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...