Promoter Polymorphisms of Cysteinyl Leukotriene Receptor 1 (CysLTR1) Influence the Effect of Leukotriene Modifier on Transcriptional Regulation of the CysLTR1 Gene Induced by IL-4

Abstract

"Background: In recent, intensive studies have attempted to elucidate the genetic background of aspirin intolerant asthma (AIA), but genetic factors associated with its pathogenesis are still not completely understood. Cysteinyl leukotrienes (CysLTs) play important roles in eosinophilic airway inflammation of AIA via CysLTR1-mediated mechanism. Leukotriene modifier MK-571, a selective CysLTR1 antagonist, is widely used in the management of asthma. We selected Cysteinyl leukotruene receptor 1 (CysLTR1) among leukotriene ？ related genes and screened for genetic variation in the 5？ upstream region of CysLTR1 gene. This study was designed to investigate whether MK-571 possesses inhibitory effect on transcriptional induction of CysLTR1 induced by IL-4 or not. Materials and methods: Genetic variation in 5？ upstream region of CysLTR1 gene were screened in 43 normal healthy controls by direct sequencing and then genetic association study of CysLTR1 genetic polymorphisms were performed in 105 aspirin intolerant asthma (AIA) patients, 111 aspirin tolerant asthma (ATA) patients, and 125 normal healthy controls (NC) . Promoter activities according to the genetic polymorphism were examined by luciferase reporter assay. Transcriptional regulation of endogeneous CysLTR1 gene induced by IL-4 was investigated using lung carcinoma cell line, A549 cells, by real time PCR and flowcytometry. Effect of MK-571 on the transcriptional induction of CysLTR1 by IL-4 was examined by luciferase reporter assay. Results: In the screening of 5？ upstream region of CysLTR1 gene in 43 normal healthy controls, three single nucleotide polymorphisms (SNPs) including C-634T, A-475C, and A-336G were identified. Though a genetic association studies of 3SNPs in 105 aspirin -intolerant asthma (AIA) patients, 111 aspirin tolerant asthma (ATA) patients, and 125 normal healthy controls (NCs), significant association of 3 SNPs with AIA were found. There were significant differences in allele frequencies of the 3 SNPs within male subjects; the common 3 SNP haplotypes, ht1 [C-A-A], was associated with decreased disease risk (p=0.031 for AIA vs. NC; p=0.022 for AIA vs. ATA), and the ht2 [T-C-G] haplotype with increased disease risk (p=0.031 for AIA vs. NC; p=0.031 for AIA vs. ATA). Moreover, 3 SNPs can modulate CysLTR1 gene expression; promoter activity was enhanced with ht2 [T-C-G] construct in comparison of ht1 [C-A-A] construct in jurkat cells, U937 cells, and A549 cells. Endogeneous level of CysLTR1 gene was remarkably increased by IL-4 in A549 cells. Promoter activity was also significantly increased with the ht2 [T-C-G] construct in comparison with ht1[C-A-A] construct in IL-4 primed A549 cells (p=0.008), which was significantly inhibited by pre-treatment with MK-571(p=0.004), not with dexamethasone in which ht1 [C-A-A] construct promoter activity was not significantly inhibited by dexamethasone. Conclusion: These results suggest that genetic variants of CysLTR1, ht2 [T-C-G], can up-regulate CysLTR1 expression and significantly induced CysLTR1 expression by IL-4. MK-571 may be more beneficial to control asthmatic symptoms, especially in patients carrying variant genotype of the CysLTR1 promoter. "