TNF-alpha increases expression of IL-6 and ICAM-1 genes through activation of NF-kappaB in osteoblast-like ROS17/2.8 cells.
Tumor necrosis factor-alpha (TNF-alpha) plays a key role in inflammatory diseases such as rheumatoid arthritis and in postmenopausal osteoporosis. In various tissues, TNF-alpha action is mediated by a transcription factor, nuclear factor-kappa B (NF-kappaB). However, little is known about how TNF-alpha exerts its action in osteoblasts. We thus examined the effect of TNF-alpha on the activation of NF-kappaB in rat osteoblast-like osteosarcoma cells (ROS17/2.8). Electrophoretic mobility shift assay revealed that the activation of the p50-p65 heterodimer NF-kappaB was induced by TNF-alpha as early as 15 minutes followed by a persistent activation for 48 h. When the binding activity of NF-kappaB in cytosol was examined using detergents that dissociate NF-kappaB from an inhibitory protein IkappaB, it decreased during the initial 30 minutes and then increased to the unstimulated level. Northern blot analysis revealed a marked increase in the mRNA levels of p105, a precursor of p50, 6 h after TNF-alpha and a gradual increase in p65 mRNA levels during the initial 1 h. Significant increase in both mRNA levels continued until 24 h after TNF-alpha. These results suggest that the rapid activation of NF-kappaB by TNF-alpha is mainly due to the nuclear translocation of NF-kappaB pre-existing in cytosol, and that the subsequent increase in the expression of p50 and p65 may result in the persistent activation of NF-kappaB during TNF-alpha stimulation. TNF-alpha also increased the mRNA levels of interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1). An antioxidant, N-acetyl-L-cysteine, significantly attenuated the TNF-alpha-dependent increase in these mRNAs, and simultaneously reduced the activation of NF-kappaB by TNF-alpha, indicating that NF-kappaB mediates the TNF-alpha-dependent expression of IL-6 and ICAM-1 in ROS17/2.8 cells. These results suggest that the activation of NF-kappaB by TNF-alpha may play an important role in the production of cytokines and cell adhesion molecules from osteoblasts, leading to the promotion of bone resorption and inflammation.
Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Japan.
K
K
Kurokouchi
F
F
Kambe
K
K
Yasukawa
R
R
Izumi
N
N
Ishiguro
H
H
Iwata
H
H
Seo
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D001859:Bone Neoplasms; D003600:Cytosol / Q000378:metabolism; D016166:Free Radical Scavengers / Q000494:pharmacology; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics*; D015850:Interleukin-6 / Q000235:genetics*; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D012516:Osteosarcoma; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016166:Free Radical Scavengers; D015850:Interleukin-6; D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D000111:Acetylcysteine