Gadd45β is an inducible coactivator of transcription that facilitates rapid liver growth in mice

Abstract

The growth arrest and DNA damage-inducible 45 (Gadd45) proteins act in many cellular processes. In the liver, Gadd45b (encoding Gadd45β) is the gene most strongly induced early during both compensatory regeneration and drug-induced hyperplasia. The latter response is associated with the dramatic and rapid hepatocyte growth that follows administration of the xenobiotic TCPOBOP (1,4-bis[2-(3,5)-dichoropyridyloxy] benzene), a ligand of the nuclear receptor constitutive androstane receptor (CAR). Here, we have shown that Gadd45b-/- mice have intact proliferative responses following administration of a single dose of TCPOBOP, but marked growth delays. Moreover, early transcriptional stimulation of CAR target genes was weaker in Gadd45b-/- mice than in wild-type animals, and more genes were downregulated. Gadd45β was then found to have a direct role in transcription by physically binding to CAR, and TCPOBOP treatment caused both proteins to localize to a regulatory element for the CAR target gene cytochrome P450 2b10 (Cyp2b10). Further analysis defined separate Gadd45β domains that mediated binding to CAR and transcriptional activation. Although baseline hepatic expression of Gadd45b was broadly comparable to that of other coactivators, its 140-fold stimulation by TCPOBOP was striking and unique. The induction of Gadd45β is therefore a response that facilitates increased transcription, allowing rapid expansion of liver mass for protection against xenobiotic insults.

Figure 1. Proliferative response to TCPOBOP in wild-type and Gadd45b^–/– mice.

(A) Histological patterns of proliferation. Early labeling of a few hepatocytes was apparent at 24 hours (top) only in the wild-type mouse, but labeling was similar at 36 hours (middle). Both early and later labeling was predominantly midzonal (P, portal; C, central). At 48 hours (bottom), labeled mitoses (M) and daughter cell pairs (arrows) were apparent in both wild-type and Gadd45b^–/– animals. These could be identified in hepatocytes of varying ploidy, including diploid cells (d). Original magnification, ×200 (top and middle); ×400 (bottom). (B) Cell counts. S-phase (BrdU-labeled) nuclei are shown as a percentage of total hepatocytes, counted from 20 random high-power fields from 3–5 livers per group per time point. (C) Ccnd1 mRNA expression. Real-time RT-PCR analysis was carried out on RNA from 3–5 livers per time point. Data are mean ± SD. *P < 0.05, **P < 0.01, Gadd45b^–/– vs. wild-type, t test.

Figure 2. Growth and transcriptional effects of TCPOBOP treatment.

(A) Average liver mass as a percentage of body weight. 3–5 livers were studied per time point. Data are mean ± SEM. *P < 0.05, **P < 0.01, Gadd45b^–/– vs. wild-type, unpaired t test. (B) Growth curves were calculated from the data in A. (C) Upregulation of a set of 186 genes (Supplemental Table 2). (D) Downregulation of a set of 86 genes (Supplemental Table 3), analyzed as in C. (E) Average total transcription. Averaging of expression from both sets of regulated changes demonstrated net upregulation that was significantly greater in the wild-type mouse at all 3 time points. (C–E) All arrays were studied using 2-color hybridization, with experimental RNA as the red probe and pooled RNA from normal wild-type livers as the green probe. For this analysis, the intensities detected on individual arrays were normalized to the average green intensity of the entire set of arrays. Hybridization intensities ranged from a low intensity threshold of 100 to a maximum of 60,000. The values of all genes in the set were averaged at each time point. Data are mean ± SEM. *P < 0.05; **P < 0.01, Gadd45b^–/– vs. wild-type, paired t test.

Figure 3. Transcriptional regulation by CAR.

(A) CAR translocation. Immunoperoxidase detected a weak diffuse reaction to CAR in the cytoplasm of untreated hepatocytes. 3 hours after treatment with TCPOBOP, there was clear demonstration of nuclear CAR. Gadd45b^–/– and wild-type genotypes were indistinguishable. Original magnification, ×400. (B) Quantification of 6 representative mRNAs. Real time RT-PCR analysis was carried out as in Figure 1C. Data are mean ± SD. *P < 0.05; **P < 0.01, Gadd45b^–/– vs. wild-type, t test.

Figure 4. Transcriptional coactivation by Gadd45β.

(A) Direct binding of CAR to Gadd45β. A plasmid expressing a GST-binding domain fused to full-length Gadd45β was expressed in E. coli. Fusion protein or a control GST-binding domain protein was bound to glutathione agarose beads and then incubated with ³⁵S-CAR prepared by cell free translation. After wash, the bound protein was eluted and resolved by acrylamide gel electrophoresis. The control lane contained a 20% input fraction. (B) Comparison of coactivation by Gadd45β and Ncoa1. Cotransfection experiments were set up with a limiting amount of CAR expression plasmid (10 ng) to display maximal coactivation. (C) Inhibition of coactivation by ketoconazole (25 μM). Transfection assays used 10 ng CAR, 100 ng Ncoa1, or Gadd45β expression plasmids. (D) Intrinsic activation by DNA-bound Gadd45β. A plasmid expressing a Gal4-DBD fused to the N terminus of full-length Gadd45β was cotransfected with a Gal4 site LUC reporter, with and without ketoconazole. The strong generic activator Gal4-VP16 (100 ng) is also shown for comparison. (B–D) Data are mean ± SD of duplicate assays in HepG2 cells.

Figure 5. Recruitment of CAR and Gadd45β to an activated gene in vivo.

Control untreated (C) and TCPOBOP-treated (T) liver segments were rapidly fixed and then subjected to ChIP analysis using Abs to CAR, Gadd45β, and HNF1α. The phenobarbital response element of Cyp2b10, an upstream enhancer, was detected as a 176-bp PCR amplimer of the gene region from –2,103 to –2,128 bp. The HNF1α-binding site of the Albumin gene promoter was detected as a 160-bp amplimer of the gene region from –210 to –51.

Figure 6. Localization of transcriptional functions to specific regions of Gadd45β.

(A) LXXLL motifs in Gadd45β. Motifs at aa 98 or 117 were mutated (M) by substituting Ala for each Leu in the motif. (B) Reporter assays of coactivation by wild-type and mutant Gadd45β. Expression plasmids were cotransfected with CAR or empty vector, in combination with the Cyp2b10 LUC reporter construct. (C) Direct activation assays. Full-length wild-type or mutant Gadd45β was fused with a Gal4-DBD, and assayed for transcriptional activation using a Gal4-binding site LUC reporter. (D) Map of deletion constructs, with deduced functional domains. Arrows show the position of LXXLL domains within the 160-aa Gadd45β peptide. (E) Mapping of the CAR-binding domain. 293T cells were transfected with expression plasmids for fusion proteins, the GAL4-DBD combined with full-length wild-type or mutated Gadd45β protein, or segments retained in deletion constructs (FL, full length). A plasmid expressing 6His-CAR was transfected into separate cells. Cell extracts were mixed, and 6His-tagged proteins were captured by passing over a Ni-affinity matrix. Eluted proteins were resolved on SDS-acrylamide gels, and products were detected by Western blot using an Ab for the Gal4-DBD. (F) Mapping of the activation domain. Transcriptional activation by Gal4-DBD fusion constructs was assayed as above. (B, C, and F) Data are mean ± SD calculated from duplicate assays in HepG2 cells.

Figure 7. Expression of Gadd45b, Gadd45a, and Gadd45g mRNA in TCPOBOP-induced hyperplasia.

Quantitative RT-PCR measurements were normalized to both untreated wild-type control liver and GAPDH mRNA (left and right y axes, respectively). Data are mean ± SD, calculated from assays of 3–5 specimens per group. *P < 0.05, Gadd45b^–/– vs. wild-type, t test.

Figure 8. Coactivator mRNA responses in TCPOBOP-induced hyperplasia.

Comparison of basal (A) and maximal (B) mRNA levels of 20 coactivators after TCPOBOP treatment in untreated wild-type and Gadd45b^–/– livers. The full time course for each mRNA is shown in Supplemental Figure 10. Data are mean ± SD, calculated from assays of 3–5 specimens per group. *P < 0.05, Gadd45b^–/– vs. wild-type, t test.