CCAAT/Enhancer Binding Protein β2 Is Involved in Growth Hormone-Regulated Insulin-Like Growth Factor-II Gene Expression in the Liver of Rainbow Trout (Oncorhynchus mykiss)

Abstract

Previously, we showed that levels of different CCAAT/enhancer binding protein (C/EBP) mRNAs in the liver of rainbow trout were modulated by GH and suggested that C/EBPs might be involved in GH-induced IGF-II gene expression. As a step toward further investigation, we have developed monospecific polyclonal antibodies to detect rainbow trout C/EBPα, -β1, -β2, and -δ2 isoform proteins. Injection of GH into adult rainbow trout resulted in a significant increase of C/EBPβ1, C/EBPβ2, and C/EBPδ2 proteins in the liver. Chromatin immunoprecipitation analysis revealed that C/EBPβ2 binds to multiple sites at the 5′ promoter/regulatory region, introns, and the 3′ untranslated region of the IGF-II gene. GH treatment reduced C/EBPβ2 binding to several of these regions at 6 h after injection. The decreased occupancy of C/EBPβ2 coincided well with an increase of histone H4 acetylation at the proximal promoter and elevation of the IGF-II mRNA level. Immunoblotting analysis showed that C/EBPβ2 existed predominately as a truncated form in the liver, and cotransfection analysis further showed that the truncated C/EBPβ2 acted as a negative regulator on IGF-II proximal promoter. GH treatment caused deacetylation of C/EBPβ2 in the liver. In addition, we observed a GH-dependent interaction of C/EBPβ2 with a complex involving histone H1. All together, these results suggest that C/EBPβ2 was regulated at multiple levels by GH, and C/EBPβ2 may play a suppressive role in mediating GH-induced IGF-II expression in the liver of rainbow trout.