Induction of Smad-dependent and -independent pathways by TGF-betas in human endometrial and endometriotic cells

Abstract

Endometriosis is a chronic pathological disorder in which endometrial-like cells are found outside the uterine cavity. TGF-betas were observed to be highly expressed in the peritoneal fluid of patients with endometriosis, as well as in endometriotic sites. Thus, TGF-beta;s may play an important role in the pathogenesis of endometriosis. In this study, our aim was to investigate the biological function and signal transduction ofTGF-betas in endometrial and endometriotic cells in vitro.Four different cell lines, endometrial epithelial and stromal cells, endometriotic epithelial and stromal cells were used in this study. Cells were treated with or without TGF-beta1 or TGF-beta2, respectively. Then the cell numbers were counted and the secretion of MMP-2, MMP-9 or PAI-1 was measured with ELISAs. Also the effect of PAI-1 on cell adhesion was tested. By using specific inhibitors targeting downstream cascades of TGF-beta signaling, both Smad-dependent and Smad-independent pathways were studied. In addition, the localization of phospho-ERK1/2 after stimulation with TGF-beta1 or TGF-beta2 was analyzed with immunofluorescence.Our results showed in the four cell lines studied: (1) TGF-betas had a dual effect on the cell numbers, with increased cell numbers when the initial cell number was low, but decreased cell numbers when the initial cell number was high. (2) Endometriotic cells secreted more MMP-2, MMP-9 and PAI-1, compared to endometrial cells. TGF-beta1 or TGF-beta2 dramatically increased MMP-2 and PAI-1 secretion in all cell lines studied. (3) A TbetaRI inhibitor completely blocked the TGF-beta-induced MMP-2 or PAI-1 secretion. An ERK1/2 inhibitor partly blocked it. The p38/MAPK inhibitor had only slight effects.(4) TGF-beta1 or TGF-beta2 enhanced accumulation of phospho-ERK1/2 in the nucleus. (5) PAI-1 reduced cell attachment in all four cell lines studied.In this study, we demonstrated the influence of TGF-betas on endometrial and endometriotic cell lines in vitro. From our data, we suppose that the TGF-beta-inducedincrease in PAI-1 and MMP-2 secretion might play a role in increased tissue breakdown during menstruation and increased invasiveness during ectopicendometrial implantation. We confirmed that the Smad pathway was the main pathway of TGF-beta signaling also in endometrial and endometriotic cells. Furthermore,83 we demonstrated for the first time the participation of the ERK pathway, a mainly Smad-independent pathway, in TGF-beta signaling in endometrial and endometriotic cells. This finding might provide new options to comprehend the role of TGF-betas in the etiology of endometriosis. However, since it is only a first glimpse into the ERK pathway in TGF-beta signaling in endometrial and endometriotic cells, more researches are required to elucidate the connection between ERKs and TGF-beta, as well as the crosstalk between Smads and ERKs.