Our strategy for the development of drugs to prevent and/or reverse chronic bronchitis is to experimentally dissect signaling pathways linking tobacco smoke to mucin overproduction in lung epithelial cells. To move most rapidly, we initially chose to establish disease models using homogeneous cell lines that were most convenient for protein and RNA analysis. In such experiments, we found that smoke triggers the generation of oxygen radicals (reactive oxygen species, ROS) by NADPH oxidase. The ROS, in turn, stimulate tumor necrosis factor alpha converting enzyme (TACE, ADAM 17 metalloproteinase) to cleave transmembrane amphiregulin, a ligand for the epidermal growth factor receptor (EGFR). The binding of amphiregulin to EGFR then stimulates receptor signaling resulting in the activation of the MAP kinase erk 1/2 and the signaling pathway culminates in activation of an AP-1 response element located about 3.5 kb upstream of the MUC 5 AC transcription start site. Although these results tentatively suggest certain drug targets, the results should be validated in more physiologically relevant systems prior to proceeding. In addition, the initial work in cell lines left certain issues unresolved. Among these is the question of which mechanisms mediate EGFR-independent mucin induction. Based on evidence shown in the proposal, we hypothesize that the calciumactivated CI channel, CLCA1 plays a role in such mechanisms. To understand the relationship between CLCA1 and MUC 5AC induction by smoke, we will perform site-directed mutagenesis of the channel to detect potential sites of protein-protein interaction. To pursue signaling partners of CLCA1, we will perform yeast 2 hybrid screens of cDNA libraries created from smoke- exposed tissue or cells. These studies will validate and extend earlier data concerning the mechanism of mucin induction by smoke.
| McNamara, Nancy; Gallup, Marianne; Sucher, Anatol et al. (2006) AsialoGM1 and TLR5 cooperate in flagellin-induced nucleotide signaling to activate Erk1/2. Am J Respir Cell Mol Biol 34:653-60 |
| Chen, Ran; Lim, Jae Hyang; Jono, Hirofumi et al. (2004) Nontypeable Haemophilus influenzae lipoprotein P6 induces MUC5AC mucin transcription via TLR2-TAK1-dependent p38 MAPK-AP1 and IKKbeta-IkappaBalpha-NF-kappaB signaling pathways. Biochem Biophys Res Commun 324:1087-94 |
