Acute lung inflammation is complicated by altered pulmonary surfactant phospholipid and protein composition. The proinflammatory cytokine tumor necrosis factor-α (TNF-α) and the phorbol ester 12- O-tetradecanoyl phorbol-13-acetate (TPA) inhibit expression of surfactant-associated proteins A and B (SP-A and SP-B), both important for normal surfactant function. The transcription factor nuclear factor-κB (NF-κB) frequently mediates regulation of gene expression by TPA and TNF-α. In the present study, electrophoretic mobility shift assays (EMSAs) and pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation, were utilized to determine the role of NF-κB activation in TPA and TNF-α inhibition of the surfactant proteins in NCI-H441 cells. Pentoxifylline (PTX), which inhibits TNF-α cellular effects without preventing NF-κB activation, was also tested. By EMSA, TPA and TNF-α increased nuclear NF-κB binding activity in temporally distinct patterns. PDTC decreased TPA- and TNF-α-induced NF-κB binding activity but did not limit their inhibition of SP-A and SP-B mRNAs. PDTC independently decreased both SP-A and SP-B mRNAs. PTX partially reversed TNF-α- but not TPA-mediated inhibition of SP-A and SP-B mRNAs without altering NF-κB binding. The effects of PDTC and PTX on NF-κB and the surfactant proteins suggest that NF-κB activation does not mediate TPA or TNF-α inhibition of SP-A and SP-B mRNA accumulation.
Pulmonary surfactant, a mixture of phospholipids and specific associated proteins, reduces surface tension at the air-liquid interface of the lung and protects the large epithelial surface of the lung from infectious organisms. Surfactant proteins, SP-A and SP-B, are required for normal surfactant function. In the current work, increased levels of oxidized glutathione (GSSG) are demonstrated at doses of pyrrolidine dithiocarbamate (PDTC) which decrease SP-A and SP-B mRNAs, suggesting that cellular oxidation reduces surfactant protein expression. Similarly, reduction of SP-A and SP-B mRNA levels following accumulation of GSSG induced by glutathione reductase inhibitor 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), supports the hypothesis that surfactant protein synthesis is reduced in response to oxidation of pulmonary epithelial glutathione. Concurrent induction of apolipoprotein J (apoJ) mRNA by PDTC demonstrates the selectivity of pulmonary gene regulation by the dithiocarbamate. In contrast, the glutathione precursor N-acetyl-l-cysteine (NAC) prevented PDTC-dependent increase in GSSG/GSH ratio, inhibition of SP-A and -B mRNAs, and induction of apoJ. Insufficiency of SP-A and -B, which occurs in inflammatory lung diseases, may result from the exposure of the pulmonary epithelium to oxidant stress and may be reversed by the antioxidant NAC.
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