Inhibition of osteoblast differentiation by tumor necrosis factor-α
Inhibition of osteoblast differentiation by tumor necrosis factor-α
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DOI：
10.1210/en.141.11.3956
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发表时间：
2000-11-01
期刊：
影响因子：
4.8
通讯作者：
Nanes, MS
中科院分区：
文献类型：
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作者：
Gilbert, L;He, XF;Nanes, MS
肿瘤坏死因子-α (TNF-α) 在骨骼疾病中发挥着关键作用，它会促进成熟成骨细胞的骨形成减少并增加破骨细胞的吸收。在这里，我们证明 TNF 抑制前体细胞分化为成骨细胞。胎儿颅盖前体细胞在 21 天内自发分化为成骨细胞表型，用 TNF-α 处理可抑制分化，表现为多层矿化结节形成减少和骨骼特异性基质蛋白骨钙素分泌减少。 TNF 的作用呈剂量依赖性，IC50 为 0.6 ng/ml，表明这些前体细胞具有高敏感性。从第2-21、2-14、7-14和7-10天添加TNF-α抑制结节形成，但在第14天后添加TNF没有效果。仅在第 7-8 天添加 TNF 就观察到分化的部分抑制，表明 TNF 可以在表型选择的关键时期发挥作用。胶原包被板上的细胞生长并不能阻止 TNF 对分化的抑制，这表明增殖细胞对胶原沉积到基质中的抑制不能单独解释 TNF 的作用。 Northern分析显示TNF抑制胰岛素样生长因子I(IGF-I)的表达。通过半定量 RT-PCR 测定，TNF 对成骨骨形态发生蛋白（BMP-2、-4 和 -6）或骨骼 LIM 蛋白（LMP-1）的表达没有影响。向胎儿颅盖前体细胞培养物中添加IGF-I或BMP-6可增强分化，但不能克服TNF抑制，表明TNF在分化途径中作用于这些蛋白质的下游。还研究了克隆性成骨细胞系 MC3T3-E1-14，它在汇合后培养物中自发获得成骨细胞表型。 TNF 抑制 MC3T3-E1-14 细胞的分化，如钙和磷酸盐存在下矿化基质形成失败所示。培养物中的持续附着和代谢、台盼蓝排除和阿拉玛蓝细胞毒性测定表明，TNF 对任何一种细胞类型都没有细胞毒性。这些结果证明 TNF-α 是成骨细胞分化的有效抑制剂，并表明 TNF 在向成骨细胞表型的进展中作用于 IGF-I、BMP 和 LMP-1 的远端。
Tumor necrosis factor-alpha (TNF-alpha) has a key role in skeletal disease in which it promotes reduced bone formation by mature osteoblasts and increased osteoclastic resorption. Here we show that TNF-inhibits differentiation of osteoblasts from precursor cells. TNF-alpha treatment of fetal calvaria precursor cells, which spontaneously differentiate to the osteoblast phenotype over 21 days, inhibited differentiation as shown by reduced formation of multilayered, mineralizing nodules and decreased secretion of the skeletal-specific matrix protein osteocalcin. The effect of TNF was dose dependent with an IC50 of 0.6 ng/ml, indicating a high sensitivity of these precursor cells. Addition of TNF-alpha from days 2-21, 2-14, 7-14, and 7-10 inhibited nodule formation but addition of TNF after day 14 had no effect. Partial inhibition of differentiation was observed with addition of TNF on only days 7-8, suggesting that TNF could act during a critical period of phenotype selection. Growth of cells on collagen-coated plates did not prevent TNF inhibition of differentiation, suggesting that inhibition of collagen deposition into matrix by proliferating cells could not, alone, explain the effect 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 the osteogenic bone morphogenic proteins (BMPs-2, -4, and -6), or skeletal LIM protein (LMP-1), as determined by semiquantitative RT-PCR. Addition of IGF-I or BMP-6 to fetal calvaria precursor cell cultures enhanced differentiation but could not overcome TNF inhibition, suggesting that TNF acted downstream of these proteins in the differentiation pathway. The clonal osteoblastic cell line, MC3T3-E1-14, which acquires the osteoblast phenotype spontaneously in postconfluent culture, was also studied. TNF inhibited differentiation of MC3T3-E1-14 cells as shown by failure of mineralized matrix formation in the presence of calcium and phosphate. TNF was not cytotoxic to either cell type as shown by continued attachment and metabolism in culture, trypan blue exclusion, and Alamar Blue cytotoxicity assay. These results demonstrate 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 progression toward the osteoblast phenotype.