Inhibition of transcription factors led to reduced IL-1RA levels
Pharmacological inhibitors were used to demonstrate that the transcription factor sites within the IL-1RA promoter are relevant for the induction of IL-1RA by GP. As expected, an inhibition of NFκB via CAPE [35] and CyA, NFAT via CyA and NFIL-6 via CHX [36] could not be overruled by GP, leading to a down-regulated binding activity of the transcription factors to the IL-1RA sites, and a reduction of IL-1RA mRNA and IL-1RA protein levels (Fig. 7). The extent of reduction in NFATP2/3 binding is exemplarily shown in an autoradiogram (Fig. 7A, left side) and graphically summarized (n = 4, Fig. 7A, right side). The results for the NFκB and NFIL-6 binding sites were similar (data not shown). Because the highest IL-1RA amount was found at 24 h, mRNA was examined following 1 h inhibition and 18 h of GP. A representative gel demonstrating a decrease in IL-1RA mRNA after inhibition with CAPE, CyA and CHX is displayed in Fig. 7B (n = 4). In addition, corresponding IL-1RA protein levels after 24 h of GP are shown in Fig. 7C (n = 3). Altogether, NFκB inhibition by CAPE was more pronounced at the binding activity and the transcription, whereas CyA and CHX mainly led to a decreased IL-1RA release.
Figure 7  Inhibition of GP-induced NFκB, NFAT and NFIL-6 DNA binding to the IL-1RA promoter. A, A representative autoradiogram (lane 1: control, lane 2: GP, lane 3: GP + unlabeled mutated NFATP2/3 oligo, lane 4: GP + CAPE, lane 5: GP + CyA, lane 6: GP + CHX, lane 7: GP + unlabeled NFATP2/3 oligo, lane 8: water) as well as a graphical summary for the NFATP2/3 site, depicting a decrease in binding following inhibition when compared to GP is displayed (* = p < 0.05). Data are shown as mean ± SEM (n = 4). B, Inhibition of NFκB, NFAT and NFIL-6 resulted in decreased IL-1RA mRNA following CHX, CAPE or CyA compared to GP. GAPDH is used as housekeeping control (representative gel, n = 4). C, Inhibition of NFκB, NFAT and NFIL-6 significantly reduced the production of IL-1RA by human PBMC following treatment with CHX, CAPE or CyA when compared to GP (* = p < 0.05; ** = p < 0.01). Data are shown as mean ± SEM (n = 3).