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Department of Dermatology (U.O.), University Hospital Hamburg-Eppendorf, University of Hamburg, D-20246 Hamburg, Germany; Department of Paediatric Haematology and Oncology (M.U.), Hannover Medical School, D-30625 Hannover, Germany; Department of Biosciences and Nutrition (J.I., J.-Å.G.), Karolinska Institute, Novum, SE-14186 Stockholm, Sweden; and Department of Dermatology (R.P.), University Hospital Schleswig-Holstein, Campus Lübeck, University of Lübeck, D-23538 Lübeck, Germany
Correspondence: Address all correspondence and reprint requests to: Ralf Paus, M.D., Department of Dermatology, University Hospital Schleswig-Holstein, Campus Lübeck, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. E-mail: ralf.paus{at}derma.uni-luebeck.de
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Abstract |
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 Top Abstract I. IntroductionII. Hair Follicle Biology:...III. Cellular and Molecular...IV. Estrogens in...V. Estrogens in Pilosebaceous...VI. Open Questions and...VII. Conclusions and...References |
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Here, we chart the recent renaissance of estrogen research in hair research; explain why the hair follicle offers an ideal, clinically relevant test system for studying the role of sex steroids, their receptors, and interactions in neuroectodermal-mesodermal interaction systems in general; and illustrate how it can be exploited to identify novel functions and signaling cross talks of ER-mediated signaling. Emphasizing the long-underestimated complexity and species-, gender-, and site-dependence of E2-induced biological effects on the hair follicle, we explore targets for pharmacological intervention in clinically relevant hair cycle manipulation, ranging from androgenetic alopecia and hirsutism via telogen effluvium to chemotherapy-induced alopecia. While defining major open questions, unsolved clinical challenges, and particularly promising research avenues in this area, we argue that the time has come to pay estrogen-mediated signaling the full attention it deserves in future endocrinological therapy of common hair growth disorders.
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I. Introduction |
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TopAbstractI. IntroductionII. Hair Follicle Biology:...III. Cellular and Molecular...IV. Estrogens in...V. Estrogens in Pilosebaceous...VI. Open Questions and...VII. Conclusions and...References |
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).
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and ERß using RT-PCR (18) and immunohistochemistry (19) have reported that ERß is the predominant receptor in human skin, with strong expression in epidermis, dermal fibroblasts, blood vessels, and hair follicle (19), and human keratinocytes are reported to express both ER and ERß, possibly including a membrane ER (20). It took surprisingly many years before these intriguing leads from the literature were picked up and pursued in appropriate models.
A. Why study the role of estrogens in hair biology?
1. Estrogens and estrogen metabolism are at least as important as androgens in male and female hair biology.
Androgens are recognized key regulators of normal human hair growth and the prerequisite for sexual hair and sebaceous gland development (21, 22). However, estrogens also profoundly alter hair growth in practically all mammalian species investigated and operate as key modulators of hair follicle biology by binding to high-affinity cognate receptors (ERs) (19, 23, 24, 25, 26, 27, 28).
After a long period of relative dormancy, estrogens have been rediscovered as hair growth modulators throughout the past decade. This development was stimulated by a seminal paper by Oh and Smart (26) in 1996, who showed that the prototypic ER agonist, E2, after topical application, is a very potent hair growth inhibitor in mice, thus calling our attention to similar effects that had already been reported many decades ago in several mammalian species (1, 2, 3, 4, 5) (Table 1
). This hair growth-inhibitory activity reported in mice strikingly contrasted with the supposedly hair growth-stimulatory topical E2 therapy long practiced in many countries for the treatment of female pattern androgenetic alopecia (29, 30) and the hair loss induced by therapy with aromatase inhibitors, which lower serum and tissue E2 levels (31, 32). This apparent contradiction already suggested that E2 effects on the mammalian hair follicle were likely to be complex, and species dependent.
Besides the fact that patients with clinical hair growth disorders will all probably profit from investigations regarding the molecular pathology of these diseases, there are several reasons to systematically reexplore the role of estrogens in human hair growth control (10, 11, 12, 25, 26, 27, 28, 33): the hair follicle 1) offers a microcosmic, prototypic tissue interaction system (12, 34, 35, 36, 37) that allows one to dissect and manipulate both classical (i.e., ER-mediated) and nonclassical pathways of estrogen signaling under physiological and pathological conditions; 2) invites one to study the cross talk of pleiotropic estrogens with multiple other signaling pathways in complex neuroectodermal-mesodermal interaction systems; and 3) offers novel insights into previously unknown estrogen functions and target genes (see below for details).
Estrogens are able to modify androgen metabolism within distinct subunits of the hair follicle (e.g., in the dermal papilla), diminishing the amount of 5-dihydrotestosterone formed after incubation with testosterone (38). It is not yet known whether this effect is mediated directly by an inhibition of 5-reductase within the hair follicle or indirectly through estrogen-induced increased conversion of testosterone to weaker androgens (38). Because aromatase, the enzyme that converts testosterone to E2 is also found at many of the sites of ER and androgen receptor expression (39), the local balance between E2 and androgen levels may serve to fine-tune E2 and androgen action in their target cells (40). This is further supported by the growing evidence that steroid receptors can cross talk with one another, showing an interdependence of estrogen-, progesterone-, and androgen-receptor signaling pathways (33, 41).
Also, many of the growth and transcription factors, cytokines, and hormones that are currently recognized to control hair growth (21, 34, 35, 36) are themselves modulated by estrogens. Thus, it will be far from easy to clearly distinguish direct from indirect E2 effects on hair growth, even when ER-null mice are used, because nonclassical E2 effects could still alter the expression of many genes that have not previously been shown to have an estrogen response element (ERE) (12).
B. The hair follicle is a prototypic and ideal test system for studying the role of sex steroids and their receptors in neuroectodermal-mesodermal interaction systems
Besides the hair follicle’s most evident function, the production of a hair shaft, it is also an attractive tool for studying basic biological issues such as cellular differentiation and neuroectodermal-mesodermal tissue interactions (34, 35). Hair follicles contain both epithelial and mesenchymal compartments (Fig. 1), which are cyclically remodeled and whose interactions drive hair shaft formation and hair follicle cycling (42). Hair follicle induction and morphogenesis depend on complex bidirectional communication events between the epithelium and the underlying mesenchyme (43). Mature hair follicle mesenchyme is organized in two communicating compartments: the surrounding connective tissue sheath, and the follicular dermal papilla. The character of both these mesenchymal regions changes dramatically over the growth cycle (44). The dermal papilla is an inductive mesodermal structure that sends and receives morphogenic signals (45, 46). Its activity depends on continuous and intimate interaction with the hair matrix epithelium via native extracellular matrix (47). Anagen dermal papilla dissected free of the epithelial follicle components and inserted into non-hair-bearing skin has been shown to induce hair follicle formation from the resident epithelium (48).
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The growth and regression phases of the hair follicle are modulated by a broad spectrum of hormones such as gonadal, thyroid, adrenal cortical, pituitary, and pineal hormones (35, 51, 52, 53, 54). Because the pilosebaceous unit (i.e., hair follicle, sebaceous gland, and arrector pili muscle) (Figs. 1 and 2) expresses all enzymes to generate androgens as well as estrogens and is able to convert testosterone to estrogen (35, 55, 56, 57, 58), it must not only be viewed as a recipient of signals from distant transmitters but rather as an organized community in which the cells emit, receive, and coordinate molecular signals from a seemingly unlimited number of distant sources including established endocrine organs (modern and classical endocrine functions), neighboring tissues (paracrine and juxtacrine functions), and the pilosebaceous unit itself (autocrine and intracrine functions) (35, 54, 59).
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); they can repopulate both the hair follicle and the interfollicular epidermis (60). Bulge cells are slowly cycling and are quiescent until they receive a signal to leave their niche and begin dividing and differentiating to support a new anagen or to repopulate a skin defect (61, 62). Each follicle develops from a single layer of ectoderm into a complex miniorgan, constituting a dynamic structure that shares developmental pathways with many ectodermally derived endocrine organs like the pituitary, adrenal gland, and pancreas (35, 37, 52, 61). Hair follicle mesenchyme is placed in two intimately communicating compartments that engage in intercompartmental fibroblast trafficking: the connective tissue sheath and the follicular dermal papilla (63). The dermal papilla is separated from the proximal follicle in telogen but is embraced by the lower follicle matrix or bulb portion of the follicle during anagen (34). Morphological changes of the dermal papilla over the cycle primarily reflect changes in its extracellular matrix: in anagen, it is rich in mucins; in catagen, the glycosaminoglycan content is decreased; and in telogen, its mucin content is scant (34, 64).
The hair follicle cycle is associated with a dramatically altered cutaneous blood vessel supply. It has been shown that in species with a synchronized hair cycle, anagen development is accompanied by an increase in skin perfusion due to a rearrangement of the skin vasculature and a genuine, substantial angiogenesis (65). Therefore, the hair follicle represents an unusually attractive model for studying how physiological angiogenesis is controlled by a complex epidermal-mesenchymal interacting system in vivo. We still do not know exactly what cellular or molecular mechanisms control these vascular changes. Besides the two major recognized angiogenesis stimulators, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) (65, 66, 67), processes of angiogenesis can generally be modulated by hormonal changes, including changes in estrogen levels (68). In fact, E2 reportedly even stimulates human hair follicle synthesis of VEGF (69).
After a period of epithelial proliferation and differentiation (anagen), follicle growth stops, and catagen begins (Fig. 3). The unknown signals that drive these changes are either inherent in or delivered to the follicle. Catagen is a highly controlled process of coordinated cell differentiation and apoptosis, involving the cessation of cell growth and pigmentation, release of the papilla from the bulb, loss of the layered differentiation of the lower follicle, substantial extracellular matrix remodeling, and vectorial shrinkage (distally) of the inferior follicle by the process of apoptosis (34, 70). Pigmentation is strictly coupled to anagen III-VI, and many factors constituting the driving forces can also operate as regulators of hair follicle cycling (71, 72). Follicular melanin synthesis and pigment transfer to bulb keratinocytes are modified by hormonal signals (72).
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D. Estrogen-related hair research is clinically, psychologically, and commercially highly relevant
Although hair growth disorders like hair loss and hirsutism are often trivialized, they can profoundly affect a patient’s quality of life (82). This is evident in women with androgenetic alopecia, who often report that the onset of hair loss is associated with considerable anxiety and feelings of diminished attractiveness and helplessness, leading to social withdrawal (82). A small minority of patients may even display dysmorphophobia (83, 84). In addition, chemotherapy-induced alopecia remains one of the most serious unsolved problems in clinical oncology and has a psychologically disastrous impact on affected patients and their social environment, for which a truly satisfactory remedy remains to be developed in clinical practice (85, 86).
A multibillion dollar industry worldwide caters to the unmet needs in managing unwanted hair loss (alopecia, effluvium) and unwanted hair growth (hirsutism, hypertrichosis) (87), advertising allegedly hair growth-stimulating products or procedures such as vitamins, trace elements, exotic herbs, amino acids, etc., which typically have not been subjected to professionally designed and executed clinical trials (88). Although topical formulations containing either 17ß- or 17-estradiol have long been successfully employed for the treatment of androgenetic alopecia, where they appear to improve the telogen/anagen ratio of scalp hair follicles, this critique also applies here. In addition, phytoestrogen-containing preparations are increasingly and aggressively advertised as hair growth-promoting agents, despite the absence of sound clinical data to support such claims.
In any case, however, there are solid clinical, psychological, and economic reasons to dissect how, when, and why estrogens modulate human hair follicle growth in defined hair follicle populations and skin regions.
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II. Hair Follicle Biology: Relevant Key Facts |
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TopAbstractI. IntroductionII. Hair Follicle Biology:...III. Cellular and Molecular...IV. Estrogens in...V. Estrogens in Pilosebaceous...VI. Open Questions and...VII. Conclusions and...References |
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) (34, 36, 37). The hair follicle is the only organ that undergoes a lifelong cyclic transformation, characterized by three distinct stages: growth (anagen), regression (catagen), and resting (telogen). Although only a minority of humans with hair growth disorders have a disturbed hair shaft production, most cases of hair loss seen in clinical practice result from alterations in hair follicle cycling (87) (Fig. 3). Even the dramatic skin appendage transformations that remodel a large terminal hair follicle into a tiny vellus hair follicle are now recognized to be hair cycle-dependent phenomena (35, 88, 89). Shortly after completion of hair follicle morphogenesis, the follicle enters into catagen. In humans, this entry occurs already in utero; in mice, it happens about 17 d after birth (35, 37, 87). This is followed by a short phase of relative quiescence (telogen). Thus, morphologically, hair follicle cycling begins with catagen, not with the actual growth phase, anagen. The hair follicle’s transformations from telogen through six stages of anagen and eight stages of catagen, followed again by telogen, are genetically determined (37, 87). All hair follicles manifest this cycle, although the duration of the cycle as well as of the individual phases, and the length of the individual shafts vary dramatically from site to site (34). In the human and guinea pig, each follicle has its own inherent rhythm, and thus the cycles are asynchronous (90), although small groups of hair follicles on the human scalp are arranged in so-called follicular units, which appear to link about three terminal and/or vellus hair follicles into functional units (it is unclear to what degree cycling within a human follicular unit is synchronized) (91). In most rodents, large collections of follicles cycle together, where synchronous follicle growth occurs in large waves (34).
B. Molecular controls of hair follicle cycling
Although the ultimate oscillator system ("hair cycle clock") that drives hair follicle cycling remains unknown, an ever-increasing list of molecules is now recognized to modulate normal hair follicle cycling (34, 35, 36) (Fig. 3). For example, the duration of anagen is prolonged by IGF-I, HGF, glial-derived neurotropic factor, and VEGF, whereas anagen is shortened and catagen is induced by fibroblast growth factor 5 (FGF5), TGFß1 and TGFß2, IL-1ß, and interferon-
(IFN-) (34, 35, 65, 66, 92). One critical question in the context of the current review, therefore, is to what extent these key hair cycle modulators are regulated by ER-mediated signaling.
The expression of these key regulators of hair follicle cycling is under the control of a number of often still undefined upstream signals, which differ between species and hair follicle subpopulations. Key examples of these upstream signals are nuclear factor-
B, members of the Wnt and TGFß/bone morphogenetic protein (BMP) families as well as their functional antagonists, Shh, and ß-catenin (34, 35, 36). Interestingly, many of the same signals that drive hair follicle induction and morphogenesis are reused during anagen development (35, 45, 93). Hair follicle pigmentation and active melanogenesis in the follicle pigmentary unit are strictly coupled to anagen III-VI (94). Besides locally generated -melanocyte-stimulating hormone and/or ACTH, controlled changes in the intrafollicular expression of stem cell factor, nerve growth factor (NGF), and/or HGF are probably the inducers of melanocyte activity in the hair follicle pigmentary unit. Some of these pigmentation-regulatory factors are also regulators of hair follicle cycling (95, 96, 97). Again, the question arises, whether and how estrogens modulate the intrafollicular expression of these agents.
In catagen, which is a stringently controlled, apoptosis- and terminal differentiation-driven process of rapid organ involution (98), there are two protagonists that regulate normal apoptosis in the hair follicle: p53 (99, 100), and the product of the hairless gene (Hr), a zinc finger transcription factor (49, 101, 102). Intriguing similarities in the phenotype of hr-defective hairless mice and of mice with loss-of-function mutations in the vitamin D receptor (VDR) or retinoid X- receptor (RXR-) suggest that Hr, VDR, and RXR- are all parts of similar pathways that are critical for activation of the (as yet undefined) key genes that control the anagen-catagen transformation (35, 103). FGF5, TGFß1, TGFß2, the neurotrophins NT-3, NT-4, and brain-derived neurotrophic factor, as well as p75NTR signaling, IFN-, prolactin, and estrogen, are recognized inducers of catagen (8, 28, 52, 104, 105, 106, 107, 108, 109, 110, 111, 112).
During the resting stage (i.e., telogen, a period of relative biochemical and proliferative quiescence), ER is maximally expressed, and E2 serves as a kind of "hair cycle brake" (26, 27). Dramatic shortening of telogen is seen when the hair follicle chooses the so-called "dystrophic catagen" damage response and recovery pathway after chemotherapy induced hair follicle dystrophy and alopecia (113, 114). The damage response pathways differ in the speed and outcome of hair follicle recovery and can be manipulated pharmacologically by application of PTH/PTHrP receptor ligands and cyclosporine A as well as by steroid hormones like dexamethasone, calcitriols, or estrogens (27, 113, 115, 116). Thus, not only hair follicle cycling is profoundly influenced by estrogens, but also hair follicle recovery from chemical damage.
A selection of factors currently recognized as prominent regulators of hair follicle cycling in men or mice is shown in Fig. 3. In the current context, the key concept is that, in principle, estrogens may have both direct hair growth-modulatory effects that target the elusive hair cycle clock and indirect ones by altering the expression of important hair growth modulatory factors such as those indicated in Fig. 3 (117, 118). It is on this hair biology background that we are exploring estrogen functions in the following sections.
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III. Cellular and Molecular Mechanisms of Estrogen Action |
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TopAbstractI. IntroductionII. Hair Follicle Biology:...III. Cellular and Molecular...IV. Estrogens in...V. Estrogens in Pilosebaceous...VI. Open Questions and...VII. Conclusions and...References |
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An alternative route is via 4-androstene-3,17-dione, which is converted to estrone by a monooxygenase (EC 1.14.13.), and then by an oxidoreductase to E2 (EC 1.14.99.). Estrone can be metabolized to E2 by 3ß (or 17ß)-hydroxysteroid dehydrogenase (Ref. 119) (EC 1.1.1.51) or estradiol 17ß-dehydrogenase (Ref. 120) (EC 1.1.1.62). The only known pathway connecting testosterone to E2 is the cytochrome P-450 enzyme aromatase (EC 1.14.14.1, CYP19A1; ARO) pathway (Fig. 2). The CYP19 gene is localized on chromosome 15. It spans nine coding exons and a few untranslated exons, upstream of exon II, namely exon I1–I5.
The fact that CYP19A1 (also called aromatase or estrogen synthetase; ARO) transcripts with specific 5'-ends were isolated from various tissues (with all transcripts belonging to the exon I variant) led to the finding of tissue-specific promoter regulation. These are under an intricate control of transcription factors in response to gonadotropins, IL-6, IL-11, and TNF-. Because exon I is not translated, all proteins are therefore identical. The flexibility of the system is exemplified by the differential regulation displayed by the adipocyte ARO promoter, compared with its bone counterpart (121). Intriguingly, ARO activity was also found in human hair follicles (122, 123), and ARO transcripts have been detected in cultured hair follicle fibroblasts and keratinocytes (69). Paracrine estrogen secretion by hair follicle cells with ARO activity may be important for hair growth control, perhaps in a manner that is comparable to the paracrine activation of ERs by E2 during mammary gland duct morphogenesis (124).
The total E2 production rate of human males has been calculated to range from 35 to 45 µg (0.0130–0.0165 µmol) per day, of which 15–20% supposedly originates from the testes (125, 126, 127). About 60% of circulating E2 is thought to arise from peripheral aromatization of testosterone, whereas 20% is formed by reduction of estrone (125, 126). Estrone is formed by peripheral aromatization of androstenedione, which partly derives from the adrenal glands and partly from peripheral conversion of testosterone. Estrone can also be directly produced and released by the adrenals (126). In general, the testicular glands control circulating estrogen levels, as evident from their rapid decline after orchiectomy (128, 129). In premenopausal women, the main biosynthesis of estrogen takes place in the corpus luteum. Small amounts are also produced by the adrenals. During pregnancy, substantial amounts of estrogens are produced in the placenta as well. Postmenopausal decline of ovarian production of estrogen is partially compensated for by nonovarian conversion of androstenedione in the adrenals, liver, adipose tissue, skeletal muscle, kidney, and brain (130). Figure 2 depicts basic biosynthesis and metabolism of estrogens.
In the current context, it should be kept in mind, however, that the pilosebaceous unit itself, which displays very substantial aromatase activity, especially in its sebaceous compartment (59), is a significant source of estrogen synthesis, both in men and women. However, it is still far from clear which percentage of circulating estrogens is provided by peripheral estrogen synthesis in human skin under physiological and pathological conditions, and how much of this intracutaneous estrogen synthesis arises from the pilosebaceous unit.
B. Estrogen receptors
The effect of estrogens on their target tissues is determined by: 1) the receptor subtype(s) expressed and their posttranslational status; 2) the balance between corepressors and coactivators present; 3) the conformational transformation of the receptor after binding of the ligand; and 4) the interaction of the final receptor-multiprotein complex with the promoter of the target genes.
Two distinct isoforms of the ER exist: ER and ERß (131) (Fig. 4). Phylogenetic analysis and theoretical reasoning suggest that both isoforms diverged from a putative ancestor protein. The long time of parallel and divergent evolution could explain the distinct biological roles of these receptors, although they still possess large sequence homology (132). As regards molecular action, they show significant differences (133, 134, 135). A structural overview of human ER and ERß is shown in Fig. 4. Interestingly, a constitutively active ER ortholog without sensitivity to estradiol or related molecules is expressed in the mollusc Aplysia (136) and may correspond to an early ancestral protein from which ER, ERß, and other steroid receptors have evolved.
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gene spans a length of more than 140 kb. ER, like ERß, acts through direct intranuclear binding to DNA after activation by a ligand. ER has eight exons. The ER protein sequence varies 77–97% between rat, human, and chicken (137). A detailed overview of the ER gene structure in combination with a proposal for a consistent nomenclature has been published (138). The putative role of tissue-specific ER gene promoter regulation in developing and adult tissue is also discussed there. ERß, like ER, has eight exons, whereas the translated protein is shorter. For an overview of the organization of the ERß gene, see Ref. 139 .
Alternative splicing accounts for various subforms of ER and ERß receptors (140). ER splicing variants differ in their untranslated 5'-ends. Recently, a 46-kDa variant of ER was cloned from endothelial cells (141). It is missing 173 amino acids in its N-terminal end, whereas the protein still has its DNA- and hormone-binding domains intact, including a functional activation function (AF)-2 domain (142). Five isoforms of human ERß mRNA were isolated from various tissues, varying in their C-terminal ends and tissue-dependent expression (143). An 18-amino acid residue ligand binding domain insertion variant of ERß in rodents acts as a dominant negative repressor of ER (144). The ER proteins are subject to ubiquitinylation and proteosomal degradation (145). ER splice variants may act as regulators of ER and/or ERß (146). These variants that lack sequences coding for nuclear translocation/nuclear localization (contained within domain C encoded by exons 2 and 3 and part of domain D encoded by exons 3 and 4) and/or sequences coding for DNA binding domain (contained within domain C encoded by exons 2 and 3) exist in numerous tissues (147, 148).
New estrogen-sensitive entities, e.g., at the plasma membrane or in the endoplasmic reticulum, have been reported, although the biological significance of these binding sites for E2 is still under critical discussion (149, 150, 151, 152, 153, 154). Stimulation by estrogens evoke rapid cellular effects that peak minutes after stimulation, even in multiple cell types and after inhibition of RNA synthesis, indicating nongenomic mechanisms (155). Signaling cascades that might be involved include second messengers such as calcium and nitric oxide, receptor tyrosine kinase signaling involving epidermal growth factor (EGF) receptor (EGFR) and IGF-I receptor, G protein-coupled receptors, phosphoinositide-3 kinase, serine-threonine kinase, MAPK, nonreceptor kinase steroid receptor coactivator (SRC), and protein kinases A, B (Akt), and C (156, 157, 158, 159).
It should also be mentioned that many studies have suggested that this nongenomic effect is important in nonreproductive tissues such as brain, bone, and the cardiovascular system (160). Interestingly, it has been demonstrated that the effects of sex steroids on prevention of osteoblast apoptosis, which allegedly are mediated by nongenomic actions involving the MAPK-signaling pathway, appear to be gender nonspecific. These effects are supposedly mediated by the ligand (rather than DNA) binding domain of ER, ERß, or androgen receptor, and can be transmitted with similar efficiency irrespective of whether the ligand is an estrogen or an androgen (161). The role of such gender nonspecific, nongenomic effects in hair follicle biology is as yet unknown.
C. Nuclear receptor superfamily
ERs are members of the nuclear hormone receptor superfamily (131). Nuclear hormone receptors exhibit a sequential organization into consecutive domains enumerated A to F. These have highly specific functions: domains A and B are required for ligand-independent transactivation; domain C, with its two zinc-fingers, for DNA-binding; and domain E for ligand-dependent transactivation, dimerization, and interaction with other proteins (162). The sequence of the ER gene is conserved in all species studied except fish (163). In fish, only the C and E domains have high homology to ERs of other species. The DNA-binding domain has the highest homology between species. The function of the F domain of ER and ERß is not fully understood.
In addition to ER and ERß, the following nuclear receptors are derived from a putative common ancestor protein: the progesterone receptor, androgen receptor, glucocorticoid receptor, and mineralocorticoid receptor. Structurally, the thyroid and retinoid receptors also belong to this receptor gene superfamily (164). In addition, so-called orphan receptors have been identified, i.e., members of the nuclear receptor gene superfamily that still lack assigned ligands seem to have important functions (165, 166). Although evolutionary analysis has invited the speculation that they may be "molecular fossils" of the prototypical transcription factors without ligand-activating function (167), we may as well just have failed to identify the appropriate ligands, as exemplified by selected retinoid orphan receptors. For example, retinoid orphan receptor- has been shown to operate as a mediator of nuclear melatonin signaling (168, 169, 170), is expressed at the gene and protein levels in murine hair follicles, and displays significant hair cycle-dependent expression changes (53).
Thus, it remains a particularly intriguing challenge to explore the functional importance of the three orphan receptors (ERR1, ERR2, and ERR3) related to ERs that have been identified (165, 166, 171, 172). Murine ERR1 is found in adipocytes, muscle, brain, and testis as well as in skin. It is highly expressed in the ossification zone of mice. Compared with ER, it shows a relatively higher level of expression in osteoblast-like cells. ERR2 reportedly is restricted to embryological stages and only very few adult tissues (171).
D. Estrogen receptor signaling pathways
The basis of differential expression of target genes is binding of transcription factors like nuclear receptors to specific DNA sequences residing within regulatory promoters (173). Alternative ways of transcriptional activation by ERs are shown in Fig. 5. Tissue-specific coregulators are believed to be important factors in tissue specific effects of nuclear receptor ligands (174), which may explain, e.g., why breast cancer cells are inhibited by tamoxifen, whereas this ligand is growth promoting in the uterus (175). These cofactor proteins are part of a transient ER-multiprotein complex (176), of which several have been identified (174, 176).
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-associated protein, template-activating factor Iß, regulates transcription of estrogen-responsive genes by modulating acetylation of ER, and may also interact with other nuclear receptors (177). Classical receptor activation is understood as the ligand binding to the receptor binding pocket, which causes conformational changes from an inactive into an active receptor state. This facilitates binding of the receptor to the DNA response motif, in the typical case a 15-bp palindromic sequence ERE, as a dimer. Alternative response elements and their proteins may also be associated with liganded receptor, including the GC-box binding protein (SP-1), nuclear factor B (178), or the bipartite c-Jun, c-Fos complex [activating protein-1 (AP-1)]. It is known that ER and ERß regulate some gene promoters with AP-1 sites in an opposite manner (179). Interestingly, ERß exerts a negative transcriptional regulation at AP-1 sites when complexed with its natural ligand E2, whereas, in this context, antiestrogens positively activate gene transcription (133). In addition to ligand activation, ERs can be regulated by phosphorylation through polypeptide growth factors such as EGF and IGF-I (180, 181). For example, murine uterus responded to cotreatment with anti-EGF antibodies with attenuated E2 response. Furthermore, administration of the ER antagonist ICI164-384 reduced the uterine response to EGF (182).
An example of the significance of activation of cytosolic signal transduction proteins is the role of ERK activation in regulating osteoblast survival and bone formation (161), or the role of ERK and phosphoinositide-3 kinase activation on nitric oxide production in endothelial cells and angiogenesis (183). Growth factors such as IGF-I, EGF, and TGF, through activation of MAPK pathway, regulate phosphorylation of ER influencing its transcriptional activity. Also, in the absence of estrogen, ER can be activated by these growth factors (184). Moreover, in different cell types, estrogen regulates the expression of EGF, IGF-I, and TGF, suggesting that these growth factors are mediators of estrogen action (181). Thus, given the well-appreciated central role of IGF-I, EGF, and TGF in hair follicle biology, the cross talk between peptide growth factors and ER signaling pathways may be highly relevant in hair growth control.
E. Estrogen-responsive genes and coregulators of ER signaling
ERs interact with numerous coregulator proteins, resulting in either enhanced or repressed gene expression. Examples are the coactivator actions on the ligand-binding AF-2 domain. Crystallographic analysis of the ER ligand-binding domain has indicated that, upon binding of an agonist, four of 12 -helices in this receptor domain are rearranged to form a hydrophobic cleft with docking sites for coactivators important for AF-2 function (185, 186, 187, 188).
According to one hypothetical model of the exchange of coregulators involved in regulation of genes by ERs (189), in the unliganded state, ER may bind to either corepressor or coactivator complexes. Intracellular signaling (e.g., ligand-induced receptor activation, posttranslational receptor modification and activation) may shift this dynamic equilibrium to favor coactivator complex interaction. When ER is ligand-stimulated, a series of coregulator complexes bind and exchange in a programmed manner until the gene is activated. This involves histone modification, like acetylation, which is carried out by CBP/p300 and SRCs, followed by formation of a complex containing BRG-1/BAF57, which unwinds DNA and changes chromatin, enhancing formation of complexes functioning to activate transcription. TRAP/DRIP multiprotein complex interacts with RNA polymerase II, driving transcription. After the process is complete, the proteins involved are ubiquitin-tagged and turned over by 26S proteosomal degradation.
One particularly interesting corepressor involved in tissue-selective effects of estrogens is the repressor of estrogen action (REA) (190, 191). It is a 37-kDa ER-selective coregulator, which directly competes with SRC1. It acts on ligand bound ER and modulates its sensitivity to agonistic as well as antagonistic ligands (190).
F. Estrogen target tissues
ER and ERß are found in many organs and cell types. Besides the skin and hair follicle (Table 2), ERs have been detected in a wide variety of tissues and cells, such as mammary gland, prostate, testis, placenta, brain pituitary, cartilage, adipocytes, osteoblasts, skin, keratinocytes, and fibroblasts (191, 192, 193, 194, 195, 196, 197, 198, 199, 200). ERs are also found in the classical steroid hormone-susceptible and -producing organs, e.g., ovarian granulosa cells and testis (201). During a study of stromal and epithelial tumors of the ovary, ER and ERß isoforms were detected by RT-PCR and Southern blot analysis (202). A wide expression in malignant and normal ovary has been described. ERß was often found, with low intensity but with high incidence in granulosa cell tumors. A C-terminally shortened variant of ERß, ERßcx, was widely expressed in all tumors studied. This variant is a ligand-independent ERß isoform with antagonistic activity to ER (203).
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E7 is a natural splice variant with exon 7 absent. It is insensitive to allosteric modulation through ligands and coregulators such as p160, SRC1, and AIB1. ER E7 heterodimerizes with ER or ERß and acts in a dominant-negative manner (204). Studies focusing on the exclusive distribution of these ER variants have revealed, e.g., the presence of ER46 in the plasma membrane, cytoplasm as well as the nucleus of estrogen-deprived endothelial cells (142). mRNA of ERß wild type, and splice variants (5) were, for example, studied in normal human breast tissues of 37 women. Sixty-two percent showed coexpression of ERß wild type and ERß 5 variants, whereas in around 30% of the specimens, exclusively ERß wild type was detectable. This study suggests an important function of ERß in normal female breast biology (205). Finally, various parts of the brain also exhibit ER. Here, ER may be involved in the feedback or general regulation of various humoral factors, including estrogens themselves, as well as in other complex higher functions such as cognition, memory, and motor control (206).
G. Interdependence of estrogen and androgen signaling pathways
Estrogens have diverse effects on many tissues in both males and females, and the majority of these effects are mediated by both subtypes of ERs: ER and ERß. However, there are striking differences with respect to the tissue distribution of the ER subtypes. For example, ER appears to play a major role in mediating estrogen action in the pituitary and the uterus, whereas a clear role of ERß has been established in the ovary, lung, prostate, immune system, and brain (207).
Given the well-appreciated importance of both androgens and estrogens in hair follicle biology, the interdependence of estrogen and androgen signaling in hair growth control deserves careful scrutiny. In recent years, it has become quite clear that the conventional concept of androgens as male hormones and estrogens as female hormones is an oversimplification. We now know, for example, that estrogens play a central role in the skeleton, the cardiovascular system, and the reproductive tract of males (208, 209), whereas androgens are important for reproductive function of females (210). The effects of sex steroids on prevention of osteoblast apoptosis appear to be gender independent (161). As mentioned above, they are mediated by the ligand (rather than DNA) binding domain of ER, ERß, or the androgen receptor. In addition, there are numerous reports demonstrating that estrogen and androgen metabolites can interact with both receptor subtypes (211, 212). Further evidence for this molecular cross talk comes from studies in mouse prostate, demonstrating that the testosterone metabolite, 5-androstane-3ß,17ß-diol, binds and activates ERß, thereby reducing androgen receptor levels (213). Thus, because both androgen receptor and ERß are prominently expressed in the hair follicle [i.e., in follicular dermal papilla cells (24)], systematic studies are required to determine whether androgen metabolites, acting via ERß, affect the hair follicle’s mesenchymal "command center," i.e., follicular dermal papilla cells [similar to what has been reported in mouse prostate (213)].
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IV. Estrogens in Dermatoendocrinology |
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TopAbstractI. IntroductionII. Hair Follicle Biology:...III. Cellular and Molecular...IV. Estrogens in...V. Estrogens in Pilosebaceous...VI. Open Questions and...VII. Conclusions and...References |
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In a murine model, topical application of estradiol significantly increased skin thickness, hyaluronic acid synthase levels, hyaluronic acid content, tissue transglutaminase, and collagen type I in SKH-1 hairless mouse skin, suggesting a therapeutic role for ER agonists in wrinkle repair (219). In rat skin, E2 has effects on mitosis and on differentiation of epidermis and sebaceous glands (5). E2 also stimulates the synthesis, maturation, and turnover of collagen in rat (220) and guinea pig skin (221). Furthermore, estrogens have been shown to increase mitotic activity in the epidermis of mice and induce epidermal thickening (222, 223). However, prolonged E2 administration reportedly reduces epidermal thickness in rats (3).
Skin color varies with the menstrual cycle (224, 225). Such variations may result from a synergistic action of estrogens and progesterone on the melanogenic activity of epidermal melanocytes. Similar mechanisms account for the hyperpigmentation during pregnancy, which is most prominent in mammilar skin (224, 225). Estrogens are capable of accelerating the synthesis of melanin; the mechanism is supposed to be a direct effect of the hormone itself, because the response occurs locally when the hormone is applied directly to the skin (226). Normal human melanocytes become enlarged and dentritic in culture after 2-d incubation with estradiol (227). The effects of estrogens on melanocyte functions, however, are as yet unclear, because estrogens can increase epidermal melanocyte cell number, while decreasing melanin content and tyrosinase activity (228). In contrast, another group observed that estrogens significantly increase melanin synthesis and tyrosinase activity in normal human skin melanocytes in vitro (229). Therefore, the actual effects of estrogens on melanocyte functions may be highly context-dependent, and may reflect the local predominance of distinct ER coregulatory elements.
Estrogens have also been suggested to be major regulators of wound repair, which may reverse age-related impaired wound healing in human and animal models (216, 230, 231). Estrogens dampen inflammation (as indicated by a suppression of the production of proinflammatory cytokines, macrophage migration inhibitory factor, and TNF- by macrophages), and enhance the deposition of collagen I in the dermis, thus increasing the breaking strength of wounds in ovariectomized mice (232). The ER complex is able to stimulate the expression of growth factors, such as IGF-I, a mitosis-enhancing protein for keratinocytes (233, 234), which may also serve as a "guardian of immune privilege" (76).
In vitro, E2 stimulates human keratinocyte proliferation by promoting the expression of cyclin D2, which induces G1 to S phase progression in the cell cycle (235); in addition, it inhibits oxidative stress-induced apoptosis in keratinocytes by promoting Bcl-2 expression (236). E2 in vitro enhances production of NGF, a growth factor, e.g., for neurons and keratinocytes, in macrophage-like differentiated THP-1 cells (237) and also induces c-Fos expression in macrophages via the GPR30/cAMP/protein kinase A signaling pathway; E2 activates NGF transcription from AP-1 elements. Furthermore, in vitro, E2 induces keratinocytes to produce an autocrine growth factor, granulocyte-macrophage colony stimulating factor by increasing both its transcription and mRNA stability (79). These effects of E2 may combine to enhance wound reinnervation and reepithelialization (81).
Other authors have suggested that estradiol effects on keratinocytes are mediated via a membrane ER that activates the MAPK pathway. It has been shown that E2 induces the proliferation of human keratinocytes and stimulates MAPK activation as well as cyclin D1 expression (238). Despite the obvious effect of estrogen on dermal collagen content (220, 221, 222), the underlying molecular mechanisms are still poorly understood. However, it has been demonstrated that both ER and ERß are expressed in human dermal fibroblasts, which is in line with the concept that E2 effects in the dermis may occur, at least in part, through direct regulation of ER-mediated fibroblast functions (239).
Reportedly, the course of a number of chronic inflammatory skin diseases is modulated by estrogens: the inflammatory infiltration in psoriatic lesions may be suppressed by estrogens (81), whereas atopic dermatitis worsens during pregnancy (240). CD-1a-positive Langerhans cells are activated in the skin lesions of estrogen dermatitis (241). The proliferation of skin hemangioma vascular endothelial cells in culture is stimulated by E2 (242). Because VEGF transcription from AP-2 elements can be enhanced by E2 in vitro via the G protein-coupled receptor 30/cAMP/protein kinase A signaling pathway, E2 may promote the development of granuloma pyogenicum (243).
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V. Estrogens in Pilosebaceous Unit Biology |
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TopAbstractI. IntroductionII. Hair Follicle Biology:...III. Cellular and Molecular...IV. Estrogens in...V. Estrogens in Pilosebaceous...VI. Open Questions and...VII. Conclusions and...References |
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-reductase, aromatase, and 17ß-hydroxysteroid dehydrogenase to control estrogen synthesis in the pilosebaceous unit. Aromatase converts the substrates androstenedione to estrone and testosterone to estradiol (Fig. 2). Interestingly, both dermal papilla cells and outer root sheath keratinocytes reportedly synthesize cytochrome-P450-aromatase (25). Estrone can be converted back and forth to estradiol by 17ß-hydroxysteroid dehydrogenase (for review, see Refs. 23 , 25 , 59 , and 245). This has initiated a shift of paradigm—from the pilosebaceous unit as a mere target organ of steroid hormone activities to the concept that the pilosebaceous unit is an important site of steroid hormone synthesis and metabolism (5). Today, the skin is understood to have established its own para- and autocrine hormonal regulation networks, with the pilosebaceous unit located at center stage; it is now recognized to be highly sensitive to an ever-expanding list of hormonal regulators that are generated and/or metabolized within or in close vicinity to the pilosebaceous unit (8, 9, 52, 53).
B. Estrogen receptor expression in the hair follicle
Until the cloning of a novel gene coding for a second ER, named ERß, from rat prostate (246) and, thereafter, from human tissue (247), the consensus was that only one ER existed: ER, cloned in 1986 from MCF-7 cells (178, 248). Both receptors bind E2 with high affinity (228) and bind to classic EREs in a similar manner (249). Both receptors are detectable in the skin of humans and rodents with distinct expression patterns (14, 15, 16, 17, 18, 19, 25, 26, 28, 250, 251, 252, 253) (Table 2).
Recently, Thornton et al. (20, 24) showed that, in human scalp skin, ERß is the predominant ER. In human hair follicles dissected from male and female nonbalding scalp skin, ERß expression was found to be localized to nuclei of outer root sheath and epithelial matrix keratinocytes as well as of dermal papilla fibroblasts. In contrast, ER and the androgen receptor were only expressed in dermal papilla cells. Serial sections also showed strong nuclear expression of ERß in the cells of the bulge, whereas neither ER nor androgen receptor was detectable. In the sebaceous gland, ERß was expressed in both basal and partially differentiated sebocytes. ER exhibited a similar pattern of expression, whereas the androgen receptor was expressed in the basal and very early differentiated sebocytes (19, 24). In this study, there were no obvious differences in the expression of either ER in male or female skin. The same group found that in cultured human, nonbalding scalp dermal papilla cells, the two ERs exhibited different expression patterns, ERß showing strong nuclear expression, and ER granular cytoplasmic expression (24). This different distribution may contribute to a variable E2 responsiveness (254).
The different expression patterns of ER and androgen receptor in the hair follicle and their potential biological relevance deserve special attention, because follicular dermal papilla cells are thought to be the primary target cells within the hair follicle that mediate the growth stimulating signal of androgens by releasing growth factors that act in a paracrine fashion on other cells of hair follicle (255, 256). The exact pattern of androgen receptor expression in the mesenchyme and epithelium of human hair follicle remains a matter of contention, with published results heavily influenced by the respective methodology employed. However, Thornton et al. and other authors have reported that no androgen receptor immunoreactivity is detected in the keratinocytes of the outer root sheath (including its bulge region) and of the inner root sheath, whereas the majority of dermal papilla cells express androgen receptor (257). In contrast, ERs are more widely expressed, and importantly, ERß is strongly expressed in the bulge region of the outer root sheath. This region contains stem cells for hair follicle keratinocytes that regenerate the follicle during the anagen phase. This suggests that these epithelial stem cells are targets for estrogen action.
The wide distribution of ERß in human pilosebaceous unit suggests that estrogens play an important role in the maintenance and the regulation of the hair follicle and provides further evidence for estrogen action in nonclassic target tissues. Recently, it was reported that in cultured dermal papilla cells from nonbalding male donors, both ER and ERß showed a consistently higher expression, both at the RNA and protein levels, in occiput dermal papilla cells compared with vertex dermal papilla cells (258). With respect to ERß immunoreactivity, we found that, in anagen VI follicles microdissected from frontotemporal skin, there was a remarkable distribution difference between male and female hair follicles from frontotemporal scalp skin: ERß immunoreactivity was found in male scalp hair follicles predominantly in the matrix keratinocytes, whereas in female hair follicles, ERß immunoreactivity was predominantly found in the dermal papilla fibroblasts (10). These data not only highlight substantial, previously underappreciated sex-dependent differences in ERß expression of an important peripheral E2 target organ, but also underscore the importance of investigating whether E2 effects on the human hair follicle are location-dependent, as is well-recognized for the paradoxical hair growth effects of androgens (64, 259, 260).
Conflicting data have been presented concerning ER expression patterns in murine hair follicles. It has been reported that ER was expressed only in the dermal papilla and outer root sheath of telogen and early anagen mouse hair follicles and that ERß was undetectable (26, 250). Recently, however, we could show that both ER and ERß as well as the splice variant ERß ins are expressed throughout the entire, depilation-induced murine hair cycle at both the protein and RNA levels (28). In addition, hair follicles in late anagen (anagen VI) were highly sensitive to regulation by topically applied E2, which rapidly induced premature catagen entry. Therefore, anagen VI mouse pelage hair follicles must express fully functional ERs (28).
ER immunoreactivity peaks in murine telogen follicles within the dermal papilla and the sebaceous gland, whereas the inner root sheath and outer root sheath show weaker immunoreactivity. In anagen VI, ER immunoreactivity (IR) is detectable in the outer root sheath and the dermal papilla, whereas in early catagen it is restricted to the dermal papilla and the secondary hair germ. In anagen VI follicles, ERß is weakly positive in hair matrix and outer root sheath, whereas in catagen and telogen follicles, ERß is expressed in the dermal papilla, inner root sheath, outer root sheath, and the sebaceous gland. By RT-PCR, ER and ERß transcripts can be detected in telogen, anagen V and VI, and late catagen skin mRNA extracts. Investigation of ERß knockout mice showed an accelerated catagen development along with an increase in the number of apoptotic hair follicle keratinocytes (28). Taken together, this suggests that the catagen-promoting properties of E2 in murine skin are mediated by ER and that ERß mainly functions as a silencer of ER action in murine hair biology. (An additional list on reported expression of estrogen signaling components is provided in Table 2).
C. Estrogen target genes in the pilosebaceous unit
The classical mechanism of estrogen action involves binding to its receptors in the nucleus, after which the receptors dimerize and bind to specific response elements known as EREs located in the promoters of target genes. However, ERs can also regulate gene expression without directly binding to DNA. This occurs through protein-protein interactions with other DNA-binding transcription factors in the nucleus (261). About one third of the genes in humans that are regulated by ERs do not contain ERE-like sequences (262). Candidates of estrogen target genes with relevance to pilosebaceous biology that are activated without ERE promoter include IGF-I, collagenase, EGF, EGFR, and cyclin D1 (261, 263). Instead, progesterone receptor, prolactin, and lactoferrin are examples of relevant target genes in the pilosebaceous unit with consensus EREs (263, 264, 265).
Zouboulis et al. (266) showed that, in sebocytes, the expression of peroxisome proliferator-activated receptor (PPAR) , postulated to be required for androgen-induced lipogenesis, was down-regulated by the phytoestrogen genistein, whereas E2 enhanced the metabolism of prostaglandin D2 to 12-prostaglandin J2, a natural PPAR ligand. Additionally, the same group found that E2 increases IGF-I synthesis and down-regulates IGF-I receptor expression (266).
Recently, we have employed cDNA microarray to screen for genes in organ-cultured human scalp hair follicles that respond to E2 stimulation with transcriptional changes, using a skin focus chip and comparing the E2 response of male and female human frontotemporal scalp hair follicles. Of 1300 genes screened, more than 60 E2-responsive genes were detected. Several genes were modulated equidirectionally in both sexes (e.g., down-regulation of osteopontin and hevin = highly expressed endothelial venule protein; up-regulation of cytokeratin type II and bone morphogenetic protein 7). Intriguingly, however, several genes showed distinct regulatory responses in male and female hair follicles: e.g., down-regulation of filaggrin and FGF receptor 2 in males; up-regulation of nuclear receptor subfamily 4, group A, member 1 in females; whereas cysteine-rich 61, fos-like antigen 2, and collagen IV A6 were up-regulated in males, yet down-regulated in females (10). This reveals that terminal human scalp hair follicles from one defined region show strikingly different, sex-dependent biological responses to stimulation with the same ER ligand, strongly advocating gender-tailored management of female vs. male pattern balding (androgenetic alopecia) (10).
D. Species-specific differences in estrogen actions on hair follicle cycling
E2 has long been recognized to profoundly modulate hair growth, acting primarily as a hair growth inhibitor in mammalian species as diverse as mice, rats, guinea pigs, and dogs (1, 2, 51, 267, 268) (Table 1). For example, injections of estrogens or topical applications of ER agonists effectively inhibits spontaneous hair growth in rats and dogs (2, 267). Hair regrowth after plucking was noted to differ between male and female rats, and spontaneous hair growth was inhibited during periods of lactation and pregnancy in mice (269). In young castrated rats, im injections of estrogens inhibited the initiation of follicle growth and prolonged the duration of the entire hair growth cycle, resulting in a fine and sparse pelage (6). Removal of the ovaries in rats accelerated the passage of the moult, increased the rate of hair growth and length of hairs, and accelerated the loss of club hairs. Treatment of ovariectomized rats with estradiol delayed the initiation of the wave, slowed its passage, reduced the rate of growth and definitive length, and delayed the loss of the telogen club hairs (7). Spayed female rats shed more than 80% of telogen club hairs within 2 wk of the start of anagen; implantation of estradiol delayed this process by 3–4 wk. Anagen initiation has been shown to be delayed by more than 5 wk in the ventral hair follicles of E2-treated rats (7). Therefore, estradiol can act as a brake on hair follicle cycling by delaying the initiation of anagen and by prolonging the duration of telogen.
Hale and Ebling (270, 271) showed that estrogens reduce the rate of growth and the ultimate length of spontaneously erupting hairs by shortening the anagen period, whereas ovariectomy of rats tended to advance the spontaneous eruption of successive generations of hairs by shortening each complete hair cycle. Oh and Smart (26) found that, in mice, topical E2 administration to clipped dorsal skin arrested hair follicles in telogen and produced a profound and prolonged inhibition of hair growth, whereas treatment with the biologically inactive stereoisomer 17-estradiol did not alter hair growth. That topical E2 arrests murine pelage hair follicles in telogen was independently confirmed (28). This represents an important rediscovery of older work that had already demonstrated a prolongation of telogen by topical E2 application in various rodent species (272, 273).
Vice versa, orchiectomy induces a premature telogen-anagen transition and an anagen wave (223, 274). Application of E2 to gonadectomized animals inhibits hair follicle growth and blocks the telogen-to-anagen transition (223). Topical treatment with the selective ER antagonist ICI182-780, a pure ER antagonist, reportedly caused premature anagen induction (26, 273). These results were later confirmed by the same group in C57BL/6 and C3H male and female mice (272). More precise analysis of hair follicle cycling in C57BL/6 mice, however, revealed that the ER antagonist ICI182-780 does not prematurely initiate anagen but accelerates anagen development and anagen wave spreading, once anagen has been initiated by independent (endogenous) signals (28).
Most recently, we could demonstrate that E2 is also a potent catagen inducer (27, 28). [This was in contrast to older studies in different rodent species, where no changes in the duration of anagen had been noted after E2 application (273)]. In line with these newly discovered catagen-inducing properties of topical E2 (27, 28, 114), ERß knockout mice display accelerated catagen development, along with an increase in the number of apoptotic hair follicle keratinocytes (28). This suggests that, contrary to previous working hypotheses (274), ERß does indeed play a significant role in murine hair growth control: whereas the catagen-promoting properties of E2 are mediated via ER, ERß may mainly function as a silencer of ER action in hair biology (28). Nevertheless, ER is thought to serve as the predominant ER in the hair follicle of animals (31, 233, 254).
In mice, ER expression is stringently hair cycle-dependent (26, 28, 33), and nuclear immunoreactivity was detected for both ER and ERß throughout all investigated hair cycle stages (telogen, anagen VI, catagen) in mice (28) (Table 2). Together with the fact that the treatment of murine anagen hair follicles in vivo induces catagen, i.e., a dramatic remodeling process of a complex miniorgan (28), this renders obsolete the old concept that only telogen follicles express ER and engage in ER-mediated signaling (26). However, it is perfectly reasonable to propose that ER-mediated signaling operates as an endogenous paracrine regulator of the hair cycle (26). Stringently controlled changes in the expression and/or activity of ER may, therefore, well be an integral component of the elusive hair cycle clock (28, 35) (Fig. 3).
Just like glucocorticoids and calcitriols, topical E2 also promotes the so-called dystrophic catagen response pathway of chemotherapy-damaged anagen hair follicles (27, 114). Topical E2 significantly alters the cycling response of murine follicles to cyclophosphamide, whereas the ER antagonist ICI182-780 exerts no such effects. Initially, topical E2 enhances chemotherapy-induced alopecia by forcing the follicles into the dystrophic catagen response pathway to hair follicle damage, which allows for a maximally fast secondary recovery by construction of a new anagen hair follicle. Instead, follicles treated with ICI182-780 or vehicle shift into the dystrophic anagen response pathway. Consequently, the regrowth of normally pigmented hair shafts after chemotherapy-induced alopecia is significantly accelerated in the E2-treated group (27).
However, these findings in mice contrast with long-standing clinical experience in the topical application of E2 to the human scalp. For human scalp hair, topical E2 has long been used in the management of telogen effluvium and androgenetic alopecia, especially in women (29, 58, 275). Although this remains to be unequivocally demonstrated in vivo, E2 has been proposed to decrease the telogen rate and to prolong the anagen phase in human scalp skin, justifying the use of topical E2 in the management of hair loss characterized premature catagen entry, such as androgenetic alopecia and telogen effluvium (29, 30, 88, 275, 276, 277).
Such a catagen-inhibitory effect of E2 in human scalp hair follicles would help to explain the well-established clinical observation that topical E2 or high systemic E2 levels during estrogen-based contraception and during pregnancy increase the telogen/anagen ratio, thus notably improving a preexisting telogen effluvium, and that a telogen effluvium occurs postpartum (supposedly due to sudden E2 withdrawal) (51, 276, 277). In pregnant women, the scalp hair shaft diameter reportedly increases compared with nonpregnant women, although it remains to be determined which pregnancy-associated factors are responsible for this phenomenon (278). In view of the complex, multiple concomitant endocrine changes during and after pregnancy and lactation (including dramatic fluctuations in gestagen and prolactin levels), it clearly is very difficult to dissociate strictly E2-based hair growth effects from those that other hormones might exert on the human scalp hair follicle in vivo during this time (10, 12).
Although rodent hair follicles generally respond to E2 stimulation with an inhibition of hair shaft formation, this is not necessarily true for human scalp hair follicles, at least in males. Studying the isolated effects of E2 on human hair growth in vitro (in organ-cultured, microdissected human anagen hair bulbs), we recently showed that, in frontotemporal male hair follicles, E2 indeed slightly prolongs anagen and stimulates hair shaft elongation (11). Corresponding studies (279, 280, 281) have reported that E2 inhibits hair shaft elongation in vitro or that E2 does not influence the decay rate of organ-cultured human anagen hair follicles from occipital scalp skin (measured by morphology and autoradiographic 3H-thymidine incorporation) (279). These partially conflicting reports may become reconciled, once larger studies with organ-cultured human scalp hair follicles have been performed that systematically distinguish between male, female, frontotemporal, and occipital follicle populations, and that correlate hair shaft elongation with hair cycle effects. In addition, these in vitro studies need to be complemented by and compared with the results of clinical trials on the scalp hair growth effects of topical E2, documented by professional phototrichogram methodology (88).
E. Gender- and location-specific differences in estrogen actions on the hair follicle
Besides the estrogen-dependent development of breasts, the hair follicle is the other most characteristic skin feature of mammals. The growth of the beard and of pubic and axillary hair is, in adults of either sex, dependent on the production of sex steroids (51). The pelage changes as a mammal grows, and that of the adult often differs markedly from that of the juvenile animal, a circumstance that may reflect the changing requirements of heat regulation, camouflage, sexual and reproductive activity, and social communication (51). As to sexual hair growth, it has been recognized that not only adrenal androgens but also ovarian hormones may play a role for the growth of pubic and axillary hair in human females. Pubic hair can still develop in the presence of preadrenarchal levels of adrenal androgens in girls with precocious puberty or primary adrenal insufficiency (282). Females with primary ovarian insufficiency have very sparse pubic and axillary hair, which can be stimulated to grow with adequate and prolonged estrogenic therapy (283).
Estrogens act, either alone or together with androgens, directly at the level of the hair follicle in pubic skin to stimulate hair growth. However, in the absence of active androgen receptors, E2 cannot promote sexual hair growth, e.g., in patients with complete testicular feminization who do not grow pubic and axillary hair, despite signs of E2 effects in other tissues (33).
In human occipital scalp hair follicles, E2 may inhibit hair shaft elongation in both males and females in vitro (279, 280). However, we found sex-dependent differences of frontotemporal scalp hair shaft elongation after E2 treatment in vitro: in females the hair shaft elongation was inhibited, whereas E2 significantly stimulated hair shaft elongation in human frontotemporal anagen hair follicles from male patients in vitro (12, 281). This corresponded to a significantly up-regulated proliferation rate of the matrix keratinocytes in the male frontotemporal scalp hair follicles compared with female hair follicles (12).
The apparent differences of E2 action on human hair growth in vivo and in vitro can be reconciled if one considers that, clinically, even a significant inhibition of the speed of hair shaft production per time unit on the human scalp after E2 administration would hardly be noticed by either the patient or the doctor (unless specifically assessed with highly sensitive methods). In contrast, if the percentage of telogen follicles among the approximately 100,000 human scalp hair follicles changes in favor of anagen follicles under the influence of topical E2, this would result in an easily apparent reduction in telogen effluvium—a phenomenon that is quickly recorded by the patient, especially when assessing the hair loss after combing or shampooing (37, 87). Thus, the major, clinically relevant effect of topical E2 on human scalp hair follicles seems to be the inhibition of catagen (88).
The observed differences in the E2 response of female vs. male frontotemporal hair follicles raise the question whether E2 exerts similarly paradoxical, site-dependent effects on human hair growth (12) as are recognized for androgens (70). Perhaps, there are location-dependent differences in defined populations of human hair follicles that react to E2 stimulation in a divergent signaling and gene expression response, similar to the response of beard hair vs. scalp follicles to androgen stimulation with respect to TGFß 1 vs. IGF-I expression in the dermal papilla (118, 259).
Due to different activities of key enzymes in dermal, epidermal, or sebaceous compartments like aromatase, 17ß-hydroxysteroid dehydrogenase, or steroid sulfatase (57, 69, 123), there may indeed be important regional differences in the extrafollicular estrogen metabolism of/in hair follicle in different integumental locations that must be carefully taken into account when estrogens are applied topically. However, possibly more important than such differences in local intracutaneous estrogen metabolism are gender-associated differences in the hair follicle response to E2 stimulation: frontotemporal female scalp hair follicles display most of their ERß-associated immunoreactivity inside the (mesenchymal) dermal papilla, whereas ER-like immunoreactivity in male frontotemporal scalp hair follicles is located in the (epithelial) hair matrix and outer root sheath (10).
Furthermore, when comparing the distribution pattern of ERß-like immunoreactivity in male and female scalp hair follicles from one defined integumental site (frontotemporal), immunohistology suggests striking differences in the preferential location of ER protein expression; although ER protein expression in female hair follicles dominated in the mesenchymal command center of the hair follicle, the dermal papilla, male scalp hair follicles exhibited a much more prominent ERß-like immunoreactivity in the hair follicle epithelium (especially the hair matrix) than in the hair follicle mesenchyme. However, dermal papilla ER expression in female scalp follicles was greatly enhanced after hair follicle stimulation with E2, whereas no such up-regulation of ERß expression was seen in E2-stimulated male scalp hair follicles (10). In the context of this study strikingly sex-dependent differences in gene regulation of frontotemporal human scalp hair follicles in response to E2 stimulation have recently surfaced from microarray analyses (10).
Although these findings remain to be followed up and their biological significance for hair growth control remains to be clarified, they may be interpreted as only the "tip of an iceberg" of very substantial, previously unknown gender differences that must be taken into account when exploring the hair follicle response to E2 stimulation. Given the reported differences in the E2 response between frontotemporal and occipital human scalp hair follicles summarized above (10, 11, 279, 280, 281), it is not unreasonable to expect that a similar claim can be made for location-dependent differences in hair follicle responses to ER activation.
F. Clinical hair growth effects of estrogens
There are only a few reports on the use of systemic estrogens for hair loss management. Estrogens have been used for topical treatment of hair diseases for more than half a century (284) and constitute a firm staple of management strategies for female pattern androgenetic alopecia in central Europe (88). Orentreich observed in 1969 a decrease in daily effluvium during therapy with systemic estrogens (285), which were reported to increase the proliferation rate, slow down differentiation, and, thus, postpone telogen effluvium (286). On this basis, even intralesional stilbene administration was once recommended for the treatment of alopecia areata (287). Some studies have reported an increased anagen and decreased telogen rate after treatment with estrogens, compared with placebo (288, 289). However, professionally executed, double-blind, placebo-controlled, randomized, prospective clinical trials on the efficacy of topical E2 in the treatment of androgenetic alopecia and non-androgen-dependent telogen effluvium are still painfully missing.
Since the studies of Hamilton, we know that androgens play a crucial role in the onset and progression of androgenetic alopecia (290). Androgenetic alopecia occurs in genetically susceptible individuals and in androgen-sensitive hair follicles when testosterone is transformed to 5-dihydrotestosterone by 5-reductase (88, 291). This conversion is inhibited inter alia by E2 (288, 292). For the treatment of androgenetic alopecia in women, solutions containing estradiol benzoate, estradiol valerate, or 17-estradiol are commercially available in Europe. Due to unwanted side effects like gynecomastia, E2 should not be used in men because very high topical doses seem to be required to obtain measurable hair growth effects (84), whereas the inactive stereoisomer 17-estradiol may also be prescribed for men.
Its claimed efficacy for male or female pattern balding (290), however, remains as yet unsupported by solid, professionally designed and executed, prospective, double-blind, placebo-controlled long-term clinical trials. However, that 17-estradiol has been reported to induce aromatase activity in organ-cultured human anagen hair follicles, with the consequence of an increased conversion of testosterone to E2 and androstenedione (293) certainly encourages one to systematically explore the use of 17-estradiol for androgenetic alopecia in men and women.
G. Relevant signaling cross talks in the hair follicle
1. ER target genes.
An exhaustive list of factors responding to estrogen and/or ER signaling is readily available in the comprehensive ERGDB database (294) 2. Table 3 shows a small selection from this database. Below, we discuss some of these factors that are already recognized as hair growth-modulatory agents.
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and TGF-ß, cathepsin D, and several protooncogenes (e.g., c-fos, c-myc, and c-jun), as well as an array of heat shock proteins (295, 296, 297). Some of these factors are capable of nonclassical ER activation through modifications such as phosphorylation (298). In cell culture, for example, the growth factors IGF-I, EGF, and TGF- increase the transcription of ER target genes (298, 299, 300, 301). In the following, we list a few E2 targets to illustrate the rather stunning complexity of E2-induced signaling, without offering exhaustive coverage of all pathways that might be relevant for E2-induced effects on the hair follicle.
2. ER and EGF.
Through binding to its membrane bound receptor (EGFR), EGF can partially mimic the E2 stimulation of uterine growth in ovariectomized mice (301). EGFR may exert this effect by recruiting ER for its downstream signaling, because ER antagonists can block it (302). Vice versa, EGF signaling can be reinforced by E2 (302). This cross-connection is interesting in view of other long-appreciated hair growth-inhibitory effects of EGF in various species including man, which includes the induction of apoptosis-driven catagen (303, 304, 305, 306, 307, 308, 309, 310, 311) (Fig. 6). Interestingly, the EGFR gene is directly estrogen responsive (312), with multiple ERE sequences upstream of the transcription start site. Also, ERBB2 is ER-regulated (313) and exhibits ERE sequences within its putative promoter region (Table 3). It is, therefore, interesting to ask to which extent ER activation results in hair growth effects that are mediated, at least in part, via the described connection to EGF signaling.
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). Studies with thyroid cells (317, 318) indicate that ER indirectly alters the cell cycle through the MAPK pathway and E2 increases the proliferation rate and enhances expression of cyclin D1 (318). Intriguingly, this effect was abolishable by coapplication of the MAPK inhibitor PD098059 to E2-treated cells. In response to E2, both MAPK isoenzymes ERK1 and ERK2 were strongly phosphorylated in benign and malignant thyroid cells. This indicates that, at least in benign and malignant thyroid cells, ER utilizes the MAPK pathway to interfere with the cell cycle via cyclin D1. This provides one plausible pathway by which ER-mediated signaling could affect hair follicle cycling via cyclin D1 (319).
4. ER and the Wnt pathway.
Exciting new connections between the ER and other important factors critically involved in hair growth control were found in recent years. This is highlighted by the convergence of estrogen signaling with the Wnt signal transduction pathway (320, 321) (Fig. 6). The morphogenetic factors involved in this pathway have been the subject of studies centered on the hair follicle, including stem cell regulation, hair follicle induction, morphogenesis, and differentiation (323, 324, 325, 326).
The Wnt pathway is composed of a class of lipid-modified diffusible para- and autocrine factors that act through various intracellular signals, including the calcium and planar cell polarity pathways (323). The best characterized is the canonical pathway, with beta-catenin in its center (324, 325). Although this protein is involved in the pathogenesis of many human cancers, under physiological conditions it has at least two major distinct functions, one as a cytoplasmic cytoskeleton-adhesion mediator protein placed adjacent to the cell membrane, within the cadherin complex in the adherens junctions (326) and one through participation in regulatory networks of a whole array of important factors for keratinocyte and hair follicle homeostasis, e.g., c-myc (295, 327, 328), cyclin D1 (319), PPAR- (328), which are documented to alter hair follicle cycling or homeostasis.
Wise is a secreted Wnt modulator that has been found to be differentially expressed in inductive dermal papilla cells vs. cells in culture (329). As ER-regulated targets include the Wnt pathway, Wise is especially intriguing to study, because it is expressed at the site where ER is dominantly located. Together with the presence of Wise in the bulge region (329), the site believed to be suppressed in telogen, the connection between ERs and the Wnt pathway is also repeatedly present in the hair follicle.
5. ER and the TGF/BMP family.
Members of the TGFß/BMP family and their functional antagonists are recognized as critical regulators of hair follicle morphogenesis and cycling (322, 330, 331, 332). Therefore, this family is likely to be critically important for understanding the role of estrogens in hair biology to follow up the increasing insight into cross-connections between ER-mediated signaling and the TGFß/BMP family; e.g., BMP genes are principal ER-responsive genes (333, 334, 335) whose transcriptional modulation could profoundly affect hair growth (Fig. 6).
Activators as well as inhibitors play essential roles in the control of postnatal skin remodeling and hair follicle growth (111, 336, 337, 338, 339). Artificial loss or gain of BMP signals induces severe alterations in skin morphogenesis. Normally high levels of BMP-6 transcripts as well as proteins are expressed in the suprabasal layer of the murine epidermis from embryonic day 15.5, whereas BMP-7 mRNA is found in the basal epidermal layers at late stages of embryonic development (340, 341, 342). BMP-2 and BMP-4 transcripts are more restricted to the hair follicle epithelium and mesenchyme (340, 343). Notably, the BMP receptor (BMPR)-1a is localized to murine epidermis at embryonic day 16.5, whereas the BMPR-1b is present in the suprabasal keratinocytes (338). Mice that lack a key BMP antagonist, noggin, have around 90% reduced hair follicles and display increased proliferation of the epidermal compartment with down-regulation of Keratin 10 and misplaced up-regulation of Keratin 14 in the upper epidermal layers (338, 339); overactivation under K14 promoter leads to increased hair follicle density with various defects in skin appendage morphogenesis and differentiation (344). Also, mature hair follicle cycling is regulated by BMPs and their antagonists. For example, the active hair growth stage, anagen, is accompanied by down-regulation of the BMP4 and increased noggin mRNA expression in the hair follicle. Inhibition of BMPs by Noggin protein induces a new hair growth phase in postnatal telogen skin in vivo, and BMP signaling regulates hair follicle cycling and differentiation in mice (337). This underscores the concept that factors contributing to hair follicle morphogenesis often also participate as factors regulating the hair cycle (338, 339).
Therefore, it is important to note in the current context that ligands of the TGF-ß group, TGFß1, as well as BMP subgroup, BMP1, -15, -2, -8a, and -8b (333) are recognized as E2 genes that are up-regulated after ER activation. BMP1 and BMP2 are directly ER-regulated (334, 335), and they have ERE sequences 1004 and 3982 nucleotides upstream of the transcription start site, respectively (294). The TGF-ß1 gene was found to be estrogen sensitive in vivo and in vitro (334), but without detectable ERE sequence in the putative promoter region (294), suggesting an ERE-independent mode of transcriptional regulation. This renders members of the TGFß/BMP superfamily and their signaling pathways other candidates as key targets of E2 regulation in hair follicle biology, and makes our observation that TGFß2 protein expression in the epithelium human scalp hair follicle is regulated by E2 in a sex-dependent manner (10) even more intriguing.
6. ER and homeobox proteins.
Hox genes represent other potentially important ER targets. Their products, homeobox (Hox) proteins, function as master regulatory transcription factors. They assign roles by selecting a subdomain of otherwise uniform cells, making an organ primordium through reiteratively shaping and patterning the embryo, and are thought to be the main factors establishing the embryo bauplan (345, 346). They are constitutive, in a gene-cluster specific manner, expressed in various tissues including the skin and its appendages as well as the hair follicle (347, 348). Depending on the organism in question (human or mouse), various Hox or Msx genes are target genes of ERs (Table 3). And indeed, there are functional ties to the control of hair follicle development, differentiation, and cycling (35, 349, 350, 351). Msx-2 is a homeodomain protein that belongs to the Msh homeobox family, and Msx-2 mutants display a cyclical alopecia (349). It is particularly intriguing that Msx-2 is directly ER-controlled (350). Since ER expression in the mesenchymal signaling center of the hair follicle, the follicular dermal papilla (26), is also strictly coupled to hair follicle cycling, one wonders whether ER expression and Msx-2 activity may be intimately interlinked regulatory elements in the elusive molecular hair cycle clock (35). A linkage of position-specific effects and ER signaling was recently suggested because, for example, the craniocaudal susceptibility for chemotherapy-induced alopecia differs under treatment with estrogens (27). Although the potential interconnections between homeodomain genes and ER proteins in hair follicle biology remain highly speculative, they certainly deserve further scrutiny, beginning, e.g., with a systematic comparison and alignment of the patterns and timing of Hox genes and ER expression in the dermal papilla at various time points of hair follicle cycling, in distinct locations of the integument, and during the occurrence of hair wave phenomena.
H. Other potentially important signaling cross talks with intrafollicularly generated hormones
The number of genes that carry an ERE, whose transcription is known to be altered by E2 stimulation, is very large (294) (Table 3). Together with the fact that E2 modifies androgen metabolism and vice versa (250, 251) and the intriguing cross talk examples discussed above, this already renders the number of potential signaling cross talks and cross-modulation events that may impact how E2 can alter the growth of a given hair follicle in a defined gender and location daunting. This gets even more complicated if one enters additional recent findings into the equation. Suffice it here to list just two selected examples of this added level of complexity: possible cross talks between E2/ER and intrafollicularly generated hormones, such as melatonin (53) and prolactin (8, 9).
It is now clear that extrapituitary prolactin is generated in the epithelium of both mouse and human hair follicles, where it appears to serve as an autocrine and/or paracrine hair growth-inhibitory, catagen-promoting factor (8, 9). At the same time, it has become appreciated that prolactin- and E2-induced signaling shows cooperative activity, both in normal mammary development and in breast cancer. This results in increased dynamic phosphorylation of ERK1/2 and c-Fos, induction of c-fos promoter activity, synergistic activation of the transcription factor, Elk-1, and ultimately enhanced AP-1 activity, which may increase the expression of many target genes that are critical for oncogenesis and/or tumor progression. Thus, it is conceivable that intrafollicularly generated E2 (e.g., via the prominent aromatase activity of the pilosebaceous unit) and prolactin cooperate in regulating, e.g., the transcription of important AP-1-dependent hair growth-modulatory genes.
Because ER activation may attenuate prolactin receptor signaling through STAT5a in various cell systems in vitro (351), the documented hair cycle-dependent changes in ER expression (28) may also exert counter-regulatory effects by down-regulating auto- or paracrine prolactin receptor-mediated signaling via the STAT5a pathway. Moreover, E2 has long been appreciated to serve as one of the major stimuli for pituitary prolactin synthesis and secretion, possibly primarily due to ER-mediated signaling (352). Therefore, it deserves to be explored whether intrafollicular E2 synthesis directly regulates the—again, strikingly hair cycle-dependent (8, 9)—intrafollicular expression of prolactin.
Recently, we have identified murine skin and human scalp hair follicles as potent sources of extrapineal melatonin synthesis (53). Because melatonin reportedly down-regulates ER expression (353) and is increasingly recognized as an antiestrogenic hormone (354), we assessed the effect of melatonin stimulation on ER expression in organ-cultured murine skin. ER expression was indeed significantly decreased after incubation with melatonin, at both gene and protein levels (including a notable decline in ER-like immunoreactivity in the dermal papilla of murine hair follicles in situ, and this in all key stages of hair follicle cycling) (53). This fits well with the recent finding that melatonin, at physiological concentrations, decreases aromatase activity and expression in MCF-7 breast cancer cells in vitro (354). Thus, locally generated melatonin may operate as an intrafollicular antiestrogenic hormone and contribute to the control of hair follicle cycling by down-regulating ER expression and E2 synthesis.
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VI. Open Questions and Unmet Clinical Challenges |
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VII. Conclusions and Perspectives |
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Although we hope to have presented a strong case for the claim that the hair follicle offers an ideal, clinically relevant test system for studying the role of sex steroids, their receptors, and interactions in neuroectodermal-mesodermal interaction systems in general, we have also charted the gaping abysses in our current understanding of how exactly estrogens impact on the hair follicle. The number of potential mechanisms of action and interactions with other signaling pathways already appreciated today is so daunting that we are threatened to drown in an ocean of possibilities. No matter, however, where the long-overdue renaissance of estrogen research in hair biology will carry us (e.g., to the identification of even more functions and signaling cross talks of ER-mediated signaling events in hair biology), there can be no doubt that estrogen-mediated signaling deserves much more attention in the future endocrinological therapy of common hair growth disorders than it is awarded to date.
Soberingly, the most important vision for the future of estrogen and ER-centered research in hair biology, quite simply, is that the key open questions summarized above need to be systematically addressed and definitively answered as soon as possible. And yet, it serves to underscore both the fascination and the importance of estrogens in hair research if one concludes this review with one selected vision, beyond this long and rather austere "to-do" list, which reflects one of the current authors’ foci of interest.
From a hair research perspective, perhaps the most intriguing aspect of estrogen biology is that these sex-steroids provide an unusually potent inhibitory mechanism for hair follicle cycling, namely by arresting the hair cycle clock in telogen (28, 35). At least in rodent models, topically applied estrogens are only rivaled by glucocorticoids (113, 355) in their potency as a telogen clamp and catagen inducer (26, 27, 28, 250, 272). Like estrogens, cortisol can even be synthesized in the hair follicle itself, where it stimulates glucocorticoid receptors (52). Intriguingly, ER receptor expression peaks in the murine follicular dermal papilla during telogen. This makes it reasonable to propose that ER-mediated signaling, possibly in conjunction with GR-mediated signaling, plays a key role in telogen induction and maintenance.
This inhibitory mechanism for the telogen-anagen transition calls to mind the "chalone hypothesis" proposed more than half a century ago by Herman B. Chase (34, 90). According to this hypothesis, hair follicle cycling is controlled by an inhibition/disinhibition system: anagen is proposed to be switched off by the gradual accumulation of an (unknown) endogenous inhibitor of proliferation (chalone), whose decay/loss of activity over time eventually releases the hair follicle from its chalone-supported block in the telogen stage. Very limited experimental support is available for this concept (356), and a number of theoretical arguments are invoked against this theory as the sole convincing explanation for hair follicle cycling. The chalone hypothesis is best integrated into more comprehensive theories of hair cycle control (35). Also, the original postulate that chalones represent species-independent, but tissue-specific negative regulators of proliferation, which are released by the differentiating cell layers in a given (e.g., epithelial) tissue so as to provide a negative feedback signal for the proliferating tissue compartment (90), has not been supported by sufficiently convincing evidence to assure this theory a place in current-day biological teaching.
And yet, although estrogens do not really meet the definition of a chalone (357), estrogens and their receptors surely are strong candidates for an inherent inhibitory control system of hair follicle cycling, whose rhythmic changes in activity/signaling may well provide a key component in the machinery of the elusive hair cycle clock (35). For example, regulated changes in ER expression and/or the controlled up-regulation and shutdown of ARO activity in cells adjacent to (or identical with) ER-positive cells in key hair follicle compartments could provide one possible mechanism of how anagen is terminated and reinitiated: i.e., by administration and removal of an ER-mediated brake on hair follicle cycling.
In this hypothetical scenario, a high level of E2 synthesis, e.g., in the (epithelial) secondary hair germ could stimulate ERs that are maximally expressed by the (mesenchymal) dermal papilla during catagen/telogen. Dermal papilla fibroblasts in turn would release additional telogen-inducing and -maintaining agents to keep the hair follicle arrested in telogen. At the same time, these follicles would be maximally receptive both to systemic E2 levels and to exogenous E2 administration. In this manner, global signals could impinge upon the cyclic activity of this estrogen-entrained system, not unlike what has already been demonstrated for other hormones (52).
Because the 5' region of certain mRNAs is critical for their stability, signaling events can change their decay rate and therefore the protein level (358). This has also been shown for ARO mRNA stability. Because the gene produces different mRNA products that vary in their untranslated region, the versatility of the regulation at the ARO promoter in the hair follicle is of major interest, because it may serve as one basis for greatly fluctuating levels of intrafollicular E2 synthesis and may also underlie the site-specific regulation of telogen in various regions of the integument (23, 24, 25, 26, 27, 28). These are logical and very attractive targets for pharmaceutical intervention in the context of managing hair growth disorders, yet remain to be fully discovered by pharmaceutical industry (88).
Along with the power of mouse genomics, employing, e.g., specific ER-isoform knockout mice to dissect the individual contribution of key elements of E2-induced signaling (28), organ culture of microdissected human scalp hair follicles provides an excellent, physiologically relevant tool for addressing these issues. The way ERs access their target genes is regulated at many cellular levels: transcriptional regulation of the receptors themselves at their promoters as well as alternative splicing of their transcribed products; activation of receptors by ligands or posttranslational modification of receptors by, e.g., phosphorylation; cofactor availability determining interaction with DNA; and stability of the expressed receptor proteins.
Beyond hair follicle cycling, the secretion of intrafollicularly generated estrogens may also play a key role in the enigmatic controls of hair waves, i.e., in the synchronized switches of very large hair follicle populations from one stage of the hair cycle to another; those hair waves are most prominently observed in mouse and rat skin, but they are also present in fetal human skin and can occur in adult human skin in the context of certain endocrine disorders (27, 28, 30). Hair waves and hair follicle cycling are quite distinct phenomena (34, 35, 50, 359, 360). In any case, as classical, relatively stable endocrine signals, the controlled secretion of estrogens released from an individual hair follicle would seem like an ideal signaling instrument for executing the synchronization of hair follicle cycling within a defined skin area limited by the range of E2 diffusion. Because hair wave phenomena in murine back skin can be altered by topical administration of selective ER ligands, and because effects on the hair wave pattern can be assessed and quantitated (e.g., by dot matrix planimetry) (27, 28, 359, 360), the manipulation of hair waves by ER ligands offers an attractive model for biologists with a general interest in wave phenomena.
From a bird’s-eye perspective on cutaneous estrogen biology, the most prominent function of estrogens in hair growth control, thus, may well be that of a molecular "brake" that entrains the hair cycle clock and contributes to hair wave phenomena. This pattern of timed self-renewal of organ compartments by cyclically halting proliferation and tissue self-renewal until estrogen activity and/or ER-mediated signaling have dropped below a threshold value is likely of great relevance to understand the regulation of many other complex tissue interaction systems and organs by estrogens. Clearly, the hair follicle offers a sterling model for the exploration of this important avenue of estrogen research.
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Acknowledgments |
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Footnotes |
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Possible conflict of interest: J-Å G is cofounder, deputy board member, consultant, grant receiver, and shareholder of KaroBio AB.
First Published Online July 28, 2006
1 U.O. and M.U. contributed equally to this review. 
Abbreviations: AF, Activation function; AP-1, Activating protein-1; BMP, bone morphogenetic protein; BMPR, BMP receptor; E2, 17ß-estradiol; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; ERE, estrogen response element; FGF, fibroblast growth factor 5; HGF, hepatocyte growth factor; IFN-, interferon-; IR, immunoreactivity; NGF, nerve growth factor; PPAR, proliferator-activated receptor; REA, repressor of estrogen action; SRC, steroid receptor coactivator; VEGF, vascular endothelial growth factor.
2 Only a subset of proteins will be presented; these were selected due to the fact that they are more or less well documented to be involved in hair follicle morphogenesis, proliferation, or cycling. It should be noted here that the original data were not collected from skin or skin-derived cell populations but are from very diverse origin and multiple different vertebrate species.
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