title: Tumor necrosis factor-alpha inhibits 1,25-dihydroxyvitamin D3-stimulated bone Gla protein synthesis in rat osteosarcoma cells (ROS 17/2.8) by a pretranslational mechanism.
abstract: Tumor necrosis factor-alpha (TNF alpha), a 17,000 mol wt protein, mediates a variety of immunological and inflammatory events. TNF alpha is a potent inhibitor of bone collagen synthesis and stimulator of osteoclastic bone resorption, the net effect of which is to cause bone loss. We have previously reported that TNF alpha inhibits the synthesis of collagen by osteoblastic cells in culture out of proportion to effects on total protein synthesis, suggesting that inhibition of bone formation by TNF alpha may be due to selective inhibition of matrix protein synthesis. To further test this hypothesis and to evaluate the mechanism of TNF alpha action, we studied the effect of TNF alpha on synthesis of the osteoblast-specific bone Gla protein (BGP) by ROS 17/2.8 cells, which have the osteoblast phenotype. Cells were cultured with 10 nM 1,25-dihydroxyvitamin D3 to stimulate BGP secretion, followed by the addition of TNF alpha (1-100 ng/ml) in 1,25-dihydroxyvitamin D3-containing medium. TNF alpha (10 ng/ml) inhibited BGP secretion to 42 +/- 5%, 19 +/- 10%, and 15 +/- 3% of control values after 24, 48, and 72 h of treatment. After 48 h, inhibition of BGP secretion was observed with 2 ng/ml TNF alpha and was maximum with 100 ng/ml. To determine the effect of TNF alpha on total protein synthesis, cells were pulse labeled with [14C]leucine during the last 4 h of TNF alpha treatment, and incorporation of radioactivity into trichloroacetic acid-precipitable protein in cell layer and medium was determined. The TNF alpha inhibition of BGP secretion was independent of changes in [14C]leucine incorporation, suggesting that TNF alpha did not have a general inhibitory effect on total protein synthesis. Cell number was not affected by TNF alpha. Northern analysis of steady state BGP mRNA revealed a dose-dependent decrease in the BGP/cyclophilin mRNA hybridization signal intensity after 24 h of treatment. The maximum inhibitory effect was 41 +/- 5% of the control value with 100 ng/ml TNF alpha. The effect of TNF alpha on steady state BGP mRNA levels was not prevented by treatment of cells with cycloheximide, suggesting that TNF-induced new protein synthesis was not required for TNF alpha action. These results suggest that the mechanism of TNF alpha inhibition of BGP synthesis includes a pretranslational site and support the hypothesis that TNF alpha inhibits bone formation by a selective inhibition of matrix protein production.
authors.affiliation: Department of Medicine, Veterans Administration Medical Center, Emory University School of Medicine, Atlanta, Georgia 30033.
authors.forename: M S
authors.initials: MS
authors.lastname: Nanes
authors.forename: J
authors.initials: J
authors.lastname: Rubin
authors.forename: L
authors.initials: L
authors.lastname: Titus
authors.forename: G N
authors.initials: GN
authors.lastname: Hendy
authors.forename: B
authors.initials: B
authors.lastname: Catherwood
mesh_terms: D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D006706:Homeostasis; D015675:Osteocalcin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D015675:Osteocalcin; D002117:Calcitriol