RESUMO
Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3(-/-) cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased. We identified Stat3 mutants that selectively restored the protein's function as a transcription factor or its functions within the ETC. In mice that do not express Stat3 in the heart, there were also selective defects in the activities of complexes I and II of the ETC. These data indicate that Stat3 is required for optimal function of the ETC, which may allow it to orchestrate responses to cellular homeostasis.
Assuntos
Respiração Celular , Mitocôndrias/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Células Cultivadas , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Homeostase , Camundongos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fosforilação Oxidativa , Fosforilação , Células Precursoras de Linfócitos B/metabolismo , Fator de Transcrição STAT3/química , Serina/metabolismo , Transdução de SinaisRESUMO
Tyk2, a member of the Jak family of protein tyrosine kinases, is critical for the biological actions of alpha/beta interferon (IFN-alpha/beta). Although Tyk2(-/-) mice are phenotypically normal, they exhibit abnormal responses to inflammatory challenges in a variety of cells isolated from Tyk2(-/-) mice. The reported phenotypic alterations in both Tyk2-null cells and mice are consistent with the possibility that the expression of this tyrosine kinase may regulate mitochondrial function. We report here that Tyk2-null pro-B cells are markedly deficient in basal oxygen consumption and exhibit a significant decrease in steady-state cellular ATP levels compared to wild-type cells. Tyk2-null cells also exhibit impaired complex I, III, and IV function of the mitochondrial electron transport chain. Reconstitution of Tyk2-null pro-B cells with either the wild type or a kinase-inactive mutant of Tyk2 restores basal mitochondrial respiration. By contrast, the kinase activity of Tyk2 is required for maintenance of both complex I-dependent mitochondrial respiration as well as induction of apoptosis in cells incubated with IFN-beta. Consistent with the role of Tyk2 in the regulation of tyrosine phosphorylation of Stat3, expression of a constitutively active Stat3 can restore the mitochondrial respiration in Tyk2-null cells treated with IFN-beta. Finally, Tyk2(-/-) mice show decreased exercise tolerance compared to wild-type littermates. Our results implicate a novel role for Tyk2 kinase and Stat3 phosphorylation in mitochondrial respiration.
Assuntos
Linfócitos B/fisiologia , Mitocôndrias/fisiologia , TYK2 Quinase/metabolismo , TYK2 Quinase/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Linfócitos B/enzimologia , Respiração Celular , Células Cultivadas , Transporte de Elétrons/genética , Fatores Imunológicos/farmacologia , Interferon beta/farmacologia , Camundongos , Camundongos Knockout , Mitocôndrias/enzimologia , Modelos Biológicos , Condicionamento Físico Animal , Transdução de Sinais , TYK2 Quinase/genética , TransfecçãoRESUMO
The growth-inhibitory effects of type 1 interferons (IFNs) (IFNalpha/beta) are complex, and the role of apoptosis in their antigrowth effects is variable and not well understood. We have examined primary murine interleukin-7-dependent bone marrow-derived pro-B cells, where IFNbeta, but not IFNalpha, induces programmed cell death (PCD). IFNbeta-stimulated apoptosis is the same in pro-B cells derived from wild type and Stat1(-/-) mice. However, in pro-B cells from Tyk2(-/-) mice, where there is normal activation of Stat1 and Stat2, IFNbeta-stimulated PCD is not observed. Loss of B cells in lymphocytic choriomeningitis virus-infected mice has been shown to be mediated through the expression of IFNalpha/beta (1). In wild type mice infected with lymphocytic choriomeningitis virus, there is a greater loss of B cells in the bone marrow and spleen than in Tyk2(-/-) mice infected with the virus, suggesting that the expression of this kinase plays an in vivo role in IFNalpha/beta-mediated PCD. In contrast to IFNbeta-stimulated tyrosine phosphorylation of Stat1 and Stat2, Stat3 tyrosine phosphorylation is defective in Tyk2(-/-) pro-B cells, suggesting that this Stat family member is required for apoptosis. In support of this hypothesis, inhibition of Stat3 activation in wild type B cells reverses the apoptotic effects of IFNbeta. Furthermore, expression of a constitutively active form of Stat3 in Tyk2(-/-) B cells partially restores IFNbeta-stimulated PCD. These results demonstrate an important role of Tyk2-mediated tyrosine phosphorylation of Stat3 in the ability of IFNbeta to stimulate apoptosis of primary pro-B cells.
Assuntos
Linfócitos B/metabolismo , Interferon beta/metabolismo , Proteínas Tirosina Quinases/química , Fator de Transcrição STAT3/metabolismo , Animais , Anexina A5/metabolismo , Apoptose , Citoplasma/metabolismo , Immunoblotting , Interleucina-7/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação , TYK2 Quinase , Tirosina/químicaRESUMO
Although the antiviral actions of interferons (IFNs) are observed in most types of cells, the antiproliferative effects of IFNalpha/beta are variable as are the mechanisms of growth inhibition that may or may not be due to the induction of apoptosis. To understand more about the mechanisms that are responsible for IFNalpha/beta-stimulated apoptosis, we have characterized a new human Jurkat T cell variant named H123 where IFNalpha activates programmed cell death (PCD). No differences in IFNalpha-stimulated, Stat-dependent gene expression were detected between H123 cells and the parental Jurkat cells, which are growth inhibited, but do not undergo apoptosis with IFNalpha. Although IFNalpha stimulates the activity of both caspase 3 and 9 in H123 cells, the general caspase inhibitor Z-VAD only partially reverses the apoptotic actions of IFNalpha. Induction of apoptosis by IFNalpha occurs through a mitochondrial-dependent pathway in H123 cells, as demonstrated by the release of cytochrome C from the mitochondria. Furthermore, IFNalpha treatment of H123 cells stimulates the release of the serine protease HtrA2/Omi from the mitochondria, suggesting that it plays a role in the apoptotic actions of this cytokine. These results provide evidence for a novel type 1 IFN-mediated pathway that regulates apoptosis of T cells through a mitochondrial-dependent and caspase-dependent and independent pathway.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Interferon-alfa/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Caspase 3 , Caspase 9 , Inibidores de Caspase , Células Cultivadas , Citocromos c/metabolismo , Citosol/metabolismo , Proteína de Domínio de Morte Associada a Fas , Inibidores do Crescimento , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Células Jurkat , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais , RNA/metabolismo , Fatores de Transcrição STAT/metabolismo , Serina Endopeptidases/metabolismo , Serpinas/metabolismo , Proteínas Virais/metabolismoRESUMO
Ferroxidases are essential for normal iron homeostasis in most organisms. The paralogous vertebrate ferroxidases ceruloplasmin (Cp) and hephaestin (Heph) are considered to have nonidentical functions in iron transport: plasma Cp drives iron transport from tissue stores while intestinal Heph facilitates iron absorption from the intestinal lumen. To clarify the function of Cp, we acutely bled Cp-/- mice to stress iron homeostasis pathways. Red cell hemoglobin recovery was defective in stressed Cp-/- mice, consistent with low iron availability. Contrary to expectations, iron was freely released from spleen and liver stores in Cp-/- mice, but intestinal iron absorption was markedly impaired. Phlebotomy of wild-type mice caused a striking shift of Cp from the duodenal epithelium to the underlying lamina propria, suggesting a critical function of Cp in basolateral iron transport. Regulated relocalization of intestinal Cp may represent a fail-safe mechanism in which Cp shares with Heph responsibility for iron absorption under stress.
Assuntos
Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Ferro/metabolismo , Animais , Proteínas de Transporte de Cátions/metabolismo , Duodeno/citologia , Duodeno/metabolismo , Enterócitos/metabolismo , Homeostase , Humanos , Absorção Intestinal , Ferro/sangue , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa/metabolismo , RNA Mensageiro/metabolismo , Baço/metabolismo , Estresse Fisiológico/metabolismo , Fatores de Tempo , Transferrina/metabolismoRESUMO
Posttranslational modification of histones by acetylation, methylation or phosphorylation has emerged as a major mechanism to modify chromatin structure and gene expression. In most cases, transcriptionally active genes display enhanced binding of acetylated histones in their promoters. Activation of histone acetyltransferases or inhibition of histone deacetylases (HDACs) allows chromatin to assume a more open state permitting transcriptional activators to form a preinitiation complex. To our surprise, treatment of cells with the HDAC inhibitor, trichostatin A (TSA), inhibits selected interferon beta (IFNbeta)-stimulated immediate early genes that are activated by the transcription factors Stat1 and Stat2. However, IFNbeta activation of IRF-1, which requires tyrosine-phosphorylated Stat1 homodimers binding to a gamma interferon activation sequence in its promoter is not affected by TSA. Exposure of cells to TSA does not alter tyrosine phosphorylation of Stat1 or Stat2. TSA treatment of cells also does not alter the binding of Stat 1 or Stat2 to the endogenous ISG54 promoter. However, IFNbeta-stimulated binding of RNA polymerase II to the ISG54 promoter is prevented by TSA. Interestingly, ectopic expression of IRF9 reverses the inhibitory actions of TSA, suggesting that IRF9 functions to recruit RNA polymerase II to the promoter of interferon-stimulated genes. This particular function of IRF9 requires the activity of histone deacetylases.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Histona Desacetilases/metabolismo , Interferons/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/fisiologia , Fatores de Transcrição/fisiologia , Linhagem Celular , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Ácidos Hidroxâmicos/farmacologia , Fator Regulador 1 de Interferon , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Interferon beta/metabolismo , Luciferases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Testes de Precipitina , Ligação Proteica , RNA Polimerase II/metabolismo , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Fatores de Tempo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Tirosina/metabolismoRESUMO
Over the past decade, a wealth of knowledge has been obtained concerning the mechanisms by which interferons (IFNs) and other cytokines activate or down-regulate immediate early genes via the Jak/Stat pathway. In contrast, little information is available on interferon-activated gene expression in naïve cells compared with cells that have been desensitized and subsequently resensitized to the actions of these cytokines. In naïve cells, the ISG54 gene is activated via IFN beta-stimulated formation of ISGF3, a heterotrimeric DNA binding complex consisting of p48 (IRF9) and tyrosine-phosphorylated Stat1 and Stat2. In contrast, in previously desensitized cells IFN beta weakly stimulates the assembly of an ISGF3-like complex that lacks Stat1, even though ISG54 mRNA induction is the same as in naïve cells. The lack of Stat1 tyrosine phosphorylation and DNA binding is due to increased activity of a protein-tyrosine phosphatase. In cells that do not express the tyrosine phosphatase Tc-PTP, the rate of Stat1 dephosphorylation is the same in naïve and previously desensitized cells. These results implicate Tc-PTP in a novel role in the regulation of type 1 interferon-stimulated gene expression.