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Adenosine up-regulation of the mucin gene, MUC2, in asthma

Biomedical Sciences Program, Cardiovascular Research Institute and Department of Anatomy, University of California San Francisco, San Francisco, California, USA

¹Correspondence: Biomedical Sciences Program, Cardiovascular Research Institute and Department of Anatomy, University of California San Francisco, 513 Parnassus, HSW 1330, San Francisco, CA 94143-0452, USA. E-mail: cbas{at}itsa.ucsf.edu

SPECIFIC AIMS

Mucus hypersecretion is an important clinical feature of asthma. While the etiology is not well understood, hypersecretion has been linked to the presence of cytokines such as IL-4, IL-5, IL-9, and IL-13 in the inflamed airway. Recent work has shown that IL-13-associated events in the asthmatic airway of IL-13 transgenic mice are dependent on adenosine accumulation and altered expression of adenosine receptors. Although the abundant adenosine present in asthmatic airways has been recognized for its contributions to airway hyperreactivity and inflammation, no studies have yet implicated adenosine in mucus hypersecretion. In this study, we examined the possibility that adenosine contributes to asthmatic mucus hypersecretion.

PRINCIPAL FINDINGS

1. Ligation of an adenosine receptor stimulates MUC2 gene expression
Although most asthma models focus on the mucin gene MUC5AC, we focus here on the mucin gene MUC2. Recent work has shown that MUC2 is up-regulated in asthmatic airways. Due to the relative insolubility of MUC2, its presence, even in small amounts, may contribute to the excessive viscosity typical of mucus from asthmatic airways.

Our model consists of homogeneous epithelial cell cultures from human bronchus (NCIH292) or colon (HM3). We used cultures of primary airway epithelial cells to verify results obtained in the cell lines. Using cells stably transfected with a 2.8 kb MUC2 reporter construct, we observed that adenosine stimulated MUC2 a maximum of 7-fold (Fig. 1 a). In contrast, maximum stimulation of MUC5AC-luciferase using a 3.75 kb reporter construct was <1.5-fold (Fig. 1b ). This difference in magnitude was seen in a PCR analysis of the endogenous genes in primary human tracheal epithelial cells (Fig. 1c,d ), confirming that MUC2 is more strongly stimulated than MUC5AC by adenosine.

View larger version (36K): [in this window] [in a new window]   Figure 1. Adenosine up-regulates MUC2 gene expression in human epithelial cells. a) MUC2 or b) MUC5AC luciferase assays in HM3 cells after incubation with adenosine for 4–6 h. Cells were lysed with reporter buffer and relative light units (RLU) of luciferase activity were read using luciferase substrate. Each experiment was repeated a minimum of 3 times. *Significantly different from unstimulated control, P< 0.01. c) RNA isolated from primary human tracheal epithelial cells stimulated (or not) with adenosine (200 µM) for 6 h was analyzed by RT-PCR using MUC2- or MUC5AC-specific primers with ribosomal 18S as an endogenous control. d) Fold increase in mucin gene expression shown in panel c as determined by densitometry (Image J 1.27, NIH).

There are four known receptors for adenosine on mammalian cells: A1, A2a, A2b, and A3. Using antagonists specific for each (DPCPX for A1, DMPX for A2a and A2b, and MRS1191 for A3), we determined that the major receptor mediating mucin induction in response to adenosine appears to be A1. Consistent with this, a survey of adenosine analogs revealed a preference for A1 agonists.

2. Up-regulation of MUC2 by adenosine requires phosphorylation of the epidermal growth factor receptor
Adenosine receptors are G-protein coupled. This class of receptor (G-protein-coupled receptor, GPCR) commonly trans-activates the epidermal growth factor receptor (EGFR), whose phosphorylation is required for mucin induction by multiple stimuli. A potential role for EGFR in the adenosine response was suggested by the fact that adenosine elicited EGFR phosphorylation. Supporting this, both AG1478 and compound 32, specific inhibitors of EGFR kinase, reduced adenosine induction of MUC2 in a dose-dependent manner. RT-PCR data reflecting behavior of the endogenous MUC2 gene in NCIH292 cells confirm that both the A1 receptor and EGFR contribute to adenosine-evoked mucin production.

3. Adenosine contributes to the MUC2-stimulating capacity of human asthmatic tracheal aspirates
We demonstrated that tracheal aspirates from asthmatic subjects contained components capable of stimulating transcription of the mucin gene MUC5AC. MUC2 is sensitive to components of these aspirates. Consistent with the earlier work, we found that samples from asthmatics had a greater potency than those from healthy subjects.

We next determined the extent to which this stimulation was due to the presence of adenosine. MUC2 induction by asthmatic tracheal aspirates was reduced 30–40% in the presence of adenosine deaminase, which deaminates adenosine to form inosine. Moreover, the A1 receptor antagonist DPCPX and the EGFR kinase inhibitor AG1478 attenuated the response similarly at saturating concentrations of each drug. Taken together, these results indicate that adenosine is a major contributor to the mucin-inducing activity in asthmatic airways and that its mechanism of action is A1 and EGFR dependent.

4. Up-regulation of MUC2 by adenosine requires niflumic acid-sensitive ion channels
Recent studies have implicated the Ca²⁺-activated Cl^– channel, CLCA1, in mucin overproduction associated with human asthma and animal models of the disease. The role of these channels is unclear. To examine their possible role in the induction of MUC2 by adenosine, we exposed cells to adenosine in the presence and absence of niflumic acid, an inhibitor of Ca²⁺-activated ion channels. The diminution of the response by this agent (Fig. 2 a) supported the idea that a channel such as CLCA1 might participate in the induction of MUC2 by adenosine.

View larger version (38K): [in this window] [in a new window]   Figure 2. Evidence for involvement of a Ca2+-activated ion channel in the induction of mucin by adenosine. a) MUC2-luciferase assay indicating that the Ca2+-activated ion channel inhibitor niflumic acid (NFA) inhibits induction of MUC2 by adenosine (200 µM). *Significantly different from adenosine alone, P< 0.01. b) Immunoprecipitation of EGFR from adenosine-stimulated or control NCIH292 cells was followed by immunoblot using anti-phosphotyrosine antibody py99. Blot was stripped and reprobed with anti-EGFR to detect differences in sample loading. Results show that NFA interferes with the ability of adenosine, but not EGF, to phosphorylate EGFR. SFM = serum-free medium. c) MUC2–luciferase assay in HM3 cells indicating that NFA (200 µM) also inhibits MUC2 induction by aspirates from asthmatic airways. *Significantly different from aspirate alone, P< 0.01.

Trans-activation of EGFR by GPCRs has been reported and been shown to be metalloproteinase dependent. Surprisingly, the trans-activation of EGFR by adenosine was not blocked by the metalloproteinase inhibitor GM6001 but was inhibited in the presence of niflumic acid (Fig. 2b ). Collectively, our data suggest that the adenosine A1 receptor, CLCA1, and EGFR act sequentially in the signal transduction pathway linking adenosine with MUC2 activation. That the same mechanism may apply to human asthma was suggested by results showing that MUC2 induction by asthmatic tracheal aspirates was inhibited by niflumic acid (Fig. 2c ) as well as by the A1 inhibitor DPCPX and the EGFR inhibitor AG1478.

CONCLUSIONS AND SIGNIFICANCE

It is established that adenosine exists in asthmatic airway fluid at a concentration of 200 µM and participates in the exaggerated bronchoconstriction seen in asthmatic airways. The results presented here show that a similar concentration of adenosine stimulates mucin production, another clinical feature of asthma. The effects on mucin occur via an epithelial cell signaling pathway initiated at an adenosine receptor. The receptor transduces signals through a Ca²⁺-activated ion channel (possibly CLCA1) and EGFR (Fig. 3 ). The specific adenosine receptor subtype appears to be A1, based on experiments with specific agonists and antagonists. The relatively high doses needed for efficacy in these experiments, however, are surprising in view of the high affinity binding properties of A1. The moderate responses to A2 and A3 ligands strongly suggest that other adenosine receptor subtypes may also contribute to mucin induction. Recent work shows that EGFR can activate MUC2 through a canonical signaling pathway involving Ras, Raf, and Erk 1/2.

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating proximal events in the adenosine/MUC2 signaling pathway. Adenosine is increased in the asthmatic airway and contributes to asthmatic inflammation and hyperreactivity. The effects of adenosine on mucin occur via an epithelial cell signaling pathway initiated at an adenosine receptor that transduces signals through a Ca2+-activated ion channel and EGFR. Ligation of an adenosine receptor can stimulate Ca2+ mobilization through activation of PLCß with G-protein subunits. We hypothesize that an adenosine-stimulated increase in [Ca2+]i causes the channel to undergo a conformational change that ultimately leads to trans-activation of EGFR and up-regulation of MUC2.

In contrast to the strong stimulation of MUC2 by adenosine (Fig. 1a, c ), the corresponding effects on MUC5AC are relatively weak (Fig. 1b, c ). Although MUC2 is frequently regarded as a minor component of airway mucus (in comparison to MUC5AC), its insolubility may mean that its presence even in small amounts is sufficient to impair mucociliary clearance. Recent findings show that RNA levels of MUC2, but not MUC5AC, are elevated in epithelial cell brushings from asthmatic vs. normal airways (unpublished data). The different adenosine sensitivities of MUC2 and MUC5AC illustrate the extent to which mucins are subject to gene-specific control mechanisms.

The importance of adenosine in the pathophysiology of asthma has been emphasized by a recent study demonstrating an asthma-like phenotype in adenosine "overexpressing" (adenosine deaminase-deficient) mice. The same group reported that the asthma-like phenotype in ADA-deficient mice was similar to that found in mice overexpressing IL-13. While the latter work provided strong evidence for adenosine’s role in asthmatic inflammation and hyperreactivity, the question of adenosine’s contribution to mucus hypersecretion was left open. Data presented here support the idea that adenosine indeed contributes to mucin induction through up-regulating MUC2.

Although adenosine itself has not been implicated in the control of mucin transcription, the nucleotide derivatives ATP and UTP have been shown to stimulate both mucin synthesis and secretion. Adenosine production increases in cells under conditions of stress and adenosine may be released directly from such cells or occur extracellularly as a breakdown product of ATP. ATP release is a common response to cell injury, and may play a central role in the defensive responses of cells to various insults (e.g., osmotic stress or bacterial flagellin). Mucin, an "all-purpose" insulating barrier, is used for host defense throughout phylogeny.

A puzzling issue in the pathophysiology of asthma concerns the role of the Ca²⁺-activated Cl^– channel CLCA1. Attention originally focused on the murine homologue of this gene (Gob5/mCLCA3) when it was found to be up-regulated in two murine models of asthma. Deletion of the gene in mice abrogated the mucus hypersecretory phenotype whereas overexpression in human epithelial cells conferred mucin overproduction. Thus, CLCA1 or a close homologue is likely to be involved in the control of mucin production, yet it is not clear how a Cl^– channel could influence (mucin) gene expression. The present data, supporting functional interposition of a channel between the A1 receptor and EGFR, raise some interesting hypotheses. For example, in some systems ligation of the A1 receptor activates phospholipase C and leads to Ca²⁺ mobilization, an event that could trigger conformational changes in CLCA1. Such changes would presumably "open" the channel to Cl^– conductance, but if, as suggested for CFTR, CLCA1 serves as a scaffolding protein, conformational changes might simultaneously activate downstream signaling molecules. This scenario could readily modulate mucin gene expression.

In any case, the present work is significant in revealing a previously unknown role for adenosine in the pathophysiology of asthma: the stimulation of mucin transcription. The mechanism by which this occurs is interesting from the standpoint of drug development, as it suggests the A1-CLCA1-EGFR axis as a novel target in the treatment of mucus hypersecretion. Studies are under way to better understand the relevant mechanisms.

FOOTNOTES

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