Inhibition Of Myostatin In A204Rhabdomyosarcoma Cells Using Follistatin-derived Peptides

Myostatin, also known as growth and differentiation factor-8, belongs to the transforminggrowth factors-β superfamily of secreted growth factors and is known to play a pivotal role in thenegative regulation of skeletal muscle mass, also is the most powerful inhibitor of muscle growthidentified to date. Inhibition of myostatin activity represents an alternative strategy for increasingmuscle mass and therapy muscular diseases. The known myostatin inhibitors include follistatin,decorin, cripto, follistatin-related proteins, myostatin propeptide, growth and differentiationfactor-associated serum protein1, human small glutamine-rich tetratricopeptide repeat-containingprotein, titin-cap and soluble activin receptor II B, all of these proteins can bind to MSTN andinhibit its activities.Follistatin is a secreted glycoprotein encoded by a single gene, and also named asFSH-suppressing protein. Follistatin was found to be bind TGF-β superfamily, such as myostatin,Activin A and bone morphogenetic protein, so it has multiple effects on numerous cell types,including muscle cells, osteoblast and erythropoiesis. Follistatin is a powerful antagonism ofmyostatin and acts as an efficient inhibitor of activin A. Because activin A has various effects onmany physiological processes, its blockade by follistatin would affect multiple tissues other thanskeletal muscles. Crystallographic analyses have revealed that mature follistatin consists of anN-terminal unique domain and three FS domains (FS I, FS II and FS III), and each FS domainmay have different ligand binding activity. The N-terminal unique domain, FS I and FS II domainsare critical for binding to activin A, and the minimal myostatin-inhibiting fragment comprises theN-terminal unique domain and FS I domain. Up-regulation expression of the FS I domain ordeletion of the FS II domain also could increase the binding of myostatin and depress the bindingof Activin A.Now a lot of work has been done on human and mice‘s follistatin domains, but the researchon domestic animals has yet to report in detail. In order to study whether up-regulation expressionof FS I domains affects myostatin activity, a triple FS I domain-expressing vector, named FS I-I-I,which contained the N-terminal domain and three consecutive FS I domains, was constructed in this study. To increase the expression of FS I-I-I in skeletal muscle, FS I-I-I was under control ofthe promoter human alpha skeletal actin and the MCK enhancer2. In order to explore the action ofFS I-I-I on the myostatin-and activin-signaling pathway, FS I-I-I was transfected into A204rhabdomyosarcoma cells. Results of biological activity showed that FS I-I-I efficiently inhibitedthe myostatin-induced transcription activities, but has no effect on the activin-inducedtranscription activities. Cell proliferation assay indicated that FS I-I-I increased the growth rate ofhuman A204rhabdomyosarcoma cells compared with the negative control. Quantitative real-timePCR showed that FS I-I-I enhanced the expression levels of MyoD gene in transgenic cells.Furthermore, FS I-I-I was identified to exhibit significant effects on the Smad signal pathway,ERK signal pathway and PI3k/Akt signal pathway.Taken together, FS I-I-I can be used as a highly specific inhibitor to antagonize myostatin,thus, cell proliferation was improved. Our study provides a foundation for the construction oftransgenic animals containing FS I-I-I and for further studing the effectof FS I-I-I on porcineskeletal muscle in vivo.