Transcriptional Regulation of the Osterix (Osx, Sp7) Promoter by Tumor Necrosis Factor Identifies Disparate Effects of Mitogen-activated Protein Kinase and NFκB Pathways

Abstract

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...

Nuclear Factor-E2-related Factor-1 Mediates Ascorbic Acid Induction of Osterix Expression via Interaction with Antioxidant-Responsive Element in Bone Cells

“…However, endogenous osterix regulatory regions may contain additional AREs and other regulatory elements that have not been fully evaluated, and the AA stimulation of osterix expression in the time course experiments was based on the nucleosomal template in which the DNA is wrapped around histones. In addition, it is likely that the expression of other stage-specific transcription factors, such as Runx2, also contribute to full activation of the osterix gene during osteoblast differentiation (43)(44)(45). Some of these genes expressed in differentiated osteoblasts may also be regulated via ARE-dependent mechanisms, since putative AREs are present in the proximal promoter regions of Runx2.…”

Role of NF-κB in the skeleton

“…Yamazaki et al [43] further demonstrated that p65 can interact with the Smad1-Smad5 complex in the nucleus and disrupt its binding to target promoters. Interestingly, TNFα-mediated inhibition of Runx2 was NF-κB dependent [41], but inhibition of Osx, a downstream target of Runx, was shown to be independent of NF-κB [44]. Overall, classical NF-κB activation downregulates OB differentiation (Figure 3).…”

Regulation of osterix (Osx, Sp7) and the Osx promoter by parathyroid hormone in osteoblasts

“…Ascorbic acid and 1,25 (OH) 2 vitamin D 3 , which have positive roles in osteoblast function, have also been shown to up-regulate Osx expression (Maehata et al 2006, Xing et al 2007. Negative regulators of osteoblastogenesis, such as tumor necrosis factor-a (TNF-a) and p53, can inhibit Osx expression (Lu et al 2006, Wang et al 2006a,b, Zambetti et al 2006.…”