The role of apoptosis in growth plate cartilage during normal and abnormal growth

Abstract

Longitudinal bone growth occurs at the growth plate where resting chondrocytes proliferate, differentiate into a hypertrophic form, and finally become terminal hypertrophic chondrocytes before giving rise to bone. Because growth occurs when proliferation exceeds cell death, alterations in cell death by apoptosis, which is essential for the maintenance of tissue homeostasis, could affect growth. Therefore, apoptosis could be an important regulator of growth plate homeostasis and maturation, and subsequently linear growth. As an example, it has been proposed that the fate of the terminal hypertrophic chondrocytes is death by apoptosis but this is still debated. Furthermore, apoptosis of proliferative chondrocytes could be a mechanism of growth retardation in growth inhibiting conditions.

We first studied apoptosis during normal growth plate maturation. Our studies revealed that apoptosis and apoptosis related proteins are developmentally regulated. When the growth plate matures and the growth rate decreases, apoptosis is increased and is observed mainly in terminal hypertrophic chondrocytes at all developmental stages. The developmental changes in apoptosis occurred concurrently with changes in the Bcl family of proteins and caspases, supporting the apoptosis data. The observed developmental changes in apoptosis of terminal hypertrophic chondrocytes and their limited number may suggest that the fate of those cells is not always death by classical apoptosis.

To investigate whether apoptosis is a mechanism of growth retardation in growth-inhibiting conditions, we chose glucocorticoid-induced growth retardation as a model. Growth plates from rats treated with dexamethasone showed increased apoptosis mainly in terminal hypertrophic chondrocytes but also in early proliferative cells. Apoptosis was associated with decreased immunoreactivity for the anti-apoptotic proteins Bcl-2 and BcI-X, and increased immunoreactivity for caspase-3, thus supporting the apoptosis data. Furthermore, because Bcl-2 lies downstream of the PTHrP signalling pathway, we immunolocalized PTHrP, which proved to be down-regulated by dexamethasone.

Our data suggest that apoptosis is a mechanism of growth retardation in dexamethasone-induced apoptosis. The premature loss of proliferative chondrocytes by apoptosis could diminish the growth potential. We used the HCS-2/8 chondrocytic cell line to examine dexamethasone-induced apoptosis of proliferative chondrocytes, the underlying mechanisms and the possible anti-apoptotic role of IGF-I. Dexamethasone induced apoptosis in a dose dependent manner after 48 and 72 hours in culture through inhibition of Akt phosphorylation. Co-culture with IGF-I protected cells from dexamethasone-induced apoptosis, most likely through signalling pathways other than Akt/Pl3K. Apoptosis was caspase-dependent, with caspase-8 activation preceding that of caspase-9.

We also studied the role of apoptosis in the growth plate in cytokine-induced growth retardation. In an organ culture of fetal rat metatarsals we found that IL- I and TNF alone severely inhibited metatarsal growth, with synergistic effects when combined. In addition to decreased proliferation, apoptosis was markedly increased in proliferative chondrocytes. Antibodies against IL-1 or TNF, and co-culture with IGF-I improved growth and decreased apoptosis. The marked loss of proliferative cells by apoptosis could explain the severe growth retardation and incomplete catch-up growth observed in chronic inflammatory conditions.