Yes-associated protein (YAP) promotes cell survival by inhibiting proapoptotic dendrin signaling

Abstract

Kidney podocytes are highly specialized terminally differentiated cells that form the final barrier to urinary protein loss. Podocytes are a target for injury by metabolic, autoimmune, hereditary, inflammatory, and other stressors. Persistence of podocyte injury leads to podocyte death and loss, which results in progressive kidney damage and ultimately kidney failure. Dendrin is a dual compartment protein with proapoptotic signaling properties. Nuclear relocation of dendrin in response to glomerular injury promotes podocyte apoptosis. Here we show that Yes-associated protein (YAP), a downstream target of Hippo kinases and an inhibitor of apoptosis, is expressed in the nucleus of podocytes. The WW domains of YAP mediate the interaction with the PPXY motifs of dendrin. This interaction is functionally relevant because YAP binding to dendrin reduces dendrin-dependent, staurosporine-induced apoptosis in co-transfected HEK293 cells. Moreover gene silencing of YAP in podocytes increases adriamycin-induced podocyte apoptosis. It also increases staurosporine-induced caspase-3/7 activity, which is rescued by dendrin depletion in YAP knockdown cells. Our findings elucidate YAP binding to dendrin as a prosurvival mechanism. The antiapoptotic signaling properties of YAP in podocytes could hold significance in the quest for targeted therapeutics aimed at preventing podocyte loss.

Keywords: Apoptosis; Caspase; Cell Death; Kidney; Podocytes.

FIGURE 1.

YAP is expressed in podocytes and interacts with dendrin. A, GFP-dendrin co-precipitates with FLAG-YAP1 and FLAG-YAP2, but not with FLAG-YAP2ΔWW. No interaction is found with FLAG-Raver (control) serving as negative control. B, GFP-dendrin and GFP-dendrinΔPPXY#1 lacking PPXY motif#1 co-precipitate with FLAG-YAP2 from co-transfected HEK293 cells. In contrast GFP-dendrinΔPPXY#2+3 lacking PPXY motifs 2 and 3 or GFP-dendrinΔPPXY#1+2+3 lacking all three PPXY motifs do not interact with FLAG-YAP2. C, Western blot analysis shows YAP protein abundance in isolated mouse glomeruli as well as differentiated and undifferentiated mouse podocyte cultures. D, confocal microscopy shows co-localization of YAP with the transcription factor WT-1 in nuclei of podocytes in mouse kidney (low and high power).

FIGURE 2.

YAP and dendrin distribution in podocyte. A, in cultured podocytes, dendrin is found in the cytoplasm and the nucleus, whereas YAP is found in the nucleus. Phalloidin labeling of actin cytoskeleton visualizes cell shape. B and C, in mouse kidney glomeruli, dendrin is found in podocyte foot processes, whereas YAP is detected in podocyte nuclei.

FIGURE 3.

YAP enhances cell survival by antagonizing dendrin-mediated apoptosis. A, staurosporine induces a 4.61 ± 0.12-fold increase in HEK cells co-transfected with GFP-dendrin and FLAG-Raver serving as negative control (Con). Co-transfection of GFP-dendrin with FLAG-YAP2 but not with FLAG-ΔWWYAP2 causes a significant reduction of staurosporine-induced increase in HEK293 cell apoptosis. N.S., not significant. B, YAP-mediated protection against dendrin-induced cell death is independent of the YAP transcriptional targets cIAP1 or survivin. Staurosporine reduces cIAP1 and survivin protein abundance to a similar degree in HEK293 cells co-transfected with GFP-dendrin and FLAG-Raver control, FLAG-YAP2, or FLAG-ΔWWYAP2. C, nonsilencing control shRNA-expressing differentiated mouse podocytes showed robust YAP protein abundance, which is abrogated in YAP shRNA-expressing cells. D, gene silencing of YAP significantly increases adriamycin-induced podocyte apoptosis. E, gene silencing of YAP significantly increases staurosporine-induced caspase 3/7 activity. No difference is seen in cells treated with the vehicle control DMSO. Error bars in panels A, D, and E indicate S.E.

FIGURE 4.

Dendrin (Den) gene silencing protects YAP-depleted podocytes from staurosporine-induced apoptosis. A, protein abundance of dendrin and YAP in dendrin shRNA, YAP shRNA, or combined dendrin and YAP shRNA-expressing mouse podocytes. Con, control. WB, Western blot. B, staurosporine increases caspase 3/7 activity in YAP knockdown podocytes but not in dendrin knockdown or YAP and dendrin double knockdown podocytes. Error bars indicate S.E. *, p < 0.05. C, working model. Binding of YAP to dendrin inhibits dendrin-mediated proapoptotic signaling.