3.3 KY19382 promotes hair regrowth in mice
To investigate the effect of KY19382 on mouse hair regrowth, KY19382 or vehicle was topically applied to the shaved dorsal skin of 7-week-old mice daily for 14 or 28 days. MNX was used as the positive control (S. H. Lee et al., 2012). After 28 days, KY19382 promoted hair regrowth more efficiently than MNX (Figure 3a). Hematoxylin and eosin (H&E) staining showed that hair follicles in the control group were still in the telogen phase, but hair follicles of skin treated with KY19382 or MNX for 28 days entered anagen phase (Figure 3b). To confirm KY19382-induced changes in proliferation or Wnt/β-catenin signaling in the bulge, IHC analyses were performed on skin tissues treated for 14 days. Proliferation markers, ki67 and PCNA, were specifically increased in keratin 15-positive bulge stem cells of the KY19382-treated group (Figure 3c and supplementary figure 4a). β-Catenin was also increased only in the KY19382-treated group. Similarly, western blot analyses showed that the levels of β-catenin and PCNA were significantly increased in the KY19382-treated group (Figure 3d). Moreover, ALP, a critical marker for hair induction, was highly expressed in the DP cells of skins treated with KY19382 for 14 days (Figure 3e). Collectively, these data suggested that KY19382 promoted hair regrowth and increased markers for hair growth promotion, such as β-catenin, PCNA and ALP more efficiently than MNX.
3.4 KY19382 enhances wound-induced hair follicle neogenesis
Considering that KY19382 stimulated hair growth in in vitro ,ex vivo and in vivo systems via activation of Wnt/β-catenin signaling, the effect of KY19382 on WIHN was tested in mice.
To confirm this, we cut 1 cm2 full thickness wounds in 3-4-week-old mice and applied the drugs daily for 14, 25, and 40 days after wounding. The KY19382-treated group increased number of newly formed follicles compared to those in the vehicle treated group as confirmed by whole mount ALP staining (Figure 4a). In addition, H&E staining showed that KY19382 induced formation of neogenic follicles 14 days post-wounding (Figure 4b). IHC analyses showed that keratin 17, a marker for intermediate filament keratin protein (Ito et al., 2007), was specifically increased in neogenic follicles of the KY19382-treated group (Figure 4c). Fgf9 involved in WIHN in wound fibroblasts (S. H. Lee et al., 2017) was also increased in the dermis of wounds treated with KY19382 (Figure 4c). β-Catenin and proliferation markers, ki67 and PCNA were increased in the KY19382-treated group, especially in the neogenic follicles of wounds (Figure 4c and supplementary figure 5a). Furthermore, western blot analyses confirmed that the markers associated with hair follicle neogenesis were increased in wounded skin treated with KY19382, but not MNX (Figure 4d). The newly formed white hairs at the wound sites were found only in the KY19382-treated group (Figure 4e). Taken together, KY19382 markedly induced WIHN and WIHN-related markers by significantly activating Wnt/β-catenin pathway.