Positive Regulation of Interleukin-2 Expression by a Pseudokinase, Tribbles 1, in Activated T Cells

Abstract

Tribbles 1 (TRB1), a member of the Tribbles family, is a pseudokinase that is conserved among species and implicated in various human diseases including leukemia, cardiovascular diseases, and metabolic disorders. However, the role of TRB1 in the immune response is not understood. To evaluate this role, we examined regulation of TRB1 expression and the function of TRB1 in interleukin-2 (IL-2) induction in Jurkat cells, a human acute T cell leukemia cell line. We found that TRB1 was strongly induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin in these cells. IL-2 expression was induced in Jurkat cells activated by PMA and ionomycin; however, knockdown of TRB1 resulted in decreased induction of IL-2. TRB1 null Jurkat cells established using the CRISPR/Cas9 system also showed reduction of IL-2 expression on PMA/ionomycin stimulation. TRB1 knockdown also markedly inhibited IL-2 promoter activation. To determine the mechanism of the stimulatory effect on IL-2 induction, we focused on histone deacetylases (HDACs), and found that HDAC1 preferentially interacts with TRB1. TRB1 suppressed the interaction of HDAC1 with nuclear factor of activated T cells 2 (NFAT2), which is a crucial transcription factor for IL-2 induction. These results indicate that TRB1 is a positive regulator of IL-2 induction in activated T cells.

Interleukin-2 (IL-2) is a central factor in the immune system that affects various lymphocyte subsets during differentiation and in immune responses and homeostasis.¹⁾ IL-2 is crucial for differentiation of CD4⁺ T cells into defined effector T cell subsets following antigen-mediated activation and for development and peripheral expansion of regulatory T (Treg) cells, which promote self-tolerance by suppressing T cell responses. For CD8⁺ T cells, IL-2 signaling optimizes effector T cell generation and differentiation into memory cells.²⁾ IL-2 is produced primarily in activated CD4⁺ T cells and is regulated at the mRNA level by signals from the T cell receptor (TCR) and CD28.³⁾ Regulation of IL-2 transcription has been well characterized, and the nuclear factor of activated T cells (NFAT) and activator protein-1 (AP-1) transcription factors have been shown to be essential for inducible IL-2 expression in activated T cells.^4–6)

Activation of NFATs is mainly regulated by calcium and calcineurin, while that of AP-1 is regulated by other pathways, including the protein kinase C (PKC) and Ras-mitogen-activated protein kinase (MAPK) pathways. In response to TCR stimulation, phospholipase C γ1 (PLCγ1) is phosphorylated and activated, and subsequent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PLCγ1 produces inositol trisphosphate (IP3) and diacylglycerol (DAG) as second messengers. IP3 mediated Ca²⁺ release from the endoplasmic reticulum (ER) and sequential entry of extracellular Ca²⁺ through calcium release-activated Ca²⁺ (CRAC) channels activates calcineurin to dephosphorylate NFATs, resulting in full activation of their DNA-binding and transcriptional functions. DAG activates PKCθ and the MAP kinase pathway, while CD28-mediated co-stimulation leads to PKCθ stimulation, which activates AP-1 through the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways.^7,8)

TRB1 is a mammalian ortholog of Tribbles, which controls cell division and cell migration during embryonic Drosophila development.^9–11) TRB1 and other Tribbles family members (TRB2 and TRB3) contain the classic substrate-binding domain of a protein kinase, but not the ATP-binding and kinase-activating domains; therefore, these proteins do not have kinase activity and are classified as pseudokinases.¹²⁾ Recent reports suggest that Tribbles family members function as adaptor or scaffold proteins to facilitate degradation of target proteins and to regulate activation of various signaling pathways. Both the extent and specificity of MAPK kinase activation is regulated by association with the C-terminal region of TRB1.¹³⁾ TRB1 also has a role in the ubiquitin–proteasome pathway as an adaptor protein for constitutive photomorphogenic protein 1 (COP1), thereby promoting ubiquitination and degradation of acetyl coenzyme A carboxylase (ACC)¹⁴⁾ and CCA AT/enhancer-binding protein (C/EBPα, C/EBPβ).^15,16)

TRB1 is implicated in diseases such as cancer and myocardial infarction, and high TRB1 expression with gene amplifications is associated with acute myeloid leukemia (AML) and myelodysplastic syndrome through promotion of ERK phosphorylation and degradation of C/EBPα.^14,17,18) There is also evidence linking TRB1 to control of plasma lipid homeostasis, which suggests that this molecule may be linked to risk factors for myocardial infarction.¹⁹⁾ TRB1 is also a critical factor for adipose tissue maintenance and suppression of metabolic disorders because it controls differentiation of tissue-resident M2-like macrophages.²⁰⁾

Members of the Tribbles family (TRB1, TRB2 and TRB3) are relatively highly expressed in immune tissues and cells,^21,22) but their functions and regulation in the immune system are not completely clear. TRB1 is a negative regulator of C/EBPβ protein expression and modulates C/EBPβ-dependent gene expression in Toll like receptor (TLR)-mediated signaling²³⁾; and a recent study showed that TRB1 is strongly expressed in Tregs, as a novel binding partner of Foxp3.²⁴⁾ In this study, we show that TRB1 functions as a positive regulator of nuclear factor of activated T cells (NFATs) to activate IL-2 expression.

MATERIALS AND METHODS

Cell Culture and Reagents

Jurkat cells were cultured in RPMI 1640 (Nacalai Tesque, Kyoto, Japan) with 10% heat-inactivated fetal bovine serum (FBS) (HyClone, South Logan, UT, U.S.A.), 100 U/mL penicillin and 100 µg/mL streptomycin (Meiji Seika Pharma, Tokyo, Japan). HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin. Phorbol 12-myristate 13-acetate (PMA) and ionomycin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were dissolved in dimethyl sulfoxide (DMSO).

Plasmids and Transfection

The green fluorescence protein (GFP) expression plasmid was purchased from Clontech (Mountain View, CA, U.S.A.). The reporter plasmid containing the IL-2 promoter region was amplified by polymerase chain reaction (PCR). The NFAT2 expression plasmid was kindly provided by Dr. Takayanagi (Department of Immunology, University of Tokyo, Japan).²⁵⁾ Expression plasmids for histone deacetylase (HDAC)1, HDAC2 and HDAC3 were kind gifts from Dr. Seto (Moffitt Cancer Center and Research Institute, Tampa, FL, U.S.A.),²⁶⁾ and those for HDAC5, HDAC6 and HDAC7 were kindly provided by Dr. Verdin (University of California, San Francisco, CA, U.S.A.).²⁷⁾ An expression plasmid for TRB1 with an N-terminal Flag-tag was amplified by PCR from a human TRB1 (NM_025195.3) encoding construct (Origene Technologies, Rockville, MD, U.S.A.). The reporter plasmid mIL-2-Luc (−303/+45) and expression plasmids for Myc-tagged TRB1, Flag-tagged NFAT2 and 6×HA-tagged HDAC1 were also generated by PCR. The GAL4 DNA binding domain (1–147) derived from the pFC-dbd plasmid (Agilent Technologies, Santa Clara, CA, U.S.A.) was introduced into a pCMV5B-Flag-NFAT2 plasmid with ClaI and SalI restriction sites. All constructs were verified by sequencing. HEK293 cells were transfected by the Chen-Okayama method²⁸⁾ and Jurkat cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.).

Antibodies

Mouse anti-β-actin (AC-15) antibodies were obtained from Sigma (St. Louis, MO, U.S.A.). Mouse anti-DYKDDDDK tag monoclonal antibody was purchased from Wako Pure Chemical Indistries, Ltd. Mouse anti-GFP (B-2) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, U.S.A.). Mouse anti-Myc antibody (9E10) and mouse anti-HA antibody (12CA5) were purchased from Roche (Basel, Switzerland). Rabbit Anti-TRB1 (09-126) antibody was purchased from Millipore (Billerica, MA, U.S.A.).

Guide RNA Design

To knockout the TRB1 gene in Jurkat cells, we performed genome editing using the CRISPR (clustered regularly interspaced short palindromic repeat)−Cas9 (Crispr-associated protein) system.^29,30) Guide RNAs (gRNAs) were obtained from Operon Biotechnologies (Tokyo, Japan). The gRNAs used for genome editing were as follows; hTRB1 gRNA #1 sense strand, 5′-CAC CGC GAA GCC GGA AGA GGC TGC-3′ and antisense strand, 5′-AAA CGC AGC CTC TTC CGG CTT CGC-3′; hTRB1 gRNA #2 sense strand, 5′-CAC CGG GGG GCT GAG GTA GTC CGG-3′ and antisense strand, 5′-AAA CCC GGA CTA CCT CAG CCC CCC-3′.

Lentivirus Infection

Human TRB1 gRNA was cloned into the pX330 vector (Addgene plasmid 42230). To create the lentiviral constructs, this gRNA was subcloned into lentivirus vector pLentiCRISPR v2 (Addgene plasmid 52961). 293FT cells (Invitrogen) were transfected with pLentiCRISPR-human TRB1 gRNA or pLentiCRISPR-empty vector, together with a packaging plasmid pLP1, pLP2 and pLP/VSVG (Invitrogen) by lipofection. After 48 h, each medium was collected and centrifuged. The viral supernatants were collected at 24 and 48 h after transfection. Jurkat cells were infected in the presence of 8 µg/mL polybrene (Sigma) using a centrifugation method (2000 rpm, 30 min).

RNA Extraction and Quantitative Real-Time PCR

Total RNA was extracted as described previously³¹⁾ and reverse transcription was performed with a High Capacity RNA-to cDNA Kit (Applied Biosystems, South San Francisco, CA, U.S.A.). Real-time PCR was performed with the use of SYBR Green chemistry on a 7300 Real Time PCR System (Applied Biosystems). The oligonucleotide sequences of specific primers (sense and antisense) were as follows: for human IL-2 5′-GCA TTT ACT GCT GGA TTT-3′ and 5′-ATG TTT CAG TTC TGT GGC-3′; for human TRB1 5′-TGC AAA TTT GTT TCC CTT AAG GA-3′ and 5′-GCG GAA CTC CCA AAT CCA-3′; for human TRB2 5′-TTT GCT TTT GAA CAA TGA GGG TTT-3′ and 5′-TCT CAG GCC ATT GCT ATC ATC TC-3′; for human TRB3 5′-TGA CAA CAC TTT TCC ATG ACC ATA G-3′ and 5′-GGA GGC CGA CAC TGG TAC AA-3′; for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (control) 5′-GAA GGT GAA GGT CGG AGT C-3′ and 5′-GAA GAT GGT GAT GGG ATT TC-3′, and for human β-actin (control) 5′-TGG CAC CCA GCA CAA TGA A-3′ and 5′-CTA AGT CAT AGT CCG CCT AGA AGC A-3′. Quantitative PCR amplification was performed using activation of DNA polymerase for 30 s at 95°C, denaturation for 15 s at 95°C, annealing and extension for 60 s at 60°C (45 cycles), and dissociation for 15 s at 95°C, 60 s at 60°C, and 15 s at 95°C.

Luciferase Assay

Jurkat cells were transiently transfected with IL-2 reporter plasmid (mIL-2-luc (−303/+45)) and pCMV-β-gal plasmid using Lipofectamine 2000 (Invitrogen). 293 cells were transiently transfected with mIL-2-luc (−303/+45) or pFR-Luc (Invitrogen), and pCMV-β-gal using Lipofectamine 2000 (Invitrogen) or the calcium phosphate precipitation method. After 24 h, cells were treated with 40 nM PMA/1 µM ionomycin for 6 h. Light emission was measured using a 1420 ARVO multilabel counter (PerkinElmer, Inc., Waltham, MA, U.S.A.). Luciferase activity is expressed after normalization based on the β-galactosidase value in the same sample.

Immunoprecipitation and Western Blotting

Cells were lysed in radio immunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, and 1% Triton X-100) supplemented with 1 mM ethylenediaminetetraacetic acid (EDTA) and protease inhibitors. Lysates were incubated with anti-Flag antibody and immunoprecipitated in the presence of protein G-Sepharose (GE Healthcare, Buckinghamshire, U.K.). Immunoprecipitates were washed five times with washing buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100 and 1 mM EDTA) and then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (10%). The gels were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) and probed with the antibodies indicated in the figure legends. Western blot membranes were developed using ECL Western blotting detection reagents (GE Healthcare) and light emission was quantified with a LAS3000 mini-Lumino Image Analyzer (GE Healthcare).

RNA Interference

The following small interfering RNAs (siRNAs) were obtained from Invitrogen: TRB1 sense strand, 5′-AGA UGA UGC UUU GUC AGA CAA ACAU-3′; and TRB1 sense strand 2, 5′-AUG UUU GUC UGA CAA AGC AUC AUC U-3′. A stealth siRNA (Stealth RNAi™ siRNA Luciferase Reporter Control, Invitrogen) was used as a control. Cells were transfected with each siRNA using Lipofectamine RNAiMAX reagent (Invitrogen).

RESULTS

TRB1 Is Upregulated in Jurkat Cells Activated by PMA and Ionomycin

We first examined the expression levels of TRB1, TRB2 and TRB3 in resting and activated Jurkat cells. Treatment with PMA and ionomycin mimics activation of protein kinase C and a rise in intracellular free calcium in response to T cell receptor stimulation.³²⁾ Expression of TRB1 mRNA in Jurkat cells was markedly augmented by treatment with 40 nM PMA and 1 µM ionomycin (Fig. 1A). This induction was also observed in Jurkat cells stimulated with PMA alone, but not with ionomycin alone. An increase in endogenous TRB1 protein was also induced by PMA/ionomycin in Jurkat cells (Fig. 1B). Given the level of redundancy among Tribbles family members, we also compared induction of TRB1 to that of TRB2 and TRB3. TRB2 mRNA expression was not affected by PMA and/or ionomycin, while ionomycin slightly but significantly increased TRB3 mRNA.

Fig. 1. Increased Expression of TRB1 mRNA and Protein Was Induced by Treatment with PMA and Ionomycin in Jurkat Cells

(A) Jurkat cells were treated for 6 h with 40 nM PMA and/or 1 µM ionomycin (Io). Total RNA was extracted and real-time PCR was performed. Expression levels of TRB1, TRB2 or TRB3 mRNA were normalized to that of GAPDH mRNA. Results are shown as mean±S.D. (B) Jurkat cells were treated with 40 nM PMA/1 µM ionomycin for 6 h. TRB1 stably transfected Jurkat cells and untransfected Jurkat cells were lysed and expression levels of TRB1 or β-actin protein were determined by immunoblotting with anti-TRB1 or anti-β-actin antibodies. (C) Jurkat cells were treated with 40 nM PMA/1 µM ionomycin for the indicated time. Total RNA was extracted and expression of TRB1, IL-2 or GAPDH mRNA was determined by real-time PCR. The expression level of TRB1 or IL-2 mRNA was normalized as in (A).

We next investigated the biological relevance of the altered TRB1 levels in activated Jurkat cells. It is well known that IL-2 is induced in T lymphocytes after antigen stimulation or PMA/ionomycin treatment, and TRB1 mRNA and IL-2 mRNA were induced in a time-dependent manner by PMA/ionomycin (Fig. 1C). These data indicate that, among the Tribbles family members, TRB1 is selectively induced by PMA/ionomycin and probably by antigen stimulation in T cells.

TRB1 Enhances Expression of IL-2 in Jurkat Cells

We next examined the effect of TRB1 on IL-2 induction in the activated Jurkat cells. To examine whether TRB1 affects expression of IL-2 mRNA, TRB1-deficient Jurkat cells were established using the CRISPR/Cas9 genome editing system.^29,30) Jurkat cells were infected with lentiviral constructs expressing Cas9 and guide RNAs (gRNAs) for exon 2 (#1) or exon 1 (#2) of human TRB1 and stable transfectants were established (Fig. 2A). Introduction of a mutation in the TRB1 gene was confirmed by detecting Cas9 expression and cleaved products in a T7E1 mismatch-sensitive nuclease assay (data not shown). Induction of IL-2 by PMA/ionomycin in the TRB1-deficient Jurkat cells was significantly reduced compared to that in control Jurkat cells (Fig. 2B).

Fig. 2. TRB1 Induced Expression of IL-2 mRNA by Activating Its Transcription

(A) Jurkat cells stably expressing Cas9 and hTRB1 gRNAs (#1 and #2) or control Jurkat cells stably expressing Cas9 were lysed. Lysates were immunoprecipitated (IP) with anti-TRB1 antibody and immunoblotted with anti-TRB1 antibody. The expression level of TRB1 protein was assessed by immunoblotting of total cell lysates with anti-TRB1 antibody. (B) Total RNA was extracted from Jurkat cells stably expressing both Cas9 and hTRB1 gRNAs (#1 and #2) or control Jurkat cells stably expressing Cas9, and real-time PCR was performed. The expression level of IL-2 mRNA was normalized to that of β-actin mRNA. Cells were treated with vehicle or 40 nM PMA/1 µM ionomycin for 6 h and total RNA was extracted. Results are shown as mean±S.D. (C) Jurkat cells were transiently transfected with mIL-2-Luc (−303/+45), pCMV-β-gal, and an expression plasmid for TRB1. After 24 h, cells were treated with vehicle or 40 nM PMA/1 µM ionomycin for 6 h and then lysed. Luciferase activity was normalized to β-galactosidase activity. Results are shown as mean±S.D. (D) 293 cells were transiently transfected with control siRNA or TRB1 siRNA, and total mRNA was extracted. The expression level of TRB1 mRNA was normalized to that of β-actin mRNA. Results are shown as mean±S.D. (E) 293 cells were transiently transfected with mIL-2-Luc (−303/+45), pCMV-β-gal, and the indicated siRNAs in the presence or absence of an expression plasmid for NFAT2. After 24 h, cells were treated with vehicle or 40 nM PMA/1 µM ionomycin for 6 h and then lysed. Luciferase activity was normalized as in (C).

We next examined the effect of TRB1 on IL-2 promoter activation by constructing a reporter plasmid containing the murine IL-2 proximal promoter region (−303 to +45) in a pGL4.27 vector. There are five potential NFAT binding sites in this promoter region.⁴⁾ A luciferase assay using reporter plasmids containing the IL-2 proximal promoter showed that the IL-2 promoter activity was augmented by overexpression of TRB1 in Jurkat cells. In addition, transient transfection of TRB1 siRNA or control (luciferase) siRNA in HEK293 cells to knockdown TRB1 expression (Fig. 2D) resulted in marked suppression of IL-2 promoter activation by PMA/ionomycin, even in the presence of exogenous NFAT2 (Fig. 2E). These results suggest that IL-2 induction in PMA/ionomycin-activated cells is elevated by TRB1, probably at the transcriptional level, and that NFAT2 may be a target of TRB1.

TRB1 Physically Interacts with HDAC1 to Suppress NFAT2-HDAC1 Binding

To explore the possibility that TRB1 affects the transcriptional activity of NFAT2, we used a heterologous transcriptional activation assay in which NFAT2 is fused to the DNA-binding domain of the yeast transcription factor GAL4. Expression of this fusion protein in 293 cells mediated induction of the heterologous promoter construct, pFR-luc, which contains five GAL4 upstream activation sequences (UASs) linked to an E1b TAT A box and a luciferase reporter gene. To determine whether TRB1 enhances the NFAT2 transactivation activity, pFR-luc was transfected into 293 cells together with a GAL4-NFAT2 expression plasmid in the presence or absence of TRB1. Co-transfection with TRB1 resulted in upregulation of NFAT2-dependent activation of the promoter in the presence or absence of PMA/ionomycin (Fig. 3A). Collectively, these data suggest that TRB1 potentiates NFAT2 transactivation activity.

Fig. 3. TRB1 Suppressed Interaction of HDAC1 with NFAT2

(A) 293 cells were transiently transfected with pFR-luc, pCMV-β-gal, an expression plasmid for TRB1, and an expression plasmid for the GAL4 DNA-binding domain fused to NFAT2. After 24 h, cells were treated with vehicle or 40 nM PMA/1 µM ionomycin for 6 h and then lysed. Luciferase activity was normalized to β-galactosidase activity. Results are shown as mean±S.D. (B) 293 cells were transiently transfected with the indicated expression plasmids for HDACs and the expression plasmid for TRB1. After 36 h, cells were lysed. Lysates were immunoprecipitated (IP) with anti-Flag antibody and immunoblotted with anti-Myc or anti-Flag antibody. The pEGFP-C1 expression plasmid was included in each transfection as a transfection efficiency control. The expression level of each protein was assessed by immunoblotting of total cell lysates with anti-Myc, anti-Flag or anti-GFP antibodies. * Immunoglobulin heavy chains. (C) 293 cells were transiently transfected with expression plasmids for HDAC1, NFAT2 and TRB1. After 24 h, cells were treated with 40 nM PMA/1 µM ionomycin for 6 h and then lysed. Lysates were immunoprecipitated (IP) with anti-Flag antibody and immunoblotted with anti-HA, anti-Myc or anti-Flag antibodies. The pEGFP-C1 expression plasmid was included in each transfection as a transfection efficiency control. The expression level of each protein was assessed by immunoblotting of total cell lysates with anti-HA, anti-Myc, anti-Flag or anti-GFP antibodies.

To examine the mechanism through which TRB1 enhances NFAT activation, we investigated the involvement of HDACs, the components of co-repressors to negatively regulate gene expression in this activation. We have examined the binding assay of TRB1 with enough expression levels of various HDACs (HDAC1, 2, 3, 4, 5, 6, and 7) in 293 cells, and TRB1 interaction with HDAC1 was apparently observed (Fig. 3B (lowest bands observed in first column), data not shown). HDAC1 interacted strongly with NFAT2 in PMA/ionomycin-activated 293 cells, and TRB1 suppressed this interaction (Fig. 3C). HDAC3 also interacted with TRB1 and NFAT2, however, TRB1 could not inhibit the formation of NFAT2-HDAC3 complex (data not shown). These results indicate that TRB1 can displace HDAC1 from NFAT2, and this displacing ability of TRB1 could explain how TRB1 potentiates the IL-2 induction in activated Jurkat cells.

DISCUSSION

In this study, we showed that stimulation with PMA and ionomycin induces TRB1 in Jurkat cells, and that knockdown or knockout of TRB1 markedly attenuates IL-2 expression in activated Jurkat cells. Treatment with PMA and ionomycin mimics activation of protein kinase C and a rise in intracellular free calcium in response to T cell receptor stimulation.³²⁾ Recently, TRB1 mRNA expression is reported to be up-regulated in regulatory T cells activated with IL-2 and anti-CD3/ anti-CD28 beads.²⁴⁾ Therefore, TRB1 may function as a positive regulator upon antigen-receptor signaling.

Among the members of the TRB family, inducers of TRB3 have been well characterized. Thus, TRB3 is induced by stressors such as endoplasmic reticulum stress,^31,33) amino acid starvation,^34,35) hypoxia,^36,37) mitochondrial stress,³⁸⁾ and insulin.^39,40) In contrast, little is known about the mechanisms regulating TRB1 induction. TRB1 is up-regulated during inflammatory events such as chronic antibody-mediated rejection of transplanted organs²¹⁾ and chronic inflammation of atherosclerotic arteries⁴¹⁾; and is also induced in response to mitochondrial dysfunction, independent of production of reactive oxygen species (ROS) or metabolic stress.⁴²⁾ This induction mainly occurs under transcriptional control with ERK1/2 activation. In the current study, we showed that TRB1 is potently induced by PMA stimulation in Jurkat cells. Numerous studies have indicated that PMA activates the Ras/ERK signaling pathway,⁴³⁾ and PMA, as well as inflammatory stimuli, may also activate TRB1 transcription via the MAPK pathway.

Previous reports have shown that TRB1 suppresses gene transcription by interacting with transcription factors such as retinoic acid receptor α/retinoic acid X receptor α (RARα/RXRα)⁴⁴⁾ and p53 (our unpublished data), or by destabilizing transcription factors such as C/EBPα and C/EBPβ.^15,16,20) In contrast, in adipocytes, TRB1 is a nuclear transcriptional co-activator for the nuclear factor κB (NF-κB) subunit RelA, thereby promoting induction of proinflammatory cytokines in these cells.⁴⁵⁾ TRB1 is also a target of inflammatory signals, which provides a molecular rationale for the amplification of proinflammatory responses in white adipose tissue.⁴⁵⁾ Similarly, we also found that TRB1 is induced and is a positive regulator of NFAT2 in activated T cells, which implicates TRB1 as an amplification factor in the IL-2-mediated immune response.

HDACs regulate chromatin remodeling, gene expression, and the functions of several transcription factors and non-histone proteins.^46,47) Calcineurin and NFAT2 are essential for upregulation of myosin heavy chain I/β isoform (MyHCI/β) promoter activity and mRNA expression, and ERK1/2-mediated phosphorylation of p300 is crucial for enhancing the transactivation function of NFAT2 by acetylation, which in turn is essential for Ca²⁺-induced MyHCI/β expression.⁴⁸⁾ Our results indicate that TRB1 enhances transactivation by NFAT2 and promotes dissociation of HDAC1 from NFAT2. This displacing activity of TRB1 may contribute to the enhancement of IL-2 promoter activation by dissociating HDAC1, one of the components of co-suppressor complex from NFAT on the IL-2 promoter. Further experiments are necessary to clarify this point.

In conclusion, our results show that TRB1 induced by TCR stimulation potentiates NFAT dependent IL-2 transcription. This finding suggests the presence of a novel regulatory pathway for the IL-2-mediated immune response.

Acknowledgments

We thank Dr. Hiroshi Takayanagi, Dr. Eric Verdin, and Dr. Edward Seto for generously providing expression plasmids, and Dr. Satoshi Sakai for discussion of the work. This study was supported by a Grant-in-Aid for Scientific Research (C) (Nos. 21590067, 24590085) from the Japan Society for the Promotion of Science (JSPS), and a Grant-in-Aid for Research in Nagoya City University.

Conflict of Interest

The authors declare no conflict of interest.

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