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Molecular Endocrinology 18 (1): 241-251
Copyright © 2004 by The Endocrine Society

Fibroblast Growth Factor Receptor 3 Gene: Regulation by Serum Response Factor

Martina I. Reinhold, Donald G. McEwen and Michael C. Naski

Department of Pathology (M.I.R., M.C.N.), University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229; and Lineberger Comprehensive Cancer Center (D.G.M.), University of North Carolina, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: Michael C. Naski, M.D., Ph.D., Department of Pathology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900. E-mail: naski{at}pathology.uthscsa.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have previously identified a cis-acting sequence in the proximal promoter of the fibroblast growth factor receptor 3 (FGFR3) gene that strongly activates transcription in chondrocytic cells. Here we report that the transcriptional activity of this sequence (FRE3) requires serum response factor and its cognate recognition motif, serum response element. Although the FRE3 contains consensus sequence motifs for several transcription factors, the serum response element is paramount for the transcriptional activity of the FRE3. Additionally, the transcriptional activity of the proximal promoter of the FGFR3 gene is suppressed by mutation of the serum response element. Serum response factor binds to the FRE3 as evidenced by gel shift experiments and antibody supershift experiments and expression of a dominant negative form of serum response factor suppresses the activity of FRE3. Additionally, serum response factor binds to the FGFR3 gene in vivo, as demonstrated by chromatin immunoprecipitation. Serum response factor is an important regulator of cardiac, skeletal, and smooth muscle gene expression; these data suggest that serum response factor is also an important determinant of chondrocyte gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE FIBROBLAST GROWTH factor receptors (FGFRs) constitute a family of four receptor tyrosine kinases (FGFR 1, 2, 3, 4) (1). The FGFRs are widely, but uniquely expressed during embryonic and postnatal development as well as in adult somatic tissues. The wide distribution of FGFRs underscores their key functions during development. For example, FGFR2 and FGFR3 have critical functions during skeletal growth and development. Mice with a hypomorphic mutation of the fgfr2 gene fail to develop limb buds (2). Activating mutations in the FGFR2 gene cause deformities of the skull and distal extremities as exemplified by the human craniosynostoses, Aperts and Crouzon Syndrome (3, 4). Targeted inactivation of the murine fgfr3 gene causes skeletal overgrowth (5), whereas gain of function mutations in the FGFR3 gene cause the human dwarfing conditions achondroplasia, thanatophoric dysplasia, and hypochondroplasia (6, 7, 8, 9). Together, these data demonstrate distinct roles for FGFR3 and FGFR2 during skeletogenesis and skeletal growth. FGFR3 regulates the growth of long bones, implying that a chief function of FGFR3 is to control endochondral ossification. FGFR2 is required for the outgrowth of the embryonic limb bud, patterning of the digits and growth of the cranial sutures.

Although the FGFRs have different functions during development, the receptors have very similar structures. FGFR 2 and 3 share 63% amino acid conservation. In the tyrosine kinase domain, the receptors have 85% amino acid identity. This suggests that although the receptors have different developmental functions, they share similar signaling pathways. This is supported by the finding that transgenic mice expressing a chimeric receptor composed of either the intracellular signaling domain of FGFR1 or FGFR3 share similar dwarfing phenotypes (10). Consequently, the distinct roles of different FGFRs during vertebrate growth and development results from the unique expression patterns of the receptors and their respective ligands.

The spatial and temporal expression of FGFR3 is distinct from FGFR2 as well as other FGFRs. For example, in tissues of the developing skeleton FGFR3 is expressed in nascent cartilage and the growth plate of long bones (11, 12), whereas FGFR2 is expressed in cranial sutures and periosteum (13, 14). This temporal/spatial difference is vital to determining the effects of the receptor during development. Consequently, understanding the pathways that control FGFR3 gene expression may yield insight into the transcriptional determinants of endochondral bone growth. Recent efforts have begun to examine the cis-acting DNA sequences that contribute to the expression of FGFR3. Members of the Sp1 family of transcription factors regulate the transcription of FGFR3 gene through interactions with cis-regulatory elements in the first intron and the 5' proximal promoter (15). In addition, a cis-acting element of the FGFR3 gene that strongly regulates gene expression in chondrocytic cells was identified (16). We showed that a 52-bp motif from -2311 to -2263 of the FGFR3 gene dramatically increased the transcriptional activity of the FGFR3 promoter as well as a heterologous minimal promoter. Here we further characterize the 52-bp regulatory motif of the FGFR3 gene. The 52-bp sequence element (hereafter referred to as FRE3) contains putative binding sites for a number of transcription factors, yet we demonstrate that a serum response element (SRE), CC(A/T)6GG is essential for the transcriptional activity of FRE3. We show that serum response factor (SRF) binds to an oligonucleotide consisting of the FRE3 sequence and that the transcriptional activity of FRE3 requires an intact SRE. Chromatin immunoprecipitation shows that SRF binds to the FGFR3 gene in vivo. In addition, we show that serum and other activators of SRF can potentiate the transcriptional activity of FRE3 as well as the proximal FGFR3 promoter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Localization of Regulatory Elements in FGFR3 Gene
Previously, we defined a cis-acting sequence of the FGFR 3 gene that confers strong expression in chondrocytic cells (16). Sequence between nucleotides -2311 and -1537 (relative to the transcriptional start site) enhance transcription of a luciferase reporter construct in the chondrocytic cell line, RCJ (Fig. 1AGo). Deletion experiments showed that cis-acting regulatory activity could be localized to nucleotides -2311 and -2263 of the fgfr3 gene (16). This 52-bp motif (denoted FRE3), when multimerized into a two-copy, head-to-tail array dramatically enhanced the transcriptional activity of the Rous sarcoma virus (RSV) minimal promoter (pRSVluc) in the chondrocytic RCJ cell line (Fig. 1BGo). The FRE3 also showed strong transcriptional activity in the chondrocytic CFK2 cell line, yet significantly less activity in NIH 3T3 (fibroblast), HeLa (cervical carcinoma), MDA231 (breast carcinoma), and PC-3 (prostate carcinoma) cells (Fig. 1CGo).



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  		Fig. 1. Sequence of the FRE3 Confers Transcriptional Activity in Chondrocytic RCJ Cells

RCJ cells (or the indicated cell line) were transiently transfected with the indicated reporter constructs and a constitutive ß-galactosidase reporter plasmid. Cell lysates were assayed for luciferase and ß-galactosidase activity. Luciferase activity is expressed relative to ß-galactosidase to normalize for transfection efficiency. A, Sequence between -2311 and -1537 (relative to the transcriptional start site) of the fgfr3 gene activate gene expression in chondrocytic RCJ cells. B, Sequence between -2311 and -2263 (-2311 to -2263 = FRE3) dramatically up-regulate the activity of the RSV (pRSVluc) minimal promoter. C, Comparison of pFRE3-RSVluc transcriptional activity in transiently transfected cells of mesenchymal (RCJ, CFK2, NIH3T3) or epithelial cell lineage (HeLa, MDA231, PC3). The data of each panel (A–C) are representative of results from three independent experiments. Error bars represent ± SEM.

 
Regulation of FRE3-Mediated Transcriptional Activity by Agents that Activate Serum Response Factor
The FRE3 contains consensus recognition sites for several transcription factors (Fig. 2Go). The presence of a putative SRF binding site (SRE) within FRE3 suggested that its activity might be regulated by serum. To test this hypothesis, cells transiently transfected with pFRE3-RSVluc were serum starved for 12 h in 1% fetal bovine serum, then induced with 15% fetal bovine serum for 1, 2, or 3 h. Indeed, stimulation with 15% fetal bovine serum caused a 2-fold (P = 0.003) increase in transcriptional activity of pFRE3-RSVluc (Fig. 3AGo). RCJ cells stably transfected with pFRE3-RSVluc were induced (2.4- to 6-fold) to an even greater extent by serum (Fig. 3BGo). We also assessed the effects of other activators of SRF on pFRE3-RSVluc. Perturbations of the actin cytoskeleton profoundly increase SRF activity on certain promoters through mechanisms that are not fully understood. However, these mechanisms of activation include kinase signaling cascades, interactions with coregulators like myocardin-related transcription factors, and activation of Rho family GTPases (17, 18, 19, 20, 21). Significantly, during chondrogenesis profound alterations in actin dynamics result from 1) changes in assembly and disassembly of intercellular contacts during cell condensation or 2) changes in contacts with the extracellular matrix (22, 23, 24, 25, 26, 27, 28, 29). Moreover, modulation of the actin cytoskeleton promotes chondrocyte differentiation as evidenced by commitment to the chondrocyte cell lineage after treatment of mesenchymal precursors with cytochalasin D (28, 29). Therefore, we were particularly interested in testing the effects of cytoskeletal modulators on the transcriptional activity of FRE3. Significantly, we found that changes in cytoskeleton actin dynamics caused by cytochalasin D (an inhibitor of actin polymerization) and the constitutively active small GTPase Rac2 Q61L strongly activated pFRE3-RSVluc (Fig. 3CGo). Similarly, RCJ cells stably transfected with pFRE3-RSVluc were induced (5- to 5.8-fold) by cytochalasin D (Fig. 3BGo).



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  		Fig. 2. Sequence of the FRE3 and Recognition Site for Putative Transcriptional Regulators

The underlined sequence indicates putative binding sites for the transcription factors SRE, serum response factor; E-box, basic helix loop helix transcription factor; YY1, CREB; C/EBP, CAAT/enhancer-binding protein. M1, M2, and M3 represent mutated forms of FRE3. The mutated sequence is indicated in lowercase bold letters.

 


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  		Fig. 3. Regulation of the FRE3 Sequence by Regulators of Serum Response Factor

A, Stimulation of pFRE3-RSVluc activity by treatment of transiently transfected RCJ cells with medium containing 15% fetal bovine serum. B, Clones of RCJ cells stably transfected with pFRE3-RSVluc were treated with 1µg/ml cytochalasin D (18 h treatment) or stimulated with medium containing 15% fetal bovine serum (18 h). The data of each panel (A–C) are representative of results from three independent experiments. Error bars represent ± SEM. C, Activation of pFRE3-RSVluc in RCJ cells after cotransfection with a constitutively active Rac Q61L or treatment with 1 µg/ml cytochalasin D.

 
To further investigate whether SRF contributes to the transcriptional activity of FRE3, we modified the cognate SRF binding site, serum response element (SRE). Mutations of the SRE within the FRE3, M1 and M2 (Fig. 2Go), were cloned as two copy repeats in the pRSVluc reporter. Both constructs were completely inactive when transfected into RCJ cells (Fig. 4Go, see inset for comparison to the basal promoter, pRSVluc). Furthermore, pM1-RSVluc and pM2-RSVluc constructs were not induced by serum or cytochalasin D (Fig. 4Go). Similar results were observed upon transient transfection of the mutant constructs into chondrocytic CFK2 cells (data not shown). Thus, whereas the FRE3 contains consensus motifs for many possible transcription factors (Fig. 2Go), these data show that the serum response element is paramount for the transcriptional activity of pFRE3-RSVluc.



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  		Fig. 4. Mutations of the SRE Abolish the Transcriptional Activity of FRE3

RCJ cells were transiently transfected with the indicated constructs and assayed for luciferase activity 18 h after stimulation with medium containing 1% fetal bovine serum (1%), 15% fetal bovine serum (15%) or 1 µg/ml cytochalasin D (cyto D). The inset shows the results for pRSVluc and the mutant constructs on an expanded y-axis. Luciferase activity was normalized to the ß-galactosidase transcribed from a constitutive, cotransfected plasmid. The data shown are representative of results from three independent experiments. Error bars represent ± SEM.

 
Characterization of Nuclear Factors Binding to the SRE of FRE3
We used EMSAs and antibody supershift analysis to characterize the trans-acting factors that bind to FRE3. The FRE3 oligonucleotide containing the SRE was radiolabeled and incubated with 2 µg of nuclear extract from cells treated with 1% and 15% fetal bovine serum. Fractionation on polyacrylamide gels resolved several complexes bound to the FRE3 sequence (Fig. 5AGo). Nuclear extracts prepared after treatment with 15% fetal bovine serum resulted in a quantitative increase in the same set of complexes bound to FRE3. To assess the specificity and identity of the FRE3-binding proteins, nuclear extracts were pre-incubated with unlabelled oligonucleotide competitors. The protein complexes with FRE3 were completely disrupted by competition with a 50-fold molar excess of unlabeled FRE3 (Fig. 5Go, A and B). A nonspecific oligonucleotide used at 100-fold molar excess failed to compete for any of the DNA-protein complexes observed (data not shown). Competition with a 50-fold excess of unlabeled M1 oligonucleotide disrupted the binding of all FRE-binding proteins, except for the slow migrating species; band X (Fig. 5BGo). Similarly, competition with a 50-fold molar excess of unlabelled M2 disrupted most complexes assembled on FRE3 with the exception of complex X and a second more rapidly migrating species, complex Y (Fig. 5BGo). To determine if complex X contains SRF, we included an anti-SRF polyclonal antibody in the binding reaction. Subsequent EMSA showed a specific and complete shift of band X to a slower migrating species (Fig. 5CGo). The supershift upon the addition of anti-SRF antibody (Ab) demonstrates that complex X contains SRF. Control antibodies (anti-Nur77, anti-SF1, anti-YY1) did not influence the SRF:FRE3 complex nor other protein:FRE3 complexes (data not shown).



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  		Fig. 5. EMSA Using Nuclear Protein Extract (Extract) Prepared from RCJ Cells and Radiolabeled FRE3

A, Comparison of the protein complexes assembled on the FRE3 using nuclear extracts from RCJ cells stimulated with medium containing 1% or 15% fetal bovine serum. Binding of nuclear proteins to the labeled FRE3 is completely inhibited by including a 50-fold molar excess of unlabeled FRE3 oligonucleotide. B, Competition experiment demonstrating that the assembly of complex X is not inhibited by a 50-fold molar excess of unlabeled oligonucleotide with a mutation in the SRE (M1 and M2). The assembly of the band Y complex is not inhibited by oligonucleotide M2 that contains mutations of both 5' and 3' ends of the SRE. C, Supershift EMSA demonstrating the complex of band X is shifted to a slower migrating species (*) after the addition of an anti-SRF antibody (0.5–2.0 µg) in the binding reaction. D, EMSA using in vitro transcribed and translated SRF. Transcription/translation reactions were done using pcDNA, pcDNA-CAP protein, or pcDNA-SRF as template. Including a 50-fold molar excess of unlabeled oligonucleotides FRE3 or M3 inhibited the binding of in vitro synthesized SRF to the radiolabeled FRE. SRF complexed to the FRE3 was shifted to a slower migrating species after the addition of an anti-SRF antibody (0.5–2.0 µg). A control antibody (anti-CD47) had no effect.

 
Specific Binding of in Vitro Translated SRF to FRE3
To confirm that the SRE identified in FRE3 functioned as a SRF recognition site, we determined the binding of in vitro transcribed and translated human SRF. SRF indeed bound to FRE3 forming a DNA-protein complex of similar electrophoretic mobility to that of the FRE3:SRF complex identified in supershift experiments (Fig. 5DGo). This DNA-protein complex was not observed when pcDNA 3 alone or a control cDNA (CAP) were used as template in the in vitro translation-transcription reaction (Fig. 5DGo). Furthermore, the SRF-FRE3 complex was supershifted after the addition of anti-SRF Ab. A supershift was not observed with a control Ab (anti-CD47). Together, these data clearly show that SRF binds to the SRE within FRE3.

Overexpression of a Dominant-Negative SRF Represses FRE3-Mediated Transcription
We showed that SRF binds to the FRE3 and that mutations of the SRE abrogate both the transcriptional activity of FRE3 and the binding of SRF. To determine if SRF bound to the FRE3 is required for transcriptional activity, a dominant-negative form of SRF was used. We used SRF{Delta}C (wherein the C-terminal trans-activation domain, amino acids 266–504, is deleted) as a repressor of SRF-dependent transcription. SRF{Delta}C suppresses transcription through specific binding to SREs (30). Cotransfection of SRF{Delta}C with pFRE3-RSVluc in RCJ cells caused a dose-dependent 90% decrease in transcriptional activity, indicating that SRF is essential for the transcriptional activity of FRE3 (Fig. 6Go).



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  		Fig. 6. Dominant-Negative SRF Suppresses the Transcriptional Activity of the FRE3

RCJ cells were transiently cotransfected with pFRE3-RSVluc and the indicated amount of dominant negative SRF expression plasmid (pSRF{Delta}C). Cell lysates were assayed for luciferase activity and normalized to the amount of ß-galactosidase activity. The data shown are representative of results from three independent experiments. Error bars represent ± SEM.

 
Mutation of the SRE within the FGFR 3 Gene Promoter Diminishes Transcriptional Activity
Because the serum response element is essential to the transcriptional activity of the FRE3, we hypothesized that the SRE also contributes to the activity of the -2311 to -27 FGFR3 promoter. To test this, a mutation (CCTTATTTGG to CCTTATTTat) was engineered in the SRE of the p(-2311/-27)luc promoter. Consistent with SRF contributing to the transcriptional activity of the FGFR3 promoter, mutation of the serum response element repressed the transcriptional activity by 60%, P = 0.001 (Fig. 7AGo). The magnitude of suppression caused by mutating the SRE, was equivalent to that observed when sequence between -2311 and -1537 were deleted from the FGFR3 promoter (Fig. 1AGo). This suggests that SRF is responsible for the transcriptional activity identified in this region. Also supporting the conclusion that SRF regulates the FGFR3 promoter, the steady-state ratio of FGFR3:ß-actin increased 1.5-fold after stimulation of cells with 15% fetal bovine serum (Fig. 7BGo). In addition, SRF binds to the FGFR3 gene in vivo. We immunoprecipitated SRF bound to native chromatin with an anti-SRF antibody. The DNA associated with SRF was recovered after extraction of the proteins. PCR with specific primers for the FGFR3 gene demonstrated that the portion of the FGFR3 gene containing the FRE3 was present in the SRF immunoprecipitates (Fig. 7CGo), but not in a negative control immunprecipitate. The input lane shows PCR amplification of the FGFR3 gene in the chromatin used for immunoprecipitation.



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  		Fig. 7. Regulation of the fgfr3 Proximal Promoter by the Serum Response Element and Regulation of the Endogenous FGFR3 Gene

A, Results obtained from RCJ cells transiently transfected with reporter plasmids containing the fgfr3 proximal promoter with (p(-2311/-27_M1)luc) or without (p(-2311/-27)luc) a mutation in the serum response element. Error bars represent ± SEM. The data shown are representative of results from three independent experiments. B, Ribonuclease protection assay of total RNA isolated from RCJ cells treated with medium containing 0.5% or 15% fetal bovine serum. The top panel shows the protected fragment using an FGFR3 riboprobe. The bottom panel shows the results using a ß-actin riboprobe. C, Immunoprecipitation of the FGFR3 gene with an anti-SRF antibody. Chromatin was immunoprecipitated with an anti-SRF antibody or a mock control. The immunoprecipitated DNA was then analyzed by PCR with primers that flank the FRE sequence.

 
Characterization of a Regulatory Site Neighboring the SRE of FRE3
We showed that a luciferase reporter construct containing mutations (M2) of the 5' and 3' end of the SRE is inactive. We also showed that the oligonucleotide containing the same mutations is unable to complex with SRF or bind to the protein(s) of band Y (as evidenced by the inability of unlabeled M2 to inhibit the binding of SRF and band Y to the radiolabeled FRE3, Fig. 5BGo). The protein:nucleic acid complex of band Y was not perturbed by an anti-SRF antibody, suggesting that it does not contain SRF. Because the mutations found in M2 are within the SRE, this suggests that the recognition motif for the protein(s) of band Y partially overlaps that of SRF. Inspection of the FRE3 sequence showed the presence of a putative steroid hormone nuclear receptor half site (5'-PuGGTCA-3') on the bottom strand, overlapping the SRE. To test the contribution of this consensus motif to the activity of FRE3, we mutated the sequence adjacent to, but not involving the core recognition motif of SRF (M3, Fig. 2Go). A tandem repeat dimer of M3 cloned into pRSVluc had 57% (P = 0.002) the activity of pFRE3-RSVluc (Fig. 8AGo).



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  		Fig. 8. Regulation of the Transcriptional Activity of the FRE3 by Nuclear Binding Proteins that Overlap the Serum Response Element

A, Luciferase results obtained from RCJ cells transiently transfected with the indicated plasmids. Error bars represent ± SEM. The data shown are representative of results from three independent experiments. B, EMSAs using nuclear protein extract from RCJ cells and radiolabeled FRE3. Competition experiments were performed with a 50-fold molar excess of the indicated unlabeled duplex oligonucleotides. Unlabeled M3 duplex oligonucleotide was used at 10-, 50-, and 100-fold molar excess. C, EMSAs using nuclear protein extract from RCJ cells and radiolabeled FRE3. Competition experiments were performed with a 50-fold molar excess of unlabeled FRE3 duplex oligonucleotides. Supershift was attempted with 1 or 2 µg of anti-CREB antibody.

 
To assess the effect of this mutation on protein-nucleic acid interactions, we performed EMSAs using M3 as an unlabeled competitor for the binding to FRE3. We observed that M3 is a potent antagonist of SRF binding to FRE3 (Fig. 5DGo and 8BGo), indicating that the M3 mutation does not prevent SRF binding. By contrast, unlabeled M3 does not inhibit the binding of the Y complex to FRE3 (Fig. 8BGo). This indicates that the GGTCA motif adjacent to and partially overlapping the SRE is required for interactions with the protein(s) of band Y. In addition, these data provide evidence that this complex contributes to the transcriptional activity of FRE3. A putative cAMP response element binding protein (CREB) binding motif partially overlaps both the SRE and putative nuclear receptor half site. To test whether CREB bind to FRE3 we performed EMSAs in the presence of an anti-CREB antibody (Fig. 8CGo). The formation of complex Y was not suppressed by anti-CREB, suggesting that CREB does not contribute to the transcriptional activity of FRE3. An E-box motif (CANNTG) neighbors the nuclear receptor site (Fig. 2Go). However, mutation of the 5' end of the E-box did not alter the transcriptional activity of FRE3 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We showed that the FGFR3 gene contains a cis-acting element (FRE3) that confers transcriptional activation to a heterologous minimal promoter and the homologous FGFR3 promoter. FRE3 contains a serum response element (CArG box) that is essential for transcriptional activity. Mutation of the SRE abrogates the transcriptional activity of FRE3 and diminishes the activity of the p(-2311) FGFR3 promoter. Additionally, the same mutation disrupts the binding of SRF to the FRE3 sequence. Finally, a dominant-negative SRF blocks the activity of the FRE3. In toto, these results demonstrate the essential contribution of SRF to the transcriptional activity of FRE3 and the FGFR3 proximal promoter. Our finding that serum induction can increase the steady-state level of FGFR3 mRNA in cultured cells and that SRF binds to the FGFR3 gene in vivo, provides further evidence for SRF-dependent control of FGFR3 expression.

SRF is an important regulator of early embryogenesis and cell differentiation. SRF-/- mice lack mesoderm and die due to gastrulation defects (31). Studies of cultured cells indicate that SRF regulates gene expression and differentiation in neuronal (32) as well as cardiac, skeletal, and smooth muscle cells (33). Unlike muscle cells, the contribution of SRF to chondrocyte gene expression and differentiation is uncharacterized. Therefore, these data provide the first evidence that SRF regulates chondrocyte gene expression and suggest that SRF may contribute to the regulation of chondrocyte differentiation. In support of this, we have found that dominant negative SRF inhibits the expression of the cartilage-specific proteoglycan, aggrecan (Reinhold, M. I., and M. C. Naski, unpublished data). SRF activity is regulated by dynamics of the actin cytoskeleton (19, 20, 21, 34) and similarly chondrogenesis is modulated by perturbations of the actin cytoskeleton (24, 28, 29, 35). It will be interesting to determine if cytoskeletal changes that activate chondrogenesis utilize pathways that require SRF. During early chondrogenesis, mesenchymal cells condense and form cell-to-cell contacts mediated by N-cadherin (36). Subsequently, the chondroblasts dissociate, secrete abundant extracellular matrix and form contacts with the extracellular matrix (37, 38). Each of these steps requires reorganization of the actin cytoskeleton. We hypothesize that SRF is activated during these processes and subsequently contributes to chondrocyte differentiation.

EMSAs show that several proteins bind to the FRE3. Interestingly, though the SRF:FRE3 complex is quantitatively less abundant than other protein:nucleic acid complexes observed in the in vitro binding reaction, SRF is essential for the in vivo transcriptional activity. This implies that the in vitro binding reaction does not quantitatively correlate with in vivo activity. For example, EMSAs demonstrated that one of the complexes results from an interaction with the E-box of the FRE3 (data not shown). However, mutation of the E-box did not alter the transcriptional activity of the FRE3. Thus, whereas the FRE3:SRF complex is quantitatively less abundant than other complexes observed in EMSAs, it is essential for the transcriptional activity of the FRE3. There are, however, other transcriptional regulators that contribute to the activity of FRE3. Mutation of the sequence (M3) that neighbors the SRE or mutation of the 5'-end of the SRE (M2) prevents interactions with the protein(s) that comprise band Y. In addition, the M3 mutation diminished the transcriptional activity of FRE3. This implies that a positive acting transcriptional regulator may act in concert with SRF to regulate the FRE3. This is consonant with 1) the frequent observation that SRF controls gene expression in combination with other transcriptional regulators (39, 40, 41, 42, 43) and 2) our finding that the activity of the FRE3 is greater in chondrocytic cells (relative to nonchondrocytic cells) even though SRF is a ubiquitous transcription factor (Fig. 1CGo). Therefore it is particularly interesting that protein(s) associate with the FRE3 adjacent to the serum response element. The factor(s) of complex Y appear to interact with a site adjacent to and partially overlapping the SRE. Serum response factor binds to the SRE through interactions in the major groove (44), thus leaving the minor groove as a potential binding site for coregulators that bind to an overlapping sequence motif. Intriguingly, the transcriptional regulator YY1 has been shown to bind in certain circumstances to sequence motifs that overlap the consensus site for serum response factor. Skeletal and cardiac {alpha}-actin genes contain repeated serum response elements that are required for the expression and tissue specificity of these genes (45, 46). In these studies YY1 acts as a transcriptional repressor. However, YY1 also acts as a transcriptional enhancer. Indeed, YY1 can facilitate the association of SRF with the c-fos SRE and enhance the transcriptional activity of an SRF-VP16 fusion protein (47). Based on these data we hypothesized that YY1 may be a component of the band Y complex that assembles on the FRE3. However, we did not observe binding of YY1 to the FRE3 by supershift assays using antisera to YY1 or EMSAs using in vitro transcribed and translated YY1 (data not shown).

The identity of protein(s) in the complex that forms band Y has remained elusive. The sequence recognized by the nuclear protein(s) of band Y includes a putative nuclear receptor site. In addition, we have shown that oligonucleotides containing either a thyroid hormone response element or a retinoic acid receptor {alpha}1 consensus site inhibit the assembly of the band Y complex (16). However, neither thyroid hormone nor retinoic acid alters the transcriptional activity of FRE3 and dominant-negative forms of the retinoic acid receptor do not alter the transcriptional activity of the FRE3 (data not shown). Examination of the sequence about the putative nuclear receptor site reveals an A/T rich stretch preceding the nuclear receptor site. Similar motifs have been observed in the binding sites for the nuclear receptors Nur77 (48) and SF1 (48, 49). We did not, however, demonstrate the presence of Nur 77 or SF1 complexed with the FRE3 in EMSA experiments (data not shown). Additional experiments will be required to identify the positive transcriptional regulator(s) found in band Y that bind adjacent to and partially overlapping the serum response element. One or more of these factors may work in concert with SRF to regulate FGFR3 gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and Reagents
The plasmid containing activated Rac2 (Rac2V12) was generously supplied by G. Bokoch (La Jolla, CA). Cytochalasin D was purchased from Sigma.

Plasmid Construction
The generation of p(-2311/-27)FR3-luc, p(-1537/-27) FR3-luc and pFRE3-RSVluc (originally named CSRh) has been previously reported (16). p(-2311_M1)luc was assembled by PCR using p(-2311/-27)FR3luc as template and the oligonucleotides 5'-GGATCCACAAATGTGACCTTATTTATAAAAAGAGT-3' and 5'-CTTTATGATGTTTTTGGCGTCTTCCA-3'. The amplified product was digested with BamHI and BglII and cloned into pGL2-basic digested with BglII. All constructs generated by PCR were verified by sequencing. FRE3 mutant constructs pM1RSVluc, pM2RSVluc and pM3RSVluc were generated using the same protocol as described previously (16) using the oligonucleotides of Fig. 2Go as well as the reverse complement. Briefly, the oligonucleotides were gel-purified and subsequently annealed. Duplex oligonucleotides were 5'-phosphorylated with T4 kinase, concatamerized through self-ligation with T4 DNA ligase, and digested with SalI and XhoI. A two-copy, head-to-tail array was subcloned into pRSVluc (50) digested with XhoI. Orientation and sequence was confirmed by sequencing analysis. The plasmid for stable transfection was constructed by blunt-end cloning the neo-resistance gene (excised with AflIII and XmnI) of pcDNA 3 into the blunted SalI site of pFRE3-RSVluc.

The 3' region of SRF was PCR amplified from ADH-SRF (kindly provided by E. Olson, University of Texas, Dallas, TX), cut with StuI and EcoRI and subcloned into pBSKII (referred to as 3'SRF). The 5' region of SRF was obtained from I.M.A.G.E. clone 2308255 (American Type Culture Collection, Manassas, VA), as a NotI and StuI fragment. A KpnI linker was added to the NotI site. These KpnI and StuI fragments were subcloned into 3'SRF cut with KpnI and StuI (referred to as pBS-SRF). The KpnI and EcoRI fragment, comprising full-length SRF was then subcloned into pcDNA3.

The dominant-negative form of SRF (SRF {Delta}C) in which the C-terminal transcription activation domain (amino acid 266–504) was deleted, was generated using primers 5'-AAGCTTGCGCCATGTTACCGACCCAAGCTGGG-3' and 5'-CGGAATTCTTATCACGCCGGCTTCAGTGTGTCCTTGGT, the later containing an EcoRI site. The amplified fragment, after sequence verification, was cut with StuI and EcoRI and subcloned into pBS-SRF, cut with StuI and EcoRI. The SRF{Delta}C construct was then subcloned into pcDNA3 as described above. All construct were verified by DNA sequencing.

Cell Culture and Transfection
CFK2 (51), RCJ (52) (clone3.1C5.18) NIH3T3 (American Type Culture Collection), HeLa (American Type Culture Collection), MDA231 and PC3 cell lines were maintained subconfluent in DMEM (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine, penicillin G (100 U/ml), and streptomycin (100 µg/ml). All cells were transfected in triplicate using a modified calcium phosphate precipitate. Briefly, cells were plated 24 h before transfection at 8 x 104 cells/ml in a 12-well plate (800 µl/well). The media were replaced 4 h before transfection. A total of 7.5 µg of each DNA construct and 0.7 µg of pCScytoß-gal (53) were diluted to a final volume of 20 µl with water. To this was added 355 µl of 0.26 M CaCl2 and 375 µl of 2x BBS (50 mM N,N-bis[2-hydroxylethyl]-2-aminoethanesulfonic acid, 280 mM NaCl, and 1.5 mM Na2HPO4). After precipitation at room temperature for 10 min, 80 µl of the precipitate was added directly to the wells. Cells were incubated at 37 C for 20 h, at which point the precipitate was washed off and replaced with complete medium for 24 h. When response to fetal bovine serum was studied, cells were serum-deprived for 12 h in DMEM supplemented as above except containing 1% fetal bovine serum. Sixty hours after transfection, cell extracts were prepared and luciferase reporter expression and ß-galactosidase activity was analyzed as described previously (16).

Nuclear Extracts and EMSAs
Nuclear extracts were prepared as previously described (16, 54). The protein concentration of extracts was estimated by the Bio-Rad (Hercules, CA) protein assay reagent. All oligonucleotides encompassing the desired binding site sequences were gel purified and subsequently annealed. Binding reactions were performed at room temperature for 20 min in a 20 µl total volume, which consisted of 0.1 pmol of probe (25,000 cpm), 3 µg BSA (Promega, Madison, WI), 1.25 µg poly(deoxyinosine-deoxycytidine) (Amersham Pharmacia Biotech, Piscataway, NJ), 2–4 µg nuclear protein extract, and buffer D [20 mM HEPES (pH 7.9 at 4 C) 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, 0.5 mM dithiothreitol]. Duplex oligonucleotide competitors (specific and nonspecific) were preincubated with the nuclear extract for 5–10 min at room temperature for competition assays. For supershift assays, antibodies were added to nuclear extract and incubated on ice for 20 min, followed by incubation at room temperature for 20 min. The resulting complexes were resolved as described (16). The sense strand of each oligonucleotide used for EMSAs are shown in Fig. 2Go. The {alpha}SRF and {alpha}CREB antibodies used for EMSA supershift experiments was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

In Vitro Transcription-Translation of SRF
In vitro transcription-translation was carried out from the T7 promoter with XbaI-linearized SRF-pcDNA3 expression plasmid using the Promega TnT Quick Coupled Reticulocyte system, after the manufacturer’s protocol. For gel shift analysis, samples were diluted to a total volume of 100 µl with 50 µl gel shift buffer D (16, 54). For gel shift and supershift analysis 5 µl of diluted SRF transcription-translation reaction were hybridized to labeled probe as described above.

Ribonuclease Protections Assay
Total cellular RNA was isolated from RCJ cells using the Rneasy kit (QIAGEN, Inc., Valencia, CA). Twenty micrograms of RNA were vacuum dried, then resuspended in 40 mM piperazine-N,N'-bis[2-ethane sulfonic acid] (pH 6.4), 0.4 M NaCl, 1 mM EDTA, 80% formamide containing 1 x 105 cpm of 32P-uridine triphosphate-labeled riboprobes. The samples were denatured then hybridized at 56 C for 8 h. The sample was digested with 14 mg ribonuclease A and 248 U of ribonuclease T1 for 30 min at 30 C. The sample was extracted with Trizol Reagent (Invitrogen Life Technologies), vacuum dried and resuspended in 80% formamide, 1 mM EDTA, 0.1% bromphenol blue. Denatured samples were resolved on a 5% denaturing polyacrylamide gel. The intensity of the signals was quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software. The sizes of the protected fragments were 291 nucleotides (rat FGFR3) and 126 nucleotides (rat ß-actin).

Chromatin Immnoprecipitation
Cells (106 RCJ) were stimulated for 12 h with 15% fetal bovine serum. Thereafter, protein DNA complexes were cross-linked with 1% formaldehyde for 10 min at 37 C. Cells were washed with PBS containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin and 1 µg/ ml pepstatin A, pelleted at 1000 x g and solubilized in 1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 50 mM Tris (pH 8.1). The lysates were sonicated using a 2-mm tip pulsed three times for 10 sec. The sonicated cell supernatant was diluted 10-fold with chromatin immunoprecipitation dilution buffer [1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris (pH 8.1), and protease inhibitors] and precleared for 2 h at 4 C 60 µl of salmon sperm DNA/protein A slurry (Pierce, Rockford, IL). Chromatin:antibody complexes were formed using 1 µg anti-SRF antibody (Santa Cruz Biotechnology, Inc.) overnight at 4 C and recovered using 60 µl of salmon sperm DNA/protein A sepharose (Pierce) slurry. The bound complexes were washed with 1) 0.1% SDS, 1.0% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris (pH 8.1); 2) 0.1% SDS, 1.0% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris (pH 8.1); and 3) 0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris (pH 8.1). The samples were then eluted with 1% SDS, 0.1 M NaHCO3; and cross-links were reversed at 65 C for 4 h and DNA was recovered by phenol/chloroform extraction and ethanol precipitation. PCR was performed using FGFR3 gene primers (5'-ACAGTGGCAGCCATGTTGGA-3' and 5'-GGACACTTGTGGTTGGATTTAGAG-3') for 35 cycles (94 C x 45 sec; 64 C x 1 min; 72 C x 45 sec).


   ACKNOWLEDGMENTS
 
We thank David Ornitz for insightful review of the manuscript.


   FOOTNOTES
 
This work was supported by NIH Grant AR47070 and the Paul Beeson Physician Faculty Scholars in Aging Research Program.

Abbreviations: CAP, Control cDNA; CREB, cAMP- response element binding protein; FGFR1–4, fibroblast growth factor receptors 1–4; FRE3, fibroblast growth factor receptor 3 response element; RSV, Rous sarcoma virus; SDS, sodium dodecyl sulfate; SRE, serum response element; SRF, serum response factor.

Received for publication August 18, 2003. Accepted for publication October 9, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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