Dexamethasone-induced Gene 2 (dig2) Is a Novel Pro-survival Stress Gene Induced Rapidly by Diverse Apoptotic Signals*
Zhengqi Wang,
Michael H. Malone,
Michael J. Thomenius,
Fei Zhong,
Fang Xu and
Clark W. Distelhorst
From the
Departments of Medicine and Pharmacology, Comprehensive Cancer Center,
Case Western Reserve University School of Medicine and University Hospitals of
Cleveland, Cleveland, Ohio 44106
Received for publication, April 9, 2003
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ABSTRACT
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Glucocorticoid hormones induce apoptosis in lymphoid cells. This process
requires de novo RNA/protein synthesis. Here we report the
identification and cloning of a novel dexamethasone-induced gene designated
dig2. Using Affymetrix oligonucleotide microarray analysis of
10,000 genes and expressed sequence tags, we found that the expression of
dig2 mRNA is significantly induced not only in the murine T cell
lymphoma lines S49.A2 and WEHI7.2 but also in normal mouse thymocytes
following dexamethasone treatment. This result was confirmed by Northern blot
analysis. The induction of dig2 mRNA by dexamethasone appears to be
mediated through the glucocorticoid receptor as it is blocked in the presence
of RU486, a glucocorticoid receptor antagonist. Furthermore, we demonstrated
that dig2 is a novel stress response gene, as its mRNA is induced in
response to a variety of cellular stressors including thapsigargin,
tunicamycin, and heat shock. In addition, the levels of dig2 mRNA
were up-regulated after treatment with the apoptosis-inducing chemotherapeutic
drug etoposide. Though the function of dig2 is unknown, dig2
appears to have a pro-survival function, as overexpression of dig2
reduces the sensitivity of WEHI7.2 cells to dexamethasone-induced
apoptosis.
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INTRODUCTION
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Glucocorticosteriod hormones are potent inhibitors of T cell proliferation
and inducers of thymocyte death
(1,
2). Hence, glucocorticoids are
frequently used as immunosuppressives to treat a broad range of autoimmune and
inflammatory disorders and prevent graft rejection following bone marrow or
organ transplantation. Also, glucocorticoids are effective agents for
treatment of lymphomas and lymphoid leukemias, including acute lymphoblastic
leukemia and chronic lymphocytic leukemia
(35).
Glucocorticoids suppress lymphocyte proliferation and survival by two
fundamental processes. First, glucocorticoids arrest proliferating lymphocytes
in the G1 phase of the cell cycle
(6). Second, glucocorticoids
induce apoptosis in immature lymphocytes
(7). The negative effects of
glucocorticoids on lymphocyte proliferation and survival are mediated through
the glucocorticoid receptor, a ligand-activated transcription factor that
induces or represses transcription of individual genes and gene networks
(8). The transactivation
activity of the glucocorticoid receptor appears essential for
glucocorticoid-induced cell death, although glucocorticoid-induced
"death genes" have not yet been identified (reviewed in Ref.
2).
Recent developments in DNA microarray technology permit a large number of
cellular transcripts to be analyzed in parallel fashion
(9). Using this technology, we
have analyzed gene expression profiles of dexamethasone
(Dex)1-treated
lymphocytes to identify genes that are potentially involved in mediating or
regulating glucocorticoid-induced apoptosis. For this purpose, we have
utilized three separate but related Dex-sensitive model systems, i.e.
the S49.A2 murine T cell lymphoma line, the WEHI7.2 murine T cell lymphoma
line, and primary murine thymocytes. Although there were many genes whose
expression was either induced or repressed by Dex in these model systems, only
seven genes or expressed sequence tags (ESTs) were induced or repressed by Dex
in all three systems. One of the ESTs was particularly intriguing, because it
was induced very rapidly. In this report we show that this EST represents a
novel stress response gene that is induced in response to Dex and a variety of
other cellular stressors. Although the function of the protein encoded by this
gene remains unknown, its overexpression inhibits apoptosis induction by Dex,
suggesting that it has a pro-survival activity.
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EXPERIMENTAL PROCEDURES
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Cell CultureS49.A2 and WEHI7.2 murine T-cell lymphoma lines
were gifts of Drs. Diane Dowd and Roger Miesfeld, respectively. Cells were
cultured in Dulbecco's modified Eagle's medium (DMEM)
(Bio-Whittaker) supplemented with 2 mM glutamine and 10% (v/v)
heat-inactivated bovine calf serum at 37 °C in a 7% CO2
atmosphere under high humidity. Dex was purchased from Sigma, and a stock
solution was prepared in 100% ethanol. S49.A2 and WEHI7.2 cells were seeded at
1 x 105 cells/ml for 12 h before treatment with 1
µM Dex for 6, 12, 18, and 24 h. The control cells at each time
point were treated with ethanol vehicle only. Cell viability was monitored by
trypan blue dye staining.
Thymus IsolationC57BL/6J female mice at 811 weeks
old (Jackson Laboratory) were sacrificed by CO2 asphyxiation.
Thymus glands were removed, rinsed in ice-cold growth medium (DMEM
supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 12.5
units/ml penicillin, and 12.5 µg/ml streptomycin), and then dispersed
through a steel wire mesh into 5 ml of fresh, cold growth medium per thymus.
The suspension of thymocytes was filtered through a tube with a cell strainer
cap (BD Discovery Labware) to remove connective tissue. For each experiment,
thymocytes were pooled from five mice, diluted to 23 x
106 cells/ml in warm growth medium, treated with 1 µM
Dex or ethanol vehicle control, and grown in a humidified 7% CO2
atmosphere at 37 °C. For the microarray experiment, thymus glands were
isolated from adrenalectomized female mice.
Oligonucleotide Array Expression AnalysisGene expression
analysis was performed essentially as described in the Affymetrix GeneChip
expression analysis technical manual. Total RNA was harvested from both
Dex-treated (1 µM) and vehicle control populations at each time
point by Trizol (Invitrogen) extraction. Trizol cell lysates were separated
into aqueous and organic phases by the addition of chloroform to a final
concentration of 20% (v/v). The aqueous phase was purified and concentrated
using an RNeasy minicolumn (Qiagen). DNA complementary to total RNA samples
were reverse transcribed using SuperScript reverse transcriptase (Invitrogen)
and a T7-(dT)24 primer (Operon). This cDNA was used as a template
for the synthesis of biotinylated cRNA using the T7 MEGAscript kit from
Ambion. Biotinylated cRNA probes were fragmented and hybridized to MG-U74A(v2)
GeneChips (Affymetrix) using an Affymetrix GeneChip Fluidics Station 400 and
standard Affymetrix protocols. Fluorescence intensities were captured with a
GeneArray Scanner (Hewlett-Packard).
GeneChip image files were processed using Microarray Analysis Suite,
version 5.0 (Affymetrix). Probe cells displaying irregular fluorescence
intensity over the area of the cell were excluded from subsequence analyses.
To facilitate comparison between samples and experiments, the trimmed mean
signal of each array was scaled to a target intensity of 1500. Comparative
analysis between treatment and control samples for each time point was
performed with the Affymetrix statistical algorithm using default parameters.
To compensate for gene expression changes occurring in the control cultures
over time, each treated sample was compared with a control sample that was
split and harvested in parallel with the treated population. Metric files from
expression and comparison analyses were exported to Microsoft Access XP for
further filtering and analysis. In this work, genes called
"significantly changed" were those that possessed a reliably
detectable signal (absolute call
"absent" and signal
500
in treatment or control samples for inductions or repressions, respectively)
and were determined by the statistical algorithm to be changed 2-fold or
greater (change call
"no change" and signal
500 in
treatment or control samples for inductions or repressions, respectively). To
increase stringency, genes meeting the above criteria were filtered further to
include only those that also were changed in the same direction (change call
no change) in at least one adjacent time point regardless of
magnitude.
cDNA Cloning of dig2Reverse transcription-PCR was performed
using 5'-CTTCTGTGCGCCTTCATTC-3' and
5'-CTCAGGTGGCTATCGTCAGT-3' as primers with first strand DNA as
templates that were reverse transcribed using total RNA purified from S49.A2
and normal mouse thymocytes. PCR products were cloned into the pGEM-T easy
vector (Promega), and DNA inserts were sequenced in both directions by
Cleveland Genomics Inc.
Northern Blot AnalysisTotal RNA was extracted from cultured
cells using the Trizol reagent (Invitrogen) followed by purification through
an RNeasy minicolumn (Qiagen). Total RNA (10 µg for the sample from
cultured cells and 2.5 µg for the sample from primary thymocytes) was
separated in a 1.0% agarose-formaldehyde gel and transferred to a GeneScreen
Plus membrane (PerkinElmer Life Sciences) in 10x SSC (contents of
1x SSC, 0.15 M NaCl, and 15 mM sodium citrate,
pH7.0) by capillary blotting. The RNA was fixed to the membrane by
cross-linking with UV (245 nm, 30 s, 1200 µJ) using a Stratalinker
(Stratagene). Membranes were hybridized with a 32P-labeled probe in
QuikHyb (Stratagene) at 65 °C. Membranes were subsequently washed at 65
°C twice for 15 min each in 2x SSC, once for 30 min in 2x SSC
0.1% SDS, and once for 10 min in 0.1x SSC 0.1% SDS. Densitometric
analysis of Northern blot data was performed using NIH ImageJ software.
Western Blot AnalysisWestern blot analysis was performed
essentially as described previously
(10). The anti-Myc monoclonal
antibody was purchased from Clontech, and the anti-
-actin antibody was
obtained from Sigma. Enhanced chemiluminescence substrate (Amersham
Biosciences) was used for antibody detection.
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RESULTS
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Cloning of a Novel Dex-induced GeneIn response to Dex,
S49.A2 and WEHI7.2 cells undergo cell growth arrest first, followed by
apoptosis. In contrast, primary murine thymocytes are already growth-arrested
and rapidly undergo apoptosis following Dex treatment
(1,
2). Because all three systems
undergo apoptosis and because apoptotic pathways are generally highly
conserved, we sought to identify Dex-induced genes common to all three cell
systems. To identify Dex-regulated gene(s), we utilized oligonucleotide
microarray technology. Total RNA was purified from S49.A2 and WEHI7.2 cells
treated with or without 1 µM Dex for 6, 12, 18, and 24 h or
isolated from primary thymocytes treated with 1 µM Dex for 2 h.
The control cells at each time point were treated with ethanol vehicle only.
Isolated RNA was then hybridized to Affymetrix MG-U74A(v2) oligonucleotide
microarrays representing
10,000 genes and ESTs. Pairwise analysis
(comparing gene expression between Dex-treated and time-matched untreated
control cells) was used to identify differentially expressed genes. The
expression of a total of 132 genes in S49.A2, 296 genes in WEHI7.2, and 59
genes in primary thymocytes were changed significantly
(10). However, only seven
genes were coordinately regulated by Dex in S49.A2, WEHI7.2, and the primary
thymocytes. The fold induction for these seven genes after Dex treatment at
each time point is listed in Table
I. One of the ESTs, AI849939
[GenBank]
, which was induced 2.3
5.7-fold
after Dex treatment in S49.A2, WEHI7.2, and the primary thymocytes, was
selected for further study.
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TABLE I Genes regulated by Dex in S49.A2 cells, WEHI7.2 cells, and normal mouse
thymocytes
RNA hybridization to oligonucleotide microarray and comparative analysis
were performed as described under "Experimental Procedures."
Regulation is expressed as the fold change between treatment and control at
each time point. NC, no change.
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The results of the microarray for AI849939
[GenBank]
as presented in hybridization
signal intensity are shown in Fig.
1A. This was further confirmed by Northern blot analysis
of RNA from Dex-treated S49.A2 cells, WEHI7.2 cells, and primary thymocytes
using AI849939
[GenBank]
as a probe (Fig.
1B). As shown in Fig.
1C, AI849939
[GenBank]
was rapidly induced as early as 30 min after
the addition of Dex to S49.A2 cells. Furthermore, AI849939
[GenBank]
was also
up-regulated when WEHI7.2 cells were treated with doses of Dex as low as 5
nM (data not shown). A BLAST search of the public data base
revealed that AI849939
[GenBank]
has 100% sequence homology with a previously
uncharacterized gene, RIKEN cDNA (AK017926
[GenBank]
), and its single open reading frame
codes for a putative protein of 229 amino acids. Therefore, we have designated
it dig2 (for dexamethasone-induced
gene 2). dig2 is transcribed as a single mRNA species
around 1.8 kb. To clone the coding region of dig2, reverse
transcription-PCR was carried out using RNA purified from Dex-treated S49.A2
and normal mouse thymus gland. The sequence of the PCR products revealed that
dig2 has two isoforms with only three nucleotides (one amino acid)
difference between them (Fig.
2A). Northern blot analysis using a mouse master blot
(Clontech) revealed that dig2 is ubiquitously expressed not only in
multiple mouse tissues in the adult but also in early developmental stages
from embryo days 7 to 17 (Fig.
2B).

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FIG. 1. Dex induced up-regulation of the EST AI849939
[GenBank]
in S49.A2 cells, WEHI7.2
cells, and normal mouse thymocytes. A, the expression of AI849939
[GenBank]
was induced after Dex treatment in S49.A2 cells, WEHI7.2 cells, and normal
mouse thymocytes as revealed by oligonucleotide microarrays. Total cellular
RNA was subjected to oligonucleotide array analysis as detailed under
"Experimental Procedures." Data shown are graphical
representations of the hybridization signal intensity of AI849939
[GenBank]
expression
with or without 1 µM Dex for the indicated hours. B,
Northern blot analysis verified the expression of AI849939
[GenBank]
in S49.A2 cells,
WEHI7.2 cells, and normal mouse thymocytes. C, Dex quickly induced
the expression of AI849939
[GenBank]
in S49.A2 cells at the indicated hours. Each
lane contains 10 µg of total RNA for the samples from S49.A2 cells
and WEHI7.2 cells or 2.5 µg of total RNA for the sample from thymocytes. 28
S ribosomal RNA or -actin was used as loading control.
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FIG. 2. Nucleotide sequence of mouse dig2 cDNA and its expression in
multiple tissues. A, the predicted amino acid sequence for
dig2 is shown by the single-letter code under the nucleotide
sequence. The numbers of amino acid positions relative to the initiating Met
are shown in parentheses under those of the nucleotide positions. The
underlined sequence (three nucleotides or one amino acid) for Dig2 is
deleted to make the short form of Dig2 (GenBankTM accession number
AY260552
[GenBank]
). B, distribution of dig2 in early developmental
stages and various mouse tissues. A master blot membrane was purchased from
Clontech (catalog no. 7771-1) and contains normalized loading of
poly(A)+ RNA from 22 different mouse tissues and seven different
control RNAs and DNAs.
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Dex-induced dig2 Expression Requires Transcriptional Activation
Mediated through the Glucocorticoid ReceptorBecause Dex rapidly
induced dig2 mRNA expression, we questioned whether this induction is
transcriptionally regulated. As shown in
Fig. 3A, actinomycin D
(Act D) treatment inhibited dig2 mRNA induction by Dex, whereas
cycloheximide (CHX) did not. This result suggests that the induction of
dig2 by Dex is transcriptionally controlled. It is also notable that
dig2 seems to have a relatively short half-life, because the
abundance of dig2 mRNA decreased to nearly undetectable levels from 3
to 12 h after actinomycin D treatment. RU486, a glucocorticoid receptor
antagonist, prevented the induction of dig2 mRNA by Dex in S49.A2
cells (Fig. 3B).
Furthermore, dig2 was not induced by Dex in S49.143R cells, which
contain defective glucocorticoid receptors (data not shown). This indicates
that the induction of dig2 mRNA expression requires a functional
glucocorticoid receptor.

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FIG. 3. Both actinomycin D and RU486 inhibited Dex-induced dig2 mRNA
expression. A, actinomycin D (Act D) inhibited but
cycloheximide (CHX) did not block Dex-induced dig2 mRNA
expression. S49.A2 cells were maintained in the presence of ethanol (control),
1 µM Dex, 1 µg/ml actinomycin D or 10 µg/ml cycloheximide,
as indicated. B, RU486, a glucocorticoid receptor antagonist,
prevented the induction of dig2 mRNA by Dex. S49.A2 cells were
treated with ethanol (control), 1 µM Dex, or 50 µg/ml of
RU486, as indicated. 10 µg of total RNA per lane was used for Northern blot
analysis. The staining of 28 S ribosomal RNA was used as a loading
control.
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dig2 Is a Novel Stress Response GeneAmong the seven genes
regulated by Dex (Table I),
herp has been reported as an endoplasmic reticular (ER) stress
response gene induced by homocysteine,
-mercaptoethanol, tunicamycin
(TU), A23187
[GenBank]
, and thapsigargin (TG)
(11). Therefore, we checked
whether Dex treatment is able to induce an ER stress response. Though the
treatment with Dex induced the expression of herp and
gadd153/chop in S49.A2 cells as confirmed by Northern blot
analysis, treatment with Dex did not induce the expression of the typical ER
stress response genes encoding the glucose-regulated proteins (Grps),
grp78 and grp94 (Fig.
4). Furthermore, the ER stress inducers, TG and TU, did cause a
rapid induction of dig2 in S49.A2 cells
(Fig. 5A). The
induction of dig2 expression by TG and TU was not dependent on a
functional glucocorticoid receptor, because dig2 mRNA was induced
dramatically in S49.143R cells, which do not have functional glucocorticoid
receptors (Fig. 5B).
This up-regulation of dig2 by TG and TU was not limited to T lymphoma
cells, as it was also induced in MDA-MB-468 cells, a human breast cancer cell
line (Fig. 5C). These
results indicated that dig2 is a novel stress response gene. The Grps
and heat shock proteins (Hsps) represent the two major groups of
stress-response proteins. In Fig.
6A, we have shown that a brief heat shock treatment (43
°C, 5 min) also up-regulates dig2 gene expression in S49.A2
cells. Furthermore, when S49.A2 cells were treated with 10 mM
-mercaptoethanol (Fig.
6B) or cultured under conditions of osmotic stress
(DMEM with 0.15 M NaCl, 5 min)
(Fig. 6C), the
expression of dig2 mRNA was also induced, although the level of
induction is not as significant as that following TU and TG treatment. These
findings further support the concept that dig2 is a stress response
gene.

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FIG. 4. Dex induced the stress response genes herp and gadd153
but did not induce grp78 and grp94 genes in S49.A2
cells. Total RNA from S49.A2 cells that were left untreated (ethanol,
control) or treated by 1 µM Dex for the indicated hours were
analyzed. Each lane contained 10 µg of total RNA. The staining of 28 S
ribosomal RNA was used as a loading control.
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FIG. 5. Induction of dig2 mRNA expression by ER stress inducers,
thapsigargin (TG), and tunicamycin (TU). Total RNA from
S49.A2 (A), S49.143R (B), and MDA-MB-468 (C) cells
that were left untreated or treated by 1 µM Dex, 100
nM TG, or 10 µg/ml TU for the indicated hours were analyzed.
Each lane contained 10 µg of total RNA. Membranes were first probed with
dig2 and then stripped and probed for grp78 and
grp94. The staining of 28 S ribosomal RNA was used as a loading
control.
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Expression of the dig2 Gene in Response to Other Apoptotic
InducersThe preceding stress inducers also mediate apoptosis.
Therefore, we tested whether the induction of dig2 expression is
associated generally with apoptosis. As revealed by Northern hybridization,
when S49.A2 cells were treated with 250 nM staurosporine (STS), a
protein kinase C inhibitor, dig2 expression was not induced
(Fig. 7A). However,
etoposide, another apoptosis inducer in S49.A2 cells, induced dig2
expression significantly (Fig.
7B). dig2 induction by Dex, TG, TU, and
etoposide was not affected by Bcl-2 overexpression
(Fig. 7C).

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FIG. 7. Dig2 mRNA expression was induced in response to etoposide, but
overexpression of Bcl-2 did not block the induction of dig2.
A and B, total RNAs from S49.A2 that were left untreated or
treated by 250 nM STS (A) or 10 µM etoposide
(B) for the indicated times were analyzed. C, WEHI B27 is a
clone of WEHI7.2 cells in which Bcl-2 was stably overexpressed. Total RNAs
from cells that were left untreated or treated with 1 µM Dex, 10
µM etoposide (Etop), 100 nM TG, or 10
µg/ml TU for 6 h were analyzed. Con, control.
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Overexpression of Dig2 Desensitizes Cells to Dex-induced
ApoptosisTo assess the effect of Dig2 on the viability of WEHI7.2
cells, we cloned the cDNA encoding Myc-tagged Dig2 into the expression vector,
pIRESneo2 (Clontech), in both sense and antisense orientations. We obtained
clones stably expressing Myc-tagged Dig2 when WEHI7.2 cells were transfected
with the sense orientation vector. Though the Dig2 protein has an estimated
molecular mass of 25 kDa, Western analysis revealed that Myc-tagged Dig2 gives
rise to a protein product with a size estimated around 35 kDa
(Fig. 8A). This
suggests that Dig2 is post-translationally modified. No clones were obtained
when WEHI7.2 cells were transfected with the antisense vector. This finding
suggests that the function of Dig2 may be essential for cell survival.
Consistent with this conclusion, we found a lower percentage of dead cells in
untreated cultures of WEHI7.2 cells that stably express Myc-tagged Dig2,
versus control cells transfected with the empty vector (5.3 x
1.9% (at 24 h) and 4.3 x 2.8% (at 36 h) versus 13.5 x
6.1% (at 24 h) and 13.8 x 5.2% (at 36 h), n = 4, p is
<0.05 (at 24 h) and <0.02 (at 36 h), respectively)
(Fig. 8B). To
determine whether Dig2 regulates cell death induction by Dex, cells stably
expressing Myc-tagged Dig2 and empty vector control cells were treated with 1
µM Dex for 24 and 36 h. The percentage of dead cells following
Dex treatment was much less in cells expressing Myc-tagged Dig2 compared with
cells transfected with empty vector (9.6 x 0.9% (24 h after Dex) and
40.7 x 3.9% (36 h after Dex) versus 28.7 x 8.5% (24 h
after Dex) and 73.0 x 6.5% (36 h after Dex), n = 4, p
is <0.02 (24 h) and <0.0001 (36 h), respectively)
(Fig. 8B). We have
examined two independently selected clones of Neo and Dig2 and obtained
essentially the same results in independent clones (data not shown). These
findings indicate that Dig2 has a pro-survival function.

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FIG. 8. Dig2 has protective action in Dex-treated cells when it is overexpressed
in WEHI7.2 cells. A, Western blot for Myc-tagged Dig2 expression
in Neo transfection control cells and cells overexpressing Dig2. B,
representative clone (Dig2 number 4) from dig2-transfected or its
vector Neo control were treated with 1 µM Dex or ethanol
(vehicle control) for 24 and 36 h. Trypan blue dye staining was used to assess
the cell death of dig2-transfected WEHI7.2 cells. Data are
representative of four separate experiments that were performed in duplicate
and analyzed with Student's t tests. dig2-transfected cells
were compared with Neo control cells in either control or Dex treatment
conditions. *, p < 0.05; ***, p < 0.0001.
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DISCUSSION
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We report the identification and cloning of a novel stress response gene,
dig2. This gene has widespread tissue distribution. Moreover, it is
strongly induced in both lymphoid and non-lymphoid cells by TG, TU, and
etoposide, as well as being moderately induced by cellular stressors including
osmotic stress,
-mercaptoethanol, and heat shock. Also, dig2
transcription is induced by Dex only in lymphoid cells
(Fig. 1), whereas it is not
induced by Dex in non-lymphoid MDA-MB-468 cells (data not shown).
dig2 is a mouse homologue of a novel hypoxia-inducible factor
1-responsive gene, rtp801, reported during the course of the present
work (12). Also, we detect an
EST corresponding to dig2 among a list of p53-regulated transcripts
(13). Collectively, these
findings indicate that dig2 is induced in response to a wide variety
of cellular stressors.
One remarkable observation about dig2 is the rapidity of its
induction. dig2 induction by thapsigargin was much more rapid than
the induction of the well characterized ER stress response genes
grp78 and grp94 (Fig.
5). This is much more rapid than most of the other Dex-induced
transcripts detected with microarrays. Furthermore, although the induction of
dig2 by Dex was mediated through the glucocorticoid receptor and
blocked by the glucocorticoid receptor antagonist RU486, the mechanism of
dig2 induction is presently uncertain. Moreover, the increase in
levels of dig2 mRNA following Dex treatment was blocked by
actinomycin D but not by cycloheximide. These findings suggest that
transcriptional up-regulation was at least in part responsible for
dig2 induction and that new protein synthesis (e.g. of an
intermediate signaling factor) was not required. Together, these findings,
including the rapidity of induction, suggest the possibility of direct
transactivation of the dig2 gene by the glucocorticoid receptor.
However, analysis of the 5' dig2 promoter sequence failed to
detect a consensus sequence corresponding to a typical glucocorticoid response
element. Moreover, an 800 base pair region 5' to the transcription
initiation site of dig2 was cloned into a luciferase reporter
plasmid. However, Dex failed to induce luciferase activity when this reporter
plasmid was expressed by transient transfection in WEHI7.2 cells (data not
shown). Thus, based on these preliminary findings, there is no evidence of
direct regulation of a dig2 promoter/enhancer region by the
glucocorticoid receptor. Further work will be needed to uncover the mechanism
of dig2 induction by Dex and other cellular stressors.
The mammalian stress response is an evolutionarily conserved mechanism that
allows cells to respond to adverse environment or metabolic conditions. A wide
range of stresses, including heat shock, inhibition of energy metabolism, and
oxidative stress induce expression of Hsps
(14). Hsps assist in the
recovery from stress either by degrading or repairing damaged proteins
(protein refolding). The term "glucose-regulated proteins"
encompasses a variety of ER chaperones that can be induced by glucose
starvation or depletion of ER calcium
(15). Perturbations in ER
function disrupt protein folding, leading to accumulation of unfolded proteins
and protein aggregates that are detrimental to cell function and survival.
Hence, the induction of Grps is referred to as the unfolded protein response.
A number of different proteins are induced by ER stress. Many of these reside
in the ER lumen (e.g. Grp78 and Grp94) or are located on the ER
transmembrane (e.g. Herp), but several are in non-ER locations,
including gadd153/CHOP (nucleus), GLUT-1 (plasma membrane) and asparagine
synthetase (cytoplasm) (15,
16).
We are interested in investigating the mechanism of apoptosis induction by
glucocorticosteroid hormones. Exposure of lymphocytes to Dex induces a unique
form of cell stress. Dex quickly up-regulates the ER stress gene herp
and also induces the expression of gadd153/chop
(Fig. 4), an ER stress and
apoptosis-related protein. Here, we have found that Dex treatment rapidly
induces a stress response gene, dig2. However, Dex does not elevate
the expression of grp78 and grp94. This suggests that the
induction of dig2 by Dex is mediated through a signaling pathway
different from that mediating induction of grp78 and grp94.
The events of cell stress and cell death are linked, such that the
molecular chaperones induced in response to stress appear to function at key
regulatory points in the control of apoptosis
(14). Hsp expression is
modulated by many conditions that lead to apoptosis. Exposure of cells to
stress activates a survival response via the induction of Hsps. Moreover,
induction of Grps is essential for maintenance of cell survival following ER
stress (15). Here, the
induction of dig2 expression may well have been a survival response
when cells were exposed to the apoptosis inducer Dex. This hypothesis is
supported by the evidence that overexpression of Dig2 did inhibit Dex-induced
apoptosis (Fig. 8). Moreover,
enforced expression of rtp801, a homologue of dig2,
inhibited hypoxia-induced apoptosis, although, paradoxically, this same gene
appeared to promote apoptosis when expressed in non-dividing cells
(12). Thus, although the
mechanism of dig2/rtp801 action is unknown, this gene
appears to play a role in regulating cell survival.
In summary, the present work identifies dig2 as a novel stress
gene that is rapidly induced in response to a variety of cellular stresses.
The induction of dig2 may serve a protective function, delaying or
inhibiting apoptosis. Its mechanism of action and potential other functions
remain to be elucidated. Moreover, the signal transduction pathway(s) that
mediate dig2 induction in response to such a wide range of cellular
stresses provides a challenge for further investigation.
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FOOTNOTES
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The nucleotide sequence(s) reported in this paper has been submitted to
the GenBankTM/EBI Data Bank with accession number(s)
AY260552
[GenBank]
.
* This work was supported by National Institutes of Health Grants R01 CA42755
and R01 CA79806 (to C. W. D.), T32 CA059366
[GenBank]
(to M. H. M.), T32 CA73515 (M. J.
T.), and P30 CA43703 (to the Comprehensive Cancer Center of Case Western
Reserve University and the University Hospitals of Cleveland). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed: Division of Hematology/Oncology,
Case Western Reserve University School of Medicine, 10900 Euclid Ave.,
Cleveland, OH 44106-4936. Tel.: 216-368-1175; Fax: 216-368-1166; E-mail:
cwd{at}po.cwru.edu.
1 The abbreviations used are: Dex, dexamethasone; dig2,
dexamethasone-induced gene 2; DMEM, Dulbecco's modified Eagle's medium; ER,
endoplasmic reticular; EST, expressed sequence tag; Grp, glucose regulated
protein; Hsp, heat shock protein; STS, staurosporine; TG, thapsigargin; TU,
tunicamycin. 
 |
ACKNOWLEDGMENTS
|
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We thank Dr. Nikki Holbrook for providing the gadd153 cDNA and Dr.
Amy Lee for giving us the cDNAs for grp78 and grp94. We
acknowledge the Microarray Core facility and Drs. Martina Veigl and Patrick
Leahy for assistance in microarray analysis. We also thank Karen McColl for
technical assistance and Huiling He for initiating the microarray
experiments.
 |
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