THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 27, Issue of September 25, pp. 18352-18357,1991 ( 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U. S. A.

Effects of Endothelins on Signal Transduction and Proliferation in Human Melanocytes*

(Received for publication, February 21, 1991)

Yukihiro Yada, Kazuhiko Higuchi, and Genji ImokawaS From the Tochigi Research Laboratories, Kao Corporation, Ichikaimachi-2606, Tochigi 321 -34, Japan

We demonstrate here that human melanocytes could be regulated by endothelin (ET) derivatives, potent vasoconstrictive peptides synthesized by endothelial cells, to stimulate their proliferation and melanization via a receptor-mediated signal transduction pathway. Receptor-binding assay using [ ’2fiI]ET indicated that unlabeled ET-1 or ET-2 competitively inhibited each binding of labeled ETs to melanocytes with a concen- tration for half-maximal inhibition (ICfi0) of 0.7 or 0.9 nM, respectively. The dissociation constant ( K d ) and the number of sites of the specific bindings of ET-1 and those of ET-2 were almost the same (Kd: 1.81 nM, binding sites: 7.0-8.0 X lo4 per cell). Upon incubation with cultured cells, the mass contents of inositol 1,4,5- trisphosphate and intracellular calcium level were sub- stantially increased by 10 nM ET-1, ET-2, and ET-3, but not by big-ET with maximal response a t 80-130-s postincubation. The addition of ET-1 and ET-2 at 1- 50 nM concentrations caused human melanocytes to significantly stimulate DNA ([3H]thymidine incorpo- ration) and melanin synthesis (3H20 release and [‘“C] thiouracil incorporation). Furthermore, ETs exhibited an additive stimulatory effect on basic fibroblast growth factor-stimulated DNA synthesis. In a long- term serum-free culture system, the strongest stimu- lation of growth by 10 nM ET-1 or ET-2 was observed in the presence of 10 nM cholera toxin and 0.2% bovine pituitary extract, resulting in a 4.5-fold increase in cell number for 12 culture days. These findings strongly suggest involvement of ET in the mechanism regulating proliferation and melanization of human melanocytes.

Melanocytes are cells which produce unique biological or- ganelles, melanosomes whose melanin pigment is synthesized by the action of a specific enzyme, tyrosinases (1). Pigmen- tation occurs primarily in the skin, hair bulbs (2), and eyes, undergoing control through melanocyte function by many intrinsic environmental factors which may be derived from surrounding cells such as fibroblasts, keratinocytes, and endo- thelial cells. In the skin, melanocytes maintain their constant population and their melanogenic activity in steady state, but respond to several stimulus such as UV radiation (3) and chemical irritation (4), to accentuate proliferation and differ- entiation, leading to hyperpigmentation of the skin. Recently, knowledge has been accumulating on endogenous factors con-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: Tochigi Research Laboratories, Kao Corp., Ichikaimachi-2606, Tochigi 321-34, Japan. Tel.: 285-83-7467; Fax: 285-83-7469.

trolling melanocyte proliferation such as basic fibroblast growth factor (FGF)’ (5) and leucotriens (LT) (6), which are found to also be producible by their surrounding cells, kera- tinocytes. While normal human melanocytes survive only when cultured in the presence of phorbol 12-myristate 13- acetate (PMA) (7), basic FGF and leukotriene have so far been the only two intrinsic endogenous substances which can substitute for PMA.

In many cells, proliferative stimulants trigger a cascade of basic physiological and biochemical mechanisms. Several growth factors cause phosphatidylinositol breakdown and pro- duce inositol phosphates (&lo), which increase intracellular calcium ([Ca”]J, eventually leading to accentuation of cell proliferation or differentiation. Despite the well-known action of PMA on protein kinase C (PKC)-related signal transduc- tion (11-13), no direct evidence for cellular events including production of inositol phosphates and [Ca2+], mobilization in basic FGF-stimulated proliferation has been reported in hu- man melanocytes (14). Thus, molecular mechanisms under- lying growth factor stimulation of melanocytes and their role in controlling the potential of melanization are poorly under- stood.

Recently, vasoconstrictive peptides, ETs, which are synthe- sized by endothelial cells, have been reported to possess hormonal regulatory activities in various cells or target organs via a receptor-mediated biochemical mechanism (15-18). TO date, four mammalian ETs, ET-1, ET-2, ET-3, and big-ET, have been reported (19-21), but little information is available about the comparative biological activities of ET derivatives. Ohnishi-Suzaki et al. (22) indicate that two fibroblast popu- lations (murine and human fibroblast cell lines) possess high affinity receptors for ET-1 and ET-2 and substantially no receptors for ET-3, and the binding of ET-1 and ET-2 in- creases [Ca2+]; in these cell lines. ET-1 was also found to stimulate the proliferation of fibroblasts through signal trans- duction associated with inositol phosphate (23). Since basic FGF is produced in abundance by endothelial cells and has a strong mitogenic potential for human melanocytes, the ques- tion arises whether ETs might also stimulate melanocytes through the above-described signal transduction pathway. In this report, using a defined serum-free culture system, we have examined whether ET derivatives cause melanocytes to increase production of receptor-mediated inositol phosphate and [Ca2+Ii mobilization and investigated the physiological role of ET derivatives for proliferation and melanization of human melanocytes.

The abbreviations used are: FGF, fibroblast growth factor; ET, endothelin; PMA, phorbol-12-myristate-13-acetate; LT, leucotrien; PKC, protein kinase C; [Ca”];, intracellular calcium level; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; BPE, bovine pituitary ex- tract; CT, cholera toxin; BSA, bovine serum albumin; PBS, phos- phate-buffered saline; UVA, ultraviolet A; PUVA, 8-methoxypsoralen + UVA.

18352

The Effects of Endothelins on Human Melanocytes 18353

FIG. 5. Ratio (340/380 nm) of im- ages of Fura-2 fluoresence induced by ET-1. Fura-2/AM-loaded cells were stimulated by ET derivatives (10 nM). These results are manifested in the ratio of fluoresence of 340 nm to that of 380 nm. a, 0 s (control); b, 90 s; e, 180 s; d, 360 s.

1:

9.33. m.20.

EXPERIMENTAL PROCEDURES AND RESULTS~

DISCUSSION

The present studies give, for the first time, insights into the role of ETs as an another mitogen for human melanocytes. The potential of ETs, especially ET-1, to survive and prolif- erate human melanocytes in pure culture was significantly higher than that of basic FGF at the same concentration (10 nM). Of the mitogens for human melanocytes, PMA is the first discovered one which is utiliid for long-term culture of human melanocytes (7,32). Since PMA is known to activate PKC, which is deeply associated with cell reproduction (33), and to induce no significant down-regulation of PKC at the concentrations used for cultures, it is conceivable that the potentiation of PKC may result in surviving human melano- cytes. By contrast, keratinocytes die in the presence of PMA (7). The precise mechanism for PMA-induced proliferation of human melanocytes is still unknown. In the present studies ET-1 and ET-2 were found to substitute for PMA as a growth factor for human melanocytes in long-term growth. Receptor- mediated stimulatory effects of ETs on DNA synthesis re- quired the addition of 10 nM CT, which is known to increase intracellular CAMP, and 0.2% BPE, but the addition of 10 ng/ml PMA to the medium was definitely inhibitory to ET- stimulated DNA synthesis in human melanocytes. This inhib- itory effect of PMA on ET stimulation can be interpreted by a deficient sensitivity to ET receptor which occurs after phosphorylation of receptors by activated PKC, as observed with vascular smooth muscle cells (34).

Basic FGF has recently been discovered as another melan-

' Portions of this paper (including part of "Experimental Proce- dures," "Results," and Figs. 1-4,6-11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

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ocyte mitogen which is also included in extracts of human melanoma (35) and bovine pituitary gland (36, 37). Though basic FGF can substitute for PMA in pure culture of human melanocytes, there is no evidence to show an accentuation of PKC in basic FGF-induced melanocyte stimulation. In other cells, the presence of receptors for basic FGF has been proven and an involvement of tyrosine kinase is suggested for the mode of action mechanism by basic FGF (38). However, in human melanocytes, the presence of receptor for basic FGF is unknown.

ET is a potent vasoconstrictor/pressor peptide which was originally identified from the culture medium of porcine aortic endothelial cells and consists of 21 amino acid residues (19). Apart from its potent vasoconstrictor and pressor action, ET is reported to exert a wide spectrum of biological effects in many cells: contraction of airway, intestinal and uterine smooth muscles (3941), positive inotripic and chronotropic effects on the myocardium (42, 43)) stimulation of artrial natriuretic peptide secretion from atrial cardiac myocytes (44, 45)) stimulation of release of aldosterone from adrenocortical glomerulosa cells (46), and modulation of norepinephrine release from sympathetic terminals (47). Specific high affinity binding sites for ET are distributed not only in vascular smooth muscles (24, 34) but also widely in various noncar- diovascular tissues including the central nervous system (48) and fibroblasts (22). Several biological actions of ETs on vascular smooth muscle cells or fibroblasts occur via a dose- dependent increase in cytosolic free calcium which results from Ins(1,4,5)P3 formation (49) and intracellular calcium mobilization (50). It is suggested that phosphoinositide turn- over and PKC activation are involved in ET-induced vaso- constriction.

By analogy, in human melanocytes, ETs, especially ET-1 and ET-2, were found to stimulate the formation of Ins(1,4,5)P3 which is accompanied by the enhancement of

18354 The Effects of Endothelins on Human Melanocytes

intracellular calcium mobilization. Since the stimulations of DNA synthesis as well as melanin synthesis by ETs are observed at the same concentrations as those required for stimulation of signal transduction pathway, and receptors for ETs were detected by binding assay in human melanocytes, i t is highly likely that the observed biological actions of ETs on human melanocytes are deeply associated with the in- creased phosphoinositide turnover and PKC activation which occur through receptor-ligand interaction. This receptor-me- diated stimulation of DNA synthesis was also corroborated by the fact that the addition of PMA reduced ET-stimulated DNA synthesis of human melanocytes because PMA is im- plicated to down-regulate ET receptors in fibroblast and smooth muscle cells (24,34). Similar mitogenic action by ETs was demonstrated for cultured vascular smooth muscle cells (51), renal mesangial cells (52), and Swiss 3T3 fibroblast (53). In the latter, as with human melanocytes, stimulation of DNA synthesis was observed in synergistic combination with agents that increase the cellular level of CAMP, accompanied by a rapid increase in the intracellular calcium concentration. Of considerable interest is the fact that ETs can be a potent mitogen for human melanocytes acting in concert with an- other, solely known melanocyte growth factor, basic FGF, suggesting that ET and basic FGF do not share a common receptor in human melanocytes.

Unlike the production of basic FGF by keratinocytes, there is no report describing the production of ETs by keratinocytes. Therefore, the physiological significance of ETs in epidermal pigmentation will remain unclarified until the discovery of ET synthesis by keratinocytes. It is speculated that ET is produced by both the endothelial cells of major blood vessels and endothelial cells in the microvasculature under the con- ditions of stress and vascular damage, leading to vasodilation and vasoconstriction (54). Because cutaneous inflammation caused by UVA (55) or 8-methoxypsoralen plus UVA (PUVA) (56) exposure to skin is documented to be accompanied by severe vascular damage, it is likely that UVA or PUVA exposure, at least in part, may stimulate synthesis and secre- tion of E T from endothelial cells and that as a result pigmen- tation is induced. Thus, better understanding for the biolog- ical behavior of ET in cutaneous inflammation or in carcin- ogenic situations of melanocytes would also provide a firm basis for pathophysiology and diagnostic approaches of many congenital and acquired pigmentary diseases as well as malig- nant melanoma.

In extravascular tissues such as lung, pancreas, spleen, and neural tissue localization of ET has recently been demon- strated (57-59). Several human cancer cell lines are also documented to produce ETs, playing a modulatory role in surrounding cells because no detectable receptors are found in those cancer cell lines (60). Thus, the important role played by E T in the cellular signaling mechanism is now extending beyond its well-implicated vasoconstrictive action. Data pre- sented here suggest a mechanism for receptor-mediated stim- ulation of human melanocytes through recognition of the signaling pathway induced by ETs. Thus, ongoing studies to characterize the behavior of ETs in melanization-stimulated situations should provide critical information for the under- standing of the regulation of melanin biosynthesis in epider- mis.

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The Effects of Endothelins on Human Melanocytes 18355

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Commun. 167,161-167

18356 The Effects of Endothelins on Human Melanocytes DNA Synthesis.

F i g . 6 Shows growth response of melanocytes to different COnCentPatlOnS Of ET dePlvatlVes. ET dePlVstiveS (€"-I. ET-2 and ET-3) induced a definlte dose-dependent ~ n c ~ e a s e in DNA synthesis even at concentrations as low as 100 pH. but big-ET was SUbStantially less effective. The DNA synthesis Pates varled with the dose of ET derivstlveS used. The strongest stlmulatlm of DNA Synthesis sa5 seen at 10-25 nM fop ET-

COPrelatmn between ET-induced DNA Synthesis and Ins(1 .U.5)PI release. 1 . 5-10 nH fo? ET-2 and 10 nY COP ET-3. It YBS thus cancelvable that there was a WSltlVe

StlmUIatOry effect an DNA synthesis. we tested the effects of ET dePivstives on DNA In order to determine an Optimal Culture condition for human melanocytes in examining

Synthesis In the s e v e ~ a l combinations with CT. BPE and PHI (Fig.?). The sdditlon of CT plus BPE vas essential f o p human melanocytes to mslntain their proliferation. while llttle DNA synthesis vas ObSePYed with CT only. In these culture Systems. the addltlon of ET dePiVatiYeS (ET-1. ET-2 and ET-3) SigniflCantly enhanced the DNA SyntheSlS In the pPeSence of CT. BPE and PHA, whereas stimulation Via ET deriYatiYeS Y B S Observed to be mope effective I n the absence of PHA. ET-enhanced IncOrPOPatlOn Of thymidine YBS dPamaticaI1y decreased Yhen CT andlor BPE "ere eliminated f ~ m the medium.

Fig. 8 shows that ET-1 exhlblted an additive SrlrnulatOlly effect (about 250 X of control) on ba31C FGF-stimulated DNA synthesis.

Cell Growth.

and BPE. end their growth Pates YaPied with the ET derivatives used. Although BYsIlable Long-term growth of human melanocytes "85 e~peclally dependent on the presence of CT

evidence ShDYed that PHI was also B EFu~ial component for maintaining long-term growth. StimulatoPy effects of ETs on the growth Of human melanocyteS did not re9UiPe the presence of PHI in the present CUltuPe system (Fig.9). The presence of 10 ng/ml PHA "8.5

The gPouth StlrnulatOPy effect exhibited by ETs was distinctly Observed wlth the definitely inhlbitoPy to ET-Stimulated cell growth in human melanocytes (data not shown).

s~multaneou~ additLon or 10 nH CT. 0.2 5 BPE. and 0-1.0 ng/ml PHI lo we^ EoncentPation than usual), The stmngest stlnulatlon of the growth was seen at 10 nH ET-1 or ET-2. resulting In a U.5-foId lnc~ease in cell number fop 12 Culture days. Thus. ET-1 and ET-2 could Substitute POP PHA in long-term Culture Of human melanOcYteS.

Melanogenesis ActiYIty. ET dePlvstlveS induced a definite increase in tYPOSlnsSe activity as demonstrated by

["Cjthiowacil LncoPporatlon (Fig. 10) and ' H z 0 release (Fig. 11) . In PartlCUlaP. ET-1 SLgnlflCantly CaUSed a 2-fold increase in both thioUPaci1 InCoPPOrBtlOn and " H z 0 Peie0Se.

4 0 endothelln- I +andolhelin- 2

ENDOTHELIN (MI

Fig. 1. Displacement of Blndlng of ["'IIET-I and ['"'IIET-2 to Human Helsnacytes by hiebeled E T - I and ET-2.

Confluent cells were incubated at U T COP U h with 10 pH I"'I1ET-1 and ["'IlET-2 i n the plle~ence Or absence of unlabeled ETs at COncentrationS BS indicated. Details W e deacrlbed under "Experimental Procedures." Data ape the means of ZPipllCate determnatmns f m m two Separate experments.

- 2 7 I T

The Effects of Endothelins on Human Melanocytes 18357

~ l C T i + r . BPEl--I.PMAi-) O C T " , . BPEl+I.PMAi+! I j C T I + ) , BPEl+l.PMAl-!

z 0 4 4

g U 0 0 I w 2 z I?

r F - 0-

' CONT bFGF ET- I ET- 2 ET-3 big-ET

Flg. 8. AddltiYe Effect of ET k r i v a t ~ v e s and bFGF on I'HJthymidine Incorparat~an. The cells were cultured FOP 2L1 h i n control medium containlng I O nH ET-1, 10 nH bFCF.

and IO nH ET-1 plus 10 DM bFGF ~n the presence Of 0.2 BPE plus 10 nH CT (0). or 0.2 I BPE. 10 nM CT plus 10 ng/mi PHA (0) Bod then ['Hithymldine vas added as deSCPlbed under "Experlmental PraceduPes." Data a r e the means? SE of trlpllcate determlnatlans from three sepamte experlments ( ' , P I 0.05. * * . P ~0.01).

5 -

4 -

3 -

2 -

1 -

r

l l ;ONT I big-ET

I ET 1 '- 3 big-ET