Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47phox, a major cytosolic component of this oxidase. Protein kinase C ζ (PKC ζ), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC ζ in p47phox phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47phox with recombinant PKC ζ induced a time- and concentration-dependent phosphorylation of p47phox with an apparent Km value of 2 μM. Phosphopeptide mapping analysis of p47phox showed that PKC ζ phosphorylated fewer selective sites in comparison to “conventional” PKCs. Serine 303/304 and serine 315 were identified as targets of PKC ζ by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC ζ that correlated to that of p47phox. A cell-permeant-specific peptide antagonist of PKC ζ inhibited both fMLP-induced phosphorylation of p47phox and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC50 of 10 μM), but not that induced by PMA. The inhibition of PKC ζ expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 μM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47phox is a substrate for PKC ζ and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.
Triethylene glycol dimethacrylate (TEGDMA) is a dentin-bonding agent and a major component of various dental restorative biomaterials. TEGDMA monomers are released from dental resins and induce dental pulp inflammation and necrosis. In this study, we have investigated the mechanism of TEGDMA-induced cytotoxicity of fibroblasts. Treatment of cultured human gingival and pulpal fibroblasts with 0.1-3 mM of TEGDMA for 24 h induced a concentration-dependent and variable cytotoxic effect. Fifty percent of toxicity (TC(50)) was obtained with 1.2 +/- 0.9 and 2.6 +/- 1.1 mM of TEGDMA for gingival and pulpal fibroblasts, respectively. Moreover, TEGDMA-induced cytotoxicity was associated with an early and drastic depletion of cellular glutathione (GSH), which started at 15-30 min and was almost complete at 4-6 h. Antioxidants, such as Trolox (0.01 mM), ascorbate (0.2 mM), and N-acetylcysteine (NAC) (5 mM) prevented the TEGDMA-induced cytotoxicity while GSH depletion was partially inhibited. Finally, a late production of reactive oxygen species (ROS) occurred in fibroblasts treated with TEGDMA for 3-4 h, as determined by 2',7'-dichlorofluorescein fluorescence, and was completely inhibited by Trolox (5 microM). The data show that TEGDMA induced a drastic GSH depletion followed by production of ROS, which may contribute to the toxicity of gingival and pulpal fibroblasts. Antioxidants, such as NAC, ascorbate, and particularly Trolox, appear useful in preventing cell damage mediated by resin-containing dental restorative materials.
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