SLIT2/ROBO2 signaling pathway inhibits nonmuscle myosin IIA activity and destabilizes kidney podocyte adhesion
Xueping Fan, … , Stephen P. Berasi, Weining Lu
Xueping Fan, … , Stephen P. Berasi, Weining Lu
Published November 17, 2016
Citation Information: JCI Insight. 2016;1(19):e86934. https://doi.org/10.1172/jci.insight.86934.
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Research Article Nephrology

SLIT2/ROBO2 signaling pathway inhibits nonmuscle myosin IIA activity and destabilizes kidney podocyte adhesion

Abstract

The repulsive guidance cue SLIT2 and its receptor ROBO2 are required for kidney development and podocyte foot process structure, but the SLIT2/ROBO2 signaling mechanism regulating podocyte function is not known. Here we report that a potentially novel signaling pathway consisting of SLIT/ROBO Rho GTPase activating protein 1 (SRGAP1) and nonmuscle myosin IIA (NMIIA) regulates podocyte adhesion downstream of ROBO2. We found that the myosin II regulatory light chain (MRLC), a subunit of NMIIA, interacts directly with SRGAP1 and forms a complex with ROBO2/SRGAP1/NMIIA in the presence of SLIT2. Immunostaining demonstrated that SRGAP1 is a podocyte protein and is colocalized with ROBO2 on the basal surface of podocytes. In addition, SLIT2 stimulation inhibits NMIIA activity, decreases focal adhesion formation, and reduces podocyte attachment to collagen. In vivo studies further showed that podocyte-specific knockout of Robo2 protects mice from hypertension-induced podocyte detachment and albuminuria and also partially rescues the podocyte-loss phenotype in Myh9 knockout mice. Thus, we have identified SLIT2/ROBO2/SRGAP1/NMIIA as a potentially novel signaling pathway in kidney podocytes, which may play a role in regulating podocyte adhesion and attachment. Our findings also suggest that SLIT2/ROBO2 signaling might be a therapeutic target for kidney diseases associated with podocyte detachment and loss.

Authors

Xueping Fan, Hongying Yang, Sudhir Kumar, Kathleen E. Tumelty, Anna Pisarek-Horowitz, Hila Milo Rasouly, Richa Sharma, Stefanie Chan, Edyta Tyminski, Michael Shamashkin, Mostafa Belghasem, Joel M. Henderson, Anthony J. Coyle, David J. Salant, Stephen P. Berasi, Weining Lu

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Figure 6

The proposed model: SLIT2/ROBO2 signaling destabilizes podocyte adhesion and attachment.

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The proposed model: SLIT2/ROBO2 signaling destabilizes podocyte adhesion...
(A) ROBO2 is predominantly localized on the basal membrane of podocytes, and SLIT2 is secreted by podocytes (7) (also see Supplemental Figure 3). Upon SLIT2 binding, ROBO2 increases its binding to adaptor protein NCK (7) which, in turn, interacts with N-WASP and Arp2/3 to inhibit actin polymerization (62) (signaling ‘a’). ROBO2 also recruits SRGAP1, which inactivates the small GTPase Cdc42 to block actin polymerization (14) (signaling ‘b’). The SH3 domain of SRGAP1 binds to ROBO2, and its F-BAR domain is associated with nonmuscle myosin IIA (NMIIA)/IIB/IIC through myosin regulatory light chain (MRLC). The complex of ROBO2/SRGAPs/MRLC/NMII results in inactivation of NMII, possibly due to structurally blocking MRLC phosphorylation (signaling ‘c’). F-actin polymerization and NMIIA activity are required for normal focal adhesion formation and maturation (2, 26). Inactivation of actin polymerization and inhibition of NMII might interfere with focal adhesion assembly and result in a decrease in podocyte attachment. Signaling ‘a’: published data from Fan et al. (7); signaling ‘b’: published data from Wong et al. (14); signaling ‘c’: data from this study. Dashed lines indicate indirect inhibition while the solid line shows the direct inhibition. Red arrows indicate enhanced or inhibited activities. CC0–3, cytoplasmic conserved regions 0–3. (B) Removal of the SLIT2/ROBO2 inhibitory signaling in podocytes (e.g., in Robo2 cKO mice) promotes focal adhesion formation and podocyte attachment, which protects against podocyte loss during injury.