Notch signaling is an important driver of renal fibrosis,19,20 and it is noteworthy that LXA4 suppressed TGF-��1 driven expression of the example Notch ligand JAG1 and its target gene, hairy and enhancer of split 1 (HES1), which is also associated with renal fibrosis (Figure 1D).21 LXA4 attenuated TGF-��1 induction of numerous profibrotic genes including FN1, COL1A1, COL1A2, and THBS1 (Figure 1D). The ALX/FPR2 receptor has been shown to couple via a pertussis toxin�Csensitive G protein.22 Here we report that pertussis toxin blocked LXA4 attenuation of TGF-��1 responses including JAG1 (Figure 1, E and F) and CDH2 protein expression (Supplemental Figure 1). Using small interfering RNA (siRNA) targeted against ALX/FPR2, LXA4 no longer suppressed TGF-��1�Cinduced JAG1 (Figure 1, G and H).

These data indicate that LXA4 attenuation of the TGF-��1�Cdriven profibrotic signal is mediated through ALX/FPR2. Figure 1. LXA4 attenuates TGF-��1 signaling in HK-2 cells. (A) Semi-quantitative PCR detection of ALX/FPR2 in HK-2 cells. ALX/FPR2 expression in human mesangial cells (HMCs) is used as a positive control; NTC, non-template control. (B) Representative Western … let-7c Is Induced by Lipoxin and Suppressed by TGF-��1 miRNA expression in HK-2 cells pretreated with vehicle (0.1% ethanol) or LXA4 (1 nM) for 30 minutes and or TGF-��1 (10 ng/ml) stimulation for 24 hours was investigated by miRNA microarray (MRA-1001, miRBase V.14). miRNAs displaying high basal expression included let-7 and miR-200 family members. Several miRNAs previously identified as highly expressed in kidney23 were also detected (miR-192, miR-194, miR-215) (Supplemental Table 1).

GSK-3 Multiple let-7 family members were prominent among the miRNAs induced 24 hours after LXA4 pretreatment (Figure 2A), and several of these (let-7a, let-7c) were subsequently validated by quantitative RT-PCR (qRT-PCR) (Figure 2B). let-7 family members are encoded in multiple regions of the human genome, and are highly conserved across multiple species with respect to sequence and function.24 Consistent with previous reports,25,26 we found downregulation of miR-192 in response to TGF-��1 (Supplemental Figure 2). Time-course analysis of let-7c expression (0�C6 hours) revealed that let-7c was induced by LXA4 and repressed by TGF-��1 at early time points (Figure 2C). We investigated let-7c responses in primary human mesangial cells and rat renal fibroblasts (NRK49F) 2 hours after stimulation with LXA4 and/or TGF-��1. We observed significant elevation of let-7c levels in human mesangial cells in LXA4-treated cells, whereas in NRK49F cells we saw a significant reduction in let-7c expression in response to TGF-��1 (Figure 2D). Figure 2. let-7c miRNA is induced by lipoxins and repressed by TGF-��1 and targets TGF��R1.