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*
Institute of Molecular Biology and Genetics and School of Biological Sciences, Seoul National University, and International Vaccine Institute, Seoul, Korea; and
Department of Molecular Cell Biology, Sung Kyun Kwan University School of Medicine, and Samsung Biomedical Research Institute, Suwon, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported that GC-induced apoptosis requires the SRG3 protein in a thymoma cell line, S49.1 (6). SRG3, a mouse homologue of yeast SWI3 and human BAF155, was isolated as a gene expressed highly in immature thymocytes, but at basal level in mature T cells (6). It is a core component of the SWI-SNF protein complex, a chromatin-remodeling complex required for the regulation of transcriptional processes (7). However, we have found that the SRG3 protein may also exist as an independent form of the mouse SWI/SNF complex in GC-sensitive thymocytes (6). Interestingly, we found a correlation between the expression level of SRG3 and GC sensitivity. The protein is expressed at an approximately three times higher level in GC-sensitive thymocytes than in GC-resistant peripheral T cells. Furthermore, lowering the expression of the protein rendered the GC-sensitive S49.1 cells resistant (6). These results suggest that SRG3 may play a critical role in controlling GC-mediated apoptosis of developing thymocytes. Thus, to fully understand the nature of GC sensitivity of T cells, we investigated how the SRG3 protein controls GC sensitivity in T cells.
GCs are produced by activation of the hypothalamic-pituitary-adrenal
(HPA) axis under stress conditions in animals (8). GCs in
the periphery have been shown to exert multiple anti-inflammatory
activities by regulating transcription factors involved in cytokine
production, such as NF-
B and AP-1 (9, 10). The
capabilities of GCs to suppress T cell proliferative responses to
specific Ags as well as those to mitogens are also well documented
(11). However, it is possible that GCs released by stress
would induce apoptotic death of peripheral T cells under certain
conditions. This would also result in an immunosuppressive state in an
animal. In this report we provide evidence for such a possibility.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FVB mice were supplied by B&K Universal (Sollentuna, Sweden) and maintained at the Institute of Molecular Biology and Genetics, Seoul National University (Seoul, Korea). The murine thymoma cell line S49.1 was maintained as previously described (6). Dexamethasone (DEX) and staurosporin were purchased from Sigma (St. Louis, MO). Biotin-conjugated H57.597, FITC-conjugated 53-6.72, PE-conjugated GK1.5, PE-conjugated streptavidin, quantum red-conjugated streptavidin, anti-Fas Ab (Jo2), and FITC-conjugated annexin V were purchased from PharMingen (San Diego, CA). BuGR-2 (12) was purchased from Affinity Bioreagents (Golden, CO). Antiserum against hBRG1, a human homologue of SWI2, was donated by H. Kwon (Kyung Hee University, Seoul, Korea).
Transgenic (Tg) mice
Full-length cDNA of SRG3 was inserted into the SmaI site of the VA-hCD2 expression vector (13) by blunt end ligation. The 15-kb KpnI/NotI fragment containing the whole expression unit (hCD2 promoter, SRG3 cDNA, poly(A), and locus control region) was purified and microinjected into fertilized pronuclei of the FVB mouse. Transgene integration was determined by Southern blotting and PCR using primers (5'-GACTAGACCAAACATCTACCTC-3' and 5'-GTCAACTGAGCGACTGGATC-3') with genomic DNA isolated from the tail.
Coimmunoprecipitation and immunoblotting
Thymocytes and lymph node (LN) cells from 4- to 6-wk-old-mice or cultured cells were extracted and immunoprecipitated with BuGR-2 Ab as previously described (14). For immunoblot analysis, proteins were subjected to 7.5% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti-hBRG1 antiserum, anti-SRG3 antiserum, BuGR-2, or anti-Xpress Ab. Specific bands were visualized by the ECL system (Pierce, Rockford, IL) and quantified by using Gel-Pro Analyzer software (Media Cybernetics, Silver Spring, MD).
Measurement of apoptosis and GC sensitivity
Mesenteric lymphocytes from hCD2-SRG3 Tg mice and
littermate control mice were incubated at 1–3 x
106 cell/ml in RPMI 1640 medium, 10% FBS, and
various concentrations of DEX. The cells were harvested at various time
points and stained with 
TCR-biotin followed by PE-conjugated
streptavidin, and finally with annexin V-FITC and propidium iodide
(PI). The 10,000–30,000 events were analyzed by flow cytometry. Two
clones of S49.1 transfectants (no. 3 and 8) expressing the 89-aa
fragment of SRG3 and vector transfectant were treated with the
indicated concentrations of DEX at three time points and stained with
annexin V-FITC and PI. Fas-mediated T cell apoptosis was performed as
previously described (15). Freshly isolated mesenteric
lymphocytes (2 x 105 cells/well) were
treated with the indicated amounts of anti-Fas mAb (Jo2;
PharMingen) for 22 h in the presence of 30 µg/ml cycloheximide
(Sigma) or were treated with the indicated concentrations of
staurosporin (Sigma) for 12 h. The percentage of specific
apoptosis was calculated by the formula previously reported
(16).
In vitro translation of the 89-aa SRG3 fragment
The pcH260 plasmid was constructed by inserting a 260-bp SacI/XbaI fragment of the SRG3 (aa residues 854–922) into the pcDNA3.1/HisB vector containing the T7 promoter/priming site, CMV promoter, N-terminal polyhistidine Taq, anti-Xpress Ab epitope tag (DLYDDDDK), and the neomycin resistance gene (Invitrogen, San Diego, CA). In vitro translation of this construct was performed using the TNT T7 quick-coupled transcription/translation system (Promega, Madison, WI) and [35S]methionine (Amersham, Arlington Heights, IL) following the manufacturer’s protocol.
Luciferase assay
The two plasmid constructs used were pRcASRG3, expressing an
antisense SRG3 transcript (6), and pcH260. The S49.1 cells
were cotransfected with 5 µg pGRE-LUC, 1 µg RSV-lacZ,
along with 11 µg of one of the plasmids described above using
DEAE-dextran sulfate as previously described (17).
Transfected cells were incubated overnight in fresh medium and then
treated with 10-6 M DEX for 24 h.
Luciferase activity was assayed according to the manufacturer’s
(Promega) protocol. -Galactosidase activity was measured to
normalize the transfection efficiencies.
Overexpression of the 89-aa fragment of SRG3 in S49.1
After transfection of pcH260 into S49.1 cells by electroporation, cells were selected with 1 mg/ml G418 (Life Technologies, Gaithersburg, MD). The expression of this fragment was tested by immunoblotting with anti-Xpress Ab (Invitrogen). Two clones (no. 3 and 8) were isolated for the study.
Restraint stress (RST)
RST was used to activate the HPA axis in 6-wk-old Tg mice and littermate controls by a method previously described (18) with some modifications. Mice were physically restrained in well-ventilated 50-ml polypropylene centrifuge tubes for one period. In the case of RST2 and RST3 groups, RST was continued for an additional one and two periods, respectively. Each period was 16 h in duration, and this restraint was initiated at 1900 h, 1 h before the onset of the dark cycle. Mice were deprived of food and water during this restraint period, but were given free access to food and water following each stress period.
Cell preparation and calculation of the number of T cell subsets
Thymocytes and splenocytes were prepared by gently teasing the
thymus and spleen with glass slides and passed through the 200-gauge
nylon mesh. RBCs in the spleen were lysed by 0.83%
NH4Cl-Tris buffer (pH 7.6). Cells were counted
using a Coulter counter (Hialeah, FL). One million cells were stained
with CD8-FITC, CD4-PE, and TCR-biotin, followed by quantum
red-conjugated streptavidin. After staining, the cells were analyzed
using a FACStarPlus and CellQuest software (BD
Biosciences, Mountain View, CA). The absolute numbers of T cells
(CountT) in each spleen were calculated by multiplying the total cell
counts and the sum of the percentage of
TCR+CD4+, and
TCR+CD8+ (SP%) lymphocytes
(CountT = total cell count x SP%/100).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SRG3 protein is expressed highly in thymocytes that are
sensitive to GC-induced apoptosis, but is expressed at low levels in
peripheral T cells that are relatively resistant to GC-induced
apoptosis. Because GR mediates the transcriptional activation of genes
involved in the apoptotic process (19), we tested the
possibility that SRG3 may regulate GR activity directly. We first
investigated whether the SRG3 protein interacts with the GR and whether
there is any difference in their interaction in the thymus and
periphery. GR was immunoprecipitated from lysates of thymocytes or LN
cells using mAb against GR, BuGR-2. Results from GR immunocomplexes
blotted with anti-SRG3 antiserum revealed that GR was associated
with SRG3 in the thymus (Fig. 1
C, lane 1). However, SRG3 and GR were
coimmunoprecipitated consistently to a much lesser extent (
10 times
less) in LN than in thymus (Fig. 1C, lane
2). These results indicate that the amount of SRG3-GR
complex may correlate with the expression level of SRG3 protein. To
examine this possibility, we produced Tg mice overexpressing
SRG3 in T lineage cells. The SRG3 cDNA was placed
under control of the human CD2 promoter and the human
CD2 poly(A) sequence followed by the CD2 locus control
region (Fig. 1A) (13, 20). Three founder mice
were identified as being positive for the transgene, and they all
showed increased expression of the SRG3 protein in LN as well as
thymus. The Tg mouse line with the highest copy number contained 10
transgenes. Western blot analysis revealed that the line expresses 2
times more SRG3 protein than control littermates in both the thymus and
periphery (Fig. 1B, lanes 1–4). However, Tg LN T
cells express SRG3 at approximately half the level of thymocytes from
control littermates. We tested whether the increased expression of SRG3
in LN T cells might induce a physical association of the SRG3 protein
with GR. Immunoprecipitation of LN extracts with BuGR-2 showed an
increased formation of the complex containing SRG3 and GR in peripheral
T cells (4 times more complex in Tg LN than wild-type (WT) LN; Fig. 1C, lane 3). When we
immunoprecipitated cell extracts from normal thymocytes, LN, and Tg LN
using BuGR-2, similar amounts of GR were detected (Fig. 1C).
These results suggest that the formation of SRG3-GR complex is
dependent on the expression level of the SRG3 protein in T cell.
Overexpression of SRG3 slightly increased (1.2 ± 0.04 times
compared with control) the expression of mBRG1 in thymocytes and
peripheral T cells, a mouse homologue of SWI2, which is another core
component of the SWI/SNF complex (Fig. 1B).
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We then examined whether the LN T cells, overexpressing SRG3
protein and now containing the SRG3-GR complex, became sensitive to
GC-induced apoptosis. Cells from peripheral LNs of the Tg and
littermate control mice were incubated in medium containing DEX. As
shown in Fig. 2A, mature T cells become more sensitive to GC-induced
apoptosis by increased expression of SRG3, whereas there were no
significant differences in apoptosis produced by anti-Fas Ab or
staurosporin treatment (Fig. 2, B and C). These
results also suggest that the expression level of SRG3 is important for
the formation of SRG3-GR and also in determining the sensitivity of T
cells to GC-induced apoptosis.
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We have found that the SacI/XbaI fragment,
an 89-aa fragment from aa 854–922 of SRG3, exhibits a dominant
negative mutant effect against SRG3. The 89-aa fragment of SRG3 was in
vitro translated using [35S]methionine. The
product of the in vitro translation was confirmed by
immunoprecipitation with SRG3 antiserum (Fig. 3A, lane 1). The product was mixed with thymic
extract and then immunoprecipitated with BuGR-2. As shown in Fig. 3A, the labeled 89-aa fragment of SRG3 was
coimmunoprecipitated with GR (lane 2). This result
indicates that the 89-aa fragment of SRG3 can form a protein complex
with GR and suggests that the fragment may act as a dominant negative
mutant of SRG3 by blocking the SRG3-GR association.
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B). The pGRE-LUC
plasmid containing two GREs was transfected into S49.1 cells. When
these cells were treated with DEX after transfection, luciferase
activity showed up to an 30-fold increase compared with that of
nontreated transfectants (Fig. 3B, lane 1).
Cotransfection of this DNA together with the pRcASRG3 plasmid,
expressing the antisense SRG3 gene under the control of a CMV promoter
(6), blocked the enhancement of luciferase activity
induced by DEX treatment (Fig. 3B, lane 2). When
the pcH260 plasmid DNA expressing the 89-aa fragment of SRG3 was
cotransfected, the enhancement of luciferase activity by DEX treatment
was also reduced (Fig. 3B, lane 3). These results
suggest that SRG3 is required for GR-mediated transcription, and the
89-aa fragment can block this process.
To test whether the 89-aa fragment can disrupt the association between
SRG3 and GR, pcH260 DNA was stably transfected into GC-sensitive S49.1
cells. Two clones (3 and 8) expressing the 89-aa fragment at different
levels were isolated by selection with G418. Western blot analysis with
cell extracts showed that clone 8 expressed a significantly higher
level (5 times higher) of the fragment compared with clone 3, whereas
the expression levels of the SRG3 protein were similar (Fig. 4A). Using total cell extracts from clones 3 and 8, SRG3-GR
association was examined by immunoblotting with antiserum against SRG3.
SRG3-GR association was significantly reduced in clone 8 (58%
reduction), which expressed higher levels of the fragment, than in
clone 3 (14% reduction; Fig. 4B). We also found that clone
8 contained significantly less SRG3-GR complex than clone 3. As
expected, clone 8 was less sensitive to DEX treatment than clone 3 and
the vector transfectants (Fig. 4C). Our results show that
the 89-aa fragment of SRG3 blocks the association of SRG3 with GR,
GR-mediated transcription, and GC-induced apoptotic cell death. The
results suggest that the level of SRG3-GR complex is a critical
parameter determining the GC sensitivity in T cells, and it is
dependent on the expression level of SRG3.
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It is well established that stress activates the HPA axis to
produce GCs, and affects the homeostasis of various organs, especially
the immune system (21). Because peripheral T cells from
the Tg mice overexpressing SRG3 are more sensitive to GC-induced
apoptosis, it is likely that these cells are more sensitive to stress.
To test this possibility, 6- to 7-wk-old mice were subjected to RST,
and total T cell numbers from the thymus and spleen of each mouse were
counted using a Coulter counter and a flow cytometer. Total thymocyte
numbers were reduced by about 18% in Tg mice (1.46 x
108) compared with littermate control mice
(1.79 x 108). As the stress intensity
increased, numbers in thymocytes decreased in a similar way in both Tg
mice and littermate control mice (Fig. 5A). In the spleen, the total numbers of T cells were similar
between Tg mice (3.84 x 107) and control
littermates (3.43 x 107) under normal
conditions. However, after 1 day of the RST, the Tg mice showed a much
greater reduction (-54.4%) in the total number of splenic T cells
compared with littermate control mice (-23.3%). RST for 3 days
resulted in a significantly greater reduction in Tg mice (-75.5%)
compared with littermate controls (-41.5%; Fig. 5B). There
was no significant difference in these results between male and female
littermate mice. This reduction in the number of splenic T cells seems
to be due to the death of cells induced by GCs released under stress
conditions, which suggests that peripheral T cells overexpressing SRG3
are much more susceptible to stress-induced cell death.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we investigated whether SRG3 is involved in the regulation of GC sensitivity in vivo as well as in vitro. We provided evidence that the level of SRG3 expression is important for formation of the SRG3-GR complex and that the level of this complex is crucial in determining the GC sensitivity of T cells. Expression of a dominant negative form of SRG3 fragment efficiently blocked the formation of the complex and, correspondingly, reduced GC-mediated apoptosis in S49.1 cells. This complex appears to be required for the GR-mediated transcription, because the same SRG3 fragment blocked the GR-mediated reporter gene transcription. We have also found that the SRG3-GR complex binds to the GRE sequence motif when analyzed by chromatin immunoprecipitation assay (22) and gel mobility supershift assay (data not shown). Therefore, it is likely that the complex containing SRG3 and GR binds to GRE sequences on the promoter of a target gene(s) to induce GC-induced apoptosis in thymocytes.
Previously, it was reported that SWI/SNF protein complex is involved in
GR-mediated transcriptional regulation as a chromatin-remodeling factor
(7, 23). The rat GR, when expressed in yeast, requires
SWI/SNF proteins for transcriptional activation of GR-responsive genes,
and GR-SWI3 complexes were coimmunoprecipitated in yeast extract
(22). In a human breast cancer cell line, interaction
between the GR and SWI/SNF complex was detected in a ligand-dependent
manner (14). It was also recently reported that the
SWI/SNF complex could potentiate the activity of GR through 
1 in
both yeast and mammalian cells (24). However, we have
previously found that SRG3 protein exists as an independent form of the
mouse SWI/SNF complex as well as a subunit of the complex in
GC-sensitive thymocytes (6). We also found that some
SRG3-GR complexes do not contain the mBRG1 protein in the thymus
(manuscript in preparation). These results suggest that the SRG3
protein may function as an independent form of the SWI/SNF complex in
controlling the GC sensitivity of thymocytes.
Our results also showed that thymocytes contained GR associated with
SRG3 even without pretreatment of exogenous GCs. This SRG3-GR complex
was detected to a much lesser extent in peripheral T cells than in
thymocytes. It is possible that endogenous GCs produced in the thymus
may induce SRG3-GR complex formation. However, Tg overexpression of
SRG3 resulted in the formation of the complex in LN T cells where such
complexes are hardly detectable under normal conditions (Fig. 1C). In addition, when we expressed SRG3 cDNA in
the NIH-3T3 cell line under control of a viral promoter, we could
detect the SRG3-GR complex (data not shown). These results suggest that
the formation of the SRG3-GR complex is dependent on the expression
level of SRG3 in a cell, and GC may not be a prerequisite for it.
However, SRG3 and GR may not interact directly, because both yeast
two-hybrid assay and coimmunoprecipitation with in vitro translated
products of the two proteins failed (data not shown).
Tg overexpression of SRG3 results in formation of the SRG3-GR complex
and renders the GC-resistant LN T cells more sensitive to GCs. However,
Tg LN T cells were still less sensitive to GC-induced apoptosis than
normal thymocytes. Tg LN T cells showed 20% specific apoptosis when
treated with 10-8 M DEX for 12 h (Fig. 4A), whereas normal thymocytes showed about 40% specific
apoptosis under the same conditions (data not shown). This discrepancy
in GC sensitivity between the two populations may be due to a limited
expression of SRG3 (see Fig. 1B) in Tg peripheral T cells.
LN T cells from the Tg mice expressed SRG3 at half the level of normal
thymocytes. We were not able to produce Tg mice with LN cells
expressing SRG3 at the level of normal thymocytes. It seems that
overexpression of SRG3 protein cause deleterious effects on cells,
because overexpression of SRG3 in the EL-4 cell line disrupted normal
progression of the cell cycle, resulting in aneuploidy and slow growth
(data not shown). The discrepancy in GC sensitivity may also be
explained by a protective mechanism provided by Bcl-2 expressed in
mature T cells (25, 26, 27). It is also possible that some
other factors may be necessary for functional SRG3-GR complex
formation, and these may be limited in peripheral T cells.
Elevated GC sensitivity of peripheral T cells from hCD2-SRG3 Tg mice allowed us to investigate the effects of stress on the immune system. Stress is a cognitive stimulus that activates the HPA axis, which leads to the secretion of GCs from the adrenal cortex (8). Because the expression level of SRG3 is important in GR-mediated responses, we speculated that the increased expression of SRG3 might change the immunomodulatory effects of GCs. Under stress conditions, splenic T cells were much more efficiently removed in Tg mice than in littermate control mice. These results imply that under conditions expressing SRG3 at levels higher than normal, T cells would become more susceptible to apoptosis by GCs released by stress. The expression level of the SRG3 protein is not regulated by GCs itself (data not shown). It appears to be strictly regulated along the developmental stages of thymocytes, and there is a possibility that this regulation may be disturbed by factors such as aging, pathological agents, and other related factors. Therefore, our results suggest a novel possibility of stress effects on the immune system, that is, an immunosuppression due to GC-induced apoptosis of peripheral T cells.
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Footnotes |
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2 R.H.S. is a BioGenomics LSRI investigator.
3 Address correspondence and reprint requests to Dr. Rho Hyun Seong, Institute of Molecular Biology and Genetics and School of Biological Sciences, Seoul National University, Kwanak-gu, Shinlim-dong, San 56-1, Building 105, Seoul 151-742, Korea. E-mail address: rhseong{at}plaza.snu.ac.kr
4 Abbreviations used in this paper: GC, glucocorticoid; DEX, dexamethasone; GR, GC receptor; HPA, hypothalamic-pituitary-adrenal; LN, lymph node; PI, propidium iodide; RST, restraint stress; Tg, transgenic; WT, wild type.
5 Y. I. Choi, S. H. Jeon, J. Jang, S. Han, J. K. Kim, H. Chung, H. W. Lee, H. Y. Chung, S. D. Park, and R. H. Seong. Notch1 confers developing thymocytes a resistance to glucocorticoid induced apoptosis by down-regulating SRG3 expression.Submitted for publication.
Received for publication February 5, 2001. Accepted for publication May 16, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B pathways. J. Exp. Med. 188:1381.1 activation domain. Mol. Cell. Biol. 20:2004.This article has been cited by other articles:
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) and littermate control mice (
) were
cultured in medium with the indicated concentrations of DEX
(A), anti-Fas Ab (Jo2) and cycloheximide
(B), or staurosporin (C). The cells were
analyzed for apoptosis using flow cytometry at the indicated time
points. To determine the amount of T cell apoptosis, cells were gated
for the expression of 
,
Vector transfectant; 
, clone 8.
) and
hCD2-SRG3 Tg (