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Am. J. Respir. Cell Mol. Biol., Volume 24, Number 6, June, 2001 720-726

Double-Stranded RNA Dependence of Nitric Oxide Synthase 2 Expression in Human Bronchial Epithelial Cell Lines BET-1A and BEAS-2B

Kohsaku Uetani, Mercedes E. Arroliga, and Serpil C. Erzurum

Department of Pulmonary, Critical Care Medicine and Allergy; and Cancer Biology, Cleveland Clinic Foundation, Lerner Research Institute, Cleveland, Ohio

The human airway epithelium expresses abundant nitric oxide synthase 2 (NOS2) in vivo. Although NOS2 is easily induced by cytokines in primary cultured human airway epithelial cells and lung adenocarcinoma cell line A549, the human bronchial epithelial cell lines BEAS-2B and BET-1A do not express NOS2 in response to cytokines. Mechanisms regulating NOS2 expression in human respiratory epithelial cells are complex, but we have recently shown that NOS2 expression in primary human airway epithelial cells occurs in response to double-stranded RNA (dsRNA) through activation of signaling proteins including nuclear factor (NF)-kappa B and interferon (IFN) regulatory factor (IRF)-1. In this context, we hypothesized that BEAS-2B and BET-1A cells may express NOS2 in response to dsRNA. Here, we show that although cytokines (IFN-gamma , tumor necrosis factor-alpha and interleukin-1beta ) do not induce NOS2 expression in BEAS-2B or BET-1A cells, addition of dsRNA to this cytokine mix enables BEAS-2B cells to express NOS2. IFN-gamma and dsRNA induction of NOS2 in BET-1A cells occurs in a serum concentration-dependent manner, with a minimum of 3 d of serum treatment necessary for BET-1A cells to acquire the potential to induce NOS2. Importantly, dsRNA strongly activates NF-kappa B and IRF-1 in BEAS-2B cells, transcription factors essential for NOS2 gene expression in other cell lines. On the basis of these results, dsRNA-activated signaling pathways are clearly important for NOS2 expression in human respiratory epithelial cells. With conditions for NOS2 expression characterized, these cell lines are a convenient in vitro system to investigate the mechanisms regulating NOS2 expression in human respiratory epithelial cells.

Abbreviations: double-stranded RNA, dsRNA; dithiothreitol, DTT; ethylenediaminetetraacetic acid, EDTA; electrophoretic mobility shift assay, EMSA; fetal calf serum, FCS; interferon, IFN; interleukin, IL; IFN regulatory factor, IRF; Lechner and LaVeck medium, LHC medium; Eagle's minimum essential medium, MEM; messenger RNA, mRNA; nuclear factor, NF; nitric oxide, NO; inducible NO synthase, NOS2; dsRNA-activated protein kinase, PKR; polyinosinic-polycytidylic acid, poly IC; signal transducer and activator of transcription, STAT; simian virus 40, SV-40; tumor necrosis factor, TNF.




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