Matrix Vesicle Plasma Cell Membrane Glycoprotein-1 Regulates Mineralization by Murine Osteoblastic MC3T3 Cells

Authors


  • This work was presented in abstract form at the November 1998 meeting of the American College of Rheumatology, San Diego, California, U.S.A.

Abstract

A naturally occurring nonsense truncation mutation of the inorganic pyrophosphate (PPi)-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) PC-1 is associated with spinal and periarticular ligament hyperostosis and cartilage calcification in “tiptoe walking” (ttw) mice. Thus, we tested the hypothesis that PC-1 acts directly in the extracellular matrix to restrain mineralization. Cultured osteoblastic MC3T3 cells expressed PC-1 mRNA and produced hydroxyapatite deposits at 12–14 days. NTPPPH activity increased steadily over 14 days. Transforming growth factor-β and 1,25-dihydroxyvitamin D3 increased PC-1 and NTPPPH in matrix vesicles (MVs). Because PC-1/NTPPPH was regulated in mineralizing MC3T3 cells, we stably transfected or infected cells with recombinant adenovirus, in order to express 2- to 6-fold more PC-1. PC-1/NTPPPH and PPi content increased severalfold in MVs derived from cells transfected with PC-1. Furthermore, MC3T3 cells transfected with PC-1 deposited ∼80–90% less hydroxyapatite (by weight) than cells transfected with empty plasmid or enzymatically inactive PC-1. ATP-dependent45Ca precipitation by MVs from cells overexpressing active PC-1 was comparably diminished. Thus, regulation of PC-1 controls the PPi content and function of osteoblast-derived MVs and matrix hydroxyapatite deposition. PC-1 may provide a novel therapeutic target in certain disorders of bone mineralization.

INTRODUCTION

Osteoblasts modulate the composition of the bone matrix, where they deposit mineral in the form of the basic calcium phosphate hydroxyapatite.1-3 The initiation of matrix calcification by osteoblasts, like chondrocytes, appears to be mediated by release of matrix vesicles (MVs), membrane-limited cell fragments.4, 5 MV constituents, including a variety of enzymes, modify the extracellular matrix.4-8 The interior of MVs also serves as a sheltered environment for hydroxyapatite crystal formation.4, 5

One pathway for the initiation of mineralization has been attributed to the ability of several enzymes abundant in MVs (alkaline phosphatase [ALP], ATPase, and inorganic pyrophosphate [PPi]-generating nucleoside triphosphate pyrophosphohydrolase [NTPPPH]) to release Pi or PPi from ATP.4, 9-11 MVs also contain or bind several molecules, including annexin V, that secure free calcium.4, 6

Mechanisms that control MV-mediated mineralization include regulatory effects on the release of MVs by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3),10 activation of protein kinase C,12 and apoptosis.13, 14 In addition, the composition of the MVs, and by extension their mineralizing potential, can be regulated by certain calciotropic hormones and cytokines, including 1,25(OH)2D3, transforming growth factor-β (TGF-β) and interleukin-1 (IL-1).4, 6-7, 10, 14 15

Changes in MV and matrix PPi also regulate initiation and extravesicular propagation of mineralization.16-20 Because PPi suppresses hydroxyapatite crystal deposition and propagation,16-20 removal or exclusion of PPi at sites of initial mineralization must occur to permit active crystal deposition.4 Thus, the ability of several growth factors to actively regulate PPi concentration may control the rate, extent, and stability of mineralization.15, 21-26

PPi is generated by multiple biosynthetic reactions,27 and in part by members of the alkaline phosphodiesterase-NTPPPH family, which hydrolyze the phosphodiester I bond in purine and pyrimidine nucleoside triphosphates (EC 3.6.1.8, EC 3.1.4.1).11, 28 Chondrocytes and osteoblasts have substantially higher levels of specific activity of NTPPPH than other tissues studied to date.29 Most but not all NTPPPH activity of human osteoblasts and articular chondrocytes is attributable to plasma cell membrane glycoprotein-1 (PC-1).15, 25 In this context, increased PC-1 expression and/or NTPPPH activity can be induced by 1,25(OH)2D3, TGF-β, basic fibroblast growth factor (bFGF), aFGF, and activation of protein kinase A.15, 22-25 PC-1 expression also is inhibited by IL-1 in osteoblastoid cells.25 Such regulatory effects suggest that PC-1 expression plays an active role in differentiation programs in osteoblasts.

PC-1/NTPPPH may function in a variety of cellular compartments in osteoblasts. Specifically, PC-1 is expressed as a class II (intracellular N terminus) disulfide-linked transmembrane homodimer, catalytically active in the lumen of the rough endoplasmic reticulum and potentially other organelles, and on the external face of the cell (i.e., ectoenzyme).28 Membrane PC-1 inhibits insulin receptor tyrosine kinase signaling.30 Intracellular PC-1/NTPPPH regulates protein N-glycosylation31 and hydrolyzes phosphoadenosine phosphosulfate, the major donor for intracellular sulfation reactions such as those in proteoglycan biosynthesis.32

NTPPPH activity becomes incorporated into MVs of mineralizing cells.4, 11 33 The factors that can regulate MV NTPPPH and the function of NTPPPH in mineralization are of particular relevance because of recent observations in mice. Specifically, autosomal recessive inheritance of a naturally occurring nonsense mutation in PC-134 was linked to the hyperostotic phenotype of ttw/ttw mice (formerly known as “tiptoe walking Yoshimura” (twy/twy) mice).35, 36 The PC-1 mutation linked to ttw/ttw mice is predicted to truncate the protein N-terminal to the EF-hand motif34 essential for the binding of divalent cations that serves as a cofactor in NTPPPH activity.28

In early life, ttw/ttw mice develop progressive, osteoblast-mediated, and chondrocyte-mediated ossification of spinal ligaments adjacent to the annulus fibrosus and of the synovium and periarticular ligaments of peripheral joints.34-36 Calcium phosphate crystals have been observed in MV-like structures in the intervertebral disks of ttw/ttw mice.35

To understand better the effects of PC-1/NTPPPH on osteoblast mineralizing function, this study directly assessed the role of PC-1 in matrix mineralization by cultured osteoblastic cells in the absence of other bone cells in vitro. We used a clonal osteoblast line from murine calvaria (MC3T3-E1).37-39 When cultured for several weeks with supplemental ascorbic acid and a source of phosphate, MC3T3 cells undergo progressive differentiation to mineralizing cells.39 Our results demonstrate that both the expression and localization in osteoblastic MVs of PC-1 are regulated processes that control both MV PPi content and the extent of matrix hydroxyapatite deposition.

MATERIALS AND METHODS

Reagents

Recombinant bFGF, TGF-β1, IL-1β, and insulin-like growth factor-I (IGF-I) were from R&D Systems (Minneapolis, MN, U.S.A.), and 1,25(OH)2D3 from Dr. M. Uskokovic (Hoffman-Laroche, Nutley, NJ, U.S.A.).

Cells and cell culture

MC3T3-E1 cells, from Dr. Marja Hurley (University of Connecticut, Farmington, CT, U.S.A.), were maintained in alpha-modified essential medium (α-MEM) supplemented with 10% heat inactivated fetal calf serum (FCS), 1% penicillin-streptomycin, and 2 mM glutamine (Omega Scientific, Tarzana, CA, U.S.A.) at 37°C. Where indicated, MC3T3 cells were plated at 80% confluence (1 × 106 cells in a 10-cm tissue culture–treated polystyrene plate in 5 ml) with 2.5 mM sodium phosphate and 50 μg/ml ascorbic acid at 37°C. The media was collected and replaced daily for up to 14 days.

MC3T3 cells were stably transfected with 5 μg or 10 μg of human PC-1 in pCNDA3 using Lipofectamine (Life Technologies, Gaithersburg, MD, U.S.A.). Cells were given 600 μg/ml of G418 sulfate (Omega Scientific) for 2 weeks. Viable cells were replated individually and colonies allowed to grow for 2 more weeks before selection of two clones, PC-1#1 and PC-1#2, expressing two and four times, respectively, the NTPPPH activity of MC3T3 cells stably transfected with pCDNA3 plasmid alone. Stable lines were maintained in complete α-MEM (as above) also containing 300 μg/ml G418 for an additional 3 weeks before removal.

Alizarin red staining, crystal extraction and analysis

After 14 days in culture, plates were stained with 0.5% alizarin red S solution, pH 4.0. To extract crystals,40 cells were washed with phosphate-buffered saline three times to remove all cellular debris and 1 ml of 1 mg/ml papain (Sigma Chemical Co., St. Louis, MO, U.S.A.) was added overnight at 37°C. The enzyme suspension (1 ml) was scraped into a microfuge tube and centrifuged at 11,000g for 1 minute. Then 0.5 ml of the supernatant was removed and the pellet resuspended in the remaining supernatant with 0.5 ml of fresh 1% (v/v) sodium hypochlorite and continuously mixed at 4°C for 10–12 h. Crystals were pelleted at 11,000g for 1 minute and resuspended in sterile distilled water. The washing was repeated five times before a final bulking of the crystals with a 98% ethanol wash. Crystals were dried at 37°C for 30 minutes and the weights of each sample recorded.

To analyze crystal composition, the crystals were embedded in Spurr epoxy resin without fixation or dehydration. Thin sections of plastic-embedded material were imaged in a Philips EM430 transmission electron microscope and analyzed by electron diffraction and energy dispersive X-Ray spectroscopy using an Oxford AN10000 X-Ray system.41, 42

MV isolation

MC3T3 cells were cultured for 10 days, as described above, and the medium was changed daily until day 7, at which time 10 ml of media was left in each plate until the 10th day of culture. Media were collected and initially centrifuged at 20,000g for 20 minutes at 4°C to pellet cellular debris followed by centrifugation at 100,000g for 1 h to isolate the MV fraction, which was resuspended in 0.4 ml of lysis buffer (1% Triton X-100 in 0.2 M Tris base with 1.6 mM MgCl2, pH 8.1).

45Ca precipitation assay

MV fractions (0.04 mg of protein in 0.025 ml), isolated at day 10 in culture as described above, were added in triplicate to 0.5 ml of calcifying medium (2.2 mM CaCl2 [1 μCi/ml of45Ca], 1.6 mM KH2PO4, 1 mM ATP disodium salt, 1 mM MgCl2, 85 mM NaCl, 15 mM KCl, 10 mM NaHCO3, 50 mM N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, pH 7.6), and vortexed and incubated at 37°C for 24 h. Samples were then centrifuged at 14,000g for 10 minutes at 4°C. The pellet was washed twice with cold calcifying medium without ATP. The45Ca in the mineral phase was solubilized in HCl and counted in 5 ml of scintillation fluid. Assay controls included the omission of ATP in the calcifying media and/or heat inactivation of the MV (60°C for 30 minutes).

Western blotting

Samples from conditioned media were concentrated 20-fold with 1% 0.4 M perchloric acid (PCA) for 10 minutes on ice, centrifuged, washed once with acetone, and resuspended in lysis buffer. All samples were treated with lysis buffer (1% Triton X-100 in 0.2 M Tris base with 1.6 mM MgCl2, pH 8.1) and the protein concentration determined with the BCA protein assay (Pierce, Rockford, IL, U.S.A.). Protein, 0.03 mg from each sample, was separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose.

A rabbit polyclonal antiserum (R1769) was made to a 141 amino acid epitope of human PC-1 highly homologous to mouse PC-1, and which included the EF hand. To generate the PC-1 peptide, cDNA encoding the C-terminal 141 amino acids of human PC-1 was generated by polymerase chain reaction (PCR) using human PC-1 cDNA as a template, and the agarose gel purified PCR product ligated into pGEX-KT following BamHI and EcoRI digestion. Induction with IPTG followed by SDS-PAGE revealed a fusion protein of the expected size, which was affinity-purified on glutathione-agarose, emulsified in complete Freund's adjuvant and used to generate the rabbit antiserum, which was subsequently verified to recognize both human and mouse PC-1. In Western blots, washed membranes were incubated with horseradish peroxidase–conjugated secondary antibody in blocking buffer for 1 h, washed again, and immunoreactive products detected using the enhanced chemiluminescence system (Amersham, Arlington Heights, IL, U.S.A.).

Reverse transcription PCR analysis

Total RNA was isolated using 0.5 ml of TriZOL (Life Technologies) per 60-mm plate, with RNA extracted in chloroform and then precipitated in isopropanol overnight at −20°C. Six hundred nanograms of total RNA was reverse transcribed using 5 mM MgCl2, 1× PCR buffer (200 mM Tris-HCl, pH 8.4), 500 mM KCl) 1.25 mM of each dNTP, 2.5 μM Oligo d(T) primer, 0.25 U/ml RNase Inhibitor (Sigma), 2.5 U/μl MuLV reverse transcription (RT) in a volume of 20 μl at 42°C for 40 minutes, 99°C for 5 minutes, and 4°C for 5 minutes. One tenth of this reaction was used for a single round of RT-PCR, using the sense primer 5′-GAAAGTACGTGCTGGGGTACA-AACAGACTC, and antisense primer 5′-ATCGGAATCTGGGTCAATGATAGCCAG as previously described to assess expression of the ribosomal “housekeeping gene” protein L30.25 An equal amount of cDNA was used for two rounds of nested PCR for PC-1 mRNA levels. The PC-1 primers in the first round (sense 5′-GCCAGGATCAGATGTGGAGAT-TG-3′ and antisense 5′-TAACCGAGCAGCA-GGTCCATAC-3′) amplified a 406 base pair product corresponding to nucleotides 1023–1428 of the published sequence for mouse PC-1.28 In the second round of PCR, the sense primer 5′-GCTACAGCTTCCTAGCCATGA-AAG-3′ and the antisense primer 5′- TTTATCCAAGCCCAGGTCCTTC-3′ amplified a 173 base pair product (nucleotides 1125–1299). The conditions for each PCR reaction were: an initial 5 minutes run at 95°C before starting, then 95°C for 30 s, 55°C for 30 s, and 72°C for 1 minute, followed by an final extension for 5 minutes at 72°C. RT-PCR gel bands were quantitatively analyzed with a digital imaging system as previously described.40

PPi, NTPPPH, ALP, cellular DNA assays

PPi was determined by differential adsorption on activated charcoal of UDP-D-[6-3H] glucose (Amersham, Chicago, IL, U.S.A.) from its PPi-catalyzed reaction product 6-phospho [6-3H] gluconate, as previously described.43 PPi was equalized for the DNA concentration in each well, determined chromogenically following precipitation in perchlorate.43 To determine specific activity of NTPPPH and ALP, by previously described assays,43 the MC3T3 cells were lysed in lysis buffer, as above.

Adenoviral PC-1 expression

We subcloned cDNAs encoding wild-type human PC-1 (the BamHI–XbaI fragment from pcDNA3.PC-1), or a mouse enzyme-deficient PC-1 mutant (released by HindIII digestion from pSVT7) that contained an alanine for threonine substitution in the active site44 into the EcoRV–BamHI and XbaI-digested, or HindIII-digested pACCMVpLpA (which contains genomic sequence of the replication-defective E1 mutant of adenovirus 5 and the SV40 poly(A) tail, from Dr. Jody Martin, UCSD). The resulting plasmids (5 μg) were each cotransfected, using Superfect (Qiagen, Valencia, CA, U.S.A.), with 5 μg of the large adenoviral plasmid pJM17 (from Dr. Tom Kipps, UCSD) into the 293 packaging cell line, which provides adenoviral E1 in trans. Infectious recombinant viral stocks were further amplified in 293 cells and media containing virus used to infect cells.

For adenoviral gene transfer, the cells were infected with 5 × 107 plaque forming units (PFU) of adenoviral human wild-type PC-1, enzyme inactive mouse mutant PC-1 (mutPC-1), or the naked pACCMVpLpA adenovirus for 6 h in 2% FCS-containing medium. Using adenoviral β-galactosidase and X-gal staining, we confirmed under these conditions that ∼75% of cells were infected.

Statistical analysis

Where indicated, error bars represent SD. Statistical analysis was performed using the Student's t-test (paired two-sample testing for means), applied on Microsoft Excel 5.0 for the Macintosh computer.

RESULTS

Mineralization, PC-1 expression and PPi metabolism in cultured MC3T3 cells

MC3T3 cells cultured with ascorbate and NaPi produced alizarin red–positive deposits at 12–14 days. We verified by energy dispersive X-Ray spectroscopy that crystals deposited were calcium phosphates, with a broad and diffuse electron diffraction pattern with rings at 3.44, 2.81, 2.78, and 2.71 Å indicating hydroxyapatite of relatively poor crystallinity3 in all samples tested.

Under these conditions, total extracellular PPi transiently increased between days 2–4 (Fig. 1). In contrast, NTPPPH activity in both cell lysates and conditioned media steadily rose over 14 days (Fig. 1). PC-1 mRNA was expressed by the mineralizing MC3T3 cells (Fig. 2).

Figure FIG. 1.

Extracellular PPi generation and cellular and extracellular NTPPPH activity in mineralizing MC3T3 cells. MC3T3 cells were plated at 80% confluence (1 × 106 cells in 5 ml) and cultured in mineralizing conditions with α-MEM (with minimum essential amino acids, 10% FCS, 2.5 mM sodium phosphate and 50 μg/ml ascorbic acid) as described in the Materials and Methods. Cells were incubated at 37°C, and the media collected and replaced daily for up to 14 days. Cell lysate NTPPPH, and conditioned media NTPPPH and PPi were measured in triplicate (means ± SD) as described in the Materials and Methods. Units used for this composite graph were: NTPPPH in conditioned media (μmol of substrate hydrolyzed/h/0.5 ml); cellular NTPPPH (μmol of substrate hydrolyzed/h/100 μg of protein); PPi in conditioned media (pmol/μg of cellular DNA). Representative of eight separate experiments.

Figure FIG. 2.

PC-1 mRNA is expressed in mineralizing MC3T3 cells. Total RNA from mineralizing MC3T3 cells, cultured as described above, was collected on the days indicated and reverse-transcribed as described in the Materials and Methods. RT-PCR for PC-1 (which amplified a 173 bp product), and the housekeeping gene L30 (which amplified a 196 bp product), was performed as described above. The intensities of the bands were quantitated by densitometry as described in the Materials and Methods, and PC-1 mRNA but not L30 mRNA, was determined to increase by 66% between days 4 and 8, and 72% between days 4 and 13.

The progressively increased activity of NTPPPH in the conditioned media was associated with increased detection of extracellular PC-1 between days 7 and 14 by Western blotting (Fig. 3). ALP-specific activity remained relatively stable (data not shown).

Figure FIG. 3.

PC-1 in the conditioned media of MC3T3 cells under mineralizing conditions. The conditioned media from mineralizing MC3T3 cells, cultured as described above, were collected on the days indicated, concentrated 20-fold with 1% 0.4 M PCA for 10 minutes on ice, centrifuged, washed once with acetone, and resuspended in lysis buffer (1% Triton X-100 in 0.2 M Tris base with 1.6 mM MgCl2, pH 8.1). An equal protein concentration of 0.03 mg from each sample was separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose and probed with a rabbit polyclonal antibody that recognizes human and mouse PC-1 in Western blotting, with detection performed as described in the Materials and Methods. The figure indicates detection of PC-1 as a 127 kDa species on the Western blot.

In cultures of explanted primary human femoral osteoblasts, NTPPPH activity is increased by incubation with TGF-β and 1,25(OH)2D3.23, 24 We observed that NTPPPH-specific activity was similarly increased in MC3T3 cells incubated with TGF-β and 1,25(OH)2D3, but not IL-1, bFGF, or IGF-I (Table 1). Furthermore, TGF-β and 1,25(OH)2D3 both increased NTPPPH activity in MVs (by 45% and 180%, respectively) in association with enhanced detection of immunoreactive PC-1 (Fig. 4). In contrast, IL-1, bFGF, and IGF-I did not increase NTPPPH activity in the MVs (data not shown). Under certain conditions, 1,25(OH)2D3 induces an increase in osteoblast MV ALP.10 However, in MC3T3 cells cultured with ascorbate and sodium phosphate under this study's conditions, MV ALP-specific activity remained relatively stable (data not shown).

Table Table 1. NTPPPH Activity Is Selectively Induced by TGF-β and 1,25(OH)2D3 in Mineralizing MC3T3 Cells
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Figure FIG. 4.

MV PC-1 in response to TGF-β and 1,25(OH)2D3 in mineralizing MC3T3 cells. Mineralizing MC3T3 cells were cultured as described above, with the medium changed daily until day 7, at which time 10 ml of media was left in each plate until the 10th day of culture. Where indicated, TGF-β (10 ng/ml) or 1,25(OH)2D3 (10 nM) were present in the media through days 1–10. Media were collected and initially centrifuged at 20,000g for 20 minutes at 4°C to pellet cellular debris followed by centrifugation at 100,000g for 1 h to isolate the MV fraction. Equal amounts of MV protein (0.03 mg) were studied by SDS-PAGE and Western blotting for PC-1, as described above. Lanes: +, positive control (purified mouse PC-1); 1, unstimulated cells (cell lysate); 2, unstimulated cells (MVs); 3, TGF-β (cell lysate); 4, TGF-β (MVs); 5, 1,25(OH)2 (cell lysate); 6, 1,25(OH)2 (MVs).

Effects of enhanced PC-1 expression on mineralization by MC3T3 cells

Because PC-1 expression and the localization of PC-1/NTPPPH activity in MVs were regulated processes in MC3T3 cells, we defined how increased PC-1 expression modulated mineralization in vitro. To do so, we first stably transfected MC3T3 cells with 5 μg or 10 μg of cDNA for human PC-1, which is 81% homologous to mouse PC-1, and is expressed in full by mouse cells.28, 45 Total cellular DNA at 14 days was comparable in each experimental group transfected with PC-1 (data not shown).

We observed that total cellular NTPPPH and to a lesser extent total extracellular NTPPPH increased in the cells stably transfected with PC-1 relative to empty plasmid (Fig. 5). Stable transfection had the potential to select for MC3T3 cell clones functionally distinct in parameters other than PC-1 expression. Thus, we also expressed PC-1 in MC3T3 cells by adenoviral gene transfer (Fig. 5).

Figure FIG. 5.

Effects of stable transfection of plasmid PC-1, or adenoviral gene transfer of wild-type and enzyme-inactive PC-1 on NTPPPH activity in mineralizing MC3T3 cells. MC3T3 cells were stably transfected using 5 μg and 10 μg of human wild-type PC-1.pCNDA3 or 10 μg of empty pCDNA3 plasmid using Lipofectamine followed by selection in G418 as described in the Materials and Methods. Two different clones (PC-1#1 and PC-1#2) that expressed two to four times more NTPPPH activity were selected. Alternatively, recombinant replication-defective adenovirus expressing wild-type PC-1 or an enzyme-deficient PC-1 mutant were generated and amplified as described in the Materials and Methods. On day 1 of culture as described above, MC3T3 cells were infected with 5 × 107 PFU of the adenoviral wild-type or mutant PC-1 or empty adenovirus, as described in the Materials and Methods. MC3T3 cells were cultured for 14 days with sodium phosphate and ascorbate as described above, and cellular NTPPPH specific activity and conditioned media NTPPPH activity were measured.

MV NTPPPH increased markedly in cells transfected or adenovirally infected with PC-1 (Table 2). Under these conditions, immunoreactive PC-1 became highly concentrated in cells and their released MVs in response to infection with either wild-type or enzyme-inactive PC-1 (Fig. 6).

Table Table 2. NTPPPH Concentrates in Matrix Vesicles (MVs) in Cells Adenovirally Infected or Transfected with Wild-Type PC-1
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Figure FIG. 6.

Adenoviral gene transfer of PC-1 enriches PC-1 in both MC3T3 cells and their released MVs. On day 1 of culture as described above, MC3T3 cells were infected with 5 × 107 PFU of the adenoviral wild-type or mutant PC-1 or empty adenovirus, as described above. Cell lysates and MVs of infected and uninfected cells were collected at day 10 of culture, and 0.03 mg of protein from each sample was analyzed for PC-1 by SDS-PAGE and Western blotting, as described above. Lanes: 1, uninfected cells (cell lysate); 2, uninfected cells (MVs); 3, empty adenovirus (cell lysate); 4, empty adenovirus (MVs); 5, adenoviral PC-1 (cell lysate); 6, adenoviral PC-1 (MVs); 7, adenoviral mutant PC-1 (cell lysate); 8, adenoviral mutant PC-1 (MVs); +, positive control, purified human PC-1.

Over 14 days of culture, no change or a modest increase (< 15%) was generally observed in total extracellular PPi in the media of cells transfected with PC-1/NTPPPH (data not shown). In contrast, the concentration of PPi in the MVs of cells transfected or adenovirally infected with wild-type PC-1 increased severalfold (Fig. 7). Furthermore, purified MVs from cells transfected or adenovirally infected with wild-type PC-1 demonstrated markedly less ATP-dependent precipitation of45Ca relative to controls (Fig. 8).

Figure FIG. 7.

PPi content is enriched in MVs derived from MC3T3 cells transfected or adenovirally infected with PC-1. MC3T3 cells were cultured as described above and with the treatments indicated. MVs were collected at day 10 in culture as described above, and PPi content per microgram of protein was measured as described above. **p < 0.001 relative to controls.

Figure FIG. 8.

Effects of increased MC3T3 cell PC-1 expression on45Ca precipitation by MVs. We purified MVs, at day 10 in culture, from MC3T3 cells treated as indicated. MV fractions (0.04 mg of protein in 0.025 ml) were added in triplicate to 0.5 ml of calcifying medium (containing 2.2 mM CaCl2, 1 μCi/ml45Ca, and 1 mM ATP, pH 7.6) at 37°C for 24 h. Samples were then centrifuged at 14,000g for 10 minutes at 4°C. The pellet was washed twice with cold calcifying medium without ATP. The45Ca in the mineral phase was solublized in HCl and counted, with results expressed as percentage of total45Ca precipitated by the MVs. *p < 0.05 relative to controls.

The crystals deposited in the matrix of MC3T3 cells transfected or infected with wild-type PC-1 were not morphologically different by transmission electron microscopy from those deposited under constitutive conditions. Furthermore, electron diffraction and energy dispersive X-ray spectroscopy confirmed the crystal phase to be hydroxyapatite of relatively poor crystallinity, qualitatively identical to crystals deposited by control cells (data not shown). However, alizarin red staining of the matrix at 14 days was qualitatively weaker in MC3T3 cells that expressed increased PC-1/NTPPPH. Thus, we quantitated the direct effect of the augmented PC-1/NTPPPH expression on hydroxyapatite deposition. To do so, we extracted hydroxyapatite from culture plates after 14 days and weighed the dried crystal deposits. Increased PC-1/NTPPPH expression was associated with ∼80–90% inhibition of the dry weight of hydroxyapatite deposition (Table 3). Thus, the level of PC-1/NTPPPH expression and PC-1 localization in MVs exerted a marked regulatory effect on the quantity of hydroxyapatite deposited by MC3T3 cells.

Table Table 3. Increased PC-1 Expression Inhibits the Amount of Hydroxyapatite Deposited by MC3T3 Cells
original image

DISCUSSION

This study demonstrated that expression and localization of PC-1/NTPPPH are regulated under mineralizing conditions in osteoblastic cells. Extracellular PC-1/NTPPPH rose prior to mineralization and remained elevated during the phase of intense alizarin red deposition (days 12–14). In contrast, the concentration of PPi transiently peaked at 2–4 days in the conditioned media. In chondrocytes, ascorbate-induced extracellular PPi generation has been attributed in part to de novo protein synthesis, and it is believed that a fraction of the extracellular PPi is cosecreted with collagen.26 A linkage of extracellular PPi generation to matrix synthetic activity, and imbalances in PPi formation and degradation,27 likely contributed to the dissociation of extracellular PPi concentration from total cellular and extracellular NTPPPH activity in MC3T3 cells.

TGF-β and 1,25(OH)2D3 increased both cellular NTPPPH activity and the localization of immunoreactive PC-1 and NTPPPH in MVs derived from MC3T3 cells. MVs appear to derive by budding from specialized regions of the plasma membrane.4 However, the composition of MVs, like that of apoptotic bodies, can partially reflect the differentiation status of the cells of origin.15 Thus, our experiments focused on the regulatory role of disregulated PC-1 expression in matrix mineralization by MC3T3 cells.

We identified a quantitative but not qualitative role of PC-1 in matrix mineralization. Specifically, MC3T3 cells treated with plasmid or adenoviral DNA in order to express increased PC-1/NTPPPH deposited hydroxyapatite crystals with the same characteristics on electron diffraction and X-ray spectroscopy possessed by the crystals deposited by control cells. However, MC3T3 cells that expressed two to six times more PC-1 deposited ∼80–90% less hydroxyapatite (by weight) than cells transfected or infected with controls that included enzymatically inactive PC-1.

Transfection or adenoviral infection with both wild-type and mutant PC-1 was associated with marked enrichment of the PC-1 in MVs. Because PC-1 exists as a class II homodimeric transmembrane protein, it will be of interest to determine if the cytosolic tail of PC-1 might possess a localization signal for plasma membrane-derived MVs.

Our results identified that PC-1 regulates mineralization by MC3T3 cells at least in part via significant regulatory effects at the level of MVs. Increases in enzymatically active PC-1 enriched MV PPi content much more than the total extracellular concentration of PPi. PC-1/NTPPPH also attenuated ATP-induced MV-mediated calcium precipitation. Our results suggest that enrichment of MV PC-1 in cells stimulated by certain cytokines and calciotropic hormones could be a physiologic control mechanism in mineralization. It is likely that PC-1 plays a direct role in the inhibition of mineralization via PPi accumulation at the level of the MV membrane and/or the MV interior.4, 20

The mechanism by which MV PPi accumulates in association with increased MV PC-1 localization requires further investigation. Matsuzawa and colleagues have demonstrated that most MV ALP is enzymatically active on the outer membrane leaflet of MVs.46 However, about 20% of MV ALP may be active at a location other than the external membrane.46, 47 The Pi generated by MV ALP is partially released.4, 46 Nonetheless, ALP and ATPase activities on the outer MV membrane have the capacity to transport Pi into the MV interior,4, 9 and MV Pi content undergoes regulation in mineralizing cells.48 It will be of interest to test if there are specialized binding or transport mechanisms in MV membranes for PPi generated locally by NTPPPH activity.

The elucidation of effects of PC-1 on mineralization at the level of the matrix and MVs helps explain the abnormal and exuberant ossification of spinal and periarticular ligaments and synovium in ttw/ttw mice.34-36 These mice express a truncated PC-1 theoretically less able to bind divalent cations, a critical cofactor in NTPPPH activity.34 Deposition of calcium phosphate crystals in association with MV-like structures occurs in the articular cartilage of ttw/ttw mice.35

The hyperostosis of ttw/ttw mice also could partially reflect influences of PC-1 expression on differentiation and matrix synthesis by skeletal cells. For example, cartilaginous metaplasia of spinal ligament cells with enhanced type XI collagen expression is observed in ttw/ttw mice.49 A decrease in proteoglycan staining and degenerated collagen in articular cartilage have been proposed to contribute to calcium phosphate crystal deposition in articular cartilage in ttw/ttw mice.35

Regional decreases in the density of trabecular bone in ttw/ttw mice50 also point to the likely complexity of the role of PC-1 in the mineralization of bone in vivo. The ttw/ttw mice demonstrate a state of increased bone turnover and their osteoblastic cells appear more PTH-responsive than controls in vitro.50, 51 The net effect of PC-1 deficiency on bone mineralization in ttw/ttw mice could reflect altered differentiation and matrix synthesis, the effects of immobilization of fused spinal joints, and effects of PPi generation on mineralization.52 The suppressive effects of PC-1 on mineralization may be relatively unopposed in regions that lack osteoclasts such as the synovium, ligaments, and articular cartilage.

In chondrocytes and articular cartilages incubated with ATP, MV NTPPPH activity has been demonstrated to actually promote deposition of not only calcium pyrophosphate crystals but also hydroxyapatite.53 Specificity of chondrocyte-derived relative to osteoblast-derived matrix collagens, noncollagenous proteins, and proteoglycans6, 8 54 probably account for the paradoxical ability of NTPPPH to both promote and suppress mineralization in the pericellular matrix of articular cartilage. It will be of interest to compare the effects of chondrocyte-specific NTPPPH11 and PC-1 on mineralization in cartilage.

NTPPPH and other ectoenzymes including ALP, ATPase, and 5′-nucleotidase appear to work in a coordinated manner to regulate mineralization in bone and cartilage.19-24 For example, ALP deficiency (hypophosphatasia) is associated with both osteomalacia and a PPi supersaturation-associated pathologic articular cartilage calcification (with calcium pyrophosphate crystals).18 Studies on nondecalcified bone and growth plate MVs from subjects with hypophosphatasia revealed that calcium phosphate crystals could form inside these MVs but that extravesicular extension was inhibited relative to control MVs.20 Our results suggest that relatively unopposed effects of PC-1–induced PPi production by MVs could have contributed to these findings.

In summary, the results of this study further identify PC-1 as a potential therapeutic target in certain disorders of bone metabolism. Because the specific activities of NTPPPH and certain other membrane ectoenzymes can be altered in aging cartilage,33 further studies are indicated to determine if PC-1 expression in bone is disregulated in aging and osteoporosis.

Acknowledgements

Supported by NIH Grant PO1AG07996, and a Biomedical Sciences Research Award from the Arthritis Foundation, a Merit Review Award from the Veterans Affairs Medical Service, an award from the Stein Institute for Research on Aging at UCSD, and awards from the Victoria Research Council of Australia and Medical Research Society of Canada.

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