0013-7227/05/$15.00/0 Printed in U.S.A.

Endocrinology 146(3):1214 1225 Copyright 2005 by The Endocrine Society doi: 10.1210/en.2004-1219

Hair Cycle Control by Estrogens: Catagen Induction via Estrogen Receptor (ER)- Is Checked by ER Signaling
Ulrich Ohnemus, Murat Uenalan, Franziska Conrad, Bori Handjiski, Lars Mecklenburg, Motonobu Nakamura, Jose Inzunza, Jan-ke 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, Charite , 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
Although 17-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 and ER, only ER had been detected in murine skin. Here we show that ER, ER, and ER 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. ER 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, ER does indeed play a significant role in murine hair growth control: whereas the catagen-promoting properties of E2 are mediated via ER, ER mainly may function as a silencer of ER 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 or - 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. (Endocrinology 146: 1214 1225, 2005)

OR DECADES, STEROID hormones, such as androgens, retinoids, thyroid hormones, calcitriols, and glucocorticosteroids, have been known to act as potent hair growth modulators (111). Whereas the profound hair growthmodulatory effects of 17-estradiol (E2) had long ago been demonstrated in many mammalian species (1217), 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 20). Orchidectomy of male ER knockout mice (ERKO) inFirst Published Online December 9, 2004 Abbreviations: 18aa, 18-Amino acid insert in the ligand-binding domain of the isoform ER ins; BERKO, ER knockout mouse; DAB, diaminobenzidine; DAPI, 4, 6-diamino-2-phenylindole; DP, dermal papilla; E2, 17-estradiol; ER, estrogen receptor; ERKO, ER 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. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

duces a synchronized anagen phase of HFs, which is inhibited by E2 treatment in wild-type and ER 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 stimulation. An ER-mediated pathway of hair growth control in mice conforms with previous reports that only ER expression is detectable in murine telogen and early anagen skin, whereas the second receptor, ER, was detectable only in human skin (18, 20, 2227). 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 (1216, 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

1214

Ohnemus et al. Hair Cycle Control by Estrogens

Endocrinology, March 2005, 146(3):1214 1225

1215

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 and ER (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, 3739). For example, both ER and ER activate transcription on classical estrogen response elements. However, in the presence of E2, ER is an activator, whereas ER is an inhibitor or silencer at activating protein-1 sites (40). Also, ERs transactivation potency is down-regulated by wild-type (wt) ER in an E2 dose-dependent manner (41). In addition, one ER splice variant, called ER ins, which has an additional 18amino acid in-frame sequence between exons 5 and 6 of wt ER, may act as a dominant-negative regulator of ER, independent of the E2 concentration (42, 43). In the uterus, E2 induces epithelial cell proliferation indirectly via the stimulation of stromal ER, which leads to growth factor secretion by stromal cells, thus making the presence of ER on epithelial cells dispensable (44, 45). In the mammary gland, a derivative of the epidermis, ER 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 in the epithelium (46). More information about the exact expression patterns of ER, ER, and ER 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, ER, and the ER variant, ER 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 and ER 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 bestestablished mouse model for hair research (C57BL/6) (13, 49, 50). Based on the observation that other steroid hormone receptor ligands, which inhibit anagen (glucocorticoids, calcitriols), also induce premature catagen (1 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. 1). 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 (5254). 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. 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 (, , and ins) 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 wildtype mice.
Materials and Methods Animals
For in vivo and immunohistochemical staining experiments, 6- to 9-wk-old syngenic female C57BL/6J mice (1520 g) were purchased from Charles River (Sulzfeld, Germany) and housed in community cages with 12-h light periods at Charite 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 ER 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 rabbit polyclonal antibody, MC-20, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Chicken anti-ER antibody 503 (entire ER molecule) and chicken antiER antibody ins (18-amino acid insert in the ligand-binding domain of the isoform ER ins (18aa), were generated in the laboratory of J.-. Gustafsson as described elsewhere (Departments of Medical Nutrition and Biosciences, Karolinska Institute, NOVUM, Stockholm, Sweden) (57). Peroxidase-conjugated secondary antibodies and goat antirabbit

1216

Endocrinology, March 2005, 146(3):1214 1225

Ohnemus et al. Hair Cycle Control by Estrogens

IgG were from Jackson ImmunoResearch (West Grove, PA). Peroxidaseconjugated 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 staining and mouse ovary for ER staining. Sections from BERKO mice served as negative control for ER staining; incubation of cryosections with preimmune rabbit serum (AgResearch) served as negative control for ER staining. Mouse uterus showed strong immunoreactivity (IR) for ER; mouse ovary was positive for ER and ER ins. The negative controls did not exert any IR for ER , and ins (data not shown). Cryosections (8 m) 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 (1:2000), ER 503 (1:1000), and ER 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, ER, and ER 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 (1:8,000), ER 503 (1:10,000), and ER 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-2phenylindole (DAPI) solution (0.1 g/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 and ER (503 and ins) was measured at two previously defined reference areas in the follicular dermal papilla (see Fig. 2C) 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).

CAC TTT CCT TT-3 (58); -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 l cDNA, 10 l 10 PCR buffer, 8 l deoxynucleotide triphosphate mixture (2.5 mm each), 1 l forward primer (50 pmol), 1 l reverse primer (50 pmol), 0.5 l TaKaRa Ex Taq polymerase (Takara, Otsu, Japan), and 79.5 l water. Amplification was performed over 32 cycles for -actin, 35 cycles for ER, and 35 cycles for ER. 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 traumainduced anagen. Mice received 10 nmol of the test substances in 200 l 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 l 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).

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 g 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 and mouse ovary for ER. The resulting cDNA was stored at 80 C until further use. Primers used are listed as follows: ER, forward, 5-AAT TCT GAC AAT CGA CGC CAG-3, reverse, 5-GTG CTT CAA CAT TCT CCC TCC TC-3; ER, forward, 5-CTT GGT CAC GTA CCC CTT AC-3, reverse, 5-GTA TCG CGT

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 (5254). The procedure is illustrated in Fig. 1.

Histology and microscopic analysis by quantitative histomorphometry

Hair cycle stages were classified according to Mu ller-Ro ver et al. (54) by the means of quantitative histomorphometry. A hair cycle score

Ohnemus et al. Hair Cycle Control by Estrogens

Endocrinology, March 2005, 146(3):1214 1225

1217

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). (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-m 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, 85117 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 MannWhitney U test for unpaired samples combined with Monte-Carlo calculation for exactness. Differences were judged as significant if P 0.05.

2a, A). In Fig. 2a, A and B, ER IR in anagen VI is demonstrated with the more sensitive TSA staining technique. ER 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. 2a, C and D), whereas ER ins IR was weaker but coexpressed with ER 503 (Fig. 2a, E and F). In early catagen, weak ER IR was seen in the dermal papilla, matrix keratinocytes, inner and outer root sheath, and the connective tissue sheath; no ER IR was seen in the epidermis, bulge region, and sebaceous gland. In late catagen, ER IR was restricted to the dermal papilla, secondary hair germ, outer root sheath, bulge region, and the sebaceous gland; (Fig. 2a, G and H and M and N). ER 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. 2a, I and J and O and P). ER ins IR was again weaker but coexpressed with ER 503 (Fig. 2a, K and L and Q and R). During the anagen-catagen transformation of hair follicles and their entry into the telogen stage, ER 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 positivity (Fig. 2a, S and T). ER (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. ER ins positivity in sebocytes was of a similar degree as ER (Fig. 2a, U and V and W and X). ER IR intensity was quantified throughout the investigated stages by NIH image (Fig. 2c). Within the follicular dermal papilla we found hair cycle-dependent differences of ER expression. Mean fluorescence intensity of ER was highest in telogen follicles and lowest in early catagen. There were no significant differences of IR intensity of ER (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 mRNA levels peak in telogen skin

Results ER, ER, and ER ins IR is present in normal mouse skin throughout the murine HF cycle

First, we studied IR for ER and ER (whole ER 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, ER 503, and ER ins IR changed during the different investigated stages of the murine HF cycle [telogen, anagen (VI), early catagen (II-III), and late catagen (VIVIII)] (Fig. 2). In actively growing HFs (anagen VI), ER 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 IR (Fig.

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 depilationinduced 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 -actin, ER 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 mRNA steady-state levels was demonstrated at d 12. During catagen (d 19), the levels increased further (Fig. 3). ER transcripts were also detected within murine skin, but by semiquantitative RT-PCR, ER mRNA levels were relatively constant throughout the whole hair cycle (Fig. 3). These findings confirm that ER and ER are expressed in

1218

Endocrinology, March 2005, 146(3):1214 1225

Ohnemus et al. Hair Cycle Control by Estrogens

FIG. 2. ER, ER, and ER ins expression patterns detected by immunohistochemistry. a, Immunoreactivity patterns of ER, ER, and ER 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)

Ohnemus et al. Hair Cycle Control by Estrogens

Endocrinology, March 2005, 146(3):1214 1225

1219

FIG. 2. (continued) and the more sensitive TSA technique (B, D, F, H, J, L, N, P, R, T, V, X). In the ABC-stained photos, ER- and ER-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- and ER-positive cells. ER 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 IR in the DP, matrix keratinocytes (MK), and inner-root sheath (IRS) and ORS, whereas the epidermis, SG, and bulge region showed no ER IR. In early catagen ER IR was seen in the DP, matrix keratinocytes (MK), IRS, ORS, and connective tissue sheath (CTS). In late catagen ER IR was restricted to the DP, GC, ORS, bulge, and SG. ER 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 ER in anagen VI. ER ins IR was weaker but coexpressed throughout the whole cycle and in same locations like ER 503. b, Schematic representation of IR patterns (based on TSA stainings). Gray markings: ER, ER, and ER ins expression. c, ER, ER, and ER ins staining intensity detected by NIH image. The expression of ER within the follicular DP was significantly hair cycle dependent. Mean fluorescence intensity of ER was highest in telogen follicles and lowest in early catagen. There were no significant differences of IR-intensity of ER (503 and ins) within the DP during the HF cycle. Based on TSA staining, the fluorescent staining intensity of ER and ER 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.

1220

Endocrinology, March 2005, 146(3):1214 1225

Ohnemus et al. Hair Cycle Control by Estrogens

FIG. 3. RT-PCR analysis of ER and ER. Left side, Semiquantitative RTPCR analysis of expression of ER, ER, and -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 actin mRNA. Error bars, 1 SEM.

murine skin, both on the protein and gene level, and that at least ER 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. 4A, 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.
Exogenous E2 accelerates catagen development in anagen hair follicles

depilation, compared with ICI 182.780 or vehicle-treated control animals (P 0.001) (Fig. 4B). 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. 5A). 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. 5B). 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.
Catagen development is accelerated in ER-deficient mice

By dotmatrix planimetry, both test and control animals showed the typical wave-like radial spreading of catagendevelopment 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

To study whether deletion of ER alters spontaneous catagen development, we examined BERKO mice and their wildtype (/) 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 agematched 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. 6A). In addition, dermal thickness was significantly lower (P 0.001) in BERKO, compared with wild-type, mice (Fig. 6B). 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. 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

Ohnemus et al. Hair Cycle Control by Estrogens

Endocrinology, March 2005, 146(3):1214 1225

1221

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 182.780treated 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 ICItreated mice. Level of significance compared with control: *, P 0.05; **, P 0.01; ***, P 0.001.

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. 6C). This further confirms a substantially accelerated apoptosis-driven HF regression in the absence of functional ER.
Discussion

Our data show that all recognized ER subtypes (, , and 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 is expressed in murine skin, whereas, probably due to the difficulty of obtaining suitable and specific antibodies at this time, ER was reported to be undetectable (18, 20). In the present study, we show that both ERs, ER and ER, are expressed at the protein and RNA levels within murine skin throughout the entire depilation-induced murine HF cycle (see Figs. 2, a and b, and 3). Furthermore, we demonstrate that the ER splice variant

1222

Endocrinology, March 2005, 146(3):1214 1225

Ohnemus et al. Hair Cycle Control by Estrogens

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 IVV), 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

ER ins (18aa), which has an alternate last exon, stains more weakly, but is coexpressed with ER and wt ER (503) in the skin (Fig. 2). ER ins does not bind E2 and has no capacity to activate transcription of an E2-sensitive reporter gene. If coexpressed with ER wt receptor, ER ins has been reported to act as a negative regulator of ER-dependent pathways by heterodimerizing more frequently with ER than ER (42, 57). The coexpression of these ER subtypes within murine skin (Fig. 2) 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-dihydrotestosterone, E2 may modulate hair growth by interfering with androgen metabolism (62). Our data (Figs. 4 and 5) 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. 4) (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. 4 and 5). Most recently it has been postulated that only ER, not ER, is involved in the regulation of anagen development in orchidectomized mice (21). ER expression is hair cycle dependent (with an expression maximum in telogen skin), whereas ER expression is more constant throughout the HF cycle (Fig. 2). Therefore, our results are consistent with the concept that stimulation of ER, not ER, functions as a molecular hair cycle brake in mice. Reportedly, the presence of ER is indicative for nonproliferating cells in the rodent mammary gland because ER 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. 5). 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 ER (Fig. 6), 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), ER, (including ER ins), functions as a quencher of ER-mediated effects. The absence of ER antagonistic signaling via ER in BERKO mice would explain uninhibited and thus accelerated catagen induction via ER. ER 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- and -glutamylcysteine synthase are

Ohnemus et al. Hair Cycle Control by Estrogens

Endocrinology, March 2005, 146(3):1214 1225

1223

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 ER 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); 85117 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.

up-regulated by transcriptional activity of ER (65). Therefore, one role of ER may be to protect tissues from oxidative stress by inducing a battery of antioxidative enzymes (64, 65). Thus, the widespread and constant expression of ER 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 in-

1224

Endocrinology, March 2005, 146(3):1214 1225

Ohnemus et al. Hair Cycle Control by Estrogens

ducers (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. Received September 14, 2004. Accepted December 3, 2004. 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@ uke.uni-hamburg.de. 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.

References
1. Paus R, Handjiski B, Czarnetzki BM, Eichmu ller S 1994 A murine model for inducing and manipulating hair follicle regression (catagen): effects of dexamethasone and cyclosporin A. J Invest Dermatol 103:143147 2. Paus R, Handjiski B, Eichmuller S, Czarnetzki B 1994 Chemotherapyinduced alopecia in mice. Induction by cyclophosphamide, inhibition by cyclosporine A, and modulation by dexamethasone. Am J Pathol 144:719 734 3. Paus R, Schilli MB, Handjiski B, Menrad A, Henz BM, Plonka P 1996 Topical calcitriol enhances normal hair regrowth but does not prevent chemotherapyinduced alopecia in mice. Cancer Res 56:4438 4443 4. Stenn KS, Paus R, Dutton T, Sarba B 1993 Glucocorticoid effect on hair growth initiation: a reconsideration. Skin Pharmacol 6:125134 5. Randall VA, Thornton MJ, Hamada K, Redfern CP, Nutbrown M, Ebling FJ, Messenger AG 1991 Androgens and the hair follicle. Cultured human dermal papilla cells as a model system. Ann NY Acad Sci 642:355375 6. Ebling FJ, Hale PA, Randall VA 1991 Hormones and hair growth. In: Goldsmith LA, ed. Physiology, biochemistry and molecular biology of the skin. 2nd ed. New York: Oxford University Press 7. Billoni N, Gautier B, Mahe YF, Bernard BA 1997 Expression of retinoid nuclear receptor superfamily members in human hair follicles and its implication in hair growth. Acta Derm Venereol 77:350 355 8. Alonso LC, Rosenfield RL 2003 Molecular genetic and endocrine mechanisms of hair growth. Horm Res 60:113 9. Itami S, Kurata S, Sonoda T, Takayasu S 1995 Interaction between dermal papilla cells and follicular epithelial cells in vitro: effect of androgen. Br J Dermatol 132:527532 10. Safer JD, Fraser LM, Ray S, Holick MF 2001 Topical triiodothyronine stimulates epidermal proliferation, dermal thickening, and hair growth in mice and rats. Thyroid 11:717724 11. Chen W, Thiboutot D, Zouboulis CC 2002 Cutaneous androgen metabolism: basic research and clinical perspectives. J Invest Dermatol 119:9921007 12. Fraser AS, Nay T 1953 Growth of the mouse coat II. Effects of sex and pregnancy. Aust J Biol Sci 6:645 656 13. Emmens CW 1942 The endocrine system and hair growth in the rat. J Endocrinol 3:64 78 14. Whitaker WL 1958 Local inhibition of hair growth in the guinea pig by the topical application of Estradiol. Anat Rec 124:447 448

15. Jackson D, Ebling FJ 1972 The activity of hair follicles and their response to estradiol in the guinea-pig Cavia porcellus L. J Anat 111:303316 16. Gardner WK, De Vita J 1940 Inhibition of hair growth in dogs receiving estrogens. Yale J Biol And Med 13:213216 17. Schumacher-Stock U 1981 Estrogen treatment of hair diseases. In: Orfanos CE, Montagna M, Stuttgen G, eds. Hair research. Berlin/Heidelberg: SpringerVerlag; 318 321 18. Oh HS, Smart RC 1996 An estrogen receptor pathway regulates the telogenanagen hair follicle transition and influences epidermal cell proliferation. Proc Natl Acad Sci USA 93:1252512530 19. Smart RC, Oh HS, Chanda S, Robinette CL 1999 Effects of 17-estradiol and ICI 182 780 on hair growth in various strains of mice. J Investig Dermatol Symp Proc 4:285289 20. Chanda S, Robinette CL, Couse JF, Smart RC 2000 17-estradiol and ICI182780 regulate the hair follicle cycle in mice through an estrogen receptor- pathway. Am J Physiol Endocrinol Metab 278:E202E210 21. Move rare S, Lindberg MK, Faergemann J, Gustafsson J-, Ohlsson C 2002 Estrogen receptor , but not estrogen receptor , is involved in the regulation of the hair follicle cycling as well as the thickness of epidermis in male mice. J Invest Dermatol 119:10531058 22. Uzuka M, Nakajima K, Mori Y 1978 Estrogen receptor in the mouse skin. Biochim Biophys Acta 544:329 337 23. Stumpf WE, Sar M, Joshi SG 1974 Estrogen target cells in the skin. Experientia 30:196 198 24. Hasselquist MB, Goldberg N, Schroeter A, Spelsberg TC 1980 Isolation and characterization of the estrogen receptor in human skin. J Clin Endocrinol Metab 50:76 82 25. Punnonen R, Lovgren T, Kouvonen I 1980 Demonstration of estrogen receptors in the skin. J Endocrinol Invest 3:217221 26. Brandenberger AW, Tee MK, Lee JY, Chao V, Jaffe RB 1997 Tissue distribution of estrogen receptors (ER-) and (ER-) mRNA in the midgestational human fetus. J Clin Endocrinol Metab 82:3509 3512 27. Thornton MJ, Taylor AH, Mulligan K, Al-Azzawi F, Lyon CC, ODriscoll J, Messenger AG 2003 Oestrogen receptor is the predominant oestrogen receptor in human scalp skin. Exp Dermatol 12:181190 28. Ohnemus U, Uenalan M, Handjiski B, Paus R 2004 Topical estrogen accelerates hair regrowth in mice after chemotherapy-induced alopecia by favoring the dystrophic catagen response pathway to damage. J Invest Dermatol 122: 713 29. Paus R 1996 Control of the hair cycle and hair diseases as cycling disorders. Curr Op Dermatol 3:248 258 30. Paus R, Mu ller-Ro ver S, Botchkarev VA 1999 Chronobiology of the hair follicle: hunting the hair cycle clock. J Investig Dermatol Symp Proc 4:338 345 31. Thornton MJ 2002 The biological actions of estrogens on skin. Exp Dermatol 11:487502 32. Conrad F, Ohnemus U, Bodo E, Bettermann A, Paus R 2004 Estrogens and human scalp hair growthstill more questions than answers. J Invest Dermatol 122:840 842 33. Conrad F, Ohnemus U, Gerstmayer B, Bosio A, Betterman A, Bodo E, Paus R 2004 Substantial sex-dependent differences in the response of human scalp hair follicles to estrogen stimulation in vitro advocate gender-tailored management of female versus male pattern balding. JDDG 2:530 (Abstract) 34. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, Enmark E, Pettersson K, Warner M, Gustafsson J- 2001 Mechanisms of estrogen action. Physiol Rev 81:15351565 35. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J- 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:59255930 36. Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V 1997 Cloning, chromosomal localization and functional analysis of the murine estrogen receptor . Mol Endocrinol 11:353365 37. Collins P, Webb C 1999 Estrogen hits the surface. Nat Med 5:1130 1131 38. Nilsson S, Gustafsson J 2002 Estrogen receptor action. Crit Rev Eukaryot Gene Expr 12:237257 39. Conrad F, Paus R 2004 Estrogens and the hair follicle. JDDG 2:412 423 40. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J-, Kushner PJ, Scanlan TS 1997 Differential ligand activation of estrogen receptors ER and ER at AP 1 sites. Science 277:1508 1510 41. Pettersson K, Delaunay F, Gustafsson J- 2000 Estrogen receptor acts as a dominant regulator of estrogen signaling. Oncogene 19:4970 4978 42. Saji S, Omoto Y, Shimizu C, Warner M, Hayashi Y, Horiguchi S, Watanabe T, Hayashi S, Gustafsson J-, Toi M 2002 Expression of estrogen receptor (ER) ()cx protein in ER ()-positive breast cancer: specific correlation with progesterone receptor. Cancer Res 62:4849 4853 43. Weihua Z, Ekman J, Almkvist A, Saji S, Wang L, Warner M, Gustafsson J- 2002 Involvement of androgen receptor in 17 -estradiol-induced proliferation in rat uterus. Biol Reprod 67:616 623 44. Weihua Z, Saji S, Makinen S, Cheng G, Jensen EV, Warner M, Gustafsson J- 2000 Estrogen receptor (ER) , a modulator of ER in the uterus. Proc Natl Acad Sci USA 97:5936 5941 45. Cooke PS, Buchanan DL, Young P, Setiawan T, Brody J, Korach KS, Taylor

Ohnemus et al. Hair Cycle Control by Estrogens

Endocrinology, March 2005, 146(3):1214 1225

1225

46. 47. 48. 49. 50.

51. 52. 53. 54.

55.

56.

57. 58.

J, Lubahn DB, Cunha GR 1997 Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium. Proc Natl Acad Sci USA 94:6535 6540 Cheng G, Zhang W, Warner M, Gustafsson J 2004 Estrogen receptors ER and ER in proliferation in the rodent mammary gland. Proc Natl Acad Sci USA 101:3739 3746 Paus R, Cotsarelis G 1999 The biology of hair follicles. N Engl J Med 341: 491 497 Wakeling AE, Dukes M, Bowler J 1991 A potent specific pure antiestrogen with clinical potential. Cancer Res 51:38673873 Chase HB 1954 Growth of the hair. Physiol Rev 34:113126 Maurer M, Handjiski B, Paus R 1997 Hair growth modulation by topical immunophilin ligands: induction of anagen, inhibition of massive catagen development, and relative protection from chemotherapy-induced alopecia. Am J Pathol 150:14331441 Schilli MB, Paus R, Menrad A 1998 Reduction of intrafollicular apoptosis in chemotherapy-induced alopecia by topical calcitriol-analogs. J Invest Dermatol 111:598 604 Slominski A, Paus R 1993 Melanogenesis is coupled to murine anagen: toward new concepts for the role of melanocytes and the regulation of melanogenesis in hair growth. J Invest Dermatol 101:90S97S Paus R, Stenn KS, Link RE 1990 Telogen skin contains an inhibitor of hair growth. Br J Dermatol 122:777784 Mu ller-Ro ver S, Handjiski B, van der Veen C, Eichmu ller S, Foitzik K, McKay IA, Stenn KS, Paus R 2001 A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol 117:315 Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson J-, Smithies O 1998 Generation and reproductive phenotypes of mice lacking estrogen receptor . Proc Natl Acad Sci USA 95:1567715682 Vidal O, Lindberg MK, Hollberg K, Baylink DJ, Andersson G, Lubahn DB, Mohan S, Gustafsson J-, Ohlsson C 2000 Estrogen receptor specificity in the regulation of skeletal growth and maturation in male mice. Proc Natl Acad Sci USA 97:5474 5479 Saji S, Sakaguchi H, Andersson S, Warner M, Gustafsson J- 2001 Quantitative analysis of estrogen receptor proteins in rat mammary gland. Endocrinology 142:31773186 Weihua Z, Andersson S, Cheng G, Simpson ER, Warner M, Gustafsson J- 2003 Update on estrogen signaling. FEBS Lett 546:1724

59. Lindner G, Botchkarev VA, Botchkareva NV, Ling G, van der Veen C, Paus R 1997 Analysis of apoptosis during hair follicle regression (catagen). Am J Pathol 151:16011617 60. Foitzik K, Lindner G, Mu ller-Ro ver S, Maurer M, Botchkareva N, Botchkarev V, Handjiski B, Metz M, Hibino T, Soma T, Dotto GP, Paus R 2000 Control of murine hair follicle regression (catagen) by TGF-1 in vivo. FASEB J 14:752760 61. Paus R, Mu ller-Ro ver S, Van der Veen C, Maurer M, Eichmu ller S, Ling G, Hoffmann U, Foitzik K, Mecklenburg L, Handjiski B 1999 A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis. J Invest Dermatol 113:523532 62. Niiyama S, Happle R, Hoffmann R 2001 Influence of estrogens on the androgen metabolism in different subunits of human hair follicles. Eur J Dermatol 11:195198 63. Plikus M, Chuong CM 2004 Making waves with hair. J Invest Dermatol 122:vii-ix 64. Montano MM, Katzenellenbogen BS 1997 The quinone reductase gene: a unique estrogen receptor-regulated gene that is activated by antiestrogens. Proc Natl Acad Sci USA 94:25812586 65. Montano MM, Deng H, Liu M, Sun X, Singal R 2004 Transcriptional regulation by the estrogen receptor of antioxidative stress enzymes and its functional implications. Oncogene 23:24422453 66. Gustafsson J- 1998 Therapeutic potential of selective estrogen receptor modulators. Curr Opin Chem Biol 2:508 511 67. Turgeon J, McDonnell D, Martin K, Wise P 2004 Hormone therapy: physiological complexity belies therapeutic simplicity. Science 304:1269 1273 68. Barman J, Astore I, Pecoraro V 1969 The normal trichogram of pregnant women. In: Montagna W, Dobson R, eds. Advances in biology of skin. IX. Hair growth. Oxford, UK: Pergamon Press; 203220 69. Wu stner H, Orfanos C 1974 Alopecia androgenetica und ihre lokalbehandlung mit o strogen und corticosteroidhaltigen externa. Z Hautkr 49:879 888 70. Nelson LD, Messenger AG, Karoo R, Thornton JW 2003 17-Estradiol, but not 17-estradiol inhibits human hair growth in whole follicle organ culture. J Invest Dermatol 121:821 (Abstract) 71. Kondo S, Hozumi Y, Aso K 1990 Organ culture of human scalp hair follicles: effect of testosterone and oestrogen on hair growth. Arch Dermatol Res 282:442 445

Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.