Gadd45β is an inducible coactivator of transcription that facilitates rapid liver growth in mice
Jianmin Tian, Haiyan Huang, Barbara Hoffman, Dan A. Liebermann, Giovanna M. Ledda-Columbano, Amedeo Columbano, Joseph Locker
Jianmin Tian, Haiyan Huang, Barbara Hoffman, Dan A. Liebermann, Giovanna M. Ledda-Columbano, Amedeo Columbano, Joseph Locker
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Research Article Hepatology

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.

Authors

Jianmin Tian, Haiyan Huang, Barbara Hoffman, Dan A. Liebermann, Giovanna M. Ledda-Columbano, Amedeo Columbano, Joseph Locker

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Figure 6

Localization of transcriptional functions to specific regions of Gadd45β.

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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.