Abstract
Phenobarbital (PB), a widely used antiepileptic drug, is known to upregulate the expression of numerous drug-metabolizing enzymes and transporters in the liver primarily via activation of the constitutive androstane receptor (CAR, NR1I3). The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays an important role in intracellular citrate homeostasis that is associated with a number of metabolic syndromes and neurological disorders. Here, we show that PB markedly elevates the expression of SLC13A5 through a pregnane X receptor (PXR)-dependent but CAR-independent signaling pathway. In human primary hepatocytes, the mRNA and protein expression of SLC13A5 was robustly induced by PB treatment, while genetic knockdown or pharmacological inhibition of PXR significantly attenuated this induction. Utilizing genetically modified HepaRG cells, we found that PB induces SLC13A5 expression in both wild type and CAR-knockout HepaRG cells, whereas such induction was fully abolished in the PXR-knockout HepaRG cells. Mechanistically, we identified and functionally characterized three enhancer modules located upstream from the transcription start site or introns of the SLC13A5 gene that are associated with the regulation of PXR-mediated SLC13A5 induction. Moreover, metformin, a deactivator of PXR, dramatically suppressed PB-mediated induction of hepatic SLC13A5 as well as its activation of the SLC13A5 luciferase reporter activity via PXR. Collectively, these data reveal PB as a potent inducer of SLC13A5 through the activation of PXR but not CAR in human primary hepatocytes.
Highlights
The solute carrier family 13 member 5 (SLC13A5), known as Na+/citrate cotransporter (NaCT) or mammalian I’m Not Dead Yet, is a member of the sodium dicarboxylate/sulfate cotransporter family [1,2], mainly expressed in the liver, followed by the testes, brain, and bone marrows at much lower levels [3,4,5]
We provide experimental evidence to show that PB treatment markedly induces the expression of SLC13A5 at both mRNA and protein levels in human primary hepatocytes (HPH), while 6-(4-chlorophenyl) imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzy l)-oxime (CITCO), a selective human CAR agonist, only marginally affects SLC13A5 expression
Utilizing a combination of in silico gene analysis, electrophoretic mobility shift assays, and luciferase reporter experiments, we identify novel pregnane X receptor (PXR) response elements in both the promoter and intron regions of the SLC13A5 gene that contribute to PB-mediated induction
Summary
The solute carrier family 13 member 5 (SLC13A5), known as Na+/citrate cotransporter (NaCT) or mammalian I’m Not Dead Yet (mINDY), is a member of the sodium dicarboxylate/sulfate cotransporter family [1,2], mainly expressed in the liver, followed by the testes, brain, and bone marrows at much lower levels [3,4,5]. Due to the relatively high levels of citrate (100–150 μM) in the human bloodstream [11], the abundance of SLC13A5 on the sinusoidal membrane of hepatocytes determines how efficiently the liver can utilize circulating citrate for energy metabolism, lipid synthesis, and gene regulation. In an attempt to identify novel nuclear receptors as potential regulators of SLC13A5, we observed a robust induction of SLC13A5 expression in human primary hepatocytes (HPH) by phenobarbital (PB), a prototypical activator of the constitutive androstane receptor (CAR, NR1I3). Utilizing a combination of in silico gene analysis, electrophoretic mobility shift assays, and luciferase reporter experiments, we identify novel PXR response elements in both the promoter and intron regions of the SLC13A5 gene that contribute to PB-mediated induction. Our results reveal that PB is a robust inducer of hepatic SLC13A5 through activation of the PXR but not CAR signaling
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