Identification of PKCα as the Upstream Activator of NF-κB in Myeloid Cells
Even though the PKC family consists of 10 members, finding that PKCα/β inhibitors and intracellular calcium inhibitors reduced M-CSF-induced NF-κB activity, suggested PKCα was involved in NF-κB activation after M-CSF treatment. To confirm the role of PKCα in NF-κB activation in macrophages, constructs for either wildtype (WT)-PKCα or kinase-deficient (KD)-PKCα was co-transfected with the pNF-κB-SEAP reporter gene and SEAP secretion was measured. As shown in Figure 7A, MDMs co-transfected with pNF-κB-SEAP and WT-PKCα had a 1.8-fold increase in NF-κB transcriptional activity after M-CSF activation compared with NS cells (p = 0.05), similar to M-CSF-treated cells expressing only pNF-κB-SEAP. Transfecting human macrophages with the KD-PKCα construct significantly reduced M-CSF-induced NF-κB activity compared to WT-PKCα transfected cells (p = 0.016). Similarly, RAW 264.7 cells transfected with WT-PKCα had 2.5-fold more NF-κB transcriptional activity after M-CSF activation compared to unstimulated RAW 264.7 cells (NS) transfected with WT-PKCα (Figure 7B). Expression of the KD-PKCα construct into RAW 264.7 cells reduced M-CSF-induced NF-κB activity to 1.5-fold (p = 0.045) compared to cells transfected with WT PKCα.
10.1371/journal.pone.0028081.g007 Figure 7  PKCα regulates phosphorylation of NF-κB p65 at Ser276.
(A) MDM or (B) RAW 264.7 cells were transiently transfected with pNF-κB-SEAP along with either WT-PKCα or the kinase-deficient (KD)-PKCα construct at a 1∶5 ratio. Cells were serum starved and stimulated with M-CSF and then SEAP secretion in the medium was measured. Data is from of three independent experiments. The p-value of cells transfected with KD compared to those transfected with WT was 0.05. (C) MDMs were removed from the plate using accutase and apoptosis of MDMs was measured by flow cytometry using Annexin V-FITC and propidium iodine (PI). (D) Whole cell lysates from the transfected RAW 264.7 cells were subjected to Western blot analysis with phospho-Ser276 or phospho-Ser536 NF-κB p65 antibodies. Blots were immunoblotted with PKCα to determine equal protein expression for the PKCα constructs. β-actin served as a loading control. Shown is a representative blot from three independent experiments. (E) MDM or (F) RAW 264.7 cells were transiently transfected with a pNF-κB-SEAP along with either 100 nM PKCα siRNA or control siRNA for 20-24 hours. Cells were serum starved for 2-4 hours and stimulated with 100 ng/ml M-CSF for 6 hours for MDM or RAW 264.7 for 2 hours and then SEAP secretion in the medium was measured. Shown is data of three independent experiments. (G) MDMs were removed from the plate using accutase and apoptosis of MDMs was measured by Annexin V-FITC and propidium iodine (PI) staining and analyzed by flow cytometry. (H) Whole cell lysates from the transfected MDM and RAW 264.7 cells were subjected to Western blot analysis with PKCα antibody. β-actin served as a loading control. Shown is a representative blot from at least three independent experiments.   Cell survival was also examined in MDMs expressing either WT-PKCα or KD-PKCα constructs by Annexin V-FITC and PI staining. As expected, M-CSF increased MDM survival as measured by the percent of Annexin V/PI negative cells. Similarly, expression of WT-PKCα protected cells from apoptosis. In contrast, expression of KD-PKCα decreased M-CSF-induced cell survival (p<0.01) (Figure 7C).
Next, we examined the effect of expressing the PKCα constructs on NF-κB phosphorylation. As shown in Figure 7D, expression of KD-PKCα in RAW 264.7 cells did not affect the constitutive phosphorylation at Ser536 of NF-κB p65, but attenuated the phosphorylation at Ser276. Expression of WT-PKCα did not effect the phosphorylation of either residue with or without M-CSF stimulation. These observations demonstrate that PKCα is important in M-CSF-regulated cell survival and NF-κB activation and likely regulated through phosphorylation of Ser276 of NF-κB p65.
To further validate the impact that PKCα played in M-CSF-induced NF-κB transcriptional activity, we next employed PKCα siRNA treatment of MDM or RAW cells. A pool of specific PKCα siRNA were transfected into MDM or Raw 264.7 cells in the presence or absence or M-CSF. Reducing native PKCα expression decreased M-CSF-induced NF-κB transcriptional activity in both MDM (Figure 7E) (p = 0.012) and Raw 264.7 cells (Figure 7F) (p = 0.01). We also examined cell survival of the MDMs by Annexin V-FITC and PI staining after PKCα siRNA transfection. As shown in Figure 7G, M-CSF-induced MDM survival was reduced in the cells transfected with PKCα siRNA compared with cells transfected with control siRNA (p = 0.047). In Figure 7H, we confirmed that PKCα siRNA transfection decreased PKCα protein expression in both MDM and Raw 264.7 cells. Our results indicated that PKCα regulated NF-κB activation and M-CSF-regulated cell survival.