Spontaneous apoptosis of neutrophils is attenuated during sepsis and inflammatory cytokines such as TNFalpha have been implicated. The pathophysiological mechanisms involved in TNFalpha-mediated attenuation of apoptosis are poorly understood but proposed to be mediated by the p60 TNF receptor (p60TNFR). In adherent neutrophils to engage beta2 integrins, TNFalpha triggers phosphorylation of the p60TNFR, activation of cell survival signaling pathways and inhibition of spontaneous apoptosis. Our studies implicate phosphatidylinositol 3-kinase (PI 3-kinase) in TNFalpha triggered NFkappaB activation and that TNFalpha activation of PI 3-kinase requires beta2 integrins. The protein kinase C isotype delta (delta-PKC) phosphorylates p60TNFR on serine residue(s) in TNFalpha activated neutrophils. Rottlerin, a delta-PKC inhibitor, suppressed the inhibitory effect of TNFalpha on neutrophil apoptosis and activation of the transcription factor NFkappaB suggesting that delta-PKC may regulate TNFalpha anti-apoptotic signaling. Our model proposes a selective role for delta-PKC in regulating anti-apoptotic signaling triggered by TNFalpha binding to the p60TNFR. The goal of this study is to establish a role for delta-PKC in TNFalpha mediated anti-apoptotic signaling through the p60TNFR using antisense technology to selectively deplete delta-PKC from HL60 cells differentiated to a neutrophilic phenotype. We will: 1: Determine whether delta-PKC regulates TNFalpha mediated anti- apoptotic signaling. Determine the effect of delta-PKC deletion on TNFalpha-mediated suppression of spontaneous apoptosis and activation of the antiapoptotic NFkappaB and the MAP kinases ERK1/2. 2: Assess the role of PI 3-kinase in TNFalpha mediated suppression of spontaneous apoptosis, and determine if delta-PKC is required for TNFalpha mediated activation of PI 3-kinase. 3: Identify the delta-PKC phosphorylation site on the p60TNFR and establish if phosphorylation is in the a) death domain, b) juxtamembrane region, or c) a novel domain. 4: Assess the role of delta-PKC in regulating the assembly of TNFalpha anti-apoptotic signaling complexes and their association with the p60TNFR.
