RESUMO
The zinc finger protein 36-like 2, ZFP36L2, is a member of a small family of RNA-binding proteins composed by ZFP36 (also known as tristetraprolin, TTP), ZFP36L1 and ZFP36L2 in humans, with corresponding murine orthologs. These proteins bind to adenine uridine-rich element (ARE) in the 3'untranslated region of target messenger RNA and stimulate target degradation. ZFP36 functions as an anti-inflammatory modulator in murine models of inflammatory diseases by down-regulating the production of inflammatory cytokines such as tumor necrosis factor-α. However, how ZFP36L1 and ZFP36L2 alter the function of CD4+ T cells is not completely understood. We addressed this issue by searching for the target genes of ZFP36L2 by comprehensive transcriptome analysis. We observed that ZFP36L2 is highly expressed in naïve CD4+ T cells; however, when CD4+ T cells are stimulated through their T cell receptors, ZFP36L2 expression is rapidly reduced in both humans and mice. Among CD4+ T cell populations, the expression levels of ZFP36L2 in regulatory T cells (Tregs) were significantly lower than those in naïve or effector CD4+ T cells. RNA-sequence analysis revealed that the forced expression of ZFP36L2 decreased Ikzf2 (encoding Helios) expression in Foxp3+ Tregs and inhibited the ability of induced Tregs (iTregs). ZFP36L2 directly bound to and destabilized the 3'untranslated region of Ikzf2 mRNA, which contains AU-rich elements. These results indicate that ZFP36L2 reduces the expression of Ikzf2 and suppresses iTreg function, raising the interesting possibility that the inhibition of ZFP36L2 in iTregs could be a therapeutic strategy for autoimmune diseases.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica/imunologia , Fator de Transcrição Ikaros/biossíntese , Linfócitos T Reguladores/imunologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/imunologia , Tristetraprolina/imunologia , Animais , Proteínas de Ligação a DNA/imunologia , Regulação para Baixo , Humanos , Fator de Transcrição Ikaros/imunologia , Camundongos , Fatores de Transcrição/metabolismo , Tristetraprolina/metabolismoRESUMO
BACKGROUND: Intestinal inflammation and epithelial injury are the leading actors of inflammatory bowel disease (IBD), causing an excessive pro-inflammatory cytokines expression. Tristetraprolin (TTP), an mRNA binding protein, plays a role in regulating the inflammatory factors, recognizing specific sequences on the 3' untranslated region of cytokine mRNAs. TTP activity depends on its phosphorylation state: the unphosphorylated TTP degrades pro-inflammatory cytokine mRNAs; on the contrary, the phosphorylated TTP fails to destabilize mRNAs furthering their expression. The phospho-TTP forms a complex with the chaperone protein 14-3-3. This binding could be one of the factors that promote intestinal inflammation as a cause of disease progression. AIM: To assess if TTP phosphorylation has a role in paediatric IBD. METHODS: The study was carried out on a cohort of paediatric IBD patients. For each patient enrolled, a specimen of inflamed and non-inflamed colonic mucosa was collected. Furthermore, the experiments were conducted on macrophages differentiated from blood samples of the same patients. Macrophages from healthy donors' blood were used as controls. Co-immunoprecipitation assay and immunoblotting analyses were performed to observe the formation of the phospho-TTP/14-3-3 complex. In the same samples TNF-α expression was also evaluated as major factor of the pro-inflammatory activity. RESULTS: In this work we studied indirectly the phosphorylation of TTP through the binding with the chaperone protein 14-3-3. In inflamed and non-inflamed colon mucosa of IBD paediatric patients immunoblot assay demonstrated a higher expression of the TTP in inflamed samples respect to the non-inflamed; the co-immunoprecipitated 14-3-3 protein showed the same trend of expression. In the TNF-α gene expression analysis higher levels of the cytokine in inflamed tissues compared to controls were evident. The same experiments were conducted on macrophages from IBD paediatric patients and healthy controls. The immunoblot results demonstrated a high expression of both TTP and co-immunoprecipitated 14-4-3 protein in IBD-derived macrophages in comparison to healthy donors. TNF-α protein levels from macrophages lysates showed the same trend of expression in favour of IBD paediatric patients compared to healthy controls. CONCLUSION: In this work, for the first time, we describe a relation between phospho-TTP/14-3-3 complex and IBD. Indeed, a higher expression of TTP/14-3-3 was recorded in IBD samples in comparison to controls.
Assuntos
Colite Ulcerativa/patologia , Doença de Crohn/patologia , Mucosa Intestinal/patologia , Tristetraprolina/metabolismo , Proteínas 14-3-3/imunologia , Proteínas 14-3-3/metabolismo , Biópsia , Criança , Estudos de Coortes , Colite Ulcerativa/imunologia , Colo/imunologia , Colo/patologia , Colonoscopia , Doença de Crohn/imunologia , Humanos , Mucosa Intestinal/imunologia , Masculino , Fosforilação/imunologia , Tristetraprolina/imunologiaRESUMO
The present study was undertaken to determine whether extracellular calreticulin (CRT) participates in the regulation of ICAM-1in rheumatoid arthritis (RA) and further explore the potential mechanism. Our results showed that ICAM-1 and VCAM-1 levels were positively correlated with CRT levels in RA serum and synovial fluid, respectively. In RA synovial tissue, increased co-expressions of CRT and ICAM-1 in vascular endothelium and perivascular areas and elevated co-location of CRT and VCAM-1 localized predominantly to lining layer were observed compared to those in OA. In in vitro HUVECs model, enhanced ICAM-1expression and increased phosphorylation levels of Akt and eNOS were detected in the presence of CRT. Increased phosphorylated eNOS was significantly inhibited by a PI3K inhibitor LY294002 and elevated ICAM-1expression was partially blocked by the inhibitors of both PI3K and eNOS (L-NAME). It has been certified that the RNA-binding protein TTP targets AU-rich elements in the ICAM-1 3'-UTR and suppresses ICAM-1 expression. Knocking down TTP in HUVECs led to an increased induction of ICAM-1 by CRT. We have currently known that activation of p38 downstream kinase MK-2 leads to phosphorylation and inactivation of human TTP. The block of p38 MAPK/MK-2 signaling led to decreased protein expression and mRNA stability of TTP and ICAM-1. Furthermore, L-NAME and/or LY294002 pre-treated HUVECs manifested decreased p38 and MK-2 phosphorylation, which was accompanied by reduced TTP and ICAM-1 protein expression as well as decreased mRNA stability. Our results suggested that CRT could promote ICAM-1 expression in endothelial cells through PI3K/Akt/eNOS/p38 MAPK signaling mediated TTP accumulation, probably in an inactive form, which may provide a possible proinflammatory mechanism of CRT in RA.
Assuntos
Artrite Reumatoide/imunologia , Calreticulina/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Tristetraprolina/imunologia , Regulação para Cima/imunologia , Artrite Reumatoide/patologia , Cromonas/farmacologia , Feminino , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Morfolinas/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/imunologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Tristetraprolin (TTP) is an RNA-binding protein that targets numerous immunomodulatory mRNA transcripts for degradation. Many TTP targets are key players in the pathogenesis of periodontal bone loss, including tumor necrosis factor-α. To better understand the extent that host immune factors play during periodontal bone loss, we assessed alveolar bone levels, inflammation and osteoclast activity in periodontal tissues, and immune response in draining cervical lymph nodes in TTP-deficient and wild-type (WT) mice in an aging study. WT and TTP-deficient (knockout [KO]) mice were used for all studies under specific pathogen-free conditions. Data were collected on mice aged 3, 6, and 9 mo. Microcomputed tomography (µCT) was performed on maxillae where 3-dimensional images were generated and bone loss was assessed. Decalcified sections of specimens were scored for inflammation and stained with tartrate-resistant acid phosphate (TRAP) to visualize osteoclasts. Immunophenotyping was performed on single-cell suspensions isolated from primary and peripheral lymphoid tissues using flow cytometry. Results presented indicate that TTP KO mice had significantly more alveolar bone loss over time compared with WT controls. Bone loss was associated with significant increases in inflammatory cell infiltration and an increased percentage of alveolar bone surfaces apposed with TRAP+ cells. Furthermore, it was found that the draining cervical lymph nodes were significantly enlarged in TTP-deficient animals and contained a distinct pathological immune profile compared with WT controls. Finally, the oral microbiome in the TTP KO mice was significantly different with age from WT cohoused mice. The severe bone loss, inflammation, and increased osteoclast activity observed in these mice support the concept that TTP plays a critical role in the maintenance of alveolar bone homeostasis in the presence of oral commensal flora. This study suggests that TTP is required to inhibit excessive inflammatory host responses that contribute to periodontal bone loss, even in the absence of specific periodontal pathogens.
Assuntos
Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/imunologia , Tristetraprolina/imunologia , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Homeostase/imunologia , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Knockout , Osteoclastos/metabolismo , Fenótipo , Organismos Livres de Patógenos Específicos , Tristetraprolina/deficiência , Microtomografia por Raio-XRESUMO
Obesity is associated with adipose tissue inflammation that contributes to insulin resistance. Zinc finger protein 36 (Zfp36) is an mRNA-binding protein that reduces inflammation by binding to cytokine transcripts and promoting their degradation. We hypothesized that myeloid-specific deficiency of Zfp36 would lead to increased adipose tissue inflammation and reduced insulin sensitivity in diet-induced obese mice. As expected, wild-type (Control) mice became obese and diabetic on a high-fat diet, and obese mice with myeloid-specific loss of Zfp36 [knockout (KO)] demonstrated increased adipose tissue and liver cytokine mRNA expression compared with Control mice. Unexpectedly, in glucose tolerance testing and hyperinsulinemic-euglycemic clamp studies, myeloid Zfp36 KO mice demonstrated improved insulin sensitivity compared with Control mice. Obese KO and Control mice had similar macrophage infiltration of the adipose depots and similar peripheral cytokine levels, but lean and obese KO mice demonstrated increased Kupffer cell (KC; the hepatic macrophage)-expressed Mac2 compared with lean Control mice. Insulin resistance in obese Control mice was associated with enhanced Zfp36 expression in KCs. Compared with Control mice, KO mice demonstrated increased hepatic mRNA expression of a multitude of classical (M1) inflammatory cytokines/chemokines, and this M1-inflammatory hepatic milieu was associated with enhanced nuclear localization of IKKß and the p65 subunit of NF-κB. Our data confirm the important role of innate immune cells in regulating hepatic insulin sensitivity and lipid metabolism, challenge-prevailing models in which M1 inflammatory responses predict insulin resistance, and indicate that myeloid-expressed Zfp36 modulates the response to insulin in mice.
Assuntos
Tecido Adiposo/metabolismo , Citocinas/genética , Fígado Gorduroso/genética , Inflamação/genética , Resistência à Insulina/genética , Obesidade/genética , Tristetraprolina/genética , Tecido Adiposo/imunologia , Tecido Adiposo/patologia , Animais , Citocinas/imunologia , Citocinas/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/imunologia , Diabetes Mellitus/metabolismo , Dieta Hiperlipídica , Fígado Gorduroso/imunologia , Fígado Gorduroso/metabolismo , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Células de Kupffer/imunologia , Células de Kupffer/metabolismo , Camundongos , Camundongos Knockout , Células Mieloides/metabolismo , Obesidade/imunologia , Obesidade/metabolismo , Tamanho do Órgão , RNA Mensageiro/metabolismo , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismo , Tristetraprolina/imunologia , Tristetraprolina/metabolismoRESUMO
The immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3' UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers.
Assuntos
Antígeno B7-H1/imunologia , Neoplasias Colorretais/imunologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/imunologia , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Tristetraprolina/imunologia , Evasão Tumoral , Animais , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Clivagem do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Transdução de Sinais , Tristetraprolina/genéticaRESUMO
RNA-binding protein tristetraprolin (TTP) plays a fundamental role in various physiological and pathological processes including differentiation, reprogramming, metabolism, proliferation, pluripotency, tumorigenesis and immunity. Due to its ability to bind and target ARE-containing mRNAs for rapid degradation, TTP down-regulates the expression of a mass of critical genes, thereby functioning as cancer suppressor gene. The loss of TTP has been reported in several human cancers and is relevant to poor prognosis. Recent research shows that TTP also has an emerging significant role in immunity. The aim of this paper is to provide an overview of various roles of TTP in human cancers and immunity. We summarize TTP deficiency in several cancers and discuss that the lack of TTP can influence tumor progression at different aspects such as promoting cancer cell proliferation; accelerating cell cycle; improving survivability and resisting cell death; inducing angiogenesis; activating invasion and metastasis; inducing epithelial-mesenchymal transition; and deregulating cellular energetics. We also pay attention to novel understanding of the relationship between TTP and immunity. Finally, due to its vital role, the disorder of TTP in both cancer and immune cells receives increasing attention and we overview current thinking about regulatory mechanisms of TTP itself expression. This knowledge may contribute to TTP becoming a diagnostic marker for cancer or immune-related diseases and a possible therapeutic target.
Assuntos
Neoplasias/patologia , Tristetraprolina/imunologia , Tristetraprolina/metabolismo , Animais , Proliferação de Células , Citocinas/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Hematopoese , Humanos , Neoplasias/genética , Neovascularização Patológica/genética , Biossíntese de Proteínas , Estabilidade de RNA , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Tristetraprolina/genéticaRESUMO
In myeloid cells, the mRNA-destabilizing protein tristetraprolin (TTP) is induced and extensively phosphorylated in response to LPS. To investigate the role of two specific phosphorylations, at serines 52 and 178, we created a mouse strain in which those residues were replaced by nonphosphorylatable alanine residues. The mutant form of TTP was constitutively degraded by the proteasome and therefore expressed at low levels, yet it functioned as a potent mRNA destabilizing factor and inhibitor of the expression of many inflammatory mediators. Mice expressing only the mutant form of TTP were healthy and fertile, and their systemic inflammatory responses to LPS were strongly attenuated. Adaptive immune responses and protection against infection by Salmonella typhimurium were spared. A single allele encoding the mutant form of TTP was sufficient for enhanced mRNA degradation and underexpression of inflammatory mediators. Therefore, the equilibrium between unphosphorylated and phosphorylated TTP is a critical determinant of the inflammatory response, and manipulation of this equilibrium may be a means of treating inflammatory pathologies.
Assuntos
Macrófagos/imunologia , Mutação , RNA Mensageiro/imunologia , Salmonelose Animal/imunologia , Tristetraprolina/imunologia , Alanina/genética , Alanina/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Citocinas/antagonistas & inibidores , Citocinas/genética , Citocinas/imunologia , Feminino , Expressão Gênica , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosforilação , Cultura Primária de Células , Estabilidade de RNA , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Salmonelose Animal/genética , Salmonelose Animal/patologia , Salmonella typhimurium/imunologia , Serina/genética , Serina/metabolismo , Tristetraprolina/genéticaRESUMO
Dual-specificity phosphatase (DUSP) 1 dephosphorylates and inactivates members of the MAPK superfamily, in particular, JNKs, p38α, and p38ß MAPKs. It functions as an essential negative regulator of innate immune responses, hence disruption of the Dusp1 gene renders mice extremely sensitive to a wide variety of experimental inflammatory challenges. The principal mechanisms behind the overexpression of inflammatory mediators by Dusp1(-/-) cells are not known. In this study, we use a genetic approach to identify an important mechanism of action of DUSP1, involving the modulation of the activity of the mRNA-destabilizing protein tristetraprolin. This mechanism is key to the control of essential early mediators of inflammation, TNF, CXCL1, and CXCL2, as well as the anti-inflammatory cytokine IL-10. The same mechanism also contributes to the regulation of a large number of transcripts induced by treatment of macrophages with LPS. These findings demonstrate that modulation of the phosphorylation status of tristetraprolin is an important physiological mechanism by which innate immune responses can be controlled.
Assuntos
Fosfatase 1 de Especificidade Dupla/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , RNA Mensageiro/imunologia , Tristetraprolina/imunologia , Animais , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Quimiocina CXCL2/genética , Quimiocina CXCL2/imunologia , Fosfatase 1 de Especificidade Dupla/genética , Regulação da Expressão Gênica , Imunidade Inata , Interleucina-10/genética , Interleucina-10/imunologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 11 Ativada por Mitógeno/genética , Proteína Quinase 11 Ativada por Mitógeno/imunologia , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/imunologia , Fosforilação , Cultura Primária de Células , Estabilidade de RNA , RNA Mensageiro/genética , Transdução de Sinais , Tristetraprolina/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Macrophages play important roles in many diseases and are frequently found in hypoxic areas. A chronic hypoxic microenvironment alters global cellular protein expression, but molecular details remain poorly understood. Although hypoxia-inducible factor (HIF) is an established transcription factor allowing adaption to acute hypoxia, responses to chronic hypoxia are more complex. Based on a two-dimensional differential gel electrophoresis (2D-DIGE) approach, we aimed to identify proteins that are exclusively expressed under chronic but not acute hypoxia (1% O2). One of the identified proteins was cathepsin B (CTSB), and a knockdown of either HIF-1α or -2α in primary human macrophages pointed to an HIF-2α dependency. Although chromatin immunoprecipitation (ChIP) experiments confirmed HIF-2 binding to a CTSB enhancer in acute hypoxia, an increase of CTSB mRNA was evident only under chronic hypoxia. Along those lines, CTSB mRNA stability increased at 48 h but not at 8 h of hypoxia. However, RNA stability at 8 h of hypoxia was enhanced by a knockdown of tristetraprolin (TTP). Inactivation of TTP under prolonged hypoxia was facilitated by c-Jun N-terminal kinase (JNK), and inhibition of this kinase lowered CTSB mRNA levels and stability. We postulate a TTP-dependent mechanism to explain delayed expression of CTSB under chronic hypoxia.
Assuntos
Catepsina B/metabolismo , Hipóxia Celular/genética , Macrófagos/metabolismo , Estabilidade de RNA/genética , Tristetraprolina/metabolismo , Catepsina B/imunologia , Linhagem Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Tristetraprolina/imunologiaRESUMO
Regulatory RNA binding proteins allow for specific control of gene expression in a very dynamic manner. In mammals ZFP36, formerly known as Tristetraprolin, controls the inflammatory response by binding to an AU-rich element located in the 3' untranslated region of its target mRNAs. The developping embryo relies on a population of primitive macrophages to ensure proper immunity. Although the role of zfp36 in adult immunity has been extensively studied, its expression in the developing immune system has been poorly documented. Here, we have used whole mount in situ hybridization with a 3' UTR specific probe to address the expression of zfp36 in developing Xenopus tropicalis embryos. We have shown that zfp36 is expressed in two distinct cellular populations. First, it is a new marker of primititive myeloid cells, being coexpressed with the myeloid marker mpo. Therefore this early expression may suggest a role for zfp36 in macrophage differentiation and activation. In addition, a second cell population was found to transiently express zfp36, but not mpo, along the fusing neural folds and may correspond to cells undergoing autophagy during neural tube closure.
Assuntos
Regiões 3' não Traduzidas/genética , Células Mieloides/metabolismo , Crista Neural/metabolismo , Tubo Neural/embriologia , Tristetraprolina/biossíntese , Animais , Autofagia/genética , Regulação da Expressão Gênica/genética , Fator Estimulador de Colônias de Granulócitos/biossíntese , Inflamação/imunologia , Interleucina-3/biossíntese , Ativação de Macrófagos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/biossíntese , Tristetraprolina/imunologia , Xenopus/embriologia , Xenopus/metabolismoRESUMO
Nicotine stimulates the cholinergic anti-inflammatory pathway and prevents excessive inflammation by inhibiting the release of inflammatory cytokines from macrophages. We have previously reported that heme oxygenase-1 (HO-1) and tristetraprolin (TTP) are induced by nicotine and mediate the anti-inflammatory function of nicotine in macrophages. However, it was not clear whether the two molecules are functionally linked. In this study, we sought to determine whether HO-1 associates with TTP to mediate the anti-inflammatory effects of nicotine. Inhibition of HO-1 activity or HO-1 expression attenuated the effects of nicotine on STAT3 activation, TTP induction, and TNF-α production in LPS-treated macrophages. Induction of HO-1 expression increased the level of TTP in the absence of nicotine. In an LPS-induced endotoxemia model, HO-1 deficiency blocked the effects of nicotine on the STAT3 phosphorylation, TTP induction, and LPS-induced TNF-α production in the liver. Downregulation of STAT3 by siRNA attenuated the effect of nicotine on TTP expression and TNF-α production but did not affect the nicotine-mediated induction of HO-1. In TTP knockout mice, nicotine treatment enhanced HO-1 expression and STAT3 activation but failed to inhibit LPS-induced TNF-α production. Our results suggest that HO-1 and TTP are functionally linked in mediating the anti-inflammatory effects of nicotine; HO-1 is necessary for the induction of TTP by nicotine. This novel nicotine-HO-1-TTP signaling pathway provides new possibilities for the treatment of inflammatory diseases.
Assuntos
Heme Oxigenase-1/imunologia , Inflamação/tratamento farmacológico , Nicotina/farmacologia , Fator de Transcrição STAT3/imunologia , Tristetraprolina/imunologia , Animais , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citocinas/imunologia , Citocinas/metabolismo , Endotoxemia/imunologia , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Inflamação/imunologia , Lipopolissacarídeos , Fígado/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Nicotina/efeitos adversos , Nicotina/imunologia , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Tristetraprolina/genética , Fator de Necrose Tumoral alfa/biossínteseRESUMO
B cell function with age is decreased in class switch recombination (CSR), activation-induced cytidine deaminase (AID), and stability of E47 mRNA. The latter is regulated, at least in part, by tristetraprolin (TTP), which is increased in aged B cells and also negatively regulates TNF-α. In this study, we investigated whether B cells produce TNF-α, whether this changes with age, and how this affects their function upon stimulation. Our hypothesis is that in aging there is a feedback mechanism of autocrine inflammatory cytokines (TNF-α) that lowers the expression of AID and CSR. Our results showed that unstimulated B cells from old BALB/c mice make significantly more TNF-α mRNA and protein than do B cells from young mice, but after stimulation the old make less than the young; thus, they are refractory to stimulation. The increase in TNF-α made by old B cells is primarily due to follicular, but not minor, subsets of B cells. Incubation of B cells with TNF-α before LPS stimulation decreased both young and old B cell responses. Importantly, B cell function was restored by adding anti-TNF-α Ab to cultured B cells. To address a molecular mechanism, we found that incubation of B cells with TNF-α before LPS stimulation induced TTP, a physiological regulator of mRNA stability of the transcription factor E47, which is crucial for CSR. Finally, anti-TNF-α given in vivo increased B cell function in old, but not in young, follicular B cells. These results suggest new molecular mechanisms that contribute to reduced Ab responses in aging.
Assuntos
Envelhecimento/imunologia , Comunicação Autócrina/imunologia , Linfócitos B/imunologia , Regulação para Baixo/imunologia , Fator de Necrose Tumoral alfa/imunologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Comunicação Autócrina/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/patologia , Células Cultivadas , Citidina Desaminase/biossíntese , Citidina Desaminase/imunologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Switching de Imunoglobulina/efeitos dos fármacos , Switching de Imunoglobulina/imunologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/imunologia , Fator 3 de Transcrição/imunologia , Fator 3 de Transcrição/metabolismo , Tristetraprolina/biossíntese , Tristetraprolina/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
For a successful yet controlled immune response, cells need to specifically destabilize inflammatory mRNAs but prevent premature removal of those still used. The regulatory circuits controlling quality and timing in the global inflammatory mRNA decay are not understood. Here, we show that the mRNA-destabilizing function of the AU-rich element-binding protein tristetraprolin (TTP) is inversely regulated by the p38 MAPK activity profile such that after inflammatory stimulus the TTP-dependent decay is initially limited to few mRNAs. With time, the TTP-dependent decay gradually spreads resulting in cumulative elimination of one third of inflammation-induced unstable mRNAs in macrophages in vitro. We confirmed this sequential decay model in vivo since LPS-treated mice with myeloid TTP ablation exhibited similar cytokine dysregulation profile as macrophages. The mice were hypersensitive to LPS but otherwise healthy with no signs of hyperinflammation seen in conventional TTP knockout mice demonstrating the requirement for myeloid TTP in re-installment but not maintenance of immune homeostasis. These findings reveal a TTP- and p38 MAPK-dominated regulatory mechanism that is vital for balancing acute inflammation by a temporally and qualitatively controlled mRNA decay.
Assuntos
Inflamação/genética , Estabilidade de RNA/imunologia , Tristetraprolina/imunologia , Animais , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Inflamação/imunologia , Lipopolissacarídeos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estabilidade de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma , Tristetraprolina/genética , Tristetraprolina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Zinc finger protein tristetraprolin (TTP) modulates macrophage inflammatory activity by destabilizing cytokine mRNAs. In this study, through a screen of TTP-bound mRNAs in activated human macrophages, we have identified CCL3 mRNA as the most abundantly bound TTP target mRNA and have characterized this interaction via conserved AU-rich elements. Compared to the wild-type cells, TTP(-/-) macrophages produced higher levels of LPS-induced CCL3. In addition, the plasma level of CCL3 in TTP(-/-) mice was markedly higher than that in wild-type mice. To determine the in vivo significance of TTP-regulated CCL3, we generated CCL3(-/-)TTP(-/-) double-knockout mice. Along with decreased proinflammatory cytokines in their paw joints, there were significant functional and histologic improvements in the inflammatory arthritis of TTP(-/-) mice when CCL3 was absent, although cachexia, reflecting systemic inflammation, was notably unaffected. Furthermore, the marked exacerbation of aortic plaque formation caused by TTP deficiency in the APOE(-/-) mouse model of atherosclerosis was also rescued by disrupting CCL3. Taken together, our data indicate that the interaction between TTP and CCL3 mRNA plays an important role in modulating localized inflammatory processes in tissues that are dissociated from the systemic manifestations of chronic inflammation.
Assuntos
Quimiocina CCL3/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Tristetraprolina/metabolismo , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Sequência de Bases , Quimiocina CCL3/genética , Quimiocina CCL3/imunologia , Feminino , Humanos , Imunoprecipitação , Inflamação/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Tristetraprolina/imunologiaRESUMO
Tristetraprolin (TTP) is a well-characterized, zinc finger-containing, RNA-binding protein. TTP targets tumor necrosis factor α for degradation via the 3' untranslated region (3'UTR). Although AU-rich elements (AREs) in the 3'UTR of interleukin-6 (IL-6) mRNA dictate mRNA degradation, the role of TTP in the post-transcriptional regulation of IL-6 gene expression is unclear. Here we used TTP-deficient mice to test the hypothesis that IL-6 expression is influenced by TTP. Genetic and siRNA-mediated knockdown of TTP resulted in increased IL-6 production and overexpression of TTP had the reverse effect. IL-6 and tumor necrosis factor α production were elevated after injection of IL-1ß in TTP-deficient mice. Further, embryonic fibroblasts from these mice (mouse embryonic fibroblasts) exhibited greater IL-6 mRNA expression and longer half-life than wild-type mouse embryonic fibroblasts. Overexpression of TTP reduced IL-6 3'UTR luciferase reporter activity in an ARE-dependent manner. Proximal and distal regions of the 3'UTR acted synergistically to produce the full repression of TTP. Mutation-based luciferase assays show that ARE2, ARE3, and ARE4 are required for TTP-mediated repression. The constitutively activated p38-MK2 pathway abrogated TTP-mediated repression of IL-6 3'UTR reporter activity. RNA immunoprecipitation assay indicated that the deficiency of p38α resulted in the increased affinity of TTP to IL-6 mRNA. Taken together, we propose that TTP downregulates IL-6 gene expression at the post-transcriptional level by targeting ARE elements in the 3'UTR region.
Assuntos
Fibroblastos/metabolismo , Interleucina-6/metabolismo , Tristetraprolina/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Células Cultivadas , Fibroblastos/imunologia , Fibroblastos/patologia , Interleucina-6/genética , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Mutação/genética , Ligação Proteica/genética , Processamento Pós-Transcricional do RNA/genética , RNA Interferente Pequeno/genética , Transgenes/genética , Tristetraprolina/genética , Tristetraprolina/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
ZFP36L1 and ZFP36L2 are RNA-binding proteins (RBPs) that interact with AU-rich elements in the 3' untranslated region of mRNA, which leads to mRNA degradation and translational repression. Here we show that mice that lacked ZFP36L1 and ZFP36L2 during thymopoiesis developed a T cell acute lymphoblastic leukemia (T-ALL) dependent on the oncogenic transcription factor Notch1. Before the onset of T-ALL, thymic development was perturbed, with accumulation of cells that had passed through the beta-selection checkpoint without first expressing the T cell antigen receptor beta-chain (TCRbeta). Notch1 expression was higher in untransformed thymocytes in the absence of ZFP36L1 and ZFP36L2. Both RBPs interacted with evolutionarily conserved AU-rich elements in the 3' untranslated region of Notch1 and suppressed its expression. Our data establish a role for ZFP36L1 and ZFP36L2 during thymocyte development and in the prevention of malignant transformation.
Assuntos
Proteínas Nucleares/deficiência , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Linfócitos T/imunologia , Timo/imunologia , Tristetraprolina/deficiência , Sequência de Aminoácidos , Animais , Fator 1 de Resposta a Butirato , Sequência Conservada , Humanos , Imunofenotipagem , Estimativa de Kaplan-Meier , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptor Notch1/genética , Receptor Notch1/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Alinhamento de Sequência , Timo/crescimento & desenvolvimento , Transcrição Gênica , Tristetraprolina/genética , Tristetraprolina/imunologiaRESUMO
Dendritic cells (DCs) serve to maintain peripheral tolerance under steady state conditions. Upon triggering by activation signals they initiate strong immune responses. The activation of DCs is accompanied by a rapid upregulation of proinflammatory cytokines, which were shown in other cell types to be regulated by mechanisms at the transcriptional and posttranscriptional level. Tristetraprolin (TTP), an important RNA binding protein, is involved in the regulation of mRNA stability of such cytokines. In this study we analyzed the significance of TTP for mouse DCs, which were derived from TTP(-/-) and WT bone marrow progenitor cells (BM-DCs). Unstimulated BM-DCs of TTP(-/-) mice expressed lower levels of mRNAs encoding the costimulatory molecules CD40 and CD86 and surprisingly also the canonical TTP targets TNF-alpha and IL-10 as compared with WT DCs. On the protein level, both DC populations expressed comparable amounts of CD80 and CD86 and of either cytokine, but TTP(-/-) DCs expressed less MHCII than WT DCs. On the other hand, TTP(-/-) DCs displayed elevated expression of other TTP target mRNAs like IL-1beta, c-fos and Mkp-1. Stimulation of BM-DCs of either genotype with lipopolysaccharide resulted in a rapid upregulation to a comparable extent of all molecules monitored so far, except for c-fos mRNA. Subsequent mRNA decay analysis revealed gene-specific differences in mRNA stability, which was influenced by the presence of TTP and the activation state of the DCs. Unstimulated TTP(-/-) DCs exerted a markedly lower allogeneic T cell stimulatory potential than WT DCs. Moreover, TTP(-/-) DCs induced an altered cytokine pattern in cocultures of DCs and T cells. However, allogeneic T cells primed by unstimulated DCs of either genotype were equally refractory to restimulation and suppressed the proliferation of naive T cells to the same extent. Thus, the findings of this study lend support to the interpretation that without external stimulation antigen presenting activity in DCs in the presence of TTP is more pronounced than in its absence and that posttranscriptional regulation contributes to the control of gene expression in DCs.
Assuntos
Células Dendríticas/imunologia , Estabilidade de RNA/imunologia , RNA Mensageiro/imunologia , Proteínas de Ligação a RNA/imunologia , Tristetraprolina/imunologia , Animais , Antígeno B7-2/biossíntese , Antígeno B7-2/imunologia , Antígenos CD40/biossíntese , Antígenos CD40/imunologia , Células Dendríticas/metabolismo , Fosfatase 1 de Especificidade Dupla/biossíntese , Fosfatase 1 de Especificidade Dupla/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/imunologia , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/imunologia , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/genética , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Tristetraprolina/genética , Tristetraprolina/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
The potential of some proinflammatory mediators to inhibit gp130-dependent STAT3 activation by enhancing suppressor of cytokine signaling (SOCS) 3 expression represents an important molecular mechanism admitting the modulation of the cellular response toward gp130-mediated signals. Thus, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by proinflammatory mediators. In this study, we investigate SOCS3 expression initiated by the proinflammatory cytokine TNF-alpha. In contrast to IL-6, TNF-alpha increases SOCS3 expression by stabilizing SOCS3 mRNA. Activation of the MAPK kinase 6 (MKK6)/p38(MAPK)-cascade is required for TNF-alpha-mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression. In fibroblasts or macrophages deficient for MAPK-activated protein kinase 2 (MK2), a downstream target of the MKK6/p38(MAPK) cascade, basal SOCS3-expression is strongly reduced and TNF-alpha-induced SOCS3-mRNA stabilization is impaired, indicating that MK2 is crucial for the control of SOCS3 expression by p38(MAPK)-dependent signals. As a target for SOCS3 mRNA stability-regulating signals, a region containing three copies of a pentameric AUUUA motif in close proximity to a U-rich region located between positions 2422 and 2541 of the 3' untranslated region of SOCS3 is identified. One factor that could target this region is the zinc finger protein tristetraprolin (TTP), which is shown to be capable of destabilizing SOCS3 mRNA via this region. However, data from TTP-deficient cells suggest that TTP does not play an irreplaceable role in the regulation of SOCS3 mRNA stability by TNF-alpha. In summary, these data indicate that TNF-alpha regulates SOCS3 expression on the level of mRNA stability via activation of the MKK6/p38(MAPK) cascade and that the activation of MK2, a downstream target of p38(MAPK), is important for the regulation of SOCS3 expression.
Assuntos
Regulação da Expressão Gênica/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro/biossíntese , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Fator de Necrose Tumoral alfa/imunologia , Células 3T3 , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase 6/imunologia , MAP Quinase Quinase 6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/imunologia , Camundongos , Proteínas Quinases/imunologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/imunologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/imunologia , Tristetraprolina/imunologia , Tristetraprolina/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Interferons (IFNs) are cytokines with pronounced proinflammatory properties. Here we provide evidence that IFNs also play a key role in decline of inflammation by inducing expression of tristetraprolin (Ttp). TTP is an RNA-binding protein that destabilizes several AU-rich element-containing mRNAs including TNFalpha. By promoting mRNA decay, TTP significantly contributes to cytokine homeostasis. Now we report that IFNs strongly stimulate expression of TTP if a costimulatory stress signal is provided. IFN-induced expression of Ttp depends on the IFN-activated transcription factor STAT1, and the costimulatory stress signal requires p38 MAPK. Within the Ttp promoter we have identified a functional gamma interferon-activated sequence that recruits STAT1. Consistently, STAT1 is required for full expression of Ttp in response to LPS that stimulates both p38 MAPK and, indirectly, interferon signaling. We demonstrate that in macrophages IFN-induced TTP protein limits LPS-stimulated expression of several proinflammatory genes, such as TNFalpha, IL-6, Ccl2, and Ccl3. Thus, our findings establish a link between interferon responses and TTP-mediated mRNA decay during inflammation, and propose a novel immunomodulatory role of IFNs.