| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 282, Issue 21, 15872-15883, May 25, 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1
¶2||23
From the
Faculty of Dentistry, McGill University, Montreal, Quebec H3A 2B2, Canada, the Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark, and the Departments of ¶Medicine and ||Anatomy and Cell Biology, Faculty of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada
Received for publication, February 6, 2007 , and in revised form, March 21, 2007.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PPi prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
In addition to the organic, mineral-binding noncollagenous proteins of bones and teeth, inorganic molecules in these tissues have also been proposed as molecular determinants of mineralization. Pyrophosphate (PPi) is a potent, mineral-binding small molecule inhibitor of crystal nucleation and growth (3), and recent studies using transgenic and naturally occurring mutant mice have identified the proteins/enzymes involved in producing and handling PPi (see references below).
PPi is a by-product of many intracellular metabolic reactions (4). PPi is also present in the extracellular matrix of most tissues and bodily fluids including plasma (5, 6), where it acts as a potent inhibitor of mineral nucleation and growth at micromolar concentrations (3, 7, 8). Although the mechanism by which PPi inhibits hydroxyapatite crystal growth is not entirely known, PPi is thought to adsorb specifically to crystal growth sites, thus preventing further apposition of mineral ions (7). Although some physicochemical studies have been conducted on the actions of PPi on crystal growth in cell-free systems, there is limited information available on putative direct effects of PPi on cells in general and, more specifically, on mineralizing cell culture models (9).
Extracellular matrix mineralization in bone is tightly linked to the Pi/PPi ratio found in this tissue (10). Extracellular PPi deficiency leads to excess hydroxyapatite formation in the skeleton (11), whereas PPi elevation results in decreased skeletal mineralization (6, 12-14) and the formation of calcium pyrophosphate dihydrate crystals in joints (15, 16). The homeostatic regulation of local PPi levels by bone cells is thus an important part of their function related to mineralization. The major ectoenzyme responsible for generation of extracellular PPi is Enpp1 (ectonucleotide pyrophosphatase/phosphodiesterase 1; also known as NPP1 and NPPS) (17); this enzyme is expressed at particularly high levels in bone, cartilage, and teeth (17-19). Intracellular PPi can also be exported into the extracellular compartment by the membrane transporter Ank (progressive ankylosis or ANKH), which is also highly expressed in bone, cartilage, and teeth (20-22). Deletion of the Ank gene in mice results in ectopic mineral formation in joint spaces and, eventually, complete joint ankylosis (23). Given the high expression levels of these two genes in mineralized tissues, removal of PPi is expected to be a prerequisite for physiologic mineralization, and thus regulation of the expression and activities of these two proteins is likely central to the control of mineralization. Tnap (tissue-nonspecific alkaline phosphatase; also known as Akp2), a well characterized marker of the osteoblast lineage (24), is capable of hydrolyzing PPi into Pi (25). In osteoblast cultures, Tnap is also responsible for the generation of Pi from phosphate esters such as -glycerophosphate (GP),4 which is commonly used as a source of organic phosphate for mineralization (26).
Another key inhibitor of mineralization found in bone is Opn (osteopontin), a highly phosphorylated glycoprotein with strong mineral binding properties (27). A link between the enzymes regulating extracellular PPi levels and nonenzymatic protein inhibitors of mineralization such as Opn has been demonstrated in Enpp1-deficient mice that show a decrease in both PPi and Opn levels and consequential hypermineralization particularly related to joints and ligaments (11). This phenotype is also observed in the naturally occurring mouse mutant ttw (tip-toe-walking mice; Ref. 28).
In view of recent findings linking PPi generators (Enpp1), transporters (Ank), and degraders (Tnap) with extracellular matrix protein inhibitors of mineralization (Opn), we have used the MC3T3-E1 murine preosteoblast cell line to examine whether there is a direct effect of PPi on Opn gene expression and the specific signaling pathways involved in this response. We report that PPi up-regulates Opn in osteoblasts via MAPK pathways, thus providing a dual mechanism for inhibition/control of hydroxyapatite crystal growth during mineralization.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2. All of the experiments were carried out at a seeding density of 50,000 cells/cm2. Cell differentiation and mineralization was initiated 24 h after plating by replacing the medium with -minimum essential medium supplemented with 10% fetal bovine serum, 50 µg/ml ascorbic acid (Sigma), and 10 mM GP (Sigma) or 5 mM sodium phosphate (Sigma). The medium was changed every 48 h. For inhibition experiments, sodium pyrophosphate tetrabasic, levamisole, kidney tissue-nonspecific alkaline phosphatase, and phosphonoformic acid (foscarnet) were purchased from Sigma. Signaling pathway inhibitors U0126 and SB202190 were obtained from VWR, and inhibitor SP600125 was purchased from SuperArray. Cytotoxicity of inhibitors and phosphate ester treatments were determined to be negligible by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay, data not shown). Quantification of Mineralization—Mineral was visualized by von Kossa staining using a 5% silver nitrate solution (Sigma) combined with exposure of the culture dishes to bright light. For quantification of insoluble calcium in the cell/matrix layer, the cultures were decalcified with 0.5 N HCl, and calcium in the supernatant was determined colorimetrically using a calcium assay kit (Diagnostic Chemicals). Following decalcification, the cultures were solubilized with 0.1 N NaOH and 0.1% SDS, and total protein content was measured with the BCA protein kit (Pierce). The calcium content of the cell/matrix layer was normalized to the protein content.
Assay for Alkaline Phosphatase and Nucleotide Pyrophosphatase Phosphodiesterase Activity—The cultures were washed three times with phosphate-buffered saline (PBS) and solubilized in 10 mM Tris, pH 7.4, 0.2% Igepal (Sigma), and 2 mM phenylmethylsulfonyl fluoride. After sonication and centrifugation, alkaline phosphatase activity in the supernatant was determined colorimetrically in a reaction mixture containing 50 mM Tris-HCl, pH 8.8, 10 mM MgCl2, and 20 mM p-nitrophenylphosphate. Calf intestinal alkaline phosphatase (Sigma) was used as a standard. One unit will hydrolyze 1 µmol of p-nitrophenylphosphate/min at 37 °C. Nucleotide pyrophosphatase phosphodiesterase activity was determined colorimetrically using p-nitrophenylthymidine monophosphate as a substrate (17).
Enzyme Inhibition Kinetics—All of the assays were performed in 50 mM Tris-HCl buffer, pH 8.8, with 10 mM MgCl2 at 37 °C and were repeated in triplicate. All of the enzymatic rates obtained are initial rates. Quantification of substrate reaction product (phosphate) was performed colorimetrically by the ammonium molybdate method. Inhibitor constants were obtained from averages of Dixon and s/v plots (29, 30).
RNA Isolation and Quantitative Real Time PCR—Total RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer's protocol and quantified in a spectrophotometer by absorbance readings at 260 nm. RNA was treated with DNase (Invitrogen), and 1 µg was used for cDNA synthesis using the Thermoscript first strand cDNA synthesis reverse transcription-PCR System (Invitrogen) according to the manufacturer's instructions. Primers (Invitrogen) for Ank, Enpp1, Tnap, and Gapdh were prepared as suggested by Foster et al. (31), and for Opn, as suggested by Ehara et al. (32). The sequences used are as follows: Ank,5'-GAACTATCTGCCGCAC-3' and 5'-AGGCGAGTAAACGCAA-3'; Enpp1,5'-CGCCACCGAGACTAAA-3' and 5'-AGGAATCATAGCGTCCG-3'; Tnap,5'-GGGGACATGCAGTATGAGTT-3' and 5'-GGCCTGGTAGTTGTTGTGAG-3'; Gapdh,5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'; and Opn,5'-CTGCTAGTACACAAGCAGACA-3' and 5'-CATGAGAAATTCGGAATTTCAG-3'.
PCR efficiency was optimized, and melting curve analysis of products were performed to ensure specificity. All of the primers were used at 0.5 µM except for those of Opn, which were used at 0.25 µM. PCR was carried out in an Applied Biosystems 7500 system using Power SYBR Green PCR Master Mix (Applied Biosystems). The sequential reaction conditions were 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Gene expression was quantified using the sequence detection software of the system (Applied Biosystems). Gapdh was used as the housekeeping gene for data normalization. Triplicate RNA samples from at least two separate experiments were used for these experiments.
|
Western Blotting—The cell/matrix layer was harvested in buffer containing 10 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 0.5 M EDTA (to release mineral-bound proteins) supplemented with protease inhibitor mixture (Sigma) containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin. Protein concentrations of cell lysates were determined by the BCA protein assay (Pierce), and 20 µg of total protein was separated by SDS-PAGE. The proteins were electrophoretically transferred to a nitrocellulose membrane (Pall Life Sciences). The membranes were blocked with 5% milk (from powder) in Tris-buffered saline/Tween 20 (TBS-T) and then probed with primary antibody in 5% bovine serum albumin (BSA) (Sigma) in TBS-T followed by the appropriate horseradish peroxidase-conjugated secondary antibody. The blots were visualized by chemiluminesence using ECL Plus (Amersham Biosciences). For densitometric analysis, the films were scanned and quantified with Quantity One software (Bio-Rad). Rabbit anti-Opn (LF-175) was kindly provided by Dr. Larry W. Fisher (National Institutes of Health, Bethesda, M). Anti-phospho-Erk1/2, phospho-p38, phospho-Jnk, pan-Erk1/2, pan-p38, pan-Jnk, and anti-rabbit horseradish peroxidase antibodies were purchased from Cell Signaling Technologies. Anti-Gapdh horseradish peroxidase was purchased from Abcam.
Immunofluorescence—The cells were cultured on 8-well chamber slides and then fixed in 3.7% formaldehyde. The cells were then first permeabilized with 0.25% Triton X-100 in PBS for 5 min or directly blocked with 2% BSA for 30 min. Actin cytoskeleton labeling was performed using phalloidin Alexa Fluor 564, and nuclei were observed using 4', 6-diamidino-2-phenylindole staining of DNA (Molecular Probes). Opn was immunolabeled using goat anti-mouse Opn from R & D Systems and chicken anti-goat Alexa Fluor 488 secondary antibody (Molecular Probes). The s1lides were mounted with Prolong Antifade (Molecular Probes).
Phosphorylated OPN and Dephosphorylation—Phosphorylated bovine milk OPN (Arla Foods, Denmark; prepared according to methods similar to those developed by E. S. Sørensen (33)) with 24 phosphorylations (of 28 potential sites) either was added directly to the cultures or was dephosphorylated at a concentration of 10 mg/ml in 50 mM Tris-HCl, pH 8.8, containing 10 mM MgCl2 by passing through an immobilized-alkaline phosphatase column (Mobitec) according to the manufacturer's protocol. Eluted protein was separated from the reaction mixture by precipitating with 12% trichloroacetic acid and washing with acetone. Dephosphorylation of OPN was confirmed using a phosphoprotein assay kit (Pierce).
Immunogold Labeling—Cells cultured in 35-mm dishes were fixed with 2% glutaraldehyde, dehydrated through a series of graded ethanol dilutions, and embedded in LR White acrylic resin (London Resin Company). Thin sections (80 nm) were then placed on Formvar-coated nickel grids. Post-embedding immunolabeling was performed on the grid-mounted sections as described previously (34) using goat anti-mouse Opn (R & D Systems) and protein A-colloidal gold complex (15 nm; Dr. G. Posthuma, University of Utrecht, Utrecht, The Netherlands). Briefly, the grids were incubated for 10 min with 1% BSA in PBS, followed by incubation with primary antibody for 1 h, blocking with 1% BSA in PBS, and then incubation with protein A-gold for 30 min. The grids were then rinsed with distilled water, air-dried, and stained conventionally for transmission electron microscopy using uranyl acetate and lead citrate. The images were recorded using a Philips FEI Tecnai 12 transmission electron microscope operating at 120 kV.
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Because PPi has previously been shown to bind to growing crystals (3), we next examined whether mineral-bound PPi (versus free PPi) could also be degraded by TNAP. PPi adsorbed onto synthetic hydroxyapatite crystals were incubated with TNAP, and Pi release was assayed biochemically. As shown in Fig. 1B, free (unadsorbed) PPi was hydrolyzed to release Pi, whereas mineral-bound PPi was resistant to TNAP hydrolysis and yielded only negligible amounts of Pi. Together, these results indicate that PPi inhibits mineralization of MC3T3-E1 osteoblast cultures, and this inhibition can be reversed by TNAP, but not if PPi has bound to mineral.
Given that PPi/Pi ratios are regulated by the activities of Tnap, Enpp1, and Ank, we examined the effect of the above PPi treatments (Fig. 1A) on their gene expression and activity. Quantitative real time PCR showed that PPi caused a slight increase in Tnap, which was significant only at the 2.5 µM dose (Fig. 1C). Elevation of Tnap levels was anticipated because PPi would be expected to up-regulate the enzyme responsible for its degradation in an attempt to normalize its concentration. Furthermore, any PPi remaining unadsorbed to mineral could be hydrolyzed to Pi, which would then stimulate Tnap expression as has been shown previously (35).
The same 2.5 µM treatment of PPi led to a decrease in Enpp1 expression (Fig. 1C), likely again reflecting an attempt by the cells to lower extracellular PPi. The addition of exogenous of TNAP resulted in increased Enpp1, presumably representing an attempt by the cells to compensate for the increased PPi degradation. These data are consistent with the increased Enpp1 activity observed in Tnap-transfected osteoblasts (36).
Unexpectedly, we found that PPi dose-dependently increased Ank expression. That Ank, a gene whose encoded transporter protein outwardly pumps PPi out into the extracellular matrix, would be up-regulated in response to increased extracellular PPi is surprising. Although the reason for this is not immediately obvious, we hypothesize that elevated Pi produced by TNAP hydrolysis of free, unadsorbed (to mineral) PPi could be responsible because Pi has been shown to elevate Ank expression in cementoblasts (31). Furthermore, the structure and function of Ank is not entirely clear (20, 37), and Wang et al. (21) have demonstrated that blocking Ank expression led to increases in both intra- and extracellular PPi levels, whereas Ank overexpression led to decreases in both intra- and extracellular PPi levels, suggesting a level of complexity in PPi handling and homeostasis that needs to be better clarified. Increased Ank expression as a result of increased exogenous PPi could simply be an attempt by the cells to return extracellular PPi levels to normal by as yet an unknown mechanism.
|
Pyrophosphate Up-regulates Opn Expression—Recent data have shown that mineralizing cell culture systems (primarily osteoblasts and cementoblasts) are sensitive to local levels of Pi and PPi (22, 31, 35, 38). The role of anions such as PPi in skeletal and dental tissues appears not to be limited to their crystal binding properties. High doses of Pi (4-10 mM) have been shown to regulate levels of the crystal growth inhibitor protein Opn (39). To determine whether PPi could elicit a similar response, we examined levels of Opn by enzyme-linked immunosorbent assay and Western blotting in PPi-treated osteoblast cultures. 0.5 mM PPi treatment for 4 days led to a marked increase in total Opn levels (Fig. 2A). Most of the Opn was found in the cell/matrix layer, and Opn in the conditioned medium was also slightly (but not significantly) increased by the PPi treatment (Fig. 2A).
To determine whether the effect of PPi was reflected at the RNA level, we examined the PPi-treated cultures by real time PCR and observed that Opn mRNA was slightly, but significantly, up-regulated (Fig. 2B). Using Western blotting to further examine the up-regulation of Opn, we observed that PPi treatment led to a gradual cumulative increase in Opn levels over 4 days of PPi treatment (Fig. 2C).
We next examined whether this up-regulation of Opn was in fact directly attributable to PPi and not to its hydrolysis end product, Pi.PPi treatment of MC3T3-E1 cultures in the presence of a specific Tnap inhibitor, levamisole, did not prevent induction of Opn, suggesting that PPi regulates Opn levels by a hydrolysis-independent mechanism (Fig. 2D). Furthermore, treatment of cultures with an equivalent dose of 0.5 mM Pi failed to increase Opn levels. This is consistent with previous work by Beck et al. (39) showing that Opn up-regulation by Pi in the MC3T3-E1 cell line requires a minimum dose of 4 mM and is maximal at 10 mM.
To further confirm that PPi and Pi regulate Opn by different mechanisms, we then treated MC3T3-E1 cultures with 0.5 mM PPi and with 10 mM Pi in the presence of a phosphate uptake inhibitor, foscarnet. 10 mM Pi was used here as a positive control for the phosphate transport-dependent (foscarnet-sensitive) system. Both Western blotting and immunofluorescence microscopy confirmed that whereas Pi-mediated up-regulation of Opn was sensitive to foscarnet, PPi-mediated induction of Opn was not (Fig. 3, A and B). Foscarnet failed to block Opn induction by PPi, and although this inhibitor caused a slight attenuation of the Opn signal in PPi-treated cultures, this was negligible as compared with its effects on the Pi-treated cultures where it completely abolished the strong Opn up-regulation by Pi. Localization of Opn to the extracellular matrix in the PPi-treated cultures is shown in Fig. 3C. Taken together, these results suggest that up-regulation of Opn by PPi is dependent on neither PPi hydrolysis nor Pi uptake.
Pyrophosphate Regulates Opn via MAPK Signaling Pathways—Osteoblast-specific gene expression has previously been shown to be regulated by the MAPK pathways (40-42). To elucidate the mechanism by which PPi regulates Opn expression, we next examined the involvement of major cell signaling pathways using specific inhibitors. MC3T3-E1 cells were treated for 4 days with 0.5 mM PPi in the presence of the selective MAPK inhibitors U0126 (Erk1/2 inhibitor), SB202190 (p38 inhibitor), or SP600125 (JNK inhibitor). Inhibition of Erk1/2 and p38 prevented induction of Opn by PPi (Fig. 4A), but Jnk inhibition had no significant effect. We then tested the ability of PPi to activate Erk1/2 and p38. Western blot analysis of Erk1/2 and p38 followed by densitometric analysis of replicate blots from three separate experiments revealed that significant activation of Erk1/2 occurred within 5 min of PPi treatment, whereas p38 activation occurred at 60 min (Fig. 4, B and C) and continued thereafter (data not shown).
|
|
|
GP and OPN—GP is a monophosphoester commonly used as an organic source of phosphate in cell cultures (43, 44) whose cleavage by TNAP yields free Pi for apatite crystal growth during mineralization (26). Mineralization of MC3T3-E1 cells can therefore be initiated with either GP or Pi, with the main difference being that GP-induced mineralization is Tnap-dependent. Although PPi inhibited mineralization in both culture systems, the dose of PPi required was differed. Whereas 5 µM PPi was sufficient to inhibit GP-induced mineralization, surprisingly, 50 µM PPi was required to block Pi-induced mineralization (Fig. 6, A and B). This 10-fold difference in potency suggests that PPi might be interfering with Tnap-mediated Pi release from GP in addition to blocking mineral growth by binding to crystal surfaces. We thus monitored gene expression levels to determine whether cultures induced by both phosphate sources behaved in a similar manner when exposed to PPi (Fig. 6C). Under both conditions, the effect of PPi was more or less analogous, with PPi causing a slight elevation of Tnap, Opn, and Ank. Enpp1 expression decreased at low doses of PPi and increased at the higher doses. This unexpected increase in Enpp1, as well as the increase in Ank, might be attributable to the Pi released from the hydrolysis of PPi that is not bound to mineral. Regardless of the source of added phosphate for mineralization, there were no major differences in gene expression between the Pi- and GP-treated cultures. The changes in Tnap and Enpp1 expression were reflected at the protein level in phosphatase and nucleotide pyrophosphatase phosphodiesterase enzyme activity assays (Fig. 6D). Although these assays reflect TNAP and Enpp1 protein levels, actual in vitro activity levels may differ because cell lysates were necessarily assayed here in the absence of culture medium, extracellular matrix, and endogenous inhibitors, which could affect activity in situ.
|
GP-induced cultures, causing decreased mineralization of the cultures, we examined the direct effect of PPi on the GPase activity of Tnap in vitro. Cell lysates from mineralized MC3T3-E1 cultures contain high Tnap activity. Incubation of these extracts with GP results in release of free Pi as measured colorimetrically (Fig. 7A). The addition of PPi to the cell extract/GP reaction mixture led to an inhibition of GP hydrolysis and Pi release (Fig. 7A). To confirm that PPi was having a direct effect on the enzymatic activity of Tnap, we performed the above experiment using purified TNAP in place of the cell lysate. As is shown in Fig. 7B, TNAP independently hydrolyzes both PPi and GP, but when the two substrates were combined, there was a reduction in Pi release from GP (see also below, Fig. 8). These results therefore suggest that inhibition of MC3T3-E1 mineralization by PPi also involves altered Tnap-mediated hydrolysis of GP.
The ability of PPi to inhibit Tnap activity may provide some insight into why Tnap present in fully differentiated osteoblasts does not enzymatically cleave the phosphates from Opn, which would in turn decrease binding of Opn to mineral. To test the hypothesis that phosphorylated Opn is at least partly protected by PPi from the phosphatase actions of TNAP, we examined whether PPi could inhibit TNAP hydrolysis of bovine OPN. As was demonstrated for GP in Fig. 7 (A and B), hydrolysis of OPN phosphate by TNAP was significantly reduced in the presence of PPi (Fig. 7C). To avoid the potential for substrate end product inhibition, all of the experiments were performed in the linear range of enzymatic activity as determined by our measuring of the enzyme kinetics (data not shown).
|
GP hydrolysis at various PPi concentrations was determined. Both the slope and intercepts increase with increasing inhibitor concentration, and thus the curves intersected to the left of the y axis and above the x axis indicate a mixed type of inhibition (Fig. 8A). The changes in both apparent Km and Vmax imply that PPi may bind to both free enzyme and to the enzyme-substrate complex.
To calculate the dissociation constant (Ki) of the PPi-TNAP complex, a Dixon plot of reciprocal velocities against PPi concentration was constructed (Fig. 8B), resulting in a Ki of 0.73 mM. The dissociation constant of the PPi-TNAP-GP complex (Ki') was calculated from a plot of s/v against PPi concentration (Fig. 8C). The intersection of the curves results in a Ki' of 1.97 mM. Kinetic parameters obtained from the plots are summarized in Fig. 8D. In summary, a molecule of PPi may bind to either the TNAP active site (competitive inhibition) or to the TNAP-GP complex at a secondary site that is only available after GP binding (noncompetitive inhibition).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Consistent with inhibition of crystal growth by PPi, in this study, we show that PPi inhibits mineralization of extracellular matrix in vitro produced by a well characterized osteoblast cell line, the MC3T3-E1 subclone M14 (45). We also show that this inhibition of mineralization by PPi is abrogated by the actions of tissue-nonspecific TNAP, which hydrolyzes PPi into its constituent Pi ions. The importance of PPi as a physiologic inhibitor of mineralization is evident from the skeletal and dental hypomineralization observed in patients with hypophosphatasia attributable to impaired TNAP activity. These patients, as well as Akp2-/- knock-out mice, demonstrate elevated plasma PPi levels in the absence of TNAP enzymatic activity (6, 13). Indeed, the bone and tooth hypomineralization of Akp2-/- mice is normalized in Akp2-/-;Enpp1-/- double-knock-out mice (36, 46), suggesting that homeostatic regulation of PPi levels by antagonistic Tnap enzyme activity modulates hydroxyapatite formation in these tissues. However, our results also indicate that there is rapid adsorption of PPi onto hydroxyapatite crystals and that once adsorbed, it is resistant to hydrolysis by TNAP (Fig. 1B). Furthermore, the dose of PPi required to inhibit GP-induced mineralization is only 5 µM, and thus any hydrolysis would yield a negligible amount of Pi relative to the 10 mM amount of GP used here and commonly by others to provide a source of Pi for mineralization. The dose of PPi that we have used is in the physiologic range as reported for human plasma (3.5 µM average PPi) by Russell et al. (6).
Our results also demonstrate that PPi is a specific signal for the induction of Opn expression in osteoblasts. Previous studies have demonstrated that PPi deficiency in Enpp1-/- mice also results in Opn deficiency (11). In our study, we have identified the involvement of specific MAPK signaling pathways responsible for Opn regulation by PPi. Recently, others have shown that Pi itself can regulate osteoblast and cementoblast gene expression (31, 35, 38, 39). However, whereas Pi regulation of Opn is dependent on intracellular uptake via Na+/Pi co-transporters (39), most notably Pit-1 (47), we demonstrate here that PPi directly regulates Opn in a phosphate uptake-independent manner. Moreover, whereas previous studies by Beck et al. (39), have shown that Pi regulation of Opn is not dependent on the p38 MAPK pathway in osteoblasts (48), we report here that this pathway is activated by PPi to regulate Opn expression. It should be noted however, that although foscarnet is widely used as an inhibitor of Pi uptake, there are indeed other mechanisms for Pi transport that may be not be foscarnet-sensitive. Nevertheless, because the inhibitor levamisole prevents Tnap-mediated PPi hydrolysis to Pi, and this treatment had no effect on the induction of Opn, then these data, plus the failure of an equivalent dose of Pi to induce Opn, collectively indicate that PPi regulates Opn by a mechanism distinct from that of Pi.
The addition of exogenous PPi resulted in an increase in Opn mRNA, which led to a gradual cumulative increase in Opn protein levels over 4 days of treatment, with 4 days representing roughly one-third of the 12 days of culture typically used to assay for full MC3T3-E1osteoblast differentiation and mineralization of the extracellular matrix. Indeed, induction of Tnap activity during differentiation by the addition of ascorbic acid and GP typically takes up to 6 days in control cultures. Compared with this 6-day induction time for Tnap, Opn induction at 4 days is consistent with this same time frame. Furthermore this might explain why a relatively high dose of PPi (0.5 mM) might be needed to induce Opn over 4 days compared with the low dose (5 µM) required to inhibit mineralization after 12 days. The 0.5 mM concentration of PPi required to induce Opn is far less than the Pi concentration required (4-10 mM) to have a similar induction (39).
The mechanism by which extracellular PPi leads to activation of intracellular signaling pathways remains unknown. Based on its size and charge, PPi likely cannot passively cross the cell membrane. In prokaryotes, chloroplasts, and mitochondria, an ATP-ADP translocator has been reported to import PPi (49-51), but there is no known importer or extracellular receptor for PPi in mammalian cells (10). The possibility that PPi acts through a plasma membrane receptor is not unreasonable. Other reported examples of small molecule signaling through osteoblast receptors include the ion-sensing Ca2+-sensing receptor (52), the nucleotide P2-purinoreceptor family (53), and PPi analogues (bisphosphonates) acting to open connexin 43 hemi-channels (54), all of which in turn activate intracellular signaling pathways. Work is ongoing in our laboratory to clarify upstream effectors of the signaling pathways involved in PPi action as well as to identify transcription factors and genes regulated by PPi.
Based on the known inhibitory actions of Opn on hydroxyapatite crystal growth in cell-free systems (55-57), in cell cultures models (this study Fig. 5A, and Wada et al. (58)), and in vivo in mineralized tissues (59), the up-regulation of Opn by PPi would likely act to further inhibit and thus regulate mineralization. Recent evidence for this in vivo is demonstrated by the elevated Opn levels in Akp2-/- mice that have elevated PPi in the absence of Akp2 enzymatic activity and by the fact that in Akp2-/-;Opn-/- double-knock-out mice, there is partial rescue of the bone hypomineralization phenotype otherwise observed in the Akp2-/- knock-out alone (60). We also propose here, as an alternative (or simultaneous) activity for this elevated Opn when PPi levels are high, that increased Opn might prevent calcium pyrophosphate crystal deposition in fluids and tissues as is commonly seen in joints in pseudogout (61). Studies are currently in progress to assess the ability of Opn to inhibit calcium pyrophosphate crystal growth and to stabilize mineral ions and putative amorphous precursor phases in solution.
Phosphorylation of Opn appears to be ordered in triplet clusters that favor its interaction with calcium in the hydroxyapatite crystal lattice (62, 63). Opn binds directly to hydroxyapatite crystal surfaces both in vivo (64) and in vitro (34), and dephosphorylation of Opn by Tnap prevents much of its mineral binding and crystal growth inhibiting activity (56, 65, 66) (Fig. 5A). Our high resolution, immunogold localization of Opn at crystal growth sites in the extracellular matrix of MC3T3-E1 osteoblast cultures is consistent with previous data demonstrating a similar, electron microscopic localization of Opn in primary rat osteoblast cultures (34). Dose-dependent inhibition of calcification in vitro by the addition of exogenous Opn has been shown previously for vascular smooth muscle cell cultures (58). Our study in osteoblast cultures confirms this finding and further demonstrates that Tnap modulation of Opn phosphorylation levels is important for inhibition of mineralization. Using a gelatin gel crystal growth system, Gericke et al. (67) likewise demonstrated the importance of phosphorylation status of Opn in inhibiting hydroxyapatite formation, and similar phosphorylation influences were observed for inhibition of hydroxyapatite and also calcium oxalate crystal growth by peptides of Opn (55, 57).
Although differentially phosphorylated forms of Opn have been observed in rat bone cell cultures (68), the amount of Opn dephosphorylation resulting from Tnap activity in vivo remains unknown. Indirect in vivo data supporting a link between Tnap activity and Opn and its phosphorylation status is evidenced by the altered distribution of Opn in bone from Akp2-/- mice (69), although direct measurement of phosphorylation level of Opn in this case has not been determined.
Another finding in our study is that the inhibition of mineralization by PPi that is abrogated by the actions of Tnap can be reduced when another Tnap substrate is present. In vitro, this second substrate is GP, but in vivo, other substrates would be present including mineral-inhibiting phosphoproteins such as Opn. Mineralization of osteoblast cultures can be initiated in vitro with a variety of phosphate esters with GP being the organic phosphate source most commonly used (26, 70-72). Although high doses of Pi have also been demonstrated to inhibit Tnap activity (73), our kinetic experiments clearly demonstrate that PPi may function as a mixed type of inhibition system. It is possible that with substrate (GP, Opn) already occupying the active site in Tnap, PPi might bind to a second site on the enzyme, causing conformational change that inhibits its activity. This possibility has previously been suggested for the Tnap activity associated with matrix vesicle-mediated mineralization (72) and is also supported by recent data demonstrating that PPi induces conformational changes in Escherichia coli alkaline phosphatase leading to inhibition of its enzymatic activity (74). Our data showing that PPi acts as an inhibitor of TNAP when another substrate such as GP (in vitro) or Opn (or other phosphoproteins, in vivo) is present also provides a hypothesis to explain why osteoblast Tnap does not inactivate the mineral-inhibiting potential of endogenous Opn in the extracellular matrix (Fig. 7). Recent evidence (75) that PPi also inhibits PHEX activity (an enzyme whose inactivating mutations cause X-linked hypophosphatasia in humans) implicates multiple additional factors affecting the Pi/PPi balance regulating mineralization, and more work in this area is required to elucidate their respective contributions.
In summary, the in vitro data presented here for PPi, Tnap, and Opn, together with our recent in vivo data in transgenic mice overexpressing Tnap in collagen extracellular matrices (18), suggest the following partial scenario for induction and regulation of extracellular matrix mineralization. Mineralization initially involves a collagen matrix and enzymatic removal of PPi. The enzymatic hydrolysis of PPi by Tnap also produces additional Pi potentially available for hydroxyapatite mineral deposition. Once hydroxyapatite crystal growth is initiated by an as yet unknown mechanism, the enzymatically (Tnap) maintained Pi/PPi balance regulates further mineralization in part by inhibiting crystal growth via multiple mechanisms that include direct binding to crystals, up-regulation of mineral-inhibiting Opn, and inhibition of Tnap enzymatic activity. Thus, mineralization is not only inhibited by enzymatic inactivation by Tnap of these two molecular determinants of mineralization, one a protein and the other a small anion inhibitor, but is further enhanced by the fact that PPi also up-regulates Opn. Both inhibitors are found at high levels in bone, both bind to mineral via negatively charged phosphate residues, and both are inactivated by Tnap. Inhibition of Tnap by PPi (in the presence of other Tnap substrates, e.g. Opn and potentially other matrix phosphoproteins) also then maintains the phosphorylated form of Opn to further regulate mineral growth. Given that skeletal and dental mineralization occurs to at least some extent in transgenic mice deficient in these and other mineral-regulating proteins, it is likely that other molecular determinants also are important in regulating crystal nucleation and growth within the extracellular matrix.
|
FOOTNOTES |
|---|
1 Supported by a graduate student award from the Canadian Institutes of Health Research Skeletal Health Training program.
2 Supported by scholarship awards from the Fonds de la Recherche en Santé du Québec. Members of the McGill Centre for Bone and Periodontal Research, the McGill Centre for Biorecognition and Biosensors, the McGill Institute for Advanced Materials, and the Fonds de la Recherche en Santé du Québec Network for Oral and Bone Health Research.
3 Member of the Canadian Arthritis Network. To whom correspondence should be addressed: Faculty of Dentistry, McGill University, 3640 University St., Montreal, Quebec H3A 2B2, Canada. Tel.: 514-398-7203, Ext. 00041; Fax: 514-398-8900; E-mail: marc.mckee{at}mcgill.ca.
4 The abbreviations used are: GP, -glycerophosphate; BSA, bovine serum albumin; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
), 0.5 mM (
), 1 mM (
), 2.5 mM (
), and 5 mM (
). B, Dixon plot of 1/v against [PPi]mM at the following fixed concentrations of