RESUMO
The HIF hydroxylase enzymes (PHD1-3 and FIH) are cellular oxygen-sensors which confer hypoxic-sensitivity upon the hypoxia-inducible factors HIF-1α and HIF-2α. Microenvironmental hypoxia has a strong influence on the epithelial and immune cell function through HIF-dependent gene expression and consequently impacts upon the course of disease progression in ulcerative colitis (UC), with HIF-1α being protective while HIF-2α promotes disease. However, little is known about how inflammation regulates hypoxia-responsive pathways in UC patients. Here we demonstrate that hypoxia is a prominent microenvironmental feature of the mucosa in UC patients with active inflammatory disease. Furthermore, we found that inflammation drives transcriptional programming of the HIF pathway including downregulation of PHD1 thereby increasing the tissue responsiveness to hypoxia and skewing this response toward protective HIF-1 over detrimental HIF-2 activation. We identified CEBPα as a transcriptional regulator of PHD1 mRNA expression which is downregulated in both inflamed tissue derived from patients and in cultured intestinal epithelial cells treated with inflammatory cytokines. In summary, we propose that PHD1 downregulation skews the hypoxic response toward enhanced protective HIF-1α stabilization in the inflamed mucosa of UC patients.
Assuntos
Colite Ulcerativa/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Western Blotting , Células CACO-2 , Imunoprecipitação da Cromatina , Colite Ulcerativa/genética , Biologia Computacional , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Imuno-Histoquímica , Inflamação/genética , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
RATIONALE: Changes in redox potentials of cardiac myocytes are linked to several cardiovascular diseases. Redox alterations are currently mostly described qualitatively using chemical sensors, which however do not allow quantifying redox potentials, lack specificity, and the possibility to analyze subcellular domains. Recent advances to quantitatively describe defined redox changes include the application of genetically encoded redox biosensors. OBJECTIVE: Establishment of mouse models, which allow the quantification of the glutathione redox potential (EGSH) in the cytoplasm and the mitochondrial matrix of isolated cardiac myocytes and in Langendorff-perfused hearts based on the use of the redox-sensitive green fluorescent protein 2, coupled to the glutaredoxin 1 (Grx1-roGFP2). METHODS AND RESULTS: We generated transgenic mice with cardiac myocyte-restricted expression of Grx1-roGFP2 targeted either to the mitochondrial matrix or to the cytoplasm. The response of the roGFP2 toward H2O2, diamide, and dithiothreitol was titrated and used to determine the EGSH in isolated cardiac myocytes and in Langendorff-perfused hearts. Distinct EGSH were observed in the cytoplasm and the mitochondrial matrix. Stimulation of the cardiac myocytes with isoprenaline, angiotensin II, or exposure to hypoxia/reoxygenation additionally underscored that these compartments responded independently. A compartment-specific response was also observed 3 to 14 days after myocardial infarction. CONCLUSIONS: We introduce redox biosensor mice as a new tool, which allows quantification of defined alterations of EGSH in the cytoplasm and the mitochondrial matrix in cardiac myocytes and can be exploited to answer questions in basic and translational cardiovascular research.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Células Cultivadas , Coração/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Oxirredução , Consumo de Oxigênio/fisiologiaRESUMO
Tumors are composed of phenotypically heterogeneous cell populations. The nongenomic mechanisms underlying transitions and interactions between cell populations are largely unknown. Here, we show that diffuse large B-cell lymphomas possess a self-organized infrastructure comprising side population (SP) and non-SP cells, where transitions between clonogenic states are modulated by exosome-mediated Wnt signaling. DNA methylation modulated SP-non-SP transitions and was correlated with the reciprocal expressions of Wnt signaling pathway agonist Wnt3a in SP cells and the antagonist secreted frizzled-related protein 4 in non-SP cells. Lymphoma SP cells exhibited autonomous clonogenicity and exported Wnt3a via exosomes to neighboring cells, thus modulating population equilibrium in the tumor.