Biological characterization of human fibroblast-derived mitogenic factors for human melanocytes

Abstract

To clarify the paracrine linkage between human fibroblasts and melanocytes in cutaneous pigmentation, we studied the effects of human fibroblast-derived factors on the proliferation of human melanocytes. In medium conditioned for 4 days with human fibroblast culture, factors were produced that markedly stimulated DNA synthesis of human melanocytes. The stimulatory effect was higher in medium conditioned with fibroblasts from aged skin than in medium conditioned with fibroblasts from young skin, and was interrupted by inhibitors of tyrosine kinase, such as tyrphostin, genistein and herbimycin, but not by inhibitors of protein kinases C and A, such as H-7 and phloretin. The conditioned medium was also capable of activating mitogen-activated protein kinase of human melanocytes, with old fibroblasts being more effective than young ones. Analysis of factors released into the conditioned medium revealed that levels of hepatocyte growth factor (HGF) and stem cell factor (SCF) were increased in old-fibroblast-conditioned medium compared with young-fibroblast-conditioned medium. In contrast, levels of basic fibroblast growth factor (bFGF) were similar in both media. When the conditioned medium was treated with HGF antibody with or without SCF antibody, the increase in DNA synthesis by human melanocytes was decreased to 20% of the elevated level, whereas antibodies to bFGF had no effect. Analysis of the medium conditioned for 4 days after cytokine application demonstrated that, of the cytokines tested, interleukin 1alpha and tumour necrosis factor alpha are highly effective in stimulating HGF secretion by old fibroblasts. HGF and SCF, but not bFGF, were markedly increased in culture medium in the presence of IL-1alpha, and this stimulatory effect was confined to young human fibroblasts. These findings suggest that SCF and HGF derived from human fibroblasts may play a part in regulating cutaneous pigmentation during inflammation and aging.

Intracellular Signaling Mechanisms Leading to Synergistic Effects of Endothelin-1 and Stem Cell Factor on Proliferation of Cultured Human Melanocytes

“…The formation of IP 3 following stimulation is a hallmark for evaluation of PKC activation because IP 3 and the protein kinase C activator, diacylglycerol, are simultaneously generated at an equimolar ratios. The time course of formation of IP 3 following SCF and ET-1 treatment revealed that there is no difference in The mechanism of tyrosine kinase receptor-stimulated mitogenic signaling involves the formation between complexes of the guanine nucleotide exchange protein SOS, and the SH2 and SH3 domain-containing adaptor protein Grb2 with another tyrosine-phosphorylated adaptor protein Shc (21-23). Recent studies have shown that some G protein-coupled receptors utilize the same effectors as the tyrosine kinase receptor pathway (e.g.…”

“…The radioactivity on the filter was directly measured using MATRIX 96 (Packard Bioscience Co.). 3 . Extraction of Cellular Proteins-Cells were incubated with ET-1 and/or SCF in 60-mm diameter culture dishes, then solubilized in 500 l of ice-cold RIPA (radioimmunoprecipitation) buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.25% Na deoxycholate, 1 mM EDTA, 50 mM NaF, 1 mM sodium orthovanadate, 50 mM phenylmethylsulfonyl fluoride, and 1 mg/ml aprotinin.…”

“…2 Furthermore, stem cell factor (SCF) plays an important role in the increased pigmentation of the epidermis overlying benign fibroblastic tumors in dermatofibroma 3 as well as in UVB-induced pigmentation. 4 We have also observed that aged fibroblasts in culture produce a larger amount of SCF than do younger ones (3), which probably provides a basis for the tendency of aged skins to be more sensitive to environmental stimuli and to be easily induced to epidermal hyperpigmentation. SCF is also known as a stimulator for epidermal hyperpigmentation in mastocytosis where mast cells undergo hyperproliferation in response to the soluble type of SCF derived from keratinocytes (4).…”

The Roles of Growth Factors in Keratinocyte Migration

“…76 GM-CSF is a potent autocrine mitogen for KCs. 77,78 GM-CSF activity results in faster wound closure, but it has not been established whether this effect is due to increased KC proliferation resulting from increased KC production of the mitogenic factors IL-6 and interferon-c, increased migration of KCs into the wound bed, or a combination of the two. 79,80 Clinically, the application of recombinant GM-CSF has been shown to be efficacious for the treatment of chronic wounds and burns but has no effect on wound healing in healthy individuals; these effects are most likely mediated through increased recruitment of macrophages and neutrophils and increased vascularization at the wound site rather than via a direct effect on KC migration.…”

UV-B Radiation Induces Macrophage Migration Inhibitory Factor–Mediated Melanogenesis through Activation of Protease-Activated Receptor-2 and Stem Cell Factor in Keratinocytes

“…A few of these cytokines are known mitogens for human melanocytes, including basic fibroblast growth factor, SCF, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, and ET-1. [42][43][44][45][46] Several cytokines, such as IL-1␣ and TNF-␣, significantly increase the expression of SCF protein by cultured human keratinocytes. 5 Accordingly, MIF may induce PAR-2 to mediate cutaneous pigmentation by increasing both the uptake of melanosomes by keratinocytes and the release of SCF, which stimulates melanogenesis.…”