Higher Levels of Steroidogenic Acute Regulatory Protein and Type I 3β-Hydroxysteroid Dehydrogenase in the Scalp of Men with Androgenetic Alopecia

Abstract

androgenetic alopecia steroid 3β-hydroxysteroid dehydrogenase cytochrome P450 17α-hydroxylase/17,20-lyase cytochrome P450 cholesterol side-chain cleavage enzyme steroidogenic acute regulatory protein TO THE EDITOR Androgenetic alopecia (AGA) is the most common form of human baldness mediated by androgens. As most of the patients show normal levels of circulating androgens, a local in situ overproduction or/and androgen hyper-response of the skin are suggested and supported by many studies, whereas higher levels of type I and type II 5α-reductase isozymes and androgen receptors have been demonstrated in frontal hair follicles than in occipital follicles (Hibberts et al., 1998Hibberts N.A. Howell A.E. Randall V.A. Balding hair follicle dermal papilla cells contain higher levels of androgen receptors than those from non-balding scalp.J Endocrinol. 1998; 156: 59-65Crossref PubMed Scopus (198) Google Scholar; Hoffmann, 2003Hoffmann R. Steroidogenic isoenzymes in human hair and their potential role in androgenetic alopecia.Dermatology. 2003; 206: 85-95Crossref PubMed Scopus (35) Google Scholar). It remains unclear, however, whether the locally overproduced testosterone and 5α-dihydrotestosterone in the bald scalp derive from the cutaneous de novo androgen synthesis from serum cholesterol or from a shortcut, using serum dehydroepiandrosterone. Both are abundant, especially cholesterol. Four “upstream” enzymes including steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) and cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17), and steroid 3β-hydroxysteroid dehydrogenase (3β-HSD) are responsible for the early steps of androgenesis from cholesterol to dehydroepiandrosterone. StAR regulates the first committed rate-limiting step by controlling the delivery of cholesterol from the outer membrane to the inner membrane of mitochondria, where cholesterol is further converted to pregnenolone by P450scc complex (Miller, 2002Miller W.L. Androgen biosynthesis from cholesterol to DHEA.Mol Cell Endocrinol. 2002; 198: 7-14Crossref PubMed Scopus (123) Google Scholar). Pregnenolone undergoes 17α-hydroxylation by microsomal P450c17 to form 17-hydroxypregnenolone and then is transformed to dehydroepiandrosterone by the 17,20-lyase activity of P450c17. Dehydroepiandrosterone is then converted by 3β-HSD to androstenedione, the substrate for testosterone. We studied regional differences of the bald frontal–parietal versus the occipital scalp in the gene expression of the foregoing four enzymes in male AGA by using a quantitative, competitive RT-PCR method (Tsai and Wiltbank, 1996Tsai S.J. Wiltbank M.C. Quantification of mRNA using competitive RT-PCR with standard-curve methodology.Biotechniques. 1996; 21: 862-866PubMed Google Scholar) (Figure 1a and b). Totally, 51 male AGA patients at stages III–VII according to the modified Norwood/Hamilton classification were recruited, with 25 patients (mean age 34.9 years, range 24–52 years) free of medication and 26 patients (mean age 37.3 years, range 21–58 years) taking 1 mg finasteride daily for at least 3 months. The study was conducted according to the Declaration of Helsinki Principles and approved by the Medical Ethical Committee of the Chang Gung Memorial Hospital, with written informed consent given by the participants. Paired data from both areas were obtained from 48, 46, and 20 of the total 51 patients for StAR, 3β-HSD, and P450scc, respectively. The estimated amount of the mRNA of each gene in the bald scalp, as normalized to the expression level of glyceraldehyde 3-phosphate dehydrogenase, was most abundant for StAR, followed by 3β-HSD, and much less for P450scc and P450c17. In 19 patients with detectable P450c17 mRNA, 10 had only expression in the bald scalp, whereas three only in the occipital scalp, and six had paired data from both areas. DNA sequencing of the PCR amplification of 3β-HSD (Tsai et al., 2001Tsai S.J. Wu M.H. Lin C.C. Sun H.S. Chen H.M. Regulation of steroidogenic acute regulatory protein expression and progesterone production in endometriotic stromal cells.J Clin Endocrinol Metab. 2001; 86: 5765-5773Crossref PubMed Scopus (104) Google Scholar) confirmed the gene expression of type I instead of type II 3β-HSD in the skin, even in the bald scalp (Dumont et al., 1992Dumont M. Luu-The V. Dupont E. Pelletier G. Labrie F. Characterization, expression, and immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase in human skin.J Invest Dermatol. 1992; 99: 415-421Abstract Full Text PDF PubMed Google Scholar). When the mRNA expression in the occipital (H) scalp was taken as 1, significantly higher levels of StAR and 3β-HSD were demonstrated in the bald parieto-frontal (B) scalp as compared to the occipital scalp, represented by elevated B/H ratio (P=0.0117 and 0.0164, respectively, paired t-test) (Figure 1c and d). No regional difference existed regarding P450scc (P=0.3752). The regional difference of the examined genes was not consistent among the examinee, that is, some AGA patients with overexpression of StAR in their bald scalps showed normal or lower levels of 3β-HSD in the same area, and vice versa. No association existed between the expressional intensity of StAR, 3β-HSD, or P450scc and the baldness severity (Pearson's product-moment correlation test) or between them and the patients' age (multiple linear regression). Correlation among the four examined genes with each other was insignificant (analysis of variance test). There was no difference between the finasteride treatment and non-treatment group regarding the regional expressional intensity (B/H) of each examined gene (paired t-test). All the statistics and analysis were performed using SAS PC software (version 9.1; SAS, Cary, NC). Immunocytochemistry performed on cultured SZ95 sebocytes showed localization of StAR mainly in the perinuclear portion of the cytoplasma (Figure 2a). The smaller rather undifferentiated sebocytes displayed more strongly the StAR protein. Immunohistochemistry showed strong expression of StAR in the basal layer of sebaceous glands (Figure 2b), epidermal keratinocytes (Figure 2c), outer root sheath of hair follicles (Figure 2d) but not dermal papilla cells (data not shown), vascular tissues (Figure 2e), and eccrine sweat ducts but not eccrine sweat glands (Figure 2f). It is noteworthy that in hair follicles, androgen receptor expression was demonstrated to be restricted to dermal papilla cells, whereas in sebaceous glands, it was observed by some authors to predominate in the basal sebocytes but by others to exist both in basal cells and differentiating sebocytes (Thornton et al., 2003Thornton M.J. Taylor A.H. Mulligan K. Al-Azzawi F. Lyon C.C. O'Driscoll J. et al.The distribution of estrogen receptor beta is distinct to that of estrogen receptor alpha and the androgen receptor in human skin and the pilosebaceous unit.J Investig Dermatol Symp Proc. 2003; 8: 100-103Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar; Pelletier and Ren, 2004Pelletier G. Ren L. Localization of sex steroid receptors in human skin.Histol Histopathol. 2004; 19: 629-636PubMed Google Scholar). Our finding of a stronger expression of StAR protein in the undifferentiated sebocytes seems to be in line with the observation that androgens stimulate the proliferation of human sebocytes (Zouboulis et al., 1999Zouboulis C.C. Seltmann H. Neitzel H. Orfanos C.E. Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).J Invest Dermatol. 1999; 113: 1011-1020Crossref PubMed Scopus (279) Google Scholar), contrary to the data from rat preputial sebocyte model, which postulated the major effect of androgens was on an early step in cell differentiation (Miyake et al., 1994Miyake K. Ciletti N. Liao S. Rosenfield R.L. Androgen receptor expression in the preputial gland and its sebocytes.J Invest Dermatol. 1994; 103: 721-725Abstract Full Text PDF PubMed Scopus (36) Google Scholar; Rosenfield et al., 1999Rosenfield R.L. Kentsis A. Deplewski D. Ciletti N. Rat preputial sebocyte differentiation involves peroxisome proliferator-activated receptors.J Invest Dermatol. 1999; 112: 226-232Crossref PubMed Scopus (126) Google Scholar). In steroidogenic cells, tropic hormones can regulate the StAR protein expression by cAMP-dependent mechanisms (Manna et al., 2003Manna P.R. Wang X.J. Stocco D.M. Involvement of multiple transcription factors in the regulation of steroidogenic acute regulatory protein gene expression.Steroids. 2003; 68: 1125-1134Crossref PubMed Scopus (123) Google Scholar). A recent study showed that prostaglandin E2-stimulated StAR gene activation is mediated by E prostanoid receptor 2-coupled cAMP–protein kinase A pathway in peripheral endocrine tissues (Sun et al., 2003Sun H.S. Hsiao K.Y. Hsu C.C. Wu M.H. Tsai S.J. Transactivation of steroidogenic acute regulatory protein in human endometriotic stromalcells is mediated by the prostaglandin EP2 receptor.Endocrinology. 2003; 144: 3934-3942Crossref PubMed Scopus (98) Google Scholar). cAMP is also required for the growth and differentiation of rat preputial sebocytes (Rosenfield et al., 2002Rosenfield R.L. Wu P.P. Ciletti N. Sebaceous epithelial cell differentiation requires cyclic adenosine monophosphate generation.In vitro Cell Dev Biol Anim. 2002; 38: 54-57Crossref PubMed Scopus (11) Google Scholar). These data including our own seem to suggest the close interaction between androgenesis and prostaglandins in regulation of the sebocyte development (Zouboulis, 2005Zouboulis C.C. Sebaceous glands and the prostaglandin pathway – key stones of an exciting mosaic.J Invest Dermatol. 2005; 125: x-xiCrossref PubMed Scopus (7) Google Scholar). Upregulation of StAR and 3β-HSD in the bald scalp of AGA patients may simply reflect marked hyperplasia of sebaceous glands contrasted with the miniaturized hair follicles or imply the active de novo biosynthesis of androgens in pilosebaceous units. Given the juxtaposition of hair follicle and sebaceous glands, a kind of paracrine effect originating from sebaceous gland-secreted androgens on the growth and development of hair follicles is possible. Separate isolation of sebaceous glands and hair follicles to determine the difference of gene expression in bald and hairy scalp would help clarify the contributory role of each steroidogenic organ. The significance of high StAR levels but limited gene expression of P450scc and P450c17 in the skin as well as in most other steroidogenic tissues is not clear (Slominski et al., 1996Slominski A. Ermak G. Mihm M. ACTH receptor, CYP11A1, CYP17 and CYP21A2 genes are expressed in skin.J Clin Endocrinol Metab. 1996; 81: 2746-2749Crossref PubMed Scopus (193) Google Scholar; Thiboutot et al., 2003Thiboutot D. Jabara S. McAllister J.M. Sivarajah A. Gilliland K. Cong Z. et al.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: 905-914Crossref PubMed Scopus (239) Google Scholar). As StAR is an acutely regulated protein with very short half-life, its regulation at the transcriptional level may be required in order to provide sufficient mRNA transcripts for synthesizing protein that carries cholesterol to the inner mitochondria. On the other hand, the P450c17 expression in diseased state may be different, as P450c17 mRNA was more commonly detected in the bald than occipital areas of our AGA patients. Enhanced expression of StAR and 3β-HSD in the bald area further implies that locally aberrant production of androgens may play a significant role in the development of the disease. Comparable data from normal controls without AGA including functional studies on the enzyme activities are needed to determine the role of de novo androgenesis in the pathogenesis of AGA. The authors state no conflict of interest. This work was supported by grants from the National Science Council Taiwan (NSC 93-2314-B-182A-021) and the Chang Gung Medicine Research Project (CMRPG-8060).