Cigarette smoke condensate induces MMP-12 gene expression in airway-like epithelia

https://doi.org/10.1016/j.bbrc.2005.02.144Get rights and content

Abstract

Cigarette smoke (CS)-induced emphysema is attributable to matrix metalloproteinase-12 (MMP-12) in mice, however, a relationship between CS and MMP-12 is absent in human emphysema. Here, we show that cigarette smoke condensate (CSC) induces MMP-12 gene expression in airway-like epithelia through a hydrogen peroxide (H2O2)-dependent pathway involving NADPH oxidase, AP-1, and TNF-α. Cigarette smoke condensate-induced H2O2 production and MMP-12 gene expression were inhibited by apocynin, a specific inhibitor of NADPH oxidases, while 3-aminobenzamide, an inhibitor of AP-1, attenuated CSC-induced MMP-12 gene expression. Messenger RNAs encoding phagocytic NADPH oxidase components and a homologue of p67phox, p51 (NOXA1), were detected, while mRNA of dual oxidase (Duox)1 was unchanged by CSC. Enbrel, an inhibitor of TNF-α function, reduced CSC-induced H2O2 production and MMP-12 expression. These findings provide novel evidence of a direct relationship between CS exposure and MMP-12 in human airway epithelia and suggest several targets for modulation of this potentially pathogenic pathway.

Section snippets

Materials and methods

Derivation of airway-like NHBEC cultures. Differentiation of primary NHBECs (passage 1) into cultures bearing airway-like characteristics, including enhanced mucin production and secretion, has been described [22] and previously performed in this laboratory [6]. Briefly, NHBECs cultured on fibrillar collagen/human fibronectin membranes were maintained at an air-liquid interface for fourteen days in serum-free NHBEC culture medium (50% BEBM, Cambrex, Walkersville, MD; 50% DMEM (high glucose),

Cigarette smoke condensate induces H2O2 production in NHBECs through activation of NADPH oxidase

To investigate whether CSC induces oxidant production in airway-like NHBEC cultures, we challenged DHR-loaded, airway-like NHBEC cultures with CSC (0.4 μg/ml) for 60 min. Although the vehicle (DMSO) failed to induce oxidant generation (Fig. 1A), CSC-induced oxidant production in airway-like NHBEC cultures, as indicated by the robust fluorescence observed in CSC-treated cultures (Fig. 1B). Co-challenge of cultures with CSC and catalase significantly reduced DHR-derived fluorescence emission (Fig. 1

Discussion

These findings directly show that a concentrated form of the primary cause of emphysema, namely CS, can regulate the expression of MMP-12, which, based on studies in mice [4], is a protein that could play a major role in CS-induced emphysema in humans. Furthermore, we provide evidence that an H2O2-dependent pathway mediates the effect of CSC in this in vitro model of airway bronchial epithelium. Cigarette smoke condensate-induced oxidant generation and MMP-12 expression that were inhibitable by

Acknowledgment

The authors thank Liping Zhang for determining nucleotide sequences of RT-PCR fragments.

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