Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts

Polycystin-1 (PC1) mutations result in proliferative renal cyst growth and progression to renal failure in autosomal dominant polycystic kidney disease (ADPKD). The transcription factor STAT3 (signal transducer and activator of transcription 3) was shown to be activated in cyst-lining cells in ADPKD and PKD mouse models and may drive renal cyst growth, but the mechanisms leading to persistent STAT3 activation are unknown. A proteolytic fragment of PC1 corresponding to the cytoplasmic tail, PC1-p30, is overexpressed in ADPKD. Here, we show that PC1-p30 interacts with the nonreceptor tyrosine kinase Src, resulting in Srcdependent activation of STAT3 by tyrosine phosphorylation. The PC1-p30-mediated activation of Src/ STAT3 was independent of JAK family kinases and insensitive to the STAT3 inhibitor suppressor of cytokine signaling 3. Signaling by the EGF receptor (EGFR) or cAMP amplified the activation of Src/STAT3 by PC1-p30. Expression of PC1-p30 changed the cellular response to cAMP signaling. In the absence of PC1-p30, cAMP dampened EGFR-or IL-6-dependent activation of STAT3; in the presence of PC1-p30, cAMP amplified Src-dependent activation of STAT3. In the polycystic kidney (PCK) rat model, activation of STAT3 in renal cystic cells depended on vasopressin receptor 2 (V2R) signaling, which increased cAMP levels. Genetic inhibition of vasopressin expression or treatment with a pharmacologic V2R inhibitor strongly suppressed STAT3 activation and reduced renal cyst growth. These results suggest that PC1, via its cleaved cytoplasmic tail, integrates signaling inputs from EGFR and cAMP, resulting in Src-dependent activation of STAT3 and a proliferative response.

Section: Discussionmentioning

confidence: 93%

The Cleaved Cytoplasmic Tail of Polycystin-1 Regulates Src-Dependent STAT3 Activation

Talbot¹,

Song²,

Wang³

et al. 2014

“…10,11 IM-PTECs were preloaded with 5-(and-6)-carboxy-29,79-dichlorodihydrofluorescein diacetate (DCF-DA), which is converted into fluorescent DCF via oxidation by radicals. 18 After hypoxia, reoxygenation was induced by addition of fresh medium to the cells. Reoxygenation induced a rapid increase in probe conversion ( Figure 1A), indicating fast accumulation of cytosolic ROS.…”

Section: Activation Of Epac By 8-pcpt-29-o-me-camp Reduces Productionmentioning

confidence: 99%

Epac-Rap Signaling Reduces Oxidative Stress in the Tubular Epithelium

Stokman

Qin

Booij

et al. 2014

Activation of Rap1 by exchange protein activated by cAMP (Epac) promotes cell adhesion and actin cytoskeletal polarization. Pharmacologic activation of Epac-Rap signaling by the Epac-selective cAMP analog 8-pCPT-29-O-Me-cAMP during ischemia-reperfusion (IR) injury reduces renal failure and application of 8-pCPT-29-O-Me-cAMP promotes renal cell survival during exposure to the nephrotoxicant cisplatin. Here, we found that activation of Epac by 8-pCPT-29-O-Me-cAMP reduced production of reactive oxygen species during reoxygenation after hypoxia by decreasing mitochondrial superoxide production. Epac activation prevented disruption of tubular morphology during diethyl maleate-induced oxidative stress in an organotypic three-dimensional culture assay. In vivo renal targeting of 8-pCPT-29-O-Me-cAMP to proximal tubules using a kidney-selective drug carrier approach resulted in prolonged activation of Rap1 compared with nonconjugated 8-pCPT-29-O-Me-cAMP. Activation of Epac reduced antioxidant signaling during IR injury and prevented tubular epithelial injury, apoptosis, and renal failure. Our data suggest that Epac1 decreases reactive oxygen species production by preventing mitochondrial superoxide formation during IR injury, thus limiting the degree of oxidative stress. These findings indicate a new role for activation of Epac as a therapeutic application in renal injury associated with oxidative stress.

“…A recent study has also implicated the MEK-ERK pathway in regulating UT-A phosphorylation in response to forskolin in rat IMCD. 28 To test whether extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase is involved in regulating UT-A phosphorylation, isolated rat IMCD samples were incubated with dDAVP in the presence or absence of 10 M MEK1/2 (map kinase kinase 1 and 2) inhibitor U0126. Immunoblotting for phosphorylated ERK (pERK) demonstrated that dDAVP treatment did not increase ERK phosphorylation and that U0126 significantly blocked ERK phosphorylation in both control and dDAVP-treated samples ( Figure 9, A and F).…”

Section: Regulation Of ␤-Catenin Phosphorylation By Ddavpmentioning

confidence: 99%

Phosphoproteomic Profiling Reveals Vasopressin-Regulated Phosphorylation Sites in Collecting Duct

Bansal

Hoffert

Pisitkun

et al. 2010

Protein phosphorylation is an important component of vasopressin signaling in the renal collecting duct, but the database of known phosphoproteins is incomplete. We used tandem mass spectrometry to identify vasopressin-regulated phosphorylation events in isolated rat inner medullary collecting duct (IMCD) suspensions. Using multiple search algorithms to identify the phosphopeptides from spectral data, we expanded the size of the existing collecting duct phosphoproteome database from 367 to 1187 entries. Label-free quantification in vasopressin-and vehicle-treated samples detected a significant change in the phosphorylation of 29 of 530 quantified phosphopeptides. The targets include important structural, regulatory, and transporter proteins. The vasopressin-regulated sites included two known sites (Ser-486 and Ser-499) present in the urea channel UT-A1 and one previously unknown site (Ser-84) on vasopressin-sensitive urea channels UT-A1 and UT-A3. In vitro assays using synthetic peptides showed that purified protein kinase A (PKA) could phosphorylate all three sites, and immunoblotting confirmed the PKA dependence of Ser-84 and Ser-486 phosphorylation. These results expand the known list of collecting duct phosphoproteins and highlight the utility of targeted phosphoproteomic approaches.