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Originally published In Press as doi:10.1074/jbc.M604416200 on September 1, 2006

J. Biol. Chem., Vol. 281, Issue 43, 32684-32693, October 27, 2006
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Up-regulation of Endogenous RGS2 Mediates Cross-desensitization between Gs and Gq Signaling in Osteoblasts*

Anju Anne Roy{ddagger}12, Caroline Nunn{ddagger}13, Hong Ming{ddagger}1, Min-Xu Zou{ddagger}3, Josef Penninger§, Lorrie A. Kirshenbaum4, S. Jeffrey Dixon{ddagger}||, and Peter Chidiac{ddagger}5

From the {ddagger}Department of Physiology and Pharmacology, ||Canadian Institutes of Health Research Group in Skeletal Development and Remodeling, the University of Western Ontario, London, Ontario N6A 5C1, Canada, the §Institute of Molecular Biotechnology of the Austrian Academy of Sciences, A-1030 Vienna, Austria, and the Department of Physiology, Institute of Cardiovascular Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada

Received for publication, May 9, 2006 , and in revised form, August 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulator of G protein signaling (RGS) proteins limit G protein signals. In this study, we investigated the role of RGS2 in the control of G protein signaling cascades in osteoblasts, the cells responsible for bone formation. Expression of RGS2 was up-regulated in primary cultures of mouse calvarial osteoblasts by parathyroid hormone-related peptide (PTHrP)-(1-34), which stimulates Gs signaling. RGS2 was also up-regulated by extracellular ATP, which selectively activates Gq, as well as by forskolin and phorbol myristate acetate, which activate targets downstream of Gs and Gq, respectively. To assess the role of endogenous RGS2, we characterized Gs and Gq signaling in osteoblasts derived from wild type and rgs2-/- mice. Under control conditions, nucleotide-stimulated calcium release, endothelin-stimulated accumulation of inositol phosphates, and PTHrP-stimulated cAMP accumulation were equivalent in osteoblasts isolated from wild type and rgs2-/- mice. Thus, basal levels of endogenous RGS2 do not appear to regulate Gs or Gq signaling in osteoblasts. Interestingly, forskolin treatment of wild type but not rgs2-/- osteoblasts suppressed both endothelin-stimulated accumulation of inositol phosphates and nucleotide-stimulated calcium release, indicating that up-regulation of RGS2 by Gs signaling desensitizes Gq signals. Furthermore, pretreatment with ATP suppressed PTHrP-dependent cAMP accumulation in wild type but not rgs2-/- osteoblasts, implying that up-regulation of RGS2 by Gq signaling desensitizes Gs signals. Our findings demonstrate that endogenously expressed RGS2 can limit Gs signaling. Moreover, up-regulation of RGS2 contributes to cross-desensitization of Gs- and Gq-coupled signals.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-coupled receptors (GPCRs)6 respond to a variety of hormones, paracrine factors, and neurotransmitters by activating heterotrimeric G proteins. Upon activation of the G protein, G{alpha} is stimulated to exchange bound GDP for cytosolic GTP and is thought to subsequently dissociate from the beta{gamma} dimer. Both G{alpha} and the beta{gamma} dimer are then capable of interacting with cellular effectors for a period of time that is limited by the intrinsic GTPase activity of the G{alpha} subunit. Regulator of G protein signaling (RGS) proteins can reduce the duration of these interactions by increasing the rate of GTP hydrolysis by the G{alpha} subunits, or by otherwise blocking interactions between G{alpha} and its target enzymes through a poorly understood process sometimes referred to as "effector antagonism" (1, 2).

G protein signaling in osteoblasts is a critical regulator of bone formation. The predominant GPCRs expressed by osteoblasts include the parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor type 1 (PTH1R), P2Y nucleotide receptors, and prostaglandin receptors (3). Other GPCRs found in osteoblasts include endothelin, adenosine, beta-adrenergic, angiotensin II, and calcium-sensing receptors (3, 4). Binding of PTH to its specific receptor, PTH1R, predominantly leads to the activation of Gs (although under certain conditions the receptor can also couple to Gq and Gi) (5, 6). Activated Gs stimulates adenylyl cyclase to generate intracellular cAMP, which results in the activation of protein kinase A. Stimulation of Gq promotes the activity of phospholipase C-beta (PLC-beta), resulting in the accumulation of inositol 1,4,5-trisphosphate and diacylglycerol, which lead, respectively, to release of calcium from intracellular stores and activation of protein kinase C. Nucleotide stimulation of P2Y receptors in osteoblasts results in PLC-beta activation and calcium mobilization, with no effect on adenylyl cyclase (3, 7).

An understanding of the role of RGS proteins in regulating GPCR signal transduction is slowly emerging. RGS proteins interact selectively with G proteins and their corresponding receptors (8). Most RGS proteins interact with the Gi family of G proteins and a subset also interact with Gq (1, 9). RGS2, RGS3, RGS13, and RGS-PX1 have been shown to regulate Gs activity in transfection studies (8, 10-16). RGS2 is unique in preferring Gq over Gi, with a relatively low affinity for Gi as compared with other RGS proteins (17).

Recently, RGS2 has been implicated in the regulation of G protein signaling in bone. Northern blot analyses of rat metaphyseal and diaphyseal bone, mouse calvarial organ cultures, and cultured osteoblasts demonstrated that levels of RGS2 mRNA are rapidly and transiently increased in response to PTH, PTHrP, and prostaglandin E2 (18, 19). RGS2 mRNA is also up-regulated in osteoblasts by promoters of protein kinase A activity, such as forskolin, cholera toxin, and cell-permeant cAMP analogues (18, 19), and to a lesser extent by the protein kinase C activator phorbol myristate acetate (PMA) and the calcium ionophore ionomycin (19). Real time PCR studies have further established that RGS2 mRNA is up-regulated by PTH and forskolin in osteoblast-like cell lines (20, 21). In contrast, there seems to be no overt bone-related phenotype in rgs2-/- mice (22), which suggests that RGS2 is not involved in bone development and/or function under basal conditions.

Functionally, RGS2 increases the rate of GTP hydrolysis on the G{alpha}q subunit (9, 23) and also inhibits GTP{gamma}S-stimulated activation of PLC-beta by G{alpha}q (24). In contrast, RGS2 has no effect on the rate of GTP hydrolysis by the G{alpha}s subunit (8, 23) but nevertheless inhibits intracellular cAMP accumulation (8, 13-16). Both Gq and Gs were found to recruit RGS2 to the plasma membrane in mammalian cells (8), and both of these G proteins appear to bind directly to RGS2 (16, 20, 21, 25).

This study demonstrates that RGS2 expression is regulated by both Gs and Gq signaling in primary cultures of osteoblasts. Furthermore, we employed an rgs2-/- model (22) to assess the effects of endogenous RGS2 in osteoblasts. Basal levels of endogenous RGS2 had no detectable effects on Gs or Gq signaling in osteoblasts. However, up-regulation of RGS2 by agents that activate the Gs signaling pathway inhibited P2Y and endothelin receptor-stimulated Gq signals. Conversely, Gq-mediated up-regulation of RGS2 inhibited PTH1R-stimulated Gs signals, revealing previously unrecognized cross-talk between these pathways.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Study Protocol—The generation and genotyping of rgs2-/- mice have been described previously (22). rgs2-/- mice were backcrossed with C57BL/6 strain from Charles River Breeding Laboratories (Canada) for six generations to ensure equivalent genetic backgrounds before performing any experiments. After this backcross, rgs2+/- mice were mated to generate mice with an rgs2-/- genotype, and all rgs2-/- mice used in this study were of the F1 and F2 generation from this cross. Mice designated as wild type (rgs2+/+) were obtained from the C57BL/6 mice that were used for back-crossing. Mice were provided with normal food and water and subjected to a standard light/dark cycle. Animals were maintained in accordance with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals. The studies were approved by the Council on Animal Care at the University of Western Ontario and complied with the guidelines of the Canadian Council on Animal Care.

Osteoblast Isolation and Cell Culture—Primary cultures of osteoblasts were isolated from mouse calvariae by sequential enzymatic digestion as described previously (26), and populations II to V were cultured in {alpha}-minimum essential medium (Invitrogen) with 10% fetal bovine serum (HyClone) and antibiotics (Invitrogen). Osteoblasts were initially seeded at a density of 1-1.5 x 106 cells/10-cm plate. Cells were cultured for 7-10 days prior to being assayed.

Recombinant Adenovirus—The cDNA encoding RGS2 was purchased from the Guthrie Institute, Danville, PA. A replication defective adenovirus encoding RGS2 was generated as described previously (27). Where indicated, osteoblasts were infected with adenovirus encoding either RGS2 or green fluorescent protein (GFP) (control) at a multiplicity of infection of 100 for 24 h. Following adenoviral infection, osteoblasts were maintained in {alpha}-minimum essential medium with 10% fetal bovine serum for an additional 24 h prior to intracellular cAMP measurements.

Real Time PCR—Osteoblasts from wild type and rgs2-/- mice were treated with PTHrP-(1-34) (human, rat; Bachem) (100 nM) (vehicle = acetic acid 5 µM, bovine albumin 1 µg/ml), forskolin (100 µM) (Tocris Biosciences) (vehicle = dimethyl sulfoxide 0.1%), ATP (10 µM) (Sigma), PMA (1 µM) (inactive control = 4-{alpha}-PMA, 1 µM), or vehicle for 2 h. After treatment, total RNA was extracted by using Trizol® reagent (Invitrogen) according to the manufacturer's instructions. 5 µg of total RNA was used for reverse transcription followed by real time PCR using SYBR Green chemistry (Sigma). The primer sequences used for RGS2 were sense primer 5'-AGTAAATATGGGCTGGCTGCATTC-3' and antisense primer 5'-GCCTCTTGGATATTTTGGGCAATC-3'. The PCR conditions were 94 °C for 10 min, followed by 35 cycles at 94 °C for 20 s, 58 °C for 20 s, and 72 °C for 30 s. Relative gene expression of RGS2 was determined based on the threshold cycles of the gene in relation to the threshold cycle of the corresponding internal standard 18 S RNA. The data were normalized by expressing the level of RGS2 mRNA in osteoblasts that were treated with test agents as a fraction of RGS2 mRNA in vehicle-treated osteoblasts.

Immunofluorescence—Primary osteoblast cultures were treated with ATP (10 µM) or forskolin (100 µM) for 3 h and then fixed with periodate/lysine/paraformaldehyde for 30 min (28). Fixed cells were permeabilized with 0.3% Triton X-100 in phosphate-buffered saline for 10 min and then blocked with 5% bovine albumin in phosphate-buffered saline for 20 min at room temperature to minimize nonspecific binding of antibodies. Cells were incubated with a polyclonal goat antibody to the first 16 amino-terminal residues of RGS2 (Santa Cruz Biotechnology, catalogue number sc-9933; dilution 1:250, overnight at 4 °C) and subsequently incubated with an anti-goat IgG conjugated to Texas Red-X (Molecular Probes; dilution 1:1000, 1 h at room temperature). Following repeated washings, coverslips were mounted on slides with Immumount (Fisher) for visualization by confocal microscopy. Slides were observed using an LSM 410 confocal microscope (Zeiss) equipped with a krypton/argon laser and x64 oil immersion lens. For each experimental condition, fluorescence distribution patterns similar to the image shown were observed in the majority (70-80%) of cells inspected.

Western Blotting—Osteoblasts from wild type and rgs2-/- mice seeded in 6-cm plates were treated with forskolin (100 µM) or vehicle (dimethyl sulfoxide, 1%) for 3 h. Cell lysates were prepared on ice by washing twice with ice-cold phosphate-buffered saline and scraping into 70 µl of lysis buffer (150 mM NaCl, 50 mM Hepes, pH 7.4, 1% Triton X-100, 2 mM EDTA, 10 mM NaF, 100 µM Na3VO4, 200 µM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 10 µg/ml aprotinin). The lysates were sheared through a 26-gauge needle and sedimented at 13,000 x g for 15 min at 4 °C. Protein concentrations of the supernatant were determined using the Bradford assay protein quantification kit (Bio-Rad). Equal amounts of cell proteins were separated by 12% SDS-PAGE and electroblotted onto polyvinylidene difluoride membrane (Pall Corp.). The membranes were incubated for 1 h in blocking buffer (Tris-buffered saline, 5% nonfat milk, 0.1% Tween 20) before overnight incubation with a polyclonal chicken antibody to full-length RGS2 (1:800 in blocking buffer, Genway Biotech catalogue number A22245F) and 1 h incubation with horseradish peroxidase-conjugated anti-chicken IgG (1:800 in blocking buffer; Promega). The immunoblots were visualized with Lumiglow Reserve chemiluminescent substrate (Kirkegaard & Perry Laboratories) using a digital camera (FluorChem 8000, AlphaEaseFC software; Alpha Innotech Corp.). Purified His6-tagged RGS2 (prepared as described previously (17)) was included on the gel as a positive control.

Measurement of Cytosolic Calcium—Calvarial osteoblasts from wild type and rgs2-/- mice were treated with forskolin (100 µM) or vehicle (0.1% dimethyl sulfoxide) for 2.5 h, and then loaded with indo-1-AM (2 µM) by incubation in serum-free {alpha}-minimum essential medium for an additional 30 min. Cells were then washed and harvested by a 1-min exposure to trypsin solution. Conditioned medium was added to inactivate trypsin; subsequently, cells were sedimented and resuspended in HCO-3-free {alpha}-minimum essential medium. Aliquots of cell suspension were sedimented and resuspended in 2 ml of continuously stirred calcium-Hepes buffer (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 20 mM Na+-Hepes, pH 7.3) in a fluorometric cuvette maintained at 37 °C. ATP (10 µM) or UTP (10 µM) was added directly to the cuvette.

Cytosolic calcium was monitored using a dual emission spectrofluorometer (QuantaMaster model QM-8/2001, from Photon Technology International, Lawrenceville, NJ) at 355 nm excitation and emission wavelengths of 405 and 485 nm. The system software was used to subtract background fluorescence and calculate the ratio, R, which is the fluorescence intensity at 405 nm divided by the intensity at 485 nm. [Ca2+] was determined from the relationship [Ca2+] = Kd ((R - Rmin)/(Rmax - R))beta, where Kd (for the indo-1-calcium complex) was taken as 250 nM, Rmin and Rmax were the values of R at low and saturating concentrations of calcium, respectively, and beta was the ratio of the fluorescence at 485 nm measured at low and saturating calcium concentrations (29).

To estimate the size of intracellular calcium stores, indo-1-loaded osteoblasts were resuspended in nominally calcium-free buffer (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 20 mM Na+-Hepes, pH 7.3) immediately prior to measurement of [Ca2+]. Residual extracellular calcium was depleted with 0.5 mM EGTA, and calcium stored in intracellular compartments was released using ionomycin (1 µM).

Measurement of Accumulation of Inositol Phosphates—Calvarial osteoblasts from wild type and rgs2-/- mice in 12-well plates were incubated overnight with 0.5 µCi/ml myo-[2-3H]inositol (PerkinElmer Life Sciences) in serum-free Dulbecco's modified Eagle's medium (Invitrogen). On the day of the experiment, forskolin (100 µM) or vehicle (dimethyl sulfoxide, 1%) was added to the wells for 1 h (forskolin had no discernible effect on cellular uptake of myo-[2-3H]inositol). Unincorporated myo-[2-3H]inositol was removed by washing the cells twice with Hanks' balanced salt solution (HBSS) (116 mM NaCl, 20 mM Hepes, 11 mM glucose, 5 mM NaHCO3, 4.7 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4, 1.2 mM KH2PO4, pH 7.4) containing 10 mM LiCl.7 Cells were then incubated for 15 min in 500 µl of HBSS containing 10 mM LiCl to inhibit inositol monophosphatase before treatment with endothelin for 15 min. The reaction was stopped by 30 min of incubation on ice with 500 µl of perchloric acid (0.8 M), and the lysate was neutralized with 400 µl of 0.72 M KOH, 0.6 M KHCO3. For each sample, total myo-[2-3H]inositol incorporated into the cells was determined by counting the radioactivity present in 50 µl of the cell lysate, and total inositol phosphates were recovered from the cell extracts by anion exchange chromatography using Dowex AG1-X8 (formate form), 200-400 dry mesh anion exchange resin. 900 µl of the cell extract was added to the columns before successive washing with distilled water and 60 mM ammonium formate. Bound inositol phosphates were eluted with 1 M ammonium formate, 0.1 M formic acid, and radioactivity was determined in a liquid scintillation counter. For each sample the percent conversion of total myo-[2-3H]inositol to [3H]inositol phosphates was determined, and data were normalized to the effect of 1 µM endothelin in vehicle-treated cells. Maximum activities ranged from 0.16 to 0.65 (wild type) and 0.12 to 0.20 (rgs2-/-) percent conversion of total myo-[2-3H]inositol to [3H]inositol phosphates.

Measurement of Intracellular cAMP—Osteoblasts were incubated with 2 µCi/ml [3H]adenine (30 Ci/mmol) for 3 h at 37 °C to allow for the accumulation of intracellular [3H]ATP. During this time, some cells were co-incubated with ATP (10 µM) to promote RGS2 expression (total cellular uptake of [3H]adenine in cells treated with ATP was 47 ± 5% of that in vehicle-treated cells, however there was no difference in uptake between wild type and rgs2-/- cells). After cells were rinsed with phosphate-buffered saline and given fresh medium, the assay was started by the addition of 0.5 mM isobutylmethylxanthine plus PTHrP-(1-34) (vehicle = acetic acid 5 µM, bovine albumin, 1 µg/ml), forskolin (vehicle = 0.1% Me2SO) or vehicle at 37 °C. After 2 min, cells were lysed with 10% trichloroacetic acid, plus 2 mM cAMP and 2 mM ATP. [3H]cAMP was recovered by sequential chromatography over Dowex and alumina (30, 31). For each sample, cellular uptake of tritium was estimated from the lysate, and recovered [3H]cAMP was calculated as a fraction of total cellular tritium to control for differences in cell number and uptake of [3H]adenine.


Figure 1
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  		FIGURE 1.
Regulation of RGS2 protein expression in osteoblasts. A, confocal microscopy. Calvarial osteoblast cultures prepared from wild type (rgs2+/+) and rgs2-/- mice were treated with vehicle (0.1% dimethyl sulfoxide; panels ii and v), 10 µM ATP (panels i, iii, and vi), or 100 µM forskolin (panel iv) for 3 h, followed by fixing and immunostaining using a goat polyclonal antibody to RGS2 (panels ii-vi). Images were captured using Zeiss LSM 410 confocal software at x64 magnification and a resolution of 512 x 521. After treatment with ATP or forskolin, osteoblasts from wild type mice expressed detectable levels of RGS2 protein. All scale bars represent 20 µm. B, Western blotting. Lysates from wild type (rgs2+/+) and rgs2-/- osteoblast cells treated with vehicle (1% dimethyl sulfoxide) or 100 µM forskolin for 3 h were separated by 12% SDS-PAGE and immunoblotted using a chicken polyclonal antibody for RGS2. Purified 6xHis-RGS2 was included on the gel as a positive control.

 
Data Analysis and Statistics—Peak calcium elevation, cAMP accumulation, and real time PCR data were calculated as means ± S.E. Concentration dependence data of cAMP and inositol phosphates were analyzed by nonlinear regression using a sigmoidal curve fit with a variable slope (GraphPad Prism 3.0). For each individual experiment, the EC50 values and maximal effect were calculated. The presented EC50 and maximal values are the means ± S.E. obtained from the individual fits to multiple independent data sets. Data were assessed using the Student's t test or analysis of variance followed by multiple comparisons test (GraphPad Prism 3.0). Statistical significance was defined as p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Regulation of RGS2 mRNA in Mouse Osteoblasts—We first evaluated the effects of activation of G protein signaling on RGS2 expression. Primary cultures of calvarial osteoblasts from wild type and rgs2-/- mice were treated with various agents, and RGS2 mRNA levels were subsequently analyzed by real time PCR (Table 1). RGS2 mRNA from wild type animals was significantly up-regulated after 2 h of treatment with PTHrP. RGS2 mRNA was even more strongly up-regulated by the adenylyl cyclase activator forskolin. No RGS2 mRNA was detected under any condition in cells from rgs2-/- animals. RGS2 mRNA was also up-regulated in wild type osteoblasts by extracellular ATP. Because ATP binds to P2Y receptors to activate Gq, osteoblasts were also treated with the phorbol ester PMA, which activates protein kinase C. PMA also induced RGS2 gene expression. Therefore, RGS2 mRNA was up-regulated by activators of both the Gs- and Gq-coupled pathways in murine osteoblasts.


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  		TABLE 1
Regulation of RGS2 mRNA in mouse osteoblasts

Osteoblasts from wild type mice were treated with PTHrP-(1-34) (100 nM), forskolin (100 µM), ATP (10 µM), or PMA (1 µM) and harvested after 2 h. Total RNA was extracted from osteoblasts and used for reverse transcription followed by real time PCR. The amount of RGS2 mRNA was calculated as a fraction of 18 S RNA. The fold induction of RGS2 mRNA was then calculated as the ratio of RGS2 mRNA expression in treated cells to RGS2 mRNA expression in vehicle-treated cells. Levels of RGS2 mRNA in vehicle-treated cells were comparable to RGS2 mRNA levels in control cells. Data represent the means ± S.E. of at least five individual experiments. Statistically significant increases (*, p < 0.05) in RGS2 mRNA expression for each treatment group compared to control were determined using one sample t test.

 
Expression of RGS2 Protein—To determine whether up-regulation of RGS2 mRNA is followed by increased protein abundance in osteoblasts, immunofluorescence was performed on fixed cells using a commercial RGS2 antibody. RGS2 protein expression was revealed by fluorescence of Texas Red-conjugated secondary antibody. Labeling was undetectable in cells from wild type mice in which RGS2 antibody was absent (Fig. 1A, panel i), as well as in vehicle-treated cells from wild type mice (Fig. 1A, panel ii). However, upon treatment with ATP or forskolin, RGS2 protein expression was easily detected in wild type osteoblasts (Fig. 1A, panels iii and iv). As expected, RGS2 staining was also undetectable in osteoblasts prepared from rgs2-/- mice (Fig. 1A, panels v and vi). Western blotting, using anti-RGS2, was also performed on lysates of osteoblasts from wild type and rgs2-/- mice (Fig. 1B). These immunoblots confirmed that treatment with forskolin (100 µM, 3 h) leads to a large increase in RGS2 protein levels that are undetectable under basal conditions. Furthermore, in lysates from rgs2-/- osteoblasts, there was no detectable RGS2 protein under either basal or forskolin-treated conditions. Taken together, these results indicate that up-regulation of RGS2 mRNA corresponds to increased expression of RGS2 protein. Furthermore, RGS2 protein expression is below detection levels under control conditions, which may simply be a technical limitation (i.e. low sensitivity of antibody) or, alternatively, could suggest that basal levels of RGS2 in these cells are low and thus may not significantly affect G protein signaling.

Effect of RGS2 on Calcium Signaling—We next tested the potential role of endogenous RGS2 on Gq-dependent calcium signaling in primary calvarial osteoblasts. Cells were prepared from wild type and rgs2-/- mice. Levels of cytosolic free calcium were monitored, and cells were stimulated with ATP. There was no significant difference in peak calcium elevation between rgs2-/- and wild type osteoblasts (Fig. 2A) (n = 5, p > 0.2). ATP binds to and activates both P2X and P2Y nucleotide receptors in osteoblasts (32). Activated P2Y receptors couple to Gq to increase cytosolic calcium, whereas P2X receptors are ligand-gated cation channels. Unlike ATP, UTP activates a subset of P2Y receptors but is not an agonist at any P2X receptor. Thus, we can be confident that responses to UTP are mediated exclusively by Gq activation of PLC-beta (32, 33). Similar to ATP, there was no significant difference in the UTP-stimulated peak calcium increase between osteoblasts isolated from wild type and rgs2-/- mice (Fig. 2B) (n = 5, p > 0.5). These results suggest that RGS2 plays a minimal role in regulating P2Y receptor-stimulated release of calcium from intracellular stores in osteoblasts expressing basal levels of RGS2.


Figure 2
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  		FIGURE 2.
Changes in cytosolic free calcium levels in response to activation of P2 nucleotide receptors by extracellular nucleotides. Calvarial osteoblast cultures from wild type and rgs2-/- mice were loaded with indo-1 for 30 min and then suspended in calcium-Hepes buffer and stimulated with 10 µM ATP (A) or 10 µM UTP (B). Calcium time plots shown are representative of six individual experiments. Bar charts represent the average peak calcium elevation (n = 6), which is defined as the difference between peak and baseline [Ca2+] following agonist stimulation. Data demonstrate no significant difference (p > 0.05) in calcium elevation in cells from wild type and rgs2-/- mice.

 


Figure 3
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  		FIGURE 3.
Effect of pretreatment with forskolin on P2 nucleotide receptor-stimulated calcium elevation. Calvarial osteoblast cultures from wild type and rgs2-/- mice were treated with 100 µM forskolin or vehicle (0.1% dimethyl sulfoxide) for 2.5 h, before loading with indo-1 for 30 min and suspension in calcium-Hepes buffer. Osteoblast suspensions were stimulated with 10 µM ATP (A) or 10 µM UTP (B). Calcium time plots shown are representative of five individual experiments. C, peak calcium elevation in forskolin-treated cells was calculated as a percentage of the peak calcium elevation in vehicle-treated cells. Bars represent mean ± S.E. of five individual experiments. The response of forskolin-treated wild type osteoblasts was significantly inhibited (p < 0.01) compared with untreated wild type cells. In contrast, forskolin had no significant effect on cells from rgs2-/- mice.

 
To determine whether up-regulation of RGS2 alters calcium signaling in osteoblasts, cells were pretreated with forskolin for 3 h to induce RGS2 expression, prior to stimulation with ATP or UTP. Pretreatment with forskolin significantly attenuated the ATP-stimulated peak calcium elevation in osteoblasts prepared from wild type mice (n = 4, p < 0.02; Fig. 3A), and a similar change was observed with UTP (n = 4, p < 0.03; Fig. 3B). Notably, these agonist responses in osteoblasts prepared from rgs2-/- mice were insensitive to forskolin pretreatment. Thus, suppression of calcium signaling after treatment of wild type osteoblasts with forskolin is likely due to induction of RGS2 expression. RGS2 therefore seems to be a critical regulator of Gq-dependent calcium signaling in osteoblasts.


Figure 4
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  		FIGURE 4.
Effect of pretreatment with forskolin on endothelin receptor-stimulated accumulation of inositol phosphates. Calvarial osteoblast cultures from wild type (A) or rgs2-/- (B) mice which were loaded with myo-[2-3H]inositol overnight were incubated with 100 µM forskolin or vehicle (1% dimethyl sulfoxide) for 1 h. After 15 min of incubation with 10 mM LiCl to inhibit inositol monophosphatase, the cells were treated with indicated concentrations of endothelin for 15 min. Formation of inositol phosphates was calculated asa%of total myo-[2-3H]inositol incorporated into each sample, and data were normalized to the maximal effect of 1 µM endothelin in vehicle-treated cells in each experiment. Data represent the mean ± S.E. of at least three individual experiments. Differences in maximum responses and EC50 values between treatment groups were assessed by t tests. A, vehicle-treated, maximum inositol phosphates = 96 ± 6%, EC50 = 4 ± 2nM; forskolin-treated, maximum inositol phosphates = 65 ± 5%, EC50 = 6 ± 4 nM. B, vehicle-treated, maximum inositol phosphates = 111 ± 12%, EC50 = 17 ± 13 nM; forskolin-treated, maximum inositol phosphates = 102 ± 9%, EC50 = 12 ± 6nM. There was no significant difference between the EC50 values from forskolin- and vehicle-treated osteoblasts in wild type or rgs2-/- mice. There was also no significant difference between the maximum inositol phosphates values between forskolin- and vehicle-treated rgs2-/- osteoblasts. The maximum inositol phosphates was significantly lower in forskolin-treated compared with vehicle-treated osteoblasts from wild type mice.

 
It has been demonstrated previously that, in pancreatic acinar cells, the calcium content of the endoplasmic reticulum in rgs2-/- cells is elevated relative to wild type cells, because of a compensatory increase in expression of sarco/endoplasmic reticulum calcium ATPase-2b (34). To test whether this also occurs in osteoblasts, we compared the intracellular calcium stores of wild type and rgs2-/- cells. Osteoblasts were loaded with indo-1 and suspended in calcium-free buffer. Addition of ionomycin released calcium from intracellular stores, giving rise to a transient elevation of cytosolic calcium. There was no significant difference in total ionomycin-stimulated calcium release in wild type and rgs2-/- osteoblasts (6.5 ± 0.3 and 7.0 ± 0.4 µM/s, respectively). Furthermore, there was no significant difference in calcium stores between wild type and rgs2-/- osteoblasts that were treated with forskolin at concentrations sufficient to induce RGS2 expression (12.6 ± 1.8 and 11.5 ± 1.4 µM/s, respectively). These results imply that loss of RGS2 did not appreciably affect intracellular calcium stores and that the observed changes in nucleotide responses are not attributable to changes in the size of these stores. Interestingly, in osteoblasts from both wild type and rgs2-/- mice, forskolin increased ionomycin-releasable stores, suggesting that forskolin alters calcium handling in osteoblasts.

Effect of RGS2 on Agonist-induced Accumulation of Inositol Phosphates—We examined the role of RGS2 on Gq-dependent accumulation of inositol phosphates in osteoblasts. Inositol phosphates are released from phosphatidylinositol 4,5-bisphosphate by the action of PLC-beta and are therefore a more proximal measurement of Gq activation than is calcium elevation. In this set of experiments, we used endothelin to stimulate Gq signaling. Endothelin and its receptors are present and functional in both osteoblasts and osteosarcoma cells, and activation of endothelin receptors leads to robust accumulation of inositol phosphates in these cells (35-37).

Up-regulation of RGS2 with forskolin significantly blunted nucleotide-stimulated cytosolic calcium increase in wild type but not rgs2-/- osteoblasts. Therefore, we examined whether its up-regulation would have a similar effect on endothelin-stimulated accumulation of inositol phosphates. In agreement with the calcium data, pretreatment with forskolin significantly attenuated maximal accumulation of inositol phosphates in osteoblasts from wild type mice (Fig. 4A), with no effect in osteoblasts isolated from rgs2-/- mice (Fig. 4B).8 There was no significant difference in EC50 between any treatment conditions (p > 0.7). These data demonstrate that up-regulation of RGS2 by forskolin attenuates endothelin-mediated accumulation of inositol phosphates, supporting the hypothesis that RGS2 is a critical regulator of signaling via Gq-coupled receptors in osteoblasts.

Effect of RGS2 on cAMP Accumulation—To test the effect of RGS2 on Gs signaling, osteoblasts prepared from wild type and rgs2-/- mice were treated with PTHrP, and cAMP accumulation was measured (Fig. 5A). There was no significant difference in the EC50 value or the maximal cAMP response to PTHrP between the two cell populations. In addition, when cells were stimulated acutely with forskolin, there was no significant difference in cAMP accumulation between osteoblasts prepared from wild type or rgs2-/- mice (Fig. 5B).


Figure 5
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  		FIGURE 5.
Effects of PTHrP and forskolin on cAMP accumulation. A, calvarial osteoblast cultures from wild type and rgs2-/- mice were incubated with [3H]adenine for 3 h, and subsequently treated with indicated concentrations of PTHrP-(1-34) for 2 min. Intracellular cAMP formation was determined from recovered [3H]cAMP and expressed as a fraction of total intracellular [3H]. Data represent the means ± S.E. of four individual experiments. There was no significant difference between the EC50 or maximum cAMP values from wild type and rgs2-/- mice, wild type EC50 = 27 ± 2nM, maximum cAMP = 0.015 ± 0.006; rgs2-/-, EC50 = 36 ± 2nM, maximum cAMP = 0.013 ± 0.006. B, calvarial osteoblast cultures from wild type and rgs2-/- mice were treated with 100 µM forskolin for 2 min. Data represent the mean ± S.E. of five individual experiments, rgs2+/+ = 0.023 ± 0.005 and rgs2-/- = 0.028 ± 0.004.

 


Figure 6
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  		FIGURE 6.
Effect of pretreatment with ATP on changes in PTHrP receptor-stimulated intracellular cAMP levels. Calvarial osteoblast cultures from wild type (A) or rgs2-/- mice (B) were incubated with [3H]adenine ± 10 µM ATP for 3 h and subsequently treated with indicated concentrations of PTHrP-(1-34) for 2 min. Intracellular cAMP formation was determined from recovered [3H] cAMP and expressed as a fraction of total intracellular [3H] incorporated into each sample, and data were normalized to the maximal effect of 300 nM PTHrP-(1-34) in control cells. Data represent the mean ± S.E. of four individual experiments. Differences in maximum cAMP responses and EC50 values between treatment groups were assessed by paired t tests. A, vehicle-treated, maximum cAMP = 104 ± 8%, EC50 = 21 ± 2nM; ATP-treated, maximum cAMP = 62 ± 17%, EC50 = 15 ± 4nM. B, vehicle-treated, maximum cAMP = 99 ± 22%, EC50 = 35 ± 3nM; ATP-treated, maximum cAMP = 97 ± 31%, EC50 = 47 ± 4nM. There was no significant difference between the EC50 values from ATP- and vehicle-treated osteoblasts in wild type or rgs2-/- mice. There was also no significant difference between the maximum cAMP values between ATP- and vehicle-treated rgs2-/- osteoblasts. The maximum cAMP was significantly lower in ATP-treated compared with vehicle-treated wild type osteoblasts.

 
To determine whether up-regulation of endogenous RGS2 regulates Gs signaling in osteoblasts, cells were treated with ATP for 3 h prior to stimulation with PTHrP. In cells prepared from wild type animals, the maximal cAMP response was significantly reduced (p < 0.05) after treatment with ATP (Fig. 6A). Correspondingly, there was no significant change in cAMP response after ATP treatment in cells obtained from rgs2-/- animals (Fig. 6B). There was also no significant difference between the EC50 value of ATP-treated wild type or rgs2-/- osteoblasts, when compared with vehicle-treated cells. The lack of inhibition of cAMP accumulation in cells prepared from rgs2-/- mice suggests that the inhibitory effect of ATP on cAMP accumulation in wild type osteoblasts is because of up-regulation of RGS2.


Figure 7
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  		FIGURE 7.
Effect of RGS2 adenoviral expression on cAMP accumulation. Calvarial osteoblast cultures from wild type mice were infected with an adenovirus encoding RGS2 or GFP (virus control). Approximately 48 h later, osteoblasts were incubated with [3H]adenine for 3 h, and subsequently treated with indicated concentrations of PTHrP-(1-34) for 2 min. Intracellular cAMP formation was determined from recovered [3H]cAMP and expressed as a fraction of total intracellular [3H] incorporated into each sample, and data were normalized to the maximal effect of 300 nM PTHrP-(1-34) in uninfected cells. Data represent the mean ± S.E. of four individual experiments. The maximum cAMP in RGS2-infected osteoblasts was significantly lower than the maximum cAMP in GFP-infected osteoblasts. There was no significant difference between the EC50 values from GFP-infected and RGS2-infected osteoblasts. GFP-infected, maximum cAMP = 93 ± 3%, EC50 = 23 µM. RGS2-infected, maximum cAMP = 64 ± 3%, EC50 = 28 µM.

 
To confirm the role of RGS2, osteoblasts prepared from wild type mice were infected with either a GFP-(virus control) or RGS2-encoding adenovirus. Compared with uninfected osteoblasts, the maximal PTHrP-stimulated cAMP accumulation was significantly decreased in osteoblasts infected with RGS2 adenovirus (p < 0.05) but not in osteoblasts infected with GFP (Fig. 7). There was no significant difference between the EC50 values of the different osteoblast populations. Taken together, the results shown in Figs. 5, 6 and 7 indicate that up-regulation of RGS2 attenuates cAMP production in response to PTHrP in primary osteoblast cultures.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Gs- and Gq-coupled signaling pathways actively regulate bone formation by osteoblasts. Here we investigated the effects of Gs- and Gq-mediated signals on RGS2 expression in osteoblasts, as well as the effect of RGS2 on those signals. PTHrP and forskolin, activators of the Gs signaling pathway in osteoblasts, induced RGS2 expression. In addition, ATP and PMA, which trigger and mimic Gq signaling, respectively, also led to up-regulation of RGS2. Although Gs and Gq signaling each were equivalent in untreated wild type and rgs2-/- osteoblasts, up-regulation of RGS2 in wild type cells altered signaling via both G proteins. Thus, RGS2 expression regulates both Gs- and Gq-coupled signaling in osteoblasts. Significantly, our results demonstrate that endogenous RGS2 attenuates Gs-mediated signaling and is responsible for cross-desensitization between Gq and Gs signaling pathways.

Regulation of Gs Signaling by RGS2—The present results indicate that, under basal conditions, endogenous RGS2 does not substantially affect Gs signaling in osteoblasts. However, ATP treatment of wild type osteoblasts, which results in up-regulation of RGS2, attenuates Gs signaling. This is likely to be due to RGS2 up-regulation and not other experimental factors (e.g. decreased uptake of [3H]adenine into cells), because ATP treatment produced no significant decrease in receptor-dependent cAMP yield in osteoblasts from rgs2-/- mice. Furthermore, overexpression of RGS2 in osteoblasts via adenoviral infection (which had no effect on [3H]adenine uptake) yielded a similar result, i.e. decreased maximal PTHrP-stimulated cAMP accumulation with no change in the EC50 values. Our results demonstrate that increased expression of RGS2 in osteoblasts inhibits Gs signaling. It follows that the induction of RGS2 may interfere with Gs-coupled signaling pathways in bone, which dynamically regulate bone metabolism.

Previous studies have shown that exogenous or overexpressed RGS2 can impede Gs-mediated signaling, yet the physiological relevance of this effect has never been clear. Notably, the present results imply that such effects actually can occur in vivo. The precise mechanism by which RGS2 inhibits Gs-stimulated cAMP production remains uncertain, although it appears to be independent of any effect on GTPase activity (8, 23). RGS2 may bind to both G{alpha}s and adenylyl cyclase (25), possibly acting as an effector antagonist. RGS2 may also bind to Gs-coupled receptors (25), which represents a further possible site of regulation. We have shown previously that co-expression of G{alpha}s recruits GFP-RGS2 from the nucleus to the plasma membrane of HEK 293 cells and that overexpression of RGS2 inhibits basal cAMP production (8). Transiently expressed RGS2 has also been shown to inhibit glucose-dependent insulinotropic peptide-induced cAMP accumulation in L293 cells (16) and both PTH- and forskolin-stimulated cAMP accumulation in UMR-106 cells (13). Furthermore, RGS2 attenuates the in vitro activity of types III, V, and VI adenylyl cyclase (14) and inhibits constitutively active G{alpha}s (G{alpha}s-Q227L)- or beta2-adrenergic receptor-stimulated cAMP accumulation in HEK 293 cells (15). Here, we show for the first time an inhibitory effect of RGS2 on Gs-coupled adenylyl cyclase activity in nontransfected cells.

Regulation of Gq Signaling by RGS2—The present results indicate that regulation of endogenous RGS2 controls Gq-dependent calcium signaling in osteoblasts. Under basal conditions, there was no difference in either accumulation of inositol phosphates or calcium elevation between osteoblasts isolated from wild type and rgs2-/- mice. However, treatment of osteoblasts with forskolin, which up-regulates RGS2 in wild type cells, attenuated both endothelin-stimulated accumulation of inositol phosphates and ATP-stimulated calcium elevation in wild type but not rgs2-/- cells. This implies that RGS2 can be up-regulated to limit Gq-dependent signaling in osteoblasts, whereas basal RGS2 levels have a minimal effect on such signals.

The fact that the effect of RGS2 up-regulation was similar for both endothelin and ATP-mediated responses suggests that RGS2 has a general effect on Gq, independent of the receptor involved. This is consistent with data from Xu et al. (38) who showed that in pancreatic acinar cells RGS1, RGS4, and RGS16 displayed receptor selectivity in the inhibition of Gq-dependent calcium signaling, whereas RGS2 inhibited the response of all receptors studied with similar potency.

Our findings stand in contrast to those of Heximer et al. (39), who studied calcium responses in aortic smooth muscle cells isolated from wild type and rgs2-/- mice. Under basal conditions, peak calcium responses were greater in cells derived from rgs2-/- as compared with wild type animals (39). It follows that the role of RGS2 in regulating Gq signals varies from one cell type to another. Its prominent expression in some tissues, such as heart, lung, lymphocytes, and vascular smooth muscle (40-42), suggests that RGS2 constitutively regulates Gq signaling in some cell types, whereas its high inducibility by Gq and Gs signals (discussed below) implies a role in agonist-dependent GPCR desensitization in cell types such as osteoblasts.

Taken together, our results imply that up-regulation of RGS2 in osteoblasts significantly inhibits Gq-dependent signaling in these cells. This inhibition may be due to an increase in the rate at which G{alpha}q hydrolyzes GTP to GDP (9, 23), an effector antagonist action on the productive coupling of Gq to PLC-beta (43), or both. Regardless of the exact mechanism, the present results are the first to demonstrate the physiological relevance of RGS2 up-regulation in osteoblasts and to establish that RGS2 mediates bidirectional cross-desensitization between Gs and Gq signaling pathways.

Up-regulation of RGS2 by G Protein-mediated Signaling—RGS2 is up-regulated in response to Gs and/or Gq signaling in osteoblasts and a variety of other cell types, possibly reflecting activation of a cAMP-response element in the RGS2 promoter region (44). Here we show that PTHrP increases the levels of RGS2 mRNA, and a previous study showed comparable effects with PTH (18). Both PTHrP and PTH activate PTH1R in osteoblasts (45). In contrast to previous studies (18-20), we found that prolonged treatment with forskolin produced a more robust increase in RGS2 mRNA levels than was observed following activation of PTH1R. This may reflect experimental differences, e.g. primary cultures versus established cell lines, although it is not attributable to the particular agonist used, as PTH-(1-34) and PTHrP produced similar effects in our system (data not shown). Also, although PTHrP and forskolin yielded similar increases in cAMP in 2-min steady-state assays (Fig. 5), further experiments pointed to differences in the duration of these responses. Specifically, PTHrP promoted increases in cAMP that tapered off within 5 min, whereas cAMP increased continually in the presence of forskolin for up to 30 min (data not shown). It follows that, on the time scale used to measure RGS2 mRNA up-regulation (2 h), cellular cAMP levels were higher with forskolin than with PTHrP.

Implications of RGS2 Regulation of Gs and Gq Signaling in Osteoblasts—Osteoblasts function in a complex and dynamic milieu in which they continually sense and respond to a variety of endocrine and paracrine factors, many of which signal through GPCRs. Regulation of cytosolic calcium levels is an important signaling pathway in osteoblasts (46). Many ligands such as PTH, endothelin, and nucleotides bind to Gq-coupled receptors to induce release of calcium from intracellular stores and activate protein kinase C, which in turn contribute to the regulation of genes such as insulin-like growth factor-binding protein 5 (47) and transforming growth factor beta1 in osteoblastic cells (48). Nucleotides act through Gq-coupled P2Y receptors on osteoblasts to stimulate proliferation (32). Recently, Pasteurella multocida toxin, which activates Gq, has been shown to inhibit osteoblast differentiation (49). Thus, suppression of Gq signaling in osteoblasts by RGS2 would be expected to regulate target gene expression and enhance osteoblast differentiation.

Gs signaling is thought to be the primary pathway mediating the effects of PTH and PTHrP on osteoblasts (50). Gs-adenylyl cyclase-protein kinase A signaling regulates the expression of multiple genes that ultimately result in the activation of bone resorption by osteoclasts and bone formation by osteoblasts. Interestingly, the net effect on bone mass depends upon the kinetics of PTH administration. It has been demonstrated recently that osteoblast-specific G{alpha}s deficiency leads to reduced bone turnover in mice (51). In humans, G{alpha}s-activating mutations suppress osteoblast differentiation, whereas inactivating mutations have the opposite effect (52). Thus, inhibition of Gs signaling in osteoblasts by RGS2 would be expected to influence the expression of multiple genes and enhance osteoblast differentiation. Therefore, heterologous or homologous up-regulation of RGS2 in osteoblasts would serve to limit excessive signaling through Gs and Gq pathways and thus may promote osteoblast differentiation.


   FOOTNOTES
 
* This work was supported in part by grants from the Canadian Institutes of Health Research (to P. C. and S. J. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this work. Back

2 Supported by studentships from the Natural Sciences and Engineering Research Council of Canada and the Heart and Stroke Foundation of Canada. Back

3 Supported by the Heart and Stroke Foundation of Ontario Program Grant in Heart Failure. Back

4 Holds a Canada Research Chair in Molecular Cardiology. Back

5 To whom correspondence should be addressed. Tel.: 519-661-3318; Fax: 519-661-3827; E-mail: Peter.Chidiac{at}schulich.uwo.ca.

6 The abbreviations used are: GPCR, G protein-coupled receptor; GFP, green fluorescent protein; HBSS, Hanks' balanced salt solution; HEK 293 cells, human embryonic kidney 293 cells; PLC-beta, phospholipase C-beta; PMA, phorbol myristate acetate; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related peptide; PTH1R, parathyroid hormone receptor type 1; RGS, regulator of G protein signaling; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate. Back

7 Cells were treated with forskolin for 1 h in these experiments because the serum-deprived cells did not tolerate forskolin for longer periods. Experiments similar to those in Fig. 1B showed significant up-regulation of RGS2 at 1 h. Back

8 Pretreatment with forskolin led to a slight increase in basal accumulation of inositol phosphates (Fig. 4). The reason is unclear but may reflect increased activity of endogenous Gq-coupled receptors (53) or indirect stimulation of PLC-{epsilon} by forskolin (54). Back



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