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. 2012 Sep;16(9):2127-39.
doi: 10.1111/j.1582-4934.2011.01520.x.

Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

Iveta Yotova  1 , Ping Quan, Aulona Gaba, Nadja Leditznig, Petra Pateisky, Christine Kurz, Walter Tschugguel
Affiliations

Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

Iveta Yotova et al. J Cell Mol Med. 2012 Sep.

Abstract

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.

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Figures

Fig 1
Fig 1
Effects of Raf-1 knockdown on hESC cell morphology. (A) Immunofluorescence analysis of cytoskeleton by vimentin (green), phalloidin (red) and merge (green and red), 48 hrs after transfection of Co-hESC, Eu-hESC and Ec-hESC cells with siRNA are shown, scale bar line = 100 μm. (B) Average number of cells with contracted phenotype (estimated by two independent investigators) out of three biological replicates are given as% of cells with contracted phenotype relative to the total cell number (set to 100%), **P < 0.005.
Fig 2
Fig 2
Raf-1 regulates hESC morphology and migration. (A) Immunofluorescence analysis of cytoskeleton by phalloidin (red), Raf-1 cellular levels (green) and merge (green and red) 48 and 144 hrs after transfection of Eu-hESC cells with siRNA are shown on the left. Average number of cells with contracted phenotype (estimated by two independent investigators) out of three biological triplicates are given on the right as % of cells with contracted phenotype relative to the total cell number (set to 100%, ± S.D.). **P < 0.005; scale bar line = 100 μm. (B) Effects of Raf-1 knockdown on cell migration in Eu-hESC 48 and 144 hrs after initial transfection with either control (transfection reagent only) or control siRNA or Raf-1 siRNA are given. Average values of biological triplicates are shown as percentage of migrating cells relative to the controls (mean value set to 100%), **P < 0.005. (C) Raf-1 knockdown does not change the migration rate of Ec-hESC. A migration assay, 48 hrs after Raf-1 knockdown in Ec-hESC, was performed. Average values of biological triplicates are shown as percentage of migrating cells relative to the siRNA controls (mean value set to 100%).
Fig 3
Fig 3
Cellular Raf-1 levels determine hESC motility. (A) Western blot analysis of Raf-1 expression levels in individual hESC cultures (numbered at the top of the immunoblot and corresponding to the sample ID number given in Table S1 (for Ec-hESC; n = 7) and in [25] for Eu-hESC; n = 7) is shown on the left and the graphical representation of the analysis on the right. The level of the protein is shown as optical density (OD) of the Western blot lanes normalized to the OD of α-tubulin. (B) Box plots of the data obtained using Western blot analysis pE/R/M (left panel; n = 8 per group) and total E/R/M levels (right panel; n = 8 per group) in Co-, Eu- and Ec-hESC are given. The levels of the proteins are shown as OD of the Western blot lanes normalized to the OD of β-actin. The corresponding P-values obtained after anova and Post hoc analysis are additionally inserted on the top of the box plots. (C) The ROCK II activity in Co-, Eu- and Ec-hESC is shown as average values of absorbance (OD 450 nm) determined in six biological replicates (mean ± S.D.) and normalized to the absorbance in Co-hESC. The statistical analysis of the data was performed with anova followed by Post-hoc test, *P < 0.05. (D) The analysis of cell migration in Co-, Eu- and Ec-hESC is given in per cent as average values of migrating cells from biological triplicates relative to that in Co-hESC (mean value set to 100%).
Fig 4
Fig 4
Raf-1 knockdown affects phosphorylation levels of downstream target proteins. (A) Total cell lysates (TCL) from Co- and Eu-hESC cells were analysed for Raf-1, pE/R/M, phospho-paxillin (pPax), E/R/M, pMYPT1, MYPT1 and α-tubulin, 48 hrs after their transfection with either Raf-1 or control siRNA. The Raf-1 and pPax levels were normalized by total α-tubulin. The pE/R/M and pMYPT1 levels were normalized by E/R/M and MYPT1 respectively. Representative blots from biological triplicates (left panel) and graphical representation of Raf-1, pE/R/M, pPax and pMYPT1 (right panel) are shown as protein levels in% (mean value ± S.D.) relative to their normalized levels (mean value set to 100%) in cells transfected with control siRNA, **P < 0.0001. (B) TCLs from Ec-hESC were analysed for Raf-1, pE/R/M, E/R/M, pPax, pMYPT1, MYPT1 and α-tubulin 48 hrs after their transfection with Raf-1 siRNA or control siRNA. The levels of the analysed proteins were normalized as described in (A). Representative blots from biological triplicates and graphical representation of Raf-1, pE/R/M, pMYPT1 and pPax as described in (A) are shown. **P < 0.0001. (C) ROCK II activity in Raf-1 knockdown Ec-hESC is shown as average values of absorbance (OD 450 nm) determined in four biological replicates (mean ± S.D.) and normalized to the absorbance in siRNA controls (mean value set to 1), *P < 0.05. (D) TCL from Eu- and Ec-hESC were analysed for pMLC, 48 hrs after their transfection with Raf-1 siRNA or control siRNA. The pMLC levels were normalized by GAPDH. Representative blots from biological triplicates and graphical representation of pMLC are shown as protein levels in% (mean value ± S.D.) relative to their normalized levels (mean value set to 100%) in cells transfected with control siRNA, *P < 0.05.
Fig 5
Fig 5
Western blot analysis of MYPT1 and E/R/M phosphorylation analysed 24 hrs after treatment of hESC with specific Raf-1 inhibitors (ZM336372 and GW5074 – 1 μM) is shown on the left. Representative blots from three independent experiments are given. Graphical representation of pMYPT1 (right panels) is shown as protein levels in% (mean value ± S.D.) relative to their normalized levels (mean value set to 1) in the non-treated controls, *P < 0.05.
Fig 6
Fig 6
(A) Western blot analysis of ROCKII knockdown in Co- and Eu-hESC, showing down-regulation of E/R/M, MYPT1, paxillin and MLC phosphorylation in knockdown cells versus respective siRNA controls. Representative blots of six independent experiments (left panel) and graphical representation of the levels of phosphorylated proteins (right panel) normalized by α-tubulin are shown as protein level in % (mean ± SD) relative to their normalized levels (set to 100%) in cells transfected with control siRNA, *P < 0.05 and **P < 0.0001. (B) Western blot analysis of ROCKII knockdown in Ec-hESC showing up-regulation of pPax and down-regulation of both pMYPT1 and pMLC in the knockdown cells versus siRNA controls. Representative blots of four independent experiments (left panel) and graphical representation of the level of pPax and pE/R/M (middle panel) normalized either by α-tubulin or E/R/M are shown. The levels are presented in% (mean ± S.D.) relative to their normalized level (set to 100%) in cells transfected with control siRNA, **P < 0.005. The analysis of cell migration in ROCKII knockdown Ec-hESC is given on the right. Average values of migrating cells from four biological replicates are shown in% relative to the values of siRNA controls (mean value set to 100), *P < 0.02. (C) The activity of ROCKII in Co-, Eu- and Ec-hESC is shown as average values of absorbance (OD 450 nm) determined in biological triplicates (mean ± S.D.) and normalized to the absorbance measured in control siRNA treated Co-hESC (mean value set to 1), **P < 0.005 (left panel). Immunofluorescence analysis of the cytoskeleton in Ec-hESC, 48 hrs after their transfection with either ROCKII siRNA or control siRNA and visualized by phalloidin (red), vimentin (green) and merge (red and green) is shown on the right. Scale bar line = 100 μm.
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