Mechanisms of decreased expression of transforming growth factor-beta receptor type I at late stages of HPV16-mediated transformation.

Transforming growth factor-beta (TGF-beta) signaling is disrupted in many cancers, including cervical cancer, leading to TGF-beta resistance. Although initially sensitive, human papillomavirus type 16 (HPV16) immortalized human keratinocytes (HKc/HPV16) become increasingly resistant to the growth inhibitory effects of TGF-beta during in vitro progression to a differentiation resistant phenotype (HKc/DR). We have previously shown that loss of TGF-beta sensitivity in HKc/DR is attributed to decreased expression of TGF-beta receptor type I (TGF-beta RI), while the levels of TGF-beta receptor type II (TGF-beta RII) remain unchanged. The present study explored molecular mechanisms leading to reduced TGF-beta RI expression in HKc/DR. Using TGF-beta RI and TGF-beta RII promoter reporter constructs, we determined that acute expression of the HPV16 oncogenes E6 and E7 decreased the promoter activity of TGF-beta RI and TGF-beta RII by about 50%. However, promoter activity of TGF-beta RI is decreased to a greater extent than TGF-beta RII as HKc/HPV16 progress to HKc/DR. Reduced TGF-beta RI expression in HKc/DR was found not to be linked to mutations within the TGF-beta RI promoter or to promoter methylation. Electrophoretic mobility shift and supershift assays using probes encompassing Sp1 binding sites in the TGF-beta 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-beta RI promoter, along with altered levels of Sp1 or Sp3, are not responsible for the reduced expression of TGF-beta RI we observe in HKc/DR. Rather the HPV16 oncogenes E6 and E7 themselves exhibit an inhibitory effect on TGF-beta receptor promoter activity.