Molecular Immunology 52 (2012) 51–60 Contents lists available at SciVerse ScienceDirect Molecular Immunology jo u rn al hom epa ge: www.elsevier.com/locate/molimm Calcium/calmodulin-dependent protein kinase II regulates IL-10 production by human T lymphocytes: A distinct target in the calcium dependent pathway Stavroula Boubali a,1 , Kassiani Liopeta a,1 , Laura Virgilio b , George Thyphronitis c , George Mavrothalassitis b , George Dimitracopoulos a , Fotini Paliogianni a,∗ a Department of Microbiology, School of Medicine, University of Patras, Asclepiou Street, 26500 Patras, Greece b School of Medicine, University of Crete and IMBB-FORTH, Voutes, Heraklion, Crete 714-09, Greece c Department of Biological Applications and Technology, University of Ioannina, 45110 Ioannnina, Greece a r t i c l e i n f o Article history: Received 7 February 2012 Accepted 13 April 2012 Available online 10 May 2012 Keywords: T cells IL-10 Calcium MEF2 CaMKII a b s t r a c t Calcium (Ca 2+ ) plays an essential role in lymphocyte activation and differentiation by affecting sig- naling pathways leading to cytokine production. Among the enzymes responding to calcium increase, Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has been involved in anergy with a still poorly characterized role. IL-10 produced by different T lymphocyte subpopulations is critical mediator of toler- ance. We tested the hypothesis that CaMKII may be involved in IL-10 production. We report that CaMKII upregulates IL-10 production by primary human T lymphocytes stimulated through the antigen receptor or bypassing that. Overexpression of constitutively active mutant forms of Calcineurin or CaMKII specif- ically increase IL-10 protein product and IL-10 mRNA accumulation in T lymphocytes. By cotransfecting constitutively active CaMKII with luciferase reporter plasmids carrying specific fragments or the whole IL-10 promoter, we show that CaMKII specifically activates IL-10 promoter activity, whereas it inhibits IL-2 and IL-4 promoter. This effect is mediated by the first 500 bp fragment, which contains binding sites for Myocyte Enhancer Factor-2 (MEF2). A constitutively active mutant of CaMKII activated a luciferase reporter plasmid under the control of MEF2, when cotransfected in T lymphocytes stimulated by Ion- omycin and PMA, whereas its inhibitor KN-62 inhibited MEF2 binding in cell lysates of the same cells. Moreover, overexpression of MEF2 enhanced by 2.5-fold IL-10 promoter activity. Our data for the first time suggest a distinct role of CaMKII in the induction of anergy in T lymphocytes, by differential regula- tion of IL-10 and IL-2 gene transcription suggest MEF2 as a molecular target which can integrate different calcium signals. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction IL-10, a pleiotropic cytokine that exerts immunomodulatory properties, has a well recognized role in the regulation of inflam- matory responses and the pathogenesis of autoimmune diseases (Saraiva and O’Garra, 2010). Although it can be secreted by a variety of cell types, the main sources of IL-10 production are mono- cytes/macrophages and subpopulations of T cells (Moore et al., 2001; Pestka et al., 2004). Regulation of IL-10 production has been extensively stud- ied in human monocytes/macrophages in response to LPS or Abbreviations: Ca 2+ , calcium; CaM, calmodulin; CaMK, Ca 2+ /calmodulin- dependent protein kinases; CaMKII, Ca 2+ /calmodulin-dependent protein kinase II; CaMKIV, Ca 2+ /calmodulin-dependent protein kinase IV; HDACs, histone deacety- lases; I, Ionomycin; I/PMA, Ionomycin/PMA; MEF2, Myocyte Enhancer Factor-2; MEF2D, Myocyte Enhancer Factor-2D. ∗ Corresponding author. Tel.: +30 2610 999660; fax: +30 2610 994922. E-mail address: fpal@upatras.gr (F. Paliogianni). 1 Authors distributed equally. catecholamines and it has been shown to involve transcription fac- tor Specificity Protein 1 (Sp1) activated by p38 MAPK, Stat3 and cAMP (Benkhart et al., 2000; Ma et al., 2001; Platzer et al., 2000, 1999). Transcriptional regulation of IL-10 by Sp1 and Sp3, has been described in murine experimental systems (Brightbill et al., 2000; Tone et al., 2000) as well as post-transcriptional, through sequences present in its 3 ′ -UTR region (Powell et al., 2000). In T cells the tran- scription factors NF-B (Mori and Prager, 1997), Jun (Wang et al., 2005), c-Maf (Xu et al., 2009) and GATA-3 (Zheng and Flavell, 1997) have been reported as regulators of IL-10 production. Epigenetic regulation of IL-10 has also been described in T cells (Im et al., 2004; Shoemaker et al., 2006). Ca 2+ regulated transcription factors, such as the NFAT family members, have been recognized to regulate IL-10 gene transcrip- tion directly (Lee et al., 2009) or in synergy with other transcription factors (Hu et al., 2002), in differentiated Th2 cells. The role of Ca 2+ has been well documented in the regulation of IL-10 production by normal T cells (Bullens et al., 2001; Rafiq et al., 2001), but the molecular mechanisms of its action remain largely unknown. 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.04.008