{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T04:53:43Z","timestamp":1791262423414,"version":"4.1.0"},"reference-count":49,"publisher":"Oxford University Press (OUP)","issue":"7","license":[{"start":{"date-parts":[[2013,2,20]],"date-time":"2013-02-20T00:00:00Z","timestamp":1361318400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2013,7,1]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n               <jats:sec>\n                  <jats:title>\u2003<\/jats:title>\n                  <jats:p>Functional ablation of tissue-nonspecific alkaline phosphatase (TNAP) (Alpl\u2212\/\u2212 mice) leads to hypophosphatasia, characterized by rickets\/osteomalacia attributable to elevated levels of extracellular inorganic pyrophosphate, a potent mineralization inhibitor. Osteopontin (OPN) is also elevated in the plasma and skeleton of Alpl\u2212\/\u2212 mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in Alpl\u2212\/\u2212 mice is unknown. Here, we generated a transgenic mouse line expressing human TNAP under control of an osteoblast-specific Col1a1 promoter (Col1a1-Tnap). The transgene is expressed in osteoblasts, periosteum, and cortical bones, and plasma levels of TNAP in mice expressing Col1a1-Tnap are 10 to 20 times higher than those of wild-type mice. The Col1a1-Tnap animals are healthy and exhibit increased bone mineralization by micro\u2013computed tomography (\u00b5CT) analysis. Crossbreeding of Col1a1-Tnap transgenic mice to Alpl\u2212\/\u2212 mice rescues the lethal hypophosphatasia phenotype characteristic of this disease model. Osteoblasts from [Col1a1-Tnap] mice mineralize better than nontransgenic controls and osteoblasts from [Col1a1-Tnap+\/\u2212; Alpl\u2212\/\u2212] mice are able to mineralize to the level of Alpl+\/\u2212 heterozygous osteoblasts, whereas Alpl\u2212\/\u2212 osteoblasts show no mineralization. We found that the increased levels of OPN in bone tissue of Alpl\u2212\/\u2212 mice are comprised of phosphorylated forms of OPN whereas wild-type (WT) and [Col1a1-Tnap+\/\u2212; Alpl\u2212\/\u2212] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [Col1a1-Tnap] osteoblasts were more dephosphorylated than nontransgenic control cells. Titanium dioxide-liquid chromatography and tandem mass spectrometry analysis revealed that OPN peptides derived from Alpl\u2212\/\u2212 bone and osteoblasts yielded a higher proportion of phosphorylated peptides than samples from WT mice, and at least two phosphopeptides, p(S174FQVS178DEQY182PDAT186DEDLT191)SHMK and FRIp(S299HELES304S305S306S307)EVN, with one nonlocalized site each, appear to be preferred sites of TNAP action on OPN. Our data suggest that the promineralization role of TNAP may be related not only to its accepted pyrophosphatase activity but also to its ability to modify the phosphorylation status of OPN.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1002\/jbmr.1901","type":"journal-article","created":{"date-parts":[[2013,2,20]],"date-time":"2013-02-20T21:25:10Z","timestamp":1361395510000},"page":"1587-1598","source":"Crossref","is-referenced-by-count":134,"title":["In Vivo Overexpression of Tissue-Nonspecific Alkaline Phosphatase Increases Skeletal Mineralization and Affects the Phosphorylation Status of Osteopontin"],"prefix":"10.1093","volume":"28","author":[{"given":"Sonoko","family":"Narisawa","sequence":"first","affiliation":[{"name":"Sanford Children's Health Research Center, Sanford-Burnham Medical Research InstituteLa Jolla, CAUSA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manisha C","family":"Yadav","sequence":"additional","affiliation":[{"name":"Sanford Children's Health Research Center, Sanford-Burnham Medical Research InstituteLa Jolla, CAUSA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jos\u00e9 Luis","family":"Mill\u00e1n","sequence":"additional","affiliation":[{"name":"Sanford Children's Health Research Center, Sanford-Burnham Medical Research InstituteLa Jolla, CAUSA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2013,2,20]]},"reference":[{"key":"2024020318380972700_jbmr1901-bib-0001","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1002\/aja.1002010306","article-title":"Stage-specific expression of alkaline phosphatase during neural development in the mouse","volume":"201","author":"Narisawa","year":"1994","journal-title":"Dev Dyn."},{"key":"2024020318380972700_jbmr1901-bib-0002","doi-asserted-by":"crossref","first-page":"432","DOI":"10.1002\/(SICI)1097-0177(199703)208:3<432::AID-AJA13>3.0.CO;2-1","article-title":"Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia","volume":"208","author":"Narisawa","year":"1997","journal-title":"Dev Dyn."},{"key":"2024020318380972700_jbmr1901-bib-0003","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1002\/1096-9896(2000)9999:9999<::AID-PATH722>3.0.CO;2-Y","article-title":"Abnormal vitamin B6 metabolism in alkaline phosphatase knock-out mice causes multiple abnormalities, but not the impaired bone mineralization","volume":"193","author":"Narisawa","year":"2001","journal-title":"J Pathol."},{"key":"2024020318380972700_jbmr1901-bib-0004","doi-asserted-by":"crossref","first-page":"2015","DOI":"10.1359\/jbmr.1999.14.12.2015","article-title":"Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia","volume":"14","author":"Fedde","year":"1999","journal-title":"J Bone Miner Res."},{"key":"2024020318380972700_jbmr1901-bib-0005","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1016\/S0002-9440(10)63172-0","article-title":"Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice","volume":"164","author":"Anderson","year":"2004","journal-title":"Am J Pathol."},{"key":"2024020318380972700_jbmr1901-bib-0006","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1002\/3527608060.ch5","volume-title":"Mammalian alkaline phosphatases\u2014from biology to applications in medicine and biotechnology","author":"Mill\u00e1n","year":"2006"},{"key":"2024020318380972700_jbmr1901-bib-0007","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1111\/j.1749-6632.2010.05387.x","article-title":"Physiological role of alkaline phosphatase explored in hypophosphatasia","volume":"1192","author":"Whyte","year":"2010","journal-title":"Ann N Y Acad Sci."},{"key":"2024020318380972700_jbmr1901-bib-0008","doi-asserted-by":"crossref","first-page":"9445","DOI":"10.1073\/pnas.142063399","article-title":"Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization","volume":"99","author":"Hessle","year":"2002","journal-title":"Proc Natl Acad Sci U S A."},{"key":"2024020318380972700_jbmr1901-bib-0009","doi-asserted-by":"crossref","first-page":"1199","DOI":"10.1016\/S0002-9440(10)63208-7","article-title":"Concerted regulation of inorganic pyrophosphate and osteopontin by Akp2, Enpp1, and Ank\u2014an integrated model of the pathogenesis of mineralization disorders","volume":"164","author":"Harmey","year":"2004","journal-title":"Am J Pathol."},{"key":"2024020318380972700_jbmr1901-bib-0010","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/ng0995-45","article-title":"Mice lacking tissue non-specific alkaline phosphatase die from seizures due to 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hypophosphatasia","volume":"49","author":"Yadav","year":"2011","journal-title":"Bone."},{"key":"2024020318380972700_jbmr1901-bib-0014","doi-asserted-by":"crossref","first-page":"1722","DOI":"10.1002\/jbmr.1619","article-title":"Enzyme replacement prevents enamel defects in hypophosphatasia mice","volume":"27","author":"Yadav","year":"2012","journal-title":"J Bone Miner Res."},{"key":"2024020318380972700_jbmr1901-bib-0015","doi-asserted-by":"crossref","first-page":"1879","DOI":"10.1359\/jbmr.2000.15.10.1879","article-title":"Functional characterization of osteoblasts and osteoclasts from alkaline phosphatase knockout mice","volume":"15","author":"Wennberg","year":"2000","journal-title":"J Bone Miner Res."},{"key":"2024020318380972700_jbmr1901-bib-0016","doi-asserted-by":"crossref","first-page":"994","DOI":"10.1359\/jbmr.2003.18.6.994","article-title":"Linked deficiencies in extracellular PPi and osteopontin mediate pathologic calcification associated with defective PC-1 and ANK 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resorption","volume":"96","author":"Yoshitake","year":"1999","journal-title":"Proc Natl Acad Sci U S A."},{"key":"2024020318380972700_jbmr1901-bib-0032","doi-asserted-by":"crossref","first-page":"1093","DOI":"10.1101\/gad.1276205","article-title":"Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone","volume":"19","author":"Murshed","year":"2005","journal-title":"Genes Dev."},{"key":"2024020318380972700_jbmr1901-bib-0033","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1679\/aohc.66.123","article-title":"Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants","volume":"66","author":"Kawamoto","year":"2003","journal-title":"Arch Histol Cytol."},{"key":"2024020318380972700_jbmr1901-bib-0034","doi-asserted-by":"crossref","first-page":"2900","DOI":"10.1016\/S0021-9258(18)61592-3","article-title":"Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone","volume":"262","author":"Prince","year":"1987","journal-title":"J Biol Chem."},{"key":"2024020318380972700_jbmr1901-bib-0035","doi-asserted-by":"crossref","first-page":"1513","DOI":"10.1038\/nprot.2009.154","article-title":"Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE","volume":"4","author":"Kinoshita","year":"2009","journal-title":"Nat Protoc."},{"key":"2024020318380972700_jbmr1901-bib-0036","doi-asserted-by":"crossref","first-page":"256","DOI":"10.2144\/01312bm03","article-title":"Vectors for high-level expression of cDNAs controlled by tissue-specific promoters in transgenic mice","volume":"31","author":"Stec","year":"2001","journal-title":"Biotechniques."},{"key":"2024020318380972700_jbmr1901-bib-0037","doi-asserted-by":"crossref","first-page":"1266","DOI":"10.1056\/NEJM200004273421707","article-title":"Evaluation of abnormal liver-enzyme results in a symptomatic patients","volume":"342","author":"Pratt","year":"2000","journal-title":"N Engl J Med."},{"key":"2024020318380972700_jbmr1901-bib-0038","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1007\/s004180050103","article-title":"Immunolocalization of tissue non-specific alkaline phosphatase in mice","volume":"107","author":"Hoshi","year":"1997","journal-title":"Histochem Cell Biol."},{"issue":"2","key":"2024020318380972700_jbmr1901-bib-0039","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1016\/j.bone.2012.12.048","article-title":"Isozyme profile and tissue-origin of alkaline phosphatases in mouse serum","volume":"53","author":"Linder","year":"2013","journal-title":"Bone."},{"key":"2024020318380972700_jbmr1901-bib-0040","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1007\/BF00334492","article-title":"Mechanism of action of \u03b2-glycerophosphate on bone cell mineralization","volume":"51","author":"Chung","year":"1992","journal-title":"Calcif Tissue 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Res."},{"key":"2024020318380972700_jbmr1901-bib-0044","doi-asserted-by":"crossref","first-page":"906","DOI":"10.1056\/NEJMoa1106173","article-title":"Enzyme-replacement therapy in life-threatening hypophosphatasia","volume":"366","author":"Whyte","year":"2012","journal-title":"N Engl J Med."},{"key":"2024020318380972700_jbmr1901-bib-0045","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1002\/jbmr.195","article-title":"Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and alkaline phosphatase function: a unified model of the mechanisms of initiation of skeletal calcification","volume":"26","author":"Yadav","year":"2011","journal-title":"J Bone Miner Res."},{"key":"2024020318380972700_jbmr1901-bib-0046","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1016\/S0022-3476(82)80061-9","article-title":"Infantile hypophosphatasia: enzyme replacement therapy by intravenous infusion of alkaline phosphatase-rich plasma from patients with Paget bone disease","volume":"101","author":"Whyte","year":"1982","journal-title":"J Pediatr."},{"key":"2024020318380972700_jbmr1901-bib-0047","doi-asserted-by":"crossref","first-page":"926","DOI":"10.1016\/S0022-3476(84)80079-7","article-title":"Enzyme replacement therapy for infantile hypophosphatasia attempted by intravenous infusions of alkaline phosphatase-rich Paget plasma: results in three additional patients","volume":"105","author":"Whyte","year":"1984","journal-title":"J Pediatr."},{"key":"2024020318380972700_jbmr1901-bib-0048","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1111\/j.1651-2227.1989.tb11297.x","article-title":"Biochemical and morphological effects of human hepatic alkaline phosphatase in a neonate with hypophosphatasia","volume":"360","author":"Weninger","year":"1989","journal-title":"Acta Paediatr Scand Suppl."},{"key":"2024020318380972700_jbmr1901-bib-0049","first-page":"S155","article-title":"Failure of hyperphosphatasemia by intravenous infusion of purified placental alkaline phosphatase to correct severe hypophosphatasia: evidence against a role for circulating ALP in skeletal mineralization","volume":"7","author":"Whyte","year":"1992","journal-title":"J Bone Miner Res."}],"container-title":["Journal of Bone and Mineral 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