ß2-Adrenergic Receptors Potentiate Glucocorticoid Receptor Transactivation via G Protein ß
-Subunits and the Phosphoinositide 3-Kinase Pathway
Peer Schmidt,
Florian Holsboer and
Dietmar Spengler
Max Planck Institute of Psychiatry D-80804 Munich, Germany
 |
ABSTRACT
|
---|
Glucocorticoid hormones influence manifold
neuronal processes including learning, memory, and emotion via the
glucocorticoid receptor (GR). Catecholamines further modulate these
functions, although the underlying molecular mechanisms are poorly
understood. Here, we show that epinephrine and norepinephrine
potentiate ligand-dependent GR transactivation in a hippocampal cell
line (HT22) via ß2-adrenergic receptors. This
enhancement was strongest at low concentrations of glucocorticoids and
was accompanied by increased GR binding to a glucocorticoid-responsive
element (GRE). ß2-Adrenergic
receptor-mediated GR enhancement was relayed via G protein
ß
-subunits, insensitive to pertussis toxin and independent of
protein kinase A (PKA). In contrast, the catecholamine-evoked GR
enhancement was strongly reduced by wortmannin, suggesting a critical
role for phosphoinositide 3-kinase (PI3-K). In agreement, epinephrine
directly activated PI3-K in vivo. Similarly, stimulation of
tyrosine kinase receptors coupled to PI3-K activation, e.g.
receptors for insulin-like growth factor I (IGF-I) or fibroblast growth
factor (FGF), increased GR transactivation. Further analysis indicated
that G protein-coupled receptor (GPCR) and tyrosine kinase receptor
signals converge on PI3-K through separate mechanisms. Blockade of GR
enhancement by wortmannin was partially overcome by expression of the
downstream-acting protein kinase B (PKB/Akt). Collectively, our
findings demonstrate that GPCRs can regulate GR transactivation by
stimulating PI3-K. This novel cross-talk may provide new insights into
the molecular processes of learning and memory and the treatment of
stress-related disorders.
 |
INTRODUCTION
|
---|
The glucocorticoid receptor (GR), NR3C1, is widely expressed in
mammalian tissues and mediates the effects of glucocorticoids released
from the adrenal cortex upon stress. This release is regulated by the
hypothalamic-pituitary-adrenocortical (HPA) system, and the GR
critically controls HPA activity by maintaining negative feedback
control at several levels (1). High concentrations of GR are expressed
in the hippocampus, an important region of the limbic system modulating
HPA function, mood, learning, and memory (2).
The GR is a member of the steroid receptor family and is closely
related to the mineralocorticoid, progesterone, androgen, and estrogen
receptors. After glucocorticoid binding, the activated GR is released
from its cytoplasmatic protein complex and translocates into the
nucleus. There, it binds to glucocorticoid-responsive elements (GREs)
located in the regulatory region of target genes and modulates their
activity by interacting with the transcriptional machinery (3).
Depending on the nature and location of the GREs and, in addition, on
the presence of coregulators, the GR displays transactivation or
transrepression. Furthermore, cross-talk between the GR and other
transcription factors has been observed (3, 4).
Ligand-independent modulation of steroid receptor function in a cell
type-specific manner is well known for the estrogen, progesterone, and
androgen receptors with regard to growth factors, cyclins, cAMP, or
other signals. In contrast, a huge number of studies agreed that the GR
can be modulated by such stimuli only after ligand activation (for
review see Refs. 5, 6). However, the mechanisms by which these
signal transduction pathways modify GR function are less well
understood. An advanced insight into these processes appears mandatory
to improve our understanding of various physiological and
pathophysiological conditions related to GR function and
dysfunction.
A number of studies indicate that adrenergic and glucocorticoid
signaling is intimately interwoven in the brain (2, 7). In particular,
memory consolidation requires activation of both glucocorticoid- and
norepinephrine-dependent pathways, and both kinds of stress hormones
have been implicated in psychiatric disorders (8, 9). Despite this
increased understanding at the systemic level, no evidence for a direct
interaction of the two stress hormone pathways within neuronal cells
has been reported so far. Hence, we set out to examine whether, at the
cellular level, neuronal GR activity is modulated by catecholamines,
and if so, to identify the relevant pathway.
The catecholamines epinephrine and norepinephrine bind to G
protein-coupled receptors (GPCRs) in the cell membrane, leading to
dissociation of the G protein
and ß
subunits. In addition to
the well known signaling of GPCRs via G
, the interest in biological
effects mediated by Gß
has increased continuously during recent
years. In particular, ß
-subunits from inhibitory
Gi proteins have been demonstrated to interact
with specific isoforms of phosphoinositide-3 kinase (PI3-K) and to
stimulate the mitogen-activated protein kinase (MAPK) pathway (10, 11).
In this study, we report that (nor)epinephrine, acting via
ß2-adrenergic receptors, potentiates GR
transactivation in hippocampal cells. Our data reveal a novel
cross-talk between endogenous adrenergic receptors and GRs in neuronal
cells via a pathway depending on PI3-K.
 |
RESULTS
|
---|
Catecholamines Potentiate Neuronal GR Activity via
ß2-AR
The hippocampus regulates HPA axis activity and contains both
glucocorticoid and adrenergic receptors (2, 12). Therefore, a murine
hippocampus-derived cell line (HT22) was used as a cellular model to
investigate the interaction between glucocorticoid and catecholamine
signaling (13). HT22 cells were transiently transfected with a reporter
plasmid containing the glucocorticoid-responsive mouse mammary tumor
virus promoter upstream of the luciferase gene (MMTV-Luc). Treatment of
transfected hippocampal cells with increasing concentrations of
dexamethasone stimulated reporter gene expression in a dose-dependent
manner, indicating the presence of functional GRs (Fig. 1A
, open circles).
Interestingly, when transfected cells were costimulated with
dexamethasone and epinephrine, a marked increase in
glucocorticoid-induced reporter activity was observed while the
catecholamine alone was ineffective. A similar enhancement was obtained
with the natural ligand corticosterone, which displays lower affinity
for the GR (Fig. 1A
).

View larger version (28K):
[in this window]
[in a new window]
|
Figure 1. Epinephrine Potentiates Glucocorticoid Signaling
through a GRE
A, Epinephrine enhances dexamethasone and corticosterone
signaling. HT22 cells were transiently transfected with MMTV-Luc,
stimulated with increasing concentrations of dexamethasone (dex,
circles) or corticosterone (cort,
diamonds), and cotreated with vehicle (open symbols) or
epinephrine (30 µM, filled symbols). **,
P < 0.01 compared with glucocorticoid treatment
alone. B, Epinephrine reduces the ED50 of
glucocorticoids. The results from panel A of treatment with
dexamethasone are plotted as per cent of maximal induction of each
condition. The strongest relative enhancement of GR transactivation is
obtained at the lowest glucocorticoid concentrations
(inset). C, Catecholaminergic
potentiation of GR transactivation is mediated by
ß2-adrenergic receptors. HT22 cells were transiently
transfected with MMTV-Luc and stimulated with dexamethasone and
epinephrine or norepinephrine. Where indicated, the antagonists RU
38486 (GR-specific, 1 µM), propranolol (ß-AR-specific,
30 µM), or ICI 118551 (ß2-AR-specific, 100
µM) were included. Results are shown as per cent
induction of treatment with dexamethasone alone (dashed
line). D, GR enhancement by catecholamines
occurs through GREs. HT22 cells were transiently transfected with
TK-Luc containing either no (-) or two (+) GREs and stimulated with
dexamethasone (10 nM) and epinephrine (30 µM)
as indicated. E, Epinephrine costimulation does not
increase GR mRNA or protein levels. Cells were treated as indicated for
6 h before analysis of 20 µg RNA by Northern blot
(top) or 50 µg cell lysate by Western blot
(bottom). F, GR DNA binding is
significantly increased after treatment with epinephrine. A
representative image of an EMSA is shown in the left
panel. Cellular extracts from HT22 cells pretreated with
vehicle (lanes 1, 3, 4) or epinephrine (lane 2) were incubated with
dexamethasone (10 nM). Specific GR-DNA complexes
(arrow) disappeared in the presence of a 150-fold excess
of unlabeled competitor (cold GRE, lane 3) but not mutated GRE (cold
mtGRE, lane 4). In the right panel, EMSA results
representing the average signal intensity + SEM of the
specific GR-DNA complexes (n = 4, P < 0.05,
Students t test) are expressed as per cent of
dexamethasone treatment alone. Results in panels A and D are shown as
fold induction. Data from transient transfections represent the
mean + SEM of at least three independent experiments
performed in duplicate.
|
|
The increase in GR transactivation is illustrated in Fig. 1B
by
presenting the data as per cent of maximal induction in the presence or
absence of epinephrine. The left-shift caused by epinephrine reflects
an enhanced effectiveness of available glucocorticoids, reducing the
ED50 from 26 nM to 12 nM.
Remarkably, catecholamine regulation was strongest at low doses of
glucocorticoid, with a 5-fold increase of the induction by 3
nM dexamethasone alone (Fig. 1B
, inset). The
extent of potentiation gradually diminished at higher dexamethasone
concentrations, but epinephrine still doubled GR transactivation at 100
nM of glucocorticoids and beyond. Hence, in
neuronal cells epinephrine strengthens GR transactivation potency
predominantly at low doses of GR agonists.
Epinephrine concentrations as low as 30 nM were sufficient
for GR enhancement (data not shown). Moreover, treatment of HT22 cells
with glucocorticoids and norepinephrine instead of epinephrine caused a
similar enhancement of GR transactivation (Fig. 1C
). Potentiation could
be blocked by the GR antagonist RU 38486, demonstrating specificity of
the transcriptional response (Fig. 1C
). Furthermore, enhancement could
be prevented by addition of the ß-adrenergic antagonist propranolol
and also by ICI 118551, suggesting that GR enhancement was mediated by
ß2-adrenergic receptors (AR) (Fig. 1C
).
To examine whether enhanced GR transactivation occurred through the
GREs or, alternatively, through unrelated regulatory elements in the
MMTV promoter, we used a minimal thymidine kinase (TK) promoter as
control. Neither stimulation with dexamethasone nor epinephrine
increased reporter gene activity in HT22 cells. However, after
insertion of two GREs adjacent to the regulatory region, this promoter
displayed both glucocorticoid induction and potentiation by epinephrine
(Fig. 1D
). We conclude that the effect of epinephrine on enhanced GR
transactivation requires a GRE and is not confined to the MMTV promoter
context.
Enhanced GR function might be due to an increase in receptor
concentration under catecholamine treatment. However, we found no
significant change in GR transcript or protein levels after 6 h or
24 h of epinephrine treatment (Fig. 1E
). Likewise, the number of
ligand binding sites and the affinity constant were unaltered after
incubation of HT22 cells with epinephrine for 0, 4, 8, or 24
h (not shown). Therefore, higher transactivation occurred despite
unaltered GR binding parameters.
Binding to the GRE is an important step before induction of
glucocorticoid-responsive genes. In nontransfected HT22 cells,
electrophoretic mobility shift assay (EMSA) revealed that specific DNA
binding of the GR is significantly stronger in extracts of cells
stimulated with epinephrine than in those of untreated cells (Fig. 1F
, lanes 1 and 2; quantification depicted in right panel).
Specific GR-DNA complexes disappeared in the presence of an excess of
cold competitor GRE oligonucleotides (Fig. 1F
, lane 3). As a negative
control, the same excess of cold mutated GRE oligonucleotides did not
compete with complex formation (lane 4), demonstrating specificity of
the complexes. These results suggest that adrenergic receptor
activation potentiates GR function, at least in part, by improving DNA
binding.
Enhanced GR Transactivation by Epinephrine Is Mediated via G
Protein ß
-Subunits
ß2-ARs are G
s
protein-coupled receptors leading to activation of protein kinase A
(PKA) (14). To investigate the role of PKA in enhanced GR
transactivation, we treated transfected HT22 cells with both
dexamethasone, epinephrine, and specific inhibitors of PKA.
Cotransfection of an expression vector encoding a PKA inhibitor (PKI)
peptide or treatment with the inactive cAMP-analog Rp-cAMP did not
reduce enhanced GR transactivation, suggesting that GR potentiation is
not mediated by PKA in HT22 cells (Fig. 2A
). Of note, pretreatment with H-89
further increased GR function (Fig. 2A
), possibly due to effects of
H-89 unrelated to PKA (15).

View larger version (27K):
[in this window]
[in a new window]
|
Figure 2. Gß Subunits Confer GR Enhancement
A, Epinephrine-dependent GR enhancement does not require
a PKA-dependent switch from Gs to Gi coupling.
HT22 cells were transiently transfected with MMTV-Luc and PKI
expression plasmid or empty vector (10 ng each) and stimulated with
dexamethasone, epinephrine, and the PKIs Rp-cAMP (30 µM)
or H-89 (1 µM) as indicated. Inset,
Transfected cells were prestimulated for 90 min with epinephrine or
vehicle. Where indicated, blockade of Gi proteins with Ptx
(100 ng/ml) was performed before costimulation with dexamethasone.
B, G protein ß -subunits can replace epinephrine
stimulation. HT22 cells were cotransfected with MMTV-Luc, Gß (30 ng)
and G (left bar), or inactive G -
(right bar) (100 ng each) and stimulated with
dexamethasone. The long dashed line indicates GR
transactivation after costimulation with epinephrine. C,
GR enhancement by G s-coupled receptors. Ectopic
expression of the cDNAs of GPCRs for dopamine (D1-R, 1
µg), pituitary adenylate cyclase-activating peptide (PAC1-R, 100 ng),
or LH (LH-R, 100 ng) enabled potentiation of GR function by the cognate
ligands comparable to epinephrine via endogenous ß2-AR.
Cells were stimulated with dexamethasone and dopamine (6
µM), PACAP-27 (20 nM), or LH (100
pM). Data are shown as per cent of dexamethasone induction
(short dashed line) and represent the mean +
SEM of at least three independent experiments performed in
duplicate.
|
|
The preferential coupling of
ß2-AR to G
s has been
reported to switch to G
i as a consequence of
receptor phosphorylation by PKA (16). In light of the above results,
such a PKA-mediated switch from Gs to
Gi proteins appeared unlikely to contribute to
enhanced GR transactivation. To further exclude the involvement of
Gi proteins, we investigated the effect of
pertussis toxin (Ptx) on GR enhancement. HT22 cells preincubated with
Ptx showed no reduction of GR enhancement after costimulation with
dexamethasone and epinephrine (Fig. 2A
, inset, right bar).
As an additional control, cells prestimulated with epinephrine for up
to 90 min were treated with vehicle or Ptx before the addition of
dexamethasone. Blockade of Gi signaling by Ptx
after prestimulation with epinephrine did not reduce the GR response
either (Fig. 2A
, inset). Finally, Ptx alone did not modulate
reporter gene induction (not shown). Together, these findings
demonstrate that 1) blockade of the cAMP-PKA pathway does not prevent
enhanced GR transactivation by ß2-adrenergic
receptors; and 2) differential coupling of
ß2-AR to Gi proteins is
not required for potentiation of GR transactivation.
Within recent years, increasing attention has been paid to the cellular
actions of Gß
-subunits upon GPCR activation (17). Whereas the
biological functions of ß
-subunits released from inhibitory
G
i have been described in some detail,
e.g. activation of PI3-K and MAPK (for review see Ref. 18),
much less is known about the role of ß
signaling in the case of
receptors coupled to stimulatory G
s. In view
of our experiments, we reasoned that the stimulatory effect of
epinephrine on the GR may be relayed by ß
-subunits in response to
stimulation by ß2-ARs. To further test this
hypothesis, we asked whether catecholamine treatment could be replaced
by cotransfection of free Gß
-subunits. In fact, expression of Gß
and G
cDNAs could substitute for epinephrine stimulation (Fig. 2B
).
In contrast, coexpression of Gß in conjunction with a mutated
G
- that fails to associate with the membrane
(10) did not increase GR transactivation, indicating that
Gß
-subunits are critical for enhanced GR transactivation by
epinephrine (Fig. 2B
). Collectively, these data strongly suggest that
ß2-adrenergic signaling to the GR is transduced
via Gß
-subunits released from G
s.
To examine whether GR enhancement was restricted to
ß2-AR, we ectopically expressed other GPCRs in
HT22 cells. Costimulation with dexamethasone and dopamine, pituitary
adenylate cyclase- activating peptide (PACAP), or LH did not
influence GR transactivation in HT22 cells (not shown). In contrast,
these ligands strongly enhanced GR activity when their cognate
receptors were cotransfected, indicating that the observed effect on
the GR is not specific to ß2-ARs but also
extends to other GPCRs coupled to G
s (Fig. 2C
).
PI3-K Relays Signaling to the GR
Several recent studies reported a cross-talk between
Gß
-subunits released from G
i and the
PI3-K cascade (11, 19). We asked whether PI3-K might also play a
role in the G
s-mediated effect of epinephrine
on GR transactivation. To test this hypothesis, we treated
cotransfected HT22 cells with wortmannin, a potent and specific
inhibitor of PI3-K (20). In fact, wortmannin decreased the enhancement
of GR transactivation by epinephrine in a dose-dependent manner (Fig. 3A
) while it was ineffective alone.
Importantly, this reduction was also obtained with LY 294002, a
structurally unrelated inhibitor of PI3-K, demonstrating specificity of
the blockade (Fig. 3A
). Inhibition by wortmannin was maximal at 100
nM, preventing approximately 80% of the
epinephrine-mediated GR potentiation. This suggests a major role of
PI3-K in GR enhancement.

View larger version (29K):
[in this window]
[in a new window]
|
Figure 3. PI3-K Is Necessary and Sufficient for Enhanced GR
Transactivation by Epinephrine and Growth Factors
A, Catecholamine-mediated GR enhancement depends on PI3-K. Transfected
cells were treated with dexamethasone, epinephrine, and increasing
concentrations of wortmannin or LY 294002 (10 µM),
respectively. Data are shown as per cent of dexamethasone induction. *,
P < 0.05; ***, P < 0.001.
B, Activation of the PI3-K pathway is sufficient to
enhance GR transactivation. Transfected cells were plated in serum-free
medium and treated with 30 ng/ml IGF-I or bFGF and other substances as
indicated. Note that the effects of epinephrine and either growth
factor on steroid receptor function are additive. Furthermore, cells
transfected with the constitutively active catalytic subunit p110* (1
µg) were plated in serum-free medium and stimulated as indicated.
Data are shown as per cent of dexamethasone induction (short
dashed line). The long dashed line indicates GR
transactivation after costimulation with epinephrine. *,
P < 0.05; **, P < 0.01; ***,
P < 0.001 compared with dexamethasone alone; ,
P < 0.05 compared with IGF-I + dexamethasone.
C, Epinephrine does not require phosphotyrosine binding
of p85. Cells were cotransfected with wild-type p85, the dominant
negative form p85, or empty vector (30 ng each) and costimulated
with dexamethasone and IGF-I or epinephrine under serum-free
conditions. Results are shown as per cent induction compared with
costimulation in the presence of empty vector. Data represent the
mean + SEM of at least three independent experiments
performed in duplicate.
|
|
Since PI3-K acts downstream of ß2-ARs in HT22
cells, we reasoned that physiological stimuli of the phosphoinositide
cascade, e.g. growth factors, might similarly modulate GR
function. To this end, we treated transfected, serum-deprived HT22
cells with insulin-like growth factor 1 (IGF-I) or with basic
fibroblast growth factor (bFGF). Similar to epinephrine, IGF-I and bFGF
alone showed no induction of the GRE-containing promoter. In contrast,
administration of either polypeptide significantly increased GR
transactivation by dexamethasone, which could be blocked by wortmannin
(Fig. 3B
). Remarkably, cotreatment with both the catecholamine and
either growth factor further enhanced the GR response in an additive
manner, indicating that both signals were transduced through separate
pathways (Fig. 3B
).
The family of PI3-Ks comprises various isoforms, and those belonging to
class I are composed of a heterodimer consisting of a regulatory and a
catalytic subunit. Different isoforms of each subunit have been
isolated, and their patterns of expression and modes of activation show
some variation (21). The crucial role of PI3-K was further underlined
by results obtained from cotransfecting a constitutively active
catalytic subunit (p110*) of PI3-K into HT22 cells. In agreement with
above data, expression of this kinase enhanced ligand-dependent GR
transactivation by 2-fold in the absence of both IGF-I and bFGF (Fig. 3B
). Consequently, PI3-K activation is necessary and sufficient for
mediating growth factor-dependent GR potentiation and, additionally,
represents a major pathway for catecholamine-mediated enhancement of GR
transactivation in HT22 cells.
One of the PI3-K-regulatory subunits, p85, binds to phosphotyrosine
residues, e.g. on receptor tyrosine kinases (RTK) upon
growth factor stimulation, and recruits the catalytic subunit p110 to
the membrane (21). To investigate whether phosphotyrosine binding of
p85 was also required for ß
-induced signaling, we cotransfected
HT22 cells with a dominant negative form of p85 (
p85) lacking the
binding site for p110.
p85 can still bind to phosphotyrosine
residues, but it can no longer recruit p110. In agreement with this
prediction, the IGF-I-induced GR potentiation could be nearly
completely blocked by cotransfection of
p85 but not wild-type p85
(Fig. 3C
). In contrast, neither
p85 nor wild-type p85 significantly
affected the enhancement conferred by epinephrine (Fig. 3C
). We
conclude that epinephrine-induced ß
signaling does not depend on
phosphotyrosine binding of the PI3-K-regulatory subunit p85 but appears
to lead to direct activation of p110. This confirms the notion that
growth factor and ß
signaling occur via distinct mechanisms
leading to stimulation of PI3-K.
Epinephrine Activates the PI3-K Cascade
To further confirm that epinephrine leads to activation of PI3-K
in vivo, we treated nontransfected HT22 cells with vehicle
or epinephrine under serum-free conditions and measured the activity of
PI3-K immunoprecipitated by anti-p85-Ab. As shown in Fig. 4A
, incubation with epinephrine
stimulated the catalytic activity of PI3-K by 2.1-fold. As a control,
PI3-K activity was also enhanced by 2-fold after incubation with IGF-I
(not shown). These results corroborate our transfection studies and
clearly demonstrate a cross-talk between adrenergic receptors and PI3-K
in neuronal cells.

View larger version (14K):
[in this window]
[in a new window]
|
Figure 4. Epinephrine Activates PI3-K and PKB Confers GR
Enhancement
A, Epinephrine activates PI3-K. HT22 cells were stimulated with vehicle
or epinephrine in vivo and PI3-K activity was measured
in vitro. Products were separated by TLC and
autoradiographed. Enhanced PI3-K activity is evidenced by, on average,
2.1-fold increased formation of radioactive
phosphatidylinositol-3-phosphate (PtdIns-3-P; n = 3). A
representative autoradiograph is shown. B, PKB enhances
GR transactivation. Cells cotransfected with empty vector or a
constitutively active form of PKB (PKB*) (10 ng each) were plated in
serum-free medium and stimulated with dexamethasone. C,
PKB can partly restore GR enhancement under wortmannin treatment. HT22
cells cotransfected with PKB*, SGK, p70S6K, or empty vector
(10 ng each) were stimulated as indicated in serum-free medium. **,
P < 0.01 compared with induction after wortmannin
blockade. Data in panels B and C are shown as per cent of dexamethasone
induction and represent the mean + SEM of at least
three independent experiments performed in duplicate.
|
|
PI3-K may stimulate several downstream kinases including MAPK, protein
kinase C (PKC), serum and glucocorticoid-inducible kinase (SGK),
p70S6K, and protein kinase B (PKB/Akt) (22). To
identify the PI3-K-dependent effectors responsible for enhanced GR
transactivation, we examined each of these pathways individually.
Activation of PKC by phorbol esters did not increase GR
transactivation. Similarly, inhibition of MAPK kinase or cotransfection
of dominant negative forms of SGK or p70S6K
failed to decrease epinephrine-induced GR potentiation, arguing against
a role of these kinases in GR enhancement (data not shown). In
contrast, cotransfection of a constitutively active form of PKB (PKB*)
strongly increased dexamethasone-induced GR transactivation under
serum-free conditions (Fig. 4B
). Next, we tested whether expression of
PKB* could overcome the impaired epinephrine-mediated GR potentiation
under wortmannin treatment. In fact, cotransfection of PKB* reversed
nearly half of the wortmannin-mediated inhibition of GR enhancement,
while SGK and p70S6K were ineffective (Fig. 4C
).
These findings strongly suggest a role for PKB in epinephrine signaling
to the GR.
 |
DISCUSSION
|
---|
In this study, we employed a neuronal, hippocampus-derived cell
line that expresses adrenergic receptors and GRs to examine the
interaction of both classes of stress hormones at the cellular level
and to elucidate the underlying signaling pathways. We observed that
epinephrine enhances ligand-dependent GR transactivation and raises the
efficiency of available glucocorticoid hormone levels. This increase in
GR activity correlated with improved DNA binding, while receptor levels
and ligand binding affinity remained constant. There was no indication
of a switch in coupling from Gs to
Gi proteins by ß2-ARs
under prolonged epinephrine treatment. Moreover,
ß2-AR activation of the GR was independent of
PKA but, instead, was relayed by Gß
-subunits.
Interestingly, we further identified the phosphoinositide-3-phosphate
signal transduction cascade as a major determinant downstream of
ß2-ARs in HT22 cells. Our findings demonstrate
that activation of PI3-K is an important step in GR enhancement. First,
wortmannin, as well as the structurally unrelated PI3-K inhibitor LY
294002, blocked increased GR transactivation by epinephrine. Second,
expression of PI3-Ks constitutively active catalytic subunit p110*
augmented GR activity. Third, epinephrine directly stimulated PI3-K
activity in neuronal cells. Our data point to a new mode of cross-talk
between G
s-coupled receptors and PI3-K via
Gß
-subunits in a neuronal cell line. In this view, our findings
further exemplify the complexity of cross-talk between GPCRs and
downstream kinases (18). Inhibition of GR potentiation by wortmannin
could be partially overcome by PKB, which acts downstream of PI3-K.
Hence, ß2-adrenergic stimulation of GR in HT22
cells is predominantly transferred by a pathway under the control of
PI3-K (Fig. 5
). In agreement with these
findings, growth factor-mediated GR potentiation in HT22 cells
could be completely blocked by wortmannin, again establishing the
crucial role of PI3-K.

View larger version (19K):
[in this window]
[in a new window]
|
Figure 5. Model of a New Cross-Talk between
ß2-AR and the GR via PI3-K
Glucocorticoids induce a conformational change of the GR and its
translocation to the nucleus. There, GR dimers can bind to GREs and
stimulate gene transcription. (Nor)epinephrine, in turn, activates G
protein-coupled ß2-adrenergic receptors. Subsequently,
catecholamines enhance GR transactivation by Gß signaling to PI3-K
and its downstream target PKB. ß2-AR,
ß2-adrenergic receptor; G s, ß, ,
GTP-binding protein subunits; GR, glucocorticoid receptor; GRE,
glucocorticoid responsive element; PI3-K, phosphoinositide 3-kinase;
PKB, protein kinase B (Akt).
|
|
ß2-AR-dependent signals are transduced mainly,
although not exclusively, through PI3-K. Approximately 20% of the
epinephrine-mediated enhancement was resistant to wortmannin, and this
is mediated by a mechanism distinct from Gß
and PI3-K (P. Schmidt,
F. Holsboer, and D. Spengler, unpublished observation). Elucidation of
this additional pathway for GR enhancement will require further
study.
Activation by G protein ß
-subunits has been
shown for PI3-K
in vivo and for PI3-Kß in
vitro (11, 23). However, only PI3-Kß is widely expressed in
various tissues (21). Our results on PI3-K activation by epinephrine
in vivo strongly suggest a role for PI3-Kß in HT22 cells,
since PI3-K
does not immunoprecipitate with the regulatory subunit
p85 (21). The additional increase in GR transactivation after
costimulation with growth factors may be mediated by another PI3-K
isoform recruited to tyrosine-phosphorylated residues, i.e.
or
. If PI3-Kß mediated both signals, a synergistic rather
than additive effect would be expected (23, 24). In agreement with the
observation that the regulatory subunit p85 is required for growth
factor signaling but does not interfere with Gß
signaling (24),
our results using a dominant negative form of p85 support the
possibility of two different mechanisms leading to PI3-K activation. In
particular, they demonstrate that epinephrine signaling does not
proceed via tyrosine phosphorylation of growth factor receptors in HT22
cells (25).
PI3-K can activate several downstream targets including PKB, PKC, MAPK,
SGK, and p70S6K (22). We found that, with respect
to these PI3-K targets, only PKB stimulated GR function in HT22 cells.
Furthermore, PKB can, in part, restore the wortmannin-inhibited effect
of catecholamines on GR function, suggesting an involvement of this
kinase in GR enhancement. PKB has been reported to increase GR function
in PC12 cells by antagonizing the inhibitory action of glycogen
synthase kinase-3 (GSK-3) (26). In HT22 cells, however, coexpression of
GSK-3 did not inhibit enhanced GR transactivation by epinephrine.
Likewise, expression of a dominant negative form of GSK-3 did not
improve GR function (P. Schmidt, F. Holsboer, and D.
Spengler, unpublished observations). Therefore, our results gave no
evidence for an involvement of GSK-3 in GR transactivation in HT22
cells, but they strongly support a critical role of the PI3-K target
PKB in relaying adrenergic signaling to the GR.
Consistent with a number of previous studies, epinephrine was unable to
activate the GR in the absence of glucocorticoids. In this respect, the
GR seems to behave differently from most other steroid hormone
receptors (5, 6). The strict ligand dependency of the
epinephrine-induced GR potentiation in our model system distinguishes
this regulation from a recent report on ligand-independent activation
of the GR by ß2-adrenergic agonists in
fibroblasts (27).
Interestingly, the highest enhancement by epinephrine was achieved at
low concentrations of glucocorticoids and led to a 5-fold increase of
GR transactivation compared with dexamethasone alone. Hence, the
strongest potentiation of GR function occurred at glucocorticoid
concentrations comparable to in vivo baseline conditions.
Furthermore, we detected an equivalent enhancement of GR
transactivation by epinephrine in the presence of the natural ligand
corticosterone. The fact that the ED50 values
observed were somewhat higher than expected from studies based on
receptor overexpression may be due to the limited, but physiological,
levels of GR expression in HT22 cells. Moreover, in this cell line the
effect of a given concentration of glucocorticoids could be nearly
doubled by blocking the membrane P-glycoprotein, which extrudes several
lipophilic molecules from the cytoplasm, with verapamil (28). This was
the case for both cortisol and dexamethasone (28), and we could
reproduce this finding for corticosterone (data not shown).
The observed cross-talk between catecholamines and steroids might play
a role under various physiological conditions. For instance, enhanced
recruitment of the GR under low glucocorticoid levels may facilitate
glucocorticoid-dependent memory storage (8). Furthermore, our findings
might represent a molecular mechanism for fine tuning the neuronal
stress response by (nor)epinephrine (2). Notably, epinephrine reduced
the dose of glucocorticoids necessary for half-maximal GR activity by
more than 50%, which might also be of relevance for stress-related
disorders. Long-term treatment with antidepressants or lithium has been
shown to influence GPCR and PI3-K signaling at various levels and might
help to restore negative feedback regulation by glucocorticoids in
depressed patients (9, 29).
The fact that both catecholamines and polypeptide growth factors
enhance GR transactivation seems to indicate redundancy in this
process. However, the complexity of these signaling systems suggests
that distinct biological responses are achieved based on
context-dependent activity profiles and cell type-specific expression
patterns. In addition, epinephrine and IGF-I probably act via different
isoforms of PI3-K, which may ensure selectivity. Thus, despite
overlapping effects on GR transactivation, either stimulus may modulate
glucocorticoid signaling in the nervous system in a specific and
context-dependent manner. The interaction between growth factors and
glucocorticoids may, for instance, play a role in neurodevelopment and
neuronal plasticity of the hippocampus (30). Moreover, additional
physiological functions in tissues other than the hippocampus might be
suggested by our finding of GR enhancement by different
G
s- coupled receptors.
In summary, this study reveals that adrenergic stress hormones enhance
glucocorticoid activity in hippocampus-derived cells via the
phosphoinositide pathway. Our findings demonstrate a novel cross-talk
between GPCRs, PI3-K, and the GR and suggest a critical role for PKB in
regulating steroid receptor function. These results imply a model of GR
activity in which constant levels of steroid receptor and ligand may
bring forth a broad range of activity in neuronal cells. This notion of
GPCR-dependent regulation of GR function appears significant in the
light of both physiological and pathophysiological processes.
 |
MATERIALS AND METHODS
|
---|
Plasmids
cDNAs for p85 and
p85 (31) have been subcloned as
BamHI-EcoRI fragments into the vector pRK7
harboring a cytomegalovirus (CMV) promoter (32). Construction of PKI
(33), Gß
(34), G
- (10), constitutively
active p110* (35), SGK (36, 37), p70S6K (38), PKB
(39), constitutively active PKB* (40), and GSK-3 constructs (41) has
been described.
Cell Culture and Transient Transfections
HT22 cells were grown in DMEM supplemented with 10% FCS
(Life Technologies, Inc., Karlsruhe, Germany). Cells
(3 x 106) were transfected with reporter
plasmid [1 µg MMTV-Luc, TK-Luc, or TK-GRE2-Luc
(42)] and, where indicated, with expression plasmids using an
electroporation system (BTX, Inc., San Diego, CA). The
ß-galactosidase expression vector pRK7-Gal [0.1 µg (32)] and
carrier DNA (pGEM4, Promega Corp., Madison, WI) up to a
final amount of 6 µg were included in each experiment.
Electroporated cells were replated in DMEM supplemented with 10%
charcoal-stripped, steroid-free FCS, except where indicated, and
incubated with substances as described in the figure legends. Unless
indicated otherwise, concentrations of 10 nM dexamethasone
and 30 µM epinephrine were used. After 24 h, cell
extracts were assayed for luciferase activity, and results were
normalized by ß-galactosidase activity as described previously (42).
ICI 118551 and PD 98059 were purchased from Tocris (Ballwin, MO)
and IGF-I and bFGF from R&D Systems (Wiesbaden, Germany). Wortmannin,
LH, and PACAP-27 were obtained from Calbiochem
(Schwalbach, Germany). All other substances were from
Sigma (Deisenhofen, Germany). Statistical evaluation was
performed using Students t test.
Northern Blot Analysis
Cells were treated with dexamethasone (300 nM),
epinephrine (30 µM), or both and total RNA was harvested
using Trizol Reagent (Life Technologies, Inc.). Blotting
was performed with 20 µg RNA by capillary transfer. As a probe, a
fragment of the hGR cDNA was used (42). Signals were quantified using a
digital image analysis system (BAS reader, Fuji Photo Film Co., Ltd.).
Western Blot Analysis
Total cellular lysates (50 µg) from cells pretreated with
vehicle or dexamethasone (10 nM) and epinephrine (30
µM) were subjected to SDS-PAGE and blotted as described
(43). Detection of GR was performed with polyclonal rabbit GR primary
antibody (PA1512, Affinity BioReagents, Inc. Golden, CO)
and a secondary antibody coupled to horseradish peroxidase
(Amersham Pharmacia Biotech, Freiburg, Germany) using
enhanced chemiluminescence (Roche Molecular Biochemicals, Mannheim, Germany).
Electrophoretic Mobility Shift Assay
Annealed GRE-oligonucleotides
(5'-GGAGCTTAGAACACAGTGTTCTCTAGAG-3' and
5'-GGAGTCCTCTAGAGAACACTGTGTTCTA-3') were labeled with
32P-
dCTP (Amersham Pharmacia Biotech) using Klenow fragment (New England Biolabs, Inc., Schwalbach, Germany). HT22 cells were homogenized in
ice-cold buffer containing 20 mM Tris-HCl pH 7.5, 600
mM KCl, 20% glycerol, and 2 mM dithiothreitol
(DTT) and centrifuged for 45 min (100,000x g, 4 C). Samples
(20 µl) contained 10 mM HEPES, pH 7.9, 4%
Ficoll, 1 mM DTT, 1 µg poly[dIdC], and 1 µl
cell extract (10 µg protein). A 150-fold excess of unlabeled
competitor GRE or mutated oligonucleotide was included in parallel
samples to confirm specificity of the GR-DNA complex. After 10 min at 0
C, 0.1 ng 32P-labeled GRE was added and
incubation was continued for 15 min at 25 C. DNA-protein complexes were
resolved on a 4% polyacrylamide gel in 0.5x Tris-borate-EDTA
(TBE), and autoradiographs were quantified with a digital image
analysis system.
PI3-K Assay
HT22 cells grown to 70% confluence were treated for 1 min with
epinephrine or vehicle before lysis in the presence of sodium
orthovanadate. Precleared lysates were immunoprecipitated with
anti-PI3K-p85 (No. 06195, Upstate Biotechnology, Inc.,
Lake Placid, NY) according to the manufacturers instructions.
Immunoprecipitates were resuspended three times in washing buffer (500
mM LiCl, 100 mM Tris-HCl, pH 7.4, 2
mM sodium orthovanadate) and twice in equilibration buffer
(100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1
mM sodium orthovanadate). Equal concentrations of PI3-K as
determined by immunoblotting were used for PI3-K assays. Reactions were
started by adding 12.5 mM MgCl2 and 5
µM ATP in the presence of 10 µg phosphatidylinositol
and 830 nM 32P-
dATP (3,000
Ci/mmol, Amersham Pharmacia Biotech). Incubation was
stopped after 20 min with 1 M HCl and samples were
extracted using chloroform-methanol (1:1). Products were separated by
TLC (silica gel 60, Merck Eurolab, Ismaning, Germany) in
chloroform-methanol-H2O- ammonium hydroxide
(43:38:7:5) and dried plates were autoradiographed.
 |
ACKNOWLEDGMENTS
|
---|
The authors are grateful to M. Greenberg, S. Gutkind, B.
Hemmings, M. Kasuga, P. Klein, A. Klippel, F. Lang, M. Lohse, R.
Maurer, R. Roth, and G. Thomas for the gift of expression plasmids and
to C. Behl for the HT22 cell line. We thank S. Heck, T. Rein, J. Reul,
and T. Trapp for valuable discussions and A. Hoffmann for excellent
technical assistance. We are indebted to J. Reul and A. Gesing for the
receptor binding data.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Florian Holsboer, Ph.D., M.D., Max Planck Institute of Psychiatry, Kraepelinstrasse 10, D-80804 Munich, Germany. E-mail: holsboer{at}mpipsykl mpg.de.
Received for publication August 14, 2000.
Revision received December 1, 2000.
Accepted for publication December 5, 2000.
 |
REFERENCES
|
---|
-
de Kloet ER, Vreugdenhil E, Oitzl MS, Joëls M 1998 Brain corticosteroid receptor balance in health and disease. Endocr Rev 19:269301[Abstract/Free Full Text]
-
de Kloet ER 1991 Brain corticosteroid receptor balance and
homeostatic control. Front Neuroendocrinol 12:95164
-
Beato M, Herrlich P, Schütz G 1995 Steroid hormone
receptors: many actors in search of a plot. Cell 83:851857[Medline]
-
McKenna NJ, Lanz RB, OMalley BW 1999 Nuclear receptor
coregulators: cellular and molecular biology. Endocr Rev 20:321344[Abstract/Free Full Text]
-
Weigel NL, Zhang Y 1998 Ligand-independent activation of
steroid hormone receptors. J Mol Med 76:469479[CrossRef][Medline]
-
Cenni B, Picard D 1999 Ligand-independent activation of
steroid receptors: new roles for old players. Trends Endocrinol Metabol 10:4146[CrossRef][Medline]
-
McEwen BS, Sapolsky RM 1995 Stress and cognitive function.
Curr Opin Neurobiol 5:205216[CrossRef][Medline]
-
Roozendaal B, Nguyen BT, Power AE, McGaugh JL 1999 Basolateral amygdala noradrenergic influence enables enhancement of
memory consolidation induced by hippocampal glucocorticoid receptor
activation. Proc Natl Acad Sci USA 96:1164211647[Abstract/Free Full Text]
-
Holsboer F, Barden N 1996 Antidepressants and
hypothalamic-pituitary-adrenocortical regulation. Endocr Rev 17:187205[Medline]
-
Crespo P, Xu N, Simonds WF, Gutkind JS 1994 Ras-dependent
activation of MAP kinase pathway mediated by G-protein ß
subunits. Nature 369:418420[CrossRef][Medline]
-
Lopez-Ilasaca M, Crespo P, Pellici PG, Gutkind JS, Wetzker R 1997 Linkage of G protein-coupled receptors to the MAPK signaling
pathway through PI3-Kinase
. Science 275:394397[Abstract/Free Full Text]
-
Asanuma M, Ogawa N, Mizukawa K, Haba K, Hirata H, Mori A 1991 Distribution of the ß2-adrenergic receptor
messenger RNA in the rat brain by in situ hybridization
histochemistry: effects of chronic reserpine treatment. Neurochem Res 16:12531256[Medline]
-
Davis JB, Maher P 1994 Protein kinase C activation inhibits
glutamate-induced cytotoxicity in a neuronal cell line. Brain Res 652:169173[CrossRef][Medline]
-
Lefkowitz RJ, Caron MG 1988 Adrenergic receptors - models for
the study of receptors coupled to guanine-nucleotide regulatory
proteins. J Biol Chem 263:49934996[Free Full Text]
-
Shoshan MC, Ljungdahl S, Linder S 1996 H-89 inhibits
collagenase induction by phorbol ester through a mechanism that does
not involve protein kinase A. Cell Signal 8:191195[CrossRef][Medline]
-
Daaka Y, Luttrell LM, Lefkowitz RJ 1997 Switching of the
coupling of the ß2-adrenergic receptor to
different G proteins by protein kinase A. Nature 390:8891[CrossRef][Medline]
-
Clapham DE, Neer EJ 1997 G protein ß
subunits. Annu
Rev Pharmacol Toxicol 37:167203[CrossRef][Medline]
-
Luttrell LM, Daaka Y, Lefkowitz RJ 1999 Regulation of tyrosine
kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol 11:177183[CrossRef][Medline]
-
Murga C, Laguinge L, Wetzker R, Cuadrado A, Gutkind JS 1998 Activation of Akt/protein kinase B by G protein-coupled receptors. A
role for
and ß
subunits of heterotrimeric G proteins
acting through phosphatidylinositol-3-OH kinase
. J Biol Chem 273:1908019085[Abstract/Free Full Text]
-
Ui M, Okada T, Hazeki K, Hazeki O 1995 Wortmannin as a unique
probe for an intracellular signalling protein, phosphoinositide
3-kinase. Trends Biochem Sci 20:303307[CrossRef][Medline]
-
Vanhaesebroeck B, Waterfield MD 1999 Signaling by distinct
classes of phosphoinositide 3-kinases. Exp Cell Res 253:239254[CrossRef][Medline]
-
Chan TO, Rittenhouse SE, Tsichlis PN 1999 AKT/PKB and other D3
phosphoinositide-regulated kinases: kinase activation by
phosphoinositide-dependent phosphorylation. Annu Rev Biochem 68:9651014[CrossRef][Medline]
-
Kurosu H, Maehama T, Okada T, Yamamoto T, Hoshino S, Fukui Y,
Ui M, Hazeki O, Katada T 1997 Heterodimeric phosphoinositide 3-kinase
consisting of p85 and p110ß is synergistically activated by the
ß
subunits of G proteins and phosphotyrosyl peptide. J Biol
Chem 272:2425224256[Abstract/Free Full Text]
-
Maier U, Babich A, Nürnberg B 1999 Roles of non-
catalytic subunits in Gß
-induced activation of class I
phosphoinositide 3-kinase isoforms ß and
. J Biol Chem 274:2931129317[Abstract/Free Full Text]
-
Maudsley S, Pierce KL, Zamah AM, Miller WE, Ahn S, Daaka Y,
Lefkowitz RJ, Luttrell LM 2000 The
ß2-adrenergic receptor mediates extracellular
signal-regulated kinase activation via assembly of a multi-receptor
complex with the epidermal growth factor receptor. J Biol Chem 275:95729580[Abstract/Free Full Text]
-
Rogatsky I, Waase CLM, Garabedian MJ 1998 Phosphorylation and
inhibition of rat glucocorticoid receptor transcriptional activation by
glycogen synthase kinase-3 (GSK-3). Species-specific differences
between human and rat glucocorticoid receptor signaling as revealed
through GSK-3 phosphorylation. J Biol Chem 273:1431514321[Abstract/Free Full Text]
-
Eickelberg O, Roth M, Lörx R, Bruce V, Rüdiger J,
Johnson M, Block L-H 1999 Ligand-independent activation of the
glucocorticoid receptor by ß2-adrenergic
receptor agonists in primary human lung fibroblasts and vascular smooth
muscle cells. J Biol Chem 274:10051010[Abstract/Free Full Text]
-
Herr AS, Wochnik GM, Rosenhagen MC, Holsboer F, Rein T 2000 Rifampicin is not an activator of glucocorticoid receptor. Mol
Pharmacol 57:732737[Abstract/Free Full Text]
-
Chalecka-Franaszek E, Chuang D-M 1999 Lithium activates the
serine/threonine kinase Akt-1 and suppresses glutamate-induced
inhibition of Akt-1 activity in neurons. Proc Natl Acad Sci USA 96:87458750[Abstract/Free Full Text]
-
MacLennan KM, Smith PF, Darlington CL 1998 Adrenalectomy-induced neuronal degeneration. Prog Neurobiol 54:481498[CrossRef][Medline]
-
Kotani K, Yonezawa K, Hara K, Ueda H, Kitamura Y, Sakaue H,
Ando A, Chavanieu A, Calas B, Grigorescu F, Nishiyama M, Waterfield MD,
Kasuga M 1994 Involvement of phosphoinositide 3-kinase in
insulin-induced or IGF-1-induced membrane ruffling. EMBO J 13:23132321[Abstract]
-
Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J,
Seeburg PH, Journot L 1993 Differential signal transduction by five
splice variants of the PACAP receptor. Nature 365:170175[CrossRef][Medline]
-
Day RN, Walder JA, Maurer RA 1989 A protein kinase inhibitor
gene reduces both basal and multihormone-stimulated prolactin gene
transcription. J Biol Chem 264:431436[Abstract/Free Full Text]
-
Quitterer U, Lohse MJ 1999 Crosstalk between
G
i- and
G
q-coupled receptors is mediated by Gß
exchange. Proc Natl Acad Sci USA 96:1062610631[Abstract/Free Full Text]
-
Klippel A, Escobedo M-A, Wachowicz MS, Apell G, Brown TW,
Giedlin MA, Kavanaugh WM, Williams LT 1998 Activation of
phosphatidylinositol 3-kinase is sufficient for cell cycle entry and
promotes cellular changes characteristic of oncogenic transformation.
Mol Cell Biol 18:56995711[Abstract/Free Full Text]
-
Park J, Leong MLL, Buse P, Maiyar AC, Firestone GL, Hemmings
BA 1999 Serum and glucocorticoid-inducible kinase (SGK) is a target of
the PI3-kinase-stimulated signaling pathway. EMBO J 18:30243033[Abstract/Free Full Text]
-
Waldegger S, Barth P, Raber G, Lang F 1997 Cloning and
characterization of a putative human serine/threonine protein kinase
transcriptionally modified during anisotonic and isotonic
alterations of cell volume. Proc Natl Acad Sci USA 94:44404445[Abstract/Free Full Text]
-
Jefferies HBJ, Fumagalli S, Dennis PB, Reinhard C, Pearson RB,
Thomas G 1997 Rapamycin suppresses 5'TOP mRNA translation through
inhibition of p70S6K. EMBO J 16:36933704[Abstract/Free Full Text]
-
Franke TF, Yang S-I, Chan TO, Datta K, Kazlauskas A, Morrison
DK, Kaplan DR, Tsichlis PN 1995 The protein kinase encoded by the Akt
proto-oncogene is a target of the PDGF-activated phosphatidylinositol
3-kinase. Cell 81:727736[Medline]
-
Kohn AD, Summers SA, Birnbaum MJ, Roth RA 1996 Expression of a
constitutively active Akt Ser/Thr kinase in 3T3L1 adipocytes
stimulates glucose uptake and glucose transporter 4 translocation.
J Biol Chem 271:3137231378[Abstract/Free Full Text]
-
Pierce SB, Kimelman D 1995 Regulation of Speman organizer
formation by the intracellular kinase XGSK-3. Development 121:755765[Abstract/Free Full Text]
-
Rupprecht R, Arriza JL, Spengler D, Reul JMHM, Evans RM,
Holsboer F, Damm K 1993 Transactivation and synergistic properties of
the mineralocorticoid receptor: relationship to the glucocorticoid
receptor. Mol Endocrinol 7:597603[Abstract]
-
Spengler D, Villalba M, Hoffmann A, Pantaloni C, Houssami S,
Bockaert J, Journot L 1997 Regulation of apopotosis and cell cycle
arrest by Zac1, a novel zinc finger protein expressed in the pituitary
gland and the brain. EMBO J 16:28142825[Abstract/Free Full Text]