title: Inhibition of osteoblast differentiation by tumor necrosis factor-alpha.
abstract: 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.
authors.affiliation: Division of Endocrinology and Metabolism, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, Georgia 30033, USA.
authors.forename: L
authors.initials: L
authors.lastname: Gilbert
authors.forename: X
authors.initials: X
authors.lastname: He
authors.forename: P
authors.initials: P
authors.lastname: Farmer
authors.forename: S
authors.initials: S
authors.lastname: Boden
authors.forename: M
authors.initials: M
authors.lastname: Kozlowski
authors.forename: J
authors.initials: J
authors.lastname: Rubin
authors.forename: M S
authors.initials: MS
authors.lastname: Nanes
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D015152:Blotting, Northern; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D001842:Bone and Bones / Q000196:embryology; D002130:Calcium Phosphates / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins; D015870:Gene Expression; D007334:Insulin-Like Growth Factor I / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D010006:Osteoblasts / Q000166:cytology*; D010710:Phosphates / Q000008:administration & dosage; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins; D002130:Calcium Phosphates; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C484380:PDLIM7 protein, human; D010710:Phosphates; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D007334:Insulin-Like Growth Factor I; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous; D001205:Ascorbic Acid