SCF-SKP2 E3 ubiquitin ligase links mTORC1/ER stress/ISR with YAP activation in murine renal cystogenesis

The Hippo pathway nuclear effector Yes-associated protein (YAP) potentiates the progression of polycystic kidney disease (PKD) arising from ciliopathies. The mechanisms underlying the increase in YAP expression and transcriptional activity in PKD remain obscure. We observed that in kidneys from mice with juvenile cystic kidney (jck) ciliopathy, the aberrant hyperactivity of mechanistic target of rapamycin complex 1 (mTORC1), driven by ERK1/2 and PI3K/AKT cascades, induced ER proteotoxic stress. To reduce this stress by reprogramming translation, the protein kinase R–like ER kinase–eukaryotic initiation factor 2α (PERK/eIF2α) arm of the integrated stress response (ISR) was activated. PERK-mediated phosphorylation of eIF2α drove the selective translation of activating transcription factor 4 (ATF4), potentiating YAP expression. In parallel, YAP underwent K63-linked polyubiquitination by SCF S-phase kinase-associated protein 2 (SKP2) E3 ubiquitin ligase, a Hippo-independent, nonproteolytic ubiquitination that enhances YAP nuclear trafficking and transcriptional activity in cancer cells. Defective ISR cellular adaptation to ER stress in eIF2α phosphorylation–deficient jck mice further augmented YAP-mediated transcriptional activity and renal cyst growth. Conversely, pharmacological tuning down of ER stress/ISR activity and SKP2 expression in jck mice by administration of tauroursodeoxycholic acid (TUDCA) or tolvaptan impeded these processes. Restoring ER homeostasis and/or interfering with the SKP2-YAP interaction represent potential therapeutic avenues for stemming the progression of renal cystogenesis.

C E ll'.

Supplemental methods
Kidney ultrasonography. Mice were anesthetized with isoflurane, the fur was removed with depilatory cream, and ultrasound Eco gel 100 was applied. The VEVO3100 Ultra High-Frequency ultrasound system was used to obtain 2D images of the kidneys in B-mode and acquired data were used to reconstruct 3D images of the organ. Kidney volume was determined from the reconstructed 3D images. For cyst analysis, uniformity was maintained between the samples by choosing the 2D image for each kidney sample where the renal artery can be distinctly identified. The diameter of individual cysts was determined, the area was calculated, and the statistical significance was determined. Data were analyzed using the VEVO3100 software.
Urine collection/analysis. Urine was collected from individual animals from each treatment group one week before euthanasia. Mice were placed individually in metabolic cages with only access to water for 4 hours. Urine volumes were recorded and stored at -80 o C freezer before osmolarity measurements. Osmolality was measured using Advanced Instruments Model 2020 Micro Osmometer. Calibration was done with solutions of 2000 mOsm/kg to 50 mOsm/kg, and a reference solution of 290 mOsm/kg. Gene expression. RNA was isolated from the tissue samples using TRIZOL reagent (Invitrogen Inc). cDNA was synthesized using TranScript one-in-all kit (TransGen Biotech, Inc), and gene expression was measured by quantitative real-time PCR using a LightCycler FastStartDNA Master SYBR Green 1 kit (Roche). Relative gene expression was normalized against β-Actin expression. The set of primers used to analyze gene expression are outlined below: (Masterflex -Item # UZ-07596-20) was also introduced serially into the system so that more than 90% of the energy associated with the pulsatile flow was lost to the volume within the dampener. Algebraic analysis showed that to achieve the desired 3 dyn/cm 2 , the flow rate required was 0.22 mL/s and this translated to 16 RPMs for the peristaltic pump.
Yap immunofluorescence -MDCKII cells or WT and jck MEFs were grown in 4-well chamber slides. After 24 hours, MDCKII cells and WT MEFs were treated either with vehicle or with 4 mM of chloral hydrate for 18 hours. All three cell types were then fixed in PLP solution containing 4% paraformaldehyde and L-Lysine.
Immunofluorescence was performed using a rabbit anti-YAP antibody (Cell Signaling Western blot analysis. 10-15 µg total protein was loaded in each slot, separated by electrophoresis, proteins transferred to a nitrocellulose membrane, and immunoblotting was performed. The intensity of the bands was measured using ImageJ software.
Sirius red and trichrome staining. Sections were stained with picro-sirius red solution for 1 hr followed by brief exposure to acidified water. After washing, sections were counterstained with hematoxylin. Trichrome staining was performed by immersing the sections in Biebrich scarlet-acid fuchsin solution for 10-15 minutes, differentiated in phosphomolybdic-phosphotungstic acid solution for 10-15 minutes. Sections were transferred to aniline blue solution and finally differentiated in acetic acid solution briefly. After washing in water, sections were dehydrated, cleared in xylene, and mounted.
Apoptosis assay. In situ labeling kit (Novus Biologicals) was used for the detection of apoptosis. Paraffin sections of kidney samples were deparaffinized, hydrated, and Br-dU-based end-labeling of DNA in the apoptotic cells was performed following the supplier's instructions.