M-CSF-dependent PKC Regulates NF-κB-targeted Genes
NF-κB induces a number of downstream genes, including the IκB family. Among the IκB molecules, IκBα is highly induced by NF-κB activation [40]. Having shown that PKC regulated NF-κB activity in M-CSF-stimulated MDMs, we next determined whether inhibition of PKC activity decreased expression of NF-κB-regulated genes. We treated both MDMs and RAW 264.7 cells with the PKC inhibitor Ro-31-8220 for 30 minutes and then stimulated with M-CSF. IκBα gene was measured by qRT-PCR. As shown in Figures 6A and 6B, M-CSF enhanced IκBα gene expression and PKC inhibition by Ro-31-8220 decreased IκBα gene expression in both MDMs and RAW 264.7 cells (p<0.01), demonstrating that PKC affected NF-κB-regulated gene expression in macrophages.
10.1371/journal.pone.0028081.g006 Figure 6  Inhibition of M-CSF-induced PKC reduces NF-κB-regulated genes in both MDMs and RAW 264.7 cells.
MDMs (A and C) and RAW 264.7 (B) cells were pretreated with Ro-31-8220 or solvent control DMSO for 30 minutes prior to M-CSF stimulation for the indicated times. Total RNA was isolated and converted to cDNA. Real-time RT PCR was performed using primers for IκBα, BCL-xl or GAPDH. Data are expressed as relative fold increase of IκBα or BCL-xl gene expression upon treatment over non-stimulated cells. Data represent the mean ± S.E.M for three independent experiments.   To further define the role of PKC in mediating human MDM survival in response to M-CSF, we examined the expression of the anti-apoptotic gene BCL-xL, which is also regulated by NF-κB. As shown in Figure 6C, Ro-31-8220 reduced M-CSF-stimulated BCL-xL expression compared to cells treated with M-CSF and the vehicle control DMSO (p<0.05).