The long term goals of this project are to understand the molecular mechanisms of cell differentiation and proliferation. The sponsor's lab has recently identified a novel signalling pathway whereby Vitamin D3 and TNF-alpha were shown to activate a neutral sphingomyelinase which cleaves sphingomyelin into Ceramide and cholinephosphate. Ceramide is capable of inhibiting cell growth and inducing differentiation. Based on our recent finding that Ceramide down regulates c-myc, the following hypothesis will be investigated: Ceramide is an intracellular mediator of TNF-alpha's activity in down-regulating c-myc, inhibiting cell proliferation, and inducing differentiation.
The specific aims of this project are therefore: 1. To define the physiologic role of Ceramide as a second messenger involved in c-myc regulation. Planned objectives include a) demonstrating Ceramide generation in response to TNF-alpha using a sensitive and quantitative enzymatic assay; b) establishing the effects of Ceramide on c- myc down-regulation using Northern Blot analysis; and c) confirming that the mechanism of Ceramide on c-myc down-regulation is analogous to that of TNF-alpha using Actinomycin D and nuclear run--on assays. 2. To delineate Ceramide's mechanism of action. Since preliminary work suggests Ceramide activates a protein phosphatase, the objectives include determining a) the effects of Ceramide on endogenous protein phosphorylation using 2D electrophoresis; b) the ability of Ceramide to activate a protein phosphatase; c) structure-function activity of Ceramide analogs in activating phosphatase and in down-regulating c-myc; and d) the effects of phosphatase inhibitors on Ceramide's and TNF-alpha's actions. It is hoped that these studies will define a novel signaling pathway that may disclose important therapeutic targets for modulation of cell proliferation and differentiation.
