RESUMEN
IκBζ (encoded by the Nfkbiz) is a member of the nuclear IκB family, which is involved in the expression of secondary response genes based on signals from TLR or IL-1R. ST2L, an IL-33R, is a member of the IL-1R family and abundantly expressed in tissue-resident immune cells, such as mast cells and innate lymphoid cells; however, its downstream signaling pathway remains unelucidated. In this study, we examined the role of IκBζ in ST2L-mediated cytokine and chemokine production in mast cells. Murine bone marrow cells were differentiated ex vivo into bone marrow-derived mast cells (BMMCs). The treatment of BMMCs with IL-33 transiently induced robust IκBζ expression. Of the 40 cytokines and chemokines examined using a cytokine and chemokine array, the concentrations of IL-6, IL-13, CCL2, CCL3, and TNF-α in the supernatant were augmented by IL-33. The deletion of IκBζ in BMMCs resulted in a significant reduction of the production of these mediators and the expression of their mRNA. NF-κB p50 but not p65 translocated to the nucleus by IL-33 and was not affected by the deletion of IκBζ. However, induction of IκBζ and the resultant cytokine and chemokine productions were significantly inhibited by pretreatment with an NF-κB inhibitor. The deletion of IκBζ did not affect the phosphorylation of ERK, p38 MAPK, or JNK by IL-33, and the treatment with inhibitors of these mitogen-activated kinases failed to abolish the expression of Nfkbiz Our findings suggest that IκBζ augments IL-33-dependent cytokine and chemokine production in BMMCs through the action of NF-κB.
Asunto(s)
Citocinas/biosíntesis , Proteínas I-kappa B/metabolismo , Interleucina-33/inmunología , Mastocitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Citocinas/inmunología , Proteínas I-kappa B/deficiencia , Mastocitos/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/metabolismoRESUMEN
The translation of capsid proteins of Plautia stali intestine virus (PSIV), encoded in its second open reading frame (ORF2), is directed by an internal ribosomal entry site (IRES) located in the intergenic region (IGR). Owing to the specific properties of PSIV IGR in terms of nucleotide length and frame organization, capsid proteins are also translated via stop codon readthrough in mammalian cultured cells as an extension of translation from the first ORF (ORF1) and IGR. To delineate stop codon readthrough in PSIV, we determined requirements of cis-acting elements through a molecular genetics approach applied in both cell-free translation systems and cultured cells. Mutants with deletions from the 3' end of IGR revealed that almost none of the sequence of IGR is necessary for readthrough, apart from the 5'-terminal codon CUA. Nucleotide replacement of this CUA trinucleotide or change of the termination codon from UGA severely impaired readthrough. Chemical mapping of the IGR region of the most active 3' deletion mutant indicated that this defined minimal element UGACUA, together with its downstream sequence, adopts a single-stranded conformation. Stimulatory activities of downstream RNA structures identified to date in gammaretrovirus, coltivirus, and alphavirus were not detected in the context of PSIV IGR, despite the presence of structures for IRES. To our knowledge, PSIV IGR is the first example of stop codon readthrough that is solely defined by the local hexamer sequence, even though the sequence is adjacent to an established region of RNA secondary/tertiary structures.
Asunto(s)
Codón de Terminación , Dicistroviridae/genética , ARN Viral/genética , Animales , Secuencia de Bases , Células COS , Sistema Libre de Células , Chlorocebus aethiops , ADN Intergénico , Células HEK293 , Células HeLa , Humanos , Sitios Internos de Entrada al Ribosoma , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Viral/química , Eliminación de SecuenciaRESUMEN
Objective- Inhibition of HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is atheroprotective primarily by decreasing plasma LDL (low-density lipoprotein)-cholesterol. However, it is unknown whether inhibition of HMGCR in myeloid cells contributes to this atheroprotection. We sought to determine the role of myeloid HMGCR in the development of atherosclerosis. Approach and Results- We generated mice with genetically reduced Hmgcr in myeloid cells ( Hmgcr m-/m-) using LysM (Cre) and compared various functions of their macrophages to those of Hmgcr fl/fl control mice. We further compared the extent of atherosclerosis in Hmgcr m-/ m- and Hmgcr fl/fl mice in the absence of Ldlr (LDL receptor). Hmgcr m-/ m- macrophages and granulocytes had significantly lower Hmgcr mRNA expression and cholesterol biosynthesis than Hmgcr fl/fl cells. In vitro, Hmgcr m-/ m- monocytes/macrophages had reduced ability to migrate, proliferate, and survive compared with Hmgcr fl/fl monocytes/macrophages. However, there was no difference in ability to adhere, phagocytose, store lipids, or polarize to M1 macrophages between the 2 types of macrophages. The amounts of plasma membrane-associated small GTPase proteins, such as RhoA (RAS homolog family member A), were increased in Hmgcr m-/ m- macrophages. In the setting of Ldlr deficiency, Hmgcr m-/ m- mice developed significantly smaller atherosclerotic lesions than Hmgcr fl/fl mice. However, there were no differences between the 2 types of mice either in plasma lipoprotein profiles or in the numbers of proliferating or apoptotic cells in the lesions in vivo. The in vivo migration of Hmgcr m-/ m- macrophages to the lesions was reduced compared with Hmgcr fl/fl macrophages. Conclusions- Genetic reduction of HMGCR in myeloid cells may exert atheroprotective effects primarily by decreasing the migratory activity of monocytes/macrophages to the lesions.
Asunto(s)
Aorta/enzimología , Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Movimiento Celular , Hidroximetilglutaril-CoA Reductasas/metabolismo , Macrófagos Peritoneales/enzimología , Monocitos/enzimología , Traslado Adoptivo , Animales , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Hidroximetilglutaril-CoA Reductasas/genética , Lípidos/sangre , Macrófagos Peritoneales/patología , Macrófagos Peritoneales/trasplante , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/patología , Proteínas de Unión al GTP Monoméricas/metabolismo , Fenotipo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de SeñalRESUMEN
The interleukin-33 (IL-33)-ST2L signaling pathway has been shown to play important roles in the field of immunology, especially as a trigger for allergic reactions such as bronchial asthma. However, coming back to the original finding that the ST2 gene is induced during initiation of the cell cycle of fibroblastic cell lines, the possible functions of the ST2 gene products and their specific ligand, IL-33, in the field of cell growth regulation are still interesting problems to be solved. In this study, we used NIH-3T3 mouse cell line and added IL-33 before and after cell proliferation assay, which revealed the dual function of IL-33. When IL-33 was added to the confluent cells before the start of cell proliferation, it suppressed the cell growth concentration-dependently. On the other hand, if IL-33 was added after the start of cell proliferation, it enhanced the cell growth. The negative effect of IL-33 on cell proliferation is a novel finding and would provide an important clue to the roles of IL-33 and ST2/ST2L in growth regulation.
Asunto(s)
Proliferación Celular , Interleucinas/farmacología , Interleucinas/fisiología , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Interleucina-33 , Ligandos , Ratones , Células 3T3 NIH , Proteínas Recombinantes/farmacologíaRESUMEN
Interleukin-33 (IL-33) is a dual-function molecule that regulates gene expression in nuclei and, as a cytokine, conveys proinflammatory signals from outside of cells via its specific receptor ST2L. There are still a lot of questions about localization and processing of IL-33 gene products. In the course of re-evaluating human IL-33 gene, we found distinct promoter usage depending on the cell type, similar to the case in the ST2 gene. Furthermore, we found a novel exon 2E in the conventional intron 2 whose open reading frame corresponded to a transmembrane protein of 131 amino acids. Dependence of exon 2E expression on differentiation of HUVEC cells is of great interest in relation to human IL-33 function.
Asunto(s)
Exones/genética , Interleucinas/genética , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interleucina-33 , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Regiones Promotoras GenéticasRESUMEN
Nitrogen-containing bisphosphonates (NCBPs) have been widely used as standard supportive therapy to reduce skeletal-related events (SREs) in myeloma patients through suppression of osteoclast activity. In various prospective randomized trials that were performed following preliminary reports concerning efficacy, NCBPs have shown a significant beneficial effect on myeloma bone disease through both suppression of bone resorption and direct antimyeloma activity. Thus, NCBPs have an influence on many types of human cells. In this study, we examined the effect of an NCBP (YM-175) on an apoptosis of a monocytic cell line and of human native monocytes/macrophages and dendritic cells (DCs). We confirmed that monocytes, monocyte-derived macrophages, DCs, and a monoblastic cell line (THP-1) showed dose-dependent and time-dependent apoptosis related to the activation of caspases after exposure to YM-175 at concentrations below that at which the apoptosis of myeloma cell lines was induced. Such apoptosis of monocytic cells was suppressed by the addition of farnesol or geranylgeraniol. These findings suggest that the inhibition of monocyte-lineage cells or DCs by NCBPs might interfere with phagocytic activity or pathogen-presenting activity.
Asunto(s)
Apoptosis/efectos de los fármacos , Difosfonatos/farmacología , Monocitos/efectos de los fármacos , Prenilación/efectos de los fármacos , Conservadores de la Densidad Ósea/farmacología , Línea Celular Tumoral , Humanos , Monocitos/citología , Monocitos/metabolismo , Mieloma MúltipleRESUMEN
Soluble ST2 (sST2) is a soluble form of the transmembrane receptor for interleukin (IL)-33, ST2L, and is a member of the IL-1 receptor family. sST2 antagonizes IL-33-ST2L signaling by competing with ST2L as a decoy receptor for IL-33. We investigated the sST2 and IL-33 levels in the sera and bullous fluid of bullous pemphigoid patients and compared these with the corresponding levels in normal healthy controls. As controls, we used the bullous fluid of burn patients and that from suction blisters induced in normal healthy volunteers. The serum sST2 concentrations of bullous pemphigoid patients were higher than those of healthy controls. Serum sST2 levels correlated with the area of skin involvement and serum lactate dehydrogenase levels, suggesting that serum sST2 levels reflect disease severity. The sST2 concentrations in bullous fluid from bullous pemphigoid patients were higher than those from controls. The concentration of IL-33 ligand was below the detectable limits in all enzyme-linked immunosorbent assay samples. Thus, our study suggested that the serum sST2 level may be a useful marker of disease severity and that sST2 functions as a negative regulator in the pathophysiology of bullous pemphigoid.
Asunto(s)
Interleucinas/sangre , Penfigoide Ampolloso/sangre , Receptores de Superficie Celular/sangre , Anciano , Anciano de 80 o más Años , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , L-Lactato Deshidrogenasa/sangre , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Penfigoide Ampolloso/fisiopatologíaRESUMEN
Although TNF inhibitors have dramatically improved the outcome of patients with rheumatoid arthritis, 30-40% of patients do not respond well to them and treatment needs to be changed. In an effort to discriminate good and poor responders, we focused on the change in serum and synovial fluid levels of interleukin (IL-) 33 before and after treatment with TNF inhibitors. They were also measured in synovial fluids from 17 TNF inhibitor-naïve patients, and fibroblast-like synoviocytes (FLS) in-culture from 6 patients and correlated with various pro-inflammatory cytokines. Serum levels of IL-33 at 6 months after treatment decreased significantly in responders, while they did not change in non-responders. Synovial fluid levels of IL-33 in 6 patients under treatment with TNF inhibitors stayed high in 3 who were refractory and slightly elevated in 2 moderate responders, while they were undetectable in one patient under remission. Among inflammatory cytokines measured in 17 synovial fluids from TNF inhibitor-naïve patients, levels of IL-33 showed a significant positive correlation only to those of IL-1ß. IL-1ß increased IL-33 expression markedly in FLS in vitro, compared to TNF-α. IL-1ß might be inducing RA inflammation through producing pro-inflammatory IL-33 in TNF inhibitor-hypo-responders. Sustained elevation of serum and/or synovial levels of IL-33 may account for a poor response to TNF inhibitors, although how TNF inhibitors affect the level of IL-33 remains to be elucidated.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Fibroblastos/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Interleucinas/metabolismo , Líquido Sinovial/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adolescente , Adulto , Anciano , Artritis Reumatoide/sangre , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Biomarcadores/metabolismo , Células Cultivadas , Femenino , Humanos , Mediadores de Inflamación/sangre , Interleucina-33 , Interleucinas/sangre , Interleucinas/genética , Japón , Masculino , Persona de Mediana Edad , Transducción de Señal , Factores de Tiempo , Insuficiencia del Tratamiento , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Adulto JovenRESUMEN
BACKGROUND: IL-33 is a dual-functional molecule; it acts as a cytokine to enhance type 2 inflammation, and as a nuclear factor. The roles of nuclear IL-33 are not yet fully understood. OBJECTIVE: We aimed to investigate the role of IL-33 in normal human epidermal keratinocytes (NHEKs). METHODS: We utilized RNA interference to knock down cellular IL-33. RESULTS: The IL-33-knockdown (KD) cells showed decreased BrdU incorporation and decreasing tendency in RhoA activity and decreased ECT2 oncogene expression, compared to the controls. Supplementation of IL-33 expression utilizing adenovirus vector recovered the BrdU incorporation in IL-33-KD cells. Increased number of G2/M phase cells and binucleated cells were observed among the KD cells. Overtime observation revealed that IL-33-KD cells could not divide properly, formed binucleated cells, and were less motile than control cells. CONCLUSION: IL-33 KD in NHEKs affected the division and motility, probably by slightly decreasing the RhoA activity by attenuating ECT2 expression.
Asunto(s)
Movimiento Celular , Citocinesis , Interleucina-33 , Queratinocitos , Células Cultivadas , Epidermis/metabolismo , Humanos , Interleucina-33/metabolismo , Queratinocitos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína de Unión al GTP rhoARESUMEN
IL-33, a member of the IL-1 family, activates MAPK and NF-kappaB through its receptor ST2L and IL-1RAcP. ST2, a member of the IL-1R superfamily, is a secreted form of ST2 gene products, which has been shown to act as a decoy receptor for IL-33 and to inhibit the IL-33/ST2L/IL-1RAcP signaling pathway. In this work, we generated ST2 transgenic mice. In control mice, intraperitoneal administration of IL-33 caused an increased number of eosinophils in blood and in peritoneal cavity, an increased number of peritoneal M Phi, splenomegaly, accumulation of periodic acid-Schiff-positive material in the lung, and high concentrations of serum IL-5 and IL-13. However, these alterations were hardly detectable in ST2 Tg mice. In peritoneal M Phi from IL-33-stimulated mice, mRNA expression of M2 M Phi marker genes were increased compared with thioglycollate-elicited peritoneal M Phi. The IL-33-stimulation also increased the secretion of IL-6 from M Phi. However, when the IL-33 was preincubated with ST2 prior to its addition to the M Phi cultures, the secretion of IL-6 was attenuated. These data suggest that, though IL-33 induced the Th2-type immune responses and infiltration of M2 type M Phi into the peritoneal cavity, ST2 can downregulate these reactions both in vivo and in vitro.
Asunto(s)
Eosinófilos/metabolismo , Interleucinas/administración & dosificación , Macrófagos Peritoneales/metabolismo , Ratones Transgénicos , Receptores de Interleucina/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Eosinófilos/patología , Femenino , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-13/biosíntesis , Interleucina-13/sangre , Interleucina-13/genética , Interleucina-33 , Interleucina-5/biosíntesis , Interleucina-5/sangre , Interleucina-5/genética , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Ratones , Ratones Endogámicos C3H , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Esplenomegalia/genética , Células Th2/inmunologíaRESUMEN
ST2 gene products that are members of IL-1 receptor family are expressed in various cells such as growth-stimulated fibroblasts and Th2 helper T-cells, and recently, IL-33, which belongs to IL-1 family, was identified as the ligand for ST2L, the receptor type product of the ST2 gene. Subsequently, IL-33 and ST2L have been reported to be involved in Th2 immunity and inflammation, however, their functions on non-immunological cells are still obscure. Among non-immunological adhesive cells, vascular endothelial cells were reported to express both ST2 gene products and IL-33, therefore, we investigated the expression manner of the ST2 gene in vascular endothelial cells and the effect of IL-33 on endothelial cells. ST2 gene was expressed in each of the vascular endothelial cell types tested, and the expression was growth-dependent and down-regulated when the cells were differentiated to form vascular structures on the extracellular membrane matrix. IL-33 scarcely affected the growth and tube formation of the endothelial cells, but induced IL-6 and IL-8 secretion from endothelial cells with the rapid activation of extracellular signal-regulated kinase (ERK) 1/2, so IL-33 is supposed to involve in inflammatory reaction of vascular endothelial cells through its receptor, ST2L.
Asunto(s)
Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Mediadores de Inflamación/metabolismo , Interleucinas/metabolismo , Receptores de Superficie Celular/genética , Células Cultivadas , Endotelio Vascular/citología , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Ligandos , Receptores de Superficie Celular/metabolismoRESUMEN
Psychoneuroimmunological studies have clearly demonstrated that both cellular and humoral immunity are related to major depression. Soluble ST2 is regarded as a key molecule regulating immune system as well as cell proliferation. Indeed, soluble ST2 is reported to reduce IL-33-induced IL-6 and TNF-α production in macrophages and IL-33-induced IL-5 and IL-13 production in type 2 innate lymphoid cells. Elevated serum concentrations of soluble ST2 have been reported in patients with neuropsychiatric disorders, suggesting pathophysiological roles of soluble ST2 in behavioral phenotypes. Nevertheless, the relation between soluble ST2 and depressive behavior remain to be uncovered. To complement this point, we performed broad behavioral phenotyping, utilizing transgenic mice with a high concentration of serum ST2 in the present study. Soluble ST2 overexpression mice (ST2 Tg mice) were generated on a C3H/HeJ background. ST2 Tg mice crossed onto the BALB/c genetic background were used. Before starting tests, each mouse was observed in a clean cage for a general health check and neurological screening tests. In Experiment I, comprehensive behavioral phenotyping was performed to reveal the role of soluble ST2 on sensorimotor functions, anxiety-like behaviors, depression-like behaviors, social behaviors, and learning and memory functions. In Experiment II, to confirm the role of soluble ST2 on depression-like behaviors, a depression test battery (two bottle choice test, forced swimming test, and tail suspension test) was applied. The general health check indicated good general health and normal gross appearance for ST2 Tg mice. Further, the neurological reflexes of all the mice were normal. We found that soluble ST2 overexpression resulted in decreased social interaction. Moreover, depression-like behaviors of ST2 Tg mice were observed in two well-established behavioral paradigms, the forced swimming test and the tail suspension test. Nevertheless, hedonic reaction to sucrose was observed in ST2 Tg mice similar to WT mice. These results suggest the depression in the ST2 Tg mice. In conclusion, through a series of experiments, we established the animal model for assessing role of soluble ST2 in neuropsychiatric disorders, and revealed the possible involvement of soluble ST2 in depressive behavior.
Asunto(s)
Conducta Animal/fisiología , Depresión/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Aprendizaje/fisiología , Memoria , Animales , Ansiedad/genética , Ansiedad/metabolismo , Ansiedad/fisiopatología , Escala de Evaluación de la Conducta , Depresión/genética , Depresión/fisiopatología , Modelos Animales de Enfermedad , Proteína 1 Similar al Receptor de Interleucina-1/genética , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Transgénicos , Actividad Motora/genética , Actividad Motora/fisiología , Conducta Social , Natación , Regulación hacia ArribaRESUMEN
Interleukin (IL)-33 is a novel member of the IL-1 family. IL-33 is primarily synthesized as a 30-kDa precursor (pro-IL-33). Pro-IL-33 is cleaved by caspase-1 into an 18-kDa mature form (mature IL-33) in vitro. Recombinant mature IL-33 has been known to induce T-helper type-2 (Th2)-associated cytokines and inflammatory cytokines via its receptor, ST2L. However, processing of pro-IL-33 in vivo has not been clarified yet. Here, we report that calpain mediates pro-IL-33 processing in vivo. Pro-IL-33 was expressed by stimulating human epithelial cells with phorbol 12-myristate 13-acetate. Calcium ionophore induced pro-IL-33 cleavage and mature IL-33 production. This cleavage was inhibited by treatment with a calcium chelator and calpain inhibitors. Moreover, short interfering RNA-mediated knockdown of calpains suppressed pro-IL-33 cleavage. These results indicate that calpains play a critical role in pro-IL-33 processing in vivo.
Asunto(s)
Calpaína/metabolismo , Interleucinas/metabolismo , Calcio/farmacología , Calpaína/genética , Línea Celular Tumoral , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Humanos , Interleucina-33 , ARN Interferente Pequeño/genética , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L. However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38, JNK and Nuclear factor-kappaB (NF-kappaB) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappaB in TRAF6 deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAF6 and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappaB activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity.
Asunto(s)
Interleucinas/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Carboxipeptidasas A/genética , Carboxipeptidasas A/metabolismo , Línea Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-33 , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucinas/farmacología , Ratones , Proteínas Mutantes/metabolismo , Transducción de Señal/efectos de los fármacos , Factor 6 Asociado a Receptor de TNF/deficiencia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
The ST2 gene was originally identified as a primary responsive gene induced by stimulation with growth factors and by oncogenic stress. The ST2 gene harbors two distinct promoters - a distal promoter and a proximal promoter. In this study, we identified a novel type of serum-responsive element in the ST2 proximal promoter using reporter gene analysis; this element includes a possible responsive element for STAT family proteins. Indeed, enforced expression of constitutively active STAT3 activated this promoter element and induced the expression of ST2 gene products. Furthermore, an oncogenic Ras (G12V) mutant also caused the expression of ST2 gene products by utilizing the proximal promoter. We also clarified that activation of the ST2 promoter by either growth stimulation or oncogenic Ras was suppressed by the inhibitors for STAT3 and ERK pathways. Our observations strongly suggest the importance of STAT family and ERK pathways for the induction of ST2 gene products by cell growth stimulation.
RESUMEN
BACKGROUND: Skin is the outermost tissue of the human body, and works as a mechanical, chemical, and biological barrier. The epidermis is the uppermost layer of the skin, and keratinocytes constitute the majority of epidermal cells. Wounds are disruptions of skin integrity, and cause tremendous disadvantages to humans; accordingly, rapid wound healing is very important. Interleukin (IL)-33 is expressed in barrier tissue cells, such as epithelial and endothelial cells. Upon injury, IL-33 is released to stimulate immune cells, functioning as an "alarmin." ST2 is a receptor for IL-33; its soluble form (s)ST2 acts as a decoy receptor and competes for IL-33 binding. OBJECTIVES: We aimed to clarify the role of IL-33 in wound healing. MATERIALS AND METHODS: Wild-type (WT), IL-33 knockout (IL33 KO) mice, and sST2 transgenic (Tg) mice were wounded with a 4-mm punch, and the wound healing process was compared. Immunohistochemical analyses were performed to detect macrophages, neutrophils, and mast cells. Total RNA was extracted from the skin samples and real-time PCR was performed. An in vitro scratch wound assay was performed. RESULTS: Wound healing was delayed in IL33 KO mice compared to WT mice, while wound healing in sST2 Tg mice was comparable to that of WT mice. A histological examination showed delayed elongation of the epidermal tongue in IL-33 KO mice. An immunohistochemical study revealed prolonged neutrophilic infiltration at a later stage in IL-33 KO mice. IL-6, IL-1ß, and CXCL1 transcripts were more abundant in the wounds of IL-33 KO mice than WT mice. Intraperitoneal administration of an NFκB inhibitor to IL-33 KO mice normalized the delayed wound healing and the enhanced expression of IL-6 in IL-33 KO mice. Epidermal keratinocytes from IL-33 KO mice showed delayed wound closure compared to those from WT mice. CONCLUSION: Our results indicate that nuclear IL-33, but not IL-33 as a cytokine, has beneficial effects on wound healing in mice, probably by suppressing NFκB to inhibit excessive inflammation and by maintaining keratinocyte proliferation or migration for epithelialization.
Asunto(s)
Núcleo Celular/metabolismo , Células Epidérmicas , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Queratinocitos/metabolismo , Cicatrización de Heridas/fisiología , Animales , Epidermis/patología , Humanos , Inmunohistoquímica , Proteína 1 Similar al Receptor de Interleucina-1/genética , Interleucina-33/genética , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Cultivo Primario de Células , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
The ST2 gene was originally identified as a primary responsive gene, and the expressions of its gene products are induced by stimulation with growth factors and by oncogenic stresses. In this study, we observed that oncogenic Ras mutant induced the expression of ST2 and ST2L proteins. Interestingly, the enforced expression of ST2 gene products in NIH-3T3 murine fibroblasts remarkably enhanced Ras (G12V)-induced cellular transformation. Furthermore, when the expression of ST2 gene products was silenced by RNA-interference technique, Ras (G12V)-induced cellular transformation was drastically suppressed. According to these observations, it was indicated that the oncogenic Ras-induced expression of ST2 gene products is required for the acceleration of cellular transformation, and this seems to be independent of the stimulation with IL-33, a ligand for ST2/ST2L. Interestingly, knockdown of ST2 gene products caused a reduction in Rb phosphorylation in transformed murine fibroblasts, suggesting the functional involvement of ST2 gene products in cell cycle progression during cellular transformation. Our current study strongly suggests the importance of ST2 gene products in cellular transformation, and the presence of novel mechanism how ST2 gene products affect the cellular transformation and cell proliferation.
RESUMEN
Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Dependovirus/genética , Edición Génica , Vectores Genéticos/administración & dosificación , Hemofilia B/terapia , Animales , Animales Recién Nacidos , Antitrombina III/metabolismo , Secuencia de Bases , ADN Complementario/genética , Exones/genética , Factor IX/metabolismo , Intrones/genética , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Fenotipo , Reparación del ADN por Recombinación/genéticaRESUMEN
ST2 is a member of the interleukin-1 receptor family and is expressed in type-2 T helper (Th2) cells. Here, we have studied the molecular mechanism responsible for the transcriptional regulation of the ST2 gene in Th2 cells using a mouse thymoma cell line, EL-4. The ST2 gene has distal and proximal promoters. ST2 mRNA was produced from the distal promoter in EL-4 cells stimulated with both phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (Bt2cAMP). The region of approximately 100 bp upstream of transcription start site, containing two GATA consensus sites, was indispensable for the activation of the distal promoter in reporter gene analysis. An electrophoretic mobility shift assay showed that transcription factor GATA-3 bound one of the GATA consensus sites (from -84 to -79) with nuclear extracts from PMA plus Bt2cAMP-stimulated EL-4 cells. The overexpression of GATA-3 enhanced the activity of the distal promoter. On the other hand, mutations of the GATA consensus site canceled out the enhancement by GATA-3. These data suggest that GATA-3 is an important transcription factor for the expression of the ST2 gene in Th2 cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Transactivadores/metabolismo , Animales , Secuencia de Bases , Western Blotting , Bucladesina/farmacología , Línea Celular , Cartilla de ADN , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Factor de Transcripción GATA3 , Componentes del Gen , Regulación de la Expresión Génica/efectos de los fármacos , Proteína 1 Similar al Receptor de Interleucina-1 , Luciferasas , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Plásmidos/genética , Receptores de Interleucina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Acetato de Tetradecanoilforbol/farmacología , Transactivadores/genética , TransfecciónRESUMEN
The ST2 gene is induced in murine fibroblast cells at the start of cell proliferation. Although IL-33 has been identified as a ligand for one of the two major gene products of ST2 - namely, the transmembrane receptor form ST2L - prompting immunological research on inflammation, the roles of the ST2 gene products in cell proliferation remain to be elucidated. Using a cell proliferation assay system with NIH-3T3 cells, a normal murine fibroblast cell line, we found that treatment with recombinant ST2 caused an acceleration of cell proliferation, suggesting that ST2 acts in an autocrine/paracrine fashion. Strikingly, shRNA-induced knockdown of both ST2 gene products, ST2 and ST2L, reduced cell proliferation. This effect was effectively canceled by the expression of shRNA-resistant ST2, but not shRNA-resistant ST2L. The novel enhancement of cell proliferation by ST2 appears to involve positive feedback. Since the ST2 level is increased in various diseases involving inflammation, future investigations into the role of ST2 gene products in relation to various diseases, including malignancies, may be warranted.