In Vivo Overexpression of Tissue‐Nonspecific Alkaline Phosphatase Increases Skeletal Mineralization and Affects the Phosphorylation Status of Osteopontin. (18th June 2013)

• Record Type: Journal Article
• Authors: Narisawa, Sonoko; Yadav, Manisha C; Millán, José Luis
• Is Part Of: Journal of bone and mineral research. Volume 28:Number 7(2013:Jul.)
• Page Start: 1587
• Page End: 1598
• Publication Date: 2013-06-18
• Subjects: Bones -- Metabolism -- Periodicals; Mineral metabolism -- Periodicals; 612.392
• DOI: 10.1002/jbmr.1901
• Languages: English
• ISSNs: 0884-0431

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>ABSTRACT</title> <sec id="jbmr1901-sec-0001" sec-type="section"> <p>Functional ablation of tissue‐nonspecific alkaline phosphatase (TNAP) (<italic>Alpl</italic><sup>−/−</sup> 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 <italic>Alpl</italic><sup>−/−</sup> mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in <italic>Alpl</italic><sup>−/−</sup> mice is unknown. Here, we generated a transgenic mouse line expressing human TNAP under control of an osteoblast‐specific <italic>Col1a1</italic> promoter (<italic>Col1a1‐Tnap</italic>). The transgene is expressed in osteoblasts, periosteum, and cortical bones, and plasma levels of TNAP in mice expressing <italic>Col1a1‐Tnap</italic> are 10 to 20 times higher than those of wild‐type mice. The <italic>Col1a1‐Tnap</italic> animals are healthy and exhibit increased bone mineralization by micro–computed tomography (µCT) analysis. Crossbreeding of <italic>Col1a1‐Tnap</italic> transgenic mice to <italic>Alpl</italic><sup>−/−</sup> mice rescues the lethal hypophosphatasia phenotype characteristic of this disease model. Osteoblasts from [<italic>Col1a1‐Tnap</italic>] mice mineralize better than nontransgenic controls and osteoblasts from [<italic>Col1a1‐Tnap</italic><sup>+/−</sup>; <italic>Alpl</italic><sup>−/−</sup>] mice are able to mineralize to the level of <italic>Alpl</italic><sup>+/−</sup> heterozygous osteoblasts, whereas <italic>Alpl</italic><sup>−/−</sup> osteoblasts show no mineralization. We found that the increased levels of OPN in bone tissue of <italic>Alpl</italic><sup>−/−</sup> mice are comprised of phosphorylated forms of OPN whereas wild‐type (WT) and [<italic>Col1a1‐Tnap</italic><sup>+/−</sup>; <italic>Alpl</italic><sup>−/−</sup>] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [<italic>Col1a1‐Tnap</italic>] osteoblasts were more dephosphorylated than nontransgenic control cells. Titanium dioxide‐liquid chromatography and tandem mass spectrometry analysis revealed that OPN peptides derived from <italic>Alpl</italic><sup>−/−</sup> bone and osteoblasts yielded a higher proportion of phosphorylated peptides than samples from WT mice, and at least two phosphopeptides, p(S<sup>174</sup>FQVS<sup>178</sup>DEQY<sup>182</sup>PDAT<sup>186</sup>DEDLT<sup>191</sup>)SHMK and FRIp(S<sup>299</sup>HELES<sup>304</sup>S<sup>305</sup>S<sup>306</sup>S<sup>307</sup>)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.</p> </sec> </abstract>