The objective of the work proposed in this application is to explore the mechanisms and significance of the activation of NF-kappaB in lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) signaling. NF-kappaB is the prototype of a family of dimers whose constituents are members of the Rel family of transcription factors. Responsive to cytokines, NF-kappaB plays an often obligatory role in the processes of inflammation, apoptosis/anti-apoptosis, and cell replication. The finding that LPA and S1P activate NF-kappaB is particularly significant, as both agonists act on cells to achieve a broad range of immediate and long-lasting effects which include ranges in cell shape and tension, chemotaxis, proliferation, and differentiation. LPA and S1P are thought to be critical in wound healing, tissue regeneration and development. The first goal of the project is to evaluate intermediate pathways employed by LPA and S1P to achieve activation of NF-kappaB, with a concentration on roles played by components of the ERK activation cascade, the Rho family of monomeric G proteins, reactive oxygen species, and pathways that converge with those employed by pro-inflammatory cytokines. Fibroblasts will constitute the primary experimental model, wherein dominant negative molecules will be introduced by microinjection or transfection. The second goal is to test the hypothesis that the activation of NF-kappaB is achieved through one or more members of the Edg family of G protein-coupled receptors. This will be carried out by reconstitution of receptors into cells in which NF-kappaB is nor normally activated by LPA or S1P. The identity of the G proteins activated by these receptors will be ascertained using a novel [35S]GTPgammaS/GDP exchange assay. The third goal is to identify G protein subunits that cause activation of NF-kappaB, using mutationally activated subunits and chimeras. The identification of relevant alpha and betagamma subunits will help forge the link between GPCRs and relevant downstream phenomena including reactive oxygen species generation. The fourth goal is to determine specific roles for NF-kappaB in LPA and S1P signaling. The hypothesis that NF-kappaB suppresses apoptosis in the course of normal or aberrant signaling will be tested, as well a role in reinitiation of DNA synthesis. We will also identify genes activated by LPA and S1P, and the subset whose activation is dependent on NF-kappaB.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Pharmacology A Study Section (PHRA)
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Spalholz, Barbara A
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University of Pennsylvania
Schools of Medicine
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Siehler, Sandra; Manning, David R (2002) Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors. Biochim Biophys Acta 1582:94-9
Chen, C A; Manning, D R (2001) Regulation of G proteins by covalent modification. Oncogene 20:1643-52
Windh, R T; Lee, M J; Hla, T et al. (1999) Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the G(i), G(q), and G(12) families of heterotrimeric G proteins. J Biol Chem 274:27351-8
Shahrestanifar, M; Fan, X; Manning, D R (1999) Lysophosphatidic acid activates NF-kappaB in fibroblasts. A requirement for multiple inputs. J Biol Chem 274:3828-33
Cowen, D S; Molinoff, P B; Manning, D R (1997) 5-hydroxytryptamine1A receptor-mediated increases in receptor expression and activation of nuclear factor-kappaB in transfected Chinese hamster ovary cells. Mol Pharmacol 52:221-6
Barr, A J; Brass, L F; Manning, D R (1997) Reconstitution of receptors and GTP-binding regulatory proteins (G proteins) in Sf9 cells. A direct evaluation of selectivity in receptor.G protein coupling. J Biol Chem 272:2223-9
Barr, A J; Manning, D R (1997) Agonist-independent activation of Gz by the 5-hydroxytryptamine1A receptor co-expressed in Spodoptera frugiperda cells. Distinguishing inverse agonists from neutral antagonists. J Biol Chem 272:32979-87
Cowen, D S; Sowers, R S; Manning, D R (1996) Activation of a mitogen-activated protein kinase (ERK2) by the 5-hydroxytryptamine1A receptor is sensitive not only to inhibitors of phosphatidylinositol 3-kinase, but to an inhibitor of phosphatidylcholine hydrolysis. J Biol Chem 271:22297-300
Butkerait, P; Zheng, Y; Hallak, H et al. (1995) Expression of the human 5-hydroxytryptamine1A receptor in Sf9 cells. Reconstitution of a coupled phenotype by co-expression of mammalian G protein subunits. J Biol Chem 270:18691-9