Observation of Niflumic Acid and AG-1478 Reduce Cigarette Smoke-Induced Mucin Synthesis

COPDThis study demonstrated that cigarette smoke exposure increases MUC5AC mRNA and mucin synthesis via a signaling pathway that involves up-regulation of EGFR and CLCA1 both in vivo and in vitro. It has been shown that cigarette smoke up-regulates EGFR expression leading to mucin production in the airway epithelium. This is the first study to show a critical role for CLCA1 in cigarette smoke-induced mucin production, which is one of the major features of COPD.

In this study, we showed that either a blocker of CLCAs, niflumic acid, or a selective EGFR tyrosine kinase inhibitor, AG-1478, are completely able to inhibit cigarette smoke-induced mucin synthesis as well as the upregulation of MUC5AC mRNA expression, and that the effect of combining both drugs is not synergistic. This indicates that both CLCA1 and EGFR are dependently affecting mucin production as a part of a single complex signaling pathway, but it does not exclude the participation of CLCAs other than CLCA1. It seems that CLCA1 is involved in the pathologic induction of mucin, while a different CLCAs are related to the baseline physiologic mucin secretion. Although niflumic acid is a known blocker of the CLCAs and has been shown to block the function of hCLCAi,- it is nonspecific to CLCA1. Therefore the contribution of its blocking of any other CLCAs cannot be accurately evaluated. In fact, despite the increasing number of discovered CLCAs both in animals and human and their seemingly important functions, understanding these channels has been limited by the absence of specific blockers and the fact that their molecular identities remain in question.

Our results indicate that CLCAs are part of the EGFR-MUC5AC pathway because niflumic acid can inhibit the EGF-induced MUC5AC expression and mucin staining in NCI-H292 cells, which was the same level of inhibition caused by AG-1478. Several previous reports have shown that activation of several EGFR-related tyrosine kinases open different types of outwardly rectifying chloride channels, which might mean that CLCA1 is probably downstream to EGFR. Furthermore, Leverkoehne and Gruber demonstrated that the mCLCA3 (mouse counterpart of hCLCA1) protein is exclusively associated with mucin granule membranes inside the goblet cells. They suggested that mCLCA3 is involved in the synthesis, condensation, or secretion of mucins. Collectively, we suggest that cigarette smoke up-regulates the EGFR and CLCA1 mRNA and protein expressions in goblet cells and it also leads to activation of the EGFR, which then activates the CLCA1 in the mucin granule membranes causing upregulation of the MUC5AC mRNA and induction of mucin synthesis.

Several reports have pointed to a link between CLCA16- or EGFR and the T-helper type 2 (Th2) cytokines: interleukin (IL)-4, IL-9, and IL-13. However, Th2 cytokines were also shown to play a role in smoke-induced COPD. Taken together, these findings support our results that suggest that the mechanism of cigarette smoke-induced mucin synthesis involves the EGFR and CLCA1 stimulation induced by these Th2 cytokines.

Tumor necrosis factorTumor necrosis factor (TNF)-a is another mediator that has been shown to induce hCLCA1 leading to upregulation of MUC5AC mRNA and mucus. Since TNF-a increases EGFR expression in airway epithelium, it is possible that the induction of hCLCA1 and mucin production by TNF-a in the airway is mediated via the EGFR signaling pathway. Cigarette smoke can induce TNF-a both in vivo and in vitro.28 Therefore, we speculated that the upregulation of the EGFR and hCLCA1 genes in the present study may be partly mediated by TNF-a.

We demonstrated in this study that DMSO partially inhibited the smoke-induced MUC5AC gene expression in NCI-H292 cells. DMSO is a known potent antioxidant agent. Cigarette smoke contains free radicals and other oxidants in abundance. It has been shown that oxidative stress causes mucin synthesis via ligand-independent EGFR activation. Taken together, it is suggested that at least some part of the mucin upregulation induced by cigarette smoke is mediated through the oxidants.

Although our results were largely similar between the study of the human cell line and that of the Sprague-Dawley rats, they cannot automatically correspond to humans without further confirmation. Interspecies or even interstrain differences in the response to cigarette smoke are well identified. We have exposed the C57BL/6 and Balb/c mice to cigarette smoke for up to 8 weeks without detecting any effect on the mCLCA3, EGFR, and MUC5AC gene expressions (A.E. Hegab, PhD; unpublished data; March 2005), while these changes were significantly detected in rats within 3 weeks. The difference is even more manifest when it comes to human development of chronic bronchitis, which requires many years of smoking and affects only a subpopulation of smokers.

In conclusion, we are showing for the first time that a blocker of the CLCAs probably acting through inhibition of hCLCA1 prevents cigarette smoke-induced MUC5AC synthesis in vivo and in vitro. More studies are needed to further understand the mechanism of actions and interactions of hCLCA1 that might reveal a key signaling pathway that can be targeted with pharmacologic interventions to treat mucus hypersecretion.


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