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Received: 9 August 2018    Revised: 5 February 2019    Accepted: 7 March 2019

DOI: 10.1111/exd.13915

VIEWPOINT

How hormones may modulate human skin pigmentation in

melasma: An in vitro perspective

Muriel Cario1,2,3,4

1
Inserm 1035, Bordeaux, France
2 Abstract
Univ. Bordeaux, Bordeaux, France
3
Aquiderm, Bordeaux, France
4
National reference center for rare skin
Diseases, Bordeaux Hospital, Bordeaux,
France
Correspondence
Muriel Cario, Univ. Bordeaux, Inserm,
BMGIC, UMR1035, Bordeaux, France.
Email: muriel.cario-andre@u-bordeaux.fr
Melasma is a common acquired hyperpigmentary disorder occurring primarily in
photo-­exposed areas and mainly affecting women of childbearing age. To decipher
the role of sex hormones in melasma, this viewpoint reviews the effects of sex hor-
mones on cutaneous cells cultured in monolayers, in coculture, in 3D models and
explants in the presence or the absence of UV. The data show that sex steroid hor-
mones, especially oestrogen, can modulate in vitro pigmentation by stimulating mel-
anocytes and keratinocyte pro-­pigmentary factors, but not via fibroblast or mast cell
activation. In vitro data suggest that oestrogen acts on endothelial cell count, which
may in turn increase endothelin-­1 concentrations. However, data on explants re-
vealed that sex steroid even at doses observed during pregnancy cannot induce mel-
anogenesis alone nor melanosome transfer but that it acts in synergy with UVB. In
conclusion, we hypothesize that in predisposed persons, sex steroid hormones initi-
ate hyperpigmentation in melasma by amplifying the effects of UV on melanogenesis
via direct effects on melanocytes or indirect effects via keratinocytes and on the
transfer of melanosomes. They also help to sustain hyperpigmentation by increasing
the number of blood vessels and, in turn, the level of endothelin-­1.

KEYWORDS
endothelial cells, fibroblasts, keratinocytes, melanocytes, oestrogen, progesterone

1 |  H O R M O N E S A N D M E L A S M A I N V I VO to solar radiation seem to be the most important triggers.[1,5,6]

Melasma is a common acquired hyperpigmentary disorder occurring
primarily in photo-­exposed areas (face, neck, forearms) and mainly
affecting women of childbearing age. It can occur in all skin types
but is more common in intermediate phototypes (Hispanic, Asiatic,
Mediterranean African, Middle-­Eastern populations) and rare in ex-
treme phototypes (I, VI).[1,2] While a clear female predominance of 9
or 10 to 1 is observed, the prevalence female/male differs according
to the ethnic group, ranging from 6:1 to 39:1. [1]
While many factors
are associated with the onset of the disease, genetic predisposition, [3]
hormonal influence, especially female sex hormone (oral contracep-
tive use, hormone replacement therapy, pregnancy), [4]
and exposure
Melasma is mainly characterized by excessive epidermal pigmenta-
tion.[6–9] There are three subtypes: epidermal, dermal and mixed.[5]
Dermal melasma is characterized by the presence of melanophages
in the superficial and deep dermis. However, melanophages are also
found in perilesional and non-­lesional skin, suggesting that pure
dermal melasma does not exist.[7,9,10] An inapparent type with der-
mal pigment deposition has been described in dark women.[5] The
epidermal type occurs in 72% of pregnancy-­associated melasma,[4]
and pregnancy-­induced melasma is associated with earlier onset.[1]
Histologically, epidermal melasma is characterized by increased
melanin throughout the layers of the epidermis, and an increase in
melanosome number and transfer.[5] However, in Caucasoid women,

Experimental Dermatology. 2019;28:709–718. wileyonlinelibrary.com/journal/exd   © 2019 John Wiley & Sons A/S. |  709
Published by John Wiley & Sons Ltd
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melanosomes are transferred more individually than as polymelano-
somes.[5,7,11] While melanocytes are enlarged and have prominent
dendrites, the change in their number is still debated.
As a photoageing disorder,[12] melasma is also characterized by
solar elastosis.[6,7,13] Other features such as damage to the basal
membrane,[6,8] an increase in blood vessels and in dermal mast
cells[6,13] and lymphohistiocytic infiltration have been described.[14]
The aetiology of melasma seems more complex than that of senile
lentigo, another photoageing disorder, which can be attributed
[15]
mainly to dysregulation of UVA-­induced fibroblasts. As summa-
[12]
rized by Passeron and Picardo, solar radiation (UVA, UVB and
visible light) can induce melanogenesis in melasma by stimulating
melanocytes directly or indirectly via secretion of melanogenic fac-
tors by keratinocytes,[16] fibroblasts,[15–18] endothelial cells[19] and
probably sebocytes. Chronic exposure to UVA1 and visible light
seems to be implicated in dermal and basal membrane damage.[12]
On the other hand, sex hormones such as oestrogens, especially
17β-­estradiol (E2) and progesterone, are factors involved in the reg-
ulation of pigmentation,[20] collagen content, skin ageing, skin mois-
ture and skin thickness.[21,22]
Estradiol, luteinizing hormone (LH) and follicle-­
s timulating
hormone (FSH) levels were found to be higher in the sera of
women with melasma than in those of control women without
melasma, whereas progesterone levels were similar in the two
groups.[23,24] In the sera of men with melasma, luteinizing hor-
mone levels were higher than in those of controls without me-
lasma, whereas FSH levels were similar and testosterone levels
were lower.[25,26] In Indian males with melasma, no or only weak
expression of oestrogen receptors, progesterone receptors and
stem cell factor was observed in lesional skin and no significant
changes in serum hormone levels were detected.[27] In a Japanese
male, oestrogen therapy for prostate cancer was incriminated in
the onset of melasma.[28] Moreover, a testosterone metabolite,
5α–androstane-­3 β–diol, competes with E2 to bind ERβ and thus
elicits an oestrogenic response in cells.[29]
Oestrogen and progesterone act through receptors expressed in
both the epidermis and dermis. In human (male and female) adult
scalp skin, only oestrogen receptor (ER) β was observed in the
epidermis, fibroblast blood vessels and hair follicles, whereas in
human neonatal foreskin both ER-­α and ER-­β were detected.[21,30]
Oestrogen and progesterone receptors are expressed differently in
melasma skin than in perilesional or non-­lesional skin (Table 1 [31–34]).
The presence of these receptors and the effect of their ligands on
cutaneous cells were assessed in vitro (Table 2).
2 | E FFEC T O F S E X S TE RO I D H O R M O N E S
O N E PI D E R M A L PI G M E NTATI O N
2.1 | Direct effects of sex steroid hormones on
melanocytes
Apart from FBS and phenol red which have a mild oestrogenic ef-
fects (Table 3),[20,35,36] inter-­
individual variations which induce a
specific response should be considered.[36,37] In women,
donor-­
estradiol and progesterone concentrations vary according to age,
menstrual cycle, during pregnancy and at menopause (Table 4A, B).
Men have lower levels of plasmatic estradiol and progesterone than
women (Table 4C).
2.1.1 | Effects of oestrogen on melanocytes
Exposure to 17β-­estradiol at concentrations found in males and non-­
pregnant females (10−11 to 10−9 mol/L) induced an increase in mela-
nin content in responsive melanocytes 3 days post-­treatment,[37,38]

TA B L E   1   Expression of oestrogen and progesterone receptors in melasma skin (M) as compared to perilesional (PL) or non-­lesional skin
(NL); nd: not detected; na: not analysed

Progesterone
Ethnic origin Technique Localization Oestrogen receptor receptor Ref.

American M/NL IHC Increase na [31]

Korean M/PL IHC Epidermis Slight Increase ERβ Increase [32]
Dermis: Increase ERβ = [66]
Around small blood vessels
Fibroblast-­like cells
(probably telocytes)
Brazilian M/NL IHC Epidermis Increase ERβ Increase [33]
Dermis = =
Transcriptomic Whole skin = =
Korean M/NL Transcriptomic Whole skin, Variable expression of ERα and ERβ na [34]
14 women 3 women: great Increase ERβ and ERα;
2 women: great Increase ERβ;
2 women: Decrease ERα;
3 women: Decrease ERβ
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TA B L E   2   Expression of oestrogen and progesterone receptors but that their expression in vitro depends on stimulation by exogenous
in cells factors.
Progesterone Activation of GPER and PAQR7 respectively stimulates or inhib-
Oestrogen receptors receptors its cAMP signalling, the same pathway used by α-­MSH to regulate

Cells ERα ERβ GPER PR PAQR7 Ref.

pigmentation, so they counteract each other.[20] In order to, oestro-
gen via fixation on ERα and in association with Sp1 can modulate the
Melanocytes yes yes yes [35,36]
level of expression of the progesterone receptor by binding to ERE/
no no yes no yes [20]
Sp1 site (oestrogen response element and sp1 sites)[45] and proges-
Keratinocytes yes yes [47]
terone can inhibit ER expression.[41]
Mast cells yes yes [81]
Working on prostate stromal cells, Smith et al hypothesized that
yes no [78]
high titres of estradiol saturate the ER, inducing in turn a decrease
Fibroblasts yes yes [62]
in ER synthesis.45,46 Thus, we hypothesize that, in vitro, physiologi-
Sebocytes yes yes [61]
cal doses of oestrogen[10,11] stimulate the production of melanin by
Endothelial yes Do yes [71]
melanocytes, but that the latter regulate transcription of their ER to
cells express
maintain their homeostasis. Since we have sometimes observed that
oestrogen has no effect, we suppose that the threshold of sensitivity
varies according to the strain of melanocytes, or that transcriptional
Telocytes yes yes [66,67]
regulation of ER is induced more or less quickly. At doses found
during pregnancy and with short-­term exposure, oestrogen induced
whereas it was decreased after 10 days.[35] Under stimulation with melanin production, whereas progesterone decreased it in vitro. On
25 nmol/L (2.5.10−8 mol/L) 17β-­estradiol, a concentration observed the other hand, no data were found on the effect on melanocytes of
during pregnancy[39] or 2.5.10−8 mol/L ethinyl estradiol, which is exposure to both oestrogen and progesterone in vitro.
often used in oral contraceptive pills, melanin synthesis was in- We hypothesize that in vivo and in basal conditions, an equilib-
creased in melanocytes cultured in monolayers.[20] Modifications rium between oestrogen and progesterone pathways maintains pig-
in melanin production were inversely correlated with melanocyte mentation at its basal level. However, during hormone replacement
proliferation.[35,37] Experiments conducted with several strains of therapy and pregnancy, levels of oestrogen and progesterone might
melanocytes showed that some melanocytes did not respond to be modified and the ratio between the two hormones might vary.
stimulation by oestrogen, even at a high dose (10−7M) [36] (Table 5). This would explain the onset of melasma in women, who are more
sensitive to sex steroid hormones or are less able to regulate the
level of their receptors.
2.1.2 | Effect of progesterone on melanocytes
Few authors have analysed the effects of progesterone on pigmen-
2.2 | Indirect effect of sex steroid hormones on
tation, and no data have been reported with progesterone concen-
pigmentation via keratinocytes
trations found in males and non-­pregnant females. Progesterone
at 5.10 −7M, a physiological concentration observed in the third
2.2.1 | Effects of oestrogen on melanocytes via
trimester of pregnancy,[39] decreased pigmentation in melanocytes
keratinocytes
cultured in monolayers[20] (Table 5). Female melanocytes derived
from iPS and male melanocytes from various body locations (facial In keratinocytes, E2 (10 −11-­10 −9M) induced ERα synthesis in a dose-­
vs foreskin) and of different age (adult vs. newborn) had a similar dependent manner, whereas E2 did not modify ERβ levels.[47] On
[20]
response to oestrogen and progesterone (n = 3 for each group). the other hand, oestrogen at concentrations found in pregnancy
upregulated ERα and ERβ (34). E2 induced proliferation of keratino-
cytes in vitro[47] via non-­genomic and genomic pathways. However,
2.1.3 | Expression of sex steroid hormone receptors
an increase in proliferation was observed at basal concentrations
in melanocytes
(10 −11-­10 −9M), whereas, according to authors and thus probably
Oestrogen and progesterone may act through several receptors. For the donors, the high concentrations found in pregnant women
example, adult and foreskin melanocytes did[35,36] or did not[20] express (10 −8-­10 -7M) have no effect on proliferation[48] or reach a plateau at
ER and PR at mRNA and protein levels. However, while Natale et al did 10 -8 mol/L.[49] Estradiol is inactivated by oestrogen sulfotransferase,
not find ER and PR, they found that melanocytes responded to oestro- which is more highly expressed in calcium-­differentiated keratino-
gen via fixation to GPER (G-­protein coupled oestrogen receptor) and to cytes than in proliferating keratinocytes, suggesting that the ac-
progesterone via fixation to PAQR7 (progestin and adipoQ receptor 7), tion of estradiol is suppressed in differentiated epidermal layers.[50]
[20]
two G protein-­coupled receptors. Since various factors such as TPA, Estradiol (10 -8 mol/L, concentration observed during pregnancy)
[40–44]
PMA, CT, insulin, IGF-­1 and EGF modulate ER and PR, we hypoth- also enhanced the production of GM-­C SF, a well-­known melano-
esize that melanocytes are able to express ER, GPER, PR and PAQR7 genic factor,[16] in keratinocytes via the PKC and ERK pathways.[51]
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712       CARIO

TA B L E   3   Summary of melanocyte culture

Seeded melanocytes/cm² Type of Time of

Origin, age and phototype support Medium Supplements [Ho] Treatment incubation Ref.

Neonatal foreskin ECM coated EMEM 2% FCS 17β-­estradiol 10 -12 3/day 3 d [37]
2.10 4 or uncoated to 10 -9 mol/L + 1 time 4 h
Iscove 0.1% BSA,
age: 1-­4 months prior assay
medium 80 μg/mL soya bean
lecithin,
30 μg/mL transferrin
Neonatal foreskin uncoated EMEM 5% chelexed FCS 17β-­estradiol 10 -12 1 24 h [38]
1-­4.10 4 to 10 -9 mol/L
Passage 4 to 6
Korean Adult foreskin nd MCDB 4% chelexed FBS, Oestrogen 1/day 6 d [36]
5.3.10 4 153 8 nM TPA, or progesterone
phototype 0.6 ng/mL bFGF, 10 -9 mol/L and
III-­V 5 μg/mL insulin, 10 -7
1 g/mL tocopherol, 10 000
units/mL penicillin,
10 000 g/mL streptomycin
Foreskin nd M 254 0.2% BPE, 2.5.10 -­8 nd 4 d [20
Lightly pigmented 0.5% FBS, 17β–estradiol
2.6.106 1 μg/mL insulin-­like growth
factor-­I ,
Foreskin heavily pigmented 5.10 -7
5 μg/mL Bovine transferrin,
2.6.106 Progesterone
3 ng/mL bFGF,
0.18 μg/mL Hydrocortisone,
3 μg/m Heparin,
10 ng/m PMA,
10 000 units/mL penicillin,
10 000 g/mL streptomycin
Foreskin Vitronectin F12 Wo 20 ng/mL EGF, 17β–estradiol nd 4 and 10 d [35]
2.10 4 coated phenol 5 μg/mL insulin, 10 -12 and
red 40 ng/ml CT, 10 -9 mol/L
5 μg/mL transferrin,
1 μmol/L Hydrocortisone,
15 μg/mL Endothelial cell
growth supplement,
1.10 -7 mol/L retinol,
85 nmol/L TPA,
1% chelexed FCS

[Ho], hormone concentration; bFGF, basic fibroblast growth factor; BPE, bovine pituitary extract; BSA, bovine serum albumin; CT, cholera toxin; EGF,
epidermal growth factor; FBS, heat-inactivated fetal bovine serum; FCS, heat-inactivated fetal calf serum; Nd, not described; PMA, Phorbol 12-myristate
13-acetate; TPA, 12-O-tetradecanoyl phorbol 13-acetate; Wo, without.
Chelexed serum has no oestrogen.

In melasma lesional skin, PDZK1 (PDZ domain protein kidney 1),
[52]
an oestrogen-­induced factor, is overexpressed in keratinocytes
[34]
and melanocytes. In a melanocyte-­keratinocyte coculture, over-
expression of PDZK1 increased the oestrogen (10 -7-­10 -8M)-­induced
tyrosinase expression, whereas knock-­down of PDZK1 reduced the
oestrogen-­induced upregulation of tyrosinase. Oestrogen induces
tyrosinase by way of MITF. This overexpression also stimulated mel-
anosome transfer via increased phosphorylation of ERM (ezrin/ra-
dixin/moesin) and Rac1 (Ras-­related C3 botulinum toxin substrate 1)
[34]
but not via PAR-­2. (Figure 1 A)
2.2.2 | Effects of progesterone on melanocytes via
keratinocytes
An increased proliferation of keratinocytes was observed with basal
concentrations of progesterone (10 -11-­10 -9M). Concentrations found
during pregnancy and luteal phase (10 -8-­10 -7M) did not have any ef-
fect on the proliferation of keratinocytes.[48] Until now, there is no
evidence indicating that progesterone stimulates the secretion of
melanogenic factors by keratinocytes.
Due to the expression of oestrogen sulfotransferase in differ-
entiated keratinocytes, the effect of oestrogen is mainly observed
in the basal layer,[50] suggesting that oestrogen mainly affects the
epidermal melanin unit. In the epidermal melanin unit, the number of
melanocytes and keratinocytes is tightly regulated,[53] so if oestro-
gen induces proliferation of the basal keratinocytes, the number of
melanocytes increases. It is well known that keratinocytes secrete
exosomes and factors that regulate melanogenesis, differentiation
and proliferation of melanocytes.[54,55] We hypothesize that oes-
trogen (10 -7-­10 -8 mol/L) induces an increase in basal keratinocytes
which, in turn, secrete factors inducing proliferation of melanocytes,
tyrosinase activity and melanosome transfer leading to melasma.
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      713

TA B L E   4   Concentration of estradiol and progesterone in females (A, B) [82–86] and males (C) [85–88]

(A)

Age (years) 0 to <6 6 to <11 11 to <15 15 to <19

-10 -10 -10
Estradiol (M) 0.73-­1.94 × 10 0.73-­2.16 × 10 0.73-­3.19 × 10 0.73-­
4.07 × 10 -10
Progesterone (M) 2.7 10 −10 -­4.7 × 10 −8

(B)

Menstrual cycle Weeks of pregnancy

Stage Follicular Ovulation Luteal <12 28 39 Menopause

−10 −9 −10 −9 −8 −8
Estradiol (M) 1.1-­3.6 × 10 0.47-­1.28 × 10 1.8-­6.6 × 10 ~8 ×10 ~4 × 10 ~8 × 10 <2.2 × 10 −10
−9 −9 −8 −8 −7 −7
Progesterone (M) 0.47-­2.2 × 10 ~8 × 10 0.6-­7.95 × 10 ~6 × 10 ~1.3 × 10 ~2.2 × 10 0.2-­5 × 10 −9

(C)

Age (years) 0 to <19 18-­3 0 20-­47 56-­71

-10 −10
Estradiol (M) 0.73-­1.46 × 10 ~2 × 10 ~1.4 × 10 −10
Progesterone (M) 2.1 × 10 −10 -­1.3 × 10 −8 ~6 × 10 −10

~, around

Nevertheless, the melanocyte/keratinocyte ratio may vary accord-
ing to the patient's sensitivity or feedback control on the oestrogen
receptor. Progesterone does not seem to play a role in the modula-
tion of pigmentation by keratinocytes in melasma.
3 | E FFEC T S O F S E X S TE RO I D H O R M O N E S
O N D E R M A L CO M PA RTM E NT A N D
I N D I R EC T E FFEC T O N E PI D E R M A L
PI G M E NTATI O N
It is interesting to examine the influence of hormones in the der-
mal compartment for two reasons. First, in melasma, the number of
blood vessels and mast cells is greater in lesional skin than in perile-
sional skin.[6] Second, dermal regulation of pigmentation has already
been demonstrated in vitro,[17,56,57] in vivo [18]
and in pigmentary
disorders such as senile lentigo[15] and vitiligo[58,59] and has been
[19]
hypothesized in melasma. Furthermore, in systemic scleroderma
(SSc) whose main skin feature is fibrosis, characteristic modifica-
tions of the fibroblast functional phenotype are thought to be due
to an interplay between damaged endothelial cells, immune cells
and their soluble mediators.[60] As in SSc, there might also be an in-
terplay in melasma. In addition, since most dermal cells possess sex
steroid hormone receptors (Table 2), dermal cells may influence epi-
[16]
dermal pigmentation directly or indirectly via other dermal cells.
3.1 | Indirect effects of sex steroid hormones on
pigmentation via regulation of fibroblast secretions
In dermis, fibroblasts express ERα and β at the mRNA and protein
level.[61,62] Oestrogen at 2-­5 10−9 (basal concentration) stimulated
collagen synthesis in culture fibroblasts from postmenopausal
women but had no effect on MMP-­2 and MMP-­9.[63] Oestrogen at
10 -7 mol/L (pregnancy concentration) did not induce the prolifera-
tion of fibroblasts from women aged between 28 and 41 years or
the production of collagen, nor activate cyclin D1 and D3. However,
it induced morphological changes via reorganization of the actin
cytoskeleton and focal adhesion.[64] Contradictory results were
observed in another study conducted on dermal fibroblasts of un-
determined age, since oestrogen (10 -8-­10 -7 mol/L), progesterone
(10 -7 mol/L) and a combination of oestrogen (10 -8) and progesterone
(10 -7 mol/L) decreased the proliferation of fibroblasts and MMP 1
and increased collagen I synthesis.[65]
We did not find any evidence that the effects of oestrogen and
progesterone on fibroblasts modulate pigmentation, but they do
modulate collagen synthesis. Thus, in melasma, oestrogen modula-
tion of fibroblasts does not seem to be involved in pigmentation.
However, we hypothesize that high doses of oestrogen in some
patients decrease the synthesis of collagen and thus facilitate the
protrusion of melanocytes and the dermal deposition of melanin.[8]
In melasma, fibroblast-­
like cells, located around small blood
vessels, expressed highly ERβ (Table 1).[32] These cells are probably
telocytes (interstitial Cajal-­like cell (ICLC), CD34+stromal cells or
CD34+ fibrocytes). Telocytes are localized in the papillary dermis
just beneath the basement membrane,[66] around blood vessels and
surrounding sebaceous glands, expressed oestrogen and progester-
one receptors in vitro and seemed to play a role in angiogenesis[66,67]
and tissue homeostasis.[67] Indeed, they secrete extracellular vesi-
cles[36,68] which allow communication with neighbouring cells such
as fibroblasts, immune cells nerve endings and endothelial cells.[69,70]
We may hypothesize that these cells participate in the increase in
the number of vessels in melasma.
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714       CARIO

TA B L E   5   Effects of estradiol and progesterone on melanocytes and melanogenesis

Tyrosine
Number of hydroxylase
Hormone cultures Concentration Proliferation activity Tyrosinase activity Melanin Ref.
−12 
17-­Estradiol 4 10 mol/L NE nd NE nd [38]
−11 
4 10 mol/L NE Slight increase
4 10 −10 and Decreasea Increase a
10 −9 mol/L
23 10 −12 to 13: Dose-­ nd 13: Dose-­ Increase (production [37]
10 −9 mol/L dependent dependent and extrusion)
decrease increase
8:NE 8:NE
nd 10 −12 and Dose-­dependent nd Dose-­dependent Dose-­dependent [35]
10 −9 mol/L increase at 10 d decrease at 4 d decrease at 10 d
3 0.5-­2.5. nd nd nd Dose-­dependent [20]
10−8 mol/L increase
Ethinyl 3 0.5-­2.5. nd nd nd Dose-­dependent [20]
estradiol 10 −8 mol/L increase
Estrone 4 10 −9 mol/L NE nd Increase NE [37]
−9 
Estriol 4 10 mol/L NE nd Increase NE [37]
Oestrogen 8 10 −9 mol/L 3: Increase, 4 (a) Increase nd nd [36]
1 (c): NE
1 (b): decrease, 4: NE
3:NE
8 10 −7 mol/L 3: Increase, 4 (a) Increase
1(c): decrease
4: NE 4: NE
Progesterone 8 10 −9 mol/L 3: Increase, 4 (a) increase nd nd [36]
1 (c): NE
1 (b): decrease, 3: 4: NE
NE
8 10 −7 mol/L 3: Increase, 4 (a) Increase
1 (c): NE
1: increase, 4:NE
1: decrease,
2: NE
3 10 −8-­ nd nd nd Dose-­dependent [20]
5.10 −7 mol/L decrease

nd, not determined; NE, no effect.

Description of results given only when results differed between strains.
(a): Represents a group of four melanocyte strains which behave similarly. (b), (c): Represent two special strains of melanocytes.
a
Similar value for 10 -10 and 10 -9 mol/L.

stimulates MITF phosphorylation, tyrosinase and TRP-­2 .[19] UVB-­

3.2 | Indirect effects of sex steroid hormones
on pigmentation via regulation of endothelial
cell secretions
Endothelial cells express ERα and GPER and should express
ERβ.[71] Regazzetti et al observed an increase in pigmenta-
[19]
tion near to vessels in various pigmentary disorders
number of vessels is increased in melasma, [6] so vessels may
be involved in hyperpigmentation in melasma. Human dermal
microvascular endothelial cell (HDMEC) secretome promotes
melanogenesis via ET-­1 /EDNRB (endothelin receptor B), which
or UVA-­irradiated HDMEC secretome stimulates melanogenesis
more strongly via an increase in SCF but not in ET-­1 , FGF-­2 and
POMC, which are well-­k nown melanogenic paracrine factors.[72]
Since UV did not induce ET-­1 in HDMEC and since ET-­1 is in-
creased in melasma lesional skin, [1] we believe that estradiol is
and the involved in the increase in ET-­1 , although no study has yet been
done on HDMEC and sex hormones. However, on other endothe-
lial cells, estradiol (2.10 −10 -­3 .7 10 −7M) did not induce any effect
on endothelin-­1 (ET-­1) secretion, nor did it inhibit its secretion
but led to an increase in eNOS.[73,74] On the other hand, E2
CARIO
F I G U R E   1   Schematic representation of effect of oestrogen
and progesterone at concentration found during pregnancy
on pigmentation in vitro and hypothesis of development of
hyperpigmentation in melasma. Effect of sex steroid hormones
on melanocytes and keratinocytes cultured in monolayer and in
coculture (A) or in 3D (B). Oestrogen stimulated melanin production
directly via increase in tyrosinase and indirectly via secretion
of GM-­C SF by keratinocytes, whereas progesterone inhibited
melanin production. Oestrogen stimulated also melanin transfer.
Effects of sex steroid hormones on explants before (C) and after
irradiation (D). Oestrogen and progesterone induced no effect,
whereas oestrogen + progesterone+ UVB induced transfer of single
melanosome. Modelization of effect of oestrogen on initiation
of hyperpigmentation (E) and sustaining of hyperpigmentation
(F) in melasma. First exposure to high level of oestrogen and
UVB induced melanin production and transfer second oestrogen
stimulated angiogenesis and thus ET-1 concentration is increased.
Green arrow activation, red arrow inhibition
induced via ERα an increase in VEGF, proliferation and migration
of endothelial cells, whereas via GPER triggered an increase in
angiogenesis.[71] Thus, E2 may be responsible for the increased
number, density and size of vessels in melasma and the effect on
pigmentation may just be due to the high number of vessels se-
creting endothelin 1 rather than to an increase in the level of ET-­1
secreted by each type of endothelial cell. In addition, endothelial
cells may also secrete NO due to an increase in eNOS and thus
75
stimulate melanogenesis.
|
      715
3.3 | Is regulation of mast cell metabolism by sex
steroid hormones involved in the modulation of
epidermal pigmentation?
Dermal mast cells secrete histamine, which can stimulate mela-
nin synthesis, proliferation and migration of melanocytes,[6] and
tryptase, which may be involved in dermal and basal membrane dam-
age.[6] Progesterone (10 -7M, concentration during pregnancy) inhib-
ited substance P-­induced release of histamine by rat mast cells.[76]
Thus, the absence of substance P in melasma may be related to pro-
gesterone.[77] E2-­induced release of LTC4 (leukotriene C4) is known
to be mitogenic for melanocytes in culture at concentrations ranging
between 10 -10 and 10 -9 mol/L; that is, basal concentrations below
the high concentrations observed during pregnancy.[78,79] Since the
main factors secreted by mast cells, histamine and tryptase, are not
released by hormone treatment but are efficiently released by UV
radiation, the involvement of mast cells in melasma may be due to
solar radiation rather than to sex steroid hormones.
3.4 | Effects of sex steroid hormones on
regulation of pigmentation in 3D model and explants
In 3D reconstructs which allow crosstalk between melanocytes
and keratinocytes, E2 at 2.5.10 -8M increased melanin levels but not
the number of melanocytes, whereas progesterone at 5.10−7 mol/L
decreased pigmentation but not the number of melanocytes[20]
(Figure 1B). 2.5.10−8 mol/L E2 and 5.10−7 mol/L progesterone are
values that occur during pregnancy,[39] so these two hormones have
an opposite effect regarding pigmentation in reconstructed epider-
mis. Thus, we hypothesize that, in the absence of other stimulation
such as UV irradiation and predisposing factors due to opposing
effect of these two molecules, pigmentation is maintained during
pregnancy.
In skin explants, which is an even more complex model, 6-­day
exposure to 10−9 mol/L and 10−7 mol/L oestrogen increased pigmen-
tation in only 2/5 explants, whereas 10 -7M progesterone did not
modify pigmentation.[36] Oestrogen and progesterone (10−9 mol/L
and 10−7 mol/L) did not induce any significant change, especially in
the formation of rete ridges on skin explants.[36] Moreover, treatment
of Caucasoid skin explants with 10 -8 mol/L oestrogen or 10 -8 mol/L
progesterone did not change the number of melanosomes or their
transfer, whereas 50 mJ.cm−² UVB induced a slight increase in
transfer. On the contrary, 10−8 mol/L oestrogen + 50 mJ.cm−² UVB
induced a dramatic increase in the number and transfer of single
melanosomes.[80] Treatment with 10 -8M progesterone + 50 mJ.cm-²
UVB was more efficient than UVB alone in increasing the transfer
of single melanosomes and seemed to be implicated in alteration
of the basal membrane,[80] which may facilitate the loss of melano-
cytes and melanin in the dermis (free melanin or melanophages).[6]
No additive effect of 10 -8 mol/L progesterone on the number and
transfer of single melanosomes was observed in explants treated
with both hormones and UVB as compared to oestrogen + UVB[80]
(Figure 1C,D). Thus, the change in the number and transfer of single
 |
716       CARIO
melanosomes may be attributed to the synergistic effect of oes-
trogen and UVB. Even though only short-­term effects such as gene
regulation and melanosome transfer may be observed, these models
are useful since they show that primary changes such as morpho-
logical changes in melanosomes occur after acute exposure to oes-
trogen and UV. Thus, long-­term exposure may maintain and amplify
changes. Experiments on explants, which are more complex than 3D
models, showed that sex steroid hormones alone are not able to in-
duce melasma.
4 |  CO N C LU S I O N
While the cells in the monolayer have receptors and can respond
to sex steroid hormones, the effects are more difficult to decipher
in three-­dimensional models. While oestrogen increases melano-
genesis in the melanocyte monolayer and induces the production
of melanogenic factors by keratinocytes, the same dose failed to
increase it in explants. Therefore, it is likely that explants are able
to maintain skin homeostasis during short-­term exposure due to
the presence of the cutaneous cells (melanocytes, keratinocytes,
fibroblasts, endothelial cells). On the other hand, this is not pos-
sible when a second trigger like UV irradiation is applied. Since UV
irradiation alone induced only a slight increase in transfer in this
model as compared to oestrogen+ UVB, the explant model con-
firms that melasma can occur only on photo-­exposed skin in pa-
tients subjected to high levels of oestrogen. Moreover, the number
of mast cells and blood vessels in explants did not increase, so we
hypothesize that mast cells and endothelial cells do not contribute
to triggering lesions in melasma. In addition, these effects were
observed with UVB, suggesting that UVA is implicated by amplify-
ing and/or sustaining the modifications observed in melasma.
In vitro studies on monolayers have shown that the effect of
sex hormones on cells, especially melanocytes and fibroblasts,
varies according to the concentrations used and to the donors. In
vitro, it seems that high-­p hototype melanocytes are more sensitive
to progesterone, whereas low-­p hototype ones are more sensitive
to oestrogen.[20] This might explain why melasma occurs earlier in
low phototypes than in higher ones. In 3D models (reconstructed
epidermis and explants), oestrogen seemed to be more involved
in regulating pigmentation than progesterone, but the effect was
again donor-­d ependent, thereby confirming that genetic predis-
position is an important factor in the effect of hormones. Both UV
irradiation alone and sex hormones alone are able to induce pig-
mentation. However, one may hypothesize that UV irradiation and
sex hormones are necessary to maintain hyperpigmentation, es-
pecially oestrogen in predisposed patients. In women, oestrogen
and progesterone levels vary during the menstrual cycle and preg-
nancy and their balance is modified.[39] Indeed, one may postulate
that a local increase in estradiol in the papillary dermis induces
the proliferation of endothelial cells. Such a local increase in blood
vessels might modify local levels of oestrogen, progesterone, ET-­1
and SCF. In addition, an increase in the number of endothelial cells
might also induce local hyperoxia that would stimulate melano-
genesis, an effect already observed in melanocytes both in mono-
layers and in 3D cultures (Cario et al, unpublished data), and would
sustain hyperpigmentation. If irradiation is chronic, this local dis-
turbance might also stimulate keratinocytes, melanocytes, mast
cells and fibroblasts, which could potentiate and maintain hyper-
pigmentation and modify the basal membrane.
Altogether, the data suggest that sex steroid hormones in me-
lasma, especially oestrogen, are not able alone to induce hyperpig-
mentation but act in synergy with UVB. However, oestrogen may
sustain hyperpigmentation by increasing the number of blood ves-
sels and thus stimulating the secretion of endothelin-­1. Since the
sensitivity of cells to sex steroid hormones varies greatly, we hy-
pothesize that sex steroid hormones are responsible for the differ-
ence in susceptibility to melasma (Figure 1E,F).
C O N FL I C T O F I N T E R E S T
The author declares no conflict of interest.
AU T H O R C O N T R I B U T I O N
Muriel Cario analysed the data and wrote the paper.
ORCID
Muriel Cario  https://orcid.org/0000-0003-0462-5684
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