Androgen Regulation of the Human Hair Follicle: the Type I Hair Keratin Hha7 Is a Direct Target Gene in Trichocytes

Androgen Regulation of the Human Hair Follicle: The Type I Hair Keratin hHa7 Is a Direct Target Gene in Trichocytes Luis F. Jave-Suarez,Ã Lutz Langbein,w Hermelita Winter,Ã Silke Praetzel,w Michael A. Rogers,Ã and Juergen SchweizerÃ ÃSection of Normal and Neoplastic Epidermal Differentiation and wDivision of Cell Biology, German Cancer Research Center, Heidelberg, Germany Previous work had shown that most members of the complex human hair keratin family were expressed in terminal scalp hairs. An exception to this rule was the type I hair keratin hHa7, which was only detected in some but not all vellus hairs of the human scalp (Langbein et al, 1999). Here we show that hHa7 exhibits constitutive expression in medullary cells of all types of male and female sexual hairs. Medullated beard, axillary, and pubic hairs arise during puberty from small, unmedullated vellus hairs under the influence of circulating androgens. This suggested an androgen-controlled expression of the hHa7 gene. Further evidence for this assumption was provided by the demonstration of androgen receptor (AR) expression in the nuclei of medullary cells of beard hairs. Moreover, homology search for the semipalindromic androgen receptor-binding element (ARE) consensus sequence A GG /TACAnnnTGTTCT in the proximal hHa7 promoter revealed three putative ARE motifs. Electrophoretic mobility shift assays demonstrated the specific binding of AR to all three hHa7 AREs. Their function as AR-responsive elements, either individually or in concert within the hHa7 promoter, could be further confirmed by transfection studies with or without an AR expression vector in PtK2 and prostate PC3-Arwt cells, respectively in the presence or absence of a synthetic androgen. Our study detected hHa7 as the first gene in hair follicle trichocytes whose expression appears to be directly regulated by androgens. As such, hHa7 represents a marker for androgen action on hair follicles and might be a suitable tool for investigations of androgen-dependent hair disorders. Key words: androgens/differentiation/hair follicle/keratins J Invest Dermatol 122:555 –564, 2004 The human hair keratin family consists of nine type I and six gene grouping in the two hair keratin gene domains is type II members, whose genes are organized as distinct biologically relevant. For example, the highly related type I clusters within the type I and type II epithelial keratin gene and type II group A genes are all expressed in the cortex of domains on chromosomes 17q21.2 and 12q13.3, respec- the hair shaft. In contrast, with one notable exception, i.e., tively (Rogers et al, 1998, 2000). A comparison of the two hHb4, the unrelated type I and type II group C genes are hair keratin gene clusters reveals striking similarities in differentially expressed in either the hair cuticle, matrix, or regard to their physical organization into distinct gene cortex. Furthermore, the type I group B hair keratin hHa8 groups. Thus, one end of each domain contains a group A displays an unusual restriction of its expression to only few, of essentially three, structurally related genes (type I, hHa1, randomly scattered cortex cells (Langbein et al, 1999, hHa4, and isoforms hHa3-I and -II; type II, hHb3, hHb6, 2001). Consistently, all these hair keratins can be demon- hHb1), while the opposite ends of the clusters harbor three strated in terminal scalp hairs. One further exception to this structurally less related group C genes (type I, hHa6, hHa5, observation exists. Up to now, neither in situ hybridization hHa2; type II, hHb2, hHb4, hHb5). Moreover, the type I hair with a specific cDNA probe nor immunocytochemistry with keratin gene cluster contains three central, structurally a specific antibody have led to the detection of the type I related group B genes, which have no counterparts within group B hair keratin hHa7 in terminal hairs of human scalp. the type II gene domain (Rogers et al, 1998, 2000). Two of Instead, this hair keratin was only found in some of the small these genes, hHa7 and hHa8, are functional while the vellus hairs (Langbein et al, 1999), which constitute a minor remaining gene, jhHaA, represents a rare form of a hair population of the healthy human scalp (Montagna transcribed pseudogene, whose defect mRNA species are and Parakkal, 1974). In these hairs, hHa7 expression was not translated into protein in humans (Winter et al, 2001). restricted to the innermost cells of the lower to upper cortex Detailed expression studies provided evidence that the forming a small, ascending column within the hHa7-negative portion of the cortex. Morphologically, these central hHa7- positive cells were not distinct from the surrounding spindle- shaped cortex cells (Langbein et al, 1999). Abbreviations: AR, androgen receptor; ARE, androgen receptor- binding element; GR, glucocorticoid receptor; EMSA, electro- In accordance with these tissue expression data, we mobility shift assay were initially unable to demonstrate the hHa7 protein in a Copyright r 2004 by The Society for Investigative Dermatology, Inc. 555 556 JAVE-SUAREZ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY keratin extract of a scalp hair sample by western blotting. from clipped beard hairs, male and female axillary and Surprisingly, however, the hHa7 hair keratin was clearly pubic hairs as well as an occipital terminal hair sample, cut demonstrable as an 54 kDa protein in extracts of beard distinctly above the scalp surface. Western blots with the hairs (Langbein et al, 1999), which are routinely used in our hHa7 antiserum revealed that this keratin was strongly laboratories as an alternative source for easily available hair present in male and female sexual hairs, while it was not samples. Beard hairs arise from small vellus hairs during detectable in the scalp hair sample analyzed (Fig 1A, lanes puberty under the influence of circulating androgens and b–g). Figure 1B shows that all sexual hairs possessed a represent particularly coarse, strongly pigmented, and medulla that was essentially continuous in beard hairs, medullated hairs (Ebling, 1987; Randall, 1994). In the mostly fragmented in pubic and axillary hairs and absent present study, we show that hHa7 is generally expressed from the analyzed occipital hairs. IIF studies using the hHa7 in sexual hairs and provide evidence that its gene antiserum on cryostat sections of human scalp confirmed transcription is directly controlled by binding of the previous results on the varying expression of this keratin in a androgen receptor to response elements in the hHa7 central column of the cortex of vellus hairs ((4); Fig 2A,B). In promoter. contrast, sections of plucked beard hair follicles showed constitutive hHa7 expression in medullary cells (Fig 2C). The hHa7 expression in the medulla began directly above the Results dermal papillae where the lowermost medulla cells have not yet adopted the typical horizontal orientation of later hHa7 and the androgen receptor are co-expressed in the medulla cells, and lasted up to the height of the upper medulla of sexual hairs To learn more about the expres- cortex region. Very rarely, we observed isolated spindle- sion characteristics of hHa7, we extracted total keratins shaped cortex cells that were also positive for hHa7 (Fig 2c, white arrowheads). In addition, we noticed few vertically oriented, rather large cells in the center of the lower medulla (Fig 2C,D, white closed arrows) which seemed to be devoid of hHa7 (Fig 2C). Double label IIF using antisera against medullary hHa7 (green color) and the cortex keratin hHa1 (red color) revealed that the expression of the two keratins was mutually exclusive in the two tissue compartments (Fig 2F–H). An exception was the vertical cells in the center of the medulla. These cells co-expressed hHa1 and hHa7, although the latter was distinctly less abundant and co- located with hHa1 in a patchy manner (merged yellow color in Fig 2F ). Moreover, we observed hHa1-positive cortex cells or cortex cell protrusions that extended laterally into the medullary cell column (Fig 2F,G, white open arrows). Compared with human vellus and beard hair follicles, the expression pattern of the chimpanzee ortholog of hHa7 was completely different in the variably medullated body hair follicles of this species (Glaister, 1931). Figure 2E shows an unmedullated scalp hair follicle of an adult male chimpanzee in which the hHa7 ortholog was strongly expressed in the entire mid- to upper cortex region. IIF studies on beard hair follicles using an antibody against the human AR clearly demonstrated that this steroid hormone receptor was strongly expressed in the nuclei of nearly all medulla cells up to the mid-cortex region while the surrounding cortex cells exhibited background staining (Fig 3A,A0). In accordance with previous studies (Bla¨ uer et al, 1991;Choudhry et al, 1992; Kimura et al, 1993; Liang et al, 1993; Hodgins et al, 1998), nuclear AR was also seen, albeit to a varying extent, in dermal papilla cells (compare Fig 3A and B) and occasionally in basal and suprabasal cells of the upper, multilayered ORS. Exposure of the AR antibody with Figure 1 blocking peptide sc-816P resulted in a complete abolish- Identification of hHa7 by western blot analysis. (A) One dimension- ment of nuclear AR staining (results not shown). Double ally resolved keratin extracts (10% SDS-PAGE) from male beard hair (lane b), male (lane c) and female axillary hair (lane d), male (lane e) and label IIF with the hHa7 antibody (red color) and the AR female pubic hair (lane f ), and an occipital hair probe (lane g) were antibody (green color) showed that the keratin and the AR subjected to western blotting with the hHa7 antiserum. Control lane (a) were co-expressed in medullary cells, with AR being shows the Coomassie stained total beard hair keratin pattern (Type I nuclear and hHa7 displaying a cytoplasmic location (Fig HKs, type I hair keratins; Type II HKs, type II hair keratins).

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