Tooth root dentin mineralization defects in a mouse model of hypophosphatasia

Tissue‐nonspecific alkaline phosphatase (TNAP) is expressed in mineralizing tissues and functions to reduce pyrophosphate (PP_i), a potent inhibitor of mineralization. Loss of TNAP function causes hypophosphatasia (HPP), a heritable disorder marked by increased PP_i, resulting in rickets and osteomalacia. Tooth root cementum defects are well described in both HPP patients and in Alpl^?/? mice, a model for infantile HPP. In Alpl^?/? mice, dentin mineralization is specifically delayed in the root; however, reports from human HPP patients are variable and inconsistent regarding dentin defects. In the current study, we aimed to define the molecular basis for changes in dentinogenesis observed in Alpl^?/? mice. TNAP was found to be highly expressed by mature odontoblasts, and Alpl^?/? molar and incisor roots featured defective dentin mineralization, ranging from a mild delay to severely disturbed root dentinogenesis. Lack of mantle dentin mineralization was associated with disordered and dysmorphic odontoblasts having disrupted expression of marker genes osteocalcin and dentin sialophosphoprotein. The formation of, initiation of mineralization within, and rupture of matrix vesicles in Alpl^?/? dentin matrix was not affected. Osteopontin (OPN), an inhibitor of mineralization that contributes to the skeletal pathology in Alpl^?/? mice, was present in the generally unmineralized Alpl^?/? mantle dentin at ruptured mineralizing matrix vesicles, as detected by immunohistochemistry and by immunogold labeling. However, ablating the OPN‐encoding Spp1 gene in Alpl^?/? mice was insufficient to rescue the dentin mineralization defect. Administration of bioengineered mineral‐targeting human TNAP (ENB‐0040) to Alpl^?/? mice corrected defective dentin mineralization in the molar roots. These studies reveal that TNAP participates in root dentin formation and confirm that reduction of PP_i during dentinogenesis is necessary for odontoblast differentiation, dentin matrix secretion, and mineralization. Furthermore, these results elucidate developmental mechanisms underlying dentin pathology in HPP patients, and begin to explain the reported variability in the dentin/pulp complex pathology in these patients. © 2013 American Society for Bone and Mineral Research     

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

Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Alkaline phosphatase (TNAP) plays a crucial role promoting mineralization of the extracellular matrix by restricting the concentration of the calcification inhibitor inorganic pyrophosphate (PP_i). Mutations in the TNAP gene cause hypophosphatasia, a heritable form of rickets and osteomalacia. Here we show that PHOSPHO1, a phosphatase with specificity for phosphoethanolamine and phosphocholine, plays a functional role in the initiation of calcification and that ablation of PHOSPHO1 and TNAP function prevents skeletal mineralization. Phospho1^?/? mice display growth plate abnormalities, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis in early life. Primary cultures of Phospho1^?/? tibial growth plate chondrocytes and chondrocyte‐derived matrix vesicles (MVs) show reduced mineralizing ability, and plasma samples from Phospho1^?/? mice show reduced levels of TNAP and elevated plasma PP_i concentrations. However, transgenic overexpression of TNAP does not correct the bone phenotype in Phospho1^?/? mice despite normalization of their plasma PP_i levels. In contrast, double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality. We conclude that PHOSPHO1 has a nonredundant functional role during endochondral ossification, and based on these data and a review of the current literature, we propose an inclusive model of skeletal calcification that involves intravesicular PHOSPHO1 function and P_i influx into MVs in the initiation of mineralization and the functions of TNAP, nucleotide pyrophosphatase phosphodiesterase‐1, and collagen in the extravesicular progression of mineralization. © 2011 American Society for Bone and Mineral Research.     

In Vivo Overexpression of Tissue‐Nonspecific Alkaline Phosphatase Increases Skeletal Mineralization and Affects the Phosphorylation Status of Osteopontin

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(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.     

The Role of Phosphatases in the Initiation of Skeletal Mineralization

Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene cause hypophosphatasia, a heritable form of rickets and osteomalacia, caused by an arrest in the propagation of hydroxyapatite (HA) crystals onto the collagenous extracellular matrix due to accumulation of extracellular inorganic pyrophosphate (PP_i), a physiological TNAP substrate and a potent calcification inhibitor. However, TNAP knockout (Alpl ^–/– ) mice are born with a mineralized skeleton and have HA crystals in their chondrocyte- and osteoblast-derived matrix vesicles (MVs). We have shown that PHOSPHO1, a soluble phosphatase with specificity for two molecules present in MVs, phosphoethanolamine and phosphocholine, is responsible for initiating HA crystal formation inside MVs and that PHOSPHO1 and TNAP have nonredundant functional roles during endochondral ossification. Double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality, despite normal systemic phosphate and calcium levels. This strongly suggests that the P_i needed for initiation of MV-mediated mineralization is produced locally in the perivesicular space. As both TNAP and nucleoside pyrophosphohydrolase-1 (NPP1) behave as potent ATPases and pyrophosphatases in the MV compartment, our current model of the mechanisms of skeletal mineralization implicate intravesicular PHOSPHO1 function and P_i influx into MVs in the initiation of mineralization and the functions of TNAP and NPP1 in the extravesicular progression of mineralization.     

Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte‐Derived Matrix Vesicles in Phospho1–/– and Phospho1/Pit1 Double‐Knockout Mice

We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue‐nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis, but that their chondrocyte‐derived and osteoblast‐derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1‐mediated intravesicular generation of inorganic phosphate (P_i) and phosphate transporter‐mediated influx of P_i. To test this hypothesis, we generated mice with col2a1‐driven Cre‐mediated ablation of Slc20a1, hereafter referred to as P_it1, alone or in combination with a Phospho1 gene deletion. P_it1^col2/col2 mice did not show any major phenotypic abnormalities, whereas severe skeletal deformities were observed in the [Phospho1^–/–; P_it1^col2/col2] double knockout mice that were more pronounced than those observed in the Phospho1^–/– mice. Histological analysis of [Phospho1^–/–; P_it1^col2/col2] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1^–/–; P_it1^col2/col2] skeleton displayed significant decreases in BV/TV%, trabecular number, and bone mineral density, as well as decreased stiffness, decreased strength, and increased postyield deflection compared to Phospho1^–/– mice. Using atomic force microscopy we found that ～80% of [Phospho1^–/–; P_it1^col2/col2] MVs were devoid of mineral in comparison to ～50% for the Phospho1^–/– MVs and ～25% for the WT and P_it1^col2/col2 MVs. We also found a significant decrease in the number of MVs produced by both Phospho1^–/– and [Phospho1^–/–; P_it1^col2/col2] chondrocytes. These data support the involvement of phosphate transporter 1, hereafter referred to as P_iT‐1, in the initiation of skeletal mineralization and provide compelling evidence that PHOSPHO1 function is involved in MV biogenesis. © 2016 American Society for Bone and Mineral Research.     

Gene Therapy Using Adeno-Associated Virus Serotype 8 Encoding TNAP-D10 Improves the Skeletal and Dentoalveolar Phenotypes in Alpl−/− Mice

Hypophosphatasia (HPP) is caused by loss-of-function mutations in the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP), whose deficiency results in the accumulation of extracellular inorganic pyrophosphate (PP_i), a potent mineralization inhibitor. Skeletal and dental hypomineralization characterizes HPP, with disease severity varying from life-threatening perinatal or infantile forms to milder forms that manifest in adulthood or only affect the dentition. Enzyme replacement therapy (ERT) using mineral-targeted recombinant TNAP (Strensiq/asfotase alfa) markedly improves the life span, skeletal phenotype, motor function, and quality of life of patients with HPP, though limitations of ERT include frequent injections due to a short elimination half-life of 2.28 days and injection site reactions. We tested the efficacy of a single intramuscular administration of adeno-associated virus 8 (AAV8) encoding TNAP-D₁₀ to increase the life span and improve the skeletal and dentoalveolar phenotypes in TNAP knockout (Alpl^−/−) mice, a murine model for severe infantile HPP. Alpl^−/− mice received 3 × 10¹¹ vector genomes/body of AAV8-TNAP-D₁₀ within 5 days postnatal (dpn). AAV8-TNAP-D₁₀ elevated serum ALP activity and suppressed plasma PP_i. Treatment extended life span of Alpl^−/− mice, and no ectopic calcifications were observed in the kidneys, aorta, coronary arteries, or brain in the 70 dpn observational window. Treated Alpl^−/− mice did not show signs of rickets, including bowing of long bones, enlargement of epiphyses, or fractures. Bone microstructure of treated Alpl^−/− mice was similar to wild type, with a few persistent small cortical and trabecular defects. Histology showed no measurable osteoid accumulation but reduced bone volume fraction in treated Alpl^−/− mice versus controls. Treated Alpl^−/− mice featured normal molar and incisor dentoalveolar tissues, with the exceptions of slightly reduced molar enamel and alveolar bone density. Histology showed the presence of cementum and normal periodontal ligament attachment. These results support gene therapy as a promising alternative to ERT for the treatment of HPP. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Enzyme replacement prevents enamel defects in hypophosphatasia mice

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by deficiency of alkaline phosphatase activity, leading to rickets or osteomalacia and to dental defects. HPP occurs from loss‐of‐function mutations within the gene that encodes the tissue‐nonspecific isozyme of alkaline phosphatase (TNAP). TNAP knockout (Alpl^?/?, aka Akp2^?/?) mice closely phenocopy infantile HPP, including the rickets, vitamin B6‐responsive seizures, improper dentin mineralization, and lack of acellular cementum. Here, we report that lack of TNAP in Alpl^?/? mice also causes severe enamel defects, which are preventable by enzyme replacement with mineral‐targeted TNAP (ENB‐0040). Immunohistochemistry was used to map the spatiotemporal expression of TNAP in the tissues of the developing enamel organ of healthy mouse molars and incisors. We found strong, stage‐specific expression of TNAP in ameloblasts. In the Alpl^?/? mice, histological, µCT, and scanning electron microscopy analysis showed reduced mineralization and disrupted organization of the rods and inter‐rod structures in enamel of both the molars and incisors. All of these abnormalities were prevented in mice receiving from birth daily subcutaneous injections of mineral‐targeting, human TNAP at 8.2 mg/kg/day for up to 44 days. These data reveal an important role for TNAP in enamel mineralization and demonstrate the efficacy of mineral‐targeted TNAP to prevent enamel defects in HPP. © 2012 American Society for Bone and Mineral Research.     

Enzyme replacement for craniofacial skeletal defects and craniosynostosis in murine hypophosphatasia

Hypophosphatasia (HPP) is an inborn-error-of-metabolism disorder characterized by deficient bone and tooth mineralization due to loss-of function mutations in the gene (Alpl) encoding tissue-nonspecific alkaline phosphatase (TNAP). Alpl^−/− mice exhibit many characteristics seen in infantile HPP including long bone and tooth defects, vitamin B6 responsive seizures and craniosynostosis. Previous reports demonstrated that a mineral-targeted form of TNAP rescues long bone, vertebral and tooth mineralization defects in Alpl^−/− mice. Here we report that enzyme replacement with mineral-targeted TNAP (asfotase-alfa) also prevents craniosynostosis (the premature fusion of cranial bones) and additional craniofacial skeletal abnormalities in Alpl^−/− mice. Craniosynostosis, cranial bone volume and density, and craniofacial shape abnormalities were assessed by microscopy, histology, digital caliper measurements and micro CT. We found that craniofacial shape defects, cranial bone mineralization and craniosynostosis were corrected in Alpl^−/− mice injected daily subcutaneously starting at birth with recombinant enzyme. Analysis of Alpl^−/− calvarial cells indicates that TNAP deficiency leads to aberrant osteoblastic gene expression and diminished proliferation. Some but not all of these cellular abnormalities were rescued by treatment with inorganic phosphate. These results confirm an essential role for TNAP in craniofacial skeletal development and demonstrate the efficacy of early postnatal mineral-targeted enzyme replacement for preventing craniofacial abnormalities including craniosynostosis in murine infantile HPP.     

Increased Osteopontin Contributes to Inhibition of Bone Mineralization in FGF23‐Deficient Mice

Excessive FGF23 has been identified as a pivotal phosphaturic factor leading to renal phosphate‐wasting and the subsequent development of rickets and osteomalacia. In contrast, loss of FGF23 in mice (Fgf23^?/?) leads to high serum phosphate, calcium, and 1,25‐vitamin D levels, resulting in early lethality attributable to severe ectopic soft‐tissue calcifications and organ failure. Paradoxically, Fgf23^?/? mice exhibit a severe defect in skeletal mineralization despite high levels of systemic mineral ions and abundant ectopic mineralization, an abnormality that remains largely unexplained. Through use of in situ hybridization, immunohistochemistry, and immunogold labeling coupled with electron microscopy of bone samples, we discovered that expression and accumulation of osteopontin (Opn/OPN) was markedly increased in Fgf23^?/? mice. These results were confirmed by qPCR analyses of Fgf23^?/? bones and ELISA measurements of serum OPN. To investigate whether elevated OPN levels were contributing to the bone mineralization defect in Fgf23^?/? mice, we generated Fgf23^?/?/Opn^?/? double‐knockout mice (DKO). Biochemical analyses showed that the hypercalcemia and hyperphosphatemia observed in Fgf23^?/? mice remained unchanged in DKO mice; however, micro‐computed tomography (µCT) and histomorphometric analyses showed a significant improvement in total mineralized bone volume. The severe osteoidosis was markedly reduced and a normal mineral apposition rate was present in DKO mice, indicating that increased OPN levels in Fgf23^?/? mice are at least in part responsible for the osteomalacia. Moreover, the increased OPN levels were significantly decreased upon lowering serum phosphate by feeding a low‐phosphate diet or after deletion of NaPi2a, indicating that phosphate levels contribute in part to the high OPN levels in Fgf23^?/? mice. In summary, our results suggest that increased OPN is an important pathogenic factor mediating the mineralization defect and the alterations in bone metabolism observed in Fgf23^?/? bones. © 2014 American Society for Bone and Mineral Research.     

Effects of pH on the Production of Phosphate and Pyrophosphate by Matrix Vesicles’ Biomimetics

During endochondral bone formation, chondrocytes and osteoblasts synthesize and mineralize the extracellular matrix through a process that initiates within matrix vesicles (MVs) and ends with bone mineral propagation onto the collagenous scaffold. pH gradients have been identified in the growth plate of long bones, but how pH changes affect the initiation of skeletal mineralization is not known. Tissue-nonspecific alkaline phosphatase (TNAP) degrades extracellular inorganic pyrophosphate (PP_i), a mineralization inhibitor produced by ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1), while contributing P_i from ATP to initiate mineralization. TNAP and NPP1, alone or combined, were reconstituted in dipalmitoylphosphatidylcholine liposomes to mimic the microenvironment of MVs. The hydrolysis of ATP, ADP, AMP, and PP_i was studied at pH 8 and 9 and compared to the data determined at pH 7.4. While catalytic efficiencies in general were higher at alkaline pH, PP_i hydrolysis was maximal at pH 8 and indicated a preferential utilization of PP_i over ATP at pH 8 versus 9. In addition, all proteoliposomes induced mineral formation when incubated in a synthetic cartilage lymph containing 1 mM ATP as substrate and amorphous calcium phosphate or calcium–phosphate–phosphatidylserine complexes as nucleators. Propagation of mineralization was significantly more efficient at pH 7.5 and 8 than at pH 9. Since a slight pH elevation from 7.4 to 8 promotes considerably more hydrolysis of ATP, ADP, and AMP primarily by TNAP, this small pH change facilitates mineralization, especially via upregulated PP_i hydrolysis by both NPP1 and TNAP, further elevating the P_i/PP_i ratio, thus enhancing bone mineralization.     

Genetic Ablation of Osteopontin in Osteomalacic Hyp Mice Partially Rescues the Deficient Mineralization Without Correcting Hypophosphatemia

PHEX is predominantly expressed by bone and tooth-forming cells, and its inactivating mutations in X-linked hypophosphatemia (XLH) lead to renal phosphate wasting and severe hypomineralization of bones and teeth. Also present in XLH are hallmark hypomineralized periosteocytic lesions (POLs, halos) that persist despite stable correction of serum phosphate (P_i) that improves bulk bone mineralization. In XLH, mineralization-inhibiting osteopontin (OPN, a substrate for PHEX) accumulates in the extracellular matrix of bone. To investigate how OPN functions in Hyp mice (a model for XLH), double-null (Hyp;Opn^−/−) mice were generated. Undecalcified histomorphometry performed on lumbar vertebrae revealed that Hyp;Opn^−/− mice had significantly reduced osteoid area/bone area (OV/BV) and osteoid thickness of trabecular bone as compared to Hyp mice, despite being as hypophosphatemic as Hyp littermate controls. However, tibias examined by synchrotron radiation micro-CT showed that mineral lacunar volumes remained abnormally enlarged in these double-null mice. When Hyp;Opn^−/− mice were fed a high-P_i diet, serum P_i concentration increased, and OV/BV and osteoid thickness normalized, yet mineral lacunar area remained abnormally enlarged. Enpp1 and Ankh gene expression were increased in double-null mice fed a high-P_i diet, potentially indicating a role for elevated inhibitory pyrophosphate (PP_i) in the absence of OPN. To further investigate the persistence of POLs in Hyp mice despite stable correction of serum P_i, immunohistochemistry for OPN on Hyp mice fed a high-P_i diet showed elevated OPN in the osteocyte pericellular lacunar matrix as compared to Hyp mice fed a control diet. This suggests that POLs persisting in Hyp mice despite correction of serum P_i may be attributable to the well-known upregulation of mineralization-inhibiting OPN by P_i, and its accumulation in the osteocyte pericellular matrix. This study shows that OPN contributes to osteomalacia in Hyp mice, and that genetic ablation of OPN in Hyp mice improves the mineralization phenotype independent of systemic P_i-regulating factors. © 2020 American Society for Bone and Mineral Research.     

Improvement of the skeletal and dental hypophosphatasia phenotype in Alpl−/− mice by administration of soluble (non-targeted) chimeric alkaline phosphatase

Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP), and accumulation of inorganic pyrophosphate, a potent inhibitor of mineralization that is also a natural substrate of TNAP, in the extracellular space. HPP causes mineralization disorders including soft bones (rickets or osteomalacia) and defects in teeth and periodontal tissues. Enzyme replacement therapy using mineral-targeting recombinant TNAP has proven effective in preventing skeletal and dental defects in TNAP knockout (Alpl^−/−) mice, a model for life-threatening HPP. Here, we show that the administration of a soluble, intestinal-like chimeric alkaline phosphatase (ChimAP) improves the manifestations of HPP in Alpl^−/− mice. Mice received daily subcutaneous injections of ChimAP at doses of 1, 8 or 16 mg/kg, from birth for up to 53 days. Lifespan and body weight of Alpl^−/− mice were normalized, and vitamin B6-associated seizures were absent with 16 mg/kg/day of ChimAP. Radiographs, μCT and histological analyses documented improved mineralization in cortical and trabecular bone and secondary ossification centers in long bones of ChimAP16-treated mice. There was no evidence of craniosynostosis in the ChimAP16-treated mice and we did not detect ectopic calcification by radiography and histology in the aortas, stomachs, kidneys or lungs in any of the treatment groups. Molar tooth development and function improved with the highest ChimAP dose, including enamel, dentin, and tooth morphology. Cementum remained deficient and alveolar bone mineralization was reduced compared to controls, though ChimAP-treated Alpl^−/− mice featured periodontal attachment and retained teeth. This study provides the first evidence for the pharmacological efficacy of ChimAP for use in the treatment of skeletal and dental manifestations of HPP.     

Enzyme Replacement Therapy for Murine Hypophosphatasia

Introduction: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss‐of‐function mutation(s) within the gene that encodes the tissue‐nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PP_i), an inhibitor of mineralization, and pyridoxal 5′‐phosphate (PLP), a co‐factor form of vitamin B₆. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B₆‐dependent seizures. There is no established medical treatment. Materials and Methods: Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one‐step purification and a deca‐aspartate sequence (D₁₀) for targeting to mineralizing tissue (sALP‐FcD₁₀). TNALP‐null mice (Akp2^?/?), an excellent model for infantile HPP, were treated from birth using sALP‐FcD₁₀. Short‐term and long‐term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP‐FcD₁₀ for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP‐FcD₁₀ activity, calcium, PP_i, and pyridoxal, as well as skeletal and dental manifestations using radiography, μCT, and histomorphometry. Results: Akp2^?/? mice receiving high‐dose sALP‐FcD₁₀ grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PP_i, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. Conclusions: Enzyme replacement using a bone‐targeted, recombinant form of human TNALP prevents infantile HPP in Akp2^?/? mice.     

The calcium‐sensing receptor and 25‐hydroxyvitamin D–1α‐hydroxylase interact to modulate skeletal growth and bone turnover

We examined parathyroid and skeletal function in 3‐month‐old mice expressing the null mutation for 25‐hydroxyvitamin D–1α‐hydroxylase [1α(OH)ase^?/?] and in mice expressing the null mutation for both the 1α(OH)ase and the calcium‐sensing receptor [Casr^?/?1α(OH)ase^?/?] genes. On a normal diet, all mice were hypocalcemic, with markedly increased parathyroid hormone (PTH), increased trabecular bone volume, increased osteoblast activity, poorly mineralized bone, enlarged and distorted cartilaginous growth plates, and marked growth retardation, especially in the compound mutants. Osteoclast numbers were reduced in the Casr^?/?1α(OH)ase^?/? mice. On a high‐lactose, high‐calcium, high‐phosphorus “rescue” diet, serum calcium and PTH were normal in the 1α(OH)ase^?/? mice but increased in the Casr^?/?1α(OH)ase^?/? mice with reduced serum phosphorus. Growth plate architecture and mineralization were improved in both mutants, but linear growth of the double mutants remained abnormal. Mineralization of bone improved in all mice, but osteoblast activity and trabecular bone volume remained elevated in the Casr^?/?1α(OH)ase^?/? mice. These studies support a role for calcium‐stimulated maturation of the cartilaginous growth plate and mineralization of the growth plate and bone and calcium‐stimulated CaSR‐mediated effects on bone resorption. PTH‐mediated bone resorption may require calcium‐stimulated CaSR‐mediated enhancement of osteoclastic activity. © 2010 American Society for Bone and Mineral Research. © 2010 American Society for Bone and Mineral Research     

Kinetic analysis of substrate utilization by native and TNAP‐, NPP1‐, or PHOSPHO1‐deficient matrix vesicles

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite seed crystals in the sheltered interior of membrane‐limited matrix vesicles (MVs). Here, we have studied phosphosubstrate catalysis by osteoblast‐derived MVs at physiologic pH, analyzing the hydrolysis of ATP, ADP, and PP_i by isolated wild‐type (WT) as well as TNAP‐, NPP1‐ and PHOSPHO1‐deficient MVs. Comparison of the catalytic efficiencies identified ATP as the main substrate hydrolyzed by WT MVs. The lack of TNAP had the most pronounced effect on the hydrolysis of all physiologic substrates. The lack of PHOSPHO1 affected ATP hydrolysis via a secondary reduction in the levels of TNAP in PHOSPHO1‐deficient MVs. The lack of NPP1 did not significantly affect the kinetic parameters of hydrolysis when compared with WT MVs for any of the substrates. We conclude that TNAP is the enzyme that hydrolyzes both ATP and PP_i in the MV compartment. NPP1 does not have a major role in PP_i generation from ATP at the level of MVs, in contrast to its accepted role on the surface of the osteoblasts and chondrocytes, but rather acts as a phosphatase in the absence of TNAP. © 2010 American Society for Bone and Mineral Research     

EP1−/− mice have enhanced osteoblast differentiation and accelerated fracture repair

As a downstream product of cyclooxygenase 2 (COX‐2), prostaglandin E₂ (PGE₂) plays a crucial role in the regulation of bone formation. It has four different receptor subtypes (EP1 through EP4), each of which exerts different effects in bone. EP2 and EP4 induce bone formation through the protein kinase A (PKA) pathway, whereas EP3 inhibits bone formation in vitro. However, the effect of EP1 receptor signaling during bone formation remains unclear. Closed, stabilized femoral fractures were created in mice with EP1 receptor loss of function at 10 weeks of age. Healing was evaluated by radiographic imaging, histology, gene expression studies, micro–computed tomographic (µCT), and biomechanical measures. EP1^?/? mouse fractures have increased formation of cartilage, increased fracture callus, and more rapid completion of endochondral ossification. The fractures heal faster and with earlier fracture callus mineralization with an altered expression of genes involved in bone repair and remodeling. Fractures in EP1^?/? mice also had an earlier appearance of tartrate‐resistant acid phosphatase (TRAcP)–positive osteoclasts, accelerated bone remodeling, and an earlier return to normal bone morphometry. EP1^?/? mesenchymal progenitor cells isolated from bone marrow have higher osteoblast differentiation capacity and accelerated bone nodule formation and mineralization in vitro. Loss of the EP1 receptor did not affect EP2 or EP4 signaling, suggesting that EP1 and its downstream signaling targets directly regulate fracture healing. We show that unlike the PGE₂ receptors EP2 and EP4, the EP1 receptor is a negative regulator that acts at multiple stages of the fracture healing process. Inhibition of EP1 signaling is a potential means to enhance fracture healing. © 2011 American Society for Bone and Mineral Research.     

Pharmacological inhibition of PHOSPHO1 suppresses vascular smooth muscle cell calcification

Medial vascular calcification (MVC) is common in patients with chronic kidney disease, obesity, and aging. MVC is an actively regulated process that resembles skeletal mineralization, resulting from chondro‐osteogenic transformation of vascular smooth muscle cells (VSMCs). Here, we used mineralizing murine VSMCs to study the expression of PHOSPHO1, a phosphatase that participates in the first step of matrix vesicles‐mediated initiation of mineralization during endochondral ossification. Wild‐type (WT) VSMCs cultured under calcifying conditions exhibited increased Phospho1 gene expression and Phospho1^–/– VSMCs failed to mineralize in vitro. Using natural PHOSPHO1 substrates, potent and specific inhibitors of PHOSPHO1 were identified via high‐throughput screening and mechanistic analysis and two of these inhibitors, designated MLS‐0390838 and MLS‐0263839, were selected for further analysis. Their effectiveness in preventing VSMC calcification by targeting PHOSPHO1 function was assessed, alone and in combination with a potent tissue‐nonspecific alkaline phosphatase (TNAP) inhibitor MLS‐0038949. PHOSPHO1 inhibition by MLS‐0263839 in mineralizing WT cells (cultured with added inorganic phosphate) reduced calcification in culture to 41.8% ± 2.0% of control. Combined inhibition of PHOSPHO1 by MLS‐0263839 and TNAP by MLS‐0038949 significantly reduced calcification to 20.9% ± 0.74% of control. Furthermore, the dual inhibition strategy affected the expression of several mineralization‐related enzymes while increasing expression of the smooth muscle cell marker Acta2. We conclude that PHOSPHO1 plays a critical role in VSMC mineralization and that “phosphatase inhibition” may be a useful therapeutic strategy to reduce MVC. © 2013 American Society for Bone and Mineral Research     

Visualization of Mineral-Targeted Alkaline Phosphatase Binding to Sites of Calcification In Vivo

A mineral-targeted form of recombinant tissue-nonspecific alkaline phosphatase (TNAP), asfotase alfa, was approved multinationally as an enzyme replacement therapy for hypophosphatasia in 2015. Two reports to date have shown evidence of binding of this drug to mineralizing tissues using histochemistry and immunohistochemistry. Here, we sought to expand on those earlier studies by directly visualizing the in vivo binding of asfotase alfa conjugated with AnaTag HiLyte Fluor 750 or Alexa Fluor 647 fluorescent dye to sites of skeletal/dental mineralization and ectopic calcification. We utilized 40-day-old Tagln-Cre; Hprt^ALPL/Y mice, a model of severe medial vascular calcification; Tie2-Cre; Hprt^ALPL/Y mice, a model of severe intimal calcification; and sibling WT Hprt^ALPL/Y mice, devoid of soft-tissue calcification. A single dose of 8 mg/kg labeled asfotase alfa was injected via the retro-orbital route. Skeletal tissues and soft organs were imaged ex vivo 2 days after the injection. Strong fluorescence signal was observed in all skeletal tissues (calvaria, vertebra, long bones, jaw, and mandibles) from mutant and WT mice. Fluorescence analysis of histological sections from bones revealed strong binding of asfotase alfa. Asfotase alfa binding to sites of ectopic calcification in the heart, aorta, and renal artery were found in both the Tagln-Cre; Hprt^ALPL/Y and Tie2-Cre; Hprt^ALPL/Y mice but not in WT mice. In addition, asfotase alfa binding was also found in the kidney stroma and brain of the Tie2-Cre; Hprt^ALPL/Y mice. Our results show that fluorescence-labeled asfotase alfa administered in vivo binds not only to sites of skeletal and dental mineralization but also to sites of ectopic calcification in these animal models. © 2020 American Society for Bone and Mineral Research.     

Rapamycin‐mediated suppression of renal cyst expansion in del34 Pkd1−/− mutant mouse embryos: An investigation of the feasibility of renal cyst prevention in the foetus

Aim: Polycystic kidney disease (PKD) in humans involves kidney cyst expansion beginning in utero. Recessive PKD can result in end‐stage renal disease (ESRD) within the first decade, whereas autosomal dominant PKD (ADPKD), caused by mutations in the PKD1 or PKD2 gene, typically leads to ESRD by the fifth decade of life. Inhibition of mTOR signalling was recently found to halt cyst formation in adult ADPKD mice. In contrast, no studies have investigated potential treatments to prevent cyst formation in utero in recessive PKD. Given that homozygous Pkd1 mutant mice exhibit cyst formation in utero, we decided to investigate whether mTOR inhibition in utero ameliorates kidney cyst formation in foetal Pkd1 homozygous mutant mice. Methods: Pregnant Pkd1^+/? female mice (mated with Pkd1^+/? male mice) were treated with rapamycin from E14.5 to E17.5. Foetal kidneys were dissected, genotyped and evaluated by cyst size as well as expression of the developmental marker, Pax2. Results: Numerous cysts were present in Pkd1^?/? kidneys, which were twice the weight of wild‐type kidneys. Cyst size was reduced by a third in rapamycin‐treated Pkd1^?/? kidney sections and kidney mass was reduced to near wild‐type levels. However, total cyst number was not reduced compared with control embryos. Pax2 expression and kidney development were unaltered in rapamycin‐treated mice but some lethality was observed in Pkd1^?/? null embryos. Conclusion: Rapamycin treatment reduces cyst formation in Pkd1^?/? mutant mice; therefore, the prevention of kidney cyst expansion in utero by mTOR inhibition is feasible. However, selective rapamycin‐associated lethality limits its usefulness as a treatment in utero.     

Depletion of annexin A5, annexin A6, and collagen X causes no gross changes in matrix vesicle–mediated mineralization,but lack of collagen X affects hematopoiesis and the Th1/Th2 response

Numerous biochemical studies have pointed to an essential role of annexin A5 (AnxA5), annexin A6 (AnxA6), and collagen X in matrix vesicle–mediated biomineralization during endochondral ossification and in osteoarthritis. By binding to the extracellular matrix protein collagen X and matrix vesicles, annexins were proposed to anchor matrix vesicles in the extracellular space of hypertrophic chondrocytes to initiate the calcification of cartilage. However, mineralization appears to be normal in mice lacking AnxA5 and AnxA6, whereas collagen X–deficient mice show only subtle alterations in the growth plate organization. We hypothesized that the simultaneous lack of AnxA5, AnxA6, and collagen X in vivo induces more pronounced changes in the growth plate development and the initiation of mineralization. In this study, we generated and analyzed mice deficient for AnxA5, AnxA6, and collagen X. Surprisingly, mice were viable, fertile, and showed no obvious abnormalities. Assessment of growth plate development indicated that the hypertrophic zone was expanded in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? newborns, whereas endochondral ossification and mineralization were not affected in 13‐day‐ and 1‐month‐old mutants. In peripheral quantitative computed tomography, no changes in the degree of biomineralization were found in femora of 1‐month‐ and 1‐year‐old mutants even though the diaphyseal circumference was reduced in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? mice. The percentage of naive immature IgM⁺/IgM⁺ B cells and peripheral T‐helper cells were increased in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? mutants, and activated splenic T cells isolated from Col10a1^?/? mice secreted elevated levels of IL‐4 and GM‐CSF. Hence, collagen X is needed for hematopoiesis during endochondral ossification and for the immune response, but the interaction of annexin A5, annexin A6, and collagen X is not essential for physiological calcification of growth plate cartilage. Therefore, annexins and collagen X may rather fulfill functions in growth plate cartilage not directly linked to the mineralization process. © 2012 American Society for Bone and Mineral Research.