title: Abnormal activation of Ras/Raf/MAPK and RhoA/ROCKII signalling pathways in eutopic endometrial stromal cells of patients with endometriosis.
abstract: BACKGROUND Enhanced proliferation and survival of eutopic endometrial cells from patients with endometriosis compared with healthy women is associated with abnormal activation of extra-cellular signal-regulated kinases 1 and 2 (ERK1/2). Given the role of Ras/Raf/mitogen-activated protein kinase (MAPK) and RhoA/ROCKII signalling pathways in the regulation of cell proliferation and migration, we analysed their possible roles in endometriosis. METHODS Primary eutopic endometrial stromal cells of patients with endometriosis (Eu-hESC, n= 16) and endometriosis-free controls (Co-hESC, n= 14) were harvested and subjected to proliferation and migration assays as well as kinase activity assays and immunoblot analysis of proteins from the Ras/Raf/MAPK and RhoA/ROCKII signalling pathways. Effects of ROCKII (Y-27632) and MAPK (U0126) inhibitors or siRNA knockdown of ROCKII, Raf-1 and B-Raf were analysed. RESULTS The proliferation rate of Eu-hESC was 54% higher than Co-hESC. Eu-hESC also displayed a 75% higher migration rate than Co-hESC. Eu-hESC displayed higher levels of ERK phosphorylation (83%) and p27 expression (61%) and lower levels of Raf-1 protein (47%) compared with controls. In addition to an inhibitory effect on cell proliferation, ROCKII knockdown led to significant down-regulation of cyclinD1 and p27 but did not affect ERK phosphorylation. Down-regulation of Raf-1 by siRNA was dispensable for cell proliferation control but led to an increase in ROCKII activity and a decrease in cell migration. B-Raf was shown to act as a regulator of hESC proliferation by modulating cellular ERK1/2 activity and cyclinD1 levels. Eu-hESC displayed 2.4-fold higher B-Raf activity compared with Co-hESC and therefore exhibit abnormally activated Ras/Raf/MAPK signalling. CONCLUSIONS We show that the same molecular mechanisms operate in Co- and Eu-hESC. The differences in cell proliferation and migration between both cell types are likely due to increased activation of Ras/Raf/MAPK and RhoA/ROCKII signalling pathways in cells from endometriosis patients.
authors.affiliation: Department of Obstetrics and Gynecology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.
authors.forename: I Y
authors.initials: IY
authors.lastname: Yotova
authors.forename: P
authors.initials: P
authors.lastname: Quan
authors.forename: N
authors.initials: N
authors.lastname: Leditznig
authors.forename: U
authors.initials: U
authors.lastname: Beer
authors.forename: R
authors.initials: R
authors.lastname: Wenzl
authors.forename: W
authors.initials: W
authors.lastname: Tschugguel
mesh_terms: D002465:Cell Movement; D049109:Cell Proliferation; D004715:Endometriosis / Q000201:enzymology*; D004717:Endometrium / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D020935:MAP Kinase Signaling System; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000378:metabolism; D017154:Stromal Cells / Q000201:enzymology*; D048490:raf Kinases / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D048490:raf Kinases; D054460:rho-Associated Kinases; D018631:ras Proteins; D020742:rhoA GTP-Binding Protein