ARTICLE
Auteur(s) : Elodie Krahn-Bertil1,2,3,5, Marie-Alexandrine Bolzinger3, Valérie Andre2, Isabelle
Orly2, Jean Kanitakis4, Patricia
Rousselle5, Odile
Damour1,5
1Laboratoire des substituts cutanés, CNRS UPR-412,
Hôpital Edouard Herriot, 5 place d’Arsonval, 69437 Lyon, France
2BASF Beauty Care Solutions, Lyon, France
3Université de Lyon, Lyon, F-69008, France; Université
Lyon 1, Institut des sciences pharmaceutiques et biologiques,
Laboratoire de dermopharmacie et cosmétologie, Lyon, F-69008,
France; CNRS, UMR 5007, Laboratoire d’automatique et de génie des
procédés (LAGEP), Villeurbanne, F-69622, France
4Département de dermatologie, Hôpital Edouard Herriot,
Lyon, France
5IFR128 BioSciences Lyon-Gerland; Institut de biologie
et chimie des protéines; UMR 5086; CNRS; Univ. Lyon1; Lyon,
France
accepté le 29 Mars 2008
Skin is a non-classical target for estrogens. Clinically,
estrogen deficiency causes the skin of aging women to become
thinner and dryer, with the appearance of wrinkles [1]. As in
reproductive organs, estrogenic effects are thought to be mediated
by Estrogen Receptors (ERs) in the skin [2]. However, whereas ERβ
cutaneous expression has been demonstrated, the presence of ERα in
human skin remains controversial [2-4].
Another family of receptors, the Estrogen Related Receptors
(ERRs), shows a strong sequence homology with ERs. Its first two
members were identified by screening cDNA banks of human heart and
kidney cells, using the ERα DNA Binding Domain, hence their name
ERRα and β [5], but they do not bind estradiol (E2) [6]. ERRs are
always described as orphan receptors as no endogenous ligand has so
far been identified. The third member of the ERR family, ERRγ has
been identified more recently [7] and is expressed in estrogen
classical target tissues like breast or endometrium [8, 9].
The ERRs distinguish themselves from the classical ERs through
their activation mechanism, which is ligand-independent [10].
Indeed, the ERRs can directly bind to target gene promotors as
monomer or dimer in the absence of any ligand. However, these two
types of receptors, ERs and ERRs, do have exogenous ligands in
common. Certain synthetic estrogens, such as 4-hydroxy-tamoxifen
and diethylstilbestrol [11, 12], are common antagonists that
disrupt ERRγ-coactivator interaction, thus inhibiting its
constitutive transcriptional activity [13]. In addition, among the
phytoestrogens, the flavonoids daidzein and genistein act as
agonists [13, 14]. In the presence of these agonist ligands, the
basal target gene transcription is significantly increased.
Growing evidence suggests that ERRs can cross-talk with ERs in
different cell types via competition for DNA sites and
coactivators. In the presence of common coactivators, such as
proteins from the SRC family and p160, ERs and ERRs recognize the
same binding sites on the target gene promoters: Estrogen Response
Elements (ERE) [15], AP-1 [14] and Sp1 [16]. More specifically,
ERRs can bind to the Steroidogenic Factor-1 Response Element (SFRE)
(thus becoming ERE-Related Response Elements, ERRE). Among the ERs,
only ERα binds to this latter site [17, 18]. Thus osteopontin, a
glycoprotein involved in bone remodeling [19], possesses a promoter
that can be controlled either by ERα, ERRα or ERRγ [20, 21].
All these results suggest that the transmission of estrogen
effects, other than those of estradiol, could involve not only the
ERs but also ERRγ.
We have recently demonstrated the expression of ERRα in normal
human skin (NHS) [22] but until now, ERRγ expression has never been
studied in human skin.
The aim of our study was to search for ERRγ expression in the
skin of young adults (31 to 48 years old). ERRγ expression was
first looked for with RT-PCR and Western Blot. Its localization was
then studied immunohistochemically in NHS and skin equivalent (SE).
The upper abdominal area was chosen because it is rarely exposed to
light and relatively distant from skin regions with high hormonal
trophicity (urogenital and breast areas).
Materials and methods
Tissue procurement
Normal human skin samples from the abdomen were obtained following
abdominoplasties with the informed consent of the 11 patients, 8
premenopausal women (31 to 42 years old) and 3 men (31 to 48 years
old) at the clinic Ste-Marie Thérèse (Bron, France). We
deliberately did not select patients with a particular hormonal
status (contraception) in order to get a global view concerning
ERRγ expression in skin.
Cell isolation
For cell isolation, the dermal and epidermal compartments were
separated by enzymatic digestion with thermolysin (overnight at
4 °C). Epidermal cells were then isolated using a trypsin
treatment (14 min at 37 °C) whereas collagenase
(overnight at 37 °C) was used for the isolation of fibroblasts
from the dermis.
Cell culture
In order to prepare keratinocyte monolayers, cells extracted from
the epidermis were amplified until second passage and plated in
24-well plates in defined keratinocyte basal medium-2 (KBM-2;
Cambrex Bio-Sciences, Emerainville, Belgium) at 90,000 per well for
molecular biology. For Western Blotting experiments, keratinocytes
were plated in the same medium in 6-well plates at 2 ×
104 cells/well, supplemented over 3 days and stopped at
subconfluence. The keratinocytes used for skin equivalent
preparation were grown in Green medium [23], a 3:1 mixture of DMEM
and Ham’s F12 (Invitrogen), respectively, supplemented with 10% FCS
(HyClone), 10 ng/mL epidermal growth factor (EGF) (Austral
Biologic, San Ramon, California, USA), 0.12 IU/mL insulin (Lilly,
Saint-Cloud, France), 0.4 μg/mL hydrocortisone (UpJohn, St
Quentin en Yvelines, France), 5 μg/mL triiodo-L-thyronine
(Sigma, St Quentin Fallavier, France), 24.3 μg/mL adenine
(Sigma) and antibiotics.
For all experiments, fibroblast monolayers were obtained after
amplification of dermal cells until passage 7 in Dulbecco’s
Modified Eagle’s Medium (DMEM with Glutamax-1, Invitrogen),
supplemented with 10% calf serum (HyClone, Logan, USA),
20 μg/mL gentamicin (Panpharma, Fougères, France), 100 IU/mL
penicillin (Sarbach, Suresnes, France) and 1 μg/mL
amphotericin B (Bristol Myers Squibb, Puteaux, France). For
molecular biology and Western Blotting experiments, fibroblasts
were then plated in defined fibroblast growth medium (FGM;
Promocell GmbH, Heidelberg, Germany) in respectively 24-well plates
(50,000 cells per well) and in Petri plate (25,000
cells/cm2).
Molecular biology
RNA extraction
Keratinocytes and dermal fibroblasts were isolated from 8
pre-menopausal female patient skins. All freshly extracted and
cultured cells were stored at –80 °C in TRI®
Reagent (Sigma-Aldrich; Saint Louis USA). Total RNA from monolayers
was extracted using the SV 96 Total RNA Isolation
System® (Promega, Madison, WI, USA) and eluted in
100 μL of nuclease-free water. Total RNA from freshly
extracted epidermal and dermal cells was extracted using
TRI® Reagent (Sigma-Aldrich) in accordance with the
method recommended by the supplier. Briefly, total RNA was
extracted with chloroform, precipitated using propanol and rinsed
with ethanol. Finally, samples were treated with DNase (Ambion,
Austin, TX USA) before use. Total RNA integrity and purity were
monitored on a 2% precast agarose gel (E-gels, Invitrogen,
Cergy-Pontoise, France) and quantities were evaluated
photometrically using Spectramax 190 (Molecular Devices, Sunnyvale,
USA) (data not shown).
Qualitative RT-PCR
RT-PCR was carried out using iScriptTM One-Step RT-PCR
Kits (Biorad, Hercules, USA) in a Tetrad (Biorad) using 50 ng
of total RNA. Primer sequences, amplified fragment sizes and
annealing temperatures are shown in table
1. RT-PCR conditions for all primers were: synthesis of
first strand cDNA at 50 °C for 10 min, followed by
denaturing at 94 °C for 5 min, 50 cycles of amplification
(94 °C for 15 s, adapted annealing temperature for
30 s, 72 °C for 30 s) and a final extension step at
72 °C for 10 min. Reaction products were visualized by
electrophoresis. Each band was quantified using gel images taken
with a digital camera and image analysis software (Phoeretix 1D,
Alphelys, Plaisin, France). Finally the amplification product for
ERRγ was sequenced for control (Millegen, Labège, France).
Table 1 RT-PCR Conditions and primer sequences
|
Primers
|
Nucleotide sequences
|
Size of the amplicon
|
Annealing temperature
|
Access number
|
|
ERR γ 5’
|
5’-ACCATGAATGGCCATCAG AA-3’
|
469 bp
|
60 °C
|
AF094518
|
|
ERR γ 3’
|
5’-ACCAGCTGAGGGTTCAGGTAT-3’
|
|
β Actin 5’
|
5′-GTGGGGCGCCCCAGGCACCA-3′
|
540 bp
|
60 °C
|
NM_001101
|
|
β Actin 3’
|
5′-CTCCTTAATGTCACGCACGATTTC-3′
|
Western blotting
Keratinocytes and fibroblasts were isolated respectively from the
skin of 2 and 4 pre-menopausal women. Cultured normal human
keratinocytes and fibroblasts were extracted with PBS, 1% Triton
X100, pH 7.4 containing 50 μM N-ethylmaleimide and 50 μM
phenylmethanesulfonyl fluoride. After centrifugation of the
extracts at 4 °C, the protein content in the supernatants was
evaluated with Advanced Protein Assay Reagent (Cytoskeleton, Tebu,
Le Perray en Yvelines, France). Proteins (150 μg/well) were
separated on a 12% SDS-PAGE gel under reducing conditions,
transferred to nitrocellulose membrane, probed with the mouse
antibody against ERRγ (10 μg/mL; R&D system, Mineapolis,
USA) and then with goat anti-mouse immunoglobulin G (IgG) linked to
peroxidase (10 μg/mL; Biorad) followed by immunodetection with
western lightning chemiluminescence reagent plus (Perkin Elmer Life
Science, Boston, MA).
Immunohistochemistry
Tissues
Samples from a total of 11 patients, including 8 females and 3
males, were fixed in Bouin solution overnight (Gifrer, Décines,
France), embebbed in paraffin and sectioned at 5 μm thickness.
Antibodies and protocol
Immunohistological studies were carried out with polyclonal
antibodies. The antibodies used, concentrations and other
characteristics are summarized in table
2. Bouin-fixed sections were bleached using a solution of
glycin (0.1 M) and NH4Cl (50 mM). After washing in Tris
buffered saline (TBS; Sigma-Aldrich), slides were incubated with
the appropriate enzyme treatment for antigen retrieval at 42°C
(10 min) (table 2). The slides were
immersed in a solution of TBS-BSA (3%) containing 6% hydrogen
peroxide (Sigma-Aldrich) for 10 min at room temperature.
Following a wash in PBS-Tween 20 (PBS, Biomerieux France; Tween
0.2%, Sigma-Aldrich), sections were blocked with TBS-BSA 3%
(10 min). Samples were rinsed in PBS-Tween 20, further blocked
with swine serum for 1 hour and incubated for 2 hours with
antibodies (table 2) in a humidified
chamber. After a further wash in PBS, samples were incubated with
the secondary antibodies EnVision+ Dual Link System Peroxidase
(DakoCytomation) for 45 min. Samples were washed in PBS and
antigen-antibodies complexes were visualized using diaminobenzidine
solution for 2 min (liquid DAB+ Substrate chromogen system;
DakoCytomation). Tissue sections were subsequently counterstained
using Harris hematoxylin (25%; Sigma-Aldrich) for 30 sec. Sections
were finally mounted using Faramount aqueous mounting medium
(DakoCytomation). After each of the last three steps, the sections
were washed in tap water for 10 min. For each specimen
treated, a negative control section was prepared using rabbit total
IgG instead of the primary antibody (Chemicon, Temecula, CA,
USA).
Table 2 Summary of primary antibodies employed in this
study
|
Primary antigen
|
Host
|
Dilution
|
References
|
Source
|
Antigen retrieval treatment
|
|
ERRγ
|
Mouse
|
10 μg/mL
|
PP-H6812-00
|
R & D systems (Mineapolis USA)
|
Pepsin ready-to use (Zymed, Montrouge, France)
|
|
Rabbit
|
5 μg/mL
|
ab12988
|
|
Ficin ready-to-use (Zymed)
|
|
Rabbit
|
10 μg/mL
|
LS-A5960
|
Lifespan Biosciences (Seattle, USA)
|
Ficin ready-to-use (Zymed)
|
Preparation of the skin equivalent
Primary keratinocyte and fibroblast cultures were isolated from
human foreskin. The skin equivalent (SE) was prepared as described
[24]. Briefly, fibroblasts were seeded at a density of 200,000
cells/cm2 onto dermal substrate made of
chitosan-cross-linked collagen-glycosaminoglycans matrix as
previously described by Collombel [25]. Dermal equivalents were
cultured for 14 days. Keratinocytes were seeded on a dermal
equivalent at day 14, at a density of 200,000 cells/cm2
[25]. After 7 days of submerged culture in the keratinocyte medium,
the SE was elevated to the air-liquid (A/L) interface and cultured
in a simplified keratinocyte medium containing DMEM supplemented
with 10% calf serum, 10 ng/mL EGF, 0.12 IU/mL insulin,
0.4 μg/mL hydrocortisone and antibiotics. The medium was
supplemented with 50 μg/mL L-ascorbic acid and changed every
day. The SE samples were harvested on day 38 of skin equivalent for
immunohistology.
Results
ERRγ mRNA expression in the epidermis and in the dermis of
premenopausal women (figure 1)
ERRγ mRNA was detected in freshly extracted epidermal and dermal
cells from all 8 donors. Moreover, ERRγ expression was also
detected in all monolayer keratinocyte and fibroblast cultures. In
parallel, amplification of a house-keeping gene, β-actin, was
performed on each RNA sample (figure 1).
ERRγ protein expression in normal human premenopausal women
keratinocytes and fibroblasts cultivated in defined media (figure 2A and B)
We found that ERRγ protein was present in female normal human
fibroblasts (figure
2A) and keratinocytes (figure 2B) grown in
defined medium. As described for other tissues, the protein
appeared in the form of a 51 kDa band by Western Blotting [18].
ERRγ immuno-localization in human skin
In normal human skin (figure 3)
In the epidermis (figures 3A and
B)
Whatever the antibody (2 polyclonal and 1 monoclonal), the same
expression profile was observed in all the skin samples tested (8
women and 3 men). Figure
3 shows ERRγ expression in two representative samples (one
male (3A) and one female (3B). No sex differences were observed
under our experimental conditions. ERRγ showed a slight cytoplasmic
expression in all epidermal layers with the stratum granulosum
being intensively stained. A nuclear staining was also present in
some keratinocytes. However, the stratum corneum always appeared
unlabelled.
ERRγ was expressed by some fibroblasts in human skin. In the
eccrine sweat glands only a subpopulation of secretory cells was
immunoreactive for ERRγ whereas the excretory part was never
stained (figure
3C). In sebaceous glands the ERRγ cytoplasmic staining was
mostly observed in the basal cells. However nuclear labeling was
also detected in some sebocytes (figure 3D). Hair follicles
showed a cytoplasmic staining for ERRγ (figure 3E).
In skin equivalents (SEs) (figure 3F)
Figure 3F shows
the expression profile of ERRγ in the epidermis of a skin
equivalent that was identical to the profile observed in normal
human skin samples. ERRγ was also expressed by fibroblasts of the
reconstructed dermis at day 45 confirming the staining detected in
normal human skin using the same antibody.
Discussion
This is the first demonstration of ERRγ expression in normal human
skin. Our results clearly show the presence of ERRγ mRNA and
protein in abdominal skin of 31 to 48 years old adults.
We first demonstrated ERRγ transcriptional expression by RT-PCR
on epidermal and dermal cells directly extracted from eight donors
in order to avoid the effect of growth factors and hormones from
culture medium on the receptor physiological state. In order to
eliminate other epidermal cell types such as Langerhans cells or
melanocytes, we also investigated ERRγ expression by RT-PCR on
defined medium-cultivated keratinocytes and fibroblasts in which
ERRγ was detected. The presence of ERRγ protein was confirmed in
both cultured human keratinocytes and fibroblasts by WB and also by
immunohistochemistry in NHS and SE. Immunohistological analysis
showed a reproducible labelling whatever the antibody (three
different antibodies for ERRγ), donor (11) or donor sex.
In the epidermis, ERRγ showed a nuclear expression in some
keratinocytes and a cytoplasmic labelling in all layers, with the
same intensity in the basal and the suprabasal layers. However the
stratum granulosum appeared intensively labelled. This staining is
different from the expression profile of ERRα observed on the same
NHS and SE samples [26]. ERRα showed a strong cytoplasmic labelling
with an intensity gradient, increasing from the basal layer to the
upper stratum spinosum layers with the strata granulosum and
corneum remaining unstained.
These results differ from those found in other organs such as
the prostate or the brain [7, 27, 28] where ERRα and γ colocalize.
They suggest that these two ERR isoforms could play different roles
in the epidermis.
In the dermis, fibroblasts expressed both ERRγ mRNA and protein
that could participate to estrogen-regulated collagen synthesis as
suggested in clinical studies [1]. However, this remains to be
demonstrated.
In eccrine sweet glands, implicated in thermoregulation, the
ERRγ protein was detected. We were not surprised to find ERRγ
expressed in these glands as one of their target genes,
osteopontin, has been previously reported to be present in sweet
glands [29].
ERRγ was also detected in basal cells of sebaceous glands and in
some sebocytes. This result was expected because of the potential
role of ERRγ in fatty acid metabolism through the interaction with
the coactivator PGC-1α [30]. Recent published data provide evidence
for the implication of ERRγ in the control of heart energy
metabolism [31]. Also ERRγ seems to be associated with a favourable
clinical course in breast and ovarian cancers [32, 33]. Our present
results are insufficient to postulate about the role of ERRγ in
skin. However, ERRγ expression in both the dermis and the epidermis
opens new research ways for the understanding of cutaneous
estrogenic effects.
Conclusion
This study shows that the orphan receptor ERRγ is expressed in
human skin where it could play regulatory roles by sharing similar
ER-mediated pathways or acting independently.
Acknowledgments
Eric Perrier, Pr. Jean-Yves Soazec, Dr Sabine Pain, Dr Zilliox, Dr
Durand Clinique Ste Marie-Thérèse Bron, Dr Van der Stegen Clinique
Pasteur St-Priest, Jan Ewert, Michael Krahn. Grant ARC (3848) was
given to P. Rousselle. Conflict of interest: none.
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