Jinpiao Lin

A critical role of Cyr61 in interleukin-17–dependent proliferation of fibroblast-like synoviocytes in rheumatoid arthritis

OBJECTIVE Fibroblast-like synoviocytes (FLS) are a major component of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Cyr61 (CCN1) is a product of a growth factor-inducible immediate early gene and is involved in cell adhesion, proliferation, and differentiation. However, the role that Cyr61 plays in FLS proliferation has remained undetermined. The aim of this study was to identify the role of Cyr61 in regulating the proliferation of FLS derived from patients with RA. METHODS Expression of Cyr61 in synovial tissue (ST) and in FLS was determined simultaneously using immunohistochemistry, real-time polymerase chain reaction, and Western blotting. Cyr61 levels in synovial fluid (SF) were determined by enzyme-linked immunosorbent assay. FLS proliferation stimulated by SF, Cyr61, and interleukin-17 (IL-17) was measured by thymidine incorporation. Activation of signal transduction pathways was determined by Western blotting and confocal microscopy. RESULTS Cyr61 was overexpressed in ST, FLS, and SF samples from RA patients as compared with samples from normal controls. Elevated levels of Cyr61 in RA SF promoted the proliferation of FLS, an effect that was abrogated by a neutralizing monoclonal antibody against human Cyr61. Furthermore, in samples from RA patients, Cyr61 was found to protect FLS from apoptosis and to sustain the expression of Bcl-2 in FLS. Most importantly, the expression of Cyr61 in FLS was regulated by IL-17 mainly via the p38 MAPK and NF-kappaB signaling pathways. Knockdown of expression of the Cyr61 gene inhibited IL-17-stimulated FLS proliferation. CONCLUSION Our findings indicate that Cyr61 plays a critical role in IL-17-mediated proliferation of FLS in RA and likely contributes to hyperplasia of synovial lining cells and eventually to joint destruction in patients with RA.

A Novel p53/microRNA-22/Cyr61 Axis in Synovial Cells Regulates Inflammation in Rheumatoid Arthritis

We previously showed that Cyr61 acts to promote fibroblast‐like synoviocyte (FLS) proliferation and Th17 cell differentiation, suggesting that Cyr61 plays an important role in mediating the joint inflammation and damage in rheumatoid arthritis (RA). The aim of this study was to investigate whether Cyr61 expression is regulated at the posttranscription level, and if so, how this regulation connects to other etiologic factors in RA.

Cyr61 Induces IL-6 Production by Fibroblast-like Synoviocytes Promoting Th17 Differentiation in Rheumatoid Arthritis

Cysteine-rich protein 61 (Cyr61)/CCN1 is a product of an immediate early gene and functions in mediating cell adhesion and inducing cell migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) promotes FLS proliferation and participates in RA pathogenesis with the IL-17–dependent pathway. However, whether Cyr61 in turn regulates Th17 cell differentiation and further enhances inflammation of RA remained unknown. In the current study, we explored the potential role of Cyr61 as a proinflammatory factor in RA pathogenesis. We found that Cyr61 treatment dramatically induced IL-6 production in FLS isolated from RA patients. Moreover, IL-6 production was attenuated by Cyr61 knockdown in FLS. Mechanistically, we showed that Cyr61 activated IL-6 production via the αvβ5/Akt/NF-κB signaling pathway. Further, using a coculture system consisting of purified CD4+ T cells and RA FLS, we found that RA FLS stimulated Th17 differentiation, and the pro-Th17 differentiation effect of RA FLS can be attenuated or stimulated by Cyr61 RNA interference or addition of exogenous Cyr61, respectively. Finally, using the collagen-induced arthritis animal model, we showed that treatment with the anti-Cyr61 mAb led to reduction of IL-6 levels, decrease of Th17 response, and attenuation of inflammation and disease progression in vivo. Taken together, our results reveal a novel role of Cyr61 in promoting Th17 development in RA via upregulation of IL-6 production by FLS, thus adding a new layer into the functional interplay between FLS and Th17 in RA pathogenesis. Our study also suggests that targeting of Cyr61 may represent a novel strategy in RA treatment.

Total glucosides of paeony inhibits Th1/Th17 cells via decreasing dendritic cells activation in rheumatoid arthritis

Total glucoside of paeony (TGP), an active compound extracted from paeony root, has been used in therapy for rheumatoid arthritis (RA). Th1 and Th17 cells are now believed to play crucial roles in the lesions of RA. However, the molecular mechanism of TGP in inhibition of Th1 and Th17 cells remains unclear. In this study, we found that TGP treatment significantly decreased percentage and number of Th1 and Th17 cells in collagen induced arthritis (CIA) mice. Consistently, treatment with TGP decreased expression of T-bet and RORγt as well as phosphorylation of STAT1 and STAT3. In particular, TGP treatment inhibited dendritic cells (DCs) maturation and reduced production of IL-12 and IL-6. Moreover, TGP-treatment RA patients showed shank population of matured DCs and IFN-γ-, IL-17-producing cells. Taken together, our results demonstrated that TGP inhibited maturation and activation of DCs, which led to impaired Th1 and Th17 differentiation in vivo.

Cyr61 is involved in neutrophil infiltration in joints by inducing IL-8 production by fibroblast-like synoviocytes in rheumatoid arthritis

IntroductionIt is well known that neutrophils play very important roles in the development of rheumatoid arthritis (RA) and interleukin (IL)-8 is a critical chemokine in promoting neutrophil migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in RA promotes FLS proliferation and Th17 cell differentiation, thus Cyr61 is a pro-inflammatory factor in RA pathogenesis. In this study, we explored the role of Cyr61 in neutrophil migration to the joints of RA patients.MethodsRA FLS were treated with Cyr61 and IL-8 expression was analyzed by real-time PCR and ELISA. The migration of neutrophils recruited by the culture supernatants was determined by the use of a chemotaxis assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as a control. Arthritis severity was determined by visual examination of the paws and joint destruction was determined by hematoxylin-eosin (H&E) staining. Signal transduction pathways in Cyr61-induced IL-8 production were investigated by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay or chromatin immunoprecipitation (ChIP) assay.ResultsWe found that Cyr61 induced IL-8 production by RA FLS in an IL-1β and TNF-α independent pathway. Moreover, we identified that Cyr61-induced IL-8-mediated neutrophil migration in vitro. Using a CIA animal model, we found that treatment with anti-Cyr61 mAb led to a reduction in MIP-2 (a counterpart of human IL-8) expression and decrease in neutrophil infiltration, which is consistent with an attenuation of inflammation in vivo. Mechanistically, we showed that Cyr61 induced IL-8 production in FLS via AKT, JNK and ERK1/2-dependent AP-1, C/EBPβ and NF-κB signaling pathways.ConclusionsOur results here reveal a novel role of Cyr61 in the pathogenesis of RA. It promotes neutrophil infiltration via up-regulation of IL-8 production in FLS. Taken together with our previous work, this study provides further evidence that Cyr61 plays a key role in the vicious cycle formed by the interaction between infiltrating neutrophils, proliferated FLS and activated Th17 cells in the development of RA.

A novel anti-Cyr61 antibody inhibits breast cancer growth and metastasis in vivo

Cysteine-rich protein 61(CCN1/Cyr61) has been implicated as an important mediator in proliferation and metastasis of breast cancer, which indicated that blockage of Cyr61 might be a potent target for breast cancer treatment. However, the antitumor effect of anti-Cyr61 antibodies on breast cancer in vivo has not been reported so far. In this study, we reported the effect and likely mechanism of generated anti-human Cyr61 monoclonal antibodies (mAb) on Cyr61 high expression line MDA-MB-231, known as a highly malignant and invasive human breast cancer cell line, at aspects of proliferation and migration in vitro and in vivo. We found the mAb, denoted as 093G9, revealed inhibitory effects on MDA-MB-231 cell proliferation, migration, and invasion through downregulation of both AKT and ERK phosphorylation in vitro compared with its isotype control. 093G9 also showed significant efficacy on suppressing primary tumor growth and spontaneous lymph node metastasis in in vivo mouse model. The specific epitope recognized by 093G9 was identified to be 140LPNLGCP146, adjacent to the VWC domain of Cyr61 by Ph.D.-C7C phage library display system. Our study provides direct evidence that Cyr61 can be a potent therapeutic target for patients who bear high Cyr61 expression breast cancer. Furthermore, the mAb, 093G9 developed in our laboratory, has shown a promising therapeutic characteristic in breast cancer.

Total glucosides of paeony attenuated functional maturation of dendritic cells via blocking TLR4/5 signaling in vivo.

It is well known that dendritic cells (DCs) play a critical role in the initiation and development of an immune response. Inhibitory effect on DC maturation alters immune-mediated inflammatory reaction in vivo. Total glucosides of paeony (TGP) are active compounds extracted from the roots of Paeonia lactiflora and have been widely used to ameliorate inflammation in therapy for autoimmune diseases. However, whether TGP act on DC maturation remains unknown. In this study, we investigated the effect of TGP on DC maturation in ovalbumin (OVA) immunized mice. Ear inflammation was inhibited by TGP (150 mgkg(-1), i.p.×11 days) obviously. The antigen presenting capacity of DC derived from TGP-treated mice was arrested. Meanwhile, OVA specific T cell proliferation was inhibited. In addition, we found that maturation of DCs was decreased by TGP treatment. Furthermore, OVA specific T cell proliferation was rescued by the adoptive transfer of mature DCs (mDCs) into TGP treated OVA-challenged mice. The research on the mechanism showed that TGP significantly inhibited activation of TLR4/5 singling. All these results demonstrated that TGP inhibited DC maturation and function by selectively blocking TLR4/5 activation in vivo, which in turn leads to reduce immune-mediated inflammation in vivo, adding a novel mechanism and therapeutic target of TGP for inflammatory and autoimmune disease treatment.

Up-regulation of Th17 cells may underlie inhibition of Treg development caused by immunization with activated syngeneic T cells.

Background Our previous work showed that mice immunized with attenuated activated syngeneic T cells (aTCV) led to damping Treg function which resulted in enhancing anti-tumor immunity. It is well known that DC plays a very important role in controlling Th cell differentiation; whether DC involves Treg attenuation in immunized mice remained unknown. In this study, we provided evidence that increased mature DC (mDC) after immunization with aTCV skewed Th17 differentiation, which resulted in inhibition of Treg differentiation through IL-6 signaling pathway. Principal Findings In the present study, we found that the frequency of mDCs increased dramatically in the immunized mice accompanied by lower Treg cells compared to the controls. Moreover, both DCs and serum derived from the immunized mice suppressed Treg differentiation in vitro, respectively. mDCs generated from bone marrow precursor cells in vitro strongly inhibited Treg development and simultaneously drove Th17 differentiation with elevated IL-6 production. However, PD-L1, a potent Treg inducer did not show effect on Treg down-regulation. Assay with transwell systems showed that cell-cell contact was necessary for IL-6 production to a threshold to activate Th17 transcriptional factor RORγt and to inhibit Treg counterpart Foxp3. Conclusions Our results implicate up-regulated Th17 development might be one of mechanisms of enhancing anti-tumor immunity induced by immunization with aTCV, which provide a novel insight in numerous mechanisms responsible for anti-tumor immunity.

Evaluation of the efficacy and safety of different Tripterygium preparations on collagen-induced arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE Tripterygium preparations (TPs), a traditional Chinese Medicines extracted from Tripterygium wilfordii Hook f., are widely used for treatment of rheumatoid arthritis (RA). However, TPs from different Pharmaceutical factory have different efficacy and side effects for RA treatment. AIM OF THE STUDY The purpose of the current study is to evaluate the efficacy and safety of four TPs from different Pharmaceutical factory in china on the treatment of collagen-induced arthritis (CIA) rats and provide a theoretical and experimental basis for the individualized use of TPs. MATERIALS AND METHODS The model of wistar rats of CIA was made, and the rats were perfused a stomach with four TPs for 3 weeks continuously. Then arthritis severity was determined by visual examination of the paws and histopathologic changes of joint, liver, kidney and testis were determined by hematoxylin-eosin (H&E) staining. The expression of inflammatory cytokines (IL-1β, TNF-α, IL-17 and IL-6) in the joint was analyzed by real-time PCR, and the count and motion parameters (sperm motility and progressive sperm) of sperm in cauda epididymis were assessed with computer-assisted sperm analysis (CASA) system. Routine blood tests were conducted using automated hematology analyzer, and the aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities, creatinine (Cr), and blood urea nitrogen (BUN) in serum of CIA rats were measured using a UniCel DxC 880i autoanalyzer. RESULTS All of tested TPs could reduce inflammatory score, histopathological arthritis severity and joint׳s inflammatory cytokines (IL-1β, TNF-α, IL-17 and IL-6) expression in CIA rats, however, TP-D showed stronger inhibitory effect for inflammatory score compared with other three TPs in vivo. All of tested TPs did not show hepatotoxicity and nephrotoxicity and also had little effect for the concentration of hemoglobin (Hb) and the count of white blood cell (WBC). Analysis of red blood cell (RBC) number showed that TP-C and TP-D could reverse lower RBC number in untreated CIA rats to normal level. Interestingly, the results showed TPs named TP-C and TP-D could decrease platelet (PLT) number which significantly increases in untreated CIA rats. Reproductive toxicity, the main side effect of TPs, assay showed that the sperm quality (density, viability, and motility) in four of TPs-treated CIA rats were decreased significantly, consistently with spermatogenic cell density reduced. However parallel analysis showed that in four TPs-treated rats, the number of sperm, motile sperm and progressive sperm were highest in TP-D group, in contrast, were lowest in TP-C group. CONCLUSIONS These findings suggested that four TPs showed significantly therapeutic effect on ameliorating inflammation of CIA rats, with no obvious hepatotoxicity and nephrotoxicity in vivo. TP-D showed advantages with its higher efficacy and less reproductive toxicity as well as increasing RBC number, decreasing PLT number in CIA treatment. Thus, in the development of individualized treatment plan for RA patients, TP-D might be considered preferentially.

Cyr61 participates in the pathogenesis of rheumatoid arthritis via promoting MMP-3 expression by fibroblast-like synoviocytes

Abstract Objectives: The aim of this study was to investigate the effect and potential mechanism of Cysteine-rich 61 (Cyr61) on stimulating MMP-3 expression by fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. Methods: Primarily cultured RA FLS were treated with exogenous Cyr61 protein or Cyr61-siRNA, then, MMP-3 expression was analyzed by real-time PCR, western blotting and ELISA. Signal transduction pathways in Cyr61-induced MMP-3 production were examined by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as control and MMP-3 in the joint was detected by IHC, real-time PCR and western blotting. Results: High expressed MMP-3 and Cyr61 were positively correlated in RA ST; Cyr61 stimulated MMP-3 production in FLS of RA patients in an IL-1β and TNF-α independent manner. Cyr61 induced MMP-3 could further enhance the invasive ability of RA FLS. Mechanistically, we found that Cyr61 promoted MMP-3 production via the P38, JNK-dependent AP-1 signaling pathway. Blockage of Cyr61 function with monoclonal antibody could decrease MMP-3 expression in the joints of CIA mice. Conclusion: This study provides new evidence that Cyr61 participates in RA pathogenesis not only as a pro-inflammatory factor but also plays a key role in bone erosion via promoting MMP-3 expression. We suggest that targeting of Cyr61 may represent a potential strategy in RA treatment.