{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T01:46:24Z","timestamp":1784598384027,"version":"3.55.0"},"posted":{"date-parts":[[2025,10,10]]},"group-title":"Developmental Biology","reference-count":49,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T00:00:00Z","timestamp":1760054400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"NIH NIAMS","award":["R01AR082460"],"award-info":[{"award-number":["R01AR082460"]}]},{"name":"NIH NIAMS","award":["R21AR083597"],"award-info":[{"award-number":["R21AR083597"]}]},{"name":"PXE International"},{"DOI":"10.13039\/501100011019","name":"National Research Development and Innovation Office of Hungary","doi-asserted-by":"crossref","award":["FK131946"],"award-info":[{"award-number":["FK131946"]}],"id":[{"id":"10.13039\/501100011019","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,10,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>ABCC6 and ANK are integral membrane proteins involved in the efflux of specific organic anions. ABCC6, primarily expressed in the liver, regulates circulating levels of the mineralization inhibitor pyrophosphate (PPi). In contrast, ANK is widely expressed and plays a dual role, maintaining extracellular PPi homeostasis and, notably, mediating cellular citrate efflux. We studied how both proteins affected extracellular metabolite levels, ectopic calcification, and bone homeostasis.<\/jats:p>\n                <jats:p>\n                  We observed that plasma PPi was reduced by \u223c36% in\n                  <jats:italic>\n                    Ank\n                    <jats:sup>ank\/ank<\/jats:sup>\n                  <\/jats:italic>\n                  mice and \u223c60% in\n                  <jats:italic>\n                    Abcc6\n                    <jats:sup>\u2212\/\u2212<\/jats:sup>\n                  <\/jats:italic>\n                  mice. However, plasma citrate levels depended primarily on ANK, dropping \u223c75% in\n                  <jats:italic>\n                    Ank\n                    <jats:sup>ank\/ank<\/jats:sup>\n                  <\/jats:italic>\n                  and double mutants, but remaining unchanged in\n                  <jats:italic>\n                    Abcc6\n                    <jats:sup>\u2212\/\u2212<\/jats:sup>\n                  <\/jats:italic>\n                  mice.\n                <\/jats:p>\n                <jats:p>\n                  MicroCT revealed extreme ectopic calcification in double mutants, far exceeding either single knockout, affecting muzzle skin and ear cartilage. Oral citrate was bioavailable and, at high doses, prevented soft tissue calcification in\n                  <jats:italic>\n                    Abcc6\n                    <jats:sup>\u2212\/\u2212<\/jats:sup>\n                  <\/jats:italic>\n                  mice, suggesting a systemic protective role.\n                <\/jats:p>\n                <jats:p>In bone, ANK was essential for incorporating both PPi and citrate, while ABCC6 mainly affected PPi. ANK deficiency led to reduced trabecular volume, cortical thickness, cortical area fraction, and mineral density, with more pronounced effects in males. Biomechanical testing showed decreased ultimate moment, bending rigidity, and energy in ANK-deficient femora, alongside increased post-yield displacement, indicating compensatory matrix changes.<\/jats:p>\n                <jats:p>Collectively, our findings identify ANK as a dual regulator of PPi and citrate, with a previously unrecognized role in preventing soft tissue calcification. This study positions ANK as a potential therapeutic target for mineralization disorders like pseudoxanthoma elasticum (caused by ABCC6 deficiency) and conditions of low bone mineral density like osteoporosis.<\/jats:p>","DOI":"10.1101\/2025.10.09.681172","type":"posted-content","created":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:50:12Z","timestamp":1760136612000},"source":"Crossref","is-referenced-by-count":1,"title":["ABCC6 and ANK regulate extracellular homeostasis of pyrophosphate and citrate and affect mineral deposition in bones and soft connective tissues"],"prefix":"10.64898","author":[{"given":"Flora","family":"Szeri","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fatemeh","family":"Niaziorimi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Celina","family":"Ng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cassandra","family":"Mikkelson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ibtesam","family":"Rajpar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qinglin","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven P.","family":"Matyus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Margery A.","family":"Connelly","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"George","family":"Tavadze","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefan","family":"Lundkvist","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6713-6047","authenticated-orcid":false,"given":"Ryan E.","family":"Tomlinson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1905-2743","authenticated-orcid":false,"given":"Koen","family":"van de Wetering","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025101405250878000_2025.10.09.681172v1.1","doi-asserted-by":"publisher","DOI":"10.1097\/01.bor.0000209431.59226.46"},{"key":"2025101405250878000_2025.10.09.681172v1.2","doi-asserted-by":"crossref","first-page":"1372","DOI":"10.1111\/brv.12614","article-title":"The \u2018biomineralization toolkit\u2019 and the origin of animal skeletons","volume":"95","year":"2020","journal-title":"Biol Rev"},{"key":"2025101405250878000_2025.10.09.681172v1.3","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1038\/s41574-021-00488-z","article-title":"The role of biomineralization in disorders of skeletal development and tooth formation","volume":"17","year":"2021","journal-title":"Nat Rev Endocrinol"},{"key":"2025101405250878000_2025.10.09.681172v1.4","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-genet-102108-134242"},{"key":"2025101405250878000_2025.10.09.681172v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajpath.2013.03.002"},{"key":"2025101405250878000_2025.10.09.681172v1.6","doi-asserted-by":"crossref","first-page":"115243","DOI":"10.1016\/j.bone.2020.115243","article-title":"Extracellular pyrophosphate: The body\u2019s \u201cwater softener\u201d","volume":"134","year":"2020","journal-title":"Bone"},{"key":"2025101405250878000_2025.10.09.681172v1.7","doi-asserted-by":"crossref","first-page":"762","DOI":"10.1016\/j.ajpath.2022.01.012","article-title":"Inorganic pyrophosphate deficiency syndromes and potential treatments for pathologic tissue calcification","volume":"192","year":"2022","journal-title":"Am. 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