hDPCs were purchased from Cefobio (Seoul, Korea) as primary cells and were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen/Gibco-BRL, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS; Invitrogen/Gibco-BRL) and 1% penicillin in a humidified environment. Human follicular DPCs in the third or fourth passage were used.

Nuclear and cytoplasmic proteins were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit following the detailed instructions provided by the manufacturer (Pierce Biotechnology, Rockford, IL, USA). Protease inhibitor tablets (Roche Diagnostics, Basel, Switzerland) and the phosphatase inhibitors, 10 mM sodium pyrophosphate and 100 µM sodium orthovanadate, were added to cytoplasmic extraction reagent I (CERI; Pierce Biotechnology) and nuclear extraction reagent (NER; Pierce Biotechnology) prior to use.

hDPCs were prepared using lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% deoxycholic acid] containing a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA). Protein was quantified using a Bio-Rad DC protein assay kit II (Bio-Rad Laboratories, Hercules, CA, USA), separated on 10% sodium dodecyl sulfate SDS-PAGE gel, and electrotransferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in Tris-buffered saline containing 0.5% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA), the blots were probed with antibodies against β-catenin (Cat. no. 610154; BD Transduction Laboratories, Lexington, KY, USA) and p-AKT (Cat. no. 3787), AKT (Cat. no. 9272), dephosphorylated β-catenin (Cat. no. 8814), p-GSK-3β (Cat. no. 9322), GSK-3β (Cat. no. 9315), p-ERK (Cat. no. 9101), ERK (Cat. no. 4695) and actin (Cat. no. 4968; all from Cell Signaling Technology, Danvers, MA, USA) and then exposed to horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies. GAPDH (Cat. no. 5174) and lamin B1 (Cat. no. 9087; both from Cell Signaling Technology) were taken as cytoplasm and nuclear extract control. Protein expression was detected using an enhanced chemiluminescence (ECL) system (Amersham Pharmacia, Piscataway, NJ, USA). Images of the blotted membranes were obtained using a Lumino Image Analyzer (LAS-1000; Fujifilm, Tokyo, Japan). The protein levels were compared to a loading control (actin or non-phosphorylated protein).

BrdU incorporation was quantified using the BrdU Cell Proliferation Assay kit (Cell Signaling Technology) according to the manufacturer's instructions. hDPCs were seeded at a density of 3×10³ cells/wells in a 96-well plate. Briefly, the cells were examined for BrdU incorporation at 48 h. For the pathway determination, the following inhibitors were used: LY294002 (a specific inhibitor of phosphatiylinositol 3-kinase (PI3K), 440206; Calbiochem, San Diego, CA, USA), BIO (a specific inhibitor of GSK-3β, B1686; Sigma-Aldrich), PD98059 (a specific inhibitor of extracellular signal-regulated kinase (ERK) 1/2), 513000; Calbiochem) for 30 min and then were treated with the MXD or HPE plus MXD. At the designated time points, BrdU (10 µM) was added to each well. After 4 h, the medium was replaced with fixing/denaturing solution, and the cells were incubated with anti-BrdU antibody for 1 h. The incorporated BrdU was determined by measuring the HRP-conjugated antibody to BrdU and 3,3′,5,5′-tetrameth-ylbenzidine (TMB) substrates. The absorbance was read at 450 nm using a SpectraMax 340 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The results are expressed as the percentage inhibition of BrdU incorporation compared to the untreated control. Values are expressed as the means ± SD of 3 separate experiments, each performed in triplicate.

ALP activity was observed using an ALP Live Stain kit (Invitrogen Life Technologies, Carlsbad CA, USA) according to the manufacturer's instructions. Briefly, the cells were washed twice in fresh medium, and an appropriate amount of 1X ALP Live Stain solution was then directly applied to the cell culture, after which the cells were incubated at 37°C for 30 min. The cells were then rinsed 3–4 times with fresh medium and observed under an Olympus BX51 fluorescence microscope (Olympus, Melville, NY, USA) using a standard FITC filter.

The DPCs at 1.0×10⁴ cells/500 µl per chamber were seeded into a chamber slide, serum-starved for 24 h, and then treated with HPE, MXD or HPE plus MXD for 48 h. Following treatment with 4% paraformaldehyde for 10 min and 0.1% Triton X-100 for 5 min, the cultured hDPCs were incubated with anti-β-catenin antibody (1:500, Cat. no. 610154; BD Transduction Laboratories) at 4°C overnight and then with FITC-labeled goat anti-mouse IgG (1:1,000, Cat. no. NB720-F; Novus Biologicals, Littleton, CO, USA). A 4′6-diamidino-2-phenylindole (DAPI) mounting medium kit (Golden Bridge International, Inc., Mukilteo, WA, USA) was used to counterstain the nuclei. The immunostained cells were mounted in medium containing DAPI and visualized using an Olympus FLUOVIEW FV10i confocal microscope (Olympus Optical Co., Ltd., Tokyo, Japan).

Total RNA was purified using the RNeasy Plus Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. A total of 1 µg of DNase-treated total RNA was used for first-strand cDNA. This reaction was performed using a random primer and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen Life Technologies). The primers used for RT-PCR were as follows: Sonic hedgehog (SHH) sense, 5′-ACC GAG GGC TGG GAC GAA GA-3′ and antisense, 5′-ATT TGG CCG CCA CCG AGT T-3′; transforming growth factor (TGF)−β1 sense, 5′-AAA TTG AGG GCT TTC GCC TTA-3′ and antisense, 5′-GAA CCC GTT GAT GTC CAC TTG-3′; TGF-β2 sense, 5′-TCC AAA GAT TTA ACA TCT CCA ACC-3′ and antisense, 5′-CAT GCT CCA GCA CAG AAG TTC G-3′; ALP sense, 5′-TGG AGC TTC AGA AGC TCA ACA CCA-3′ and antisense, 5′-ATC TCG TTG TCT GAC TAC CAG TCC-3′; and versican sense, 5′-GAC GAC TGT CTT GGT GG-3′ and antisense, 5′-ATA TCC AAA CAA GCC TG-3′. The PCR cycling conditions were as follows: 30–35 cycles at 94°C for 2–10 min, 94°C for 30 sec to 3 min, 50–58°C for 30 sec to 1 min, 72°C for 30 sec to 1 min, and 72°C for 4–7 min. The PCR products were run on 0.75–1.5% agarose gels. Images of the gels were analyzed using Quantity One software (Bio-Rad Laboratories).

All procedures involving animals were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Chung-Ang University in Korea (institutional review board no. 14–0058). The isolation of rat vibrissa follicles was performed as previously described (21). Briefly, rat vibrissa follicles were harvested from 23-day-old male Wistar rats. The rats were sacrificed using carbon dioxide (CO₂). Subsequently, both the left and right mystacial pads were removed from the rats and placed in a 1:1 (v/v) solution of Earle's balanced salts solution (EBSS; Sigma-Aldrich). Anagen vibrissa follicles were then dissected using a stereomicroscope (Olympus) from the posterior sections of the mystacial pads, with considerable care being taken to remove the surrounding connective tissue without damaging the vibrissa follicle. Using this method, we were able to isolate <30 follicles from each animal. The isolated follicles were then placed in separate wells in 12-well plates that contained 1 ml Williams' medium E supplemented with 2 mM L-glutamine (both from Gibco-BRL), 10 µg/ml insulin (Sigma-Aldrich), 50 nM hydrocortisone (Sigma-Aldrich), 100 unit/ml penicillin and 100 µg/ml streptomycin at 37°C and cultivated in an atmosphere comprised of 5% CO₂ and 95% air. The isolated follicles were then treated with the vehicle (Williams' medium E) as a control and 20% HPE. The hydrolysate of HPE (Laennec; GCJBP Corp., Yongin, Korea) was provided by GCJBP. MXD (Sigma-Aldrich) was used as a positive control in the culture systems. The culture medium was changed every 3 days, and photographs of the cultured rat vibrissae follicles were acquired using a stereomicroscope for 3 weeks. The lengths of the hair follicles were measured using a DP controller (Olympus).

Cryosections (7 µm) were cut from the rat vibrissae follicles embedded in Tissue-Tek (Miles Diagnostic Division, Elkhart, IN, USA) and frozen in isopentane cooled by liquid nitrogen. Prior to staining, the cryosections were fixed in acetone for 10 min at −20°C. Some sections were stained for immunohistochemical markers using monoclonal antibodies against anti-β-catenin (1:500, Cat. no. 610154; BD Transduction Laboratories), anti-TGF-β1 (1:100, Cat. no. ab92486; Abcam, Cambridge, MA, USA) and anti-TGF-β2 (1:30, Cat. no. ab66045; Abcam). Immunohistochemical analysis was performed using a high-temperature antigen unmasking technique. Briefly, the sections were heated in an unmasking solution (citrate buffer, pH 6.0), washed, and then incubated with primary monoclonal antibodies at room temperature for 1 h. This procedure was followed by incubation with secondary antibodies (EnVision Detection kit K5007; Dako, Glostrup, Denmark). Reaction products were developed using diaminobenzidine solution as a chromogen. After the chromogen reactions, the sections were rinsed and counterstained with hematoxylin. After rinsing, the sections were dehydrated and covered with permount (Thermo Fisher Scientific, Inc., Fair Lawn, NJ, USA). Counterstained sections were then examined by light microscopy using an Olympus BX40 microscope (Olympus America Inc., Melville, NY, USA) in order to assess histological changes. Two independent, blinded pathologists evaluated each section. Each pathologist assigned every section a score according to the following scale: 0, negative control; +, moderately increased staining; ++, considerably increased staining, based on the percentages of stained cells in each category.

Statistical analyses of the data were performed using the Student's t-test or multivariate analysis of variance (ANOVA). The results are expressed as the means ± standard deviation of at least 3 independent experiments, and a P-value <0.05 was considered to indicate a statistically significant difference.

To evaluate the effects of HPE and MXD on DPCs, we examined BrdU and ALP activation. The cells were treated with various concentrations of HPE (5, 10 and 20%), MXD (0.1, 0.5 and 1 µM), or HPE plus MXD for 48 h. The BrdU incorporation assay confirmed that hDPC proliferation was increased following treatment with HPE and MXD, with a significant increase observed following treatment with 20% HPE and 20% HPE plus 0.5 µM MXD (Fig. 1A). To directly examine the effects of HPE on cell proliferation, we measured ALP expression in the hDPCs, as ALP is a well-established DP marker (22). As shown in Fig. 1B, there was a marked increase in ALP expression at 72 h of culture, when the cells were treated with a combination of 10% HPE and 0.5 µM MXD compared to treatment with 10% HPE alone. These results suggest that HPE and MXD effectively influence ALP expression. We then investigated the changes in the expression of DP signature genes by performing RT-PCR on hDPCs treated with 5 and 10% HPE, 0.5 µM MXD, or HPE plus MXD for 30 min or 24 h. Notably, the SHH and versican expression levels were increased in a dose-dependent manner when the cells were treated with HPE (Fig. 1C). These results suggest that HPE, which upregulates the gene expression of SHH and versican in DPCs, targets proliferation effectors in hDPCs. Based on the collective results, we hypothesized that combined treatment with HPE and MXD significantly increased the proliferation of hDPCs.

The cells were treated with various concentrations of HPE (0, 1, 2, 5, 10 and 20%) for 48 h, and the expression of β-catenin, and GSK-3β was measured by western blot analysis. Treatment with HPE increased the total level of β-catenin protein in a dose-dependent manner. In particular, the levels of dephospho-β-catenin, an active form of β-catenin protein, were elevated. The p-GSK-3β (Ser⁹) levels began to markedly increase following treatment with 10% HPE, whereas the total GSK-3β protein levels did not increase (Fig. 2A and B). GSK-3β is inactivated by phosphorylation at Ser⁹; thus, these results suggested that the increase in the β-catenin protein levels in the hDPCs induced by HPE was due to the decreased destruction caused by GSK-3β inactivation. These results suggest that treatment with 10% HPE induces cell proliferation through the activation of β-catenin and the inactivation of GSK-3β in hDPCs. As shown in Fig. 2C, the whole cell or tissue lysates were separated into nuclear and cytoplasmic fractions, and western blot analysis was performed with anti-β-catenin antibody. We found that treatment with HPE induced the nuclear translocation of β-catenin, since an increasing amount of β-catenin was detected in the nuclear fraction 48 h after the cells were treated with HPE in a dose-dependent manner. These results suggest that HPE induces β-catenin and p-GSK-3β (Ser⁹) activation in hDPCs.

MXD induces the nuclear accumulation of β-catenin and increases the phosphorylation of GSK-3β (23). Thus, we examined the effects of HPE and MXD on proteins related to cell growth in the hDPCs by western blot analysis. Treatment with MXD (0.5 µM) plus HPE (10%) significantly increased the protein expression of GSK-3β and β-catenin compared with the cells treated with HPE or MXD alone for 12 h. The protein expression was increased more significantly following treatment with 0.5 µM MXD in combination with 10% HPE compared to treatment with MXD alone (Fig. 3A and B). Moreover, we observed that treatment with HPE plus MXD effectively promoted β-catenin and p-GSK-3β (Ser⁹) activation in a time-dependent manner (Fig. 3C and D). These results suggest that treatment with a combination of HPE and MXD is more effective than treatment with HPE or MXD alone.

To elucidate the mechanisms underlying the induction of β-catenin activation in the cells treated with HPE and HPE plus MXD, we performed immunocytochemistry on the hDPCs. The localization of β-catenin was examined after treating the hDPCs with HPE, MXD or HPE plus MXD for 24 h. β-catenin was mainly associated with the cell membrane and was weakly detected in the nuclei. In the HPE- or MXD-treated cells, the increased expression of β-catenin was noted. Treatment with HPE plus MXD induced the nuclear translocation of β-catenin in the hDPCs (Fig. 4). These results suggest that treatment with HPE plus MXD causes the significant induction of GSK-3β phosphorylation at the Ser⁹ residue. GSK-3β inactivation was synchronized with the accumulation of β-catenin after 48 h. Thus, the combination of HPE and MXD may be important for the crosstalk involved in β-catenin/GSK-3β signaling.

Growth factor signaling has previously been implicated in the regulation of the GSK-3β/β-catenin axis in hair morphogenesis and growth signaling (20). Recently, it was shown that Wnt/β-catenin signaling is involved in regulating the expression of extracellular-signal-regulated kinase (ERK) and phosphatidylinositol-3-kinase (PI3K)/AKT as well as in the proliferation of hDPCs (24,25). In this study, to elucidate the molecular mechanisms involved in the HPE-associated acceleration of MXD-induced hair growth in hDPCs, we examined the role of the ERK, PI3K/AKT and GSK-3β signaling pathway in the proliferation of these cells using the specific mitogen-activated protein kinase (MAPK) inhibitors, PD98059 (20 µM, for ERK1/2), LY294002 (20 µM, for PI3K) and 6-bromoindirubin-3′-oxime (BIO; 1.5 µM, for GSK-3β). As shown in Fig. 5A, DNA synthesis at 48 h increased by 1.3-fold in the cells treated with 0.5 µM MXD in combination with 10% HPE compared to those treated with HPE alone. Treatment with HPE significantly influenced proliferation through the induction of the GSK-3β pathway. We also observed a similar reduction in the GSK-3β protein expression levels at 48 h by western blot analysis (Fig. 3D). The effects of pre-treatment with each inhibitor were examined by western blot analysis in order to determine the concentrations required to significantly decrease HPE-induced dephospho-β-catenin and p-GSK-3β in these cells (Fig. 5B). Consistently, PD98059 and LY294002, but not BIO blocked the HPE-induced activation of dephospho-β-catenin. Soma et al (16), reported that Wnt/β-catenin activation results in the maintenance of the hair-inducing ability of hDPCs. Taken together, these results indicate that pGSK-3β activation plays a pivotal role in the HPE stimulation of MXD-induced hair growth.

To determine whether HPE stimulates MXD-induced hair fiber elongation, we examined the effects of HPE and MXD using an organ culture of the rat vibrissa follicle. The rat vibrissae follicles were treated with 20% HPE, 10 µM MXD or 10% HPE plus 5 µM MXD for 21 days (Fig. 6). The hair fiber lengths of the vibrissa follicles treated with 5 µM MXD plus 10% HPE (123.14±9.23) were similar to the lengths of the follicles treated with 10 µM MXD (119.78±8.33; Fig. 6A and B). As a result, no synergy was observed following treatment with 5 µM MXD plus 10% HPE compared to treatment with 10 µM MXD alone. Thus, it can be concluded that a lower concentration is required in combination treatment than in individual treatment in order to produce a similar effect. We then investigated the mechanisms through which HPE stimulates MXD-induced hair fiber elongation by measuring the expression levels of β-catenin, TGF-β1 and TGF-β2 in the cultured rat vibrissae follicles. After 21 days, the cultured vibrissa follicles, which were expected to be in the anagen-catagen transition phase, were positive for TGF-β2, whereas the vibrissae follicles in the bulb region treated with 20% HPE for 21 days were negative for TGF-β2. These results support the conclusion that treatment with HPE decreases the expression of TGF-β2, thereby preventing apoptosis in the bulb region (Fig. 6C). Collectively, these data demonstrate that HPE in combination with a low concentration of MXD exerts a positive effect on hair growth and delays catagen progression.