IGF-I in common carp: Gene structure, promoter characterization, regulation of gene expression and cloning of receptor subtypes.

摘要

Insulin-like growth factors (IGFs) are potent mitogens for many different cell types and play a central role in growth and development. A multitude of regulatory principles modulate the biological effects of IGFs. For instance, the number of receptors expressed on target cells determines the strength of the IGF signal, the interaction with large soluble proteins, the IGF binding proteins, interferes the biological activity of the IGFs, a vast array of hormones and other signaling molecules regulate the levels of bioactive IGF-I in an endocrine or autocrine fashion.; In spite of the importance of IGF-I for growth and development in fish, knowledge about the regulation of its expression in non-salmonid species is rather limited. Our laboratory started such studies on a local tropical fish, the common carp (Cyprinus carpio). The common carp IGF-I gene of a size of 13 kb was obtained by genomic library screening. The gene contains 5 exons and 4 introns. Compared to the human, rat, chicken and salmon counterpart, the common carp IGF-I gene is the smalles. Liver is found to be the major site of IGF-I expression in common carp with a similar pattern of expression between juvenile and adult fish. IGF-I mRNA was also found in other tissues of the common carp with relatively low expression levels.; To understand the molecular mechanism which controls the transcription of the IGF-I gene in common carp, we have cloned the 5'-flanking region of the common carp IGF-I gene and characterized its promoter activity. Activation of the reporter gene by human growth hormone (hGH) added to the medium of the transfected 293hi cells was observed. The carp IGF-I promoter contains two putative liver-enriched transcription factor-binding sites for HNF-1alpha and STAT5. We have demonstrated that these two transcription factors enhanced the activity of IGF-I promoter and acted synergistically in the presence of hGH. Mutations of the HNF-1alpha and STAT5 binding sites completely abolished such enhancing effects. Results of the mutation studies also support the notion that STAT5 plays an intermediate role between the GH receptor and IGF-I. GH cannot stimulate IGF-I transcription without STAT5 binding to the IGF-I promoter.; In teleosts, the GH dependence of liver IGF expression was observed, whereas the GH-dependency of IGF-II expression in teleosts is conflicting. Moreover, most of the studies on bony fish were focused on the liver, and data on the GH-dependence of other tissues are not available. In this study, differential expression pattern of IGF-I and IGF-II by GH stimulation in various tissues of the common carp was observed using real-time quantitative PCR.; In order to study the molecular action of IGF-I in common carp, we have cloned and characterized the IGF-I receptor in carp. The receptor in common carp was found to exist in two subtypes, IGF-I receptor subtype a and IGF-I receptor subtype b, which were structurally conserved in vertebrates. However, there were differences in their cellular localization and relative abundance in defined cells/regions suggesting they might play distinct roles in regulating growth and development in common carp.