Changes in the expression of annexin A5 gene during in vitro chondrocyte differentiation: Influence of cell attachment

Several lines of evidence indicate that annexin A5, a membrane-associated p rotein with calcium-channel activity, plays a key role in cartilage calcifi cation during endochondral ossification. As a major constituent of cartilag e matrix vesicles, which are released from microvilli of hypertrophic chond rocytes, it is involved in calcium uptake necessary for the initial stages of cartilage calcification. Little is known, however, concerning transcript ional regulation of the annexin A5 gene during chondrocyte differentiation. Here, we report on changes in annexin A5 expression by measuring mRNA and protein levels during in vitro differentiation of chick sternal chondrocyte s to tile hypertrophic phenotype. Terminal differentiation of mature sterna l chondrocytes was achieved in the presence of sodium ascorbate in high-den sity cultures growing either in monolayer or over agarose as cell aggregate s. Differentiation of chondrocytes to hypertrophic cells was followed by mo rphological analysis and by the onset of type X collagen expression. High e xpression levels of annexin A5 mRNA were detected in chondrocytes freshly i solated from the sterna by enzymatic digestion and subsequently in cells gr owing in monolayer, but annexin A5 gene transcription was rapidly downregul ated when cells were grown in suspension as aggregates over agarose. Howeve r, protein levels did not decrease probably due to its low turnover rate. I n suspension culture, annexin A5 mRNA reappeared after 3 weeks concomitantl y with segregation of the aggregates into single cells and onset of chondro cyte hypertrophy. Tile downregulation of annexin A5 expression in cells gro wing as matrix-rich aggregates was reverted when extracellular matrix compo nents were removed and cells were reseeded onto tissue culture plastic, sug gesting that cull spreading, formation of focal contacts and stress fibers stimulated annexin A5 expression in proliferating as well as in hypertrophi c chondrocytes. (C) 2001 Wiley-Liss, Inc.