Cultured dermal papilla cells from androgen-dependent human hair follicles (e.g. beard) contain more androgen receptors than those from non-balding areas of scalp V. A.
Randall, M. J. Thornton and A. G. Messenger
Department of Biomedicai Sciences, University of Bradford, Bradford bd7 1dp, U.K. *Department of Dermatology, Royal Hallamshire Hospital, Sheffield sio2JF, U.K. revised manuscript received 23 September 1991 ABSTRACT
Androgens stimulate hair growth in many areas, e.g. the beard; they also induce regression and balding on the scalp with increasing age in genetically disposed individuals. The cause(s) of this biological conundrum is unknown but age-related; androgen-potentiated changes also occur in the prostate. The mesenchymederived dermal papilla situated at the base of the hair follicle is thought to play an important role in regulating the growth and development of the follicular epithelium. Since androgens probably act on the hair follicle via the dermal papilla, cultures of dermal papilla cells from human hair follicles with differing responses to androgens in vivo have been established and their ability to bind androgens assessed. Receptor binding was assayed by saturation analysis (0\m=.\05\p=n-\ 10 nmol/l) using the synthetic non-metabolizable androgen, [3H]mibolerone. Shionogi 115 cells were also assayed as a positive control.
INTRODUCTION
It is well known that androgens regulate the human hair follicle (Anonymous, 1970; for review see Randall, 1992). The response of human hair follicles to andro¬ gens is highly dependent on the location of the follicle; for example, beard follicles produce longer, thicker and darker hairs, eyelashes have no response and scalp follicles may regress in a relatively precise sequence in genetically sensitive individuals (Hamilton,
1942, 1958).
Follicles in some regions are stimulated by female levels of androgens, e.g. axilla and female pubic escutcheon, while others, such as the beard and chest, require male androgen levels (Marshall & Tanner, 1969, 1970). The responses of many follicles to andro¬ gens is gradual, increasing with age despite steady
Specific high-affinity low-capacity androgen receptors identified in 12 dermal papilla primary cell lines with similar characteristics to established androgen receptors. Cells from androgen-sensitive follicles (beard, scrotum and pubis) contained higher levels of androgen receptors than those derived from relatively androgeninsensitive non-balding scalp follicles whether the receptor content was calculated in relation to cell number, protein were
or
DNA content of the cells. These results support the
hypothesis that androgens act on hair follicles via the dermal papilla in vivo and demonstrate that dermal papilla cells exhibit an altered phenotype in culture which depends on the body site from which they were derived. Cultured human dermal papilla cells should
prove a useful model system for studies of the mechanism of androgen action, and further investigations may elucidate the paradox of why bald men can grow beards. Journal of Endocrinology (1992) 133, 141\p=n-\147
circulating androgens, e.g. the development ofterminal chest hair and male pattern baldness (Hamilton, 1946). The mechanisms by which androgens cause such diverse and progressive effects in the same tissues within the same individual are unknown. However, changes also occur in the prostate with increasing age, resulting in benign prostatic hypertrophy and prostatic carcinoma which are believed to be strongly influenced by androgens (Griffiths, Davies, Eaton et al. 1987). The hair follicle consists of a tube of epithelial cells protruding into the dermis and enclosing in its base the mesenchyme-derived dermal papilla. Hairs do not grow continuously; each follicle undergoes a repetitive sequence of growth and rest known as the hair growth cycle (Kligman, 1959). During the growth phase (anagen), epithelial cells derived from a highly proliferative region at the base of the follicle (the hair
bulb matrix) move distally and differentiate to form the hair shaft and the inner root sheath surrounding the hair. After a certain period determined by the body site, the follicle enters a resting phase (telogen) where mitotic activity ceases and the complex hair bulb structure is changed. After an interval, the follicle re-enters anagen, a new hair is formed and the old hair shed. During early anagen the hair follicle appears partially to recapitulate its embryonic
development. Although the molecular mechanisms responsible for controlling hair growth are not well understood, the dermal papilla is believed to play an essential role in inducing and maintaining epithelial differentiation (Oliver, 1966, 1967; Oliver & Jahoda, 1989); it may also exert a controlling influence on the hair growth cycle and determine other characteristics such as hair diameter (Van Scott & Ekel, 1958; Ibrahim & Wright, 1982) and cultured rat dermal papilla cells have been shown to retain their inductive capacity when re¬ implanted in vivo (Jahoda, Home & Oliver, 1984). Mesenchymal cells are also known to influence devel¬ oping epithelia in other hormone-dependent tissues including the mammary gland (Oka & Yoshimura, 1986) and prostate (Cunha, Taguchi, Shannon et al.
1984). Androgen receptors are required in the mesenchyme for prostate development (Cunha et al. 1984), and it, therefore, seems likely that androgens will influence the
hair follicle via the dermal papilla. Autoradiographic studies of [3H]testosterone have shown that dermal papillae, but not follicular epithelial cells, take up androgens (Stumpf & Sar, 1976). Intracellular androgen-receptor proteins are considered a pre¬ requisite for any cell to respond to androgens (Mainwaring, 1977) and testicular feminization patients, who lack functional androgen receptors, do not develop axillary, pubic or beard terminal hair (Keenan, Meyer, Hadjian et al. 1974). We have, there¬ fore, established primary cell lines of dermal papilla cells from human hair follicles obtained from body sites with varying responses to androgens in vivo and investigated whether they contain specific, saturable androgen receptors and whether the levels of receptors present varied with the androgen sensitivity of the follicle in vivo. MATERIALS AND METHODS
Cell culture
Primary cultures of dermal papilla cells from human hair follicles were established and grown as described by Messenger (1984). Briefly, hair follicles were iso¬ lated from normal skin samples obtained during routine clinical excisions. Dermal papillae were
microdissected from the base of anagen hair follicles, transferred to 35-mm sterile tissue-culture-treated Petri dishes and cultured in Eagles medium 199 (El99; Flow Laboratories Ltd, Irvine, Strathclyde, U.K.) supplemented with 20% (v/v) fetal bovine serum (Globefarm Ltd, Esher, Surrey, U.K.) and antibiotics at 37 °C in a humidified atmosphere containing 5% C02. The cell outgrowths were subcultured after 2-4 weeks into single 25-cm2 flasks in the same medium and conditions and then passaged into three flasks when confluent at intervals of 1-2 weeks. Tissue culture plastics were supplied by Flow Laboratories.
Androgen receptor assay The ability of cultured dermal papilla cells to bind the radiolabelled synthetic non-metabolizable androgen,
7a, 17a-dimethyl-19-nortestosterone (mibolerone) mibolerone; specific activity
([17a-methyl-3H]
2-74-3-33 TBq/mmol; Amersham International pic, Amersham, Bucks, U.K.) was determined by satu¬ ration analysis in 12 primary cell lines at passage numbers 3 to 6. Cells were seeded into 100 mm tissueculture-treated Petri dishes (Falcon Labware, Becton Dickenson, Cowley, Oxon, U.K.) at a density of about 2xl05 cells/dish and grown almost to con¬ fluence in normal medium. Preliminary experiments demonstrated that it was not possible to obtain good reproducible data when cells were cultured in 35 mm dishes. The cells were washed with Dulbecco's phosphatebuffered saline (PBS) and incubated in 5 ml E199 without serum for 24 h to reduce endogenous androgens. This medium was then replaced with 5 ml serum-free medium containing a range of concen¬ trations of [3H]mibolerone (005-10nmol/1; 9-10 concentration points) in the presence or absence of a 100-fold excess of 17ß-hydroxy-5a-androstanexcess of 3-one (5a-dihydrotestosterone); 1000
9-fluoro-16-hydroxyprednisolone-16a,17a-acetonide
also added to all of [3H]mibolerone to progesterone receptors. After 2 h at 37 GC the incubations were terminated by removal of the medium and washing the cells four times with PBS at 4°C; the cells were scraped from the dishes into cold PBS and collected by centrifugation. The super¬ natant was aspirated and the radioactive content ofthe cell pellets extracted with chloroform-methanol (1:1, v/v). The cells were collected again by centrifugation and the radioactivity in the supernatant was counted, after evaporation of the solvents, on an LKB liquid scintillation spectrophotometer for 10 min with a counting efficiency of 50%. Aliquots of the medium were also counted to obtain the amount of 'free' radioactivity remaining, i.e. not bound.
(triamcinolone acetonide)
was
incubations to prevent any
possible binding
0-5 1-0 2-0 3-0 4-0 5-0 Concentration of [3H]mibolerone (nmol/1)
10-0
5 10 Mibolerone bound
15
20
(fmol/mg protein)
1. Saturation analysis of [3H]mibolerone binding to the androgen receptor of cultured dermal papilla cells derived from human male beard follicles (passage 4). (a) Specific binding of mibolerone with increasing concentrations of 3H-labelled steroid (005-10 nmol/1) was determined as the difference between total (3H-labelled steroid alone) and non-specific binding ( -labelled steroid plus a 100-fold excess of unlabelled steroid). (¿>) Scatchard analysis of the data of specific binding as described above. The maximum binding capacity was 18-5 fmol/mg protein and the binding affinity 0-35 nmol/1. figure
The protein and DNA content of the remaining cell pellets from the individual Petri dishes were deter¬ mined by the methods of Lowry, Rosebrough, Farr & Randall (1951) and Burton (1956) respectively. The
number of cells present in one dish at the time of assay counted six times using a haemocytometer. The androgen-responsive mouse mammary carcinoma cell line Shionogi 115 (Jung-Testas & Baulieu, 1987) was assayed as a positive control. was
Scatchard plots (Fig. 1 b) were calculated for each cell line to provide an estimation of the affinity of the receptor for the ligand, i.e. the dissociation constant (Kd) and the number of binding sites available (Bmax). Radioligand binding by Shionogi 115 cells showed the expected high concentrations of high-affinity saturable androgen receptors (Figs 2 and 3) (Kd 0-29 nmol/1; Bmax 1-43 fmol/104 cells; 638-5 fmol/mg protein; 5-07 fmol/ pg DNA); this supports the validity of the assay.
Specificity of steroid binding To confirm that the radiolabelled steroid was actually binding to specific androgen receptors, the ability of various unlabelled steroids to compete for the binding was investigated. Cells were prepared as described for the receptor assay and dishes of cells from five cell lines were incubated in triplicate with 1 nmol[3H]mibolerone or in triplicate or duplicate with 1 nmol [3H]mibolerone plus a range of radioinert steroids (100 nmol/1) for 2 h at 37 °C. Binding was assessed as described above. Statistics
Differences between the types of cell line were analysed for significance using Student's /-test and MannWhitney U-test.
0-4r • P4
0-3
I«
o
I
0-2 •P5
p4
.P5 •P4
SP6
Beard
Genital
*P6
0-1 P4 •P5 •
*P5 Scalp
figure 2. Binding affinity (Ká nmol/1) for [3H]mibolerone in cultured dermal papilla cells derived from human scalp, beard and sexual skin hair follicles. passage number. =
RESULTS
Androgen receptor assay Specific binding of [3H]mibolerone was saturated at around 1 nmol/1 in all cells examined (Fig. la);
Androgen binding by dermal papilla cells from various body sites Saturable high-affinity binding was detected in all dermal papilla cells regardless of origin (Figs 2 and 3).
·4
50
40
S. 30
·3
I
o
I
20 »P5
E
PQ
10
,4 6 :, 4 ·5 ·4
·6
,P5 :p5 •P4
Beard Scalp figure 3. Concentration of androgen receptors (i.e. binding capacity (Bmax) for
Genital
pH]mibolerone) in cultured dermal papilla cells derived from human scalp, beard and sexual skin hair follicles.
=
passage number.
Dermal papilla cells derived from androgen-responsive hair follicles in beard, moustache, pubis and scrotum had a similar affinity for [3H]mibolerone to the Shionogi 115 cells (Fig. 2); the affinity of cells from androgeninsensitive non-balding scalp areas was significantly (P
Randall, M. J. Thornton and A. G. Messenger
Department of Biomedicai Sciences, University of Bradford, Bradford bd7 1dp, U.K. *Department of Dermatology, Royal Hallamshire Hospital, Sheffield sio2JF, U.K. revised manuscript received 23 September 1991 ABSTRACT
Androgens stimulate hair growth in many areas, e.g. the beard; they also induce regression and balding on the scalp with increasing age in genetically disposed individuals. The cause(s) of this biological conundrum is unknown but age-related; androgen-potentiated changes also occur in the prostate. The mesenchymederived dermal papilla situated at the base of the hair follicle is thought to play an important role in regulating the growth and development of the follicular epithelium. Since androgens probably act on the hair follicle via the dermal papilla, cultures of dermal papilla cells from human hair follicles with differing responses to androgens in vivo have been established and their ability to bind androgens assessed. Receptor binding was assayed by saturation analysis (0\m=.\05\p=n-\ 10 nmol/l) using the synthetic non-metabolizable androgen, [3H]mibolerone. Shionogi 115 cells were also assayed as a positive control.
INTRODUCTION
It is well known that androgens regulate the human hair follicle (Anonymous, 1970; for review see Randall, 1992). The response of human hair follicles to andro¬ gens is highly dependent on the location of the follicle; for example, beard follicles produce longer, thicker and darker hairs, eyelashes have no response and scalp follicles may regress in a relatively precise sequence in genetically sensitive individuals (Hamilton,
1942, 1958).
Follicles in some regions are stimulated by female levels of androgens, e.g. axilla and female pubic escutcheon, while others, such as the beard and chest, require male androgen levels (Marshall & Tanner, 1969, 1970). The responses of many follicles to andro¬ gens is gradual, increasing with age despite steady
Specific high-affinity low-capacity androgen receptors identified in 12 dermal papilla primary cell lines with similar characteristics to established androgen receptors. Cells from androgen-sensitive follicles (beard, scrotum and pubis) contained higher levels of androgen receptors than those derived from relatively androgeninsensitive non-balding scalp follicles whether the receptor content was calculated in relation to cell number, protein were
or
DNA content of the cells. These results support the
hypothesis that androgens act on hair follicles via the dermal papilla in vivo and demonstrate that dermal papilla cells exhibit an altered phenotype in culture which depends on the body site from which they were derived. Cultured human dermal papilla cells should
prove a useful model system for studies of the mechanism of androgen action, and further investigations may elucidate the paradox of why bald men can grow beards. Journal of Endocrinology (1992) 133, 141\p=n-\147
circulating androgens, e.g. the development ofterminal chest hair and male pattern baldness (Hamilton, 1946). The mechanisms by which androgens cause such diverse and progressive effects in the same tissues within the same individual are unknown. However, changes also occur in the prostate with increasing age, resulting in benign prostatic hypertrophy and prostatic carcinoma which are believed to be strongly influenced by androgens (Griffiths, Davies, Eaton et al. 1987). The hair follicle consists of a tube of epithelial cells protruding into the dermis and enclosing in its base the mesenchyme-derived dermal papilla. Hairs do not grow continuously; each follicle undergoes a repetitive sequence of growth and rest known as the hair growth cycle (Kligman, 1959). During the growth phase (anagen), epithelial cells derived from a highly proliferative region at the base of the follicle (the hair
bulb matrix) move distally and differentiate to form the hair shaft and the inner root sheath surrounding the hair. After a certain period determined by the body site, the follicle enters a resting phase (telogen) where mitotic activity ceases and the complex hair bulb structure is changed. After an interval, the follicle re-enters anagen, a new hair is formed and the old hair shed. During early anagen the hair follicle appears partially to recapitulate its embryonic
development. Although the molecular mechanisms responsible for controlling hair growth are not well understood, the dermal papilla is believed to play an essential role in inducing and maintaining epithelial differentiation (Oliver, 1966, 1967; Oliver & Jahoda, 1989); it may also exert a controlling influence on the hair growth cycle and determine other characteristics such as hair diameter (Van Scott & Ekel, 1958; Ibrahim & Wright, 1982) and cultured rat dermal papilla cells have been shown to retain their inductive capacity when re¬ implanted in vivo (Jahoda, Home & Oliver, 1984). Mesenchymal cells are also known to influence devel¬ oping epithelia in other hormone-dependent tissues including the mammary gland (Oka & Yoshimura, 1986) and prostate (Cunha, Taguchi, Shannon et al.
1984). Androgen receptors are required in the mesenchyme for prostate development (Cunha et al. 1984), and it, therefore, seems likely that androgens will influence the
hair follicle via the dermal papilla. Autoradiographic studies of [3H]testosterone have shown that dermal papillae, but not follicular epithelial cells, take up androgens (Stumpf & Sar, 1976). Intracellular androgen-receptor proteins are considered a pre¬ requisite for any cell to respond to androgens (Mainwaring, 1977) and testicular feminization patients, who lack functional androgen receptors, do not develop axillary, pubic or beard terminal hair (Keenan, Meyer, Hadjian et al. 1974). We have, there¬ fore, established primary cell lines of dermal papilla cells from human hair follicles obtained from body sites with varying responses to androgens in vivo and investigated whether they contain specific, saturable androgen receptors and whether the levels of receptors present varied with the androgen sensitivity of the follicle in vivo. MATERIALS AND METHODS
Cell culture
Primary cultures of dermal papilla cells from human hair follicles were established and grown as described by Messenger (1984). Briefly, hair follicles were iso¬ lated from normal skin samples obtained during routine clinical excisions. Dermal papillae were
microdissected from the base of anagen hair follicles, transferred to 35-mm sterile tissue-culture-treated Petri dishes and cultured in Eagles medium 199 (El99; Flow Laboratories Ltd, Irvine, Strathclyde, U.K.) supplemented with 20% (v/v) fetal bovine serum (Globefarm Ltd, Esher, Surrey, U.K.) and antibiotics at 37 °C in a humidified atmosphere containing 5% C02. The cell outgrowths were subcultured after 2-4 weeks into single 25-cm2 flasks in the same medium and conditions and then passaged into three flasks when confluent at intervals of 1-2 weeks. Tissue culture plastics were supplied by Flow Laboratories.
Androgen receptor assay The ability of cultured dermal papilla cells to bind the radiolabelled synthetic non-metabolizable androgen,
7a, 17a-dimethyl-19-nortestosterone (mibolerone) mibolerone; specific activity
([17a-methyl-3H]
2-74-3-33 TBq/mmol; Amersham International pic, Amersham, Bucks, U.K.) was determined by satu¬ ration analysis in 12 primary cell lines at passage numbers 3 to 6. Cells were seeded into 100 mm tissueculture-treated Petri dishes (Falcon Labware, Becton Dickenson, Cowley, Oxon, U.K.) at a density of about 2xl05 cells/dish and grown almost to con¬ fluence in normal medium. Preliminary experiments demonstrated that it was not possible to obtain good reproducible data when cells were cultured in 35 mm dishes. The cells were washed with Dulbecco's phosphatebuffered saline (PBS) and incubated in 5 ml E199 without serum for 24 h to reduce endogenous androgens. This medium was then replaced with 5 ml serum-free medium containing a range of concen¬ trations of [3H]mibolerone (005-10nmol/1; 9-10 concentration points) in the presence or absence of a 100-fold excess of 17ß-hydroxy-5a-androstanexcess of 3-one (5a-dihydrotestosterone); 1000
9-fluoro-16-hydroxyprednisolone-16a,17a-acetonide
also added to all of [3H]mibolerone to progesterone receptors. After 2 h at 37 GC the incubations were terminated by removal of the medium and washing the cells four times with PBS at 4°C; the cells were scraped from the dishes into cold PBS and collected by centrifugation. The super¬ natant was aspirated and the radioactive content ofthe cell pellets extracted with chloroform-methanol (1:1, v/v). The cells were collected again by centrifugation and the radioactivity in the supernatant was counted, after evaporation of the solvents, on an LKB liquid scintillation spectrophotometer for 10 min with a counting efficiency of 50%. Aliquots of the medium were also counted to obtain the amount of 'free' radioactivity remaining, i.e. not bound.
(triamcinolone acetonide)
was
incubations to prevent any
possible binding
0-5 1-0 2-0 3-0 4-0 5-0 Concentration of [3H]mibolerone (nmol/1)
10-0
5 10 Mibolerone bound
15
20
(fmol/mg protein)
1. Saturation analysis of [3H]mibolerone binding to the androgen receptor of cultured dermal papilla cells derived from human male beard follicles (passage 4). (a) Specific binding of mibolerone with increasing concentrations of 3H-labelled steroid (005-10 nmol/1) was determined as the difference between total (3H-labelled steroid alone) and non-specific binding ( -labelled steroid plus a 100-fold excess of unlabelled steroid). (¿>) Scatchard analysis of the data of specific binding as described above. The maximum binding capacity was 18-5 fmol/mg protein and the binding affinity 0-35 nmol/1. figure
The protein and DNA content of the remaining cell pellets from the individual Petri dishes were deter¬ mined by the methods of Lowry, Rosebrough, Farr & Randall (1951) and Burton (1956) respectively. The
number of cells present in one dish at the time of assay counted six times using a haemocytometer. The androgen-responsive mouse mammary carcinoma cell line Shionogi 115 (Jung-Testas & Baulieu, 1987) was assayed as a positive control. was
Scatchard plots (Fig. 1 b) were calculated for each cell line to provide an estimation of the affinity of the receptor for the ligand, i.e. the dissociation constant (Kd) and the number of binding sites available (Bmax). Radioligand binding by Shionogi 115 cells showed the expected high concentrations of high-affinity saturable androgen receptors (Figs 2 and 3) (Kd 0-29 nmol/1; Bmax 1-43 fmol/104 cells; 638-5 fmol/mg protein; 5-07 fmol/ pg DNA); this supports the validity of the assay.
Specificity of steroid binding To confirm that the radiolabelled steroid was actually binding to specific androgen receptors, the ability of various unlabelled steroids to compete for the binding was investigated. Cells were prepared as described for the receptor assay and dishes of cells from five cell lines were incubated in triplicate with 1 nmol[3H]mibolerone or in triplicate or duplicate with 1 nmol [3H]mibolerone plus a range of radioinert steroids (100 nmol/1) for 2 h at 37 °C. Binding was assessed as described above. Statistics
Differences between the types of cell line were analysed for significance using Student's /-test and MannWhitney U-test.
0-4r • P4
0-3
I«
o
I
0-2 •P5
p4
.P5 •P4
SP6
Beard
Genital
*P6
0-1 P4 •P5 •
*P5 Scalp
figure 2. Binding affinity (Ká nmol/1) for [3H]mibolerone in cultured dermal papilla cells derived from human scalp, beard and sexual skin hair follicles. passage number. =
RESULTS
Androgen receptor assay Specific binding of [3H]mibolerone was saturated at around 1 nmol/1 in all cells examined (Fig. la);
Androgen binding by dermal papilla cells from various body sites Saturable high-affinity binding was detected in all dermal papilla cells regardless of origin (Figs 2 and 3).
·4
50
40
S. 30
·3
I
o
I
20 »P5
E
PQ
10
,4 6 :, 4 ·5 ·4
·6
,P5 :p5 •P4
Beard Scalp figure 3. Concentration of androgen receptors (i.e. binding capacity (Bmax) for
Genital
pH]mibolerone) in cultured dermal papilla cells derived from human scalp, beard and sexual skin hair follicles.
=
passage number.
Dermal papilla cells derived from androgen-responsive hair follicles in beard, moustache, pubis and scrotum had a similar affinity for [3H]mibolerone to the Shionogi 115 cells (Fig. 2); the affinity of cells from androgeninsensitive non-balding scalp areas was significantly (P
