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

Abstract

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.

Keywords: ATP secretion; ENPP1; MRP6; ectopic calcification.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Effect of human and rat ABCC6-conditioned medium on in vitro mineralization of ATDC5 cells. ATDC5 cells were seeded in a 24-well plate (2.8 × 104 cells/well) and cultured in propagation medium for 4 d and in differentiation medium for 7 d, before the addition of mineralization medium supplemented with 50% conditioned medium from HEK293/GFP (control), HEK293/rABCC6, or HEK293/hABCC6 cells (three different batches). After 2 d, calcium phosphate deposits were visualized using alizarin red staining (A), followed by solubilization in cetylpyridinium and quantitation at 550 nm (B). Data are mean ± SD (n = 6).

Fig. 2.

Effect of ABCC6 expression and ectonucleotidase inhibition on nucleotide and pyrophosphate levels in culture medium. Control cells (HEK293, ○-dashed line and HEK293/GFP, □-dashed line) and ABCC6-overexpressing cells (HEK293/hABCC6, ▲-solid line and HEK293/rABCC6, ▼-solid line) were seeded in a six-well plate (7.5 × 105 cells/well) and grown to confluence overnight. Medium was replaced with control medium (A–C) or medium containing 1 mM ARL67156 plus 0.5 mM AMP-PCP (D–F), and samples were collected for LC-MS metabolomics and PPi (A and D) quantification. Metabolite levels were determined semiquantitatively by accurate mass extraction from the LC-MS data. Data are mean ± SD (n = 3). AU, arbitrary units.

Fig. 3.

Effect of induced rABCC6, hABCC6, or hABCC6V1289F expression on PPi levels in culture medium. Control cells (Flp-In T-REx 293) and Flp-In T-REx 293 containing inducible rABCC6 (A), hABCC6, or hABCC6V1298F (B) constructs were seeded in 24-well plates (2 × 105 cells/well) and grown to confluence overnight. ABCC6 expression was induced by adding 1 µg/mL of doxycyclin (+dox). Each day, the medium was collected for PPi determination and replaced with fresh medium. Data are mean ± SD (n = 3). LLQ, lower limit of quantitation of the assay.

Fig. 4.

Effect of the Abcc6 genotype on PPi levels in mouse plasma. Platelet-free plasma was collected and analyzed with a specific PPi assay based on the PPi-dependent conversion of glucose-1-phosphate to glucuronic acid-6-phosphate. Data are mean ± SD. **P < 0.01; ***P < 0.001, one-way ANOVA (n = 7, 8, and 6 for females; n = 7, 6, and 5 for males).