1 INTRODUCTION
Hair loss or alopecia is a highly prevalent dermatological disorder
which can impair the quality of life and cause a lifetime of mental
suffering. Hair loss is influenced by multiple factors such as genetic
factors, hormonal dysfunction, sebum content, aging, mental disorders
and environmental factors (Jang et al., 2013; Patel, Harrison, &
Sinclair, 2013; Pratt, King, Messenger, Christiano, & Sundberg, 2017).
Despite enormous effort in developing drugs to treat alopecia, only
minoxidil and
finasteride
have been approved by the US Food and Drug Administration (FDA) for
clinical practice (Libecco & Bergfeld, 2004; Linas & Nies, 1981; Van
Neste et al., 2000). Minoxidil is an antihypertensive vasodilator (Sica,
2004). Finasteride can block the conversion of testosterone to
dihydrotestosterone and is used to treat alopecia patients unresponsive
to minoxidil (Bolduc & Shapiro, 2000). Both drugs have limited efficacy
in alopecia treatment and possess significant adverse effects. The
effect of both drugs is not long lasting and require prolonged use.
Patients using finasteride may have serious side effects on sexual and
reproductive health. Minoxidil can produce pruritus, irritant
dermatitis, and dizziness during treatment. Therefore, there is an
urgent need to develop drugs with novel mechanisms of action for the
treatment of alopecia.
The
Wnt/β‐catenin
signaling pathway plays a crucial role in hair morphogenesis, hair
growth, hair cycling and regeneration (Andl, Reddy, Gaddapara, &
Millar, 2002; Ouji et al., 2008; Zhou et al., 2016). The substantial
stabilization of the free β-catenin is indispensable for the
Wnt/β-catenin pathway, which is controlled by a destruction complex. In
this destruction complex, the scaffolding protein Axin interacts and
takes into proximity to the other components comprising the adenomatosis
polyposis coli protein (APC), the casein kinase 1 (CK1) and the glycogen
synthetase kinase 3β enzyme (GSK3β). The β-catenin protein is
phosphorylated by GSK3β, resulting in its ubiquitination-mediated
degradation. The Wnt/β-catenin pathway is triggered when the Wnt
proteins bind to their receptors Frizzled (Fzd) and low-density
lipoprotein receptor-related proteins 5/6 (LRP5/6). A critical step in
Wnt pathway stimulation is the recruitment of the Axin complex towards
the LRP5/6 receptor complex(Clevers & Nusse, 2012). The Axin complex
peculiarly interacts with LRP5/6 cytoplasmic tail, facilitating the
phosphorylation of LRP5/6, which hinders β-catenin phosphorylation and
constitutive degradation (Tamai et al., 2000; Zeng et al., 2005).
Consequently, the stabilized β-catenin hoards in the cytoplasm and is
translocated into the nucleus, where it binds with the T-cell
factor/lymphoid enhancer factor 1 (TCF/LEF1) transcription factors to
trigger the expression of Wnt signaling target genes, including Axin2,
LEF1, Fibronectin and Survivin (Nusse & Clevers, 2017).
Previous studies found that nuclear β-catenin existed in chick feather
prior to placode formation, and LEF1 highly expressed in mouse dermis
during vibrissa follicle formation, indicating the involvement of
Wnt/β-catenin signaling in follicular development (Kratochwil, Dull,
Farinas, Galceran, & Grosschedl, 1996; Noramly, Freeman, & Morgan,
1999). Subsequent studies further revealed that multiple Wnt molecules
could stimulate hair follicle stem cells and promote hair regeneration
via activating β-catenin signaling (Hu et al., 2021). Dong et al
demonstrated that treatment with Wnt1a-enriched conditioned media could
accelerate the progression of hair follicle from telogen to anagen,
upregulate the expression of hair induction-associated genes, and
enhance the amount of hair in depilated mouse skin (Dong et al., 2014).
Wnt10b has been shown to increase the growth of hair follicle by
enhancing telogen to anagen switch via β-catenin stabilization (Wu, Zhu,
Liu, Liu, & Li, 2020). Wnt3a has been found to activate β-catenin
signaling and promote hair growth in nude mice receiving skin
reconstitution (Xing, Yi, Miao, Su, & Lei, 2018).
Rajendran
et al reported that treatment with Wnt3a and Wnt7b-enriched
macrophage-extracellular vesicle significantly increased the
proliferation and migration of dermal papilla cells via enhancing the
Wnt/β-catenin pathway (Rajendran et al., 2020). In contrast, the Wnt
signaling antagonist DKK1 could trigger anagen-to-catagen transition
when injected into the hypodermis of C57BL/6 mice, while neutralizing
DKK-1 antibody delayed catagen progression in mice (Kwack, Kim, Kim, &
Sung, 2012). Overall, these studies highlight the Wnt/β‐catenin
signaling pathway as a promising therapeutic target for hair loss.
The small molecule isoxazole-9 [ISX9,
N-cyclopropyl-5-(thiophen-2-yl)isoxazole-3-carboxamide] has been
identified as a neurogenesis inducer through high-throughput screening
of chemical libraries in stem cell-based assays (Schneider et al.,
2008). ISX9 was found to potentiate cell proliferation and increase the
number or differentiation of neurons in the hippocampal dentate gyrus in
a myocyte-enhancer factor 2 (MEF2)-dependent manner (Bettio et al.,
2016). ISX9 could also induce intracellular Ca2+influx which activates phosphorylated CaMK and regulates nuclear export
of the MEF2 modulator HDAC5, thus allowing neuronal differentiation (Koh
et al., 2015). In other tissues, ISX9 was shown to induce
differentiation of cardiac and islet endocrine progenitors (Tsakmaki,
Fonseca Pedro, Pavlidis, Hayee, & Bewick, 2020; Xuan et al., 2018).
Dioum et al demonstrated that ISX9 increased the expression and
secretion of insulin in islets and upregulated the expression of
neuronal differentiation 1 (NeuroD1) and other regulators of islet
differentiation (Dioum et al., 2011). Kalwat et al also reported that
ISX9 could alter β-cell metabolites, protect glucose-responsive
signaling pathways under lipotoxic conditions, and improve glycemia in a
mouse model of β-cell regeneration (Kalwat et al., 2016). However, the
molecular mechanism of ISX9 action remains unclear. In the present
study, ISX9 was identified as a novel activator of the Wnt/β-catenin
signaling pathway. We further investigated the mechanism involved in
activation of Wnt/β-catenin signaling by ISX9. Finally, our results
demonstrated that ISX9 could stimulate the proliferation of hair
follicle and promote hair growth in mice through activating the Wnt
signaling pathway.