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Journal of Undergraduate Research

Keywords

fibroblast proliferation, treatment, novel TGF-beta pathway inhibitor

College

Life Sciences

Department

Exercise Sciences

Abstract

Fibroblasts play a key role in repairing injured tissue by secreting collagen and growth factors into the tissue. In states of disease and overuse, fibroblast activity (i.e. greater fibroblast content and collagen secreting activity) can lead to skeletal muscle fibrosis, in which myofibers are replaced by collagen, decreasing strength and muscle elasticity. It has been shown in previous studies that TGF-beta signaling directly increases fibroblast proliferation and collagen secretion. It has also been shown that inhibition of TGF-beta signaling reduces known symptoms of muscular dystrophies including decreased fibrosis and increased muscular strength. SGI-1252 is a novel small molecule that our lab has developed over the past 2 years. SGI-1252 inhibits a pathway important for muscle growth called myostatin (TGF-beta member). The purpose of my original project was to determine how treatment of cultured fibroblasts with the novel small molecule SGI-1252 affects proliferation and collagen expression. Shortly after submission a concurrent project in our lab showed that treatment of mice with SGI-1252 showed no significant growth in muscle, contrary to our hypothesis based on our knowledge of the myostatin pathway. Based on the results in muscle growth that we saw in the mice treated with SGI-1252, we decided to consider the alternative pathways that could be affecting the muscle growth. This lead us to the JAK/STAT pathway. Recent studies have highlighted a role for the JAK/STAT signaling pathway in the regulation of muscle satellite cell behavior and differentiation. Stimulation of the JAK/STAT pathway could cause a decrease in the differentiation of muscle fibers and explain the results found during our myostatin experiment. Based on this knowledge, my mentor, Dr. Hyldahl, and I decided to change the aims of my research to test the effects of SGI-1252 on the JAK/STAT pathway. To test these effects, the compound, SGI-1252, was administered orally (400mg/kg) in a 10% dextrose solution to wild type mice (n=6) 3 times per week for 8 weeks. A control group (n=6) received only the dextrose solution. After the 8 weeks, mice that were treated with SGI-1252 showed a significant increase in the number of Pax7+ satellite cells relative to controls (1852 ± 387 vs 2780 ± 657 SC/mm-3, p=0.02)(Figure 1B and C). This lead us to infer that SGI- 1252 effectively shut down the differentiation of these satellite cells. To further test our hypothesis, we looked at the effect of SGI-1252 on the expression of MyoD and Myogenin(Figure 2). The loss and replacement of Pax7 with MyoD and Myogenin instructs the dividing satellite cells to differentiate, whereas inhibition of STAT3 activity (JAK/STAT pathway) attenuates myogenic differentiation. We found a significant decrease in the expression of MyoD and Myogenin in SGI-1252 treated myoblasts, further supporting the idea that SGI-1252 causes an attenuation in satellite cell differentiation.

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