PMID- 11089525
OWN - NLM
STAT- MEDLINE
DCOM- 20001222
LR  - 20220309
IS  - 0013-7227 (Print)
IS  - 0013-7227 (Linking)
VI  - 141
IP  - 11
DP  - 2000 Nov
TI  - Inhibition of osteoblast differentiation by tumor necrosis factor-alpha.
PG  - 3956-64
AB  - 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.
FAU - Gilbert, L
AU  - Gilbert L
AD  - Division of Endocrinology and Metabolism, Emory University School of Medicine and 
      Veterans Affairs Medical Center, Atlanta, Georgia 30033, USA.
FAU - He, X
AU  - He X
FAU - Farmer, P
AU  - Farmer P
FAU - Boden, S
AU  - Boden S
FAU - Kozlowski, M
AU  - Kozlowski M
FAU - Rubin, J
AU  - Rubin J
FAU - Nanes, M S
AU  - Nanes MS
LA  - eng
GR  - 1R01-AR44228/AR/NIAMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Endocrinology
JT  - Endocrinology
JID - 0375040
RN  - 0 (Adaptor Proteins, Signal Transducing)
RN  - 0 (Bone Morphogenetic Proteins)
RN  - 0 (Calcium Phosphates)
RN  - 0 (Carrier Proteins)
RN  - 0 (Cytoskeletal Proteins)
RN  - 0 (Intracellular Signaling Peptides and Proteins)
RN  - 0 (LIM Domain Proteins)
RN  - 0 (PDLIM7 protein, human)
RN  - 0 (Phosphates)
RN  - 0 (RNA, Messenger)
RN  - 0 (Tumor Necrosis Factor-alpha)
RN  - 0 (alpha-tricalcium phosphate)
RN  - 0 (tetracalcium phosphate)
RN  - 67763-96-6 (Insulin-Like Growth Factor I)
RN  - 701EKV9RMN (calcium phosphate, monobasic, anhydrous)
RN  - 97Z1WI3NDX (calcium phosphate)
RN  - L11K75P92J (calcium phosphate, dibasic, anhydrous)
RN  - PQ6CK8PD0R (Ascorbic Acid)
SB  - IM
MH  - Adaptor Proteins, Signal Transducing
MH  - Animals
MH  - Ascorbic Acid/pharmacology
MH  - Blotting, Northern
MH  - Bone Morphogenetic Proteins/genetics
MH  - Bone and Bones/embryology
MH  - Calcium Phosphates/metabolism
MH  - Carrier Proteins/genetics
MH  - *Cell Differentiation/drug effects
MH  - Cell Line
MH  - Cells, Cultured
MH  - Cytoskeletal Proteins
MH  - Gene Expression
MH  - Insulin-Like Growth Factor I/genetics
MH  - Intracellular Signaling Peptides and Proteins
MH  - LIM Domain Proteins
MH  - Osteoblasts/*cytology
MH  - Phosphates/administration & dosage
MH  - RNA, Messenger/analysis
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Reverse Transcriptase Polymerase Chain Reaction
MH  - Tumor Necrosis Factor-alpha/*pharmacology
EDAT- 2000/11/23 11:00
MHDA- 2001/02/28 10:01
CRDT- 2000/11/23 11:00
PHST- 2000/11/23 11:00 [pubmed]
PHST- 2001/02/28 10:01 [medline]
PHST- 2000/11/23 11:00 [entrez]
AID - 10.1210/endo.141.11.7739 [doi]
PST - ppublish
SO  - Endocrinology. 2000 Nov;141(11):3956-64. doi: 10.1210/endo.141.11.7739.