28 29 30 31 Keywords: 32 extracellular citrate, extracellular pyrophosphate, ATP release, ANKH/ANK, ectopic 33 mineralization, bone 34 35 36 37 2 Abstract 38 The membrane protein Ankylosis homologue (ANKH, mouse orthologue: ANK) prevents 39 mineralization of joint-space and articular cartilage. The accepted view is that ANKH mediates 40 cellular release of inorganic pyrophosphate (PPi), a strong physiological inhibitor of 41 mineralization. Using global metabolite profiling, we identified citrate as the most prominent 42 metabolite leaving HEK293 cells in an ANKH-dependent manner. Although PPi levels were 43 increased in culture medium of HEK293-ANKH cells, PPi was formed extracellularly after release 44 of ATP and other nucleoside triphosphates. Ank ank/ank mice, which lack functional ANK, had 45 substantially reduced concentrations of citrate in plasma and urine, while citrate was undetectable 46 in urine of a human patient lacking functional ANKH. Bone hydroxyapatite of Ank ank/ank mice also 47 contained markedly reduced levels of citrate and PPi and displayed diminished strength.
48Together, our data show that ANKH is a crucial factor in extracellular citrate and PPi homeostasis 49 that is essential for normal bone development.