Molecular And Functional Characterization Of The Zebrafish Clusterin Gene And Investigation Of The Mechanism Under The IGFs Regulation In Myoblast

(1)Clusterin is a highly conserved disulfide-linked heterodimer glycoprotein with nearly ubiquitous tissue distribution, apparently involved in many biological prosesses and diverse pathological states. Recent genome wide association studies have implicated the human Clusterin gene as one of the factors in the etiology of Alzheimer’s disease. The in vivo function of the Clusterin gene and its possible role in the pathogenesis of Alzheimer’s disease, however, is poorly understood. Despite a large amount of in vitro data on various possible cellular actions of Clusterin in a wide variety of cultured mammalian cells, the in vivo physiological functions of this protein remain unclear. Clusterin knockout mice develop and live normally, leaving in question the functional role of Clusterin during fetal development.Here we identified and characterized the Clusterin gene from zebrafish. The structure of zebrafish Clusterin gene and protein is similar to its human orthologue. Biochemical assays show that zebrafish Clusterin is a secreted protein. In adult zebrafish, Clusterin mRNA is detected in many tissues. During early development, Clusterin mRNA becomes detectable at 12 hpf and its levels gradually increase as embryos grow. In situ hybridization analysis indicates that Clusterin mRNA is specifically expressed in the developing diencephalic and myelencephalic choroid plexus (CP). Among various stresses tested, heat shock, but not hypoxic or ionic stresses, increases the levels of Clusterin mRNA. Specific inhibition the IGF-1 receptor-mediated signaling and overexpression of IGF ligands did not change Clusterin mRNA levels. Inhibition or targeted knockdown of Notch signaling significantly increased Clusterin mRNA expression in CP. These results suggest that Clusterin is a marker of CP and its expression in the developing CP is under the regulation of Notch, but not IGF signaling. (2)The insulin-like growth factor (IGF) signaling pathway is a conserved pathway. Studies in a variety of animal and cellular systems suggest that the IGF signaling pathway plays a key role in regulating skeletal muscle growth, differentiation, and in maintaining homeostasis of the adult muscle tissues. Despite many advances in our understanding of the expression and role of IGFs in myogenesis, and the potential therapeutic role of IGFs in treatment of muscle-related diseases, such as muscle atrophy, muscular dystrophy and neuromuscular disorders, however, there are no reports on exogenous IGFs regulation of endogenous IGFs in myogenesis, not to mention its regulatory mechanisms.In our study, we use C2C12 myoblast as a model of skeletal myogenesis to investigate the endogenous regulation of IGFs. Firstly, we found that exogenous IGF-2 time-dependently decreases the expression of IGF-1 mRNA level and increases IGF-2 mRNA level. Then we did dose-effect experiment using insulin, IGF-1 and IGF-2 respectively, and speculated that insulin receptor (IR), IGF-1 receptor (IGF-1R) and IGF-1R/IR hybrid receptor may all mediate the regulation, and IR is the predominant one. Through a variety of inhibition and overexpression experiments for the downstream pathways, we showed that various pathways are involved.