Comparison of Myostatin-Inhibitory Capacity of Various Myostatin-Binding Proteins Using a Luciferase Gene Reporter Assay System
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2014-01-15
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University of Hawaii at Manoa
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Myostatin (MSTN) is a negative muscle growth regulator, thus MSTN-binding molecules may be developed as agents to treat muscle-wasting conditions in humans and to improve muscle growth in meat-producing animals. The objectives of the research project were 1) to optimize the HEK293 (CAGA)12-luciferase gene reporter assay system, an in vitro system for the measurement of MSTN bioactivity, and 2) to compare the inhibitory capacities of three MSTN- binding proteins using a HEK293 (CAGA)12-luciferase gene reporter assay system. The three MSTN-inhibitory proteins used in this study included MSTN prodomain, follistatin (FST), and follistatin related gene product (FLRG). Based on optimization results of the system, a standard amount of MSTN, cell concentration, and cell incubation time were used to test the MSTN- inhibitory capacity of the three proteins. Various concentrations of the above proteins were examined for their MSTN-inhibitory capacity, then the half maximal inhibition concentration of each molecule was determined. Results of the optimization study showed that an appropriate cell concentration was 4X10^4 cells per well in a 96-well plate in overnight incubation. The optimal time for luciferase activity measurement was determined to be between 12 and 24 hours after MSTN addition. In conclusion, FST was the most potent inhibitor of MSTN; followed by FLRG MSTN prodomain, with the half-maximal inhibition concentrations of 0.36, 0.91, and 3.39 nM, respectively. The results of this study demonstrated that the HEK293 (CAGA)12-luciferase gene reporter assay system is useful in screening the capacity of MSTN inhibition of MSTN-binding molecules rapidly in vitro. Use of this system can contribute to future studies on the identification and function of various MSTN-binding molecules.
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54 pages
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