Effect of tumor necrosis factor-α on the expression of insulin-like growth factor I and insulin-like growth factor binding protein 4 in mouse osteoblasts

Tumor necrosis factor-α (TNF-α) is a cytokine produced by immune cells, which has multiple effects on bone cells and is therefore thought to mediate changes in bone metabolism occurring during inflammation. In the present study we have investigated the effect of TNF-α on the secretion of insulin-like growth factor I (IGF-I) and IGF binding protein 4 (IGFBP-4) by clonal mouse osteoblasts (MC3T3-E1 cells) using subconfluent in vitro cultures and serum-free conditions. The IGF-I was determined by radioimmunoassay under conditions eliminating the interference of IGFBPs. Treatment of MC3T3-E1 cultures with TNF-α for 24h resulted in a dose-dependent decrease in IGF-I secretion (maximally to 34 ± 9.7% of control with 60 pmol/l TNF-α; mean ± SD). The TNF-α treatment also resulted in decreased messenger ribonucleic acid (mRNA) levels of IGF-I at 4 and 24h, as detected by Northern analysis. Because basal secretion of IGFBPs is very low in MC3T3-E1 cells, effects of TNF-α on IGFBP secretion were studied in cultures in which IGFBP-4 expression was increased by calcitriol (1,25(OH)₂D₃) treatment. The presence of TNF-α (600 pmol/l) inhibited this calcitriol-induced stimulation of IGFBP-4 mRNA levels from 4h onwards, with complete inhibition of the calcitriol effect occurring at 24 h. We also observed a dose-dependent inhibition of calcitriol-stimulated IGFBP-4 secretion into the culture medium (as detected by Western ligand blot}, with the maximal inhibition occurring with 600 pmol/l TFN-α to 25 ± 7% of control levels. These TNF-α effects were not prevented by indomethacin treatment, suggesting that they are not dependent on prostaglandins. The DNA synthesis was reduced to 62 ± 8% of the control value by 600 pmol/l TNF-α. We conclude that secretion of IGFs and IGFBPs by osteoblasts can be modulated by TNF-α, which in turn may be responsible for some of the known effects of TNF-α on osteoblastic cell proliferation and differentiation.