PAI-1 secretion of endometrial and endometriotic cells is Smad2/3- and ERK1/2-dependent and influences cell adhesion

Abstract

In the endometrium transforming growth factor-betas (TGF-βs) are involved mainly in menstruation and endometriosis. After binding of the ligands to the high-affinity receptors, TGF-β receptors (TBR1 and TBR2), TGF-βs activate Smad signaling to modulate gene expression and cellular functions. However, recently also Smad-independent pathways have been studied in more details. To evaluate both pathways, we have analyzed TGF-β signaling in human endometrial and endometriotic cells. Although endometrial and endometriotic cells secrete TGF-β1, secretion by stromal cells was higher compared to epithelial cells. In contrast, secretion of TGF-β2 was higher in endometriotic stromal and endometriotic epithelial cells compared to normal endometrial cells. Treatment of endometrial and endometriotic stromal and epithelial cells with TGF-β1 or TGF-β2 increased Smad-dependent secretion of plasminogen activator inhibitor-1 (PAI-1) dramatically in all three cell lines. Of note, endometriotic cells secreted clearly higher levels of PAI-1 compared to endometrial cells. Whereas a TBR1 kinase inhibitor completely blocked the TGF-β1 or TGF-β2-induced PAI-1 secretion, an ERK1/2 inhibitor only partially reduced PAI-1 secretion. This inhibition was not dependent on epidermal growth factor receptor (EGFR) activation by phosphorylation but on kinase activity of the TBR1. Finally, treatment of endometrial and endometriotic cell lines with recombinant PAI-1 showed reduced cell adhesion, especially of the endometrial cells. In summary, our results demonstrate that both Smad-dependent and TBR1-dependent ERK1/2 pathways are necessary for TGF-β-dependent high level secretion of PAI-1, which might increase cellular deadhesion.

Keywords: ERK; PAI-1; SMAD; TGF-betas; endometriosis.

Figure 1

Quantification of TGF-β1, TGF-β2 and PAI-1 secretion by all cell lines. Stromal cell lines T-HESC and 22B secreted higher levels of TGF-β1 compared to epithelial cells 12Z (A). Endometriotic cell lines 22B and 12Z secreted more TGF-β2 compared to endometrial cells T-HESC (B). Endometriotic stromal cells 22B secreted 4-fold higher levels compared to endometrial stromal cells T-HESC. Endometriotic epithelial cells 12Z secreted modest amounts of PAI-1 (C). Each experiment was repeated three times in duplicates (A+B); each experiment was repeated nine times in duplicates (C).

Figure 2

Treatment of cells with TGF-β1 or TGF-β2 (10 ng/ml), respectively, induced PAI-1 secretion in endometrial stromal T-HESC cells (A), endometriotic stromal 22B cells (B) and endometriotic epithelial 12Z cells (C). The TBR1 inhibitor (LY364947) blocked TGF-β-induced PAI-1 secretion of all cell lines studied completely to control levels (***P<0.001; **P<0.01; *P≤0.05). Each experiment was repeated three times in duplicates. Ctrl, control; TBRi; TBR1 inhibitor; B1, TGF-β1; B2, TGF-β2.

Figure 3

TGF-β1 or TGF-β2 induced PAI-1 secretion was moderately but significantly inhibited by the ERK inhibitor (ERKi) in endometrial stromal T-HESC cells (A), endometriotic stromal 22B cells (B), and endometriotic epithelial 12Z cells (C) by 50%, 30%, and 40%, respectively (**P<0.001, **P<0.01; *P<0.05). Each experiment was repeated three times in duplicates. Ctrl, control; B1, TGF-β1; B2, TGF-β2.

Figure 4

PAI-1 dose dependently inhibited cell adhesion of endometrial stromal T-HESC cells (A), endometriotic stromal 22B cells (B), and endometriotic epithelial 12Z cells (C). The numbers of attached cells decreased in a range of 2-40% after treatment for four hours with various concentrations of recombinant PAI-1 (***P<0.001, **P<0.01, *P<0.05). Each experiment was repeated three times in triplicates.