Expression and Functional Assessment of an Alternatively Spliced

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Endocrinology 146(12):5294 –5303 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2005-0256

Expression and Functional Assessment of an Alternatively Spliced Extracellular Ca2ⴙ-Sensing Receptor in Growth Plate Chondrocytes Luis Rodriguez, Chialing Tu, Zhiqiang Cheng, Tsui-Hua Chen, Daniel Bikle, Dolores Shoback, and Wenhan Chang Endocrine Research Unit, Department of Medicine, Department of Veterans Affairs Medical Center, University of California, San Francisco, California 94121 The extracellular Ca2ⴙ-sensing receptor (CaR) plays an essential role in mineral homeostasis. Studies to generate CaRknockout (CaRⴚ/ⴚ) mice indicate that insertion of a neomycin cassette into exon 5 of the mouse CaR gene blocks the expression of full-length CaRs. This strategy, however, allows for the expression of alternatively spliced CaRs missing exon 5 [Exon5(ⴚ)CaRs]. These experiments addressed whether growth plate chondrocytes (GPCs) from CaRⴚ/ⴚ mice express Exon5(ⴚ) CaRs and whether these receptors activate signaling. RT-PCR and immunocytochemistry confirmed the expression Exon5(ⴚ) of CaR in growth plates from CaRⴚ/ⴚ mice. In Chinese hamster ovary or human embryonic kidney-293 cells, recombinant human Exon5(ⴚ)CaRs failed to activate phospholipase C likely due to their inability to reach the cell surface as assessed by intact-cell ELISA and immunocytochemistry. Human Exon5(ⴚ)CaRs, however, trafficked normally to the cell

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ROWTH PLATE DEVELOPMENT is a critical component in endochondral bone formation (1, 2). Studies of patients with rickets due to Ca2⫹ and/or vitamin D deficiency and vitamin D receptor-knockout mice suggest that a sufficient quantity of Ca2⫹ is required for normal chondrocyte differentiation and growth plate development (3–7). Studies with cultured growth plate chondrocytes (GPCs) (8) and the rat chondrogenic cell line RCJ3.1C5.18 (or C5.18 cells) further indicate that different levels of extracellular [Ca2⫹] ([Ca2⫹]e) mediate chondrocyte differentiation directly (9 – 11). It is, however, unclear what signaling mechanisms are responsible for coupling changes in [Ca2⫹]e to downstream cellular responses in chondrocytes. In 1993 Brown et al. (12) identified cDNA from a bovine parathyroid cDNA library encoding a G protein-coupled receptor that mediates responsiveness of the parathyroid gland to Ca2⫹ that they termed the extracellular Ca2⫹-sensFirst Published Online September 15, 2005 Abbreviations: ADH, Autosomal dominant hypoparathyroidism; Agg, aggrecan; ␣1(II), ␣-subunit of type II collagen; [Ca2⫹]e, extracellular [Ca2⫹]; CaR, Ca2⫹-sensing receptor; CaR⫺/⫺, CaR knockout; CHO, Chinese hamster ovary; CIM, chondrocyte isolation medium; DAB, 3, 3⬘diaminobenzidine; FBS, fetal bovine serum; GP, growth plate; GPC, GP chondrocyte; h, human; HEK, human embryonic kidney; HPTH, hyperparathyroidism; InsP, inositol phosphate; OP, osteopontin; PLC, phospholipase C; q-PCR, quantitative real-time PCR; wt, wild type. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

surface when overexpressed in wild-type or CaRⴚ/ⴚ GPCs. Immunocytochemistry of growth plate sections and cultured GPCs from CaRⴚ/ⴚ mice showed easily detectable cell-membrane expression of endogenous CaRs (presumably Exon5(ⴚ) CaRs), suggesting that trafficking of this receptor form to the membrane can occur in GPCs. In GPCs from CaRⴚ/ⴚ mice, high extracellular [Ca2ⴙ] ([Ca2ⴙ]e) increased inositol phosphate production with a potency comparable with that of wild-type GPCs. Raising [Ca2ⴙ]e also promoted the differentiation of CaRⴚ/ⴚ GPCs as indicated by changes in proteoglycan accumulation, mineral deposition, and matrix gene expression. Taken together, our data support the idea that expression of Exon5(ⴚ)CaRs may compensate for the loss of fulllength CaRs and be responsible for sensing changes in [Ca2ⴙ]e in GPCs in CaRⴚ/ⴚ mice. (Endocrinology 146: 5294 –5303, 2005)

ing receptor (CaR). CaRs are expressed in a remarkably diverse range of tissues in addition to the parathyroid, including the kidney, brain, intestine, stomach, skin, breast, bone, and cartilage (13, 14). Both human genetic studies and the CaR knockout (CaR⫺/⫺) mouse model developed by Ho et al. (15) support the essential nonredundant roles of CaRs in controlling PTH secretion, parathyroid gland hyperplasia, and renal Ca2⫹ excretion (16, 17). The exact role of CaRs in many of the other tissues in which they are expressed in vivo remains an unsettled issue. Our work, using antisense oligonucleotides and expression of dominant-negative CaR cDNA constructs, has implicated CaRs in coupling changes in [Ca2⫹]e to matrix synthesis, mineral deposition, and gene expression in C5.18 cells (9, 10). Observations from CaR double-knockout mouse models, however, argue against a physiological function of CaRs in growth plate (GP) cartilage (16, 17). The phenotype of the CaR⫺/⫺ mouse, initially developed by Ho et al. (15), included severe hypercalcemia, parathyroid hyperplasia, and hypocalciuria, confirming a critical role for CaRs in parathyroid and kidney function. Interestingly, these mice also showed severe rickets and very low bone formation rates (15, 18). The early death of CaR⫺/⫺ mice within 2–3 wk of birth precluded the assessment of their cartilage and bone beyond the neonatal period. For this reason, rescue models were developed by Tu et al. (16) and Kos et al. (17). These rescue models were created by breeding CaR⫺/⫺ mice with PTH⫺/⫺ mice (17) or gcm2⫺/⫺ mice (16). In both of these double-

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Rodriguez et al. • Alternatively Spliced CaRs in Chondrocytes

knockout models, hyperparathyroidism (HPTH) cannot develop. PTH is not made in the case of the PTH⫺/⫺ mouse, and the parathyroid glands do not develop in the case of the gcm2⫺/⫺ mouse (17) because gcm2 is a transcription factor essential for parathyroid development. Studies of the double-knockout mice [CaR⫺/⫺/PTH⫺/⫺ or CaR⫺/⫺/gcm2⫺/⫺] indicate that the skeletal defects, originally thought to be related to the absence of CaRs, were healed when HPTH was prevented (16, 17). This work led to the conclusion that wild-type (wt) CaRs are nonessential in the development and function of the GP and bone. That conclusion, however, conflicted with many studies on the role of CaRs in bone cells and chondrocytes in vitro by several groups (19 –22). These findings led us to look more closely at the CaR⫺/⫺ mouse model. The knockout of CaRs in the original CaR⫺/⫺ mouse (15) is incomplete. These animals can express an alternatively spliced CaR [Exon5(⫺)CaR] lacking peptide sequence encoded by exon 5 because of the gene targeting strategy. Exon5(⫺)CaR transcripts have been found in skin (23, 24) and kidney (25) from CaR⫺/⫺ mice and may be present in other tissues. This study addressed the hypothesis that GPCs in CaR⫺/⫺ mice express Exon5(⫺)CaRs and that they may compensate for the absence of full-length CaRs in sensing changes in [Ca2⫹]e. We examined the expression of Exon5(⫺) CaRs in GPCs by RT-PCR, immunoblotting, and immunocytochemistry. We assessed signaling and expression properties of Exon5(⫺)CaRs in transfected Chinese hamster ovary (CHO) and human embryonic kidney (HEK)-293 cells and GPCs. Finally, we tested the ability of GPCs from CaR⫺/⫺ mice to activate signal transduction and modulate differentiation in response to changes in [Ca2⫹]e and other known CaR agonists. Our findings support the idea that these alternatively spliced CaR transcripts may play a functional role in cartilage under conditions in which wt CaRs are absent. Materials and Methods Materials Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Atlanta, GA) and Tissue Culture Biologicals (Tulare, CA). Primers and probes for quantitative real-time PCR (q-PCR) and RT-PCR were custom made by Integrated DNA Technologies (Skokie, IL) according to published nucleotide sequences. Other supplies were from previously noted sources (8 –10, 26, 27)

Cloning of CaR cDNA Poly A⫹ RNA was extracted from epiphyseal growth plate cartilage from 2-d-old CaR⫺/⫺ mice and their wt (CaR⫹/⫹) and heterozygous (CaR⫹/⫺) littermates that were genotyped as described (24, 27). CaR cDNA was amplified with sense (5⬘-CAAGGTCATTGTCGTTTTCTCCAGC) and antisense (5⬘-GCAATGCAGGAAGTGTAGTTCTCAT) primers (11), subcloned into pCR 2.1 plasmid by TOPO-TA cloning kit (Invitrogen, Carlsbad, CA), and sequenced.

GPC cultures Epiphyseal growth plates from the limbs of newborn CaR⫹/⫹, CaR⫹/⫺, and CaR⫺/⫺ mice (2– 4 d old) were dissected free of soft tissues, and chondrocytes were released by enzymatic digestion (8). After three washes with a chondrocyte isolation medium [CIM: DMEM ⫹ penicillin (100 ␮g/ml), streptomycin (100 U/ml), NaHCO3 (45 mm), HEPES (20 mm, pH 7.4), Fungizone (0.25 ␮g/ml), and gentamicin (0.15 mg/ml)],

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cartilage was incubated with trypsin (0.25%, wt/vol) in CIM (10 ml per 1 g tissue) at 37 C for 15 min. After two washes with CIM supplemented with FBS (10%, vol/vol) to inactivate the trypsin, cartilage was incubated with an enzyme mixture containing (wt/vol) collagenase type IA (0.18%), hyaluronidase (0.1%), and DNase II (0.01%) for 20 –30 min at 37 C with agitation. GPCs in suspension were collected by centrifugation at 400 ⫻ g for 10 min and washed three times with chondrocyte maintenance medium [DMEM ⫹ penicillin (100 ␮g/ml), streptomycin (100 U/ml), and fetal calf serum (10%, vol/vol)] at 37 C. Undigested cartilage was continuously digested with fresh enzymes until completion. All cell harvests but the first were combined and plated (105 cells/cm2). To test the effects of Ca2⫹, GPCs were grown to confluence and switched to chondrocyte differentiation medium [␣MEM containing Mg2⫹ (0.5 mm) plus ascorbic acid (50 ␮g/ml) and ␤-glycerol phosphate (5 mm)] with different [CaCl2] (0.5 to 3.0 mm) for different times before sample analyses. All protocols involving mice were approved by the Animal Care Subcommittee of the San Francisco Department of Veterans Affairs Medical Center.

Expression of human CaRs in CHO, HEK-293 cells, and mouse GPCs Plasmids encoding the full-length wt human (h)-CaR (wt-hCaR/ pcDNA3.1-hygro) and the alternatively spliced CaR missing exon 5 [Exon5(⫺)hCaR/pcDNA3.1-hygro] were constructed as described and transfected into CHO and HEK-293 cells using Lipofectamine (Invitrogen) or Ca phosphate methods (27). For studies of cells transiently expressing these receptors, assays were performed 48 –72 h after transfection. In studies of cells stably expressing CaRs, CHO cells transfected with wt-hCaR/pcDNA3.1-hygro or Exon5(⫺)hCaR /pcDNA3.1-hygro were treated with hygromycin B (200 ␮g/ml) for 4 – 6 wk, and cell clones were isolated by limiting dilution. For dual-fluorescence immunocytochemistry, HEK-293 cells and subconfluent (50 –70% confluence) GPCs from CaR⫺/⫺ and CaR⫹/⫹ mice were transfected with cDNA encoding wt-hCaR or Exon5(⫺)hCaR tagged with V5 epitope (GKPIPNPLLGLDST) at their C termini. Cells were transfected as above and cultured for 72 h before samples were prepared for Western analysis and immunocytochemistry (27).

Measurement of inositol phosphates (InsPs) and cAMP content Levels of total InsPs in transfected CHO and HEK-293 cells and GPCs from CaR⫹/⫹ and CaR⫺/⫺ mice were determined after incubating the cells at different [Ca2⫹]e (0.5–30 mm) or other CaR agonists, including Sr2⫹ (0.5–30 mm), Mn2⫹ (0.3–10 mm), Mg2⫹ (0.5–30 mm), and neomycin (0.1–3 mm), for 60 min according to previous protocols (28 –30). Effects on total InsP accumulation is presented as the fold increase over the basal levels in cells maintained at 0.5 mm Ca2⫹ and 0.5 mm Mg2⫹. cAMP accumulation was determined after incubating cells with assay medium [MEM with BSA (0.1%, wt/vol), 3-isobutyl-1-methylxanthine (0.4 mm), MgSO4 (0.5 mm)] containing different concentrations of Ca2⫹ (0.5, 2.5, 5.0, 10.0, or 30 mm) in the presence of forskolin (10⫺5 m) (11, 28). cAMP content at each [Ca2⫹] is expressed in comparison with control levels at 0.5 mm Ca2⫹.

ELISA for CaRs in transfected CHO and HEK-293 cells Cell surface expression of recombinant CaRs was quantified by an intact-cell ELISA as previously described (31–33) with an anti-hCaR 7F8 antiserum that was raised against the entire extracellular domain of the human CaR kindly provided by Drs. Allen Spiegel and Paul Goldsmith (National Institute of Diabetes and Digestive and Kidney Diseases). Briefly, CHO cells stably transfected with full-length and Exon5(⫺)hCaRs cDNAs or control vectors and HEK-293 cells transiently transfected with the same constructs were incubated with anti-hCaR 7F8 (1 ␮g/ml) in ice-cold MEM (106 cells per 100 ␮l) containing 10% FBS for 60 min. After three washes with PBS, cells were incubated with peroxidase-conjugated goat antimouse IgG antibodies for 60 min at 4 C, followed by three more washes with PBS. Immunoreactivity was detected by a peroxidase substrate 2,2-azinobis-3-ethylbenzthiazoline-6-sulfonic acid (Sigma, St. Louis, MO) and quantified by absorbance at 410 nm.

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Immunocytochemistry and Immunoblotting Detection of CaR immunoreactivity in growth plates from 4-d-old CaR⫹/⫹ and CaR⫺/⫺ mice was performed with anti-CaR antiserum 21825A (500 nm) in combination with either peroxidase- or fluoresceinconjugated antirabbit IgG antibodies (11, 27). Signals were detected by 3, 3⬘-diaminobenzidine (DAB)-substrate and visualized by light microscopy or confocal fluorescence microscopy. Specificity was confirmed by the lack of signals in cartilage sections treated with peptide-preabsorbed antisera (11). Localization of endogenous CaRs and the intrinsic membrane protein CD44 in cultured CaR⫺/⫺ and CaR⫹/⫹ mouse GPCs was performed with anti-CaR (21825A) and anti-CD44 antisera and dual-fluorescence confocal microscopy as described (11). Expression of V5-tagged wthCaRs and Exon5(⫺)hCaRs and endogenous CD44 in transfected CaR⫺/⫺ GPCs and HEK-293 cells was assessed with anti-V5 (Invitrogen) and anti-CD44 antisera, respectively. Briefly, HEK-293 cells and CaR⫺/⫺ mouse GPCs grown on coverslips were transfected with cDNAs for 48 –72 h, fixed with paraformaldehyde (4%) for 20 min, and permeabilized with methanol (80%). Cells were coincubated with anti-V5 and anti-CD44 antisera overnight at 4 C, followed by fluorescein- and Texas red-conjugated anti-IgG antibodies for 60 min at room temperature. After washing, coverslips were mounted on glass slides and examined by confocal microscopy as previously described (11). Paired pseudocolored fluorescent images were obtained sequentially, and their overlays are presented. Lysates and membrane proteins prepared from CHO and HEK-293 cells and GPCs were electrophoresed on 5 or 7% SDS-PAGE gels in the presence of 100 mm dithiothreitol and then transferred onto Immobilon-P nitrocellulose membrane (28). Membranes with proteins were immunoblotted with anti-CaR antiserum (21825A) or anti-V5 antiserum for the recombinant human CaRs as previously described (11, 28). Specificity of the antisera was confirmed by the absence of signal when antisera were preincubated with peptides against which they were raised.

Alcian green, alizarin red, and von Kossa staining Proteoglycan and mineral accumulation in cultures was assessed by Alcian green 2GX and alizarin red S staining and quantified by absorbance at 340 and 563 nm, respectively (9, 10).

q-PCR Total RNA was isolated using a RNA-Stat reagent (Tel-Test Inc., Friendswood, TX) (11). First-strand cDNAs were synthesized from RNA with Moloney murine leukemia virus reverse transcriptase (Invitrogen). Gene expression was quantified using probe-based TaqMan q-PCR kits, ABI PRISM 7900HT sequence detection system, and SDS software (Applied Biosystems, Foster City, CA). A cycle threshold (minimal PCR cycles required to generate a fluorescent signal exceeding a preset threshold) was determined for each gene of interest and normalized to a cycle threshold for a housekeeping gene (L19) determined in parallel. L19 is a ribosomal protein whose expression was not changed by growth at different [Ca2⫹]e (data not shown).

Expression of CaR⫺/⫺ mice

Results CaRs in growth plates from

Exon5(⫺)

To determine whether chondrocytes in wt and CaR⫺/⫺ mice express CaR transcripts, we performed RT-PCR on RNAs isolated from 2- to 4-d-old GPs with primers flanking exons 4 and 6 of the mouse CaR gene. These primers enable us to amplify and distinguish by size products from wt vs. Exon5(⫺) CaRs. From the GP and kidney of CaR⫹/⫹ mice (as a positive control), we amplified a product of the expected size (⬃1007 bp) for a fragment of the full-length CaR (Fig. 1A). Both the nucleotide and deduced amino acid sequences of this putative mouse growth plate CaR (11) were conserved

FIG. 1. Cloning of mouse CaR cDNAs. A, Ethidium bromide gel electrophoresis of RT-PCR products amplified from cartilage and kidney RNAs from CaR⫹/⫹, CaR⫹/⫺, and CaR⫺/⫺ mice. B, Alignments of predicted peptide sequences for exon 5-deficient CaR from CaR(⫺/⫺) GP and a full-length GP CaR from wt littermates (AF159565). Results shown are representative of two independent experiments from two different mice for each phenotype. RT, Reverse transcription.

(99.6 and 99.8%, respectively) with sequences encoding residues 265–599 in the extracellular domain of the mouse CaR (25), confirming that this product is derived from the CaR gene. Using the same primer pair, a smaller cDNA (776 bp) was amplified from reversed-transcribed cDNA isolated from GPs of CaR⫺/⫺ mice (Fig. 1A). Nucleotide (data not shown) and deduced amino acid sequences of this PCR product (Fig. 1B) indicated that it was part of a transcript missing exon 5 of the mouse CaR gene. From CaR⫹/⫺ GPCs, both PCR products were amplified. In this PCR amplification, the product representing the wt CaR fragment was fainter than the fragment encoding the spliced CaR (Fig. 1A), but in other amplifications, equal intensity was detected for both wt and spliced CaRs. To assess CaR protein expression in the GP, we performed immunocytochemistry using anti-CaR antiserum (no.



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serum in CaR⫺/⫺ GPs (Fig. 2F) when compared with CaR⫹/⫹ GPs. Under higher-power views, CaR immunoreactivity was strongly localized by DAB staining to intracellular organelles (arrows) and to the cell surface (arrowheads) of chondrocytes from both CaR⫹/⫹ and CaR⫺/⫺ mice (Figs. 2, C and G) as well as by fluorescence confocal microscopy (Fig. 2, D and H). The presence of many unstained cells in these sections (Fig. 2, A–C and E–G) argues in favor of specificity. We also tested antiserum, preabsorbed with bovine CaR peptides 214 –235, and little if any immunoreactivity was seen in these sections (data not shown).

21825A) raised against a highly conserved epitope (amino acids 214 –235) in the extracellular domain of most CaRs. As indicated by brown DAB staining, CaRs were detected in maturing chondrocytes in the GP from CaR⫹/⫹ mice, and their expression increased across the GP into the hypertrophic zone (Fig. 2, A and B). This pattern of expression was comparable with that previously described in bovine and rat GPs (11). A similar pattern of CaR protein expression was also demonstrated in GPs from CaR⫺/⫺ mice (Figs. 2, E and F). It is, however, notable that the hypertrophic zones of CaR⫺/⫺ growth plates were significantly expanded (Fig. 2E), when compared with their wt counterparts (Fig. 2A), confirming the rachitic phenotype of CaR⫺/⫺ mice reported previously (18). We also observed larger numbers of proliferating chondrocytes immunoreactive with anti-CaR anti-

Signaling and expression properties of and HEK-293 cells

FIG. 2. Immunocytochemical detection of CaRs in GPs from CaR⫹/⫹ (A–D) and CaR⫺/⫺ (E–H) mice, using anti-CaR antiserum (no. 21825A, 500 nM). Expression of CaRs, depicted by brown DAB staining (A–C and E–G), or fluorescent signals by confocal microscopy (D and H) were mainly found in maturing and hypertrophic chondrocytes. Arrowheads and arrows depict immunoactivity in the cell membranes and intracellular organelles, respectively. Magnification (A and E), ⫻20; (B and F), ⫻40; (C and D), and (G and H) ⫻63. Results shown are representative of three experiments from two different mice for each phenotype. PZ, Proliferation zone; MZ, maturation zone; HZ, hypertrophic zone.

To test whether CaRs lacking exon 5 remain responsive to changes in [Ca2⫹]e and activate signal transduction, we stably expressed cDNAs encoding either full-length or exon 5-deficient human keratinocyte CaRs in CHO cells and tested the effects of different [Ca2⫹]e on these cells. In a CHO clone expressing high levels of wt-hCaRs, raising [Ca2⫹]e from 0.5 to 10 or 30 mm increased InsP levels (P ⬍ 0.001) by 24- or 26-fold, respectively (Fig. 3A). Three independent clones expressing wt-hCaRs produced similar signaling responses (data not shown). High [Ca2⫹]e also suppressed forskolin (10⫺5 m)-induced cAMP accumulation in these cells by up to 76% (P ⬍ 0.001) with an IC50 approximately 3 mm Ca2⫹ (Fig. 3B). Raising [Ca2⫹]e from 0.5 up to 30 mm, however, did not increase InsP production or lower cAMP levels in cells expressing Exon5(⫺)hCaRs (Figs. 3, A and B). There was a slight but significant (P ⬍ 0.05) increase (⬃30%) in cAMP levels at [Ca2⫹]e 2.5 mm or more in Exon5(⫺)hCaR-expressing cells, but this was also seen in control cells expressing vector (Fig. 3B). Immunoblotting confirmed that wt or Exon5(⫺)hCaRs were expressed in lysates from cells separately transfected with these constructs (Fig. 3C). Lysates from cells expressing wthCaRs demonstrated two glycosylated CaR proteins of approximately 140 and 160 kDa and larger aggregates of more than 214 kDa. There was, however, only one prominent band of approximately 130 kDa likely representing a glycosylated form of the receptor (23), which was detected in lysates from Exon5(⫺) hCaRs-expressing CHO cells. The predicted size of a CaR protein lacking exon 5 is approximately 110 kDa. There were other higher molecular mass (⬎214 kDa) bands that are typically detected in CaR immunoblots (11, 28, 34, 35) and interpreted as receptor aggregates that were absent from these immunoblots. These data suggest differences in posttranslational modifications between wt-hCaRs and Exon5(⫺) hCaRs in CHO cells. To determine whether Exon5(⫺)hCaRs are detectable on the membranes of CHO cells, we performed an intact-cell ELISA. In cells stably expressing wt-hCaRs, membrane immunoreactivity specific for CaRs was clearly evident, compared with the background signals detected in vector-transfected control cells (Fig. 3D). CaR antibody binding was not, however, detectable on Exon5(⫺)hCaR-expressing cells (Fig. 3D). This observation was confirmed with another Exon5(⫺)hCaR-expressing clone (data not shown). These data suggest that the lack of sufficient and/or stable cell surface expression of

Exon5(⫺)

CaRs in CHO

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FIG. 4. CaR expression in HEK-293 cells transiently transfected with vector (Cont), wt-hCaR (Wt), or Exon 5(⫺)hCaR [Exon5(⫺)] cDNAs. A, Intact-cell ELISA with anti-hCaR 7F8 antiserum in the transfected cells. B, Lysates (50 ␮g protein/lane) from transfected cells were blotted with anti-CaR antisera (no. 21825A; 50 nM) or anti-CaR antisera preincubated with peptides (25 ␮M) (⫹PEP). C and D, Confocal fluorescence microscopy of transiently transfected cells expressing wt-hCaRs and Exon 5(⫺)hCaRs. Cells were stained with anti-CaR antisera (21825A, 500 nM) and detected with Texas red-conjugated goat antirabbit IgG antisera (left panels) and anti-CD44 antisera detected by goat antirat IgG antisera conjugated with fluorescein isothiocyanate (middle panels). Overlays of doubly stained cells are shown in the panels on the far right. Results shown are representative of more than 40 cells from two separate transfections. FIG. 3. Signal transduction and protein expression of wt-hCaR and Exon 5(⫺) hCaRs in stably transfected CHO cells. Total 3H-InsPs (A) and forskolin (10⫺5 M, B)-induced cAMP production at different [Ca2⫹]e in cells expressing vector (open bars), wt-hCaR (black bars), or Exon 5(⫺)hCaRs (gray bars) (n ⫽ 3 independent experiments in triplicate). *, P ⬍ 0.01. C, Immunoblotting of lysates (50 ␮g protein/ lane) from cells expressing vector (Cont), wt-hCaR (Wt]), or Exon5(⫺)hCaR [Exon5(⫺)] cDNAs with anti-CaR antiserum (no. 21825A; 50 nM). Results shown are representative of six immunoblots from three independent transfections. B, Detection of cell surface expression of CaRs by intact-cell ELISA with anti-hCaR 7F8 antiserum in the transfected CHO cells (see Materials and Methods for detailed protocols) (n ⫽ 3 independent transfections). Exon5(⫺)

hCaRs may explain the inability of CHO cells expressing these receptors to respond to changes in [Ca2⫹]e. To rule out the possibility that the selection of specific cell clones and/or the CHO cell line itself might explain our inability to detect cell surface expression of the Exon5(⫺)hCaRs in the cells, we transiently expressed this receptor in HEK293 cells. In HEK-293 cells expressing wt-hCaRs, abundant receptors were detected in the membrane by ELISA. Little, if any, CaR antibody binding was detected in HEK-293 cells expressing Exon5(⫺)hCaRs (Fig. 4A). Immunoblots confirmed that substantial quantities of both wt-hCaRs and

Exon5(⫺)

hCaRs were translated in both populations of cells (Fig. 4B). All protein bands shown on the immunoblot appear to be authentic CaRs because their signals were absent when antisera were preabsorbed with peptide (Fig. 4B). Impaired surface expression of Exon5(⫺)hCaRs was further confirmed by dual-fluorescence immunocytochemistry (Fig. 4C). In HEK293 cells transiently expressing wt-hCaRs, CaR immunoreactivity was localized to intracellular compartments as well as the cell membranes (Fig. 4C, anti-CaR). In the latter instance, another membrane receptor CD44 (Fig. 4C, antiCD44) was coexpressed. An overlay of the images confirmed abundant colocalization of CaR and CD44 in wt-hCaR-expressing HEK-293 cells (Fig. 4C, overlay). In Exon5(⫺)hCaRexpressing cells, however, CaR immunoreactivity was also detected in intracellular organelles (Fig. 4D, anti-CaR) but was undetectable in cell membranes as indicated by the lack of colocalization with CD44 (Fig. 4D, overlay). At least 30 HEK-293 cells expressing Exon5(⫺) hCaRs were analyzed in two independent experiments supporting these results. These data document the impaired cell surface expression of Exon5(⫺) hCaR in CHO and HEK-293 cells.


Expression of

Exon5(⫺)

CaRs in GPCs

As shown above, CaR immunoreactivity was present in the membranes of chondrocytes in the GPs of CaR⫺/⫺ mice (Fig. 2, G and H). We further confirmed this by staining GPCs cultured from CaR⫺/⫺ mice with CaR antisera. In those cells, the expression of CaRs localized to both intracellular compartments and cell membranes, the latter confirmed by colocalization with the membrane marker CD44 (Fig. 5A). Because the CaR⫺/⫺ mouse can make only the Exon5(⫺) CaR (15), CaR immunoreactivity present in these cells is presumed to be due to expression of CaR proteins lacking exon 5. Taken together, these observations indicate that chondrocytes, unlike CHO and HEK-293 cells, provide a permissive cellular context for maintaining detectable quantities of exon 5-deficient CaRs in the membrane. To further test this hypothesis, we examined whether recombinant Exon5(⫺)hCaRs are efficiently expressed on the surface of GPCs after transient transfection of GPCs from CaR⫺/⫺ mice. To distinguish recombinant CaRs from endogenous receptors, we introduced a V5-epitope at the C terminus of the Exon5(⫺)hCaRs and wt-hCaRs. Adding the V5 epitope does not affect the cell membrane expression and function of wt-hCaRs in HEK-293 cells as determined by the ability to activate phospholipase C (PLC) (our unpublished data). In GPCs from CaR⫺/⫺ mice expressing V5-tagged wt-hCaRs or Exon5(⫺)hCaRs, anti-V5 antisera localized both forms of these recombinant receptors to intracellular organelles and the cell membrane (Fig. 5B). Immunoblots with anti-V5 antisera further confirmed the expression of both wt-hCaRs and Exon5(⫺)hCaRs in GPCs from CaR⫺/⫺ mice (Fig. 5C). Comparable results were obtained with GPCs from CaR⫹/⫹ mice (data not shown), suggesting that the absence

FIG. 5. Dual-fluorescence immunocytochemistry and immunoblotting of GPCs isolated from CaR⫺/⫺ mice. A, Expression of endogenous CaRs, presumably Exon5(⫺)CaRs, and CD44 was assessed by anti-CaR (no. 21825A, 500 nM) and anti-CD44 antisera and confocal microscopy at ⫻100 magnification. B, Expression of V5-tagged hCaRs and CD44s in CaR⫺/⫺ GPCs transiently expressing wt-hCaR and Exon5(⫺)hCaR cDNAs was assessed using anti-V5 and anti-CD44 antisera. Expression of CaR and CD44 is indicated by the signals from fluoresceinconjugated (green) and Texas red-conjugated (red) anti-IgG, respectively. Overlays of paired images for CaR and CD44 from the same cells are shown. C, Lysates (50 ␮g protein/lane) from CaR⫺/⫺ GPCs expressing hCaR (Wt) or Exon 5(⫺)hCaR [Exon5(⫺)] cDNAs were blotted with anti-V5 antisera.


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of residues encoded by exon 5 of the mouse CaR gene did not block the cell surface expression of the Exon5(⫺)hCaRs in chondrocytes. Effects of changes in [Ca2⫹]e on signaling and differentiation of CaR⫺/⫺ GPCs

To test whether GPCs from CaR⫺/⫺ mice, which express CaR transcripts and likely protein, respond to changes in [Ca2⫹]e, we examined the ability of high [Ca2⫹]e and various CaR agonists to activate signaling responses in these cells. In GPCs from wt mice, raising [Ca2⫹]e from 0.5 to 5, 10, 20, or 30 mm stimulated the production of total InsPs by 0.3-, 1.1-, 2.1-, or 1.8-fold (P ⬍ 0.01), respectively (Fig. 6A, Œ). The effect of Ca2⫹ on these cells was mimicked by a variety of divalent cations with a rank order of potency of Mn2⫹ greater than Ca2⫹ greater than Sr2⫹ greater than Mg2⫹ (Fig. 6A). Interestingly, Mn2⫹ elicited a maximal response, which was significantly lower (P ⬍ 0.001) than those stimulated by Ca2⫹ and Sr2⫹, despite its greater potency (ED50 ⬃0.6 mm) when compared with the other two ions (⬃10 mm for Ca2⫹ and ⬃15 mm for Sr2⫹). Incubating wt GPCs with a nonpermeable CaR ligand neomycin also increased InsP production by up to 60% with an ED50 of approximately 300 ␮m (Fig. 6B). Comparable signaling responses were also observed in GPCs from CaR⫺/⫺ mice (Fig. 6, C and D). Raising [Ca2⫹]e from 0.5 to 5, 10, 20, or 30 mm increased total InsPs by about 0.3-, 0.8-, 1.6-, or 1.7-fold (P ⬍ 0.01), respectively (Fig. 6C, ‚). These responses are comparable with those seen with wt GPCs (Fig. 6A, Œ). Furthermore, Mn2⫹, Sr2⫹, and neomycin also stimulated InsP production in the CaR⫺/⫺ Exon5(⫺)

FIG. 6. Effects of different concentrations of divalent cations (Ca2⫹, Mg2⫹, Sr2⫹, and Mn2⫹) (A and C) and neomycin (B and D) on total 3 H-InsPs in GPCs from CaR⫹/⫹ (A and B) and CaR⫺/⫺ mice (C and D). Total 3H-InsP production in GPCs in response to CaR agonists are presented as fold increase over basal levels at 0.5 mM Ca2⫹ without other ligands. [n ⫽ 3 (A and B) and 2 (C and D) independent experiments in triplicate or quadruplicate].

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GPCs (Figs. 6, C and D) with a rank order of potency similar to that in wt GPCs, i.e. neomycin greater than Mn2⫹ greater than Ca2⫹ greater than Sr2⫹. Together, these data support the idea that Exon5(⫺)CaRs expressed in CaR⫺/⫺ GPCs could activate downstream effectors as efficiently as wt CaRs. Our previous studies showed that changing [Ca2⫹]e modulated the pace of murine GPC differentiation (8). Raising [Ca2⫹]e promoted cell differentiation by blocking proteoglycan accumulation, enhancing mineral deposition, and altering expression of cartilage marker genes (8). We tested whether mouse GPCs expressing CaRs, missing the amino acids encoded by exon 5, were less able to respond to changes in the [Ca2⫹]e and regulate the above steps in GPC differentiation. As expected, in GPCs from CaR⫹/⫹ mice, increasing [Ca2⫹]e suppressed PG accumulation (Fig. 7A) in a dosedependent manner with an ID50 approximately 2 mm (Fig. 7B). High [Ca2⫹]e also promoted mineral deposition (Fig. 7C) with an ED50 approximately 2–3 mm in these cells (Fig. 7D). In cultured CaR⫺/⫺ GPCs, raising [Ca2⫹]e also decreased proteoglycan accumulation (Figs. 7, A and B) and increased mineral deposition (Fig. 7, C and D) with a potency similar to that of cells from CaR⫹/⫹ mice. High [Ca2⫹]e also induced changes in matrix gene expression in CaR⫺/⫺ GPCs at concentrations similar to those that were active in GPCs from wt mice with ED50s of approximately 1–2 mm for aggrecan (Agg), the ␣-subunit of type II collagen [␣1(II)], and osteopontin (OP) (8). Raising [Ca2⫹]e from 0.5 to 3.0 mm for 14 d decreased RNA levels for Agg and ␣1(II) and increased expression of OP in these cells (Fig. 7E), indicating that high [Ca2⫹]e advanced key steps in the differentiation of CaR⫺/⫺ GPCs. Discussion 2⫹

Ca serves many functions in cartilage. Ca2⫹-containing minerals in the matrix contribute to strength and structural integrity (36). Ca2⫹ also mediates cell-matrix and cell-cell interactions in cartilage (36) and is a critical intracellular messenger in signaling. Studies of rickets, due to Ca2⫹-deficiency, indicate that an adequate supply of Ca2⫹ and phosphate is critical to skeletal development and especially to the differentiation of GP cartilage (4, 6, 37, 38). When the systemic [Ca2⫹] is low, rickets develops. In rickets, the GP is expanded and disorganized. This results from aberrant matrix production and the lack of Ca2⫹ for adequate mineralization (39, 40). The histologic appearance of the GP in rickets suggests a delay in chondrocyte maturation and differentiation and a failure to progress to a stage capable of mineralization (39, 40). Studies in cultured chondrocytes and bone explants support a direct action of extracellular Ca2⫹ on chondrocyte function (9 –11, 41, 42). Our studies show that raising [Ca2⫹]e activates PLC, mobilizes intracellular Ca2⫹, and promotes cell differentiation in GPCs (8) and C5.18 cells (9 –11). The molecules responsible for coupling changes in [Ca2⫹]e to downstream cellular responses in chondrocytes have not been elucidated. In the parathyroid and kidney, the evidence is clear that CaRs are the molecules critical for sensing changes in [Ca2⫹]e (13, 43). When activated by high [Ca2⫹]e, CaRs activate multiple signaling responses that eventually lead to the inhibi-

FIG. 7. Effects of incubating cells with different [Ca2⫹]e for 14 d on proteoglycan levels by Alcian green (AG) staining (A and B) , mineral deposition by alizarin red (AR) (C and D), and gene expression of chondrogenic markers by q-PCR (E) in chondrocytes from CaR⫹/⫹ and/or CaR⫺/⫺ mice. B and D, Quantification of absorbance of eluted stains at 340 (AG) and 562 (AR) nm, respectively. E, RNA levels for early chondrogenic markers, Agg and ␣1(II), and the terminal differentiation marker OP was expressed as percent of expression of a ribosomal protein L19.

tion of PTH secretion, parathyroid cell proliferation, and the urinary excretion of Ca2⫹ (13, 43). CaR⫺/⫺ mice demonstrate dramatic hypercalcemia, parathyroid gland hyperplasia, and hypocalciuria, the end-products of defective Ca2⫹-sensing in the parathyroid and kidney (15, 16, 18). Whereas many observations in vitro support a role for parathyroid-like CaRs in mediating key steps in chondrocyte differentiation, defini-


tive in vivo evidence implicating parathyroid-like CaRs in cartilage has lagged. The initial GP phenotype in CaR⫺/⫺ mice developed by Ho et al. (15) was obscured by the severe HPTH and mineral imbalance in these animals. Because alternatively spliced CaRs can be made in CaR⫺/⫺ mice, this animal is not a good CaR null model for tissues like cartilage. Observations in patients with disorders of CaR function support a role for CaRs in cartilage development. First, certain patients with neonatal severe HPTH (due to homozygous CaR inactivation) are reported to have skeletal abnormalities of their chest and head with dysmorphic facies (44 – 46). Although their severe HPTH and hypercalcemia could be important in the pathogenesis of these abnormalities, only a subset of patients with neonatal severe HPTH have these phenotypic features, and all have the same metabolic disturbances. Thus, the skeletal anomalies may be the result of markedly reduced CaR expression and/or function during embryonic cartilage and bone development. Second, individuals in a kindred with autosomal dominant hypoparathyroidism (ADH) were reported to have short stature and premature osteoarthritis (47). Although these individuals have hypoparathyroidism, which could be contributing to their growth and joint problems, it is also possible that constitutively active CaRs in GP and articular chondrocytes may be involved in the pathogenesis of their phenotype. These features are not expected because of hypocalcemia or hypoparathyroidism alone. It is of note that other ADH kindreds have no such skeletal phenotype. Clearly, only certain ADH mutants can affect cartilage function. Given that cell context affects the expression and perhaps function of mutated/ truncated CaRs as we demonstrated in this study, it is conceivable that some tissues may be more susceptible to CaR mutation than others. This may explain the greater impact of CaR mutations on the parathyroid and kidney than other tissues like cartilage and bone. Studies with newer mouse models, bred onto the background of the CaR⫺/⫺ mouse developed by Ho et al. (15), raise further questions as to the role of CaRs in skeletal development. The skeletal phenotype of CaR⫺/⫺ mice (15) was prevented by blocking the development of HPTH by breeding CaR⫺/⫺ mice with gcm2⫺/⫺ and PTH⫺/⫺ mice to get double knockouts (16, 17). The conclusion that severe HPTH is the cause of the cartilage and bone defects in CaR⫺/⫺ mice was reached with the underlying assumption that the expression of all CaR forms are blocked in this knockout model (15). Our studies suggest this assumption is incorrect, insofar as cartilage is concerned. Alternatively spliced CaR transcripts [Exon5(⫺)CaRs] are also expressed in the skin and kidneys of CaR⫺/⫺ mice (24, 25). In generating the CaR⫺/⫺ mouse model, a neomycin-cassette was introduced into exon 5 of the CaR gene to disrupt the expression of full-length CaRs (15). This manipulation clearly causes exon 5 to be deleted from nascent transcripts, but at least in certain cells, it allows for the expression of Exon5(⫺)CaRs. Of interest, Exon5(⫺)CaRs are also expressed in normal human keratinocytes (23), indicating that the splicing out of exon 5 can occur in normal cells. It is unclear whether Exon5(⫺)CaR transcripts are normally expressed in cartilage in vivo and have a physiological role. According to our RT-PCR data,


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full-length CaRs remain the dominant form of the receptor in the GP in normal animals. Our studies with ELISA and immunocytochemistry indicate that the deletion of the 77 amino acids encoded by exon 5 interferes with the ability of CaRs to be stably expressed in the membranes of CHO and HEK-293 cells and, as expected, any signaling responses to changes in [Ca2⫹]e. Impaired receptor trafficking and/or receptor instability may be due to aberrant receptor glycosylation (23). Our analysis indicates that there are two potential N-linked glycosylation sites at residues Asn-468 and Asn-488 within the domain encoded by exon 5 that would be deleted in Exon5(⫺)CaRs. It was previously shown that mutating these two residues to Gln decreased receptor glycosylation and partially blocked (by ⬃50%) cell surface expression and the ability to activate PLC (31). This observation suggests that deletion of these two sites and their surrounding residues en masse may contribute to the phenotype of Exon5(⫺)hCaRs, which in CHO and HEK-293 cells is largely due to defective surface expression. Our results support this. Having been guided in past studies by results with CHO and HEK-293 cells expressing recombinant CaRs, we were initially surprised to find normal cell surface expression of Exon5(⫺) hCaRs in GPCs from CaR⫺/⫺ mice. This suggests that the domain encoded by exon 5 is not a key determinant for CaR trafficking or stability on the membrane in these cells. Apparently, stable cell surface expression of Exon5(⫺)CaRs depends on the specific cellular context. We speculate that this property may allow Exon5(⫺)CaRs to function in key anatomical sites like the GP but not in tissues in which cellmembrane expression of the receptor does not occur. Whether this is due to altered glycosylation or another membrane localizing mechanism remain to be determined. The ability of wt and CaR⫺/⫺ GPCs to interact with the well-defined agonists of CaR (Mn2⫹, Ca2⫹, Sr2⫹, Mg2⫹, and neomycin) (28 –30, 48 –52) supports the idea that wt and alternatively spliced CaRs are likely responsible for sensing changes in [Ca2⫹]e and coupling them to cellular responses in normal and CaR⫺/⫺ GPCs, respectively. We observed clear shifts to the right in the dose-response curves for these ligands in GPCs when compared with the responses in parathyroid cells or heterologous cells expressing recombinant receptors (28, 48 –52). This is likely due to differences in the number and posttranslational modification of the receptors, availability of downstream signaling molecules, and/or the ability of these receptors to interact with unknown modulators of CaR. It is also possible that the negatively charged matrix produced by chondrocytes may limit the access of ligands to the cells. Further characterization of the properties of the CaRs in the context of GPCs is required to address these possibilities. Nonetheless, it appears that the cell context of mouse GPCs is equally permissible for the activation of both wt and Exon5(⫺)CaRs because both normal and CaR⫺/⫺ GPCs respond to the same ligands with little, if any, difference in their pharmacology. This further supports the ability of Exon5(⫺)CaRs to compensate for the loss of fulllength CaRs in CaR⫺/⫺ growth plates. Our data, however, do not rule out the possibility that other Ca2⫹-sensing molecules, encoded by totally different genes, may be involved in Ca2⫹-sensing in chondrocytes. Such molecules remain elu-

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sive. To further distinguish these possibilities will require studies using chondrocyte models and cartilage-specific knockout animals in which the expression of all forms of the CaR are blocked. Acknowledgments The authors acknowledge the technical support of Dr. Yuko Oda and Ms. Stacy A. Pratt and the gift of anti-CD44 antisera by Dr. Lilly Bourguignon of the Endocrine Research Unit of the San Francisco Veterans Affairs Medical Center. The authors also thank Drs. Allen M. Spiegel and Paul K. Goldsmith of National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases for the gift of antibody for the ELISA and their critical review and advice on the manuscript. Received March 4, 2005. Accepted September 7, 2005. Address all correspondence and requests for reprints to: Wenhan Chang, Endocrine Research Unit, 111N, San Francisco Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, California 94121. E-mail: [email protected]. This work was supported by National Institutes of Health Grants AG-21353 and AR-050662 and a Veterans Affairs Merit Review.


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