Rheumatoid arthritis (RA) is a chronic progressive inflammatory disease of multi-factorial aetiology and a systemic disorder characterized by synovium inflammation and subsequent destruction and deformity of joints.1,2 Signaling pathways that regulate the production of cytokines and destructive enzymes have been implicated in its pathogenesis and represent potential therapeutic targets. Heterotrimeric guanine nucleotide binding proteins (G protein) pathway and mitogen-activated protein kinases (MAPKs) pathway play important roles in chronic synovitis in RA.3
G proteins are biochemical transducers operating allosteric regulatory elements. G proteins-adenylyl cyclase (AC)-cyclic adenosine 3’,5’-monophosphate (cAMP) signaling pathway is an important mediator of extracellular signals from prokaryotes to higher eukaryotes. The signaling cascade of MAPK kinase (MEK)/extracellular signal-related kinase (ERK) is a ubiquitously expressed intracellular signaling pathway that controls cellular functions such as proliferation, differentiation and apoptosis through a series of sequential phosphorylation events.4 Raf (a serine-threonine kinase) phosphorylates and activates MEK1/2, which, in turn, phosphorylates ERK1/2.5,6 MAPK cascades are involved in inflammation and tissue destruction in RA. In particular, ERK1/2 is highly activated in RA fibroblast-like synoviocytes (FLS). Activation of ERK1/2 significantly contributes to destructive arthritis in transgenic mice.7
There are cross-talks between MAPKs signaling pathway and G proteins-AC-cAMP signaling pathway. MAPK cascades can be activated by G proteins-coupled receptors (GPCRs)/G-protein components. G-protein-coupled receptor kinase (GRK) phosphorylate receptors, which result in receptor/G-protein uncoupling via subsequent binding of arrestin proteins. GRK2 and MEK were in the same multimolecular complex. GRK2 regulated the activity of ERK, which involves a direct or coordinate interaction with MEK at the level of the MEK-ERK interface. ERK1/2 is activated by Gi protein in FLS of patients with RA. Activation of ERK1/2 is inhibited by pertussis toxin (PT, a Gi-protein inhibitor).8
Collagen-induced arthritis (CIA) in rats is an experimental model that shares some features with human RA, such as swelling, cartilage degradation and loss of joint function.9 Here we evaluated interactions and cross-talks between MEK1/2-ERK1/2 signaling and G protein-couple signaling in synoviocytes of CIA rats stimulated with interleukin 1 (IL-1, an inflammatory factor), U0126 (a selective inhibitor of MEK1/2), isoprenaline hydrochloride (an agonist of beta adrenergic receptor) and aminophyline (a non-selective inhibitor of phosphodiesterases (PDEs)), respectively.
Thirty male Sprague-Dawley (SD) rats ((180±20) g) were purchased from Shanghai BK Experimental Animal Center (Grade II, Certificate No D-65) (10 in normal group and 20 in CIA group). All rats were acclimatized under standard laboratory conditions. During the experimental period, all rats were housed five per cage and fed with a standard laboratory feed and water. The rats were also kept on a 12-hour dark vs 12-hour light cycle at a constant temperature of (20±5)°C. The experimental protocols in this study were reviewed and passed by the Ethics Review Committee for Animal Experimentation of Institute of Clinical Pharmacology, Anhui Medical University.
Induction of CIA
Chicken type II collagen (Institute of Bencao Biological Medicine, Shanghai, China) was dissolved in acetic acid 0.1 mmol/L and emulsified with an equal volume of complete Freund’s adjuvant (CFA) in ice-bath. The final concentration of type II collagen and Bacillus Calmette Guerin (BCG, Shanghai Biological Products Factory) were both 1 g/L. Emulsion 1 ml was injected intradermally into the base of the tail, back and left hind paw of rats. On day 7 after immunization, rats were given a booster injection. Control group was received the injection of an equal volume of 0.1 mmol/L acetic acid at the same location.10
The legs and paws of rats were removed at day 28 after immunization and fixed with 10% formaldehyde in PBS. Then the samples were decalcified for 10 days with EDTA and embedded in paraffin for histological analysis. The paraffin sections were stained with hematoxylin and eosin.
Preparation and culture of synoviocytes
Synovium tissues were digested in RPMI-1640 medium containing 0.4% (w/v) collagenase type II and 0.25% (w/v) trypsinase for 2 hours at 37°C in 5% CO2 atmosphere. After incubation, the tissue was filtrated into a sterile centrifuge tube through sterile 108 μm2 nylon mesh. Cells (1×109 cells/L) were washed three times and incubated in 5% CO2 atmosphere at 37°C for 5-7 days. Finally primary FLS was obtained.
Rats with CIA were anaesthetized and decapitated on day 0, day 20, day 28 and day 35 respectively. Synoviocytes were isolated according to the method above. The cells were suspended in RPMI-1640 containing 10% FBS at a density of 1×109 cells/L and cultured for 24 hours. Western blot was applied to examine the expressions of Gi, p-MEK1/2 and p-ERK1/2 in synoviocytes.
Effects of MEK1/2-ERK1/2 signaling on Gi protein-couple signaling
To investigate the effects of MEK1/2-ERK1/2 signaling on Gi protein-couple signaling, synoviocytes (1×109 cells/L) of CIA rats and rIL-1α (10 ng/ml) were incubated for 30 minutes, and U0126 (25 μmol/L) was added. The cultures were incubated for 30 minutes again. Western blot was used to examine the changes of Gi, p-MEK1/2 and p-ERK1/2. The level of cAMP was measured by CPBA. Protein kinase A (PKA) activity was assessed by kinase-glo® luminescent kinase assay kit (Promaga, USA).
Effects of Gi protein-couple signaling on MEK1/2-ERK1/2 signaling
To examine the effects of Gi protein-couple signaling on MEK1/2-ERK1/2 signaling, synoviocytes of CIA rats were suspended in RPMI-1640 containing 10% FBS at a density of 1×109 cells/L. U0126 (25 μmol/L), isoprenaline hydrochloride (100 μmol/L) and aminophyline (100 μmol/L) were added respectively. Then the cultures were incubated at 37°C in 5% CO2 for 1 hour. Western blotting was used to examine the changes of Gi, p-MEK1/2 and p-ERK1/2. The level of cAMP and PKA activity were measured according to the methods above.
Western blotting of Gi, p-MEK1/2 and p-ERK1/2 in synoviocytes
Synoviocytes were suspended in 100 μl lysis buffer. The soluble fraction was obtained by centrifugation at 14 000 r/min for 15 minutes. Protein samples (20 μg per lane) were run on 12% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) microporous membranes (Millipore, Beijing, China) at 50 mA in transfer buffer containing 25 mmol/L trisbase, 192 mmol/L glycine and 20% methanol. Western blotting analysis was performed. Briefly, blots were blocked with PBS saline plus 0.05% Tween 20 and 5% milk for 1 hour. Then the blots were incubated with anti-Gi antibody (1:2500, rat antibody, Santa Cruz, USA) in 4°C overnight. The membranes was washed 3 times and incubated with horseradish peroxidase-conjugated secondary antibody (Zhongshan Golden Bridge Biotechnology Co. LTD., Beijing, China) for 2 hours at room temperature. The proteins were visualized by an enhanced chemi-luminescence system.11
The procedure for Western blotting of p-MEK1/2 and p-ERK1/2 was the same as one described for Western blotting of Gi, except for using antibodies to detect p-MEK1/2 (Ser218/Ser22212-B, goat polyclonal antibody, Santa Cruz Biotechnology, USA) and p-ERK1/2 (E-10, goat polyclonal antibody, Santa Cruz). The Hema GSM-3.0 gel graph analyzing system was used to calculate the numerical value of every blot. The mean values of Gi, p-MEK1/2 and p-ERK1/2 from three different experiments were depicted as bar graphs. All the experiments reported in this study were performed three times and the results were reproducible.
Analysis of the level of cAMP and PKA activity in synoviocytes
Synoviocytes were isolated and suspended in RPMI-1640 containing 10% FBS at a density of 1×109 cells/L. U0126, isoprenaline hydrochloride and aminophyline were added respectively. Then the cultures were incubated at 37°C in 5% CO2 for 2 hours. The reactions were terminated by centrifugation for 20 seconds. The supernatant was quickly removed and the pellet was re-suspended in 250 μl Tris-EDTA (50 mmol) buffer at pH 7.5. The samples were placed in a boiling water bath for 5 minutes and frozen. After thawing, samples were sonicated. The protein flocculate was pelleted by centrifugation. The supernatants were collected and stored at -80°C for measurement. The level of cAMP was conducted by using competitive protein binding assay (CPBA) according to the previous procedures.12 PKA activity was assessed by kinase-glo® luminescent kinase assay kit. The kinase reactions were mixed at room temperature, and luminescence was measured 10 minutes later on a GLOMAXTM 96 Microplate luminoteter (Promega).
Data were expressed as means ± standard deviation (SD). Analysis of variance (ANOVA) and non-parametric tests from the program Sigma Stat were used to determine significant differences among the groups. A P value less than 0.05 was considered statistically significant. The Western blotting was repeated at least three times for each sample and subjected to semiquantitative analysis to ensure maximal accuracy of the conclusions drawn from these data.
Histopathology changes in knee joint of CIA rats
There was remarkable inflammation in CIA rats accompanied by swelling paws. The slides of histological pathology demonstrated the fact that the synovium of healthy rats had no inflammatory cells. Pannus and cartilage erosion were not found yet. Compared to those of normal (Figure 1A), the synovium of CIA rats was hyperplastic. Synoviocytes increased. Pannus and cartilage erosion were formed. Inflammatory cells infiltrated into synovium (Figure 1B).
Changes of the expression of Gi, p-MEK1/2 and p-ERK1/2 proteins in synoviocytes of CIA rats
The expression of Gi, p-MEK1/2 and p-ERK1/2 proteins in synoviocytes of CIA rats was analyzed on day 0, day 20, day 28 and day 35 respectively. Results showed that the peak content of Gi was on day 28. The peak content of p-MEK1/2 was on day 20. The expression of p-ERK1/2 was high from day 20 to day 28 (Figure 2).
MEK1/2-ERK1/2 signaling regulated Gi protein-couple signaling
rIL-1α increased the expression of Gi, p-ERK1/2 and p-MEK1/2 in synoviocytes of CIA rats. U0126 inhibited the expression of Gi, p-MEK1/2 and p-ERK1/2 in synoviocytes stimulated by rIL-1α (Figure 3). rIL-1α also enhanced the level of cAMP and PKA activity in synoviocytes of CIA rats. U0126 could suppress cAMP and PKA in synoviocytes stimulated by rIL-1α (Table).
Gi protein-couple signaling regulated MEK1/2-ERK1/2 signaling
It showed that isoprenaline hydrochloride increased the expression of Gi protein and had no effects on p-MEK1/2 and p-ERK1/2 expression. Aminophyline had no effect on Gi protein, but inhibited the expression of p-MEK1/2 and p-ERK1/2 (Figure 4). Isoprenaline hydrochloride and aminophyline improved cAMP level and PKA activity (Table 1).
The role of synoviocytes in mediating inflammation and joint destruction in RA has been investigated extensively in recent years. A hallmark of RA was the pseudo-tumoral expansion of FLS, and FLS had therefore been proposed as a therapeutic target.13 The normal synovium consists of a lining that was two- to four-cell layers thick. In RA, inflammatory synovium was found characterized by accumulation of T cells, plasma cells, macrophages and pannus.14 CIA in rat was a classic model in investigating the mechanism of RA. In our study, paws of CIA were swelling. Synovial tissue was hyperplasia accompanied by cartilage erosion, inflammatory cells and pannus.
Signaling pathways were changed with the increased function of synoviocytes.15 RA patients showed a significantly increased cAMP production in lymphocytes by beta2-adrenergic stimulation.16 Our previous studies showed that Gs mRNA and cAMP level of synoviocytes decreased in CIA rats, but Gi mRNA increased. The functions of Gi and Gs proteins were imbalanced.17,18 In the present study, Gi protein expression was enhanced. cAMP level and PKA activity were reduced. It suggests that the Gi protein-AC-cAMP signal pathway is probably one of the important mechanisms of inflammatory and immune response of CIA.
Expression of MAPKs had been at high levels in the RA synovium. MEK and ERK were activated by phosphorylation at specific sites in synoviocytes. Activation of the MEK/ERK signaling pathway promoted the proliferation and survival of cells. In this study, we found the fact that the expression of p-MEK1/2 and p-ERK1/2 in synoviocytes of CIA rats increased. rIL-1α induced phosphorylation of MEK1/2 and ERK1/2 in synoviocytes of normal rats. These results suggested that MAPKs signal transduction is involved in joint damage and synovitis.
There were cross-talks between G protein-coupled signaling and MAPK cascades. MAPKs cascades in FLS were induced by pro-inflammatory cytokines and could be regulated by G proteins-coupled signaling in RA. Beta-PDGF receptor used a classical GPCR-mediated pathway to induce efficient activation of p42/p44 MAPK in response to PDGF. RGS12 domain could reduce the PDGF-induced activation of p42/p44 MAPK.19 Pertussis toxin (PT) and cholera toxin (CT) inhibited phosphorylation of ERK, c-Jun N-terminal kinase (JNK), and p38. The phosphorylation was induced by rIL-1α in AA-derived rat FLS.20 Beta-adrenergic receptors belong to the GPCR family whose function was turned off by GRK. Isoproterenol is a beta1-adrenergic receptors agonist. Gs alpha mediated isoproterenol, induced the stimulation of AC, and activated cAMP synthesis. The present studies showed that isoprenaline hydrochloride increased Gi protein expression, cAMP level and PKA activity, while had no effects on p-MEK1/2 and p-ERK1/2 expression. Aminophyline, which is a non-selective inhibitor of PDEs, elevated cAMP level and PKA activity and inhibited the expression of p-MEK1/2 and p-ERK1/2. But aminophyline had no effect on Gi protein.
U0126 and SB203580, which were inhibitor of MEK1/2 and p38 respectively, decreased the expressions of Gi1, Gi2, Gi3, Gs and the level of cAMP induced by rIL-1α in AA-derived rat FLS.20 cAMP-responsive element binding protein (CREB) mediated CRE binding via ERK1/2 and PKA pathway respectively.21 Vasodilator-stimulated phosphoprotein (VASP) was a major substrate of PKA. Gα13-induced VASP phosphorylation was dependent on activation of RhoA and MEKK1, leading to the stimulation of the nuclear factor kappa B signaling pathway.22 rIL-1α was an important pro-inflammation cytokine which mediated bone resorption and cartilage destruction by inducing MAPK signal transduction in RA.23 U0126, which was one of tyrosine-kinase blockers and inhibited MEK1/2, was also able to block inflammatory responses and the proliferation of synoviocytes.24 Results of the study showed that rIL-1α induced the expression of Gi, p-MEK1/2 and p-ERK1/2 and increased cAMP level and PKA activity. U0126 decreased cAMP level and PKA activity in synoviocytes stimulated by rIL-1α. Meanwhile U0126 inhibited Gi, p-MEK1/2 and p-ERK1/2.
These results suggest that the functional responses of Gs-couple-beta-adrenergic receptors were decreased in pathological symptom with CIA and Gi activity was enhanced. These changes inhibited AC activity resulting in the lower level of cAMP and PKA activity. Activation of PKA down-regulated Ras activity,25 but PKA activity was decreased with the change of cAMP level in synoviocytes of CIA rats. Therefore the suppressed effects of Gs-cAMP-PKA pathway on Ras-MAPKs were impaired. Furthermore, activation of PKC was up-regulated by Gi-coupled PLC activation in synoviocytes of CIA rats. PKC could activate the small G protein Ras and phosphorlate c-Raf directly, and then mediated MAPKs signaling pathway.26 Therefore MAPKs signaling pathway was regulated by G protein-couple signaling pathway. IL-1 activated Ras/Raf/MEK/ERK cascades and stimulated the translocation of NF-kappaB from the cytosol to the nucleus of synovial fibroblasts. In the end PGE2 was released. Furthermore, IL-1 might induce EP2 and EP4 mRNA expression in synovial fibroblasts. EP2 and EP4, which were PGE2 receptors, activated AC via Gs proteins and increased cAMP level. These results also suggest that cAMP was produced via PGE2. PGE2 might block IL-1-stimulated ERK activation and attenuate cytokine-induced inflammatory responses via regulation of the localization of NF-kappaB dimmers.27 U0126 decreased cAMP and PKA in FLS stimulated by IL-1 and inhibited Gi, p-MEK1/2 and p-ERK1/2.
In summary, our results suggest that signaling mediated by G-protein-AC-cAMP and MAPKs play critical roles in the inflammation and joint destruction in RA. These findings also reveal cross-talks between G protein-AC-cAMP and MAPKs signal transduction pathways in FLS of CIA rats. Activation of MAPKs was regulated by at least two G protein-coupled signaling such as Gi and Gs signal transduction pathway. Inhibition of MAPKs cascades show a negative feedback to G protein associated transmembrane signaling in CIA-derived rat FLS.
This work was also supported by the Key Laboratory of Anti-inflammatory and Immune-pharmacology in Anhui Province, Key Laboratory of Research and Development of Chinese Medicine in Anhui Province, Engineering Technology Research Center of Anti-inflammatory and Immune-pharmacology in Anhui Province and Scientific and Technological Team of Anti-inflammatory and Immune-pharmacology of Chinese Medicine in Anhui Province. We are grateful to Professor WANG Yuan, ZHOU Qing and YANG Feng.
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Keywords:© 2008 Chinese Medical Association
arthritis; signaling; cross-talk; G protein; MAP kinases