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Effects of estrogens on human melanocytes in vitro
Pergamon
0960-0760(94)E0002-3
J. Steroid Biochem. Molec. Biol. Vol. 49, No. 1, pp. 9-14, 1994 Copyright © 1994 Elsevier Science L t d Printed in Great Britain. All rights reserved 0960-0760/94 $7.00 + 0.00
Effects o f E s t r o g e n s on H u m a n In v i t r o
Melanocytes
Stephen D. McLeod, Marie Ranson and Rebecca S. Mason* Department of Physiology, University of Sydney, New South Wales 2006, Australia S u b j e c t s with e l e v a t e d s e r u m e s t r o g e n c o n c e n t r a t i o n s , such as t hose who a r e p r e g n a n t or ingesting e s t r o g e n - c o n t a i n i n g c o n t r a c e p t i v e m e d i c a t i o n , m a y devel op i n c r e a s e d skin p i g m e n t a t i o n . As little i n f o r m a t i o n is a v a i l a b l e on t he m e c h a n i s m ( s ) u n d e r l y i n g this r e l a t i o n s h i p , t h e in vitro effects o f e s t r o g e n s on m e l a n o c y t e s c u l t u r e d f r o m n o r m a l h u m a n skin w e r e e x a m i n e d . P h y s i o l o g i c a l c o n c e n t r a t i o n s o f 17fl-estradiol (10 -11 to 10-9M) si gni fi cant l y i n c r e a s e d t he a c t i v i t y o f t y r o s i n a s e in m e l a n o c y t e s f r o m 15 o f 23 subjects. T h e o b s e r v e d i n c r e a s e s r a n g e d f r o m 1.2- to 2.4-fold. M e l a n i n synthesis, w h i c h c o r r e l a t e d w i t h t y r o s i n a s e a c t i v i t y (r = 0.98, P < 0.001) was i n c r e a s e d to a s i m i l a r e x ten t. M e l a n i n e x t r u s i o n was also i n c r e a s e d by 17fl-estradiol (10-9M). T h e e s t r o g e n s , e strio l (10 -9 M ) a n d e s t r o n e (10 -9 M ) s t i m u l a t e d t y r o s i n a s e a c t i v i t y a n d m e l a n i n e x t r u s i o n to a lesser e x t e n t t h a n 17~-estradiol. T h e a n a l o g u e 17~-estradiol (10 -9 M) was shown to have effects on m e l a n o c y t e t y r o s i n a s e a c t i v i t y a n d m e l a n i n e x t r u s i o n t h a t w e r e e q u i v a l e n t to those o f 17/~-estradiol. T h e p u r e e s t r o g e n a n t a g o n i s t ICI 164384 (10-6M) also s t i m u l a t e d t y r o s i n a s e activity. C y c l o h e x i m i d e (50 p g / m l ) i n h i b i t e d 1 7 p - e s t r a d i o l - i n d u c e d t y r o s i n a s e s t i m u l a t i o n (P < 0.001). T h e s e r e s u l t s i n d i c a t e t h a t s e v e r a l as p e c t s o f m e l a n o c y t e f u n c t i o n r e s p o n d d i r e c t l y to e s t r o g e n i c s t i m u l a t i o n . T h e e q u i v a l e n t effects o f th e 170t-analogue a n d a " p u r e " a n t i - e s t r o g e n suggest t h a t t he 17/~-estradiol r e s p o n s e m a y be m e d i a t e d t h r o u g h a n o n - c l a s s i c a l m e c h a n i s m w hi ch is s i m i l a r to t h a t d e s c r i b e d in o t h e r tissues o f n e u r a l c r e s t origin.
J. Steroid Biochem. Molec. Biol., Vol. 49, No. 1, pp. 9-14, 1994
INTRODUCTION High estrogen levels have been reported to be associated with an increase in skin pigmentation [1]. This effect is particularly evident in pregnancy and in individuals ingesting estrogen-containing oral contraceptives, though many factors have been implicated in the development of melasma including genetic predisposition, elevated progesterone or thyroid hormone concentrations, and solar exposure [2, 3]. We have previously reported that physiological concentrations of 17fl-estradiol enhanced tyrosinase activity (E.C.1.14.18.1) of cultured melanocytes from normal human skin [4]. In the current study, the effects of 17fl-estradiol and other estrogens on tyrosinase activity were further examined, as well as the effect of 17fl-estradiol on melanin synthesis and melanin extrusion, a process whereby formed melanin is secreted into the matrix surrounding the melanocyte [5]. An attempt was made to determine whether the observed effects were mediated through *Correspondence to R. S. Mason. Received 22 Sept. 1993; accepted 20 Dec. 1993.
the classical estrogen receptor [6], using the 17~t-analogue of 17fl-estradiol and two anti-estrogenic compounds.
EXPERIMENTAL
Materials All chemicals, including steroid hormones, were obtained from Sigma Chemical Co. (St Louis, MO) except for ICI 164384 which was provided by Dr A. Wakeling (Imperial Chemical Industries, Macclesfield, England). [3H]- and [14C]tyrosine and [3H]17fl-estradiol were obtained from Amersham Australia Pty Ltd (Sydney, Australia). Eagles minimum essential medium with Earle's salts (EMEM), Iscove's medium and foetal calf serum (FCS) were obtained from Flow Laboratories Inc. (Sydney, Australia). Plastic culture plates coated with bovine corneal endothelium-derived extracellular matrix (ECM) were obtained from International Biotechnologies Ltd (Jerusalem, Israel), Wood Scientific Ltd. (Sydney, Australia) and Cell Dynamics (Sydney, Australia).
10
Stephen D. McI.eod c: ~d.
Culture conditions H u m a n melanocytes were grown from neonatal fl)reskin samples according to the method of Eisinger and Marko [7] with minor modifications as described previously [8]. T h e cultures were maintained on uncoated plastic in E M E M with 5% FCS (v/v), pH 7.2, containing phorbol 12-myristate 13-acetate (PMA, 2 0 n M ) , cholera toxin (CT, 10 nM), penicillin (60/2g/ml) and streptomycin (100 ~tg/ml). T h e cells used in this study had been cultured for 1-4 months.
Experimental conditions Melanocytes near confluence were detached from the culture flasks with trypsin (0.05%) and ethylenediaminetetraacetic acid ( E D T A , 0.02%) in phosphatebuffered saline and seeded at a density of 2 x 104cells/cm 2 on to E C M - c o a t e d 24 well plates unless otherwise stated. T w e n t y - f o u r to 48 h after seeding, the growth medium was replaced with E M E M containing 5% FCS as the only mitogen. After overnight incubation, this medium was changed to E M E M containing 2°i, FCS, or Iscove's medium containing bovine serum albumin (0.1% v/v), soybean lecithin (80/~g/ml) and transferrin (30/tg/ml). Melanocytes were then treated with the estrogens or other agents, added 3 times/day for up to 3 days and once on the day of assay, with daily changes of experimental medium.
Tyrosinase activity Tyrosinase activity was measured by a modification of the method of Pomerantz [9], as described previously [8]. This method measures the amount of [3H]H20 released from [3H]tyrosine as a result of the tyrosine hydroxylase activity of the tyrosinase enzyme. All values were corrected for non-enzymic hydroxylation by subtracting the amount of [3H]H20 formed by cell extracts containing 1 m M phenylthiourea (PTU), a specific inhibitor of the tyrosinase enzyme [10]. Cell numbers from replicate wells were counted in a Coulter Counter or haemocytometer.
Melanin synthesis [laC]Tyrosine incorporation into TCA-precipitable melanin by live, whole cells was measured by a modification of the method of Chen and Chavin [11], as described previously [8]. The counts obtained in P T U inhibited controls were subtracted from all values obtained. U n d e r these conditions, melanin formation was linear with cell number, with substrate concentration up to 8.0/~Ci/ml, and with time up to 6 h.
Melanin extrusion T h e extrusion of [14C]melanin into the medium was measured by a slight modification of the melanin synthesis method. After 8 h in the presence or absence of estrogens, [HC]tyrosine (2/,Ci/ml) and dihydroxy-
phenylalanine (DOPA, 10Hg/ml) wcrc added to the wells, which were incubated for a further 16 tl. 'l'he medium was then removed and the cells washed and lysed with Triton X-100 (0.05°o v/v) in phosphate buffered saline. Melanin in the medium or cell extracts was precipitated with trichloroacetic acid as described previously [8]. Correction fi)r non-specific precipitation of counts was made by subtracting the counts obtained from cells incubated in the presence of P T U .
Estrogen receptor assay T h e estrogen receptor immunocytochemical assay ( E R - I C A ) kit was obtained from Abbott Labs (North Chicago, II). Melanocytes and frozen sections of a human breast cancer specimen were fixed and incubated with anti-estrogen receptor monoclonal antibody according to the method described by Bilous et al. [12]. In an attempt to induce the estrogen receptor, melanocytes were also preincubated with 17fl-estradiol ( 1 0 - l ° tO 10 sin) for 2 4 h before staining with antibody. Uptake binding studies of [3Hll7fl-estradiol were carried out in whole cells as described previously for another steroid, 1,25-dihydroxyvitamin D~ [4].
Statistical analysis Statistical comparisons of the data were made using Student's t-test for unpaired observations and by Analysis of Variance where appropriate. Where results were expressed as ratios such as tyrosinase activity/number of cells, the standard deviations of the ratios were calculated using the method of Colquhoun [13] before application of the t-test. RESULTS Incubation with 17//-estradiol significantly increased tyrosinase activity in melanocyte strains from 15 of 23 donors. In melanocytes from those subjects responsive to estradiol, the maximal increases ranged from 1.2- to 2.4-fold with a mean increase of 1.6 + 0.1 (SEM)-fold. T h e dose-response relationship for 3 donors is shown in Fig. 1. When tyrosinase activity was unaltered by 17fl-estradiol treatment, there was also no change in the cell numbers (data not shown). In contrast, significant increases in tyrosinase activity were accompanied by decreases in cell numbers. There was, however, no correlation between the magnitude of the increase in tyrosinase activity and the magnitude of the corresponding decrease in cell numbers (n = 12, r = 0.255, P > 0.3). T h e stimulatory effect of 17fl-estradiol was only seen when melanocytes were plated on E C M and not when they were maintained on uncoated plastic. Tyrosinase activity and melanin synthesis were positively correlated under basal conditions [8], and when tyrosinase activity and melanin synthesis were compared before and after treatment with various concentrations of 17fl-estradiol, there was a strong positive correlation between these parameters (r = 0.98,
Estrogens and Pigmentation
Table 1. Effects of estrogens ( I 0 - 9 M ) on melanocyte tyrosinase activity cell numbers and melanin extrusion (mean + S E M )
Key
400
-A'-cellline 240589 -e--cell line 010393 "-~--eellline 110789
Estrogen Control fl-estradiol Estrone Estriol
~ , , / * *
*
300 . ..-'°
~ 100 u~
$ [-
Control
i -12
i -11
i
i -I0
i -9
17 beta-estradiol (log molar concentration / dose) Fig. 1. L i n e a r d o s e - r e s p o n s e r e l a t i o n s h i p b e t w e e n 1 7 / J - e s t r a diol a n d m e l a n o c y t e t y r o s i n a s e a c t i v i t y . D a t a a r e t h e me.an + SEM (bars) of triplicate determinations. Signific a n t l y d i f f e r e n t f r o m c o n t r o l v a l u e s , * P < 0.05; * * P < 0.025; * * * P < 0.0l; * * * * P < 0.0025.
280 ~
240
~ 20o o
160 .~
120
=
80
"~
40
. / ..,/.
:/
/
I I I 40 80 120 160 200 240 T y r o s i n a s e activity (% o f control v a l u e s )
Tyrosinase activity Cells/well Melaninextruded (cpm/100cells/4 h) (in duplicate) (cpm/100 cells/16 h) 548 + 21 1015 + 60*** 615 + 29* 733 + 58**
29774 16350 26700 24720
6.4 + 2.0 22.0 + 0.1"** 8.7 + 1.1 3.8 + 2.4
Significantly different from control values; *P < 0.02; **P < 0.01; ***P < 0.001.
****
200
0
11
I 280
Fig. 2. L i n e a r r e g r e s s i o n a n a l y s i s o f t y r o s i n a s e a c t i v i t y v e r s u s m e l a n i n s y n t h e s i s a f t e r i n c u b a t i o n w i t h 1 7 / ~ - e s t r a d i o l (10 12 to 1 0 - 9 M ) . T h e r e s p o n s e s o f m e l a n o c y t e s t r a i n s f r o m 3 d i f f e r e n t d o n o r s to 1 7 / ~ - e s t r a d i o l w e r e c a l c u l a t e d a s p e r c e n t ages of control values. Data are the mean of triplicate determinations for each parameter.
P < 0.001; Fig. 2). In addition to increased melanogenesis, melanin extrusion into the medium was also enhanced by 17fl-estradiol. T h e relative effects of 17fl-estradiol, estrone and estradiol on tyrosinase activity, cell numbers and melanin extrusion are shown in Table 1. Preliminary results indicate that the catechol estrogen 2-hydroxyestradiol at 1 0 - 9 M increased tyrosinase activity and melanin synthesis by 1.13 + 0.04 (SEM)-fold (P < 0.05) and 1.25 + 0.04 (SEM)-fold (P < 0.05), respectively without affecting cell numbers. T h e presence of cycloheximide, in a dose shown to inhibit [3H] leucine incorporation [14], abolished the stimulatory effect of 17fl-estradiol on tyrosinase activity [Figure 3(A)]. Cycloheximide markedly increased melanin extrusion into the medium in otherwise untreated cells from 754 + 23 to 3192 + 540 cpm/104 cells/16 h (P < 0.001). N o radiolabelled melanin was detected in either control or estradiol-treated melanocytes in the presence of cycloheximide. Incubation of melanocytes with the pure estrogen antagonist I C I 164384 (10 -6 M) also increased tyrosinase activity [Fig. 3(B)]. When both 17fl-estradiol and I C I 164384 were preincubated with the cells, tyrosinase activity was no higher than with either agent alone. In each of three experiments, the stimulation of tyrosinase by 17fl-estradiol was mimicked by 17~estradiol (Table 2). In an experiment where treatment with 1 0 - 9 M 17fl-estradiol increased melanin extrusion 3.4 + 0.01 (SEM)-fold (P < 0.001), treatment with 1 0 - 9 M 17~-estradiol also increased melanin extrusion 2.0 + 0.3 (SEM)-fold (P < 0.001). Cell numbers were similar after both treatments. Nuclear magnetic resonance studies indicated that the spectra of the 17fl- and 17~-estradiol compounds were consistent with the expected stereochemical configurations (data not shown). N o estrogen receptors were found using the Abbott monoclonal antibody estrogen receptor kit, which showed clearly positive results with breast cancer cells. Since it has been proposed that apparent estrogen binding to pigment cells may be an artefact due to [3H]H20 release from tritiated estradiol by tyrosinase [29], specific binding of [3H] 17fl-estradiol was tested in the presence of the tyrosinase inhibitor P T U . Specific binding was not demonstrated under these conditions.
I2
Stephen 1). e\lcl,eod cl a/. A
200
175
7,
150
Z Z Z Z Z
125
z/
Z
g
lOO
i7` 75
z z
7, Z
Z Z 7, Z
7, 50
Z
Pa
y., 25
z1 t 9,
7,
Z
,'/
+.
g
+g
Fig. 3. (A) T y r o s i n a s e a c t i v i t y in m e l a n o c y t e s a f t e r t r e a t m e n t w i t h 1 7 ~ - e s t r a d i o l (10 9M) in t h e p r e s e n c e o r a b s e n c e o f cyeloheximide (50/*g/ml) (B) Tyrosinase activity in m e l a n o c y t e s a f t e r t r e a t m e n t w i t h 1 7 # - e s t r a d i o l (10 ~ M ) in t h e p r e s e n c e or a b s e n c e o f ICI 164384 (10 ~ M). D a t a a r e t h e m e a n _+_S E M ( b a r ) o f t r i p l i c a t e d e t e r m i n a t i o n s . S i g n i f i c a n t l y different from control values, *P < 0.025; **P < 0.01; ***P < 0.0025; ****P < 0.001.
DISCUSSION
Skin pigmentation in vivo is d e t e r m i n e d by genetic, environmental, local and endocrine factors which influence both melanin synthesis within each melanocyte and the distribution of melanin t h r o u g h o u t the epidermis [15]. In a previous report, we demonstrated that 17fl-estradiol increased melanocyte tyrosinase activity in vitro [4]. T h e s e studies have now been extended to show that responsiveness to 17fi-estradiol is d o n o r - d e p e n d e n t 7"able 2. Comparison between the effects of 17fi-estradiol (I0 9M) and 17~-estradiol (10 9M) on melanocyte tyrosinase activity in cpm/lO0 cells~4 h (mean + S E M ) Experiment 1 Experiment 2 Experiment 3
Control
fl-Estradiol
:~-Estradiol
548 + 2l 671 ± 12 1079 + 42
1015 + 60** 965 ± 68* 1473 + 28**
988 + 78** 900 _+ 57* 1750 + 47**
Significantly' different from control values; *P < 0.005; **P < 0.001.
and that melanin synthesis and extrusion is als~ ll> creascd by the hormone. Significant stimulation of tyrosinase activity by l - f i estradiol was seen in 65",, of donors. M e l a n o c y i c strains from 8 out of 23 donors showed no significant alteration in tyrosinase activity in response 1o lhe h o r m o n e . Despite the reproducibility of the stimulatory effect of 17fi-estradiol in most donors, both thc basal tyrosinase activity and the degree of stimulation varied noticeably in melanocytes from different donors, as would be expected from in vivo studies. Estrogen responsiveness could not be predicted by basal tvrosinase activity or plating/proliferative capacity as assessed by cell n u m b e r s in vehicle-treated wells. A response to h o r m o n e s was only observed in melanocytes plated on E C M in the absence of P M A and C T . T h e E C M is u n d e r s t o o d to resemble the basal lamina of skin where melanocytes normally reside, and it maintains melanocytes in a relatively quiescent but highly differentiated state in the absence of mitogens which might otherwise interfere with h o r m o n e signal processing [8]. T h e authors are also aware that, whilst most studies on melanocytes have d e p e n d e d on cells from neonatal skin, some caution m u s t be exercised in extrapolating any of the results to the adult situation. T h e strong correlation between tyrosinase activity and melanin synthesis in cells treated with 17fi-cstradiol is not surprising since melanin content in m a m malian p i g m e n t cells is quantitatively dependent on tyrosinase activity [4, 16, 17]. T h e subsequent transfer of melanin from melanocytes to keratinocytcs is bclieved to occur by two processes. T h e first process involves the contact of a melanocytic dendrite with the cell m e m b r a n e of a keratinocyte, which then pinches off the dendritic tip containing m e l a n o s o m c s and encloses it within a phagocytic vacuole prior to melanin dispersion [15]. T h e second process inw)lves the extracellular secretion of melanosomes from melanocvtic dendrites, and their subsequent engulfment by the n e i g h b o u r i n g keratinocytes [5]. O u r studies with cvcloheximide, which inhibited the increase in tvrosinasc induced by 17fl-estradiol and caused almost complete melanin extrusion u n d e r basal conditions, is consistent with the suggestion that melanin extrusion may be tonically inhibited by a protein with a relatively short half-life. T h a t 17fi-estradiol directly enhances melanogenesis and melanin extrusion in melanocytes in vitro provides some evidence to s u p p o r t a role for estrogens in m o d u l a t i n g skin pigmentation and in the development o f melasma. T h e biological effects of estrogens in classical target tissues, such as the uterus and breast, are believed to be mediated by the b i n d i n g of estrogen to an intracellular steroid receptor protein and the subsequent alteration of gene expression [6]. T h e affinities of estrone and estradiol for the estrogen receptor in uterine tissues are approx. 20 and 5"<,, respectively of that of 17fiestradiol [6]. T h e reduced activity of estrone and estriol
Estrogens and Pigmentation on the tyrosinase activity of melanocytes, together with t h e a b o l i t i o n o f t h e e s t r o g e n i c effects in t h e p r e s e n c e o f c y c l o h e x i m i d e , a r e c o n s i s t e n t w i t h a classical e s t r o g e n r e c e p t o r - m e d i a t e d m e c h a n i s m , b u t a r e also c o n s i s t e n t with other mechanisms. However, despite the reports t h a t I C I 1 6 4 3 8 4 is a p u r e r a n t i - e s t r o g e n t h a n t a m o x i f e n a n d has little a g o n i s t a c t i v i t y [ 1 8 - 2 0 ] , a s t i m u l a t o r y e f f e c t o f t h i s a g e n t w a s o b s e r v e d in o u r m e l a n o c y t e system. M o r e s t r i k i n g l y , t h e c o n c e p t o f a classic r e c e p t o r m e d i a t e d m e c h a n i s m is i n c o n s i s t e n t w i t h o u r o b s e r v a t i o n t h a t t h e effects o f 1 7 ~ - e s t r a d i o l w e r e v e r y s i m i l a r to t h o s e o f 1 7 f l - e s t r a d i o l . S i m i l a r effects o f 17c¢ a n d 1 7 f l - e s t r a d i o l h a v e p r e v i o u s l y b e e n r e p o r t e d in t h e estrogen receptor negative MDA-MB-330 human b r e a s t c a n c e r cell l i n e [21]. W h i l e it is p o s s i b l e t h a t 1 7 ~ - e s t r a d i o l c a n b e i s o m e r i z e d to 1 7 f l - e s t r a d i o l in v i t r o [22], o t h e r e x p e r i m e n t s i n d i c a t e t h a t in s o m e tissues of neural crest origin the phenolic 17fl-estradiol c a n b e c o n v e r t e d b y 2- a n d 4 - h y d r o x y l a s e e n z y m e s to c o m p o u n d s w h i c h r e s e m b l e c a t e c h o l a m i n e s [23, 24]. T h e s e c a t e c h o l e s t r o g e n s e x h i b i t r e d u c e d affinity f o r t h e e s t r o g e n r e c e p t o r [25] b u t , b y v i r t u e o f t h e i r c a t e c h o l s t r u c t u r e , h a v e b i o l o g i c a l a c t i v i t y in s e v e r a l in v i t r o s y s t e m s [26, 27]. M o r e o v e r , 1 7 ~ - e s t r a d i o l c a n b e c o n v e r t e d to c a t e c h o l e s t r o g e n s in a s i m i l a r m a n n e r to 1 7 f l - e s t r a d i o l [28], a n d is e q u i p o t e n t to 1 7 f l - e s t r a d i o l in i n h i b i t i n g t y r o s i n e h y d r o x y l a s e a n d c a t e c h o l - o m e t h y l t r a n s f e r a s e a c t i v i t y in r a b b i t a n d rat b r a i n in v i t r o [25, 28]. T h e r e l a t i v e l y m o d e s t effects o f 2 - h y d r o x y e s t r a d i o l in t h i s p r e l i m i n a r y s t u d y m a y b e d u e to t h e l i k e l y r a p i d m e t a b o l i s m o f t h i s m e t a b o l i t e w h e n it is p r o v i d e d as s i n g l e d o s e s r a t h e r t h a n b e i n g s y n t h e s i z e d o v e r a p e r i o d o f t i m e f r o m its s u b s t r a t e [29]. E v i d e n c e for t h e p r e s e n c e o f c a t e c h o l a m i n e b i o s y n t h e t i c p a t h w a y s w i t h i n m e l a n o m a cells has b e e n p r e s e n t e d b y M c E w a n a n d P a r s o n s [30]. F u r t h e r m o r e , it has b e e n s h o w n t h a t t y r o s i n a s e c a n u t i l i z e e s t r a d i o l as a s u b s t r a t e a n d h y d r o x y l a t e it to a c a t e c h o l - l i k e c o m p o u n d [31]. T h i s m a y b e p o s s i b l e b e c a u s e t h e A r i n g o f e s t r a d i o l has s t r u c t u r a l r e s e m b l a n c e to t h e p h e n o l i c group of tyrosine and thus may substitute for tyrosine. T h e p r e l i m i n a r y o b s e r v a t i o n in o u r l a b o r a t o r y o f m e l a n o c y t e t y r o s i n a s e s t i m u l a t i o n b y t h e c a t e c h o l estrogen 2-hydroxyestradiol suggests the possible regulation of tyrosinase by catechol-like compounds. However, the precise mechanism by which estrogens m a y affect m e l a n o c y t e f u n c t i o n t h r o u g h c a t e c h o l i n t e r m e d i a t e s is as y e t u n k n o w n . Acknowledgements--This work was supported by the National
Health and Medical Research Council of Australia, the University of Sydney Cancer Research Fund, the New South Wales State Cancer Council, the Leo and Jenny Leukemia and Cancer Foundation of Australia and the Government Employees Assistance to Medical Research Fund. NMR studies were performed by Dr A. Cheung in the Department of Pharmacology, University of Sydney. Estrogen receptor studies with the Abbot monoclonal antibody were performed by Dr A. M. Bilous, Department of Anatomical Pathology, Westmead Hospital, Sydney, Australia.
13
REFERENCES 1. Snell R. S.: The pigmentary changes occurring in the breast skin during pregnancy and following estrogen treatment. J. Invest. Dermat. 43 (1964) 181-186. 2. Sanchez N. P., Pathak M. A., Sato S., Fitzpatrick T. B., Sanchez J. L. and Mihm M. C.: Melasma: a clinical, light microscopic, ultrastructural and immunofluorescence study. J. Am. Acad. Dermat. 4 (1981) 698-704. 3. Lufti R. J., Fridmanis M., Misiunas A. L., Pafume O., Gonzalez E. A., Villemur J. A., Mazzini M. A. and Niepomniszcze H.: Association of melasma with thyroid autoimmumity and other thyroidal abnormalities and their relationship to the origin of the melasma. J. Clin. Endocr. Metab. 61 (1985) 28-31. 4. Ranson M., Posen S. and Mason R. S.: Human melanocytes as a target tissue for hormones: In vitro studies with lc~-25, dihydroxyvitamin D3, ~-melanocyte stimulating hormone and betaestradiol. J. Invest. Dermat. 91 (1988) 593-598. 5. Wolff K.: Melanocyte-keratinocyte interactions in vivo: the fate of melanosomes. Yale J. Biol. Med. 46 (1973) 384-388. 6. Grody W. W., Schrader W. T. and O'Malley B. W.: Activation, transformation, and subunit structure of steroid hormone receptors. Endocrine Rev. 3 (1982) 141-163. 7. Eisinger M. and Marko O.: Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc. Natn. Acad. Sci. U.S.A. 79 (1982) 2018-2022. 8. Ranson M., Posen S. and Mason R. S.: Extracellular matrix modulates the function of human melanocytes but not melanoma cells. J. Cell. Physiol. 136 (1988) 281-288. 9. Pomerantz S. H.: The tyrosine hydroxylase activity of mammalian tyrosinase. J. Biol. Chem. 241 (1966) 161-168. 10. Whittaker J. R.: Loss of melanogenic phenotype in vitro by differentiated retinal pigment cells: demonstration of mechanisms involved. Devl. Biol. 15 (1967) 553-574. 11. Chen Y. M. and Chavin W.: Radiometric assay oftyrosinase and theoretical considerations of melanin formation. Analyt. Biochem. 13 (1965) 234-258. 12. Bilous A. M., Tyler J. P., Toppila M. and Milliken J.: Whither cytosolic estrogen receptor assays? A comparison of commercially available kits for estrogen receptor assay. Pathology 19 (1987) 223-228. 13. Colquohoun D.: Lectures in Biostatistics--An Introduction to Statistics with Applications in Biology and Medicine. Clarendon Press, Oxford (1971) p. 41. 14. Ranson M.: Human pigment cells in vitro. PhD Thesis, The University of Sydney, 2006, Australia (1989). 15. Quevedo Jr W. C., Fitzpatrick T. B., Szabo G. and Jimbow K.: Biology of melanocytes. In Dermatology in General Medicine (Edited by T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg and K. F. Austen). McGraw-Hill Inc, New York (1987) pp. 224-251. 16. Halaban R., Pomerantz S. H., Marshall S., Lambert D. T. and Lerner A. B.: Regulation of tyrosinase in human melanocytes grown in culture. J. Cell. Biol. 97 (1983) 480--488. 17. Hearing V. J. and Jimenez M.: Mammalian tyrosinase--the critical regulatory control point in melanocyte pigmentation. Int J. Biochem. 19 (1987) 1141-1147. 18. Wakeling A. E. and Bowler J.: Steroid pure antioestrogens. J. Endocri. 112 (1987) R7-R10. 19. Wakeling A. E. and Bowler J.: Novel antioestrogens without partial agonist activity. J. Steroid Biochem. 31 (1988) 645-653. 20. Wakeling A. E., Newboult E. and Peters S. W.: Effects of antioestrogens on the proliferation of MCF-7 human breast cancer cells. J. Molec Endocr. 2 (1989) 225-234. 21. Reddel R. R. and Sutherland R. L.: Effects of pharmacological concentrations of estrogens on proliferation and cell cycle kinetics of human breast cancer cell lines in vitro. Cancer Res. 47 (1987) 5323-5329. 22. Papendorp J. T., Schatz R. W., Soto A. M. and Sonnenschein C.: On the role of 17 alpha-estradiol and 17 beta-estradiol in the proliferation of MCF7 and T47D-A11 human breast tumor cells. J. Cell Physiol. 125 (1985) 591-595. 23. Fishman J. and Norton B.: Catechol estrogen formation in the central nervous system of the rat. Endocrinology 96 (1975) 1054-1059. 24. Mondschein J. S., Hersey R. M. and Weisz J.: Purification and
14
S t e p h e n 1). M c L e o d
characterization of estrogen-2/4-hydroxylase activity from rabbit hypothalami: peroxidase-mediated catechol estrogen formation. Endocrinology 119 (1986) 1105-1111. 25. Merriam G. R., M a c L u s k y N. J., J o h n s o n L. A. and Naftolin F.: 2-Hydroxyestradiol-17~ and 4-hydroxyestradiol-17~, catechol estrogen analogs with reduced estrogen receptor affinity. Steroids 36 (1980) 13-16. 26. Lloyd T. and Weisz J.: Direct inhibition of tyrosine hydroxylase activity by catechol estrogens. J. Biol. Chem. 253 (1978) 4841-4843. 27. T h e r o n C. N., Russell V. A. and Taljaard J. J.: Estrogen-2/4-hydroxylase activities in rat brain and liver microsomes exhibit different substrate preferences and sensitivities to inhibition. J. Steroid Biochem. 28 (1987) 533-541.
ct a/.
28. Hersey R. M., Lloyd T., MacI,usky N. J. and \Vcisz J.: T h e catechol estrogen, 2-hydroxyestradiol-17z is t'ormcd from estradiol-I 7~ by hypothalamic tissuc iH ~,itr~, and inhibits tyrosine hydroxylase. Endocrinoloa, v 111 1 9 8 2 1734-36. 29. Schneider J., H u h M. M., Bradlow H. L. and Fishman J.: Antiestrogen action of 2-hydroxyestrone on ,\ICF-7 h u m a n breast cancer cells..~'. Biol. Chem. 259 (1984) 4840--1845. 30. M c E w a n M. and Parsons P. G.: Inhibition of melanization in h u m a n melanoma cells by a serotonin uptake inhibitor. ,)r ltn,est. Dermat.89 (1987) 82-86. 31. Zava D. T. and Goldhirsch A.: Estrogen receptor in malignant melanoma: fact or artefact? Eur. J. Cancer. Ch'n. Oncol. 19 :"1983) 1151-1159.
0960-0760(94)E0002-3
J. Steroid Biochem. Molec. Biol. Vol. 49, No. 1, pp. 9-14, 1994 Copyright © 1994 Elsevier Science L t d Printed in Great Britain. All rights reserved 0960-0760/94 $7.00 + 0.00
Effects o f E s t r o g e n s on H u m a n In v i t r o
Melanocytes
Stephen D. McLeod, Marie Ranson and Rebecca S. Mason* Department of Physiology, University of Sydney, New South Wales 2006, Australia S u b j e c t s with e l e v a t e d s e r u m e s t r o g e n c o n c e n t r a t i o n s , such as t hose who a r e p r e g n a n t or ingesting e s t r o g e n - c o n t a i n i n g c o n t r a c e p t i v e m e d i c a t i o n , m a y devel op i n c r e a s e d skin p i g m e n t a t i o n . As little i n f o r m a t i o n is a v a i l a b l e on t he m e c h a n i s m ( s ) u n d e r l y i n g this r e l a t i o n s h i p , t h e in vitro effects o f e s t r o g e n s on m e l a n o c y t e s c u l t u r e d f r o m n o r m a l h u m a n skin w e r e e x a m i n e d . P h y s i o l o g i c a l c o n c e n t r a t i o n s o f 17fl-estradiol (10 -11 to 10-9M) si gni fi cant l y i n c r e a s e d t he a c t i v i t y o f t y r o s i n a s e in m e l a n o c y t e s f r o m 15 o f 23 subjects. T h e o b s e r v e d i n c r e a s e s r a n g e d f r o m 1.2- to 2.4-fold. M e l a n i n synthesis, w h i c h c o r r e l a t e d w i t h t y r o s i n a s e a c t i v i t y (r = 0.98, P < 0.001) was i n c r e a s e d to a s i m i l a r e x ten t. M e l a n i n e x t r u s i o n was also i n c r e a s e d by 17fl-estradiol (10-9M). T h e e s t r o g e n s , e strio l (10 -9 M ) a n d e s t r o n e (10 -9 M ) s t i m u l a t e d t y r o s i n a s e a c t i v i t y a n d m e l a n i n e x t r u s i o n to a lesser e x t e n t t h a n 17~-estradiol. T h e a n a l o g u e 17~-estradiol (10 -9 M) was shown to have effects on m e l a n o c y t e t y r o s i n a s e a c t i v i t y a n d m e l a n i n e x t r u s i o n t h a t w e r e e q u i v a l e n t to those o f 17/~-estradiol. T h e p u r e e s t r o g e n a n t a g o n i s t ICI 164384 (10-6M) also s t i m u l a t e d t y r o s i n a s e activity. C y c l o h e x i m i d e (50 p g / m l ) i n h i b i t e d 1 7 p - e s t r a d i o l - i n d u c e d t y r o s i n a s e s t i m u l a t i o n (P < 0.001). T h e s e r e s u l t s i n d i c a t e t h a t s e v e r a l as p e c t s o f m e l a n o c y t e f u n c t i o n r e s p o n d d i r e c t l y to e s t r o g e n i c s t i m u l a t i o n . T h e e q u i v a l e n t effects o f th e 170t-analogue a n d a " p u r e " a n t i - e s t r o g e n suggest t h a t t he 17/~-estradiol r e s p o n s e m a y be m e d i a t e d t h r o u g h a n o n - c l a s s i c a l m e c h a n i s m w hi ch is s i m i l a r to t h a t d e s c r i b e d in o t h e r tissues o f n e u r a l c r e s t origin.
J. Steroid Biochem. Molec. Biol., Vol. 49, No. 1, pp. 9-14, 1994
INTRODUCTION High estrogen levels have been reported to be associated with an increase in skin pigmentation [1]. This effect is particularly evident in pregnancy and in individuals ingesting estrogen-containing oral contraceptives, though many factors have been implicated in the development of melasma including genetic predisposition, elevated progesterone or thyroid hormone concentrations, and solar exposure [2, 3]. We have previously reported that physiological concentrations of 17fl-estradiol enhanced tyrosinase activity (E.C.1.14.18.1) of cultured melanocytes from normal human skin [4]. In the current study, the effects of 17fl-estradiol and other estrogens on tyrosinase activity were further examined, as well as the effect of 17fl-estradiol on melanin synthesis and melanin extrusion, a process whereby formed melanin is secreted into the matrix surrounding the melanocyte [5]. An attempt was made to determine whether the observed effects were mediated through *Correspondence to R. S. Mason. Received 22 Sept. 1993; accepted 20 Dec. 1993.
the classical estrogen receptor [6], using the 17~t-analogue of 17fl-estradiol and two anti-estrogenic compounds.
EXPERIMENTAL
Materials All chemicals, including steroid hormones, were obtained from Sigma Chemical Co. (St Louis, MO) except for ICI 164384 which was provided by Dr A. Wakeling (Imperial Chemical Industries, Macclesfield, England). [3H]- and [14C]tyrosine and [3H]17fl-estradiol were obtained from Amersham Australia Pty Ltd (Sydney, Australia). Eagles minimum essential medium with Earle's salts (EMEM), Iscove's medium and foetal calf serum (FCS) were obtained from Flow Laboratories Inc. (Sydney, Australia). Plastic culture plates coated with bovine corneal endothelium-derived extracellular matrix (ECM) were obtained from International Biotechnologies Ltd (Jerusalem, Israel), Wood Scientific Ltd. (Sydney, Australia) and Cell Dynamics (Sydney, Australia).
10
Stephen D. McI.eod c: ~d.
Culture conditions H u m a n melanocytes were grown from neonatal fl)reskin samples according to the method of Eisinger and Marko [7] with minor modifications as described previously [8]. T h e cultures were maintained on uncoated plastic in E M E M with 5% FCS (v/v), pH 7.2, containing phorbol 12-myristate 13-acetate (PMA, 2 0 n M ) , cholera toxin (CT, 10 nM), penicillin (60/2g/ml) and streptomycin (100 ~tg/ml). T h e cells used in this study had been cultured for 1-4 months.
Experimental conditions Melanocytes near confluence were detached from the culture flasks with trypsin (0.05%) and ethylenediaminetetraacetic acid ( E D T A , 0.02%) in phosphatebuffered saline and seeded at a density of 2 x 104cells/cm 2 on to E C M - c o a t e d 24 well plates unless otherwise stated. T w e n t y - f o u r to 48 h after seeding, the growth medium was replaced with E M E M containing 5% FCS as the only mitogen. After overnight incubation, this medium was changed to E M E M containing 2°i, FCS, or Iscove's medium containing bovine serum albumin (0.1% v/v), soybean lecithin (80/~g/ml) and transferrin (30/tg/ml). Melanocytes were then treated with the estrogens or other agents, added 3 times/day for up to 3 days and once on the day of assay, with daily changes of experimental medium.
Tyrosinase activity Tyrosinase activity was measured by a modification of the method of Pomerantz [9], as described previously [8]. This method measures the amount of [3H]H20 released from [3H]tyrosine as a result of the tyrosine hydroxylase activity of the tyrosinase enzyme. All values were corrected for non-enzymic hydroxylation by subtracting the amount of [3H]H20 formed by cell extracts containing 1 m M phenylthiourea (PTU), a specific inhibitor of the tyrosinase enzyme [10]. Cell numbers from replicate wells were counted in a Coulter Counter or haemocytometer.
Melanin synthesis [laC]Tyrosine incorporation into TCA-precipitable melanin by live, whole cells was measured by a modification of the method of Chen and Chavin [11], as described previously [8]. The counts obtained in P T U inhibited controls were subtracted from all values obtained. U n d e r these conditions, melanin formation was linear with cell number, with substrate concentration up to 8.0/~Ci/ml, and with time up to 6 h.
Melanin extrusion T h e extrusion of [14C]melanin into the medium was measured by a slight modification of the melanin synthesis method. After 8 h in the presence or absence of estrogens, [HC]tyrosine (2/,Ci/ml) and dihydroxy-
phenylalanine (DOPA, 10Hg/ml) wcrc added to the wells, which were incubated for a further 16 tl. 'l'he medium was then removed and the cells washed and lysed with Triton X-100 (0.05°o v/v) in phosphate buffered saline. Melanin in the medium or cell extracts was precipitated with trichloroacetic acid as described previously [8]. Correction fi)r non-specific precipitation of counts was made by subtracting the counts obtained from cells incubated in the presence of P T U .
Estrogen receptor assay T h e estrogen receptor immunocytochemical assay ( E R - I C A ) kit was obtained from Abbott Labs (North Chicago, II). Melanocytes and frozen sections of a human breast cancer specimen were fixed and incubated with anti-estrogen receptor monoclonal antibody according to the method described by Bilous et al. [12]. In an attempt to induce the estrogen receptor, melanocytes were also preincubated with 17fl-estradiol ( 1 0 - l ° tO 10 sin) for 2 4 h before staining with antibody. Uptake binding studies of [3Hll7fl-estradiol were carried out in whole cells as described previously for another steroid, 1,25-dihydroxyvitamin D~ [4].
Statistical analysis Statistical comparisons of the data were made using Student's t-test for unpaired observations and by Analysis of Variance where appropriate. Where results were expressed as ratios such as tyrosinase activity/number of cells, the standard deviations of the ratios were calculated using the method of Colquhoun [13] before application of the t-test. RESULTS Incubation with 17//-estradiol significantly increased tyrosinase activity in melanocyte strains from 15 of 23 donors. In melanocytes from those subjects responsive to estradiol, the maximal increases ranged from 1.2- to 2.4-fold with a mean increase of 1.6 + 0.1 (SEM)-fold. T h e dose-response relationship for 3 donors is shown in Fig. 1. When tyrosinase activity was unaltered by 17fl-estradiol treatment, there was also no change in the cell numbers (data not shown). In contrast, significant increases in tyrosinase activity were accompanied by decreases in cell numbers. There was, however, no correlation between the magnitude of the increase in tyrosinase activity and the magnitude of the corresponding decrease in cell numbers (n = 12, r = 0.255, P > 0.3). T h e stimulatory effect of 17fl-estradiol was only seen when melanocytes were plated on E C M and not when they were maintained on uncoated plastic. Tyrosinase activity and melanin synthesis were positively correlated under basal conditions [8], and when tyrosinase activity and melanin synthesis were compared before and after treatment with various concentrations of 17fl-estradiol, there was a strong positive correlation between these parameters (r = 0.98,
Estrogens and Pigmentation
Table 1. Effects of estrogens ( I 0 - 9 M ) on melanocyte tyrosinase activity cell numbers and melanin extrusion (mean + S E M )
Key
400
-A'-cellline 240589 -e--cell line 010393 "-~--eellline 110789
Estrogen Control fl-estradiol Estrone Estriol
~ , , / * *
*
300 . ..-'°
~ 100 u~
$ [-
Control
i -12
i -11
i
i -I0
i -9
17 beta-estradiol (log molar concentration / dose) Fig. 1. L i n e a r d o s e - r e s p o n s e r e l a t i o n s h i p b e t w e e n 1 7 / J - e s t r a diol a n d m e l a n o c y t e t y r o s i n a s e a c t i v i t y . D a t a a r e t h e me.an + SEM (bars) of triplicate determinations. Signific a n t l y d i f f e r e n t f r o m c o n t r o l v a l u e s , * P < 0.05; * * P < 0.025; * * * P < 0.0l; * * * * P < 0.0025.
280 ~
240
~ 20o o
160 .~
120
=
80
"~
40
. / ..,/.
:/
/
I I I 40 80 120 160 200 240 T y r o s i n a s e activity (% o f control v a l u e s )
Tyrosinase activity Cells/well Melaninextruded (cpm/100cells/4 h) (in duplicate) (cpm/100 cells/16 h) 548 + 21 1015 + 60*** 615 + 29* 733 + 58**
29774 16350 26700 24720
6.4 + 2.0 22.0 + 0.1"** 8.7 + 1.1 3.8 + 2.4
Significantly different from control values; *P < 0.02; **P < 0.01; ***P < 0.001.
****
200
0
11
I 280
Fig. 2. L i n e a r r e g r e s s i o n a n a l y s i s o f t y r o s i n a s e a c t i v i t y v e r s u s m e l a n i n s y n t h e s i s a f t e r i n c u b a t i o n w i t h 1 7 / ~ - e s t r a d i o l (10 12 to 1 0 - 9 M ) . T h e r e s p o n s e s o f m e l a n o c y t e s t r a i n s f r o m 3 d i f f e r e n t d o n o r s to 1 7 / ~ - e s t r a d i o l w e r e c a l c u l a t e d a s p e r c e n t ages of control values. Data are the mean of triplicate determinations for each parameter.
P < 0.001; Fig. 2). In addition to increased melanogenesis, melanin extrusion into the medium was also enhanced by 17fl-estradiol. T h e relative effects of 17fl-estradiol, estrone and estradiol on tyrosinase activity, cell numbers and melanin extrusion are shown in Table 1. Preliminary results indicate that the catechol estrogen 2-hydroxyestradiol at 1 0 - 9 M increased tyrosinase activity and melanin synthesis by 1.13 + 0.04 (SEM)-fold (P < 0.05) and 1.25 + 0.04 (SEM)-fold (P < 0.05), respectively without affecting cell numbers. T h e presence of cycloheximide, in a dose shown to inhibit [3H] leucine incorporation [14], abolished the stimulatory effect of 17fl-estradiol on tyrosinase activity [Figure 3(A)]. Cycloheximide markedly increased melanin extrusion into the medium in otherwise untreated cells from 754 + 23 to 3192 + 540 cpm/104 cells/16 h (P < 0.001). N o radiolabelled melanin was detected in either control or estradiol-treated melanocytes in the presence of cycloheximide. Incubation of melanocytes with the pure estrogen antagonist I C I 164384 (10 -6 M) also increased tyrosinase activity [Fig. 3(B)]. When both 17fl-estradiol and I C I 164384 were preincubated with the cells, tyrosinase activity was no higher than with either agent alone. In each of three experiments, the stimulation of tyrosinase by 17fl-estradiol was mimicked by 17~estradiol (Table 2). In an experiment where treatment with 1 0 - 9 M 17fl-estradiol increased melanin extrusion 3.4 + 0.01 (SEM)-fold (P < 0.001), treatment with 1 0 - 9 M 17~-estradiol also increased melanin extrusion 2.0 + 0.3 (SEM)-fold (P < 0.001). Cell numbers were similar after both treatments. Nuclear magnetic resonance studies indicated that the spectra of the 17fl- and 17~-estradiol compounds were consistent with the expected stereochemical configurations (data not shown). N o estrogen receptors were found using the Abbott monoclonal antibody estrogen receptor kit, which showed clearly positive results with breast cancer cells. Since it has been proposed that apparent estrogen binding to pigment cells may be an artefact due to [3H]H20 release from tritiated estradiol by tyrosinase [29], specific binding of [3H] 17fl-estradiol was tested in the presence of the tyrosinase inhibitor P T U . Specific binding was not demonstrated under these conditions.
I2
Stephen 1). e\lcl,eod cl a/. A
200
175
7,
150
Z Z Z Z Z
125
z/
Z
g
lOO
i7` 75
z z
7, Z
Z Z 7, Z
7, 50
Z
Pa
y., 25
z1 t 9,
7,
Z
,'/
+.
g
+g
Fig. 3. (A) T y r o s i n a s e a c t i v i t y in m e l a n o c y t e s a f t e r t r e a t m e n t w i t h 1 7 ~ - e s t r a d i o l (10 9M) in t h e p r e s e n c e o r a b s e n c e o f cyeloheximide (50/*g/ml) (B) Tyrosinase activity in m e l a n o c y t e s a f t e r t r e a t m e n t w i t h 1 7 # - e s t r a d i o l (10 ~ M ) in t h e p r e s e n c e or a b s e n c e o f ICI 164384 (10 ~ M). D a t a a r e t h e m e a n _+_S E M ( b a r ) o f t r i p l i c a t e d e t e r m i n a t i o n s . S i g n i f i c a n t l y different from control values, *P < 0.025; **P < 0.01; ***P < 0.0025; ****P < 0.001.
DISCUSSION
Skin pigmentation in vivo is d e t e r m i n e d by genetic, environmental, local and endocrine factors which influence both melanin synthesis within each melanocyte and the distribution of melanin t h r o u g h o u t the epidermis [15]. In a previous report, we demonstrated that 17fl-estradiol increased melanocyte tyrosinase activity in vitro [4]. T h e s e studies have now been extended to show that responsiveness to 17fi-estradiol is d o n o r - d e p e n d e n t 7"able 2. Comparison between the effects of 17fi-estradiol (I0 9M) and 17~-estradiol (10 9M) on melanocyte tyrosinase activity in cpm/lO0 cells~4 h (mean + S E M ) Experiment 1 Experiment 2 Experiment 3
Control
fl-Estradiol
:~-Estradiol
548 + 2l 671 ± 12 1079 + 42
1015 + 60** 965 ± 68* 1473 + 28**
988 + 78** 900 _+ 57* 1750 + 47**
Significantly' different from control values; *P < 0.005; **P < 0.001.
and that melanin synthesis and extrusion is als~ ll> creascd by the hormone. Significant stimulation of tyrosinase activity by l - f i estradiol was seen in 65",, of donors. M e l a n o c y i c strains from 8 out of 23 donors showed no significant alteration in tyrosinase activity in response 1o lhe h o r m o n e . Despite the reproducibility of the stimulatory effect of 17fi-estradiol in most donors, both thc basal tyrosinase activity and the degree of stimulation varied noticeably in melanocytes from different donors, as would be expected from in vivo studies. Estrogen responsiveness could not be predicted by basal tvrosinase activity or plating/proliferative capacity as assessed by cell n u m b e r s in vehicle-treated wells. A response to h o r m o n e s was only observed in melanocytes plated on E C M in the absence of P M A and C T . T h e E C M is u n d e r s t o o d to resemble the basal lamina of skin where melanocytes normally reside, and it maintains melanocytes in a relatively quiescent but highly differentiated state in the absence of mitogens which might otherwise interfere with h o r m o n e signal processing [8]. T h e authors are also aware that, whilst most studies on melanocytes have d e p e n d e d on cells from neonatal skin, some caution m u s t be exercised in extrapolating any of the results to the adult situation. T h e strong correlation between tyrosinase activity and melanin synthesis in cells treated with 17fi-cstradiol is not surprising since melanin content in m a m malian p i g m e n t cells is quantitatively dependent on tyrosinase activity [4, 16, 17]. T h e subsequent transfer of melanin from melanocytes to keratinocytcs is bclieved to occur by two processes. T h e first process involves the contact of a melanocytic dendrite with the cell m e m b r a n e of a keratinocyte, which then pinches off the dendritic tip containing m e l a n o s o m c s and encloses it within a phagocytic vacuole prior to melanin dispersion [15]. T h e second process inw)lves the extracellular secretion of melanosomes from melanocvtic dendrites, and their subsequent engulfment by the n e i g h b o u r i n g keratinocytes [5]. O u r studies with cvcloheximide, which inhibited the increase in tvrosinasc induced by 17fl-estradiol and caused almost complete melanin extrusion u n d e r basal conditions, is consistent with the suggestion that melanin extrusion may be tonically inhibited by a protein with a relatively short half-life. T h a t 17fi-estradiol directly enhances melanogenesis and melanin extrusion in melanocytes in vitro provides some evidence to s u p p o r t a role for estrogens in m o d u l a t i n g skin pigmentation and in the development o f melasma. T h e biological effects of estrogens in classical target tissues, such as the uterus and breast, are believed to be mediated by the b i n d i n g of estrogen to an intracellular steroid receptor protein and the subsequent alteration of gene expression [6]. T h e affinities of estrone and estradiol for the estrogen receptor in uterine tissues are approx. 20 and 5"<,, respectively of that of 17fiestradiol [6]. T h e reduced activity of estrone and estriol
Estrogens and Pigmentation on the tyrosinase activity of melanocytes, together with t h e a b o l i t i o n o f t h e e s t r o g e n i c effects in t h e p r e s e n c e o f c y c l o h e x i m i d e , a r e c o n s i s t e n t w i t h a classical e s t r o g e n r e c e p t o r - m e d i a t e d m e c h a n i s m , b u t a r e also c o n s i s t e n t with other mechanisms. However, despite the reports t h a t I C I 1 6 4 3 8 4 is a p u r e r a n t i - e s t r o g e n t h a n t a m o x i f e n a n d has little a g o n i s t a c t i v i t y [ 1 8 - 2 0 ] , a s t i m u l a t o r y e f f e c t o f t h i s a g e n t w a s o b s e r v e d in o u r m e l a n o c y t e system. M o r e s t r i k i n g l y , t h e c o n c e p t o f a classic r e c e p t o r m e d i a t e d m e c h a n i s m is i n c o n s i s t e n t w i t h o u r o b s e r v a t i o n t h a t t h e effects o f 1 7 ~ - e s t r a d i o l w e r e v e r y s i m i l a r to t h o s e o f 1 7 f l - e s t r a d i o l . S i m i l a r effects o f 17c¢ a n d 1 7 f l - e s t r a d i o l h a v e p r e v i o u s l y b e e n r e p o r t e d in t h e estrogen receptor negative MDA-MB-330 human b r e a s t c a n c e r cell l i n e [21]. W h i l e it is p o s s i b l e t h a t 1 7 ~ - e s t r a d i o l c a n b e i s o m e r i z e d to 1 7 f l - e s t r a d i o l in v i t r o [22], o t h e r e x p e r i m e n t s i n d i c a t e t h a t in s o m e tissues of neural crest origin the phenolic 17fl-estradiol c a n b e c o n v e r t e d b y 2- a n d 4 - h y d r o x y l a s e e n z y m e s to c o m p o u n d s w h i c h r e s e m b l e c a t e c h o l a m i n e s [23, 24]. T h e s e c a t e c h o l e s t r o g e n s e x h i b i t r e d u c e d affinity f o r t h e e s t r o g e n r e c e p t o r [25] b u t , b y v i r t u e o f t h e i r c a t e c h o l s t r u c t u r e , h a v e b i o l o g i c a l a c t i v i t y in s e v e r a l in v i t r o s y s t e m s [26, 27]. M o r e o v e r , 1 7 ~ - e s t r a d i o l c a n b e c o n v e r t e d to c a t e c h o l e s t r o g e n s in a s i m i l a r m a n n e r to 1 7 f l - e s t r a d i o l [28], a n d is e q u i p o t e n t to 1 7 f l - e s t r a d i o l in i n h i b i t i n g t y r o s i n e h y d r o x y l a s e a n d c a t e c h o l - o m e t h y l t r a n s f e r a s e a c t i v i t y in r a b b i t a n d rat b r a i n in v i t r o [25, 28]. T h e r e l a t i v e l y m o d e s t effects o f 2 - h y d r o x y e s t r a d i o l in t h i s p r e l i m i n a r y s t u d y m a y b e d u e to t h e l i k e l y r a p i d m e t a b o l i s m o f t h i s m e t a b o l i t e w h e n it is p r o v i d e d as s i n g l e d o s e s r a t h e r t h a n b e i n g s y n t h e s i z e d o v e r a p e r i o d o f t i m e f r o m its s u b s t r a t e [29]. E v i d e n c e for t h e p r e s e n c e o f c a t e c h o l a m i n e b i o s y n t h e t i c p a t h w a y s w i t h i n m e l a n o m a cells has b e e n p r e s e n t e d b y M c E w a n a n d P a r s o n s [30]. F u r t h e r m o r e , it has b e e n s h o w n t h a t t y r o s i n a s e c a n u t i l i z e e s t r a d i o l as a s u b s t r a t e a n d h y d r o x y l a t e it to a c a t e c h o l - l i k e c o m p o u n d [31]. T h i s m a y b e p o s s i b l e b e c a u s e t h e A r i n g o f e s t r a d i o l has s t r u c t u r a l r e s e m b l a n c e to t h e p h e n o l i c group of tyrosine and thus may substitute for tyrosine. T h e p r e l i m i n a r y o b s e r v a t i o n in o u r l a b o r a t o r y o f m e l a n o c y t e t y r o s i n a s e s t i m u l a t i o n b y t h e c a t e c h o l estrogen 2-hydroxyestradiol suggests the possible regulation of tyrosinase by catechol-like compounds. However, the precise mechanism by which estrogens m a y affect m e l a n o c y t e f u n c t i o n t h r o u g h c a t e c h o l i n t e r m e d i a t e s is as y e t u n k n o w n . Acknowledgements--This work was supported by the National
Health and Medical Research Council of Australia, the University of Sydney Cancer Research Fund, the New South Wales State Cancer Council, the Leo and Jenny Leukemia and Cancer Foundation of Australia and the Government Employees Assistance to Medical Research Fund. NMR studies were performed by Dr A. Cheung in the Department of Pharmacology, University of Sydney. Estrogen receptor studies with the Abbot monoclonal antibody were performed by Dr A. M. Bilous, Department of Anatomical Pathology, Westmead Hospital, Sydney, Australia.
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