TGF-betas stimulate PAI-1 secretion of endometrial and endometriotic cells via Smad2/3 and ERK1/2

Objectives: In clinical trials, transforming growth factor betas (TGF-betas) and plasminogen activator inhibitor-1 (PAI-1) were observed to be highly expressed at endometriotic sites as well as in the peritoneal fluid or serum of patients who suffer from endometriosis. PAI-1 is one of the most important genes to test for upregulation of mRNA expression after treatment of cells with TGF-betas. Furthermore, PAI-1 might contribute to activation of latent TGF-betas via plasmin or is misregulated in cancer tissue, e.g. breast cancer. Thus, we have analyzed PAI-1 secretion in endometrial and endometriotic cells after treatment with TGF-betas and have measured PAI-1 serum levels in patients suffering from endometriosis.

Methods and Materials: Inhibitors for Smad2/3, p38/MAPK and ERK1/2 were used, which target Smad- and non-Smad-dependent pathways after stimulations with TGF-betas. Immortalized endometrial and endometriotic cells representative for stroma and epithelium were used for TGF-beta treatments in various combinations with the inhibitors. After three days collected supernatants were measured with PAI-1 ELISAs and cell numbers were determined.

Results: Secretion of PAI-1 was found only in stromal endometrial cells but not in epithelial cells. In contrast, endometriotic stromal and epithelial cells secreted higher amounts of PAI-1 compared with endometrial cells. In all cell lines secretion of PAI-1 was strongly upregulated by treatment with TGF-betas (TGF-beta1 to TGF-beta3). However, this increase was completely blocked by the Smad2/3 inhibitors in all cell lines. In general, the ERK1/2 inhibitor was less effective but also showed significant inhibiting effects on PAI-1 levels. In serum of endometriosis patients (n=35) we found no significantly different values compared to patients without endometriosis (n=34). Of note, in the secretory phase, values were lower compared with the proliferative phase or menstruation.

Conclusions: In our study, endometriotic cells secreted more PAI-1 than normal endometrial cells, which indicates a possible role of PAI-1 in the pathogenesis of endometriosis. Furthermore, PAI-1 secretion was stimulated by TGF-betas in both endometrial and endometriotic cells mainly via Smad2/3 and to a lesser extent via ERK1/2. Although PAI-1 serum levels were not higher in patients suffering from endometriosis, we are currently investigating tissue levels. We suggest that TGF-betas by upregulating secretion of PAI-1 might contribute to the pathogenesis of endometriosis.

Keywords: TGF-betas, PAI-1, Smad2/3, ERK, endometriosis