Ionizing Radiation Stimulates Expression of Pro-Osteoclastogenic Genes in Marrow and Skeletal Tissue

Abstract

Exposure to ionizing radiation can cause rapid mineral loss and increase bone-resorbing osteoclasts within metabolically active, cancellous bone tissue leading to structural deficits. To better understand mechanisms involved in rapid, radiation-induced bone loss, we determined the influence of total body irradiation on expression of select cytokines known both to stimulate osteoclastogenesis and contribute to inflammatory bone disease. Adult (16 week), male C57BL/6J mice were exposed to either 2 Gy gamma rays ((137)Cs, 0.8 Gy/min) or heavy ions ((56)Fe, 600MeV, 0.50-1.1 Gy/min); this dose corresponds to either a single fraction of radiotherapy (typical total dose is ≥10 Gy) or accumulates over long-duration interplanetary missions. Serum, marrow, and mineralized tissue were harvested 4 h-7 days later. Gamma irradiation caused a prompt (2.6-fold within 4 h) and persistent (peaking at 4.1-fold within 1 day) rise in the expression of the obligate osteoclastogenic cytokine, receptor activator of nuclear factor kappa-B ligand (Rankl), within marrow cells over controls. Similarly, Rankl expression peaked in marrow cells within 3 days of iron exposure (9.2-fold). Changes in Rankl expression induced by gamma irradiation preceded and overlapped with a rise in expression of other pro-osteoclastic cytokines in marrow (eg, monocyte chemotactic protein-1 increased by 11.9-fold, and tumor necrosis factor-alpha increased by 1.7-fold over controls). The ratio, Rankl/Opg, in marrow increased by 1.8-fold, a net pro-resorption balance. In the marrow, expression of the antioxidant transcription factor, Nfe2l2, strongly correlated with expression levels of Nfatc1, Csf1, Tnf, and Rankl. Radiation exposure increased a serum marker of bone resorption (tartrate-resistant acid phosphatase) and led to cancellous bone loss (16% decrement after 1 week). We conclude that total body irradiation (gamma or heavy-ion) caused temporal elevations in the concentrations of specific genes expressed within marrow and mineralized tissue related to bone resorption, including select cytokines that lead to osteoclastogenesis and elevated resorption; this is likely to account for rapid and progressive deterioration of cancellous microarchitecture following exposure to ionizing radiation.

FIG. 1.

2 Gy iron irradiation caused acute bone loss in the tibial metaphysis by 7 days through removal of trabecular struts. (A) Bone volume fraction (BV/TV), (B) trabecular number (Tb.N), (C) trabecular thickness (Tb.Th), and (D) trabecular spacing (Tb.Sp). Data are mean±SD, with * denoting P<0.05 versus sham.

FIG. 2.

2 Gy iron irradiation effects on cytokine gene expression in tibial marrow cells on day 3 (expression level normalized to L19). Radiation exposure increased the gene expression levels of (A) Rankl. Gene expression levels of (B) Tnf were unchanged. Data are mean±SD, with ** denoting P<0.01 versus sham.

FIG. 3.

2 Gy iron irradiation effects on gene expression in tibial tissue (sans marrow) by day 3. Comparison of expression levels of (A) Rankl, (B) Opg, (C) Rankl/Opg, (D) Acp5, and (E) Ctk genes after iron irradiation compared with controls (expression level normalized to L19). Data are mean±SD, with ** denoting P<0.01 versus sham.

FIG. 4.

2 Gy gamma radiation increased expression of pro-osteoclastic and resorption-related genes in pooled tibial and femoral marrow (expression level normalized to L19). Time course (+1, +3, and+7 days post-irradiation) for the following genes compared with sham control: (A) Rankl, (B) Opg, (C) Rankl/Opg, (D) Csf1, (E) Nfatc1, (F) Tnf, (G) Mcp1, (H) Il6, and (I) Nfe2l2. Data are mean±SD, with * denoting P<0.05 and **P<0.01 versus sham and # denoting P=0.061 for the ANOVA.

FIG. 5.

2 Gy gamma irradiation increased gene expression in femoral and tibial tissue (sans marrow) by day 3. Comparison of expression levels of (A) Rankl, (B) Opg, (C) Rankl/Opg, (D) Acp5, (E) Ctk, (F) Tnf, and (G) Nfe2l2 genes after iron irradiation compared with controls (expression level normalized to L19). Data are mean±SD, with * denoting P<0.05 and ** denoting P<0.01 versus sham.