A study on the growth hormone-regulated gene expression of C/EBP isoforms and their relationship with IGF-II gene expression in the liver of rainbow trout (Oncorhynchus mykiss)

Insulin-like growth factor I and II (IGF-I and IGF-II) are mitogenic peptides primarily produced in the liver and play important roles in development, growth, and differentiation in vertebrates. In rainbow trout (Oncorhynchus mykiss), the hepatic expression of both IGF-I and IGF-II is induced by growth hormone (GH). Previous studies conducted in our laboratory suggested that CCAAT/enhancer binding protein (C/EBP) may be involved in mediating GH-regulated IGF-II gene expression in the trout liver. In this dissertation, focus was placed on investigating the functional correlations among GH, C/EBPs, and IGF-II in the liver of rainbow trout. Since only one C/EBP isoform was identified in rainbow trout before this study, this project started from isolation and characterization of rainbow trout C/EBP complementary DNA (cDNA). A total of five C/EBP isoforms were cloned from trout liver. These C/EBP isoforms have highly conserved C-terminal DNA binding and leucine zipper dimerization domains as well as N-terminal functional motifs across different vertebrates. Tissue-specific mRNA expression of C/EBP isoforms revealed distinct patterns when compared with those of other vertebrates. In vivo and in vitro studies demonstrated that these C/EBP isoforms responded differently to GH treatment in trout hepatocytes. C/EBPβ1, β2 and δ2 mRNAs were induced early by GH treatment, while C/EBPα mRNA expression was suppressed by GH until 6 h post GH treatment in vivo. In an effort to identify the C/EBP isoform involved in mediating GH-induced IGF-II expression, several C/EBP isoform-specific antibodies were developed. Time course profiles of hepatic C/EBP isoform protein expression in response to GH were determined by immunoblotting assays with the antibodies, and the data were in good agreement with the mRNA data. Chromatin immunoprecipitation analysis demonstrated that C/EBPβ2 interacted with multiple regions of the IGF-II gene. GH treatment caused decreased C/EBPβ2 occupancy and increased histone H4 acetylation at the IGF-II proximal promoter region. The truncated C/EBPβ2 was observed as the predominant form in trout liver and it played a negative regulatory role on the IGF-II proximal promoter. Additionally, GH-dependent C/EBPβ2 deacetylation and dissociation from histone H1 complex were also observed. Results of this dissertation established the GH/C/EBPβ2/ IGF-II axis in teleost fish and revealed diverse regulatory effects of GH on C/EBP62 in trout hepatocytes.