4 DISCUSSION
The Wnt/β-catenin signaling pathway is crucial for neurogenesis (Boitard et al., 2015; McQuate, Latorre-Esteves, & Barria, 2017). The components of Wnt signaling have been involved in neuronal development, neuronal maturation, neuronal differentiation, and proliferation (Varela-Nallar & Inestrosa, 2013). Lie et al reported that the expression of Wnt3 induced neurogenesis in the adult rat dentate gyrus (Lie et al., 2005). The dominant-negative LEF1 could reduce neuronal differentiation induced by co-culture with hippocampal astrocytes (Armenteros, Andreu, Hortigüela, Lie, & Mira, 2018). Wnt7a has been identified as an endogenous regulator of hippocampal neurogenesis that mediates proliferation and neuronal differentiation (Qu et al., 2013). Kuwabara et al found that Wnt/β-catenin signaling could mediate activation of NeuroD1 and play an important role in balancing self-renewal of neural stem cells and neuronal differentiation in adult dentate gyrus (Kuwabara et al., 2009). ISX9 has been identified as a neurogenesis inducer which can upregulate the expression of NeuroD1, promote neurogenesis and enhance neuronal differentiation (Schneider et al., 2008). However, the molecular mechanism underlying ISX9 action in neurogenesis remains unclear. In this study, ISX9 was identified as a potent activator of Wnt/β-catenin signaling. ISX9 could promote the association of LRP6 with Axin1, resulting in the accumulation of β-catenin and the upregulation of Wnt target genes and stemness marker genes. The Wnt agonistic actions of ISX9 may contribute to its functions in neurogenesis.
Axin is a critical scaffolding protein of in the Wnt/β-catenin pathway (Kim et al., 2013). Upon binding of a Wnt ligand to its specific receptor complex, the Wnt co-receptor LRP5/6 is immediately phosphorylated, which generates a docking site for Axin1. Subsequently, the Axin-GSK3β complex is recruited to the receptor, which facilitates GSK3β-induced LRP5/6 phosphorylation and inhibits β-catenin phosphorylation, resulting in the stabilization of β-catenin (Liu et al., 2022). Axin1 has been identified as a cellular target for small molecular compound HLY78. HLY78 could bind to the DIX domain of Axin1 and potentiates the Axin1-LRP6 association, thus activating the Wnt signaling transduction (Wang et al., 2013). Gwak et al reported that the small molecular compound SKL2001 could stabilize intracellular β-catenin via blocking the Axin/β-catenin interaction (Gwak et al., 2012). In our study, ISX9 was found to potently induce the accumulation of intracellular β-catenin without influencing the expression and activity of LRP6 and GSK3β. By covalently binding to the N-terminal region (aa1-300) of Axin1, ISX9 abrogated the association of Axin1 with β-catenin. Meanwhile, ISX9 exhibited a strong promoting effect on the interaction between LRP6 and Axin1, thus leading to stabilizing intracellular β-catenin. Together, our results uncover a novel mechanism by which ISX9 induces the accumulation of β-catenin and activates the Wnt/β-catenin cascade.
The periodic growth of hair follicles includes the three phases of telogen, anagen, and catagen (Lin, Zhu, & He, 2022; Müller-Röver et al., 2001). The cessation of these cyclic phases is the major mechanism contributing to the pathogenesis of alopecia (Petukhova et al., 2010). Accumulating evidence has demonstrated that the hair follicle stem cells are located in the bulge area of the hair follicle and are required for the growth of hair follicles (Liu et al., 2021; Schneider, Schmidt-Ullrich, & Paus, 2009). As a subpopulation of adult stem cells with self-renewal ability, the hair follicle stem cells are able to activate and differentiate into various hair follicle cell types for hair follicle regeneration (Mistriotis & Andreadis, 2013). Increasing evidence showed that hair regeneration and growth heavily depends on the Wnt/β‐catenin signaling activation in the hair follicle (Andl et al., 2002). Some Wnt/β‐catenin signaling activators have been reported to stimulate hair follicle development. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has been found to accelerate hair cycle and stimulate hair growth. VPA could activate the Wnt/β-catenin signaling pathway by inhibiting GSK3β (Lee et al., 2012). Xing et al demonstrated that baicalin, a natural bioactive flavonoid extracted fromScutellaria baicalensis Georgi, could promote the growth of hair follicles via activation of Wnt/β-catenin signaling in mice (Xing et al., 2018). KY19382 is one of the newly synthesized analogues of GSK3β inhibitor indirubin‐3′‐monoxime. It exhibited hair growth-promoting effect by inactivating GSK3β and blocking the interaction between CXXC‐type zinc finger protein 5 (CXXC5) and dishevelled (DVL) (Ryu et al., 2021). Ahmed et al. examined the effect of a tocotrienol-rich formulation (TRF) on hair growth. TRF could reduce the expression of epidermal E-cadherin, and increase nuclear translocation of β-catenin and its interaction with TCF3. Topical application of TRF markedly induced epidermal hair follicle development and early anagen induction (Ahmed et al., 2017). Currently, the drugs for promoting hair growth by targeting Wnt/β‐catenin signaling are not yet commercially available. Here, our results revealed that ISX9 could potently activate Wnt/β‐catenin signaling and upregulate the expression of stemness marker genes LEF1, LGR5, Twist1, SOX2, OCT4 and Keratin17. Importantly, ISX9 exerted strong hair growth-promoting activity without causing skin abnormalities in mice. These results indicated that ISX9 may have a great potential to be developed as a hair growth-promoting agent for the treatment of alopecia.
In summary, we identified ISX9 as a novel activator of the Wnt/β-catenin signaling pathway. This compound activated Wnt/β-catenin signaling via elevating the level of intracellular β‐catenin. ISX9 was found to target Axin1 through covalently binding to the N-terminal region of Axin1. The binding of ISX9 to Axin1 induced the release of Axin1 from the β-catenin destruction complex, and meanwhile triggered the interaction between Axin1 and LRP6, leading to the stabilization of β‐catenin. We further showed that ISX9 could upregulate the expression of Wnt signaling target genes and stemness marker genes. Finally, ISX9 demonstrated potent hair growth-promoting activity in mice, accompanied by the upregulation of the Wnt signaling pathway in vivo . These results suggested that ISX9 may be a promising therapeutic agent for alopecia.
REFERENCES
Ahmed, N. S., Ghatak, S., El Masry, M. S., Gnyawali, S. C., Roy, S., Amer, M., . . . Khanna, S. (2017). Epidermal E-Cadherin Dependent β-Catenin Pathway Is Phytochemical Inducible and Accelerates Anagen Hair Cycling. Mol Ther, 25 (11), 2502-2512. doi:10.1016/j.ymthe.2017.07.010
Andl, T., Reddy, S. T., Gaddapara, T., & Millar, S. E. (2002). WNT signals are required for the initiation of hair follicle development.Dev Cell, 2 (5), 643-653. doi:10.1016/s1534-5807(02)00167-3
Armenteros, T., Andreu, Z., Hortigüela, R., Lie, D. C., & Mira, H. (2018). BMP and WNT signalling cooperate through LEF1 in the neuronal specification of adult hippocampal neural stem and progenitor cells.Scientific Reports, 8 (1), 9241-9241. doi:10.1038/s41598-018-27581-0
Bettio, L. E. B., Patten, A. R., Gil-Mohapel, J., O’Rourke, N. F., Hanley, R. P., Kennedy, S., . . . Christie, B. R. (2016). ISX-9 can potentiate cell proliferation and neuronal commitment in the rat dentate gyrus. Neuroscience, 332 , 212-222. doi:https://doi.org/10.1016/j.neuroscience.2016.06.042
Boitard, M., Bocchi, R., Egervari, K., Petrenko, V., Viale, B., Gremaud, S., . . . Kiss, Jozsef Z. (2015). Wnt Signaling Regulates Multipolar-to-Bipolar Transition of Migrating Neurons in the Cerebral Cortex. Cell Reports, 10 (8), 1349-1361. doi:https://doi.org/10.1016/j.celrep.2015.01.061
Bolduc, C., & Shapiro, J. (2000). Management of androgenetic alopecia.Am J Clin Dermatol, 1 (3), 151-158. doi:10.2165/00128071-200001030-00002
Chai, M., Jiang, M., Vergnes, L., Fu, X., de Barros, S. C., Doan, N. B., . . . Huang, J. (2019). Stimulation of Hair Growth by Small Molecules that Activate Autophagy. Cell Rep, 27 (12), 3413-3421.e3413. doi:10.1016/j.celrep.2019.05.070
Clevers, H., & Nusse, R. (2012). Wnt/β-catenin signaling and disease.Cell, 149 (6), 1192-1205. doi:10.1016/j.cell.2012.05.012
Curtis, M. J., Alexander, S., Cirino, G., Docherty, J. R., George, C. H., Giembycz, M. A., . . . Ahluwalia, A. (2018). Experimental design and analysis and their reporting II: updated and simplified guidance for authors and peer reviewers. British journal of pharmacology, 175 (7), 987-993. doi:10.1111/bph.14153
Dioum, E. M., Osborne, J. K., Goetsch, S., Russell, J., Schneider, J. W., & Cobb, M. H. (2011). A small molecule differentiation inducer increases insulin production by pancreatic β cells. Proc Natl Acad Sci U S A, 108 (51), 20713-20718. doi:10.1073/pnas.1118526109
Dong, L., Hao, H., Xia, L., Liu, J., Ti, D., Tong, C., . . . Han, W. (2014). Treatment of MSCs with Wnt1a-conditioned medium activates DP cells and promotes hair follicle regrowth. Scientific Reports, 4 (1), 5432. doi:10.1038/srep05432
Gwak, J., Hwang, S. G., Park, H.-S., Choi, S. R., Park, S.-H., Kim, H., . . . Oh, S. (2012). Small molecule-based disruption of the Axin/β-catenin protein complex regulates mesenchymal stem cell differentiation. Cell Research, 22 (1), 237-247. doi:10.1038/cr.2011.127
Hu, X. M., Li, Z. X., Zhang, D. Y., Yang, Y. C., Fu, S. A., Zhang, Z. Q., . . . Xiong, K. (2021). A systematic summary of survival and death signalling during the life of hair follicle stem cells. Stem Cell Res Ther, 12 (1), 453. doi:10.1186/s13287-021-02527-y
Jang, W. S., Son, I. P., Yeo, I. K., Park, K. Y., Li, K., Kim, B. J., . . . Hong, C. K. (2013). The annual changes of clinical manifestation of androgenetic alopecia clinic in korean males and females: a outpatient-based study. Ann Dermatol, 25 (2), 181-188. doi:10.5021/ad.2013.25.2.181
Kalwat, M. A., Huang, Z., Wichaidit, C., McGlynn, K., Earnest, S., Savoia, C., . . . Cobb, M. H. (2016). Isoxazole Alters Metabolites and Gene Expression, Decreasing Proliferation and Promoting a Neuroendocrine Phenotype in β-Cells. ACS Chem Biol, 11 (4), 1128-1136. doi:10.1021/acschembio.5b00993
Kim, S. E., Huang, H., Zhao, M., Zhang, X., Zhang, A., Semonov, M. V., . . . He, X. (2013). Wnt stabilization of β-catenin reveals principles for morphogen receptor-scaffold assemblies. Science, 340 (6134), 867-870. doi:10.1126/science.1232389
Koh, S. H., Liang, A. C., Takahashi, Y., Maki, T., Shindo, A., Osumi, N., . . . Lo, E. H. (2015). Differential Effects of Isoxazole-9 on Neural Stem/Progenitor Cells, Oligodendrocyte Precursor Cells, and Endothelial Progenitor Cells. PLoS One, 10 (9), e0138724. doi:10.1371/journal.pone.0138724
Kratochwil, K., Dull, M., Farinas, I., Galceran, J., & Grosschedl, R. (1996). Lef1 expression is activated by BMP-4 and regulates inductive tissue interactions in tooth and hair development. Genes Dev, 10 (11), 1382-1394. doi:10.1101/gad.10.11.1382
Kuwabara, T., Hsieh, J., Muotri, A., Yeo, G., Warashina, M., Lie, D. C., . . . Gage, F. H. (2009). Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis. Nature neuroscience, 12 (9), 1097-1105. doi:10.1038/nn.2360
Kwack, M. H., Kim, M. K., Kim, J. C., & Sung, Y. K. (2012). Dickkopf 1 promotes regression of hair follicles. J Invest Dermatol, 132 (6), 1554-1560. doi:10.1038/jid.2012.24
Lee, S. H., Yoon, J., Shin, S. H., Zahoor, M., Kim, H. J., Park, P. J., . . . Choi, K. Y. (2012). Valproic acid induces hair regeneration in murine model and activates alkaline phosphatase activity in human dermal papilla cells. PLoS One, 7 (4), e34152. doi:10.1371/journal.pone.0034152
Libecco, J. F., & Bergfeld, W. F. (2004). Finasteride in the treatment of alopecia. Expert Opin Pharmacother, 5 (4), 933-940. doi:10.1517/14656566.5.4.933
Lie, D. C., Colamarino, S. A., Song, H. J., Désiré, L., Mira, H., Consiglio, A., . . . Gage, F. H. (2005). Wnt signalling regulates adult hippocampal neurogenesis. Nature, 437 (7063), 1370-1375. doi:10.1038/nature04108
Lilley, E., Stanford, S. C., Kendall, D. E., Alexander, S. P. H., Cirino, G., Docherty, J. R., . . . Ahluwalia, A. (2020). ARRIVE 2.0 and the British Journal of Pharmacology: Updated guidance for 2020.177 (16), 3611-3616. doi:https://doi.org/10.1111/bph.15178
Lin, X., Zhu, L., & He, J. (2022). Morphogenesis, Growth Cycle and Molecular Regulation of Hair Follicles. 10 . doi:10.3389/fcell.2022.899095
Linas, S. L., & Nies, A. S. (1981). Minoxidil. Ann Intern Med, 94 (1), 61-65. doi:10.7326/0003-4819-94-1-61
Liu, F., Zhang, X., Peng, Y., Zhang, L., Yu, Y., Hua, P., . . . Zhang, L. (2021). miR-24 controls the regenerative competence of hair follicle progenitors by targeting Plk3. Cell Reports, 35 (10), 109225. doi:https://doi.org/10.1016/j.celrep.2021.109225
Liu, J., Xiao, Q., Xiao, J., Niu, C., Li, Y., Zhang, X., . . . Yin, G. (2022). Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduction and Targeted Therapy, 7 (1), 3. doi:10.1038/s41392-021-00762-6
McQuate, A., Latorre-Esteves, E., & Barria, A. (2017). A Wnt/Calcium Signaling Cascade Regulates Neuronal Excitability and Trafficking of NMDARs. Cell Rep, 21 (1), 60-69. doi:10.1016/j.celrep.2017.09.023
Mistriotis, P., & Andreadis, S. T. (2013). Hair follicle: a novel source of multipotent stem cells for tissue engineering and regenerative medicine. Tissue Eng Part B Rev, 19 (4), 265-278. doi:10.1089/ten.TEB.2012.0422
Mosimann, C., Hausmann, G., & Basler, K. (2009). Beta-catenin hits chromatin: regulation of Wnt target gene activation. Nat Rev Mol Cell Biol, 10 (4), 276-286. doi:10.1038/nrm2654
Müller-Röver, S., Handjiski, B., van der Veen, C., Eichmüller, S., Foitzik, K., McKay, I. A., . . . Paus, R. (2001). A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol, 117 (1), 3-15. doi:10.1046/j.0022-202x.2001.01377.x
Noramly, S., Freeman, A., & Morgan, B. A. (1999). beta-catenin signaling can initiate feather bud development. Development, 126 (16), 3509-3521. doi:10.1242/dev.126.16.3509
Nusse, R., & Clevers, H. (2017). Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities. Cell, 169 (6), 985-999. doi:10.1016/j.cell.2017.05.016
Ouji, Y., Yoshikawa, M., Moriya, K., Nishiofuku, M., Matsuda, R., & Ishizaka, S. (2008). Wnt-10b, uniquely among Wnts, promotes epithelial differentiation and shaft growth. Biochem Biophys Res Commun, 367 (2), 299-304. doi:10.1016/j.bbrc.2007.12.091
Patel, M., Harrison, S., & Sinclair, R. (2013). Drugs and hair loss.Dermatol Clin, 31 (1), 67-73. doi:10.1016/j.det.2012.08.002
Petukhova, L., Duvic, M., Hordinsky, M., Norris, D., Price, V., Shimomura, Y., . . . Christiano, A. M. (2010). Genome-wide association study in alopecia areata implicates both innate and adaptive immunity.Nature, 466 (7302), 113-117. doi:10.1038/nature09114
Pratt, C. H., King, L. E., Jr., Messenger, A. G., Christiano, A. M., & Sundberg, J. P. (2017). Alopecia areata. Nat Rev Dis Primers, 3 , 17011. doi:10.1038/nrdp.2017.11
Qu, Q., Sun, G., Murai, K., Ye, P., Li, W., Asuelime, G., . . . Shi, Y. (2013). Wnt7a regulates multiple steps of neurogenesis. Mol Cell Biol, 33 (13), 2551-2559. doi:10.1128/mcb.00325-13
Rajendran, R. L., Gangadaran, P., Seo, C. H., Kwack, M. H., Oh, J. M., Lee, H. W., . . . Ahn, B. C. (2020). Macrophage-Derived Extracellular Vesicle Promotes Hair Growth. Cells, 9 (4). doi:10.3390/cells9040856
Ring, A., Kim, Y.-M., & Kahn, M. (2014). Wnt/catenin signaling in adult stem cell physiology and disease. Stem cell reviews and reports, 10 (4), 512-525. doi:10.1007/s12015-014-9515-2
Ryu, Y. C., Lee, D.-H., Shim, J., Park, J., Kim, Y.-R., Choi, S., . . . Choi, K.-Y. (2021). KY19382, a novel activator of Wnt/β-catenin signalling, promotes hair regrowth and hair follicle neogenesis.British journal of pharmacology, 178 (12), 2533-2546. doi:10.1111/bph.15438
Schneider, J. W., Gao, Z., Li, S., Farooqi, M., Tang, T. S., Bezprozvanny, I., . . . Hsieh, J. (2008). Small-molecule activation of neuronal cell fate. Nat Chem Biol, 4 (7), 408-410. doi:10.1038/nchembio.95
Schneider, M. R., Schmidt-Ullrich, R., & Paus, R. (2009). The hair follicle as a dynamic miniorgan. Curr Biol, 19 (3), R132-142. doi:10.1016/j.cub.2008.12.005
Sica, D. A. (2004). Minoxidil: an underused vasodilator for resistant or severe hypertension. J Clin Hypertens (Greenwich), 6 (5), 283-287. doi:10.1111/j.1524-6175.2004.03585.x
Stenn, K. S., & Paus, R. (2001). Controls of hair follicle cycling.Physiol Rev, 81 (1), 449-494. doi:10.1152/physrev.2001.81.1.449
Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama, Y., . . . He, X. (2000). LDL-receptor-related proteins in Wnt signal transduction. Nature, 407 (6803), 530-535. doi:10.1038/35035117
Tao, F., Soffers, J., Hu, D., Chen, S., Gao, X., Zhang, Y., . . . Li, L. (2020). β-Catenin and Associated Proteins Regulate Lineage Differentiation in Ground State Mouse Embryonic Stem Cells. Stem Cell Reports, 15 (3), 662-676. doi:10.1016/j.stemcr.2020.07.018
Tsakmaki, A., Fonseca Pedro, P., Pavlidis, P., Hayee, B., & Bewick, G. A. (2020). ISX-9 manipulates endocrine progenitor fate revealing conserved intestinal lineages in mouse and human organoids. Mol Metab, 34 , 157-173. doi:10.1016/j.molmet.2020.01.012
Van Neste, D., Fuh, V., Sanchez-Pedreno, P., Lopez-Bran, E., Wolff, H., Whiting, D., . . . Kaufman, K. D. (2000). Finasteride increases anagen hair in men with androgenetic alopecia. Br J Dermatol, 143 (4), 804-810. doi:10.1046/j.1365-2133.2000.03780.x
Varela-Nallar, L., & Inestrosa, N. (2013). Wnt signaling in the regulation of adult hippocampal neurogenesis. 7 . doi:10.3389/fncel.2013.00100
Wang, S., Yin, J., Chen, D., Nie, F., Song, X., Fei, C., . . . Li, L. (2013). Small-molecule modulation of Wnt signaling via modulating the Axin-LRP5/6 interaction. Nat Chem Biol, 9 (9), 579-585. doi:10.1038/nchembio.1309
Waters, J. M., Richardson, G. D., & Jahoda, C. A. (2007). Hair follicle stem cells. Semin Cell Dev Biol, 18 (2), 245-254. doi:10.1016/j.semcdb.2007.02.003
Wu, Z., Zhu, Y., Liu, H., Liu, G., & Li, F. (2020). Wnt10b promotes hair follicles growth and dermal papilla cells proliferation via Wnt/β-Catenin signaling pathway in Rex rabbits. Bioscience Reports, 40 (2). doi:10.1042/bsr20191248
Xing, F., Yi, W. J., Miao, F., Su, M. Y., & Lei, T. C. (2018). Baicalin increases hair follicle development by increasing canonical Wnt/β‑catenin signaling and activating dermal papillar cells in mice.Int J Mol Med, 41 (4), 2079-2085. doi:10.3892/ijmm.2018.3391
Xuan, W., Wang, Y., Tang, Y., Ali, A., Hu, H., Maienschein-Cline, M., & Ashraf, M. (2018). Cardiac Progenitors Induced from Human Induced Pluripotent Stem Cells with Cardiogenic Small Molecule Effectively Regenerate Infarcted Hearts and Attenuate Fibrosis. Shock, 50 (6), 627-639. doi:10.1097/shk.0000000000001133
Zeng, X., Huang, H., Tamai, K., Zhang, X., Harada, Y., Yokota, C., . . . He, X. (2008). Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions. Development, 135 (2), 367-375. doi:10.1242/dev.013540
Zeng, X., Tamai, K., Doble, B., Li, S., Huang, H., Habas, R., . . . He, X. (2005). A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature, 438 (7069), 873-877. doi:10.1038/nature04185
Zhou, L., Xu, M., Yang, Y., Yang, K., Wickett, R. R., Andl, T., . . . Zhang, Y. (2016). Activation of β-Catenin Signaling in CD133-Positive Dermal Papilla Cells Drives Postnatal Hair Growth. PLoS One, 11 (7), e0160425. doi:10.1371/journal.pone.0160425