Research Article

Metabolic acidosis regulates RGS16 and G protein signaling in osteoblasts

Abstract

Chronic metabolic acidosis stimulates cell-mediated net Ca2+ efflux from bone mediated by increased osteoblastic cyclooxygenase 2, leading to prostaglandin E2-induced stimulation of receptor activator of NF-κB ligand-induced osteoclastic bone resorption. Ovarian cancer G protein-coupled receptor-1 (OGR1), an osteoblastic H+-sensing G protein-coupled receptor, is activated by acidosis and leads to increased bone resorption. As regulator of G protein signaling (RGS) proteins limit GPCR signaling, we tested whether RGS proteins themselves are regulated by metabolic acidosis. Primary osteoblasts were isolated from neonatal mouse calvariae and incubated in physiological neutral or acidic (MET) medium. Cells were collected, and RNA was extracted for real-time PCR analysis with mRNA levels normalized to ribosomal protein L13a. RGS1, RGS2, RGS3, RGS4, RGS10, RGS11, and RGS18 mRNA did not differ between MET and neutral medium; however, by 30 min, MET decreased RGS16, which persisted for 60 min and 3 h. Incubation of osteoblasts with the OGR1 inhibitor CuCl2 inhibited the MET-induced increase in RGS16 mRNA. Gallein, a specific inhibitor of Gβγ signaling, was used to determine if downstream signaling by the βγ-subunit was critical for the response to acidosis. Gallein decreased net Ca2+ efflux from calvariae and cyclooxygenase 2 and receptor activator of NF-κB ligand gene expression from isolated osteoblasts. These results indicate that regulation of RGS16 plays an important role in modulating the response of the osteoblastic GPCR OGR1 to metabolic acidosis and subsequent stimulation of osteoclastic bone resorption.

NEW & NOTEWORTHY The results presented in this study indicate that regulation of regulator of G protein signaling 16 and G protein signaling in the osteoblast plays an important role in modulating the response of osteoblastic ovarian cancer G protein-coupled receptor 1 (OGR1) to metabolic acidosis and the subsequent stimulation of osteoclastic bone resorption. Further characterization of the regulation of OGR1 in metabolic acidosis-induced bone resorption will help in understanding bone loss in acidotic patients with chronic kidney disease.

INTRODUCTION

In humans, chronic metabolic acidosis (MET), an increase in systemic H+ concentration with a reduced bicarbonate concentration, leads to greater urine Ca2+ excretion without a significant increase in intestinal Ca2+ absorption (1). The source of this additional urinary Ca2+ appears to be H+-mediated dissolution and resorption of bone as the additional H+ is buffered by the mineral phases of bone (2). Using neonatal mouse calvariae cultured in medium acidified to model physiological MET, through a reduction of the medium bicarbonate concentration, we have demonstrated that MET directly stimulates net Ca2+ efflux from bone (3, 4). Within 3 h, this model of MET induces physicochemical mineral dissolution resulting in a release of Ca2+ and buffering of the medium acidity (3, 5, 6). However, by 24 h, MET stimulates cell-mediated Ca2+ efflux through inhibition of osteoblastic bone formation and stimulation of osteoclastic bone resorption (3, 4, 7). This MET-induced Ca2+ efflux is mediated primarily through increased osteoblastic cyclooxygenase 2 (COX2), leading to prostaglandin E2-induced stimulation of receptor activator of NF-κB ligand (RANKL) and increased osteoclastic bone resorption (810). We and others have demonstrated that osteoblasts express ovarian cancer G protein-coupled receptor-1 (OGR1; also termed GPR68), an H+-sensing G protein-coupled receptor (GPCR) (11, 12). We have found that H+ activation of OGR1 results in increased osteoblastic intracellular Ca2+ signaling and that the OGR1 inhibitor CuCl2, which directly stabilizes histidine residues in OGR1, inhibits acid-induced stimulation of bone resorption in cultured neonatal mouse calvariae (12). In addition, pharmacological inhibition of intracellular Ca2+ release with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate or 2-aminoethoxydiphenyl borate blocks the acid-induced stimulation of COX2 and RANKL in primary osteoblasts and acid-induced bone resorption in neonatal mouse calvariae, confirming that OGR1 activation by MET requires activation of Gαq (13). Thus, OGR1 appears to be the primary H+ sensor that detects the increase in H+ concentration during MET and initiates a subsequent osteoblastic signaling cascade leading to increased osteoclastic bone resorption. Growing mice generate large amounts of endogenous acid (14). In mice that lack OGR1, we found increased bone density (15), suggesting that they are unable to respond to their acid environment, lending further support for an important role for OGR1 in the response of bone to MET.

There are several mechanisms known to regulate GPCRs such as OGR1. One mechanism uses regulators of G protein signaling (RGS), which act as GTPase-activating proteins (16, 17). These negative regulatory molecules limit GPCR signaling by enhancing the hydrolysis of GTP on the α-subunit and returning the GPCR to its inactive heterotrimeric state (18); this may be a means of determining signaling specificity (19). Alternatively, regulation could occur through Gβγ downstream signaling once the α-subunit and βγ-subunit are uncoupled after receptor activation.

Over 30 RGS proteins have been identified in mammalian systems (16, 20, 21), with many of them exhibiting selective activity toward different Gi or Gq α-subunits (22, 23). As OGR1 is coupled to activation of Gq (11, 24), in this study we tested the hypothesis that MET modulates specific RGS proteins in primary osteoblasts. It is possible that both the α- and βγ-subunits of the GPCR OGR1 could activate downstream responses to MET. To also determine whether regulation of this GPCR signaling by MET involves activation of Gβγ signaling, we tested whether gallein, a selective inhibitor of Gβγ signaling activity (25, 26), blocked downstream effects of MET in the osteoblast.

METHODS

Primary Bone Cell Culture

Primary bone cells, which are almost exclusively osteoblasts (27, 28), were isolated from neonatal CD-1 mouse calvariae immediately after dissection as previously described (27). Bones were washed in PBS containing 4 mM EDTA for 10 min at 37°C and then incubated in HEPES buffer solution [25 mM HEPES (pH 7.4), 70 mM NaCl, 30 mM KCl, 10 mM NaHCO3, 1.5 mM K2HPO4, 1 mM CaCl2, 60 mM sorbitol, 27.8 mM d(+)-glucose, and 1 mg/mL BSA] containing 2 mg/mL collagenase (Wako Pure Chemicals, Richmond, VA) and 90 µM Nα-tosyl-l-lysyl chloromethyl ketone for three sequential 20-min digestion periods at 37°C in a shaking water bath. At the end of each digestion, released cells were collected and resuspended in HEPES buffer also containing 1 mM MgSO4, and all three digests were pooled for plating on 60-mm Primaria culture dishes (Falcon, Corning, Corning, NY). Medium was changed every 2–3 days. Confluent, quiescent cells were then cultured in preequilibrated medium simulating physiological neutral (NTL; pH ∼7.4, Pco2: ∼39 mmHg, HCO3 concentration: ∼25 mM) or MET (pH ∼7.15, Pco2: ∼39 mmHg, HCO3 concentration: ∼15 mM) for the indicated times. Only the physiological HCO3/Pco2 buffer system was used in these experiments. Medium pH and Pco2 were determined with a blood gas analyzer (ABL30, Radiometer), and the concentration of medium HCO3 was calculated from pH and Pco2 as previously described (7, 29).

Bone Organ Culture

Neonatal mouse calvariae were cultured in DMEM containing heat-inactivated horse serum (15%) with or without 20 µM gallein (Sigma-Aldrich, St. Louis, MO) in NTL medium (pH ∼7.4, Pco2: ∼39 mmHg, HCO3 concentration: ∼25 mM) or MET medium (pH ∼7.15, Pco2: ∼39 mmHg, HCO3 concentration: ∼15 mM) preequilibrated at 37°C for >3 h in 35-mm Petri dishes. After equilibration, medium was removed to determine the initial medium pH, Pco2, and total Ca2+ concentration, and two calvariae were placed in each dish on a stainless steel wire grid. Bones were cultured at 37°C for 48 h; after 24 h, calvariae were transferred to preincubated fresh medium. After each 24-h period, medium was removed and analyzed for pH, Pco2, and Ca2+. Medium pH and Pco2 were determined, and medium HCO3 concentration calculated as aforementioned. Ca2+ was measured using a Nova 10 electrolyte analyzer (Nova Biomedical, Waltham, MA). Net Ca2+ flux was calculated as Vm × ([Ca2+]f − [Ca2+]i), where Vm is the medium volume (1.8 mL) and [Ca2+]f and [Ca2+]i are the final and initial medium Ca2+ concentrations, respectively.

RNA Isolation and Quantitative Real-Time PCR

Primary osteoblasts were incubated for indicated times in a CO2 incubator modified with gloves inserted into the door to facilitate medium changes inside the incubator while maintaining constant Pco2. At each indicated time point, cells were washed with cold PBS and then lysed in RLT buffer according to the Qiagen protocol using a Qiashredder (Qiagen, Hilden, Germany). Total RNA was isolated from primary CD-1 calvarial cells using an RNeasy kit (Qiagen) as previously described (30). RNA (1 µg) was reverse transcribed to first-strand cDNA using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA), and initial characterization of RNA expression was done by PCR amplification using iQ SYBR green in an iCycler thermocycler (Bio-Rad). PCR products were resolved by electrophoresis in agarose (Invitrogen), and the migration rate was compared with a standard size (1-kb Plus DNA ladder, Invitrogen, Thermo Fisher Scientific, Waltham, MA). Primers were synthesized by Integrated DNA Technologies (Coralville, IA), and the sequences are shown in Table 1.

Table 1. Primers for real-time PCR

Primers
RGS1Forward: 5′-TTGAATTCTGGTTGGCTTGTG-3′
Reverse: 5′-TTGATTTCAGGAACCTGGGATAA-3′
RGS2Forward: 5′-GACCCGTTTGAGCTACTTCTTG-3′
Reverse: 5′-CCGTGGTGATCTGTGGCTTTTTAC-3′
RGS3Forward: 5′-TCCCGGAAGAGAAAGAGCAAAAA-3′
Reverse: 5′-ATGCCTGGATCGCGATGTATTCA-3′
RGS4Forward: 5′-TGCAAGCAACAAAAGAGGTGAA-3′
Reverse: 5′-CCCCGCAGCTGGAAGGAT-3′
RGS10Forward: 5′-GAGCCTTAAGAGCACAGCCAAGTG-3′
Reverse: 5′-GTGGCTCTTCCAGAATCTTTTCAG-3′
RGS11Forward: 5′-TCAGTGCGGAAAACCTCA-3′
Reverse: 5′-CCGCAAGAATGGAAATG-3′
RGS12Forward: 5′-ATCGAAATGTTAGAAAGACCAAAGAAGAC-3′
Reverse: 5′-ATGGAGAACCCGGACTTGACAGCA-3′
RGS16Forward: 5′-TGCCGCACCCTAGCCACCTTC-3′
Reverse: 5′-TTCGCTGCGGATGTACTCGTCAAA-3′
RGS18Forward: 5′-GCCAAAATCAGAGCGAAAGA-3′
Reverse: 5′-GTGCCGTATCAAAACTGTGGAG-3′
COX2Forward: 5′-GGGTTGCTGGGGGAAGAATGTG-3′
Reverse: 5′-GGTGGCTGTTTTGGTAGGCTGTG-3′
RANKLForward:5′-CCAAGATCTCTAACATGACG-3′
Reverse: 5′-CACCATCAGCTGAAGATAGT-3′
β-ActinForward: 5′-GGCCGCCCTAGGCACCAG-3′
Reverse: 5′-GGGTCATCTTTTCACGGTTGGC-3′
RPL13AForward: 5′-GGATCCCTCCACCCTATGACA-3′
Reverse: 5′-CTGGTACTTCCAGACCTC-3′

COX2, cyclooxygenase 2; RANKL, receptor activator of NF-κB ligand; RGS, regulator of G protein signaling; RPL13A, ribosomal protein L13A.

Subsequent specific transcript levels were analyzed by quantitative real-time PCR using iQ SYBR green in an iCycler thermocycler and analyzed with MyIQ optical system software (Bio-Rad). For the quantitative PCR, standard curves were generated for each primer tested. Relative expression levels were normalized to β-actin or ribosomal protein L13a RNA levels, as indicated, by the comparative threshold cycle (CT) method (31, 32).

Immunoblot Analysis

Primary osteoblasts were incubated for the indicated times in NTL or MET medium. At the conclusion of the experiment, cells were washed with cold PBS, collected in Laemmli buffer, and boiled for 5 min. Replicate plates were used to determine protein concentration by the BCA method (Pierce Endogen, Thermo Fisher Scientific). Equal amounts of protein were then separated on 10% polyacrylamide gels, transferred to PVDF membranes, and immunoblotted with primary antibody to RGS16 (Proteintech Group, Rosemont, IL). Specific binding was detected using secondary antibody coupled to horseradish peroxidase and the SuperSignal West Dura chemiluminescent detection system (Pierce Endogen) and analyzed in a FluorChem 8800 image analyzer (AlphaInnotech, Protein Simple, San Jose, CA). After detection, blots were stripped and reprobed with primary antibody to β-actin (Santa Cruz Biotechnology, Dallas, TX).

Statistical Analyses

All tests of significance were calculated using ANOVA with a Bonferroni correction for multiple comparisons using a conventional computer program (Statistica, StatSoft, TIBCO Software, Palo Alto, CA) on a personal computer. All values are expressed as means ± SE. P < 0.05 was considered significant.

RESULTS

RNA from confluent, quiescent primary osteoblastic cells was analyzed by PCR for the expression of specific RGS proteins. The specific RGS proteins chosen for analysis (RGS1, RGS2, RGS3, RGS4, RGS10, RGS11, RGS12, RGS16, and RGS18) were those that have been shown to be coupled to Gq (23). A number of different RGS proteins are present in these cells, including RGS1, RGS2, RGS3, RGS4, RGS10, RGS11, RGS16, and RGS18, whereas there was no detected signal for RGS12 (Fig. 1).

Figure 1.

Figure 1.RNA expression of specific regulator of G protein signaling (RGS) proteins in primary osteoblasts. Primary calvarial cells were cultured to confluence, and RNA was isolated. RNA was reverse transcribed to first-strand cDNA and resolved on an agarose gel. Specific transcripts were identified using the primers shown in Table 1. stds, standard.


We next determined whether the levels of any of these RGS proteins were regulated by acidosis. Primary osteoblastic cells were incubated in physiological NTL medium (pH 7.48 ± 0.01, Pco2: 37.8 ± 0.4 mmHg, HCO3 concentration: 27.7 ± 0.3 mM) or physiological acidic (MET) medium (pH 7.15 ± 0.01, Pco2: 38.1 ± 0.4 mmHg, HCO3 concentration: 11.8 ± 0.1 mM). At the indicated time points, cells were collected and RNA was extracted for real-time PCR analysis. There was no difference in the amount of RGS16 in MET compared with NTL at 20 min of incubation; however, at 30 min, MET induced a significant decrease in RGS16. Similar inhibition of RGS16 was observed at 60 min and 3 h; however, at later time periods (6–24 h), there was no difference in the response to MET compared with NTL (Fig. 2). In contrast, there was no change in mRNA levels for RGS2, RGS4, and RGS10 in response to MET compared with NTL at any time period (Fig. 3). Similarly, there was no change in mRNA levels of RGS1, RGS3, RGS11, or RGS18 in response to MET (data not shown).

Figure 2.

Figure 2.Effect of acid on regulator of G protein signaling 16 (RGS16) mRNA expression in primary calvarial osteoblasts. Primary cells were incubated in neutral medium (NTL; pH ∼7.45) or acidic medium (MET; pH ∼7.15) for the indicated times. RNA was isolated and analyzed by real-time PCR for changes in RGS16. Data are means ± SE; n =6−30 bones/group. *P < 0.05 compared with baseline; °P < 0.001 compared with NTL at the same time point. rel, relative; ′, min.


Figure 3.

Figure 3.Lack of effect of acid on other regulator of G protein signaling (RGS) mRNA expression in primary calvarial osteoblasts. Primary cells were incubated in neutral medium (NTL; pH ∼7.45) or acidic medium (MET; pH ∼7.15) for the indicated times. RNA was isolated and analyzed by real-time PCR for changes in RGS2 (A), RGS4 (B), or RGS10 (C), as indicated. Data are means ± SE; n =6−20 bones/group. MET was not different from NTL at any of the same time points. rel, relative; ′, min.


We have shown in previous studies that the H+ sensor for signaling in response to MET in the osteoblast is the GPCR OGR1 (12, 13) and that signaling in response to this receptor can be inhibited by CuCl2 (12). Incubation of primary osteoblasts in the presence of 100 µM CuCl2 significantly prevented MET inhibition of RGS16 mRNA expression (Fig. 4).

Figure 4.

Figure 4.CuCl2 (Cu) blocks acidosis inhibition of regulator of G protein signaling 16 (RGS16) RNA expression. Primary cells were incubated in neutral medium (NTL; pH ∼7.45) or acidic medium (MET; pH ∼7.15) in the absence or presence of 100 µM CuCl2. RNA was isolated and analyzed by real-time PCR for changes in RGS16 at 30 min (′). Data are means ± SE; n =6−8 cultures/group. *P < 0.001 vs. NTL; °P < 0.05 vs. 30′ MET. rel, relative.


The effect of MET on the level of RGS16 protein in osteoblasts was determined by immunoblot analysis. A decrease in the amount of RGS16 protein was observed within 3 h of incubation of primary osteoblasts in MET medium compared with incubation in NTL medium (representative experiment shown in Fig. 5).

Figure 5.

Figure 5.Decreased regulator of G protein signaling 16 (RGS16) protein in primary calvarial osteoblasts by 3 h. Primary calvarial cells were incubated in neutral medium (N; pH ∼7.45) or acidic medium (M; pH ∼7.15) for the indicated times. Cellular protein was collected, and immunoblot analysis was performed with equal amounts of total protein using a specific antibody to RGS16. β-actin was used to normalize the data. rel, relative; ′, min.


RGS16 interacts with the α-subunit and is regulated by MET. It is also possible that the βγ-subunit is involved in the downstream response to MET. To determine whether regulation of OGR1 by MET involves activation of Gβγ signaling, we tested whether gallein, a selective inhibitor of Gβγ signaling activity (25, 26), blocked downstream effects of MET in the osteoblast. We cultured neonatal mouse calvariae in NTL or MET medium for 48 h in the absence or presence of gallein. At 48 h, MET induced an increase in net Ca2+ flux from the calvariae, which was significantly decreased in the presence of 20 µM gallein (Fig. 6). When primary osteoblasts were cultured for 6 h in NTL or MET medium in the absence or presence of 20 µM gallein, MET stimulated cox2 gene expression and gallein inhibited this response to MET (Fig. 7A). Similarly, when primary osteoblasts were incubated for 48 h in NTL or MET medium in the absence or presence of gallein, MET stimulated rankl gene expression and gallein again significantly inhibited the response to MET (Fig. 7B). We have previously shown that MET stimulation of COX2 and RANKL in the osteoblast is critical for MET to induce an increase in net Ca2+ flux from bone in organ culture (30, 33).

Figure 6.

Figure 6.Effect of gallein (gal) on acid-induced net Ca2+ efflux from calvariae in organ culture. Neonatal mouse calvariae were incubated in neutral medium (NTL; pH ∼7.45) or acidic medium (MET; pH ∼7.20) in the absence or presence of 20 µM gallein for 48 h. Data are means + SE; n =8 pairs of bones/group. *P < 0.05 vs. NTL; °P < 0.05 vs. MET alone; #P < 0.05 vs. NTL + gallein.


Figure 7.

Figure 7.Gallein (gal) inhibits acidosis stimulation of cyclooxygenase 2 (COX2; A) and receptor activator of NF-κB ligand (RANKL; B) RNA expression. Primary osteoblasts were incubated in neutral medium (NTL; pH ∼7.45) or acidic medium (MET; pH ∼7.20) in the absence or presence of 20 µM gallein for 6 or 48 h as indicated. RNA was isolated and analyzed by real-time PCR normalized to ribosomal protein L13A. Data are means ± SE; n =6−10. *P < 0.001 vs . NTL; °P < 0.05 vs. MET. rel, relative.


DISCUSSION

Chronic MET induces Ca2+ loss from bone mineral in the process of buffering systemic acidity. Using an in vitro physiological model of MET, we found that MET decreases osteoblastic collagen synthesis and mineralization and stimulates osteoclastic bone resorption (7). MET-induced bone resorption is mediated by an initial increase in intracellular Ca2+ signaling (12, 13, 34) and stimulation of osteoblastic COX2 (33, 35), leading to a prostaglandin E2-mediated increase in RANKL expression (9, 30). We have previously shown that OGR1, found in many tissues (36, 37), including bone (11), is important in the regulation of bone resorption by MET (12, 15, 38). We subsequently found that MET increases intracellular Ca2+ in primary neonatal mouse calvarial osteoblasts through activation of OGR1 (34). We also demonstrated that growing mice with a global lack of OGR1 have increased bone density that appears to be due primarily to increased osteoblastic activity (15). However, we also found that there is a direct response of osteoclastic OGR1 to MET (38).

The results of the present study address regulation of the GPCR OGR1 by MET. We demonstrate that a model of MET inhibits RGS16 mRNA expression in osteoblasts within 1 h and protein expression by 3 h. No significant changes were observed for other RGS proteins, including RGS2, RGS4, and RGS10. Inhibition of RGS16 by MET is blocked by concomitant incubation with the known OGR1 inhibitor CuCl2, suggesting that RGS16 is important in modulating the signaling response of the osteoblast to acid activation by OGR1. MET inhibition of RGS16 would prolong the acid-stimulated signaling response through OGR1 by preventing GTP hydrolysis that returns the G protein to an inactive state. The ability of gallein, which inhibits Gβγ signaling (25, 26), to prevent MET stimulation of net Ca2+ flux from calvariae, as well as blocking MET stimulation of cox2 and rankl gene expression in primary osteoblasts, further supports the importance of this GPCR in the response of the osteoblast to MET.

RGS16 is a member of the R4 family of RGS proteins based on sequence homology (16, 20). As a class, RGS proteins function as GTPase-activating proteins to accelerate GTP hydrolysis of Gα subunits and enhance G protein deactivation. RGS proteins can decrease signaling through Gα-GTP by direct GTPase-activating protein activity or by acting as an antagonist to inhibit the interaction of the Gα subunit with its downstream effector (22). A conserved RGS domain in these proteins specifically interacts with α-subunits of G proteins, which results in decreased signaling from the activated GPCR (39). Although there are hundreds of GPCRs as well as multiple Gα, Gβ, and Gγ subunits and many cells can express more than one subtype of each component, there is a precise combination of GPCR, G protein, and RGS protein that will determine a specific response to a given stimulus (18). For example, RGS16 has pronounced receptor selectivity for inhibition of phospholipase Cβ and Ca2+ signaling in pancreatic acinar cells, preferentially inhibiting the response to carbachol compared with cholecystokinin (19). In human embryonic kidney cells, RGS16 has been shown to interact with 14-3-3 protein to form a signaling scaffold that inhibits its availability or GTPase-activating protein activity and prolong GPCR signaling (40). Studies in yeast transfected with human Gαq subunits have suggested that RGS16 acts predominantly through its effect on GTPase activity (18).

RGS16 was initially cloned in the retina and has also been found in heart, brain, liver, and hematopoietic cells (16). Using human Gαq expressed in a yeast system, RGS16 has been shown to selectively inhibit Gαq signaling (18). Although RGS16 appears to be relatively specific for its interaction with Gαq and not non-GPCR signaling components, it has been shown that tyrosine phosphorylation of RGS16 by epidermal growth factor receptor activation can enhance its GTPase activity (16, 41). In addition, RGS16 has been shown to attenuate p38 MAPK but not ERK activation by platelet-activating factor in Chinese hamster ovary cells (42) and to suppress phosphatidylinositol 3-kinase activity in breast tumor cells (43) in addition to its GTPase activity. Overexpression of RGS16 has been shown to decrease proinflammatory cytokines in human monocytes in vitro (44).

There has been limited study of the role of RGS proteins in regulation of the response of bone cells to various stimuli (17). Using a microarray analysis to examine runt-related transcription factor 2 (Runx2)-dependent genes that may be involved in the regulation of osteoblast proliferation, RGS16 was identified as one of several components of G protein-coupled signaling modulated by Runx2, along with RGS2, RGS4, and RGS5 (45). Validation by quantitative real-time PCR demonstrated significant downregulation of RGS16 when Runx2 was transfected into Runx2-null osteoblasts compared with empty vector. RGS2 increased as a function of osteoblast differentiation but was downregulated with Runx2 expression. In contrast, parathyroid hormone was found to upregulate RGS2 in primary mouse calvarial osteoblasts (46, 47), suggesting that RGS2 plays a significant role in modulating the response to parathyroid hormone receptor signaling. RGS18 is expressed in osteoclasts, and treatment of osteoclast precursors with RANKL reduced RGS18 expression (48). RGS10-deficient mice exhibit severe osteopetrosis and impaired osteoclast differentiation (49). In this model, RGS10 appeared to regulate RANKL-induced Ca2+ signaling in the osteoclast. RGS12 has also been found to be expressed in osteoclasts and interact with N-type Ca2+ channels, also regulating RANKL-induced Ca2+ oscillations and osteoclast differentiation (50). More recently, RGS12 has been shown to play a role in osteoblast maturation and mineralization, and its loss leads to impaired Ca2+ oscillations (51).

For optimal functioning, GPCRs rely on the interaction of Gα with Gβγ subunits (52). Our results demonstrate that MET stimulates OGR1 by decreasing the ability of RGS16 to inactivate the Gαq subunit and prevent its interaction with downstream effectors, but the stimulation can also be blocked by the inhibition of Gβγ signaling by gallein. Further studies will be necessary to determine the relative importance of each of these subunits in the OGR1-mediated response to MET in the osteoblast.

The results presented here demonstrate that regulation of G protein signaling through modulation of RGS16 and/or Gβγ activation is important in determining the OGR1-mediated response of the osteoblast to MET and subsequent stimulation of bone resorption. Further understanding of the interplay between regulation of RGS16 and regulation of Gβγ by acidosis will advance our insight into H+-mediated intracellular signaling in the osteoblast and its subsequent regulation of osteoclastic bone resorption. These data suggest that the response of OGR1 in both osteoblasts and osteoclasts is critical for MET to stimulate bone resorption. Understanding the mechanism by which MET leads to increased bone resorption may help to improve bone content and quality in patients, especially those with chronic kidney disease.

GRANTS

This work was supported by National Institutes of Health Grants RO1AR46289, DK075462, and DK75462 (to D.A.B.) and grants from the Renal Research Institute (to N.S.K.).

DISCLOSURES

D. A. Bushinsky consults for, is on the medical advisory board, and has stock and stock options from Tricida, a company that is developing a gastric binder of acid to treat metabolic acidosis. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

N.S.K. conceived and designed research; N.S.K. analyzed data; N.S.K. and D.A.B. interpreted results of experiments; N.S.K. prepared figures; N.S.K. drafted manuscript; N.S.K. and D.A.B. edited and revised manuscript; N.S.K. and D.A.B. approved final version of manuscript.

ACKNOWLEDGMENTS

The authors acknowledge the expert technical assistance of Stephanie Yee, Christopher Culbertson, and Kelly Kyker-Snowman.

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