Non-genomic effects of 17beta-estradiol in activation of the ERK1/ERK2 pathway induces cell proliferation through upregulation of cyclin D1 expression in bovine artery endothelial cells.
OBJECTIVE
Growing evidence indicates that estrogen's non-genomic effects play important roles in cellular functions and backs up the hypothesis of the existence of a membrane estrogen receptor (mER) in a number of cell types, but little is known about the complementary effects between traditional genomic and novel non-genomic effects of estrogen. The aim of the present study was to explore the non-genomic activation of ERK1/2 mitogen-activated protein kinase (MAPK) by 17beta-estradiol (E(2)) through mER and its role in cell proliferation.


METHODS
On cultured bovine artery endothelial cells (BAECs) we used the [(3)H]thymidine incorporation assay to evaluate the influence of E(2) on cell proliferation and fluorescence microscopy to show the presence of mER on the cell membrane. Scatchard analysis was performed to identify and characterize the mER on a purified membrane fraction of BAECs.


RESULTS
E(2) upregulated cyclin D1 protein expression and enhanced cell proliferation. Inhibition of the MAPK cascade with PD98059 or of G protein with pertussis toxin (PTX) completely abolished the above effects, while the estrogen receptor antagonist tamoxifen attenuated E(2)-dependent upregulation of cyclin D1 and cell proliferation. Accordingly, E(2) rapidly led to ERK1/ERK2 activation, which was prevented by tamoxifen or PTX and was entirely reproduced by membrane-impermeable estradiol-bovine serum albumin conjugate (E(2)coBSA). Immunofluorescent staining with E(2)coBSA-fluorescein isothiocyanate resulted in a punctuate staining pattern of the plasma membrane and Scatchard analysis of the E(2)-binding protein in a purified membrane fraction of BAECs showed that E(2) binds to the membrane fraction with a dissociation constant of 0.2394 nmol/l.


CONCLUSIONS
Our findings showed that E(2) induces cell proliferation through upregulation of cyclin D1 via non-genomic activation of the ERK1/ERK2 pathway mediated by mER and G protein.
Fu
Xiao-Dong
XD
Molecular and Cellular Gynecological Endocrinology Laboratory, Department of Reproductive Medicine and Child Development, University of Pisa, Pisa, Italy.
Cui
Yu-Hong
YH
Lin
Gui-Ping
GP
Wang
Ting-Huai
TH
D000818:Animals; D001158:Arteries / Q000166:cytology; D002417:Cattle; D049109:Cell Proliferation; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism*; D042783:Endothelial Cells / Q000201:enzymology*; D004958:Estradiol / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D011960:Receptors, Estrogen / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D015854:Up-Regulation
D011960:Receptors, Estrogen; D019938:Cyclin D1; D004958:Estradiol; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019204:GTP-Binding Proteins