Abstract
Previous studies showed that high levels of placenta growth factor (PlGF) correlated with increased plasma levels of endothelin-1 (ET-1), a potent vasoconstrictor, in sickle cell disease (SCD). PlGF-mediated transcription of the ET-1 gene occurs by activation of hypoxia inducible factor 1α (HIF-1α) and posttranscriptionally by microRNA 199a2 (miR-199a2), which targets the 3′ untranslated region (UTR) of HIF-1α mRNA. However, relatively less is known about how PlGF represses the expression of miR-199a2 located in the DNM3 opposite strand (DNM3os) transcription unit. Here, we show that PlGF induces the expression of activated transcription factor 3 (ATF3), which, in association with accessory proteins (c-Jun dimerization protein 2 [JDP2], ATF2, and histone deacetylase 6 [HDAC6]), as determined by proteomic analysis, binds to the DNM3os promoter. Furthermore, we show that association of HDAC6 with ATF3 at its binding site in this promoter was correlated with repression of miR-199a2 transcription, as shown by DNM3os transcription reporter and chromatin immunoprecipitation (ChIP) assays. Tubacin, an inhibitor of HDAC6, antagonized PlGF-mediated repression of DNM3os/pre-miR-199a2 transcription with a concomitant reduction in ET-1 levels in cultured endothelial cells. Analysis of lung tissues from Berkeley sickle (BK-SS) mice showed increased levels of ATF3 and increased expression of ET-1. Delivery of tubacin to BK-SS mice significantly attenuated plasma ET-1 and PlGF levels. Our studies demonstrated that ATF3 in conjunction with HDAC6 acts as a transcriptional repressor of the DNM3os/miR-199a2 locus.
ACKNOWLEDGMENTS
We thank Michael Kilberg (University of Florida) for kindly providing ATF3-luciferase reporter constructs. Mass spectrometry was performed by the USC Proteomics Core. We thank the technical assistance of Emiliano Huesca in some of the experiments.
This work was supported by grant number RO1-HL111372 (V.K.K. and P.M.) from the National Heart, Lung, and Blood Institute and by Analytical-Metabolic Instrumentation Core, University of Southern California Research Center for Liver Disease grant P30-DK048522 (to Neil Kaplowitz)
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We declare that we have no conflicts of interest with the contents of this article.
C.L. performed all in vitro experiments and data analysis. P.M. and A.L. designed and performed in vivo studies with tubacin and provided mouse lung tissues and plasma for analysis. Y.Z. performed proteomic analysis. V.K.K. and S.M.T. designed experiments, supervised analysis, and interpreted data. C.L., V.K.K., S.M.T., and P.M. wrote the manuscript.