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.

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.

Inhibition of terminal differentiation and matrix calcification in cultured avian growth plate chondrocytes by Rous sarcoma virus transformation.

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.

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).

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.

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.

Physicochemical characterization of the nucleational core of matrix vesicles.

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.

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.

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.

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.

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.

Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: evidence for cellular processing of Ca2+ and Pi prior to matrix 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.

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.

Characterization, cloning and expression of the 67-kDA annexin from chicken growth plate cartilage matrix 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.

Characterization of the nucleational core complex responsible for mineral induction by growth plate cartilage matrix vesicles.

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.

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.

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.

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.