Abstract
Slit guidance ligand 2 (SLIT2) is a large, secreted protein that binds roundabout (ROBO) receptors on multiple cell types, including neurons and kidney podocytes. SLIT2-ROBO-mediated signaling regulates neuronal migration and ureteric bud (UB) outgrowth during kidney development as well as glomerular filtration in adult kidneys. Additionally, SLIT2 binds Gremlin, an antagonist of bone morphogenetic proteins (BMPs), and BMP-Gremlin signaling also regulates UB formation. However, direct cross-talk between the ROBO2-SLIT2 and BMP-Gremlin signaling pathways has not been established. Here, we report the discovery of negative feedback between the SLIT2 and BMP-Gremlin signaling pathways. We found that the SLIT2-Gremlin interaction inhibited both SLIT2-ROBO2 signaling in neurons and Gremlin antagonism of BMP activity in myoblasts and fibroblasts. Furthermore, BMP2 down-regulated SLIT2 expression and promoter activity through canonical BMP signaling. Gremlin treatment, BMP receptor inhibition, and SMAD family member 4 (SMAD4) knockdown rescued BMP-mediated repression of SLIT2. BMP2 treatment of nephron progenitor cells derived from human embryonic stem cells decreased SLIT2 expression, further suggesting an interaction between the BMP2-Gremlin and SLIT2 pathways in human kidney cells. In conclusion, our study has revealed direct negative cross-talk between two pathways, previously thought to be unassociated, that may regulate both kidney development and adult tissue maintenance.
Highlights
Slit guidance ligand 2 (SLIT2) is a large, secreted protein that binds roundabout (ROBO) receptors on multiple cell types, including neurons and kidney podocytes
BMP2 treatment of posterior intermediate mesoderm cells resulted in a statistically significant decrease in SLIT2 mRNA expression, and Gremlin treatment rescued SLIT2 mRNA down-regulation by BMP2 (Fig. 7B). Because both SLIT2–ROBO2 and BMP2–Gremlin signalings regulate the expression and activity of glial cell-derived neurotrophic factor (GDNF) [14, 36, 37, 52] and the formation of a complex of GDNF with RET and GDNF family receptor ␣1 (GFR␣1) is the initiating factor in ureteric bud (UB) outgrowth during kidney development [51], we investigated the effects of BMP2 treatment on GDNF, RET, and GFR␣1 mRNA expression in posterior intermediate mesenchyme-like cells
We have confirmed that SLIT2 results in an N-terminal fragment (SLIT2N) binds Gremlin, and we determined that this interaction occurs via the D2 domain of SLIT2
Summary
It had been previously reported that rat Gremlin interacts directly with the repulsive guidance cue SLIT2 [30]. Because BMP2 regulates Id1 promoter activity in C2C12BRE myoblasts, we measured ID1 gene expression in MRC5 fibroblasts to confirm the effects of SLIT2N on Gremlin antagonism of BMP2 signaling (Fig. 3C). To determine the effects of BMP2 and Gremlin treatment on SLIT2 expression in human nephron progenitor cells, we treated these cells while they were in the posterior intermediate mesoderm stage (day 7) with differentiation media (ARPMI ϩ 10 ng/ml FGF9) [69] containing BMP2, Gremlin, or BMP2 ϩ Gremlin for 48 h until they were in the metanephric mesenchyme stage of development (day 9) (Fig. 7A). BMP2 promoted SMAD1 phosphorylation in posterior intermediate mesoderm-like cells (day 7) (Fig. 7J), as well as in metanephric mesoderm-like cells (day 9) (Fig. 7K), confirming that canonical BMP2 signaling pathways are active in these nephron progenitor cells Taken together, these data suggest that BMP–Gremlin signaling regulates SLIT2 expression, as well as the expression of GDNF and RET, in human nephron progenitor-like cells differentiated from ES cells
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