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.

A GEF-to-phospholipase molecular switch caused by PA, RAC and jak tyrosine kinase, that explains leukocyte cell migration

“…In addition, Rac2 regulates actin cytoskeletal machinery during the formation of lamellipodia and membrane ruffles, cell migration and adhesion of immune cells (GomezCambronero, 2011). Abnormal activation of Rho GTPases is implicated in diseases, such as tumorigenesis (Aznar and Lacal, 2001;Benitah et al, 2003;Karlsson et al, 2009), endometriosis (Yotova et al, 2011) and Fragile X syndrome (Chen et al, 2010). Hence, activation of Rho GTPases and the upstream guaninenucleotide exchange factors (GEFs) is tightly regulated (Pai et al, 2010).…”

Hexane extract of aged black garlic reduces cell proliferation and attenuates the expression of ICAM-1 and VCAM-1 in TNF-α-activated human endometrial stromal cells

“…These MAPK signaling pathways are also present in endometrial cells, and MAPKs play pathophysiological roles in the development of endometriosis (14,27). It has been shown that MAPK signaling pathways are abnormally activated in HESCs, and that MAPK acts upon the regulation of HESC proliferation (28,29).…”

Estrogen signaling in the proliferative endometrium: implications in endometriosis

“…45 More recently, in an attempt to understand the molecular mechanisms that control not only the baseline proliferation of endometrial human cells but also their migration, it was demonstrated for the first time that the protein rho-associated kinase II (ROCKII) acts as a point of integration between cell proliferation and migration, and that the protein Rapidly accelerated fibrosarcoma-1 (Raf-1) regulates negatively the activity of ROCKII in endometrial cells. 46 Still, since levels of Raf-1 are lower and B-Raf activity is higher in eutopic endometrial cells than in normal cells, the B-Raf-MAPK and Rho/ROCKII pathways are abnormally activated, leading to a greater proliferation ability and increased migratory potential, which explains the incomplete transition in the damaged endometrium and the high proliferative migratory phenotype (Figure 1e*).…”

“…7 One of the main features of the eutopic endometrium in patients with endometriosis has been the incomplete transition of the proliferative (estrogen-dependent) to the secretory (progesterone-dependent) endometrium, leading to a persistent expression of genes involved in the synthesis of DNA and cellular mitosis in endometriotic lesions. 8,9 In addition, latest research shows that the proliferative phase is not a uniform period of endometrial growth. 10 This represents without any doubt a great challenge in the search for the understanding of the mechanisms of cellular signaling by which estrogen regulates the human endometrium.…”