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, 16 (3), R112

Liver X Receptors Alpha Gene (NR1H3) Promoter Polymorphisms Are Associated With Systemic Lupus Erythematosus in Koreans

Liver X Receptors Alpha Gene (NR1H3) Promoter Polymorphisms Are Associated With Systemic Lupus Erythematosus in Koreans

Ja-Young Jeon et al. Arthritis Res Ther.

Abstract

Introduction: Liver X receptors are established sensors of lipid and cholesterol homeostasis. Recent studies have reported that these receptors are involved in the regulation of inflammation and immune responses. We attempted to identify single nucleotide polymorphisms (SNPs) of the NR1H3 gene associated with the susceptibility to systemic lupus erythematosus (SLE).

Methods: SNPs were genotyped using SNaPSHOT assay in 300 Korean patients with SLE and 217 normal controls (NC), and in replication samples (160 SLE patients and 143 NC). Also, the functional effects of NR1H3 gene promoter polymorphisms were analyzed using a luciferase assay, real-time polymerase chain reaction, B cell proliferation assay and an electrophoretic mobility shift assay.

Results: We identified five polymorphisms: -1851 T > C (rs3758673), -1830 T > C (rs3758674), -1003 G > A (new), -840 C > A (rs61896015) and -115 G > A (rs12221497). There was a significant and reproducible difference in the -1830 T > C, -1003 G > A and -115 G > A polymorphisms between the SLE and the NC. Luciferase activity of the structure containing -1830 C was less enhanced compared to the structure containing -1830 T in basal, GW3965 and T0901317 treated Hep3B cells (P = 0.009, P = 0.034 and P <0.001, respectively). Proliferation of the -1830 TC type was increased compared to the -1830 TT type in basal, GW3965 and T0901317 treated B cells from SLE patients (P = 0.011, P = 0.040 and P = 0.017, respectively). Transcription factor GATA-3 preferentially bound the -1830 T allele in the promoter.

Conclusions: NR1H3 genetic polymorphisms may be associated with disease susceptibility and clinical manifestations of SLE. Specifically, -1830 T > C polymorphism within NR1H3 promoter region may be involved in regulation of NR1H3 expression.

Figures

Figure 1
Figure 1
Functional effect of the polymorphisms on transcriptional activity. (A, B) Relative luciferase production (ratio of luciferase production driven by the reporter construct to that by the promoter-less control vector, pGL3-Basic) in Hep3B cells (A) and COS-7 cells (B). (C, D) Relative luciferase production by stimulation with 200 ng/mL LPS, 3 μmol/L GW3965 and 5 μmol/L T0901317 in Hep3B cells (C) and COS-7 cells (D). The P value was determined by a paired t test. *P <0.05. LPS, lipopolysaccharide.
Figure 2
Figure 2
Results of an EMSA for the -1830 T > C polymorphism using nuclear extracts obtained from Hep3B cells. (A) Nucleotide sequences of oligonucleotides used as probes and competitors. (B) Differential binding of a nuclear protein to -1830 T > C alleles. (C) Identification of the transcription factor binding to 1830 T > C probe using competitive binding assay. EMSA, electrophoretic mobility shift assay.
Figure 3
Figure 3
Effect of the -1830 T > C polymorphism on NR1H3 mRNA expression and B cells proliferation assay. (A) RT PCR products were separated by electrophoresis on 1% agarose gel and stained with EtBr. Gels were examined by ultraviolet illumination. (B) The mRNA levels were determined by quantitative-PCR normalized to β-actin mRNA expression. Each experiment was conducted in duplicate for each sample, and the results are expressed as mean ± SD for three independent experiments. The P value was determined by a paired t test. (C) The effect of the -1830 T > C polymorphism on the NR1H3 gene on the B cells proliferation assay. This is a colorimetric assay containing a tetrazolium compound. When the tetrazolium is reduced, it produces a colored formazan product that is soluble in cell culture medium and maximally absorbed at 490 nm. Assays were conducted in triplicate, and the experiments were repeated at least three times. Values are mean ± SD; *P <0.05. (D) Effect of the -1830 T > C polymorphism on the NR1H3 gene on B cells proliferation by stimulation with 200 ng/mL LPS, 3 μmol/L GW3965 and 5 μmol/L T0901317. Values are mean ± SD. The P value was determined by a paired t test. *P <0.05. LPS, lipopolysaccharide; SD, standard deviation.

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