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Molecular and Cellular Biology, December 2000, p. 9009-9017, Vol. 20, No. 23
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mice with an Increased Glucocorticoid Receptor Gene
Dosage Show Enhanced Resistance to Stress and Endotoxic Shock
Holger M.
Reichardt,
Thorsten
Umland,
Anton
Bauer,
Oliver
Kretz, and
Günther
Schütz*
Division of Molecular Biology of the Cell I,
German Cancer Research Center, 69120 Heidelberg, Germany
Received 8 May 2000/Returned for modification 13 June 2000/Accepted 1 September 2000
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ABSTRACT |
Targeted mutagenesis of the glucocorticoid receptor has revealed an
essential function for survival and the regulation of multiple
physiological processes. To investigate the effects of an increased
gene dosage of the receptor, we have generated transgenic mice carrying
two additional copies of the glucocorticoid receptor gene by using a
yeast artificial chromosome. Interestingly, overexpression of the
glucocorticoid receptor alters the basal regulation of the
hypothalamo-pituitary-adrenal axis, resulting in reduced expression of
corticotropin-releasing hormone and adrenocorticotrope hormone and a
fourfold reduction in the level of circulating glucocorticoids. In
addition, primary thymocytes obtained from transgenic mice show an
enhanced sensitivity to glucocorticoid-induced apoptosis. Finally,
analysis of these mice under challenge conditions revealed that
expression of the glucocorticoid receptor above wild-type levels leads
to a weaker response to restraint stress and a strongly increased
resistance to lipopolysaccharide-induced endotoxic shock. These results
underscore the importance of tight regulation of glucocorticoid
receptor expression for the control of physiological and pathological
processes. Furthermore, they may explain differences in the
susceptibility of humans to inflammatory diseases and stress, depending
on individual prenatal and postnatal experiences known to influence the
expression of the glucocorticoid receptor.
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INTRODUCTION |
The glucocorticoid receptor (GR) is
a ligand-dependent transcription factor which belongs to the nuclear
hormone receptor superfamily. Due to its almost ubiquitous expression,
GR plays an important role during development and in many physiological and pathological processes. These include regulation of energy homeostasis, adaptation to stress, and modulation of central nervous system functions (16). In addition, GR is a major modulator of the immune system due to its proficient antiinflammatory and immunosuppressive activities, thus serving a function which is frequently put to use in the treatment of inflammatory diseases, autoimmune disorders, and leukemia with glucocorticoids (2).
To study the role of GR in more detail, several mutant mouse strains
have been generated by gene targeting (24, 32). The analysis
of GR knockout mice has revealed a pivotal role for the receptor both
in lung maturation and as the negative feedback control of the
hypothalamo-pituitary-adrenal (HPA) axis (5, 14, 23).
Furthermore, analysis of mice selectively lacking GR in the nervous
system has revealed an important function of the GR in the brain for
processes such as reacting to anxiety (31). A gene targeting
approach was also taken to study the relative importance of different
modes of action of the receptor. Specifically, mice that carried a
point mutation in one of the dimerization domains of the GR, resulting
in a DNA-binding-defective receptor, were generated (22).
Surprisingly, analysis of these mice has shown that
DNA-binding-dependent transactivation was dispensable not only for
survival but also for the regulation of many physiological processes,
such as thymocyte apoptosis.
In addition to a large number of loss-of-function experiments, there is
a growing body of evidence suggesting that an increased gene dosage may
also have profound effects on physiology and development. This was
exemplified for the Pax-6 (28) and the Zipro-1
(36) genes. In the case of Pax-6, overexpression from a
yeast artificial chromosome (YAC) led to abnormalities of the eyes,
whereas additional copies of Zipro-1 expressed from a bacterial
artificial chromosome caused a proliferation defect in cerebellum and
skin. The latter observation was particularly unexpected given that
Zipro-1 knockout mice lack an obvious phenotype (36). In the
case of GR, evidence to date also suggests a gene dosage effect.
Specifically, the magnitude of the transcriptional response elicited by
GR in vitro was shown to be proportional to the number of receptor
molecules per cell (34). Furthermore, heterozygous GR
knockout mice show differences in the control of the HPA axis
(5). Collectively, these data suggest that overexpression of
GR by introduction of additional alleles into mice may lead to
alterations in gene expression and physiological responses.
Expression of classical plasmid transgenes in mice is often variable
and low and does not necessarily reflect the endogenous expression
pattern of the gene. These limitations can be circumvented by using
YACs (27). YACs span up to 1 Mb of genomic sequences and
allow transfer of a transgene within an almost natural chromosomal context due to the large stretches of flanking sequences which protect
the gene from position effects at the integration site (3, 17,
26). Therefore, this approach usually guarantees expression of
the transgene in a copy number-dependent and position-independent manner (27). Furthermore, due to their large size, YACs are an ideal vector system for introduction of genes such as that for the
GR, which spans at least 110 kb (29). Consequently, we have
used a 290-kb YAC which covers the entire Gr locus to generate GR-overexpressing mice. Significantly, neuroendocrine regulation, the sensitivity of thymocytes to glucocorticoid-induced apoptosis, and the responses to stress and inflammation are severely altered in these transgenic mice. Thus, our results allow new insights
into the mechanisms of GR in physiological and pathological processes.
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MATERIALS AND METHODS |
Isolation, characterization, and modification of Gr
YAC.
A YAC library from C57BL/6 mouse DNA in the yeast strain
AB1380 (Research Genetics, Huntsville, Ala.) was screened by PCR using
two primers specific for Gr exon 2. This resulted in the isolation of three independent Gr YAC clones. One clone,
designated YGR4, with an insert length of 620 kb, was transferred from
the library's host strain to YPH925 by kar cross. The relative
location of the Gr gene on the YAC could then be determined
after electroporation with the fragmentation vector pHIS3-TelGR and
subsequent analysis of His-autotroph colonies by pulsed-field gel
electrophoresis (PFGE). The size of the homologously recombined YACs
was compared to the size of YGR4 and from that comparison the relative
position of the Gr locus was deduced. To shorten the YAC, a
YPH925 clone carrying YGR4 was electroporated with the B1
element-containing fragmentation vector pCEN/HIS3-TelB1, and
histidine-autotroph clones were analyzed by PFGE. Two clones contained
a 290-kb YAC, and one of the two was chosen for microinjection.
Generation of transgenic mice.
YAC DNA was purified for
microinjection as previously described (10). Conditions for
preparative PFGE were 1% agarose in 0.5× Tris-agarose-EDTA buffer,
14°C, 6 V/cm, 120°, and a time ramp of 10 to 60 s for 20 h (CHEF-DR III system; Bio-Rad).
RNA analysis.
Total RNA was isolated after guanidinium
isothiocyanate extraction according to standard procedures. cDNA
synthesis for reverse transcription (RT)-PCR analysis (20)
and in situ hybridization (22) was performed as described previously.
Restriction fragment length polymorphism (RFLP) analysis.
An
additional RsaI site in the 3' region of the Gr
gene due to the different origins of the YAC DNA (C57BL/6 mouse strain) and the oocytes used for microinjection (FVB/N mouse strain) was identified in the FVB/N allele. To distinguish between the
endogenous Gr allele and the one derived from the YAC,
either genomic DNA or a DNA fragment obtained by RT-PCR was digested
with RsaI and analyzed by Southern blotting using a probe
specific for the GR 3' untranslated region.
Protein analysis.
For Western blot analysis, proteins were
extracted in radioimmunoprecipitation assay buffer and 30 µg of the
whole-cell extracts was resolved on a denaturing sodium dodecyl
sulfate-7.5% polyacrylamide gel. Proteins were transferred to a
polyvinylidene difluoride membrane and stained with a GR-specific
antibody (M-20; Santa Cruz Biotechnology, Santa Cruz, Calif.), and
immunoreactive bands were visualized by enhanced chemiluminescence.
Hormone and cytokine measurements.
Prior to the analyses,
mice were kept in a quiet place under a constant dark-light cycle.
Blood was quickly collected from the trunk after decapitation (at 9:00
a.m. for basal hormone measurements). The serum was isolated by
centrifugation and stored in aliquots at 
80°C. Concentrations of
corticosterone and adenocorticotrope hormone (ACTH) were determined
using commercially available radioimmunoassay kits (ICN, Meckenheim,
Germany) according to the manufacturer's instructions. Titers of
interleukin-6 (IL-6) were analyzed using an enzyme-linked immunosorbent
assay kit (Endogen, Woburn, Mass.). Synthetic ACTH1-24 stimulation was
performed by intraperitoneal injection of vehicle (phosphate-buffered
saline [PBS]) or ACTH (10 µg/kg of body weight). Blood was
collected 20 min after the injection by tail phlebotomy.
Immunohistochemistry.
Tissues were fixed in
phosphorate-buffered 4% paraformaldehyde overnight at 4°C,
dehydrated through an ascending ethanol series, and embedded in
paraffin. Immunostaining was performed on 6-µm-wide sections using polyclonal antibodies against ACTH or
corticotropin-releasing hormone (CRH). Antibody reactivity was
visualized using a goat anti-rabbit immunoglobulin G conjugated with
horseradish peroxidase and 3,3'-diaminobenzidine.
Quantification of in situ hybridization and
immunohistochemistry.
The area positively immunostained or
occupied by silver grains was taken as a measure for peptide and mRNA
expression. Quantitative analysis of the signals was performed using an
image processing system as described previously (14).
Thymocyte apoptosis.
Culturing of primary thymocytes as well
as detection and evaluation of apoptosis was performed as previously
described (18, 22). Briefly, aliquots of cells were
cultivated at a concentration of 4 × 106 cells/ml in
complete RPMI medium containing 10% heat-inactivated fetal calf serum
plus the respective concentrations of dexamethasone (10
5
to 109 M) and ZK112,339 (106 M)
(35) for 9 and 24 h each. Thymocytes were harvested and resuspended in 300 µl of a propidium iodide (PI) solution (50 µg/ml) containing 0.1% Triton X-100-0.1% sodium citrate. The
samples were incubated overnight at 4°C and subsequently analyzed on
a FACScalibur instrument (Becton Dickinson, San Jose, Calif.). Gated cells (excluding duplets) were evaluated by their FL-2 area versus side
scatter (SSC) pattern. Based on low PI fluorescence and high SSC, cells
considered sub-G1 and apoptotic were gated in region 2 and
the percentage of apoptotic cells was calculated from the total number
of gated cells.
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RESULTS |
Generation of YAC transgenic mice carrying two additional copies of
the Gr gene.
YAC YGR4 was isolated from a mouse
C57BL/6 library using GR-specific primers. Briefly, YGR4 was 620 kb in
length and contained the entire Gr locus of 110 kb,
according to a genomic organization scheme based upon previously
published results (Fig. 1A and B) (29). Using fragmentation analysis, the Gr gene
was mapped close to the short arm of YGR4 (Fig. 1A). To facilitate
microinjection, YGR4 was shortened. This was accomplished using
homologous recombination of the YAC insert with a replacement vector
targeting B1 elements frequently distributed over the mouse genome.
Thereby we generated YACs 500 kb and 290 kb in length (Fig. 1A and C).
YAC YGR290, which contained approximately 150 kb of sequence upstream
of the Gr gene and 25 kb of sequence downstream of the
Gr gene, was used to generate transgenic mice.
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FIG. 1.
Isolation of a YAC carrying the Gr gene. (A)
Structure of the unmodified YAC YGR4 and the shortened YAC YGR290. The
position of the Gr gene on the YAC and its exon-intron
structure (enlarged) is indicated. (B) Restriction analysis of YGR4 by
digestion with the rare-cutting enzymes SfiI and
NotI, PFGE, and Southern blotting with probes for GR exons 2 and 8. Alignment of fragments is shown in panel A. (C) PFGE and
Southern blot analysis of YACs obtained after B1 fragmentation. The
position of the original YAC YGR4 and its shortened derivative YGR290
are indicated by an arrow.
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DNA from YGR290 was purified by PFGE and used for microinjection into
FVB/N oocytes. Two founder mice were obtained, one of
which, designated
YGR, was analyzed in detail. An RFLP identified
in the 3' region of the
Gr gene allowed the endogenous
Gr allele
to be
distinguished from the YAC-derived one. Based on Southern
blot analysis
of tail DNA, the copy number of the YAC in YGR mice
was determined to
be two (Fig.
2A).
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FIG. 2.
Expression analysis of YGR transgenic mice. (A)
Determination of the YAC copy number in YGR mice by Southern blot
analysis of tail DNA from WT (wt) mice and one transgenic mouse from
each generation (F1 and F2). (B) Analysis of
transgene expression in the hippocampus of a C57BL/6 mouse, an FVB/N
mouse, and a YGR mouse by RT-PCR and RFLP. PCR products were cut with
RsaI and analyzed by Southern blotting. (C) Analysis of
transgene expression in various tissues by RT-PCR and RFLP. br, whole
brain; hyp, hypothalamus; co, cortex; pit, pituitary; ad, adrenal; thy,
thymus; sp, spleen; li, liver; lu, lung; w, wild type; Y, YGR
transgenic mice. (D) Western blot analysis of GR protein expression in
the hippocampus. wt, wild type.
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RT-PCR followed by RFLP analysis was used to demonstrate expression of
YAC-encoded GR mRNA in YGR mice. Based on this experiment
we conclude
that both YAC-derived and endogenous GR mRNA were
expressed at
equivalent levels in the hippocampus (Fig.
2B). Similar
results were
obtained in other organs and tissues known to contain
GR, demonstrating
faithful expression of GR from the YGR290 transgene
(Fig.
2C).
Since autoregulation of GR has been reported to occur at the level of
transcription (
11,
12,
19), we also determined
the total GR
mRNA expression in several organs from YGR mice.
Interestingly, the
expression level of GR did not reach the twofold
elevation predicted if
the four alleles of GR were fully transcribed
in transgenic mice. The
highest level of overexpression in YGR
mice was achieved in the brain
and the pituitary, in which the
GR mRNA was elevated by 60 and 43%,
respectively, whereas in the
spleen, thymus, and liver, GR was
overexpressed by 20 to 24% (data
not shown). This demonstrates that
mRNA expression of GR is subject
to autoregulatory
downmodulation.
Finally, we tested whether the increase in GR mRNA expression was
paralleled by higher receptor levels in transgenic mice.
We found that
YGR mice contained about 50% more GR protein in
the hippocampus than
the wild-type controls (Fig.
2D). This clearly
shows that the higher
copy number of the GR gene in YGR mice not
only leads to increased
transcription but also results in higher
expression of GR
protein.
Overexpression of GR causes multiple neuroendocrine changes and
results in a blunted response to restraint stress.
GR plays an
important role in the negative feedback control of the HPA axis
(8). This mechanism ensures proper regulation of serum
glucocorticoid levels as well as a quick return to homeostasis after
challenges such as stress. The two major targets for the repressive
effect of GR in the regulation of the HPA axis are CRH in the
hypothalamus and pro-opiomelanocortin (POMC) in the anterior pituitary.
As a measure of CRH synthesis by the hypothalamus, the content of
immunoreactive CRH in the median eminence was determined (14). YGR mice displayed a more-than-twofold reduction of
CRH immunoreactivity (Fig. 3A and B and
Table 1), suggesting that overexpression
of GR in the brain leads to increased repression of CRH production. In
addition, expression of POMC mRNA and its major peptide product, ACTH,
in the anterior pituitary was reduced almost threefold in YGR mice
(Fig. 3C to F and Table 1). This demonstrates that a higher GR level in
the pituitary, possibly combined with reduced stimulation by CRH, leads
to suppression of POMC and ACTH expression.
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FIG. 3.
Expression analysis of genes involved in HPA axis
regulation. (A) Immunohistochemistry of CRH in the median eminence of
WT mice; (B) immunohistochemistry of CRH in the median eminence of YGR
mice; (C) in situ hybridization of POMC in the anterior pituitary of WT
mice; (D) in situ hybridization of POMC in the anterior pituitary of
YGR mice; (E) immunohistochemistry of ACTH in the anterior pituitary of
WT mice; (F) immunohistochemistry of ACTH in the anterior pituitary of
YGR mice. 3rd, third ventricle; ME, median eminence; AL, anterior lobe
of the pituitary; NIL, intermediate lobe of the pituitary; PL,
posterior lobe of the pituitary.
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Next, we analyzed whether the observed changes in gene expression would
translate into altered corticosterone and ACTH levels
in serum.
Corticosterone levels in YGR mice were more than fourfold
lower than
those in wild-type (WT) controls, whereas ACTH levels
were elevated
almost threefold (Table
1). Thus, basal secretion
of the two hormones
is altered in opposite directions. In addition,
mRNA expression of the
mineralocorticoid receptor was also reduced
by 33% (
P < 0.05,
n = 5), indicating reciprocal regulation of
the two GRs
in the brain (
6).
To further study the divergent regulation of basal corticosterone and
ACTH secretion, an ACTH stimulation test was performed.
Injections of
exogenous ACTH (10 µg/kg) into WT mice led to a
level of circulating
corticosterone that was increased fourfold
(88 ± 27 ng/ml)
compared to the level in vehicle-injected controls
(23 ± 4 ng/ml). In YGR mice a fivefold increase in serum corticosterone
levels
was observed after ACTH injection (9 ± 2 ng/ml for
vehicle-injected
controls versus 46 ± 16 ng/ml for ACTH-injected
mice). Thus, following
appropriate stimulation, a similar increase in
glucocorticoid
secretion is achieved in WT and transgenic mice. This
clearly
indicates that YGR mice are able to mount a normal
glucocorticoid
response. Finally, histological analysis of the adrenal
gland
did not reveal any abnormalities, such as a hypotrophy of the
cortex (data not
shown).
Since a major function of GR in the control of the HPA axis is its
downregulation after challenges such as stress (
15,
31),
we
analyzed the response of YGR mice to acute restraint stress.
Immobilization for 40 min led to a strong elevation of serum
corticosterone
levels in mice of both genotypes (Fig.
4A); 20 min after the relief
of stress
the corticosterone levels were significantly decreased.
Despite the
qualitatively similar response in YGR mice, the elevation
of the
corticosterone levels during stress was significantly smaller
and the
serum corticosterone concentrations also declined faster,
demonstrating
a weaker stress response in YGR mice. In addition,
immobilization
stress caused a strong increase in ACTH secretion,
which returned to
moderate levels after 20 min (Fig.
4B). Interestingly,
at all time
points measured, ACTH levels were higher in YGR mice
than in WT mice.
Again, this confirms the divergence of ACTH and
corticosterone
secretion in YGR mice.
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FIG. 4.
Consequences of restraint stress on hormone secretion in
WT (wt) and YGR mice. (A) Serum corticosterone levels. Basal levels,
levels after 40 min of restraint stress, and levels 20 min after the
removal of the stressor are shown. (B) Serum ACTH levels at the same
time points as described for panel A. Statistical significance was
determined by the Student t test (n  5).
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Increased sensitivity of primary thymocytes to
glucocorticoid-induced apoptosis in YGR mice.
A well-studied
function of GR is the induction of apoptosis in response to
glucocorticoid exposure (1, 4, 21). This GR action is
thought to be involved in modulating positive and negative selection
during T-cell maturation in the thymus, thereby influencing the
responsiveness of the immune system to autoantigens (13,
30). Importantly, induction of thymocyte apoptosis was demonstrated to require DNA-binding-dependent gene activation by GR
(22).
To study the impact of increased GR expression on apoptosis, we
cultivated primary thymocytes for 9 h in the presence of the
GR
agonist dexamethasone, with concentrations ranging from
10
9 to 10
5 M (Fig.
5). Interestingly, WT cells started to
undergo apoptosis
at 3 · 10
8 M, whereas thymocytes
from YGR mice did so at 3 · 10
9 M. Furthermore,
maximal apoptosis in WT cells was reached at
around 3 · 10
7 M, compared to 3 · 10
8 M in YGR
cells. Finally, the maximal level of apoptosis achieved
in YGR cells
was higher than the one in controls (Fig.
5). Qualitatively
similar
results were obtained after 24 h of incubation (data not
shown).
Taken together, the data indicate that overexpression
of GR in
thymocytes results in a shift of the dose-response curve
to the right
and an increase of the maximal level of apoptosis.
Consequently, the
expression level of GR critically determines
the magnitude of
activation-dependent events such as thymocyte
apoptosis.
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FIG. 5.
Glucocorticoid-induced apoptosis of primary thymocytes
of WT (wt) and YGR mice. (A) Flow cytometric analysis of thymocytes
cultivated for 9 h in the presence of 108 M
dexamethasone (dex) and stained with PI. Analysis by gating the cells
in region 2 on the basis of their DNA content (FL2-A) and the
granulation (SSC) pattern is exemplified. (B) Dose-response curves of
dexamethasone-treated thymocytes from four individual mice, two WT and
two YGR, are depicted. Cells were cultivated for 9 h in the
absence (con) or presence of various concentrations of the GR agonist
dexamethasone or after treatment with the GR antagonist ZK112,339
(106 M). The degree of apoptosis was determined as
described in Materials and Methods and plotted against the
concentration of dexamethasone.
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Reduced inflammatory responses in YGR mice.
Glucocorticoids
are powerful immunosuppressive and antiinflammatory drugs which achieve
their effects by repression of cytokine synthesis and downregulation of
other immune functions via GR (2). A suitable model to study
this GR function in vivo is injection of lipopolysaccharides (LPS) into
mice. Whereas low doses of LPS are used to study regulation of immune
mediators such as cytokines (33), injection of high doses is
considered a good paradigm for the pathogenesis of endotoxic shock
(9).
Injection of WT mice with a comparatively low concentration of LPS (4 mg/kg) caused a strong increase of serum IL-6 levels,
which peaked at
around 3 h after injection and returned to basal
levels after
15 h (Fig.
6A). In YGR mice the same
kinetics were
observed, although with reduced intensity. At both 3 and
6 h after
injection, serum IL-6 levels in YGR mice were 40 to 50%
lower
than those observed in WT mice. Importantly, this difference
cannot
be attributed to differences in glucocorticoid secretion, since
corticosterone was strongly induced to a level of 700 to 800 ng/ml
in
both genotypes and similarly returned to moderate levels after
15 h (data not shown). This suggests that overexpression of GR
in cells
mediating systemic inflammation, notably macrophages
and neutrophils,
leads to an increased sensitivity of these cells
to repression by
glucocorticoids.
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FIG. 6.
LPS-induced inflammation and endotoxic shock in WT (wt)
and YGR mice. (A) LPS injection (4 mg/kg intraperitoneally) and
analysis of IL-6 levels in the serum at the given time points. The
difference at 3 h is highly significant. (B) Survival after
injection of a high dose of LPS (40 mg/kg intraperitoneally). The
percentage of surviving mice was determined at 24-h intervals (for WT
mice, n = 6; for YGR mice, n = 8).
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To study the possible impact of the altered cytokine response on
pathology, we analyzed the resistance of YGR mice to the
lethal effects
of endotoxic shock (
9). To this end, mice were
injected with
a high dose of LPS (40 mg/kg) and the percentage
of survivors was
determined (Fig.
6B). Within the first 24 h 67%
of the WT mice
but only 13% of the YGR mice died. After 4 days
all WT animals were
dead, whereas 75% of the YGR mice were still
alive, without any
obvious signs of illness. In contrast, mock
injections of vehicle did
not cause death in any case. We conclude
that overexpression of GR
leads to a strongly increased resistance
to endotoxic shock,
underscoring the importance of GR signaling
for protection against an
overshooting inflammatory
response.
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DISCUSSION |
The presence of GR is essential for survival at birth, and its
function is important for proper regulation of many physiological processes. This is known from human pathology (16), in vitro and in vivo experiments, and the analysis of genetically manipulated mice (5, 22, 31). However, in contrast to the large body of
data describing the physiological consequences of loss of GR function,
to our knowledge, analyses of the physiological impact of ubiquitous GR
overexpression have not yet been undertaken. We previously established
that position-independent and copy number-dependent expression of a
transgene can be achieved by the use of YACs (27). Consequently, to address the question of GR overexpression, we have
introduced two additional Gr alleles on YACs into the germ line of mice. As predicted, the YAC-derived GR is faithfully expressed in YGR mice in all expected tissues at levels equivalent to the endogenous GR. However, despite the presence of four functional Gr alleles, the absolute expression level in YGR mice varied
in different organs, suggesting tissue-specific autoregulatory
mechanisms (11, 12, 19).
Both prenatal and postnatal experiences are known to influence the
regulation of the HPA axis and the response to stress (15, 25). Prenatal immune challenges were shown to result in increased corticosterone secretion under basal as well as stress conditions, which was correlated with a reduction of GR expression in the hippocampus (25). Conversely, postnatal handling of mice
results in reduced glucocorticoid secretion during immobilization
stress, an effect which is associated with an increase in GR expression in the hippocampus (15). Taken together, these data suggest a relationship between HPA axis activity, stress responsiveness, and
central GR expression. This model is now strongly supported by our
findings in GR-overexpressing mice. YGR mice display a strongly reduced
expression of the main components of the HPA axis, a fourfold lower
secretion of corticosterone, and a significantly weaker response to
restraint stress. Thus, an altered expression of GR represents a
potential mechanism by which prenatal and postnatal experiences might
influence the regulation of the HPA axis and responsiveness to stress
in adults. Given the importance of a disturbed control of the HPA axis
for the development of psychiatric disorders, our data also point
towards a possible explanation for the influence of brain development
on the pathogenesis of diseases such as major depression.
Stimulation of the HPA axis usually results in parallel changes in
glucocorticoid and ACTH secretion. Consequently, immobilization stress
and ACTH injection led to a strong increase in both serum corticosterone and ACTH levels, irrespective of the presence of the
transgene. However, under basal conditions both hormones were altered
in opposite directions in YGR mice. This divergence of the regulation
of the two hormones might be explained by developmental influences
causing a new equilibrium of basal hormone secretion due to altered GR
expression. This interpretation is supported by the finding that the
components of the HPA axis are fully responsive to glucocorticoids
already by day 16.5 of fetal development (23). Furthermore,
stimulation of the adrenal gland by extrapituitary factors, including
neuronal input, e.g., via the splanchnic nerve, might also participate
in this phenomenon. Interestingly, a similar divergence has been
observed in nervous system-specific GR knockout mice (31)
and in patients suffering from major depression. Thus, our finding
appears to be of general significance.
Thymocyte apoptosis can be induced by glucocorticoid treatment via
GR-dependent gene activation. This function is thought to play an
important role in determining the T-cell repertoire by modulating
positive and negative selection of thymocytes during the maturation
process in the thymus (1, 21). Notably, mice expressing
reduced amounts of GR in the thymus show decreased apoptosis of
thymocytes following glucocorticoid treatment and a leftward shift of
the dose-response curve (13). Furthermore, mice deficient in
DNA-binding-dependent gene activation by GR are completely resistant to
thymocyte apoptosis (22). Taken together, these findings are
in line with the observation that higher GR expression in YGR mice
causes an increased sensitivity of thymocytes to glucocorticoid-induced
apoptosis. Since the shift of the dose-response curve to the right and
the overall elevated level of apoptosis after 9 h are just the
opposite of what was observed in mice with reduced GR expression
(13), the magnitude of activation-dependent processes
appears to be linked to the expression level of GR.
The strength of the inflammatory response is at least in part
determined by the HPA axis and the cellular sensitivity to
glucocorticoids. From animal studies as well as clinical observations
it is known that increased glucocorticoid signaling confers strong
resistance to the development of TH1-mediated diseases (7).
Fisher rats which have a hyperactive stress system are less prone to
develop experimental allergic encephalomyelitis, and women in the third trimester of pregnancy, who have increased cortisol levels, frequently experience remission of rheumatoid arthritis and multiple sclerosis (7). Taken together, this suggests that enhanced GR
signaling protects the organism from an overshooting immune response.
The finding that an increased gene dosage of GR enhances the resistance of mice to inflammation provides strong genetic support for this hypothesis. Significantly, YGR mice are less prone to the deleterious effects of endotoxin, which, in its most extreme form, leads to endotoxic shock. Whereas in WT mice, LPS injection results in a strong
increase in IL-6 secretion and at high concentrations causes death, YGR
mice secrete significantly less IL-6 during the inflammatory response
and are highly resistant to endotoxic shock. Thus, the ability of
glucocorticoids to repress expression and secretion of cytokines and
other factors involved in inflammation and the pathogenesis of
endotoxic shock depends on the level of GR and therefore on the
strength of glucocorticoid signaling.
Collectively, our results argue that higher expression of GR is likely
to be of significant adaptive value due to the increased resistance to
stress and inflammation. Given that prenatal and postnatal experiences
seem to influence the expression level of GR and that this in turn
alters physiological responses in the adult, individual differences in
humans with regard to their susceptibility to infections, inflammatory
diseases, and stress may, at least in part, be accounted for by
differences in GR levels.
| |
ACKNOWLEDGMENTS |
We thank Brenda Stride for careful reading of the manuscript and
Heike Glaser and Nadine Sold for expert technical assistance.
This work was supported by the Deutsche Forschungsgemeinschaft through
SFB 405; by the Fonds der Chemischen Industrie; by the European
Community, through the grants PL 96 0179 and Marie Curie
QLK2-CD-1999-51404; by the BMBF through the HGP grant 01 KW 9606/7; by
the Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren
(HGF); by the Alexander von Humboldt-Stiftung; by the
Volkswagen-Stiftung; and by Boehringer Ingelheim.
H.M.R. and T.U. contributed equally to this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Molecular Biology of the Cell I (A0200), German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Phone:
0049-6221-423411. Fax: 0049-6221-423470. E-mail:
g.schuetz{at}dkfz.de.
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Abstract
Introduction
