Regulation Of HIF-1 Research Articles

Intermittent Hypoxia Mediates Cancer Development and Progression Through HIF-1 and miRNA Regulation

IntroductionThere is still a debate for the link between obstructive sleep apnoea (OSA) and cancer. The mechanisms underlying this causality are poorly understood. Several miRNAs are involved in cancer development and progression with expression being influenced by hypoxia.The aims of this work were (i) to compare miRNAs expression in controls versus patients affected by OSA without or with cancer (ONCO-OSA) and (ii) in colorectal cancer cells exposed to intermittent hypoxia (IH), to evaluate miRNAs impact on tumor progression in vitro. MethodsWe detected miRNAs by qRT-PCR in patients’ sera and in CaCo2 cells exposed to 2–32h of IH with or without acriflavine (ACF), a HIF-1 inhibitor. Viability and transwell invasion test were applied to investigate the proliferation and migration of CaCo2 exposed to IH and treated with miRNA inhibitors or acriflavine. HIF-1α activity was evaluated in CaCo2 cells after IH. ResultsThe levels of miR-21, miR-26a and miR-210 increased in OSA and ONCO-OSA patients compared to controls. MiR-23b increased in ONCO-OSA patients, and miR-27b and miR-145 increased in OSA but not ONCO-OSA patients. MiR-21, miR-26a, miR-23b and miR-210 increased in cells after IH. IH stimulated cell proliferation and migration. This effect was reduced after either miRNA inhibition or acriflavine treatment. MiRNA inhibition reduces HIF-1α gene expression. Conversely, acriflavine reduced the expression of these miRNAs. ConclusionsWe identified a signature of miRNAs, induced by the IH environment. They could be implicated in cancer development and progression through a regulatory loop involving HIF-1.

HIF and MYC signaling in adrenal neoplasms of the neural crest: implications for pediatrics.

Pediatric neural crest-derived adrenal neoplasms include neuroblastoma and pheochromocytoma. Both entities are associated with a high degree of clinical heterogeneity, varying from spontaneous regression to malignant disease with poor outcome. Increased expression and stabilization of HIF2α appears to contribute to a more aggressive and undifferentiated phenotype in both adrenal neoplasms, whereas MYCN amplification is a valuable prognostic marker in neuroblastoma. The present review focuses on HIF- and MYC signaling in both neoplasms and discusses the interaction of associated pathways during neural crest and adrenal development as well as potential consequences on tumorigenesis. Emerging single-cell methods together with epigenetic and transcriptomic analyses provide further insights into the importance of a tight regulation of HIF and MYC signaling pathways during adrenal development and tumorigenesis. In this context, increased attention to HIF-MYC/MAX interactions may also provide new therapeutic options for these pediatric adrenal neoplasms.

Integrating UPLC-Q-Exactive Orbitrap/MS, network pharmacology and experimental validation to reveal the potential mechanism of Tibetan medicine Rhodiola granules in improving myocardial ischemia-reperfusion injury

Ethnopharmacology relevanceRhodiola granules (RG) is a traditional Tibetan medicine prescription that can be used to improve the symptoms of ischemia and hypoxia in cardiovascular and cerebrovascular diseases. However, there is no report on its use to improve myocardial ischemia/reperfusion (I/R) injury, and its potential active ingredients and mechanism against myocardial ischemia/reperfusion (I/R) injury remain unclear. Aim of the studyThis study aimed to reveal the potential bioactive components and underlying pharmacological mechanisms of RG in improving myocardial I/R injury through a comprehensive strategy. Materials and methodsUPLC-Q-Exactive Orbitrap/MS technology was used to analyze the chemical components of RG, the potential bioactive components and targets were tracked and predicted by the SwissADME and SwissTargetPrediction databases, and the core targets were predicted through the PPI network, as well the functions and pathways were determined by GO and KEGG analysis. In addition, the molecular docking and ligation of the anterior descending coronary artery-induced rat I/R models were experimentally validated. ResultsA total of 37 ingredients were detected from RG, including nine flavones, ten flavonoid glycosides, one glycoside, eight organic acids, four amides, two nucleosides, one amino acid, and two other components. Among them, 15 chemical components, such as salidroside, morin, diosmetin, and gallic acid were identified as key active compounds. Ten core targets, including AKT1, VEGF, PTGS2, and STAT3, were discovered through the analysis of the PPI network constructed from 124 common potential targets. These possible targets were involved in the regulation of oxidative stress and HIF-1/VEGF/PI3K-Akt signaling pathways. Furthermore, molecular docking confirmed that the potential bioactive compounds in RG have good potential binding abilities to AKT1, VEGFA, PTGS2, STAT3, and HIF-1α proteins. Then, the animal experiments showed that RG could significantly improve the cardiac function of I/R rats, reduce the size of myocardial infarction, improve the myocardial structure, and reduce the degree of myocardial fibrosis, inflammatory cell infiltration, and myocardial cell apoptosis rate in I/R rats. In addition, we also found that RG could decrease the concentration of AGE, Ox-LDL, MDA, MPO, XOD, SDH, Ca2+, and ROS, and increase the concentration of Trx, TrxR1, SOD, T-AOC, NO, ATP, Na+k+-ATPase, Ca2+-ATPase, and CCO. Moreover, RG could significantly down-regulate the expressions of Bax, Cleaved-caspase3, HIF-1α, and PTGS2, as well up-regulate the expressions of Bcl-2, VEGFA, p-AKT1, and p-STAT3. ConclusionIn summary, we revealed for the first time the potential active ingredients and mechanisms of RG for myocardial I/R injury therapy through a comprehensive research strategy. RG may synergistically improve myocardial I/R injury through anti-inflammatory, regulating energy metabolism, and oxidative stress, improving I/R-induced myocardial apoptosis, which may be related to the HIF-1/VEGF/PI3K-Akt signaling pathway. Our study provides new insights into the clinical application of RG and also provides a reference for the development and mechanism research of other Tibetan medicine compound preparations.

Abstract 4804: p38 MAPK-dependent regulation of HIF-1 and expression of the hypoxia-inducible gene IGFBP-3 in leiomyosarcoma cells

Abstract Hypoxia is a common feature of tumor microenvironments and stimulates a variety of responses in a cell. Tumor hypoxia is also associated with poor prognosis in soft tissue sarcomas (STS), including leiomyosarcomas. Genes upregulated under hypoxia contribute to the adaptation of tumor cells and promote tumor progression. Using transcriptome-wide gene expression analysis, we identified IGFBP-3 as one of the most highly upregulated genes in SK-LMS-1 leiomyosarcoma cells in hypoxic conditions. IGFBP-3 has been studied in several tumor types, including STS, and is associated with their altered pathogenesis. Mechanisms responsible for the hypoxia-induced expression of IGFBP-3 in STS have not been previously well defined. Hypoxia results in the stabilization of the hypoxia-inducible transcription factor, HIF-1, and production of reactive oxygen species (ROS) in other cell types. Here, we examined HIF-1 and ROS-dependent mechanisms to determine their roles in the hypoxia-induced expression of IGFBP-3 and other genes in SK-LMS-1 cells. Our data indicate that both ROS production and HIF-1 activation are required for the enhanced IGFBP-3 mRNA expression in hypoxic SK-LMS-1 cells. We also tested the roles of the stress activated protein kinase p38 and the tumor suppressor p53 for their potential to contribute to the regulation of IGFBP-3 expression in these cells. Inhibition of p53 tumor suppressor activity only partially reduced IGFBP-3 expression. We also determined that neither hypoxia nor inhibition of p53 affected the expression of the p53 target gene, CDKN1A (p21), in these cells. In contrast, inhibition of p38 MAPK completely prevented the hypoxia-induced increase in IGFBP-3 expression. Additionally, we found that p38 MAPK inhibition reduced HIF-1 protein accumulation and activity but had no effect on its mRNA expression under hypoxic conditions. Together these results suggest that ROS-dependent activation of p38 MAPK stabilizes HIF-1 and promotes the downstream expression of IGFBP-3. In contrast, p53 appears to have a limited effect on the expression of IGFBP-3 in SK-LMS-1 cells. Our results clarify the signaling pathway involved in the response of soft tissue sarcoma cells to hypoxia and may provide new avenues for intervention in cancer progression. Citation Format: Nuha Haque, Eric Shelden. p38 MAPK-dependent regulation of HIF-1 and expression of the hypoxia-inducible gene IGFBP-3 in leiomyosarcoma cells. [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 4804.

Tissue Inhibitors of Metalloproteinases 1 Confers 5-Fluorouracil Resistance and Stemness of Gastric Cancer Cells via the Up-Regulation of Hypoxia-Inducible Factor-1α

The role and potential mechanism of TIMP1 in resistance to 5-Fluorouracil (5-Fu) and stem properties in gastric cancer (GC) were investigated. The expressions of HIF-1α and TIMP1, as well as the chemosensitivity of the 5-Fu in GC cell lines (GCCL) (e.g., MGC-803, BGC-823, SGC-7901, HGC-27 and AGS) upon normoxia or hypoxia were analyzed by means of RT-PCR and CCK-8 assay, respectively. Meanwhile, the population of stem cells was determined by using sphere formation assay, while stem cell markers (SCM) (Oct4 and CD44) were detected by western blot to evaluate stem properties. Hypoxia led to upregulated expression of HIF-1α and TIMP1, and enhanced resistance to 5-Fu, sphere formation capability, and expression of SCM in GC cells (GCCs). Indeed, the expressions of TIMP1 and HIF-1α were positively related to each other. The protein levels of both HIF-1α and TIMP1 were increased and decreased by overexpressing and silencing TIMP1, respectively. Under hypoxia conditions, overexpression of TIMP1 conferred 5-Fu-resistance and stem properties to MGC-803 and AGS cells, as revealed by increased IC50 value of 5-Fu, enhanced sphere formation, and up-regulation of Oct4 and CD44; silencing TIMP1 caused the contrary results. TIMP1 is an effective regulator of HIF-1 and plays a critical role in resistance to 5-Fu and stem properties in GCCs upon hypoxia.

The hypoxia response pathway promotes PEP carboxykinase and gluconeogenesis in C. elegans

Actively dividing cells, including some cancers, rely on aerobic glycolysis rather than oxidative phosphorylation to generate energy, a phenomenon termed the Warburg effect. Constitutive activation of the Hypoxia Inducible Factor (HIF-1), a transcription factor known for mediating an adaptive response to oxygen deprivation (hypoxia), is a hallmark of the Warburg effect. HIF-1 is thought to promote glycolysis and suppress oxidative phosphorylation. Here, we instead show that HIF-1 can promote gluconeogenesis. Using a multiomics approach, we reveal the genomic, transcriptomic, and metabolomic landscapes regulated by constitutively active HIF-1 in C. elegans. We use RNA-seq and ChIP-seq under aerobic conditions to analyze mutants lacking EGL-9, a key negative regulator of HIF-1. We integrate these approaches to identify over two hundred genes directly and functionally upregulated by HIF-1, including the PEP carboxykinase PCK-1, a rate-limiting mediator of gluconeogenesis. This activation of PCK-1 by HIF-1 promotes survival in response to both oxidative and hypoxic stress. Our work identifies functional direct targets of HIF-1 in vivo, comprehensively describing the metabolome induced by HIF-1 activation in an organism.

The impact of COVID-19 on populations living at high altitude: Role of hypoxia-inducible factors (HIFs) signaling pathway in SARS-CoV-2 infection and replication.

Cases of coronavirus disease 2019 (COVID-19) have been reported worldwide. However, one epidemiological report has claimed a lower incidence of the disease in people living at high altitude (>2,500 m), proposing the hypothesis that adaptation to hypoxia may prove to be advantageous with respect to SARS-CoV-2 infection. This publication was initially greeted with skepticism, because social, genetic, or environmental parametric variables could underlie a difference in susceptibility to the virus for people living in chronic hypobaric hypoxia atmospheres. Moreover, in some patients positive for SARS-CoV-2, early post-infection ‘happy hypoxia” requires immediate ventilation, since it is associated with poor clinical outcome. If, however, we accept to consider the hypothesis according to which the adaptation to hypoxia may prove to be advantageous with respect to SARS-CoV-2 infection, identification of the molecular rational behind it is needed. Among several possibilities, HIF-1 regulation appears to be a molecular hub from which different signaling pathways linking hypoxia and COVID-19 are controlled. Interestingly, HIF-1α was reported to inhibit the infection of lung cells by SARS-CoV-2 by reducing ACE2 viral receptor expression. Moreover, an association of the rs11549465 variant of HIF-1α with COVID-19 susceptibility was recently discovered. Here, we review the evidence for a link between HIF-1α, ACE2 and AT1R expression, and the incidence/severity of COVID-19. We highlight the central role played by the HIF-1α signaling pathway in the pathophysiology of COVID-19.

Abstract 120: HIF-1α regulation by Smurf2-mediated protein degradation in a VHL-independent manner

Abstract HIF-1α is the main mediator of hypoxic response in cells and contributes to angiogenesis, metastasis and therapy resistance in cancer. The canonical pathway of HIF-1α degradation in normoxia is through the Von Hippel-Lindau (VHL) E3 ubiquitin ligase. We previously found that knockdown of CDK4 decreased HIF-1α expression in a VHL-independent manner. To explore the mechanism of CDK4-induced HIF-1α stabilization, we performed a proteomic analysis on HIF-1α-interacting proteins with palbociclib treatment, a bioinformatic prediction of HIF-1α-targeting E3 ubiquitin ligases and identified SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) to be involved in a non-canonical HIF-1α regulation mechanism. Overexpression of Smurf2 enhanced the ubiquitination of HIF-1α and reduced expression of HIF-1α in HCT116 colorectal cancer cells. Smurf2 overexpression downregulated HIF-1α target gene SLC2A1 but not HIF1A mRNA level in hypoxia. Knockdown of SMURF2 increased HIF-1α expression in normoxia even when CDK4 was knocked-down. TCGA analysis showed correlation between high SMURF2 mRNA and better prognosis in kidney renal clear cell carcinoma (KIRC). In KIRC, VHL loss is a frequent mutational event that leads to high constitutive expression of HIF-1α and HIF-2α. Thus, we investigated a role for Smurf2 in HIF regulation in this cancer type. Overexpression of Smurf2 suppressed HIF-1α expression in RCC4 VHL-deficient clear cell renal cancer line in normoxia. Knockdown of SMURF2 increased HIF-1α expression in RCC4 cells. Treatment of RCC4 cells with Smurf2 inhibitor Heclin also increased HIF-1α expression in normoxia. HIF-2α expression was reduced upon Smurf2 overexpression in RCC4 cells under normoxia and in SW480 colorectal cancer cells under hypoxia. In summary, Smurf2 mediates VHL-independent HIF-1α degradation, which provides new insights for HIF-1α targeting in cancers especially with VHL deficiency, and targeting of Smurf2 to increase HIF activity may have functional consequences in non-neoplastic disorders. Citation Format: Shuai Zhao, Wafik S. El-Deiry. HIF-1α regulation by Smurf2-mediated protein degradation in a VHL-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 120.

The multifaceted role of EGLN family prolyl hydroxylases in cancer: going beyond HIF regulation

EGLN1, EGLN2 and EGLN3 are proline hydroxylase whose main function is the regulation of the HIF factors. They work as oxygen sensors and are the main responsible of HIFα subunits degradation in normoxia. Being their activity strictly oxygen-dependent, when oxygen tension lowers, their control on HIFα is released, leading to activation of systemic and cellular response to hypoxia. However, EGLN family members activity is not limited to HIF modulation, but it includes the regulation of essential mechanisms for cell survival, cell cycle metabolism, proliferation and transcription. This is due to their reported hydroxylase activity on a number of non-HIF targets and sometimes to hydroxylase-independent functions. For these reasons, EGLN enzymes appear fundamental for development and progression of different cancer types, playing either a tumor-suppressive or a tumor-promoting role, according to EGLN isoform and to tumor context. Notably, EGLN1, the most studied isoform, has been shown to have also a central role in tumor micro-environment modulation, mediating CAF activation and impairing HIF1α -related angiogenesis, thus covering an important function in cancer metastasis promotion. Considering the recent knowledge acquired on EGLNs, the possibility to target these enzymes for cancer treatment is emerging. However, due to their multifaceted and controversial roles in different cancer types, the use of EGLN inhibitors as anti-cancer drugs should be carefully evaluated in each context.

RHY‐1 Promotes Hydrogen Sulfide Tolerance

Once known best for its toxic effects, recent work has shown that hydrogen sulfide (H2 S) has many roles as a cellular messenger. For example, both endogenously produced and exogenously supplied H2 S protect against cellular damage and death associated with ischemia/reperfusion injury in mammals. The mechanistic relationship between beneficial and toxic effects of H2 S is not understood. We have developed C. elegans as a model to understand the mechanistic basis of H2 S effects in animals. In addition to facile genetic and genomic tools, using C. elegans provides the ability to precisely control both genotype and cellular H2 S exposure. C. elegans grown in 50 ppm H2 S are long-lived and resistant to hypoxia-induced disruption of protein homeostasis. The early transcriptional response to H2 S requires the C. elegans orthologue of the hypoxia-inducible transcription factor, hif-1. HIF-1 promotes survival in H2 S, at least in part, by upregulating expression of sqrd-1, which encodes the sulfide-quinone oxidoreductase. SQRD-1 catalyzes the first step in the mitochondrial oxidation of H2 S. In an unbiased forward genetic screen, we found that expression of rhy-1 suppresses lethality of hif-1 mutant animals in 50 ppm H2 S. RHY-1 is an integral-membrane ER protein with predicted acyltransferase (ACYL3) activity. The ACYL3 family is large and conserved across species from bacteria to primates, but the function of these enzymes is largely unstudied. RHY-1 was first characterized as a negative regulator of HIF-1, and our data indicate that it also promotes survival in H2 S independently of HIF-1. To understand RHY-1 function we have used biotinylation by antibody recognition (BAR) to identify proteins that interact with RHY-1. We show that RHY-1 directly associates with CYSL proteins, which are orthologues of cystathionine β-synthase. Mutations in cysl-1 abrogate RHY-1 function to regulate HIF-1 and to promote survival independently of HIF-1. We have also identified a novel methyltransferase, RIPS-1, that is required for RHY-1 to promote survival in H2 S but which is dispensable for the proper regulation of HIF-1. Together, our data show that RHY-1 promotes survival in H2 S through a novel mechanism which can bypass the only known catabolic pathway for H2 S in vivo. Understanding the mechanism of RHY-1 activity in H2 S will begin to elucidate the relationship between H2 S signaling and toxicity, and could lead to new therapeutic strategies to modulate endogenous H2 S levels.

Intermittent Hypoxia Destabilizes Histone Deacetylase 5 via Reactive Oxygen Species Dependent Dephosphorylation

We recently identified histone deacetylase (HDAC5) protein as an early epigenetic regulator of HIF‐1 mediated transcription of pro‐oxidants resulting in intermittent hypoxia (IH)‐induced sympathetic activation (Wang et al Frontiers Physiology 2021). However, underlying molecular mechanisms of decreased HDAC5 protein by IH remain unknown. In this study, we show that reactive oxygen species (ROS) generated by NADPH oxidases contribute to IH‐induced HDAC5 destabilization in pheochromacytoma 12 (PC12) cells. Treating IH exposed cells with phosphatase but not kinase inhibitors stabilized HDAC5 protein. Furthermore, overexpression of mutant HDAC5 by substituting S259 with alanine decreased HDAC5 protein expression. Pharmacological treatment with Calyculin A, a serine/threonine protein phosphatase (PP1 and PP2A) inhibitor or overexpression of NIPP1 (endogenous nuclear PP1 inhibitor) restored HDAC5 protein in IH exposed cells. Taken together these data suggest that dephosphorylation of S259 by PP1 destabilizes HDAC5 protein in response to IH. Our results demonstrate that ROS‐dependent activation of phosphatases constitute another layer of cellular responses to gene activation by IH distinct from previously identified protein kinase mechanisms contributing to IH induced autonomic morbidities.

USP33 deubiquitinates and stabilizes HIF-2alpha to promote hypoxia response in glioma stem cells.

Hypoxia regulates tumor angiogenesis, metabolism, and therapeutic response in malignant cancers including glioblastoma, the most lethal primary brain tumor. The regulation of HIF transcriptional factors by the ubiquitin-proteasome system is critical in the hypoxia response, but hypoxia-inducible deubiquitinases that counteract the ubiquitination remain poorly defined. While the activation of ERK1/2 also plays an important role in hypoxia response, the relationship between ERK1/2 activation and HIF regulation remains elusive. Here, we identified USP33 as essential deubiquitinase that stabilizes HIF-2alpha protein in an ERK1/2-dependent manner to promote hypoxia response in cancer cells. USP33 is preferentially induced in glioma stem cells by hypoxia and interacts with HIF-2alpha, leading to its stabilization through deubiquitination. The activation of ERK1/2 upon hypoxia promoted HIF-2alpha phosphorylation, enhancing its interaction with USP33. Silencing of USP33 disrupted glioma stem cells maintenance, reduced tumor vascularization, and inhibited glioblastoma growth. Our findings highlight USP33 as an essential regulator of hypoxia response in cancer stem cells, indicating a novel potential therapeutic target for brain tumor treatment.

HIF-pathway genes prognostic for progression-free and overall survival in metastatic clear cell renal cell carcinoma (mccRCC).

370 Background: Clear cell renal cell carcinoma (ccRCC) is characterized by defects in the Von Hippel-Lindau/hypoxia-inducible factor (VHL/HIF) pathway. Several FDA-approved RCC therapies target the products of HIF-response genes. HIF2A is a promising new target and drugs targeting HIF2A are undergoing clinical testing. A better understanding of HIF-related genes may reveal useful biomarkers and drug targets. Methods: Gene expression was determined from 324 archival pretreatment nephrectomy specimens available from the Cancer and Leukemia Group B (CALGB) 90206, a phase III trial of patients treated with INF-α vs. INF-α plus bevacizumab. TaqMan RT-qPCR was performed on the OpenArray platform using RNA extracted from tumor tissue macrodissected based on review of H&E staining by a genitourinary pathologist. HIF pathway genes were assessed across both treatment arms, utilizing the proportional hazards model as predictors of OS and PFS, with time from randomization to death or disease progression as endpoints. Results: A total of 28 HIF-related genes (involved either in canonical or non-canonical HIF regulation) were assessed in univariate PH. Nine of these genes were associated with OS (i.e., HIF2A , VEGFC, VEGFD, TGFA, VHL, CCND1, EGFR, EGLN3 and HSP90AA1); and 6 of them were also associated with PFS (i.e., HIF2A , TGFA, VHL, CCND1 and EGLN3, and HSP90AA1). The HIF isotypes HIF1A and HIF3A were not prognostic, likewise VEGFA and VEGFB. Prolyl hydroxylase domain (PHD) proteins efficiently hydroxylate HIFα in normoxic conditions. PHD isotypes EGLN (1-3) were evaluated and only EGLN3 was associated with OS and PFS. HIF is also regulated by non-canonical pathways that function independent of oxygen concentration. HSP90AA1 was the only non-canonical pathway gene that was prognostic, and it predicted both OS and PFS. When the 9 genes that were prognostic in univariate analysis were used in a backward stepwise multivariate cox regression, VEGFD, TGFA and EGLN3 predicted OS while HIF2A, VHL and TGFA predicted PFS. Conclusions: VHL, HIF2A (but not HIF1A and HIF3A), EGLN3 (but not EGLN1 and EGLN2) and some of the HIF-response genes were prognostic of both OS and PFS. HSP90AA1 was prognostic for OS and PFS, suggesting that the non-canonical HIF pathway also plays a role in disease progression. Future studies should consider these HIF pathway genes as potential drug targets, and as predictors of response to treatments targeting the hypoxia pathway and angiogenesis.[Table: see text]

Inactivation of mouse transmembrane prolyl 4-hydroxylase increases blood brain barrier permeability and ischemia-induced cerebral neuroinflammation

Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the hypoxic induction of >300 genes required for survival and adaptation under oxygen deprivation. Inhibition of HIF-P4H-2 has been shown to be protective in focal cerebral ischemia rodent models, while that of HIF-P4H-1 has no effects and inactivation of HIF-P4H-3 has adverse effects. A transmembrane prolyl 4-hydroxylase (P4H-TM) is highly expressed in the brain and contributes to the regulation of HIF, but the outcome of its inhibition on stroke is yet unknown. To study this, we subjected WT and P4htm−/− mice to permanent middle cerebral artery occlusion (pMCAO). Lack of P4H-TM had no effect on lesion size following pMCAO, but increased inflammatory microgliosis and neutrophil infiltration was observed in the P4htm−/− cortex. Furthermore, both the permeability of blood brain barrier and ultrastructure of cerebral tight junctions were compromised in P4htm−/− mice. At the molecular level, P4H-TM deficiency led to increased expression of proinflammatory genes and robust activation of protein kinases in the cortex, while expression of tight junction proteins and the neuroprotective growth factors erythropoietin and vascular endothelial growth factor was reduced. Our data provide the first evidence that P4H-TM inactivation has no protective effect on infarct size and increases inflammatory microgliosis and neutrophil infiltration in the cortex at early stage after pMCAO. When considering HIF-P4H inhibitors as potential therapeutics in stroke, the current data support that isoenzyme-selective inhibitors that do not target P4H-TM or HIF-P4H-3 would be preferred.

Regulation of Hypoxia Inducible Factors HIF-1 and HIF-2 by Calcidiol in Hepatocellular Carcinoma Cells Under Normoxia and Hypoxia

Regulation of Hypoxia Inducible Factors HIF-1 and HIF-2 by Calcidiol in Hepatocellular Carcinoma Cells Under Normoxia and Hypoxia

Reciprocal regulation of LOXL2 and HIF1\u03b1 drives the Warburg effect to support pancreatic cancer aggressiveness

Hypoxic microenvironment is common in solid tumors, particularly in pancreatic ductal adenocarcinoma (PDAC). The Warburg effect is known to facilitate cancer aggressiveness and has long been linked to hypoxia, yet the underlying mechanism remains largely unknown. In this study, we identify that lysyl oxidase-like 2 (LOXL2) is a hypoxia-responsive gene and is essential for the Warburg effect in PDAC. LOXL2 stabilizes hypoxia-inducible factor 1α (HIF1α) from prolyl hydroxylase (PHD)-dependent hydroxylation via hydrogen peroxide generation, thereby facilitating the transcription of multiple glycolytic genes. Therefore, a positive feedback loop exists between LOXL2 and HIF1α that facilitates glycolytic metabolism under hypoxia. Moreover, LOXL2 couples the Warburg effect to tumor growth and metastasis in PDAC. Hijacking glycolysis largely compromises LOXL2-induced oncogenic activities. Collectively, our results identify a hitherto unknown hypoxia-LOXL2-HIF1α axis in regulating the Warburg effect and provide an intriguing drug target for PDAC therapy.

Exploring the Effect of Jiawei Buguzhi Pills on TGF-β-Smad Pathway in Postmenopausal Osteoporosis Based on Integrated Pharmacological Strategy.

Objective To explore the effect of Jiawei Buguzhi Pills (JWBGZP) on the TGF-β-Smad pathway in postmenopausal osteoporosis (PMO) based on integrated pharmacological strategy. Method The ETCM database was used to collect JWBGZP. GeneCards and OMIM databases were utilized to obtain PMO-related genes. Cytoscape was used for network construction and analysis, and DAVID was used for GO and KEGG enrichment analysis of key targets. Animal experiments and cell experiments were conducted to further explore the mechanism. The bone mass density was detected by dual-energy X-ray bone densitometer. The TGF-β1 and Smad4 mRNA in bone tissue were detected by RT-qPCR. The TGF-β1 and Smad4 protein in bone tissue were detected by the western blot. The TGF-β1 and Smad4 protein in osteoblasts were determined by immunohistochemistry. Result A total of 721 JWBGZP potential targets and 385 PMO-related genes were obtained. The enrichment analysis showed that JWBGZP may regulate the TGF-beta signaling pathway, oxidation-reduction process, aging, response to hypoxia, response to ethanol, negative regulation of cell proliferation, PI3K-Akt, HIF-1, and other signaling pathways. The animal experiments showed that compared with the model group, the femoral bone mineral density and lumbar bone mineral density of the JWBGZP group increased (P < 0.05); the expression levels of TGF-β1 and Smad mRNA and proteins in the JWBGZP group were significantly higher (P < 0.05). The cell experiment results showed a large number of osteoblast stained blue-purple and orange-red calcified nodules. The expression levels of TGF-β1 and Smad proteins in the JWBGZP group were significantly higher than those in the blank control group and the sham operation group, and the protein expression levels in the model group were the lowest (P < 0.05). Conclusion JWBGZP may be involved in PI3K-Akt, HIF-1, estrogen, prolactin, and other signaling pathways and regulate MAPK1, AKT1, PIK3CA, JAK2, and other gene targets, regulate bone metabolism, and thereby treat PMO.

A secondary role for hypoxia and HIF1 in the regulation of (IFN\u03b3-induced) PD-L1 expression in melanoma

Cancer cells are able to escape immune surveillance by upregulating programmed death ligand 1 (PD-L1). A key regulator of PD-L1 expression is transcriptional stimulation by the IFNγ/JAK/STAT pathway. Recent studies suggest that hypoxia can induce PD-L1 expression. As hypoxia presents a hallmark of solid tumor development, hypoxic control of PD-L1 expression may affect the efficacy of cancer immunotherapy. This study aims to explore the hypoxic regulation of PD-L1 expression in human melanoma, and its interaction with IFNγ-induced PD-L1 expression. Analysis of the cutaneous melanoma dataset from the cancer genome atlas revealed a significant correlation of the HIF1-signaling geneset signature with PD-L1 mRNA expression. However, this correlation is less pronounced than other key pathways known to control PD-L1 expression, including the IFNγ/JAK/STAT pathway. This secondary role of HIF1 in PD-L1 regulation was confirmed by analyzing single-cell RNA-sequencing data of 33 human melanoma tissues. Interestingly, PD-L1 expression in these melanoma tissues was primarily found in macrophages. However, also in these cells STAT1, and not HIF1, displayed the most pronounced correlation with PD-L1 expression. Moreover, we observed that hypoxia differentially affects PD-L1 expression in human melanoma cell lines. Knockdown of HIF1 expression indicated a minor role for HIF1 in regulating PD-L1 expression. A more pronounced influence of hypoxia was found on IFNγ-induced PD-L1 mRNA expression, which is controlled at a 952 bp PD-L1 promoter fragment. These findings, showing the influence of hypoxia on IFNγ-induced PD-L1 expression, are relevant for immunotherapy, as both IFNγ and hypoxia are frequently present in the tumor microenvironment.

HIF-1α is transcriptionally regulated by NF-κB in acute kidney injury.

Oxygen homeostasis disturbances play a critical role in the pathogenesis of acute kidney injury (AKI). The transcription factor hypoxia-inducible factor-1 (HIF-1) is a master regulator of adaptive responses to hypoxia. Aside from posttranslational hydroxylation, the mechanism of HIF-1 regulation in AKI remains largely unclear. In this study, the mechanism of HIF-α regulation in AKI was investigated. We found that tubular HIF-1α expression significantly increased at the transcriptional level in ischemia-reperfusion-, unilateral ureteral obstruction-, and sepsis-induced AKI models, which was closely associated with macrophage-dependent inflammation. Meanwhile, NF-κB, which plays a central role in the inflammation response, was involved in the increasing expression of HIF-1α in AKI, as evidenced by pharmacological modulation (NF-κB inhibitor BAY11-7082). Mechanistically, NF-κB directly bound to the HIF-1α promoter and enhanced its transcription, which occurred not only under hypoxic conditions but also under normoxic conditions. Moreover, the induced HIF-1α by inflammation protected against tubular injury in AKI. Thus, our findings not only provide novel insights into HIF-1 regulation in AKI but also offer to understand the pathophysiology of kidney diseases.NEW & NOTEWORTHY Here, the mechanism of hypoxia-inducible factor-α (HIF-α) regulation in acute kidney injury (AKI) was investigated. We found that tubular HIF-1α expression significantly increased at the transcriptional level, which was closely associated with macrophage-dependent inflammation. Meanwhile, NF-κB was involved in the increasing expression of HIF-1α in AKI. Mechanistically, NF-κB directly bound to the HIF-1α promoter and enhanced its transcription. Our findings not only provide novel insights into HIF-1 regulation in AKI but also offer to understand the pathophysiology of kidney diseases.

Blood Plasma Metabolic Profile of Newborns with Hypoxic-Ischaemic Encephalopathy by GC-MS.

Background Early diagnosis of hypoxic-ischaemic encephalopathy (HIE) is crucial in preventing neurodevelopmental disabilities and reducing morbidity and mortality. The study was to investigate the plasma metabolic signatures in the peripheral blood of HIE newborns and explore the potential diagnostic biomarkers. Method In the present study, 24 newborns with HIE and 24 healthy controls were recruited. The plasma metabolites were measured by gas chromatography-mass spectrometry (GC-MS), and the raw data was standardized by the EigenMS method. Significantly differential metabolites were identified by multivariate statistics. Pathway enrichment was performed by bioinformatics analysis. Meanwhile, the diagnostic value of candidate biomarkers was evaluated. Result The multivariate statistical models showed a robust capacity to distinguish the HIE cases from the controls. 52 metabolites were completely annotated. 331 significantly changed pathways were enriched based on seven databases, including 33 overlapped pathways. Most of them were related to amino acid metabolism, energy metabolism, neurotransmitter biosynthesis, pyrimidine metabolism, the regulation of HIF by oxygen, and GPCR downstream signaling. 14 candidate metabolites showed great diagnostic potential on HIE. Among them, alpha-ketoglutaric acid has the potential to assess the severity of HIE in particular. Conclusion The blood plasma metabolic profile could comprehensively reflect the metabolic disorders of the whole body under hypoxia-ischaemic injury. Several candidate metabolites may serve as promising biomarkers for the early diagnosis of HIE. Further validation based on large clinical samples and the establishment of guidelines for the clinical application of mass spectrometry data standardization methods are imperative in the future.