Phytocomponent 4-hydroxy-3-methoxycinnamaldehyde ablates T-cell activation by targeting protein kinase C-θ and its downstream pathways Uroos Akber a , Bo-Ra Na a , You-Seung Ko a , Hyun-Su Lee a , Hye-Ran Kim a , Min-Sung Kwon a , Zee-Yong Park a , Eun-Ju Choi b , Weon-Cheol Han c , Seung-Ho Lee d , Hyun-Mee Oh e, ⁎, Chang-Duk Jun a, ⁎⁎ a School of Life Sciences, Immune Synapse Research Center and Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea b Division of Sport Science, College of Natural Sciences, Konkuk University, Chungbuk 380-702, Korea c Department of Pathology, Wonkwang University School of Medicine, Iksan, Chonbuk 570-749, Korea d College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Korea e Bioindustrial Process Research Center, KRIBB, Jeongeup 580-185, Korea abstract article info Article history: Received 19 October 2014 Received in revised form 17 January 2015 Accepted 21 January 2015 Available online 28 January 2015 Keywords: 4-Hydroxy-3-methoxycinnamaldehyde Immunosuppression PKCθ IL-2 T-cell activation MAP kinase Autoreactive T-cell responses have a crucial role in the pathology and clinical course of autoimmune diseases. Therefore, controlling the activation of these cells is an important strategy for developing therapies and therapeu- tics. Here, we identified that 4-hydroxy-3-methoxycinnamaldehyde (4H3MC) has a therapeutic potential for T- cell activation by modulating protein kinase C-θ (PKCθ) and its downstream pathways. Pre- and post-treatment with 4H3MC prevented IL-2 release from human transformed and untransformed T cells at the micromolar con- centrations without any cytotoxic effects, in fact more efficiently than its structural analogue 4-hydroxycinnamic acid—a previously reported T-cell inhibitor. In silico analysis showed that 4H3MC is a potential inhibitor of PKC isotypes, including PKCθ—a crucial PKC isotype in T cells. Consistently, 4H3MC significantly blocked PKC activity in vitro and also inhibited the phosphorylation of PKCθ in T cells. 4H3MC had no effect on TCR-mediated membrane-proximal-signalling events such as phosphorylation of Zap70. Instead, it attenuated the phosphory- lation of mitogen-activated protein kinases (ERK and p38) and promoter activities of NF-κB, AP-1 and NFAT. Taken together, our results provide the evidences that 4H3MC may have curative potential as a novel immune modulator in a broad range of immunopathological disorders by modulating PKCθ activity. © 2015 Elsevier B.V. All rights reserved. 1. Introduction T-cell-mediated immunity is an adaptive method to develop antigen-specific T cells for eradication of pathogens and malignant cells. T-cell-mediated immunity can also include atypical recognition of self-antigens, leading to autoimmune inflammatory diseases [1]. These autoreactive T cells lead to target organ and tissue damages, which are exaggerated by elevated T-cell cytokines even after antigen clearance [2]. Therefore, modulating the T-cell response is a central ap- proach to develop the therapeutics for autoimmune diseases. T cells communicate with antigen-presenting cells (APCs), thus decoding external signals, through highly structured intracellular signalling pathways, into specific T-cell effector responses. A prompt event in T-cell receptor (TCR) activation is lymphocyte protein tyrosine kinase (Lck)-mediated phosphorylation of immunoreceptor tyrosine- based activation motifs (ITAMs) on the cytosolic side of the TCR/CD3 complex. Consequently ζ-chain associated protein kinase (Zap-70) is recruited to the TCR/CD3 complex, stimulating recruitment and phos- phorylation of downstream adaptor or scaffold proteins [3]. It results in activation of PKCθ and the MAPK/ERK pathways, both stimulating NF-κB activation. Impaired function of Zap-70 function induces severe combined immunodeficiency (SCID) in both mice and human, charac- terized by an insufficiency of functional T cells [4]. The role of NF-κB in the regulation of proinflammatory genes comprising chemokines, cyto- kines and membrane adhesion proteins has extensively been reported to be involved in immune response [5–7]. Lignin plays a key role in establishing the resistance of plants to biot- ic and abiotic stresses [8], and cinnamic acid is thought to be a key pre- cursor in the biosynthesis of lignin, including 4H3MC as an intermediate derivative [9]. 4H3MC has been reported to have antifungal [10] and cytotoxic activity for various cancer cell lines [11]. In addition, 4H3MC was recently reported to prevent the melanin production in mouse International Immunopharmacology 25 (2015) 130–140 ⁎ Correspondence to: H.-M. Oh, Bioindustrial Process Research Center, Bio-Materials Research Institute, KRIBB, 1404 Sinjeong-dong, Jeongeup 580-185, Korea. Tel.: +82 63 570 5233; fax: +82 63 570 5239. ⁎⁎ Correspondence to: C.-D. Jun, School of Life Sciences, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Korea. Tel.: +82 62 715 2506; fax: +82 62 715 2546. E-mail addresses: ohhm@kribb.re.kr (H.-M. Oh), cdjun@gist.ac.kr (C.-D. Jun). http://dx.doi.org/10.1016/j.intimp.2015.01.020 1567-5769/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp