Regulation of the Mouse Cartilage-derived Retinoic Acid-sensitive Protein Gene by the Transcription Factor AP-2*

Wei-Fen XieDagger , Seiji KondoDagger , and Linda J. SandellDagger §

From the Departments of Orthopaedics and Biochemistry, University of Washington and Veterans Administration Puget Sound Health Care Systems, Seattle, Washington 98108

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The expression of cartilage-derived retinoic acid-sensitive protein (CD-RAP) is initiated at the beginning of chondrogenesis and continues throughout the cartilage development. In chondrocytes, CD-RAP is down-regulated by retinoic acid. To understand the molecular mechanism underlying this regulation and the cell-specific expression, the deletion constructs of the mouse CD-RAP promoter were transfected into chondrocytes and a melanoma cell line. The results revealed a domain that demonstrated high levels of expression specifically in chondrocytes. In this functional domain, we show that a cis-acting element, 5'-GCCTGAGGC-3', binds to the trans-acting factor protein AP-2. Mutation of the AP-2 site on the CD-RAP promoter led to decreased transcription in C5.18 chondrocytes, indicating that this site may act as an activator of transcription. In contrast, increased concentration of AP-2, stimulated by retinoic acid, led to decreased transcription of the CD-RAP promoter, an effect that was abolished by mutation of the AP-2 binding site. The effect of AP-2 was further examined by co-transfection of C5.18 and HepG2 cells with the CD-RAP promoter constructs and an AP-2 expression plasmid. In a dose-dependent manner, cotransfection with AP-2 elevated and then decreased CD-RAP promoter activity. Taken together, these results suggest that AP-2 is involved in the biphasic regulation of CD-RAP transcription.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Cartilage-derived retinoic acid-sensitive protein (CD-RAP)1 is a newly discovered secretory protein cloned from chondrocytes (1). Its human homologue, melanoma inhibitory activity (MIA), was originally isolated from the media of a highly metastatic melanoma and exerts autologous growth-inhibitory effects on melanoma cells in vitro (2, 3). CD-RAP is co-regulated with type II collagen by retinoic acid (RA) and is expressed exclusively by chondrocytes in adult rat and fetal bovine tissues (1). CD-RAP protein inhibits DNA synthesis in primary mature chondrocytes (4), but its normal function during chondrogenesis remains unknown.

CD-RAP was originally isolated as an mRNA down-regulated by RA in primary bovine articular chondrocyte cultures (1). Retinoids affect target genes by interacting with two families of ligand-dependent nuclear receptors, the RA receptors (RARalpha , -beta , and -gamma ) and retinoid X receptors (RXRalpha , -beta , and -gamma ) (5). Among the target genes identified for RA signaling are RARalpha 3, RARbeta isoforms, RARalpha 2, and cellular retinoic acid-binding protein I and II genes. Synthesis of the transcription factor AP-2 is increased in response to RA with a lag period of 24-48 h in NT2 cells (6).

Eukaryotic gene expression is subject to the combined action of multiple DNA-binding proteins interacting with specific DNA motifs present in the promoters and enhancers. The transcription factor AP-2 recognizes the palindromic sequence 5'-GCCNNNGGC-3' (7). AP-2 has been shown to play a crucial role in the control of gene expression in response to cell differentiation signals within neural crest and epidermal cell lineages (8). So far, three isoforms of the AP-2 gene have been identified from humans and mice that recognize the same binding motif (9, 10). Transcriptional regulation by AP-2 may involve both positive and negative regulatory effects on gene expression (11-13). Two independent groups have recently generated the mouse lines where the AP-2 gene has been disrupted by homologous recombination (14, 15). The AP-2 null mice died perinatally with cranio-abdominoschisis and severe dismorphogenesis of the face and skull, suggesting an AP-2 effect on the skeletal development.

Recent studies have shown that the human MIA/CD-RAP promoter directs a high level of gene expression specifically in human and murine melanoma cells (16). We have recently cloned and analyzed the mouse CD-RAP gene (17). In the present study, we evaluated the functional regulatory domains in the mouse CD-RAP promoter by transient transfection into chondrocyte and melanoma cell lines and demonstrated that an AP-2 binding motif may be a positive regulatory domain for chondrocyte-specific promoter activity. In contrast, the increase in AP-2 plays an important role in transcriptional reduction of CD-RAP in response to RA in chondrocytes. CD-RAP is the first gene specifically expressed in the skeletal development found to be regulated by AP-2. As CD-RAP is expressed during the chondrogenesis phase of endochondral bone formation, these findings may provide insight into the severe skeletal deformation observed in the AP-2-deficient animals.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The materials used in this work were purchased as follows: Dulbecco's modified Eagle's medium and Eagle's minimum essential medium from Bio-Whittaker; alpha -modified Eagle's minimum essential medium and restriction enzymes from Life Technologies, Inc.; fetal calf serum (FCS) from HyClone Laboratories, Inc.; pCMV-beta -Gal from CLONTECH Laboratories, Inc.; luciferase expression vectors pGL3-Basic and pGL3-Control, beta -galactosidase enzyme assay system, luciferase assay reagent, gel shift assay systems, and the reverse transcription system from Promega; [32P]dCTP and [gamma -32P]ATP (3000 Ci/mmol) from NEN Life Science Products; Hybond-N membrane from Amersham Corp.; anti-AP-2 and anti-Sp1 antibodies from Santa Cruz Biotechnology; all-trans-RA from Sigma; poly(dI-dC)·(dI-dC) double-stranded DNA from Pharmacia Biotech Inc.; DOTAP liposomal transfection reagent from Boehringer Mannheim; and B16 (ATCC CRL 6322), HepG2 (ATCC HB-8065), and BALB/3T3 (ATCC CCL163) from ATCC. The rat calvaria chondrogenic cell line RCJ 3.1 C5.18 was kindly provided by Drs. Jane Aubin and Johan Heerche. The rat chondrosarcoma (RCS) cell line was provided by Dr. James H. Kimura. Human AP-2alpha A expression plasmid in pCMX-PL1 vector was provided by Dr. Reinhard Buettner. Human AP-2 expression plasmid SP-AP-2 and the control vector SP-NN were from Dr. Trevor Williams (7).

Cell Culture-- RCJ 3.1 C5.18 cells were maintained in alpha -modified Eagle's minimum essential medium supplemented with 10% heat-inactivated FCS and 10 nM dexamethasone to preserve their cartilage phenotype (18). Primary chondrocytes were prepared from bovine articular cartilage as described by Kuettner et al. (19) and cultured in Dulbecco's modified Eagle's medium with 10% FCS as were B16 (mouse melanoma cell line), BALB/3T3 (mouse fibroblast), and RCS cells. HepG2 (human liver hepatoblastoma) cells were cultured in Eagle's minimum essential medium with 2.0 mM L-glutamine, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, Earle's buffered salt solution, and 10% FCS.

To investigate the RA effect on CD-RAP expression and protein binding, all-trans-RA was dissolved in absolute ethanol to obtain the 1000 × stock solution at various concentrations. An aliquot of the stock solution was added to the prewarmed medium, which was then added to the culture. The ethanol vehicle served as a control and was added to the medium to a final concentration of 0.1%.

Plasmid Constructs-- To construct the CD-RAP promoter 5'-deletion constructs, the mouse CD-RAP template was amplified by polymerase chain reaction (PCR) with a 3'-antisense primer that bound at -3 relative to the CD-RAP translational start site in conjunction with different 5'-sense primers that bound at varying distances within the CD-RAP upstream flanking sequence. To facilitate subcloning of the amplified fragments, the antisense primer contained a HindIII restriction site adaptor, and the sense primer contained a SmaI site. The PCR fragments and the luciferase expression vector pGL3-Basic were digested separately with SmaI and HindIII before ligation. The nomenclature used for each deletion construct (shown in Fig. 1) indicates the number of base pairs of the upstream 5'-flanking sequence with respect to the ATG translation start codon. Site-directed mutagenesis within the CD-RAP promoter was performed by PCR (20). The wild type and the mutated DNA constructs were verified by DNA sequencing.

Transient Transfection and Luciferase Assay-- DNA transfection of RCS cells was performed by electroporation as described by Mukhopadhyay et al. (21). C5.18, B16, HepG2, and 3T3 were transfected by the lipofection method. Briefly, the cells were cultured in the 35-mm dishes. Each cationic lipid/plasmid DNA suspension was prepared by mixing 2 µg of the luciferase reporter plasmid and 0.5 µg of the internal control plasmid pCMV-beta -Gal with a solution of DOTAP in Hepes (20 mM, pH 7.4) according to the manufacturer's instructions. The cells were harvested 48 h later, and the lysate was analyzed for luciferase activity with a Turner TD 20e luminometer using Promega luciferase assay reagent. The galactosidase activity was measured with 50 µl of the lysate using the colormetric assay as described by the manufacturer. The luciferase activities were normalized to the beta -galactosidase value. At least three independent transfection experiments were carried out for each construct. Data are presented as the mean ± S.E.

Preparation of Nuclear Extracts-- Nuclear extracts were prepared from the cultured cells by the method of Dignam et al. (22), except that all of the buffers were supplemented with protease inhibitors (1 µM phenylmethylsulfonyl fluoride, 1 mM each of leupeptin and pepstatin). For RA stimulation, the C5.18 cells were harvested after treatment with 1 µM of RA for 2 days.

Electrophoretic Mobility Shift Assay-- Fragment A (between -401 and -548 relative to the mouse CD-RAP translational start site) was amplified by PCR. All oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer. The fragment A was end-labeled with T4 polynucleotide kinase and [gamma -32P]ATP. Band shifts were performed by incubating 4 µg of the extract in the mobility shift buffer (2.5 µg of poly(dI-dC)·(dI-dC), 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 50 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5) with the DNA probe. For the competition studies, the cold oligonucleotides were added at a 100-fold molar excess and incubated for 10 min at room temperature before adding the DNA probe. DNA protein complexes were resolved on a 6% nondenaturing polyacrylamide gel at 120 V for 2-3 h. For the antibody interference experiments, the antibody or preimmune serum was added to the reaction mixture and incubated for 30 min at room temperature before being analyzed on a polyacrylamide gel. The AP-2 antibody from Santa Cruz Biotechnology reacts with AP-2alpha but not with AP-2beta or AP-2gamma .

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Northern Blot Analysis-- Total RNA was isolated from C5.18 and B16 cells using Qiagen RNeasy Midi Kits. For the study of the RA effect on AP-2 expression, the C5.18 cells were treated with 1 µM of RA for 2 days before they were harvested. RT-PCR was performed as described previously (10). 2 µg of total RNA was used to synthesize the first strand. Primers 5'-CTGTTCAGTTCCGGGTCGCC-3' and 5'-CGGTCCTGAGCCAGCAGG-3' were added to amplify the AP-2alpha A and expected to yield a 426-base pair product. 10 µl of PCR products was electrophoresed on a 1.5% agarose gel. To facilitate quantification, DNA was transferred to Hybond-N membrane and hybridized with the 32P-labeled specific human AP-2alpha A cDNA. Amplification of the beta -actin mRNA served as a control as described by Covert and Splitter (23).

Northern blot was carried out as described previously (10). 8 µg of total RNA was loaded on 1.0% agarose formaldehyde gels and blotted onto nylon membranes. Hybridization was performed using radiolabled specific AP-2alpha A cDNA probe. The bands of the autoradiograph were quantitated by densitometry using the ISS SepraScan 2001TM 1-D system (ISS-Enprotech).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Mouse CD-RAP Is Expressed in Chondrocytes and Melanoma Cells-- The 5'-flanking sequence of the mouse CD-RAP gene was fused to a promoterless luciferase reporter plasmid pGL3-Basic and tested for its ability to generate luciferase activity in the transiently transfected cells (Table I). As expected, the construct m2251 generated expression in melanoma cell line B16 and no expression in BALB/3T3. In addition, m2251 also demonstrated relatively high luciferase activity in chondrocytes and chondrosarcoma cells, suggesting that this CD-RAP upstream sequence contains the elements necessary for chondrocyte activity. Subsequent experiments were performed using RCJ 3.1C5.18 cells as the representative chondrocytes.

                              
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Table I
Comparison of the mouse CD-RAP promoter activity in different cell types
The 2251-base pair mouse CD-RAP upstream flanking sequence was cloned into pGL3-B and transiently transfected into various cultured cells. The pGL3-Basic vector lacks eukaryotic promoter and enhancer sequences. The construct pGL3-C contains the SV40 promoter and enhancer sequences. The luciferase values are presented as mean ± S.E. C5.18, RCJ 3.1 C5.18 chondrogenic cell line; B16, melanoma cell line; 3T3, Balb/3T3 mouse fibroblasts.

Deletion Analysis of the Mouse CD-RAP Promoter-- The rat calvaria chondrogenic cell line RCJ 3.1 C5.18 cells maintain their cartilage phenotype under the appropriate conditions (18). The synthesis of type II collagen, aggrecan, and CD-RAP mRNA by C5.18 cells was confirmed by Northern blot analysis (data not shown). To identify the cis-acting elements mediating the mouse CD-RAP expression in chondrocytes, we transfected a series of the nested deletion CD-RAP promoter-luciferase expression plasmids into C5.18 and B16 cells and measured the luciferase activities generated in these cells. In B16 melanoma cells, the construct m277 generated the highest activity, while the activities of longer promoter constructs were consistently lower (Fig. 1A). In C5.18 cells, although the construct m277 could confer high activity, the longer constructs were also active, for example the construct m475 generated a 75-fold higher activity compared with the promoterless luciferase vector (Fig. 1B).


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Fig. 1.   Nested deletion analysis of the mouse CD-RAP promoter. The mouse CD-RAP promoter-luciferase reporter plasmids were cotransfected with pCMV-beta -Gal control vector into B16 (A) and C5.18 cells (B). The luciferase activities were assayed 48 h later and normalized to the beta -galactosidase value. The basal activity of the pGL3-Basic is set at 1.

The luciferase activity was altered when upstream sequences of the CD-RAP promoter were deleted and transfected into the C5.18 cells, suggesting the presence of several positive and negative regulatory elements (Fig. 1B). Deletion of the promoter sequence from -1051 to -898 resulted in increased expression approximately 2-fold, identifying one or more silencer(s). The comparison of promoter activities generated by the constructs m657 and m548 revealed at least one negative regulatory element in this region. Deletion of the promoter sequence from -401 to -277 suggested another negative element. In contrast, truncation of the promoter sequences from -898 to -657, from -475 to -401, and from -277 to -216 showed a reduction of the promoter activity, suggesting the presence of at least three positive cis-acting sequences among these sites.

Identification of AP-2 Protein Binding Sites on CD-RAP Promoter-- According to the transient transfection analysis, the addition of the sequences from -401 to -475 relative to the ATG protein start codon showed a remarkable increase in the promoter activities in C5.18 cells, implying that this region contained the positive regulatory elements for chondrocyte expression. In contrast, the luciferase activity of the constructs m401 and m475 varied slightly in the melanoma cells, and both of them expressed lower activities in B16 compared with the construct m277. We amplified a fragment (fragment A, -401 to -548) by PCR and performed gel mobility shift analyses to test whether this fragment binds to any protein factor that might regulate the CD-RAP promoter activity in chondrocytes. One strong DNA protein complex was observed with the B16 cell lysate (Fig. 2B, lane 2). An identical but fainter band was observed in the nuclear extracts of bovine articular cartilage and C5.18 cells (data not shown). To more precisely localize the DNA binding site, we carried out mobility shift assays using a series of synthetic oligonucleotides to compete against the labeled fragment A for protein binding. Fig. 2A shows the relative position of the five 30- or 20-mer oligonucleotide competitors. The competition was performed using the nuclear extract prepared from B16 cells. As shown in Fig. 2B, a 30-base pair competitor designated oligo 3 effectively inhibited the binding of the probe. The oligo 3 contains an AP-2 cis-acting motif as shown in Fig. 2A. The AP-2 site is located between -456 and -463 relative to the ATG protein start codon. To determine whether the protein binding could be competed by the known cis-acting sequences, we carried out gel shift analysis with the consensus oligonucleotides. The bandshift was inhibited by the consensus AP-2 oligonucleotide but not by the oligonucleotides containing AP1, Sp1, and OCT1 consensus sequences (data not shown), suggesting that AP-2 or a related protein bound to the CD-RAP promoter.


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Fig. 2.   Competition experiments to identify the binding site of fragment A. A, the relative location of various competitive oligonucleotides. The AP-2 motif in oligo 3 is underlined. B, competition was performed with different synthetic oligonucleotides using lysate from B16.

To determine which nucleotides of CD-RAP were important for the protein binding, mutations were introduced into the oligonucleotide competitors. Fig. 3A shows the sequences of oligo 3 with the wild type and the mutated AP-2 motifs. In the competition study, the mutations of GCC residues in the AP-2 core sequence (MuA and MuB) partially abolished the binding properties of the oligonucleotides, and they were not able to act as the effective competitors (Fig. 3B). In contrast, the oligo 3 still competed for the protein binding when mutated at T and A residues (MuC and MuD). This result is consistent with the AP-2 core recognition element for the AP-2 binding site, 5'-GCCNNNGGC-3', where the internal 3 nucleotides are not critical for the binding (7). To confirm the binding of AP-2, a supershifted DNA-protein complex was obtained by incubating the labeled fragment A with the B16 extract and the antibody directed against AP-2. The preimmune serum did not alter the electrophoretic mobility pattern of the complex (Fig. 3C).


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Fig. 3.   Identification of the AP-2 binding on CD-RAP promoter. A, the sequences of the wild type and mutant oligo 3. The wild type (WT) oligo 3 contains the AP-2 binding motif (in italics). The mutations of AP-2 site are underlined. B, mutation analysis of the AP-2 binding site. Competitive electrophoretic mobility shift analysis of B16 extract was performed using fragment A as a labeled probe. The wild type (lane 3) and the mutant oligo 3 (lanes 4-7) were added to interfere with the protein binding. Lane 1, no nuclear extract; lane 2, no competitor. C, supershift assay with AP-2 antibodies. The preimmune serum (lane 3) or the antibodies against AP-2 were incubated together with B16 lysate and the labeled fragment A. Antibodies were used at two different dilutions: 1:10 (lane 4) and 1:50 (lane 5). Lane 1 (no nuclear extract) and lane 2 (containing nuclear extract) were incubated without serum. The complex was supershifted as indicated by the arrow.

Functional Activity of AP-2 Sites-- To investigate the function of the cis-acting AP-2 motif in the CD-RAP promoter, we introduced the same mutations that abolished the binding activities in mobility shift experiments (MuA and MuB) by site-directed mutagenesis within the context of the strongly expressing reporter plasmid m475 and the longest promoter construct m2251. As shown in Fig. 4, the activities generated by the AP-2 mutant plasmid m2251-MuA decreased approximately 2-fold in comparison with its wild type promoter construct in C5.18 cells. In addition, the same mutation A within the construct m475 (m475-MuA) resulted in reduction of the promoter activity almost 3-fold. The construct m475-MuB also decreased the promoter activity. These results suggest that the AP-2 motif may act as a positive regulatory element necessary for the full expression of CD-RAP in chondrocytes under normal culture conditions. The remaining levels of CD-RAP promoter activity suggest that additional elements also contribute to its constitutive expression in chondrocytes.


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Fig. 4.   Mutation analysis of the AP-2 binding motif. A, schematic diagram of the wild type and the mutated DNA constructs. The AP-2 binding site is indicated as an open circle. The mutated AP-2 motif is shown as a cross (mutation A) or solid square (mutation B), respectively. B, the luciferase activities conferred by the mutated plasmids in transiently transfected 3T3 (open bar) and C5.18 cells (hatched bar) were compared with the wild type constructs. The activity of promoterless plasmid pGL3-B was set at 1.

Induction of AP-2 Expression by RA in C5.18-- Since the previous experiment suggested that inhibition of the binding of AP-2 by mutagenesis of the AP-2 site reduced the CD-RAP transcription, and the binding of AP-2 in chondrocytes was faint in the gel shift assay, we expected that the constitutive concentration of AP-2 would be very low in C5.18. AP-2 gene expression is up-regulated in many cell types by the developmental morphogen, RA (6). We therefore speculated that RA might stimulate the AP-2 expression in chondrocytes, resulting in more AP-2 binding to the CD-RAP promoter. Semiquantitative RT-PCR was performed to investigate the effect of RA on AP-2 gene expression. Equal amounts of total RNA harvested from the C5.18 cells treated with ethanol control or RA were used. As shown in Fig. 5A, AP-2alpha A transcript levels were increased after 48 h of exposure to RA. Northern blot confirmed the stimulation of AP-2 expression in C5.18 cells by RA (Fig. 5B). Densitometric analysis revealed low levels of AP-2 RNA in the untreated C5.18 cells that increased 6-fold at 48 h of RA treatment. The AP-2 level in B16 cells was about 10 times higher than that in the untreated C5.18 cells.


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Fig. 5.   The RA effect on AP-2 in C5.18 cells. A, total RNA was isolated from C5.18 cells incubated for 48 h with ethanol (-) or with 1 µM RA (+). RT-PCR was conducted using specific primers for AP-2alpha A. The PCR cycle is 28. The PCR products were separated on an agarose gel and transferred to nylon membranes. Hybridization was performed with AP-2alpha A cDNA probe. Amplification of beta -actin served as a control for mRNA concentration (shown stained with ethidium bromide). B, total RNA isolated from C5.18 cells after 48 h of exposure to ethanol (lane 1) or 1 µM of RA (lane 2) and from B16 cells (lane 3) was analyzed by Northern blot hybridization with a radiolabled specific AP-2alpha A cDNA probe. As a control for RNA loading, hybridization was performed with a cDNA probe for ribosomal elongation factor 1 (ELF1).

We then conducted a gel shift assay using the C5.18 lysates derived from the cells treated with RA for 2 days. A DNA-protein complex appeared at the AP-2 corresponding position (Fig. 6). This complex was inhibited by the oligo 3, which contains the AP-2 binding site and a consensus AP-2 oligo, but not by the mutant A of the oligo 3 (CC right-arrow AA). A supershift complex was specifically induced by the antibody against AP-2. As a control, the Sp1 antibody was unable to shift the DNA-protein complex. These experiments confirmed that RA increased the AP-2 expression in C5.18 cells, leading to more binding to the CD-RAP promoter in the gel shift assay.


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Fig. 6.   Induction of AP-2 binding by RA in C5.18 cells. Mobility shift analysis was carried out with the extract from C5.18 treated with RA for 2 days. The specificity of AP-2 was confirmed by competition with oligo 3 spanning the AP-2 site on CD-RAP (lane 3), consensus oligo AP-2 (lane 4), and mutant A of oligo 3 (CC right-arrow AA, lane 5). Sp1 (lane 6) and AP-2 (lane 7) antibodies were added to supershift to the complex.

RA Reduces AP-2-dependent CD-RAP Gene Transcription-- We have previously shown that RA down-regulates the CD-RAP expression (1). Since we now have shown that RA increases the AP-2 expression in C5.18 cells, we sought to determine whether AP-2 plays a role in the regulation of CD-RAP expression by RA. The wild type construct m2251, the mutated construct m2251-MuA, and the AP-2 deletion construct m455 were employed. RA or ethanol was added just prior to the transfection. RA caused a reduction of promoter activity in the construct m2251 in a concentration ranging from 10 nM to 1 µM (Fig. 7). Mutation of the AP-2 site (construct m2251-MuA) and deletion of the AP-2 site (construct m455) abolished the ability of RA to inhibit CD-RAP gene expression. In addition, RA also inhibited the promoter activity of the construct m475 containing the AP-2 site but had no effect on the construct m415 lacking the AP-2 site (data not shown). This experiment strongly suggests that the AP-2 binding site is involved in the RA-induced reduction of the CD-RAP transcription.


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Fig. 7.   Inhibition of the CD-RAP expression by RA. The CD-RAP promoter constructs were transfected into C5.18 cells. The constructs were wild type (m2251), mutated at AP-2 site (m2251-MuA), and deletion of AP-2 (m455). Different concentrations of RA or ethanol were added at the beginning of transfection. The luciferase activity was assayed 48 h later. The luciferase value without RA was taken as 100%.

Functional Effect of AP-2 on CD-RAP Promoter-- We have shown that AP-2 may stimulate CD-RAP transcription, since mutation of the AP-2 motif reduces its promoter activity in C5.18 cells. On the other hand, high levels of AP-2 expression apparently inhibit the transcriptional activation of CD-RAP. The RT-PCR result revealed that the AP-2 transcript levels were significantly elevated upon RA treatment, and the increase in AP-2 with RA treatment is correlated with a decrease in CD-RAP promoter activity. These results suggest that AP-2 may have a biphasic effect on CD-RAP transcription dependent on the AP-2 concentration. To test this hypothesis, HepG2 cells that are considered to be AP-2-deficient (24) were transfected with the construct m2251 and a human AP-2 expression plasmid AP-2alpha A in pCMX-PL1 vector. In the absence of the AP-2 expression vector, the activity of m2251 was low in HepG2 cells. In contrast, CD-RAP activity was induced by AP-2 in a dose-dependent fashion (Fig. 8A). As control, AP-2 had no effect on the promoterless construct pGL3-B and the construct m455, which does not include the AP-2-binding site (data not shown).


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Fig. 8.   AP-2 effect on CD-RAP expression. HepG2 (A) and C5.18 (B) cells were cotransfected with 2 µg of the construct m2251 and various amount of the human AP-2 expression plasmid AP-2alpha A in pCMX-PL1 vector. The differences in AP-2 DNA concentration were compensated for by control vector. The promoter activity was assayed 48 h later. The luciferase value of the control (without AP-2) was set as 1 in HepG2 cells and as 100% in C5.18 cells.

To determine whether expression of AP-2 affects the transcriptional activity of CD-RAP in C5.18 cells, the plasmid AP-2alpha A was cotransfected with the construct m2251. Fig. 8B shows that AP-2 slightly increased CD-RAP expression at low doses. Maximal activity was obtained at a dose of 0.25 µg of DNA/dish (35 mm). The CD-RAP expression was inhibited by high doses of AP-2. AP-2 reduced the CD-RAP promoter activity approximately 2-fold at a dose of 2.0 µg of AP-2alpha A DNA/dish. Similarly, another AP-2 expression plasmid (SP-AP2) also inhibited the CD-RAP promoter activities at high concentrations (data not shown). These results demonstrate that high expression of AP-2 results in the reduction of the CD-RAP promoter activity in C5.18 cells.

Last, to confirm that the exogenous AP-2 protein binds to the AP-2 DNA motif, we cotransfected the wild type and mutated CD-RAP promoter constructs into C5.18 cells along with 2 µg of human AP-2alpha A plasmid. 2 µg of AP-2 plasmid was shown above to decrease CD-RAP expression. As shown in Table II, the transcription of the wild type constructs m2251 and m475, both containing the AP-2 motif, was down-regulated more than 50% by AP-2. In contrast, mutation of the AP-2 binding site (m2251-MuA and m475-MuA) and deletion of AP-2 site (m455) abolished the AP-2 effect. This experiment confirms that the AP-2 motif is the primary site for AP-2 binding and is responsible for the reduction of the mouse CD-RAP transcription in C5.18 cells.

                              
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Table II
AP-2 effect on the CD-RAP expression: effects of the mutated AP-2 cis-acting sequences
A concentration of AP-2 shown to be inhibitory (Fig. 8) was used. The wild type constructs (m2251 and m475), the mutated constructs (m2251-MuA and m475-MuA), and the AP-2 deletion constructs (m455) were cotransfected into C5.18 cells with 2 µg of the human AP-2alpha A plasmid or a control plasmid, pCMX-PL1.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

CD-RAP is thus far known to be expressed in chondrocytes (1, 17) and melanoma cells (2, 16). The function of CD-RAP is unclear, although it is thought to be involved in the regulation of DNA synthesis and cell shape (2). In this report, we present evidence for the involvement of the trans-acting factor, AP-2, in the regulation of CD-RAP transcription. These results show, for the first time, a functional role of AP-2 in cartilage differentiation and a potentially physiologically relevant RA effect mediated through AP-2 binding. The results are particularly significant in light of the recent reports of a severe skeletal phenotype resulting from disruption of the mouse AP-2 gene (14, 15).

AP-2 functions in mediating the regulation of gene expression in response to a number of different signal transduction pathways. Phorbol esters and cyclic AMP induce AP-2 activity independent of protein synthesis (25). RA induces AP-2 activity by increasing AP-2 mRNA levels in human teratocarcinoma cells (6) and P19 embryonic carcinoma cells (9). In addition, AP-2 is associated with the programmed gene expression during retinoid-controlled murine embryogenesis (8). Restricted spatial and temporal expression patterns of AP-2 have been detected in several embryonic tissues, in particular in neural crest-derived cell lineages and in limb bud mesenchyme during the developmental stage when they are known to be retinoid-sensitive. Analyses of AP-2 expression in the embryonic and adult Xenopus tissues suggest a role for AP-2 in regulation of keratin gene expression during skin differentiation (26). Our deletion analysis of the CD-RAP promoter identified a fragment that includes an AP-2 motif and generates a high level of expression specifically in chondrocytes. Mutation or deletion of this AP-2 site led to decreased CD-RAP transcription in the transiently transfected chondrocytes, indicating that AP-2 may act as an activator of transcription for CD-RAP. Since mutation of the AP-2 site did not completely inhibit CD-RAP transcription, factors other than AP-2 may also be important in chondrocyte expression. Sox9 has recently been shown to be involved in the control of the cell-specific activation of COL2A1 in chondrocytes and to directly regulate the type II collagen gene in vivo (27, 28). Gel shift and mutation analysis have identified a potential protein binding site for Sox9 at -409, and an Sp1 binding site at -100. The role of these sites in CD-RAP transcription is currently under investigation.

RA plays an important role in the regulation of growth during embryonic development and cell differentiation. RA is involved in induction and morphogenesis of limbs (29) and, in excess, is a potent teratogen predominantly deforming limbs, craniofacial structures, and the central nervous system (30-32). RA also participates in the regulation of chondrocyte metabolism during endochondral ossification of the growth plate. Pacifici and colleagues (33) have shown that 10-100 nM RA stimulates the maturation of chondrocytes including causing growth plate chondrocytes in culture to flatten, thus favoring cell adhesion and spreading (33). RA clearly suppresses CD-RAP expression in chondrocytes at the mRNA level (1), and we show here that it inhibited the CD-RAP gene transcription. Mutation or deletion of the AP-2 site abolished the RA effect on CD-RAP expression, suggesting that AP-2 plays a critical role in the RA-induced down-regulation of CD-RAP in chondrocytes.

AP-2 can both activate and inhibit gene expression in genes other than CD-RAP. For example, a biphasic response has been observed in the insulin-like growth factor-binding protein-5 gene after cotransfection of AP-2 expression plasmid with the insulin-like growth factor-binding protein-5 promoter construct. Our results showed that exogenously added AP-2 expression vector increased transcription of the transfected CD-RAP promoter in AP-2-deficient HepG2 cells. In chondrocytes, due to endogenous AP-2 expression, the addition of low levels of AP-2 exerted only slight activation of the CD-RAP promoter, while higher amounts of AP-2 inhibited CD-RAP promoter activity. Altogether, these results suggest that AP-2 may play a dual role in the control of CD-RAP expression. At low levels, as occur constitutively in chondrocytes, it may activate CD-RAP expression; at high levels, as occur with RA treatment, it suppressed CD-RAP expression.

AP-2 has been shown to inhibit gene transcription by three mechanisms. First, an alternative splice product of AP-2alpha A, called AP-2alpha B, contains the activation domain of AP-2 and part of the DNA binding domain but lacks the dimerization domain necessary for DNA binding. AP-2alpha B is a potent inhibitor of transactivation by AP-2alpha A by interfering with binding of AP-2alpha A with DNA (34). Second, AP-2 can inhibit transactivation of Myc by competing with an overlapping binding site as well as binding directly to Myc and impairing DNA binding of the Myc-Max complex (11). Third, high levels of AP-2 can down-regulate gene expression by the mechanism of "self-interference" (12). Self-interference is thought to occur when excess AP-2 molecules interact with one or more putative AP-2 cofactors, making them unavailable for AP-2 function (12). Since there is no AP-2alpha B as detected by RT-PCR in the chondrocytes (data not shown) and no obvious overlapping cis-acting sequence and the RT-PCR and Northern blot analysis revealed that the AP-2 expression was significantly increased after RA treatment, we speculate that down-regulation of the CD-RAP promoter activity in chondrocytes by RA is due to the "self-interference" of AP-2.

AP-2 recently, and unexpectedly, has been associated with the lethal skeletal defects in mice (14, 15). The AP-2 knockout mice exhibited anencephaly, craniofacial defects, and thoraco-abdominoschisis. In the trunk and head, many bones were deformed or absent. The expression of skeletal patterning genes Pax-3, twist, and Msx-1 (14) was normal, indicating that they were unaffected by removal of AP-2. Taken together with the involvement of AP-2 in the regulation of the CD-RAP gene expression, it is possible that this cartilage gene is abnormally expressed in the AP-2-deficient animals. We have generated a transgenic mouse line harboring the 2.2-kb mouse CD-RAP promoter used in the present studies, which shows chondrocyte-specific expression.2 Mutations of the AP-2 motif in the CD-RAP promoter will be made using transgenic mice to clarify the role of AP-2 in CD-RAP expression in vivo.

    ACKNOWLEDGEMENTS

We thank Xin Zhang for expert technical assistance and Reinhard Buettner and Trevor Williams for providing expert advice and the human AP-2 expression vectors. We also thank Dr. Sherri Davies for helpful comments and suggestions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R0136994 and a merit review grant from the Department of Veterans Affairs.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 Present address: Washington University School of Medicine, Dept. of Orthopaedic Surgery and Dept. of Cell Biology and Physiology, 216 S. Kingshighway, Yalem Research Bldg., Room 704, St. Louis, MO 63110. 

§ To whom correspondence should be addressed: Washington University School of Medicine, Dept. of Orthopaedic Surgery, Yalem Research Bldg., Room 704, 216 S. Kingshighway, St. Louis, MO 63110. Tel.: 314-454-7800; Fax: 314-454-5900; E-mail: sandelll{at}msnotes.wustl.edu.

1 The abbreviations used are: CD-RAP, cartilage-derived retinoic acid-sensitive protein; MIA, melanoma inhibitory activity; RA, retinoic acid; FCS, fetal calf serum; RCS, rat chondrosarcoma; PCR, polymerase chain reaction; RT, reverse transcription.

2 W.-F. Xie and L. J. Sandell, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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