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Androgen Receptors in Dermal Papilla Cells of Scalp Hair Follicles in Male Pattern Baldness M. B. HODGINS," R. CHOUDHRY," G. PARKER," R. F. OLIVER? C. A. B. JAHODAb, A. P. WITHERS? A. 0. BRINKMA",' T. H. VAN DER KWAST,' W. J. A. BOERSMA,d K. M. LAMMERS,'T. K. WONG,' C. J. W A W R Z Y N W AND R. WARRENe ODermatology Department University of Glasgow Glasgow GI I 6NU, Scotland bDepartment of Biological Sciences University of Dundee, Dundee, Scotland CErasmus University, Rotterdam, the Netherlands 'MBL -TNO Rijswijk, the Netherlands eThe Procter & Gamble Company Miami Valley Laboratories Cincinnati, Ohio 45237-8707
Androgens may influence hair growth by modulating tissue interactions between the dermal papilla and germinative matrix of hair follicles. Our previous studies' demonstrated that human scalp and pubic dermal papilla cells in culture expressed androgen receptors and were active in androgen metabolism. The possibility that variation in papilla cell androgen receptors might be a factor in pattern baldness has now been investigated. Vertex and occipital scalp skin specimens were obtained from healthy men (ages 22-73 years, 9 balding, 11 nonbalding). Four nonbalding donors provided specimens from both sites. Dermal papillae were cultured in Chang medium.2 Androgen receptors were assayed in dermal papilla cells (passage 3-6) by a modification of methods used for skin fibroblasts with the synthetic androgen [3H]mibolerone as labeled ligand.3 A specific monoclonal antibody directed against the N-terminal region of human androgen receptofl was used to stain cryostat sections of human skin. Papilla cells from terminal hairs on the occipital region of nonbalding men and the vertex of balding men expressed similar, low levels of androgen receptors in culture, but cells from the vertex of nonbalding men were more variable (FIG.1). Receptor levels were higher in vertex than occipital in 3/4paired cultures. Immuno448
HODGINS ef al.: MALE PATTERN BALDNESS
:
449
40
P
t
0
r
f 30rn 0
I /
;20P r 0
t e
10-
i
n
-
0
I
bald
non-bald
FIGURE 1. Quantitation of androgen receptor sites in human scalp dermal papilla cells. A saturating concentration (2.0 nmol/l) of the synthetic androgen [3H]mibolerone (t 200 nmol nonradioactive mibolerone/l to correct for nonspecific binding) was used to measure specific androgen receptor sites in dermal papilla cells growing in plastic multiwell plates. Receptors are expressed as fmol [3H]mibolerone bound/mg cell protein. Open symbols represent vertex and closed symbols occipital cultures. Each point is the mean of triplicate assays on a single dermal papilla cell line. Assays were carried out between passages 3 and 6. The method is a modification of that previously described for skin fibroblast^.^
cytochemical staining revealed androgen receptors in nuclei of dermal papilla cells of a proportion of follicles from scalp and beard skin. Staining in the hair follicle was restricted to the dermal papilla (FIG.2). These observations strengthen the view that the dermal papilla is an important site of androgen action on the hair follicle. As large terminal follicles in anagen are selected for dissection of dermal papillae, cultures from balding scalps represent hairs that may be relatively unresponsive to androgens. This is consistent with rather uniformly low androgen receptor expression in cultures from the vertex of bald men and in occipital follicles of nonbald men. In contrast, vertex cultures from certain nonbalding men may have been derived from hair follicles of potentially greater sensitivity to androgens. The precise relationship between receptor expression in situ and in culture is unknown, but our results suggest that variation in androgen receptor expression in dermal papillae, both across the scalp and between individuals, may be related to development of baldness. However, the androgen receptor may not be the sole determinant of androgen action on scalp hair follicles, as a high level of receptor was found in a dermal papilla culture from the vertex of a nonbald man of 73 years.
450
ANNALS NEW YORK ACADEMY OF SCIENCES
FIGURE 2. Specific androgen receptor staining in nuclei of dermal papilla cells of an anagen hair follicle in human skin. Cryostat sections of skin were stained with a specific mouse monoclonal antibody directed against the N-terminal domain of human androgen receptor.4 Standard avidin-biotin complex immunoperoxidase technique with DAB as chromogen was used to visualize the bound antibody. Cell nuclei were lightly counterstained with hematoxylin. Nonspecific controls included normal mouse serum, fetal calf serum, and irrelevant mouse monoclonal antibodies. Note that antibody staining is exclusively nuclear and limited to cells of the dermal papilla (arrow). Magnification: lo00 x.
HODGINS ef al.: MALE PATTERN BALDNESS
451
REFERENCES 1. MURAD, S. M., M. B. HODGINS, R. F. OLIVER & C. A. B. JAHODA. 1986. J. Invest. Dermatol.
87: 158. 2. WARREN, R., T. K. WONG,& K. M. LAMMERS. 1990. J. Invest. Dermatol. 9 4 589. 1. J., D. A. ROWNEY & M. B. HODGINS. 1989. J. Steroid Biochem. 32: 789-795. 3. MCEWAN, 4. ZEGERS, N. D., E. CLASSEN, C. NEELEN, E. MULDER, J. H. VANLAAR, M. M. VOORHORST, C. A. BERREVETS, A. 0. BRINKMANN, T. VAN DER KWAST,J. A. RUIZEVELD DE WINTER, J. TRAPMAN & W. J. A. BOERSMA. 1991. Biochim. Biophys. Acta 1073: 23-32.
Androgen Receptors in Dermal Papilla Cells of Scalp Hair Follicles in Male Pattern Baldness M. B. HODGINS," R. CHOUDHRY," G. PARKER," R. F. OLIVER? C. A. B. JAHODAb, A. P. WITHERS? A. 0. BRINKMA",' T. H. VAN DER KWAST,' W. J. A. BOERSMA,d K. M. LAMMERS,'T. K. WONG,' C. J. W A W R Z Y N W AND R. WARRENe ODermatology Department University of Glasgow Glasgow GI I 6NU, Scotland bDepartment of Biological Sciences University of Dundee, Dundee, Scotland CErasmus University, Rotterdam, the Netherlands 'MBL -TNO Rijswijk, the Netherlands eThe Procter & Gamble Company Miami Valley Laboratories Cincinnati, Ohio 45237-8707
Androgens may influence hair growth by modulating tissue interactions between the dermal papilla and germinative matrix of hair follicles. Our previous studies' demonstrated that human scalp and pubic dermal papilla cells in culture expressed androgen receptors and were active in androgen metabolism. The possibility that variation in papilla cell androgen receptors might be a factor in pattern baldness has now been investigated. Vertex and occipital scalp skin specimens were obtained from healthy men (ages 22-73 years, 9 balding, 11 nonbalding). Four nonbalding donors provided specimens from both sites. Dermal papillae were cultured in Chang medium.2 Androgen receptors were assayed in dermal papilla cells (passage 3-6) by a modification of methods used for skin fibroblasts with the synthetic androgen [3H]mibolerone as labeled ligand.3 A specific monoclonal antibody directed against the N-terminal region of human androgen receptofl was used to stain cryostat sections of human skin. Papilla cells from terminal hairs on the occipital region of nonbalding men and the vertex of balding men expressed similar, low levels of androgen receptors in culture, but cells from the vertex of nonbalding men were more variable (FIG.1). Receptor levels were higher in vertex than occipital in 3/4paired cultures. Immuno448
HODGINS ef al.: MALE PATTERN BALDNESS
:
449
40
P
t
0
r
f 30rn 0
I /
;20P r 0
t e
10-
i
n
-
0
I
bald
non-bald
FIGURE 1. Quantitation of androgen receptor sites in human scalp dermal papilla cells. A saturating concentration (2.0 nmol/l) of the synthetic androgen [3H]mibolerone (t 200 nmol nonradioactive mibolerone/l to correct for nonspecific binding) was used to measure specific androgen receptor sites in dermal papilla cells growing in plastic multiwell plates. Receptors are expressed as fmol [3H]mibolerone bound/mg cell protein. Open symbols represent vertex and closed symbols occipital cultures. Each point is the mean of triplicate assays on a single dermal papilla cell line. Assays were carried out between passages 3 and 6. The method is a modification of that previously described for skin fibroblast^.^
cytochemical staining revealed androgen receptors in nuclei of dermal papilla cells of a proportion of follicles from scalp and beard skin. Staining in the hair follicle was restricted to the dermal papilla (FIG.2). These observations strengthen the view that the dermal papilla is an important site of androgen action on the hair follicle. As large terminal follicles in anagen are selected for dissection of dermal papillae, cultures from balding scalps represent hairs that may be relatively unresponsive to androgens. This is consistent with rather uniformly low androgen receptor expression in cultures from the vertex of bald men and in occipital follicles of nonbald men. In contrast, vertex cultures from certain nonbalding men may have been derived from hair follicles of potentially greater sensitivity to androgens. The precise relationship between receptor expression in situ and in culture is unknown, but our results suggest that variation in androgen receptor expression in dermal papillae, both across the scalp and between individuals, may be related to development of baldness. However, the androgen receptor may not be the sole determinant of androgen action on scalp hair follicles, as a high level of receptor was found in a dermal papilla culture from the vertex of a nonbald man of 73 years.
450
ANNALS NEW YORK ACADEMY OF SCIENCES
FIGURE 2. Specific androgen receptor staining in nuclei of dermal papilla cells of an anagen hair follicle in human skin. Cryostat sections of skin were stained with a specific mouse monoclonal antibody directed against the N-terminal domain of human androgen receptor.4 Standard avidin-biotin complex immunoperoxidase technique with DAB as chromogen was used to visualize the bound antibody. Cell nuclei were lightly counterstained with hematoxylin. Nonspecific controls included normal mouse serum, fetal calf serum, and irrelevant mouse monoclonal antibodies. Note that antibody staining is exclusively nuclear and limited to cells of the dermal papilla (arrow). Magnification: lo00 x.
HODGINS ef al.: MALE PATTERN BALDNESS
451
REFERENCES 1. MURAD, S. M., M. B. HODGINS, R. F. OLIVER & C. A. B. JAHODA. 1986. J. Invest. Dermatol.
87: 158. 2. WARREN, R., T. K. WONG,& K. M. LAMMERS. 1990. J. Invest. Dermatol. 9 4 589. 1. J., D. A. ROWNEY & M. B. HODGINS. 1989. J. Steroid Biochem. 32: 789-795. 3. MCEWAN, 4. ZEGERS, N. D., E. CLASSEN, C. NEELEN, E. MULDER, J. H. VANLAAR, M. M. VOORHORST, C. A. BERREVETS, A. 0. BRINKMANN, T. VAN DER KWAST,J. A. RUIZEVELD DE WINTER, J. TRAPMAN & W. J. A. BOERSMA. 1991. Biochim. Biophys. Acta 1073: 23-32.
