The JNK/NFκB pathway is required to activate murine lymphocytes induced by a sulfated polysaccharide from Ecklonia cava Ginnae Ahn a , So Jin Bing b , Sung-Myung Kang c , Won-Woo Lee c , Seung-Hong Lee d , Hiroshi Matsuda e , Akane Tanaka a , Ik-Hyun Cho f , You-Jin Jeon c, ⁎ , 1 , Youngheun Jee b, ⁎⁎ , 1 a Laboratory of Comparative Animal Medicine, Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan b College of Veterinary Medicine and Applied Radiological Science Institute, Jeju National University, Jeju 690-756, Republic of Korea c Department of Marine Life Science, Jeju National University, Jeju 690-756, Republic of Korea d School of Medicine, Jeju National University, Jeju 690-756, Republic of Korea e Laboratory of Veterinary Molecular Pathology and Therapeutics, Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan f Department of Anatomy, College of Oriental Medicine, and Institute of Oriental Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea abstract article info Article history: Received 5 June 2012 Received in revised form 21 November 2012 Accepted 10 December 2012 Available online 20 December 2012 Keywords: Ecklonia cava (E. cava) Sulfated polysaccharide (SP) Lymphocytes JNK NFκB p65 Background: The proven immunomodulatory and immune system activating properties of Ecklonia cava (E. cava) have been attributed to its plentiful polysaccharide content. Therefore, we investigated whether the sulfated polysaccharide (SP) of E. cava specifically activates the protein kinases (MAPKs) and nuclear factor-κB (NFκB) to incite immune responses. Methods: To assess immune responsiveness, lymphocytes were isolated from spleens of ICR mice and cul- tured with SP and its inhibitors. Assays included 3 H-thymidine incorporation, flow cytometry, real time po- lymerase chain reaction (rtPCR), enzyme linked immunosorbent assay (ELISA), intracellular cytokine assay, Western blot, and electrophoretic mobility shift assay (EMSA). Results: SP dose-dependently increased the proliferation of lymphocytes without cytotoxicity. In particular, SP markedly enhanced the proliferation and differentiation of CD3 + mature T cells and CD45R/B220 + pan B cells. Additionally, SP increased the expression and/or production of IL-2, IgG 1a , and IgG 2b compared to that in untreated cells. The subsequent application of JNK (SP600125), NFκB (PDTC), and serine protease (TPCK) inhibitors significantly inhibited the proliferation and IL-2 production of SP-treated lymphocytes as well as the phosphorylation of JNK and IκB, the activation of nuclear NFκB p65, and binding of NFκB p65 DNA. Moreover, co-application of both JNK and NFκB inhibitors completely blocked the proliferation of lym- phocytes even in the presence of SP. Conclusion: These results suggest that SP induced T and B cell responses via both JNK and NFκB pathways. General significance: The effect of SP on splenic lymphocyte activation was assayed here for the first time and indicated the underlying functional mechanism. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. 1. Introduction Algal polysaccharides including agar, carrageenans, alginates, laminaran, rhamnan sulfate, and fucoidan isolated from seaweeds are popular materials for the food, agriculture, and related industries due to their beneficial biological effects. These effects include immunomodu- latory, anti-inflammatory, anti-coagulant, anti-cancer, anti-angiogenic, and anti-adhesive activities [1–8]. Consequently, the important role of algal polysaccharides as modulators and activators of the immune sys- tem has evoked widespread attention [1,9–12]. Recently, the brown seaweed Ecklonia cava Kjellman (class, Phaeophyceae; family, Lessoniaceae; order, Laminariales, E. cava) has been the subject of considerable research because of its beneficial effects on oxygen-radical scavenging, radio-protection, anti-coagulation, and matrix metalloproteinase inhibition [5,6,13–20]. We have demonstrated Biochimica et Biophysica Acta 1830 (2013) 2820–2829 Abbreviations: CFSE, carboxy fluorescein diacetate succinimidyl ester; Con A, conca- navalin A; E. cava, Ecklonia cava; ERK, extracellular signal-regulated kinases; DMSO, dimethylsulfoxide; ELISA, enzyme linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; IL, inter- leukin; HRP, horseradish peroxidase; JNK, Jun-NH 2 -terinal kinases; mAb, mouse anti- body; MAPKs, mitogen-activated protein kinases; NFκB, nuclear factor-κB; PDTC, pyrrolidine dithiocarbamate; PE, phycoerythrin; PMA, phorbol 12-myristate 13-acetate; rtPCR, real-time polymerase chain reaction; SDS, sodium dodecyl sulfate; SDS–PAGE, SDS polyacrylamide gel electrophoresis; SP, sulfated polysaccharide; SPSS, statistical package for the social sciences; TPCK, N-p-Tosyl-L-phenylalanine chloromethyl ketone ⁎ Correspondence to: Y.J. Jeon, Department of Marine Life Science, Jeju National University, Jeju 690-756, Republic of Korea. Tel.: +82 64 754 3475; fax: +82 64 756 3493. ⁎⁎ Correspondence to: Y. Jee, College of Veterinary Medicine and Applied Radiological Science Institute, Jeju 690-756, Republic of Korea. Tel.: +82 64 754 3374; fax: +82 64 756 3354. E-mail addresses: youjinj@jejunu.ac.kr (Y.-J. Jeon), yhjee@jejunu.ac.kr (Y. Jee). 1 Contributed equally to this study. 0304-4165/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2012.12.008 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen