Quercetin Promotes Human Epidermal Stem Cellproliferation Through Estrogen Receptor/?-catenin/c-Myc Signaling Pathway

Objective Skin epidermal stem cells(EpSCs)play important roles in skin homeostasis and wound healing.EpSCs are important seed cells for skin tissue engineering to develop skin equivalents for skin grafting.The methods to expand EpSCs are required.The search for compounds that promote epidermal cell proliferation will be helpful for getting more seed cells for preparation of engineering skin tissue in vitro.Quercetin is a phytoestrogens which has been reported topromote keratinocyte proliferation,migration,and accelerate skin wound healing.But its effect on proliferation EpSCs is not clear.Therefore,to investigate the effect of quercetin on proliferation of human EpSCs and explore the underlying mechanisms.Method EpSCs were isolated form human skin tissue.The expression of biomarkers of EpSC(CK19,CD71,?6 integrin and ?1 integrin)was examined by flow cytometry analysis,immunofluorescence and immunohistochemical staining.Cell proliferation was evaluated by BrdU incorporation assay,MTT assay,Ki-67 immunofluorescence staining and proliferating cell nuclear antigen(PCNA)immunohistochemical staining.Cell cycle phase distribution was analyzed by flow cytometry.The expression of ?-catenin,c-Myc and cyclins were examined by RT-PCR and Western blot.Results BrdU incorporation assay showed that quercetin promoted human EpSC proliferation in a time-and dose-dependent manner.Ki-67 immunofluorescence staining confirmed the pro-proliferative effectof quercetin on EpSCs.Cell cycle analysis showed that quercetin treatment significantly increased the cell number of EpSCs in the S phase.Mechanistic studies showed that quercetin significantly upregulated the expression of ?-catenin,c-Myc,and cyclin A2/E1 which are critical for G1/Sphase transition.The ?-catenin inhibitor XAV-939 and c-Myc inhibitor 10058-F4 both significantly inhibited quercetin-induced proliferation of EpSCs.The ?-catenin inhibitor suppressed quercetin-induced expression of ?-catenin,c-Myc and cyclin A2/E1.The c-Myc inhibitor inhibited the upregulation of c-Myc and cyclin A2 by quercetin.Pretreatment of EpSCs with estrogen receptor(ER)antagonist ICI182780,but notthe G protein-coupled ER1 antagonist G15,reversed quercetin-induced cell proliferation and upregulation of ?-catenin,c-Myc and cyclin A2.Furthermore,immunohistochemical staining of cell proliferation marker PCNA and EpSC markers(?6 integrin and ?1 integrin)showed that incubation of skin tissue with quercetin significantly increased the thickness of the epidermis and increased the number of PCNA-,integrin ?1-and integrin ?6-positive cellsat the basal layer of the epidermis.Conclusions Quercetin promotes EpSC proliferation through ER mediated upregulation of ?-catenin,c-Myc and cyclin A2 expression.Quercetin can be used to expand EpSCs for generating epidermal autografts and engineered skin equivalents.