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Endocrinology Vol. 142, No. 10 4379-4388
Copyright © 2001 by The Endocrine Society

ARTICLES

Matrix {gamma}-Carboxyglutamic Acid Protein Is a Key Regulator of PTH-Mediated Inhibition of Mineralization in MC3T3-E1 Osteoblast-Like Cells

Rajaram Gopalakrishnan, Hongjiao Ouyang, Martha J. Somerman, Laurie K. McCauley and Renny T. Franceschi

Department of Periodontics/Prevention/Geriatrics (R.G., H.O., M.J.S., L.K.M., R.T.F.) and Department of Cariology, Restorative Sciences, and Endodontics (H.O.), School of Dentistry, and Departments of Pharmacology (M.J.S.) and Biological Chemistry (R.T.F.), School of Medicine, University of Michigan, Ann Arbor, Michigan 48109-1078

Address all correspondence and requests for reprints to: Renny T. Franceschi, Ph.D., Department of Periodontics/Prevention/Geriatrics, University of Michigan Dental School, Ann Arbor, Michigan 48109-1078. E-mail: rennyf{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
As part of its overall function as a major regulator of calcium homeostasis, PTH stimulates bone resorption and inhibits osteoblast-mediated biomineralization. To determine the basis for the inhibitory actions of this hormone, we compared the time course of PTH-dependent inhibition of mineralization in MC3T3-E1 osteoblast-like cells with changes in mRNA levels for several extracellular matrix proteins previously associated either with induction or inhibition of mineralization. Mineralizing activity was rapidly lost in PTH-treated cells (~30% inhibition after 3 h, 50% inhibition at 6 h). Of the proteins examined, changes in matrix {gamma}-carboxyglutamic acid protein were best correlated with PTH-dependent inhibition of mineralization. Matrix {gamma}-carboxyglutamic acid protein mRNA was rapidly induced 3 h after PTH treatment, with a 6- to 8-fold induction seen after 6 h. Local in vivo injection of PTH over the calvaria of mice also induced a 2-fold increase in matrix {gamma}-carboxyglutamic acid protein mRNA. Warfarin, an inhibitor of matrix {gamma}-carboxyglutamic acid protein {gamma}-carboxylation, reversed the effects of PTH on mineralization in MC3T3-E1 cells, whereas vitamin K enhanced PTH activity, as would be expected if a {gamma}-carboxyglutamic acid-containing protein were required for PTH activity. Levels of the other mRNAs examined were not well correlated with the observed changes in mineralization. Osteopontin, an in vitro inhibitor of mineralization, was induced approximately 4-fold 12 h after PTH addition. Bone sialoprotein mRNA, which encodes an extracellular matrix component most frequently associated with mineral induction, was inhibited by 50% after 12 h of PTH treatment. Osteocalcin mRNA, encoding the other known {gamma}-carboxyglutamic acid protein in bone, was also inhibited by PTH, but, again, with a significantly slower time course than was seen for mineral inhibition. Taken together, these results show that the rapid inhibition of osteoblast mineralization induced by in vitro PTH treatment is at least in part explained by induction of matrix {gamma}-carboxyglutamic acid protein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH AND PTH-RELATED protein (PTHrP) have similar actions on classic PTH target organs, such as bone and kidneys, which lead to an increase in blood calcium levels (1). PTH normally functions as a systemic regulator of calcium homeostasis, whereas PTHrP is a local regulator of cell differentiation that has systemic actions equivalent to those of PTH when it is produced by tumor cells (1, 2). The in vivo actions of PTH on bone can be either catabolic or anabolic depending on dose, method of administration, and frequency of treatment (3). The catabolic actions of PTH involve an increase in osteoblast-mediated resorption via secretion of RANK ligand, direct activation of osteoclasts, and inhibition of osteoblastic activity (4, 5, 6). Suppression of mineralization is observed after in vitro exposure to PTH in primary calvarial osteoblasts, calvarial explants, and osteoblast-like cell lines (7, 8, 9, 10). A similar response to PTHrP is also seen in cementoblasts, the cells lining the tooth root surface that produce a mineralized extracellular matrix, cementum (11). In contrast, numerous in vivo studies have reported an increase in bone mass after intermittent low dose administration of PTH and PTHrP to animals, a response believed to be due to both an increase in differentiation and inhibition of osteoblast apoptosis (12, 13).

The basis for the inhibitory actions of PTH and PTHrP on mineralization are not well understood. Because mineralization is probably controlled by the interplay between various nucleators and inhibitors of hydroxyapatite (HA) crystal formation, two models can be envisioned to explain the mechanism used by these peptides to inhibit mineralization. These models, which are not mutually exclusive, involve either the suppression of inducing molecules and/or the induction of mineralization inhibitors.

Of the known bone extracellular matrix (ECM) proteins, bone sialoprotein (BSP) has properties most consistent with a role in mineral induction. BSP is a highly acidic phosphoprotein that binds HA and calcium with high affinity and through its RGD sequence is involved in cell attachment (14). It has an in vivo distribution that parallels the distribution of mineralization foci and can function as a potent inducer of HA crystal nucleation in vitro (15, 16, 17). Moreover, we previously showed that overexpression of BSP in nonmineralizing osteoblast subclones can stimulate mineralization (18). PTH can down-regulate BSP and inhibit the synthesis of a collagenous matrix by osteoblasts (9, 19). In related studies we showed that treatment of cementoblasts with PTHrP inhibits both BSP expression and mineralization (11). However, in no case has it been possible to establish a causal relationship between inhibition of BSP expression and the decreased ability of cells to mineralize. Furthermore, current evidence for BSP having a direct role in mineralization in vivo is equivocal in that there are no significant mineralization defects in mice harboring a targeted ablation of the BSP gene (20). Although this does not preclude a function for BSP in the mineralization process, it does imply that additional factors must be required. These factors could be inducers or inhibitors of mineralization.

A number of bone-related ECM proteins have been reported to inhibit mineralization in vitro, including osteocalcin (OCN), matrix {gamma}-carboxyglutamic acid (Gla) protein (MGP), osteopontin (OPN), and the large aggregating proteoglycans, albumin and fibronectin (17, 21, 22, 23, 24, 25, 26). Of these, OPN, MGP, and OCN have been most extensively studied.

OPN is a secreted, glycosylated protein normally expressed in mineralized tissues such as bones and teeth. It is also found in calcifications of the aortic valve and atherosclerotic plaques (27). OPN inhibits de novo mineral formation by blocking crystal growth rather than HA nucleation (16, 17, 23) and can also promote cell adhesion and migration (28). As was the case for the BSP gene, targeted deletion of OPN does not grossly alter the mineralization process in mice (29). However, bones from OPN-deficient mice are resistant to both ovariectomy and PTH-induced bone resorption, indicating a requirement for OPN in bone remodeling (30, 31).

Both MGP and OCN contain the modified amino acid, Gla. Gla residues are produced in a posttranslational {gamma}-carboxylation reaction requiring vitamin K as a cofactor (32, 33). MGP is produced by a variety of cell types in culture, including osteoblasts, chondrocytes, cardiac myocytes, and vascular endothelial cells. In contrast, in situ hybridization studies reported to date localized MGP mRNA only to vascular smooth muscle cells, proliferating and late hypertrophic chondrocytes and epithelial cells lining the cortical and medullary tubules of kidney (34, 35, 36). Due to its structural similarities with osteocalcin, MGP is thought to function as a regulator/inhibitor of mineralization by binding through its Gla residues to calcium and hydroxyapatite (32). Major in vivo evidence for its role as a physiological inhibitor of mineralization comes from studies with MGP-null mice, which exhibit extensive mineralization of arteries and cartilage (35). More recently, mutations leading to the production of a nonfunctional MGP protein were identified in Keutel syndrome, an autosomal recessive disorder associated with abnormal cartilage calcification, short terminal phalanges, peripheral pulmonary stenosis, and midfacial hyoplasia (37). Recent elegant studies by Yagami et al. (38) using retrovirus-mediated overexpression of MGP in chick limb bud showed that this molecule could inhibit both cartilage mineralization and endochondral ossification. These studies also suggested that MGP is regulated in a very controlled manner during development. However, the factors and mechanisms required for this regulation remain unknown. OCN is another Gla-containing protein with a potential function as an inhibitor of osteoblast activity. It is the most abundant noncollagenous protein of bone ECM and is selectively produced by osteoblasts (39). Mice deficient in OG1 and OG2 (two genes that encode OCN) exhibit an increase in bone formation without any apparent defect in resorption (40). More detailed analysis using Fourier transform infrared microspectroscopy revealed a mineral maturation defect in bones from OCN-deficient mice, suggesting a role in bone remodeling (41). In addition, it was shown that OCN inhibits de novo mineralization by delaying nucleation of HA in vitro (21, 22).

In the present study we compare PTH-dependent changes in the expression of BSP, OPN, OCN, and MGP with hormone-dependent loss of mineralizing activity in osteoblasts with the goal of determining whether changes in any of these major bone constituents can explain the observed inhibition of mineral formation. As will be shown, the actions of PTH on mineralization can at least in part be explained by its ability to rapidly and specifically up-regulate MGP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
{alpha}MEM and FBS were obtained from Life Technologies, Inc. (Gaithersburg, MD) and Summit Laboratories (Fort Collins, CO), respectively. Penicillin/streptomycin was also obtained from Life Technologies, Inc. PTH-(1–34), PTH-(7–34), and PTHrP-(1–34) were purchased from Bachem (King of Prussia, PA). Cycloheximide, warfarin, vitamin K, and ascorbic acid (AA) were obtained from Sigma (St. Louis, MO).

Cell culture
A highly mineralizing subclone of MC3T3-E1 cells (MC-4 cells) was cultured and maintained in AA-free {alpha}MEM containing 10% FBS as described previously (42). Cells were passaged every 4–5 d and were not used beyond passage 15. In all experiments cells were grown in AA-containing medium for 6–8 d before use. This is sufficient time for cells to differentiate into mature osteoblast-like cells that express high levels of osteoblast markers, including the PTH-1 receptor, BSP, and OCN. These cells can readily form a mineralized extracellular matrix after the addition of either inorganic phosphate (Pi) or ß-glycerol phosphate to cultures (42, 43).

For studies that examined effects of vitamin K and warfarin on PTH action (Fig. 6Go), MC-4 cells were grown in AA-containing medium for 6 d, at which time cells were treated with PTH (10-7 M) or vitamin K3 (10 µM). For warfarin-treated cultures (20 µM), pretreatment began on d 4 (i.e. 2 d before PTH or vitamin K3 addition). On d 8, all cultures were allowed to mineralize by incubation with Pi for 24 h. Mineralization was detected by von Kossa staining. Separate cultures were harvested for extraction of total RNA and for indirect assessment of osteocalcin {gamma}-carboxylation using an HA binding assay (see below).



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  		Figure 6. Role of {gamma}-carboxylation in PTH-mediated inhibition of mineralization. MC-4 cells were grown under differentiating conditions for 6 d, at which time cells were treated as indicated. In the case of warfarin-treated cultures, pretreatment began on d 4 (i.e. 2 d before PTH or vitamin K3 treatment). On d 8, all cultures were allowed to mineralize by incubation with Pi (final concentration, 4 mM) for 24 h. Mineralization was detected by von Kossa staining (A). B, Densitometric measurements (mean ± SEM) of von Kossa-stained cultures using NIH image software. a, significantly different from untreated control group (P < 0.001); b, significantly different from group treated only with PTH (P < 0.05); c, significantly different from group treated with PTH and vitamin K (P < 0.001). C, Northern blot analysis for MGP and OCN mRNA expression in MC4 cells after treatment with vitamin K3 and warfarin.

 
In vitro mineralization assays
Mineralization of MC3T3-E1 cells was visually evaluated by von Kossa staining as previously described (43). Densitometric analysis of stained cultures was carried out using NIH Image 1.61 software. Initial mineral formation was measured by 45Ca uptake as described by Bellows and co-workers (44). Differentiated cell cultures were transferred to medium containing 1.0 µCi/ml 45CaCl2 (NEN Life Science Products-DuPont, Boston, MA) and the indicated concentrations of Pi. After various times, cell layers were washed six times with Earle’s balanced salt solution to remove unbound radioactivity, harvested in 10 mM Tris-Cl, pH 7.4, and mixed with an equal volume of 30% trichloroacetic acid to dissolve mineral-associated 45Ca. After incubation overnight at 4 C, samples were vortexed vigorously and centrifuged, and the radioactivity in the supernatant was determined by liquid scintillation counting. The 45Ca accumulation in each sample was normalized with the corresponding sample protein content. Proteins were allowed to dissolve in 0.5% SDS by incubating them at 55 C for several hours for each sample and were measured using the DC protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA).

Measurement of OCN binding to HA
As an indirect means of assessing the extent of protein {gamma}-carboxylation after vitamin K and warfarin treatments (Fig. 6Go), the ability of secreted OCN to bind to HA in vitro was measured as previously described (45). Conditioned medium was prepared from MC-4 cells by culturing them in {alpha}MEM/0.1% FBS for 24 h. Total immunoreactive OCN was then measured using a commercially available RIA kit (Biomedical Technologies, Stoughton, MA). Conditioned medium was diluted to give a final OCN concentration of 50 ng/ml, and HA slurry was added to give final concentrations of 5, 20, and 40 mg/ml. After incubation at 4 C for 5 h, HA was pelleted by centrifugation, and OCN in the supernatant was measured by RIA. Previous studies showed that {gamma}-carboxylated OCN preferentially binds to HA relative to OCN from warfarin-treated cells in which {gamma}-carboxylation was effectively blocked (45). Thus, the fraction of total OCN remaining in the supernatant after HA treatment is an index of the amount of undercarboxylated OCN.

Northern blot analysis
Total RNA was isolated from cells using TRIzol reagent (Life Technologies, Inc.) as described by the manufacturer and quantified by UV spectroscopy. Fifteen-microgram aliquots of total RNA were fractionated on 1.2% agarose-formaldehyde gels and blotted onto Duralon-UV nitrocellulose membranes (Stratagene, La Jolla, CA) as described by Thomas (46), after which they were UV cross-linked using Stratalinker (Stratagene). The mouse cDNA probes used were obtained from the following sources: bone sialoprotein (47) and osteopontin (48) from Dr. Marion Young (NIDCR, NIH), osteocalcin (49) from Dr. John Wozney (Genetics Institute, Cambridge, MA), and MGP (34) from Dr. Gerard Karsenty (Baylor College of Medicine, Houston, TX). The membranes were hybridized with a cDNA probe labeled with [{alpha}-32P]deoxy-CTP (NEN Life Science Products-DuPont) using a random primer labeling kit (Roche Molecular Biochemicals, Indianapolis, IN). All cDNA inserts were previously excised from plasmid DNA with the appropriate restriction enzymes and purified by agarose gel electrophoresis. Hybridizations were performed as previously described using an Autoblot hybridization oven (Bellco Glass Inc., Vineland, NJ), quantitatively scanned using an InstantImager (model A2024, Packard Instrument Co., Downers Grove, IL), and subsequently exposed to Kodak BIOMAX film (Eastman Kodak Co., Rochester, NY) at -70 C. All blots were normalized for RNA loading by stripping and reprobing with cDNA to 18S rRNA (50).

In vivo injection of PTH
Five C57BL/6 mice per group were injected sc over the calvarial region with 20 µg PTH or PBS vehicle as previously described (51). After 8 h, whole calvaria were dissected free of loose connective tissue, and total RNA was extracted from five mice in each group by homogenization in TRIzol reagent (Life Technologies, Inc.). MGP expression in calvaria was then analyzed by Northern blot analysis.

Statistical analysis
All statistical analyses were performed using Instat 3.0 (GraphPad Software, Inc., San Diego, CA). Unless indicated otherwise, each value reported is the mean and SEM of triplicate independent samples. Studies examining PTH inhibition of 45Ca accumulation (Fig. 3AGo) and PTH/warfarin/vitamin K interactions (Fig. 6Go) were analyzed using ANOVA, followed by Tukey-Kramer multiple comparison’s test to determine statistically significant differences between groups. Statistical significance for the time course and Pi concentration dependence for 45Ca accumulation (Fig. 2Go) as well as the in vitro expression of ECM protein mRNA levels in MC-4 cells (Fig. 3Go) and in vivo MGP mRNA analysis (Fig. 7Go) were assessed using an unpaired t test.



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  		Figure 3. Comparison of time course of mineralization inhibition by PTH-(1–34) and changes in mRNA levels for bone ECM proteins. A, Time course for inhibition of mineralization after PTH-(1–34) treatment. After differentiation for 8 d in AA-containing medium, MC-4 cells were treated with vehicle for 48 h (•) or 10-7 M PTH-(1–34) for the times indicated ({blacktriangleup}). For each sample, initial mineralization was measured by a 3-h incubation in medium containing 4 mM Pi and 45Ca as described in Materials and Methods. {circ}, Basal 45Ca accumulation in the absence of added Pi. All values are the mean ± SEM for triplicate samples. B, Northern blot analysis of bone ECM protein mRNAs in MC-4 cells after PTH-(1–34) treatment. MC-4 cells were allowed to differentiate as described in A and treated with PTH-(1–34) or vehicle for the times indicated. Total RNA was isolated from triplicate independent cultures, and mRNA was quantified by Northern blot analysis. Blots were imaged, and values were normalized for loading and blotting efficiency using 18S rRNA. C and D, Graphical representation of Northern blots showing fold induction (percentage of control ± SD) for MGP and OPN (C) and fold decrease (percentage of control ± SD) for BSP and OCN (D) expression after normalization with 18S rRNA. a, P < 0.05; b, P < 0.01 (significantly different from vehicle control).

 


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  		Figure 2. Measurement of initial mineralization by 45Ca accumulation in MC-4 cells. A, Time course of 45Ca accumulation after initiation of mineralization. MC-4 cells were grown for 8 d in the presence (•) or absence ({circ}) of AA. Mineralization was then initiated by transfer into medium containing 4.0 mM Pi and 1 µCi/ml 45Ca. Cells were harvested at the times indicated for measurement of 45Ca accumulation in cell layers as described in Materials and Methods. The inset shows an enlargement of 45Ca accumulation for early time points. B, Effect of Pi concentration on 45Ca accumulation. Cells were grown as described in A and incubated with 45Ca and the indicated concentration of Pi for 3 h before harvesting. All values in A and B are the mean ± SEM for triplicate samples. *, Values for AA-treated cultures that are significantly different from controls (P < 0.01).

 


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  		Figure 7. In vivo induction of MGP mRNA expression in calvaria after sc PTH administration. C57BL/6 mice (total of five animals per group) were sc injected with saline vehicle or 20 µg PTH-(1–34) over the calvaria. After 8 h, calvaria were isolated, and total RNA was prepared and analyzed for MGP mRNA expression by Northern blot analysis. A, A representative Northern blot of RNA from three control and three PTH-treated animals that was probed for MGP and 18S RNA (for normalization). B, Normalized MGP mRNA levels (mean ± SEM) for all five animals in each group. *, Significantly different from controls (P < 0.001).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Development of conditions for measuring initial mineral formation
All cell culture experiments in this study used MC-4 cells, a previously described highly differentiating subclone of the murine MC3T3-E1 preosteoblast cell line (42). These cells form an extensively mineralized ECM and express osteoblast-related genes, including BSP, OCN, pro-{alpha}2(I) collagen, Cbfa1 and the PTH-1 receptor mRNA. Of the osteoblast-like cell lines described to date, MC3T3-E1 cells are most like primary osteoblast in that they express osteoblast differentiation markers and mineralize only after growth in AA-containing medium, which is necessary for secretion of a collagen-containing ECM. We previously showed that MC3T3-E1 cells are a good model system for studying osteoblast-mediated mineralization (43). Specifically, mineral in MC3T3-E1 cultures is exclusively associated with collagen fibrils, has an x-ray diffraction pattern characteristic of a weak apatite, and can be distinguished from Ca/P precipitates that form at high Ca2+-PO43- ion products. Furthermore, comparison of a number of mineralizing and nonmineralizing MC3T3-E1 subclones revealed that there is a good correlation between the ability of a given subclone to mineralize in cell culture and its ability to form bone in vivo after implantation into immunodeficient mice (42).

In agreement with previous reports using other osteoblast culture systems, treatment of differentiated MC-4 cells with 10-7 M PTH-(1–34) for 48 h inhibited the ability of cells to mineralize, as measured by the accumulation of a von Kossa-positive matrix (Fig. 1Go). In contrast, PTH-(7–34), an antagonist for PTH-(1–34), was totally inactive. Similar results were obtained using PTHrP-(1–34) (data not shown). The relatively long times required to obtain visible Ca/P deposits using von Kossa assays (12–24 h) precluded studies aimed at correlating early changes in gene expression with inhibition of mineralization. To examine more rapid effects of PTH on mineralization, it was first necessary to develop a method for detecting early mineral formation. To accomplish this, we used a modification of the 45Ca uptake assay developed by Bellows et al. (44). This assay was optimized for both 45Ca uptake time and medium phosphate concentration. As shown in Fig. 2AGo, differentiated MC-4 cells, but not undifferentiated cells, grown in the absence of AA begin depositing acid-extractable 45Ca into their ECM after as little as 3 h, with accumulation continuing to increase for an additional 12–24 h. Initial 45Ca accumulation measured using a 3-h uptake time was stimulated in differentiated cells as the total medium Pi concentration was increased above 3 mM (Fig. 2BGo). In contrast, 45Ca uptake in undifferentiated cells (grown in the absence of AA) only slightly increased as medium phosphate was increased. In subsequent experiments mineralization was initiated by the addition of phosphate to a final concentration of 4.0 mM, and 45Ca accumulation was measured after 3 h.



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  		Figure 1. Effect of PTH-(1–34) on mineralization. MC-4 cells were grown for 8 d in the presence of AA to induce differentiation. Cells were then treated with vehicle, 10-7 M PTH-(1–34) or 10-7 PTH-(7–34). After 48 h, all cultures were transferred to medium containing 4.0 mM Pi and incubated for 24 h to induce mineralization, which was detected by von Kossa staining.

 
PTH inhibition of mineralization
The 45Ca uptake assay described above was used to measure the time course of PTH effects on MC-4 cell mineralization. Cells were grown in AA-containing medium for 8 d to induce differentiation. PTH (10-7 M) was then added for various times up to 48 h before mineralization was initiated by addition of phosphate and 45Ca. Samples were harvested after an additional 3 h. All values indicated on the x-axis of Fig. 3AGo refer to the period of PTH pretreatment (i.e. before addition of phosphate and 45Ca). PTH significantly inhibited mineralization after as little as 3 h (~30% inhibition; P < 0.05), with inhibition increasing to approximately 50% after 6 h (P < 0.01). The extent of inhibition gradually increased with time, such that at 48 h, mineralization was reduced to background levels.

Effects of PTH on osteoblast gene expression
To provide insight into the mechanism of mineral inhibition after PTH treatment, we compared the results shown in Fig. 3AGo with the temporal pattern of gene expression for several ECM proteins previously associated with the mineralization process (Fig. 3Go, B–D). Cells were grown as described in Fig. 3AGo and exposed to either vehicle or 10-7 M PTH for 0.5, 1, 3, 6, 12, 24, or 48 h. Total RNA was isolated from triplicate independent cultures, and Northern blots were probed for BSP, OCN, MGP, and OPN mRNA. All values are expressed relative to those for time-matched controls. As shown in Fig. 3BGo, control mRNA levels remained relatively constant for the duration of the experiments (variance <20%) with the exception of OPN, which gradually increased such that the 48 h value was 2-fold greater than the value at the beginning of the experiment. PTH dramatically up-regulated MGP mRNA levels after as little as 1–3 h (4-fold induction at 3 h; P < 0.01), with highest levels seen at 6 h (~6.5-fold induction; P < 0.01). MGP mRNA levels remained elevated for up to 24 h, but dropped to basal levels after 48 h. In contrast, BSP mRNA, which is normally present in high levels after 8 d of growth in AA-containing medium, was significantly reduced only after 12 h of PTH treatment (50% inhibition at 12 h, P < 0.01); the small apparent drop at 3 and 6 h was not significant at P < 0.05, with nearly complete loss of expression at 24 h. OCN mRNA was also significantly inhibited at 12 h (40% inhibition; P < 0.05), with nearly complete loss of expression at 24 h. We also evaluated the expression of another inhibitor of mineralization, OPN, which was induced by PTH. The time course of OPN mRNA up-regulation was slow, peaking at 12 h (1.5-fold induction at 6 h and 4-fold induction at 12 h; P < 0.01) and returning to baseline levels after 24 h. Thus, of the four mRNAs examined, the time course of MGP induction was most closely correlated with initial PTH-dependent inhibition of mineralization.

PTH-(1–34) induced MGP mRNA in a dose-dependent manner, with an inductive effect detected at concentrations as low as 10-10–10-9 M (Fig. 4Go). Maximal induction measured at 6 h was observed at a concentration of 10-8 M, with mRNA levels remaining constant at higher PTH concentrations (up to 10-6 M; not plotted). In contrast, PTH-(7–34) was totally inactive in terms of its ability to both induce MGP expression (Fig. 4Go) and inhibit mineralization (Fig. 1Go). In studies not shown, PTHrP-(1–34) also stimulated MGP mRNA expression with a time course and concentration dependence that paralleled results obtained with PTH. Although previous studies suggested a potential link between cell proliferation and MGP expression (52, 53), we did not observe any alteration in cell numbers over the time frame of our experiments (data not shown). Also, PTH-dependent alterations in osteoblast cell number were not observed in a previous study from this laboratory (10).



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  		Figure 4. Concentration-dependent and peptide-specific induction of MGP mRNA by PTH-(1–34). Differentiated MC-4 cells were treated with either vehicle or the indicated amounts of PTH-(1–34) (•) or PTH-(7–34) ({blacktriangleup}) for 6 h. MGP mRNA levels were measured by Northern blot analysis (A), and results are graphically represented after normalization with 18S RNA (B).

 
Effect of cycloheximide on PTH-mediated up-regulation of MGP mRNA expression
Cycloheximide (10 µg/ml), a protein synthesis inhibitor, was used to assess whether MGP mRNA induction by PTH requires a protein intermediate. Control studies showed that this concentration of inhibitor reduced total protein synthesis by greater than 95% as measured by [3H]leucine incorporation (result not shown). Similar to previous experiments, MC-4 cells were plated and allowed to differentiate for 8 d by the addition of AA. Cells were then treated with vehicle, PTH, PTH plus cycloheximide, or cycloheximide alone for 3 h. As shown in Fig. 5Go, cycloheximide completely blocked the inductive effects of PTH on MGP mRNA. Cycloheximide also blocked the induction of MGP mRNA by PTHrP (data not shown). This short 3-h exposure to cycloheximide had no adverse effect on either cell number or morphology (data not shown). Furthermore, cycloheximide alone did not affect MGP expression in control cultures (not treated with PTH). These experiments suggest that actions of PTH on MGP expression are indirect, requiring the rapid induction of a protein intermediate.



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  		Figure 5. Effect of cycloheximide on PTH-mediated MGP mRNA up-regulation. Differentiated MC-4 cells were treated for 3 h with either vehicle or PTH-(1–34) in the presence or absence of cycloheximide. MGP mRNA levels were measured by Northern blot analysis.

 
Effect of vitamin K and the vitamin K antagonist, warfarin, on PTH-mediated inhibition on mineralization
More direct evidence for MGP being involved in the inhibition of mineralization by PTH was provided by experiments using the vitamin K antagonist, warfarin. To be functional, MGP requires a vitamin K derivative as a cofactor for {gamma}-carboxylation, producing Gla residues that bind to calcium and hydroxyapatite. There are five Gla residues in MGP, two of which are adjacent to a disulfide bond, thus facilitating interaction with hydroxyapatite (54). Addition of warfarin inhibits the production of Gla residues by reducing the amount of vitamin K available for {gamma}-carboxylation. If MGP is required for PTH-dependent inhibition of mineralization, increasing the level of {gamma}-carboxylation would be expected to further inhibit mineralization, whereas blocking {gamma}-carboxylation with warfarin should allow mineralization to proceed even in the presence of PTH. These predictions are borne out by the results shown in Fig. 6Go. Thus, the inhibitory activity of PTH was significantly enhanced by the addition of 10 µM vitamin K3 to cultures (PTH alone inhibited mineralization by 75% vs. 96% inhibition by PTH plus vitamin K3). In contrast, treatment with 20 µM warfarin in the presence of vitamin K3 reduced PTH-dependent inhibition of mineralization to 42%. However, vitamin K3 or warfarin alone did not affect mineralization in the absence of PTH.

Two control studies were carried out to confirm whether the effects of vitamin K3 and warfarin are explained by changes in protein {gamma}-carboxylation rather than nonspecific alterations in MGP or OCN mRNA levels. First, to confirm that warfarin specifically blocked {gamma}-carboxylation, we measured changes in the HA-binding activity of OCN derived from control, vitamin K3-treated, and warfarin-treated cells. OCN rather than MGP was selected for analysis because of the availability of an RIA kit that specifically recognizes the murine protein. Previous studies showed that {gamma}-carboxylated OCN binds to HA with high affinity, whereas undercarboxylated OCN produced by warfarin-treated cells exhibits lower affinity for HA (55). Cells were grown as described in Fig. 6AGo, except that on d 8, cultures were transferred to low serum medium which was then harvested after 24 h for HA binding assays and OCN RIAs. As expected, PTH lowered total immunoreactive OCN from 3.19 ± 0.16 to 0.74 ± 0.04 ng/µg DNA. In contrast, treatment with either vitamin K3 or warfarin caused only small fluctuations in levels of total secreted OCN (2.67 ± 0.20 and 3.47 ± 0.09 ng/µg DNA for vitamin K3- and warfarin-treated groups, respectively). On the other hand, 96.1 ± 0.8% and 98.6 ± 0.5% of OCN in control and vitamin K3-treated cultures, respectively, bound HA (20 mg/ml), whereas only 12.7 ± 5.5% of OCN from warfarin-treated cultures was bound. Similar results were obtained when HA binding assay was performed using 5 and 40 mg/ml HA (data not shown). Thus, vitamin K3 increased the already high levels of carboxylated OCN, whereas warfarin effectively blocked {gamma}-carboxylation. We also assessed whether vitamin K3 or warfarin could alter levels of MGP and OCN mRNAs (Fig. 6CGo). Cultures were treated as described in Fig. 6AGo, except that cells were exposed to PTH for only 6 h to produce the maximal induction of MGP. No significant alterations in MGP or OCN mRNA levels were observed in cultures treated with vitamin K3 alone or warfarin alone. Furthermore, MGP mRNA was induced to a similar extent in cultures treated with PTH alone, PTH plus vitamin K3, and PTH plus vitamin K3 plus warfarin (Fig. 6CGo). Based on these studies, we conclude that warfarin and vitamin K3 treatments specifically altered levels of protein {gamma}-carboxylation, and furthermore, a vitamin K-dependent protein, such as MGP, mediates the inhibitory effects of PTH on mineralization.

Regulation of MGP expression by PTH in vivo
To determine whether the up-regulation of MGP mRNA observed after PTH treatment in cell culture is also seen in vivo, we sc administered 20 µg PTH or saline vehicle above the calvaria of C57BL/6 mice (five mice per group). Calvarial RNA was isolated 8 h after PTH injection, and MGP mRNA expression was evaluated by Northern blot analysis. As shown in Fig. 7Go, MGP mRNA levels were up-regulated approximately 2-fold after a single injection of PTH. For vehicle-treated samples, the MGP/18S mRNA ratio was 2.5 ± 0.035 (mean ± SEM) compared with 4.3 ± 0.036 (mean ± SEM) for the PTH-treated group. Values were significantly different at P < 0.001.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PTH and PTHrP are two related peptide hormones that cause an elevation in serum calcium levels by acting on bone to increase resorption and inhibit osteoblast activity. In the present study we demonstrate that the inhibitory effects of PTH on mineralization are at least in part explained by up-regulation of MGP. Induction of MGP was rapid, being seen after as little as 3 h, was specific to active PTH peptide, and was closely correlated with PTH-dependent inhibition of mineralization. As would be expected if actions of PTH were mediated by a Gla-containing protein, PTH inhibited mineralization to a greater degree in the presence of added vitamin K3. In contrast, inhibition was greatly reduced by the vitamin K antagonist, warfarin. In vivo sc administration of PTH-(1–34) over the calvaria of mice also induced a 2-fold increase in MGP mRNA. Although PTH treatment of differentiated osteoblasts also increased OPN and inhibited BSP and OCN mRNAs, none of these changes was well correlated with changes in mineralizing activity. Thus, induction of MGP by PTH provides a viable explanation for how this hormone might inhibit the mineralizing activity of osteogenic cells during bone remodeling. However, our results do not completely exclude the participation of OPN, BSP, and OCN in the PTH response.

The catabolic actions of PTH on bone are accompanied by a number of specific changes in osteoblast activity, including inhibition of type I collagen and alkaline phosphatase expression and induction of collagenase and tissue plasminogen activator (5). Together with RANK ligand-mediated stimulation of osteoclast formation, these responses may all contribute to the bone resorption response. A number of studies also showed that PTH can inhibit osteoblast and cementoblast-mediated mineralization in cell culture, although the basis for this effect is not well understood (7, 9, 11). One possibility is that PTH suppresses the synthesis of a mineral nucleator such as BSP. In this regard, Wang et al. (9) reported that a transient activation of the PKA pathway by low dose PTH-(1–34) or short-term cAMP analog treatment caused a reversible reduction in the deposition of BSP into the extracellular matrix secreted by UMR 106–01 BSP osteosarcoma cells as well as a loss of mineralizing activity. Similar to our results, they also found a decrease in BSP mRNA after 12 h of PTH treatment. In previously reported studies with cementoblasts, we reported a similar decrease in BSP expression after 6–12 h of PTH treatment (11). As neither of these studies examined the early effects of PTH on mineralization, it was not possible to correlate observed changes in BSP expression with PTH-dependent loss of mineralizing activity.

In the present study we used 45Ca accumulation into cell layers as a means of detecting rapid mineral deposition and PTH responsiveness of cultures. Using this approach, we detected a PTH-dependent decrease in mineralizing activity within 3–6 h of hormone addition. Although the degree of inhibition continued to increase for 24–48 h, the largest effects of PTH (50% inhibition) were seen within 6–9 h. Of the four bone ECM protein mRNAs examined after PTH treatment, only MGP message levels changed over a time frame that was well correlated with the observed inhibition of mineralization. Peak induction of MGP mRNA was seen after 3–6 h. Levels remained high for 12–24 h and returned to basal levels after 48 h. OPN mRNA, which encodes another bone ECM protein with the ability to inhibit HA formation in vitro, was also induced by PTH. In this case peak induction (4-fold) was seen 12 h after PTH addition, with mRNA levels returning to control values by 24 h. Thus, peak OPN mRNA induction is observed after the major PTH-dependent drop in mineralizing activity. OCN, the other major Gla-containing protein of bone, was initially proposed to also be an inhibitor of bone formation based on the finding that OCN knockout mice have greater bone mass than wild-type animals (40). More recent studies of OCN-deficient bones showed a defect in mineral maturation, suggesting a role for OCN in bone remodeling (41). Although the relationship between these studies and our cell culture experiments is not clear, PTH-induced changes in OCN are not likely to account for the effects of this hormone on mineralization, because maximal inhibition was seen after 12–24 h, a time course that lags significantly behind that seen for the inhibition of mineralization. As was the case for OCN, a significant decrease in BSP mRNA was not detected until 12 h after PTH addition. Although protein levels for BSP, OPN, OCN, or MGP were not measured in this study, previous work from this and other laboratories showed a good correlation between protein and mRNA levels for BSP and OCN in MC3T3-E1 cells, although PTH regulation was not specifically examined (42, 56). A suitable antibody is not currently available for detection of mouse MGP. However, when taken together with studies showing that PTH-dependent inhibition of mineralization requires {gamma}-carboxylation, we believe that our work provides compelling evidence that MGP is required for the inhibitory actions of PTH on mineral formation. This is not to say that other molecules, including BSP, OCN, and OPN, could also have secondary roles in controlling mineralizing activity.

Our demonstration that in vivo administration of PTH is able to induce MGP mRNA provides clear evidence that this hormonal response is not restricted to cultured cells. However, the specific calvarial cell types responding to PTH in vivo remain to be identified. Previous in situ hybridization analysis failed to detect osteoblast-associated MGP mRNA in calvaria of 17.5 mouse embryos, although the effects of PTH treatment were not examined (34). Further in situ hybridization studies are being carried out to resolve this issue and will be reported at a later date.

The role of MGP induction in the catabolic actions of PTH also remains a matter for speculation at this time. Because of its many similarities with OCN, MGP is considered to function as a calcium- and hydroxyapatite-binding protein (32). Both proteins contain Gla and phosphoserine, are localized predominantly in mineralized matrixes, and bind strongly to hydroxyapatite through a process requiring Gla residues (33). MGP differs from OCN in that it binds tightly to the ECM in the absence of HA, probably because of its highly hydrophobic nature. Furthermore, comparison of OCN and MGP gene deletion studies revealed major differences in the apparent functions of these two proteins. Thus, OCN gene knockout mice exhibit a subtle overall increase in bone mass and a decrease in remodeling, whereas MGP-null mice exhibit a dramatic phenotype involving extensive cartilage and arterial calcification (35, 40). This major role for MGP as a mineralization inhibitor suggests that it could function during PTH-induced resorption to prevent remineralization in resorption lacunae. In this way it might facilitate the removal of calcium and phosphate liberated from the bone matrix by osteoclast activity while, at the same time, suppressing any new mineral formation until the resorption phase of the bone remodeling cycle was complete.

In our studies PTHrP had similar activity to PTH in stimulating MGP expression and inhibiting osteoblast mineralization. Unlike PTH, which has systemic effects on calcium homeostasis, PTHrP normally has highly localized actions, particularly in growth plate cartilage, where it suppresses chondrocyte hypertrophy and mineralization (57). Although we have not yet examined whether PTHrP can induce MGP expression in chondrocytes, it is tempting to speculate that some of the effects of PTHrP on cartilage mineralization are mediated through MGP. Of note are the striking similarities between PTHrP-null and MGP-null mice. PTHrP-deficient mice exhibit a characteristic chondrodysplasia related to overall acceleration of chondrocyte differentiation with premature hypertrophy and mineralization (58). As noted above, Mgp-null mice also exhibit premature mineralization of cartilage (35). In contrast, mice overexpressing PTHrP in cartilage have an overall defect in cartilage maturation and mineralization (59). Similarly, retrovirus-induced overexpression of MGP in chick limb bud markedly reduces mineralization (38).

We report here for the first time that PTH and PTHrP can regulate MGP gene expression in osteoblasts. Previously, only 1,25-dihydroxyvitamin D3 and retinoic acid were known to induce this gene (60, 61). The actions of both PTH and PTHrP are mediated through a single G protein-coupled receptor, the PTH-1 receptor, also known as the PTH/PTHrP receptor, which is expressed in all known PTH target tissues (62). PTH or PTHrP stimulation of cells expressing the PTH-1 receptor activates two well defined signal transduction pathways, the adenylate cyclase/PKA and PLC/PKC pathways (5). Although the mechanism of MGP induction by PTH is not known, the involvement of one or both of these pathways is likely. Previous studies with osteosarcoma cells as well as our experiments with MC3T3-E1 cells, primary calvarial cells, and cementoblasts concluded that the PKA pathway is required for PTH/PTHrP-dependent inhibition of mineralization (9, 10, 11). In the present study we showed that PTH-dependent induction of MGP mRNA is blocked by cycloheximide pretreatment. Thus, the actions of hormone on MGP expression are indirect and probably require the induction of an early PTH-responsive protein intermediate. Currently, we are in the process of conducting more detailed studies aimed at understanding the mechanisms involved in PTH and PTHrP regulation of MGP.


   Acknowledgments
 
The authors thank Ms. Amy J. Koh-Paige and Mr. Eben Alsberg for technical assistance.


   Footnotes
 
This work was supported by NIH Grants DE-12211 (to R.T.F.), DE-09532 (to M.J.S.), and DK-53904 (to L.K.M.), a postdoctoral fellowship from the Center for Organogenesis (to R.G.), and the Center for Biorestoration of Oral Health at the University of Michigan.

Abbreviations: AA, Ascorbic acid; BSP, bone sialoprotein; ECM, extracellular matrix; Gla, {gamma}-carboxyglutamic acid; HA, hydroxyapatite; MGP, matrix Gla protein; OCN, osteocalcin; OPN, osteopontin; Pi, inorganic phosphate; PTHrP, PTH-related protein.

Received January 17, 2001.

Accepted for publication June 5, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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