The role of ecdysteroids on Myostatin and mTOR signaling gene expression in molt-dependent growth and atrophy of skeletal muscle in Gecarcinus lateralis and Carcinus maenas
Date
2016
Authors
Cosenza, Kathy S., author
Mykles, Donald L., advisor
Quackenbush, Sandra L., committee member
Kanatous, Shane, committee member
DeLuca, Jennifer G., committee member
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Abstract
During premolt, increasing ecdysteroid levels are correlated with increased protein synthesis in the claw of decapod crustaceans. Increased protein synthesis is necessary to remodel the claw muscle in preparation for rapid hypertrophy immediately after ecdysis. Ecdysteroids are negatively correlated with G. lateralis-Myostatin (Gl-Mstn) mRNA levels, allowing upregulation of protein synthesis. Conversely, steroids upregulate Mstn expression in mammals. Glucocorticoids inhibit protein synthesis in mammals through downregulation of the mechanistic Target of Rapamycin (mTOR)-dependent protein synthesis pathway, and upregulation of Mstn. Here, we look at the relationship between ecdysteroids and the mRNA levels of Mstn and mTOR pathway components in different skeletal muscles of G. lateralis and C. maenas. The claw muscle and limb bud muscles respond to increasing premolt ecdysteroid levels with increased protein synthesis, while thoracic muscle does not change in protein synthesis. Our first hypothesis is that ecdysteroid levels will be negatively correlated with Mstn mRNA levels in the claw muscle and the limb bud muscles (muscles with increased protein synthesis), but not in thoracic muscle (no change in protein synthesis). Our second hypothesis is that Gl-Rheb mRNA levels of will be positively correlated with ecdysteroid levels, and negatively correlated with Mstn levels in the claw and limb bud muscles, but not in thoracic muscle. Our third hypothesis is that ecdysteroids directly regulate Gl-Mstn promoter expression through an ecdysone response element (EcRE) in the Gl-Mstn promoter. Through molt manipulations, or allowing natural molts, we showed that ecdysteroid levels were negatively correlated with Gl-Mstn mRNA levels, but not correlated with C. maenas-Mstn (Cm-Mstn) mRNA levels, in claw muscle. In limb bud muscle, there was no correlation between ecdysteroid levels and Gl-Mstn levels. Gl-Mstn levels remained very low, whether limb buds were growing or growth had been suspended. There were no correlations between ecdysteroids and Mstn mRNA levels in the thoracic muscle of either species. These data indicate that ecdysteroid regulation of Mstn is not consistent between species, and that ecdysteroid regulation is muscle specific. Contrary to our second hypothesis, Mstn was positively correlated with mTOR signaling components in the claw muscle of both G. lateralis and C. maenas, not negatively correlated. There was no correlation between Gl-Mstn and mTOR component mRNA levels in the limb buds. This indicates that in specific situations with dramatically changing protein synthesis, Mstn as a chalone that has a modulating effect, to prevent excessive protein synthesis. Using DNA walking, a putative EcRE was located in the promoter region of the Gl-Mstn gene. A heterologous ecdysteroid cell system was developed in mammalian cells to determine Gl-Mstn promoter activity in response to precisely controlled ecdysteroid levels. The heterologous system showed that the Gl-Mstn promoter was functional in this system, but we were unable to demonstrate direct regulation of the Gl-Mstn promoter by ecdysteroids. Further work with the heterologous cell system may determine whether the putative EcRE in the Gl-Mstn is functional.
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Subject
decapod
Ecdysteroids
mTOR
Rheb
crustaceans
Myostatin