Restricted accessResearch articleFirst published online 2017-01
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
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.
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References
1.
SennettR and RendlM. (2012). Mesenchymal-epithelial interactions during hair follicle morphogenesis and cycling. Semin Cell Dev Biol, 23:917–927.
2.
RompolasP and GrecoV. (2014). Stem cell dynamics in the hair follicle niche. Semin Cell Dev Biol, 25–26:34–42.
3.
TrempusCS, MorrisRJ, BortnerCD, CotsarelisG, FairclothRS, ReeceJM and TennantRW. (2003). Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol, 120:501–511.
4.
Nishikawa-TorikaiS, OsawaM and NishikawaS. (2011). Functional characterization of melanocyte stem cells in hair follicles. J Invest Dermatol, 131:2358–2367.
5.
BajpaiVK, MistriotisP and AndreadisST. (2012). Clonal multipotency and effect of long-term in vitro expansion on differentiation potential of human hair follicle derived mesenchymal stem cells. Stem Cell Res, 8:74–84.
6.
YuH, KumarSM, KossenkovAV, ShoweL and XuX. (2010). Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles. J Invest Dermatol, 130:1227–1236.
7.
LiP, LiuF, WuC, JiangW, ZhaoG, LiuL, BaiT, WangL, JiangY, et al. (2015). Feasibility of human hair follicle-derived mesenchymal stem cells/CultiSpher®-G constructs in regenerative medicine. Cell Tissue Res, 362:69–86.
8.
LiuJY, PengHF and AndreadisST. (2008). Contractile smooth muscle cells derived from hair-follicle stem cells. Cardiovasc Res, 79:24–33.
9.
LiuJY, PengHF, GopinathS, TianJ and AndreadisST. (2010). Derivation of functional smooth muscle cells from multipotent human hair follicle mesenchymal stem cells. Tissue Eng Part A, 16:2553–2564.
10.
LiuZ, LuSJ, LuY, TanX, ZhangX, YangM, ZhangF, LiY and QuanC. (2015). Transdifferentiation of human hair follicle mesenchymal stem cells into red blood cells by OCT4. Stem Cells Int, 2015:389628.
11.
AmohY, LiL, HamadaY, KatsuokaK and HoffmanRM. (2005). Multipotent nestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons. Proc Natl Acad Sci U S A, 102:5530–5534.
12.
YashiroM, MiiS, AkiR, HamadaY, ArakawaN, KawaharaK, HoffmanRM and AmohY. (2015). From hair to heart: nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells differentiate to beating cardiac muscle cells. Cell Cycle, 14:2362–2366.
GaoY, LiuF, ZhangL, SuX, LiuJY and LiY. (2014). Acellular blood vessels combined human hair follicle mesenchymal stem cells for engineering of functional arterial grafts. Ann Biomed Eng, 42:2177–2189.
15.
AmohY, LiL, KatsuokaK and HoffmanRM. (2008). Multipotent hair follicle stem cells promote repair of spinal cord injury and recovery of walking function. Cell Cycle, 7:1865–1869.
16.
WuC, LiuF, LiP, ZhaoG, LanS, JiangW, MengX, TianL, LiG, LiY and LiuJY. (2015). Engineered hair follicle mesenchymal stem cells overexpressing controlled-release insulin reverse hyperglycemia in mice with type L diabetes. Cell Transplant, 24:891–907.
17.
LaiWT, KrishnappaV and PhinneyDG. (2011). Fibroblast growth factor 2 (Fgf2) inhibits differentiation of mesenchymal stem cells by inducing Twist2 and Spry4, blocking extracellular regulated kinase activation, and altering Fgf receptor expression levels. Stem Cells, 29:1102–1111.
18.
DombrowskiC, HelledieT, LingL, GrunertM, CanningCA, JonesCM, HuiJH, NurcombeV, van WijnenAJ and CoolSM. (2013). FGFR1 signaling stimulates proliferation of human mesenchymal stem cells by inhibiting the cyclin-dependent kinase inhibitors p21(Waf1) and p27(Kip1). Stem Cells, 31:2724–2736.
19.
ZhangX, WangY, GaoY, LiuX, BaiT, LiM, LiL, ChiG, XuH, et al. (2013). Maintenance of high proliferation and multipotent potential of human hair follicle-derived mesenchymal stem cells by growth factors. Int J Mol Med, 31:913–921.
FergusonKM. (2008). Structure-based view of epidermal growth factor receptor regulation. Annu Rev Biophys, 37:353–373.
22.
OdaK, MatsuokaY, FunahashiA and KitanoH. (2005). A comprehensive pathway map of epidermal growth factor receptor signaling. Mol Syst Biol, 1:2005.0010.
23.
KnudsenSL, MacAS, HenriksenL, van DeursB and GrovdalLM. (2014). EGFR signaling patterns are regulated by its different ligands. Growth Factors, 32:155–163.
24.
HuQ, ZhangL, WenJ, WangS, LiM, FengR, YangX and LiL. (2010). The EGF receptor-sox2-EGF receptor feedback loop positively regulates the self-renewal of neural precursor cells. Stem Cells, 28:279–286.
25.
TamamaK, FanVH, GriffithLG, BlairHC and WellsA. (2006). Epidermal growth factor as a candidate for ex vivo expansion of bone marrow-derived mesenchymal stem cells. Stem Cells, 24:686–695.
26.
ChenJX, XuLL, WangXC, QinHY and WangJL. (2011). Involvement of c-Src/STAT3 signal in EGF-induced proliferation of rat spermatogonial stem cells. Mol Cell Biochem, 358:67–73.
27.
Garcia-HernandezV, Flores-MaldonadoC, Rincon-HerediaR, Verdejo-TorresO, Bonilla-DelgadoJ, Meneses-MoralesI, GariglioP and ContrerasRG. (2015). EGF regulates claudin-2 and-4 expression through Src and STAT3 in MDCK cells. J Cell Physiol, 230:105–115.
28.
PardeeAB. (1989). G1 events and regulation of cell proliferation. Science, 246:603–608.
29.
BrackenAP, CiroM, CocitoA and HelinK. (2004). E2F target genes: unraveling the biology. Trends Biochem Sci, 29:409–417.
30.
SherrCJ and RobertsJM. (1999). CDK inhibitors: positive and negative regulators of G(1)-phase progression. Genes Dev, 13:1501–1512.
31.
PavletichNP. (1999). Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors. J Mol Biol, 287:821–828.
32.
ZhaoG, LiuF, LanS, LiP, WangL, KouJ, QiX, FanR, HaoD, et al. (2015). Large-scale expansion of Wharton's jelly-derived mesenchymal stem cells on gelatin microbeads, with retention of self-renewal and multipotency characteristics and the capacity for enhancing skin wound healing. Stem Cell Res Ther, 6:38.
33.
LevitzkiA and GazitA. (1995). Tyrosine kinase inhibition: an approach to drug development. Science, 267:1782–1788.
34.
FavataMF, HoriuchiKY, ManosEJ, DaulerioAJ, StradleyDA, FeeserWS, Van DykDE, PittsWJ, EarlRA, et al. (1998). Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem, 273:18623–18632.
35.
VlahosCJ, MatterWF, HuiKY and BrownRF. (1994). A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem, 269:5241–5248.
36.
LiuD, SiH, ReynoldsKA, ZhenW, JiaZ and DillonJS. (2007). Dehydroepiandrosterone protects vascular endothelial cells against apoptosis through a Galphai protein-dependent activation of phosphatidylinositol 3-kinase/Akt and regulation of antiapoptotic Bcl-2 expression. Endocrinology, 148:3068–3076.
37.
RamehLE and CantleyLC. (1999). The role of phosphoinositide 3-kinase lipid products in cell function. J Biol Chem, 274:8347–8350.
38.
SongH, WangR, WangS and LinJ. (2005). A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells. Proc Natl Acad Sci U S A, 102:4700–4705.
39.
HoogduijnMJ, GorjupE and GeneverPG. (2006). Comparative characterization of hair follicle dermal stem cells and bone marrow mesenchymal stem cells. Stem Cells Dev, 15:49–60.
40.
KramperaM, PasiniA, RigoA, ScupoliMT, TecchioC, MalpeliG, ScarpaA, DazziF, PizzoloG and VinanteF. (2005). HB-EGF/HER-1 signaling in bone marrow mesenchymal stem cells: inducing cell expansion and reversibly preventing multilineage differentiation. Blood, 106:59–66.
41.
MarquesMM, MartinezN, Rodriguez-GarciaI and AlonsoA. (1999). EGFR family-mediated signal transduction in the human keratinocyte cell line HaCaT. Exp Cell Res, 252:432–438.
42.
BressanRB, MeloFR, AlmeidaPA, BittencourtDA, VisoniS, JeremiasTS, CostaAP, LealRB and TrentinAG. (2014). EGF-FGF2 stimulates the proliferation and improves the neuronal commitment of mouse epidermal neural crest stem cells (EPI-NCSCs). Exp Cell Res, 327:37–47.
43.
TamamaK, FanVH, GriffithLG, BlairHC and WellsA. (2006). Epidermal growth factor as a candidate for ex vivo expansion of bone marrow-derived mesenchymal stem cells. Stem Cells, 24:686–695.
44.
ParkJH and HanHJ. (2009). Caveolin-1 plays important role in EGF-induced migration and proliferation of mouse embryonic stem cells: involvement of PI3K/Akt and ERK. Am J Physiol Cell Physiol, 297:C935–C944.
45.
LiuW, RenH, RenJ, YinT, HuB, XieS, DaiY, WuW, XiaoZ, YangX and XieD. (2013). The role of EGFR/PI3K/Akt/cyclinD1 signaling pathway in acquired middle ear cholesteatoma. Mediators Inflamm, 2013:651207.
46.
HanW and HeYY. (2009). Requirement for metalloproteinase-dependent ERK and AKT activation in UVB-induced G1-S cell cycle progression of human keratinocytes. Photochem Photobiol, 85:997–1003.
47.
HarashimaM, SekiT, ArigaT and NiimiS. (2013). Role of p16 (INK4a) in the inhibition of DNA synthesis stimulated by HGF or EGF in primary cultured rat hepatocytes. Biomed Res, 34:269–273.
48.
KamiyaA, ItoK, YanagidaA, ChikadaH, IwamaA and NakauchiH. (2015). MEK-ERK activity regulates the proliferative activity of fetal hepatoblasts through accumulation of p16/19 (cdkn2a). Stem Cells Dev, 24:2525–2535.
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