Characterization of a tumor necrosis factor-responsive element which down-regulates the human osteocalcin gene.

Abstract

Tumor necrosis factor (TNF) down-regulates the production of bone matrix proteins by osteoblasts, thereby inhibiting bone formation. Osteocalcin, the major noncollagenous protein in bone, is inhibited by TNF at the transcriptional level. Mapping studies were undertaken to characterize the TNF-responsive element (TNFRE) in the osteocalcin promoter. Deletion analysis localized the TNFRE to the -522/-511 region, which contains a 9-bp palindromic motif (AGGCTGCCT). Promoter segments containing this sequence down-regulated a heterologous simian virus 40 promoter. Site-specific mutagenesis of the TNFRE eliminated TNF downregulation. Mobility shift assays demonstrated that a constitutively expressed nuclear factor bound to the TNFRE; this factor was tentatively identified as the p50 homodimer of NF-KB. TNF stimulation induced a second TNFRE-binding protein which displaced the constitutive factor. The TNF-induced protein was not inhibitable by the NF-KB consensus sequence and was unreactive with anti-NF-cB antiserum. DNase footprinting demonstrated that both factors protected the -522/-501 portion of the promoter, consistent with the results of mapping studies and competitive mobility shift assays. It is hypothesized that the generalized catabolic activities of TNF in infectious and malignant diseases may be regulated via this novel element.Tumor necrosis factor (TNF) is a highly pleiotropic cytokine which induces many proinflammatory effects, including fever, shock, cytokine and prostaglandin synthesis, and bone and cartilage resorption (3, 4). TNF-stimulated upregulation of many genes, including cytokines, cytokine receptors, immunoglobulins, major histocompatibility antigens, and modulation of human immunodeficiency virus 1 replication, involves induction of the nuclear transcription factor KB (NF-KB) (9,13,19,20,27,34,36,39). TNF receptor binding results in the phosphorylation and subsequent dissociation of the inhibitor IKB from cytoplasmic NF-KB, freeing NF-KB to bind to specific enhancer sites on DNA (2,39,47).TNF also has significant inhibitory effects on the expression of other genes. TNF plays a key role in the cachexia (wasting) secondary to malignancy, extensive burns, and chronic infections, including AIDS (28,33,43). Cachexia involves the consumption of whole-body lipid reserves, a consequence of the down-regulation of lipoprotein lipase and other lipogenic enzymes (5), as well as the decreased synthesis of many structural proteins of muscle and bone, including actin, myosin, collagen, and osteocalcin (3, 15). In contrast to inductive phenomena, the mechanism(s) by which TNF negatively regulates these various proteins has not yet been elucidated.Osteocalcin is an osteoblast-specific polypeptide which constitutes the major noncollagenous bone matrix protein (18). Osteocalcin production correlates with histomorphometric parameters of bone formation, and levels of this protein in serum have been used clinically to evaluate whole-body bone formation rates (11). Osteocalcin has therefore been widely used as ...

Genetic confirmation for a central role for TNFα in the direct action of thyroid stimulating hormone on the skeleton

“…Tnfα has been shown to inhibit osteoblast differentiation to a mineralizing phenotype and to stimulate apoptosis in vitro (15,25). Specifically, Tnfα inhibits osteoblastic function by activating NF-κB and repressing transcription of the osteocalcin gene (26,27). However, these effects cannot explain the high bone formation noted in Tnfα transgenic mice (20).…”

Tumor necrosis factor-alpha eliminates binding of NF-Y and an octamer-binding protein to the lipoprotein lipase promoter in 3T3-L1 adipocytes.

“…The loss of multiple protein-DNA interactions suggests that TNFa does not work through a distinct "response element" of its own on the LPL gene, such as those that have been found for other genes, including the MHC class I or osteocalcin genes (17,22 (24). This could also occur in adipocytes.…”

Regulation of endothelial thrombomodulin expression by inflammatory cytokines is mediated by activation of nuclear factor-kappa B

“…[32][33][34] To dissect the mechanism by which NF-B mediates cytokine-induced transcriptional repression of TM, the ability of NF-B to bind to the TM promoter was first investigated. Sequence analysis of the 5Ј untranslated region did not reveal the presence of a classic NF-B consensus site (GGGRNNYYCC) within the TM promoter (Ϫ1539 to ϩ28, relative to the transcriptional start site).…”