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.     

Induction of human methionine adenosyltransferase 2A expression by tumor necrosis factor alpha. Role of NF-kappa B and AP-1

Two genes (MAT1A and MAT2A) encode for methionine adenosyltransferase (MAT), an essential cellular enzyme responsible for S-adenosylmethionine biosynthesis. MAT1A is expressed mostly in the liver, whereas MAT2A is widely distributed. We showed a switch from MAT1A to MAT2A expression in human hepatocellular carcinoma (HCC), which facilitates cancer cell growth. Using DNase I footprinting analysis, we previously identified a region in the MAT2A promoter protected from DNase I digestion in HCC. This region contains NF-kappa B and AP-1 elements, and the present study examined whether they regulate MAT2A promoter activity. We found nuclear binding of NF-kappa B and AP-1 to the MAT2A promoter increased in HCC. Tumor necrosis factor alpha (TNFalpha), which activates both NF-kappa B and AP-1, increased MAT2A expression in a dose- and time-dependent manner, binding of both NF-kappa B and AP-1 to the MAT2A promoter and MAT2A promoter activity, with the latter effect blocked by site-directed mutagenesis of the NF-kappa B and AP-1 binding sites. Blocking NF-kappa B with I kappa B super-repressor or AP-1 with dominant-negative c-Jun led to decreased basal MAT2A expression and prevented the TNF alpha-induced increase in MAT2A expression. Although blocking NF-kappa B had no influence on the ability of TNF alpha to increase AP-1 nuclear binding, blocking AP-1 with dominant-negative c-Jun prevented the TNF alpha-mediated increase in NF-kappa B binding. In conclusion, both NF-kappa B and AP-1 are required for basal MAT2A expression in HepG2 cells and mediate the increase in MAT2A expression in response to TNF alpha treatment. Increased trans-activation of these two sites also contributes to MAT2A up-regulation in HCC.