RESUMEN
T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.
Asunto(s)
Proteínas de Ciclo Celular , Metiltransferasas , Adenosina/análogos & derivados , Animales , Proteínas de Ciclo Celular/metabolismo , Metilación , Metiltransferasas/genética , Ratones , Factores de Empalme de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies.
Asunto(s)
Autoinmunidad , Citotoxicidad Inmunológica , Inmunoterapia Adoptiva , Melanoma Experimental/terapia , Proteínas Represoras/metabolismo , Ribonucleasas/metabolismo , Neoplasias Cutáneas/terapia , Linfocitos T/trasplante , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Femenino , Células HEK293 , Células HeLa , Humanos , Inmunidad Humoral , Masculino , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Fenotipo , Unión Proteica , Proteínas Represoras/genética , Ribonucleasas/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Microambiente Tumoral , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
ERI1 is a 3'-to-5' exoribonuclease involved in RNA metabolic pathways including 5.8S rRNA processing and turnover of histone mRNAs. Its biological and medical significance remain unclear. Here, we uncover a phenotypic dichotomy associated with bi-allelic ERI1 variants by reporting eight affected individuals from seven unrelated families. A severe spondyloepimetaphyseal dysplasia (SEMD) was identified in five affected individuals with missense variants but not in those with bi-allelic null variants, who showed mild intellectual disability and digital anomalies. The ERI1 missense variants cause a loss of the exoribonuclease activity, leading to defective trimming of the 5.8S rRNA 3' end and a decreased degradation of replication-dependent histone mRNAs. Affected-individual-derived induced pluripotent stem cells (iPSCs) showed impaired in vitro chondrogenesis with downregulation of genes regulating skeletal patterning. Our study establishes an entity previously unreported in OMIM and provides a model showing a more severe effect of missense alleles than null alleles within recessive genotypes, suggesting a key role of ERI1-mediated RNA metabolism in human skeletal patterning and chondrogenesis.
Asunto(s)
Exorribonucleasas , Histonas , Humanos , Exorribonucleasas/genética , Histonas/genética , Mutación Missense/genética , ARN Ribosómico 5.8S , ARN , ARN Mensajero/genéticaRESUMEN
Humoral autoimmunity paralleled by the accumulation of follicular helper T cells (T(FH) cells) is linked to mutation of the gene encoding the RNA-binding protein roquin-1. Here we found that T cells lacking roquin caused pathology in the lung and accumulated as cells of the T(H)17 subset of helper T cells in the lungs. Roquin inhibited T(H)17 cell differentiation and acted together with the endoribonuclease regnase-1 to repress target mRNA encoding the T(H)17 cell-promoting factors IL-6, ICOS, c-Rel, IRF4, IκBNS and IκBζ. This cooperation required binding of RNA by roquin and the nuclease activity of regnase-1. Upon recognition of antigen by the T cell antigen receptor (TCR), roquin and regnase-1 proteins were cleaved by the paracaspase MALT1. Thus, this pathway acts as a 'rheostat' by translating TCR signal strength via graded inactivation of post-transcriptional repressors and differential derepression of targets to enhance T(H)17 differentiation.
Asunto(s)
Caspasas/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Ribonucleasas/metabolismo , Células Th17/citología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/inmunología , Diferenciación Celular/inmunología , Línea Celular , Genes rel/genética , Células HEK293 , Humanos , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Factores Reguladores del Interferón/genética , Interleucina-6/genética , Péptidos y Proteínas de Señalización Intracelular , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas Nucleares/genética , Proteínas/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Células Th17/inmunología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17â¼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.
Asunto(s)
Colitis/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Proteínas Represoras/inmunología , Serina-Treonina Quinasas TOR/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Diferenciación Celular , Colitis/genética , Colitis/patología , Modelos Animales de Enfermedad , Femenino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/inmunología , Regulación de la Expresión Génica , Centro Germinal/inmunología , Centro Germinal/patología , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , MicroARNs/inmunología , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/inmunología , Fosfatidilinositol 3-Quinasas/genética , Cultivo Primario de Células , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Transducción de Señal , Bazo/inmunología , Bazo/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Serina-Treonina Quinasas TOR/genética , Células Th17/inmunología , Células Th17/patología , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Constitutive activation of the MALT1 paracaspase in conventional T cells of Malt1TBM/TBM (TRAF6 Binding Mutant = TBM) mice causes fatal inflammation and autoimmunity, but the involved targets and underlying molecular mechanisms are unknown. We genetically rendered a single MALT1 substrate, the RNA-binding protein (RBP) Roquin-1, insensitive to MALT1 cleavage. These Rc3h1Mins/Mins mice showed normal immune homeostasis. Combining Rc3h1Mins/Mins alleles with those encoding for constitutively active MALT1 (TBM) prevented spontaneous T cell activation and restored viability of Malt1TBM/TBM mice. Mechanistically, we show how antigen/MHC recognition is translated by MALT1 into Roquin cleavage and derepression of Roquin targets. Increasing T cell receptor (TCR) signals inactivated Roquin more effectively, and only high TCR strength enabled derepression of high-affinity targets to promote Th17 differentiation. Induction of experimental autoimmune encephalomyelitis (EAE) revealed increased cleavage of Roquin-1 in disease-associated Th17 compared to Th1 cells in the CNS. T cells from Rc3h1Mins/Mins mice did not efficiently induce the high-affinity Roquin-1 target IκBNS in response to TCR stimulation, showed reduced Th17 differentiation, and Rc3h1Mins/Mins mice were protected from EAE. These data demonstrate how TCR signaling and MALT1 activation utilize graded cleavage of Roquin to differentially regulate target mRNAs that control T cell activation and differentiation as well as the development of autoimmunity.
Asunto(s)
Autoinmunidad , Encefalomielitis Autoinmune Experimental , Ratones , Animales , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Inflamación/metabolismo , Diferenciación Celular , Encefalomielitis Autoinmune Experimental/genética , Receptores de Antígenos de Linfocitos T/genética , Ubiquitina-Proteína LigasasRESUMEN
According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2-16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8(+) T cell immunity.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Muromegalovirus/inmunología , Perforina/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Virus Vaccinia/inmunología , Vaccinia/inmunología , Animales , Señalización del Calcio , Comunicación Celular , Células Cultivadas , Citotoxicidad Inmunológica , Humanos , Evasión Inmune , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía de Fluorescencia por Excitación Multifotónica , Perforina/genética , Subgrupos de Linfocitos T/virología , Linfocitos T Citotóxicos/virologíaRESUMEN
The RNA-binding proteins Roquin-1/2 and Regnase-1 exert essential regulation by controlling pro-inflammatory mRNA expression to prevent autoimmune disease. More recently, inhibition of this post-transcriptional gene regulatory program has been demonstrated to enable enhanced anti-tumor responses by tumor antigen-specific CD8+ T cells. In this review, we describe the functions of these RNA-binding proteins and the phenotypes that arise in association with genetic inhibition or inactivation. We discuss how inducible inactivation of the system reprograms CD4+ and CD8+ T cell fates by changing cell metabolism, activation, differentiation or effector/memory decisions. We furthermore outline what we need to know to precisely modulate this system in order to dampen autoimmune reactions or boost the efficacy of adoptively transferred T cells or chimeric antigen receptor (CAR) T cells in cancer immunotherapies.
Asunto(s)
Enfermedades Autoinmunes , Regulación de la Expresión Génica , Humanos , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/terapia , Diferenciación Celular , Endorribonucleasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
During persistent antigen stimulation, CD8(+) T cells show a gradual decrease in effector function, referred to as exhaustion, which impairs responses in the setting of tumors and infections. Here we demonstrate that the transcription factor NFAT controls the program of T cell exhaustion. When expressed in cells, an engineered form of NFAT1 unable to interact with AP-1 transcription factors diminished T cell receptor (TCR) signaling, increased the expression of inhibitory cell surface receptors, and interfered with the ability of CD8(+) T cells to protect against Listeria infection and attenuate tumor growth in vivo. We defined the genomic regions occupied by endogenous and engineered NFAT1 in primary CD8(+) T cells and showed that genes directly induced by the engineered NFAT1 overlapped with genes expressed in exhausted CD8(+) T cells in vivo. Our data show that NFAT promotes T cell anergy and exhaustion by binding at sites that do not require cooperation with AP-1.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Anergia Clonal/genética , Factores de Transcripción NFATC/fisiología , Proteínas Recombinantes/farmacología , Factor de Transcripción AP-1/metabolismo , Animales , Células Cultivadas , Anergia Clonal/efectos de los fármacos , Regulación de la Expresión Génica/genética , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Listeriosis/microbiología , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Factores de Transcripción NFATC/genética , Neoplasias/inmunología , Regiones Promotoras Genéticas/genética , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes/genéticaRESUMEN
Viral infection induces a number of cytokines that shape T cell responses. In this issue of Immunity, Ray et al. (2014) describe how CD4(+) T cells decide on T follicular helper (Tfh) or T helper 1 (Th1) cell skewed gene expression during acute viral infection.
Asunto(s)
Diferenciación Celular , Interferón Tipo I/metabolismo , Factor de Transcripción STAT3/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , AnimalesRESUMEN
Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m6A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m1A, m5C, m7G, 2'-OMe, and Ψ were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abundance of the modification, particularly on mRNAs, generated data sets might resemble noise rather than specific modification patterns. Therefore, it is critical that the affinity and specificity is rigorously tested using complementary approaches. Here, we provide an experimental toolbox that allows for testing antibody performance prior to their use.
Asunto(s)
Anticuerpos/genética , Ribonucleótidos/genética , Nucleótidos/genética , ARN/genética , ARN Mensajero/genéticaRESUMEN
The molecular mechanism by which roquin controls the expression of inducible costimulator (ICOS) to prevent autoimmunity remains unsolved. Here we show that in helper T cells, roquin localized to processing (P) bodies and downregulated ICOS expression. The repression was dependent on the RNA helicase Rck, and roquin interacted with Rck and the enhancer of decapping Edc4, which act together in mRNA decapping. Sequences in roquin that confer P-body localization were essential for roquin-mediated ICOS repression. However, this process did not require microRNAs or the RNA-induced silencing complex (RISC). Instead, roquin bound ICOS mRNA directly, showing an intrinsic preference for a previously unrecognized sequence in the 3' untranslated region (3' UTR). Our results support a model in which roquin controls ICOS expression through binding to the 3' UTR of ICOS mRNA and by interacting with proteins that confer post-transcriptional repression.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , ARN Helicasas DEAD-box/inmunología , MicroARNs/genética , Proteínas Proto-Oncogénicas/inmunología , ARN Mensajero/metabolismo , Transcripción Genética , Ubiquitina-Proteína Ligasas/metabolismo , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación de Linfocitos T/genética , Autoinmunidad/genética , Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , ARN Helicasas DEAD-box/genética , Regulación de la Expresión Génica , Proteína Coestimuladora de Linfocitos T Inducibles , Ratones , Ratones Mutantes , Ratones Transgénicos , MicroARNs/inmunología , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , ARN Mensajero/inmunología , Ubiquitina-Proteína Ligasas/inmunologíaRESUMEN
The Roquin-1 protein binds to messenger RNAs (mRNAs) and regulates gene expression posttranscriptionally. A single point mutation in Roquin-1, but not gene ablation, increases follicular helper T (Tfh) cell numbers and causes lupus-like autoimmune disease in mice. In T cells, we did not identify a unique role for the much lower expressed paralog Roquin-2. However, combined ablation of both genes induced accumulation of T cells with an effector and follicular helper phenotype. We showed that Roquin-1 and Roquin-2 proteins redundantly repressed the mRNA of inducible costimulator (Icos) and identified the Ox40 costimulatory receptor as another shared mRNA target. Combined acute deletion increased Ox40 signaling, as well as Irf4 expression, and imposed Tfh differentiation on CD4(+) T cells. These data imply that both proteins maintain tolerance by preventing inappropriate T cell activation and Tfh cell differentiation, and that Roquin-2 compensates in the absence of Roquin-1, but not in the presence of its mutated form.
Asunto(s)
Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , ARN Mensajero/metabolismo , Receptores OX40/metabolismo , Proteínas Represoras/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Antígenos CD4/metabolismo , Diferenciación Celular/genética , Células HEK293 , Humanos , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Unión Proteica , Receptores OX40/genética , Proteínas Represoras/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The expression of proteins during inflammatory and immune reactions is coordinated by post-transcriptional mechanisms. A particularly strong suppression of protein expression is exerted by a conserved translational silencing element (TSE) identified in the 3' UTR of NFKBIZ mRNA, which is among the targets of the RNA-binding proteins Roquin-1/2 and MCPIP1/Regnase-1. We present evidence that in the context of the TSE MCPIP1, so far known for its endonuclease activity toward mRNAs specified by distinct stem-loop (SL) structures, also suppresses translation. Overexpression of MCPIP1 silenced translation in a TSE-dependent manner and reduced ribosome occupancy of the mRNA. Correspondingly, MCPIP1 depletion alleviated silencing and increased polysomal association of the mRNA. Translationally silenced NFKBIZ or reporter mRNAs were mostly capped, polyadenylated and ribosome associated. Furthermore, MCPIP1 silenced also cap-independent, CrPV-IRES-dependent translation. This suggests that MCPIP1 suppresses a post-initiation step. The TSE is predicted to form five SL structures. SL4 and 5 resemble target structures reported for MCPIP1 and together were sufficient for MCPIP1 binding and mRNA destabilization. Translational silencing, however, required SL1-3 in addition. Thus the NFKBIZ TSE functions as an RNA element in which sequences adjacent to the site of interaction with MCPIP1 and dispensable for accelerated mRNA degradation extend the functional repertoire of MCPIP1 to translational silencing.
Asunto(s)
Silenciador del Gen , Proteínas I-kappa B/genética , Proteínas Nucleares/genética , Biosíntesis de Proteínas , Secuencias Reguladoras de Ácido Ribonucleico , Ribonucleasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Sitios de Unión , Células HeLa , Humanos , Extensión de la Cadena Peptídica de Translación , Dominios Proteicos , ARN Mensajero/metabolismo , Receptor EphB3 , Ribonucleasas/química , Ribosomas/metabolismo , Factores de Transcripción/químicaRESUMEN
Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4(+) T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T-cell-expressed miRNAs in naive mouse CD4(+) T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR-100, miR-99a and miR-10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR-99a cooperated with miR-150 to repress the expression of the Th17-promoting factor mTOR. The comparably low expression of miR-99a was strongly increased by the Treg cell inducer "retinoic acid", and the abundantly expressed miR-150 could only repress Mtor in the presence of miR-99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs.
Asunto(s)
MicroARNs/genética , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/fisiología , Serina-Treonina Quinasas TOR/genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/fisiología , Diferenciación Celular/genética , Células Cultivadas , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Tretinoina/farmacologíaRESUMEN
Treatment for fibrosis represents a critical unmet need, because fibrosis is the leading cause of death in industrialized countries, and there is no effective therapy to counteract the fibrotic process. The development of fibrosis relates to the interplay between vessel injury, immune cell activation, and fibroblast stimulation, which can occur in various tissues. Immunotherapies have provided a breakthrough in the treatment of immune diseases. The glycoprotein OX40-OX40 ligand (OX40L) axis offers the advantage of a targeted approach to costimulatory signals with limited impact on the whole immune response. Using systemic sclerosis (SSc) as a prototypic disease, we report compelling evidence that blockade of OX40L is a promising strategy for the treatment of inflammation-driven fibrosis. OX40L is overexpressed in the fibrotic skin and serum of patients with SSc, particularly in patients with diffuse cutaneous forms. Soluble OX40L was identified as a promising serum biomarker to predict the worsening of lung and skin fibrosis, highlighting the role of this pathway in fibrosis. In vivo, OX40L blockade prevents inflammation-driven skin, lung, and vessel fibrosis and induces the regression of established dermal fibrosis in different complementary mouse models. OX40L exerts potent profibrotic effects by promoting the infiltration of inflammatory cells into lesional tissues and therefore the release of proinflammatory mediators, thereafter leading to fibroblast activation.
Asunto(s)
Ligando OX40/antagonistas & inhibidores , Ligando OX40/sangre , Fibrosis Pulmonar/prevención & control , Esclerodermia Sistémica/sangre , Piel/patología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Biomarcadores/sangre , Bleomicina , Estudios de Casos y Controles , Células Cultivadas , Evaluación Preclínica de Medicamentos , Fibrosis , Antígeno 2 Relacionado con Fos/genética , Humanos , Hipertensión Pulmonar/prevención & control , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Terapia Molecular Dirigida , Fibrosis Pulmonar/genética , Esclerodermia Sistémica/tratamiento farmacológico , Piel/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Post-transcriptional gene regulation by RNA-binding proteins is a fast and effective way to adapt gene expression and change cellular responses. These trans-acting factors have been involved in a number of cell fate decisions, and their mutation is often associated with the development of disease. The RNA-binding protein Roquin-1 has been found to be crucial in the maintenance of peripheral tolerance and the prevention of autoimmune disease. This review describes the molecular role of Roquin family proteins in the control of follicular T-helper cell differentiation. Here, we discuss the redundant regulation of Icos and Ox40 costimulatory receptor mRNAs by Roquin-1 and Roquin-2 proteins. A major focus is placed on the distinct activity of Roquin-1 or Roquin-2 proteins in the mouse models of conditional gene targeting. These recent data are then integrated into an interpretation of altered Roquin protein function in the sanroque mouse that expresses the Roquin-1 protein with just one amino acid substitution and, different from the Roquin-1-deficient mouse, develops lupus-like autoimmune disease.
Asunto(s)
Diferenciación Celular , Lupus Eritematoso Sistémico/inmunología , Proteínas Represoras/fisiología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Transactivadores/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Tolerancia Inmunológica/genética , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/terapia , Ratones , Ratones Noqueados , Terapia Molecular Dirigida , Mutación/genética , Ligando OX40/metabolismo , Proteínas Represoras/genética , Linfocitos T Colaboradores-Inductores/inmunología , Transactivadores/genética , Transactivadores/inmunología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The Carma1-Bcl10-Malt1 (CBM) complex bridges T-cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF-κB pathway. NF-κB activation is triggered by PKCθ-dependent phosphorylation of Carma1 after TCR/CD28 co-stimulation. PKCθ-phosphorylated Carma1 was suggested to function as a molecular scaffold that recruits preassembled Bcl10-Malt1 complexes to the membrane. We have identified the serine-threonine protein phosphatase PP2A regulatory subunit Aα (PPP2R1A) as a novel interaction partner of Carma1. PPP2R1A is associated with Carma1 in resting as well as activated T cells in the context of the active CBM complex. By siRNA-mediated knockdown and in vitro dephosphorylation, we demonstrate that PP2A removes PKCθ-dependent phosphorylation of Ser645 in Carma1, and show that maintenance of this phosphorylation is correlated with increased T-cell activation. As a result of PP2A inactivation, we find that enhanced Carma1 S645 phosphorylation augments CBM complex formation, NF-κB activation and IL-2 or IFN-γ production after stimulation of Jurkat T cells or murine Th1 cells. Thus, our data define PP2A-mediated dephosphorylation of Carma1 as a critical step to limit T-cell activation and effector cytokine production.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Linfocitos T CD4-Positivos/fisiología , Guanilato Ciclasa/metabolismo , Activación de Linfocitos/fisiología , Complejos Multiproteicos/metabolismo , FN-kappa B/metabolismo , Proteína Fosfatasa 2/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Cartilla de ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Guanilato Ciclasa/genética , Células HEK293 , Humanos , Inmunoprecipitación , Células Jurkat , Luciferasas , Ratones , Ratones Transgénicos , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción GenéticaAsunto(s)
Inflamación/inmunología , FN-kappa B/metabolismo , Proteínas Represoras/fisiología , Transducción de Señal/inmunología , Ubiquitina-Proteína Ligasas/fisiología , Ubiquitina/metabolismo , Animales , Proteínas de Unión al ADN , Regulación hacia Abajo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Represoras/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3' extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3' processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3' rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.