Background and Purpose:
ISX9 is a neurogenesis-promoting small molecule compound which can
upregulate the expression of NeuroD1 and induce differentiation of
neuronal, cardiac and islet endocrine progenitors. So far, the molecular
mechanisms underlying the action of ISX9 still remain elusive.
Experimental Approach: To identify a novel agonist of the
Wnt/β‐catenin,
a cell-based SuperTOPFlash reporter system was used to screen
known-compound libraries. An activation effect of ISX9 on the
Wnt/β‐catenin pathway was analysed with the SuperTOPFlash or
SuperFOPFlash reporter system. Effects of ISX9 on Axin1/LRP6 interaction
were examined using a mammalian two-hybrid system,
co‐immunoprecipitation, microscale thermophoresis (MST), emission
spectra and mass spectrometry (MS) assays. The expression of Wnt target
and stemness marker genes were evaluated with real‐time PCR and
immunoblotting. In vivo hair regeneration abilities of ISX9 were
analysed by immunohistochemical staining, real‐time PCR and
immunoblotting in hair regrowth model using C57BL/6J mice.
Key Results: In this study, ISX9 was identified as a novel
agonist of the
Wnt/β‐catenin
pathway. ISX9 targeted Axin1 by covalently binding to its N-terminal
region and potentiated the LRP6-Axin1 interaction, thereby resulting in
the stabilization of
β‐catenin
and upregulation of Wnt target genes and stemness marker genes.
Moreover, the topical application of ISX9 markedly promoted hair
regrowth in C57BL/6J mice and induced hair follicle transition from
telogen to anagen via enhancing
Wnt/β‐catenin
pathway.
Conclusions and Implications: Taken together, our study
unraveled that ISX9 could activate
Wnt/β‐catenin
signaling by potentiating the association between LRP6 and Axin1, and
may be a promising therapeutic agent for alopecia treatment.
Key words: ISX9, Wnt/β-catenin signaling, LRP6/Axin1
interaction, alopecia