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.