title: Annexin-mediated Ca2+ influx regulates growth plate chondrocyte maturation and apoptosis.
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.
authors.affiliation: Department of Orthopaedics, University of Maryland School of Medicine, Baltimore 21201, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Jinping
authors.initials: J
authors.lastname: Xu
authors.forename: Thorsten
authors.initials: T
authors.lastname: Kirsch
mesh_terms: D000818:Animals; D017302:Annexins / Q000502:physiology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D002642:Chick Embryo; D017931:DNA Primers; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D006132:Growth Plate / Q000166:cytology / Q000187:drug effects / Q000254:growth & development*; D017136:Ion Transport; D013841:Thiazepines / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology
chemical_list: D017302:Annexins; D002614:Chelating Agents; D017931:DNA Primers; D013841:Thiazepines; C109183:K201 compound; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D014212:Tretinoin; D002118:Calcium