Recombinant rat CC16 protein inhibits LPS-induced MMP-9 expression via NF-κB pathway in rat tracheal epithelial cells

Abstract

Clara cell protein (CC16) is a well-known anti-inflammatory protein secreted by the epithelial Clara cells of the airways. It is involved in the development of airway inflammatory diseases such as chronic obstructive pulmonary disease and asthma. Previous studies suggest that CC16 gene transfer suppresses expression of interleukin (IL)-8 in bronchial epithelial cells. However, its role in the function of these cells during inflammation is not well understood. In this study, we evaluated the effect of CC16 on the expression of matrix metalloproteinase (MMP)-9 in lipopolysaccharide (LPS)-stimulated rat tracheal epithelial cells and its underlying molecular mechanisms. We generated recombinant rat CC16 protein (rCC16) which was bioactive in inhibiting the activity of phospholipase A2. rCC16 inhibited LPS-induced MMP-9 expression at both mRNA and protein levels in a concentration-dependent (0–2 µg/mL) manner, as demonstrated by real time RT-PCR, ELISA, and zymography assays. Gene transcription and DNA binding studies demonstrated that rCC16 suppressed LPS-induced NF-κB activation and its binding of gene promoters as identified by luciferase reporter and gel mobility shift assays, respectively. Western blotting and immunofluorescence staining analyses further revealed that rCC16 concentration dependently inhibited the effects of LPS on nuclear increase and cytosol reduction of NF-κB, on the phosphorylation and reduction of NF-κB inhibitory IκBα, and on p38 MAPK-dependent NF-κB activation by phosphorylation at Ser276 of its p65 subunit. These data indicate that inhibition of LPS-mediated NF-κB activation by rCC16 involves both translocation- and phosphorylation-dependent signaling pathways. When the tracheal epithelial cells were pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, cellular uptake of rCC16 and its inhibition of LPS-induced NF-κB nuclear translocation and also MMP-9 production were significantly abolished. Taken together, our data suggest that clathrin-mediated uptake of rCC16 suppresses LPS-mediated inflammatory MMP-9 production through inactivation of NF-κB and p38 MAPK pathways in tracheal epithelial cells.