Meeting Abstract
63.4 Sunday, Jan. 5 14:15 Effect of molting hormones (ecdysteroids) on myostatin and mTOR expression in skeletal muscle and limb regenerates in the blackback land crab, Gecarcinus lateralis COSENZA, K. S.*; KIM, K. S.; CHANG, E. S.; MYKLES, D. L.; Colorado State Univ; Colorado State Univ; UC Davis Bodega Marine Lab; Colorado State Univ kcosenza@rams.colostate.edu
During premolt, increasing ecdysteroid levels cause claw muscle atrophy in Gecarcinus lateralis, allowing withdrawal of the claw at ecdysis. Myostatin (Gl-Mstn) is negatively correlated to ecdysteroids, while protein synthesis is up-regulated to allow myofibril remodeling during premolt. Our hypothesis is that ecdysteroids inhibit Gl-Mstn expression through the ecdysteroid receptor. Gl-Mstn, in turn, inhibits protein synthesis via Smad transcription factors. Using DNA walking, an ecdysone receptor response element (EcRE) was located near the 5’ end of the Gl-Mstn promoter, suggesting that Gl-Mstn expression is directly regulated by ecdysteroids. Gl-Mstn, Gl-EF2, Gl-mTOR, Gl-Rheb, Gl-Akt, and Gl-S6K mRNAs were quantified by qPCR. After two weeks of daily 20-hydroxyecdysone (20E) injections, Gl-Mstn mRNA levels were significantly decreased in claw muscle. By contrast, a single 20E injection had no effect on gene expression over a 24-hour period. Limb bud autotomy, which suspends premolt by lowering hemolymph ecdysteroids levels, increased Gl-Mstn mRNA levels in claw muscle. Unexpectedly, expression of Gl-mTOR, Gl-Rheb, Gl-Akt, and Gl-S6K was increased by LBA. The data suggest that Gl-Mstn expression is regulated by ecdysteroids. However, there was no consistent linkage between expression of Gl-Mstn and expression of mTOR signaling components in claw muscle. However, Gl-Rheb and Gl-S6K expression was decreased in growth-suspended limb buds, compared to growing limb buds. The Gl-Mstn promoter was fused to luciferase and transfected into HeLa cells, along with Uca pugilator EcR (Up-EcR) and Up-RXR, to determine whether the EcRE is functional. Supported by NSF (IBN-0618203).
