Egr-1 Mediates Transcriptional Repression of COL2A1 Promoter Activity by Interleukin-1beta *

Lujian TanDagger , Haibing PengDagger , Makoto OsakiDagger , Bob K. ChoyDagger , Philip E. AuronDagger , Linda J. Sandell§, and Mary B. GoldringDagger

From the Dagger  Rheumatology Division, Beth Israel Deaconess Medical Center and New England Baptist Bone & Joint Institute, Harvard Institutes of Medicine, Boston, Massachusetts 02115 and the § Department of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, September 25, 2002, and in revised form, February 17, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Following induction and activation of the early growth response (Egr)-1 transcription factor in human chondrocytes, interleukin-1beta (IL-1beta ) suppresses the expression of the type II collagen gene (COL2A1), associated with induction of Egr-1 binding activity in nuclear extracts. The COL2A1 proximal promoter contains overlapping binding sites for Egr-1 and Sp1 family members at -119/-112 bp and -81/-74 bp. Mutations that block binding of Sp1 and Sp3 to either site markedly reduce constitutive expression of the core promoter. IL-1beta -induced Egr-1 binds strongly to the -119/-112 bp site, and mutations that block Egr-1 binding prevent inhibition by IL-1beta . Cotransfection with pCMV-Egr1 potentiates the inhibition of COL2A1 promoter activity by IL-1beta , whereas overexpression of dominant-negative Egr-1 mutant, Wilm's tumor-1 (WT1)/Egr1, Sp1, or Sp3 reverses the inhibition by IL-1beta . Cotransfection of pGL2-COL2/Gal4, in which we substituted the critical residue for Egr-1 binding with a Gal4 binding domain and a pCMV-Gal4-Egr1 chimera permits an inhibitory response to IL-1beta that is reversed by overexpression of Gal4-CBP. Our results indicate that IL-1beta -induced activation of Egr-1 binding is required for inhibition of COL2A1 proximal promoter activity and suggest that Egr-1 acts as a repressor of a constitutively expressed collagen gene by preventing interactions between Sp1 and the general transcriptional machinery.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Type II collagen, encoded by the COL2A1 gene, is the major collagen of the extracellular matrix of mature articular cartilage. Together with the other cartilage-specific type IX and XI collagens, the highly cross-linked fibrils of triple helical type II collagen molecules form a fibrillar network that confers tensile strength to articular surfaces (1). Chondrocytes comprise the single cellular component of adult hyaline cartilage and are responsive to a number of growth factors and cytokines that either stimulate or inhibit type II collagen synthesis (2). Interleukin-1 (IL-1)1 is an inflammatory cytokine that plays a critical role in cartilage destruction in arthritis. IL-1beta is known to induce or activate members of the NF-kappa B, Jun/Fos, C-EBP, and ETS families of transcription factors, which may then induce or modulate the expression of MMPs, COX-2, and other genes involved in inflammatory and destructive processes (3). IL-1beta also contributes to cartilage depletion by suppressing the expression of cartilage-specific genes, including types II, IX, and XI collagens and aggrecan (4-6).

The early growth response (Egr)-1 transcription factor (also known as Tis8, Krox24, NHFI-A, and Zif268) is a serum-inducible zinc finger protein that is a critical upstream regulator of proliferation, differentiation, and apoptosis (7-10). Egr-1 gene expression is rapidly induced by mitogens, hypoxia, shear stress, or mechanical injury in fibroblasts, endothelial cells, and other cell types via mitogen- and stress-activated protein kinases (see Ref. 11 for review). These protein kinases, including ERK, JNK, and p38 MAPK, may also modulate the phosphorylation state of Egr-1 and its ability to bind DNA and other transcription factors. Egr-1 contains both transactivation and repression domains and regulates gene transcription either positively or negatively depending upon other DNA binding sites near the Egr-1 site (12). In human fibrosarcoma and glioblastoma cells, Egr-1 directly transactivates the genes encoding fibronectin, transforming growth factor (TGF)-beta 1, and plasminogen activator-1 (13, 14). During vascular remodeling, Egr-1 is involved in transactivation of multiple genes including platelet-derived growth factor A and B, tissue factor, TGF-beta , and membrane type 1 matrix metalloproteinase (11, 15, 16). Less commonly, Egr-1 may be involved in transcriptional repression of genes such as the TGF-beta type II receptor (TGF-beta RII), which has a role in restraining vascular repair (17).

Induction of Egr-1 mRNA by IL-1beta , tumor necrosis factor (TNF)-alpha , oncostatin M, ionizing, or UV radiation, retinoic acid, and prostaglandin E2 (PGE2) has been demonstrated in a variety of cell types, including dermal and synovial fibroblasts, osteoblasts, and chondrocytes (18-25). A recent study identified Egr-1 as a critical transcription factor involved in the induction of CD44 by IL-1beta in human endothelial cells (26). Paradoxically, given the role of cytokines in cartilage destruction, Egr-1 is differentially expressed at lower levels in osteoarthritic than in normal adult human articular cartilage (27). In contrast, Egr-1 is overexpressed in rheumatoid synovium, which is characterized by increased cell proliferation and expression of high levels of inflammatory cytokines (28).

Structural and functional analyses of type II collagen genes from different species have revealed multiple potential regulatory elements within both the promoter and first intron regions (29-36). These regions may interact with positive or negative transcription factors that determine developmental stage- and tissue-specific expression during chondrogenesis (37, 38). Sp1 binding motifs were among the first identified in type II collagen genes (29, 35, 39), and recent work has shown that Sp3 represses Sp1-mediated transactivation of the promoter activity (40). E-box sites, which are consensus binding sites for basic helix-loop-helix proteins, are present in both promoter and enhancer regions and have been proposed as important regulators of type II collagen gene expression during chondrocyte differentiation (32, 41, 42). A conserved E-box site (CAGGTG) in the promoter also interacts with the zinc finger homeodomain protein, delta EF1, which represses constitutive activity of the rat Col2a1 promoter (42). The zinc finger protein, cKrox, activates Col2a1 transcription in differentiated chondrocytes but inhibits constitutive activity in subcultured cells via the -266 bp promoter (43). Sox9, the first transcription factor to specify the chondrogenic lineage, activates type II collagen gene transcription by binding to the first intron enhancer through its high mobility group (HMG) DNA binding domain and acts cooperatively with L-Sox5 and Sox6 to regulate chondrogenesis in vivo (44, 45). It has been proposed that down-regulation of Sox9 expression by IL-1 or up-regulation by fibroblast growth factors or bone morphogenetic proteins (BMPs) determines the regulation of type II collagen gene transcription by these cytokines (44, 46, 47). The homeobox protein, Dlx-2, which is stimulated by BMP-2, also acts via the intronic enhancer to increase Col2a1 expression (48).

In our previous studies, we showed that IL-1beta decreases the levels of type II collagen mRNA by inhibiting COL2A1 gene transcription in chondrocytes in a manner that did not require the intronic enhancer (49). We also showed that IL-1beta induces the expression of immediate early gene mRNAs encoding c-Fos, c-Jun, Jun B, and Egr-1 preceding down-regulation of COL2A1 mRNA levels and up-regulation of matrix metalloproteinase mRNAs in human chondrocytes (20). Recently, we reported that the COL2A1 core promoter, which is highly GC-rich and contains a TATA box, expresses constitutively in chondrocytes and responds to negative regulation by IFN-gamma (50). In the present study, we have identified two potential Egr-1 sites within the proximal COL2A1 promoter spanning -131 to +125 bp that overlap with functional Sp1 binding sites. Since Egr-1 has been shown to regulate the activities of certain promoters by displacing Sp1 from overlapping binding motifs (11, 15), we sought to determine whether Egr-1 could interact functionally with the COL2A1 promoter and account partially for the inhibitory effect of IL-1beta . Our results indicate that Egr-1, when activated by IL-1beta , functions as a transcriptional repressor of COL2A1 promoter activity in cells that constitutively express this chondrocyte-specific gene and acts through displacement of Sp1 family members from at least one of the overlapping binding sites in the proximal promoter.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- The immortalized human chondrocyte cell line C-28/I2 (20) was cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 (1/1, v/v; Invitrogen) containing 10% fetal calf serum (Biowhitaker) and passaged using trypsin-EDTA solution (Invitrogen) at >95% confluency every 5-6 days. For experiments, confluent cultures were changed to medium containing 1% Nutridoma-SP (Roche Applied Science) for 24 h prior to incubation in the absence and presence of IL-1beta at 200-500 pg/ml for the times indicated.

RNA Extraction and Analysis-- Total RNA was isolated by a one-step extraction procedure using the TRIzol reagent (Invitrogen), and 0.9 µg was reverse-transcribed in 20 µl containing final concentrations of 2.4 IU/µl of MuLv Reverse Transcriptase (RT), 2.5 µM of oligo d(T)16, and 1 unit/µl of RNase inhibitor (all obtained from PerkinElmer Life Sciences), as described previously (51). The primers for human Egr-1 were 5'- GCTGCAGATCTCTGACCCGTTCG-3' (sense) and 5'-GCCGCTGGAGATGGTGCTGAG-3' (antisense) derived from the mRNA sequence (GenBankTM accession X52541). The primers for COL2A1 and glyceraldehyde-3-phosphate dehydrogenase were as described previously (51). The PCR amplification mixture contained 5 µl of the RT product in a final volume of 50 µl containing 1 mM MgCl2, 200 µM dNTPs, 0.2 µM of each sense and antisense primers, and 2.5 units of Taq DNA polymerase (Promega). Semiquantitative PCR was performed at 30-35 cycles such that all products could be assayed in the exponential phase of the amplification curve in the MJR Research PTC-200 Peltier thermal cycler. Following an initial step at 95 °C for 2 min, each cycle consisted of 30 s of denaturation at 95 °C, 30 s of annealing at 60 °C (COL2A1) or 55 °C (Egr-1), and 30 s of extension at 72 °C, with a final extension at 72 °C for 7 min. The PCR products, 30 µl of PCR reaction per well, were separated on 1.5% agarose gels.

Western Blot Analysis-- Equivalent amounts of protein (20 µg) in nuclear extracts were resolved on 7.5% SDS-polyacrylamide gels, electroblotted on 0.45-µm immobilon-P membranes (Millipore), and immunoblotted with anti-Egr-1 rabbit polyclonal antibody (1:200 dilution; from Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Blots were then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000 dilution) for 1 h at room temperature and the Egr-1/antibody complexes were visualized by chemiluminescence according to the manufacturer's recommendations (Amersham Biosciences).

Electrophoretic Mobility Shift Assays (EMSA) and Supershift Analysis-- For preparation of nuclear extracts, C-28/I2 cells were passaged and grown to confluence, changed to medium containing 1% Nutridoma-SP overnight, and treated with IL-1beta for 0.5, 1, 4, 14, and 18 h. The cells were lysed in hypotonic buffer with Nonidet P-40 at a final concentration of 0.5%, as described (52). Nuclear proteins were extracted in buffer C according to the modified method of Dignam et al. (53), diluted with low salt buffer D, and used directly for analysis of binding to DNA. Double-stranded synthetic DNA oligonucleotides were purchased from Operon Technologies, Inc. (Alameda, CA) and end-labeled using T4 polynucleotide kinase and [alpha -32P]dATP. Binding reactions were carried out for 30 min at room temperature using 5 µg of nuclear extract and 0.8 pmol (~10,000 cpm) of labeled probe in a final volume of 20 µl containing 12 mM HEPES-KOH (pH 7.9), 0.94 mM EDTA, 4.65 mM MgCl2, 50 mM KCl, 0.85 mM dithiothreitol, 12.5% glycerol, 0.5 mg/ml bovine serum albumin, and 1.25 µg of poly(dI-dC). The protein-DNA complexes were separated in low ionic strength 4% polyacrylamide gels using Tris/glycine-EDTA buffer (TGE) or Tris borate-EDTA buffer (TBE, 45, mM Tris borate, pH 8.3, and 1 mM EDTA), as indicated, and autoradiographed. The wild-type and mutant COL2A1 promoter sequences spanning -141 to -102 bp (wt1) and -93/-62 bp (wt2), listed in Fig. 1, and the consensus and mutant Egr-1 and Sp1 oligonucleotides (Santa Cruz Biotechnology) were used as labeled probes and as competitors at 50-fold excess. For supershift analysis, antibodies specific for Egr-1, Egr-2, Sp1, Sp2, Sp3, and Sp4 (Santa Cruz Biotechnology) were incubated with the binding reaction mixture for 30 min at room temperature before electrophoresis.

Plasmid Constructions and Mutagenesis-- For preparation of COL2A1 reporter constructs the region spanning -577/+3426 bp from pCAT/B4.0 (29, 49) was cloned into the pGL2-Basic luciferase vector (Promega). The pGL2B-577/+125 construct was prepared by enzyme digestion of pGL2B4.0 using SmaI and PstI to remove the +126/+3426 bp fragment followed by religation. The deletion construct containing -131/+125 bp was prepared by enzyme digestion of pGL2B-577/+125 using HindIII (cloning site in vector) and ApaI, followed by ligation. The pGL2B-131/+125 construct was used as a template to generate point mutations by PCR mutagenesis employing the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA). The pGL2-COL2/Gal4 reporter vector was constructed using pGL2B-131/+125 as template, the primers 5'-gccctccgaggggcgggcggttcaggttac-3' (forward) and 5'-tgtcacccgcggagccccgcctgggccctgc-3' (reverse), to substitute the Gal4 DNA binding domain (DBD, underlined) (54) in place of the Egr-1 site (see Fig. 1), and the ExSite PCR-based site-directed mutagenesis kit (Stratagene). Luciferase reporter plasmids were prepared for transfection using the EndoFree plasmid maxi kit (Qiagen).


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Fig. 1.   Structure and sequence of the proximal region of the COL2A1 promoter. A, the overlapping Egr-1/Sp1 sites, identified by MatInspector (www.gsf.de/biodv/matinspector.html) and TESS (www.cbil.upenn.edu/tess), and previously identified E-box and TATA-box sites are represented schematically. The positions of nucleotides upstream of the transcription start site (+1) are indicated above. B, the wild-type sequences, wt1 (-141/-102 bp) and wt2 (-93/-62 bp), the mutants (m1-m9), and the sequences containing the consensus and mutant Egr-1 sites and the Sp1 consensus site used in this study are listed below. The Egr-1 sites are underlined and the overlapping Sp1 sites are double-underlined.

The expression vectors used for cotransfections were pCMV-Egr1wt, pCMV-Egr1Delta mt, from which amino acids 331-374 containing the entire first zinc finger and part of the second were removed, and the dominant-negative pCMV-WT1-Egr1 chimera, containing the Wilm's tumor 1 (WT1) activation domain and the Egr-1 zinc finger DBD (kind gifts from Dr. Vikas P. Sukhatme, Beth Israel Deaconess Medical Center, Boston, MA) (55). The expression vectors for Sp1 and Sp3 were obtained, respectively, from Dr. J. Madri (Yale University, New Haven, CT) (56) and Dr. G. Suske (Marburg, Germany) (57). The pCMV-Gal4-Egr1 expression vector, containing the Gal4 (amino acids 1-95) DBD and the Egr-1 (amino acids 1-337) activation domain, was made by PCR using pCMV-Egr1wt as template and the primers 5'-cgggatccatggccgcggccaaggccgagatgc-3' (forward) and 5'-gctctagactaagggcgttcgtgggggggcgtc-3' (reverse), which created, respectively, BamH1 and XbaI sites (underlined). The PCR products were purified by the Concert Rapid PCR purification system (Invitrogen), digested with BamH1 and XbaI, and ligated into pCMV-Gal4. The pEF6-Egr1ZnR1 (amino acids 337-427) and pEF6-Egr1ZnR2 (amino acids 337-533) expression vectors were generated using pCMV-Egr1wt as template, the forward primer, 5'-aagatggcgcccccccacgaacgcccttacgc-3', and the reverse primers, 5'-tttgtctgctttcttgtccttc-3' for Egr1ZnR1 and 5'-gcaaatttcaattgtcctgg-3' for Egr1ZnR2. The PCR products were ligated into the pEF6/V5-His-TOPO vector (Invitrogen).

Expression vectors were constructed to generate recombinant and fusion proteins for analysis of binding to specific DNA sequences by EMSA. PCR products for Gal4 (1-95) DBD and the Gal4 (1-95)/Egr1 (1-337) were made using pCMV-Gal4-Egr1 as template, the forward primer, 5'-gaaatggggaagctactgtcttctat-3', and the reverse primers, 5'-tgttaacaatgcttttatatcctg-3' for Gal4 and 5'-gctctagactaaggggcgttcgtggggggcgtc-3' for Gal4-Egr1 and ligated into the pEF6/V5-His-TOPO vector (Invitrogen). The PCR product for Sp3 was made using pCMV-Sp3 as template, the forward primer 5'-gaaatggccggggcccccgccgccgccgg-3', and the reverse primer 5'-ctccattgtctcatttccagaaactgtg-3' and ligated into pCR2.1 (Invitrogen). Proteins were produced by in vitro translation using the TNT Quick-coupled Transcription/Translation System (Promega). The Gal4-CBP expression vector was provided by A. E. Goldfeld (Center for Blood Research, Harvard Medical School) with the permission of Dr. D. Thanos (Columbia University).

The sequences of all constructs were confirmed by DNA sequencing performed at the Beth Israel Deaconess Medical Center DNA sequencing facility using ABI PRISM® BigDyeTM primer cycle sequencing kit (Applied Biosystems, Foster City, CA) and the Automatic DNA Sequencer Model 373A (Applied Biosystems).

Transient Transfections and Luciferase Assays-- Transient transfection experiments were carried out in C-28/I2 cells using LipofectAMINE PLUSTM Reagent (Invitrogen). Cells were seeded in 6-well tissue culture plates at 3.5 × 105 cells/well in DMEM/F12 containing 10% fetal calf serum 24 h prior to transfection. For each well, COL2A1-luciferase construct (maximum of 1 µg of DNA), 6 µl of PLUS reagent and 92 µl of serum-free DMEM/F12 were mixed and incubated for 15 min at room temperature. LipofectAMINE+ reagent (4 µl) in 100 µl of serum-free medium was then added to each reaction mixture, and incubation was continued for an additional 30 min at room temperature. Finally, the transfection mixture was combined with 800 µl of serum-free medium and the lipid-nucleic acid complex was transferred to the washed cell monolayer in each well. After incubation for 4 h at 37 °C, the transfection mixture was diluted with an equal volume of DMEM/F12 containing 2% Nutridoma-SP, IL-1beta was added 2 h later, and incubation was continued for 18 h. For cotransfections, the expression vectors and the empty vectors were first titrated at amounts ranging from 10 to 200 ng per well; 100 ng was found to be optimal, whereas 200 ng produced nonspecific effects because of the CMV promoter. After cotransfection using 750 ng of COL2A1-luciferase vector and 50 or 100 ng of expression vector per well, the cells were incubated for 24 h to permit expression of recombinant proteins prior to treatment with IL-1beta for a further 18 h. Cell lysates were prepared by extraction with 200 µl of Reporter Lysis Buffer (Promega), and the protein content was determined using the Coomassie Plus Protein Assay Reagent (Pierce Chemical Company). Luciferase activities were determined by chemiluminescence assay using the Autolumat LB953 luminometer (EG&G Berthold, Oak Ridge, TN), normalized to the amount of protein, and expressed as relative activities against that of untreated pGL2B-131/+125 in each experiment. Each experiment was repeated at least three times, and each data point was calculated as the mean of the results (3-6 wells/experiment) ± S.D. The relative activities of the wild type and mutant constructs were checked by the Dual-Luciferase Reporter Assay (Promega) using 750 ng of COL2A1-luciferase vector and 20 ng of the pRL-TK Renilla luciferase control vector. The levels of luciferase activity expressed as RLU/µg of protein ranged from ~1 × 103 to 1 × 105 for COL2A1 promoter activity in untreated C-28/I2 cells.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Time Course of IL-1beta -induced Expression of Egr-1 mRNA and Inhibition of COL2A1 mRNA in Human Chondrocytes-- The C-28/I2 human chondrocyte cell line was selected as a reproducible model for this study because it expresses chondrocyte-specific matrix proteins, including type II collagen and aggrecan and responds to IL-1beta , as described previously (20, 58, 59). We first verified that IL-1beta stimulates Egr-1 mRNA levels and suppresses COL2A1 mRNA levels in the C-28/I2 cells, as we had reported previously in primary and immortalized chondrocyte cultures (4, 49). The C-28/I2 cells were preincubated in serum-free medium for 24 h prior to the addition of IL-1beta for periods of time between 15 min and 24 h (Fig. 2A). Compared with untreated controls at each time point, IL-1beta increased the levels of Egr-1 mRNA within 15 min, with peak induction by 1 h, and the IL-1beta -stimulated levels declined thereafter. Incubation with the protein synthesis inhibitor cycloheximide increased the levels of Egr-1 mRNA in either the absence or presence of IL-1beta and stabilized expression beyond 4 h. The apparent increases in Egr-1 mRNA levels in the untreated controls at each time point were not observed in every experiment, but could be due to a stress response after 24-48 h in serum-free medium. Higher constitutive levels of Egr-1 mRNA were observed on Northern blots in a previous study when a medium change was done at the time of addition of IL-1beta (20)


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Fig. 2.   Time course of IL-1beta -induced expression of Egr-1 mRNA in immortalized human chondrocytes. The C-28/I2 cells were plated in serum-containing medium and cultured for 5 days, and confluent cultures were changed to serum-free medium containing 1% Nutridoma 24 h before treatment with IL-1beta for the times indicated. A, cycloheximide (10 µg/ml) or B, emetine (10 µg/ml) was added in the absence (-) or presence (+) of IL-1beta . Total RNA was extracted using TRIzol and Egr-1, COL2A1, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels were analyzed by semiquantitative RT-PCR. DNA ladders are shown in the left lane for each mRNA. PCR products are 500, 359, and 346 bp for Egr-1, COL2A1, and glyceraldehyde-3-phosphate dehydrogenase, respectively.

Constitutive levels of COL2A1 mRNA increased with time throughout the 24 h time course, whereas suppression by IL-1beta was observed as early as 4 h (Fig. 2A). Cycloheximide appeared to prevent this inhibition at the earlier time points, although it had no effect on COL2A1 mRNA levels by 24 h in either the absence or the presence of IL-1beta . These results are consistent with our previous studies, in which COL2A1 mRNA was found to have a half-life of 17.5 h, and inhibition by IL-1beta was ascribed to decreased gene transcription but not to decreased COL2A1 mRNA stability (20, 49).

The increase in Egr-1 mRNA levels in response to cycloheximide alone may be explained by its capacity to activate stress pathways and act as nuclear signaling agonist to induce the expression of immediate early genes such as c-Fos and c-Jun mRNA. Thus, we compared the action of emetine, a protein synthesis inhibitor that does not act as a signaling agonist (60). As shown in Fig. 2B, emetine alone, similar to cycloheximide was able to increase Egr-1 mRNA as early as 15 min and to cause a "superinduction" in the presence of IL-1beta . In this experiment, unlike that shown in Fig. 2A, constitutive expression of Egr-1 was evident at 15 min and declined after 1 h; thus, the up-regulation by IL-1beta was not evident until the 1-h time point. This result suggests that the immortalized cells may constitutively express early growth response genes because they are in a constant proliferative state and supports the idea that a stress response may occur in these cells after serum deprivation. The sustained up-regulation of Egr-1 mRNA by emetine was remarkably similar to that in the presence of cycloheximide and indicates that both agents are acting as translational inhibitors in this cellular context.

Time Course of IL-1beta -induced Expression of Egr-1 Protein and Egr-1 Binding Activity in Human Chondrocytes-- To determine whether IL-1beta treatment of chondrocytes could also induce activation of Egr-1, we examined DNA binding activity to the Egr-1 consensus sequence. Nuclear extracts were prepared after incubation of the C-28/I2 cells with IL-1beta for times between 15 min and 18 h, and Western blotting analysis was performed using a polyclonal antibody against Egr-1. As shown in Fig. 3A, Egr-1 protein was present in untreated nuclear extracts and IL-1beta increased the level of Egr-1 protein (82 kDa) by 15 min, which peaked by 1 h and remained stable up to 18 h. When these nuclear extracts were analyzed in the EMSA shown in Fig. 3B, binding to the Egr-1 consensus oligonucleotide appeared within 15 min after addition of IL-1beta , peaked by 1 h, and began to decline by 4 h. Furthermore, the IL-1beta -induced binding activity was supershifted by the Egr-1 antibody, but not by the Egr-2 antibody (Fig. 3C). These findings indicate that IL-1beta increases both the levels of Egr-1 protein and its activation manifested by increased binding to the Egr-1 consensus DNA element.


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Fig. 3.   Time course of Egr-1 protein expression in IL-1beta -treated chondrocytes and activation of binding to the Egr-1 consensus sequence. Nuclear extracts were prepared after incubation of C-28/I2 cells in the absence (0) or presence of IL-1beta for the times indicated. A, equivalent amounts of total protein were fractionated on 7.5% polyacrylamide Tris-glycine/EDTA gels, transferred to nitrocellulose and probed with Egr-1 antibody. B, the double-stranded oligonucleotide containing the Egr-1 consensus site was end-labeled and incubated with the nuclear extracts shown in A. C, nuclear extracts from cells incubated in the absence (0) or presence of IL-1beta for 1 h were incubated with the Egr-1 consensus (cons) probe and binding of Egr-1 was identified by supershift analysis using the Egr-1 antibody (E1). Note that the Egr-2 antibody (E2) did not produce a supershift.

IL-1beta Increases Binding of Egr-1 to COL2A1 Proximal Promoter Elements-- Computer-assisted analysis of the proximal region of the COL2A1 promoter identified two potential binding sites for Egr-1, one at position -119 to -112 bp (CCGGGGGCGGGCGGGCGG) and the other at position -81 to -74 bp (CTGGGGGCAGGGGGCGG), both of which also contain previously identified Sp1 core binding sites (see Fig. 1) (29). In initial screens, the fragment spanning -93 to -62 bp bound an Egr-1-like factor in IL-1beta -treated nuclear extracts, but less strongly than the -141/-102 bp fragment (data not shown). As shown in Fig. 4 (left panel), nuclear factors from IL-1beta -treated cells formed a major complex with the labeled Egr-1 consensus sequence that disappeared in the presence of excess unlabeled self-competitor or the wild-type COL2A1 fragment (-141/-102 bp). When the labeled -141/-102-bp oligonucleotide was used as probe, a more complex pattern of binding could be observed. IL-1beta -induced binding activity appeared that was ablated by competition with either the Egr-1 consensus or the -141/-102 bp sequence (Fig. 4, right panel). The identity of the Egr-1 binding activity was further verified by the supershifts with Egr-1 antibody on both probes. However, upon competition with the self-fragment (wt) on the -141/-102 bp probe, several additional bands disappeared that may be attributable to other factors that recognize GC-rich sequences, such as Sp1 family members (see below), cKrox, or Ap2.


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Fig. 4.   Competition and supershift analysis of nuclear protein binding to the Egr-1 consensus and COL2A1 (-141/-102 bp) promoter sequences. End-labeled probes containing the Egr-1 consensus (left panel) and -141/-102 bp of COL2A1 (right panel) were incubated with nuclear extracts from untreated (-) or IL-1beta -treated (+) C-28/I2 cells in the absence or presence of 50× excess unlabeled competitor oligonucleotides, including Egr-1 consensus (cons), COL2A1 wild-type (wt), or mutants (m) 1, 2, or 3 (see sequences in Fig. 1). The identity of the Egr-1 complex was confirmed by supershift analysis using a specific antibody (Ab) against Egr-1 (open arrows).

To characterize the critical sequences required for Egr-1 binding, we performed mutation analysis using double-stranded oligonucleotides containing mutations within (m1 = CtaGGGCGGGCGG) and outside (m2 CGGGGGCGGCta) the Egr-1 core-binding site within the -141/-102 bp fragment, and at both sites (m3 = CtaGGGCGGCta) (see Fig. 1). As shown in Fig. 4, excess unlabeled m1 and m3 oligonucleotides, which contained the GG to TA mutation in the Egr-1 site, did not compete for the IL-1beta -induced binding activity attributable to Egr-1 on either the Egr-1 consensus or the -141/-102-bp sequence. In contrast, the m2 oligonucleotide with a mutation outside the Egr-1 site produced competition approximately equivalent to wild type competitor except for a binding activity that overlapped with one of the nonspecific bands. These results indicate that the Egr-1 binding activity on the COL2A1 probe migrates closely with at least one other specific binding activity.

Analysis of Binding Activities of Sp1 Family Members in Nuclear Extracts from C-28/I2 Cells-- EMSA competition and supershift experiments using a labeled Sp1 consensus probe confirmed the presence of Sp1 family members in nuclear extracts from C-28/I2 cells (Fig. 5A). The formation of at least 3 complexes of different mobilities was consistent with the expected binding patterns of Sp1 family members and identification was aided by supershift analysis. The Sp1 antibody produced a supershift of at least one of the bands. The Sp2 and Sp4 antibodies did not produce discernible supershifts, but decreased the intensities of all of the bands in this region, especially that of the upper band that was also recognized by the Sp1 and Sp3 antibodies. The Sp3 antibody produced a strong supershift and ablated the binding of the three specific complexes. Addition of the Egr-1 antibody to the binding reaction did not alter the pattern of binding to the Sp1 probe. However, the excess unlabeled Egr-1 consensus sharpened or decreased the intensities of the three Sp1/3-specific bands, suggesting that Egr-1 may be able to interact nonspecifically with the G-rich Sp1 consensus under the conditions of the in vitro assay. The Sp1 consensus, which is recognized by all family members, competed completely for the binding of all three of the specific complexes (Fig. 5A).


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Fig. 5.   Supershift analysis of DNA binding of Sp1 family members in nuclear extracts from C-28/I2 cells. A, end-labeled consensus Sp1 oligonucleotide was incubated with nuclear extracts from IL-1beta -treated (+) C-28/I2 cells. Supershift analysis was performed using antibodies against Egr-1, Sp1, Sp2, Sp3, or Sp4. Competition analysis with 50× molar excess of Egr-1 or Sp1 consensus sequence confirmed the specificity of binding of the Sp1 family members. B, the COL2A1 (-141/-102 bp) probe was incubated with nuclear extracts from untreated (-) or IL-1beta -treated (+) C-28/I2 cells. Incubation with antibodies against Egr-1, Sp1, Sp2, Sp3, or Sp4, decreased specific binding activities (upper panel) and produced supershifts that are better observed on the lower panel.

The pattern of binding of nuclear factors to the labeled -141/-102 bp COL2A1 probe was more complex than that observed using the Sp1 or Egr-1 probe (Fig. 5B). The binding activities of Sp1 family members were identified by their relative mobilities compared with those on the Sp1 probe in Fig. 5A and by the loss of these bands in the presence of antibodies. The major band for Egr-1 (82 kDa) was very close to one of the bands attributable to Sp3 (90 kDa) (Fig. 5B, upper panel). This may explain our inability to cleanly dissociate Egr-1 binding on the -141/-102 bp probe. The presence of Egr-1 binding activity in untreated nuclear extracts may represent some degree of autoactivation during storage or handling during the assay, since the same nuclear extracts were used at an earlier time for the experiment in Fig. 4. In the presence of extracts from IL-1beta -treated cells, however, the intensity of the Egr-1 supershift increased, whereas the Sp1 and Sp3 supershifts decreased, as visualized on an overexposed autoradiograph (Fig. 5B, lower panel). This loss of binding was probe-specific, since IL-1beta did not decrease the amount of Sp1 and Sp3 recognized by the Sp1 probe (Fig. 5A). Taken together, these results suggest that IL-1beta -induced Egr-1 may interfere with the binding of Sp1 family members, to the overlapping site in the -141/-102 bp sequence, and that the extent of binding may depend upon the balance between Egr-1 and Sp1 family members in the nuclear extracts.

Mutation Analysis of Egr-1 and Sp1 Binding Sites in the -141/-102 bp COL2A1 Promoter Fragment-- To dissociate the binding site for Egr-1 from that for Sp1 family members on the -141/-102 bp fragment, we compared nuclear binding activities on the Egr-1 and Sp1 consensus probes using a series of mutant oligonucleotides as competitors. Since the m1 mutation in the Egr-1 binding site (see Fig. 4) might also be expected to affect binding to the overlapping Sp1 site (underlined), we used additional mutant oligonucleotides: m4, atGGGGCGGGCGG; m5, CGGGGGCGatCGG; m6, aGGGGGCGGGCGG; and m7, CGaGGGCGGGCGG (see Fig. 1). The capacity of each mutant probe to bind nuclear proteins from IL-1beta -treated C-28/I2 cells was assessed by competition assays using Sp1 or Egr-1 as probe. As shown in Fig. 6, A and B, m1 and m3 bound neither Sp1 nor Egr-1, whereas m2 and m6 bound both. In contrast, m4 bound Sp1 but not Egr-1, whereas m5 bound Egr-1 but not Sp1. However, m7 prevented Sp1 binding, but was a partial competitor against Egr-1 binding. Comparison of m4 and m6 indicates that the G at position -119 bp is critical for binding of Egr-1. Comparison of m5 and m7 indicates that the G nucleotides at positions -118 bp and -112 bp are critical for binding of Sp1. EMSA analysis using the -141/-102 bp probe showed similar patterns of competition and confirmed the identities of the binding activities (Fig. 6C). Egr-1 binding activity was present only in the IL-1beta -treated nuclear extracts in this experiment, whereas Sp1-like binding was present in untreated cells but increased after IL-1beta treatment. Thus, these results lend further support to the notion that both Sp1 family members and activated Egr-1 bind to this overlapping site but that the extent of binding depends upon the relative concentrations of the different factors.


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Fig. 6.   Mutation analysis of binding of nuclear proteins to the Sp1 and Egr-1 consensus sequences using wild type and mutant COL2A1 sequences. End-labeled oligonucleotides containing (A) the Sp1 consensus, (B) the Egr-1 consensus, and (C) COL2A1 (-141/-102 bp) sequences were incubated with nuclear extracts from untreated (-) or IL-1beta -treated (+) C-28/I2 cells. Unlabeled competitor oligonucleotides containing mutations (m1-7, see Fig. 1) in the -141/-102 bp COL2A1 promoter fragment were added at 50× molar excess, as indicated.

Functional Analysis of Egr-1 and Sp1 Sites in the COL2A1 Promoter Region in Transient Transfections-- To determine the functional consequences of mutations in the Egr-1 and Sp1 binding sites, mutations were generated in the COL2A1 promoter construct, pGL2B-131/+125, and transfections were performed in the C-28/I2 cells. IL-1beta treatment suppressed the activity of the wild-type construct by 40-60% (Fig. 7A). The m1 mutation in the major overlapping Sp1/Egr-1 site decreased constitutive activity of the -131-bp promoter by ~20%, while the extent of inhibition by IL-1beta was decreased to 10-35% compared with the untreated mutant promoter (Fig. 7A). The m2 and m3 mutations were not studied, since m2 bound both Egr-1 and Sp1, and m3, similar to m1, bound neither (see Figs. 4 and 6). The m4 mutation in the Egr-1 binding site had no effect on constitutive activity, while the response to IL-1beta was reduced (Fig. 7A). In contrast, the m5 mutation, which mutated the Sp1 binding site, decreased constitutive activity, but did not prevent the inhibition by IL-1beta . The m6 mutation, which permitted binding of both factors, had no effect on constitutive promoter activity, nor did it affect the IL-1beta response. In contrast, the m7 mutation, which did not bind Sp1 but partially blocked Egr-1 binding, decreased constitutive activity to a similar extent as m5 and reduced the inhibition by IL-1beta to around 25% (Fig. 7A).


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Fig. 7.   Functional analysis of Egr-1 and Sp1 sites in the COL2A1 promoter in transient transfections. A, C-28/I2 cells constructs were transfected with the wild type pGL2B-131/+125 or mutant construct (m1-m9, see Fig. 1) and IL-1beta was added for 18 h prior to harvest for luciferase assay. The results are expressed as the means ± S.D. derived from 3 (m1, m7-m9) or 6 (m4-m6) separate transfection experiments, each done in triplicate. B, double-stranded oligonucleotides containing mutations m4 and m5 and wt1 (-141/-102 bp) were end-labeled and incubated alone (0) or with nuclear extracts from IL-1beta -treated C-28/I2 cells. Note that the wt1 competitor added at 50× molar excess prevented the specific binding of nuclear proteins. C, end-labeled Egr-1 and Sp1 consensus oligonucleotides were incubated with nuclear extracts from IL-1beta -treated C-28/I2 cells in the absence (-) or presence of unlabeled wt2 (-93/-62 bp), m8, or m9 competitors at 50× molar excess.

The m4 and m5 mutants were the only competitors that were able to dissociate the binding of Egr-1 and Sp1 to the -141/-102 bp sequence, as well as to distinguish constitutive activity and the IL-1beta response in transient transfections. We, therefore, compared the binding of nuclear factors to labeled m4 and m5 oligonucleotides to determine whether they were indeed able to bind Sp1 and Egr-1, respectively. In the EMSA analysis shown in Fig. 7B, binding activities consistent with Sp1 family members were observed on the m4 probe, whereas m5 bound Egr-1 only, consistent with the competition analysis shown in Fig. 6. These results also suggest that the loss of Egr-1 binding activity may account for the lack of IL-1beta response by the m4 construct and that loss of Sp1 binding at the m5 site may decrease constitutive activity.

Despite the consistencies in the binding and functional analyses of the mutations within the -141/-102 bp site, it was of interest to examine the other potential Egr-1/Sp1 site at -83/-73 bp. Transient expression analysis showed that COL2A1 promoter constructs containing deletions at either Delta (-121/-108) or Delta (-83/-73) expressed at 20-50% of the level of wild type constructs (data not shown), suggesting that both regions are required for full constitutive activity of the COL2A1 promoter. Deletion of the intervening sequence Delta (-98/-88) also decreased constitutive activity (not shown), possibly because it shortens the distance between the two Sp1-binding regions or because it contains a nonconserved E-box site (CAGCTG at -92/-87 bp). To determine the contribution of the downstream site at -81/-74 bp to either constitutive activity or the IL-1beta response, we examined the consequences of the m8 and m9 mutations (see Fig. 1), which analogous to m4 and m7 were predicted to affect Egr-1 and Sp1 binding, respectively. However, both mutations decreased constitutive activity and the response to IL-1beta to a similar extent (Fig. 7A). These functional results were consistent with the EMSA analyses using nuclear extracts, in which both m8 and m9 were ineffective competitors against Sp1/3 and Egr-1 binding to consensus probes (Fig. 7C), as well as the binding of recombinant Egr-1, Sp1, or Sp3 to the COL2A1 (-93/-62 bp) probe (data not shown). These findings may also account for the failure of mutations in the upstream Egr-1/Sp1 site to completely block constitutive activity or the response to IL-1beta , since the -83/-73 bp site would still be available for binding to Egr-1 and Sp1.

Overexpression of Egr-1, Sp1, and Sp3 Modulates COL2A1 Promoter Activity and the IL-1beta Response in Cotransfection Assays-- To determine whether overexpression of recombinant Egr-1, Sp1, and Sp3 proteins could modify constitutive COL2A1 promoter activity or the response to IL-1beta , cotransfection experiments were performed using the expression vectors shown in Fig. 8A. The expression vectors driven by the CMV promoter and the empty vector (pCMV) were first titrated at amounts ranging from 10 to 200 ng per well to determine the optimal amount that would avoid nonspecific effects. In the experiments shown in Fig. 8, the C-28/I2 cells were cotransfected with pGL2B-131/+125 and 50 ng of expression vector. Cotransfection with 50 ng of pCMV-Egr1wt did not change constitutive activity of the COL2A1 promoter, but the inhibitory effect of IL-1beta was potentiated compared with cotransfection with the empty vector (Fig. 8B). These results suggest that IL-1beta is required to activate the overexpressed Egr-1 protein.


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Fig. 8.   Effects of Egr-1, Sp1, and Sp3 expression vectors on COL2A1 promoter activity and IL-1beta responses in cotransfection assays. A, wild type and truncated Egr-1 proteins and the WT1-Egr1 fusion protein expressed by the vectors used in this study are shown schematically. B, C-28/I2 cells were cotransfected with pGL2B-131/+125 and the empty vector, pCMV, or the expression vector for Egr1wt, Egr1(Delta 331-374), WT1-Egr1, Sp1, or Sp3 at 50 ng of vector per well. C, cotransfections were performed with pGL2B-131/+125 and 100 ng of pEF6 (empty vector), pEF6-Egr1ZnR1, or pEF6-Egr1ZnR2. Cotransfections were performed with the mutant constructs pGL2B-131/m1 (D), pGL2B-131/m4 (E), and pGL2B-131/m5 (F) together with the Egr1wt, Egr1(Delta 331-374), WT1-Egr1, Sp1, and Sp3 expression vectors. After transfections, the cells were incubated for 24 h prior to further incubation for 18 h in the absence (solid bars) or presence (hatched bars) of IL-1beta , followed by luciferase assay. The results show the means ± S.D. of triplicate wells from one representative of 5 (B, C) or 3 (D-F) separate transfection experiments.

To determine the roles of the different Egr-1 domains, Egr1Delta mt, and WT1-Egr-1 were overexpressed and found to have no effect on constitutive activity of pGL2B-131/+125. The pCMVEgr1Delta mt vector, containing the transactivation domain and one-half of the zinc fingers in the DBD, reversed completely the inhibitory effect of IL-1beta (Fig. 8B). The pCMV-WT1-Egr-1 vector, a chimera of the WT1 activation domain and the entire Egr-1 zinc finger DBD, which acts in a dominant-negative manner against Egr-1 activity in other systems (55), also reversed the suppression by IL-1beta without affecting constitutive activity (Fig. 8B). In contrast, overexpression of Sp1 increased constitutive activity of the -131-bp promoter by 1.5-fold. Overexpression of Sp3 had no effect on constitutive activity in this experiment, although we frequently observed inhibition of up to 50%. However, overexpression of Sp1 or Sp3 reversed the inhibitory effect of IL-1beta . These results confirm the suggestion from the binding data in Fig. 5 that IL-1beta -induced Egr-1 may decrease constitutive activity by competing with endogenous Sp1 family members, and further suggest that increasing the ratio of Sp1 or Sp3 to Egr-1 may attenuate the action of IL-1beta -induced Egr-1.

To further substantiate the role of the Egr-1 DBD, cotransfections were performed using expression vectors encoding the Egr-1 zinc finger region alone (pEF6-Egr1ZnR1) or with the C-terminal protein interaction region (pEF6-Egr1ZnR2) (see Fig. 8A). Overexpression of Egr1ZnR1 had no effect on constitutive activity but reversed the inhibition by IL-1beta . In contrast, overexpression of Egr1ZnR2 decreased constitutive activity by 30%, and almost completely reversed the inhibition by IL-1beta (Fig. 8C). These results confirm that binding of Egr-1 to the COL2A1 core promoter is essential for the inhibitory effect of IL-1beta .

To further explore the sequences required for the Egr-1-mediated IL-1beta response, we performed similar cotransfections using the mutant reporters, pGL2-131/m1 (Fig. 8D), pGL2-131/m4 (Fig. 8E), and pGL2-131/m5 (Fig. 8F). Overexpression of the Egr1wt, Egr1-Delta mt, or WT1-Egr1 expression vector down-regulated the activity of the m1 construct to a similar extent, around 25%, and the extent of inhibition by IL-1beta remained similar to that in presence of the empty pCMV vector. In contrast, overexpression of Sp1 increased m1 activity by 2.5-fold, whereas addition of IL-1beta decreased the Sp1-stimulated expression to the pCMV control level. Sp3, on the other hand decreased m1 activity to a slightly greater extent than Egr1wt either in the absence or presence of IL-1beta .

Analysis of the responses of the m4 and m5 constructs dissociated responses to Egr-1 and Sp1/3 expression vectors. The m4 reporter did not respond to the wild type or dominant-negative Egr-1 expression vectors, whereas the Sp1 expression vector increased m4 activity, but only by 20%, and Sp3 had no effect. The dominant-negative effects of Egr1Delta mt and WT1-Egr1 suggest that the 24-30% inhibition by IL-1beta in the presence of empty vector, Sp1, or Sp3 may be mediated via the downstream Egr-1/Sp1 site. On the other hand, in the absence of the upstream Sp1/3 binding site, the wild type and mutant Egr-1 expression vectors seemed to be permissive for increased constitutive m5 activity, and WT1-Egr1 had the expected dominant-negative effect on the IL-1beta response. Sp1 only slightly increased m5 activity, Sp3 had no apparent effect, but both Sp1 and Sp3 reversed the inhibitory effect of IL-1beta . Although difficult to interpret, the combined results using the m1, m4, and m5 promoters suggest that in the absence of the binding site for Egr-1 or Sp1/3 in the -141/-102 bp region, the binding sites in the -92/-62 bp region may take over the functional responses to Sp1 and Sp3. However, the absence of regulation by the Egr-1 wild type and mutant expression vectors is consistent with the weak Egr-1 binding activity in the -92/-62 bp region and suggests that the IL-1beta response is also mediated by other regulators that interact directly or indirectly with the COL2A1 proximal promoter.

The Gal4-Egr-1 Chimera Acts as a Transcriptional Activator of pGL2-COL2/Gal4 and Interferes with Binding of Sp1 or Sp3 to the Adjacent Site; Reversal of IL-1beta -induced Inhibition by CBP-- To determine the nature of the interaction between Egr-1 and Sp1, the Egr-1 binding site was "knocked out" by substituting the critical G for Egr-1 binding at -119 bp in the pGL2B(-131/+125) construct with 18 bp of nucleotides encompassing a Gal4 binding site (Fig. 9A). We also constructed an expression vector for a chimeric protein containing the Gal4 (1-95) DBD and the Egr-1 activation domain (1-337) (Fig. 9A). Neither the empty pCMV vector nor pCMV-Gal4 had any effect on pGL2-COL2-Gal4 activity in either the absence or presence of IL-1beta (Fig. 9B). Thus, binding of Gal4 to the substituted Gal4 binding site was, by itself, not sufficient to induce promoter activity. However, overexpression of the chimeric Gal4-Egr1 protein increased pGL2-COL2-Gal4 activity in a dose-dependent manner (Fig. 9B), but to a level that was 25% of the activity of the wild-type pGL2-COL2-131/+125 reporter. Furthermore, IL-1beta inhibited this expression by more than 50% (Fig. 9B). These results suggest that the presence of the adjacent Sp1-binding site is not sufficient for full activation of this artificial promoter, which also contains the downstream Sp1 site at -81/-74 bp. Furthermore, Egr-1 (amino acids 1-337) appears to serve as an activator in this context, but requires induction by IL-1beta to produce an inhibitory response. This is similar to the response of the m5 promoter (Fig. 8F), where Egr-1 is an apparent activator in the absence of the Sp1 site. Additional support for the validity of this mechanism is provided by the experiment in Fig. 9C, in which cotransfection with the CBP expression plasmid reversed the IL-1beta -induced inhibitory response.


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Fig. 9.   The Gal4-Egr-1 chimera acts as a transcriptional activator of pGL2-COL2/Gal4 and interferes with binding of Sp1 or Sp3 to the adjacent site. A, pCMV-Gal4-Egr1 expression vector contains the Gal4 DBD fused to the Egr-1 activation domain (1-337). The pGL2-COL2/Gal4 sequence contains the -131/+125 bp promoter with a Gal4 binding sequence (underlined) inserted in place of the nucleotide at -119 bp and adjacent to the Sp1 site (double-underlined). B, C-28/I2 cells were cotransfected with the pGL2-COL2/Gal4 reporter and pCMV (50 ng), pCMV-Gal4 (50 ng), or pCMV-Gal4-Egr1 at 25-150 ng, as indicated. Note that cotransfection with 25, 100, or 150 ng of pCMV-Gal4 produced activity similar to that shown for 50 ng of this vector. C, cotransfections were performed using 25 ng of each expression plasmid, including the vector expressing full-length CBP fused to the Gal4 DBD (Gal4/CBP). (D) For EMSA analysis, the COL2/Gal4 oligonucleotide shown in A was labeled and incubated alone (0) or with the Gal4-Egr1 fusion protein at 0.5 µg (+) or increasing amounts (left panel, 0.5, 1, 1.5, 2, 2.5 µg, or right panel, 0.5, 1, 1.5 µg). In vitro translated Sp1 was added at 0.5 µg (left panel) or 1 µg (right panel) and Sp3 was added at 1 µg (right panel).

Since pGL2-COL2/Gal4 was constructed with the Gal4 site adjacent to rather than overlapping the Sp1 site as in the wild type promoter, we performed EMSA analysis of the Gal4-substituted COL2A1 sequence to determine whether Gal4-Egr1 could influence binding of Sp1 or Sp3 to the adjacent site. The results in Fig. 9D show that recombinant Sp1 and Sp3, as well as the fusion protein Gal4-Egr-1, are all capable of binding independently to the COL2/Gal4 sequence. When increasing amounts of recombinant Gal4-Egr1 were added, the binding of constant amounts Sp1 (left panel) or Sp3 alone or together with Sp1 (right panel) was decreased and disappeared. However, when the ratio of Gal4-Egr1 to Sp1 or Sp3 was increased to 2:1 or greater, the binding of Sp1 or Sp3 was displaced. Recombinant Egr-1 alone did not bind to this sequence (data not shown). These results suggest that binding of Gal4-DBD with the attached Egr-1 activation domain is able to interfere with Sp1 or Sp3 binding to the immediately adjacent site.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We showed previously that IL-1beta inhibits COL2A1 mRNA expression and synthesis of type II collagen, resulting in accelerated dedifferentiation and loss of phenotype in primary cultures of human chondrocytes (4). Treatment of chondrocytes with IL-1beta also increases the levels of the immediate early gene mRNAs, including c-Fos, c-Jun, and Jun B, as well as Egr-1, preceding down-regulation of COL2A1 mRNA (20). However, binding sites directly responsive to nuclear protein complexes containing Fos/Jun proteins or other IL-1beta -induced transcription factors have not been identified previously in the COL2A1 promoter. Our present results show that Egr-1 mediates, in part, the inhibition by IL-1beta of expression of the proximal COL2A1 promoter by interacting with an overlapping binding site for Sp1 family members and thereby interfering with constitutive gene transcription. Egr-1 is coregulated with c-Fos during skeletal development in the perichondrial interface and interstitial cells of opposing cartilaginous elements and at sites destined for endochondral ossification (61). However, the absence of skeletal phenotype in Egr-1 knockout mice indicates that this transcription factor is not essential for normal cartilage and bone development (62). Thus, these findings are relevant primarily to chondrocyte function in adult cartilage. Similar to other immediate early genes, Egr-1 is induced by serum and acts primarily as a transcriptional inducer of gene expression. Certain growth factors present in serum may suppress the expression of COL2A1-reporter gene activity (49). Thus, the ability of Egr-1 to repress transcription in the context of the strongly expressed COL2A1 promoter may reflect decreased expression of this matrix gene observed in proliferating chondrocytes that are undergoing dedifferentiation in vitro.

The COL2A1 proximal promoter region studied here is particularly GC-rich and contains binding sites for zinc finger factors, such as Sp1, E box/bHLH proteins, and cKrox, which may positively or negatively regulate constitutive expression in a cell-type specific manner (39, 42, 43). In this study, we identified two binding sites for another zinc finger protein, Egr-1, one at -119/-112 bp and the other at -81/-74 bp, both of which also contain the previously identified Sp1 core motifs (29, 39, 40). Many previous studies have shown that Egr-1 can activate gene transcription by displacing Sp1 in promoters where Sp1 serves as a weaker activator. However, interaction of Egr-1 with an Ets-like site was shown to serve a negative regulatory function in the TGFbeta RII promoter (17). Mechanisms by which Egr-1 could suppress transcription include direct repression of promoter activity via DNA binding (12, 17) or "squelching" transcription through interactions with Sp1, independent of Sp1 binding to DNA (63).

Our experiments indicate that a reduction in COL2A1 transcription is associated with increased binding of Egr-1 to the -119/-112 bp site and displacement of Sp1, rather than binding of these factors to adjacent sites in a noncompetitive manner. The extent of binding depends upon the balance of Egr-1 and Sp1 family members in the nuclear extracts. The results of the mutation analyses indicate that the Egr-1 and Sp1 binding sites overlap and encompass the core Sp1-binding site, GGGGCG. Egr-1 binding requires the sequence, GGGGGCG, with the critical G at the 5'-end. Both 5' and 3' nucleotides are critical for Sp1 binding, since mutation to GGGGGCGat (m5) or to GaGGGCGGG (m7) prevents competition for binding to the Sp1 consensus. The interaction of Egr-1 with the overlapping Sp1 site is consistent with known binding activities of these zinc finger transcription factors (11, 64).

Our functional analysis of the -131/+125 bp promoter supports the findings of the binding studies, which indicate that Egr-1 binding to the overlapping site at -119/-112 bp mediates IL-1beta -induced suppression of promoter activity and that Sp1 binding is important for constitutive expression. In fact, all mutations except m4 and m6 decrease constitutive activity, corresponding with the loss of Sp1 binding activity. The results of experiments using m4 and m5 as competitors, where the binding activities of Sp1 and Egr-1 are dissociated, also correlate with the functional data. The constitutive activity of -131/+125-bp promoter is sensitive to the m5 mutation, which does not permit Sp1 binding, whereas the IL-1beta response is sensitive to the m4 mutation that blocks Egr-1 binding. Furthermore, the m7 reporter construct is less responsive to IL-1beta possibly because it binds Egr-1 less strongly. Thus, in the absence of elements upstream of -131 bp, binding of Egr-1 to the -119/-112-bp site is required for the IL-1beta response. However, both the m8 and m9 mutations in the downstream site at -81/-74 bp, although they are analogous to m4 and m5, respectively, decrease both Egr-1 and Sp-1 binding and their functional activities are similar. Thus, the binding of Sp1 to the downstream site at -81/-74 bp is required for full constitutive activity of the proximal promoter, but the upstream site at -119/-112 is required for the full response to IL-1beta , as also suggested by the relative nonresponsiveness of the m4 promoter to Egr-1 overexpression (Fig. 8E). The different responses of the mutant promoters to Sp3 overexpression indicate that this Sp1 family member may act as a repressor, especially in the absence of Egr-1 binding to the upstream site.

The cotransfection data further indicate that IL-1beta -induced Egr-1 can suppress COL2A1 transcription when overexpressed in chondrocytes. This effect requires activation of the overexpressed Egr-1, since suppression of -131/+125 bp promoter activity is not observed in the absence of IL-1beta . Additional support for the involvement of Egr-1 as a transcriptional suppressor is provided by the results showing reversal of the IL-1beta -induced suppression by overexpression of pCMV-WT1-Egr1. This fusion protein, which contains the Egr-1 zinc finger DBD, has been shown to act in a dominant-negative manner in other systems (55, 65). The Egr1Delta mt, with deletion of 1.5 of the zinc fingers, also acts in a dominant-negative manner, possibly due to its ability to interfere with activation of the endogenous Egr-1. Neither construct, however, has any effect on constitutive activity. Further support for the DNA-binding requirement for the Egr-1-mediated IL-1beta response is provided by the results showing reversal of IL-1beta -induced inhibition by overexpression of Egr1ZnR1 and Egr1ZnR2.

Since residual Sp3 binding activity persists in IL-1beta -treated nuclear extracts and overexpression of Sp3 decreases expression of the -131 bp promoter, Sp3 may play a negative role among the Sp1 family members in chondrocytes. In contrast, overexpression of Sp1 increases COL2A1 promoter activity. However, overexpression of either Sp3 or Sp1 blocks the inhibition by IL-1beta . These results suggest that the Sp1/Sp3 ratio determines the level of constitutive activity of the COL2A1 promoter, but that increasing the ratio of either Sp1 or Sp3 to Egr-1 may prevent Egr-1 binding. Our results contrast somewhat with those from previous studies in which Sp1 binding activities to the COL2A1 promoter either decreased (43) or increased (39) in dedifferentiated chondrocytes that expressed low levels of COL2A1. However, our findings agree with those of Ghayor et al. (40), who showed that Sp1 transactivates the Col2a1 promoter (-266/+121 bp) independent of the differentiation state of chondrocytes and that Sp3 blocks Sp1-induced activity. Thus, increasing the ratio of Sp3 to Sp1 may promote chondrocyte dedifferentiation manifested by decreased COL2A1 promoter expression. Although the presence of endogenous Sp1 and Sp3 proteins may obscure the interpretation of the cotransfection experiments, the lack of any effect after a 6-h expression time following transfection (data not shown) compared with the significant changes observed when the expression time is extended to 24 h prior to IL-1beta treatment indicates that the observed changes are specific. Our findings are consistent with the general concept that, in mammalian cells, Sp3 is a DNA binding-dependent repressor that may abrogate Sp1-driven transcription from promoters containing GC boxes by displacing Sp1 and preventing its interaction with the general transcription machinery (57). Similarly, Sp1 acts as a transcriptional activator of the type I collagen gene, COL1A2, whereas Sp3 overexpression blocks the promoter activity induced by TGF-beta (66). Constitutive nuclear factor binding to Sp1 sites in the TGF-beta RI promoter, for example, could then represent an additional level at which the TGF-beta response is mediated (67).

Our finding that the chimeric Gal4-Egr1 protein is capable of activating the -131/+125 bp promoter with the Egr-1 site knocked out by substitution with a Gal4 binding site, suggests that Egr-1 does not mediate repression of COL2A1 via direct interaction with Sp1 or Sp3. The Gal4 substitution only removes the upstream G critical for Egr-1 binding leaving the Sp1 site intact, such that it is possible to observe binding of recombinant Sp1 and Sp3 proteins in the presence of the Gal4-Egr1 fusion protein when added at equivalent concentrations. However, increasing the relative concentration of Gal4-Egr1 to Sp1and/or Sp3 prevents binding of the latter proteins. Nevertheless, the Gal4-Egr1 fusion protein is able to mediate an inhibitory response to IL-1beta on the artificial COL2/Gal4 promoter construct, possibly due to the presence of the transcription activation domain (TAD) of Egr-1. This model suggests that Egr-1 could serve as an activator of the COL2A1 promoter by cooperating with Sp1 or Sp3 bound to the adjacent site, but in the presence of IL-1beta , Egr-1 serves as a repressor.

IL-1beta induces many cellular responses via the stress-activated kinases, p38 MAPK and JNK, which have been shown to modify the phosphorylation state of preformed Egr-1 protein and activate DNA binding (11, 68). Induction of Egr-1 promoter activity is also mediated by these kinases through activation of the ternary complex factor, Elk-1 (68). On the other hand, casein kinase II, which also mediates phosphorylation of Sp1, may act as a negative regulator of DNA binding activity of Egr-1 and also modify protein-protein interactions between Egr-1 and Sp1 (63, 69). Further work will be required to determine the stress-activated signaling pathways involved in inhibition of COL2A1 expression by IL-1beta , as suggested for p38 MAP kinase in our previous study (51).

The enhanced sensitivity of differentiated chondrocytes to inflammatory cytokines suggests that indirect regulation of promoter activity may occur through suppression of chondrocyte-specific constitutive factors such as Sox9 that bind to the intronic enhancer. A recent report indicates that IL-1beta decreases Sox9 mRNA and protein levels via stimulation of NF-kappa B, which then binds to the Sox9 promoter and inhibits its activity (46). However, the levels of L-Sox5, Sox6, and Sox9 mRNAs, which are all expressed by the C-28/I2 cells used in this study (50), are not decreased by IL-1beta treatment and the response of the COL2A1 promoter to IL-1beta occurs in the presence or absence of the intronic enhancer.2 The early time course of the response in our study and the lack of dependence on protein synthesis for the later suppression of COL2A1 mRNA levels also argue against a primary effect via inhibition of Sox9 expression, although sustained suppression in vivo would require inhibition of further Sox9 protein synthesis. Similarly, we found that interferon-gamma causes a rapid decrease in activity of the proximal COL2A1 promoter without affecting Sox9 expression (50). Thus, our studies are consistent with the roles of Sox9 and related HMG factors as architectural proteins that function to bend and unwind DNA and thereby maintain an open chromatin network surrounding the constitutively active COL2A1 promoter (70).

It has been proposed that Sp1 binding to sites in the COL2A1 core promoter and first intron enhancer regions may result in the formation of a DNA loop that could mediate protein-protein interactions at a distance (29, 35). Although Sp1 is also required for constitutive promoter activities of type I collagen genes (66), whether there are functional Egr-1 binding sites in those promoters has not been studied. Thus, in the context of the COL2A1 promoter the role of Egr-1 as a transcriptional suppressor via interference with Sp1 may be unique among the interstitial collagen genes. Recent reports suggest that Sp1 may act in concert with Sox9 and cKrox, another zinc finger protein and positive regulator of COL2A1 expression that binds to GC-rich sites overlapping with the Egr-1 sites studied here (40, 43). The role of the coactivator CBP/p300 in transcriptional activation by Egr-1 (11, 71), as well as by some SOX proteins (72), has been established. IL-1beta has been shown to stimulate CBP-dependent histone H4 acetylase activity associated with stimulation of GM-CSF promoter activity (73). Our results showing that CBP overexpression reverses the inhibitory effect of IL-1beta suggest that coactivators such as CBP play a positive role in maintaining constitutive activity of the COL2A1 promoter. Thus, Egr-1 may prevent interactions among CBP, Sp1 and TATA-binding proteins and thereby permit transcriptional repression by other IL-1beta -induced factors that bind to upstream promoter sequences. Candidate factors are C/EBP and ETS family members that are known to be involved in cytokine regulation of other genes (74-77). Our results support the notion that there are multiple pathways and transcription factors mediating the effects of IL-1beta on COL2A1 gene expression. These findings provide new insights into a mechanism whereby Egr-1, when activated by IL-1beta , can serve as a transcriptional suppressor of a constitutively expressed collagen gene by preventing interaction of positive regulators such as Sp1 with the general transcriptional machinery.

    ACKNOWLEDGEMENTS

We thank Drs. V. Sukhatme, J. Madri, G. Suske, and A. E. Goldfeld for generously providing expression vectors.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants AR45378 and AG22021 (to M. B. G.) and CA68544 (to P. E. A.) and a Biomedical Science Grant from the Arthritis Foundation (to M. B. G.).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.

To whom correspondence should be addressed: Harvard Institutes of Medicine, Room 246, New England Baptist Bone & Joint Institute, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-667-0742; Fax: 617-975-5299; E-mail: mgoldrin@bidmc.harvard.edu.

Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M301676200

2 R. Yamin, L. Tan, M. Osaki, B. K. Choy, L. J. Sandell, and M. B. Goldring, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: IL-1, interleukin-1; BMP, bone morphogenetic protein; Egr-1, early growth response transcription factor; TGF, transforming growth factor; TNF, tumor necrosis factor; PGE2, prostaglandin E2; DMEM, Dulbecco's modified Eagle's medium; EMSA, electrophoretic mobility shift assay; DBD, DNA-binding domain; WT1, Wilm's tumor 1; HMG, high mobility group; CBP, CREB-binding protein; CMV, cytomegalovirus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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