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.

Recombinant CC16 protein inhibits the production of pro-inflammatory cytokines via NF-κB and p38 MAPK pathways in LPS-activated RAW264.7 macrophages

“…rCC16 protein was prepared according to published procedures [13]. Briefly, the open reading frame of rat CC16 gene was amplified and cloned into the pET-30a(+) vector (Novagen, Darmstadt, Germany).…”

Label-free LC-MS/MS shotgun proteomics to investigate the anti-inflammatory effect of rCC16

“…To further determine the differential expression patterns of these proteins in RTE cells cultured in the absence or presence of LPS and rCC16, RT-qPCR was performed for transcriptional analyses of the differentially expressed proteins at the mRNA level, (with the exception of MMP-9, the expression of which has been previously described by our laboratory) (7). As presented in Fig.…”

“…Consequently, administration of CC16 may be an effective treatment for COPD. Our previous study demonstrated that rCC16 inhibited the expression of COPD-contributing MMP-9 in LPS-treated RTE cells via the attenuation of NF-κB activation (7). The present study has identified a network of proteins involved in the mechanisms underlying the anti-inflammatory activity of rCC16.…”

“…The cells were washed with phosphate-buffered saline (PBS) and cultured in serum-free media (control group), serum-free media supplemented with 0.1 µg/ml LPS (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) for 24 h (LPS treatment group) or pretreated with 2.0 µg/ml rCC16 in serum-free media for 2 h prior to 0.1 µg/ml LPS treatment for a further 24 h (rCC16 treatment group) (7). …”

“…Induction of CC16 expression by gene transfection inhibits interleukin (IL)-1β-induced IL-8 expression in BEAS-2B bronchial epithelial cells by suppressing the transcriptional activity of nuclear factor (NF)-κB (6). Our previous study demonstrated that recombinant rat CC16 suppressed lipopolysaccharide (LPS)-mediated inflammatory matrix metalloproteinase (MMP)-9 production through inactivation of nuclear factor κB (NF-κB) and p38 mitogen-activated protein kinase signaling pathways in rat tracheal epithelial (RTE) cells (7). The anti-inflammatory properties of CC16 may render it a useful strategy for treatment of inflammatory respiratory disorders; This may be verified by the elucidation of the underlying mechanism via protein profile analysis of CC16-treated cells.…”