PERSPECTIVE
The Leukotriene C₄ Synthase Gene and Asthma

Timothy D. Bigby

Pulmonary and Critical Care Section, Department of Medicine, San Diego VA Healthcare System and the University of California, San Diego, California

The cysteinyl leukotrienes (LT), LTC₄, LTD₄, and LTE₄, are oxygenated metabolites of arachidonic acid derived from the 5-lipoxygenase (5-LO) pathway (Figure 1) (1). The term leukotriene was originally proposed by Samuelsson as a trivial name to describe these compounds that are derived from leukocytes and are conjugated trienes (2). 5-LO is an iron-containing enzyme most often found free in the cytosol of cells of myeloid origin (3). With cellular activation, this enzyme translocates to the nuclear envelope (4). Once translocated, it is responsible for the first committed steps of the 5-LO pathway in association with the 5-LO-activating protein (FLAP). The precise role of FLAP remains obscure; however, it is required for appreciable 5-LO activity in intact inflammatory cells (5). 5-LO inserts molecular oxygen at the carbon 5 position of arachidonic acid forming the hydroperoxy-intermediate, 5-hydroperoxyeicosatetraenoic acid. This intermediate is further metabolized by 5-LO via a dehydration reaction to form the epoxy intermediate, LTA₄. LTA₄ can be further metabolized to LTB₄ by the enzyme LTA₄ hydrolase, an epoxide hydrolase enzyme with a ubiquitous distribution (6). The role of LTB₄ in asthma remains to be clarified. LTA₄ can also be metabolized to LTC₄ by the enzyme LTC₄ synthase (7). This enzyme is a microsomal glutathione-S-transferase that belongs to a larger family of membrane-bound proteins involved in eicosanoid and glutathione metabolism (8). LTC₄ synthase appears to have a more restricted distribution than 5-LO and appears to be limited to eosinophils, mast cells, basophils, mononuclear phagocytes, and some leukemic cells lines such as THP-1 (9) and KG-1 (10).

View larger version (23K): [in this window] [in a new window]   Figure 1.   5-Lipoxygenase (5-LO) pathway of arachidonic acid metabolism. 5-LO and FLAP are responsible for the first two enzymatic steps in this pathway leading to the formation of the epoxide intermediate, LTA4. LTC4 synthase (shaded) is responsible for the first committed step in the synthesis of the cysteinyl leukotrienes (rectangle).

The concept that lipid soluble mediators may play a role in asthma is not new. Van Leeuwen and Zeydner demonstrated that alcohol extracts of blood obtained from subjects with asthma, but not healthy individuals, contained a substance that caused contraction of smooth muscle (11). Moreover, Harkavy reported in 1930 that alcohol extracts of sputum obtained from asthmatics during exacerbation also contained a substance that could induce sustained smooth muscle contraction, which he termed "spasm-producing substance" (12). In studies performed by Feldberg and Kellaway (13, 14), and subsequently by Kellaway and Trethewie (15), a substance that could induced sustained smooth muscle contraction was present in the blood of antigen-challenged guinea pigs. These authors coined the term "slow-reacting smooth-muscle stimulating substance in anaphylaxis," which ultimately was refined to "slow-reacting substance of anaphylaxis" (SRS-A) by Brocklehurst (16). The subsequent forty years of research and major efforts of many investigators culminated in the characterization of SRS-A as the cysteinyl leukotrienes C₄, D₄, and E₄ (17). These compounds mediate sustained smooth muscle contraction, mucus hypersecretion, airway edema (18), and eosinophil recruitment (19). Since 1979 a substantial body of evidence has accumulated demonstrating that the cysteinyl LTs are increased in the sputum (20), lung lining fluid (21), blood, and urine (22) of asthmatics when compared with healthy subjects.

Aspirin-intolerant asthma (AIA) is a syndrome of aspirin sensitivity, asthma, and nasal polyposis (23). Individuals with this syndrome often have profound systemic reactions when challenged with aspirin. Reactions to aspirin may include cutaneous and gastrointestinal manifestations, in addition to potentially life-threatening acute airway obstruction. The onset of AIA is usually beyond the third decade, and these individuals commonly have severe asthma, which may require systemic corticosteroids. We now recognize that this is not an allergically mediated phenomenon, but is instead a disorder that can occur with any nonsteroidal anti-inflammatory drug that acts via inhibition of cyclo-oxygenase. However, the molecular mechanism(s) involved in the pathogenesis of AIA remains unclear (23). The initial hypothesis suggested that AIA was due to a shunt of arachidonic acid from the cyclo-oxygenase pathway to the lipoxygenase pathway (24). We now understand that the pathogenesis of AIA is more complex. Utilization of arachidonic acid within the cell is a highly ordered process with multiple regulatory steps, and simple shunting of substrate does not appear to play a major role.

Additional hypotheses have been put forth to explain AIA. For example, studies suggest that a failure of cyclo-oxygenase-2 expression in the nasal mucosa of patients with AIA may be a feature (25). The largest body of evidence, however, supports a central role for cysteinyl LT synthesis and an enzyme responsible for this synthesis, LTC₄ synthase, in AIA. AIA patients have markedly elevated cysteinyl LT levels at baseline that increase dramatically with aspirin challenge (26). Moreover, studies have demonstrated that LTC₄ synthase is strikingly increased in eosinophils within the airways of aspirin-intolerant asthmatics (27). In 1996 the LTC₄ synthase gene was cloned and assigned to the long arm of chromosome 5 by two groups simultaneously (28, 29). This region of chromosome 5 (q35) is adjacent to a region of substantial interest (q31-33), which includes a variety of candidate asthma genes, including the -adrenergic receptor and a cluster of cytokine genes involved in the allergic phenotype. This region of chromosome 5 has been implicated in asthma by linkage analysis (30). Moreover, the proximity of the LTC₄ synthase gene to this region and the potential error in localization by linkage analysis could also suggest that LTC₄ synthase should be considered a gene candidate for asthma. Therefore, studies examining the LTC₄ synthase gene for mutations and studies examining the regulation of this gene have been performed by several laboratories.

Early studies examining regulation, examined the effects of cytokines on cysteinyl LT release or LTC₄ synthase activity (31) before more sophisticated molecular tools were available. However, studies have now been reported that examine the molecular regulation of LTC₄ synthase. Initial sequence analysis of the regulatory elements of this gene suggested that it was similar to a house-keeping gene and might be constitutively active (28, 29). However, this is inconsistent with our knowledge of the distribution of the LTC₄ synthase in a limited list of inflammatory cells, including eosinophils, mast cells, basophils, and mononuclear phagocytes. In mononuclear phagocytes, only transforming growth factor (TGF)- has been shown to induce expression of LTC₄ synthase, and it does this, at least in part, by inducing transcription of the gene (9). Detailed analysis of the 5'-untranslated portion of the LTC₄ synthase gene has revealed that the ubiquitous transcription factor Sp1 plays a key role in the transcription of this gene (34), but this factor does not impart the striking tissue specificity observed (Figure 2). Additional studies have found a similar role for Sp1 and also provide evidence for a tissue-specific element upstream of the Sp1 site (35, 36).

View larger version (9K): [in this window] [in a new window]   Figure 2.   Schematic of the LTC4 synthase gene. LTC4 is a small gene (2.5 kb) consisting of five small exons and four introns. A single nucleotide polymorphism exists 444 nucleotides upstream of the translation start site (366 bp upstream of the transcription start site), in which a cytosine is substituted for an adenine at this position. The regulatory elements of this gene discovered thus far reside in the first 100 bp upstream of the transcription start site. A consensus binding site for Sp1 plays a pivotal role but does not impart tissue-specific expression.

In 1997 Sanak and colleagues reported a study in which they screened the 5'-untranslated region of the LTC₄ synthase gene for mutations (37). They found an adenine (A) to cytosine (C) transversion 444 base pair upstream (444 nucleotides) from the translation start site (37) and 366 bp (366 nucleotides) upstream of the transcription start site (28). Moreover, Sanak and colleagues also reported that the frequency of the C allele was greater in AIA. They suggested that the C allele of this single nucleotide polymorphism (SNP) was responsible for a gain in function of the regulatory elements of this gene, which in turn resulted in increased transcription of the LTC₄ synthase gene. Previous studies have demonstrated that mutations in regulatory elements of genes can result in a change in expression of the involved gene (38). In fact, it has been reported that an SNP was responsible for a gain in activity of the regulatory element and, in turn, overexpression of the matrix metalloproteinase-1 gene (38).

In the current issue of the American Journal of Respiratory Cell and Molecular Biology, Sanak and colleagues report a particularly interesting extension of their prior work on relationships between the LTC₄ synthase gene and AIA. One of the most exciting findings of this study is that peripheral blood eosinophils obtained from all asthmatics have increased mRNA encoding for LTC₄ synthase and that this mRNA is particularly increased in eosinophils obtained from AIA patients. These data provide strong support for the hypothesis that overexpression of the enzyme LTC₄ synthase occurs in AIA, and in turn appears to play a role in pathogenesis. These authors also provide evidence that eosinophils from all asthmatics overexpress LTC₄ synthase relative to healthy subjects. The authors again present evidence in this report that the C allele is more frequent in AIA than in aspirin-tolerant asthmatic or normal subjects and that this difference correlates with disease severity. However, the C allele is common, making an absolute disease association less likely. The reported sample size is also small for an association study, thus limiting the strength of the correlation. Moreover, other investigators have not found an association of the C allele with AIA (41). Sanak and colleagues also report herein that they have begun studies examining the relationship between allelic variation at the 444 nucleotide of LTC₄ synthase and the function of the gene. They have found that the presence of the C allele in promoter-reporter gene constructs results in an increase in transcriptional activity, although the detailed data are not included in this report. In contrast, studies by other investigators have found that the C allele is associated with an approximately 50% loss of transcriptional activity in cells that normally express LTC₄ synthase (42). Sanak and colleagues also provide electrophoretic mobility shift assay (EMSA) data that the transcription factor AP-2 and histone H4 transcription factor bind to the region involving the A444C single nucleotide polymorphism. Others have not found similar binding with EMSAs (42). Finally, they have found that aspirin challenge of eosinophils obtained from patients that have the C allele make more LTC₄, supporting their conclusion that the A444C transversion is associated with increased expression of LTC₄ synthase.

In summary, the report by Sanak and colleagues contained in this issue is exciting and provocative. They provide evidence that a single nucleotide polymorphism in the regulatory element of the LTC₄ synthase gene may play a role in AIA. As always, more data are needed to clarify the role of the A444C transversion. Additional patients with AIA will need to be studied, and other investigators will need to confirm their observations. However, difficult questions remain. For example, why does AIA have an onset in adulthood if genetic polymorphisms play a major role? If supported by the findings of other investigators, the A444C transversion would explain only a limited proportion of AIA cases. What is the etiology of the other cases? Is there another mutation in the regulatory elements of this gene? Are there environmental cofactors that induce the expression of this gene in adulthood? What is the relationship of this mutation to the cyclo-oxygenase pathway? Do cyclo-oxygenase pathway products play an important regulatory role in the expression of this gene? All of these questions are important to answer. Studies examining the pathogenesis of AIA are important because of the substantial numbers of individuals with AIA that are severely afflicted and poorly controlled (23). Moreover, studies of AIA are perhaps even more important because of the insights they may provide into the pathogenesis of asthma in general. This is especially true as we begin to understand the molecular defect(s) in AIA in greater detail.

	Footnotes

Address correspondence to: Timothy D. Bigby, Mail Code 111-J, San Diego VA Healthcare System, 3350 La Jolla Village Drive, San Diego, CA 92161. E-mail: tbigby{at}UCSD.edu

(Received in original form July 10, 2000).

Acknowledgments: Supported in part by a grant from the Merit Review Board of the Department of Veterans Affairs and the University of California Tobacco-Related Disease Research Program (7RT-0097).

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