Binding activities of NFκB, NFIL-6 and NFAT from human PBMC to the TNFα, IL-6 and IFNγ promoters following an in vitro stimulation (1 h) with LPS, TSST-1, GP, GP + LPS and GP + TSST-1
GP induced band shifts, indicating binding of NFκB as well as of NFIL-6 to the corresponding DNA oligonucleotides (κ consensus, κ1, κ2, κ3 sites from the TNFα promoter; κ consensus from the IL-8 promoter; NFIL-6consensus from the IL-6 promoter; Table 1; Fig. 1A). The extent of the band shifts induced by GP was not statistically different when compared to LPS or TSST-1 (Fig. 1B). A supershift, conducted for an NFκB consensus site from the IL-8 promoter, demonstrated a GP-induced predominant binding of NFκB p65 and to a lesser degree of p50 (Fig. 1A). Accordingly, an immunoblot of nuclear extracts from GP-treated PBMC showed a strong binding of NFκB p65 and a weaker reaction of p50, whereas p52 was negative (Fig. 2). Simultaneous co-treatment of PBMC with GP did not change the LPS-induced NFκB binding to oligos from the TNFα promoter significantly (Fig. 1B), but substantially decreased the TSST-1-induced NFκB binding when compared to TSST-1 or GP (100 μg) and thus differed completely from the theoretical value of GP/TSST-1 calc. (n = 4; p < 0.05 vs. GP/TSST-1 calc. and p = 0.07 vs. TSST-1; Fig. 1B). GP was also able to induce band shifts indicative of binding of particular variants of NFAT to an oligo from the IFNγ promoter (Fig. 1A). An immunoblot for two NFAT subunits demonstrated a GP-induced binding of NFATc2 and to a lesser degree of NFATc1 (Fig. 2).
Table 1  Sense strand of oligonucleotides used in EMSAs.
Oligonucleotidesa  Base sequences (5'- XXX -3')  binding site/cytokine gene promoter (position of the cytokine gene from the TSS)b
NFκBcS  GAT CCT CAG AGG GGA CTT TCCc GAT G  NFκB consensus sequence/TNFα promoter [27]
NFκB1S  GAT CCT GGG ACA GCC CAG  NFκB1 sequence/TNFα promoter [27]
NFκB2S  GAT CCG GGG TAT CCT G  NFκB2 sequence/TNFα promoter [27]
NFκB3S  GAT CCT GGG TTT CTC CG  NFκB3 sequence/TNFα promoter [27]
IL-8κBcS  ATC GTG GAA TTT CCT CTG A  NFκB consensus sequence/IL-8 promoter [28]
NFATP2S  GAT CTA AAA TTT CCA GTC CTT GA  NFATP2 sequence/IFNγ promoter [29]
NFIL-6S  TGC AGA TTG CGC AAT CTG CA  NFIL-6 consensus sequence/IL-6 promoter [13]
NFκBcS  GCG AGG AGG GTA TTT CCG CTT  between -80 and -100 NFκB consensus/IL-1RA promoter [30]
NFκB3S  ACA ACA GCA AGG GTT TCT CTT TTT GGA AAT  between -100 and -130 NFκB3/IL-1RA promoter
NFκBcS  AGT AGG GAG TTT GGT  between -266 and -280 NFκB consensus sequence/IL-1RA promoter
NFκB2/3S  ACT CTG GGT ACC TGT  between -288 and -302 NFκB2/3 sequence/IL-1RA promoter
NFATP2/3S  GGC GCA CAA AAC CTA AAA TAT TTA CTA TCT  between -471 and -500 NFATP2/3 sequence/IL-1RA promoter
NFIL-6S  TTA CAA CAC TCC ATT GCG ACA CTT AGT GGG  between -140 and -170 NFIL-6 sequence/IL-1RA promoter [31]
Oct-1 DNAS  AAT TGC ATT GCC TGC AGG TCG ACT CTA GAG GAT CCA TGC AAA TGG ATC CCC GGG TAC CGA GCT C  exclusion of unspecific bindings, unrelated DNA, (Amersham Pharmacia)
(a: S = sense; b: TSS = transcription start site;c: ___ = main binding motif).
Figure 1  A, GP led to DNA binding of NFAT, NFκB and NFIL-6. Human PBMC were incubated with medium control or GP (1 and 100 μg) for 1 h at 37°C. Nuclear extracts were incubated with a 32P-labeled NFAT, NFκB, NFIL-6 or NFκB-IL-8 oligonucleotide probe corresponding to the IFNγ, TNFα, IL-6 and IL-8 gene promoters (Table 1). Arrows with the black head indicate migrational location of the NFAT-DNA, NFκB-DNA, NFIL-6-DNA or NFκB-IL-8-DNA complex compared to free probe (no shift). Arrow with the open head indicates a supershift of NFκB-IL-8-DNA-anti-p65 and -p50, respectively. An autoradiogram from a representative experiment is shown (n = 7). B, Decreased DNA binding of NFκB following GP + TSST-1, compared to TSST-1, GP, or TSST-1/GP. Human PBMC were incubated with medium control, LPS, TSST-1, GP (100 μg), GP + TSST-1 and GP + LPS for 1 h at 37°C. An autoradiogram from a representative experiment is shown. Schematic representation of NFκB-DNA binding activity following GP (1 and 100 μg) or TSST-1 treatment (250 ng) as well as simultaneous administration of both compounds and the theoretical value (GP/TSST-1 calc.). Medium control levels were set equal to 1± SEM and significances (* = p < 0.05) are shown with respect to medium control (n = 4).
Figure 2  NFκB p65/p50 and NFATc2/c1 were involved in GP-induced DNA binding. In order to determine whether the DNA binding proteins activated by GP were related to NFκB and NFAT, an immuno(dot) blot was performed. A representative dot blot showing staining of negative and positive controls (recombinant proteins NFκB p52, c-rel; nuclear extracts of PMA-treated Jurkat cells positive for NFκB p50, p65 and for NFATc1, c2) in the upper panel and staining of nuclear extracts of GP-treated PBMC (positive for NFκB p65, p50 and NFAT c2, c1) in the lower panel is shown (n = 3).