Abstract
Transforming growth factor‐beta (TGF‐β), a potent inhibitor of normal melanocyte growth, does not significantly suppress growth of melanoma cells. The mechanism of melanocyte desensitization to TGF‐β in the transformation process remains largerly unknown. We investigated whether the tumor promoting phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) may induce melanocyte resistance to TGF‐β. Cell proliferation and DNA synthesis of normal human melanocytes were strongly inhibited by TGF‐β, whereas in the presence of TPA remained largerly unaffected. The inactive phorbol ester 4α‐phorbol 12,13 didecanoate did not modify the TGF‐β antiproliferative effect, whereas the diacylglycerol analog 1‐oleoyl‐2‐acetyl‐sn‐glycerol counteracted TGF‐β effects. Protein kinase C (PKC) is the major cellular receptor of tumor promoting phorbol esters. PKC‐α expression and phosphorylation were almost completely downregulated under combined treatment with TGF‐β + TPA at 24 and 72 h, as shown by immunoblots. Confocal microscopy demonstrated that TGF‐β‐induced nuclear accumulation of PKC‐α was abolished in the presence of TPA at the same time points. The selective PKC inhibitor Ro‐31‐8220 weakened the TGF‐β antiproliferative effect. Smads are central mediators for TGF‐β signal transduction. Smad‐dependent transcriptional activity was suppressed in TGF‐β‐treated melanocytes in the presence of TPA, as well as in ALK5 (constitutively active type I TGF‐β receptor)‐ or Smad3 + Smad4‐transfected melanocytes in the presence of Ro‐31‐8220. In addition, an antisense oligodeoxynucleotide against PKC‐α abolished TGF‐β‐driven Smad‐mediated transcription. These findings show that tumor promoting phorbol esters induce melanocyte resistance to TGF‐β, associated with downregulation of PKC‐α and suppression of Smad‐dependent transcription. This may represent an important mechanism for expansion of melanocytes exposed to PKC‐targeting tumor promoters. J. Cell. Physiol. 214: 363–370, 2008. © 2007 Wiley‐Liss, Inc.