PKC is Essential in Regulating NF-κB Activity
M-CSF-dependent PKC activity facilitated NF-κB p65 phosphorylation at Ser276 but not Ser536. To confirm the role of PKC and p65 Ser276 phosphorylation on NF-κB activity, we co-transfected the NF-κB-SEAP construct with either WT NF-κB p65 or NF-κB p65 276S/A constructs in Raw 264.7 cells, then treated cells with the PKC inhibitor Ro-31-8220 in the absence or presences of M-CSF. As expected in cells transfected with vector control, inhibiting PKC reduced M-CSF-stimulated NF-κB activity compared to cells treated with M-CSF and the vehicle DMSO (p = 0.001) (Figure 8A). In contrast, expressing WT NF-κB p65 (p65 WT) increased NF-κB activity, while the general PKC inhibitor Ro-31-8220 decreased this NF-κB activity (p = 0.001). Notably, the introduction of NF-κB p65 276 S/A construct significantly reduced NF-κB activity compared with the WT NF-κB p65 construct (p = 0.005) and M-CSF treatment was unable to overcome this inhibition.
10.1371/journal.pone.0028081.g008 Figure 8  NF-κB p65 Ser276 is essential in regulating NF-κB activity.
(A) Raw 264.7 cell line was transiently transfected with pNF-κB-SEAP along with empty vector or plasmid encoding either NF-κB p65 WT or NF-κB p65 276S/A. The cells were transfected for 18-24 hours, serum starved for 4 hours, and then incubated with 10 µM of Ro-31-8220 for 30 minutes prior to treatment with 100 ng/ml of M-CSF for 2 hours and SEAP secretion in the medium was measured. (B) NF-κB p65−/− cell line was transiently transfected with pNF-κB-SEAP along with empty vector or plasmid encoding either NF-κB p65 WT, NF-κB p65 276S/A, or NF-κB p65 536S/A. The cells were cultured for 24 hours and then serum starved for 4 hours. Cells were then incubated in fresh DMEM medium for 2 hours and SEAP secretion in the medium was measured. The results shown are fold change over empty vector + pTAL-SEAP. (C) NF-κB p65−/− cell line was transiently transfected with pNF-κB-SEAP with either empty vector or plasmid encoding either NF-κB p65 WT or NF-κB p65 276S/A. The cells were transfected for 24 hours and serum starved for 4 hours, and then incubated with 10 µM of Ro-31-8220 for 30 minutes prior to treatment with 10 ng/ml of TNFα. The supernatant were collected after 2 hours of treatment and SEAP secretion in the medium was measured. The results shown are the fold change over empty vector + pNF-κB-SEAP. Data shown are mean ± S.E.M for at least three independent experiments performed in duplicate.   Furthermore, we co-transfected the NF-κB-SEAP construct along with either the WT NF-κB p65, NF-κB p65 276S/A or NF-κB p65 536S/A constructs into a NF-κB p65−/− murine fibroblast cell line and measured NF-κB activity. As predicted, expression of WT NF-κB p65 (p65 WT) in NF-κB p65−/− cell line constitutively activated NF-κB compared to cells transfected with vector control without any stimulation (Figure 8B). In comparison, transfecting the NF-κB p65 276S/A construct reduced NF-κB activity by 5-fold (p = 0.006, WT NF-κB p65 vs. NF-κB p65 276S/A), while expressing the NF-κB p65 536S/A construct increased NF-κB activity in the p65−/− cells to levels similar to WT NF-κB p65 transfected cells.
Since M-CSF-induced PKC activation regulated NF-κB activity via Ser276 residue of NF-κB p65 in primary human MDMs and RAW 264.7 cells, we next examined if this occurred in NF-κB p65−/− fibroblasts in response to a native stimulus for these cells, TNFα. NF-κB activity was measured in the WT NF-κB p65 or NF-κB p65 276S/A transfected cells treated with TNFα in the absence or presence of Ro-31-8220 (Figure 8C). As expected, TNFα increased NF-κB activity in NF-κB p65−/− cells expressing WT p65 and (p = 0.001) PKC inhibitors decreased NF-κB activity to the non-stimulated (NS) level (p = 0.007). Introducing NF-κB p65 276 S/A constructs significantly reduced NF-κB activity compared with the WT NF-κB p65 construct (p = 0.001). Notably, TNFα failed to increase NF-κB activity in the NF-κB p65−/− cells expressing NF-κB p65 276S/A constructs. These observations are similar to macrophages overexpressing the NF-κB p65 276S/A (Figure 8A). Our data demonstrate that Ser276 of NF-κB p65 is essential in regulating NF-κB activity and suggests that PKC regulates NF-κB activity by modulating the phosphorylation of NF-κB p65 at Ser276 residue.