ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release.

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease characterized by progressive ectopic mineralization of the skin, eyes, and arteries, for which no effective treatment exists. PXE is caused by inactivating mutations in the gene encoding ATP-binding cassette sub-family C member 6 (ABCC6), an ATP-dependent efflux transporter present mainly in the liver. Abcc6(-/-) mice have been instrumental in demonstrating that PXE is a metabolic disease caused by the absence of an unknown factor in the circulation, the presence of which depends on ABCC6 in the liver. Why absence of this factor results in PXE has remained a mystery. Here we report that medium from HEK293 cells overexpressing either human or rat ABCC6 potently inhibits mineralization in vitro, whereas medium from HEK293 control cells does not. Untargeted metabolomics revealed that cells expressing ABCC6 excrete large amounts of nucleoside triphosphates, even though ABCC6 itself does not transport nucleoside triphosphates. Extracellularly, ectonucleotidases hydrolyze the excreted nucleoside triphosphates to nucleoside monophosphates and inorganic pyrophosphate (PPi), a strong inhibitor of mineralization that plays a pivotal role in several mineralization disorders similar to PXE. The in vivo relevance of our data are demonstrated in Abcc6(-/-) mice, which had plasma PPi levels <40% of those found in WT mice. This study provides insight into how ABCC6 affects PXE. Our data indicate that the factor that normally prevents PXE is PPi, which is provided to the circulation in the form of nucleoside triphosphates via an as-yet unidentified but ABCC6-dependent mechanism.

ABCG2 functions as a general phytoestrogen sulfate transporter in vivo.

ABCG2 is an ATP-dependent efflux transporter that limits the systemic exposure of its substrates. The preferred substrates of ABCG2 in vivo are largely unknown. We aimed to identify the compounds transported by ABCG2 under physiological conditions. In vitro, ABCG2 transports several sulfate conjugates at high rates. We therefore used targeted metabolomics, specifically detecting compounds conjugated to sulfate, to search in plasma, urine, and bile samples of wild-type and Abcg2-/- mice for differentially present compounds, which are likely to represent in vivo ABCG2 substrates. Levels of many sulfate conjugates were up to 15-fold higher in plasma and urine of Abcg2-/- than of wild-type mice, with the opposite effect seen in bile. These differentially present compounds were identified as the sulfate conjugates of phytoestrogens, compounds with weak pro- or antiestrogenic properties. We confirmed that these sulfate conjugates were ABCG2 substrates using transportomics, a method that uses vesicular transport assays to screen for substrates of ABC transporters in body fluids. In conclusion, our results show that ABCG2 limits the systemic exposure to many different phytoestrogens, a class of compounds to which mammals are exposed on a daily basis via food of plant origin, by directing their sulfate conjugates for excretion via the feces.

Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays.

The ATP-binding cassette (ABC) genes encode the largest family of transmembrane proteins. ABC transporters translocate a wide variety of substrates across membranes, but their physiological function is often incompletely understood. We describe a new method to study the substrate spectrum of ABC transporters: We incubate extracts of mouse urine with membrane vesicles prepared from Spodoptera frugiperda Sf9 insect cells overproducing an ABC transporter and determine the compounds transported into the vesicles by LC/MS-based metabolomics. We illustrate the power of this simple "transportomics" approach using ABCC2, a protein present at sites of uptake and elimination. We identified many new substrates of ABCC2 in urine. These included glucuronides of plant-derived xenobiotics, a class of compounds to which humans are exposed on a daily basis. Moreover, we show that the excretion of these compounds in vivo depends on ABCC2: compared to wild-type mice, the urinary excretion of several glucuronides was increased up to 20-fold in Abcc2(-/-) mice. Transportomics has broad applicability, as it is not restricted to urine and can be applied to other ATP-dependent transport proteins as well.