Purification and characterization of an allergy-induced melanogenic stimulating factor in brownish guinea pig skin

We have demonstrated recently that phenylazonaphthol (PAN) allergy-induced hyperpigmentation in brownish guinea pig skin is associated with the concomitant appearance of a melanogenic soluble factor(s) that activates the intracellular signal transduction system, including phosphatidylinositol turnover subsequent to ligand-receptor binding in cultured guinea pig melanocytes. In this study we have purified and characterized the PAN-induced melanogenic stimulating factor (PIMSF) that occurs in allergy-associated hyperpigmented skin. By successive column chromatography on TSH 2000SW, Mono Q, and octadecyl-NPR, the PIMSF was purified 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 original skin homogenate. In cultured guinea pig melanocytes, this purified PIMSF had the potential of activating an intracellular signal transduction system such as inositol 1,4,5-trisphosphate formation and intracellular calcium levels through a pertussis toxin-sensitive G protein-coupled receptor. PIMSF consistently caused a rapid translocation of cytosolic protein kinase C (PRC) to membrane-bound PKC within 5 min of treatment with a return to the basal level after 120 min, The stimulating effects of PIMSF on proliferation and melanization of cultured guinea pig melanocytes were abolished completely by a PKC down-regulating agent (phorbol 12,13-dibutyrate), PIMSF was similar in molecular mass 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 blotting analysis, Further, the stimulatory effect of purified PIMSF on DNA synthesis of cultured guinea pig melanocytes was suppressed markedly by the addition of anti-rat GRO-alpha antibody, implying that the PIMSF is apparently identical to GRO-alpha. These findings suggest that PAN allergy provides a new mechanism of hyperpigmentation in which biological factors such as the GRO-alpha superfamily generated within allergy-induced skin stimulate melanocytes through activation of the PKC-related signal transduction pathway.