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• W2104465349 abstract "Dentin matrix protein 1 (DMP1) is highly expressed in osteocytes and is mechanically responsive. To study osteocyte-specific and mechanically regulated DMP1 gene expression, the transcriptional activity of three cis-regulatory regions was first examined in an osteoblast differentiation model in vitro using a green fluorescent protein (GFP) reporter. Expression of the -9624 to +1996 bp (10 kb) and -7892 to +4439 bp (8 kb) DMP1 cis-regulatory regions dramatically increased in areas of mineralized matrix, in dendritic, osteocyte-like cells. Mineralizing cultures expressing the 8-kb construct show dramatic GFP increases in response to loading in cells with a dendritic morphology. Transgenic mice expressing the 8-kb DMP1-GFP and -2433 to +4439 bp (2.5 kb) DMP1-LacZ were generated. Osteocyte-specific expression was found with the 8 kb but not with the 2.5 kb in postnatal animals. However, the 2.5 kb could support expression in rapidly forming osteoblasts and pre-osteocytes in the embryo. Primary calvarial osteoblast cultures demonstrated that the 2.5 kb supports weak expression in a subset of osteoblasts and pre-osteocytes, but not in mature osteocytes. However, the 8 kb supports robust expression in primary bone marrow cultures. Therefore the region -7892 to -2433 bp, termed a 5.5-kb “Osteocyte Enhancer Module,” appears to be required for osteocyte specificity. Ulnae of mice with the 8-kb DMP1-GFP were subjected to mechanical loading where GFP expression increased selectively and locally in osteocytes, distal to the mid-shaft and near the surface of the bone. Thus, the 8-kb region of the DMP1 gene is a target for mechanotransduction in osteocytes, and its cis-regulatory activity may be correlated to local strain in bone. Dentin matrix protein 1 (DMP1) is highly expressed in osteocytes and is mechanically responsive. To study osteocyte-specific and mechanically regulated DMP1 gene expression, the transcriptional activity of three cis-regulatory regions was first examined in an osteoblast differentiation model in vitro using a green fluorescent protein (GFP) reporter. Expression of the -9624 to +1996 bp (10 kb) and -7892 to +4439 bp (8 kb) DMP1 cis-regulatory regions dramatically increased in areas of mineralized matrix, in dendritic, osteocyte-like cells. Mineralizing cultures expressing the 8-kb construct show dramatic GFP increases in response to loading in cells with a dendritic morphology. Transgenic mice expressing the 8-kb DMP1-GFP and -2433 to +4439 bp (2.5 kb) DMP1-LacZ were generated. Osteocyte-specific expression was found with the 8 kb but not with the 2.5 kb in postnatal animals. However, the 2.5 kb could support expression in rapidly forming osteoblasts and pre-osteocytes in the embryo. Primary calvarial osteoblast cultures demonstrated that the 2.5 kb supports weak expression in a subset of osteoblasts and pre-osteocytes, but not in mature osteocytes. However, the 8 kb supports robust expression in primary bone marrow cultures. Therefore the region -7892 to -2433 bp, termed a 5.5-kb “Osteocyte Enhancer Module,” appears to be required for osteocyte specificity. Ulnae of mice with the 8-kb DMP1-GFP were subjected to mechanical loading where GFP expression increased selectively and locally in osteocytes, distal to the mid-shaft and near the surface of the bone. Thus, the 8-kb region of the DMP1 gene is a target for mechanotransduction in osteocytes, and its cis-regulatory activity may be correlated to local strain in bone. Osteocytes are terminally differentiated cells derived from osteoblasts which first become embedded and surrounded by osteoid matrix that subsequently mineralizes (1Marotti G. Ferretti M. Muglia M.A. Palumbo C. Palazzini S. Bone. 1992; 13: 363-368Crossref PubMed Scopus (93) Google Scholar, 2Marotti G. Ital. J. Anat. Embryol. 1996; 101: 25-79PubMed Google Scholar, 3Knothe Tate M.L. J. Biomech. 2003; 36: 1409-1424Crossref PubMed Scopus (252) Google Scholar, 4Knothe Tate M.L. Adamson J.R. Tami A.E. Bauer T.W. Int. J. Biochem. Cell Biol. 2004; 36: 1-8Crossref PubMed Scopus (268) Google Scholar). The large cuboidal osteoblasts begins to produce the stellate shape of the osteocyte and produce large numbers of long cell processes that are in the 5- to 30-μm length range. In parallel the preosteocyte is being surrounded by an osteoid matrix and begins production of its own local matrix that is rich in molecules that will later prevent the osteocyte and its processes from direct contact with mineral and define the structure of the osteocyte lacunae and canalicular walls, pericellular matrix, and the unique cytoskeleton in the cell processes of the osteocyte (5Tanaka-Kamioka K. Kamioka H. Ris H. Lim S.S. J. Bone Miner. Res. 1998; 13: 1555-1568Crossref PubMed Scopus (166) Google Scholar, 6Hughes D.E. Salter D.M. Simpson R. J. Bone Miner. Res. 1994; 9: 39-44Crossref PubMed Scopus (100) Google Scholar, 7Nakamura H. Kenmotsu S. Sakai H. Ozawa H. Cell Tissue Res. 1995; 280: 225-233PubMed Google Scholar, 8You L.D. Weinbaum S. Cowin S.C. Schaffler M.B. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 2004; 278: 505-513Crossref PubMed Scopus (271) Google Scholar, 9Han Y. Cowin S.C. Schaffler M.B. Weinbaum S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16689-16694Crossref PubMed Scopus (380) Google Scholar). Osteocytes survive in the bone matrix through a mutually connected network of long dendritic processes surrounded by bone fluid within canaliculi that can communicate through gap junctions (10Doty S.B. Calcif Tissue Int. 1981; 33: 509-512Crossref PubMed Scopus (382) Google Scholar, 11Cheng B. Zhao S. Luo J. Sprague E. Bonewald L.F. Jiang J.X. J. Bone Miner Res. 2001; 16: 249-259Crossref PubMed Scopus (175) Google Scholar). It is well known that bone tissue has the capacity to alter its mass and structure in response to mechanical strain. Osteocytes are regarded as the mechanosensory cells that send signals to other bone cells to initiate bone formation and remodeling (12Burger E.H. Klein-Nulend J. FASEB J. 1999; 13: S101-S112Crossref PubMed Scopus (740) Google Scholar, 13Huiskes R. Ruimerman R. van Lenthe G.H. Janssen J.D. Nature. 2000; 405: 704-706Crossref PubMed Scopus (884) Google Scholar). Osteocytes are the most abundant cells in bone, but unique regulatory pathways controlling osteocyte biology have not been identified. Also, little is known of the processes responsible for osteocyte differentiation and formation from a mature osteoblast. DMP1 1The abbreviations used are: DMP1, dentin matrix protein 1; GFP, green fluorescent protein; MEM, minimal essential medium; MOPS, 4-morpholinepropanesulfonic acid; BMP2, bone morphogenic protein 2; PBS, phosphate-buffered saline; dpc, days postcoitum; NLS, nuclear localization signal peptide. 1The abbreviations used are: DMP1, dentin matrix protein 1; GFP, green fluorescent protein; MEM, minimal essential medium; MOPS, 4-morpholinepropanesulfonic acid; BMP2, bone morphogenic protein 2; PBS, phosphate-buffered saline; dpc, days postcoitum; NLS, nuclear localization signal peptide. is an osteocyte-selective, extracellular matrix-associated phosphoprotein also found in other mineralizing tissues such as enamel, dentin, cementum, and bone (14George A. Gui J. Jenkins N.A. Gilbert D.J. Copeland N.G. Veis A. J. Histochem. Cytochem. 1994; 42: 1527-1531Crossref PubMed Scopus (80) Google Scholar, 15MacDougall M. Gu T.T. Luan X. Simmons D. Chen J. J. Bone Miner Res. 1998; 13: 422-431Crossref PubMed Scopus (135) Google Scholar). In cellular cementum, DMP1 is localized in cementocytes similar to osteocytes in bone. In odontogenesis or dentinogenesis, DMP1 is expressed in the early polarized odontoblast as the dental tubules are beginning to form (16Hao J. Zou B. Narayanan K. George A. Bone. 2004; 34: 921-932Crossref PubMed Scopus (79) Google Scholar). DMP1 belongs to the SIBLING (small integrin binding ligand N-linked glycoprotein) family of extracellular matrix proteins that includes osteopontin (Spp1), bone sialoprotein (IBsp), dentin sialophosphoprotein (DSPP), and MEPE/osteogenic factor 45 (Mepe). The SIBLING genes are found in a gene cluster on human chromosome 4q21, and inclusion of DMP1 in this group supports a potential role of this molecule in the process of mineralization of dentin and bone (17Aplin H.M. Hirst K.L. Crosby A.H. Dixon M.J. Genomics. 1995; 30: 347-349Crossref PubMed Scopus (47) Google Scholar, 18Fisher L.W. Torchia D.A. Fohr B. Young M.F. Fedarko N.S. Biochem. Biophys. Res. Commun. 2001; 280: 460-465Crossref PubMed Scopus (522) Google Scholar, 19MacDougall M. Simmons D. Gu T.T. Dong J. Connect Tissue Res. 2002; 43: 320-330Crossref PubMed Google Scholar, 20Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Crossref PubMed Scopus (190) Google Scholar). DMP1 is highly expressed and therefore a good marker for the osteocyte lineage (21Toyosawa S. Shintani S. Fujiwara T. Ooshima T. Sato A. Ijuhin N. Komori T. J. Bone Miner. Res. 2001; 16: 2017-2026Crossref PubMed Scopus (236) Google Scholar, 22Toyosawa S. Okabayashi K. Komori T. Ijuhin N. Bone. 2004; 34: 124-133Crossref PubMed Scopus (40) Google Scholar) and is specifically expressed along and in the canaliculi of osteocytes within the bone matrix suggesting a role for DMP1 in osteocyte function. The DMP1 gene is activated in response to mechanical loading in osteocytes in alveolar bone in response to tooth movement (23Gluhak-Heinrich J. Ye L. Bonewald L.F. Feng J.Q. MacDougall M. Harris S.E. Pavlin D. J. Bone Miner. Res. 2003; 18: 807-817Crossref PubMed Scopus (141) Google Scholar). Potential roles for DMP1 in osteocytes have been suggested and are related to the post-translational processing and modifications of the protein as a highly phosphorylated protein and regulator of hydroxyapatite formation (24He G. George A. J. Biol. Chem. 2004; 279: 11649-11656Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). It has been suggested that DMP1, depending on proteolytic processing and phosphorylation state, regulates local mineralization processes that are carried out within the lacunae and canaliculi of osteocytes in mature bone, thus keeping the lacunae and canaliculi open to allow bone fluid flow (23Gluhak-Heinrich J. Ye L. Bonewald L.F. Feng J.Q. MacDougall M. Harris S.E. Pavlin D. J. Bone Miner. Res. 2003; 18: 807-817Crossref PubMed Scopus (141) Google Scholar). Complex networks of canaliculi, containing osteocyte dendritic processes, penetrate bone; therefore, increases or decreases in canalicular volume or changes in canalicular structural integrity could alter the dynamics of fluid flow thereby altering responses of osteocytes under various physiological or pathological load conditions. A related function for DMP1 in osteocyte biology may be to define the structural, mechanical, and material properties of the canalicular and lacunae wall. The stiffness of this wall could play important roles in detecting and transmitting mechanical signals. Recently, we generated a mouse model containing a DMP1 region, -7892 to +4439 bp (8 kb), driving GFP and thus directing expression to osteocytes (30Kalajzic I. Braut D. Guo D. Jiang X. Kronenberg M.S. Mina M. Harris M.A. Harris S.E. Rowe D.W. Bone. 2004; 35: 74-82Crossref PubMed Scopus (177) Google Scholar). Our hypothesis is that a limited region of the DMP1 gene contains an early and late osteocyte-specific control module that also regulates responses to mechanical loading in an osteocyte-specific manner. Mechanical stimulation of mouse bones will activate the endogenous DMP1 gene through the cis-regulatory region containing the osteocyte-specific control regions. Moreover, osteoblasts containing the 8-kb DMP1 region driving a reporter, such as GFP or LacZ, should become active as they begin to differentiate into osteocytes and produce stellate morphology. Culture of Osteoblast 2T3 Cells—The osteoblast 2T3 cell line used in these experiments (25Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar, 26Chen D. Ji X. Harris M.A. Feng J.Q. Karsenty G. Celeste A.J. Rosen V. Mundy G.R. Harris S.E. J. Cell Biol. 1998; 142: 295-305Crossref PubMed Scopus (340) Google Scholar) is BMP2-responsive and produces a well organized mineralized matrix. The structure and mineral composition of the mineralized bone matrix produced by 2T3 cells is similar to that of normal woven bone and to mineralized bone matrix produced by primary cultures of fetal rat calvarial osteoblasts (27Bonewald L.F. Harris S.E. Rosser J. Dallas M.R. Dallas S.L. Camacho N.P. Boyan B. Boskey A. Calcif. Tissue Int. 2003; 72: 537-547Crossref PubMed Scopus (270) Google Scholar). 2T3 cells were plated into T-150 flasks (Costar Corp., Cambridge, MA) at a density of 1 × 104 cells/cm2 for RNA extraction or plated on 6-well plates (Costar Corp.) at density of 12 × 104 cells per well for matrix formation studies. These cells were cultured in α-minimal essential medium (Invitrogen) containing 7% fetal bovine serum (Invitrogen). Differentiation media, added at day 0 or at confluency, contained 5 mm β-glycerophosphate (glycerol 2-phosphate) and 80 μg/ml ascorbic acid. Recombinant BMP2 (human), a gift of Dr. John Wozney and the Genetics Institute, Inc./Wyeth Inc., was added at 40 ng/ml on days 0, 3, and 7 of the culture. Northern Analysis and in Situ Hybridization of the Endogenous DMP1 mRNA—Total RNA was isolated from 2T3 cells using RNAzol B reagent (Tel-test Inc., Houston, TX). Enrichment of polyadenylated RNA (poly(A)) was obtained using oligo(dT) cellulose columns. 0.5 μg of poly(A) RNA was denatured in RNA sample buffer with ethylene bromide (Sigma) and loaded onto a 1% agarose MOPS/EDTA/acetate gel containing 2.2 m formaldehyde. The gel was transferred to a Nytran Plus filter (Schleicher & Schüll) by capillary blotting with 20 × SSC for 16 h or overnight. The filter was then cross-linked by a UV cross-linker (Stratalinker, Stratagene, La Jolla, CA). Hybridization was carried out at 42 °C in 5× SSC, 50% formamide, 1× PE (recipe from Stratagene), and 150 μg/ml denatured salmon sperm DNA. After 1–2 h of prehybridization, DNA probes (DMP1, Osteocalcin, and glyceraldehyde-3-phosphate dehydrogenase) were added to the hybridization solution. Hybridization was carried out for 16 h or overnight at 42 °C. The filters were washed twice with 2× SSC, 0.1% SDS for 30 min each, once with 0.5× SSC, 0.1% SDS, and with 0.1× SSC, 0.1% SDS at 15 min each. All washes were done at 56 °C. The filter images were collected using a Cyclone phosphor system and analyzed using Optiquant software (PerkinElmer Life Sciences). The data were analyzed using Optiquant and transferred to Microsoft Excel for final calculation. For in situ hybridization studies, 2T3 cultures were allowed to form a mineralized matrix in the presence and absence of BMP2, as described above. Abundant mineralization occurs by 10–15 days in the BMP2-treated cultures. At different time points, the media was removed, and the cells were washed with diethylpyrocarbonate-treated PBS. The cells and matrix were allowed to remain in the PBS for 20–30 min on ice. The entire matrix and cell sheets were then gently removed from the plastic tissue culture well and placed in a 15-ml polypropylene tube with 5 ml of cold RNase-free 4% paraformaldehyde in PBS for 10 min. After the cells and matrix were washed and centrifuged for 5 min at 600 × g using cold PBS, the samples were then resuspended in 15% EDTA with 0.5% paraformaldehyde and allowed to decalcify for 3 days. The cell samples were washed with PBS, and the pellet after centrifugation was washed two times with 70% ethanol. The matrix and cells were resuspended in 70% ethanol, carried to 100% ethanol, and processed for paraffin embedding. Sections were then cut perpendicular to the plane of the matrix and cells and then processed for in situ hybridization as previously described for DMP1 (23Gluhak-Heinrich J. Ye L. Bonewald L.F. Feng J.Q. MacDougall M. Harris S.E. Pavlin D. J. Bone Miner. Res. 2003; 18: 807-817Crossref PubMed Scopus (141) Google Scholar). Stable Transfection of Cis-regulatory Regions of DMP1-GFPtopaz Constructs into 2T3 Osteoblasts—Based on DNA sequence conservation in non-coding regions of the human and mouse DMP1 genes (Fig. 2A), three constructs were designed using either GFP or LacZ as a reporter. These constructs were designed based on the hypothesis that the major cis-regulatory regions specific for osteocytes and for response to mechanical strain would be found in one or all of these DNA regions of the DMP1 gene. The constructs were: 1) -9624 bp of the 5′-flanking region, exon 1, and 1900 bp of the intron 1 (10 kb); 2) -7892 to +4439 bp, 5′-flanking region, exon 1 and having all of intron 1 and part of exon 2 (8 kb); and 3) -2433 to +4439 bp (2.5 kb). All three constructs were ligated to the pGFPtopaz reporter (28Kalajzic I. Kalajzic Z. Kaliterna M. Gronowicz G. Clark S.H. Lichtler A.C. Rowe D. J. Bone Miner. Res. 2002; 17: 15-25Crossref PubMed Scopus (316) Google Scholar). Construct 3 with the 2.5-kb region was also ligated to an NLS-containing LacZ reporter (20Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Crossref PubMed Scopus (190) Google Scholar). Construct 1 required that a splice acceptor site be placed 5′ of the GFP reporter before inserting the -9624 to +1996 bp region (10 kb) of the DMP1 gene. For comparative expression studies, we used the DMP1 knock-in mice with the LacZ reporter (20Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Crossref PubMed Scopus (190) Google Scholar), as a sensitive read-out of endogenous DMP1 expression. The DMP1 -9624 to +1996 bp (10 kb), -7892 to +4439 bp (8 kb), and -2433 to +4439 bp (2.5 kb) cis-regulatory regions driving GFP in combination with a 10-fold excess pCDNA3 were stably transfected into 2T3 cells using the lipofection method (29Strauss W.M. Methods Mol Biol. 1996; 54: 307-327PubMed Google Scholar) with G418 sulfate (Geneticin, Invitrogen) selection. Cells were plated and transfected using Lipofectamine Plus (Invitrogen). After 24 h, the cells were replated on culture dishes, 100 × 20 mm, at a density of 2–4 × 103 cells per dish. After 24 h, 400 μg/ml G418 was added every other day. Colonies appeared after 2 weeks in the pCDNA3 transfection group. Initially, a large number of clones where evaluated for fluorescence. Three to six clones were then selected based on detectable fluorescence expression. In some cases, to assure clonality, clones were subcloned. At least three clones with the -7892 to +4439 bp (8 kb) DMP1-GFPtopaz or the -9624 to +1996 bp (10 kb) DMP1-GFPtopaz were evaluated for changes in spatial GFP expression patterns during differentiation. A Nikon Eclipse TE300 inverted microscope with a fluorescein isothiocyanate-Texas Red dual fluorescent filter cube was used to evaluate GFP expression and recorded by taking Fluorescent Images using a charge-coupled device camera (Optronics Co., Goleta, CA) every other day and imaged using AnalySIS software (Soft Imaging System Co., Lakewood, CO). Fluid Flow Experiments—Mineralized bone nodules form between 8 and 13 days in which GFP expression was found associated with the osteoid and mineralized matrix and presumed osteocytes at different stages of differentiation. Recombinant BMP2 was used to accelerate the formation of the mineralized structures and was added to the cultures on Days 0 and 3 at 40 ng/ml. Addition at this level of BMP2 on only these two early days rather than throughout the culture period establishes a slower rate of mineralized nodule formation and assures well organized mineralized structures. The fluid shear responses of the DMP1 cis-regulatory regions in stably transfected mineralizing 2T3 cultures were then analyzed, after exposure to 16 dynes/cm2 (1.6 pascals) shear stress for 2 h using a computer-driven Flexcell Streamer Gold strain unit (Flexcell International Co., Hillsborough, NC). Post-incubation and evaluation after the 2 h of fluid flow was at 2 and 24 h. RNA was then extracted from the cultures and prepared as described above for Northern analysis. Generation of the 8-kb DMP1-GFP Transgenic Mice—Mouse DMP1 DNA sequences were used for generation of the GFP reporter line as described and characterized previously (30Kalajzic I. Braut D. Guo D. Jiang X. Kronenberg M.S. Mina M. Harris M.A. Harris S.E. Rowe D.W. Bone. 2004; 35: 74-82Crossref PubMed Scopus (177) Google Scholar). Generation of the 2.5-kb DMP1-GFP Transgenic Mice—The 2.5-kb DMP1 region was ligated to a nuclear localization signal containing a β-galactosidase cassette (LacZ) (20Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Crossref PubMed Scopus (190) Google Scholar). The 2.5-kb regulatory sequences of DMP1, consisting of 2433-bp promoter region, 95-bp non-coding exon I, the entire 4326-bp intron I, and the first 17 bp of the exon II (4 bp upstream of the translation initiation codon ATG) were cloned into the SalI site, upstream of the bacterial (NLS) galactosidase cDNA in pPD-LacZ-MH vector. The 2.5-kb DMP1-LacZ transgene was released by restriction endonuclease enzyme PmeI and purified by using a Qiaquick gel extraction kit (Qiagen). The purified transgene was microinjected into fertilized CD1 eggs to generate transgenic mice, as above. Tail DNA was isolated to screen transgenic mice by using PCR analysis (described below). The transgenic lines were maintained on a CD1 background. DMP1-LacZ Knock-in Heterozygote—An NLS LacZ cassette was inserted into the DMP1 knock-out/in allele and replaced Exon 6 as described previously (20Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Crossref PubMed Scopus (190) Google Scholar). The heterozygotes of these mice serve as a standard for endogenous expression of DMP1 in various embryos and tissues. PCR Genotyping—Transgenic mice/embryos and DMP1 knock-in heterozygous mice/embryos were genotyped by PCR analysis of genomic DNA extracted from tail samples. For the transgenic mice, the 2.5-kb DMP1-LacZ transgene-specific primers were used. The forward primer (5′-ACCTGGCTTCTTTTTAACCTG-3′) is located at +4178∼+4198, relative to the transcription start site of DMP1 gene. The reverse primer (5′-GGCAATATCGCGGCTCAGTTC-3′) is located at +61∼+81, relative to the ATG site of the NLS-galactosidase reporter gene (LacZ). The PCR product is 364 bp long. For the DMP1 heterozygous mice, DMP1-LacZ knock-in targeting gene-specific primers were used. The forward primer (5′-CTTGACTTCAGGCAAATAGTGACC-3′) is located at +9010 to +9033, relative to the transcription start site of DMP1 gene; the reverse primer (5′-GCGGAATTCGATAGCTTGGCTG-3′) is located at the 5′-junction of DMP1-LacZ knock-in targeting gene. The PCR product is 280 bp in length. The primers for the 8-kb DMP1-GFP mice are as described in Ref. 30Kalajzic I. Braut D. Guo D. Jiang X. Kronenberg M.S. Mina M. Harris M.A. Harris S.E. Rowe D.W. Bone. 2004; 35: 74-82Crossref PubMed Scopus (177) Google Scholar. LacZ Assay and Tissue Processing—LacZ staining was performed to determine the spatial and temporal activity of the transgene. To prepare the sectioned tissues from the DMP1 heterozygous and the transgenic mice, tissue samples were dissected and washed in cold PBS, and fixed in ice-cold 4% paraformaldehyde for 30 min to 1 h. The samples were then washed three times with PBS for 15–30 min each, cryoprotected in 15% sucrose in PBS at 4 °C until they moved to the bottom, and then placed in 30% sucrose in PBS overnight at 4 °C. They were then embedded in Tissue-Tek OCT at 60 °C. Blocks were cryosectioned at 10–12 μm, collected onto positively charged slides (Shandon Microscope Slides Superfrost (Plus)), and dried for 2 to 4 h at room temperature. For 1-month-old mice, the tissue samples were decalcified in 10% EDTA, pH 7.2–7.4, at 4 °C for 7 days after fixation and then processed for frozen section as described above. The LacZ-staining procedure has been previously described (31Lobe C.G. Koop K.E. Kreppner W. Lomeli H. Gertsenstein M. Nagy A. Dev. Biol. 1999; 208: 281-292Crossref PubMed Scopus (447) Google Scholar). Briefly, the slides were washed in PBS and then stained at 37 °C overnight (12–16 h) in freshly prepared LacZ-staining solution containing 0.02% Nonidet P-40, 0.01% sodium deoxycholate, 10 mm Tris (pH 8.0), 2 mm magnesium chloride, 5 mm potassium ferricyanide, 5 mm potassium ferrocyanide, and 0.5 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, Gold BioTechnology, Inc.) in PBS, protected from light. When the staining was complete, the slides were washed extensively in PBS. The slides were then counterstained with hematoxylin/eosin or eosin only, mounted with Permount, and photographed under light microscopy. Bone Marrow Culture—4-week-old mice were sacrificed by CO2 asphyxiation. Femurs and tibiae were removed, and soft tissue was dissected away from the bone. The epiphyseal growth plates were removed, and the marrow was collected by flushing with α-minimal essential medium culture medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum (Invitrogen) with a 25-gauge needle. Cell suspensions with single or double cells were prepared by passing the cell suspension through an 18-gauge needle several times. The suspension was then filtered through a 70-μm cell strainer (Falcon #2350, Fisher Scientific). Cells were plated at a density 1 × 106 cells/cm2 in 35-mm culture plates (Falcon, #3046, Fisher Scientific). Differentiation was induced as previously described (28Kalajzic I. Kalajzic Z. Kaliterna M. Gronowicz G. Clark S.H. Lichtler A.C. Rowe D. J. Bone Miner. Res. 2002; 17: 15-25Crossref PubMed Scopus (316) Google Scholar). Isolation and Culture of Primary Osteoblasts from 3-Day-old Mouse Calvariae—Osteoblasts from 3-day-old mice were isolated as described by Harris et al. (32Harris S.E. Feng J.Q. Harris M.A. Ghosh-Choudhury N. Wozney J. Mundy G.R. Mol. Cell. Different. 1995; 3: 137-155Google Scholar), with several modifications. Briefly, the calvariae was dissected free of soft tissue and incubated in the digestion solution containing 0.1% Collagenase, type I (Sigma, C-0130) and 0.2% Dispase II (Roche Applied Science, 165-859) in PBS at 37 °C with gentle shaking. The first 5-min digest was discarded, and the following five sequential 10-min digests were combined and plated in α-minimal essential medium containing 10 mm β-glycerophosphate (Sigma, G9891), 50 μg/ml l-ascorbic acid (Sigma, A4403), and 15% fetal bovine serum. The cells were grown to confluency over the next 5–6 days, trypsinized, counted, and frozen in liquid nitrogen for storage. Tail samples were then genotyped for the 2.5-kb DMP1-LacZ and the DMP1-LacZ knock-in animals. For bone nodule formation assay, the frozen cells were thawed and plated into T75 flasks and grown to confluency over the next 3 days in growth medium containing 10% fetal bovine serum in α-minimal essential medium. The cells were then trypsinized and plated in 12-well plates at an initial density of 6 × 104 cells per well. The cells were allowed to grow to confluency over the next 4 days. The medium was then changed to differentiation medium containing 5 mm β-glycerophosphate, 100 μg/ml fresh l-ascorbic acid, and 10% fetal bovine serum in α-minimal essential medium. The media was changed every 2 days. Recombinant BMP2 was added to increase the rate of nodule formation during the first 4 days at 20 ng/ml. Mechanical Loading of 8-kb DMP1-GFP Transgenic Mice—Under avertin-induced anesthesia (0.015 ml of 2.5% avertin/g of body weight), the right forelimbs of 4- to 7-month-old female mice were subjected to axially applied compressive loads of 2.4 Newtons using a haversine waveform (peak stress of 2.4 newtons at 2 Hz for 60 cycles, 30 s). Non-invasive loading was applied such that mechanical forces were transmitted from the flexed carpus to the olecranon (elbow) process. Due to the natural curvature of the ulna, this loading protocol results in bending-induced peak compressive and tensile strains on the medial and lateral surfaces, respectively, distal to the ulna mid-shaft (33Torrance A.G. Mosley J.R. Suswillo R.F. Lanyon L.E. Calcif. Tissue Int. 1994; 54: 241-247Crossref PubMed Scopus (244) Google Scholar). All procedures in the loading experiment were conducted in accordance with Institutional Animal Care and Use Committee guidelines. Histological Evaluation of GFP Expression—Bone tissue from 4-week-old male mice were dissected free of surrounding tissue, and then fixed in 4% paraformaldehyde/PBS (adjusted to pH 7.4 with 10 n NaOH) at 4 °C for 1–2 days. Bones were decalcified in 15% EDTA (adjusted to pH 7.1 with NH4OH) for 4 days and then placed in 30% sucrose overnight. Other tissues were processed in a similar manner without decalcification. The tissues were then embedded in tissue-embedding medium (Cryomatrix, Termo Shandon, Pittsburgh, PA) on dry ice. Bones and other tissues were cryosectioned in 5-μm-thin sections longitudinally. GFP expression in histological sections was directly observed and photographed using fluorescein isothiocyanate-Texas Red dual fluorescent filter cube of a Zeiss Axiovert 200M microscope and photographed by using an Axiocam digital camera. This cube allows the green GFP signal to be distinguished from the light red-yellow autofluorescent background located in the marrow space and minimizes the strong background fluorescence of decalcified bone. The mechan" @default.
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