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Deacetylase inhibitor trichostatin A down-regulates Foxp3 expression and reduces CD4+CD25+ regulatory T cells
Biochemical and Biophysical Research Communications 400 (2010) 409–412
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Deacetylase inhibitor trichostatin A down-regulates Foxp3 expression and reduces CD4+CD25+ regulatory T cells Zhijian Liu a, Chenquan Zhang a, Jian Sun a,b,⇑ a Laboratory of B Cell and Autoantibody, Institute of Health Sciences, Shanghai Institutes for Biological Sciences & Shanghai JiaoTong University School of Medicine, Chinese Academy of Sciences, Shanghai 200025, China b Shanghai Institute of Immunology, Institutes of Medical Sciences, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China
a r t i c l e
i n f o
Article history: Received 11 August 2010 Available online 27 August 2010 Keywords: Trichostatin A (TSA) Foxp3 Regulatory T cells (Treg)
a b s t r a c t The forkhead transcription factor Foxp3 is essential for the development and function of CD4+CD25+ regulatory T (Treg) cells, which act to maintain immune tolerance and prevent autoimmunity. Lysine acetylation that is regulated by lysine acetyltransferases and lysine deacetylases plays an important role in gene transcription and protein function. Lysine deacetylase inhibitor trichostatin A (TSA) is reported to up-regulate Foxp3 expression and increase the generation of CD4+CD25+ Treg cells in vivo. In contrast, we found that TSA dramatically reduced the levels of Foxp3 mRNA and protein in vitro. Moreover, TSA enhanced the activity of the Foxp3 promoter but increased the decay of Foxp3 mRNA. Furthermore, administration of TSA significantly impaired the expression of Foxp3 and reduced the number of CD4+CD25+Foxp3+ Treg cells in C57BL/6J mice. Thus, our results show that TSA reduces the expression of Foxp3 through induction of mRNA degradation in vitro. Accordingly, TSA decreases Foxp3 expression and reduces the number of Treg cells in vivo. Our results are not in agreement with previous reports, which are discussed. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Forkhead transcription factor Foxp3 is predominantly expressed in CD4+CD25+ regulatory T (Treg) cells and programs their development and function [1,2]. Lysine acetylation of proteins is the result of a balance between lysine acetyltransferases and lysine deacetylases and is essential for gene transcription and protein function [3,4]. It has been reported that Foxp3 is associated with lysine acetylation-modified enzymes, leading to Foxp3 acetylation and the regulation of Foxp3-mediated functions [5]. Lysine deacetylase inhibitor TSA is extensively studied in many aspects, such as acetylation, inflammation and tumors [6,7]. It is known that TSA affects the expression of about 2–10% of genes, where similar numbers of genes are either up- or down-regulated [6,7]. Recent studies showed that TSA promoted the expression of Foxp3 and increased the generation of CD4+CD25+ Treg cells in vivo [8,9]. In contrast to the results, we found that TSA dramatically reduced the expression of Foxp3 mRNA and protein in vitro. Moreover, TSA did not increase but instead impaired the expression of Foxp3 and reduced the number of CD4+CD25+Foxp3+ Treg cells in C57BL/6J mice.
2.1. Cells and cell culture
⇑ Corresponding author at: Laboratory of B Cell and Autoantibody, Institute of Health Sciences, 225 South Chongqing Road, Shanghai 200025, China. Fax: +86 21 63852729. E-mail address: [email protected] (J. Sun). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.08.090
Splenic T cells and CD4+ cells were isolated from C57BL/6 mice by negative selection using cell isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany), and cells were cultured at 37 °C and 5% CO2 in RPMI 1640 medium supplemented with 10% FBS and antibiotics. C57BL/6 mice were purchased from the Shanghai Laboratory Animal Center of Chinese Academy of Sciences (SLACCAS). 2.2. RT-PCR Separated cells were cultured with or without TSA (Sigma–Aldrich, St. Louis, MO). In some experiments, T cells were activated by anti-CD3 antibody (Southern Biotech, Birmingham, AL) in the presence or absence of TSA. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA), and cDNA was synthesized using a cDNA synthesis kit (Promega, Madison, WI). The mRNA levels were measured by RT-PCR using the following gene-specific primers: Foxp3 (50 -AGT GCC CCT AGT CAT GGT GG-30 ; 50 -GAT CTG CTT GGC AGT GCT TG-30 ), TGFb1 (50 -TGA GTG GCT GTC TTT TGA CG-30 ; 50 -TGG TTG TAG AGG GCA AGG AC-30 ), IL-2 (50 -AAC CTG AAA CTC CCC AGG AT-30 ; 50 -TCC ACC ACA GTT GCT GAC TC-30 ), and b-actin
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Z. Liu et al. / Biochemical and Biophysical Research Communications 400 (2010) 409–412
(50 -TGT TAC CAA CTG GGA CGA CA-30 ; 50 -TCT CAG CTG TGG TGG TGA AG-30 ).
2.7. Flow cytometric analysis
Splenic CD4+ cells treated with or without TSA were lysed using RIPA buffer. The proteins in the supernatant were separated by SDS–PAGE. Foxp3 protein was blotted with anti-Foxp3 antibody (eBioscience, San Diego, CA).
CD4+CD25+Fop3+ cells were determined using regulatory T cell staining kit (eBioscience). Briefly, splenic cells were treated with anti-CD16/32 antibody to block surface Fc receptor, followed by staining of FITC anti-CD4 and APC anti-CD25 antibodies. After fixation/permeabilization, cells were stained with PE anti-Fopx3 antibody (clone: FJK-16s) or PE rat IgG2a isotype control and analyzed by flow cytometry.
2.4. Luciferase assay
2.8. Statistical analysis
Foxp3 reporter plasmids were constructed by PCR amplification of 1788 bp or 1212 bp of 50 upstream sequence of Foxp3, and ligation into the XhoI and HindIII sites of pGL3-basic. 293T cells were transfected with Foxp3 reporter constructs by Lipofectamine 2000 (Invitrogen) and cultured with or without the addition of TSA. Proteins were collected 24 h after transfection, and luciferase activity was measured using Promega luciferase assay reagents. Results were normalized to the protein concentration.
Statistical differences were determined with a Student’s twotailed t-test using GraphPad Prism software (San Diego, CA, USA). A p value <0.05 was considered statistically significant.
2.3. Western blot
2.5. mRNA decay assay Cellular transcription was blocked by actinomycin D (Sigma–Aldrich), followed by the addition of TSA or vehicle after 4 h. The levels of Foxp3 and b-actin mRNA at the indicated times were measured by RT-PCR. 2.6. TSA treatment in vivo C57BL/6 mice were injected i.p. with different doses of TSA for varied time periods, as indicated in each experiment. The RNA samples were collected from splenic cells and used for RT-PCR. Splenic cells were stained with antibodies and analyzed by flow cytometry.
3. Results and discussion Our results showed that the expression of Foxp3 mRNA was greatly inhibited in splenic T cells treated with TSA (Fig. 1A and B). TSA (10 ng/ml) markedly reduced the level of Foxp3 mRNA (Fig. 1A). Moreover, Foxp3 mRNA levels were severely diminished within 8 h in the presence of 50 ng/ml of TSA (Fig. 1B). To confirm the specificity of these results, we measured the mRNA levels of some Foxp3-related genes, such as TGF-b and IL-2, in TSA-treated cells. No significant decrease in the mRNA levels was observed. In contrast, TSA slightly increased the expression of IL-2 mRNA (Fig. 1C). We further examined the effect of TSA on the expression of Foxp3 mRNA in activated splenic T cells. Although T cells activated with anti-CD3 antibody displayed increased levels of Foxp3 mRNA, TSA severely impaired Foxp3 mRNA levels in the activated T cells (Fig. 1D). Consistent with these results, the level of Foxp3 protein was markedly decreased following TSA treatment (Fig. 1E). These results show that TSA dramatically reduced the
Fig. 1. TSA reduces the expression of Foxp3 by inducing mRNA degradation in vitro. (A and B) Splenic T cells were isolated from C57BL/6J mice. T cells (5 106/ml) were treated either with different doses of TSA for 24 h (A) or with 50 ng/ml TSA for different time periods (B). Foxp3 mRNA levels were measured by RT-PCR. b-Actin was used as a control. (C) The mRNA level of the indicated genes in splenic T cells treated with TSA was determined by RT-PCR. (D) Splenic T cells were stimulated with anti-CD3 antibody for 24 h in the absence or presence of TSA, and the Foxp3 mRNA levels are shown. (E) Splenic CD4+ cells were treated with or without TSA for 24 h. The level of Foxp3 protein was analyzed by western blot. GAPDH was used as a control. (F) 293T cells were transfected with 1.6 lg of Foxp3 reporter constructs as indicated and cultured for 24 h with or without the addition of TSA. Luciferase activity was measured. Bars indicate the mean ± SE of three independent experiments. (G) Splenic cells were treated with 5 lg/ml actinomycin D, followed by the addition of 50 ng/ml TSA or vehicle after 4 h. The levels of Foxp3 mRNA at the indicated times were measured by RT-PCR. The mRNA ratios of Foxp3 to b-actin at each time are shown. Data represent the mean ± SE of three independent experiments.
Z. Liu et al. / Biochemical and Biophysical Research Communications 400 (2010) 409–412
Fig. 2. TSA impairs the expression of Foxp3 and reduces the number of CD4+CD25+Foxp3+ Tregs in vivo. (A and B) C57BL/6 mice were injected i.p. with 5 mg/kg TSA (n = 3) or vehicle (n = 3) once a day for three consecutive days (A) or the mice were injected i.p. with 1 mg/kg/day of TSA (n = 5) or vehicle (n = 5) for seven consecutive days (B). The RNA samples were extracted 12 h after the last injection. The levels of Foxp3 and b-actin mRNA were measured by RT-PCR. Data are expressed as the mRNA ratio of Foxp3 to b-actin. Bars indicate the mean ± SE of each group. (C and D) Flow cytometric analysis of CD4+Foxp3+ (C) and CD4+CD25+ (D) cells. Splenic cells were prepared from the mice treated with TSA (n = 13) or vehicle (n = 14) as described in (B), and stained with anti-CD4-FITC and anti-CD25APC, followed by intracellular staining of anti-Foxp3-PE. Representative dot plots are given. Bars indicate the mean ± SE of each group. The p value represents the statistical difference between the compared groups.
411
pared to vehicle-treated controls. The Foxp3 mRNA was almost completely degraded by the addition of TSA within 8 h, which is consistent with the results shown in Fig. 1B. Together, these results show that TSA caused degradation of Foxp3 mRNA and thereby contributed to the reduction of Foxp3 mRNA levels. The findings above are not in agreement with the previously published in vivo results [8,9], which prompted us to examine the effect of TSA on Foxp3 expression in vivo. We injected C57BL/6 mice i.p. with TSA at a dose of 5 mg/kg body weight once daily for three days. As shown in Fig. 2A, the TSA-treated group exhibited a markedly reduced level of Foxp3 mRNA compared to that of the vehicle-treated control mice. We next treated the mice with 1 mg/kg/day of TSA for seven days, a treatment schedule followed in the previous reports [8,9]. The level of Foxp3 mRNA was also decreased in TSA-treated mice (Fig. 2B), although the degree of reduction was less than that observed for mice treated with 5 mg/kg/day of TSA. Furthermore, we analyzed splenic Foxp3 expression by flow cytometry. Administration of TSA, as indicated in Fig. 2B, significantly decreased the percentage of CD4+Foxp3+ cells (Fig. 2C). The absolute number of CD4+Foxp3+ cells was also reduced in TSA-treated mice (Fig. 2C). Accordingly, the percentage and number of CD4+CD25+ Treg cells were significantly decreased in the TSA-treated group (Fig. 2D). These results showed that TSA impaired Foxp3 expression and reduced the number of CD4+CD25+Foxp3+ Tregs in vivo. Our results indicated that TSA reduces the expression of Foxp3 in vitro, which is supported by the results obtained from T cells activated with anti-CD3 and anti-CD28 antibodies [10]. Unfortunately, the previous two studies did not provide in vitro data regarding the effect of TSA on Foxp3 expression [8,9]. Importantly, we failed to observe the result that TSA increases Foxp3 expression and CD4+CD25+Foxp3+ Treg cells in vivo, as reported in the two previous studies. In contrast, our results showed that TSA decreases Foxp3 expression and reduces the number of CD4+CD25+Foxp3+ Tregs in vivo using the same procedure as the previous studies. Our in vivo results are in line with our in vitro results. It is known that TSA reduces the mRNA levels of many genes. Although the mechanism of the TSA-mediated mRNA reduction is not completely clear, we have proposed that the down-regulation of mRNA levels by TSA could be independent of the inhibition of lysine deacetylases and suggest that there are non-specific target(s) of TSA [4]. Our study provides evidence that TSA down-regulates Foxp3 expression and reduces CD4+CD25+ regulatory T cells, which is significant for the understanding of the regulation of CD4+CD25+Foxp3+ Tregs by the deacetylase inhibitor TSA. TSA has potent effects against cell proliferation and inflammation in a time- and dose-dependent manner [6,7]. Therefore, it is not surprising that TSA reduces inflammatory bowel disease and kidney inflammation [8,9]. However, our results challenge that the effects of TSA on tissue inflammation are mediated by an increase in the number of regulatory T cells and, instead, indicate the need to evaluate the underlying mechanism for these observations. Acknowledgments
level of Foxp3 mRNA, resulting in a substantial decrease in Foxp3 protein expression in vitro. To decipher the mechanism of the TSA-induced reduction in Foxp3 mRNA, we investigated the effect of TSA on Foxp3 gene transcription. Fig. 1F shows that TSA did not reduce the activity of the two Foxp3 promoter constructs in 293T cells. In contrast, the promoter activities were increased by TSA in a dose-dependent manner. These results suggest that the TSA-induced reduction in Foxp3 mRNA could be caused by degradation. Therefore, we blocked cell transcription with actinomycin D and determined the effect of TSA on the decay of Foxp3 mRNA. As shown in Fig. 1G, TSA treatment indeed resulted in the increased decay of Foxp3 mRNA as com-
We thank members of the Sun Laboratory for technical assistance. This work was supported by grants from the National Natural Science Foundation of China (30872314), the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX1YW-R-45), and the Shanghai Science and Technology Committee (074319112 and 08JC1421200). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.08.090.
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Z. Liu et al. / Biochemical and Biophysical Research Communications 400 (2010) 409–412
References [1] J.D. Fontenot, M.A. Gavin, A.Y. Rudensky, Foxp3 programs the development and function of CD4+CD25+ regulatory T cells, Nat. Immunol. 4 (2003) 330–336. [2] J.H. Buckner, S.F. Ziegler, Functional analysis of FOXP3, Ann. NY Acad. Sci. 1143 (2008) 151–169. [3] T. Kouzarides, Chromatin modifications and their function, Cell 128 (2007) 693–705. [4] Z.J. Liu, A. Mai, J. Sun, Lysine acetylation regulates Bruton’s tyrosine kinase in B cell activation, J. Immunol. 184 (2010) 244–254. [5] B. Li, A. Samanta, X. Song, et al., FOXP3 interactions with histone acetyltransferase and class II histone deacetylases are required for repression, Proc. Natl Acad. Sci. USA 104 (2007) 4571–4576.
[6] D.C. Drummond, C.O. Noble, D.B. Kirpotin, Z. Guo, G.K. Scott, C.C. Benz, Clinical development of histone deacetylase inhibitors as anticancer agents, Annu. Rev. Pharmacol. Toxicol. 45 (2005) 495–528. [7] S.V. Chittur, N. Sangster-Guity, P.J. McCormick, Histone deacetylase inhibitors: a new mode for inhibition of cholesterol metabolism, BMC Genom. 9 (2008) 507. [8] R. Tao, E.F. de Zoeten, E. Ozkaynak, et al., Deacetylase inhibition promotes the generation and function of regulatory T cells, Nat. Med. 13 (2007) 1299–1307. [9] C.M. Reilly, M. Thomas, R. Gogal Jr., et al., The histone deacetylase inhibitor trichostatin A upregulates regulatory T cells and modulates autoimmunity in NZB/W F1 mice, J. Autoimmun. 31 (2008) 123–130. [10] C. Moon, S.H. Kim, K.S. Park, et al., Use of epigenetic modification to induce FOXP3 expression in naive T cells, Transplant. Proc. 41 (2009) 1848–1854.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Deacetylase inhibitor trichostatin A down-regulates Foxp3 expression and reduces CD4+CD25+ regulatory T cells Zhijian Liu a, Chenquan Zhang a, Jian Sun a,b,⇑ a Laboratory of B Cell and Autoantibody, Institute of Health Sciences, Shanghai Institutes for Biological Sciences & Shanghai JiaoTong University School of Medicine, Chinese Academy of Sciences, Shanghai 200025, China b Shanghai Institute of Immunology, Institutes of Medical Sciences, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China
a r t i c l e
i n f o
Article history: Received 11 August 2010 Available online 27 August 2010 Keywords: Trichostatin A (TSA) Foxp3 Regulatory T cells (Treg)
a b s t r a c t The forkhead transcription factor Foxp3 is essential for the development and function of CD4+CD25+ regulatory T (Treg) cells, which act to maintain immune tolerance and prevent autoimmunity. Lysine acetylation that is regulated by lysine acetyltransferases and lysine deacetylases plays an important role in gene transcription and protein function. Lysine deacetylase inhibitor trichostatin A (TSA) is reported to up-regulate Foxp3 expression and increase the generation of CD4+CD25+ Treg cells in vivo. In contrast, we found that TSA dramatically reduced the levels of Foxp3 mRNA and protein in vitro. Moreover, TSA enhanced the activity of the Foxp3 promoter but increased the decay of Foxp3 mRNA. Furthermore, administration of TSA significantly impaired the expression of Foxp3 and reduced the number of CD4+CD25+Foxp3+ Treg cells in C57BL/6J mice. Thus, our results show that TSA reduces the expression of Foxp3 through induction of mRNA degradation in vitro. Accordingly, TSA decreases Foxp3 expression and reduces the number of Treg cells in vivo. Our results are not in agreement with previous reports, which are discussed. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Forkhead transcription factor Foxp3 is predominantly expressed in CD4+CD25+ regulatory T (Treg) cells and programs their development and function [1,2]. Lysine acetylation of proteins is the result of a balance between lysine acetyltransferases and lysine deacetylases and is essential for gene transcription and protein function [3,4]. It has been reported that Foxp3 is associated with lysine acetylation-modified enzymes, leading to Foxp3 acetylation and the regulation of Foxp3-mediated functions [5]. Lysine deacetylase inhibitor TSA is extensively studied in many aspects, such as acetylation, inflammation and tumors [6,7]. It is known that TSA affects the expression of about 2–10% of genes, where similar numbers of genes are either up- or down-regulated [6,7]. Recent studies showed that TSA promoted the expression of Foxp3 and increased the generation of CD4+CD25+ Treg cells in vivo [8,9]. In contrast to the results, we found that TSA dramatically reduced the expression of Foxp3 mRNA and protein in vitro. Moreover, TSA did not increase but instead impaired the expression of Foxp3 and reduced the number of CD4+CD25+Foxp3+ Treg cells in C57BL/6J mice.
2.1. Cells and cell culture
⇑ Corresponding author at: Laboratory of B Cell and Autoantibody, Institute of Health Sciences, 225 South Chongqing Road, Shanghai 200025, China. Fax: +86 21 63852729. E-mail address: [email protected] (J. Sun). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.08.090
Splenic T cells and CD4+ cells were isolated from C57BL/6 mice by negative selection using cell isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany), and cells were cultured at 37 °C and 5% CO2 in RPMI 1640 medium supplemented with 10% FBS and antibiotics. C57BL/6 mice were purchased from the Shanghai Laboratory Animal Center of Chinese Academy of Sciences (SLACCAS). 2.2. RT-PCR Separated cells were cultured with or without TSA (Sigma–Aldrich, St. Louis, MO). In some experiments, T cells were activated by anti-CD3 antibody (Southern Biotech, Birmingham, AL) in the presence or absence of TSA. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA), and cDNA was synthesized using a cDNA synthesis kit (Promega, Madison, WI). The mRNA levels were measured by RT-PCR using the following gene-specific primers: Foxp3 (50 -AGT GCC CCT AGT CAT GGT GG-30 ; 50 -GAT CTG CTT GGC AGT GCT TG-30 ), TGFb1 (50 -TGA GTG GCT GTC TTT TGA CG-30 ; 50 -TGG TTG TAG AGG GCA AGG AC-30 ), IL-2 (50 -AAC CTG AAA CTC CCC AGG AT-30 ; 50 -TCC ACC ACA GTT GCT GAC TC-30 ), and b-actin
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Z. Liu et al. / Biochemical and Biophysical Research Communications 400 (2010) 409–412
(50 -TGT TAC CAA CTG GGA CGA CA-30 ; 50 -TCT CAG CTG TGG TGG TGA AG-30 ).
2.7. Flow cytometric analysis
Splenic CD4+ cells treated with or without TSA were lysed using RIPA buffer. The proteins in the supernatant were separated by SDS–PAGE. Foxp3 protein was blotted with anti-Foxp3 antibody (eBioscience, San Diego, CA).
CD4+CD25+Fop3+ cells were determined using regulatory T cell staining kit (eBioscience). Briefly, splenic cells were treated with anti-CD16/32 antibody to block surface Fc receptor, followed by staining of FITC anti-CD4 and APC anti-CD25 antibodies. After fixation/permeabilization, cells were stained with PE anti-Fopx3 antibody (clone: FJK-16s) or PE rat IgG2a isotype control and analyzed by flow cytometry.
2.4. Luciferase assay
2.8. Statistical analysis
Foxp3 reporter plasmids were constructed by PCR amplification of 1788 bp or 1212 bp of 50 upstream sequence of Foxp3, and ligation into the XhoI and HindIII sites of pGL3-basic. 293T cells were transfected with Foxp3 reporter constructs by Lipofectamine 2000 (Invitrogen) and cultured with or without the addition of TSA. Proteins were collected 24 h after transfection, and luciferase activity was measured using Promega luciferase assay reagents. Results were normalized to the protein concentration.
Statistical differences were determined with a Student’s twotailed t-test using GraphPad Prism software (San Diego, CA, USA). A p value <0.05 was considered statistically significant.
2.3. Western blot
2.5. mRNA decay assay Cellular transcription was blocked by actinomycin D (Sigma–Aldrich), followed by the addition of TSA or vehicle after 4 h. The levels of Foxp3 and b-actin mRNA at the indicated times were measured by RT-PCR. 2.6. TSA treatment in vivo C57BL/6 mice were injected i.p. with different doses of TSA for varied time periods, as indicated in each experiment. The RNA samples were collected from splenic cells and used for RT-PCR. Splenic cells were stained with antibodies and analyzed by flow cytometry.
3. Results and discussion Our results showed that the expression of Foxp3 mRNA was greatly inhibited in splenic T cells treated with TSA (Fig. 1A and B). TSA (10 ng/ml) markedly reduced the level of Foxp3 mRNA (Fig. 1A). Moreover, Foxp3 mRNA levels were severely diminished within 8 h in the presence of 50 ng/ml of TSA (Fig. 1B). To confirm the specificity of these results, we measured the mRNA levels of some Foxp3-related genes, such as TGF-b and IL-2, in TSA-treated cells. No significant decrease in the mRNA levels was observed. In contrast, TSA slightly increased the expression of IL-2 mRNA (Fig. 1C). We further examined the effect of TSA on the expression of Foxp3 mRNA in activated splenic T cells. Although T cells activated with anti-CD3 antibody displayed increased levels of Foxp3 mRNA, TSA severely impaired Foxp3 mRNA levels in the activated T cells (Fig. 1D). Consistent with these results, the level of Foxp3 protein was markedly decreased following TSA treatment (Fig. 1E). These results show that TSA dramatically reduced the
Fig. 1. TSA reduces the expression of Foxp3 by inducing mRNA degradation in vitro. (A and B) Splenic T cells were isolated from C57BL/6J mice. T cells (5 106/ml) were treated either with different doses of TSA for 24 h (A) or with 50 ng/ml TSA for different time periods (B). Foxp3 mRNA levels were measured by RT-PCR. b-Actin was used as a control. (C) The mRNA level of the indicated genes in splenic T cells treated with TSA was determined by RT-PCR. (D) Splenic T cells were stimulated with anti-CD3 antibody for 24 h in the absence or presence of TSA, and the Foxp3 mRNA levels are shown. (E) Splenic CD4+ cells were treated with or without TSA for 24 h. The level of Foxp3 protein was analyzed by western blot. GAPDH was used as a control. (F) 293T cells were transfected with 1.6 lg of Foxp3 reporter constructs as indicated and cultured for 24 h with or without the addition of TSA. Luciferase activity was measured. Bars indicate the mean ± SE of three independent experiments. (G) Splenic cells were treated with 5 lg/ml actinomycin D, followed by the addition of 50 ng/ml TSA or vehicle after 4 h. The levels of Foxp3 mRNA at the indicated times were measured by RT-PCR. The mRNA ratios of Foxp3 to b-actin at each time are shown. Data represent the mean ± SE of three independent experiments.
Z. Liu et al. / Biochemical and Biophysical Research Communications 400 (2010) 409–412
Fig. 2. TSA impairs the expression of Foxp3 and reduces the number of CD4+CD25+Foxp3+ Tregs in vivo. (A and B) C57BL/6 mice were injected i.p. with 5 mg/kg TSA (n = 3) or vehicle (n = 3) once a day for three consecutive days (A) or the mice were injected i.p. with 1 mg/kg/day of TSA (n = 5) or vehicle (n = 5) for seven consecutive days (B). The RNA samples were extracted 12 h after the last injection. The levels of Foxp3 and b-actin mRNA were measured by RT-PCR. Data are expressed as the mRNA ratio of Foxp3 to b-actin. Bars indicate the mean ± SE of each group. (C and D) Flow cytometric analysis of CD4+Foxp3+ (C) and CD4+CD25+ (D) cells. Splenic cells were prepared from the mice treated with TSA (n = 13) or vehicle (n = 14) as described in (B), and stained with anti-CD4-FITC and anti-CD25APC, followed by intracellular staining of anti-Foxp3-PE. Representative dot plots are given. Bars indicate the mean ± SE of each group. The p value represents the statistical difference between the compared groups.
411
pared to vehicle-treated controls. The Foxp3 mRNA was almost completely degraded by the addition of TSA within 8 h, which is consistent with the results shown in Fig. 1B. Together, these results show that TSA caused degradation of Foxp3 mRNA and thereby contributed to the reduction of Foxp3 mRNA levels. The findings above are not in agreement with the previously published in vivo results [8,9], which prompted us to examine the effect of TSA on Foxp3 expression in vivo. We injected C57BL/6 mice i.p. with TSA at a dose of 5 mg/kg body weight once daily for three days. As shown in Fig. 2A, the TSA-treated group exhibited a markedly reduced level of Foxp3 mRNA compared to that of the vehicle-treated control mice. We next treated the mice with 1 mg/kg/day of TSA for seven days, a treatment schedule followed in the previous reports [8,9]. The level of Foxp3 mRNA was also decreased in TSA-treated mice (Fig. 2B), although the degree of reduction was less than that observed for mice treated with 5 mg/kg/day of TSA. Furthermore, we analyzed splenic Foxp3 expression by flow cytometry. Administration of TSA, as indicated in Fig. 2B, significantly decreased the percentage of CD4+Foxp3+ cells (Fig. 2C). The absolute number of CD4+Foxp3+ cells was also reduced in TSA-treated mice (Fig. 2C). Accordingly, the percentage and number of CD4+CD25+ Treg cells were significantly decreased in the TSA-treated group (Fig. 2D). These results showed that TSA impaired Foxp3 expression and reduced the number of CD4+CD25+Foxp3+ Tregs in vivo. Our results indicated that TSA reduces the expression of Foxp3 in vitro, which is supported by the results obtained from T cells activated with anti-CD3 and anti-CD28 antibodies [10]. Unfortunately, the previous two studies did not provide in vitro data regarding the effect of TSA on Foxp3 expression [8,9]. Importantly, we failed to observe the result that TSA increases Foxp3 expression and CD4+CD25+Foxp3+ Treg cells in vivo, as reported in the two previous studies. In contrast, our results showed that TSA decreases Foxp3 expression and reduces the number of CD4+CD25+Foxp3+ Tregs in vivo using the same procedure as the previous studies. Our in vivo results are in line with our in vitro results. It is known that TSA reduces the mRNA levels of many genes. Although the mechanism of the TSA-mediated mRNA reduction is not completely clear, we have proposed that the down-regulation of mRNA levels by TSA could be independent of the inhibition of lysine deacetylases and suggest that there are non-specific target(s) of TSA [4]. Our study provides evidence that TSA down-regulates Foxp3 expression and reduces CD4+CD25+ regulatory T cells, which is significant for the understanding of the regulation of CD4+CD25+Foxp3+ Tregs by the deacetylase inhibitor TSA. TSA has potent effects against cell proliferation and inflammation in a time- and dose-dependent manner [6,7]. Therefore, it is not surprising that TSA reduces inflammatory bowel disease and kidney inflammation [8,9]. However, our results challenge that the effects of TSA on tissue inflammation are mediated by an increase in the number of regulatory T cells and, instead, indicate the need to evaluate the underlying mechanism for these observations. Acknowledgments
level of Foxp3 mRNA, resulting in a substantial decrease in Foxp3 protein expression in vitro. To decipher the mechanism of the TSA-induced reduction in Foxp3 mRNA, we investigated the effect of TSA on Foxp3 gene transcription. Fig. 1F shows that TSA did not reduce the activity of the two Foxp3 promoter constructs in 293T cells. In contrast, the promoter activities were increased by TSA in a dose-dependent manner. These results suggest that the TSA-induced reduction in Foxp3 mRNA could be caused by degradation. Therefore, we blocked cell transcription with actinomycin D and determined the effect of TSA on the decay of Foxp3 mRNA. As shown in Fig. 1G, TSA treatment indeed resulted in the increased decay of Foxp3 mRNA as com-
We thank members of the Sun Laboratory for technical assistance. This work was supported by grants from the National Natural Science Foundation of China (30872314), the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX1YW-R-45), and the Shanghai Science and Technology Committee (074319112 and 08JC1421200). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.08.090.
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References [1] J.D. Fontenot, M.A. Gavin, A.Y. Rudensky, Foxp3 programs the development and function of CD4+CD25+ regulatory T cells, Nat. Immunol. 4 (2003) 330–336. [2] J.H. Buckner, S.F. Ziegler, Functional analysis of FOXP3, Ann. NY Acad. Sci. 1143 (2008) 151–169. [3] T. Kouzarides, Chromatin modifications and their function, Cell 128 (2007) 693–705. [4] Z.J. Liu, A. Mai, J. Sun, Lysine acetylation regulates Bruton’s tyrosine kinase in B cell activation, J. Immunol. 184 (2010) 244–254. [5] B. Li, A. Samanta, X. Song, et al., FOXP3 interactions with histone acetyltransferase and class II histone deacetylases are required for repression, Proc. Natl Acad. Sci. USA 104 (2007) 4571–4576.
[6] D.C. Drummond, C.O. Noble, D.B. Kirpotin, Z. Guo, G.K. Scott, C.C. Benz, Clinical development of histone deacetylase inhibitors as anticancer agents, Annu. Rev. Pharmacol. Toxicol. 45 (2005) 495–528. [7] S.V. Chittur, N. Sangster-Guity, P.J. McCormick, Histone deacetylase inhibitors: a new mode for inhibition of cholesterol metabolism, BMC Genom. 9 (2008) 507. [8] R. Tao, E.F. de Zoeten, E. Ozkaynak, et al., Deacetylase inhibition promotes the generation and function of regulatory T cells, Nat. Med. 13 (2007) 1299–1307. [9] C.M. Reilly, M. Thomas, R. Gogal Jr., et al., The histone deacetylase inhibitor trichostatin A upregulates regulatory T cells and modulates autoimmunity in NZB/W F1 mice, J. Autoimmun. 31 (2008) 123–130. [10] C. Moon, S.H. Kim, K.S. Park, et al., Use of epigenetic modification to induce FOXP3 expression in naive T cells, Transplant. Proc. 41 (2009) 1848–1854.