0021.972W96/$03.00/0
Journal
of Clinical
Endocrinology
and Metabolism
Copyright
0 1996 by The Endocrine
Society
Human
skin contains luteinizing
Vol. 61, No. 7
Printed
rn U.S.A.
hormonekhorionic
gonadotropin
J. E. PABON, J. S. BIRD, X. LI, Z-H. HUANG, Z. M. LEI, J. S. SANFILIPPO,
receptors
M. A. YUSSMAN
AND Ch.V. RAO*
Laboratory of Molecular ReproductiveBiology and Medicine. Departmentof Obstetricsand Gynecology,
Universiry of Louisville Medical Center, Louisville, KY 40292
Abstract
Normal human skin contains a major 4.5 kb and several minor mRNA transcripts and a 66 kDa protein of luteinizing
hormone (LH)/chorionic gonadotropin (hCG) receptors which are capable of binding exogenous “‘I-hCG. The distribution
of receptor transcripts and receptor protein are the highest in epidermis followed by hair follicles, sebaceous and sweat glands.
LH/hCG rece@ors are co-local&d with androgen receptors in all the skin appendages. These data are the first demonstration
of skin containing LH5CG receptors and would suggest that LH and hCG may regulate skin functions.
Introduction
Hirsutism, excessive skin oiliness and acne are common in
chronic anovulatory women (1). Hirsutism, dry skin and
skin atrophy are common in postmenopausal women (2).
The skin changes in chronic anovulatory women have been
attributed to increased circulatory or local androgen levels
(36). The local androgens are derived from the conversion
of
weak
androgen
such as testosterone
or
dehydroepiandrosterone (DHEA) or its sulfated form into a
more active androgen, dihydrotestosterone (DHT). These
conversions are catalyzed by type I 5-a reductase and 3-B
hydroxysteroid dehydrogenase present in various skin
appendages (7,8). The skin changes in postmenopausal
women have been thought to be due to decreased circulatory
estrogen levels (2). Skin is also an estrogen target organ and
contains functional estradiol receptors (9). The LH levels
are elevated in chronic anovulatory as well as in
postmenopausal women. However, the relationship between
elevated LH levels and skin changes seen in these women
has not previously been investigated because the presence of
LH/hCG receptors in skin was unknown. Therefore, we
investigated whether normal human skin contains LH/hCG
receptors.
Materials
and Methods
Sixteen skin samples were obtained from pre-menopausal
women. Thirteen of them were from Pfmensteil skin
incisions as a portion of the discarded specimens from scar
revision. The remaining three were paraffin embedded
blocks from the pathology department. One of the blocks
was from lower abdominal skin removed at abdominoplasty
while the other two were from skin removed at breast
reduction. The use of these tissues was approved by our
University Human Studies Committee.
*Correspondence and reprint requests
Northern blotting was performed with 25 c(g total RNA
isolated from skin samples from five patients (10). The
membranes were probed with 1 X lo6 cpm/ml of 210 base
“P-labeled antisense riboprobe transcribed from human
LH/hCG receptor cDNA using an in vitro transcription kit
from Promega Corp. (Madison, WI). The 210 base probe
was prepared from 200 to 1029 bp receptor cDNA in
pBSK-SK’ plasmid (courtesy of Dr. A. Hsueh, Stanford
Univ.) by digesting with Hint II and religating the 3.1 kb
fragment containing vector and receptor cDNA from 200
to 410 bp.
A nonradioactive method of in situ hybridization was
performed on skin samples from three patients with 200
rig/ml of 210 base fluorescein II-UTP labeled antisense and
sense LHlhCG receptor riboprobes and a kit purchased
from Amersham Searle Co. (Arlington Heights, IL). The
procedure was optimized from the kit manufacturer
mm&ations. The hybridization signals were detected
CT using an anti-fluorescein alkaline phosphatase conjugate and 4-nitroblue tetrazolium/5-bromo-4-&loro-3indolylphosphate.
Western immunoblotting was performed with 40 pg
protein of the supernates from the 10,000 X g centrifugation
of the skin homogenates of five patients (11). The rest of
the details, including the presence of three different
protease inhibitors in the homogenization buffer and the use
of 1: 1,000 dilution of polyclonal LHlhCG receptor antibody
(cowtesty of Dr. P. Roche, Mayo Clinic) and an enhanced
chemiluminescence detection system, are the same as
previously described (12). For a procedural control,
rece@or antiiy preabsorbed with excess receptor peptide
was used.
Immunocytochemistry was performed on skin samples
from ten patients by an avidin biotin immunoperoxidase
method (13). The receptor antibody was used at 1:350
dilution. The receptor antibody, preabsorbed with the
corresponding excess receptor peptide, served as a
procedural control. Double immunostaining for androgen
receptors (polyclonal antibody purchased from Affinity
2738
RAPID
A
B
C-
t66
kDa
2.54
kb
Fig. 1. Nortbem (A), western (B) and l&and (C) blotting
forLH/hCGmce@orsinhumanskin.
Lane2 isanantibody
pm&sorption control and lane 4 is competition of ir%hCG
binding with excess unlabeled hCG.
BioReagents) and LIUhCG receptors was performed on skin
samples from three patients using 1:25 and 1:3.50 dilution,
respectively (14). The sections were processed, including
exposure to microwave radiation, according to the
instructions from Affinity BioReagents prior to immunostaining for the androgen receptors by an an avidm biotin
immunoperoxidase method. Diaminobenzidine, used as the
substrate for the enzyme, gave a brown color. Then the
same sections were then immunostained for LH/hCG
receptors using an avidin-biotin complex conjugated to
alkaline phosphatase. The 5-bromo&.hloro-3-indolylphosphate/4nitroblue
tetraxolium, used as substrate for the
enzyme, gave a blue color.
Ligad blotting was performed with 60 pg protein from the
supem
pmpad for western blotting (15). The rest of the
details including the use of 2 X lo6 cpm/ml of “‘1-hCG
prepared by the lactoperoxidase technique (sp.act. 85.7
pCilpg) and 20 ~glml excess unlabeled
hCG in the
comp&tion expeheds are the same as previously described
(12).
2739
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ResUlt.5
Northern blotting demonstrated that skin contains a major
4.5 kb and several minor transcripts (Fig. 1, A). Western
blotting showed that skin also contains a 66 kDa receptor
protein (Fig. lB, lane 1) which was not detectable when the
receptor antibody was pm&sorbed with excess receptor
pqtide @me 2). Ligand blotting demonstrated that %hCG
can bind to the 66 kDa protein (Fig. lC, lane 3) and this
binding was inhibited by excess unlabeled hCG (lane 4).
Non radioactive in situ hybridization with antisense
riboprobe revealed that epidermis, hair follicles, sebaceous
ad sweat glads ad blood vessels contain LH/hCG receptor
transcripts (Fig. 2A & B). The hybridization signals are
absent in the sense controls (Fig. 2C & D).
Immunocytochemistry showed that epidermis, hair follicles,
sebaceous and sweat glands and blood vessels also contain
LH/hCG receptor protein (Fig. 2 E to G). This receptor
immunostainiug in all the skin appendages dramatically
decreased following the preabsorlxion of the receptor antibody
with excess receptor peptide (Fig. 2H to I). Both in situ
hybridization
and immunocytochemistry
showed that
epidemis cmtains the highest ieceptor levels followed by hair
follicles, sebaceous and sweat glands. Dermal collagen and
fibroblasts contain very few or no receptors.
Since skin is an androgen target organ and contains
androgen receptors (16), we used double immunostaining to
determine whether LH/hCG receptors are co-localized with
androgen receptors in various skin appendages. Figs. 2K to
2N indeed show that the blue color representing LH/hCG
receptors and the brown color representing androgen
receptors, are present in the same cells in epidermis, hair
follicles, sebaceous and sweat glands.
Discussion
The skin problems in chronic anovulatory and
postmenopausal women have not previously been considered
to be due to elevated LH levels. Rather, they were thought to
be due to excess andmgeus or diminished circulating estrogen
levels, mspectively (1,2). The therapies designed to decrease
androgen levels in chronic anovulatory women and estrogen
replacement in postmenopausal women seem to result in
symptomatic relief (17,18). Whether these therapies could
have altered LH levels and whether such alterations may have
played a role in skin changes have never previously been
investigated. ‘Ihis was primady due to the lack of knowledge
that nod
skin contains LHlhCG receptors. The possibility
that skin may contain these receptors came from the recent
data which demonstrated that LH/hCG receptors are widely
distributed and that these gonadotropins may have pervasive
actions in the body (12-14,19). There was a recent study,
however, indicating that normal skin does not contain,
whereas skin derived kaposi sarcomas contain hCG/LH
nzceptors (20). This finding was erroneously based on using
an antibody to hCG instead of hCG/LH receptors in
immunostaining experiments.
Due to the lack of availability, we were unable to investigate
the skin LH/hCG receptors in chronic anovulatory or
postmenopausal women.
Nevertheless, the presence of
LH/hCG receptors in skin may possibly explain skin changes
seen in these women.
LHhCG receptors are co-local&cl with androgen receptors
in all the skin appendages. This co-localization suggests a
funchoaal relationship between the two receptor systems. In
fact, our pmhminary data has shown that in vitro treatment of
skin tissue with exogenous hCG results in a change of
androgen receptor levels (Bird et al, unpublished data).
Recently, the presence of LHlhCG receptors and in vitro
In situ hybridization
(A-D)
and immunocytochemistry
(B to J) for LH/hCG receptors and double immumstaiuing
Q
iavarjousharrrnskin~.
CandDareseaeinsituhybridizatkmcoWols.
H, I and J am antiiy
pm&sorption
immunosw
controls. Epidermis is indicakxl by solid arrows, sebamms glands by
arrows, sweat glands by brge arrow heals, hair follicles by asterisks and blood vessela by small arrow heads. In K to
RAPID
and in vivo inhibitory
effects of hCG on growth,
development
and metastasis of Kaposi sarcomas has been described
(20).
In addition
to chronic
anovulatory
patients
and postmenopausal
women,
skin LH/hCG
receptors
may also be
functionally
relevant
in pregnancy
in which hCG is present;
the preovulatory
period during which the LH surge occurs
and; precocious
puberty
due to constitutive
activation
of
LH/hCG
receptors and/or due to hCG producing
brain tumors
(21,22).
Perhaps LH and hCG present in the circulation
may serve
as ligands for skin receptors.
Whether
skin is also capable of
producing
LH or hCG or related peptides which can bind to
the receptors and function in an autocrine
or paracrine
manner
is not known.
This possibility,
as well as a better
understanding
of the functional
relevance
of LHlhCG
receptors
in normal
skin and in the skin of chronic
anovulatory
and postmenopausal
women and the therapeutic
value of the gonadotropins
in skin diseases, require further
studies.
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