PAI-1 secretion and matrix deposition in human peritoneal mesothelial cell cultures: transcriptional regulation by TGF-beta 1.
BACKGROUND
Plasminogen activator inhibitor-1 (PAI-1), the main inhibitor of plasminogen activators in plasma and in peritoneum, impairs plasmin formation that is essential for the repair processes of the mesothelium damaged by peritoneal dialysis fluids and peritonitis. The fibrogenetic 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 metalloproteinase-9 in peritoneal mesothelial cells. In this study, we analyzed the effects of TGF-beta 1 on PAI-1 production and deposition in extracellular matrix.


METHODS
We used primary cultured mesothelial cells and a recently established human peritoneal mesothelial cell line (HMrSV5). Cell-associated and secreted plasminogen activators and their inhibitors were detected and characterized by substrate gel zymography. PAI-1 was 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, that is, by the release of paranitroaniline from the plasmin synthetic substrate 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, the subcellular distribution of PAI-1 was analyzed by immunofluorescence and by immunogold silver staining.


RESULTS
TGF-beta 1 increased PAI-1 antigen in the conditioned media of HMrSV5 cells, in a time- and concentration-dependent manner. This induced a dramatic decrease of free tPA in the cell medium and of membrane-bound uPA, and a parallel increase of high molecular weight PA-PAI complexes. Consequently, secreted and cell-associated plasminogen activator activities were considerably reduced. In primary cultured peritoneal mesothelial cells, TGF-beta 1 also induced PAI-1 secretion and the shift of tPA toward high molecular weight complexes. TGF-beta 1 increased PAI-1 mRNA in a time- and concentration-dependent manner. This effect was at least in part transcriptional since an approximately threefold increase in the rate of PAI-1 gene transcription was observed in nuclei sampled after a four-hour cell exposure to 5 ng/ml TGF-beta 1. Finally, TGF-beta 1 substantially increased the amount of intracellular and matrix-associated PAI-1.


CONCLUSIONS
These results suggest that excessive TGF-beta 1 stimulated PAI-1 could prevent appropriate peritoneal healing by impairing the degradation of fibrin and of unorganized matrix components, and by interfering with cell migration.
Rougier
J P
JP
Instiut National de la Santé et de la Recherche Médicale, Unité 489, Paris, France.
Guia
S
S
Hagège
J
J
Nguyen
G
G
Ronco
P M
PM
D015152:Blotting, Northern; D002461:Cell Line, Transformed / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D010537:Peritoneum / Q000166:cytology; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D010960:Plasminogen Activators / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D016622:Silver Staining; D015533:Transcriptional Activation / Q000187:drug effects*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D016326:Extracellular Matrix Proteins; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D005337:Fibrin; D010960:Plasminogen Activators