Tappei Takada

Common Defects of ABCG2, a High-Capacity Urate Exporter, Cause Gout: A Function-Based Genetic Analysis in a Japanese Population

Dysfunctional genotype combinations of polymorphic adenosine 5′-triphosphate–binding cassette transporter gene ABCG2/BCRP, which encodes a high-capacity urate secretion transporter in human gut and kidney, are major causes of gout. Gout, the “Disease of Kings” as it is often known, is a painful medical condition characterized by sharp acute pain in bone joints, due to the high deposition of uric acid crystals from the blood serum into the surrounding cartilage. It affects approximately 1% of the U.S. population and remains a significant public health concern. The prevalence of gout is much higher in certain Asian ethnic groups, and is also reportedly rising in African Americans. Current medical treatments are aimed at ameliorating pain severity, but as the underlying genetic etiology of the disease unfolds, new targets for future therapies are likely to be found. Although genome-wide association studies (GWAS) have enabled the calculation of risk predispositions for a wide variety of complex diseases, the relation of gene function to the causality of disease-related mutations has remained largely unclear. A recent U.S. population–based study supported an association between urate levels and gout in individuals carrying variants in a multifunctional transporter gene, ABCG2. This study identified Q141K as a high-risk variant in nearly 10% of gout cases in Caucasians. Now, a team led by Hirotaka Matsuo report that in a Japanese population, another risk variant in ABCG2, namely the Q126X nonfunctional mutation, confers an even higher risk associated with an increase in uric acid deposition in the blood and may cause gout in Asians. Because this gene is responsible for giving rise to a protein that transports harmful waste products and metabolites out of the kidney and gut, they extensively validate the biological activity of ABCG2 using functional assays in vitro that effectively recapitulate human data obtained from Japanese individuals afflicted with the disease. These findings lend weight to previously reported GWAS; moreover, these newly identified specific high-risk variants that block urate secretion may serve as potential intervention points for quelling the disease. Gout based on hyperuricemia is a common disease with a genetic predisposition, which causes acute arthritis. The ABCG2/BCRP gene, located in a gout-susceptibility locus on chromosome 4q, has been identified by recent genome-wide association studies of serum uric acid concentrations and gout. Urate transport assays demonstrated that ABCG2 is a high-capacity urate secretion transporter. Sequencing of the ABCG2 gene in 90 hyperuricemia patients revealed several nonfunctional ABCG2 mutations, including Q126X. Quantitative trait locus analysis of 739 individuals showed that a common dysfunctional variant of ABCG2, Q141K, increases serum uric acid. Q126X is assigned to the different disease haplotype from Q141K and increases gout risk, conferring an odds ratio of 5.97. Furthermore, 10% of gout patients (16 out of 159 cases) had genotype combinations resulting in more than 75% reduction of ABCG2 function (odds ratio, 25.8). Our findings indicate that nonfunctional variants of ABCG2 essentially block gut and renal urate excretion and cause gout.

Niemann-Pick C1-Like 1 Mediates α-Tocopherol Transport

Dietary lipids and fat-soluble micronutrients are solubilized in mixed micelles and absorbed in the small intestine. Based on an assumption that cholesterol and other fat-soluble molecules share a number of transport mechanisms and the fact that Niemann-Pick C1-like 1 (NPC1L1) is critical for intestinal cholesterol absorption, we hypothesized that some fat-soluble molecules may be transported by NPC1L1. To investigate this hypothesis, we compared the cellular uptake and inhibitory effects of ezetimibe, the molecular target of which is NPC1L1, between cholesterol and some fat-soluble molecules using rat NPC1L1-overexpressing Caco-2 cells. The in vitro analysis suggested that NPC1L1 mediates the uptake of α-tocopherol (vitamin E) in an ezetimibe-sensitive manner as well as the uptake of cholesterol but does not mediate the uptake of retinol (vitamin A) or cyclosporin A. To confirm the ezetimibe-sensitive uptake of α-tocopherol in vivo, we performed an in vivo absorption study using rats and the results suggested a physiologically significant role of NPC1L1-mediated α-tocopherol absorption. Furthermore, using human NPC1L1 overexpression system, we demonstrated that both cholesterol and α-tocopherol uptake was also significantly increased by the overexpression of human NPC1L1 and ezetimibe inhibited their uptake. Mutual inhibition studies of cholesterol and α-tocopherol in human NPC1L1-mediated uptake revealed the inhibitory effect of cholesterol and the stimulatory effect of α-tocopherol on the NPC1L1-mediated transport of both substrates. The present data suggest, for the first time, that NPC1L1 has the ability to transport α-tocopherol and that ezetimibe is able to inhibit the intestinal absorption of α-tocopherol.

Niemann-Pick C1-Like 1 Overexpression Facilitates Ezetimibe-Sensitive Cholesterol and β-Sitosterol Uptake in CaCo-2 Cells

Previous in vivo studies including those with knockout mice suggested that Niemann-Pick C1-like 1 (NPC1L1) plays an essential role in the intestinal absorption of cholesterol. To characterize the mechanism of cholesterol uptake mediated by NPC1L1, an in vitro system reflecting the function of this transporter needs to be established. In the present study, we constructed NPC1L1 overexpressing CaCo-2 cells as an in vitro model and characterized the transport properties of NPC1L1. Immunohistochemical staining revealed that CaCo-2 cells express NPC1L1 on the apical membrane. It was also demonstrated that the uptakes of both cholesterol and β-sitosterol are increased by NPC1L1 overexpression. In addition, the uptake of cholesterol was increased in a dose-dependent manner by an increase in the content of taurocholate in micelles, whereas micellar phosphatidylcholine showed a negative correlation with cholesterol uptake. Furthermore, it was confirmed that sterol uptake increased by NPC1L1 overexpression was inhibited by ezetimibe. We could thus establish an in vitro intestinal model to study the mechanism of NPC1L1-dependent sterol uptake and to screen drug candidates whose target is NPC1L1.

Two common PFIC2 mutations are associated with the impaired membrane trafficking of BSEP/ABCB11.

Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by a mutation in the bile salt export pump (BSEP/ABCB11) gene. However, the mechanisms for the deficiency in the function of two mutations (E297G and D482G), which are frequently found in European patients, have not yet been identified. In the present study, we examined the transport activity and cellular localization of these two mutants in human embryonic kidney 293 and Madin‐Darby canine kidney II cells, respectively. Introduction of E297G and D482G mutations into the human BSEP gene by site‐directed mutagenesis resulted in a significant reduction in the BSEP expression level, which was associated with impaired membrane trafficking. Most of the D482G BSEP and some of the E297G BSEP underwent only core glycosylation and appeared to be predominantly located in the endoplasmic reticulum. The inhibition of proteasome function by MG132 resulted in the cellular accumulation of the core glycosylation form of the two mutants. In contrast, transport studies for taurocholate and glycocholate with membrane vesicles isolated from complementary DNA–transfected cells indicated that both mutations did not significantly affect the transport function of BSEP per se. In conclusion, E297G and D482G mutations result in impaired membrane trafficking, whereas the transport functions of these mutants remain largely unchanged. (HEPATOLOGY 2005;41:916–924.)

NPC1L1 over-expression facilitates ezetimibe-sensitive cholesterol and β-sitosterol uptake in CaCo-2 cells

Previous in vivo studies including those with knockout mice suggested that Niemann-Pick C1-like 1 (NPC1L1) plays an essential role in the intestinal absorption of cholesterol. To characterize the mechanism of cholesterol uptake mediated by NPC1L1, an in vitro system reflecting the function of this transporter needs to be established. In the present study, we constructed NPC1L1 overexpressing CaCo-2 cells as an in vitro model and characterized the transport properties of NPC1L1. Immunohistochemical staining revealed that CaCo-2 cells express NPC1L1 on the apical membrane. It was also demonstrated that the uptakes of both cholesterol and β-sitosterol are increased by NPC1L1 overexpression. In addition, the uptake of cholesterol was increased in a dose-dependent manner by an increase in the content of taurocholate in micelles, whereas micellar phosphatidylcholine showed a negative correlation with cholesterol uptake. Furthermore, it was confirmed that sterol uptake increased by NPC1L1 overexpression was inhibited by ezetimibe. We could thus establish an in vitro intestinal model to study the mechanism of NPC1L1-dependent sterol uptake and to screen drug candidates whose target is NPC1L1.

ABCG2 dysfunction causes hyperuricemia due to both renal urate underexcretion and renal urate overload

Gout is a common disease which results from hyperuricemia. We have reported that the dysfunction of urate exporter ABCG2 is the major cause of renal overload (ROL) hyperuricemia, but its involvement in renal underexcretion (RUE) hyperuricemia, the most prevalent subtype, is not clearly explained so far. In this study, the association analysis with 644 hyperuricemia patients and 1,623 controls in male Japanese revealed that ABCG2 dysfunction significantly increased the risk of RUE hyperuricemia as well as overall and ROL hyperuricemia, according to the severity of impairment. ABCG2 dysfunction caused renal urate underexcretion and induced hyperuricemia even if the renal urate overload was not remarkable. These results show that ABCG2 plays physiologically important roles in both renal and extra-renal urate excretion mechanisms. Our findings indicate the importance of ABCG2 as a promising therapeutic and screening target of hyperuricemia and gout.

Common dysfunctional variants in ABCG2 are a major cause of early-onset gout

Gout is a common disease which mostly occurs after middle age, but more people nowadays develop it before the age of thirty. We investigated whether common dysfunction of ABCG2, a high-capacity urate transporter which regulates serum uric acid levels, causes early-onset gout. 705 Japanese male gout cases with onset age data and 1,887 male controls were genotyped, and the ABCG2 functions which are estimated by its genotype combination were determined. The onset age was 6.5 years earlier with severe ABCG2 dysfunction than with normal ABCG2 function (P = 6.14 × 10−3). Patients with mild to severe ABCG2 dysfunction accounted for 88.2% of early-onset cases (twenties or younger). Severe ABCG2 dysfunction particularly increased the risk of early-onset gout (odds ratio 22.2, P = 4.66 × 10−6). Our finding that common dysfunction of ABCG2 is a major cause of early-onset gout will serve to improve earlier prevention and therapy for high-risk individuals.

Itraconazole-Induced Cholestasis: Involvement of the Inhibition of Bile Canalicular Phospholipid Translocator MDR3/ABCB4

Biliary secretion of bile acids and phospholipids, both of which are essential components of biliary micelles, are mediated by the bile salt export pump (BSEP/ABCB11) and multidrug resistance 3 P-glycoprotein (MDR3/ABCB4), respectively, and their genetic dysfunction leads to the acquisition of severe cholestatic diseases. In the present study, we found two patients with itraconazole (ITZ)-induced cholestatic liver injury with markedly high serum ITZ concentrations. To characterize the effect of ITZ on bile formation in vivo, biliary bile acids and phospholipids were analyzed in ITZ-treated rats, and it was revealed that biliary phospholipids, rather than bile acids, were drastically reduced in the presence of clinically relevant concentrations of ITZ. Moreover, by using MDR3-expressing LLC-PK1 cells, we found that MDR3-mediated efflux of [14C]phosphatidylcholine was significantly reduced by ITZ. In contrast, BSEP-mediated transport of [3H]taurocholate was not significantly affected by ITZ, which is consistent with our in vivo observations. In conclusion, this study suggests the involvement of the inhibition of MDR3-mediated biliary phospholipids secretion in ITZ-induced cholestasis. Our approach may be useful for analyzing mechanisms of drug-induced cholestasis and evaluating the cholestatic potential of clinically used drugs and drug candidates.

Genome-wide association study of clinically defined gout identifies multiple risk loci and its association with clinical subtypes

Objective Gout, caused by hyperuricaemia, is a multifactorial disease. Although genome-wide association studies (GWASs) of gout have been reported, they included self-reported gout cases in which clinical information was insufficient. Therefore, the relationship between genetic variation and clinical subtypes of gout remains unclear. Here, we first performed a GWAS of clinically defined gout cases only. Methods A GWAS was conducted with 945 patients with clinically defined gout and 1213 controls in a Japanese male population, followed by replication study of 1048 clinically defined cases and 1334 controls. Results Five gout susceptibility loci were identified at the genome-wide significance level (p<5.0×10−8), which contained well-known urate transporter genes (ABCG2 and SLC2A9) and additional genes: rs1260326 (p=1.9×10−12; OR=1.36) of GCKR (a gene for glucose and lipid metabolism), rs2188380 (p=1.6×10−23; OR=1.75) of MYL2-CUX2 (genes associated with cholesterol and diabetes mellitus) and rs4073582 (p=6.4×10−9; OR=1.66) of CNIH-2 (a gene for regulation of glutamate signalling). The latter two are identified as novel gout loci. Furthermore, among the identified single-nucleotide polymorphisms (SNPs), we demonstrated that the SNPs of ABCG2 and SLC2A9 were differentially associated with types of gout and clinical parameters underlying specific subtypes (renal underexcretion type and renal overload type). The effect of the risk allele of each SNP on clinical parameters showed significant linear relationships with the ratio of the case–control ORs for two distinct types of gout (r=0.96 [p=4.8×10−4] for urate clearance and r=0.96 [p=5.0×10−4] for urinary urate excretion). Conclusions Our findings provide clues to better understand the pathogenesis of gout and will be useful for development of companion diagnostics.

Molecular mechanisms of membrane transport of vitamin E.

Vitamin E is an essential fat-soluble micronutrient for higher mammals and functions as an antioxidant for lipids and also as a regulator of gene expression and a modulator of cell signaling and proliferation. To exert its physiological functions, vitamin E must achieve an appropriate disposition throughout the body via several processes, such as intestinal absorption, uptake and efflux in peripheral tissues and biliary secretion. In this review, we mainly discuss membrane proteins involved in these transport processes (ATP-binding cassette transporter A1, scavenger receptor class B type I, Niemann-Pick C1-like 1 and multidrug resistance 3) and vitamin E-mediated regulation of their expression.