RESUMO
Epithelial-mesenchymal transition (EMT)-a fundamental process in embryogenesis and wound healing-promotes tumor metastasis and resistance to chemotherapy. While studies have identified signaling components and transcriptional factors responsible in the TGF-ß-dependent EMT, whether and how intracellular metabolism is integrated with EMT remains to be fully elucidated. Here, we showed that TGF-ß induces reprogramming of intracellular amino acid metabolism, which is necessary to promote EMT in non-small cell lung cancer cells. Combined metabolome and transcriptome analysis identified prolyl 4-hydroxylase α3 (P4HA3), an enzyme implicated in cancer metabolism, to be upregulated during TGF-ß stimulation. Further, knockdown of P4HA3 diminished TGF-ß-dependent changes in amino acids, EMT, and tumor metastasis. Conversely, manipulation of extracellular amino acids induced EMT-like responses without TGF-ß stimulation. These results suggest a previously unappreciated requirement for the reprogramming of amino acid metabolism via P4HA3 for TGF-ß-dependent EMT and implicate a P4HA3 inhibitor as a potential therapeutic agent for cancer.
Assuntos
Aminoácidos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Metabolômica , Camundongos , Pró-Colágeno-Prolina Dioxigenase/fisiologiaRESUMO
BACKGROUND: Extracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. METHODS: Amino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. RESULTS: Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 103 M-1 s-1. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. CONCLUSIONS: Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. GENERAL SIGNIFICANCE: These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.
Assuntos
Peróxidos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Ácido Úrico/análogos & derivados , Domínio Catalítico , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Sobrevivência Celular/fisiologia , Cromatografia Líquida/métodos , Cisteína/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , Oxirredução , Peroxidases/metabolismo , Agregação Plaquetária , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Isomerases de Dissulfetos de Proteínas/fisiologia , Compostos de Sulfidrila/metabolismo , Espectrometria de Massas em Tandem/métodos , Trombose/metabolismo , Ácido Úrico/metabolismoRESUMO
Prolyl hydroxylase 3 (PHD3) has initially been reported to hydroxylase hypoxia-inducible factor α (HIFα) and mediate HIFα degradation. More recent studies have shown that, in addition to HIFα, PHD3 has also other substrates. Moreover, pHD3 is believed to act as a tumor suppressor, but the underlying mechanism remains to be elucidated. Here, we demonstrate that PHD3 stabilizes p53 in a hydroxylase-independent manner. We found that PHD3 overexpression increases and PHD3 knockdown decreases p53 levels. Mechanistically, PHD3 bound MDM2 proto-oncogene (MDM2) and prevented MDM2 from interacting with p53, thereby inhibiting MDM2-mediated p53 degradation. Interestingly, we found that PHD3 overexpression could enhance p53 in the presence of the prolyl hydroxylase inhibitor dimethyloxalylglycine, and the prolyl hydroxylase activity-deficient variant PHD3-H196A also inhibited the p53-MDM2 interaction and stabilized p53. Genetic ablation of PHD3 decreased p53 protein levels in mice intestinal epithelial cells, but a genetic knockin of PHD3-H196A did not affect p53 protein levels in vivo These results suggest that the prolyl hydroxylase activity of PHD3 is dispensable for its ability to stabilize p53. We found that both PHD3 and PHD3-H196A suppress the expression of the stem cell-associated gene NANOG and inhibited the properties of colon cancer stem cells through p53. Our results reveal an additional critical mechanism underlying the regulation of p53 expression and highlight that PHD3 plays a role in the suppression of colon cancer cell stemness in a hydroxylase-independent manner.
Assuntos
Neoplasias do Colo/patologia , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Células-Tronco Neoplásicas/patologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/química , Animais , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neoplásicas/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-mdm2/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , UbiquitinaçãoRESUMO
Hypoxia in the embryo is a frequent cause of intra-uterine growth retardation, low birth weight, and multiple organ defects. In the kidney, this can lead to low nephron endowment, predisposing to chronic kidney disease and arterial hypertension. A key component in cellular adaptation to hypoxia is the hypoxia-inducible factor pathway, which is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3. In the adult kidney, PHD oxygen sensors are differentially expressed in a cell type-dependent manner and control the production of erythropoietin in interstitial cells. However, the role of interstitial cell PHDs in renal development has not been examined. Here we used a genetic approach in mice to interrogate PHD function in FOXD1-expressing stroma during nephrogenesis. We demonstrate that PHD2 and PHD3 are essential for normal kidney development as the combined inactivation of stromal PHD2 and PHD3 resulted in renal failure that was associated with reduced kidney size, decreased numbers of glomeruli, and abnormal postnatal nephron formation. In contrast, nephrogenesis was normal in animals with individual PHD inactivation. We furthermore demonstrate that the defect in nephron formation in PHD2/PHD3 double mutants required intact hypoxia-inducible factor-2 signaling and was dependent on the extent of stromal hypoxia-inducible factor activation. Thus, hypoxia-inducible factor prolyl-4-hydroxylation in renal interstitial cells is critical for normal nephron formation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/fisiologia , Rim/crescimento & desenvolvimento , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Insuficiência Renal/genética , Anemia/sangue , Anemia/tratamento farmacológico , Anemia/etiologia , Animais , Hipóxia Celular/fisiologia , Ensaios Clínicos Fase III como Assunto , Modelos Animais de Doenças , Inibidores Enzimáticos/uso terapêutico , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Hidroxilação/fisiologia , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Rim/citologia , Rim/metabolismo , Nefropatias/complicações , Nefropatias/tratamento farmacológico , Camundongos , Terapia de Alvo Molecular/métodos , Mutação , Tamanho do Órgão/fisiologia , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/genética , Insuficiência Renal/mortalidade , Insuficiência Renal/patologia , Células Estromais/metabolismoRESUMO
Sustained exposure to low oxygen concentration leads to profound changes in gene expression to restore oxygen homeostasis. Hypoxia-inducible factors (HIFs) comprise a group of transcription factors which accumulate under hypoxia and contribute to the complex changes in gene expression. Under normoxic conditions HIFs are degraded by prolyl-hydroxylases (PHD), however during hypoxia this degradation is inhibited causing HIF accumulation and subsequent changes in gene expression. Pulmonary neuroepithelial bodies (NEB) are innervated serotonin (5-HT)-producing cells distributed throughout the airway epithelium. These putative O(2) sensors are hypothesized to contribute to the ventilatory response to hypoxia. NEB dysfunction has been implicated in several paediatric lung diseases including neuroendocrine cell hyperplasia of infancy and sudden infant death syndrome, both characterized by a marked NEB hyperplasia with unknown functional significance. We have previously reported striking NEB hyperplasia in PHD1(-/-) mice making these mice a potential model to study the role of NEBs in paediatric lung diseases. Here we report in vitro studies on 5-HT release from NEB using this model.
Assuntos
Pneumopatias/etiologia , Corpos Neuroepiteliais/metabolismo , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Serotonina/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos KnockoutRESUMO
Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.
Assuntos
Mucosa Intestinal/fisiologia , Ocludina/fisiologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Animais , Colite/genética , Colite/prevenção & controle , Deleção de Genes , Células HEK293 , Humanos , Camundongos , Camundongos TransgênicosRESUMO
On a molecular level, cells sense changes in oxygen availability through the PHDs, which regulate the protein stability of the α-subunit of the transcription factor HIF. Especially, PHD3 has been additionally associated with apoptotic cell death. We hypothesized that PHD3 plays a role in cell-fate decisions in macrophages. Therefore, myeloid-specific PHD3(-/-) mice were created and analyzed. PHD3(-/-) BMDM showed no altered HIF-1α or HIF-2α stabilization or increased HIF target gene expression in normoxia or hypoxia. Macrophage M1 and M2 polarization was unchanged likewise. Compared with macrophages from WT littermates, PHD3(-/-) BMDM exhibited a significant reduction in TUNEL-positive cells after serum withdrawal or treatment with stauro and SNAP. Under the same conditions, PHD3(-/-) BMDM also showed less Annexin V staining, which is representative for membrane disruption, and indicated a reduced early apoptosis. In an unbiased transcriptome screen, we found that Angptl2 expression was reduced in PHD3(-/-) BMDM under stress conditions. Addition of rAngptl2 rescued the antiapoptotic phenotype, demonstrating that it is involved in the PHD3-mediated response toward apoptotic stimuli in macrophages.
Assuntos
Macrófagos/citologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/biossíntese , Angiopoietinas/genética , Angiopoietinas/farmacologia , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Células da Medula Óssea/citologia , Hipóxia Celular , Células Cultivadas , Regulação da Expressão Gênica , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/enzimologia , NF-kappa B/metabolismo , Pró-Colágeno-Prolina Dioxigenase/deficiência , Pró-Colágeno-Prolina Dioxigenase/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/farmacologia , S-Nitroso-N-Acetilpenicilamina/farmacologia , Estaurosporina/farmacologia , Transcrição Gênica , TranscriptomaRESUMO
BACKGROUND: Recent studies suggest that the oxygen-sensing pathway consisting of transcription factor hypoxia-inducible factor and prolyl hydroxylase domain proteins (PHDs) plays a critical role in glucose metabolism. However, the role of adipocyte PHD in the development of obesity has not been clarified. We examined whether deletion of PHD2, the main oxygen sensor, in adipocytes affects diet-induced obesity and associated metabolic abnormalities. METHODS AND RESULTS: To delete PHD2 in adipocyte, PHD2-floxed mice were crossed with aP2-Cre transgenic mice (Phd2(f/f)/aP2-Cre). Phd2(f/f)/aP2-Cre mice were resistant to high-fat diet-induced obesity (36.7±1.7 versus 44.3±2.0 g in control; P<0.01) and showed better glucose tolerance and homeostasis model assessment-insulin resistance index than control mice (3.6±1.0 versus 11.1±2.1; P<0.01). The weight of white adipose tissue was lighter (epididymal fat, 758±35 versus 1208±507 mg in control; P<0.01) with a reduction in adipocyte size. Macrophage infiltration into white adipose tissue was also alleviated in Phd2(f/f)/aP2-Cre mice. Target genes of hypoxia-inducible factor, including glycolytic enzymes and adiponectin, were upregulated in adipocytes of Phd2(f/f)/aP2-Cre mice. Lipid content was decreased and uncoupling protein-1 expression was increased in brown adipose tissue of Phd2(f/f)/aP2-Cre mice. Knockdown of PHD2 in 3T3L1 adipocytes induced a decrease in the glucose level and an increase in the lactate level in the supernatant with upregulation of glycolytic enzymes and reduced lipid accumulation. CONCLUSIONS: PHD2 in adipose tissue plays a critical role in the development of diet-induced obesity and glucose intolerance. PHD2 might be a novel target molecule for the treatment of obesity and associated metabolic abnormalities.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Intolerância à Glucose/etiologia , Intolerância à Glucose/fisiopatologia , Obesidade/etiologia , Obesidade/fisiopatologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo Branco/irrigação sanguínea , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Glucose/metabolismo , Intolerância à Glucose/patologia , Transportador de Glucose Tipo 4/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia , Metabolismo dos Lipídeos/fisiologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Neovascularização Fisiológica/fisiologia , Obesidade/patologia , Consumo de Oxigênio/fisiologia , Pró-Colágeno-Prolina Dioxigenase/deficiência , Pró-Colágeno-Prolina Dioxigenase/genéticaRESUMO
Hypoxia is a prominent feature in the maintenance of hematopoietic stem cell (HSC) quiescence and multipotency. Hypoxia-inducible factor (HIF) prolyl hydroxylase domain proteins (PHDs) serve as oxygen sensors and may therefore regulate this system. Here, we describe a mouse line with conditional loss of HIF prolyl hydroxylase 2 (PHD2) in very early hematopoietic precursors that results in self-renewal of multipotent progenitors under steady-state conditions in a HIF1α- and SMAD7-dependent manner. Competitive bone marrow (BM) transplantations show decreased peripheral and central chimerism of PHD2-deficient cells but not of the most primitive progenitors. Conversely, in whole BM transfer, PHD2-deficient HSCs replenish the entire hematopoietic system and display an enhanced self-renewal capacity reliant on HIF1α. Taken together, our results demonstrate that loss of PHD2 controls the maintenance of the HSC compartment under physiological conditions and causes the outcompetition of PHD2-deficient hematopoietic cells by their wild-type counterparts during stress while promoting the self-renewal of very early hematopoietic progenitors.
Assuntos
Células-Tronco Hematopoéticas/citologia , Hipóxia/fisiopatologia , Células-Tronco Multipotentes/citologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Estresse Fisiológico , Animais , Transplante de Medula Óssea , Ciclo Celular , Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Multipotentes/metabolismo , Proteína Smad7/metabolismoRESUMO
Hypoxia-inducible factors (HIFs) are key transcriptional regulators that play a major role in oxygen homeostasis. HIF activity is tightly regulated by oxygen-dependent hydroxylases, which additionally require iron and 2-oxoglutarate as cofactors. Inhibition of these enzymes has become a novel target to modulate the hypoxic response for therapeutic benefit. Inhibition of prolyl-4-hydroxylase domains (PHDs) have been shown to delay neuronal cell death and protect against ischemic injury in the hippocampus. In this study we have examined the effects of prolyl hydroxylase inhibition on synaptic transmission and plasticity in the hippocampus. Field excitatory postsynaptic potentials (fEPSPs) and excitatory postsynaptic currents (EPSCs) were elicited by stimulation of the Schaffer collateral pathway in the CA1 region of the hippocampus. Treatment of rat hippocampal slices with low concentrations (10 µM) of the iron chelator deferosoxamine (DFO) or the 2-oxoglutarate analogue dimethyloxalyl glycine (DMOG) had no effect on fEPSP. In contrast, application of 1 mM DMOG resulted in a significant decrease in fEPSP slope. Antagonism of the NMDA receptor attenuated the effects of DMOG on baseline synaptic signalling. In rat hippocampal slices pretreated with DMOG and DFO the induction of long-term potentiation (LTP) by tetanic stimulation was strongly impaired. Similarly, neuronal knockout of the single PHD family member PHD2 prevented murine hippocampal LTP. Preconditioning of PHD2 deficient hippocampi with either DMOG, DFO, or the PHD specific inhibitor JNJ-42041935, did not further decrease LTP suggesting that DMOG and DFO influences synaptic plasticity primarily by inhibiting PHDs rather than unspecific effects. These findings provide striking evidence for a modulatory role of PHD proteins on synaptic plasticity in the hippocampus.
Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/enzimologia , Potenciação de Longa Duração/fisiologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Aminoácidos Dicarboxílicos/farmacologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/enzimologia , Desferroxamina/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp/instrumentação , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
Prolyl hydroxylase domain protein 2 (PHD2, also known as Egg Laying Defective Nine homolog 1) is a key oxygen-sensing protein in metazoans. In an oxygen-dependent manner, PHD2 site-specifically prolyl hydroxylates the master transcription factor of the hypoxic response, hypoxia-inducible factor-α (HIF-α), thereby targeting HIF-α for degradation. In this report we show that the heat shock protein 90 (HSP90) co-chaperones p23 and FKBP38 interact via a conserved Pro-Xaa-Leu-Glu motif (where Xaa = any amino acid) in these proteins with the N-terminal Myeloid Nervy and DEAF-1 (MYND)-type zinc finger of PHD2. Knockdown of p23 augments hypoxia-induced HIF-1α protein levels and HIF target genes. We propose that p23 recruits PHD2 to the HSP90 machinery to facilitate HIF-1α hydroxylation. These findings identify a link between two ancient pathways, the PHD:HIF and the HSP90 pathways, and suggest that this link was established concurrent with the emergence of the PHD:HIF pathway in evolution.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Hipóxia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Espectrometria de Massas/métodos , Modelos Biológicos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/química , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND & AIMS: MicroRNAs (miRNAs) have been shown to be involved in many biological processes by affecting their target gene expression. miR-122 has been extensively studied in hepatocarcinogenesis. However, the role of miR-122 in liver fibrosis remains unknown. METHODS: The mRNA expression levels of miR-122, prolyl 4-hydroxylase subunit alpha-1 (P4HA1), and CCAAT/enhancer binding protein alpha (C/EBPα) were assessed by real-time PCR. The protein expression levels of P4HA1, C/EBPα and collagen, type I, alpha 1 (COL1A1) were analyzed by Western blot and immunofluorescence. MTT assay was used to assess cell proliferation. Chromatin immunoprecipitation (ChIP) assay was used to examine the binding activity of C/EBPα to miR-122 promoter. RESULTS: miR-122 expression was significantly reduced in transactivated HSCs and in the livers of mice treated with CCl(4). Overexpression of miR-122 inhibited the proliferation of LX2 cells. We also demonstrated that P4HA1 was a target gene of miR-122. The mRNA expression level of PAHA1 inversely correlated with that of miR-122 in HSCs and in the mouse liver. Overexpression of miR-122 markedly attenuated the expression of P4HA1 via targeting a binding site located at 3'-UTR of P4HA1 mRNA. We further showed that miR-122 overexpression led to decreased collagen maturation and ECM production. Finally, the binding activity of C/EBPα to miR-122 promoter was significantly decreased in activated HSCs. CONCLUSIONS: Our study suggests that miR-122 may play an important role in negatively regulating collagen production in HSCs and that targeted expression of miR-122 in HSCs may represent a new strategy for the treatment of liver fibrosis.
Assuntos
Colágeno/biossíntese , Células Estreladas do Fígado/fisiologia , MicroRNAs/fisiologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Proliferação de Células , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/fisiologia , Humanos , Masculino , Pró-Colágeno-Prolina Dioxigenase/genética , Ratos , Ratos Sprague-DawleyRESUMO
Pulmonary NEB, widely distributed within the airway mucosa of mammalian lungs, are presumed hypoxia sensitive airway O(2) sensors responding to changes in airway gas concentration. NEB cell hyperplasia has been reported after exposure to chronic hypoxia and in a variety of paediatric and adult lung disorders. Prolyl hydroxylases (PHD 1-3) regulate the stability of hypoxia-inducible factors (HIF's) in an O(2)-dependent manner and function as intrinsic oxygen sensors. To determine a possible role of PHD-1in NEB cells we have quantitated NEB's in lungs of neonatal (P2) and adult (2 months) PHD-1-deficient mice and compared them to wild type (WT) control mice. Lung tissues fixed in formalin and embedded in paraffin were processed for immunoperoxidase method and frozen sections for multilabel immunoflourescence using antibodies for NEB markers synaptophysin, synaptic vesicle protein 2 and the peptide CGRP. The frequency and size of NEB in lungs of PHD-1 deficient neonatal mice (P2) and at 2 months was increased significantly compared to WT controls (p < 0.01). The present data suggests an important role for PHD enzymes in NEB cell biology deserving further studies. Since the PHD-1 deficient mouse appears to be the first animal model showing NEB cell hyperplasia it may be useful for studies of NEB physiology and pathobiology.
Assuntos
Pulmão/patologia , Corpos Neuroepiteliais/patologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Animais , Imunofluorescência , Hiperplasia , Técnicas Imunoenzimáticas , Pulmão/enzimologia , Camundongos , Camundongos KnockoutRESUMO
PURPOSE: Liver regeneration after partial hepatectomy (PH) occurs in conditions of reduced oxygen supply. HIF prolyl hydroxylase enzymes (PHD1, PHD2, and PHD3) are oxygen sensors involved in adaptive response to hypoxia. Specific functions of these PHD enzymes in liver regeneration have, however, remained enigmatic. Here, we investigated the significance of PHD1 in liver regeneration following hepatectomy. METHODS: Liver regeneration was studied in PHD1-deficient (PHD1(-/-)) and wild type (WT) mice subjected to 80% hepatectomy. For in vitro analyses, hepatocytes were isolated from PHD1(-/-) and WT livers. Cell cycle progression was studied via FACS-based analysis of nuclear DNA profile. Transcription factor binding assays, qRT-PCR, and immunoblotting were applied to study the relevance of PHD1 downstream effectors during liver regeneration. RESULTS: Liver regeneration was significantly enhanced in PHD1(-/-) mice compared to WT littermates. This effect was due to enhanced proliferation rather than to hypertrophy of liver cells. Cell cycle progression was significantly enhanced, and transcriptional activity of the cell cycle regulator c-Myc was increased in PHD1-deficient hepatocytes. These changes coincided with increased expression of cyclin D2, a cell cycle-promoting c-Myc target, and decreased expression of the cell cycle-delaying c-Myc target p21. CONCLUSIONS: Loss of PHD1 enhances liver regeneration by boosting hepatocyte proliferation in a c-Myc-dependent fashion. PHD1 might, therefore, represent a potential target to facilitate liver regeneration after surgical resection.
Assuntos
Hepatectomia , Hepatócitos/metabolismo , Regeneração Hepática/fisiologia , Pró-Colágeno-Prolina Dioxigenase/deficiência , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Western Blotting , Ciclo Celular , Proliferação de Células , Células Cultivadas , Hepatócitos/citologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
An endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm(-/-) mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm(-/-) mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497-treated Hif-p4h-2 hypomorphic (Hif-p4h-2(gt/gt)) and Hif-p4h-3(-/-) mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm(-/-) and wild-type mice, but caused higher increases in both values in the Hif-p4h-2(gt/gt) mice and in hematocrit value in the Hif-p4h-3(-/-) mice than in the wild-type. Hif-p4h-2(gt/gt)/P4h-tm(-/-) double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2(gt/gt) or P4h-tm(-/-) mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.
Assuntos
Eritropoese/fisiologia , Eritropoetina/sangue , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Western Blotting , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hematócrito , Hemoglobinas/metabolismo , Hepcidinas , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hypoxia inducible factor (HIF) is an oxygen-sensitive transcription factor that enables aerobic organisms to adapt to hypoxia. This is achieved through the transcriptional activation of up to 200 genes, many of which are critical to cell survival. Under conditions of normoxia, the hydroxylation of HIF by prolyl hydroxylase domain-containing (PHD) enzymes targets it for polyubiquitination and proteosomal degradation by the von Hippel-Lindau protein (VHL). However, under hypoxic conditions, PHD activity is inhibited, thereby allowing HIF to accumulate and translocate to the nucleus, where it binds to the hypoxia-responsive element sequences of target gene promoters. Experimental studies suggest that HIF may act as a mediator of ischemic preconditioning, and that the genetic or pharmacological stabilization of HIF under normoxic conditions, may protect the heart against the detrimental effects of acute ischemia-reperfusion injury. The mechanisms underlying the cardioprotective effect of HIF are unclear, but it may be attributed to the transcriptional activation of genes associated with cardioprotection such as erythropoietin, heme oxygenase-1, and inducible nitric oxide synthase or it may be due to reprogramming of cell metabolism. In this review article, we highlight the role of HIF in mediating both adaptive and pathological processes in the heart, as well as focusing on the therapeutic potential of the HIF-signaling pathway as a target for cardioprotection.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Adiponectina/fisiologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Terapia Genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Precondicionamento Isquêmico Miocárdico , Proteínas com Domínio LIM/fisiologia , MicroRNAs/fisiologia , Mitocôndrias/fisiologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Sirtuínas/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologiaRESUMO
BACKGROUND: Since mammalian cells rely on the availability of oxygen, they have devised mechanisms to sense environmental oxygen tension, and to efficiently counteract oxygen deprivation (hypoxia). These adaptive responses to hypoxia are essentially mediated by hypoxia inducible transcription factors (HIFs). Three HIF prolyl hydroxylase enzymes (PHD1, PHD2 and PHD3) function as oxygen sensing enzymes, which regulate the activity of HIFs in normoxic and hypoxic conditions. Many of the compensatory functions exerted by the PHD-HIF system are of immediate surgical relevance since they regulate the biological response of ischemic tissues following ligation of blood vessels, of oxygen-deprived inflamed tissues, and of tumors outgrowing their vascular supply. PURPOSE: Here, we outline specific functions of PHD enzymes in surgically relevant pathological conditions, and discuss how these functions might be exploited in order to support the treatment of surgically relevant diseases.
Assuntos
Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Dioxigenases/genética , Dioxigenases/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Vísceras/irrigação sanguínea , Vísceras/cirurgia , Neoplasias Abdominais/irrigação sanguínea , Neoplasias Abdominais/genética , Trifosfato de Adenosina/metabolismo , Animais , Morte Celular/genética , Morte Celular/fisiologia , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Homeostase/genética , Homeostase/fisiologia , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Imunidade Inata/genética , Imunidade Inata/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/fisiopatologiaRESUMO
Prolyl hydroxylases belong to the family of iron- and 2-oxoglutamate-dependent dioxygenase enzyme. Several distinct prolyl hydroxylases have been identified. The hypoxia-inducible factor (HIF) prolyl hydroxylase termed prolyl hydroxylase domain (PHD) enzymes play an important role in oxygen regulation in the physiological network. There are three isoforms that have been identified: PHD1, PHD2 and PHD3. Deletion of PHD enzymes result in stabilization of HIFs and offers potential treatment options for many ischemic disorders such as peripheral arterial occlusive disease, myocardial infarction, and stroke. All three isoforms are oxygen sensors that regulate the stability of HIFs. The degradation of HIF-1α is regulated by hydroxylation of the 402/504 proline residue by PHDs. Under hypoxic conditions, lack of oxygen causes hydroxylation to cease HIF-1α stabilization and subsequent translocation to the nucleus where it heterodimerizes with the constitutively expressed ß subunit. Binding of the HIF-heterodimer to specific DNA sequences, named hypoxia-responsive elements, triggers the transactivation of target genes. PHD regulation of HIF-1α-mediated cardioprotection has resulted in considerable interest in these molecules as potential therapeutic targets in cardiovascular and ischemic diseases. In recent years, attention has been directed towards identifying small molecule inhibitors of PHD. It is postulated that such inhibition might lead to a clinically useful strategy for protecting the myocardium against ischemia and reperfusion injury. Recently, it has been reported that the orally absorbed PHD inhibitor GSK360A can modulate HIF-1α signaling and protect the failing heart following myocardial infarction. Furthermore, PHD1 deletion has been found to have beneficial effects through an increase in tolerance to hypoxia of skeletal muscle by reprogramming basal metabolism. In the mouse liver, such deletion has resulted in protection against ischemia and reperfusion. As a result of these preliminary findings, PHDs is attracting increasing interest as potential therapeutic targets in a wide range of diseases.
Assuntos
Cardiotônicos/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Dioxigenases/fisiologia , Inibidores Enzimáticos/uso terapêutico , Proteínas Nucleares/fisiologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Animais , Cardiotônicos/administração & dosagem , Doenças Cardiovasculares/enzimologia , Dioxigenases/antagonistas & inibidores , Dioxigenases/genética , Inibidores Enzimáticos/administração & dosagem , Regulação Enzimológica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/genéticaRESUMO
BACKGROUND: Pancreatic cancer is 1 of the most common and poorly treated tumors. In search of new therapeutic approaches, the oxygen sensors prolyl hydroxylases (PHD) are potential targets. PHD2 is considered the key oxygen sensor-regulating hypoxia-inducible factor (HIF). Currently, there is conflicting evidence regarding the exact role of PHD2 in tumorigenesis. The objective of this study was to investigate the role of PHD2 in pancreatic cancer growth and progression. METHODS: PHD2 expression was analyzed by quantitative real-time polymerase chain reaction analysis and immunohistochemistry in human tissue specimens and cell lines. Knockdown of PHD2 was done by using short-interfering RNAs (siRNAs) specific against PHD2, and PHD2 overexpression was achieved by stable combinational DNA transfection. In vivo, an orthotopic murine model was used. Angiogenic cytokines were assessed with enzyme-linked immunosorbent assays, and invasion was studied with Matrigel assays. RESULTS: PHD2 expression was not altered substantially in cancer tissues and their metastases. Lymph node-negative tissues had higher levels of PHD2 than lymph node-positive tissues. PHD2 was hypoxia-inducible in pancreatic cancer cell lines and regulated cell growth through cyclin D1 down-regulation samples with PHD2 suppression and through p21 up-regulation in samples with of PHD2 overexpression. In vivo, PHD2 caused tumor growth retardation and reduced tumor invasion by inhibiting angiogenesis. This observation was caused by the suppression of angiogenic cytokines and tumor invasion. CONCLUSIONS: The current results indicated that PHD2 plays an important role in pancreatic tumorigenesis. In summary, the authors concluded that PHD2 may function as a tumor suppressor gene in pancreatic cancer and, thus, may define a potential target for the treatment of pancreatic cancer.