TGF-ß regulation of gene expression at early and late stages of HPV16-mediated transformation of human keratinocytes.

In our in vitro model for HPV16-mediated transformation, HPV16-immortalized human keratinocytes (HKc/HPV16) give rise to differentiation resistant, premalignant cells (HKc/DR). HKc/DR, but not HKc/HPV16, are resistant to growth inhibition by transforming growth factor beta (TGF-ß), due to a partial loss of TGF-ß receptor type I. We show that TGF-ß activates a Smad-responsive reporter construct in HKc/DR to about 50% of the maximum levels of activation observed in HKc/HPV16. To investigate the functional significance of residual TGF-ß signaling in HKc/DR, we compared gene expression profiles elicited by TGF-ß treatment of HKc/HPV16 and HKc/DR on Agilent 44k human whole genome microarrays. TGF-ß altered the expression of cell cycle and MAP kinase pathway genes in HKc/HPV16, but not in HKc/DR. However, epithelial-mesenchymal transition (EMT) responses to TGF-ß were comparable in HKc/HPV16 and HKc/DR, indicating that the signaling pathways through which TGF-ß elicits growth inhibition diverge from those that induce EMT in HPV16-transformed cells.

Partial loss of Smad signaling during in vitro progression of HPV16-immortalized human keratinocytes.

BACKGROUND: Disruption of the transforming growth factor-beta (TGF-ß) signaling pathway is observed in many cancers, including cervical cancer, resulting in TGF-ß resistance. While normal human keratinocytes (HKc) and human papillomavirus type 16-immortalized HKc (HKc/HPV16) are sensitive to the growth inhibitory effects of TGF-ß, HKc/HPV16 develop resistance to TGF-ß1 as they progress in vitro to a differentiation resistant phenotype (HKc/DR). The loss of sensitivity to the antiproliferative effects of TGF-ß1 in HKc/DR is due, at least partially, to decreased expression of the TGF-ß receptor type I. In the present study, we explored in detail whether alterations in Smad protein levels, Smad phosphorylation, or nuclear localization of Smads in response to TGF-ß could contribute to the development of TGF-ß resistance during in vitro progression of HKc/HPV16, and whether TGF-ß induction of a Smad-responsive reporter gene was altered in HKc/DR. METHODS: Western blot analysis was used to assess Smad protein levels. In order to study Smad nuclear localization we performed indirect immunofluorescence. In addition, we determined Smad-mediated TGF-ß signaling using a luciferase reporter construct. RESULTS: We did not find a decrease in protein levels of Smad2, Smad3 or Smad4, or an increase in the inhibitory Smad7 that paralleled the loss of sensitivity to the growth inhibitory effects of TGF-ß1 observed in HKc/DR. However, we found diminished Smad2 phosphorylation, and delayed nuclear Smad3 localization in response to TGF-ß1 in HKc/DR, compared to normal HKc and TGF-ß sensitive HKc/HPV16. In addition, we determined that TGF-ß1 induction of a Smad responsive promoter is reduced by about 50% in HKc/DR, compared to HKc/HPV16. CONCLUSIONS: These results demonstrate that alterations in Smad protein levels are not associated with the loss of response to the antiproliferative effects of TGF-ß in HKc/DR, but that diminished and delayed Smad phosphorylation and nuclear localization, and decreased Smad signaling occur in response to TGF-ß in HKc/DR.