Nucleotide Pyrophosphatase Phosphodiesterase Research Articles

Spectral, quantum chemical, X-ray crystallographic, Hirshfeld surface, energy framework, and molecular docking investigations of 4-acetylphenyl 4-methylbenzenesulfonate (APMBS)

4-acetylphenyl 4-methylbenzenesulfonate (C15H14O4S), a tosyl ester derivative, has been synthesized and characterized by spectroscopic and single crystal X-ray diffraction (SCXRD) techniques. The Hartree-Fock (HF) and the Density functional theory (DFT) methods [B3LYP/6-311 ++ G (d, p) basis set] have been employed to obtain the optimized structural geometry. The structure exhibits C—H···O type of intra- and intermolecular interactions, besides C—O···π and C—H···π interactions. The DFT calculation facilitated the exploration of frontier molecular orbitals (FMOs), Mulliken charges, density of states (DOS), and molecular electrostatic potential (MEP). A detailed Hirshfeld surface analysis has been made. Bader's atoms-in-molecules quantum theory (QTAIM) and non-covalent interaction index analysis as based on reduced density gradient (NCI-RDG) led to a quantitative description of the nature of interactions present in the crystal structure. The molecular docking has been performed against Nucleotide pyrophosphatase/phosphodiesterase (NPP) enzyme.

ENPP1’s regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating STING signaling

The metazoan innate immune second messenger 2′3′-cGAMP is present both inside and outside cells. However, only extracellular cGAMP can be negatively regulated by the extracellular hydrolase ENPP1. Here, we determine whether ENPP1’s regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating stimulator of interferon genes (STING) signaling. We identified ENPP1H362A, a point mutation that cannot degrade the 2′-5′ linkage in cGAMP while maintaining otherwise normal function. The selectivity of this histidine is conserved down to bacterial nucleotide pyrophosphatase/phosphodiesterase (NPP), allowing structural analysis and suggesting an unexplored ancient history of 2′-5′ cyclic dinucleotides. Enpp1H362A mice demonstrated that extracellular cGAMP is not responsible for the devastating phenotype in ENPP1-null humans and mice but is responsible for antiviral immunity and systemic inflammation. Our data define extracellular cGAMP as a pivotal STING activator, identify an evolutionarily critical role for ENPP1 in regulating inflammation, and suggest a therapeutic strategy for viral and inflammatory conditions by manipulating ENPP1 activity.

Structure of a nucleotide pyrophosphatase/phosphodiesterase (NPP) from Euphorbia characias latex characterized by small-angle X-ray scattering: clues for the general organization of plant NPPs.

Little information is available concerning the structural features of nucleotide pyrophosphatase/phosphodiesterases (NPPs) of plant origin and the crystal structures of these proteins have not yet been reported. The aim of this study was to obtain insight into these aspects by carrying out a comparative analysis of the sequences of two different fragments of an NPP from the latex of the Mediterranean shrub Euphorbia characias (ELNPP) and by studying the low-resolution structure of the purified protein in solution by means of small-angle X-ray scattering. This is the first structure of a plant NPP in solution that has been reported to date. It is shown that the ELNPP sequence is highly conserved in many other plant species. Of note, the catalytic domains of these plant NPPs have the same highly conserved PDE-domain organization as mammalian NPPs. Moreover, ELNPP is a dimer in solution and this oligomerization state is likely to be common to other plant enzymes.

Synthesis, biological evaluation, and molecular docking study of sulfonate derivatives as nucleotide pyrophosphatase/phosphodiesterase (NPP) inhibitors.

A new series of sulfonate derivatives 1a-zk were synthesized and evaluated as inhibitors of nucleotide pyrophosphatases. Most of the compounds exhibited good to moderate inhibition towards NPP1, NPP2, and NPP3 isozymes. Compound 1m was a potent and selective inhibitor of NPP1 with an IC50 value of 0.387 ± 0.007 µM. However, the most potent inhibitor of NPP3 was found as 1x with an IC50 value of 0.214 ± 0.012 µM. In addition, compound 1e was the most active inhibitor of NPP2 with an IC50 value of 0.659 ± 0.007 µM. Docking studies of the most potent compounds were carried out, and the computational results supported the in vitro results.

Hypothyroidism and hyperthyroidism change ectoenzyme activity in rat platelets.

The purinergic system has an important role in the regulation of vascular functions. The interference of thyroid hormones in this system and in cardiovascular events has been studied in recent years. However, the mechanisms involved in vascular, purinergic, and oxidative changes in thyroid disorders are not completely understood. Therefore, the present study aimed to assess purinergic enzyme activity in platelets from rats with hypothyroidism and hyperthyroidism induced, respectively, by continuous exposure to methimazole (MMI) at 20 mg/100 mL or L-thyroxine at 1.2 mg/100 mL in drinking water for 1 month. Results showed that rats exposed to L-thyroxine had a significant decrease in NTPDase activity, wherein ATP hydrolysis was 53% lower and ADP hydrolysis was 40% lower. Moreover, ecto-5'-nucleotidase activity was decreased in both groups, by 39% in the hypothyroidism group and by 52% in the hyperthyroidism group. On the other hand, adenosine deaminase (ADA) activity was increased in hyperthyroidism (75%), and nucleotide pyrophosphatase/phosphodiesterase (NPP) activity was increased in animals with hypothyroidism (127%) and those with hyperthyroidism (128%). Our findings suggest that changes in purinergic enzyme and purine levels could contribute to the undesirable effects of thyroid disturbances. Moreover, oxidative stress and, in particular, a high level of ROS production, showed a causal relation with changes in ectonucleotidase activity and nucleotide and nucleoside levels.

Intraperitoneal pyrophosphate treatment reduces renal calcifications in Npt2a null mice.

Mutations in the proximal tubular sodium-dependent phosphate co-transporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis, however the relative contribution of genotype, dietary calcium and phosphate, and modifiers of mineralization such as pyrophosphate (PPi) to the formation of renal mineral deposits is unclear. In the present study, we used Npt2a-/- mice to model the renal calcifications observed in these disorders. We observed elevated urinary excretion of PPi in Npt2a-/- mice when compared to WT mice. Presence of two hypomorphic Extracellular nucleotide pyrophosphatase phosphodiesterase 1 (Enpp1asj/asj) alleles decreased urine PPi and worsened renal calcifications in Npt2a-/- mice. These studies suggest that PPi is a thus far unrecognized factor protecting Npt2a-/- mice from the development of renal mineral deposits. Consistent with this conclusion, we next showed that renal calcifications in these mice can be reduced by intraperitoneal administration of sodium pyrophosphate. If confirmed in humans, urine PPi could therefore be of interest for developing new strategies to prevent the nephrocalcinosis and nephrolithiasis seen in phosphaturic disorders.

Crystal structure of the human alkaline sphingomyelinase provides insights into substrate recognition

Absorption of dietary sphingomyelin (SM) requires its initial degradation into ceramide, a process catalyzed by the intestinal enzyme alkaline sphingomyelinase (alk-SMase, NPP7, ENPP7). alk-SMase belongs to the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, the members of which hydrolyze nucleoside phosphates, phospholipids, and other related molecules. NPP7 is the only paralog that can cleave SM, and its activity requires the presence of bile salts, a class of physiological anionic detergents. To elucidate the mechanism of substrate recognition, we determined the crystal structure of human alk-SMase in complex with phosphocholine, a reaction product. Although the overall fold and catalytic center are conserved relative to other NPPs, alk-SMase recognizes the choline moiety of its substrates via an NPP7-specific aromatic box composed of tyrosine residues. Mutational analysis and enzymatic activity assays identified features on the surface of the protein-a cationic patch and a unique hydrophobic loop-that are essential for accessing SM in bile salt micelles. These results shed new light on substrate specificity determinants within the NPP enzyme family.

Golgi-to-plastid trafficking of proteins through secretory pathway: Insights into vesicle-mediated import toward the plastids

ABSTRACTThe diversity of protein targeting pathways to plastids and their regulation in response to developmental and metabolic status is a key issue in the regulation of cellular function in plants. The general import pathways that target proteins into and across the plastid envelope with changes in gene expression are critical for plant development by regulating the response to physiological and metabolic changes within the cell. Glycoprotein targeting to complex plastids involves routing through the secretory pathway, among others. However, the mechanisms of trafficking via this system remain poorly understood. The present article discusses our results in site-specific N-glycosylation of nucleotide pyrophosphatase/phosphodiesterases (NPPs) glycoproteins and highlights protein delivery in Golgi/plastid pathway via the secretory pathway. Furthermore, we outline the hypotheses that explain the mechanism for importing vesicles trafficking with nucleus-encoded proteins into plastids.

N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate that trans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice.

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1–NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)–Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2–GFP and NPP6–GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER–Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.

1,25-Dihydroxyvitamin D3 and extracellular calcium promote mineral deposition via NPP1 activity in a mature osteoblast cell line MLO-A5

While vitamin D supplementation is common, the anabolic mechanisms that improve bone status are poorly understood. Under standard mineralising conditions including media ionised calcium of 1.1 mM, 1,25-dihydroxyvitamin D3 (1,25D) enhanced differentiation and mineral deposition by the mature osteoblast/pre-osteocyte cell line, MLO-A5. This effect was markedly increased with a higher ionised calcium level (1.5 mM). Gene expression analyses revealed that 1,25D-induced mineral deposition was associated with induction of Enpp1 mRNA, coding for nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) and NPP1 protein levels. Since MLO-A5 cells express abundant alkaline phosphatase that was not further modified by 1,25D treatment or exposure to increased calcium, this finding suggested that the NPP1 production of pyrophosphate (PPi) may provide alkaline phosphatase with substrate for the generation of inorganic phosphate (Pi). Consistent with this, co-treatment with Enpp1 siRNA or a NPP1 inhibitor, PPADS, abrogated 1,25D-induced mineral deposition. These data demonstrate that 1,25D stimulates osteoblast differentiation and mineral deposition, and interacts with the extracellular calcium concentration. 1,25D regulates Enpp1 expression, which presumably, in the context of adequate tissue non-specific alkaline phosphatase activity, provides Pi to stimulate mineralisation. Our findings suggest a mechanism by which vitamin D with adequate dietary calcium can improve bone mineral status.

Ecto-nucleotidase inhibitors: recent developments in drug discovery.

Ecto-nucleotidases are nucleotide metabolizing enzymes that are divided into four different families; nucleoside triphosphate diphosphohydrolases (NTPDases), ecto-5'-nucleotidase (ecto-5'-NT), nucleotide pyrophosphatase/phosphodiesterases (NPPs), and alkaline phosphatases (APs). These enzymes are responsible for the hydrolysis of nucleotidases (nucleoside 5'-triphosphates, 5'-diphosphates and 5'-monophosphates). Ecto-nucleotidases modulate P1- and P2-receptor-mediated signaling. Alterations in extracellular nucleotide and adenosine level can increase or decrease P1 and P2 activity. Potent and selective ligands for certain ectonucleotidase are important as pharmacological tools to investigate the (patho)physiological roles of these enzymes. Furthermore, such ligands are required to study their potential as novel drugs, e.g., as immunomodulatory agents, for the treatment of cancer, cardiovascular or central nervous system disorders. Hence, this review aims to provide an overview of ecto-nucleotidases inhibitors developed so far.

Changes of activity and isoforms of alkaline sphingomyelinase (nucleotide pyrophosphatase phosphodiesterase 7) in bile from patients undergoing endoscopic retrograde cholangiopancreatography.

BackgroundAlkaline sphingomyelinase (NPP7) is an ecto-enzyme expressed in intestinal mucosa, which hydrolyses sphingomyelin (SM) to ceramide and inactivates platelet activating factor. It is also expressed in human liver and released in the bile. The enzyme may have anti-tumour and anti-inflammatory effects in colon and its levels are decreased in patients with colon cancer and ulcerative colitis. Active NPP7 is translated from a transcript of 1.4 kb, whereas an inactive form from a 1.2 kb mRNA was found in colon and liver cancer cell lines. While the roles of NPP7 in colon cancer have been intensively studied, less is known about the function and implications of NPP7 in the bile. The present study examines the changes of NPP7 in bile of patients with various hepatobiliary diseases.MethodsBile samples were obtained at endoscopic retrograde cholangiopancreatography (ERCP) in 59 patients with gallstone, other benign disease, tumour, and primary sclerosing cholangitis (PSC). The NPP7 activity was determined. The appearance of the 1.4 and 1.2 kb products in the bile was examined by Western blot. The results were correlated to the diseases and also plasma bilirubin and alkaline phosphatase.ResultsNPP7 activity in the tumour group was significantly lower than in the gallstone group (p < 0.05). The activity in the tumour plus PSC group was also lower than in gallstone plus other benign disease group (p < 0.05). Within the tumour group NPP7 activity was lowest in cholangiocarcinoma patients, being only 19% of that in gallstone patients. Bilirubin correlated inversely to NPP7 and was higher in the tumour than in the gallstone group. Western blot identified both the 1.4 kb and the 1.2 kb products in most bile samples. The density ratio for the 1.4/1.2 kb products correlated to NPP7 activity significantly. Two patients (one PSC and one cholangiocarcinoma) lacking NPP7 activity had only the 1.2 kb form in bile.ConclusionNPP7 activity and the ratio of 1.4/1.2 kb products in bile are significantly decreased in malignancy, particularly in cholangiocarcinoma. The implications of the finding in diagnosis of cholangiocarcinoma and 1.2 kb product in hepatobiliary diseases require further investigation.

Site-Directed Mutagenesis Maps Interactions That Enhance Cognate and Limit Promiscuous Catalysis by an Alkaline Phosphatase Superfamily Phosphodiesterase

Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >10⁶-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 10⁴-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH₃). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²⁺ bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition of side chains that interact with the substrate atoms and substituents that project away from the Zn²⁺ bimetallo core.

Rolofylline, an adenosine A1 receptor antagonist, inhibits osteoclast differentiation as an inverse agonist

Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5'-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A1 receptors (A1 R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A1 R agonists neither affect basal osteoclastogenesis nor do they reverse A1 R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A1 R antagonist-mediated inhibition, and, when we saw no effect, determined whether A1 R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation. Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand-macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa. Rolofylline inhibited osteoclast formation in a dose-dependent manner (IC50 = 20-70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold. Based on these findings, we hypothesize that the A1 R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A1 R antagonists act as inverse agonists to release A1 R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A1 R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation.

Nucleotide Pyrophosphatase/Phosphodiesterase 1 Exerts a Negative Effect on Starch Accumulation and Growth in Rice Seedlings under High Temperature and CO2 Concentration Conditions

Nucleotide pyrophosphatase/phosphodiesterase (NPP) is a widely distributed enzymatic activity occurring in both plants and mammals that catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides. Unlike mammalian NPPs, the physiological function of plant NPPs remains largely unknown. Using a complete rice NPP1-encoding cDNA as a probe, in this work we have screened a rice shoot cDNA library and obtained complete cDNAs corresponding to six NPP genes (NPP1–NPP6). As a first step to clarify the role of NPPs, recombinant NPP1, NPP2 and NPP6 were purified from transgenic rice cells constitutively expressing NPP1, NPP2 and NPP6, respectively, and their enzymatic properties were characterized. NPP1 and NPP6 exhibited hydrolytic activities toward ATP, UDP-glucose and the starch precursor molecule, ADP-glucose, whereas NPP2 did not recognize nucleotide sugars as substrates, but hydrolyzed UDP, ADP and adenosine 5′-phosphosulfate. To gain insight into the physiological function of rice NPP1, an npp1 knockout mutant was characterized. The ADP-glucose hydrolytic activities in shoots of npp1 rice seedlings were 8% of those of the wild type (WT), thus indicating that NPP1 is a major determinant of ADP-glucose hydrolytic activity in rice shoots. Importantly, when seedlings were cultured at 160 Pa CO2 under a 28°C/23°C (12 h light/12 h dark) regime, npp1 shoots and roots were larger than those of wild-type (WT) seedlings. Furthermore, the starch content in the npp1 shoots was higher than that of WT shoots. Growth and starch accumulation were also enhanced under an atmospheric CO2 concentration (40 Pa) when plants were cultured under a 33°C/28°C regime. The overall data strongly indicate that NPP1 exerts a negative effect on plant growth and starch accumulation in shoots, especially under high CO2 concentration and high temperature conditions.

Hits of a High-Throughput Screen Identify the Hydrophobic Pocket of Autotaxin/Lysophospholipase D As an Inhibitory Surface

Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and ∼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC₅₀ vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5'-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1-3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.

Association of NPP1 polymorphism with postoperative progression of ossification of the posterior longitudinal ligament in Chinese patients

Ossification of the posterior longitudinal ligament (OPLL) is a condition of the spine that can cause paralysis by compressing the spinal cord. The aim of this study was to evaluate the possible role of nucleotide pyrophosphatase phosphodiesterase 1 gene (NPP1) polymorphism in the etiology and pathology of the OPLL in Chinese patients. DNA from patients with OPLL (N = 95) and without OPLL (N = 90) were genotyped for 4 NPP1 single-nucleotide polymorphisms (SNPs): A533C, C973T, IVS15-14T→C, and IVS20-11delT. An association study evaluated the relationship between specific SNP genotypes and susceptibility. We also evaluated whether genotypes of these SNPs were associated with disease severity and the probability of disease progression after surgery. The C973T and IVS15-14T SNPs were associated with the existence of the disease. The TT genotypes of C973T and IVS15→14T as well wild-type IVS20 (lack of deletion) were associated with more severe disease. Patients with the T deletion of IVS20 or the AA genotype of A533C had an approximately 3 times greater chance of not having than having disease progression after surgery. We concluded that the 4 SNPs analyzed appeared to have different effects on the etiology and pathology of OPLL. To our knowledge, this study is the first to investigate the relationship between these SNPs and disease progression after surgery. Our findings suggest that the presence of specific genotypes of the IVS20-11delT and A533C SNPs may predict disease outcome after surgical intervention.

Correction: Altered Bone Development and an Increase in FGF-23 Expression in Enpp1−/−Mice

Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PPi), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1 mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1 mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1 mice (P,0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1 tibiae and femurs from 22-week-old mice (P,0.05). Circulating phosphate and calcium levels were reduced (P,0.05) in the Enpp1 null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P,0.05) in Enpp1 mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1 mice at 22 weeks of age (P,0.05). Fgf23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1 mice compared to controls. These results indicate that Enpp1 mice are characterized by severe disruption to the architecture and mineralization of longbones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization. Citation: Mackenzie NCW, Zhu D, Milne EM, van ’t Hof R, Martin A, et al. (2012) Altered Bone Development and an Increase in FGF-23 Expression in Enpp1 Mice. PLoS ONE 7(2): e32177. doi:10.1371/journal.pone.0032177 Editor: Marlon R. Schneider, University of Munich, Germany Received October 11, 2011; Accepted January 22, 2012; Published February 16, 2012 Copyright: 2012 Mackenzie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an Institute Strategic Programme Grant and Institute Career Path Fellowship funding from the Biotechnology and Biological Sciences Research Council (BBSRC; www.bbsrc.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: vicky.macrae@roslin.ed.ac.uk

Decreased levels of nucleotide pyrophosphatase phosphodiesterase 1 are associated with cartilage calcification in osteoarthritis and trigger osteoarthritic changes in mice

ObjectiveTo analyse the function of nucleotide pyrophosphatase phosphodiesterase (NPP1), a member of the pyrophosphate pathway, in osteoarthritis (OA).MethodsmRNA expression of NPP1, ANK ankylosing protein and tissue non-specific alkaline phosphatase was...

Altered bone development and an increase in FGF-23 expression in Enpp1(-/-) mice.

Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PPi), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1−/−) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1−/− mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1−/− mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1−/− mice (P<0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1−/− tibiae and femurs from 22-week-old mice (P<0.05). Circulating phosphate and calcium levels were reduced (P<0.05) in the Enpp1−/− null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P<0.05) in Enpp1−/− mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps™) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1−/− mice at 22 weeks of age (P<0.05). Fgf-23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1−/− mice compared to controls. These results indicate that Enpp1−/− mice are characterized by severe disruption to the architecture and mineralization of long-bones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization.