PMC:3245220 / 10513-13203 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3245220","sourcedb":"PMC","sourceid":"3245220","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3245220","text":"PKC Inhibition Reduces NF-κB Activity in Human MDMs and RAW 264.7 Cells\nSince NF-κB is activated by PKC in several cell types [36], we next determined if M-CSF-induced NF-κB transcriptional activity was dependent on PKC activation and if calcium, a co-factor for conventional PKC isoform activation, was important. In addition, because of the number of conventional PKCs existing within mononuclear cells, pharmacological inhibitors of PKC family activation was used to determine the relationship of PKC activation to M-CSF-induced cellular survival. MDMs were transfected with the pNF-κB-SEAP reporter and then treated with the general PKC inhibitor, Ro-31-8220; the conventional PKCα/β inhibitor Gö-6976; or the intracellular calcium blocker BAPTA/AM. Ro-31-8220 significantly suppressed M-CSF-induced NF-κB activity compared to cells treated with M-CSF alone (Figure 2A). In addition, Gö-6976 and BAPTA/AM also blocked NF-κB activity in M-CSF-treated MDMs. Trypan blue exclusion analysis did not indicate cell death suggesting that this suppression was not due to non-specific toxicity. These data indicate that M-CSF mediated NF-κB activation through calcium-dependent conventional PKC activation.\n10.1371/journal.pone.0028081.g002 Figure 2 Inhibition of PKC reduces NF-κB activity in M-CSF stimulated macrophages.\n(A) Human MDMs transfected with pNF-κB-SEAP were pre-incubated with inhibitors; Ro-31-8220, Gö-6976, or BAPTA/AM for 30 minutes prior to 6 hours M-CSF stimulation. (B) RAW 264.7 cells transfected with the pNF-κB-SEAP construct were pre-incubated with inhibitors for 30 minutes prior to 2 hours of stimulation with mouse recombinant M-CSF. The NF-κB activity was analyzed by measuring SEAP production in the medium and data are expressed as fold increase of SEAP activity over that in pTAL-SEAP transfected resting cells. The graph represents mean ± S.E.M for three independent experiments. *The p-values of cells treated with inhibitors/M-CSF compared to vehicle/M-CSF were ≤0.05. We next investigated whether PKC inhibition affected NF-κB activity in the mouse macrophage cell line, RAW 264.7. Similar to MDMs, PKC inhibitors Ro-31-8220, Gö-6976 and BAPTA/AM reduced M-CSF-induced NF-κB activity in a dose-dependent manner in RAW 264.7 macrophages (Figure 2B). To ensure that PKC specifically regulated NF-κB p65 and not the closely related family member, c-Rel, we co-transfected c-Rel and NF-κB-SEAP constructs into the Raw 264.7 cell line and measured NF-κB activity in response to M-CSF. There was no increased NF-κB activity in cells expressing c-Rel (Figure S1). These observations indicate that PKC functioned upstream of NF-κB p65 in MDMs and RAW 264.7 cells.","divisions":[{"label":"Title","span":{"begin":0,"end":71}},{"label":"Figure 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