Chondrocyte death is linked to development of a mitochondrial membrane permeability transition in the growth plate

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Abstract
In the companion article, we reported that the local phosphate (Pi) concentration triggers apoptosis in epiphyseal chondrocytes. The goal of the current investigation was to evaluate the apoptotic process in relationship to the energy status of cells in the growth plate. For these studies, we used sections of the adolescent growth plate, as well as cells isolated from the tissue. We found that there was a maturation‐dependent loss of mitochondrial function in growth plate chondrocytes and these cells generated energy by glycolysis. Since treatment with the uncoupler 2,4‐dinitrophenol as well as the site‐specific inhibitors antimycin A and rotenone failed to elicit a further increase in the activity of the glycolytic pathway, we concluded that oxidative metabolism was minimum in these cells. Flow cytometric studies of growth plate cells and confocal microscopy of growth plate sections using the mitochondrial probes Rh123 and DiOC6(3) provided unequivocal evidence that there was loss of mitochondrial membrane potential in hypertrophic cells. Furthermore, the intrinsic fluorescence of the flavoprotein lipoamide dehydrogenase complex of the electron transport chain revealed that the mitochondria were in an oxidized state. Finally, we assessed Bcl‐2 expression in these cells. Although immunohistochemical and Western blot analysis showed that the chick cells contained a low level of the anti‐apoptotic protein Bcl‐2, reverse transcription‐polymerase chain reaction (RT‐PCR) analysis indicated that transcripts were present in chondrocytes. Based on these observations, we suggest that terminally differentiated chondrocytes undergo a maturation‐dependent loss of mitochondrial function. In concert with the low expression of Bcl‐2, they become sensitive to signals for programmed cell death. We hypothesize that Pi triggers apoptosis in these energy‐compromised cells by promoting a mitochondrial membrane transition, thereby inducing the death process. J. Cell. Physiol. 179:287–296, 1999.