Mechanism Of Different Activators Of PPAR α Regulating Plasminogen Activator Inhibitor-1 Expression

Plasminogen activator inhibitor-1(PAI-1) is a main inhibitor with plasma fibrinolysis activity in vivo, which regulating and maintaining the dynamic balance between the system of fibrinolysis and thrombosis. High plasma level of PAI-1 is closely associated with many thromboembolic diseases such as artherosclerosis, coronary heart disease, myocardial infarction and cerebral infarction and so on.. Peroxisome proliferator-activated receptor alpha (PPARα ) is a family of nuclear transcriptional factors that activated by ligand. They have various biological effects and play a key role in many process of physiology and physiology. Fenofibrate and linoleic acid are specific exogenous and exogenous activators of PPAR α , but the mechanism how they regulate PAI-1 and PPAR α are less known. So we use molecular biology and cell biology techniques to investigate the mechanism and the study include three parts. 1. Effect of different activators of PPAR α on PAI-1 and PPAR α in HepG2 cellsObjective: To observe the effects of fenofibrate and linoleic acid which was the different activators of PPAR α on the expression of PAI-1 and PPAR α in HepG2 cells. Methods: HepG2 cells were exposed to fenofibrate and linoleic acid in varying concentrations, RT-PCR was used to determine the mRNA expression of PAI-1 and PPAR α . Results: 1. Fenofibrate could remarkably decreased PAI-1 mRNA expression in HepG2 cells (P <0.05 or P<0.01), but linoleic acid could significant increased PAI-1 mRNA level (P <0.05 or P<0.01), and they were all in a concentration-dependent manner. 2. Fenofibrate and linoleic acid could raise the mRNA level of PPARa (P<0.05 or P<0.01), which were also in a concentration-dependent way. Conclusions: Fenofibrate and linoleic acid couldincrease the mRNA level of PPARa , and they regulat the synthesis of PAI-1 from transcriptional level, which was concerned with the activated of PPAR a by Fenofibrate and linoleic acid. The effects on expression of PAI-1 were very inconsistently, so there might be other mechanisms involved. 2. Effect and mechanism of different activators of PPAR a on the expression of PAI-1 in HepG2 cellsObjective: To investigate the relationship and mechanisms between the promoter and expresstion of PAI-1 that how the fenofibrate and linoleic acid to act on PAI-1. And to observe if PPAR a involved in this procession. Methods: Several luciferase reporter gene recombinant plasmid containing different length sequences of human PAI-1 gene promoter from -804 to +17bp were constructed and transiently transfected into HepG2 cells.At the same time, co-transfected with PPAR a -pSG5 expression plasmid, then different stimulating factors were added to induce the transfected cells. Transcriptional activity of PAI-1 was demonstrated by the measure of luciferase activity. Results: 1. The PAI-1 transcriptional activity were significantly suppressed by fenofibrate ( P <0.05 ) , but induced by linoleic acid ( P<0.01 ) In HepG2 cells transfected with PAI-pGL3 total length promoter constructs. 2. When co-transfected with PPAR a -pSG5, fenofibrate could suppress the level of PAI-1 transcription further more( P<0.05), while increased y linoleic acid( PO.Ol). 3. The PAI-1 transcriptional activity were very inconsistent When transfected with the plasmid containing different length sequences of human PAI-1 gene promoter from -804 to +17bp. Conclusions:!. Fenofibrate could suppress the gene transcription of PAI-1 in HepG2 cells , but induced by linoleic acid, they could both regulate the gene expression of PAI-1 from transcriptional level. 2. PPARa involved in the regulation of PAI-1 gene expression induced by fenofibrate and linoleic acid. 3. The sequences that could regulate the expression of PAI-1 gene induced by fenofibrate might exist in the areas from -804 to -636 and -636 to -449 of PAI-1 promoter.and existed in the areas rom -804 to -636 and -449 to -276 induced bylinoleic acid. 4.There was a specific sequence CAGA named Smad binding element (SBE) located in the promotor of PAI-1, the sequence and Smad signaling pathway might be involved in the regulation of PAI-1 gene expression by fenofibrate and linoleic acid..3. Role of Smad signaling pathway in the regularion of PAI-1 induced by different activators of PPAR a in HepG2 cellsObjective: To confirm if the Smad binding element (SBE) and Smads protein /Smad signaling pathway involved in the regulation of PAI-1 gene expression by fenofibrate and linoleic acid. Methods:The site-directed mutants plasmids of SBE in PAI-1 promoter were constructed using overlap extention PCR and transiently transfected into HepG2 cells.Transcriptional activity of PAI-1 was demonstrated by the luciferase activity. Protein levels of Smad3 and 4 were measured by Western blot. Results: 1. The level of PAI-1 transcription was gradually increased induced by fenofibrate when transfected the plasmid of dl-PAI-pGL3, d2-PAI-pGL3 and d3-PAI-pGL3 to HepG2 cells. Compared between A-PAI-pGL3 and dl-PAI-pGL3 plasmid, transcriptional activity of PAI-1 changed remarkably and it was the same when Compared between d2-PAI-pGL3 and dl-PAI-pGL3 palsmids. 2. The level of PAI-1 transcription was decreased obviously induced by linoleic acid when transfected plasmid of dl-PAI-pGL3. 3. Fenofibrate could suppress the protein levels of Smad3 and 4 in HepG2 cells, but linoleic acid was conversely. Conclusions: 1. The SBEs sited in -734/-731 and -585/-582 of PAI-1 promotor could regulate gene expression of PAI-1 induced by fenofibrate, but only the SBE sited in -734/-731 might play its role induced by linoleic acid.. 2. Smads protein and Smad signaling pathway involved in the regulation of PAI-1 gene expression by fenofibrate and linoleic acid.Summary1. Fenofibrate and linoleic acid could regulate the expression of PAI-1 from transcriptional level in HepG2 cells and playconversely role, fenofibrate was to supress,while linoleic acid was upregulation.2. Fenofibrate and linoleic acid could activate PPARa by up-regulating the mRNA level of PPAR a in HepG2 cells, PPAR a involved in the regulation of PAI-1 gene expression induced by fenofibrate and linoleic acid.3. The specific sequences CAGA located in PAI-1 promotor, which named Smad binding element (SBE) were involved in the regulation of PAI-1 gene expression by fenofibrate and linoleic acid, and both Smads protein and Smad signaling pathway acted main role in this procession.