Transforming growth factor-beta (TGF-β) signaling is disrupted in many cancers, including cervical cancer, leading to TGF-β resistance. Although initially sensitive, human papillomavirus type 16 (HPV16) immortalized human keratinocytes (HKc/HPV16) become increasingly resistant to the growth inhibitory effects of TGF-β during in vitro progression to a differentiation resistant phenotype (HKc/DR). We have previously shown that loss of TGF-β sensitivity in HKc/DR is attributed to decreased expression of TGF-β receptor type I (TGF-β RI), while the levels of TGF-β receptor type II (TGF-β RII) remain unchanged. The present study explored molecular mechanisms leading to reduced TGF-β RI expression in HKc/DR. Using TGF-β RI and TGF-β RII promoter reporter constructs, we determined that acute expression of the HPV16 oncogenes E6 and E7 decreased the promoter activity of TGF-β RI and TGF-β RII by about 50%. However, promoter activity of TGF-β RI is decreased to a greater extent than TGF-β RII as HKc/HPV16 progress to HKc/DR. Reduced TGF-β RI expression in HKc/DR was found not to be linked to mutations within the TGF-β RI promoter or to promoter methylation. Electrophoretic mobility shift and supershift assays using probes encompassing Sp1 binding sites in the TGF-β RI promoter found no changes between HKc/ HPV16 and HKc/DR in binding of the transcription factors Sp1 or Sp3 to the probes. Also, Western blots determined that protein levels of Sp1 and Sp3 remain relatively unchanged between HKc/ HPV16 and HKc/DR. Overall, these results demonstrate that mutations in or hypermethylation of the TGF-β RI promoter, along with altered levels of Sp1 or Sp3, are not responsible for the reduced expression of TGF-β RI we observe in HKc/DR. Rather the HPV16 oncogenes E6 and E7 themselves exhibit an inhibitory effect on TGF-β receptor promoter activity.

Human papillomavirus type 16 (HPV16) is the primary etiologic agent for greater than 50% of all cervical carcinomas. Expression of the HPV16 E6 and E7 oncoproteins is under control of the upstream regulatory region (URR), which contains a myriad of transcription factor binding sites, including 7 half sites for NFI. These NFI binding sites were used as probes in electrophoretic mobility shift assays (EMSAs), and mutational analysis of individual and multiple NFI binding sites was performed in order to demonstrate the relative importance of particular NFI sites to URR activity. By using 5 NFI half sites as an enhancer, we were able to detect a 4-fold increase in URR activity. Our results define the role and relative contribution of NFI binding sites to the basal activity of the HPV16 promoter, and demonstrate that NFI binding sites can act independently to enhance HPV16 URR activity in immortalized keratinocytes.

Human papillomavirus type 16 (HPV16) is the primary etiologic agent for greater than 50% of all cervical carcinomas. Expression of the HPV16 E6 and E7 oncoproteins is under control of the upstream regulatory region (URR), which contains a myriad of transcription factor binding sites, including 7 half sites for NFI. These NFI binding sites were used as probes in electrophoretic mobility shift assays (EMSAs), and mutational analysis of individual and multiple NFI binding sites was performed in order to demonstrate the relative importance of particular NFI sites to URR activity. By using 5 NFI half sites as an enhancer, we were able to detect a 4-fold increase in URR activity. Our results define the role and relative contribution of NFI binding sites to the basal activity of the HPV16 promoter, and demonstrate that NFI binding sites can act independently to enhance HPV16 URR activity in immortalized keratinocytes.