The cysteinyl leukotrienes (CysLTs) sub-family has been shown to play an important role in the pathophysiology of many inflammatory disorders including asthma (Drazen, 1998). Thus, these molecules and their receptors constitute major potential therapeutic targets. Two receptors have been identified so far: CysLT receptor 1 (CLTR1; Lynch et al., 1999; Sarau et al., 1999) and CysLT receptor 2 (CLTR2; Heise et al., 2000). Little is known about transcriptional control of their expression (Brink et al., 2003). The aims of this study are to define the genomic organisation of CLTR1 and the mechanisms underlying the regulation of its expression.

5’rapid amplification of cDNA 5’ends (5’RACE; GeneRacer kit, Invitrogen) was used on total RNA extracted from human airway smooth muscle (HASM) isolated from 3 donors. Starting from around the most downstream putative transcription start site (TSS) of the main transcript species (TSS1+94bp), a fragment of ~200bp followed by 4 others made in increasing steps of ~200bp up to a length of ~1kb were cloned into vectors expressing firefly luciferase (PGL3 enhancer, Promega). Two other constructs of ~2 and ~3kb were also made. HASM cells were transfected using Fugene 6 (Roche) and the luciferase signal was measured 48h later using the Bright-Glo luciferase assay system (Promega) and a luminescence counter (TopCount NXT, Packard).

Although the only transcript species of CLTR1 previously published represented 90% of all transcript species identified and so is probably the most functionally relevant transcript under basal conditions, two other species were found with a frequency of 6% and 4%. The genomic location of the TSSs also suggested that there are two putative CLTR1 promoters, the main one regulating the synthesis of the main transcript species and a splice variant. The majority of the transcriptional activity of the main putative promoter was detected in a region between 400 and 800bp upstream of TSS1+94bp, with constructs containing ~200, 400, 600, 800 and 1000bp of this region showing respectively a 3.67±1.11, 6.52±2.01, 9.82±1.95 (p<0.05 vs 200bp and 1kb clones, anova), 6.22±1.42 and 3.64±0.48 increase fold in luciferase expression compared to the empty vector. A 1.52±0.08 fold reduction in luciferase expression was seen between the ~1kb and ~2kb constructs and a further 1.53±0.18 fold reduction in activity was also observed between the ~2kb and ~3kb constructs. These data suggest that the majority of CLTR1 transcripts in airway smooth muscle arise from a single promoter located immediately upstream of TSS1, although rarer species can also occur arising from more 5’TSS.

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