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1.  Hypophosphatasia - pathophysiology and treatment 
Actualizaciones en osteologia  2012;8(3):164-182.
English Summary
Hypophosphatasia (HPP) is the inborn-error-of-metabolism caused by loss-of-function mutation(s) in the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNAP). The disease has been classified according to patient age when the first signs and symptoms manifest; i.e., perinatal, infantile, childhood, adult HPP. Other types include odonto HPP and perinatal benign. Babies with the perinatal/infantile forms of HPP often die with severe rickets and respiratory insufficiency and sometimes hypercalcemia and vitamin B6-responsive seizures. The primary biochemical defect in HPP is a deficiency of TNAP activity that leads to elevated circulating levels of substrates, in particular inorganic pyrophosphate (PPi), a potent calcification inhibitor. To-date, the management of HPP has been essentially symptomatic or orthopedic. However, enzyme replacement therapy with mineral-targeting TNAP (sALP-FcD10, also known as ENB-0040 or asfotase alfa) has shown promising results in a mouse model of HPP (Alpl−/− mice). Administration of mineral-targeting TNAP from birth increased survival and prevented the seizures, rickets, as well as all the tooth abnormalities, including dentin, acellular cementum, and enamel defects in this model of severe HPP. Clinical trials using mineral-targeting TNAP in children 3 years of age or younger with life-threatening HPP was associated with healing of the skeletal manifestations of HPP as well as improved respiratory and motor function. Improvement is still being observed in the patients receiving continued asfotase alfa therapy, with more than 3 years of treatment in some children. Enzyme replacement therapy with asfotase alfa has to-date been successful in patients with life-threatening HPP.
PMCID: PMC4171060  PMID: 25254037
2.  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 10-20 times higher than those of wild-type mice. The Col1a1-Tnap animals are healthy and exhibit increased bone mineralization by microCT 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 non-transgenic controls and osteoblasts from [Col1a1-Tnap+/−; Alpl−/−] mice are able to mineralize to the level of Alpl+/− heterozygous osteoblasts, while 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 while WT and [Col1a1-Tnap+/−; Alpl−/−] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [Col1a1-Tnap] osteoblasts were more phosphorylated than non-transgenic 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 non-localized site each, appear to be preferred sites of TNAP action on OPN. Our data suggest that the pro-mineralization 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.
doi:10.1002/jbmr.1901
PMCID: PMC3688694  PMID: 23427088
hypophosphatasia; phosphorylation; phosphopeptides; mineralization; bone mass; transgenic mice; knockout mice
3.  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, 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.
doi:10.1002/jbmr.1733
PMCID: PMC3562655  PMID: 22887744
High-throughput screening; small-molecules; pharmacological inhibitors; alkaline phosphatase; kinetic studies
4.  Enzyme replacement prevents enamel defects in hypophosphatasia mice 
Journal of Bone and Mineral Research  2012;27(8):1722-1734.
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−/−, a.k.a. 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 (sALP-FcD10, a.k.a. ENB-0040) 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.
doi:10.1002/jbmr.1619
PMCID: PMC3395779  PMID: 22461224
5.  Dose response of bone-targeted enzyme replacement for murine hypophosphatasia 
Bone  2011;49(2):250-256.
Hypophosphatasia (HPP) features rickets or osteomalacia from tissue-nonspecific alkaline phosphatase (TNSALP) deficiency due to deactivating mutations within the ALPL gene. Enzyme replacement therapy with a bone-targeted, recombinant TNSALP (sALP-FcD10, renamed ENB-0040) prevents manifestations of HPP when initiated at birth in TNSALP knockout (Akp2−/−) mice. Here, we evaluated the dose-response relationship of ENB-0040 to various phenotypic traits of Akp2−/− mice receiving daily subcutaneous (SC) injections of ENB-0040 from birth at 0.5, 2.0, or 8.2 mg/kg for 43 days. Radiographs, μCT, and histomorphometric analyses documented better bone mineralization with increasing doses of ENB-0040. We found a clear, positive correlation between ENB-0040 dose and prevention of mineralization defects of the feet, rib cage, lower limbs, and jaw bones. According to a dose-response model, the ED80 (the dose prevents the bone defects in 80% of mice) was 3.2, 2.8 and 2.9 mg/kg/day for these sites, respectively. Long bones seemed to respond to lower daily doses of ENB-0040. There was also a positive relationship between ENB-0040 dose and survival. Median survival, body weight, and bone length all improved with increasing doses of ENB-0040. Urinary PPi concentrations remained elevated in all treatment groups, indicating that while this parameter is a good biochemical marker for diagnosing HPP, it may not be a good follow up marker for evaluating response to treatment when administering bone-targeted TNSALP. These dose-response relationships strongly support the pharmacological efficacy of ENB-0040 for HPP, and provide the experimental basis for the therapeutic range of ENB-0040 chosen for clinical trials.
doi:10.1016/j.bone.2011.03.770
PMCID: PMC3117961  PMID: 21458605
alkaline phosphatase; calcification; ENB-0040; rickets; osteomalacia
6.  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 (PPi). 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 PPi concentrations. However, transgenic overexpression of TNAP does not correct the bone phenotype in Phospho1−/− mice despite normalization of their plasma PPi 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 Pi 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.
doi:10.1002/jbmr.195
PMCID: PMC3179344  PMID: 20684022
OSTEOMALACIA; OSTEOIDOSIS; SCOLIOSIS; CALCIFICATION; BIOMINERALIZATION; HYPOPHOSPHATASIA; AKP2; TNAP
7.  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 PPi 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 PPi in the MV compartment. NPP1 does not have a major role in PPi 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.
doi:10.1359/jbmr.091023
PMCID: PMC3153326  PMID: 19874193
biomineralization; knockout mice; calcification; pyrophosphatases; atpases
8.  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 (PPi), 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, PPi, 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, PPi, 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.
doi:10.1359/JBMR.071213
PMCID: PMC2652241  PMID: 18086009
alkaline phosphatase; calcification; epilepsy; osteomalacia; rickets
9.  Reference point indentation is not indicative of whole mouse bone measures of stress intensity fracture toughness 
Bone  2014;69:174-179.
Bone fragility is a concern for aged and diseased bone. Measuring bone toughness and understanding fracture properties of the bone are critical for predicting fracture risk associated with age and disease and for preclinical testing of therapies. A reference point indentation technique (BioDent) has recently been developed to determine bone's resistance to fracture in a minimally invasive way by measuring the indentation distance increase (IDI) between the first and last indentations over cyclic indentations in the same position. In this study, we investigate the relationship between fracture toughness KC and reference point indentation parameters (i.e. IDI, total indentation distance (TID) and creep indentation distance (CID)) in bones from 38 mice from six types (C57Bl/6, Balb, oim/oim, oim/+, Phospho1−/− and Phospho1 wild type counterpart). These mice bone are models of healthy and diseased bone spanning a range of fracture toughness from very brittle (oim/oim) to ductile (Phospho1−/−). Left femora were dissected, notched and tested in 3-point bending until complete failure. Contralateral femora were dissected and indented in 10 sites of their anterior and posterior shaft surface over 10 indentation cycles. IDI, TID and CID were measured. Results from this study suggest that reference point indentation parameters are not indicative of stress intensity fracture toughness in mouse bone. In particular, the IDI values at the anterior mid-diaphysis across mouse types overlapped, making it difficult to discern differences between mouse types, despite having extreme differences in stress intensity based toughness measures. When more locations of indentation were considered, the normalised IDIs could distinguish between mouse types. Future studies should investigate the relationship of the reference point indentation parameters for mouse bone in other material properties of the bone tissue in order to determine their use for measuring bone quality.
Highlights
•We investigated fracture toughness and reference point indentation parameters from BioDent microindentation in six different mouse bone types.•Fracture toughness clearly distinguished mouse phenotypes.•BioDent parameters at mid-diaphysis across mouse types were close to each other making it difficult to discern between groups.•Increasing the number of indentations along the bone shaft increases the standard deviations within the data reflecting bone's heterogeneity.•BioDent parameters values are not indicative of stress intensity fracture toughness in mouse bone.
doi:10.1016/j.bone.2014.09.020
PMCID: PMC4228060  PMID: 25280470
Bone fracture; Bone quality; Bone toughness; Mouse bone; Reference point indentation; BioDent
10.  Mineralisation of collagen rich soft tissues and osteocyte lacunae in Enpp1−/− mice 
Bone  2014;69:139-147.
Ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyse nucleotide triphosphates to the corresponding nucleotide monophosphates and the mineralisation inhibitor, pyrophosphate (PPi). This study examined the role of NPP1 in osteocytes, osteoclasts and cortical bone, using a mouse model lacking NPP1 (Enpp1−/−). We used microcomputed tomography (μCT) to investigate how NPP1 deletion affects cortical bone structure; excised humerus bones from 8, 15 and 22-week old mice were scanned at 0.9 μm. Although no changes were evident in the cortical bone of 8-week old Enpp1−/− mice, significant differences were observed in older animals. Cortical bone volume was decreased 28% in 22-week Enpp1−/− mice, whilst cortical porosity was reduced 30% and 60% at 15 and 22-weeks, respectively. This was accompanied by up to a 15% decrease in closed pore diameter and a 55% reduction in the number of pores. Cortical thickness was reduced up to 35% in 15 and 22-week Enpp1−/− animals and the endosteal diameter was increased up to 23%. Thus, the cortical bone from Enpp1−/− mice was thinner and less porous, with a larger marrow space. Scanning electron microscopy (SEM) revealed a decrease in the size and number of blood vessel channels in the cortical bone as well as a 40% reduction in the mean plan area of osteocyte lacunae. We noted that the number of viable osteocytes isolated from the long bones of Enpp1−/− mice was decreased ≤ 50%. In contrast, osteoclast formation and resorptive activity were unaffected by NPP1 deletion. μCT and histological analysis of Enpp1−/− mice also revealed calcification of the joints and vertebrae as well as soft tissues including the whisker follicles, ear pinna and trachea. This calcification worsened as the animals aged. Together, these data highlight the key role of NPP1 in regulating calcification of both soft and skeletal tissues.
Highlights
•We examine the role of NPP1 in osteocytes, osteoclasts and cortical bone.•Osteocyte lacunae are reduced in size and number in NPP1 knockout mice.•Sclerostin levels are increased in NPP1 knockout mice.•Osteoclast formation and resorptive activity are unaffected by NPP1 deletion.•Mice lacking NPP1 display widespread calcification of collagen rich soft tissues.
doi:10.1016/j.bone.2014.09.016
PMCID: PMC4228085  PMID: 25260930
NPP1; Osteocytes; Osteoclasts; Soft tissue mineralisation; Pyrophosphate
11.  Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia 
Periodontology 2000  2013;63(1):10.1111/prd.12029.
As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth – where calcium phosphate crystals are deposited and grow within an extracellular matrix – is essential to dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition such that teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibres (Sharpey's fibres) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins (SIBLINGs), and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here with clinical examples given, namely tissue-nonspecific alkaline phosphatase (TNAP) and phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX). Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia (HPP) and X-linked hypophosphatemia (XLH), respectively, where levels of local and systemic circulating mineralization determinants are perturbed. In XLH, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating SIBLING proteins such as matrix extracellular phosphoglycoprotein (MEPE) and osteopontin (OPN), and the phosphorylated peptides proteolytically released from them such as the acidic serine- and aspartate-rich motif (ASARM) peptide, may accumulate locally to impair mineralization in this disease.
doi:10.1111/prd.12029
PMCID: PMC3766584  PMID: 23931057
12.  The Role of Phosphatases in the Initiation of Skeletal Mineralization 
Calcified tissue international  2012;93(4):299-306.
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 (PPi), 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 Pi 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 PHOS-PHO1 function and Pi influx into MVs in the initiation of mineralization and the functions of TNAP and NPP1 in the extravesicular progression of mineralization.
doi:10.1007/s00223-012-9672-8
PMCID: PMC3594124  PMID: 23183786
Biomineralization; Bone and cartilage development; Metabolic bone disease; Animal model
13.  Alkaline phosphatases contribute to uterine receptivity, implantation, decidualization and defense against bacterial endotoxin in hamsters 
Reproduction (Cambridge, England)  2013;146(5):419-432.
Alkaline phosphatase (AP) activity has been demonstrated in the uterus of several species, but its importance in the uterus, in general and during pregnancy, is yet to be revealed. In this study, we focused on identifying AP isozyme types, and their hormonal regulation, cell-type and event-specific expression and possible functions in the hamster uterus during the cycle and early pregnancy. Our RT-PCR and in situ hybridization studies demonstrated that among the known Akp2, Akp3, Akp5 and Akp6 murine AP isozyme genes, hamster uteri express only Akp2 and Akp6; and both genes are co-expressed in luminal epithelial cells. Studies in cyclic and ovariectomized hamsters established that while progesterone is the major uterine Akp2 inducer, both progesterone and estrogen are strong Akp6 regulators. Studies in preimplantation uteri showed induction of both genes and the activity of their encoded isozymes in luminal epithelial cells during uterine receptivity. However, at the beginning of implantation, Akp2 showed reduced expression in luminal epithelial cells surrounding the implanted embryo. In contrast, expression of Akp6 and its isozyme was maintained in luminal epithelial cells adjacent to, but not away from, the implanted embryo. Following implantation, stromal transformation to decidua was associated with induced expressions of only Akp2 and its isozyme. We next demonstrated that uterine APs dephosphorylate and detoxify endotoxin lipopolysaccharide at their sites of production and activity. Taken together, our findings suggest that uterine APs contribute to uterine receptivity, implantation, and decidualization in addition to their role in protection of the uterus and pregnancy against bacterial infection.
doi:10.1530/REP-13-0153
PMCID: PMC3805369  PMID: 23929901
14.  Isozyme profile and tissue-origin of alkaline phosphatases in mouse serum 
Bone  2013;53(2):399-408.
Mouse serum alkaline phosphatase (ALP) is frequently measured and interpreted in mammalian bone research, however; little is known about the circulating ALPs in mice and their relation to human ALP isozymes and isoforms. Mouse ALP was extracted from liver, kidney, intestine, and bone from vertebra, femur and calvaria tissues. Serum from mixed strains of wild-type (WT) mice and from individual ALP knockout strains were investigated, i.e., Alpl−/− (a.k.a. Akp2 encoding tissue-nonspecific ALP or TNALP), Akp3−/− (encoding duodenum-specific intestinal ALP or dIALP), and Alpi−/− (a.k.a. Akp6 encoding global intestinal ALP or gIALP). The ALP isozymes and isoforms were identified by various techniques and quantified by high-performance liquid chromatography. Results from the WT and knockout mouse models revealed identical bone-specific ALP isoforms (B/I, B1, and B2) as found in human serum, but in addition mouse serum contains the B1x isoform only detected earlier in patients with chronic kidney disease and in human bone tissue. The two murine intestinal isozymes, dIALP and gIALP, were also identified in mouse serum. All four bone-specific ALP isoforms (B/I, B1x, B1, and B2) were identified in bone tissues from mice, in good correspondence with those found in human bones. All mouse tissues, except liver and colon, contained significant ALP activities. This is a notable difference as human liver contains vast amounts of ALP. Histochemical staining, Northern and Western blot analysis confirmed undetectable ALP expression in liver tissue. ALP activity staining showed some positive staining in the bile canaliculi for BALB/c and FVB/N WT mice, but not in C57Bl/6 and ICR mice. Taken together, while the main source of ALP in human serum originates from bone and liver, and a small fraction from intestine (<5%), mouse serum consists mostly of bone ALP, including all four isoforms, B/I, B1x, B1, and B2, and two intestinal ALP isozymes dIALP and gIALP. We suggest that the genetic nomenclature for the Alpl gene in mice (i.e., ALP liver) should be reconsidered since murine liver has undetectable amounts of ALP activity. These findings should pave the way for the development of user-friendly assays measuring circulating bone-specific ALP in mice models used in bone and mineral research.
doi:10.1016/j.bone.2012.12.048
PMCID: PMC3593980  PMID: 23313280
alkaline phosphatase; bone; glycosylation; hypophosphatasia; knockout mice; mineralization
15.  Exonic splicing signals impose constraints upon the evolution of enzymatic activity 
Nucleic Acids Research  2014;42(9):5790-5798.
Exon splicing enhancers (ESEs) overlap with amino acid coding sequences implying a dual evolutionary selective pressure. In this study, we map ESEs in the placental alkaline phosphatase gene (ALPP), absent in the corresponding exon of the ancestral tissue-non-specific alkaline phosphatase gene (ALPL). The ESEs are associated with amino acid differences between the transcripts in an area otherwise conserved. We switched out the ALPP ESEs sequences with the sequence from the related ALPL, introducing the associated amino acid changes. The resulting enzymes, produced by cDNA expression, showed different kinetic characteristics than ALPL and ALPP. In the organism, this enzyme will never be subjected to selection because gene splicing analysis shows exon skipping due to loss of the ESE. Our data prove that ESEs restrict the evolution of enzymatic activity. Thus, suboptimal proteins may exist in scenarios when coding nucleotide changes and consequent amino acid variation cannot be reconciled with the splicing function.
doi:10.1093/nar/gku240
PMCID: PMC4027185  PMID: 24692663
16.  Osteoconductivity and Osteoinductivity of NanoFUSE® DBM 
Cell and tissue banking  2012;14(1):33-44.
Bone graft substitutes have become an essential component in a number of orthopedic applications. Autologous bone has long been the gold standard for bone void fillers. However, the limited supply and morbidity associated with using autologous graft material has led to the development of many different bone graft substitutes. Allogeneic demineralized bone matrix (DBM) has been used extensively to supplement autograft bone because of its inherent osteoconductive and osteoinductive properties. Synthetic and natural bone graft substitutes that do not contain growth factors are considered to be osteoconductive only. Bioactive glass has been shown to facilitate graft containment at the operative site as well as activate cellular osteogenesis. In the present study, we present the results of a comprehensive in vitro and in vivo characterization of a combination of allogeneic human bone and bioactive glass bone void filler, NanoFUSE® DBM. NanoFUSE® DBM is shown to be biocompatible in a number of different assays and has been approved by the FDA for use in bone filling indications. Data are presented showing the ability of the material to support cell attachment and proliferation on the material thereby demonstrating the osteoconductive nature of the material. NanoFUSE® DBM was also shown to be osteoinductive in the mouse thigh muscle model. These data demonstrate that the DBM and bioactive glass combination, NanoFUSE® DBM, could be an effective bone graft substitute.
doi:10.1007/s10561-012-9297-1
PMCID: PMC3463676  PMID: 22323112
demineralized bone matrix; bioactive glass; osteoconductivity; osteoinductivity
17.  Catalytic Signature of a Heat-Stable, Chimeric Human Alkaline Phosphatase with Therapeutic Potential 
PLoS ONE  2014;9(2):e89374.
Recombinant alkaline phosphatases are becoming promising protein therapeutics to prevent skeletal mineralization defects, inflammatory bowel diseases, and treat acute kidney injury. By substituting the flexible crown domain of human intestinal alkaline phosphatase (IAP) with that of the human placental isozyme (PLAP) we generated a chimeric enzyme (ChimAP) that retains the structural folding of IAP, but displays greatly increased stability, active site Zn2+ binding, increased transphosphorylation, a higher turnover number and narrower substrate specificity, with comparable selectivity for bacterial lipopolysaccharide (LPS), than the parent IAP isozyme. ChimAP shows promise as a protein therapeutic for indications such as inflammatory bowel diseases, gut dysbioses and acute kidney injury.
doi:10.1371/journal.pone.0089374
PMCID: PMC3933536  PMID: 24586729
18.  High-Throughput Screening of Tissue-Nonspecific Alkaline Phosphatase for Identification of Effectors with Diverse Modes of Action 
Nature protocols  2010;5(8):1431-1439.
A protocol for the identification of effectors of tissue-nonspecific alkaline phosphatase (TNAP) is described. It is based on highly sensitive method for detecting TNAP activity. A dioxetane-based substrate after dephosphorylation by TNAP undergoes a series of chemical transformations resulting in light production. The light intensity serves as a quantitative measure of the velocity of the TNAP catalysed reaction in the steady state. The protocol includes guidelines for the optimization of the assay and execution of the high-throughput screening in multiwell plates. The assay is sensitive to the influence of diverse effectors of TNAP as long as the assay optimization steps are repeated for each new batch of the enzyme; full optimization is accomplished in under two days. Depending on the available equipment 10,000-100,000 compounds could be screened in 8-hour period. This protocol provides thousands-fold more sensitive and tenfold faster way of screening TNAP, when compared with a conventional colorimetric assay with p-nitrophenyl phosphate.
doi:10.1038/nprot.2010.86
PMCID: PMC3908691  PMID: 20671726
alkaline phosphatase; chemiluminescent assay; enzyme assay; functional assay; high-throughput screening
19.  Tissue-Nonspecific Alkaline Phosphatase Acts Redundantly with PAP and NT5E to Generate Adenosine in the Dorsal Spinal Cord 
The Journal of Neuroscience  2013;33(27):11314-11322.
Prostatic acid phosphatase (PAP) and ecto-5′-nucleotidase (NT5E) hydrolyze extracellular AMP to adenosine in dorsal root ganglia (DRG) neurons and in the dorsal spinal cord. Previously, we found that adenosine production was reduced, but not eliminated, in Pap−/−/Nt5e−/− double knock-out (dKO) mice, suggesting that a third AMP ectonucleotidase was present in these tissues. Here, we found that tissue-nonspecific alkaline phosphatase (TNAP, encoded by the Alpl gene) is expressed and functional in DRG neurons and spinal neurons. Using a cell-based assay, we found that TNAP rapidly hydrolyzed extracellular AMP and activated adenosine receptors. This activity was eliminated by MLS-0038949, a selective pharmacological inhibitor of TNAP. In addition, MLS-0038949 eliminated AMP hydrolysis in DRG and spinal lamina II of dKO mice. Using fast-scan-cyclic voltammetry, we found that adenosine was rapidly produced from AMP in spinal cord slices from dKO mice, but virtually no adenosine was produced in spinal cord slices from dKO mice treated with MLS-0038949. Last, we found that AMP inhibited excitatory neurotransmission via adenosine A1 receptor activation in spinal cord slices from wild-type, Pap−/−, Nt5e−/−, and dKO mice, but failed to inhibit neurotransmission in slices from dKO mice treated with MLS-0038949. These data suggest that triple elimination of TNAP, PAP, and NT5E is required to block AMP hydrolysis to adenosine in DRG neurons and dorsal spinal cord. Moreover, our data reveal that TNAP, PAP, and NT5E are the main AMP ectonucleotidases in primary somatosensory neurons and regulate physiology by metabolizing extracellular purine nucleotides.
doi:10.1523/JNEUROSCI.0133-13.2013
PMCID: PMC3718384  PMID: 23825434
20.  Effects of pH on the Production of Phosphate and Pyrophosphate by Matrix Vesicles' Biomimetics 
Calcified tissue international  2013;93(3):222-232.
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 (ePPi), a mineralization inhibitor produced by ectonucleotide pyrophosphatase/ phosphodiesterase-1 (NPP1), while contributing Pi from ATP to initiate mineralization. TNAP and NPP1, alone or combined, were reconstituted in dipalmitoylphosphatidylcholine (DPPC) liposomes to mimic the microenvironment of MVs. The hydrolysis of ATP, ADP, AMP and PPi 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, PPi hydrolysis was maximal at pH 8 and indicated a preferential utilization of PPi over ATP, at pH 8 versus 9. In addition, all proteoliposomes induced mineral formation when incubated in a synthetic cartilage lymph (SCL) containing 1 mM ATP as substrate and amorphous calcium phosphate (ACP) or calciumphosphate- phosphatidylserine complexes (PS-CPLX) as nucleators. Propagation of mineralization was significantly more efficient at pHs 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 PPi hydrolysis by both NPP1 and TNAP, further elevating the Pi/PPi ratio, thus enhancing bone mineralization.
doi:10.1007/s00223-013-9745-3
PMCID: PMC3752608  PMID: 23942722
biomineralization; alkaline pH; microenvironment; proteoliposomes; pyrophosphate; ATP
21.  Ablation of TNAP function compromises myelination and synaptogenesis in the mouse brain 
Cell and tissue research  2012;349(2):459-471.
Mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene can result in skeletal and dental hypomineralization and severe neurological symptoms. TNAP is expressed in the synaptic cleft and the node of Ranvier in normal adults. Using TNAP knockout (KO) mice (Akp2−/−), we studied synaptogenesis and myelination with light- and electron microscopy during the early postnatal days. Ablation of TNAP function resulted in a significant decrease of the white matter of the spinal cord accompanied by ultrastructural evidence of cellular degradation around the paranodal regions and a decreased ratio and diameter of the myelinated axons. In the cerebral cortex, myelinated axons, while present in wild-type, were absent in the Akp2−/− mice and these animals also displayed a significantly increased proportion of immature cortical synapses. The results suggest that TNAP deficiency could contribute to neurological symptoms related to myelin abnormalities and synaptic dysfunction, among which epilepsy, consistently present in the Akp2−/− mice and observed in severe cases of hypophosphatasia.
doi:10.1007/s00441-012-1455-z
PMCID: PMC3415568  PMID: 22696173
Alkaline phosphatase; Electron microscopy; Node of Ranvier; Hypophosphatasia; Epilepsy
22.  Soluble Ecto-5′-nucleotidase (5′-NT), Alkaline Phosphatase, and Adenosine Deaminase (ADA1) Activities in Neonatal Blood Favor Elevated Extracellular Adenosine*  
The Journal of Biological Chemistry  2013;288(38):27315-27326.
Background: Newborns have elevated plasma adenosine levels, which may influence their immunological function.
Results: Compared with adults, newborns have elevated plasma 5′-NT and alkaline phosphatase activities and lower adenosine deaminase activity.
Conclusion: Soluble enzymes significantly influence extracellular purine metabolism in blood, and the levels of these enzymes in newborns promote elevated adenosine.
Significance: Higher adenosine generation in newborn blood may promote an anti-inflammatory immunological status.
Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5′-nucleotidase (5′-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5′-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5′-NT enhanced Toll-like receptor-mediated TNF-α production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.
doi:10.1074/jbc.M113.484212
PMCID: PMC3779727  PMID: 23897810
Adenosine; Adenosine Receptor; ADP; AMP; ATP; Immunology; Infectious Diseases; Innate Immunity; Purine; Purinergic Agonists
23.  Mechanical and biocompatible characterization of a cross-linked collagen-hyaluronic acid wound dressing 
Biomatter  2013;3(4):e25633.
Collagen scaffolds have been widely employed as a dermal equivalent to induce fibroblast infiltrations and dermal regeneration in the treatment of chronic wounds and diabetic foot ulcers. Cross-linking methods have been developed to address the disadvantages of the rapid degradation associated with collagen-based scaffolds. To eliminate the potential drawbacks associated with glutaraldehyde cross-linking, methods using a water soluble carbodiimide have been developed. In the present study, the glycosaminoglycan (GAG) hyaluronic acid (HA), was covalently attached to an equine tendon derived collagen scaffold using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) to create ntSPONGE™. The HA was shown to be homogeneously distributed throughout the collagen matrix. In vitro analyses of the scaffold indicated that the cross-linking enhanced the biological stability by decreasing the enzymatic degradation and increasing the thermal denaturation temperature. The material was shown to support the attachment and proliferation of mouse L929 fibroblast cells. In addition, the cross-linking decreased the resorption rate of the collagen as measured in an intramuscular implant model in rabbits. The material was also shown to be biocompatible in a variety of in vitro and in vivo assays. These results indicate that this cross-linked collagen-HA scaffold, ntSPONGE™, has the potential for use in chronic wound healing.
doi:10.4161/biom.25633
PMCID: PMC3866196  PMID: 23896569
collagen sponge; hyaluronic acid; cross-linking; histology; wound repair
24.  Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures 
Bone  2013;53(2):478-486.
Studies on various compounds of inorganic phosphate, as well as on organic phosphate added by post-translational phosphorylation of proteins, all demonstrate a central role for phosphate in biomineralization processes. Inorganic polyphosphates are chains of orthophosphates linked by phosphoanhydride bonds that can be up to hundreds of orthophosphates in length. The role of polyphosphates in mammalian systems, where they are ubiquitous in cells, tissues and bodily fluids, and are at particularly high levels in osteoblasts, is not well understood. In cell-free systems, polyphosphates inhibit hydroxyapatite nucleation, crystal formation and growth, and solubility. In animal studies, polyphosphate injections inhibit induced ectopic calcification. While recent work has proposed an integrated view of polyphosphate function in bone, little experimental data for bone are available. Here we show in osteoblast cultures producing an abundant collagenous matrix that normally shows robust mineralization, that two polyphosphates (PolyP5 and PolyP65, polyphosphates of 5 and 65 phosphate residues in length) are potent mineralization inhibitors. Twelve-day MC3T3-E1 osteoblast cultures with added ascorbic acid (for collagen matrix assembly) and β-glycerophosphate (a source of phosphate for mineralization) were treated with either PolyP5 or PolyP65. Von Kossa staining and calcium quantification revealed that mineralization was inhibited in a dose-dependent manner by both polyphosphates, with complete mineralization inhibition at 10 μM PolyP. Cell proliferation and collagen assembly were unaffected by polyphosphate treatment, indicating that polyphosphate inhibition of mineralization results not from cell and matrix effects but from direct inhibition of mineralization. This was confirmed by showing that PolyP5 and PolyP65 bound to synthetic hydroxyapatite in a concentration-dependent manner. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) efficiently hydrolyzed the two PolyPs as measured by Pi release. Importantly, at the concentrations of polyphosphates used in this study which inhibited bone cell culture mineralization, the polyphosphates competitively saturated TNAP, thus potentially interfering with its ability to hydrolyze mineralization-inhibiting pyrophosphate (PPi) and mineralizing-promoting β-glycerophosphate (in cell culture). In the biological setting, TNAP may regulate mineralization by shielding the essential inhibitory substrate pyrophosphate from TNAP degradation, and in the same process, delay the release of phosphate from this source. In conclusion, the inhibition of mineralization by polyphosphates is shown to occur via direct binding to apatitic mineral and by mixed inhibition of TNAP.
doi:10.1016/j.bone.2013.01.020
PMCID: PMC3712787  PMID: 23337041
polyphosphates; phosphate; bone; biomineralization; extracellular matrix; osteoblasts
25.  Successful Gene Therapy in Utero for Lethal Murine Hypophosphatasia 
Human Gene Therapy  2011;23(4):399-406.
Abstract
Hypophosphatasia (HPP), caused by mutations in the gene ALPL encoding tissue-nonspecific alkaline phosphatase (TNALP), is an inherited systemic skeletal disease characterized by mineralization defects of bones and teeth. The clinical severity of HPP varies widely, from a lethal perinatal form to mild odontohypophosphatasia showing only dental manifestations. HPP model mice (Akp2−/−) phenotypically mimic the severe infantile form of human HPP; they appear normal at birth but die by 2 weeks of age because of growth failure, hypomineralization, and epileptic seizures. In the present study, we investigated the feasibility of fetal gene therapy using the lethal HPP model mice. On day 15 of gestation, the fetuses of HPP model mice underwent transuterine intraperitoneal injection of adeno-associated virus serotype 9 (AAV9) expressing bone-targeted TNALP. Treated and delivered mice showed normal weight gain and seizure-free survival for at least 8 weeks. Vector sequence was detected in systemic organs including bone at 14 days of age. ALP activities in plasma and bone were consistently high. Enhanced mineralization was demonstrated on X-ray images of the chest and forepaw. Our data clearly demonstrate that systemic injection of AAV9 in utero is an effective strategy for the treatment of lethal HPP mice. Fetal gene therapy may be an important choice after prenatal diagnosis of life-threatening HPP.
Sugano and colleagues investigate the feasibility of fetal gene therapy for hypophosphatasia (HPP), which is caused by mutations in tissue-nonspecific alkaline phosphatase (TNALP). Mice that phenotypically mimic the severe infantile form of human HPP underwent transuterine intraperitoneal injection of adeno-associated viral vector type 9 (AAV9) expressing bone-targeted TNALP. Treated mice showed normal weight gain and seizure-free survival for at least 8 weeks after birth, with consistently high levels of ALP activity in plasma and bone.
doi:10.1089/hum.2011.148
PMCID: PMC3327603  PMID: 22133046

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