Chronic Restraint Stress Modulates Expression of Genes
Y. Wang1, Y. Zhang2, D. Yin2, J. Moorman2, Xiao Zeng1
1SuperArray Bioscience Corporation, Frederick, MD,
2Department of Internal Medicine, East Tennessee State University, Johnson City, TN.
Materials and Methods
Restraint stress mouse model: 6-8 week old male Balb/c mice from Charles
River Laboratories were maintained in the Division of Laboratory Animal Resources
at East Tennessee State University (ETSU). The mice were subjected to a chronic
physical restraint protocol (3). Briefly, mice were placed in a 50-ml conical centrifuge
tube filled with multiple punctures to allow ventilation. Mice were held horizontally in
the tubes for a continuous 12 h followed by a 12-h rest, while food and water were
provided ad libitum. Control mice were kept in their original cages and in the same
diet schedule as the experimental group. Mice were physically restrained for two
cycles. After physical restraint, the spleens were rapidly frozen in liquid nitrogen and
stored at -80ºC for later isolation of total RNA. Genomic DNA interference was
eliminated by DNase I treatment.
Figure 3: Validation of Microarray Data Using Real-Time PCR
RealFigure 1: Microarray Analysis of Apoptotic Genes in Restraint Stress
Physical restraint stress alters apoptotic
gene expression profiles in the spleen.
Panel A displays representative images
of the Oligo GEArray® Mouse Apoptosis
Microarrays. The experiments were
repeated three times. As an example, the
up-regulation of Bnip3 is circled in red.
Panel B lists the 23 genes whose
expression levels are significantly
changed in stressed versus unstressed
RNA using the TrueLabeling-AMP™ 2.0 Kit (Catalog No. GA-030). The resulting
biotin-labeled cRNA probe was allowed to hybridize overnight to the Oligo GEArray®
microarray in a standard hybridization oven at 60ºC. After blocking, washing and
chemiluminescent detection steps, images were acquired by a Chemi-Doc imaging
system (CCD camera). Raw signal intensity for individual genes on the microarray
was extracted using web-based GEArray® Expression Analysis Suite software and
normalized against signal intensities from housekeeping genes.
Real-time RT-PCR: cDNA was synthesized from total RNA using the
ReactionReady™ First Strand cDNA Synthesis Kit (Catalog No. C-01). Real-time
PCR was performed using RT2 Real-Time™ SYBR Green Fluorescein PCR Master
Mix (Catalog No. PA-011) on the Bio-Rad iCycler real-time PCR detection system.
GAPDH and β-Actin were chosen as housekeeping genes for normalization.
Threshold cycle numbers (Ct) were used for “∆∆Ct” analysis. Fold-changes between
samples were then calculated as 2^(-∆∆Ct). The statistical significance was
determined by one-way ANOVA and Bonferroni tests (p<0.05).
Physical restraint stress alters the p53signaling pathway in the spleen.
Panel A displays representative images
of the Oligo GEArray® Mouse p53
Signaling Pathway Microarrays. The
experiments were repeated three times.
As an example, the up-regulation of p21
is shown in red circles. Panel B lists the
24 genes whose expression levels are
significantly increased in stressed versus
unstressed mice, as well as Stat1 whose
gene expression decreased.
Up-regulation of Fas, FADD,
p53, and p21 genes in
spleens from mice with
physical restraint stress was
confirmed by quantitative
real-time PCR. The data are
presented as the relative
fold-changes normalized to
GAPDH as the internal
control. The data are
representative of three
experiments where the
asterisk (*) denotes p < 0.01
when compared to control.
By focusing on apoptosis and p53 signaling pathways, we have
found that 22.3% of the examined genes showed significant upregulated expression in splenocytes between stressed and
The up-regulation of FADD, FAS, p53 and p21 is further confirmed
through real time PCR.
Apaf1: Apoptotic peptidase activating factor 1
Apex1: Apurinic/apyrimidinic endonuclease 1
Aurkb: Aurora kinase B
Wdr39: WD repeat domain 39
Bak1: BCL2-antagonist/killer 1
Bap1: Brca1 associated protein 1
Bax: Bcl2-associated X protein
Bid: BH3 interacting domain death agonist
Birc5: Baculoviral IAP repeat-containing 5
Brca1: Breast cancer 1
Brca2: Breast cancer 2
Btg2: B-cell translocation gene 2, anti-proliferative
Ccng2: Cyclin G2
Ccnh: Cyclin H
Cdc25a: Cell division cycle 25 homolog A (S.
Cdc25c: Cell division cycle 25 homolog C (S. cerevisiae)
Cdc2a: Cell division cycle 2 homolog A (S. pombe)
Cdk7: Cyclin-dependent kinase 7 (homolog of Xenopus
MO15 cdk-activating kinase)
Cdkn1a: Cyclin-dependent kinase inhibitor 1A (P21)
Chek1: Checkpoint kinase 1 homolog (S. pombe)
Chek2: CHK2 checkpoint homolog (S. pombe)
Cyr61: Cysteine rich protein 61
Daxx: Fas death domain-associated protein
Gadd45a: Growth arrest and DNA-damage-inducible 45
Stat1: Signal transducer and activator of transcription 1
Figure 2: Microarray Analysis of p53-Mediated Signaling in Restraint Stress
Oligo GEArray® System: Two cataloged Oligo GEArray® microarrays from
SuperArray were used in this study. The Oligo GEArray® Mouse Apoptosis
Microarray (Catalog No. OMM-012) contains 112 genes involved in apoptosis. The
Oligo GEArray® Mouse p53 Signaling Pathway Microarray (Catalog No. OMM-027) is
designed to profile the expression of 113 key genes involved in the p53 signaling
Microarray processing: Biotinylated cRNA target was synthesized from total
Akt1: Thymoma viral proto-oncogene 1
Api5: Apoptosis inhibitor 5
Bax: Bcl2-associated X protein
Bnip3l: BCL2/adenovirus E1B interacting protein 325
Birc1e: Baculoviral IAP repeat-containing 1e
Birc2: Baculoviral IAP repeat-containing 2
Bnip2: BCL2/adenovirus E1B interacting protein 1,
Bnip3: BCL2/adenovirus E1B interacting protein 1,
Casp3: Caspase 3
Bok: Bcl-2-related ovarian killer protein
Casp2: Caspase 2
Casp8: Caspase 8
Dsip1: TSC22 domain family 3
Cradd: CASP2 and RIPK1 domain containing adaptor
with death domain
Fadd: Fas (TNFRSF6)-associated via death domain
Ltbr: Lymphotoxin B receptor
Nfkb1: Nuclear factor of kappa light chain gene
enhancer in B-cells 1, p105
Zc3hc1: Zinc finger, C3HC type 1
Rnf7: Ring finger protein 7
Tnfrsf21: Tumor necrosis factor receptor superfamily,
Fas: Fas (TNF receptor superfamily member)
109 Trp53: Transformation related protein 53
Trp53inp1: Transformation related protein 53
inducible nuclear protein 1
F o ld C h a n g e
Psychological and physical stress can alter the immune system in both
human and animals (1). It has been reported that mice subjected to
chronic 12-hour daily physical restraint for two days showed dramatic
apoptosis in splenocytes (2). To identify genes that contribute to the
splenocyte apoptosis, we compared gene expression in the spleens of
restraint stressed mice with that in the spleens of unstressed mice
using oligo microarrays consisting of 225 genes. We report here that
mice subjected to chronic 12-hour daily physical restraint for two days
exhibited significantly altered expression of 48 out of 225 genes. These
genes included pro-apoptotic genes. We selected four genes of
interest, and confirmed the microarray results by real time PCR.
Although these findings are not specific for restraint stress-induced
apoptosis in splenocytes, they identify a potentially important
component of pro-apoptotic activity in restraint stress and suggest a
possible target for anti-apoptotic therapy in stress-induced apoptosis
in splenocytes. Our mouse model and identified biomarkers would be
very useful for further study of the intercommunication of the immune
and the nervous systems.
Our data provide additional evidence that apoptotic machinery
underlines lymphopenia during stress.
It remains to be further investigated whether the apoptotic genes
provide a marker for individuals with chronic stress.
Pathway-focused microarray analysis is a simple and easy
approach to screen for genes of interest.
1. Padgett,D.A. and Glaser,R. (2003). How stress influences the immune response. Trends
Immunol. 24, 444-448.
2. Yin,D., Tuthill,D., Mufson,R.A., and Shi,Y. (2000). Chronic restraint stress promotes
lymphocyte apoptosis by modulating CD95 expression. J. Exp. Med. 191, 1423-1428.
3. Sheridan,J.F., Dobbs,C., Jung,J., Chu,X., Konstantinos,A., Padgett,D., and Glaser,R.
(1998). Stress-induced neuroendocrine modulation of viral pathogenesis and immunity.
Ann. N. Y. Acad. Sci. 840, 803-808.