Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
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ABSTRACT |
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We have previously
demonstrated that glucocorticoids increased Gq/11
protein expression and phospholipase C activity in the rat osteosarcoma
cell line UMR 106-01. In this study, we demonstrated that
G11
is the primary Gq-subtype family member
expressed in UMR cells. Dexamethasone treatment increased the
expression of G11
protein in both a time- and a
dose-dependent manner. Glucocorticoid treatment significantly increased
the half-life of G11
protein from 20.3 to 63 h.
Steady-state G11
mRNA level was also increased by
glucocorticoid treatment by ~70%. This change was not the result of
changes in RNA stability but rather the result of increased transcription, because the glucocorticoid-mediated upregulation of
G11
mRNA was blocked by the transcription inhibitor
actinomycin D. The dexamethasone induction of G11
mRNA
occurred after a time lag of 12-24 h and was blocked by the
protein synthesis inhibitor cycloheximide. These results suggest that
the dexamethasone-induced rise in G11
protein results
primarily from changes in the degradation rate of the protein, whereas
changes in G11
mRNA play a smaller role and require de
novo synthesis of regulatory protein(s).
glucocorticoids; G protein; G11; osteosarcoma
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INTRODUCTION |
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HETEROTRIMERIC G
PROTEINS are a family of guanine nucleotide-binding proteins
composed of three subunits: ,
, and
. Because G proteins are
crucial for a large array of transmembrane signaling (7),
any alteration in protein levels of these three subunits in the cell
could potentially affect its ability to respond to external signals. In
addition, both the amount and the type of G proteins expressed in
individual cells may change the overall cellular response to external
stimuli. Very little is known, however, about how the cellular levels
of each G protein subtype are regulated to allow the G proteins to
fulfill the needs of particular cells. In vivo, the status of various
hormones has been reported to affect the steady-state levels of several
G protein subunits. Hypothyroidism has been reported to decrease the
steady-state level of Gs
in reticulocytes
(28) but to increase the steady-state levels of Gi
(12), Go
(13), and G
1/2 (21) in adipose
tissue. Subsequently, G
1/2 expression has been shown to
be regulated at the mRNA level (19). Therefore, G protein
subunits are differentially regulated by thyroid hormones in fat cells
at both the protein and mRNA levels. Glucocorticoids have also been
shown to modulate the steady-state levels of various G proteins.
Adrenalectomy increases the amount of Gi
while
decreasing the level of Gs
in liver (6) and
G
1/2 in adipose tissue (22). Alteration in
the mRNA level of Gi
, Gs
(24), and G
1/2 (23) has
subsequently been shown to be responsible for the changes in the
steady-state level of G protein subunits after adrenalectomy.
Glucocorticoid administration increases the protein and mRNA levels of
Gs
but decreases those of Gi
in rat
cerebral cortex (24). In vitro treatment with
dexamethasone, a synthetic glucocorticoid analog, has also been shown
to increase the protein and mRNA levels of Gs
in rat
pituitary cells (3) and G
1/2 in rat fat
cells (23). Therefore, the biochemical mechanism
underlying the change in G protein levels by glucocorticoids may be
altered transcription of mRNA encoding the G protein subunits.
Most of the work performed to date has examined the
effect of hormone status on regulation of components of the adenylyl
cyclase system. To understand the effect of glucocorticoids on the
phospholipase C (PLC) system, our laboratory has investigated the
effect of dexamethasone on the expression of Gq/11
protein and the hormone stimulation of PLC activity in rat bone cells.
We examined the effect of glucocorticoids on parathyroid hormone (PTH)
activation of two signal transduction pathways: PLC and adenylyl
cyclase (AC), in rat osteosarcoma cells UMR 106-01 and found that
glucocorticoids are more potent regulators of the PLC pathway than of
the AC pathway in UMR cells (16). Dexamethasone increased
PTH-activated PLC activity correlated with an elevated
Gq/11
protein expression. Glucocorticoid administration
to rats has also been reported to increase Gq/11
levels
and vasopressin-stimulated PLC activity in the pituitary
(18). In an effort to gain better insight into the
molecular mechanism by which glucocorticoids regulate the Gq-mediated
signaling system, we examined the effects of dexamethasone treatment on
the steady-state expression and stability of Gq/11
protein and mRNA.
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EXPERIMENTAL PROCEDURES |
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Materials.
UMR-106-01 rat osteosarcoma cells were a kind gift from Dr.
N. C. Partridge, St. Louis University School of Medicine (St. Louis, MO). Dexamethasone and Tran-[35S]methionine (1,000 Ci/mmol) were purchased from ICN Biochemicals (Aurora, OH). Pansorbin
was purchased from Calbiochem (San Diego, CA). Recombinant
G11 protein was purchased from Chemicon (Temecula, CA).
Antibodies raised to peptides corresponding to the amino acid sequence
within the amino-terminal domain of Gq
and
G11
, or to the carboxy terminus of Gq/11
,
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Full-length mouse G11
cDNA was generously provided by
Dr. M. I. Simon, California Institute of Technology Division of
Biology (Pasedena, CA). [
-32P]dCTP (3,000 Ci/mmol) was
purchased from Perkin-Elmer (Markham, ON). TRIzol Reagent, M-MLV
reverse transcriptase, and all tissue culture media were from GIBCO-BRL
Life Technologies (Burlington, ON). Actinomycin D was purchased from
Biomol (Plymouth Meeting, PA). Cycloheximide and
5,6-dichlorobenzimidazole riboside (DRB) were purchased from Sigma
(Oakville, ON).
Cell culture. UMR-106-01 cells were maintained in DMEM-Ham's F-12 (50:50) supplemented with 5% fetal bovine serum (FBS), 1 U/ml penicillin, and 1 µg/ml streptomycin and grown at 37°C in a humidified 95% air-5% CO2 atmosphere. Cells were treated with dexamethasone or vehicle (0.001% ethanol) in medium containing 5% FBS, with replacement of medium every 24 h.
Western blot analysis.
After treatment with dexamethasone or vehicle, cells were harvested
from 75-cm2 flasks using 0.1% trypsin and resuspended in a
lysis buffer containing 20 mM Tris (pH 7.5), 1 mM EGTA, and 1 mM
dithiothreitol. Cells were homogenized, and protein concentrations were
determined by amido black protein assay (25). For Western
immunoblotting analysis, 20, 40, and 60 µg of protein were run on
11% acrylamide gels by use of the Laemmli method (10) as
described previously (16). G11 was
identified on immunoblots as a band migrating at the same position as a
recombinant G11
protein standard run on the same gel.
This band was not seen when immunoblots were incubated with
anti-G11
antisera in the presence of a
G11
-blocking peptide (Santa Cruz, CA).
Protein stability.
Metabolic labeling of the UMR cells was performed as described by Shah
et al. (26). Cells were seeded in 6-well plates and grown
until 60% confluent. Tran-35S label (150 µCi) was then
added and cells were incubated for 24 h. The radioactive medium
was removed, and the cells were washed with normal medium. The labeled
cells were then incubated in 2 ml/well of normal medium in the presence
or absence of 100 nM dexamethasone. At appropriate times, the medium
was removed, and 200 µl of RIPA buffer [50 mM Tris, 150 mM NaCl, 1%
NP-40, 0.5% deoxycholate (DOC), and 0.1% SDS] were added to
each well to dissolve cells. The cell extract was then boiled for 20 min to denature it. After centrifugation (15,000 g,
5 min, 4°C), the supernatant was collected. Pansorbin beads (50 µl)
were added to the denatured cell extract and incubated at 4°C for
1 h with constant rotation. After centrifugation (15,000 g, 1 min, 4°C), the supernatant was collected and
immunoprecipitated by addition of 3 µl of Gq/11 antiserum and incubated at 4°C with constant rotation for 1 h. Pansorbin beads (50 µl) were subsequently added to the immune complex
and incubated at 4°C overnight with constant rotation. After
centrifugation (15,000 g, 1 min, 4°C), the beads were
washed three times with 65 µl of wash buffer (50 mM Tris, 150 mM
NaCl, and 1% NP-40), resuspended in Laemmli sample buffer, and boiled at 100°C for 5 min. After a final centrifugation (15,000 g, 1 min, 4°C), the supernatant was collected and
subjected to SDS-PAGE (13% acrylamide gel). After resolution of the
proteins, the gel was stained with Coomassie blue, dried, and exposed
to phosphor screen and scanned with PhosphoImager (Molecular Dynamics,
Sunnyvale, CA). The intensity of the signal was quantified using
ImageQuant software.
Northern blot analysis.
Total RNA was extracted from UMR cells after dexamethasone treatment
using TRIzol reagent in accordance with the manufacturer's protocol
(Life Technologies, Burlington, ON). The concentration of RNA was
determined by absorbance at 260 nm, and RNA integrity was assessed by
gel electrophoresis on 2% agarose gel. For Northern blot analysis, 2.5 µg of denatured total RNA were separated on a 1.2% agarose gel
containing 3.7% formaldehyde and transferred to Hybond-N nylon
membrane (Amersham Pharmacia Biotech, Oakville, ON) by capillary
transfer with sodium phosphate (100 mM) transfer buffer. The
transferred RNAs were immobilized by ultraviolet cross-linking and
prehybridized overnight at 42°C in a solution containing 100 mM
sodium phosphate, 1 mM EDTA, 7% SDS, 10 mg/ml BSA, and 0.045 mg/ml
salmon sperm DNA. The G11 cDNA probe was obtained by
cutting the plasmid pCISG
11 (29) with ClaI
and XhoI to yield a 1,200-bp fragment that represented the
full-length G11
cDNA. A probe specific to 18S rRNA was
made by PCR amplifying the first-strand cDNA with 18S-specific primer
pairs (Ambion, Austin, TX). The amplified product (350 bp) was
electrophoresed on agarose gel, excised, and gel purified (Qiagen,
Mississauga, ON). These probes were labeled using the random priming
method with [
-32P]dCTP and purified using G-50 sizing
columns (Roche Molecular Biochemicals, Laval, QC). Radiolabeled probes
(17 × 106
counts · min
1 · µg
1) were
added to the prehybridization solution and hybridized overnight at
42°C. The membranes were then washed for 20 min in each of the
following solutions: 2× SSC-0.1% SDS-0.5× SSC-0.1% SDS and 0.1×
SSC-0.1% SDS. The membranes were exposed to phosphor screen, and the
signals were quantified by use of ImageQuant software.
Presentation of data. The results presented were obtained from at least two independent experiments performed on cell cultures between passages 22 and 28. Results of some experiments are expressed as values (means ± SE) of separate experiments. Statistical significance was determined using Student's t-test.
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RESULTS |
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Characterization of Gq and G11
protein expression in UMR 106-01 cells.
Our laboratory has previously demonstrated that dexamethasone increased
Gq/11
protein expression and PLC activity
(16). Because the antiserum used previously for
quantitation recognized both Gq
and G11
proteins, we wanted to determine which one of the two G protein
subtypes was expressed in our cells and upregulated by dexamethasone to
increase PLC activity. Using antisera raised against sequences unique
to Gq
and G11
subunits, we found that G11
is the primary PLC-activating G protein expressed in
UMR cells and that Gq
was not detected (Fig.
1A). On the basis of this
finding, we used the G11
-specific antibody to reevaluate the effect of dexamethasone on G11
protein expression.
Treatment with 100 nM dexamethasone for 3 days increased
G11
protein expression fivefold (Fig. 1B),
indicating that dexamethasone is a potent regulator for
G11
expression in these cells.
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Effect of dexamethasone on G11 protein stability.
To investigate whether the elevated steady-state G11
protein level was due to increased stability of the protein, UMR cells were incubated with 35S-labeled methionine for 24 h,
and the loss of radio-labeled G11
protein with time was
monitored in cells that were either untreated or treated with 100 nM
dexamethasone. Antiserum CQ2, which recognized both Gq
and G11
, was used to immunoprecipitate
G11
from the UMR cell extracts. The rate of loss of
35S-labeled G11
protein was slower in cells
treated with dexamethasone (Fig. 3). In
untreated UMR cells, the half-life (t1/2) for
G11
protein was estimated to be ~20.3 h, whereas the
glucocorticoid-treated cells demonstrated a threefold increase in
t1/2 for G11
to 63 h.
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Effect of dexamethasone on steady-state G11 mRNA
level.
To assess whether changes in G11
mRNA expression could
also contribute to the glucocorticoid-induced increase in steady-state G11
protein expression, the effect of dexamethasone on
G11
mRNA level was determined by Northern blot analysis.
With use of a 32P-labeled mouse G11
cDNA
probe, a single mRNA transcript of ~4 kb was detected in UMR cells,
as reported previously in brain (29). Incubation of the
cells with 100 nM dexamethasone induced a significant 70% increase in
G11
mRNA expression after 24 h of hormone treatment
(Fig. 4A). Longer exposure to
glucocorticoids in UMR cells produced no further increase in
G11
mRNA level, but levels remained significantly higher
than those of the untreated cells. The increase in G11
mRNA by glucocorticoids was also found to be dose dependent, with
increased levels seen after 24-h treatment with 1 nM dexamethasone and
a maximal increase seen with 100 nM dexamethasone (Fig. 4B).
The dose dependence of increases in steady-state G11
mRNA level was more sensitive to low concentrations of dexamethasone (1 nM) compared with its effect on steady-state protein level.
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Mechanism of dexamethasone-induced G11 mRNA
induction.
The steady-state mRNA level can be regulated by altering the
transcription of the gene and/or the stability of the RNA transcript. To determine whether the stimulatory effect of glucocorticoid on
G11
mRNA levels was due to changes in mRNA transcript
stability, UMR cells were pretreated with or without 100 nM of
dexamethasone for 24 h and then exposed to the transcriptional
inhibitor DRB (150 µM) in the absence or presence of the
glucocorticoid for 0.5-48 h. The G11
mRNA
transcript remained stable for the first 8 h and then began to
decrease; the t1/2 for G11
mRNA
was ~37.5 h, and dexamethasone treatment did not affect the stability
of the transcript in these cells (Fig.
5A).
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Characterization of dexamethasone-mediated response on
G11 mRNA.
As shown in Fig. 4A, there were no significant changes in
G11
mRNA expression by dexamethasone for the first
12 h, but it was increased by 70% after 24 h. In addition,
cycloheximide alone decreased control levels of G11
mRNA
expression by ~40% and completely abolished the
dexamethasone-induced stimulation of G11
mRNA expression (Fig. 6). Together, these results
indicate that the glucocorticoid effect on G11
transcription was a secondary response.
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DISCUSSION |
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PTH stimulation of the PTH/PTHrP receptor on osteoblastic cells
leads to stimulation of both Gs and
Gq/11
, activating both the AC and PLC pathways
(11). Our previous work in the UMR cell line has
demonstrated that glucocorticiods increased PTH activation of both of
these pathways, although the effect on the PLC pathway was greater than
the increase in AC (16). The ability of glucocorticoids to
regulate the AC pathway has been well documented in many different tissues and cell lines, and it appears to result from increased expression of Gs
as well as of receptors (3, 4,
20, 24). In contrast, the effect of glucocorticoids on the PLC
pathway has not been well studied. Our own study in the UMR cells
was the first to demonstrate an increase in receptor-stimulated PLC activity and Gq/11
proteins by glucocorticoids
(16). This was followed by the demonstration of a similar
upregulation of Gq/11
and vasopressin-stimulated PLC in
rat pituitary glands after glucocorticoid administration in vivo to
rats (18). These studies suggested that glucocorticoid
regulation of hormone-stimulated PLC signal transduction may occur in
many different tissues, similar to their effects on the AC system, and
therefore we continued to explore the molecular mechanism by which the
levels of Gq/11
proteins are increased.
In the present report, we demonstrate that the predominant
PLC-stimulating G protein expressed in the UMR cell line was
G11. Gq
protein was not detected in our
assays; however, we have been able to detect low levels of mRNA
encoding Gq
in these cells by RT-PCR, suggesting that
there may be very low levels of the Gq
protein that were
undetectable in our immunoblotting assays. Clearly, the
G11
protein is the major protein of the Gq
family expressed in these cells and accounts for all of the protein
detected by the Gq/11
antibody.
After incubation with the synthetic glucocorticioid dexamethasone,
G11 protein levels increased approximately fivefold
within 72 h. This increase in G11
protein was
accompanied by a more modest increase (~70%) in G11
mRNA level. This increase was not the result of changes in the
stability of G11
mRNA but likely was the result of a
stimulation of G11
transcription, because it was not
seen in the presence of the transcription inhibitor actinomycin D. The
glucocorticoid effect on G11
steady-state mRNA levels
appeared to be a secondary response to stimulation of the synthesis of
some other regulatory protein, because the increase in
G11
mRNA was not seen in the presence of the protein synthesis inhibitor cycloheximide. Furthermore, the time course of
dexamethasone-induced increases in G11
mRNA is more
consistent with a secondary response. Primary glucocorticoid responses
involving binding of agonist to the glucocorticoid receptor, nuclear
translocation, and binding to the glucocorticoid response element have
all been shown to occur within 30 min (2). Therefore, the
long delay period of 12-24 h before dexamethasone-mediated
induction of G11
mRNA is not consistent with a primary response.
The magnitude of the increase in G11 mRNA that we have
seen in UMR cells was far less than that previously reported for the effect of dexamethasone on Gs
mRNA in GH3
cells, which were increased fivefold after 72 h (3).
This suggested that the increase in G11
mRNA may not
have been the primary mechanism regulating G11
protein
levels in the UMR cells. Indeed, further investigation demonstrated a
profound effect of glucocorticoids on the stability of
G11
protein, increasing the t1/2
of the protein from 20.3 to 63 h. This is the first report of
glucocorticoid-induced changes in G protein stability. Previous studies
have reported an increase in the Gs
protein stability
with changes in t1/2 from 50 to 72 h after
triiodothyronine treatment of neonatal rat ventricular myocytes
(1). Several studies have also demonstrated that G protein
stability can be decreased after prolonged activation of cells by
hormones (9, 15, 17, 26, 30, 31) or direct activation of
Gs
by cholera toxin (3, 14). Together,
these reports suggest that the rate of degradation of G proteins is regulated by the cellular hormonal environment and as a consequence may
determine the responsiveness of G protein-coupled systems.
The effect of glucocorticoids on protein turnover is tissue specific. In muscle, glucocorticoids have significant proteolytic effects associated with inhibition of protein translation initiation (27), as well as increased proteasome-dependent and calcium-dependent proteolytic pathways (5, 32). In liver, on the other hand, glucocorticoids stimulate gluconeogenesis in part by increasing the production of key hepatic enzymes (8). In the osteoblastic cells used in our study, dexamethasone increased total protein content of the cells by ~30% over 3 days, suggesting a general anabolic effect. This increase in total protein content, as well as the larger increase in specific G protein subunits, may reflect effects of the steroids on cellular proteolytic pathways. There are no reports in the literature of which degradative pathways govern heterotrimeric G protein subunit life spans; therefore, it is difficult to speculate how this could be altered by glucocorticoids. We are currently pursuing studies to determine which proteolytic pathways degrade G proteins in the osteoblastic cells and how these are influenced by glucocorticoids.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. I. Simon for the G11 cDNA.
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FOOTNOTES |
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This research was supported by a research grant from the Canadian Institute of Health Research.
Address for reprint requests and other correspondence: J. Mitchell, Rm. 4342, Dept. of Pharmacology, Medical Science Bldg., 1 King's College Circle, Univ. of Toronto, Toronto, ON, Canada M5S 1A8 (E-mail: jane.mitchell{at}utoronto.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 June 2001; accepted in final form 23 August 2001.
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