Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria.
J Cell Mol Med. 2012 Sep;16(9):2127-39. doi: 10.1111/j.1582-4934.2011.01520.x.
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.
子宫内膜异位症是一种以子宫内膜组织在子宫腔外定位为特征的疾病。据推测，与健康对照组相比，从子宫内膜异位症患者中获得的人子宫内膜基质细胞（hESC）的迁移差异与 Raf-1/ROCKII 信号通路的异常激活有关。为了评估 Raf-1 调节细胞骨架重排和运动的机制，我们使用了来自子宫内膜异位症患者的原发性在位（Eu，n=16）和异位（Ec，n=8；从卵巢囊肿中分离）hESC 以及无子宫内膜异位症对照组（Co-hESC，n=14）。Raf-1 siRNA 敲低在 Co- 和 Eu-hESC 中导致收缩和迁移减少，与 siRNA 对照相比。在这些细胞中重新表达 Raf-1 后，这种表型得到逆转。在 Ec-hESC 中 Raf-1 水平最低与 ROCKII 和 ezrin/radixin/moesin（E/R/M）的过度激活、迁移受损和收缩表型有关，类似于 Co- 和 Eu-hESC 中的 Raf-1 敲低。我们进一步表明，Raf-1 在 hESC 中介导迁移的机制包括直接肌球蛋白轻链磷酸酶（MYPT1）磷酸化和通过 ROCKII 激酶的过度激活间接调节 E/R/M、桩蛋白、MYPT1 和肌球蛋白轻链（MLC）磷酸化的水平。此外，我们提出，与 Co- 和 Eu-hESC 不同，其中细胞 Raf-1 水平调节迁移率，Ec-hESC 中细胞 Raf-1 含量低可能通过保持收缩的细胞表型来确保其受限的迁移。总之，我们的发现表明，细胞水平的 Raf-1 调整了子宫内膜异位症中 hESC 迁移的阈值。
