Differential effects on melanocyte growth and melanization of low vs. high calcium keratinocyte-conditioned medium

Epidermal keratinocytes secrete several growth factors that stimulate melanocyte proliferation and melanin pigment synthesis in vitro. As the epidermis is formed of two distinct layers, i.e. basal cell layer and suprabasal cell layers, and both are functionally and biologically different compartments, it was interesting to investigate which type of epidermal keratinocytes modulate melanocyte proliferation and function most. Normal human epidermal melanocytes (HMel) were incubated with melanocyte-conditioned medium (M-CM) and low Ca2+ and high Ca2+ keratinocyte-conditioned medium (K-CM) obtained from the same skin source of melanocytes. The morphology, proliferation rate and melanin synthesis were evaluated at days 3, 6 and 12 of incubation. The results showed no evidence of major morphological changes in the epidermal melanocytes with any of the conditioned media, although marked dendrite formation was observed in coculture of melanocytes and differentiated keratinocytes. On the other hand, low Ca2+ K-CM induced a mild but statistically significant stimulation of melanocyte growth in a time-dependent manner. The significant percentage increase was evident on day 6 (124.6%, P < 0.05) and on day 12 (138.1%, P < 0.01) of incubation. In contrast, high Ca2+ K-CM showed no significant effect on melanocyte proliferation (P > 0.05). Both low Ca2+ and high Ca2+ K-CM stimulated melanin synthesis, although synthesis induced by low Ca2+ K-CM was higher than that of high Ca2+ K-CM. The significant percentage increase induced by low Ca2+ K-CM was evident on day 6 (117.9%, P < 0.05) and on day 12 (127.8%, P < 0.05) of incubation, whereas it was evident with high Ca2+ K-CM only on day 12 (119.7%, P < 0.05) of incubation. It is concluded from the above data that keratinocytes grown at a low Ca2+ level release factors that stimulate melanocyte proliferation as well as melanin synthesis, whereas keratinocytes grown at a high Ca2+ level release factors that only stimulate melanin synthesis. This may provide an explanation of the anatomical position of melanocytes and may play a part in the pigmentary changes following injury to epidermal cells.