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\begin{document}

\title{ISX9, a small molecule targeting Axin, activates Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling
and promotes hair regrowth\selectlanguage{english}}



\author[1]{Sapna Sayed}%
\author[1]{Jiaxing  Song}%
\author[1]{Ling Wang}%
\author[1]{Boxin Liu}%
\author[1]{Zijie  Su}%
\author[1]{Li Huan}%
\author[1]{Vivian Weiwen  Xue}%
\author[1]{Shan-Shan  Liu}%
\author[1]{Xianxiong Chen}%
\author[1]{Guangqian   Zhou}%
\author[1]{Qi Sun}%
\author[1]{Desheng Lu}%
\affil[1]{Shenzhen University}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
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/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin, a cell-based SuperTOPFlash reporter system was used to
screen known-compound libraries. An activation effect of ISX9 on the
Wnt/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}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 spectroscopy assays. The expression of Wnt target and
stemmness 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/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}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 \selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin and upregulation of Wnt
target genes and stemmness 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/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin pathway. Conclusions and Implications: Taken together, our
study unraveled that ISX9 could activate Wnt/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin signaling by
potentiating the association between LRP6 and Axin1, and may be a
promising therapeutic agent for alopecia treatment%
\end{abstract}%



\sloppy


\selectlanguage{greek}\textbf{ISX9, a small molecule targeting Axin, activates Wnt/β-catenin
signaling and promotes hair regrowth}\selectlanguage{english}

\textbf{Running Title:} ISX9 activates Wnt/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin Signaling

Sapna Sayed\textsuperscript{1}, Jiaxing Song\textsuperscript{1 2}, Ling
Wang\textsuperscript{1}, Boxin Liu\textsuperscript{1}, Zijie
Su\textsuperscript{1}, Huan Li\textsuperscript{1}, Vivian Weiwen
Xue\textsuperscript{1}, Shanshan Liu\textsuperscript{1}, Xianxiong
Chen\textsuperscript{3}, Guangqian Zhou\textsuperscript{3}, Qi
Sun\textsuperscript{1}* and Desheng Lu\textsuperscript{1}*

\textsuperscript{1} Guangdong Provincial Key Laboratory of Regional
Immunity and Diseases, International Cancer Center, Department of
Pharmacology, Shenzhen University Health Science Center, Shenzhen, China

\textsuperscript{2} Medical Scientific Research Center, Life Sciences
Institute, Guangxi Medical University, Nanning, China

\textsuperscript{3} Department of Physiology, School of Medicine, Health
Science Center, Shenzhen University, Shenzhen, China

* To whom correspondence may be addressed. Qi Sun or Desheng Lu,
Shenzhen University Health Science Center, Shenzhen 518060, Guangdong,
China.

Email: sunqi@szu.edu.cn or delu@szu.edu.cn.

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study are available from the
corresponding authors upon reasonable request.

Words Count (Excluding Abstract/ Methods/References/Figure Legends):
3396

\textbf{ACKNOWLEDGEMENTS}

We would like to acknowledge the support provided by International
Science and Technology Cooperation: Carson Cancer Stem Cell Vaccines
R\&D Center, Shenzhen University. This work was supported by the
National Natural Science Foundation of China (31970739, 81802662,
31900881, and U21A20421), the Natural Science Foundation of Guangdong
Province (2020A1515010340, 2020A1515010543 and 2022A1515010598), the
Shenzhen Key Basic Research Program (JCYJ20200109105001821), the
Shenzhen Natural Science Fund (the Stable Support Plan Program)
(20200826134656001).

\textbf{DECLARATION OF CONFLICT OF INTERESTS}

The authors declare no competing interests.

\textbf{AUTHORS CONTRIBUTIONS}

D L designed the research; S S, J S, L W, B L, Z S, H L, V W X, S L, X C
and Q S performed experiments; S S, G Z, Q S, and D L carried out the
data analysis. D L, L W, S S, Q S, and G Z made critical comments on the
manuscript. S S, Q S and D L wrote and edited the manuscript.

\textbf{ABSTRACT}

\textbf{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.

\textbf{Experimental Approach:} To identify a novel agonist of the
Wnt/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin, a cell-based SuperTOPFlash reporter system was used to
screen known-compound libraries. An activation effect of ISX9 on the
Wnt/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}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. \emph{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.

\textbf{Key Results:} In this study, ISX9 was identified as a novel
agonist of the Wnt/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}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 \selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}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/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin pathway.

\textbf{Conclusions and Implications:} Taken together, our study
unraveled that ISX9 could activate Wnt/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin signaling by
potentiating the association between LRP6 and Axin1, and may be a
promising therapeutic agent for alopecia treatment.

\textbf{Key words:} ISX9, Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling, LRP6/Axin1
interaction, alopecia

\textbf{Bullet point summary}

\textbf{What is already known}

\begin{itemize}
\tightlist
\item
  ISX9 has been identified as a neurogenesis inducer
\item
  ISX9 could induce differentiation of neuronal, cardiac and islet
  endocrine progenitors.\textbf{What this study adds}
\item
  ISX9 is a novel activator of Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling and targets Axin1
  via its N-terminal region.
\item
  ISX9 potentiates the LRP6-Axin1 association and thereby, resulting in
  stabilization of \selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin.\textbf{What is the clinical significance}
\item
  ISX9 may be a promising therapeutic agent for alopecia treatment via
  upregulating Wnt/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin signaling
\end{itemize}

\textbf{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/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}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 \selectlanguage{greek}β-\selectlanguage{english}catenin is indispensable for the
Wnt/\selectlanguage{greek}β-\selectlanguage{english}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 3\selectlanguage{greek}β \selectlanguage{english}enzyme (GSK3\selectlanguage{greek}β\selectlanguage{english}). The \selectlanguage{greek}β-\selectlanguage{english}catenin protein is
phosphorylated by GSK3\selectlanguage{greek}β, \selectlanguage{english}resulting in its ubiquitination-mediated
degradation. The Wnt/\selectlanguage{greek}β-\selectlanguage{english}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 \selectlanguage{greek}β-\selectlanguage{english}catenin phosphorylation and
constitutive degradation (Tamai et al., 2000; Zeng et al., 2005).
Consequently, the stabilized \selectlanguage{greek}β-\selectlanguage{english}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 \selectlanguage{greek}β-\selectlanguage{english}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/\selectlanguage{greek}β-\selectlanguage{english}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 \selectlanguage{greek}β-\selectlanguage{english}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 \selectlanguage{greek}β-\selectlanguage{english}catenin stabilization (Wu, Zhu,
Liu, Liu, \& Li, 2020). Wnt3a has been found to activate \selectlanguage{greek}β-\selectlanguage{english}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/\selectlanguage{greek}β-\selectlanguage{english}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/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}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 Ca\textsuperscript{2+}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 \selectlanguage{greek}β-\selectlanguage{english}cell
metabolites, protect glucose-responsive signaling pathways under
lipotoxic conditions, and improve glycemia in a mouse model of \selectlanguage{greek}β-\selectlanguage{english}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/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling pathway. We further
investigated the mechanism involved in activation of Wnt/\selectlanguage{greek}β-\selectlanguage{english}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.

\textbf{2 METHODS}

\textbf{2.1 Cell culture}

The human embryonic kidney 293T (HEK293T) cells (ATCC Cat\#CRL-3216;
RRID: CVCL-0063), the human keratinocyte (HaCaT) cells (ATCC
Cat\#CRL-2309; RRID: CVCL-0038)\emph{,} and the mouse embryonic
fibroblasts (NIH3T3) cells (ATCC Cat\# CRL-1658, RRID:CVCL\_0594) were
acquired from American Type Culture Collection (ATCC, Manassas, VA, USA)
and sustained in Dulbecco's Modified Eagle's Medium (DMEM) (Thermo
Fisher Scientific, Cat\#C11995500BT, Waltham, MA, USA) accompanied with
10\% fetal bovine serum (FBS) (Thermo Fisher Scientific, Cat\#
10270-106, Shanghai, China). Cells were cultured with 120 \selectlanguage{greek}µ\selectlanguage{english}g/mL
penicillin along with 200 \selectlanguage{greek}µ\selectlanguage{english}g/mL streptomycin (Thermo Fisher Scientific,
Cat\# 15-140-122, Shanghai, China) at 37 \selectlanguage{ngerman}°C in an incubator with 5\%
CO\textsubscript{2}. ISX9 was dissolved in dimethyl sulfoxide (DMSO) for
provision of a stock solution at a final concentration of 20 mM. For
treating cells, 20 mM stock solution was diluted with the specific media
with respect to cells, and the final DMSO concentration was
\textless{}0.1\%.

\textbf{2.2} \textbf{Plasmids} \textbf{and screening of drug library}

Following the manufacturer's instruction, the transfection of HEK293T
and HaCat cells were performed using Lipofectamine 2000 transfection
reagent (Roche Diagnostics GmbH, Cat\#11668-019, Mannheim, Germany). The
reporter plasmids SuperTOPFlash was modestly gift of Karl Willert
(University of California, San Diego, CA, USA) while SuperFOPFlash was
bought from Beyotime (Beyotime Biotechnology, Cat\# D2503, Shenzhen,
China). For the construction of C-terminally V5-tagged LRP6 expression
plasmid, the cDNA encoding LRP6 was amplified by RT-PCR and cloned into
the pcDNA 3.1/V5-His mammalian expression vector. The N-terminally
tagged Flag-Axin1 expression plasmid was built by inserting the
conforming cDNA into the pFlag-CMV2 expression vector.

For drugs screening, HEK293T cells were fully-fledged in 10 cm plates.
After 24 h, achieving 50\% convergence, cells were transfected with 5 \selectlanguage{greek}µ\selectlanguage{english}g
of SuperTOPFlash or SuperFOPFlash reporters, hauler DNA pcDNA3 and 1 \selectlanguage{greek}µ\selectlanguage{english}g
of control-plasmid pCMX-\selectlanguage{greek}β-\selectlanguage{english}gal (\selectlanguage{greek}β-\selectlanguage{english}galactosidase, \selectlanguage{greek}β-\selectlanguage{english}gal) amassing a total
of 10 \selectlanguage{greek}µ\selectlanguage{english}g/plate. After 24 h-transfection, cells were placid and
disseminated in 96-well microtiter plates. Finally, cells were treated
with different drugs for the preliminary screen. Exploiting a microtiter
plate luminometer, the 24 h-incubated cells were lysed and activities of
luciferase were detected using a luciferase assay kit (Promega Cat\#
E1501, Madison, WI, USA). Making use of the \selectlanguage{greek}β-\selectlanguage{english}galactosidase as an
internal control, the values from luciferase assay were normalized for
distinctions in transfection efficiency.

\textbf{2.3 Protein purification}

The expression plasmids for His-tagged intracellular domain of
LRP6/1394-1613 (His-LRP6-ICD), GST-tagged Axin1 1-300 (GST-Axin1-300),
GST-tagged Axin1 301-521 (GST-Axin301-521), and GST-tagged Axin1 522-862
(GST-Axin522-862) were constructed and expressed in \emph{Escherichia
coli} (\emph{E. coli} ) BL21. The expression of fusion proteins was
induced with IPTG (Sigma-Aldrich, Shanghai, China) at 16 or 28 \selectlanguage{ngerman}°C for
8-12\selectlanguage{english} h. His-LRP6-ICD was purified by NiNTA Purification System, while
GST and GST fusion proteins were purified by affinity chromatography
with glutathione-sepharose 4B beads (GE Healthcare, Piscataway, NJ,
USA). The factor Xa protease was used to remove GST and His tag from
fusion proteins.

\textbf{2.4 RNA extraction and real-time PCR analyses}

Following manufacturing's guideline, RNA was extracted employing RNAiso
Plus (TaKaRa Cat\# 9109, Kusatsu, Shiga, Japan). The isolated total RNA
is then converted into cDNA by means of the Primescript RT Reagent Kit
(TaKaRa Cat\# RR037A, Kusatsu, Shiga, Japan) following the
manufacturer's practice. The cDNA was next utilized for quantitative PCR
analysis by an ABI Prism 7300 Real\selectlanguage{english}-Time PCR System (Applied Biosystems,
Foster City, CA, USA) via qPCR Master Mix (Bimake Cat\# B21203, Houston,
TX, USA). Quantification of mRNA levels of human and mouse genes i.e.,
Axin2, LEF1, Fibronectin, Survivin, OCT4, Twist1, LGR5, and SOX2 were
carried out. The ``comparative Ct method'' was accomplished to evaluate
relative countenance of genes. The data are obtained as the fold change.
The fold change was premeditated as 2-\selectlanguage{greek}ΔΔ\selectlanguage{english}Ct (\selectlanguage{greek}ΔΔ\selectlanguage{english}Ct =\selectlanguage{greek}Δ\selectlanguage{english}Cttreated -
\selectlanguage{greek}Δ\selectlanguage{english}Ctcontrol., Ct is the cycle figure at spot where fluorescence first
surpasses the threshold). The \selectlanguage{greek}Δ\selectlanguage{english}Ct values from each target gene were
obtained by subtracting the values for GAPDH Ct from the illustration
Ct. The data of three technical replicates/sample are presented. The
primer sequences are shown in supplementary tables (Supplementary Table
S1 and S2).

\textbf{2.5 Immunoblot Analysis}

For extraction of proteins, cells or mice-skin tissues were treated with
RIPA buffer (20 mM Tris-HCl -pH 7.4, 150 mM NaCl, 1 \% Triton X-100, 0.1
mM DTT, 1 mM EDTA, 1 mM EGTA, and 1 mM PMSF) containing the protease and
phosphatase inhibitor cocktail (Topscience) trailed by sonication. The
protein extraction was then fractionated by SDS-PAGE and passed-on to
PVDF membranes (Cat\# ISEQ00005, Millipore, Burlington, MA, USA).
Western blotting was accomplished with the subsequent primary
antibodies: anti-\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin (1:2,000, Santa Cruz Biotechnology Cat\#
sc-7963, RRID:AB\_626807), anti-LGR5 (1:1,000, Abcam Cat\# ab75732,
RRID:AB\_1310281), anti-SOX2 (1:1,000, Cell Signaling Technology Cat\#
23064, RRID:AB\_2714146), anti-Flag (1:5,000, Sigma-Aldrich Cat\# F1804,
RRID:AB\_262044), anti-GAPDH (1:5,000, Transgen Biotech Cat\# HC301--02,
RRID:AB\_2629434), anti-LRP6 antibody (1:1000 Cell Signaling Technology
Cat\# 2560, RRID:AB\_2139329), anti-CK1\selectlanguage{greek}ε \selectlanguage{english}antibody (1:1000 Cell Signaling
Technology Cat\# 12448, RRID:AB\_2797919), anti-active \selectlanguage{greek}β-\selectlanguage{english}catenin
antibody (1:1000 Cell Signaling Technology Cat\#8814,
RRID:AB\_11127203), anti-LEF1 antibody (1:1000 Cell Signaling Technology
Cat\#76010, RRID:AB\_2799877), anti-Axin2 antibody (1:1000 Cell
Signaling Technology Cat\# 2151, RRID:AB\_2062432), anti-V5 antibody
(1:1000 Cell Signaling Technology Cat\#13202, RRID:AB\_2687461),
anti-Survivin antibody (1:1000 Santa Cruz Biotechnology Cat\# sc-17779,
RRID:AB\_628302), anti-pLRP6 antibody (1:1000 Cell Signaling Technology
Cat\# 2568 RRID:AB\_2139327), anti-GSK-3-beta (1:1000 Cell Signaling
Technology Cat\#9315S RRID:AB\_490890), anti-Twist1 (1:1000 Cell
Signaling Technology Cat\#46702S RRID:AB\_2799308), anti-Phospho-GSK-3\selectlanguage{greek}β
\selectlanguage{english}(1:1000 Cell Signaling Technology Cat\#9322S RRID:AB\_2115196), and Anti
cytokeratin 17 (1:1000, Abcam Cat\# ab53707, RRID:AB\_869865). Western
blotting was carried out with the chosen primary antibodies with nurture
of 4 \selectlanguage{ngerman}°C overnight. Next, the PVDF membranes were mounting with HRP
(Horseradish Peroxidase) conjugated goat anti\selectlanguage{english}-rabbit (1:10,000, Thermo
Fisher Scientific Cat\# A16096, RRID:AB\_2534770) or anti-mouse
(1:10,000, Thermo Fisher Scientific Cat\# A16066, RRID:AB\_2534739) or
IgG at room temperature for 1 h. After incubating with substrate of ECL
Plus-Western-Blotting (Thermo Fisher Scientific, Cat\# 32132), the
immunoblots were established by Chemiluminescent Imaging System (Tanon
5200, Shanghai, China).

\textbf{2.6 Co-immunoprecipitation assays}

HEK293T and HaCaT cells (0.5 x 10\textsuperscript{6} cells per well)
were scattered in six-well plates, and treated with the indicated
concentrations of ISX9 or DMSO for 24 h. The pooled of 100 \selectlanguage{greek}µ\selectlanguage{english}g
protein-lysates were accrued by using 500 \selectlanguage{greek}μ\selectlanguage{english}L RIPA initially (phosphatase
inhibitor cocktails and protease inhibitor freshly added), after that
centrifugation is executed (12,000 rpm for 15 min at 4 \selectlanguage{ngerman}°C). After
quantification, the lysates were established with respective antibodies
at 4 °C overnight. By using anti-Flag agarose bead (Bimake), we then
enacted an immunoprecipitation. The immunocomplexes were washed five
times with RIPA buffer and boiled in the SDS-loading buffer. They were
then resolute by 10\% SDS-PAGE and relocated to PVDF membranes, stalked
by immunoblotting with the stipulated antibodies.

\textbf{2.7 Microscale thermophoresis}

Microscale thermophoresis (MST) experiments were conducted on a Monolith
NT.115 system (NanoTemper Technologies GmbH, Germany), which was used to
quantify the interaction between Axin1 fragments (Axin1 1-300, Axin1
301-521, Axin1 522-862) and ISX9, and the interaction between LRP6-ICD
and ISX9, respectively. Axin1 fragments or LRP6-ICD were labeled with
the manufacturer's labeling kits (Monolith\textsuperscript{TM} RED-NHS,
NanoTemper Technologies GmbH, Germany). During the measurement of Axin1
fragments with ISX9, the concentration of labeled Axin1 fragments or
LRP6-ICD is 100 nM. Unlabeled ISX9 was prepared in 10 mM PBS dimethyl
sulfoxide (DMSO), with the final concentrations of ISX9 ranging from
0.61 nM to 5 \selectlanguage{greek}μ\selectlanguage{english}M, the samples were added to the monolith capillaries (MO
L022, NanoTemper Technologies) and subsequently subjected to MST
analysis. The values obtained were normalized and plotted against the
Axin1 fragments or LRP6-ICD and ISX9 concentration, respectively. The
dissociation constant was then determined using a single-site model to
fit the curve.

\textbf{2.8 Emission spectra}

Axin1 protein fragment was titrated into ISX9, and the mixtures were
allowed to equilibrate at room temperature for 2 min. Emission spectra
were recorded in the 520-680 nM range using an excitation wavelength of
465 nM. The change of luminescence intensity was observed by a
fluorescence spectrometer (Edinburgh FS5, UK).

\textbf{2.9 Mass spectrometry assays}

10 \selectlanguage{greek}µ\selectlanguage{english}L ISX9 at 1 mM in PBS was incubated with 40 \selectlanguage{greek}µ\selectlanguage{english}L purified flag-tagged
Axin1 1-300 (1 mg/mL) overnight at 4 . All analyses were performed
using a Thermo Scientific Ultimate 3000 Binary UHPLC system (Thermo
Fisher Scientific, Germering, Germany) coupled online to a Thermo
Scientific Ultra High Mass Range Q Exactive hybrid quadrupole-Orbitrap
mass spectrometer using an IonMax API source with a HESI-II probe
(Thermo Fisher Scientific, Bremen, Germany).

The separation of protein-ligand in ACQUITY UPLC Protein BEH C4 (300A,
21 mm\selectlanguage{ngerman}×150 mm, 1.7 \selectlanguage{greek}μ\selectlanguage{english}m) was performed by using 0.1\% TFA water and 0.1\%
TFA in ACN as mobile phases A and B, respectively. A gradient of 20\% to
60\% B in 8 min, followed by 60\% to 80\% B in 0.1 min was applied.
Following the analytical gradient, an isocratic step at 80\% B for 3 min
was used to wash the column, followed by 4 min of column
re-equilibration at 20\% B. The flow rate was 0.2 mL/min, column
temperature 60 \selectlanguage{ngerman}°C and injection volume 4 \selectlanguage{greek}µ\selectlanguage{english}L (1 mg/mL sample material).
The UV acquisition wavelength was 214 nm.

Full MS spectra were acquired in positive polarity in a scan range of
500--4,000 m/z. The resolution was set to 17,500 at 400 m/z, with an AGC
target of 3e6 ions and 5 microscans were performed. The maximum
injection time was 200 ms. In-source trapping desolvation was set to 20
V, and the trapping gas pressure was set to 7.0. Sheath gas was set to
35 arbitrary units (AU) and auxiliary gas was set to 10 AU. The spray
voltage was 3.8 kV, the capillary temperature was 300 \selectlanguage{ngerman}°C, the S-lens RF
was set to 80 V and the auxiliary gas heater temperature was 300 °C.

Mass spectra were acquired using Thermo Scientific Xcalibur version
4.1.31.9. The analysis of the acquired mass spectra was carried out
using the Thermo Scientific BioPharma Finder software version 3.1.

\textbf{2.10 Animal model studies}

All animal care and experimental techniques for anesthetics, hair
regrowth experiments and assays were accomplished with the permission of
Animal Research Ethics Committee of Shenzhen University (permit number
AEWC-201412003). Animal studies are stated in amenability with the
ARRIVE guidelines and with the commendations made by the British Journal
of Pharmacology (Lilley et al., 2020) Specific pathogen-free C57BL/6
mice (6 weeks old) were purchased from Nanjing Biomedical Research
Institute of Nanjing University, (Nanjing, China) and acclimatized for 1
week. After anesthesia via an intraperitoneal injection of
2,2,2-tribromoethanol (Sigma-Aldrich, St. Louis, USA), the skin hairs on
the dorsal side of 7-week-old mice (whose hair follicles were in the
telogen phase) were clean-shaven by a hair clipper. The mice were
randomly separated into three groups(Lee et al., 2012).. For every
mouse, 200 \selectlanguage{greek}μ\selectlanguage{english}L of each drug was topically applied at an appropriate
concentration everyday (as described in figure legends) for 25 days. The
control group received vehicle alone, the ISX9 group received vehicle
containing 1\% of ISX9, and the minoxidil group received 2\% of
minoxidil. All reagents used for the hair growth test were dissolved in
a vehicle composed of 50\% (v/v) ethanol, 30\% water, and 20\% propylene
glycol. The back skin of the mice was photographed at 0, 7, 14, 21, 25
days, and analyses was done regarding the weight of regrown hair in
different groups treated for 25 days. After killing mice, skin tissues
were gathered and saved at -80 \selectlanguage{ngerman}°C.

\textbf{2.11 Histology and immunohistochemistry analyses}

Samples were immobile in 4\% formaldehyde in PBS for 24 h, then
dehydrated and implanted in paraffin, sectioned at 5 \selectlanguage{greek}μ\selectlanguage{english}m before
haematoxylin and eosin (H\&E) staining and immunohistochemistry were
accomplished. The primary antibodies exploited for immunohistochemistry
include anti-Ki-67 (1:200, Cell Signaling Technology Cat\# 12202,
RRID:AB\_2620142), anti-LGR5 (1:100, Abcam Cat\# ab75732,
RRID:AB\_1310281), Anti-active \selectlanguage{greek}β-\selectlanguage{english}catenin antibody (1:100 Cell Signaling
Technology Cat\#8814, RRID:AB\_11127203) and anti-\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin (1:100 Cell
Signaling Technology Cat\# 9562, RRID:AB\_331149).

\textbf{2.12 Hair cycle analyses}

The analyses of hair cycle were centered on level of skin pigmentation
and hair shaft growth, as earlier reported (Stenn \& Paus, 2001).
Briefly, clean-shaved hair-coat-recovery values were premeditated based
on skin pigmentation levels from 0 to 100\% (Chai et al., 2019). For
illustration, 0\% implied no hair growth (and no pigmentation); 50\%
couched full-length just grown hairs casing half of the dorsal
designated skin (or pigmentation level on 100\% of the dorsal definite
skin area without any hair shafts); 70\% implicit full-length-hair
progress on \textasciitilde{}70\% of the dorsal skin (or pigmentation
level covering 100\% of dorsal skin along with 30--40\% hair shafts);
100\% inferred afresh full-length hair growth.

\textbf{2.13} \textbf{Hair follicles count}

The H\&E stained slides were photographed using a digital
photomicrograph and all of the images were cropped in a fixed width of
300 pixels. We counted hair follicles manually in a fixed area
(0.09 mm\textsuperscript{2}). Digital photomicrographs were taken from
representative areas at a fixed magnification of 100X.

\textbf{2.14 Data and statistical analyses}

All experiments were equal sized, randomized and blinded. No data values
were omitted in any statistical analysis. The data and statistical
analysis according to the recommendations of the \emph{British Journal
of Pharmacology} on experimental design and analysis in pharmacology
(Curtis et al., 2018). Statistical analyses were acquired with GraphPad
prism7.00 software (GraphPad, RRID: SCR\_000306). The data were
evaluated by Student's t test or one- way ANOVA followed by Dunnett's t
test. Results are presented as mean \selectlanguage{ngerman}± SD. Differences at P \textless{}
0.05, were considered statistically significant.

\textbf{2.15 Materials}

ISX9 was obtained from TargetMol (Cat\#T2003 CAS 832115625, Shanghai,
China). Minoxidil was also purchased from TargetMol (Shanghai, China).
Reagents for cell culture were bought from GIBCO. Compound libraries
consist of antifungal, antibacterial and antiviral agents, were acquired
from MedChemExpress (MCE, Monmouth Junction, NJ, USA). All additional
chemical reagents were attained from Sigma-Aldrich (St. Louis, MO, USA)
apart from reagents with special instructions.

\textbf{3 RESULTS}

\selectlanguage{greek}\textbf{3.1 ISX9 activates the Wnt/β-catenin signaling pathway}\selectlanguage{english}

In order to identify novel small molecules that modulate the
Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling pathway, a cell-based SuperTOPFlash reporter
system was used to screen a known-compound library. Our screening
identified ISX9, a small molecule with the isoxazole scaffold, as a
potential activator of Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling (Figure 1A and B). To
determine the agonistic effect on the Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin pathway, the
luciferase reporter SuperTOPFlash was transfected into HEK293T cells.
Treatment with 2.5-40 \selectlanguage{greek}µ\selectlanguage{english}M ISX9 strongly increased the transcriptional
activity of the SuperTOPFlash reporter in a dose-dependent manner. ISX9
at 40 \selectlanguage{greek}µ\selectlanguage{english}M could induce the reporter activity \textasciitilde{}2700-fold
above the basic levels (Figure 1C). However, ISX9 had little effect on
the activity of negative control reporter SuperFOPFlash, which contains
mutated TCF-binding elements. As anticipated, lithium chloride (LiCl), a
well-known activator of Wnt signaling, remarkably increased the activity
of the SuperTOPFlash reporter (Figure 1D).

To further confirm the effect of ISX9 on Wnt signaling, we performed the
SuperTOPFlash reporter assay in human keratinocyte HaCaT cells.
Similarly, ISX9 dose-dependently enhanced the activity of the
SuperTOPFlash reporter in HaCat, while it did not affect the
SuperFOPFlash reporter activity (Figure 1E). Together, these results
demonstrated that ISX9 is capable of activating the Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin
pathway.

\selectlanguage{greek}\textbf{3.2 ISX9 induces the accumulation of β-catenin without affecting
the expression and activity of LRP6 and GSK3β}

\selectlanguage{english}To explore the potential target of ISX9 action in the Wnt signaling
cascade, we examine the effect of ISX9 on key components in Wnt
signaling cascade, including active \selectlanguage{greek}β-\selectlanguage{english}catenin, total \selectlanguage{greek}β-\selectlanguage{english}catenin,
phosphorylated LRP6, total LRP6, Ser9 phosphorylated GSK3\selectlanguage{greek}β, \selectlanguage{english}total GSK3\selectlanguage{greek}β,
\selectlanguage{english}and CK1e \selectlanguage{english}in HEK293T, HaCat and NIH3T3 cells. As shown in Figure 2, ISX9
treatment dramatically upregulated protein levels of active \selectlanguage{greek}β-\selectlanguage{english}catenin
and total \selectlanguage{greek}β-\selectlanguage{english}catenin in a concentration-dependent fashion, whereas it had
little effect on the levels of phosphorylated LRP6, total LRP6, Ser9
phosphorylated GSK3\selectlanguage{greek}β, \selectlanguage{english}total GSK3\selectlanguage{greek}β, \selectlanguage{english}and CK1e \selectlanguage{english}in all three cell lines. We
also checked ISX9 effect on mRNA expression of \selectlanguage{greek}β-\selectlanguage{english}catenin in HEK293T
cells. There was no significant change in mRNA expression of \selectlanguage{greek}β-\selectlanguage{english}catenin
in response to ISX9 treatment (Supplementary Figure S1). Collectively,
these results imply that ISX9 could activate Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling
mainly through inducing the accumulation of intracellular \selectlanguage{greek}β-\selectlanguage{english}catenin
without affecting the expression and activity of LRP6 and GSK3\selectlanguage{greek}β.

\selectlanguage{english}\textbf{3.3 ISX9 potentiates the association of LRP6 with Axin1}

It has been known that Wnt stimulation-induced LRP6 phosphorylation
promotes the association of LRP6 with Axin, a key step to regulate
\selectlanguage{greek}β--\selectlanguage{english}catenin stability (Xin Zeng et al., 2008). We therefore examined the
effect of ISX9 on interaction between LRP6 and Axin1 using a mammalian
two-hybrid assay consisted of UAS-TK-Luc reporter and the expression
vectors for Gal4-LRP6 intracellular domain (Gal4-LRP6-ICD) and
VP16-Axin1. The interaction between LRP6-ICD and Axin1 was observed in
HEK293T cells transfected with both Gal4-LRP6-ICD and VP16-Axin1, and
treatment with ISX9 dose-dependently potentiated the interaction between
LRP6-ICD and Axin1. However, little signal was detected when UAS-TK-Luc
reporter was co-expressed with either Gal4-LRP6-ICD or VP16-Axin in the
presence or absence of ISX9 (Figure 3A). Notably, LiCl treatment exerted
little effect on the association of Gal4-LRP6 ICD with VP16-Axin1
(Figure 3B). These results suggest that ISX9 may potentiate the
association of LRP6 with Axin1. To further confirm the effect of ISX9 on
the interaction of LRP6 with Axin1, a co-immunoprecipitation assay was
performed using anti-Flag beads in HEK293T cells transfected with
LRP6-V5 and Flag-Axin1 expression vectors. Figure 3C showed that
increasing amounts of LRP6-V5 protein were immunoprecipitated using
anti-Flag beads in the presence of ISX9 (Figure 3C). Treatment with ISX9
also promoted endogenous interaction between LRP6 and Axin1 in HEK293T
and HaCat cells (Figure 3D and E). These results demonstrated that ISX9
is capable of promoting the interaction of LRP6 with Axin1.

\textbf{3.4 ISX9 enhances the interaction of LRP6-ICD with Axin1 through
covalently binding to the N-terminal region of Axin}

To explore the mechanism through which ISX9 potentiates the
protein-protein interaction between LRP6 and Axin1, a microscale
thermophoresis (MST) analysis was performed to determine whether ISX9
could directly bind to LRP6 and Axin1. We expressed and purified the
three truncated forms of Axin1, Axin1-300 (aa 1--300), Axin301-521 (aa
301-521) and Axin522-862 (aa 522-862), and LRP6-ICD domain as His-tagged
fusion proteins. The results showed the titration of ISX9 to
fluorescently labeled Axin1-300, yielded dose-dependent upward-shifted
thermophoresis profiles with a Kd value of 595 nM (Figure 4A),
suggesting the direct binding between ISX9 and Axin1-300. However, no
detectable binding was observed between ISX9 and Axin301-521(Figure 4B),
Axin522-862 (Figure 4C) or LRP6-ICD (Figure 4D). The fluorescence
emission spectra also showed that ISX9 could interact with purified
Axin1-300 protein (Figure 4E).

Next, mass spectrometry was used to verify ISX9 binding mode with
Axin1-300. The molecular weight of recombinant Axin1-300 is about
31417.38 Da. We detected an extra peak at 31650.81 Da after incubating
purified Axin1-300 fragment with ISX9 (233.44 Da) (Figure 4F),
indicating that covalent binding was formed between ISX9 and Axin1-300.

\selectlanguage{greek}\textbf{3.5 ISX9 inhibits the association of Axin1 with β-catenin}\selectlanguage{english}

Axin is a vital component in the \selectlanguage{greek}β-\selectlanguage{english}catenin destruction complex, which
provides a scaffold for recruitment of GSK3\selectlanguage{greek}β, \selectlanguage{english}CK1\selectlanguage{greek}α, \selectlanguage{english}and \selectlanguage{greek}β-\selectlanguage{english}catenin,
facilitating the phosphorylation and degradation of \selectlanguage{greek}β-\selectlanguage{english}catenin. We
therefore examined the effect of ISX9 binding to Axin1 on the
interaction of Axin1 and \selectlanguage{greek}β-\selectlanguage{english}catenin. An immunoprecipitation assay was
performed in HEK293T cells. As seen in Figure S2, treatment with ISX9
dramatically inhibited the interaction between Axin and \selectlanguage{greek}β-\selectlanguage{english}catenin, but
had little effect on the association of \selectlanguage{greek}β-\selectlanguage{english}catenin with GSK3\selectlanguage{greek}β
\selectlanguage{english}(Supplementary Figure S2). These results indicated that ISX9 binding to
Axin1 could facilitate the release Axin1 from the \selectlanguage{greek}β-\selectlanguage{english}catenin destruction
complex.

\textbf{3.6 ISX9 upregulates the expression of Wnt target genes Axin2,
LEF1, Fibronectin, and Survivin}

Considering that activation of Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling will upregulate
the transcription of Wnt target genes (Mosimann, Hausmann, \& Basler,
2009), we assessed the effect of ISX9 on mRNA expression of Wnt target
genes Axin2, LEF1, Fibronectin, and Survivin. HEK293T, HaCat and NIH3T3
cells were treated with increasing concentrations of ISX9 (2.5-40 \selectlanguage{greek}µ\selectlanguage{english}M)
for 24 h. Real-time PCR analysis showed that ISX9 dose-dependently
enhanced mRNA expression of Axin2, LEF1, Fibronectin, and Survivin in
all three cell lines (Figure 5A-C). Consistent with mRNA data, we
detected increased protein levels of Axin2, LEF1, and Survivin in
response to ISX9 treatment in all three cell lines (Figure 5D-F). These
results confirmed the activation of Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling in HEK293T,
HaCat and NIH3T3 cells treated with ISX9.

\textbf{3.7 ISX9 promotes the expression of stemness marker genes SOX2,
LGR5, Twist1, and OCT4}

Concerning the importance of the Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin pathway in the
self-renewal and expansion of various stem cell populaces (Ring, Kim, \&
Kahn, 2014; Tao et al., 2020), the expression of stemness marker genes
SOX2, LGR5, Twist1, and OCT4 was examined after treatment with ISX9 by
real-time PCR and immunoblotting. As seen in Figure 5, ISX9 treatment
significantly enhanced the mRNA of SOX2, LGR5, Twist1, OCT4, and protein
levels of SOX2, LGR5, and Twist1 in HEK293T, HaCat and NIH3T3 cells
(Figure 5G-L), suggesting that ISX9 may have a potential to promote the
stemness property via activating the Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin pathway.

\selectlanguage{greek}\textbf{3.8 ISX9 promotes hair regrowth in mice via the activation of
Wnt/β-catenin signaling}\selectlanguage{english}

The Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin pathway has been shown to play a crucial role in the
development of new hair follicles and initiation of hair growth. We next
evaluated the effect of ISX9 on the hair regrowth in mice. Minoxidil was
exploited as a positive control. ISX9 and minoxidil were topically
applied daily to the shaved dorsal skin of 7-week-old C57BL/6 mice. On
day 14 and day 21, ISX9 was more effective than minoxidil in promoting
hair regrowth in mice. After treatment for 25 days, both ISX9 and
minoxidil exhibited strong hair regrowth-promoting activity compared to
control mice (Figure 6A and B). Similar results were obtained by
measuring the weight of freshly grown hair (Figure 6C). Histopathology
with H\&E staining showed significantly increased numbers of hair
follicles in the anagen phase on day 25 in ISX9 and minoxidil group
compared with that in control mice (Figure 6D), while hair follicles in
control mice were still in the telogen phase. Consistently, quantitative
analysis of hair follicles also confirmed that the number of hair
follicles were markedly increased in ISX9 and minoxidil-treated group
compared with control mice (Figure 6E). These results suggest that
treatment with ISX9 or minoxidil induces hair follicle transition from
telogen to anagen.

We next examined the effect of ISX9 on the Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin pathway in
mice. The results of immunohistochemical staining indicated that ISX9
remarkedly increased the expression of active \selectlanguage{greek}β-\selectlanguage{english}catenin, total \selectlanguage{greek}β-\selectlanguage{english}catenin
and the Wnt signaling target gene LGR5, concomitant with expression of
the proliferation marker Ki-67 (Figure 6F). Moreover, real-time PCR
analysis showed that ISX9 treatment significantly upregulated the
expression of Wnt target genes (LEF1, Axin2, Fibronectin and Survivin)
and stemness marker genes (LGR5, Twist1, SOX2 and OCT4) (Figure 7A and
B). Consistent with the above results, ISX9-treated mice exhibited
obviously increased protein levels of active \selectlanguage{greek}β-\selectlanguage{english}catenin, total \selectlanguage{greek}β-\selectlanguage{english}catenin,
LEF1, Axin2, Survivin, LGR5, and SOX2, while no apparent differences in
protein levels of phosphorylated LRP6, total LRP6, CK1\selectlanguage{greek}ε, \selectlanguage{english}phosphorylated
GSK3\selectlanguage{greek}β \selectlanguage{english}and total GSK3\selectlanguage{greek}β \selectlanguage{english}were observed in ISX9, minoxidil and control
groups (Figure 7C). Interestingly, we noted that Keratin17 (Waters,
Richardson, \& Jahoda, 2007), a known-marker of cells with stem cell
properties in hair follicle, was also upregulated in response to ISX9
treatment (Figure 7C).

\textbf{4 DISCUSSION}

The Wnt/\selectlanguage{greek}β-\selectlanguage{english}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,
Hortig\selectlanguage{ngerman}üela, 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/\selectlanguage{greek}β-\selectlanguage{english}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/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling. ISX9 could promote the association of LRP6 with
Axin1, resulting in the accumulation of \selectlanguage{greek}β-\selectlanguage{english}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/\selectlanguage{greek}β-\selectlanguage{english}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-GSK3\selectlanguage{greek}β \selectlanguage{english}complex is recruited to the receptor, which facilitates
GSK3\selectlanguage{greek}β-\selectlanguage{english}induced LRP5/6 phosphorylation and inhibits \selectlanguage{greek}β-\selectlanguage{english}catenin
phosphorylation, resulting in the stabilization of \selectlanguage{greek}β-\selectlanguage{english}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 \selectlanguage{greek}β-\selectlanguage{english}catenin
via blocking the Axin/\selectlanguage{greek}β-\selectlanguage{english}catenin interaction (Gwak et al., 2012). In our
study, ISX9 was found to potently induce the accumulation of
intracellular \selectlanguage{greek}β-\selectlanguage{english}catenin without influencing the expression and activity
of LRP6 and GSK3\selectlanguage{greek}β. \selectlanguage{english}By covalently binding to the N-terminal region
(aa1-300) of Axin1, ISX9 abrogated the association of Axin1 with
\selectlanguage{greek}β-\selectlanguage{english}catenin. Meanwhile, ISX9 exhibited a strong promoting effect on the
interaction between LRP6 and Axin1, thus leading to stabilizing
intracellular \selectlanguage{greek}β-\selectlanguage{english}catenin. Together, our results uncover a novel mechanism
by which ISX9 induces the accumulation of \selectlanguage{greek}β-\selectlanguage{english}catenin and activates the
Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin cascade.

The periodic growth of hair follicles includes the three phases of
telogen, anagen, and catagen (Lin, Zhu, \& He, 2022; M\selectlanguage{ngerman}üller-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/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin signaling activation in the hair follicle (Andl et al.,
2002). Some Wnt/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}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/\selectlanguage{greek}β-\selectlanguage{english}catenin
signaling pathway by inhibiting GSK3\selectlanguage{greek}β \selectlanguage{english}(Lee et al., 2012). Xing et al
demonstrated that baicalin, a natural bioactive flavonoid extracted
from\emph{Scutellaria baicalensis} Georgi, could promote the growth of
hair follicles via activation of Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling in mice (Xing
et al., 2018). KY19382 is one of the newly synthesized analogues of
GSK3\selectlanguage{greek}β \selectlanguage{english}inhibitor indirubin-3'-monoxime. It exhibited hair
growth-promoting effect by inactivating GSK3\selectlanguage{greek}β \selectlanguage{english}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 \selectlanguage{greek}β-\selectlanguage{english}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/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}catenin signaling are not
yet commercially available. Here, our results revealed that ISX9 could
potently activate Wnt/\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}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/\selectlanguage{greek}β-\selectlanguage{english}catenin
signaling pathway. This compound activated Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling via
elevating the level of intracellular \selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}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 \selectlanguage{greek}β-\selectlanguage{english}catenin
destruction complex, and meanwhile triggered the interaction between
Axin1 and LRP6, leading to the stabilization of \selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}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 \emph{in vivo} . These results suggested that
ISX9 may be a promising therapeutic agent for alopecia.

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Amer, M., . . . Khanna, S. (2017). Epidermal E-Cadherin Dependent
\selectlanguage{greek}β-\selectlanguage{english}Catenin Pathway Is Phytochemical Inducible and Accelerates Anagen Hair
Cycling. \emph{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
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\textbf{FIGURES LEGENDS}

\selectlanguage{greek}\textbf{Figure 1. ISX9 activates Wnt/β-catenin signaling in HEK293T
cells.}\selectlanguage{english} (A) Screening of chemical compounds that activate Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin
signaling. HEK293T cells carrying a Wnt responsive luciferase reporter
(SuperTOPFlash) were treated with LiCl and individual small molecule
compounds. The cells were lysed and luciferase activities were
quantified. (B) Chemical structure of ISX9. (C) HEK293T cells were
transfected with SuperTOPFlash or SuperFOPFlash reporter constructs as
indicated. After transfection for 24 h, the cells were treated with
vehicle control (DMSO) or increasing concentrations of ISX9 (2.5-40 \selectlanguage{greek}µ\selectlanguage{english}M)
for another 24 h, then luciferase values were normalized to \selectlanguage{greek}β\selectlanguage{english}\textbf{-}
gal activities. (D) HEK293T cells were transfected with SuperTOPFlash or
SuperFOPFlash reporter constructs as indicated. After transfection for
24 h, the cells were treated with vehicle control or LiCl (20 and 40 mM)
for another 24 h, then luciferase values were normalized to \selectlanguage{greek}β\selectlanguage{english}\textbf{-}
gal activities. (E) HaCat cells were transfected with SuperTOPFlash or
SuperFOPFlash reporter plasmids for 24 h. The cells were then treated
with vehicle control or increasing concentrations of ISX9 (2.5-40 \selectlanguage{greek}µ\selectlanguage{english}M)
for another 24 h and finally, the luciferase values were normalized to
\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}gal activities. Each treatment was performed in six replicates (n =
6).

\selectlanguage{greek}\textbf{Figure 2. ISX9 increases the expression levels of active
β-catenin and total β-catenin, but had little effect on expression and
activity of LRP6 and GSK3β.} \selectlanguage{english}HEK293T (A), HaCat (B), and NIH3T3 (C)
cells were treated with either vehicle control (DMSO) or increasing
doses of ISX9 (2.5-40 \selectlanguage{greek}µ\selectlanguage{english}M) for 24 h. The endogenous expression levels of
active \selectlanguage{greek}β-\selectlanguage{english}catenin, total \selectlanguage{greek}β-\selectlanguage{english}catenin, phosphorylated LRP6, total LRP6,
phosphorylated GSK3\selectlanguage{greek}β, \selectlanguage{english}total GSK3\selectlanguage{greek}β, \selectlanguage{english}and CK1\selectlanguage{greek}ε, \selectlanguage{english}were detected by western
blotting.

\textbf{Figure 3} : \selectlanguage{greek}\textbf{ISX9 potentiates Axin1/LRP6 association to
activate the Wnt/β-catenin pathway.}\selectlanguage{english} (A) (Upper panel) Schematic
representation of the mammalian two-hybrid system used in this study.
(Lower panel). The reporter plasmid UAS-TK-Luc was transfected into
HEK293T cells with a VP16-Axin1 expression plasmid, either alone or with
VP16-Axin1 and Gal4-LRP6. Transfected cells were treated with different
ISX9 concentrations (5 and 10 \selectlanguage{greek}μ\selectlanguage{english}M) as indicated. The cells were harvested
at 24 h after treatment, and then luciferase activities were determined.
(B) The reporter plasmid UAS-TK-Luc was transfected into HEK293T cells
with a VP16-Axin1 expression plasmid, either alone or with VP16-Axin1
and Gal4-LRP6. Transfected cells were treated with different LiCl
concentrations (20 and 40 mM) as indicated. The cells were harvested at
24 h after treatment, and then luciferase activities were determined.
(C) Exogenous immunoblotting was performed using HEK293T cells. The
cells were co-transfected with LRP6-V5 and Axin1-Flag expression
plasmids for 24 h. The cells were incubated with the indicated treatment
of vehicle control (DMSO) or ISX9 (5 and 10 \selectlanguage{greek}µ\selectlanguage{english}M) for another 24 h. The
lysates were immunoprecipitated with anti-flag beads. Endogenous
immunoblotting was performed using (D) HEK293T and HaCat (E) cells.
Immunoprecipitation of cell lysates were performed using control IgG or
anti-Axin1 antibody. Immunoblotting was carried out using the indicated
antibodies. The images shown are representative of data generated in at
least three independent experiments.

\textbf{Figure 4} . \textbf{ISX9 promotes the association of LRP6-ICD
with Axin1 through covalently binding to the N-terminal region of
Axin1.} The binding of fluorescently labeled Axin1-300 (A), Axin301-521
(B), Axin522-862 (C), LRP6-ICD (D) to ISX9 were analyzed with a MST
assay. The ISX9 is titrated from 0.61 nM to 5 \selectlanguage{greek}μ\selectlanguage{english}M. The change in the
thermophoretic signal leads to a Kd of 595 nM. (E) The emission spectra
of the ISX9 in the presence of increasing concentrations of Axin1-300
fragment (0-9 mg/mL) (\selectlanguage{greek}λ\selectlanguage{english}ex = 405 nm). (F) MS spectra of protein-ligand
complex formed between Axin1-300 and ISX9. The blue arrow indicated
molecular weight of protein, red arrow indicated molecular weight of the
protein-ligand complex. The molecular weight of ligand ISX9 was
calculated as 233.44.

\textbf{Figure 5. ISX9 upregulates the expression of Wnt target genes
and stemness marker genes.} HEK293T (A), HaCat (B), and NIH3T3 (C) cells
were incubated with vehicle control (DMSO) or the indicated
concentrations of ISX9 (2.5-40 \selectlanguage{greek}µ\selectlanguage{english}M) for 24 h. Total RNA was extracted and
cDNAs were obtained by reverse transcription, and then subjected to
real-time PCR analysis to detect the mRNA expression of Axin2, LEF1,
Fibronectin and Survivin. The results were shown as mean \selectlanguage{ngerman}± SD from three
independent experiments. HEK293T (D), HaCat (E), and NIH3T3 (F) cells
were treated with vehicle control (DMSO) or the indicated concentrations
of ISX9 (2.5-40 \selectlanguage{greek}µ\selectlanguage{english}M) for 24 h. Protein expression of Axin2, LEF1 and
Survivin were detected by immunoblotting. Data shown were representative
of three independent experiments. The relative expression levels of
Axin2, LEF1, Fibronectin and Survivin, were quantified after
normalization to GAPDH. *p \textless{} 0.05, compared to vehicle
control. HEK293T (G), HaCat (H) and NIH3T3 (I) cells were incubated with
vehicle control (DMSO) or the indicated amounts of ISX9 (2.5-40 \selectlanguage{greek}µ\selectlanguage{english}M) for
24 h. Total RNA was extracted and then reverse-transcribed into cDNA.
Prepared cDNAs were subjected to quantitative PCR analysis to detect the
mRNA expression of stemness marker genes SOX2, LGR5, Twist1, and OCT4.
The protein levels of stemness marker genes SOX2, LGR5, and Twist1 were
measured with immunoblotting after treatment with the indicated doses of
ISX9 in HEK293T (J), HaCat (K) and NIH3T3 cells (L)
respectively\textbf{.} Data shown were representative of three
independent experiments. The relative expression levels of SOX2, LGR5,
Twist1, and OCT4 were quantified after normalization to GAPDH. *p
\textless{} 0.05, compared to vehicle control.

\textbf{Figure 6. ISX9 promotes hair regrowth \emph{in vivo}.} C57BL/6J
mice in the telogen phase (7 weeks old) were depilated. Vehicle
(50\%{[}v/v{]} ethanol, 30\% water and 20\% propylene glycol), 1\% ISX9,
or 2\% minoxidil were topically applied daily to the dorsal skin for 25
days (n = 5 per group) (A) Representative photos of mice showed hair
regrowth on mentioned days treated with each drug. (B) Quantitative
measurements of hair coat recovery at the designated area from 0-25
days. (C) Gross analyses of weight of regrown hair in different groups
treated for 25 days. (D) Haematoxylin and eosin (H\&E) staining to
assess the hair follicles of skins in different groups treated (vertical
and transverse view) (E) Quantitative evaluation of the comparative
number of hair follicles of HE staining images (n \textbf{=} 5) (F)
Immunohistochemistry (IHC) staining for active and total \selectlanguage{greek}β-\selectlanguage{english}catenin, Ki67
and LGR5 using the dorsal skin of mice treated with each drug for 25
days. Scale bars \textbf{=} 50 \selectlanguage{greek}μ\selectlanguage{english}m. Values are expressed as means \selectlanguage{ngerman}± SEM.
*p \textless{} 0.05 and **p \textless{} 0.01, significantly different,
ns, not significant.

\textbf{Figure 7. ISX9 promotes the expression of hair regrowth-related
markers in C57BL/6J mice.} The vehicle control, 1\% ISX9, or 2\%
minoxidil were topically applied daily to the dorsal skin for 25 days
after depilation. (n = 5 per group). (A) The expression of the indicated
genes, LEF1, Axin2, Fibronectin, and Survivin in each treated mouse was
quantified by real-time PCR analysis on day 25. (B) The expression of
the indicated gene, LGR5, Twist1, SOX2 and OCT4 in each treatment group
was measured using real-time PCR analysis on day 25. (C) Immunoblotting
analyses for phosphorylated LRP6, total LRP6, CK1\selectlanguage{greek}ε, \selectlanguage{english}phosphorylated
GSK3\selectlanguage{greek}β, \selectlanguage{english}total GSK3\selectlanguage{greek}β, \selectlanguage{english}active \selectlanguage{greek}β-\selectlanguage{english}catenin, total \selectlanguage{greek}β-\selectlanguage{english}catenin, LEF1, Axin2,
Survivin, LGR5, SOX2, and Keratin17 on day 25. Values are expressed as
means \selectlanguage{ngerman}± SEM. *p \textless{} 0.05 and **p \textless{} 0.01, significantly
different; ns, not significant.

\textbf{SUPPLEMENTAL FIGURES}

\selectlanguage{greek}\textbf{Figure S1. Treatment with ISX9 does not affect the mRNA
expression of β-catenin in HEK293T and HaCat cells.}\selectlanguage{english} (A) HEK293T and (B)
HaCat cells were treated with the indicated concentrations of ISX9 for
24 h. The relative mRNA level of \selectlanguage{greek}β-\selectlanguage{english}catenin was measured by real-time
PCR. The data from three independent experiments are presented.

\selectlanguage{greek}\textbf{Figure S2. ISX9 inhibits the interaction of Axin1 with
β-catenin.}\selectlanguage{english} HEK293T cells were incubated with the indicated
concentrations of ISX9 for 24 h. Cell lysates were extracted and
subjected to immunoprecipitation (IP) using anti-\selectlanguage{greek}β-\selectlanguage{english}catenin antibody
followed by Western blotting using specific antibodies as indicated.

\textbf{SUPPLEMENTARY INFORMATION}

Supplementary Information includes two figures and 2 tables\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-7/Figure-7}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-S1/Figure-S1}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-S2/Figure-S2}

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\end{figure}

\textbf{Hosted file}

\verb`Supplemertary table-.docx`  available at \url{https://authorea.com/users/503180/articles/582965-isx9-a-small-molecule-targeting-axin-activates-wnt-%CE%B2-catenin-signaling-and-promotes-hair-regrowth}

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