The long term goal of this laboratory is to elucidate the mechanisms underlying the formation of keloids, which are benign tumors that develop during an abnormal wound healing program. Our laboratory has reported an abnormal pattern of responses of keloid fibroblasts in vitro that may help to elucidate the underlying mechanism of abnormal growth and matrix metabolism of keloids, in vivo. In cultures derived from keloid and normal dermal fibroblasts, TGFbeta partially reverses the stimulation of DNA synthesis by EGF, PDGF, and SmC in normal, but not keloid cells. It has been reported that, in mink epithelial cells, growth inhibition by TGFbeta is linked to decreased phosphorylation of the retinoblastoma protein during Gl progression and induction of transcription of genes encoding certain inhibitors of cyclin dependent kinases. Therefore, the specific aims of the studies described herein are to 1) determine if the differential effects of TGFbeta on the response of keloid and normal fibroblasts to EGF are accompanied by differential phosphorylation of the retinoblastoma (Rb) protein, and 2) determine if these effects are accompanied by differential expression of the cyclin dependent kinase inhibitors p15 and p21. Confluent cultures from both keloid and normal fibroblasts in 1 percent serum will be exposed to either TGFbeta alone, EGF in the presence or absence of TGFbeta, or neither growth factor. Cells from each condition described will be harvested at various times for immunoblotting with antibodies against either Rb, p15 or p21. Intensity of the bands will estimate protein levels and relative electrophoretic mobility will vary with the phosphorylation status of Rb. The outcomes of these studies will contribute to an understanding of the pathology underlying the formation of keloids, the regulatory events governing cell cycle progression, and the effects of perturbation of those events on biological systems.
