RESUMO
Macrophage activation is a complex process with multiple control elements that ensures an adequate response to the aggressor pathogens and, on the other hand, avoids an excess of inflammatory activity that could cause tissue damage. In this study, we have identified RND3, a small GTP-binding protein, as a new element in the complex signaling process that leads to macrophage activation. We show that RND3 expression is transiently induced in macrophages activated through Toll receptors and potentiated by IFN-γ. We also demonstrate that RND3 increases NOTCH signaling in macrophages by favoring NOTCH1 expression and its nuclear activity; however, Rnd3 expression seems to be inhibited by NOTCH signaling, setting up a negative regulatory feedback loop. Moreover, increased RND3 protein levels seem to potentiate NFκB and STAT1 transcriptional activity resulting in increased expression of proinflammatory genes, such as Tnf-α, Irf-1, or Cxcl-10. Altogether, our results indicate that RND3 seems to be a new regulatory element which could control the activation of macrophages, able to fine tune the inflammatory response through NOTCH.
Assuntos
Macrófagos , Transdução de Sinais , Proteínas rho de Ligação ao GTP , Macrófagos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Camundongos , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
IL-13 signaling polarizes macrophages to an M2 alternatively activated phenotype, which regulates tissue repair and anti-inflammatory responses. However, an excessive activation of this pathway leads to severe pathologies, such as allergic airway inflammation and asthma. In this work, we identified NOTCH4 receptor as an important modulator of M2 macrophage activation. We show that the expression of NOTCH4 is induced by IL-13, mediated by Janus kinases and AP1 activity, probably mediated by the IL-13Rα1 and IL-13Rα2 signaling pathway. Furthermore, we demonstrate an important role for NOTCH4 signaling in the IL-13 induced gene expression program in macrophages, including various genes that contribute to pathogenesis of the airways in asthma, such as ARG1, YM1, CCL24, IL-10, or CD-163. We also demonstrate that NOTCH4 signaling modulates IL-13-induced gene expression by increasing IRF4 activity, mediated, at least in part, by the expression of the histone H3K27me3 demethylase JMJD3, and by increasing AP1-dependent transcription. In summary, our results provide evidence for an important role of NOTCH4 signaling in alternative activation of macrophages by IL-13 and suggest that NOTCH4 may contribute to the increased severity of lesions in M2 inflammatory responses, such as allergic asthma, which points to NOTCH4 as a potential new target for the treatment of these pathologies.
Assuntos
Asma , Interleucina-13 , Humanos , Macrófagos/metabolismo , Inflamação/metabolismo , Transdução de Sinais/genética , Receptor Notch4/metabolismoRESUMO
NOTCH4 is a member of the NOTCH family of receptors whose expression is intensively induced in macrophages after their activation by Toll-like receptors (TLR) and/or interferon-γ (IFN-γ). In this work, we show that this receptor acts as a negative regulator of macrophage activation by diminishing the expression of proinflammatory cytokines, such as IL-6 and IL-12, and costimulatory proteins, such as CD80 and CD86. We have observed that NOTCH4 inhibits IFN-γ signaling by interfering with STAT1-dependent transcription. Our results show that NOTCH4 reprograms the macrophage response to IFN-γ by favoring STAT3 versus STAT1 phosphorylation without affecting their expression levels. This lower activation of STAT1 results in diminished transcriptional activity and expression of STAT1-dependent genes, including IRF1, SOCS1 and CXCL10. In macrophages, NOTCH4 inhibits the canonical NOTCH signaling pathway induced by LPS; however, it can reverse the inhibition exerted by IFN-γ on NOTCH signaling, favoring the expression of NOTCH-target genes, such as Hes1. Indeed, HES1 seems to mediate, at least in part, the enhancement of STAT3 activation by NOTCH4. NOTCH4 also affects TLR signaling by interfering with NF-κB transcriptional activity. This effect could be mediated by the diminished activation of STAT1. These results provide new insights into the mechanisms by which NOTCH, TLR and IFN-γ signal pathways are integrated to modulate macrophage-specific effector functions and reveal NOTCH4 acting as a new regulatory element in the control of macrophage activation that could be used as a target for the treatment of pathologies caused by an excess of inflammation.
Assuntos
Interferon gama/metabolismo , Ativação de Macrófagos/genética , Macrófagos Peritoneais/imunologia , Receptor Notch4/metabolismo , Transdução de Sinais/genética , Receptor 4 Toll-Like/metabolismo , Animais , Doadores de Sangue , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Células RAW 264.7 , Receptor Notch4/genética , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Macrophage activation by Toll receptors is an essential event in the development of the response against pathogens. NOTCH signaling pathway is involved in the control of macrophage activation and the inflammatory processes. In this work, we have characterized NOTCH signaling in macrophages activated by Toll-like receptor (TLR) triggering and determined that DLL1 and DLL4 are the main ligands responsible for NOTCH signaling. We have identified ADAM10 as the main protease implicated in NOTCH processing and activation. We have also observed that furin, which processes NOTCH receptors, is induced by TLR signaling in a NOTCH-dependent manner. NOTCH3 is the only NOTCH receptor expressed in resting macrophages. Its expression increased rapidly in the first hours after TLR4 activation, followed by a gradual decrease, which was coincident with an elevation of the expression of the other NOTCH receptors. All NOTCH1, 2 and 3 contribute to the increased NOTCH signaling detected in activated macrophages. We also observed a crosstalk between NOTCH3 and NOTCH1 during macrophage activation. Finally, our results highlight the relevance of NOTCH3 in the activation of NF-κB, increasing p65 phosphorylation by p38 MAP kinase. Our data identify, for the first time, NOTCH3 as a relevant player in the control of inflammation.
Assuntos
Inflamação/imunologia , Macrófagos/imunologia , Receptor Notch3/fisiologia , Animais , Regulação da Expressão Gênica , Humanos , Ativação de Macrófagos , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , NF-kappa B/imunologia , Células RAW 264.7 , Transdução de Sinais , Receptores Toll-Like/imunologiaRESUMO
The NOTCH family of receptors and ligands is involved in numerous cell differentiation processes, including adipogenesis. We recently showed that overexpression of each of the four NOTCH receptors in 3T3-L1 preadipocytes enhances adipogenesis and modulates the acquisition of the mature adipocyte phenotype. We also revealed that DLK proteins modulate the adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells in an opposite way, despite their function as non-canonical inhibitory ligands of NOTCH receptors. In this work, we used multipotent C3H10T1/2 cells as an adipogenic model. We used standard adipogenic procedures and analyzed different parameters by using quantitative-polymerase chain reaction (qPCR), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), luciferase, Western blot, and metabolic assays. We revealed that C3H10T1/2 multipotent cells show higher levels of NOTCH receptors expression and activity and lower Dlk gene expression levels than 3T3-L1 preadipocytes. We found that the overexpression of NOTCH receptors enhanced C3H10T1/2 adipogenesis levels, and the overexpression of NOTCH receptors and DLK (DELTA-like homolog) proteins modulated the conversion of cells towards a brown-like adipocyte phenotype. These and our prior results with 3T3-L1 preadipocytes strengthen the idea that, depending on the cellular context, a precise and highly regulated level of global NOTCH signaling is necessary to allow adipogenesis and determine the mature adipocyte phenotype.
Assuntos
Tecido Adiposo Marrom/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Receptores Notch/metabolismo , Animais , Diferenciação Celular , Células HEK293 , Humanos , Camundongos , TransfecçãoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
The role of NOTCH signaling in adipogenesis is highly controversial, with data indicating null, positive or negative effects on this differentiation process. We hypothesize that these contradictory results could be due to the different global NOTCH signaling levels obtained in different experimental settings, because of a specific modulation of NOTCH receptors' activity by their ligands. We have previously demonstrated that DLK1 and DLK2, two non-canonical NOTCH1 ligands that inhibit NOTCH1 signaling in a dose-dependent manner, modulate the adipogenesis process of 3T3-L1 preadipocytes. In this work, we show that over-expression of any of the four NOTCH receptors enhanced adipogenesis of 3T3-L1 preadipocytes. We also determine that DLK proteins inhibit not only the activity of NOTCH1, but also the activity of NOTCH2, 3 and 4 receptors to different degrees. Interestingly, we have observed, by different approaches, that NOTCH1 over-expression seems to stimulate the differentiation of 3T3-L1 cells towards a brown-like adipocyte phenotype, whereas cells over-expressing NOTCH2, 3 or 4 receptors or DLK proteins would rather differentiate towards a white-like adipocyte phenotype. Finally, our data also demonstrate a complex feed-back mechanism involving Notch and Dlk genes in the regulation of their expression, which suggest that a precise level of global NOTCH expression and NOTCH-dependent transcriptional activity of specific targets could be necessary to determine the final phenotype of 3T3-L1 adipocytes.
RESUMO
Inhibition of Notch signalling in T cells attenuates the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Growing evidence indicates that myeloid cells are also key players in autoimmune processes. Thus, the present study evaluates the role of the Notch1 receptor in myeloid cells on the progression of myelin oligodendrocyte glycoprotein (MOG)35-55 -induced EAE, using mice with a myeloid-specific deletion of the Notch1 gene (MyeNotch1KO). We found that EAE progression was less severe in the absence of Notch1 in myeloid cells. Thus, histopathological analysis revealed reduced pathology in the spinal cord of MyeNotch1KO mice, with decreased microglia/astrocyte activation, demyelination and infiltration of CD4+ T cells. Moreover, these mice showed lower Th1 and Th17 cell infiltration and expression of IFN-γ and IL-17 mRNA in the spinal cord. Accordingly, splenocytes from MyeNotch1KO mice reactivated in vitro presented reduced Th1 and Th17 activation, and lower expression of IL-12, IL-23, TNF-α, IL-6, and CD86. Moreover, reactivated wild-type splenocytes showed increased Notch1 expression, arguing for a specific involvement of this receptor in autoimmune T cell activation in secondary lymphoid tissues. In summary, our results reveal a key role of the Notch1 receptor in myeloid cells for the initiation and progression of EAE.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Células Mieloides/imunologia , Receptor Notch1/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Expressão Gênica/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/imunologia , Medula Espinal/metabolismo , Células Th1/metabolismo , Células Th17/metabolismoRESUMO
The involvement of NOTCH signaling in macrophage activation by Toll receptors has been clearly established, but the factors and pathways controlling NOTCH signaling during this process have not been completely delineated yet. We have characterized the role of TSPAN33, a tetraspanin implicated in a disintegrin and metalloproteinase (ADAM) 10 maturation, during macrophage proinflammatory activation. Tspan33 expression increases in response to TLR signaling, including responses triggered by TLR4, TLR3, and TLR2 activation, and it is enhanced by IFN-γ. In this study, we report that induction of Tspan33 expression by TLR and IFN-γ is largely dependent on NOTCH signaling, as its expression is clearly diminished in macrophages lacking Notch1 and Notch2 expression, but it is enhanced after overexpression of a constitutively active intracellular domain of NOTCH1. TSPAN33 is the member of the TspanC8 tetraspanin subgroup more intensely induced during macrophage activation, and its overexpression increases ADAM10, but not ADAM17, maturation. TSPAN33 favors NOTCH processing at the membrane by modulating ADAM10 and/or Presenilin1 activity, thus increasing NOTCH signaling in activated macrophages. Moreover, TSPAN33 modulates TLR-induced proinflammatory gene expression, at least in part, by increasing NF-κB-dependent transcriptional activity. Our results suggest that TSPAN33 represents a new control element in the development of inflammation by macrophages that could constitute a potential therapeutic target.
Assuntos
Ativação de Macrófagos , Macrófagos/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Tetraspaninas/metabolismo , Receptores Toll-Like/metabolismo , Animais , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células RAW 264.7 , Tetraspaninas/genética , Células U937RESUMO
Macrophages activated through Toll receptor triggering increase the expression of the A(2A) and A(2B) adenosine receptors. In this study, we show that adenosine receptor activation enhances LPS-induced pfkfb3 expression, resulting in an increase of the key glycolytic allosteric regulator fructose 2,6-bisphosphate and the glycolytic flux. Using shRNA and differential expression of A(2A) and A(2B) receptors, we demonstrate that the A(2A) receptor mediates, in part, the induction of pfkfb3 by LPS, whereas the A(2B) receptor, with lower adenosine affinity, cooperates when high adenosine levels are present. pfkfb3 promoter sequence deletion analysis, site-directed mutagenesis, and inhibition by shRNAs demonstrated that HIF1α is a key transcription factor driving pfkfb3 expression following macrophage activation by LPS, whereas synergic induction of pfkfb3 expression observed with the A(2) receptor agonists seems to depend on Sp1 activity. Furthermore, levels of phospho-AMP kinase also increase, arguing for increased PFKFB3 activity by phosphorylation in long term LPS-activated macrophages. Taken together, our results show that, in macrophages, endogenously generated adenosine cooperates with bacterial components to increase PFKFB3 isozyme activity, resulting in greater fructose 2,6-bisphosphate accumulation. This process enhances the glycolytic flux and favors ATP generation helping to develop and maintain the long term defensive and reparative functions of the macrophages.
Assuntos
Adenosina/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/enzimologia , Fosfofrutoquinase-2/biossíntese , Receptor 4 Toll-Like/agonistas , Adenosina/genética , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Frutosedifosfatos/genética , Frutosedifosfatos/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Glicólise/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoenzimas/biossíntese , Isoenzimas/genética , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/fisiologia , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosfofrutoquinase-2/genética , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina , Deleção de Sequência , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
The protein DLK2, highly homologous to DLK1, belongs to the EGF-like family of membrane proteins, which includes NOTCH receptors and their DSL-ligands. The molecular mechanisms by which DLK proteins regulate cell differentiation and proliferation processes are not fully established yet. In previous reports, we demonstrated that DLK1 interacts with itself and with specific EGF-like repeats of the NOTCH1 extracellular region involved in the binding to NOTCH1 canonical ligands. Moreover, the interaction of DLK1 with NOTCH1 caused an inhibition of basal NOTCH signaling in preadipocytes and mesenchymal multipotent cells. In this work, we demonstrate, for the first time, that DLK2 interacts with itself, with DLK1, and with the same NOTCH1 receptor region as DLK1 does. We demonstrate also that the interaction of DLK2 with NOTCH1 similarly results in an inhibition of NOTCH signaling in preadipocytes and Mouse Embryo fibloblasts. In addition, we demonstrate that a membrane DLK1 variant, lacking the sequence recognized by the protease TACE, also inhibits NOTCH signaling. Furthermore, both DLK1 and DLK2 are able to decrease NOTCH activity also when triggered by specific NOTCH ligands. However, the decrease in NOTCH signaling induced by overexpression of Dlk2 is reversed by the overexpression of Dlk1, and viceversa. We conclude that DLK1 and DLK2 act as inhibitory non-canonical protein ligands for the NOTCH1 receptor that modulate NOTCH signaling.
Assuntos
Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Células 3T3 , Células 3T3-L1 , Proteínas Adaptadoras de Transdução de Sinal , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Ligação Competitiva , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Células HEK293 , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ligação Proteica , Receptor Notch1/genética , Proteínas Serrate-Jagged , Técnicas do Sistema de Duplo-HíbridoRESUMO
Macrophages present different Notch receptors and ligands on their surface. Following macrophage activation by LPS or other TLR ligands, Notch1 expression is upregulated. We report here that Notch signaling increases both basal and LPS-induced NF-kappaB activation, favoring the expression of genes implicated in the inflammatory response, such as the cytokines TNF-alpha and IL-6, or enzymes, such as iNOS. Delta4 seems to be the most effective ligand to induce Notch activation and increasing NF-kappaB transcriptional activity in macrophages. We show that Notch1 signaling promotes NF-kappaB translocation to the nucleus and DNA binding by increasing both phosphorylation of the IkappaB kinase alpha/beta complex and the expression of some NF-kappaB family members. Treatment of macrophages with the gamma-secretase inhibitor DAPT, which prevents the cleavage and activation of Notch receptors, inhibits all these processes, diminishing NF-kappaB activity following LPS stimulation. Additionally, we show that the active intracellular Notch fragment can directly interact with TNF-alpha and iNOS promoters. Our results suggest that Notch signaling results in an amplification of the macrophage-dependent inflammatory response by enhancing NF-kappaB signaling.
Assuntos
Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , NF-kappa B/imunologia , Receptor Notch1/imunologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/imunologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , NF-kappa B/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
The Dlk1 gene appears to function as a regulator of adipogenesis. Adult Dlk1-deficient mice are obese, but adipose tissue still develops in transgenic mice overexpressing an Fc-dlk1 fusion protein, and neither type of genetically modified mice displays serious abnormalities. It was therefore possible that one yet unidentified gene might either compensate or antagonize for the absence or for overexpression, respectively, of Dlk1 in those animals. In database searches, we found a novel gene, EGFL9, encoding for a protein whose structural features are virtually identical to those of dlk1, suggesting it may function in a similar way. As dlk1 does, the protein encoded by EGFL9/Dlk2 affects adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells; however, it does so in an opposite way to that of dlk1. In addition, expression levels of both genes appear to be inversely correlated in both cell lines. Moreover, enforced changes in the expression of one gene affect the expression levels of the other. Our data suggest that adipogenesis may be modulated by the coordinated expression of Dlk1 and EGFL9/Dlk2.
Assuntos
Adipogenia/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio , Células Cultivadas , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , TransfecçãoRESUMO
Notch signaling has been extensively implicated in cell-fate determination along the development of the immune system. However, a role for Notch signaling in fully differentiated immune cells has not been clearly defined. We have analyzed the expression of Notch protein family members during macrophage activation. Resting macrophages express Notch-1, -2, and -4, as well as the Notch ligands Jagged-1 and -2. After treatment with LPS and/or IFN-gamma, we observed a p38 MAPK-dependent increase in Notch-1 and Jagged-1 mRNA and protein levels. To study the role of Notch signaling in macrophage activation, we forced the transient expression of truncated, active intracellular Notch-1 (Notch-IC) proteins in Raw 264.7 cells and analyzed their effects on the activity of transcription factors involved in macrophage activation. Notch-IC increased STAT-1-dependent transcription. Furthermore, Raw 264.7 Notch-IC stable transfectants increased STAT1-dependent transcription in response to IFN-gamma, leading to higher expression of IFN regulatory factor-1, suppressor of cytokine signaling-1, ICAM-1, and MHC class II proteins. This effect was independent from an increase of STAT1 Tyr or Ser phosphorylation. However, inducible NO synthase expression and NO production decreased under the same conditions. Our results show that Notch up-regulation and subsequent signaling following macrophage activation modulate gene expression patterns known to affect the function of mature macrophages.
Assuntos
Apresentação de Antígeno/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Inflamação/imunologia , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Interferon gama/biossíntese , Proteína Jagged-1 , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , Receptor Notch1/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Proteínas Serrate-Jagged , Fator de Transcrição AP-1/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis.
Assuntos
Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linhagem Celular , DNA Antissenso/genética , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Receptor Notch1 , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Aminoácidos , Fatores de Transcrição HES-1 , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical liver dysfunction. We have previously shown that PGE1 preadministration protects against NO-dependent cell death induced by D-galactosamine (D-GalN) through a rapid increase of nuclear factor kappaB (NF-kappaB) activity, inducible NO synthase (NOS-2) expression, and NO production. The present study investigates whether PGE1-induced NO was able to abolish NF-kappaB activation, NOS-2 expression, and apoptosis elicited by D-GalN. Rat hepatocytes were isolated following the classical method of collagenase perfusion of liver. PGE1 (1 micromol/L) was administered 2 hours before D-GalN (5 mmol/L) in primary culture rat hepatocytes. PGE1 reduced inhibitor kappaBalpha degradation, NF-kappaB activation, NOS-2 expression, and apoptosis induced by D-GalN. The administration of an inhibitor of NOS-2 abolished the inhibitory effect of PGE1 on NF-kappaB activation and NOS-2 expression in D-GalN-treated hepatocytes. Transfection studies using different plasmids corresponding to the NOS-2 promoter region showed that D-GalN and PGE1 regulate NOS-2 expression through NF-kappaB during the initial stage of hepatocyte treatment. PGE1 was able to reduce the promoter activity induced by D-GalN. In addition, a NO donor reduced NOS-2 promoter activity in transfected hepatocytes. In conclusion, administration of PGE1 to hepatocytes produces low levels of NO, which inhibits its own formation during D-GalN-induced cell death through the attenuation of NF-kappaB-dependent NOS-2 expression. Therefore, a dual role for NO in PGE1-treated D-GalN-induced toxicity in hepatocytes is characterized by a rapid NO release that attenuates the late and proapoptotic NOS-2 expression.