1 Molecular and Cellular Toxicology, Department of Toxicology, and 2 Department of Anatomy, Physiological Sciences and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27695; and 3 Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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Estradiol (E2) applied
topically twice weekly to mouse skin at doses as low as 1 nmol
inhibited hair growth by blocking the transition of the hair follicle
from the resting phase (telogen) to the growth phase (anagen). In
contrast, application of 10 nmol of other steroids produced limited
inhibition. Topical treatment with the estrogen receptor (ER)
antagonist ICI-182780 reversed the effects of E2, and when
applied alone, ICI-182780 caused a telogen-to-anagen transition. Both
E2 and ICI-182780 were highly effective at their site of
application but not at distant sites, indicating the direct rather than
secondary systemic nature of their effects. Western analysis detected a
65-kDa ER-
immunoreactive dermal protein, and Northern analysis
revealed the presence of a 6.7-kb ER-
mRNA. A ribonuclease
protection assay confirmed the presence of ER-
transcripts but
failed to detect ER-
transcripts. These findings implicate a
skin-specific ER-
pathway in the regulation of the hair follicle cycle.
skin; hair growth; hormones; androgens
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INTRODUCTION |
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THE OVARY IS A MAJOR SOURCE of 17-estradiol
(E2), and E2's function is classically viewed
with regard to its role in the process of reproduction. However,
E2 is also produced at many nonovarian sites (adipose
tissues, testes, various brain regions, and adrenals) (42) and can
exert profound effects in nonreproductive organs in both females and
males. For example, E2 was found to modify arterial smooth
muscle response (1, 22), bone formation and growth (39, 43), brain
function (3, 4), and levels of low- and high-density lipoproteins (24,
37). More recently, E2 has also been demonstrated to be
involved in the regulation of the hair follicle cycle in mice (29).
The hair follicle cycle is characterized by a period of follicle growth (anagen), followed by a period of regression and remodeling (catagen), and finally by a resting period (telogen) (5, 11). The hair follicle is a self-renewing system and is considered to be governed by a slowly cycling stem cell (7). The bulge activation hypothesis has been proposed, which states that the follicular stem cell resides in the bulge area of the permanent portion of the hair follicle and that this stem cell is stimulated during early anagen to divide and produce transient amplifying stem cells (7). A small group of highly specialized mesenchymal cells, known as the dermal papilla, resides at the base of the epithelial portion of the follicle, and these cells are thought to provide the signal that initiates anagen and instructs the bulge follicular stem cell to divide (19, 27, 30). The transient amplifying stem cells or matrix cells proliferate and then differentiate under the influence of unidentified morphogens into the inner root sheath cells and the cells that terminally differentiate into the mature hair fiber (13). At some point, the follicle enters catagen, and after degeneration of the lower follicle, the follicle enters telogen and remains in telogen until the dermal papilla signals the bulge stem cells to divide, and the hair follicle cycle begins again. The actual dermal papilla signals that initiate and terminate the cycles of the hair follicle remain poorly understood.
Using an immunohistochemical approach, our laboratory found that an
estrogen receptor (ER) immunoreactive protein is expressed in the
nuclei of the dermal papilla cells and that topical treatment of mouse
skin with E2 arrests follicles in telogen, whereas topical treatment with the ER antagonist ICI-182780 causes the telogen follicle
to enter anagen and initiate hair growth (29). These results indicate
that an ER pathway within the dermal papilla regulates the
telogen-anagen transition of the hair follicle. However, two ERs have
now been identified. Recently a novel ER cDNA was cloned from rat
prostate, and this ER cDNA was named ER- (23), to distinguish it
from the highly homologous ER cDNA previously cloned in mouse, rat,
human, and chicken (now referred to as ER-
). Rat ER-
encodes a
54-kDa protein that is highly homologous to the 65-kDa ER-
protein,
with ~90% amino acid identity in the DNA binding domain and ~55%
amino acid identity in the COOH-terminal ligand binding domain (23).
Both ER-
and ER-
are expressed in numerous tissues (8, 32). In
light of this, we wanted to determine whether ER-
and/or ER-
is
expressed in mouse skin.
Although it appears that the topical application of E2 and
ICI-182780 is directly producing their effects locally within the skin,
it is also possible that these compounds are absorbed into the general
circulation and are acting in a systemic manner to indirectly alter the
hair follicle cycle. In support of such a notion are studies
demonstrating that multiple subcutaneous injections of high levels of
estradiol benzoate retard hair growth in rats (15) and mice (17) and
that gonadectomy and adrenalectomy alter hair growth (17). In general,
it has been difficult to sort out the action of local vs. systemic
factors that regulate the hair follicle cycle. This is further
complicated by the fact that numerous steroid-synthesizing enzymes,
such as aromatase, 17-hydroxysteroid dehydrogenase,
3
-hydroxysteroid dehydrogenase, 3
-hydroxysteroid dehydrogenase,
and 5
-reductase, are expressed within the hair follicle (33). In
addition, the androgen receptor is also expressed in skin, and
androgens are considered to influence the hair follicle cycle (2). Thus
the objectives of this study were to examine 1) the specificity
and potency of E2 compared with other steroid hormones on
the follicle cycle, 2) the potency of ICI-182780 compared
with other ER antagonists, 3) whether ER-
and/or ER-
is
expressed in mouse skin, and 4) whether the effects of
topically applied E2 and ICI-182780 on the regulation of
the hair follicle cycle are due to their local effects within the skin
or to a secondary systemic effect resulting from their cutaneous absorption.
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MATERIALS AND METHODS |
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Chemicals. All the chemicals, unless otherwise mentioned, were purchased from Sigma Chemical (St. Louis, MO). ICI-182780 was a kind gift from Zeneca Pharmaceuticals (Wilmington, DE).
Animals.
Female CD-1 mice (5 wk old) with identical dates of birth were obtained
from Charles River Laboratories (Raleigh, NC) and kept in our animal
facility for 1 wk before use. They were fed rodent chow and water ad
libitum, were kept on corncob bedding, and were placed on a
12:12-h light-dark cycle.
Animal treatment. Dorsal or both dorsal and ventral (depending on the experiment) surfaces were clipped once in the beginning of the study and from then on treated with the respective chemicals in 200 µl acetone or acetone alone twice weekly until full hair regrowth occurred. Full hair regrowth is defined as the complete growth of hair over the entire clipped dorsal surface. For the ovariectomy study, mice were ovariectomized as previously described (26) by use of halothane anesthesia, and they were treated twice a week with either 1 nmol E2 or acetone from the day of ovariectomy.
Western analysis.
Skin from the dorsal surface was removed and homogenized on ice with a
polytron homogenizer in 500 µl of buffer [10 mM
Tris · HCl (pH 7.5)-1.5 mM EDTA-10 mM
-mercaptoethanol] containing (in mM) 0.25 antipain, 0.25 leupeptin, 0.25 aprotinin, and 1 phenylmethylsulfonyl fluoride. Tissue
homogenates were centrifuged at 4°C for 50 min in an
ultracentrifuge at 105,000 g, and the supernatants were used
for the electrophoresis. Protein concentrations of the samples were
determined by the method of Lowry et al. (25). The samples were
electrophoresed in an 8% polyacrylamide Novex (San Diego, CA)
mini-gel, transferred to a polyvinylidene fluoride membrane, blocked
with 5% milk, and then incubated overnight with the primary monoclonal
antibody to ER-
(either H222 or H226, dilutions 1:2,500; generous
gifts from Dr. Geoffrey Greene, University of Chicago). The membranes
were washed and incubated with a secondary antibody conjugated to
horseradish peroxidase (Amersham, Arlington Heights, IL; dilution
1:5,000) and finally, signals were detected by Amersham enhanced
chemiluminescence detection reagent. H222 binds to an ER-
epitope
within the carboxy F domain; H226 binds to an epitope within
NH2-terminal transactivation domain of ER-
.
Ribonuclease protection assay.
The ribonuclease protection assay (RPA) for ER- and ER-
mRNA was
carried out as previously described (8). Antisense riboprobes were
generated from linearized templates by use of the Maxiscript kit
(Ambion, Austin, TX) and the incorporation of
[32P]CTP (Amersham) according to the
manufacturer's protocol. The mouse ER-
antisense riboprobe protects
a specific fragment of 366 nucleotides (nt), whereas the mouse ER-
antisense riboprobe protects a specific fragment of 262 nt. To equate
loading among lanes, all reactions included an antisense riboprobe
specific for mouse cyclophilin, generated from the template
pTRI-Cyclophilin (Ambion) and producing a protected fragment of 103 nt.
For all RPA reactions, 5 x 104 counts/min (cpm) of each
probe, a 10-µg sample RNA, and yeast tRNA (for a final total RNA
equal to 25 µg) were mixed and ethanol precipitated at
70°C for 3 h to overnight. The resulting pellets were then
processed through the RPA with the Hybspeed kit (Ambion) according to
the manufacturer's protocol. Final analysis of protected fragments was
carried out by electrophoresis on a 1.5-mm thick 6% bis-acrylamide-8.3
M urea-1X TBE gel (National Diagnostics, Atlanta, GA), which was then
fixed, dried, and exposed to Hyperfilm (Amersham) X-ray film.
RIA.
Blood for the RIA was collected by cardiac puncture while the mice were
maintained under halothane anesthesia. After the blood was allowed to
clot at room temperature, the samples were centrifuged at 1,550 g for 15 min, and the serum was harvested for storage at
20°C until the assay was performed. A modification of the method
previously reported by Cox et al. (9) was used for the RIA. Briefly,
after extraction of 25-50 µl of serum once with 4 ml ethyl
acetate (carbonyl free grade, Burdick Jackson, Muskegon, MI), the
aqueous phase was frozen in a dry ice-methanol bath, and the ethyl
acetate was decanted into a test tube for evaporation at 37°C in a
dry heat block under a stream of nitrogen. The samples were then
reconstituted with 200 µl PBS-gel buffer (0.01 M PBS, 0.1% gelatin,
pH 7.0) and incubated overnight at 4°C with 200 µl antibody
(diluted 1:1,500,000 with PBS-gel). On the next day, 100 µl of tracer
diluted with PBS-gel {~8,000 cpm
estradiol-6-(O-carboxymethyl)oximino-(2-[125I]iodohistamine;
Amersham)} were added and incubated at 4°C for 6 h.
Dextran-coated charcoal (500 µl containing 0.05% dextran and 0.05%
charcoal in PBS-gel) was then added to each sample, vortexed, and
incubated for 45 min to adsorb the unbound hormone. Centrifugation at
1,550 g for 15 min was then used to precipitate the charcoal.
The supernatant was decanted, and radioactivity was measured using a
gamma counter (1272 Clinigamma, Wallac Instruments, Gaithersburg, MD).
The amount of estradiol in the samples was determined from a standard
curve that was generated using known amounts of estradiol ranging from
0.098 to 12.5 pg per tube. Extraction efficiency (generally >85%)
was monitored by determining the amount of labeled estradiol that was
recovered from representative serum samples after ethyl acetate
extraction. The values reported have been corrected for these recoveries.
Northern analysis.
RNA was extracted by the method of Chomczynski and Sacchi (6). Briefly,
fresh tissue was homogenized in 4 M guanidine thiocyanate-25 mM lithium
citrate-0.5% sarcosyl-0.12% -mercaptoethanol-0.1% antifoam A (2 ml/0.1 g tissue). The homogenate was filtered through spectramesh (a
macroporous polypropylene filter, 210 µm) into sterile polypropylene
tubes to get rid of the hair residuals, and the volume was adjusted
back to the original volume with the buffer. After this, 0.1 vol of 2 M
sodium acetate (pH 4.0), 1 vol of diethyl pyrocarbonate
water-saturated phenol, and 0.2 vol of chloroform:isoamyl alcohol
(49:1) were added and vortexed for 15 s. The mixture was allowed to
stand on ice for 15 min and then was centrifuged at 10,000 g
for 20 min at 4°C (Sorvall RC-5 high-speed centrifuge with rubber
adopter). The clear aqueous phase was then carefully taken, an equal
volume of cold isopropanol was added, and the mixture was stored at
20°C for 2 h. Samples were then spun at 11,000 g for 20 min at 4°C. The pellet was then resuspended in homogenization
buffer and stored overnight at
20°C with an equal volume of cold
isopropanol to precipitate RNA. Next day the tubes were centrifuged for
20 min at 4°C. The pellets were then washed with 75% and then 95%
cold ethanol and dried in a speed-vacuum centrifuge for 10 min. The RNA
concentration was determined by ultraviolet absorption at 260 nm.
Samples of total RNA and molecular weight markers (GIBCO-BRL,
Gaithersberg, MD) were denatured and subjected to size fractionation by
a 1% agarose-formaldehyde gel; the RNA was then transferred to a
positively charged nylon membrane (Zeta Probe GT; Bio-Rad, Hercules,
CA) by use of a Turbo Blotter System (Schleicher and Schuell, Keene,
NH). The filter was baked at 80°C for 1 h. After prehybridization
at 65°C for 10 min, the membrane was hybridized at 65°C
overnight by use of a 32P-labeled mouse ER-
cDNA probe
in a hybridization buffer containing 0.25 M sodium phosphate and 7%
SDS. The [32P]cDNA probe was prepared by using
a random priming kit from GIBCO-BRL with
deoxy-[32P]CTP (New England Nuclear, Boston,
MA). The specific activities of the cDNA probe were >109
cpm/µg. The filter was washed several times after hybridization with
0.1X standard sodium citrate (1X SSC: 0.15 mol/l sodium chloride and
0.015 mol/l sodium citrate)-0.1% SDS, and then autoradiographed on
Kodak XAR-5 film (Eastman Kodak, Rochester, NY) with an intensifying screen at ~80°C for 36 h.
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RESULTS |
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Effect of topical application of various steroids and ER antagonists
on hair growth.
The hair follicle cycle of mice is highly synchronized from birth to 12 wk of age, with fixed periods of anagen, telogen, and catagen. The
second telogen phase in CD-1 mice begins at ~6 wk of age and lasts
until ~9 wk of age, at which time the hair follicles enter the third
anagen synchronously. Topically applied E2 (10 nmol/200
µl acetone, twice a week) to mouse skin has been shown to block hair
growth by arresting hair follicles in telogen, whereas
17-E2 was without effect (29). To further determine the
specificity and potency of E2, we examined the effect of
equimolar doses of a variety of other steroid hormones on hair growth.
E2, dihydrotestosterone (DHT), androstenedione,
testosterone, or progesterone was applied topically twice weekly at a
dose of 10 nmol/200 µl acetone vehicle to the clipped dorsal skin of
CD-1 female mice beginning at 6 wk and continuing to the 17th wk of
age. As shown in Fig. 1, vehicle control
(acetone-treated) mice grew a full coat of hair by 13 wk of age,
whereas mice treated with E2 did not demonstrate any hair
growth. Compared with the acetone-treated control mice, testosterone
treatment produced a slight inhibitory effect on hair growth, whereas
DHT delayed full hair growth by 3-4 wk. Interestingly,
androstenedione and progesterone stimulated hair growth. These results
demonstrate that, of the steroid hormones examined, E2 is
the most effective at blocking hair growth.
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Dose-response relationship of E2 and ICI-182780 on hair
growth, and effect of coadministration of ICI-182780 with
E2.
Dose-response studies were conducted to determine whether lower doses
of E2 and ICI-182780 were capable of modulating hair growth. Different doses (1, 5, and 10 nmol) of E2 or
ICI-182780 were applied topically in 200 µl acetone, twice weekly, to
the clipped dorsal surface of 6-wk-old CD-1 female mice. As shown in
Fig. 2A, the 1-nmol dose of
E2 was found to be inhibitory to hair growth; however, hair
growth inhibition was attenuated with time, and by 18 wk of age, the
mice had developed a full coat of hair. In contrast, only 20% of the
mice treated with 10 nmol E2 developed a full coat of hair
by 21 wk of age. For the 5-nmol dose, 100% of the mice had a full coat
of hair by 20 wk of age. As shown in Fig. 2B, a dose-dependent
stimulation of hair growth by ICI-182780 was apparent at the beginning
of the treatment period, but the dose dependency was attenuated at
later time points. By 10.5 wk of age, all of the mice treated with all
three doses of ICI-182780 developed a full coat of hair. To determine
whether ICI-182780 could antagonize the inhibitory action of topically applied E2, mice were treated topically twice weekly with 1 nmol E2 along with 10 nmol ICI-182780 (ICI-182780 was
applied first, and then E2 was applied after 1 h). As shown
in Fig. 2C, 10 nmol ICI-182780 reversed the inhibitory effect
of 1 nmol E2 on hair growth, further supporting the idea
that the effects of E2 and ICI-182780 on hair growth are
mediated through the ER.
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ER- but not ER-
is expressed in
mouse skin.
Using an immunohistochemical approach, Oh and Smart (29) demonstrated
the presence of an ER immunoreactive protein in the nuclei of the
dermal papilla cells of the mouse telogen hair follicle. In the present
study, we detected a 6.7-kb ER-
mRNA in RNA isolated from 6-wk-old
female mouse skin by using a mouse ER-
cDNA probe (Fig.
3A). In addition, Western analysis,
using monoclonal antibody H222 to the carboxy-terminal F region of
ER-
, or monoclonal antibody H226 to the NH2-terminal A/B
transactivation region of ER-
, revealed a 65-kDa ER-
immunoreactive protein in mouse skin protein extracts from 6-wk-old
mice (Fig. 3B). A protein of similar size was detected in
uterine extracts with these antibodies (data not shown). These results
demonstrate that ER-
is expressed in mouse skin. Next, we wanted to
determine whether ICI-182780, E2, or the hair follicle cycle could alter the expression of ER-
protein. Six-wk-old female mice were treated topically twice weekly with acetone vehicle, 10 nmol
ICI-182780, or 10 nmol E2 in 200 µl of acetone. At 8 wk of age, mice treated with ICI-182780 had entered anagen (data not
shown), and skin protein extracts from these mice demonstrated decreased levels (>50% as demonstrated by laser densitometry) of
ER-
H226 immunoreactive protein compared with the vehicle-treated mice, whereas the mice treated with E2 demonstrated
1.5-fold increased levels of the protein (Fig. 3C). At 10 wk of
age, the hair follicles of the mice treated with acetone were in anagen
(data not shown), and skin protein extracts from these mice had levels
of the ER-
immunoreactive protein similar to those of the
ICI-treated group, whereas the protein extracts from
E2-treated mice demonstrated greater than 3-fold elevated
levels of ER-
, and their follicles remained in telogen. These
results indicate that the level of ER-
expression is modulated by
E2 and ICI-182780 treatment and by the phase of the hair
follicle. To determine whether ER-
is expressed in female mouse
skin, we conducted an RPA on RNA isolated from mouse skin containing
predominantly anagen or telogen hair follicles. As shown in Fig.
4, RPA analysis of mouse skin RNA confirmed
the presence of ER-
transcripts in RNA isolated from mouse skin
containing predominantly telogen or anagen hair follicles; however,
ER-
transcripts were not detectable in mouse skin. In addition,
ER-
could not be detected using an ER-
- specific antibody and
Western analysis (data not shown).
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Effect of ventral application of 17-estradiol and
ICI-182780 on dorsal hair growth.
To begin to determine whether the effects of E2 on hair
growth are mediated within the skin itself or due to a systemic effect of cutaneously absorbed topically applied E2, we injected
1, 5, or 10 nmol E2 in corn oil intraperitoneally (ip) into
6-wk-old female mice twice weekly for 7 wk. Intraperitoneal
administration of E2 had no inhibitory effect on hair
growth. Previously, we reported that intraperitoneal administration of
10 nmol ICI-182780 at the same dosing frequency was also without effect
on hair growth (29). These results suggest that the effects of
E2 and ICI-182780 are produced locally within the skin;
however, it is possible that these compounds are rapidly metabolized
and/or eliminated after intraperitoneal administration. Therefore, in a
further effort to determine whether E2 and ICI-182780 are
functioning directly within the skin, 6-wk-old mice were treated
topically twice weekly with either 1 nmol or 10 nmol E2 or
10 nmol ICI-182780 on the clipped ventral surface (Fig.
5). Hair growth was monitored on both the
dorsal and ventral clipped surfaces of the treated mice, with the
intention that if the effects were of a systemic nature, modulation of
hair growth would be seen irrespective of the site of application.
Application of 10 nmol of ICI-182780 promoted hair growth on the
ventral surface (site of application) (Fig. 5A) but had no
effect on the hair growth on the dorsal surface (Fig. 5B).
E2 (1 nmol) inhibited hair growth on the ventral surface (Fig. 5A) but not on the dorsal surface (Fig. 5B).
E2 (10 nmol) inhibited hair growth on both the dorsal and
the ventral surfaces, but the inhibition of hair growth on the dorsal
surface (site away from the site of application) was much attenuated
compared with the ventral surface (site of application). Similar
results were found in experiments in which E2 and
ICI-182780 were applied on the clipped dorsal surface and hair growth
was monitored on the clipped ventral surface (data not shown).
Collectively, these results indicate that the hair growth-modulating
effects of topically applied E2 and ICI-182780 are
occurring locally rather than systemically.
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Effect of ovariectomy on hair growth.
It is evident that the topical application of exogenous E2
arrests hair follicles in telogen. Therefore, we wanted to determine whether removal of the ovaries, the major source of endogenous estrogen, would alter the hair follicle cycle. To this end, 6-wk-old female mice whose follicles were in the telogen phase of the hair follicle cycle were subjected to ovariectomy, and hair growth was
monitored in this group and compared with the sham-ovariectomized group. As shown in Fig. 6, ovariectomy
caused a profound and rapid telogen-to-anagen transition, with
accompanying full hair growth. To determine whether the effect of
ovariectomy on hair growth could be prevented by topical application of
E2, ovariectomized mice were treated twice weekly with 1 nmol E2. As shown in Fig. 6, topical application of
E2 prevented the hair growth in ovariectomized mice.
Although this dose of E2 was without a systemic effect on hair growth in the dorsal-ventral experiment, we wanted to assure that
cutaneous absorption of E2 did not result in high sustained serum E2 levels in ovariectomized mice. RIA analysis
revealed that after topical application of 1 nmol E2, serum
estradiol increased rapidly within 2 h to levels comparable with those
found during proestrous in CD-1 mice (40) but declined to a low
physiological range by 12 h (Fig. 7). This
rapid decline resulted inan abbreviated exposure to E2
compared with the normal cycling mouse (40). The uterus was capable of
responding to these levels of estradiol with less than a half-maximal
increase in wet weight (data not shown), whereas under similar topical
dosing, hair growth was unaffected in the dorsal-ventral experiment.
Therefore, these data further support the notion that the effect of
E2 on the hair follicle cycle is due to its direct action
in the skin.
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DISCUSSION |
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Estrogen receptors, as detected by Scatchard analysis, have been found
in human (14, 31) and mouse skin (38) and, on the basis of
[3H]estrogen binding, are present in rat skin
(36). Utilizing a monoclonal antibody (H222) to ER, we have previously
found that an ER immunoreactive protein was extensively expressed in
the nuclei of the dermal papilla of the telogen follicles in mice (29).
These results provided direct evidence that the ER is expressed and
that its expression is localized to specific cells thought to be
important in regulating the hair follicle cycle. However, two ERs have
now been identified, and both ER- and ER-
are expressed in
numerous tissues (8, 32). Therefore, it was necessary to determine
whether ER-
and/or ER-
is expressed in mouse skin. We found that
monoclonal antibodies H226 and H222, directed toward either the
NH2-terminal transactivation A/B domain or to the
carboxy-terminal F domain of ER-
, respectively, recognize a
65-kDa protein in skin protein extracts. This 65-kDa size corresponds to the known size of the mouse ER-
protein (12, 16). In addition, Northern blot analysis of RNA isolated from mouse skin probed with a
mouse ER-
cDNA revealed a 6.7-kb mRNA that corresponds to the size
of the mouse ER-
mRNA (41). RPA analysis confirmed that ER-
transcripts are present in mouse skin and also demonstrated that ER-
transcripts are not detectable in mouse skin. These findings, coupled
with the previous immunohistochemical localization studies, in which
monoclonal antibody H222 detected an ER immunoreactive protein
predominantly if not exclusively in the nuclei of the dermal papilla
cells, indicate that it is ER-
that is expressed in the nuclei of
dermal papilla cells of mouse skin.
Earlier studies from our laboratory demonstrated that the topical
application of estrogen to mouse skin blocks hair growth by arresting
the hair follicle in telogen and that the topical application of the ER
antagonist ICI-182780 initiates hair growth by causing the telogen
follicle to enter anagen. Although these data are supportive of a skin
ER pathway, it is also possible that the effects of topically applied
E2 and ICI-182780 were due to a secondary systemic effect
subsequent to dermal absorption. Our current results demonstrate that
the ventral topical twice weekly application of either 10 nmol
ICI-182780 or 1 nmol E2 produces hair growth effects at the
site of application but not at remote sites away from the site of
application. These results indicate that the effects of E2
and ICI-182780 on hair growth are mediated locally within the skin and
are not due to a secondary systemic effect. Furthermore, the fact that
topically applied ICI-182780 can prevent the hair growth-inhibitory
action of topically applied E2 provides additional evidence
for a skin ER- pathway that regulates the telogen-anagen hair
follicle transition in mice.
We found that topical application of 1 nmol E2 to mouse
skin resulted in a rapid increase in serum E2 levels that
returned to low physiological levels within 12 h, indicating that
E2 is rapidly absorbed through mouse skin and rapidly
cleared from the serum. Whereas the serum levels of E2 are
not sustained after topical application, a single E2 dose
every 3-4 days is sufficient to maintain the hair follicle in
telogen. Therefore, it appears that each E2 treatment has a
prolonged local inhibitory effect on the hair follicle cycle. Perhaps
E2, through ER-, is inducing the expression of stable
proteins that are sufficiently long-lived to block the telogen-anagen
transition, or perhaps E2 is sequestered in the skin. We
found that topical E2 treatment increases the amount of
ER-
protein expression in skin, suggesting that E2 itself may be involved in the regulation of ER-
expression in skin.
It is possible that this increase in ER-
protein is sufficient to
induce or maintain the putative telogen regulatory signal. In support
of this notion are results demonstrating that the levels of ER-
in
skin are decreased in ICI-182780-treated mice as well as in the skin of
control mice whose follicles have cycled into anagen. In addition,
previous studies have demonstrated the absence of the ER immunoreactive
protein in the dermal papilla of anagen follicles (29).
The fact that E2 and estrone are produced by the hair
follicle itself (35) and that within the skin the ER is predominantly expressed in the dermal papilla (29) suggests that estrogen may
function as a paracrine regulator of the hair follicle cycle. However,
we found that ovariectomy does induce a rapid telogen-anagen transition
with accompanying hair growth and that this effect can be blocked by
twice weekly topical application of 1 nmol E2, a dose that
has hair growth effects only at the site of treatment. These results
suggest that the skin is dependent on serum estrogen or ovarian
precursor steroids for the regulation of the telogen-anagen transition.
However, it does not appear that the estrous cycle alters or controls
the hair follicle cycle in mice. For example, 6-wk-old female mice are
reproductively mature and actively cycling, yet the hair follicles of
these mice are arrested in telogen from the age of 6 wk to 9 wk. Thus,
over this 3-wk period, these mice have undergone ~5 estrous cycles,
indicating that the hair follicle cycle is independent of the high and
low levels of serum E2 that occur during proestrus and
diestrus, respectively. Although it is possible that the effects of
ovariectomy are due to events other than or in addition to the decrease
in serum estrogen, it is clear that topical treatment with estrogen can
prevent the effects of ovariectomy on hair growth. Because ER- can
be activated via an epidermal growth factor receptor pathway in a
ligand-independent manner in reproductive tissues (18), additional
pathways could be operative in the hair follicle as well. Collectively,
our results suggest that estrogen production and uptake in skin are
complex and that further investigation will be required to clarify the pathways that regulate the presence of dermal estrogens and link them
with inhibition of hair growth.
Although androgens have been extensively studied as regulators of hair
follicle growth and differentiation (34), estrogens have received
comparatively little attention. Recently, inhibitors of 5-reductase
have hair growth-promoting properties, indicating an inhibitory role
for DHT on hair growth (10, 21). Our current results in mice
demonstrate that among the steroids examined, which included
E2, testosterone, DHT, progesterone, and androstenedione, E2 is the most effective at blocking hair growth. Based on
the hair growth-inhibitory action of 1 nmol E2, it appears
that E2 is
10-fold more potent than DHT. ER-binding
proteins have been identified in human skin (14, 31) and may represent
potential targets for hair growth modulation by E2 or
ICI-182780. Among the ER antagonists examined, ICI-182780 is the most
potent at inducing hair growth. All of the ER antagonists examined
promoted the telogen-anagen transition and hair growth to some extent; however, higher doses actually inhibited hair growth. The decreased activity of these ER antagonists, as well as their inhibitory action at
higher doses, is consistent with the fact that these compounds are
partial agonists of ER (20).
In summary, the growth and cyclicity of a hair follicle are controlled
through complex and intricate interactions between the epithelial cells
of the follicle and mesenchymal cells of the dermal papilla. Although
it has been suggested that diffusible factors derived from the dermal
papilla cells regulate the follicle cycle, the exact nature of these
factors is still unknown. The identification of regulatory molecules
such as estrogen and ER antagonists that modulate the telogen-to-anagen
transition could allow for the identification of the downstream
effectors of ER-. Identification of the effector pathways may allow
for the development of effective therapy to treat hair-related
abnormalities like alopecia and hirsutism.
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ACKNOWLEDGEMENTS |
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This research was supported by Grant ES-08127 (to R. C. Smart and C. L. Robinette) from the National Institute of Environmental Health Sciences and Grant CA-46637 (to R. C. Smart) from the National Cancer Institute.
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FOOTNOTES |
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Current address for S. Chanda: Cerus Corporation, 2525 Stanwell Drive, Suite 300, Concord, CA 94520.
Address all correspondence and requests for reprints to: R. C. Smart, Molecular and Cellular Toxicology, Department of Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (E-mail: rcsmart{at}unity.ncsu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 14 June 1999; accepted in final form 20 September 1999.
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