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Role of the α2-Integrin in Osteoblast-specific Gene Expression and Activation of the Osf2 Transcription Factor

1998; Elsevier BV; Volume: 273; Issue: 49 Linguagem: Inglês

10.1074/jbc.273.49.32988

ISSN

1083-351X

Autores

Guozhi Xiao, Dian Wang, M. Douglas Benson, Gérard Karsenty, Renny T. Franceschi,

Tópico(s)

S100 Proteins and Annexins

Resumo

Extracellular matrix molecules such as type I collagen are required for the adhesion, migration, proliferation, and differentiation of a number of cell types including osteoblasts. Matrix components often affect cell function by interacting with members of the integrin family of cell surface receptors. Previous work showed that collagen matrix synthesis, induced by addition of ascorbic acid to cells, precedes and is essential for the expression of osteoblast markers and induction of the osteocalcin promoter in murine MC3T3-E1 cells. This later response requires OSE2, the promoter element recognized by Osf2 (also called Cbfa1/AML3/PEBP2αA), a recently identified osteoblast-specific transcription factor. Osteoblasts express several integrins including α2β1 which is a major receptor for type I collagen. This paper examines the role of the α2-integrin subunit in osteocalcin promoter activation and osteoblast differentiation. Disruption of α2-integrin-ECM interactions with a blocking antibody or DGEA peptide containing the cell-binding domain of type I collagen blocked activation of the mouse osteocalcin gene 2 promoter by ascorbic acid as well as induction of endogenous osteocalcin mRNA and mineralization. Furthermore, anti-α2-integrin blocking antibody or peptide reduced ascorbic acid-dependent binding of Osf2 to OSE2 without affecting levels of transcription factor mRNA. Time course studies revealed that ascorbic acid-dependent binding of Osf2 to OSE2 preceded increases in osteocalcin and bone sialoprotein expression and this increase in Osf2 binding was not accompanied by comparable changes in levels of transcription factor mRNA or protein. Taken together, these studies demonstrate that an α2-integrin-collagen interaction is required for activation of Osf2 and induction of osteoblast-specific gene expression. Furthermore, matrix signals may regulate Osf2 through a post-translational pathway or via an accessory factor. Extracellular matrix molecules such as type I collagen are required for the adhesion, migration, proliferation, and differentiation of a number of cell types including osteoblasts. Matrix components often affect cell function by interacting with members of the integrin family of cell surface receptors. Previous work showed that collagen matrix synthesis, induced by addition of ascorbic acid to cells, precedes and is essential for the expression of osteoblast markers and induction of the osteocalcin promoter in murine MC3T3-E1 cells. This later response requires OSE2, the promoter element recognized by Osf2 (also called Cbfa1/AML3/PEBP2αA), a recently identified osteoblast-specific transcription factor. Osteoblasts express several integrins including α2β1 which is a major receptor for type I collagen. This paper examines the role of the α2-integrin subunit in osteocalcin promoter activation and osteoblast differentiation. Disruption of α2-integrin-ECM interactions with a blocking antibody or DGEA peptide containing the cell-binding domain of type I collagen blocked activation of the mouse osteocalcin gene 2 promoter by ascorbic acid as well as induction of endogenous osteocalcin mRNA and mineralization. Furthermore, anti-α2-integrin blocking antibody or peptide reduced ascorbic acid-dependent binding of Osf2 to OSE2 without affecting levels of transcription factor mRNA. Time course studies revealed that ascorbic acid-dependent binding of Osf2 to OSE2 preceded increases in osteocalcin and bone sialoprotein expression and this increase in Osf2 binding was not accompanied by comparable changes in levels of transcription factor mRNA or protein. Taken together, these studies demonstrate that an α2-integrin-collagen interaction is required for activation of Osf2 and induction of osteoblast-specific gene expression. Furthermore, matrix signals may regulate Osf2 through a post-translational pathway or via an accessory factor. extracellular matrix ascorbic acid osteocalcin osteoblast-specific factor 2 core binding factor α mouse osteocalcin gene 2 monoclonal antibody Dulbecco's α-modified Eagle's medium fetal bovine serum phosphate-buffered saline bone sialoprotein mitogen-activated protein kinase fluorescein isothiocyanate. As a cell primarily devoted to matrix production, the osteoblast must have the ability to monitor the composition of the extracellular matrix (ECM)1 it is secreting as well as adapt matrix composition to the changing mechanical needs of bone. Consistent with the concept that there is a dialogue between the osteoblast and its ECM, osteoblast precursors must secrete a collagenous matrix before they will differentiate. Inhibition of collagen synthesis by growing cells in the absence of ascorbic acid (AA) or through the use of specific inhibitors totally blocks osteoblast differentiation (1Gerstenfeld L.C. Chipman S.D. Glowacki J. Lian J.B. Dev. Biol. 1987; 122: 49-60Crossref PubMed Scopus (357) Google Scholar, 2Owen 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 (1377) Google Scholar, 3Franceschi R.T. Iyer B.S. J. Bone Miner. Res. 1992; 7: 235-246Crossref PubMed Scopus (479) Google Scholar, 4McCauley L.K. Koh A.J. Beecher C.A. Cui Y. Rosol T.J. Franceschi R.T. J. Cell. Biochem. 1996; 61: 638-647Crossref PubMed Scopus (81) Google Scholar, 5Ibaraki K. Termine J.D. Whitson S.W. Young M.F. J. Bone Miner. Res. 1992; 7: 743-754Crossref PubMed Scopus (137) Google Scholar, 6Quarles L.D. Yohay D.A. Lever L.W. Caton R. Wenstrup R.J. J. Bone Miner. Res. 1992; 7: 683-692Crossref PubMed Scopus (816) Google Scholar). In vivo, both bone formation and osteoblast differentiation, as assessed by expression of osteocalcin (OCN) and alkaline phosphatase mRNAs, are also severely reduced in vitamin C-deficient animals (7Togari A. Arai M. Nakagawa S. Banno A. Aoki M. Matsumoto S. Jpn. J. Pharmacol. 1995; 68: 255-261Crossref PubMed Scopus (24) Google Scholar, 8Mahmoodian F. Gosiewska A. Peterkofsky B. Arch Biochem. Biophys. 1996; 336: 86-96Crossref PubMed Scopus (33) Google Scholar). Thus, the ECM is an important, but poorly understood regulator of the osteoblast differentiation pathway. Integrins are the principle mediators of the molecular dialogue between a cell and its ECM environment (for reviews, see Refs 9Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar and 10Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2929) Google Scholar). These transmembrane receptors convey information from the ECM to the intracellular compartment utilizing several signal transduction pathways including those involving focal adhesion kinase and mitogen-activated protein kinases (MAPK) (11Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2816) Google Scholar). The unique combinations of integrin subunits determines which ECM molecules will be recognized by a cell. For example, type I collagen interacts with α1β1, α2β1, and α3β1-integrins while fibronectin binds α3β1, α4β1, and α5β1 dimers. Osteoblasts express several integrin species including α1, α2, α3, α4, α5, αv, β1, β2, and β3 subunits (12Clover J. Dodds R. Gowen M. J. Cell Sci. 1992; 103: 267-271Crossref PubMed Google Scholar, 13Grzesik W.J. Robey P.G. J. Bone Miner. Res. 1994; 9: 487-496Crossref PubMed Scopus (324) Google Scholar, 14Hughes D. Salter S. Dedhar S. Simpson R. J. Bone Miner. Res. 1993; 8: 527-533Crossref PubMed Scopus (230) Google Scholar, 15Sinha R. Tuan R. Bone. 1996; 18: 451-457Crossref PubMed Scopus (177) Google Scholar, 16Gronthos S. Stewart K. Graves S.E. Hay S. Simmons P.J. J. Bone Miner. Res. 1997; 12: 1189-1197Crossref PubMed Scopus (215) Google Scholar) and their presence may be important in regulating the response of these cells to the ECM. Of particular interest, blocking α2-integrin-type I collagen interactions with specific peptides and antibodies prevents AA-dependent induction of alkaline phosphatase and differentiation-dependent down-regulation of transforming growth factor-β receptors in MC3T3-E1 preosteoblast cells suggesting that this integrin participates in the differentiation process (17Takeuchi Y. Nakayama K. Matsumoto T. J. Biol. Chem. 1996; 271: 3938-3944Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Osteoblast-specific factor 2 (Osf2), also known as AML3 (18Banerjee C. McCabe L.R. Choi J.Y. Hiebert S.W. Stein J.L. Stein G.S. Lian J.B. J. Cell. Biochem. 1997; 66: 1-8Crossref PubMed Scopus (398) Google Scholar) or PEBP2αA1/Cbfa1 (19Ogawa E. Maruyama M. Kagoshima H. Inuzuka M. Lu J. Satake M. Shigesada K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6859-6863Crossref PubMed Scopus (562) Google Scholar), is the bone-specific product of theCbfa1 gene (20Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3646) Google Scholar) and a possible mediator of the response of osteoblasts to ECM. Osf2 is one of three mammalian transcription factors related to the Drosophila protein, runt (21Levanon D. Negreanu V. Bernstein Y. Bar A. Avivi L. Groner Y. Genomics. 1994; 23: 425-432Crossref PubMed Scopus (381) Google Scholar).In vivo gene inactivation studies and in vitroand in vivo expression experiments indicate that Osf2 is a major regulator of the osteoblast phenotype and necessary for osteoblast-specific expression of the OCN gene (20Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3646) Google Scholar, 22Komori 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. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3651) Google Scholar, 23Otto 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. Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2412) Google Scholar, 24Lee B. Thirunavukkarasu K. Zhou L. Pastore L. Baldini A. Hecht J. Geoffroy V. Ducy P. Karsenty G. Nat. Genet. 1997; 16: 307-310Crossref PubMed Scopus (493) Google Scholar, 25Frendo J.-L. Xiao G. Fuchs S. Franceschi R.T. Karsenty G. Ducy P. J. Biol. Chem. 1998; 273: 30509-30516Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). We recently showed that AA increases mouse OCN gene 2 (mOG2) promoter activity approximately 20-fold in MC3T3-E1 preosteoblast cells. Significantly, this response, like the overall differentiation response of osteoblasts, requires collagen matrix synthesis and OSE2, the downstream promoter-binding site for Osf2. Induction of matrix synthesis is also accompanied by a dramatic increase in the binding of a protein in nuclear extracts (presumably Osf2) to OSE2, suggesting that ECM synthesis up-regulates and/or activates Osf2 (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar). The present study was undertaken to evaluate the role of α2-integrin-type I collagen interactions in the control of osteoblast-specific transcription and differentiation. As will be shown, blocking integrin-type I collagen binding prevents activation of the OCN promoter by AA and suppresses binding of Osf2 to OSE2 DNA. Interestingly, the dramatic stimulation of Osf2 activity and osteoblast-specific gene expression by AA is not accompanied by comparable increases in transcription factor mRNA or protein suggesting that the ECM activates this factor through either a post-translational pathway or via an accessory factor. Tissue culture medium and fetal bovine serum were obtained from HyClone (Logan, UT). [γ-32P]ATP and [α-32P]dCTP (3000 Ci/mmol) were purchased from Amersham (Arlington Heights, IL). Peptides (DGEA, RGDS, and RGES) were purchased from Advanced ChemTech (Louisville, KY) while the KDGE peptide was synthesized by the core facility of the University of Michigan. Blocking monoclonal antibodies (mAbs) to the mouse α2-integrin subunit (clone HMα2) or α5subunit (clone HMα5) and FITC-labeled anti-mouse α2-integrin antibody were purchased from PharMingen (San Diego, CA). FITC-labeled IgG (Hamster) was purchased from KPL (Gaithersburg, MA). Normal hamster IgG was obtained from Sigma. Mouse Osf2 antiserum was prepared in rabbits using a synthetic peptide having the following sequence: SFFWDPSTSRRFSPPS (amino acids 84–99 of the Osf2 sequence (20Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3646) Google Scholar)). This antiserum exhibited no detectable cross-reactivity with other members of the runt family of transcription factors on either gels retardation assays or Western blots (see Figs. 3 C and 5 A). All other chemicals were of analytical grade.Figure 5Time course of immunoreactive Osf2 protein levels and DNA binding activity. A, specificity of Osf2 antiserum. Whole cell extracts were prepared from the following cell lines as described under “Experimental Procedures”: 3T3L1 (L1), F9 (F9), C2C12 (C2), C3H10T1/2 (10T1/2), Jurkat (Jurkat), control MC3T3-E1 subclone 4 (4C), AA-treated subclone 4 (4AA), ROS 17/2.8 (ROS), and UMR106-01 (UMR). Aliquots containing 25 μg of protein were used for Western blotting. The blot was probed with Osf2 antiserum at a dilution of 1/1000. B, Osf2 protein levels. Subclone 4 cells were cultured in the presence (+) or absence (−) of AA for the indicated times. Whole cell extracts containing 2 μg of DNA equivalents were probed with Osf2 antiserum as in panel A. C, gel retardation assay. A separate group of cells were treated as in panel B and used as a source of nuclear extracts. Position of the major shifted species is noted by thearrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This study used two previously described subclones of the murine MC3T3-E1 preosteoblast cell line. Subclone 4 cells express high levels of osteoblast marker mRNAs and form a mineralized ECM after growth in AA-containing medium. Subclone 42 cells similarly exhibit a high differentiation/mineralization potential in the presence of AA and contain stably integrated copies of a 1.3-kilobase mouse osteocalcin gene 2 promoter fragment-firefly luciferase fusion gene (p1.3OG2-luc) that is induced by AA with a time course that parallels endogenous OCN mRNA (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar). Both subclones were plated at a density of 25,000 cells/cm2. Medium was changed after 48 h and cells cultured in the presence or absence of 50 μg/ml AA as described previously (27Franceschi R.T. Nutr. Rev. 1992; 50: 65-70Crossref PubMed Scopus (116) Google Scholar). Subclones were cultured in AA-free α-modified Eagle's medium (MEMα) containing 10% fetal bovine serum (FBS). UMR106-01 osteosarcoma cells, a generous gift from Dr. Ronald Midura (Cleveland Clinic, Cleveland, OH), were maintained in MEM, 10% FBS, 20 mm HEPES with 1 × non-essential amino acids. C2C12 myoblasts, a gift from Dr. Daniel Goldman (University of Michigan, Ann Arbor, MI), C3H10T1/2 mesenchymal cells (American Type Culture Collection), and 3T3-L1 mouse preadipocytes (American Type Culture Collection) were maintained in DMEM, 10% FBS. F9 teratocarcinoma cells (American Type Culture Collection) and ROS 17/2.8 osteosarcoma cells, a gift from Dr. Laurie McCauley (University of Michigan School of Dentistry), were grown in MEM, 10% FBS. The Jurkat human T-cell line was grown in RPMI 1640 medium containing 2.0 mml-glutamate, 1.5 g/liter sodium bicarbonate, 4.5 g/liter l-glucose, 10 mm HEPES. For immunofluorescence measurements, subclone 4 cells were cultured on glass slides for 2 days, fixed with 2% paraformaldehyde in PBS for 30 min, washed 3 × for 2 min with PBS containing 0.1% goat serum, and permeabilized with PBS containing 0.2% Triton X-100 for 20 min at 4 °C. Slides were blocked with DMEM containing 5% calf serum, 10% normal goat serum for 45 min at room temperature and washed 3 × for 2 min with PBS containing 0.1% goat serum. 50 μl of FITC-labeled anti-mouse α2-integrin Ab (0.1 μg/μl) or the same concentration of FITC-labeled control IgG in 5% FBS/DMEM was then added and slides were incubated for 1 h at room temperature. Slides were then washed 3 × 5 min with PBS containing 0.1% goat serum, mounted with anti-fade mountant, and examined with a Nikon Eclipse E800 fluorescence microscope and Optronics Digital Camera DEI-470 imaging system. For Western blots, whole cell extracts were prepared by rinsing cell layers in phosphate-buffered saline containing 100 μl of Sigma protease inhibitor mixture/107 cells. Inhibitor mixture contained 4-(2-aminoethyl)-benzenesulfonyl fluoride, pepstatin A,trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane, bestatin, leupeptin, and aprotinin and 1.0 mmphenylmethylsulfonyl fluoride. Cells were then harvested in 1 × SDS-polyacrylamide gel electrophoresis loading buffer (2% SDS, 2m urea, 10 mm dithiothreitol, 10% glycerol, 10 mm Tris-HCl, 0.002% bromphenol blue, 1.0 mmphenylmethylsulfonyl fluoride). Failure to use a combination of protease inhibitors led to the generation of proteolytic fragments of Osf2, particularly in AA-treated cells. To compensate for the increased protein in AA-treated samples, aliquots of whole cell extracts equivalent to 2 μg of DNA were subjected to SDS-polyacrylamide gel electrophoresis on 10% gels. Separated proteins were electrophoretically transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). Primary antibody (rabbit anti-mouse Osf2 antiserum) was used at a dilution of 1:1,000. Second antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG) was used at a dilution of 1:10,000. Blocking and reaction with antibodies was conducted as described previously (28Winnard R.G. Gerstenfeld L.C. Toma C.D. Franceschi R.T. J. Bone Miner. Res. 1995; 10: 1969-1977Crossref PubMed Scopus (69) Google Scholar). Immunoreactivity was determined using the ECL chemiluminescence reaction (Amersham). Films were scanned and subjected to image analysis using NIH Image version 1.61 software. Total RNA was isolated from cell layers according to the method of Chomczynski and Sacchi (29Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63189) Google Scholar). Poly(A)-containing RNA was purified by oligo(dT) cellulose chromatography (30Aviv H. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 1408-1412Crossref PubMed Scopus (5183) Google Scholar). Aliquots were fractionated on 1.0% agarose-formaldehyde gels and blotted onto nitrocellulose paper as described by Thomas (31Thomas P.S. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5201-5205Crossref PubMed Scopus (5861) Google Scholar). Mouse cDNA probes used for hybridization were obtained from the following sources: OCN (32Celeste A.J. Rosen V. Bueker J.L. Kriz R. Wang E.A. Wozney J.M. EMBO J. 1986; 5: 1885-1890Crossref PubMed Scopus (354) Google Scholar) from Dr. John Wozney (Genetics Institute, Boston, MA), bone sialoprotein (BSP) (33Young M.F. Ibaraki K. Kerr J.M. Lyu M.S. Kozak C.A. Mamm. Genome. 1994; 5: 108-111Crossref PubMed Scopus (94) Google Scholar) from Dr. Marion Young (NIDR, National Institutes of Health, Bethesda, MD), and mouse α2-integrin (34Wu J.E. Santoro S.A. Dev. Dyn. 1994; 199: 292-314Crossref PubMed Scopus (69) Google Scholar) from Dr. Samuel Santoro (Washington University, St. Louis, MO). The mouse Osf2 cDNA was recently described (20Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3646) Google Scholar). All cDNA inserts were excised from plasmid DNA with the appropriate restriction enzymes and purified by agarose gel electrophoresis before labeling with α-[32P]dCTP using a random primer kit (Boehringer-Mannheim). Hybridizations were performed as described previously using a Bellco Autoblot hybridization oven (35Franceschi R.T. Iyer B.S. Cui Y. J. Bone Miner. Res. 1994; 9: 843-854Crossref PubMed Scopus (389) Google Scholar) and quantitatively scanned using a Packard A2024 InstantImager. All values were normalized for RNA loading by probing blots with cDNA to 18 S rRNA (36Renkawitz R. Gerbi S.A. Glatzer K.H. Mol. Gen. Genet. 1979; 173: 1-13Crossref PubMed Scopus (59) Google Scholar). Subclone 42 cells were plated in 24-well plates and, after 2 days, cultures were treated with the indicated concentrations of antibodies or peptides in the presence or absence of AA. Medium was replenished every other day and cells harvested at day 6. Luciferase assays were performed using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI) and reagents and protocols from Promega (Madison, WI). The DNA content of each sample was then measured for normalization (37Schneider W.C. Methods Enzymol. 1957; 3: 680-684Crossref Scopus (1740) Google Scholar). To determine the mineralization potential of cultures, cells were grown with the indicated additions for 8 days. Inorganic phosphate was then added to a final concentration of 3.5 mm and, after 24 h, samples were stained using the Von Kossa method (38Marsh M.E. Munne A.M. Vogel J.J. Cui Y. Franceschi R.T. J. Bone Miner. Res. 1995; 10: 1635-1643Crossref PubMed Scopus (70) Google Scholar). Nuclear extracts were prepared and gel retardation assays conducted as described previously (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar). Each reaction contained 2 μg of DNA equivalents of nuclear extract. For supershift assays, anti-mouse Osf2 serum was incubated with nuclear extracts at 4 °C for 30 min prior to addition of labeled probe. All values are reported as mean ± S.D. based on triplicate independent cell cultures from a representative experiment. All experiments were repeated at least twice, and qualitatively identical results were obtained. Previous work from this laboratory showed that cell-collagen interactions are crucial for osteoblast differentiation and tissue-specific gene expression. Induction of osteoblast markers by AA is blocked by collagen synthesis inhibitors or digestion of the ECM with purified collagenase (35Franceschi R.T. Iyer B.S. Cui Y. J. Bone Miner. Res. 1994; 9: 843-854Crossref PubMed Scopus (389) Google Scholar). In a similar manner, AA-dependent mOG2 promoter activation also requires collagen synthesis (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar). Little is known concerning the mechanism of this regulation. One possibility explored in the experiments described below is that osteoblast differentiation requires an α2-integrin-type I collagen interaction. This integrin subunit is a component of the α2β1-integrin, a major binding site for collagens that has previously been detected in osteoblasts (14Hughes D. Salter S. Dedhar S. Simpson R. J. Bone Miner. Res. 1993; 8: 527-533Crossref PubMed Scopus (230) Google Scholar). As shown in Fig. 1, MC3T3-E1 cells clearly produce the α2-integrin subunit as detected by both immunofluorescence (panel A) and Northern blotting (panel B). Levels of a 5-kilobase α2-integrin mRNA increased with time in culture (result not shown), but were not substantially changed by AA treatment. To determine whether an α2-integrin-type I collagen interaction is required for activation of the osteocalcin promoter, specific peptides and antibodies were used to disrupt this and related interactions. These experiments used subclone 42 cells, a previously described, highly differentiating MC3T3-E1 subclone containing stably integrated copies of the mOG2 promoter driving expression of firefly luciferase. As previously reported, growth of these cells in the presence of AA induces OCN promoter activity as well as endogenous OCN and BSP mRNAs (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar). Induction of osteocalcin promoter activity was selectively inhibited by a DGEA peptide (Fig. 2 A) which is a component of the α2β1-integrin-binding domain of type I collagen (39Staatz W.D. Fok K.F. Zutter M.M. Adams S.P. Rodriguez B.A. Santoro S.A. J. Biol. Chem. 1991; 266: 7363-7367Abstract Full Text PDF PubMed Google Scholar) or a specific α2-integrin blocking antibody (Fig. 2 B). Inhibition was dose-dependent and highly specific in that a control peptide with the sequence, KDGE, an RGDS peptide which mimics the cell-binding domain of fibronectin and related proteins, the inactive fibronectin cell-binding domain variant, RGES, or an anti-α5-integrin blocking antibody were all totally inactive over the concentration range used for active inhibitors. Both the DGEA peptide and α2-integrin antibody were also found to be highly effective and selective inhibitors of mineralization, the end point for osteoblast differentiation (Fig. 2 C). Cells treated with inhibitors showed no signs of toxicity such as visible cell detachment or significant reduction in the amount of DNA per culture dish (control, 4.7 ± 0.2 μg of DNA/dish; 1.0 mm DGEA, 4.5 ± 0.1 μg of DNA/dish; 12 μg/ml of anti-α2 antibody, 4.4 ± 0.5 μg of DNA/dish). In an earlier study, we showed that AA-dependent activation of the OCN promoter requires OSE2, the cis-acting element in this promoter that is regulated by Osf2. Furthermore, nuclear extracts from AA-treated cells were found to contain more OSE2 binding activity (presumably Osf2) than controls (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar) (also see Fig. 5, below). Because peptide or antibody blocking of α2-integrin function inhibited osteoblast differentiation and transcriptional activity of the OCN promoter, we considered it important to examine the requirement for this integrin species in Osf2 activation as measured by its ability to bind OSE2 (Fig. 3). This and subsequent experiments used subclone 4 MC3T3-E1 cells grown for various times in the presence or absence of AA. Like the stably transfected subclone 42 cells described above, this subclone also readily differentiates after treatment with AA (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar). Cells were grown for 3 days in the presence of AA and the indicated peptides or antibodies before RNA or nuclear extracts were prepared. DGEA peptide or anti-α2-integrin antibody selectively suppressed expression of endogenous OCN mRNA (panel A) without affecting the Osf2 message. In contrast, binding of nuclear extracts to OSE2 was clearly inhibited by both of these treatments (panel B). Control peptide, IgG, and anti-α5-integrin antibody were all devoid of inhibitory activity. The shifted species generated when subclone 4 nuclear extracts were reacted with OSE2 did, in fact, contain Osf2 because it was supershifted by an anti-Osf2 antiserum (panel C). The specificity of the anti-Osf2 antiserum was confirmed by demonstrating that it was not able to interact with the shifted species generated between a Jurkat cell nuclear extract and OSE2 (also see Fig. 5 for further information on the specificity of this antiserum). Jurkat cells contain AML1 and -2, two related mammalian runt domain proteins that are also capable of binding the OSE2 core sequence (21Levanon D. Negreanu V. Bernstein Y. Bar A. Avivi L. Groner Y. Genomics. 1994; 23: 425-432Crossref PubMed Scopus (381) Google Scholar). Taken together, these experiments demonstrate that AA-dependent induction of the OCN gene, differentiation/mineralization of MC3T3-E1 preosteoblast cells, and stimulation of Osf2 transcription factor binding to OSE2 all require an α2-integrin-collagen interaction. As noted above, although inhibition of α2-integrin binding to collagen blocked osteoblast-specific gene expression and binding of Osf2 to OSE2, these treatments did not affect Osf2 mRNA levels. Assuming Osf2 mRNA and protein levels are related, this suggests that an integrin-mediated signal increases the transcriptionalactivity of Osf2 without affecting its actual levels. This hypothesis was explored further in the experiments shown in Figs. 4 and 5which compared the temporal relationship between Osf2 mRNA and protein with OSE2 binding activity and osteoblast marker mRNA expression during AA-induced differentiation. As previously reported, several days were required for full induction of OCN and BSP mRNAs (3Franceschi R.T. Iyer B.S. J. Bone Miner. Res. 1992; 7: 235-246Crossref PubMed Scopus (479) Google Scholar, 26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar, 35Franceschi R.T. Iyer B.S. Cui Y. J. Bone Miner. Res. 1994; 9: 843-854Crossref PubMed Scopus (389) Google Scholar) (Fig. 4). The first detectable increase in BSP message was seen after 2 days while 4 days were required for induction of the OCN mRNA. At day 6, AA treatment increased OCN and BSP mRNA levels approximately 12-fold (Fig. 4 B). Interestingly, levels of Osf2 mRNA were only slightly affected by AA treatment over the same time period. After 4 and 6 days, AA increased Osf2 mRNA only 1.5–2-fold. Western blots were next used to assess Osf2 protein levels in whole cell extracts (Fig. 5). In agreement with a previous report (18Banerjee C. McCabe L.R. Choi J.Y. Hiebert S.W. Stein J.L. Stein G.S. Lian J.B. J. Cell. Biochem.

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