Effects of Calphostin C, Specific PKC Inhibitor on TPA‐Induced Normal Human Melanocyte Growth, Morphology and Adhesion

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Abstract
Normal human melanocytes, which rarely undergo mitosis in vivo, require many growth factors and growth-stimulating agents in vitro, such as basic fibroblast growth factor (bFGF) and cyclic adenosine monophosphate-stimulating agents or 12-0-tetradecanoylphorbol 13-acetate (TPA), to proliferate. TPA, known as a protein kinase C (PKC)-activator, supports normal human melanocyte growth and influences on melanocyte dendrite formation. We have further confirmed the role of the PKC-mediated pathway in the TPA-dependent melanocyte functions-i.e., proliferation, morphology, and adhesion-using Calphostin C (CPC), a highly specific PKC inhibitor. Melanocytes require the continual presence of TPA for growth in culture. Addition of 8 nM TPA to the medium increased melanocyte growth by 198.4 +/- 2.3% of that without TPA. The growth induction by TPA was suppressed by the addition of 10 nM CPC at the level comparable to that without TPA without any morphological alterations. Significant levels of PKC were detected in melanocytes chronically exposed to TPA as determined by Western blotting. A long-term exposure to TPA (more than 5 days) resulted in marked reduction of melanocyte adhesion to plastic cell culture dishes, both uncoated and coated with type IV collagen. By the addition of 10 nM CPC in the adhesion assay, the melanocyte adhesion was further inhibited in both conditions. These results indicated the critical involvement of PKC activation in the TPA-dependent melanocyte functions. Continuous activation of PKC by TPA is implicated in melanocyte growth stimulation. TPA also has effects on melanocyte morphology, causing the formation of long extended dendrites with little cytoplasm. However, inhibition of PKC activation by CPC does not affect the melanocyte morphology, and CPC reduces melanocyte adhesion to uncoated or type IV collagen coated plastic cell culture dishes.