行政院國家科學委員會專題研究計畫成果報告

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1 行政院國家科學委員會專題研究計畫成果報告 性別決定基因於男性雄性禿患者之表現 計畫類別 : 個別型計畫計畫編號 : NSC B 執行期間 : 91 年 08 月 01 日至 92 年 07 月 31 日執行單位 : 國立成功大學醫學系皮膚科 計畫主持人 : 陳文杰 計畫參與人員 : 許漢銘, 蔡仁雨, 楊朝鈞 報告類型 : 精簡報告 處理方式 : 本計畫可公開查詢 中華民國 92 年 10 月 1 日

2 性別決定基因於男性雄性禿患者之表現 The expression of sex-determining genes in male androgenetic alopecia 計畫編號 : NSC B 執行年限 :91 年 8 月 1 日至 92 年 7 月 31 日主持人 : 陳文杰助理教授國立成功大學醫學院皮膚科共同主持人 : 蔡仁雨醫師台北醫學大學萬芳醫院雷射中心許漢銘教授國立成功大學醫學院皮膚科計畫參與人員 : 楊朝均醫師國立成功大學醫學院皮膚科 中文摘要皮膚與性腺及腎上腺類似為一合成類固醇之器官 它具有所有性賀爾蒙合成與代謝所需之酵素 有關性別決定與性腺分化的分子控制在過去十年被廣泛的研究 本實驗欲了解性別決定基因在皮膚的表現以及在雄性禿致病機轉的角色 RT-PCR 的分析顯示 SF1 WT1 SRY SOX9 與 DAX1 在人類頭皮有表現, 而免疫組織學研究偵測到 DAX1 與 SOX9, 其中 SOX9 特別存在於汗腺 在西方墨點試驗的分析中, 男性雄性禿患者之禿髮部位與頭髮部位相較, 前者 SRY 與 DAX1 的表現較強 SRY 與 DAX1 的量似乎隨著禿頭程度嚴重而增加, 而 DAX1 的表現卻隨年齡增加而下降 有關 SRY 與 DAX1 在雄性禿的意義需要進一步大規模雄性禿族群的研究 關鍵詞 : 雄性禿 頭髮 性別決定基因 Abstract The skin is a steroidogenic organ similar to gonads and adrenal cortex. It possesses all the enzymes required for steroid sex-hormone synthesis and metabolism. The molecular control of sex determination and gonadal differentiation has been extensively investigated over the past ten years. This study aims to understand the expression of sex-determining genes in human skin and their roles in the pathogenesis of androgenetic alopecia (AGA). The expression of SF1, WT1, SRY, SOX9 and DAX1 was demonstrated in human scalp by RT-PCR, while immunohistochemical studies detected DAX1 and SOX9 with SOX9 especially localized in the eccrine sweat glands. In male patients with AGA, densitometric evaluation showed enhanced protein expression of SRY and DAX1 in bald fronto-parietal scalp as compared to hair-donating occipital scalp. SRY and DAX1 appeared to increase their expression as baldness advanced according to the Norwood-Hamilton scale, while the expression of DAX1 diminished seemingly with increasing age. Further studies on larger patient population are needed to elucidate the functional significance of SRY and DAX1 in androgenetic alopecia. Keywords: androgenetic alopecia, hair, sex-determining genes Background 2

3 The human skin, especially the pilosebaceous unit, can synthesize androgens from cholesterol de novo or by locally converting weaker circulating androgens to more potent ones. Like other classical steroidogenic organs, such as the adrenals and the gonads, the skin has been shown to express the major androgen-metabolizing enzymes, namely steroid acute regulatory protein (StAR), P450 side chain cleavage enzyme (P450scc), P hydroxylase, steroid sulfatase, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase, steroid 5α-reductase, 3α-hydroxysteroid dehydrogenase, and P450 aromatase (1,2). The locally produced androgens, especially 5α-dihydrotestosterone, are considered to play a key pathogenic role in most androgen-dependent skin diseases, such as acne, androgenetic alopecia (AGA) and hirsutism; local overproduction is crutial since most patients exhibit normal circulating androgen levels (3). This hypothesis is supported by the findings of enhanced expression of many of the androgen-metabolizing enzymes as well as the regional level of the androgen receptors in diseased tissues (2). However, linkage analysis of the genes encoding the two steroid 5α-reductase isoenzymes failed to show significant associations (4,5). Since the cloning of the Y-located testis-determining factor, sex-determining region Y (SRY) in 1990, several other genes have been identified in the process of mammalian sex determination (6). In brief, the Wilm s tumor 1 gene (WT1) and steroidogenic factor 1 (SF1) have been demonstrated to be involved in the formation of the gonads prior to their differentiation to testes or ovaries. Subsequent sex-specific gonadal differentiation appears to be mediated by SRY and SRY-related HMG-box-transcription factor (SOX9) genes in the testis, and the dosage-sensitive sex reversal locus (DSS)-X-linked adrenal hypoplasia congenital (AHC) critical region on the X, gene 1 (DAX1) in the ovary. Although the number of genes known to be involved in sex determination and gonadal development is not large, protein-protein interactions and regulatory relationships among their protein products are complex (7). DAX1 and SF1, members of the orphan nuclear receptor superfamily,are critical regulatory components of the hypothalamic-pituitary-adrenal-gonadal axis. In adrenal and gonadal tissues they regulate the expression of the cytochrome P450 steroid hydroxylase genes, the key mediators of steroidogenesis. Based on the common function of steroid hormone-producing tissues and homologous regulation by the hypothalamo-pituitary axis the classical steroidogenic organs, the adrenal cortex and the gonads, have been suggested to have an intimate ontogenic relationship (8). The molecular mechanism of the tissue-specific and pituitary hormone-regulated expression of the genes encoding P-450 enzymes in the steroidogenic tissues is the central issue of long-term regulation of steroidogenesis (9). We hypothesized that common transcription factors may also be implicated in the differentiation of the peripheral steroidogenic tissues, e.g. the skin (10). In this work, we characterized the expression of SF1, WT1, SRY, SOX9 and DAX1 in scalp specimens from male patients with androgenetic alopecia as well as in 3

4 cultured human sebocytes. Differences between bald fronto-parietal and hair-donating occipital scalp of the same individual patients were evaluated. Materials and Methods Collection of Human specimens Skin specimens from bald frontal-parietal (B) and hair-donating occipital scalp areas (H) were obtained from 21 males with AGA (aged 23-50, mean 33 years) at stages II-IV (Norwood/Hamilton), during a hair transplantation procedure. Because of the tiny sample size, specimens from patients 1-8 were used completely for reverse transcription-polymerase chain reaction (RT-PCR), while those from patients 9-21 for western blot analysis. Specimens from the fronto-parietal scalp area of 7 male individuals without AGA at the time of examination were also included for RT-PCR (n=2) and western blot analysis (n=5). RT-PCR Total RNA was extracted from the whole scalp specimens and cultured SZ95 sebocytes using a commercial kit (StrataPrep, Statagene, La Jolla, CA), including a specific DNA removal step with DNAse. 2 µg of total RNA was reverse transcribed using oligo(dt) primers and M-MLV reverse transcriptase (Promega, Madison, WI, USA). The oligonucleotide primers used for RT-PCR (synthesized by GibcoBRL, Tokyo, Japan), are listed in Table 1. PCR amplification of the complementary DNA was carried out for 35 consecutive cycles in 50 µl of amplification buffer (Promega) containing 2.5 U Tag polymerase (Promega) per reaction, and 5 and 3 primers each in concentration of 0.5 µm. The thermal profile was initially 94 C for 5 min, followed by 35 cycles at 94 C for 30 sec, at hybridization temperature 58 C (SF1), 50 C (WT1), 60 C (SRY), 58 C (SOX9), 60 C (DAX1) for 45 sec, at 72 C for 45 sec and finally at 72 C for 10 min in a programmable thermal cycler (GeneAmp PCR system 2400, Applied Biosystems, Fostercity, CA, USA). Western Blot Analysis Protein extraction was performed using M-PER TM mammalian protein extraction reagent (Pierce, Rockford, IL, USA). Aliquots (10 µg) of total protein isolated from whole scalp specimens and the SZ95 sebocytes were heated for 15 min at 95 C in a buffer to denaturize proteases. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of each sample was performed on 12.5% gels. Proteins were transferred to a PVDF transfer membrane (PolyScreen, NEN, Boston, USA), using a semi-dry blotting system (Bio-Rad, Hercules, CA, USA). The blots were primarily hybridized with goat polyclonal IgG (anti-human SRY and WT1) and rabbit polyclonal IgG (anti-human DAX1 and SOX9) (all from Santa Cruz, CA, USA) at concentration of 1:250 and were visualized using a chromophore-conjugated anti-igg antibody at a concentration of 1:5000, and enhanced chemiluminescence reagents (ECL Western blotting luminol reagents, Santa Cruz) followed 4

5 by exposure to BioMax light film (Kodak, Rochester, NY, USA). The results were analyzed by densitometry (Bio-1D Version 99, Vilber Lourmat, France). Immunohistochemical studies Frozen sections (6 µm thick) of skin specimens were fixed for 15 min in cold acetone, followed by treatment in 0.3% (vol/vol) H 2 O 2 in methanol for 15 min to block endogenous peroxidase activity, and then incubated with 0.1% BSA for 30 min to block nonspecific staining. Incubation with the aforementioned primary antibodies diluted to 1:100 for DAX1, SRY, SOX-9 and WT1 in antibody diluent (DAKO, Glostrup, Denmark) was carried out at room temperature for 1 h, followed by sequential 10-min incubation steps with the respective biotinylated link antibodies and peroxidase-labeled streptavidine (DAKO). Staining was completed after incubation with diaminobenzidine/chromogen solution (DAKO) and mounted in aqueous mounting medium. Results Expression of sex-determining genes in human scalp mrna expression of SF1, WT1, SRY, SOX9 and DAX1 was detected in normal human scalp skin as well as in the scalp skin of AGA patients. In 5/8 patients, the expression of SRY and SOX9 appeared to be stronger in bald fronto-parietal areas as compared to that in occipital areas. The mrna expression of DAX1 and WT1 were not consistent. Densitometry of the western blot analysis exhibited an enhanced expression of SRY protein in bald (B) vs. hair-donating (H) scalp areas, with B/H ratio 1.1 in 9/13 patients (69%) and 1.4 in 4/13 patients (30%), while enhanced expression of DAX1 with B/H ratio 1.1 was found in 7 /13 (54%) patients. Parallel enhancement between SRY and DAX1 was seen in 6/13 (46%) patients, with 4 of them (4/6) aging < 30 years. The expression of WT1 and SOX9 in bald scalp was rather heterogenous, with B/H ratio 1.1 in 4/13 (30%) and 2/13 (15%) patients, respectively, B/H ratio > 0.9 in 3/13 (23%) and 5/13 (38%) patients, and no significant change in 6/13 (46%) and 6/13 (46%) patients, respectively. The expressional intensities of SRY and DAX1 appeared to increase as baldness advanced, although no statistical significance could be established. The expression of DAX1, but not that of SRY, WT1 or SOX9, seemed to diminish with increasing age. The SRY expression in fronto-parietal scalp from normal subjects appeared to be higher than in fronto-parietal scalp from AGA patients and was nearly 1.5-fold the intensity in occipital scalp from AGA patients. Expression of DAX1, SOX9, WT1 and SRY in normal human scalp Immunohistochemistry showed strong expression of DAX1 in the nucleus of basal cells of sebaceous glands, inter-follicular epidermal basal keratinocytes and keratinotyes in outer root sheath of hair follicles. Strong expression of SOX9 was found in the nuclear as well as cytoplasmic portion 5

6 of epidermal as well as follicular keratinocytes and eccrine sweat glands, while its expression in sebaceous glands was faint. The in vivo expression of SRY was very faint by the antibody in use and WT1 could not be detected. Discussion The action of sex-determining genes on androgen homeostasis is complicated and closely interactive. SF1 and SOX9 seem to potentiate the upstream androgenesis by upregulating StAR. DAX1 exerts multifaceted anti-androgen effect by not only antagonizing upstream androgenic enzymes such as StAR, P450scc and 3β-HSD, but also downregulating the AR. SRY seems to have no direct regulatory effect on the expression of androgenic enzymes. In the current study, we showed mrna expression of SF1, WT1, SRY, SOX9 and DAX1 in male scalp. We failed to detect SF1 mrna in cultured human sebocytes, which is compatible with the data obtained in another recent study (12). Whether the missing SF1 mrna expression in skin specimens and human sebocytes could be explained by a delay in transportation and processing of the specimens as previously suggested is unclear (13). rong expression of DAX1 in the cultured cells. Previous studies of human skin revealed prominent nuclear immunostaining for DAX1 confined to the epidermis, sebaceous glands, sweat glands, and outer root sheath of the hair follicle (13). Cytoplasmic staining of DAX1 was also observed in the basal layer of the epidermis. SF1 was detected in the epidermis but displayed a scattered nuclear pattern across all layers (12), which was stronger than DAX1 in the inner root sheath, matrix cells, and dermal papilla cells. The DAX1 protein seems to be able to shuttle between nucleus and cytoplasm in human adrenal cortex and mouse Leydig tumor cells (14) The discrepancy of the detection of WT1 mrna but not protein in cultured sebocytes may result from low protein amounts or the antibody used. Our finding of strong expression of SOX9 in human skin is interesting. Noteworthy is the recent identification of another subfamily of the SOX gene family: SOX18, whose mutations were found to underlie cardiovascular and hair follicle defects in ragged mice, suggesting a critical role of Sox18 for cardiovascular and hair follicle formation (15). DAX1 and SF1 are two members of the orphan nuclear receptor superfamily known to play an important role in mediating transcriptional regulation of several steroid hydroxylase genes (9). SF1 controls basal and camp-stimulated transcription of the StAR gene, a procedure which represents the first and rate-limiting step in steroid biosynthesis, as well as many other steroidogenic genes, including P450 aromatase (16). SF-1 is in part responsible for the tissue-specific expression of these genes. SF-1 expression has recently been described in human skin where it is speculated to play a role in cutaneous steroidogenesis (13). DAX1 was shown to block the SF1-mediated induction of steroid biosynthesis by repressing StAR expression as well as the expressions of P450scc and 3β-HSD (17). Previous work has established a co-regulatory role of DAX1 because of its inhibitory effect on the activity of 6

7 SF1, androgen receptor (AR) as well as of the two estrogen receptors (ER), ERα and ERβ (18). On the other hand, sexually dimorphic expression of Dax1 was demonstrated in the mouse adrenal cortex, suggesting that Dax1 gene transcription is suppressed by androgens and AR (19). The current understanding of androgenetic alopecia focuses on the excessive in situ conversion of testosterone to 5α-DHT, which involves the hyperactivity of 5α-reductase and 17β-HSD coupled with hypoactivity of aromatase and overexpression of androgen receptors in balding versus non-balding scalps (2). However, the molecular mechanism controlling the dysregulation of local enzyme expression in diseased tissue is poorly delineated. In the present study, enhanced expression of SRY protein in bald scalp was observed in nearly 70% of the AGA patients and the disease severity (baldness staging) appears to positively correlate to the levels of SRY and DAX1. No specific correlation could be found between WT1 and SRY, or WT1 and DAX1. However, since SRY and DAX1 were shown to act synergistically in negatively regulating the AR activity in androgen-dependent cells (20), the significance of their enhanced co-expression in bald scalp is not known, in light of the prevailing evidences showing hyperactivity of AR (21). However, the high expression of SRY in normal subjects without AGA seems to have a protective function, since SRY was shown to oppose the effect of endogenous AR in androgen-dependent cells (20). On the other hand, the diminished levels of DAX1 by aging may predispose aged people to AGA, partially due to the less braking effect of DAX1 on androgen generation (17). Further studies enrolling more patients, especially normal people without AGA as control, is required to clarify our preliminary observation. It would be also interesting to perform a comparative study on female AGA patients. In conclusion, the current study demonstrates the expression of sex-determining genes in human skin and their possible association with cutaneous steroidogenesis and androgen-dependent skin diseases like AGA. Further studies on their functional roles in regulating the expression of steroidogenic enzymes can be performed by using cultured SZ95 sebocytes as an in vitro model. References 1. Zouboulis CC. Human skin: an independent peripheral endocrine organ. Horm Res 2000; 54: Chen W, Thiboutot D, Zouboulis CC. Cutaneous androgen metabolism: basic research and clinical perspectives. J Invest Dermatol 2002; 119: Deplewski D, Rosenfield RL. Role of hormones in pilosebaceous unit development. Endocr Rev 2000; 21: Ellis JA, Stebbing M, Harrap SB. Genetic analysis of male pattern baldness and the 5α-reductase genes. J Invest Dermatol 1998; 110: Sreekumar GP, Pardinas J, Wong CQ et al. Serum androgens and genetic linkage 7

8 analysis in early onset androgenetic alopecia. J Invest Dermatol 1999; 113: Vilain E, McCabe ER. Mammalian sex determination: from gonads to brain. Mol Genet Metab 1998; 65: Bowles J, Koopman P. New clues to the puzzle of mammalian sex determination. Genome Biol 2001; 2: Morohashi K. The ontogenesis of the steroidogenic tissues. Genes Cells 1997; 2: Peter M, Dubuis JM. Transcription factors as regulators of steroidogenic P-450 enzymes. Eur J Clin Invest 2000; 30 (Suppl 3): Corticotropin-releasing hormone: an autocrine hormone that promotes lipogenesis in human sebocytes. Zouboulis CC, Seltmann H, Hiroi N et al. Proc Natl Acad Sci U S A. 2002; 99: Zouboulis CC, Seltmann H, Neitzel H, Orfanos CE. Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95). J Invest Dermatol. 1999;113: Thiboutot D, Jabara S, McAllister JM, Sivarajah A, Gilliland K, Cong Z, Clawson G. Human skin is a steroidogenic tissue: Steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1). J Invest Dermatol. 2003; 120: Patel MV, McKay IA, Burrin JM. Transcriptional regulators of steroidogenesis, DAX-1 and SF-1, are expressed in human skin. J Invest Dermatol 2001; 117: Lalli E, Ohe K, Hindelang C, Sassone-Corsi P. Orphan receptor DAX-1 is a shuttling RNA binding protein associated with polyribosomes via mrna. Mol Cell Biol 2000; 20: Pennisi D, Gardner J, Chambers D et al. Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice. Nat Genet 2000; 24: Sugawara T, Saito M, Fujimoto S. Sp1 and SF-1 interact and cooperate in the regulation of human steroidogenic acute regulatory protein gene expression. Endocrinology 2000; 141: Lalli E, Melner MH, Stocco DM, Sassone-Corsi P. DAX-1 blocks steroid production at multiple levels. Endocrinology 1998; 139: Holter E, Kotaja N, Makela S, et al. Inhibition of androgen receptor (AR) function by the reproductive orphan nuclear receptor DAX-1. Mol Endocrinology 2002; 16: Mukai T, Kusaka M, Kawabe K, et al. Sexually dimorphic expression of Dax-1 in the adrenal cortex. Genes Cells 2002; 7: Yuan X, Lu ML, Li T, Balk SP. SRY interacts with and negatively regulates androgen receptor transcriptional activity. J Biol Chem 2001; 276:

9 21. Sawaya ME, Shalita AR. Androgen receptor polymorphisms (CAG repeat lengths) in androgenetic alopecia, hirsutism, and acne. J Cutan Med Surg 1998; 3: