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Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, IrelandDepartment of Rheumatology, St. Vincent's University Hospital, University College Dublin, Dublin, Ireland
Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, IrelandDepartment of Rheumatology, St. Vincent's University Hospital, University College Dublin, Dublin, Ireland
Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, IrelandDepartment of Rheumatology, St. Vincent's University Hospital, University College Dublin, Dublin, Ireland
Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, IrelandDepartment of Rheumatology, St. Vincent's University Hospital, University College Dublin, Dublin, Ireland
UCD Veterinary Sciences Centre, University College Dublin, Belfield, IrelandConway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland
We examined thrombospondin-1 (THBS1, alias TSP-1) expression in human synovial tissue (ST) during the resolution phase of chronic inflammation and elucidated its transcriptional regulation by the orphan receptor 4A2 (NR4A2). In vivo, rheumatoid arthritis (RA) serum and ST revealed altered expression levels and tissue distribution of TSP-1. After anti–tumor necrosis factor therapy, a reciprocal relationship between TSP-1 and NR4A2 expression levels was measured in patients with clinical and ST responses to biological treatment. In vitro, primary RA fibroblast-like synoviocytes (FLSs) expressed minimal TSP-1 mRNA levels with high transcript levels of NR4A2, vascular endothelial growth factor (VEGF), and IL-8 measured. Hypoxic modulation of RA FLSs resulted in inverse expression levels of TSP-1 compared with NR4A2, IL-8, and VEGF. Ectopic NR4A2 expression led to reduced TSP-1 mRNA and protein levels with concomitant increases in proangiogenic mediators. NR4A2 transcriptional activity, independent of DNA binding, repressed the hTSP-1 promoter leading to reduced mRNA and protein release in immortalized K4IM FLSs. Bioinformatic and deletion studies identified a 5′ region of the TSP-1 promoter repressed by NR4A2 and proangiogenic transcription factors, including NF-κB and Ets1/2. Stable depletion of NR4A2 levels resulted in a shift in the TSP-1/VEGF expression ratio. Thus, modulation of TSP-1 expression is achieved through anti–tumor necrosis factor therapy effects on specific transcriptional networks, suggesting that enhanced TSP-1 expression may help restore tissue homeostasis during resolution of inflammation.
Thrombospondin-1 (THBS1, alias TSP-1) is a large oligomeric extracellular matrix glycoprotein that mediates cell-cell and cell-matrix interactions by binding cell-surface receptors, including integrins, integrin-associated protein/CD47, CD36, heparin sulfate proteoglycans, low-density lipoprotein–related protein 1, and very low-density lipoprotein receptor, in addition to other extracellular matrix proteins and cytokines.
As the first identified naturally occurring angiogenic inhibitor, TSP-1 has been shown to play a critical role in inhibiting angiogenesis, resulting in inhibition of tumor growth and metastases.
In addition to TSP-1, several angiostatic factors, including TSP-2, angiostatin, IL-4, and IL-13, are produced by rheumatoid arthritis (RA) synovial tissue (ST).
Although these mediators have been shown to negatively affect angiogenesis through endothelial cell proliferation, activation, and rate of apoptosis, they are not produced to sufficient levels to limit synovial angiogenesis and inflammation driven by growth factors [vascular endothelial growth factor (VEGF) and Ang-1/-2], cytokines [tumor necrosis factor (TNF-α), IL-1β, IL-6, and IL-18], chemokines (IL-8, Gro-α, and SDF-1), and cell adhesion molecules (E-selectin, α1/β3 integrins, and platelet endothelial cell adhesion molecule 1).
Targeted overexpression of TSP-1 in mice suppresses wound healing and tumorigenesis, whereas lack of functional TSP-1 results in increased vascularization of selected tissues.
Direct intra-articular administration of adenoviral vectors encoding TSP-1 significantly ameliorates the clinical course of collagen-induced arthritis and experimental osteoarthritis through reduced microvessel density and inflammation.
TSP-1–derived peptide treatment shows similar effects in the PG-PS model of erosive arthritis, suggesting that TSP-1 peptide mimetics may be an effective therapeutic strategy for the treatment of diseases driven by excessive angiogenesis.
In contrast, transcriptional regulation that alters the production of angiostatic proteins leading to inhibition of angiogenesis is less studied. Regulatory networks controlled by the transcriptional activity of Smad2/4, JunD, and the nuclear receptors peroxisome proliferator-activated receptor alpha/gamma have been shown to interfere with VEGF-mediated angiogenesis through the enhancement of TSP-1 levels.
The profiling of early response genes induced by VEGF-A has identified novel transcription factors, including orphan nuclear receptor 4A (NR4A) subfamily members NR4A1, NR4A2, and NR4A3.
Vascular endothelial growth factor-regulated gene expression in endothelial cells: kDR-mediated induction of Egr3 and the related nuclear receptors Nur77. Nurr1, and Nor1.
The transcriptional function of these constitutively active NR4A receptors can be controlled at the level of expression and nuclear localization, and NR4A subfamily members are aberrantly expressed in inflamed human ST, colorectal cancer, psoriatic, and atherosclerotic lesions compared with normal tissue.
NR4A1 transcriptional activity is both necessary and sufficient for VEGF-A–induced proliferation and survival of cultured endothelial cells and for angiogenesis in vivo, whereas depletion of NR4A2 expression attenuates VEGF-A–induced endothelial proliferation, migration, and in vivo Matrigel (BD Biosciences, Franklin Lakes, NJ) angiogenesis.
NR4A1 transcriptional activity also regulates vascular permeability by enhancing endothelial nitric oxide synthase and down-regulating several endothelial cell junction proteins that are required to maintain vascular homeostasis.
In primary endothelial and fibroblast-like synoviocyte (FLS) cell populations isolated from RA ST, differential regulation of NR4A expression has also been confirmed, suggesting that the NR4A2 receptor may have a distinct role in regulating gene expression in response to inflammatory and growth factor signals.
Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5′-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.
In vivo, the aberrant expression of NR4A2 in FLSs of the lining layer confirms that NR4A2 is expressed in cells at the leading edge of invading pannus.
Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5′-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.
Although NR4A receptors induce transcription of target genes by binding directly to NGFI-β–responsive elements in promoter regions, these receptors can also act as transcriptional repressors through mechanisms involving protein-protein interactions with other transcription factors or co-regulatory molecules.
In FLSs, NR4A2 cooperates with NF-κB/p65 to robustly promote IL-8 transcription, revealing novel transcriptional responses to TNF-α signaling that act to promote cell migration and angiogenesis.
The clinical efficacy of anti–TNF-α (TNFi) therapy is established in RA and other diseases, including psoriasis and psoriatic arthritis, characterized by increased angiogenesis.
In psoriatic lesions, the effect of etanercept suggests that this agent drives the resolution of inflammation through significant up-regulation of TSP-1 and time-dependent reduction in VEGF-A levels, leading to reduced dermal blood vessel numbers over time.
Detailed tissue analysis indicates that a combined decrease in NF-κB and increase in TSP-1 levels in vivo during etanercept treatment may be important in limiting angiogenesis.
A similar reciprocal association between NF-κB and TSP-1 levels has been observed in rat granulation tissue where NF-κB blockade leads to TSP-1 up-regulation.
Similar to the TNF-α inhibitory effect on NF-κB signaling, NR4A2 mRNA and protein levels in involved psoriatic skin are significantly decreased and cytoplasmic distribution is restored after treatment with infliximab or etanercept.
In RA, the effect of TNFi therapy on NF-κB activity is established. Therefore, we hypothesized that TNFi may also promote ST changes through altered NR4A2 and TSP-1 expression.
The present study was undertaken to elucidate the molecular mechanisms regulating TSP-1 expression in human inflammatory arthritis. These data indicate that in patients with RA, expression of TSP-1 is significantly reduced, and these levels can be restored in patients responding to TNFi treatment. Furthermore, we identify the orphan receptor NR4A2 as a novel transcriptional repressor of TSP-1 expression. Collectively, these data suggest that targeting TSP-1 expression early in disease may be beneficial in ameliorating the clinical course of human inflammatory arthritis.
Materials and Methods
Patients and Tissue Collection
All the research was performed in accordance with the Declaration of Helsinki, and approval for this study was granted by the St. Vincent’s University Hospital (Dublin, Ireland) medical and research ethics committee. Patients with active RA (n = 12) who fulfilled the American College of Rheumatology criteria were recruited from the Rheumatology Clinics, St. Vincent’s University Hospital. At baseline, 50% of patients were naive for disease-modifying antirheumatic drugs and corticosteroids; however, all patients, including those taking disease-modifying antirheumatic drugs [methotrexate alone, 35%; methotrexate + salazopyrine, 5%; and hydroxychloroquine (Plaquenil, Sanofi Aventis, Bridgewater, NJ) alone, 5%], were biological naive, had active disease, had at least one inflamed knee joint, and were due to commence biological therapy. All the patients provided fully informed written consent and underwent arthroscopy at baseline and 3 months after commencement of TNF blocking therapy. Clinical and laboratory assessments included tender and swollen joint counts, rheumatoid factor level, anti-CCP antibody level, erythrocyte sedimentation rate, C-reactive protein (CRP) level, and global health visual analog scale score obtained the day before arthroscopy.
Disease activity score (DAS28-CRP) is a measure of disease burden using a general health assessment, tender joint counts, swollen joint counts (up to 28), and CRP levels. Under local anesthetic, arthroscopy of the inflamed knee was performed using a Wolf 2.7-mm needle (Richard Wolf Medical Instruments Corp., Vernon Hills, IL) as previously described.
ST biopsy samples were obtained by using 2-mm grasping forceps under direct visualization and were subsequently embedded in ornithine carbamoyltransferase or established as ex vivo synovial explant cultures.
Normal synovium (n = 4) was obtained from consenting patients undergoing interventional arthroscopy for ligament tears. After receiving informed consent, serum (n = 12) and synovial fluid (n = 16) were obtained from patients with active inflammatory arthritis and diagnosed as having RA, whereas normal serum samples were collected from healthy volunteers (n = 5). Ex vivo RA synovial explant cultures (n = 5) were established to determine expression levels of TSP-1 and VEGF at the site of inflammation. These explant cultures maintain the synovial architecture and cell-cell contact and, therefore, more closely reflect the in vivo environment.
Cyclooxygenase 2-derived prostaglandin E2 production by corticotropin-releasing hormone contributes to the activated cAMP response element binding protein content in rheumatoid arthritis synovial tissue.
Synovial explant tissues were cultured in 96-well plates (Falcon, BD Biosciences, Franklin Lakes, NJ) in RPMI 1640 medium supplemented with 100 U/mL of streptomycin and 100 U/mL of penicillin and were incubated for 24 hours at 37°C in 5% CO2. Supernatants were harvested and assayed for spontaneous secretion of TSP-1 and VEGF by enzyme-linked immunosorbent assay (ELISA).
Primary and Immortalized Cell Culture
Primary human FLSs were derived from patients with RA and were cultured as described previously.
Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5′-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.
Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5′-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.
Primary FLSs were exposed to hypoxic conditions by incubating them in an atmosphere composed of 3% O2 and 5% CO2, created using a hypoxic chamber. The medium was changed to serum-free medium before RA FLSs were subjected to the hypoxic conditions, and for each experiment, parallel cultures were grown in serum-free medium under normoxic conditions. After culturing the FLSs under these conditions for 2 to 24 hours, the cells were harvested for subsequent experiments.
Stable NR4A2-Overexpressing Clones
K4IM FLSs were stably transfected using a pUB6/NR4A2 expression vector or a pUB6/β-galactosidase (lacZ) control vector (Cell Trends Inc., Middletown, MD) using a nonliposomal technique. Forty-eight hours after transfection, the medium was replaced with RMPI 1640 medium supplemented with 10 μg/mL of blasticidin. The selective medium was then replaced every 3 to 4 days until blasticidin-resistant colonies were detected. Single colonies overexpressing NR4A2 or the pUB6/β-galactosidase (lacZ) vector were then picked and propagated in selection medium.
The selected clones were subsequently screened to ensure overexpression of NR4A2 mRNA and protein. Three overexpressing NR4A2 clones (clones 1, 2, and 3) were selected for further analysis (Supplemental Figure S1).
TSP-1, VEGF, and IL-8 ELISAs
Primary FLSs and K4IM FLS NR4A2 clones were seeded at 2.5 × 105 cells per well of a 6-well culture plate and were incubated overnight. Cells were grown in serum-free RPMI 1640 medium for 24 hours, at which time the supernatant was collected. TSP-1, VEGF, and IL-8 protein levels were measured in cell supernatants, synovial fluid, and human plasma using Quantikine human TSP-1 or VEGF ELISAs (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. IL-8 protein levels were measured by ELISA as previously described.
Total RNA was isolated from all cell types using TRIzol solution (Invitrogen, Carlsbad, CA). RNA concentrations were determined using a NanoDrop (NanoDrop Products, Wilmington, DE) and were integrity checked by gel electrophoresis. One microgram of total RNA was reverse transcribed into cDNA using an oligo(dT) primer and Moloney Murine Leukemia Virus Reverse Transcriptase (Invitrogen). Real-time PCR was conducted using SYBR Green master mix (Applied Biosystems, Foster City, CA) using an ABI 7300 thermocycler (Applied Biosystems). Primers used to amplify NR4A1, NR4A2, NR4A3, TSP-1, VEGF, IL-8, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were previously described.
Relative expression levels of target genes were determined using the 2−ΔΔCt method, with GAPDH as a control.
Immunohistochemical and Microscopic Analyses
Cryostat sections (7 μm) were stained overnight at 4°C with either a monoclonal mouse anti–TSP-1 primary antibody (dilution 1:100) (Abcam Inc., Cambridge, MA) or a polyclonal rabbit anti-NR4A2 primary antibody (dilution 1:600) (Santa Cruz Biotechnology, Santa Cruz, CA) that recognizes the amino terminus (N20) of human NR4A2.
Serial sections were also incubated with primary antibodies against mouse monoclonal anti-CD4, anti-CD8, anti-CD68, anti-CD19, and anti-CD20. Appropriate biotinylated secondary antibodies were added the following day, and sections were incubated using a VECTASTAIN elite avidin-biotin-peroxidase complex kit; Vector Laboratories, Burlingame, CA). Color was developed in a solution containing diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO) and 0.5% H202 in PBS (pH 7.6). Slides were counterstained with hematoxylin and mounted. Isotype-matched IgGs for each antibody yielded negative staining in all tissue sections examined (Supplemental Figure 2A).
Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5′-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.
Cyclooxygenase 2-derived prostaglandin E2 production by corticotropin-releasing hormone contributes to the activated cAMP response element binding protein content in rheumatoid arthritis synovial tissue.
Quantitative microscopic analysis of inflammation in rheumatoid arthritis synovial membrane samples selected at arthroscopy compared with samples obtained blindly by needle biopsy.
Modulation of orphan nuclear receptor NURR1 expression by methotrexate in human inflammatory joint disease involves adenosine A2A receptor-mediated responses.
Microscopic analysis was conducted on randomly coded slides by two blinded assessors (J.P.M. and M.G.). Only ST sections in which the lining layer was identifiable were included in the analysis. Scoring was performed in randomly selected high-power fields (HPFs) (×400 magnification); a minimum of 17 HPFs from three separate sections were scored using a 6-point scoring system (0 = <1% positive cells, 1 = 1% to 10% staining, 2 = 11% to 25%, 3 = 26% to 50%, 4 = 51% to 75%, and 5 = 76% to 100% staining). A minimum of two matched biopsy samples for each patient were averaged to give a median (range) score. Lining layer, sublining layer, and blood vessels in each section were scored separately. Positive cells for nuclear NR4A2 and cytoplasmic NR4A2 were scored separately. Where differences in scoring occurred, slides were reviewed by both assessors and a consensus score was obtained. These scoring techniques have been previously validated and reported.
Quantitative microscopic analysis of inflammation in rheumatoid arthritis synovial membrane samples selected at arthroscopy compared with samples obtained blindly by needle biopsy.
Modulation of orphan nuclear receptor NURR1 expression by methotrexate in human inflammatory joint disease involves adenosine A2A receptor-mediated responses.
The primary antibody for factor VIII/von Willebrand factor (clone F8/26; Dako, Carpinteria, CA) mouse monoclonal antibody was diluted 1:100 and was incubated on tissue sections for 1 hour (Supplemental Figure S2B). Biotinylated secondary antibodies were added the following day, and sections were incubated with avidin-biotin-peroxidase complex (VECTASTAIN). Color was developed in a solution containing diaminobenzidine tetrahydrochloride (Sigma-Aldrich) and 0.5% H202 in PBS (pH 7.6). Slides were counterstained with hematoxylin and mounted. Counting of blood vessels was performed in 10 randomly selected HPFs (×100 magnification) from three separate sections, and the median (range) value was counted.
Modulation of orphan nuclear receptor NURR1 expression by methotrexate in human inflammatory joint disease involves adenosine A2A receptor-mediated responses.
NR4A2 RNA interference was achieved using a Mission lentiviral packaging mix (Sigma-Aldrich) as per instructions. For controls, cells were transduced with scrambled shRNA. Cells were seeded at 2.5 × 105 cells in 6-well plates and were incubated overnight. Hexadimethrine bromide (8 μg/mL) and lentiviral particles (25 μL) were added, and cells were incubated at 37°C. Medium containing 5 μg/μL of puromycin was changed 3 to 4 days until resistant colonies were identified.
K4IM NR4A2-overexpressing clones were cultured in 75 cm2 tissue culture flasks and were grown to 80% confluency. Cells were harvested and washed with cold PBS. Nuclear extracts were prepared using the NE-PER extraction kit (Pierce Biotechnology, Rockford, IL), as previously described.
Cyclooxygenase 2-derived prostaglandin E2 production by corticotropin-releasing hormone contributes to the activated cAMP response element binding protein content in rheumatoid arthritis synovial tissue.
Nuclear protein (10 μg) was separated on 12% SDS–polyacrylamide gel and was transferred to nitrocellulose membrane. A mouse monoclonal anti-human NR4A2 IgG2a (clone N1404, dilution 1:1000; R&D Systems), followed by a horseradish peroxidase–conjugated anti-mouse antibody (dilution 1:1000; Cell Signaling Technology Inc., Beverly, MA) was applied to the membrane. Signal detection was performed using SuperSignal west dura extended substrate (Pierce Biotechnology) according to the manufacturer's instructions. ProSieve color marker (Cambrex Corp., East Rutherford, NJ) served to verify protein molecular weights.
Immunofluorescence
Wild-type K4IM synoviocyte cells and K4IM NR4A2–overexpressing clones were seeded at 1 × 105 cells per well in an 8-well Lab-Tek chamber slide (Nunc, Roskilde, Denmark) for 24 hours. After serum starvation for 24 hours, wild-type K4IM cells were stimulated for 1 hour with 10−6 mol/L prostaglandin E2. Subsequently, NR4A2 detection was conducted using an anti-NR4A2 rabbit polyclonal antibody (N-20; Santa Cruz Biotechnology) and isotype-matched nonimmune IgG, as previously described.
Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5′-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.
Cells were washed in ice-cold PBS and were mounted using VECTASHIELD mounting media containing DAPI (Vector Laboratories).
Promoter Studies and Analysis
Plasmids possessing the −2033/+750 bp of the human TSP-1 promoter driving a luciferase reporter gene were a gift from Dr. Paul Bornstein (University of Washington, Seattle, WA).
Deletion mutations were constructed using PCR amplification of the regions of interest and were subcloned into a TOPO cloning vector (Invitrogen). Subsequently, the promoter regions were cloned into the pGL3-Basic vector (Promega Corp., Madison, WI) and were sequenced to confirm deletion and the correct orientation of the insertion. The Genomatix software suite program MatInspector (Genomatix Software GmbH, Munich, Germany) was used to predict and analyze human TSP-1 promoter regions. The first test used was the core similarity test, and the maximum core similarity score of 1.0 is reached only when the highest conserved bases of a matrix match exactly in the sequence. The second test was the matrix similarity test, and the score must be >0.80. A perfect match to the matrix has a score of 1.00 (each sequence position corresponds to the highest conserved nucleotide at that position in the matrix), and a good match to the matrix usually has a similarity score >0.80.
Transfections
For reporter assays, K4IM synoviocyte cells were transfected using GeneJuice transfection reagent (Merck KGaA, Darmstadt, Germany). Cells were seeded in a 24-well plate at a density of 0.5 × 105 cells, left overnight, and transfected with 500 ng of endotoxin-free plasmid DNA. Cells were transfected with various combinations of the following plasmids: TSP-1 promoter constructs, VEGF promoter construct (−1005 bp) [a gift from Dr. Gregg Semenza (University of Maryland, Baltimore, MD], IL-8 promoter, pCMX-NR4A2 [a gift from Dr. Thomas Perlmann (Ludwig Institute for Cancer Research Ltd, Stockholm, Sweden)], pCMV-NR4A2:C293G binding mutant,
Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5′-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.
pCMV-Ets1, and pCMV-Ets2 [a gift from Dr. Leonie Young (Royal College of Surgeons in Ireland, Dublin, Ireland)]. Twenty-four hours after transfection, cells were washed with PBS and protein was harvested using passive lysis buffer (Promega Corp.). Transfection efficiencies >70% were routinely obtained (Supplemental Table S1). Luciferase activity was measured using a luminometer and luciferase assay reagents (Promega Corp.). Results are expressed as means ± SEM relative luciferase units. Experiments were repeated multiple times in triplicate with similar results achieved. Transfection efficiencies >70% were routinely obtained. Luciferase activity was measured using a luminometer and luciferase assay reagents (Promega Corp.). Experiments were repeated multiple times in triplicate with similar results achieved. Primary RA FLSs are limited by low transfection efficiencies. Using state-of-the-art Nucleofector technology (Lonza Inc., Walkersville, MD), the highest transfection efficiency we can achieve in these cells is approximately 10% to 15% (Supplemental Figure S3A and Supplemental Table S1). In K4IM synoviocytes we can achieve transfection efficiencies >70%. Representative images of enhanced green fluorescent protein–transfected RA FLSs and immortalized K4IM cells and percentage of cells transfected are shown in Supplemental Figure 3A and Supplemental Table S1.
Statistical Analysis
Nonparametric data were analyzed using the Wilcoxon signed rank test and the Spearman correlation coefficient. Student’s t-test was used in GraphPad Prism software version 4.0 (GraphPad Software Inc., San Diego, CA) to determine statistically significant differences between the control and treatment groups. P ≤ 0.05 was considered significant.
Results
TSP-1 and VEGF Expression Levels in Local and Peripheral Tissues
Microarray analysis has established that ectopic expression of NR4A2 in human immortalized K4IM FLSs leads to increased expression of proinflammatory genes, including IL-8.
Bioinformatic analysis also shows consistent and statistical differences in the expression of VEGF (2.8-fold) and angiopoietin-1 (2.7-fold), which is consistent with studies that have established the NR4A receptors as important transcriptional regulators of proangiogenic mediators in vitro and in vivo.
The present analysis further identifies TSP-1 as the most statistically meaningful gene whose expression was suppressed (−9.1-fold, P < 0.005) on the array. Enhanced NR4A2 expression in K4IM FLS cell lines (clones 1, 2, and 3) results in reduced TSP-1 with enhanced VEGF mRNA levels. Ectopic NR4A2 localized to the nucleus and expression levels are comparable with levels induced by the proinflammatory mediator prostaglandin E2 (Supplemental Figure S1).
Having confirmed altered mRNA levels of TSP-1 in in vitro NR4A2-expressing K4IM FLS cell lines (Supplemental Figure S1), we sought to determine protein localization of TSP-1 and NR4A2 in normal and inflamed human ST (Figure 1A). Immunohistochemical analysis of normal ST (n = 5) revealed abundant TSP-1 staining in the lining layer and vascular endothelial cells of the sublining capillary vessels (Figure 1A). Serial sections co-localized NR4A2 and TSP-1 in the cells of the lining and vascular endothelial cells in the sublining region. However, in contrast to high TSP-1 levels, NR4A2 immunostaining of normal ST was weak and localized to the cytoplasmic compartment of positive cells. In RA ST (n = 12), TSP-1 was found predominantly in cells of the sublining layer, with highest staining intensity associated with endothelial cells. Compared with normal ST, RA lining layer cells showed significantly (P < 0.05) less staining for TSP-1. As shown in Figure 1A, and consistent with previous studies,
Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5′-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.
Modulation of orphan nuclear receptor NURR1 expression by methotrexate in human inflammatory joint disease involves adenosine A2A receptor-mediated responses.
abundant NR4A2 expression is observed throughout the lining and sublining regions of inflamed ST, confirming reciprocal expression patterns for TSP-1 and NR4A2 in normal and inflamed ST.
Figure 1Expression levels of orphan receptor 4A2 (NR4A2), thrombospondin-1 (TSP-1), and vascular endothelial growth factor (VEGF) produced by normal and rheumatoid arthritis (RA) synovial tissue (ST), serum, explanted ST, and synovial fluid (SF). A: Representative tissue sections from normal human ST (n = 5) and inflamed ST (n = 12) were stained with anti–TSP-1 or anti-NR4A2 primary antibodies and were hematoxylin counterstained. Positive cells stained light/dark brown. Original magnification, ×200. B: Range of TSP-1 and VEGF protein levels measured in conditioned media from explanted ST (n = 5) and SF (n = 16) isolated from patients with inflammatory arthritis. C: Circulating TSP-1 serum levels measured in healthy volunteers (n = 5) and patients with inflammatory arthritis (n = 12). TSP-1 and VEGF protein levels were measured by ELISA. Values are given as means ± SEM. ∗∗∗P < 0.005.
To further examine TSP-1 protein levels produced by RA synovium, we established an ex vivo culture system using RA ST biopsy samples (n = 5). After 48 hours of incubation with explanted ST, levels of TSP-1 and VEGF protein released into the culture media were measured (Figure 1B). Means ± SEM TSP-1 (50.7 ± 12.4 μg/mL, n = 5) secreted from explanted ST were comparable with levels detected in isolated synovial fluid (52.5 ± 6.2 μg/mL, n = 16), indicating local production of TSP-1 by RA ST.
Having established that normal and inflamed STs demonstrate altered patterns of TSP-1 and NR4A2 expression, we investigated TSP-1 levels in serum from healthy volunteers (n = 5) and patients with joint disease (n = 12) (Figure 1B). The means ± SEM range of TSP-1 protein levels measured in normal serum (16.54 ± 3.12 μg/mL) was comparable with those measured in previous studies.
The present analysis showed significantly altered (P < 0.005) means ± SEM TSP-1 protein levels in circulating serum of patients (7.06 ± 1.00 μg/mL) compared with healthy volunteers.
TSP-1 Expression in Inflamed ST and Serum before and after TNFi Treatment
To study the effects of reduced ST inflammation on TSP-1 and NR4A2 expression levels, we extended the analysis to the cohort of patients receiving TNFi therapy (n = 12). Clinical and ST characteristics at baseline and at 12 weeks of treatment are summarized in Table 1. Significant reductions in all the clinical parameters were demonstrated after treatment (Table 1). At baseline arthroscopy (week 0), all the patient ST (n = 12) demonstrated macroscopic features of synovial inflammation (synovitis), with microscopic analysis confirming inflammatory cell infiltrates and increased lining layer thickness compared with normal ST (Table 1 and Figure 2A).
Table 1Clinical and ST Responses before and during TNFi Therapy
Characteristic
Baseline
12 weeks
P value
Age (years)
56 (29–82)
NA
NA
Disease diagnosis
RA
NA
NA
Disease duration (months)
150 (7–554)
NA
NA
Clinical features
Tender joint count
6.0 (0–28.0)
2.0 (0–5.0)
0.0106
Swollen joint count
6.0 (1–19.0)
2.0 (0–6.0)
0.0008
ESR (mm/hour)
25.0 (8.0–80.0)
16.0 (6.0–53.0)
0.0044
CRP (mg/L)
22.5 (4.0–110.0)
22.5 (4.0–110.0)
0.0082
DAS28-CRP
4.05 (2.76–6.52)
3.05 (1.15–4.32)
0.0008
Synovial tissue analysis
Composite synovitis score
Collective group
67.5 (4.0–100.0)
38.5 (15.0–82.0)
0.0403
DAS28-CRP ≤3.2
80.0 (32.0–100.0)
23.0 (17.0–82.0)
0.0313
DAS28-CRP >3.2
49.3 (4.0–99.0)
50.0 (15.0–92.8)
NS
Blood vessels (No./10 HPFs)
Collective group
9.8 (6–24)
9.1 (4.8–16.1)
NS
DAS28-CRP ≤3.2
10.2 (5.6–13.4)
10.4 (5.2–16.1)
NS
DAS28-CRP >3.2
8.0 (5–15.8)
8.8 (4.8–14.6)
NS
Values are given as median (range). P values were assessed using the nonparametric Wilcoxon signed rank test. DAS28-CRP is a composite score of tender joint count, swollen joint count, CRP level, and a general health assessment on a visual analog scale. Cell-specific synovitis score of ST is a composite score for CD4, CD8, CD68, CD19, and CD20 expression levels. Patients were further categorized according to their response during 12 weeks of biological therapy. Using the DAS28-CRP cutoff value of 3.2, patients with a DAS28 using the CRP level (DAS28-CRP) ≤3.2 at 12 weeks were defined as responders, and those with a DAS28-CRP >3.2 were defined as nonresponders. No significant differences between responders and nonresponders in disease duration or in clinical markers of inflammation, including CRP, ESR, swollen joint count, tender joint count, and DAS28, were found at baseline.
ESR, erythrocyte sedimentation rate; RA, rheumatoid arthritis; NA, not applicable; NS, not significant.
Figure 2Reciprocal thrombospondin-1 (TSP-1) and orphan receptor 4A2 (NR4A2) expression in synovial tissue (ST) and peripheral blood correlated with inflammatory resolution during anti–TNF-α (TNFi) therapy. A: ST was isolated from patients with inflammatory disease (n = 12) at baseline (week 0) and after 12 weeks of TNFi therapy (week 12). Patients were subdivided into two categories based on clinical response to therapy: clinical responders [disease activity score (DAS) reduced at week 12 by >0.5] and nonresponders (DAS not reduced at week 12 by >0.5). Serial tissue sections were stained with anti–TSP-1 or anti-NR4A2 primary antibodies and were hematoxylin counterstained. Positive cells stained light/dark brown. Negative cells were stained blue. Original magnification, ×200. B: Serum TSP-1 protein levels were measured in the same patient cohort (n = 15). Changes in serum TSP-1 protein levels at weeks 0 and 12 in the responder versus nonresponder patient groups. P = 0.013. C: TSP-1 protein levels in the responder group versus the nonresponder group at weeks 0 (W0) and 12 (W12) during treatment. P = 0.047. Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90th percentiles. D: Correlations between changes in DAS (Δ DAS) and changes in TSP-1 protein levels (Δ TSP-1) were calculated. P < 0.040; r = −0.536.
Modified disease activity scores that include twenty-eight-joint counts: development and validation in a prospective longitudinal study of patients with rheumatoid arthritis.
Acute-phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy.
Patients were divided into two categories, responders and nonresponders, based on changes in DAS28-CRP. Patients who did not achieve a DAS28-CRP of <3.2 after 12 weeks of TNFi therapy were considered nonresponders (n = 4), whereas patients with a DAS28-CRP of ≤3.2 were considered responders (n = 8).
Acute-phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy.
No significant differences between responder and nonresponder patients in disease duration or in clinical markers of inflammation, including CRP, erythrocyte sedimentation rate, swollen joint count, tender joint count, and DAS28, were found at baseline.
Cell-specific markers of T cells (CD4 and CD8), macrophages (CD68), B cells (CD19 and CD20), and blood vessel number were quantified in the whole patient cohort and in patient groups determined by responses to biological therapy (Table 1). During treatment, the composite synovitis score was decreased significantly in the responder group; in contrast, no significant difference was observed in the nonresponder group. The number of blood vessels counted per 10 HPFs remained unchanged from baseline during treatment in both groups (Table 1).
In the nonresponder group of patients, immunostaining in the lining layer and sublining regions demonstrated no significant changes in TSP-1 levels or in distribution after 12 weeks of treatment. TSP-1 protein levels at 12 weeks were similar to, and in some cases lower than, expression levels observed at baseline before treatment (Figure 2A). TSP-1 levels in the ST lining layer of patients who responded well to treatment were significantly (P < 0.005) restored to levels observed in normal ST after 12 weeks of TNFi therapy. Higher protein levels are measured in the lining layer cells, vascular endothelium, and perivascular areas (Figure 2A). These results indicate that TSP-1 expression is reduced in the ST lining layer and endothelial cells of inflamed ST and that after effective treatment, clinical and ST responses and TSP-1 levels and distribution are restored to comparable expression levels observed in noninflamed ST.
In the same cohort of patients (n = 12), ST immunostaining for NR4A2 revealed intense staining in the lining layer and synovial vascular endothelium at baseline RA ST compared with normal synovium (Figure 2A). High-power views of synovial sections localized NR4A2 expression in the nucleus and cytoplasmic compartments of the cells, with nuclear staining being predominant (data not shown). After 12 weeks of treatment with TNFi therapy, NR4A2 protein distribution was significantly (P < 0.005) altered in patients who responded to therapy (DAS-CRP ≤3.2; n = 8). The predominant localization of NR4A2 protein noted in patients at baseline shifted from the nucleus to the cytoplasmic compartment. In contrast, intense NR4A2 nuclear staining in the lining and sublining regions remained in patients who had a poor response to treatment (DAS-CRP >3.2, n = 4) (Figure 2A). Collectively in the responder group after therapy, an enhanced TSP-1/NR4A2 ratio was observed due to reduced NR4A2 levels in lining and sublining regions, whereas TSP-1 levels were significantly increased in the lining layer cells.
To ascertain whether ST alterations of TSP-1 expression observed during treatment were reflected in peripheral serum, TSP-1 protein levels were quantified at week 0 and after 12 weeks of TNFi therapy (n = 15). Similar to ST analysis, patients were divided into two categories: responders (patients whose DAS-CRP reduced by >0.5 after 12 weeks of treatment) and nonresponders/poor responders (DAS-CRP unchanged by >0.5), and changes in serum TSP-1 expression levels (Δ TSP-1) in responders versus nonresponders were measured. The data revealed significant (P = 0.013) alterations in TSP-1 protein levels between the two groups (Figure 2B). The increased change in TSP-1 expression levels in responders versus nonresponders was further confirmed when we measured TSP-1 protein levels in patients before and after TNF blockade (Figure 2C). We observed no significant differences in TSP-1 protein levels of patients in the nonresponder subgroup after 12 weeks of TNFi therapy. However, the responder subgroup revealed a significant (P < 0.047) increase in TSP-1 levels after 12 weeks of treatment (Figure 2C). The increase in serum TSP-1 levels achieved in patients who responded to treatment was comparable with TSP-1 levels measured in healthy volunteers (Figure 1B). At baseline, the nonresponder group mean serum TSP-1 level of 8.7 μg/mL compared with a mean level of 13.6 μg/mL in the responder group. However, the differences between groups did not reach statistical significance owing to the broad TSP-1 range (±5.4 μg/mL) in the responder group.
Having demonstrated altered TSP-1 protein expression in ST of patients who responded to TNFi therapy, we then assessed whether a correlation existed between changes in TSP-1 expression (Δ TSP-1) and changes in DAS (Δ DAS) in patient serum. Analysis revealed a significant inverse correlation between Δ TSP-1 and Δ DAS (n = 15; r = −0.536; P < 0.04), confirming that an improvement in DAS promotes increased TSP-1 expression levels in ST and circulating plasma in patients with RA (Figure 2D).
Expression and Modulation of NR4A Receptors and Angiogenic Mediators in Primary Human FLSs
Having established altered NR4A2 and TSP-1 protein levels in ST lining layer cells, primary FLS cell lines (n = 9) were established from RA ST biopsy samples to extend gene expression analysis. During early culture (passage <4), RA FLSs are highly activated, producing large quantities of proteoglycans, cytokines, growth factors, matrix metalloproteinases, and prostaglandin E2.
After ex vivo culture and by passage >4, RA FLSs becomes more quiescent, and the production of inflammatory factors gradually declines; however, primary FLSs can be maintained in culture for extended periods and remain viable up to passage 8.
NR4A1-3 mRNA expression was measured by quantitative RT-PCR (RT-qPCR) in early/activated (passage ≤4, n = 5) and late/quiescent (passage ≥5, n = 4) primary FLS cell lines (Figure 3A). Compared with normal human synoviocyte cells (K4IM FLSs), increased NR4A1 (mean, 380-fold), NR4A2 (mean, 400-fold), and NR4A3 (mean, 28-fold) mRNA levels were measured in activated FLSs (Figure 3A). In quiescent FLSs, NR4A2 and NR4A1 levels decreased significantly (P < 0.005), and NR4A3 levels remained unchanged (Figure 3A).
Figure 3Inverse expression and modulation of orphan receptor (NR4)A1, NR4A2, and NR4A3 receptors and angiogenic mediators in primary human fibroblast-like synoviocytes (FLSs). Primary human FLSs (n = 9) were isolated from inflamed synovial tissue (ST) biopsy samples. Total RNA was harvested from activated primary FLSs (low passage ≤4; n = 5) or more quiescent primary FLSs (high passage ≥5; n = 4). NR4A1, NR4A2, and NR4A3 (A) and IL-8 and thrombospondin-1 (TSP-1) (B) transcript levels were measured by RT-qPCR analysis. C: Primary human FLSs (n = 4) were grown under normal or hypoxic (3%) oxygen levels for 24 hours; mRNA levels of NR4A2, TSP-1, vascular endothelial growth factor (VEGF), and IL-6 were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH); and the relative expression of each gene was calculated using the 2−ΔΔCt method. Results are graphed as means ± SEM fold change relative to control and represent four independent experiments.
To examine potential links between NR4A2 expression levels in primary FLSs and mediators of angiogenesis, RT-qPCR analysis of TSP-1, VEGF, and IL-8 was undertaken. Consistent with elevated NR4A2 expression, and with previous results confirming that IL-8 expression is transcriptionally regulated by NR4A2,
high IL-8 mRNA levels were measured in low-passage FLSs (means ± SEM, 900 ± 20-fold; P < 0.0005), whereas higher-passage FLSs expressed significantly reduced IL-8 mRNA levels (Figure 3B). TSP-1 mRNA levels were found to be negligible in activated/low-passage FLSs, with significantly increased levels measured in quiescent/higher-passage cells (means ± SEM, 6.8 ± 2.5-fold; P < 0.005) (Figure 3B). VEGF mRNA levels in low- and high-passage FLSs were not significantly altered (data not shown). Thus, changes in TSP-1 mRNA levels in early- and advanced-passage primary FLSs are consistent with protein changes as measured in normal and inflamed RA ST (Figures 1 and 2). Taken together, data from primary FLSs corroborate the immunohistochemical analysis where intense TSP-1 staining in normal ST and reduced levels in lining layer FLSs of inflamed ST were measured.
Several studies confirm that inadequate oxygenation (3% hypoxia) drives cytokine production and the proangiogenic responses in RA ST.
Thus, modulation of proangiogenic/antiangiogenic mediators was further monitored in primary FLSs grown under normal and 3% hypoxic conditions, and changes in gene expression over 24 hours were analyzed. Hypoxia rapidly induces NR4A1-3 family members with maximal induction of NR4A2 mRNA measured within 2 to 4 hours (Figure 3C). Under similar hypoxic growth conditions, 60% reduction (means ± SEM, 0.41 ± 0.1-fold) in TSP-1 mRNA levels was measured, with significant (P < 0.005) increases in means ± SEM VEGF (8.1 ± 1.8-fold) and IL-6 (39 ± 9.9-fold) mRNA levels relative to mRNA levels measured under normoxia (Figure 3C). Consistent with previous studies, relative IL-8 mRNA levels also increased (means ± SEM, 200 ± 11.2-fold) under reduced oxygen conditions (data not shown).
Immortalized K4IM FLSs express endogenous levels of TSP-1, VEGF, and IL-8, and we investigated the effects of enhanced NR4A2 activity on these genes. To study the impact of NR4A2 on TSP-1 and VEGF transcription, NR4A2 was stably and transiently overexpressed in normal human K4IM FLSs (Figure 4). Several stable NR4A2-overexpressing clones were isolated, and three cell lines (lines 1, 2, and 3) were selected for subsequent analysis (Figure 4 and Supplemental Figures S1 and S2). To confirm gene expression analysis using primary RA FLSs, we measured TSP-1 mRNA and protein expression levels in the three NR4A2-expressing cell lines. Consistent with this primary FLS analysis, TSP-1 mRNA and protein levels were significantly reduced in NR4A2-overexpressing cells as measured by RT-qPCR and ELISA (Figure 4, A and B). In NR4A2-expressing K4IM cell lines, the effects of enhanced NR4A2 activity on IL-8 and VEGF mRNA levels, promoter activity, and protein secretion were also quantified, and the results indicate significantly enhanced expression of both proteins (Supplemental Figure S4, A–D).
Figure 4Ectopic orphan receptor 4A2 (NR4A2) expression reduced thrombospondin-1 (TSP-1) mRNA levels, secreted protein levels, and promoter activity in K4IM fibroblast-like synoviocytes (FLSs). A: Western blot analysis of immortalized human FLSs (K4IM) stably expressing lacZ cDNA (control) or NR4A2 cDNA (clones 1, 2, and 3) using NR4A2 antibody. mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the relative expression of each gene was calculated using the 2−ΔΔCt method. B: Secreted TSP-1 protein levels measured in NR4A2-overexpressing clones by ELISA. K4IM FLSs were co-transfected with human TSP-1 promoter (−2033/+750 bp) (C) or human TSP-1 promoter deletion −1178/+750 bp (D), together with pCMV–β-gal (lacZ), pCMX-NR4A2, or pCMV–NF-κB/p65. Transfected cells were harvested after 24 hours, and luciferase activity was measured. RLUs, relative luciferase units. Results are graphed as means ± SEM fold change relative to lacZ control, and data are representative of four independent experiments. ∗∗∗P < 0.005.
NR4A2 Modulates the Transcriptional Activity of the TSP-1 Promoter Independent of DNA Binding
Transient transfection of the human −2033/+750-bp TSP-1 promoter construct reveals high basal promoter activity in K4IM FLSs, which reflects high endogenous TSP-1 mRNA and protein levels produced by these cells (Figure 4C and Supplemental Figure 1A). Enhanced expression of NR4A2 significantly represses the −2033/+750-bp TSP-1 promoter region in transiently transfected K4IM FLSs (Figure 4C). The −2033/+750-bp TSP-1 promoter region was consistently repressed 50% to 60% by NR4A2, consistent with the effects of stable NR4A2 overexpression on endogenous TSP-1 mRNA and protein levels (Figure 4, A and B). Equivalent levels of ectopic NR4A2 is capable of transactivating the human IL-8 (−1508 bp) and VEGF (−1005 bp) promoter regions, leading to increased IL-8 and VEGF mRNA levels while demonstrating promoter-specific effects of NR4A2 in K4IM FLSs (Supplemental Figure S4).
Inhibition of TSP-1 expression by NF-κB has recently been suggested to control proangiogenic responses in human psoriasis and rat granulation tissue.
We have identified several putative κB binding sites in the −2033-bp hTSP-1 promoter region (Supplemental Figure S3). Thus, in addition to NR4A2, we tested the ability of NF-κB/p65 to regulate TSP-1 promoter activity using transient transfection assays. In K4IM FLSs, expression of NF-κB/p65 significantly repressed TSP-1 promoter activity comparable with the effects of NR4A2 (Figure 4C). In contrast, NF-κB potently induced the −1508-bp IL-8 promoter region with subsequent increases in IL-8 protein release (Supplemental Figure S4, E and F), which is consistent with previously described studies.
To determine the region of the TSP-1 promoter that is important for repression by NR4A2, we generated several 5′ deletions of the human TSP-1 promoter and co-transfected each with NR4A2 or NF-κB/p65. The present data reveal that in a shorter promoter construct (−1178/+750 bp), no significant changes in basal promoter activity were measured; however, NR4A2 and NF-κB lost the ability to repress the −1178-bp TSP-1 promoter fragment (Figure 4D). These results consistently revealed that removal of the 5′ distal promoter region changes NR4A2 and NF-κB/p65 transcriptional capacity from repression to transactivation. Both proteins significantly enhanced the truncated promoter region (Figure 4D). Previous promoter studies have identified that 5′ regions of the hTSP-1 promoter are required for transcriptional repression by several transcription factors, including YY1, Id1 (via Ets binding sites), and AP1 (Supplemental Figure S5).
From the detailed analysis performed on this region of the TSP-1 promoter, we could identify an NR4A2 consensus binding site (NGFI-β–responsive element) to permit NR4A2 binding in this region. Thus, to test whether direct binding to the TSP-1 promoter is required for NR4A2-dependent repression, we analyzed the ability of an NR4A2 DNA-binding mutant protein (B mut) to drive repression of TSP-1 transcription (Figure 5A). The introduction of C283G mutation in the DNA-binding domain of NR4A2 prevents receptor binding and transactivation of a consensus NGFI-β–responsive element sequence in various cell types, including K4IM FLSs.
Transrepression of the human TSP-1 promoter (−2033/+750 bp) and subsequent inhibition of TSP-1 protein release in K4IM FLSs was not affected by the introduction of this mutation into the NR4A2 protein (Figure 5, A and B). The magnitude of the NR4A2 mutant effects is similar to wild-type NR4A2 at all concentrations tested (Figure 5, C and D). Taken together, the NR4A2 DNA-binding mutant is capable of driving repression of the −2033/+750-bp TSP-1 promoter region with significant (P < 0.005) reductions in protein levels and release. In addition to monitoring NR4A2 and NF-κB/p65 transcriptional effects, and based on bioinformatic analysis (Supplemental Figure S5), we further tested the ability of Ets-1 and Ets-2 to regulate −2033/+750-bp TSP-1 promoter activity and protein release (Figure 5, E and F). Ets-1 and Ets-2 transcription factors strongly and significantly (P < 0.005) repress TSP-1 promoter activity (>85% and >70%, respectively) with robust changes in protein release (Figure 5F) that are comparable with NR4A2- and NF-κB–mediated effects.
Figure 5Transcriptional repression of TSP-1 promoter activity resulted in reduced levels of secreted thrombospondin-1 (TSP-1). K4IM fibroblast-like synoviocytes (FLSs) were transiently co-transfected in triplicate with 500 ng of human TSP-1 promoter (−2033/+750 bp) combined with pCMV–β-gal (lacZ) and pCMX- orphan receptor 4A2 (NR4A2) DNA-binding mutant (B mut) (A); pCMV–β-gal (lacZ) or pCMX-NR4A2 (C); or pCMV–β-gal (lacZ), pCMV–NF-κB/p65, pCMV–Ets-1, or pCMV–Ets-2 (E). Transfected cells were harvested after 24 hours, and luciferase activity was measured. Results are graphed as means ± SEM fold change relative to lacZ control and represent four independent experiments. B, D, and F: For each transfection experiment represented in A, C and E, levels of secreted TSP-1 protein in conditioned media were measured by ELISA. RLUs, relative luciferase units. ∗∗∗P < 0.005, ∗∗∗∗P < 0.0005.
Collectively, we identified a 5′ region of TSP-1 promoter that is repressed by NR4A2 and other transcription factors known to mediate proinflammatory/proangiogenic signaling pathways. Furthermore, these data indicate that NR4A2-dependent regulation of TSP-1 gene transcription and concomitant changes in TSP-1 protein secretion do not require direct NR4A2 binding to the repressor region in the 5′ region of hTSP-1 promoter.
Depletion of NR4A2 Enhances TSP-1 Expression
To further evaluate NR4A2-dependent regulation of TSP-1 expression, we sought to modulate NR4A2 expression and to monitor the effects on TSP-1 expression. We and others have previously demonstrated that NR4A2 is potently modulated by adenosine and a stable analog of adenosine, NECA, in primary RA and K4IM FLSs.
Modulation of orphan nuclear receptor NURR1 expression by methotrexate in human inflammatory joint disease involves adenosine A2A receptor-mediated responses.
Consistent with these studies, we observed significant modulation of NR4A2 mRNA and protein levels in response to 1 μmol/L NECA treatment (Figure 6, A and B). Next, we generated stable cell lines transduced with shRNA directed against NR4A2 leading to significantly reduced receptor expression levels. The efficiency of depletion was evaluated by RT-qPCR and Western blot analysis (Figure 6, A and B). Endogenous and NECA-inducible NR4A2 mRNA levels were significantly reduced (P < 0.001) in cells expressing NR4A2 shRNA compared with levels in control transduced cells. Moreover, consistent with mRNA data, protein levels measured by Western blot analysis showed that basal and NECA-induced NR4A2 levels were also reduced in cells stably transduced compared with cells transduced with scrambled shRNA (Figure 6, A and B).
Figure 6Depletion of orphan receptor 4A2 (NR4A2) receptor levels enhanced endogenous and inducible thrombospondin-1 (TSP-1) expression. NR4A2-depleted cells were generated using a control scrambled shRNA construct or an shRNA construct specific for NR4A2. Control and shNR4A2 cells were left untreated (NT) or were treated with 1 μmol/L NECA for 2 to 4 hours and were analyzed for NR4A2 mRNA by RT-qPCR (A), NR4A2 protein levels by Western blot analysis (B), TSP-1 (C), and TSP-1/vascular endothelial growth factor (VEGF) mRNA expression ratios (D) in shRNA scrambled and shRNA NR4A2-expressing cells. Inset: VEGF mRNA levels in control and shRNA NR4A2-expressing cells measured by RT-qPCR. Results represent means ± SEM from three independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005. †P < 0.05 compared with NT NR4A2 shRNA cells, †††P < 0.005 compared with NECA treated shRNA scrambled cells.
Depletion of NR4A2 expression levels resulted in significantly increased expression of TSP-1 mRNA levels (Figure 6C). After NECA treatment of NR4A2-depleted cells, we observed a further increase in TSP-1 modulation within 2 hours (Figure 6C). In contrast, endogenous VEGF mRNA levels were significantly reduced (>40%, P < 0.01) in NR4A2-depleted cells (Figure 6D), further confirming the extent of NR4A2 knockdown in our cell system. Consequently, in NR4A2-depleted cells, the ratio of endogenous TSP-1/VEGF mRNA expression is altered, resulting in a significant (P < 0.01) shift to elevated levels (Figure 6D).
Discussion
To gain an understanding of the role of mediators that control chronic inflammation, we evaluated the expression of TSP-1 in healthy and inflamed ST and monitored tissue and serum changes in TSP-1 during inflammatory resolution. Transcriptional control of TSP-1 by the orphan nuclear receptor NR4A2 and additional transcription factors, which are established effector molecules of proinflammatory signaling, was also assessed. Collectively, these data indicate that in human inflammatory arthritis, the expression pattern of TSP-1 is significantly altered, and these levels can be restored in patients responding to TNFi therapy. Furthermore, we identified NR4A2 as a novel transcriptional repressor of TSP-1 in human ST, leading to reduced TSP-1 expression. These results identify TSP-1 as a target of NR4A2 activity, complementing and strengthening recent studies proposing a proangiogenic role for the NR4A subfamily of orphan nuclear receptors in vivo.
Data from the present study demonstrate that patients with active inflammatory joint disease express low TSP-1 protein in free-circulating plasma and ST compared with levels of TSP-1 in healthy individuals. TNFi therapy is capable of restoring TSP-1 protein in patients to equivalent levels measured in healthy tissues. Furthermore, this analysis shows a significant correlation between altered TSP-1 expression and changes in disease activity and synovitis scores for patients with active inflammatory arthritis. Thus, this study suggests that restitution of TSP-1 levels during treatment may facilitate changes to limit ST inflammation to help ameliorate repair and promote tissue homeostasis. Similar changes in TSP-1 expression are observed in cancer biology: TSP-1 is frequently decreased or lost during progression of human cancers, and TSP-1 loss/down-regulation has been demonstrated in a variety of cancer models. Reexpression of TSP-1 can lead to delayed cancer progression, decreased microvascular density, and tumor cell apoptosis.
During the acute phase of inflammation, TSP-1 is strongly expressed, promoting resolution of the inflammatory process and protecting tissue from excessive damage. TSP-1 modulates inflammation by altering the production of cytokine, including IL-10 and IL-12.
Interaction of TSP-1 with CD47 is also crucial for activation of CD4+ CD25+ regulatory T cells to maintain self-tolerance and induce a suppressive phenotype.
the abundant TSP-1 expression we observed in healthy synovium are in contrast to the reduced levels measured in chronically inflamed RA ST. This analysis further shows that TSP-1 expression levels are minimal in early-passage FLSs isolated from RA ST biopsy samples. As RA FLSs are cultured and become less activated, significant increases in TSP-1 mRNA levels are measured. This analysis confirms that altered TSP-1 levels expressed by low- and high-passage primary FLSs may help explain differences that exist, whereas individual studies demonstrated up- and down-regulation of TSP-1 expression in RA FLSs.
Because TSP-1 participates in diverse biological processes, it is not surprising that TSP-1 has been shown to have both anti-inflammatory and proinflammatory effects in several diseases and animal models.
These contrasting functions may be due to dose-dependent interactions with multiple receptors, matricellular proteins, and the local tissue environment. TSP-1 at high concentrations (>100 nmol/L) becomes proangiogenic, possibly by engaging integrin clustering via CD47. On the other hand, lower TSP-1 concentrations (1 to 5 nmol/L) are angioinhibitory owing to high-affinity ligand-receptor interactions with CD36. By implanting human TSP-1–containing pellets (0.1, 0.5, or 2 μg) into the ankle of rats with adjuvant-induced arthritis, Koch et al
showed that TSP-1 exhibited a biphasic modulatory effect on synovial vessel density, with an early phase of increased vessel density and a later phase of decreased vessel formation. Direct intra-articular administration of adenoviral vectors encoding TSP-1 significantly ameliorated the clinical course of collagen-induced arthritis, demonstrating clinical efficacy.
Furthermore, increased transforming growth factor β expression and decreased production of VEGF was measured in adenoviral vector encoding mouse TSP-1–treated ankle joints.
The NR4A subfamily of orphan nuclear receptors has emerged as transcriptional regulators of proangiogenic growth factor and cytokine action in several inflammatory diseases.
We recently showed that enhanced NR4A2 activity induces a phenotypic shift in normal FLSs that parallels the cellular transformation and hyperplasia observed during the progression of inflammatory arthritis.
Through the generation of stable and transient NR4A2-expressing FLSs, we created an in vitro cell system that reflects the characteristics of activated primary FLSs.
Using such cell systems, we observed that NR4A2-overexpressing clones and low-passage primary FLSs express high NR4A2, VEGF-A, and IL-8 mRNA levels, with significantly reduced TSP-1 mRNA and secreted protein levels. Such gene expression changes complement and closely mirror the pattern of expression levels measured in RA ST and lend further support to previous studies that demonstrate that NR4A2 activity may promote angiogenesis by increasing endothelial proliferation, survival, and migration.
The present study is the first to establish that enhancement or depletion of NR4A2 levels in FLSs leads to inverse changes in TSP-1 mRNA and protein levels. The identification of TSP-1 as a transcriptional target downstream of NR4A2 activity provides further evidence that NR4A nuclear receptors may promote inflammation through the inhibition of angiostatic mediators. Another member of this nuclear receptor family, peroxisome proliferator-activated receptor, has been shown to modulate TSP-1 during early inflammation. Peroxisome proliferator-activated receptor alpha−/− in leukocytes results in enhanced TSP-1 expression, whereas peroxisome proliferator-activated receptor gamma ligands greatly augment the proapoptotic effects of the TSP-1–derived peptide ABT510.
Thrombospondin-1 mimetic peptide inhibitors of angiogenesis and tumor growth: design, synthesis, and optimization of pharmacokinetics and biological activities.
There is evidence suggesting that during chronic inflammation an angiogenic switch occurs, which shuts off angiogenic inhibitors, such as TSP-1, TSP-2, and angiostatin, leading to increased production of proangiogenic mediators, including, VEGF, Ang-1, IL-6, and IL-8.
Although it has been established that overexpression of TSP-2 in human RA models suppresses angiogenesis and alters immune stimulation and inflammation, it remains to be established whether TSP-2 levels are altered in vivo in response to treatment(s).
However, detailed molecular analyses confirming this association remain to be explored. Therefore, this molecular analysis confirming NF-κB–dependent regulation of TSP-1 promoter activity, mRNA, and protein expression provides compelling evidence that NF-κB/p65 is a transcriptional repressor of TSP-1 expression in human FLSs. The role(s) of TSP-1 in tumor progression is closely associated with its regulation by tumor suppressors and oncogenes. Tumor suppressors, including p53, PTEN, and Smad4, up-regulate TSP-1 expression to maintain high levels of TSP-1, but the oncogenes, including c-jun, v-src, and c-myc, down-regulate TSP-1 expression.
The transcription factor Id1 regulates tumor neovascularization via transcriptional repression of TSP-1. Id1 acts indirectly on the mouse TSP-1 promoter by interacting with Ets transcriptional activation sites.
Detailed bioinformatic analysis of the human TSP-1 promoter reveals a complex promoter composed of many consensus-binding sites, including several putative NF-κB and Ets-1/2 binding sites, and the presence of AP-1 and HIF-1 consensus motifs (Supplemental Figure S5). These sites are located in the 5′ distal −2033/−1178-bp promoter region, which we show is repressed by NR4A2, NF-κB, and Ets1/2. A similar repressible region has also been described in the mouse TSP-1 promoter between −2800 and −1120 bp.
The present data show that NR4A2 does not require direct DNA binding to regulate the TSP-1 promoter. Studies have demonstrated that NR4A receptors interact with other transcription factors and co-regulatory proteins to modulate gene transcription.
Direct binding to the IL-8 promoter by NR4A2 is not required to transactivate IL-8 gene transcription, and it seems that protein-protein interactions between NF-κB/p65 and NR4A2 can enhance IL-8 transcription.
Thus, NR4A2 may repress TSP-1 expression through similar protein-protein interactions that converge on the distal 5′ promoter region.
Several studies provide evidence to suggest that effective TNFi therapy in RA may halt active angiogenesis but has little effect on the expanded mature vascular bed.
Consistent with these studies, we found no profound decrease in ST blood vessel numbers after treatment. The present observations are further supported by the results of a study by Izquierdo et al
that demonstrated that mature vessel numbers do not alter during treatment and that decreases in immature vessels are closely related to patient response to TNF inhibition. In patients refractory to treatment, immature vascular development seemed insufficiently targeted by TNFi therapies.
Thus, to accurately assess TNFi effects on angiogenesis/vasculogenesis, parallel histologic and functional studies are required to evaluate the contribution of immature/mature vessels during the RA disease course and in response to established and emerging anticytokine treatments. Selective regression of immature vessels by targeting VEGF has been shown to promote vascular normalization rather than global vascular reduction. The TSP-1 mimetic ABT-510 reduces abnormal immature vasculature and increases the presence of mature vessels, promoting the uptake of chemotherapeutic drugs in an in vivo model of ovarian cancer.
Therefore, it is possible that therapeutic interventions that enhance the TSP-1/VEGF balance (via altered NR4A2 activity) may facilitate the elimination of immature vessels and promote endothelial function by inducing normalization of vessels.
In conclusion, these results reveal molecular mechanisms regulating TSP-1 expression and identify TSP-1 as a novel downstream mediator of NR4A2 activity in human inflammatory arthritis. Modulation of TSP-1 expression in RA ST can be achieved in vivo through TNFi therapy effects on proinflammatory transcriptional networks, suggesting that altered TSP-1 levels early in disease may be beneficial, leading to inflammatory resolution.
Acknowledgments
We thank Hanna Harant for providing the plasmids used in this study and members of the Murphy Laboratory for helpful suggestions and discussions.
Enhanced orphan receptor 4A2 (NR4A2) expression in K4IM fibroblast-like synoviocytes (FLSs) alters thrombospondin-1 (TSP-1) and vascular endothelial growth factor (VEGF) mRNA levels. A: Total RNA was harvested from control FLSs (K4IM-LacZ) and NR4A2 stable-expressing K4IM FLSs (clones 1, 2, and 3) and endpoint real-time PCR analysis was performed to amplify NR4A2, TSP-1, VEGF, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Real-time PCR analysis was also performed in the absence of reverse transcriptase (Neg). B: Quantification of NR4A2 protein levels in human K4IM FLSs stably expressing NR4A2 (clones 1, 2, and 3) compared with control K4IM FLSs stably expressing lacZ cDNA. C: Immunofluorescence localization of NR4A2 (red) in K4IM-lacZ FLSs and K4IM NR4A2 clones 1, 2, and 3. Endogenous NR4A2 levels were measured in K4IM FLSs left untreated or treated with 1 μmol/L prostaglandin E2 (PGE2) for 1 hour. Cells were counterstained with DAPI (blue) and isotype-matched IgG. Original magnification, ×200.
Nonimmune IgG staining controls and factor VIII staining on normal and rheumatoid arthritis (RA) ST. Representative normal and RA ST sections were incubated with nonimmune mouse and rabbit IgG and were counterstained with hematoxylin (A) and with immune serum directed against von Willebrand factor (factor VIII) (B). Positive endothelial cells are indicated by dark brown staining. The stromal lining layer is indicated by arrows. Nuclei are counterstained with hematoxylin.
Transfection efficiencies of K4IM fibroblast-like synoviocytes (FLSs) and rheumatoid arthritis (RA) FLSs. Representative images of K4IM FLSs and RA FLSs transfected with green fluorescent protein expression vector. Cell nuclei are counterstained with DAPI (blue).
Enhanced orphan receptor 4A2 (NR4A2) activity promoted the transcription of proangiogenic mediators. IL-8 (A) and vascular endothelial growth factor (VEGF) (B) mRNA levels were measured in K4IM fibroblast-like synoviocytes (FLSs) stably overexpressing NR4A2 (clones 1, 2, and 3) by RT-qPCR analysis. mRNA levels were normalized to GAPDH, and the relative expression of each gene was calculated using the 2−ΔΔCt method. K4IM FLSs were transiently co-transfected in triplicate with pCMV–β-gal (lacZ), pCMX-NR4A2, or pCMV–NF-κB/p65 combined with 500 ng of human IL-8 promoter (–1508 bp) (C and E) or VEGF promoter (–1071 bp) luciferase reporter constructs (D). Transfected cells were harvested after 24 hours, and luciferase activity was measured. Results are graphed as means ± SEM fold change relative to lacZ control and represent four independent experiments. F: IL-8 protein levels released from K4IM FLSs transiently co-transfected in triplicate with pCMV–β-gal (lacZ) or pCMV–NF-κB/p65 were measured by ELISA and expressed as means ± SEM. **P < 0.005, ***P < 0.0005.
Bioinformatic modeling of transcription factor binding sites in the human –2033/+750-bp TSP-1 promoter region. Identification and location of transcription factor binding sites in the human TSP-1 promoter region (–2033/+750 bp) using the Genomatix software suite program MatInspector. The promoter region –2033/–1178 bp upstream from the transcription start site and repressed by orphan receptor 4A2 (NR4A2) and NF-κB/p65 activity is highlighted in red. All transcription factor binding consensus sequences highlighted scored >0.90 in the first and second testing.
Vascular endothelial growth factor-regulated gene expression in endothelial cells: kDR-mediated induction of Egr3 and the related nuclear receptors Nur77. Nurr1, and Nor1.
Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5′-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.
Cyclooxygenase 2-derived prostaglandin E2 production by corticotropin-releasing hormone contributes to the activated cAMP response element binding protein content in rheumatoid arthritis synovial tissue.
Quantitative microscopic analysis of inflammation in rheumatoid arthritis synovial membrane samples selected at arthroscopy compared with samples obtained blindly by needle biopsy.
Modulation of orphan nuclear receptor NURR1 expression by methotrexate in human inflammatory joint disease involves adenosine A2A receptor-mediated responses.
Modified disease activity scores that include twenty-eight-joint counts: development and validation in a prospective longitudinal study of patients with rheumatoid arthritis.
Acute-phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy.
Thrombospondin-1 mimetic peptide inhibitors of angiogenesis and tumor growth: design, synthesis, and optimization of pharmacokinetics and biological activities.
Supported by Science Foundation Ireland grants SFI/BCIF259/2007 and SFI/IN.1/B26613/2009 (E.P.M.) and Health Research Board, Ireland, grant HRB/RP/162/2007 (E.P.M.).
Current address of P.P.T., GlaxoSmithKline, Stevenage, UK.
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