In vitro hair growth‐promoting effect of Lgr5‐binding octapeptide in human primary hair cells

Hair loss occurs due to various biological and environmental causes, which can have psychosocial consequences. The Wnt/β‐catenin signaling is well‐known for its role in hair growth and regeneration, as it induces the proliferation and differentiation of hair cells. When the leucine‐rich G protein‐coupled receptor 5 (Lgr5) interacts with the R‐spondins, the frizzled receptor (FZD), a Wnt receptor, becomes stabilized, resulting in an increased β‐catenin activity.

growth stimulation signaling is the canonical Wnt signaling.The receptors of secreted Wnt proteins are low-density lipoprotein-related protein 5/6 (LRP5/6) and its coreceptor, FZD.In the absence of Wnt, β-catenin is constitutively targeted for proteasomal degradation after being phosphorylated and ubiquitinated by the destruction complex consisting of glycogen synthase kinase-3β (GSK-3β), casein kinase-1 (CKI), adenomatous polyposis coli (APC), and axin.By contrast, β-catenin is released from the destruction complex when secreted Wnts bind to receptors.[19][20][21] R-spondin1-4 are proteins that have been shown to significantly increase Wnt/β-catenin signaling by binding to their receptors, leucine-rich G protein-coupled receptor 4/5/6 (Lgr4/5/6). 22,23When R-spondin1-4 binds to Lgr4/5/6, the zinc and ring finger 3 (ZNRF3)/ ring finger protein 43 (RNF43), which are E3 ubiquitin ligases that induce ubiquitination-proteasomal degradation of FZD, are internalized and results in the activation of Wnt signaling. 24,25ptides, chains of 2 to 50 amino acids, are actively developed in cosmetics and medicines due to their high bioavailability and production efficiency.Generally, active peptides are derived from natural proteins, synthetic libraries, or gene recombination libraries. 26,27 previously screened our library of synthetic peptides consisting of 3-13 amino acids using SPR system, Biacore™ to find peptides that have the binding ability to Lgr5, and we discovered the octapeptide.In this study, we confirmed the binding affinity of octapeptide to Lgr5 and observed an increase in the Wnt/β-catenin signaling following octapeptide treatment.Additionally, the promoting effects of octapeptide on cell proliferation and differentiation were confirmed in human primary hair cells, including HHFDPCs, HHFORSCs, HHFGMCs, and HHFSCs.

Conclusion:
We found that octapeptides activated the Wnt/β-catenin signaling and induced the proliferation and differentiation of human primary hair cells by acting as an exogenous ligand for Lgr5.In addition, octapeptides recovered inhibited hair regeneration characters by DHT in androgenetic alopecia-mimic in vitro model.These findings suggest that octapeptides may be a promising therapeutic option for treating hair loss.

| Peptide synthesis
Octapeptide was synthesized by the solid phase peptide synthesis (SPPS) method using 2-chlorotrityl chloride (2-CTC) resin (BeadTech).The detailed process is as follows.2-CTC resin (0.54 mmoL/g) was swelled in DMF for 15 min.Then, we added Fmoc-Val-OH (3 equivalents) and DIEA (2.5 equivalents) into the reactor and let the mixture react for 2 h at room temperature.After coupling reaction, the resin was washed with DMF.For removal of the protecting group, Fmoc, resin was reacted with deprotection solution (Piperidine: DMF = 2: 8) for 20 min.Subsequently, the following process was repeated according to the amino acid sequence in the direction of C terminal to N terminal (Table 1).
We added Fmoc-amino acid (2 equivalents), HBTU (2 equivalents), HOBT (2 equivalents), and DIEA (2.5 equivalents) into the reactor and let the mixture react for 2 h.The termination of the coupling step was confirmed with the Kaiser test, and the resin was washed with DMF.For removal of Fmoc, resin was reacted with deprotection solution for 20 min.
After the final synthesis was completed, the cleavage cocktail solution (trifluoroacetic acid: water: triisopropylsilane = 95: 2.5: 2.5) was added to separate the peptide from the resin.The peptide was precipitated with diethyl ether, and air-drying was performed to recover the peptide product.
The crude peptide was purified by C-815 MPLC system (BUCHI) with C18 column (YMC).For characterization of the purified peptide, the purity was analyzed by U-3000 HPLC system (Thermo Fisher Scientific) with C18 column (Agilent) and the molecular mass was analyzed by 3200 Q-trap LC-MS/MS system (AB SCIEX) with C18 column (Agilent) (Table 1, Figure 1).

| Surface plasmon resonance (SPR) analysis
The interaction between octapeptide and Lgr5 was measured by surface plasmon resonance (SPR) using Biacore T200 system (Cytiva).Human recombinant Lgr5-His protein was immobilized on the surface of a CM5 sensor chip at 1800 RU by using EDC/NHS amine coupling kit according to the manufacturer's instructions.
The octapeptide was injected into the flow cell at a rate of 30 μL/ min at 25°C with HBS-EP buffer as a running buffer.Data analysis was performed using Biacore T200 evaluation software ver.3.1 (Cytiva).

| Cell culture
We purchased HHFDPC, HHFORSC, and HHFGMC from Sciencell Research Laboratories, and the cells were incubated in MSCM media containing 5% heat-inactivated FBS and 1% penicillin/streptomycin at 37°C at 5% CO 2 .We purchased HHFSC from Celprogen and the cells were incubated in human hair follicle stem cell media at the same condition described above.

| Cell proliferation assay
HHFDPCs, HHFORSCs, HHFGMCs, and HHFSCs were seeded in a 96-well plate at a density of 2 × 10 3 cells/well.After overnight incubation they were then treated with various concentrations of octapeptide for 72 h.Formazan crystals formed by 0.4 mg/mL MTT treatment were dissolved with DMSO.Absorbance was measured at 540 nm using a microplate reader SpectraMax M2e (Molecular Devices).

| Gene expression analysis (RT-PCR)
For the gene expression analysis in HHFDPCs, HHFORSCs, and HHFGMCs, cells were seeded in a 6-well plate at a density of 4 × 10 5 cells/well.After overnight incubation they were then treated with various concentrations of octapeptide for 24 h in serum-free media.
For the gene expression analysis in HHFSCs, cells were seeded in a 6-well plate at a density of 3 × 10 5 cells/well.After overnight incubation they were then treated with various concentrations of octapeptide for 72 h.
We isolated RNA from harvested cells using an RNA extraction kit (Qiagen), and we performed cDNA synthesis and subsequent PCR using RT-PCR premix and PCR premix (iNtRON Biotechnology) each.We performed the processes using a gradient PCR machine (Mastercycler® nexus, Eppendorf), and the expression patterns were analyzed by agarose gel electrophoresis.

| Western blot analysis
For the analysis of β-catenin nuclear translocation, HHFDPCs were seeded in a 6-well plate at a density of 4 × 10 5 cells/well.After overnight incubation they were then treated with various concentrations of octapeptide for 24 h in serum-free media.Nuclear and cytoplasmic proteins were isolated with Nuclear and Cytoplasmic Extraction Reagents (Thermo).ImageQuant™ 800 (Cytiva, USA).

| Wnts ELISA
HHFDPCs were seeded in a 6-well plate at a density of 4 × 10 5 cells/well.After overnight incubation they were then treated with various concentrations of octapeptide for 24 h in serum-free media.The released Wnt-5a and Wnt-10b proteins were detected with ELISA kit for each protein according to the manufacturer's instruction.

| HHFDPC spheroids (3D) culture
For HHFDPC spheroids culture, 2 × 10 5 cells/well were seeded in 24-well plate coated with 5% PVA and incubated for 72 h.They were then treated with various concentrations of octapeptide with or without 100 nM DHT for 72 h.For HHFDPC monolayer culture, 2 × 10 5 cells/well were seeded in 24-well plate and incubated for 6 days.After incubation, the cells were used for analysis of Wnts mRNA expression, and culture media were used for analysis of secreted Wnts protein and HHFSCs differentiation assay.
The released Wnt-5a and Wnt-10b proteins were detected with ELISA kit.

| HHFSCs differentiation assay using HHFDPC spheroids culture media
HHFSCs were seeded in a 6-well plate at a density of 3 × 10 5 cells/ well and incubated for 16 h.They were then treated with 70% of HHFDPC spheroids culture or monolayer culture media for 24 h.
After incubation, the cells were used for gene expression analysis of differentiation-related markers.

| Statistics
The experiments were repeated three times, and the data were expressed as mean ± standard deviation (SD).Statistical analysis was performed using Student's t-test, and we considered statistically significant when the p-value was less than 0.05 compared to the negative control (NC) (*p < 0.05, **p < 0.01, ***p < 0.001).

| Octapeptide bound to Lgr5
We confirmed octapeptide binding to Lgr5 through surface plasmon resonance (SPR) analysis using the Biacore T200 (GE Healthcare).
We obtained SPR sensorgrams from the injection of octapeptide, at six different concentrations (ranging from 15.625 to 500 μM), on the Lgr5-immobilized CM5 chip.As a result, although relatively rapid association and dissociation were observed, an increase in the response unit (RU), which is concentration-dependent, was observed (Figure 2A).To analyze the binding affinity, fitting the sensorgrams with the 1:1 binding model using the Biacore T200 evaluation software 3.1 was performed.The K D value was found to be 260.4± 20.3 μM (Figure 2B).

| Octapeptide induced HHFDPC proliferation through the Wnt/β -catenin signaling activation
To determine whether the octapeptide could activate the Wnt/βcatenin signal, HHFDPC, a critical cell that controls the periodic regeneration of HF, was treated with octapeptide, and cell proliferation was observed (Figure 3A).As a result, a concentrationdependent cell proliferation increase of up to 30% was confirmed.
Since activated β-catenin moves from the cytoplasm to the nucleus, we did Western blotting to see whether octapeptide promotes the nuclear translocation of β-catenin (Figure 3B).When treated with octapeptide, the β-catenin protein level showed a significant decrease in the cytoplasm in a concentration-dependent manner and a marked increase in the nucleus.Additionally, we observed whether octapeptide promoted the expression of β-catenin downstream genes.As a result, octapeptide increased the expression levels of β-catenin downstream genes such as LEF1, CCND1, and MYC by up to 3.4 times, 9.6 times, and 2 times, respectively (Figure 3C).To determine whether the octapeptide also has inducing effects on Wnts, we conducted the expression analysis for Wnt-5a and Wnt-10b.As a result, octapeptide increased the expression levels of WNT5A and WNT10B genes by up to 2.9 and 3.7 times, respectively (Figure 3D).The protein levels of Wnt-5a and Wnt-10b were also increased by octapeptide in a concentration-dependent manner (Figure 3E,F).The maximum increases of Wnt-5a and Wnt-10b expressions were 1.9 and 2.6 times higher than the control, respectively.

| Octapeptide induced the expression of hair growth-stimulating factors in HHFDPCs
9][30] We investigated whether octapeptide increased the expression of these genes, as the Wnt/β-catenin signal is known to induce the expression of growth factors 31,32 (Figure 4A).
As a result, it was confirmed that the FGF2, FGF7, HGF, and VEGFA gene expressions were markedly increased up to 5.6-fold, 1.6-fold, 1.8-fold, and 4.8-fold, respectively, by octapeptide treatment.To determine whether the HHFDPC proliferation-inducing effect of octapeptide also affected from these growth factors, we confirmed the phosphorylation levels of Akt and ERK, which are representative proliferation-related signaling molecules (Figure 4B).Octapeptide treatment resulted in a 4.4-fold increase in phosphorylated Akt (p-Akt) levels and a 1.6-fold increase in phosphorylated ERK (p-ERK) levels, as measured by the ratios of p-Akt/total Akt (t-Akt) and p-ERK/total ERK (t-ERK), respectively.

| Octapeptide activated other human hair cells, including HHFORSCs and HHFGMCs
4][35] Thus, we conducted the proliferation assay and analyzed the transcriptional

| Octapeptide induced the proliferation and differentiation of HHFSCs
7][38] We examined proliferation and differentiation of HHFSCs to confirm whether the octapeptide affects the activity of HHFSCs.
Octapeptide increased the proliferation of HHFSCs with up to a 2.3-fold in a concentration-dependent manner (Figure 6A).To examine its effect on differentiation, we measured mRNA expression levels of stem cell markers (e.g., CD34, PROM1, KRT15, SOX9, and LHX2 genes) after octapeptide treatment (Figure 6B).In case of CD34 and PROM1, significant reductions were observed for all groups treated with octapeptide compared to the control group.
The mRNA expression levels of other genes, including KRT15, SOX9, and LHX2 were also significantly decresed by 100 μM octapeptide.In the same treatment group, mRNA expression levels of differentiation markers, including ACTA2, SNX1, CORIN, and VIM genes, were observed (Figure 6C).We confirmed that expressions of ACTA2, SNX1, CORIN, and VIM genes were significantly increased by octapeptide treatment, up to 30-fold, 1.7-fold, 20-fold, and 1.7-fold, respectively.In addition, protein expression levels of stem cell markers and differentiation markers were examined.
As a result, protein expression levels of stem cell markers, including CD34, CD133, and Keratin 15, showed a significant decrease, as seen from the changes in mRNA expression levels (Figure 6D).
Protein expression levels of differentiation markers, such as α-SMA, Versican, and K6hf, increased up to 2.3-fold, 3.6-fold, and 2.3-fold, respectively (Figure 6E).leading to abrupt changes in the relevant transcriptional profiles. 30,39,40To overcome these limitations relating to the 2D culture model, the DPC spheroids (3D) culture model is being studied, as it restores hair-inducing properties by creating an in vivo tissue microenvironment. 40,41In this 3D culture model, the octapeptide increased the expression of Wnt-5a and Wnt-10b, similar to the results in the 2D culture model shown in Figure 3D-F (Figure S1A-C).

| Octapeptide overcame androgen inhibition in
To examine whether octapeptide restores the DPC activity which is inhibited by DHT in spheroids (3D) culture model, and thereby improves the differentiation of HHFSCs, we performed the experiment according to the study of Leirós GJ et al. with a few modifications. 42As a result, WNT5A and WNT10B mRNA expression levels increased more in 3D culture conditions than in 2D culture conditions.These mRNA expression levels decreased significantly
Wnt/β-catenin signaling is then activated, increasing cell proliferation and the expression of differentiation-related factors, resulting in induction of HF regeneration. 26,27,46evious studies have reported the hair growth-promoting effects of Lgr5/R-spondins signaling.When R-spondin1 was treated in cultured bulge stem cells, Wnt/β-catenin signaling was activated, and precocious anagen entry was observed when exogenous R-spondin1 was intradermally injected into the dorsal skin of midtelogen phase mice. 47Additionally, R-spondin2/3 promoted the proliferation of hair follicle dermal stem cells (hfDSCs) and epithelial progenitors in vitro and induced precocious anagen via intradermal injection into the skin of midtelogen phase mice in vivo. 48A different study reported that, in mice injected with R-spondin2 intradermally, the onset of catagen phase was delayed and the anagen phase was prolonged, increasing overall hair shaft length and thickness. 49sides, the watercress extract fraction increased the expression of endogenous R-spondin1, inducing hair shaft elongation in ex vivo tests using human hair follicles, and increased hair thickness and density during a 6-month clinical trial in 44 male subjects with androgenetic alopecia (AGA). 50 this study, we found that cell proliferation was promoted via inducing β-catenin signaling activation and expression of GFs when the octapeptide, binds to Lgr5, was treated to HHFDPC.
It was also confirmed that the octapeptide promoted the pro- Based on our in vitro test results, in vivo studies will be conducted to see whether the octapeptide, similar to R-spondins that are endogenous ligands of Lgr5, can promote hair regeneration by inducing anagen phase entry while inhibiting hair retardation by inhibiting the anagen-catagen transition.
In conclusion, we developed an octapeptide that acts as an exogenous ligand for Lgr5 to promote the Wnt/β-catenin signal.

For
the analysis of Akt and Erk phosphorylation, HHFDPCs were seeded in a 6-well plate at a density of 3 × 10 5 cells/well.After overnight incubation they were then treated with various concentrations of octapeptide for 30 min in serum-free media.For the analysis of HHFSCs differentiation, cells were seeded in a 6-well plate at a density of 3 × 10 5 cells/well.After overnight incubation they were then treated with various concentrations of octapeptide for 72 h.The cell lysates were separated by 8% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes.Membranes were blocked in 5% w/v skim milk prepared with phosphate buffered saline containing 0.5% Tween-20, and probed with each primary antibodies overnight at 4°C.The appropriate secondary antibodies were incubated with the membranes for 1 h at room temperature, and the protein bands were detected via a Western detection reagent (Elpis Biotech) with Amersham™

F I G U R E 1 F I G U R E 3
Characterization of synthesized octapeptide used in this study.(A) The purity of octapeptide was analyzed by high performance liquid chromatography (HPLC) system.(B) The molecular weight of octapeptide was analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS).The peak at m/z of 1056.4 (marked with arrowhead) that corresponds to octapeptide.F I G U R E 2 SPR analysis of the binding between LGR5 and octapeptide.(A) The sensorgrams of the binding curves (colored lines) and the global fitted curves (black lines) in a 1:1 binding model for octapeptide binding to LGR5-immobilized chip.The concentrations of octapeptide were 15.63, 31.25,62.5, 125, 250, and 500 μM (from bottom to top).(B) Steady state fitting of the sensorgrams.level of differentiation-related factors to examine whether octapeptide activates both cells.As a result, the octapeptide significantly increased the proliferation of HHFORSCs and HHFGMCs up to 28% and 44% each (Figure 5A,B).It was confirmed that the expression of differentiation markers of HHFORSCs (i.e., KRT5, KRT14, KRT19, and KRT33B genes) resulted in marked increases of up to Effects of the octapeptide on proliferation and β-catenin activation in HHFDPCs.(A) HHFDPCs were treated with the indicated concentration of octapeptide for 72 h.Cell proliferation was determined by MTT assay.(B) HHFDPCs were treated with the indicated concentration of octapeptide for 24 h.The β-catenin protein levels in cytoplasm and nucleus were determined by Western blotting.HHFDPCs were treated with the indicated concentration of octapeptide for 24 h.(C) The mRNA expression levels of β-catenin downstream target genes were determined by RT-PCR.LEF1: Lef-1, CCND1: Cyclin D1, MYC: c-Myc.(D) The mRNA expression levels of Wnts were determined by RT-PCR.WNT5A: Wnt-5a, WNT10B: Wnt-10b.(E) The Wnt-5a level and (F) the Wnt-10b level included in the culture media obtained under the same conditions as above were measured by ELISA.1.3-fold,10.9-fold, 2-fold, and 24.7-fold, respectively, by octapeptide treatment (Figure5C).Additionally, the octapeptide treatment in HHFGMCs was found to significantly increase the transcription factors involved in hair shaft differentiation, that is, MSX2, FOXN1, and HOXC13 expressions, by up to 5.3-fold, 3-fold, and 3.2-fold, respectively (Figure5D).

F I G U R E 4
HHFDPCs-mediated HHFSCs differentiation DPC exhibits an aggregative behavior, and this characteristic induces new hair follicle formation.According to previous studies, when DPCs isolated from HF are cultured in a standard monolayer (2D), the hair follicle-inductive capacity is impeded significantly, Effects of the octapeptide on the expression of GFs and phosphorylation of Akt and Erk in HHFDPCs.(A) HHFDPCs were treated with the indicated concentration of octapeptide for 24 h.The mRNA expression levels of GFs were determined by RT-PCR.FGF2: bFGF, FGF7: KGF, HGF: HGF, VEGFA: VEGF-A.(B) HHFDPCs were treated with the indicated concentration of octapeptide for 30 min.The phosphorylation levels of Akt and Erk were determined by Western blotting.

F I G U R E 5 F I G U R E 6
Figure7A.The maximum increases of Wnt-5a and Wnt-10b expressions were 2.6 and 2.2 times higher than the negative control, respectively.After confirming that octapeptide can restore the decreased levels of Wnts by DHT treatment in DPCs, the same DPC culture media from the above experiment was treated with HHFSCs to examine its effect on stem cell differentiation.As a result, mRNA expression levels of stem cell markers (e.g., CD34, PROM1, and KRT15) were decreased as differentiation was induced by the DPC 3D culture media treatment, which was reversed upon treatment with DPC 3D culture media containing DHT.This increase turned into a decrease after being treated with DHT and octapeptide-containing DPC 3D culture media (Figure7D).In addition, mRNA expression levels of differentiation markers (e.g., ACTA2, CORIN, KRT75, VCAN, and VIM) were significantly increased when treated with DPC 3D culture media, which was reversed upon treatment with DPC 3D culture media containing DHT.In the group treated with DPC 3D culture media containing DHT and octapeptide, the mRNA expression levels of ACTA2, CORIN, KRT75, VCAN, and VIM were significantly increased up to 2.2-fold, 4.7fold, 2-fold, 2.4-fold, and 1.5-fold, respectively than the negative control (Figure7E).
liferation and differentiation of other human primary hair cells, including HHFORSCs and HHFGMCs.Additionally, when octapeptide was treated to HHFSCs, which are known for Lgr5 expression and involvement in hair regeneration, the proliferation was increased in a dose-dependent manner.Upon octapeptide treatment in HHFSCs, the expressions of stem cell markers (e.g., CD34, CD133, Keratin 15, SOX9, and LHX2) were decreased, and the expressions of differentiation markers (e.g., α-SMA, Nexin-1, Corin, Vimentin, Versican, and Keratin 75) were increased.This means that octapeptide can have a differentiation-promoting effect on HHFSCs, which are initially present in the bulge region and move down during anagen entry and differentiate into hair cells.Subsequently, we conducted 3D culture of HHFDPCs with aggregative growth characteristics to mimic the in vivo microenvironment of cultured cells in vitro.We found that octapeptide induced the Wnts expression which was decreased by DHT in HHFDPCs spheroids, as well as restored the differentiation suppressed by HHFDPCs 3D culture media containing DHT in HHFSCs.We speculate that these results were caused by two mechanisms.First, it appears that octapeptide acted directly on Lgr5-expressing HHFSCs, restored the Wnt/β-catenin signaling that was inhibited by a canonical Wnt/β-catenin inhibitor, DKK-1, which was induced by DHT.Second, by increasing Wnts secretion in HHFDPCs, octapeptide indirectly restored the Wnt/β-catenin signal in HHFSCs.
Octapeptide has been shown to significantly induce proliferation and differentiation in human primary hair cells, such as HHFDPCs, HHFORSCs, HHFGMCs, and HHFSCs.It also showed restorative effects on the suppressed differentiation of HHFSCs in DHT-treated condition.Therefore, it is expected that the octapeptide of this study can be used as a material for promoting hair growth and suppressing androgenic hair retardation.AUTH O R CO NTR I B UTIO N S E.J. Lee, Y.J. Chung, and E.M. Kim were responsible for the conception and design of the study.E.J. Lee, M.W. Kim, and H. Gil performed the experiments.E.J. Lee and E.M. Kim analyzed research data.E.J. Lee wrote the original draft.E.J. Lee and E.M. Kim were responsible for the review and revision of the manuscript.