Meeting Abstract
P1.59 Saturday, Jan. 4 15:30 Ecdysteroid regulation of the Gecarcinus laterals myostatin gene: a heterologous cell expression system to test the function of an ecdysteroid response element DONOVAN, M.W.*; TROWBRIDGE, R.L.; COSENZA, K.S.; MYKLES, D.L.; Colorado State University, Fort Collins ; Colorado State University, Fort Collins; Colorado State University, Fort Collins ; Colorado State University, Fort Collins matt_donovan@live.com
Myostatin (Mstn) is a potent inhibitor of protein synthesis in mammalian skeletal muscle. In G. laterals claw muscle there is an inverse relationship between hemolymph ecdysteroid levels and Gl-Mstn expression. The promoter region of the Gl-Mstn gene contains a putative ecdysteroid response element (EcRE). These data suggest that ecdysteroid represses Gl-Mstn expression. As a ligand, ecdysteroids, such as 20-hydroxyecdysone (20E), bind to a heterodimeric nuclear receptor, consisting of the Ecdysone Receptor (EcR) and the Retinoid X Receptor (RXR). We hypothesize that this ligand/receptor complex directly inhibits transcription of the Gl-Mstn gene via the EcRE in the promoter region. A heterologous cell expression system was used to determine if the putative EcRE is functional. We cloned Uca pugilator EcR (Up-EcR) cDNA encoding the EcR open reading frame (ORF) into the pKH3-CMV and Up-RXR cDNA encoding the RXR ORF into the p3XFLAG-CMV plasmid vectors. The pKH3-CMV has a triple hemagglutinin (HA) tag and the p3XFLAG-CMV has a FLAG tag. Initial results indicate the fusion HA-EcR protein was expressed from the pKH3 vector in HeLa cells. The Gl-Mstn promoter sequence was cloned into the pGL3-Basic plasmid, which contains a luciferase reporter gene. HeLa cells will be transfected with the plasmid constructs encoding the HA-EcR and FLAG-RXR fusion proteins and the Mstn promoter-luciferase sequence. We hypothesize that 20E will suppress luciferase expression in cells expressing both EcR and RXR and not in cells expressing only EcR or RXR. This experiment will determine if ecdysteroids directly regulate Mstn expression in crustaceans. Supported by NSF (IBN-0618203).
