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Receptors Regulate Phosphorylation of Calcium/Cyclic Adenosine 3',5'-Monophosphate Response Element-Binding Protein and Activation of p42/p44 Mitogen-Activated Protein Kinase
Parke-Davis Neuroscience Research Center, Cambridge, United Kingdom CB2 2QB
Address all correspondence and requests for reprints to: Dr. S. McNulty, Parke-Davis Neuroscience Research Center, Robinson Way, Cambridge, United Kingdom CB2 2QB. E-mail: shaun.mcnulty{at}wl.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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receptors.
Signaling pathways were investigated using immunocytochemical, Western
blot, and imaging techniques. Treatment with sauvagine increased
phosphorylation of p42/p44, but not of p38 or stress-activated protein
kinase (SAPK)/JUN N-terminal kinase (JNK) mitogen-activated protein
(MAP) kinases correlating with increased p42/p44 MAP kinase activity.
Mobilization of intracellular Ca2+ stores was observed in
cells treated with high concentrations (100 nM, 1
µM) of sauvagine. A time- and dose-dependent increase in
phosphorylation of the transcription factor CREB was observed in
cultures treated with sauvagine. Phosphorylation of CREB occurred at
lower concentrations of sauvagine than those required to mobilize
intracellular calcium stores, and phosphorylation was not blocked by
the mitogen-activated protein kinase kinase inhibitor PD98059 at a
concentration (1 µM) that fully inhibited phosphorylation
of MAP kinase. Cotreatment of cultures with the protein kinase A
inhibitor H89 (10 µM) blocked fully the stimulatory
actions of sauvagine (0.1 nM, 1 nM) on
phosphorylation of CREB, but not those on phosphorylation of MAP
kinase. Phosphorylation of MAP kinase was partially blocked by the
phosphoinositide 3-kinase inhibitor LY294002 (5 µM) and
by the phosphoinositide-phospholipase C inhibitor U73122 (10
µM). These data demonstrate that cAMP-,
Ca2+-, and MAP kinase-dependent signaling pathways are
activated by stimulation of CRF-1 and CRF-2 receptors. However, in
these cells, only protein kinase A-dependent pathways contribute
significantly to enhanced phosphorylation of CREB. These represent the
first reported observations of CRF receptor-mediated phosphorylation of
the transcription factor CREB and activation of MAP kinase signal
transduction pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, CRF-2ß,
and CRF-2
(7, 8, 9). In addition to the anterior and intermediate lobes
of the pituitary (3), CRF receptors are located in a wide variety of
locations, including cerebellum, cerebral cortex, olfactory bulb,
amygdala, and spleen (10, 11, 12, 13). The wide spread distributions of both
CRF-1 and CRF-2 receptors in the central nervous system are
consistent with a general neuromodulatory role within the brain in
addition to control of the endocrine stress response. The intracellular signaling pathways used by CRF in the anterior pituitary have been extensively studied (14, 15, 16). In this tissue, receptor activation by CRF causes Gs-mediated stimulation of adenylyl cyclase leading to increased levels of the intracellular second messenger cAMP. Further studies have shown that activation of CRF receptors within the central nervous system stimulates cAMP production (17, 18). In addition to increased production of cAMP, activation of CRF receptors has also been shown to increase hydrolysis of phosphatidylinositol 4,5-bisphosphate (19, 20), leading to elevated levels of intracellular free calcium ([Ca2+]i) (20). The hydrolysis of inositol lipids leads to the formation of several distinct second messengers, including diacylglycerol (21), raising the possibility of simultaneous CRF-mediated activation of multiple signaling pathways. CRF-stimulated production of cAMP has also been shown to increase [Ca2+]i indirectly through protein kinase A (PKA) modulation of voltage-sensitive calcium channels (22), demonstrating CRF-mediated cross-talk between distinct signaling pathways. However, the mechanisms by which CRF receptor agonists regulate cellular actions downstream from control of second messenger levels are poorly understood.
Increased cytosolic levels of cAMP and/or Ca2+ lead to the activation of several intracellular kinases, including PKA and Ca2+/calmodulin-dependent kinase II, which are able to phosphorylate the calcium/cAMP response element-binding protein (CREB) at Ser133 and thereby cause activation. This phosphorylated form of the transcription factor CREB (pCREB) is then able to regulate the transcription of genes containing the calcium/cAMP response element (CRE) (23, 24), including c-fos (25). In addition to cAMP- and calcium-dependent signaling pathways, activation of mitogen-activated protein (MAP) kinase pathways increases phosphorylation of CREB through activation of a distinct CREB kinase, RSK2 (26, 27). Activation of MAP kinase also leads to phosphorylation of the transcription factor Elk1. Modulation of gene expression by CREB may involve it binding to and interacting with additional nuclear proteins (28), including the CREB-binding protein. Therefore, both cAMP-dependent and cAMP-independent signaling pathways converge to modulate CREB phosphorylation (29). As CRF has been proven to activate signaling pathways that converge on cellular kinases known to phosphorylate CREB, it is possible that CREB provides a link between CRF receptor activation and control of changes in gene expression.
The aim of the present study was to investigate the effect of
activation of both CRF-1 and CRF-2 receptors on phosphorylation of
CREB and activation of MAP kinases and to characterize the possible
signal transduction pathways responsible for mediating phosphorylation
of CREB. Phosphorylation of CREB was examined using antisera raised
against a synthetic peptide containing the phospho-Ser133
residue to perform Western blot and immunocytochemistry. Changes in
[Ca2+]i were estimated by fluorescence
imaging, and activation of MAP kinase was assessed using Western blot
analysis. Experiments using binding studies and functional assays of
cAMP production had confirmed the validity of the CHO-CRF-1 and
CHO-CRF-2 cell lines. Competition binding studies using the
radioligand [125I-Tyr0]sauvagine revealed
rank orders of potency for the CRF-1 receptor of sauvagine =
astressin = urocortin = rat/human CRF = ovine CRF
> -helical CRF, and for the CRF-2 receptor of sauvagine =
astressin = urocortin > -helical CRF > rat/human
CRF > ovine CRF. Assays of receptor-mediated stimulation of cAMP
demonstrated a rank order of potency for the CRF-1 receptor of
sauvagine = rat/human CRF = ovine CRF, and for the CRF-2
receptor of sauvagine > rat/human CRF > ovine CRF,
confirming the utility of the rat CRF-1 and rat CRF-2 cell
lines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptors and were maintained in MEM medium supplemented with 10%
FCS at 37 C in an atmosphere containing 5% CO2. For
immunocytochemical analysis and measurements of intracellular free
calcium concentration, cells were seeded at a density of 1.5 x
105 onto sterile coverslips contained in six-well plates
and incubated overnight at 37 C with 5% CO2 and 95%
humidity to permit the cells to adhere. Cells were used after 48 h
in culture. For Western blot analysis, cells were plated at a density
of 2.5 x 105 into six-well plates and incubated
overnight at 37 C with 5% CO2 and 95% humidity. Cells
were used after 48 h in culture.
Western blot analysis of CREB and MAP kinase phosphorylation
Western blots for CREB immunoreactivity (CREB-ir) were performed
using a validated, commercially available kit (New England Biolabs, Inc., Beverley, MA). Cells were maintained as
described, then starved of serum by washing three times in serum-free
medium and incubated in 2 ml medium for 30 min before experimentation.
Stock solutions of agonists and antagonists were added to a given well,
and cells were incubated for the required experimental period at 37 C
and 5% CO2. At the end of this period medium was
aspirated, and cells were washed once in PBS containing 1
mM NaF before extraction in 200 µl SDS-PAGE sample
buffer. Extracts were then sonicated for 5 sec and centrifuged at 4000
rpm to remove debris. Samples (15 µl) were loaded onto the stacking
gel lanes of 10% SDS-PAGE minigels (Novex, San Diego,
CA), and proteins were separated using a Novex XCell II
Mini-Cell electrophoresis system for 2 h at 100 V. Proteins were
then transferred (25 V, for 2 h) to nitrocellulose membranes
(Novex) before detection. Membranes were incubated for
1 h at 20 C in blocking solution (10 ml; Tris-buffered saline
containing 5% dried milk and 0.1% Tween-20) and then incubated
overnight at 4 C in primary antibody solution [10 ml; pCREB, 1:1000;
total CREB, 1:2000; phospho-MAP kinase, 1:1000; total MAP kinase
(phosphorylation state-independent antiserum, raised against residues
345–358 of rat p42 MAP kinase), 1:2000; phospho-Elk1, 1:1000 in TBS
containing 5% BSA and 0.1% Tween-20]. The next day, antiserum was
removed, and blots were washed three times in TBS (10 ml) containing
0.1% Tween before incubation with secondary antibody (10 ml; 1:1000
horseradish peroxidase-linked goat antirabbit, in blocking solution,
New England Biolabs, Inc.) for 30 min at room temperature.
Blots were then rinsed three times for 5 min each time in TBS (10 ml)
and a further three times in washing buffer (10 ml; New England Biolabs, Inc.) before development using a modified enhanced
chemiluminescence detection system (Amersham, Arlington Heights,
IL).
Western blot analysis of MAP kinase assay
Measurement of activity was carried out using the p44/42 MAP
kinase immunoprecipitation assay kit (New England Biolabs, Inc.). After agonist stimulation, cells were rinsed with PBS,
and 0.5 ml cell lysis buffer [20 mM Tris (pH 7.5), 150
mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
ß-glycerolphosphate, 1 mM sodium orthovanadate, and 1
µg/ml leupeptin] was added. The cells were incubated on ice for 5
min, then harvested. Cell lysates were transferred to tubes and
sonicated four times for 5 sec each time. Cell debris was removed by
centrifugation (12,000 x g, 4 C, 10 min). Phospho-MAP
kinase p44/42 antiserum (Thr202, Thr204
phosphorylation site-specific antiserum, 1:100 dilution) was added to
200 µl supernatant and incubated overnight at 4 C with gentle
shaking. Protein A-Sepharose beads were added (20 µl of a 50% bead
solution in PBS), and the mixture was incubated for a further 3 h
at 4 C. Samples were centrifuged (2400 x g, 1 min) and
washed twice with 0.5 ml lysis buffer. The pellet was then washed twice
with kinase buffer [25 mM Tris (pH 7.5), 5
mM ß-glycerolphosphate, 2
mM dithiothreitol, 0.1 mM
sodium orthovanadate, and 10 mM
MgCl2], and the pellet was suspended in 50 µl
kinase buffer containing 100 µM ATP and 1 µg Elk1
fusion protein. Samples were incubated for 30 min at 30 C, and the
reaction was terminated with 2 x SDS-PAGE sample buffer
(Novex). Samples were Western blotted and probed using
phospho-Elk1 antiserum specific for the
Ser383-phosphorylated form of Elk1.
Analysis of Western blot immunoreactivity
Analysis was undertaken essentially as described previously
(30). Briefly, individual bands were viewed on an image analysis system
consisting of a monochrome video camera (Dage MTI CCD 72; MTI, Michigan
City, IN) connected to an Inter Focus Ltd. (MCID Imaging Research Inc.,
St. Catharines, Ontario, Canada) image analysis system. To
quantify the relative intensity of a given band, the entire band was
selected, and the average intensity of signal above mean background was
expressed as a relative optical density (ROD) value. Further
measurements were made at other points on the lane free from
identifiable immunoreactivity, and these values were subtracted from
the measurements obtained for individual bands to assess the intensity
of specific immunoreactivity.
Immunocytochemical analysis of CREB phosphorylation
Immunocytochemistry and analysis were undertaken as described
previously (31, 32). Briefly, cultures were starved of serum for 30
min. To begin treatment, 2 ml of agonist were added to given wells.
Incubations were terminated by rinsing coverslips twice with PBS and
then fixing with 1 ml 4% paraformaldehyde for 30 min at room
temperature. The cells were then washed twice for 5 min each time in 2
ml glycine solution (10 mM glycine in PBS) and then placed
in 2 ml blocking solution (PBS containing 2% normal goat serum and
0.3% Triton X-100) for 30 min. Blocking solution was replaced with 1
ml primary antibody solution (pCREB, 1:1000; New England Biolabs, Inc.; in PBS containing 5% BSA and 0.3% Triton X-100).
Coverslips were incubated overnight at 25 C, washed twice
with PBS for 10 min, and 1 ml biotinylated secondary antibody
(Vector Laboratories, Inc., Burlingame, CA) was added to
each well for 60 min at room temperature. The coverslips were washed
twice for 10 min each time in PBS, and immunoreactivity was detected by
a standard Vectastain ABC reaction using diaminobenzidine
as chromogen (Vector Laboratories, Inc.).
Densitometry of nuclear immunoreactivity in cell populations
Total nuclear pCREB-ir was quantified using image analysis and
densitometry as described previously (30, 31, 32). Coverslips were viewed
on an image analysis system consisting of a monochrome video camera
(Dage MTI CCD 72) connected to an Inter Focus Ltd. MCID image analysis
system. To quantify the relative degree of immunostaining, ROD
measurements were made of the staining found in individual nuclei
chosen at random from the total cell population on the coverslip. In
all cases, the level of illumination was initially set so that the
background measure through the coverslip in an area free of cells was
consistent, in order that measurements between coverslips were
comparable. The relative intensity of the reaction product in any given
nucleus that passed through a random transect line was recorded from a
total of 50 nuclei from any given coverslip. Measurements were also
made along transect lines at further points free of cells to calculate
an average background mean for each coverslip. These background values
were subtracted from the individual measurements obtained from each
nucleus sampled on a given coverslip. The final values obtained from
experimental coverslips were expressed as mean (ROD) values ±
SEM from a single representative experiment. All control
and experimental cultures were processed identically and simultaneously
for ICC so that direct experimental comparisons could be made.
Experiments were undertaken at least four times with different cell
preparations and produced ROD values that were consistent between as
well as within studies and gave similar experimental outcomes. The
effects of various treatments were determined by t test and
ANOVA, and differences between experimental groups were assessed by
post-hoc Dunnett’s t test. For a given result, *
indicates significance at the P < 0.05 level, and **
corresponds to significance at the P < 0.01 level.
Analysis of changes in
[Ca2+]i
Coverslips containing cells were prepared and maintained as
described. Cultured cells (grown attached to 22-mm diameter coverslips)
were washed twice in a Krebs-HEPES extracellular medium buffer (EM;
NaCl, 118 mM; KCl, 4.7 mM; MgSO4,
1.2 mM; CaCl2, 1.2 mM;
KH2PO4, 1.2 mM; HEPES, 10
mM; glucose, 11 mM; BSA, 0.1%; pH 7.2 at 20 C)
(33) and then loaded with fura-2 (34) by incubation for 3 h at 20
C with EM containing fura-2/AM (2 µM; Molecular Probes, Inc., Eugene, OR). This procedure enables the cells to
load with fura-2/AM, which becomes hydrolyzed to the free acid form
once inside the intact cells. After loading, coverslips were mounted
into imaging chambers and perfused with EM to remove extracellular
fura-2/AM and to allow hydrolysis of intracellular fura-2/AM to occur.
Measurements of changes in the free [Ca2+]i
in individual cells were made from the fluorescence ratio (excitations,
340 nm/380 nm; emission, >510 nm) using a spectral Wizard
monochromator, cooled integrating CCD camera, and a dedicated suite of
software (Merlin, Life Sciences Resources, Cambridge, UK). Data are
expressed as the ratio of 340/380 nm units.
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Results |
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(a–f). Basal unstimulated levels of pCREB-ir were low (Fig. 1a);
however, stimulation of CHO-CRF-1 cultures with sauvagine (1
µM, 10 min) caused a large increase in immunoreactivity
that was restricted to the cell nucleus (Fig. 1b). Identical results
were observed after treatment of CHO-CRF-2 cultures with sauvagine
(data not shown). Analysis of nuclear immunoreactivity demonstrated
that sauvagine caused both a time (Fig. 1c)- and dose (Fig. 1e)-dependent increase in pCREB-ir in CHO-CRF-1 cultures and also in
CHO-CRF-2 cultures (Fig. 1, d and f).
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) as the phosphorylated form of the protein CREB (Fig. 2, a–d). Blots probed using an antiserum
that recognizes both phosphorylated and nonphosphorylated forms of CREB
did not show a systematic increase in immunoreactivity on treatment of
CHO-CRF-1 and CHO-CRF-2 cultures with 1 µM sauvagine
for varying time intervals (Fig. 2, c and d). These results demonstrate
that de novo CREB synthesis does not account for the
observed increase in pCREB-ir. The consistency of the total CREB-ir
band between cell extracts from a given experiment provided an index of
equal extraction and preparation of samples and was used as an internal
control in all experiments. Sauvagine caused a time-dependent increase
in pCREB-ir in two major bands with apparent molecular masses of 43 and
38 kDa, consistent with published reports (31). The 43-kDa band was
recognized by both CREB- and pCREB-specific antiserum, confirming its
identity as native CREB; the lower molecular mass band probably
represented an additional unidentified CREB family member, potentially
phospho-ATF-1. The increase in phosphorylation of CREB was apparent by
1 min of stimulation, becoming maximal between 15–30 min, with
immunoreactivity remaining elevated after 60 min of stimulation (Fig. 2, a and b). Treatment with sauvagine for different time periods was
without effect on either CREB phosphorylation or total CREB protein in
the parental CHO-Pro5 cell line, confirming the specificity of
sauvagine-induced phosphorylation of CREB in CRF-1 and CRF-2
cultures (Fig. 2, e and f). Treatment of CHO-CRF-1 and CHO-CRF-2
cultures with various concentrations of sauvagine for 15 min caused a
dose-dependent increase in phosphorylation of CREB (Fig. 3, a and b). Analysis of the
dose-dependent increase in pCREB-ir from three independent experiments
revealed EC50 values of 0.106 ± 079 and 0.04 ±
0.073 nM for CRF-1 and CRF-2 receptors, respectively
(Fig. 3, c and d). These were comparable to the EC50 values
for sauvagine stimulation of cAMP production of 1.27 nM
(range, 0.69–2.85 nM) for CHO-CRF-1 and 0.16
nM (range, 0.14–0.23 nM) for CHO-CRF-2
(n = 3 independent experiments; data not shown). The stimulatory
effect of sauvagine (1 µM) was not apparent in the
CHO-Pro5 parental line from which the CHO-CRF-1 and CHO-CRF-2
cultures were derived (Fig. 4a). However,
forskolin (1 µM), 12-O-tetradecanoylphorbol
13-acetate (TPA; 100 nM), and ionomycin (1
µM) were all able to increase significantly the
phosphorylation of CREB in the CHO-Pro5 line, confirming the ability of
CREB to undergo phosphorylation in this cell type (Fig. 4a). In
addition to sauvagine, TPA (100 nM), forskolin (1
µM), and ionomycin (1 µM) all significantly
increased phosphorylation of CREB in CHO-CRF1 and CHO-CRF-2 cultures
(Fig. 4, b and c, respectively).
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cultures were blocked by the CRF
receptor antagonist astressin at a concentration (1 µM)
known to fully inhibit the binding of [125I]sauvagine to
both receptor subtypes (Fig. 4, d and e). The CRF-1 receptor antagonist
PD171729 fully inhibited the stimulatory actions of sauvagine, but not
those of forskolin, in the CHO-CRF-1 cell line. However, PD171729 was
without effect on sauvagine-stimulated phosphorylation of CREB in
CHO-CRF-2 cultures, confirming the receptor subtype-specific nature
of its action.
Effects of sauvagine on the MAP kinase signaling pathways
Activation of the MAP kinase signal transduction pathways is a
significant route by which phosphorylation of CREB can occur (27).
Sauvagine (1 µM) stimulation of CHO-CRF-1 cultures caused
a time-dependent increase in phospho-MAP kinase p42/p44-ir in two
bands, with p42 appearing to be the predominant form. However, no
increase was apparent in the total amount of p42/p44 MAPK-ir identified
using antiserum that recognized both nonphosphorylated and
phosphorylated forms of p42/p44 MAP kinase (Fig. 5, a and b). Results similar to these
were observed in CHO-CRF-2 cultures (Fig. 5, c and d). Sauvagine was
without effect on phosphorylation of p38 and SAP-JNK kinases in
CHO-CRF-1 and CHO-CRF-2 cultures (data not shown). Increased
phospho-p42/p44-ir was apparent 1 min after stimulation, became maximal
between 5–10 min, and fell to unstimulated levels by 60 min of
treatment (Fig. 5, b and d). Dose-dependent increases in
phosphorylation of MAP kinase p42/p44 were observed in CHO-CRF-1 and
CHO-CRF-2 cultures stimulated with sauvagine (Fig. 5, e and f,
respectively).
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). Both astressin and PD98059 were able
to significantly inhibit the stimulatory actions of sauvagine in both
cell lines at a concentration (1 µM) previously shown to
strongly stimulate phosphorylation of p44/p42 MAP kinase (Fig. 5, e and
f). The CRF1 antagonist PD171729 fully inhibited the stimulatory
actions of sauvagine in CHO-CRF-1 cultures, but was without effect in
CHO-CRF-2 cultures. Simultaneous analysis of the experimental
samples for the phosphorylated form of CREB confirmed the effects of
astressin and PD171729 observed previously (Fig. 4). However, the MEK
inhibitor PD98059 was not able to modulate phosphorylation of CREB at a
dose that fully inhibited the phosphorylation of MAP kinase (Fig. 6b).
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). However treatment with TPA or
ionomycin increased phosphorylation of MAP kinase, confirming the
existence of functional MAP kinase signaling pathways in the parental
line. Sauvagine, TPA, and ionomycin were all found to increase
phosphorylation of MAP kinase p42/p44 CREB in CHO-CRF1 and CHO-CRF-2
cultures (Fig. 7, b and c). The ability of sauvagine to stimulate MAP
kinase p42/p44 activity was confirmed using an immunoprecipitation
Western blot assay (New England Biolabs, Inc.), which uses
the ability of immunoprecipitated phospho-MAP kinase p42/p44 to
phosphorylate Elk1 fusion protein in vitro (see
Materials and Methods; Fig. 7, d and e). The nonspecific
higher molecular mass band represents IgG peptide (
55 kDa) derived
from the immunoprecipitation reaction (Fig. 7, d and e). Treatment with
forskolin was without effect on phospho-Elk1 levels. However, treatment
with TPA (100 nM), ionomycin (1 µM), or
sauvagine (1 µM) strongly increased phosphorylation of
Elk1, confirming that phosphorylation of p44/p42 MAP kinase correlates
with activation.
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cells loaded with the calcium-sensitive
dye fura-2 (2 µM) and treated with sauvagine at
concentrations of 100 nM and 1 µM, but not 10
nM, gave a transient increase in R340:380, indicative of an
increase in [Ca2+]i (Fig. 8, a and b). Sauvagine at a concentration
of 1 µM was without effect on the R340:380 ratio of the
parental CHO-Pro5 line (Fig. 8c). Experiments were undertaken in the
absence of extracellular calcium (nominally calcium-free EM with 1
mM EGTA added) to investigate whether the increase in
intracellular calcium elicited by sauvagine occurred as a consequence
of mobilization from intracellular stores and/or of calcium entry
across the plasma membrane in CHO-CRF-1 cultures (Fig. 8d). Exchanging
normal EM for calcium-free EM (with 1 mM EGTA) caused a
reduction in [Ca2+]i shown by R340:380 values
(Fig. 8d). Stimulation of CHO-CRF-1 cells with sauvagine (1
µM) after a 5-min period in calcium-free medium caused an
increase in R340:380 above basal values, with a reduction in peak width
characteristic of a loss of a secondary calcium entry component.
Reintroduction of calcium to the perfusate led to an increase in basal
R340:380 values (Fig. 8d). A third sauvagine treatment period in the
presence of extracellular calcium gave a characteristic broad peak
indicative of both a mobilization and an entry component
contributing to the observed increase in intracellular calcium (Fig. 8d). To confirm that treatment of CHO-CRF-1 cultures with
sauvagine caused mobilization of intracellular calcium stores,
experiments were undertaken using U73122 (10 µM), a
potent inhibitor of phospholipase C. Treatment of cultures with
sauvagine alone (1 µM) increased R340:380 (Fig. 8e).
However, in cells treated with sauvagine in the presence of U73122, no
increase in R340:380 values was observed (Fig. 8e). No differences were
observed between the patterns of responses observed for CHO-CRF-1
cultures and those observed for CHO-CRF-2 cultures (data not
shown).
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cultures treated
with sauvagine (0.1 nM, 1 nM) or forskolin (10
µM; Fig. 9, a and b,
respectively). Cells were pretreated with H89 for a total of 90 min
before stimulation with agonist. Sauvagine at concentrations of 0.1 and
1 nM and forskolin at 10 µM all increased
phosphorylation of CREB in untreated cells. The stimulatory effects of
both sauvagine and forskolin were inhibited fully by pretreatment with
H89 (10 µM, 1.5 h). Inhibition of PKA by H89 was
without effect on the total level of CREB protein (data not shown).
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) and CHO-CRF-2 (Fig. 9d) cultures
treated with sauvagine (100 nM for 10 min). Sauvagine
increased phosphorylation of p44/42 MAP kinase above control levels in
all experiments. Inhibition of PKA by H89 at a concentration (10
µM) that fully inhibited phosphorylation of CREB was
without effect on the total level of phospho-p44/p42 MAP kinase.
Inhibition of PI-specific PLC with U73122 at a concentration (10
µM) that fully inhibited sauvagine-mediated mobilization
of intracellular calcium caused a slight, but consistent, inhibition of
MAP kinase phosphorylation. Inhibition of PI3 kinase (LY294002, 10
µM) also partially inhibited sauvagine-stimulated
phosphorylation of MAP kinase in both CHO-CRF-1 and CHO-CRF-2
cultures. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, expressing the rat CRF
type 1 and the rat CRF type 2 receptors, respectively. Treatment
with sauvagine caused a time- and dose- dependent increase in
phosphorylation of nuclear CREB and of p42/p44 MAP kinase in both
CHO-CRF-1 and CHO-CRF-2 cultures. Sauvagine was without effect on
the parental CHO-Pro5 cell line. Phosphorylation was blocked by the CRF
receptor antagonist astressin, demonstrating the requirement for
activation of the CRF receptor for phosphorylation to occur. The
EC50 values for sauvagine stimulation of cAMP and for
phosphorylation of CREB are consistent with phosphorylation occurring
as a consequence of activation of adenylyl cyclase. Inhibition of PKA
fully blocked the stimulatory effects of sauvagine on phosphorylation
of CREB; however, inhibition of MEK did not affect CREB,
demonstrating that phosphorylation of CREB requires activation of PKA,
but not of MAP kinase. Inhibition of PI3 kinase or of PI-specific PLC
reduced phosphorylation of MAP kinase. Treatment of cultures with
sauvagine increased intracellular levels of Ca2+ through
both mobilization of intracellular stores and influx across the plasma
membrane. In human epidermoid A-431 cells, sauvagine (EC50
of 1.4 fM) increases [Ca2+]i by
calcium influx through G protein-coupled channels and by mobilization
from IP3-sensitive stores (20). However, the increase in
[Ca2+]i observed in the present study was not
apparent when cultures were treated with 10 nM sauvagine, a
dose that caused maximal stimulation of CREB phosphorylation, and it is
possible that the stimulatory effect of sauvagine on
[Ca2+]i may occur as a consequence of
promiscuous G protein coupling. These results demonstrate for the first
time CRF receptor-mediated phosphorylation of CREB and activation of
MAP kinase signaling pathways.
The data presented in the present study indicate that activation of
either CRF-1 or CRF-2 receptor type leads to phosphorylation and
activation of p42/p44 MAP kinase. The mechanisms by which sauvagine
causes activation of MAP kinase in CHO-CRF1 and CHO-CRF-2 cultures
are not fully understood. However, forskolin was without effect on
p42/p44 MAP kinase activity in the present study, and inhibition of PKA
did not modulate sauvagine stimulation of MAP kinase phosphorylation.
Therefore, sauvagine does not activate MAP kinase through increased
intracellular levels of cAMP, and it is unlikely that
sauvagine-stimulated activation of PKA inhibits MAP kinase as could
have been expected from the observations of Crespo et al.
(39). In COS-7 cells, activation of MAP kinase is mediated by the
ß-subunits of heterotrimeric G proteins through interaction with
Ras-dependent pathways (35, 36). Additional studies have
demonstrated the ability of ß-subunits to increase MAP kinase
activity in both Rat-1 fibroblasts stimulated with insulin-like growth
factor I (37) and in signaling pathways mediated by activation of the
ß-adrenergic receptor (38, 39), confirming the general significance
of ß-subunit modulation of MAP kinase activity (reviewed in Ref.
40). One mechanism by which ß-subunit modulation of MAP kinase
activity may occur is through PI3 kinase activation of cellular protein
tyrosine kinases. The ability of the PI3 kinase inhibitor LY294002 to
partially inhibit sauvagine-stimulated phosphorylation of MAP kinase in
CRF-1 and CRF-2 cultures demonstrates that PI3 kinase may provide a
link between CRF receptor activation and MAP kinase phosphorylation in
our cells. In addition, U73122 was found to inhibit
sauvagine-stimulated phosphorylation of MAP kinase in CRF-1 and
CRF-2 cultures, potentially through inhibition of calcium-sensitive
Pyk-2-mediated activation of protein tyrosine kinases. It is possible
that PI3 kinase and calcium activate Pyk-2 in concert to stimulate MAP
kinase activation in response to sauvagine. Activation of the small G
protein Ras by ß causes recruitment and activation of Raf, which
phosphorylates MEK, the p42/p44 MAP kinase kinase (reviewed in Refs. 39, 41). Phosphorylation of p42/p44 by MEK leads to activation and
translocation to the cell nucleus. Activated p42/p44 MAP kinase
phosphorylates RSK2, leading to phosphorylation and activation of CREB
(27), providing a mechanism by which MAP kinase can modulate gene
expression through the CRE. In addition, MAP kinase activates the
transcription factor Elk1, which binds to the serum response element
(SRE) together with the serum response factor protein to cause
increased transcription of immediate early genes containing the SRE.
These pathways provide mechanisms by which activation of either CRF-1
or CRF-2 receptor type can alter gene transcription.
Three signaling pathways known to cause phosphorylation of CREB, those
sensitive to cAMP, Ca2+, and MAP kinase, are stimulated by
treatment of CHO-CRF-1 and CHO-CRF-2 cultures with sauvagine. The
major pathway by which sauvagine regulates phosphorylation of CREB is
not through calcium-mediated activation of calmodulin- dependent
kinase, as changes in intracellular calcium are not apparent after
treatment with sauvagine at a concentration (10 nM) that
causes maximal phosphorylation of CREB in both cell types. The MEK
inhibitor PD98059 at a concentration (10 µM) that fully
inhibited the phosphorylation of MAP kinase was without effect on
phosphorylation of CREB, raising the possibility that phosphorylation
was mediated entirely by cAMP-dependent pathways. In support of this,
treatment with the PKA inhibitor H89 (10 µM) fully
inhibited the effects of sauvagine on CREB at a concentration (1
nM) known to maximally phosphorylate CREB. This
demonstrates that cAMP-, PKA-dependent signaling pathways are
responsible for sauvagine-stimulated phosphorylation of CREB. However,
cooperative or synergistic effects of p42/p44 MAP kinase signaling
through activation of the transcription factor Elk1, and PKA-dependent
signaling through activation of CREB may be apparent at the level of
the control of gene expression. One example of this may be control of
transcription of the immediate early gene c-fos, which under
certain circumstances can be modulated by Elk1 acting at the SRE site
and CREB acting at the CRE site (27).
The molecular mechanisms by which CRF exerts control on the expression of the POMC gene encoding ACTH in the anterior pituitary are currently under investigation. CRF has been shown to stimulate POMC promoter activity by 3.5-fold (42), and this effect was dependent on PKA activity. However, activation of PKA, although required for POMC gene expression, may not be sufficient to cause maximal stimulation, and other PKA- and protein kinase C-independent signaling pathways may be required (43). One intermediate between kinase activation and increased POMC gene expression may be activation of the immediate early gene c-fos (44, 45). However, at least one novel transcription factor, PCRH-REB-1, has been identified that responds to CRF and increases activation of the POMC promoter (46), emphasizing the complexity of control of the POMC promoter. Although the POMC human gene does not possess the classical CRE, it has been shown to have a novel POMC-CRE and that CREB proteins can bind to this POMC-CRE site (47). This raises the possibility that phosphorylated activated CREB may directly modulate POMC gene expression. In addition to a direct effect of CREB on POMC gene expression, CRF may, through modulation of MAP kinase and Elk1 activity, increase nuclear levels of c-Fos protein and thereby increase POMC gene expression.
The current study suggests that PKA-mediated phosphorylation of the
transcription factor CREB may be an important intermediary step in the
transduction pathways arising from activation of CRF-1 and CRF-2
receptors and leading to modulation of gene transcription within the
nucleus of target cells. In addition, receptor stimulation causes
increased activation of p44/42 MAP kinase independently of cAMP and
phosphorylation of CREB. These observations raise the possibility that
PKA and MAP kinase may act in concert to control gene transcription in
CRF-responsive cells. Therefore, it is possible that both
phosphorylation of CREB and activation of MAP kinase signaling pathways
modulate POMC gene expression and adaptive responses to stress in
vivo in response to increased CRF levels.
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Acknowledgments |
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Received December 19, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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