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In vivo overexpression of tissue-nonspecific alkaline phosphatase increases skeletal mineralization and affects the phosphorylation status of osteopontin.

ABSTRACT: Functional ablation of tissue-nonspecific alkaline phosphatase (TNAP) (Alpl?/? 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?/? mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in Alpl?/? 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-computed tomography (µCT) analysis. Crossbreeding of Col1a1-Tnap transgenic mice to Alpl?/? 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?/?; Alpl?/?] mice are able to mineralize to the level of Alpl?/? heterozygous osteoblasts, whereas Alpl?/? osteoblasts show no mineralization. We found that the increased levels of OPN in bone tissue of Alpl?/? mice are comprised of phosphorylated forms of OPN whereas wild-type (WT) and [Col1a1-Tnap?/?; Alpl?/?] 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?/? bone and osteoblasts yielded a higher proportion of phosphorylated peptides than samples from WT mice, and at least two phosphopeptides, p(S¹??FQVS¹??DEQY¹?²PDAT¹??DEDLT¹?¹)SHMK and FRIp(S²??HELES³??S³??S³??S³??)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.

SUBMITTER: Narisawa S

PROVIDER: S-EPMC3688694 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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In vivo overexpression of tissue-nonspecific alkaline phosphatase increases skeletal mineralization and affects the phosphorylation status of osteopontin.

Narisawa Sonoko S Yadav Manisha C MC Millán José Luis JL

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 20130701 7

Functional ablation of tissue-nonspecific alkaline phosphatase (TNAP) (Alpl⁻/⁻ 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⁻/⁻ mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in Alpl⁻/⁻ mice is unknown. Here, we generat ...[more]

PMID: 23427088

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Project description:Tissue-nonspecific alkaline phosphatase (TNAP) is necessary for skeletal mineralization by its ability to hydrolyze the mineralization inhibitor inorganic pyrophosphate (PPi), which is mainly generated from extracellular ATP by ectonucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Since children with TNAP deficiency develop bone metaphyseal auto-inflammations in addition to rickets, we hypothesized that TNAP also exerts anti-inflammatory effects relying on the hydrolysis of pro-inflammatory adenosine nucleotides into the anti-inflammatory adenosine. We explored this hypothesis in bone metaphyses of 7-day-old Alpl+/- mice (encoding TNAP), in mineralizing hypertrophic chondrocytes and osteoblasts, and non-mineralizing mesenchymal stem cells (MSCs) and neutrophils, which express TNAP and are present, or can be recruited in the metaphysis. Bone metaphyses of 7-day-old Alpl+/- mice had significantly increased levels of Il-1? and Il-6 and decreased levels of the anti-inflammatory Il-10 cytokine as compared with Alpl+/+ mice. In bone metaphyses, murine hypertrophic chondrocytes and osteoblasts, Alpl mRNA levels were much higher than those of the adenosine nucleotidases Npp1, Cd39 and Cd73. In hypertrophic chondrocytes, inhibition of TNAP with 25 ?M of MLS-0038949 decreased the hydrolysis of AMP and ATP. However, TNAP inhibition did not significantly modulate ATP- and adenosine-associated effects in these cells. We observed that part of TNAP proteins in hypertrophic chondrocytes was sent from the cell membrane to matrix vesicles, which may explain why TNAP participated in the hydrolysis of ATP but did not significantly modulate its autocrine pro-inflammatory effects. In MSCs, TNAP did not participate in ATP hydrolysis nor in secretion of inflammatory mediators. In contrast, in neutrophils, TNAP inhibition with MLS-0038949 significantly exacerbated ATP-associated activation and secretion of IL-1?, and extended cell survival. Collectively, these results demonstrate that TNAP is a nucleotidase in both hypertrophic chondrocytes and neutrophils, and that this nucleotidase function is associated with autocrine effects on inflammation only in neutrophils.

Project description:Tissue-nonspecific alkaline phosphatase (TNALP) is a glycoprotein expressed by osteoblasts that promotes bone mineralization. TNALP catalyzes the hydrolysis of the mineralization inhibitor inorganic pyrophosphate and ATP to provide inorganic phosphate, thus controlling the inorganic pyrophosphate/inorganic phosphate ratio to enable the growth of hydroxyapatite crystals. N-linked glycosylation of TNALP is essential for protein stability and enzymatic activity and is responsible for the presence of different bone isoforms of TNALP associated with functional and clinical differences. The site-specific glycosylation profiles of TNALP are, however, elusive. TNALP has 5 potential N-glycosylation sites located at the asparagine (N) residues 140, 230, 271, 303, and 430. The objective of this study was to reveal the presence and structure of site-specific glycosylation in TNALP expressed in osteoblasts. Calvarial osteoblasts derived from Alpl+/- expressing SV40 Large T antigen were transfected with soluble epitope-tagged human TNALP. Purified TNALP was analyzed with a lectin microarray, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and liquid chromatography with tandem mass spectrometry. The results showed that all sites (n = 5) were fully occupied predominantly with complex-type N-glycans. High abundance of galactosylated biantennary N-glycans with various degrees of sialylation was observed on all sites, as well as glycans with no terminal galactose and sialic acid. Furthermore, all sites had core fucosylation except site N271. Modelling of TNALP, with the protein structure prediction software ColabFold, showed possible steric hindrance by the adjacent side chain of W270, which could explain the absence of core fucosylation at N271. These novel findings provide evidence for N-linked glycosylation on all 5 sites of TNALP, as well as core fucosylation on 4 out of 5 sites. We anticipate that this new knowledge can aid in the development of functional and clinical assays specific for the TNALP bone isoforms.

Project description:Hypophosphatasia is a rare heritable disorder caused by inactivating mutations in the gene (Alpl) that encodes tissue nonspecific alkaline phosphatase (TNAP). Hypophosphatasia with onset in infants and children can manifest as rickets. How TNAP deficiency leads to bone hypomineralization is well explained by TNAP's primary function of pyrophosphate hydrolysis when expressed in differentiated bone forming cells. How TNAP deficiency leads to abnormalities within endochondral growth plates is not yet known. Previous studies in hypophosphatemic mice showed that phosphate promotes chondrocyte maturation and apoptosis via MAPK signaling. Alpl-/- mice are not hypophosphatemic but TNAP activity does increase local levels of inorganic phosphate. Therefore, we hypothesize that TNAP influences endochondral bone development via MAPK. In support of this premise, here we demonstrate cranial base bone growth deficiency in Alpl-/- mice, utilize primary rib chondrocytes to show that TNAP influences chondrocyte maturation, apoptosis, and MAPK signaling in a cell autonomous manner; and demonstrate that similar chondrocyte signaling and apoptosis abnormalities are present in the cranial base synchondroses of Alpl-/- mice. Micro CT studies revealed diminished anterior cranial base bone and total cranial base lengths in Alpl-/- mice, that were prevented upon injection with mineral-targeted recombinant TNAP (strensiq). Histomorphometry of the inter-sphenoidal synchondrosis (cranial base growth plate) demonstrated significant expansion of the hypertrophic chondrocyte zone in Alpl-/- mice that was minimized upon treatment with recombinant TNAP. Alpl-/- primary rib chondrocytes exhibited diminished chondrocyte proliferation, aberrant mRNA expression, diminished hypertrophic chondrocyte apoptosis and diminished MAPK signaling. Diminished apoptosis and VEGF expression were also seen in 15 day-old cranial base synchondroses of Alpl-/- mice. MAPK signaling was significantly diminished in 5 day-old cranial base synchondroses of Alpl-/- mice. Together, our data suggests that TNAP is essential for the later stages of endochondral bone development including hypertrophic chondrocyte apoptosis and VEGF mediated recruitment of blood vessels for replacement of cartilage with bone. These changes may be mediated by diminished MAPK signaling in TNAP deficient chondrocytes due to diminished local inorganic phosphate production.

Dataset Information

In vivo overexpression of tissue-nonspecific alkaline phosphatase increases skeletal mineralization and affects the phosphorylation status of osteopontin.

Publications

In vivo overexpression of tissue-nonspecific alkaline phosphatase increases skeletal mineralization and affects the phosphorylation status of osteopontin.

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