David B. Donner - US grants

Tumor necrosis factor (TNF) is a monokine produced by macrophages in response to invasive stimulation. Tumor necrosis factor can induce hemorrhagic necrosis and regression of some tumors in experimental animals and is cytotoxic to tumor cells in culture. TNF may also be responsible for the wasting (cachexia) often observed in chronic disease states including cancer. My goal is to understand how tumor necrosis factor produces its actions in cells. The initiating step in TNF action is binding to membrane receptors. Affinity labeling has identified TNF binding sites in rat liver that are structurally different from those identified on other cells. It is essential to relate specific cellular responses to interaction of TNF with its different binding sites on various target cells. Elucidation of the molecular basis for TNF action will also require the isolation and purification of the TNF receptor. We wish to understand how TNF binding is regulated and transduced into cellular responses. Binding induces cooperative interactions among TNF receptors. Also, receptors are continuously removed from (down-regulation) and added to (up- regulation) the plasma membrane of target cells. Cooperative interactions among binding sites and down- and up-regulation of the TNF receptor will be characterized and related to changes of cellular responsiveness to TNF. Coupling of cytokine-receptor complexes to the membrane cytoskeleton and changes in the phosphorylation state of specific target proteins are two mechanisms through which binding is transduced into biologic actions. The role of these events in TNF action will be characterized.

THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OR AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEND ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE ABSTRACT BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. DESCRIPTION: The long-term objective of the proposed research is to develop an understanding of how tumor necrosis factor (TNF) promotes its biological effects in its target cells. TNF is a cytokine with potential for the treatment of cancer that also promotes immunity, anti- viral responses, metabolic changes that accompany various diseases, the insulin-resistance of non-insulin dependent diabetes and inflammatory processes, including those that lead to arthritis. To promote the potential of TNF as a therapeutic agent and to abrogate its pathological activities requires insight into the mechanisms of TNF action. Two different TNF receptors promote cellular responses but neither contains intrinsic tyrosine kinase activity nor any motif that suggests how a signal is transmitted into the cell. Receptors without tyrosine kinase activity often bind accessory proteins that mediate interactions with signaling cascades thereby promoting biological responses. However, genes for TNF receptor-associated proteins (TRAPS) and the sequences of the proteins that they encode have not been previously identified and this may be the most significant gap in our understanding of how TNF brings about its effects. The applicant has used the yeast based two- hybrid system to discover three potentially novel genes that encode TRAPS. By analyzing which cellular responses are promoted by each TRAP, and by characterizing how TRAPS mediate signal transduction, the applicant hopes to gain fundamental insight into the molecular basis for TNF action. To accomplish this goal, the applicant will overexpress each TRAP or TRAP antisense in cells from which TNF elicits diverse responses. Control and TNF-stimulated cells will then be assayed for activation of various second messenger systems and cellular responses, such as cytotoxicity and activation of NF-kappaB.

DESCRIPTION (Adapted from Investigator's Abstract): Vascular endothelial cell growth factor (VEGF) is a mitogen that promotes angiogenesis associated with physiological and pathological processes (cancer, rheumatoid arthritis, retinopathies). The first step in VEGF action is binding to either of two receptor tyrosine kinases, Flk-1 and Flt-1. The roles of these receptors in mediating cellular responses to VEGF have not been well defined nor have the signaling pathways that promote the activities of either receptor. Using ribozyme/antisense constructs that specifically target and degrade Flk-1 and Flt-1, they will isolate endothelial cells in which one or the other VEGF receptor has been knocked out. Such cells will be used to define the responses mediated by Flk-1 and Flt-1. From among the transduction pathways associated with VEGF action, they have found that phosphatidylinositol 3-kinase and mitogen activated protein kinases are especially important. The role of these systems in promoting diverse responses induced by VEGF will be defined. Finally, they have found that protein tyrosine phosphatases can positively and negatively regulate VEGF action. The role of such phosphatases, two in particular- SYP and LAR, in modulating VEGF signaling and cellular responses to VEGF will be defined.