liritish lournal of Dermatology {\ 992) 126. 118-124.

Effects of staurosporine, PMA and A23187 on human melanocyte cultures with dibutyryl cyclic AMP K.MAEDA.*t Y.TOMITA.* M.FUKUDAt AND H.TAGAMI 'Department of Dermatologii. Tohoku iJniversitii School of Medicine. Sendai. Japati 'tSliisetdo Research Center. Yokohama, /d/wn Accepted for publication 5 September 1991

Summary

Staurosporine. a protein kinase (I'K) inhibitor, phorbol-12-myristate-l 3-acetate (PMA). a PKC activator and A2 3187 calcium ionophore were added to human melanocyte cultures with or without dibutyryl cyclic AMP (dbcAMP). After 2 days' incubation, changes in various melanogenic factors were examined such as tyrosinase activity and the amount of tyrosinase-related protein (TRP) as well as the morphology ofthe meianocytes. dbcAMP stimulated all the melanogenic factors. Staurosporine increased tyrosinase activity and amount of TRP and caused morphological changes with the formation of numerous dendrites. regardless of the presence of dbcAMP. In contrast. PMA did not signiiicantly affect tyrosinase activity. TRP content or dendrite formation, with or without dbcAMi'. The effects of staurosporine on tyrosinase activity and TRP content were completely inhibited by PMA. but PMA did not significantly affect the staurosporine-induced morphological changes. A2J187 inhibited both tyrosinase activity and TRP content, regardless ofthe presence of dbcAMP. but did not affect the morphology of meianocytes. These findings suggest that tyrosinase activity and TRP content are regulated by adenylate cyclase and Ca- ' and partly by PKC. while the morphological features of meianocytes are affected by intracellular cAMP accumulation and by the inhibition of PKC.

Cyclic AMP (cAMP), the first example of a second messenger for hormone action in various cells, is well iiuown to accelerate meianogenesis in mouse melanoma cells' * and normal human meianocytes in vitro.^ Similar iindings have been reported regarding cultured human meianocytes when the intracellular cAMP level is enhanced by theophylline. 3-isobutyl-1 -methylxanthine (IBMX) and other phosphodiesterase inhibitors.' Another major messenger system in cells is one based on an inositol lipid. a structural component of the plasma membrane. During receptor activation, the inositol lipid is cleaved into its two constituent parts: inositol 1.4,5-triphosphate functioning as a caiciummohllizer and diacylglycerol. an activator of protein kinase C (PKC).''" Recently, it has been suggested that PKC and calcium In the second-messenger transduction mechanism play some role in meianogenesis. Gordon and Gilchrest^ reported that I-oleoyl 2-acetyI glycerol Correspondence: Dr Yasusbi Tomita. l>;partment of Dermatology. Toboku University Scbciol of Medicine. 1-1 Seiryo-machi. Aoba-ku. Sendai 980, Japan.

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(OAG). a synthetic analogue of diacylglycerol. increased melanin synthesis in human meianocytes mediated through the activation ofthe PKC pathway. Also OAG caused a significant augmentation of melanin content in normal human meianocytes. and 12-0-tetradecanoyi phorbol 13-acetate (TPA) and di-octanoyi glycerol (DOG), which activate PKC, caused a small increase of melanin."^ In contrast, meianogenesis. in Bid mouse melanoma cells was inhibited by TPA.'" OAG'' and by the calcium ionophore A23187.^~ These differing effects of PKC activators and Ca^^ in various pigment cells suggest a complex involvement of PKC and Ca-' in meianogenesis. To clarify Ihe possible participation of the signal transduction system based on the inositol lipids in the pigmentation of human skin, we examined the effect of a PK inhibitor, staurosporine,''''' a PKC activator. PMA,'^ and calcium ionophore, A23187."' on the cAMP-mediated induction of tyrosinase and tyrosinaserelated protein (TRP) as well as on the morphology of cells from cultures of normal human meianocytes.

HUMAN MELANOCYTE CULTURES

Methods Chemicals The medium MCDB15 3 was obtained from Kyokuto Pharmaceutical Co., Tokyo. Japan. Bovine pituitary extract (BPE) was from Clonetics Corporation. San Diego. CA. U.S.A. Recombinant human epidermal growth factor (EGF) was irom Wakunaga Pharmaceutical Co.. Hiroshima, japan. Staurosporine was from Calbiochem. La Jolla. CA, U.S.A. Other chemicals were from Sigma. St Louis. MO. U.S.A. Melanocyte culture Meianocytes were isolated from the forearms of healthy lapane.se men (20-24 years of age) using the suctionblister method'"" and were incubated in MCDBI5 3 medium containing 0-15 mM CaCi2, 10 ng/ml EGF, 5 /(g/ml insulin. 50 ^(g/ml bovine pituitary extract (BPE), 10% foetal calf serum (FCS) and 20 ng/ml of phorbol-12myristate-13-acetate (PMA) for 4 days, followed by 3 days in the medium without PMA. This treatment resulted in the virtual elimination of all contaminating cells such as keratinocytes. Cells cultured in this medium exhibited bipolar and polydendritic phenotypes and continued to proliferate. In studies of intracellular signal transduction systems in metanocytes, BPE is always eliminated because it contains a potent melanocyte mitogen that stimulates the formation of dendrites and pigmentation. The cells were cultured in glass LabTek chamber slides (Miles Laboratories. Naperville, IL, U.S.A.) for 2 days with MCDBl 5 3 medium without BPE and PMA, to which various combinations of N''-2' 0-dibutyryl adenosine 3':5'-cyclic monophosphate (dbcAMP) and second-messenger activator and/or inhibitor were added. After the addition of the agents and following incubation for 2 days the cultures were fixed.

Imnnmofluorescence staining and morphometry Cultured melanocytes were fixed with methanol and acetone and stained immunocylologically with a rat monoclonal antibody (TMIl-1)"* against TRP (brown (b) locus protein)''' and a secondary antibody conjugated with tluorescein isothiocyanate (FITC: TAGO Inc. Burlingame. CA. U.S.A.).^" The intensity of fluorescence relative to the amount of antigen in the melanocyte was measured using a microphotometer (model P-1. Nikon. Tokyo, japan). Cellular features such as the number of dendrites. perimeter and area were determined on fluorescence microscopy and with an image analyser

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(model PC-fN 502. NEC, Tokyo. Japan) connected to a personal computer (model PC-9801VM, NEC, Tokyo. Japan) and with the appropriate software as previously described.'' The perimeter of a meianocyte was tneasured as a substitute for the sum of the length of all dendrites. although the perimeter of the cell reflects not only the length of each dendrite but also the size of the cell. Assay for tyrosinase activity Human melanocytes were seeded at a density of 2500 cells per well in 100 fi\ medium in 9fi-well plates. After 24 h. dbeAMP and other agents were added to the wells. The cells were incubated for 2 days at 3 V^C. washed with PBS and lysed with 45 /d of 1% Triton-X/PBS. After vibration. 5 /il of 10 mM L-DOPA was added to the wells and foiiowing incubation of the plates at 37''C for 30 min. the absorbance measured at 475 nm in a MODH^3550 ELISA Reader (Bio-Rad Lab., Richmond, CA. U.S.A.). The absorbance values were compared with a standard curve obtained using tnushroom tyrosinase (Sigma. St Louis, MO. U.S.A.): the standard curve was linear within the range of experimental values. The coefficient of correlation was determined as 0-999. Statistical analysis Thirty individual cells per field were selected by random sampling and tneasured in the immunofluorescence study. Student's (-test was used to compare the means (± SD) of the determinations.

Results Effect ofcijclic AMP enhancing agents on normal human melanocytes In vitro The effects of various cAMP-enhancing agents on TRP content and the morphology of human melanocytes are shown in Figure 1. The addition of various concentrations of adenosine, dbcAMP. and forskolin.--. an adenylate cyclase activator, to the medium resulted in increased TRP content as demonstrated by immunoreactive TRP and a notable cellular enlargement. Their effects on TRP levels were dose-dependent and significant effects were observed at 10 //M and 100 /tM (P

Effects of staurosporine, PMA and A23187 on human melanocyte cultures with dibutyryl cyclic AMP.

Staurosporine, a protein kinase (PK) inhibitor, phorbol-12-myristate-13-acetate (PMA), a PKC activator and A23187 calcium ionophore were added to huma...
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