RESUMEN
OBJECTIVE: Increased expression of insulin-like growth factor 2 (IGF2) by fibroblast-like synoviocytes (FLS) was associated with low inflammatory synovium of patients with rheumatoid arthritis (RA). The aim of this study was to analyse whether the differential expression of IGF2, whose expression is normally restricted to one allele, is due to activation of the normally suppressed allele. METHODS: IGF2 gene expression of RA FLS was quantified by quantitative real-time PCR. FLS heterozygous for a 3'-untranslated region IGF2 polymorphism were selected to measure the relative contribution of the allelic transcripts by allele-specific transcript quantification assay. Proliferation was determined by [(3)H]thymidine incorporation. RESULTS: IGF2 was shown to contribute to RA FLS proliferation. FLS could be classified in IGF2 high and IGF2 low-expressing cell lines. Allelic IGF2 transcript quantification analysis revealed that in part of the RA FLS the normally suppressed allele was activated, resulting in biallelic expression of the IGF2 gene. Biallelic expression was associated with increased levels of IGF2 mRNA production. CONCLUSION: The findings indicate that the imprinting status of IGF2 might underlie the increased expression of IGF2, which may contribute to autonomous growth of RA FLS of low inflammatory synovial tissues.
Asunto(s)
Artritis Reumatoide/metabolismo , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Membrana Sinovial/metabolismo , Artritis Reumatoide/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Regulación de la Expresión Génica , Impresión Genómica , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/farmacología , ARN Mensajero/genética , Membrana Sinovial/patologíaRESUMEN
Cytokine secretion profiles of activated T cells are critical for maintaining the immunologic balance between protection and tolerance. In mice, several cytokines have been reported to exhibit monoallelic expression. Previously, we found that the human interleukin-1 alpha (IL1A) gene exhibits a stable allele-specific expression pattern in CD4+ T-cell clones. We investigated whether DNA methylation is involved in the allele-specific expression of IL-1alpha. Here, we show that differential methylation of CpGs in the proximal promoter region is associated with allele-specific expression of IL-1alpha in CD4+ T cells. The differential methylation pattern is already observed in naive T cells. In keratinocytes, which constitutively produce IL-1alpha, the proximal promoter is hypomethylated. CpGs located further upstream and in intron 4 were almost all methylated, irrespective of expression. Treatment of nonexpressing cells and of T-cell clones with 5-aza-2'deoxycytidine induced IL-1alpha expression in the nonexpressing cells and induced expression of the formerly silent allele in T-cell clones. In addition, electrophoretic mobility shift assays showed that methylation of CpGs in the proximal promoter resulted in direct inhibition of binding of nuclear factor(s). Taken together, these results suggest that allele-specific expression of IL-1alpha in CD4+ cells is achieved, at least in part, by differential methylation of the promoter.
Asunto(s)
Alelos , Linfocitos T CD4-Positivos/metabolismo , Regulación de la Expresión Génica , Interleucina-1/genética , Interleucina-1/metabolismo , Regiones Promotoras Genéticas , Animales , Azacitidina/análogos & derivados , Azacitidina/farmacología , Islas de CpG , Metilación de ADN , Decitabina , Inhibidores Enzimáticos/farmacología , Humanos , Linfocitos/metabolismo , RatonesRESUMEN
A number of reports have described the monoallelic expression of murine cytokine genes. Here we describe the monoallelic expression of the human IL-1alpha gene in CD4+ T cells. Analysis of peripheral blood T cell clones derived from healthy individuals revealed that the IL-1alpha gene shows predominantly monoallelic expression. Monoallelic expression was observed in Th0, Th1, and Th2 cell clones. In addition, we demonstrate monoallelic expression in T cell clones from rheumatoid arthritis patients derived from synovial fluid of the knee joint, suggesting that the occurrence of this phenomenon is not different from that in clones derived from healthy individuals. The finding of monoallelic expression of a cytokine gene in human CD4+ T cell clones provides evidence for allele-specific silencing/activation as another layer of regulation of IL-1alpha gene expression.