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1.  Vascular calcification is dependent on plasma levels of pyrophosphate 
Kidney international  2014;85(6):1351-1356.
Plasma levels of pyrophosphate, an endogenous inhibitor of vascular calcification, are reduced in end-stage renal disease and correlate inversely with arterial calcification. However, it is not known whether the low plasma levels are directly pathogenic or are merely a marker of reduced tissue levels. This was tested in an animal model in which aortas were transplanted between normal mice and Enpp1−/− mice lacking ectonucleotide pyrophosphatase phosphodiesterase, the enzyme that releases extracellular pyrophosphate. Enpp1−/− mice had very low plasma pyrophosphate and developed aortic calcification by 2 months that was greatly accelerated with a high-phosphate diet. Aortas of Enpp1−/− mice showed no further calcification after transplantation into wild type mice fed a high phosphate diet. Aorta allografts of wild type mice calcified in Enpp1−/− mice but less so than the adjacent recipient Enpp1−/− aorta. Donor and recipient aortic calcium contents did not differ in transplants between wild type and Enpp1−/− mice, demonstrating that transplantation per se did not affect calcification. Histology revealed medial calcification with no signs of rejection. Thus, normal levels of extracellular pyrophosphate are sufficient to prevent vascular calcification and systemic Enpp1 deficiency is sufficient to produce vascular calcification despite normal vascular extracellular pyrophosphate production. This establishes an important role for circulating extracellular pyrophosphate in preventing vascular calcification.
PMCID: PMC4308968  PMID: 24717293
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.
PMCID: PMC3688694  PMID: 23427088
hypophosphatasia; phosphorylation; phosphopeptides; mineralization; bone mass; transgenic mice; knockout mice
3.  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.
PMCID: PMC3593980  PMID: 23313280
alkaline phosphatase; bone; glycosylation; hypophosphatasia; knockout mice; mineralization
4.  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.
PMCID: PMC3463676  PMID: 22323112
demineralized bone matrix; bioactive glass; osteoconductivity; osteoinductivity
5.  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.
PMCID: PMC3562655  PMID: 22887744
High-throughput screening; small-molecules; pharmacological inhibitors; alkaline phosphatase; kinetic studies
6.  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.
PMCID: PMC3395779  PMID: 22461224
7.  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.
PMCID: PMC3866196  PMID: 23896569
collagen sponge; hyaluronic acid; cross-linking; histology; wound repair
8.  Successful Gene Therapy in Utero for Lethal Murine Hypophosphatasia 
Human Gene Therapy  2011;23(4):399-406.
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.
PMCID: PMC3327603  PMID: 22133046
9.  Rescue of Severe Infantile Hypophosphatasia Mice by AAV-Mediated Sustained Expression of Soluble Alkaline Phosphatase 
Human Gene Therapy  2011;22(11):1355-1364.
Hypophosphatasia (HPP) is an inherited disease caused by a deficiency of tissue-nonspecific alkaline phosphatase (TNALP). The major symptom of human HPP is hypomineralization, rickets, or osteomalacia, although the clinical severity is highly variable. The phenotypes of TNALP knockout (Akp2-/-) mice mimic those of the severe infantile form of HPP. Akp2-/- mice appear normal at birth, but they develop growth failure, epileptic seizures, and hypomineralization and die by 20 days of age. Previously, we have shown that the phenotype of Akp2-/- mice can be prevented by enzyme replacement of bone-targeted TNALP in which deca-aspartates are linked to the C-terminus of soluble TNALP (TNALP-D10). In the present study, we evaluated the therapeutic effects of adeno-associated virus serotype 8 (AAV8) vectors that express various forms of TNALP, including TNALP-D10, soluble TNALP tagged with the Flag epitopes (TNALP-F), and native glycosylphosphatidylinositol-anchored TNALP (TNALP-N). A single intravenous injection of 5×1010 vector genomes of AAV8-TNALP-D10 into Akp2-/- mice at day 1 resulted in prolonged survival and phenotypic correction. When AAV8-TNALP-F was injected into neonatal Akp2-/- mice, they also survived without epileptic seizures. Interestingly, survival effects were observed in some animals treated with AAV8-TNALP-N. All surviving Akp2-/- mice showed a healthy appearance and a normal activity with mature bone mineralization on X-rays. These results suggest that sustained alkaline phosphatase activity in plasma is essential and sufficient for the rescue of Akp2-/- mice. AAV8-mediated systemic gene therapy appears to be an effective treatment for the infantile form of human HPP.
Matsumoto and colleagues evaluate the therapeutic effects of adeno-associated virus serotype 8 (AAV8) vectors that express various forms of tissue-nonspecific alkaline phosphatase (TNALP) in a mouse model of hypophosphatasia (HPP). A single intravenous injection of 5 × 1010 vector genomes of AAV8-TNALP in neonatal HPP mice results in prolonged survival and phenotypic correction.
PMCID: PMC3225041  PMID: 21388343
10.  Identification and Characterization of Novel Tissue-Nonspecific Alkaline Phosphatase Inhibitors with Diverse Modes of Action 
Journal of Biomolecular Screening  2009;14(7):824-837.
Tissue-nonspecific alkaline phosphatase (TNAP) plays a major role in maintaining a ratio of phosphate to inorganic pyrophosphate (Pi/PPi) in biological fluids that is conducive to controlled skeletal mineralization while preventing inappropriate ectopic calcification. Medial calcification associated with Enpp1 or Ank deficiency or with end–stage renal disease is associated with an increase in TNAP activity in arteries that leads to reduced levels of PPi and increased vascular calcification. Here, we describe in detail a high-throughput screening (HTS) campaign to identify inhibitors of TNAP, performed within the Molecular Library Screening Center Network (MLSCN). A homogeneous luminescent TNAP assay was developed and optimized for identification of compounds with diverse mechanism of action (MOA). The MLSCN compound collection, containing 64,394 molecules at the time of screening, was tested in the assay. Several novel inhibitory scaffold classes were identified and demonstrated to have diverse selectivity and mode of inhibition (MOI) profiles. Representatives of the novel scaffolds exhibited nanomolar potency surpassing the inhibitors known to date.
This paper sets a successful example in which pharmacologically active compounds, with outstanding selectivity in a panel of more than 200 assays, are identified from high throughput screening. Integral to the success of the project were a well-designed compound collection, an industrial-level screening facility and a deep knowledge of target biology that were brought together through the NIH-sponsored Roadmap Initiative.
PMCID: PMC3403534  PMID: 19556612
NIH Roadmap Initiatives; MLSCN; TNAP inhibitors; diverse MOA; compound selectivity
11.  Central Role of Pyrophosphate in Acellular Cementum Formation 
PLoS ONE  2012;7(6):e38393.
Inorganic pyrophosphate (PPi) is a physiologic inhibitor of hydroxyapatite mineral precipitation involved in regulating mineralized tissue development and pathologic calcification. Local levels of PPi are controlled by antagonistic functions of factors that decrease PPi and promote mineralization (tissue-nonspecific alkaline phosphatase, Alpl/TNAP), and those that increase local PPi and restrict mineralization (progressive ankylosis protein, ANK; ectonucleotide pyrophosphatase phosphodiesterase-1, NPP1). The cementum enveloping the tooth root is essential for tooth function by providing attachment to the surrounding bone via the nonmineralized periodontal ligament. At present, the developmental regulation of cementum remains poorly understood, hampering efforts for regeneration. To elucidate the role of PPi in cementum formation, we analyzed root development in knock-out (−/−) mice featuring PPi dysregulation.
Excess PPi in the Alpl−/− mouse inhibited cementum formation, causing root detachment consistent with premature tooth loss in the human condition hypophosphatasia, though cementoblast phenotype was unperturbed. Deficient PPi in both Ank and Enpp1−/− mice significantly increased cementum apposition and overall thickness more than 12-fold vs. controls, while dentin and cellular cementum were unaltered. Though PPi regulators are widely expressed, cementoblasts selectively expressed greater ANK and NPP1 along the root surface, and dramatically increased ANK or NPP1 in models of reduced PPi output, in compensatory fashion. In vitro mechanistic studies confirmed that under low PPi mineralizing conditions, cementoblasts increased Ank (5-fold) and Enpp1 (20-fold), while increasing PPi inhibited mineralization and associated increases in Ank and Enpp1 mRNA.
Results from these studies demonstrate a novel developmental regulation of acellular cementum, wherein cementoblasts tune cementogenesis by modulating local levels of PPi, directing and regulating mineral apposition. These findings underscore developmental differences in acellular versus cellular cementum, and suggest new approaches for cementum regeneration.
PMCID: PMC3366957  PMID: 22675556
12.  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.
PMCID: PMC3179344  PMID: 20684022
13.  Prolonged Survival and Phenotypic Correction of Akp2−/− Hypophosphatasia Mice by Lentiviral Gene Therapy 
Hypophosphatasia (HPP) is an inherited systemic skeletal disease caused by mutations in the gene encoding the tissue-nonspecific alkaline phosphatase (TNALP) isozyme. The clinical severity of HPP varies widely, with symptoms including rickets and osteomalacia. TNALP knockout (Akp2−/−) mice phenotypically mimic the severe infantile form of HPP; that is, TNALP-deficient mice are born with a normal appearance but die by 20 days of age owing to growth failure, hypomineralization, and epileptic seizures. In this study, a lentiviral vector expressing a bone-targeted form of TNALP was injected into the jugular vein of newborn Akp2−/− mice. We found that alkaline phosphatase activity in the plasma of treated Akp2−/− mice increased and remained at high levels throughout the life of the animals. The treated Akp2−/− mice survived for more than 10 months and demonstrated normal physical activity and a healthy appearance. Epileptic seizures were completely inhibited in the treated Akp2−/− mice, and X-ray examination of the skeleton showed that mineralization was significantly improved by the gene therapy. These results show that severe infantile HPP in TNALP knockout mice can be treated with a single injection of lentiviral vector during the neonatal period. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179312  PMID: 20687159
Alkaline phosphatase; Lentiviral vector; Enzyme replacement; Epilepsy; Calcification
14.  Intestinal Alkaline Phosphatase Has Beneficial Effects in Mouse Models of Chronic Colitis 
Inflammatory bowel diseases  2011;17(2):532-542.
The brush border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and is protective against dextran sulfate sodium (DSS)-induced acute injury in rats. The present study evaluated the potential therapeutic role for orally administered calf IAP (cIAP) in two independent mouse models of chronic colitis: (1) DSS-induced chronic colitis, and (2) chronic spontaneous colitis in Wiskott-Aldrich Syndrome protein (WASP) deficient (knockout) mice that is accelerated by irradiation.
The wild-type (WT) and IAP knockout (IAP-KO) mice received 4 cycles of 2% DSS ad libitum for 7 days. Each cycle was followed by a 7-day DSS-free interval during which mice received either cIAP or vehicle in the drinking water. The WASP-KO mice received either vehicle or cIAP for 6 weeks beginning on the day of irradiation.
Microscopic colitis scores of DSS-treated IAP-KO mice were higher than DSS-treated WT mice (52 ± 3.8 vs. 28.8 ± 6.6, respectively, P < 0.0001). cIAP treatment attenuated the disease in both groups (KO = 30.7 ± 6.01, WT = 18.7 ± 5.0, P < 0.05). In irradiated WASP-KO mice cIAP also attenuated colitis compared to control groups (3.3 ± 0.52 vs. 6.2 ± 0.34, respectively, P < 0.001). Tissue myeloperoxidase activity and pro-inflammatory cytokines were significantly decreased by cIAP treatment.
Endogenous IAP appears to play a role in protecting the host against chronic colitis. Orally administered cIAP exerts a protective effect in two independent mouse models of chronic colitis and may represent a novel therapy for human IBD.
PMCID: PMC3154118  PMID: 20645323
DSS-induced chronic colitis; WASP-KO and spontaneous chronic colitis; inflammatory bowel disease; gut mucosal defense; lipopolysaccharides
15.  Inhibition of PHOSPHO1 activity results in impaired skeletal mineralization during limb development of the chick 
Bone  2010;46(4):1146-1155.
PHOSPHO1 is a bone specific phosphatase implicated in the initiation of inorganic phosphate generation for matrix mineralization. The control of mineralization is attributed to the actions of tissue-non specific alkaline phosphatase (TNAP). However, matrix vesicles (MVs) containing apatite crystals are present in patients with hypophosphatasia as well as TNAP null (Akp2-/-) mice. It is therefore likely that other phosphatases work with TNAP to regulate matrix mineralization. Although PHOSPHO1 and TNAP expression is associated with MVs, it is not known if PHOSPHO1 and TNAP are co-expressed during the early stages of limb development. Furthermore the functional in-vivo role of PHOSPHO1 in matrix mineralization has yet to be established. Here, we studied the temporal expression and functional role of PHOSPHO1 within chick limb bud mesenchymal micromass cultures and also in wild-type and talpid3 chick mutants. These mutants are characterized by defective hedgehog signalling and the absence of endochondral mineralization. The ability of in-vitro micromass cultures to differentiate and mineralize their matrix was temporally associated with increased expression of PHOSPHO1 and TNAP. Comparable changes in expression were noted in developing embryonic legs (developmental stages 23–36HH). Micromass cultures treated with lansoprazole, a small-molecule inhibitor of PHOSPHO1 activity, or FGF2, an inhibitor of chondrocyte differentiation, resulted in reduced alizarin red staining (P<0.05). FGF2 treatment also caused a reduction in PHOSPHO1 (P<0.001) and TNAP (P<0.001) expression. Expression analysis by whole mount RNA in-situ hybridization, correlated with qPCR micromass data and demonstrated the existence of a tightly regulated pattern of Phospho1 and Tnap expression which precedes mineralization. Treatment of developing embryos for 5-days with lansoprazole completely inhibited mineralization of all leg and wing long bones as assessed by alcian blue/alizarin red staining. Furthermore, long bones of the talpid3 chick mutant did not express Phospho1 or Tnap whereas flat bones mineralized normally and expressed both phosphatases. In conclusion, this study has disclosed that PHOSPHO1 expression mirrors that of TNAP during embryonic bone development and that PHOSPHO1 contributes to bone mineralization in developing chick long bones.
PMCID: PMC2842458  PMID: 20053388
PHOSPHO1; Alkaline Phosphatase; chondrocyte differentiation; mineralization; talpid3
16.  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.
PMCID: PMC3153326  PMID: 19874193
biomineralization; knockout mice; calcification; pyrophosphatases; atpases
17.  Alkaline Phosphatase Knock-Out Mice Recapitulate the Metabolic and Skeletal Defects of Infantile Hypophosphatasia* 
Hypophosphatasia is an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and skeletal disease due to impaired mineralization of cartilage and bone matrix. We investigated two independently generated TNSALP gene knock-out mouse strains as potential models for hypophosphatasia. Homozygous mice (−/−) had < 1% of wild-type plasma TNSALP activity; heterozygotes had the predicted mean of ~50%. Phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5′-phosphate are putative natural substrates for TNSALP and all were increased endogenously in the knock-out mice. Skeletal disease first appeared radiographically at ~10 days of age and featured worsening rachitic changes, osteopenia, and fracture. Histologic studies revealed developmental arrest of chondrocyte differentiation in epiphyses and in growth plates with diminished or absent hypertrophic zones. Progressive osteoidosis from defective skeletal matrix mineralization was noted but not associated with features of secondary hyperparathyroidism. Plasma and urine calcium and phosphate levels were unremarkable. Our findings demonstrate that TNSALP knock-out mice are a good model for the infantile form of hypophosphatasia and provide compelling evidence for an important role for TNSALP in postnatal development and mineralization of the murine skeleton.
PMCID: PMC3049802  PMID: 10620060
18.  Discovery and Validation of a Series of Aryl Sulfonamides as Selective Inhibitors of Tissue-Nonspecific Alkaline Phosphatase (TNAP) 
Journal of medicinal chemistry  2009;52(21):6919-6925.
We report the characterization and optimization of drug-like small molecule inhibitors of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme critical for the regulation of extracellular matrix calcification during bone formation and growth. High-throughput screening (HTS) of a small molecule library led to the identification of arylsulfonamides as potent and selective inhibitors of TNAP. Critical structural requirements for activity were determined, and the compounds were subsequently profiled for in vitro activity and bioavailability parameters including metabolic stability and permeability. The plasma levels following subcutaneous administration of a member of the lead series in rat was determined, demonstrating the potential of these TNAP inhibitors as systemically active therapeutic agents to target various diseases involving soft tissue calcification. A representative member of the series was also characterized in mechanistic and kinetic studies.
PMCID: PMC2783186  PMID: 19821572
19.  Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms 
Bone  2009;45(5):987-993.
Three circulating human bone alkaline phosphatase (BALP) isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. The present study was designed to correlate differing glycosylation patterns of each BALP isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant isoforms of the tissue-nonspecific ALP (TNALP) isozyme, i.e., TNALP-flag, used extensively for mutation analysis of hypophosphatasia mutations and sALP-FcD10, a chimeric enzyme recently used as therapeutic drug in a mouse model of infantile hypophosphatasia.
The BALP isoforms were prepared from human osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-nitrophenylphosphate (pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic pyrophosphate (PPi), pyridoxal 5′-phosphate (PLP), and phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by lectin binding and precipitation.
The kcat/KM was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD10 was similar to the activity of the human BALP isoforms. The BALP isoforms differed in their lectin-binding properties and dose-dependent lectin precipitation, which also demonstrated differences between native and denatured BALP isoforms. The observed differences in lectin specificity were attributed to N-linked carbohydrates.
In conclusion, we demonstrate significantly different catalytic properties among the BALP isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP isoforms or for the recombinant TNALP isoforms. The TNALP-flag and the sALP-FcD10 isoforms faithfully mimic the biological properties of the human BALP isoforms in vivo validating the use of these recombinant enzymes in studies aimed at dissecting the pathophysiology and treating hypophosphatasia.
PMCID: PMC2753715  PMID: 19631305
bone turnover; glycosylation; hypophosphatasia; kinetics; pyrophosphate
20.  Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics* 
The Journal of Biological Chemistry  2010;285(10):7598-7609.
We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5′-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5′-monophosphate, and PPi by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5′-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PPi were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PPi by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.
PMCID: PMC2844207  PMID: 20048161
Calcium/ATPase; Cell/Compartmentation; Enzymes/ATPases; Membrane/Enzymes; Membrane/Reconstitution; Methods/Liposomes; Subcellular Organelles/Vesicles
21.  Design and synthesis of pyrazole derivatives as potent and selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP) 
Tissue-nonspecific alkaline phosphatase (TNAP) plays a central role in regulating extracellular matrix calcification during bone formation and growth. High throughput screening (HTS) for small molecule TNAP inhibitors led to the identification of hits in the sub-micromolar potency range. We report the design, synthesis and in vitro evaluation of a series of pyrazole derivatives of a screening hit which are potent TNAP inhibitors exhibiting IC50 values as low as 5 nM. A representative of the series was characterized in kinetic studies and determined to have a mode of inhibition not previously observed for TNAP inhibitors.
PMCID: PMC2752324  PMID: 19038545
22.  Enzyme Replacement Therapy for Murine Hypophosphatasia* 
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.
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.
Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2−/− mice.
PMCID: PMC2652241  PMID: 18086009
alkaline phosphatase; calcification; epilepsy; osteomalacia; rickets
23.  Role of Cytochrome c in Apoptosis: Increased Sensitivity to Tumor Necrosis Factor Alpha Is Associated with Respiratory Defects but Not with Lack of Cytochrome c Release▿  
Molecular and Cellular Biology  2007;27(5):1771-1783.
Although the role of cytochrome c in apoptosis is well established, details of its participation in signaling pathways in vivo are not completely understood. The knockout for the somatic isoform of cytochrome c caused embryonic lethality in mice, but derived embryonic fibroblasts were shown to be resistant to apoptosis induced by agents known to trigger the intrinsic apoptotic pathway. In contrast, these cells were reported to be hypersensitive to tumor necrosis factor alpha (TNF-α)-induced apoptosis, which signals through the extrinsic pathway. Surprisingly, we found that this cell line (CRL 2613) respired at close to normal levels because of an aberrant activation of a testis isoform of cytochrome c, which, albeit expressed at low levels, was able to replace the somatic isoform for respiration and apoptosis. To produce a bona fide cytochrome c knockout, we developed a mouse knockout for both the testis and somatic isoforms of cytochrome c. The mouse was made viable by the introduction of a ubiquitously expressed cytochrome c transgene flanked by loxP sites. Lung fibroblasts in which the transgene was deleted showed no cytochrome c expression, no respiration, and resistance to agents that activate the intrinsic and to a lesser but significant extent also the extrinsic pathways. Comparison of these cells with lines with a defective oxidative phosphorylation system showed that cells with defective respiration have increased sensitivity to TNF-α-induced apoptosis, but this process was still amplified by cytochrome c. These studies underscore the importance of oxidative phosphorylation and apoptosome function to both the intrinsic and extrinsic apoptotic pathways.
PMCID: PMC1820455  PMID: 17210651
24.  Accelerated Fat Absorption in Intestinal Alkaline Phosphatase Knockout Mice† 
Molecular and Cellular Biology  2003;23(21):7525-7530.
Intestinal alkaline phosphatase (IAP) is the most ancestral of the tissue-specific members of the AP gene family. Several studies have suggested an absorptive function for IAP, but in vivo data to this effect have been lacking. We inactivated the mouse IAP gene in embryo-derived stem cells and generated mice homozygous for the null mutation. The mice were macroscopically and histologically normal and fertile and showed no difference from the wild-type controls under normal laboratory conditions. However, when maintained long-term on a high-fat diet, the IAP-deficient mice showed faster body weight gain than did control animals. Histological examination revealed an accelerated transport of fat droplets through the intestinal epithelium and elevation of serum triglyceride levels in the IAP-deficient mice compared to wild-type mice. Our study suggests that IAP participates in a rate-limiting step regulating fat absorption.
PMCID: PMC207564  PMID: 14560000
25.  Testis-Specific Cytochrome c-Null Mice Produce Functional Sperm but Undergo Early Testicular Atrophy 
Molecular and Cellular Biology  2002;22(15):5554-5562.
Differentiating male germ cells express a testis-specific form of cytochrome c (Cyt cT) that is distinct from the cytochrome c expressed in somatic cells (Cyt cS). To examine the role of Cyt cT in germ cells, we generated mice null for Cyt cT. Homozygous Cyt cT−/− pups were statistically underrepresented (21%) but developed normally and were fertile. However, spermatozoa isolated from the cauda epididymis of Cyt cT-null animals were less effective in fertilizing oocytes in vitro and contain reduced levels of ATP compared to wild-type sperm. Sperm from Cyt cT-null mice contained a greater number of immotile spermatozoa than did samples from control mice, i.e., 53.1% ± 13.7% versus 33.2% ± 10.3% (P < 0.0001) for vas deferens sperm and 40.1% ± 9.6% versus 33.2% ± 7.5% (P = 0.0104) for epididymal sperm. Cyt cT-null mice often exhibit early atrophy of the testes after 4 months of age, losing germ cells as a result of increased apoptosis. However, no difference in the activation of caspase-3, -8, or -9 was detected between the Cyt cT−/− testes and controls. Our data indicate that the Cyt cT-null testes undergo early atrophy equivalent to that which occurs during aging as a consequence of a reduction in oxidative phosphorylation.
PMCID: PMC133957  PMID: 12101247

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