NRBP1 modulates uric acid transporter ABCG2 expression by activating the Wnt/β-catenin pathway in HK-2 cells

Background: Nuclear receptor binding protein 1 (NRBP1) and ATP-binding cassette subfamily G member 2 (ABCG2) was the gout risk gene and high-capacity urate exporter respectively. However, the relationship between NRBP1 and ABCG2 and the underlying molecular mechanism contributing to these associations are unknown. Methods: Firstly, the efficiency of the overexpression and knockdown of NRBP1 was confirmed by western blot. Next, the effect of NRBP1 overexpression and knockdown on the expression of ABCG2, organic anion transporter 1 (OAT1), glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) was detected by qRT-PCR and western blot. At the same time, the cellular location of ABCG2 and its expression after NRBP1 overexpression and knockdown was tested by immunofluorescence (IF) staining. Then, the mechanism of NRBP1 modulates ABCG2 expression was evaluated by western blot with or without the β-catenin inhibitor (21H7). Results: The lentivirus system was used to generate stable NRBP1 overexpression, while the plasmids carrying a NRBP1 siRNA was generated to knockdown NRBP1 expression in HK-2 cells. Meanwhile, the overexpression of NRBP1 significantly decreased the mRNAs and proteins expression of GLUT9 and URAT1, while the knockdown of NRBP1 increased the mRNAs and proteins expression of ABCG2 significantly. In addition, the NRBP1 modulates the expression of ABCG2 was by ctivating the Wnt/β-catenin pathway in HK-2 cells according to the IF and western blot results. Conclusion: Taken together, our study demonstrated that NRBP1 inhibition played an essential role in attenuating hyperuricemia and gout by upregulation of ABCG2 via Wnt/β-catenin signaling pathway in HK-2 cells. Resumen: Antecedentes: La proteína de unión al receptor nuclear 1 (NRBP1) y el miembro G de la subclase ATP binding Box 2 (ABCG2) son los genes de riesgo de gota y los genes de salida de urato de alto rendimiento, respectivamente. Sin embargo, se desconoce la relación entre NRBP1 y ABCG2, y los posibles mecanismos moleculares que conducen a estas asociaciones. Métodos: En primer lugar, la sobreexpresión y el knockout de NRBP1 fueron confirmados por Western-blot. Los efectos de la sobreexpresión y knockout de NRBP1 en la expresión de ABCG2, transportador de aniones orgánicos 1 (OAT1), transportador de glucosa 9 (GLUT9) y transportador de ácido úrico 1 (URAT1) fueron detectados por qRT-PCR y Western-blot. Mientras tanto, la localización y expresión de ABCG2 después de la sobreexpresión y knockout de NRBP1 fueron detectadas por inmunofluorescencia (IF). Luego, el efecto regulador de NRBP1 sobre la expresión de ABCG2 fue estudiado por Western-blot y comparado con el inhibidor de la β-catenina (21H7). Resultados: El sistema lentiviral indujo una sobreexpresión estable de NRBP1, mientras que el plásmido portador de SiRNA NRBP1 inhibió la expresión de NRBP1 en las células HK-2. Mientras tanto, la sobreexpresión de NRBP1 redujo significativamente la expresión de ARNm y proteínas de GLUT9 y URAT1, mientras que el knockout de NRBP1 aumentó significativamente la expresión de ARNm y proteínas de ABCG2. Además, de acuerdo con los resultados de IF y Western-blot, NRBP1 regula la expresión de ABCG2 activando la vía Wnt/β-catenina en las células HK-2. Conclusión: La inhibición del NRBP1 aumenta la regulación de ABCG2 a través de la vía de señalización Wnt/β-catenina, que desempeña un papel importante en la reducción de la hiperuricemia y la gota

DNA hypomethylation of a transcription factor binding site within the promoter of a gout risk gene NRBP1 upregulates its expression by inhibition of TFAP2A binding

Abstract Background Genome-wide association studies (GWASs) have identified dozens of loci associated with gout, but for most cases, the risk genes and the underlying molecular mechanisms contributing to these associations are unknown. This study sought to understand the molecular mechanism of a common genetic variant, rs780093, in the development of gout, both in vitro and in vivo. Results Nuclear receptor binding protein 1 (NRBP1), as a gout risk gene, and its regulatory region, 72 bp upstream of the transcription start site, designated as B1, were identified through integrative analyses of genome-wide genotype and DNA methylation data. We observed elevated NRBP1 expression in human peripheral blood mononuclear cells (PBMCs) from gout patients. In vitro luciferase reporter and protein pulldown assay results showed that DNA methylation could increase the binding of the transcription factor TFAP2A to B1, leading to suppressed gene expression. There results were further confirmed by in vivo bisulfite pyrosequencing showing that hypomethylation on B1 is associated with increased NRBP1 expression in gout patients. Conclusions Hypomethylation at the promoter region of NRBP1 reduces the binding of TFAP2A and thus leads to elevated NRBP1 expression, which might contribute to the development of gout

Additional file 2: File 1. of DNA hypomethylation of a transcription factor binding site within the promoter of a gout risk gene NRBP1 upregulates its expression by inhibition of TFAP2A binding

Detailed experimental methods. a Quantitative real-time PCR. b Generation of methylated DNA and luciferase reporter. c Protein pulldown assay. (DOCX 13 kb

Additional file 1: Figure S1. of DNA hypomethylation of a transcription factor binding site within the promoter of a gout risk gene NRBP1 upregulates its expression by inhibition of TFAP2A binding

Work flow diagram. Figure S2. Serum uric acid is regulated by B1 methylation level and NRBP1 expression. a A marginal significant negative association between DNA methylation of B1 and serum uric acid (P value = 0.08). b A significant positive association between NRBP1 expression and serum uric acid (P value = 0.03). Figure S3. Decreased DNA methylation at the promoter region of NRBP1 in gout patients. a The DNA sequence at the promoter region of NRBP1 gene. The CpG sites, designated as B1 to B6, are highlighted in red. b The methylation level for each CpG site indicated in a, was investigated by bisulfite pyrosequencing. Data are represented as mean ± SEM. (*P value <0.01, **P value <0.001, Student’s t test, unpaired, two-sided). (PPTX 1077 kb

Carbonate weathering-related carbon sink fluxes under different land uses: A case study from the Shawan Simulation Test Site, Puding, Southwest China

In the study of global climate change, a major focus of research into the carbon cycle is to determine the fate of missing carbon sinks. Carbonate weathering-related carbon sinks as a result of water-carbonate-CO2-aquatic phototroph interactions may make a major contribution. Establishing optimal land uses, which determine soil CO2 concentrations and water supplies for carbonate dissolution, may be a feasible and effective way to increase the sink potential. Elucidating both the hydrological and the hydrochemical behavior under different land uses is critical for rational planning of land use changes to increase the carbon sink. Given the complexity within natural karst catchments, the Shawan Simulation Test Site was established at Puding, Southwest China, to simulate the influence of land uses with controlled carbonate test beds - bare rock, bare soil, crop land, grass land, shrub land. Soil CO2 concentrations, hydrochemical parameters (pH, major ion concentrations) and the 'spring' (artificial drain) discharge were intensively measured from Sept. 1, 2015 to Aug. 31, 2016 to investigate the carbon and water responses to different land uses in different seasons. In the vegetated land uses (crop, grass or shrub), DIC increased due to the increase of soil CO2 resulting from stronger microbial activities and root respiration in summer and autumn growing seasons. In the bare rock and soil cases, there was also an increase in DIC in summer and autumn, due to decomposition of prior organic matter within soils and/or rock pores. The average DIC concentration ranking, high to low, was grass land, shrub land, crop land, bare soil, bare rock. Soil CO2 concentration was thus the dominant of DIC concentration, which is a key multiplier of carbon sink fluxes (CSF = 0.5 * [DIC] * RD, where RD is depth of runoff, [DIC] is DIC concentration, and 0.5 because in carbonate dissolution, only half of the [HCO3-] is of atmospheric carbon origin). However, runoff depth ranked almost in reverse order, i.e., from high to low, bare rock, bare soil, crop land, shrub land, grass land. The CSF ranking, from high to low, was grass, crop, shrub, bare rock, bare soil. A new parameter, LCIC (Land use Change Impact on CSF) is defined to compare the impacts of land use change on [DIC] and RD, and evaluate their combined effects on CSF. Compared to bare rock, the absolute values of LCIC (| LCIC| s) were&gt; 1, and CSFs were larger for the three tanks with vegetation cover; CSF is smaller for bare soil, | LCIC| &lt; 1. Finally, it was found that grass land may constitute an optimal land-use for increasing the carbonate weathering-related carbon sink that is critical for carbon management to counter global warming

DNA methylation mediates genotype and smoking interaction in the development of anti-citrullinated peptide antibody-positive rheumatoid arthritis

Abstract Background Multiple factors, including interactions between genetic and environmental risks, are important in susceptibility to rheumatoid arthritis (RA). However, the underlying mechanism is not fully understood. This study was undertaken to evaluate whether DNA methylation can mediate the interaction between genotype and smoking in the development of anti-citrullinated peptide antibody (ACPA)-positive RA. Methods We investigated the gene-smoking interactions in DNA methylation using 393 individuals from the Epidemiological Investigation of Rheumatoid Arthritis (EIRA). The interaction between rs6933349 and smoking in the risk of developing ACPA-positive RA was further evaluated in a larger portion of the EIRA (1119 controls and 944 ACPA-positive patients with RA), and in the Malaysian Epidemiological Investigation of Rheumatoid Arthritis (MyEIRA) (1556 controls and 792 ACPA-positive patients with RA). Finally, mediation analysis was performed to investigate whether DNA methylation of cg21325723 mediates this gene-environment interaction on the risk of developing of ACPA-positive RA. Results We identified and replicated one significant gene-environment interaction between rs6933349 and smoking in DNA methylation of cg21325723. This gene-smoking interaction is a novel interaction in the risk of developing ACPA-positive in both Caucasian (multiplicative P value = 0.056; additive P value = 0.016) and Asian populations (multiplicative P value = 0.035; additive P value = 0.00027), and it is mediated through DNA methylation of cg21325723. Conclusions We showed that DNA methylation of cg21325723 can mediate the gene-environment interaction between rs6933349 and smoking, impacting the risk of developing ACPA-positive RA, thus being a potential regulator that integrates both internal genetic and external environmental risk factors

Additional file 1: of DNA methylation mediates genotype and smoking interaction in the development of anti-citrullinated peptide antibody-positive rheumatoid arthritis

Supplementary materials include five supplementary figures and a supplementary table. (PDF 440 kb