PAI-1 SECRETION AND MATRIX DEPOSITION IN HUMAN PERITONEAL MESOTHELIALCELL-CULTURES - TRANSCRIPTIONAL REGULATION BY TGF-BETA-1

Background. Plasminogen activator inhibitor-1 (PAI-1), the main inhibi tor of plasminogen activators in plasma and in peritoneum, impairs pla smin formation that is essential for the repair processes of the mesot helium damaged by peritoneal dialysis fluids and peritonitis. The fibr ogenetic cytokine transforming growth factor-beta (TGF-beta) displays variable effects on extracellular matrix remodeling enzymes and their inhibitors depending on tissues and cell lines. We previously found an unexpected stimulating effect of TGF-beta 1 on matrix metalloproteina se-9 in peritoneal mesothelial cells. In this study, we analyzed the e ffects of TGF-beta 1 on PAI-1 production and deposition in extracellul ar matrix. Methods. We used primary cultured mesothelial cells and a r ecently established human peritoneal mesothelial cell line (HMrSV5). C ell-associated and secreted plasminogen activators and their inhibitor s were detected and characterized by substrate gel zymography. PAI-1 w as identified by reverse zymography and by Western blotting, and total PAI-1 was measured by ELISA. Secreted and cell-associated PA activity was measured by its ability to activate plasminogen into plasmin, tha t is! by the release of paranitroaniline from the plasmin synthetic su bstrate S-2251. PAI-1 mRNA accumulation was assessed by Northern blot. In vitro nuclear run-on assays were carried out to determine whether TGF-beta 1 had transcriptional effects on PAI-1 expression. Finally, t he subcellular distribution of PAI-1 was analyzed by immunofluorescenc e and by immunogold silver staining. Results. TGF-beta 1 increased PAI -1 antigen in the conditioned media of HMrSV5 cells, in a time- and co ncentration-dependent manner. This induced a dramatic decrease of free tPA in the cell medium and of membrane-bound uPA, and a parallel incr ease of high molecular weight PA-PA1 complexes. Consequently, secreted and cell-associated plasminogen activator activities were considerabl y reduced. In primary cultured peritoneal mesothelial cells, TGF-beta 1 also induced PAI-1 secretion and the shift of tPA toward high molecu lar weight complexes. TGF-beta 1 increased PAI-1 mRNA in a time- and c oncentration-dependent manner. This effect was at least in part transc riptional since an similar to threefold increase in the rate of PAI-1 gene transcription was observed in nuclei sampled after a four-hour ce ll exposure to 5 ng/ml TGF-beta 1. Finally, TGF-beta 1 substantially i ncreased the amount of intracellular and matrix-associated PAI-1. Conc lusions. These results suggest that excessive TGF-beta 1-stimulated PA I-1 could prevent appropriate peritoneal healing by impairing the degr adation of fibrin and of unorganized matrix components, and by interfe ring with cell migration.