Dexamethasone, a Synthetic Glucocorticoid, Induces the Activity of Androgen Receptor in Human Dermal Papilla Cells

Abstract

Psychosocial stress stimulates the secretion of glucocorticoids (GCs), which are stress-related neurohormones. GCs are secreted from hair follicles and promote hair follicle regression by inducing cellular apoptosis. Moreover, the androgen receptor (AR) is abundant in the balding scalp, and androgens suppress hair growth by binding to AR in androgenetic alopecia. First, by using immunofluorescence, we investigated whether the treatment of dermal papilla (DP) cells with dexamethasone (DEX), a synthetic GC, causes the translocation of the glucocorticoid receptor (GR) into the nucleus. DEX treatment causes the translocation of the GR into the nucleus. Next, we investigated whether stress-induced GCs affect the AR, a key factor in male pattern baldness. In this study, we first assessed that DEX increases the expression of AR mRNA in non-balding DP cells, which rarely express AR without androgen. RU486, a GR antagonist, attenuated DEX-inducible AR mRNA expression and AR activation in human non-balding DP cells. In addition, AR translocated into the nucleus after DEX treatment. Furthermore, we indeed showed that the expression of AR was induced in the nucleus by DEX in DP cells of human and mouse hair follicles. Our results first suggest that stress-associated hair loss may be due to increased AR expression and activity induced by DEX. These results demonstrate that hair loss occurs in non-balding scalps with low AR expression.

Introduction

Stressful conditions activate the hypothalamic-pituitary-adrenal axis and elevate the levels of stress-related neurohormones, including glucocorticoids (GCs). GCs are secreted by human hair follicles via activation of the hypothalamic-pituitary-adrenal axis [1]. GC signaling is primarily mediated by the glucocorticoid receptor (GR). When GCs bind to the GR, they translocate into the nucleus where they control various effector mechanisms [2]. A recent study showed that GR is expressed in human hair follicles [3], and dexamethasone (DEX), a synthetic GC, inhibits the proliferation of human hair dermal papilla (DP) cells by decreasing the expression of growth factors required for hair growth [4]. In addition, DEX promotes catagen, which is known as the hair follicle regression phase [3, 5, 6].

Androgenetic alopecia (AGA) is a common form of hair loss induced by androgens. Although the specific mechanism underlying AGA is unknown, androgens are thought to act on the androgen receptor (AR) to induce hair follicle miniaturization and hair loss [7]. Circulating androgens enter the DP capillaries and bind to the AR. The androgen-AR complex then translocates into the nucleus, resulting in the activation or repression of target genes. The balding scalp of patients with AGA exhibits high levels of dihydrotestosterone (DHT), a potent androgen, and increased expression of the AR [7, 8, 9].

Recently, we reported that DEX, a known stress-related neurohormone, increases the expression of dickkopf-1 (DKK1) in non-balding DP cells. Interestingly, DKK-1 which is induced by DHT promotes the regression of the hair cycle [3, 9, 10]. These findings prompted us to investigate whether stress modulates AR activity and causes hair loss in a non-balding scalp.

In this study, we examined whether DEX induces AR expression and translocation into the nucleus of human non-balding DP cells, and increases the activation of the AR element of AR signaling. In addition, AR activation in response to DEX was investigated by immunostaining in cultured human hair follicles and in mouse skin.

Materials and Methods

Isolation and Culture of Human Hair Follicles and DP

This study was approved by the Medical Ethics Committee of Kyungpook National University Hospital (IRB Number KNUH 2021-01-013-001). Non-balding scalp specimens were obtained from male patients undergoing hair transplantation surgery with the patients' consent at the Kyungpook National University Hospital (Daegu, Korea). Hair follicles were isolated and cultured according to the method described previously [3, 11]. Briefly, the hair follicles were isolated and dissected under a binocular microscope before culturing in Williams E media without phenol red (Sigma, St. Louis, MO, USA) and with or without DEX (Sigma) for 24 h at 37°C in a humidified atmosphere of 5% CO_2.

Non-balding DPs were isolated from the bulbs of dissected hair follicles, and cultured in a type 1 collagen-coated dish in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Gaithersburg, MD, USA) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and 20% heat-inactivated fetal bovine serum. After cell outgrowth, the cells were subcultured in DMEM supplemented with 10% fetal bovine serum at a split ratio of 1:4.

Immunofluorescence Staining

DP cells were seeded at 5,000 cells per well on an eight-chamber slide (Nunc Lab-Tek, Roskilde, Denmark) for 24 h, and cultured in serum-free DMEM in the presence of DEX (Sigma) or DHT (Sigma) for 24 h. The cells were fixed and permeabilized with 0.1% Triton X-100 in 4% paraformaldehyde for 10 min and blocked with 5% normal donkey serum (Abcam, Cambridge, UK) for 1 h at room temperature. The cells were then incubated with mouse anti-GR antibody (1:100 dilution; R&D Systems, Minneapolis, CA, USA) or rabbit anti-AR antibody (1:100 dilution; Abcam) at 4°C overnight. Slides were washed three times with PBS and incubated with Alexa Fluor 488-labeled donkey anti-mouse or anti-rabbit secondary antibody (1:100 dilution; Molecular Probes, Eugene, OR, USA) for 1 h, respectively. The slides were washed with PBS and counterstained with 4,6-diamidino-2-phenylindole for 10 min. Normal mouse or rabbit IgG (R&D Systems) was used as the negative controls.

The tissues were embedded in a cryomold (OCT compound, Tissue-Tek; Miles, Napierville, IL, USA) and placed in a freezer at −80°C. The tissue block was cut into 7-μm-thick sections using a cryostat (Leica CM3050 S; Heidelberg, Germany). The slides were immunostained with rabbit anti-AR antibody (1:100 dilution; Abcam) as described above.

RT-PCR and Real-Time PCR Analysis

The total RNA was extracted using the RNeasy Mini Kit (Qiagen, Houston, TX, USA), and cDNA was synthesized from 3 μg of total RNA using a cDNA synthesis kit and oligo-dT primer containing the ImProm-II^TM reverse transcriptase (Promega, Madison, WI, USA). cDNA (1 μL) was amplified using forward and reverse primers. For the detection of AR, 30 cycles (1 min at 94°C, 45 s at 60°C, and 45 s at 72°C) of amplification were performed with 5′-GGTAAGGGAAGTAGGTGGAA-3′ and 5′-CCTTCTAGCCCTTTGGTGTA-3′. For the detection of β-actin, 20 cycles (1 min at 94°C, 45 s at 58°C, and 45 s at 72°C) of amplification were performed with 5′-GGGAAATCGTGCGT GACATT-3′ and 5′-GGAGTTGAAGGTAGT TTCGTG-3′. The PCR products were separated by electrophoresis on a 1% agarose gel and visualized under UV light.

For real-time PCR analysis, 100 ng of cDNA and 10-μM primers were amplified using Power SYBR Green premix and OnePlus Real-Time PCR system (Applied Biosystems). The AR primers (Hs_AR_1_SG QuantiTect Primer) were purchased from Qiagen. Amplification was performed under the following cycling conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 60 s.

Reporter Assay

Human DP cells were transfected with 0.5 μg of a pAR (Qiagen)-binding consensus sequence, followed by the luciferase gene using a microporator (Invitrogen). At 24 h after transfection, the cells were treated with DEX in the absence and presence of the GR antagonist RU486 for 24 h. A luciferase assay was performed using dual luciferase assay reporter kit according to the manufacturer's instructions (Promega).

Animal Study

Five-week-old female C57BL/6 mice at the anagen stage of the hair cycle were purchased from Orient Bio Inc. (Seongnam, Korea) and shaved with clippers. DEX (100 nM) in 100 μL of propylene glycol (Sigma) was spread on the dorsal skin shaved mice for 4 days. On the following day, the mice were sacrificed and the treated region of the dorsal skin was collected for immunofluorescence staining.

Results and Discussion

GR and AR are transcription factors with hormone activity and belong to the subfamily of steroid receptors (SRs). The SR family comprises three main functional domains: the N-terminal transactivation domain, central DNA-binding domain, and C-terminal ligand-binding domain. After the steroid binds to the ligand-binding domain, it translocates to the nucleus and binds to the steroid response elements to control the expression of target genes [12]. Among the SRs, GR and AR share a significant number of chromatin binding sites, which are associated with genes regulated by androgens and GCs [13, 14]. Recently, several reports suggested that GCs can activate androgen signaling by interacting with AR [15, 16, 17, 18]. First, we showed the expression of GR in human non-balding DP cells. Immunostaining showed that GR is expressed in the cytoplasm. Moreover, GR translocated into the nucleus within 24 h after treatment with 100 nM DEX. These results show that human DP cells respond to DEX. GR was strongly expressed and responded to DEX (Fig. 1a; online suppl. Fig. 1; for all online suppl. material, see www.karger.com/doi/10.1159/000525067), whereas AR was hardly expressed in non-balding DP cells [8, 19].

Fig. 1.

Effect of DEX on the expression of AR in human non-balding DP cells. a Cells were incubated in the absence or presence of DEX for 24 h and immunostained with anti-GR antibody (left panel). Corresponding 4,6-diamidino-2-phenylindole (DAPI) nuclear staining is also shown (right panel). b, d Cells were treated with 100 nM DEX for varying times and analyzed by RT-PCR and real-time PCR. c, e Cells were also treated with varying concentrations of DEX for 24 h and analyzed by RT-PCR and real-time PCR. f, g DP cells were treated with 100 nM DEX in the presence or absence of 100 nM RU486 for 24 h and subsequently analyzed by RT-PCR and real-time PCR. Relative levels of AR are shown as mean ± SD from three independent experiments (*p < 0.05).

Based on previous reports, we investigated whether DEX induced in a stressful environment affects the expression and activity of AR, a key factor in male pattern hair loss. Consistent with the results of previous studies [7, 8, 20], we observed that AR expression was very low in non-balding DP cells. Surprisingly, the expression of AR mRNA increased in a time- and dose-dependent manner after DEX treatment. RT-PCR (Fig. 1b, c; online suppl. Fig. 2) and real-time PCR (Fig. 1d, e) results revealed that AR expression was highly upregulated in non-balding DP cells (n = 3) treated with 100 nM DEX for 24 h. Furthermore, RU486, a synthetic GR antagonist, significantly attenuated DEX-induced AR mRNA expression in DP cells (n = 3; Fig. 1f, g), demonstrating that DEX-induced AR expression is GR-dependent. Interestingly, we observed no induction of AR mRNA in hair keratinocytes, mouse fibroblasts, or periodontal ligament stem cells. In particular, periodontal ligament stem cells are the cells that we previously reported in response to DEX [4].

Next, to assess the effect of DEX on AR activity, we transiently transfected non-balding DP cells with an AR reporter plasmid. DEX significantly stimulated transcriptional AR activity. Consistent with the above data, AR transcriptional activity was suppressed by RU486 treatment (Fig. 2a). As mentioned in several reports, DEX activates androgen signaling by interacting with AR. To confirm that the DEX induces AR activity, we examined AR translocation after DEX treatment in non-balding DP cells. Immunofluorescence staining showed that AR expression was very low in the absence of DEX. In contrast, we observed that the AR was strongly expressed in the nucleus 24 h after DEX treatment (Fig. 2b; online suppl. Fig. 3). These results suggest that DEX not only increases AR expression but also activates AR signaling.

Fig. 2.

Effect of DEX on the activity of AR in hair follicles of human and mouse. a Cells were transfected with pARE-luciferase plasmid and treated with or without 100 nM DEX and 100 nM RU486 for 24 h. Data are expressed as means ± SD of two determinations per experiment from three independent experiments (*p < 0.05). b Cells were incubated in the absence or presence of 100 nM DEX for 24 h and immunostained with anti-AR antibody (upper panels). Corresponding DAPI nuclear staining is shown in the lower panels. c Human non-balding hair follicles were treated with 100 nM DEX for 48 h, followed by immunostaining to examine the induction of AR (upper panels). DAPI nuclear staining was also performed (lower panels). White stars indicate DP in hair follicles. d The dorsal skin of C57BL/6 mice was treated with 100 nM DEX for 4 days, and the expression of AR was examined (upper panels). DAPI nuclear staining was also performed (lower panels). White stars indicate the DP in hair follicles.

The hair follicle is a multilayered organ that contains DP and dermal sheath cells that are derived from the mesenchymal and outer root sheath, and inner root sheath, matrix, and hair shaft that are derived from the epithelium [21]. Circulating androgens in the blood vessels affect various cells around the DP by regulating the expression of various factors after binding to AR in the DP [22]. However, in vitro experiments are limited in their ability to study the effects of various cells present in the hair follicle. Therefore, we next assessed whether DEX activated AR and increased its expression in hair follicles in vivo. First, we treated non-balding human hair follicles with 100 nM DEX for 96 h and performed KI67 immunostaining and TUNEL assay to observe cell proliferation and apoptosis (online suppl. Fig. 4). DEX sharply decreased KI67-positive cells and increased TUNEL-positive cells compared to the control. In addition, hair follicles exhibited catagen-like morphology and decreased DP size. To observe AR activity in the DP, we reduced the DEX treatment time. Next, we treated non-balding human hair follicles with 100 nM DEX for 48 h followed by AR immunostaining. Upon DEX treatment, the expression of the AR significantly increased in the DP of human hair follicles and was expressed in the nucleus of DP cells (Fig. 2c; online suppl. Fig. 5).

Moreover, to observe the expression of AR in mouse hair follicles, we performed an in vivo study. Several groups have reported that DEX induces catagen stage in mice and humans. In addition, it has been reported to cause apoptosis in human DP cells and inhibit the expression of growth factors related to hair growth [3, 4, 6]. More recently, treatment with DHT in a mouse model showed hair growth inhibition and induction of AR translocation in mice hair follicles similar to human hair follicles [19]. We conducted a mouse experiment with the above references to observe the phenomenon that appears in hair blocks of the skin rather than a single human hair follicle. The dorsal area of 5-week-old female C57BL/6 mice was shaved during the anagen stage of the hair cycle and treated with various concentrations of DEX for 4 days (online suppl. Fig. 6). Catagen promotion was dependent on DEX concentration. To observe the expression of AR in the DP of mouse hair follicles, we treated the skin with 100 nM DEX for 4 days. The skin samples were stained with an anti-AR antibody. Consistent with the results of AR staining of the human hair follicles, the AR was strongly expressed in the nucleus of DP cells in DEX-treated mouse hair follicles (Fig. 2d; online suppl. Fig. 7). Previous studies demonstrated that DEX premature the stage of hair regression by inducing DKK1 secretion, which is induced by DHT and causes hair regression as a male pattern hair loss inducer. Interestingly, DKK1 is secreted by both androgen (DHT) and GC (DEX) and induces hair regression [3, 9, 10]. DEX production induced by a stressful environment is expected to cause hair loss by activating the AR and increasing AR expression, even in non-balding DP cells with low AR expression.

In summary, we observed for the first time that DEX increased the expression of AR in non-balding DP cells. Furthermore, DEX activated AR signaling by translocating AR into the nucleus in hair follicles of both human and mouse. Taken altogether, our data suggest that stress-associated hair loss may be due, at least in part, to AR expression and activation, increased by DEX.

Statement of Ethics

This study was conducted in accordance with the Declaration of Helsinki Principles. Informed written consent was obtained from all patients. The Medical Ethical Committee of the Kyungpook National University Hospital (Daegu, Korea) approved all of the described studies (IRB Number KNUH 2021-01-013-001). Animal care and treatment protocols were in accordance with the guidelines of the use of laboratory animals. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Kyungpook National University (IRB No. KNU 2019-0171). This study was carried out in compliance with the ARRIVE guidelines.

Conflict of Interest Statement

The authors state no conflicts of interest.

Funding Sources

This Study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1A2C4002222).

Author Contributions

All authors contributed to the conception of the study. Mi Hee Kwack and Ons Ben Hamida performed the experiments. Mi Hee Kwack, Moon Kyu Kim, Jung Chul Kim, and Young Kwan Sung contributed new reagents and analyzed the data. Mi Hee Kwack supervised the research and wrote the manuscript. All authors have read and approved the manuscript.

Data Availability Statement

All data generated or analyzed during this study are included in this article and its online supplementary material. Further inquiries can be directed to the corresponding author.

Supplementary Material

Supplementary data

Supplementary data

Funding Statement

This Study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1A2C4002222).