MOLECULAR MECHANISMS OF SYNERGISTIC TRANSCRIPTIONAL REGULATION OF INDOLEAMINE 2,3-DIOXYGENASE
Interferon (IFN)-gamma-induced indoleamine 2,3-dioxygenase (IDO) activity inhibits the growth of some intracellular pathogens by catalyzing the oxidative cleavage of the indole ring of L-tryptophan and depleting pools of this essential amino acid. Tumor necrosis factor (TNF)-alpha synergistically enhances the IDO activity induced by IFN at the level of transcription in human epithelial cells. The purpose of this study was to characterize the molecular mechanisms responsible for synergistic gene expression in response to IFN and TNF. It was found that IFN-induced binding of STAT-1 to gamma activation sequences (GAS) and IFN responsive factor (IRF)-1 to IFN-stimulated response elements (ISRE), is more highly activated following treatment with interferon and TNF. This enhanced signal transduction is due to the increase in IFN receptor expression following combined cytokine stimulation. CCAAT enhancer binding protein (C/EBP)-beta binds to one of three consensus C/EBP sites in the IDO regulatory region in response to TNF alone or in combination with IFN. A transcriptional reporter construct consisting of green fluorescent protein expressed from the IDO regulatory region was utilized to understand which enhancer regions are responsible for synergistic IDO gene expression in response to IFN and TNF. Mutation of other individual enhancers and large deletions within the regulatory region showed that increased binding of IFN-specific factors to GAS and ISRE sites is alone responsible for synergistic transcriptional activation. An indirect requirement for NF-KB in synergistic IDO expression by regulating IRF-1 expression was also explored. A gamma activated sequence and a KB site, respectively, reside in the IRF-1 regulatory region. It was important to identify whether increased translocation of NF-KB to the nucleus and binding to the kB site upstream of the IRF-1 gene in response to IFN and TNF, is rate-limiting in enhanced IRF-1 expression. Limiting NF-KB translocation to the nucleus with a proteasome inhibitor reduced synergistic IRF-1 expression comparable to that achieved in response to IFN alone, and subsequently blocked synergistic IDO transcription. This defines an indirect requirement for NF-KB in synergistic IDO expression in response to IFN and TNF by regulating IRF-1 expression.
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:transcriptional regulation interferon tumor necrosis factor indoleamine 2 3 dioxygenase stat 1 irf c ebp beta nf kb
Date of Publication:01/01/2004