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The plasma membrane protein Ankylosis Homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints. We recently showed that when overproduced in HEK293 cells, ANKH mediates release of large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. ATP is converted extracellularly into PPi and AMP by the ectoenzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANKH. We now addressed the question if PPi leaves cells via ANKH using HEK293 cells that completely lack ENPP1. Introduction of ANKH in these ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi seen in ENPP1-proficient cells. Ank-activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1(−/−) mice contained < 2.5% of the PPi found in bones of wild type mice, showing that Enpp1-activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60–70 % of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification, therefore do so by extruding NTPs rather than PPi itself.

Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe an PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (Abcc6-/- rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory.In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.

The plasma membrane protein Ankylosis Homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints, but we recently showed that ANKH releases large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. This ATP is converted extracellularly into PPi and AMP by the ectoenzyme Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANK. We now addressed this question by determining the effect of a complete absence of ENPP1 on ANKH-dependent extracellular PPi concentrations. Introduction of ANKH in ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi seen in ENPP1-proficient cells.Ank-activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1-/- mice contained < 2.5% of the PPi found in bones of wild type mice, showing that Enpp1-activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60-70 % of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification, therefore do so by extruding NTPs rather than PPi itself.

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality., Author summary Biallelic inactivating mutations in the progressive ankylosis gene (ANKH/Ank, humans/mice) cause a severe form of arthritis, primarily affecting joints of hands, feet and the spine. Low extracellular concentrations of the mineralization inhibitor pyrophosphate underlie the joint calcification in mice and humans that are devoid of functional Ank or ANKH, respectively. It was previously thought that ANKH mediates the release of pyrophosphate from the cytosol to the extracellular environment. We here show, however, that PPi is formed in the extracellular environment by Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1), from ATP released via ANKH. Untargeted metabolomics further revealed that ANKH provides a pathway for the release of nucleoside triphosphates in general and, unexpectedly, the Krebs-cycle intermediate citrate. Mice lacking Ank activity (Ankank/ank mice), have substantially reduced concentrations of citrate in their plasma and urine. Citrate was even undetectable in urine of a human patient lacking functional ANKH. Bones of Ankank/ank mice have reduced mechanical strength and their bone matrix contains dramatically reduced levels of PPi and citrate. Our data demonstrate that ANKH/Ank crucially contributes to the disposition of citrate and PPi in vivo and profoundly affects bone quality. Extracellular citrate is ubiquitous, and additional phenotypes are therefore expected to be associated with absence of Ank activity.

The membrane protein Ankylosis homologue (ANKH, mouse orthologue: ANK) prevents mineralization of joint-space and articular cartilage. The accepted view is that ANKH mediates cellular release of inorganic pyrophosphate (PPi), a strong physiological inhibitor of mineralization. Using global metabolite profiling, we identified citrate as the most prominent metabolite leaving HEK293 cells in an ANKH-dependent manner. Although PPi levels were increased in culture medium of HEK293-ANKH cells, PPi was formed extracellularly after release of ATP and other nucleoside triphosphates.Ankank/ankmice, which lack functional ANK, had substantially reduced concentrations of citrate in plasma and urine, while citrate was undetectable in urine of a human patient lacking functional ANKH. Bone hydroxyapatite ofAnkank/ankmice also contained markedly reduced levels of citrate and PPi and displayed diminished strength. Together, our data show that ANKH is a crucial factor in extracellular citrate and PPi homeostasis that is essential for normal bone development.