M-CSF Induces NF-κB Transcriptional Activity in Human Monocyte–Derived Macrophages (MDMs) and Mouse Macrophage Cell Line, RAW 264.7
To determine if M-CSF induced NF-κB DNA binding in human macrophages, we performed EMSA analysis on nuclear lysates from M-CSF-treated MDMs. Similar to previous reports [11], nuclear NF-κB constitutively bound DNA in non-stimulated monocytes (Figure 1A). Interestingly, adding M-CSF did not alter NF-κB DNA binding by EMSA. In contrast, after transiently transfecting human MDMs with pNF-κB-SEAP constructs containing four NF-κB consensus binding sequences, M-CSF treatment of the transfected cells resulted in a 2.3-fold increase in SEAP release in the culture media compared to PBS (vehicle)-treated transfected MDMs (Figure 1B). As a control, the pTAL-SEAP construct lacking NF-κB binding sites was used. Cells transfected with the pTAL-SEAP construct did not produce SEAP in the absence or presence of M-CSF (Figure 1B).
10.1371/journal.pone.0028081.g001 Figure 1  M-CSF induces NF-κB transcriptional activity in macrophages.
(A) Nuclei were extracted from human MDMs treated without or with M-CSF for 15 or 30 minutes. NF-κB DNA binding activity was analyzed by EMSA Shown is a representative blot from three independent experiments. NS: non-stimulated. (B) Human MDMs transiently transfected with pTAL-SEAP or pNF-κB-SEAP constructs were treated with M-CSF in X-vivo medium and incubated for 6 hours before the collection of medium. NF-κB activity was analyzed by measuring the amount of SEAP secreted into the medium and data are expressed as fold increase of SEAP activity over that in pTAL-SEAP transfected resting cells. (C) RAW 264.7 cells were transiently transfected with pTAL-SEAP or pNF-κB-SEAP construct. Cells were serum starved for 4 hours prior to 2 hours stimulation with mouse recombinant M-CSF (100 ng/ml). Culture media was collected to measure SEAP production. Data are expressed mean ± S.E.M, for three independent experiments.   We next investigated whether M-CSF induced NF-κB activity in the mouse macrophage cell line, RAW 264.7. RAW 264.7 cells were transfected with either the NF-κB-SEAP reporter or control pTAL-SEAP construct. As shown in Figure 1C, M-CSF treatment of RAW 264.7 cells increased NF-κB reporter activity by 2.5-fold over that of non-treated cells. Together, our data demonstrate that M-CSF induced NF-κB transcriptional activity in macrophages.