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INTRODUCTION |
Galectin-3 (Mac-2,
BP, IgE-binding protein, CBP35, CBP30, L-29,
and L-34) is one of ten members of the protein family of
-galactoside specific lectins (1). It was first identified as an
antigen on murine thioglycollate-elicited peritoneal macrophages (2).
The molecular mass of galectin-3 ranges between 30 and 42 kDa in
different species (3). 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 (4-6). Extracellular galectin-3 has been
associated with modulation of cell adhesion in organogenesis, immunological processes, and tumorigenesis (7-9). Intracellular galectin-3 has been implicated to play a role in pre-mRNA splicing (10).
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 (3, 11-14).
Galectin-3 is expressed in several skeletal tissues. Fowlis et
al. (13) 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 (15).
Aubin et al. (16) reported galectin-3 expression in rat
osteoblasts. In addition, its expression was shown in the epiphyseal
cartilage and bone of neonatal mice (17).
The human galectin-3 gene LGALS3 (lectin,
galactoside binding, soluble 3) was
mapped to chromosome 14 at band 14q21-22. The human and murine
LGALS3 genes are organized in six exons, with the
translation start site located in exon 2 (18, 19). Genomic fragments
encompassing nucleotides
836 to +141 relative to the transcription
start site of human LGALS3 show significant promoter activity in reporter assays (18). 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' (20-23). Runx2 is a key regulator of osteoblast differentiation. The
expression of Runx2 is restricted to osteoblasts, epiphyseal cartilage,
nucleus pulposus, and mammary gland (24). Mice deficient in Runx2
expression are devoid of osteoblasts and bone (24, 25). Expression of
several osteoblast-specific genes is regulated by Runx2 (26).
Furthermore, the transcription factor has been implicated to play a
role in chondrocyte maturation (27, 28). 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 situ
hybridization that in contrast to wild type mice, Runx2-deficient mice
lack expression of galectin-3 in long bones.
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MATERIALS AND METHODS |
Cell Culture and Transfection--
The murine embryonic calvaria
cell line MC3T3-E1 was obtained from the Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany)
(29). The cells were maintained in
-modified minimal essential
medium supplemented with 10% fetal calf serum and 2 mM
L-glutamine (Invitrogen). The conditions for osteogenic
differentiation were adopted from those reported for differentiation of
fetal rat calvaria cells (30). When cells reached confluency, medium
supplemented with 10 mM
-glycerophosphate and 50 µg/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
10% fetal calf serum and 2 mM L-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 µg/ml (Sigma-Aldrich). Stably transfected C3H10T1/2
clones with inducible Runx2 expression were grown with the addition of
150 µg/ml zeocin and 200 µg/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, 105
C3H10T1/2 cells were incubated with 2.5 µg of plasmid DNA and 5 µl
of FuGENE 6 reagent (Roche Molecular Biochemicals). After transfection
with pSwitch, the clones were selected with 200 µg/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
-galactosidase. The cells were fixed in 2% formaldehyde and 0.2%
glutardialdehyde for 5 min at room temperature and subsequently
stained using 5 mM K3Fe[CN]6, 5 mM K4Fe[CN]6, 2 mM
MgCl2, 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 µg/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.
Cloning the Galectin-3 Promoter and cDNAs for Hybridization
and Expression--
All cDNAs were obtained by reverse
transcriptase-PCR using total RNA purified from limbs of newborn mice.
Reverse transcription was carried out using 2 µg 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
-actin primers were adopted from Gessner et
al. (31), glyceraldehyde-3-phosphate dehydrogenase primers from a
PCR-Select cDNA Subtraction kit (BD
Clontech) and osteocalcin primers from Desbois
et al. (32). PCR products were cloned into TA cloning vector
pCR2.1 (Invitrogen), and inserts were sequenced by a Taq
DyeDeoxy Sequencing system (ABI, Weiterstadt, Germany). Runx2 cDNA
was subcloned into expression vector pCMV
(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 lacZ
gene. The following primers were used: LGALS3p2000for, 5'-CTCTGCGAGCTTGTAAGTCTATCCTA-3', and LGALS3rev,
5'-CGCTCACCTGATTAGTGCTCC-3'.
Sequence Analysis of Murine and Human LGALS3 Promoter
Fragments--
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 (21-23) with special
respect on structure data (33). Thus the sequence was searched for the
motif ACCPuCPu, and positions 2 and 3 (CC) were considered to be most important.
Northern Blot Analysis--
Total RNA (15 µg/lane) was
resolved on a 1% formaldehyde-agarose gel and transferred onto a
Hybond N+ nylon membrane (Amersham Biosciences) using 10× SSC. The
probes were labeled with [
-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, 7%
(w/v) SDS, 1 mM EDTA, 100 µg/ml salmon sperm DNA, adapted from Church et al. (34)). The blots were washed in 2× SSC,
0.1% SDS at room temperature for 15 min and twice in 0.1× SSC, 0.1% SDS at 60 °C for 20 min. The blots were exposed to Kodak XAR-5 film
at
70 °C using two intensifier screens.
SDS-Polyacrylamide Gel Electrophoresis and Western
Blotting--
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 10%
polyacrylamide gels (35). After electrophoresis, the protein was
transferred onto Hybond P membranes (Amersham Biosciences) by semidry
electroblotting (36). Afterward the membranes were blocked in 5%
skimmed milk in TBST buffer (10 mM Tris-HCl, 15 mM NaCl, 0.05% Tween 20, pH 8.0) overnight. For
immunodetection of Runx2 protein, the blots were incubated in 5%
skimmed milk in TBST buffer with 70 µg/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 5% 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 mM
NaCl, pH 8.0) specific protein was visualized by chemiluminiscence
using the ECLplus system (Amersham Biosciences) according
to the manufacturer's instructions.
In Vitro Translation of Runx2 Protein and Electromobility Shift
Assays (EMSA)--
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
[
-32P]dCTP, and oligonucleotide Oligo A for binding
control was adopted from Tahirov et al. (33). 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-µl samples were
prepared containing 3 µl in vitro translation reaction, 5 µl of labeled double-stranded oligonucleotide (20000-25000 cpm), 1 or 4 µl of respective unlabeled competitor oligonucleotide (resulting
in 15- or 60-fold molar excess of competitor, respectively), 1 µl of
pepstatin, and 1 µl of poly(dI-dC) (1 A260/µl) in 2% glycerol, 5 mM Tris-HCl, 0.2 mM EDTA, 0.01% Nonidet-P 40, 0.1 mM dithiothreitol, 17.5 mM NaCl, 10 µg/ml
bovine serum albumin, pH 7.5. The binding reactions were incubated for
30 min at room temperature and resolved on a 5% polyacrylamide gel in
0.5× TBE. The gels were dried and then exposed to Kodak XAR-5 film at
70 °C using intensifier screens.
RNA in Situ hybridization--
In situ hybridizations
using 33P-labeled antisense riboprobes were carried out as
previously described (37). Briefly, the sections were deparaffinized,
rehydrated, pretreated with proteinase K (3 min, 10 µg/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.
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RESULTS |
Galectin-3 Gene Expression Is Up-regulated during Osteogenic
Differentiation of Mouse Calvaria MC3T3-E1 Cells--
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 (29). 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
-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.

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Fig. 1.
Expression of galectin-3 is up-regulated
during osteogenic differentiation of MC3T3-E1 mouse calvarial
cells. The cells were grown under conditions inducing
differentiation toward a mature osteoblastic phenotype. A,
alkaline phosphatase activity of MC3T3-E1 cells during osteogenic
differentiation. B, Northern blot analysis of galectin-3,
osteocalcin (as marker for osteogenic differentiation), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression
during osteoblastic differentiation in vitro.
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Analysis of the Galectin-3 Promoter Sequence--
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 (18, 19). We
identified additional putative binding sites for AP1, AP4,
C/EBP
, CDP CR, c-Ets, CP2, c-Rel, GATA proteins, GFI1, HNF3b,
Ikaros factors, Lmo2 complex, MyoD, MZF1, NF1, NF-AT, NF-
B, 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/EBP
, 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/EBP
are bone-related transcription factors that control the transcription of several bone-specific genes (26, 38, 39). Osterix was recently identified as a
zinc finger transcription factor involved in osteogenesis and was shown
to bind to the SP1 consensus motif (40). Hence, galectin-3 expression
in osseous tissues may be regulated by the transcription factor Runx2
and modulated by Ets-1, C/EBP
, and osterix.
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Table I
Putative transcription factor binding sites detected in both the
murine and the human LGALS3 promoter
region
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Fig. 2.
Identification 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. (19) are
indicated as (GenBankTM accession number
AY130769).
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Forced Stable and Inducible Expression of Runx2 in Mesenchymal
Progenitor C3H10T1/2 Cells Leads to Osteogenic
Differentiation--
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 (41-43).
C3H10T1/2 cells have already been used successfully in the
characterization of known Runx2 target genes (26). 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 mediates osteogenic
differentiation (Fig. 3B) (26). C3H10T1/2-Runx2 cells grew
more slowly than wild type cells and exhibited a similar but more
spindle-like morphology (data not shown).

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Fig. 3.
C3H10T1/2-Runx2 cells constitutively express
biologically active Runx2. Wild type C3H10T1/2 (WT)
cells and C3H10T1/2-Runx2 were cultured as described under "Materials
and Methods." A, Western blot of cell lysates using
anti-Runx2 antiserum. C3H10T1/2-Runx2 cells express Runx2 protein of
the expected size (57 kDa). After immunodetection the blotting membrane
was stained with Coomassie Brilliant Blue for loading and transfer
control. Molecular mass marker sizes are indicated. B, Runx2
protein produced by C3H10T1/2-Runx2 cells is biologically active;
Northern blot of wild type and C3H10T1/2-Runx2 cells hybridized with
Runx2, galectin-3, osteocalcin, and -actin. mRNA levels of
osteocalcin, a previously described Runx2 target, indicate the
functional activity of recombinantly expressed Runx2.
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To investigate whether or not gelectin-3 is directly regulated by
Runx2, we generated C3H10T1/2 cells with an inducible expression system
for Runx2 ("Gene Switch" system, Invitrogen; "Materials and
Methods"). After selection, the clones were analyzed for the inducible expression of Runx2 with 30 nM mifepristone for
38 h or with the solvent ethanol as a control. Functional analysis of clone 18/17 is shown in Fig. 4.
Northern blot analysis showed the presence of Runx2 mRNA in cells
stimulated with mifepristone (Fig. 4B). Cells treated
with ethanol alone did not express detectable amounts of Runx2
mRNA. To confirm that protein of correct size was being translated,
Western blotting was performed. A band at 57 kDa was present in protein
lysate of induced cells (Fig. 4A). Osteocalcin gene
expression is known to be directly controlled by Runx2 (26). Therefore,
Northern blot analysis for osteocalcin expression was used to verify
biological activity of transgenic Runx2. The results shown in Fig.
4B reveal high levels of osteocalcin mRNA in induced as
compared with noninduced cells. Taken together, these results confirm
that the transfectants inducibly express biologically active Runx2. The
morphology of these cells did not change after induction with
mifepristone up to 5 days (data not shown). Likewise, a change in
growth rate could not be observed (data not shown). A stepwise increase
in mifepristone concentration from 5 to 80 nM did not lead
to a further elevation in Runx2 expression levels when assessed by
Western blot (data not shown). After induction Runx2 expression could
be maintained for 5 days, with maximum expression levels at 24 h
(data not shown). To enhance the expression from the Runx2 transgene in
cells treated with mifepristone, we investigated the effect of the
histone acetyl transferase inhibitor TSA. TSA has been reported to
derepress silenced genes by elevating the overall acetylation status of
histones, thus increasing the accessibility of genomic DNA to
DNA-binding factors. The pretreatment of cells with TSA led to an
elevated Runx2 expression in induced clones. The Runx2 expression level
was dependent on the concentration of supplemented TSA (Fig.
5A). Both Runx2 and
osteocalcin expression levels were higher in C3H10T1/2 cells
constitutively expressing Runx2 than in those with inducible expression
(data not shown).

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Fig. 4.
Inducible expression of Runx2 in
C3H10T1/2 cells leads to an up-regulation of galectin-3
transcription. Cells from one clone 18/17 were treated with 30 nM mifepristone (+) or the respective volume of ethanol
( ) for 38 h. A, mifepristone-induced
Runx2-transfected C3H10T1/2 cells express Runx2 protein of correct size
(57 kDa). Western blot with Runx2 antiserum and staining with Coomassie
Brilliant Blue for loading and transfer control is shown. B,
Runx2 protein expressed after induction is biologically active.
Northern blot analysis from induced and noninduced cells was performed
with the probes indicated. Runx2 expression is confirmed after
induction with mifepristone. Osteocalcin mRNA levels were measured
to investigate the functional activity of recombinant Runx2. Galectin-3
gene synthesis is up-regulated after induction of Runx2 expression.
mRNA sizes are indicated for orientation. Mif.,
mifepristone.
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Fig. 5.
Pretreatment with TSA increases the inducible
expression of Runx2 and galectin-3 in C3H10T1/2 cells.
A, cells were grown as described under "Materials and
Methods." Induction of Runx2 expression by the addition of 30 nM mifepristone after preincubation with different
concentrations of TSA. B, Runx2-dependent
synthesis of galectin-3 mRNA can be stimulated by pretreatment of
cells with TSA. The molecular masses are indicated. Mif.,
mifepristone.
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Forced Stable and Inducible Expression of Runx2 in
Mesenchymal Progenitors C3H10T1/2 Leads to
Up-regulation of Galectin-3 mRNA Synthesis--
The two
expression systems described above were used to study the
influence of Runx2 on galectin-3 gene expression. Galectin-3 expression in C3H10T1/2-Runx2 cells was compared with that in wild type
C3H10T1/2 cells. The two cell lines were cultured in parallel
experiments for Northern blot analysis. Galectin-3 mRNA levels were
found to be dramatically higher in C3H10T1/2-Runx2 cells as compared
with wild type cells (Fig. 3B). Hybridization with a
Runx2 cDNA probe confirmed that Runx2 was only expressed in
transgenic C3H10T1/2-Runx2 cells (Fig. 3B).
To exclude the possibility that the observed increase in galectin-3
expression is a result of differences in differentiation states,
galectin-3 expression was also investigated in C3H10T1/2 cells with
inducible Runx2 expression. A moderate up-regulation of galectin-3 gene
expression was noted in C3H10T1/2 cells expressing Runx2, 38 h
after induction with mifepristone (Fig. 4B). Note that the stronger hybridization signal of noninduced cells (Fig. 4B) compared with that of wild type cells (Fig.
3B) is a result of longer film exposure. As a control for
Runx2 expression, the same blot was hybridized to a Runx2 cDNA
probe. This showed tight repression of transgenic Runx2 in noninduced
cells and potent induction of gene expression after the addition of
mifepristone (Fig. 4). These results were confirmed in three individual
experiments, using two different cell clones (11/2 and 18/17).
However, a more striking difference in galectin-3 expression between
induced and noninduced cells had been expected. Therefore, we
investigated whether the stimulation of galectin-3 expression in the
inducible Runx2 cell system could be increased by pretreatment with
TSA. Inducible C3H10T1/2 cells were pretreated with TSA 20 h prior
to induction with 30 nM mifepristone. Under these
conditions, a further increase in galectin-3 mRNA was monitored in
Northern blot analyses.
Stimulation of galectin-3 expression was dependent on the concentration
of TSA used (Fig. 5B). This experiment was reproduced three
times with different clones and resulted in comparable induction levels. In conclusion, these experiments provide strong evidence for
transcriptional control of the galectin-3 gene by Runx-2 in C3H10T1/2 cells.
Runx2 Binds to Sequences Identified in the Galectin-3
Promoter--
To verify that putative sites in the galectin-3 promoter
region identified by in silico analysis are able to interact
with the Runx2, protein electromobility shift assays were performed. Using an in vitro transcription and translation system,
Runx2 protein was produced (Fig.
6B). Double-stranded
oligonucleotides representing the four putative Runx-binding sites as
well as two additional sequences that had not been identified by
in silico analysis but are very similar to the Runx
consensus sequence were incubated with Runx2 protein after
radiolabeling (Fig. 6A). Unlabeled oligonucleotides were
used as competitor to show specific binding (Fig. 6C). As
control, a mutant Runx2 protein carrying a mutation identified in
patients with cleidocranial dysplasia were used (data not shown). In a
second set of experiments, a labeled standard oligonucleotide with
known Runx2 binding capacity was used. Sequences identified
in the galectin-3 promoter were used as competitors (Fig.
6D). Both sets of experiments pointed to sites 2 and 3 as having the strongest Runx2 binding ability.

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Fig. 6.
Runx2 protein binds to sequences in the
promoter of the LGALS3 gene. A, oligonucleotides used
for EMSA experiments. The putative binding sites are shaded.
B, Western blot showing in vitro translated Runx2
protein used in EMSA experiments (pGEM-Runx2). The pGEM vector
containing CBF was used as control (pGEM). C, EMSA
performed using the oligonucleotides derived from the LGALS3
promoter. N.S. denotes a nonspecific band. D,
EMSA using a labeled standard oligonucleotide with unlabeled
oligonucleotides derived from the LGALS3 promoter as
competitors.
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Galectin-3 Expression Is Absent in Chondrocytes of
Runx2-deficient Mice--
The results described above demonstrate
the involvement of Runx2 in the transcriptional regulation of
galectin-3 in cell culture systems. To determine the relevance of Runx2
for galectin-3 transcription under in vivo conditions, we
studied its expression in limbs of wild type and Runx2-deficient
embryos. Paraffin sections of 17.5 dies post coitum
embryos were submitted to RNA in situ hybridization using a
33P labeled antisense RNA probe for galectin-3 (Fig.
7). In wild type animals the antisense
probe revealed a strong galectin-3 expression in prehypertrophic
chondrocytes and skin. Posthypertrophic cartilage exhibited no
galectin-3 expression, whereas moderate hybridization signals could be
detected in bone. In Runx2-deficient mice osteoblast development as
well as chondrocyte maturation are disturbed. These animals are devoid
of osteoblasts, and cartilage differentiation is severely impaired.
Some hypertrophic cartilage, however, develops at embryonic day 17.5 in
the anlagen of radius and ulna (24). We used sections through embryonic
day 17.5 forearms (radius and ulna) to test for galectin-3 expression
in Runx2
/
mice. We were not able to detect galectin-3 expression
in chondrocytes of Runx2-deficient animals. Galectin-3 expression
levels in the skin of Runx2-deficient mice were similar to wild type
animals. These data support the in vitro and cell culture
data presented in this study and propose a role for Runx2 in the
transcriptional regulation of the LGALS3 gene in skeletal
tissues.

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Fig. 7.
Galectin-3 expression in long bones is
severely reduced in Runx2-deficient mice. RNA in situ
hybridization for glalectin-3 was performed on paraffin sections from
the forelimbs of wild type (A) and Runx2-deficient
(C) mouse embryos (embryonic day 17.5). An in
vitro transcribed antisense RNA probe, derived from a cloned
cDNA fragment for galectin-3 (see "Materials and Methods") was
labeled with 33P and applied for hybridization. The
sections were counterstained with Toluidine-O (B and
D). b, bone; c, cartilage;
hc, hypertrophic cartilage; pc, prehypertropic
cartilage; po, posthypertrophic cartilage.
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DISCUSSION |
In this study, we identified LGALS3, the gene coding
for the
-galactoside-specific lectin galectin-3, as a
transcriptional target of Runx2. Overexpression of Runx2, a
transcription factor with a key role in skeletal development, leads to
an increase in galectin-3 expression in a mesenchymal progenitor cell
line. In contrast, galectin-3 expression is severely reduced in
Runx2-deficient mice at sites where both genes are coexpressed in wild
type animals.
Of all of the putative Runx-binding sites in the galectin-3 promoter,
the sequence 5'-AACCACA-3' (site 2) shows the strongest affinity for
Runx2 protein in our EMSA experiments. This sequence may indeed
represent the preferred element for Runx2 binding, because a number of
proven Runx2-binding sites (e.g. in the osteocalcin and
Runx2 promoters) exactly match this sequence (38, 44). Interestingly,
site 2 is located in close proximity to a C/EBP
-binding site. A
cooperative binding of Runx2 and C/EBP
transcription factors with
physical interaction of the two proteins has been demonstrated for the
osteocalcin promoter where the respective binding sites show virtually
identical spacing (38).
Chondrocytes in the growth plate of long bones mature in a defined
sequence. From the resting zone they enter the proliferation zone and
emerge to mature and undergo hypertrophy. Eventually they undergo
apoptosis in the posthypertrophic zone to be replaced by osteoblasts.
Chondrocytic expression of galectin-3 starts in the proliferation zone
and persists through maturation to hypertrophy. In the posthypertrophic
zone galectin-3 protein is still present at low levels, but no more
galectin-3 mRNA is produced by posthypertrophic chondrocytes. The
physiological role of galectin-3 in chondrocyte maturation is evident
from mice deficient in its expression. These animals are characterized
by disturbances in growth plate architecture. This effect is most
pronounced in the posthypertrophic zone where an increased number of
empty lacunae point to enhanced apoptosis of terminally differentiated
chondrocytes (45). The transcription factor Runx2, which was shown in
this study to enhance galectin-3 expression, is present in all stages
of chondrocyte maturation that are characterized by galectin-3
expression (24). This is consistent with a positive regulatory effect
of Runx2 on galectin-3 transcription in these cells. The Runx2
expression domain in the growth plate exceeds that of galectin-3
slightly in addition to comprising the posthypertrophic zone. Members
of the TLE family of transcriptional corepressors (e.g.
TLE-1 and TLE-3) are expressed in the developing skeleton and have been
shown to interact with runt proteins, thereby converting their
transactivating properties to active transcriptional repression (46,
47). Thus TLE proteins might interact with Runx2 to down-regulate
galectin-3 expression in posthypertrophic chondrocytes.
The two other members of the Runt family of transcription factors,
i.e. Runx1 and Runx3, are also expressed in growth plate chondrocytes, and their expression domains overlap that of Runx2 (48).
All three mammalian Runt proteins bind to the same consensus sequence
in their respective target gene promoters (20-23). Hence it is
possible that galectin-3 expression in growth plate cartilage is
controlled by all three Runt proteins. The lack of galectin-3 expression in the growth plates of mice deficient in Runx2 expression, however, indicates that there is no redundancy in this respect of Runt
factor function. Interestingly, the expression pattern of Runx1 and
Runx3 overlaps at several nonskeletal sites with that of galectin-3. In
hematopoietic cells, notably including macrophages where galectin-3 was
initially identified, both transcription factors are expressed, and
Runx1 was shown to be essential for mature hematopoiesis (2, 48, 49).
Other sites of overlapping expression are skin and olfactory epithelium
(13, 14, 48). Recently dorsal root ganglia neurons have been identified
as dependent on Runx3 in their development (48). Dorsal root ganglia
cells also express significant amounts of galectin-3 (50). Therefore, it is tempting to speculate that Runt factors control galectin-3 expression at these extraskeletal sites.
Galectin-3 is expressed by cells of the osteoblastic lineage as
shown by us and others. Its expression increases significantly at
the matrix maturation stage. Culture conditions for MC3T3-E1 cells
involving ascorbic acid have been shown to enhance the binding affinity
of Runx2 to its binding site (51). Runx2 is phosphorylated via the
mitogen-activated protein kinase pathway in MC3T3-E1 cells. Phosphorylation of Runx2 in these cells leads to increased binding to
and expression from the promoter of the known Runx2 target gene
osteocalcin (52). This is in agreement with our finding that galectin-3
expression increases, whereas Runx2 mRNA and protein levels are
stable during differentiation of MC3T3-E1 cells in the presence of
ascorbic acid.
Further experimental work will have to provide direct evidence for a
functional interaction of different transcription factors on the
galectin-3 promoter in skeletal tissue. Moreover, an analysis of the
transcriptional regulation of the galectin-3 gene in nonskeletal tissues may show this gene to be a first example of a common target for
the different Runt factors.