The Roles of Annexins and Types II and X Collagen in Matrix Vesicle-mediated Mineralization of Growth Plate Cartilage

Abstract

Annexins II, V, and VI are major components of matrix vesicles (MV), i.e. particles that have the critical role of initiating the mineralization process in skeletal tissues. Furthermore, types II and X collagen are associated with MV, and these interactions mediated by annexin V stimulate Ca 2؉ uptake and mineralization of MV. However, the exact roles of annexin II, V, and VI and the interaction between annexin V and types II and X collagen in MV function and initiation of mineralization are not well understood. Annexins are a family of proteins that has in common the ability to bind to acidic phospholipids in the presence of Ca 2ϩ (1, 2). The family consists of at least 12 members, and three of them, annexins II, V, and VI, are highly expressed in calcifying cartilage and bone (3, 4). Annexin II and V each contain four 70 -80 amino acids repeats with an annexin consensus sequence. Annexin VI contains eight such repeats. These four or eight repeats form the conserved core region, which is responsible for the Ca 2ϩ -dependent binding of the proteins to phospholipids. In contrast, the N-terminal regions of the annexins are highly variable and may contribute to the specific functions of the various annexins (1, 2).Annexins II, V, and VI are major components of matrix vesicles (MV), 1 which are particles that, after being released from the plasma membrane of hypertrophic chondrocytes or osteoblasts, have the critical role of initiating the mineralization process in cartilage and perhaps in bone (3, 5). Three independent lines of evidence indicate that annexin II, V, and VI exhibit distinct Ca 2ϩ ion channel properties. First, when inserted into artificial phosphatidylserine bilayers they form voltage-dependent Ca 2ϩ ion channels (6 -8). Second, the crystal structures of these annexins are largely ␣-helical with parallel barrels of ␣-helical domains forming a hydrophilic, charged pore through the center of the protein (6, 8, 9). Third, annexin II, V, and VI are able to mediate Ca 2ϩ influx into artificial liposomes (10;11). It was shown that annexin-mediated Ca 2ϩ influx into liposomes is rapid during the first 20 min and then reaches a plateau after 20 min (10, 11).The initial phase of MV-mediated mineralization is characterized by the uptake of mineral ions by these particles and the formation and growth of the first mineral phase inside the vesicles (5). Because MV are enclosed by a membrane, channel proteins are required to mediate the influx of mineral ions into these particles. Previous findings from our and other laboratories, showed that chymotrypsin treatment, which removes most of the annexins from MV, and zinc treatment, which inhibits annexin-mediated Ca 2ϩ influx into phosphatidylserine (PS)-enriched liposomes, diminished MV Ca 2ϩ uptake (12-15), suggesting that annexins II, V, and VI serve as ion channels in MV, enabling Ca 2ϩ loading of the vesicles during the initial phase of mineralization.Previous studies have revealed that collagen types II and X are associated with the outer surface of MV (16). We...

Proteomic Analysis of Human Osteoblastic Cells: Relevant Proteins and Functional Categories for Differentiation

“…46 ANXA2 is a calcium-dependent phospholipid binding protein located in the ECM and in MV 14,43 where they are thought to be important for Ca 2+ uptake. 47 The fact that we observed the highest expression of ANXA2 in the premineralization phase might be associated with increasing ALP activity verified at this stage and the start of Ca 2+ uptake into the MV. Interestingly, ANXA2 has been shown to be an autocrine factor for osteoclasts, increasing osteoclastogenesis and resorption.…”

Syndecan-3 Is a Selective Regulator of Chondrocyte Proliferation

“…Proteins and Antibodies-Recombinant extracellular region of syndecan-3 (amino acids 215-313) was prepared using the pGEX expression vector (Amersham Biosciences) as we described previously (27). That region was chosen because it shows no homology to other syndecans and because it has been suggested to have important roles in function (16).…”

Biophysical aspects of biomineralization

“…A distinct characteristic of each Anx is the amino-terminal region which varies in length and primary sequence and may influence the cellular functions. It has been described that AnxA2, AnxA5 and AnxA6 are involved in Ca 2+ homeostasis by mediating Ca 2+ influx into MVs (Kirsch et al 2000;Wang and Kirsch 2002;Wang et al 2005).…”