3.5 KY19382 promotes hair follicle neogenesis in patch assays
To investigate the therapeutic effect of KY19382, we utilized a hair patch assay system. Mouse dermal cells were treated with 5 μM KY19382 for 72 hours prior to transplantation, then mixed with epithelial cells for injection into hairless mice.
After 14 days post-injection, reconstituted hair follicles were observed on the skin of hairless mice where cells were injected (Figure 5a). The number and density of neogenic hair follicles generated in the tissue injected with the KY19382-treated cells were significantly higher than those injected with the non-treated control cells (Figure 5b). Moreover, the expression levels of β-catenin and Ki67 were greatly increased in neo-generated hair follicles induced by KY19382 as demonstrated by IHC analysis (Figure 5c).
DISSCUSSION
Currently available drugs for treating alopecia are limited due to their inability to regenerate hair follicles. MNX and finasteride can promote hair growth when the hair follicle is present, but they are not effective in patients with severe alopecia (Libecco & Bergfeld, 2004; Messenger & Rundegren, 2004; Price, 1999; Rossi et al., 2016). Existing drugs that control the proliferation of hair cells can be difficult for treating patients with miniaturized or absent hair follicles (Han et al., 2004). Therefore, we aimed to develop a drug that is effective in promoting hair regrowth and hair follicle neogenesis by inducing markers for hair induction such as ALP and activating hair follicle stem cells via Wnt/β-catenin pathway.
Hair follicle neogenesis could be an important strategy for alopecia treatment (Ito et al., 2007). Previous studies have shown that hair follicle neogenesis highly depends on the activation of Wnt/β-catenin signaling in DP cells and keratinocytes, along with activation of hair follicle stem cells (Enshell-Seijffers, Lindon, Kashiwagi, & Morgan, 2010; Huelsken et al., 2001; Ito et al., 2007; Waters, Richardson, & Jahoda, 2007). Wnt/β-catenin signaling plays essential roles in maintaining the hair-inducing ability of DP cells and promoting hair follicles to the anagen phase (Andl et al., 2002; Kishimoto et al., 2000; Sick, Reinker, Timmer, & Schlake, 2006). In addition, previous literature has suggested that Wnt/β-catenin signaling activators, such as valproic acid (S. H. Lee et al., 2012), Aconiti ciliare tuberextract (Park et al., 2012), and Malva verticillata seed extract (E. Y. Lee et al., 2016), are potential candidates for alternative hair growth treatments as they induce the expression of hair inducing markers in DP cells. Therefore, it is important to use Wnt/β-catenin signaling activators that activate both hair induction markers and stem cells to effectively promote hair growth and regeneration.
Although direct Wnt/β-catenin signaling activators such as valproic acid promote hair growth, but they fail to sustain hair growth and often show marginal effects in clinical tests (Jo et al., 2013; Jo et al., 2014). This marginal and limited effect may be attributed to functions of negative feedback regulators such as CXXC5 or DKK1 (Lee et al., 2017; Kwack et al., 2012). We found that CXXC5, a negative feedback regulator of Wnt/β-catenin signaling, is specifically increased in the miniaturized follicles of bald scalps, and CXXC5 knock out mice exhibited enhanced hair growth (Lee et al., 2017). PTD-DBM, a peptide that interfered with CXXC5-Dvl protein interaction, enhanced hair growth, and the combinatory treatment of PTD-DBM and valproic acid synergistically increased hair growth and the WIHN (Lee et al., 2017). Similarly, KY19382, which strongly activated the Wnt/β-catenin signaling via interference of the CXXC5-Dvl interaction and inhibited GSK-3β (Choi et al., 2019), critically enhanced hair re-growth as well as WIHNin vivo . The increased CXXC5 in bald scalps and the effectiveness of PTD-DBM or KY19382 on hair growth in mice suggest potential for use of KY19382 as hair growth treatment in the clinic.
In this study, we confirmed that the levels of β-catenin, p-GSK3β (S9) and proliferation marker, PCNA, were increased in human DP cells treated with KY19382. Increased ALP activity after KY19382 treatment suggested that KY19382 increased the hair inducing ability in human DP cells. Compared with the non-treated control, mouse vibrissa follicles treated with KY19382 for 6 days, significantly promoted elongation of hair shaft. The levels of β-catenin, Ki67, and PCNA were increased in the keratin 15-positive bulge of mouse skin treated with KY19382, suggesting a positive effect of KY19382 on hair follicle stem cell activity. Moreover, KY19382 regenerated a number of neogenic follicles in the WIHN assay, and histological images showed higher expression levels of not only β-catenin and proliferation markers but also markers for hair follicle neogenesis, fgf 9 and keratin 17. These results indicate that KY19382 treatment may be a possible therapy for baldness. In the hair patch assay, KY19382 regenerated a greater number of neogenic hair follicles, and histological evaluation revealed higher expression levels of β-catenin and Ki67 than control, demonstrating that pretreatment with KY19382 enhanced the hair-inducing ability of dermal cells. Therefore, our results suggest that KY19382 may be useful for treatment of hair loss and baldness via its effective dual targeting ability to inhibit both GSK-3β and CXXC5-Dvl interaction.