Epidermal Growth Factor Induces Proliferation of Hair Follicle-Derived Mesenchymal Stem Cells Through Epidermal Growth Factor Receptor-Mediated Activation of ERK and AKT Signaling Pathways Associated with Upregulation of Cyclin D1 and Downregulation of p16

Abstract

The maintenance of highly proliferative capacity and full differentiation potential is a necessary step in the initiation of stem cell-based regenerative medicine. Our recent study showed that epidermal growth factor (EGF) significantly enhanced hair follicle-derived mesenchymal stem cell (HF-MSC) proliferation while maintaining the multilineage differentiation potentials. However, the underlying mechanism remains unclear. Herein, we investigated the role of EGF in HF-MSC proliferation. HF-MSCs were isolated and cultured with or without EGF. Immunofluorescence staining, flow cytometry, cytochemistry, and western blotting were used to assess proliferation, cell signaling pathways related to the EGF receptor (EGFR), and cell cycle progression. HF-MSCs exhibited surface markers of mesenchymal stem cells and displayed trilineage differentiation potentials toward adipocytes, chondrocytes, and osteoblasts. EGF significantly increased HF-MSC proliferation as well as EGFR, ERK1/2, and AKT phosphorylation (p-EGFR, p-ERK1/2, and p-AKT) in a time- and dose-dependent manner, but not STAT3 phosphorylation. EGFR inhibitor (AG1478), PI3K-AKT inhibitor (LY294002), ERK inhibitor (U0126), and STAT3 inhibitor (STA-21) significantly blocked EGF-induced HF-MSC proliferation. Moreover, AG1478, LY294002, and U0126 significantly decreased p-EGFR, p-AKT, and p-ERK1/2 expression. EGF shifted HF-MSCs at the G1 phase to the S and G2 phase. Concomitantly, cyclinD1, phosphorylated Rb, and E2F1expression increased, while that of p16 decreased. In conclusion, EGF induces HF-MSC proliferation through the EGFR/ERK and AKT pathways, but not through STAT-3. The G1/S transition was stimulated by upregulation of cyclinD1 and inhibition of p16 expression.

Introduction

Hair follicle is one of the appendages of the human skin, derived from the interaction between epidermal and mesenchymal compartments during embryogenesis [1]. Accumulating studies show that hair follicle contains multiple stem cell sources, including keratinocytes, melanocyte stem cells, mesenchymal stem cells, and neural crest stem cells. These cells synergistically interact, subsequently regulating hair follicle development and functionality and maintaining hair follicle self-renewal and cycles, which consist of growth, regression, and rest phases (anagen, catagen, and telogen) throughout the adult life in a temporal and spatial manner [2 –6]. In addition, these cells can be sequentially isolated, highly expanded, and used as cell sources in stem cell-based regenerative medicine [7,8].

Among these cell sources, hair follicle-derived mesenchymal stem cells (HF-MSCs) not only express surface markers of mesenchymal stem cells but also display multiple lineage differentiation potentials toward adipocytes, osteoblasts, chondrocytes, hematopoietic cells, smooth muscle cells, neuron cells, and cardiac muscle cells under the appropriate induction, holding promises in stem cell-based regenerative medicine [9 –12]. Indeed, HF-MSCs demonstrated tremendous capacities in the reconstitution of the hematopoietic system, in situ blood vessels, and neuronal system repair and regeneration as well as engineering of small-diameter functional vascular grafts by using smooth muscle cells derived from HF-MSCs as cell sources [9,13 –15]. Transgenic HF-MSCs overexpressing the release-controlled insulin gene were generated and exhibited remarkable functionality in reversing hyperglycemia and decreasing the death rate of streptozotocin-induced diabetic mice [16]. The aforementioned properties of HF-MSCs in regenerative medicine, together with intrinsic properties of easily accessible rich source of autologous stem cells, endowed HF-MSCs with significant potentials in stem cell-based regenerative medicine.

Self-renewal and multilineage differentiation potential are necessary features for stem cells to function properly in stem cell-based regenerative medicine. Our and other research groups demonstrated that basic fibroblast growth factor (bFGF) plays a critical role in maintaining self-renewal and multilineage differentiation potential of mesenchymal stem cells [17 –19]. We recently determined that epidermal growth factor (EGF) not only significantly promotes proliferation of HF-MSCs but also dramatically maintains the multilineage differentiation potentials of the cells [19]. However, the molecular mechanism underlying this phenomenon remains unclear. Stem cell treatment relies on a number of highly proliferative and multipotent seed cells. Thus, understanding the mechanisms by which growth factors induce the proliferation of MSCs is of vital importance.

By binding to the EGF receptor (EGFR), EGF phosphorylates EGFR, activating downstream signaling pathways and subsequently affecting the cell's biological behavior [20,21]. Although the major downstream signaling pathways of EGF-EGFR are known and include the ERK, PI3K-AKT, and JAK/STAT signaling pathways [22,23], these signaling pathways are cell specific and the pathway by which EGF affects HF-MSCs has not been reported [24 –27].

Activation of downstream pathways of EGFR by EGF leads to cell cycle transition from G1 to S phase. According to the classical cell cycle model, G1-phase cyclins bind to and activate relevant cyclin-dependent kinases (CDKs), which leads to the phosphorylation of retinoblastoma protein (Rb), resulting in the release of E2F, a transcription factor, which regulates the expression of genes encoding proteins necessary for the G1 to S transition [28,29]. There are also two classes of CDK inhibitory proteins (CDKIs) that negatively regulate the CDK activation, including the inhibitors of CDK4 (INK4) family, consisting of p15, p16, p18, and p19, and the CDK interacting protein/kinase inhibitory protein (CIP/KIP) family, consisting of p21, p27, and p57. CDKIs bind to free CDKs or cyclin/CDK complexes, inhibiting their activities and preventing the phosphorylation of Rb, leading to the inhibition of cell cycle progression [30,31]. In this study, we attempted to elucidate the mechanistic role of EGF/EGFR and its downstream molecules in G1/S progression of HF-MSCs. These findings would benefit the development of HF-MSC therapeutics.

Materials and Methods

Isolation and culture of HF-MSCs

All protocols of human tissue handling were approved by the Ethics Committee of Basic College of Medicine, Jilin University. The isolation of HF-MSCs was performed as described previously [16,19]. Briefly, at least 20 hairs with complete hair follicles in the anagen phase were physically plucked from the occipital region of the scalps of two volunteers (a 27-year-old female and a 49-year-old male). Hairs were intensively rinsed with phosphate-buffered saline (PBS) containing 1% penicillin/streptomycin (P/S) solution (100 IU/mL penicillin, 100 IU/mL streptomycin; HyClone, Victoria, Australia) three times. After rinsing, hair shafts were cut off and hair follicles were transferred onto the bottom of a 24-well plate (Corning, Tewksbury, MA), one follicle per well, and maintained in Dulbecco's modified Eagle's medium: nutrient mixture F-12 (DMEM/F-12; Life Technologies, Madison, WI) supplemented with 10% fetal bovine serum (FBS; Hyclone), 10 ng/mL bFGF (PeproTech, London, UK) in a 37°C/5% CO₂ incubator. The medium was replaced every 3 days. Five to 10 days later, fibroblast-like cells migrated out of the dermal sheath or papilla. When the fibroblast-like cells proliferated to confluency in the well of a 24-well plate, they were passaged and passages 5–7 cells were used in the following experiments.

Differentiation experiments

Multilineage differentiation potential of HF-MSCs was analyzed as described previously [7,32]. Briefly, for adipogenic differentiation, HF-MSCs were induced in high-glucose DMEM (HG-DMEM; Life Technologies) containing 10% FBS (Hyclone),1 mM dexamethasone, 0.5 mM isobutylmethylxanthine, 10 mM insulin, and 200 mM indomethacin (all four from Sigma-Aldrich, St Louis, MO). After incubation for 14 days, the cells were stained with Oil red O (Sigma-Aldrich) to evaluate the formation of intracellular lipid droplets.

For osteogenic differentiation, cells were induced in HG-DMEM, 10% FBS, 0.1 mM dexamethasone, 50 mM ascorbate-2-phosphate, and 10 nM β-glycerophosphate (last four from Sigma-Aldrich). After 4 weeks of culture, Alizarin red S (Sigma-Aldrich) staining was performed to observe the formation of mineralized nodules.

For chondrogenic differentiation, 20 μL of a suspension of 8 × 10⁶ HF-MSCs/mL was allowed to form a sphere by hanging-drop culture and spheres were cultured in HG-DMEM, 10% FBS, 6.25 μg/mL insulin, 10 ng/mL transforming growth factor-beta 1 (PeproTech), and 50 nM of ascorbate-2-phosphate (Sigma-Aldrich) for 3 weeks. After induction, spheres were fixed in 10% buffered formaldehyde, embedded in paraffin, sectioned at 5 μm, and stained with toluidine blue (Dingguo, Beijing, China).

Immunofluorescence staining

Fibroblast-like cells from hair follicle were seeded on coverslips and cultured in DMEM/F-12 supplemented with 10% FBS. When cells grew to 70% confluence on the coverslips, they were fixed with 4% paraformaldehyde for 15 min at room temperature, blocked with 1% bovine serum albumin (BSA; Roche Diagnostics, Mannheim, Germany), and incubated with primary mouse/rabbit anti-human antibodies against CD90, CD105, CD31 (all from eBioscience, San Diego, CA), CD44 (R&D Systems, Abingdon, UK), CD73 (Life Technologies), and EGFR (Cell Signaling Technology, Beverly, MA) at a dilution of 1:100 at 4°C overnight, followed by Alexa Fluor^® 488-conjungated goat anti-mouse/rabbit antibody (1:500 dilution; Cell Signaling Technology) at room temperature for 1 h in the dark. After staining with Hoechst 33342 (Life Technologies) for 5 min to visualize the nuclei, cells were imaged by fluorescence microscopy (Olympus, Tokyo, Japan).

Flow cytometry assays

Cultured cells were collected by centrifugation and aliquoted into Eppendorf tubes, 0.5–1 × 10⁶ cells per tube. Cells were fixed with paraformaldehyde, blocked with BSA, incubated with antibodies as described for the immunofluorescence staining, and subjected to flow cytometry (FACSCalibur flow cytometer; BD Biosciences, San Jose, CA) to assess the expression of CD markers.

Inhibitors

The EGFR inhibitor, AG1478 (Sigma-Aldrich), is a specific EGFR tyrosine kinase inhibitor [33]. The EKR1/2 inhibitor, U0126 (Promega, Madison, WI), is a chemically synthesized organic compound that inhibits the activation of ERK 1/2 by inhibiting the kinase activity of MAP kinase kinase (MAPKK or MEK 1/2) [34]. The PI3K-AKT inhibitor, LY294002 (Promega), is a potent and specific cell-permeable inhibitor of PI3K, which inhibits ATP binding to the catalytic subunit of PI3K and blocks PI3K-dependent Akt phosphorylation and kinase activity [35 –37]. The STAT3 inhibitor, STA-21 (Santa Cruz Biotechnology, Santa Cruz, CA), is a small molecule that blocks STAT3 signaling by impeding STAT3 DNA binding activity, STAT3 dimerization, and STAT3-dependent luciferase activity [38].

Cell proliferation assays

HF-MSCs were first trypsinized into single-cell suspension, seeded into 24-well plates (4,000 cells per well), and cultured in basic culture medium (DMEM/F-12, 10% FBS) overnight. For the time-course experiment in the presence of EGF, cells were cultured in basic culture medium with or without EGF (10 ng/mL) for 1–6 days. For the EGF dose-dependence experiment, HF-MSCs were cultured in basic culture medium supplemented with EGF at concentrations ranging from 1 to 50 ng/mL for 72 h. For the dose-dependence experiment using inhibitors of the EGFR signaling pathway, cells were pretreated with basic culture medium containing AG1478 (0–5 μM), U0126 (0–50 μM), LY294002 (0–50 μM), or STA-21 (0–50 μM) for 1 h, and then, EGF (10 ng/mL) was added into the medium and the cells were further cultured for 72 h. After culture, cells were harvested from each well by trypsinization and stained with trypan blue. The total number of living cells per well was calculated using a hemocytometer.

Cell cycle assays

HF-MSCs were seeded on 6-cm culture dish and cultured in DMEM/F-12 containing 10% FBS without any growth factors overnight. The next day, inhibitors together with EGF (10 ng/mL) were added to the culture medium and cells were cultured for 24 h. After culture, cells were collected by trypsinization and centrifugation, washed twice with cold PBS, and fixed with 70% ice-cold ethanol at 4°C overnight. After three washes in PBS, the cells were incubated with RNaseA (Beyotime, Shanghai, China) at 37°C for 30 min and then stained with propidium iodide (Dingguo) for 30 min at 4°C in the dark. After incubation, cell cycle progression of HF-MSCs was analyzed by flow cytometry and the cell proliferation index (PI) was calculated using the formula: PI = (S + G2/M)/(G0/1 + S + G2/M) × 100%.

Western blot analysis

HF-MSCs were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Beyotime) containing 1 mM PMSF (Beyotime) and 1% protein phosphatase inhibitor mixture (Solarbio, Beijing, China) for 30 min on ice and then centrifuged at 16,000g for 10 min at 4°C to obtain total proteins. Equal amounts of protein samples were separated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis gel and electrotransferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Then, the membranes were blocked in 5% nonfat milk for 1 h at room temperature, followed by incubation with the specific primary antibodies overnight. After three washes with TBST buffer, the membranes were incubated with goat anti-rabbit/mouse IgG-HRP secondary antibody (dilution 1:3,000; Sanjian, Beijing, China) for 1 h at room temperature. Membranes were incubated with ECL reagent (Beyotime), and proteins were visualized using GENE GNOME (Gene Company Ltd., Hong Kong, China). The gray-scale intensities of the results were analyzed by Gene Tools software. The primary antibodies used for western blotting are listed in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/scd).

Statistical analysis

SPSS version17.0 (SPSS, Chicago, IL) was used for statistical analysis and all quantitative data are presented as mean ± standard deviation. All data are from at least three independent experiments. Comparisons between two groups were tested by Student's t-test. Multiple group comparisons were tested using one-way analysis of variance. P < 0.05 was considered statistically significant.

Results

Isolation and characteristics of human HF-MSCs

The isolated hair follicles adhered to the 24-well plate and cells migrated out of the hair follicle dermal sheath or papilla in 7–10 days, exhibiting typical fibroblast-like shape in morphology (Fig. 1A). Under adipogenic, chondrogenic, and osteogenic culture conditions, these fibroblast-like cells differentiated into adipocytes, chondrocytes, and osteoblasts, respectively, as shown by the presence of intracellular lipid droplets (Oil red O staining; Fig. 1B), cartilage formation (Toluidine blue staining; Fig. 1C), and calcium nodule formation (Alizarin red staining; Fig. 1D). Immunofluorescence staining and flow cytometry assay showed that these fibroblast-like cells expressed surface markers of mesenchymal stem cells. They were positive for CD73, CD44, CD90, and CD105, but negative for CD31 (Fig. 1E, F). As these hair follicle-derived fibroblast-like cells exhibited surface markers of mesenchymal stem cells and display trilineage differentiation potentials toward adipocytes, chondrocytes, and osteoblasts, they were defined as HF-MSCs.

FIG. 1.

Isolation and characterization of HF-MSCs. (A) Cells, which exhibited fibroblast-like morphology, migrated and spread out from the hair follicle. (B–D) Multipotent differentiation potential of the fibroblast-like cells. After culture under adipogenic, osteogenic, and chondrogenic conditions, Oil red O, Alizarin red, and Toluidine blue were used to evaluate adipogenesis (B), osteogenesis (C), and chondrogenesis (D), respectively. (E, F) Immunofluorescence and flow cytometry assays were used to measure the expression of various cell surface markers on fibroblast-like cells. (G, H) Immunofluorescence staining and flow cytometry assays to assess the expression of EGFR on HF-MSCs. Cells stained without primary antibodies served as a negative control. Hoechst 33342 was used to track nuclear localization. Scale bars: 40 μm (A), 20 μm (B, C, E, G), and 100 μm (D). EGFR, epidermal growth factor receptor; HF-MSCs, hair follicle-derived mesenchymal stem cells.

EGF stimulates proliferation and activates EGFR signaling in HF-MSCs

Immunofluorescence staining and flow cytometry assay were performed to investigate the EGFR expression levels in HF-MSCs. EGFR-specific signal was detected in cultured human HF-MSCs by immunofluorescence (Fig. 1G). Flow cytometry analysis further demonstrated that more than 98% of HF-MSCs expressed EGFR (Fig. 1H). To investigate the effect of EGF on the proliferation of HF-MSCs, HF-MSCs were treated with EGF (10 ng/mL) for 1–6 days. HF-MSCs did not show any significant increase in cell numbers during the first two days of EGF treatment compared with untreated HF-MSCs. However, from day 3 on, HF-MSCs treated with EGF grew significantly compared to nontreated cells as shown by a higher cell number (Fig. 2A). To determine whether the effect of EGF on cell proliferation was dose dependent, HF-MSCs were treated with various concentrations of EGF for 72 h. No dose-dependent relationship between HF-MSC proliferation and EGF was detected. However, EGF at the range of 1–50 ng/mL significantly enhanced the proliferation of HF-MSCs (Fig. 2B).

FIG. 2.

Effect of EGF on the proliferation of human HF-MSCs. (A) Cell proliferation assays were performed to determine the promoting effects of EGF (10 ng/mL) on the proliferation of HF-MSCs for 1–6 days of treatment. (B) EGF increased the proliferation of HF-MSCs in a dose-dependent manner after 72 h of treatment. (C, D) Western blot analysis of EGFR phosphorylation in HF-MSCs exposed to EGF (10 ng/mL) at different time points or various concentrations of EGF for 5 min. GAPDH was used as an endogenous control for equal loading. The bar graphs represent p-EGFR levels, which were normalized to GAPDH. *P < 0.05, **P < 0.01, ***P < 0.001. EGF, epidermal growth factor.

To investigate whether EGF could induce EGFR phosphorylation in HF-MSCs and to determine which time point was the most appropriate to assess EGFR phosphorylation, HF-MSCs were incubated with EGF (10 ng/mL) at different time points from 0 to 6 h and EGFR phosphorylation was examined by western blotting. EGFR phosphorylation was at its maximum at 5 min after incubation with EGF (10 ng/mL), then decreased with time, reaching basal level 30 min post-treatment with EGF (Fig. 2C). Based on this result, HF-MSCs were incubated with EGF at 0, 1, 5, 10, 20, and 50 ng/mL in the culture medium for 5 min. Western blot data showed that EGF upregulated EGFR phosphorylation levels in a dose-dependent manner (Fig. 2D). Since 10 ng/mL of EGF was sufficient to induce the proliferation of HF-MSCs and upregulated EGFR phosphorylation, this EGF concentration was used in the following experiments.

Inhibition of the EGFR signaling pathway reduces the EGF-induced proliferation of HF-MSCs

To test whether the effect of EGF on the proliferation of HF-MSCs depends upon EGFR-mediated signaling, HF-MSCs were treated with AG1478, an inhibitor for EGFR, and the proliferation of HF-MSCs was assessed. As expected, AG1478 treatment significantly reduced the proliferation of HF-MSCs in a dose-dependent manner (Fig. 3A). To further dissect the effects of the downstream signaling molecules, ERK1/2, PI3K, and STAT3, on the proliferation of HF-MSCs, HF-MSCs were treated with various doses of U0126, an inhibitor of ERK1/2; LY294002, an inhibitor for PI3K; or STA-21, an inhibitor for STAT3. As expected, U0126, LY294002, and STA-21 significantly reduced EGF-induced proliferation of HF-MSCs (Fig. 3B–D). Based on these data, AG1478 at 2 μM, U0126 at 10μM, LY294002 at 10 μM, and STA-21 at 20 μM were chosen in the following experiments.

FIG. 3.

Different inhibitors of the EGFR signaling pathway attenuated the EGFR-mediated proliferation of HF-MSCs. (A–D) Dose-dependent inhibition effects of EGFR inhibitor, AG1478, ERK1/2 inhibitor, U0126, PI3K inhibitor, LY294002, or STAT3 inhibitor, STA-21, on EGF-induced proliferation of HF-MSCs. EGF: 10 ng/mL. **P < 0.01, ***P < 0.001.

EGF induces the phosphorylation of ERK1/2 and AKT

The above results showed that both EGFR inhibitor and inhibitors of EGFR downstream signaling molecules could block EGF-induced proliferation of HF-MSCs, indicating that EGF might promote the proliferation of HF-MSCs through EGFR-mediated ERK1/2, PI3K, and STAT3 signaling pathways. To assess this hypothesis, HF-MSCs were incubated with EGF (10 ng/mL) for different time points and phosphorylation of ERK1/2, AKT, and STAT3 was examined by western blotting. In accordance with the time-course of EGFR phosphorylation (Fig. 2C), ERK1/2 and AKT phosphorylation peaked at 5 min, decreased with time, and was back to basal level 1 h after incubation with EGF (10 ng/mL). However, EGF (10 ng/mL) did not promote STAT3 phosphorylation at any time point within 6 h of incubation with EGF (Fig. 4A). In addition, HF-MSCs were pretreated with AG1478 (2 μM), U0126 (10 μM), or LY294002 (10 μM) for 1 h and then incubated with EGF (10 ng/mL) for 5 min. Western blot analysis indicated that the EGFR-inhibitor, AG1478, decreased EGF-induced phosphorylation of EGFR, ERK1/2, and AKT. The ERK1/2-inhibitor, U0126, and PI3K-inhibitor, LY294002, abolished the phosphorylation of ERK1/2 and AKT induced by EGF, respectively, without affecting the levels of EGFR phosphorylation (Fig. 4B), suggesting that EGF induced the proliferation of HF-MSCs through the EGFR/ERK and AKT pathways, but not STAT3 signaling.

FIG. 4.

Phosphorylation of ERK1/2, AKT, and STAT3 induced by EGF in HF-MSCs. (A) Phosphorylation of EGFR downstream molecules, ERK1/2, AKT, and STAT3, at different time points in HF-MSCs. (B) EGF-induced upregulation of ERK1/2 and AKT phosphorylation was inhibited by AG1478 (2 μM). HF-MSCs were pretreated with AG1478 (2 μM), U0126 (10 μM), or LY294002 (10 μM) for 1 h and then incubated with EGF (10 ng/mL) for 5 min. The bar graphs represent the phosphorylation levels of different molecules, which were normalized to GAPDH.*P < 0.05, **P < 0.01, ***P < 0.001.

Effect of EGF on the cell cycle phase distribution of HF-MSCs

Since cell proliferation is regulated by the cell cycle, we next determined whether EGF-induced HF-MSC proliferation involved cell cycle changes. We tested the cell cycle phase distribution by flow cytometry in HF-MSCs before and after treatment with EGF (10 ng/mL) and EGFR inhibitors (AG1478: 2 μM, U0126: 10 μM, LY294002: 10 μM, and STA-21: 20 μM). As shown in Fig. 5A, EGF treatment of HF-MSCs for 24 h induced the entry of the cells into S and G2/M phases of the cell cycle, whereas inhibitors of EGFR and its downstream molecules (2 μM AG1478, 10 μM U0126, 10 μM LY294002, and 20 μM STA-21) significantly prevented HF-MSCs from entering the S and G2/M phases. For the cell cycle analysis, the cell PI was calculated using the formula: PI = (S + G2/M)/(G0/1 + S + G2/M) × 100%. Results showed that the cell PI increased markedly in cells treated with EGF (10 ng/mL) compared with that of untreated cells. Moreover, in accordance with the cell cycle phase distribution, inhibitors of EGFR signaling (2 μM AG1478, 10 μM U0126, 10 μM LY294002, and 20 μM STA-21) significantly prevented this effect (Fig. 5B).

FIG. 5.

Effect of EGF on the cell cycle phase distribution of HF-MSCs. HF-MSCs were treated with AG1478 (2 μM), U0126 (10 μM), or LY294002 (10 μM) and stimulated with EGF (10 ng/mL) for 24 h. (A) Distribution percentages of cells in G0/G1, S, and G2/M phases of the cell cycle in control, EGF only, and EGF with inhibitors of the EGFR signaling pathway-treated cells. (B) Cell proliferation index of control, EGF only, and EGF with inhibitors of the EGFR signaling pathway-treated cells. **P < 0.01, ***P < 0.001.

Effect of EGF on the expression of G1/S transition regulatory proteins in HF-MSCs

Since G1/S transition is necessary for cell cycle control, to elucidate the mechanistic role of EGF in G1/S progression, we examined the effect of EGF on the expression of G1/S transition regulatory proteins in HF-MSCs, including cyclins (cyclin D1 and cyclin E1), cyclin-dependent protein kinases (CDK2, CDK4, and CDK6), cyclin kinase inhibitors (p16, p18, p19, p21, and p27), phosphorylation of Rb, and E2F1. As shown in Fig. 6A, HF-MSCs were treated with EGF (10 ng/mL) for 0, 8, and 24 h, and western blot results demonstrated that EGF (10 ng/mL) upregulated cyclin D1, Rb, phosphorylation of Rb, and E2F1 expression, whereas it downregulated p16 expression at 24 h. There was no change in the expression levels of other G1/S transition regulatory proteins mentioned above between the treated and untreated cells at 8 and 24 h (Fig. 6B). Therefore, the time point of 24 h was chosen to further dissect the effects of EGFR and ERK1/2, AKT, and STAT3 on G1/S transition regulatory proteins in HF-MSCs by western blotting.

FIG. 6.

Effect of EGF on the expression of G1/S transition regulatory proteins in HF-MSCs. (A) EGF (10 ng/mL) upregulated cyclin D1, Rb, phosphorylation of Rb, and E2F1 expression, while it downregulated p16 expression at 24 h. (B) Expression levels of other G1/S transition regulatory proteins were not changed after EGF treatment for 8 or 24 h.

Inhibitors of EGFR signaling (2 μM AG1478, 10 μM U0126, 10 μM LY294002, and 20 μM STA-21) were added to the culture medium with EGF (10 ng/mL) and western blot results showed that EGF-induced upregulation of cyclin D1, Rb, phosphorylation of Rb, and E2F1 was inhibited by AG1478, U0126, LY294002, and STA-21. However, EGF-induced downregulation of p16 was only inhibited by AG1478 and U0126. P27 and p21 expression was not affected by EGF in the presence of AG1478, U0126, and LY294002 in HF-MSCs (Supplementary Figure S1). These results indicated that EGF upregulated cyclin D1, Rb, phosphorylation of Rb, and E2F1 expression through the EGFR/ERK and PI3K-AKT signaling pathways, but downregulated p16 by activating the EGFR/ERK signaling pathway. STAT3 inhibitor downregulated the EGF-induced expression of cyclin D1, Rb, phosphorylation of Rb, and E2F1 (Fig. 7). However, since EGF could not induce the phosphorylation of STAT3 (Fig. 4A), these changes were not mediated by the EGFR signaling pathway.

FIG. 7.

Effects of EGFR and inhibitors of its downstream molecules on G1/S transition regulatory proteins in HF-MSCs. The bar graphs represent the data after normalization to GAPDH. *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

In this study, we dissected the signaling pathways and key molecules involved in the EGF-induced proliferation of HF-MSCs. We confirmed that EGF induced the proliferation of HF-MSCs through the EGFR/ERK and AKT pathways, but not through STAT-3. ERK and AKT activation by EGF/EGFR signaling induced G1-S cell cycle progression of HF-MSCs by upregulating cyclinD1 expression and inhibiting p16 expression.

Mesenchymal stem cells isolated from hair follicles were fibroblastic in appearance, expressing CD73, CD44, CD90, and CD105, and were capable of adipogenic, osteogenic, and chondrogenic differentiation. Thus, HF-MSCs share similar morphology, cell-surface markers, and differentiation potential with bone marrow mesenchymal stem cells (BM-MSCs) [39]. In addition, HF-MSCs expressed EGFR, which is also expressed in BM-MSCs [25,40].

EGF, a ligand of EGFR, promotes proliferation of various cell types, including epithelial, stromal, endothelial cells, and even some stem cells. For example, EGF-induced proliferation was observed in the keratinocyte cell line, HaCaT, epidermal neural crest stem cells, and human BM-MSCs, as well as in the mouse neural precursor cells [24,25,41,42]. Our results indicated that 1–50 ng/mL EGF could induce the proliferation of HF-MSCs, but dose dependency was not detected when the cells were cultured with EGF for 72 h. This may be because the maximum stimulation of HF-MSC proliferation was achieved with 1 ng/mL EGF in 72 h under our culture conditions, and increasing the dose of EGF would not affect the proliferative effect, which is consistent with results obtained in BM-MSCs showing that changing the concentration of EGF from 10 nM to 100 nM (from 1.6 to 16 ng/mL) did not increase the proliferation of BM-MSCs [43].

Expression of EGFR and activation of the EGF/EGFR downstream signaling showed species and tissue specificity. EGF could promote the proliferation of immortalized human BM-MSCs. However, it was not observed in rat BM-MSCs. It may be due to the low level of EGFR in rat BM-MSCs. EGF induced the phosphorylation of ERK and AKT, but weakly induced the phosphorylation of EGFR in rat BM-MSCs [43]. In contrast, in human BM-MSCs and mouse neural precursor cells, EGFR, ERK, and AKT were strongly phosphorylated [24], which was consistent with our observations in HF-MSCs. STAT3, which is also considered as a downstream molecular target of EGF to promote cellular proliferation, could be phosphorylated and activated by EGF in rat spermatogonial stem cells and Madin–Darby canine kidney cells [26,27]. However, our data showed that it was not in HF-MSCs. A report showed that STAT3 was not activated in both human and rat BM-MSCs treated with EGF, which is in agreement with our results [43].

Our cell cycle assay results further demonstrated that EGF/EGFR downstream signaling positively regulated the proliferation of HF-MSCs. Since G1/S transition is essential for cell cycle progression, we determined which proteins related to G1/S transition were involved in EGF-induced proliferation of HF-MSCs. Our results indicated that EGF/EGFR signaling regulated the levels of cyclinD1 and p16 to control the phosphorylation of Rb, so as to facilitate cell cycle progression through E2F-related transcriptional mechanisms in HF-MSCs. Similar to mouse embryonic stem cells and primary external auditory canal keratinocytes, ERK and PI3K/AKT pathways were involved in EGF-induced increase of cyclinD1 expression in HF-MSCs [44,45]. Likewise, in keratinocytes, metalloproteinase mediates the EGFR/ERK/AKT/cyclinD1 pathways and G1-S cell cycle progression induced by UVB radiation [46]. P16, a CDK inhibitor, plays a significant role in the inhibition of DNA synthesis stimulated by HGF or EGF in primary cultured rat hepatocytes [47]. Our study also indicated that EGF/EGFR signaling induced the downregulation of p16. However, these data are not consistent with a previous study, which showed that MEK/ERK signaling induced cell cycle arrest through accumulation of p16/19 in mouse hepatoblasts, a type of somatic progenitor cells in the fetal liver [48].

Interestingly, STA-21, an inhibitor of STAT3, reduced EGF-mediated cell proliferation and downregulated EGF-induced expression of proteins related to G1/S transition. However, as EGF could not induce STAT3 phosphorylation, these changes may be independent of the EGFR signaling pathway. The exact mechanism needs further study.

Our study presents some limitations. In fact, our results were obtained by in vitro culture. Since HF-MSCs are located within the dermal papilla and dermal sheath, further study could be performed to assess the function of EGFR signaling on HF-MSCs in vivo.

In conclusion, our results indicate that EGFR is functionally expressed in HF-MSCs. Exogenous EGF could induce the proliferation of HF-MSCs through EGFR-mediated activation of ERK and AKT, but not STAT3 signaling. EGFR signaling in HF-MSCs promotes cell cycle progression, which is associated with upregulation of cyclin D1 and downregulation of p16. This mechanism will facilitate large-scale in vitro expansion of HF-MSCs, which is required for initiation of stem cell-based therapy.

Footnotes

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (81573067), the Frontier Interdiscipline Program of Norman Bethune Health Science Center of Jilin University (2013101007), and the Specialized Research Fund for the Doctoral Program of Higher Education (20130061110077).

Author Disclosure Statement

No competing financial interests exist.

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