RESUMEN
Periodontitis is a common chronic inflammatory condition that results in increased levels of inflammatory cytokines and inflammatory mediators. In addition to oral disease and tooth loss, it also causes low-grade systemic inflammation that contributes to development of systemic conditions including cardiovascular disease, pre-term birth, diabetes and cancer. Chronic inflammation is associated with epigenetic change, and it has been suggested that such changes can alter cell phenotypes in ways that contribute to both ongoing inflammation and development of associated pathologies. Here we show that exposure of human gingival fibroblasts to IL-1ß increases expression of maintenance methyltransferase DNMT1 but decreases expression of de novo methyltransferase DNMT3a and the demethylating enzyme TET1, while exposure to PGE2 decreases expression of all three enzymes. IL-1ß and PGE2 both affect global levels of DNA methylation and hydroxymethylation, as well as methylation of some specific CpG in inflammation-associated genes. The effects of IL-1ß are independent of its ability to induce production of PGE2, and the effects of PGE2 on DNMT3a expression are mediated by the EP4 receptor. The finding that exposure of fibroblasts to IL-1ß and PGE2 can result in altered expression of DNA methylating/demethylating enzymes and in changing patterns of DNA methylation suggests a mechanism through which inflammatory mediators might contribute to the increased risk of carcinogenesis associated with inflammation.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Dinoprostona/metabolismo , Fibroblastos/metabolismo , Interleucina-1beta/metabolismo , Oxigenasas de Función Mixta/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , ADN Metiltransferasa 3A , Encía/citología , Humanos , Oxigenasas de Función Mixta/genética , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Matrix metalloproteinases (MMPs) have both protective and pathological roles in inflammation, and transcriptional mechanisms are important in regulating physiological levels to maintain health. Zinc-binding protein-89 (ZBP-89) is a transcription factor with roles in regulating vital cellular processes, acting through complex interactions with other proteins to ensure appropriate expression of tightly regulated genes. ZBP-89 binds the MMP-3 promoter at a polymorphic (5A/6A) site along with NF-κB. This polymorphism affects MMP-3 protein levels in tissues. In disease association studies, both over- and under-expression has negative consequences to health, and this promoter element is important in maintaining balanced expression. There is evidence that effects of the polymorphism vary under different conditions, but the role of ZBP-89 in these differences is not known. ZBP-89 was stably knocked-down in MG-63 osteosarcoma cells in order to study its role in regulation of MMP-3 expression in response to cytokines, and evaluate the functionality of a putative binding site in the MMP-1 promoter. Results show ZBP-89 is needed for maximal induction of both genes by IL-1ß and TNFα. Binding of both ZBP-89 and NF-κB to both promoters was decreased in the knock-down cells under basal and TNF-induced conditions, and protein interactions between ZBP-89 and NF-κB were suggested. These data provide the first evidence of a role for ZBP-89 in regulation of MMP-1 expression, and suggest the possibility of a larger role for ZBP-89 in inflammation through interactions with NF-κB.
Asunto(s)
Citocinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Metaloproteinasas de la Matriz/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Inmunoprecipitación , Inflamación/genética , Inflamación/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genéticaRESUMEN
Matrix metalloproteinase-3 (MMP-3) over-expression is associated with tissue destruction in the context of chronic inflammation. Previous studies showed that IL-4 inhibits induction of MMP-3 by IL-1ß, and suggested that AP-1 might be involved. Here we show that IL-1 induced binding of transcription factor AP-1 to the MMP-3 promoter consists primarily of c-Jun, JunB, and c-Fos and that binding of c-Jun and c-Fos is inhibited by the combination of cytokines while binding of Jun B is not. Mutation of the AP-1 site in the MMP-3 promoter decreased the ability of IL-4 to inhibit its transcription in transfected MG-63 cells. Western blotting showed that both cytokines activate Jun N-terminal kinase (JNK), but with somewhat different kinetics, and that activation of JNK by both cytokines individually is inhibited by the combination. These results indicate that IL-4 inhibition of MMP-3 expression is associated with reduction of IL-1 induced binding of active forms of the AP-1 dimer, while less active JunB-containing dimers remain, and suggest that these changes are associated with decreased activation of JNK.
Asunto(s)
Interleucina-1/farmacología , Interleucina-4/farmacología , Metaloproteinasa 3 de la Matriz/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Western Blotting , Línea Celular Tumoral , Activación Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Prepucio/citología , Humanos , Masculino , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Proteína Quinasa 8 Activada por Mitógenos/genética , Periodontitis/enzimología , Periodontitis/metabolismo , Periodontitis/patología , Regiones Promotoras Genéticas , Unión Proteica , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Activación Transcripcional , TransfecciónRESUMEN
A 5T/6T polymorphism in the human MMP-3 promoter affects gene expression and impacts the risk and/or severity of various pathological conditions. Chromatin immunoprecipitation (ChIP) in human fibroblasts homozygous for the 6T site demonstrate that it is bound by NF-kappaB and ZBP-89 transcription factors in its native chromatin. ChIP in COS-1 cells transfected with plasmids containing the 5T and 6T sites in the context of 2kb of the MMP-3 promoter showed that NF-kappaB p50 binds preferentially to the 6T site, while more ZBP-89 binding is detected to the 5T site. Over-expressed ZBP-89 increased transcription from the 5T promoter but not from the 6T, while NF-kappaB decreased transcription from both promoters, even in the presence of excess ZBP-89. A model is suggested in which the physiological impact of the polymorphism is dependent on the relative levels and activities of these competing factors in various cell types and conditions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Metaloproteinasa 3 de la Matriz/genética , FN-kappa B/metabolismo , Factores de Transcripción/metabolismo , Animales , Células COS , Chlorocebus aethiops , Humanos , Polimorfismo Genético , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Zinc-binding protein-89 (ZBP-89; ZNF148, BERF-1, BFCOL-1) is a zinc-finger transcription factor of the Kruppel family. It has been shown to regulate the expression of a number of genes, acting as either an activator or repressor of gene expression, depending on the context. It is over-expressed in several cancers, but has been shown to be involved in apoptosis and to have a negative influence on cell growth in part by interactions with p53. Previously, ZBP-89 was shown to activate transcription of the matrix metalloproteinase-3 (MMP-3) gene by binding to a polymorphic promoter element in competition with nuclear factor kappaB (NF-kappaB). NF-kappaB is known to be a key regulator of the inflammatory response, but relatively little is known about regulation of ZBP-89. In order to ascertain whether ZBP-89 is regulated during inflammation, we designed experiments to determine whether and to what extent ZBP-89 levels are affected by inflammatory cytokines. Here we show that ZBP-89 mRNA and protein expression are significantly inhibited in human fibroblasts by the inflammatory cytokine interleukin-1beta. Since any change in the levels of ZBP-89 would presumably impact the regulation of MMP-3 and other ZBP-89 target genes, these results provide important insight into mechanisms involved in fine-tuning the immune response.
RESUMEN
BACKGROUND: Interleukin 4 (IL-4) has been shown to suppress interleukin-1 (IL-1) induced expression of matrix metalloproteinase-3 (MMP-3) in human synovial and gingival fibroblasts, but the mechanism of suppression has not been determined. Activators of peroxisome proliferator-activated receptor-gamma (PPARgamma) have been shown to inhibit cytokine induced expression of MMPs in other cell types, and IL-4 has been shown to activate PPARgamma by stimulating production of ligands through the lipoxygenase pathway. It has been suggested that PPARgamma may inhibit expression of MMPs by competing with transcription factor AP-1 for binding to a putative composite binding element in the promoters. The objective of this study was to determine whether the suppressive effects of IL-4 on the IL-1 induced expression of MMP-3 involve activation of lipoxygenase and/or PPARgamma. RESULTS: Western blotting revealed the presence of PPARgamma in nuclear extract of HGF. IL-1 induced binding of nuclear extract to the putative composite PPRE/AP-1 site was diminished in the presence of pioglitazone, but there was no evidence of any change in the composition of the retarded complexes, and no evidence of PPARgamma binding to this site. Nordihydroguaiaretic acid (NDGA), a non-selective lipoxygenase inhibitor, and MK886, a specific inhibitor of 5-lipoxygenase, induced MMP-3 expression synergistically with IL-1. However IL-4 was still able to inhibit MMP-3 expression in the presence of NDGA or MK886 and IL-1. Activation of PPARgamma with pioglitazone not only failed to inhibit IL-1 induced expression of MMP-3 mRNA, but rather super-induced MMP-3 in the presence of IL-1. PPARgamma antagonist GW9662 failed to abolish the suppressive effects of IL-4. Another PPARgamma activator, 15-deoxy-Delta12,14prostaglandin J2 (15dPGJ2), also super-induced MMP-3 mRNA, and this was due at least in part to increased transcription. CONCLUSION: IL-4 suppression of IL-1-induced MMP-3 expression in HGF is independent of lipoxygenase activity and activation of PPARgamma. Super-induction of MMP-3 by pioglitazone may have important implications for patients using pioglitazone to treat type II diabetes in the presence of chronic inflammation.
Asunto(s)
Encía/metabolismo , Interleucina-1/metabolismo , Interleucina-4/farmacología , Lipooxigenasa/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , PPAR gamma/metabolismo , Sitios de Unión , Núcleo Celular/metabolismo , Células Cultivadas , Activación Enzimática , Fibroblastos/enzimología , Fibroblastos/metabolismo , Encía/citología , Encía/enzimología , Humanos , Metaloproteinasa 3 de la Matriz/genética , Pioglitazona , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Tiazolidinedionas/farmacología , Factor de Transcripción AP-1/genética , TransfecciónRESUMEN
A 5T/6T polymorphic site in the matrix metalloproteinase-3 (MMP-3) promoter has been identified as a repressor element involved in inhibiting induction of MMP-3 transcription by interleukin 1; and the 6T allele has been associated with decreased expression of MMP-3 as compared to the 5T allele. Zinc-binding protein-89 (ZBP-89) was cloned from a yeast one-hybrid assay via its ability to interact with this site, but when the protein was over-expressed, it resulted in activation of the MMP-3 promoter rather than repression. Here we show that in nuclear extracts isolated from human gingival fibroblasts stimulated with IL-1, this site is bound by p50 and p65 components of NF-kappaB in addition to ZBP-89, and that recombinant p50 binds preferentially to the 6T binding site. These results are consistent with a role for NF-kappaB in limiting the cytokine induced expression of MMP-3.
Asunto(s)
Metaloproteinasa 3 de la Matriz/genética , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Represión Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Encía/citología , Humanos , Interleucina-1/farmacología , Subunidad p50 de NF-kappa B , Polimorfismo Genético , Elementos de Respuesta , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Although nitric oxide (NO) synthesis is increased in periodontal disease (PD), little is known about the possible sources of production by gingival tissues. In fact, gingival tissues from patients with periodontitis demonstrate greater levels of inducible nitric oxide (iNOS) expression than healthy tissue. Macrophages are the source of the iNOS expression, with endothelial cells also contributing. In the present study, our hypothesis has been that human gingival fibroblasts (HGF) also have the ability to produce NO. We have established for the first time that HGF express increased levels of iNOS and modulate NO synthesis in response to proinflammatory cytokines that act synergistically. METHODS: NO production under basal conditions or following incubation with tumor necrosis factor (TNF-alpha), interleukin (IL)-1beta, and interferon (IFN)-gamma was assessed by measurement of stable NO metabolites, nitrite, and nitrate, in a microplate adaptation of the Griess assay. Total RNA was isolated from HGF for determination of iNOS mRNA levels. RESULTS: We have shown that NO production is elevated in HGF that are stimulated simultaneously by TNF-alpha, IL-1beta, and IFN-gamma. Northern blot analysis confirmed that the production of iNOS mRNA by HGF is upregulated in the presence of these cytokines. Addition of mercaptoethyl guanidine (MEG), a specific inhibitor of iNOS, profoundly reduced the production of NO in HGF. Non specific inhibitors of iNOS, L-NG-monomethyl arginine (L-NMMA), and L-arginine-methyl ester (L-NAME) had little or no effect on NO produced in HGF. CONCLUSIONS: These results suggest that elevated NO production could be important in the pathogenesis of PD, and also suggest the ability of an iNOS inhibitor to modulate the disease. Treatments with drugs to block the production of nitric oxide or block its effects might be therapeutically valuable.