Abstract
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.
Highlights
Evidence has accumulated demonstrating that chemical mediators such as arachidonic metabolites (3, 4) or growth factorlike substances (5) secreted during inflammation could be responsible for stimulating the function and population of melanocytes, leading to hyperpigmentation induced by several stimuli including ultraviolet (UV) light
Purification of Melanogenic Soluble Factors—In previous studies we reported that DNA synthesis and melanogenesis of cultured guinea pig melanocytes were stimulated by the addition of skin extracts obtained on days 7, 16, 19, and especially on day 28 postchallenge with PAN (2)
Recent studies suggest that paracrine linkage between melanocytes and surrounding cells in skin tissue such as keratinocytes and fibroblasts plays an important role in stimulating proliferation and melanogenesis of melanocytes, leading to epidermal hyperpigmentation (5, 7, 9)
Summary
Evidence has accumulated demonstrating that chemical mediators such as arachidonic metabolites (3, 4) or growth factorlike substances (5) secreted during inflammation could be responsible for stimulating the function and population of melanocytes, leading to hyperpigmentation induced by several stimuli including ultraviolet (UV) light. Few of these mediators are known as intrinsic cytokines responsible for hyperpigmentation in vivo. We have extended our previous work and have purified a 7.9-kDa polypeptide from guinea pig skin, and we have characterized its effects on melanocytes We identified this factor to be a member of the growth-related oncogene ␣ (GRO-␣) family or very close to it. It seems likely that specific melanogenic factors, such as GRO-␣, produced in allergic cutaneous inflammation are responsible for the induction of late appearing hyperpigmentation by PAN allergy
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