The role of autocrine growth factors in radiation damage to the epiphysealgrowth plate

Radiation therapy plays an important role as part of the multimodality trea tment for a number of childhood malignancies. Dose-limiting complications o f radiotherapy include skeletal abnormalities and disturbances in skeletal development within the irradiated field. The current study was undertaken t o investigate the molecular mechanisms involved in radiation-induced arrest of bone growth. Our hypotheses were: (1) Expression of autocrine growth fa ctors that regulate chondrocyte proliferation is inhibited by radiation in a specific pattern; (2) the disparity in radiosensitivity of growth plate c hondrocytes and epiphyseal chondrocytes is due to differential modulation o f autocrine growth factor expression by radiation. Given the important role these cells play in skeletal growth and development, we examined the compa rative effects of radiation on expression of specific mitogenic growth fact ors in growth plate chondrocytes. The effect of radiation on the expression of autocrine/paracrine growth factors was examined in an established avian model of epiphyseal growth plate maturation. Exposure of growth plate chon drocytes to radiation resulted in a specific pattern of biochemical and mor phological alterations that were dependent on dose and were progressive ove r time. While radiation did not affect the mRNA expression of some of the a utocrine and paracrine factors important in endochondral ossification (such as FGF2 and TGFB isoforms), it did lead to a decrease in the mRNA expressi on of PTHrP, a critically important mitogen in growth plate chondrocytes, a nd a dose-dependent decrease in the PTH/PTHrP receptor mRNA. Interestingly, PTHrP mRNA levels were not affected in irradiated epiphyseal chondrocytes, the main source of PTHrP. Given evidence indicating a role for intracellul ar calcium levels in regulating PTHrP expression, basal calcium levels in i rradiated growth plate chondrocytes;md epiphyseal chondrocytes were examine d 24 h after treatment. While cytosolic calcium levels were significantly h igher in irradiated growth plate chondrocytes, they were not significantly affected in irradiated epiphyseal chondrocytes. The importance of calcium i n mediating radiation damage to growth plate chondrocytes was further demon strated by the finding that the addition of 4.0 mM EGTA (a calcium chelator ) to the cell cultures before irradiation prevented the decrease in PTHrP m RNA levels. Since PTHrP up-regulates BCL2 levels and prevents growth plate chondrocyte maturation and apoptosis, BCL2 mRNA levels were examined in irr adiated growth plate chondrocytes, and a dose-dependent decrease was found. An increase in apoptosis was further confirmed by a fivefold increase in c aspase 3 levels in irradiated growth plate chondrocytes. The results of the current study suggest that radiation may interfere with proliferation of g rowth plate chondrocytes in part by causing an increase in cytosolic calciu m levels which in turn leads to a decrease in PTHrP mRNA. Growth plate chon drocyte PTHrP receptor mRNA expression is also inhibited by radiation, furt her decreasing PTHrP signaling. Despite subtle differences between the chic k and mammalian growth plates, further studies should provide an enhanced u nderstanding of the mechanism(s) of radiation injury to the growth plate, a s well as possibilities for new therapeutic strategies to protect the growi ng skeleton from the detrimental effects of radiotherapy. (C) 2001 by Radia tion Research Society.