Melatonin increases growth properties in human dermal papilla spheroids by activating AKT/GSK3β/β-Catenin signaling pathway

Cell culture and reagents

Three to seven human dermal papilla (HDP) passage cells (PromoCell, Heidelberg, Germany) were grown in 5% CO₂ at 37 °C using a follicle DP cell growth medium kit (PromoCell). For experiments, cells were seeded in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% (v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Human epidermal keratinocyte (HEK) cells (CELLnTEC, Bern, Switzerland) were maintained in CnT-Prime Epithelial Culture Medium (CELLnTEC) in 5% CO₂ at 37 °C. Human dermal fibroblast (HDF) cells (Lonza, Basel, Switzerland) were maintained in DMEM (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin (Thermo Fisher Scientific). 293T cells (American Type Culture Collection, Manassas, VA, USA) were maintained in DMEM supplemented with 10% (v/v) FBS (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Antibodies against lamin A/C (sc-7293) and β-tubulin (sc-5274) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). MTNR1A (ab203038), MTNR1B (ab203346), bone morphogenetic protein 2 (BMP2; ab14933), versican (VCAN) (ab19345), and alkaline phosphatase (ALP) (ab108337) were purchased from Abcam (Cambridge, UK). Antibodies against protein kinase B (AKT) (#9272), phosphorylated AKT at serine 473 residue (#9271), phosphorylated AKT at threonine 308 residue (#9275), protein kinase A (PKA) (#4782), phosphorylated PKA at threonine 198 residue (#4781), extracellular signal-regulated kinase (ERK) (#9102), phosphorylated ERK at threonine 202/tyrosine 204 residues (#9101), p38 (#9212), phosphorylated p38 at threonine 180/tyrosine 182 residues (#9211), SRC (#2108), phosphorylated SRC at tyrosine 416 residue (#2101), glycogen synthase kinase 3β (GSK-3β) (#9315), phosphorylated GSK-3β at serine 9 residue (#9323), β-catenin (#9562S), phosphorylated β-catenin at serine 33/37/threonine 41 residues (#9561S), and WNT5a (#2392) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against β-actin were purchased from Sigma-Aldrich (Darmstadt, Germany USA). TCF/LEF luciferase reporter plasmid was purchased from Promega (Madison, WI, USA). The selective phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, melatonin, and the melatonin receptor antagonist luzindole were purchased from Sigma-Aldrich. In the present study, melatonin was prepared as a 500 mM stock solution with dimethyl sulfoxide (DMSO) solvent and was used at DMSO concentrations up to 0.2%.

Cell viability analysis

Cell viability analysis was performed using EZ-Cytox (ITSbio, Seoul, South Korea) and a water-soluble tetrazolium salt (WST-1) assay kit, according to the manufacturer’s protocol. Eighty thousand HDP cells were seeded in clear 96-well flat-bottom ultra-low attachment microplates (Corning, Corning, NY, USA) and further cultured for 24 h. The cells were treated with the indicated concentrations of melatonin (0, 0.1, 0.25, 0.5, 0.75, 1, 2.5, and 5 mM) and incubated for 48 h. The WST-1 solution was diluted to 10% in medium and subsequently added to each well. Cell viability was assessed by measuring absorbance at 450 nm using a Synergy™ HTX Multi-Mode Microplate Reader (Bioteck, Winooski, VT, USA).

3D spheroid culture of HDP cells

3D spheroid culture of HDP cells was performed as previously described (Choi et al., 2017; Lee et al., 2019). Fifty thousand HDP cells were seeded in clear 96-well round-bottom ultra-low attachment microplates (Corning, Glendale, AZ, USA) and maintained for 24 h to obtain one spherical cell structure. After incubation, spheroid cells were treated with the indicated concentrations of melatonin (0, 0.5, 0.75, and 1 mM) and further incubated for 48 and 96 h. The diameters of the spheroids were measured using phase-contrast microscopy.

Luciferase reporter assay

293T cells (1  × 10⁵ cells/well) were seeded and cultured for 24 h. The cells were then co-transfected with the TCF/LEF promoter-driven luciferase reporter plasmid and pSV- β-galactosidase (pSV-β-gal) plasmid using Lipofectamine reagent (Thermo Fisher Scientific). Four hours post-transfection, cells were treated with melatonin (0, 0.75, and 1 mM) alone or with LY294002 (Sigma-Aldrich). At 0, 12, and 24 h post-treatment, transfected cells were lysed using passive lysis buffer (Promega). To determine luminescence, the cell lysates were incubated with d-luciferin (Sigma-Aldrich) and luciferase activity was measured using a Synergy HTX Multi-Mode Microplate Reader (Bioteck). Beta-galactosidase activity was measured using the Luminescent β-galactosidase detection kit II (Takara Bio Inc., Tokyo, Japan). Relative luciferase activity was calculated by normalizing luciferase activity to β-galactosidase activity.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis

Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific), and cDNA was synthesized from 2 µg of the total RNA using oligo dT primers, 1.5 mM dNTPs, 0.1 M DTT, 5X First-Strand Buffer, and M-MLV reverse transcriptase (Thermo Fisher Scientific). qRT-PCR was carried out using a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) and detected using SYBR Green PCR Master Mix (Thermo Fisher Scientific). The expression levels of mRNA were quantified using the 2^−ΔΔCt method and normalized to the expression level of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene, an internally expressed reference gene. The following primers were used for the amplification of specific genes: WNT family member 5A (WNT5A), 5′-TTGAAGCCAATTCTTGGTGGTCGC-3′(forward) and 5′-TGGTCCTGATACAAGTGGCACAGT-3′(reverse); ALP, 5′-CAAACCGAGATACAAGCACTCCC-3′(forward) and 5′-CGAAGAGACCCAATAGGT AGTCCAC-3′(reverse); VCAN, 5′-GGCAATCTATTTACCAGGACCTGAT-3′(forward) and 5′-TGGCACACAGGTGCATACGT-3′(reverse); BMP2, 5′-GGAACGGACATTCGG TCCTT-3′(forward) and 5′-CACCATGGTCGACCTTTAGGA-3′(reverse); MTNR1A, 5′- GCCACAGTCTCAAGTACGACA-3′(forward) and 5′- CTGGAGAACCAGGATCCATAAT-3′(reverse); MTNR1B, 5′- TACGACCCACGCATCTATTCC- 3′(forward) and 5′- AGGTAGCAGAAGGACACGACA- 3′(reverse); and GAPDH, 5′-CGGAGTCAACGGATTT GGTCGTAT-3′(forward) and 5′-AGCCTTCTCCATGGTGAAGAC-3′(reverse).

Cell fractionation analysis

Total cell lysates were prepared and protein extracts were obtained after lysis in RIPA buffer (25 mM Tris-Cl, 5 mM ethylenediaminetetraacetic acid , 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.025% sodium dodecyl sulfate) containing 200 mM phenylmethylsulfonyl fluoride. Nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) were used for nuclear and cytoplasmic fractionation of total cell lysates according to the manufacturer’s protocol.

Statistical analysis

All statistical analyses were performed in triplicate. All data are presented as the mean ± standard deviation. Normally distributed data were analyzed using GraphPad Prism (v8.4) (GraphPad Software, San Diego, CA, USA). For the results, the p-value is from a one-way analysis of variance (ANOVA) followed by Turkey’s post hoc test or two-way ANOVA followed by Turkey’s post hoc test. Cell viability data were analyzed using a two-tailed Student’s t-test. Statistical significance was set at P < 0.05.

Differential expressions of melatonin receptors in human DP cells cultured in conventional 2D monolayer culture and 3D spheroid culture

We examined the expression of melatonin receptors in primary skin cells to study melatonin-induced intracellular signaling in skin cells, including HDP cells. We analyzed the expression levels of MTNR1A and MTNR1B in HEK, HDF, and HDP cells. As shown in Fig. 1A, the transcriptional expression levels of MTNR1A and MTNR1B in HDP cells were higher than that in HEK and HDF cells. Similarly, immunoblotting showed that protein expression was higher in HDP cells (Fig. 1B). We further compared the expression of melatonin receptors in HDP cells under conventional 2D monolayer culture and 3D sphere culture conditions, which mimics the actual in vivo environment. As shown in Fig. 1C, the transcriptional expression of melatonin receptors, especially MTNR1B, was higher in the 3D sphere culture condition than in the 2D monolayer culture condition. These results show that 3D sphere culture conditions upregulated the expression of melatonin receptor in HDP cells.

Melatonin increases the volume of 3D spheroid of human DP cells

As the expression of melatonin receptors was examined in 3D sphere-cultured HDP cells, we analyzed the effect of melatonin on the toxicity and proliferation of 3D sphere-cultured HDP cells using the WST-1 assay. Sphere-cultured HDP cells were treated with 0.1, 0.25, 0.5, 0.75, 1, 2.5, and 5 mM melatonin for 48 h, and cell viability was determined using the WST-1 assay. As shown in Fig. 2A, cell viability was not significantly affected by melatonin at concentrations up to 1 mM in sphere-cultured HDP cells. However, treatment with 2.5 and 5 mM melatonin significantly reduced HDP cell viability to 12.7% and 21.9%, respectively, suggesting that melatonin at a concentration of 1 mM or higher affected the viability of HDP cells (Fig. 2A). We then measured and compared the diameters of spheroids at 48 h and 96 h to analyze the effect of melatonin on spheroid proliferation (Fig. 2B). It has been reported that sphere formation of DP cells is closely related to their hair-inductive properties; DP sphere size is directly linked to hair shaft diameter (Morgan, 2014; Shimizu et al., 2011). Figures 2B and 2C show that after 96 h, the size of sphere-cultured HDP cells increased by 12.25%, 20.29%, and 20.86% after treatment with 0.5, 0.75, and 1 mM melatonin, respectively. These data suggest that melatonin has proliferation and formation abilities in the HDP spheroids.

Melatonin induced hair growth properties are mediated by melatonin receptor pathway

Several studies have indicated that DP signature genes, including ALP, VCAN, BMP2, and wingless-type MMTV integration site family member 5A (WNT5A), are important regulators of hair inductivity in vivo (Iida, Ihara & Matsuzaki, 2007; McElwee et al., 2003; Ohyama et al., 2010; Reddy et al., 2001; Rendl, Polak & Fuchs, 2008; Soma, Tajima & Kishimoto, 2005; Yang & Cotsarelis, 2010). Therefore, we examined whether melatonin affects the expression of DP signature genes. Sphere-cultured HDP cells were treated with 0.75, and 1 mM melatonin for 48 h, and the expression levels of DP signature genes were determined using qRT-PCR. As shown in Figs. 3A,3B,3C, and 3D, melatonin increased the mRNA expression of DP signature genes ALP, VCAN, BMP2, and WNT5A in a dose-dependent manner compared with the untreated control. Moreover, immunoblotting showed that the protein expression levels of these genes were increased with melatonin treatment (Fig. 3E). To determine whether the increase in the expression of DP signature genes by melatonin is due to melatonin receptor signaling, we investigated the effect of melatonin-induced expression of DP signature genes following treatment with the melatonin receptor antagonist luzindole. Sphere-cultured HDP were cotreated with 1 mM melatonin alone or 50 µM luzindole for 48 h and the expression of BMP2 and WNT5A was examined by qRT-PCR and immunoblotting. The expression of WNT5A and BMP2, which increased following melatonin treatment, decreased at the transcriptional (Figs. 3F and 3G) and protein levels (Fig. 3H) following cotreatment with luzindole. Collectively, these results suggest that melatonin has the potential to effectively upregulate hair-inductive properties by promoting the expression of DP signature genes that regulate melatonin receptor signaling in HDP spheroids.

Melatonin activates the WNT/β-catenin signaling pathway in 3D HDP spheroids

WNT/β-catenin signaling is a master regulator of hair-inductive properties of DP cells (Enshell-Seijffers et al., 2010; Kishimoto, Burgeson & Morgan, 2000; Lowry et al., 2005; Soma et al., 2012). Therefore, we investigated whether melatonin treatment activated WNT/β-catenin signaling in HDP spheroids. As shown in Fig. 4A, melatonin decreased the phosphorylation of β-catenin at serine 33/37 residues and increased the total protein level of β-catenin in a dose-dependent manner. Moreover, increase in β-catenin protein levels and TCF/LEF reporter activity was also observed when DP spheroids were treated with 10 nM and 10 µM melatonin for a longer duration (72 h), suggesting that the effects of melatonin on the growth of DP spheroids is not the result of high treatment concentration (1 mM) (Fig. S1). Cell fractionation showed that β-catenin translocation into the nucleus increased with melatonin treatment in HDP spheroids compared to that in DMSO-treated controls (Fig. 4B). Next, we analyzed whether TCF/LEF transcriptional activity was upregulated by melatonin treatment in 293T cells expressing melatonin receptors (Chan et al., 1997). Luminescence analysis showed that melatonin upregulated TCF/LEF-driven luciferase activity in a dose-and time-dependent manner (Fig. 4C).

AKT signaling mediated the activation of melatonin-induced GSKSβ/β-catenin signaling pathway in 3D HDP spheroids

GSK3β is a major regulator of the canonical WNT signaling pathway (Wu & Pan, 2010). Therefore, we investigated the effect of melatonin on kinase that regulate GSK3β. After HDP spheroids were treated with 1 mM melatonin for 24 h, protein kinases related to the GSK3β signaling pathway were analyzed by immunoblotting (Fig. 5A). Phosphorylation of PKA, ERK, p38, and SRC was not affected by melatonin treatment. However, melatonin treatment increased phosphorylation of AKT at serine 473 in a dose-dependent manner (Figs. 5A and 5B). Threonine 308 and serine 473 residue is present in the activation loop and C-terminal hydrophobic motif of AKT, respectively (Manning & Toker, 2017). Therefore, we further analyzed the phosphorylation of threonine 308 residue and found that both threonine 308 and serine 473 residues on AKT were phosphorylated following melatonin treatment (Fig. S2).

To investigate whether melatonin-mediated phosphorylation of AKT is associated with activation of AKT, we used a lentivirus-mediated fluorescent translocation reporter system (HeLa/FoxO1-Clover) composed of fluorescent Clover protein fused with a modified FoxO1 protein, a well-characterized substrate of AKT (Gross & Peter, 2015). Cells were treated with melatonin (0, 200, 400 µM, and 2 mM) under serum starvation conditions for 24 h to inhibit AKT. Consequentially, melatonin treatment induced the subcellular localization of FoxO1-Clover from the nucleus to the cytoplasm (Fig. S3). These results suggest that melatonin enhanced the activation of AKT signaling, followed by the induction of the inhibitory phosphorylation of GSK3β at the serine 9 residue.

We further investigated whether AKT activation by melatonin induces phosphorylation of GSK3β at the serine 9 residue and regulates the expression of β-catenin using the selective PI3K/AKT inhibitor LY294002. Fig. 5C shows that treatment with LY294002 restored phosphorylation of AKT at serine 473, phosphorylation of GSK3β at serine 9, and increased β-catenin levels in sphere-cultured HDP cells. Furthermore, we confirmed that LY294002 treatment abolished melatonin-induced TCF/LEF-driven luciferase activity (Fig. 5D). Collectively, these results suggest that melatonin-induced inhibition of GSK3β and increase in β-catenin levels are mediated through the activation of the AKT signaling pathway in sphere-cultured HDP cells (Fig. 6).