Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes

Epiphyseal chondrocytes end their life cycle through apoptosis, While this event provides a mechanism for the removal of terminally differentiated cel ls from cartilage, agents that promote this physiological process have not been defined. To address this issue, using a cell culture technique that mo dels events that take place in the growth plate, we asked the following que stions: Can agents that promote chondrocyte maturation and cartilage minera lization serve as specific triggers for cell death? Are chondrocytes suscep tible to apoptogens at a singular developmental stage? Treatment of embryon ic tibial chondrocytes with inorganic phosphate (Pi) induced death in a dos e- and time-dependent manner. Within 48 hr, 3 mM Pi increased chondrocyte d eath by 30%; lower concentrations of Pi induced death after 48 hr. To ascer tain if death was due to apoptosis, Lye evaluated Pi-induced death by a num ber of different methods and compared the results to those induced by the a poptogen, staurosporine. Analysis of the death process indicated that carti lage cells shared many of the common biological Features of the apoptotic p rocess. Thus, there was DNA fragmentation, terminal deoxynucleotidyl transf erase (TUNEL) labeling, an increase in cells in the sub-C, fraction of the cell cycle, and morphological-evidence of apoptosis. To explore the specifi city of the Pi effect, the experiment was repeated using embryonic sternal cephalic and caudal chondrocytes, cells that are at an earlier developmenta l stage than the terminally differentiated tibial cells. We noted that thes e cells remained vital despite a major increase in the medium Pi content. R esults of this study suggest that Pi is a stage-specific inducer of apoptos is in maturing chondrocytes and that this role may be linked to chondrocyte maturation and mineralization of the extracellular matrix. (C) 1999 Wiley- Liss, Inc.