TNFα-Mediated Induction of PAI-1 Restricts Invasion of HTR-8/SVneo Trophoblast Cells

The pro-inflammatory cytokine TNFα has numerous effects on placental trophoblasts. Here, we investigated the effects of the cytokine on gene expression and function of the extravillous trophoblast cell line HTR-8/SVneo. Wound healing and Matrigel invasion assays demonstrate that TNFα impairs motility and invasiveness. In contrast, counting of cumulative cell numbers and FACS analyses revealed that the cytokine did neither affect proliferation nor distribution of cell cycle phases. Immunocytochemistry of the cytokeratin 18 neo-epitope suggests that TNFα did not induce apoptosis in HTR-8/SVneo cells. Gelatine zymography and enzyme activity assays of supernatants of TNFα-treated cells demonstrate elevation of the pro- and active form of MMP-9 suggesting that increased expression of the protease cannot overcome the TNFα-inhibitory effect on cell invasion. Semi-quantitative RT-PCR analyses suggest that the cytokine may not alter mRNA levels of uPA and tPA. However, elevated expression of PAI-1 was detected by RT-PCR, as well as by Northern and Western blot analyses. Supplementation of PAI-1-blocking antibodies restored invasion of TNF-α-incubated HTR-8/SVneo cells through Matrigel-coated transwells. In addition, immunocytochemistry revealed nuclear accumulation of the p65 subunit of NFκB in the presence of the cytokine. EMSA indicated TNFα-induced binding of the inflammatory transcription factor to an NFκB consensus sequence and to the NFκB recognition site located in the PAI-1 promoter. The data suggest that TNFα restricts trophoblast invasion mainly by increasing the expression of PAI-1. Induction of the inhibitor may involve TNFα-stimulated activation of NFκB.