Transcription coactivator peroxisome proliferator-activated receptor-binding protein/mediator 1 deficiency abrogates acetaminophen hepatotoxicity

Abstract

Peroxisome proliferator-activated receptor-binding protein (PBP), also known as thyroid hormone receptor-associated protein 220/vitamin D receptor-interacting protein 205/mediator 1, an anchor for multisubunit mediator transcription complex, functions as a transcription coactivator for nuclear receptors. Disruption of the PBP gene results in embryonic lethality around embryonic day 11.5 by affecting placental and multiorgan development. Here, we report that targeted deletion of PBP in liver parenchymal cells (PBP(Liv-/-)) results in the abrogation of hypertrophic and hyperplastic influences in liver mediated by constitutive androstane receptor (CAR) ligands phenobarbital (PB) and 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene, and of acetaminophen-induced hepatotoxicity. CAR interacts with the two nuclear receptor-interacting LXXLL (L, leucine; X, any amino acid) motifs in PBP in a ligand-dependent manner. We also show that PBP interacts with the C-terminal portion of CAR, suggesting that PBP is involved in the regulation of CAR function. Although the full-length PBP only minimally increased CAR transcriptional activity, a truncated form of PBP (amino acids 487-735) functioned as a dominant negative repressor, establishing that PBP functions as a coactivator for CAR. A reduction in CAR mRNA and protein level observed in PBP(Liv-/-) mouse liver suggests that PBP may regulate hepatic CAR expression. PBP-deficient hepatocytes in liver failed to reveal PB-dependent translocation of CAR to the nucleus. Adenoviral reconstitution of PBP in PBP(Liv-/-) mouse livers restored PB-mediated nuclear translocation of CAR as well as inducibility of CYP1A2, CYP2B10, CYP3A11, and CYP7A1 expression. We conclude that transcription coactivator PBP/TRAP220/MED1 is involved in the regulation of hepatic CAR function and that PBP deficiency in liver abrogates acetaminophen hepatotoxicity.

Fig. 1.

Liver weight and hepatocellular proliferation. (A) Liver weight/body weight ratios in PBP^+/+ and PBP^Liv-/- mice treated i.p. with PB (100 mg/kg), or TCPOBOP (TC) (3 mg/kg) daily for 3 days, compared with controls. PBP^+/+ mice treated with PB (P = 0.003) or TC (P = 0.0001) had significantly larger livers, compared with similarly treated PBP^Liv-/- mice. (B) Liver cell proliferation. Control and TC-treated mice (as above) were given BrdUrd in drinking water (0.5 mg/ml). Liver nuclear labeling in PBP^+/+ mice treated with TC was significantly higher, compared with PBP^Liv-/- mice (P = 0.0001). (C and D) Representative photomicrographs illustrate random distribution of BrdUrd-labeled nuclei in the TC-treated wild-type liver (C) but mostly restricted to centrilobular regions in PBP^Liv-/- livers (D). (E and F) Liver sections immunohistochemically stained for PBP reveal nuclear staining in all hepatocytes in wild-type mouse liver (E) but staining was limited to a few hepatocytes in PBP^Liv-/- mouse liver that represent cells escaping gene deletion (F).

Fig. 2.

The role of PBP in APAP hepatotoxicity. (A) Northern blot analysis of total liver RNA prepared from control, PBP^+/+, and PBP^Liv-/- mice and those treated with either PB or TC for 3 days. cDNA probes used for analysis are indicated; GAPDH serves as RNA loading control. (B) Immunoblot analysis of liver proteins of control, PB-, and TC-treated PBP^+/+ and PBP^Liv-/- mice for CAR, GSTπ, and catalase (CAT). (C and D) Serum ALT and hepatic GSH levels are shown. Mice were pretreated for 3 days with PB or PBS and given APAP (250 mg/kg i.p) 24 h before being killed. Serum ALT levels (C) and hepatic GSH levels (D) were determined at the time of being killed. ALT levels in PB + APAP and TC + APAP-treated PBP^+/+ mice were significantly high, compared with PBP^Liv-/- mice (P = 0.0021 and P = 0.0222, respectively). (E) Histological examination of livers from different groups. Note the centrilobular hepatic necrosis in PBP^+/+ mice pretreated with PB or TC before APAP administration and abrogation of necrosis in the livers of PBP^Liv-/- mice. PBS and APAP alone showed no hepatic necrosis.

Fig. 3.

CAR-PBP interactions. (A-D) GST pull-down assays. (A) Interaction of in vitro-translated ³⁵S-labeled PBP with bacterially expressed GST-CAR in the presence (+) and absence (-) of TCPOBOP (TC). (B and C) Binding of in vitro-translated ³⁵S-labeled CAR to bacterially expressed GST-PBP. CAR does not bind to PBP fragments consisting of amino acids 1-335, 740-1170, and 980-1370 (no LXXLL motifs) but does bind to amino acids 440-740 (contains both LXXLL motifs). Binding of CAR to PBP fragments with two LXXLL motifs is enhanced in the presence of ligand TC. (D) ³⁵S-labeled in vitro-translated PBP (amino acids 1-689) binds to GST-CAR in the presence of ligand. PBP mutants devoid of LXXLL motif NR1 or NR2 bound to CAR but PBP lacking both LXXLLs did not bind to CAR. (E) Electrophoretic gel mobility-shift analysis. In vitro-translated CAR, RXR, truncated mouse SRC-1 (amino acids 576-775), glucocorticoid receptor-interacting protein 1 (amino acids 617-769), and PBP (amino acids 487-751) proteins were used in the assay. CAR and RXR were allowed to bind radiolabeled oligonucleotide as the probe in the presence of ligand (TC). To this complex, coactivator proteins containing the LXXLL motifs were added as shown. The complex was separated on nondenaturing 5% PAGE. (F) Chromatin immunoprecipitation assays to show recruitment of PBP, peroxisome proliferator-activated receptor-interacting protein, SRC-1, and CREB-binding protein to the PB response unit in PBP^+/+ mouse liver. No detectable coactivator recruitment was found in PBP^Liv-/- mouse liver. (G) Repression of CAR-mediated transactivation by truncated PBP (PBPT). PB-responsive element-TK-LUC was cotransfected with pCMV-CAR along with pCMV-PBPT (amino acids 487-739) and pCMV into CV-1 cells in the presence or absence of TC. For control transfections, pCMV-FLAG2 plasmid was used instead of pCMV-PBPT. Luciferase activity is presented as percent, where induced CAR activity in the presence of TC is arbitrarily set at 100% (4).

Fig. 4.

The role of PBP in nuclear translocation of CAR by PB in mouse liver. (A-C) Immunohistochemical staining of liver sections for CAR of PBP^+/+ control (A), PBP^+/+ mouse treated with PB (WT + PB) (B), and PBP^Liv-/- mouse treated with PB (PBP^Liv-/- + PB) (C). Few hepatocytes in wild-type control liver show nuclear localization of CAR, whereas CAR is nuclear in almost all hepatocytes after PB treatment in wild-type mice. A PBP^Liv-/- mouse treated with PB for 3 days revealed only a few cells with nuclear CAR. (D-F) Adenoviral expression of PBP. Adjacent liver sections of adenovirally reexpressed PBP in PBP^Liv-/- mice were immunostained for PBP (D) and CAR (E). Note that reexpression of PBP leads to PB-induced nuclear translocation of CAR. (F) CAR translocation to nucleus was not seen in the absence of PB treatment in Ad/PBP-injected PBP^Liv-/- mouse liver. (G) Northern blot of liver RNA obtained from wild-type (PBP^+/+) and PBP liver-null (PBP^-/-) mice on control or PB-treated mice and from PBP liver-conditional mice infected with adenoviral vector expressing PBP (Ad/PBP). Reexpression of PBP in the liver of PBP-null mice restores PBP inducibility of CYP1A2 and CYP2B10 and, to a certain extent, increases the CAR mRNA level.