Licia N.Y. Wu

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 ∼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.

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 β‐glycerophosphate‐containing, serum‐free (HL‐1) and β‐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 ∼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.

Changes in Phospholipid Extractability and Composition Accompany Mineralization of Chicken Growth Plate Cartilage Matrix Vesicles

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

In vitro modeling of matrix vesicle nucleation: synergistic stimulation of mineral formation by annexin A5 and phosphatidylserine.

Annexins A5, A2, and A6 (Anx-A5, -A2, and -A6) are quantitatively major proteins of the matrix vesicle nucleational core that is responsible for mineral formation. Anx-A5 significantly activated the induction and propagation of mineral formation when incorporated into synthetic nucleation complexes made of amorphous calcium phosphate (ACP) and Anx-A5 or of phosphatidylserine (PS) plus ACP (PS-CPLX) and Anx-A5. Incorporation of Anx-A5 markedly shortened the induction time, greatly increasing the rate and overall amount of mineral formed when incubated in synthetic cartilage lymph. Constructed by the addition of Ca2+ to PS, emulsions prepared in an intracellular phosphate buffer matched in ionic composition to the intracellular fluid of growth plate chondrocytes, these biomimetic PS-CPLX nucleators had little nucleational activity. However, incorporation of Anx-A5 transformed them into potent nucleators, with significantly greater activity than those made from ACP without PS. The ability of Anx-A5 to enhance the nucleation and growth of mineral appears to stem from its ability to form two-dimensional crystalline arrays on PS-containing monolayers. However, some stimulatory effect also may result from its ability to exclude Mg2+ and HCO–3 from nucleation sites. Comparing the various annexins for their ability to activate PS-CPLX nucleation yields the following: avian cartilage Anx-A5 > human placental Anx-A5 > avian liver Anx-A5 ≥ avian cartilage Anx-A6 >> cartilage Anx-A2. The stimulatory effect of human placental Anx-A5 and avian cartilage Anx-A6 depended on the presence of PS, since in its absence they either had no effect or actually inhibited the nucleation activity of ACP. Anx-A2 did not significantly enhance mineralization.

Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens.

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

RETINOIC ACID TREATMENT ELEVATES MATRIX METALLOPROTEINASE-2 PROTEIN AND MRNA LEVELS IN AVIAN GROWTH PLATE CHONDROCYTE CULTURES

Matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling. In growth plate (GP) cartilage, extensive remodeling occurs at the calcification front. To study the potential involvement of MMPs in retinoic acid (RA) regulation of skeletal development, we studied the effect of all‐trans‐RA on MMPs levels in mineralizing chicken epiphyseal chondrocyte primary cultures. When treated for 4 day periods on days 10 and 17, RA increased levels of an ∼70 kDa gelatinase activity. The N‐terminal sequence of the first 20 amino acid residues of the purified enzyme was identical to that deduced from chicken MMP‐2 cDNA. Time‐course studies indicated that RA elevated MMP‐2 activity levels in the cultures within 16 h. This increase was inhibited by cycloheximide and was enhanced by forskolin. The increase in MMP‐2 activity induced by RA was accompanied by an increase in MMP‐2 mRNA levels and was abolished by treatment with cycloheximide. This upregulation of MMP levels by RA in GP chondrocytes is consistent with its effects on osteoblasts and osteosarcoma cells and opposite its inhibitory effects on fibroblasts and endothelial cells. It may well be related to the breakdown of the extracellular matrix in the GP and would be governed by the availability of RA at the calcification front where extensive vascularization also occurs. J. Cell. Biochem. 68:90–99, 1998.

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). J. Cell. Biochem. 65:209–230.

Effects of CaPi ratio, Ca2+ × Pi ion product, and pH of incubation fluid on accumulation of 45Ca2+ by matrix vesicles in vitro

The capacity of matrix vesicles (MV) to induce mineralization under various electrolyte conditions has not been explored. Accordingly, we examined the ability of isolated MV to induce calcification using synthetic lymphs with ranges of Ca/Pi ratio, Ca2+ x Pi ion product, and pH relevant to both normal and pathological conditions. At a fixed ion product of 2.84 mM2, 45Ca2+ uptake was supported at all Ca/Pi ratios tested, with ratios of 1.3-1.4 being optimal. Rapid ion uptake became saturated at levels greater than 2.7 mM2 when studied at a fixed Ca/Pi = 1.3, indicating a rate-limiting membrane ion porter. However, treatment of MV with non-ionic detergent did not destroy their ability to induce mineralization. At constant Ca/Pi of 1.3 and Ca2+ x Pi of 2.63 mM2, maximal uptake rates occurred at pH 7.6-7.8 over a pH range of 7.0-8.0, with significant uptake being supported only over the narrow range of pH 7.4-7.8. Studies showing that the effects of pH on amorphous calcium phosphate (ACP)-mediated calcification were very similar to those of MV, indicate that a stabilized form of internal ACP may induce crystalline mineral formation during MV-mediated calcification.

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 β‐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 (∼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 decrease 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.

Analysis and Molecular Modeling of the Formation, Structure, and Activity of the Phosphatidylserine-Calcium-Phosphate Complex Associated with Biomineralization

The nucleational core of matrix vesicles contains a complex (CPLX) of phosphatidylserine (PS), Ca2+, and inorganic phosphate (Pi) that is important to both normal and pathological calcification. Factors required for PS-CPLX formation and nucleational activity were studied using in vitro model systems and molecular dynamic simulations. Ca2+ levels required for and rates of PS-CPLX formation were monitored by light scattering at 340 nm, assessing changes in amount and particle size. Fourier transform infrared spectroscopy was used to explore changes in chemical structure and composition. Washing with pH 5 buffer was used to examine the role of amorphous calcium phosphate in CPLX nucleational activity, which was assessed by incubation in synthetic cartilage lymph with varied pH values. Addition of 4 Ca2+/PS was minimally required to form viable complexes. During the critical first 10-min reaction period, rapid reduction in particle size signaled changes in PS-CPLX structure. Fourier transform infrared spectroscopy revealed increasing mineral phosphate that became progressively deprotonated to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{PO}_{4}^{3-}\) \end{document}. This Ca2+-mediated effect was mimicked in part by increasing the Ca2+/PS reaction ratio. Molecular dynamic simulations provided key insight into initial interactions between Ca2+ and Pi and the carboxyl, amino, and phosphodiester groups of PS. Deduced interatomic distances agreed closely with previous radial distribution function x-ray-absorption fine structure measurements, except for an elongated Ca2+–N distance, suggesting additional changes in atomic structure during the critical 10-min ripening period. These findings clarify the process of PS-CPLX formation, reveal details of its structure, and provide insight into its role as a nucleator of crystalline calcium phosphate mineral formation.