Figure 5
Figure 5. C3aR-mediated signal transduction in monocytes involves ERK1/2 phosphorylation. (A) C3aR stimulation induces ERK1/2 phosphorylation. Monocytes were either left in untreated media or preincubated 30 minutes in media with 1 μM MEK inhibitor (a reagent that prevents ERK1/2 phosphorylation) and then activated as indicated for 2, 4, 10, and 30 minutes. ERK1/2 phosphorylation was assessed in cell lysates by western blot analyses comparing nonphosphorylated ERK1/2 (ERK1/2) vs phosphorylated ERK1/2 (p-ERK1/2) (shown is the 10-minute time point, when ERK1/2 phosphorylation peaked). (B) Inhibition of ERK phosphorylation decreases IL-1β production. Monocytes were activated as described in panel A and IL-1β secretion measured at 18 hours postactivation. (C) Inhibition of ERK1/2 phosphorylation prevents pannexin-1 channel expression/opening upon monocyte activation. Experiments were performed as described in panel A and pannexin-1 channel expression was measured by western blot analysis at 8 hours postactivation. (D) Inhibition of pannexin-1 channel function impairs IL-1β production. Monocytes were activated as depicted in the presence or absence of 3 mM Probenecid and IL-1β secretion was measured 18 hours postactivation. (E-F) ERK1/2 or pannexin-1 channel inhibition results in reduced cellular efflux. Cells were activated as shown and retention of Yo-PRO dye measured 4 hours postactivation by FACS analysis. Results shown in panels A,C are representative of 2 separately performed experiments and results shown in panels B,D-F represent mean ± SD derived from 3 independent experiments (n = 3). *P < .05; **P < .001; ***P < .0005, determined by the paired t test with Bonferroni correction. (G) Model schematic of TLR and C3aR-mediated crosstalk leading to increased IL-1β production by human monocytes. TLR-mediated signals involving NF-κB translocation activate the NLRP3 inflammasome, which leads to subsequent caspase-1 activation and cleavage of inactive IL-1β into its active form. C3a generation during infection or inflammation engages the C3aR which induces an ERK1/2 phosphorylation-dependent increase in extracellular ATP availability. This in turn potentiates P2X7 signaling and caspase-1 activation cumulating in substantially increased IL-1β secretion. FACS, fluorescence-activated cell sorter.

C3aR-mediated signal transduction in monocytes involves ERK1/2 phosphorylation. (A) C3aR stimulation induces ERK1/2 phosphorylation. Monocytes were either left in untreated media or preincubated 30 minutes in media with 1 μM MEK inhibitor (a reagent that prevents ERK1/2 phosphorylation) and then activated as indicated for 2, 4, 10, and 30 minutes. ERK1/2 phosphorylation was assessed in cell lysates by western blot analyses comparing nonphosphorylated ERK1/2 (ERK1/2) vs phosphorylated ERK1/2 (p-ERK1/2) (shown is the 10-minute time point, when ERK1/2 phosphorylation peaked). (B) Inhibition of ERK phosphorylation decreases IL-1β production. Monocytes were activated as described in panel A and IL-1β secretion measured at 18 hours postactivation. (C) Inhibition of ERK1/2 phosphorylation prevents pannexin-1 channel expression/opening upon monocyte activation. Experiments were performed as described in panel A and pannexin-1 channel expression was measured by western blot analysis at 8 hours postactivation. (D) Inhibition of pannexin-1 channel function impairs IL-1β production. Monocytes were activated as depicted in the presence or absence of 3 mM Probenecid and IL-1β secretion was measured 18 hours postactivation. (E-F) ERK1/2 or pannexin-1 channel inhibition results in reduced cellular efflux. Cells were activated as shown and retention of Yo-PRO dye measured 4 hours postactivation by FACS analysis. Results shown in panels A,C are representative of 2 separately performed experiments and results shown in panels B,D-F represent mean ± SD derived from 3 independent experiments (n = 3). *P < .05; **P < .001; ***P < .0005, determined by the paired t test with Bonferroni correction. (G) Model schematic of TLR and C3aR-mediated crosstalk leading to increased IL-1β production by human monocytes. TLR-mediated signals involving NF-κB translocation activate the NLRP3 inflammasome, which leads to subsequent caspase-1 activation and cleavage of inactive IL-1β into its active form. C3a generation during infection or inflammation engages the C3aR which induces an ERK1/2 phosphorylation-dependent increase in extracellular ATP availability. This in turn potentiates P2X7 signaling and caspase-1 activation cumulating in substantially increased IL-1β secretion. FACS, fluorescence-activated cell sorter.

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