Modulation of cellular tryptophan metabolism in human fibroblasts by transforming growth factor-β: Selective inhibition of indoleamine 2,3- dioxygenase and tryptophanyl-tRNA synthetase gene expression

Alterations in the rate of cellular tryptophan metabolism are involved in mediating important biological activities associated with cytokines and growth factors. Indoleamine 2,3-dioxygenase (IDO) and tryptophanyl-tRNA synthetase are enzymes of tryptophan metabolism whose expression in a variety of cells and tissues is highly inducible by interferon-γ (IFN-γ). Transforming growth factor-β (TGF-β) antagonizes many cellular responses to IFN-γ. The interaction of these two cytokines plays an important role in maintaining homeostasis during inflammation and repair. In human skin and synovial fibroblasts in vitro, TGF-β caused time-and dose-dependent abrogation of IFN-γ-stimulated expression of IDO and tryptophanyl-tRNA synthetase mRNAs. The inhibition was selective and did not appear to be due to down-regulation of IFN-γ signaling by TGF-β. In parallel with its effect on IDO mRNA expression, TGF-β caused a marked reduction in intracellular IDO protein levels and abrogated IDO activity and tryptophan catabolism in these cells induced by IFN-γ. IFN-γ caused a rapid and striking increase in the amount of IDO heterogeneous nuclear pre-mRNA and induced transcription of the IDO gene, as demonstrated by transient transfection assays. TGF-β partially reversed this stimulation. IFN regulatory factor (IRF)-1 and stat1 are cellular intermediates in IFN signaling. Both are implicated in activation of IDO transcription in response to IFN-γ. The stimulation by IFN-γ of IRF-1 protein and mRNA expression was not prevented by treatment of fibroblasts with TGF-β. Furthermore, gel mobility shift assays indicated that TGF-β did not inhibit the induction of stat1 and IRF-1 binding activity to their cognate DNA recognition sites in the IDO gene promoter. In contrast, the stability of IDO mRNA transcripts was reduced in fibroblasts treated with TGF-β, as shown by determination of mRNA half-lives following blockade of transcription with 5,6-dichlorobenzimidazole riboside. The findings indicate that TGF-β prevents the induction of IDO and tryptophanyl-RNA synthetase gene expression in fibroblasts. The repression of IDO expression by TGF-β is mediated at both transcriptional and posttranscriptional levels. These results implicate TGF-β in the negative regulation of tryptophan metabolism, provide evidence for the molecular basis of this regulation, and indicate that cellular tryptophan metabolism is under tight immunological control.