PMC:1472690 / 16202-19656 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1472690","sourcedb":"PMC","sourceid":"1472690","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1472690","text":"Role of NFκB, NFIL-6 and NFAT binding in LPS-, TSST-1-, GP-, GP + LPS- and GP + TSST-1-induced IL-1RA expression by human PBMC\nBecause GP induced band shifts to NFκB, NFIL-6 and NFAT sites, but no TNFα, IL-6 or IFNγ, we hypothesized that the activated transcription factors might bind to sites in the IL-1RA promoter. We investigated previoulsy described and newly discovered transcription factor binding sites of the IL-1RA promoter (Table 1) using the program OMIGA (v.1.1.3, Oxford Molecular, Oxford, UK). The LPS responsive κB consensus site of LRE-1 (between -84 and -93) [30] displayed band shifts, but no significant alterations of its binding activity, irrespective of the treatment (data not shown). A new κB3 site (-100 and -130) within the LRE-1 [30] showed increased binding induced by TSST-1 (+30% vs. control), but GP co-treatment up-regulated the LPS (+65% vs. control)- and the TSST-1 (+110% vs. control)-induced binding in a superadditive synergistic fashion, when compared to medium control as well as to LPS, TSST-1 or GP or the theoretical values of GP/LPS calc. or GP/TSST-1 calc. (all p \u003c 0.05; Fig. 4). Binding to a new κB consensus site (between -266 and -280) was significantly elevated for GP + TSST-1 (p \u003c 0.05; data not shown). The LPS-, TSST-1- and GP + LPS-induced binding to the new κB2/3 site (-288 and -302) was down-regulated by about 20–35% in comparison with medium control (p \u003c 0.05; data not shown). The NFIL-6 site (-147 and -170) close to LRE-2 [31,32] showed an up-regulation of binding for all compounds and combinations vs. medium control, ranging from 25–110% (p \u003c 0.05). Further, GP + TSST-1 induced an increased binding to the NFIL-6 site in comparison with TSST-1 (1.3fold) or GP (1.6fold; p \u003c 0.05), (data not shown). A new binding site for NFATP2/3 was found more distal of the transcription start site, between -471 and -490, displaying again a substantial increase of binding for all compounds and combinations, when compared to medium control (p \u003c 0.05). In addition, binding to the NFATP2/3 site following both, GP + LPS (1.4fold of GP) and GP + TSST-1 (1.5fold of GP, 1.25fold of TSST-1) was elevated (p \u003c 0.05; Fig. 5), but only in an additive synergistic manner, i.e. the experimental results were not different from the theoretical values (calc.).\nFigure 4 GP modulated LPS- and TSST-1-induced binding to a new NFκB3 site in the IL-1RA promoter. Binding was enhanced for GP + LPS and GP + TSST-1, when compared to LPS, TSST-1, GP, GP/LPS calc. or GP/TSST-1 calc., respectively (* = p \u003c 0.05; ** = p \u003c 0.01). Medium control levels were set equal to 1 ± SEM (n = 6). DNA binding was assessed by EMSA (see methods).\nFigure 5 GP modulated TSST-1-induced DNA binding to a new NFATP2/3 site in the IL-1RA promoter. Binding was up-regulated for GP + LPS and GP + TSST-1, when compared to GP and, in the latter case, to both GP and TSST-1 (* = p \u003c 0.05). Human PBMC were incubated with medium control, LPS, TSST-1, GP, GP + TSST-1 and GP + LPS for 1 h at 37°C. Schematic representation of the elevated NFAT-DNA binding activity to the site in the IL-1RA promoter. Bars depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are added. Medium control levels were set equal to 1 ± SEM (n = 6). An autoradiogram of a representative experiment is shown in the lower panel (lane 1: water; lane 2: medium control; lane 3: GP; lane 4: LPS; lane 5: TSST-1, lane 6: GP + LPS; lane 7: GP + 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