Ligand-dependent and -independent regulation of human hepatic sphingomyelin phosphodiesterase acid-like 3A expression by pregnane X receptor and crosstalk with liver X receptor

Biochem Pharmacol. 2017 Jul 15;136:122-135. doi: 10.1016/j.bcp.2017.04.013. Epub 2017 Apr 13.

Abstract

Pregnane X receptor (PXR) mainly regulates xenobiotic metabolism and detoxification. Additionally, it exerts pleiotropic effects on liver physiology, which in large parts depend on transrepression of other liver-enriched transcription factors. Based on the hypothesis that lower expression levels of PXR may reduce the extent of this inhibition, an exploratory genome-wide transcriptomic profiling was performed using HepG2 cell clones with different expression levels of PXR. This screen and confirmatory real-time RT-PCR identified sphingomyelin phosphodiesterase acid-like (SMPDL) 3A, a novel nucleotide phosphodiesterase and phosphoramidase, as being up-regulated by PXR-deficiency. Transient siRNA-mediated knock-down of PXR in HepG2 cells and primary human hepatocytes similarly induced mRNA up-regulation, which translated into increased intracellular and secreted extracellular protein levels. Interestingly, ligand-dependent PXR activation also induced SMPDL3A in HepG2 cells and primary human hepatocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated binding of PXR to the previously identified liver X receptor (LXR)-binding DR4 motif as well as to an adjacent ER8 motif in intron 1 of SMPDL3A. Constitutive binding of the unliganded receptor to the intron 1 chromatin indicated ligand-independent repression of SMPDL3A by PXR. Transient transfection and reporter gene analysis confirmed the specific role of these motifs in PXR- and LXR-dependent activation of the SMPDL3A intronic enhancer. PXR inhibited LXR mainly by competition for binding sites. In conclusion, this study describes that a decrease in PXR expression levels and ligand-dependent activation of PXR and LXR increase hepatic SMPDL3A levels, which possibly connects these receptors to hepatic purinergic signaling and phospholipid metabolism and may result in drug-drug interactions with phosphoramidate pro-drugs.

Keywords: GW3965 hydrochloride (PubChem CID: 16078973); Gene regulation; Hepatocytes; Ligand-independent repression; Liver X receptor; Pregnane X receptor; Rifampin (PubChem CID: 6243627); T0901317 (PubChem CID: 447912).

MeSH terms

  • Adult
  • Aged
  • Cells, Cultured
  • Female
  • Gene Expression
  • Hep G2 Cells
  • Hepatocytes / metabolism*
  • Humans
  • Ligands
  • Liver X Receptors / metabolism*
  • Male
  • Middle Aged
  • Pregnane X Receptor
  • Receptor Cross-Talk / physiology*
  • Receptors, Steroid / metabolism*
  • Sphingomyelin Phosphodiesterase / biosynthesis*
  • Sphingomyelin Phosphodiesterase / genetics

Substances

  • Ligands
  • Liver X Receptors
  • Pregnane X Receptor
  • Receptors, Steroid
  • SMPDL3A protein, human
  • Sphingomyelin Phosphodiesterase