Abstract 12804: HIF-1a/HuR Pathway Regulates the Increased Expression of PAI-1 Induced by Sphingosine 1-phosphate in HepG2 Cells Rendered Hypoxic: A Novel Linkage between Thrombosis and Hypoxia
Hypoxia is a prothrombotic risk. The mechanisms are not fully described. We have recently shown that in human HepG2 liver cell line sphingosine 1-phosphate (S1P), a bioactive lipid richly endowed in platelets and released upon activation, increases transcription of plasminogen activator inhibitor type-1 (PAI-1), the physiologic inhibitor of fibrinolysis, within 4 hours. In this study we elucidated the molecular mechanisms responsible. S1P (500 nM) increased PAI-1 promoter activity by 2 fold (luciferase assay). Hypoxia and S1P had additive effects and increased PAI-1 promoter activity by 10 fold. S1P did not increase the activity of promoters lacking the hypoxia responsive element (HRE) 2. S1P transiently increased the concentration of hypoxia inducible factor (HIF)-1α, a transcriptional factor capable of binding to HRE (Western blot). When HIF-1α was knocked down by siRNA, the induction of transcription of PAI-1 by S1P was no longer observed, suggesting that S1P and hypoxia increase PAI-1 in relation to HRE2. Hypoxia increased enzymatic activity of sphingosine kinase, a key enzyme for S1P synthesis from sphingosine. Inhibitor of sphingosine kinase, SKI, attenuated the increases of HIF-1α induced by hypoxia. Increases in concentrations of the HIF-1α protein induced by hypoxia were prevented by S1P1/3 receptor antagonists, VPC-23019.
Knockdown of Hu-antigen R (HuR), an RNA binding protein that can promote HIF-1α translation under conditions of hypoxia, decreased the induction of HIF-1α protein by S1P and hypoxia. HuR overexpression increased HIF-1α and PAI-1 protein. S1P increased the binding to HuR to HIF-1α mRNA (immunoprecipitation). Hypoxia and S1P induced shuttling of HuR from nuclei to cytoplasm (immunofluorescence microscopy), which was prevented by VPC-23019. Thus, hypoxia and S1P promoted binding of HuR to HIF-1α mRNA. These results suggest that the HIF-1α/HuR pathway is a necessary component of the pathway that leads to increased transcription of PAI-1 induced by hypoxia. Modification of induction of HIF-1α by S1P leading to increased transcription of PAI-1 may be an attractive therapeutic target to diminish thrombosis exacerbated by elaboration of PAI-1 and consequent inhibition of fibrinolysis associated with hypoxia.
- © 2013 by American Heart Association, Inc.