ABCC6-mediated ATP secretion by the liver is the main source of the mineralization inhibitor inorganic pyrophosphate in the systemic circulation-brief report

Abstract

Objective: Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PPi). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PPi, and whether human PXE ptients have low plasma PPi concentrations.

Approach and results: Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PPi. The absence of functional ABCC6 in patients with PXE leads to strongly reduced plasma PPi concentrations.

Conclusions: Hepatic ABCC6-mediated ATP release is the main source of circulating PPi, revealing an unanticipated role of the liver in systemic PPi homeostasis. Patients with PXE have a strongly reduced plasma PPi level, explaining their mineralization disorder. Our results indicate that systemic PPi is relatively stable and that PXE, generalized arterial calcification of infancy, and other ectopic mineralization disorders could be treated with PPi supplementation therapy.

Keywords: multidrug resistance-associated proteins; nucleotides; pathologic calcification; pyrophosphatases; vascular calcification.

Figure 1.

HEK293 and HeLa cells overproducing rat ATP-binding cassette subfamily C member 6 (rABCC6) release ATP. A, Flp-In T-REx 293 control (squares) or Flp-In T-REx 293 rABCC6 (circles) cells were grown in the presence (filled symbols) or absence (open symbols) of 1 μg/mL doxycycline to induce rABCC6 expression. Two days later, ATP efflux was followed in real-time for 2 hours using the ATP detection reagent BactiterGlo. B, ATP efflux from Flp-In T-REx HeLa control (squares) or Flp-In T-REx HeLa rABCC6 (circles) cells grown in the presence (filled symbols) or absence (open symbols) of 1 μg/mL doxycycline was followed for 2 hours in real-time. Data (n=12) represent mean±SEM.

Figure 2.

Hepatic ATP-binding cassette subfamily C member 6 (ABCC6) raises inorganic pyrophosphate (PP_i) levels via ATP release. Released ATP is rapidly converted into AMP and PP_i within the liver vasculature. A, PP_i levels in culture medium of sandwich-cultured primary wild-type (WT) and Abcc6^−/− hepatocytes (n=3 for WT, n=4 for Abcc6^−/−); total amount of (B) PP_i, (C) AMP, and (D) ATP in mouse liver perfusates collected from WT and Abcc6^−/− livers during 30 minutes (n=5 for WT, n=6 for Abcc6^−/−). E, PP_i levels in platelet-free plasma samples from healthy subjects (n=14) and patients with PXE (n=12). Patient and control characteristics are given in the online-only Data Supplement. Data are presented as mean±SD. **P<0.01, ***P<0.001. Note that AMP and PP_i levels are in nmoles, whereas ATP levels are in pmoles and close to background levels.

Figure 3.

Proposed model for hepatic ATP-binding cassette subfamily C member 6 (ABCC6)–mediated pyrophosphate generation and ectopic mineralization. ATP released from the liver by an ABCC6-dependent mechanism is converted into the mineralization inhibitor pyrophosphate (inorganic pyrophosphate [PP_i]) by hepatic ectonucleotide pyrophosphatase-phosphodiesterase 1 (ENPP1). In the periphery, PP_i is hydrolyzed by tissue-nonspecific alkaline phosphatase (TNAP). Inactive ABCC6 classically causes pseudoxanthoma elasticum (PXE), whereas inactive ENPP1 causes generalized arterial calcification of infancy (GACI). Nonfunctional ecto-5′-nucleotidase results in arterial calcification due to deficiency of CD73 (ACDC), and inactive TNAP causes hypophosphatasia (HOPS). Local PP_i levels also depend on the transmembrane protein progressive ankylosis protein homolog (ANKH), a protein postulated to be a PP_i channel/efflux transporter. Mutations in ANKH can result in chon-drocalcinosis type 2 (CCAL2) or craniometaphyseal dysplasia (CMD).