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Endocrinology Vol. 146, No. 3 1214-1225
Copyright © 2005 by The Endocrine Society

Hair Cycle Control by Estrogens: Catagen Induction via Estrogen Receptor (ER)-{alpha} Is Checked by ERß Signaling

Ulrich Ohnemus, Murat Uenalan, Franziska Conrad, Bori Handjiski, Lars Mecklenburg, Motonobu Nakamura, José Inzunza, Jan-Åke Gustafsson and Ralf Paus

Department of Dermatology (U.O., F.C., L.M., R.P.), University Hospital Hamburg-Eppendorf, University of Hamburg, 20246 Hamburg, Germany; Department of Clinical Endocrinology (M.U.), Hannover Medical School, 30623 Hannover, Germany; Center of Biomedical Research (B.H.), Department of Internal Medicine, Charité, 10117 Berlin, Germany; Department of Dermatology (M.N.), Graduate School of Medicine, 606-8507 Kyoto, Japan; and Department of Medical Nutrition (J.I., J.-A.G.), Karolinska Institute, Novum, 14157 Huddinge, Sweden

Address all correspondence to: Ralf Paus, M.D., Department of Dermatology, University Hospital Hamburg-Eppendorf, University of Hamburg, Martinistr. 52, D-20246 Hamburg, Germany. E-mail: paus{at}uke.uni-hamburg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although 17ß-estradiol (E2) is recognized as a potent hair growth modulator, our knowledge of estrogen function, signaling, and target genes in hair biology is still very limited. Between the two recognized estrogen receptors (ERs), ER{alpha} and ERß, only ER{alpha} had been detected in murine skin. Here we show that ER{alpha}, ERß, and ERß ins are all expressed throughout the murine hair cycle, both at the protein and RNA level, but show distinct expression patterns. We confirm that topical E2 arrests murine pelage hair follicles in telogen and demonstrate that E2 is a potent inducer of premature catagen development. The ER antagonist ICI 182.780 does not induce anagen prematurely but accelerates anagen development and wave spreading in female mice. ERß knockout mice display accelerated catagen development along with an increase in the number of apoptotic hair follicle keratinocytes. This suggests that, contrary to previous concepts, ERß does indeed play a significant role in murine hair growth control: whereas the catagen-promoting properties of E2 are mediated via ER{alpha}, ERß mainly may function as a silencer of ER{alpha} action in hair biology. These findings illustrate the complexity of hair growth modulation by estrogens and suggest that one key to more effective hair growth manipulation with ER ligands lies in the use of selective ER{alpha} or -ß antagonists/agonists. Our study also underscores that the hair cycling response to estrogens offers an ideal model for studying the controls and dynamics of wave propagation in biological systems.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOR DECADES, STEROID hormones, such as androgens, retinoids, thyroid hormones, calcitriols, and glucocorticosteroids, have been known to act as potent hair growth modulators (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). Whereas the profound hair growth-modulatory effects of 17ß-estradiol (E2) had long ago been demonstrated in many mammalian species (12, 13, 14, 15, 16, 17), interest in the role of estrogens in hair biology was only recently revived by the finding that topically applied E2 arrests murine pelage hair follicle (HF) in telogen and that the estrogen receptor (ER) antagonist ICI 182.780 reportedly induces premature anagen development (18, 19). Furthermore, gonadectomy of female mice resulted in a profound and rapid telogen to anagen transition (20). Therefore, exogenous and endogenous ER agonists may control HF cycling by stimulation of ER in the follicular dermal papilla (DP), mainly by arresting the HF in its resting stage (telogen) (18, 19, 20).

Orchidectomy of male ER{alpha} knockout mice (ERKO) induces a synchronized anagen phase of HFs, which is inhibited by E2 treatment in wild-type and ERß knockout mice (BERKO) but not in ERKO and ER double-knockout mice (21). This suggests that the telogen to anagen transition is inhibited by ER{alpha} stimulation. An ER{alpha}-mediated pathway of hair growth control in mice conforms with previous reports that only ER{alpha} expression is detectable in murine telogen and early anagen skin, whereas the second receptor, ERß, was detectable only in human skin (18, 20, 22, 23, 24, 25, 26, 27).

Most recently we demonstrated that topical E2 enhances chemotherapy-induced massive hair loss (alopecia) in mice by forcing HFs into the dystrophic catagen response pathway to HF damage (2). Instead, follicles treated by the selective E2 antagonist ICI 182.780 or vehicle shift into the dystrophic anagen response pathway (28). Therefore, the regrowth of normally pigmented hair shafts after chemotherapy-induced alopecia is significantly accelerated after E2 treatment. This encouraged us to further dissect the role of estrogens in normal catagen control, one key to hair cycle control and perhaps the most promising target for therapeutically desired hair growth manipulation under clinical conditions (29, 30).

In mice, rats, guinea pigs, and dogs, estrogens exhibit inhibitory properties on hair growth (12, 13, 14, 15, 16, 31). In humans, however, E2 effects are more complex: E2 prolongs the anagen phase of scalp hair follicles in vivo (17), whereas it stimulates hair shaft elongation in frontotemporal male scalp skin in vitro (32). Recently we confirmed that E2 inhibits hair shaft elongation and prolongs anagen duration in female occipital scalp hair follicles in vitro (33). These contradictory results suggest that the control of hair growth by estrogens is much more complex than previously appreciated and differs among distinct integumental sites, genders, and species.

Estrogens act through their nuclear receptors, ER{alpha} and ERß (34, 35), which belong to a superfamily of ligand-activated transcription factors, comprising receptors for steroids, thyroid hormones, retinoids, prostanoids, and vitamin D (36). Estrogen action depends on multiple mechanisms of staggering complexity, involving not only the two receptors but also likely other, non-ER-related proteins (34, 37, 38, 39).

For example, both ER{alpha} and ERß activate transcription on classical estrogen response elements. However, in the presence of E2, ER{alpha} is an activator, whereas ERß is an inhibitor or silencer at activating protein-1 sites (40). Also, ER{alpha}’s transactivation potency is down-regulated by wild-type (wt) ERß in an E2 dose-dependent manner (41). In addition, one ERß splice variant, called ERß ins, which has an additional 18-amino acid in-frame sequence between exons 5 and 6 of wt ERß, may act as a dominant-negative regulator of ER{alpha}, independent of the E2 concentration (42, 43). In the uterus, E2 induces epithelial cell proliferation indirectly via the stimulation of stromal ER{alpha}, which leads to growth factor secretion by stromal cells, thus making the presence of ER{alpha} on epithelial cells dispensable (44, 45). In the mammary gland, a derivative of the epidermis, ER{alpha} is exclusively expressed in epithelial cells, and E2 causes proliferation in the mammary epithelium in BERKO mice. Therefore, E2 likely exerts its mammary effects by acting directly on ER{alpha} in the epithelium (46).

More information about the exact expression patterns of ER{alpha}, ERß, and ERß ins is, therefore, necessary when the role of estrogens in skin and hair biology is to be investigated. To better understand potential interactions among the distinct ERs in stromal and epithelial cells in the hair follicle, we reevaluated the expression of ER{alpha}, ERß, and the ERß variant, ERß ins, during the spontaneous anagen VI, catagen, telogen transformation of normal murine hair follicles in situ by light microscopic immunohistology and immunofluorescence. Expression of ER subtypes ER{alpha} and ERß was examined semiquantitatively on the RNA levels by RT-PCR.

Because the most frequent hair growth disorders encountered in clinical practice represent disorders of HF cycling (29, 47), it was of paramount interest to elucidate how exogenous ER ligands (agonists and antagonists) affect HF cycling in vivo. Previous studies focused on the influence of E2 on telogen and subsequent anagen development (18, 19), whereas it remained to be determined whether and how E2 affects catagen development under physiological conditions. Given the crucial role of catagen control in clinically relevant hair growth disorders (29), we explored whether topically applied E2 and the ER antagonist ICI 182.780 (48) stimulate or inhibit spontaneous catagen development in the best-established mouse model for hair research (C57BL/6) (1, 2, 3, 49, 50). Based on the observation that other steroid hormone receptor ligands, which inhibit anagen (glucocorticoids, calcitriols), also induce premature catagen (1, 2, 3, 4, 51), we hypothesized that E2 should be a potent catagen inducer. For quantifying the results, quantitative histomorphometry of catagen development in anagen HFs was applied, whereas the macroscopic spreading of anagen/catagen waves was assessed with a recently developed planimetric assay (dotmatrix analysis) (28) (Fig. 1Go). This facilitated the accurate, quantitative, macroscopic assessment of HF cycling in C57BL/6 mice, which is based on the strict coupling of skin color switches to defined hair cycle stages (52, 53, 54). We purposely examined the effects of exogenous ER ligands in female, nonovariectomized mice, i.e. in the presence of full endogenous estrogen levels, to obtain physiologically relevant data. This was further supported by the argument that any clinical exploitation of the results from these animal studies would usually occur in a similar setting, i.e. administration of exogenous ER ligands in the presence of ovarian, adrenal, and/or intracutaneously generated endogenous estrogens.



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  		FIG. 1. Dotmatrix planimetry. Schematic representation of dotmatrix planimetry used for the macroscopic, quantitative assessment of HF cycling and wave propagation by measuring the exact percentages of distinct HF stages in individual mice. (for details, see Ref.28 ). A, Transparency on which the appropriate areas that were in different hair cycle stages (pink = telogen, gray = catagen, black = anagen; see also C) are marked. First, the transparency was put on a polaroid photo of a mouse back to mark the areas that were in different stages. Afterward a dotmatrix (sheet with a uniform defined dot pattern) was placed under the marked foil to calculate the percentages of the regions of interest by counting the dots. In this calculation example, a total of 282 dots = 100% was determined. The exact percentages of the different hair cycle stages can be calculated by dividing the dot counts of the corresponding areas by the whole dot number (see B).

 
Finally, spontaneous HF cycle development and HF phenotype were investigated in BERKO mice (55). We focused on catagen development (HF regression) and catagen associated HF keratinocyte apoptosis of BERKO mice and checked their differences to age-matched corresponding wild-type mice.

In summary, the objectives of this study were to examine: 1) which estrogen receptor subtypes ({alpha}, ß, and ßins) are detectable at the protein and/or mRNA level during the murine HF cycle, 2) how E2 and the ER antagonist ICI 182.780 influence the spontaneous and depilation-induced HF cycle, and 3) whether BERKO mice show any abnormalities in their HF phenotype or cycling, compared with age-matched wild-type mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
For in vivo and immunohistochemical staining experiments, 6- to 9-wk-old syngenic female C57BL/6J mice (15–20 g) were purchased from Charles River (Sulzfeld, Germany) and housed in community cages with 12-h light periods at Charité Animal Facilities, Campus Virchow Hospital, Berlin, Germany. They were fed water and mouse chow ad libitum (n = 106).

In C57BL/6J black skin, all melanin pigmentation is coupled to the HF, which makes it easy to correlate macroscopic skin colors to the underlying hair cycle stage: active hair growth (anagen = black); hair follicle regression (catagen = gray); and resting of the hair production (telogen = pink) (54).

To study hair cycling abnormalities and phenotypic differences in ER-deficient mice wild-type and BERKO female mice from d 19 after birth were used (n = 5 per group). BERKO mice were generated by targeted disruption of the ERß gene as described before (55). These mice represent the offspring of ER heterozygous mice with a mixed C57BL/6 J/129 background (55). Genotyping of tail DNA was performed as described elsewhere (56).

All animal experimentation was conducted in accord with accepted standards of humane animal care and approved by the local ethics committee.

Chemicals and antibodies
E2 was purchased from Sigma (St. Louis, MO). ICI 182.780 was from Astra Zeneca (Hamburg, Germany). ER{alpha} rabbit polyclonal antibody, MC-20, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Chicken anti-ERß antibody 503 (entire ERß molecule) and chicken anti-ERß antibody ins (18-amino acid insert in the ligand-binding domain of the isoform ERß ins (18aa), were generated in the laboratory of J.-Å. Gustafsson as described elsewhere (Departments of Medical Nutrition and Biosciences, Karolinska Institute, NOVUM, Stockholm, Sweden) (57). Peroxidase-conjugated secondary antibodies and goat antirabbit IgG were from Jackson ImmunoResearch (West Grove, PA). Peroxidase-conjugated secondary antibodies and rabbit antichicken IgG were obtained from Vector Laboratories (Burlingame, CA). Signal visualization was performed with diaminobenzidine (DAB) substrate kit from Vector Laboratories. Tyramide signal amplification (TSA) kit (Rhodamine system staining) was purchased from NEN Life Science Products (Boston, MA).

Immunohistochemistry
Dorsal skin from untreated mice was excised and cryopreserved at d 0 (unmanipulated telogen skin), d 12 (anagen VI), d 17 (catagen III), d 19 (predominantly catagen VII), and d 25 (telogen) after depilation (53, 54). Mouse uterus was used as positive control for ER{alpha} staining and mouse ovary for ERß staining. Sections from BERKO mice served as negative control for ERß staining; incubation of cryosections with preimmune rabbit serum (AgResearch) served as negative control for ER{alpha} staining. Mouse uterus showed strong immunoreactivity (IR) for ER{alpha}; mouse ovary was positive for ERß and ERß ins. The negative controls did not exert any IR for ER {alpha}, ß and ß ins (data not shown). Cryosections (8 µm) were fixed in acetone for 10 min at –20 C. After inhibition of endogenous peroxidase with 3% H2O2 in PBS for 15 min, the slides were incubated with 10% of the normal serum of the host for the secondary antibody and Triton X-100, 0.3%, at room temperature for 20 min. Subsequently the cryostat sections were incubated overnight at 4 C with the primary antibodies to ER{alpha} (1:2000), ERß 503 (1:1000), and ERß ins 18aa (1:1000). This was followed by an incubation of 45 min with a biotinylated goat antirabbit IgG [1:200 in Tris-buffered saline, Jackson ImmunoResearch] together with 2% normal goat serum and 4% normal mouse serum. Afterward we performed a routine detection with the avidin biotin complex, labeled with peroxidase. The signal was visualized with DAB (Vector Laboratories), and the sections were finally counterstained with methylene green (Dako A/S, Glostrup, Denmark). Slides were mounted in Eukitt (O. Kindler GmbH & Co., Freiburg, Germany).

In parallel, we performed a TSA (fluorescein system staining, NEN Life Science Products) for ER{alpha}, ERß, and ERß ins. Acetone-fixed frozen sections were incubated for 15 min in PBS with 3% H2O2 for inhibition of endogenous peroxidase. This was followed by preincubation with TNB (Tris-NaCl blocking reagent) blocking buffer for 30 min. Then the slides were incubated overnight with the primary antibody to ER{alpha} (1:8,000), ERß 503 (1:10,000), and ERß ins (1:10,000) followed by incubation for 30 min with a biotinylated secondary antibody (1:200 in TNB, Jackson ImmunoResearch). Subsequently slides were washed, and streptavidin-conjugated horseradish peroxidase (1:100 in TNB) was added for another 30 min. Finally the sections were incubated in fluorophore tyramide stock solution (1:50 in amplification diluent) for 30 min. After incubation, the sections were dipped in 4', 6-diamino-2-phenylindole (DAPI) solution (0.1 µg/ml) in PBS) for 30 sec at room temperature to delineate nuclei and mounted in Fluoromount (Southern Biotechnology, Birmingham, AL). Slides were examined under an Axiophot fluorescence microscope (C. Zeiss, Jena, Germany). Images were recorded as computer files via a Hamamatsu digital camera (Hamamatsu Photonics, Hamamatsu City, Japan) and analyzed by using an Openlab program (Improvision, Coventry, UK). Based on TSA staining, the mean fluorescence intensity of ER{alpha} and ERß (503 and ins) was measured at two previously defined reference areas in the follicular dermal papilla (see Fig. 2CGo) by National Institutes of Health image system, and the average mean fluorescence intensity was calculated (n = 24 HFs/hair cycle stage, derived from more than three individual mice).




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  		FIG. 2. ER{alpha}, ERß, and ERß ins expression patterns detected by immunohistochemistry. a, Immunoreactivity patterns of ER{alpha}, ERß, and ERß ins IR during the depilation-induced murine hair cycle stained by ABC method using DAB as substrate (A, C, E, G, I, K, M, O, Q, S, U, W) and the more sensitive TSA technique (B, D, F, H, J, L, N, P, R, T, V, X). In the ABC-stained photos, ER{alpha}- and ERß-positive cells are brown. Follicular melanin is stained by the dark brown to black color, hair shafts are stained turquoise. In the TSA staining, rhodamine was used as fluorophore (red). Cell nuclei were counterstained with DAPI (blue). Pink-red and light purple (overlay of DAPI and rhodamine) indicate ER{alpha}- and ERß-positive cells. ER{alpha} IR had its peak in telogen follicles within the DP and the sebaceous gland (SG), whereas the outer-root sheath (ORS), epithelial strand (ES), germ capsule (GC), and bulge region showed weaker IR. In anagen VI we observed ER{alpha} IR in the DP, matrix keratinocytes (MK), and inner-root sheath (IRS) and ORS, whereas the epidermis, SG, and bulge region showed no ER{alpha} IR. In early catagen ER{alpha} IR was seen in the DP, matrix keratinocytes (MK), IRS, ORS, and connective tissue sheath (CTS). In late catagen ER{alpha} IR was restricted to the DP, GC, ORS, bulge, and SG. ERß 503 IR was intense and ubiquitously seen in the DP, IRS and ORS, MK, SG, bulge region, and epidermis throughout all investigated stages; the CTS was positive for ERß in anagen VI. ERß ins IR was weaker but coexpressed throughout the whole cycle and in same locations like ERß 503. b, Schematic representation of IR patterns (based on TSA stainings). Gray markings: ER{alpha}, ERß, and ERß ins expression. c, ER{alpha}, ERß, and ERß ins staining intensity detected by NIH image. The expression of ER{alpha} within the follicular DP was significantly hair cycle dependent. Mean fluorescence intensity of ER{alpha} was highest in telogen follicles and lowest in early catagen. There were no significant differences of IR-intensity of ERß (503 and ins) within the DP during the HF cycle. Based on TSA staining, the fluorescent staining intensity of ER{alpha} and ERß was measured at two previously defined reference areas in the follicular DP by NIH image, and the average mean fluorescence intensity was calculated (n = 24 HFs/hair cycle stage, derived from more than three individual mice). Given are the mean values + 1 SEM. Level of significance: **, P ≤ 0.01; ***, P ≤ 0.001.

 
RT-PCR
RNA from full-thickness back skin of C57BL/6 mice, homogenized under liquid nitrogen, was isolated by using Qiagen RNeasy minikit (Qiagen, Hilden, Germany) according to the protocol provided by the manufacturer. Subsequently, cDNA was synthesized from 1 µg of total RNA using a first-strand cDNA synthesis kit for RT-PCR (AMV) (Roche Molecular Biochemicals, Mannheim, Germany). Mouse uterus was used as positive control for ER{alpha} and mouse ovary for ERß. The resulting cDNA was stored at –80 C until further use. Primers used are listed as follows: ER{alpha}, forward, 5'-AAT TCT GAC AAT CGA CGC CAG-3', reverse, 5'-GTG CTT CAA CAT TCT CCC TCC TC-3'; ERß, forward, 5'-CTT GGT CAC GTA CCC CTT AC-3', reverse, 5'-GTA TCG CGT CAC TTT CCT TT-3' (58); ß-actin, forward, TGT TAC CAA CTG GGA CGA CA; reverse, TCT CAG CTG TGG TGG TGA AG. The PCR was run on a program temp control system (Astec, Fukushima, Japan). The reaction consisted of 1 µl cDNA, 10 µl 10 x PCR buffer, 8 µl deoxynucleotide triphosphate mixture (2.5 mM each), 1 µl forward primer (50 pmol), 1 µl reverse primer (50 pmol), 0.5 µl TaKaRa Ex Taq polymerase (Takara, Otsu, Japan), and 79.5 µl water. Amplification was performed over 32 cycles for ß-actin, 35 cycles for ER{alpha}, and 35 cycles for ERß. Each cycle consisted of the following steps: denaturation at 94 C, annealing at 55 C, and extension at 72 C. The PCR products were analyzed by agarose gel electrophoresis. Densitometry was performed by using a color imaging ES-2200 (Epson, Tokyo, Japan) and NIH image for assessing staining results.

Apoptosis assay
For evaluation of apoptotic cells within the skin of P19 BERKO and age-matched wild-type mice, we used an established, commercially available terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) kit (Apop Tag, Intergen, Purchase, NY) with antidigoxigenin fluorescein isothiocyanate-conjugated antibodies. Negative controls were performed by omitting terminal dideoxy transferase; thymus of infantile mice served as positive controls. For more detailed description, see previous publications (59, 60).

Hair cycle manipulation by ER ligands: influence on spontaneous anagen development
To evaluate whether topical E2 arrests C57BL/6 HFs in prolonged telogen stage, we tested in three identical experiments a total of 66 animals. Twenty-two animals were treated topically with acetone as vehicle control, 22 animals with E2, and 22 animals with ICI 182.780. Hair shafts of age-matched mice with all follicles in telogen (postnatal d 42 mice) were carefully clipped with electric clippers to avoid trauma-induced anagen. Mice received 10 nmol of the test substances in 200 µl vehicle (acetone) over the clipped area of the dorsum on d 1, 5, 8, 12, 15, 19, 22, and 26 after clipping. Controls received vehicle alone. Under general ketamine-hydrochloride anesthesia, spontaneous anagen development was evaluated macroscopically twice a week. After 33 d, mice were killed by cervical dislocation. Skin was embedded according to previously published procedures (61).

Hair cycle manipulation by ER ligands: influence on catagen development
To explore whether catagen development is influenced by E2 and ICI 182.780 treatment, anagen was induced by hair shaft depilation, as described elsewhere (1). In two identical experiments, a total of 40 test animals received once daily (beginning 9 d after anagen induction) either 10 nmol E2 (n = 16) or ICI 182.780 (n = 8), each diluted in 200 µl acetone. Sixteen control animals received the vehicle (acetone) alone. The compounds were applied topically with a pipette and distributed evenly on the upper half of the depilated back skin on d 9, 11, 13, and 15 after depilation. Catagen development, recognizable through characteristic skin color changes, was photodocumented and analyzed via dotmatrix planimetry. At the end of the experiment on d 18 post depilation, mice were killed, and the skin was embedded according to previously published procedures (61). The hair cycle stage of follicles in back skin was assessed by histomorphometry (54).

Macroscopic analysis by dotmatrix planimetry
For macroscopic in vivo analysis of hair cycle staging, we used our recently developed planimetric assay (dotmatrix analysis) as described previously (28). It enables one to measure macroscopically the exact percentages of HF cycling stages in individual mice, which are based on the strict coupling of skin color switches to defined hair cycle stages (52, 53, 54). The procedure is illustrated in Fig. 1Go.

Histology and microscopic analysis by quantitative histomorphometry
Hair cycle stages were classified according to Müller-Röver et al. (54) by the means of quantitative histomorphometry. A hair cycle score (HCS) was calculated with the formula presented by Maurer et al. (50). Morphometry was performed on formalin-fixed, paraffin-embedded, hematoxylin and eosin, or alkaline phosphatase-stained 8-µm sections that were taken from defined back skin regions. A minimum of 50 back skin HFs was analyzed for each test and control mouse.

The distance between the basement membrane of the epidermis and the panniculus carnosus muscle was measured for assessing the dermal thickness in BERKO mice and corresponding wild-type animals. In total, 85–117 such measurements were performed, deriving from five animals per mutant and wild-type group.

Statistical analysis
Data derived from identical experiments, mean values, and SEMs were calculated from pooled data. With the statistical analysis software SPSS (SPSS Inc, Chicago, IL), significance was assessed with the Mann-Whitney U test for unpaired samples combined with Monte-Carlo calculation for exactness. Differences were judged as significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ER{alpha}, ERß, and ERß ins IR is present in normal mouse skin throughout the murine HF cycle
First, we studied IR for ER{alpha} and ERß (whole ERß molecule 503 and splice variant ins 18aa) throughout the murine HF cycle, using conventional and TSA immunohistochemistry. Within the murine HF, intensity and distribution of ER{alpha}, ERß 503, and ERß ins IR changed during the different investigated stages of the murine HF cycle [telogen, anagen (VI), early catagen (II-III), and late catagen (VI–VIII)] (Fig. 2Go).

In actively growing HFs (anagen VI), ER{alpha} IR was observed in the dermal papilla, matrix keratinocytes, the inner root sheath, and the outer root sheath, whereas the epidermis, bulge region, and sebaceous gland showed no ER{alpha} IR (Fig. 2aGo, A). In Fig. 2aGo, A and B, ER{alpha} IR in anagen VI is demonstrated with the more sensitive TSA staining technique. ERß 503 IR was intense and ubiquitously seen in the dermal papilla, matrix keratinocytes, inner root sheath, outer root sheath, connective tissue sheath, the sebaceous gland, bulge region, and the epidermis of anagen VI follicles (Fig. 2aGo, C and D), whereas ERß ins IR was weaker but coexpressed with ERß 503 (Fig. 2aGo, E and F).

In early catagen, weak ER{alpha} IR was seen in the dermal papilla, matrix keratinocytes, inner and outer root sheath, and the connective tissue sheath; no ER{alpha} IR was seen in the epidermis, bulge region, and sebaceous gland. In late catagen, ER{alpha} IR was restricted to the dermal papilla, secondary hair germ, outer root sheath, bulge region, and the sebaceous gland; (Fig. 2aGo, G and H and M and N). ERß IR became visible in cells of the outer root sheath, inner root sheath, bulb matrix, dermal papilla, epidermis, and bulge region and in the sebaceous gland, both in early and late catagen stage (Fig. 2aGo, I and J and O and P). ERß ins IR was again weaker but coexpressed with ERß 503 (Fig. 2aGo, K and L and Q and R).

During the anagen-catagen transformation of hair follicles and their entry into the telogen stage, ER{alpha} IR had its peak in telogen follicles and was intensely visible in the dermal papilla and cells of the sebaceous gland. Keratinocytes of the outer root sheath, bulge, and germ capsule showed weaker ER{alpha} positivity (Fig. 2aGo, S and T). ERß (503 and ins) expression in telogen follicles was observed in the dermal papilla, keratinocytes of the germ capsule, remaining outer root sheath, and bulge region. ERß ins positivity in sebocytes was of a similar degree as ERß (Fig. 2aGo, U and V and W and X).

ER IR intensity was quantified throughout the investigated stages by NIH image (Fig. 2cGo). Within the follicular dermal papilla we found hair cycle-dependent differences of ER{alpha} expression. Mean fluorescence intensity of ER{alpha} was highest in telogen follicles and lowest in early catagen. There were no significant differences of IR intensity of ERß (503 and ins) within the dermal papilla during the HF cycle (level of significance: **, P ≤ 0.01; ***, P ≤ 0.001; n = 24 HFs/hair cycle stage, derived from more than three individual mice).

ER{alpha} mRNA levels peak in telogen skin
To correlate these receptor protein expression patterns with ER transcription, the ER mRNA levels in full-thickness back skin homogenates were analyzed by semiquantitative RT-PCR. We investigated skin samples of depilation-induced HF cycling from d 0 (unmanipulated telogen), 8 (anagen V), 12 (anagen VI), 19 (catagen VII), and 25 (telogen) after depilation. When receptor levels were normalized against ß-actin, ER{alpha} mRNA levels appeared to be hair cycle dependent and peaked at d 0 and 25 (telogen skin). They declined during anagen development at d 8, when the lowest transcript levels were noted. Densitometrically, an increase in ER{alpha} mRNA steady-state levels was demonstrated at d 12. During catagen (d 19), the levels increased further (Fig. 3Go).



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  		FIG. 3. RT-PCR analysis of ER{alpha} and ERß. Left side, Semiquantitative RT-PCR analysis of expression of ER{alpha}, ERß, and ß-actin in murine skin during the depilation-induced hair cycle on d 0 (nondepilated telogen skin), d 8 (midanagen), d 12 (late anagen), d 19 (late catagen), and d 25 (telogen). Right side, The graphs show the intensity of each band evaluated by digital image analysis based densitometry. The x-axis lists the days after anagen induction by depilation. Experiments were generated from three individual animals per time point. The samples were normalized according to the expression of ß-actin mRNA. Error bars, 1 SEM.

 
ERß transcripts were also detected within murine skin, but by semiquantitative RT-PCR, ERß mRNA levels were relatively constant throughout the whole hair cycle (Fig. 3Go). These findings confirm that ER{alpha} and ERß are expressed in murine skin, both on the protein and gene level, and that at least ER{alpha} expression is hair cycle dependent.

ICI 182.780 does not induce premature initiation of anagen but accelerates anagen development and anagen wave propagation
Topically applied E2 and ICI 182.780 had profound effects on anagen development in C57BL/6 mice. After shaving the backs of telogen mice (postnatal d 42), hair cycle progression (anagen/catagen wave) was followed for a duration of 33 d. E2-treated mice showed a substantially retarded progression of anagen waves after clipping when compared with the control group. In all groups similar anagen hair wave formation was observed, whereas the estrogen-treated animals more often displayed an irregular patchy pattern. These spots appeared early and confluted only with considerable delay into a wave pattern.

In contrast, the ER antagonist-treated group often displayed rapid anagen development with a diffuse pattern, reminiscent of the macroscopic anagen pattern reported after ovariectomy (20). As shown in Fig. 4AGo, the absolute hair regrowth differed significantly between the different groups after clipping (control: n = 22; ICI182.780: n = 22; E2: n = 22). When analyzed by dotmatrix planimetry, significantly larger anagen areas were observed in the ICI 182.780-treated animals, compared with the vehicle group (P < 0.01), on d 29 as well as d 33 after depilation (P < 0.001). However, ICI182.780 did not induce premature anagen initiation.



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  		FIG. 4. Macroscopic dotmatrix analysis of HF cycling in murine back skin. A, The graph shows the back skin percentage of female C57BL/6 mice in anagen. Animals in telogen were shaved on d 0 and subsequently treated with E2, ICI 182.780, and vehicle on d 1, 5, 8, 12, 15, 19, 22, and 26. Compared with the control and ICI 182.780-treated group, E2 significantly inhibits anagen development, whereas ICI 182.780 treatment promotes anagen development and wave propagation significantly, compared with the control and E2-treated group, yet does not initiate anagen prematurely. Given are the mean values ± SEM of pooled data from three experiments, representing a total of 22 animals per group. Level of significance compared with control: **, P ≤ 0.01; ***, P ≤ 0.001. B, Catagen wave progression. Dotmatrix analysis of the induction of premature catagen by exogenous applied E2 in C57BL/6 mice 16 and 18 d after anagen induction by depilation (note that catagen develops spontaneously in control mice). The panel shows that E2 significantly accelerates catagen development, compared with control or ICI 182.780-treated animals. Given are the means ± 1 SEM of pooled data from two independent experiments, representing a total of 16 vehicle- or E2-treated mice each, and eight ICI-treated mice. Level of significance compared with control: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

 
Exogenous E2 accelerates catagen development in anagen hair follicles
By dotmatrix planimetry, both test and control animals showed the typical wave-like radial spreading of catagen-development at day 16 after hair shaft depilation. Catagen development always began in the neck area and ectopic foci of catagen development did not occur. In E2-treated mice, catagen occurred earlier and the total area of catagen skin was significantly larger. This was still evident on day 18 after depilation, compared with ICI 182.780 or vehicle-treated control animals (P < 0.001) (Fig. 4BGo).

This observation was verified by quantitative histomorphometry: on d 18 after depilation, mice treated with E2 showed significantly fewer HFs in early catagen stages and more HFs in late catagen stages than control animals (P < 0.001) (Fig. 5AGo). When the HCS was calculated (50), E2 treatment was found to be associated with a significantly higher score (P < 0.001) than the control and the ICI-treated group. Morphological abnormalities were not seen (Fig. 5BGo). Taken together, this provides definitive proof that topical E2 is a potent inducer of apoptosis-driven, premature HF regression (catagen development) in anagen VI mouse back skin pelage HFs in vivo.



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  		FIG. 5. Histomorphometry of catagen development on d 18 post depilation. The graph shows three different groups of catagen follicles to better visualize the mean cycling activity of the already classified catagen follicles. Early catagen (catagen I-III), midcatagen (catagen IV+V), and late catagen (catagen VI-VIII). Given are the means ± SEM of pooled data from two experiments, representing a total of 16 vehicle- and E2-treated animals and eight ICI-treated animals. A minimum of 50 back skin HFs was analyzed for each test and control mouse. Level of significance compared with control: **, P ≤ 0.01; ***, P ≤ 0.001. B, HCS on d 18 post depilation. The numbers indicate the HCSs of vehicle-, E2-, or ICI-treated regressing back skin. Higher HCSs indicate progression of catagen. E2 treatment lead to a significant 2-fold progression in the HCS (~200), whereas the vehicle- and ICI-treated skin show nearly identical scores around 120. These data further confirm the potent catagen-enhancing properties of E2. Given are the means +1 SEM. Level of significance: **, P ≤ 0.01

 
Catagen development is accelerated in ERß-deficient mice
To study whether deletion of ERß alters spontaneous catagen development, we examined BERKO mice and their wild-type (+/+) littermates during initiation of the first hair cycle. After the completion of HF morphogenesis, the HF begins its lifelong cycling activity by entering spontaneously into the first catagen stage between postnatal d 17 and 19 (29, 54, 59).

Comparing the hair cycle stages of BERKO and age-matched wild-type animals by quantitative histomorphometry, a significant difference became evident (P < 0.05): about 85% of the HFs of BERKO mice were already in late catagen (VII-VIII), whereas only 40% of the wild-type animals showed HFs in these stages (Fig. 6AGo). In addition, dermal thickness was significantly lower (P < 0.001) in BERKO, compared with wild-type, mice (Fig. 6BGo). Because dermal thickness is strictly coupled to synchronized HF cycling and because catagen skin is much thinner than anagen skin, (49, 53, 54), this serves as an independent, confirmatory marker, which documents that BERKO mice show a much faster anagen-catagen-telogen transition of HF at this time point.



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  		FIG. 6. Histomorphometry of spontaneous catagen development on d 19 after birth in BERKO and wt mice. The graph shows percentages of HFs in different hair cycle stages in the back skin of BERKO mice and the age matched wild-type animals (n = 5/group) at d 19 after birth. x-axis, Hair cycle stages; y-axis, percent of follicles (mean +1 SEM). Fifty follicles per mouse were counted and hair cycle stages were assessed. Follicles of BERKO mice were predominantly in late catagen (85%), whereas there were no follicles in early catagen or anagen at p19. The difference proved to be significant, compared with wild-type animals, in which almost as many follicles were in midcatagen as in late catagen. In wild-type animals, 8% of the follicles were still in early catagen stage. Level of significance compared with control: *, P ≤ 0.05. B, Skin thickness of back skin (neck region) from BERKO and wild-type mice. For investigation of catagen development, skin thickness of ERß knockout and wild-type animals has been compared. Thickness was measured in the neck region between the basement membrane of the epidermis and distal margin of the panniculus carnosus muscle in murine subcutis. The graph shows mean values of skin thickness from the neck region (+1 SEM); 85–117 measurements were performed per group (n = 5 animals/group). At d 19 post partum, skin of BERKO mice had been significantly reduced, compared with wild-type animals, confirming a faster catagen progression in BERKO animals. Level of significance compared with control: ***, P ≤ 0.001. C, Apoptosis within hair matrix keratinocytes of BERKO and wt littermates on d 19 after birth. Hair bulb matrix keratinocytes in BERKO mouse back skin contain more apoptotic cells than wild-type follicles at the same time point. Quantitative analysis of TUNEL+ cells shows an increase in HFs of BERKO mice, compared with wild-type littermates. The difference between the two groups (n = 5 / group) proved to be significant. The graph shows mean values (+1 SEM). Level of significance compared with control: *** = P ≤ 0.001.

 
Finally, we examined the differences in apoptosis during catagen development between these mice. Performing TUNEL staining, we found that there were much more TUNEL+, apoptotic nuclei in the hair follicles of BERKO compared with wild-type mice. For statistical analysis, we counted TUNEL+ keratinocytes in the HF matrix of the different phenotypes and detected significantly more apoptosis in BERKO mice (P < 0.001) (Fig. 6CGo). This further confirms a substantially accelerated apoptosis-driven HF regression in the absence of functional ERß.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data show that all recognized ER subtypes ({alpha}, ß, and ß ins) are detectable at the protein and mRNA level during the murine HF cycle in a hair cycle-dependent manner. We confirm that E2 arrests murine pelage HFs in telogen and show that E2 is a potent inducer of premature catagen development. On the other hand, the ER antagonist does not induce premature initiation of anagen but is able to accelerate anagen development and anagen wave propagation in female mice. In addition, BERKO mice display an accelerated catagen development along with an increase in the number of apoptotic HF keratinocytes.

Previously it has been reported that only ER{alpha} is expressed in murine skin, whereas, probably due to the difficulty of obtaining suitable and specific antibodies at this time, ERß was reported to be undetectable (18, 20). In the present study, we show that both ERs, ER{alpha} and ERß, are expressed at the protein and RNA levels within murine skin throughout the entire depilation-induced murine HF cycle (see Figs. 2Go, a and b, and 3Go).

Furthermore, we demonstrate that the ERß splice variant ERß ins (18aa), which has an alternate last exon, stains more weakly, but is coexpressed with ER{alpha} and wt ERß (503) in the skin (Fig. 2Go). ERß ins does not bind E2 and has no capacity to activate transcription of an E2-sensitive reporter gene. If coexpressed with ER wt receptor, ERß ins has been reported to act as a negative regulator of ER-dependent pathways by heterodimerizing more frequently with ER{alpha} than ERß (42, 57). The coexpression of these ER subtypes within murine skin (Fig. 2Go) suggests that intrinsic counterregulatory controls of ER-mediated signaling are installed in and around the murine hair follicle.

So far, there is only indirect evidence for the significance of endogenous E2 and ER signaling in hair growth control under physiological and pathological circumstances (28, 32). Also, it is still unknown how ER expression is controlled and which are the key target genes for ER signaling in skin (39). For example, by inhibition of the synthesis of 5{alpha}-dihydrotestosterone, E2 may modulate hair growth by interfering with androgen metabolism (62).

Our data (Figs. 4Go and 5Go) support the concept that there is an ER-dependent pathway that contributes to the control of HF cycling and confirm that E2 blocks murine hair growth and arrests HFs in the telogen phase (Fig. 4Go) (18, 19). In contrast to these previous reports (18, 19), we did not observe that the ER antagonist ICI 182.780 induces an earlier onset of anagen when more refined and sensitive quantitative macroscopic (dotmatrix planimetry) and microscopic (quantitative histomorphometry) techniques for assessing hair follicle cycling are employed. However, using these techniques, we show that treatment with the ER antagonist accelerates anagen development and anagen wave propagation, once anagen has been initiated, whereas anagen development is retarded by ER agonists (E2) (Figs. 4Go and 5Go).

Most recently it has been postulated that only ER{alpha}, not ERß, is involved in the regulation of anagen development in orchidectomized mice (21). ER{alpha} expression is hair cycle dependent (with an expression maximum in telogen skin), whereas ERß expression is more constant throughout the HF cycle (Fig. 2Go). Therefore, our results are consistent with the concept that stimulation of ER{alpha}, not ERß, functions as a molecular hair cycle brake in mice. Reportedly, the presence of ER{alpha} is indicative for nonproliferating cells in the rodent mammary gland because ER{alpha} is not colocalized in nuclei with proliferation markers (46). Because telogen is the hair cycle stage of relative quiescence (49), this concept may also apply to the HF.

The key hair cycle finding in the current study is that E2-treated murine skin displays a significantly accelerated catagen wave progression (Fig. 5Go). Consequently, E2-treated mice had significantly more back skin HFs in catagen than controls or ICI 182.780-treated animals, suggesting a direct catagen-inductive activity of E2. This is in line with our previous finding that E2 promotes the so-called dystrophic catagen pathway of HFs to chemical damage, using cyclophosphamide-induced alopecia in mice as a model (28). However, application of the ER antagonist ICI 182.780 had no decelerating effect on catagen development. The present model offers an excellent research tool for further dissecting the complex, ill understood mechanisms of hair wave formation (63).

Catagen progression is accelerated in mice lacking ERß (Fig. 6Go), and BERKO mice show significant up-regulation of apoptotic HF keratinocytes as well as a significantly reduced dermal thickness. This supports the concept that, as in other organs, e.g. the uterus and mammary gland (43, 57), ERß, (including ERß ins), functions as a quencher of ER{alpha}-mediated effects. The absence of ER{alpha} antagonistic signaling via ERß in BERKO mice would explain uninhibited and thus accelerated catagen induction via ER{alpha}.

ERß stimulation by antiestrogens may mediate antioxidant actions on activator protein-1 sites by inducing quinone reductase (64), and the antioxidative stress enzymes glutathione S-transferase-{pi} and {gamma}-glutamylcysteine synthase are up-regulated by transcriptional activity of ERß (65). Therefore, one role of ERß may be to protect tissues from oxidative stress by inducing a battery of antioxidative enzymes (64, 65). Thus, the widespread and constant expression of ERß within the pilosebaceous unit may also serve to protect the skin and its appendages, which are continuously more exposed to oxidative damage than most other organs.

If reproducible in the human system, the observations reported here suggest that topically administered ER modulators may be clinically exploited as powerful catagen inducers (e.g. for treating hirsutism). The growing number of selective ER modulators (66, 67) and our improved understanding of ER signaling (39) make it increasingly attractive to explore the use of estrogens or antiestrogens in the modulation of hair growth. Today topical E2 administration is traditionally employed in the treatment of female pattern androgenetic alopecia in many countries. The limited trichogram evidence that is currently available suggests that, in androgen-sensitive areas of female scalp, topical E2 decreases the telogen rate and prolongs the anagen phase of human scalp hair follicles (17, 68, 69). Also, E2 inhibits hair shaft elongation in human occipital scalp hair follicles in vitro (32, 33, 70, 71). However, E2 effects on human frontotemporal scalp HFs show significant differences between the sexes (stimulation of hair shaft elongation in males and inhibition in females) (32). Therefore, not only species-, but also sex-, and location-dependent differences in the hair follicle response to estrogens must be taken into account (32).


   Acknowledgments
 
The authors are grateful to Ruth Pliet, Monika Dietrich, Silvia Wegerich, Gundula Pillnitz-Stolze, and Patricia Humire for excellent technical assistance. We thank Margaret Warner for scientific advice.


   Footnotes
 
This work was supported in part by grants from Cutech Srl., Venice, Italy, and Deutsche Forschungsgemeinschaft (Pa 345/8–4) (to R.P.) and grants from the Swedish Cancer Fund and KaroBio AB.

First Published Online December 9, 2004

Abbreviations: 18aa, 18-Amino acid insert in the ligand-binding domain of the isoform ERß ins; BERKO, ERß knockout mouse; DAB, diaminobenzidine; DAPI, 4', 6-diamino-2-phenylindole; DP, dermal papilla; E2, 17ß-estradiol; ER, estrogen receptor; ERKO, ER{alpha} knockout mouse; HCS, hair cycle score; HF, hair follicle; ICI 182.780, Imperial Chemical Industries compound 182.780, a selective ER antagonist; IR, immunoreactivity; TSA, tyramide signal amplification; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; wt, wild-type.

Received September 14, 2004.

Accepted for publication December 3, 2004.


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