Concerted Regulation of Inorganic Pyrophosphate and Osteopontin by Akp2, Enpp1, and Ank
2004; Elsevier BV; Volume: 164; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63208-7
ISSN1525-2191
AutoresDympna Harmey, Lovisa Hessle, Sonoko Narisawa, Kristen Johnson, Robert Terkeltaub, José Luís Millán,
Tópico(s)Bone and Dental Protein Studies
ResumoTissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Deletion of the TNAP gene (Akp2) in mice results in hypophosphatasia characterized by elevated levels of PPi and poorly mineralized bones, which are rescued by deletion of nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) that generates PPi. Mice deficient in NPP1 (Enpp1−/−), or defective in the PPi channeling function of ANK (ank/ank), have decreased levels of extracellular PPi and are hypermineralized. Given the similarity in function between ANK and NPP1 we crossbred Akp2−/− mice to ank/ank mice and found a partial normalization of the mineralization phenotypes and PPi levels. Examination of Enpp1−/− and ank/ank mice revealed that Enpp1−/− mice have a more severe hypermineralized phenotype than ank/ank mice and that NPP1 but not ANK localizes to matrix vesicles, suggesting that failure of ANK deficiency to correct hypomineralization in Akp2−/− mice reflects the lack of ANK activity in the matrix vesicle compartment. We also found that the mineralization inhibitor osteopontin (OPN) was increased in Akp2−/−, and decreased in ank/ank mice. PPi and OPN levels were normalized in [Akp2−/−; Enpp1−/−] and [Akp2−/−; ank/ank] mice, at both the mRNA level and in serum. Wild-type osteoblasts treated with PPi showed an increase in OPN, and a decrease in Enpp1 and Ank expression. Thus TNAP, NPP1, and ANK coordinately regulate PPi and OPN levels. The hypomineralization observed in Akp2−/− mice arises from the combined inhibitory effects of PPi and OPN. In contrast, NPP1 or ANK deficiencies cause a decrease in the PPi and OPN pools that leads to hypermineralization. Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Deletion of the TNAP gene (Akp2) in mice results in hypophosphatasia characterized by elevated levels of PPi and poorly mineralized bones, which are rescued by deletion of nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) that generates PPi. Mice deficient in NPP1 (Enpp1−/−), or defective in the PPi channeling function of ANK (ank/ank), have decreased levels of extracellular PPi and are hypermineralized. Given the similarity in function between ANK and NPP1 we crossbred Akp2−/− mice to ank/ank mice and found a partial normalization of the mineralization phenotypes and PPi levels. Examination of Enpp1−/− and ank/ank mice revealed that Enpp1−/− mice have a more severe hypermineralized phenotype than ank/ank mice and that NPP1 but not ANK localizes to matrix vesicles, suggesting that failure of ANK deficiency to correct hypomineralization in Akp2−/− mice reflects the lack of ANK activity in the matrix vesicle compartment. We also found that the mineralization inhibitor osteopontin (OPN) was increased in Akp2−/−, and decreased in ank/ank mice. PPi and OPN levels were normalized in [Akp2−/−; Enpp1−/−] and [Akp2−/−; ank/ank] mice, at both the mRNA level and in serum. Wild-type osteoblasts treated with PPi showed an increase in OPN, and a decrease in Enpp1 and Ank expression. Thus TNAP, NPP1, and ANK coordinately regulate PPi and OPN levels. The hypomineralization observed in Akp2−/− mice arises from the combined inhibitory effects of PPi and OPN. In contrast, NPP1 or ANK deficiencies cause a decrease in the PPi and OPN pools that leads to hypermineralization. Bone mineralization is a tightly controlled process, the initial stages of which begin in chondrocyte- and osteoblast-derived matrix vesicles (MVs).1Anderson HC Molecular biology of matrix vesicles.Clin Orthop Res. 1995; 314: 266-280PubMed Google Scholar, 2Boskey AL Boyan BD Schwartz Z Matrix vesicles promote mineralization in a gelatin gel.Calcif Tissue Int. 1997; 60: 309-315Crossref PubMed Scopus (51) Google Scholar MVs contain calcium and inorganic phosphate ions (Pi) and it is within the MVs that the first crystals of hydroxyapatite are formed. These crystals grow and are exposed to the extracellular environment because of protrusion through the MV membrane. Exposure of the hydroxyapatite crystals to the extracellular milieu further enables growth and proliferation of the crystals.3Anderson HC Vesicles associated with calcification in the matrix of epiphyseal cartilage.J Cell Biol. 1969; 41: 59-72Crossref PubMed Scopus (750) Google Scholar, 4Ali SY Sajdera SW Anderson HC Isolation and characterization of calcifying matrix vesicles from epiphyseal cartilage.Proc Natl Acad Sci USA. 1970; 67: 1513-1520Crossref PubMed Scopus (437) Google Scholar, 5Akisaka T Gay CV The plasma membrane and matrix vesicles of mouse growth plate chondrocytes during differentiation as revealed in freeze-fracture replicas.Am J Anat. 1985; 173: 269-286Crossref PubMed Scopus (18) Google Scholar Inorganic pyrophosphate (PPi) antagonizes the ability of Pi to crystallize with calcium to form hydroxyapatite and thereby suppresses hydroxyapatite crystal propagation. For normal mineral deposition to proceed, a tight balance is required between the levels of extracellular Pi and PPi. Three molecules have been identified as central regulators of extracellular PPi and Pi levels (Table 1), ie, tissue-nonspecific alkaline phosphatase (TNAP), which hydrolyzes PPi,6Moss DW Eaton RH Smith JK Whitby LG Association of inorganic-pyrophosphatase activity with human alkaline-phosphatase preparations.Biochem J. 1967; 102: 53-57Crossref PubMed Scopus (144) Google Scholar, 7Majeska RJ Wuthier RE Studies on matrix vesicles isolated from chick epiphyseal cartilage.. Association of pyrophosphatase and ATPase activities with alkaline phosphatase.Biochim Biophys Acta. 1975; 391: 51-60Crossref PubMed Scopus (232) Google Scholar, 8Meyer JL Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase.Arch Biochem Biophys. 1984; 231: 1-8Crossref PubMed Scopus (177) Google Scholar, 9Rezende A Pizauro J Ciancaglini P Leone F Phosphodiesterase activity is a novel property of alkaline phosphatase from osseous plate.Biochem J. 1994; 301: 517-522Crossref PubMed Scopus (64) Google Scholar, 10Hessle L Johnsson KA Anderson HC Narisawa S Sali A Goding JW Terkeltaub R Millán JL Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization.Proc Natl Acad Sci USA. 2002; 99: 9445-9449Crossref PubMed Scopus (688) Google Scholar nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), which generates PPi from nucleoside triphosphates,11Terkeltaub R Rosenbach M Fong F Goding J Causal link between nucleotide pyrophosphohydrolase overactivity and increased intracellular inorganic pyrophosphate generation demonstrated by transfection of cultured fibroblasts and osteoblasts with plasma cell membrane glycoprotein-1.Arthritis Rheum. 1994; 37: 934-941Crossref PubMed Scopus (73) Google Scholar, 12Johnson K Vaingankar S Chen Y Moffa A Goldring M Sano K Jin-Hua P Sali A Goding J Terkeltaub R Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes.Arthritis Rheum. 1999; 42: 1986-1997Crossref PubMed Scopus (108) Google Scholar, 13Johnson K Moffa A Chen Y Pritzker K Goding J Terkeltaub R Matrix vesicle plasma membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells.J Bone Miner Res. 1999; 14: 883-892Crossref PubMed Scopus (113) Google Scholar and the multiple-pass transmembrane protein ANK, which mediates intracellular to extracellular channeling of PPi.14Hakim FT Cranley R Brown KS Eanes ED Harne L Oppenheim JJ Hereditary joint disorder in progressive ankylosis (ank/ank) mice. I. Association of calcium hydroxyapatite deposition with inflammatory arthropathy.Arthritis Rheum. 1994; 27: 1411-1420Crossref Scopus (71) Google Scholar, 15Ho AM Johnson MD Kingsley DM Role of mouse ank gene in control of tissue calcification and arthritis.Science. 2000; 289: 265-269Crossref PubMed Scopus (550) Google ScholarTable 1Glossary of the Genes and Proteins Studied in This ArticleGene symbolProtein symbolCommon name; synonymsAkp2TNAPTissue-nonspecific alkaline phosphatase; TNSALPEnpp1NPP1Nucleotide pyrophosphatase phosphodiesterase-1; PC-1Ank (wild-type allele) and ank (mutated allele)ANKAnkylosis proteinOpnOPNOsteopontin Open table in a new tab TNAP is an important promoter of mineralization because it catalyzes the hydrolysis of PPi thereby decreasing the concentrations of this calcification inhibitor, while concomitantly increasing the levels of Pi. Mice in which the TNAP gene has been inactivated (Akp2−/−) mimic the most severe form of hypophosphatasia, a disease characterized by rickets, osteomalacia, spontaneous bone fractures, and increased PPi levels.16Whyte MP Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization.Endocrine Rev. 1994; 15: 439-461PubMed Google Scholar, 17Whyte MP The Metabolic and Molecular Bases of Inherited Disease.in: Scriver CR Beaudet AL Sly WS Valle D Childs B Kinzler KW Vogelstein B MacGraw-Hill, New York2001: 5313-5329Google Scholar Akp2−/− skeletal preparations show poor mineralization in the parietal bones, scapulae, vertebral bones, and ribs.18Waymire KG Mahuren JD Jaje JM Guilarte TR Coburn SP McGregor GR Mice lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B-6.Nat Genet. 1995; 11: 45-51Crossref PubMed Scopus (339) Google Scholar, 19Narisawa N Frölander N Millán JL Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia.Dev Dyn. 1997; 208: 432-446Crossref PubMed Scopus (326) Google Scholar, 20Fedde KN Blair L Silverstein J Coburn SP Ryan LM Weinstein RS Waymire K Narisawa S Millán JL MacGregor GR Whyte MP Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia.J Bone Miner Res. 1999; 14: 2015-2026Crossref PubMed Scopus (323) Google Scholar NPP1 serves as a physiological inhibitor of calcification, at least in part by generating PPi.11Terkeltaub R Rosenbach M Fong F Goding J Causal link between nucleotide pyrophosphohydrolase overactivity and increased intracellular inorganic pyrophosphate generation demonstrated by transfection of cultured fibroblasts and osteoblasts with plasma cell membrane glycoprotein-1.Arthritis Rheum. 1994; 37: 934-941Crossref PubMed Scopus (73) Google Scholar, 12Johnson K Vaingankar S Chen Y Moffa A Goldring M Sano K Jin-Hua P Sali A Goding J Terkeltaub R Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes.Arthritis Rheum. 1999; 42: 1986-1997Crossref PubMed Scopus (108) Google Scholar, 13Johnson K Moffa A Chen Y Pritzker K Goding J Terkeltaub R Matrix vesicle plasma membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells.J Bone Miner Res. 1999; 14: 883-892Crossref PubMed Scopus (113) Google Scholar, 21Terkeltaub R Inorganic pyrophosphate generation and disposition in pathophysiology.Am J Physiol. 2001; 281: C1-C11Google Scholar In human infants, severe NPP1 deficiency states were recently linked to a syndrome of spontaneous infantile arterial and periarticular calcification.22Rutsch R Vaingankar S Johnson K Goldfine I Maddux B Schauerte P Kalhoff H Sano K Boisvert WA Superti-Furga A Terkeltaub R PC-1 Nucleoside triphosphate pyrophosphohydrolase deficiency in idiopathic infantile arterial calcification.Am J Pathol. 2001; 158: 543-554Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 23Rutsch F Ruf N Vaingankar S Toliat MR Suk A Höhne W Schauer G Lehmann M Roscioli T Schnabel D Epplen JT Knisely A Superti-Furga A McGill J Filippone M Sinaiko AR Vallance H Hinrichs B Smith W Ferre M Terkeltaub R Nürnberg P Mutations in ENPP1 are associated with ‘idiopathic’ infantile arterial calcification.Nat Genet. 2003; 34: 379-381Crossref PubMed Scopus (479) Google Scholar NPP1 knockout mice (Enpp1−/−) also known as tiptoe walking (ttw/ttw) mice, spontaneously develop progressive ankylosing intervertebral and peripheral joint hyperostosis and articular cartilage calcification.24Hashimoto S Ochs RL Komiya S Lotz M Linkage of chondrocyte apoptosis and cartilage degradation in human osteoarthritis.Arthritis Rheum. 1998; 41: 1632-1638Crossref PubMed Scopus (465) Google Scholar, 25Okawa A Nakamura I Goto S Moriya H Nakamura Y Ikegawa S Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine.Nat Genet. 1998; 19: 271-273Crossref PubMed Scopus (358) Google Scholar, 26Sali A Favaloro JM Terkeltaub R Goding JW Germline deletion of the nucleoside triphosphate pyrophosphohydrolase (NTPPPH) plasma cell membrane glycoprotein-1 (PC-1) produces abnormal calcification of periarticular tissues.in: Vanduffel L Lemmems R Ecto-ATPases and Related Ectoenzymes. Shaker Publishing BV, Maastricht1999: 267-282Google Scholar, 27Johnson K Pritzker K Goding J Terkeltaub R The nucleoside triphosphate pyrophosphohydrolase isozyme PC-1 directly promotes cartilage calcification through chondrocyte apoptosis and increased calcium precipitation by mineralizing vesicles.J Rheumatol. 2001; 28: 2681-2691PubMed Google Scholar, 28Johnson K Hashimoto S Lotz M Pritzker K Goding J Terkeltaub R Up-regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC-1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification.Arthritis Rheum. 2001; 44: 1071-1081Crossref PubMed Scopus (139) Google Scholar, 29Johnson K Goding J Van Etten D Sali A Hu SI Farley D Krug H Hessle L Millán JL Terkeltaub R Linked deficiencies in extracellular inorganic pyrophosphate (PPi) and osteopontin expression mediate pathologic ossification in PC-1 null mice.J Bone Miner Res. 2003; 18: 994-1004Crossref PubMed Scopus (172) Google Scholar Despite the different manner in which NPP1 and ANK supply PPi to bone matrix, a similar phenotype is associated with a naturally occurring truncation mutation of the C-terminal cytosolic domain of ANK that appears to attenuate PPi channeling in ank/ank mutant mice.14Hakim FT Cranley R Brown KS Eanes ED Harne L Oppenheim JJ Hereditary joint disorder in progressive ankylosis (ank/ank) mice. I. Association of calcium hydroxyapatite deposition with inflammatory arthropathy.Arthritis Rheum. 1994; 27: 1411-1420Crossref Scopus (71) Google Scholar, 15Ho AM Johnson MD Kingsley DM Role of mouse ank gene in control of tissue calcification and arthritis.Science. 2000; 289: 265-269Crossref PubMed Scopus (550) Google Scholar, 30Nürnberg P Theile H Chandler D Höhne W Cunningham ML Ritter H Leschik G Uhlmann K Mischung C Harrop K Goldblatt J Borochowitz ZU Kotzot D Westermann F Mundlos S Braun HS Laing N Tinschert S Heterozygous mutations in ANKH, the human ortholog of the mouse progressive ankylosis gene, result in craniometaphyseal dysplasia.Nat Genet. 2001; 28: 37-41PubMed Google Scholar The TNAP-, NPP1-, and ANK-deficient mice all have altered levels of PPi and thus, these mice are valuable tools to further understand the function of PPi in the process of bone mineralization. Central to our understanding of this process is not only how osteoblasts and chondrocytes make and dispose of PPi, but what downstream effects PPi has on bone forming cells, in particular osteoblast gene expression. We have previously shown that crossbreeding the Akp2−/− and the Enpp1−/− mice rescues the PPi levels of the single-deficient animals, resulting in a correction of the mineralization defects.10Hessle L Johnsson KA Anderson HC Narisawa S Sali A Goding JW Terkeltaub R Millán JL Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization.Proc Natl Acad Sci USA. 2002; 99: 9445-9449Crossref PubMed Scopus (688) Google Scholar Given the similarity in function of NPP1 and ANK, and also the similarity in phenotype of the deficient mice, here we investigated whether simultaneously affecting TNAP and ANK function would also ameliorate the mineralization defects of the single-deficient mice as previously observed for the [Akp2−/−; Enpp1−/−] double deficiency. We also examined the effects of PPi on osteoblastic gene expression. It is well established that during the process of normal bone differentiation and subsequent deposition of mineral, a number of osteoblast marker genes are expressed in a defined spatial and temporal manner. We have recently shown that osteopontin (OPN), a putative inhibitor of mineralization,31Boskey AL Maresca M Ulrich W Doty SB Butler WT Prince CW Osteopontinhydroxyapatite interactions in vitro. Inhibition of hydroxyapatite formation and growth in a gelatin gel.Bone Miner. 1993; 22: 147-159Abstract Full Text PDF PubMed Scopus (390) Google Scholar, 32Hunter GK Kyle CL Goldberg HA Modulation of crystal formation by bone phosphoproteins; structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation.Biochem J. 1994; 300: 723-728Crossref PubMed Scopus (374) Google Scholar, 33Sodek J Ganss B McKee MD Osteopontin.Crit Rev Oral Biol Med. 2000; 11: 279-303Crossref PubMed Scopus (936) Google Scholar is down-regulated in the hypermineralized Enpp1 and ank/ank mice.29Johnson K Goding J Van Etten D Sali A Hu SI Farley D Krug H Hessle L Millán JL Terkeltaub R Linked deficiencies in extracellular inorganic pyrophosphate (PPi) and osteopontin expression mediate pathologic ossification in PC-1 null mice.J Bone Miner Res. 2003; 18: 994-1004Crossref PubMed Scopus (172) Google Scholar Here we investigated the changes in OPN expression in the single- and double-knockout mice and the effects of TNAP-, ANK-, and NPP1-mediated alterations in PPi levels on both OPN expression and hydroxyapatite deposition. Our data have enabled us to build a model of the concerted action of these three molecules in controlling normal mineralization that also explains the pathological abnormalities in hypo- and hypermineralization disorders. All routine chemicals were of an analytical grade from Sigma (St. Louis, MO), unless otherwise indicated. The generation and characterization of Akp2−/−, Enpp1−/−, ank/ank, and [Akp2−/−; Enpp1−/−] mice has been reported previously.10Hessle L Johnsson KA Anderson HC Narisawa S Sali A Goding JW Terkeltaub R Millán JL Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization.Proc Natl Acad Sci USA. 2002; 99: 9445-9449Crossref PubMed Scopus (688) Google Scholar, 19Narisawa N Frölander N Millán JL Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia.Dev Dyn. 1997; 208: 432-446Crossref PubMed Scopus (326) Google Scholar, 20Fedde KN Blair L Silverstein J Coburn SP Ryan LM Weinstein RS Waymire K Narisawa S Millán JL MacGregor GR Whyte MP Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia.J Bone Miner Res. 1999; 14: 2015-2026Crossref PubMed Scopus (323) Google Scholar, 26Sali A Favaloro JM Terkeltaub R Goding JW Germline deletion of the nucleoside triphosphate pyrophosphohydrolase (NTPPPH) plasma cell membrane glycoprotein-1 (PC-1) produces abnormal calcification of periarticular tissues.in: Vanduffel L Lemmems R Ecto-ATPases and Related Ectoenzymes. Shaker Publishing BV, Maastricht1999: 267-282Google Scholar Mice carrying the ank mutation14Hakim FT Cranley R Brown KS Eanes ED Harne L Oppenheim JJ Hereditary joint disorder in progressive ankylosis (ank/ank) mice. I. Association of calcium hydroxyapatite deposition with inflammatory arthropathy.Arthritis Rheum. 1994; 27: 1411-1420Crossref Scopus (71) Google Scholar, 15Ho AM Johnson MD Kingsley DM Role of mouse ank gene in control of tissue calcification and arthritis.Science. 2000; 289: 265-269Crossref PubMed Scopus (550) Google Scholar were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred into the Akp2−/− strain to generate [Akp2−/−; ank/ank] double-deficient mice. To determine genotypes, genomic DNA was isolated from tails and analyzed using polymerase chain reaction (PCR) protocols.10Hessle L Johnsson KA Anderson HC Narisawa S Sali A Goding JW Terkeltaub R Millán JL Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization.Proc Natl Acad Sci USA. 2002; 99: 9445-9449Crossref PubMed Scopus (688) Google Scholar, 29Johnson K Goding J Van Etten D Sali A Hu SI Farley D Krug H Hessle L Millán JL Terkeltaub R Linked deficiencies in extracellular inorganic pyrophosphate (PPi) and osteopontin expression mediate pathologic ossification in PC-1 null mice.J Bone Miner Res. 2003; 18: 994-1004Crossref PubMed Scopus (172) Google Scholar Southern blots were used to confirm the double-knockout genotypes. Whole-mount skeletal preparations were prepared by removal of skin and viscera of mice followed by a 1-week immersion in 100% ethanol, followed by 100% acetone. Samples were then transferred to a 100% ethanol solution containing 0.01% Alizarin Red S, 0.015% Alcian Blue 8GX, and 0.5% acetic acid for 3 weeks. Samples were then destained with 1% (v/v) KOH/50% glycerol solution. Cleared samples were stored in 100% glycerol. For analysis of mineral deposition, the lumbar spines from 10-day-old mice were fixed in 10% neutral buffered formalin for 5 days. After washing in phosphate-buffered saline-based sucrose solutions, samples were embedded in optimal cutting temperature (OCT) solution. Sections (16 μm) were stained using the von Kossa procedure as previously described.34Narisawa S Wennberg C Millán JL Abnormal vitamin B6 metabolism in alkaline phosphatase knock-out mice causes multiple abnormalities, but not the impaired bone mineralization.J Pathol. 2001; 193: 125-133Crossref PubMed Scopus (93) Google Scholar The degree of mineralization of the spines was quantified by examining the vertebral apophyses of the von Kossa-stained lumbar sections from each respective genotype. The number of ossification centers containing visible mineral deposits (stained black/brown) versus those containing no deposits, was determined using low (×16) magnification. Three sections, each containing a minimum of five vertebrae, were counted per mouse and the percentage of mineralized apophyses was plotted as a function of the Akp2 and ANK genotypes. The number of mice examined for each genotype were: [Akp2+/+; Ank/Ank], n = 2; [Akp2+/+; Ank/ank], n = 7; [Akp2+/+; ank/ank], n = 2; [Akp2+/−; Ank/Ank], n = 8; [Akp2+/−; Ank/ank], n = 9; [Akp2+/−; ank/ank], n = 3; [Akp2−/−; Ank/Ank], n = 2; [Akp2−/−; Ank/ank], n = 9; and [Akp2−/−; ank/ank], n = 2. For immunohistochemical analysis, mouse skeletal tissues were dissected and fixed in 10% neutral buffered formalin for 2 days and then decalcified in 4% hydrochloric acid, processed for histology, and embedded in paraffin. For detection of OPN, sections were deparaffinized, blocked with 10% goat serum for 20 minutes, and incubated overnight at 4°C with rabbit polyclonal antibody to OPN (Chemicon, Temecula, CA). Washed sections were incubated for 1 hour at 22°C with biotinylated goat anti-rabbit IgG followed by a 1-hour incubation with peroxidase-conjugated avidin. Peroxidase activity was detected using the Fast DAB staining kit (Sigma), according to the manufacturer's instructions. Mouse calvarial cells were isolated from 3-day-old mice through sequential collagenase digestion, as previously described.10Hessle L Johnsson KA Anderson HC Narisawa S Sali A Goding JW Terkeltaub R Millán JL Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization.Proc Natl Acad Sci USA. 2002; 99: 9445-9449Crossref PubMed Scopus (688) Google Scholar Calvarial cells of the same genotype were pooled and plated at a density of 4 × 104 cells/cm2 in α-MEM (Life Technologies, Inc., Grand Island, NY), supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 1% l-glutamine. The cells were incubated in a humidified atmosphere of 5% CO2 in air at 37°C. The medium was completely replaced every third day. For studies under mineralizing conditions, media were supplemented with β-glycerophosphate (10 mmol/L) and l-ascorbic acid (50 μg/ml). PPi levels were determined by differential adsorption on activated charcoal of UDP-D-[6-3H]glucose (Amersham Biosciences, Piscataway, NJ) from the reaction product of 6-phospho[6-3H]gluconate, as previously described.12Johnson K Vaingankar S Chen Y Moffa A Goldring M Sano K Jin-Hua P Sali A Goding J Terkeltaub R Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes.Arthritis Rheum. 1999; 42: 1986-1997Crossref PubMed Scopus (108) Google Scholar, 13Johnson K Moffa A Chen Y Pritzker K Goding J Terkeltaub R Matrix vesicle plasma membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells.J Bone Miner Res. 1999; 14: 883-892Crossref PubMed Scopus (113) Google Scholar To determine intracellular PPi, washed cells were heated at 65°C for 45 minutes, washed again, and lysed in 1% Triton X-100, 1.6 mmol/L MgCl2, 0.2 mol/L Tris, pH 8.1 (lysis buffer).12Johnson K Vaingankar S Chen Y Moffa A Goldring M Sano K Jin-Hua P Sali A Goding J Terkeltaub R Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes.Arthritis Rheum. 1999; 42: 1986-1997Crossref PubMed Scopus (108) Google Scholar Extracellular PPi was determined from conditioned media treated in the same manner.12Johnson K Vaingankar S Chen Y Moffa A Goldring M Sano K Jin-Hua P Sali A Goding J Terkeltaub R Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes.Arthritis Rheum. 1999; 42: 1986-1997Crossref PubMed Scopus (108) Google Scholar We determined cell protein, and specific activity of nucleosidetriphosphate pyrophosphohydrolase and alkaline phosphatase activities as described.13Johnson K Moffa A Chen Y Pritzker K Goding J Terkeltaub R Matrix vesicle plasma membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells.J Bone Miner Res. 1999; 14: 883-892Crossref PubMed Scopus (113) Google Scholar Primary calvarial osteoblasts were treated with mineralization media for 14 days and the cell-associated MVs were collected by collagenase digestion for 2 hours at 37°C. The supernatant was collected and initially centrifuged at 20,000 × g for 20 minutes at 4°C to pellet cellular debris. This was followed by centrifugation at 100,000 × g for 1 hour to isolate the MV fraction, which was resuspended in Hanks' balanced salt solution. Fifty μg of protein was used for Western blotting from both the MV fraction as well as the cell lysates collected in the first centrifugation. Western blot analysis was performed as previously described using rabbit anti-mouse ANK,35Johnson KA Hessle L Wennberg C Mauro S Narisawa S Goding J Sano K Millán JL Terkeltaub R Tissue-nonspecific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) act as selective and mutual antagonists of mineralizing activity by murine osteoblasts.Am J Physiol. 2000; 279: R1365-R1377Google Scholar and rabbit anti-mouse NPP1.15Ho AM Johnson MD Kingsley DM Role of mouse ank gene in control of tissue calcification and arthritis.Science. 2000; 289: 265-269Crossref PubMed Scopus (550) Google Scholar Total RNA was isolated from osteoblasts using 0.5 ml of Trizol (Life Technologies)/35-mm dish and was reverse-transcribed using the Titanium One-Step RT-PCR kit (Invitrogen, San Diego, CA) according to the manufacturer's instructions. The primers used were as follows: Mouse Opn (5′-CTCCCGGAGAAAGTGACTGA-3′, 5′-GACCTCAGAAGATGAACTAT-3′), mouse Akp2 (5′-AGTCCGTGGGCATTGTGACTA-3′, 5′-TGCTGCTCCACTCACGTC GAT-3′), mouse Ank (5′-CTAGCAGGGTTTGTGGGAGAA-3′, 5-TTTATGAAGCAGGGG CGTGAA-3), mouse Enpp1(5′-GCTACAGCTTTCTAGCCATGA-3′, TTAATCCAAGCC CAGGTCCTT-3′). Mouse β-actin primers were used as a loading control. The PCR products were separated by electrophoresis on 1% agarose gels and werevisualized by ethidium bromide staining with UV light illumination. To measure OPN protein levels, we developed an enzyme-linked immunosorbent assay based on a previously published method36Bautista DS Saad Z Chambers AF Tonkin KS O'Malley FP Singhai H Tokmakejian S Bramwell V Harris JF Quantification of osteopontin in human plasma with an ELISA: basal levels in pre- and postmenopausal women.Clin Biochem. 1996; 29: 231-239Crossref PubMed Scopus (61) Google Scholar using plates coated with a monoclonal antibody to native OPN (Chemicon) using a 1:2500 dilution of OPN antibody in 100 μl/well 0.1 mol/L sodium bicarbonate, pH 9.0, at 4°C overnight. Wells were blocked with 10 mmol/L Tris, 150 mmol/L NaCl, and 0.05% Tween-20, pH 8.0, for 1 hour at 22°C. Samples, diluted in 1% bovine serum albumin, 10 mmol/L Tris, and 150 mmol/L NaCl, pH 8.0, were added to the wells and incubated for 1 hour at 37°C. Recombinant murine OPN was used as standard. Washed wells were incubated sequentially for 1 hour at 37°C with rabbit anti-OPN (1:1000, Chemicon), biotinylated goat anti-rabbit IgG (1:1000), and streptavidin conjugated with AP (1:500 dilution). Color was developed using p-nitrophenylphosphate and read at 405 nm. We have previously shown that the simultaneous deletion of the Akp2 and Enpp1 genes can rescue the mineralization defects of both the Akp2 and Enpp1 single-knockout mice.10Hessle L Johnsson KA Anderson HC Narisawa S Sali A Goding JW Terkeltaub R Millán JL Tissue-nonspecific alkaline ph
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