Modulation of human papillomavirus type 16 early gene expression by transforming growth factor-beta

The cytokine, transforming growth factor-beta (TGF-β) is a potent growth inhibitor of epithelial cells. Loss of sensitivity to TGF-β is characteristic of carcinomas, including cervical cancer. Human papillomaviruses (HPV) are present in virtually all cervical cancers, and TGF-β has been shown to inhibit the transcription of the E6 and E7 early genes in HPV type 16 immortalized, TGF-β sensitive, human foreskin keratinocytes (HKc/HPV16). Expression of E6 and E7, the HPV16 oncoproteins, is under control of the upstream regulatory region (URR). To investigate elements responsible for TGF-β inhibition of HPV16 early gene expression, we transfected HKc/HPV16 with a URR/luciferase reporter construct. Luciferase expression was inhibited up to 85% upon TGF-β treatment. The URR contains a myriad of transcription factor binding sites, including 7 sites for nuclear factor 1 (NFI). To identify potential transcription factors involved in TGF-β modulation of the URR, we performed DNase I footprint analysis on the HPV16 URR using HKc/HPV16 nuclear extracts treated with or without TGF-β. Differentially protected regions were found to be located around NFI binding sites. Electrophoretic mobility shift assays, using NFI binding sites as probes, showed decreased binding upon TGF-β treatment, which was not due to reduced NFI protein or mRNA levels. Mutational analysis of individual and multiple NFI binding sites in the URR defined their role in TGF-β sensitivity and the relative contribution of each NFI site to basal activity of the promoter. Overexpression of the NFI family members decreased the ability of TGF-β to inhibit URR activity. Since the oncoprotein Ski has been shown to interact with and increase the transcriptional activity of NFI, and since Ski levels are decreased by TGF-β, we explored the possibility that Ski may provide a link between TGF-β signaling and NFI activity. Anti-NFI antibodies co-immunoprecipitated endogenous Ski in HKc/HPV16 nuclear extracts, confirming that NFI and Ski interact in these cells. Ski levels dramatically decrease upon TGF-β treatment, and overexpression of Ski abrogated the ability of TGF-β to inhibit URR activity. Overall, these studies suggest that TGF-β inhibition of E6/E7 expression is mediated by a decrease in Ski levels, which in turn reduces NFI activity.