Redundant enhancement of mouse constitutive androstane receptor transactivation by p160 coactivator family members

Abstract

Constitutive androstane receptor (CAR) transactivation is enhanced by p160 coactivators, which include three members, SRC-1, SRC-2, and SRC-3. Each of the p160 coactivators enhanced mouse CAR (mCAR) transactivation of the CYP2B1 phenobarbital (PB)-responsive enhancer in transfected cultured cells and mouse hepatocytes in vivo. The cellular localization of the p160 coactivators in hepatocytes in vivo was not altered by PB treatment, nor did any of the p160 coactivators selectively colocalize with mCAR in the nucleus. Exogenous expression of each p160 coactivator mediated the PB-independent nuclear accumulation of mCAR in hepatocytes in vivo. Induction of Cyp2b10 gene expression by PB was equivalent or greater in mice null for each of the p160 coactivators than in wild type mice. These results indicate that the p160 coactivators are redundant with regard to enhancing CAR-mediated induction of cytochrome P450 genes. SRC-3 alone of the p160 coactivators enhanced CAR transactivation in hepatic cells without PB treatment.

Figure 1

Transactivation by mCAR and p160 coactivators of the CYP2B2 PBRU in transiently transfected HepG2 and Cos1 cells. HepG2 and Cos1 cells were transfected with expression vectors for GFPCAR and individual SRC coactivators (pCMV160), a reporter plasmid containing the CYP2B2 PBRU fused to the promoter of CYP2C1 and the firefly luciferase gene, and pRLSV40 containing the SV40 enhancer/promoter and the Renilla luciferase gene as an internal standard. Luciferase activities were determined as described under “Materials and Methods,” and the activity was calculated as a ratio of firefly luciferase activity to Renilla luciferase activity. Triplicate transfections in two separate experiments were done, and the standard errors are indicated.

Figure 2

Transactivation by mCAR and p160 coactivators of the CYP2B2 PBRU in transiently transfected hepatocytes in vivo. Expression and reporter plasmids that were used are described in the legend to Fig.1. Hepatocytes were transfected by injection of DNA via the tail veins of mice. Twenty-four h after injection of DNA, the mice were sacrificed, and the liver extracts were prepared as described in “Materials and Methods.” Luciferase activities were determined as described in the legend to Fig. 1. Standard errors are shown with n values of 5 or 6

Figure 3

Cellular localization in vivo of CAR-GFP chimera in hepatocytes expressing exogenous p160 coactivators. Hepatocytes of untreated mice were transfected in vivo by tail vein injection of expression vectors for mCAR and individual p160 coactivators as indicated and were imaged by confocal microscopy as described under “Materials and Methods”. Forty to 100 cells were randomly selected for each of the control mice and mice injected with p160 expression vectors, and the RNF was calculated as described in “Materials and Methods”. The mean and standard deviation of the RNF are shown. * p < 0.01 compared to the control.

Figure 4

Subcellular distribution of p160 coactivators in mouse hepatocytes in vivo. Expression vectors for GFP chimera of the p160 coactivators were injected into mice by tail vein injection. Two h after injection, the mice were treated intraperitoneally with saline (control) or 100 μg/g body weight PB and were sacrificed 4 h later. Frozen tissues were prepared and DNA was stained with propidium iodide. Fluorescence was detected with a laser confocal microscope. Two representative cells are shown for each SRC group. For each cell, both red fluorescence from propidium iodide and green fluorescence from GFP are shown in the left panel, and only green fluorescence is shown in the right panel.

Figure 5

Coexpression of GFP chimera of p160 coactivators and RFPCAR in mouse hepatocytes in vivo. Ten μg of expression vectors for GFP chimera of p160 coactivators and HcREDCAR were injected into the tail vein of mice. Two h after injection, the mice were treated intraperitoneally with 100 μg/g body weight PB and were sacrificed 4 h later. Frozen tissues were prepared and fluorescence was detected with a laser confocal microscope. Red fluorescence is from HcREDCAR and green fluorescence is from GFP chimera of p160 coactivators (GFPp160). To better illustrate the localization, only a magnified portion of a nucleus of a representative cell simultaneously expressing a GFP chimera of a p160 coactivator and RFPCAR is shown.

Figure 6

PB-mediated Cyp2b10 mRNA induction in p160-null mice. Total RNA was isolated from mouse liver 16 h after injection i.p. with isotonic saline (C) or 100 μg/g PB from either congenic wild type mice (WT) or from p160-null (Null) mice. The null gene for both (A) and (B) is indicated at the bottom of the figure. Quantitative RT-PCR (A) or semi-quantitative RT-PCR (B) was performed using total liver RNA. The amount of Cyp2b10 DNA was normalized to that of β-actin included as an internal control. The numbers inside the panels indicate the fold-induction. The standard error of the mean is indicated for an n of 3.

Figure 7

Expression of p160 coactivator mRNA in p160-null mice. Total RNA was isolated from the livers of wild type (WT) mice and mice null (Null) for SRC-1 (A), SRC-2 (B), or SRC-3 (C) 16 h after injection i.p. with isotonic saline (C) or 100 μg/g PB (P). The level of mRNA was determined by quantitative RT-PCR using primers specific for the indicated p160 coactivator. The amount of p160 coactivator mRNA was normalized to that of β-actin included as internal standard. The standard error of the mean is indicated for an n of 3.