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Endocrinology, doi:10.1210/en.2007-0653
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Endocrinology Vol. 148, No. 10 4984-4992
Copyright © 2007 by The Endocrine Society

Type B {gamma}-Aminobutyric Acid Receptors Modulate the Function of the Extracellular Ca2+-Sensing Receptor and Cell Differentiation in Murine Growth Plate Chondrocytes

Zhiqiang Cheng, Chialing Tu, Luis Rodriguez, Tsui-Hua Chen, Melita M. Dvorak, Marta Margeta, Martin Gassmann, Bernhard Bettler, Dolores Shoback and Wenhan Chang

Endocrine Research Unit (Z.C., C.T., L.R., T.-H.C., M.M.D., D.S., W.C.), Department of Veterans Affairs Medical Center, Department of Medicine, University of California, San Francisco, California 94121; Department of Physiology (M.G., B.B.), Biozentrum/Pharmazentrum, University of Basel, CH-4056 Basel, Switzerland; and Department of Pathology (M.M.), University of California, San Francisco, California 94143

Address all correspondence and requests for reprints to: Wenhan Chang, Endocrine Research Unit, 111N, Department of Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, California 94121. E-mail: Wenhan.Chang{at}ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Extracellular calcium-sensing receptors (CaRs) and metabotropic or type B {gamma}-aminobutyric acid receptors (GABA-B-Rs), two closely related members of family C of the G protein-coupled receptor superfamily, dimerize in the formation of signaling and membrane-anchored receptor complexes. We tested whether CaRs and two GABA-B-R subunits (R1 and R2) are expressed in mouse growth plate chondrocytes (GPCs) by PCR and immunocytochemistry and whether interactions between these receptors influence the expression and function of the CaR and extracellular Ca2+-mediated cell differentiation. Both CaRs and the GABA-B-R1 and -R2 were expressed in the same zones of the growth plate and extensively colocalized in intracellular compartments and on the membranes of cultured GPCs. The GABA-B-R1 coimmunoprecipitated with the CaR, confirming a physical interaction between the two receptors in GPCs. In vitro knockout of GABA-B-R1 genes, using a Cre-lox recombination strategy, blunted the ability of high extracellular Ca2+ concentration to activate phospholipase C and ERK1/2, suppressed cell proliferation, and enhanced apoptosis in cultured GPCs. In GPCs, in which the GABA-B-R1 was acutely knocked down, there was reduced expression of early chondrocyte markers, aggrecan and type II collagen, and increased expression of the late differentiation markers, type X collagen and osteopontin. These results support the idea that physical interactions between CaRs and GABA-B-R1s modulate the growth and differentiation of GPCs, potentially by altering the function of CaRs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FAMILY C OF THE G protein-coupled receptor (GPCR) superfamily includes the two type B or metabotropic {gamma}-aminobutyric acid (GABA) receptors (GABA-B-Rs) (R1 and R2), the extracellular Ca2+-sensing receptor (CaR), the metabotropic glutamate receptors (mGluRs), and a large number of taste and odorant receptors (1, 2). Growing evidence supports the idea that family C receptors function in multimeric complexes, either as homomeric or heteromeric signaling receptors (2, 3). For example, the heterodimerization of GABA-B-R1 and -R2 is an essential step in forming functional GABA-B-Rs that traffic to the cell surface, bind ligand, and mediate signaling responses (4). Similarly, CaRs are thought to function as homodimers (or even larger-order complexes) in their key target tissues, the parathyroid and kidney (5, 6). CaRs can heterodimerize with the mGluR1 or -R5 in specific populations of neurons as well as in HEK-293 cells transfected with cDNAs encoding these receptors (7). We recently demonstrated coimmunoprecipitation of CaRs with the GABA-B-R1 and -R2 in HEK-293 cells, suggesting that these receptors can form heteromeric complexes (8). The biological significance of receptor multimerization (e.g. between CaRs and mGluRs or CaRs and GABA-B-Rs) has not been investigated, especially in cells that normally express several members of family C such as parathyroid and kidney cells, neurons, and growth plate chondrocytes (GPCs).

Studies in other receptor systems have shown that, compared with pure populations of purinergic P2Y1 or adenosine A1 receptors, heterodimers of P2Y1 and A1 receptors display distinct sensitivity to antagonists and activate different G proteins (9, 10). The hypothesis that complexing CaRs with either mGluRs or GABA-B-Rs alters their pharmacological and signaling properties in cells that coexpress these receptors is plausible but requires definitive testing.

There is scant information on the expression and activities of family C receptors in growth plate cartilage. We and others have demonstrated that extracellular Ca2+ sensing is a property of chondrocytes including those derived from the growth plate (11, 12, 13, 14, 15, 16, 17, 18). High extracellular Ca2+ concentration ([Ca2+]e) activates G protein-coupled signaling pathways and alters matrix synthesis, mineralization, and gene expression in GPCs, all in directions indicative of terminal differentiation (14, 15). Manipulating the expression and function of CaRs in chondrocyte cell lines alters matrix production, mineralization, and gene expression, also in a direction supporting the role of CaR signaling in promoting the phenotype of terminal differentiation (14, 15, 16, 17, 18). GABA-B-Rs have also been implicated in regulating the proliferation of mouse chondrogenic ATDC5 cells (19), but it is not known whether GABA-B-Rs interact with CaRs and alter their signal transduction in this system.

Using quantitative real-time PCR (qPCR), we assessed the expression in GPCs of several family C receptors that are known to have Ca2+-sensing functions in other cell systems. The relative abundance of receptor expression was as follows: GABA-B-R1 > CaR >> mGluR1 >> mGluR5. Both CaRs and GABA-B-R1s and -R2s were closely associated in both intracellular compartments and on the cell membranes of GPCs by immunocytochemistry and coimmunoprecipitation. Reducing GABA-B-R1 expression profoundly affected the proliferation and apoptosis of GPCs. These changes were accompanied by reduced ability of high [Ca2+]e both to activate phospholipase C (PLC) and ERK1/2 and promote terminal differentiation of GPCs. Such changes in CaR-mediated signal transduction and in the growth, survival, and differentiation of GPCs indicate putative roles for extracellular Ca2+ and for CaRs, GABA-B-Rs, and their interactions in the differentiation program of cartilage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Fetal bovine serum was purchased from Atlanta Biologicals (Atlanta, GA). Primers and probes for qPCR were custom-made by Integrated DNA Technologies (Skokie, IL) according to published nucleotide sequences (14, 15) or purchased from Applied Biosystems (Foster City, CA). A guinea pig anti-GABA-B-R2 antibody (catalog no. G1015-50) was obtained from U.S. Biological (Swampscott, MA). Rabbit antisera specific to the phosphorylated (pERK1/2) and common form (tERK1/2) of ERK1/2 were obtained from Cell Signaling Technology (Beverly, MA). Other supplies were from previously noted sources (14, 15).

Culture and adenovirus infection of GPCs
Epiphyseal growth plates from wild-type Black Swiss or floxed-GABA-B-R1 (BALB/C strain) (20) newborn mice (2–4 d old) were obtained as described (14). The floxed-GABA-B-R1 mouse has both alleles of its GABA-B-R1 genes flanked with loxP sites (20), which allows these genes to be excised in vitro via DNA recombination in the presence of bacterial Cre recombinase carried by a commercially available adenovirus (Microbix Biosystems Inc., Toronto, Ontario, Canada). GPCs were released by enzymatic digestion with a mixture of (wt/vol) collagenase IA (0.18%), hyaluronidase (0.1%), and DNase II (0.01%) in a chondrocyte isolation medium [DMEM plus penicillin (100 µg/ml), streptomycin (100 U/ml), NaHCO3 (45 mM), HEPES (20 mM, pH 7.4), fungizone (0.25 µg/ml), and gentamicin (0.15 mg/ml)] as previously described (14, 15). Cells were plated (105 cells/cm2) in a chondrocyte maintenance medium (CMM) [DMEM plus penicillin (100 µg/ml), streptomycin (100 U/ml), and fetal bovine serum (10%, vol/vol)] and grown at 37 C with 5% CO2.

In vitro knockout of the GABA-B-R1 gene was achieved by infecting confluent GPCs (3–4 d after isolation) from mice carrying floxed-GABA-B-R1 genes with replication-deficient adenoviruses (~16 pfu/cell) carrying the cDNA encoding the bacterial Cre recombinase (Ad-Cre) or no insert (control vector; Ad-Cont). Adenoviruses were prepared and titered (14, 15), and cells were infected for 48–72 h.

To examine the effects of changing [Ca2+]e on the expression of genes that mark different stages of differentiation, GPCs from mice with floxed-GABA-B-R1 genes were infected with either Ad-Cre or Ad-Cont for 72 h and cultured in chondrocyte differentiation medium [{alpha}-MEM containing Mg2+ (0.5 mM) plus ascorbic acid (50 µg/ml) and ß-glycerol phosphate (5 mM)] containing different concentrations of CaCl2 (0.5–3.0 mM) for an additional 7 d before RNA isolation.

Cloning of GABA-B-R1a, -R1b, and -R2 cDNAs
Total RNA was extracted from wild-type GPCs cultured in CMM for 5 d (15). cDNAs encoding GABA-B-R1a and -R1b, two alternatively spliced forms of the GABA-B-R1 (21), and GABA-B-R2 were amplified with primer pairs listed in Table 1Go, cloned into pcDNA3.1 using the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA), and sequenced to confirm their identities. The GABA-B-R1b is 116 amino acids shorter than GABA-B-R1a due to different promoter usage and alternative splicing of exons encoding the N-terminal domains of the receptor (21, 22).


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  		TABLE 1. Primers used in cloning GABA-B-R1a, -R1b, and -R2 cDNAs from mGPCs

 
Immunocytochemistry
Immunocytochemical detection of the CaR and GABA-B-R1 and -R2 in growth plates from 1- to 3-wk-old Black Swiss mice was performed with anti-CaR antisera 21825A (500 nM) (23), a guinea pig anti-GABA-B-R1 antibody (100 nM) directed against a peptide domain (CPSEPPDRLSSDGSRVHLLYK) at the C terminus of either the GABA-B-R1a or -R1b, and a commercial guinea pig anti-GABA-B-R2 antibody (100 nM), respectively, in combination with peroxidase-conjugated secondary antibodies. Signals were detected by 3,3'-diaminobenzidine (DAB)-substrate and visualized by light microscopy. Specificity was confirmed by the absence of signals in cartilage sections treated with peptide-preabsorbed antisera or with secondary antibodies alone.

Subcellular localization of the CaR and GABA-B-R1 and -R2 in cultured GPCs was determined with the specific antisera noted above. Dual-fluorescence confocal microscopy was used for detection, and paired pseudo-colored fluorescent images were obtained sequentially as described (15, 24).

Immunoprecipitation and immunoblotting
Crude membranes were prepared from cultured GPCs as described (15, 18). These membrane preparations were extracted with the nonionic detergent Nonidet P-40 (NP-40; 1%) in PBS. Protein extracts (500 µg) were incubated with anti-GABA-B-R1 antibodies (5 µg) in NP-40/PBS (500 µl) for 1 h, followed by addition of protein A/G-conjugated beads (20 µl) and overnight incubation at 4 C. Beads containing bound antibodies were washed with NP-40/PBS (1 ml) five times and pelleted by centrifugation. Antibody-protein complexes were eluted with a sample buffer [300 mM Tris-HCl (pH 6.8), 10% SDS, 0.01% bromophenol blue, 50% glycerol, and 100 µM dithiothreitol] at 37 C for 30 min. Control experiments included immunoprecipitations without anti-GABA-B-R1 antibodies. Input lysates and immunoprecipitated proteins were electrophoresed on SDS-PAGE gels and transferred to nitrocellulose membranes (23, 25, 26). Membranes were blotted with anti-CaR (50 nM) and anti-GABA-B-R1 (10 nM) plus the appropriate peroxidase-conjugated secondary antibodies (23, 25, 26). Signals were detected with a SuperSignal chemiluminescence substrate and Kodak x-ray film.

Total protein lysates (50 µg) from floxed-GABA-B-R1 GPCs infected with Ad-Cre or Ad-Cont were blotted with anti-CaR antisera (50 nM) and anti-ß-actin (10 nM) as described above.

qPCR
Total RNA was isolated from infected floxed-GABA-B-R1 or noninfected wild-type GPCs, and the first-strand cDNAs were reverse-transcribed with Maloney murine leukemia virus reverse transcriptase (Invitrogen) (14). Expression of the CaR, GABA-B-R1, mGluRs, and differentiation markers was quantified using probe-based TaqMan qPCR kits and ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) and is presented as percentage of expression of the control gene encoding ribosomal L19 protein. Expression of L19 was not affected by the duration of culture or varying the [Ca2+]e during culture (data not shown). Fluorescent probes and PCR primers for each gene were designed based on published sequences or purchased (see Table 2Go); sequences of commercial primers are not available due to proprietary protection (Applied Biosystems).


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  		TABLE 2. Sequences of qPCR primers and probes

 
Measurements of proliferation and apoptosis of virally infected GPCs
Cell number, indicated by total DNA content, in cultures infected with Ad-Cre or Ad-Cont for 72 h was determined by crystal violet (CV) staining and quantified by absorbance at 590 nm as described (14). Cell proliferation was assessed by the incorporation of bromodeoxyuridine (BrdU) into genomic DNA for 24 h using a colorimetric ELISA kit (catalog no. 11-647-229-001; Roche Applied Science, Indianapolis, IN) according to the manufacturer’s instructions. Signals were detected with a DAB substrate and quantified by absorbance at 450 nm. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) using a commercial Apoptag kit (Oncor, Gaithersburg, MD) as described (14). All staining determinations were done in triplicate in at least two independent experiments.

Measurement of PLC and ERK1/2 activation
Levels of total inositol phosphates (InsPs) including all isomers of InsP3, InsP2, and InsP1 as an index of PLC activation were determined in GPCs infected with Ad-Cre and Ad-Cont after incubating the cells with different [Ca2+]e (0.5–10 mM) for 60 min. Prelabeling of membrane polyphosphoinositides with [3H]myoinositol was done before stimulating the cells by raising the [Ca2+]e as described (15, 23). Total InsP accumulation is presented as the fold increase over basal levels in cells maintained at 0.5 mM Ca2+.

ERK1/2 activation in GPCs infected with Ad-Cre and Ad-Cont were determined after incubating the cells with different [Ca2+]e (0.5–20 mM) for 10 min in a serum-free medium by Western blotting of cell lysates with antisera specific to pERK1/2 and tERK1/2. Intensities of pERK1/2 and tERK1/2 signals were determined by densitometry and presented as ratios of pERK1/2 over tERK1/2.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of family C receptors in GPCs and the growth plate
To determine whether GPCs express transcripts that encode for members of family C of GPCRs, we performed qPCR using RNA extracted from confluent cultured GPCs. We focused on family C members that have been reported to respond to changes in [Ca2+]e in other cells. As shown in Table 3Go, we detected a substantial level of GABA-B-R1 mRNA (0.61% of L19) that was about 60-fold higher than that of the CaR (0.01% of L19). High levels of GABA-B-R1 expression support a role for this receptor in growth plate function. In contrast, the much lower levels of mGluR1 and -R5 RNA expression (<2% of CaR and <0.3% of CaR mRNA levels) argue against major roles for these receptors in GPCs.


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  		TABLE 3. RNA expression of members of GPCR-C in mGPCs by qPCR (n = 3 experiments)

 
To examine the expression and distribution of GABA-B-Rs in chondrocytes in the highly specialized zones of the growth plate, we performed immunocytochemistry on sections of mouse growth plate cartilage with anti-CaR, anti-GABA-B-R1, and anti-GABA-B-R2 antisera. GABA-B-R1 immunoreactivity was scattered in chondrocytes in the proliferation zone (Fig. 1AGo, red arrowheads), present at higher levels in chondrocytes in the maturation zone (red arrows), and expressed at the highest levels in hypertrophic chondrocytes (black arrowheads). This expression pattern mirrors that of the GABA-B-R2 (Fig. 1BGo) and the CaR (Fig. 1CGo). Immunoreactivity for GABA-B-R1 and -R2 and CaRs was judged to be specific because it was absent in bone and cartilage sections treated with antisera preabsorbed with the corresponding peptides or when only the secondary antibody was included in the incubation (Fig. 1DGo, GABA-B-R1 plus peptide, and data not shown).


Figure 1
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  		FIG. 1. Immunocytochemical detection of the GABA-B-R1, GABA-B-R2, and the CaR in the growth plates of 1- to 3-wk-old Black Swiss mice, using anti-GABA-B-R1, anti-GABA-B-R2, and anti-CaR antisera. Expression of the GABA-B-R1 (A), R2 (B), and the CaR (C), depicted by brown DAB staining, is present in scattered proliferating chondrocytes (red arrowheads) and is more prominent in maturing (red arrows) and hypertrophic (black arrowheads) chondrocytes. Magnification, x63. Results are representative of growth plates from two mice. A control for specificity of the GABA-B-R1 antisera is shown, in which the antisera were preabsorbed with the antigenic peptide before immunostaining (D) (14 18 ). E, Cloning of cDNAs encoding the two splice forms of the GABA-B-R1 (R1a and R1b) and the GABA-B-R2. RT-PCR products amplified from RNA prepared from GPCs cultured in CMM for 5 d were separated by gel electrophoresis and visualized by staining with ethidium bromide. Sizes of DNA markers (ladder) are indicated in kb.

 
To confirm the expression of GABA-B-R transcripts in GPCs, we performed RT-PCR with primers derived from published cDNA sequences for the murine GABA-B-R1a, -R1b, and -R2 (see Table 1Go). As shown in Fig. 1EGo, we consistently amplified cDNAs from GPC RNA (n = 3) with sizes of about 3.2, 2.6, and 2.8 kb that encode the murine cartilage GABA-B-R1a, -R1b, and -R2, respectively, as confirmed by DNA sequencing of these PCR products (data not shown).

Colocalization and physical interaction between CaR and GABA-B-R1 in GPCs
We performed dual-fluorescence confocal microscopy to further examine the cellular distribution of the CaR and GABA-B-R in GPCs. GABA-B-R1s (Fig. 2AGo, green) and GABA-B-R2s (Fig. 2BGo, green) extensively colocalized with CaRs (Fig. 2Go, A and B, red) in intracellular organelles (arrows) as well as on the cell membranes (arrowheads) (Fig. 2Go, A and B, overlay images). As expected, GABA-B-R1s (Fig. 2CGo, green) and GABA-B-R2s (Fig. 2CGo, red), proteins required to associate to form a signaling GABA-B-R complex, were also closely colocalized in the same intracellular compartments and on the cell surface, as demonstrated by overlay images. These data support the colocalization of the CaR with both the GABA-B-R1 and -R2 as well as colocalization of the GABA-B-R1 and R2, which has been previously described in rat growth plates and chondrogenic ATDC5 cells (19).


Figure 2
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  		FIG. 2. Expression of GABA-B-Rs and immunoprecipitation of CaRs and GABA-B-R1s in GPCs from newborn Black Swiss Mice. A–C, Dual-fluorescence immunocytochemistry of GPCs isolated from newborn Black Swiss mice was visualized by confocal microscopy at x100 magnification. A, GPCs were immunostained with antisera against the GABA-B-R1 (green) and CaR (red); B, GPCs were immunostained with antisera against the GABA-B-R2 (red) and CaRs (green); C, GPCs were immunostained with antisera against GABA-B-R1 (green) and GABA-B-R2 (red). Individual images, overlays of paired images, and bright-field images from the same cells are shown. D and E, Immunoblotting of crude membrane proteins in the input (lane 1) and of immunoprecipitates from GPCs, pulled down with (lane 2) or without (lane 3) anti-GABA-B-R1 antisera, with anti-GABA-B-R1 (D) or anti-CaR (E) antisera. Immunocytochemistry (A–C) is representative of three experiments. Immunoprecipitations (D and E) were repeated in two experiments.

 
To test whether the CaR is associated with the GABA-B-R1 in GPCs, we performed immunoprecipitation with anti-GABA-B-R1 antisera. In membrane preparations before immunoprecipitation, we detected a dominant protein band of about 120 kDa, two fainter bands of about 100 and 130 kDa, and larger aggregates of about 200 kDa that reacted with anti-GABA-B-R1 antisera (Fig. 2DGo, lane 1). Anti-GABA-B-R1 antisera immunoprecipitated GABA-B-R1s from GPC membrane preparations with sizes of about 100, 120, and 130 kDa as well as two distinct larger forms at about 190 and 210 kDa (Fig. 2DGo, lane 2). The immunoprecipitation was judged specific because no GABA-B-R1 was pulled down when anti-GABA-B-R1 antisera were omitted from the reaction (Fig. 2DGo, lane 3). In membrane preparations before immunoprecipitation designated as input (Fig. 2EGo, lane 1), the anti-CaR antisera detected three predominant forms of CaRs with sizes of about 120, 130, and 140 kDa and larger aggregates of more than 200 kDa. In the immunoprecipitates pulled down by anti-GABA-B-R1 antisera (Fig. 2EGo, lane 2), anti-CaR antisera detected a predominant band with a size of about 120 kDa and a fainter band at 130 kDa. These signals were absent when immunoprecipitation was done without anti-GABA-B-R1 antisera (Fig. 2EGo, lane 3). These data support a physical interaction between the CaR and the GABA-B-R1 in GPCs.

Function of GABA-B-R1 in GPCs
To determine whether GABA-B-Rs contribute to cellular functions in GPCs, we performed an in vitro knockout of the GABA-B-R1 genes by Cre-lox recombination. We infected GPCs carrying floxed-GABA-B-R1 genes with adenoviruses carrying Cre recombinase (Ad-Cre) or with control viral constructs without inserts (Ad-Cont). In the cells infected with Ad-Cre, the expression of all GABA-B-R1 transcripts including their spliced forms was reduced by more than 90% at 72 h after infection, compared with cells infected with Ad-Cont (P < 0.001; n = 3 independent experiments; Fig. 3AGo). GABA-B-R1 protein expression, as assessed by fluorescent immunocytochemistry, was also markedly reduced in cells infected with Ad-Cre (Fig. 3BGo, bottom) compared with cells infected with Ad-Cont (Fig. 3BGo, top) as expected. These data indicate that the knockout of the GABA-B-R1 genes in this system was essentially complete.


Figure 3
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  		FIG. 3. Expression of the GABA-B-R1, proliferation, and apoptosis in GPCs from floxed-GABA-B-R1 mice infected with control viruses (Ad-Cont) or viruses carrying cre recombinase (Ad-Cre). A, RNA levels were determined by qPCR using specific primers as described in the Materials and Methods and presented as percentage of the expression of the housekeeping gene L19. *, P < 0.001. B, Fluorescent immunocytochemical detection of the GABA-B-R1 in cells infected with Ad-Cont (top) or Ad-Cre (bottom) was performed with anti-GABA-B-R1. C–E, CV staining (C), BrdU incorporation (D), and TUNEL staining (E) were performed as described in Materials and Methods in GPCs infected with viral constructs for 72 h before harvesting cells for assays. CV staining and BrdU incorporation were quantified by absorbance at wavelengths 590 and 540 nm, respectively (n = 3–4 independent experiments in triplicate; *, P < 0.001). TUNEL-positive cells are depicted by brown DAB staining (red arrows).

 
Because GABA-B-Rs have been implicated in the control of cell proliferation and apoptosis in chondrogenic cell line ATDC5 cells (19), we investigated the impact of deleting GABA-B-R1 genes on the growth and death of GPCs in culture. We used two methods to assess growth and proliferation of adenovirus-infected GPCs: CV staining, which reflects the quantity of DNA and is used as an index of cell number, and BrdU incorporation as an index of DNA synthesis. In GPCs infected with Ad-Cre, CV staining decreased by about 40% within 3 d of infection compared with cultures infected with Ad-Cont (P < 0.001; n = 9 from three independent cell isolations; Fig. 3CGo). BrdU incorporation also decreased by about 48% in cultures infected with Ad-Cre vs. Ad-Cont (P < 0.001; n = 6 for cell preparations; Fig. 3DGo). Furthermore, cultures infected with Ad-Cre had qualitatively more TUNEL-positive cells (Fig. 3EGo), indicating an increase in apoptosis of GPCs. These data support a critical role for the GABA-B-R1 in the growth and survival of GPCs.

Deletion of the GABA-B-R1 alters the extracellular Ca2+-sensing properties of GPCs
In studies with HEK-293 cells, we found that the GABA-B-R1 and -R2 can physically interact with the CaR and that coexpression of the GABA-B-R1 results in suppression of CaR protein levels (8). We used cells from floxed-GABA-B-R1 mice to test whether deleting the GABA-B-R1 gene and, consequently, the R1 protein in GPCs affects the expression of endogenous CaRs. In GPCs from floxed-GABA-B-R1 mice that were infected with Ad-Cre, the expression of CaR protein, quantified by Western analysis and densitometry, and the electrophoretic mobilities of protein bands immunoreactive with CaR antiserum did not change when compared with those of cells infected with Ad-Cont (Fig. 4AGo; n = 3 lysates). CaR mRNA levels were not different in cells from either infection (Ad-Cre vs. Ad-cont infected; n = 4 PCR from three RNA preparations; Fig. 4BGo).


Figure 4
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  		FIG. 4. Expression of CaRs and high [Ca2+]e-induced signaling responses in floxed-GABA-B-R1 GPCs infected with Ad-Cont (~16 pfu/cell) or Ad-Cre (~16 pfu/cell) for 72 h. A, Immunoblotting of total lysates (50 µg) from virally infected cells with an anti-CaR antibody or with an anti-ß-actin antibody (loading controls). B, RNA levels were determined by qPCR with specific primers and presented as percentage of L19 expression. C, Effects of [Ca2+]e (0.5–10 mM) on the accumulation of total [3H]InsPs in floxed-GABA-B-R1 GPCs infected with Ad-Cont or Ad-Cre for 72 h. Total [3H]InsPs in response to different [Ca2+]e are presented as the percent increase over the basal level at 0.5 mM Ca2+. *, P < 0.01 when compared with basal levels at 0.5 mM Ca2+; n = 3 experiments in triplicate. D, Effects of raising [Ca2+]e (0.5–20 mM) on ERK1/2 activation in floxed-GABA-B-R1 GPCs infected with Ad-Cont or Ad-Cre for 72 h. Total lysates were collected after the cells were exposed to different [Ca2+]e for 10 min, an optimized time point from pilot studies, and subjected to immunoblotting with antisera against pERK1/2 and tERK1/2 forms of ERK1/2 MAPK. E, Intensities of protein bands shown in D and other blots were quantified by densitometry and presented as ratios of pERK1/2 over tERK1/2 (n = 6 blots from three experiments).

 
To test whether the knockdown of the GABA-B-R1 affects cellular responsiveness to extracellular Ca2+, we compared the ability of high [Ca2+]e to activate PLC and ERK1/2 phosphorylation in lysates from floxed-GABA-B-R1 GPCs infected with Ad-Cre vs. Ad-Cont. In control cells (infected with Ad-Cont), raising [Ca2+]e from 0.5 to 2.5, 5.0, and 10.0 mM increased [3H]InsP production by 16 ± 4, 24 ± 2, and 51 ± 4%, respectively (P < 0.01; n = 6 for two cell preparations). The same changes in [Ca2+]e increased [3H]InsP production by 16 ± 4, 19 ± 2, and 31 ± 3%, respectively, in GPCs in which GABA-B-R1 genes were knocked out (P < 0.001; Fig. 4CGo). It is also noteworthy that the ability of high [Ca2+]e (10.0 mM) to increase [3H]InsP production was significantly blunted in GPCs with GABA-B-R1 genes knocked out (P < 0.01), indicating a decrease in sensitivity of these cells to [Ca2+]e.

Knocking out GABA-B-R1 genes in GPCs also blunted the ability of high [Ca2+]e to induce ERK1/2 activation as evidenced by ERK1/2 autophosphorylation. In control GPCs infected with Ad-Cont, raising [Ca2+]e from 0.5 to 20 mM dose-dependently increased the proportion of pERK1/2 (compared with total ERK1/2) with an ED50 of about 3.5 mM (Fig. 4Go, D and E). In GPCs infected with Ad-Cre, the ED50 of [Ca2+]e for the activation of ERK1/2 was significantly shifted from about 3.5 to about 4.5 mM (P < 0.01; n = 6 blots from three independent experiments) without affecting the maximal responses to high [Ca2+]e (Fig. 4Go, D and E).

Deletion of the GABA-B-R1 alters the expression of differentiation markers
We next tested whether deleting the GABA-B-R1 gene, which blunts the ability of high [Ca2+]e to activate PLC and ERK1/2, affected the expression of markers of chondrocyte differentiation. Blocking GABA-B-R1 expression in GPCs significantly reduced the expression of early chondrocyte markers, aggrecan (Agg) and type II collagen [{alpha}1(II)], and increased the expression of markers of maturing and terminally differentiated chondrocytes, type X collagen [{alpha}1(X)] and osteopontin (OP), respectively, at all [Ca2+]e tested (0.5, 1.5, and 3.0 mM) when compared with cells infected with Ad-Cont (Fig. 5Go, A–D) (P < 0.01; n = 4 PCR from three RNA preparations). To further determine whether the ability of high [Ca2+]e to regulate the expression of these genes changed in GPCs lacking GABA-B-R1 expression, we normalized RNA levels at different [Ca2+]e to the basal levels at 0.5 mM Ca2+ in GPCs infected with Ad-Cont or with Ad-Cre (Fig. 5Go, E–H). In GPCs infected with Ad-Cre, raising [Ca2+]e from 0.5 to 3.0 mM blocked the expression of Agg and {alpha}1(II) more profoundly than in cells infected with Ad-Cont. In contrast, the same changes in [Ca2+]e had less of an impact on the expression of {alpha}1(X) and OP in the Ad-Cre-infected cells than in the control cells. For example, in GPCs infected with Ad-Cre, raising [Ca2+]e to 3.0 mM suppressed Agg RNA levels by 74 ± 4% (vs. 60 ± 4% in control cells) (P < 0.01) and {alpha}1(II) by 77 ± 4% (vs. 54 ± 6% in control cells) (P < 0.01). In contrast, raising the [Ca2+]e from 0.5 to 3.0 mM during the culture of the Ad-Cre-infected GPCs produced an augmentation in {alpha}1(X) RNA levels of 1.71 ± 0.06-fold (vs. 2.81 ± 0.11 in control cells) (P < 0.01) and in OP RNA by 3.56 ± 0.22-fold (vs. 5.79 ± 0.25-fold in control cells) (P < 0.01). Taken together, these data indicate that GPCs in which GABA-B-R1 was acutely knocked down exhibit an advanced state of cell differentiation and altered sensitivity to changes in [Ca2+]e.


Figure 5
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  		FIG. 5. Gene expression in floxed-GABA-B-R1 GPCs infected with Ad-Cont ({blacksquare}) or Ad-Cre ({square}) for 72 h and subsequently cultured in chondrocyte differentiation medium at different [Ca2+]e (0.5, 1.5, and 3.0 mM) for an additional 7 d. Levels of RNA for Agg, {alpha}1(II), {alpha}1(X), and OP were determined by qPCR and expressed as a percentage of L19 expression (A, B, C, and D, respectively) and as normalized to the RNA levels at 0.5 mM (E, F, G, and H, respectively). Statistical significance between the values obtained from Ad-Cont-infected vs. Ad-Cre-infected cells at each specified [Ca2+]e is indicated (n = 3 experiments in hextuplicate; *, P < 0.01 for all).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the central nervous system, GABA receptors of the A, B, and C subtypes respond to the amino acid GABA, the brain’s major inhibitory neurotransmitter. Type A and C GABA-Rs are ionotropic receptors, comprised of multiple subunits that form ligand-gated ion channels. In contrast, type B GABA-Rs are metabotropic receptors that use G proteins to couple GABA binding to downstream signaling pathways. In addition to strong expression in neurons, GABA-B-Rs are widely expressed in peripheral tissues including the heart, lungs, liver, kidneys, adrenals, testes, ovaries, pancreas, bone, and cartilage where their functions remain unclear (19, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36).

Our studies in GPCs reveal for the first time an interaction between the GABA-B-R1 and the CaR, two closely related members of family C of GPCRs, in mouse GPCs. Antisera specific for the GABA-B-R1 coimmunoprecipitated the CaR from chondrocyte lysates, and both receptors were expressed in and colocalized to specific chondrocyte subpopulations in the growth plate and to specific cellular compartments. Our data further indicate that manipulating expression of the endogenous GABA-B-R1 in GPCs could alter signal transduction by the CaR and the growth, survival, and differentiation of GPCs. Because CaRs and GABA-B-R1s are coexpressed in many other tissues including brain (21), intestine (27, 29), bone (28), parathyroid (8), and kidney (8), potential interactions and cross-talk between these two receptors could influence a variety of functions in these systems.

This notion is supported by our recent finding that the GABA-B-R1 coimmunoprecipitated with the CaR from brain lysates and that the CaR expression increased in brain lysates of GABA-B-R1 knockout mice and in cultured hippocampal neurons lacking GABA-B-R1 expression (manuscript in review). These data support a role for the GABA-B-R1 in modulating CaR protein expression in neurons and other cells in the nervous system. In contrast to the findings in neurons, we did not observe changes in steady-state levels of CaR protein and RNA in GPCs when their GABA-B-R1 genes were knocked out, suggesting that cell context and other differences between chondrocytes and neurons are critical in determining ultimate effects on CaR expression and function.

Our data, however, demonstrate that the interaction and/or cross-talk between the CaRs and GABA-B-Rs have clear functional consequences in GPCs. The presence of both GABA-B-R1s and CaRs vs. only CaRs produced a distinct pharmacology of extracellular Ca2+ sensing in GPCs. Knocking down GABA-B-R1 expression inhibited high [Ca2+]e-induced activation of PLC and ERK1/2 in these cells. It is, however, unclear whether GABA-B-R1s modulate signaling of CaRs directly, by forming complexes with them, or indirectly, by altering the availability and/or binding of signaling molecules required for CaR signaling. We favor the former idea based on our recent observations that the CaR and GABA-B-R1 and -R2 form complexes and their coexpression alters the ability of the CaR to sense changes in [Ca2+]e in HEK-293 cells expressing these receptors (8).

How might the interaction between the GABA-B-R1 and CaR affect the ability of GPCs to sense changes in [Ca2+]e? One potential pathway is that GABA-B-Rs mediate the trafficking of CaRs to the cell surface. Several groups have noted that the dimerization of the GABA-B-R1 with GABA-B-R2 is an essential step in the efficient cell-surface expression of receptor complexes (21, 37, 38, 39). It is possible that the GABA-B-Rs modulate the processing and trafficking of CaRs endogenously and, therefore, the availability of the CaRs on the cell surface to signal to the cell’s interior. Alternatively, direct interactions between GABA-B-Rs and CaRs produce protein complexes that have distinct Ca2+-binding properties and specialized abilities to activate downstream signaling pathways. Our signaling assays support the latter scenario, because they show that blocking the GABA-B-R1 expression in GPCs reduced cellular sensitivity of extracellular Ca2+ to activate ERK1/2 without affecting the maximal level of ERK1/2 phosphorylation.

It is of interest that blocking GABA-B-R1 expression also suppressed high [Ca2+]e-induced activation of PLC in GPCs, supporting the possibility that endogenous GABA-B-R1s may have a role to enhance CaR-mediated signaling in these cells. These findings in GPCs differ from our observations in transiently transfected HEK-293 cells in which coexpressing high levels of the GABA-B-R1 with the CaR suppressed the ability of high [Ca2+]e to activate PLC (8). Clearly, different cell contexts (chondrocytes vs. HEK-293 cells), the relative abundance of the receptors (endogenous expression levels vs. targeted overexpression), altered stoichiometry, and different downstream signaling elements (G protein subunits and PLC isoforms) could readily contribute to the different signaling responses. Furthermore, the ability of the CaR to homodimerize and heterodimerize with other family C receptors in chondrocytes, such as the GABA-B-R2 and mGluRs, could also contribute to the unique Ca2+-sensing properties in chondrocytes vs. HEK-293 cells. Additional studies in other cell types will help to illuminate this issue.

The role of GABA-B-Rs in chondrocyte differentiation in vivo is uncertain. Although three generalized GABA-B-R1 knockout mouse models [GABA-B-R1(–/–)] have been developed to investigate the role of the GABA-B-R1 in the brain (40, 41, 42), the dramatic neurological phenotype (recurrent seizures, severe behavioral disturbances, and early death in two of three strains) makes it difficult to study primary effects of the GABA-B-R1 on cartilage function and growth dynamics in vivo. The body weights of the GABA-B-R1(–/–) in the murine background C57B16/J were significantly reduced, suggesting a growth problem. The cause for this, however, is uncertain because of the severe neurological abnormalities in these mice (40). It will be essential to examine the consequences of deletion of the GABA-B-R1 gene, restricted to the growth plate in vivo, to determine the effects of GABA-B-R1 knockout on CaR protein, gene expression, and proliferation in vivo.

Might the changes induced by altering GABA-B-R1 expression in GPCs be due to direct interruption in GABA signaling? This must be considered. Tamayama et al. (19) reported that agonists of GABA-B-Rs, such as baclofen and GABA, promoted the proliferation of chondrogenic ATDC5 cells in culture and that their effects can be blocked by CGP-35348, a GABA-B-R1 antagonist. We showed that knocking down the expression of the GABA-B-R1 in GPCs reduced cell proliferation, induced apoptosis, and promoted the expression of marker genes indicative of terminal differentiation at all [Ca2+]e tested, suggesting a CaR-independent action of GABA-B-R1. We and others have detected the presence of GABA and the expression of glutamic acid decarboxylase 65 and 67 (GAD65/67), enzymes that convert glutamate into GABA, in the growth plate and cultured GPCs (43) (unpublished data). These observations suggest that the GABA-B-R1s and their ligands may constitute an auto/paracrine system that modulates GPC differentiation. Because knocking out GABA-B-R1s profoundly alters CaR signaling, it is difficult to determine definitely whether putative modulation of differentiation by GABA-B-R1s is direct or indirect via alterations in CaR signaling. The fact that knocking down the GABA-B-R1 expression impacted on the ability of high [Ca2+]e to activate signaling responses and gene expression suggests that GABA-B-R1 could mediate chondrocyte function at least in part via CaR-dependent pathways. Chondrocyte models that permit the selective and timed manipulation of CaR expression, such as GPCs derived from a floxed CaR mouse, will be required to further distinguish these possibilities.

Previous work has raised the possibility that other members of family C might function as Ca2+ sensors in GPCs (44, 45). The mGluR1 and -R5 were initially strong candidates, because they can sense [Ca2+]e and activate some of the same signaling pathways that CaRs do (e.g. PLC activation and the inhibition of adenylate cyclase activity). Their low levels of expression, however, argue against that possibility. Additional experiments, however, will be required to definitely exclude them and other mGluR subtypes. The robust, although right-shifted, sensitivity to changes in [Ca2+]e of GPCs lacking GABA-B-R1s compared with control cells argues against a role for the GABA-B-R1 as a critical Ca2+-sensing molecule in GPCs, despite its strong expression in those cells. The CaR, thus far, remains the most legitimate candidate extracellular Ca2+ sensor in GPCs. We speculate that the distinctive signaling responses to high [Ca2+]e and other CaR agonists, which set GPCs apart from other Ca2+-sensing cells like parathyroid cells, may be due to unique posttranslational modifications and/or interactions of CaRs with other molecules in GPCs. One such interaction may pair CaRs and other members of family C or other receptor or signaling molecules yet to be identified.


   Acknowledgments
 
We gratefully acknowledge helpful discussions of Drs. Lily Y. Jan (University of California, San Francisco, Departments of Physiology and Biochemistry and Biophysics), Robert Nissenson, and Daniel Bikle (University of California, San Francisco, Endocrine Unit) in the planning and completion of these studies.


   Footnotes
 
This work was supported by the Department of Veterans Affairs Merit Review (D.S.) and Research Education Advancement Program in Bone Disease (W.C. and D.S.), by National Institutes of Health RO1 AG 21353 and R21 AR054441 (W.C.), and by the Swiss Science Foundation Grant 3100-067100.01 (B.B.).

Disclosure Statement: All authors have nothing to disclose.

First Published Online July 5, 2007

Abbreviations: {alpha}1(II), Type II collagen; {alpha}1(X), type X collagen; Ad-Cont, control adenovirus carrying no insert; Ad-Cre, adenovirus carrying bacterial Cre recombinase; Agg, aggrecan; BrdU, bromodeoxyuridine; [Ca2+]e, extracellular Ca2+ concentration; CaR, Ca2+-sensing receptor; CMM, chondrocyte maintenance medium; CV, crystal violet; DAB, 3,3'-diaminobenzidine; GABA, {gamma}-aminobutyric acid; GABA-R, GABA receptor; GPC, growth plate chondrocytes; GPCR, G protein-coupled receptor; InsP, inositol phosphate; mGluR, metabotropic glutamate receptor; NP-40, Nonidet P-40; OP, osteopontin; pERK, phosphorylated ERK; PLC, phospholipase C; qPCR, quantitative real-time PCR; tERK, common ERK; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Received May 17, 2007.

Accepted for publication June 20, 2007.


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