Abstract 13238: Simvastatin Upregulates Endothelial Cell Integrin β4 Expression by Promoter Activation and Epigenetic Modification
Introduction: We previously reported that protection conferred by simvastatin in murine acute lung injury (ALI) is mediated by the upregulation of endothelial cell integrin β4 (ITGB4). However, the mechanisms underlying these effects are unknown. To explore this further, we investigated ITGB4 promoter regulation and epigenetic modifcations by simvastatin.
Methods: Human pulmonary artery EC were transfected with luciferase reporter promoter deletion constructs prior to treatment with simvastatin (5 μM) to identify specific 5’ regions within the ITGB4 gene responsible for statin-mediated ITGB4 expression. Regions of interest were then subjected to chromatin immuoprecipitation (ChIP) to identify candidate transcription factors (TFs). In separate experiments, ECs were pretreated with either a histone deacteylation inhibitor, trichostain (TSA, 5 μM, 2h), or a histone methylation inhibitor, 5-aza-2’-deoxycytidine (5-Aza, 5 μM, 2h) prior to treatment with simvastatin (5 μM, 16 h) followed by ITGB4 mRNA and protein assay, respectively. Finally, ChIP assays were conducted to assess simvastatin effects on ITGB4 promoter methylation as well as specific histone modifications including H3K9 acetylation (H3K9ac) and H3K4 trimethylation (H3K4me3), and trimethylation of H3K27 (H3K27me3).
Results: ChIP assays identified simvastatin-induced NF-kB binding to the ITGB4 promoter. Subsequently, we confirmed that silencing of NF-kB2 specifically inhibits ITGB4 upregulation by simvastatin. In addition, inhibition of histone deacetylation (TSA) resulted in decreased simvastatin-induced ITGB4 mRNA and protein levels while inhibition of histone methylation (5-Aza) was associated with increased ITGB4 mRNA and protein levels in response to simvastatin. Finally, simvastatin treatment induced significant ITGB4 promoter demethylation in addition to both increased H3K9ac and H3K4me3 as well as decreased H3K27me3.
Conclusion: These data confirm ITGB4 upregulation by simvastatin is mediated by both NF-kB activation of the ITGB4 promoter and specific epigenetic modifications. Our findings identify mechanisms of ITGB4 expression regulation which may ultimately lead to novel therapeutic strategies in patients with ALI or with cancer.
Author Disclosures: W. Chen: None. X. Ni: None. L. Xie: None. V. Elagovan: None. J.R. Jacobson: None.
- © 2014 by American Heart Association, Inc.