Cbfa1 Acts Downstream of Annexin-mediated Alteration of Calcium Homeostasis in Regulating Growth Plate Chondrocyte Maturation

INTRODUCTION: Endochondral bone formation processes through cartilage formation and replacement of mineralizin g growth plate cartilage by bone. Growth plate chondrocytes undergo a series of differentiation events, including proliferation, hypertrophy, terminal differentiation and subsequent programmed cell death (apoptosis). Terminal differentiation of growth plat e chondrocytes is a crucial process, because it activates the invasion of blood vessels into mineralized cartilage and its replacement by bone. Previously, we have shown that retinoic acid, which stimulates terminal differentiation events, acts by increasing the transcription of three annexin proteins (annexins II, V, VI) and causing them to form calcium channels in the plasma membrane (1). The formation of annexins into channels in the plasma membrane further boosts cytosolic calcium thereby regulating terminal differentiation events, including upregulation cbfa1 gene expression (1, 2). Cbfa1, a member of the runtdomain family of transcription factors, is expressed by osteoblasts and hypertrophic chondrocytes. Cbfa1-deficient mice lack hypertrophic and terminally differentiated growth plate chondrocytes, whereas overexpression of cbfa1 in immature chondrocytes induced their terminal differentiation (3, 4). Therefore, it is possible that annexin -mediated alteration of Ca homeostasis might control terminal differentiation events through the regulation of cbfa1 expression. To test this hypothesis, we over-expressed annexin V or cbfa1 in growth plate chondrocytes by using a retroviral expression vector (RCASBP) and treated infected cells with a specific annexin channel blocker K-201 (5). METHODS: Full-length annexin V cDNA was cloned into RCASBP with a 10amino acid epitope of human c-myc tag fused to the C-terminal end of the recombinant protein. Chicken cbfa1 cDNA cloned into RCASBP was a gift from Dr. Iwamoto, Thomas Jefferson University, Philadelphia. Primary chondrocytes isolated from hypertrophic zone of day-19 chicken embryonic tibia growth plate were incubated with a high titer viral stock for 30-60 min. Thereafter, cells were cultured in Dulbecco’s modified Eagle’s medium containing 5% fetal calf serum until ~90% of the cells were infected. The protein expression levels were determined by immunoblot analysis using antibodies specific for annexin V or cbfa1. Infected growth plate chondrocytes were cultured in the absence or presence of K-201 (2 μM) for 6 days. Gene expression of annexin V, cbfa1, alkaline phosphatase (APase), and osteocalcin (OC) were determined in these cells using quantitative real time PCR and SYBR Green. The degree of mineralization in chondrocyte cultures was determined using alizarin red S staining and the released alizarin red was quantified as described previously (1). RESULTS: Hypertrophic growth plate chondrocytes infected with RCASBP containing cDNA encoding full-length annexin V or cbfa1 showed 5to 6-fold increased levels of annexin V or cbfa1 mRNA expression compared to the levels of uninfected or RCASBP alone infected cells (Fig. 1). Real time PCR analysis revealed that over-expression of annexin V led to markedly up -regulation of cbfa1, APase, and osteocalcin gene expression. These genes are involved in terminal differentiation and mineralization events. Treatment of annexin V overexpressing chondrocytes with the annexin Ca channel blocker K-201 prevented up -regulation of these genes (Fig. 1). Over-expression of cbfa1 in growth plate chondrocytes led to up-regulation of APase and osteocalcin gene expression, whereas annexin V gene expression was not affected. Treatment of cbfa1 over-expressing cells with K-201 did not alter up -regulation of APase and osteocalcin gene expression (Fig. 1). Annexin V and cbfa1 over-expressing growth plate chondrocytes showed a notable increase in the degree of mineralization compared to the degree of mineralization in uninfected or RCASBP-infected cells (Fig. 2). K-201 treatment only inhibited the increase in the degree of mineralization in annexin V over-expressing chondrocytes but not in cbfa1 over-expressing cells (Fig. 2). DISCUSSION: Previously we have shown that annexins alter Ca homeostasis in growth plate chondrocytes thereby regulating terminal differentiation of these cells (1, 2). In addition, we have demonstrated that chelating cytosolic Ca with BAPTA-AM or blocking annexin Ca channel activities prevented terminal differentiation of growth plate chondrocytes (1, 2). Annexin -mediated alteration of Ca homeostasis leads to an up-regulation of cbfa1 gene expression. In this study, we provide evidence that cbfa1 acts downstream of annexin action. Upregulated expression of cbfa1 via annexin-mediated Ca influx into growth plate chondrocytes results in up-regulation of mineralizationand terminal differentiation-related genes, such as APase and osteocalcin, and stimulation of mineralization. Increases in the cytosolic calcium concentration have been shown to activate various signaling pathways that lead to the expression of differentiation specific genes. Ca influx has been also demonstrated to activate transcription factors such as CREB and MEF-2 (6). Therefore, our findings suggest that annexin-mediated Ca influx into hypertrophic growth plate chondrocytes activates cbfa1 gene expression. Cbfa1 then activates and regulates the expression of mineralizationand terminal differentiationrelated genes, including APase and osteocalcin, leading to matrix mineralization and terminal differentiation of growth plate chondrocytes.