Inhibitor of Kappa B Epsilon (IkBe) Is a Non-Redundant Regulator of c-Rel-Dependent Gene Expression in Murine T and B Cells Joanna M. Clark1, Karolina Aleksiyadis2, Alex Martin2, Kay McNamee2, Tharsana Tharmalingam1, Richard O. Williams 2 , Sylvie Me´ met3,4, Andrew P. Cope1* 1 Academic Department of Rheumatology, Centre for Molecular and Cellular Biology of Inflammation, Division of Immunology, Infection and Inflammatory Diseases, School of Medicine, King’s College London, University of London, London, United Kingdom, 2 The Kennedy Institute of Rheumatology Division, Imperial College London, London, United Kingdom, 3 Institut Pasteur, Unite´ de Mycologie Mole´culaire, Paris, France, 4 Centre National de la Recherche Scientifique, Unite´ de Recherche Associe´e 3012, Paris, France Abstract Inhibitors of kappa B (IkBs) -a, -b and -e effect selective regulation of specific nuclear factor of kappa B (NF-kB) dimers according to cell lineage, differentiation state or stimulus, in a manner that is not yet precisely defined. Lymphocyte antigen receptor ligation leads to degradation of all three IkBs but activation only of subsets of NF-kB-dependent genes, including those regulated by c-Rel, such as anti-apoptotic CD40 and BAFF-R on B cells, and interleukin-2 (IL-2) in T cells. We report that pre-culture of a mouse T cell line with tumour necrosis factor-a (TNF) inhibits IL-2 gene expression at the level of transcription through suppressive effects on NF-kB, AP-1 and NFAT transcription factor expression and function. Selective upregulation of IkBe and suppressed nuclear translocation of c-Rel were very marked in TNF-treated, compared to control cells, whether activated via T cell receptor (TCR) pathway or TNF receptor. IkBe associated with newly synthesised c-Rel in activated cells and, in contrast to IkBa and -b, showed enhanced association with p65/c-Rel in TNF-treated cells relative to controls. Studies in IkBe-deficient mice revealed that basal nuclear expression and nuclear translocation of c-Rel at early time-points of receptor ligation were higher in IkBe2/2 T and B cells, compared to wild-type. IkBe2/2 mice exhibited increased lymph node cellularity and enhanced basal thymidine incorporation by lymphoid cells ex vivo. IkBe2/2 T cell blasts were primed for IL-2 expression, relative to wild-type. IkBe2/2 splenic B cells showed enhanced survival ex vivo, compared to wild-type, and survival correlated with basal expression of CD40 and induced expression of CD40 and BAFF-R. Enhanced basal nuclear translocation of c-Rel, and upregulation of BAFF-R and CD40 occurred despite increased IkBa expression in IkBe2/2 B cells. The data imply that regulation of these c-Rel-dependent lymphoid responses is a non- redundant function of IkBe. Citation: Clark JM, Aleksiyadis K, Martin A, McNamee K, Tharmalingam T, et al. (2011) Inhibitor of Kappa B Epsilon (IkBe) Is a Non-Redundant Regulator of c-Rel- Dependent Gene Expression in Murine T and B Cells. PLoS ONE 6(9): e24504. doi:10.1371/journal.pone.0024504 Editor: Ben C.B. Ko, Chinese University of Hong Kong, Hong Kong Received October 8, 2010; Accepted August 12, 2011; Published September 6, 2011 Copyright: 2011 Clark et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by (1) The Wellcome Trust: http://www.wellcome.ac.uk/ (2) Arthritis Research UK: http://www.arthritisresearchuk.org/ (3) The European Community’s Seventh Framework Programme FP7 under grant agreement no. HEALTH-F2-2008-223404 (http://www.Masterswitchproject.eu). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: andrew.cope@kcl.ac.uk Introduction Members of the nuclear factor of kappa B (NF-kB) transcription factor family, p50, p52, p65 (RelA), c-Rel and RelB, are maintained as inactive homo- or heterodimers in the cytoplasm by inhibitors of kappa B (IkBs). Interactions are via ankyrin repeats of IkB and Rel homology domains (RHD) of NF-kB [1,2]. The RHDs contain dimerisation, nuclear localisation and DNA- binding regions, and c-Rel, p65 and RelB, but not p50 and p52, also have transactivation domains which confer transcriptional activity. c-Rel, p65 and p50 are regulated by IkBs -a, -b and -e whose phosphorylation, ubiquitination and degradation are effected following activation of the IkB kinase (IKK) complex, particularly IKK2, when the classical NF-kB pathway is activated [3]. c-Rel, p65 and p50 also associate with the intramolecular IkB domains of p105 and p100, precursor proteins of p50 and p52 respectively. p50 is generated through constitutive processing, and p52 upon activation of the alternative, IKK1-dependent pathway, which also involves RelB. IkB degradation permits nuclear translocation of NF-kB and induction of genes responsible for many cellular responses including inflammation, survival and differentiation [4,5,6]. Activated NF-kB itself induces resynthesis of all three IkBs. How, then, are signal-, cell- and subunit-specific regulation of NF- kB dimers and their target genes brought about? Studies suggest that the necessary specificity may be achieved via differential expression - in space and time - of IkB isoforms. Thus, the rate of degradation and resynthesis of each isoform may vary with stimulus and cell-type [7], and according to the differentiation status of a cell. IkBe, for example, is upregulated in HL60 cells as they undergo terminal differentiation and IkBe is expressed at high levels in both na¨ıve murine splenic B cells and IgM+ mouse B cell lines, relative to IgG+ cells [8,9]. IkBe differs from IkBs -a and -b structurally [8]; and IkBs differ from one another in preferential PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24504