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.