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Expression of Galectin-3 in Skeletal Tissues Is Controlled by Runx2

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m207631200

1083-351X

Michael Stock, Henning Schäfer, Sigmar Stricker, Gerhard Groß, Stefan Mundlos, Florian Otto,

RNA modifications and cancer

The औ-galatoside-specific lectin galectin-3 is expressed in vivo in osteoblasts as well as in epiphyseal cartilage. Here we show that in vitro, galectin-3 expression is up-regulated in the preosteoblastic cell line MC3T3-E1 during the matrix maturation stage of the osteoblast developmental sequence. Expression persists into late differentiation stages when the mature osteoblastic phenotype is established. The skeletal expression pattern of galectin-3 overlaps at many sites with that of the transcription factor Runx2. Runx2 is a key regulator of osteoblast development and necessary for chondrocyte differentiation in the growth plate. Both human and mouse galectin-3 promoters contain putative Runx-binding sites. The constitutive or inducible forced expression of Runx2 is sufficient for the onset of galectin-3 transcription in the mesenchymal precursor cell line C3H10T1/2. Moreover, Runx2 is able to bind to at least two sites in the galectin-3 promoter region. The crucial role of Runx2 was confirmed in Runx2-deficient mice, which are devoid of galectin-3 expression in skeletal cells. The overlapping expression pattern of galectin-3 with the other two members of the Runt family of transcription factors (Runx1 and Runx3) points to a potential regulation of the galectin-3 gene (LGALS3) by these factors in hematopoietic, skin, and dorsal root ganglial cells. The औ-galatoside-specific lectin galectin-3 is expressed in vivo in osteoblasts as well as in epiphyseal cartilage. Here we show that in vitro, galectin-3 expression is up-regulated in the preosteoblastic cell line MC3T3-E1 during the matrix maturation stage of the osteoblast developmental sequence. Expression persists into late differentiation stages when the mature osteoblastic phenotype is established. The skeletal expression pattern of galectin-3 overlaps at many sites with that of the transcription factor Runx2. Runx2 is a key regulator of osteoblast development and necessary for chondrocyte differentiation in the growth plate. Both human and mouse galectin-3 promoters contain putative Runx-binding sites. The constitutive or inducible forced expression of Runx2 is sufficient for the onset of galectin-3 transcription in the mesenchymal precursor cell line C3H10T1/2. Moreover, Runx2 is able to bind to at least two sites in the galectin-3 promoter region. The crucial role of Runx2 was confirmed in Runx2-deficient mice, which are devoid of galectin-3 expression in skeletal cells. The overlapping expression pattern of galectin-3 with the other two members of the Runt family of transcription factors (Runx1 and Runx3) points to a potential regulation of the galectin-3 gene (LGALS3) by these factors in hematopoietic, skin, and dorsal root ganglial cells. trichostatin A electromobility shift assay CCAAT/enhancer-binding protein Galectin-3 (Mac-2, εBP, IgE-binding protein, CBP35, CBP30, L-29, and L-34) is one of ten members of the protein family of औ-galactoside specific lectins (1Leffler H. Masiarz F.R. Barondes S.H. Biochemistry. 1989; 28: 9222-9229Crossref PubMed Scopus (138) Google Scholar). It was first identified as an antigen on murine thioglycollate-elicited peritoneal macrophages (2Ho M.K. Springer T.A. J. Immunol. 1982; 128: 1221-1228PubMed Google Scholar). The molecular mass of galectin-3 ranges between 30 and 42 kDa in different species (3Nishiyama J. Kobayashi S. Ishida A. Nakabayashi I. Tajima O. Miura S. Katayama M. Nogami H. Am. J. Pathol. 2000; 157: 815-823Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The protein can exhibit intranuclear, cytoplasmatic, or extracellular localization. Lacking a signal peptide extracellular deposition of galectin-3 is mediated by a nonclassical pathway where it can bind to the membrane or associate with the extracellular matrix (4Barondes S.H. Cooper D.N. Gitt M.A. Leffler H. J. Biol. Chem. 1994; 269: 20807-20810Abstract Full Text PDF PubMed Google Scholar, 5Lindstedt R. Apodaca G. Barondes S.H. Mostov K.E. Leffler H. J. Biol. Chem. 1993; 268: 11750-11757Abstract Full Text PDF PubMed Google Scholar, 6Sato S. Burdett I. Hughes R.C. Exp. Cell Res. 1993; 207: 8-18Crossref PubMed Scopus (168) Google Scholar). Extracellular galectin-3 has been associated with modulation of cell adhesion in organogenesis, immunological processes, and tumorigenesis (7Bao Q. Hughes R.C. J. Cell Sci. 1995; 108: 2791-2800Crossref PubMed Google Scholar, 8Liu F.T. Immunol. Today. 1993; 14: 486-490Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 9Raz A. Lotan R. Cancer Metastasis Rev. 1987; 6: 433-452Crossref PubMed Scopus (294) Google Scholar). Intracellular galectin-3 has been implicated to play a role in pre-mRNA splicing (10Dagher S.F. Wang J.L. Patterson R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1213-1217Crossref PubMed Scopus (364) Google Scholar). The expression pattern of galectin-3 comprises various tissues and developmental stages. High levels of galectin-3 expression have been reported for blood cells such as activated macrophages, basophils, and mast cells as well as epithelial structures, e.g. skin, renal tubule cells, and human olfactory epithelium (3Nishiyama J. Kobayashi S. Ishida A. Nakabayashi I. Tajima O. Miura S. Katayama M. Nogami H. Am. J. Pathol. 2000; 157: 815-823Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 11Liu F.T. Frigeri L.G. Gritzmacher C.A. Hsu D.K. Robertson M.W. Zuberi R.I. Immunopharmacology. 1993; 26: 187-195Crossref PubMed Scopus (30) Google Scholar, 12Sato S. Hughes R.C. J. Biol. Chem. 1994; 269: 4424-4430Abstract Full Text PDF PubMed Google Scholar, 13Fowlis D. Colnot C. Ripoche M.A. Poirier F. Dev. Dyn. 1995; 203: 241-251Crossref PubMed Scopus (69) Google Scholar, 14Heilmann S. Hummel T. Margolis F.L. Kasper M. Witt M. Histochem. Cell Biol. 2000; 113: 241-245Crossref PubMed Scopus (23) Google Scholar). Galectin-3 is expressed in several skeletal tissues. Fowlis et al. (13Fowlis D. Colnot C. Ripoche M.A. Poirier F. Dev. Dyn. 1995; 203: 241-251Crossref PubMed Scopus (69) Google Scholar) reported the presence of galectin-3 mRNA and protein in the notochord and developing bones of the murine postimplantation embryo. Notochord expression was confirmed in an analysis of human embryos. Moreover, galectin-3 protein was detected in the human nucleus pulposus, the notochordal remnant within the intervertebral disc, and in chordoma, a tumor thought to originate from notochordal tissue (15Gotz W. Kasper M. Miosge N. Hughes R.C. Differentiation. 1997; 62: 149-157Crossref PubMed Google Scholar). Aubin et al. (16Aubin J.E. Gupta A.K. Bhargava U. Turksen K. J. Cell. Physiol. 1996; 169: 468-480Crossref PubMed Scopus (42) Google Scholar) reported galectin-3 expression in rat osteoblasts. In addition, its expression was shown in the epiphyseal cartilage and bone of neonatal mice (17Colnot C. Sidhu S.S. Poirier F. Balmain N. Cell Mol. Biol. 1999; 45: 1191-1202PubMed Google Scholar). The human galectin-3 gene LGALS3 (lectin,galactoside binding, soluble 3) was mapped to chromosome 14 at band 14q21–22. The human and murineLGALS3 genes are organized in six exons, with the translation start site located in exon 2 (18Kadrofske M.M. Openo K.P. Wang J.L. Arch. Biochem. Biophys. 1998; 349: 7-20Crossref PubMed Scopus (101) Google Scholar, 19Rosenberg I.M. Iyer R. Cherayil B. Chiodino C. Pillai S. J. Biol. Chem. 1993; 268: 12393-12400Abstract Full Text PDF PubMed Google Scholar). Genomic fragments encompassing nucleotides −836 to +141 relative to the transcription start site of human LGALS3 show significant promoter activity in reporter assays (18Kadrofske M.M. Openo K.P. Wang J.L. Arch. Biochem. Biophys. 1998; 349: 7-20Crossref PubMed Scopus (101) Google Scholar). Putative binding sites for transcription factors within the promoter region have been identified by sequence analysis in the same study. Nevertheless, factors regulating galectin-3 expression in vivo are still to be defined. Runx2 is one of three mammalian members of the runt family of transcription factors that bind to the consensus motif 5′-ACCPuCPu-3′ (20Levanon D. Negreanu V. Bernstein Y. Bar-Am I. Avivi L. Groner Y. Genomics. 1994; 23: 425-432Crossref PubMed Scopus (381) Google Scholar, 21Kamachi Y. Ogawa E. Asano M. Ishida S. Murakami Y. Satake M. Ito Y. Shigesada K. J. Virol. 1990; 64: 4808-4819Crossref PubMed Google Scholar, 22Melnikova I.N. Crute B.E. Wang S. Speck N.A. J. Virol. 1993; 67: 2408-2411Crossref PubMed Google Scholar, 23Zaiman A.L. Lewis A.F. Crute B.E. Speck N.A. Lenz J. J. Virol. 1995; 69: 2898-2906Crossref PubMed Google Scholar). Runx2 is a key regulator of osteoblast differentiation. The expression of Runx2 is restricted to osteoblasts, epiphyseal cartilage, nucleus pulposus, and mammary gland (24Otto 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 (2436) Google Scholar). Mice deficient in Runx2 expression are devoid of osteoblasts and bone (24Otto 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 (2436) Google Scholar, 25Komori 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 (3693) Google Scholar). Expression of several osteoblast-specific genes is regulated by Runx2 (26Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3683) Google Scholar). Furthermore, the transcription factor has been implicated to play a role in chondrocyte maturation (27Enomoto H. Enomoto-Iwamoto M. Iwamoto M. Nomura S. Himeno M. Kitamura Y. Kishimoto T. Komori T. J. Biol. Chem. 2000; 275: 8695-8702Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 28Kim I.S. Otto F. Zabel B. Mundlos S. Mech. Dev. 1999; 80: 159-170Crossref PubMed Scopus (400) Google Scholar). Therefore, we hypothesized that galectin-3 expression in these tissues might be controlled by Runx2. The data presented in this study support the finding of an involvement of galectin-3 in bone and cartilage development. We provide evidence for an up-regulation of galectin-3 transcription after constitutive and inducible forced expression of Runx2 in C3H10T1/2 cells. Furthermore we show the presence of several Runx consensus binding sites in the galectin-3 promoter and the ability of Runx2 to physically interact with some of these sites. Finally we show by in situhybridization that in contrast to wild type mice, Runx2-deficient mice lack expression of galectin-3 in long bones. The murine embryonic calvaria cell line MC3T3-E1 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) (29Sudo H. Kodama H.A. Amagai Y. Yamamoto S. Kasai S. J. Cell Biol. 1983; 96: 191-198Crossref PubMed Scopus (1501) Google Scholar). The cells were maintained in α-modified minimal essential medium supplemented with 107 fetal calf serum and 2 mml-glutamine (Invitrogen). The conditions for osteogenic differentiation were adopted from those reported for differentiation of fetal rat calvaria cells (30Owen 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 (1385) Google Scholar). When cells reached confluency, medium supplemented with 10 mm औ-glycerophosphate and 50 ॖg/ml ascorbic acid was used (Sigma-Aldrich). The medium was changed every 2 days. Osteogenic differentiation was monitored by alkaline phosphatase staining using Naphtol AS-TR phosphate and Fast Red RC tablets (Sigma-Aldrich), according to the manufacturer's instructions. C3H10T1/2 embryonic fibroblasts were obtained from the American Type Culture Collection (Manassas, VA). The C3H10T1/2 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 107 fetal calf serum and 2 mml-glutamine. The C3H10T1/2 cells were subcultured before reaching confluency. For stably transfected C3H10T1/2 cells (C3H10T1/2-Runx2) medium was supplemented with G418 at 600 ॖg/ml (Sigma-Aldrich). Stably transfected C3H10T1/2 clones with inducible Runx2 expression were grown with the addition of 150 ॖg/ml zeocin and 200 ॖg/ml hygromycin (both from Invitrogen). Cell clones with inducible expression of Runx2 were generated using the mifepristone-regulated expression system GeneSwitch (all of components were from Invitrogen). For stable transfections, 105C3H10T1/2 cells were incubated with 2.5 ॖg of plasmid DNA and 5 ॖl of FuGENE 6 reagent (Roche Molecular Biochemicals). After transfection with pSwitch, the clones were selected with 200 ॖg/ml hygromycin. Individual clones expressing the regulatory protein encoded by pSwitch were identified by transient transfection with pGene/V5 His/lacZ. 24 h after incubation in the presence of 30 nm mifepristone, the cells were stained for औ-galactosidase. The cells were fixed in 27 formaldehyde and 0.27 glutardialdehyde for 5 min at room temperature and subsequently stained using 5 mm K3Fe[CN]6, 5 mm K4Fe[CN]6, 2 mmMgCl2, and 1 mg/ml X-Gal. Two of the clones that proved to be inducible were stably transfected with pGene-Runx2 as described with the addition of 150 ॖg/ml Zeocin. The clones were screened for inducible expression of Runx2 by Western blotting after induction with 30 nm mifepristone for 24 h. For expression analysis in induced C3H10T1/2 clones, the medium was supplemented with 30 nm mifepristone (dissolved in ethanol), and the cells were harvested after 24–40 h. Control cells were grown for the same period in medium supplemented with the respective volume of ethanol. Pretreatment of inducible C3H10T1/2 cells with trichostatin A (TSA)1 was performed in standard medium supplemented with 25–200 nm TSA. After 20 h mifepristone was added to a final concentration of 30 nm to induce cells. All cDNAs were obtained by reverse transcriptase-PCR using total RNA purified from limbs of newborn mice. Reverse transcription was carried out using 2 ॖg of total RNA, random hexamer primers, and Superscript II reverse transcriptase (Invitrogen). Prior to PCR the cDNA was treated with ribonuclease H (Invitrogen). PCR reagents were from Qiagen. The primers used for reverse transcriptase-PCR were as follows: Runx2expressfor, 5′-TCACTACCAGCCACCCAGACCAA-3′; Runx2expressrev, 5′-CACTTATGAAAACAGACCAGACAACACCTT-3′; Runx2hybfor, 5′-AACCCACGGCCCTCCCTGAACTCT-3′; Runx2hybrev, 5′-TGACGTGACTGGCGGGGTGTAGGT-3′; Gal3for, 5′-TGGGAAAAGGAAGAAAGACAGTC-3′; and Gal3rev, 5′-GTTTCCCACTCCTAAGGCACAC-3′. Sequences of औ-actin primers were adopted from Gessner et al. (31Gessner A. Schroppel K. Will A. Enssle K.H. Lauffer L. Rollinghoff M. Infect. Immun. 1994; 62: 4112-4117Crossref PubMed Google Scholar), glyceraldehyde-3-phosphate dehydrogenase primers from a PCR-Select cDNA Subtraction kit (BD Clontech) and osteocalcin primers from Desboiset al. (32Desbois C. Hogue D.A. Karsenty G. J. Biol. Chem. 1994; 269: 1183-1190Abstract Full Text PDF PubMed Google Scholar). PCR products were cloned into TA cloning vector pCR2.1 (Invitrogen), and inserts were sequenced by a TaqDyeDeoxy Sequencing system (ABI, Weiterstadt, Germany). Runx2 cDNA was subcloned into expression vector pCMVऔ (BD Clontech) replacing the lacZ gene to generate pCMV-Runx2. Likewise, Runx2 cDNA was subcloned into inducible expression vector pGene/V5-His A (GeneSwitch System, Invitrogen). This vector is referred to as pGene-Runx2. The murine galectin-3 promoter from −1867 to +50 nucleotides relative to the transcription start site was amplified from Fvb murine genomic DNA and cloned into pBlue-TOPO (Invitrogen) 5′ to the lacZgene. The following primers were used: LGALS3p2000for, 5′-CTCTGCGAGCTTGTAAGTCTATCCTA-3′, and LGALS3rev, 5′-CGCTCACCTGATTAGTGCTCC-3′. Screening of DNA sequences for putative transcription factor-binding sites was performed using the web-based prediction programs MatInspector (Transfac; transfac.gbf.de/) and TFSEARCH (www.cbrc.jp/research/db/TFSEARCH.html). The MatInspector thresholds for core similarity and matrix similarity were set to 0.85 and 0.90, respectively. The TFSEARCH minimum score was set to 90.0 points. Putative Runx-binding sites were identified by searching for sequences matching the published consensus motif (21Kamachi Y. Ogawa E. Asano M. Ishida S. Murakami Y. Satake M. Ito Y. Shigesada K. J. Virol. 1990; 64: 4808-4819Crossref PubMed Google Scholar, 22Melnikova I.N. Crute B.E. Wang S. Speck N.A. J. Virol. 1993; 67: 2408-2411Crossref PubMed Google Scholar, 23Zaiman A.L. Lewis A.F. Crute B.E. Speck N.A. Lenz J. J. Virol. 1995; 69: 2898-2906Crossref PubMed Google Scholar) with special respect on structure data (33Tahirov T.H. Inoue-Bungo T. Morii H. Fujikawa A. Sasaki M. Kimura K. Shiina M. Sato K. Kumasaka T. Yamamoto M. Ishii S. Ogata K. Cell. 2001; 104: 755-767Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Thus the sequence was searched for the motif ACCPuCPu, and positions 2 and 3 (CC) were considered to be most important. Total RNA (15 ॖg/lane) was resolved on a 17 formaldehyde-agarose gel and transferred onto a Hybond N+ nylon membrane (Amersham Biosciences) using 10× SSC. The probes were labeled with [α-32P]dCTP (3000 Ci/mmol;Amersham Biosciences) using a Megaprime DNA labeling system (Amersham Biosciences). The blots were prehybridized and hybridized at 65 °C in Church buffer (500 mm phosphate buffer, pH 7.2, 77 (w/v) SDS, 1 mm EDTA, 100 ॖg/ml salmon sperm DNA, adapted from Church et al. (34Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7275) Google Scholar)). The blots were washed in 2× SSC, 0.17 SDS at room temperature for 15 min and twice in 0.1× SSC, 0.17 SDS at 60 °C for 20 min. The blots were exposed to Kodak XAR-5 film at −70 °C using two intensifier screens. For Western blot analysis of Runx2 expression, the cells were washed in phosphate-buffered saline and lysed in Laemmli buffer. The protein samples were resolved by SDS-PAGE through denaturing 107 polyacrylamide gels (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207843) Google Scholar). After electrophoresis, the protein was transferred onto Hybond P membranes (Amersham Biosciences) by semidry electroblotting (36Maniatis T.F. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). Afterward the membranes were blocked in 57 skimmed milk in TBST buffer (10 mm Tris-HCl, 15 mm NaCl, 0.057 Tween 20, pH 8.0) overnight. For immunodetection of Runx2 protein, the blots were incubated in 57 skimmed milk in TBST buffer with 70 ॖg/ml rabbit polyclonal anti-Runx2 antibody (anti-AML3 Ab-1; Calbiochem Corp., San Diego, CA) for 90 min. The blots were washed in TBST and incubated in 57 skimmed milk in TBST buffer with a 1:2000 dilution of secondary antibody goat anti-rabbit IgG-horseradish peroxidase conjugate (Santa Cruz Biotechnology, Santa Cruz, CA) for 60 min. After washing with TBST and Tris-buffered saline (10 mm Tris-HCl, 15 mmNaCl, pH 8.0) specific protein was visualized by chemiluminiscence using the ECLplus system (Amersham Biosciences) according to the manufacturer's instructions. For in vitro translation Runx2 cDNA was subcloned into pGEM-3Zf(+). In vitro translation was carried out using TnT-coupled wheat germ Extract system (Promega) according to the supplier's instructions. In vitro translated protein was analyzed by Western blot. Double-stranded galectin-3 promoter-derived oligonucleotides were designed with 5′-G overhangs for labeling with [α-32P]dCTP, and oligonucleotide Oligo A for binding control was adopted from Tahirov et al. (33Tahirov T.H. Inoue-Bungo T. Morii H. Fujikawa A. Sasaki M. Kimura K. Shiina M. Sato K. Kumasaka T. Yamamoto M. Ishii S. Ogata K. Cell. 2001; 104: 755-767Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). For oligonucleotide sequences, please refer to Fig. 6A. Labeling reactions were performed using a Megaprime DNA labeling system (Amersham Biosciences). Labeled oligonucleotides were purified by column chromatography using Sephadex G25 Quickspin columns (Roche Molecular Biochemicals). For binding reactions, 20-ॖl samples were prepared containing 3 ॖl in vitro translation reaction, 5 ॖl of labeled double-stranded oligonucleotide (20000–25000 cpm), 1 or 4 ॖl of respective unlabeled competitor oligonucleotide (resulting in 15- or 60-fold molar excess of competitor, respectively), 1 ॖl of pepstatin, and 1 ॖl of poly(dI-dC) (1A260/ॖl) in 27 glycerol, 5 mm Tris-HCl, 0.2 mm EDTA, 0.017 Nonidet-P 40, 0.1 mm dithiothreitol, 17.5 mm NaCl, 10 ॖg/ml bovine serum albumin, pH 7.5. The binding reactions were incubated for 30 min at room temperature and resolved on a 57 polyacrylamide gel in 0.5× TBE. The gels were dried and then exposed to Kodak XAR-5 film at −70 °C using intensifier screens. In situ hybridizations using 33P-labeled antisense riboprobes were carried out as previously described (37Vortkamp A. Lee K. Lanske B. Segre G.V. Kronenberg H.M. Tabin C.J. Science. 1996; 273: 613-622Crossref PubMed Scopus (1669) Google Scholar). Briefly, the sections were deparaffinized, rehydrated, pretreated with proteinase K (3 min, 10 ॖg/ml at room temperature), and hybridized overnight at 70 °C. The next day the slides were washed and dipped in photoemulsion (Kodak), dried, and exposed for 2–8 days at 4 °C. The slides were counterstained with Toluidine blue-O mounted with entellan. MC3T3-E1 is a pre-osteoblastic cell line derived from murine calvaria cells. These cells have been demonstrated to undergo osteogenic differentiation in response to prolonged confluent culture (29Sudo H. Kodama H.A. Amagai Y. Yamamoto S. Kasai S. J. Cell Biol. 1983; 96: 191-198Crossref PubMed Scopus (1501) Google Scholar). To investigate galectin-3 gene expression during osteogenesis, we performed an in vitro differentiation assay based on MC3T3-E1 cells. The cells were grown in nonsupplemented medium until confluency. (The first day of confluent culture is referred to as day 0.) From day 0, osteogenic differentiation was supported by medium supplemented with औ-glycerophosphate and ascorbic acid as described under "Materials and Methods." The cells were harvested at days 0, 4, 8, 12, 16, and 20, and the total RNA was isolated for Northern blot analysis. In parallel, the cells were stained for alkaline phosphatase protein as a marker for the intermediate stage of osteogenic differentiation. Histochemically distinct alkaline phosphatase-positive cells were seen as early as day 4 post-confluence (Fig.1A). As specific marker for late stages of osteogenic differentiation, osteoclacin mRNA synthesis was assessed by Northern blot. Osteocalcin mRNA became detectable at very low levels already at day 4 post-confluence and is expressed at highest levels on day 20 post-confluence, indicating efficient development of the mature osteoblastic phenotype at this late stage of cultivation (Fig. 1B). Galectin-3 mRNA was expressed in vitro at any time point during the differentiation kinetics in MC3T3-E1 cells. However, galectin-3 mRNA levels increased continuously during ongoing osteoblastic differentiation up to day 8 post-confluence when expression levels reached a maximum during central matrix maturation stage of the osteoblast developmental sequence. Thereafter, galectin-3 mRNA expression persists at considerable levels into later stages of osteogenic differentiation (day 20 post-confluence) (Fig.1B). Thus galectin-3 mRNA expression paralleled the transition of the cell line MC3T3-E1 from the fibroblastic to a distinct osteoblastic phenotype as assessed by the expression of markers typical for osteoblastic differentiation in these cells. Although already present at the preosteoblastic stage, galectin-3 expression increases during ongoing osteogenic differentiation and persists at high levels late into the osteoblast developmental sequence in vitro. To elucidate the mechanism by which galectin-3 expression is up-regulated during osteogenic differentiation in MC3T3-E1 cells, we directly sequenced 2.0 kb of the promoter region of the murine galectin-3 (Fvb mouse strain; pGal3–2000) and screened for transcription factor-binding sites in the promoter region that might be involved in establishing or maintaining the osteoblastic lineage. To elucidate biologically relevant transcription factor-binding sites, we scanned 900 bp of the 5′-flanking region of the murine and 836 bp of the corresponding 5′-flanking region of the human LGALS3 gene (GenBankTM accession number AF031421.1). The sequences were examined using the prediction programs MatInspector and TFSEARCH and were compared with find binding motifs conserved between both the human and murine promoters. As reported previously, the promoter regions of either species do not contain a TATA box, and CAAT motifs are missing. A GC-rich region harboring an SP1-binding site is located immediately upstream the transcription start site in both murine and human promoters, which is typical for TATA-less promoters (18Kadrofske M.M. Openo K.P. Wang J.L. Arch. Biochem. Biophys. 1998; 349: 7-20Crossref PubMed Scopus (101) Google Scholar, 19Rosenberg I.M. Iyer R. Cherayil B. Chiodino C. Pillai S. J. Biol. Chem. 1993; 268: 12393-12400Abstract Full Text PDF PubMed Google Scholar). We identified additional putative binding sites for AP1, AP4, C/EBPऔ, CDP CR, c-Ets, CP2, c-Rel, GATA proteins, GFI1, HNF3b, Ikaros factors, Lmo2 complex, MyoD, MZF1, NF1, NF-AT, NF-κB, NFY, Nkx-2.5, Runx factors, S8, and USF in the LGALS3 promoter regions of both species (Table I and Fig. 2). Of special interest with respect to osteogenesis are five binding sites for Runx, one for Ets factors, four for C/EBPऔ, and three for SP1 in the murine promoter. In the murine promoter one additional Runx-binding site was identified further upstream at −1477 nucleotides relative to the transcription start. Runx2, Ets-1, and C/EBPऔ are bone-related transcription factors that control the transcription of several bone-specific genes (26Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3683) Google Scholar, 38Gutierrez S. Javed A. Tennant D.K. van Rees M. Montecino M. Stein G.S. Stein J.L. Lian J.B. J. Biol. Chem. 2002; 277: 1316-1323Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 39Sato M. Morii E. Komori T. Kawahata H. Sugimoto M. Terai K. Shimizu H. Yasui T. Ogihara H. Yasui N. Ochi T. Kitamura Y. Ito Y. Nomura S. Oncogene. 1998; 17: 1517-1525Crossref PubMed Scopus (239) Google Scholar). Osterix was recently identified as a zinc finger transcription factor involved in osteogenesis and was shown to bind to the SP1 consensus motif (40Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2857) Google Scholar). Hence, galectin-3 expression in osseous tissues may be regulated by the transcription factor Runx2 and modulated by Ets-1, C/EBPऔ, and osterix.Table IPutative transcription factor binding sites detected in both the murine and the human LGALS3 promoter region Open table in a new tab Figure 2Identification of putative transcription factor-binding sites in the promoter sequence of the murine (strain Fvb) galectin-3 gene (LGALS3).Consensus binding sites for factors with a potential role in skeletal tissues are underlined. The transcribed sequence of exon1 is indicated as a shaded box. The positions of transcription initiation sites as described by Rosenberg et al. (19Rosenberg I.M. Iyer R. Cherayil B. Chiodino C. Pillai S. J. Biol. Chem. 1993; 268: 12393-12400Abstract Full Text PDF PubMed Google Scholar) are indicated as ♦ (GenBankTM accession numberAY130769).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the functional relevance of Runx2-binding sites in the promoter of the LGALS3 gene, the influence of Runx2 on the expression of galectin-3 was determined in cellular differentiation systems in vitro. A recombinant murine C3H10T1/2 cell line was established harboring an expression vector mediating constitutive Runx2 expression (C3H10T1/2-Runx2). The murine cell line C3H10T1/2 has properties of mesenchymal stem cells. These cells differentiate into adipocytes, myoblasts, chondrocytes, and osteoblasts dependent on the culture conditions (41Ahrens M. Ankenbauer T. Schroder D. Hollnagel A. Mayer H. Gross G. DNA Cell Biol. 1993; 12: 871-880Crossref PubMed Scopus (312) Google Scholar, 42Konieczny S.F. Emerson Jr., C.P. Cell. 1984; 38: 791-800Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 43Taylor S.M. Jones P.A. Cell. 1979; 17: 771-779Abstract Full Text PDF PubMed Scopus (901) Google Scholar). C3H10T1/2 cells have already been used successfully in the characterization of known Runx2 target genes (26Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3683) Google Scholar). Here, the stable expression of Runx2 in C3H10T1/2-Runx2 cells was confirmed by Northern and Western blot analysis (Fig. 3,A and B). In contrast to untransfected cells, C3H10T1/2-Runx2 cells expressed osteocalcin mRNA as shown by Northern blot, indicating that the biological activity of transgenic Runx2