Epidermal growth factor promotes stromal cells migration and invasion via up-regulation of hyaluronate synthase 2 and hyaluronan in endometriosis

OBJECTIVE: To investigate the role(s) of hyaluronan synthase 2 (HAS2) and hyaluronan in disease progression of endometriosis and epidermal growth factor (EGF)-induced motility changes of endometriotic cells. DESIGN: A case-control experimental study and in vitro primary cell culture study. SETTING: University hospital-affiliated research centers. PATIENTS: A total of 21 women with stage I/II endometriosis, 33 women with stage III/IV endometriosis with endometrioma, and 32 women without endometriosis were included in our study. INTERVENTIONS: Serum, eutopic endometrial tissues, and/or ectopic endometriotic tissues were collected. Primary eutopic endometrial stromal cells (EuESCs) and ectopic ovarian endometriotic stromal cells (OvESCs) were isolated and cultured from women with ovarian endometrioma, and then treated with or without EGF. MAIN OUTCOME MEASURES: The concentrations of EGF and hyaluronan in serum were analyzed by enzyme-linked immunosorbent assay. The expressions and localizations of EGF receptor (EGFR), phosphorylated-(p)EGFR, HAS2, and hyaluronan receptor CD44 in tissues were examined by immunohistochemistry. The mRNA and protein levels of HAS2 in EuESCs and OvESCs were examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot, respectively, and the concentrations of hyaluronan in conditioned medium were examined by enzyme-linked immunosorbent assay (ELISA). Cell motility was evaluated by transwell migration/invasion assays. RESULTS: Serum EGF and hyaluronan concentrations were higher in women with stage III/IV endometriosis than in women with stage I/II or without endometriosis. EGFR, pEGFR, HAS2, and CD44 were immunolocalized in eutopic endometrium and ectopic endometriotic lesions, and the expressions of pEGFR and HAS2 were elevated in ectopic endometriotic lesions compared to eutopic endometrium. Treatment with EGF upregulated HAS2 and hyaluronan expression as well as cell migration and invasion in both EuESCs and OvESCs, and pharmaceutical blocking of EGFR abolished these effects. In addition, knockdown of HAS2 by small interfering RNA attenuated both basal and EGF-induced hyaluronan expression and cell motility changes. Notably, ERK1/2 and AKT signaling pathways were shown to be downstream of EGF in regulating HAS2 and hyaluronan expression as well as cell migration and invasion. CONCLUSION: EGF increased the expression of endometriosis-associated hyaluronan and its synthase HAS2, both of which mediated EGF-induced stromal cell migration and invasion in women with endometriosis.