High mobility group box-1 protein acts as a coactivator of nuclear factor of activated T cells-2 in promoting interleukin-2 transcription

The International Journal of Biochemistry & Cell Biology 41 (2009) 641–648

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High mobility group box-1 protein acts as a coactivator of nuclear factor of activated T cells-2 in promoting interleukin-2 transcription Hui Liu c , Yong-ming Yao b,∗ , Li-hua Ding a , Hao Zhang a , Bing Yuan a , Qing Song c , Qi-nong Ye a , Cui-fen Huang a , Zhi-yong Sheng b a

Beijing Institute of Biotechnology, 27 Tai-Ping Lu Road, Beijing 100850, PR China Department of Microbiology and Immunology, Burns Institute, First Hospital Affiliated to the Chinese PLA General Hospital (formerly 304th Hospital), Beijing 100037, PR China c Surgical Intensive Care Unit, Chinese PLA General Hospital, Beijing 100853, PR China b

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Article history: Received 3 May 2008 Received in revised form 6 July 2008 Accepted 22 July 2008 Available online 26 July 2008 Keywords: HMGB1 NFAT2 IL-2 Immune SRNAi

a b s t r a c t High mobility group box-1 protein, an abundant and conserved constituent of vertebrate nuclei, has recently been reported to be an endogenous immune signal [Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B, Catalanotti F, Giazzon M, et al. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Reports 2004;5:825–30]. High mobility group box-1 protein can trigger the release of interleukin-2 and interleukin-12 from lymphocytes. However, at present the underlying mechanism remains unknown. It has been clarified that nuclear factor of activated T cells-2 transduces most immunological signals in T cells and modulates the production of interleukin-2. So it is natural that we asked whether high mobility group box-1 protein could promote production of interleukin-2 in a nuclear factor of activated T cells-2-dependent way. Our experiments firstly showed that high mobility group box-1 protein could bind to nuclear factor of activated T cells-2 in vivo and in vitro. High mobility group box-1 protein cotransfection markedly upregulated the transcription activity of nuclear factor of activated T cells-2 in promoting interleukin-2 reporter gene transcription, which was demonstrated to be dose-dependent. Cotransfection of high mobility group box-1 protein and nuclear factor of activated T cells-2 induced an 18.4-time increase of interleukin-2 activity in 293T cells and a 117.7-time increase in Hela cells. Moreover, inhibition of either high mobility group box-1 protein or nuclear factor of activated T cells -2 expression by sRNAi led to significant decrease of transcription activity of interleukin-2 reporter gene, suggesting that high mobility group box-1 protein and nuclear factor of activated T cells-2 both take important roles in facilitating interleukin-2 transcription, and high mobility group box-1 protein could act as a coactivator for nuclear factor of activated T cells-2 in enhancing transcription of interleukin-2. This discovery has not been reported elsewhere, and helps to understand the newly highlighted immunological role of high mobility group box-1 protein. © 2008 Elsevier Ltd. All rights reserved.

1. Introduction Interleukin (IL)-2 is an important cytokine involved in Th1/Th2 imbalance in sepsis (Yang et al., 2005), and it has long been recognized as pivotal in maintaining normal immunologic function and defense against infection after burns and trauma. IL-2 is also the cytokine which plays a role synergistically with IL-12 in the induction of lymphokine-activated killer cells and cytotoxic T lymphocytes. It is also characterized as a stimulatory factor of natural killer (NK) cell by enhanc-

∗ Corresponding author. Tel.: +86 10 66867394; fax: +86 10 68989955. E-mail address: c [email protected] (Y.-m. Yao). 1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2008.07.009

ing cytotoxicity and synthesis of interferon (IFN)-gamma by NK cells. High mobility group box-1 protein (HMGB1) was reported to trigger the production of IL-2 in allogeneic T cells (Kiani et al., 2001; Bianchi and Manfredi, 2007). However, at present the underlying mechanism is unknown. It has been reported that IL-2 expression could be regulated by nuclear factor of activated T cells (NFAT) family. It is well known to all that NFATs family is thought to regulate the expression of a variety of inducible genes such as IL-2, IL-4, and tumor necrosis factor-␣. NFATs was originally described as a nuclear protein that bound to the human IL-2 promoter upon activation of Jurkat T leukemia cells (Messmer et al., 2004). Multiple NF-AT binding sites containing the core sequence A/TGGAAAA are part of numerous lymphokine promoters. The human and murine

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IL-2 promoters/enhancers harbor two of those sites to which NF-AT factors bind with high affinity (Shaw et al., 1988). HMGB1 possibly promotes IL-2 transcription through interaction with NFAT. Our preliminary experiments showed that HMGB1 especially influenced the NFAT2 activity (data not shown), so we focused on NFAT2 in the present study, through which we hope to promote understanding of the molecular mechanism of HMGB1 in immunological regulation. 2. Materials and methods 2.1. DNA constructs and recombinant proteins Human PSH140C-Flag-NFATC1 eukaryotic expression plasmid (Randak et al., 1990) was kindly presented by Dr. Feng Chen (School of Medicine, Washington University in St. Louis, Missouri); NFATLUC (IL-2 enhancer luciferase reporter) (Northrop et al., 1994), a kind gift from Dr. Cockerill PN (Institute for Medical and Veterinary Science, Adelaide 5000, Australia). To construct pcDNA3-HMGB1, full-length human HMGB1 cDNA was obtained by standard PCR amplification from an ovary twohybrid cDNA library (Clontech). Full-length human HMGB1 cDNA was obtained using recombinant PCR (Palumbo et al., 2004). The amplified HMGB1 cDNA was cloned into pcDNA3 vector, and the constructs were confirmed by sequencing. 2.2. Cell culture and transfection Hela and HEK293T cells were purchased from ATCC and cultured in Dulbecco’s modified minimum essential medium (DMEM) supplemented with 10% FBS (fetal bovine serum) and 100 ␮g/ml penicillin and 100 ␮g/ml streptomycin. 24 h before transient transfection, cells were cultured in medium without penicillin or streptomycin. The cells were transfected using Lipofectamine 2000 (Invitrogen) with 0.1 ␮g of NFAT-LUC reporter plasmid, 0.1 ␮g of NFAT2 or HMGB1 expression vector, 0.1 ␮g of ␤-galactosidase reporter, and the respective empty vector was used to adjust the total amount of DNA. 4 h after transient transfection, 25 ng/ml phorbol myristate acetate (PMA) and 0.5 ␮mol/l ionomycin were used to stimulate cells. Reporter gene activity was measured 24 h after stimulation. Cells were harvested, and luciferase and ␤galactosidase activities were determined as described previously (Müller et al., 2001; Ye et al., 2000). ␤-Galactosidase activity was used as an internal control for transfection efficiency. All experiments were repeated at least 3 times.

2.4. Co-immunoprecipitation HEK293T or Hela cells were transfected using Lipofectamine 2000 (Invitrogen). At 24 h post-transfection, cells were washed twice with phosphate-buffered saline (PBS) and lysed in 0.5 ml of lysis buffer (50 mM Tris at pH 8.0, 500 mM NaCl, 0.5% NP-40, 1 mM DTT and protease inhibitor tablets from Roche). After brief sonication, the lysate was centrifuged at 14,000 rpm for 15 min at 4 ◦ C. The supernatant was used for subsequent co-immunoprecipitation. A 15 ␮l aliquot of 50% slurry of the anti-FLAG agarose beads (Sigma–Aldrich) was used in each immunoprecipitation. Immunoprecipitation was performed overnight at 4 ◦ C. The beads were centrifuged at 3000 rpm for 2 min, and washed 4 times with the washing buffer (the same as the lysis buffer), with each wash lasting at least 30 min. The precipitates were then eluted in 30 ␮l of 2× SDS − PAGE sample buffer and loaded on SDS − polyacrylamide gels, followed by Western blotting according to the standard procedures. A 4 ␮l aliquot of the input crude extract was used for detecting protein expression levels. The ERa proteins were detected using an anti-ERa polyclonal antibody (HC-20; Santa Cruz Biotechnology). 2.5. GST pull-down assay GST and GST-NFAT2 fusion protein were expressed and purified according to the manufacturer’s instructions (Pharmacia), with the induction of protein expression performed at 20 ◦ C overnight. The expression vector for HMGB1 and HMGB1 B-box was used for in vitro transcription and translation in the TNT Reticulocyte Lysate System (Promega). The 35 S labeled HMGB1 or HMGB1 B-box was mixed with 10 ␮g of GST derivatives bound to glutathione − Sepharose beads in 0.5 ml of binding buffer [50 mM Tris − HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.3 mM dithiothreitol (DTT), 0.1% NP-40 and protease inhibitor tablets from Roche]. The binding reaction was performed at 4 ◦ C overnight and the beads were subsequently washed 4 times with the washing buffer (the same as the binding buffer), 30 min each time. The beads were eluted in 10 ␮l of 2 × SDS − PAGE sample buffer and the proteins were resolved on a 10% denaturing gel. The gel was then dried and exposed to X-ray films overnight. 2.6. Statistical analysis The values are expressed as means ± S.D. Statistical significance in the luciferase activity experiments among constructs was determined by Student’s t-test. A P-value < 0.05 was considered statistically significant.

2.3. Transfection with siRNA plasmid

3. Results

HMGB1 short interfering RNA (siRNA) with the sequence sense 5 -GAT CCA TCA AAG GAG AAC ATC CTG TTC AAG AGA CAG GAT GTT CTC CTT TGA TTT TTT TGG AAA-3 , antisense 5 -AGC TTT TCC AAA AAA ATC AAA GGA GAA CAT CCT GTC TCT TGA ACA GGA TGT TCT CCT TTG ATG-3 , and NFAT2 siRNA with the sequence sense 5 -GAT CCG AAC ACT ATG GCT ATG CAT TTC AAG AGA ATG CAT AGC CAT AGT GTT CTT TTT TGG AAA-3 , antisense 5 -AGC TTT TCC AAA AAA GAA CAC TAT GGC TAT GCA TTC TCT TGA AAT GCA TAG CCA TAG TGT TCG-3 were synthesized (Ambion, Inc. USA). siRNA plasmids were constructed using 2.1-U6 neo vector (Ambion, Inc. USA). Lipofectamine 2000 (Invitrogen) was used according to the manufacture’s protocol with HMGB1 or NFAT2 siRNA (Ambion, Inc. USA) (with various concentrations of 10–50 nM) for 24 h. Medium was removed and cells were collected for Western blot analysis and luciferase activity measurement.

3.1. HMGB1 could bind to NFAT2 in vitro To determine the interaction between HMGB1 and NFAT2 in vitro, translated [35 S]methionine-labeled full-length HMGB1 was incubated with GST-NFAT2 or GST alone. Data showed that HMGB1 specifically bound to GST-NFAT2, but not GST. We further constructed HMGB1 B-box (88–166) (Li et al., 2003) and A-box (1–97aa) (Najima et al., 2005) recombinant plasmids for assessment, and results showed that HMGB1 GST-B-box specifically bound to NFAT2, but not GST-A-box or GST (Fig. 1A–C). 3.2. HMGB1 could bind to NFAT2 in vivo To determine the interaction between HMGB1 and NFAT2 in vivo, Flag-NFAT2 and HMGB1 were co-transfected into

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Fig. 1. Protein–protein interaction between HMGB1 and NFAT2 in vitro. (A) GST-pull down assay was performed as described above. Full-length GST-NFAT2 fusion proteins, immobilized on beads, were fixed with in vitro translation reaction mixtures of HMGB1. The bound proteins were subjected to SDS-PAGE followed by autoradiography. (B) Recombinant plasmids of GST-A box and GST-B box were designed and constructed. (C) A box and B box of HMGB1 were constructed. Purified GST-A box or GST-B box fusion proteins were fixed with in vitro translation reaction of NFAT2. Data of GST-pull down showed that GST-B box fusion proteins could bind to NFAT2.

HEK293T cells. After 24 h, cell lysate was collected for co-immune precipitation measurement. Proteins were precipitated with anti-Flag antibody and subsequently detected with anti-HMGB1 antibody. Results showed that HMGB1 could bind to NFAT2 in vivo, which could be markedly enhanced by PMA (25 ng/ml) + ionomycin (0.5 ␮mol/l) stimulation.

3.3. HMGB1 significantly increased NFAT2 transcription activity We co-transfected HMGB1 and NFAT2 into HEK293T cells (Fig. 3A), as well as Hela cells (Fig. 3B). Data showed that there was no statistical increase of reporter gene activity in groups without stimulation of PMA + Ionomycin. After stimulation with PMA + Ionomycin, activity of reporter gene increased 11–12 times when co-transfected with HMGB1 and NFAT2 in 293T cells and about 117 times in Hela cells. As transfection plasmid of HMGB1 rose from 0 to 0.4 ␮g/plate, activity of reporter gene increased from 2.5 to 148.2 times in Hela cells (Fig. 3C).

3.4. Inhibition of HMGB1 expression with siRNA down-regulated the activity of IL-2 reporter gene To further examine the indispensability of HMGB1 in promoting transcription activity of IL-2, siRNA plasmid for HMGB1 was constructed. Exogenous HMGB1 in HEK293T cells (Fig. 4A) and endogenous HMGB1 in Hela cells (Fig. 4B) were inhibited respectively, and activities of reporter gene were measured. Data showed that siRNA plasmid for HMGB1 inhibited activity of IL-2 reporter gene dose-dependently, which was supported by Western blotting assessments of HMGB1 protein levels (Fig. 4).

3.5. Inhibition of NFAT2 expression with siRNA down-regulated the activity of IL-2 reporter gene To examine further the indispensability of NFAT2 in promoting transcription activity of IL-2, siRNA plasmid for NFAT2 was constructed. Exogenous (Fig. 5A) and endogenous (Fig. 5B) NFAT2 in HEK293T cells were inhibited respectively, and activities of reporter gene were measured. Data showed that siRNA plasmid for NFAT2 inhibited activity of IL-2 reporter gene in a dose-dependent manner, and the results were corroborated by Western blotting assessments of NFAT2 protein levels (Fig. 5).

4. Discussion HMGB1, a nuclear and cytoplasm protein, was originally identified as an intranuclear factor with an important structural function in chromatin organization. HMGB1 has at present been identified as a proinflammatory cytokine that mediates immunological process, endotoxin lethality, local inflammation, and macrophage activation (Rovere-Querini et al., 2004; Wang et al., 1999). After exposure to lipopolysaccharide (LPS), tumor necrosis factor (TNF)-␣, IFN-␥, or IL-1␤, and as result of tissue damage, HMGB1 is released by activated inflammatory cells and other damaged cells. More recently, HMGB1 was reported to be an endogenous immune regulatory molecule. HMGB1 protein can trigger the release of IL-2 from T cells and IL-12 from dendritic cells (DC). However, the underlying mechanism of IL-2 production triggered by HMGB1 remains to be elucidated. It is well known that NFAT family transduces major immunological signals in T cells. NFAT translocates into nucleus and regulates gene transcription. Five members of NFAT are identified now, namely NFAT1 (NFATp, NFATc2), NFAT2 (NFATc1, NFATc), NFAT3 (NFATc4), NFAT4 (NFATc3, NFATx) and NFAT5. NFAT1 and NFAT2 are highly expressed in peripheral T cells, and they strongly acti-

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Fig. 2. Interaction between HMGB1 and NFAT2 in vivo. 293T cells were cotransfected with the expression vectors for FLAG-tagged NFAT2 and HMGB1 as indicated. Lysates from the transfected cells were immunoprecipitated (IP) using anti-FLAG antibody and the immunoprecipitates were probed with an anti-HMGB1 antibody. HMGB1 levels in lysates inputs were also detected to be used as endogenous control for standardization of IP results.

vate the IL-2 production and release (Barlic et al., 2004). It behooves us to ask whether transcription of IL-2 as promoted by HMGB1 is in a NFAT dependent manner. Therefore, interaction between NFAT2 and HMGB1 is studied here. In vitro interaction between purified GST-NFAT2 and whole length HMGB1 was examined by GST-pull down assay. As shown in Fig. 1A, in vitro translated [35 S] methionine labeled HMGB1 could bind to NFAT2. To further determine which region of HMGB1 bound to NFAT2, HMGB1 A box (1–97aa) and B box (88–166aa) plasmids with GST label were constructed and their proteins were purified. Data showed that HMGB1 B box (88–166aa) could bind to whole length of NFAT2, but HMGB1 A box (1–97aa) did not. Previous studies have verified that A box and B box are basic domains of HMGB1. Both A and B box adopt a very similar L-shaped structure, formed by two short and one long ␣-helix, which is known as the HMG box domain (McCauley et al., 2007). HMGB1 protein binds in the minor groove of DNA fragment. Both A and B boxes can bend and relax DNA structure, facilitating transcription factors binding to gene promoters on DNA fragment. It has been reported that HMGB1 B box could trigger strong inflammatory response, and treatment with antibody against B box protein could effectively protect septic mice challenged by LPS injection or bacterial peritonitis (Li et al., 2003). The above results suggested that HMGB1 could bind to NFAT2 with B box region in vitro. To determine whether HMGB1 interacts with NFAT2 in vivo, HEK293T cells were co-transfected with HMGB1 and Flag-tagged NFAT2 with or without ionomycin (0.5 ␮mol/l) + PMA (25 ng/ml) stimulation. Flag-NFAT2 was immunoprecipitated from cell lysate by an anti-Flag antibody and analyzed for HMGB1 binding by Western blotting analysis. The results showed that HMGB1 could be co-immunoprecipitated in the presence, but not in the absence of FLAG-NFAT2. Consistent with this data, stimulation with ionomycin + PMA could strengthen the interaction between HMGB1 and NFAT2 in vivo (Fig. 2). Based on the above experiments, effects of HMGB1 on transcription activity of NFAT2 were investigated. First, cooperation between HMGB1 and NFAT2 on potentiation of IL-2 reporter gene transcription activity was examined. HEK293T cells and Hela cells were incubated, and transfected with HMGB1 or NFAT2. Data showed that cotransfection with HMGB1 + NFAT2 could increase IL-2 transcription dramatically, about at least 4.2 times more than that of transfection either with HMGB1 or NFAT2 alone in HEK293T cells, and at least 2.5 times more than that of transfection either with

HMGB1 or NFAT2 alone in Hela cells. The magnitude of activity of IL-2 reporter gene induced by cotransfection of HMGB1 + NFAT2 was markedly higher than the sum of that by HMGB1 plus that by NFAT2, which strongly supported that HMGB1 and NFAT2 were synergistic in stimulating IL-2 transcription. Moreover, activity of IL-2 reporter gene rose steadily as the transfection dose of HMGB1 increased from 0 to 0.4 ␮g/plate, and the result was corroborated by Western blotting measurements of HMGB1 protein levels (Fig. 3C). Second, siRNA plasmids were constructed to inhibit HMGB1 or NFAT2 protein levels respectively and activity of IL-2 reporter gene was detected. In this way, the indispensability of HMGB1 or NFAT2 in enhancing IL-2 transcription was examined. HMGB1 protein level was higher in Hela cell than in HEK293T cell (data not shown), so exogenous HMGB1 in HEK293T cells (Fig. 4A) and endogenous HMGB1 in Hela cells (Fig. 4B) were inhibited respectively, and activities of reporter gene were measured. The results showed that activity of IL-2 reporter gene was lowered significantly by addition of HMGB1 siRNA plasmids, and HMGB1 protein level was attenuated dose-dependently while dose of siRNA plasmids increased. In addition, Exogenous (Fig. 5A) and endogenous (Fig. 5B) NFAT2 in HEK293T cells were inhibited respectively, Data showed that siRNA plasmid for NFAT2 inhibited activity of IL-2 reporter gene dose-dependently, which was corroborated by Western blotting assessments of NFAT2 protein levels (Fig. 5). These results demonstrated that HMGB1 and NFAT2 were both necessary in facilitating IL-2 transcription. To our knowledge, this is the first report that HMGB1 could act as a coactivator of NFAT2 in promoting IL-2 transcription. In fact, HMGB1, as a traditional nonhistone DNA-binding protein, is the coactivator for many transcription factors (Melvin and Edwards, 1999). HMGB1 can enhance DNA binding of receptors of estrogen, androgen and glucocorticoid. HMGB1 prefers to bind to specific DNA structures, such as prebent DNA or the sharp angles at fourway junction DNA (Assenberg et al., 2008). It appears that HMGB1 could form ternary complex with transcription factor and specific DNA site, and in this way HMGB1 enhances interaction between transcription factor and DNA segment, and also facilitates gene transcription markedly (Webb et al., 2001). Additionally, experiments in vitro had demonstrated that HMGB1 could bind with some steroid receptors transiently, which strongly indicated that HMGB1 might act as a kind of “scaffold protein” or “chaperone”, in manipulating the structures of target DNA and transcription factor, and holding them in functional position. Once gene transcription is activated, HMGB1 could dissociate from the ternary complex, probably utilizing a “hit-and-run” mechanism (Ner et al., 1994; Bustin, 1999; Ross et al., 2001; Mitsouras et al., 2002). For example, HMGB1 could enhance the interaction between estrogen receptor (ER) and estrogen response element (ERE), in this way facilitating transcription activity of ER (Das et al., 2004). At the same time, binding specificity between ER and ERE was relaxed by HMGB1 treatment. In the presence of HMGB1, ER could bind to ERE half-sites, and in contrast the binding would not occur without HMGB1. HMGB1 proteins increase the binding affinity of an HMGB-sensitive subset of factors, including steroid hormone receptors and their DNA-binding domains (DBDs) (Romine et al., 1998; Boonyaratanakornkit et al., 1998), p53 (Jayaraman et al., 1998), HOX homeodomain proteins (Zappavigna et al., 1996), Oct proteins (Butteroni et al., 2000) and the Rel family of proteins (Brickman et al., 1999). However, another possibility is that HMGB1 is recruited into the receptor–DNA complex, resulting in additional protein–DNA and/or protein–protein contacts. For example, HMGB1 has no detectable capability to bind progesterone receptor element (PREs) in the absence of receptor, even at much higher concentrations than that are required to enhance steroid receptor-DNA binding. HMGB1 association with PREs was detected only in the presence of PR, suggesting that PR

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Fig. 3. Activity of IL-2 reporter gene was increased significantly by NFAT2 in the presence of HMGB1. (A) 293T cells were transfected with 0.2 ␮g pcDNA3-HMGB1 or 0.05 ␮g pcDNA3-Flag-NFAT2, or co-transfected with both of them. pcDNA3 and pcDNA3-Flag vectors were used as the control plasmids. ** P < 0.01 compared with the control group stimulated by PMA + ionomycin. (B) Hela cells were transfected with 0.2 ␮g pcDNA3-HMGB1 or 0.1 ␮g pcDNA3-Flag-NFAT2, or co-transfected with both of them. pcDNA3 and pcDNA3-Flag vectors were used as the control plasmids. ** P < 0.01 compared with the control group stimulated by PMA + ionomycin. (C) Hela cells were transfected with 0.1 ␮g pcDNA3-Flag-NFAT2, or co-transfected with increasing dose (0, 0.05 and 0.4 ␮g) of pcDNA3-HMGB1. pcDNA3 and pcDNA3-Flag vectors were used as the control plasmids. ** P < 0.01 compared with the control group stimulated by PMA + ionomycin. Every experiment was repeated 4 times.

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Fig. 4. Activity of IL-2 reporter gene could be inhibited by HMGB1 sRNAi. (A) Exogenous HMGB1 was inhibited by increasing dose of sRNAi (0, 0.2, 0.6 and 1.2 ␮g) in 293T cells, which was confirmed to positively correlate with the activity of IL-2 reporter gene. Cell lysate was collected for the detection of HMGB1 protein level by Western blotting. ** P < 0.01 compared with the control group stimulated by PMA + ionomycin, # P < 0.05 or ## P < 0.01 compared with co-transfection group by HMGB1 + NFAT2 and stimulated by PMA + ionomycin. (B) Endogenous HMGB1 was inhibited by increasing dose of sRNAi (0, 0.2, 0.6 and 1.2 ␮g) in Hela cells, which was confirmed to positively correlate with the activity of IL-2 reporter gene. Cell lysate was collected for the detection of HMGB1 protein level by Western blotting. ** P < 0.01 compared with the control group stimulated by PMA + ionomycin, ## P < 0.01 compared with NFAT2-transfected group stimulated by PMA + ionomycin. Every experiment was repeated 4 times.

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Fig. 5. Activity of IL-2 reporter gene could be inhibited by NFAT2 sRNAi. (A) Exogenous NFAT2 was inhibited by increasing dose of sRNAi (0, 1.2, 2.4 and 4.8 ␮g) in 293T cells, which was confirmed to positively correlate with the activity of IL-2 reporter gene. Cell lysate was collected for the detection of NFAT2 protein level by Western blotting. ** P < 0.01 compared with the control group stimulated by PMA + ionomycin, ## P < 0.01 compared with co-transfection group by HMGB1 + NFAT2 and stimulated by PMA + ionomycin. (B) Endogenous NFAT2 was inhibited by increasing dose of sRNAi (0, 1.2, 2.4 and 4.8 ␮g) in 293T cells, which was confirmed to positively correlate with the activity of IL-2 reporter gene. Cell lysate was collected for the detection of NFAT2 protein level by Western blotting. ** P < 0.01 compared with the control group stimulated by PMA + ionomycin, ## P < 0.01 compared with HMGB1-transfected group stimulated by PMA + ionomycin. Every experiment was repeated 4 times.

recruits HMGB1 into the complex (Romine et al., 1998). Collectively, a common feature in both regulatory roles is the ability of HMGB1 to mediate a stronger binding affinity of the transcription factor for its cognate site, which in many cases correlates with increased transcriptional activity. However, exact mechanism required more investigation. In summary, our results provided the evidence which indicated that HMGB1 could facilitate transcription activity of NFAT2, and in this way, promote IL-2 production. Our study would help to advance the knowledge of immunological role of HMGB1.

Acknowledgments This work was supported in part, by grants from the National Natural Science Foundation of China (No. 30672178), the National Basic Research Program of China (No. 2005CB522602), and National Natural Science Outstanding Youth Foundation of China (No. 30125020). We thank Mr. Jie-zhi Li (Beijing Institute of Biotechnology) for help in preparing the reagents, and Dr. Xiao-hui Wang (Beijing Institute of Biotechnology) for help in fluorescence intensity assay.

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