Journal of Dermatological Science 27 Suppl. 1 (2001) S68– S75
www.elsevier.com/locate/jdermsci

Effects of all-trans retinoic acid on melanogenesis inpigmented skin equivalents and monolayer culture ofmelanocytes
Kotaro Yoshimura a,*, Katsuhiko Tsukamoto b, Mutsumi Okazaki a,
Victoria M. Virador c, Tie-Chi Lei c, Yasutoshi Suzuki a, Gentaro Uchida a,
Yukie Kitano a, Kiyonori Harii aaDepartment of Plastic and Reconstructi6e Surgery, Graduate School of Medicine, Uni6ersity of Tokyo, 7 -3 -1, Hongo,
Bunkyo-Ku, Tokyo 113 -8655, Japanb
Department of Dermatology, Yamanashi Prefectural Central Hospital, Yamanashi, Japanc
Laboratory of Cell Biology, National Institute of Health, Bethesda, MA, USA

Abstract
The effects of all-trans retinoic acid (RA) on melanogenesis and the mechanism of its action in topical treatment
have not been elucidated. The purpose of this study was to determine the effects of RA on melanogenesis in thepigmented skin equivalent as well as in monolayer culture of melanocytes, and to determine whether RA,
hydroquinone (HQ), and hydrocortisone (HC) show synergistic depigmenting effects in combined treatments of eachother. The suppressing effect of RA on melanogenesis was not observed in pigmented skin equivalents and monolayer
culture of murine and human melanocytes, although HQ showed strong inhibition of melanogenesis. The synergistic
effects between RA, HQ, and HC were not particularly seen. The results suggested that RA neither has direct
inhibitory effects on melanogenesis of melanocytes, nor influences the cell– cell interactions between melanocytes,
keratinocytes and fibroblasts, such as paracrine actions with regard to melanin production. The role of RA inbleaching treatments appears to be in other specific actions, such as promotion of keratinocytes proliferation andacceleration of epidermal turnover. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: All-trans retinoic acid; Melanogenesis; Pigmented skin equivalents; Melanocytes

1. Introduction* Corresponding author. Tel.: + 81-3-5800-8948; fax: + 813-5800-6929.
E-mailaddress:
yoshimura-pla@h.u-tokyo.ac.jp(K.
Yoshimura).

All-trans retinoic acid (tretinoin; RA) has beenclinically used for acne and photoaged skin for
more than two decades. Topical application of
RA is also known to be effective for melasma andsome other skin hyperpigmentation in a single[1–4] or combined use [5–8] with some other0923-1811/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 9 2 3 - 1 8 1 1 ( 0 1 ) 0 0 1 1 6 - 5

K. Yoshimura et al. / Journal of Dermatological Science 27 Suppl. 1 (2001) S68 – S75

reagents such as hydroquinone (HQ) and corticosteroids. Since Kligman et al. [5] proposed a combined use of RA, HQ and dexamethasone as a
topical depigmenting formula, a number of products based on the formula have become commercially available and widely used for bleaching of
hyperpigmented skin lesions.
Hydroquinone is the most widely used depigmenting agent at present and has been shown to
inhibit tyrosinase-mediated conversion of tyrosine
to dopa [9] and dopa to dopaquinone [10]. Corticosteroids are also known to have a depigmenting
effect in single use [11], and this is one of thereasons why Kligman et al. [5] employed corticosteroid in his formula.
Although it is known that topical RA improvesa variety of skin hyperpigmentation, the mechanism underlying these depigmenting effects of RA
has not been elucidated. Furthermore, although anumber of studies have been performed on effectsof RA on pigment cells, the experimental results
remain contradictory [12]. Some studies withmouse melanoma cell lines and humanmelanocytes indicated a pigmentation-promoting
effect of RA [13– 16]. On the other hand, there
was no melanogenic effect on K-1735P ultraviolet-irradiated, transformed murine melanoma
cells, which remained amelanotic following RA
treatment [17]. Under conditions which supported
proliferation, RA at concentrations of 0.25– 0.1
mg/ml inhibited growth of human melanocytes,
while there was no significant change in theamount of melanin per cell or in tyrosinase activity [18]. It is known that tyrosinase gene expression and activity, if already stimulated by MSH, isinhibited by RA in murine and hamster
melanoma cells [19], whereas RA treatment
caused a marked increase in MSH binding capacity for both cell surface and internal MSH bindingsites [20]. Another report indicated that RA significantly decreased the UVB-stimulated melanogenesis through suppression of tyrosinase andTRP-1 synthesis at the post-transcriptional levelin mouse melanoma cells and human melanocytes[21]. Thus, it is difficult to establish an unequivocal effect of RA on melanogenesis in pigment
cells.

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Several growth factors have been identified as
the paracrine factors from keratinocytes andfibroblasts, showing effects on melanocyte proliferation and functions [22– 25]. Three-dimensional
pigmented skin equivalent is a useful strategy toinvestigate the cell–cell interactions in the regulation of in vivo melanogenesis, and the effects ofRA has never been examined with the pigmented
skin equivalent. The purpose of this study is todetermine the effects of RA on melanocytes inmonolayer culture and also in pigmented skinequivalents consisting of melanocytes and keratinocytes grown on a dermal equivalent, and to
determine whether RA and/or HC show synergistic depigmenting effects in combined use with
HQ.

2. Materials and method

2.1. Melanocyte culture
The murine melanocyte cell lines, melan-a, andconditions for its culture have been described
previously [26]. Melan-a was cultured in Dulbecco’s modified Eagle’s minimum essential medium(DMEM) supplemented with 5% fetal calf serum(FCS), 100 mM b-mercaptoethanol, 2 mM L-gultamine, and 200 nM TPA (tumor promoter 12-Otetradecanoylphorbol acetate). Normal humanmelanocytes obtained from Asian foreskin andserum-free growth medium were purchased from
Morinaga Institute of Biological Science (Yokohama, Japan). Human melanocytes were cultured
in MM-4 medium [27] supplemented with 10 mg/l
phorbol-12-myristate 13-acetate, 10 mg/l cholera
toxin, and 150 mg/l bovine pituitary extract.
RA was obtained from Sigma (St. Louis, MO),dissolved at 10 − 3 M in ethanol, and kept in
foil-wrapped containers protected from light at
4 °C. HQ and hydrocortisone (HC) were obtained from Wako (Osaka, Japan).
Murine melanocytes (melan-a) or humanmelanocytes were seeded in 3.5 cm dishes (1.5×
105 cells/dish) for melanin and protein assay. Human melanocytes were cultured in MM-4 with
0.5% serum for 24 h, after which the medium was
exchanged for MM-4 without serum. Melan-a wasS70

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cultured in DMEM with TPA for 2 days, after
which the medium was exchanged for DMEM
without TPA. After murine or humanmelanocytes had been cultured for 4 days, every
assay was performed. RA (10 − 6 M), HQ (10 − 5M), and HC (10 − 4 M) were individually or in
combination of each other added to each medium
for 4 days, and seven kinds of culture groups wereprepared as follows; none (control), RA alone(RA), HQ alone (HQ), HC alone (HC), RA+
HQ, RA + HC, HQ + HC, and RA + HQ +HC.
At least four data were collected for each group.

2.2. Pigmented skin equi6alents
Human keratinocytes and fibroblasts were isolated from skin sections obtained from skin
surgery for young patients. Human keratinocytes
were grown in a modified serum-free KGM(Kyokuto Seiyaku, Tokyo), which consists of
MCDB153 with high concentrations of aminoacids, transferrin (final concentration, 10 mg/ml),
insulin (5 mg/ml), hydrocortisone (0.5 mg/ml),
phosphorylethanolamine (14.1 mg/ml) and bovine
pituitary extract (40 mg/ml). Human fibroblasts
were grown in DMEM supplemented with 10%
FCS. Keratinocytes and fibroblasts at population
doubling levels of 2– 4 and 10– 15, respectively,
were used for experiments.
Human keratinocytes and murine melanocytes
were cultured in a three-dimensional fashion at
the air –liquid interface on top of a dermal equivalent consisting of cultured fibroblasts (106/gel)and type I collagen. The dermal equivalents (contracted collagen gels) were prepared according to
the method described by Tsunenaga et al. [28].
Murine melanocytes were plated at 2×105/cm2
inside a glass ring (12 mm diameter) on the surface of the dermal equivalent, which was thenplaced on a stainless steel mesh. Melanocytes weregrown in DMEM plus 10% FCS on day 1. On
day 2, keratinocytes were additionally plated at
8× 105/cm2 inside the glass ring on the dermal
equivalent, and the medium was changed to 1:1
mixture of KGM and DMEM plus 10% FCS, inwhich the Ca2 + concentration was adjusted to
0.18 mM. After day 4, the medium in the glass
ring was removed and the surface of the dermal

equivalentonwhichkeratinocytes
andmelanocytes were cultured were allowed to face
the air. The discharged medium in the ring was
aspired twice a day after day 4. The medium inthe dish was changed every other day. The
medium including the designated compounds wasused from day 4. On day 9, skin equivalents were
fixed with 4% paraformaldehyde in PBS.

2.3. Measurement of pigment on the skinequi6alents
The melanin pigments of each sample were
photographed and each digital image was analyzed with Image-Pro Plus (version 3.0, Media
Cybernetics, Silver Spring, MA). Difference between an original image and its extracted background was estimated as the melanin pigments,
and area and intensity of each pigment were
measured. Total sum of melanin pigments data ofeach sample was referred to as ‘total melanin’ of
individual samples in this paper.

2.4. Melanin and protein assay
Melanin content was determined according to
the method described by Oikawa and Nakayasu[29], which we modified. Briefly, melan-a or human melanocytes were cultured at 37 °C for 4days with the various types of medium. Cell pellets were lysed in 1.0 ml extraction buffer (50 mM
Tris buffer; pH 7.5, 2 mM EDTA, 150 mM NaCl,and 1% Triton-X). Supernatants were used forprotein assay, which was performed with a BCA
protein assay kit (Pierce, Rockford, IL). After
resuspending melanin pellets in 1.0 ml extraction
buffer and centrifuging, the melanin pellets wereincubated with 0.5 ml 2 N NaOH containing
melanin 20% DMSO for 30 min. The opticalabsorbance of each sample was measured at 470
nm with an ELISA plate reader (model 550 microplate reader, Bio-Rad Laboratories, CA).

2.5. Tyrosinase assay
Compounds were tested for direct effects on
tyrosinase activity using a modified radiometrictyrosinase assay as previously described [30].

K. Yoshimura et al. / Journal of Dermatological Science 27 Suppl. 1 (2001) S68 – S75

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Briefly, the melanogenic tyrosinase assay was performed in quadruplicate in 96-well microtiterplates by adding 10 mm compound and 20 ml
purified murine or human tyrosinase, in that order. After 30 min preincubation at 23 °C, 10 ml
14
L-[ C]tyrosine was added along with 10 ml 0.25
mM L-dopa cofactor in 1 M sodium phosphatebuffer, pH 7.2, containing 0.01% albumin. Reactions were incubated for 1 h at 37 °C, after which
100 ml 0.1 M HCl with excess unlabelled L-tyrosine was added to each well. The contents ofeach well were removed with a multichannel
pipettor to a dot-blot apparatus (Bio-Rad, Hercules, CA) and acid-insoluble radioactive melanin
and melanin precursors were bound to ZetaProbeblotting membranes (Pharmacia, Piscataway, NJ)
for 15 min at 23 °C. The membranes were thendried under vacuum and washed three times with
250 ml 0.1 M HCl with excess unlabelled tyrosine;
they were then removed from the apparatus andwashed three more times for 20 min each with 100
ml 0.1 M HCl. Membranes were then air-dried
and exposed to a Storm phosphor screen; quantitation of radioactive melanin production on thoseblots was performed using a Strom 860 PhosphorImager and ImageQuant software (Molecular
Dynamics).

Total melanin was significantly reduced in HQ-,
RA + HQ-, HQ + HC-, and RA + HQ + HCtreated groups (Fig. 3). Total melanin of RAtreated group was elevated by 10% in average, but
the difference was not statistically significant. Total melanin was significantly less in HQ-, HC-,RA + HQ-, RA + HC-, HQ + HC-, and RA +HQ + HC-treated groups than in RA-treatedgroup.

2.6. Statistics

Total protein in any group was not significantly
different from that in the control (Fig. 5). In the
RA-treated group, the absolute and relativemelanin was elevated by 20–30% in average

Significant differences were sought using two
way analysis of variance (ANOVA). Post hoccomparison of individual group was performed
only if the F ratio for the overall ANOVA wassignificant; appropriate Bonferonni corrections
were applied to all post hoc comparisons. Differences were considered significant when P-values
were B 0.05.

3.2. Melanin content and total protein in murine
melanocytes
Total protein in any group except for the
RA +HQ +HC-treated group was not significantly different from that in the control (Fig. 4).
Total melanin (absolute value) did not show alarge change after RA-, HC-, or RA+ HC-treatment. The relative melanin in RA-, HC-, andRA +HC-treated groups was elevated by 10–35%
in average value, but the difference was not statistically significant. The absolute and relative valuesof melanin were significantly reduced in HQ-,
RA + HQ-, and RA + HQ +HC-treated groups.

3.3. Melanin content and total protein in humanmelanocytes

3. Results

3.1. Melanin pigments on pigmented skinequi6alents
The representative histology of the control sample was shown in Fig. 1, and representative samples of each group were demonstrated in Fig. 2.

Fig. 1. Histological view of pigmented skin equivalents (one of
the control samples) at the site of intense pigmentation. Bar =50 mm.

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K. Yoshimura et al. / Journal of Dermatological Science 27 Suppl. 1 (2001) S68 – S75

Fig. 3. Melanin production on pigmented skin equivalents
measured with a computed image analyser. Values were shownas mean + S.E. *PB 0.05 to control, c PB 0.05 to RA.

3.4. Tyrosinase acti6ity in murine melanocytes
HQ had an obvious inhibition on tyrosinase
activity up to 70% (Fig. 6). RA and HC also had
also slight inhibition on tyrosinase activity. Synergistic effects of three compounds were not seen inthis study.

3.5. Tyrosinase acti6ity in human melanocytes
RA appeared to show no suppressing effects(Fig. 7). HQ showed a 60% decrease inmelanogenic activity. HC also showed a decrease
in melanogenic activity, but no synergistic effects
in the three compounds.

Fig. 2. Representative samples of pigmented skin equivalents.
Pigmented spots were produced on the dermal equivalents
inside glass rings (12 mm in diameter).

value, nevertheless without statistical significance.
The absolute and relative values of melanin weresignificantly reduced in HQ- and RA+ HQtreated groups.

Fig. 4. Melanin and protein assay in monolayer culture ofmurine melanocytes (melan-a). The relative values to control
were shown as mean +S.E. Relative melanin was calculated as(absolute melanin)/(protein). *P B0.05 to control.

K. Yoshimura et al. / Journal of Dermatological Science 27 Suppl. 1 (2001) S68 – S75

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Fig. 7. Effects on tyrosinase activity in monolayer culture ofhuman melanocytes.
Fig. 5. Melanin and protein assay in monolayer culture ofhuman melanocytes. The relative values to control were shownas mean +S.E. Relative melanin was calculated as (absolutemelanin)/(protein). *P B 0.05 to control.

4. Discussion
The mechanisms by which RA and HQ act in
the combined bleaching protocols such as in Kligman’s regimen and others are still to be elucidated. Although a number of studies have been
performed concerning the effects of RA on skin invivo or in vitro, there are some contradictory
results the reasons for which remain unknown[31– 33]. Although it is reported that even the
topical application of RA alone has a clinicallydepigmenting effect [1– 3], the suppressive effectsof RA on melanocyte growth and melanogenesis
have not been established in vitro [12]. Welsh etal. [34] reported that the topical application of
RA to mouse skin increases the number of activated epidermal melanocytes and makes

Fig. 6. Effects on tyrosinase activity in monolayer culture ofmurine melanocytes (melan-a).

melanocytes more sensitive to activation by ultraviolet B radiation.
In the present study, suppressing effects of RA
on melanogenesis of melanocytes were not observed both in monolayer culture and organotypic
culture. Melanin production per cell was not significantly changed in the monolayer culture of themurine melanocyte cell line and humanmelanocytes,
althoughproliferationofmelanocytes and tyrosinase activity were somewhat suppressed by RA treatment in the monolayer culture of murine melanocytes. In pigmentedorganotypic culture consisting of murine
melanocytes and human keratinocytes grown on adermal equivalent, melanogenesis was even promoted, although the difference in total melanin
production between the control and RA-treatedgroups was not statistically significant. The results
suggested that RA neither has direct inhibitory
effects on melanogenesis of melanocytes, nor influences the cell–cell direct and indirect interactions between melanocytes, keratinocytes andfibroblasts in a paracrine manner with regard tomelanin production.
In the monolayer culture of murine and humanmelanocytes, all experimental groups with treatment of HQ suppressed melanin production andtyrosinase activity. HC did not show apparent
suppressing effects on melanogenesis andtyrosinase activity in the monolayer culture. Neither RA nor HC show synergistic suppressing
effects of HQ on melanogenesis and tyrosinase
activity.

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K. Yoshimura et al. / Journal of Dermatological Science 27 Suppl. 1 (2001) S68 – S75

The results of the present study suggested
that RA does not have suppressing effects on
melanogenesis of melanocytes in spite of its
clinical whitening efficacy on some pigmented
skin lesions, nor synergistic suppressive effects
with HQ. Therefore, it is suggested that the depigmenting effects in vivo of RA in a single or
combined use with HQ and/or HC are derived
from other mechanisms than direct effects on
melanocytes. A recent report suggested a role forcellular retinoic acid binding protein (CRABP)-I
in mediating RA effects on melanogenesis andinvolvement of keratinocytic and dermal influences on CRABP activity in melanocytes [35].
However, in our pigmented skin equivalents, inwhichinteraction
betweenkeratinocytes,
melanocytes and fibroblasts can be examined, inhibitory effects of RA on melanogenesis were notobserved.
RA promotes not only proliferation but also
turnover of keratinocytes in vivo, and compactionof the horny layer and hyperplasia of the epidermis are characteristic changes after the topicalapplication of RA [31]. It is also known that RA
can promote collagenogenesis in dermis andwound healing [36,37]. On the other hand, skinbecomes atrophic after the application of corticosteroid [38,39] and corticosteroid suppresses collagenogenesis and wound healing [40]. Thus,
corticosteroid appears to be antagonistic in skin
to retinoids in some aspects [36,37,39,41].
In our clinical experiences, depigmenting effects ofa combined treatment of RA and HQ were
suppressed by corticosteroid [7,8], although corticosteroids are known to have a depigmenting
effect with single use [11]. Taken together,
it is speculated that the promotion of keratinocyte proliferation and acceleration of keratinocyte
differentiation are most likely to be the roles of
RA in the depigmenting treatment. Melanin granules may be washed out of the epidermis by thefast and strong stream of keratinocytes in theepidermis induced by the mechanism above.
These effects could not be reproduced in ourpigmented skin equivalents, which could be cultured for only 1 week after they were exposed toair.

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