A Role for Adipocyte-Enriched Transcription Factor, PPAR-γ, in Up-Regulation of PAI-1 Gene Expression during Adipogenesis.

Obese patients are at risk for development of cardiovascular disease, which can in part be explained by disturbances in the haemostatic and fibrinolytic systems. Recently, it has been demonstrated that the adipocyte itself is able to produce a primary fibrinolytic inhibitor, plasminogen activator inhibitor-1 (PAI-1), possibly explaining the high levels found in obesity. We have previously shown that pioglitazone, a selective ligand for peroxisome proliferator-activated receptor (PPAR)-γ, and insulin not only enhances adipocyte differentiation, but also up-regulates PAI-1 gene transcription in 3T3-L1 adipocytes (Ihara et al. FASEB J., 2001). These results suggest that PAI-1 gene expression during adipogenesis can be regulated by a direct action of PPAR-γ. In the present study, we investigate a role of an adipocyte-enriched transcription factor, PPAR-γ, in up-regulation of PAI-1 gene expression in adipose tissues. To identify cis-acting genetic elements required for induction of PAI-1 gene transcription, we constructed the plasmids containing a 5′ deletion series of the PAI-1 promoter. When sequences between -762 and -723 were deleted, the level of induction significantly fell. By sequence homology search in this region, we found an atypical PPAR-responsive element (aPPRE) at positions −752 to −733. Gel mobility shift and supershift assays using oligonucleotides of the aPPRE overlapping C/EBP binding site indicated that the aPPRE is important for PPAR-γ binding. Chromatin immunoprecipitation assay demonstrated that PPAR-γ is physically associated with PAI-1 gene promoter region including the aPPRE in vivo. Furthermore, mutation of the aPPRE abrogated the increase in transcriptional activities of PAI-1 promoter. Functional assay revealed that besides PPAR-γ/RXR-α expression vectors, co-transfection of CREB-binding protein expression vector increased the luciferase activity to 7.5 fold and 11 fold of control value in COS-1 cells and 3T3-L1 preadipocytes, respectively. These data indicate that the transcriptional induction of PAI-1 gene during adipogenesis is mediated through the binding of PPAR-γ/RXR-α to the functional aPPRE in the PAI-1 gene promoters.