Identification of transcriptional regulatory networks in acute liver damage and regeneration

Acute liver injury triggers a myriad of signaling and transcriptional events, including stress and inflammatory transcriptional regulatory networks (TRN). These pathways may exacerbate damage or promote regeneration, depending on factors such as timing, duration, intensity and crosstalk. To identify TRN induced early and late after hepatotoxic exposure, we analyzed mouse liver tissue after a single CCl4 administration (1.6 g/kg) using Affymetrix gene arrays and bioinformatics, combined with histological and western blot analyses, at early (2h, 8h), intermediate (1, 2, 4 days) and late time points (8 and 16 days). The time-resolved analysis revealed a highly dynamic gene expression response upon hepatotoxicity, with 1,469 genes deregulated (p < 0.05, FDR adjusted). Bioinformatics analyses allowed the identification of five time-dependent biological motifs. The first gene expression wave consisted of genes with maximal induction at 2h with enrichment of MAPK-associated genes such as c-Jun and Fos. Western blot analyses confirmed the early activation of MAPK with ERK and JNK phosphorylation at 2h. Two subsequent clusters, with maximal inductions at 8 and 24h contained an overrepresentation of “protein processing in the endoplasmic reticulum” motifs, suggesting activation of ER-stress pathways. Analysis of Xbp1 splicing and induction of ER-stress markers such as the transcription factor CHOP and p-eIF2α confirmed activation of ER-stress between 2h and day 1 after CCl4 intoxication. In parallel, we also observed a massive downregulation of metabolism related genes with maximal repression at 8h and day 1. These clusters included p450 enzymes such as CYP2E1 and CYP3A4. Liver-enriched transcription factors were also downregulated including FXR, SHP and PPARA. Analysis of overrepresented transcription factor binding sites identified HNF1 and HNF4 in the downregulated gene clusters. The last two gene clusters, containing genes maximally upregulated at day 2 and day 3 represented the proliferation response that occurs after damage, including genes such as cyclins and cyclin-dependent kinases. Histological analyses of c-Jun and CHOP demonstrated that the initial burst of stress signaling occurs in pericentral regions. Interestingly, proliferation-associated genes such as c-Myc also show a pericentral expression, suggesting that stress and regeneration signaling propagate as a wave from the pericentral area. In contrast, the expression of HNF4a was downregulated in all liver parenchyma, suggesting that some TRN are zonally restricted while others occur in all hepatocytes. Inflammation-associated genes were observed in all gene clusters, including chemokines such as Cxcl1 (2h), Ccl2 (8h), and acute phase response genes such as Lcn2 (8h). In conclusion, the basic blueprint of the TRN identified in this study will serve as an excellent basis to unveil the precise contribution of further signaling and transcriptional responses induced in acute liver injury.

Corresponding author: Campos, Gisela

E-Mail: [e-mail]