title: The role of autocrine growth factors in radiation damage to the epiphyseal growth plate.
abstract: Radiation therapy plays an important role as part of the multimodality treatment for a number of childhood malignancies. Dose-limiting complications of radiotherapy include skeletal abnormalities and disturbances in skeletal development within the irradiated field. The current study was undertaken to investigate the molecular mechanisms involved in radiation-induced arrest of bone growth. Our hypotheses were: (1) Expression of autocrine growth factors that regulate chondrocyte proliferation is inhibited by radiation in a specific pattern; (2) the disparity in radiosensitivity of growth plate chondrocytes and epiphyseal chondrocytes is due to differential modulation of autocrine growth factor expression by radiation. Given the important role these cells play in skeletal growth and development, we examined the comparative effects of radiation on expression of specific mitogenic growth factors 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 chondrocytes to radiation resulted in a specific pattern of biochemical and morphological alterations that were dependent on dose and were progressive over time. While radiation did not affect the mRNA expression of some of the autocrine and paracrine factors important in endochondral ossification (such as FGF2 and TGFB isoforms), it did lead to a decrease in the mRNA expression of PTHrP, a critically important mitogen in growth plate chondrocytes, and 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 intracellular calcium levels in regulating PTHrP expression, basal calcium levels in irradiated growth plate chondrocytes and epiphyseal chondrocytes were examined 24 h after treatment. While cytosolic calcium levels were significantly higher in irradiated growth plate chondrocytes, they were not significantly affected in irradiated epiphyseal chondrocytes. The importance of calcium in mediating radiation damage to growth plate chondrocytes was further demonstrated 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 mRNA levels. Since PTHrP up-regulates BCL2 levels and prevents growth plate chondrocyte maturation and apoptosis, BCL2 mRNA levels were examined in irradiated growth plate chondrocytes, and a dose-dependent decrease was found. An increase in apoptosis was further confirmed by a fivefold increase in caspase 3 levels in irradiated growth plate chondrocytes. The results of the current study suggest that radiation may interfere with proliferation of growth plate chondrocytes in part by causing an increase in cytosolic calcium levels which in turn leads to a decrease in PTHrP mRNA. Growth plate chondrocyte PTHrP receptor mRNA expression is also inhibited by radiation, further decreasing PTHrP signaling. Despite subtle differences between the chick and mammalian growth plates, further studies should provide an enhanced understanding of the mechanism(s) of radiation injury to the growth plate, as well as possibilities for new therapeutic strategies to protect the growing skeleton from the detrimental effects of radiotherapy.
authors.affiliation: Department of Orthopaedic Surgery, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
authors.forename: D B
authors.initials: DB
authors.lastname: Pateder
authors.forename: R A
authors.initials: RA
authors.lastname: Eliseev
authors.forename: R J
authors.initials: RJ
authors.lastname: O'Keefe
authors.forename: E M
authors.initials: EM
authors.lastname: Schwarz
authors.forename: P
authors.initials: P
authors.lastname: Okunieff
authors.forename: L S
authors.initials: LS
authors.lastname: Constine
authors.forename: J E
authors.initials: JE
authors.lastname: Puzas
authors.forename: R N
authors.initials: RN
authors.lastname: Rosier
mesh_terms: D000818:Animals; D001483:Base Sequence; D002645:Chickens; D017931:DNA Primers; D016222:Fibroblast Growth Factor 2 / Q000502:physiology*; D006132:Growth Plate / Q000378:metabolism / Q000502:physiology / Q000528:radiation effects*; D066298:In Vitro Techniques; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011878:Radiotherapy / Q000009:adverse effects; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000502:physiology*
chemical_list: D017931:DNA Primers; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D012333:RNA, Messenger; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D016212:Transforming Growth Factor beta; D016222:Fibroblast Growth Factor 2