RESUMO
Keratinocytes are important for the acute phase of HSV-1 infection and subsequent persistence in sensory nervous tissue. In this study, we showed that keratinocytes (HEL-30) were refractory to IFN-gamma induction of an antiviral state to HSV-1 infection, while IFN-gamma did induce an antiviral state in fibroblasts (L929). This led us to examine the possible role of suppressor of cytokine signaling-1 (SOCS-1) in this refractiveness. RT-PCR analysis of SOCS-1 mRNA expression in HSV-1-infected cells showed a 4-fold increase for keratinocytes while having a negligible effect on fibroblasts. A similar pattern was observed at the level of SOCS-1 protein induction. Activation of STAT1alpha in keratinocytes was inhibited by HSV-1 infection. A direct effect of HSV-1 on the SOCS-1 promoter was shown in a luciferase reporter gene assay. We have developed a small peptide antagonist of SOCS-1, pJAK2(1001-1013), that had both an antiviral effect in keratinocytes against HSV-1 as well as a synergistic effect on IFN-gamma induction of an antiviral state. HSV-1 ICP0 mutant was inhibited by IFN-gamma in HEL-30 cells and was less effective than wild-type virus in induction of SOCS-1 promoter. We conclude that SOCS-1 plays an important role in the inhibition of the antiviral effect of IFN-gamma in keratinocytes infected with HSV-1. The use of SOCS-1 antagonist to abrogate this refractiveness could have a transformational effect on therapy against viral infections.
Assuntos
Herpesvirus Humano 1/imunologia , Queratinócitos/virologia , Proteínas Supressoras da Sinalização de Citocina/genética , Linhagem Celular Tumoral , Herpesvirus Humano 1/patogenicidade , Humanos , Imunidade , Fator Gênico 3 Estimulado por Interferon/antagonistas & inibidores , Interferon gama/imunologia , Queratinócitos/metabolismo , Peptídeos/farmacologia , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidoresRESUMO
Leishmania alternates between two morphologically different stages, promastigotes and amastigotes. While the majority of reports focused on how the promastigote form can alter macrophage (Mphi) signaling and function, fewer reports investigated signaling alterations mediated by amastigotes, and there is a lack of comparative studies. In this study, we performed a comparison between the ability of both forms of the parasite to alter Mphi signaling and functions. Here, we show that both promastigotes and amastigotes were able to rapidly activate host protein tyrosine phosphatases (PTPs), importantly the Src homology 2 domain-containing PTP (SHP-1). However, we found that PTP-1B is specifically activated by promastigote but not amastigote infection and that lmcpb(-/-) promastigotes were no longer able to activate PTP-1B. We also show a similarity in the way promastigotes and amastigotes inactivate the transcription factors (TFs) STAT-1alpha and AP-1, but we show differences in the modulation of NF-kappaB, with promastigotes cleaving the p65 subunit, generating a smaller p35 subunit, and amastigotes fully degrading the p65 subunit with no p35 production. Importantly, we show that the cysteine proteinase LmCPb plays a key role in the alteration of NF-kappaB, STAT-1alpha, and AP-1 by promastigote and amastigote infections, ultimately leading to the inability of these TFs to translocate to the nucleus in response to gamma interferon (IFN-gamma) stimulation and thus contributing to the ability of both parasite forms to effectively block IFN-gamma-mediated nitric oxide (NO) production in Mphis.
Assuntos
Leishmania mexicana/imunologia , Leishmania mexicana/patogenicidade , Macrófagos/imunologia , Macrófagos/parasitologia , Transdução de Sinais , Animais , Linhagem Celular , Cisteína Proteases/metabolismo , Humanos , Fator Gênico 3 Estimulado por Interferon/antagonistas & inibidores , Interferon gama/imunologia , Camundongos , Óxido Nítrico/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas de Protozoários/metabolismo , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição RelA/metabolismoRESUMO
Chondrocytes are important for the development and maintenance of articular cartilage. However, both in osteoarthritis (OA) and rheumatoid arthritis (RA) chondrocytes are involved in the process of cartilage degradation and synthesize important immunomodulatory mediators, including nitric oxide (NO) generated by the inducible NO synthase (iNOS). To uncover the role of iNOS in the pathomechanisms of OA and RA, we analyzed the regulation of iNOS expression using immortalized human chondrocytes as a reproducible model. In C-28/I2 chondrocytes, iNOS expression was associated with the expression of the chondrocyte phenotype. Peak induction by a cytokine cocktail occurred between 6 and 8h and declined by 24h. Inhibition of p38MAPK, NF-kappaB and the JAK2-STAT-1alpha pathways resulted in a reduction of iNOS expression. In contrast to other cell types, the cytokine-mediated induction of the human iNOS promoter paralleled the induction rate of the iNOS mRNA expression in C-28/I2 chondrocytes. However, in addition post-transcriptional regulation of iNOS expression by the RNA binding protein KSRP seems to operate in these cells. As seen in other chondrocyte models, glucocorticoids were not able to inhibit cytokine-induced iNOS expression in C-28/I2 cells, due to the lack of the glucocorticoid receptor mRNA expression. In this model of glucocorticoid-resistance, the new fungal anti-inflammatory compound S-curvularin was able to inhibit cytokine-induced iNOS expression and iNOS-dependent NO-production. In summary, we demonstrate for the first time that differentiated human immortalized C-28/I2 chondrocytes are a representative cell culture model to investigate iNOS gene expression in human joint diseases.
Assuntos
Condrócitos/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Óxido Nítrico Sintase Tipo II/genética , Processamento Pós-Transcricional do RNA/genética , Anti-Inflamatórios/farmacologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/enzimologia , Linhagem Celular Transformada , Condrócitos/efeitos dos fármacos , Citocinas/farmacologia , Indução Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Fator Gênico 3 Estimulado por Interferon/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/biossíntese , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transativadores/genética , Transativadores/metabolismo , Zearalenona/análogos & derivados , Zearalenona/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors that exert anti-inflammatory effects. IFN-gamma induction of class II MHC expression, which requires the class II transactivator (CIITA), is inhibited by statins; however, the molecular basis for suppression is undetermined. We describe that statins inhibit IFN-gamma-induced class II MHC expression by suppressing CIITA gene expression, which is dependent on the HMG-CoA reductase pathway. In addition, CIITA expression is inhibited by GGTI-298 or Clostridium difficile Toxin A, specific inhibitors of Rho family protein prenylation, indicating the involvement of small GTPases. Rac1 is involved in IFN-gamma inducible expression of CIITA, and statins inhibit IFN-gamma-induced Rac1 activation, contributing to the inhibitory effect of statins. IFN-gamma induction of the CIITA gene is regulated by the transcription factors STAT-1alpha, interferon regulatory factor (IRF)-1 and upstream stimulatory factor (USF)-1. We previously reported that statins inhibit constitutive STAT-1alpha expression. IRF-1, a STAT-1 dependent gene, is also inhibited by statins. Therefore, statin treatment results in decreased recruitment of STAT-1alpha and IRF-1 to the endogenous CIITA promoter IV (pIV). The recruitment of USF-1 to CIITA pIV is also reduced by statins, as is the recruitment of RNA polymerase II (Pol II), p300 and Brg-1. These data indicate that statins inhibit the transcriptional program of the CIITA gene.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Interferon gama/farmacologia , Proteínas Nucleares/genética , Sinvastatina/farmacologia , Transativadores/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Genes MHC da Classe II , Fator Regulador 1 de Interferon/antagonistas & inibidores , Fator Gênico 3 Estimulado por Interferon/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Fatores Estimuladores Upstream/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1alpha (STAT1alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse osteoblastic MC3T3-E1 cells; thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1alpha protein in PKR mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1alpha and/or Runx2 expression.
Assuntos
Osso e Ossos/fisiologia , Calcificação Fisiológica , Osteoblastos/metabolismo , eIF-2 Quinase/fisiologia , Fosfatase Alcalina , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Genes Dominantes , Técnicas In Vitro , Fator Gênico 3 Estimulado por Interferon/antagonistas & inibidores , Fator Gênico 3 Estimulado por Interferon/genética , Fator Gênico 3 Estimulado por Interferon/metabolismo , Camundongos , Fosforilação , Transporte Proteico , RNA de Cadeia Dupla/fisiologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Transativadores , Transcrição GênicaRESUMO
We have previously characterized a novel tyrosine kinase inhibitor peptide (Tkip) that is a mimetic of suppressor of cytokine signaling 1 (SOCS-1) and inhibits JAK2 phosphorylation of the transcription factor STAT1alpha. We show in this study that Tkip protects mice against experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis. Mice are immunized with myelin basic protein (MBP) for induction of disease. Tkip (63 mug) administered every other day suppressed the development of acute EAE in 75% of New Zealand White (NZW) mice. Furthermore, Tkip completely protected SJL/J mice, which where induced to get the relapsing/remitting form of EAE, against relapses compared with control groups in which >70% of the mice relapsed after primary incidence of disease. Protection of mice by Tkip was similar to that seen with the type I IFN, IFN-tau. Protection of mice correlated with lower MBP Ab titers in Tkip-treated groups as well as suppression of MBP-induced proliferation of splenocytes taken from EAE-afflicted mice. Cessation of Tkip and IFN-tau administration resulted in SJL/J mice relapsing back into disease. Prolonged treatment of mice with Tkip produced no evidence of cellular toxicity or weight loss. Consistent with its JAK2 inhibitory function, Tkip also inhibited the activity of the inflammatory cytokine TNF-alpha, which uses the STAT1alpha transcription factor. The data presented in this study show that Tkip, like the type I IFN, IFN-tau, inhibits both the autoreactive cellular and humoral responses in EAE and ameliorates both the acute and chronic relapsing/remitting forms of EAE.