American Journal of Respiratory and Critical Care Medicine

The airway epithelium is increasingly recognized as a major contributor to the pathogenesis of asthma (1). One of the most highly induced genes in epithelial cells in asthma and experimental allergic airway disease is gob-5, which is predominantly expressed by mucus-containing goblet cells (25). The systematic name for human gob-5 is calcium-activated chloride channel 1 (hCLCA1), but, confusingly, mouse gob-5 is named mCLCA3. In 2001, Nakanishi and colleagues reported that downregulation of mCLCA3 in airways of allergen-sensitized mice by antisense RNA suppressed mucus production, airway hyperreactivity, and eosinophilic airway inflammation, whereas overexpression of mCLCA3 caused more severe disease (2). In this issue of AJRCCM (pp. 1216–1221), Nakano and colleagues (6) present the latest in a series of studies from several laboratories that investigate the role of CLCAs and explore the therapeutic potential of chloride channel inhibitors in asthma. This work suggests that changes in airway epithelial cell chloride channel expression and function may be important for asthma pathogenesis. However, a number of fundamental questions remain unanswered.

Are CLCAs critical for pathogenesis of allergic airway disease? If mCLCA3 plays a critical role in allergic airway disease, one might expect that disruption of the mCLCA3 gene would prevent or reduce disease. Two recent reports demonstrate that gene-targeted mCLCA3-deficient mice have intact responses in models of allergic and virally induced airway disease (7, 8). A possible explanation for the apparent discrepancy between these results and those obtained with antisense RNA (2) is that the gene-targeted mice could develop other mechanisms that compensate for loss of mCLCA3. It has recently been suggested that another CLCA family member, mCLCA5, may be involved in compensation (8). Overexpression of either mCLCA3 or mCLCA5 produced increases in mucin gene expression, although the magnitude of these increases was modest compared with what is seen in allergen or viral infection models. Taken together, available data indicate that mCLCA3 is not required for allergic airway disease, but suggest that this molecule, and perhaps other CLCA family members, may make important contributions. Genetic analysis also suggested an association between certain hCLCA1 haplotypes and asthma (9), although the functional effects, if any, of hCLCA1 polymorphisms are not known.

Are CLCAs chloride channels? Initial functional studies showed that heterologous expression of hCLCA1 produced an increase in calcium-sensitive chloride currents, suggesting that hCLCA1 might itself function as a channel, or a component of a channel (10). However, two recent studies used structure prediction and biochemical approaches to conclude that hCLCA1 and mCLCA3 are unlikely to be ion channels (11, 12). This suggests that the effects of CLCAs on chloride currents are attributable to as yet unidentified direct or indirect regulatory activities of these proteins.

How does the chloride channel inhibitor, niflumic acid, work? The fenamate compound niflumic acid is a small molecule inhibitor of calcium-activated chloride channel function. In one sense, niflumic acid may be considered as a relatively selective inhibitor of chloride channels, because other inhibitors affect a broader range of chloride channels. However, niflumic acid and other fenamates can also affect other types of channels, including potassium, calcium, and nonselective cation channels, as well as gap junctions (13). Furthermore, niflumic acid inhibits cyclooxygenases at doses lower than those used to inhibit chloride channels, and is used clinically as a nonsteroidal antiinflammatory agent in some countries. The broad range of effects of niflumic acid, and a lack of knowledge about which molecules are directly and indirectly affected by it, complicate interpretation of experiments that involve this inhibitor.

In this issue of the Journal, Nakano and colleagues assess the effects of niflumic acid on IL-13–induced airway disease (6). IL-13 is produced during allergic responses, and direct effects of IL-13 on airway epithelial cells are sufficient to induce mCLCA3 expression, mucus overproduction, and airway hyperreactivity (14). IL-13 and the related cytokine, IL-4, signal by binding to a heterodimeric receptor, leading to phosphorylation of Janus kinases (JAKs), which, in turn, phosphorylate signal transducer and activator of transcription 6 (STAT6) (15). STAT6 then translocates to the nucleus, where it binds to promoters, thereby altering gene transcription. The induction of mCLCA3 is STAT6 dependent (14), although it is not clear whether STAT6 directly increases mCLCA3 transcription by binding to its promoter.

Nakano and colleagues found that niflumic acid suppressed airway inflammation, goblet cell metaplasia, and mucus overproduction induced by intratracheal administration of IL-13 (6). Similar niflumic acid effects had previously been reported in an allergic model (16). The effects of niflumic acid on inflammation may relate to suppression of expression of the chemokine eotaxin. Niflumic acid also inhibits IL-13–induced mucus and eotaxin production in cultured epithelial cells (6, 16), suggesting that these cells are at least one of the important sites of niflumic acid action in vivo. An obvious hypothesis would seem to be that niflumic acid–sensitive epithelial CLCA-regulated channels induce signals that somehow lead to increased expression of mucins, eotaxin, and other genes important in airway disease. A prediction of this hypothesis is that niflumic acid would affect steps in the pathway downstream, but not upstream, of CLCA induction. The most novel and surprising aspect of the new report is that it suggests that this hypothesis may be wrong—or at least incomplete.

The new finding is that niflumic acid suppresses two proximal steps in IL-13 signaling. Niflumic acid blocked IL-13–induced JAK2 and STAT6 phosphorylation in airway epithelial cells. Inhibition of JAK2 phosphorylation was apparent by 15 min after IL-13 stimulation, suggesting that niflumic acid exerted its effects before there was much hCLCA1 expression. The mechanism by which niflumic acid inhibits JAK2 and STAT6 activation is unknown, but, based on studies with other inhibitors, it is likely to involve chloride channels other than cyclooxygenases. In Nakano's studies (6), cells were pretreated with niflumic acid for 24 h before IL-13 stimulation, which raises the possibility that the effects of this inhibitor could be indirect. In any case, the new finding raises intriguing questions. Do chloride channels really participate in proximal IL-13 signaling events, or does niflumic acid block JAK2 and STAT6 activation by inhibiting other targets? Might niflumic acid also inhibit JAK and STAT6 activation induced by IL-13 and IL-4 in other cell types that are important in allergy? Do similar mechanisms apply to different JAKs and STATs that are required for responses to other cytokines? The answers to these questions promise to expand our understanding of allergic airway disease, and may help us devise new therapies for asthma.

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