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.

Effect of metal ions on calcifying growth plate cartilage chondrocytes.

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

Induction and characterization of metallothionein in chicken epiphyseal growth plate cartilage chondrocytes.

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

The influence of trace elements on calcium phosphate formation by matrix vesicles.

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

Metallothionein induction in calcifying growth plate cartilage chondrocytes.

Metallothionein (MT) induction was studied in mineralizing cultures of chicken growth plate chondrocytes and quantitated using a Cd-saturation assay. In serum free media, MT induction was observed for Cd concentrations of 0.1 microM and greater and at Zn concentrations of 100 microM and greater. Supplementation of culture media with cysteine and/or methionine resulted in higher levels of MT induction and reduced toxicity during Cd exposure. Maximum MT induction appeared to coincide with the earliest culture stages during which important enzymes and matrix components are being synthesized. Of non-metal MT inducers tested, sodium butyrate caused a low level induction of MT while interleukin-1 had no effect on basal MT levels. 1,25-dihydroxyvitamin D increased MT induction. The steroid hormone dexamethasone caused a reduction in basal and induced MT levels. These findings suggest that MT regulation in growth plate chondrocytes differs significantly from what is known in other cell types and that this difference may be related to the mineralization of this tissue.

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.

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.

Fourier transform Raman spectroscopy of synthetic and biological calcium phosphates.

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

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.

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.

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.

Ultrastructural and histochemical aspects of zinc accumulation by fish scales.

The effect of zinc exposure on the ultrastructure of the scales and scale associated cells of the estuarine teleost, Fundulus heteroclitus was investigated in laboratory experiments. The Timm sulfide silver stain indicated that in the calcified region of the scales, Zn was colocalized with the calcium phosphate mineral crystals. X-ray diffraction analysis showed that Zn did not have an effect on crystal structure. The scale osteoblasts of Zn-exposed fish showed an increase in the number of lysosome-like structures contained by the cytoplasm. In Zn-exposed animals, X-ray microanalysis revealed that these structures contain greatly increased levels of zinc and sulfur relative to controls. In all specimens, the lysosomes contained higher levels of Zn than either the surrounding cytoplasm or adjacent scales. The findings suggest that osteoblast lysosomes may be involved in the accumulation of Zn and other metals by fish scales by the enzymatic degradation of metallothioneins or other metal-binding proteins. This could represent an important mechanism for the detoxification of excess heavy metal ions taken up from the environment and the metabolism of essential metals by calcified tissues.

Correlation between loss of alkaline phosphatase activity and accumulation of calcium during matrix vesicle-mediated mineralization.

Activity of the bone/liver/kidney isozyme of alkaline phosphatase (AP) is known to be critical for mineralization in developing bone, although its role is unclear. The work now reported explores changes in the activity of this Zn2+-containing enzyme that occur during Ca2+ accumulation by matrix vesicles (MV). A marked loss (up to 65-70%) in AP activity was found to accompany Ca2+ accumulation by MV. These two events were highly correlated, both temporally and quantitatively. Investigation into possible causes revealed that the decline in AP activity during Ca2+ uptake was not due to action of proteases but rather resulted from interaction with the developing mineral phase, loss of metal ions (Zn2+ and Mg2+) from the active site of the enzyme, and concomitant irreversible denaturation of the enzyme. Protease inhibitors did not protect AP from loss of activity during mineralization; in contrast, protease treatments, which progressively destroyed the ability of MV to accumulate Ca2+ actually reduced loss of AP activity. These findings clearly demonstrate that AP is present at the site of MV mineralization and that its catalytic activity is profoundly reduced by the mineralization process.

Fourier transform infrared characterization of mineral phases formed during induction of mineralization by collagenase-released matrix vesicles in vitro.

Fourier transform infrared spectroscopy (FTIR) was used to characterize the organic and mineral phases present during the induction of mineral formation by collagenase-released matrix vesicles (CRMV) during incubation in a synthetic cartilage lymph in vitro. CRMV mineralization, which occurs in the absence of alkaline phosphatase organic phosphate substrates, is characterized by an initial short lag period of limited Ca2+ accumulation, followed by a period of rapid Ca2+ uptake, and finally, by a plateau period during which Ca2+ accumulation continued at a slower rate. FTIR spectra taken at timed intervals during the induction of mineralization revealed the presence of absorptions characteristic of protein, phospholipid, and mineral components in the CRMV. These became progressively more intense with time. To reveal underlying changes occurring during the successive stages of Ca2+ accumulation, FTIR spectra of nascent (or demineralized) CRMV were computer-subtracted from subsequent spectra, nulling on the C-H stretch modes characteristic of the lipid acyl chains. These difference spectra showed little change during early Ca2+ loading, revealing that mineral ions initially accumulated in a form similar to that present in nascent matrix vesicles (MV). During the period of rapid Ca2+ uptake prior to appearance of crystalline mineral, difference spectra revealed subtle changes in the carbonyl and amide nitrogen stretch modes indicative of protein conformational changes. The first definable mineral phase appeared late in the rapid Ca2+ uptake period and was a distinct, crystalline octacalcium phosphate (OCP)-like phase. With time, the OCP-like precursor became more apatitic in character. There was no evidence that any amorphous calcium phosphate phase formed during the MV mineralization sequence. The mature MV mineral phase closely resembled hydroxyapatite formed via an OCP precursor and was similar to other biological apatites that show a substantial incorporation of carbonate.