Biochemical and Biophysical Research Communications
Cigarette smoke condensate induces MMP-12 gene expression in airway-like epithelia
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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