EXTENDED REPORT Inhibition of Notch signalling ameliorates experimental inflammatory arthritis Jong-Sung Park, 1 Seol-Hee Kim, 1,2 Kwangmeyung Kim, 3 Cheng-Hao Jin, 4 Ki Young Choi, 5 Jiyeon Jang, 1 Yuri Choi, 1 A-Ryeong Gwon, 1 Sang-Ha Baik, 1 Ui Jeong Yun, 1 Su Young Chae, 1 Seulki Lee, 6 Young Mo Kang, 7 Kang Choon Lee, 1 Thiruma V Arumugam, 1,8 Mark P Mattson, 9,10 Jae Hyung Park, 2 Dong-Gyu Jo 1 Handling editor Tore K Kvien ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ annrheumdis-2013-203467). For numbered affiliations see end of article. Correspondence to Professor Dong-Gyu Jo, School of Pharmacy, Sungkyunkwan University, 300 Cheoncheon-Dong, Suwon 440-746, Korea; jodg@skku.edu, or Professor Jae Hyung Park, Departments of Polymer Science and Chemical Engineering, Sungkyunkwan University, 300 Cheoncheon- Dong, Suwon 440-746, Korea; jhpark1@skku.edu JSP, SHK and KK contributed equally. Received 15 February 2013 Revised 9 August 2013 Accepted 26 October 2013 To cite: Park J-S, Kim S-H, Kim K, et al. Ann Rheum Dis Published Online First: [ please include Day Month Year] doi:10.1136/ annrheumdis-2013-203467 ABSTRACT Objective To test the hypothesis that Notch signalling plays a role in the pathogenesis of rheumatoid arthritis (RA) and to determine whether pharmacological inhibition of Notch signalling with γ-secretase inhibitors can ameliorate the RA disease process in an animal model. Methods Collagen-induced arthritis was induced in C57BL/6 or Notch antisense transgenic mice by immunisation with chicken type II collagen (CII). C57BL/ 6 mice were administered with different doses of inhibitors of γ-secretase, an enzyme required for Notch activation, at disease onset or after onset of symptoms. Severity of arthritis was monitored by clinical and histological scores, and in vivo non-invasive near-infrared fluorescence (NIRF) images. Micro-CT was used to confirm joint destruction. The levels of CII antibodies and cytokines in serum were determined by ELISA and bead-based cytokine assay. The expression levels of cytokines were studied by quantitative PCR in rheumatoid synovial fibroblasts. Results The data show that Notch signalling stimulates synoviocytes and accelerates their production of proinflammatory cytokines and immune responses involving the upregulation of IgG1 and IgG2a. Pharmacological inhibition of γ-secretase and antisense- mediated knockdown of Notch attenuates the severity of inflammatory arthritis, including arthritis indices, paw thickness, tissue damage and neutrophil infiltration, and reduces the levels of active NF-κB, ICAM-1, proinflammatory cytokines and matrix metalloproteinase- 3 activity in the mouse model of RA. Conclusions These results suggest that Notch is involved in the pathogenesis of RA and that inhibition of Notch signalling is a novel approach for treating RA. INTRODUCTION The Notch-1 (Notch) signalling pathway regulates cell development, differentiation, proliferation, sur- vival and apoptosis. 12 In mammalian cells, there are four Notch receptors (Notch-1 through Notch-4) and five Notch ligands (Delta-like [DLL]-1/-3/-4 and Jagged-1/-2), which are all trans- membrane proteins. Binding of a ligand to Notch initiates a two-step proteolytic cleavage of Notch, first by an extracellular ADAM family protease, and then by the intramembrane γ-secretase complex that releases Notch intracellular domain (NICD). 34 NICD then translocates into the nucleus, where it forms a transcriptional activator complex with the CSL family of transcription factors (C-promoter binding factor 1/recombination signal sequence binding protein Jκ, suppressor of hairless, and Lag-1) and modulates the expressions of target genes such as Hes family of transcription factors members and p21. 1 Rheumatoid arthritis (RA) is a complex chronic, progressive inflammatory disease involving hyper- plasia of synovial tissues and destruction of joint architecture such as cartilage, bone and liga- ments. 56 Data support the notion that Notch sig- nalling is involved in the pathogenesis of RA. The expression and activation of Notch occur in osteo- arthritis cartilage and RA synoviocytes. 7–10 Notch signalling is involved in the TNFα-induced prolifer- ation of RA synoviocytes, 10 and Jagged-1 can modulate collagen-induced arthritis (CIA) progres- sion by affecting CD8 + T cell responses. 11 T helper (Th) cells from RA patients exhibit an altered expression profile of Notch receptors and enhanced activation of Notch signalling. 12 Recently, the inter- actions between the Notch signalling pathway and angiogenesis have been described in RA. Notch signalling mediates hypoxia and vascular endothe- lial growth factor (VEGF)/angiopoietin2 (Ang2)-induced angiogenesis in RA. 13 14 Notch sig- nalling is also involved in the inflammatory responses of multiple sclerosis, glomerular disease and stroke. 15–19 The amount of NICD is increased in diabetic nephropathy and focal segmental glo- merulosclerosis. 16 In addition, Notch signalling contributes to neuronal death after cerebral ischae- mia by enhancing apoptotic cascades in neurons and by microglia-mediated inflammatory response. 17–19 Although the functional involvement of the Notch signalling in severe inflammatory diseases has been previously suggested, Notch signalling has not been established as a therapeutic target for RA. Moreover, the mechanisms by which Notch inhib- ition might slow or stop the progression of RA are unknown. Here we report that suppression of Notch signalling using antisense technology and low-molecular-weight γ-secretase inhibitors ameli- orate the disease process in a mouse model of RA. MATERIALS AND METHODS Detailed materials and methods can be found in the online supplementary data. Park J-S, et al. Ann Rheum Dis 2013;0:1–8. doi:10.1136/annrheumdis-2013-203467 1 Basic and translational research ARD Online First, published on November 19, 2013 as 10.1136/annrheumdis-2013-203467 Copyright Article author (or their employer) 2013. Produced by BMJ Publishing Group Ltd (& EULAR) under licence. group.bmj.com on November 21, 2013 - Published by ard.bmj.com Downloaded from