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I.Y. Yotova, P. Quan, N. Leditznig, U. Beer, R. Wenzl and W. Tschugguel*Department of Obstetrics and Gynecology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria *Correspondence address. E-mail: walter.tschugguel{at}meduniwien.ac.atReceived August 30, 2010. Revision received December 29, 2010. Accepted January 10, 2011. 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. © The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com This ArticleHum. Reprod. (2011) 26 (4): 885-897. doi: 10.1093/humrep/der010 First published online: February 7, 2011Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.
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