Figure 4
Figure 4. miR-21 expression is positively regulated downstream of CSF-1R pTyr-721 to decrease the abundance of proinflammatory molecules. (A) Downstream effector analysis performed by IPA on differentially regulated CSF-1 transcripts (FC > 1.5, P < .05), predicts miR-21 as the top statistically significant miRNA (supplemental Table 5) that is activated in a pTyr-721–dependent manner (z score >2, gray dotted line). The number of coexpressed mRNAs predicted as miR-21 targets (bold) and the statistical P values (italics) are indicated for each cell line at the top of each bar. (B) Validation of miR-21 as a pTyr-721–regulated molecule and of a miR-21 inhibitor. For miR-21 validation, qRT-PCR measurements were performed on complementary DNA prepared from M−/−.WT and M−/−.Y721F macrophages and from the M−/−.3ABY721 and M−/−.3AB cells that were either CSF-1–starved overnight, then treated with CSF-1 (120 ng/mL) for the indicated time points, or constitutively grown in CSF-1 (C). For inhibitor validation, cells were constitutively grown in CSF-1. The miR-21 inhibitor (C+I), or mismatch inhibitor control (C+M), were added 48 hours before harvesting the cells and determining the miR-21 levels by qRT-PCR. (C) Cytoscape representation of the IPA-predicted miR-21 targets that are regulated in a pTyr-721–dependent manner. Note that 80% of these molecules are associated with macrophage polarization toward an inflammatory phenotype (red circles), whereas only 10% are associated with the M2 phenotype (blue circles). (D-E) CSF-1 negatively regulates IL-1β mRNA levels and IL-1β secretion in a pTyr-721– and miR-21–dependent manner. (D) qRT-PCR measurements of IL-1β mRNA in Tyr-721– and Tyr-721F–expressing macrophages. Cells were treated as described in panel B. (E) Conditioned media from Tyr-721– and Tyr-721F–expressing macrophages treated as described in panel B were used to measure the amount of soluble mature IL-1β released in the medium, by enzyme-linked immunosorbent assay. (B,D,E) Key for cell lines as in panel A. Five biological replicates; error bars, ±SD; *P ≤ .05.

miR-21 expression is positively regulated downstream of CSF-1R pTyr-721 to decrease the abundance of proinflammatory molecules. (A) Downstream effector analysis performed by IPA on differentially regulated CSF-1 transcripts (FC > 1.5, P < .05), predicts miR-21 as the top statistically significant miRNA (supplemental Table 5) that is activated in a pTyr-721–dependent manner (z score >2, gray dotted line). The number of coexpressed mRNAs predicted as miR-21 targets (bold) and the statistical P values (italics) are indicated for each cell line at the top of each bar. (B) Validation of miR-21 as a pTyr-721–regulated molecule and of a miR-21 inhibitor. For miR-21 validation, qRT-PCR measurements were performed on complementary DNA prepared from M−/−.WT and M−/−.Y721F macrophages and from the M−/−.3ABY721 and M−/−.3AB cells that were either CSF-1–starved overnight, then treated with CSF-1 (120 ng/mL) for the indicated time points, or constitutively grown in CSF-1 (C). For inhibitor validation, cells were constitutively grown in CSF-1. The miR-21 inhibitor (C+I), or mismatch inhibitor control (C+M), were added 48 hours before harvesting the cells and determining the miR-21 levels by qRT-PCR. (C) Cytoscape representation of the IPA-predicted miR-21 targets that are regulated in a pTyr-721–dependent manner. Note that 80% of these molecules are associated with macrophage polarization toward an inflammatory phenotype (red circles), whereas only 10% are associated with the M2 phenotype (blue circles). (D-E) CSF-1 negatively regulates IL-1β mRNA levels and IL-1β secretion in a pTyr-721– and miR-21–dependent manner. (D) qRT-PCR measurements of IL-1β mRNA in Tyr-721– and Tyr-721F–expressing macrophages. Cells were treated as described in panel B. (E) Conditioned media from Tyr-721– and Tyr-721F–expressing macrophages treated as described in panel B were used to measure the amount of soluble mature IL-1β released in the medium, by enzyme-linked immunosorbent assay. (B,D,E) Key for cell lines as in panel A. Five biological replicates; error bars, ±SD; *P ≤ .05.

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