Functional differences between growth plate apoptotic bodies and matrix vesicles.

Mineralization often occurs in areas of apoptotic changes. Our findings indicate that physiological mineralization is mediated by matrix vesicles. These matrix vesicles use mechanisms to induce mineralization that are different from the mechanisms used by apoptotic bodies released from apoptotic cells. Therefore, different therapeutic approaches must be chosen to inhibit pathological mineralization depending on the mechanism of mineralization (matrix vesicles versus apoptotic bodies). INTRODUCTION: Physiological mineralization in growth plate cartilage is restricted to regions of terminally differentiated and apoptotic chondrocytes. Pathological mineralization of tissues also often occurs in areas of apoptosis. We addressed the question of whether apoptotic changes control mineralization events or whether both events are regulated independently. METHODS: To induce mineralization, we treated growth plate chondrocytes with retinoic acid (RA); apoptosis in these cells was induced by treatment with staurosporine, anti-Fas, or TNFalpha. The degrees of mineralization and apoptosis were determined, and the structure and function of matrix vesicles and apoptotic bodies were compared. RESULTS: Release of matrix vesicles and mineralization in vivo in the growth plate occurs earlier than do apoptotic changes. To determine the functional relationship between apoptotic bodies and matrix vesicles, growth plate chondrocytes were treated with RA to induce matrix vesicle release and with staurosporine to induce release of apoptotic bodies. After 3 days, approximately 90% of staurosporine-treated chondrocytes were apoptotic, whereas only 2-4% of RA-treated cells showed apoptotic changes. RA- and staurosporine-treated chondrocyte cultures were mineralized after 3 days. Matrix vesicles isolated from RA-treated cultures and apoptotic bodies isolated from staurosporine-treated cultures were associated with calcium and phosphate. However, matrix vesicles were bigger than apoptotic bodies. Furthermore, matrix vesicles but not apoptotic bodies contained alkaline phosphatase and Ca2+ channel-forming annexins II, V, and VI. Consequently, matrix vesicles but not apoptotic bodies were able to take up Ca2+ and form the first mineral phase inside their lumen. Mineralization of RA-treated cultures was inhibited by antibodies specific for annexin V but not mineralization of staurosporine-treated cultures. CONCLUSION: Physiological mineralization of growth plate chondrocytes is initiated by specialized matrix vesicles and requires alkaline phosphatase and annexins. In contrast, mineral formation mediated by apoptotic bodies occurs by a default mechanism and does not require alkaline phosphatase and annexins.     

Establishment of the primary structure of the major lipid-dependent Ca2+ binding proteins of chicken growth plate cartilage matrix vesicles: identity with anchorin CII (annexin V) and annexin II.

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.     

Matrix Vesicles Promote Mineralization in a Gelatin Gel

Extracellular matrix vesicles (MVs) are associated with initial calcification in a variety of tissues, but the mechanisms by which they promote mineralization are not certain. In this study, MVs isolated from fourth passage rat growth plate chondrocyte cultures were included within a gelatin gel into which calcium and phosphate ions diffused from opposite ends. In this gel, apatite formation occurs by 3.5 days in the absence of mineralization promoters, allowing measurement of the ability of different factors to ``nucleate' apatite before this time or to assess the effects of molecules which modulate the rate and extent of mineral deposition. Mineral ion accumulation and crystal type are assayed at 5 days. In this study, MV protein content in the central band of a 10% gelatin gel was varied by including 100 μl of a Tris-buffered solution containing 0–300 μg/ml MV protein. There was a concentration-dependent increase in mineral accretion. Whereas 10 μg MV protein in the gel did not significantly promote apatite formation as compared with vesicle-free gels, 20 and 30 μg MV protein in the gel did promote apatite deposition. Inclusion of 10 mM β-glycerophosphate in the gels, along with MVs, did not significantly increase apatite formation despite the demonstrable alkaline phosphatase activity of the MVs. In contrast, MVs at all concentrations significantly increased apatite accumulation when proteoglycan aggregates or ATP, inhibitors of apatite formation and proliferation, were included in the gel. Slight increases in calcium, but not phosphate accumulation, were also noted when an ionophore was included with the MVs to facilitate Ca ion transport into the vesicles. FT-IR analysis of the mineral formed in the vesicle-containing gels revealed the presence of a bone-like apatite. These data suggest that MVs facilitate mineralization by providing enzymes that modify inhibitory factors in the extracellular matrix, as well as by providing a protected environment in which mineral ions can accumulate. Received: 28 January 1996 / Accepted: 9 August 1996     

Ultrastructure of matrix vesicles in chick growth plate as revealed by quick freezing and freeze substitution

Summary The ultrastructure of extracellular membrane-bound matrix vesicles (MVs), their biogenesis, and the surrounding matrix in chick tibial growth plate were studied after quick freezing and freeze substitution (FS) in an organic solvent. There were several notable differences in the ultrastructural preservation of cartilage when FS was used as compared with conventional fixation. The ultrastructural appearance of MVs after FS was extremely variable. Within the MVs, intravesicular filaments, amorphous material, and membrane-associated undercoat structures were observed. Intravesicular filaments, similar in diameter to microfilaments seen in the cytoplasm, were attached to the inside of MV membranes. This observation indicates the similarity of MV membranes and the plasma membrane. In some MVs in the proliferative zone an electron-dense material was present along the inner side of the MV membrane. In the prehypertrophic zone, crystalline material often appeared within the electron-dense material, which may be a precursor form of hydroxyapatite. The earliest crystals observed were in MVs but not in the extracellular matrix. Regarding MV formation, in addition to budding from cell surfaces and to cellular disintegration, this study also indicates that a sequential process of extrusion of preformed cytoplasmic structures may occur. Also, small MVs measuring 25–40 nm seem to arise from the disruption of large MVs. This is a previously unreported observation on MV biogenesis. FS preserves proteoglycans in the cartilage matrix as a fine, filamentous network. Initial extracellular calcification was not associated with this network.     

Functional involvement of PHOSPHO1 in matrix vesicle-mediated skeletal mineralization.

PHOSPHO1 is a phosphatase highly expressed in bone. We studied its functional involvement in mineralization through the use of novel small molecule inhibitors. PHOSPHO1 expression was present within matrix vesicles, and inhibition of enzyme action caused a decrease in the ability of matrix vesicles to calcify. INTRODUCTION: The novel phosphatase, PHOSPHO1, belongs to the haloacid dehalogenase superfamily of hydrolases and is capable of cleaving phosphoethanolamine (PEA) and phosphocholine to generate inorganic phosphate. Our aims in this study were to examine the expression of PHOSPHO1 in murine mineralizing cells and matrix vesicles (MV) and to screen a series of small-molecule PHOSPHO1-specific inhibitors for their ability to pharmacologically inhibit the first step of MV-mediated mineralization. MATERIALS AND METHODS: q-PCR and immunohistochemistry were used to study the expression and localization profiles of PHOSPHO1. Inhibitors of PHOSPHO1's PEA hydrolase activity were discovered using high-throughput screening of commercially available chemical libraries. To asses the efficacy of these inhibitors to inhibit MV mineralization, MVs were isolated from TNAP-deficient (Akp2(-/-)) osteoblasts and induced to calcify in their presence. RESULTS: q-PCR revealed a 120-fold higher level of PHOSPHO1 expression in bone compared with a range of soft tissues. The enzyme was immunolocalized to the early hypertrophic chondrocytes of the growth plate and to osteoblasts of trabecular surfaces and infilling primary osteons of cortical bone. Isolated MVs also contained PHOSPHO1. PEA hydrolase activity was observed in sonicated MVs from Akp2(-/-) osteoblasts but not intact MVs. Inhibitors to PHOSPHO1 were identified and characterized. Lansoprazole and SCH202676 inhibited the mineralization of MVs from Akp2(-/-) osteoblasts by 56.8% and 70.7%, respectively. CONCLUSIONS: The results show that PHOSPHO1 localization is restricted to mineralizing regions of bone and growth plate and that the enzyme present within MVs is in an active state, inhibition of which decreases the capacity of MVs to mineralize. These data further support our hypothesis that PHOSPHO1 plays a role in the initiation of matrix mineralization.     

The Role of Phosphatases in the Initiation of Skeletal Mineralization

Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene cause hypophosphatasia, a heritable form of rickets and osteomalacia, caused by an arrest in the propagation of hydroxyapatite (HA) crystals onto the collagenous extracellular matrix due to accumulation of extracellular inorganic pyrophosphate (PP_i), a physiological TNAP substrate and a potent calcification inhibitor. However, TNAP knockout (Alpl ^–/– ) mice are born with a mineralized skeleton and have HA crystals in their chondrocyte- and osteoblast-derived matrix vesicles (MVs). We have shown that PHOSPHO1, a soluble phosphatase with specificity for two molecules present in MVs, phosphoethanolamine and phosphocholine, is responsible for initiating HA crystal formation inside MVs and that PHOSPHO1 and TNAP have nonredundant functional roles during endochondral ossification. Double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality, despite normal systemic phosphate and calcium levels. This strongly suggests that the P_i needed for initiation of MV-mediated mineralization is produced locally in the perivesicular space. As both TNAP and nucleoside pyrophosphohydrolase-1 (NPP1) behave as potent ATPases and pyrophosphatases in the MV compartment, our current model of the mechanisms of skeletal mineralization implicate intravesicular PHOSPHO1 function and P_i influx into MVs in the initiation of mineralization and the functions of TNAP and NPP1 in the extravesicular progression of mineralization.     

Selective enrichment of microRNAs in extracellular matrix vesicles produced by growth plate chondrocytes

Matrix vesicles (MVs) are membrane organelles found in the extracellular matrix of calcifying cells, which contain matrix processing enzymes and regulate the extracellular environment via action of these enzymes. It is unknown whether MVs are also exosomic mediators of cell–cell communication via transfer of RNA material, and specifically, microRNA (miRNA). We investigated the presence of RNA in MVs isolated from cultures of costochondral growth zone chondrocytes. Our results showed that the average yield of MV RNA was 1.93 ± 0.78 ng RNA/10⁴ cells, which was approximately 0.1% of the parent cell's total RNA. MV RNA was well-protected from RNase by the lipid membrane and was highly enriched in small RNA molecules compared to cells. Moreover, coding and non-coding small RNAs in MVs were in proportions that differed from parent cells. Enrichment of specific miRNAs was consistently observed in all three miRNA detection platforms that we used, suggesting that miRNAs are selectively packaged into MVs. MV-enriched miRNAs were related to different signaling pathways associated with bone formation. This study suggests a significant role for MVs as “matrisomes” in cell–cell communication in cartilage and bone development via transfer of specific miRNAs.     

Primary culture of rat growth plate chondrocytes: an in vitro model of growth plate histotype, matrix vesicle biogenesis and mineralization

During endochondral ossification (EO), cartilage is replaced by bone. Chondrocytes of growth plate undergo proliferation, maturation, hypertrophy, matrix vesicle (MV) biogenesis and programmed cell death (PCD, apoptosis). The in vitro system presented here provides a potential experimental model for studying in vitro differentiation and MV biogenesis in chondrocyte cultures. Chondrocytes were obtained from collagenase-digested tibial and femoral growth plate cartilage of 7-week-old rachitic rats. The isolated chondrocytes were plated as monolayers at a density of 0.5 × 10⁶ cells per 35-mm plate and grown for 17 days in BGJ_b medium supplemented with 10% fetal bovine serum, 50 μg/ml ascorbic acid. Light microscopy revealed Sirius red-positive, apparent bone matrix in layers at the surfaces of cartilaginous nodules that developed in the cultures. The central matrix was largely alcian blue staining thus resembling cartilage matrix. Electron microscopy revealed superficial areas of bone like matrix with large banded collagen fibrils, consistent with type I collagen. Most of the central matrix was cartilaginous, with small fibrils, randomly arranged consistent with type II collagen. The presence of peripheral type I and central type II and type X collagen was confirmed by immunohistochemical staining. Immunohistochemistry with anti-Bone morphogenetic proteins 2, 4 and 6 showed that BMP expression is associated with maturing hypertrophic central chondrocytes, many of which were TUNEL positive and undergoing cell death with plasma membrane breaks, hydropic swelling and cell fragmentation. During early mineralization, small radial clusters of hydroxyapatite-like mineral were associated with matrix vesicles. Collagenase digestion-released MVs from the cultures showed a high specific activity for alkaline phosphatase and demonstrated a pattern of AMP-stimulated nonradioactive ⁴⁰Calcium deposition comparable to that observed with native MVs. These studies confirm that primary cultures of rat growth plate chondrocytes are a reasonable in vitro model of growth plate histotype, MV biogenesis and programmed cell death.     

Matrix Vesicles Mediate Mineralization of Human Thyroid Cartilage

Mineralization and ossification of human thyroid cartilage first starts after the end of adolescence when the previously cartilaginous human skeleton has become ossified and the epiphyseal discs are in the process of closing. However, the mechanisms involved in mineralization and ossification of human thyroid cartilage are not well understood. Ultrastructural analysis of human thyroid cartilage revealed that mineralization started close to cartilage canals in a matrix containing gigantic collagen fibers (asbestoid fibers). Matrix vesicles were detected in mineralized areas and were often associated with needle-like crystals. For the first time we were able to isolate matrix vesicles from human thyroid cartilage by mild enzymatic digestions and ultracentrifugation. These particles were oval and varied in size; some were heavily calcified. They were enriched in alkaline phosphatase, calcium, and inorganic phosphate, suggesting that the particles contain Ca²⁺-P_i complexes. Immunoblot analysis of these vesicles revealed the presence of annexins II, V, and VI, membrane-associated, channel-forming proteins, which allow influx of Ca²⁺ into the vesicles and intralumenal crystal growth. In addition, the vesicles were associated with types II and X collagen, suggesting that this association not only anchors the vesicles to the extracellular matrix, but, as shown previously, also stimulates Ca²⁺ influx into these particles. In conclusion, matrix vesicles isolated from human thyroid cartilage contain all the components, enabling them to initiate and mediate the mineralization process in human thyroid cartilage. Received: 21 July 1999 / Accepted: 2 November 1999     

Matrix vesicles are carriers of bone morphogenetic proteins (BMPs), vascular endothelial growth factor (VEGF), and noncollagenous matrix proteins

Matrix vesicles (MVs) are well positioned in the growth plate to serve as a carrier of morphogenetic information to nearby chondrocytes and osteoblasts. Bone morphogenetic proteins (BMPs) carried in MVs could promote differentiation of these skeletal cells. Vascular endothelial growth factor (VEGF) in MVs could stimulate angiogenesis. Therefore, a study was undertaken to confirm the presence of bone morphogenetic protein (BMP)-1 through-7, VEGF, and the noncollagenous matrix proteins, bone sialoprotein (BSP), osteopontin (OPN), osteocalcin (OC), and osteonectin (ON) in isolated rat growth plate MVs. MVs were isolated from collagenase-digested rachitic rat tibial and femoral growth plates. The presence of BMP-1 through BMP-7, VEGF, BSP, ON, OPN, and OC was evaluated by Western blot, plus ELISA analyses for BMP-2 and-4 content. The alkaline phosphatase-raising ability of MV extracts on cultured rat growth plate chondrocytes was measured as a reflection of MV ability to promote chondroosseous differentiation. BMP-1 through-7, VEGF, BSP, ON, OPN, and OC were all detected by Western blot analyses. Chondrocytes treated with MV extracts showed a two-to threefold increase in alkaline phosphatase activity over control, indicating increased differentiation. Significant amounts of BMP-2 and BMP-4 were detected in MVs by ELISA. Combined, these data suggest that MVs could play an important morphogenetic role in growth plate and endochondral bone formation.     

G proteins regulate calcium channels in the luminal membranes of the rabbit nephron

The filtered calcium (Ca2+) is reabsorbed by the luminal membrane of the proximal and distal nephron. Ca2+ enters cells across apical plasma membranes along a steep electrochemical gradient, through Ca2+ channels. Regulation by various hormones implies several steps, including binding of these hormones to the basolateral membrane, interaction with G proteins, liberation of messengers, activation of kinases and finally opening of the channels at the opposite pole of the cells. In the present study, we examined whether the Ca2+ entry through the luminal membranes of proximal and distal tubules is also regulated by G proteins, by a membrane-limited process. Luminal membranes were purified from rabbit proximal and distal tubule suspensions, and their vesicles were loaded with GTPgammas or the carrier. Then, the 45Ca2+ uptake by these membrane vesicles was measured in the presence and absence of 100 mM NaCl. In the absence of Na+, intravesicular GTPgammas significantly enhanced 0.5 mM Ca2+ uptake by the proximal membrane vesicles from 0.53 +/- 0.06 to 0.72 +/- 0.06 pmol/microg/10 s (p < 0.05). In the presence of Na+, however, this effect disappeared. In the distal tubules, intravesicular GTPgammas increased 0.5 mM Ca2+ uptake in the absence (from 0.57 +/- 0.02 to 0.79 +/- 0.02 pmol/microg/10 s, p < 0.02) and in the presence (from 0.36 +/- 0.03 to 0.55 +/- 0.03 pmol/microg/10 s, p < 0.02) of Na+. The action of GTPgammas, when present, was dose dependent with a half-maximal effect at 20 microM. The distal luminal membrane is the site of two Ca2+ channels with different kinetics parameters. GTPgammas increased the Vmax value of the low-affinity component exclusively, in the presence as in the absence of Na+. Finally, Ca2+ uptake by the membranes of the two segments was differently influenced by toxins: cholera toxin slightly stimulated transport by the proximal membrane, but had no influence on the distal membrane, whereas pertussis toxin decreased the cation uptake by the distal tubule membrane exclusively. We conclude that the nature of Ca2+ channels differs in the proximal and distal luminal membranes: Ca2+ channels present in the proximal tubule and the low-affinity Ca2+ channels present in the distal tubule membranes are directly regulated by Gs and Gi proteins respectively, whereas the high-affinity Ca2+ channel in the distal tubule membrane is insensitive to any of them.     

A phorbol ester induces secretion of alkaline phosphatase activity in human osteosarcoma cells

Summary The phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) blocked the growth of, and induced the appearance of processes in the human osteosarcoma cell line U-2 OS. The phorbol ester decreased the intracellular level of alkaline phosphatase (APase) activity (as measured per mg cell protein) and caused a marked increase in the APase activity secreted from the cells into the culture medium. The secretion of APase appeared after a lag period of 4–6 hours of TPA treatment, and it could also be visualized with histological staining. Differential ultracentrifugation of the culture media showed that the APase was released to the media in the form of vesicles. The vesicles were studied by electron microscopy and appeared similar to matrix vesicles isolated from cartilage and chondrocytes. It is thus concluded that TPA is able to induce the primary steps of mineralization in these cells.     

Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD

Patients with ESRD have a high circulating calcium (Ca) x phosphate (P) product and develop extensive vascular calcification that may contribute to their high cardiovascular morbidity. However, the cellular mechanisms underlying vascular calcification in this context are poorly understood. In an in vitro model, elevated Ca or P induced human vascular smooth muscle cell (VSMC) calcification independently and synergistically, a process that was potently inhibited by serum. Calcification was initiated by release from living VSMC of membrane-bound matrix vesicles (MV) and also by apoptotic bodies from dying cells. Vesicles released by VSMC after prolonged exposure to Ca and P contained preformed basic calcium phosphate and calcified extensively. However, vesicles released in the presence of serum did not contain basic calcium phosphate, co-purified with the mineralization inhibitor fetuin-A and calcified minimally. Importantly, MV released under normal physiologic conditions did not calcify, and VSMC were also able to inhibit the spontaneous precipitation of Ca and P in solution. The potent mineralization inhibitor matrix Gla protein was found to be present in MV, and pretreatment of VSMC with warfarin markedly enhanced vesicle calcification. These data suggest that in the context of raised Ca and P, vascular calcification is a modifiable, cell-mediated process regulated by vesicle release. These vesicles contain mineralization inhibitors derived from VSMC and serum, and perturbation of the production or function of these inhibitors would lead to accelerated vascular calcification.     

Phospholipid composition and Ca2+ uptake properties of plasma membrane isolated from canine stomach smooth muscle

The Ca2+ uptake and phospholipids influencing it in plasma membrane isolated from canine stomach smooth muscle was investigated. The major phospholipids in plasma membrane were phosphatidylethanolamine (PE, 26%), phosphatidylcholine (PC, 20%) and phosphatidylserine + phosphatidylinositol (PS + PI, 15%). The cholesterol/phospholipid ratio in the membrane was 0.64. ATP-dependent Ca2+ uptake was inhibited by exogenous PE, PC and PS, but increased by PI. Saponin and phospholipase C inhibited ATP-dependent Ca2+ uptake and accelerated release of Ca2+ from actively loaded plasma membrane vesicles. In the absence of ATP, PE slightly and PI significantly increased Ca2+ uptake, but PC and PS did not affect the Ca2+ uptake. Release of Ca2+ from actively loaded vesicles was 5 times greater in the presence of PI than in its absence. Results suggest PI to be the most active of the tested phospholipids in influencing Ca2+ movement across plasma membrane.     

Kinetic analysis of substrate utilization by native and TNAP‐, NPP1‐, or PHOSPHO1‐deficient matrix vesicles

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite seed crystals in the sheltered interior of membrane‐limited matrix vesicles (MVs). Here, we have studied phosphosubstrate catalysis by osteoblast‐derived MVs at physiologic pH, analyzing the hydrolysis of ATP, ADP, and PP_i by isolated wild‐type (WT) as well as TNAP‐, NPP1‐ and PHOSPHO1‐deficient MVs. Comparison of the catalytic efficiencies identified ATP as the main substrate hydrolyzed by WT MVs. The lack of TNAP had the most pronounced effect on the hydrolysis of all physiologic substrates. The lack of PHOSPHO1 affected ATP hydrolysis via a secondary reduction in the levels of TNAP in PHOSPHO1‐deficient MVs. The lack of NPP1 did not significantly affect the kinetic parameters of hydrolysis when compared with WT MVs for any of the substrates. We conclude that TNAP is the enzyme that hydrolyzes both ATP and PP_i in the MV compartment. NPP1 does not have a major role in PP_i generation from ATP at the level of MVs, in contrast to its accepted role on the surface of the osteoblasts and chondrocytes, but rather acts as a phosphatase in the absence of TNAP. © 2010 American Society for Bone and Mineral Research     

Matrix vesicles isolated from apical pulp of rat incisors: Crystal formation in low Ca×Pi ion-product medium containing β-glycerophosphate

Summary The ultrastructure of crystal formationin vitro associated with extracellular membranebound matrix vesicles (MV) isolated from rat incisor pulp was studied in Dulbecco's modified Eagle's medium (DMEM) supplemented with an organic phosphate, Na-β-glycerophosphate (BGP). Matrix vesicles were isolated from basal regions of the pulps using a collagenase digestion and ultracentrifugation method. Isolated MV contained alkaline phosphatase (ALP) activity and had diameters of 30–200 nm. Membrane structures of the isolated MV were well preserved. Incubation of MV in DMEM in the presence of BGP caused the development of bilaminar electron densities associated with the vesicle membrane. These preceded crystal deposition which was observed in the culture medium after 3 days. Both heat-inactivated MV incubated with BGP, and fresh MV incubated in the absence of BGP failed to show crystal formation, even after 3 days. Staining of demineralized sections of mineralized MV with uranyl acetate and lead citrate, revealed numerous needle-like structures similar in shape to the untreated crystals. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite. The requirement of BGP for mineralization of these MV and the long lag time before crystal formation is probably due to the low calcium (Ca)×inorganic phosphate (Pi) ion product in the original medium. The requirement of ALP activity which would cause hydrolysis of BGP and a rise in Pi would favor the precipitation of biologic apatite from the culture medium.     

Effects of selective prostaglandin EP4 receptor antagonist on osteoclast formation and bone resorption in vitro.

Prostaglandin estradiol (PGE(2)) stimulates bone resorption by a cyclic AMP (cAMP)-dependent mechanism that involves prostaglandin E receptors of the EP2 and EP4 subtypes. We tested a potent selective EP4 antagonist (EP4RA), which blocks PGE(2) binding to EP4 receptors. We examined the effects of EP4RA on osteoclastogenesis in murine marrow cultures, on cAMP production in primary osteoblastic (POb) cell cultures, and on bone resorption in organ cultures. EP4RA (1 micromol/L) decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP(+) MNC) by 46%-48% in cultures treated with 0.1-1.0 micromol/L PGE(2) and by 96% in cultures treated with 0.01 micromol/L PGE(2). EP4RA also decreased TRAP(+) MNC formation by 60% in 1,25-dihydroxyvitamin D (1,25D)-treated cultures and by 62% in parathyroid hormone (PTH)-treated cultures. A chemically related analog of EP4RA that lacks antagonist activity did not inhibit TRAP(+) MNC formation. EP4RA decreased cAMP production in PGE(2)-treated POb by 44% but did not block cAMP response to PTH. EP4RA inhibited the increase in receptor activator of NF-kappaB ligand (RANKL) mRNA levels produced by PGE(2). In fetal rat long bone cultures, EP4RA decreased 45Ca release from control, unstimulated cultures by 12%-25% and from PGE(2)-stimulated cultures by 22%-37%. Because EP4RA partially inhibited osteoclastogenesis not only in response to PGE(2) but also in response to 1,25D and PTH, these results suggest that activation of the EP4 receptor may play a general role in osteoclastic bone resorption. EP4RA showed partial inhibition of PGE(2)-stimulated osteoclastogenesis at 1 micromol/L, but almost complete inhibition at 0.01 micromol/L PGE(2). This could be due to the limited efficacy of the antagonist at high concentrations of PGE(2), or an alternative pathway, such as activation of the EP2 receptor.     

Production of monoclonal antibody to chicken epiphyseal cartilage matrix vesicle metalloprotease to determine its localization

Previous reports demonstrated that metalloprotease was isolated from matrix vesicles (MVs) of chicken epiphyseal cartilage. In this study, we produced the monoclonal antibody against MV metalloprotease (MVPase) and its location was studied by immunohistochemical technique. Isolated MVs were injected into Balb/c mice and spleen cells were collected for fusion with myeloma cells. A monoclonal hybrid cell producing antibody against MVPase was isolated. The immunoglobulin isotype was IgG1. On western blots, this antibody reacted with a single band protein of 33KDa which corresponded with purified MVPase. In the light immunohistochemical study, immuno reactions were observed in the mesenchymal cells of bone marrow at the lowest area in hypertrophic zone of the epiphyseal cartilage. Ultrastructural immunolocalization of protein A-gold particles revealing MVPase showed that MVs contained this enzyme, and in the calcifying area, gold particles were observed at the apatite needles of ruptured MVs. According to these observations, it is suggested that MVPase was already synthesized in undifferentiated marrow bone cells and this enzyme promotes the initial MV calcification and relates closely to the growth crystalline needles of apatite.     

Matrix vesicle plasma cell membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells.

A naturally occurring nonsense truncation mutation of the inorganic pyrophosphate (PPi)-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) PC-1 is associated with spinal and periarticular ligament hyperostosis and cartilage calcification in "tiptoe walking" (ttw) mice. Thus, we tested the hypothesis that PC-1 acts directly in the extracellular matrix to restrain mineralization. Cultured osteoblastic MC3T3 cells expressed PC-1 mRNA and produced hydroxyapatite deposits at 12-14 days. NTPPPH activity increased steadily over 14 days. Transforming growth factor-beta and 1,25-dihydroxyvitamin D3 increased PC-1 and NTPPPH in matrix vesicles (MVs). Because PC-1/NTPPPH was regulated in mineralizing MC3T3 cells, we stably transfected or infected cells with recombinant adenovirus, in order to express 2- to 6-fold more PC-1. PC-1/NTPPPH and PPi content increased severalfold in MVs derived from cells transfected with PC-1. Furthermore, MC3T3 cells transfected with PC-1 deposited approximately 80-90% less hydroxyapatite (by weight) than cells transfected with empty plasmid or enzymatically inactive PC-1. ATP-dependent 45Ca precipitation by MVs from cells overexpressing active PC-1 was comparably diminished. Thus, regulation of PC-1 controls the PPi content and function of osteoblast-derived MVs and matrix hydroxyapatite deposition. PC-1 may provide a novel therapeutic target in certain disorders of bone mineralization.