EFFECTS OF CALPHOSTIN-C, SPECIFIC PKC INHIBITOR ON TPA-INDUCED NORMALHUMAN MELANOCYTE GROWTH, MORPHOLOGY AND ADHESION

Normal human melanocytes, which rarely undergo mitosis in vivo, requir e many growth factors and growth-stimulating agents in vitro, such as basic fibroblast growth factor (bFGF) and cyclic adenosine monophospha te-stimulating agents or 12-0-tetradecanoylphorbol 13-acetate (TPA), t o proliferate. TPA, known as a protein kinase C (PKC)-activator suppor ts normal human melanocyte growth and influences on melanocyte dendrit e formation. me have further confirmed the role of the PKC-mediated pa thway in the TPA-dependent melanocyte functions-i.e., proliferation, m orphology, and adhesion-using Calphostin C (CPC), a highly specific PK C inhibitor. Melanocytes require the continual presence of TPA for gro wth in culture. Addition of 8 nM TPA to the medium increased melanocyt e growth by 198.4 +/- 2.3% of that without TPA. The growth induction b y TPA was suppressed by the addition of 10 nM CPC at the level compara ble to that without TPA without any morphological alterations. Signifi cant levels of PKC were detected in melanocytes chronically exposed to TPA as determined by Western blotting. A long-term exposure to TPA (m ore than 5 days) resulted in marked reduction of melanocyte adhesion t o plastic cell culture dishes, both uncoated and coated with type IV c ollagen. By the addition of 10 nM CPC in the adhesion assay, the melan ocyte adhesion was further inhibited in both conditions. These results indicated the critical involvement of PKC activation in the TPA-depen dent melanocyte functions. Continuous activation of PKC by TPA is impl icated in melanocyte growth stimulation. TPA also has effects on melan ocyte morphology, causing the formation of long extended dendrites wit h little cytoplasm. However inhibition of PKC activation by CPC does n ot affect the melanocyte morphology, and CPC reduces melanocyte adhesi on to uncoated or type IV collagen coated plastic cell culture dishes.