Annexin-mediated Ca2+ Influx Regulates Growth Plate Chondrocyte Maturation and Apoptosis

Wei Wang,Jinping Xu,Thorsten Kirsch

doi:10.1074/jbc.m208868200

Wei Wang, Jinping Xu + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m208868200

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Abstract

Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca(2+) influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca(2+), whereas K-201, a specific annexin Ca(2+) channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca(2+) influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca(2+) influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes.

Highlights

Maturation of epiphyseal growth plate chondrocytes, which plays an important role during endochondral ossification, is accompanied by major changes of chondrocyte morphology, biosynthetic activities, and energy metabolism
We have shown that retinoic acid (RA) treatment led to a 3-fold increase in [Ca2ϩ]i of growth plate chondrocytes compared with the concentration of untreated cells
In this study we show that RA triggers a whole series of terminal differentiation events, including up-regulation of terminal differentiation marker genes (APase, cbfa1, osteocalcin), release of mineralization-competent matrix vesicles, subsequent mineralization, and apoptosis

Summary

Introduction

Maturation of epiphyseal growth plate chondrocytes, which plays an important role during endochondral ossification, is accompanied by major changes of chondrocyte morphology, biosynthetic activities, and energy metabolism. The present study addressed the hypothesis that annexin-mediated Ca2؉ influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. Treatment of hypertrophic growth plate chondrocytes with RA induced terminal differentiation of these cells, as indicated by up-regulation of terminal differentiation marker genes, including cbfa1 (Fig. 1A), APase (Fig. 1B), and osteocalcin (Fig. 1C), compared with the expression levels in untreated cells.

Results

Conclusion