Sphingosine-1-phosphate (SPP) is a biologically active sphingolipid, which stimulates glioma cell proliferation and invasion. SPP signals through activation of members of the EDG family of G protein-coupled receptors. Glioma cells produce large amounts of SPP, and express multiple EDG receptors suggesting the potential for autocrine stimulation. The broad, long-term objective of this project is to identify which receptors mediate the mitogenic and migratory effects of SPP on glioma cells, and determine the role of SPP and EDG receptors in glioma growth and invasiveness. The hypothesis is that SPP stimulates glioma cell proliferation and invasion by cooperative signaling through multiple EDG receptors.
In Specific Aim 1, receptors mediating glioma cell proliferation will be identified by overexpressing EDG receptors in cells that are not responsive to SPP, and by inhibiting EDG receptor expression using antisense in glioma cells that are responsive to SPP.
In Specific Aim 2, we will use EDG antisense-transfected glioma cells to determine which EDG receptors mediate SPP-stimulated glioma cell motility and invasiveness, and examine signaling pathways involved.
In Specific Aim 3, the role of EDG receptor dimerization in mitogenic SPP signaling will be investigated using mutant receptors that no longer dimerize.
In Specific Aim 4, the expression of EDG receptors will be examined by in situ hybridization and laser capture microdissection along with quantitative RT-PCR analysis in tissue derived from human astrocytic gliomas of various grades of malignancy to determine if gliomas express mitogenic and invasion-stimulating combinations of EDG receptors in vivo. Cultured glioma cells express EDG receptors, and SPP levels are high in both normal brain tissue and cultured glioma cells. Thus, delineation of the receptors and signaling pathways mediating SPP-induced glioma cell proliferation and migration could extend our knowledge of the molecular mechanisms involved in uncontrolled glioma growth and invasiveness. Therefore, determination of EDG receptor expression in human gliomas could have considerable diagnostic and/or prognostic value and could provide targets for future development of novel therapies.
