Histone Methyltransferase G9a Regulates Expression of Nuclear Receptors and Cytochrome P450 Enzymes in HepaRG Cells at Basal Level and in Fatty Acid Induced Steatosis

Abstract

Obesity and non-alcoholic fatty liver disease (NAFLD) affect expression and function of cytochrome P450 genes (P450s). The increased expression of inflammatory cytokines is a major driver of the down regulation of P450 expression in NAFLD. Decrease in P450 expression could potentially lead to drug-drug interaction, inefficient pharmacological effect of a drug or hepatotoxicity. An epigenetic modifier, a histone methyl transferase enzyme G9a, known to increase histone 3 lysine 9 (H3K9) methylation, is down regulated in diet induced obesity animal models. In a liver specific G9a knockout animal model, expression of P450s was down regulated. Currently, the role of G9a in regulation of P450s in steatosis is unknown. Our hypothesis is that, in steatosis G9a plays a role in down regulation of P450 expression. In this study, we used HepaRG cells to induce steatosis using a combination of free fatty acids oleic acid and palmitic acid. The G9a was knocked down and overexpressed using small interfering RNA (siRNA) and adenovirus mediated approaches, respectively. Knockdown and overexpression of G9a in the absence of steatosis decreased and increased expression of nuclear receptors constitutive androstane receptor (CAR), pregnane x receptor (PXR), small heterodimer partner (SHP), and CYP2B6, 2E1, 2C8, 2C9, and 3A4, respectively. In steatotic conditions, overexpression of G9a prevented fatty acid mediated decreased expression of CAR, CYP2C19, 2C8, 7A1 and 3A4. Our current study suggests that G9a might serve as a key regulator of P450 expression at both the basal level and in early steatotic conditions. Single nucleotide polymorphism of G9a leading to loss/ gain of function could lead to the poor metabolizer or ultra-rapid metabolizer phenotypes.

The Long Noncoding RNA Hepatocyte Nuclear Factor 4α Antisense RNA 1 Negatively Regulates Cytochrome P450 Enzymes in Huh7 Cells via Histone Modifications

“…The involvement of histone modifications in the regulation of CYPs has been proved by more and more studies (Yan et al, 2017;Wang et al, 2019a;Pande et al, 2020). For instance, H3K27ace was reported to be involved in HNF4A-mediated CYP2C9 expression.…”

G9a promotes inflammation in Streptococcus pneumoniae induced pneumonia mice by stimulating M1 macrophage polarization and H3K9me2 methylation in FOXP1 promoter region

“…Epigenetic changes such as DNA methylation and histone modification are known to modulate gene expression changes and impact cellular function (28,29). As an epigenetic modifier, G9a has been documented to participate in many diseases (30,31) but its expression pattern and mechanism in Spn is largely unknown. This study investigated the mechanism of macrophage polarization mediated by histone methylase G9a in Spn mice, and eventually supported that G9a promoted M1 macrophage polarization by promoting H3K9me2 methylation in the FOXP1 promoter region, stimulating inflammation in Spn mice (Figure 8).…”

“…The ChIP experiment showed the enrichment of H3K9me2 in the FOXP1 promoter region in RAW264.7 macrophages was decreased after G9a knockdown, and after addition of Bix01294 [a specific inhibitor of G9a (H3K9me2)] to cells, the enrichment of H3K9me2 in the FOXP1 promoter region was evidently inhibited. An epigenetic modifier, G9a is known to increase H3K9me2 (30,38), and RT-qPCR showed that FOXP1 transcription was increased clearly after G9a depletion. FOXP1 expression was also decreased in the Spn group but increased after G9a depletion.…”

Effects of Overexpression of Fibroblast Growth Factor 15/19 on Hepatic Drug Metabolizing Enzymes

“…1 In vitro Treatment with Recombinant FGF19 Protein. HepaRG cells were cultured as previously described (Hart et al, 2010;Pande et al, 2020). PHHs were from a cryopreserved pool (5 donors) of human hepatocytes (PHH8007A, IVAL, Columbia, MD).…”