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. In this study, we demonstrate that annexin II, V, or VI mediate Ca(2+) influx into phosphatidylserine (PS)-enriched liposomes, liposomes containing lipids extracted from authentic MV, and intact authentic MV. The annexin Ca(2+) channel blocker, K-201, not only inhibited Ca(2+) influx into fura-2-loaded PS-enriched liposomes mediated by annexin II, V, or VI, but also inhibited Ca(2+) uptake by authentic MV. Types II and X collagen only bound to liposomes in the presence of annexin V but not in the presence of annexin II or VI. Binding of these collagens to annexin V stimulated its Ca(2+) channel activities, leading to an increased Ca(2+) influx into the liposomes. These findings indicate that the formation of annexin II, V, and VI Ca(2+) channels in MV together with stimulation of annexin V channel activity by collagen (types II and X) binding can explain how MV are able to rapidly take up Ca(2+) and initiate the formation of the first crystal phase.
Highlights
Annexins are a family of proteins that has in common the ability to bind to acidic phospholipids in the presence of Ca2ϩ [1, 2]
Previous findings from our and other laboratories, showed that chymotrypsin treatment, which removes most of the annexins from matrix vesicles (MV), and zinc treatment, which inhibits annexin-mediated Ca2ϩ influx into phosphatidylserine (PS)-enriched liposomes, diminished MV Ca2ϩ uptake [12,13,14,15], suggesting that annexins II, V, and VI serve as ion channels in MV, enabling Ca2ϩ loading of the vesicles during the initial phase of mineralization
Annexins and Collagens in Biomineralization tested whether annexin II, V, and VI are able to mediate Ca2ϩ influx into MV and whether binding of type II or X collagen to annexin V is able to stimulate its Ca2ϩ channel activity by measuring Ca2ϩ influx into fura-2-loaded liposomes comprised of extracted MV lipids, or PS and phosphatidylethanolamine (PE) in a molar ratio of 9:1, in the absence or presence of annexin II, V, or VI, or complexes of annexin V and type II or Isolation of Matrix Vesicles—MV were isolated from growth plate cartilage of 6- to 8-week-old broiler strain chickens and from cultures of non-mineralizing hypertrophic and mineralizing post-hypertrophic chondrocytes as described previously [12]
Summary
Annexins are a family of proteins that has in common the ability to bind to acidic phospholipids in the presence of Ca2ϩ [1, 2]. Binding of types II and X collagen to annexin V would anchor MV to the extracellular matrix, and it might further activate the annexin V channel properties This would explain the rapid influx of Ca2ϩ into MV that is required for the formation and growth of the first crystal phase within the vesicles. To test this hypothesis, we isolated MV from growth plate cartilage and measured Ca2ϩ uptake by these particles in the absence or presence of K-201 (JTV519), a specific annexin channel inhibitor [19, 20]. Annexins and Collagens in Biomineralization tested whether annexin II, V, and VI are able to mediate Ca2ϩ influx into MV and whether binding of type II or X collagen to annexin V is able to stimulate its Ca2ϩ channel activity by measuring Ca2ϩ influx into fura-2-loaded liposomes comprised of extracted MV lipids, or PS and phosphatidylethanolamine (PE) in a molar ratio of 9:1, in the absence or presence of annexin II, V, or VI, or complexes of annexin V and type II or
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