Transforming Growth Factor-β1 Inhibits Basal Melanogenesis in B16/F10 Mouse Melanoma Cells by Increasing the Rate of Degradation of Tyrosinase and Tyrosinase-related Protein-1

Abstract

Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-␤1 (TGF-␤1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-␤1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (Dopa) oxidase activities, to less than 30% of the control values but had no effect on dopachrome tautomerase activity (TRP-2). The inhibition affected to similar extents the Dopa oxidase activity associated to tyrosinase-related protein-1 (TRP-1) and tyrosinase. This inhibition was noticeable between 1 and 3 h after the addition of the cytokine, and maximal after 6 h of treatment. The decrease in the enzymatic activity was paralleled by a decrease in the abundance of the TRP-1 and tyrosinase proteins. TGF-␤1 mediated this effect by increasing the rate of degradation of tyrosinase and TRP-1. Conversely, after 48 h of treatment, the expression of the tyrosinase gene decreased only slightly, while TRP-1 and TRP-2 gene expression was not affected. An increased rate of proteolytic degradation of TRP-1 and tyrosinase seems the main mechanism accounting for the inhibitory effect of TGF-␤1 on the melanogenic activity of B16/F10 cells.Melanin pigmentation in mammals is a highly regulated and complex event occurring within specialized cells, the melanocytes (1-3). The pathway starts by two reactions catalyzed by tyrosinase (monophenol monooxygenase, EC 1.14.18.1), the hydroxylation of L-tyrosine to L-Dopa, 1 and the oxidation of LDopa to dopaquinone. In the absence of thiolic compounds, dopaquinone evolves spontaneously to L-dopachrome, and then to melanin. Two other enzymes, TRP-1 and TRP-2, have been shown to participate in the process. TRP-2, also called DCT (EC 5.3.2.3.), catalyzes the non-decarboxylative rearrangement of L-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (4 -6). This stable diphenol is oxidized by TRP-1 to an unstable oquinone that undergoes further polymerization reactions to yield melanin (7,8). Moreover, other melanosomal proteins such as pmel 17 could also be involved in the control of the distal melanogenic reactions (9, 10). Tyrosinase itself could also participate in other oxidative steps, thus enhancing the polymerization rate of melanogenic intermediates (11,12).Due to the high reactivity of the melanogenic intermediates, melanogenesis is confined in specialized organelles called melanosomes. Epidermal melanization involves not only the synthesis of the pigment but also the transfer of melanized melanosomes to the keratinocytes surrounding the dendritic processes of melanocytes. Thus, melanocytes are in intimate physical and functional contact with other epidermal cells, which could contribute to the control of the melanogenic status of melanocytes through the secretion of several cytokines and paracrine factors. In keeping with this view, the skin contains a variety of chemical messengers with the potential to affect the prolifer...

Novel Screening Method for Potential Skin-Whitening Compounds by a Luciferase Reporter Assay

“…The treatment time for compounds with this reporter assay system was 24 h, this being much shorter than the time needed for the usual melanin production assay using B16 melanoma cells. 2,4,5) This reporter assay system was more highly responsive than the usual method and proved to be suitable for a high-throughput screening assay. We then compared the effect on tyrosinase promoter activity of 56 compounds, subsequently finding with this reporter assay system that carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity.…”

“…Since the over-production and accumulation of melanin might occur in melasma, freckles, and other forms of melanin hyper-pigmentation, melanogenesis inhibitors have been developed as agents for use in medications and cosmetics; [1][2][3] for example, such tyrosinase inhibitors as arbutin, hydroquinone, and ellagic acid have been included in some cosmetic products. 14,15) Although we could also find sulforaphane with the usual method by measuring the melanin content using B16 melanoma cells, 5) this method took a long time and was troublesome for high-throughput screening.…”

“…[1][2][3] Over-production and accumulation of melanin, however, may cause various skin disorders. The use of skin-whitening agents, particularly by women, has been growing in recent years.…”

“…However, this method suffers from such disadvantages as the time taken, its sensitivity, and troublesomeness; for example, the usual method to measure melanin production needs 48-96 h upon treating with the compound being tested. 2,4,5) Furthermore, the procedure requires culturing cells at the scale of at least a 35-mm dish or well in a 6-well plate, and involves such troublesome steps as cell counting and rendering melanin soluble. As an alternative method for screening skin-whitening agents, we developed a reporter assay system for measuring the promoter activity of the tyrosinase gene, the most important regulator of melanin synthesis.…”

Cyclohexanediol Bis-Ethylhexanoate Inhibits Melanogenesis of Murine B16 Melanoma and UV-Induced Pigmentation in Human Skin

“…Hence, many natural and synthetic substances have been developed for cosmetic and pharmaceutical purposes as tyrosinase inhibitors. 4) These inhibitors act by regulating tyrosinase catalytic activity, 5,6) mRNA transcription, 7,8) or maturation. 9) Indeed, unsaturated fatty acids, such as oleic acid, linoleic acid, and α-linoleic acid, have recently been shown to regulate melanogenesis by modifying tyrosinase activity at the post-transcriptional levels.…”