Tumor necrosis factor-alpha (TNF-α) 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-α on the human osteosarcoma cell line Saos2. We used RT-PCR to examine the effects of TNF-α on bone sialoprotein (BSP), core binding factor a1 (Cbfa1), osterix, α1 (I) collagen, cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), cathepsin B, cathepsin L and tissue inhibitors of metalloproteinase-1 (TIMP-1). TNF-α (10 ng/ml) increased BSP, IL-6 and COX-2 mRNA levels after 3 h, 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-α. On the other hand, α1 (I) collagen mRNA expression was suppressed by TNF-α at 24 h.
Transient transfection analyses were performed using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. TNF-α (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-NFκB oligonucleotides revealed an increase in the binding of nuclear proteins from TNF-α-stimulated Saos2 cells. These studies, therefore, showed that TNF-α indirectly increased BSP expression, and that it could be mediated through COX-2 and Cbfa1 expression in Saos2 osteoblast-like cells.