MRCKα/CDC42BPA Is a Suppressor of Gef-H1/RhoA/MRTF Signaling in Tubular Cells

Pro-fibrotic mediators derived from tubular cells have a central role in he development of kidney fibrosis. Our previous studies revealed that GEF-H1 (ARHGEF2)/RhoA signalling is a crucial regulator of tubular mediator release. Fibrotic stimuli elevate GEF-H1 expression and activity, but the molecular mechanisms controlling tubular GEF-H1 activity during fibrotic reprogramming are incompletely explored. Here we used immunoprecipitation and proximity ligation assay to show that GEF-H1 interacts with Myotonic Dystrophy Kinase-related Cdc42-binding kinase (MRCK)α in porcine and human tubular cells. Using GEF-H1 mutants we mapped the interacting domain to the N-terminus of GEF-H1 and showed the requirement for an intact DH domain. MRCKα silencing elevated GEF-H1 activity, induced GEF-H1-dependent RhoA activation and augmented stress fibre formation and phospho-cofilin levels. Interestingly, TNFα or TGFβ1 addition rapidly increased binding between GEF-H1 and MRCKα, suggesting a negative feedback role. Indeed, the effect of TNFα or TGFβ1 on GEF-H1 activation was augmented in the absence of MRCKα. Using an mRNA array, we found that MRCKα depletion elevated basal and TGFβ1-induced expression of key fibrosis-related genes. MRCKα silencing also promoted nuclear translocation of the profibrotic transcriptional co-activator Myocardin- related Transcription Factor (MRTF). Depletion of MRTF-A and B prevented the increase in ACTA-2 (smooth muscle actin) and transgelin (TAGLN), key markers of fibrotic reprogramming, induced by MRCKα-silencing and TGFβ1 treatment. Taken together, we identified MRCKα as a new suppressor of GEF-H1/RhoA/MRTF signaling and tubular fibrotic gene expression. Cytokines augment binding between the two proteins, thereby mitigating GEF-H1 activation in a negative feedback cycle. These effects could be crucial for preventing RhoA overactivation.