The inflammatory mediator TNF-alpha elicits secretion by adherent neutrophils and the extracellular release of oxygen radicals and granule contents contributes to tissue damage. The investigator hypothesizes that activation of different protein kinase C isotypes by lipid mediators plays both positive and negative regulatory roles in these events. TNF-alpha triggers activation of effector enzymes PLC-gamma and PI3 kinase, leading to production of such lipid mediators as diglyceride and PIP3 and to activation of PKC isotypes. Positive signalling for superoxide generation is mediated through phosphorylation of p47-phox by DG-dependent PKC isotypes. Negative signalling by PKC elicits down regulation of TNF-alpha binding, for example by phosphorylation of the p60 TNF receptor by DG-independent, PIP3-dependent zeta PKC. The investigator further proposes that PKC-based signalling requires 1) access of cytosolic PLC-gamma and PI3 kinase to membrane associated lipids, 2) targeting of cytosolic PKC isotypes to PLC and PI3 kinase and thus to lipid mediators DG and PIP3, and 3) targeting of PKC isotypes to their substrates. This model will be tested through experiments focusing on four specific aims: 1) Determine if zeta PKC catalyzes phosphorylation of p60 TNFR in the death domain and thereby down regulates the receptor. 2) Use selective deletion of DG-dependent PKC isotypes to define roles for alpha, beta, and delta PKC in superoxide generation. 3) Establish TNF-alpha elicited activation of PI3 kinase and phospholipase C gamma and differential activation of PKC isotypes by PIP3 and DG. 4) Define a role for RACK (receptor for activated C kinase) in co-localization of PLC and DG-dependent PKC isotypes and activation of PKC and superoxide generation. The long term objective is to define positive and negative signalling for TNF-alpha induced activation of superoxide generation by neutrophils, thereby potentially leading to targeted molecular strategies for control of inflammation and tissue damage.
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