Cbfa1 Contributes to the Osteoblast-specific Expression of type I collagen Genes*

Britt Kern, Jianhe Shen, Michael Starbuck, and Gerard KarsentyDagger

From the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030

Received for publication, July 13, 2000, and in revised form, November 29, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Type I collagen is composed of two chains, alpha 1(I) and alpha 2(I), encoded by two distinct genes, the alpha 1(I) and alpha 2(I) collagen genes, that are highly expressed in osteoblasts. In most physiological situations, alpha 1(I) and alpha 2(I) collagen expression is coregulated, suggesting that identical transcription factors control their expression. Here, we studied the role of Cbfa1, an osteoblast-specific transcription factor, in the control of alpha 1(I) and alpha 2(I) collagen expression in osteoblasts. A consensus Cbfa1-binding site, termed OSE2, is present at the same location in the alpha 1(I) collagen promoter at approximately -1347 base pairs (bp) of the rat, mouse, and human genes. Cbfa1 can bind to this site, as demonstrated by electrophoretic mobility shift assay (EMSA) and supershift experiments using an anti-Cbfa1 antibody. Mutagenesis of the alpha 1(I) collagen OSE2 at -1347 bp reduced the activity of a alpha 1(I) collagen promoter fragment 2- to 3-fold. Moreover, multimers of this OSE2 at -1347bp confer osteoblast-specific activity to a minimum alpha 1(I) collagen promoter fragment in DNA transfection experiments as well as in transgenic mice. An additional Cbfa1-binding element is present in the alpha 1(I) collagen promoter of mouse, rat, and human at approximately position -372. This site binds Cbfa1 only weakly and does not act as a cis-acting activator of transcription when tested in DNA transfection experiments. Similar to alpha 1(I) collagen, the mouse alpha 2(I) collagen gene contains multiple OSE2 sites, of which one is conserved across multiple species. In EMSA, Cbfa1 binds to this site and multimers of this alpha 2(I) OSE2 element confer osteoblast-specific activity to the minimum alpha 1(I) collagen promoter in DNA transfection experiments. Thus, our results suggest that Cbfa1 is one of the positive regulators of the osteoblast-specific expression of both type I collagen genes.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Type I collagen is the most abundant protein of the bone extracellular matrix, accounting for 90% of the matrix protein content (1). It is a heterotrimer made of two alpha 1(I) chains and one alpha 2(I) chain (2). The alpha 1(I) and alpha 2(I) chains are encoded by two distinct genes that are expressed most highly in two cell types: the fibroblast and the osteoblast. Moreover, the expression of these two genes is often regulated by identical transcription factors (3-7). The type I collagen genes are expressed in osteoblastic cells at all stages during development and throughout life (8), suggesting that the factor(s) controlling their expression in these cells could also control osteoblast differentiation and function. Another possibility, which is not exclusive of the previous one, is that different transcription factors may control their expression in osteoblasts at various stages of development and of postnatal life. Thus, the elucidation of the molecular mechanisms controlling alpha 1(I) and alpha 2(I) collagen gene expression in osteoblasts is of critical importance in understanding how osteoblast differentiation and, thereby, bone matrix deposition by differentiated osteoblasts is regulated. Ultimately, these studies may shed light on the pathogenesis of genetically acquired bone diseases and help design appropriate therapies for some of these diseases.

The critical role that Cbfa1, a Runt-related osteoblast-specific transcription factor, plays in osteoblast differentiation and function has been demonstrated in mouse and in human using both molecular and genetic approaches (9-14). Cbfa1 was identified as a key regulator of osteoblast-specific gene expression through its binding to the OSE2 element of the mouse Osteocalcin genes 1 and 2 (OG1 and OG2) (9) and other genes expressed in osteoblasts. The early and cell-specific expression of this gene together with its biological role in vivo as a factor required for osteoblast differentiation (9-11), indicate that Cbfa1 must control the expression of multiple target genes that are expressed earlier than Osteocalcin. Conceivably, these target genes could include the alpha 1(I) and alpha 2(I) collagen genes that are expressed early during development. This hypothesis was confirmed indirectly by the observation that expression of a dominant negative form of Cbfa1 in differentiated osteoblasts leads to a decrease in expression of the type I collagen genes in vivo (14). To date, no osteoblast-specific cis-acting elements to which Cbfa1 may bind have been identified in these genes.

Two groups have extensively studied the regulation of expression of the alpha 1(I) collagen gene in osteoblasts and have identified a region in the promoter of the rat and mouse alpha 1(I) collagen gene that plays an important role in this regulation of expression (15, 16). The sequence of this region bears no homology to a Cbfa1-binding site, and several homeobox-containing proteins can bind to this sequence and affect alpha 1(I) collagen expression. However, a cell-specific transcription factor binding to this region has not yet been identified. Moreover, no osteoblast-specific cis-acting element has yet been identified in the alpha 2(I) collagen promoter. Given the large size of these genes and their expression at multiple stages of osteoblast differentiation, it is likely that several distinct osteoblast-specific cis-acting elements, besides those already described (15, 16), contribute to the expression of the type I collagen genes in osteoblast progenitors and/or in fully differentiated osteoblasts. Consistent with this hypothesis, we noticed the existence of two Cbfa1-binding sites (OSE2s) in the mouse alpha 1(I) collagen promoter and one OSE2 in the mouse alpha 2(I) collagen gene that are conserved among multiple species. The functional importance of these sites has never been studied before. Here we present evidence suggesting that Cbfa1 is one of the factors controlling osteoblast-specific expression of both type I collagen genes.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA Constructs-- For DNA transfection and generation of transgenic mice, multimers of double-stranded oligonucleotides (see Table I) were cloned into the SmaI site upstream of a chimeric pK1-luc reporter plasmid containing the alpha 1(I) collagen -86 minimal promoter (4) fused to a luciferase gene. The expression plasmid was pCMV-Osf2 (9).

Site-specific mutations were created in the intact promoter by using polymerase chain reaction-directed mutagenesis (17) on a construct containing 2.4 kb1 of the alpha 1(I) collagen promoter upstream of a luciferase reporter gene. Sole presence of the desired mutations was verified by sequencing.

Cell Culture and DNA Transfection-- COS7, NIH 3T3, HeLa, C2C12, and 10T1/2 cells were cultured in Dulbecco's minimal essential medium (Life Technologies, Inc.), 10% fetal bovine serum (Life Technologies, Inc.). ROS 17/2.8 cells were cultured in Dulbecco's minimal essential medium-F12 medium (Life Technologies, Inc.), 10% fetal bovine serum (Life Technologies, Inc.). Twenty hours before transfection, cells were plated at a density of 5 × 105 cells/dish and allowed to grow under normal culture conditions. For cotransfection experiments, we used 5 µg of Cbfa1 expression vector or empty vector, 5 µg of reporter plasmid or empty vector, and 2 µg of pSVbeta -gal vector using the calcium phosphate coprecipitation procedure (17). Transfection conditions were identical to those used in the cotransfection experiments, except that 5 µg of reporter plasmid were used. Twenty hours following transfection, cells were washed in phosphate-buffered saline and incubated in medium an additional 24 h. C2C12 cells were changed to media containing 10% horse serum (Life Technologies, Inc.) and allowed to incubate for 48 h. Cells were collected by scraping into 0.25 M Tris-HCl, pH 7.8, and lysed by three freeze-thaw cycles. beta -Galactosidase and luciferase assays were carried out as described previously (18). beta -Galactosidase assay results were used to normalize the luciferase assay results for transfection efficiency. All DNA transfection experiments were repeated at least three times in triplicate.

Electrophoretic Mobility Shift Assays-- Nuclear extract from ROS 17/2.8 cells, primary osteoblasts, and other tissues were prepared as described previously (19) from 4-day-old wild-type mice and stored at -80 °C until use. Glutathione S-transferase-Cbfa1 was purified from transformed Escherichia coli bacteria using glutathione beads as described previously (17). Double-stranded oligonucleotides (see Table I) were end-labeled and purified as previously described (19). 5 fmol of labeled oligonucleotide was incubated with 7 µg of ROS 17/2.8 nuclear extract or 0.1 µg of recombinant Cbfa1 protein. alpha 2(I) collagen EMSA experiments used twice the amount of extract or protein. The incubation mix for nuclear extract binding assays consisted of binding buffer (100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 4 mM EDTA, 2 mM DTT, 0.2% Nonidet P-40, 10% glycerol, 5 µg/ml leupeptin, 5 µg/ml pepstatin) (20), 2 µg of poly(dI-dC), and 0.5 fmol of single-stranded bottom strand oligonucleotide. Incubation took place at room temperature for 5 min. Supershift experiments were carried out as described above, except that the ROS17/2.8 nuclear extract was preincubated for 10 min at room temperature with an antibody against Cbfa1 in binding buffer prior to their incubation with the labeled oligonucleotide for 10 min at room temperature.

For recombinant protein binding assays, the incubation mix consisted of binding buffer (20 mM Tris-HCl, pH 8.0, 10 mM NaCl, 3 mM EGTA, 5 mM DTT, 0.05% Nonidet P-40) and 1 µg of bovine serum albumin. Incubation took place at room temperature for 5 min, followed by the addition of 1 µl of loading buffer (2 mM Tris-HCl, pH 8.0, 5% glycerol, 0.025% xylene cyanol, 0.025% bromphenol blue).

The reactions were run on 5% polyacrylamide gel, 0.25× TBE (89 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.0) for 90 min at 160 V. The gels were then dried and exposed to film at -80 °C.

Generation and Analysis of Transgenic Mice-- The plasmids described above containing multimers of the alpha 1AB or alpha 1mutAmutB oligonucleotides were digested, and the insert was purified by two rounds of agarose gel electrophoresis. Linear DNA inserts were injected into the pronuclei of fertilized B6D2F1 (Charles River Laboratory) mouse eggs, which were reimplanted in the oviduct of pseudo-pregnant CD1 foster mothers (Jackson Laboratories). Transgenic animals were identified by Southern blots of tail genomic DNA. The transgenic mice expressing the p4alpha 1AB-luc construct were analyzed as follows: Organs from 4-week-old F1 animals were dissected and homogenized on ice in a buffer containing 100 mM potassium phosphate (pH 7.8) and 1 mM dithiothreitol (DTT). Protein homogenates were centrifuged, and supernatants were assayed for luciferase activity according to standard procedures (19). Protein levels were measured using the Bio-Rad protein assay. Relative luciferase activities were expressed as luciferase light units per 100 µg of protein expressed as a percentage of the activity in bone.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The alpha 1(I) collagen Promoter Contains Two Conserved OSE2 Sites-- DNA sequence inspection identified three potential OSE2 sites in the promoter of the murine alpha 1(I) collagen gene. Two are located side-by-side at positions -1347 and -1338 in the mouse gene (Fig. 1A), and a third site is found at -372 in the mouse gene (Fig. 1A). These potential OSE2 sites were termed alpha 1A, alpha 1B, and alpha 1C, respectively (Fig. 1B). The presence of alpha 1A and alpha 1C, but not of alpha 1B, at approximately the same location in the alpha 1(I) collagen promoter sequences of rat and human (Fig. 1A) suggested a biological role for these sites and led us to study these two regions. To determine whether Cbfa1 could bind to the OSE2-like sequences in the mouse alpha 1(I) collagen promoter, we generated double-stranded oligonucleotides to be used in DNA binding assays (Table I). One of them, called alpha 1AB, contains the alpha 1A and alpha 1B sites and their surrounding sequences. A second one, termed alpha 1wtAmutB, contains the wild-type alpha 1A site and a mutated alpha 1B site. A third oligonucleotide, called alpha 1mutAwtB, carries a mutated alpha 1A sequence and a wild-type alpha 1B sequence. A fourth oligonucleotide, alpha 1mutAmutB, contains mutations in both the alpha 1A and the alpha 1B sites. Two other oligonucleotides, termed alpha 1C and alpha 1mutC, were generated to test the binding activity of the alpha 1C site. The mutations introduced into all the oligonucleotides mentioned above have previously been shown to abolish binding of nuclear extract or recombinant Cbfa1 to the OSE2 sequence present in the Osteocalcin (OG2) promoter (OSE2OG2) (19). These double-stranded oligonucleotides were then used as probes in electrophoretic mobility shift assays (EMSA) using either ROS 17/2.8 nuclear extract or recombinant Cbfa1 as a source of protein.



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Fig. 1.   Type I collagen OSE2 sites are well conserved across species. A, three OSE2 sites are present in the mouse alpha 1(I) collagen promoter, but only the alpha 1A site and the alpha 1C site are conserved across species. B, diagram of the alpha 1(I) and alpha 2(I) collagen promoters and the OSE2 sites of each. The arrow indicates the start site of transcription (+1). The position indicated for each element is relative to the start site of transcription. C, alpha 2(I) collagen OSE2 sequence is conserved across vertebrate species for which the sequence is available: h, human; m, mouse; r, rat; c, chicken; C.f., Canis familiaris; B.t., Bos taurus; R.c., Rana catesbeiana.


                              
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Table I
Oligonucleotides used in this study
Boldface letters indicate mutated nucleotides.

Cbfa1 Binds to an OSE2 Site in the Mouse alpha 1(I) collagen Promoter-- The complex formed upon incubation of ROS 17/2.8 nuclear extract with alpha 1AB migrated at the same location as the complex formed upon incubation of ROS 17/2.8 nuclear extract with the OSE2OG2 oligonucleotide (Fig. 2A, lanes 1 and 2), although it was of weaker intensity. In contrast, no protein-DNA complex was observed when using alpha 1mutAmutB as a probe (Fig. 2A, lane 5).



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Fig. 2.   Cbfa1 binds to the alpha 1A site in the alpha 1(I) collagen promoter. DNA binding was analyzed by EMSA. A, labeled oligonucleotides OSE2OG2 (lane 1), alpha 1AB (lane 2), alpha 1wtAmutB (lane 3), alpha 1mutAwtB (lane 4), and alpha 1mutAmutB (lane 5) were incubated with ROS 17/2.8 nuclear extract. The arrow indicates the complex of interest. B, labeled alpha 1AB was used as a probe and incubated with nuclear extract from primary osteoblasts (lane 1), brain (lane 2), kidney (lane 3), lung (lane 4), muscle (lane 5), and spleen (lane 6). The arrow indicates the complex containing Cbfa1. C, supershift EMSA was performed using an antiserum against Cbfa1 (lane 2), or nonspecific antiserum (lane 1) using alpha 1AB as a probe. The arrow indicates the complex of lower mobility observed after incubation with the anti-Cbfa1 antibody. D, EMSA using recombinant Cbfa1 as a source of protein and alpha 1AB (lane 1), alpha 1wtAmutB (lane 2), alpha 1mutAwtB (lane 3), and alpha 1mutAmutB (lane 4) oligonucleotides as probes.

To determine which of the two upstream OSE2 sites was binding to this factor, we used alpha 1wtAmutB oligonucleotide or alpha 1mutAwtB oligonucleotide as probes in EMSA. Incubation of labeled alpha 1wtAmutB with ROS 17/2.8 nuclear extract generated a protein-DNA complex that had the same mobility as the one observed when using alpha 1AB oligonucleotide as a probe and was of stronger intensity (Fig. 2A, lane 3). In contrast, when using alpha 1mutAwtB oligonucleotide as a probe, we observed only a weak binding of ROS 17/2.8 nuclear extract to the DNA (Fig. 2A, lane 4).

To determine whether this osteoblast-specific factor binding to the alpha 1A OSE2-like element was indeed Cbfa1, we performed three types of experiments. First, we asked whether the factor present in ROS 17/2.8 nuclear extract and binding to the alpha 1A site was expressed only in osteoblasts. For that purpose, we prepared nuclear extract from primary osteoblasts and several other tissues and used them in EMSA. As shown in Fig. 2B, the factor binding to the alpha 1A oligonucleotide was present only in primary osteoblast nuclear extract and not in nuclear extract of other tissues. Next we performed supershift experiments using anti-Cbfa1 antibody or a nonspecific antiserum. Incubation of the nuclear extract with an antibody against Cbfa1 prior to addition of labeled alpha 1AB oligonucleotide led to the formation of a second protein-DNA complex of slower mobility (Fig. 2C, lane 2), whereas a nonspecific serum had no effect (Fig. 2C, lane 1), demonstrating that the protein-DNA complex formed upon incubation of the labeled alpha 1AB with ROS 17/2.8 nuclear extract contains Cbfa1, because this antibody is specific for Cbfa1 (14). Third, we asked whether recombinant Cbfa1 could bind to alpha 1AB but not to alpha 1mutAmutB (Fig. 2D, lanes 1 and 4). Incubation of labeled alpha 1wtAmutB oligonucleotide with recombinant Cbfa1 resulted in the formation of a protein-DNA complex (Fig. 2D, lane 2), whereas incubations using labeled alpha 1mutAwtB oligonucleotide did not (Fig. 2D, lane 3). Taken together, these results indicate that the alpha 1A site, a site conserved in multiple species, is the major binding site for Cbfa1 in this region of the alpha 1(I) collagen promoter.

The alpha 1A Site Acts as an Osteoblast-specific Activator of Transcription in Tissue Culture Experiments and in Vivo-- We further addressed the functional relevance of the alpha 1A and alpha 1B sites using two additional approaches. First, to examine the effect of the alpha 1A site on activity of a 2.4-kb promoter fragment, a site-specific mutation was generated in the alpha 1A site via polymerase chain reaction and introduced into a 2.4-kb alpha 1(I) collagen promoter-luc chimeric gene. These 2-bp mutations resulted in a 54% decrease in promoter activity when tested in DNA transfection experiments in ROS17/2.8 cells (Fig. 3A). The same mutations did not reduce the activity of this 2.4-kb alpha 1(I) collagen promoter-luc chimeric gene in other cell lines of nonosteoblastic nature of mesenchymal and nonmesenchymal origin (Fig. 3A). This result further suggests that this cis-acting element is active only in osteoblasts. Mutations in the alpha 1B site did not affect the activity of the 2.4-kb fragment of the alpha 1(I) collagen promoter used in this study (Fig. 3A).



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Fig. 3.   The alpha 1A site can activate transcription and is required for the full activity of the alpha 1(I) collagen promoter. A, constructs containing 2.4 kb of the alpha 1(I) collagen promoter with or without mutations were transfected into several cell types. alpha 1AB (black bars), alpha 1mutAwtB (gray bars), and alpha 1wtAmutB (white bars) promoters were cloned upstream of a luciferase reporter gene and used in DNA transfection experiments in ROS 17/2.8 cells, C2C12 cells, 10T1/2 cells, NIH 3T3 cells, and HeLa cells. Values represent percentage activity compared with the wild-type promoter. alpha 1mutAwtB displays lower transcriptional activity only in ROS 17/2.8 cells. B, multimers of the oligonucleotides used for EMSA were placed upstream of a minimal alpha 1(I) collagen promoter fused to a luciferase reporter gene. These constructs were transfected into COS7 cells in the presence of a recombinant Cbfa1 expression construct (dark bars), or an empty vector (open bars). Values represent -fold activation in relation to an empty reporter vector and are the average of at least three experiments done in triplicate. C, wild-type (dark bars) and double-mutant (open bars) constructs from DNA transfection experiments were used to generate transgenic mice. Luciferase activity per 100 µg of protein was determined for several tissues, and the data are expressed in terms of percentage activity compared with that of bone.

Second, in DNA cotransfection assays performed in COS7 cells, a cell line that does not express Cbfa1 (21), exogenous Cbfa1 transactivated a construct containing four copies of the alpha 1AB oligonucleotide fused to a minimal alpha 1(I) collagen promoter-luc chimeric gene, p4alpha 1AB-luc (Fig. 3B). Indeed, cotransfection of p4alpha 1AB-luc with a recombinant Cbfa1-expressing vector resulted in a 180-fold activation, whereas an empty expression vector had no effect. This level of transactivation is similar to what we observed when using a vector containing six copies of the OSE2OG2 as a reporter (Fig. 3B). The minimal alpha 1(I) collagen promoter fragment has virtually no transactivation ability on its own (4). When using a vector containing multimers of alpha 1wtAmutB oligonucleotides cloned upstream of the minimal alpha 1(I) collagen promoter fragment, we observed a 120-fold increase in luciferase activity, indicating that the alpha 1A site is the main contributor to the transactivating function of this region of the alpha 1(I) collagen promoter. This is consistent with the observation that only the alpha 1A site is able to bind Cbfa1 in vitro. The slight decrease in activity seen with the loss of the alpha 1B site may indicate a synergistic effect of these two sites in this type of experiment. The activity of constructs containing four copies of alpha 1mutAmutB, or of alpha 1mutAwtB upstream of the minimal alpha 1(I) collagen promoter-luc chimeric gene (Fig. 3B) could not be increased upon cotransfection with the Cbfa1-expressing vector, thus demonstrating the specificity of the effect observed. In this set of experiments, we used multimers of the alpha 1(I) collagen OSE2 sites, because Cbfa1 does not transactivate the alpha 1(I) collagen promoter fragment in this type of assay. This is a consistent feature of Cbfa1 biology, indeed, we observed weak transactivation of the OG2 promoter when cotransfected with Cbfa1 (9), compared with the strong transactivating effect of Cbfa1 observed when using 6OSE2-luc (9).

Third, we asked whether these OSE2 sites could confer bone-specific expression to a reporter gene in vivo. For that purpose, we generated transgenic mice containing two of the constructs used in the above transfections, p4alpha 1AB-luc and p4alpha 1mutAmutB-luc. In transgenic mice harboring p4alpha 1AB-luc, luciferase activity could be detected in bone but neither in other tissues expressing type I collagen, nor in tissues which do not express type I collagen (Fig. 3C). As expected, given their respective sizes, the expression of p4alpha 1AB-luc was considerably lower than that of alpha 1(I) collagen (data not shown). The p4alpha 1mutAmutB-luc construct was not expressed in bone or any other tissue (Fig. 3C). Taken together with the results of the mutagenesis of the alpha 1A site and of the alpha 1B site, these results indicate that Cbfa1 contributes to the expression of alpha 1(I) collagen in osteoblasts through the alpha 1A site.

Cbfa1 Binds to the OSE2 Site Located at -372 bp in the Mouse alpha 1(I) collagen Promoter-- As mentioned at the beginning of "Results," there is a third OSE2 sequence in the alpha 1(I) collagen promoter, located at -372bp in mouse (site alpha 1C, Fig. 1A). This site is also conserved across species, and we first asked whether this OSE2-like sequence could be bound by Cbfa1 in EMSA. Labeled alpha 1C oligonucleotides were incubated with ROS 17/2.8 nuclear extract as described above, leading to the formation of a protein-DNA complex that migrated at the same location as that formed upon incubation of ROS 17/2.8 nuclear extract with labeled OSE2OG2 (Fig. 4A, lanes 1 and 3). However, the protein-DNA complex was of weak intensity compared with that we observed when using OG2OG2 or even alpha 1wtAmutB oligonucleotides as probes (Fig. 4A, lanes 1 and 2). No complex of this size was observed after incubation of labeled alpha 1mutC with ROS 17/2.8 extract (Fig. 4A, lane 3).



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Fig. 4.   The alpha 1C site is bound by Cbfa1 but is unable to activate a minimal alpha 1(I) collagen promoter. A, EMSA was performed using labeled OSE2OG2 (lane 1), alpha 1C (lane 3), or alpha 1mutC (lane 4) oligonucleotides and ROS 17/2.8 nuclear extract as a source of protein. The arrow indicates complex of interest. Supershift EMSA was performed using an antiserum against Cbfa1 (lane 6) or nonspecific antiserum (lane 5). The arrow indicates the complex of lower mobility formed after incubation with the anti-Cbfa1 antibody. B, EMSA using recombinant Cbfa1 as a source of protein and alpha 1C (lane 1) or alpha 1mutC (lane 2) oligonucleotides as a probe. C, multimers of alpha 1C and alpha 1mutC oligonucleotides were placed upstream of a minimal alpha 1(I) collagen promoter. These constructs were used in cotransfection assays in COS7 cells with a Cbfa1-expressing vector (dark bars) or an empty vector (open bars). Values represent -fold activation in relation to an empty reporter vector and are an average of at least three experiments done in triplicate.

To show that Cbfa1 was part of this protein-DNA complex, supershift experiments were performed using an anti-Cbfa1 antibody, a labeled alpha 1C oligonucleotide, and ROS17/2.8 nuclear extract as a source of protein. The incubation of ROS 17/2.8 nuclear extract with an antibody against Cbfa1 prior to the addition of labeled oligonucleotide led to the formation of a slower mobility complex. This complex was specific, because it was not observed when using a nonspecific antiserum (Fig. 4A, lanes 5 and 6). EMSA experiments using recombinant Cbfa1 provided further evidence that this site could bind, albeit weakly, Cbfa1, because Cbfa1 was able to bind to alpha 1C oligonucleotide but not to alpha 1mutC oligonucleotide (Fig. 4B, lanes 1 and 2). These results indicate that Cbfa1 is able to bind only weakly to the alpha 1C site in the mouse alpha 1(I) collagen promoter, suggesting that this OSE2 site may not play a critical role. To test this hypothesis, we cloned four copies of wild-type or mutated alpha 1C oligonucleotides upstream of the minimal alpha 1(I) collagen promoter fragment-luciferase chimeric gene used in Fig. 3A. As seen in Fig. 4C, in DNA transfection experiments in COS7 cells, neither wild-type nor mutant alpha 1C constructs could increase the activity of this reporter gene upon cotransfection with a Cbfa1 expression vector. This lack of an overt role for the alpha 1C site is consistent with the rather poor binding of Cbfa1 to this site. Taken together, these results indicate that the alpha 1C site is not a critical cis-acting element in controlling the osteoblast-specific expression of the mouse alpha 1(I) collagen gene.

Cbfa1 Binds to an OSE2 Site in the Mouse alpha 2(I) collagen Gene-- Because alpha 1(I) and alpha 2(I) collagen genes are often coregulated, we next asked whether Cbfa1 was also regulating the expression of alpha 2(I) collagen. Sequence analysis of the alpha 2(I) collagen promoter uncovered the existence of several potential OSE2 sites. Only one of these, located in the first exon of the gene, is present at the same location in the alpha 2(I) collagen gene of multiple vertebrate species (Fig. 1C). For this reason, this site was studied further. First, DNA binding was studied. EMSA was performed using a labeled double-stranded oligonucleotide containing the OSE2 element (alpha 2A) as a probe and ROS 17/2.8 nuclear extract as a source of protein. Incubation of labeled alpha 2A oligonucleotide with ROS 17/2.8 nuclear extract resulted in the generation of a protein-DNA complex migrating at the same location as the protein-DNA complex formed upon incubation of ROS 17/2.8 nuclear extract with labeled OSE2OG2 and alpha 1wtAmutB (Fig. 5A, lanes 1 and 2). This protein-DNA complex was specific, because it did not form upon incubation of ROS 17/2.8 nuclear extract with an oligonucleotide containing a mutation in this OSE2 sequence (alpha 2mutA) (Fig. 5A, lane 3). Because the binding of nuclear extract to alpha 2A oligonucleotide was weak, despite using a 2-fold higher amount of ROS 17/2.8 nuclear extract, we also used recombinant Cbfa1 protein in EMSA. The incubation of recombinant Cbfa1 with alpha 2A oligonucleotide, again using a 2-fold higher amount of Cbfa1 compared with that used to see binding of Cbfa1 to alpha 1wtAmutB, resulted in the formation of a protein-DNA complex (Fig. 5B, lane 1). This complex did not form upon incubation of Cbfa1 with alpha 2mutA oligonucleotide (Fig. 5B, lane 2). These data show that the conserved OSE2 sequence present in the alpha 2(I) collagen gene can bind Cbfa1, albeit more weakly than the alpha 1A site.



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Fig. 5.   Cbfa1 binds an OSE2 site in alpha 2(I) collagen, and this site can activate transcription. A, EMSA was performed using labeled OSE2OG2 (lane 1), alpha 2A (lane 2), or alpha 2mutA (lane 3) oligonucleotides and ROS 17/2.8 nuclear extract. B, EMSA using recombinant Cbfa1 as a source of protein and alpha 2A (lane 1) or alpha 2mutA (lane 2) oligonucleotides as a probe. C, multimers of alpha 2A and alpha 2mutA oligonucleotides were placed upstream of the minimal promoter constructs used for alpha 1(I) collagen transfections. These constructs were used in cotransfection assays in COS7 cells with a Cbfa1-expressing vector (dark bars) or with an empty vector (open bars). Values represent -fold activation in relation to an empty reporter vector and are an average of at least three experiments done in triplicate.

Cbfa1 Can Activate Transcription through the alpha 2A Site-- To study the function of the alpha 2A site, cotransfection assays were performed in COS7 cells. In this assay, exogenous Cbfa1 transactivated a construct containing a multimer of four alpha 2A oligonucleotides fused to a minimal alpha 1(I) collagen promoter-luc chimeric gene, p4alpha 2A-luc (Fig. 5C), producing an ~15-fold increase in luciferase activity. This effect was specific, because the construct containing a multimer of six alpha 2mutA oligonucleotides (Fig. 5C) produced no activity. The relatively weak increase in luciferase activity compared with the effect observed with multimers of the alpha 1wtAmutB oligonucleotide is consistent with the weaker binding of Cbfa1 to the alpha 2A sequence.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Taken together, our data provide evidence indicating that Cbfa1 is one of the transcription factors contributing to the expression of the two Type I collagen genes in osteoblasts in vivo. Moreover, along with CBF (3, 4, 18) and Sp1 (5, 7), Cbfa1 belongs to a growing group of transcription factors accounting for the coordinated regulation of both genes. The finding that Cbfa1 favors type I collagen expression in osteoblasts is consistent with the absence of bone extracellular matrix in Cbfa1-deficient mice and with the marked decrease of Type I collagen expression in transgenic mice overexpressing a dominant negative form of Cbfa1 in osteoblasts (10, 11, 14). These results broaden the spectrum of transcription factors able to regulate type I collagen gene expression at various stages of osteoblast differentiation and overall increase our understanding of type I collagen genes' regulation.

The data presented here indicate that there is a clear functional hierarchy between the different Cbfa1-binding sites, or OSE2 sites, present in the alpha 1(I) collagen promoter. Clearly, the alpha 1A element is the most potent activator of expression of all the OSE2 elements we studied in this promoter. These findings do not exclude the possibility that OSE2 sites present further upstream in the promoter and/or elsewhere in the gene may also contribute to the osteoblast-specific expression of the alpha 1(I) collagen gene. Conceivably, one of these as of yet uncharacterized OSE2 sites may bind Cbfa1 with a higher affinity and act as a more powerful osteoblast-specific cis-acting element. We did not identify any conserved consensus OSE2 sites by examining the DNA sequence of the region located between -1540 and -1656 that has been previously shown to be required for osteoblast expression (25). This reinforces the hypothesis that other cell-specific transcription factors must contribute to osteoblastic expression of the type I collagen genes.

Although Cbfa1 can bind to a site present in the alpha 2(I) collagen gene and the expression of this gene is decreased in transgenic mice expressing a dominant negative form of Cbfa1, the level of activation observed in cotransfection experiments with the alpha 2A construct was lower than that seen when using the alpha 1AB constructs based on the alpha 1(I) collagen promoter. At least two explanations could account for this observation. First, and most importantly, the binding of ROS 17/2.8 nuclear extract to the alpha 2A site was weaker than its binding to the alpha 1A site of alpha 1(I) collagen, indicating that this site has a lower affinity to Cbfa1. Second, considering the numerous OSE2 sites present in the alpha 2(I) collagen promoter, it is likely that, for this gene and for the alpha 1(I) collagen gene as well, some of the other OSE2 sites act alone or in concert with the conserved OSE2 site to control its expression in osteoblasts in vivo.

If Cbfa1 is one positive regulator of type I collagen expression in osteoblasts, it is clear from the above data that it is not the only one. Indeed, Cbfa1 expression is initiated in osteoblast progenitors after type I collagen expression can be noticed in mesenchymal cells. Moreover, at least one other cis-acting element has been shown to be implicated in osteoblast-specific expression of the alpha 1(I) collagen gene in mouse and rat (15, 16). Members of the DLX family of homeobox proteins are able to bind to this sequence and to activate transcription (22). Another homeobox-related protein, MSX2, can bind to this sequence and repress expression of the alpha 1(I) collagen gene (23), and recent genetic evidence has demonstrated that MSX2 is upstream of Cbfa1 (24). These homeobox proteins are likely to be expressed earlier than Cbfa1 and may even control its expression, directly or indirectly. It is tempting to speculate that these homeobox proteins, possibly with other regulatory proteins, act early during the specification of mesenchymal progenitor cells to the osteoblast lineage and that Cbfa1 is required for osteoblast differentiation and for the maintenance of the osteoblast phenotype. This hypothesis will be more easily testable when mice deficient for several DLX proteins are available. Regardless, the observation that Cbfa1 binds to and regulates the activity of both type I collagen genes in osteoblasts further illustrates how important Cbfa1 is in osteoblast physiology.


    ACKNOWLEDGEMENTS

We thank Dr. P. Ducy and Dr. T. Schinke for critically reading the manuscript.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants R01AR45548 and R01DE11290, March of Dimes Grant F-198-0082, and a grant from Eli Lilly and Co. (to G. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030. Tel.: 713-798-5489; Fax: 713-798-1465; E-mail karsenty@bcm.tmc.edu.

Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M006215200


    ABBREVIATIONS

The abbreviations used are: kb, kilobase(s); DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; bp, base pair(s).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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