Acute endotoxemia prolongs the survival of rat lung neutrophils in response to 12-O-tetradecanoyl-phorbol 13-acetate

Acute endotoxemia is associated with prolonged survival of adherent neutrophils in the lung vasculature. In the present studies, the effects of inflammatory mediators on signaling pathways regulating neutrophil survival were examined. We found that the protein kinase C activator, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), but not interferon-gamma (IFN-gamma), prolonged the survival of adherent vasculature lung neutrophils from endotoxemic rats, a response that was correlated with reduced apoptosis. Although endotoxin administration to rats induced the expression of the anti-apoptotic protein Mcl-1 in lung neutrophils, TPA had no effect on this response. Endotoxin administration also induced expression of total p38 and p44/42 mitogen activated protein kinases (MAPK) in neutrophils, as well as phosphatidyl inositol 3 kinase (PI3K) and its downstream target protein kinase B (PKB). Treatment of the cells with TPA increased p38 MAPK expression in cells from both control and endotoxin treated animals. Cells from endotoxin treated, but not control animals, were found to exhibit constitutive binding activity of nuclear factor kappa B (NF-kappaB) which was blocked by TPA. In contrast, constitutive CCAAT/enhancer binding protein (C/EBP) nuclear binding activity evident in neutrophils from control animals was reduced following endotoxin administration. Moreover, this response was independent of TPA. These data suggest that NF-kappaB plays a role in TPA-induced signaling leading to prolonged survival of adherent vascular neutrophils in the lung during acute endotoxemia.     

Induction of NF-kappa B-like activity by platelet-derived growth factor in mouse fibroblasts.

Nuclear factor kappa B (NF-kappa B) modulates the expression of numerous genes via interaction with a specific DNA sequence termed the kappa B site. Its activity is modulated by a cytosolic inhibitor protein termed I kappa B, and its activation occurs in response to a variety of agents in a variety of cell types, most notably B and T lymphocytes. Data presented here show that an activity (designated complex I) that binds specifically to the kappa B site is induced in density-arrested Balb/c-3T3 mouse fibroblasts by platelet-derived growth factor (PDGF), a potent mitogen for these cells. Increased levels of complex I, as evaluated by electrophoretic mobility shift assays of nuclear extracts, were observed in cells treated for 1-4 h (but not 15 min) with the BB isoform of PDGF. 12-O-tetradecanoylphorbol 13-acetate (TPA) and the AA isoform of PDGF also stimulated this response and both isoforms, but not TPA, were effective in cells depleted of protein kinase C. Complex I most likely is authentic NF-kappa B, a p50-p65 heterodimer, or a closely related factor because it exhibited properties characteristic of those previously described for NF-kappa B including inducibility by deoxycholate and cycloheximide and sensitivity to I kappa B. A second kappa B binding activity (complex II), which apparently contained p50 homodimers, displayed limited induction by PDGF, whereas a third complex (complex III) migrated faster than but behaved similarly to complex I. These studies suggest that NF-kappa B or an NF-kappa B-like factor may participate in the expression of PDGF-inducible genes.     

Induction of cIAP-2 in human colon cancer cells through PKC delta/NF-kappa B

Activation of protein kinase C (PKC) prevents apoptosis in certain cells; however, the mechanisms are largely unknown. Inhibitors of apoptosis (IAP) family members, including NAIP, cIAP-1, cIAP-2, XIAP/hILP, survivin, and BRUCE, block apoptosis by binding and potently inhibiting caspases. Activation of NF-kappa B contributes to cIAP-2 induction; however, the cellular mechanisms regulating cIAP-2 expression have not been entirely defined. In this study, we examined the role of the PKC and NF-kappa B pathways in the regulation of cIAP-2 in human colon cancers. We found that cIAP-2 mRNA levels were markedly increased in human colon cancer cells by treatment with the phorbol ester, phorbol-12-myristate-13-acetate (PMA), or bryostatin 1. Inhibitors of the Ca2+-independent, novel PKC isoforms, but not inhibitors of MAPK, PI3-kinase, or PKA, blocked PMA-stimulated cIAP-2 mRNA expression, suggesting a role of PKC in PMA-mediated cIAP-2 induction. Pretreatment with the PKC delta-selective inhibitor rottlerin or transfection with an antisense PKC delta oligonucleotide inhibited PMA-induced cIAP-2 expression, whereas cotransfection with a PKC delta plasmid induced cIAP-2 promoter activity, which, taken together, identifies a role for PKC delta in cIAP-2 induction. Treatment with the proteasome inhibitor, MG132 or inhibitors of NF-kappa B (e.g. PDTC and gliotoxin), decreased PMA-induced up-regulation of cIAP-2. PMA-induced NF-kappa B activation was blocked by either GF109203x, MG132, PDTC, or gliotoxin. Moreover, overexpression of PKC delta-induced cIAP-2 promoter activity and increased NF-kappa B transactivation, suggesting regulation of cIAP-2 expression by a PKC delta/NF-kappa B pathway. In conclusion, our findings demonstrate a role for a PKC/NF-kappa B-dependent pathway in the regulation of cIAP-2 expression in human colon cancer cells. These data suggest a novel mechanism for the anti-apoptotic function mediated by the PKC delta/NF-kappa B/cIAP-2 pathway in certain cancers.