Identification of 15d-PGJ2 as an antagonist of farnesoid X receptor: molecular modeling with biological evaluation

Abstract

15-Deoxy-Δ(12,14)-PGJ2 (15d-PGJ2) is one of the major metabolites from prostaglandin D2 in arachidonic acid (AA) metabolic pathway. It was determined as a ligand of peroxisome proliferator-activated receptor γ (PPARγ) functioning potently in adipocyte development. However, the fact that 15d-PGJ2 exerts also PPARγ-independent biological actions has highly addressed its multi-target behavior. Here, we identified that 15d-PGJ2 was an antagonist of farnesoid X receptor (FXR), as investigated by surface plasmon resonance, fluorescence quenching and homo time-resolved fluorescence based analyses, and the coactivator-recruitment and luciferase-reporter related investigation. Assay of 15d-PGJ2 regulation on hFXRα target genes revealed that treatment of HepG2 cells with 15d-PGJ2 resulted in the stimulation of mRNA expressions of bile-salt export pump (BSEP), and the decrease of cholesterol 7a-hydroxylase (CYP7a1). In addition, functional assays indicated that 15d-PGJ2 promoted the conversion of cholesterol to bile acids in HepG2 cells. Moreover, molecular docking combined with molecular dynamics simulation was applied to develop the possible model of 15d-PGJ2 binding to hFXRα ligand binding domain (LBD) at atomic level, and the responsible residues for 15d-PGJ2/hFXRα-LBD interaction were thereby determined, which were further confirmed by SPR assays against hFXRα-LBD site-directed mutations. Given that hFXRα functions potently in the regulation of hepatic bile acid metabolism and lipid/glucose homeostasis, our current work is expected to help better understand the multi-target features of this PGD2 metabolite in biological pathways, and 15d-PGJ2 as a new discovered FXR antagonist might find its potential application in further anti-hypercholesterol research.

Keywords: 15-Deoxy-Δ(12,14)PGJ(2); 15-deoxy-Δ(12,14)-PGJ(2); 15d-PGJ(2); 50% inhibitory concentration; 6-ECDCA; 6α-ethyl-chenodeoxycholic acid; AA; AF-2; Antagonist; BSEP; CDCA; CYP7a1; DMEM; DMSO; DTT; Dulbecco’s modified eagle’s medium; FBS; FQ; FXR; FXR elements; FXRE; Farnesoid X receptor; GS; Guggulsterone; HTRF; IC(50); IPTG; IκB kinase (IKK), Erk; K(D); Keap1; Kelch-like ECH-associated protein 1; LBD; LXR; MD; Molecular dynamics simulation; NR; PGD(2); PGH(2); PLA(2); PPARα; PPARγ; ROS; RUs; RXRα; SPR; TNF-α; arachidonic acid; bile-salt export pump; chenodeoxycholic acid; cholesterol 7a-hydroxylase; dimethyl sulfoxide; dithiothretiol; extracellular signal-regulated kinase; farnesoid X receptor; fetal bovine serum; fluorescence quenching; homo time-resolved fluorescence; iNOS; inducible NO synthase; isopropyl β-D-thiogalactoside; k(off); k(on); ligand binding domain; liver X receptor; molecular dynamics; nuclear receptor; peroxisome proliferator-activated receptor γ; phopholipase A(2); proliferator-activated receptor α; prostaglandin D(2); prostaglandin H(2); response units; retinoid X receptor α; rosiglitazone; surface plasmon resonance; the association rate constants; the dissociation rate constants; the equilibrium dissociation constant; transactivation function 2 domain; tumor necrosis factor α.