RESUMEN
Cellular injury caused by stimuli plays an important role in the progression of various diseases including acute and chronic kidney diseases. The dynamic transcriptional regulation responding to stimuli underlies the important mechanism of injury. In this study, we investigated the regulatory elements and their dynamic activities in kidney tubular epithelial cells. We captured the chromatin accessibility and gene expression with ATAC-seq and RNA sequencing under a variety of extracellular stimuli including H2 O2 , TGF-ß1, and FG4592 which is an agonist of hypoxia-inducible factor. Our results revealed both condition-specific and condition-shared transcription regulation. Interestingly, the shared regulation program revealed that the key transcription factor HNF1B-mediated cellular reprogramming leads to a common change among the stimuli. We found the HNF1B regulatory network was significantly disrupted in various kidney diseases.
Asunto(s)
Lesión Renal Aguda/genética , Factor Nuclear 1-beta del Hepatocito/genética , Riñón/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Insuficiencia Renal Crónica/genética , Lesión Renal Aguda/patología , Cromatina/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica/genética , Humanos , Peróxido de Hidrógeno/farmacología , Factor 1 Inducible por Hipoxia/agonistas , Factor 1 Inducible por Hipoxia/genética , Riñón/patología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Insuficiencia Renal Crónica/patología , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Early in 2020, racehorse doping cases revolved around the hypoxia-inducible factor (HIF) activator IOX-2. While the composition of IOX-2 has also been known and monitored in human doping controls for several years, the testing capability of routine sports drug testing methods was revisited for this newly surfaced doping agent. IOX-2 and the analytically well-established HIF activator roxadustat (FG-4592) share identical precursor/product ion pairs, enabling their co-detection in existing initial testing procedures in routine doping controls for the intact unconjugated analytes. In addition, hydroxylated IOX-2 and the corresponding glucuronic acid conjugates were identified as major metabolites in a microdose elimination study, contributing to enhanced initial testing and confirmation procedures.
Asunto(s)
Doping en los Deportes/prevención & control , Factor 1 Inducible por Hipoxia/agonistas , Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/orina , Isoquinolinas/orina , Detección de Abuso de Sustancias/normas , Doping en los Deportes/métodos , Glicina/administración & dosificación , Glicina/análogos & derivados , Glicina/orina , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/administración & dosificación , Isoquinolinas/administración & dosificación , Masculino , Persona de Mediana Edad , Proyectos Piloto , Estudios Retrospectivos , Detección de Abuso de Sustancias/métodosRESUMEN
The protective role of hypoxia-inducible factor-1 (HIF-1) against liver ischemia-reperfusion injury has been well proved. However its role in liver donation and preservation from donation after cardiac death (DCD) is still unknown. The objective of this study was to test the hypothesis that pharmaceutical stabilization of HIF-1 in DCD donors would result in a better graft liver condition. Male SD rats (6 animals per group) were randomly given the synthetic prolyl hydroxylase domain inhibitor FG-4592 (Selleck, 6 mg/kg of body weight) or its vehicle (dimethylsulfoxide). Six hours later, cardiac arrest was induced by bilateral pneumothorax. Rat livers were retrieved 30 min after cardiac arrest, and subsequently cold stored in University of Wisconsin solution for 24 h. They were reperfused for 60 min with Krebs-Henseleit bicarbonate buffer in an isolated perfused liver model, after which the perfusate and liver tissues were investigated. Pretreatment with FG-4592 in DCD donors significantly improved graft function with increased bile production and synthesis of adenosine triphosphate, decreased perfusate liver enzyme release, histology injury scores and oxidative stress-induced cell injury and apoptosis after reperfusion with the isolated perfused liver model. The beneficial effects of FG-4592 is attributed in part to the accumulation of HIF-1 and ultimately increased PDK1 production. Pretreatment with FG-4592 in DCD donors resulted in activation of the HIF-1 pathway and subsequently protected liver grafts from warm ischemia and cold-stored injury. These data suggest that the pharmacological HIF-1 induction may provide a clinically applicable therapeutic intervention to prevent injury to DCD allografts.
Asunto(s)
Factor 1 Inducible por Hipoxia/agonistas , Trasplante de Hígado/métodos , Hígado/efectos de los fármacos , Hígado/fisiología , Preservación de Órganos/métodos , Perfusión/métodos , Adenosina/metabolismo , Alopurinol/metabolismo , Animales , Muerte , Glutatión/metabolismo , Supervivencia de Injerto/efectos de los fármacos , Factor 1 Inducible por Hipoxia/metabolismo , Insulina/metabolismo , Masculino , Soluciones Preservantes de Órganos/metabolismo , Rafinosa/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Mimosine, which is a natural plant amino acid present in the Leucaena genus, is able to induce hypoxiainducible factors (HIFs). Previous evidence has indicated that HIF regulates angiogenesisosteogenesis coupling in bone metabolism, and it has previously been reported that mimosine inhibits prostaglandin (PG)F2αinduced osteoprotegerin (OPG) synthesis without affecting interleukin6 (IL6) production in osteoblastlike MC3T3E1 cells. In addition, PGE1 has been demonstrated to induce OPG synthesis via activation of p38 mitogenactivated protein (MAP) kinase and stressactivated protein kinase/cJun Nterminal kinase (SAPK/JNK) in these cells, and PGE1 stimulates IL6 production via the activation of protein kinase A. In the present study, the effects of mimosine on the PGE1stimulated synthesis of OPG and IL6 were investigated in osteoblastlike MC3T3E1 cells. The concentrations of OPG and IL6 were measured using relevant ELISA kits. OPG mRNA was measured by semiquantitative reverse transcription polymerase chain reaction. The phosphorylation of p38 MAP kinase and SAPK/JNK was analyzed by western blotting. Mimosine significantly reduced PGE1induced release of OPG and OPG mRNA expression levels without affecting the release of IL6. In addition, deferoxamine, which is also a normoxic HIF inducer, significantly inhibited PGE1induced OPG release and OPG mRNA expression levels; however, it had little effect on IL6 release. Furthermore, mimosine and deferoxamine failed to affect PGE1stimulated phosphorylation of p38 MAP kinase or SAPK/JNK. These results strongly suggest that normoxic HIF inducers attenuate PGE1stimulated OPG synthesis without affecting IL6 production in osteoblasts.
Asunto(s)
Alprostadil/metabolismo , Deferoxamina/farmacología , Factor 1 Inducible por Hipoxia/agonistas , Mimosina/farmacología , Osteoblastos/efectos de los fármacos , Osteoprotegerina/metabolismo , Animales , Línea Celular , Fabaceae/química , Factor 1 Inducible por Hipoxia/metabolismo , Interleucina-6/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Ratones , Mimosina/química , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoprotegerina/genética , Fosforilación/efectos de los fármacos , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In the course of our screening for activators of hypoxia-inducible factor (HIF), A-503451 A and virantmycin were isolated from the cultured broth of an actinomycete strain, Streptomyces sp. SANK 60101. From the same culture, the non-active homologs A-503451 B and D were also isolated. A-503451 A and virantmycin activated HIF-dependent reporter gene expression with EC50 values of 8 and 17 ng ml-1, respectively. They are highly potent activators of HIF and thus may be therapeutically useful for erythropoiesis and neural cell protection.
Asunto(s)
Fermentación , Factor 1 Inducible por Hipoxia/agonistas , Quinolinas/química , Streptomyces/metabolismo , Genes Reporteros , Células Hep G2 , Humanos , Estructura Molecular , Streptomyces/genéticaRESUMEN
BACKGROUND: Pharmacological induction of hypoxia-inducible factor (HIF), a global transcriptional regulator of the hypoxic response, by prolyl hydroxylase inhibitors (PHDi) is protective in murine models of colitis, and epithelial cells are critical for the observed therapeutic efficacy. Because systemic HIF activation may lead to potentially negative off-target effects, we hypothesized that targeting epithelial HIF through oral delivery of PHDi would be sufficient to protect against colitis in a mouse model. METHODS: Using a chemically induced trinitrobenzene sulfonic acid murine model of colitis, we compared the efficacy of oral and intraperitoneal (i.p.) delivery of the PHDi; AKB-4924 in preventing colitis, as measured by endoscopy, histology, barrier integrity, and immune profiling. Furthermore, we measured potential off-target effects, examining HIF and HIF target genes in the heart and kidney, as well as erythropoietin and hematocrit levels. RESULTS: Oral administration of AKB-4924 exhibited mucosal protection comparable i.p. dosing. Oral delivery of PHDi led to reduced colonic epithelial HIF stabilization compared with i.p. delivery, but this was still sufficient to induce transcription of downstream HIF targets. Furthermore, oral delivery of PHDi led to reduced stabilization of HIF and activation of HIF targets in extraintestinal organs. CONCLUSIONS: Oral delivery of PHDi therapies to this intestinal mucosa protects against colitis in animal models and represents a potential therapeutic strategy for inflammatory bowel disease, which also precludes unwanted extraintestinal effects.
Asunto(s)
Colitis/tratamiento farmacológico , Modelos Animales de Enfermedad , Membrana Mucosa/efectos de los fármacos , Piperazinas/administración & dosificación , Inhibidores de Prolil-Hidroxilasa/administración & dosificación , Piridonas/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos , Administración Oral , Animales , Colitis/inducido químicamente , Colitis/patología , Femenino , Factor 1 Inducible por Hipoxia/agonistas , Factor 1 Inducible por Hipoxia/metabolismo , Técnicas para Inmunoenzimas , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos BALB C , Membrana Mucosa/metabolismo , Piperazinas/farmacología , Inhibidores de Prolil-Hidroxilasa/farmacología , Piridonas/farmacología , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
Pharmacological stabilization of hypoxia-inducible factor (HIF) through prolyl hydroxylase (PHD) inhibition limits mucosal damage associated with models of murine colitis. However, little is known about how PHD inhibitors (PHDi) influence systemic immune function during mucosal inflammation or the relative importance of immunological changes to mucosal protection. We hypothesized that PHDi enhances systemic innate immune responses to colitis-associated bacteremia. Mice with colitis induced by trinitrobenzene sulfonic acid were treated with AKB-4924, a new HIF-1 isoform-predominant PHDi, and clinical, immunological, and biochemical endpoints were assessed. Administration of AKB-4924 led to significantly reduced weight loss and disease activity compared with vehicle controls. Treated groups were pyrexic but did not become subsequently hypothermic. PHDi treatment augmented epithelial barrier function and led to an approximately 50-fold reduction in serum endotoxin during colitis. AKB-4924 also decreased cytokines involved in pyrogenesis and hypothermia, significantly reducing serum levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α while increasing IL-10. Treatment offered no protection against colitis in epithelial-specific HIF-1α-deficient mice, strongly implicating epithelial HIF-1α as the tissue target for AKB-4924-mediated protection. Taken together, these results indicate that inhibition of prolyl hydroxylase with AKB-4924 enhances innate immunity and identifies that the epithelium is a central site of inflammatory protection afforded by PHDi in murine colitis.
Asunto(s)
Colitis/inmunología , Colitis/metabolismo , Inmunidad Innata/efectos de los fármacos , Inmunidad Mucosa/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Prolil Hidroxilasas/metabolismo , Inhibidores de Prolil-Hidroxilasa/farmacología , Animales , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Modelos Animales de Enfermedad , Endotoxemia/tratamiento farmacológico , Femenino , Factor 1 Inducible por Hipoxia/agonistas , Subunidad alfa del Factor 1 Inducible por Hipoxia/agonistas , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Permeabilidad/efectos de los fármacos , Piperazinas/administración & dosificación , Piperazinas/farmacología , Piridonas/administración & dosificación , Piridonas/farmacología , Ácido Trinitrobencenosulfónico/efectos adversos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is a major regulator of energy homeostasis and cellular adaptation to low oxygen stress. HIF-1 is also activated in response to bacterial pathogens and supports the innate immune response of both phagocytes and keratinocytes. In this work, we show that a new pharmacological compound AKB-4924 increases HIF-1 levels and enhances the antibacterial activity of phagocytes and keratinocytes against both methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus in vitro. AKB-4924 is also effective in stimulating the killing capacity of keratinocytes against the important opportunistic skin pathogens Pseudomonas aeruginosa and Acinetobacter baumanii. The effect of AKB-4924 is mediated through the activity of host cells, as the compound exerts no direct antimicrobial activity. Administered locally as a single agent, AKB-4924 limits S. aureus proliferation and lesion formation in a mouse skin abscess model. This approach to pharmacologically boost the innate immune response via HIF-1 stabilization may serve as a useful adjunctive treatment for antibiotic-resistant bacterial infections.
Asunto(s)
Antibacterianos/uso terapéutico , Factor 1 Inducible por Hipoxia/inmunología , Inmunidad Innata/efectos de los fármacos , Piperazinas/uso terapéutico , Piridonas/uso terapéutico , Enfermedades Cutáneas Bacterianas/prevención & control , Piel/microbiología , Animales , Línea Celular , Femenino , Humanos , Factor 1 Inducible por Hipoxia/agonistas , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Queratinocitos/microbiología , Ratones , Fagocitos/efectos de los fármacos , Fagocitos/inmunología , Fagocitos/microbiología , Piperazinas/farmacología , Piridonas/farmacología , Piel/efectos de los fármacos , Piel/inmunología , Enfermedades Cutáneas Bacterianas/inmunología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/inmunologíaAsunto(s)
Productos Biológicos/farmacología , Factor 1 Inducible por Hipoxia/agonistas , Factor 1 Inducible por Hipoxia/fisiología , Hipoxia/fisiopatología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/agonistas , Translocador Nuclear del Receptor de Aril Hidrocarburo/fisiología , Productos Biológicos/química , Humanos , Hipoxia/prevención & control , Subunidad alfa del Factor 1 Inducible por Hipoxia/agonistas , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiologíaRESUMEN
Abstract Ischemic stroke is a major cause of death worldwide, and current therapeutic options are very limited. Preconditioning with an ischemic or hypoxic insult is beneficial in experimental models of ischemic stroke. Ischemia/hypoxia results in activation of numerous transcription factors, including hypoxia inducible factor (HIF), which is a master regulator of oxygen homeostasis. HIF activation induces a diverse range of target genes, encompassing a wide variety of cellular processes; including angiogenesis, energy metabolism, cell survival, radical production/scavenging, iron metabolism, stem cell homing, and differentiation. Inhibition of HIF prolyl hydroxylase domain (PHD) enzymes results in activation of HIF and is likely to mimic, at least in part, the effects of hypoxia preconditioning. A caveat is that not all consequences of HIF activation will be beneficial and some could even be deleterious. Nevertheless, PHD inhibitors may be therapeutically useful in the treatment of stroke. Prototype PHD inhibitors have shown promising results in preclinical models.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Citoprotección , Inhibidores Enzimáticos/farmacología , Factor 1 Inducible por Hipoxia/agonistas , Fármacos Neuroprotectores/farmacología , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Barrera Hematoencefálica/enzimología , Encéfalo/irrigación sanguínea , Isquemia Encefálica/fisiopatología , Bloqueadores de los Canales de Calcio/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Depuradores de Radicales Libres/farmacología , Humanos , Precondicionamiento Isquémico , Ratones , Estrés Oxidativo/efectos de los fármacos , Ratas , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Accidente Cerebrovascular/enzimología , Accidente Cerebrovascular/patologíaRESUMEN
Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor regulating cellular responses to hypoxia and is composed of alpha and beta subunits. During normoxia, factor inhibiting HIF-1 (FIH-1) inhibits the activity of HIF-1 by preventing HIF-1alpha binding to p300/CBP via modification of the Asn(803) residue. However, it is not known whether FIH-1 activity can be regulated in an oxygen-independent manner. In this study, we survey possible binding proteins to FIH-1 and identify Mint3/APBA3, which has been reported to bind Alzheimer beta-amyloid precursor protein. Purified Mint3 binds FIH-1 and inhibits the ability of FIH-1 to modify HIF-1alpha in vitro. In a reporter assay, the activity of HIF-1alpha is suppressed because of endogenous FIH-1 in HEK293 cells, and expression of Mint3 antagonizes this suppression. Macrophages are known to depend on glycolysis for ATP production because of elevated HIF-1 activity. FIH-1 activity is suppressed in macrophages by Mint3 so as to maintain HIF-1 activity. FIH-1 forms a complex with Mint3, and these two factors co-localize within the perinuclear region. Knockdown of Mint3 expression in macrophages leads to redistribution of FIH-1 to the cytoplasm and decreases glycolysis and ATP production. Thus, Mint3 regulates the FIH-1-HIF-1 pathway, which controls ATP production in macrophages and therefore represents a potential new therapeutic target to regulate macrophage-mediated inflammation.
Asunto(s)
Proteínas Portadoras/fisiología , Macrófagos/metabolismo , Proteínas Represoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfato/biosíntesis , Glucólisis , Humanos , Factor 1 Inducible por Hipoxia/agonistas , Factor 1 Inducible por Hipoxia/metabolismo , Oxigenasas de Función Mixta , Unión Proteica , Transporte de Proteínas , Proteínas Represoras/antagonistas & inhibidoresAsunto(s)
Enfermedades Renales , Nefrología/tendencias , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Animales , Progresión de la Enfermedad , Diseño de Fármacos , Eritropoyetina/uso terapéutico , Fibrosis , Humanos , Factor 1 Inducible por Hipoxia/agonistas , Riñón/patología , Enfermedades Renales/etiología , Enfermedades Renales/patología , Enfermedades Renales/fisiopatología , Enfermedades Renales/terapia , Sistema Renina-Angiotensina/fisiologíaRESUMEN
The heterodimeric transcription factor HIF (hypoxia-inducible factor), consisting of a labile alpha-subunit and a stable beta-subunit, is a master regulator of genes involved in acute or chronic adaptation to low oxygen. Studies performed over the past 5 years revealed that HIFalpha-subunits are enzymatically hydroxylated in an oxygen-dependent manner. Hydroxylation of either of two conserved prolyl residues targets HIFalpha for destruction by a ubiquitin ligase containing the von Hippel-Lindau tumor suppressor protein whereas hydroxylation on a C-terminal asparagine affects HIF transactivation function. Pharmacological manipulation of HIF activity might be beneficial in diseases characterized by abnormal tissue oxygenation including myocardial infarction, cerebrovascular disease, and cancer.