Linked Deficiencies in Extracellular PPi and Osteopontin Mediate Pathologic Calcification Associated With Defective PC-1 and ANK Expression

Authors


Abstract

Osteopontin and PPi both suppress hydroxyapatite deposition. Extracellular PPi deficiency causes spontaneous hypercalcification, yet unchallenged osteopontin knockout mice have only subtle mineralization abnormalities. We report that extracellular PPi deficiency promotes osteopontin deficiency and correction of osteopontin deficiency prevents hypercalcification, suggesting synergistic inhibition of hydroxyapatite deposition.

Nucleotide pyrophosphatase phosphodiesterase (NPP) isozymes including PC-1 (NPP1) function partly to generate PPI, a physiologic calcification inhibitor. PPi transport is modulated by the membrane channel protein ANK. Spontaneous articular cartilage calcification, increased vertebral cortical bone formation, and peripheral joint and intervertebral ossific ankylosis are associated with both PC-1 deficiency and expression of truncated ANK in ank/ank mice. To assess how PC-1, ANK, and PPi regulate both calcification and cell differentiation, we studied cultured PC-1−/− and ank/ank mouse calvarial osteoblasts. PC-1−/− osteoblasts demonstrated ∼50% depressed NPP activity and markedly lowered extracellular PPi associated with hypercalcification. These abnormalities were rescued by transfection of PC-1 but not of the NPP isozyme B10/NPP3. PC-1−/− and ank/ank cultured osteoblasts demonstrated not only comparable extracellular PPi depression and hypercalcification but also marked reduction in expression of osteopontin (OPN), another direct calcification inhibitor. Soluble PC-1 (which corrected extracellular PPi and OPN), and OPN itself (≥15 pg/ml), corrected hypercalcification by PC-1−/− and ank/ank osteoblasts. Thus, linked regulatory effects on extracellular PPi and OPN expression mediate the ability of PC-1 and ANK to regulate calcification.

INTRODUCTION

TWO NUCLEOTIDE PYROPHOSPHATASE PHOSPHODIESTERASE (NPP) family isozymes, PC-1 (or NPP1) and B10 (or NPP3), are strongly expressed by mature chondrocytes and osteoblasts.(1–4) Each of these NPP family ecto-enzymes hydrolyzes the phosphodiester I bond in a variety of substrates with a purine and pyrimidine nucleoside monophosphate backbone (EC 3.1.4.1).(5) However, the recent linkage of altered mineralization with defective PC-1 expression in “tiptoe walking” (or ttw/ttw) mice has suggested a unique role for PC-1 in regulating skeletal remodeling and calcification.(6) Specifically, ttw/ttw mice are homozygous for a stop codon between the catalytic site and EF hand in PC-1.(6) The ttw/ttw mouse phenotype includes postnatal development of progressive ankylosing intervertebral and peripheral joint hyperostosis, as well as spontaneous arterial and articular cartilage calcification and increased vertebral cortical bone formation.(6–10)

Products released by NPP catalytic activity on nucleoside triphosphates (EC 3.6.1.8) include PPi, a direct calcification inhibitor.(5, 11) PPi potently antagonizes the ability of Pi to crystallize with calcium to form hydroxyapatite and PPi suppresses hydroxyapatite crystal propagation.(11) Certain observations suggest that PC-1 and PPi may further regulate calcification by modulating function of cells in PC-1-expressing tissues.(11) Specifically, marked, systemic extracellular PPi lowering in the ttw/ttw mouse PC-1 deficiency state is associated with organized periarticular and spine ligament calcification with features of both membranous and enchondral ossification, rather than simply dystrophic calcification.(6–10) In addition, ∼50% depression of plasma and fibroblast NPP activity and extracellular PPi was recently linked to human PC-1 deficiency, but pathological calcifications seemed limited to large arteries and periarticular tissues in the affected infant.(12)

PC-1 expression, NPP activity, PPi-degrading pyrophosphates activities, and extracellular PPi are regulated by several growth factors, calciotropic hormones, and cytokines.(1–3, 11, 13–16) However, altered balance between PPi generation and degradation is not the sole mechanism determining extracellular PPi.(11) Specifically, the multiple-pass membrane protein ANK promotes PPi channeling to the cell exterior, thereby regulating both intracellular and extracellular PPi.(17) “Gain of function” of wildtype ANK normally stimulates increased extracellular PPi.(17) However, putative PPi channeling by ANK is abrogated by truncation of the C-terminal ANK intracellular domain.(17) Significantly, fibroblasts cultured from mice homozygous for the truncation mutant ank demonstrated decreased extracellular PPi.(17) Moreover, the phenotypes of ank/ank mice and ttw/ttw mice are remarkably similar, including shared and pathologically comparable enthesopathy and ankylosing hyperostosis of the spine, as well as spontaneous articular cartilage calcification and peripheral joint fusion.(6–10, 17)

Here, we examined ex vivo calcification by PC-1−/− mice, whose phenotype is nearly identical to ttw/ttw mice.(18) We tested the hypothesis that deficient extracellular PPi generation by ecto-nucleoside triphosphate pyrophosphohydrolase (NTPPPH) PC-1 activity mediates both increased calcification and altered osteoblast gene expression. We studied primary calvarial osteoblasts from PC-1−/− mice and tested for shared findings in ank/ank mice. Prompted by recent mRNA differential display analyses revealing altered expression of the mineralization regulator OPN in ear cartilages from ttw/ttw mice,(19) we examined osteoblast OPN expression. OPN mediates osteoclastic bone resorption and can promote skeletal remodeling.(20–24) Like PPi, OPN also is a potent, direct inhibitor of bone mineral formation and hydroxyapatite crystal growth.(25–27) Our results reveal linked regulatory effects on calcification by PC-1 and ANK through regulation of extracellular PPi and OPN.

MATERIALS AND METHODS

Reagents

All chemical reagents were from Sigma (St Louis, MO, USA), unless otherwise indicated. Bovine serum albumin (BSA) and recombinant murine myeloma cell line-expressed, His-tagged OPN of >95% purity, in 50 μg BSA/μg OPN (catalog 499260), were from Calbiochem (La Jolla, CA, USA).

PC-1 null and ank/ank mouse models and genotyping

Generation and initial characterization of PC-1 null mice were described previously, and herein we used mice on a 129/Sv background.(18) To determine PC-1 genotypes, genomic DNA was isolated from tails or from cultured primary osteoblasts and analyzed using a 3-primer polymerase chain reaction (PCR) protocol. Primers for the targeting region in wildtype PC-1 genomic DNA sequence were 5′-CCCTTTGTGGTACAAAGGACAG-3′ and 5′-GCATGACCCATTATACACTTTGT-3′; the primer for the targeting vector PGK-PolyA was 5′-GGGTGAGAACAGAGTACCTAC-3′. This PCR reaction generated distinct products (1.2-kb PC-1 null allele, 750-bp wildtype PC-1 allele). Initial Southern blots were used to confirm reproducibility of PCR screening for each genotype.

The ank/ank mouse colony used was on a hybrid background (derived originally from crossing a C3H and C57BL/6 hybrid male with BALB/c female).(28) ANK genotypes were analyzed by PCR.(17) Heterozygote breeders were used to generate distinct litters containing PC-1 null and ank/ank mice and their respective heterozygotic and wildtype littermates.

Isolation, culture, and transfection of primary osteoblasts and reverse transcriptase-PCR analyses

Primary cultures of osteoblasts were isolated from intramembranous bone (calvariae) of 0- to 3-day-old pups through sequential collagenase digestion, as described.(4) Calvariae of the same genotype were pooled and an enriched cell population of osteoblastic phenotype seeded at ∼4 × 104 cells/cm2 in α-MEM (Gibco-BRL, Grand Island, NY, USA), containing 10% heat inactivated FCS, glutamine (2 mM), penicillin (50 U/ml), and streptomycin (0.5 mg/ml). For studies under mineralizing conditions, media were supplemented with β-glycerophosphate (2.5 mM) and L-ascorbic acid-PO4 (25 μg/ml) every third day (medium A). We transfected cDNA expression constructs (2 μg DNA) for wildtype human PC-1 and B10/NPP3 in pcDNA3.1(2, 4) into primary cells using Lipofectamine Plus (Life Technologies, Grand Island, NY, USA) with transfection efficiency >40%.(1) To further verify osteoblastic differentiation, mouse osteoblasts were confirmed to express osteocalcin and type I collagen, but not aggrecan or type II collagen, by reverse transcriptase (RT)-PCR, as described.(2, 29)

For RT-PCR, total RNA was isolated from osteoblasts using 0.5 ml TriZOL (Life Technologies)/35-mm dish. RNA was extracted and reverse transcribed as described.(1) Mouse OPN primers(30) were sense, 5′-CTCCCGGAGAAAGTGACTGA-3′, which amplified a 831-bp product. Mouse matrix Gla protein (MGP) primers(31) were sense, 5′-TGCGCTGGCCGTGGCAACCCT-3′; and antisense, 5′-CCTCTCTGTTGATCTCGTAGGCA-3′, which amplified a 181-bp product. Mouse cbfa1 primers(32) were sense, 5′-GAGGCACAAGTTCTATCTGGA-3′; and antisense, 5′-GGTGGTCCGCGATGATCTC-3′, which amplified a 336-bp product. Mouse osteoprotogerin (OPG) primers(33) were sense, 5′-CTGGACATCATTGAATGGAC-3′; and antisense 5′-AATTAGCAGGAGGCCAAATG-3′, which amplified a 495-bp product. Primers for the housekeeping gene loading control, the ribosomal protein L30, were as previously described.(1)

Assessment of calcification

To quantify calcification by osteoblasts, we used a previously described Alizarin red S binding assay using cells that reached confluency.(34) Osteoblasts (5000 cells/well) were plated in 0.1 ml of medium A in a flat bottom 96-well B-D Falcon 353075 culture plate (B-D Falcon, Bedford, MA, USA). At each time point, media were removed, and 0.05 ml of diluted Alizarin red S (0.5% vol/vol Alizarin red S, pH 5.0) was added for 10 min.(34) Data were expressed as micromoles of bound Alizarin red S per microgram DNA in each well, determined chromogenically at OD570.(34)

Histology and immunohistochemistry

Mouse skeletal tissues were isolated by dissection and fixed in10% neutral buffered formalin for 2 days and then decalcified in 4% hydrochloric acid, processed for histology, and embedded in paraffin. For immunohistologic analysis of OPN, sections were deparaffinized, blocked with 10% goat serum for 20 min, and incubated overnight at 4°C with rabbit polyclonal antibody to OPN (Chemicon, Temecula, CA, USA). Washed sections were incubated for 1 h at 22°C with biotinylated goat anti-rabbit IgG followed by a 1-h incubation with peroxidase-conjugated avidin. Peroxidase activity was detected using the Sigma Fast DAB staining kit, according to the manufacturer's instructions.

Preparation of soluble PC-1

Stable expression of a soluble PC-1 cDNA construct in the vector pSecTag2A (Invitrogen, Carlsbad, CA, USA) was established in 293 cells. The construct encoding soluble PC-1 was generated by introducing a BamH1 site through PCR at the junction of the transmembrane and extracellular domains of PC-1, as described.(2) This site was used to ligate PC-1 cDNA encoding the extracellular domain starting with the amino acid sequence PSCAKK into the BamHI siteof pSEcTag2A, allowing PC-1 to be cleaved by signal peptidase and secreted by transfected 293 cells. To isolate soluble PC-1, 700 ml volume of conditioned cell media containing soluble PC-1 was dialyzed overnight in 20 mM Tris, pH 8.0, followed by additional dialysis for 2 h in fresh buffer. The dialyzed medium was fractionated on a Q-Sepharose, Fast Flow column (Pharmacia, Piscataway, NJ, USA). The column was washed with 70 ml of 20 mM Tris, pH 8.0, 50 mM NaCl, and soluble PC-1 eluted with 20 mM Tris and 150 mM NaCl, pH 8.0. Soluble PC-1 was concentrated from fractions most enriched in NPP activity (and the preparation was verified to be PC-1-enriched by SDS-PAGE/Western blotting).

PPi, NPP, and alkaline phosphatase assays

PPi concentrations were measured radiometrically as described.(2) We determined specific activity of NPP colorimetrically using p-nitropheylthymidine monophosphate as substrate for nucleotide phosphodiesterase I activity at alkaline pH.(2) Specific activity of alkaline phosphatase (AP) was measured as described.(4) One unit of NPP (or AP) was defined as 1 μmol substrate hydrolyzed per hour (per microgram protein/sample).

OPN ELISA

To measure OPN protein levels, we developed an ELISA based on a previously published method,(35) using plates coated with a monoclonal antibody to native OPN (Chemicon; using a 1:2500 dilution of OPN antibody in 100 μl/well 0.1 M sodium bicarbonate, pH 9.0 at 4°C overnight). Wells were blocked with 10 mM Tris, 150 mM NaCl, and 0.05% Tween-20, pH 8.0, for 1 h at 22°C. Samples, diluted in 1% BSA, 10 mM Tris, and 150 mM NaCl, pH 8.0, were added to the wells and incubated for 1 h at 37°C (with dilutions of 1:2–1:8 quantified). Recombinant murine OPN was used as standard. Washed wells were incubated sequentially for 1 h at 37°C with rabbit anti-OPN (1:1000; Chemicon), biotinylated goat anti-rabbit IgG (1:1000), and streptavidin conjugated with AP (1:500 dilution), and color was developed using p-nitrophenyl phosphate and read at 405 nM.

Statistics

Where indicated, error bars represent SD. Statistical analyses were performed using Student's t-test (paired two-sample testing for means).

RESULTS

Skeletal pathology in PC-1−/− mice

PC-1+/− mice were phenotypically indistinguishable from PC-1+/+ mice. In PC-1−/− mice, we observed features essentially identical to the previously described phenotype of ttw/ttw mice.(6–10) These included the development of hyperostosis, starting at approximately 3 weeks of age, in a progressive process that culminated in ossific intervertebral fusion (Figs. 1B and 1D, and 1F) and peripheral joint ankylosis, as well as Achilles tendon calcification (data not shown).

Figure FIG. 1..

Morphological changes in PC-1 null mice. (A-D) X-rays of 100-day-old female (A and C)wildtype mice or (B and D) PC-1−/− mice. Exposures were under identical conditions (mammography apparatus, soft X-rays). C and D are magnified from A and B, respectively. In the PC-1−/− mice, there was heavy calcification adjacent to the intervertebral disks, while the vertebral bodies, arches, and spines, and the shafts of long bones show reduced bone density. (E and F) Macroscopic view of the anterior aspect of the lumbar vertebrae of 100-day-old female (E) wildtype mice and (F) PC-1−/− mice. Fine forceps and a metal brush were used to remove muscle tissue surrounding the vertebrae. Note the heavy calcification in the region of the intervertebral disks and growth plates of the PC-1−/− mice.

PC-1 and PPi in osteoblast calcification

We studied cultured primary calvarial osteoblasts to identify basic mechanisms involved in abnormal mineralization in PC-1−/− mice. PC-1 is one of several NPP family isozymes expressed in the skeleton, yet PC-1 plays a disproportionately large role in supporting chondrocyte extracellular PPi relative to other NPP isozymes.(34) We observed that extracellular PPi, intracellular PPi, and cell-associated NPP specific activity (measured as alkaline nucleotide phosphodiesterase I) all were relatively lower by approximately 50% at early time points in culture in PC-1−/− osteoblasts relative to PC-1+/+ littermate control cells (Figs. 2A–2C). NPP activity and intracellular and extracellular PPi levels rose in association with time in culture in osteoblasts of all genotypes (Figs. 2A–2C). However, absolute differences between PPi levels associated with PC-1+/+ and PC-1−/− osteoblasts remained similar over the same time in culture (Figs. 2A and 2B). In the PC-1+/− cells, levels of PPi and NPP were intermediate between those of wildtype and PC-1+/+ cells (Figs. 2A–2C). The extent and rapidity of calcification also were markedly increased in PC-1−/− primary calvarial osteoblasts relative to controls (Fig. 2D). In these studies, done on cells that had reached confluency, cell numbers at confluency were comparable between genotypes and relative changes were the same whether the calcification was measured per well or per cell DNA (data not shown).

Figure FIG. 2..

Depression of intracellular and extracellular PPi and NPP activity and augmented calcification associated with cultured osteoblasts of PC-1-deficient mice. Primary calvarial osteoblasts from PC-1+/+, PC-1+/−, and PC-1−/− mice were plated in 35-mm dishes. At confluency (designated day 1), 2.5 mM β-glycerophosphate and 25 μg/ml of ascorbic acid-PO4 were added to complete α-MEM growth medium to stimulate mineralization (as described in the Materials and Methods section); the medium was changed every 3 days. (A-C) Conditioned media and cell lysates were collected at each time point indicated for assays of PPi and of the specific activity of NPP (for which 1 U was defined as 1 μmol of substrate hydrolyzed per hour), as described in the Materials and Methods section. (D) For studies of calcification, primary calvarial osteoblasts (5000 cells per well) from PC-1+/+, PC-1+/−, and PC-1−/− mice were plated in 96-well plates, and at confluency, 2.5 mM β-glycerophosphate and 25 μg/ml of ascorbic acid-PO4 were added to complete α-MEM growth medium and designated day 1 (as described in the Materials and Methods section). Media were changed every 3 days, and at each designated time point, the media was removed and calcification measured as micromoles of bound Alizarin red S normalized per microgram cell DNA, as described in the Materials and Methods section. (A-C) Ten mice from each genotype were studied in triplicate, and results shown are pooled from all experiments. (D) Results pooled from studies on eight mice of each genotype studied in replicates of eight. (A) p < 0.05 relative to PC-1+/+ cells for extracellular PPi levels on days 4–17 in PC-1+/− cells and days 1–17 in PC-1−/− cells. (B and C) *p < 0.05 relative to PC-1+/+ cells. (D) *p < 0.05 relative to wildtype cells.

Transfection of PC-1 into PC-1−/− osteoblasts restored intracellular and extracellular NPP activity levels and PPi, as well as the hypercalcification to approximately baseline levels for wildtype cells (Fig. 3). Significantly, the restorative effects of PC-1 transfection on NPP activity and extracellular PPi were sustained at up to 10 days after transfection (Fig. 3). To assess if the correction of hypercalcification was selective for PC-1, we compared the effects of transfection of the NPP family isozyme B10/NPP3. Transfected B10/NPP3 significantly elevated NPP activity in the PC-1−/− osteoblasts, but the induced NPP activity remained predominantly intracellular (Fig. 3). B10/NPP3 also significantly elevated intracellular but not extracellular PPi in the PC-1−/− osteoblasts (Fig. 3). In contrast to PC-1, B10/NPP3 induced a small decrease in hypercalcification relative to control cells (Fig. 3).

Figure FIG. 3..

NPP family isozyme-specific rescue of matrix hypercalcification by PC-1 in mouse osteoblasts lacking PC-1 expression: Primary calvarial osteoblasts from PC-1−/− and control untransfected PC-1+/+ mice were isolated, and 5 × 105 cells were plated in 60-mm dishes. The PC-1−/− cells were transfected with 2 μg of empty plasmid DNA or plasmid DNA expression constructs for PC-1 or the NPP isozyme B10/NPP3, and aliquots of 5000 cells were studied in 96-well plates as described in the Materials and Methods section. At confluency, 2.5 mM β-glycerophosphate and 25 μg/ml of ascorbic acid-PO4 were added to complete α-MEM growth medium, which was changed every 3 days. (A) At each time point indicated, calcification was measured as micromoles of bound Alizarin red S normalized per microgram cellular DNA, as described above. The results for (B) PPi and (C) NPP on day 10. Five mice from each genotype were studied in replicates of eight. Results shown are pooled from all experiments. *p < 0.05 relative to vector-transfected PC-1−/− osteoblasts in A-C.

PC-1−/− cells also demonstrated a marked decrease in expression of OPN mRNA relative to wildtype cells (Fig. 4A). OPN expression was not appreciably distinct between the cells from PC-1+/+ and PC-1+/− mice (data not shown). The selectivity of the altered OPN expression was tested by evaluating expression in osteoblasts of the mineralization-regulating genes OPG, MGP, and cbfa1.(31–33) In contrast to OPN, only modest differences in expression of OPG, MGP, and cbfa1 mRNA were observed in PC-1−/− osteoblasts relative to wildtype cells (Fig. 4A).

Figure FIG. 4..

Diminished OPN expression by primary calvarial osteoblasts from PC-1−/− mice. We cultured PC-1−/− and PC-1+/+ osteoblasts for the number of days indicated under mineralizing conditions in complete α-MEM growth medium supplemented with 2.5 mM β-glycerophosphate and 25 μg/ml of ascorbic acid-PO4 every 3 days, as described above. (A) We analyzed the mRNA expression of selected osteoblast genes, including OPN, OPG, MGP, and cbfa1 by RT-PCR in cell culture, as described in the Materials and Methods section. Assay of the ribosomal “housekeeping gene” L30 served as the control for RNA loading. (B) To assess for potential reversal of the above-described changes in OPN expression by extracellular PPi supplementation, PC-1−/− and PC-1+/+ osteoblasts were cultured for the number of days indicated in complete α-MEM growth medium supplemented with 2.5 mM β-glycerophosphate and 25 μg/ml of ascorbic acid-PO4 every 3 days. Analyses of OPN and L30 mRNA expression by RT-PCR were performed. Where indicated (as PC-1−/− + PPi), the PC-1−/− cells were pulsed on days 0, 2, and 5 with exogenous sodium PPi (1 pmol added to 2.0 ml medium to elevate extracellular PPi by approximately 500 pM, as described above). Four mice from each genotype were studied. Results shown are representative of all mice studied.

Next, we assessed if depressed OPN expression was attributable to extracellular PPi deficiency in PC-1−/− osteoblasts. Predicated on a difference in extracellular PPi concentrations of approximately 500 pM between PC-1−/− cells and PC-1+/+ cells at multiple time points (Fig. 2), we optimized pulsing of the extracellular medium with sodium PPi to intermittently restore extracellular PPi in PC-1−/− cells to the approximate level of PC-1+/+ cells. Specifically, we determined that adding 0.05 pmol of sodium PPi per 0.1 ml volume at days 3, 5, and 7 to the cultured PC-1−/− cells elevated extracellular PPi to within 5% of the levels of PC-1+/+ cells for a 24-h period (data not shown). Accordingly, we intermittently pulsed aliquots of mineralizing PC-1−/− cells at early time points in culture with 0.05 pmol of sodium PPi per 0.1 ml (Fig. 4B). The decreased OPN expression in PC-1−/− osteoblasts at day 1 was reversed by the pulsing with sodium PPi (Fig. 4B). This effect of PPI was not demonstrable at later time points in culture.

Reversal of hypercalcification in both PC-1−/− and ank/ank osteoblasts by soluble PC-1 and OPN

Similar to PC-1−/− cells, the ank/ank osteoblasts had marked depression of extracellular PPi relative to wildtype cells (Fig. 5). However, intracellular PPi was significantly elevated in the ank/ank osteoblasts (Fig. 5), similar to the case in ank/ank fibroblasts.(17) Calcification was significantly accelerated and more extensive in cultured ank/ank osteoblasts relative to both wildtype ANK/ANK and heterozygotic ANK/ank littermate cells (Fig. 5).

Figure FIG. 5..

Altered PPi levels associated with hypercalcification in ank/ank primary calvarial osteoblasts. (A) Primary calvarial osteoblasts from ANK/ANK, ANK/ank, and ank/ank mice, plated in 35-mm dishes, treated with medium supplemented with 2.5 mM β-glycerophosphate and 25 μg/ml ascorbic acid-PO4, were assessed for intracellular and extracellular PPi concentrations, and the specific activity of NPP at the time points indicated, as described above for Fig. 3. Ten mice from each genotype were studied in triplicate. Results shown are pooled from all experiments. *p < 0.05 relative to ANK/ANK. (B) Confluent primary calvarial osteoblasts from ANK/ANK, ANK/ank, and ank/ank mice were treated with 2.5 mM β-glycerophosphate and 25 μg/ml ascorbic acid-PO4 in complete α-MEM growth medium (as described in the Materials and Methods section), and matrix calcification was measured as bound Alizarin red S, as above. Results pooled from studies on eight mice of each genotype studied in replicates of eight. *p < 0.05 relative to wildtype cells.

We focused on OPN mRNA expression at early time points in culture in ank/ank osteoblasts (Fig. 6). Although OPN mRNA levels fluctuated widely between days 1–6 in the ank/ank osteoblasts, there was depression of OPN mRNA expression at days 1 and 6 relative to ANK/ANK cells (Fig. 6A). By comparison, there was little change in OPG, MGP, and cbfa1 mRNA expression in ank/ank relative to the wildtype osteoblasts (Fig. 6A), as was the case for PC-1−/− cells above. As for the PC-1−/− cells above, the decrease in OPN expression in ank/ank osteoblasts, was reversible by PPi supplementation at day 1 in culture (Fig. 6B), although this effect of PPI was not clearly demonstrable at later time points in culture.

Figure FIG. 6..

PPi-reversible decrease in OPN expression by primary calvarial osteoblasts from ank/ank mice. Using the same experimental methods as described for Fig. 3, we cultured osteoblasts of the indicated genotypes. (A) Analyses of mRNA for OPN, OPG, MGP, cbfa1, and L30 by RT-PCR, as described above. (B) Assessment for potential reversal of the decreased OPN expression in ank/ank cells by extracellular PPi supplementation. As described above, osteoblasts of the indicated genotypes were studied. Where indicated (as ank/ank + PPi), the ank/ank cells had been pulsed on days 0, 2, and 5 with exogenous sodium PPi (1 pmol added to 2.0 ml medium to elevate extracellular PPi by approximately 500 pM). Four mice from each genotype were studied. Results shown are representative of all mice studied.

Next, we examined for functional linkage between defective PC-1 and ANK expression and OPN expression in hypercalcification. First, we analyzed OPN expression qualitatively by immunohistochemistry of PC-1−/− mouse spines. OPN staining was only weakly detectable in the disorganized trabecular bone in these samples, in contrast to robust OPN staining in trabecular bone in wildtype mice (Fig. 7, panels at 25×). We incidentally noted thinning of the nucleus pulposus in PC-1−/− mouse spines (Fig. 7, panels at 25×). OPN expression was detectable in the nucleus pulposus, as well as in the intervertebral ligaments (Fig. 7, panels at 6.25× and 13×).

Figure FIG. 7..

OPN expression in the spines of PC-1−/− mice. Deparaffinized sections of lumbar vertebrae from 4-month-old PC-1+/+ and PC-1−/− mice were analyzed for immunohistochemical staining of OPN using rabbit polyclonal anti-OPN (1:100), biotinylated goat anti-rabbit IgG, and peroxidase-conjugated avidin, as described in the Materials and Methods section. For the negative staining control, an equal dilution of rabbit serum was used in place of the primary antibody. Note particularly the staining for OPN associated with trabecular bone of wildtype mice (arrows in upper left 25× panel) in contrast with the disorganized trabecular bone in the PC-1−/− mouse associated with weak OPN staining. T, trabecular bone; E, endochondral ossification center of the vertebral body; N, nucleus pulposus.

Quantitative assessment of OPN expression in unfractionated newborn calvarial tissue by OPN ELISA demonstrated depression (>50%) in OPN levels in PC-1−/− relative to PC-1+/+ mice (Fig. 8A). Comparable in situ depression of OPN was observed in calvarial extracts of ank/ank relative to ANK/ANK mice (Fig. 8A). Correspondingly, OPN levels were significantly depressed in vitro (by ≥15 pg/ml) in conditioned media of both cultured PC-1−/− and ank/ank calvarial osteoblasts relative to their wildtype controls (Fig. 8B). Next, we supplemented the culture media of PC-1−/− and ank/ank osteoblasts with commercial recombinant murine OPN purified from murine myelomatous cells and stabilized in BSA. OPN supplementation (at 1.5, 15, and 150 pg/ml) did not significantly alter extracellular PPi (data not shown). However, OPN supplementation, at ≥15 pg/ml, prevented the hypercalcification by both PC-1−/− and ank/ank osteoblasts (Fig. 9). We verified that addition of equivalent amounts of BSA to those in the OPN preparation (up to 7.5 ng/ml) had no effect under these conditions (data not shown).

Figure FIG. 8..

Depressed OPN levels in situ in calvariae and in vitro in cultured osteoblasts from both PC-1−/− and ank/ank mice: (A) Calvariae were isolated from newborn PC-1−/−, PC-1+/+, ANK/ANK, and ank/ank mice and homogenized before the sequential digestion in 0.5% Triton X-100 in PBS. Diluted aliquots of the tissue extracts (containing 100 ng-1 μg protein) were added to plates precoated with monoclonal anti-OPN antibody for 1 h, and the OPN ELISA was performed as described in the Materials and Methods section. n = 8 calvariae of each genotype, studied in replicates of three at three dilutions (1:2, 1:4, and 1:8), *p < 0.01. (B) OPN protein levels, in the conditioned media of cultured PC-1−/− and ank/ank calvarial osteoblasts relative to their wildtype littermate controls, were measured by ELISA. n = 8 of each genotype, *p < 0.01.

Figure FIG. 9..

Reconstitution with OPN prevents hypercalcification by both PC-1−/− and ank/ank osteoblasts: We tested if correction of OPN levels (back to the OPN levels of wildtype controls, which were ≥15 pg/ml higher in vitro) would alter hypercalcification by cultured PC-1−/− and ank/ank osteoblasts. To do so, we supplemented the culture media of PC-1−/− and ank/ank osteoblasts with recombinant murine OPN (at 1.5, 15, or 150 pg/ml where indicated). We carried the cells under mineralizing conditions for up to 10 days in culture and then measured extent of calcification by Alizarin red S binding. (n = 6, *p < 0.05 for the correction of matrix hypercalcification by OPN added at 15 and 150 pg/ml).

Last, we prepared soluble PC-1 and optimized restoration of depressed NPP specific activity of PC-1−/− primary calvarial osteoblasts by titrated addition of soluble PC-1. Specifically, addition of 4.92 U NPP/ml raised extracellular NPP and extracellular PPi of PC-1−/− cells to within 5% of their respective values in PC-1+/+ cultures at each time point studied (data not shown). We demonstrated that addition of soluble PC-1 in this manner largely corrected the depression of extracellular PPi, the depression of secreted OPN levels, and the hypercalcification in not only PC-1−/− but also ank/ank osteoblasts (Fig. 10).

Figure FIG. 10..

Corrective effects of soluble PC-1 on extracellular PPi, OPN levels, and hypercalcification by cultured PC-1−/− and ank/ank osteoblasts. Primary calvarial osteoblasts from PC-1−/−, PC-1+/+, ANK/ANK, and ank/ank mice were isolated and cultured under mineralizing conditions. Where indicated, we added aliquots of purified soluble PC-1 to achieve 4.92 U of NPP activity to the cells on days 1, 3, 5, 7, 9, 11, and 14. At each time point indicated, the medium was removed and the wells analyzed for calcium precipitation by the Alizarin red S assay. The conditioned media were analyzed for extracellular PPi levels and OPN levels. Results are from replicates of eight samples derived from osteoblasts pooled from 12 mice of each genotype. *p < 0.05. (A) Addition of soluble PC-1 in this manner corrected the depression of extracellular PPi in both PC-1 null and ank/ank osteoblasts. (B) Addition of soluble PC-1 in this manner significantly corrected the depression of secreted OPN levels in both PC-1 null and ank/ank osteoblasts by day 14. (C) Addition of soluble PC-1 in this manner corrected the matrix hypercalcification by both PC-1−/− osteoblasts and ank/ank osteoblasts.

DISCUSSION

In this study, we tested the functional significance in regulating calcification of PPi generation by PC-1 and PPi transport by ANK. We directly demonstrated that deficient NPP-catalyzed PPi generation in PC-1−/− mice contributes to pathological calcification. Specifically, osteoblasts from PC-1−/− mice demonstrated ∼50% depression of NPP-specific activity and intracellular and extracellular PPI at day 1 in culture. Support of extracellular PPi levels was a NPP family isozyme-specific function of PC-1 with unique implications for regulation of calcification. Specifically, “gain of function” of NPP activity in PC-1−/− osteoblasts induced by transfection of B10/NPP3 restored intracellular PPi levels to normal. However, B10/NPP3, unlike PC-1, failed to restore depressed extracellular PPI to normal. Concurrently, the capacity of B10/NPP3 to reduce hypercalcification by PC-1−/− cells was modest relative to PC-1.

Our findings suggested thresholds for both PC-1 and extracellular PPi deficiency to heighten calcification by osteoblasts to lie between the 25% and 50% depression of PC-1 NPP activity and extracellular PPi relative to wildtype cells seen at early time points in culture. Specifically, osteoblasts from phenotypically normal PC-1+/− mice demonstrated extracellular PPi levels (and NPP levels) intermediate between PC-1+/+ and PC-1−/− mice. However, cultured osteoblasts from PC-1+/− and PC-1+/+ mice did not show significant quantitative differences in calcification. Parallel findings for extracellular PPi and calcification were seen comparing ank/ANK cells from phenotypically normal mice with ank/ank mouse-derived cells.

Cultured osteoblasts of PC-1−/− and ank/ank mice demonstrated decreased expression of OPN relative to wildtype cells. Changes in the OPN expression were at least partially selective, because the mineral resorption regulator OPG,(33) the osteoblastic transcription factor cbfa1,(32) and the physiologic mineralization inhibitor MGP(31) demonstrated only modest differences in expression in cultured PC-1−/− and ank/ank osteoblasts relative to wildtype cells. Importantly, the depressed OPN expression preceded the marked rise in calcification seen in both PC-1−/− and ank/ank osteoblasts in culture and therefore was not likely to be simply secondary to heightened calcification.

Taken together, our results suggested that the remarkable phenotypic similarities between ank/ank and PC-1−/− mice at least partly reflect the common depression of extracellular PPi and OPN. First, hypercalcification and depression of extracellular PPI and OPN expression were strikingly comparable in cultured primary calvarial osteoblasts from ank/ank mice and PC-1−/− mice. Second, OPN protein levels were diminished in PC-1−/− and ank/ank calvariae in situ. Third, soluble extracellular PC-1, which was added to provide a stable source of continuing PPi generation and which was validated to effectively restore of PPi by over 0–14 days in culture, jointly corrected extracellular PPi levels and OPN levels in both PC-1−/− and ank/ank osteoblasts. Furthermore, such restoration by soluble PC-1 of extracellular PPi and OPN levels corrected hypercalcification in osteoblasts from both PC-1−/− and ank/ank mice.

In this study, a single pulse of exogenous PPI reversed the depression in OPN mRNA expression by both PC-1−/− and ank/ank osteoblasts at day 1 in culture. However, the capacity of a pulse of exogenous PPi to reverse the decreased OPN expression was attenuated with further time in culture under pro-mineralizing conditions. It is possible that metabolism and disposition of pulsed exogenous PPi by pyrophosphatases and other factors may alter in association with changes in differentiation of osteoblasts with time in culture. Thus, identification of the microenvironment(s) in which PC-1 and ANK both act to regulate PPi levels may be pertinent to further understanding of the regulation of OPN expression and other functionally significant events in mineralizing cells. In this regard, intracellular PPi(17) was elevated in ank/ank mouse osteoblasts but was depressed in PC-1−/− cells. We speculate that such differences in intracellular PPi may contribute to subtle distinctions between PC-1−/− and ank/ank mice. These include a longer time to initial development of peripheral and spinal joint fusion in ank/ank relative to PC-1−/− mice.(6, 7, 28, 36)

Focal upregulation of OPN expression, already documented in arterial calcification and most forms of pathological “soft tissue” calcification described to date, is believed to play a physiologic restraining role in calcification.(20–27, 37) Post-translationally phosphorylated OPN,(20, 27, 38) like PPi, binds to hydroxyapatite crystals and inhibits hydroxyapatite crystal propagation, and exogenous OPN potently inhibits calcification by cultured cells.(20, 21, 26, 27) Thus, the downregulation of OPN demonstrated in PC-1−/− and ank/ank osteoblasts in vitro in this study was unexpected.

When challenged by ovariectomy or administration of parathyroid hormone (PTH), OPN-deficient tissues clearly show impaired bone resorption.(21, 22) However, unchallenged OPN knockout mice show only subtle changes in bone mineralization, such as greater hydroxyapatite crystal size and crystallinity detectable by sensitive evaluation.(25) Indeed, the grossly normal OPN null mouse phenotype stands in contrast to the phenotype of extracellular PPi-depleted PC-1−/− and ank/ank mice.(6–10, 17, 28, 36) Thus, the capacity of restoration of OPN levels to normal using exceedingly small doses (≥15pg/ml) of OPN to prevent the marked hypercalcification by cultured PC-1−/− and ank/ank osteoblasts was unexpected in this study. We speculate that the direct inhibitory effect of OPN on hydroxyapatite deposition becomes substantially more significant at sites where actively mineralizing cells are challenged by extracellular depletion of PPi. In essence, the inhibitory effects of PPi and OPN on hydroxyapatite crystal deposition may be synergistic. Decreased OPN expression seems to be a permissive factor for hypercalcification associated with extracellular PPi deficiency, and remarkably, extracellular PPi deficiency promoted OPN deficiency. It will be of interest to determine if extracellular PPi affects additional activities of OPN, such as mobilization of macrophages,(39) stimulation of activation of several metalloproteinases,(40) modulation of osteoclast-mediated bone resorption,(21–23) and promotion of connective tissue turnover in certain inflammatory states.(41)

The basic cell signaling mechanisms by which decreased PC-1 expression and extracellular PPi levels promoted both increased calcification and decreased OPN expression will be of interest to define. There is no documentation of plasma membrane receptor recognition of PPi or of active uptake of extracellular PPi in mammalian cells.(11) However, extracellular PPi is a precursor of inorganic phosphate (Pi), including tissue nonspecific AP (TNAP)-catalyzed PPi hydrolysis.(11) TNAP is an essential promoter of bone mineralization that also acts as a PC-1 antagonist.(4, 16) Although Pi stimulates calcification,(11, 42) Pi uptake and signaling triggered by Pi also promote OPN expression.(43) Given the mutual correction of mineralization abnormalities by crossing PC-1−/− and TNAP−/− mice,(16) we speculate that PC-1, ANK, and TNAP, through their effects on extracellular PPi, critically interface in the function of cells involved in calcification.

In conclusion, our observations support the potential role of therapeutic modulation of extracellular PPi for control of some forms of dysregulated calcification. The PPi analogue phosphocitrate(44) was previously ascertained to markedly suppress calcification in vivo in ank/ank mice.(36) The discovery that either soluble PC-1 or OPN rescue hypercalcification of both PC-1−/− and ank/ank osteoblasts in vitro suggests the potential therapeutic utility of these molecules to regulate calcification.

Acknowledgements

We gratefully acknowledge the technical assistance of Andrew Wigg in preparation of the soluble PC-1. This study was supported by grants from the Department of Veterans Affairs, National Institutes of Health (P01AGO7996, AR47908, AR40770, CA42595, DE12889), Monash University, Novartis, and the National Health and Medical Research Council of Australia.

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