Treatment of a variety of cultured human cells with type I interferon (IFNs) induces a rapid increase in the rate of transcription of several cellular genes. This lab is interested in the mechanism by which IFNs are capable of activating and then subsequently inhibiting the expression of such genes. Both ouabain, a specific inhibitor of the Na/K ATPase and phorbol esters which activate protein kinase C's inhibit the IFN-induced transcription of ISG-54K. Investigations are now being pursued to understand the mechanisms by which ouabain and phorbol esters inhibit IFN-induced gene expression. Phorbol ester-mediated inhibition of IFN-induced ISG-54K expression: The following facts concerning inhibitory effects of phorbol esters on IFN-activated ISG-54K expression have been established. The actions of phorbol esters are reversed by inhibitors of protein synthesis. The mechanisms by which long term IFN treatment of cells down regulates ISG-54K gene expression are distinct from those by which phorbol esters inhibit ISG-54K expression. We have mapped the region within the ISG-54K gene that allows phorbol esters to repress IFN-activated gene expression. Preliminary gel mobility shift assays detect protein(s) which bind to those sequences in the ISG-54K promotor that mediate the inhibitory effects of phorbol esters. Ouabain is known to be a specific inhibitor of the Na/K ATPase. Pre- incubation of a variety of cultured human cells with ouabain inhibits IFN-- induced expression of several RNAs. The inhibitory effects of ouabain occur at the level of transcription for ISG-54K. Experiments suggest that the actions of ouabain are relatively specific. Recently an ouabain-resistant Daudi cell variant has also been selected which shows decreased induction of a variety of IFN-alpha induced cellular genes suggesting that IFN-regulated gene expression is either directly or indirectly coupled to the presence of a functional Na/K ATPase. Gel mobility shift assays are presently being done to determine whether IFN-induced proteins that bind the interferon-stimulated response element (ISRE) are being altered-in cells treated with ouabain.
