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1.
J Cell Sci ; 129(19): 3675-3684, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27505895

RESUMO

Mutation of PKD1, encoding the protein polycystin-1 (PC1), is the main cause of autosomal dominant polycystic kidney disease (ADPKD). The signaling pathways downstream of PC1 in ADPKD are still not fully understood. Here, we provide genetic evidence for the necessity of Gα12 (encoded by Gna12, hereafter Gα12) for renal cystogenesis induced by Pkd1 knockout. There was no phenotype in mice with deletion of Gα12 (Gα12-/-). Polyinosine-polycytosine (pI:pC)-induced deletion of Pkd1 (Mx1Cre+Pkd1f/fGα12+/+) in 1-week-old mice resulted in multiple kidney cysts by 9 weeks, but the mice with double knockout of Pkd1 and Gα12 (Mx1Cre+Pkd1f/fGα12-/-) had no structural and functional abnormalities in the kidneys. These mice could survive more than one year without kidney abnormalities except multiple hepatic cysts in some mice, which indicates that the effect of Gα12 on cystogenesis is kidney specific. Furthermore, Pkd1 knockout promoted Gα12 activation, which subsequently decreased cell-matrix and cell-cell adhesion by affecting the function of focal adhesion and E-cadherin, respectively. Our results demonstrate that Gα12 is required for the development of kidney cysts induced by Pkd1 mutation in mouse ADPKD.


Assuntos
Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Rim/metabolismo , Rim/patologia , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/metabolismo , Animais , Caderinas/metabolismo , Junções Célula-Matriz , Células Epiteliais/metabolismo , Deleção de Genes , Técnicas de Inativação de Genes , Fígado/metabolismo , Fígado/patologia , Camundongos , Modelos Biológicos , Fibras de Estresse/metabolismo
2.
Physiol Genomics ; 47(2): 24-32, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25492927

RESUMO

Interaction of polycystin-1 (PC1) and Gα12 is important for development of kidney cysts in autosomal dominant polycystic kidney disease (ADPKD). The integrity of cell polarity and cell-cell adhesions (mainly E-cadherin-mediated adherens junction) is altered in the renal epithelial cells of ADPKD. However, the key signaling pathway for this alteration is not fully understood. Madin-Darby canine kidney (MDCK) cells maintain the normal integrity of epithelial cell polarity and adherens junctions. Here, we found that deletion of Pkd1 increased activation of Gα12, which then promoted the cystogenesis of MDCK cells. The morphology of these cells was altered after the activation of Gα12. By using liquid chromatography-mass spectrometry, we found several proteins that could be related this change in the extracellular milieu. E-cadherin was one of the most abundant peptides after active Gα12 was induced. Gα12 activation or Pkd1 deletion increased the shedding of E-cadherin, which was mediated via increased ADAM10 activity. The increased shedding of E-cadherin was blocked by knockdown of ADAM10 or specific ADAM10 inhibitor GI254023X. Pkd1 deletion or Gα12 activation also changed the distribution of E-cadherin in kidney epithelial cells and caused ß-catenin to shift from cell membrane to nucleus. Finally, ADAM10 inhibitor, GI254023X, blocked the cystogenesis induced by PC1 knockdown or Gα12 activation in renal epithelial cells. Our results demonstrate that the E-cadherin/ß-catenin signaling pathway is regulated by PC1 and Gα12 via ADAM10. Specific inhibition of this pathway, especially ADAM10 activity, could be a novel therapeutic regimen for ADPKD.


Assuntos
Caderinas/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Canais de Cátion TRPP/metabolismo , Proteínas ADAM/antagonistas & inibidores , Animais , Dipeptídeos/farmacologia , Cães , Células Epiteliais/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Deleção de Genes , Humanos , Ácidos Hidroxâmicos/farmacologia , Rim/citologia , Rim/metabolismo , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/metabolismo , Células Madin Darby de Rim Canino/patologia , Camundongos Knockout , Camundongos Transgênicos , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/genética , beta Catenina/metabolismo
3.
Proc Natl Acad Sci U S A ; 109(17): 6680-5, 2012 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-22493269

RESUMO

The epithelial cell tight junction separates apical and basolateral domains and is essential for barrier function. Disruption of the tight junction is a hallmark of epithelial cell damage and can lead to end organ damage including renal failure. Herein, we identify Gα12 activation by H(2)O(2) leading to tight junction disruption and demonstrate a critical role for Gα12 activation during bilateral renal ischemia/reperfusion injury. Madin-Darby canine kidney (MDCK) cells with inducible Gα12 (Gα12-MDCK) and silenced Gα12 (shGα12-MDCK) were subjected to ATP depletion/repletion and H(2)O(2)/catalase as models of tight junction disruption and recovery by monitoring transepithelial resistance. In ATP depleted cells, barrier disruption and recovery was not affected by Gα12, but reassembly was accelerated by Gα12 depletion. In contrast, silencing of Gα12 completely protected cells from H(2)O(2)-stimulated barrier disruption, a response that rapidly occurred in control cells. H(2)O(2) activated Src and Rho, and Src inhibition (by PP2), but not Rho (by Y27632), protected cells from H(2)O(2)-mediated barrier disruption. Immunofluorescent and biochemical analysis showed that H(2)O(2) led to increased tyrosine phosphorylation of numerous proteins and altered membrane localization of tight junction proteins through Gα12/Src signaling pathway. Gα12 and Src were activated in vivo during ischemia/reperfusion injury, and transgenic mice with renal tubular QLα12 (activated mutant) expression were delayed in recovery and showed more extensive injury. Conversely, Gα12 knockout mice were nearly completely protected from ischemia/reperfusion injury. Taken together, these studies reveal that ROS stimulates Gα12 to activate injury pathways and identifies a therapeutic target for ameliorating ROS mediated injury.


Assuntos
Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Peróxido de Hidrogênio/farmacologia , Junções Intercelulares/efeitos dos fármacos , Traumatismo por Reperfusão/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Cães , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Inativação Gênica , Junções Intercelulares/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação
4.
Cell Adh Migr ; 4(3): 372-6, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20424510

RESUMO

Integrins regulate cell attachment and migration through interactions with specific proteins in the extra-cellular matrix. Heterotrimeric G proteins are essential signal transduction proteins that intersect with integrin signaling to regulate fundamental cellular behaviors. Although integrin and G protein signaling often act in concert, how these mechanisms interact in epithelial cells has not been extensively studied. We recently reported Gα12 regulation of epithelial cell attachment and migration on collagen-I through α2ß1 integrins (Kong et al. Mol Biol Cell 2009). Activated Gα12 inhibited α2ß1 integrin functions through an inside-out signaling mechanism that involved Rho, Src and protein phosphatases without affecting α2 or ß1 expression. Activated Gα12 prevented tubulogenesis in 3D-MDCK cell cultures and promoted the formation of cystic structures. Herein, we extend these findings to show Gα12-stimulated transcriptional changes in integrin expression that affect MDCK cell attachment. Based on results from a microarray with MDCK cells expressing constitutively active Gα12 (QLα12), we confirmed with real time PCR that expressing QLα12 led to a 4-fold inhibition of α6 mRNA expression. Cell surface expression and total α6 protein was reduced by FACS and immunofluorescence. QLα12 expressing MDCK cells also revealed less attachment to laminin-5, an α6 integrin ligand. Taken together, G proteins regulate integrins through canonical signaling pathways and potentially regulate integrin expression levels to modulate cellular responses in a variety of pathophysiologic conditions including polycystic kidney disease.


Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Integrinas/metabolismo , Animais , Adesão Celular , Linhagem Celular , Cães , Humanos , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Transdução de Sinais
5.
J Biol Chem ; 285(14): 10243-51, 2010 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-20106977

RESUMO

Mutations in PKD1 are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1 (polycystin-1 (PC1)) is a large transmembrane protein with a short intracellular C terminus that interacts with numerous signaling molecules, including Galpha(12). Cyst formation in ADPKD results from numerous cellular defects, including abnormal cilia, changes in polarity, and dysregulated apoptosis and proliferation. Recently, we reported increased apoptosis in Madin-Darby canine kidney (MDCK) cells through Galpha(12) stimulation of JNK and degradation of the anti-apoptotic protein Bcl-2 (Yanamadala, V., Negoro, H., Gunaratnam, L., Kong, T., and Denker, B. M. (2007) J. Biol. Chem. 282, 24352-24363). Herein, we confirm this pathway in Galpha(12)-silenced MDCK cells and utilize MDCK cell lines harboring either overexpressed or silenced PC1 to demonstrate that PC1 expression levels determine activity of the JNK/Bcl-2 apoptosis pathway. PC1-overexpressing MDCK cells were resistant to thrombin/Galpha(12)-stimulated apoptosis, JNK activation, and Bcl-2 degradation. In contrast, PC1-silenced MDCK cells displayed enhanced thrombin-induced apoptosis, JNK activity, and Bcl-2 degradation. In pulldown experiments, PC1 bound to Galpha(12), but not the related Galpha(13) subunit, and thrombin-stimulated MDCK cells led to increased interaction of Galpha(12) with the PC1 C terminus. In transient transfection assays, a PC1 C-terminal mutant lacking the G protein-binding domain was uncoupled from PC1-inhibited apoptosis. PC1 expression levels may be increased or decreased in ADPKD, and these findings suggest a mechanism in which levels of PC1 expression modulate Galpha(12)/JNK-stimulated apoptosis. Taken together, these findings are consistent with a set point model in which PC1 expression levels regulate specific G protein signaling pathways important to cyst development.


Assuntos
Apoptose , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Canais de Cátion TRPP/metabolismo , Animais , Western Blotting , Células Cultivadas , Cães , Citometria de Fluxo , Rim Policístico Autossômico Dominante/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
6.
Mol Biol Cell ; 20(21): 4596-610, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19776354

RESUMO

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that G alpha 12 inhibits interaction of MDCK cells with collagen-I, the major ligand for alpha2 beta1 integrin. Activating G alpha 12 (QL point mutation or stimulating endogenous G alpha 12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with G alpha 12-regulated attachment to collagen-I, G alpha 12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in G alpha 12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. G alpha 12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of alpha2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated G alpha 12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, G alpha 12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for G alpha 12-integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.


Assuntos
Adesão Celular/fisiologia , Movimento Celular/fisiologia , Colágeno Tipo I/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Integrina alfa2beta1/metabolismo , Túbulos Renais/crescimento & desenvolvimento , Animais , Linhagem Celular , Colágeno Tipo I/genética , Cães , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Humanos , Integrina alfa2beta1/genética , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Paxilina/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo
7.
Blood ; 113(1): 224-32, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18812468

RESUMO

Hypoxia is common to several inflammatory diseases, where multiple cell types release adenine-nucleotides (particularly adenosine triphosphate/adenosine diphosphate). Adenosine triphosphate/adenosine diphosphate is metabolized to adenosine through a 2-step enzymatic reaction initiated by CD39 (ectonucleoside-triphosphate-diphosphohydrolase-1). Thus, extracellular adenosine becomes available to regulate multiple inflammatory endpoints. Here, we hypothesized that hypoxia transcriptionally up-regulates CD39 expression. Initial studies revealed hypoxia-dependent increases in CD39 mRNA and immunoreactivity on endothelia. Examination of the human CD39 gene promoter identified a region important in hypoxia inducibility. Multiple levels of analysis, including site-directed mutagenesis, chromatin immunoprecipitation, and inhibition by antisense, revealed a critical role for transcription-factor Sp1 in hypoxia-induction of CD39. Using a combination of cd39(-/-) mice and Sp1 small interfering RNA in in vivo cardiac ischemia models revealed Sp1-mediated induction of cardiac CD39 during myocardial ischemia. In summary, these results identify a novel Sp1-dependent regulatory pathway for CD39 and indicate the likelihood that CD39 is central to protective responses to hypoxia/ischemia.


Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Apirase/genética , Apirase/metabolismo , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Fator de Transcrição Sp1/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Hipóxia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno
8.
J Immunol ; 179(3): 1934-41, 2007 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-17641060

RESUMO

Central to the process of inflammation are hypoxic conditions that lead to the binding of circulating leukocytes to the endothelium. We have previously shown that such binding is mediated by monocytes being able to directly sense hypoxic conditions and respond by inducing their surface expression of the beta(2) integrin family of adhesion molecules. In this study, we show that coordinated induction of the beta(2) integrins during direct hypoxia-sensing occurs through transcriptional activation of each of the genes by which they are encoded. Certain of the molecular mechanisms that mediate this activation in transcription are dependent upon hypoxia-inducible factor-1 (HIF-1), whereas others are HIF-1 independent. In search of these HIF-1-independent mechanisms, we identified Pur alpha as a new hypoxia-response factor. Binding of Pur alpha to the HIF-1-independent beta(2) integrin promoters is induced by hypoxia and mutagenesis of these Pur alpha-binding sites almost completely abolishes the ability of the promoters to respond to hypoxic conditions. Additional studies using siRNA directed against Pur alpha also revealed a loss in the hypoxic response of the beta(2) integrin promoters. Taken together, our findings demonstrate that hypoxia induces a coordinated up-regulation in beta(2) integrin expression that is dependent upon transcriptional mechanisms mediated by HIF-1 and Pur alpha.


Assuntos
Antígenos CD18/biossíntese , Antígenos CD18/genética , Proteínas de Ligação a DNA/fisiologia , Fator 1 Induzível por Hipóxia/fisiologia , Hipóxia/metabolismo , Fatores de Transcrição/fisiologia , Antígenos CD11/biossíntese , Antígenos CD11/genética , Antígeno CD11b/biossíntese , Antígeno CD11b/genética , Antígeno CD11c/biossíntese , Antígeno CD11c/genética , Antígenos CD18/metabolismo , Adesão Celular/genética , Adesão Celular/imunologia , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Humanos , Hipóxia/genética , Hipóxia/imunologia , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Família Multigênica , Regiões Promotoras Genéticas , Ligação Proteica/genética , Ligação Proteica/imunologia , RNA Mensageiro/biossíntese , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células U937 , Regulação para Cima/genética , Regulação para Cima/imunologia
9.
J Biol Chem ; 282(33): 24352-63, 2007 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-17565996

RESUMO

Apoptosis is an essential mechanism for the maintenance of somatic tissues, and when dysregulated can lead to numerous pathological conditions. G proteins regulate apoptosis in addition to other cellular functions, but the roles of specific G proteins in apoptosis signaling are not well characterized. Galpha12 stimulates protein phosphatase 2A (PP2A), a serine/threonine phosphatase that modulates essential signaling pathways, including apoptosis. Herein, we examined whether Galpha12 regulates apoptosis in epithelial cells. Inducible expression of Galpha12 or constitutively active (QL)alpha12 in Madin-Darby canine kidney cells led to increased apoptosis with expression of QLalpha12, but not Galpha12. Inducing QLalpha12 led to degradation of the anti-apoptotic protein Bcl-2 (via the proteasome pathway), increased JNK activity, and up-regulated IkappaBalpha protein levels, a potent stimulator of apoptosis. Furthermore, the QLalpha12-stimulated activation of JNK was blocked by inhibiting PP2A. To characterize endogenous Galpha12 signaling pathways, non-transfected MDCK-II and HEK293 cells were stimulated with thrombin. Thrombin activated endogenous Galpha12 (confirmed by GST-tetratricopeptide repeat (TPR) pull-downs) and stimulated apoptosis in both cell types. The mechanisms of thrombin-stimulated apoptosis through endogenous Galpha12 were nearly identical to the mechanisms identified in QLalpha12-MDCK cells and included loss of Bcl-2, JNK activation, and up-regulation of IkappaBalpha. Knockdown of the PP2A catalytic subunit in HEK293 cells inhibited thrombin-stimulated apoptosis, prevented JNK activation, and blocked Bcl-2 degradation. In summary, Galpha12 has a major role in regulating epithelial cell apoptosis through PP2A and JNK activation leading to loss of Bcl-2 protein expression. Targeting these pathways in vivo may lead to new therapeutic strategies for a variety of disease processes.


Assuntos
Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/fisiologia , Proteínas I-kappa B/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose , Linhagem Celular , Cães , Células Epiteliais/citologia , Humanos , Inibidor de NF-kappaB alfa , Proteína Fosfatase 2 , Proteínas Proto-Oncogênicas c-bcl-2/análise
10.
FASEB J ; 21(10): 2613-21, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17412998

RESUMO

Increased tissue permeability is commonly associated with hypoxia of many origins. Since hypoxia-inducible factor (HIF) represents a predominant hypoxia signaling mechanism, we compared hypoxia-elicited changes in tissue barrier function in mice conditionally lacking intestinal epithelial hypoxia-inducible factor-1alpha (hif1a). Somewhat surprisingly, these studies revealed that mutant hif1a mice were protected from hypoxia-induced increases in intestinal permeability in vivo. Guided by microarray analysis of tissues derived from these mutant hif1a mice, we identified HIF-1-dependent repression of vasodilator-stimulated phosphoprotein (VASP), a molecule known to be important in the control of cytoskeletal dynamics, including barrier function. Studies at the mRNA and protein level confirmed hypoxia-elicited repression of VASP in murine tissue, cultured epithelia and endothelia, as well as human saphenous vein ex vivo. Targeted repression of VASP by siRNA recapitulated our findings with hypoxia and directed overexpression of VASP abolished hypoxia-induced barrier dysfunction. Studies in the cloned human VASP promoter revealed hypoxia-dependent transcriptional repression, and functional studies by chromatin immunoprecipitation (ChIP) and site-directed mutagenesis revealed hypoxia-dependent binding of HIF-1alpha to the human VASP promoter. These studies identify HIF-1-dependent repression of VASP as a control point for hypoxia-regulated barrier dysfunction.


Assuntos
Moléculas de Adesão Celular/fisiologia , Hipóxia Celular/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Endotélio Vascular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Proteínas dos Microfilamentos/fisiologia , Fosfoproteínas/fisiologia , Animais , Moléculas de Adesão Celular/genética , Primers do DNA , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Fosfoproteínas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
FASEB J ; 20(13): 2242-50, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17077301

RESUMO

Adenosine has been widely associated with hypoxia of many origins, including those associated with inflammation and tumorogenesis. A number of recent studies have implicated metabolic control of adenosine generation at sites of tissue hypoxia. Here, we examine adenosine receptor control and amplification of signaling through transcriptional regulation of endothelial and epithelial adenosine receptors. Initial studies confirmed previous findings indicating selective induction of human adenosine A2B receptor (A2BR) by hypoxia. Analysis of the cloned human A2BR promoter identified a functional hypoxia-responsive region, including a functional binding site for hypoxia-inducible factor (HIF) within the A2BR promoter. Further studies examining HIF-1alpha DNA binding and HIF-1alpha gain and loss of function confirmed strong dependence of A2BR induction by HIF-1alpha in vitro and in vivo mouse models. Additional studies in endothelia overexpressing full-length A2BR revealed functional phenotypes of increased barrier function and enhanced angiogenesis. Taken together, these results demonstrate transcriptional coordination of A2BR by HIF-1alpha and amplified adenosine signaling during hypoxia. These findings may provide an important link between hypoxia and metabolic conditions associated with inflammation and angiogenesis.


Assuntos
Hipóxia Celular/fisiologia , Endotélio Vascular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Mucosa Intestinal/fisiologia , Receptor A2B de Adenosina/genética , Sequência de Bases , Linhagem Celular , Células Cultivadas , Cromatina/genética , Clonagem Molecular , Primers do DNA , Endotélio Vascular/citologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mucosa Intestinal/citologia , Dados de Sequência Molecular , Neovascularização Fisiológica , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética
12.
Am J Physiol Cell Physiol ; 291(2): C282-9, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16571862

RESUMO

Tissue edema is commonly associated with hypoxia. Generally, such episodes of fluid accumulation are self-limiting. At present, little is known about mechanisms to compensate excessive fluid transport. Here we describe an adaptive mechanism to dampen fluid loss during hypoxia. Initial studies confirmed previous observations of attenuated electrogenic Cl- secretion after epithelial hypoxia. A screen of known ion transporters in Cl- -secreting epithelia revealed selective downregulation of Na-K-2Cl cotransporter NKCC1 mRNA, protein, and function. Subsequent studies identified transcriptional repression of NKCC1 mediated by hypoxia-inducible factor (HIF). Chromatin immunoprecipitation analysis identified a functional HIF binding site oriented on the antisense strand of genomic DNA downstream of the transcription start site corresponding to the NKCC1 5'-untranslated region. Additional in vivo studies using conditional Hif1a-null mice revealed that the loss of HIF-1alpha in Cl- -secreting epithelia results in a loss of NKCC1 repression. These studies describe a novel regulatory pathway for NKCC1 transcriptional repression by hypoxia. These results suggest that HIF-dependent repression of epithelial NKCC1 may provide a compensatory mechanism to prevent excessive fluid loss during hypoxia.


Assuntos
Cloro/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Mucosa Intestinal/metabolismo , Oxigênio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Transcrição Genética/genética , Animais , Hipóxia Celular/fisiologia , Linhagem Celular , Regulação para Baixo/fisiologia , Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout , Simportadores de Cloreto de Sódio-Potássio/genética , Membro 1 da Família 12 de Carreador de Soluto
13.
J Cell Biochem ; 95(6): 1264-75, 2005 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15962286

RESUMO

Hypoxia-inducible factor (HIF) remains the central focus of oxygen sensing during hypoxia. HIF is a heterodimeric transcription factor consisting of an oxygen-regulated alpha- and a constitutively expressed beta subunit. The von Hippel-Lindau tumor suppressor (pVHL) is a component of the E3 ubiquitin ligase complex and targets HIF-alpha to proteasomal degradation, but also is known to exert a significant control on HIF transactivation activity. However, the understanding of the full interaction between HIF and pVHL has been hindered by a lack in the understanding of pVHL regulation. Here, we report that pVHL itself is induced in prolonged hypoxia in a kinetic that parallels the observed downregulation of HIF-1alpha protein under such conditions. In addition, we document direct HIF-1alpha binding to the VHL promoter and identify a functional hypoxia response element (HRE) within the VHL promoter. Such induction of pVHL in hypoxia furthermore has functional implications for the HIF dependent hypoxic response, implicating a physiologically relevant feedback mechanism. These results provide an intriguing model, whereby HIF self-regulates expression through VHL and highlight the role of pVHL as a unifying mechanism of HIF regulation.


Assuntos
Hipóxia Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Linhagem Celular Tumoral , Humanos , Cinética , Regiões Promotoras Genéticas/genética , Fatores de Tempo , Ativação Transcricional/genética
14.
FASEB J ; 19(8): 950-9, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15923405

RESUMO

Sites of inflammation are associated with dramatic shifts in tissue metabolism. Inflammation can result in significant tissue hypoxia, with resultant induction of hypoxia-responsive genes. Given this association, we hypothesized that neutrophil (PMN) ligands expressed on epithelial cells may be regulated by hypoxia. Initial studies confirmed earlier results that epithelial hypoxia enhances PMN transepithelial migration and promotes apical clearance of PMN from the epithelial surface. A screen of known PMN ligands revealed a surprisingly stable expression pattern in hypoxia. However, this screen identified one gene, CD55, as a highly hypoxia-inducible molecule expressed on the apical membrane of mucosal epithelia. Subsequent studies verified the induction of CD55 mRNA and protein expression by hypoxia. Overexpression of CD55 by transfection in nonhypoxic epithelia resulted in a similar pattern of apical PMN clearance, and peptide mimetics corresponding to the PMN binding site on DAF blocked such apical clearance of PMN. Studies directed at understanding molecular pathways of hypoxia inducibility revealed that a approximately 200 bp region of the CD55 gene conferred hypoxia inducibility for CD55. These studies identified a functional binding site for the transcriptional regulator hypoxia-inducible factor (HIF). Taken together, these results identify HIF-dependent induction of epithelial CD55 in the resolution of ongoing inflammation through clearance of apical PMN.


Assuntos
Antígenos CD55/genética , Expressão Gênica , Fator 1 Induzível por Hipóxia/fisiologia , Neutrófilos/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Biotinilação , Antígenos CD55/análise , Células CACO-2 , Adesão Celular , Linhagem Celular , Membrana Celular/metabolismo , Movimento Celular , Sequência Consenso , Epitélio/fisiologia , Humanos , Hipóxia/genética , Inflamação/patologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese
15.
Proc Natl Acad Sci U S A ; 101(28): 10440-5, 2004 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-15235127

RESUMO

Inflammatory responses are associated with significant changes in tissue metabolism. In particular, metabolic shifts during inflammation can result in significant tissue hypoxia, with resultant induction of hypoxia-responsive genes. Given this association, we hypothesized that leukocyte functional responses are influenced by hypoxia. Initial experiments revealed that exposure of the promonocytic cell line U937 to hypoxia resulted in increased adhesion to activated endothelia. Such increases were transcription-dependent and were blocked by antibodies directed against beta2, but not beta1, integrins. Analysis of beta2 integrin mRNA and protein in U937 cells revealed a 5- to 6-fold increase with hypoxia. Extension of this analysis to hypoxic human whole blood revealed prominent induction of beta2 integrin mRNA and protein ex vivo. Furthermore, murine beta2 integrin mRNA was found to be significantly induced during hypoxia in vivo. Subsequent studies identified a binding site for hypoxia-inducible factor 1 (HIF-1) in the CD18 gene. This gene encodes the subunit common to all four known types of beta2 integrin heterodimer. HIF-1 binding was demonstrated in vivo, and mutational analysis of the HIF-1 site within the CD18 promoter resulted in a loss of hypoxia inducibility. Taken together, these results demonstrate that hypoxia induces leukocyte beta2 integrin expression and function by transcriptional mechanisms dependent upon HIF-1.


Assuntos
Antígenos CD18/genética , Adesão Celular/imunologia , Proteínas de Ligação a DNA/metabolismo , Hipóxia/fisiopatologia , Leucócitos/citologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição , Sequência de Bases , Antígenos CD18/metabolismo , Endotélio Vascular/citologia , Expressão Gênica/imunologia , Humanos , Hipóxia/imunologia , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Leucócitos/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Ativação Transcricional/imunologia , Células U937
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