RESUMO
Th17-type autoreactive T cells contribute to pathogenicity in autoimmune diseases, including autoimmune uveitis. However, the mechanisms of regulation of Th17 cell activities remain unsolved and are likely to be tissue- and disease specific. In this review, we have summarized our studies from the murine model of experimental autoimmune uveitis (EAU). The resultsdemonstrate that γδ T cells have a regulatory effect on Th17 response. The regulatory effects of γδ T cells depend on their action state. Activated γδ T cells express significantly high levels of adenosine receptor A2 (A2AR) but low CD73. Both molecules are crucially involved in adenosine generation, thus modifying T cell responses. While the increased expression of A2AR-allows activated γδ T cells to bind adenosine more effectively than other immune cells, the decreased CD73 restricts their ability to convert AMP to adenosine. Adenosine affects Th1 and Th17 autoimmune responses differently. Its activation of γδ T cells shifts the Th1/Th17 balance towards the Th17 autoreactivity.
Assuntos
Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Células Th17/imunologia , Uveíte/imunologia , Animais , Humanos , Linfócitos T Reguladores/imunologia , Células Th1/imunologiaRESUMO
Regulatory effects of γδ T-cells on immune responses have been studied for years. We have investigated the regulatory effect of γδ T-cells on Th1 and Th17 autoimmune responses, and have studied molecular and cellular mechanisms by which γδ T-cells enhance or inhibit immune responses, exploiting a well-characterized murine model of experimental autoimmune uveitis (EAU). Our results show that (1) aberrant γδ T-cell activation is an important pathogenic event in EAU; (2) γδ T-cells have a unique regulatory effect on Th17 autoimmune responses, which is shaped by the activation status of γδ T-cells; and (3) γδ-mediated immunoregulation is closely linked with the extracellular adenosine metabolism. Reciprocal interactions between γδ T-cells and extracellular adenosine partially determine the development of EAU.
Assuntos
Doenças Autoimunes/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Uveíte/imunologia , Adenosina , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Modelos Animais , Receptores de Antígenos de Linfócitos T gama-delta/genéticaRESUMO
We have previously shown that activated γδ T cells have a much stronger proinflammatory effect in the development of experimental autoimmune uveitis than their nonactivated counterparts. Our present study explored γδ T cell subsets are functionally distinct in autoimmune pathogenesis and determined the pathogenic contribution of biased Vγ4+ γδ T cell activation in this disease. By systematically comparing two major peripheral γδ T cell subsets, the Vγ1+ and the Vγ4+ cells, we found that the Vγ4+ cells were readily activated in B6 mice during experimental autoimmune uveitis development, whereas Vγ1+ cells remained nonactivated. Cytokines that were abundantly found in the serum of immunized mice activated Vγ4+, but did not activate Vγ1+, cells. The Vγ4+ cells had a strong proinflammatory activity, whereas the Vγ1+ cells remained nonactivated when tested immediately after isolation from immunized mice. However, when the Vγ1+ cells were activated in vitro, they promoted inflammation. Our results demonstrated that activation is a major factor in switching the enhancing and inhibiting effects of both Vγ1+ and Vγ4+ γδ T cell subsets, and that γδ T cell subsets differ greatly in their activation requirements. Whether the enhancing or inhibiting function of γδ T cells is dominant is mainly determined by the proportion of the γδ T cells that are activated versus the proportion not activated.
Assuntos
Doenças Autoimunes/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T gama-delta , Subpopulações de Linfócitos T/imunologia , Uveíte/imunologia , Animais , Doenças Autoimunes/fisiopatologia , Citocinas/sangue , Citocinas/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Adenosine is an important regulator of the immune response, and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies showed that adenosine receptor agonists can be anti- or proinflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoimmune uveitis (EAU) induced by immunizing EAU-prone mice with a known uveitogenic peptide, IRBP1-20. Our results showed that the effective time to administer a single dose of ADA to suppress induction of EAU was 8-14 d postimmunization, shortly before EAU expression; however, ADA treatment at other time points exacerbated disease. ADA preferentially inhibited Th17 responses, and this effect was γδ T cell dependent. Our results demonstrated that the existing immune status strongly influences the anti- or proinflammatory effects of ADA. Our observations should help to improve the design of ADA- and adenosine receptor-targeted therapies.
Assuntos
Adenosina Desaminase/administração & dosagem , Doenças Autoimunes/tratamento farmacológico , Fatores Imunológicos/administração & dosagem , Células Th17/efeitos dos fármacos , Uveíte/tratamento farmacológico , Animais , Doenças Autoimunes/imunologia , Células Cultivadas , Proteínas do Olho/imunologia , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Terapia de Alvo Molecular , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Proteínas de Ligação ao Retinol/imunologia , Células Th17/imunologia , Uveíte/imunologiaRESUMO
Adenosine is a key endogenous signaling molecule that regulates a wide range of physiological functions, including immune system function and inflammation. Studies have shown that adenosine receptor (AR) agonists can be either anti-inflammatory or proinflammatory in immune responses and in inflammation, and the clarification of the mechanisms causing these opposing effects should provide a better guide for therapeutic intervention. Whereas previous studies mostly examined the effects of AR agonists on Th1-type immune responses, in this study, we compared their effect on Th17 and Th1 autoimmune responses in experimental autoimmune uveitis, a mouse model of human uveitis induced by immunization with the human interphotoreceptor retinoid-binding protein peptides 1-20. We showed that injection of mice with a nonselective AR agonist, 5'-N-ethylcarboxamidoadenosine (NECA), at an early stage after immunization had an inhibitory effect on both Th1 and Th17 responses, whereas injection of the same amount of NECA at a late stage inhibited the Th1 response but had an enhancing effect on the Th17 response. We also showed that the effects of NECA on Th1 and Th17 responses were completely dissociated, that the enhancing effect of NECA on Th17 responses was modulated by γδ T cells, and that the response of γδ T cells to NECA was determined by their activation status. We conclude that the inflammatory environment has a strong impact on converting the effect of AR agonist on the Th17 autoimmune response from anti-inflammatory to proinflammatory. Our observation should help in the designing of better AR-targeted therapies.
Assuntos
Adenosina-5'-(N-etilcarboxamida)/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Doenças Autoimunes/imunologia , Mediadores da Inflamação/administração & dosagem , Agonistas do Receptor Purinérgico P1/administração & dosagem , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Células Th1/imunologia , Células Th17/imunologia , Uveíte/imunologia , Animais , Autoantígenos/imunologia , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/terapia , Células Cultivadas , Proteínas do Olho/imunologia , Feminino , Humanos , Imunomodulação/efeitos dos fármacos , Imunomodulação/genética , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Proteínas de Ligação ao Retinol/imunologia , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Uveíte/induzido quimicamente , Uveíte/terapiaRESUMO
The proinflammatory and pathogenic function of Th17 cells in autoimmune diseases have been established but the mechanism by which such cells cause disease remains to be determined. Inflammatory cytokines produced by Th17 cells may either promote or inhibit disease development. The major cytokines produced by the uveitogenic T cells, such as IL-17 and IL-22, are not always pathogenic, and the disease-inducing ability of pathogenic T cells is not immediately correlated to the amount of cytokine they produce. Future studies identifying factors causing increased Th17 responses and determining the types of cells that regulating Th17 autoreactive T cells should facilitate our effort of understanding Th17-mediated disease pathogenesis.
Assuntos
Doenças Autoimunes/imunologia , Doenças Autoimunes/fisiopatologia , Interleucinas/imunologia , Células Th17/imunologia , Uveíte/imunologia , Animais , Modelos Animais de Doenças , Humanos , Interleucina-23/imunologia , Interleucinas/metabolismo , Linfócitos T Reguladores/imunologia , Uveíte/etiologia , Uveíte/patologia , Interleucina 22RESUMO
We have previously reported that, depending on their activation status, mouse γδ T cells can either enhance or inhibit the activity of IL-17(+) autoreactive T cells in experimental autoimmune uveitis. In this study, we showed that γδ T cells in naive C57BL/6 (B6) mouse do not express the IL-23R, whereas in immunized mice, it is expressed on >50% of γδ T cells. In vitro studies showed that IL-23R expression on γδ T cells is modulated by their state of activation, as weakly activated γδ T cells expressed the IL-23R, but highly activated γδ T cells did not. Functional studies showed that IL-23R(+) γδ T cells had the strongest suppressive effect on IL-17(+) autoreactive T cells, and that this effect was inhibited when the IL-23R was blocked by anti-IL-23R Ab or in the presence of excessive amounts of exogenous IL-23. We conclude that the balance between the enhancing and inhibitory effects of γδ T cells is regulated by their level of IL-23R expression. The expression of variable IL-23R levels allows γδ T cells to have different regulatory effects on adaptive immune responses, conceivably as a result of αß and γδ T cells competing for IL-23.
Assuntos
Doenças Autoimunes/imunologia , Interleucina-17/metabolismo , Interleucina-23/farmacologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Interleucina/imunologia , Linfócitos T/imunologia , Uveíte/imunologia , Animais , Anticorpos/imunologia , Autoimunidade , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Interleucina/biossíntese , Linfócitos T/metabolismo , Uveíte/induzido quimicamenteRESUMO
IL-17, the hallmark cytokine of Th17 cells, plays a pivotal role in the pathogenesis of autoimmune diseases, including encephalomyelitis. In the central nervous system, neuroglial cells are the main residents that express IL-17R and respond to IL-17 by producing chemokines/cytokines and boosting local inflammation. Factors that influence the IL-17R expression in neuroglial cells can also exert their impacts on the outbreak, progression and outcome of encephalomyelitis. Here, we reported that Toll-like receptor signaling has its bias for promoting the IL-17RA, but not the IL-17RC, expression in mouse neuroglial cells in a T cell infiltration independent manner. Elevated IL-17R functionally responded to IL-17 by secreting more chemokines and accelerating CD4 cell migration. First, real-time PCR confirmed that the expression of Il-17ra, but not Il-17rc, was significantly increased in the brain and spinal cord of EAE-induced mice. This effect was elicited by something in complete Freund's adjuvant (CFA), because markedly increased IL-17R was detected in mice immunized with CFA only, even though no evidence of EAE was found. Furthermore, in Rag1(-/-) mice, it was confirmed that CFA could augment the IL-17RA expression in the CNS in the absence of T cell infiltration. In vivo immunization with TLR ligands and in vitro treatment of purified neuroglial cells demonstrated that TLR ligands directly and effectively evoke the IL-17RA expression in the CNS and in cultured astrocytes, microglia and oligodendrocytes. LPS was the most effective inducer of the IL-17RA expression in astrocytes, and polyIC was superior to LPS for microglia and oligodendrocytes. Activated CD4 cells can also promote the secretion of chemokines by LPS pre-treated astrocytes, and hence accelerate the migration of CD4 cells, which was blocked by the neutralization of IL-17RA on the surface of the astrocyte. Taken together, we concluded that TLR signaling can directly stimulate the expression of IL-17RA, but not IL-17RC, in neuroglial cells, which functionally respond to IL-17A by secreting chemokines, accelerating CD4 cell migration, and contributing to the pathogenesis of encephalomyelitis.
Assuntos
Regulação da Expressão Gênica/fisiologia , Interleucina-17/metabolismo , Neuroglia/metabolismo , Transdução de Sinais/fisiologia , Receptores Toll-Like/fisiologia , Animais , Quimiocinas/genética , Quimiocinas/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Interleucina-17/genética , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
CD73 is intensively involved in the regulation of immune responses through the conversion of pro-inflammatory ATP to immunosuppressive adenosine. Herein, we clarified whether cells in the retina express CD73 and participate in the regulation of inflammatory eye diseases such as experimental autoimmune uveitis (EAU). First, immunofluorescence staining was performed to compare the distribution of CD73(+) cells in the retinas of EAU-induced and normal B10RIII mice. The results revealed that a layer of cells in the normal retina that was consistent with the location of retinal pigment epithelial (RPE) cells strongly expressing CD73, and the expression was markedly reduced in the presence of EAU. Thereafter, EAU was also induced in C57BL/6 mice by active immunization or adoptive transfer. CD73 expression in isolated RPE cells was assessed by real-time RT-PCR and western blotting, and the catalytic abilities of the cells to convert AMP to adenosine were determined using HPLC analyses. Compared to the normal control, significantly decreased CD73 expression and AMP catalytic ability were found in the RPE cells isolated from inflamed eyes. CD73 expression and activity were also studied in cultured RPE cells treated with different stimuli, such as Toll-like receptor ligands and cytokines. Highly varied functional CD73 expression was observed in RPE cells through cytokines or Toll-like receptor agonist treatments. Finally, whether RPE cells could regulate the immune response, particularly the proliferation of CD4 cells, through surface-expressed CD73 was determined using a two-chamber assay. The robust inhibition of conventional T-cell proliferation was uniquely observed when CD73(+) RPE cells in the upper chamber were in the presence of AMP. To further confirm the function of CD73 in RPE cells, Cd73(-/-) RPE cells were isolated, and CD73-rescued control cells were constructed. CD73(+)Cd73(-/-) RPE, not Cd73(-/-) RPE, significantly suppressed interacted CD4 cells proliferation and cytokine production. Taken together, these data suggest that naive RPE cells suppressed the immune response through their high expression of CD73. The expression of CD73 in RPE cells could be regulated through many factors, and down-regulated CD73 expression attenuated the suppressive effect of RPE on the proliferation of conventional CD4 cells.
Assuntos
5'-Nucleotidase/genética , Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica/fisiologia , Terapia de Imunossupressão , Ativação Linfocitária/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Transferência Adotiva , Animais , Doenças Autoimunes/imunologia , Western Blotting , Proliferação de Células , Modelos Animais de Doenças , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Uveíte/imunologia , VacinaçãoRESUMO
We have previously identified a novel Trß isoform (TrßΔ) in the rat, in which a novel exon N (108 bps) was found between exon 3 and exon 4 of TrßΔ, which represents the only difference between TrßΔ and Trß1. In this study, we searched for an elongated Trß2-like subtype with one additional exon N. We successfully isolated the entire mRNA/cDNA of a novel elongated Trß2 isoform via PCR in the rat pituitary gland. The mRNA/cDNA was only 108 bps (exon N) longer than that Trß2, and the extension of the sequence was between exon 3 and 4 of Trß. The whole sequence of this novel Trß isoform has been published in NCBI GenBank (HM043807.1); it is named TRbeta2Delta (Trß2Δ). In adult rat pituitary tissue, quantitative real-time RT-PCR analysis showed that the mRNA levels of Trß2Δ and Trß2 were roughly equal (P > 0.05). We cloned, expressed, and purified the His-Trß2Δ protein [recombinant TRß2Δ (rTRß2Δ)]. SDS-PAGE and western blotting revealed that the molecular weight of rTRß2Δ was 58.2 kDa. Using a radioligand binding assay and an electrophoretic mobility shift assay, rTRß2Δ-bound T3 with high affinity and recognized thyroid hormone response element (TRE) binding sites. Finally, in vitro transfection experiments further confirmed that rTRß2Δ binding T3 significantly promotes the transcription of target genes via the TRE. Here, we have provided evidence suggesting that rTRß2Δ is a novel functional TR isoform.
Assuntos
Hipófise/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores beta dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/metabolismo , Animais , Sítios de Ligação/genética , Células COS , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/genética , Ligantes , RNA Mensageiro/genética , Ratos , Transcrição Gênica/genética , Transfecção/métodosRESUMO
In the current study, we showed that in vivo administration of an anti-CD25 Ab (PC61) decreased the Th17 response in C57BL/6 mice immunized with the uveitogenic peptide interphotoreceptor retinoid-binding protein, while enhancing the autoreactive Th1 response. The depressed Th17 response was closely associated with decreased numbers of a splenic dendritic cell (DC) subset expressing CD11c(+)CD3(-)CD25(+) and decreased expansion of γδ T cells. We demonstrated that ablation of the CD25(+) DC subset accounted for the decreased activation and the expansion of γδ T cells, leading to decreased activation of IL-17(+) interphotoreceptor retinoid-binding protein-specific T cells. Our results show that an enhanced Th17 response in an autoimmune disease is associated with the appearance of a DC subset expressing CD25 and that treatment of mice with anti-CD25 Ab causes functional alterations in a number of immune cell types, namely DCs and γδ T cells, in addition to CD25(+)αßTCR(+) regulatory T cells.
Assuntos
Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Células Th17/imunologia , Uveíte/imunologia , Uveíte/patologia , Animais , Doenças Autoimunes/metabolismo , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Feminino , Subunidade alfa de Receptor de Interleucina-2/fisiologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células Th17/patologia , Uveíte/metabolismoRESUMO
BACKGROUND: Immunological dysfunction-induced low-grade inflammation is regarded as one of the predominant pathogenetic mechanisms in post-infectious irritable bowel syndrome (PI-IBS). γδ T cells play a crucial role in innate and adaptive immunity. Adenosine receptors expressed on the surface of γδ T cells participate in intestinal inflammation and immunity regulation. AIM: To investigate the role of γδ T cell regulated by adenosine 2A receptor (A2AR) in PI-IBS. METHODS: The PI-IBS mouse model has been established with Trichinella spiralis (T. spiralis) infection. The intestinal A2AR and A2AR in γδ T cells were detected by immunohistochemistry, and the inflammatory cytokines were measured by western blot. The role of A2AR on the isolated γδ T cells, including proliferation, apoptosis, and cytokine production, were evaluated in vitro. Their A2AR expression was measured by western blot and reverse transcription polymerase chain reaction (RT-PCR). The animals were administered with A2AR agonist, or A2AR antagonist. Besides, γδ T cells were also injected back into the animals, and the parameters described above were examined, as well as the clinical features. Furthermore, the A2AR-associated signaling pathway molecules were assessed by western blot and RT-PCR. RESULTS: PI-IBS mice exhibited elevated ATP content and A2AR expression (P < 0.05), and suppression of A2AR enhanced PI-IBS clinical characteristics, indicated by the abdominal withdrawal reflex and colon transportation test. PI-IBS was associated with an increase in intestinal T cells, and cytokine levels of interleukin-1 (IL-1), IL-6, IL-17A, and interferon-α (IFN-α). Also, γδ T cells expressed A2AR in vitro and generated IL-1, IL-6, IL-17A, and IFN-α, which can be controlled by A2AR agonist and antagonist. Mechanistic studies demonstrated that the A2AR antagonist improved the function of γδ T cells through the PKA/CREB/NF-κB signaling pathway. CONCLUSION: Our results revealed that A2AR contributes to the facilitation of PI-IBS by regulating the function of γδ T cells via the PKA/CREB/NF-κB signaling pathway.
Assuntos
Síndrome do Intestino Irritável , Triquinelose , Camundongos , Animais , NF-kappa B/metabolismo , Interleucina-17/metabolismo , Interleucina-6 , Citocinas/metabolismo , Transdução de Sinais , Triquinelose/complicações , Inflamação/complicações , Interleucina-1RESUMO
The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1(-/-) colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.
Assuntos
MAP Quinase Quinase 1/metabolismo , Proteínas Quinases/metabolismo , Serina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/efeitos dos fármacos , Catálise , Colo/citologia , Células Epiteliais , Proteínas de Escherichia coli , Camundongos , Fosforilação , Especificidade por SubstratoRESUMO
BACKGROUND: To study the expression of the RIZ1 (Retinoblastoma protein-interacting zinc-finger gene 1) gene and investigate the promoter region methylation status of RIZ1 gene in the human esophageal squamous cell carcinoma (ESCC) cell lines of KYSE150, KYSE510, TE13, EC9706, CaEsl7, and EC109. To investigate the influence of DNMT (DNA methyltransferase) 5-aza-CdR(5-aza-2'-deoxycytidine) on the transcription of the RIZ1 gene in one cell line whose RIZ1 gene promoter region methylation was detected, and to investigate its influence on the cell proliferation. METHODS: Real-time PCR (Real-time quantitative PCR) and an immunohistochemistry technique was used to get the expression of RIZ1 in specimens from 6 human ESCC cell lines and 28 ESCC patients (tumor tissues and adjacent non-cancerous tissues). MSP (Methylation-specific PCR) was used to investigate the promoter region methylation status of the RIZ1 gene in the 6 ESCC cell lines. One cell line, whose RIZ1 gene promoter region methylation was detected, was chosen for the next studies in which it was treated it by with 5-aza-CdR. Real-time PCR was used to investigate its influence on the transcription of RIZ1 gene and MTT (methyl thiazolyl tetrazolium) was used to detect if 5-aza-CdR inhibits the proliferation of the cell line. RESULTS: In the 28 ESCC patient samples, RIZ1 expression was significantly lower in the tumor tissues than that in their adjacent non-cancerous tissues (p < 0.05). Consistently, immunohistochemistry analyses of RIZ1 protein expression showed that in the ESCC tissues RIZ1 protein expression was also significantly lower than in the adjacent tissues. In the human ESCC tissues the rate of expression accounts for 0% (0/12), and in the adjacent noncancerous tissues the rate of expression was 66.7% (8/12), the correlation was highly significant (chi2 = 12.000, p < 0.05). Promoter methylation of the RIZ1 gene was detected in TE13, CaEsl7, EC109. The cell line TE13 was chosen for the next studies. The expression of RIZ1 mRNA in TE-13 was up-regulated after having been treated with 5-aza-CdR. 5-aza-CdR inhibited cell proliferation of TE-13 in a time and concentration-dependent manner. CONCLUSIONS: Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression. Methylation of the RIZ1 promoter and loss of RIZ1 expression in human ESCC are independent biomarkers. Their determination may offer guidance for selecting appropriate diagnoses and treatments. RIZ1 may be a potential tumor suppressor in human ESCC.
Assuntos
Carcinoma de Células Escamosas/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Histona-Lisina N-Metiltransferase/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Decitabina , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
When studying the altered expression of genes associated with cartilage regeneration by quantitative real-time RT-PCR (RT-qPCR), reference genes with highly stable expression during different stages of chondrocyte developmental are necessary to normalize gene expression accurately. Until now, no reports evaluating expression changes of commonly used reference genes in rabbit articular cartilage have been published. In this study, defects were made in rabbit articular cartilage, with or without insulin-like growth factor 1 (IGF-1) treatment, to create different chondrocyte living environments. The stability and intensity of the expressions of the candidate reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S Ribosomal RNA (18S rRNA), cyclophilin (CYP), hypoxanthine phosphoribosyl transferase (HPRT1), and beta-2-microglobulin (B2M) were evaluated. The data were analyzed by geNorm and NormFinder. B2M and 18S rRNA were identified to be suitable reference genes for rabbit cartilage tissues.
Assuntos
Cartilagem/metabolismo , Perfilação da Expressão Gênica , Cicatrização/genética , Ferimentos e Lesões/genética , Animais , Modelos Animais , Coelhos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
TNF and epidermal growth factor (EGF) are well-known stimuli of cyclooxygenase (COX)-2 expression, and TNF stimulates transactivation of EGF receptor (EGFR) signaling to promote survival in colon epithelial cells. We hypothesized that COX-2 induction and cell survival signaling downstream of TNF are mediated by EGFR transactivation. TNF treatment was more cytotoxic to COX-2(-/-) mouse colon epithelial (MCE) cells than wild-type (WT) young adult mouse colon (YAMC) epithelial cells or COX-1(-/-) cells. TNF also induced COX-2 protein and mRNA expression in YAMC cells, but blockade of EGFR kinase activity or expression inhibited COX-2 upregulation. TNF-induced COX-2 expression was reduced and absent in EGFR(-/-) and TNF receptor-1 (TNFR1) knockout MCE cells, respectively, but was restored upon expression of the WT receptors. Inhibition of mediators of EGFR transactivation, Src family kinases and p38 MAPK, blocked TNF-induced COX-2 protein and mRNA expression. Finally, TNF injection increased COX-2 expression in colon epithelium of WT, but not kinase-defective EGFR(wa2) and EGFR(wa5), mice. These data indicate that TNFR1-dependent transactivation of EGFR through a p38- and/or an Src-dependent mechanism stimulates COX-2 expression to promote cell survival. This highlights an EGFR-dependent cell signaling pathway and response that may be significant in colitis-associated carcinoma.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Células Epiteliais/metabolismo , Receptores ErbB/fisiologia , RNA Mensageiro/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Linhagem Celular , Sobrevivência Celular , Colo/citologia , Colo/metabolismo , Ciclo-Oxigenase 2/deficiência , Ciclo-Oxigenase 2/genética , Células Epiteliais/efeitos dos fármacos , Receptores ErbB/deficiência , Receptores ErbB/genética , Mucosa Gástrica/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Estômago/citologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família src/metabolismoRESUMO
BACKGROUND & AIMS: The ErbB4 receptor tyrosine kinase regulates cell growth, survival, and differentiation in several tissues, but its role in the gastrointestinal tract has not been reported. We tested the hypothesis that ErbB4 promotes intestinal cell survival and restitution following injury or inflammation. METHODS: ErbB4 expression in human inflammatory bowel disease was determined by immunohistochemistry. Mice were subjected to dextran sulfate sodium (DSS, 3%) colitis or injected with tumor necrosis factor (TNF), and ErbB4 expression was quantified by immunohistochemistry and Western blot. Cultured young adult mouse colon (YAMC) cells were exposed to TNF, and ErbB4 messenger RNA, protein, and phosphorylation levels were measured. Cells transfected with ErbB4 small interfering RNA (siRNA), or over expressing ErbB4, were subjected to wound healing and apoptosis assays. RESULTS: ErbB4 levels increased in Crohn's colitis and the colon epithelium of mice with DSS colitis or injected with TNF. In YAMC cells, TNF induced ErbB4 messenger RNA, protein, and phosphorylation; nuclear factor kappaB activation also stimulated ErbB4 accumulation. ErbB4 siRNA sensitized cells to TNF-stimulated apoptosis, while over expression blocked apoptosis induced by TNF plus cycloheximide. Additionally, ErbB4 siRNA decreased YAMC cell wound healing. ErbB4 knockdown attenuated, while over expression elevated, phosphorylation of Akt in response to TNF. Inhibition of the phosphatidylinositol 3-kinase/Akt signaling cascade reversed the ability of ErbB4 over expression to protect from cytokine-induced apoptosis. CONCLUSIONS: ErbB4 expression and signaling are key elements for TNF responses in vivo and in cell culture, protecting intestinal epithelial cells from apoptosis in the inflammatory environment, possibly through Akt activation.
Assuntos
Colo/citologia , Receptores ErbB/fisiologia , Mucosa Intestinal/citologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Sobrevivência Celular , Doença de Crohn/metabolismo , Receptores ErbB/análise , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-4 , Transdução de Sinais , CicatrizaçãoRESUMO
OBJECTIVE: To explore the effects of thyroid hormone on the expression of homeobox gene Nkx2.1 mRNA in child rat by supplying their hypothyroidism pregnant mother with different dose of levothyroxine (L-thyroxine, L-T(4)) in different times. METHODS: 120 female Wistar rats were randomly divided into eight groups according to the body weight: control group, non-treatment hypothyroidism group, hypothyroidism groups supplied with L-T(4) in high, medium and low dosage in early stage (1st-17th day of pregnancy) and in late stage (18th day of pregnancy-20th day after childbirth). According to 100 grams of body weight, the concentrations of L-T(4) were 3.5, 2.0, 0.5 µg/d in high, medium and low dosage group. All the rats were fed with low-iodine food. The control group was given 200 µg/L potassium iodate solution as drinking water and the other groups were given deionized water. After three months, the rats were mated with normal male rats. After the pregnancy was confirmed, hypothyroidism groups were supplied with L-T(4) of different concentrations. Brain samples were taken from the 17-day fetal rats, new-born and 20-day old offsprings and the levels of Nkx2.1 mRNA in brain tissue were analyzed by real-time fluorescence quantitative PCR techniques. RESULTS: The levels of TT(3) in hypothyroidism groups supplied with L-T(4) in high, medium and low dosages in early and late pregnant stages, non-treatment hypothyroidism group and control group were (0.85 ± 0.17), (0.81 ± 0.18), (0.86 ± 0.21), (0.85 ± 0.20), (0.89 ± 0.18), (0.85 ± 0.20), (0.86 ± 0.20), (1.08 ± 0.07) nmol/L (F = 4.08, P < 0.01); the levels of TT(4) in each group were (0.43 ± 0.16), (0.39 ± 0.11), (0.39 ± 0.13), (0.43 ± 0.17), (0.51 ± 0.19), (0.43 ± 0.16), (0.41 ± 0.15), (39.43 ± 14.16) nmol/L (F = 31.99, P < 0.01); the levels of FT(3) in each group were (3.29 ± 0.61), (3.29 ± 0.61), (3.24 ± 0.61), (3.28 ± 0.63), (3.31 ± 0.59), (3.28 ± 0.50), (3.24 ± 0.49), (4.93 ± 0.46) pmol/L (F = 5.79, P < 0.01); the levels of FT(4) in each group were (3.38 ± 0.80), (3.31 ± 0.67), (3.29 ± 0.73), (3.27 ± 0.71), (3.48 ± 0.81), (3.56 ± 0.66), (3.29 ± 0.61), (27.29 ± 4.53) pmol/L (F = 26.34, P < 0.01). The expression of Nkx2.1 mRNA in non-treatment hypothyroidism group (9.15 × 10(-5) ± 9.17 × 10(-5)) was lower than control group (65.1 × 10(-5) ± 40.90 × 10(-5)) in 17th day of pregnancy (t = 66.224, P < 0.05); the expression of Nkx2.1 mRNA in non-treatment hypothyroidism group (3.16 × 10(-5) ± 0.142 × 10(-5)) was lower than control group (55.6 × 10(-5) ± 51.05 × 10(-5)) in new-born (t = 102.225, P < 0.05); the expression of Nkx2.1 mRNA in non-treatment hypothyroidism group (8.09 × 10(-5) ± 8.21 × 10(-5)) was lower than control group (13.9 × 10(-5) ± 7.43 × 10(-5)) in 20th day after birth (t = 9.235, P < 0.05). The trend of Nkx2.1 mRNA in hypothyroidism groups was decreased in group supplied with L-T(4) in medium dosage in early stage descends in 17th day of pregnancy, new-born and 20th day after birth (57.1 × 10(-5) ± 22.90 × 10(-5)), (30.8 × 10(-5) ± 27.20 × 10(-5)), (17.1 × 10(-5) ± 0.623 × 10(-5)) (F = 13.394, P < 0.01). The expression of Nkx2.1 mRNA in hypothyroidism groups supplied with L-T(4) in medium dosage in early stage in 17th day of pregnancy, new-born and 20th day after childbirth was closest to the control group in every period (t values were 0.225, 0.336, 0.345, all P values > 0.05). CONCLUSION: The difference in the expression of homeobox gene Nkx2.1 mRNA is highly related to the level of thyroid hormone.
Assuntos
Encéfalo/metabolismo , Hipotireoidismo/tratamento farmacológico , Proteínas Nucleares/genética , RNA Mensageiro/genética , Tiroxina/farmacologia , Fatores de Transcrição/genética , Animais , Animais Recém-Nascidos/genética , Animais Recém-Nascidos/metabolismo , Feminino , Gravidez , Prenhez , Ratos , Ratos Wistar , Fator Nuclear 1 de TireoideRESUMO
Previously, we had demonstrated that insufficient intracellular unpacking of exogene from its chitosan carrier contributes towards the restricted transfection efficiency of CS/DNA complex. In order to enhance intracellular unpacking and thus improve the transfection efficiency, our present work has addressed a novel strategy of chitosanase gene (csn) co-transfection. An Aspergillus fumigatuscsn gene was semi-synthesized and cloned into a prokaryotic expression vector, plasmid pGEX-3X, meanwhile a mutant csn gene encoding an inactive Asp129-Asn chitosanase was generated by site-directed mutagenesis. Both active csn (acCSN) and inactive csn (inCSN) genes were expressed in bacteria cells and chitosan degradation activities of those purified recombinant proteins were tested. These csn genes were further subcloned into an eukaryotic expression vector, plasmid pTracer-CMV/Bsd, containing a gfp reporter gene. Recombinant plasmid pTracer-accsn or pTracer-incsn was co-transfected with plasmid pTracer/Bsd/LacZ, which contains an additional lacZ reporter gene, into C2C12 myoblast cells by CS/DNA complex. The expression of gfp reporter gene was determined by fluorescence microscope, while the expression of lacZ reporter was evaluated quantitatively by beta-galactosidase activity. All together, findings indicate that during the exogene being delivered into mammalian cells by CS/DNA complex, the csn co-transfection is beneficial for the exogene expression.