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 TSK 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 (PKC) 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.
Imokawa
G
G
Biological Science Laboratories, Kao Corporation, Tochigi, Japan. 073733@kastanet.Kao.co.jp
Higuchi
K
K
Yada
Y
Y
D000485:Allergens / Q000009:adverse effects*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D054360:Chemokine CXCL1; D018925:Chemokines / Q000737:chemistry; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000737:chemistry; D017449:Dermatitis, Allergic Contact / Q000150:complications*; D006133:Growth Substances / Q000737:chemistry; D006168:Guinea Pigs; D036341:Intercellular Signaling Peptides and Proteins; D008548:Melanosis / Q000209:etiology* / Q000378:metabolism; D009284:Naphthols / Q000009:adverse effects*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D012867:Skin / Q000737:chemistry*; D012995:Solubility
D000485:Allergens; D054360:Chemokine CXCL1; D018925:Chemokines; D019743:Chemokines, CXC; D002630:Chemotactic Factors; C513297:Cxcl1 protein, rat; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D009284:Naphthols; C024336:1-phenylazo-2-naphthol; D011493:Protein Kinase C; D002118:Calcium