RESUMO
MicroRNAs (miRNAs) have emerged as key players in the regulation of expression of target mRNAs expression. They have been associated with diverse biological processes, and recent studies have demonstrated that miRNAs play a role in inflammatory responses. We reported previously that LPS-activated fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients express IL-18 mRNA but they do not release IL-18. Based on the observation that this inhibition was due to a rapid degradation of IL-18 mRNA, our group has conducted a study to identify miRNAs that could play a role in the "antiinflammatory" response of LPS-activated RA FLS. LPS challenge modulated the expression of 63 miRNAs as assessed by microarray analysis. Fifteen miRNAs were up-regulated, including miR-346, for which overexpression upon LPS treatment was validated by quantitative RT-PCR. We then transfected FLS with an antisense oligonucleotide targeting miR-346 and found that, in these conditions, IL-18 release could be measured upon LPS activation of FLS. Moreover, we also demonstrated that miR-346 indirectly regulated IL-18 release by indirectly inhibiting LPS-induced Bruton's tyrosine kinase expression in LPS-activated RA FLS. These findings suggest that miRNAs function as regulators that help to fine-tune the inflammatory response in RA.
Assuntos
Artrite Reumatoide/metabolismo , Regulação da Expressão Gênica/genética , Interleucina-18/metabolismo , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , Proteínas Tirosina Quinases/metabolismo , Membrana Sinovial/metabolismo , Tirosina Quinase da Agamaglobulinemia , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/genética , Sequência de Bases , Linhagem Celular , Células Cultivadas , Humanos , Interleucina-18/biossíntese , Interleucina-18/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Tirosina Quinases/genética , Interferência de RNA , RNA Mensageiro/genética , Membrana Sinovial/efeitos dos fármacosRESUMO
TNF-α is a major cytokine implicated in rheumatoid arthritis. Its expression is regulated both at the transcriptional and posttranscriptional levels and recent data demonstrated that miRNAs are implicated in TNF-α response in macrophages. LPS-activated FLS isolated from RA patients express TNF-α mRNA but not the mature protein. This prompted us to look for miRNAs which could be implicated in this anti-inflammatory effect. Using a microarray, we found two miRNAs, miR-125b and miR-939 predicted to target the 3'-UTR of TNF-α mRNA, to be up-regulated in RA FLS in response to LPS, but their repression did not restore mature TNF-α expression in FLS. We showed previously that miR-346, which is upregulated in LPS-activated FLS, inhibited Btk expression that stabilized TNF-α mRNA. Blocking miR-346 reestablished TNF-α expression in activated FLS. Interestingly, transfection of miR-346 in LPS-activated THP-1 cells inhibited TNF-α secretion. We also demonstrated that TTP, a RNA binding protein which inhibited TNF-α synthesis, is overexpressed in activated FLS and that inhibition of miR-346 decreases its expression. Conversely, transfection of miR-346 in LPS-activated THP-1 cells increased TTP mRNA expression and inhibited TNF-α release. These results indicate that miR-346 controls TNF-α synthesis by regulating TTP expression.
Assuntos
Artrite Reumatoide/genética , MicroRNAs/metabolismo , Estabilidade de RNA , Tristetraprolina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Tirosina Quinase da Agamaglobulinemia , Artrite Reumatoide/patologia , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , Modelos Biológicos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Estabilidade de RNA/efeitos dos fármacos , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Líquido Sinovial/citologia , Transfecção , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
MyD88 and focal adhesion kinase (FAK) are key adaptors involved in signaling downstream of TLR2, TLR4, and integrin alpha5beta1, linking pathogen-associated molecule detection to the initiation of proinflammatory response. The MyD88 and integrin pathways are interlinked, but the mechanism of this cross-talk is not yet understood. In this study we addressed the involvement of Etk, which belongs to the Tec family of tyrosine kinases, in the cross-talk between the integrin/FAK and the MyD88 pathways in fibroblast-like synoviocytes (FLS) and in IL-6 synthesis. Using small interfering RNA blockade, we report that Etk plays a major role in LPS- and protein I/II (a model activator of FAK)-dependent IL-6 release by activated FLS. Etk is associated with MyD88, FAK, and Mal as shown by coimmunoprecipitation. Interestingly, knockdown of Mal appreciably inhibited IL-6 synthesis in response to LPS and protein I/II. Our results also indicate that LPS and protein I/II induced phosphorylation of Etk and Mal in rheumatoid arthritis FLS via a FAK-dependent pathway. In conclusion, our data provide support that, in FLS, Etk and Mal are implicated in the cross-talk between FAK and MyD88 and that their being brought into play is clearly dependent on FAK.
Assuntos
Comunicação Celular/imunologia , Quinase 1 de Adesão Focal/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Proteínas da Mielina/fisiologia , Fator 88 de Diferenciação Mieloide/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteolipídeos/fisiologia , Transdução de Sinais/imunologia , Tirosina Quinase da Agamaglobulinemia , Proteínas de Bactérias/fisiologia , Fibroblastos/enzimologia , Fibroblastos/patologia , Quinase 1 de Adesão Focal/metabolismo , Humanos , Integrina alfa5/metabolismo , Integrina alfa5/fisiologia , Integrina beta1/metabolismo , Integrina beta1/fisiologia , Ativação Linfocitária/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Família Multigênica/imunologia , Proteínas da Mielina/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteolipídeos/metabolismo , Receptor Cross-Talk/imunologia , Membrana Sinovial/enzimologia , Membrana Sinovial/patologia , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/fisiologiaRESUMO
OBJECTIVE: It was recently demonstrated that synoviocytes (FLS) from rheumatoid arthritis (RA) patients express BAFF transcripts that are up-regulated by tumor necrosis factor alpha (TNFalpha) and interferon-gamma (IFNgamma). Thus, BAFF increases in RA target cells might be related to activation of the receptors of innate immunity. The purpose of this study was to determine whether ligands of Toll-like receptor 2 (TLR-2), TLR-4, TLR-9, and alpha5beta1 integrin are able to induce BAFF synthesis by RA FLS. METHODS: Quantitative reverse transcription-polymerase chain reaction analyses and enzyme-linked immunosorbent assays were performed to evaluate BAFF messenger RNA induction and BAFF release from FLS after stimulation by ligands for TLR-2, TLR-4, TLR-9, alpha5beta1 integrin (bacterial lipopeptide [BLP] palmitoyl-3-cysteine-serine-lysine-4, lipopolysaccharide [LPS], CpG, and protein I/II, respectively), TNFalpha, and IFNgamma. RESULTS: In contrast to IFNgamma, neither TNFalpha, LPS, BLP, nor CpG induced the de novo synthesis and release of BAFF by FLS. Priming of cells with IFNgamma did not have a synergistic effect on BAFF synthesis by FLS stimulated with bacterial products known as pathogen-associated molecular patterns. Moreover, we found that IFNgamma-induced BAFF synthesis is inhibited by simultaneous stimulation with either TLR ligands or TNFalpha. We also showed that interplay between TLRs, TNF receptors, and IFNgamma signaling induces the expression of suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 and reduces IFNgamma-dependent STAT-1 phosphorylation, which might explain this inhibition. In contrast, we demonstrated that stimulation of alpha5beta1 integrin can induce BAFF synthesis and release per se and that stimulation of this pathway has no inhibitory effect on IFNgamma-induced BAFF synthesis. CONCLUSION: Our findings indicate that BAFF secretion by resident cells in target organs of autoimmunity is tightly regulated by innate immunity, with positive and negative controls, depending on the receptors and the pathways triggered.