate that targeting HIF-1α in EPCs may constitute a novel therapeutic approach for the treatment of SONFH.

Systematic pan-cancer analysis of the potential tumor diagnosis and prognosis biomarker P4HA3.

Purpose: Prolyl 4-hydroxylase subunit alpha 3 (P4HA3) is implicated in several cancers' development. However, P4HA3 has not been reported in other cancers, and the exact mechanism of action is currently unknown. Materials and methods: First, the expression profile of P4HA3 was analyzed using a combination of the University of California Santa Cruz (UCSC) database, Cancer Cell Line Encyclopedia (CCLE) database, and Genotype-Tissue Expression (GTEx) database. UniCox and Kaplan-Meier were used to analyze the predictive value of P4HA3. The expression of P4HA3 was analyzed in clinical staging, immune subtypes, and Molecular subtypes. Secondly, the correlation of P4HA3 with immunomodulatory genes, immune checkpoint genes, RNA modification genes, immune cell infiltration, cancer-related functional status, tumor stemness index, DNA mismatch repair (MMR) genes and DNA Methyltransferase was examined. The role of P4HA3 in DNA methylation, copy number variation (CNV), mutational status, tumor mutational burden (TMB), and microsatellite instability (MSI) was also analyzed. In addition, gene set enrichment analysis (GSEA) was used to explore the potential functional mechanisms of P4HA3 in pan-cancer. Finally, P4HA3-related drugs were searched in CellMiner, Genomics of Drug Sensitivity in Cancer (GDSC), and Cancer Therapeutics Response Portal (CTRP) databases. Results: P4HA3 is significantly overexpressed in most cancers and is associated with poor prognosis. P4HA3 is strongly associated with clinical cancer stage, immune subtypes, molecular subtypes, immune regulatory genes, immune checkpoint genes, RNA modifier genes, immune cell infiltration, cancer-related functional status, tumor stemness index, MMR Gene, DNA Methyltransferase, DNA methylation, CNV, mutational status, TMB, and MSI are closely related. Available enrichment analysis revealed that P4HA3 is associated with the epithelial-mesenchymal transition and immune-related pathways. There are currently 20 drugs associated with P4HA3. Conclusion: In human pan-cancer, P4HA3 is associated with poor patient prognosis and multiple immune cells and may be a novel immunotherapeutic target. It may act on tumor progression through the epithelial-mesenchymal transition (EMT) pathway.

Drugs That Mimic Hypoxia Selectively Target EBV-Positive Gastric Cancer Cells.

Latent infection of Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cell cancers, including 10% of gastric carcinomas. We previously reported that hypoxia inducible factor-1α (HIF-1α) induces EBV's latent-to-lytic switch and identified several HIF-1α-stabilizing drugs that induce this viral reactivation. Here, we tested three classes of these drugs for preferential killing of the EBV-positive gastric cancer AGS-Akata cell line compared to its matched EBV-negative AGS control. We observed preferential killing with iron chelators [Deferoxamine (DFO); Deferasirox (DFX)] and a prolyl hydroxylase inhibitor (BAY 85-3934 (Molidustat)), but not with a neddylation inhibitor [MLN4924 (Pevonedistat)]. DFO and DFX also induced preferential killing of the EBV-positive gastric cancer AGS-BDneo and SNU-719 cell lines. Preferential killing was enhanced when low-dose DFX (10 μM) was combined with the antiviral prodrug ganciclovir. DFO and DFX induced lytic EBV reactivation in approximately 10% of SNU-719 and 20-30% of AGS-Akata and AGS-BDneo cells. However, neither DFO nor DFX significantly induced synthesis of lytic EBV proteins in xenografts grown in NSG mice from AGS-Akata cells above the level observed in control-treated mice. Therefore, these FDA-approved iron chelators are less effective than gemcitabine at promoting EBV reactivation in vivo despite their high specificity and efficiency in vitro.

The role of hypoxia-inducible factor 1 alpha (HIF-1α) modulation in heavy metal toxicity.

Hypoxia-inducible factor 1 (HIF-1) is an oxygen-sensing transcriptional regulator orchestrating a complex of adaptive cellular responses to hypoxia. Several studies have demonstrated that toxic metal exposure may also modulate HIF-1α signal transduction pathway, although the existing data are scarce. Therefore, the present review aims to summarize the existing data on the effects of toxic metals on HIF-1 signaling and the potential underlying mechanisms with a special focus on prooxidant effect of the metals. The particular effect of metals was shown to be dependent on cell type, varying from down- to up-regulation of HIF-1 pathway. Inhibition of HIF-1 signaling may contribute to impaired hypoxic tolerance and adaptation, thus promoting hypoxic damage in the cells. In contrast, its metal-induced activation may result in increased tolerance to hypoxia through increased angiogenesis, thus promoting tumor growth and contributing to carcinogenic effect of heavy metals. Up-regulation of HIF-1 signaling is mainly observed upon Cr, As, and Ni exposure, whereas Cd and Hg may both stimulate and inhibit HIF-1 pathway. The mechanisms underlying the influence of toxic metal exposure on HIF-1 signaling involve modulation of prolyl hydroxylases (PHD2) activity, as well as interference with other tightly related pathways including Nrf2, PI3K/Akt, NF-κB, and MAPK signaling. These effects are at least partially mediated by metal-induced ROS generation. Hypothetically, maintenance of adequate HIF-1 signaling upon toxic metal exposure through direct (PHD2 modulation) or indirect (antioxidant) mechanisms may provide an additional strategy for prevention of adverse effects of metal toxicity.

Roxadustat (FG-4592) abated lipopolysaccharides-induced depressive-like symptoms via PI3K signaling.

Despite its role in inflammation and the redox system under hypoxia, the effects and molecular mechanisms of hypoxia-inducible factor (HIF) in neuroinflammation-associated depression are poorly explored. Furthermore, Prolyl hydroxylase domain-containing proteins (PHDs) regulate HIF-1; however, whether and how PHDs regulate depressive-like behaviors under Lipopolysaccharides (LPS)-induced stress conditions remain covered. To highlight the roles and underlying mechanisms of PHDs-HIF-1 in depression, we employed behavioral, pharmacological, and biochemical analyses using the LPS-induced depression model. Lipopolysaccharides treatment induced depressive-like behaviors, as we found, increased immobility and decreased sucrose preference in the mice. Concurrently, we examined increased cytokine levels, HIF-1 expression, mRNA levels of PHD1/PHD2, and neuroinflammation upon LPS administration, which Roxadustat reduced. Furthermore, the PI3K inhibitor wortmannin reversed Roxadustat-induced changes. Additionally, Roxadustat treatment attenuated LPS-induced synaptic impairment and improved spine numbers, ameliorated by wortmannin. Lipopolysaccharides-dysregulates HIF-PHDs signaling may contribute to neuroinflammation-coincides depression via PI3K signaling.

CEBPB upregulates P4HA2 to promote the malignant biological behavior in IDH1 wildtype glioma.

Temozolomide (TMZ), the primary drug for glioma treatment, has limited treatment efficacy. Additionally, considerable evidence shows that isocitrate dehydrogenase 1 mutation-type (IDH1 mut) gliomas have a better response to TMZ than isocitrate dehydrogenase 1 wildtype (IDH1 wt) gliomas. Here, we aimed to identify potential mechanisms underlying this phenotype. Herein, the Cancer Genome Atlas bioinformatic data and 30 clinical samples from patients were analyzed to reveal the expression level of cytosine-cytosine-adenosine-adenosine-thymidine (CCAAT) Enhancer Binding Protein Beta (CEBPB) and prolyl 4-hydroxylase subunit alpha 2 (P4HA2) in gliomas. Next, cellular and animal experiments, including cell proliferation, colony formation, transwell, CCK-8, and xenograft assays, were performed to explore the tumor-promoting effects of P4HA2 and CEBPB. Then, chromatin immunoprecipitation (ChIP) assays were used to confirm the regulatory relationships between them. Finally, a co-immunoprecipitation (Co-IP) assay was performed to confirm the effect of IDH1-132H to CEBPB proteins. We found that CEBPB and P4HA2 expression was significantly upregulated in IDH1 wt gliomas and associated with poor prognosis. CEBPB knockdown inhibited the proliferation, migration, invasion, and temozolomide resistance of glioma cells and hindered the growth of glioma xenograft tumors. CEBPE, as a transcription factor, exerted its function by transcriptionally upregulating P4HA2 expression in glioma cells. Importantly, CEBPB is prone to ubiquitin-proteasomal degradation in IDH1 R132H glioma cells. We also demonstrated that both genes are related to collagen synthesis, as confirmed by in vivo experiments. Thus, CEBPE promotes proliferation and TMZ resistance by inducing P4HA2 expression in glioma cells and offers a potential therapeutic target for glioma treatment.

An optimized reporter of the transcription factor hypoxia-inducible factor 1α reveals complex HIF-1α activation dynamics in single cells

Immune cells adopt a variety of metabolic states to support their many biological functions, which include fighting pathogens, removing tissue debris, and tissue remodeling. One of the key mediators of these metabolic changes is the transcription factor hypoxia-inducible factor 1α (HIF-1α). Single-cell dynamics have been shown to be an important determinant of cell behavior; however, despite the importance of HIF-1α, little is known about its single-cell dynamics or their effect on metabolism. To address this knowledge gap, here we optimized a HIF-1α fluorescent reporter and applied it to study single-cell dynamics. First, we showed that single cells are likely able to differentiate multiple levels of prolyl hydroxylase inhibition, a marker of metabolic change, via HIF-1α activity. We then applied a physiological stimulus known to trigger metabolic change, interferon-γ, and observed heterogeneous, oscillatory HIF-1α responses in single cells. Finally, we input these dynamics into a mathematical model of HIF-1α-regulated metabolism and discovered a profound difference between cells exhibiting high versus low HIF-1α activation. Specifically, we found cells with high HIF-1α activation are able to meaningfully reduce flux through the tricarboxylic acid cycle and show a notable increase in the NAD+/NADH ratio compared with cells displaying low HIF-1α activation. Altogether, this work demonstrates an optimized reporter for studying HIF-1α in single cells and reveals previously unknown principles of HIF-1α activation.

Alteration of antioxidant status, glucose metabolism, and hypoxia signal pathway in Eirocheir sinensis after acute hypoxic stress and reoxygenation

Dissolved oxygen (DO) is crucial for the survival of Chinese mitten crab (Eirocheir sinensis); low DO levels adversely affect the health of these crabs. In this study, we evaluated the underlying response mechanism of E. sinensis to acute hypoxic stress by analyzing antioxidant parameters, glycolytic indicators, and hypoxia signaling factors. The crabs were exposed to hypoxia for 0, 3, 6, 12, and 24 h and reoxygenated for 1, 3, 6, 12, and 24 h. The hepatopancreas, muscle, gill, and hemolymph were sampled at different exposure times to detect the biochemical parameters and gene expression. The results showed that the activity of catalase, antioxidants, and malondialdehyde in tissues significantly increased under acute hypoxia and gradually decreased during the reoxygenation phase. Under acute hypoxic stress, glycolysis indices, including hexokinase (HK), phosphofructokinase, pyruvate kinase (PK), pyruvic acid (PA), lactate dehydrogenase (LDH), lactic acid (LA), succinate dehydrogenase (SDH), glucose, and glycogen in the hepatopancreas, hemolymph, and gills increased to varying degrees but recovered to the control levels after reoxygenation. Gene expression data showed that hypoxia signaling pathway-related genes, including hypoxia-inducible factor-1α/β (HIF1α/β), prolyl hydroxylase (PHD), factor inhibiting hypoxia-inducible factor (FIH), and glycolysis-related factors (HK and PK) were upregulated, showing that the HIF signaling pathway was activated under hypoxic conditions. In conclusion, acute hypoxic exposure activated the antioxidant defense system, glycolysis, and HIF pathway to respond to adverse conditions. These data contribute to elucidating the defense and adaptive mechanisms of crustaceans to acute hypoxic stress and reoxygenation.

E3 ligase MAEA-mediated ubiquitination and degradation of PHD3 promotes glioblastoma progression

Glioblastoma (GBM) is the most common malignant glioma, with a high recurrence rate and a poor prognosis. However, the molecular mechanism behind the malignant progression of GBM is still unclear. In the present study, through the tandem mass tag (TMT)-based quantitative proteomic analysis of clinical primary and recurrent glioma samples, we identified that aberrant E3 ligase MAEA was expressed in recurrent samples. The results of bioinformatics analysis showed that the high expression of MAEA was related to the recurrence and poor prognosis of glioma and GBM. Functional studies showed that MAEA could promote proliferation, invasion, stemness and temozolomide (TMZ) resistance. Mechanistically, the data indicated that MAEA targeted prolyl hydroxylase domain 3 (PHD3) K159 to promote its K48-linked polyubiquitination and degradation, thus enhancing the stability of HIF-1α, thereby promoting the stemness and TMZ resistance of GBM cells through upregulating CD133. The in vivo experiments further confirmed that knocking down MAEA could inhibit the growth of GBM xenograft tumors. In summary, MAEA enhances the expression of HIF-1α/CD133 through the degradation of PHD3 and promotes the malignant progression of GBM.