RESUMO
The transcription factor ThPOK (encoded by the Zbtb7b gene) controls homeostasis and differentiation of mature helper T cells, while opposing their differentiation to CD4+ intraepithelial lymphocytes (IELs) in the intestinal mucosa. Thus CD4 IEL differentiation requires ThPOK transcriptional repression via reactivation of the ThPOK transcriptional silencer element (SilThPOK). In the present study, we describe a new autoregulatory loop whereby ThPOK binds to the SilThPOK to maintain its own long-term expression in CD4 T cells. Disruption of this loop in vivo prevents persistent ThPOK expression, leads to genome-wide changes in chromatin accessibility and derepresses the colonic regulatory T (Treg) cell gene expression signature. This promotes selective differentiation of naive CD4 T cells into GITRloPD-1loCD25lo (Triplelo) Treg cells and conversion to CD4+ IELs in the gut, thereby providing dominant protection from colitis. Hence, the ThPOK autoregulatory loop represents a key mechanism to physiologically control ThPOK expression and T cell differentiation in the gut, with potential therapeutic relevance.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Linfócitos Intraepiteliais/citologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Reguladores/citologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/imunologia , Colite/imunologia , Colite/prevenção & controle , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
Repression of peroxisome proliferator-activated receptor γ (PPARγ)-dependent transcription by the nuclear receptor corepressor (NCoR) is important for homeostatic expression of PPARγ target genes in vivo. The current model states that NCoR-mediated repression requires its direct interaction with PPARγ in the repressive conformation. Previous studies, however, have shown that DNA-bound PPARγ is incompatible with a direct, high-affinity association with NCoR because of the inherent ability of PPARγ to adopt the active conformation. Here we show that NCoR acquires the ability to repress active PPARγ-mediated transcription via G protein pathway suppressor 2 (GPS2), a component of the NCoR corepressor complex. Unlike NCoR, GPS2 can recognize and bind the active state of PPARγ. In GPS2-deficient mouse embryonic fibroblast cells, loss of GPS2 markedly reduces the corepressor function of NCoR for PPARγ, leading to constitutive activation of PPARγ target genes and spontaneous adipogenesis of the cells. GPS2, however, is dispensable for repression mediated by unliganded thyroid hormone receptor α or a PPARγ mutant unable to adopt the active conformation. This study shows that GPS2, although dispensable for the intrinsic repression function of NCoR, can mediate a novel corepressor repression pathway that allows NCoR to directly repress active PPARγ-mediated transcription, which is important for the optimal corepressor function of NCoR for PPARγ. Interestingly, GPS2-dependent repression specifically targets PPARγ but not PPARα or PPARδ. Therefore, GPS2 may serve as a unique target to manipulate PPARγ signaling in diseases.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , PPAR gama/genética , Ativação Transcricional , Adipogenia , Sequência de Aminoácidos , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Dados de Sequência Molecular , Mutação , Correpressor 1 de Receptor Nuclear/genética , PPAR gama/química , PPAR gama/metabolismo , Ligação Proteica , Conformação Proteica , Receptores alfa dos Hormônios Tireóideos/metabolismoRESUMO
How multipotential cells initiate distinct gene expression programs in response to external cues to instruct cell fate choice remains a fundamental question in biology. Establishment of CD4 and CD8 T cell fates during thymocyte development is critically regulated by T cell receptor (TCR) signals, which in turn control expression of the CD4-determining transcription factor ThPOK. However, the mechanism whereby differential TCR signals are molecularly interpreted to promote or antagonize ThPOK expression, and thereby CD4 versus CD8 lineage fates remains unknown. Here we show, using reverse genetic and molecular approaches that an autonomous, position-independent TCR-sensing switch is embedded within the ThPOK locus. Further, using an in vivo mutagenesis approach, we demonstrate that differential TCR signals are interpreted during lineage commitment by relative binding of EGR, NFAT and Ebox factors to this bistable switch. Collectively our study reveals the central molecular mechanism whereby TCR signaling influences differential lineage choice. Ultimately, these findings may provide an important new tool for skewing T cell fate to treat cancer and autoimmune diseases.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Proteínas de Homeodomínio/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Anticorpos Monoclonais , Técnicas Biossensoriais , Regulação da Expressão Gênica/fisiologia , Inativação Gênica , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Most ribosomal proteins (RP) are regarded as essential, static components that contribute only to ribosome biogenesis and protein synthesis. However, emerging evidence suggests that RNA-binding RP are dynamic and can influence cellular processes by performing "extraribosomal," regulatory functions involving binding to select critical target mRNAs. We report here that the RP, Rpl22, and its highly homologous paralog Rpl22-Like1 (Rpl22l1 or Like1) play critical, extraribosomal roles in embryogenesis. Indeed, they antagonistically control morphogenesis through developmentally regulated localization to the nucleus, where they modulate splicing of the pre-mRNA encoding smad2, an essential transcriptional effector of Nodal/TGF-ß signaling. During gastrulation, Rpl22 binds to intronic sequences of smad2 pre-mRNA and induces exon 9 skipping in cooperation with hnRNP-A1. This action is opposed by its paralog, Like1, which promotes exon 9 inclusion in the mature transcript. The nuclear roles of these RP in controlling morphogenesis represent a fundamentally different and paradigm-shifting mode of action for RP.
Assuntos
Morfogênese , Precursores de RNA/genética , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Embrião não Mamífero/metabolismo , Éxons/genética , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Camundongos Endogâmicos C57BL , Morfogênese/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Proteína Smad2/metabolismo , Frações Subcelulares/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Tumor necrosis factor (TNF) is an important cytokine with multiple biological effects, including cell growth, differentiation, apoptosis, immune regulation and induction of inflammation. The effects of TNF are mediated by two receptors, TNF-R1 and TNF-R2. The major signal transduction pathways triggered by TNF include those that lead to apoptosis, activation of transcription factor NF-kappa B and protein kinase JNK. This review will discuss the molecular mechanisms of these signaling pathways.
RESUMO
T-cell responses may be shaped by sterile "danger signals" that are constituted by damage-associated molecular patterns (DAMP). However, whether and what type of adaptive immune responses are triggered in vivo by DAMPs induced by tumor progression are not well characterized. In this study, we report that the production of HMGB1, an established DAMP released by dying cells, was critical for tumor progression in an established mouse model of prostate cancer. HMGB1 was required for the activation and intratumoral accumulation of T cells that expressed cytokine lymphotoxinα(1)ß(2) (LT) on their surface. Intriguingly, these tumor-activated T cells recruited macrophages to the lesion and were essential to promote the preneoplasia to invasive carcinoma in an LTß receptor (LTßR)-dependent manner. Taken together, our findings suggest that the release of HMGB1 as an endogenous danger signal is important for priming an adaptive immune response that promotes malignant progression, with implications for cancer prevention and therapy.
Assuntos
Imunidade Adaptativa , Progressão da Doença , Proteína HMGB1/metabolismo , Lesões Pré-Cancerosas/patologia , Neoplasias da Próstata/imunologia , Linfócitos T/imunologia , Animais , Humanos , Linfotoxina-beta/metabolismo , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Transgênicos , Transdução de SinaisRESUMO
Tumor necrosis factor receptor-associated factor 6 (TRAF6) associates with the cytoplasmic domain of receptor activator of NF-kappaB (RANK) and is an essential component of the signaling complex mediating osteoclastogenesis. However, the osteoclastic activity of TRAF6 is blunted by its association with four and half LIM domain 2 (FHL2), which functions as an adaptor protein in the cytoplasm and transcriptional regulator in the nucleus. We find that TRAF6 also localizes in the nuclei of osteoclasts but not their bone marrow macrophage precursors and that osteoclast intranuclear abundance is specifically increased by RANK ligand (RANKL). TRAF6 nuclear localization requires FHL2 and is diminished in fhl2(-/-) osteoclasts. Suggesting transcriptional activity, TRAF6 interacts with the transcription factor RUNX1 in the osteoclast nucleus. FHL2 also associates with RUNX1 but does so only in the presence of TRAF6. Importantly, TRAF6 recognizes FHL2 and RUNX1 in osteoclast nuclei, and the three molecules form a DNA-binding complex that recognizes and transactivates the RUNX1 response element in the fhl2 promoter. Finally, TRAF6 and its proximal activator, RANKL, polyubiquitinate FHL2, prompting its proteasomal degradation. These observations suggest a feedback mechanism whereby TRAF6 negatively regulates osteoclast formation by intracytoplasmic sequestration of FHL2 to blunt RANK activation and as a component of a transcription complex promoting FHL2 expression.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Musculares/metabolismo , Osteoclastos/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Animais , Células COS , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlorocebus aethiops , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Homeodomínio LIM , Camundongos , Células Precursoras de Monócitos e Macrófagos/citologia , Células Precursoras de Monócitos e Macrófagos/metabolismo , Complexos Multiproteicos/genética , Proteínas Musculares/genética , Osteoclastos/citologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Elementos de Resposta/fisiologia , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/genética , Fatores de Transcrição/genética , Ubiquitinação/fisiologiaRESUMO
IFN regulatory factor-3 is a transcription factor that is required for the rapid induction of type I IFNs in the innate antiviral response. Two noncanonical IkappaB kinase (IKK) family members, IKKepsilon and TRAF family-associated NF-kappaB activator-binding kinase-1, have been shown to phosphorylate IFN regulatory factor-3 and are critically involved in virus-triggered and TLR3-mediated signaling leading to induction of type I IFNs. In yeast two-hybrid screens for potential IKKepsilon-interacting proteins, we identified Ret finger protein (RFP) as an IKKepsilon-interacting protein. Coimmunoprecipitation experiments indicated that RFP interacted with IKKepsilon and TRAF family-associated NF-kappaB activator-binding kinase-1 as well as the two canonical IKK family members, IKKbeta and IKKalpha. RFP inhibited activation of the IFN-stimulated response element and/or NF-kappaB mediated by the IKK family members and triggered by TNF, IL-1, polyinosinic-polycytidylic acid (ligand for TLR3), and viral infection. Moreover, knockdown of RFP expression by RNA interference-enhanced activation of IFN-stimulated response element and/or NF-kappaB triggered by polyinosinic-polycytidylic acid, TNF, and IL-1. Taken together, our findings suggest that RFP negatively regulates signaling involved in the antiviral response and inflammation by targeting the IKKs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Quinase I-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Humanos , Técnicas In Vitro , Fator Regulador 3 de Interferon/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Interferência de RNA , Vírus Sendai/patogenicidade , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
Members of the RIP serine/threonine kinase family are involved in activation of NF-kappaB, JNK, and p38, and induction of apoptosis. Here we report the identification of a novel RIP-homologous protein designated as RIP5. The C-terminus of RIP5 contains a kinase domain, which is mostly homologous with the kinase domain of RIP. RIP5 also contains a large unconserved N-terminal domain. Overexpression of RIP5 induces cell death with characteristic apoptotic morphology. Overexpression of RIP5 also induces DNA fragmentation and this is blocked by the caspase inhibitor crmA. However, RIP5-induced apoptotic morphology is not blocked by crmA. These findings suggest that RIP5 may induce both caspase-dependent apoptosis and caspase-independent cell death.