Common carp (Cyprinus carpio) IGF-II: Gene structure, promoter and gene expression studies

Insulin-like growth factors (IGFs) belong to a family of growth factors with structural homology to proinsulin. In fish, the expression of IGF-I exhibits growth hormone (GH) dependence. Whether the expression of IGF-II is GH-dependent or not depends on the species. We have previously demonstrated that in common carp both the expression of IGF-I and IGF-II show GH-dependence in the brain and liver of the juvenile common carps when the fish were treated in vivo with GH for a short time. In this study, we have investigated the chronic effects of GH elevation by treating juvenile carps with cysteamine, a somtostatin-inhibiting agent, for 63 days. Enhanced growth rate of the treated fish was noticed. A dose-dependent increase in the index of muscle weight over length and a decrease in hepatosomatic index was observed in the treatment groups as compared with the control group. IGF-I and IGF-II mRNA levels in liver and muscle of the treated fish were measured by real-time PCR. It was found that both IGF-I and IGF-II mRNA levels were elevated in the muscle of common carp. However, IGF-I mRNA level was decreased while IGF-II mRNA level was increased in the liver after chronic cysteamine treatment. Under the experimentally induced catabolic states of fasting, both IGF-I and IGF-II expression were reduced significantly and IGF-I expression was found to rebound significantly after refeeding.;With the elucidation of common carp IGF-II gene structure, the regulation of IGF-II promoter was investigated in a reporter gene system using transient transfection assay. The common carp IGF-II promoter was found to be a strong promoter with tissue-specific regulation of expression. Detailed analyses of the putative cis-acting elements including deletion mutant study, in situ site-directed mutagenesis, electrophoretic mobility shift assay and yeast one-hybrid screening were also carried out. Region -301 to -62 of the common carp IGF-II promoter was identified to be responsible for the basal promoter activity. Both site-directed mutagenesis and co-transfection revealed that liver-enriched activating protein could not activate the common carp IGF-II promoter in fish cell lines. These studies pave the way for the future identification and characterization of the transcription complex assembly of the common carp IGF-II promoter upon GH activation. (Abstract shortened by UMI.).