ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release Robert S. Jansena, Aslı Küçükosmanoglu a, Marcel de Haasb, Sunny Sapthua, Jon Andoni Oteroc, Ilse E. M. Hegmana, Arthur A. B. Bergend,e,f, Theo G. M. F. Gorgelsd, P. Borsta,1, and Koen van de Weteringa,1 Divisions of aMolecular Oncology and bGene Regulation, Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands; cDepartment of Biomedical Sciences-Physiology, University of Leon, 24071 Leon, Spain; dDepartment of Molecular Ophthalmogenetics, Netherlands Institute for Neuroscience (NIN-KNAW), 1105 BA, Amsterdam, The Netherlands; and Departments of eClinical Genetics and fOphthalmology, Academic Medical Center, 1105 AZ, Amsterdam, The Netherlands Contributed by P. Borst, October 21, 2013 (sent for review July 2, 2013) − − Pseudoxanthoma elasticum (PXE) is an autosomal recessive dis- a metabolic disease (13–16). Abcc6 / muzzle skin that miner- − − ease characterized by progressive ectopic mineralization of the alizes in Abcc6 / mice does not mineralize when grafted onto skin, eyes, and arteries, for which no effective treatment exists. WT mice, and muzzle skin of WT mice mineralizes only when − − PXE is caused by inactivating mutations in the gene encoding ATP- grafted onto Abcc6 / mice (17). Moreover, surgically joining the − − binding cassette sub-family C member 6 (ABCC6), an ATP-dependent systemic circulation of Abcc6 / mice with that of WT mice halts fl Abcc6−/− − − ef ux transporter present mainly in the liver. mice have mineralization in Abcc6 / mice (18). Thus, PXE is caused not by been instrumental in demonstrating that PXE is a metabolic dis- a lack of functional ABCC6 in the affected tissues, but rather by ease caused by the absence of an unknown factor in the circula- the absence of a factor that is normally provided to the circu- tion, the presence of which depends on ABCC6 in the liver. Why lation by an ABCC6-dependent mechanism. The nature of this absence of this factor results in PXE has remained a mystery. Here factor has remained elusive. we report that medium from HEK293 cells overexpressing either In vitro vesicular uptake experiments have confirmed that human or rat ABCC6 potently inhibits mineralization in vitro, fl MEDICAL SCIENCES whereas medium from HEK293 control cells does not. Untargeted ABCC6, like its close homolog ABCC1, is an ef ux transporter, metabolomics revealed that cells expressing ABCC6 excrete large transporting a handful of glutathione-conjugates and the cyclic fi amounts of nucleoside triphosphates, even though ABCC6 itself peptide BQ-123 (10, 19, 20). The short list of identi ed ABCC6 does not transport nucleoside triphosphates. Extracellularly, ecto- substrates does not include any compound obviously relevant for nucleotidases hydrolyze the excreted nucleoside triphosphates to PXE, and the only substrate shared by human ABCC6 (hABCC6) nucleoside monophosphates and inorganic pyrophosphate (PPi), and rat ABCC6 (rABCC6) is not an endogenous compound, but a strong inhibitor of mineralization that plays a pivotal role in rather the synthetic cyclic peptide BQ-123. Based on the clinical several mineralization disorders similar to PXE. The in vivo rele- similarities of PXE to other diseases, vitamin K conjugates (21) vance of our data are demonstrated in Abcc6−/− mice, which had and adenosine (22) have been proposed as substrates, but none of plasma PPi levels <40% of those found in WT mice. This study these are actually transported (23, 24). provides insight into how ABCC6 affects PXE. Our data indicate Thus, despite all efforts, the mechanism by which ABCC6 that the factor that normally prevents PXE is PPi, which is provided prevents ectopic mineralization remains a mystery, and no at- to the circulation in the form of nucleoside triphosphates via an as- tractive new hypotheses about the nature of the physiological fi yet unidenti ed but ABCC6-dependent mechanism. ABCC6 substrate have been proposed. In the present study, we used untargeted metabolomics to screen for ABCC6-dependent ATP secretion | ectopic calcification | MRP6 | ENPP1 Significance seudoxanthoma elasticum (PXE) is an autosomal recessive Pdisease characterized by late-onset and progressive ectopic Pseudoxanthoma elasticum (PXE) is a heritable disease char- mineralization of skin, eyes, and arteries (1). This prototypical ∼ acterized by mineralization of the skin, eyes, and arteries, for connective tissue disease affects 1 in 50,000 persons worldwide, which no effective treatment exists. PXE is caused by inacti- for whom there is no effective therapy (2). Along with relatively vating mutations in the gene encoding the transporter ABCC6. benign (but stigmatizing) skin lesions, PXE causes progressive loss PXE is a metabolic disease caused by the absence of an un- of vision and cardiovascular complications (2). The severity of these known factor in the circulation. Our study indicates that the symptoms is highlyvariable among patients, even within families (3). factor that normally prevents PXE is pyrophosphate, which is In 2000, multiple research groups reported that PXE is caused provided to the circulation in the form of nucleoside triphos- by inactivating mutations in the ATP-binding cassette sub-family phates via an ABCC6-dependent mechanism. Our findings C member 6 (ABCC6) gene (4–7). ABCC6, also known as mul- provide leads for the treatment of this intractable disease. tidrug resistance-associated protein 6 (MRP6), is a member of the C branch of the superfamily of ATP-binding cassette (ABC)- Author contributions: P.B. and K.v.d.W. designed research; R.S.J., A.K., M.d.H., S.S., J.A.O., transporters, which use the energy provided by the hydrolysis of I.E.M.H., T.G.M.F.G., and K.v.d.W. performed research; A.A.B.B. contributed new reagents/ ATP to transport substrates across a membrane (8). ABCC6 analytic tools; R.S.J. analyzed data; and R.S.J., P.B., and K.v.d.W. wrote the paper. expression is high in the liver, lower in the kidney, and very low The authors declare no conflict of interest. or absent in other tissues, including affected skin, eyes, and Data deposition: Untargeted metabolomics data are available through MetaboLights, www.ebi.ac.uk/metabolights (accession no. MTBLS61) and XCMS Online, https:// arteries (5, 9). In the liver, ABCC6 is present in the sinusoidal xcmsonline.scripps.edu/jobs.php?action=listpublicshares (ID 1009444–1009446). (basolateral) membrane of hepatocytes, facing the central cir- 1To whom correspondence may be addressed. E-mail: [email protected] or k.vd.wetering@ culation (10–12). nki.nl. −/− Abcc6 mice faithfully recapitulate most of the symptoms of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. PXE and have been indispensable for showing that PXE is 1073/pnas.1319582110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1319582110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 factors that inhibit mineralization, to shed more light on the basic ribonucleosides, which could explain why they were not mechanism by which ABCC6 prevents pathological mineralization. detected. Even though nucleosides, nucleotides, and nucleotide sugars account for the major differences, we also found differ- Results ences (both increases and decreases) in the levels of compounds ABCC6-Conditioned Medium Inhibits Mineralization in Vitro. PXE is that we could not identify. All raw data are available at the a metabolic disease caused by the absence of a factor, the MetaboLights repository, and the processed data are accessible presence of which depends on ABCC6 in the liver (17). Jiang on XCMS Online (25, 26). and coworkers (14, 18) previously showed that in an in vitro + + system using vascular smooth muscle cells, serum from Abcc6 / ABCC6 Increases Extracellular Pyrophosphate Levels. None of the mice inhibits mineralization to a greater extent than serum from compounds found to be increased in medium of ABCC6-con- − − Abcc6 / mice. We took a similar approach to test whether a taining cells could be linked directly to mineralization. A differ- mineralization-inhibiting factor is also present in medium from entially present compound with an exceptionally high abundance HEK293 cells overexpressing human ABCC6 (HEK293/hABCC6) and fold change in medium of ABCC6-containing cells was or rat ABCC6 (HEK293/rABCC6). Both HEK293/hABCC6- and adenosine monophosphate (AMP). Although AMP itself is not HEK293/rABCC6-conditioned medium potently inhibited miner- known to affect mineralization, adenosine triphosphate (ATP) alization of ATDC5 cells, whereas medium from control cells does have a role in the prevention of ectopic mineralization. ATP (HEK293/GFP) did not (Fig. 1). These results indicate that the can be hydrolyzed to AMP and the mineralization inhibitor in- factor that prevents mineralization in vivo is also extruded from organic pyrophosphate (PPi) by ectonucleotide pyrophosphatase- cultured HEK293 cells containing ABCC6 in vitro. phosphodiesterase (ENPP)-type ectonucleotidases (27). PPi was not detected by our untargeted metabolomics method, however, ABCC6-Conditioned Medium Contains High Levels of Nucleosides and owing to the chosen mass range. Thus, we tested whether PPi Nucleotides. To identify the ABCC6-dependent factor responsible levels were affected by the presence of ABCC6 using a specific for inhibition of mineralization, we generated global metabolite enzymatic assay. The PPi levels in HEK293/hABCC6 and profiles of plasma samples as well