Regulation Of Plasminogen Activator Inhibitor-1 Expression During 3T3-L1 Adipocytes Differentiation

Plasminogen activator inhibitor-1 (PAI-1) is a primary physiological inhibitor of both tissue-type and urokinase-type plasminogen activators. Deficient fibrinolysis is due primarily to increased PAI-1 concentration in the blood. Low fibrinolytic activity in blood is a well-recognized major risk factor not only for thrombosis, but also for atherosclerosis. PAI-1 is expressed and secreted by adipose tissue (mainly adipocytes) which may mediate the pathogenesis of obesity- associated cardiovascular complications.The regulation of PAI-1 gene expression has been well studied. PAI-1 expression is induced by tumor necrosis factor α (TNFα) through a distal TNFα-responsive region or partly through a proximal NGFI-B/Nur77 responsive element (NBRE) mediated by Nur77 transcriptional factor. Analytical studies of PAI-1 promoter regulatory region have also allowed the identification of other transcriptional response sites, including CAGA box for transforming growth factor-β (TGF-β) responsiveness, a hypoxia-responsive element (HRE), an estrogen receptor responsive site, a very-low-density lipoprotein responsive site and two Sp1 sites for glucose/glucosamine and angiotensin responsiveness.Evidence is presented in this paper that PAI-1 is not expressed by preadipocytes, but significantly induced during 3T3-L1 adipocyte differentiation and the PAI-1 expression correlates with the induction of peroxisome proliferators-activated receptory (PPARγ) as well as one of the PPARγ target gene 422/aP2.PPARγ plays a central regulatory role in adipogenesis, the expression of PPARγ mRNA and protein are induced early in the adipogenesis. There have several reports that forced expression of PPARγ or treatment with its ligand can up-regulate the PAI-1 expression in adipocytes, but the mechanism has yet to be determined. PPARγ is a ligand-activated transcription factor. Pioglitazone is one specific PPARγ ligand; it is a member of Thiazolidinediones (TZDs) which are insulin sensitization compounds widely used for treatment of insulin resistance such as type 2 diabetes mellitus.A set of deletions of the PAI-1 promoter were constructed and analyzed for the ability to drive luciferase gene expression by PPARγ and its ligand Pioglitazone.The responsive region was determined in between -235bp to -178bp of mouse PAI-1 promoter which is responsive to PPARγ activation. A peroxisome proliferator responsive element (PPRE)-like m-element located between -206bp and -194bp (TCCCCCATGCCCT) was identified in the mouse PAI-1 promoter by electrophoretic mobility shift assay (EMSA) combined with transient transfection experiments; the PPRE-like element forms a specific DNA-protein complex only with adipocyte nuclear extracts, not with preadipocyte nuclear extracts; the DNA-protein complex can be totally competed away by none-labeled consensus PPRE, and can be supershifted with PPARγ antibody. Mutation of this PPRE-like ds-element can abolish the transactivation of mouse PAI-1 promoter mediated by PPARγ. Further research shows that specific PPARγ ligand Pioglitazone can not only significantly induce the PAI-1 expression, also stimulate the secretion of PAI-1 into medium.There exists a very similar sequence in human PAI-1 gene promoter between -987bp to -974bp (TCCCCTCACCCCCT). This may explain why obesity people have a high risk of thrombolytic complications.However clinical studies show that TZDs treatment lowers plasma PAI-1 levels in insulin resistant patients. The discrepancy is possible concerned with the receptor-independent pathway exerted anti-inflammatory activity by reduction of the TNFα expression and improvement of lipid and carbohydrate metabolism thus increasing insulin sensitivity in peripheral tissue, which results in suppression of PAI-1 expression, consequently counteracting transactivation of PAI-1 expression by PPARγ activation. In our study further investigation shows that the PPARγ is degraded and PAI-1 secretion also decreased significantly from day 10 adipocytes treated by Pioglitazone with insulin longer than 48 hours. It may explain how the TZDs activate PPARγ and lower plasma PAI-1 level.