Annexin V and terminal differentiation of growth plate chondrocytes

Abstract

Terminal differentiation and mineralization are the final events in endochondral bone formation and allow the replacement of cartilage by bone. Retinoic acid (RA) stimulates these events, including upregulation of expression and activity of alkaline phosphatase (APase), expression of annexins II, V, and VI proteins, which bind to membranes and form Ca²⁺ channels, expression of osteocalcin and runx2, another mineralization-related protein and terminal differentiation-related transcription factor, and ultimately mineralization. Chelating cytosolic Ca²⁺ with BAPTA-AM, interfering with annexin Ca²⁺ channel activities using K-201, a specific annexin Ca²⁺ channel blocker, or suppression of annexin V expression using siRNA inhibited these events. Overexpression of annexin V in embryonic chicken growth plate chondrocytes resulted in an increase of cytoplasmic Ca²⁺ concentration, [Ca²⁺]_i similar to [Ca²⁺]_i increase in RA-treated cultures. Overexpression of annexin V also resulted in upregulation of annexin II, annexin VI, osteocalcin, and runx2 gene expression, expression and activity of APase, and ultimately stimulation of mineralization. K-201 inhibited upregulation of osteocalcin and runx2 gene expression, APase expression and activity, and mineralization in annexin V-overexpressing growth plate chondrocytes. These findings indicate that annexins II, V, and VI alter Ca²⁺ homeostasis in growth plate chondrocytes thereby regulating terminal differentiation and mineralization events. Overexpression of annexin V is sufficient to stimulate these terminal differentiation events in growth plate chondrocytes, whereas suppression of annexin V expression inhibits these events.

Introduction

During endochondral ossification, bone structures are first cartilaginous. Chondrocytes in these cartilage anlagen organize in columns and form the growth plate. The growth plates are responsible for linear skeletal growth. Growth plate chondrocytes undergo a series of complex differentiation events eventually leading to the replacement of cartilage by bone. These differentiation events include proliferation, hypertrophy, and terminal differentiation [1]. Terminal differentiation includes upregulation of terminal differentiation and mineralization marker genes, release of mineralization-competent matrix vesicles, matrix vesicle-mediated mineralization, and programmed cell death (apoptosis) [2], [3]. These terminal differentiation events are crucial because they allow the invasion of blood capillaries and osteoblastic precursor cells into cartilage and ultimately the replacement of cartilage by bone. Previous studies have demonstrated that interfering with chondrocyte hypertrophy and terminal differentiation results in shorter and thicker limbs [4], [5]. We and others have shown that retinoic acid (RA) plays a key role in the regulation of chondrocyte hypertrophy and terminal differentiation. RA induces hypertrophic differentiation in immature proliferating chondrocytes and hypertrophic growth plate chondrocytes treated with RA undergo terminal differentiation, including mineralization and apoptosis [6], [7], [8]. In addition, we found high levels of RA in the perichondrium/periosteum surrounding growth plate cartilage and the expression of retinoic acid receptors (RARs) in prehypertrophic and hypertrophic growth plate chondrocytes, suggesting that RA may diffuse from the perichondrium/periosteum into growth plate cartilage and stimulate hypertrophic and terminal differentiation events in vivo [9].

We have previously demonstrated that RA treatment results in an increase of cytoplasmic Ca²⁺ concentration, [Ca²⁺]_i [7]. Interestingly, our previous studies also suggested that RA treatment results in an increase of annexins II, V, and VI gene expression, relocalization, and insertion of these annexins into the plasma membrane leading to annexin channel formation in growth plate chondrocytes [7]. Interestingly, chelating cytosolic Ca²⁺ with the cell-permeable cytosolic Ca²⁺ chelator BAPTA or decreasing annexin Ca²⁺ channel activities with the specific annexin channel inhibitor K-201 resulted in inhibition of terminal differentiation events in RA-treated growth plate chondrocytes, suggesting that annexin-mediated Ca²⁺ influx into growth plate chondrocytes may be a major regulator of terminal differentiation events of growth plate chondrocytes [7], [8].

Annexins II, V, and VI are also major components of matrix vesicles. Matrix vesicles are small-membrane enclosed particles, which are released from the plasma membrane of mineralization-competent growth plate chondrocytes and osteoblasts. Once these vesicles are released into the extracellular matrix, annexins enable the influx of Ca²⁺ into these particles enabling the formation of the first mineral phase inside the vesicles and initiation of mineralization [10], [11]. Furthermore, annexin V binds to types II and X collagen and these interactions stimulate its Ca²⁺ channel activities leading to increased influx of Ca²⁺ into liposomes and matrix vesicles [12]. These findings suggest that annexin V may not only form Ca²⁺ channels in growth plate chondrocytes and matrix vesicles but may also mediated cell/vesicle/matrix interactions.

To further establish the role of annexins, especially annexin V, in terminal differentiation and mineralization of growth plate chondrocytes, we overexpressed annexin V in embryonic chicken non-mineralizing hypertrophic growth plate chondrocytes using a retroviral expression vector and compared the effect of annexin V overexpression on terminal differentiation and mineralization with those stimulated by RA. Furthermore, we interfered with annexin function using a specific inhibitor of annexin channel activities or by suppressing annexin V expression using siRNA.