SLIT2/ROBO2 signaling pathway inhibits nonmuscle myosin IIA activity and destabilizes kidney podocyte adhesion
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
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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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 4

SLIT2-ROBO2 signaling destabilizes podocyte focal adhesions.

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SLIT2-ROBO2 signaling destabilizes podocyte focal adhesions. (A) Express...
(A) Expression of ROBO2 in control and ROBO2-YFP overexpressing mouse podocytes detected by Western blot with α-tubulin as a loading control. The lanes were run on the same gel but were noncontiguous. (B) Quantification by ATPlite assay of mouse podocytes attached to collagen-coated plates in media containing buffer control (CTL) or 10 nM recombinant SLIT2 N-terminus measured after 30 minutes. Data are presented as the mean ± SD, n = 4. *P < 0.05 compared with the cells as indicated by 1-way ANOVA. Data are representative of at least 3 separate experiments and various time points between 30 and 90 minutes. (C) Representative images of immunostaining of focal adhesion markers vinculin and paxillin in differentiated vector control cells (left 2 columns) or ROBO2-overexpressing mouse podocytes (right 2 columns) treated with buffer (CTL) or SLIT2 N-terminus. The whole cells were labeled by CellMask staining. Scale bar: 61 μm. (D and E) Quantification of vinculin (D) and paxillin (E) spot staining areas divided by the cell spread areas. Data are presented as the mean ± SD, n = 7. *P < 0.05 compared with the cells as indicated by 1-way ANOVA.