EFFECT OF FORCE ACTIVATION OF NOTCH SIGNALING ON MTOR

Abstract

PURPOSE: Notch signaling is a prominent pathway necessary for repair of injured muscle. The interactions of Notch with other signaling pathways, specifically mechanistic/ mammalian target of rapamycin (mTOR), in regulating myogenesis is not well known. Studies have been conducted on Notch inhibition, but little research has been performed on activated Notch and the interactions with mTOR. This study was implemented to develop a Notch force activation protocol and to measure the effect of overexpressed Notch on C2C12 proliferation, differentiation and mTOR signaling. METHODS: Notch signaling was force activated via suspension or adhesion. For suspension, Notch-1 antibody was introduced to a 12-well plate with C2C12 cells at a concentration of 1:10. For adhesion, Notch ligand, Delta-like Ligand 1 (DLL1), was mixed with Extracellular Matrix (ECM) and coated on a 12-well plate at different concentrations (control, 2.5ug, 5ug, and 10 ug) for 12 hours. C2C12 cells were seeded at a concentration of 15,000 cells/well and differentiated for 96 hours. Following designated time period, lysates were collected for Western Blots. Primary antibodies probed for Notch (Hes1), total mTOR signaling (TmTOR), and β-actin. RESULTS: Preliminary data for suspension was analyzed via a two-way ANOVA (time x treatment). There is a significant difference in percent change of proliferation at 48 hours (p=0.02). Preliminary data of adhesion was analyzed via one-way ANOVA. There is a trend between Hes1 concentration in ligand concentration of 2.5 ug (p=0.0739). It shows no significant difference in TmTOR concentrations between the various Notch force activation concentrations in proliferating C2C12 cells (p=.4298). CONCLUSIONS: Preliminary data shows a stronger effect of suspension over adhesion for force activating Notch. A concentration of 2.5ug of ligand is possibly sufficient to force activate Notch, any higher appears too concentrated to activate Notch. Preliminary data also suggests that force activating Notch does not affect mTOR signaling. Experiments are currently testing the ideal DLL1 to activate Notch signaling, as well as examining the effect of force activating Notch on proliferation, differentiation and other markers of mTOR signaling. Different time points for differentiation should be tested in future research.