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.
Kurokouchi
K
K
Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Japan.
Kambe
F
F
Yasukawa
K
K
Izumi
R
R
Ishiguro
N
N
Iwata
H
H
Seo
H
H
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