Figure 5
Figure 5. CSF-1R pTyr-721/PI3K signaling regulates the amplitude and duration of ERK1/2 and NF-κB p65 activation and induces miR-21 expression. CSF-1–starved macrophages were stimulated with CSF-1 (120 ng/mL) for the indicated times and processed for western blotting (WB) or RNA extraction. (A) M−/−.WT and M−/−.Y721F macrophages and (B) M−/−.3ABY721 and M−/−.3AB macrophages were subjected to WB analysis with antibodies to the activated phosphorylated forms of ERK1/2 and NF-κB p65 and to the total ERK1/2 and NF-κB p65. (C) Relative qRT-PCR quantitation of miR-21 levels in M−/−.WT macrophages treated with PI3K inhibitor LY 2940002 alone (100 μM), in combination with either the ERK1/2 inhibitor PD98059 (50 μM) or the IKK inhibitor PS1145 (2 μM), or vehicle alone (1% dimethylsulfoxide). (D) miR-155 levels in M−/−.WT macrophages. Experiments performed as in panel A. Relative expression values in panels C-D indicate the fold-change of miRNA levels in CSF-1–treated cells relative to CSF-1–starved cells at the indicated times. Data are representative of 3 independent experiments (error bars, ±SD; *P ≤ .05). (E) M−/−.WT and M−/−.3ABY721 macrophages, treated for 48 hours with LNA-miR-21 inhibitor (I), or (F), an inhibitor mismatch control (M), were CSF-1–starved before CSF-1 stimulation for the indicated times and processed as described in panels A-B. Comparisons between matching cell lines (panels A-B; top 2 panels and bottom 2 panels of panels E-F) were made on the same blots (loading control, β-actin). (G) Schematic representation of pTyr-721–mediated signaling events leading to induction of miR-21 and suppression of inflammatory networks. Filled lines, relationships demonstrated in the present study; dashed lines, suggested interactions from the literature; arrows, activation; blunt arrows, inhibition; point arrows, kinetically regulated activation and inhibition. Ovals indicate molecule activity, not expression level. SIRPb1 and IL-1β are directly suppressed by miR-21 (supplemental Table 8), but are not necessarily the exclusive mediators of macrophage miR-21 effects on ERK1/2 and NF-κB. ERK1/2 activation is positively regulated through CSF-1R tyrosines 544, 559, and 807.28

CSF-1R pTyr-721/PI3K signaling regulates the amplitude and duration of ERK1/2 and NF-κB p65 activation and induces miR-21 expression. CSF-1–starved macrophages were stimulated with CSF-1 (120 ng/mL) for the indicated times and processed for western blotting (WB) or RNA extraction. (A) M−/−.WT and M−/−.Y721F macrophages and (B) M−/−.3ABY721 and M−/−.3AB macrophages were subjected to WB analysis with antibodies to the activated phosphorylated forms of ERK1/2 and NF-κB p65 and to the total ERK1/2 and NF-κB p65. (C) Relative qRT-PCR quantitation of miR-21 levels in M−/−.WT macrophages treated with PI3K inhibitor LY 2940002 alone (100 μM), in combination with either the ERK1/2 inhibitor PD98059 (50 μM) or the IKK inhibitor PS1145 (2 μM), or vehicle alone (1% dimethylsulfoxide). (D) miR-155 levels in M−/−.WT macrophages. Experiments performed as in panel A. Relative expression values in panels C-D indicate the fold-change of miRNA levels in CSF-1–treated cells relative to CSF-1–starved cells at the indicated times. Data are representative of 3 independent experiments (error bars, ±SD; *P ≤ .05). (E) M−/−.WT and M−/−.3ABY721 macrophages, treated for 48 hours with LNA-miR-21 inhibitor (I), or (F), an inhibitor mismatch control (M), were CSF-1–starved before CSF-1 stimulation for the indicated times and processed as described in panels A-B. Comparisons between matching cell lines (panels A-B; top 2 panels and bottom 2 panels of panels E-F) were made on the same blots (loading control, β-actin). (G) Schematic representation of pTyr-721–mediated signaling events leading to induction of miR-21 and suppression of inflammatory networks. Filled lines, relationships demonstrated in the present study; dashed lines, suggested interactions from the literature; arrows, activation; blunt arrows, inhibition; point arrows, kinetically regulated activation and inhibition. Ovals indicate molecule activity, not expression level. SIRPb1 and IL-1β are directly suppressed by miR-21 (supplemental Table 8), but are not necessarily the exclusive mediators of macrophage miR-21 effects on ERK1/2 and NF-κB. ERK1/2 activation is positively regulated through CSF-1R tyrosines 544, 559, and 807.28 

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