NF-κB and PKC(s) Mediate Human MDM Survival in Response to M-CSF Stimulation
Since PKC and NF-κB are critical in cell survival [37], [38], we hypothesized that M-CSF promoted cell survival through PKC in human MDMs. To detect apoptosis, the expression of cleaved caspase-3, a marker of apoptosis, was analyzed in the presence or absence of M-CSF and PKC inhibitors. In MDMs pretreated with PKC inhibitors (Ro-31-8220, Gö-6976, or BAPTA/AM) and stimulated with M-CSF, cleaved caspase-3 was elevated to levels of cells treated with vehicle alone (Figure 3A). In contrast, cells incubated with M-CSF and vehicle had less cleaved caspase-3 than cells incubated with vehicle alone or M-CSF with PKC inhibitors. These data supported the hypothesis that M-CSF-induced NF-κB activity was regulated by conventional PKCs, not novel PKCs.
10.1371/journal.pone.0028081.g003 Figure 3  Inhibition of PKC or NF-κB induces apoptosis in MDMs.
(A) MDMs were pre-incubated in RPMI medium containing inhibitors (Ro-31-8220: 5 µM, Gö-6976: 5 µM, BAPTA/AM: 2.5 µM) for 30 minutes prior to the addition of M-CSF. As a control, untreated cells were incubated with dimethyl sulfoxide (DMSO). Cell lysates were resolved by SDS-PAGE and immunoblotted with antibody recognizing the active cleaved form of caspase-3. The blots were reblotted with β-actin that served as a loading control. The ratio of active caspase-3 bands (17 kD and 19 kD) to β-actin control was determined by densitometry analysis (bottom panel). Data represents the mean ± S.E.M from two independent donors. (B) Apoptosis of the treated MDMs was also measured by Annexin V-FITC and propidium iodine (PI) staining and analyzed by flow cytometry. The percentage of surviving cells (Annexin V/PI negative) for the cells treated with vehicle/M-CSF was arbitrarily set as 100. Data shown represent the mean ± S.E.M from three independent experiments. *The p-values of inhibitors/M-CSF compared to vehicle/M-CSF were ≤0.05.   To confirm that PKC inhibition decreased cell survival, cells were treated with M-CSF in the absence or presence of PKC inhibitors and then also examined for apoptosis by Annexin V/PI staining. PKC inhibitors in the presence of M-CSF reduced the number of Annexin V/PI negative MDMs compared to cells treated with M-CSF alone (Figure 3B). These observations further suggested that MDM survival is promoted by M-CSF and critically involves conventional PKCs and NF-κB activity.