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PTH-CALCITONIN-VITAMIN D-BONE |
Endocrine Research Unit, Department of Veterans Affairs Medical Center, Department of Medicine, University of California, San Francisco, California 94121
Address all correspondence and requests for reprints to: Dolores Shoback, Endocrine Research Unit, 111N, San Francisco VA Medical Center, 4150 Clement Street, San Francisco, California 94121. E-mail: . dolores{at}itsa.ucsf.edu
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Abstract |
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Introduction |
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Chondrocytes in different zones of the growth plate produce highly specific matrix proteins and modulators of differentiation (1, 3). Resting and proliferating chondrocytes express high levels of type II collagen (4) and the cartilage-specific proteoglycan aggrecan (5). Both type II collagen and aggrecan are early genes in cartilage differentiation. Their expression falls in the lower hypertrophic zone, as chondrocytes approach terminal differentiation (4, 5, 6). Chondrocytes in the lower proliferative zone express the type I PTH/PTHrP receptor (PTHrP-R) whose activation delays chondrocyte maturation (7). Chondrocytes in the maturing and upper hypertrophic zones actively take up Ca2+, which is released in the lower hypertrophic zone to support matrix mineralization (8, 9). In this zone, markers of terminally differentiated chondrocytes (10, 11), including osteocalcin (OC) (12), osteopontin (OP) (11), and osteonectin (ON) (13), are increased. Changes in the composition of the matrix are crucial in producing an environment conducive to bone formation.
The pace of chondrocyte differentiation is tightly controlled to maintain an orderly rate of bone formation. An important local feedback mechanism in pacing the maturation and differentiation of growth plate chondrocytes involves PTHrP, Indian hedgehog (Ihh), the bone morphogenetic proteins (BMPs), and their receptors (14). Targeted ablation of the PTH/PTHrP-R gene in mice leads to accelerated chondrocyte differentiation, premature cartilage mineralization, shortened growth plates, and ultimately short-limbed dwarfism (15). Growth plates in PTHrP knockout (-/-) mice have a smaller proliferative zone and are more advanced in their differentiation (16). These observations demonstrate not only the importance of PTHrP and its receptor in slowing chondrocyte differentiation but also point to the existence of undefined regulatory mechanisms (both local and systemic) that may counteract the PTHrP/Ihh/BMP feedback mechanism and promote chondrocyte differentiation.
We hypothesize that extracellular Ca2+ serves a regulatory role in cartilage development. In nutritional rickets due to Ca2+ and or vitamin D deficiency (17, 18), the growth plate is expanded and disorganized. Bone metaphyses soften due to delayed or absent matrix mineralization. Inadequate production of matrix proteins by hypertrophic chondrocytes and the lack of Ca2+ for mineralization are postulated to cause these abnormalities (19). Supplementation of the diet with Ca2+ and vitamin D—and in some cases with Ca2+ alone—completely heals these defects (18, 20). A high Ca2+ diet also reverses the rachitic changes in bone and cartilage in vitamin D receptor knockout mice (17). These observations support a role for extracellular Ca2+ in regulating cartilage mineralization.
Extracellular Ca2+-sensing receptors (CaRs) are strongly expressed in both C5.18 cells and several types of growth plate chondrocytes (21, 22). Recent studies in CaR knockout mice indicate that CaRs are essential to prenatal growth plate and bone development (23). The present study addressed the hypothesis that high [Ca2+]o alter the expression of terminal differentiation markers and deposition of matrix mineral in C5.18 cells via CaRs. We found that high [Ca2+]o promoted the expression of cartilage markers and the production of a heavily mineralized matrix in this system. Overexpressing wild-type (wt) CaRs inhibited proteoglycan accumulation and aggrecan mRNA levels and increased OP expression in C5.18 cells, indicative of enhanced chondrocyte differentiation. Overexpression of wt and signaling-deficient CaRs in C5.18 cells led to changes in the expression of aggrecan and OP and in matrix protein production that support a role for CaRs in mediating high [Ca2+]o-induced differentiation in this chondrocyte model system.
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Materials and Methods |
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Cell culture
C5.18 cells were grown in a maintenance medium (MM) containing 
-MEM with 1.8 mM CaCl2 and 0.8 mM MgSO4 and supplemented with dexamethasone (10-7 M), nucleosides (0.004%, wt/vol), FBS (15%, vol/vol), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 C with 5% CO2 (22). Cells were plated (5 x 104 cells/cm2) on 48- or 96-well plates for alcian green and alizarin red staining, 10- or 15-cm plates for RNA and protein isolation, or chamber-slides for morphological studies. After the cells reached confluence (24–48 h), the medium was switched to a differentiation medium (DM). This medium was made from Ca2+- and Mg2+-free MM supplemented with MgSO4 (0.5 mM), ascorbic acid (50 µg/ml), ß-glycerol phosphate (10 mM), and different [CaCl2] (0.1–6.0 mM). Ionized Ca2+ concentrations in DM were determined using a Nova CRT8 analyzer (Nova Biochemical, Waltham, MA) and presented as the [Ca2+]o (22). In these experiments, culture media were replaced every 3 d.
Stable transfections
C5.18 cells were transfected with wt or mutant CaR or vector cDNAs using Lipofectamine as previously described (22). Receptor constructs (wt CaR/pcDNA3.1 and F707W CaR/pcDNA3.1) were generated by ligating a KpnI-XbaI fragment from CaR/pcDNA1/Amp or F707W CaR/pcDNA1/Amp plasmid DNA (25) containing the full-length CaR or mutant receptor cDNA, respectively, into the KpnI-XbaI digested pcDNA3.1/hygro(+) vector. After selection with hygromycin B (200 µg/ml) for at least 4 wk, transfected cells were plated and cultured as described above. After culturing in the differentiation media at basal (1.6 mM Ca2+) or different [Ca2+]o (0.6–4.2 mM) for 9 d, CaR expression was assessed by immunoblotting, RNA was extracted and analyzed by Northern blotting, and proteoglycan accumulation was determined by alcian green staining.
Generation of adenoviral stocks and infection of C5.18 cells
A cDNA encoding the CaR fused to the N terminus of the green fluorescent protein (GFP) was generated by an in-frame ligation of the full-length CaR cDNA to the 5'-end of GFP cDNA. Briefly, site-directed mutagenesis was performed on CaR/pBS constructs (25) to remove the stop codon in CaR cDNA and introduce a DraI site immediately after the coding sequence. After these mutations were confirmed by sequencing, a SalI-DraI fragment containing this mutated CaR cDNA was ligated into a pEGFP-N3 vector, precut with SalI and SmaI, to generate the CaR/GFP construct. The latter construct was used to generate recombinant viral particles using an adenovirus expression kit (PanVera Corp., Madison, WI) according to manufacturer’s instructions. Briefly, a SmaI-DraI fragment, containing CaR/GFP fusion protein cDNA, was cut from CaR/GFP plasmid and ligated into cosmid pAxCAwt at an SwaI site. Because the Ad5 adenovirus genome in the cosmid lacks E1 and E3 genes, the resulting recombinant virus is replication-deficient. This cosmid also contains a cytomegalovirus enhancer, the chicken -actin promoter, and the rabbit ß-globulin poly(A+) signal sequence to promote gene expression.
The resulting cosmid was cotransfected into human embryonic kidney (HEK)-293 cells with a DNA-terminal protein complex (DNA-TPC) that contains partial adenovirus genomic DNA with a 55-kDa terminal protein at both ends. The DNA-TPC used in the transfections was digested by an enzyme at 7 sites to minimize homologous recombination of this DNA with the adenovirus E1 gene expressed by HEK-293 cells.
After homologous recombination between cosmid and DNA-TPC, a recombinant adenovirus particle containing the cDNA encoding CaR/GFP fusion protein cDNA was generated and propagated, owing to the expression of the E1 protein by HEK-293 cells. Viral particles, containing the full-length gene, were isolated, and orientation of the construct was verified. Viral particles were mass-produced and titered. The replication deficiency of the resulting viral particles was confirmed by the lack of propagation in HeLa cells. Any potential contamination of the viral stocks with wt adenovirus was verified by PCR amplification of an E1 gene fragment (26, 27) before these stocks were used to infect C5.18 cells.
C5.18 cells were infected in suspension with different titers of recombinant viral particles (10, 50, 100, and 200 pfu/cell) containing CaR/GFP or vector for 2 h and cultured in MM for 24–48 h. After the cells reached confluence, medium was switched to the DM containing different [Ca2+]o, and cells were cultured for varying times (1–21 d) before analyses.
Northern blotting
Total RNA was extracted from C5.18 cells and subjected to Northern analysis with 32P--deoxy-CTP-labeled cDNA probes for aggrecan, OP, ON, OC, and cyclophilin (CP) as described (22). Hybridization signals were detected by BIOMAX-MR imaging film (Eastman Kodak Co., Rochester, NY) and quantified densitometrically by an optical scanner and a microcomputer.
Alizarin red, von Kossa, and alcian green staining
The mineral content of cultures was determined by alizarin red S staining. Briefly, cells were fixed in neutral buffered formalin (10%, wt/vol) for 10 min, washed with PBS 3 times, and stained with alizarin red S (2%, wt/vol) for 10 min. After washing 3 times with distilled H2O, the stain was eluted by cetylpyridinium chloride (10%, wt/vol) for 1 h with gentle agitation and quantified by A562 readings.
Mineral deposits were examined by von Kossa staining. Cell layers were fixed in neutral buffered formalin (10%, wt/vol) for 10 min, washed 3 times with distilled water, and incubated with silver nitrate (2%, wt/vol) with UV illumination for 30 min. After washing with distilled water, the cell layers were treated with sodium thiosulfate (2.5%, wt/vol) for 5 min. After three washes with water, cells were counterstained with aqueous hematoxylin, mounted, and examined.
The accumulation of proteoglycans in cultures was assessed by staining with alcian green 2GX and quantified by A340 readings (22).
Immunoblotting
Crude membrane proteins from stably transfected C5.18 cells, and total cell lysates from virus-infected cells were immunoblotted (21, 22). Briefly, membrane proteins (25 µg) or cell lysates (25 µg) were electrophoresed on 5% SDS-PAGE gels and transferred to nitrocellulose membranes. After blocking with Blotto [150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 5% (vol/vol) nonfat dry milk, 0.05% (vol/vol) Tween-20], the membranes were incubated with an anti-CaR antiserum (21825A, 50 nM). After incubation with peroxidase-conjugated goat antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA; 1:4000), standard ECL assay kits were used for signal detection (Amersham Pharmacia Biotech, Arlington Heights, IL).
InsP production in transfected HEK-293 cells
HEK-293 cells were grown in DMEM with FBS (10%, vol/vol) and transfected with wt or mutant (F707W) CaR constructs or vector DNA using CaCl2 precipitation (25). After 48–72 h, total inositol phosphate (InsP) production was assessed in triplicate (25).
Statistics
Statistical significance was assessed by single-factor ANOVA with f-test using Microsoft Corp. Excel (Microsoft Corp., Seattle, WA).
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Results |
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6 d), the cells form cartilage nodules and express the early chondrogenic genes type II collagen and aggrecan. Type X collagen is strongly expressed after >6 d. After 
12 d in culture, C5.18 cells begin to deposit alizarin red-stainable mineral and increase their expression of the terminal differentiation markers OP, OC, and ON (22, 28). We previously reported that culturing C5.18 cells at high [Ca2+]o for 12 d suppressed the accumulation of alcian green-stainable proteoglycans in a dose-dependent manner with an ID50 (dosage for 50% inhibition) approximately 2 mM Ca2+ (22). High [Ca2+]o also reduced RNA levels for the cartilage-specific markers aggrecan, type II and X collagen, and alkaline phosphatase (ALP) in a dose-dependent manner with ID50s (dosages for 50% inhibition) of 1–4 mM Ca2+ (22). These findings suggest that high [Ca2+]o either suppresses an early cartilage phenotype or promotes terminal differentiation in C5.18 cells.
To distinguish between these possibilities, we examined the effects of Ca2+ on the expression of terminal differentiation markers and mineral deposition in C5.18 cells—both late events in differentiation. Growth of cells at 1.6 and 1.0 mM Ca2+ led to markedly increased RNA levels for OP and OC, respectively, compared with 0.4 mM Ca2+. The ED50s for the ability of Ca2+ to stimulate OC and OP steady-state RNA levels were approximately 1–2 mM (Fig. 1
, A and B). The response of ON expression to [Ca2+]o was biphasic. ON RNA levels were significantly increased by 1.6 mM Ca2+ (compared with 0.4 mM Ca2+) and declined slightly in cells grown at 2.9 or 4.2 mM Ca2+ (Fig. 1, A and B). High [Ca2+]o accelerated the deposition of mineral by these cultures (Fig. 2). For example, in cells maintained at low [Ca2+]o (0.6 mM; Fig. 2A, top row), alizarin red-stainable mineral appeared only in cultures 24 d old. In cells grown at 2.9 mM Ca2+, however, mineral deposits appeared more quickly—between 8 and 12 d (Fig. 2A, bottom row). In cultures grown at different [Ca2+]o for 12–24 d, the ED50s for the effects of [Ca2+]o on mineral deposition were approximately 1–2 mM (Fig. 2B). This is comparable to the sensitivity of matrix gene expression to raising [Ca2+]o (Fig. 1B). von Kossa staining demonstrated that there was increased mineral deposition in cells cultured at 2.9 mM Ca2+ (Fig. 2C, middle panel) compared with 0.4 mM Ca2+ (left panel). High-power views of these cultures revealed that mineral was mainly deposited in proximity to cells (Fig. 2C, right panel). These data, together with our previous findings (22), strongly suggest that high [Ca2+]o accelerate the differentiation of C5.18 cells by suppressing early chondrogenic gene expression and proteoglycan synthesis and by promoting the expression of terminal differentiation markers and mineral deposition.
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After culturing the transfected cells in DM containing 1.6 mM Ca2+ for 9 d, basal aggrecan RNA levels were reduced by >80% in cells expressing wt CaRs, whereas OP expression was increased by >140% compared with vector controls (Fig. 3A). Suppression of proteoglycans by high [Ca2+]o was enhanced in wt CaR-transfected cells, compared with vector DNA-expressing controls (Fig. 4C), particularly at the [Ca2+]o between 0.6 and 2.9 mM (Fig. 3C). Immunoblotting confirmed that CaR protein was increased in wt CaR-transfected cells (Fig. 3B). The increased inhibitory effects by [Ca2+]o suggested that steady-state aggrecan expression and proteoglycan accumulation are extracellular Ca2+-dependent processes and regulated by signaling through CaRs.
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Trp mutation at residue 707 in the second intracellular loop of the receptor (25). When coexpressed with wt CaRs in HEK-293 cells, F707W CaRs suppressed high [Ca2+]o-induced increases in InsP production by >50% (Table 1).
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), compared with vector-expressing cells. Proteoglycan accumulation was significantly increased (P < 0.01) in cells stably expressing the F707W-CaR mutant, compared with vector controls at all [Ca2+]o tested (Fig. 3C). The inhibitory effects of raising [Ca2+]o from 0.6–4.2 mM on proteoglycan accumulation were blunted in cells transfected with this mutant receptor cDNA (Fig. 3C) in two experiments. Immunoblotting confirmed that CaR protein was increased in mutant CaR-transfected cells (Fig. 3B). This indicated that the ability to sense changes in [Ca2+]o in C5.18 cells had been interrupted by overexpression of this dominant negative CaR mutant and that blocking endogenous CaR function altered the expression of specific markers and promoted a less differentiated cell phenotype. Taken together, findings in the stably transfected cells implicate CaRs in mediating changes in gene expression indicative of a differentiated chondrocyte phenotype.
2) Adenoviral infection of C5.18 cells with CaR constructs. To confirm that the impact of wt and mutant CaRs on the stably transfected C5.18 cells was not due to the prolonged antibiotic selection (>4 wk), we infected C5.18 cells with an adenoviral cDNA construct encoding wt CaR-GFP fusion protein to test the effects of acutely increasing CaR number. The GFP was used to evaluate the efficiency of the exogenous CaR expression. We previously found that this fusion protein is well expressed in HEK-293 cells and mediates high [Ca2+]o-induced increases in InsPs comparable to wt CaRs (data not shown). Confocal microscopy showed that the CaR-GFP fusion protein, detected by fluorescent signals, was uniformly present in >90% of the cells infected with this construct at either 100 or 200 pfu/cell (Fig. 4A and data not shown). Expression of the fusion protein was evident at 48 h post infection and for at least 21 d (Fig. 4A). Infecting C5.18 cells with CaR-GFP viral constructs (50–200 pfu/cell) dose dependently increased CaR protein expression by approximately 4-fold, compared with cells infected with viruses carrying vector alone (Fig. 4B). Expression of CaR protein in these cells (200 pfu/cell) was increased by approximately 90% as early as 3 d post infection, peaked between 6 and 9 d, and persisted for at least 18 d (Fig. 4C). These results were confirmed in an additional experiment.
Infection of C5.18 cells with CaR/GFP viral constructs (50, 100, and 200 pfu/cell) reduced basal proteoglycan production by 12, 15, and 20%, respectively, in cells cultured at 1.0 mM Ca2+ (P < 0.01; Fig. 5A). Infection with these same titers of viral constructs in cultures grown at 1.6 mM Ca2+ increased matrix mineralization by 15, 28, and 36%, respectively, in a concentration-dependent manner (P < 0.05; Fig. 5B). Both parameters were unchanged in cells infected with adenoviruses carrying vector constructs (Figs. 5, A and B), arguing against nonspecific cytopathic effects. The ability of high [Ca2+]o to suppress proteoglycan accumulation (Fig. 5C) and promote mineral deposition (Fig. 5D) was significantly enhanced in cultures infected with CaR/GFP viruses, compared with those infected with control viruses, as indicated by the left-shifted dose-response curves (P < 0.01). Infecting C5.18 cells with CaR/GFP viral constructs also affected the expression of aggrecan and OP (Fig. 6). For example, in the presence of 1.6 mM Ca2+, infection with viruses carrying CaR/GFP DNA (200 pfu/cell) suppressed aggrecan RNA levels by approximately 55% and increased OP RNA levels by approximately 2-fold. The impact of overexpressing CaR/GFP constructs on aggrecan or OP expression was also evident at 0.4 and 2.9 mM Ca2+. The ability of high [Ca2+]o to reduce aggrecan RNA levels and to increase OP expression was preserved in cells infected with control vectors (Fig. 6). These results indicate that increasing CaR number leads to enhanced cellular responsiveness to changes in the [Ca2+]o.
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Discussion |
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Studies in humans and animals with rickets suggest that the availability of Ca2+ plays an important role in chondrocyte differentiation. When the Ca2+ supply is inadequate, growth plate development becomes disorganized. The resulting cartilage is undermineralized, and growth is stunted (17, 18). Supplementing the diet with Ca2+ reverses these defects and corrects the growth retardation. Although the skeletal anomalies in these instances are influenced by concomitant metabolic disturbances that include hyperparathyroidism, hypophosphatemia, and vitamin D deficiency (17, 18), the possibility that Ca2+ itself has a direct impact on chondrocyte function has been proposed. Our studies with cultured chondrogenic cells support this idea. Growth of C5.18 cells at high [Ca2+]o suppressed early chondrogenic genes, including aggrecan and type II collagen, and decreased synthesis of proteoglycans (22). High [Ca2+]o also enhanced the expression of terminal differentiation markers and mineral deposition. Taken together, these observations support a role for high [Ca2+]o in advancing the state of differentiation of these chondrogenic C5.18 cells.
Evidence in support of the idea that membrane CaRs are involved in the responses of C5.18 cells to changes in [Ca2+]o is substantial. 1) Changes in [Ca2+]o affect gene expression and matrix production rapidly (within an hour) and reversibly (22), compatible with membrane receptor-induced signaling. 2) High [Ca2+]o modulate signaling pathways known to couple to CaRs in parathyroid cells (InsP production, increases in [Ca2+]i, and decreases in cAMP) (21). 3) CaR transcripts and proteins are strongly expressed in C5.18 cells (22). 4) The [Ca2+]o affecting production and mineralization of matrix proteins and gene expression in C5.18 cells are within the range (1–4 mM) known to activate CaRs (22, 24). 5) Finally, modulating the function and or number of CaRs alters the sensitivity of cellular responses to changes in [Ca2+]o. These data strongly support our hypothesis that CaRs are critical modulators of C5.18 cell differentiation.
Do growth plate chondrocytes respond to changes in [Ca2+]o like C5.18 cells? Our preliminary studies showed that increases in [Ca2+]o suppress the expression of aggrecan and proteoglycan accumulation, increase [Ca2+]i, and promote mineral deposition in cultured bovine growth plate chondrocytes, similar to C5.18 cells (unpublished data). CaR transcripts and protein are expressed in the growth plate at low levels in maturing chondrocytes and at higher levels in the more differentiated hypertrophic cells (21). This anatomic distribution supports a role for CaRs in mediating terminal differentiation of growth plate chondrocytes (21).
Studies of CaR knockout (-/-) mice support a role for this molecule in cartilage function. In addition to profound hypercalcemia and hyperparathyroidism (30), CaR -/- mice have dramatic demineralization of cartilage and bone similar to the histopathology of rickets. The growth plate defects in CaR-/- mice differ from those of transgenic mice overexpressing PTH/PTHrP receptors (23). This suggests that hyperparathyroidism alone is not responsible. In CaR(-/-) mice, cartilage and bone mineralization is delayed, and the rate of bone formation is decreased (23). Their growth plates have expanded hypertrophic zones resembling the growth plates seen in rickets due to Ca2+ and or vitamin D efficiency (17). This supports an important role for CaRs in cartilage development.
Based on the available data, we propose a model for how [Ca2+]o and CaRs modulate differentiation of growth plate chondrocytes (Fig. 7). 1) Feedback pathways involving PTHrP, Ihh, and BMPs control the pace of chondrocyte maturation. 2) As chondrocytes mature, they begin to express CaRs. Activation of these receptors by high [Ca2+]o initiates signaling that counteracts the actions of PTHrP, Ihh, and BMPs and promotes differentiation. 3) As differentiation proceeds, CaR expression in the upper hypertrophic zone increases. This promotes the synthesis of ALP and type X collagen and the production of other matrix constituents required for mineral deposition. 4) When chondrocytes approach terminal differentiation, increased expression of OP, OC, and ON—as a result of higher CaR expression and other factors—promotes the deposition of minerals in the calcified hypertrophic zone. Increased activity of CaRs in chondrocytes in the calcified hypertrophic zone contributes to reduced expression of early chondrogenic markers—type II and X collagen and aggrecan—by these cells. These steps lead to the orderly deposition of a mineralized cartilage matrix containing the appropriate proteins and mineral composition that initiate new bone formation. Future experiments to test this hypothesis will require an approach that selectively targets the deletion of CaR function to specific zones in growth plate.
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Acknowledgments |
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Footnotes |
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Abbreviations: ALP, Alkaline phosphatase; BMPs, bone morphogenetic proteins; C5.18 cells, RCJ3.1C5.18 cell; CaR, Ca2+-sensing receptor; CP, cyclophilin; DM, differentiation medium; [Ca2+]o, extracellular Ca2+ concentration; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HPT, hyperparathyroidism; Ihh, Indian hedgehog; InsP, inositol phosphates; [Ca2+]i, intracellular Ca2+ concentration; MM, maintenance medium; OC, osteocalcin; ON, osteonectin; OP, osteopontin; PTHrP-R, PTHrP receptor; wt, wild-type.
Received September 17, 2001.
Accepted for publication December 6, 2001.
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