Introduction
Human rheumatoid arthritis (RA) is characterized by chronic inflammation and destruction of multiple joints, perpetuated by an invasive pannus tissue. Activated synovial fibroblasts (SFBs), whether irreversibly altered [
1] or reversibly stimulated by the inflammatory microenvironment [
2], are major components of the pannus and contribute to joint destruction by secretion of pro-inflammatory cytokines and tissue-degrading enzymes [
3].
Recently, microarray techniques employing hybridization of biological samples to immobilized cDNA probes or oligonucleotide probe sets (for example, Affymetrix
®) have been increasingly used to study genome-wide gene expression profiles and to perform initial screening for genes of potential pathogenetic interest. In the meantime, there are some studies available of differential gene expression between RA and osteoarthritis (OA) synovial membranes (SMs) [
4‐
6], RA and OA SFBs [
7] or about the effects of mediators with a central role in RA, for example, tumor necrosis factor-α and IL-1β, on SFBs [
8‐
10]. In order to identify sets of constitutively regulated genes that can be classified into well-known pathways, differential gene expression between early passage RA and OA SFBs was investigated using Affymetrix
® oligonucleotide arrays and analyzed using the Gene Set Enrichment Analysis (GSEA) tool [
11]. The differential expression of such pathway components in RA and OA SFBs may then indicate a more pronounced potency for further activation by the respective cytokines or growth factors, for example, transforming growth factor (TGF)-β. To enhance the significance of the array analysis, the mRNA data of the most important molecules were validated by real-time reverse transcriptase (RT)-PCR and the respective proteins were analyzed by western blots or immunohistochemistry. In addition, stimulation of SFBs with TGF-β1 was performed to prove the functional relevance of the enhanced expression of TGF-β pathway-related molecules in RA.
Materials and methods
Patients and samples
Synovial tissue was obtained from open joint replacement surgery or arthroscopic synovectomy at the Clinic of Orthopedics, Waldkrankenhaus "Rudolf Elle" (Eisenberg, Germany). Patients with RA or OA (
n = 6 each for gene expression analysis and further patients for validation experiments; total of 7 RA and 9 OA patients) were classified according to the American Rheumatology Association (ARA, now American College of Rheumatology (ACR)) criteria [
12] (Table
1). SFBs were purified from synovial tissue as previously published [
13]. Briefly, the tissue samples were minced, digested with trypsin/collagenase P, and the resulting single cell suspension cultured for seven days. Non-adherent cells were removed by medium exchange. SFBs were then negatively purified using Dynabeads
® M-450 CD14 and subsequently cultured over 2 passages in DMEM containing 100 μg/ml gentamycin, 100 μg/ml penicillin/streptomycin, 20 mM HEPES and 10% FCS (all from PAA Laboratories, Cölbe, Germany).
Table 1
Clinical data of patients
Patient | Gender/age (years) | Disease duration (years) | RF | ESR (mm/h) | CRP (mg/ml) | No. of ARA criteria | Concurrent treatment |
EB 73 | M/68 | 8 | + | 90 | 48.1 | 6 | MTX |
EB 74 | F/71 | 17 | + | 45 | 26.9 | 6 | NSAIDs |
EB 87 | F/65 | 12 | + | 50 | 106.7 | 5 | NSAIDs |
EB 88 | F/62 | 10 | + | 90 | 169.5 | 6 | NSAIDs |
EB 96 | M/67 | 4 | + | 68 | 75.7 | 4 | NSAIDs |
EB 108 | F/72 | 45 | + | 70 | 70.7 | 7 | NSAIDs, steroids |
EB 141 | F/73 | 1 | + | 57 | 14.3 | 5 | NSAIDs, steroids |
Osteoarthritis
| | | | | | | |
EB 77 | F/66 | 5 | - | 2 | <5.0 | 0 | None |
EB 81 | F/56 | 3 | - | 14 | <5.0 | 0 | None |
EB 90 | F/61 | 2 | - | 18 | <5.0 | 0 | None |
EB 102 | F/73 | 8 | - | 20 | <5.0 | 0 | NSAIDs |
EB 115 | F/56 | 3 | - | 11 | 8.2 | 0 | NSAIDs |
EB 118 | M/72 | 2 | - | 4 | 9.3 | 0 | NSAIDs |
EB 135 | M/57 | 4 | - | 12 | <5.0 | 0 | NSAIDs |
EB 165 | F/63 | 3 | - | 11 | 6.1 | 0 | NSAIDs |
EB 172 | M/61 | 3 | - | 5 | <5.0 | 0 | NSAIDs |
Culturing of cells and isolation of total RNA
At the end of the 2nd passage, the SFBs were starved with medium containing 1% FCS for 72 h to minimize stimulating effects by serum components. After washing with PBS, the cells were lysed with RLT buffer (Qiagen, Hilden, Germany) and frozen at -70°C. Total RNA was isolated using the RNeasy Kit (Qiagen) according to the supplier's recommendation.
Microarray data analysis
RNA probes were labeled according to the supplier's instructions (Affymetrix
®, Santa Clara, CA, USA). Analysis of gene expression was carried out using U95A oligonucleotide arrays. Hybridization and washing of gene chips was performed according to the supplier's instructions and microarrays were analyzed by laser scanning (Hewlett-Packard Gene Scanner). Background-corrected signal intensities were determined using the MAS 5.0 software (Affymetrix
®). Subsequently, signal intensities were normalized among arrays to facilitate comparisons between different patients. For this purpose, arrays were grouped according to patient groups (OA versus RA,
n = 6 each). The arrays in each group were normalized using quantile normalization [
14]. Original data from microarray analysis have been deposited in NCBIs Gene Expression Omnibus [
15] and are accessible through GEO series accession number GSE7669.
Gene set enrichment analysis
GSEA was performed using the software described in [
11]. Briefly, GSEA searches for the enrichment of up- or downregulated genes in pre-defined pathways and subsequently performs a correction for multiple-hypotheses testing. Pathways were ranked with respect to the score values (normalized enrichment scores), which indicate differential expression. GSEA was run with default settings by performing 500 random mutations for the determination of statistical significance. The pre-defined pathways contained two variants of the TGF-β pathway (called 'TGF_Beta_Signaling_Pathway' and 'tgfbPathway'). Both pathways were among the top 30 ranking pathways (out of 259). A merged pathway was created by combining the genes from the two pre-defined pathways (TGF_joint) and GSEA was re-run including this new pathway.
Quantitative real-time PCR analysis
cDNA was prepared from total RNA using oligo-dT primers and SuperScript reverse transcriptase (Invitrogen, Karlsruhe, Germany). For the genes of interest and the housekeeping aldolase gene, specific mRNA sequences were cloned using the TOPO-TA cloning kit (Invitrogen) and employed for the generation of external standard curves. Real-time PCR was performed on a LightCycler
® (Roche Diagnostics, Mannheim, Germany) using LightCycler
® FastStart DNA Master SYBR Green I (Roche) as previously described [
16] with the primer pairs presented in Table
2. The amount of cDNA in each sample was normalized using the expression of the housekeeping aldolase gene, which showed the lowest variability over all oligonucleotide arrays. The general amplification protocol (50 cycles) was set as follows: initial denaturation for 3 minutes at 95°C; denaturation for 5 s at 95°C; specific primer annealing temperature for 10 s; amplification at 72°C for the indicated time period (Table
2). The general settings for the melting curve protocol (1 cycle) were as follows: denaturation at 95°C; cooling to 5°C above the primer annealing temperature; heating to 95°C (speed 0.1°C/s); final cooling for 5 minutes at 40°C. The fluorescence emitted by double-stranded DNA-bound SYBR-Green was measured once at the end of each additional heating step and continuously during the melting curve program. The concentrations of cDNA present in each sample were calculated by the LightCycler
®-software using the external standard curves. Product specificity was confirmed by melting curve analysis and initial cycle sequencing of the PCR products.
Table 2
Primer and product sizes of real-time PCR validated genes
Aldolase | TCATCCTCTTCCATgAGACACTCTA | ATTCTgCTggCAgATACTggCATAA | 314 | 58°C/30 s |
TGF-β1 | gTTCAAgCAgAGTACACACAgC | gTATTTCTggTACAgCTCCACg | 157 | 60°C/20 s |
TGF-β2 | ATgCggCCTATTgCTTTAgA | TAAgCTCAggACCCTgCTgT | 185 | 60°C/20 s |
TGF-β3 | CAgggAgAAAATCCAggTCA | CCTggAAggCgTCTAACCAAg | 179 | 58°C/20 s |
THBS1 | gATCCTggACTCgCTgTAgg | CCgAgTATCCCTgAgCCCTC | 202 | 60°C/20 s |
TGFBR1 | ATCACCTggCCTTggTCCTgTgg | GgTCCTCTTCATTTggCACTCgATg | 140 | 54°C/20 s |
SkiL | CAgTggAAACTgATggAgAgC | ggAAgAggCAgAAATACAgTAgg | 193 | 55°C/20 s |
MMP-11 | ggTgTACgACggTgAAAAgCC | CAgggTCAAACTTCCAgTAgAgg | 353 | 64°C/30 s |
Western blotting
SFBs from RA patients (n = 6 for TGF-β1; n = 5 for TGF-β receptor (TGFBR)) and OA patients (n = 4) were cultured and starved as above. Cell lysis was performed after washing with PBS using NP-40 lysis buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM Na3VO4, as well as 1 μg/ml of aprotinin, leupeptin, and pepstatin). The protein content was determined using the BCA assay (Pierce, Rockford, IL, USA) following acetone precipitation of a 25 μl sample aliquot. Proteins were resolved by reducing SDS-PAGE of 40 μg lysate and subsequently detected by immunoblotting, using the following primary antibodies: anti-TGF-β1 (A75-2, BD Biosciences, Heidelberg, Germany), anti-TGFBR 1 (#3712, CellSignal, Beverly, MA, USA), as well as goat anti-mouse IgG horseradish peroxidase (HRP; A-3682, Sigma-Aldrich, Steinheim, Germany) or goat anti-rabbit IgG HRP (sc-2004, St Cruz Biotechnology, Heidelberg, Germany) as secondary antibodies. The blots were then stripped and re-probed with mouse anti-human β-actin (clone AC-15, Sigma-Aldrich, Deisenhofen, Germany) and goat anti-mouse IgG HRP to ensure equal loading. In the case of TGF-β1, attempts to quantify protein levels by ELISA in the supernatants of cultured SFBs were not successful, possibly due to its association with the extracellular matrix surrounding the cells.
Immunohistochemistry
SFBs from RA and OA patients (n = 3 each) were cultured and starved in chamber slides (104 cells per well) as above. After washing with PBS and fixing in 10% formalin in PBS for 10 minutes, the antigen was unmasked by treating the cells with citrate buffer (10 mM; pH 6.0 with NaOH, 0.05% Tween20) and heating for 5 minutes in a microwave oven (300 W). After cooling and washing with PBS, the slides were blocked with 5% goat serum in PBS for 30 minutes, followed by incubation for 30 minutes with the primary antibody (mouse anti-human thrombospondin (THBS)1, clone A6.1, LabVision c/o Dunn Labortechnik, Asbach, Germany) diluted at 4 μg/ml in 1% goat serum. HRP-conjugated rabbit anti-mouse IgG (in PBS/1% goat serum) was added for 30 minutes. The peroxidase was revealed using diaminobenzidine for 5 minutes, and the slides were washed and covered with Aquatex (Merck, Darmstadt, Germany). A mouse IgG1 monoclonal antibody (MOPC21, Sigma; 4 μg/ml) served as control and yielded negative results. Positively stained cells were scored semi-quantitatively by two observers (DP and RWK) in a blinded manner (0 = no; 1 = weak; 2 = medium; 3 = strong staining).
Stimulation with TGF-β1
SFBs from RA patients (n = 3) and OA patients (n = 4) were cultured and starved as above. Recombinant human TGF-β1 (Peprotech, London, UK) was added at 10 ng/ml for 4 h. After washing and lysing, RNA isolation, cDNA synthesis, and quantitative real-time PCR (qPCR) for aldolase and matrix-metalloproteinase (MMP)-11 were performed as described above. Protein expression of MMP-11 was assessed by intracellular staining of stimulated cells (10 ng/ml TGF-β1, 48 h) by flow cytometry on a FACScan cytometer (BD, Heidelberg, Germany). The cells were trypsinized, washed with PBS/1% FCS and fixed with 4% paraformaldehyde in PBS for 15 minutes at 4°C. After permeabilization with 0.5% saponin in PBS/1% FCS, the cells were incubated with anti-human-MMP-11 antibody (clone 135421, R&D Systems, Wiesbaden, Germany), followed by goat anti-mouse IgG FITC (Dako, Hamburg, Germany). A mouse-anti-keyhole limpet hemocyanin (KLH) antibody (IgG2b, clone 20116, R&D Systems) served as isotype control.
Statistical analysis
The non-parametric Mann-Whitney U test was applied for the comparison of differences between RA and OA in qPCR, western blots, immunohistochemistry, and flow cytometry assays. Statistically significant differences were accepted for p ≤ 0.05. For correlations between gene expression and clinical parameters, the Spearman Rank Test was used (p ≤ 0.01).
Discussion
The aim of the present study was to systemically analyze differentially expressed pathways in purified, early passage SFBs derived from RA and OA patients. For this purpose, gene expression was measured with oligonucleotide array technology and validated by other, low-throughput methods.
SFBs showed a differential, constitutive expression of genes involved in various cellular pathways (Table
3). Using the software tool GSEA for pathway scanning [
11], components of the TGF-β pathway were found to be over-represented among these genes. As well as TGF-β1 and its receptor TGFBRI, the following molecules were also upregulated in RA SFBs: LTBP1 and LTBP2, both components of the large latent TGF-β complex binding TGF-β to the extracellular matrix [
17]; THBS1, known to release active TGF-β from its latent form [
18]; and SARA, which recruits the TGF-β-signal-transducing smads to the membrane in the close vicinity of the receptor [
19] (Figure
1). This interesting finding provides detailed analysis of individual components of the TGF-β pathway and parallels recent reports on the existence of two distinct gene expression profiles in SFBs, one of which is characterized by the expression of TGF-β/activin A-inducible genes [
7]. Although hierarchical clustering of the data in the present study did not reveal such distinct profiles in purified SFBs (data not shown; possibly due to the low number of samples), the overexpression of TGF-β-related genes supports the importance of this pathway in synovial pathology. This is further underlined by significant correlations between the constitutive TGF-β1 and THBS1 mRNA expression in SFBs and the C-reactive protein levels or the number of fulfilled ARA criteria, that is, clinical markers of disease activity and/or severity.
TGF-β1 mRNA was expressed to a significantly higher degree in RA SFBs than OA SFBs, in parallel with previous reports showing a significantly higher expression of TGF-β1 in the RA SM than in the OA SM [
20‐
22]. Specific assignment of TGF-β1 production to fibroblast-like synoviocytes in the RA SM [
21] or to fibroblasts in synovial regions with pronounced fibrosis provides evidence for a pro-fibrotic role of TGF-β1 in RA [
22]. Also, the expression of TGF-β1 directly at the cartilage-pannus junction during the most severe phase of rat collagen-induced arthritis [
23] suggests TGF-β1 has an important pro-destructive role in experimental arthritis.
The observed discrepancy between the expression of TGF-β1 mRNA and protein in SFBs may be due to the fact that blot analysis with the present monoclonal antibody underestimates the total amount of protein by detecting only the active form of TGF-β1. In fact, newly synthesized TGF-β1 is predominantly secreted as the latent proform, as also observed in irradiated rat mesangial cells [
24]. On the other hand, known post-transcriptional regulation of the TGF-β1 gene
via the 5' untranslated region may prevent its proportional translation into protein [
25]. Secretion of TGF-β1 into the supernatant of the cells was excluded as a possible reason for the observed discrepancy, since no signal was obtained by ELISA.
In contrast to the levels of TGF-β1 mRNA (upregulated in RA SFBs), the amounts of TGF-β2 and TGF-β3 mRNA, which share the same receptors, were significantly increased in OA SFBs by qPCR. This is in agreement with results showing on the one hand predominant effects of TGF-β2/3 versus TGF-β1 in an age model of OA [
26], but on the other hand a strong immunoreactivity at the protein level only for TGF-β1 but not for TGF-β2/3 in the RA SM [
27]. In addition, there is strong evidence that the isoforms have different functions, as demonstrated by the non-overlapping phenotypes of the isoform-specific null mice [
27]. Together, these findings argue for a pivotal and differential role of TGF-β1 in the pathogenesis of RA.
In addition to TGF-β itself, its receptor TGFBRI was upregulated in RA SFBs, providing the basis for an enhanced autocrine effect of locally present TGF-β1 on SFBs in the RA SM. Whereas this study provides the first report concerning the expression of TGFBR1 in human arthritis, the type II receptor [
22] and endoglin (a receptor for TGF-β1 and 3) [
20] have been reported to be more strongly expressed in RA SM than in OA SM or normal SM, showing the relevance of TGF-β-signaling in RA.
The present study shows a constitutive upregulation of THBS1 (mRNA/protein) in RA SFBs. A constitutively higher expression of THBS1 has previously been described in RA synovium compared to OA and joint trauma [
28] and the synovial expression of this molecule has been assigned to endothelial cells, macrophages and synovial lining cells [
29]. Furthermore, it has been demonstrated that implantation of THBS1-containing pellets into the ankle joints of rats aggravates adjuvant arthritis [
30], showing the importance of THBS1 in arthritis. TGF-β is initially produced in its latent form [
31,
32], that is, covalently linked with the latency-associated proteins and attached to LTBPs, which are cross-linked to the extracellular matrix (reviewed in [
17]). In order to activate TGF-β, the mature molecule has to be released from the large latent complex by plasmin or cathepsins [
33] or, as previously described, with a high efficiency by THBS1 [
18]. The abundance of THBS1 may, therefore, lead to enhanced activation of latent TGF-β1 in RA (see above), resulting in more TGF-β1 activity in the arthritic joint. Indeed, increased levels of active TGF-β1 have recently been reported in RA synovial fluid in comparison to OA synovial fluid [
34]. This TGF-β1 activity may then contribute to enhanced proliferation of SFBs [
35] or enhanced production of MMPs [
36].
In the present study, the induction of MMP-11 (stromelysin-3) at the mRNA and protein levels by TGF-β1 was restricted to RA SFBs. This effect has been originally described for mouse fibroblasts and osteoblasts and was based on both stimulation of gene transcription and stabilization of mRNA transcripts [
37]. The rapid upregulation of mRNA after 4 h suggests a direct activation of gene transcription rather than an indirect induction via other factors, that is, platelet-derived growth factor [
38], which is also a known inducer of MMP-11 [
39]. Like other MMPs, MMP-11 requires proteolytic removal of propeptides for activation. Whereas for other MMPs this process occurs via extracellular proteases following secretion, MMP-11 is intracellularly processed by furin and secreted as an active protease (in analogy to membrane-type MMP) [
40]. Therefore, the presence of increased intracellular levels observed in the present study represents the basis for functional MMP-11 outside the cell. Although MMP-11 does not directly participate in the degradation of extracellular matrix, it is able to inactivate protease inhibitors, resulting in enhanced proteolytic activity [
41] and controls cell proliferation by processing the insulin-like growth factor-binding protein-1 [
42]. MMP-11 is, therefore, involved in matrix turnover and proliferation, both processes with implications for RA. Its specific upregulation by TGF-β further supports a functional relevance of the constitutively upregulated TGF-β pathway in RA.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DP performed the real-time PCR, the western blots, the immunohistochemistry, as well as the respective data analyses and participated in writing the manuscript. AB analyzed the microarray data and participated in writing the manuscript. DK performed the microarray experiments, HJT and TW participated in the coordination of the study, and RWK contributed to the design of the study and participated in the layout, writing, and finalization of the manuscript.