The transcription factor RUNX2 (Cbfa1/AML3/Pebp-2␣A) is a critical regulator of osteoblast differentiation. We investigated the effect of the inflammatory cytokine tumor necrosis factor ␣ (TNF) on the expression of RUNX2 because TNF is known to inhibit differentiation of osteoblasts from pluripotent progenitor cells. TNF treatment of fetal calvaria precursor cells or MC3T3-E1 clonal pre-osteoblastic cells caused a dose-dependent suppression of RUNX2 steady state mRNA as measured by reverse transcription-PCR. The IC 50 for TNF inhibition was 0.6 ng/ml. TNF suppression of RUNX2 mRNA was confirmed using Northern analysis. The effect of TNF was studied using isoform-specific primers that flanked unique regions of two major RUNX2 isoforms. TNF suppressed expression of the mRNA coding for the shorter MRIPV isoform by >90% while inhibiting expression of the mRNA for the longer MASNS isoform by 50%. RUNX2 nuclear content was evaluated by electrophoretic mobility shift assay using a rat osteocalcin promoter binding sequence as probe and by Western analysis. TNF reduced nuclear RUNX2 protein. Inhibition of new protein synthesis with cycloheximide failed to prevent TNF inhibition of RUNX2 mRNA, suggesting that a newly translated protein did not mediate the TNF effect. RUNX2 mRNA half-life was 1.8 h and reduced to 0.9 h by TNF. The effect of TNF on RUNX2 gene transcription was evaluated using a 0.6-kb RUNX2 promoter-luciferase reporter in MC3T3-E1 cells. TNF caused a dose-dependent inhibition of transcription to 50% of control values. The inhibitory effect of TNF was preserved with deletions to nucleotide ؊108 upstream of the translational start site; however, localization downstream of nucleotide ؊108 was obscured by loss of basal activity. Our results indicate that TNF regulates RUNX2 expression at multiple levels including destabilization of mRNA and suppression of transcription. The disproportionate inhibition of RUNX2 nuclear protein suggests that additional post-transcriptional mechanisms may be occurring. Suppression of RUNX2 by TNF may decrease osteoblast differentiation and inhibit bone formation in TNF excess states.The inflammatory cytokine tumor necrosis factor-␣ (TNF) 1 has been shown to contribute to bone loss through a variety of mechanisms that increase bone resorption and decrease bone formation. TNF has a major role as an inflammatory mediator in rheumatoid arthritis where increased bone resorption causes periarticular bone loss, and in postmenopausal osteoporosis in which there is generalized bone loss (1-5). In addition to the effects of TNF on bone resorption, TNF also inhibits the bone-forming function of osteoblasts. In mature osteoblasts TNF inhibits the expression of the skeletal matrix proteins type I collagen and osteocalcin, causes resistance to the genomic action of 1,25-dihydroxyvitamin D 3 , and increases the production of matrix metalloproteinases and pathologic paracrine factors (6 -11). We have shown previously that TNF inhibits the differentiation of new osteoblasts from precursor cells (1...

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.

Osteoblast (OB) differentiation is suppressed by tumor necrosis factor-␣ (TNF-␣), an inflammatory stimulus that is elevated in arthritis and menopause. Because OB differentiation requires the expression of the transcription factor osterix (Osx), we investigated TNF effects on Osx. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life. Inhibition was independent of new protein synthesis. Analysis of the Osx promoter revealed two transcription start sites that direct the expression of an abundant mRNA (Osx1) and an alternatively spliced mRNA (Osx2). Promoter fragments driving the expression of luciferase were constructed to identify TNF regulatory sequences. Two independent promoters were identified upstream of each transcription start site. TNF potently inhibited transcription of both promoters. Deletion and mutational analysis identified a TNF-responsive region proximal to the Osx2 start site that retained responsiveness when inserted upstream of a heterologous promoter. The TNF response region was a major binding site for nuclear proteins, although TNF did not change binding at the site. The roles of MAPK and NFB were investigated as signal mediators of TNF. Inhibitors of MEK1 and ERK1, but not of JNK or p38 kinase, abrogated TNF inhibition of Osx mRNA and promoter activity. TNF action was not prevented by blockade of NFB nuclear entry. The forced expression of high levels of NFB uncovered a proximal promoter enhancer; however, this site was not activated by TNF. The inhibitory effect of TNF on Osx expression may decrease OB differentiation in arthritis and osteoporosis. Osteoblasts (OBs)2 are derived from pluripotent precursor cells of mesenchymal origin that are capable of differentiation to chondrocytes, myocytes, adipocytes, or fibroblasts (1). Bone formation in the embryo and remodeling in the adult require that a sufficient number of precursor cells differentiate to functional OBs. New OBs are continuously required for the synthesis of bone matrix and replacement of cells becoming osteocytes or undergoing apoptosis. A coordinated expression of transcription factors determines the commitment of precursor cells toward the OB phenotype under the control of autocrine, paracrine, and hormonal stimuli. Two of these transcription factors, RUNX2 (Cbfa1/AML3/Pebp2␣A) and Osx, are required for differentiation to the OB lineage. In mice, RUNX2 gene knock-out causes a lethal mutation with a cartilaginous skeleton. RUNX2 is presumed to function as an organizer on promoters of skeletal-specific genes (2). A phenotype similar to the RUNX2 knock-out is observed with knock-out of Osx. Here the arrest in development occurs slightly later but also results in a cartilaginous skeleton (3). In addition, Osx induces OB differentiation of dispersed embryonic cells (4). RUNX2 is expressed in Osx knock-outs, suggesting that Osx functions downstream of RUNX2 in the differentiation pathway.Differentiation of precursor cells to OBs in adult bone is impaired by inflammatory stimuli. In rheumatoid arthri...

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.