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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 </
