CXXC5 Mediates DHT-Induced Androgenetic Alopecia via PGD2

The number of people suffering from hair loss is increasing, and hair loss occurs not only in older men but also in women and young people. Prostaglandin D2 (PGD2) is a well-known alopecia inducer. However, the mechanism by which PGD2 induces alopecia is poorly understood. In this study, we characterized CXXC5, a negative regulator of the Wnt/β-catenin pathway, as a mediator for hair loss by PGD2. The hair loss by PGD2 was restored by Cxxc5 knock-out or treatment of protein transduction domain–Dishevelled binding motif (PTD-DBM), a peptide activating the Wnt/β-catenin pathway via interference with the Dishevelled (Dvl) binding function of CXXC5. In addition, suppression of neogenic hair growth by PGD2 was also overcome by PTD-DBM treatment or Cxxc5 knock-out as shown by the wound-induced hair neogenesis (WIHN) model. Moreover, we found that CXXC5 also mediates DHT-induced hair loss via PGD2. DHT-induced hair loss was alleviated by inhibition of both GSK-3β and CXXC5 functions. Overall, CXXC5 mediates the hair loss by the DHT-PGD2 axis through suppression of Wnt/β-catenin signaling.


Introduction
The hair follicle is one of the few regenerative organs [1], which undergoes a cycle of anagen (growth phase), catagen (regression phase), and telogen (resting phase) [2]. The major features of hair loss include a shortened anagen phase, a rapid transition to the catagen phase, and eventual miniaturization of hair follicles [3]. Dihydrotestosterone (DHT) and prostaglandin D 2 (PGD 2 ) are known as major factors that cause alopecia while inducing hair follicle miniaturization [4]. The PGD 2 is elevated in alopecia patients and reduces hair regrowth as well as wound-induced hair follicle neogenesis (WIHN) [5,6]. It is known that PGD 2 inhibits hair growth through its receptor Gpr44 [6]. However, the mechanism of PGD 2 in hair loss is poorly understood.
The Wnt/β-catenin signaling pathway is one of the most important signaling pathways that regulate hair follicles [7,8]. The Wnt/β-catenin pathway is essential for the development, maintenance, and WIHN of hair follicles [9][10][11]. The decrease in the Wnt/βcatenin signaling is a key feature in the onset of the catagen phase and alopecia [3,12]. Previously, we identified that CXXC-type zinc finger protein 5 (CXXC5) is a BMP signaling target gene and a negative regulator of the Wnt/β-catenin signaling functioning by interacting with Dishevelled (Dvl) [13,14]. Similar to other alopecia factors, Cxxc5 increases in the late anagen phase to induce the catagen phase and the CXXC5 level is elevated in alopecia patients [12]. Since CXXC5 suppresses the Wnt/β-catenin signaling, both hair regrowth and WIHN are increased in Cxxc5 knock-out mice [12]. Furthermore, treatment with protein transduction domain-Dvl binding motif (PTD-DBM), a peptide that interferes with CXXC5-Dvl binding function [14], promotes hair regrowth and WIHN, as in Cxxc5 knock-out mice [12]. Although the role of CXXC5 in hair loss has been identified, the upstream event inducing CXXC5 has not yet been elucidated.
In this study, we identified that expressions of prostaglandin D 2 synthase (Ptgds) and p-smad1/5/9, a transcription factor of BMP signaling, had similar patterns to those of Cxxc5 during the hair cycle. We also found that PGD 2 induced CXXC5 through BMP signaling in vitro. PTD-DBM treatment or Cxxc5 knock-down attenuated the suppressive effects of PGD 2 in vitro. Both PTD-DBM-treated and Cxxc5 knock-out mice improved hair regrowth ex vivo and in vivo by restoring Wnt/β-catenin signaling and proliferation. Inhibition of CXXC5 function also restored the suppressed WIHN by PGD 2 . Moreover, DHT was revealed to cause alopecia via suppression of the Wnt/β-catenin signaling pathway by activating GSK-3β [15,16], and PTGDS is induced by the androgen receptor (AR) [17,18]. Our study confirmed that DHT increased PTGDS and subsequently induced CXXC5. We also found that DHT-induced alopecia was alleviated by treatment with KY19382, a small molecule mimetic of PTD-DBM which induces hair growth by simultaneous inhibition of the functions of CXXC5 and GSK-3β [19]. Taken together, we identified that DHT and PGD 2, the major inducers of androgenetic alopecia [16] , cause hair loss by Wnt/β-catenin signaling suppression via CXXC5 overexpression. Therefore, we suggest that inhibition of both CXXC5 and GSK-3β functions is an effective approach for male pattern androgenetic alopecia (AGA) treatment.

Mice
Wild-type C57BL/6N mice were obtained from Koatech Co. (Gyeonggido, Korea). The generation of the Cxxc5 knock-out mouse was described previously [20]. Cxxc5 heterozygous mice were crossed to obtain wild-type and Cxxc5 knock-out mice.

Depilation-Induced Hair Cycle Progression
Seven-week-old wild-type mice were anesthetized by intraperitoneal injection of 2,2,2,tribromoethanol (400 mg/kg, IP; Sigma-Aldrich, St. Louis, MO, USA). The dorsal skins of mice were plucked to induce hair cycle synchronization [21]. After the experiments were completed, the mice were euthanized using CO 2 gas.

Hematoxylin and Eosin (H&E) Staining
Skin tissues were fixed with 10% formalin overnight at 4 • C. The tissues were paraffinized and sliced into pieces 4 µm in thickness. The slides were deparaffinized and rehydrated. The slides were incubated in hematoxylin for 5 min and eosin for 1 min. The number of follicles was measured by counting the hair follicles in H&E staining images. Dermal thickness was measured by using ImageJ software V1.48.

Reverse Transcription and Quantitative Real-Time PCR (qRT-PCR)
The RNA was separated by using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The total RNA (2 µg) was reversely transcribed using reverse transcriptases (Invitrogen) at 42 • C for 1 h. The resulting cDNA was amplified using a reaction mixture containing 10 pmol of the primer set (Bioneer, Daejeon, Republic of Korea) and iQ SYBR Green Supermix (QIAGEN, Hilden, Germany). The primer sequences are described in Supplementary Table S1.

Immunocytochemistry
HaCaT cell lines were seeded in a 12-well plate on coverslips. Cultured cells were washed with PBS and fixed with 10% formalin (Sigma-Aldrich), and then permeabilized with 0.2% Triton X-100. After blocking with 5% BSA in PBS, the cells were blotted with primary antibodies: mouse anti-CXXC5 (Santa Cruz Biotechnology, 1:20) or rabbit anti-βcatenin (Abcam, 1:20) overnight at 4 • C. After washing with PBS, the cells were blotted with appropriate secondary antibodies: Alexa Fluor 488-conjugated goat anti-mouse or Alexa Fluor 555-conjugated goat anti-rabbit (Molecular Probes, Leiden, the Netherlands) for 1 h at room temperature and counterstained with DAPI. Images were taken using an LSM510 confocal microscope (Carl Zeiss Inc.). The fluorescence intensity was quantified using Zen software V3.1 (Carl Zeiss Inc.)

In Vivo Hair Regrowth Test
For evaluations of hair regrowth by PGD 2 and PTD-DBM, respectively, the back skins of 7-week-old mice were depilated and topically treated with 300 µL of 10 µg PGD 2 or 10 mM PTD-DBM every other day from 8 days to 12 or 20 days [5]. To measure hair regrowth, regrown hairs were collected by using a hair clipper and weighed by using a precision balance. The hair shaft length was measured by using ImageJ software V1.48. For the hair regrowth test for DHT or KY19382, the back skins of 8-week-old mice were shaved and topically applied daily with 300 µL of 100 µM DHT or 2 mM KY19382 for 42 days.

Wound-Induced Hair Follicle Neogenesis Assay
The dorsal skins of 3-week-old mice received 1 cm 2 full-thickness wounds and were treated topically with 20 µL of 10 µg PGD 2 or 10 mM PTD-DBM from 7 days to 17 or 25 days daily [6].
For whole-mount ALP staining, the wounded skin tissues were placed in 5 mM EDTA in PBS. The dermis layer was separated, fixed in 4% paraformaldehyde, washed with PBS, and then incubated in NBT/BCIP solution. The images of the dermis were taken by using a stereomicroscope (Nikon). The number of ALP-positive neogenic follicles was calculated by counting dark blue dots.

Statistical Analysis
In vivo and in vitro experiments were designed to establish randomization, equal size, and blinding. The statistical analyses were performed only for experiments with group sizes (n) ≥ 5. All group sizes represent the numbers of experimental independent values used to determine statistical analyses. All statistical data were expressed as means ± standard error of the mean. Comparisons between two unpaired groups were assessed with Student's t-test. For comparisons between multi-group studies, one-way ANOVA with Tukey's test was performed and post hoc tests were conducted only when the F of ANOVA obtained the required level of statistical significance (p < 0.05). There was no significant variance inhomogeneity. Prism software V5.01 (GraphPad, CA) was used for statistical analyses. The statistical significance is shown in the figures as follows: * p < 0.05, ** p < 0.005, *** p < 0.0005, NS: not significant.

The Expression Patterns of Ptgds Correlate and Inversely Correlate with Those of Cxxc5 and B-Catenin, Respectively
As revealed by immunohistochemical (IHC) analyses, the expression profile of hair inhibitory factor Cxxc5 is oppositely regulated with that of β-catenin during the mouse hair cycle [12]. The expression patterns of Ptgds are correlated with those of Cxxc5 and inversely correlated with β-catenin, respectively, during the mouse hair cycle (Figure 1a,b and Supplementary Figures S1 and S2). Ptgds and Cxxc5 proteins started to increase in the late anagen phase and reached a maximum level in the catagen phase [5,12]. Contrastively, β-catenin, a hair promotion factor, was decreased in the catagen phase [22]. The correlative expression patterns of Ptgds and Cxxc5 were also confirmed by quantitative RT-PCR (qRT-PCR) and immunoblotting analyses (Figure 1c,d). Overall, Ptgds, which generates the hair growth inhibitory factor PGD 2 , and Cxxc5 correlate in their expression patterns during the mouse hair growth cycle. statistical analyses. The statistical significance is shown in the figures as follows: * p < 0.05, ** p < 0.005, *** p < 0.0005, NS: not significant.

The Expression Patterns of Ptgds Correlate and Inversely Correlate with Those of Cxxc5 and Β-Catenin, Respectively
As revealed by immunohistochemical (IHC) analyses, the expression profile of hair inhibitory factor Cxxc5 is oppositely regulated with that of β-catenin during the mouse hair cycle [12]. The expression patterns of Ptgds are correlated with those of Cxxc5 and inversely correlated with β-catenin, respectively, during the mouse hair cycle (Figure 1a,b and Supplementary Figures S1 and S2). Ptgds and Cxxc5 proteins started to increase in the late anagen phase and reached a maximum level in the catagen phase [5,12]. Contrastively, β-catenin, a hair promotion factor, was decreased in the catagen phase [22]. The correlative expression patterns of Ptgds and Cxxc5 were also confirmed by quantitative RT-PCR (qRT-PCR) and immunoblotting analyses (Figure 1c,d). Overall, Ptgds, which generates the hair growth inhibitory factor PGD2, and Cxxc5 correlate in their expression patterns during the mouse hair growth cycle.  IHC staining for Ptgds, p-smad1/5/9, Cxxc5, and β-catenin. (c) Immunoblotting assay of whole skin for Ptgds, p-smad1/5/9, Cxxc5, β-catenin, and total Erk. (d) qRT-PCR of whole skin for Ptgds, Cxxc5, Hes1, and Jag1 (n = 5). Scale bars = 100 µm. Dashed lines indicate hair follicles. Values are expressed as the means ± SEM. Student's t-test: * p < 0.05, ** p < 0.005, *** p < 0.0005, N.S: not significant.

PGD 2 Suppresses Hair Growth via CXXC5
Previous studies found that CXXC5 is induced by BMP signaling in the brain and bone [13,23]. Since the BMP signaling is involved in the catagen phase [24], we examined the relationship between CXXC5 and BMP signaling in hair cells. The p-smad1/5/9, a transcription factor of BMP signaling, showed similar expression patterns to Cxxc5 and Ptgds (Figure 1b and Supplementary Figure S2b). In addition, mRNA expression patterns of Ptgds and Cxxc5 were similar to those of the p-smad target genes, Hes1 and Jag1 [25,26] ( Figure 1d). Moreover, we confirmed the mRNA and protein expression levels of CXXC5 were increased by treatment of BMP4 in HaCaT cell lines (Supplementary Figure S3a-d).
To confirm the relationship between PGD 2 signaling and CXXC5, we tested effects of PGD 2 treatment on expressions of Cxxc5 and the BMP signaling target genes in HaCaT cell lines. As HES1 and JAG1, the mRNA level of CXXC5 was increased by treatment of PGD 2 (Figure 2a). The levels of CXXC5 and p-smad1/5/9 were dose-dependently increased with the decrement in β-catenin and PCNA (Figure 2b-e). To further confirm the induction of CXXC5 by PGD 2 via BMP signaling, we tested the effects of Noggin, a BMP signaling inhibitor, on the PGD 2 -induced CXXC5 induction. Here, the increased CXXC5 and decreased β-catenin levels by PGD 2 were mostly abolished, respectively, by inhibiting BMP signaling by Noggin (Supplementary Figure S3e-h).
To investigate whether PGD 2 reduces Wnt/β-catenin signaling via CXXC5, we treated with PTD-DBM to inhibit the CXXC5 function. We confirmed that the reduced Wnt/βcatenin signaling and PCNA by PGD 2 were recovered by PTD-DBM treatment (Figure 2f-i). Correspondingly, the knock-down of CXXC5 also restored the decreased Wnt/β-catenin signaling with the proliferation (Figure 2j-m). Taken together, these results show that PGD 2 induced CXXC5 via BMP signaling, and the increased CXXC5 suppressed the Wnt/βcatenin signaling followed by suppression of the hair cell proliferation.

Blockade of CXXC5 Function Recovers Hair Growth Suppressed by PGD 2
To identify the recovery effects of blockade of CXXC5 function on hair growth suppressed by PGD 2 , we adapted the ex vivo vibrissa follicle culture system [27]. The inhibition in vibrissa elongation by PGD 2 treatment was restored by treatment of PTD-DBM (Supplementary Figure S4a,b). The restoration of the PGD 2 -mediated suppressions of βcatenin expression and proliferation by PTD-DBM treatment in vibrissa hair follicles was confirmed (Supplementary Figure S4c,d). To further confirm the effects of Cxxc5 knock-out on the decrement in vibrissa length by PGD 2 , we separated the vibrissa follicles from Cxxc5 wild-type or knock-out mice. Differently from the vibrissa hair follicles obtained from the Cxxc5 wild-type mice, those from Cxxc5 knock-out mice were not suppressed in vibrissa follicle elongation by PGD 2 (Supplementary Figure S4e,f). The ineffectiveness of PGD 2 treatment in the growth of vibrissa hair follicles correlated with no effect on β-catenin and PCNA in Cxxc5 knock-out mice (Supplementary Figure S4g (Figure 2a). The levels of CXXC5 and p-smad1/5/9 were dose-dependently increased with the decrement in β-catenin and PCNA (Figure 2b-e). To further confirm the induction of CXXC5 by PGD2 via BMP signaling, we tested the effects of Noggin, a BMP signaling inhibitor, on the PGD2-induced CXXC5 induction. Here, the increased CXXC5 and decreased β-catenin levels by PGD2 were mostly abolished, respectively, by inhibiting BMP signaling by Noggin (Supplementary Figure S3e-h).

Inhibition of CXXC5 Function Restores WIHN Reduced by PGD2
To confirm the restorative effects of inhibiting CXXC5 function in hair follicle regeneration, we used the WIHN experimental model [11]. The growth of neogenic hair follicles, which were suppressed by PGD2 [6], was recovered by treatment of PTD-DBM as shown by ALP staining (Figure 4a,b) as well as by hematoxylin and eosin (H&E) staining (Figure 4c). We revealed induction of fgf9 and keratin 17, WIHN markers [28,29] as well as the β-catenin and PCNA by the PTD-DBM treatment in the PGD2-mediated suppression of the neogenic hair follicular formation (Figure 4d-f). Corresponding to the effects of PTD-DBM treatment, we confirmed that Cxxc5 knock-out mice restored hair follicle regeneration after wounding (Figure 4g-i). Moreover, β-catenin, PCNA, and WIHN markers were not affected by PGD2 in the Cxxc5 knock-out mice (Figure 4j-l). Overall, the inhibition of the CXXC5 function restored the reduction of WIHN by PGD2.

Inhibition of CXXC5 Function Restores WIHN Reduced by PGD 2
To confirm the restorative effects of inhibiting CXXC5 function in hair follicle regeneration, we used the WIHN experimental model [11]. The growth of neogenic hair follicles, which were suppressed by PGD 2 [6], was recovered by treatment of PTD-DBM as shown by ALP staining (Figure 4a,b) as well as by hematoxylin and eosin (H&E) staining (Figure 4c). We revealed induction of fgf9 and keratin 17, WIHN markers [28,29] as well as the β-catenin and PCNA by the PTD-DBM treatment in the PGD 2 -mediated suppression of the neogenic hair follicular formation (Figure 4d-f). Corresponding to the effects of PTD-DBM treatment, we confirmed that Cxxc5 knock-out mice restored hair follicle regeneration after wounding (Figure 4g-i). Moreover, β-catenin, PCNA, and WIHN markers were not affected by PGD 2 in the Cxxc5 knock-out mice (Figure 4j-l). Overall, the inhibition of the CXXC5 function restored the reduction of WIHN by PGD 2 .

Efficient Improvement of the Androgenetic Alopecia by Inhibition of Functions of Both GSK-3β and CXXC5
Previously, DHT was reported to cause male pattern AGA via suppressing Wnt/βcatenin signaling by activating GSK-3β, one of the components of the β-catenin destruction complex [15,16]. Moreover, PTGDS was identified as the target gene for androgen receptors [17,30]. In this study, we identified that CXXC5 was induced by DHT treatment via PGD2 (Supplementary Figure S6a,b). To further characterize DHT-induced changes in the Wnt/β-catenin pathway, we tested the effects of PTD-DBM, valproic acid (VPA), a small molecular inhibitor of GSK-3β which directly activates Wnt/β-catenin signaling [31], and KY19382, a small molecule that inhibits both CXXC5 and GSK-3β functions [19]. In contrast to VPA or PTD-DBM treatment, KY19382 completely restored β-catenin suppression by DHT (Supplementary Figure S6c). Moreover, only KY19382 completely improved vibrissa hair elongation (Figure 5a,b and Supplementary Figure S6d,e). These results

Efficient Improvement of the Androgenetic Alopecia by Inhibition of Functions of Both GSK-3β and CXXC5
Previously, DHT was reported to cause male pattern AGA via suppressing Wnt/βcatenin signaling by activating GSK-3β, one of the components of the β-catenin destruction complex [15,16]. Moreover, PTGDS was identified as the target gene for androgen receptors [17,30]. In this study, we identified that CXXC5 was induced by DHT treatment via PGD 2 (Supplementary Figure S6a,b). To further characterize DHT-induced changes in the Wnt/β-catenin pathway, we tested the effects of PTD-DBM, valproic acid (VPA), a small molecular inhibitor of GSK-3β which directly activates Wnt/β-catenin signaling [31], and KY19382, a small molecule that inhibits both CXXC5 and GSK-3β functions [19]. In contrast to VPA or PTD-DBM treatment, KY19382 completely restored β-catenin suppression by DHT (Supplementary Figure S6c). Moreover, only KY19382 completely improved vibrissa hair elongation (Figure 5a,b and Supplementary Figure S6d,e). These results revealed that hair loss caused by DHT was restored by simultaneous inhibitions of CXXC5 and GSK-3β functions. revealed that hair loss caused by DHT was restored by simultaneous inhibitions of CXXC5 and GSK-3β functions. In addition, we found that KY19382 recovered the DHT-mediated suppression of hair growth by restoration of Wnt/β-catenin signaling and ALP expression (Figure 5c,d and Supplementary Figure S6f). The recovery of the DHT-mediated hair growth suppression by KY19382 was also revealed by hair regrowth assays and further confirmed by ALP and H&E staining (Figure 5e-g). Quantitative measurements of H&E staining indicated that more hair follicles in the KY19382-treated groups progressed to the anagen phase compared to the hair follicles remaining in the telogen, exogen, and kenogen phases in the DHT-treated group (Figure 5h,i). IHC staining showed that KY19382 treatment promoted hair growth as much as that shown by vehicle treatment through recovery of Wnt/β-catenin signaling (Figure 5j and Supplementary Figure S6g). Taken together, DHT-induced hair inhibition could occur via suppression of Wnt/β-catenin signaling by PGD2-induced CXXC5. In addition, we found that KY19382 recovered the DHT-mediated suppression of hair growth by restoration of Wnt/β-catenin signaling and ALP expression (Figure 5c,d and Supplementary Figure S6f). The recovery of the DHT-mediated hair growth suppression by KY19382 was also revealed by hair regrowth assays and further confirmed by ALP and H&E staining (Figure 5e-g). Quantitative measurements of H&E staining indicated that more hair follicles in the KY19382-treated groups progressed to the anagen phase compared to the hair follicles remaining in the telogen, exogen, and kenogen phases in the DHT-treated group (Figure 5h,i). IHC staining showed that KY19382 treatment promoted hair growth as much as that shown by vehicle treatment through recovery of Wnt/β-catenin signaling (Figure 5j and Supplementary Figure S6g). Taken together, DHT-induced hair inhibition could occur via suppression of Wnt/β-catenin signaling by PGD 2 -induced CXXC5.

Discussion
AGA occurs in both men and women who are genetically susceptible to androgens [32]. One of the main phenomena in AGA patients is an increase in 5α-reductase, an enzyme that converts testosterone to DHT. Elevated DHT causes hair loss in AGA patients [33]. In addition, PGD 2 and CXXC5, which were found to be increased in scalps of male pattern AGA patients, inhibit hair growth and WIHN [5,6,12]. However, the interrelationship between them has not yet been investigated [34]. In this study, we found that PGD 2 induced CXXC5 via activation of BMP signaling, and the increased CXXC5 mediated PGD 2 -induced hair loss. We also confirmed that DHT augmented PTGDS and subsequently induced CXXC5. These results suggest a model for an action mechanism for male pattern AGA by DHT involving the suppression of Wnt/β-catenin signaling through the PGD 2 -CXXC5 axis ( Figure 6).

Discussion
AGA occurs in both men and women who are genetically susceptible to androgens [32]. One of the main phenomena in AGA patients is an increase in 5α-reductase, an enzyme that converts testosterone to DHT. Elevated DHT causes hair loss in AGA patients [33]. In addition, PGD2 and CXXC5, which were found to be increased in scalps of male pattern AGA patients, inhibit hair growth and WIHN [5,6,12]. However, the interrelationship between them has not yet been investigated [34]. In this study, we found that PGD2 induced CXXC5 via activation of BMP signaling, and the increased CXXC5 mediated PGD2-induced hair loss. We also confirmed that DHT augmented PTGDS and subsequently induced CXXC5. These results suggest a model for an action mechanism for male pattern AGA by DHT involving the suppression of Wnt/β-catenin signaling through the PGD2-CXXC5 axis ( Figure 6). In alopecia, the Wnt/β-catenin signaling in hair cells is reduced, but the topical agent minoxidil (MNX) does not affect Wnt/β-catenin signaling [3,35]. Due to the effectiveness of Wnt/β-catenin signaling on hair growth, including neogenic hair growth, Wnt/βcatenin signaling activators including natural products and small molecules have been tested for hair growth [8,31,36,37]. However, the direct Wnt/β-catenin signaling activators have not shown satisfactory results in clinical trials [38,39]. This could be attributed to the functioning of the negative feedback regulator CXXC5 [12]. Previously, we found that CXXC5 is induced by BMP-Smad signaling and interacts with Dvl to suppress Wnt/βcatenin signaling [13,14]. CXXC5 is overexpressed in scalps of male pattern AGA [12], and the male pattern AGA inducers, DHT and PGD2, decreased Wnt/β-catenin signaling via the negative regulator, CXXC5. The role of CXXC5 as a mediator for DHT-and PGD2- In alopecia, the Wnt/β-catenin signaling in hair cells is reduced, but the topical agent minoxidil (MNX) does not affect Wnt/β-catenin signaling [3,35]. Due to the effectiveness of Wnt/β-catenin signaling on hair growth, including neogenic hair growth, Wnt/β-catenin signaling activators including natural products and small molecules have been tested for hair growth [8,31,36,37]. However, the direct Wnt/β-catenin signaling activators have not shown satisfactory results in clinical trials [38,39]. This could be attributed to the functioning of the negative feedback regulator CXXC5 [12]. Previously, we found that CXXC5 is induced by BMP-Smad signaling and interacts with Dvl to suppress Wnt/β-catenin signaling [13,14]. CXXC5 is overexpressed in scalps of male pattern AGA [12], and the male pattern AGA inducers, DHT and PGD 2 , decreased Wnt/β-catenin signaling via the negative regulator, CXXC5. The role of CXXC5 as a mediator for DHT-and PGD 2induced male pattern AGA was further indicated by the recovery effects of inhibition of CXXC5's function, by PTD-DBM or KY19382.
The role of CXXC5 in male pattern AGA is further supported by various studies which have shown that the involvement of Wnt/β-catenin signaling in male pattern AGA is related to DHT; DHT causes male pattern AGA by decreasing Wnt/β-catenin signaling [3]. DHT activates GSK-3β, which down-regulates the Wnt/β-catenin pathway, by inhibiting phosphorylation at Ser-9 of GSK-3β [15]. DHT-induced reduction of Wnt-3a decreases keratinocyte proliferation [16]. Moreover, DHT modulates Wnt agonists and antagonists; decreasing the Wnt agonists, Wnt-10b and Wnt-5a, while increasing DKK-1, a Wnt antagonist [40,41]. In this study, we found that CXXC5 is a mediator for DHT-induced male pattern AGA through PGD 2 . Overall, the Wnt/β-catenin signaling inhibitor CXXC5 plays a role in DHT-induced PGD 2 signaling activation and subsequent male pattern AGA. Here, we also found that enhanced activation of Wnt/β-catenin signaling by simultaneous inhibitions of GSK-3β activity and CXXC5-Dvl PPI by KY19382 is much more effective than that acquired by single inhibition by VPA or PTD-DBM. Therefore, a chemical approach effectively activating Wnt/β-catenin signaling by blockade of the functions of CXXC5 and GSK-3β provides an ideal approach for the treatment of male pattern AGA.
Although use of the mouse system to investigate the mechanism related to the hair loss and treatments of alopecia involving DHT is not ideal due to the differences between mouse and human systems, studies using mice have been used frequently [42][43][44][45][46]. In addition, mice have also been used in the applicaiton of clinical treatment [38,39]. Overall, considering the role of CXXC5 as an important mediator of DHT action and phenotypical outcomes, the inhibition of CXXC5 function is a potential treatment for androgenetic alopecia.

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
Conflicts of Interest: K-Y.C. is the CEO of CK Regeon Inc. (Seoul, Korea), which has a license to develop and use the Wnt/beta-catenin pathway activator disclosed in the publication. The authors have no conflicts of interest to declare. The company had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results.