Degraded CGN Induce IκB Degradation and NF-κB Activation
The expression of genes encoding for ICAM-1 and TNF-α is controlled by the nuclear factor NF-κB. Site-specific phosphorylation of the inhibitor IκB leads to its degradation by proteasome and to a consequential activation of the NF-κB pathway. Using a reporter plasmid for NF-κB activation, it was confirmed that dCGN induced a strong activation of NF-κB, as reflected by an increase in luciferase activity (Fig. 7A). Both forms of dCGN used induced NF-κB activation in a dose dependent manner. However, the effect was more strongly induced by the 40 kDa dCGN (Fig. 7A). These results were further confirmed by directly detecting NF-κB in the cell nucleus by Western blotting (Fig. 7C) and by FACS (Fig. 7D). These assays also allowed us to determine what NF-κB subunits were activated by dCGN. Both forms (10 or 40 kDa) of dCGN induced activation of the p50 and p65 subunits of NF-κB. This nuclear factor was present in low levels in the cell nucleus and increased considerably after treatment with dCGN. Western blots suggested the the 40 kDa form of dCGN induced a stronger activation of NF-κB (Fig. 7C). A more sentive assay for nuclear factor activation is flow cytometry of nuclei stained with specific antibodies for the nuclear factor of interest. In agreement with the previous data, FACS analysis of nuclei from THP-1 cells showed that there was a basal level of nuclear NF-κB (Fig. 7D). Again, both forms (10 or 40 kDa) of dCGN induced an increase of the p50 and p65 subunits of NF-κB in the nucleus of these cells. The 40 kDa degraded CGN gave a stronger increase of NF-κB (Fig. 7D). These data strongly suggest that the heterodimer p50/p65 is the NF-κB isoform activated by degraded CGN in monocytes. In addition, degradation of the inhibitor IκBα was also observed in cells treated with dCGN (Fig. 7B). No significant IκBα degradation was detected within two hours of dCGN treatment, but IκBα was markedly degraded by four hours of dCGN treatment (Fig. 7B). We focused on IκBα subunit, since it masks the nuclear localisation sequence of p65, it is the most rapidly degraded subunit and the most studied one.
10.1371/journal.pone.0008666.g007 Figure 7  Degraded CGN activated the NF-kB pathway in monocytes.
A: THP-1 cells were transfected with a NF-κB reporter plasmid driving expression of luciferase. Cells were then treated with various concentrations of 10 kDa (triangles), or 40 kDa dCGN (squares). B: THP-1 cells treated with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) were lysed after various periods of time. Proteins in cell extracts were resolved by SDS-PAGE and then Western blotted for IκBα or α−tubulin as loading control. C: Degraded carrageenans (dCGN) induced activation of NF-κB. THP-1 cells were treated with nothing (control), or with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) for 30 minutes at 37°C. Nuclei were isolated and lysed. Proteins in nuclear extracts were resolved by SDS-PAGE and then Western blotted for NF-κB p50 subunit (p50) or NF-κB p65 subunit (p65). Lower panels show Western blots of nuclear ERK revealing equivalent amount of protein in each sample. Data are representative of three separate experiments. D: Degraded carrageenan (dCGN) induced activation of NF-κB. Nuclei isolated from THP-1 cells were fluorescence-stained for NF-κB p50 subunit or NF-κB p65 subunit before (filled area) or after cells were treated with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) for 30 minutes at 37°C. Dashed line corresponds to nuclei stained only with secondary fluorescence antibody. Fluorescence intensity was analyzed by flow cytometry as described.