Inhibition of osteoblast differentiation by tumor necrosis factor-alpha

Tumor necrosis factor-alpha (TNF-alpha) has a key role in skeletal disease in which it promotes reduced bone formation by mature osteoblasts and incre ased osteoclastic resorption. Here we show that TNF-inhibits differentiatio n of osteoblasts from precursor cells. TNF-alpha treatment of fetal calvari a precursor cells, which spontaneously differentiate to the osteoblast phen otype over 21 days, inhibited differentiation as shown by reduced formation of multilayered, mineralizing nodules and decreased secretion of the skele tal-specific matrix protein osteocalcin. The effect of TNF was dose depende nt with an IC50 of 0.6 ng/ml, indicating a high sensitivity of these precur sor cells. Addition of TNF-alpha from days 2-21, 2-14, 7-14, and 7-10 inhib ited nodule formation but addition of TNF after day 14 had no effect. Parti al inhibition of differentiation was observed with addition of TNF on only days 7-8, suggesting that TNF could act during a critical period of phenoty pe selection. Growth of cells on collagen-coated plates did not prevent TNF inhibition of differentiation, suggesting that inhibition of collagen depo sition into matrix by proliferating cells could not, alone, explain the eff ect of TNF. Northern analysis revealed that TNF inhibited the expression of insulin-like growth factor I(IGF-I). TNF had no effect on expression of th e osteogenic bone morphogenic proteins (BMPs-2, -4, and -6), or skeletal LI M protein (LMP-1), as determined by semiquantitative RT-PCR. Addition of IG F-I or BMP-6 to fetal calvaria precursor cell cultures enhanced differentia tion but could not overcome TNF inhibition, suggesting that TNF acted downs tream of these proteins in the differentiation pathway. The clonal osteobla stic cell line, MC3T3-E1-14, which acquires the osteoblast phenotype sponta neously in postconfluent culture, was also studied. TNF inhibited different iation of MC3T3-E1-14 cells as shown by failure of mineralized matrix forma tion in the presence of calcium and phosphate. TNF was not cytotoxic to eit her cell type as shown by continued attachment and metabolism in culture, t rypan blue exclusion, and Alamar Blue cytotoxicity assay. These results dem onstrate that TNF-alpha is a potent inhibitor of osteoblast differentiation and suggest that TNF acts distal to IGF-I, BMPs, and LMP-1 in the progress ion toward the osteoblast phenotype.