Radiation-induced suppression of the Bmp2 signal transduction pathway in the pluripotent mesenchymal cell line C2C12: an in vitro model for prevention of heterotopic ossification by radiotherapy.
Heterotopic ossification is a common complication after total hip replacement. Clinical studies showed the effectiveness of radiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to study an analogue model showing a time- and dose-dependent effect of radiation. Using cells of the defined embryonic mouse cell line C2C12, the influence of ionizing radiation on the Bmp-induced signal cascade leading to osteogenic differentiation was analyzed. Binding of iodinated Bmp2 to the receptors, Smad1 activation, and alkaline phosphatase (ALP) activity were determined in cells with or without irradiation. The cytotoxic effect of radiotherapy was evaluated using viability tests. Radiotherapy reduced formation of the Bmp2/Bmp receptor complex. This effect was dependent on dose. The phosphorylation (activation) of Smad1 decreased after irradiation in a time-dependent manner, whereas the level of total Smads was not influenced by radiotherapy. The ALP activity decreased after radiotherapy. A dose of 7 Gy delivered 6 h before or after incubation with Bmp resulted in about a 30% decrease in ALP activity. No signs of cytotoxic effects were observed within the time window studied using doses of 0 to 20 Gy. The time- and dose-dependent effect of radiotherapy for prevention of heterotopic ossification known from the results of clinical studies has an analogue in the C2C12 cell model. The primary mechanism of radiotherapy seems to be an influence on cellular responsiveness to the Bmp2-induced osteoblastic differentiation. The results suggest a down-regulation of the Bmp2/receptor complex.
Pohl
F
F
Department of Radiooncology, University of Würzburg, Würzburg, Germany. pohl@strahlentherapie.uni-wuerzburg.de
Hassel
S
S
Nohe
A
A
Flentje
M
M
Knaus
P
P
Sebald
W
W
Koelbl
O
O
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D003035:Cobalt; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D008648:Mesoderm / Q000528:radiation effects*; D051379:Mice; D008954:Models, Biological; D009999:Ossification, Heterotopic; D010006:Osteoblasts / Q000378:metabolism; D011233:Precipitin Tests; D015398:Signal Transduction / Q000528:radiation effects*; D051785:Smad Proteins; D051898:Smad1 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta
C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D051785:Smad Proteins; D051898:Smad1 Protein; C497401:Smad1 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D003035:Cobalt; D000469:Alkaline Phosphatase