The pregnane X receptor down‐regulates organic cation transporter 1 (SLC22A1) in human hepatocytes by competing for (“squelching”) SRC‐1 coactivator

Abstract

BACKGROUND AND PURPOSEThe organic cation transporter 1 (OCT1) transports cationic drugs into hepatocytes. The high hepatic expression of OCT1 is controlled by the HNF4α and USF transcription factors. Pregnane X receptor (PXR) mediates induction of the principal xenobiotic metabolizing enzymes and transporters in the liver. Here, we have assessed the down-regulation of OCT1 expression by PXR activation. EXPERIMENTAL APPROACHWe used primary human hepatocytes and related cell lines to measure OCT1 expression and activity, by assaying MPP + accumulation. Western blotting, qRT-PCR, the OCT1 promoter gene reporter constructs and chromatin immunoprecipitation assays were also used. KEY RESULTSOCT1 mRNA in human hepatocytes was down-regulated along with reduced [ 3 H]MPP + accumulation in differentiated HepaRG cells after treatment with rifampicin. Rifampicin and hyperforin as well as the constitutively active PXR mutant T248D suppressed activity of the 1.8 kb OCT1 promoter construct in gene reporter assays. Silencing of both PXR and HNF4α in HepaRG cells blocked the PXR ligand-mediated down-regulation of OCT1 expression. The mutation of HNF4α and USF1 (E-box) responsive elements reversed the PXR-mediated inhibition in gene reporter assays. Chromatin immunoprecipitation assays indicated that PXR activation sequestrates the SRC-1 coactivator from the HNF4α response element and E-box of the OCT1 promoter. Consistent with these findings, exogenous overexpression of the SRC-1, but not the PGC1α coactivator, relieved the PXR-mediated repression of OCT1 transactivation. CONCLUSIONS AND IMPLICATIONSPXR ligands reduced the HNF4α-mediated and USF-mediated transactivation of OCT1 gene expression by competing for SRC-1 and decreased delivery of a model OCT1 substrate into hepatocytes. AbbreviationsCAR, constitutive androstane receptor; ChiP , chromatin immunoprecipitation assay; CML, chronic myeloid leukaemia; DR-2, direct repeat separated by two nucleotides; HNF4α, hepatocyte nuclear factor-4-α; OCT1, organic cation transporter 1; PXR, pregnane X receptor; SRC-1, steroid receptor coactivator; PGC1α, PPARγ coactivator 1α; USFs, upstream stimulating factors

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

“…Therefore, a functional cross-talk between PXR and HNF-4α, a key hepatic regulator of genes involved in bile acid synthesis including the cholesterol 7-alpha hydroxylase (CYP7A1) and sterol 12-alpha hydroxylase (CYP8B1) genes, has been studied. It was shown that PXR interacts with the coactivator PGC-1α through its C-terminal ligand binding domain in a rifampicin-dependent manner and that PGC-1α coactivates PXR transactivation (Bhalla et al, 2004;Chiang, 2005, 2006;Hyrsova et al, 2016). Consistently, endogenous Pgc-1α from mouse liver extracts was found to bind to PXR, and recombinant PGC-1α directly interacts with PXR.…”

“…PGC-1α at the same time coactivates and enhances the transcriptional activity of HNF-4α in the regulation of several liver-specific genes, including CYP7A1, CYP8A1, SHP, OCT1 (SLC22A1), and PEPCK1 (Bhalla et al, 2004;Chiang, 2005, 2006;Hyrsova et al, 2016). This PGC-1α coactivation of HNF4α was reported to be suppressed by PXR ligands in an SHP-independent manner.…”

“…Coactivation by SRC-1 stimulated after PXR activation has not been confirmed in some rigorous biophysical studies (Navaratnarajah et al, 2012). It was also demonstrated that PXR and SRC-1 interact in the absence of a PXR ligand, and further, that the interaction is strengthened by rifampicin (Saini et al, 2005;Li and Chiang, 2006;Rulcova et al, 2010;Krausova et al, 2011;Hyrsova et al, 2016). Moreover, no ligand activated release, or even stronger interaction with SMRTα has been reported for PXR (Takeshita et al, 2002;Mani et al, 2005;Li et al, 2009;Navaratnarajah et al, 2012;Hirooka-Masui et al, 2013).…”

“…Activated PXR competes and deplete ("squelches") the SRC-1 coactivator from HNF4-mediated transactivation of OCT1 (SLC22A1) gene. OCT1 is dominantly regulated by HNF4α in the liver (Hyrsova et al, 2016)…”

“…SRC-1 interacts with many NRs including PXR, LXRα, CAR, FXR, HNF4α, GR etc. It was shown that PXR and SRC-1 interact in the absence of a PXR ligand and the interaction is strengthened by rifampicin (Saini et al, 2005;Li and Chiang, 2006;Rulcova et al, 2010;Krausova et al, 2011;Hyrsova et al, 2016), although SRC-1 was proposed to bind to PXR much weaker that PGC-1α (Li and Chiang, 2005).…”