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Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, PL-02093 Warsaw, PolandUniversité de Lyon, Lyon, F-69003, FranceUniversité Lyon 1, Villeurbanne, F-69622, FranceINSA-Lyon, Villeurbanne, F-69622, FranceCPE Lyon, Villeurbanne, F-69616, FranceICBMS CNRS UMR 5246, Villeurbanne, F-69622, France
Address correspondence and reprint requests to: Dr René Buchet, Ph.D., Université Lyon 1, Bâtiment Chevreul, 43 Boulevard du 11 Novembre 1918, F-69 622 Villeurbanne Cedex, France. Tel: 33-4-72-43-13-20; Fax: 33-4-72-43-15-43.
Pathological mineralization is induced by unbalance between pro- and anti-mineralization factors. In calcifying osteoarthritic joints, articular chondrocytes undergo terminal differentiation similar to that in growth plate cartilage and release matrix vesicles (MVs) responsible for hydroxyapatite (HA) or calcium pyrophosphate dihydrate (CPPD) deposition. Inorganic pyrophosphate (PPi) is a likely source of inorganic phosphate (Pi) to sustain HA formation when hydrolyzed but also a potent inhibitor preventing apatite mineral deposition and growth. Moreover, an excess of PPi can lead to CPPD formation, a marker of pathological calcification in osteoarthritic joints. It was suggested that the Pi/PPi ratio during biomineralization is a turning point between physiological and pathological mineralization. The aim of this work was to determine the conditions favoring either HA or CPPD formation initiated by MVs.
Methods
MVs were isolated from 17-day-old chicken embryo growth plate cartilages and subjected to mineralization in the presence of various Pi/PPi ratios. The mineralization kinetics and the chemical composition of minerals were determined, respectively, by light scattering and infrared spectroscopy.
Results
The formation of HA is optimal when the Pi/PPi molar ratio is above 140, but is completely inhibited when the ratio decreases below 70. The retardation of any mineral formation is maximal at Pi/PPi ratio around 30. CPPD is exclusively produced by MVs when the ratio is below 6, but it is inhibited for the ratio exceeding 25.
Conclusions
Our findings are consistent with the Pi/PPi ratio being a determinant factor leading to pathological mineralization or its inhibition.
. In the prenatal and early postnatal life, biomineralization is the last essential event in the endochondral and intramembranous bone formation leading to the replacement of cartilaginous skeleton and craniofacial fibrous tissue by the definitive bone skeleton. Throughout life, the mineralization process continues to play a crucial role in bone remodeling and repair. The regulation of physiological mineralization is mediated at molecular, cellular and tissue levels
, can occur during aging, degenerative joint diseases, or genetic and various metabolic disorders. This causes an excessive mineral deposition in articular cartilages
that leads to joint inflammation and the progression of osteoarthritis. Several calcific diseases are characterized by the deposit of calcium pyrophosphate dihydrate (CPPD) or of hydroxyapatite (HA) in degenerative joints
During endochondral ossification, chondrocytes undergo a series of differentiation: cell proliferation, hypertrophy, terminal differentiation and cell apoptosis
. These chondrocytes do not proliferate and produce extracellular matrix components such as chondroitin-4-sulfate, chondroitin-6-sulfate, keratansulfate, as well as types II, III, VI, IX and XI collagen
in degenerative joints. MVs from osteoarthritic cartilage own similar protein machinery than MVs from growth plate cartilage, necessary for Ca2+ uptakes into MV lumen: annexin A2 (AnxA2), AnxA5 and AnxA6
Up-regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC-1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification.
. In addition to these proteins, osteoarthritis articular chondrocytes express type X collagen (a marker of hypertrophic chondrocytes), osteonectin, bone morphogenetic proteins (which induce new bone formation) and RUNX2 (a transcription factor regulating hypertrophic chondrocyte differentiation)
Sustained osteomalacia of long bones despite major improvement in other hypophosphatasia-related mineral deficits in tissue nonspecific alkaline phosphatase/nucleotide pyrophosphatase phosphodiesterase 1 double-deficient mice.
on mineral formation due to their opposing activities: production of Inorganic pyrophosphate (PPi) by NPP1 or its hydrolysis by TNAP. TNAP provides Pi from various phosphate substrates during mineralization
, supplies PPi from adenosine triphosphate (ATP) or uridine triphosphate (UTP) hydrolysis. At low concentrations, PPi prevents the seeding of calcium phosphate minerals
Comparison of characteristics of patients with and without calcium pyrophosphate dihydrate crystal deposition disease who underwent total knee replacement surgery for osteoarthritis.
Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders.
, and NPP1, are overexpressed in chondrocytes of osteoarthritic articular cartilage, contributing to increase PPi, where CPPD crystal formation could occur
Up-regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC-1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification.
Upregulated ank expression in osteoarthritis can promote both chondrocyte MMP-13 expression and calcification via chondrocyte extracellular PPi excess.
Up-regulated expression of cartilage intermediate-layer protein and ANK in articular hyaline cartilage from patients with calcium pyrophosphate dihydrate crystal deposition disease.
. Since osteoarthritic MVs and growth plate MVs exhibit similar structural and functional properties, we selected MVs isolated from chick embryo growth plate cartilage to determine the effect of the Pi/PPi ratio on HA and CPPD depositions.
Materials and methods
Purification of MVs
MVs were isolated from growth plate and epiphyseal cartilage slices of 17-day-old chicken embryos by collagenase digestion
. Seventeen-day-old chicken embryo leg bones were cut into 1–3-mm thick slices and washed five times in a synthetic cartilage lymph (SCL) containing 100 mM NaCl, 12.7 mM KCl, 0.57 mM MgCl2, 1.83 mM NaHCO3, 0.57 mM Na2SO4, 1.42 mM NaH2PO4, 5.55 mM d-glucose, 63.5 mM sucrose and 16.5 mM N-tris(hydroxymethyl)methyl 2-aminoethane sulfonic acid (TES) (pH 7.4). Growth plate and epiphyseal cartilage slices were digested at 37°C for 3.5–4 h in the SCL buffer with 1 mM Ca2+ and collagenase (500 units/g of tissue, type IA, Sigma). It was vortexed and filtered through a nylon membrane. The suspension was centrifuged at 600× g for 10 min to pellet intact hypertrophic chondrocytes. The supernatant was centrifuged at 13,000× g for 20 min. The pellet was discarded and the supernatant was submitted to a third centrifugation at 70,000× g for 1 h. The final pellet containing MVs was suspended in 300 μL of SCL buffer and stored at 4°C. The protein concentration in the MV fraction was determined using the Bradford assay kit (Bio-Rad). Proteins of MVs were separated in 7.5 or 10% (w/v) sodium dodecyl sulfate (SDS)-polyacrylamide gels
. The gels were stained with Coomassie Brilliant Blue R-250.
Transmission electron microscopy
A 20 μL aliquot of MV fraction was transferred to carbon-coated grids. The grids were negatively stained with 2% uranyl acetate and dried. The grids were viewed with an electron microscope Philips CM140 at 80 kV accelerating voltage.
Treatment of MVs by phosphatidylinositol specific phospholipase C
MVs (1 μg of MV proteins/μL) were incubated in SCL with 10 mM Mg2+, 5 μM Zn2+ and 1 unit of phosphatidylinositol specific phospholipase C (PI-PLC) per mL for 7 h at 37°C under gentle agitation. The supernatant of MVs (sMVs) treated by PI-PLC containing MV glycosylphosphatidylinositol (GPI)-anchored proteins and the pellet (pMV) were separated by centrifugation at 90,000× g for 30 min. The pellet of MVs treated by PI-PLC (pMV) was resuspended in the same volume of SCL as before the centrifugation.
Immunodetection of chicken caveolin-1
Proteins of MVs were separated in 12% (w/v) SDS-polyacrylamide gels
. The nitrocellulose membranes were blocked with 5% (w/v) milk in a buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl) for 1 h at room temperature, then incubated with 3% (w/v) milk and 0.1% (v/v) mouse monoclonal IgG against chicken caveolin-1 (BD Biosciences) in tween 20 tris buffered saline (TTBS) buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween 20) at 4°C overnight. The nitrocellulose membranes were washed several times with TTBS and incubated with 3% (w/v) milk and 0.05% (v/v) goat anti-mouse IgG conjugated with alkaline phosphatase (Immuno-Blot Assay Kit, Bio-Rad) in TTBS buffer. The membranes were washed, and bands were visualized by addition of color-developing solution according to the manufacturer's instructions.
Specific revelation of alkaline phosphatase
MV proteins were incubated under mild denaturing conditions (without heating before the gel migration) in the Tris buffer containing 2% SDS but no β-mercaptoethanol to preserve the TNAP activity. After the migration, SDS-polyacrylamide gels were incubated in a solution containing 0.1 M Tris–HCl (pH 9.6), 0.1 M NaCl, 5 mM MgCl2, 0.24 mM bromo-chloro-indolyl phosphate (BCIP), a TNAP substrate and 0.25 mM nitroblue tetrazolium (NBT) until the blue band associated to alkaline phosphatase was visible
, in 25 mM piperazine and 25 mM glycylglycine buffer, by monitoring the release of p-nitrophenolate at 420 nm (ɛ=9.2 cm−1mM−1 at pH 7.4; ɛ=18.5 cm−1mM−1 at pH 10.4, M−1). One unit of PME activity corresponds to the amount of enzyme hydrolyzing 1 μmol of p-NPP per minute at 37°C. The phosphodiesterase (PDE) activity of MVs was measured at pH 7.4 or at 9, with 2 mM bis-p-nitrophenyl phosphate (bis-p-NPP) as substrate in 25 mM piperazine and 25 mM glycylglycine buffer, and monitoring the release of p-nitrophenolate at 420 nm (ɛ=9.2 cm−1mM−1 at pH 7.4; ɛ=17.8 cm−1mM−1 at pH 9)
. One unit of PDE activity corresponds to the amount of enzymes hydrolyzing 1 μmol of bis-p-NPP per minute at 37°C. To determine the pyrophosphatase activity, MVs were incubated in 25 mM piperazine and 25 mM glycylglycine buffer (at the indicated pH) containing 0.25–2 mM PPi, for 20 min at 37°C. The reaction was stopped by adding 10 mM levamisole and stored at 4°C. Aliquots of the reaction mixture were collected to determine PPi concentrations with the Sigma reagent kit. One unit of pyrophosphatase activity corresponds to the amount of enzymes hydrolyzing 1 μmol of PPi per minute at 37°C.
Determination of mineralization kinetics
Aliquots of the MV stock solution were diluted to a final concentration of 20 μg of MV proteins/mL in the SCL buffer containing 2 mM Ca2+ and different concentrations of ions (Pi, PPi) or phosphate substrates [adenosine monophosphate (AMP), ATP], as indicated in the figure legends. They were incubated at 37°C and their absorbances at 340 nm were measured at 15-min intervals with Uvikon spectrophotometer model 932 (Kontron Instruments). When MVs were incubated in SCL containing 2 mM Ca2+ but not Pi, PPi and other phosphate substrates, there were no changes in turbidity. Thus, the increase in turbidity was due to mineral formation
Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens.
Identification of minerals by infrared spectroscopy
The minerals were determined by infrared spectroscopy (Nicolet 510M FTIR spectrometer). They were centrifuged at 3,000× g for 10 min and washed several times with water. They were dried and incorporated by pressing into 100 mg of KBr. Standard CPPD was prepared by incubating stoichiometric proportions of Ca2+ and PPi at 37°C for 2 weeks. Standard HA was purchased from Sigma.
Results
Biochemical characterization of MVs
The MVs extracted from chicken embryo growth plate cartilages were round structures with a diameter ranging from 100 to 250 nm (Fig. 1), in agreement with Anderson et al.
. Caveolin-1, a marker of caveolae, present in the plasma membrane of hypertrophic chondrocytes [26 kDa, Fig. 2(B), lane 2], was absent in MVs [Fig. 2(B), lane 1], indicating that isolated MVs are not contaminated by the fragments of plasma membrane. TNAP, a marker enzyme of MVs
, was enriched in the MV fractions. The PME activity associated with TNAP of MVs at pH 10.4 was 25.0±3.4 units/mg of proteins, approximately five times higher in comparison to hypertrophic chondrocytes (Table I), indicating a high degree of purity of MV preparations.
Fig. 1Electron microscope view of MVs. MVs exhibit spherical shapes with a 100–250 nm diameter (magnifications: A, ×53,000; B, ×100,000; C, ×75,000; D, ×100,000).
Fig. 2(A) Protein pattern of MVs in a 10% SDS-polyacrylamide gel. Lane 1, MVs. (B) Western-Blot of MVs and hypertrophic chondrocytes for the detection of caveolin-1. Lane 1, MVs; Lane 2, co-isolated chondrocytes.
Table IPreparation of MVs from femoral and tibial growth plate cartilages of 17-day-old chick embryos. Growth plate cartilages were digested by collagenase. Hypertrophic chondrocytes were obtained by a centrifugation at 600× g for 10 min, the second pellet by a centrifugation at 13,000× g for 20 min and MVs by a last centrifugation at 70,000× g for 60 min. PME activity is expressed as μmol of p-NPP hydrolyzed per minute, per mg of MV proteins at pH 10.4
To delineate the importance of TNAP in PME and pyrophosphatase activities of MVs, the enzyme was digested out from MVs by a cleavage of its GPI anchor with PI-PLC. GPI-anchored TNAP in untreated MVs exhibited an apparent molecular weight of 118 kDa (Fig. 3, lane 1). After centrifugation, TNAP without GPI anchor was detected in the supernatant (sMV, Fig. 3, lane 2), but not in the pellet containing MVs devoid of GPI-anchored proteins (pMV, Fig. 3, lane 3). The specific PME activity of MVs amounted to 0.62±0.10 units/mg of MV proteins at physiological pH, e.g., 40 times lower than at pH 10.4 (Table II). The percentage of total PME activity of the sMV was 92±3.7% at pH 10.4 and 91±3% at pH 7.4 (Table II), indicating that more than 91% of PME activity is associated with TNAP in MVs. The PDE activity of MVs reflecting both TNAP
was 2.66±0.30 units/mg of MV proteins at optimal conditions (pH 9). It was 0.52±0.08 units/mg of MV proteins at pH 7.4. When the substrate concentration was reduced from 2 to 0.5 mM, the optimal pH for the activity shifted from 8.8 to 8.2 (Fig. 4). In the presence of 2 mM PPi, the pyrophosphatase activity of MVs was 3.70±0.31 units/mg of MV proteins at pH 8.8, and 1.00±0.08 units/mg of MV proteins at physiological pH (Table II). The apparent Km of PPi hydrolysis at physiological pH was identical for MVs, sMV and pMV, and amounted to 355±6 μM. The pyrophosphatase activity of all these samples was also inhibited in the same competitive manner by Pi; Ki amounted to 3.63±0.14 mM. Over 96±5.2% of the pyrophosphatase activity was attributed to the sMV at pH 7.4 (Table II), indicating that the ability of MVs to hydrolyze PPi was due to TNAP.
Fig. 3BCIP–NBT visualization of TNAP in a 7.5% SDS-polyacrylamide gel. Lane 1, MVs; Lane 2, sMV (supernatant of MVs treated by PI-PLC); Lane 3, pMV.
Table IIHydrolysis of p-NPP and PPi by MVs, the supernatant fraction (sMV) and the pellet fraction (pMV) of MVs treated by PI-PLC. PME activity was measured by hydrolysis of p-NPP at pH 10.4 (PME10.4) and at pH 7.4 (PME7.4). Pyrophosphatase activity was determined by hydrolysis of PPi at pH 8.8 (PPi8.8) and at pH 7.4 (PPi7.4). The activities of MVs are expressed as μmol of substrate hydrolyzed per minute, per mg of MV proteins, under described conditions. The activities of sMV and pMV are expressed as percentages of total MV activities
Fig. 4The effect of pH on the PPi hydrolysis by MVs. The pyrophosphatase activity of MVs was measured at different pH from 7 to 11, in the presence of different concentrations of PPi: (■) 2 mM, (●) 1 mM, and (▴) 0.5 mM.
The isolated MVs, incubated in the SCL buffer with 2 mM Ca2+ were able to induce mineral formation, after a short lag period of 3.5–4 h, corresponding to the time of accumulation of Ca2+ and Pi within MVs
Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens.
. Then, the mineral formation increased rapidly and reached saturation (Fig. 5). MVs in SCL medium without Ca2+ were not able to mineralize, indicating that the presence of 0.57 mM Mg2+ in SCL medium containing MVs cannot induce mineral formation. No mineral was formed in the SCL buffer with 2 mM Ca2+ devoid of MVs, indicating that MVs are essential to initiate mineralization.
Fig. 5Kinetics of mineral formation by MVs. MVs were incubated at 37°C in SCL buffer containing 2 mM Ca2+ and 1.42 mM Pi with additional Pi or phosphate substrates, as follows: (▾) without additional substrates, (▶) total Pi=2.42 mM, (▴) total Pi=3.42 mM, (□) 1 mM AMP, and (♢) 0.33 ATP. Mineral formation was assessed by light scattering at 340 nm.
The MV-induced mineral was identified by infrared spectroscopy. The infrared spectrum of mineral formed by MVs in SCL buffer exhibited five peaks at 1090 cm−1, 1030–1034 cm−1, 960–961 cm−1, 600–602 cm−1 and 561–562 cm−1 (Fig. 6, spectrum: SCL), corresponding to the peaks of HA (Fig. 7, spectrum: HA)
Fourier transform infrared characterization of mineral phases formed during induction of mineralization by collagenase-released matrix vesicles in vitro.
, indicating the ability of MVs to produce HA. Addition of 1 mM or 2 mM Pi (corresponding, respectively, to a total Pi concentration of 2.42 mM or 3.42 mM in SCL) into the mineralization medium reduced the lag period of mineral formation induced by MVs from 3.5–4 h to 1.5–2 h or to 0.5 h, respectively (Fig. 5). In both cases, the minerals formed by MVs were identified as crystalline HA (Fig. 6, spectrum: Pi). Addition of 1 mM AMP reduced the induction phase from 3.5–4 h to 2.5–3 h (Fig. 5), i.e., to a lower extent as compared with the addition of 1 mM Pi, due to the time required for hydrolysis of AMP by TNAP. The mineral formed was also HA (Fig. 6, spectrum: AMP). However, addition of 0.33 mM ATP, increased the time delay of mineral formation from 3.5–4 h to 18–20 h (Fig. 5). This retardation was due to the inhibitory effect of ATP on HA deposition
. The mineral phase produced by MVs in the presence of 0.33 mM ATP revealed HA and a small amount of other minerals, as suggested by the presence of a broad contour in the 1200–1000 cm−1 region (Fig. 6, spectrum: ATP), and as reported elsewhere
Fig. 6Infrared spectra of minerals formed by MVs in the presence of different concentrations of Pi or different phosphate substrates. MVs were incubated at 37°C in SCL buffer containing 2 mM Ca2+ and 1.42 mM Pi with additional Pi or phosphate substrates: without additional substrates as a control (spectrum SCL), total Pi=3.42 mM (spectrum Pi), 1 mM AMP (spectrum AMP), 0.33 ATP (spectrum ATP).
To identify the conditions to produce HA or other minerals, MVs were incubated in the SCL buffer with 2 mM Ca2+, Pi at 1.42–3.42 mM concentration range and PPi at 0.01–2.41 mM concentration range. Pi/PPi ratio was calculated initially and during the induction phase of mineralization since PPi was continuously hydrolyzed and both Pi and PPi were involved in the mineral formation. The final Pi/PPi ratio was determined for each initial Pi/PPi ratio. The initial Pi/PPi ratio (within 1.42–3.42 mM Pi and 0.01–2.41 mM PPi) predetermined the type of mineral formed by MVs. Without PPi, the period of induction phase was about 3 h when MVs were incubated in the SCL buffer with 2 mM Ca2+ [Fig. 8(A) ], and 0.5 h when the SCL buffer was supplemented by 2 mM Ca2+ and 2 mM Pi corresponding to a total amount of 3.42 mM Pi in SCL [Fig. 8(B)]. A higher amount of Pi decreased the induction time of mineral formation. MVs incubated in the presence of Ca2+ and Pi formed crystalline HA (Fig. 9, spectrum I). The induction time of mineralization increased from 3 to 6 h [Fig. 8(A)] after addition of 0.01 mM PPi into the SCL medium with 2 mM Ca2+ (since SCL medium contained 1.42 mM Pi, the initial Pi/PPi ratio was 142±47 and final Pi/PPi ratio was 198.3±65.6, Table III). Addition of 0.024 mM PPi and 2 mM Pi in SCL (total Pi was 3.42 mM in SCL medium; initial Pi/PPi ratio was 142±47 and final Pi/PPi ratio was 198.3±65.6), increased the induction time of mineralization from 0.5 to 2.5 h [Fig. 8(B)] and the mineral formed was crystalline HA (Fig. 9, spectrum II). At the initial Pi/PPi ratio between infinite and 142±47, the induction time increased (Table III), but the mineral formed was always HA (Table III). The turning point where the mineral phase contained a mixture of poorly crystalline HA and other minerals, was reached with an initial Pi/PPi ratio of 71±14.2 (Table III). The maximal induction time of mineral formation occurred upon addition of 0.05±0.01 mM PPi in SCL with 1.42 mM total Pi concentration [18 h, Fig. 8(A)] or 0.12 mM PPi in SCL with 3.42 mM total Pi concentration [10 h, Fig. 8(B)], corresponding for both to an initial Pi/PPi ratio of 28.4±5.7 [Fig. 8(C)] and to a final Pi/PPi ratio of 102.9±20.7 (Table III). The minerals formed under these conditions were not HA as evidenced by the absence of characteristic HA bands at 960–961 cm−1, 600–601 cm−1 and 560–562 cm−1. Amorphous mixtures were produced (Fig. 9, spectrum III). The induction time of mineral formation decreased with the diminution of initial Pi/PPi ratio from 28.4±5.7, indicating faster mineral formation. Addition of 0.1 mM PPi in SCL with 1.42 mM total Pi concentration (initial Pi/PPi ratio of 14.2±1.4 and final Pi/PPi ratio of 24.5±2.4) reduced the induction time of mineral formation to 9 h. It was further reduced to 7 h with the addition of 0.5 mM PPi (initial Pi/PPi ratio of 2.8±0.3 and final ratio of 6.2±0.7) and to 5 h with 1 mM PPi (initial Pi/PPi ratio of 1.4±0.1 and final ratio of 2.8±0.3) [Fig. 8(A)]. We observed also a decrease of induction time of mineral formation at the same Pi/PPi ratio but with higher PPi concentrations in SCL medium containing 3.42 mM Pi [Fig. 8(B)]. Due to the higher amount of PPi and Pi, there was a higher amount of mineral formed as evidenced by the larger turbidity and the kinetics [Fig. 8(B) vs Fig. 8(A)]. Although the mineral formation in MVs was stimulated with higher concentrations of PPi, the nature of mineral deposits was different. At the initial Pi/PPi ratios between 14.2±1.4 and 2.8±0.3, the mineral phase was composed of a mixture of minerals, including CPPD (Fig. 9, spectrum IV), as characterized by the appearance of the characteristic CPPD bands at 1140 cm−1, 925 cm−1, 725 cm−1 and 555 cm−1 (Fig. 7, spectrum: CPPD). At the initial Pi/PPi ratio lower than 2.8±0.3, the spectrum of the mineral formed by MVs resembled to CPPD (Fig. 9, spectrum V). CPPD mineral was exclusively produced by MVs when the initial Pi/PPi ratio was lower than 1.4±0.1 (Fig. 9, spectrum VI). Under the same conditions, no CPPD was formed in SCL medium without MVs.
Fig. 8Retardation of PPi-initiated mineral formation. (A) MVs were incubated at 37°C in SCL buffer containing 2 mM Ca2+, 1.42 mM Pi and PPi at various concentrations: (▾) without additional PPi, (▶) 0.01 mM, (▴) 0.05 mM, (□) 0.1 mM, (♢) 0.5 mM and (○) 1 mM PPi corresponding to an initial Pi/PPi ratio of 142, 28.4, 14.2, 2.8, and 1.4, respectively. (B) MVs were incubated at 37°C in SCL buffer containing 2 mM Ca2+, 3.42 mM Pi and different concentrations of PPi: (▾) without additional PPi, (▶) 0.024 mM, (▴) 0.12 mM, (□) 0.24 mM, (♢) 1.2 mM and (○) 2.41 mM PPi, corresponding to the same Pi/PPi ratio as in panel A. (C) All results were combined and normalized as percentages of the maximal retardation of mineralization induced by PPi.
Fig. 9Infrared spectra of minerals produced by MVs at different Pi/PPi molar ratios: (I) no PPi, (II) 142, (III) 28.4, (IV) 14.2, (V) 2.8 and (VI) 1.4. MVs were incubated at 37°C in SCL buffer containing 2 mM Ca2+, 3.42 mM Pi and PPi at 0, 0.024 mM, 0.12 mM, 0.24 mM, 1.2 mM or 2.41 mM.
Table IIIThe effect of the Pi/PPi ratio on the mineralization mediated by MVs. MVs were incubated in the SCL buffer containing 2 mM Ca2+, Pi at 1.42–3.42 mM concentration range and PPi at 0.01–2.41 mM concentration range. Initial Pi/PPi ratios and Pi/PPi ratios prior to the onset of calcification were calculated. The kinetics of mineralization was followed by light scattering at 340 nm and the minerals formed by MVs were identified by infrared spectroscopy (the numbering of spectra corresponded to the infrared spectra in Fig. 9 and to the minerals formed at a specific Pi/PPi ratio as indicated in the table). Induction time was the longest (100%) at initial [Pi]/[PPi]=28.4±5.7 and the lowest (12.5±1.4) in the absence of PPi
Our report focused on the conditions favoring HA and CPPD minerals induced by MVs from growth plate cartilage. MVs were used to mimic pathological calcification, since the initiation of mineral formation mediated by MVs during endochondral calcification is similar to that which appears in a variety of pathologic calcification
. Although MV model has the disadvantage that matrix and cellular issues cannot be addressed, it provides an easily quantifiable and well-characterized model to analyze the initiation of HA or CPPD formation
. MVs served to model arthritic crystal deposition characterized by HA or CPPD deposits in joint cartilage. In the absence of inhibitors, Ca2+/Pi ratio and the Ca2+×Pi product are critical factors affecting the kinetics of the biomineralization process
. Increasing the Pi concentration (Fig. 5) and addition of phosphomonoester substrates of TNAP, such as AMP, reduced the induction time of HA formation (Fig. 5). However, addition of ATP, another source of Pi after its hydrolysis, led to a high retardation of the induction phase of mineralization (Fig. 5) and to a mixture of poorly crystalline HA and other minerals (Fig. 3, spectrum: ATP), consistent with Zhang et al.
, on the HA formation. ATP is a source of Pi (after its hydrolysis by TNAP, ATPases and other PME enzymes) but also a source of PPi after its hydrolysis by NPP1 and TNAP
. PPi, when hydrolyzed, provides Pi for HA formation but inhibits the seeding of calcium–phosphate minerals itself. In addition, high concentrations of PPi led to the precipitation of immature CPPD mineral. Alternatively, metastable equilibrium between Ca2+, Pi and PPi can be disturbed, inducing mineral formations without MVs. Cheng and Pritzker
reported that HA was formed in aqueous solution when Pi/PPi was higher than 100, while CPPD was produced when Pi/PPi was less than 3. MVs from growth plate cartilages are able to produce CPPD minerals
Further characterization of ATP-initiated calcification by matrix vesicles isolated from rachitic rat cartilage. Membrane perturbation by detergents and deposition of calcium pyrophosphate by rachitic matrix vesicles.
. Since osteoarthritic MVs and growth plate MVs own similar protein machinery associated with mineralization, these findings underline a mechanism of CPPD pathological deposit. Our data emphasize that not only PPi concentration affected the nature of the formed mineral but also the Pi/PPi ratio is a key parameter to favor HA or CPPD formation as proposed previously
. The Pi/PPi ratio is a determinant factor leading to pathological mineralization or its inhibition. Initial Pi/PPi ratio higher than 140 led to HA deposition, mimicking conditions during endochondral bone formation or arthritic crystal deposition. When Pi/PPi ratio was lower than 70, it inhibited the MV-induced seeding of HA, which corresponds to the conditions where mineralization is inhibited. An initial Pi/PPi ratio lower than 2.8 led to deposits of pathological CPPD, while initial Pi/PPi ratio higher than 28.4 inhibited CPPD formation. The Pi/PPi ratio could reflect somehow the overall differentiation states of chondrocytes (mature vs hypertrophic), the levels of expression of TNAP, NPP1 or other proteins affecting Pi and PPi concentrations as well as the balance between pro- and anti-calcification factors and may serve as an indicator of calcification process.
Conflict of interest
None of the authors of this paper have any financial and personal relationships with people or organization that could inappropriately influence (bias) their work.
Acknowledgments
We thank Dr Laurence Bessuelle for her help with electron microscopy and Dr John Carew for correcting the English. This work was supported by a Polonium grant (05819NF), CNRS (France) and by a grant N301 025 32-1120 from Polish Ministry of Science and Higher Education.
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Extracellular ATP and its effects on physiological and pathological mineralization.
Up-regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC-1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification.
Sustained osteomalacia of long bones despite major improvement in other hypophosphatasia-related mineral deficits in tissue nonspecific alkaline phosphatase/nucleotide pyrophosphatase phosphodiesterase 1 double-deficient mice.
Comparison of characteristics of patients with and without calcium pyrophosphate dihydrate crystal deposition disease who underwent total knee replacement surgery for osteoarthritis.
Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders.
Upregulated ank expression in osteoarthritis can promote both chondrocyte MMP-13 expression and calcification via chondrocyte extracellular PPi excess.
Up-regulated expression of cartilage intermediate-layer protein and ANK in articular hyaline cartilage from patients with calcium pyrophosphate dihydrate crystal deposition disease.
Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens.
Fourier transform infrared characterization of mineral phases formed during induction of mineralization by collagenase-released matrix vesicles in vitro.
Further characterization of ATP-initiated calcification by matrix vesicles isolated from rachitic rat cartilage. Membrane perturbation by detergents and deposition of calcium pyrophosphate by rachitic matrix vesicles.
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