Serum response elements activate and cAMP responsive elements inhibit expression of transcription factor Egr-1 in synovial fibroblasts of rheumatoid arthritis patients

Wilhelm K. Aicher, Adelheid Dinkel, Bodo Grimbacher, Christian Haas, Elisabeth v. Seydlitz-Kurzbach, Hans H. Peter and Hermann Eibel

Clinical Research Unit for Rheumatology and Department of Rheumatology and Clinical Immunology, University Hospital Freiburg, 79106 Freiburg, Germany

Correspondence to: H. Eibel, Clinical Research Unit for Rheumatology, University Hospital Freiburg, Breisacher Strasse 64, 79106 Freiburg, Germany


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Analyzing the induction kinetics and promoter elements regulating the expression of the transcription factor Egr-1, we found elevated levels of Egr-1-encoding mRNA in synovial fibroblasts of rheumatoid arthritis (RA) patients when compared to controls. By contrast, synovial lymphocytes and macrophages do not show an elevated Egr-1 transcription. Therefore, the overexpression of Egr-1 may serve as a diagnostic marker to characterize synovial fibroblasts of RA patients. To study the regulatory mechanisms controlling Egr-1 expression we analyzed the function of transcription factor binding sites located in the Egr-1 promoter. Individual transcription factor binding sites within the Egr-1 promoter were specifically mutated and Egr-1 promoter activity was tested using reporter gene constructs. Our experiments demonstrate that serum response elements are the main positive regulators and binding to a cAMP responsive element represents the major negative regulator for Egr-1 expression in synovial fibroblasts. In addition, we functionally defined a new element, which was not yet described in the human Egr-1 promoter and which serves as a second negative regulatory element for Egr-1 expression. Therefore increased serum response factor activity or failure of Egr-1 repressing signals may account for Egr-1 overexpression in RA synovial fibroblasts.

Keywords: autoimmune disease, c-fos, Egr-1, rheumatoid arthritis, transcription factor


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is characterized by a chronic inflammatory process which may result in destruction of cartilage and loss of articular function (1). To date, knowledge on etiology and underlying pathomechanisms remains incomplete. The articular process in RA is characterized by expansion of synovial lining cells, invasive growth of the fibroblasts, and by infiltration of lymphocytes, macrophages and granulocytes into the joint (2). Histologically, two populations of synovial lining cells can be distinguished. (i) First the macrophage-like cells which express MHC class II antigens, Fc receptors and characteristic surface markers such as CD14 (3). (ii) The fibroblast-like cells which lack the typical surface markers of macrophages or lymphocytes but are characterized by a granular endoplasmic reticulum (3) and the expression of VCAM at high levels (4). Both the macrophage-like cells and the fibroblast-like cells are capable of producing constitutively inflammatory cytokines and tissue degrading enzymes which contribute to the inflammatory degradation of the articular cartilage in RA (5). Compared to non-RA diseases, in RA the articular tissue destruction proceeds more aggressively, possibly due to chronic overexpression of inflammatory cytokines and matrix degrading proteases such as collagenase or cathepsin-L (6,7). In this process the activated synovial fibroblasts invade into adjacent cartilage and bone (8).

Our previous studies showed activation of immediate early (IE) growth response gene Egr-1 in fibroblasts derived from RA synovium (9). IE genes are transcribed in cells preincubated with cycloheximide immediately after stimulation, indicating that all proteins for signal transduction and transcription are stored within the cells and translation is not required for generation of the respective mRNA. Egr-1 encodes a C2–H2 zinc-finger transcription factor which was cloned independently by several groups from different cells and therefore is also called Krox 24, TIS8, Z-225, NGFI-A or zif268 respectively (1013). Transcription of Egr-1 is rapidly induced after stimulation of the cells and Egr-1 expression is actively switched off soon thereafter by factors including c-fos (14). Therefore, as a marker for cellular activation we analyzed expression of Egr-1 in comparison to c-fos. Earlier reports stated that both Egr-1 and c-fos are constitutively co-activated in RA synoviocytes, and that their regulation was impaired in cells obtained from RA patients (15). Indeed, Egr-1 and c-fos are transiently co-activated in most experimental systems or in limb anlagen and tissues during embryonal development (1618). In our previous studies we showed that Egr-1-encoding transcripts were detected in situ in zones of the synovial membrane where synovial macrophages and fibroblasts predominate. In the lymphoid follicles, Egr-1 mRNA was not detected (9). In primary cultures of RA synovial tissue fibroblasts elevated transcription of Egr-1 was seen in comparison to controls including osteoarthritis (OA)-derived fibroblasts. Interestingly, in some cases elevated transcription of Egr-1 persisted for several passages in vitro (9). In all of these studies, however, the lineage of the Egr-1high synoviocytes and the expression of Egr-1 in synovial fluid-derived cells remained obscure. Others reported elevated co-expression of c-fos and junB in RA and in OA synovial lining cells (19). Again, as we showed for Egr-1, the lymphoid follicles or the disperse lymphoid infiltrates did not express jun/fos gene products (20), although ample clinical and experimental evidence suggests that the synovial T and B cell compartments of RA patients are activated and antigen driven (21,22).

In order to gain more information on the regulation of expression of the IE transcription factors Egr-1 and c-fos in RA we compared their spontaneous expression in synovial fluid fibroblasts and synovial membrane fibroblasts ex vivo and in primary cultures. Skin fibroblasts and synovial fibroblasts from non-RA patients served as controls. The induction of Egr-1 and c-fos transcription by addition of FCS or medium harvested from primary culture RA synoviocytes was also analyzed in later passages of RA synovial fibroblasts (passage 6–8), autologous RA skin fibroblasts and in SV40 T antigen (TAg) immortalized synovial fibroblasts from a healthy donor (23). In addition, the regulatory potential of transcription factors such as p68 serum response factor (SRF) or AP-1 (jun/fos) in the Egr-1 promoter was studied by reporter gene assays using Egr-1 promoter reporter vectors. In conclusion, our data show that Egr-1 expression is induced in RA synovial fibroblasts at higher levels and for an extended time period than c-fos. Factors binding to the AP-1 (jun/fos) recognition sequence do not repress Egr-1 transcription which may contribute to the characteristic Egr-1high phenotype. The major negative regulatory elements of Egr-1 expression in synovial fibroblasts are the cAMP responsive elements (cAMP RE), especially the proximal cAMP RE located ~125 bp 3' of the transcription start.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patient samples and cell culture
All patient specimens were obtained from the Division of Rheumatology, Freiburg University Hospital or Department of Orthopedic Surgery, Tübingen University Hospital. The patients were diagnosed according to the ACR (American College of Rheumatology) revised criteria for RA (24). Surgical samples were obtained from knee joints. Eleven out of 21 patients were diagnosed as having active RA. Five of them were rheumatoid factor (RF) positive. Control specimens from patients with reactive arthritis (reA), osteoarthritis (OA), plasmocytoma, ancylosing spondylitis and from a healthy donor (meniscectomy) were included (23).

Synovial tissue samples, also referred to as pannus, were obtained by surgical synovectomy or from arthroplastic surgery. Cell suspensions of synovial tissue fibroblasts were prepared as described before (9). Synovial fluid cells were obtained by aseptic tapping of rheumatic joint effusions. The synovial fluid was diluted 1:1 with PBS and subjected to a Ficoll-Hypaque gradient centrifugation at 1000 g for 20 min at 20°C. The low-density fraction (P <= 1.077) was enriched for lymphocytes and mononuclear phagocytes, and the high-density fraction contained mainly polymorphonucleocytes in addition to macrophages and fibroblast-like cells.

Synovial fluid fibroblasts were enriched by Ficoll gradient centrifugation in the high-density fraction (P >= 1.077). The fibroblasts were washed and grown as a monolayer culture in the presence or absence of autologous non-adherent synovial cells, i.e. mainly T cells and monocytes and rarely some B cells. For some experiments, the non-adherent cells were removed and fibroblasts were harvested at ~70–80% confluence using trypsin–EDTA. Cells were diluted and seeded at a starting density of 3x105 cells/260 ml flask in Iscove's complete medium containing 10% FCS.

Human synovial fibroblasts were immortalized by use of an SV40 TAg expression vector (23). In brief, the SV40 TAg-encoding DNA fragment was subcloned into a pGEM plasmid (Promega). For transfection, 5 µg of the TAg expression vector was linearized and synovial fibroblasts were transfected by a liposome method. TAg-transformed cells were enriched through their enhanced mitotic activity and by frequent passaging of the cells (23).

Analysis of cells by flow cytometry
Cells directly isolated from synovial fluid or harvested from in vitro cultures were washed twice with cold PBS containing 3% FCS and 0.1% azide. Cell density and viability were determined in a standard hemocytometer using the Trypan blue exclusion method. Cells (3–5x105) cells were seeded in a 96-well plate and flow cytometry was performed as described before (25): For T cells, anti-CD3 mAb OKT3 or UCHT1 (Immunotech, Marseilles, France) were used; for macrophages, anti-CD11b (Mac-1, ATCC, HB-248) was used. Fibroblasts were stained with the mAb F15-42-1, a reagent to a Thy-1-related 26 kDa antigen present on human fibroblasts (26,27), and mAb AS02, an antibody-reactive with human fibroblasts from different tissues (28) (Dianova, Hamburg, Germany). Dead cells were excluded using propidium iodide staining. For flow cytometry, cells were gated according to their forward/side scatter and 104 viable cells were acquired for flow cytometry. Adherently growing fibroblasts were harvested by treatment with 5 mM EDTA/PBS. Cells were washed and analyzed by flow cytometry as described above or stained directly as adherent cells as described before (6,15).

Preparation of plasmid DNA
Plasmid DNA and probes for Northern blotting were prepared as described (9). In brief, the actin cDNA-containing plasmid was digested with PstI restriction endonuclease to yield a 550 bp DNA fragment. The c-fos plasmid was cut with EcoRI and SalI restriction endonuclease to yield a 651 bp fragment (15). The Egr-1 plasmid, clone Z-225, was cut with EcoRI and a 2 kb fragment was used as probe (9). The respective cDNA fragments were purified and labeled by the oligonucleotide priming method for use as probes (29).

RNA isolation and Northern blot analysis
RNA was extracted using guanidinium isothiocyanate (30). Yield and purity were determined by UV spectrophotometry (29). For Northern blotting, 20 µg/slot of total RNA was subjected to electrophoresis in a 1% (w/v) agarose gel containing 6.7% (v/v) formaldehyde. The RNA was transferred to nylon filters (Schleicher & Schuell, Dassel, Germany) and fixed by UV cross-linking. Filters were prehybridized overnight at 42°C (31). For detection of specific transcripts, [{alpha}-32P]dATP-labeled denatured DNA probes were added and incubated for 24–48 h in the same solution. Filters were washed in 0.1xSSC/0.1% SDS at 50°C and exposed to X-ray films (Kodak X-AR) with an intensifier screen at –70°C for various times to obtain optimal exposures.

cDNA synthesis and PCR analysis
To generate a cDNA substrate for PCR analysis, 10 µg of total RNA was subjected to a reverse transcription reaction using SuperScript enzyme (Gibco/BRL, Gaithersburg, MD) and oligo(dT) as primer. For PCR amplification, one-fifth of the cDNA sample (2 µg total RNA equivalent) was amplified using SuperTaq DNA polymerase (Staehlin, Basel, Switzerland) in a rapid capillary thermocycler. For Egr-1 analysis, we used the PCR primers 5'-ACAAGAAAGCAGACAAAAGTG and 5'-GGGAAGTGGGCAGAAAGGATT, resulting in a 531 bp PCR product corresponding to position 1520–2051 of the Egr-1 cDNA (12,32). PCR temperature profiles were 30 s at 94°C, 30 s at 57°C and 60 s at 72°C for the first cycles, reducing the process time to a 5/5/30 s cycling profile in cycles 6–35. For analysis of c-fos transcripts, 5'-GCCTAACCGCCACGATGATGT and 5'-GCCCCTCCTGCCAATGCTCTG primers were used resulting in a 395 bp fragment corresponding to position 275–669 of the cDNA sequence (32). Temperature profiles were 30 s at 94°C, 30 s at 60°C and 60 s at 72°C for the first cycles, reducing the process time to a 5/5/30 s cycling profile in cycles 6–35. Analysis of ß-actin cDNA (5'-ACTCTTCCAGCCTTCCTTCC and 5'-TGTCACCTTCACCGTTCCAG, position 821–1335) served as PCR control.

For quantitative analysis of steady-state mRNA levels by RT-PCR, an internal standard DNA was generated (33). For Egr-1 a 420 bp DNA internal standard and for c-fos a 556 bp DNA internal standard were constructed (34). To establish the method of quantitative PCR, increasing amounts of the respective recombinant internal standard DNA were co-amplified in one tube with known amounts of a Egr-1- or c-fos-encoding plasmid or serial dilutions thereof. Analogously, for quantitative RT-PCR of the mRNA samples, co-amplifications of increasing amounts of internal standard DNA and serial dilutions of the sample cDNA were performed. Amplified DNAs were separated and visualized on agarose gels containing ethidium bromide, and a good correlation of amplified products was found ranging from 0.1 pg/ml to 1 ng/ml internal standard. To quantify the PCR products, the gel was recorded by a video device (Herolab, Munich, Germany) and the cDNA concentration was calculated from the scanning data using NIH Image software comparing the signal intensities of the internal standard of the PCR reaction to the signals generated by the cDNA sample (33). Defined amounts of marker DNA were used as reference in each gel to equilibrate the signal intensities of the PCR products generated. Since for all experiments the internal standards were used from the same stock solutions, all quantitative PCR experiments of different samples used in this study could directly be compared and quantified (34). In some samples, the data obtained by quantitative PCR of cDNA were compared to a Northern blot and an excellent correlation of the results was found.

Egr-1 and c-fos promoter analysis
To analyze the regulatory potential of transcription factors binding to the Egr-1 promoter (11) (Fig. 1Go), we ligated the 780 bp SmaI–BamHI promoter fragment of the human Egr-1 genomic DNA clone TIS8 (generous gift of Drs Sakamoto and Gasson, UCLA) into the pGL-2 basic luciferase-encoding reporter vector (Promega, Madison, WI). The following transcription factor binding sites were deleted and replaced for cloning purposes by a SalI site (GTCGAC) using PCR and specific oligonucleotides. Correct deletion and cloning were confirmed by restriction site mapping and DNA sequencing (29). A FASTA search (EMBL) confirmed that other regulatory promoter elements had not been generated by these mutations. We deleted cAMP RE at position –623 to –630 (cAMP1) and position –127 to –134 (cAMP2), the AP-1 binding site (AP-1 BS) at position –602 to –609, the Egr-1 binding site at position –589 to –597 (EBS2), and the serum response elements (SRE) at positions –394 to –410, –326 to –376 and –89 to –108 (11). Recombinant DNA spacers to replace the deleted binding sites were not introduced into the promoter to avoid unspecific regulatory effects, although the different spacing of the remaining promoter elements might play a role for promoter activity. In addition, upon sequencing the Egr-1 promoter we found two additional transcription factor binding sites which have not been functionally described so far. At position –685 to–695 we found an additional distal Egr-1 binding site (EBS1) and at position –198 to –211 a NF{kappa}B-like binding site which were also deleted as described above (Fig. 1Go). Luciferase-encoding reporter plasmids containing the human Egr-1 promoter or the respective deletion mutants thereof were prepared in at least two independent plasmid preparations by either the CsCl/EtBr method or Sephadex chromatography (29). To transfect equal amounts of plasmids for reporter analysis, purity and concentration of plasmids were determined by UV spectrophotometry and by gel electrophoresis followed by EtBr staining (29). A human growth hormone (HGH)-encoding expression vector was used as standard for transfection efficiency and 2 µg of HGH expression vector were co-transfected in each experiment with the luciferase plasmid (35). For c-fos promoter analysis the XhoI–HincII fragment of the human c-fos gene-encoding plasmid (generous gift of P. Shaw, University of Nottingham, England) was used to generate a promoter reporter vector in the pGL-2 basic plasmid. For promoter reporter analysis of human synovial fibroblasts the SV40 TAg-immortalized synovial fibroblast lines were used (23). Fibroblasts were transfected using a liposome method (DOTAP) as recommended by the supplier (Boehringer, Mannheim, Germany). In brief, 2 µg RSV-HGH plasmid, 2 µg luciferase reporter plasmid and 10 µl DOTAP/well were premixed to form the DNA–liposome complex and added to the fibroblasts in six-well plates at 80% confluence as described (23). The pGL-2 basic plasmid containing no promoter element and a pGL-2 vector containing the RSV promoter served as controls. After incubation with transfection mix for 8–16 h, medium was changed and cells were incubated for 2 days with Iscove's complete medium containing FCS ranging from 0.5 to 10% or complete medium conditioned by primary culture of RA synoviocytes respectively. Cell supernatant was removed and HGH content was determined in the supernatant using anti-HGH-coated beads in a standard assay (Nichols Institute). Cells were washed 3 times with cold PBS and lysed with 250 µl/well of a buffer containing 1% Triton (36). The Egr-1 or c-fos promoter activities were determined as enzymatic activity of firefly luciferase in the cytoplasmic extract. Luciferase reagent contained 25 mM glycylglycine buffer, MgSO4, DTT, EGTA, ATP and coenzyme A. The injection mix contained glycylglycine and luminol as substrate (36). For luminometric analysis a luminometer (Berthold, Tübingen, Germany) was employed. Determining the HGH activity in each sample allowed us to standardize the assay for, for example, transfection efficiency or variations in cell density. Promoter activity is given as relative light units delivered by mixing 25/250 µl cytoplasmic extract with 300 µl reaction mix and 300 µl injection mix for a 10 s time period. In some experiments, Egr-1 wild-type promoter activity was set as 100% in comparison to the deletion mutants. All luciferase assays were performed in duplicate at least 3 times using different plasmid stocks. The results given represent mean value ± SD. The significance of the value differences and probability of error were calculated for Egr-1 promoter activity and the respective mutants using JMP software according to Dunnett's method (P values, post hoc t-test).



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Fig. 1. Transcription factor binding sites in the human Egr-1 promoter. A SmaI–BamH I DNA fragment containing the Egr-1 promoter was prepared from genomic DNA clone TIS8 (11) and ligated into pGL-2 luciferase reporter plasmid. Binding sites for regulatory transcription factors listed on top (square symbols) were deleted individually as indicated in the diagram below. Since SP1 confers basic transcription, SP1 binding sites (oval dots) were not mutated. All deletions in the promoter were confirmed by restriction mapping and DNA sequence analysis.

 
Immunoblot analysis
Cytoplasmic Egr-1 expression was detected by immunoblotting as described (23). In brief, 106 fibroblasts were washed with PBS and harvested by aid of trypsin–EDTA. The cells were sedimented and lysed in 100 µl buffer containing NaCl, Tris–HCl, NP-40 and PMSF. Cell debris were removed by centrifugation (12,000 g, 4°C, 15 min) and an aliquot of the extract was separated by SDS–PAGE. Proteins were transferred onto a Nylon membrane and the Egr-1 protein was detected using the C19 anti-Egr-1 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA), followed by horseradish peroxidase-labeled anti-rabbit serum (Dianova, Hamburg, Germany) and visualized on X-ray film using the ECL detection reagents (Amersham, Amersham, UK).

Mobility shift analysis
Binding of transcription factors to the SRE or the NF{kappa}B-like element in the Egr-1 promoter in comparison to consensus SRE and NF{kappa}B binding sites was analyzed by a gel retardation assay (36,37). The following DNA oligonucleotides with consensus SRE or NF{kappa}B or the respective motifs derived from the Egr-1 promoter were used (binding element bold, complementary stretch underlined):

con SRE:5'-TTGGATGTCCATATTAGGACATCT (Santa Cruz)

Egr-1 SRE:5'-TTCGGAACAACCCTTATTTGGGCAGCACCT [position –381 ff (11)]

con NFkB:5'-TTAGTTGAGGGGACTTTCCCAGGC (SantaCruz)

NFkB-like:5'-TTGGGCGCCTGGGATGCGGGCCGGGCC-GGG[position –211 ff (11)]

The complementary oligonucleotides were mixed, annealed and 12.5 pmol of the double-stranded oligonucleotides was radiolabeled with Klenow enzyme and 50 µCi [{alpha}-32P]dATP using the 5' TT overhang on both sides of the annealed DNA fragments. The oligonucleotides were purified by chromatography (NAP-5 columns; Pharmacia, Freiburg, Germany) and extracted in 1 ml phosphate buffer. Nuclear extracts were prepared from 106 cells. The synovial fibroblasts were washed with PBS and harvested by aid of trypsin–EDTA. The cells were washed twice in PBS to remove the trypsin. Then 106 cells were resuspended in 400 µl hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and 0.5 mM PMSF), incubated on ice for 15 min and then lysed by addition of 25 µl 10% NP-40. Cells were mixed (vortex, 30 s). Nuclei were sedimented in a microcentrifuge (12,000 g, 30 s). The nuclear pellets were resuspended in 50 µl extraction buffer containing 20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF, and nuclear proteins were extracted by incubation on ice for 20 min. The extracts were sedimented in a cold microcentrifuge (12,000 g, 4°C, 5 min), and aliquots were frozen and stored at –70°C for later use. For the mobility shift assay using the SRE the reaction was carried out in reaction buffer (40 mM NaCl, 10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM ß-mercaptoethanol and 4% glycerol), 2 µg annealed poly(dI–dC) competitor (Boehringer Mannheim), 1 µl radiolabeled oligonucleotide and 1 µl nuclear extract. For the NF{kappa}B and NF{kappa}B-like elements the reaction was carried out in 10 mM HEPES, pH 7.2, 40 mM KCl, 0.05 mM EDTA, 0.0025% NP-40, 2% Ficoll 400, 2% glycerol, 1 mg/ml BSA, 1 mM DTT, 1 mM PMSF, 2 µg poly(dI–dC), 1 µl radiolabeled oligonucleotide and 1 µl nuclear extract. The DNA and nuclear extracts were incubated at room temperature for 30 min, mixed with DNA gel loading buffer and separated on a 4% PAGE using 0.5xTBE as buffer (37). Gels were transferred onto Whatman paper, dried in vacuo at 80°C and exposed to X-ray films.


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 Methods
 Results
 Discussion
 References
 
Characterization of Egr-1 expressing cells in RA synovial tissue and RA synovial fluid cells
Steady-state levels of Egr-1-encoding mRNA were analyzed by Northern blotting. To demonstrate the specific induction of the Egr-1-encoding gene we compared steady-state Egr-1 transcript levels to the mRNA levels encoding c-fos (Fig. 2Go). RNA extracted from RA synovial explants immediately after surgery showed high levels of Egr-1 mRNA (Fig. 2Go, lane 1). In contrast, the lymphocyte/monocyte fraction isolated from synovial fluid obtained immediately prior to surgery from the same patient exhibited barely detectable levels of Egr-1-encoding mRNA when examined ex vivo (Fig. 2Go, lane 2). When incubated in the presence of synovial fibroblasts, the lymphocyte/monocyte fraction isolated from the synovial fluid of the same RA patient showed only basal levels of Egr-1 transcripts (Fig. 2Go, lane 3). Skin fibroblasts from RA patients served as controls and did not show elevated Egr-1 expression (Fig. 2Go, lane 4). Reprobing the filter with a c-fos-encoding cDNA did not show enhanced transcription of c-fos in the fibroblasts and only faint signals were seen after extended exposure in the monocyte fraction from primary culture supernatants (Fig. 2Go). Rehybridization with a ß-actin cDNA probe documented that the amounts of RNA loaded in all lanes were sufficient to detect even low abundancy transcripts (Fig. 2Go).



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Fig. 2. Transcription of Egr-1 is elevated in fibroblast-like synoviocytes of RA patients. RNA was extracted from frozen synovial tissue (lane 1) and from the freshly isolated low-density mononuclear cell fraction of synovial fluid (T cells, macrophages, lane 2). When the mononuclear cells were collected as non-adherent cells from the primary cultures of synovial fluid cells of the same RA patient they exhibited a low degree of Egr-1 expression (lane 3). Skin fibroblasts grown in Iscove's complete medium served as controls (lane 4). RNA (20 µg) was separated in a denaturing agarose gel, blotted onto a filter and probed with Egr-1 (top). After exposure the filter was stripped, exposed for control to X-ray film and then incubated with a c-fos-encoding probe (middle). Rehybridization with a ß-actin probe shows sufficient amounts of RNA in all lanes (bottom).

 
In comparison to the RA synoval tissue fibroblasts we also analyzed RA synovial fluid fibroblasts for Egr-1-encoding mRNA. In six of eight primary cultures of synovial fluid fibroblasts from RA patients elevated Egr-1-encoding mRNA levels were found. Whenever synovial fluid and synovial tissue fibroblasts were obtained in sufficient amounts for analysis (four of four), primary culture RA synovial fluid fibroblasts transcribed Egr-1 mRNA at elevated levels comparable to synovial tissue-derived fibroblasts (Table 1Go). By contrast, four Epstein–Barr virus B cell lines established from HLA-DR4+ RA patients failed to show Egr-1 transcripts (data not shown).


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Table 1. Egr-1 transcription in fibroblasts
 
We then expanded the fibroblast-like cells from primary cultures and analyzed spontaneous Egr-1 steady-state mRNA levels in early passage fibroblasts (passage < 4). In eight of 10 cases we found higher Egr-1 mRNA levels in RA synovial fibroblasts than in the control fibroblasts. We confirmed that the elevated Egr-1 mRNA levels are a specific feature of RA synovial fibroblasts by analyzing synovial fibroblast isolates from different chronic inflammatory diseases such as reA, psoriatic arthritis, ancylosing spondylitis or osteoarthritis cultured under identical conditions. In these cases we did not detect elevated Egr-1-encoding transcripts (Table 1Go) nor did fibroblasts isolated from the synovial membrane of a healthy donor exhibit elevated expression of Egr-1-encoding mRNA (Table 1Go). However, low levels of Egr-1 transcription could be detected in these cells by extended exposure (not shown).

In a second set of experiments primary culture RA synovial fibroblasts were expanded and Egr-1 protein expression was detected by immunoblot using an antiserum specific for the C-terminal domain of the human Egr-1 (Fig. 3Go). Egr-1 protein was detected in five of seven early passage RA synovial fibroblasts (passage 1–4) (Fig. 3Go). The Egr-1 protein was not detected in two of the RA synovial fibroblasts. Staining of the membrane with Coomassie blue dye showed that comparable amounts of protein were loaded in each lane (Fig. 3Go). The data document for the first time that the Egr-1 protein was detected in early passage RA synovial fibroblasts and confirmed the experiments which documented elevated Egr-1 steady-state mRNA levels by in situ hybridization (9) or Northern blotting (Fig. 2Go and Table 1Go). Histological analysis and staining with the fibroblast-specific mAb F15-42-1 and mAb AS02 confirmed that the cells tested belong to the synovial fibroblast population (Fig. 4Go). Therefore, in contrast to previous reports (9,15), this study for the first time shows directly that the Egr-1high synoviocytes derived from RA synovial tissue or synovial fluid belong to the fibroblast lineage. Since elevation of Egr-1 expression was found in cells isolated from early stages of RA and from long-term patients under different therapeutic regimen, our data suggest that elevated expression of Egr-1 is specific for disease and neither due to the influence of external factors nor to the presence of RF (Table 2Go).



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Fig. 3. Detection of Egr-1 protein in RA synovial fibroblasts. Primary culture or early passage RA synovial fibroblasts were grown to confluence in complete medium containing 10% FCS. Cells were harvested and lysed in buffer containing NP-40 and PMSF. Proteins were separated by SDS–PAGE, and Egr-1 protein was detected by immunoblotting using anti-Egr-1 serum C-19 (Santa Cruz) followed by peroxidase-labeled goat anti-rabbit reagent (Dianova) and developed by ECL reagents. Egr-1 protein was detected in five out of seven RA synovial fibroblast extracts (top). Direct Coomassie blue staining of the membrane documents comparable amounts of proteins in each lane (bottom).

 


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Fig. 4. Characterization of primary culture synovial fluid fibroblast-like cells from a RA patient. Adherent cells were grown, washed with cold PBS and harvested by EDTA/PBS omitting trypsin. Samples of 105 cells were stained with the following reagents: mAb F15-42-1, anti-CD11b or anti-CD3. All cells significantly stained for the fibroblast-associated 26 kDa antigen, detectable by staining with mAb F15-42-1. Macrophages or T cells were not detected.

 

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Table 2. Egr-1 expression in relation to disease parameters and therapy in RA patients
 
Quantification of early growth response gene transcripts ex vivo and in vitro in RA synovial fibroblasts
A direct comparison of Egr-1-encoding steady-state mRNA in freshly isolated RA synovial tissue and parallel in primary culture RA synovial fibroblasts from the same patient by Northern blot was limited due to numbers and size of biopsies. Since activation of Egr-1 transcripts may have occurred at least partially during expansion of fibroblasts by in vitro culture, we developed a quantitative PCR approach to analyze steady-state mRNA levels encoding Egr-1 in cDNA prepared directly from RA synovial biopsies in comparison to primary culture synovial fibroblasts. RNA from snap-frozen samples of synovial tissue of an RA patient was analyzed by RT-PCR for Egr-1 transcripts. A dilution series ranging from 1 µg to 3.3 ng of cDNA was subjected to PCR amplification in the presence of increasing amounts of internal standard DNA (1–300 pg/ml; Fig. 5AGo). Co-amplification of cDNA and internal standard DNA resulted in comparable band intensities at 10 pg/ml. Evaluation of the signal intensities revealed a relative frequency of Egr-1-encoding mRNA molecules within the total mRNA pool of 4x10–5 (Fig. 5AGo). When a synovial explant from an OA patient was analyzed for transcripts of Egr-1-encoding message, a 100 times lower relative frequency (4x10–7) was found (not shown). RNA isolated from primary culture synovial fluid fibroblasts obtained by arthroscopy prior to surgery from the same RA patient revealed a frequency of 1.2x10–5 Egr-1 transcripts (not shown). In non-RA synovial fibroblasts and skin fibroblasts of RA patients serving as controls, Egr-1 transcripts were quantified at 4x10–6 or lower (Fig. 5BGo). RA skin fibroblasts were induced for transient Egr-1 transcription by addition of 20% FCS and served as positive control. Egr-1 transcript frequencies of 1.2x10–5 or more were detected (not shown). The relative cDNA frequencies were calculated as described recently (34).



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Fig. 5. (A) Analysis of Egr-1-encoding mRNA in RA synovial tissue by quantitative RT-PCR. RNA was extracted from freshly isolated RA synovial tissue. cDNA was generated from 10 µg of total RNA. Samples of a serial 1:3 dilution of aliquots of the cDNA (1.0 µg to 3.3 ng) were co-amplified in the presence of an inverse serial dilution of internal standard ranging from 1 to 300 pg/ml (25–7500 fg total DNA). Equal signals were observed in the reaction containing 100 ng RNA equivalent and 250 fg internal standard indicating that 1:25,000 cDNA molecules represented a Egr-1 gene product. This corresponds to a relative mRNA frequency of 4x10–5. The relative mRNA frequencies in the different samples were computed as described (34). (B) Analysis of Egr-1-encoding mRNA in reA primary culture synovial fibroblasts by quantitative RT-PCR. RNA was extracted from primary culture synovial tissue fibroblasts of a reA patient. RNA extraction, cDNA synthesis and quantitative PCR were performed as described in (A). Only a weak signal was obtained in the reaction representing 1 µg RNA equivalent and 25 fg internal standard. The relative frequency of Egr-1-encoding mRNA therefore is <4x10–6.

 
In RA synovial tissue or synovial fibroblasts only low amounts of c-fos-encoding mRNA were detected by Northern blot (Fig. 2Go). We therefore quantified the c-fos-encoding message by a more sensitive RT-PCR in RA synovial fibroblast-like cells. Message coding for c-fos was determined in RA synoviocytes ex vivo and in primary cultures at relative frequencies of <1.4x10–6 to 1.4xl0–5. Primary culture fibroblasts from reA patients transcribed c-fos mRNA at distinctly lower levels. Again, FCS-stimulated fibroblasts served as positive controls and transcribed c-fos-encoding mRNA up to 100-fold higher, i.e. in the range of 2–3x10–4, when compared to the respective not-induced controls (2x10–6). In quiescent skin fibroblasts steady-state mRNA frequencies encoding c-fos were below the signal range of the internal standard DNA as analyzed by quantitative RT-PCR (not shown).

Taken together, these data document (i) that in RA synovial tissue in comparison to the corresponding primary culture cells the steady-state Egr-1 mRNA levels correlate well and that c-fos was not transcribed at a similar elevated level as observed for Egr-1, (ii) that the expression of c-fos in resting primary culture RA fibroblasts was less prominent than the expression of Egr-1, and (iii) that primary culture synoviocytes of non-RA patients express only basal levels of c-fos and Egr-1 mRNA. Characteristically, high spontaneous expression levels of transcription factor Egr-1 were found only in RA fibroblast samples (Figs 2–5GoGoGoGo).

Induction of Egr-1 and c-fos transcripts by RA-conditioned medium
Freshly isolated RA synovial fibroblasts expressed Egr-1 at elevated levels. However, in most cases the elevated steady-state mRNA levels encoding Egr-1 faded over four to six passages in RA synovial fibroblasts, whereas the steady-state mRNA levels encoding c-fos transcripts were gradually increasing with time in culture. We speculated that supernatants from fresh or early passage RA synovial fibroblast cultures contained factors which preferentially promote Egr-1 expression but induced c-fos gene products at lower rates. Therefore, we analyzed induction kinetics for Egr-1 and c-fos transcripts. Addition of FCS to Egr-1low RA skin fibroblasts (derived from RA patient GSE, Table 1Go) induced maximal transcription of c-fos at 30–60 min; maximal Egr-1 transcription was found 60–90 min after stimulation (data not shown). Thus, a 60 min induction time was chosen to study activation kinetics of both genes. When the RA skin fibroblasts were activated with their own supernatants harvested at earlier passages neither Egr-1 nor c-fos transcription were induced (Fig. 6Go). In contrast, supernatant collected from RA synovial tissue fibroblasts and synovial fluid fibroblasts of the same patient (GSE) transiently induced strong Egr-1 but weak c-fos transcription. It is important to note that the synovial fibroblast supernatant did not induce a sustained Egr-1 transcription since 4 h after induction Egr-1 transcripts were reduced again to normal levels (Fig. 6Go). To demonstrate that the differences between these Egr-1 and c-fos mRNA signals result from transcriptional activation due to different promoter activities of these genes, we Egr-1 and c-fos promoters compared in a promoter reporter assay (Fig. 7Go). Since in native synovial fibroblasts the sensitivity of the luciferase assay was too low for the Egr promoter analysis SV40 TAg-immortalized synovial fibroblasts were used. In these immortalized cells the luciferase synthesis was sufficient for Egr-1 promoter analysis. SV40 TAg expression did not induce or repress Egr-1 or c-fos expression in all synovial fibroblast lines tested [n = 4 (34)]. Egr-1 promoter activity was induced 3- to 8-fold above the activity obtained in quiescent cells upon addition of supernatant from primary culture synoviocytes of an Egr-1high RA patient (SOZ). This patient had recently developed RA, showed active synovitis (24) and did not receive any anti-rheumatic treatment before obtaining a biopsy specimen (Tables 1 and 2GoGo, and Fig. 7Go). As expected from the Northern blot and RT-PCR experiments, induction of c-fos promoter activity in quiescent fibroblasts after addition of RA-conditioned medium showed only an overall increase of ~2- to 3-fold over baseline activity. Comparable induction levels were obtained for the Egr-1 and c-fos promoter using other SV40 TAg-expressing synovial fibroblasts and the same conditioned medium from patient SOZ (data not shown). These data complement well the short time induction experiments (Fig. 6Go). We conclude that in contrast to other stimuli (e.g. FCS), RA synovial fluid or RA synovial fibroblast-conditioned medium contain factors which preferentially induce Egr-1 but only to a lesser extent c-fos transcription in fibroblasts.



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Fig. 6. Egr-1 transcription is significantly but transiently induced by RA synovial cell-conditioned medium. Egr-1low skin fibroblasts of RA patient GSE were grown in medium containing 10% FCS and analyzed for spontaneous steady-state mRNA levels encoding Egr-1 (upper panel, lane 1) and for Egr-1 transcription upon stimulation with 20% FCS (lanes 2 and 6), after incubation with conditioned medium of autologous skin fibroblasts (lanes 3 and 7), with autologous primary culture synovial tissue fibroblast supernatant (lanes 4 and 8) and by autologous primary culture synovial fluid fibroblasts (lanes 5 and 9). RNA was isolated after 1 h (lanes 2 –5) and 4 h (lanes 6–9) stimulation, and analyzed for transcription of Egr-1 (top) and c-fos (below) by Northern blot analysis. Reprobing of the blot with ß-actin cDNA served as internal control (bottom).

 


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Fig. 7. Analysis of Egr-1 and fos promoter activity in SV40 TAg-immortalized human synovial fibroblasts. Cells were seeded in six-well plates and co-transfected with RSV-HGH reporter plasmid and either with Egr-1 wild-type promoter reporter (on the left) or with c-fos wild-type promoter reporter plasmids (on the right). Each set of cells was incubated with medium containing 0.5% FCS, 5% FCS or 5% FCS enriched by addition of 1 volume of primary culture RA synoviocyte-conditioned medium (patient SOZ). Luciferase activity was determined in each sample and corrected by a factor using the expression of HGH. The graph shows a representative experiment using SV40 TAg-immortalized synovial fibroblasts of patient HSE (Table 2Go). Co-transfection with pGL-2 basic reporter plasmid served as control.

 
Analysis of Egr-1 promoter activity in SV40 TAg-transformed synovial fibroblast-like cells
To identify the promoter elements that regulate the Egr-1 promoter activity in synovial fibroblasts we generated a set of luciferase reporter vectors based on the pGL-2 basic plasmid equipped with the promoter region of the human Egr-1 gene. To characterize the functional role of the different transcription factor binding sites individual sites were deleted by in vitro mutagenesis (Fig. 1Go). SV40 TAg-immortalized human synovial fibroblasts (23) were co-transfected with the respective Egr-1 promoter reporter and the HGH expression vector as internal standard. After transfection the cells were incubated in complete medium containing 10% FCS for 2 days, and HGH activity was determined to standardize transfection efficiency and cell density in each well. For the Egr-1 wild-type promoter activity, we found a 14-fold increase of luciferase activity over pGL-2 basic background levels (P < 0.001, Fig. 8Go). Deletion of the proximal Egr-1 binding site ({delta}EBS2) did not change the promoter activity, whereas deletion of both Egr-1 binding sites reduced the Egr-1 promoter activity by ~30% ({delta}EBS1+2). When the fos/jun (AP-1) binding site in the Egr-1 promoter was deleted, luciferase activity was reduced to 83% ({delta}AP1). The mutation missing the distal cAMP binding site enhanced the Egr promoter activity ({delta}cAMP1). None of these mutations, however, showed highly significant changes of Egr-1 promoter activity (Fig. 8Go). Most interestingly, removal of the proximal cAMP RE (position –130) showed an increase of Egr-1 promoter activity (197%, P = 0.173, {delta}cAMP2, Fig. 8Go). Deletion of the NF{kappa}B-like element at position –200 also activated the luciferase expression (130%, P = 0.477, {delta}NF{kappa}B, Fig. 8Go). As expected from the Egr-1 induction experiments using FCS (Fig. 6Go) the SRE contributed activation signals for Egr-1 transcription; thus, Egr-1 promoter activity was reduced to 65% in the deletion mutant missing the distal SRE (position –400, {delta}SRE1). Deletion of a cluster of three SRE at position–320 to –380 upstream of the transcription start ({delta}SRE2–4) reduced promoter activity to 20% (P < 0.001) and deletion of the proximal SRE to 30% (P < 0.001, {delta}SRE5, Fig. 8Go). Expression of SV40 TAg probably did not bias the luciferase assay since in murine fibroblasts comparable patterns of Egr-1 promoter activation were observed (unpublished results). In addition, mobility shift assay using nuclear extracts from native and SV40 TAg-immortalized synovial fibroblasts resulted in identical binding patterns in SV40 TAg-expressing and native synovial fibroblasts (Fig. 9Go). In summary, our data suggest that factors binding to the SRE are the major factors activating Egr-1 in synovial fibroblasts. Binding of transcription factors at the cAMP RE and at the NF{kappa}B-like element cause reduction of Egr-1 transcription. Interestingly, binding of AP-1 proteins to the Egr-1 promoter may activate Egr-1 transcription in synovial fibroblasts. The regulatory effect of binding of the Egr-1 zinc-finger protein to the Egr-1 promoter itself seems to activate expression.



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Fig. 8. Analysis of Egr-1 promoter activity in human synovial fibroblasts. SV40 TAg-immortalized synovial fibroblasts K4IM (23) were seeded in six-well plates and transiently transfected with 2 µg/well pGL-2 reporter plasmid, reporter plasmid containing the human Egr-1 wild-type promoter or the different deletion mutants. HGH expression vector at 2 µg/well was co-transfected as internal standard. Cells were incubated in normal medium containing 10% FCS. After 48 h incubation, HGH activity was determined in the supernatant to standardize the different transfections, at the same time Egr-1 wild-type promoter activity or promoter activities delivered by the deletion mutants were determined in the cytoplasmic extracts. Luciferase activity observed by Egr-1 wild-type promoter was set as 100% of relative promoter activity and effects of mutating the Egr-1 promoter on total luciferase production were computed accordingly. The data represent the calculated average of three independent experiments. It can be seen that deletions of the SRE greatly diminished luciferase activity, suggesting that these binding sites are essential for enhancement of Egr-1 promoter activity. By contrast, deletion of the cAMP and NF{kappa}B-like binding sites increased Egr-1 promoter activity, suggesting that these sites serve for reduction of the Egr-1 promoter activity.

 


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Fig. 9. Mobility shift assay using SRE. Nuclear extracts were prepared from normal K4IM (lanes 1 and 9) or serum stimulated K4IM cells (lanes 2–4 and 10 –12) and RA early passage synovial fibroblasts (lanes 5–8). Extracts were incubated with radiolabeled consensus SRE oligonucleotide (lanes 1–8) or Egr-1 promoter-derived SRE (lanes 9–12). For competitive inhibition of the interaction 0.5 or 2 µl cold Egr-1 promoter-derived oligonucleotide was added in lanes 3, 4, 6 or 8, and cold consensus SRE oligonucleotide was added in lanes 11 and 12, as indicated. A specific signal was detected with extracts from normal and serum-activated cells (lanes 1, 2, 5, 7 and 9–11). Addition of cold oligonucleotides reduced the signals of the consensus SRE binding (left) and Egr-1 promoter-derived SRE (right). SV40 TAg-expressing K4IM showed a shift pattern (lanes 1–4 and 9–12) comparable to native synovial fibroblasts RA1 and RA2 (lanes 5–8).

 
Mobility shift assay of Egr-1 promoter binding proteins
To document specific promoter–transcription factor interactions at the SRE and at the novel NF{kappa}B-like element in the human Egr-1 promoter we performed mobility shift assays (Figs 9 and 10GoGo). Nuclear extracts from immortalized and native synovial fibroblasts were prepared and incubated with radiolabeled oligonucleotides corresponding to a consensus SRE or consensus NF{kappa}B site (Santa Cruz). In parallel, the nuclear extracts were also incubated with oligonucleotides derived from a SRE (position –381) and the NF{kappa}B-like binding site (position –221) in the human Egr-1 promoter (Fig. 1Go). Mobility shift assay showed a specific interaction of K4IM nuclear proteins with the consensus SRE (Fig. 9Go, lanes 1 and 2). The signal was reduced by addition of non-labeled Egr-1 promoter-derived SRE oligonucleotides (Fig. 9Go, lanes 3 and 4). Using native early passage RA synovial fibroblasts, the same mobility shift signals were obtained (Fig. 9Go, lanes 5–8). Radiolabeled consensus SRE generated shift signals (Fig. 9Go, lane 5 and 7) and addition of cold Egr-1 promoter-derived SRE oligonucleotides reduced the signal (Fig. 9Go, lanes 6 and 8). Radiolabeled SRE oligonucleotides derived from the Egr-1 promoter incubated with nuclear extracts from K4IM resulted in prominent shift signals as well (Fig. 9Go, lanes 9 and 10). Addition of excess amounts of cold consensus SRE to labeled SRE from the Egr-1 promoter reduced the shift signals confirming that both oligonucleotides interacted with the same nuclear proteins (Fig. 9Go, lane 12). Incubation of the Egr-1 promoter-derived NF{kappa}B-like oligonucleotide with K4IM nuclear extracts showed a mobility shift pattern which was not identical to the signals obtained with the consensus NF{kappa}B DNA (Fig. 10Go). Addition of a surplus of the consensus NF{kappa}B oligonucleotide to the Egr-1 promoter-derived radiolabeled NF{kappa}B-like nucleotide did not influence the shift signals confirming that different proteins interacted with the respective DNA elements. Using consensus NF{kappa}B oligonucleotides a prominent mobility shift was seen in K4IM synovial fibroblasts (Fig. 10Go). Compensation with the Egr-1 promoter-derived NF{kappa}B-like oligonucleotide did not weaken this signal indicating that different proteins interacted with the consensus NF{kappa}B site and the Egr-1 promoter-derived NF{kappa}B-like binding site. Activation of the cells by preincubation with TNF-{alpha} resulted in identical mobility shift signals. Signal intensities or electrophoretic mobility were not different in activated cells when compared to non-induced controls (not shown). A detailed analysis of the protein complexes interacting with this NF{kappa}B-like element is beyond the scope of this study and is part of future investigations.



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Fig. 10. Mobility shift assay using SRE. Nuclear extracts were prepared from synovial fibroblasts K4IM (lanes 1–8). The nuclear extracts were incubated with radiolabeled NF{kappa}B-like oligonucleotides derived from the Egr-1 promoter (lanes 1–4) or consensus NF{kappa}B binding site oilgonucleotides (lane 5–8). A shift signal was obtained with the Egr-1 promoter-derived NF{kappa}B-like binding site (lanes 1 and 5) which was not compensated by oligonucleotides representing a consensus NF{kappa}B site (lanes 2–4). The consensus NF{kappa}B oligonucleotide also generated specific signals (lanes 5–8), which differed in mobility from the Egr-1 promoter-derived NF{kappa}B-like oligonucleotide–protein complex (lanes 1–4) and was not reduced by addition of a surplus of Egr-1 promoter-derived NF{kappa}B-like oligonucleotide (lanes 6–8).

 
In summary, the Egr-1 promoter reporter analysis and the mobility shift assay showed that in the synovial fibroblasts transcription of Egr-1 is regulated by SRE, cAMP RE and a novel NF{kappa}B-like element. For activation of Egr-1 transcription the SRE are the most important segments since deletion of SRE reduced Egr-1 promoter activity significantly (Fig. 8Go). Interaction of nuclear proteins with a consensus SRE and an Egr-1 promoter-derived SRE confirmed that this regulatory complex is active in synovial fibroblasts (Fig. 9Go). Egr-1 transcription is negatively regulated by a cAMP RE at position –130 and a novel NF{kappa}B-like element since deletion of cAMP RE or the NF{kappa}B-like element at position –200 activated the Egr-1 promoter (Fig. 8Go). Mobility shift assay confirmed a specific interaction of nuclear proteins with this NF{kappa}B-like promoter element, which most probably is not identical to the consensus NF{kappa}B (Fig. 10Go). Finally, the mobility shift assay signals did not differ in SV40 TAg-expressing K4IM versus native synovial fibroblasts, suggesting that at least with respect to main regulatory factors, the regulation of Egr-1 transcription was not biased by SV40 TAg.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Activation of synovial fibroblasts, their elevated expression of cytokines, matrix proteases and the formation of the hypertrophic RA synovium is a characteristic hallmark of RA (1). These fibroblasts contribute to the tissue degradation process which is underlined by reports that demonstrate articular cartilage destruction also in the absence of macrophages and/or T cells (8,3840). Therefore, activation of synovial fibroblasts seems to be highly important for matrix degradation in RA. Comparable conclusions were drawn from histological examination of synovial tissue of early stage RA patients (41).

As a characteristic indicator for the stage of activation of the synovial fibroblasts in RA, we focused on the analysis of Egr-1 and c-fos transcription. Egr-1 and c-fos are among the first genes transcribed after stimulation by a variety of different growth signals (42) or cytokines (43,44). Using synovial tissue and synovial fluid fibroblasts of RA patients we could demonstrate directly that synovial fibroblasts expressing surface markers characterized by the F15-42-1 or AS02 antibodies (Fig. 4Go) express Egr-1 at elevated levels. These results extend our previous studies that showed elevated Egr-1 steady-state mRNA levels in joints of RA patients (9). High Egr-1 mRNA levels contrast with c-fos mRNA levels which were found to be low in primary cultures or early passages of RA synovial fibroblasts (Fig. 2Go). This difference in Egr-1 and c-fos steady-state mRNA levels argues against unspecific activation of Egr-1 by cell isolation or culture conditions. By in situ hybridization experiments we found previously that c-fos was activated only in synovial lining cells located at the immediate site of synovial invasion into cartilage and bone (15,19,20). The majority of synoviocytes, including fibroblasts, throughout the hyperplastic RA synovium did not show reactivity with anti-Fos antibodies (15). Therefore, c-fos seems not to be expressed homogeneously throughout the synovium. By contrast, synoviocytes transcribing Egr-1 at elevated levels were found at different sites including the deeper layers of the synovium but not in lymphoid infiltrates (9). In addition, as the synovial lining layer comprises macrophage-like cells and fibroblast-like synoviocytes (3), it cannot be excluded that the jun/fos staining as detected by in situ hybridization (20) is mainly also derived from macrophage-like cells. Based on primary culture synoviocytes stained with mAb (F15-42-1 and AS02) in combination with Northern blot and RT-PCR analysis steady-state mRNA levels encoding these IE genes, we conclude that Egr-1 is predominantly activated in fibroblast-like synoviocytes. This concept is supported by the induction experiments in resting skin fibroblasts (Fig. 6Go). Using autologous skin fibroblast supernatants, elevation of Egr-1 or c-fos transcription was not seen (Fig. 6Go). Interestingly, using culture supernatants from autologous RA primary synovial fibroblasts a significant induction of Egr-1 was seen, whereas only weak signals encoding c-fos mRNA were detected (Fig. 6Go). The induction of Egr-1 was transient, since 4 h after stimulation Egr-1 transcription returned to baseline levels. The differences of transcriptional response to RA-conditioned medium are most probably based on differential binding of transcription factors to the Egr-1 and c-fos promoters respectively (11,45) (Fig. 1Go). In the Egr-1 promoter, a cluster of three SRE and two additional SRE are located at some distance to other transcription factor binding sites (11) (Fig. 1Go). By contrast, in the c-fos promoter a single SRE adjoining the AP-1 binding site is found (45). Therefore moderate activation of SRF-dependent signaling pathways may be responsible for the induction differences seen for Egr-1 and c-fos transcripts upon stimulation with RA-conditioned medium (Fig. 6Go); irrespective of the fact that both genes respond well to FCS stimulation (Fig. 6Go). Although Fos may down-regulate transcription of Egr-1 (46,47) our experiments show that in synovial fibroblasts engagement of transcription factors at AP-1 (TRE, fos/jun binding site) failed to modulate strongly Egr-1 promoter activity (Fig. 8Go). Since elevated levels of c-fos-encoding mRNA were not present in RA synovial fibroblasts (Fig. 2Go) nor induced by RA-conditioned medium at higher rates (Figs 6 and 7GoGo) the Fos protein actually cannot be considered a major factor for Egr-1 regulation. Nevertheless, promoter reporter analysis showed a moderate fos activation in response to RA-conditioned medium (Fig. 7Go). The RA synovial lining additional factors including extracellular matrix and/or adjacent cells should be taken into account for the enhanced induction of the c-fos transcription as found in situ.

The luciferase reporter analysis of the Egr-1 promoter and the mobility shift assay clearly showed that the principle Egr-1 induction is mediated by factors binding to the SRE, possibly the p68 SRF proteins, since deletion of the SRE significantly reduced (P < 0.001) Egr-1 promoter activity. A corresponding oligonucleotide showed a strong signal in the mobility shift assay (Figs 8 and 9GoGo). The organization of the Egr-1 promoter suggests that three clusters of transcription factor binding regions control the Egr-1 gene expression (Fig. 1Go); the SRE elements are concentrated in the proximal region (–100 to –200, SRE5) and at –300 to –400 of the transcription start. Activation of SRE binding proteins, e.g. p68 SRF, requires phosphorylation and dimerization, which again depends on signals transmitted by a kinase/phosphatase cascade (48). Phosphatase inhibitors induced a sustained Egr-1 transcription and prolonged half-life time of Egr-1 mRNA in fibroblasts (49). Activation of PKC by addition of phorbol ester induced Egr-1 expression in synovial fibroblasts (unpublished results). Phosphorylation of Egr-1 protein modulates nuclear transport, transcription factor activity and DNA binding of Egr-1 (50,51). Cytokines which use this signal transduction pathway and may induce Egr-1 expression include epidermal growth factor, fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) (43,44,48,5254). Our findings in the context of these reports raise the question whether extended autocrine activation of Egr-1 in RA synovial fibroblasts was induced by soluble factors including cytokines such as FGF or PDGF. Binding sites for Egr-1 were found in the promoter regions of these cytokine genes or their corresponding receptor genes (55). The Egr-1 induction experiments using RA-conditioned medium support this hypothesis (Figs 6 and 7GoGo). Therefore it cannot be excluded that the elevated expression of Egr-1 in RA synovial membrane and primary culture fibroblasts is the result of ongoing re-stimulation which ceases during cell passages due to dilution effects or differentiation of the fibroblasts. Alternatively, synovial macrophages may secrete factors which induce Egr-1 by paracrine mechanisms. Since synovial macrophages have a limited live span and are not proliferating in vitro, paracrine induction of IE genes such as Egr-1 will be thinned out over time. However, macrophages or T cells were not detected in primary culture RA synovial fibroblast populations (Fig. 4Go) and in situ Egr-1 was not expressed at the rim of myeloid or lymphoid infiltrates in RA synovial biopsies (9). Therefore, an autocrine Egr-1 stimulation mechanism seems to be more likely. The nature of this Egr-1 induction factor has not been analyzed to date. Since in fibroblasts PDGF and FGF rather induce collagen synthesis but not protease expression, overexpression of Egr-1 in synovial fibroblasts may compensate the effects that are induced by other proto-oncogenes including fos, ras and myc, which are known to primarily induce protease expression in fibroblasts (6,15,56). Along those lines, it is of interest to note that preliminary data obtained recently in our laboratory indicate that Egr-1 plays an important role in activation of metalloproteinase inhibitor TIMP-1 but failed to activate expression of cathepsin L or metalloproteinases (manuscript in preparation).

One of the factors clearly contributing to the matrix degradation pathway is macrophage-derived tumor necrosis factor (TNF)-{alpha}, which was shown to induce collagenase expression in fibroblasts (57) and promote an RA-like disease in mice expressing human TNF-{alpha} as transgene (38). Activation of Egr-1 via protein kinase C (PKC)-dependent phosphorylation of p68 SRF may be able to compensate for TNF-{alpha}-induced collagenase expression in synovial fibroblasts. Indeed PKC activation was shown to reduce the sensitivity of cells to TNF receptor signaling (58,59). Although the deletion of the NF{kappa}B-like element did not show a highly significant activation of the Egr-1 promoter, the mechanisms of NF{kappa}B- and/or TNF-{alpha}-dependent Egr-1 regulation requires more detailed studies, especially in the light of Egr-1 induction studies in T cells using recombinant tax protein, which interfered with the NF{kappa}B signal transduction and activated maximal Egr-1/Z225 transcription (12). A direct interaction of nuclear proteins extracted from synovial fibroblasts K4IM documented that indeed a specific promoter–transcription factor interaction occurs at this site in the human Egr-1 promoter, since a weak but specific signal was obtained in a mobility shift assay (Fig. 10Go). The weak signal obtained by mobility shift assay may also explain in part the regulatory effects at the NF{kappa}B-like site in comparison to the Egr-1 wild-type construct. As determined by promoter reporter analysis, the deletion of the NF{kappa}B-like site resulted in a somewhat variable activation of Egr-1 promoter activity (Fig. 8Go) which was not statistically highly significant (130%, P = 0.477). However, in murine fibroblasts a comparable pattern of promoter activation was seen using the {delta}NF{kappa}B-like construct in comparison to the Egr-1 wild-type plasmid (unpublished results). Interestingly, the protein or the protein complex binding to the NF{kappa}B-like element is not exactly identical with NF{kappa}B, since shift signals were detected at different heights and the binding was not mutually competitively inhibited using the respective oligonucleotides. Detailed analysis of this NF{kappa}B-like transcription complex must await future experiments.

As seen with the deletion mutant of the NF{kappa}B-like element, the deletion of the cAMP RE increased Egr-1 promoter activity in synovial fibroblasts indicating that in these cells cAMP RE binding protein (CREB) represses Egr-1 expression (Fig. 8Go). Activation or inactivation of adenylate cyclase which determines the cytoplasmic cAMP levels is dependent of G-proteins coupled to the respective receptor. Therefore, cytokines utilizing such cAMP-dependent pathways may act as negative regulators for Egr-1. By contrast, in macrophages it was recently shown that cAMP induced activation of Egr-1 (60), whereas in PC12 cells cAMP did not induce Egr-1 but rather induced c-fos (61). Thus the role of increased cAMP levels for regulation of Egr-1 in synovial fibroblasts and macrophages also awaits further analysis.

In summary, we show that in the RA synovium and in primary synovial tissue and synovial fluid fibroblasts elevated steady-state Egr-1-encoding mRNA levels are found when compared to non-RA synovial fibroblasts, synovial fluid-derived macrophages or lymphoid cells. Elevated amounts of Egr-1 protein were detected in RA synovial fibroblasts. Induction experiments suggest a autocrine stimulation mechanism of elevated Egr-1 transcription in RA. Paracrine mechanisms may also contribute to the activation of the Egr-1 expression. The enhanced Egr-1 expression may be used as a specific diagnostic marker to characterize synovial fibroblasts of RA patients. The overexpression of Egr-1 fades away after extended passages of the cells but it can be transiently re-induced by factors present in RA synovial fluid and supernatants harvested from primary cultures of RA synovial fibroblasts. In contrast to Egr-1, induction of c-fos by RA-conditioned medium was less prominent, which corresponds well with the low c-fos expression in primary culture synovial fibroblast. The major Egr-1-activating protein in synovial fibroblasts is p68 SRF, whereas the cAMP RE and the NF{kappa}B-like binding site serve as negative regulators for Egr-1 expression.


    Acknowledgments
 
We thank Dr Gasson and Dr Sakamoto, UCLA, Los Angeles for the TIS8 plasmid, Dr Shaw, University of Nottingham for the c-fos reporter plasmid, Dr Küsswetter, CRONA, Tübingen for synovial tissue samples, Dr Selbmann for statistical evaluation and P. Fiedler for excellent technical support. The project was supported by DFG grant Ei 235/3-1, by a Hans-Hench Foundation fellowship to B. G. and by the Bode Foundation (W. K. A.).


    Abbreviations
 
AP-1activator protein 1, jun/fos binding site TRE
cAMP REcAMP responsive element
CREBcAMP response element binding protein
Egr- 1human early growth response gene 1
Egr BSEgr-1 binding site
FGFfibroblast growth factor
HGHhuman growth hormone
IE geneimmediate early growth response gene
OAosteoarthritis
PDGFplatelet-derived growth factor
PKCprotein kinase C
RArheumatoid arthritis
reAreactive arthritis
RFrheumatoid factor
SREserum response element
SRFserum response factor
TAgT antigen
TNFtumor necrosis factor

    Notes
 
Transmitting editor: K. Eichmann

Received 20 February 1998, accepted 29 September 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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