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Control and Counter-Control of TGF-ß Activity through FAST and Runx (CBFa) Transcriptional Elements in Osteoblasts

Departments of Surgery (Plastic Surgery Section) (C.J., T.L.M., M.C.) and Pathology (O.E.), Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Michael Centrella, Ph.D., Department of Surgery, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520-8041. E-mail: michael.centrella{at}yale.edu

	Abstract

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FAST and Runx (CBFa) transcription factors, which are expressed during specific phases of embryogenesis and tissue patterning, bind directly to Smad proteins and integrate effects induced by various TGF-ß gene family members. The DNA binding sequences for FAST and Runx differ only minimally. The isoform Runx2 (previously termed CBFa1) is highly expressed by osteoblasts and regulates expression of the TGF-ß receptor I in these cells. Here we show that FAST-dependent transcription is endogenously restricted in osteoblasts but can be significantly enhanced by disruption of Runx2 expression. Native and synthetic Runx2 bind to both Runx and FAST binding sequences, whereas FAST-1 efficiently binds only to the FAST binding sequence. However, overexpression of FAST-1 potently suppresses TGF-ß receptor I gene expression in osteoblasts and thereby reduces TGF-ß activity independently of competing for Runx2 at the level of DNA binding. These results provide a new example of how nuclear factors associated with specific developmental states or tissue lineages may influence TGF-ß-dependent events in restricted ways.

	Introduction

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NUCLEAR EVENTS INDUCED by TGF-ß gene family members occur through multiple intracellular mediators (1, 2). In vitro studies reveal that certain transcription factors interact directly with members of one group of these mediators, termed Smads. This interaction appears to integrate and focus signals generated by TGF-ß or activin-A in specific ways (3, 4, 5). A consensus DNA sequence, 5'-AATCCACA-3', binds two forkhead transcription factors, expressed during early embryogenesis and designated as FAST proteins, that act in this manner (3, 4). This sequence differs only slightly from a strong DNA binding sequence, 5'-XAACCACA-3', for members of the Runt homology domain transcription factor family (AML, CBFa, PEBP2), now termed Runx factors (6). These similarities suggest that transcriptional effects currently ascribed to either a FAST or a Runx protein might also be regulated under conditions where a member of the other nuclear factor gene family is preferentially expressed.

In bone, the Runx isoform Runx2 (previously termed CBFa1) controls the expression of several genes associated with differentiated osteoblasts (7, 8, 9, 10, 11, 12). In this regard, the TGF-ß type I receptor (TßRI), whose gene promoter contains several Runx binding sites (9, 13), is retained on differentiated osteoblasts when other TßR levels decline (14). Forced expression of Runx protein enhances TßRI gene promoter activity in undifferentiated bone cells (13). Furthermore, loss of Runx2 by exposure to high levels of glucocorticoid reduces TßRI expression by fetal rat osteoblasts, and in so doing, suppresses TGF-ß-dependent biochemical effects (9).

Due to strong similarities between the cognate Runx and FAST binding sequences, Runx may therefore bind to both cis-acting elements in cells like osteoblasts that express preferentially high levels of Runx protein. In these cases, Runx-dependent genes may be controlled in appropriate ways, whereas the expression of genes that require FAST protein complexes may be either induced or disrupted. Similarly, and independently of its own cognate effects, high levels of FAST may interact directly with Runx cis-acting elements, or may indirectly regulate TGF-ß activity through changes in Runx-dependent TßRI expression. In this study, we assessed whether variations in the expression of Runx2 and FAST-1 alter gene expression directed by their individual cis-acting elements in differentiated osteoblasts, where an abundance of Runx2 preexists. Because Runx2 regulates TßRI expression by these cells, we also examined whether a high level of FAST-1 alters TßRI gene promoter activity and TGF-ß-dependent gene expression in these cells. Our results provide a new example of how nuclear events induced by TGF-ß may rely on contextual changes in transcription factors during specific developmental states or within individual tissue lineages.

	Materials and Methods

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Cell cultures
Osteoblast-enriched cell cultures were prepared from parietal bones of 22- d-old Sprague Dawley rat fetuses (Charles River Breeding Laboratories, Raleigh, NC) by methods approved by Yale Animal Care and Use Committee. Sutures were removed by dissection, and cells were released from bone fragments by five sequential digestions with collagenase (15). Cells from the last three digestions exhibit high levels of PTH receptors and type I collagen synthesis, an increase in osteocalcin expression in response to vitamin D₃, and high alkaline phosphatase specific activity (15, 16, 17). Differential sensitivity to TGF-ß, bone morphogenic proteins, various PGs, expression of nuclear factor Runx2, mineralized nodule formation in vitro further distinguish osteoblast-enriched cultures from less differentiated periosteal cells released during the initial collagenase digestion interval (13, 14, 18, 19). Cells were plated at 4,000/cm² in DMEM supplemented with 10% FBS (Life Technologies, Inc.). COS-7 (CRL 1651) and LLC-PK₁ (CRL1392), obtained from the ATCC (Manassas, VA), were plated at 10,000/cm² in identical culture medium.

Transfection constructs
Reporter plasmids SBE4, SBE, FBE, and FSBE, which contain Smad, FAST, or composite FAST/Smad binding elements (3, 20), and an expression construct encoding the human FAST-1 gene (3), were obtained from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). Reporter plasmid 3TPLux, which contains three AP1-related response elements and a minimal fragment of the plasminogen activator inhibitor 1 gene promoter and is highly sensitive to stimulation by TGF-ß in many cells, including in primary osteoblast cultures (9, 20, 21, 22), was obtained from Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center, New York, NY). Reporter plasmid ARE, which contains a FAST-dependent TGF-ß response element within the context of a Mix.2 gene promoter fragment (23) was obtained from Dr. Malcolm Whitman (Harvard University, Boston, MA). Reporter plasmids pSXN1C, with two Runx binding elements (9, 13), pTßRI/0.4, with three Runx binding elements within the context of a rat TßRI gene promoter fragment (13), and expression constructs encoding murine Runx2 (initially designated as PEBP2-A1) (24) were described previously. Antisense to Runx2 was produced by ligating a 2.25-kb restriction fragment of the murine gene in reversed orientation into vector pSV7d. For some studies, full-length FAST-1 in vector pcDNA3 (Invitrogen) was modified to include a carboxyl-terminal hemophilus influenza hemagglutinin (HA) epitope tag.

Transfections
Promoter/reporter or gene expression plasmid constructs, or empty vectors that were pretitrated for optimal expression efficiency, were transfected using LT1 (Mirus Corp., Madison, WI). Briefly, cultures at 50–70% confluence were exposed to plasmids for 16 h in serum-depleted medium, and supplemented to obtain a final concentration of 5% serum. For reporter gene assays, cultures were expanded for 48 h, treated in serum-free medium, rinsed, and lysed. Nuclear-free supernatants were analyzed for reporter gene activity and corrected for protein content. To account for competition among plasmids for limiting transcriptional components, control cultures were transfected with equivalent amounts of empty expression vectors. Transfection efficiency was assessed in parallel in cells transfected with positive and negative reporter plasmids, as in previous studies (9, 13).

Cytoplasmic and nuclear protein extracts
Cells were rinsed, harvested by scraping and centrifugation, and lysed in hypotonic buffer supplemented with phosphatase and protease inhibitors and 1% Triton X-100. Nuclei and cytoplasm were separated by centrifugation. Nuclei were resuspended in hypertonic buffer with glycerol and phosphatase and protease inhibitors. Released nuclear proteins were separated from insoluble material by centrifugation (9, 13).

Western blot analysis
Cell extracts were fractionated by electrophoresis on a 12% SDS polyacrylamide gel, blotted onto Immobilon P membranes (Millipore Corp., Bedford, MA), probed with antibody to Runx2, TßRI or TßRII, and visualized with secondary antibody, ECL reagents (Amersham Pharmacia Biotech, Piscataway, NJ) and chemiluminescence (9, 13, 25).

EMSA
Commercial double-stranded DNA probes (Table 2) were radiolabeled by annealing complementary oligonucleotides. Overhangs were filled with dNTPs and [-³²P]dATP using the Klenow fragment of DNA polymerase I. Five to 10 µg of nuclear extract protein was preincubated on ice without or with unlabeled specific or nonspecific competitor DNA, supplemented with [³²P]-labeled probe (0.1–0.2 ng at 5 x 10⁴ cpm). In some samples, nuclear extract was preincubated with antiserum for 30 min before adding [³²P]-labeled probe. Radioactive complexes were resolved on a 5% nondenaturing polyacrylamide gel and visualized by autoradiography (9, 13).

Reagents
Recombinant TGF-ß1 identical in sequence to human TGF-ß1 was used in collaboration with Bristol-Myers Squibb Co. (Seattle, WA). Antiserum directed against human Runx2 (initially designated AML-3) was generously provided by Dr. Scott W. Hiebert (Vanderbilt University, Nashville, TN) (25). Nonimmune rabbit serum and antibodies directed against TßRI, TßRII, and HA were from Santa Cruz Biotechnologies (Santa Cruz, CA).

Statistical analysis
Statistical differences were assessed by one-way ANOVA, and the Student’s-Newman-Keuls method was used for post hoc analysis, with SigmaStat software (Jandel Corp., San Rafael, CA). A significant difference was assumed by a P value of <0.05.

	Results

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TGF-ß-dependent gene promoter usage in osteoblasts
Basal gene expression directed by the promoter/reporter transfection construct SBE4 (Fig. 1A), which contains four Smad binding elements, is relatively high in untreated osteoblasts by comparison to constructs SBE, FBE, or FSBE (Fig. 1B), which contain single Smad or FAST DNA binding elements, or composite FAST and Smad binding elements, respectively (see Table 1). Basal gene expression by the promoter/reporter construct 3TPLux (Fig. 1C), which contains three AP1 binding elements and a minimal fragment of the plasminogen activator inhibitor promoter and is a widely used indicator of TGF-ß-dependent gene expression (9, 20, 21, 22), is also relatively low in osteoblasts. A maximally effective amount of TGF-ß1 induced a 2-fold increase in gene expression by SBE4 (Fig. 1A) and a 20-fold increase by 3TPLux (Fig. 1C). Although gene expression by 3TPLux increases in osteoblasts in response to growth regulators that activate protein kinase C, the extent and duration of reporter expression by these factors is limited by comparison to treatment with TGF-ß (data not shown). However, similar to results in transformed colon and keratinocyte cell cultures (3, 4, 5), TGF-ß1 did not significantly affect the activity of the gene reporter constructs SBE or FBE in osteoblasts. In contrast to those systems, however, TGF-ß induced a 2-fold increase in FSBE gene promoter activity without the need to express FAST-1 transgenically (Fig. 1B). Therefore, trans-acting factors that use Smad, or combined Smad and FAST binding elements are present in differentiated osteoblasts, but appear surprisingly limited in their ability to respond to TGF-ß by comparison to the transcriptional factors that drive gene promoter activity by 3TPLux. In analogous studies in LLC-PK₁ porcine kidney cells (ATCC CRL 1392), each reporter plasmid construct propagated a similar relative basal activity. However, TGF-ß1 induced a more striking 10- to 20-fold stimulatory effect on reporter gene expression by SBE4 and had virtually no effect on FSBE activity (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 1. TGF-ß-dependent gene promoter activity in osteoblasts. Osteoblast-enriched cultures were transfected for 48 h with 200 ng/0.5 ml of promoter/reporter plasmid SBE4 (four Smad DNA binding elements) in A; with SBE (one Smad binding element), FBE (one FAST binding element), or FSBE (contiguous FAST and Smad binding elements) in B; or with 3TPLux (three AP1 binding elements upstream of a minimal plasminogen activator inhibitor promoter sequence) in C. Transfected cells were treated for 24 h with vehicle (-) or 120 pM TGF-ß1 (+) in serum-free medium, and reporter gene activity was measured and corrected for relative protein content. Data are means ± SE from 9 to 18 replicate cultures per condition and 3 to 6 experiments. TGF-ß significantly increased reporter gene expression in cells transfected with SBE4, FSBE, and 3TPLux plasmids.

View larger version (47K): [in this window] [in a new window]   Figure 2. Runx2 differentially regulates Runx and FAST-dependent gene expression. In A, osteoblast-enriched cultures were transfected with 300 ng/0.5 ml of empty vector or Runx2 antisense expression construct, and visualized after 2 d at subconfluence (S) or after 5 d at confluence (C). In B, 50 µg of total nuclear protein from confluent untransfected (0) cells, or cells transfected for 48 h with 300 ng/0.5 ml of empty vector (V) or Runx2 antisense (S) expression plasmid was examined by Western blot analysis with anti-Runx2 specific antibody. In C, cells were cotransfected for 48 h with 200 ng of promoter/reporter plasmid SXN1C (containing two Runx2 binding elements) and 300 ng of empty vector or Runx2 antisense expression plasmid in 0.5 ml, as indicated. In D, cells were cotransfected for 48 h with 200 ng of promoter/reporter plasmid FSBE and 300 ng total of empty vector or Runx2 antisense expression plasmid in 0.5 ml, as indicated. In E, cells were cotransfected for 48 h with 200 ng of promoter/reporter plasmid FSBE and 50 ng of empty vector or Runx2 expression plasmid in 0.5 ml, as indicated. In F, cells were cotransfected for 48 h with 1 µg of promoter/reporter plasmid ARE and 50 ng of empty vector or Runx2 expression plasmid in 0.5 ml, as indicated. In D–F, cells were then treated for 24 h with vehicle (control) or 120 pM TGF-ß1 in serum-free medium. In C–F, reporter gene activity was measured and corrected for relative protein content. Data are means ± SE from 9–18 replicate cultures per condition and 3–6 experiments. Treatment with TGF-ß significantly increased reporter gene expression in cells transfected with plasmid FSBE in D and E, and with plasmid ARE in F. Transfection with Runx2 antisense significantly reduced reporter gene expression in cells transfected with plasmid SXN1C in C, as well as the stimulatory effects of TGF-ß in cells transfected with plasmid FSBE in E and plasmid ARE in F. In contrast, transfection with Runx2 antisense at 0.3 µg significantly increased reporter gene expression in cells transfected with plasmid FSBE in D.

View larger version (26K): [in this window] [in a new window]   Figure 3. Relative binding by Runx2 and FAST-1 to Runx and FAST consensus DNA binding elements. In A, nuclear extract from osteoblast enriched cultures was combined with 32P-labeled probe Runx CS2 (encoding a consensus Runx2 binding sequence in the TßRI gene promoter), without (0) or with 50- or 200-fold excess of unlabeled CS2 (R2), FAST (F), or Runx (R1) consensus oligonucleotide, as indicated. In B, nuclear extract from osteoblast-enriched cultures, or from COS-7 cells transfected to express Runx2 (COS/Runx2) or FAST-1 (COS/FAST-1) was combined with 32P-labeled consensus probes encoding FAST (FAST CS) or Runx (Runx CS1) binding sequences. Reactions included no addition (0) or 200-fold excess of unlabeled Runx (R1) or FAST (F) consensus probe, as indicated. In C, extract from COS-7 cells cotransfected to express HA-tagged FAST-1 and Runx2 (COS/HA-F+R2) was combined with 32P-labeled FAST CS probe, without or with anti-HA (HA) or anti-Runx2 (R2) specific antibody. Complexes were resolved on 5% nondenaturing polyacrylamide gels and visualized by autoradiography. Similar results were observed in two to three studies.

To assess whether Runx2 could still interact with the FAST consensus sequence in the presence of high levels of FAST-1, nuclear extract was prepared from COS cells transfected to express both Runx2 and HA-tagged FAST-1. Complex R formed with the FAST consensus probe was specifically reduced by anti-Runx2 antibody, whereas a fourth complex (designated HA-F) was reduced by anti-HA antibody (Fig. 3C). However, consistent with little or no binding by FAST-1 to RUNX consensus sequences, no anti-HA antibody reactive complex formed with this extract and Runx2 probe (data not shown). Therefore, Runx2 can bind to both the FAST and Runx consensus DNA sequences to form complex R, and FAST-1 predominantly binds the FAST consensus sequence to form complex F.

Loss of Runx2 limits TßRI expression and TGF-ß activity in osteoblasts
Consistent with multiple Runx binding sites in the TßRI gene promoter and high levels of Runx2 in osteoblasts, transfection with the Runx2 antisense expression construct dose dependently decreased TßRI gene promoter activity in these cells. Runx2 antisense expression also specifically decreased the amount of TßRI protein in these cells, whereas it had no effect on TßRII (Fig. 4, A and B). Loss of Runx2 in this way did not alter basal collagen synthesis in untreated cultures (102 ± 9% of control). However, in agreement with its ability to reduce TßRI synthesis, expression of Runx2 antisense limited the stimulatory effect of TGF-ß on collagen synthesis as well as on gene expression through the promoter/reporter construct 3TPLux (Figs. 4, C and D).

View larger version (47K): [in this window] [in a new window]   Figure 4. Loss of Runx2 suppresses TßRI gene expression and TGF-ß activity. In A, osteoblast-enriched cultures were cotransfected for 48 h with 100 ng of promoter/reporter plasmid TßRI/0.4 and 300 ng total of empty vector or Runx2 antisense (S) expression plasmid in 0.5 ml. In B, 100 µg of total cytoplasmic protein from confluent untransfected cells (0), or cells transfected for 48 h with 300 ng/0.5 ml of empty vector (V) or Runx2 S expression plasmid, was examined by Western blot analysis with anti-TßRI or TßRII specific antibody. In C, cells were transfected for 48 h with 300 ng/0.5 ml of empty vector or Runx2 S expression plasmid. Transfected cells were treated for 24 h with the amounts of TGF-ß1 indicated in serum-free medium, and pulsed with [3H]proline for the last two h of incubation. Collagenase digestible protein was determined in the total cell extract. Data are means ± SE from 12–16 replicate cultures per condition and 3–4 experiments. In D, cells were cotransfected for 48 h with 200 ng of promoter/reporter plasmid 3TPLux and 300 ng of empty vector or Runx2 S expression plasmid in 0.5 ml. Transfected cells were treated for 24 h with 120 pM TGF-ß1 in serum-free medium. In A and D, reporter gene activity was measured and corrected for relative protein content. Data are means ± SE from 9–15 replicate cultures per condition and 3–5 experiments. Transfection with the Runx2 S expression plasmid significantly suppressed TßRI gene promoter activity in A, and the stimulatory effect of TGF-ß on collagen synthesis in C and the 3TPLux reporter plasmid in D.

View larger version (35K): [in this window] [in a new window]   Figure 5. High levels of FAST-1 suppress Runx-dependent gene expression. Osteoblast-enriched cultures were cotransfected for 48 h with 100 ng of empty vector or FAST-1 expression plasmid and in A, with 200 ng of promoter/reporter plasmid SXN1C; in B, with 100 ng of promoter/reporter plasmid TßRI/0.4; in C, with 200 ng promoter/reporter plasmid 3TPLux; or in F, with 300 ng total of empty vector or Runx2 antisense expression plasmid and 200 ng of promoter/reporter plasmid FSBE in 0.5 ml, as indicated. In C–E, transfected cells were treated for 24 h with vehicle (control) or 120 pM TGF-ß1 in serum-free medium. In A–C and F, reporter gene activity was measured and corrected for relative protein content. Data are means ± SE from 9–24 replicate cultures per condition and 3–8 experiments. In D and E, cells were pulsed with [3H]proline for the last 2 h of incubation, and incorporation into collagen and noncollagen protein was determined in the total cell extract. Data are means ± SE from 9 replicate cultures per condition and 3 experiments. Transfection with the FAST-1 expression plasmid significantly suppressed Runx-dependent gene expression in A, TßRI gene promoter activity in B, and stimulatory effects of TGF-ß on the 3TPLux and FSBE reporter plasmids, in C and F. Transfection with the FAST-1 expression plasmid also significantly reduced the stimulatory effects of TGF-ß on collagen synthesis and noncollagen protein synthesis in D and E.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
TGF-ß produces diverse effects on gene expression that relate to tissue and cell specificity and to cellular differentiation status. This is thought to require different or different combinations of intracellular effectors and transcription factor complexes (1, 2, 3). In this study, we provide new evidence for counter-regulatory effects by members of two transcription factor gene families, FAST and Runx, previously associated with discrete aspects of TGF-ß activity. Links between these factors occur in part through competition for a common transcriptional element, and in part through changes in TßRI expression. We found that FAST-dependent gene expression was minimal in cells where high levels of Runx2 preexist, and that Runx2 effectively competed for and associated with a consensus FAST binding sequence. We also found that forced expression of FAST-1 could decrease Runx2 activity. In this case, however, FAST-1 did not compete for consensus Runx binding sequences, although it limited TßRI gene promoter activity in osteoblasts and suppressed downstream effects by TGF-ß treatment. Therefore, effects by FAST-1 appeared focused on other elements required for Runx transcriptional activity. A model for these events is shown in Fig. 6.

View larger version (30K): [in this window] [in a new window]   Figure 6. FAST and Runx transcription factors cosuppress gene expression in osteoblasts. As shown on the left, Runx may suppress FAST-dependent gene expression in osteoblasts by competing directly for FAST-specific cis-acting DNA binding elements. As shown on the right, FAST and Runx may also compete for Smad coregulators of gene transcription. By contrast, FAST does not compete well for binding at Runx-specific cis-acting elements, whereas at sufficiently high levels it can compete with Runx for Smads and thereby suppress Runx-dependent gene expression. In this way, it also decreases TßRI synthesis and reduces TGF-ß activity in these cells, where Runx helps to maintain a high level of TßRI.

Runx was either more abundant in osteoblast-derived extracts, or exhibited a higher DNA binding affinity relative to FAST protein, when tested with either Runx or FAST consensus binding sequences. We were only able to assess the presence of endogenous FAST in fetal rat osteoblasts by its functional effects on gene expression with synthetic and native promoter fragments, because cross-reactive antibody to rat FAST protein was unavailable for our studies (Malcolm Whitman, Harvard University; personal communication). However, recombinant FAST-1 prepared from transfected COS-7 cells readily associated with a consensus FAST binding sequence, but bound only poorly to a Runx consensus element. We earlier showed that oligonucleotide with an A to T substitution, positioned as it occurs in the FAST binding sequence used here, effectively competed for complex formation by two radiolabeled consensus Runx probes (13). Therefore, stimulatory effects by FAST may be focused to a more limited set of gene promoters by comparison to the growing number now associated with Runx proteins. In this regard, the slightly different FAST binding sequence that occurs in the Mix.2 gene promoter (5), where a single nucleotide difference disrupts the Runx binding core, also shows minimal activity in osteoblasts. Moreover, in initial studies we found that treatment with bone morphogenetic protein 2, which increases Runx2 expression by osteoblasts, suppresses Mix.2 gene promoter activity in osteoblasts, whereas TGF-ß does not (unpublished results). Nevertheless, forced expression of FAST-1 did not overcome the inhibitory effect of Runx on FAST-dependent gene expression, but suppressed Runx-dependent gene expression by osteoblasts. Importantly, FAST and Runx family members each directly associate with specific Smads to integrate the expression of genes induced by various TGF-ß gene family members (1, 2, 3, 4, 5, 26, 27, 28). Therefore, FAST appears to suppress Runx-dependent gene expression as well as some aspects of TGF-ß activity in osteoblasts by competition for a limiting level of Smads. Alternately, at sufficiently high levels, FAST proteins may bind to and directly suppress Runx activity. We are now assessing whether the ability of these factors to compete in restricted ways for common and discrete genomic elements or transcription integrators, or to form complexes between themselves, distinguishes direct stimulatory effects on some gene promoters and indirect inhibitory effects on others.

In summary, our study provides new examples of mechanisms to account for restricted gene expression in response to TGF-ß treatment. Some occur at the level of transcription factor expression, some on competition between factors for common transcription elements, and some on downstream effects on TßRI expression. A better understanding of the interactions between FAST and Runx gene family members for common cis- and trans-acting genomic elements, and the relatively tissue-restricted expression of Runx proteins by hematopoietic and skeletal cells, may help to define transitional effects by TGF-ß during development and tissue specification.

	Acknowledgments

 
We are grateful for promoter/reporter and expression plasmid constructs from Dr. Bert Vogelstein (Baltimore, MD), Dr. Joan Massague (New York, NY), and Dr. Malcolm Whitman (Boston, MA); for anti-Runx2 (human AML-3) specific antiserum from Dr. Scott Hiebert (Nashville, TN); for a Runx2 expression construct (murine PEBP2A1) from Dr. Yoshiaki Ito (Kyoto, Japan); for recombinant TGF-ß1 used in collaboration with Bristol-Myers Squibb Co. (Seattle, WA); and for helpful discussions with Dr. Whitman, and with Dr. Peter Gergen (Stonybrook, NY).

	Footnotes

 
These studies were supported by National Institute of Musculoskeletal and Skin Diseases Award AR-39201 and the Arthritis Foundation Biomedical Science Research Program.

Abbreviations: HA, Hemagglutinin; TßRI, TGF-ß type I receptor.

Received February 26, 2001.

Accepted for publication May 26, 2001.

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