PURIFICATION AND CHARACTERIZATION OF AN ALLERGY-INDUCED MELANOGENIC STIMULATING FACTOR IN BROWNISH GUINEA-PIG SKIN

We have demonstrated recently that phenylazonaphthol (PAN) allergy-ind uced hyperpigmentation in brownish guinea pig skin is associated with the concomitant appearance of a melanogenic soluble factor(s) that act ivates the intracellular signal transduction system, including phospha tidylinositol turnover subsequent to ligand-receptor binding in cultur ed guinea pig melanocytes. In this study we have purified and characte rized the PAN-induced melanogenic stimulating factor (PIMSF) that occu rs in allergy-associated hyperpigmented skin. By successive column chr omatography on TSH 2000SW, Mono Q, and octadecyl-NPR, the PIMSF was pu rified to homogeneity with a single band of apparent molecular mass of 7.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, The specific bioactivity of PIMSF increased by 5,195-fold over the or iginal skin homogenate. In cultured guinea pig melanocytes, this purif ied PIMSF had the potential of activating an intracellular signal tran sduction system such as inositol 1,4,5-trisphosphate formation and int racellular calcium levels through a pertussis toxin-sensitive G protei n-coupled receptor. PIMSF consistently caused a rapid translocation of cytosolic protein kinase C (PRC) to membrane-bound PKC within 5 min o f treatment with a return to the basal level after 120 min, The stimul ating effects of PIMSF on proliferation and melanization of cultured g uinea pig melanocytes were abolished completely by a PKC down-regulati ng agent (phorbol 12,13-dibutyrate), PIMSF was similar in molecular ma ss to rat growth-related oncogene alpha (GRO-alpha; molecular mass of 7.9 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had immunocross-reactivity with GRO-alpha upon Western immune blot ting analysis, Further, the stimulatory effect of purified PIMSF on DN A synthesis of cultured guinea pig melanocytes was suppressed markedly by the addition of anti-rat GRO-alpha antibody, implying that the PIM SF is apparently identical to GRO-alpha. These findings suggest that P AN allergy provides a new mechanism of hyperpigmentation in which biol ogical factors such as the GRO-alpha superfamily generated within alle rgy-induced skin stimulate melanocytes through activation of the PKC-r elated signal transduction pathway.