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Targeted Disruption of the Osteoblast/Osteocyte Factor 45 Gene (OF45) Results in Increased Bone Formation and Bone Mass

2003; Elsevier BV; Volume: 278; Issue: 3 Linguagem: Inglês

10.1074/jbc.m203250200

1083-351X

Lori C. Gowen, Donna N. Petersen, Amy L. Mansolf, Hong Qi, Jeffrey L. Stock, George T. Tkalcevic, Hollis A. Simmons, David T. Crawford, Kristen L. Chidsey-Frink, Hua Zhu Ke, John D. McNeish, Thomas A. Brown,

Bone Metabolism and Diseases

We have previously described osteoblast/osteocyte factor 45 (OF45), a novel bone-specific extracellular matrix protein, and demonstrated that its expression is tightly linked to mineralization and bone formation. In this report, we have cloned and characterized the mouse OF45 cDNA and genomic region. Mouse OF45 (also called MEPE) was similar to its rat orthologue in that its expression was increased during mineralization in osteoblast cultures and the protein was highly expressed within the osteocytes that are imbedded within bone. To further determine the role of OF45 in bone metabolism, we generated a targeted mouse line deficient in this protein. Ablation ofOF45 resulted in increased bone mass. In fact, disruption of only a single allele of OF45 caused significantly increased bone mass. In addition, knockout mice were resistant to aging-associated trabecular bone loss. Cancellous bone histomorphometry revealed that the increased bone mass was the result of increased osteoblast number and osteoblast activity with unaltered osteoclast number and osteoclast surface in knockout animals. Consistent with the bone histomorphometric results, we also determined thatOF45 knockout osteoblasts produced significantly more mineralized nodules in ex vivo cell cultures than did wild type osteoblasts. Osteoclastogenesis and bone resorption in ex vivo cultures was unaffected by OF45 mutation. We conclude that OF45 plays an inhibitory role in bone formation in mouse. We have previously described osteoblast/osteocyte factor 45 (OF45), a novel bone-specific extracellular matrix protein, and demonstrated that its expression is tightly linked to mineralization and bone formation. In this report, we have cloned and characterized the mouse OF45 cDNA and genomic region. Mouse OF45 (also called MEPE) was similar to its rat orthologue in that its expression was increased during mineralization in osteoblast cultures and the protein was highly expressed within the osteocytes that are imbedded within bone. To further determine the role of OF45 in bone metabolism, we generated a targeted mouse line deficient in this protein. Ablation ofOF45 resulted in increased bone mass. In fact, disruption of only a single allele of OF45 caused significantly increased bone mass. In addition, knockout mice were resistant to aging-associated trabecular bone loss. Cancellous bone histomorphometry revealed that the increased bone mass was the result of increased osteoblast number and osteoblast activity with unaltered osteoclast number and osteoclast surface in knockout animals. Consistent with the bone histomorphometric results, we also determined thatOF45 knockout osteoblasts produced significantly more mineralized nodules in ex vivo cell cultures than did wild type osteoblasts. Osteoclastogenesis and bone resorption in ex vivo cultures was unaffected by OF45 mutation. We conclude that OF45 plays an inhibitory role in bone formation in mouse. matrix glutamic acid protein tartrate-resistant acid phosphatase rapid amplification of cDNA ends reverse transcriptase embryonic stem peripheral quantitative computerized tomography wild type knockout bone volume tissue volume bone surface bone formation rate Chinese hamster ovary heterozygote bacterial artificial chromosome matrix extracellular phosphoglycoprotein phosphate-regulating endopeptidose, X-linked The constant modulation of the balance between skeletal strength and mineral availability in bone is effected by competing cell types in response to physiological needs. Osteoblasts produce, organize, and mineralize bone matrix in forming bone. Osteoclasts break down matrix by forming a lytic pocket in which bone is degraded and calcium is released. The generation and activity of these cell types is tightly regulated to provide equilibrium between formation and resorption and, thereby, an appropriate balance of strength and mineral release. Under certain conditions, such as aging, postmenopausal estrogen deficiency, or some pathophysiological states, there can exist an imbalance between bone resorption and bone formation. As a result, skeletal mass and strength are compromised and osteoporotic fractures can occur in the afflicted individuals. Bone is produced by the organization and mineralization of the extracellular matrix produced by osteoblasts. The major component of the extracellular matrix of these cells is Type I collagen, which functions as a scaffold for new bone. In addition, non-collagenous matrix proteins have been identified that influence the operations of bone turnover, formation, and repair. These proteins are generally acidic and highly post-translationally modified by phosphorylation, glycosylation, or sulfation (1McKee M.D. Zalzal S. Nanci A. Anat. Rec. 1996; 245: 293-312Crossref PubMed Scopus (128) Google Scholar). Targeted deletion of extracellular matrix genes in mice has been a useful method to determine the in vivo functions of several matrix proteins. For example, osteocalcin is an abundant gamma carboxyl glutamic acid-containing bone matrix protein shown to be highly expressed in osteoblasts and is a biochemical marker of the bone remodeling process. Osteocalcin deletion in mice results in increased cortical bone thickness due to increased osteoblast activity, indicating that osteocalcin has negative effects in vivo on osteoblasts and bone formation (2Ducy P. Desbois C. Boyce B. Pinero G. Story B. Dunstan C. Smith E. Bonadio J. Goldstein S. Gundberg C. Bradley A. Karsenty G. Nature. 1996; 382: 448-452Crossref PubMed Scopus (1430) Google Scholar). Another gene of this category, matrix glutamic acid protein (known as “MGP”),1 was deleted in mice, resulting in extensive cartilage calcification and in the inappropriate calcification of arteries, leading to blood vessel rupture, and lethality in homozygotes (3Luo G. Ducy P. McKee M.D. Pinero G.J. Loyer E. Behringer R.R. Karsenty G. Nature. 1997; 386: 78-81Crossref PubMed Scopus (1794) Google Scholar). Ablation of the extracellular matrix protein osteonectin causes reduced bone formation with a decrease in both osteoblasts and osteoclasts resulting in a net loss of trabecular bone compared with wild type controls (4Delaney A.M. Amling M. Priemel M. Howe C. Baron R. Canalis E. J. Clin. Invest. 2000; 105: 915-923Crossref PubMed Scopus (243) Google Scholar). Targeted disruption of osteopontin, an RGD-containing protein, results in a defect in the in vitro differentiation of osteoclasts; increased mineral content and maturity in long bones; and resistance to ovariectomy-induced, parathyroid hormone-induced, and mechanical unloading-induced bone resorption in mice (5Rittling S.R. Matsumoto H.N. McKee M.D. Nanci A., An, X.R. Novick K.E. Kowalski A.J. Noda M. Denhardt D.T. J. Bone Miner. Res. 1998; 13: 1101-1111Crossref PubMed Scopus (371) Google Scholar, 6Liaw L. Birk D.E. Ballas C.B. Whitsitt J.S. Davidson J.M. Hogan B.L. J. Clin. Invest. 1998; 101: 1468-1478Crossref PubMed Google Scholar, 7Yoshitake H. Rittling S.R. Denhardt D.T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8156-8160Crossref PubMed Scopus (321) Google Scholar, 8Boskey A.L. Spevak L. Paschalis E. Doty S.B. McKee M.D. Calcified Tissue Int. 2002; 71: 145-154Crossref PubMed Scopus (256) Google Scholar). The functions of matrix proteins can be further elucidated through the characterization of mice deficient in combinations of genes. For example, mice deficient in both osteopontin and MGP exhibit more vascular calcification than the MGP knockout mice, demonstrating that osteopontin can be an inhibitor of ectopic calcification in vivo (9Speer M.Y. McKee M.D. Guildberg R.E. Liaw L. Yang H.-Y. Tung E. Karsenty G. Giachelli C.M. J. Exp. Med. 2002; 196: 1047-1055Crossref PubMed Scopus (290) Google Scholar). In an effort to further characterize the process of bone metabolism and identify the proteins involved, we screened RNA of rat bone marrow cell cultures for novel transcripts specific to bone mineralization (10Petersen D.N. Tkalcevic G.T. Mansolf A.L. Rivera-Gonzalez R. Brown T.A. J. Biol. Chem. 2000; 275: 36172-36180Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). This screen produced a novel clone that we designated osteoblast/osteocyte factor 45 (OF45). Rat OF45encodes an RGD protein having 45% homology to the recently cloned human MEPE as well as loose homology toAG-1/DMP1. In rat, OF45 is highly expressed in the tibial shaft and metaphysis as well as in osteoblasts of induced bone marrow cultures, calvaria, and the UMR106 osteoblastic cell line. Immunohistochemistry in rat tibia revealed abundant OF45 protein in the osteocytes. Newly embedded rat osteocytes were also positive for OF45 in a marrow ablation model. Interestingly, the temporal expression pattern of OF45 differs from other well-characterized bone markers such as osteocalcin, osteonectin, and osteopontin in both in vitro and in vivo bone growth models (10Petersen D.N. Tkalcevic G.T. Mansolf A.L. Rivera-Gonzalez R. Brown T.A. J. Biol. Chem. 2000; 275: 36172-36180Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In this report we have cloned and characterized the mouse OF45 cDNA. With the exception of some additional sequence we report at the 5-prime end, the sequence reported here was identical to that recently published as MEPE (11Argiro L. Desbarats M. Glorieux F.H. Ecarot B. Genomics. 2001; 74: 342-351Crossref PubMed Scopus (145) Google Scholar). To demonstrate the functional significance of an elimination or reduction of OF45 expression, we have cloned the mouse OF45genomic DNA, generated a mouse line with a targeted disruption of theOF45 gene, and subjected these mice to phenotypic analysis. A 1255-bp probe encoding most of the rat OF45 cDNA sequence, excluding the 3′-untranslated repeat, was used to screen a mouse 129 genomic lambda phage library (12Sambrook J.F. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A 15-kb clone found to include exon 3 was used to generate PCR primers to clone the complete cDNA, the BAC clone, as well as the targeting construct. The 5′-end of the mRNA was determined by RNA ligase-mediated RACE as described (13Frohman M.A. PCR Methods and Applications. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: S40-S58Google Scholar). Briefly, 50 μg of total RNA from mouse tibia was dephosphorylated with calf intestinal alkaline phosphatase (Roche Molecular Biochemicals, Indianapolis, IN) and the 5′-cap was removed using tobacco acid pyrophosphatase (Epicentre Technologies, Madison, WI). The RNA linker generated from the plasmid pGbx-1 (Gift from Dr. M. A. Frohman) was ligated to the decapped RNA using T4 RNA ligase (Epicentre Technologies, Madison, WI). The RNA was converted to cDNA by using Superscript II reverse transcriptase and p(dT)12–18 primer (Invitrogen, Gaithersburg, MD). PCR was used to amplify the 5-end of the cDNA using primer pair NRC-1–288A and 36387.233B and primer pair NRC-1–288B and 36387.233A in the first and second round, respectively. A single PCR product of ∼550 bp was obtained in the second round PCR. A control sample in which the pyrophosphatase step was omitted did not yield a PCR product, indicating that the ∼550-bp product likely resulted from full-length mRNA that had been 5′-capped. The second round PCR fragment was isolated and ligated using a TA cloning kit (Invitrogen, Carlsbad, CA). Insert-containing plasmids were sequenced. The 3′-end of the mRNA was determined using a 3′ RACE kit (Invitrogen, Gaithersburg, MD) as suggested by the manufacturer. Briefly, 5 μg of mouse tibia total RNA was reverse-transcribed using the AP primer provided. The PCR reaction amplified the sequence between OF45 nucleotide 527 (primer 36387.233E) and the AUAP primer provided. Two fragments close in size were cloned with the TA cloning kit (Invitrogen) and sequenced. The sequences of the two fragments were identical except for a short stretch of additional sequence at the 3′-end. The gene-specific primers were as follows: 36387.233A, 5′-TGTGTCAGGTAGTGAGTGCTCC-3′; 36387.233B, 5′-ACTGCCACCATGTCCTTCTC-3′; 36387.233C, 5′-CCAGCAGATGTCAATGATGC-3′; 36387.233D, 5′-TTGGCAGCATCTGTGTATCC-3′; 36387.233E, 5′-CCCAAGAGCAGCAAAGGTAG-3′; and 36387.233F, 5′-TGCGTGATATTTCTGAGGAGG-3′. The pGbx-1 RNA linker primers were, NRC-1–288A, 5′-CCAAGACTCACTGGGTACTGC-3′; NRC-2–288B, 5′-CTAGAGGGGCCTGTTGAACC-3′; NRC-3–288C, 5′-GGGAGAGGCCAGCGTATTCC-3′; RC NRC-1–30A, 5′-GCAGTACCCAGTGAGTCTTGG-3′; RC NRC-2–30B, 5′-GGTTCAACAGGCCCCTCTA-3′; and RC NRC-3–30C, 5′-GGAATACGCTGGCCTCTCCC-3′. A 124-bp probe for the 5′-end of the cDNA was generated by PCR with primers to amplify bases 12–135 of the cDNA sequence using primers 36393.80A (5′-TTTCAGCAAATGCCCAGAG-3′) and 36393.80B (5′-CCAGGTCATACTGAAGAGGAGC-3′). This probe was sent to Genome Systems, Inc. (St. Louis, MO) for screening of a mouse ES-129/SVIII BAC library. A single clone was identified and characterized. Chromosomal localization was determined by fluorescence in situhybridization using the BAC clone by Genome Systems, Inc. according to their protocols. Intron/exon boundaries were determined by alignment of BAC clone sequence with the OF45 cDNA sequence. The 1340 bp containing the coding region of the mouse OF45 cDNA (bases 53–1392) was amplified using primers 36393.44C (5′-TTTCCTGAAGGTGAATGACG-3′) and 36393.44H (5′-CTAGTCACCATGACTCTCACTAG-3′) and subcloned in the cytomegalovirus mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA) for transfection into CHO cells using LipofectAMINE Plus (Invitrogen). High titer polyclonal antiserum was generated by immunization of rabbits with bacterial expressed full-length OF45 (Zymed Laboratories Inc., South San Francisco, CA). OF45 antibody was affinity-purified by chromatography on OF45-coupled agarose beads by standard methods (14Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY1988Google Scholar). Protein samples were separated by electrophoresis on Novex (San Diego, CA) 10% NuPAGE gels and transferred to nitrocellulose using a semi-dry transfer. Blots were blocked using 1% Western Blocking Reagent from Roche Molecular Biochemicals (Mannheim, Germany) for 1 h and 1oantibody diluted in 0.5% block for 1 h. After washing, goat anti-rabbit peroxidase 2o antibody (Roche Molecular Biochemicals) diluted into 0.5% block was applied to the blot for 1 h. Signal was detected using an ECL detection kit (AmershamBiosciences, Buckinghamshire, England). Multiple tissue Northern blots (Clontech, Palo Alto, CA) were hybridized with the full-length OF45 cDNA. RNA isolated from mouse tissues was converted to cDNA by using Superscript II reverse transcriptase and poly(dT)12–18 primer (Invitrogen, Gaithersburg, MD). PCR was used to amplify an internal 483 bp from OF45mRNA bases 986–1468 using primers 36499–143A (5′-ACTATCCACAAGTGGCCTCG-3′) and 36499–143B (5′-CTGTTGGCTTGCTCAGTTCC-3′). The cDNA was then hybridized with a radiolabeled cDNA probe specific to bases 160–1020. Genomic DNA fragments from the 15-kb genomic clone were subcloned into the JNS2 targeting plasmid (15Dombrowicz D. Flamand V. Brigman K.K. Koller B.H. Kinet J.P. Cell. 1993; 75: 969-976Abstract Full Text PDF PubMed Scopus (342) Google Scholar) E14Tg2a embryonic stem (ES) cell line derived from the 129sj mouse strain was used for gene targeting of theOF45 locus (16Hooper M. Hardy K. Handyside A. Hunter S. Monk M. Nature. 1987; 326: 292-295Crossref PubMed Scopus (968) Google Scholar). Electroporation, selection, expansion, and microinjection of ES cells into C57BL/6 embryos were as described previously (17Roach M.L. Stock J.L. Byrum R. Koller B.H. McNeish J.D. Exp. Cell Res. 1995; 221: 520-525Crossref PubMed Scopus (50) Google Scholar). Out of 70 neomycin and gancyclovir-resistant clones, four were positive for the desired recombination event, resulting in a targeting frequency of 1 in 17. Chimeric animals were mated with C57BL/6 males and females. An 850-bp SpeI/MscI probe, homologous to a region directly 3′ of the targeted mutation, was used to genotype agouti offspring by Southern blot analysis. Digestion of tail DNA with BamHI/BglII double digest resulted in a 10-kb hybridizing band in the wild type allele and a 9-kb hybridizing band in the targeted allele. F2 or F3 129/Bl6 mix heterozygote mice were bred to generate successive populations of littermates used for in vivo experimentation at either 4 months or 1 year of age. The same parent animals were used to generate both 4-month and 1-year age groups. 10-month-old males backcrossed onto a C57/Bl6 background for nine generations were used for dynamic histomorphometry studies. Animals were maintained on a 12-h light/12-h dark cycle and were provided food and water ad libitum. 12 and 2 days prior to sacrifice, mice were given subcutaneous injection of the fluorochrome calcein at 10 mg/kg (Sigma Chemical Co., St. Louis, MO). Animals were euthanized by cervical dislocation, and the femurs were placed in 70% ethanol for later pQCT and histomorphometric analysis. The experiment was conducted according to Pfizer Animal Care and Use-approved protocols, and the animals were maintained in accordance with the ILAR (Institute of Laboratory Animal Research) Guide for the Care and Use of Laboratory Animals. Calcium and inorganic phosphate levels were measured from blood serum at DNX Transgenic Sciences (Cranbury, NJ) using the ACE Clinical Chemistry System (Alfa Wassermann Inc., West Caldwell, NJ) by the manufacturer's protocols. One-year-old animals were used for this study. Sera from 12 male animals of each genotype were used. Seven wild type and seven knockout females were also studied. Femurs were examined at 2× and 3× magnification on a Faxitron model MX-20 Specimen Radiography System (Buffalo Grove, IL) with Kodak Min-R 2000 Mammography film in Min-R 2000 cassettes with an intensifying screen. Magnification was calculated by SID/SOD = IS/OS where SID is source to image distance, SOD is source to object distance, IS is image size, and OS is object size. The femurs of 12 to 27 animals of each sex, age, and genotype were examined. Excised femurs were scanned by a pQCT x-ray machine (Stratec XCT Research M, Norland Medical Systems, Fort Atkinson, WI) with software version 5.40. A 1-mm-thick cross-section of the femur metaphysis was taken at 2.5 mm proximal from the distal end with a voxel size of 0.07 mm. Cortical bone was defined and analyzed using contour mode 2 and cortical mode 4. An outer threshold setting of 340 mg/cm3 was used to distinguish the cortical shell from soft tissue and an inner threshold of 529 mg/cm3 to distinguish cortical bone along the endocortical surface. Trabecular bone was determined using peel mode 4 with a threshold setting of 655 mg/cm3 to distinguish (sub)cortical from cancellous bone. An additional concentric peel of 1% of the defined cancellous bone was used to ensure (sub)cortical bone was eliminated from the analysis. Volumetric content, density, and area were determined for both trabecular and cortical bone. Using the above setting, we have determined that the ex vivo precision of volumetric content, density, and area of total bone, trabecular, and cortical regions ranged from 0.99% to 3.49% with repositioning (18Ke H.Z., Qi, H. Chidsey-Frink K.L. Crawford D.T. Thompson D.D. J. Bone Miner. Res. 2001; 16: 765-773Crossref PubMed Scopus (81) Google Scholar). Number of animals examined: females, 4 months, 12 WT, 20 Het, 18 KO; females, 1 year, 12 WT, 17 Het, 27 KO; males, 4 months, 18 WT, 21 Het, 18 KO; and males, 1 year, 21 WT, 19 Het, 21 KO. Following pQCT analysis, the distal half of the femur from each animal of 4-month-old and 1-year-old animals was dehydrated and embedded in methyl methacrylate. 4-μm longitudinal sections were prepared with a Reichert-Jung Polycut S microtome (Leica, Deerfield, IL) and stained with modified Masson's Trichrome stain. Trabecular bone volume, trabecular number, and trabecular thickness were determined on the distal femoral metaphysis for these 4-month-old and 1-year-old animals (19Parfitt A.M. Drezner M.K. Glorieux F.H. Kanis J.A. Malluche H. Meunier P.J. Ott S.M. Recker R.R. J. Bone Miner. Res. 1987; 2: 595-610Crossref PubMed Scopus (4953) Google Scholar, 20Jee W.S.S. Li X.J. Inoue J. Jee K.W. Haba T. Ke H.Z. Setterberg R.B. Ma Y.F. Takahashi H. Handbook of Bone Morphology. Nishimusa, Niigata City, Japan1997: 87-112Google Scholar). To understand the changes in osteoblasts and osteoclasts on the cancellous bone, a group of wild type (n = 8) and knockout (n = 10) male mice were necropsied at 10 months of age. The left distal half of the femur from these 10-month-old animals was decalcified, and 4-μm longitudinal sections were prepared and stained with Toluidine Blue (Sigma, St. Louis, MO). These decalcified sections were used to determine the trabecular bone volume (BV/TV), percent osteoblast surface (Ob.S/BS), number of osteoblast per millimeter of bone surface (N.Ob/BS), percent osteoclast surface (Oc.S/BS), and number of osteoclast per millimeter of bone surface (N.Oc/BS) on the same area of the distal femoral metaphysis as described above (19Parfitt A.M. Drezner M.K. Glorieux F.H. Kanis J.A. Malluche H. Meunier P.J. Ott S.M. Recker R.R. J. Bone Miner. Res. 1987; 2: 595-610Crossref PubMed Scopus (4953) Google Scholar, 20Jee W.S.S. Li X.J. Inoue J. Jee K.W. Haba T. Ke H.Z. Setterberg R.B. Ma Y.F. Takahashi H. Handbook of Bone Morphology. Nishimusa, Niigata City, Japan1997: 87-112Google Scholar). The right distal half of the femur from 10-month-old animals was dehydrated and embedded in methyl methacrylate. 10-μm longitudinal sections were prepared and left unstained for the determination of mineral apposition rate, bone formation rate/bone surface referent (BFR/BS), and bone formation rate/tissue volume referent (BFR/TV) on the distal femoral metaphysis (19Parfitt A.M. Drezner M.K. Glorieux F.H. Kanis J.A. Malluche H. Meunier P.J. Ott S.M. Recker R.R. J. Bone Miner. Res. 1987; 2: 595-610Crossref PubMed Scopus (4953) Google Scholar, 20Jee W.S.S. Li X.J. Inoue J. Jee K.W. Haba T. Ke H.Z. Setterberg R.B. Ma Y.F. Takahashi H. Handbook of Bone Morphology. Nishimusa, Niigata City, Japan1997: 87-112Google Scholar). An Image Analysis System (Osteomeasure, Inc., Atlanta, GA) was used for all histomorphometric analysis. Histomorphometric measurements were performed on cancellous bone tissue of the distal femoral metaphyses between 0.5 and 2 mm proximal to the growth plate-epiphyseal junction and extended to the endocortical surface in the lateral dimension (21Ke H.Z. Brown T.A. Chidsey-Frink K.L. Qi H. Crawford D.T. Simmons H.A. Petersen D.N. Allen M.R. McNeish J.D. Thompson D.D. J. Musculoskel. Neuron. Interact. 2002; 2: 479-488PubMed Google Scholar). Long bones were isolated from 3- to 6-month-old animals. Femur and tibia bone marrow cells harvested by centrifugation were plated at a density of 15 × 106 cells per 100-mm plate in α-minimal essential medium (Invitrogen), with 10% fetal bovine serum and 50 μg/ml Gentamicin (Invitrogen). 50 μg/ml l-ascorbic acid, and 10 mm β-glycerophosphate were added at day 10 of culture. Cultures were fed three times per week, maintained for 4 weeks, and then stained by the Von Kossa method (22Kossa J.von Beitr. Pathol. Anat. Allg. Pathol. 1901; 29: 163-202Google Scholar). Calvaria were dissected from postnatal day 3 mice. 0.2 mg/ml collagenase P/0.25% Trypsin digestions were performed as described previously (23Owen T.A. Aronow M. Shalhoub V. Barone L.M. Wilming L. Tassinari M.S. Kennedy M.B. Pockwinse S. Lian J.B. Stein G.S. J. Cell. Physiol. 1990; 143: 420-430Crossref PubMed Scopus (1411) Google Scholar). The cells liberated in the second digest were plated at 2 × 104cells/cm2. At confluence, cultures were supplemented with 50 μg/ml l-ascorbic acid and 10 mmβ-glycerophosphate. Cells were later harvested for RNA or Alizarin Red staining (24Stanford C.M. Jacobson P.A. Eanes E.D. Lembke L.A. Midura R.J. J. Biol. Chem. 1995; 270: 9420-9428Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Stain solubilization and quantitation were as described (25Schiller P.C. D'Ippolito G. Roos B.A. Howard G.A. J. Bone Miner. Res. 1999; 14: 1504-1512Crossref PubMed Scopus (100) Google Scholar). Bone marrow cultures were established as described above, plated at 1 × 106 cells per well in a 24-well plate. Cells were stimulated with 10 nm1,25-dihydroxyvitamin D3 and TRAP stained as described (26Takahashi N. Yamana H. Yoshiki S. Roodman G.D. Mundy G.R. Jones S.J. Boyde A. Suda T. Endocrinology. 1988; 122: 1373-1382Crossref PubMed Scopus (702) Google Scholar). Bone resorption was assessed by culturing bone marrow cells on bone slices for 21 days and counting the resorption pits as has been described by Grasser et al. (27Grasser W.A. Pan L.C. Thompson D.D. Paralkar V.M. J. Cell. Biochem. 1997; 65: 159-171Crossref PubMed Scopus (18) Google Scholar). Statistical significance of in vivo and in vitro parameters was determined by two-tailed Student's t test. A p value of less than 0.05 was considered statistically significant. MouseOF45 was cloned using low stringency hybridization of a ratOF45 sequence to a murine 129 strain lambda phage genomic library. The full-length cDNA was then cloned by RT-PCR and 5′- and 3′-rapid amplification of cDNA ends (RACE) from mouse tibia RNA. The complete cDNA was 1741 bp in length. An additional 1679-bp cDNA was cloned, which was identical to the longer, more abundant message with the exception of a shorter 3′-extension. Three early methionine codons allow alternative start sites for the OF45 protein, the first of which would result in a 441-amino acid protein. A Kyte-Doolittle hydrophilicity plot revealed that the peptide sequence contained a hydrophobic leader sequence followed by a hydrophilic protein (Fig. 1 A). Analysis of the amino acid sequence with PSORTII software, which predicts the subcellular localization sites of proteins from their amino acid sequences, indicated that this cDNA likely encodes an extracellular protein (28Nakai K. Kanehisa M. Genomics. 1992; 14: 897-911Crossref PubMed Scopus (1377) Google Scholar, 29McGeoch D.J. Virus Res. 1985; 3: 271-286Crossref PubMed Scopus (161) Google Scholar, 30von Heijne G. Nucleic Acids Res. 1986; 14: 4683-4690Crossref PubMed Scopus (4155) Google Scholar). The predicted site of cleavage would be between Ala-24 and Ala-25, yielding a 417-amino acid secreted peptide with a calculated molecular mass of 44247 Da. Experimental confirmation of this analysis was obtained by Western blot analysis of media from transiently transfected CHO cells. An OF45-specific antibody detected a secreted protein of ∼44 kDa (Fig. 1 B). An OF45 peptide product of lower molecular weight was also detected in variable amounts, suggesting that proteolytic processing or degradation occurred in the media. The predicted amino acid composition of the basic OF45 peptide (predicted pI 9.17) was rich in serine, glycine, and charged residues. Several consensus protein kinase C, casein kinase II, tyrosine kinase, and cAMP-dependant kinase phosphorylation sites, oneN-glycosylation site, and a SDGD glycosaminoglycan binding site offer the potential for post-translational modifications to increase the acidic character of the protein. Based on sequence and expression analysis, we believe that the mouse OF45cDNA reported here represents the mouse orthologue of the ratOF45 gene (10Petersen D.N. Tkalcevic G.T. Mansolf A.L. Rivera-Gonzalez R. Brown T.A. J. Biol. Chem. 2000; 275: 36172-36180Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) (GenBankTM accession number 260922). Alignment with the rat OF45 amino acid sequence demonstrated 67% identity. Importantly, key structural features were conserved. Both proteins were rich in serine, glycine, and charged amino acids and included an amino-terminal hydrophobic signal sequence that targeted the peptides for secretion. The RGD sequence at amino acids 183–185 is an element traditionally involved in cell-matrix interactions through integrin binding and signaling and was also conserved between species. Comparison of the OF45 cDNA sequence to GenBankTM using the BLAST 1.4 algorithm (31Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (73997) Google Scholar) showed 45% amino acid identity to the recently identified human matrix extracellular phosphoglycoprotein (MEPE) gene (32Rowe P.S. de Zoysa P.A. Dong R. Wang H.R. White K.E. Econs M.J. Oudet C.L. Genomics. 2000; 67: 54-68Crossref PubMed Scopus (323) Google Scholar). The macaque MEPE gene showed similar identity to OF45(GenBankTM accession number AB05025). The homology between mouse OF45 and human MEPE was distributed throughout the sequences and the RGD motif was conserved. MEPE was cloned as a candidate gene for a tumor-secreted phosphaturic factor responsible for tumor-induced osteomalacia. Like MEPE, OF45 shares structural features common to a class of extracellular matrix phosphoglycoproteins that includes osteopontin, dentin sialophosphoprotein, dentin matrix protein 1, and bone sialoprotein II. These genes are highly expressed in bone or dentin and play significant roles in mineralization (33Robey P.G. Connect. Tisssue Res. 1996; 35: 131-136Crossref PubMed Scopus (128) Google Scholar, 34Boskey A.L. Connect. Tissue Res. 1996; 35: 357-363Crossref PubMed Scopus (179) Google Scholar, 35D'Souza R.N. Cavender A. Sunavala G. Alvarez J. Ohshima T. Kulkarni A.B. MacDougall M. J. Bone Miner. Res. 1997; 12: 2040-2049Crossref PubMed Scopus (309) Google Scholar). The mouse OF45 sequence was also 100% identical to the mouseMEPE cDNA recently published by Argiro et al.(11Argiro L. Desbarats M.