Investigation into white spots in the carapace of a moribund mud crab (Scylla serrata) from a white spot syndrome virus (WSSV) positive zone in Moreton Bay, Australia.

BACKGROUND: A freshly deceased mud crab (Scylla serrata) exhibiting multiple white spots under the carapace was found in Pumicestone Passage, northern Moreton Bay in May 2018. This crab was taken from within a biosecurity zone established due to a recent incursion of White Spot Syndrome Virus (WSSV) into populations of wild penaeids (Penaeus spp., Metapenaeus spp.) and crabs (Thalamita crenata) in the area. Because grossly visible white spots have been previously observed under the carapace of moribund S. serrata with white spot disease (WSD) in India, an investigation into the cause of death was undertaken. CASE REPORT: The affected S. serrata was negative for WSSV DNA when gill samples were tested by real-time PCR. Histopathology found no evidence of WSD lesions in the form of basophilic hypertrophied intranuclear inclusions in any tissues of ectodermal or mesodermal origin. Histopathology of the affected carapace showed that the white spots consisted of multiple lighter coloured foci in the exocuticle formed from concentric crystalline-like rings, which extended into the endocuticle. These were interpreted as evidence of mineral mobilisation within the carapace during the pre-moult (D1 or D2) stage of the moult cycle. The cause of death in this case therefore may have been due to moult-related complications. CONCLUSION: These observations confirm that formation of grossly visible white spots under the carapace of S. serrata are not pathognomonic for infection with WSSV. Similar observations in previous studies where WSSV was detected by PCR in this same host may have been incidental findings.

Defect in formation of functional matrix vesicles by growth plate chondrocytes in avian tibial dyschondroplasia: evidence of defective tissue vascularization.

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.

Thyroid hormone inhibits growth and stimulates terminal differentiation of epiphyseal growth plate chondrocytes.

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.

Effects of calcitonin and parathyroid hormone on calcification of primary cultures of chicken growth plate chondrocytes.

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.

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.

Effects of 24R,25- and 1alpha,25-dihydroxyvitamin D3 on mineralizing growth plate chondrocytes.

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.

Association between proteoglycans and matrix vesicles in the extracellular matrix of growth plate cartilage.

Matrix vesicles (MV) are microstructures localized to the extracellular matrix of developing hard tissues that induce mineral formation. MV proteins are not well characterized, and little is known of how they interact with the surrounding matrix. However, recent electron microscopic studies indicate that MV interact with matrix proteins in growth plate cartilage. In the studies now reported, procedures developed for dissecting various components from isolated MV led to the discovery that two major vesicle proteins (38 and 46 kDa) are readily released from MV by low ionic strength solutions. These low ionic strength-soluble proteins (LISSP) were shown to be major fragments of the link protein (LP) and hyaluronic acid-binding region (HABR) of matrix proteoglycans: they react immunologically with highly specific monoclonal antibodies to LP and HABR, and the NH2-terminal sequence of the 38-kDa LISSP is essentially identical to residues 40-78 of chicken cartilage LP and that the 46-kDa LISSP represents HABR. Release of both LISSP is enhanced by hyaluronidase treatment, indicating anchorage by a hyaluronate-mediated mechanism. Both LP and HABR are firmly attached to MV in either isotonic or hypertonic solutions. In contrast, our other studies show that dissociation of type II collagen from MV occurs only with hypertonic salts which do not release the LISSP. Thus, strong interactions occur under physiological conditions between MV and both the proteoglycans and collagens, but these take place by different mechanisms.

Identification of phospholipid-dependent calcium-binding proteins as constituents of matrix vesicles.

Uptake of mineral ions by isolated matrix vesicles (MV) incubated in synthetic cartilage lymph follows a consistent pattern. After an initial lag period, MV rapidly accumulate large amounts of Ca2+ and Pi before the appearance of crystalline mineral. The ability of MV to accumulate Ca2+ is readily destroyed by proteases, indicating that proteins are important in Ca2+ accumulation. Since MV contain significant amounts of phosphatidylserine (PS), an acidic phospholipid with affinity for Ca2+, it seemed probable that this lipid might also contribute to Ca2+ binding. The development of methods for reproducible isolation of pure active MV enabled us to search for factors responsible for the rapid accumulation of Ca2+. Reported here are studies which reveal that a set of intensely staining MV proteins, extractable with EGTA, selectively bind to Ca2+, but only in the presence of acidic phospholipids. These 30-36-kDa proteins form readily sedimentable insoluble ternary complexes of protein, Ca2+, and lipid in the presence of low levels of Ca2+. With liposomes composed of PS, alone or in combination with phosphatidylethanolamine, submicromolar levels of Ca2+ or certain other divalent cations, but not Mg2+, are sufficient to form the complexes. The physical and chemical properties of these MV proteins appear to be like those of the calpactin family of membrane-associated proteins. In fact, these MV proteins were found to cross-react with antibodies to calpactin II. Thus, calpactins appear to be important protein constituents of avian growth plate MV. This finding helps explain the enrichment in PS previously noted in MV and may also point to the mechanism by which MV rapidly accumulate Ca2+.

Differential fractionation of matrix vesicle proteins. Further characterization of the acidic phospholipid-dependent Ca2(+)-binding proteins.

Matrix vesicles (MV) initiate de novo mineralization in a variety of vertebrate-calcifying tissues. In recent studies, a quantitatively major group of MV proteins, the acidic phospholipid-dependent Ca2(+)-binding proteins (APD-CaBP) were found to be immunologically related to the annexin family of proteins that possess phospholipase A2 inhibitory activity. This finding helped explain the enrichment of phosphatidylserine as well as the presence of large amounts of complexed Ca2+ noted previously in these structures. To characterize further these annexin-like proteins, preparations of both collagenase-released MV and MV-enriched microsomes were subjected to a differential fractionation process that led to the isolation and purification to homogeneity of two of the MV APD-CaBP, a 33-kDa protein and a 36-kDa calpactin II-like protein. Polyclonal antibodies raised to each pure protein were found not to cross-react with the other, thus indicating two distinctive proteins. Measurement of the phosphatidylserine-dependent Ca2(+)-binding properties of the proteins revealed apparent Kd values of 2.5 x 10(-7) and 5.0 x 10(-7) M for the 36- and 33-kDa proteins, respectively. Such high affinities indicate that both proteins would be normally bound to the membrane of MV. Immunological studies revealed the presence of both APD-CaBP in cultured growth plate chondrocytes but not in vesicles released into the culture medium. The finding of the 33-kDa but not the 36-kDa protein in vesicles released from the calcifying matrix of the chondrocyte cultures by collagenase digestion may indicate a role for this protein in MV mineralization.

Matrix vesicle annexins exhibit proteolipid-like properties. Selective partitioning into lipophilic solvents under acidic conditions.

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.

Induction of mineral deposition by primary cultures of chicken growth plate chondrocytes in ascorbate-containing media. Evidence of an association between matrix vesicles and collagen.

A serum-free primary culture system for chicken growth plate chondrocytes has been developed which consistently undergoes mineral deposition. Upon attainment of confluency, the chondrocytes develop locally into multilayer cellular nodules leading to matrix calcification. Mineralization first occurs in matrix vesicles (MV) that are abundant in the extraterritorial matrix between the hypertrophic cells. Studies with 45Ca reveal that significant accumulation of Ca2+ occurs as early as day 12, continuing progressively throughout the culture period. By day 24, the nodules become densely calcified. Fourier transform infrared spectroscopy reveals the mineral to be similar to apatite, with features essentially identical to those of mineral formed by MV in vitro. The presence of ascorbate is critical to the culture system; in its absence, calcification is rarely observed. Ascorbate stimulates MV formation and synthesis of cellular protein, alkaline phosphatase, and especially types II and X collagens. In addition, there is strong evidence that the types II and X collagens are associated with MV. 1) Electron microscopy reveals MV embedded in a type II collagenous network; 2) Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MV using monospecific antibodies to types X and II collagen indicate that both collagens are present in specific MV fractions; 3) sucrose gradient purification of MV does not remove associated collagens; 4) graded salt extraction selectively releases type II collagen from MV; and 5) incubation of radiolabeled types II and X collagens with MV leads to their cosedimentation upon subsequent centrifugation. Taken together, the data suggest that coordinated synthesis of the collagens, alkaline phosphatase, MV formation, and Ca2+ accumulation by the cultures combine to induce mineral deposition in the multilayer nodules.

Modulation of cultured chicken growth plate chondrocytes by transforming growth factor-beta 1 and basic fibroblast growth factor.

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.

Retinoic acid stimulates matrix calcification and initiates type I collagen synthesis in primary cultures of avian weight-bearing growth plate chondrocytes.

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).

Similarity in calcium channel activity of annexin V and matrix vesicles in planar lipid bilayers.

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.

In situ levels of intracellular Ca2+ and pH in avian growth plate cartilage.

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.

A facilitative role for carbonic anhydrase activity in matrix vesicle mineralization.

Carbonic anhydrase (CA) which catalyzes the reversible hydrolysis of carbon dioxide is known to be important in osteoclastic bone resorption, however, suggested roles in calcium phosphate mineral formation have not been previously demonstrated. Biochemical evidence is provided for the presence of CA in growth plate matrix vesicles (MV) and the level of activity determined by enzyme assay. Inhibition of CA activity with the specific inhibitor acetazolamide resulted in reduced rates of MV mineralization. Other inhibitor studies showed that MV mineralization was also impaired by 4,4-diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS), a blocker of membrane bicarbonate channels. No evidence was found for the presence of any proton pumps or channels. When acetazolamide and DIDS were combined, their inhibitory effects on MV mineralization were additive. These findings suggest that MV possess a pH regulation system composed of carbonic anhydrase and a putative bicarbonate channel. This system may function in the MV by providing intraluminal buffering capacity. The control of intravesicular pH is important for the stabilization of the acid-labile nucleational core complex and in preventing the build-up of protons during calcium phosphate phase transformations.

Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: modulation by retinoic acid.

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.

Collagen-binding proteins in collagenase-released matrix vesicles from cartilage. Interaction between matrix vesicle proteins and different types of collagen.

Recent evidence indicates that matrix vesicles (MV) interact with cartilage-specific collagens and other matrix proteins. Both type II and X collagens bind to and cosediment with MV. Our companion study shows that MV also are tightly coupled to proteoglycan link proteins (LP) and hyaluronic acid-binding region (HABR) in cartilage matrix. Here we sought to identify proteins responsible for the nexus between MV and matrix collagens using affinity chromatography with types I, II, and X collagen-Sepharose columns. Elution with NaCl step-gradients in the presence of nonionic detergent was used to assess the affinity between the MV proteins and the covalently attached collagens. Several MV proteins were found to bind to native type I, II, and X collagens but none bound to denatured type I collagen. Alkaline phosphatase, proteoglycan LP and HABR, and the 33- and 67-kDa annexins, bound with varying affinities to the native type I, II and X columns. In particular, LP and HABR, the 67-kDa annexin, and alkaline phosphatase bound with high affinity to the cartilage-specific collagens, although LP, HABR, and a 37-kDa protein also bound less tightly to native type I collagen. Thus, several MV proteins bind specifically to native type II and X collagens and should promote interaction between MV and the extracellular matrix. Such interactions may be important in MV formation, or in MV-mediated mineralization.

Regulatory effect of endogenous zinc and inhibitory action of toxic metal ions on calcium accumulation by matrix vesicles in vitro.

Matrix vesicles (MV) isolated from chicken growth plate by collagenase digestion and incubated in 45Ca-labelled synthetic cartilage lymph (SCL) rapidly induce mineral formation. 45Ca uptake occurs in three distinct stages: (1) an initial lag period of limited accumulation, (2) a period of rapid ion uptake and (3) an extended period of slower uptake. Treatment of MV with buffered aqueous 1,10-phenanthroline (OP), a metal ion chelator, eliminated the lag period, promoting immediate, enhanced Ca2+ uptake. Analysis of MV for trace metals showed them to contain relatively high concentrations of Zn (1.58 mumol/g MV) and lesser amounts of Cu (0.07 mumol/g MV). At least 30-40% of the Zn was readily extractable in isosmotic buffers. Addition of Zn to SCL at levels as low as 5 microM completely inhibited MV mineralization; addition of OP to Zn-inhibited MV restored their ability to mineralize. The findings suggest that Zn2+ ions act as an endogenous regulator of MV Ca2+ uptake and that the normal lag period results from a competition between Zn2+ and Ca2+ for high affinity Ca2+ binding sites in the MV membrane or within the MV lumen. Other metals tested included Cu2+, Pb2+ and Cd2+ which had little or no effect on MV mineralization, Mn2+, which had an intermediate effect, and Al3+, which was found to be almost as inhibitory as Zn2+. This finding may have implications for aluminum-associated osteomalacia.

Characterization and reconstitution of the nucleational complex responsible for mineral formation by growth plate cartilage matrix vesicles.

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.