Myocyte Enhancer Factor 2 Mediates Calcium-Dependent Transcription of the Interleukin-2 Gene in T Lymphocytes --A calcium signaling module that is distinct from but collaborates with NFAT

The second messenger calcium plays an essential role in the T cell receptor-mediated signal transduction pathways leading to transcription of the interleukin-2 gene. A key mechanism of calcium signaling has been shown to be mediated by calcineurin and NFAT. We report herein that the transcription factor myocyte enhancer factor (MEF)-2 is another calcium signal transducer involved in the regulation of the IL-2 promoter. A MEF2-binding site was identified in proximity to the TATA box of the IL-2 promoter. This site was shown to be bound by MEF2 in both resting and activated T cells. Overexpression of MEF2 enhanced, while overexpression of a dominant negative form of MEF2 or the MEF2-specific transcriptional corepressors Cabin1 and histone deacetylase 4 inhibited, the TCR-dependent activation of an IL-2 reporter gene. Downregulation of MEF2 by RNA interference in primary human T cells led to the inhibition of endogenous IL-2 transcription. These results suggest that MEF2 is required for the transcriptional activation of IL-2 and likely other cytokine genes in response to calcium signaling and may serve as a novel target for development of immunosuppressants.


INTRODUCTION
Activation of peripheral T cells through the engagement of TCR with MHC-peptide complexes on antigen presenting cells leads to the transcriptional activation of a number of cytokine genes that are involved in the orchestration of the cellular immune response. Prominent among the cytokines secreted by activated T cells is IL-2, which plays an essential role in subsequent T cell proliferation and homeostasis (1). The intracellular signal transduction pathway linking TCR and the IL-2 promoter has been used as a paradigm to elucidate mechanisms of signaling from cell surface to the nucleus. The second messenger calcium has been shown to be an essential mediator of the TCR signaling pathway. Through the use of the immunosuppressive natural products CsA and FK506, both of which inhibit a calcium-dependent signaling step leading to IL-2 transcription, a key downstream transducer of calcium signaling was identified as the calcium, and calmodulindependent protein phosphatase calcineurin (2)(3)(4)(5)(6)(7)(8) (also known as protein phosphatase 2B). The primary calcineurin substrates were revealed to be members of the nuclear factor of activated T cells (NFAT) protein family (9)(10)(11). NFAT exists in its latent form as a phosphoprotein in the cytosol in resting T cells. Upon activation of TCR, an increase in the cytosolic calcium concentration leads to the activation of calcineurin by calmodulin, which dephosphorylates NFAT, leading to its translocation into the nucleus where it binds to the IL-2 promoter and activates its transcription.
Another calcium signaling module, mediated by the transcription factor MEF2 (12), was recently identified and shown to be involved in the regulation of the pro-apoptotic gene Nur77 during thymocyte apoptosis (13)(14)(15)(16). Unlike NFAT, MEF2 is constitutively bound to its cognate DNA-binding elements in the nucleus independent of the intracellular calcium concentration. In unactivated thymocytes, MEF2 recruits a family of functionally redundant transcriptional repressors including Cabin1 (also known as cain) (17,18), MEF2 interacting transcriptional repressor (MITR), and HDAC4, 5, & 7 (14,19,20). These MEF2-specific corepressors recruit histone deacetylases to MEF2-associated promoter region, which remodel the chromatin structure, silencing the promoter by guest on March 22, 2020 http://www.jbc.org/ Downloaded from activity. Upon calcium influx, these MEF2 repressors are removed from MEF2 by activated calmodulin, enabling it to bind to such coactivators as p300, turning on transcription of the target genes (14,21). The binding of some MEF2 transcriptional repressors has also been shown to be subject to regulation by calmodulin-dependent kinases, providing another mechanism of activation of MEF2 by calcium (22,23).
In an attempt to ascertain the role of Cabin1 in thymocyte apoptosis in vivo, we generated a partial Cabin1 knockout mouse (Cabin1∆C) in which the C-terminal calcineurin-and MEF2binding domains are deleted (24

EXPERIMENTAL PROCEDURES
Cell culture, transfection and reporter gene assay. Jurkat T cells were cultured and transfected as described previously (17). The Cabin1 deletion mutant (with the calcineurin-binding domain deleted) was subcloned into pSG5 mammalian expression vector (Stratagene) and the resultant plasmid was named as pSG5-Cabin1∆CNBD. The mutant of pIL-2 luciferase (pIL2-Luc/Mut) reporter was made using a site-directed mutagenesis kit (Stratagene).
EMSA. Nuclear extracts from Jurkat T cells treated with PMA and ionomycin were prepared as described (17). The probe used was derived from the IL-2 promoter (-70 to -45) and contained the minimal putative MEF2-binding site (5'-ATTTTGACACCCCCATAATATTTTTC-3').
Construction of siRNA expression vector. The procedure for plasmid construction is similar to that described previously with slight modifications (25). To generate pBS/U6-siMEF2D, two oligonucleotides were synthesized. Oligo1 (sense): The first 20-nucleotide of Oligo 1 corresponds to 273-288 of human MEF2D cDNA sequence, followed by 9 nucleotides linker (small letter) and the inverted sequence containing five T's.
Positive clones containing the appropriate inserts were confirmed by DNA sequencing.

Construction of Lentivirus Vector.
To generate the lentiviral siRNA vector, pBS/U6-siMEF2D was digested with BamHI to release the U6 promoter along with the MEF2D siRNA targeting sequence. The BamHI fragment was subsequently blunt ended with Klenow fragment and inserted into the unique PacI site FUGW vector, affording pFUP-siMEF2D (26). For the control, the BamHI fragment from pBS/U6 was inserted into FUGW to give pFUP. Of the two possible orientations, the plasmid in which the U6 promoter was in opposite orientation to the LTR was chosen, as it gives slightly higher viral titer.
Lentivirus production. Recombinant lentiviruses were generated using a three-plasmid system as previous described (27). Viruses were harvested at 48 and 72 h after transfection and titer was determined based on percentages of GFP-positive Jurkat T cells after transduction with serially diluted viral supernatant. The titer, calculated as transducing units (TU) per milliliter of supernatant, was in the range of 2 × 10 6 to 8 × 10 6 TU/ml. The virus-containing supernatant was concentrated using Amicon Ultra Concentrator (Millipore) and stored at -80 °C as previously described (27,28).

RESULTS AND DISCUSSION
A MEF2-binding site exists in close proximity to the TATA box of the IL-2 promoter--As deletion of the C-terminal MEF2-binding domain along with the calcineurin-binding domain in Cabin1 resulted in upregulation of the expression of IL-2 and other cytokine genes, we hypothesized that MEF2 may be involved in the regulation of not only Nur77 expression during thymocyte apoptosis, but also transcription of IL-2 and other cytokines during activation of peripheral T cells (24). Thus, we searched the IL-2 promoter region for a potential MEF2-binding site, and a contiguous AT-rich sequence composed of eleven A or T nucleotides was found between -46 and -55 in the proximal IL-2 promoter ( Figure 1A). Although this AT-rich sequence deviates from the consensus MEF2binding sequence [CAT(A/T) 4 TAG] (30), the core AT nucleotides are conserved. The putative single nucleotide change from an "A" in the human sequence to a "T" in the mouse sequence, but this does not disrupt the contiguous "A/T" feature required for MEF2 binding.
To test whether the putative MEF2-binding site in the IL-2 promoter is capable of binding to MEF2, we prepared a [ 32 P]-labeled double-stranded DNA probe spanning the site, and performed electrophoretic gel mobility shift assay (EMSA). Using nuclear extracts of Jurkat T cells, we observed a protein-DNA complex ( Figure 1B Indeed, MEF2D, the most abundant isoform of MEF2 in T cells, is bound to the promoter of IL-2 ( Figure 2A). Similar to its association with the Nur77 promoter in T cell hybridoma, the binding of MEF2D to the endogenous IL-2 promoter is independent of the activation state of T cells ( Figure   2A, Lane 2 and 3). As a negative control, we also attempted to determine whether anti-MEF2 antibodies also pulled down the promoter of the house-keeping gene glyceraldehyde-6-phosphate dehydrogenase (GAPDH) using a pair of specific primers (32). Anti-MEF2D antibodies failed to pull down the GAPDH promoter (data not shown), suggesting that the binding of MEF2 to the IL-2 promoter was specific.
It has been previously shown that the MEF2-specific transcriptional co-repressors Cabin1 and HDAC4 are bound to MEF2 in a calcium-dependent fashion (14,21). These repressors are released from MEF2 upon calcium signaling. We therefore determined if Cabin1 and HDAC4 could also associate with the IL-2 promoter via MEF2. Both Cabin1 and HDAC4 were found to be associated with the IL-2 promoter in non-stimulated T cells ( promoter. Thus, the MEF2-binding site in the IL-2 promoter plays a critical role in its activation. If MEF2 is required for IL-2 promoter activation, it is predicted that downregulation of MEF2 activity or its protein level should cause inhibition of IL-2 reporter gene expression. As a preliminary assessment of the importance of MEF2 in IL-2 activation, we expressed a dominant negative form of MEF2D (dnMEF2) missing the C-terminal transactivation domain (33,34), and determined its effect on the IL-2 reporter gene activation stimulated by PMA and ionomycin.
Overexpression of the dnMEF2 mutant significantly inhibited the activation of the IL-2 reporter gene (Figure 3), suggesting that MEF2 is required for IL-2 promoter activation. Purified CD4+ T cells from human peripheral blood were cultured with IL-7 and transduced by either vector. The gene transduction efficiency by both vectors was estimated to be around 80%, based on GFP expression in flow cytometric analysis (not shown). As determined by

Downregulation of MEF2 by RNA interference blocks transcription of endogenous IL-2 gene in response to stimulation by TCR agonists--To
Western blot analysis, the MEF2 siRNA construct significantly decreased the expression of MEF2D ( Figure 5A, upper panel). The effect of the siRNA construct appeared to be specific for MEF2D, as it had no effect on the protein level of endogenous tubulin ( Figure 5A, lower panel).

When the virally transduced human primary T cells (including untransduced T cells) were
stimulated with a mixture of anti-CD3 and anti-CD28 antibodies, IL-2 transcription was seen in cells transduced with the empty vector pFUP ( Figure 5B). A significant decrease in IL-2 mRNA synthesis was observed in the T cell population transduced with the MEF2D siRNA viruses. In agreement with the decrease in RNA level, the level of IL-2 protein secreted into the media was also dramatically reduced in human T cells transduced with MEF2D siRNA viruses ( Figure 5C). In    Wild type IL-2 luciferase reporter plasmid (pIL2-Luc/WT) and a plasmid with mutations on the putative MEF2-binding site (pIL2-Luc/Mut) were transfected into Jurkat T cells along with other expression plasmids as shown. Cells were treated with PMA plus ionomycin for 8 h before they were lysed for measurement of luciferase and β-galactosidase activities.