Mechanisms of regulation of G11alpha protein by dexamethasone in osteoblastic UMR 106-01 cells

Ricky Cheung and Jane Mitchell

Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5S 1A8


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously demonstrated that glucocorticoids increased Gq/11alpha protein expression and phospholipase C activity in the rat osteosarcoma cell line UMR 106-01. In this study, we demonstrated that G11alpha is the primary Gq-subtype family member expressed in UMR cells. Dexamethasone treatment increased the expression of G11alpha protein in both a time- and a dose-dependent manner. Glucocorticoid treatment significantly increased the half-life of G11alpha protein from 20.3 to 63 h. Steady-state G11alpha 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 G11alpha mRNA was blocked by the transcription inhibitor actinomycin D. The dexamethasone induction of G11alpha 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 G11alpha protein results primarily from changes in the degradation rate of the protein, whereas changes in G11alpha mRNA play a smaller role and require de novo synthesis of regulatory protein(s).

glucocorticoids; G protein; G11alpha ; osteosarcoma


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HETEROTRIMERIC G PROTEINS are a family of guanine nucleotide-binding proteins composed of three subunits: alpha , beta , and gamma . 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 Gsalpha in reticulocytes (28) but to increase the steady-state levels of Gialpha (12), Goalpha (13), and Gbeta 1/2 (21) in adipose tissue. Subsequently, Gbeta 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 Gialpha while decreasing the level of Gsalpha in liver (6) and Gbeta 1/2 in adipose tissue (22). Alteration in the mRNA level of Gialpha , Gsalpha (24), and Gbeta 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 Gsalpha but decreases those of Gialpha 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 Gsalpha in rat pituitary cells (3) and Gbeta 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/11alpha 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/11alpha protein expression. Glucocorticoid administration to rats has also been reported to increase Gq/11alpha 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/11alpha protein and mRNA.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 G11alpha protein was purchased from Chemicon (Temecula, CA). Antibodies raised to peptides corresponding to the amino acid sequence within the amino-terminal domain of Gqalpha and G11alpha , or to the carboxy terminus of Gq/11alpha , were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Full-length mouse G11alpha cDNA was generously provided by Dr. M. I. Simon, California Institute of Technology Division of Biology (Pasedena, CA). [alpha -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). G11alpha was identified on immunoblots as a band migrating at the same position as a recombinant G11alpha protein standard run on the same gel. This band was not seen when immunoblots were incubated with anti-G11alpha antisera in the presence of a G11alpha -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/11alpha 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 G11alpha cDNA probe was obtained by cutting the plasmid pCISGalpha 11 (29) with ClaI and XhoI to yield a 1,200-bp fragment that represented the full-length G11alpha 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 [alpha -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of Gqalpha and G11alpha protein expression in UMR 106-01 cells. Our laboratory has previously demonstrated that dexamethasone increased Gq/11alpha protein expression and PLC activity (16). Because the antiserum used previously for quantitation recognized both Gqalpha and G11alpha 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 Gqalpha and G11alpha subunits, we found that G11alpha is the primary PLC-activating G protein expressed in UMR cells and that Gqalpha was not detected (Fig. 1A). On the basis of this finding, we used the G11alpha -specific antibody to reevaluate the effect of dexamethasone on G11alpha protein expression. Treatment with 100 nM dexamethasone for 3 days increased G11alpha protein expression fivefold (Fig. 1B), indicating that dexamethasone is a potent regulator for G11alpha expression in these cells.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1.   A: relative levels of Gqalpha and G11alpha protein in UMR 106-01 cells. Brain (B), liver (L), or UMR (U) cell membranes were subjected to SDS-PAGE and then immunoblotted using Gqalpha , G11alpha , and Gq/11alpha -specific antiserum as described in EXPERIMENTAL PROCEDURES. B: effect of dexamethasone on expression of G11alpha protein in UMR 106-01 cells. Cells were incubated with 100 nM dexamethasone (Dex) or 0.001% ethanol (EtOH, vehicle) for 72 h. Membranes (40 µg) were subjected to SDS-PAGE and immunoblotted using a G11alpha -specific antipeptide antiserum, as described in EXPERIMENTAL PROCEDURES. Bars represent means ± SE of 3 experiments (representative blot shown in inset). Values significantly different from control, *P < 0.05.

As shown in Fig. 2A, dexamethasone elevated the level of G11alpha in a time-dependent fashion. Significant increase in G11alpha protein was observed after 48 h of incubation with dexamethasone and continued to increase after 72 h. No further increases in G11alpha protein were seen after longer incubations with dexamethasone (data not shown). Dexamethasone stimulation of G11alpha protein expression was dose dependent (Fig. 2B), with maximal stimulation of G11alpha protein expression seen with 100 nM dexamethasone treatment, which did not increase further with higher concentrations.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Characterization of dexamethasone effects on G11alpha protein expression in UMR 106-01 cells. Time (A) and concentration (B) dependence of dexamethasone on G11alpha protein expression in UMR cells. Cells were incubated with 100 nM dexamethasone or 0.001% ethanol (vehicle) for indicated times (for time course experiment) or with indicated concentrations of dexamethasone for 72 h (for concentration effect experiment). Increasing amounts (20, 40, and 60 µg) of UMR cell membranes were subjected to SDS-PAGE and immunoblotted using a G11alpha -specific antipeptide antiserum as described in EXPERIMENTAL PROCEDURES. Bars represent means ± SE of triplicate determinations (representative blot shown in inset). Values significantly different from control, *P < 0.05.

Effect of dexamethasone on G11alpha protein stability. To investigate whether the elevated steady-state G11alpha 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 G11alpha protein with time was monitored in cells that were either untreated or treated with 100 nM dexamethasone. Antiserum CQ2, which recognized both Gqalpha and G11alpha , was used to immunoprecipitate G11alpha from the UMR cell extracts. The rate of loss of 35S-labeled G11alpha protein was slower in cells treated with dexamethasone (Fig. 3). In untreated UMR cells, the half-life (t1/2) for G11alpha protein was estimated to be ~20.3 h, whereas the glucocorticoid-treated cells demonstrated a threefold increase in t1/2 for G11alpha to 63 h.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of dexamethasone on stability of G11alpha protein in UMR 106-01 cells. Cells were labeled with Tran-[35S]methionine for 24 h and then incubated with 100 nM dexamethasone () or 0.001% ethanol (open circle ) for indicated times. Membranes were prepared and immunoprecipitated with Gq/11alpha -specific antiserum. Isolated proteins were subjected to SDS-PAGE, and the resulting gel was exposed to a phosphor plate for 72 h. Bands were quantified using ImageQuant software as described in EXPERIMENTAL PROCEDURES. Each point represents mean ± SE of 2 experiments (representative blot shown in inset).

Effect of dexamethasone on steady-state G11alpha mRNA level. To assess whether changes in G11alpha mRNA expression could also contribute to the glucocorticoid-induced increase in steady-state G11alpha protein expression, the effect of dexamethasone on G11alpha mRNA level was determined by Northern blot analysis. With use of a 32P-labeled mouse G11alpha 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 G11alpha mRNA expression after 24 h of hormone treatment (Fig. 4A). Longer exposure to glucocorticoids in UMR cells produced no further increase in G11alpha mRNA level, but levels remained significantly higher than those of the untreated cells. The increase in G11alpha 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 G11alpha mRNA level was more sensitive to low concentrations of dexamethasone (1 nM) compared with its effect on steady-state protein level.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4.   Characterization of dexamethasone effects on G11alpha mRNA expression in UMR 106-01 cells. Time (A) and concentration (B) dependence of dexamethasone on G11alpha mRNA expression in UMR cells. Cells were incubated with 100 nM dexamethasone or 0.001% ethanol (vehicle) for indicated times (for time course experiment) or with indicated concentrations of dexamethasone for 72 h (for concentration effect experiment). Extracted total RNA (2.5 µg) was loaded and resolved by 2% agarose gel electrophoresis. Transferred RNA blots were hybridized with 32P-labeled rat G11alpha and 18S cDNA and visualized by autoradiography. Bands were quantified using ImageQuant software as described in EXPERIMENTAL PROCEDURES. Bars represent means ± SE of 3 different RNA preparations (representative blot shown in inset). Values significantly different from control, *P < 0.05.

Mechanism of dexamethasone-induced G11alpha 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 G11alpha 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 G11alpha mRNA transcript remained stable for the first 8 h and then began to decrease; the t1/2 for G11alpha mRNA was ~37.5 h, and dexamethasone treatment did not affect the stability of the transcript in these cells (Fig. 5A).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5.   Mechanism of dexamethasone-induced G11alpha mRNA induction. A: effect of dexamethasone on stability of G11alpha mRNA in UMR 106-01 cells. Cells were incubated with 100 nM dexamethasone () or 0.001% ethanol (open circle ) for 24 h, followed by addition of 150 µM DRB for indicated times. B: effect of transcription inhibitor actinomycin D (AD) on dexamethasone (Dex)-mediated response to G11alpha mRNA. Cells were treated for 24 h with 100 nM dexamethasone or 0.001% ethanol (vehicle) in the absence or presence of 3 ng/ml actinomycin D. Total RNA was extracted at the indicated time points and subjected to Northern blot analysis. RNA (2.5 µg) was loaded and resolved by 2% agarose gel electrophoresis. Transferred RNA blots were hybridized with 32P-labeled rat G11alpha and 18S cDNA and visualized by autoradiography. Bands were quantified using ImageQuant software as described in EXPERIMENTAL PROCEDURES. Each point represents mean ± SE of 2 individual experiments (representative blot shown in inset). Values significantly different from control, *P < 0.05.

A transcription inhibitor, actinomycin D, was used to investigate whether de novo RNA synthesis was required for the observed increase in steady-state G11alpha mRNA levels by dexamethasone. UMR cells were treated for 24 h with dexamethasone (100 nM) in the presence or absence of 3 ng/ml of actinomycin D. Under this condition, actinomycin D effectively inhibited transcription without having toxic effects on the cells. As shown in Fig. 5B, actinomycin D alone had no inhibitory effect on G11alpha mRNA expression in control cells, but it completely blocked the dexamethasone-induced stimulation of G11alpha mRNA expression.

Characterization of dexamethasone-mediated response on G11alpha mRNA. As shown in Fig. 4A, there were no significant changes in G11alpha 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 G11alpha mRNA expression by ~40% and completely abolished the dexamethasone-induced stimulation of G11alpha mRNA expression (Fig. 6). Together, these results indicate that the glucocorticoid effect on G11alpha transcription was a secondary response.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of cycloheximide (CX) on dexamethasone-induced increases in G11alpha mRNA expression in UMR 106-01 cells. Cells were treated for 24 h with 100 nM dexamethasone or 0.001% ethanol (vehicle) in the absence or presence of 1 µM CX. Total RNA was extracted and subjected to Northen blot analysis. RNA (2.5 µg) was loaded and resolved by 2% agarose gel electrophoresis. Transferred RNA blots were hybridized with 32P-labeled rat G11alpha and 18S cDNA and visualized by autoradiography. Bands were quantified using ImageQuant software as described in EXPERIMENTAL PROCEDURES. Each point represents means ± SE of 3 individual experiments (representative blot shown in inset). Values significantly different from control, *P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PTH stimulation of the PTH/PTHrP receptor on osteoblastic cells leads to stimulation of both Gsalpha and Gq/11alpha , 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 Gsalpha 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/11alpha proteins by glucocorticoids (16). This was followed by the demonstration of a similar upregulation of Gq/11alpha 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/11alpha proteins are increased.

In the present report, we demonstrate that the predominant PLC-stimulating G protein expressed in the UMR cell line was G11alpha . Gqalpha protein was not detected in our assays; however, we have been able to detect low levels of mRNA encoding Gqalpha in these cells by RT-PCR, suggesting that there may be very low levels of the Gqalpha protein that were undetectable in our immunoblotting assays. Clearly, the G11alpha protein is the major protein of the Gq family expressed in these cells and accounts for all of the protein detected by the Gq/11alpha antibody.

After incubation with the synthetic glucocorticioid dexamethasone, G11alpha protein levels increased approximately fivefold within 72 h. This increase in G11alpha protein was accompanied by a more modest increase (~70%) in G11alpha mRNA level. This increase was not the result of changes in the stability of G11alpha mRNA but likely was the result of a stimulation of G11alpha transcription, because it was not seen in the presence of the transcription inhibitor actinomycin D. The glucocorticoid effect on G11alpha steady-state mRNA levels appeared to be a secondary response to stimulation of the synthesis of some other regulatory protein, because the increase in G11alpha mRNA was not seen in the presence of the protein synthesis inhibitor cycloheximide. Furthermore, the time course of dexamethasone-induced increases in G11alpha 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 G11alpha mRNA is not consistent with a primary response.

The magnitude of the increase in G11alpha mRNA that we have seen in UMR cells was far less than that previously reported for the effect of dexamethasone on Gsalpha mRNA in GH3 cells, which were increased fivefold after 72 h (3). This suggested that the increase in G11alpha mRNA may not have been the primary mechanism regulating G11alpha protein levels in the UMR cells. Indeed, further investigation demonstrated a profound effect of glucocorticoids on the stability of G11alpha 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 Gsalpha 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 Gsalpha 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.


    ACKNOWLEDGEMENTS

We thank Dr. M. I. Simon for the G11alpha cDNA.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Bahouth, SW. Thyroid hormone regulation of transmembrane signalling in neonatal rat ventricular myocytes by selective alteration of the expression and coupling of G-protein alpha-subunits. Biochem J 307: 831-841, 1995[ISI][Medline].

2.   Beato, M, Kalimi M, and Feigelson P. Correlation between glucocorticoid binding to specific liver cytosol receptors and enzyme induction in vivo. Biochem Biophys Res Commun 47: 1464-1472, 1972[ISI][Medline].

3.   Chang, FH, and Bourne HR. Dexamethasone increases adenylyl cyclase activity and expression of the alpha-subunit of Gs in GH3 cells. Endocrinology 121: 1711-1715, 1987[Abstract].

4.   Davies, AO, and Lefkowitz RJ. Regulation of beta-adrenergic receptors by steroid hormones. Annu Rev Physiol 46: 119-130, 1984[ISI][Medline].

5.   Du, J, Mitch WE, Wang X, and Price SR. Glucocorticoids induce proteasome C3 subunit expression in L6 muscle cells by opposing the suppression of its transcription by NF-kappa B. J Biol Chem 275: 19661-19666, 2000[Abstract/Free Full Text].

6.   Garcia-Sainz, JA, Huerta-Bahena ME, and Malbon CC. Hepatocyte beta -adrenergic responsiveness and guanine nucleotide-binding regulatory proteins. Am J Physiol Cell Physiol 256: C384-C389, 1989[Abstract/Free Full Text].

7.   Gilman, AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56: 615-649, 1987[ISI][Medline].

8.   Hornbrook, KR, Burch HB, and Lowry OH. The effects of adrenalectomy and hydrocortisone on rat liver metabolites and glycogen synthetase activity. Mol Pharmacol 2: 106-116, 1966[Abstract].

9.   Kai, H, Fukui T, Lassegue B, Shah A, Minieri CA, and Griendling KK. Prolonged exposure to agonist results in a reduction in the levels of the Gq/G11 alpha subunits in cultured vascular smooth muscle cells. Mol Pharmacol 49: 96-104, 1996[Abstract].

10.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

11.   Lowik, CW, van Leeuwen JP, van der Meer JM, van Zeeland JK, Scheven BA, and Herrmann-Erlee MP. A two-receptor model for the action of parathyroid hormone on osteoblasts: a role for intracellular free calcium and cAMP. Cell Calcium 6: 311-326, 1985[ISI][Medline].

12.   Malbon, CC, Rapiejko PJ, and Mangano TJ. Fat cell adenylate cyclase system. Enhanced inhibition by adenosine and GTP in the hypothyroid rat. J Biol Chem 260: 2558-2564, 1985[Abstract].

13.   Milligan, G, Spiegel AM, Unson CG, and Saggerson ED. Chemically induced hypothyroidism produces elevated amounts of the alpha subunit of the inhibitory guanine nucleotide binding protein (Gi) and the beta subunit common to all G-proteins. Biochem J 247: 223-227, 1987[ISI][Medline].

14.   Milligan, G, Unson CG, and Wakelam MJ. Cholera toxin treatment produces down-regulation of the alpha-subunit of the stimulatory guanine-nucleotide-binding protein (Gs). Biochem J 262: 643-649, 1989[ISI][Medline].

15.   Mitchell, FM, Buckley NJ, and Milligan G. Enhanced degradation of the phosphoinositidase C-linked guanine-nucleotide-binding protein Gq alpha/G11 alpha following activation of the human M1 muscarinic acetylcholine receptor expressed in CHO cells. Biochem J 293: 495-499, 1993[ISI][Medline].

16.   Mitchell, J, and Bansal A. Dexamethasone increases Galpha q-11 expression and hormone-stimulated phospholipase C activity in UMR-106-01 cells. Am J Physiol Endocrinol Metab 273: E528-E535, 1997[Abstract/Free Full Text].

17.   Mullaney, I, Caulfield MP, Svoboda P, and Milligan G. Activation, cellular redistribution and enhanced degradation of the G proteins Gq and G11 by endogenously expressed and transfected phospholipase C-coupled muscarinic m1 acetylcholine receptors. Prog Brain Res 109: 181-187, 1996[ISI][Medline].

18.   Rabadan-Diehl, C, and Aguilera G. Glucocorticoids increase vasopressin V1b receptor coupling to phospholipase C. Endocrinology 139: 3220-3226, 1998[Abstract/Free Full Text].

19.   Rapiejko, PJ, Watkins DC, Ros M, and Malbon CC. Thyroid hormones regulate G-protein beta-subunit mRNA expression in vivo. J Biol Chem 264: 16183-16189, 1989[Abstract/Free Full Text].

20.   Rodan, SB, and Rodan GA. Dexamethasone effects on beta-adrenergic receptors and adenylate cyclase regulatory proteins Gs and Gi in ROS 17/2.8 cells. Endocrinology 118: 2510-2518, 1986[Abstract].

21.   Ros, M, Northup JK, and Malbon CC. Steady-state levels of G-proteins and beta-adrenergic receptors in rat fat cells. Permissive effects of thyroid hormones. J Biol Chem 263: 4362-4368, 1988[Abstract/Free Full Text].

22.   Ros, M, Northup JK, and Malbon CC. Adipocyte G-proteins and adenylate cyclase. Effects of adrenalectomy. Biochem J 257: 737-744, 1989[ISI][Medline].

23.   Ros, M, Watkins DC, Rapiejko PJ, and Malbon CC. Glucocorticoids modulate mRNA levels for G-protein beta-subunits. Biochem J 260: 271-275, 1989[ISI][Medline].

24.   Saito, N, Guitart X, Hayward M, Tallman JF, Duman RS, and Nestler EJ. Corticosterone differentially regulates the expression of Gs alpha and Gi alpha messenger RNA and protein in rat cerebral cortex. Proc Natl Acad Sci USA 86: 3906-3910, 1989[Abstract].

25.   Schaffner, W, and Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem 56: 502-514, 1973[ISI][Medline].

26.   Shah, BH, MacEwan DJ, and Milligan G. Gonadotrophin-releasing hormone receptor agonist-mediated down-regulation of Gq alpha/G11 alpha (pertussis toxin-insensitive) G proteins in alpha T3-1 gonadotroph cells reflects increased G protein turnover but not alterations in mRNA levels. Proc Natl Acad Sci USA 92: 1886-1890, 1995[Abstract].

27.   Shah, OJ, Kimball SR, and Jefferson LS. Acute attenuation of translation initiation and protein synthesis by glucocorticoids in skeletal muscle. Am J Physiol Endocrinol Metab 278: E76-E82, 2000[Abstract/Free Full Text].

28.   Stiles, GL, Stadel JM, De Lean A, and Lefkowitz RJ. Hypothyroidism modulates beta adrenergic receptor adenylate cyclase interactions in rat reticulocytes. J Clin Invest 68: 1450-1455, 1981[ISI][Medline].

29.   Strathmann, M, and Simon MI. G protein diversity: a distinct class of alpha subunits is present in vertebrates and invertebrates. Proc Natl Acad Sci USA 87: 9113-9117, 1990[Abstract].

30.   Svoboda, P, Kim GD, Grassie MA, Eidne KA, and Milligan G. Thyrotropin-releasing hormone-induced subcellular redistribution and down-regulation of G11alpha: analysis of agonist regulation of coexpressed G11alpha species variants. Mol Pharmacol 49: 646-655, 1996[Abstract].

31.   Wise, A, Lee TW, MacEwan DJ, and Milligan G. Degradation of G11 alpha/Gq alpha is accelerated by agonist occupancy of alpha 1A/D, alpha 1B, and alpha 1C adrenergic receptors. J Biol Chem 270: 17196-17203, 1995[Abstract/Free Full Text].

32.   Yeh, JY, Ou BR, and Forsberg NE. Effects of dexamethasone on muscle protein homeostasis and on calpain and calpastatin activities and gene expression in rabbits. J Endocrinol 141: 209-217, 1994[Abstract].


Am J Physiol Endocrinol Metab 282(1):E24-E30
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society