Search

Matrix vesicles (MVs) induce calcification during endochondral bone formation. Experimental methods for structural, compositional, and functional analysis of MVs are reviewed. MV proteins, enzymes, receptors, transporters, regulators, lipids and electrolytes are detailed. MV formation is considered from both structural and biochemical perspectives. Confocal imaging of Ca(2+) and H(+) were used to depict how living chondrocytes form MVs. Biochemical studies revealed that coordinated mitochondrial Ca(2+) and Pi metabolism produce MVs containing a nucleational complex (NC) of amorphous calcium phosphate, phosphatidylserine and annexin A5--all critical to the mechanism of mineral nucleation. Reconstitution of the NC and modeling with unilamellar vesicles reveal how the NC transforms into octacalcium phosphate, regulated by Mg(2+), Zn(2+) and annexin A5. Extravasation of intravesicular mineral is mediated by phospholipases and tissue-nonspecific alkaline phosphatase (TNAP). In the extravesicular matrix, hydroxyapatite crystal propagation is enhanced by cartilage collagens and TNAP, which destroys inhibitory PPi, and by metalloproteases that degrade proteoglycans. Other proteins also modulate mineral formation. Recent findings from single and multiple gene knockouts of TNAP, NPP1, ANK, PHOSPHO1, and Annexin A5 are reviewed.

The differential accumulation of fluorescent molecules in tumorigenic versus normal cells is a well-reported phenomenon and is the basis for photodiagnostic therapy. Through the use of confocal microscopy, the kinetic uptake and accumulation of fusarochromanone (FC101) was determined in two lines of living tumorigenic cells of mesenchymal-epithelial origin and normal fibroblast cells. Like other fluorescent cationic molecules, FC101 showed increased accumulation in tumorigenic cells; however, unlike other molecules, it appeared to be accumulated in a time-dependent manner. Also, unlike traditional fluorescent cationic molecules, FC101, a potent inhibitor of cell growth, showed preferential inhibition of tumorigenic B-16 melanoma cells and MCF7 cells derived from breast cancer adenocarcinoma when compared to normal cardiac fibroblasts. Further analysis of FC101's physicochemical properties using both experimentally obtained and simulated values revealed the likelihood of membrane permeation and oral bioavailability of the compound. These physicochemical properties of FC101 were also used to predict its intracellular localization lending credence to data observed by confocal microscopy.

Mg(2+) and Zn(2+) are present in the mineral of matrix vesicles (MVs) and biological apatites, and are known to influence the onset and progression of mineral formation by amorphous calcium phosphate (ACP) and hydroxyapatite (HAP). However, neither has been studied systematically for its effect on mineral formation by phosphatidylserine-Ca(2+)-Pi complexes (PS-CPLX), an important constituent of the MV nucleation core. Presented here are studies on the effects of increasing levels of Mg(2+) and Zn(2+) on the process of mineral formation, either when present in synthetic cartilage lymph (SCL), or when incorporated during the formation of PS-CPLX. Pure HAP and PS-CPLX proved to be powerful nucleators, but ACP took much longer to induce mineral formation. In SCL, Mg(2+) and Zn(2+) had significantly different inhibitory effects on the onset and amount of mineral formation; HAP and PS-CPLX were less affected than ACP. Mg(2+) and Zn(2+) caused similar reductions in the rate and length of rapid mineral formation, but Zn(2+) was a more potent inhibitor on a molar basis. When incorporated into PS-CPLX, Mg(2+) and Zn(2+) caused significantly different effects than when present in SCL. Even low, subphysiological levels of Mg(2+) altered the inherent structure of PS-CPLX and markedly reduced its ability to induce and propagate mineral formation. Incorporated Zn(2+) caused significantly less effect, low (<20 microM) levels causing almost no inhibition. Levels of Zn(2+) present in MVs do not appear to inhibit their nucleational activity.

Matrix vesicles (MVs) in the growth plate bind to cartilage collagens and initiate mineralization of the extracellular matrix. Native MVs have been shown to contain a nucleational core responsible for mineral formation that is comprised of Mg(2+)-containing amorphous calcium phosphate and lipid-calcium-phosphate complexes (CPLXs) and the lipid-dependent Ca(2+)-binding proteins, especially annexin-5 (Anx-5), which greatly enhances mineral formation. Incorporation of non-Ca(2+)-binding MV lipids impedes mineral formation by phosphatidylserine (PS)-CPLX. In this study, nucleators based on amorphous calcium phosphate (with or without Anx-5) were prepared with PS alone, PS + phosphatidylethanolamine (PE), or PS + PE and other MV lipids. These were incubated in synthetic cartilage lymph containing no collagen or containing type II or type X collagen. Dilution of PS with PE and other MV lipids progressively retarded nucleation. Incorporation of Anx-5 restored nucleational activity to the PS:PE CPLX; thus PS and Anx-5 proved to be critical for nucleation of mineral. Without Anx-5, induction of mineral formation was slow unless high levels of Ca(2+) were used. The presence of type II collagen in synthetic cartilage lymph improved both the rate and amount of mineral formation but did not enhance nucleation. This stimulatory effect required the presence of the nonhelical telopeptides. Although type X collagen slowed induction, it also increased the rate and amount of mineral formation. Both type II and X collagens markedly increased mineral formation by the MV-like CPLX, requiring Anx-5 to do so. Thus, Anx-5 enhances nucleation by the CPLXs and couples this to propagation of mineral formation by the cartilage collagens.

The nucleational core of matrix vesicles contains a complex (CPLX) of phosphatidylserine (PS), Ca(2+), and inorganic phosphate (P(i)) that is important to both normal and pathological calcification. Factors required for PS-CPLX formation and nucleational activity were studied using in vitro model systems and molecular dynamic simulations. Ca(2+) levels required for and rates of PS-CPLX formation were monitored by light scattering at 340 nm, assessing changes in amount and particle size. Fourier transform infrared spectroscopy was used to explore changes in chemical structure and composition. Washing with pH 5 buffer was used to examine the role of amorphous calcium phosphate in CPLX nucleational activity, which was assessed by incubation in synthetic cartilage lymph with varied pH values. Addition of 4 Ca(2+)/PS was minimally required to form viable complexes. During the critical first 10-min reaction period, rapid reduction in particle size signaled changes in PS-CPLX structure. Fourier transform infrared spectroscopy revealed increasing mineral phosphate that became progressively deprotonated to PO(4)(3-). This Ca(2+)-mediated effect was mimicked in part by increasing the Ca(2+)/PS reaction ratio. Molecular dynamic simulations provided key insight into initial interactions between Ca(2+) and P(i) and the carboxyl, amino, and phosphodiester groups of PS. Deduced interatomic distances agreed closely with previous radial distribution function x-ray-absorption fine structure measurements, except for an elongated Ca(2+)-N distance, suggesting additional changes in atomic structure during the critical 10-min ripening period. These findings clarify the process of PS-CPLX formation, reveal details of its structure, and provide insight into its role as a nucleator of crystalline calcium phosphate mineral formation.

Annexins A5, A2, and A6 (Anx-A5, -A2, and -A6) are quantitatively major proteins of the matrix vesicle nucleational core that is responsible for mineral formation. Anx-A5 significantly activated the induction and propagation of mineral formation when incorporated into synthetic nucleation complexes made of amorphous calcium phosphate (ACP) and Anx-A5 or of phosphatidylserine (PS) plus ACP (PS-CPLX) and Anx-A5. Incorporation of Anx-A5 markedly shortened the induction time, greatly increasing the rate and overall amount of mineral formed when incubated in synthetic cartilage lymph. Constructed by the addition of Ca(2+) to PS, emulsions prepared in an intracellular phosphate buffer matched in ionic composition to the intracellular fluid of growth plate chondrocytes, these biomimetic PS-CPLX nucleators had little nucleational activity. However, incorporation of Anx-A5 transformed them into potent nucleators, with significantly greater activity than those made from ACP without PS. The ability of Anx-A5 to enhance the nucleation and growth of mineral appears to stem from its ability to form two-dimensional crystalline arrays on PS-containing monolayers. However, some stimulatory effect also may result from its ability to exclude Mg(2+) and HCO(-)(3) from nucleation sites. Comparing the various annexins for their ability to activate PS-CPLX nucleation yields the following: avian cartilage Anx-A5 > human placental Anx-A5 > avian liver Anx-A5 > or = avian cartilage Anx-A6 >> cartilage Anx-A2. The stimulatory effect of human placental Anx-A5 and avian cartilage Anx-A6 depended on the presence of PS, since in its absence they either had no effect or actually inhibited the nucleation activity of ACP. Anx-A2 did not significantly enhance mineralization.

Fusarochromanone is a toxic metabolite produced by Fusarium equiseti, a fungus present in decaying cereal plants in northern latitudes; it has been detected in various food grains. Fusarochromanone has been shown to have both stimulatory and inhibitory effects on various mammalian cells, depending on the concentration used. Whether these cytotoxic effects can be used in the clinical treatment of tumors remains to be established. Here, we evaluated the effects of fusarochromanone on the growth of human melanoma cells both in vitro and in vivo. In vitro, low concentrations (0.1-1 nmol/l) of fusarochromanone were found to be cytotoxic to many melanoma cell lines. In contrast, growth of normal melanocytes was inhibited only at much higher fusarochromanone concentrations (100-200 nmol/l). In vivo, the growth of melanoma cells implanted subcutaneously in immuno-compromised mice was significantly (P<0.05) reduced by daily administration of fusarochromanone. Immunohistological analyses indicated a significant (P<0.05) increase in the expression of active caspase-3 in tumor masses of mice treated with fusarochromanone, compared with controls. Together, these observations show that fusarochromanone increased apoptosis of tumor cells and reduced tumor growth in vivo. Therefore, the effects of fusarochromanone warrant further investigation as an adjuvant molecule to prevent growth and recurrence of melanomas.

Matrix vesicles (MVs) are involved in de novo mineral formation by nearly all vertebrate tissues. The driving force for MV mineralization is a nucleational core composed of three principal constituents: (i) amorphous calcium phosphate (ACP), complexed in part with phosphatidylserine (PS) to form (ii) calcium-phosphate-lipid complexes (CPLX), and (iii) annexin A5 (AnxA5), the principal lipid-dependent Ca(2+)-binding protein in MVs. We describe methods for reconstituting the nucleational core using a biomimetic approach and for analyzing the kinetics of its induction of mineral formation. The method is based on light scattering by the nascent crystallites at 340 nm and monitors mineral formation at regular intervals without disturbing the system using an automated plate reader. It yields precise replicate values that typically agree within less than 5%. As with MVs, mineral formation by the synthetic complex follows a sigmoidal pattern; following a quiescent induction period, rapid formation ensues for a limited time, followed by a distinct decline in rate that continues to slow, ultimately reaching a maximal asymptotic value. Key to quantization of mineral formation is the use of first-derivative analysis, which defines the induction time, the rate and the amount of initial mineral formation. Furthermore, using a five-parameter logistic curve-fitting algorithm, the maximal amount of mineral formation can be predicted accurately. Using these methods, we document the dramatic finding that AnxA5 synergistically activates PS-CPLX, transforming it from a very weak nucleator of mineral formation to a potent one. The methods presented should enable systematic study of the effects of numerous other factors thought to contribute to mineral formation.

Time- and dosage-dependent effects of 1,25(OH)(2)D(3) and 24,25(OH)(2)D(3) on primary cultures of pre- and post-confluent avian growth plate (GP) chondrocytes were examined. Cultures were grown in either a serum-containing culture medium designed to closely mimic normal GP extracellular fluid (DATP5) or a commercially available serum-free media (HL-1) frequently used for studying skeletal cells. Hoechst DNA, Lowry protein, proteoglycan (PG), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) activity and calcium and phosphate mineral deposition in the extracellular matrix were measured. In preconfluent cultures grown in DATP5, physiological levels of 24,25(OH)(2)D(3) (0.10-10 nM) increased DNA, protein, and LDH activity significantly more than did 1,25(OH)(2)D(3) (0.01-1.0 nM). However, in HL-1, the reverse was true. Determining ratios of LDH and PG to DNA, protein, and each other, revealed that 1,25(OH)(2)D(3) specifically increased PG, whereas 24,25(OH)(2)D(3) increased LDH. Post-confluent cells were generally less responsive, especially to 24,25(OH)(2)D(3). The positive anabolic effects of 24,25(OH)(2)D(3) required serum-containing GP-fluid-like culture medium. In contrast, effects of 1,25(OH)(2)D(3) were most apparent in serum-free medium, but were still significant in serum-containing media. Administered to preconfluent cells in DATP5, 1,25(OH)(2)D(3) caused rapid, powerful, dosage-dependent inhibition of Ca(2+) and Pi deposition. The lowest level tested (0.01 nM) caused >70% inhibition during the initial stages of mineral deposition; higher levels of 1,25(OH)(2)D(3) caused progressively more profound and persistent reductions. In contrast, 24,25(OH)(2)D(3) increased mineral deposition 20-50%; it required >1 week, but the effects were specific, persistent, and largely dosage-independent. From a physiological perspective, these effects can be explained as follows: 1,25(OH)(2)D(3) levels rise in hypocalcemia; it stimulates gut absorption and releases Ca(2+) from bone to correct this deficiency. We now show that 1,25(OH)(2)D(3) also conserves Ca(2+) by inhibiting mineralization. The slow anabolic effects of 24,25(OH)(2)D(3)are consistent with its production under eucalcemic conditions which enable bone formation. These findings, which implicate serum-binding proteins and accumulation of PG in modulating accessibility of the metabolites to GP chondrocytes, also help explain some discrepancies previously reported in the literature., (Copyright 2006 Wiley-Liss, Inc.)

We report here a comparative study of the development and behavior of chondrocytes isolated from normal growth plate tissue, tibial dyschondroplasic lesions, and from articular cartilage. The objective of these studies was to determine whether the properties exhibited by chondrocytes in dysplasic lesions or in articular cartilage were due to their cellular phenotype, their environment, or both. We had previously analyzed the electrolytes and amino acid levels in the extracellular fluid of avian growth plate chondrocytes. Using these data, we constructed a culture medium (DATP5) in which growth plate cells essentially recapitulate their normal behavior in vivo. Here, we used DATP5 to examine the behavior of chondrocytes isolated from lesions of tibial dyschondroplasia (TD). We found that once isolated from lesion and grown in this supportive medium, dysplasic chondrocytes behaved essentially like normal growth plate cells. These findings suggest that the cause of TD is local factors operating in vivo to prevent these cells from developing normally. With respect to articular chondrocytes, our data indicate that they more closely retain normal protein and proteoglycan synthesis when grown in serum-free media. These cells readily induced mineral formation in vitro, both in the presence and absence of serum. However, in serum-containing media, mineralization was significantly enhanced when the cells were exposed to retinoic acid (RA) or osteogenic protein-1 (OP-1). Our studies support previous work indicating the presence of autocrine factors produced by articular chondrocytes in vivo that prevent mineralization and preserve matrix integrity. The lack of inhibitory factors and the presence of supporting factors are likely reasons for the induction of mineralization by articular chondrocytes in vitro., (2005 Wiley-Liss, Inc.)

Matrix vesicles (MV) are lipid bilayer-enclosed nanoscale structures that initiate extracellular mineral formation in most vertebrate species. Little attention has been given to differences between species in membrane lipid composition or to how new mineral is formed in MV. To explore more precisely the lipids of MV isolated from avian and bovine species, we developed a new high-performance liquid chromatography (HPLC) method used in combination with evaporative light scattering detection (ELSD) to quantify their lipid composition. HPLC analyses were performed on a Lichrosorb silica column using separate binary gradient elution systems for analyzing polar and nonpolar lipids. Standard mixtures of both phospholipids and nonpolar lipids were used to prepare calibration curves for each lipid, which were analyzed mathematically by polynomial regression for accurate quantitation. This new methodology provides high-resolution separations and quantitation of both the polar and the nonpolar lipids typically present in biological membranes, including lyso- (monoacyl-) phospholipids. We have applied this method to quantitate the phospholipid and nonpolar lipid composition of MV isolated from chicken and bovine growth plate cartilage. We also compared the ability of these MV to induce mineral formation. While the ability to induce mineralization and the lipid composition were generally similar, some significant differences between MV from these two disparate species were seen.

Matrix vesicles released by epiphyseal growth plate chondrocytes are known to contain a significant quantity of labile Zn(2+). Zonal analysis of chicken metatarsal bones showed that the resting/proliferative region of the growth plate contained high levels of Zn(2+) with significantly lower levels in the hypertrophic cartilage suggesting a loss of cellular Zn(2+) as the chondrocytes mature. Intracellular labile Zn(2+) was measured in primary cultures of growth plate chondrocytes by assay with the fluorescent Zn-chelator toluenesulfonamidoquinoline (TSQ) and imaged by multi-photon laser scanning microscopy (MPLSM) with the TSQ derivative zinquin. Short-term exposure to Zn(2+), both in the presence and absence of pyrithione resulted in significant increases in cytosolic Zn(2+). Treatment with the membrane-permeant Zn(2+) chelator TPEN rapidly reduced the levels of labile Zn(2+) and triggered apoptosis. Cytosolic Zn(2+) levels were significantly reduced following 24-h incubations with known inducers of chondrocyte apoptosis. The loss of intracellular Zn(2+) was accompanied by a significant reduction in the cytosolic metal-binding protein metallothionein. Examination of Zn(2+)-treated cells with MPLSM showed uniformly higher zinquin fluorescence. Treatment of Zn(2+)-loaded cells with TPEN quenched zinquin fluorescence confirming that the observed fluorescence in chondrocytes is due to the presence of intracellular Zn(2+). A dose-dependent increase in zinquin fluorescence was observed in cells treated with a range of Zn(2+) concentrations. Short-term treatment of cultured chondrocytes with apoptosis-inducing chemicals resulted in transient increases in intracellular labile Zn(2+). These results indicate that Zn(2+) is mobilized from intracellular binding sites in the early stages of chondrocyte apoptosis and is subsequently lost from the cells. The early mobilization of Zn(2+) provides a mechanism for its movement to matrix vesicles and the extracellular matrix., (Copyright 2003 Wiley-Liss, Inc.)

The mechanism of matrix vesicle (MV) mineralization was studied using MVs isolated from normal growth plate tissue, as well as several putative intermediates in the MV mineralization pathway--amorphous calcium phosphate (ACP), calcium phosphate phosphatidylserine complex (CPLX) and hydroxyapatite (HAP). Radionuclide uptake and increase in turbidity were used to monitor mineral formation during incubation in synthetic cartilage lymph (SCL). Inhibitors of phosphate (Pi) metabolism, as well as replacing Na(+) with various cations, were used to study MV Pi transport, which had been thought to be Na(+)-dependent. MVs induced rapid mineralization approximately 3 h after addition to SCL; CPLX and HAP caused almost immediate induction; ACP required approximately 1 h. Phosphonoformate (PFA), a Pi analog, potently delayed the onset and reduced the rate of mineral formation of MV and the intermediates with IC(50)'s of 3-6 microM and approximately 10 microM, respectively. PFA:Pi molar ratios required to reduce the rate of rapid mineralization by 50% were approximately 1:30 for ACP, approximately 1:20 for HAP, approximately 1:3.3 for CPLX, and approximately 1:2.0 for MVs. MV mineralization was not found to be strictly Na(+)-dependent: substitution of Li(+) or K(+) for Na(+) had minimal effect; while N-methyl D-glucamine (NMG(+)) was totally inhibitory, choline(+) was clearly stimulatory. Na(+) substitutions had minimal effect on HAP- and CPLX-seeded mineral formation. However with ACP, NMG(+) totally blocked and choline(+) stimulated, just as they did MV mineralization. Thus, kinetic analyses indicate that ACP is a key intermediate, nevertheless, formation of CPLX appears to be the rate-limiting factor in MV mineralization., (Copyright 2003 Elsevier Science Inc.)

Stable, large unilamellar vesicles (LUV) have been constructed that model matrix vesicles (MV) in inducing de novo mineral formation when incubated in synthetic cartilage lymph (SCL). Using a dialysis method for incorporation of predetermined pure lipid, electrolyte and protein constituents, the detergent n-octyl beta-D-glucopyranoside enabled formation of stable, impermeable LUV with a diameter ( approximately 300 nm), lipid composition (phosphatidylcholine-phosphatidylserine-cholesterol, 7:2:2, molar ratio) and enclosed inorganic phosphate level (25-100 mM) similar to that of native MV. Mineral formation by these LUVs was measured by 45Ca(2+) uptake and FTIR analysis following incubation in SCL. Addition of the ionophore A23187 to SCL enabled 45Ca(2+) uptake comparable to that of native MV. FTIR analysis revealed that crystalline mineral formed in the LUV during incubation in SCL, but not in the absence of ionophore. This mineral had an IR absorption spectrum like that of the acid-phosphate-rich, octacalcium phosphate-like mineral formed by native MV. Perturbing the LUV membrane with either detergents or phospholipase A(2) following prior incubation in SCL enabled egress of mineral crystallites from the vesicle lumen, stimulating further mineral formation. Annexin V, a major protein in native MV with known Ca(2+) channel activity, incorporated into the LUV lumen or added to the external medium, induced only limited 45Ca(2+) uptake. This indicates that additional factors are required for annexin V to form Ca(2+) channels. Nevertheless for the first time, stable LUVs have been constructed with MV-like lipid, electrolyte, and protein composition and size that induce formation of mineral like that formed by native MV.

Matrix vesicles are lipid bilayer-enclosed structures that initiate extracellular mineral formation. Little attention has been given to how newly formed mineral interacts with the lipid constituents and then emerges from the lumen. To explore whether specific lipids bind to the incipient mineral and if breakdown of the membrane is involved, we analyzed changes in lipid composition and extractability during vesicle-induced calcification. Isolated matrix vesicles were incubated in synthetic cartilage lymph to induce mineral formation. At various times, samples of the lipids were taken for analysis, extracted both before and after demineralization to remove deposited mineral. Phosphatidylserine and phosphatidylinositol both rapidly disappeared from extracts made before decalcification, indicating rapid degradation. However, extracts made after demineralization revealed that phosphatidylserine had become complexed with newly forming mineral. Concomitantly, its levels actually increased, apparently by base-exchange with phosphatidylethanolamine. Though partially complexed with the mineral, phosphatidylinositol was nevertheless rapidly broken down. Sphingomyelin and phosphatidylethanolamine also underwent rapid breakdown, but phosphatidylcholine was degraded more slowly, all accompanied by a buildup of free fatty acids. The data indicate that phosphatidylserine forms complexes that accompany mineral formation, while degradation of other membrane phospholipids apparently enables egress of crystalline mineral from the vesicle lumen.

Sonic hedgehog (Shh) is a key signal protein in early embryological patterning of limb bud development. Its analog, Indian hedgehog (Ihh), primarily expressed during early cartilage development in prehypertrophic chondrocytes, regulates proliferation and suppresses terminal differentiation of postnatal growth plate (GP) chondrocytes. We report here for the first time that both Shh and Ihh mRNA are expressed in the GP of rapidly growing 6-week-old broiler-strain chickens. They are also expressed in other tissues such as articular chondrocytes, kidney, and bone. In situ hybridization and RT-PCR analyses reveal Shh in all zones of the GP, with peak expression in late hypertrophy. Using primary cultures of GP chondrocytes in serum-containing medium, we followed the patterns of Shh and Ihh mRNA expression as the cultures matured and mineralized. We find a cyclical expression of both hedgehog genes during the early period of culture development between day 10 and 14; when one is elevated, the other tended to be suppressed, suggesting that the two hedgehogs may play complementary roles during GP development. Retinoic acid (RA), a powerful modulator of gene expression in cell differentiation, stimulates GP chondrocytes toward terminal differentiation, enhancing mineral formation. We find that RA strongly suppresses Ihh, but enhances expression of Shh in this system. While Ihh suppresses maturation of GP chondrocytes to hypertrophy, we hypothesize that Shh acts to push these cells toward hypertrophy., (Copyright 2002 Wiley-Liss, Inc.)

This report describes Pi transport activity in chondrocytes isolated from the growth plate (GP) of normal adolescent chickens grown in primary cell culture. Our recent work showed that Pi transport in matrix vesicles (MV) isolated from normal GP cartilage was not strictly Na+-dependent, whereas previously characterized Pi transport from rachitic GP cartilage MV was. This Na+-dependent Pi transporter (NaPiT), a member of the Type III Glvr-1 gene family, is expressed only transiently during early differentiation of GP cartilage, is enhanced by Pi-deficiency, and is most active at pH 6.8. Since GP mineralization requires abundant Pi and occurs under slightly alkaline conditions, it seemed unlikely that this type of Pi transporter was solely responsible for Pi uptake during normal GP development. Therefore we asked whether the lack of strict Na+-dependency in Pi transport seen in normal MV was also evident in normal GP chondrocytes. In fact, cellular Pi transport was found not to be strictly Na+-dependent, except for a brief period early in the culture. Choline could equally serve as a Na+ substitute. Activity of choline-supported Pi transport was optimum at pH 7.6-8.0. In addition, prior exposure of the cells to elevated extracellular Pi (2-3 mM) strongly enhanced subsequent Pi uptake, which appeared to depend on prior loading of the cells with mineral ions. Prevention of Pi loading by pretreatment with Pi transport inhibitors not only inhibited subsequent cellular Pi uptake, it also blocked mineral formation. Treatment with elevated extracellular Pi did not induce apoptosis in these GP chondrocytes., (Copyright 2002 Wiley-Liss, Inc.)

As a continuation of our studies on mineralization in epiphyseal growth plate (GP) chondrocyte cultures, the effects of tri-iodothyronine (T3) in both beta-glycerophosphate-containing, serum-free (HL-1) and beta-glycerophosphate-free, serum-containing medium (DATP5) were studied. The GP cells responded to T3 in a serum-, stage-, and dosage-dependent manner. Added at graded levels (0.1-10.0 nM) to preconfluent cultures (from day 7) in both HL-1 and DATP5, T3 caused progressive decreases in protein, collagen, and DNA synthesis but increased mineral deposition. In postconfluent cultures, these effects of T3 were generally muted. In preconfluent cultures, proteoglycan (PG) levels were not significantly affected in DATP5, although in HL-1 they were decreased by approximately 50%. In postconfluent cultures, T3 increased PG levels in DATP5 but had no effect in HL-1. In HL-1, alkaline phosphatase (ALP) activity was progressively increased by 200-500% in both pre- and postconfluent cultures. In DATP5 in preconfluent cultures, T3 initially stimulated but later suppressed ALP; in postconfluent cultures, T3 also transiently increased ALP but did not suppress activity upon longer exposure. The inhibitory effects of T3 on protein, PG, and DNA levels of GP chondrocytes suggest that in vivo its effects on bone growth must occur primarily after cellular proliferation. Apparently by binding to the 50 kDa thyroxine-binding globulin, which cannot penetrate the PG barrier, accessibility of T3 to GP chondrocytes is limited until the time of vascular penetration when its stimulatory effects on ALP and mineral deposition become critical for continued bone development.

Endochondral bone formation involves the progression of epiphyseal growth plate chondrocytes through a sequence of developmental stages which include proliferation, differentiation, hypertrophy, and matrix calcification. To study this highly coordinated process, we infected growth plate chondrocytes with Rous sarcoma virus (RSV) and studied the effects of RSV transformation on cell proliferation, differentiation, matrix synthesis, and mineralization. The RSV-transformed chondrocytes exhibited a distinct bipolar, fibroblast-like morphology, while the mock-infected chondrocytes had a typical polygonal morphology. The RSV-transformed chondrocytes actively synthesized extracellular matrix proteins consisting mainly of type I collagen and fibronectin. RSV-transformed cells produced much less type X collagen than was produced by mock-transformed cells. There also was a significant reduction of proteoglycan levels secreted in both the cell-matrix layer and culture media from RSV-transformed chondrocytes. RSV-transformed chondrocytes expressed two- to- threefold more matrix metalloproteinase, while expressing only one-half to one-third of the alkaline phosphatase activity of mock infected cells. Finally, RSV-transformed chondrocytes failed to calcify the extracellular matrix, while mock-transformed cells deposited high levels of calcium and phosphate into their extracellular matrix. These results collectively indicate that RSV transformation disrupts the preprogrammed differentiation pattern of growth plate chondrocytes and inhibit chondrocyte terminal differentiation and mineralization. They also suggest that the expression of extracellular matrix proteins, type II and type X collagens, and the cartilage proteoglycans are important for chondrocyte terminal differentiation and matrix calcification.

The effects of the trace metals zinc (Zn), manganese (Mn), and cadmium (Cd) on the metabolism of growth plate chondrocytes was examined using a mineralizing culture system. Supplementation of serum-free primary cultures of growth plate chondrocytes with 10-100 mu m Zn resulted in an increase in cell protein and greatly increased alkaline phosphatase (AP) activity; however, above 25 mu m Zn mineralization of the cultures was reduced. The effects of Zn on cellular protein and AP activity were enhanced by the addition of the albumin to the culture media. Removal of Zn from basal culture media resulted in recoverable reductions in cellular protein and AP activities. Cadmium was acutely toxic to chondrocyte cell cultures at concentrations above 5 mu m. Even at very low concentrations (0.25 mu m) Cd caused significant reductions in DNA, cellular protein, and matrix protein synthesis. In contrast, Cd had negligible effects on AP activity or culture mineralization. Manganese treatment (50 mu m) resulted in reduced levels of proteoglycan, cell protein, DNA synthesis, and collagen synthesis, although AP specific activity did not change. At 10 mu m, Mn significantly reduced mineralization but had only minor influence on other culture parameters. Both Zn (200 mu m) and Cd (0.1 mu m), but not Mn, induced the synthesis of metallothionein. The physiological and biochemical effects of specific metal ions is largely dependent on their physicochemical properties, especially their ligand affinities. Knowledge of these properties allows predictions to be made regarding whether the organic or the mineral phase are most likely to be affected in a mineralized tissue.

A phosphatidyl serine-amorphous calcium phosphate complex has been synthesized as a model of the matrix vesicle system that is associated with the induction of mineral deposition in bone, cartilage and dentine. The complex has been studied using a novel technique of subtractive extended X-ray absorption fine structure (EXAFS). This enables spectra of the components of the molecules to be subtracted from the complex so as to identify the sites of interaction. The results suggest there is a movement in the nitrogen atom of the phosphatidyl serine which approaches the calcium atom in the mineral phase. This interpretation would link the membrane structure of the vesicle to the structure of the mineral in a way that could explain some of its roles in biomineralization.

Following exposure to cadmium or zinc, chickens were sacrificed and the liver, kidney, and bone epiphyseal growth plates harvested. When cytosolic extracts of the growth plate cartilage were fractionated by gel filtration chromatography, a protein with high metal-binding capacity and low ultraviolet (UV) absorbance eluted in the same position as liver metallothionein (MT) and a MT standard. Cd or Zn treatment resulted in a 25-fold or 5-fold induction in growth plate MT, respectively. In liver the greatest level of MT induction was seen with short-term Cd exposures. In contrast, MT levels in the growth plate increased as the duration of Cd exposure increased. Induction of MT in growth plate chondrocyte cell cultures was observed for media Cd concentrations of > or = 0.1 microM and Zn concentrations of > or = 100 microM. Basal and inducible levels of MT declined through the culture period and were lowest in the terminally differentiated mineralized late stages of the culture. Alkaline phosphatase activity was also lowest in the late-stage cultures, while total cellular protein increased throughout the culture period. Treatment of chondrocytes with Zn prior to Cd exposure resulted in a protective induction of MT. Pre-treatment of chondrocytes with dexamethasone resulted in suppressed synthesis of MT upon Cd exposure and greater Cd toxicity. Both Cd and Zn resulted in significantly increased levels of MT mRNA in chondrocyte cell cultures. Dexamethasone treatment resulted in an approximate 2- to 3-fold increase in MT mRNA. This is contrary to the finding that MT protein levels were decreased by dexamethasone. The findings suggest that an increased rate of MT degradation in dexamethasone-treated and late-stage chondrocyte cultures may be associated with the terminally differentiated phenotype.

Matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling. In growth plate (GP) cartilage, extensive remodeling occurs at the calcification front. To study the potential involvement of MMPs in retinoic acid (RA) regulation of skeletal development, we studied the effect of all-trans-RA on MMPs levels in mineralizing chicken epiphyseal chondrocyte primary cultures. When treated for 4 day periods on days 10 and 17, RA increased levels of an approximately 70 kDa gelatinase activity. The N-terminal sequence of the first 20 amino acid residues of the purified enzyme was identical to that deduced from chicken MMP-2 cDNA. Time-course studies indicated that RA elevated MMP-2 activity levels in the cultures within 16 h. This increase was inhibited by cycloheximide and was enhanced by forskolin. The increase in MMP-2 activity induced by RA was accompanied by an increase in MMP-2 mRNA levels and was abolished by treatment with cycloheximide. This upregulation of MMP levels by RA in GP chondrocytes is consistent with its effects on osteoblasts and osteosarcoma cells and opposite its inhibitory effects on fibroblasts and endothelial cells. It may well be related to the breakdown of the extracellular matrix in the GP and would be governed by the availability of RA at the calcification front where extensive vascularization also occurs.

Osteogenic protein-1 (OP-1), a member of the TGF-beta family of proteins, induces endochondral bone formation. Here we studied the effect of OP-1 on the development of primary cultures of avian growth plate (GP) chondrocytes in either serum-free or serum-containing medium, in the absence or presence of retinoic acid (RA). OP-1 was added on day 7 of culture and continued for 7 days, or until the cultures were harvested, typically on day 21. Alone, OP-1 caused approximately 2-fold increase in proteoglycan synthesis into both the medium and the cell:matrix layer. Additionally, OP-1 caused a dosage-dependent increase in alkaline phosphatase (ALP) activity, and an increase in protein, when given from days 7-14 and examined on day 14. This stimulation was greater in cells grown in serum-free than in serum-containing media (3-5-fold vs. 2-3-fold increase in ALP; approximately 40% vs. approximately 20% increase in protein). Such stimulation of ALP activity and proteoglycan (PG) synthesis in cultured GP cells indicates that OP-1 elicits differentiation of chondrocytes. OP-1 minimally affected cell division (DNA content); however, a slight increase was seen when examined early in the culture. Alone, OP-1 increased mineral (Ca and Pi) content of the cultures by approximately 2-fold in both types of media. As early as day 14, clusters of mineral encircled many of the OP-1 treated cells. Thus, as in vivo, OP-1 strongly promoted mineral formation by the cultured GP chondrocytes. When present together, OP-1 and RA generally blocked the action of the other. Separately OP-1 and RA each stimulated protein synthesis, ALP activity, and Ca2+ deposition; together they were inhibitory to each. Also, RA blocked the stimulation of PG synthesis induced by OP-1; whereas OP-1 decreased cell division engendered by RA. Thus, this GP chondrocyte culture system is a good model for studying factors that influence differentiation and mineral deposition during bone growth in vivo.

The effect of retinoic acid (RA) on primary cultures of growth plate chondrocytes obtained from weight-bearing joints was examined, Chondrocytes were isolated from the tibial epiphysis of 6- to 8-week-old broiler-strain chickens and cultured in either serum-containing or serum-free media. RA was administered at low levels either transiently or continuously after the cells had become established in culture. Effects of RA on cellular protein levels, alkaline phosphatase (AP) activity, synthesis of proteoglycan (PG), matrix calcification, cellular morphology, synthesis of tissue-specific types of collagen, and level of matrix metalloproteinase (MMP) activity were explored. RA treatment generally increased AP activity and stimulated mineral deposition, especially if present continuously. RA also caused a shift in cell morphology from spherical/polygonal to spindle-like. This occurred in conjunction with a change in the type of collagen synthesized: type X and II collagens were decreased, while synthesis of type I collagen was increased. There was also a marked increase in the activity of MMP. Contrasting effects of continuous RA treatment on cellular protein levels were seen: they were enhanced in serum-containing media, but decreased in serum-free HL-1 media. Levels of RA as low as 10 nM significantly inhibited PG synthesis and caused depletion in the levels of PG in the medium and cell-matrix layer. Thus, in these appendicular chondrocytes, RA suppressed chondrocytic (PG, cartilage-specific collagens) and enhanced osteoblastic phenotype (cell morphology, type I collagen, alkaline phosphatase, and mineralization).

Few studies have been directed toward elucidating the action of calcitonin (CT) and parathyroid hormone (PTH) on growth plate chondrocytes, cells directly involved in longitudinal bone growth and provisional calcification. In this study, primary cultures of avian growth plate chondrocytes that calcify without the supplement of beta-glycerophosphate were used to investigate the effects of synthetic human CT and 1-34 bovine PTH on (1) cell division and growth; (2) the deposition of Ca2+ and inorganic phosphate (Pi); (3) the activity of alkaline phosphatase (AP), an enzyme long associated with the mineralization process; (4) the levels of proteoglycans; and (5) the synthesis of collagens. Added continually to preconfluent cultures from day 6 until harvest, CT (1-30 nM) and PTH (0.1-1.0 nM) increased mineral deposition; the maximal increase was seen between days 18-21 at 10 nM CT (175-260%) and 0.5 nM PTH (approximately 170-280%), both p < 0.001. CT had no significant effect on cellular protein, or AP-specific activity, whereas PTH increased cellular protein, DNA, proteoglycan, and collagen content of the cultures in a dosage-dependent manner. AP activity and levels of Type II and X collagens and fibronectin in the culture medium showed a biphasic response to PTH; maximal increases were seen at 0.5 nM between days 15-18. Longer exposure (days 21-27) to PTH at higher levels (5-10 nM) caused a marked decreased in AP activity but a lesser decrease in the collagens. These results indicate that CT and PTH can act directly on chondrocytes to stimulate mineralization, but that PTH specifically stimulated cell division and synthesis of cellular and extracellular proteins by growth plate chondrocytes. The implications of these findings with regard to Ca2+ homeostasis and bone formation are discussed.

While previous studies revealed that matrix vesicles (MV) contain a nucleational core (NC) that converts to apatite when incubated with synthetic cartilage lymph, the initial mineral phase present in MV is not well characterized. This study explored the physicochemical nature of this Ca2+ and Pi-rich NC. MV, isolated from growth plate cartilage, were analyzed directly by solid-state 31P NMR, or incubated with hydrazine or NaOCl to remove organic constituents. Other samples of MV were subjected to sequential treatments with enzymes, salt solutions, and detergents to expose the NC. We examined the NC using transmission electron microscopy, energy-dispersive analysis with x-rays, and electron and x-ray diffraction, Fourier transform-infrared spectroscopy, high performance thin-layer chromatographic analysis, and SDS-polyacrylamide gel electrophoresis. We found that most of the MV proteins and lipids could be removed without destroying the NC; however, NaOCl treatment annihilated its activity. SDS-polyacrylamide gel electrophoresis showed that annexin V, a phosphatidylserine (PS)-dependent Ca2+-binding protein, was the major protein in the NC; high performance thin-layer chromatographic analysis revealed that the detergents removed the majority of the polar lipids, but left significant free cholesterol and fatty acids, and small but critical amounts of PS. Transmission electron microscopy showed that the NC was composed of clusters of approximately 1.0 nm subunits, which energy-dispersive analysis with x-rays revealed contained Ca and Pi with a Ca/P ratio of 1.06 +/- 0. 01. Electron diffraction, x-ray diffraction, and Fourier transform-infrared analysis all indicated that the NC was noncrystalline. 1H-Cross-polarization 31P NMR indicated that the solid phase of MV was an HPO42--rich mixture of amorphous calcium phosphate and a complex of PS, Ca2+, and Pi. Taken together, our findings indicate that the NC of MV is composed of an acid-phosphate-rich amorphous calcium phosphate intermixed with PS-Ca2+-Pi, annexin V, and other proteins and lipids.

Interactions with the extracellular matrix, accumulation of Ca2+, formation of matrix vesicles, and regulation of tissue pH by growth plate chondrocytes all appear to be vital to endochondral calcification. Thus, the activities of Ca2+ and H+ ions in these cells, while still embedded in their organic matrix, are of great interest. Using laser confocal imaging and sensitive Ca2+ (Indo 1) and pH (BCECF) probes, cellular Ca2+ and pH were analyzed in thin sections of freshly isolated cartilage. Mean values of cytosolic Ca2+ in cells from the various zones of the growth plate were quite similar, but levels in individual cells and subcellular compartments varied significantly. Ca2+ was elevated intensely near the periphery of cells in the zones of maturation and hypertrophy, and many Ca2+ rich particles were seen in the matrix near these cells. Levels of Ca2+ within the cells varied with time. In the proliferative region, cyclical increases and decreases in Ca2+ were seen, but there was little shedding of Ca2+ rich particles. However, after repeated Ca2+ cycling, in the zones of maturation and hypertrophy, Ca2+ rich particles were shed from the cell surface, forming what appeared to be matrix vesicles. Intracellular pH levels also varied significantly within the chondrocytes and between the cells and zones. Numerous focal elevations in pH (> 8.0) were seen in central regions of the maturing and early hypertrophic cells, with lower pH (6.5-7.2) near the cell periphery of the late hypertrophic and calcifying cells. This pattern of cytoplasmic alkalinization and subsequent acidification appears to contribute to loading of Ca2+ and Pi into matrix vesicles during their formation by the chondrocytes.

The effects of two inhibitors, fluoride (F-) and zinc (Zn2+), were studied on the formation of mineral by matrix vesicles (MV) in an in vitro system. Kinetically, mineral formation by MV incubated in a synthetic cartilage lymph (SCL) is characterized by three phases: a lag period, a period of rapid uptake, and finally a period of slow uptake. Zn2+ at > or = 5 microM completely inhibited MV mineralization; at < or = 1 microM, it had little effect on rate of ion uptake, but delayed conversion of an OCP-like intermediate into hydroxyapatite (OHAp). F- at > or = 10 microM reduced the rate of rapid uptake by MV and caused the OCP-like precursor to convert to OHAp. When synthetic OCP was seeded into SCL, mineralization ensued and OHAp became the dominant phase. With Zn2+ present, OCP-like features persisted longer; with F-, the OCP-like features were lost more rapidly. When ACP was seeded into SCL, OHAp formed; Zn2+ at < or = 1 microM caused OCP-like mineral to form. Our findings indicate that Zn2+ stabilizes a noncrystalline precursor in MV regulating the length of the lag period; Zn2+ also favors the formation of an OCP-like intermediate whose growth accounts for the rapid uptake phase. This OCP-like phase appears to nucleate formation of OHAp by MV.

Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation in growing bone, are rich in annexin V. When MVs are fused with planar phospholipid bilayers, a multiconductance Ca2+ channel is formed, with activity essentially identical to that observed when annexin V is delivered to the bilayer with phosphatidylserine liposomes. Ca2+ currents through this channel, from either MV or annexin V liposomes, are blocked by Zn2+, as is Ca2+ uptake by MV incubated in synthetic cartilage lymph. Blockage by Zn2+ was most effective when applied to the side containing the MV or liposomes. ATP and GTP differentially modulated the activity of this channel: ATP increased the amplitude of the current and the number of conductance states; GTP dramatically reduced the number of events and conductance states, leading to well-defined Ca2+ channel activity from either MV or the annexin V liposomes. In the distinctive effects of ATP, GTP, and Zn2+ on the Ca2+ channel activity observed in both the MV and the liposome systems, the common factor was the presence of annexin V. From this we conclude that Ca2+ entry into MV results from the presence of annexin V in these membrane-enclosed structures.

Previous studies revealed that matrix vesicles (MV) have an acid-labile nucleationally active core (ALNAC) essential for mineral formation; current studies were aimed at characterizing and reconstituting ALNAC. SDS-PAGE and FTIR analyses revealed the presence of lipids, proteins and amorphous calcium phosphate (ACP) in ALNAC. Extraction with chloroform-methanol reduced, but did not destroy MV calcification; treatment with chloroform-methanol-HCl destroyed all activity. This acidic solvent extracted the annexins, (phosphatidylserine (PS)-dependent Ca(2+)-binding proteins), and dissociated PS-Ca(2+)-Pi complexes present in the MV. Attempts to reconstitute ALNAC, centered on the Ca(2+)-PS-Pi complex. Various pure lipids, electrolytes and proteins were combined to form a synthetic nucleationally active complex (SNAC), analyzing the rate of Ca2+ uptake. Inclusion of phosphatidylethanolamine (PE) or sphingomyelin (SM) with PS, or Mg2+ or Zn2+ with Ca2+, strongly inhibited activity; incorporation of annexin V increased SNAC activity. Thus, approaching from either deconstruction or reconstruction, it appears that ALNAC is composed of ACP complexed with PS and the annexins. Other lipids, proteins and electrolytes modulate its activity. These findings also indicate how ALNAC must be formed in vivo.

Avian tibial dyschondroplasia (ATD), a disease characterized by an almost total lack of mineralization in affected areas of growth plate cartilage, may involve defective matrix vesicle (MV) mineralization. To explore the biochemical defect in ATD, both normal and diseased tissue were analyzed for the amount of isolatable MVs, their chemical composition, and their ability to induce mineral formation. We found significantly fewer MVs in ATD tissue, and in contrast to normal MVs, which rapidly mineralized when incubated in synthetic cartilage lymph, those isolated from ATD lesions induced only limited mineralization even after prolonged incubation. Analysis by detergent extraction revealed a nearly dysfunctional nucleational core in ATD MVs. Thus, in ATD tissue, there is a defect in the formation of MVs, and those that form are nearly inactive. There were also alterations in the lipid-dependent Ca2+(-)binding proteins (annexins) in ATD MVs. There were lower levels of annexins II and VI in endogenously produced collagenase-released matrix vesicles (CRMVs), but not in matrix vesicle-enriched microsomes (MVEMs) produced by tissue homogenization. These findings indicate that there is insufficient Ca2+ in ATD cells to enable incorporation of the annexins into MVs. Finally, there was evidence of phospholipid breakdown in ATD MVs, as well as in ATD tissue generally. This indicated that the ATD lesions were becoming necrotic. Taken together, these findings indicate that there is a defect in tissue vascularization such that the supply of mineral ions and nutrients to ATD cartilage is inadequate to support normal MV formation and subsequent mineralization.

Advances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone-related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca2+ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca2+ in their periphery and X-ray microanalytical mapping revealed the presence of many Ca(2+)- and Pi-rich cell surface structures ranging from filamentous processes 0.14 +/- 0.02 microns by 0.5-2.0 microns, to spherical globules 0.70 +/- 0.27 microns in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 +/- 0.19 micron) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca(2+)-rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca2+ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca2+ and Pi is directly involved in endochondral calcification.

Matrix vesicles (MV) were shown to initiate mineralization in cartilage and other vertebrate tissues. However, the factors that drive this process remain to be fully elucidated. Recent studies have shown that a preformed nucleational core consisting mainly of a Ca(2+)-phosphatidylserine-Pi complex, is necessary for the accumulation of Ca2+ by MV. In addition, the collagens attached to the MV surface were shown to play an important role in stimulating Ca2+ uptake. In this study, we extend this knowledge by showing that both, the nucleational core and the collagens (types II and X), are co-requirements for rapid influx of Ca2+ into intact MV. MV to which collagen fragments were attached were released from hypertrophic chicken cartilage by trypsin and collagenase digestion (trypsin/collagenase-released MV (TCRMV), while "collagen-free" MV were released by hyaluronidase and collagenase digestion (hyaluronidase/collagenase-released MV (HCRMV). In contrast to TCRMV which showed active uptake of Ca2+, HCRMV showed only little uptake. However, binding of native type II collagen to HCRMV stimulated uptake of Ca2+. Sucrose gradients separated TCRMV and HCRMV into three different density fractions: a low density top fraction (SI), an intermediate density middle fraction (SII), and a high density pellet fraction (SIII). The SIII fractions of TCRMV and HCRMV contained significantly higher levels of mineral ions than did the SI and SII fractions. Only the SIII fraction of TCRMV which contained a stable nucleational core and surface-attached collagens, showed active Ca2+ uptake; all other sucrose fractions of TCRMV and HCRMV showed little or no uptake. Detergent treatment to purposely rupture the membrane greatly enhanced Ca2+ uptake by the SIII fraction of HCRMV, presumably by exposing the internal nucleational core. Addition of either native type II or type X collagen to the intact SIII fraction of HCRMV stimulated Ca2+ uptake to a level similar to that of the SIII fraction of TCRMV; however, incubation of the SI and SII fractions of either TCRMV or HCRMV with type II or X collagen did not activate Ca2+ uptake. These findings indicate that both a functional nucleational core and surface-attached collagens need to be present to support active mineralization of MV.

Fourier-transform (FT) Raman spectroscopy was used to characterize the organic and mineral components of biological and synthetic calcium phosphate minerals. Raman spectroscopy provides information on biological minerals that is complimentary to more widely used infrared methodologies as some infrared-inactive vibrational modes are Raman-active. The application of FT-Raman technology has, for the first time, enabled the problems of high sample fluorescence and low signal-to-noise that are inherent in calcified tissues to be overcome. Raman spectra of calcium phosphates are dominated by a very strong band near 960 cm-1 that arises from the symmetric stretching mode (v1) of the phosphate group. Other Raman-active phosphate vibrational bands are seen at approximately 1075 (v3), 590 (v4), and 435 cm-1 (v2). Minerals containing acidic phosphate groups show additional vibrational modes. The different calcium phosphate mineral phases can be distinguished from one another by the relative positions and shapes of these bands in the Raman spectra. FT-Raman spectra of nascent, nonmineralized matrix vesicles (MV) show a distinct absence of the phosphate v1 band even though these structures are rich in calcium and phosphate. Similar results were seen with milk casein and synthetic Ca-phosphatidyl-serine-PO4 complexes. Hence, the phosphate and/or acidic phosphate ions in these noncrystalline biological calcium phosphates is in a molecular environment that differs from that in synthetic amorphous calcium phosphate. In MV, the first distinct mineral phase to form contained acidic phosphate bands similar to those seen in octacalcium phosphate. The mineral phase present in fully mineralized MV was much more apatitic, resembling that found in bones and teeth.(ABSTRACT TRUNCATED AT 250 WORDS)

Matrix vesicles (MV), microstructures which rapidly accumulate Ca2+ and induce mineral formation in vitro, are linked to type II and X collagens and proteoglycans in the hypertrophic cartilage. However, the roles of these matrix proteins on MV function are not known. This led us to investigate the influence of type II and X collagen binding on Ca2+ uptake by MV. MV isolated from chicken growth plate cartilage were treated with pure bacterial collagenase and 1 M NaCl in synthetic cartilage lymph to selectively and completely remove associated type II and X collagens. Uptake of 45Ca2+ by these collagen-depleted vesicles was markedly reduced. Further treatment with detergent, which disrupted the membrane, restored Ca2+ uptake, indicating that the vesicle membrane structure and the nucleational core inside the vesicle lumen were still intact after the collagenase and 1 M NaCl treatments. Readdition of either native type II or X collagen to the collagenase, 1 M NaCl-treated MV stimulated their Ca2+ uptake to levels similar to those of untreated vesicles. Pepsin-treated type II and X collagens were less effective in stimulating Ca2+ uptake, indicating that non-triple helical domains of these collagens were involved. The pepsin treatment of these collagens also decreased their binding to annexin V (anchorin CII), one of three annexins found in MV, suggesting that annexin V is involved in mediating the binding of type II and X collagens to the MV surface. Furthermore, treatment of collagenase, 1 M NaCl-treated MV with chymotrypsin, which damaged annexin V as well as many other MV proteins, prevented the stimulation of Ca2+ uptake by these collagens. Thus, the interaction between type II and X collagens with MV activates the influx of Ca2+ into MV and may play an important role in calcification of the vesicles.

Analysis of a 67 kDa lipid-dependent Ca(2+)-binding protein from avian matrix vesicles revealed amino acid sequences homologous to mammalian annexin VI. PCR methods were used to identify a clone from an avian cDNA library that contained a full length copy of the 67-kDa annexin cDNA. This was restriction mapped, subcloned and sequenced. The cDNA sequence of the open reading frame showed 70 percent identity to that of murine annexin VI; the predicted amino acid sequence, 78 percent identity. There was no homology in the 5'- and 3'-untranslated regions. A plasmid was constructed that overexpresses the intact chicken 67-kDa matrix vesicle annexin in E. coli DH5 alpha in high yield; the physicochemical properties and the amino terminal sequence of the expressed protein exactly matched those of the native protein.

The factors that drive mineralization of matrix vesicles (MV) have proven difficult to elucidate; in the present studies, various detergent, chemical, and enzyme treatments were used to reveal the nature of the nucleational core. Incubation with detergents that permeabilized the membrane enhanced calcification of treated MV incubated in synthetic cartilage lymph. While detergents removed most of the membrane lipid, they left significant amounts of the MV annexins and nearly all of the Ca2+, Pi, and Zn2+. Extraction with 1 M NaCl removed much of the Ca2+ and Pi present in MV, markedly reducing Ca2+ accumulation; these effects could be prevented by low levels of Ca2+ and Pi in the NaCl extractant. Treatment with chymotrypsin appeared to damage proteins required for MV mineralization; further treatment with detergents to bypass the membrane reactivated MV mineralization. Treatment of MV with pH 6 citrate removed Ca2+ and Pi, destroying their ability to mineralize; subsequent treatment with detergents did not reactivate these MV. Incubation of the detergent-resistant core with o-phenanthroline complexed Zn2+ and stimulated mineralization; addition of Zn2+ to synthetic cartilage lymph blocked the ability of the core to mineralize. These studies show that once the nucleational core complex is formed, the membrane-enclosed domain is no longer essential for MV calcification. Our findings indicate that the MV core contains two main components as follows: a smaller membrane-associated complex of Ca2+, Pi, phosphatidylserine, and the annexins that nucleates crystalline mineral formation, and a larger pool of Ca2+ and Pi bound to lumenal proteins. These proteins appear to bind large amounts of mineral ions, stabilize the nucleational complex, and aid its transformation to the first crystalline phase. Once nucleated, the crystalline phase appears to feed on protein-bound mineral ions until external ions enter through the MV ion channels. Zn2+ appears to regulate gating of the ion channels and conversion of the nucleational complex to the crystalline state.

Past work established that matrix vesicles (MV) are primary initiators of extracellular mineral deposition in endochondral calcification. Reviewed here are studies on how direct cellular metabolism of Ca2+ and inorganic phosphate (Pi), and cellular interaction with the matrix, are involved in the formation of calcifiable MV. Presented is a working model of how chondrocytes in growth plate (GP) cartilage are envisioned to induce the formation of calcifiable MV. In part, this model is based on recent laser confocal imaging of living cartilage tissue sections with Indo-I AM, a fluorescent permeant Ca2+ probe. These studies indicate that GP chondrocytes actively acquire Ca2+, concentrate it to the cell periphery and exfoliate it as Ca(2+)-rich MV. Data from direct chemical analysis and 31P-NMR studies on freshly isolated cells show that GP chondrocytes are depleted of ATP and have elevated cytosolic Pi, a condition prerequisite to formation of Ca(2+)-acidic phospholipid (APL)-Pi complex-primed MV. Chondrocyte cell membrane processes from which MV arise have been found to be tightly linked to the cartilage-specific extracellular matrix collagens and proteoglycans. Annexins V and VI, APL-dependent Ca(2+)-binding proteins that form Ca2+ channels in chondrocytes and MV membranes, also bind to the matrix collagens and may serve as mechano-transducers in GP cartilage, gating Ca2+ entrance into the cells and MV. This interaction between the extracellular matrix and chondrocytes appears to facilitate Ca2+ loading of chondrocytes, formation of Ca2+ and Pi-primed MV and rapid induction of mineralization in GP cartilage.

Electron microscopic studies of calcifying vertebrate tissues reveal the locus of de novo mineral formation within matrix vesicles (MV). The direct involvement of MV in the initiation of mineral formation is supported by the fact that MV isolated from avian growth plate cartilage rapidly accumulate large amounts of Ca2+ and P(i) and induce mineral formation. Exploration of the constituents of MV has revealed two major protein components, a 33 and a 36 kD protein, the former of which binds to cartilage-specific collagens. These annexin-like proteins bind to acidic phospholipids in the presence of submicromolar levels of Ca2+. Antibodies raised against both the purified 33 and the 36 kD MV annexin do not cross-react with the other, indicating that they are distinct proteins. Reported here are studies elucidating the primary structure of both MV proteins using both conventional protein and molecular biologic methods. These studies establish that the 33 kD protein is nearly identical to anchorin CII (annexin V) and that the 36 kD protein is identical to avian annexin II. Immunolocalization studies show that hypertrophic chondrocytes at the calcification front of avian growth plate contain the highest level of these annexins. Further, immunogold labeling indicates that the annexins are localized within MV isolated from the growth plate. Recent studies indicate that annexin V is a new type of ion-selective Ca2+ channel protein that possesses selective collagen binding properties. Since MV are tightly associated with the collagen- and proteoglycan-rich matrix, it is tempting to speculate that this MV protein may be a component of stretch-activated ion channels that enhance Ca2+ uptake during mechanical stress.

Expression of several cellular and matrix proteins which increase significantly during the maturation of growth plate cartilage has been shown to be affected by various endocrine and autocrine factors. In the studies reported here, transforming growth factor-beta (TGF-beta 1) and basic fibroblast growth factor (bFGF) were administered to primary cultures of avian growth plate chondrocytes at pre- or post-confluent stages to study the interplay that occurs between these factors in modulating chondrocytic phenotype. Added continuously to pre-confluent chondrocytes, TGF-beta 1 stimulated the cells to produce abundant extracellular matrix and multilayered cell growth; cell morphology was altered to a more spherical configuration. These effects were generally mimicked by bFGF, but cell shape was not affected. Administered together with TGF-beta 1, bFGF caused additive stimulation of protein synthesis, and alkaline phosphatase (AP) activity was markedly, but transiently enhanced. During this pre-confluent stage, TGF-beta 1 also increased fibronectin secretion into the culture medium. Added to post-confluent cells, TGF-beta 1 alone caused a dosage-dependent suppression of AP activity, but bFGF alone did not. Under these conditions, TGF-beta 1 and bFGF had little effect on general protein synthesis, but TGF-beta 1 alone caused large, dosage-dependent increases in synthesis of fibronectin, and to some extent type II and X collagens. Given together with bFGF, TGF-beta 1 synergistically increased secretion of fibronectin. These findings reveal that regulation of phenotypic expression in maturing growth plate chondrocytes involves complex interactions between growth factors that are determined by timing, level, continuity, and length of exposure.

With the use of the whole cell arrangement of the patch-clamp technique, an outward-directed time-dependent potassium current was identified in cultured chicken growth plate chondrocytes. This delayed rectifier potassium current (IK) activated with a sigmoidal time course during voltage steps to potentials positive to -40 mV. The half-maximal voltage required for current activation was determined to be -8 mV. The reversal potential (Erev) for IK, measured using deactivating tail currents, was -72 mV in the presence of 140 mM internal and 5 mM external [K+] solutions. Changes in external [K+] caused Erev to shift in a manner expected for a potassium-selective channel. In addition, increasing external [K+] from 5 to 50 mM caused the slope conductance of the tail currents to increase twofold. The chondrocyte IK was inhibited by the potassium-channel blocker 4-aminopyridine (4-AP) at concentrations of 0.5-4 mM and by the scorpion venom toxin charybdotoxin (CTX; 10 nM) but was unaffected by 10 mM tetraethylammonium (TEA). Addition of 20 microM ZnCl2 reduced IK in a voltage-dependent manner with the greatest inhibition found to occur at potentials near the threshold for current activation. Reduction of IK by ZnCl2 was accompanied by a slowing in the kinetics of IK activation. On the basis of the gating and pharmacological properties of this current, it is suggested that the chondrocyte channel belongs to a superfamily of K+ channels found in bone and immune system cells. The chondrocyte K+ channel may contribute to the unusually high [K+] found in the extracellular fluid of growth plate cartilage.

A previously described chondrocyte alkaline phosphatase induction factor (CAP-IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS-PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye-ligand affinity (Affi-Gel Blue and Reactive Green-19 agarose) and hydroxyapatite column chromatographies. As determined by immunoprecipitation of [35S]methionine-labeled cellular proteins after 3 day treatment, this highly purified CAP-IF increases the level of AP and certain other membrane proteins 2- to 3-fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N-terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)-inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP-IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn2+ did not reactivate the EDTA-treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8-fold during 3 day exposure. Because of the very high homology between fetuin and the A-chain of alpha 2-HS glycoprotein, we also tested and found that alpha 2HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP-inducing activity resides in a labile, cystatin/Zn(2+)-binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal growth plate cartilage differentiation.

Calcifiable proteolipids present in mineralizing tissues have been postulated to enhance apatite deposition by structuring membrane phosphatidylserine molecules into a conformation conducive to mineral formation. To examine whether proteolipid-like molecules are present in mineralizable matrix vesicles (MV), the vesicles were first extracted with chloroform/methanol (2:1, v/v), and then with chloroform/methanol/HCl (200:100:1, v/v) and the organic-soluble proteins subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Protein fractions were analyzed by Coomassie Blue staining and by immunoblot analysis of electrophoretically transferred MV protein with antisera to the 33- and 36-kDa annexins. We found that several MV proteins selectively partitioned into the lipophilic milieu under acidic conditions; however, very little protein did so at neutral pH. The principal organic-soluble MV proteins had molecular masses of 14, 33, and 36 kDa, with lesser bands at 28, 30, and 68 kDa. Immunological analyses revealed that the 33- and 36-kDa proteins were the MV annexins; the 14-kDa protein appeared to be hemoglobin, based on NH2-terminal sequencing. Our findings indicate that under acidic conditions the 33- and 36-kDa MV annexins undergo a conformational change which imparts a marked increase in the hydrophobicity of the proteins. While these observations reveal that the annexins possess proteolipid-like properties, radiolabeling and immunoprecipitation studies using [3H]myristic acid in chondrocyte cultures indicate that the MV annexins are not myristylated. Amino-terminal sequence analysis of the peptides generated by site-specific cleavage of the 33- and the 36-kDa MV annexins at tryptophan residues indicate that the 33 kDa is highly homologous to anchorin CII, a protein known to bind type II collagen, while the 36-kDa protein shares close homology with endonexin II, a tyrosine kinase substrate.