Analysis of transcription factors regulating induction of indoleamine 2,3-dioxygenase by IFN-gamma.
IFN-gamma treatment of the human carcinoma cell line ME180 causes cell death due to induction of indoleamine 2,3-dioxygenase (IDO) and resulting starvation for tryptophan. A mutant cell line 3B6A derived from ME180 was resistant to IFN-gamma because of loss of IDO activity. Cotransfecting an IDO promoter-chloramphenicol acetyl transferase (CAT) construct with IFN regulatory factor-1 (IRF-1) resulted in induction of CAT activity in both ME180 and 3B6A cells even in the absence of IFN-gamma. This induction was reduced by cotransfection with IRF-2. However, IRF-1 was not able to restore IDO activity, suggesting a possible repressor site outside the IDO promoter region. Stat1alpha (p91) restored both CAT and IDO activities in 3B6A cells following IFN-gamma treatment. 3B6A cells doubly treated with IFN-gamma and IFN-alpha or IFN-beta restored IDO activity, although neither cytokine on its own could induce IDO. Western blot analysis showed that both constitutive expression and induction of Stat1alpha by IFN-gamma were reduced in 3B6A cells, and double treatment of IFN-gamma with IFN-alpha or IFN-beta restored the expression level of Statla. Electrophoretic mobility shift assays indicated that Stat1 binds to the IFN-gamma-activated sequence (GAS) region in the IDO promoter in ME180 cells following IFN-gamma treatment. Our results indicated that the defect in 3B6A cells was reduced expression of Stat1alpha and that IRF-1, NF-kappaB, and PKR were all involved to some extent in the induction of IDO following IFN-gamma treatment.
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Indiana University Institute for Molecular and Cellular Biology, Department of Biology, Indiana University, Bloomington 47405, USA.
Sotero-Esteva
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Taylor
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MW
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; D007370:Interferon Type I / Q000494:pharmacology; D050832:Interferon-Stimulated Gene Factor 3; D007371:Interferon-gamma / Q000494:pharmacology*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014366:Tryptophan Oxygenase / Q000096:biosynthesis* / Q000235:genetics; D014407:Tumor Cells, Cultured; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; C494229:IRF2 protein, human; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; D007370:Interferon Type I; D050832:Interferon-Stimulated Gene Factor 3; D016328:NF-kappa B; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C079868:gamma interferon activation factor; D007371:Interferon-gamma; D004247:DNA; D014366:Tryptophan Oxygenase; D015500:Chloramphenicol O-Acetyltransferase; D019892:eIF-2 Kinase