The Cross Talk Between G Protein-AC-cAMP Signaling And Ras-Raf-MEK-ERK Signaling In Fibroblasts Like Synoviocytes Of Rats With Collagen-Induced Arthritis And The Regulation Of Paeoniflorin

Rheumatoid arthritis (RA) is a chronic progressive inflammatory disease of multi-factorial aetiology and is a systemic disorder characterized by synovial inflammation, subsequent destruction and deformity of joints. The pivotal role of pro-inflammatory cytokines such as interlukin-1 (IL-1), tumor necrosis factor (TNF)-alpha and inflammatory mediators like prostaglandin E2 (PGE2) had been demonstrated in basic research. 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, mitogen-activated protein kinases (MAPKs) pathway and so on play a number of important roles in RA chronic synovitis. 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. In our research group, we found that the mRNA expression of stimulatory subunit of G protein (Gs) and cAMP level of synoviocytes of rats with adjuvant arthritis (AA) decreased, and mRNA expression of inhibitory subunit of G protein (Gi) was enhanced. The expressions of Gi1, Gi2 and Gi3 in synoviocytes of AA rats increased abnormally and Gs expression of synoviocytes decreased. The expressions of EP2 and EP4, which were the receptors of PGE2, reduced also. Membrane location ofβ-arrestins and G protein-coupled receptor kinase2 (GRK2) of synoviocytes decreased. These results suggest that G protein-coupled receptors (GPCRs), the regulatory proteins of GPCRs, G protein and G proteins-coupled signaling were abnormal in synoviocytes of AA rats. In synoviocytes of rats with collagen-induced arthritis (CIA), mRNA expression of Gs and cAMP level of synoviocytes decreased, and mRNA expression of Gi was enhanced. Pertussis toxin (PT)–catalyzed ADP-ribosylation of Gαincreased and cholera toxin (CT) catalyzed ADP-ribosylation of Gαreduced. The expressions ofβ-arrestins, GRK2, GRK5 and GRK6 were also enhanced. The results suggest that the functions of Gi and Gs proteins in synoviocytes of CIA rats were imbalance, and G protein coupled signaling was abnormal. The signaling cascade of Ras- Raf- 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. Ras is a monomeric GTP-binding protein that is involved in transmission of mitogen signals from the plasma membrane to the nucleus through the Raf/MEK/ERK kinase cascade. Raf (a serine-threonine kinase) phosphorylates and activates MEK1/2, which, in turn, phosphorylates ERK1/2. MAPK cascades are involved in inflammation and tissue destruction in RA. In particular, ERK1/2 is highly activated in RA fibroblast-like synoviocytes (FLS). In period research, we found that the phosphorylation of ERK, c-Jun N-terminal kinase (JNK) and p38 increased in AA rat, and rIL-1αcould phosphorylated ERK, JNK and p38 rapidly. These results suggest that MAPKs signaling were abnormal in synoviocytes of AA rats.Reports showed that GRK2 and MEK were in the same multimolecular complex in human embryonic kidney (HEK) 293 cell, GRK2 regulated the activity of ERK, and the presence of increased levels of GRK2 in HEK 293 cells produced a significant reduction of the ERK response to CCL2. In mouse cardiomyocytes, stimulation of beta-adrenergic receptor (beta AR) activates MAPKs, particularly ERK1/2 which is involved in the regulation of a multitude of cellular processes. In N18TG2 neuroblastoma cells, Gi protein could activate ERK1/2, the activation of ERK1/2 was inhibited by PT, which was a Gi protein inhibitor. In HEK 293 and airway smooth muscle cells, platelet derived growth factor (PDGF) receptor uses a classical GPCR-mediated pathway to induce efficient activation of ERK1/2 in response to PDGF. RGS12 reduced the PDGF-induced activation of ERK1/2. In our group, we found that CT inhibited phosphorylation of ERK1/2, JNK, and p38 induced by rIL-1αin AA rat -derived FLS. U0126 and SB203580, which was inhibitor of MEK1/2 and p38 respectively, decreased the expressions of G i1, G i2, G i3, G s and the level of cAMP induced by rIL-1αin AA rat -derived FLS. These researches suggest that there are cross -talks between MAPKs signaling pathway and G proteins- AC -cAMP signaling pathway in neuroblastoma, cardiomyocytes and synoviocytes.Paeoniflorin (Pae), a monoterpene glucoside, is one of the main bioactive components of total glucosides of paeony (TGP) extracted from the root of Paeonia lactiflora. As a disease-modifying drug, TGP was approved for marketing in 1998. TGP has both anti-inflammatory and immune-regulatory effects, and has been used in the treatment of RA. It ameliorates clinical symptoms and signs of RA. It also plays an important role in restoring and regulating the abnormal immune function of RA. Studies had showed that the clinical effects of TGP were as good as that of methotrexate (MTX). But the adverse drug reactions of TGP were less than that of MTX. TGP also inhibited acute inflammation of rat induced by carrageenan, and inhibited secondary inflammatory reaction, bone destruction and ultrastructure changes of synoviocytes in AA rats. Furthermore, TGP inhibited cell proliferation and MMPs expression in FLS in AA rats. TGP also decreased the increased phosphorylation of ERK1/2, p38 and JNK, and increased the expression of Gs, EP2 and EP4 expression.TGP contains more than 90% of Pae. Pae was extracted and purified by methods of solvent extraction, column chromatography, and its structure was identified by physicochemical properties and spectroscopic analysis. The content was determined by high performance liquid chromatography (HPLC). In vivo, Pae significantly diminished the secondary hind paw swelling and arthritis scores, and further decreased the lowered proliferation of mesenteric lymph node (MLN) lymphocytes in AA rats. In vitro, Pae restored the previously increased level of cAMP of MLN lymphocytes. Meanwhile, Pae increased protein expressions of beta 2-AR and GRK2, and decreased that of beta-arrestin 1/2 of MLN lymphocytes in AA rats.Previous studies in our research group showed that the correlation between MAPKs and G proteins- AC -cAMP signaling pathway in experimental arthritis rats. However, Which link does MAPKs signaling regulate on G proteins- AC -cAMP signaling by? Which link does G proteins- AC -cAMP signaling regulate on MAPKs signaling by? where is the key site of the cross talks between the two signaling pathways? Does Pae regulate the cross talks between the two signaling pathways in treatment RA? If it is true, where is the regulatory link? These questions have not been defined. In this study, CIA rats were induced, FLS of CIA rats were isolated and cultured, first time to investigate the core site of the cross -talks between Ras-Raf-MEK1/2- ERK1/2 signaling and G protein - couple signaling in FLS of CIA rats by the stimulation of rIL-1α(an inflammatory cytokine), U0126 (a selective inhibitor of MEK1/2), isoprenaline hydrochloride (Iso, an agonist of beta adrenergic receptor) and aminophyline (Ami, a non-selective inhibitor of phosphodiesterases (PDEs)) respectively, and to illuminate the effect of Pae on the correlation between the two pathways and the regulatory link.Objectives1. According to the changes of Ras, Raf, p-MEK1/2 and p-ERK1/2, the effect of G proteins- AC -cAMP signal pathways on Ras-MAPKs signal pathways was observed in FLS of CIA rats by the stimulation of Iso and Ami respectively. According to the changes of the expression of Gi, cAMP level and PKA activity, to investigate the effect of Ras- MAPKs signal pathways on G proteins- AC -cAMP signal pathways in FLS stimulated by rIL-1αor U0126 respectively, and to find the regulatory key site between the two signaling.2. According to the changes of the expression of Ras, Raf, p-MEK1/2, p-ERK1/2, Gi proteins, cAMP level and PKA activity in FLS, to observe the regulation of Pae on the cross -talks between Ras- MAPKs and G proteins- AC -cAMP signaling pathway and the regulatory link in FLS of CIA rats stimulated by Iso, Ami, rIL-1αor U0126.MethodsCIA was induced with chicken type II collagen (CII) in Sprague-Dawley (SD) rats. CIA rats were fed with Pae (25, 50, 100 mg·kg^-1) after initiation of arthritis, once a day. Weight of rats was measured every 4 days. Right hind paw volume was determined with MK-550 volume meter. AI was graded on a scale of 0-4. Histological pathology of knee joints of rats was analyzed. FLS were isolated and cultured. FLS were stimulated by different reagants such as Iso, Ami, rIL-1α, U0126 or Pae. IL-1 activity was measured by ³H-TdR - intake method. TNF alpha and PGE₂ production were measured according to the TNF- alpha and PGE₂ ¹²⁵I RIA kit. The expressions of Gi, Ras, Raf, p-MEK1/2 and p-ERK1/2 were analyzed by immunohistochemistriy and Western blotting respectively. The level of cAMP was conducted by using competitive protein binding assay (CPBA), PKA activity was assessed by Kinase-Glo? Luminescent Kinase Assay kit.Resultsâ… .The cross -talks and the regulatory key site between Ras-MAPKs signaling pathway and G proteins- AC -cAMP signaling pathway in FLS of CIA rats1. Induction and evaluation of CIA in rats. Inflammatory responses were found obviously in CIA rats. The weight of CIA rats decreased, paws were swelling, AI increased, accompanying with hyperplastic synovium, pannus and cartilage erosion in joints. IL-1 activity increased. PGE₂ and TNF- alpha production were enhanced.2. The expressions of Gi, Ras, Raf, p-MEK1/2 and p-MEK1/2 in FLS of CIA rats. The expressions of Gi, Ras, Raf, p-MEK1/2 and p-MEK1/2 in FLS of CIA rats increased. The peak contents of Gi, Ras, Raf and p-ERK1/2 were from day 20 to day 28. The peak content of p-MEK1/2 was on day 20.3. Effects of Ras-MAPKs on G proteins- AC -cAMP signal pathways and the regulatory key site in FLS. rIL-1αpromoted the expression of Ras, Raf, p-ERK1/2 and p-MEK1/2 in FLS of CIA rats, and enhanced Gi expression, cAMP level and PKA activity. U0126 inhibited the expression of Gi, p-MEK1/2 and p-ERK1/2, the level of cAMP and PKA activity in FLS stimulated by rIL-1α. Ras and Raf were enhanced at 15 min and Gi increased at 30 min after the stimulation of rIL-1α. p-MEK1/2 reduced at 30 min, cAMP level and PKA activity decreased at 60 min after the stimulation of U0126.These results suggest the activation of Ras-MAPKs signaling promote G proteins coupled signaling, inhibition of Ras-MAPKs signaling might down-regulate on G proteins coupled signaling.4. G proteins- AC -cAMP signal pathway effects on Ras-MAPKs signal pathway and the regulatory key site in FLS of CIA rats. Iso enhanced Gi protein expression, cAMP level and PKA activity, but had no effects on Ras, Raf, p-MEK1/2 and p-ERK1/2. Ami had no effect on Gi protein, but inhibited the expression of Ras, Raf, p-MEK1/2 and p-ERK1/2, and promoted cAMP level and PKA activity. cAMP level and PKA activity were enhanced at 15 min and Ras reduced at 30 min after the stimulation of Ami.These results suggest to heighten cAMP level and PKA activity down- regulate on Ras-MAPKs signaling, PKA inhibites Ras, and down- regulate on Ras-MAPKs signaling may be the key site of G proteins- AC -cAMP signaling regulates on Ras-MAPKs signaling.â…¡. Pae regulates the cross -talks between G proteins- AC -cAMP signaling and Ras-MAPKs signaling and the regulatory link in FLS of CIA rats.1. Pae suppressed inflammatory response and inflammatory mediator production by FLS in CIA rats. Pae restored the weight of rats with CIA, inhibted paws swelling, diminished AI and ameliorated the changes of histopathology of rats with CIA. Pae had also significant inhibitory effects on IL-1, TNFαand PGE₂ production in FLS of CIA rats.2. Pae regulates G proteins - AC -cAMP signal pathways in FLS of CIA rats. The expression of Gi protein in FLS of CIA was elevated. The level of cAMP and PKA activity decreased. Pae suppressed Gi proteins, and enhanced the level of cAMP and PKA activity in FLS of CIA in vivo and in vitro.3. Pae regulates Ras-MAPKs in FLS synoviocytes of CIA rats. The expressions of Ras, Raf, p- MEK1/2 and p- ERK1/2 in FLS of CIA increased. The Ras, Raf, p- MEK_1/2 and p- ERK_1/2 expressions in FLS of CIA diminished after treatment of Pae in vivo and in vitro.4. Pae effects on the cross -talks between Ras- MAPKs and G proteins- AC -cAMP signaling pathway and the regulatory link in FLS stimulated by rIL-1α, U0126 or Ami.4.1 Gi protein was inhibited at 30 min and Ras reduced at 60 min after the stimulation of Pae. These results suggest Pae suppresse Gi protein first, and enhance PKA activity. PKA down-regulates Ras and inhibites Ras- MAPKs signaling.4.2 rIL-1αimproved the expression of Gi, Ras, Raf, and enhanced cAMP level and PKA activity in vitro. Pae (2.5mg·L^-1) inhibited the expression of Gi, Ras, Raf, and promoted cAMP level and PKA activity further. It suggests that the activation of Ras-MAPKs signaling promote G proteins coupled signaling, and Pae could inhibite the promotion.4.3 U0126 could inhibit the expression of Gi, p-MEK1/2 and p-ERK1/2, and suppressed cAMP level and PKA activity. Pae (2.5mg·L^-1) down-regulated on Gi, p-MEK1/2 and p-ERK1/2, but resumed cAMP level and PKA activity. It suggests that inhibition of Ras-MAPKs signaling might down-regulate on G proteins coupled signaling, Pae suppresse Gi protein, then enhance cAMP level and PKA activity.4.4 Ami significantly suppressed the expression of Ras and Raf, and enhanced cAMP level and PKA activity in vitro. Pae (2.5mg·L^-1) inhibited Ras and Raf further, and improved cAMP level and PKA activity further. These results suggest that to heighten PKA activity may inhibit Ras-MAPKs signaling.These results above-mentioned suggest Pae regulates the cross -talks between G proteins- AC -cAMP signaling and Ras-MAPKs signaling in FLS of CIA rats, and the regulatory link may be Pae inhibit Gi protein, up-regulate cAMP level and PKA activity and inhibite Ras-MAPKs signaling.Conclusion 1. CIA model is a chronic immune-inflammatory model for RA. CIA model has been extensively used to elucidate the pathogenic mechanisms of RA and to evaluate anti-arthritic drugs.2. There are cross talks between Ras–MAPKs and G protein - AC-cAMP signaling in FLS of rats with CIA.The activation of Ras-MAPKs signaling promote G proteins coupled signaling, inhibition of Ras-MAPKs signaling might down-regulate on G proteins coupled signaling. To heighten cAMP level and PKA activity down- regulate on Ras-MAPKs signaling, PKA inhibites Ras, and down- regulate on Ras-MAPKs signaling may be the key site of G proteins- AC -cAMP signaling regulates on Ras-MAPKs signaling.3. Pae has anti-inflammatory and immune regulatory effects on CIA rats. Pae suppressed inflammatory response and inflammatory mediator production by FLS of rats with CIA.4. Pae regulates G proteins- AC -cAMP signaling pathway in FLS of rats with CIA, inhibits Gi protein expression, and enhances cAMP level and PKA activity. Pae regulates Ras- MAPKs signaling pathway in FLS of rats with CIA and suppresses the activation of Ras- MAPKs cascades. Pae regulates the cross -talks between G proteins- AC -cAMP signaling and Ras-MAPKs signaling in FLS of CIA rats, and the regulatory link may be Pae inhibit Gi protein, up-regulate cAMP level and PKA activity and inhibite Ras-MAPKs signaling. This is one of mechanisims of anti-inflammatory and immune regulatory effects of Pae.5. Pae is a new monomer, which has bioactivity of anti-inflammatory and immuno-regulatory. This indicates that Pae has therapeutic potential for future use in RA.