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• W2047496722 abstract "Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I α1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of β-catenin distribution at the cell cortex and within the nucleus. Diminished β-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-β-catenin and active glycogen synthase kinase 3β, a kinase known to target β-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the β-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with β-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and β-catenin. We also determined that cell-cell contacts and cortically associated EB1/β-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation.Background: EB1 is a microtubule (MT) plus-end-binding protein known to influence MT stability.Results: EB1 is up-regulated in osteoblasts and is required for bone cell differentiation.Conclusion: EB1 affects β-catenin stability and cooperates at cell-cell adhesion sites to influence gene expression.Significance: Learning how peripherally targeted proteins interact at cell-cell contact sites is crucial for understanding developmental processes. Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I α1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of β-catenin distribution at the cell cortex and within the nucleus. Diminished β-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-β-catenin and active glycogen synthase kinase 3β, a kinase known to target β-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the β-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with β-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and β-catenin. We also determined that cell-cell contacts and cortically associated EB1/β-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation. Background: EB1 is a microtubule (MT) plus-end-binding protein known to influence MT stability. Results: EB1 is up-regulated in osteoblasts and is required for bone cell differentiation. Conclusion: EB1 affects β-catenin stability and cooperates at cell-cell adhesion sites to influence gene expression. Significance: Learning how peripherally targeted proteins interact at cell-cell contact sites is crucial for understanding developmental processes. The dynamic nature of bone involves an interplay between bone formation (osteogenesis) and born resorption (osteolysis). It is through constant bone remodeling that vertebrates are able to maintain constant bone mass in disease-free states. The tightly regulated balance of remodeling of bone involves osteoblasts that form new bone and osteoclasts that remove bone. Osteoclast ontogeny and the mechanisms that regulate bone resorption have been studied intensely (1.Asagiri M. Takayanagi H. The molecular understanding of osteoclast differentiation.Bone. 2007; 40: 251-264Crossref PubMed Scopus (1041) Google Scholar, 2.Boyle W.J. Simonet W.S. Lacey D.L. Osteoclast differentiation and activation.Nature. 2003; 423: 337-342Crossref PubMed Scopus (4897) Google Scholar), with much of the therapeutic treatments for bone-wasting disorders targeted toward osteoclasts (3.Edwards J.R. Weivoda M.M. Osteoclasts. Malefactors of disease and targets for treatment.Discov. Med. 2012; 13: 201-210PubMed Google Scholar). In light of the capability to target bone resorption mechanisms, there has been a more recent movement toward understanding how the osteoblastic differentiation mechanism can be targeted to enhance bone formation (4.Canalis E. Giustina A. Bilezikian J.P. Mechanisms of anabolic therapies for osteoporosis.N. Engl. J. Med. 2007; 357: 905-916Crossref PubMed Scopus (531) Google Scholar, 5.Hoeppner L.H. Secreto F.J. Westendorf J.J. Wnt signaling as a therapeutic target for bone diseases.Expert Opin. Ther. Targets. 2009; 13: 485-496Crossref PubMed Scopus (200) Google Scholar). Thus, understanding the molecular mechanisms that control osteoblast differentiation is paramount to identify therapeutic targets in bone wasting disorders. Mature osteoblasts originate from multipotent mesenchymal stem cells that are induced to differentiate toward an osteoblastic lineage by various factors. The canonical Wnt signaling plays a major role in osteoblast differentiation (6.Lin G.L. Hankenson K.D. Integration of BMP, Wnt, and notch signaling pathways in osteoblast differentiation.J. Cell. Biochem. 2011; 112: 3491-3501Crossref PubMed Scopus (346) Google Scholar), with a pool of soluble and highly unstable β-catenin transducing the Wnt signal in this pathway (7.Davis E.K. Zou Y. Ghosh A. Wnts acting through canonical and noncanonical signaling pathways exert opposite effects on hippocampal synapse formation.Neural Dev. 2008; 3: 32Crossref PubMed Scopus (113) Google Scholar). The signaling pathway is initiated by the ligation of the Wnt ligand to the Frizzled and LDL receptor-related protein 5/6 (LRP5/6) coreceptors, which initiates a signaling cascade that leads to an intracellular accumulation and nuclear recruitment of β-catenin (8.MacDonald B.T. Tamai K. He X. Wnt/beta-catenin signaling. Components, mechanisms, and diseases.Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (4151) Google Scholar). In the absence of Wnt ligands, cytoplasmic β-catenin is recruited into a destruction complex, which induces β-catenin phosphorylation, ubiquitination, and subsequent proteasomal degradation. Within the canonical pathway, Wnt stimulation promotes dissociation of the destruction complex, inhibits β-catenin degradation, and promotes the translocation of β-catenin to the nucleus and subsequent interaction with T-cell factor/lymphoid enhancer factor (TCF/LEF) 4The abbreviations used are: TCFT-cell factorLEFlymphoid enhancer factorMTmicrotubuleAPCadenomatous polyposis coliGSK-3βglycogen synthase kinase 3βAAascorbic acidIPimmunoprecipitationALPalkaline phosphatase. to initiate transcription of target genes (6.Lin G.L. Hankenson K.D. Integration of BMP, Wnt, and notch signaling pathways in osteoblast differentiation.J. Cell. Biochem. 2011; 112: 3491-3501Crossref PubMed Scopus (346) Google Scholar, 7.Davis E.K. Zou Y. Ghosh A. Wnts acting through canonical and noncanonical signaling pathways exert opposite effects on hippocampal synapse formation.Neural Dev. 2008; 3: 32Crossref PubMed Scopus (113) Google Scholar). The necessity of β-catenin in osteoblast differentiation and bone development is well established (9.Day T.F. Guo X. Garrett-Beal L. Yang Y. Wnt/β-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis.Dev. Cell. 2005; 8: 739-750Abstract Full Text Full Text PDF PubMed Scopus (1340) Google Scholar, 10.Hill T.P. Später D. Taketo M.M. Birchmeier W. Hartmann C. Canonical Wnt/β-catenin signaling prevents osteoblasts from differentiating into chondrocytes.Dev. Cell. 2005; 8: 727-738Abstract Full Text Full Text PDF PubMed Scopus (907) Google Scholar, 11.Hu H. Hilton M.J. Tu X. Yu K. Ornitz D.M. Long F. Sequential roles of Hedgehog and Wnt signaling in osteoblast development.Development. 2005; 132: 49-60Crossref PubMed Scopus (561) Google Scholar), wherein osteoblast precursors that lack β-catenin do not differentiate (10.Hill T.P. Später D. Taketo M.M. Birchmeier W. Hartmann C. Canonical Wnt/β-catenin signaling prevents osteoblasts from differentiating into chondrocytes.Dev. Cell. 2005; 8: 727-738Abstract Full Text Full Text PDF PubMed Scopus (907) Google Scholar). T-cell factor lymphoid enhancer factor microtubule adenomatous polyposis coli glycogen synthase kinase 3β ascorbic acid immunoprecipitation alkaline phosphatase. The differentiation of osteoblasts involves the sequential activation of at least two osteoblast-specific transcription factors, RUNX2 and osterix, which acts further downstream from RUNX2 (12.Nakashima K. de Crombrugghe B. Transcriptional mechanisms in osteoblast differentiation and bone formation.Trends Genet. 2003; 19: 458-466Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). RUNX2, also known as CBFA1, is considered a master regulator of transcription for early osteoblast genes (6.Lin G.L. Hankenson K.D. Integration of BMP, Wnt, and notch signaling pathways in osteoblast differentiation.J. Cell. Biochem. 2011; 112: 3491-3501Crossref PubMed Scopus (346) Google Scholar). The presence of a TCF regulatory element on the Runx2 promoter indicates that the canonical Wnt signaling pathway directly regulates Runx2 gene expression in pluripotent mesenchymal and osteoprogenitor cells via the recruitment of β-catenin to the Runx2 gene and thus contributes to osteoblast maturation (13.Gaur T. Lengner C.J. Hovhannisyan H. Bhat R.A. Bodine P.V. Komm B.S. Javed A. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression.J. Biol. Chem. 2005; 280: 33132-33140Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). Runx2 knock-out mice have a severe defect in intramembranous and endochondral ossification (14.Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts.Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3633) Google Scholar, 15.Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2403) Google Scholar). RUNX2 is expressed in early stages and throughout osteoblast differentiation and has been shown to bind to and regulate the expression of many osteoblast genes, with RUNX2 binding regions present in the promoter regions of osteocalcin, collagen, and bone sialoprotein genes (16.Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Osf2/Cbfa1. A transcriptional activator of osteoblast differentiation.Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3630) Google Scholar). Interestingly, the ectopic expression of RUNX2 in fibroblasts that are not committed to the osteoblast lineage induces the gene expression of the osteoblast-specific markers, including collagen, bone sialoprotein, and osteocalcin (16.Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Osf2/Cbfa1. A transcriptional activator of osteoblast differentiation.Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3630) Google Scholar). Aside from the role of β-catenin in the Wnt signaling pathway, β-catenin also has a secondary function at sites of cell-cell contacts at adherens junctions. The transmembrane cell adhesion molecule, E-cadherin, is a major component of adherens junctions in epithelial and other cell types (17.Boller K. Vestweber D. Kemler R. Cell-adhesion molecule uvomorulin is localized in the intermediate junctions of adult intestinal epithelial cells.J. Cell Biol. 1985; 100: 327-332Crossref PubMed Scopus (291) Google Scholar, 18.Collares-Buzato C.B. Jepson M.A. McEwan G.T. Simmons N.L. Hirst B.H. Junctional uvomorulin/E-cadherin and phosphotyrosine-modified protein content are correlated with paracellular permeability in Madin-Darby canine kidney (MDCK) epithelia.Histochemistry. 1994; 101: 185-194Crossref PubMed Scopus (29) Google Scholar, 19.Collares-Buzato C.B. McEwan G.T. Jepson M.A. Simmons N.L. Hirst B.H. Paracellular barrier and junctional protein distribution depend on basolateral extracellular Ca2+ in cultured epithelia.Biochim. Biophys. Acta. 1994; 1222: 147-158Crossref PubMed Scopus (72) Google Scholar) that recruits β-catenin and results in the coupling of E-cadherin to the Wnt pathway. The binding of β-catenin to type I cadherins renders a stable pool of membrane-bound β-catenin that regulates and stabilizes these cell-cell contacts (20.Nelson W.J. Nusse R. Convergence of Wnt, β-catenin, and cadherin pathways.Science. 2004; 303: 1483-1487Crossref PubMed Scopus (2234) Google Scholar, 21.Chigita S. Sugiura T. Abe M. Kobayashi Y. Shimoda M. Onoda M. Shirasuna K. CD82 inhibits canonical Wnt signalling by controlling the cellular distribution of β-catenin in carcinoma cells.Int. J. Oncol. 2012; 41: 2021-2028Crossref PubMed Scopus (18) Google Scholar). High resolution analysis has allowed understanding of the elaborate cell adhesion complex that includes cadherins, catenins, and the F-actin network (22.Ishiyama N. Ikura M. The three-dimensional structure of the cadherin-catenin complex.Subcell. Biochem. 2012; 60: 39-62Crossref PubMed Scopus (29) Google Scholar). Adherens junctions also have a microtubule (MT) component, wherein dynamic MTs recruit and control the regional distribution of cadherins at cell-cell contacts (23.Stehbens S.J. Paterson A.D. Crampton M.S. Shewan A.M. Ferguson C. Akhmanova A. Parton R.G. Yap A.S. Dynamic microtubules regulate the local concentration of E-cadherin at cell:cell contacts.J. Cell Sci. 2006; 119: 1801-1811Crossref PubMed Scopus (142) Google Scholar). MT plus-end binding proteins have been observed to target these adherens junctions (23.Stehbens S.J. Paterson A.D. Crampton M.S. Shewan A.M. Ferguson C. Akhmanova A. Parton R.G. Yap A.S. Dynamic microtubules regulate the local concentration of E-cadherin at cell:cell contacts.J. Cell Sci. 2006; 119: 1801-1811Crossref PubMed Scopus (142) Google Scholar, 24.Bellett G. Carter J.M. Keynton J. Goldspink D. James C. Moss D.K. Mogensen M.M. Microtubule plus-end and minus-end capture at adherens junctions is involved in the assembly of apico-basal arrays in polarised epithelial cells.Cell Motil. Cytoskeleton. 2009; 66: 893-908Crossref PubMed Scopus (51) Google Scholar, 25.Ligon L.A. Holzbaur E.L. Microtubules tethered at epithelial cell junctions by dynein facilitate efficient junction assembly.Traffic. 2007; 8: 808-819Crossref PubMed Scopus (64) Google Scholar, 26.Shaw R.M. Fay A.J. Puthenveedu M.A. von Zastrow M. Jan Y.N. Jan L.Y. Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions.Cell. 2007; 128: 547-560Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). The end-binding protein, EB1, is one of the best studied MT plus-end binding proteins that stabilizes MTs (27.Wen Y. Eng C.H. Schmoranzer J. Cabrera-Poch N. Morris E.J. Chen M. Wallar B.J. Alberts A.S. Gundersen G.G. EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration.Nat. Cell Biol. 2004; 6: 820-830Crossref PubMed Scopus (463) Google Scholar, 28.Zhang T. Zaal K.J. Sheridan J. Mehta A. Gundersen G.G. Ralston E. Microtubule plus-end binding protein EB1 is necessary for muscle cell differentiation, elongation and fusion.J. Cell Sci. 2009; 122: 1401-1409Crossref PubMed Scopus (45) Google Scholar) by forming comet-like structures at the tips of growing microtubules (29.Tirnauer J.S. Bierer B.E. EB1 proteins regulate microtubule dynamics, cell polarity, and chromosome stability.J. Cell Biol. 2000; 149: 761-766Crossref PubMed Scopus (217) Google Scholar, 30.Morrison E.E. Wardleworth B.N. Askham J.M. Markham A.F. Meredith D.M. EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle.Oncogene. 1998; 17: 3471-3477Crossref PubMed Scopus (194) Google Scholar). In conjunction with the EB3 family member, EB1 promotes continuous MT growth in cells by inhibiting MT catastrophes (31.Komarova Y. De Groot C.O. Grigoriev I. Gouveia S.M. Munteanu E.L. Schober J.M. Honnappa S. Buey R.M. Hoogenraad C.C. Dogterom M. Borisy G.G. Steinmetz M.O. Akhmanova A. Mammalian end binding proteins control persistent microtubule growth.J. Cell Biol. 2009; 184: 691-706Crossref PubMed Scopus (279) Google Scholar). Dynamic MT ends are required for the lateral movement and clustering of E-cadherin but are not necessary for E-cadherin surface display (23.Stehbens S.J. Paterson A.D. Crampton M.S. Shewan A.M. Ferguson C. Akhmanova A. Parton R.G. Yap A.S. Dynamic microtubules regulate the local concentration of E-cadherin at cell:cell contacts.J. Cell Sci. 2006; 119: 1801-1811Crossref PubMed Scopus (142) Google Scholar). EB1 has been shown to target to β-catenin puncta at the cell surface (24.Bellett G. Carter J.M. Keynton J. Goldspink D. James C. Moss D.K. Mogensen M.M. Microtubule plus-end and minus-end capture at adherens junctions is involved in the assembly of apico-basal arrays in polarised epithelial cells.Cell Motil. Cytoskeleton. 2009; 66: 893-908Crossref PubMed Scopus (51) Google Scholar, 26.Shaw R.M. Fay A.J. Puthenveedu M.A. von Zastrow M. Jan Y.N. Jan L.Y. Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions.Cell. 2007; 128: 547-560Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar) and co-localize with cadherins (23.Stehbens S.J. Paterson A.D. Crampton M.S. Shewan A.M. Ferguson C. Akhmanova A. Parton R.G. Yap A.S. Dynamic microtubules regulate the local concentration of E-cadherin at cell:cell contacts.J. Cell Sci. 2006; 119: 1801-1811Crossref PubMed Scopus (142) Google Scholar, 24.Bellett G. Carter J.M. Keynton J. Goldspink D. James C. Moss D.K. Mogensen M.M. Microtubule plus-end and minus-end capture at adherens junctions is involved in the assembly of apico-basal arrays in polarised epithelial cells.Cell Motil. Cytoskeleton. 2009; 66: 893-908Crossref PubMed Scopus (51) Google Scholar, 25.Ligon L.A. Holzbaur E.L. Microtubules tethered at epithelial cell junctions by dynein facilitate efficient junction assembly.Traffic. 2007; 8: 808-819Crossref PubMed Scopus (64) Google Scholar). The adenomatous polyposis coli (APC) tumor suppressor protein, which is also an MT plus-end protein, stabilizes complexes with the axin scaffolding protein and the two kinases, glycogen synthase kinase 3β (GSK-3β) and casein kinase 1α, to form the destruction complex and regulate β-catenin protein levels (32.Brocardo M. Henderson B.R. APC shuttling to the membrane, nucleus and beyond.Trends Cell Biol. 2008; 18: 587-596Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). EB1 has been identified in a binding screen for APC (33.Su L.K. Burrell M. Hill D.E. Gyuris J. Brent R. Wiltshire R. Trent J. Vogelstein B. Kinzler K.W. APC binds to the novel protein EB1.Cancer Res. 1995; 55: 2972-2977PubMed Google Scholar), and thus EB1 may target APC to MT plus-ends and thus enable the interactions of APC with cortical targets (29.Tirnauer J.S. Bierer B.E. EB1 proteins regulate microtubule dynamics, cell polarity, and chromosome stability.J. Cell Biol. 2000; 149: 761-766Crossref PubMed Scopus (217) Google Scholar). In addition, overexpression of EB1 has been found to promote cellular growth in cancer models via the β-catenin/TCF pathway (34.Fujii K. Kondo T. Yokoo H. Yamada T. Iwatsuki K. Hirohashi S. Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye.Proteomics. 2005; 5: 1411-1422Crossref PubMed Scopus (83) Google Scholar, 35.Wang Y. Zhou X. Zhu H. Liu S. Zhou C. Zhang G. Xue L. Lu N. Quan L. Bai J. Zhan Q. Xu N. Overexpression of EB1 in human esophageal squamous cell carcinoma (ESCC) may promote cellular growth by activating β-catenin/TCF pathway.Oncogene. 2005; 24: 6637-6645Crossref PubMed Scopus (75) Google Scholar, 36.Nishigaki R. Osaki M. Hiratsuka M. Toda T. Murakami K. Jeang K.T. Ito H. Inoue T. Oshimura M. Proteomic identification of differentially expressed genes in human gastric carcinomas.Proteomics. 2005; 5: 3205-3213Crossref PubMed Scopus (111) Google Scholar, 37.El-Rifai W. Frierson Jr., H.F. Harper J.C. Powell S.M. Knuutila S. Expression profiling of gastric adenocarcinoma using cDNA array.Int. J. Cancer. 2001; 92: 832-838Crossref PubMed Scopus (91) Google Scholar). Given the importance of the Wnt signaling cascade in osteoblast differentiation, in the present study, we identify how osteoblast differentiation is influenced by cytoskeletal elements, namely EB1, the MT plus-end-binding protein. We utilized the MC3T3-E1 mouse preosteoblast cell line to allow molecular manipulation of EB1 protein levels. We show that EB1 is significantly up-regulated in ascorbic acid (AA)-stimulated osteoblasts and that EB1 knockdown significantly impairs the osteoblast differentiation program. Through cell biology analysis, we determine that EB1 interacts with and influences the stability of β-catenin and identify EB1 as an important regulator of cell-cell adhesion-induced osteoblast differentiation. Fetal bovine serum was purchased from Wisent Inc. (St-Bruno, Canada). α-Minimal essential medium, Alexa Fluor 488, Oligofectamine, and Lipofectamine 2000 were purchased from Invitrogen. Rat and mouse monoclonal antibodies against EB1 were purchased from Abcam (Cambridge, UK) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. β-Catenin mouse monoclonal antibody was purchased from BD Transduction Laboratories (Mississauga, Canada). Mouse monoclonal active β-catenin antibody was purchased from Millipore (Billerica, MA). Phospho-β-catenin (Ser-33/37/Thr-41) rabbit polyclonal, GSK-3β, phospho-GSK-3α/β rabbit polyclonal (Ser-21/9), and GAPDH HRP rabbit polyclonal antibodies were purchased from Cell Signaling Inc. (Danvers, MA). Anti-E-cadherin rat monoclonal blocking antibody (ECCD-1) was purchased from Calbiochem. Phalloidin was purchased from Invitrogen. Anti-acetylated mouse monoclonal tubulin and mouse monoclonal actin antibodies were purchased from Sigma-Aldrich. Collagen type I α1 rabbit polyclonal antibody was purchased from Cedarlane (Burlington, Canada). All fluorescently labeled and horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The Pierce chemiluminescence kit was obtained from Thermo Fisher Scientific (Rockford, IL). l-Ascorbic acid powder and control isotype IgG from rat serum were purchased from Sigma-Aldrich. MC3T3-E1 subclone 4 was acquired from ATCC (American Type Culture Collection, Manassas, VA). The MC3T3-E1 preosteoblast cell line was routinely cultured in α-minimal essential medium without AA, supplemented with heat-inactivated 10% fetal bovine serum, and incubated in a humidified atmosphere at 37 °C with 5% CO2. For all experiments, osteoblasts grown to 80% confluence were passed using 0.05% trypsin and then plated onto 6-well plates (Starstedt Inc.) with 25-mm glass coverslips for immunofluorescent studies. For Western blotting and RNA extraction, cells were grown in 6-well plates without coverslips. For differentiation, osteoblasts were stimulated with AA (50 μg/ml) or 100 ng/ml rhBMP-2 (PeproTech, Rocky Hill, NJ), over a 5-day period, with medium and AA being replenished every second day. For primary cell analysis, cells were isolated from postnatal day 0 C57BL/6J mouse calvaria using a modification of a method described previously (38.Aubin J.E. Heersche J.N. Merrilees M.J. Sodek J. Isolation of bone cell clones with differences in growth, hormone responses, and extracellular matrix production.J. Cell Biol. 1982; 92: 452-461Crossref PubMed Scopus (182) Google Scholar, 39.Nabavi N. Urukova Y. Cardelli M. Aubin J.E. Harrison R.E. Lysosome dispersion in osteoblasts accommodates enhanced collagen production during differentiation.J. Biol. Chem. 2008; 283: 19678-19690Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). For plasmid transfection, osteoblasts were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For comparative experiments between low and high density growth, cells were plated at 10,000 and 100,000 cells/ml, respectively, and treated with AA for 5 days. For experiments involving E-cadherin-blocking antibody, cells were treated with isotype control IgG or a blocking antibody against E-cadherin (20 μg/ml) at days 2 and 4 during a 5-day stimulation with AA. Microarray data were obtained from Ref. 40.Nabavi N. Pustylnik S. Harrison R.E. RabGTPase mediated procollagen trafficking in ascorbic acid stimulated osteoblasts.PLoS One. 2012; 7: e46265Crossref PubMed Scopus (17) Google Scholar. MC3T3-E1 osteoblasts were grown in 6-well plates in triplicates and differentiated with AA (50 μg/ml) for 5 days. Total RNA from control and AA-treated osteoblasts was purified from cultured cells using the RNeasy minikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Samples were hybridized to MOE430.20 GeneChips (Affymetrix, Santa Clara, CA) at the Center for Applied Genomics at SickKids (Toronto, Canada) and analyzed as described previously (40.Nabavi N. Pustylnik S. Harrison R.E. RabGTPase mediated procollagen trafficking in ascorbic acid stimulated osteoblasts.PLoS One. 2012; 7: e46265Crossref PubMed Scopus (17) Google Scholar). -Fold changes in gene expression levels were analyzed using Student's unpaired t tests. A critical p value of 0.01 was considered as the criterion to select a significant -fold change in gene expression. Of the total 45,103 genes identified by (40.Nabavi N. Pustylnik S. Harrison R.E. RabGTPase mediated procollagen trafficking in ascorbic acid stimulated osteoblasts.PLoS One. 2012; 7: e46265Crossref PubMed Scopus (17) Google Scholar), EB1 and components of Wnt signaling were selected for further investigation in the present study. MC3T3-E1 cells treated with AA for 2 days were transfected with Oligofectamine according to the manufacturer's instructions using MISSION siRNA (SASI_Mm02_00312684, Sigma-Aldrich) directed against EB1 at a concentration of 20 μm for 72 h in addition to treatment with AA. Control cells were transfected with a MISSION® siRNA universal negative (scrambled) control (Sigma-Aldrich). Protein levels of siRNA-treated cells were analyzed by immunoblotting, immunofluorescence, and quantitative PCR 3 days after transfection. All expression constructs were created with standard PCR-based cloning strategies. Total RNA was isolated from cultured MC3T3-E1 cells using the RNeasy minikit from Qiagen (Hilden, Germany). The cDNA sequences of full-length EB1 and β-catenin were amplified with Superscript One-Step RT-PCR with a platinum Taq kit using the following primer pairs: EB1-F, 5′-aatgctagcaccatggcagtgaat-3′; EB1-R, 5′-attgaattcgatactcttcttgttcctc-3′; β-catenin-F, 5′-aagctagcaccatggctactcaagctgacc-3′; β-catenin-R, 5′-aagctagcaccatggctactcaagctgacc-3′. The resulting PCR products were cloned in frame to pRetroQ-mcherry-N1 and peGFP-N1 vectors, respectively, using NheI and EcoRI restriction sites for EB1 and NheI and BamHI for β-catenin. Quantitative RT-PCR was performed following a time course of ascorbic acid stimulation, in addition to measuring differences in mRNA levels of cells grown in low or high density environments. For quantitative RT-PCR, total mRNA from MC3T3-E1 cells grown in 6-well plates was extracted using the RNeasy kit (Qiagen, Hilden, Germany). The purity and quantity of RNA were confirmed by a NanoDrop® ND-100 spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized from 1 μg of total RNA in 50-μl reaction mixtures, using the SuperScript III First-Strand Synthesis SuperMix for quantitative RT-PCR from Invitrogen. A control cDNA serial dilution series of known concentration was constructed for each gene to establish a standard curve. Identical volumes of cDNA were loaded for all samples, and samples were run in triplicate. GAPDH was chosen as the reference gene for normalization of the results. The 25-μl real-time qua" @default.
• W2047496722 created "2016-06-24" @default.
• W2047496722 creator A5031900884 @default.
• W2047496722 creator A5038691946 @default.
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• W2047496722 date "2013-07-01" @default.
• W2047496722 modified "2023-10-15" @default.
• W2047496722 title "EB1 Levels Are Elevated in Ascorbic Acid (AA)-stimulated Osteoblasts and Mediate Cell-Cell Adhesion-induced Osteoblast Differentiation" @default.
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