Objective To examine the effect of hypoxia on Signal Transducer and Activator of Transcription 3 (STAT3)-induced pro-inflammatory pathways in rheumatoid arthritis (RA).
Methods Detection of phospho-STAT3 was assessed in RA synovial tissue and fibroblasts (RASFC) by immunohistology/immunofluorescence. Primary RASFCs and a normal synoviocyte cell line (K4IM) were cultured under hypoxic and normoxic conditions±Stat3-siRNA, HIF-siRNA or WP1066 (JAK2-inhibitor). HIF1α, p-STAT3, p-STAT1 and Notch-1IC protein expression were analysed by western blot. Functional mechanisms were quantified by invasion chamber, matrigel and migration assays. IL-6, IL-8, IL-10 and matrixmetalloproteinases (MMP)-3 were quantified by ELISA. Notch-1 receptor, its DLL-4 ligand and downstream target genes (hrt-1, hrt-2) were quantified by real-time PCR. The effect of WP1066 on spontaneous secretion of pro/anti-inflammatory cytokines and Notch signalling was examined in RA synovial explants ex vivo.
Results p-STAT3 was increased in RA synovium compared with control (p<0.05). Hypoxia induced p-STAT3, p-STAT1 and HIF1α expression, an effect blocked by Stat3-siRNA and WP1066. Hypoxia-induced cell invasion, migration and cytokine production were inhibited by Stat3-siRNA (p<0.05) and WP1066 (p<0.05). While HIF1α siRNA inhibited hypoxia-induced p-STAT3 detection, Stat3-siRNA also inhibited hypoxia-induced HIF1α. Furthermore, hypoxia-induced Notch-1IC, DLL4, hrt-1 and -2 expression were significantly inhibited by WP1066 (p<0.05). Finally, in RA synovial explant cultures ex vivo, WP1066 decreased spontaneous secretion of IL-6, IL-8 and MMP3 (p<0.05), Notch-1 mRNA (p<0.05) and induced IL-10 (p<0.05).
Conclusions This is the first study to provide evidence of a functional link between HIF1α, STAT3 and Notch-1 signalling in the regulation of pro-inflammatory mechanisms in RA, and further supports a role for STAT blockade in the treatment of RA.
- Rheumatoid Arthritis
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Rheumatoid arthritis (RA) is an autoimmune disease characterised by synovial proliferation and leukocyte extravasation leading to joint destruction.1 ,2 RA synovial fibroblasts (RASFC) adapt to the adverse microenvironment of the inflamed joint, manifesting an abnormal phenotype characterised by increased invasiveness and impaired apoptotic pathways.3–7 Additionally, profound hypoxia occurs in the inflamed synovial joint,3 ,8 associated with synovitis, dysfunctional vascularity and increased expression of signalling pathways, hypoxia-inducible factor 1α (HIF1α) and NFκβ.4 ,6 ,7 Hypoxia is a powerful trigger for RASFC and endothelial cell (EC) activation, proliferation and survival, and can induce key growth factors, chemokines and matrixmetalloproteinases (MMP).3 ,4 ,6 ,8
Signal Transducer and Activator of Transcription (STAT) are cytoplasmic transcription factors activated by tyrosine phosphorylation in response to interferons (IFN) and IL-6 cytokines acting via the gp130 receptor.5 ,9 ,10 STAT3 is associated with human malignancies,11 oncogenic transformation,12 angiogenesis13 and invasion by tumour metastases.14 Furthermore, STAT3-dependent IL-22 signalling regulates keratinocyte activation,15 and IL-6 trans-signalling mediates TLR4-induced inflammation via STAT3 activation in animal models.16 In RA, STAT3 expression correlates with synovitis, modulates Th17 differentiation17 and induces RASFC survival.18 STAT3 inhibition suppresses disease severity in collagen-induced arthritis,19 mediates RANKL-dependent osteoclastogenesis,20 and chemokine expression in RA synoviocytes.21 A selective inhibitor of the JAK/STAT pathway CP-690550 has been developed for RA treatment, with Phase II/III clinical trials demonstrating significant ACR50/70 responses.22 ,23
While hypoxia is a key driving force for joint inflammation, little is known about its effect on JAK-STAT signalling in RA. Previous studies have shown HIF1α can facilitate the binding of STAT3 to the haptoglobin promoter in HepG2 human hepatoma cells,24 STAT3 can inhibit HIF1α degradation in tumour cells,25 and is a potential regulator of HIF1α-mediated vascular endothelial growth factor (VEGF) expression in renal carcinoma cells.26 Furthermore, studies have demonstrated that HIF1α requires the interaction of the Notch signalling pathway with STAT3 for gene transcription and cell survival in tumour cells.27 Notch-1, its ligands and downstream target genes are expressed in the inflamed synovium and inversely associated with in vivo pO2,28 with gene silencing studies demonstrating that Notch mediates pro-inflammatory and angiogenic mechanisms.27–29 The aim of this study is to investigate the effect of hypoxia on STAT3 signalling and downstream pro-inflammatory mechanisms in RA.
Material and methods
Patient recruitment and arthroscopy
RA patients (n=12, 5M:7F) and healthy controls (n=9, 7M:2F) were recruited from the Rheumatology Department, St. Vincent's University Hospital. RA patients fulfilled the American College of Rheumatology criteria. The median age of RA patients was 63.42 (33.25–74.08) years and healthy control was 44.51 (26.91–74.92) years. The median DAS28 for RA patients was 5.205 (2.95–7.13); 58% of RA patients were naive for DMARDS (disease modifying antirheumatic drugs and steroids), 42% had failed at least one DMARDS test. All patients gave fully informed written consent approved by the institutional ethics committee, and research was performed in accordance with the Declaration of Helsinki. Synovial tissue biopsies were obtained at arthroscopy under local anaesthetic using a Wolf 2.7 mm telescope (RWolf, Illinois, USA) as previously described.7 Biopsies were either OCT embedded (Tissue-Tek, Zoeterwoude, The Netherlands) for immunohistochemical analysis, established as ex vivo RA whole-tissue synovial explant cultures, or snap-frozen in liquid nitrogen for protein analysis.
Immunohistology/immunofluorescence (see online supplementary file 1 material and methods)
Culture of synovial fibroblasts
Primary RASFC were isolated from RA biopsies by digestion with 1 mg/mL collagenase type 1 (Worthington biochemical, Freehold, New Jersey, USA) in RPMI 1640 containing 10% fetal bovine serum (FCS) (Life Technologies-BRL, Paisley, UK) for 4 h at 37°C in humidified air with 5% CO2. Dissociated cells were grown to confluence and used between passages 4 and 8. K4IM were cultured in RPMI 1640 supplemented with 10% FCS, penicillin (100 units/mL; Bio-sciences, Dublin, Ireland), streptomycin (100 units/mL; Bio-sciences), fungizone (0.25 µg/mL; Bio-sciences) and HEPES (20 mM; Life Technologies-BRL). K4IM were grown to confluence, and and used between passages 35 and 38.
Functional assaysTranswell Invasion assays, ELISA, Network Formation, Cell Migration, Cell viability and Protein preparation (see online supplementary file 1 material and methods).
RNAi gene-silencing studies
For each 25 cm2 flask of RASFC or K4IM to be transfected, 5 µL of 50pmol gene-specific siRNA duplexes (Stat-3 or Scramble) and 5 µL of Lipofectamine 2000 Reagent (Invitrogen, BioSciences, Ireland) were mixed gently with 0.99 mL serum/antibiotic free OPTI-MEM (Invitrogen, BioSciences, Ireland) and incubated at room temperature for 20–30 min in the dark. The combination was mixed with RPMI 1640 containing 10% FCS, added to the cells and incubated for 48 h. The siRNA duplexes for STAT3 (NM-139276), HIF1α (NM_001530) and Scrambled control (a nonsense siRNA of the target sequence) were sourced from Sigma. Nuclear protein and whole cell lysates were extracted to assess knockdown efficiency of HIF1α and STAT3 in RASFC and K4IM by western blot.
Western blot analysis
Protein (20–50 µg) was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (5% stacking, 10% resolving), gels were then transferred onto nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK) prior to 1 h blocking in wash buffer containing 5% non-fat milk, with gentle agitation at room temperature (RT). Membranes were incubated with rabbit polyclonal anti-p-STAT3 (1:500; Cell Signalling Technology), STAT3 (1:500; Cell Signalling Technology), anti-p-STAT1 (1:500; Cell Signalling Technology), STAT1 (1:500; Cell Signalling Technology), rabbit polyclonal anti-Notch-1 (1:500; Millipore, Temecula, California, USA) or mouse monoclonal anti-HIF1α (1:500, BD Biosciences, Oxford, UK) diluted in 5% non-fat milk containing 0.1% Tween 20 at 4°C overnight with gentle agitation. β-actin (1:5000, Sigma) was used as a loading control. Following three 15 min washes, membranes were incubated with appropriate horseradish peroxidase conjugated secondary antibodies (1:1000) for 3 h at RT. The signal was detected using SuperSignal West Pico Chemiluminescent Substrate (Amersham Biosciences), and density of the bands was analysed using EDAS 120 system from Kodak (Kodak, Rochester, New York, USA).
mRNA Purification and real-time PCR analysis
Total RNA from K4IM was isolated using RNeasy Mini Kit (Qiagen, Crawley, UK) and from RA synovial explant biopsies using miRNeasy Mini Kit (Qiagen) according to manufacturer's instructions. The purity of the RNA was measured by spectrophotometry, a ratio over 1.8 (260:280 nm) of samples was used and 1–2 µg total RNA was transcribed to cDNA. Relative quantification of gene expression was analysed using Lightcycler-480 PCR technology (Roche Diagnostics, Lewes, UK) with preoptimised conditions. Specific primers for Notch-1 (Hs00413187_m1), DLL-4 (Hs00184092_m1), hrt-1 (Hs01114113_m1) and hrt-2 (Hs00232622_m1) were acquired from Applied Biosystems (Applied Biosystems, Cheshire, UK). Primers for 18S ribosomal RNA (Hs99999901_m1) and β-actin (Hs99999903_m1) were used as an endogenous control.
RA synovial tissue explant culture model ex vivo
To investigate the effect of WP1066 on cytokine and MMP-3 production in the arthritic joint, an ex vivo RA synovial explant model was established. This system maintains the synovial architecture and cell-cell contact, and spontaneously releases pro-inflammatory mediators.30 ,31 RA synovial biopsies were sectioned into 1 mm cubes and cultured immediately from arthroscopy (to maintain maximal activity of the inflamed synovium) in 96-well plates (Falcon, Franklin Lakes, New Jersey, USA) in RPMI 1640 (Life Technologies-BRL) supplemented with streptomycin (100 units/mL; Bio-sciences) and penicillin (100 units/mL; Bio-sciences). Explants were cultured in the presence of 2.5, 5 or 10 µM WP1066 or dimethyl sulfoxide (DMSO) vehicle control for 72 h. Following culture, wet weight of each biopsy was obtained and biopsies were stored in RNA later, and supernatants were harvested.
SPSS15 system for Windows was used for statistical analysis. Parametric Student t tests were used to analyse normally distributed data and non-parametric Wilcoxon Signed Rank test and non-parametric analysis of variance (ANOVA) were used for analysis of RA synovial tissue; p<0.05 was statistically significant.
Localisation and detection of p-STAT3 in the inflammatory joint
Immunohistology analysis was performed to detect p-STAT3 localisation in synovial tissues biopsies from RA patients (n=12) and healthy control synovial tissue (n=9) (figure 1A,B). p-STAT3 was detected in synovial tissue lining, sublining and vascular regions (figure 1A, i–iv) with minimal detection observed in normal healthy control tissue (figure 1A, v and vi). No expression was observed for IgG control. Figure 1C shows representative blots of p-STAT3 protein in RA synovial biopsy lysates demonstrating a marked increase in p-STAT3 in RA synovial tissue compared to healthy control tissue, with no differences observed for global expression of total STAT3.
STAT3 and HIF1α interaction
A stable human synoviocyte line (K4IM) from a healthy donor immortalised with SV40T antigen (TAg) was also used. This line maintains many of the phenotypic properties of RASFC.32 Previous studies have shown that they retain the ability to proliferate in response to physiological signals, and that overexpression of specific genes can further potentiate proliferation of these cells and promote cell division.7 ,33 Therefore, the K4IM cell line represents a valuable tool, in parallel with RASFC, to study mechanisms that induce or maintain synoviocyte activation. Initial experiments demonstrated that 3% hypoxia dramatically increased the level of p-STAT3 protein detected compared with normoxic control in K4IM; phosphorylation of STAT3 was completely inhibited by the presence of WP1066 (figure 2A). Although the STAT3β of total STAT3 protein was reduced by WP1066, the major isoform STAT3α remained unaltered. In parallel, p-STAT1 protein detection was also increased under hypoxic condition, the phosphorylation of STAT1 was inhibited by WP1066 (figure 2A). Additionally, hypoxia-induced HIF1α expression was decreased in the presence of WP1066 (figure 2A). Figure 2B shows representative images of increased cell invasion following exposure to 3% hypoxia compared to normoxic control (*p<0.05). Quantification of hypoxia-induced invasion is graphically illustrated, an effect which was significantly blocked in the presence of WP1066 (*p<0.05). Furthermore, in culture supernatants 3% hypoxia significantly increased IL-6 and IL-8 expression (*p<0.05), an effect which was significantly inhibited by WP1066 (*p<0.05) (figure 2C). These cytokines are endogenously expressed under basal conditions, as is p-STAT3, therefore, WP1066 also inhibited basal production of IL-6 and IL-8. The effect of WP1066 on hypoxia-induced cytokine production was similar to the level of inhibition observed under basal conditions. Further experiments transiently transfected RASFC and K4IM with HIF1α siRNA or scrambled control for 48 h, and showed that hypoxia-induced p-STAT3 protein was significantly inhibited with HIF1α siRNA compared with scrambled control in RASFC and K4IM (figure 2D). Hypoxia-induced RASFC HIF1α protein expression was also markedly decreased in the presence of STAT3 siRNA (figure 2E). STAT3 knockdown transfection had no effect on cell viability (online supplementary figure 1D).
Hypoxia-induced synovial fibroblast invasion and migration is STAT3 dependent
Figure 3A shows a representative image of p-STAT3 nuclear staining in primary RASFC following exposure to 3% hypoxia. Furthermore, hypoxia-induced p-STAT3 levels in RASFC was inhibited in the presence of WP1066 (figure 3B). To further investigate the effect of STAT3 blockade, RASFC were transiently transfected with Stat3 siRNA or scrambled control for 48 h (figure 4A) and invasion and migration was examined. Transient transfection of RASFC with Stat3 siRNA significantly inhibited hypoxia-induced migration (*p<0.05) (figure 4B). Representative images showing inhibition of RASFC migration are shown in online supplementary figure 1A. Furthermore, 3% hypoxia significantly induced RASFC network formation on matrigel (figure 4C) (p<0.05) an effect that was significantly inhibited in the presence of Stat3 siRNA (p<0.05). Therefore, these experiments clearly provide evidence that STAT3 does mediate hypoxia-induced effects in RASFC.
STAT3 inhibition regulates Notch signalling pathways
To further explore the mechanisms involved in hypoxia/STAT3 signalling interactions, we examined the effect of Stat3 siRNA and WP1066 on the Notch signalling pathway; 3% hypoxia-induced Notch-1IC expression, an effect that was completely inhibited by Stat3 siRNA and WP1066 (figure 5A,B). Furthermore, 3% hypoxia-induced gene expression of Notch-1 receptor, its ligand DLL-4 and downstream target genes hrt-1 and hrt-2 were inhibited by WP1066 (*p<0.05) (figure 5C). While gene expression of Notch-1 receptor was inhibited to near-basal levels following WP1066, hypoxia-induced DLL-4 and hrt-1 and hrt-2 were only partially inhibited, similar to that observed under basal conditions. Notch components mRNA levels were also examined in RA explants ex vivo cultured with WP1066. Notch-1 receptor gene expression was significantly decreased in the presence of WP1066 (figure 5D) (*p<0.05), however, no significant differences were observed for hrt-1 and hrt-2 genes. DLL-4 expression was undetectable (figure 5D).
Inhibition of pro-inflammatory cytokines in RA synovial explant culture ex vivo
RA synovial explants were established, cultured with WP1066, and spontaneous cytokine secretion was assessed. WP1066 significantly inhibited spontaneous release of IL-6, IL-8 and MMP3 from RA explant cultures (*p<0.05) (figure 6A–C). By contrast, IL-10, was significantly increased in response to WP1066 (*p<0.05) (figure 6D).
In this study, we examine the role of STAT3 in mediating key pro-inflammatory pathways involved in the pathogenesis of RA. We demonstrated increased p-STAT3 nuclear staining in RA synovial tissue compared with control tissue. We showed that 3% hypoxia induced p-STAT3/p-STAT1 protein levels in RASFC and K4IM, an effect that is blocked by HIF1α siRNA. We demonstrated that hypoxia-induced cytokine production, cell migration and invasion were inhibited by siRNA STAT3 or WP1066. Blockade of STAT3 signalling also inhibited hypoxia-induced HIF1α expression. Furthermore, hypoxia-induced expression of Notch-1IC, its ligand DLL4 and downstream target genes hrt-1 and hrt-2 were inhibited by WP1066. Finally, using RA synovial tissue explants ex vivo, STAT3 blockade significantly inhibited IL-6, IL-8, MMP3 and Notch-1 receptor mRNA in contrast with IL-10 which was significantly induced. Together, these results provide evidence of a functional link between HIF1α, STAT3 and Notch-1 signalling pathways in the regulation of pro-inflammatory mechanisms in RA.
Increased nuclear p-STAT3 protein was detected in RA-inflamed synovium compared with normal control synovium, localised to sublining and lining layer regions. This is consistent with previous studies which have demonstrated increased STAT3 in RA synovial tissue associated with severity of synovitis,16 transfection of RA synovial fluid mononuclear cells with Stat3 siRNA inhibits Th17 differentiation,17 and STAT3-mutated RA synoviocytes fail to grow in culture.18 A highly selective JAK2 inhibitor has been shown to ameliorate disease in CIA and CAIA models of arthritis, with decreased paw STAT3 expression.34 In gp130 mutant mice, development of lymphocyte-mediated RA is mediated through STAT3/IL-7-dependent homeostatic proliferation of CD4 T cells.35
We also showed in these studies, that 3% hypoxia induced nuclear translocation of p-STAT3 in RASFC which are the invasive cells that destroy adjacent cartilage and bone. WP1066, is a tyrphostin analogue of AG 490 which is a selective JAK2 inhibitor.36 Studies have shown that WP1066 inhibits p-STAT3 in PBMCs and increases T-cell cytotoxicity by inhibition of Tregs in melanoma.37 Furthermore, WP1066 can alter proliferation/apoptotic mechanisms in vivo and ex vivo,38–41 and is thought to have a more potent effect on STAT3.36 We showed that hypoxia-induced p-STAT3 was inhibited in the presence of WP1066. In parallel, hypoxia-induced p-STAT1 was also inhibited by WP1066 consistent with previous studies.39 ,42–44 Furthermore, we showed that Stat3 siRNA or WP1066 inhibited hypoxia-induced cell migration, invasion and expression of pro-inflammatory mediators, however, the inhibitory effects of Stat3 siRNA on hypoxia-induced function was greater than that of WP1066, suggesting that WP1066 may only partly mediate hypoxia-induced pathways. Interestingly, while HIF1α siRNA blocked hypoxia-induced p-STAT3 in RASFC and K4IM, we also demonstrated that Stat3 siRNA inhibited hypoxia-induced HIF1α expression in RASFC, suggesting HIF/STAT bidirectional interactions.
The important role of hypoxia in driving inflammatory responses in RA has been shown by several in vitro and in vivo studies. We have previously demonstrated, in vivo, that the inflamed synovial joint is profoundly hypoxic,7 HIF1α expression has been demonstrated in RA synovium, and hypoxia induces key pro-inflammatory mediators involved in RA pathogenesis.8 ,45 ,46 The interaction between HIF1α and STAT3 is supported by studies showing that HIF1α facilitates the binding of STAT3 to the haptoglobin promoter in HepG2 human hepatoma cells.24 STAT3 inhibits HIF1α degradation through competition with Von Hippel–Lindau tumor suppressor (pVHL) for binding to HIF1α, thus stabilising HIF1α protein levels in tumour cells,25 and p-STAT3 is a potential regulator of HIF1α-mediated VEGF expression in renal carcinoma cells.26
Consistent with our previous studies, we demonstrated that 3% hypoxia induced Notch-1IC, Dll-4, Hrt-1 and Hrt-2 expression,28 and is supported by studies showing that Notch/HIF1α interactions mediate hypoxia-induced EC function in vitro.27–29 Crosstalk between Notch-HIF1α signalling has been demonstrated in studies showing protein–protein interaction between Notch-1 and HIF1α.29 Additionally, in this study, we showed that Stat3 siRNA or WP1066 inhibited hypoxia-induced Notch signalling suggesting a functional link between these pathways. While hypoxia-induced Notch-1 receptor mRNA was inhibited to near-basal levels following WP1066, DLL-4, hrt-1 and hrt-2 were only partially inhibited. WP1066 also inhibited Notch-1 receptor in the RA explants. The interactions between Notch and its ligands are complex, and lateral inhibition can occur. Activation of Notch signalling requires binding of Notch to its ligands DLL or Jagged on the adjacent cell, which initiate proteolytic cleavage of notch intracellular domain (NICD), and translocation into the nucleus where it activates downstream transcription. Once activated, lateral inhibition to suppress the signal in the primary activating cell occurs.47 Furthermore, studies have shown that Jagged-1 can antagonise DLL-mediated Notch signalling.48 Therefore, negative and positive feedback processes are involved.47 ,48
In animal models, p-STAT3 mediates LIF-induced neural stem progenitor expansion through activation of Notch-1 following hypoxia/ischemia conditions.49 STAT3 inhibition reduces the hypoxia-induced Notch signalling in glioblastoma stem cells.50 Hes/STAT3 interactions have been shown to regulate HIF1α expression.27 Jagged1/Notch interactions regulate inflammatory responses through the NFκB and Jak/STAT/SOCS signalling pathways,51 and STAT-3 is required for hypoxia-induced VEGF expression.26 These studies, therefore, suggest that depending on the cell type and the inflammatory milieu, Notch and STAT signalling interactions may cooperate in the regulation of HIF1α.
To further examine the potential role of p-STAT3 in the pathogenesis of RA, we investigated the effect of WP1066 on pro-inflammatory mediators using an RA whole-tissue synovial explant model ex vivo, which closely reflects the in vivo joint environment.30 ,31 WP1066 significantly inhibited spontaneous secretion of IL-6, IL-8 and MMP3, and induced IL-10 secretion in RA explants. Previous studies have demonstrated the inhibitory effects of WP1066 mainly in tumour models, where it prevents the proliferative effect and neointima formation,52 induces apoptosis in malignant glioma cells,36 ,38 and inhibits RANTES, MCP-1, VEGF in melanoma models.53 More recent data has shown that CP690550 inhibits TNF-induced expression of IP-10, RANTES and MCP-1 in RASFC,54 and inhibits IL-4-dependent Th2 cell differentiation and Th17 cell differentiation.55 Tofacitinib has now been approved for the treatment of RA.56
In conclusion, this is the first study to provide evidence of a functional link between HIF1α, STAT3 and Notch-1 signalling in the regulation of pro-inflammatory mechanisms in RA. The inhibitory effects on cytokine and MMP production in the synovial explant model further support a role for blockade of Jak/STAT signalling pathways in the treatment of RA.
Handling editor Tore K Kvien
Contributors All authors contributed to this manuscript. UF had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design; acquisition of data; analysis and interpretation of data: WG, JMC, MC, EB, DJV and UF.
Funding This work was supported by the Health Research Board of Ireland and EU IMI ‘BeTheCure’ programme.
Competing interests None.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the St Vincent's University Hospital Ethics Committee.
Provenance and peer review Not commissioned; externally peer reviewed.