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

Disruption of the transforming growth factor-beta (TGF-β) signaling pathway is observed in many cancers, including cervical cancer, resulting in TGF-β resistance. While normal human keratinocytes (HKc) and human papillomavirus type 16-immortalized HKc (HKc/HPV16) are sensitive to the growth inhibitory effects of TGF-β, HKc/HPV16 develop resistance to TGF-β1 as they progress in vitro to a differentiation resistant phenotype (HKc/DR). The loss of sensitivity to the antiproliferative effects of TGF-β1 in HKc/DR is due, at least partially, to decreased expression of the TGF-β 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-β could contribute to the development of TGF-β resistance during in vitro progression of HKc/HPV16, and whether TGF-β induction of a Smad-responsive reporter gene was altered in HKc/DR.

These results demonstrate that alterations in Smad protein levels are not associated with the loss of response to the antiproliferative effects of TGF-β in HKc/DR, but that diminished and delayed Smad phosphorylation and nuclear localization, and decreased Smad signaling occur in response to TGF-β in HKc/DR.

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-β signaling using a luciferase reporter construct.

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-β1 observed in HKc/DR. However, we found diminished Smad2 phosphorylation, and delayed nuclear Smad3 localization in response to TGF-β1 in HKc/DR, compared to normal HKc and TGF-β sensitive HKc/HPV16. In addition, we determined that TGF-β1 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-β in HKc/DR, but that diminished and delayed Smad phosphorylation and nuclear localization, and decreased Smad signaling occur in response to TGF-β in HKc/DR.