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.

Reduced hepatobiliary bile acid transport due to loss of BSEP function leads to increased hydroxylation of bile acids in the liver. Metabolic preconditioning with a hydrophilic bile pool protects the BSEP mice from acquired cholestatic liver disease.

Background and aimsInterruption of the enterohepatic circulation of bile acids (BAs) may protect against BA-mediated cholestatic liver and bile duct injury. BA sequestrants are established to treat cholestatic pruritus, but their impact on the underlying cholestasis is still unclear. We aimed to explore the therapeutic effects and mechanisms of the BA sequestrant colesevelam in a mouse model of sclerosing cholangitis.MethodsMdr2−/− mice received colesevelam for 8 weeks. Gene expression profiles of BA homeostasis, inflammation and fibrosis were explored in liver, intestine and colon. Hepatic and faecal BA profiles and gut microbiome were analysed. Glucagon-like peptide 1 (GLP-1) levels in portal blood were measured by ELISA. Furthermore, Mdr2−/− mice as well as wild-type 3,5-diethoxy-carbonyl-1,4-dihydrocollidine-fed mice were treated with GLP-1-receptor agonist exendin-4 for 2 weeks prior to analysis.ResultsColesevelam reduced serum liver enzymes, BAs and expression of proinflammatory and profibrogenic markers. Faecal BA profiling revealed increased levels of secondary BAs after resin treatment, while hepatic and biliary BA composition showed a shift towards more hydrophilic BAs. Colonic GLP-1 secretion, portal venous GLP-1 levels and intestinal messenger RNA expression of gut hormone Proglucagon were increased, while ileal Fgf15 expression was abolished by colesevelam. Exendin-4 treatment increased bile duct mass without promoting a reactive cholangiocyte phenotype in mouse models of sclerosing cholangitis. Microbiota analysis showed an increase of the phylum δ-Proteobacteria after colesevelam treatment and a shift within the phyla Firmicutes from Clostridiales to Lactobacillus.ConclusionColesevelam increases faecal BA excretion and enhances BA conversion towards secondary BAs, thereby stimulating secretion of GLP-1 from enteroendocrine L-cells and attenuates liver and bile duct injury in Mdr2−/− mice.

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Endometriosis is a disease characterized by the localization of endometrial tissue outside the uterine cavity. The differences observed in migration of human endometrial stromal cells (hESC) obtained from patients with endometriosis versus healthy controls were proposed to correlate with the abnormal activation of Raf-1/ROCKII signalling pathway. To evaluate the mechanism by which Raf-1 regulates cytoskeleton reorganization and motility, we used primary eutopic (Eu-, n = 16) and ectopic (Ec-, n = 8; isolated from ovarian cysts) hESC of patients with endometriosis and endometriosis-free controls (Co-hESC, n = 14). Raf-1 siRNA knockdown in Co- and Eu-hESC resulted in contraction and decreased migration versus siRNA controls. This phenotype was reversed following the re-expression of Raf-1 in these cells. Lowest Raf-1 levels in Ec-hESC were associated with hyperactivated ROCKII and ezrin/radixin/moesin (E/R/M), impaired migration and a contracted phenotype similar to Raf-1 knockdown in Co- and Eu-hESC. We further show that the mechanism by which Raf-1 mediates migration in hESC includes direct myosin light chain phosphatase (MYPT1) phosphorylation and regulation of the levels of E/R/M, paxillin, MYPT1 and myosin light chain (MLC) phosphorylation indirectly via the hyperactivation of ROCKII kinase. Furthermore, we suggest that in contrast to Co-and Eu-hESC, where the cellular Raf-1 levels regulate the rate of migration, the low cellular Raf-1 content in Ec-hESC, might ensure their restricted migration by preserving the contracted cellular phenotype. In conclusion, our findings suggest that cellular levels of Raf-1 adjust the threshold of hESC migration in endometriosis.