CELL CYCLE 2016, VOL. 15, NO. 15, 1956–1960 http://dx.doi.org/10.1080/15384101.2016.1189042
EXTRA VIEW
Genetic insights into Map3k-dependent proliferative expansion of T cells Tesha Suddason and Ewen Gallagher Department of Medicine, Imperial College London, London, UK
ABSTRACT
ARTICLE HISTORY
Mapks are important regulators of T cell proliferative expansion and cell cycle progression. Detailed genetic analysis of unconventional iNKT cells in both Map3k1DKD and LckCre/CMap3k1f/f mice demonstrated that Mekk1 (encoded by Map3k1) signaling activates Mapks to regulate Cdkn1b (encoding p27Kip1) expression and p27Kip1-dependent proliferative expansion in response to antigen. Mekk1 signaling and activation of E3 ubiquitin ligase Itch, by a phosphorylation-dependent conformational change, is also an important regulatory mechanism for the control of T helper cell cytokine production. Cdkn1b expression is regulated by Mekk1-dependent signaling in differentiated Th17 cells. Mekk1 is one of the 19 Ste11-like Map3ks, and Mekk1 signaling regulates iNKT cell proliferative expansion in response to glycolipid antigens and T cell homeostasis in the liver. Tak1 (encoded by Map3k7), a related Map3k to Mekk1, similarly regulates the proliferative expansion and homeostasis of T cells in the liver, and this illustrates the importance of multiple Map3ks for mammalian Mapk signaling.
Received 2 March 2016 Revised 6 May 2016 Accepted 6 May 2016
Introduction Signal transduction by Mitogen-activated protein kinases (Mapks) emerged a quarter of a century ago as a potential mechanism of regulation for the cell cycle in response to extracellular stimuli.1,2 But, after the initial biochemical characterization of Mapks, it only proved possible to carefully clarify our understanding of the many proteins that constitute Mapk cascades by intricate analyses using mice that harbor genetic mutations of these signaling proteins that, in turn, alter the biochemical function of Mapks.2 By these means mammalian genetic analysis bridged the gap in our knowledge derived from simple in vitro enzyme-substrate reactions toward the greater complexity found within cells of the whole organism.2 Mapk kinase (Map2k) kinases (Map3ks) form critical points for Mapk regulation in response to mitogenic stimuli, and consequently the genetic analyses of Mapk signaling has proved informative in defining the role of Mapks in the cell cycle process.3 Of these, Map3k1 (encoding Mek kinase 1, Mekk1) has now been analyzed in mice by germline knockout (Map3k1DKD and Map3k1-/- mice), knockin (Map3k1mPHD mice) and very recently by tissue-specific deletion in T cells (LckCre/CMap3k1f/f mice).3-5 Out of these important genetic investigations the cell cycle component p27Kip1 (encoded by Cyclin-dependent kinase inhibitor 1B, Cdkn1b) has emerged from global gene expression analysis as a c-Jun N-terminal kinase (Jnk) and Mekk1 signaling target, and p27Kip1 forms a signaling mechanism to control proliferative expansion of unconventional invariant natural killer T (iNKT) cells.3 The genetic roles of Jnk isoforms as precise mechanisms for controlling mouse embryonic fibroblast (MEF) proliferation and T cells are well established.6-8 Upstream of Jnks, the analysis of
KEYWORDS
Cdkn1b; iNKT; Mapk; Mekk1; proliferation; T cell; th17
lymphocyte signaling using kinase-deficient Map3k1DKD mice has shown important roles for Mekk1 in controlling both B and T cell proliferation.5,9 Mekk1 has been shown to be of importance in T helper (Th) 2 differentiation, though its potential role in Th17 differentiation has not been analyzed to date.5,10,11 In the case of B cells, engagement of CD40 by CD40 ligand critically initiates p38 and Jnk activation to phosphorylate Jun family and other transcription factors, and these, in turn, regulate proliferation by the upregulation of Cyclin D2 transcription.9,12 Both CD4C and CD8C Map3k1DKD T cells display aberrant proliferation following T cell receptor (TCR) engagement by crosslinking with antibodies or in response to antigen presented by cells.5,9 Mechanistically the identification by global gene expression analysis of p27Kip1 as a downstream Mekk1 signaling protein has revealed a divergence between Mapk signaling and the control of cell cycle between B and T cells.3,9 Mekk1 is rapidly recruited to cytokine (e.g. CD40 and TGF-b receptors) and antigen receptors, along with the rest of the Mapk singling module, in a low phosphorylation state, and binds to upstream receptor components by the Mekk1 N-terminal regulatory domain, containing a plant homeodomain (PHD) that can act as an E3 ubiquitin (Ub) ligase.4,13,14 High-throughput protein microarray screening precisely elucidated the Mekk1 PHD substrates in depth, and these include TGF-b activated kinase 1/MAP3K7 binding protein 1 (Tab1), TNF receptor associated factor 2 (Traf2) and CARD-containing MAGUK protein 1 (Carma1).3,4,14 The Mekk1 PHD, along with other E3 Ub ligases, facilitates the non-canonical ubiquitination that occurs at TGF-b receptors, CD40 and antigen receptors as the Mapk module switches into its active form.4,12-14 Ubiquitination facilitates
CONTACT Ewen Gallagher [email protected] Department of Medicine, Imperial College London, Du Cane Road, London, W12 ONN, UK. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kccy. © 2016 Taylor & Francis
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both the activation of the Mapk pathway and the translocation of the Mapk signaling complex from the receptor and onto its downstream targets.4,12,13
Results and discussion
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expression and real-time PCR analyses (Fig. 1D and E) identified Cdkn1b as a Mekk1-regulated cell cycle gene.3 Thus, Mekk1 signaling regulates Cdnk1b expression and the cell cycle in several important T cell effector responses, both Th17 differentiation and iNKT cell expansion in response to glycolipid antigen.3
Mekk1 signaling regulates Cdkn1b expression during T cell differentiation Since Cdkn1b expression is critical for iNKT cell expansion we investigated whether a similar regulatory mechanism might also occur in conventional CD4C T cells undergoing differentiation.3,5,15 Map3k1DKD T cells are defective in Interleukin (IL) -17 production when undergoing differentiation into Th17 cells (Fig. 1A and B). Similarly, inhibition of Jnks, Ubiquitinconjugating enzyme E2N (Ube2n) or TGF-b receptor signaling reduced IL-17 production in Th17 cells (Fig. 1C).4 Global gene
Map3k1 and Map3k7 regulate iNKT cell proliferative expansion in the liver Map3k1DKD and LckCre/CMap3k1f/f mice display elevated numbers of iNKT cells within the liver.3 Both Mekk1 and Tak1 (encoded by Map3k7) have an important role in T cell Mapk signaling,5,16 and to assess this further we compared the numbers of iNKT cells in Map3k1DKD and LckCre/CMap3k1f/f mice with LckCre/CMap3k7f/f mice and found elevated numbers of
Figure 1. Mekk1 regulates Cdkn1b expression in Th17 cells. (A) Na€ıve CD4CT cells were extracted from WT and Map3k1DKD mice and differentiated under Th17 conditions. FACS analysis was performed with the indicated antibodies (4 mice per experiment). (B) Quantitation of WT and Map3k1DKD IL-17CCD4CT cells (& WT and & Map3k1DKD) differentiated under Th17 conditions (n D 3). (C) Na€ıve CD4C T cells were extracted from WT and Map3k1DKD mice and incubated with SP600125, NSC697923 or SB431542 inhibitors while differentiating under Th17 conditions (4 mice per experiment). FACS analysis was performed with the indicated antibodies. (D) Global gene expression analysis of Th17 cells. Na€ıve CD4C T cells were extracted from WT and Map3k1DKD mice and differentiated into Th17 cells. RNA was isolated from WT and Map3k1DKD splenic Th17 cells, processed and hybridized onto Affymetrix arrays. Bioinformatics analysis was performed and a heat map comparing gene hits between WT and Map3k1DKD Th17 cell microarray screen hits was constructed. The data is from 3 independent experiments (4 mice per experiment). (E) Real-time PCR analysis of Map3k1DKD for Cdkn1b undergoing Th17 differentiation. Na€ıve CD4C T cells from the spleen of WT and Map3k1DKD mice were isolated, differentiated under Th17 conditions and their RNA was analyzed by real-time PCR as indicated (& WT and & Map3k1DKD). The average relative expression (§ SEM) of genes from 3 independent experiments was statistically analyzed, where appropriate, by 2-tailed Student’s t test (, p 0.05; , p 0.01; , p 0.001).
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Figure 2. Mekk1 and Tak1 regulate iNKT cell proliferative expansion in the liver. (A) Liver cells from WT, Map3k1DKD, LckCre/CMap3k1f/f and LckCre/CMap3k7f/f mice were isolated and stained with CD1d tetramer and anti-CD3 antibody and analyzed by flow cytometry as shown. Data is representative of at least 5 mice. (B) Aberrant iNKT liver cell expansion in LckCre/CMap3k1f/fand LckCre/CMap3k7f/f( ) mice. Map3k1DKD/C or LckCre/CMap3k7f/C ( ), Map3k1DKD ( ) or LckCre/CMap3k7f/f ( ) were i.p. injected with a-GalCer over a 6-day timecourse as shown. Liver cells were harvested at days 0, 3, and 6, stained with CD1d tetramer and anti-TCRb antibody and analyzed by flow cytometry as shown. Data was representative of at least 3 mice.
iNKT cells in both Map3k1 and Map3k7 conditionally deleted T cells (Fig. 2A). The iNKT cells from both Map3k1DKD and LckCre/CMap3k7f/f mice overexpand following stimulation by glycolipid antigen (Fig. 2B). Thus, both Map3k1 and Map3k7 perform similar roles in regulating liver iNKT cell proliferative expansion in response to antigen.3 At the molecular level Mekk1 can regulate Tak1 signaling by the non-canonical ubiquitination of Tab1.4,14
Perspectives Detailed genetic analyses of T cell proliferative expansion and differentiation have revealed that Mekk1-dependent regulation of Jnks and Cdkn1b expression is an important mechanism that controls lymphocyte cell cycle progression (Fig. 3).3 Future investigations will apply our Mekk1 signaling mechanisms to the regulation of proliferative expansion in other cell types and in cancers.
Figure 3. Schematic illustrating Mekk1 signaling and the regulation of Cdkn1b expression in T cells. Following antigen receptor engagement Mekk1 signaling activates the Jnk Mapk pathway to regulate Cdkn1b transcription. p27Kip1 negatively regulates T cell proliferative expansion.
CELL CYCLE
Materials and methods
Funding
Cell culture, mice and cell harvest DK
Cre/C
1959
f/f
Cre/C
f/f
WT, Map3k1 D, Lck Map3k1 and Lck Map3k7 mice (C57BL/6 background) were bred and maintained as previously described.3,17 T cell differentiation assays were performed as previously described.5,15 For T cell differentiation assays, na€ıve T cells were activated with anti-CD3 (3 g/ml, BD Biosciences), anti-IL-4 (10 mg/ml, BD Biosciences), anti-IFNg (10 mg/ml, BD Biosciences) and anti-CD28 (2 g/ml, BD Biosciences) antibodies and polarizing cytokines (IL-6 at 25 ng/ml, IL-1b at 10 ng/ml and TGF-bat 2.5 ng/ml, all from PeproTech).15 T cells were collected for analysis at day 5 of culture. Harvested liver and spleen were passed through 70 mm and 100 mm strainers, resuspended in RPMI 1640 medium (Invitrogen) supplemented with 10 % FBS (ThermoScientific), gently pelleted in a centrifuge, treated with RBC lysis buffer (Sigma), washed and resuspended in RPMI 1640 medium. Liver cells were resuspended in 38 % Percoll (GE Healthcare) and centrifuged at 500 g for 20 minutes at room temperature. Cell pellets were treated with RBC lysis buffer, washed and resuspended in RPMI 1640 medium. The isolation of iNKT cells was performed using PE-conjugated and a-GalCer-loaded CD1d tetramer as previously described.3,18 Flow cytometry and real-time PCR Cell staining for flow cytometry was performed as previously described.3 Stained cells were then analyzed using a Cyan ADP (DakoCytomation) flow cytometer and FlowJo bioinformatics software (TreeStar). Na€ıve CD4C T cells were isolated using MACS LS columns (Miltenyi Biotech) according to the manufacturers protocols. Th17 cell RNA was purified using a RNeasy kit (Qiagen) and the RNA (500 mg) was converted to cDNA using a High Capacity cDNA RT-Kit (Applied Biosystems). Real-time PCR was performed as previously described.3 Global gene expression and bioinformatics analyses RNA from Th17 cells was reverse transcribed using a RNA amplification kit (Ambion) as previously described.3 RNA samples were hybridized onto Mouse Gene 1.0 ST arrays (Affymetrix).4 GeneSpringX software was used to generate heat maps.3 Statistical data was analyzed, where appropriate, as standard error of the mean (SEM) and statistical significance was assessed by 2-tailed Student’s t test (GraphPad). ArrayExpress accession number: E-MTAB-4499.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Acknowledgments We would like to thank Shashi Prajapati (Biogen) for thoughts on the manuscript. The authors have no conflicting financial interests.
Research was supported by grants from the Wellcome Trust (WT090939MA) and Cancer Research UK (C26616/A12679).
References [1] Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cellproliferation and transformation. Biochimica et biophysica acta 1991; 1072:129-57; PMID:1751545 [2] Karin M, Gallagher E. From JNK to pay dirt: jun kinases, their biochemistry, physiology and clinical importance. IUBMB Life 2005; 57:283-95; PMID:16036612; http://dx.doi.org/10.1080/ 15216540500097111 [3] Suddason T, Anwar S, Charlaftis N, Gallagher E. T-Cell-Specific Deletion of Map3k1 Reveals the Critical Role for Mekk1 and Jnks in Cdkn1b-Dependent Proliferative Expansion. Cell Rep 2016; 14:449-57; PMID:26774476; http://dx.doi.org/10.1016/j.celrep. 2015.12.047 [4] Charlaftis N, Suddason T, Wu X, Anwar S, Karin M, Gallagher E. The MEKK1 PHD ubiquitinates TAB1 to activate MAPKs in response to cytokines. EMBO J 2014; 33:2581-96; PMID:25260751; http://dx.doi.org/10.15252/embj.201488351 [5] Gao M, Labuda T, Xia Y, Gallagher E, Fang D, Liu YC, Karin M. Jun turnover is controlled through JNK-dependent phosphorylation of the E3 ligase Itch. Science 2004; 306:271-5; PMID:15358865; http:// dx.doi.org/10.1126/science.1099414 [6] Sabapathy K, Hochedlinger K, Nam SY, Bauer A, Karin M, Wagner EF. Distinct roles for JNK1 and JNK2 in regulating JNK activity and c-Jun-dependent cell proliferation. Mol Cell 2004; 15:713-25; PMID:15350216; http://dx.doi.org/10.1016/j.molcel.2004.08.028 [7] Sabapathy K, Kallunki T, David JP, Graef I, Karin M, Wagner EF. cJun NH2-terminal kinase (JNK)1 and JNK2 have similar and stagedependent roles in regulating T cell apoptosis and proliferation. J Exp Med 2001; 193:317-28; PMID:11157052; http://dx.doi.org/ 10.1084/jem.193.3.317 [8] Sabapathy K, Hu Y, Kallunki T, Schreiber M, David JP, Jochum W, Wagner EF, Karin M. JNK2 is required for efficient T-cell activation and apoptosis but not for normal lymphocyte development. Curr Biol 1999; 9:116-25; PMID:10021384; http://dx.doi.org/10.1016/ S0960-9822(99)80065-7 [9] Gallagher E, Enzler T, Matsuzawa A, Anzelon-Mills A, Otero D, Holzer R, Janssen E, Gao M, Karin M. Kinase MEKK1 is required for CD40-dependent activation of the kinases Jnk and p38, germinal center formation, B cell proliferation and antibody production. Nat Immunol 2007; 8:57-63; PMID:17143273; http://dx.doi.org/10.1038/ ni1421 [10] Venuprasad K, Elly C, Gao M, Salek-Ardakani S, Harada Y, Luo JL, Yang C, Croft M, Inoue K, Karin M, et al.. Convergence of Itchinduced ubiquitination with MEKK1-JNK signaling in Th2 tolerance and airway inflammation. J Clin Invest 2006; 116:1117-26; PMID:16557301; http://dx.doi.org/10.1172/JCI26858 [11] Enzler T, Chang X, Facchinetti V, Melino G, Karin M, Su B, Gallagher E. MEKK1 binds HECT E3 ligase Itch by its amino-terminal RING motif to regulate Th2 cytokine gene expression. J Immunol 2009; 183:3831-8; http://dx.doi.org/10.4049/jimmunol.0803412 [12] Karin M, Gallagher E. TNFR signaling: ubiquitin-conjugated TRAFfic signals control stop-and-go for MAPK signaling complexes. Immunological reviews 2009; 228:225-40; PMID:19290931; http:// dx.doi.org/10.1111/j.1600–065X.2008.00755.x [13] Matsuzawa A, Tseng PH, Vallabhapurapu S, Luo JL, Zhang W, Wang H, Vignali DA, Gallagher E, Karin M. Essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex. Science 2008; 321:663-8; PMID:18635759; http://dx.doi.org/10.1126/science.1157340 [14] Suddason T, Gallagher E. A RING to rule them all? Insights into the Map3k1 PHD motif provide a new mechanistic understanding into the diverse roles of Map3k1. Cell Death Differ 2015; PMID:25613373
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[15] Chang X, Liu F, Wang X, Lin A, Zhao H, Su B. The kinases MEKK2 and MEKK3 regulate transforming growth factor-betamediated helper T cell differentiation. Immunity 2011; 34:20112; PMID:21333552; http://dx.doi.org/10.1016/j.immuni. 2011.01.017 [16] Wan YY, Chi H, Xie M, Schneider MD, Flavell RA. The kinase TAK1 integrates antigen and cytokine receptor signaling for T cell development, survival and function. Nat Immunol 2006; 7:851-8; PMID:16799562; http://dx.doi.org/10.1038/ni1355
[17] Sato S, Sanjo H, Tsujimura T, Ninomiya-Tsuji J, Yamamoto M, Kawai T, Takeuchi O, Akira S. TAK1 is indispensable for development of T cells and prevention of colitis by the generation of regulatory T cells. Int Immunol 2006; 18:1405-11; PMID:16940043; http:// dx.doi.org/10.1093/intimm/dxl082 [18] Nagaleekar VK, Sabio G, Aktan I, Chant A, Howe IW, Thornton TM, Benoit PJ, Davis RJ, Rincon M, Boyson JE. Translational control of NKT cell cytokine production by p38 MAPK. J Immunol 2011; 186:4140-6; http://dx.doi.org/10.4049/jimmunol.1002614
EXTRA VIEW
Genetic insights into Map3k-dependent proliferative expansion of T cells Tesha Suddason and Ewen Gallagher Department of Medicine, Imperial College London, London, UK
ABSTRACT
ARTICLE HISTORY
Mapks are important regulators of T cell proliferative expansion and cell cycle progression. Detailed genetic analysis of unconventional iNKT cells in both Map3k1DKD and LckCre/CMap3k1f/f mice demonstrated that Mekk1 (encoded by Map3k1) signaling activates Mapks to regulate Cdkn1b (encoding p27Kip1) expression and p27Kip1-dependent proliferative expansion in response to antigen. Mekk1 signaling and activation of E3 ubiquitin ligase Itch, by a phosphorylation-dependent conformational change, is also an important regulatory mechanism for the control of T helper cell cytokine production. Cdkn1b expression is regulated by Mekk1-dependent signaling in differentiated Th17 cells. Mekk1 is one of the 19 Ste11-like Map3ks, and Mekk1 signaling regulates iNKT cell proliferative expansion in response to glycolipid antigens and T cell homeostasis in the liver. Tak1 (encoded by Map3k7), a related Map3k to Mekk1, similarly regulates the proliferative expansion and homeostasis of T cells in the liver, and this illustrates the importance of multiple Map3ks for mammalian Mapk signaling.
Received 2 March 2016 Revised 6 May 2016 Accepted 6 May 2016
Introduction Signal transduction by Mitogen-activated protein kinases (Mapks) emerged a quarter of a century ago as a potential mechanism of regulation for the cell cycle in response to extracellular stimuli.1,2 But, after the initial biochemical characterization of Mapks, it only proved possible to carefully clarify our understanding of the many proteins that constitute Mapk cascades by intricate analyses using mice that harbor genetic mutations of these signaling proteins that, in turn, alter the biochemical function of Mapks.2 By these means mammalian genetic analysis bridged the gap in our knowledge derived from simple in vitro enzyme-substrate reactions toward the greater complexity found within cells of the whole organism.2 Mapk kinase (Map2k) kinases (Map3ks) form critical points for Mapk regulation in response to mitogenic stimuli, and consequently the genetic analyses of Mapk signaling has proved informative in defining the role of Mapks in the cell cycle process.3 Of these, Map3k1 (encoding Mek kinase 1, Mekk1) has now been analyzed in mice by germline knockout (Map3k1DKD and Map3k1-/- mice), knockin (Map3k1mPHD mice) and very recently by tissue-specific deletion in T cells (LckCre/CMap3k1f/f mice).3-5 Out of these important genetic investigations the cell cycle component p27Kip1 (encoded by Cyclin-dependent kinase inhibitor 1B, Cdkn1b) has emerged from global gene expression analysis as a c-Jun N-terminal kinase (Jnk) and Mekk1 signaling target, and p27Kip1 forms a signaling mechanism to control proliferative expansion of unconventional invariant natural killer T (iNKT) cells.3 The genetic roles of Jnk isoforms as precise mechanisms for controlling mouse embryonic fibroblast (MEF) proliferation and T cells are well established.6-8 Upstream of Jnks, the analysis of
KEYWORDS
Cdkn1b; iNKT; Mapk; Mekk1; proliferation; T cell; th17
lymphocyte signaling using kinase-deficient Map3k1DKD mice has shown important roles for Mekk1 in controlling both B and T cell proliferation.5,9 Mekk1 has been shown to be of importance in T helper (Th) 2 differentiation, though its potential role in Th17 differentiation has not been analyzed to date.5,10,11 In the case of B cells, engagement of CD40 by CD40 ligand critically initiates p38 and Jnk activation to phosphorylate Jun family and other transcription factors, and these, in turn, regulate proliferation by the upregulation of Cyclin D2 transcription.9,12 Both CD4C and CD8C Map3k1DKD T cells display aberrant proliferation following T cell receptor (TCR) engagement by crosslinking with antibodies or in response to antigen presented by cells.5,9 Mechanistically the identification by global gene expression analysis of p27Kip1 as a downstream Mekk1 signaling protein has revealed a divergence between Mapk signaling and the control of cell cycle between B and T cells.3,9 Mekk1 is rapidly recruited to cytokine (e.g. CD40 and TGF-b receptors) and antigen receptors, along with the rest of the Mapk singling module, in a low phosphorylation state, and binds to upstream receptor components by the Mekk1 N-terminal regulatory domain, containing a plant homeodomain (PHD) that can act as an E3 ubiquitin (Ub) ligase.4,13,14 High-throughput protein microarray screening precisely elucidated the Mekk1 PHD substrates in depth, and these include TGF-b activated kinase 1/MAP3K7 binding protein 1 (Tab1), TNF receptor associated factor 2 (Traf2) and CARD-containing MAGUK protein 1 (Carma1).3,4,14 The Mekk1 PHD, along with other E3 Ub ligases, facilitates the non-canonical ubiquitination that occurs at TGF-b receptors, CD40 and antigen receptors as the Mapk module switches into its active form.4,12-14 Ubiquitination facilitates
CONTACT Ewen Gallagher [email protected] Department of Medicine, Imperial College London, Du Cane Road, London, W12 ONN, UK. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kccy. © 2016 Taylor & Francis
CELL CYCLE
both the activation of the Mapk pathway and the translocation of the Mapk signaling complex from the receptor and onto its downstream targets.4,12,13
Results and discussion
1957
expression and real-time PCR analyses (Fig. 1D and E) identified Cdkn1b as a Mekk1-regulated cell cycle gene.3 Thus, Mekk1 signaling regulates Cdnk1b expression and the cell cycle in several important T cell effector responses, both Th17 differentiation and iNKT cell expansion in response to glycolipid antigen.3
Mekk1 signaling regulates Cdkn1b expression during T cell differentiation Since Cdkn1b expression is critical for iNKT cell expansion we investigated whether a similar regulatory mechanism might also occur in conventional CD4C T cells undergoing differentiation.3,5,15 Map3k1DKD T cells are defective in Interleukin (IL) -17 production when undergoing differentiation into Th17 cells (Fig. 1A and B). Similarly, inhibition of Jnks, Ubiquitinconjugating enzyme E2N (Ube2n) or TGF-b receptor signaling reduced IL-17 production in Th17 cells (Fig. 1C).4 Global gene
Map3k1 and Map3k7 regulate iNKT cell proliferative expansion in the liver Map3k1DKD and LckCre/CMap3k1f/f mice display elevated numbers of iNKT cells within the liver.3 Both Mekk1 and Tak1 (encoded by Map3k7) have an important role in T cell Mapk signaling,5,16 and to assess this further we compared the numbers of iNKT cells in Map3k1DKD and LckCre/CMap3k1f/f mice with LckCre/CMap3k7f/f mice and found elevated numbers of
Figure 1. Mekk1 regulates Cdkn1b expression in Th17 cells. (A) Na€ıve CD4CT cells were extracted from WT and Map3k1DKD mice and differentiated under Th17 conditions. FACS analysis was performed with the indicated antibodies (4 mice per experiment). (B) Quantitation of WT and Map3k1DKD IL-17CCD4CT cells (& WT and & Map3k1DKD) differentiated under Th17 conditions (n D 3). (C) Na€ıve CD4C T cells were extracted from WT and Map3k1DKD mice and incubated with SP600125, NSC697923 or SB431542 inhibitors while differentiating under Th17 conditions (4 mice per experiment). FACS analysis was performed with the indicated antibodies. (D) Global gene expression analysis of Th17 cells. Na€ıve CD4C T cells were extracted from WT and Map3k1DKD mice and differentiated into Th17 cells. RNA was isolated from WT and Map3k1DKD splenic Th17 cells, processed and hybridized onto Affymetrix arrays. Bioinformatics analysis was performed and a heat map comparing gene hits between WT and Map3k1DKD Th17 cell microarray screen hits was constructed. The data is from 3 independent experiments (4 mice per experiment). (E) Real-time PCR analysis of Map3k1DKD for Cdkn1b undergoing Th17 differentiation. Na€ıve CD4C T cells from the spleen of WT and Map3k1DKD mice were isolated, differentiated under Th17 conditions and their RNA was analyzed by real-time PCR as indicated (& WT and & Map3k1DKD). The average relative expression (§ SEM) of genes from 3 independent experiments was statistically analyzed, where appropriate, by 2-tailed Student’s t test (, p 0.05; , p 0.01; , p 0.001).
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Figure 2. Mekk1 and Tak1 regulate iNKT cell proliferative expansion in the liver. (A) Liver cells from WT, Map3k1DKD, LckCre/CMap3k1f/f and LckCre/CMap3k7f/f mice were isolated and stained with CD1d tetramer and anti-CD3 antibody and analyzed by flow cytometry as shown. Data is representative of at least 5 mice. (B) Aberrant iNKT liver cell expansion in LckCre/CMap3k1f/fand LckCre/CMap3k7f/f( ) mice. Map3k1DKD/C or LckCre/CMap3k7f/C ( ), Map3k1DKD ( ) or LckCre/CMap3k7f/f ( ) were i.p. injected with a-GalCer over a 6-day timecourse as shown. Liver cells were harvested at days 0, 3, and 6, stained with CD1d tetramer and anti-TCRb antibody and analyzed by flow cytometry as shown. Data was representative of at least 3 mice.
iNKT cells in both Map3k1 and Map3k7 conditionally deleted T cells (Fig. 2A). The iNKT cells from both Map3k1DKD and LckCre/CMap3k7f/f mice overexpand following stimulation by glycolipid antigen (Fig. 2B). Thus, both Map3k1 and Map3k7 perform similar roles in regulating liver iNKT cell proliferative expansion in response to antigen.3 At the molecular level Mekk1 can regulate Tak1 signaling by the non-canonical ubiquitination of Tab1.4,14
Perspectives Detailed genetic analyses of T cell proliferative expansion and differentiation have revealed that Mekk1-dependent regulation of Jnks and Cdkn1b expression is an important mechanism that controls lymphocyte cell cycle progression (Fig. 3).3 Future investigations will apply our Mekk1 signaling mechanisms to the regulation of proliferative expansion in other cell types and in cancers.
Figure 3. Schematic illustrating Mekk1 signaling and the regulation of Cdkn1b expression in T cells. Following antigen receptor engagement Mekk1 signaling activates the Jnk Mapk pathway to regulate Cdkn1b transcription. p27Kip1 negatively regulates T cell proliferative expansion.
CELL CYCLE
Materials and methods
Funding
Cell culture, mice and cell harvest DK
Cre/C
1959
f/f
Cre/C
f/f
WT, Map3k1 D, Lck Map3k1 and Lck Map3k7 mice (C57BL/6 background) were bred and maintained as previously described.3,17 T cell differentiation assays were performed as previously described.5,15 For T cell differentiation assays, na€ıve T cells were activated with anti-CD3 (3 g/ml, BD Biosciences), anti-IL-4 (10 mg/ml, BD Biosciences), anti-IFNg (10 mg/ml, BD Biosciences) and anti-CD28 (2 g/ml, BD Biosciences) antibodies and polarizing cytokines (IL-6 at 25 ng/ml, IL-1b at 10 ng/ml and TGF-bat 2.5 ng/ml, all from PeproTech).15 T cells were collected for analysis at day 5 of culture. Harvested liver and spleen were passed through 70 mm and 100 mm strainers, resuspended in RPMI 1640 medium (Invitrogen) supplemented with 10 % FBS (ThermoScientific), gently pelleted in a centrifuge, treated with RBC lysis buffer (Sigma), washed and resuspended in RPMI 1640 medium. Liver cells were resuspended in 38 % Percoll (GE Healthcare) and centrifuged at 500 g for 20 minutes at room temperature. Cell pellets were treated with RBC lysis buffer, washed and resuspended in RPMI 1640 medium. The isolation of iNKT cells was performed using PE-conjugated and a-GalCer-loaded CD1d tetramer as previously described.3,18 Flow cytometry and real-time PCR Cell staining for flow cytometry was performed as previously described.3 Stained cells were then analyzed using a Cyan ADP (DakoCytomation) flow cytometer and FlowJo bioinformatics software (TreeStar). Na€ıve CD4C T cells were isolated using MACS LS columns (Miltenyi Biotech) according to the manufacturers protocols. Th17 cell RNA was purified using a RNeasy kit (Qiagen) and the RNA (500 mg) was converted to cDNA using a High Capacity cDNA RT-Kit (Applied Biosystems). Real-time PCR was performed as previously described.3 Global gene expression and bioinformatics analyses RNA from Th17 cells was reverse transcribed using a RNA amplification kit (Ambion) as previously described.3 RNA samples were hybridized onto Mouse Gene 1.0 ST arrays (Affymetrix).4 GeneSpringX software was used to generate heat maps.3 Statistical data was analyzed, where appropriate, as standard error of the mean (SEM) and statistical significance was assessed by 2-tailed Student’s t test (GraphPad). ArrayExpress accession number: E-MTAB-4499.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Acknowledgments We would like to thank Shashi Prajapati (Biogen) for thoughts on the manuscript. The authors have no conflicting financial interests.
Research was supported by grants from the Wellcome Trust (WT090939MA) and Cancer Research UK (C26616/A12679).
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