OR23-5 A Model “Obesogen”, Tributyltin, Promotes Steatosis in Human Liver Cells by Upregulating Lipogenic Gene Expression as a Consequence of Alterations in Both Genomic and Non-Genomic Signaling

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a worldwide problem. In addition to known risk factors, such as consumption of high-fat diets, animal data suggests a clear causal link between exposure to obesogenic chemicals, such as tributyltin (TBT), and NAFLD development. TBT can directly bind and potently activate peroxisome proliferator activator gamma (PPARγ) and retinoid-x-receptor alpha (RXRα) at nanomolar concentrations relevant to human exposures. However, exactly how TBT binding to PPAR-RXR heterodimers may induce lipid accumulation in human liver cells is not understood. To address this, we examined the molecular actions of an acute TBT exposure to the human hepatoma cell line HepaRG on lipid accumulation, the transcriptome, kinase signaling pathways, and the recruitment of epigenetic coregulator complexes to PPRE-containing target genes. From microscopy of LipidTox stained cells, we found that a 72 hour exposure to 50 nM TBT induced lipid accumulation as compared to DMSO vehicle treatment. Consistent with this observation, upon analysis of RNA-sequencing data of vehicle versus TBT treated cells (for 6 hours only), we found 813 genes to be differentially expressed (q less than 0.05; fold change 1.25). Many lipogenic genes were found to be TBT induced, including known PPAR-RXR targets (e.g., ANGPLT4, PLIN2, and ACSL3). Importantly, we further validated that these select TBT-induced genes are also induced in TBT-treated primary human hepatocytes. Small-interfering RNA knockdown of ANGPLT4 or PLIN2 significantly reduced the number and size of lipid droplets in TBT-treated HepaRG cells. Upon immunoblot screening of kinase pathway activation, we found many pathways are rapidly activated (within 5 to 30 minutes) in HepaRG cells, including the mitogen-activated protein kinases (ERK1/2, JNK, and p38), protein kinase D and AMP activated kinase. We further tested the effect of pharmacological inhibitors to each kinase on validated TBT-induced genes, which revealed significantly reduced expression of only ANGPLT4 with ERK kinase inhibition. Using HepaRG nuclear extracts in a peroxisome proliferator activator response element (PPRE) DNA pulldown assay coupled with mass spectrometry, we found that TBT, either added cell-free or to live HepaRG cells for 30 minutes, induced the recruitment of distinct epigenetic coregulators to the 3xPPRE-containing template. Specifically, we revealed enhanced binding of steroid receptor coactivator 2, MLL3/4 histone H3 lysine 4 methyltransferases, the H3K27 methyltransferase EZH2, the histone demethylase LSD2, and the histone deacetylase HDAC3. We are currently testing how TBT exposure may affect the histone post-translational modifications created/removed by each of these complexes on our above-validated endogenous target genes.This work was supported by grants from National Institutes of Health (NIEHS and NIDDK).