Dihydrotestosterone Regulates Hair Growth Through the Wnt/β-Catenin Pathway in C57BL/6 Mice and In Vitro Organ Culture

Abstract

Dihydrotestosterone (DHT) is the most potent androgen that regulates hair cycling. Hair cycling involves cross-talk between the androgen and Wnt/β-catenin pathways. However, how DHT regulates hair follicle (HF) growth through the Wnt/β-catenin pathway has not been well investigated. This study aimed to investigate the roles of DHT in hair growth in vivo and in vitro. Human scalp HFs were treated with different concentrations of DHT (10^-5, 10^-6, 10^-7, 10^-8, and 10^-9 mol/L) for 10 days. The effects of DHT on hair shaft elongation, the proliferation of hair matrix cells, and the levels of β-catenin, GSK-3β, and phosphorylated GSK-3β (ser9) were evaluated in the cultured HFs. The effects of DHT were further investigated in C57BL/6 mice. Moreover, the growth of cultured human HFs was observed after interfering with the β-catenin pathway through inhibitors or activators in the presence or absence of DHT. We found that different concentrations of DHT had different effects on human HFs in vitro and C57BL/6 mice. At 10^-6 mol/L, DHT inhibited HF growth and β-catenin/p-GSK-3β expression, whereas 10^-7 mol/L DHT induced HF growth and β-catenin/p-GSK-3β expression. In addition, a β-catenin inhibitor (21H7) inhibited HF growth in vitro, while a β-catenin activator (IM12) promoted HF growth in vitro and antagonized the inhibition of HFs by high levels of DHT. These results suggest that DHT plays a pivotal role in region-specific hair growth, which may be related to the Wnt/β-catenin pathway.

Keywords: Wnt/β-catenin pathway; androgenetic alopecia; dihydrotestosterone; hair follicle; hair growth; β-catenin activator.

Figure 1

Effects of different concentrations of dihydrotestosterone (DHT) on hair shaft growth of human HFs in vitro. Isolated anagen human HFs were cultured with DHT at various concentrations or vehicle for 10 days. (A) Mean growth rates of hair shafts. Hair shaft length was measured every 2 days (n = 18). (B) Changes in hair bulb morphology during the 10-day experimental period and immunofluorescence staining of Ki67 in HFs cultured with various concentrations of DHT (0, 10^-6 and 10^-7 mol/L) at day 10. (C) Percentages of Ki-67-positive cells in the matrix area (n = 6). The data are presented as the mean ± SD. Each bar represents the mean of three independent experiments performed in triplicate. Compared with the vehicle-treated control group, ^*P < 0.05.

Figure 2

Effects of topical dihydrotestosterone (DHT) on hair regeneration in C57BL/6 mice. The back skin was treated with DHT (10^-6, 10^-7, or 10^-8 mol/L) every day for 18 days in the DHT groups, while the vehicle-treated control group was treated with 40% DMSO. The back skin was photographed at days 0, 3, 6, 9, 12, 14, and 17 after treatment initiation.

Figure 3

Effects of different concentrations of dihydrotestosterone (DHT) on hair follicle morphology in C57BL/6 mice. The back skin of C57BL/6 mice at 18 days after treatment was shaved and then stained with hematoxylin and eosin. Representative HE staining images from mice treated with different concentrations of DHT are shown. The red line refers to back skin, while the blue line represents gray skin.

Figure 4

Effects of different concentrations of dihydrotestosterone (DHT) on the expression of β-catenin, GSK3β, and p-GSK3β (ser9) in human HFs. The hair follicles (HFs) were fixed and labeled with anti-β-catenin, anti-GSK3β, and anti-p-GSK3β (ser9) primary antibodies, followed by incubation with fluorescent secondary antibodies. (A) The HFs were visualized: green, β-catenin and p-GSK3β (ser9); red, GSK3β (magnification: 200× and 400×). (B, C) The mean fluorescence intensity of β-catenin, GSK3β, and p-GSK3β (ser9) in the matrix area. The data are presented as the mean ± SD (n = 6). Each bar represents the mean of three independent experiments performed in triplicate. Compared with the control group, ^*P < 0.05. Scale bar 50 μm (200×).

Figure 5

The expression pattern of AR in the normal human hair follicles (HFs). AR is mainly expressed in dermal papilla cells and hair matrix cells. Scale bar 50 μm (200×).

Figure 6

Dihydrotestosterone (DHT) regulates human hair follicles (HFs) growth through the Wnt/β-catenin pathway. HFs were cultured with IM12 (50, 100, or 500 nM), 21H7 (1, 2, or 4 µM), DHT (10^-6 mol/L), IM12 (500 nM), or 10^-6 mol/L DHT and 500 nM IM12. (A, B) Mean growth rates of hair shafts treated with IM12 and 21H7 (n = 10). (C) Mean growth rates of hair shafts treated with DHT and IM12 (n = 10). (D, E) Immunofluorescence staining and mean fluorescence intensity of β-catenin in HFs cultured with DHT and IM12 (n = 6). Magnification: 200× and 400×. The data are presented as the mean ± SD. Each bar represents the mean of three independent experiments performed in triplicate. Compared with the control (0 ) group, *P < 0.05 , ^&P < 0.05 and ^#P < 0.05. Scale bar 50 μm (200×).

Figure 7

Effects of different concentrations of dihydrotestosterone (DHT) on the expression of β-catenin, GSK3β, and p-GSK3β (ser9) in C57BL/6 mice. The nucleoproteins and total proteins of the back skin were extracted for Western blot. (A) Western blot analysis of the expression of nuclear β-catenin, total β-catenin (t-β-catenin), total GSK3β (t-GSK3β), and p-GSK3β (ser9) (p-GSK3β) in the back skin. (B) The statistical results of Western blot analysis. The data are presented as the mean ± SD (n = 5). Each bar represents the mean of three independent experiments performed in triplicate. Compared with the control group, ^*P < 0.05 and ^#P < 0.05.