Role of Membranes in De Novo Calcification - American Chemical

Chapter 4

Role of Membranes in De Novo Calcification 1

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Downloaded by NORTH CAROLINA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: November 26, 1991 | doi: 10.1021/bk-1991-0444.ch004

B. D.Boyan ,Z.Schwartz ,and L. D. Swain 1

Department of Orthopaedics, University of Texas Health Sciences Center at San Antonio, San Antonio, TX 78284-7823 Hebrew University Hadassah Faculty of Dental Medicine, Jerusalem, Israel 2

The association of lipids with hydroxyapatite deposition has been well characterized. This is particularly true of the acidic phospholipids, which require mineralized tissues to be decalcified before they can be extracted. In addition, phospholipid films can serve as nucleating sites for crystal formation. Mineral deposits in calcifying cartilage are initially observed in membranebound extracellular matrix vesicles. The earliest mineral crystals are, in fact, formed in association with the vesicle membrane. Specific membrane proteins, termed proteolipids, have been shown to structure phosphatidylserine, which has an extremely high affinity for Ca , in a conformation conducive to hydroxyapatite deposition. Proteolipids have also been shown to be involved in ion transport, possibly facilitating the export of protons and import of Ca and Pi required for crystal formation. It is increasingly clear that phospholipid metabolism in the growth plate is important to the regulation of matrix vesicle-dependent mineralization. 2+

Initial hydroxyapatite formation in cartilage has been associated with matrix vesicles (1,2,3). Matrix vesicles are membrane bound extracellular organelles present in the territorial matrix of the cartilage cells and are derived from the plasma membrane of these cells (1). Transmission electron micrographs of the matrix vesicles have demonstrated that they are approximately 20-50 nm in diameter and contain an electron dense, amorphous interior (4,5). They appear to form by budding from the plasma membrane (6) in a process that has been termed non-inflammatory cell deletion (7). This implies that the process of matrix vesicle production is regulated by the cell and that it does not occur in response to inflammation but is a normal cellular event. The mechanisms by which the cells regulate events in their matrix are complex. While it is now well accepted that mineral formation begins in or on matrix 0097-6156/91/0444-0042$06.00/0 © 1991 American Chemical Society In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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vesicles (8), it is clear that the bulk phase of hydroxyapatite deposition does not require the matrix vesicle or its constituents (9,10). To ensure that calcification of cartilage is under cellular control, the initial events are tightly regulated. This paper will describe the important role matrix vesicles play in this process.


The Growth Plate The growth plate of the epiphyseal cartilage (Figure 1) is a highly organized structure which permits the longitudinal expansion of the bone under tremendous load. At the top of the growth plate, the cartilage cells (chondrocytes) are arranged in a random fashion, dispersed in a proteoglycan aggregate-rich extracellular matrix. This area is called the reserve zone or resting zone cartilage because these cells are the reservoir for the chondrocytes which will populate the growth plate. In response to a number of differentiation signals (Π), the resting zone cells begin to proliferate and align into columns which are oriented parallel to the axis of the long bone. After a set number of divisions, the cells begin to hypertrophy. The effect of this series of events is to increase the length of the cartilage. The cells in the lower proliferative cell zone and in the hypertrophic cell zone produce matrix vesicles which have an amorphous, granular appearance. In the lower hypertrophic zone, hydroxyapatite crystals are seen along the inner leaflet of the matrix vesicle membrane. At the base of the growth plate, the intercellular matrix is calcified. The crystals extend out of the matrix vesicles and are deposited in the collagenous matrix. We now have considerable information about the biochemical events which are associated with the differentiation of the growth plate. As the cells hypertrophy, the proteoglycan aggregate is degraded (12). In addition to the action of chondroitinases (13) and hyaluronidases (14), a number of proteases break down the proteoglycan core protein and link protein (15). Collagenase degrades the collagen as well (16). At the same time, the activity of alkaline phosphatase, an enzyme associated with calcification, increases. Alkaline phosphatase is enriched in matrix vesicles (17) and is often used as a marker enzyme to monitor their purification. There is a concomitant increase in phospholipase A 2 activity (18), suggesting that this enzyme is involved in the breakdown of the matrix vesicles. Once the cartilage begins to calcify, a new group of proteins is found in the matrix including the C-propeptide of cartilage-specific type II (19) and type X collagen (20) and osteocalcin (21,22), which is considered to be a bone matrix protein. Lipids in Cartilage Calcification Early studies by Irving and Wuthier (23) indicated that acidic lipids were involved in mineralization of the growth plate. Histologic analyses of the tissue using Sudan Black Β to stain the acidic lipids demonstrated that the stain was localized to the cells in the resting zone but was present in the extracellular matrix in the

In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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hypertrophic zone. It was necessary to repeatedly demineralize the tissue before all of the stainable material could be extracted with chloroformimethanol 2:1 suggesting that it was intimately associated with the mineral phase. More recently, Boskey and Posner (24) demonstrated that calcium:phospholipid:phosphate complexes (CPLX), consisting of 1 mole of calcium per mole of total phosphate (phospholipid phosphate plus inorganic phosphate), increase in content in the hypertrophic zone of the growth plate. CPLX concentration is greatest just before bulk phase mineralization occurs in the calcifying zone. In a related series of studies, Boyan and Boskey (25,26) found that CPLX formation is related to the presence of specific proteolipids, a class of membrane proteins involved in ion transport (27). The concentration of these proteins also increases in the hypertrophic zone of the growth plate (28) and both CPLX (24) and proteolipids (29) have been isolated from matrix vesicles. It is becoming increasingly clear that phospholipid metabolism in the growth plate is important to the regulation of matrix vesicle-dependent mineralization. Shapiro et al. (30) have observed that the oxidative metabolism of fatty acids in the growth plate varies with the zone of maturation. Matrix vesicle phosphatidylethanolamine content decreases whereas phosphatidylserine content is increased (31), probably through selective susceptibility to resident phospholipases. Phosphatidylethanolamine is a substrate for phospholipase A 2 (32) while CPLX containing phosphatidylserine is resistant to phospholipase A 2 (Boskey, personal communication). Numerous investigators have shown that phospholipids (33) and proteolipids (34) can play a structural role in promoting hydroxyapatite formation. Liposomes formed from phosphatidylinositol-4,5-bisphosphate and egg white lysozyme (35) serve as excellent substrates for mineral deposition in vitro. When liposomes are constructed from phosphatidylcholine, dicetyl phosphate and cholesterol (36) and pre-loaded with phosphate, they do not support hydroxyapatite formation unless an ionophore is used to permit uptake of ions. The paper by Mann et al. in this volume (37) also shows that lipid films can serve as nucleating sites for crystal formation. Perhaps the most conclusive experiment demonstrating the role of lipids in mineral formation is that performed by Raggio et al. (38). CPLX, proteolipids and non-proteolipid phospholipids were extracted from rabbit bone, placed in diffusion chambers and implanted in rabbit peritoneum. CPLX and proteolipids supported hydroxyapatite deposition whereas the non- proteolipid phospholipids bound calcium but did not calcify. In addition, CPLX formed on the proteolipidassociated phospholipids. Experiments performed in collaboration with Dr. David Howell at the University of Miami School of Medicine demonstrate that a phospholipid-containing material can be isolated from micropuncture fluid obtained from rat hypertrophic cartilage which supports hydroxyapatite formation in vitro. Most calcification studies are performed under buffered conditions where the evolution of protons during hydroxyapatite crystal formation is not an issue.

In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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However, in vivo, the lipid macromolecules described above are found in matrix vesicles which are delimited by a membrane. Thus, proteolipids may have two functions; to permit influx of calcium and phosphate ions and efflux of protons (39), and to serve as a structural entity in CPLX formation and hydroxyapatite deposition (40).


Regulation of Matrix Vesicle Lipids Since matrix vesicles are originally derived from chondrocyte plasma membranes, their production and maturation are under cellular regulation. As shown in Figure 2, there are a number of changes that take place in the matrix vesicles over time once they are released into the matrix. Cellular control of these events may occur at various points. There may be genomic regulation resulting in new gene transcription. Messenger RNA levels for matrix vesicle proteins may be differentially regulated and there may be post-translational regulation of these proteins as well. In addition, the cell may release factors into the matrix which influence activity of matrix vesicle enzymes in the matrix itself. To determine whether one or more of the mechanisms function in cartilage, we developed a chondrocyte culture model for assessing the regulation of matrix vesicles at two different stages of chondrogenic maturation. Resting zone and growth zone chondrocytes are cultured separately in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and the factor of interest. These chondrocytes retain their phenotypic markers in culture including production of matrix vesicles with distinctive lipid compositions and enzyme activities (41). The resting zone cells respond primarily to the vitamin D3 metabolite, 24,25-(OH)2D3. Non-collagenase digestible protein synthesis is stimulated (42). Matrix vesicle alkaline phosphatase is increased but phospholipase A2 activity is decreased. In contrast, growth zone chondrocytes respond primarily to 1,25(OH)2D3. Collagen synthesis and matrix vesicle alkaline phosphatase and phospholipase A2 activities are increased. In culture, plasma membrane alkaline phosphatase and phospholipase A2 activity are not changed by either metabolite in either cell type. This suggested that the vitamin D metabolites might be acting directly on the matrix vesicle membrane. To test this, we isolated matrix vesicles and plasma membranes and incubated them in vitro with l,25-(OH) D3 and 24,25-

Role of Membranes in De Novo Calcification - American Chemical

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