Effect of TNF-alpha on human osteosarcoma cell line Saos2--TNF-alpha regulation of bone sialoprotein gene expression in Saos2 osteoblast-like cells.
Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of inflammatory response in many diseases. It inhibits bone formation and stimulates bone resorption. To determine the molecular mechanisms involved in the regulation of gene expression of osteoblast-like cells, we analyzed the effects of TNF-alpha on the human osteosarcoma cell line Saos2. We used RT-PCR to examine the effects of TNF-alpha on bone sialoprotein (BSP), core binding factor a1 (Cbfa1), osterix, alpha 1 (I) collagen, cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), cathepsin B, cathepsin L and tissue inhibitors of metalloproteinase-1 (TIMP-1). TNF-alpha (10ng/ml) increased BSP, IL-6 and COX-2 mRNA levels after 3h, reaching maximal levels at 12 h. Cbfa1 mRNA levels increased after 3 h, but decreased by 24 h. Osterix, cathepsin B, cathepsin L and TIMP-1 mRNA levels did not change after stimulation with TNF-alpha. On the other hand, alpha 1 (I) collagen mRNA expression was suppressed by TNF-alpha at 24 h. Transient transfection analyses were performed using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. TNF-alpha (10 ng/ml) had no effect on the promoter activities of BSP transfected into Saos2 cells. The results of gel mobility shift assays using radiolabeled double-stranded cAMP response element (CRE) and FGF2 response element (FRE) oligonucleotides in the proximal promoter of the rat BSP gene showed increased binding of nuclear proteins at 6 h. Gel mobility shift assays with radiolabelled COX-2-CRE and COX-2-NF kappa B oligonucleotides revealed an increase in the binding of nuclear proteins from TNF-alpha-stimulated Saos2 cells. These studies, therefore, showed that TNF-alpha indirectly increased BSP expression, and that it could be mediated through COX-2 and Cbfa1 expression in Saos2 osteoblast-like cells.
Nakayama
Youhei
Y
Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan.
Kato
Naoko
N
Nakajima
Yu
Y
Shimizu
Emi
E
Ogata
Yorimasa
Y
D045744:Cell Line, Tumor; D050659:Core Binding Factor Alpha 1 Subunit; D050660:Core Binding Factors; D051546:Cyclooxygenase 2; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D010006:Osteoblasts / Q000378:metabolism*; D053495:Osteopontin; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D050659:Core Binding Factor Alpha 1 Subunit; D050660:Core Binding Factors; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D053495:Osteopontin; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases