This article describes polymorphisms located 5′ of ARG1 that regulate gene expression and are associated with BDR in three populations with asthma. In the 5′ region spanning approximately 1.7 kb, we identified a total of 14 polymorphisms via resequencing of ARG1. The most common haplotype (denoted 1) at this locus is associated with higher BDR in all trials, whereas the two less frequent haplotypes correlate with low BDR. Furthermore, these haplotypes were shown to differentially regulate luciferase expression in our reporter gene assays with the major haplotype increasing reporter gene expression. Thus, our results demonstrate that a combination of alleles at multiple 5′ SNPs regulate gene expression and modulate an individual's response to β2-agonists. Specifically, haplotype 1 was shown to increase gene expression by 50% compared with the other two haplotypes and was associated with a 25–118% greater likelihood of a high response to β2-agonists.
Although increased arginase activity was first detected among patients with asthma nearly two decades ago (8
), its role in asthma susceptibility remains poorly understood and the mechanism whereby genetic variants in the arginase pathway might lead to interindividual differences in BDR is unknown. However, earlier studies have demonstrated that increased expression of arginase results in increased production of l
-ornithine and decreased nitric oxide (NO) levels as a result of competitive binding of arginase and nitric oxide synthase (NOS) to the common substrate l
-ornithine is known to be a key element in tissue repair and airway remodeling (24
), whereas NO is known to regulate inflammation and smooth muscle relaxation (9
). Decreased NO levels have been shown to induce production of peroxynitrites, which are proinflammatory, cytotoxic, and stimulate airway smooth muscles to contract (25
). These results were consistent with other studies showing that reduced NO levels promote increased airway reactivity to methacholine (26
) and cytokines (11
), and increased response to repeated allergen challenge (28
). Not surprisingly, elevated arginase activity has also been correlated with reduced lung function (10
). Nevertheless, the link between β2
-agonist bronchodilation and arginase 1 activity remains undefined.
In our transfected cells, we found little or no haplotype-dependent effects of β2
-agonist on luciferase expression, suggesting that there are no receptor-activated changes in transcription factors that regulate ARG1
expression under these experimental conditions. Other conditions or cell types might reveal differential effects of β2
-agonist on promoter activity. Indeed, the complement of transcription factors, and their regulation by β2
-agonist, differs substantially between airway epithelial cells and smooth muscle cells (21
). However, cells containing haplotype 1 expressed significantly greater reporter gene activity compared with cells containing the other two haplotypes. Furthermore, haplotype 1 showed the strongest association with high BDR in this manuscript.
The potential interaction between the arginase 1 and β2
-adrenergic receptors pathways may be primarily at the physiologic level, with NO and cyclic adenosine monophosphate both having independent effects on smooth muscle relaxation. If so, one might expect subjects with haplotype 1 to have less NO, increased smooth muscle tone, and thus a greater change in β2
-agonist–mediated flow as a consequence of the increased tone. However, the FEV1%
predicted for patients with haplotype 1 was not decreased, as would be expected in this scenario (Table E4). Finally, we recognize that unexpected regulation of seemingly “unrelated” pathways is often found using unbiased approaches, such as expression microarrays. We have observed such crosstalk when specific signal transduction proteins are knocked-down or overexpressed in airway smooth muscle cells (29
) so we cannot exclude an interaction between the two pathways at this time.
A limitation of our study was the small sample sizes of our trial populations, especially for LOCCS (n = 159) and LODO (n = 155), which precluded integration of low-frequency haplotypes identified through the ARG1 sequencing. In future studies, the integration of large populations that homogeneously span a wide range of BDR may help to improve the understanding of the effects of ARG1 haplotypes on BDR.
The haplotypes we have identified in ARG1
have the potential to facilitate the development of genetic tests for BDR prediction in those with asthma. In addition, this study suggests that arginase 1 may be an important therapeutic target for asthma treatment. It has been suggested that l
-arginine supplementation would benefit patients with asthma by increasing substrate availability for NOS to elevate the bronchodilator NO (9
). Alternatively, previous studies have also suggested that arginase inhibition would be beneficial to patients with asthma. For example, a loss of function of arginase 1 by RNA interference resulted in decreased airway response to methacholine and decreased response to IL-13 (11
), whereas the use of an arginase inhibitor led to a decrease in airway hyperresponsiveness after an allergic reaction (32
). In contrast to these findings, Ckless and coworkers (33
) reported an increase in airway hyperresponsiveness among mice administered with a different arginase inhibitor. Moreover, Niese and colleagues (34
) demonstrated that arginase is not required for regulation of inflammation and hyperresponsiveness of the lungs in chimeric mice deficient of arginase 1. These conflicting results may be explained by the different methods used for arginase inhibition (i.e., RNAi, inhibitors, genetic ablation of bone marrow arginase); nonspecific inhibition of arginase via these approaches; and the different animal models used (i.e., human vs. mice vs. guinea pigs). Collectively, these earlier reports and the current study suggest that further investigation to gain a better understanding of arginase regulation is necessary to determine how modifying this pathway would improve asthma therapy.
In conclusion, we have identified haplotypes in the ARG1 locus that are associated with BDR to β2-agonists in three independent asthma cohorts. In vitro studies with transfected cells revealed differential reporter gene expression by haplotype. Depending on ethnicity, the lower-responding haplotypes may represent approximately 25–50% of the population, which is sufficient to consider ARG1 promoter haplotyping as part of a collection of pharmacogenetic tests to individualize asthma therapy. However, the treatment options for those with the unfavorable haplotypes are not known and require specific clinical trials.