Androgen Regulation of the Human Hair Follicle: The Type I Hair hHa7 Is a Direct Target Gene in Trichocytes

Luis F. Jave-Suarez,à Lutz Langbein,w Hermelita Winter,à Silke Praetzel,w Michael A. Rogers,à and Juergen Schweizerà 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 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/ 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 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 chimpan- zee 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). (B) Light microscopic examination of (from left to right) beard hair, axillary hair, 3C). The rather weak hHa7 staining in medullary cells was pubic hair, and occipital hair for the presence of a medulla. due to the formaldehyde fixation of the section. While being 122 : 3 MARCH 2004 ANDROGEN RECEPTOR TARGET GENES IN THE HUMAN HAIR FOLLICLE 557

Figure 2 Demonstration of hHa7 in vellus and sexual hair follicles by IIF. Longitudinal cryostat sections of two human scalp vellus hairs from the temple region (A,B), beard hairs (C,D,F–H), and a chimpanzee hair (E) are shown. Sections A–C, E, IIF study with the hHa7 antiserum; sections F–H, double label IIF study with the hHa7 antiserum (green) and the hHa1 antibody (red). Note the lack of hHa7 expression in one of the vellus hairs (A). The white arrowheads in C indicate single hHa7-positive cortex cells. Section D is a phase-contrast micrograph of a beard hair. The white closed arrows in C, D, and G denote vertically oriented cells in the center of the medulla, which appear negative for hHa7 but show minor expression of this keratin upon double label IIF with the hHA1 antibody (F ). The white open arrows in G indicate hHa1-positive cortex cells extending into the network of medullary cells. The broken line in C, F–H indicates the boundary between the dermal papilla and the precortex. Sections A–C and E were stained with DAPI. med, medulla; co, cortex; dp, dermal papilla. Scale bars ¼ 100 mm.

a prerequisite for optimal AR demonstration, this fixation promoters (Jave-Suarez et al, 2002). Homology search for method visibly compromised the detection of the keratin. the semipalindromic AR consensus binding sequence A GG /TACAnnnTGTTCT (Roche et al, 1992) revealed the Identification of three putative AREs in the hHa7 presence of three putative AREs, designated as ARE-1, promoter Figure 4a shows schematically an about 1.4 kb ARE-2, and ARE-3 (Fig 4A). We did not detect further sequence region upstream of the TATA box of hair keratin putative ARE sequences upstream of the 1.4 kb promoter. gene hHa7 whose numbering refers to the published gene Compared with the ARE consensus sequence, the putative sequence (Rogers et al, 1998). Like the proximal promoters hHa7 AREs deviated mainly in their second half side, in of type I hair keratin genes hHa1–hHa6 and hHa8 (Rogers which ARE-3 differed in three positions, while ARE-1 and et al, 1998), the hHa7 promoter contains a LEF-1 binding ARE-2 differed in four positions. In addition, ARE-1 site (arrowheads), as well as a HOXC13 binding site (white exhibited a G ! A change in the second position of the boxes) which is also present in the hHa5 and hHa2 first half side (Fig 4B). 558 JAVE-SUAREZ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 3 Demonstration of AR expression in beard hair follicles by IIF. Longitudinal cryostat sections of beard hair follicles probed with an AR antibody (A,B). Sections A0 and B0 are the corresponding DAPI stainings. Section C shows a double label IIF study with the AR antibody (green) and the hHa7 antiserum (red). The lack of AR staining in the dermal papilla region was due to the loss of the lower bulb region below the dotted line during plucking of this follicle. Note that all sections were fixed with formaldehyde, which weakens the staining of the hHa7 keratin in panel C. med, medulla; co, cortex; dp, dermal papilla. Scale bars ¼ 100 mm.

visualization of the normally weak ARE-1/AR complex (Fig 5A, third lanes). Most probably, the prominent uppermost shifted complex originated from a reaction of the antibody with dimeric AR/oligonucleotide complexes while the lower and weaker shifted band may stem from a few monomeric AR/oligonucleotide complexes. Identical results were ob- tained with nuclear extracts of PC3-ARwt cells, exhibiting constitutive AR expression (results not shown). The functionality of the three hHa7 AREs was further assessed by transient transfection studies. As in our studies on the role of HOXC13 in hair keratin gene regulation (Jave- Suarez et al, 2002), we were faced with the problem that at present, trichocytes, i.e., hair-forming cells of the central Figure 4 hair follicle, can neither be cultured nor do immortalized hHa7 promoter fragments and hHa7 AREs. (A) Schematic presenta- trichocyte lines exist. Therefore, we decided to perform the tion of the 1.4 kb hHa7 promoter and various deletion fragments transfection experiments in both AR-expressing PC3-Arwt thereof. The numbering of the promoter refers to the published gene sequence (Rogers et al, 1998; accession number Y16793). The prostate cells and AR-deficient PtK2 cells (Jave-Suarez positions of the three androgen-responsive elements ARE-1, ARE-2, et al, 2002). To this purpose, we first generated several AR- and ARE-3 are indicated. The white box denotes the position of the responsive control constructs. Thus, construct pMMTV-gal LEF-1 site, while the arrowhead indicates that of the HOXC13 binding site (Rogers et al, 1998; Jave-Suarez et al, 2002). (B) hHa7 ARE contained 235 bp of the AR-responsive MMTV promoter, sequences compared with the ARE consensus sequence (Roche et al, cloned in front of the -galactosidase gene of vector pNass 1992) and the ARE-III sequence of the prostate-specific antigen, PSA (Fig 6). This MMTV promoter region comprised the long (Riegman et al, 1991; Cleutjens et al, 1997; Zhang et al, 1997a, b). terminal repeat (LTR), which harbors several steroid hormone-responsive elements (HREs), out of which the most upstream lying motif was previously found to function as the most active AR-responsive element (Cato et al, 1988; AR binds to the individual hHa7 AREs and enhances Gowland and Buetti, 1998). Based on these data, we deleted transcription In order to determine AR binding to the three 39 bp of the most 50 lying LTR sequences and cloned the putative hHa7 AREs, we performed electrophoretic mobility resulting DMMTV promoter variant (Fig 6) into the pNass shift assays (EMSA) using double-stranded oligonucleo- vector (construct pDMMTV-gal). In addition, we generated tides, each containing one of the individual hHa7 AREs in a construct pPSA-ARE-III-DMMTV-gal, containing a chi- central position. The oligonucleotides were assayed with meric promoter obtained by cloning two copies of PSA- nuclear extracts of PtK2 cells, which had been transiently ARE-III (Fig 4b), i.e., the strongest ARE of the prostate- transfected with the human AR expression vector pSG5- specific antigen promoter (Riegman et al, 1991; Cleutjens AR. While rather strong DNA/protein complexes were et al, 1997; Zhang et al, 1997a, b), in front of DMMTV in formed with the ARE-2 and ARE-3 oligonucleotides, the construct pDMMTV-gal. As expected, transfection of these DNA/protein complex obtained with the ARE-1 oligonucleo- constructs into PC3-ARwt cells in the presence of the tide was extremely weak (Fig 5A, second lanes; 5B, first synthetic androgen R1881 showed that relative to the lanes). In each case, DNA/protein complex formation could strongly activating intact MMTV promoter, the transactivat- be abolished by an excess of the respective unlabeled ing capacity of variant DMMTV was reduced by about 80% oligonucleotide (Fig 5B, second lanes), but remained (Fig 7A). Almost intact MMTV promoter transactivation unaffected by an unrelated DNA sequence (Fig 5B, third could, however, be restored upon transfection of construct lanes). Moreover, supplementation of the binding assays pPSA-ARE-III-DMMTV-gal (Fig 7A). Essentially identical with an AR antibody not only led to a supershift of the -gal activity profiles were obtained upon co-transfection of various DNA/protein complexes but also to a better the various constructs with the AR expression vector pSG5- 122 : 3 MARCH 2004 ANDROGEN RECEPTOR TARGET GENES IN THE HUMAN HAIR FOLLICLE 559

Figure 5 The androgen receptor binds to the three hHa7 AREs. (A) Double-stranded, end-labeled oligonucleotides containing the individual hHa7 ARE-1, ARE-2, and ARE-3 sequences were incubated with nuclear protein extracts of untransfected (first lanes) or pSG5-AR-transfected PtK2 cells (second lanes). The indicated oligo- nucleotides were further incubated with nuclear protein extracts of pSG5-AR-trans- fected PtK2 cells, to which a polyclonal antibody against the human AR was added (third lanes). (B) End-labeled hHa7 ARE oligonucleotides, incubated with nuclear extracts of pSG5-AR-transfected PtK2 cells (first lanes), were competed with a 100-fold excess of the corresponding unlabeled oligonucleotides (second lanes) or a random oligonucleotide (third lanes). DNA/protein binding complexes are mar- ked by arrowheads; the two supershifted complexes are denoted by asterisks.

Figure 6 Mouse mammary tumor virus, MMTV, promoter sequence. The shown sequence corresponds to the proximal MMTV promoter region, ranging from nucleotide 1 (transcription start site) to nucleotide À235 (large bent arrow) (Cato et al, 1988; Gowland and Buetti, 1998). Sequences 236–264 are part of the cloning site of the pNass vector. Underlined HRE indicates steroid hormone-responsive elements lo- cated within the long terminal repeat, LTR, which is indicated between small bent arrows. DMMTV denotes a deletion variant of the promoter.

AR into PtK2 cells (results not shown). This demonstrated that the functionality of putative AREs can be assessed by substituting the most 50 lying HRE motif of the MMTV-LTR with the elements in question. Therefore, we generated Figure 7 Transient transfection experiments. (A) The prostate cell line PC3- constructs pARE-1-DMMTV-gal, pARE-2-DMMTV-gal, ARwt, exhibiting constitutive AR expression, was transiently trans- and pARE-3-DMMTV-gal by replacing the duplicated fected with pNass reporter constructs pMMTV-gal (MMTV ), PSA-ARE-III sequence of construct pPSA-ARE-III- pDMMTV-gal (DMMTV ), pPSA-ARE-III-DMMTV-gal (PSA-DMMTV ), and the pNass vector alone (pNass), either in the absence (gray DMMTV-gal with duplicated versions of the three hHa7 columns) or presence of the synthetic androgen R1881 (white columns). AREs (Fig 4B). Transfection of these constructs into PC3- (B) PC3-ARwt cells were transiently transfected with pNass reporter ARwt cells in the presence of R1881 showed that all three constructs pMMTV-gal (MMTV), pARE-1-DMMTV-gal (ARE-1), hHa7 AREs displayed distinct transactivating capacities in pARE-2-DMMTV-gal (ARE-2), pARE-3-DMMTV-gal (ARE-3), and pARE-1as-DMMTV-gal (ARE-1as), either in the absence (gray col- the order ARE-3 > ARE-2 ffi ARE-1 (Fig 7B). In each case umns) or the presence of the synthetic androgen R1881 (white the observed -gal activity exceeded that of control columns). Results are given as x fold -galactosidase activity relative construct pMMTV-gal and was also much higher than that to control (average of three experiments Æ SD). obtained with the pPSA-ARE-III-DMMTV-gal construct (Fig 7A). As expected for transcriptional enhancers, con- structs containing the three hHa7 AREs in reverse orienta- The endogenous hHa7 promoter is activated by the tion also yielded a strong activation of the reporter gene. AR The 1.4 kb region of the endogenous hHa7 promoter, This is representatively shown for construct pARE-1as- containing all three AREs, as well as four progressively DMMTV-gal, which exhibited an even stronger reporter deleted hHa7 promoter fragments with sizes of 1.0, 0.6, 0.3 gene activation than its sense analog (Fig 7B). and 0.2 kb, respectively (see Fig 4A), were cloned into 560 JAVE-SUAREZ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 8 Transient co-transfection experi- ments. (A) PtK2 cells were transiently co-transfected with expression vector pSG5-AR and pNass reporter con- structs of the hHa7 promoter frag- ments p0.2a7-N-p1.4a7-N indicated in Fig 4. Construct p3D1.4a7-N con- tains a 1.4 kb promoter version in which the three AREs were specifically deleted. Constructs p0.9a2-N and p0.9a5-N contain the proximal pro- moter regions of hair keratin genes hHa2 and hHa5, respectively (Rogers et al, 1998). MMTV represent control construct pMMTV--gal. Transfections were performed either in the absence (gray columns) or presence of R1881 (white columns). (B) The co-transfec- tion series described in A were re- peated with the GR expression vector pSG5-AR either in the absence (gray columns) or presence of dexametha- sone (white columns). Results are given as x fold -galactosidase activity relative to control (average of three experiments Æ SD).

vector pNass and designated as p1.4a7-gal through ated with an expression vector for the human glucocorticoid p0.2a7-gal (Fig 8A). Surprisingly, upon transfection into (GR) receptor in the absence or presence of dexamatha- AR-expressing PC3-ARwt cells, control construct MMTV- sone. As shown in Fig 8B, except for a generally slightly gal, but virtually none of the hHa7 promoter constructs, higher -gal activity, the GR/dexamethasone-mediated was able to transactivate the reporter gene in the presence transactivation profile of the hHa7 promoter variants of R1881. As these negative results were also obtained was an exact duplicate of that obtained with AR and upon transfection into LNCaP cells (results not shown), the R1881 (Fig 8A). hHa7 promoter analysis was performed in PtK2 cells. Figure 8A shows that co-transfection of the various hHa7 promoter constructs with AR expression vector pSG5-AR in the Discussion presence of R1881 led to a -gal activation profile that mirrored those obtained with the individual AREs in the Out of the 15 members of the human hair keratin multigene MMTV promoter context (Figs 7B, and 8A). While we family (Rogers et al, 1998, 2000), the type I hair keratin hHa7 observed only a minor reporter gene activation for the was particular in that we failed to demonstrate its expres- ARE-1- and ARE-1/ARE-2-containing fragments, the sion in terminal scalp hair follicles but were able to detect it fragment harboring ARE-3 in addition exhibited a signifi- in the center of the cortex of some, but not all, vellus hairs of cantly higher transactivating capacity (Fig 8A). As expected, the scalp (Langbein et al, 1999). In this paper we have the effect of the three hHa7 AREs could completely shown that besides its sporadic expression in scalp vellus be abolished by their specific deletion in construct hairs, hHa7 is constitutively expressed in human sexual p3D1.4a7-gal, which behaved like constructs p0.9a2-gal hairs. In beard hairs, which we have chosen as an easily and p0.9a5-gal, containing the ARE-less promoter re- available model for this hair type, hHa7 is a constituent of gions of hair keratin genes hHa2 and hHA5, respectively the cells of the medulla which is generally present in sexual (Fig 8A). Since AREs of the imperfect palindromic repeat hairs. type are also potential binding sites for other steroid The medulla of mammalian hair represents a vertical cell hormone receptors (Roche et al, 1992), the co-transfec- column in the center of the hair cortex, which begins directly tion studies of the hHa7 promoter constructs were repe- above the apex of the dermal papilla. Morphologically, it 122 : 3 MARCH 2004 ANDROGEN RECEPTOR TARGET GENES IN THE HUMAN HAIR FOLLICLE 561 consists of large, loosely connected cells that are arranged functional ARs, exhibit normal scalp hairs but do not horizontally in a ring-like fashion between the vertically develop sexual hairs (Randall et al, 1991). While numerous oriented cortex cells. When almost fully formed, cells of the investigations on AR expression in human hair follicles of medulla develop large intra- and intercellular spaces filled various body sites differ considerably with regard to AR with air that provide insulating capacities to animal hairs expression in the ORS, they are generally consistent about while this function has largely been lost in humans. its expression in the specialized fibroblasts of the dermal Moreover, medullary cells become wedged between cell papillae (Bla¨ uer et al, 1991; Choudhry et al, 1992; Kimura projections obviously originating from adjacent cortical cells et al, 1993; Liang et al, 1993; Hodgins et al, 1998). This AR (Mahrle and Orfanos, 1971; Montagna and Parakkal, 1974). expression pattern has led to the current concept that Indeed, we were able to show that these cells expressed androgen actions in the hair follicle proceed predominantly the cortex keratin hHa1 instead of hHa7. In most beard via hormone/receptor complex-mediated alterations of hairs, we also noticed spindle-shaped vertical hHa1- gene expression in dermal papilla cells, leading to paracrine positive cells in the center of the medulla, which exhibited factors that appropriately modify gene expression and a weak co-expression with hHa7. Most probably, these cells properties of both follicular keratinocytes of the root with essentially cortical properties have partially escaped sheaths and trichocytes of the hair-forming compartment. the molecular signals which, in the course of the gradual Examples for such paracrine factors are the insulin-like transformation of facial vellus hairs into terminal beard hairs growth factor IGF-I and TGF-1 (Philpott et al, 1994; Itami during puberty, switched the commitment of the basal et al, 1995; Inui et al, 2003). trichocytes above the apex of the dermal papillae from a The novel finding in beard hairs that, besides the nuclei cortical to a medullary program of differentiation. of dermal papillae cells, AR is also expressed in the nuclei of At present, the few molecular constituents known to be lower medullary cells suggested to us that the medulla- expressed in medullary cells comprise trichohyalin (Tarcsa specific expression of the hHa7 gene might be regulated by et al, 1997), peptidyl-arginine deaminase III (Nishijyo et al, AR. Indeed, unlike the promoters of virtually all other hair 1997), transglutaminases 1 and 3 (Tarcsa et al, 1997), and keratin genes, the hHa7 promoter contained three se- corneodesmosin. While early electron microscopic studies, quence motifs, ARE-1, ARE-2, ARE-3, with a high homology A as well as X-ray and biochemical analyses, suggested the to the semipalindromic ARE consensus sequence GG /T- occurrence of only few, if any, a-keratins in the medulla of ACAnnnTGTTCT (Roche et al, 1992). EMSA with ARE-1, human hair (Rudall, 1947; Swift, 1969; Mahrle and Orfanos, ARE-2, and ARE-3 oligonucleotides using nuclear extracts 1971), subsequent IIF studies with type I and type II keratin from PC3-ARwt or pSGR5-AR-transfected PtK2 cells antibodies clearly revealed the presence of a variety of consistently revealed one specific band of retarded mobility, epithelial keratins of both types (Heid et al, 1988; McGowan which could be supershifted by an AR antibody. Insertion of and Coulombe 2000). Our present study identified the type I the individual ARE sequences in front of a modified MMTV hair keratin hHa7 as a further constituent of medullary cells promoter with a drastically reduced androgen-mediated of sexual hairs. Moreover, in our previous hair keratin transactivating capacity and transient transfection of the expression studies, the type II hair keratins hHb1, hHb5, corresponding -gal reporter gene constructs into AR- and hHb6 could be detected in the medulla of beard hairs containing or -co-transfected cell lines in the presence of (Langbein et al, 2001). The expression of these hair keratins the synthetic androgen R1881 yielded reporter gene activity solves the problem of putative type II partners of hHa7 for IF profiles whose extent mirrored the degree of deviation of formation in medullary cells. Collectively, these data indicate the individual hHa7 AREs from the ARE consensus that out of the various tissue compartments of the human sequence. Surprisingly, while the individual AREs in the hair follicle, the medulla is unique in the co-expression of MMTV promoter context enhanced the expression of the epithelial and hair keratins. However, while the epithelial and reporter gene in PC3-ARwt prostate cells, the endogenous hair keratins identified so far in the medulla are consistently hHa7 promoter containing all three AREs completely failed expressed in other compartments of the hair follicle, hHa7 to activate the reporter gene upon transfection into both seems to be specific for the medulla. PC3-Arwt and LNCaP cells. Considering that the prostate In humans, the coarse, strongly pigmented, and medul- represents another organ in which AR plays a crucial gene- lated sexual hairs arise gradually during puberty from fine, regulatory role, we speculate that the inability to activate the uncolored vellus hairs devoid of a medulla. This ‘‘vellus- hHa7 promoter through R1881 in prostate cell lines might terminal hair switch’’ is precipitated by an increase in be due to a putative inhibitor or the lack of a cofactor in both circulating androgens either bound to sex hormone binding prostate and prostate tumors, which prevents aberrant hair globulin or albumin (Ebling, 1987; Randall, 1994; Kaufman, keratin expression. In contrast, co-transfection of consecu- 1996; Stenn et al, 1996). The intimate relationship between tive ARE deletion variants of the hHa7 promoter and an the androgen-dependent development of terminal sexual AR-expression vector into PtK2 cells showed that in the hairs and the formation of a medulla is testified by the presence of R1881, ARE-3 contributed most to the observation that treatment of women suffering from activation of the reporter gene. The transactivation profiles idiopathic hirsutism with antiandrogenic steroids drastically of the three hHa7 AREs were strikingly reminiscent of those reduces the abnormal size of facial and body hairs observed previously for the promoter of the prostate- concomitant with the reduction of their extent of medullation specific antigen gene, PSA. In both cases, two proximal (Ebling, 1987). The androgen-mediated growth of sexual AREs possessed minor transactivating capacities (Riegman hairs requires binding of testosterone or dihydrotestoster- et al, 1991; Zhang et al, 1997a,b), while a significant reporter one to the AR. Testicular feminization patients, who lack gene activation was only exerted by the most distal ARE 562 JAVE-SUAREZ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

(Cleutjens et al, 1997; Zhang et al, 1997a,b). In line with the hHa7 is present in variable amounts in about 10% of these known interaction of semipalindromic ARE sequences with terminal hair samples (unpublished data). The presence of a other steroid hormone receptors, the endogenous hHa7 small, seemingly androgen-mediated vellus and terminal promoter could also be activated by the glucocorticoid hair population in the normal human scalp bears consider- receptor, GR. Confirming previous GR expression studies able significance in regard to hair follicle miniaturization however (Karstila et al, 1994), this steroid hormone receptor during androgenetic alopecia. It would therefore be inter- could only be demonstrated in upper ORS cells of beard esting to further investigate hHa7 expression patterns hair follicles (data not shown). Furthermore, the complete in the various hair types of the scalp during and after insensitivity of the hHa7 promoter to R1881 in AR- puberty as well as in various stages of androgenetic transfected PtK2 cells observed after the specific deletion alopecia. of the three ARE sequences definitely characterized them as functional AR regulatory elements. In conclusion, while the prevailing concept of androgen Materials and Methods action on hair follicles suggests AR target genes primarily in Antibodies Primary mono- or polyclonal antibodies/sera against fibroblasts of the dermal papilla, our study has identified the the following antigens were used: hHa7, guinea-pig polyclonal hHa7 gene as the first AR target gene in trichocytes of antibody, Enhanced Chemo Luminiscence (ECL) dilution 1:2000, sexual hairs. We do, however, not exclude that, following AR indirect immuno fluorescence (IIF) dilution 1:1000 (Langbein et al, induction, further transcription factors might be involved in 1999); hHa1, mouse monoclonal antibody LHTric-1, IIF 1:50 the control of medullary hHa7 expression. We have (Westgate et al, 1997; Langbein et al, 1999); human glucocorticoid receptor (GR), mouse monoclonal IgG antibody NCL-GCR (Novo previously shown that in beard hairs the homeobox protein 2a Castra, Dossenheim, Germany), IIF dilution 1:20. Among several HOXC13 is present in the nuclei of matrix, precortex and commercially available antibodies against the human androgen lower medulla cells. In vitro, HOXC13 is able to activate the receptor (AR) tested, we selected the rabbit polyclonal antibody hHa7 promoter through binding to highly specific response AR(N-20) from Santa Cruz Biotechnology (Heidelberg, Germany), elements (Jave-Suarez et al, 2002). Whether the LEF-1 site which was used for IIF studies at a dilution of 1:50. The specificity in the hHa7 promoter (Rogers et al, 1998) is functional in of the AR antibody in IIF studies was confirmed by competition vivo is questionable. Recent investigations in our laboratory with the commercially available blocking peptide sc-816P accord- ing to the instructions of the manufacturer (Santa Cruz Biotechnol- have not provided evidence for nuclear LEF-1 in the medulla 1 ogy). For supershift analyses (see below), a rabbit polyclonal of human hair. Moreover, although the three AREs are fully antiserum raised against the first 20 amino acids of the human AR preserved in the promoter of the chimpanzee ortholog of the was used. This antiserum was kindly provided by Dr Frank hHa7 gene (data not shown), its expression in the cortex of Claessens (Leuven, Belgium). The secondary antibodies were the androgen-independent body hairs of this species shows anti-mouse, anti-rabbit and anti-guinea-pig coupled to Cy3 (IIF that the gene is not necessarily androgen-dependent and dilution 1:500), Cy2 (IIF dilution 1:100) (Dianova, Hamburg, that its sensitivity to androgens has been acquired in the Germany), Alexa 568 or Alexa 488 (IIF dilution 1:200) (MoBiTec, Go¨ ttingen, Germany). Chemoluminescence (Amersham Pharmacia human lineage. Biotech) goat and guinea-pig IgG(H þ L) were used as secondary The identification of medullary hHa7 as a molecular antibodies at a dilution of 1:10,000 (Dianova). marker for androgen action on human hairs cannot leave the initial observation of sporadic hHa7 expression in a Cell lines Rat kangaroo kidney epithelial cells PtK2 (ATCC medulla-like structure within the cortex of vellus hairs of the number, CCL-56); PC3-ARwt cells (derived from the androgen- human scalp uncommented on. It is known that vellus hairs independent prostate cell line PC3 by stable transfection of the human AR), kindly provided by Dr A. Cato, Institute of Genetics and constitute a minor hair population of the healthy human Toxicology, Nuclear Research Center, Karlsruhe, Germany; LNCaP adult scalp (Montagna and Parakkal, 1974). Interestingly, cells (ATCC number, CRL-1740). vellus hairs are invariably seen also in the scalp of children. It has been observed that these juvenile vellus hairs become One-dimensional Western blotting Hair keratins were extracted rare in late puberty, suggesting that they are transformed from clipped beard hair as well as from male and female axillary into terminal hairs. Indicating the reverse pathway, however, and pubic hair as described (Langbein et al, 1999, 2001). One- dimensional sodium dodecylsulfate polyacrylamide electro- vellus hairs slowly reappear in the twenties and seem to phoresis (SDS-PAGE, 10% polyacrylamide) and western blot remain constant in the healthy adult scalp (Montagna and procedures using the hHa7 antiserum were performed essentially Parakkal, 1974). Based on their timing, most probably these as described (Langbein et al, 1999, 2001). processes occur under the influence of androgens. This would imply that besides vellus hairs, hHa7 expression IIF IIF studies were performed on cryostat sections of plucked should also be sporadically seen in adult terminal scalp human beard hair follicles or adult chimpanzee skin (kindly provided by Dr Peter Heidt, Primate Research Center, Rijskwijk, hairs either originating from the observed vellus-terminal The Netherlands). Detection of hair keratins using the hHa7 and hair switch or representing the targets of its later reversal. hHa1 antiserum/body was carried out essentially as described Indeed, contrary to our initial findings in a few adult terminal (Langbein et al, 1999, 2001). Briefly, tissue sections were fixed for hair samples, we were recently able to show, by a large- 10 min in À201C acetone. After briefly rinsing in phosphate- scale hHa7 western blot analysis of keratin extracts of more buffered saline (PBS), the samples were permeabilized for than 70 individual adult distal hair probes of both sexes, that 1 minute in PBS containing 0.1% Triton X100 and rinsed in PBS. Primary and secondary antibodies were applied for 30 min with intermittent washing steps using TBST. For demonstration of the 1Cribier B, Peltre B, Langbein L, Grosshans E, Schweizer J: steroid receptors, the cryosections were exposed to a 5 min Regulation of hair keratin gene expression: Lessons from studies in fixation step in freshly prepared phosphate-buffered formaldehyde pilomatricomas. In press. (2% p-formaldehyde), followed by a 5 min blocking step with 122 : 3 MARCH 2004 ANDROGEN RECEPTOR TARGET GENES IN THE HUMAN HAIR FOLLICLE 563

50 mM ammonium chloride for 5 min, both at room temperature. forward primers: The treatment of the samples with detergent was omitted. It should 50-ccattgaattcatgctggtctcgaactcctgac-30 (1.4 kb fragment), be noted that the optimal demonstration of hair keratins and 50-tcatcgaattcttgaggcaatgagtaaccacct-30 (1.0 kb fragment), steroid receptors required distinctly different fixation procedures 50-tgtttgaattcgttattgcttctcaagggagta-30 (0.6 kb fragment), (see also Results). IIF results were documented with a photo- 50-agagcgaattcgggcctgaatcaactgcggtg-30 (0.3 kb fragment), microscope (Axiophot 2, Carl Zeiss, Jena/Oberkochen, Germany). 50-aagaggaattcagatttgtcaacattgctttaatt-30 (0.2 kb fragment). Common reverse primer: Preparation of nuclear extracts PC3-ARwt cells and PtK2 were 50-ggctgctcgagtgcttcagatcagctgggaaggc-30. cultured in RPMI-1640 and DMEM medium (Sigma, Frieburg, Germany), respectively. Ptk2 cells were transfected (see ‘‘Transient transfections’’) with the human AR, expression vector pSG5-AR, The primer pairs used for the generation of 0.9 kb promoter kindly provided by Dr Claessens (University of Leuven, Belgium). fragments of hair keratin genes hHa5 and hHa2 have been Cells were harvested 48 h after transfection. One hour before described elsewhere (Jave-Suarez et al, 2002). After EcoRI/XhoI harvesting, both PC3-Arwt and PtK2 cells were supplemented digestion of the PCR products, the gel-purified fragments were with 100 nM of the synthetic androgen methyltrienolone, R1881 cloned into the EcoRI/XhoI digested vector pNass. The cloned (Invitrogen, Karlsruhe, Germany). The extraction of nuclear PCR fragments and the promoter/vector junctions were verified by has been described in detail (Jave-Suarez et al, 2002). Nuclear DNA sequencing. Subsequently, construct p1.4a7-gal was used extracts were subjected to protein quantification and stored in for the specific deletion of the three putative ARE sequences. aliquots at À801C. Primer pairs for the deletion of the three AREs were ARE-1: Electrophoretic mobility shift and supershift assays Oligonu- 50-ccattgaattcatgctggtctcgaactcctgac-30, cleotides, each containing one of the putative androgen receptor- 50-actatgggcccaacaaacaccagcagttgattc-30, binding elements (AREs) of the hHa7 promoter were synthesized 50-tctgagggcccaccaacagctaacctgatatgg-30, as overhanging complementary strands, annealed and end-labeled 50-ggctgctcgagtgcttcagatcagctgggaaggc-30. with [g-32P]ATP and T4 polynucleotide kinase. Oligos for ARE-1 ARE-2: were 50-ttgttgagcagaaacagtgagtctctgactt-30 and 50-tgggaagtcaga- 50-ccattgaattcatgctggtctcgaactcctgac-30, gactcactgtttctgctca-30; oligos for ARE-2 were 50-tgatgaacagggaa- 50-gttccggtaccatcaggagtaaacaatgcctc-30, cattgcgtgtccaggaa-30 and 50-ctggttcctggacacgcaatgttccctgttc-30; 50-gtcctggtaccagcacactgcctgagttatggg-30, oligos for ARE-3 were 50-ttcctttgctggaacacaaagaccttcagta-30 and 50-ggctgctcgagtgcttcagatcagctgggaaggc-30. 50-aaattactgaaggtctttgtgttccagcaaa-30. The conditions for electro- phoretic mobility shift assays have been described previously in ARE-3: 0 0 detail (Jave-Suarez et al, 2002). Gels were dried and autoradio- 5 -ccattgaattcatgctggtctcgaactcctgac-3 , 0 0 graphed. To ascertain binding specificity, a 100-fold excess of 5 -tgtgatccggaaaaggaaaagatttaaatacta-3 , 0 0 unlabeled specific or unspecific competitor oligonucleotides were 5 -gaccttccggaattttcttttcaccacaatttc-3 , 0 0 incubated with the protein extract prior to the addition of the 5 -ggctgctcgagtgcttcagatcagctgggaaggc-3 . labeled oligonucleotide. For supershift assays, 1 mL of a rabbit The resulting construct was designated as p3D1.4a7-gal. polyclonal AR antiserum (see ‘‘Antibodies’’) was preincubated with nuclear extracts in binding buffer for 30 min on ice prior to Transient transfections Due to the lack of both primary tricho- the addition of the labeled oligonucleotides in order to allow the cytes and trichocyte cell lines, transient transfection assays were formation of protein–antibody complexes. carried out either in AR-expressing cell lines PC3-ARwt and LNCaP MMTV control constructs and chimeric hHa7 ARE-MMTV or in AR-negative PtK2 cells (Jave-Suarez et al, 2002). Prostate constructs Vector pHCwt, kindly provided by Dr A. Cato (Institute cells were cultured in RPMI-1640 medium (Sigma), and PtK2 cells of Genetics and Toxicology, Nuclear Research Center, Karlsruhe, were cultured in DMEM medium (Sigma). In both cases, fetal calf Germany), was used to generate the AR-responsive control serum (FCS) deprived of steroids by charcoal filtration was used. 5 construct pMMTV-gal. The construct comprised a 235 bp Consistently, cells were plated at a density of 3 Â 10 cells/35-mm TM sequence region upstream of the transcription start of the mouse dishes. The FuGENE 6 transfection protocol (Roche Molecular mammary tumor virus gene, MMTV (Fig 6) (Cato et al, 1988), which Biochemicals, Mannheim, Germany) used for all cell lines has was cloned into the EcoRI/XhoI site of the -galactosidase reporter recently been described in detail for PtK2 cells (Jave-Suarez et al, vector pNass (Clontech, Heidelberg, Germany). Forward primer 2002). Briefly, 1 d after plating, PtK2 cells were co-transfected with 50-gatctgggcccaaggatgtgagacaagtggtttcc-30 and reverse primer 0.5 mg of the various reporter plasmids and 0.5 mg of either 50-tcctgaaaatctgccaagctc-30 were used to amplify a deleted androgen receptor expression vector pSG5-AR or glucocorticoid version of the proximal MMTV promoter, which lacked bp À163 receptor expression vector pSG5-GR (kindly provided by Dr F. to À235 (Fig 6). The PCR product was cloned into the ApaI/XhoI Claessens, University of Leuven, Belgium) or 0.5 mg pcDNA3, to sites of a modified pNass vector (containing an extended cloning bring the total amount of DNA to 1.0 mg. PC3Arwt and LNCaP cells site) to yield construct pDMMTV-gal. Constructs pARE-1-DMMTV- were only transfected with 1.0 mg of the various reporter plasmids. TM gal, pARE-1as-DMMTV-gal, pARE-2-DMMTV-gal, pARE-3- Each DNA mixture contained 2 mL of FuGENE 6 transfection DMMTV-gal, and pPSA-ARE-III-DMMTV-gal were generated by reagent. The synthetic androgen R1881 was added at a concen- cloning two copies of each of the three putative hHa7 AREs tration of 1 nM; dexamethasone was added at a concentration of or ARE-III of the prostate-specific antigen promoter (Riegman et al, 100 nM. Two days after transfection, cells were harvested, washed 1991; Cleutjens et al, 1997; Zhang et al, 1997a, b) in front of in PBS and incubated for 10 min in 200 mL of lysis buffer (60 mM pDMMTV-gal. Each of the individual AREs contained five endo- Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1mM MgSO4, 2.5 mM genous nucleotides flanking the ARE core sequences. EDTA, pH 8.3, 0.25% Nonidet P-40). Protein concentrations were determined using 10 mL of lysate. Each protein sample (20 mg) hHa7 reporter plasmids and hHa7 deletion constructs Promo- was incubated for 5 min in 200 mL of buffer A (60 mM Na2HPO4, ter fragments of the type I hair keratin gene hHa7 (GenBank, 40 mM NaH2PO4, 10 mM KCl, 1mM MgSO4, 2.5 mM EDTA, pH 8.3, accession number Y16793) were generated by PCR, using the 50 mM 2-mercaptoethanol) and supplemented with 50 ml of sub- previously described PAC3 clone (Rogers et al, 1998) as template strate solution (8 mM chlorophenol red–galactopyranoside (Roche DNA as well as forward primers containing an EcoRI site and Molecular Biochemicals) in buffer A). -Galactosidase activity was reverse primers containing a XhoI site. Primer pairs used for the determind at 570 nm. Transfections were performed in triplicate in generation of the various hHa7 promoter fragments were at least three independent experiments. 564 JAVE-SUAREZ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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