ABSTRACT
Our group has recently reported that in the immortal human HepG2 liver cell line, sphingosine 1phosphate (S1P) increases transcription of plasminogen activator inhibitor type1 (PAI1), the major physiological inhibitor of fibrinolysis, within 4 h. The present study aimed to elucidate the molecular mechanisms underlying this effect. PAI1 expression was measured by reverse transcriptionquantitative polymerase chain reaction and immunoblotting. It was demonstrated that S1P increased PAI1 promoter activity but did not increase the activity of promoters lacking the hypoxia responsive element (HRE) 2. In addition, S1P transiently increased the concentration of hypoxia inducible factor (HIF)1α, a transcription factor capable of binding to HRE. When HIF1α was knocked down, the induction of transcription of PAI1 by S1P was no longer observed. Sphingosine kinase (SPHK) activity is increased by hypoxia. It was demonstrated that increases in the concentration of the HIF1α protein induced by hypoxia were prevented by treatment with SPHK inhibitor or S1P receptor antagonists. Thus, modification of the induction of HIF1α by S1P, leading to increased transcription of PAI1, may be an attractive therapeutic target for thrombosis and consequent inhibition of fibrinolysis associated with hypoxia.
Subject(s)
Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/genetics , Hypoxia/metabolism , Lysophospholipids/biosynthesis , Plasminogen Activator Inhibitor 1/genetics , Sphingosine/analogs & derivatives , Autocrine Communication , Hep G2 Cells , Humans , Paracrine Communication , Promoter Regions, Genetic , Receptor, ErbB-2/metabolism , Sphingosine/biosynthesis , Transcriptional ActivationABSTRACT
Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a major physiologic inhibitor of fibrinolysis, is implicated in the progression of atherosclerosis. Sphingosine 1-phosphate (S1P) regulates expression of diverse genes and alters expression of PAI-1 in several types of cells. However, the nature of posttranscriptional regulation of expression of PAI-1 by S1P has not yet been thoroughly elucidated. The present study was undertaken to determine whether S1P has important effects on the posttranscriptional regulation of PAI-1 expression. To evaluate this possibility, we determined promoter activity, mRNA levels, 3'-untranslated region (UTR) activity, and protein levels of PAI-1 in HepG2 cells. S1P increased PAI-1 promoter activity and the expression of PAI-1 mRNA within 4h of exposure. It decreased the expression of PAI-1 mRNA and the accumulation of PAI-1 protein into the media in 24h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2kb). S1P decreased the baseline luciferase activity of the 1kb fragment of the 3' terminus (+2177 to 3176nt) of the 3'-UTR of the 3.2kb PAI-1 mRNA [3'-UTR (+2177-3176)]. S1P decreased expression of PAI-1 protein, presumably by regulating PAI-1 expression at the posttranscriptional level thereby affecting mRNA stability. SERPINE1 mRNA binding protein (SERBP1) and ARE3 in the 3'-UTR were involved in the posttranscriptional regulation by S1P. Our data suggest that S1P can destabilize 3.2kb PAI-1 mRNA through specific effects on the 3'-UTR. These effects appear to involve SERBP1 leading to decreased expression of PAI-1 protein.