The acquisition of macrophage anti-tumor function results, at least in part, from alterations in gene expression which occur in response to activating stimuli such as IFN(gamma) or LPS. The kappa(B) sequence motif is an important component of transcriptional control of inducible macrophage gene expression. The kappa(B) transcription control system is a highly complex collection of stimulus-sensitive kappa(B) specific DNA binding factors, inhibitors of kappa(B) DNA binding activities, and distinct kappa(B) sequence elements. In recent work, both published and unpublished, we have demonstrated that two distinct kappa(B) sites regulate transcription of the IP-10 gene in murine macrophages in response to LPS of IFN(gamma). Results from functional analysis as well as preliminary findings regarding the nuclear proteins which recognize these sites in stimulated macrophages lead to the following hypotheses: That IFN(gamma) and LPS differentially activate distinct sets of kappa(B) binding and/or transcriptional regulatory proteins (including p50/c- Rel/p65) which promote or diminish transcription of selected genes through interaction with at least two classes of kappa(B) motif. Furthermore, kappa(B)-dependent transcriptional activity is negatively regulated by one or more inhibitory factors and these may act in stimulus-dependent fashion. Negative regulation may be diminished during activation from a resting state and enhanced during restoration of activated cells to a resting state. These hypotheses will be tested through performance of three specific aims: 1. The identity and function of Rel gene products involved in kappa(B) recognition in IFN(gamma)- or LPS-stimulated macrophages will be examined. The specific Rel gene products present in vivo or in DNA- protein complexes formed in vitro will be determined. In addition, the functional significance of relevant family members will be assessed through selected analysis of wild-type and mutant forms of such genes using gene transfer in macrophage cell lines. 2. The function of I(kappa)B (and other negative regulators of kappa(B) binding activity) will be carried out through specific mutagenesis followed by in vitro analysis of in vitro DNA binding activity and in vivo function using gene transfer in macrophage cell lines. 3. the role of the JAK family of protein tyrosine kinases in IFN(gamma) or LPS-stimulated activation of kappa(B) binding activity and transactivating function in macrophages will be evaluated.
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