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.     

Interleukin 1 induces HIV-1 expression in chronically infected U1 cells: blockade by interleukin 1 receptor antagonist and tumor necrosis factor binding protein type 1.

BACKGROUND: Cytokines and cytokine antagonists modulate human immunodeficiency virus (HIV) replication in vitro and may be involved in HIV disease pathogenesis. An understanding of these cytokine networks may suggest novel treatment strategies for HIV-seropositive persons. MATERIALS AND METHODS: U1 cells, a chronically infected promonocytic cell line, were stimulated with interleukin 1 alpha (IL-1 alpha), IL-1 beta or tumor necrosis factor (TNF) for 24 hr. The effects of these cytokines, and of anti-IL-1 receptor type 1 and type 2 (IL-1RI and II) antibody, IL-1 receptor antagonist (IL-1Ra), and recombinant human TNF binding protein type 1 (rhTBP-1, a form of TNF receptor p55), on HIV-1 replication, as measured by ELISA for HIV-1 p24 antigen, were determined. The effects of IL-1 and IL-1Ra on nuclear factor-kappa B (NF-kappa B) DNA binding activity, as measured by electrophoretic mobility shift assays, were also determined. RESULTS: IL-1 alpha and IL-1 beta increased p24 antigen production in a concentration-dependent manner. IL-1Ra completely, and rhTBP-1 partially, suppressed IL-1-induced p24 antigen production. IL-1 increased NF-kappa B DNA binding activity and IL-1Ra blocked this effect. Since IL-1Ra blocks IL-1 from binding to both the IL-1RI and Il-1RII, monoclonal antibodies directed against each receptor were used to ascertain which IL-1R mediates IL-1-induced HIV-1 expression. Antibody to the IL-1RI reduced IL-1-induced p24 antigen production. Although anti-IL-1RII antibody blocked the binding of 125IL-1-1 alpha to U1 cells by 99%, this antibody did not affect IL-1-induced p24 antigen production. IL-1 beta enhanced TNF alpha-induced HIV expression when added before or simultaneously with TNF alpha. CONCLUSIONS: IL-1 induces HIV-1 expression (via the IL-1RI) and NF-kappa B activity in U1 cells. These effects are blocked by IL-1Ra and partially mediated by TNF. IL-1 enhances TNF alpha-induced HIV replication in U1 cells.     

Activation of interleukin-6 gene expression through the NF-kappa B transcription factor.

The promoter region of the interleukin-6 (IL-6) gene has a putative NF-kappa B-binding site. We found that a fragment of the IL-6 promoter containing the site specifically binds highly purified NF-kappa B protein and the NF-kappa B protein in nuclear extracts of phorbol ester-induced Jurkat cells. Mutations of the NF-kappa B site abolished complex formation with both purified NF-kappa B and the nuclear extract protein. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing the IL-6 promoter revealed very little activity of the promoter in U-937 monocytic cells and in HeLa cells before stimulation. However, stimulation of U-937 and HeLa cells by inducers of NF-kappa B led to a dramatic increase in CAT activity. Mutations in the NF-kappa B-binding site abolished inducibility of IL-6 promoter-cat constructs in U-937 cells by lipopolysaccharide, tumor necrosis factor alpha, the double-stranded RNA poly(IC), or phytohemagglutinin and in HeLa cells by tumor necrosis factor alpha and drastically reduced but did not completely eliminate inducibility in HeLa cells stimulated by double-stranded RNA poly(IC) or phorbol 12-myristate 13-acetate. These results suggest that NF-kappa B is an important mediator for activation of the IL-6 gene by a variety of IL-6 inducers in both U-937 and HeLa cells and that alternative inducible enhancer elements contribute in a cell-specific manner to IL-6 gene induction. Because NF-kappa B is involved in the control of a variety of genes activated upon inflammation, NF-kappa B may play a central role in the inflammatory response to infection and tissue injury.     

Overexpression of thioredoxin reductase 1 regulates NF-kappa B activation

Thioredoxin reductase (TrxR) is a flavoprotein that contains a C-terminal penultimate selenocysteine (Sec) and has an ability to reduce thioredoxin (Trx), which regulates the activity of NF-kappa B. To date, three TrxR isozymes, TrxR1, TrxR2, and TrxR3, have been identified. In the present study, we found that among these isozymes only TrxR1 was induced by tumor necrosis factor-alpha (TNF alpha) in vascular endothelial cells. Furthermore, the overexpression of TrxR1 enhanced TNF alpha-induced DNA-binding activity of NF-kappa B and NF-kappa B-dependent gene expression. The catalytic Sec residue of TrxR1, which is essential for reducing Trx, was required for this NF-kappa B activation, and aurothiomalate, an inhibitor of TrxR, suppressed TNF alpha-induced activation of NF-kappa B and the expression of NF-kappa B-targeted proinflammatory genes such as E-selectin and cyclooxygenase-2. These results suggest that TrxR1 may act as a positive regulator of NF-kappa B and may play an important role in the cellular inflammatory response.