Nucleotide binding and nucleotide hydrolysis properties of the ABC transporter MRP6 (ABCC6)

Mutations in the MRP gene family member MRP6 cause pseudoxanthoma elasticum (PXE) in humans, a disease affecting elasticity of connective tissues. The normal function of MRP6, including its physiological substrate(s), remains unknown. To address these issues, recombinant rat Mrp6 (rMrp6) was expressed in the methylotrophic yeast Pichia pastoris. The protein was expressed in the membrane fraction as a stable 170 kDa protein. Its nucleotide binding and hydrolysis properties were investigated using the photoactive ATP analogue 8-azido-[alpha-(32)P]ATP and compared to those of the drug efflux pump MRP1. rMrp6 can bind 8-azido-[alpha-(32)P]ATP in a Mg(2+)-dependent and EDTA-sensitive fashion. Co(2+), Mn(2+), and Ni(2+) can also support 8-azido-[alpha-(32)P]ATP binding by rMrp6 while Ca(2+), Cd(2+), and Zn(2+) cannot. Under hydrolysis conditions (at 37 degrees C), the phosphate analogue beryllium fluoride (BeF(x)()) can stimulate trapping of the 8-azido-[alpha-(32)P]adenosine nucleotide in rMrp6 (and in MRP1) in a divalent cation-dependent and temperature-sensitive fashion. This suggests active ATPase activity, followed by trapping and photo-cross-linking of the 8-azido-[alpha-(32)P]ADP to the protein. By contrast to MRP1, orthovanadate-stimulated nucleotide trapping in rMrp6 does not occur in the presence of Mg(2+) but can be detected with Ni(2+) ions, suggesting structural and/or functional differences between the two proteins. The rMrp6 protein can be specifically photolabeled by a fluorescent photoactive drug analogue, [(125)I]-IAARh123, with characteristics similar to those previously reported for MRP1 (1), and this photolabeling of rMrp6 can be modulated by several structurally unrelated compounds. The P. pastoris expression system has allowed demonstration of ATP binding and ATP hydrolysis by rMrp6. In addition to providing large amounts of active protein for detailed biochemical studies, this system should also prove useful to identify potential rMrp6 substrates in [(125)I]-IAARh123 photolabeling competition studies, as well as to study the molecular basis of PXE mutations, which are most often found in the NBD2 of MRP6.     

Expression and in vivo rescue of human ABCC6 disease-causing mutants in mouse liver

Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application.     

Analysis of ABCC6 (MRP6) in normal human tissues

To determine the tissue distribution of the ABC transporter ABCC6 in normal human tissues, we analyzed tissue arrays for the presence of ABCC6 mRNA by in situ hybridization and ABCC6 protein by immunohistochemistry using the polyclonal antibody HB-6. We detected ABCC6 mRNA and protein in various epithelial cells of exocrine and endocrine tissues, such as acinar cells in the pancreas, mucosal cells of the intestine and follicular epithelial cells of the thyroid. We obtained a very strong immunostaining for enteroendocrine G cells in the stomach. In addition, ABCC6 mRNA and protein were present in most neurons of the brain, in alveolar macrophages in the lungs and lymphocytes in the lymph node. Immunohistochemisty using the monoclonal antibody M₆II-31 confirmed the widespread tissue distribution of ABCC6. The physiological substrate(s) of ABCC6 are yet unknown, but we suggest that ABCC6 fulfills multiple functions in different tissues. The strong immunostaining for ABCC6 in G cells suggests that it plays an important role in these endocrine cells.     

A spectrum of ABCC6 mutations is responsible for pseudoxanthoma elasticum

To better understand the pathogenetics of pseudoxanthoma elasticum (PXE), we performed a mutational analysis of ATP-binding cassette subfamily C member 6 (ABCC6) in 122 unrelated patients with PXE, the largest cohort of patients yet studied. Thirty-six mutations were characterized, and, among these, 28 were novel variants (for a total of 43 PXE mutations known to date). Twenty-one alleles were missense variants, six were small insertions or deletions, five were nonsense, two were alleles likely to result in aberrant mRNA splicing, and two were large deletions involving ABCC6. Although most mutations appeared to be unique variants, two disease-causing alleles occurred frequently in apparently unrelated individuals. R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Putative disease-causing mutations were identified in approximately 64% of the 244 chromosomes studied, and 85.2% of the 122 patients were found to have at least one disease-causing allele. Our results suggest that a fraction of the undetected mutant alleles could be either genomic rearrangements or mutations occurring in noncoding regions of the ABCC6 gene. The distribution pattern of ABCC6 mutations revealed a cluster of disease-causing variants within exons encoding a large C-terminal cytoplasmic loop and in the C-terminal nucleotide-binding domain (NBD2). We discuss the potential structural and functional significance of this mutation pattern within the context of the complex relationship between the PXE phenotype and the function of ABCC6.     

Clustering of disease-causing mutations on the domain-domain interfaces of ABCC6

Mutations in ABCC6 are responsible for pseudoxanthoma elasticum (PXE), a rare genetic disease affecting the elastic tissues of the body. ABCC6 encodes a 1503 amino acid long ABC transporter, ABCC6/MRP6. The functional link between the impaired activity of the protein and the disease is not known. We have built a homology model of this transporter, and analyzed the distribution of the known 119 missense PXE-associated mutations within the predicted structure. Significant clustering of the missense mutations has been found at complex domain-domain interfaces: at the transmission interface that involves four intracellular loops and the two ABC domains as well as at the ABC-ABC interacting surfaces. The mutations affecting these regions are 2.75 and 3.53-fold more frequent than the average mutational rate along the transporter protein sequence. These data provide a genetic proof of the importance of these domain-domain interactions in the ABCC6 transporter.     

Novel mutations of ABCC6 gene in Japanese patients with Angioid streaks

Angioid streaks (AS) are eye abnormalities caused by breaks in Bruch’s membrane. The condition is often associated with pseudoxanthoma elasticum (PXE). The ATP-binding cassette, sub-family C (CFTR/MRP), member 6 (ABCC6) is reported to be the causal gene for PXE, although there have been no reports on whether the ABCC6 gene is the causal gene for AS. The aims of this study are to isolate the causal mutations for AS using a haplotype-based case-control study. We genotyped 54 Japanese AS patients and 150 controls for 5 single-nucleotide polymorphisms (SNPs). A simple association study using each SNP and a haplotype-based case-control study were performed. Twelve patients with special haplotypes for AS were selected, and were then subjected to gene sequencing. Six variants were successfully identified as causal mutations for AS (p.R419Q, p.E422K, c.2542delG, Del_Exon23, c.3774-3775insC and p.E1427K), and 4 of these were novel. This method can be applied to both identifying susceptibility variants of multifactorial diseases and isolating mutations in single-gene diseases.     

RNA interference-triggered reversal of ABCC2-dependent cisplatin resistance in human cancer cells

The adenosine triphosphate binding cassette (ABC)-transporter ABCC2 (MRP2/cMOAT) can mediate resistance against the commonly used anticancer drugs cisplatin and paclitaxel. To overcome the ABCC2-depending drug resistance, two specific anti-ABCC2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS. Since both siRNAs showed biological activity, for stable inhibition of ABCC2 a corresponding short hairpin RNA (shRNA)-encoding expression vector was designed. By treatment of A2780RCIS cells with this construct, the expressions of the targeted ABCC2 encoding mRNA and transport protein were inhibited. These effects were accompanied by reversal of resistance against cisplatin and paclitaxel. Thus, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that siRNA- and shRNA-mediated RNAi-based gene therapeutic approaches may be applicable in preventing and reversing ABCC2-depending drug resistance.     

The multidrug resistance protein ABCC1 drug-binding domains show selective sensitivity to mild detergents

The multidrug resistance protein (ABCC1 or MRP1) causes resistance to multiple drugs through reduced drug accumulation. We have previously demonstrated direct interaction between MRP1 and unmodified drugs using photoreactive drug analogues. In this study, we describe the use of [125I]iodoaryl azido-rhodamine123 (IAARh123)-a photoactive drug analogue of rhodamine 123, to study the effects of mild detergents and denaturing agents on MRP1-drug binding in membrane vesicles prepared from HeLa cells transfected with the MRP1 cDNA. Our results show that the zwitterionic detergent CHAPS and a nonionic detergent Brij35 inhibited the photolabeling of MRP1 with IAARh123. Sodium deoxycholate (SDC) and octyl-beta-glucoside (OG), structurally similar to CHAPS and Brij35 and disrupting the lipid bilayer, showed a modest increase of MRP1 photolabeling with IAARh123. Proteolytic digestion of IAARh123 photolabeled MRP1 labeled in the presence or absence of various detergents (CHAPS, SDC, or OG) revealed identical photolabeled peptides. Consistent with the drug-binding results, non-toxic concentrations of CHAPS and Brij35 reversed vincristine and etoposide (VP16) toxicity in MRP1 expressing cells. Taken together, the results of this study show that MRP1-drug interaction can occur outside the lipid bilayer environment. However, this interaction is inhibited with certain mild detergents.     

Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6)

Mutations in the ABCC6 (MRP6) gene cause pseudoxanthoma elasticum (PXE), a rare heritable disorder resulting in the calcification of elastic fibers. In the present study a cDNA encoding a full-length normal variant of ABCC6 was amplified from a human kidney cDNA library, and the protein was expressed in Sf9 insect cells. In isolated membranes ATP binding as well as ATP-dependent active transport by ABCC6 was demonstrated. We found that glutathione conjugates, including leukotriene C(4) and N-ethylmaleimide S-glutathione (NEM-GS), were actively transported by human ABCC6. Organic anions (probenecid, benzbromarone, indomethacin), known to interfere with glutathione conjugate transport of human ABCC1 and ABCC2, inhibited the ABCC6-mediated NEM-GS transport in a specific manner, indicating that ABCC6 has a unique substrate specificity. We have also expressed three missense mutant forms of ABCC6, which have recently been shown to cause PXE. MgATP binding was normal in these proteins; ATP-dependent NEM-GS or leukotriene C(4) transport, however, was abolished. Our data indicate that human ABCC6 is a primary active transporter for organic anions. In the three ABCC6 mutant forms examined, the loss of transport activity suggests that these mutations result in a PXE phenotype through a direct influence on the transport activity of this ABC transporter.     

Subcellular localization and N-glycosylation of human ABCC6, expressed in MDCKII cells

Mutations in the gene coding for a human ABC transporter protein, ABCC6 (MRP6), are responsible for the development of pseudoxanthoma elasticum. Here, we demonstrate that human ABCC6, when expressed by retroviral transduction in polarized mammalian (MDCKII) cells, is exclusively localized to the basolateral membrane. The human ABCC6 in MDCKII cells was found to be glycosylated, in contrast to the underglycosylated form of the protein, as expressed in Sf9 cells. In order to localize the major glycosylation site(s) in ABCC6, we applied limited proteolysis on the fully glycosylated and underglycosylated forms, followed by immunodetection with region-specific antibodies for ABCC6. Our results indicate that Asn15, which is located in the extracellular N-terminal region of human ABCC6, is the only N-glycosylation site in this protein. The polarized mammalian expression system characterized here provides a useful tool for further examination of routing, glycosylation, and function of the normal and pathological variants of human ABCC6.     

Novel mutations in the ABCC6 gene of German patients with pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a heritable disorder of the connective tissue affecting the skin, eyes, and cardiovascular system. Recently, the PXE candidate gene ABCC6 was identified and a limited number of ABCC6 mutations were observed in different PXE cohorts. To identify novel PXE-causing ABCC6 mutations in German patients with PXE, we investigated a cohort of 54 German PXE patients and 23 family members from 49 apparently nonconsanguineous families. From the mutational analysis we found 27 different ABCC6 sequence variations. Among these, 11 were polymorphisms or neutral alterations and 16 were PXE-causing mutations. The most common mutation in our PXE cohort was the nonsense mutation p.R1141X, which occurred with an allele frequency of 25.9%. Furthermore, we found nine missense, one additional nonsense, and two putative splice site mutations as well as three single-nucleotide deletions. Most of these mutations were unique and occurred in cytoplasmic regions of the MRP6 protein; these mutations are proposed to be critical for the physiological function of the MRP6 protein. In these regions we also found the three novel PXE-causing mutations p.R1114C, p.Y1239H, and p.G1311E, which were identified in three alleles from patients with PXE and were absent in 200 healthy control subjects. In addition, the first genotype-phenotype correlation was observed. By obtaining these genetic mutation data, we are contributing to an overview of all ABCC6 mutations leading to PXE and the pathogenetics of this disease.     

Regulation of ABCC6 Trafficking and Stability by a Conserved C-terminal PDZ-Like Sequence

Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE). The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein.     

Role of the MRP1/ABCC1 multidrug transporter protein in cancer

Multidrug resistance is a major obstacle to cancer treatment and leads to poor prognosis for the patient. Multidrug resistance-associated protein 1 (MRP1) transports a wide range of therapeutic agents as well as diverse physiological substrates and may play a role in the development of drug resistance in several cancers including those of the lung, breast and prostate, as well as childhood neuroblastoma. The majority of patients with neuroblastoma present with widely disseminated disease at diagnosis and despite intensive treatment, the prognosis for such patients is dismal. There is increasing evidence that MRP1 is a MYCN target gene involved in the development of multidrug resistance in neuroblastoma. Given the importance of MRP1 overexpression in neuroblastoma, MRP1 inhibition may be a clinically relevant approach to improving patient outcome in this disease.     

Effects of plant sterols on human multidrug transporters ABCB1 and ABCC1

The effects of dietary plant sterols on human drug efflux transporters P-glycoprotein (P-gp, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-gp-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural phytosterols found in foods, herbs, and dietary supplements such as β-sitosterol, campesterol, stigmasterol, fucosterol, and z-guggulsterone were investigated. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-gp, increased in the presence of guggulsterone in KB-C2 cells. The efflux of rhodamine 123 from KB-C2 cells was inhibited by guggulsterone. Guggulsterone also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activities of P-gp and MRP1 were stimulated by guggulsterone. These results suggest that guggulsterone, a natural dietary hypolipidemic agent have dual inhibitory effects on P-gp and MRP1 and the potencies to cause food-drug interactions.     

COX-1 is coupled with mPGES-1 and ABCC4 in human cervix cancer cells

Cyclooxygenases are key enzymes in the arachidonic acid metabolism. Their unstable intermediate, prostaglandin H₂, is further metabolized to bioactive lipids by various downstream enzymes. In this study, utilizing short hairpin RNAs, we prepared a cell line of human cervix carcinoma with stable down-regulated cyclooxygenase-1 (COX-1) to assess the impact of COX-1 reduction on the downstream enzymes. We found a significant microsomal prostaglandin E synthase-1 (mPGES-1) suppression. In addition, mRNA expression of multidrug resistance protein 4 (MRP4, ABCC4), supposed to take part in antiviral and anticancer drug transport from cells, was up-regulated after COX-1 down-regulation. Our findings indicate that mPGES-1, believed to be coexpressed preferentially with cyclooxygenase-2, may be coupled to COX-1. ABCC4 up-regulation further supports the assumption of its involvement in prostanoid transport.