AIMTo investigate the molecular mechanism underlying the effect of linoleic acid on plasminogen activator inhibitor type-1 (PAI-1) expression in HepG2 cells.

METHODSHepG2 cells were exposed to different concentrations of linoleic acid and PAI-1 expression was determined by RT-PCR and colorimetric assay. Luciferase reporter gene plasmids containing four sequentially truncated fragments of the PAI-1 promoter region (-804 to +17) were constructed, and plasmids carrying constructs of Smad binding element (SBE)-site directed deletions in PAI-1 promoter were also generated using overlap extention PCR and transiently transfected into HepG2 cells, the transcriptional activity of PAI-1 was demonstrated by the luciferase activity.The effect of linoleic acid on Smad3 and Smad4 protein levels in cultured HepG2 cells was measured by Western blot analysis.

RESULTS(1) Linoleic acid remarkably increased PAI-1 mRNA expression and transcription in varying concentrations. (2) The level of PAI-1 transcription was gradually decreased induced by linoleic acid when transfected the SBE- site directed-deletions plasmids in PAI-1 promoter at -734/-731. (3) Protein levels of both Smad3 and 4 in HepG2 cells were increased by linoleic acid.

CONCLUSIONLinoleic acid regulated the expression of PAI-1 from transcriptional level in HepG2 cells and SBE involved in the regulation, and both Smads protein and Smad signaling pathway acted main role in this procession.