Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking

Bogum, Jana; Faust, Dörte; Zühlke, Kerstin; Eichhorst, Jenny; Moutty, Marie Christine; Furkert, Jens; El-Dahshan, Adeeb; Neuenschwander, Martin; Kries, Jens Peter von; Wiesner, Burkhard; Trimpert, Christiane; Deen, Peter M.T.; Valenti, Giovanna; Rosenthal, Walter; Klußmann, Jens Peter

doi:10.1681/asn.2012030295

Cited by 25 publications

(28 citation statements)

References 56 publications

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“…PKA activity was measured by applying the PepTag protein kinase assay (Promega) according to the manufacturer's instructions (8). Porcine coronary artery SMCs (passage 2) were cultured in 100-mm culture dishes in SmGM medium for 2 days.…”

Section: Methodsmentioning

confidence: 99%

G protein-coupled estrogen receptor 1 mediates relaxation of coronary arteries via cAMP/PKA-dependent activation of MLCP

Klussmann

et al. 2014

American Journal of Physiology-Endocrinology and Metabolism

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Yu X, Li F, Klussmann E, Stallone JN, Han G. G proteincoupled estrogen receptor 1 mediates relaxation of coronary arteries via cAMP/PKA-dependent activation of MLCP. Am J Physiol Endocrinol Metab 307: E398 -E407, 2014. First published July 8, 2014 doi:10.1152/ajpendo.00534.2013.-Activation of GPER exerts a protective effect in hypertension and ischemia-reperfusion models and relaxes arteries in vitro. However, our understanding of the mechanisms of GPER-mediated vascular regulation is far from complete. In the current study, we tested the hypothesis that GPER-induced relaxation of porcine coronary arteries is mediated via cAMP/PKA signaling. Our findings revealed that vascular relaxation to the selective GPER agonist G-1 (0.3-3 M) was associated with increased cAMP production in a concentration-dependent manner. Furthermore, inhibition of adenylyl cyclase (AC) with SQ-22536 (100 M) or of PKA activity with either Rp-8-CPT-cAMPS (5 M) or PKI (5 M) attenuated G-1-induced relaxation of coronary arteries preconstricted with PGF2␣ (1 M). G-1 also increased PKA activity in cultured coronary artery smooth muscle cells (SMCs). To determine downstream signals of the cAMP/PKA cascade, we measured RhoA activity in cultured human and porcine coronary SMCs and myosin-light chain phosphatase (MLCP) activity in these artery rings by immunoblot analysis of phosphorylation of myosin-targeting subunit protein-1 (p-MYPT-1; the MLCP regulatory subunit). G-1 decreased PGF2␣-induced p-MYPT-1, whereas Rp-8-CPT-cAMPS prevented this inhibitory effect of G-1. Similarly, G-1 inhibited PGF2␣-induced phosphorylation of MLC in coronary SMCs, and this inhibitory effect was also reversed by Rp-8-CPT-cAMPS. RhoA activity was downregulated by G-1, whereas G36 (GPER antagonist) restored RhoA activity. Finally, FMP-API-1 (100 M), an inhibitor of the interaction between PKA and A-kinase anchoring proteins (AKAPs), attenuated the effect of G-1 on coronary artery relaxation and p-MYPT-1. These findings demonstrate that localized cAMP/PKA signaling is involved in GPER-mediated coronary vasodilation by activating MLCP via inhibition of RhoA pathway.

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Section: Methodsmentioning

confidence: 99%

G protein-coupled estrogen receptor 1 mediates relaxation of coronary arteries via cAMP/PKA-dependent activation of MLCP

Klussmann

et al. 2014

American Journal of Physiology-Endocrinology and Metabolism

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“…Mouse collecting duct (MCD)4 cells, [5,26,27] stably expressing human AQP2, were seeded in 384-well ultrathin optical bottom (190 µm) microtiter plates (GREINER, µclear). The MCD4 cells were transfected with the siRNAs using Lipofectamine 2000 (Thermo Fisher; https://www.thermofisher.com).…”

Section: Kinome Screening and Automated Immunofluorescence Microscopymentioning

confidence: 99%

“…Kinase and pNPP Phosphatase Activity Assays PKA activity was monitored using the PepTag Assay (Promega, Walldorf, Germany) as described. [5,26] For non-radioactive evaluation of CDK18 kinase activity, Wt, constitutively active and kinase dead forms (see above) and cyclin A were transiently expressed in HEK293 cells. The CDK18 forms were immunoprecipitated 48 h later with anti-FLAG-coupled magnetic beads (Sigma-Aldrich; 1 h, 4 • C).…”

Section: Cell Lysis Immunoprecipitation Camp Pull-down Western Blomentioning

confidence: 99%

“…They represent a cellular model for AQP2 trafficking. The cells stably express human AQP2 and when stimulated with the adenylyl cyclase activator, forskolin, cAMP levels increase, and trigger the AQP2 redistribution predominantly to the basolateral plasma membrane [5,26,27]. The AQP2 redistribution was monitored by automated immunofluorescence microscopy.…”

Section: Kinome Knockdown Identifies Candidates Controlling the Redismentioning

confidence: 99%

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Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP

Dema

Faust

Lazarow

et al. 2020

Cells

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Arginine-vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells through regulation of the water channel aquaporin-2 (AQP2). The hormone binds to vasopressin V2 receptors (V2R) on the surface of the cells and stimulates cAMP synthesis. The cAMP activates protein kinase A (PKA), which initiates signaling that causes an accumulation of AQP2 in the plasma membrane of the cells facilitating water reabsorption from primary urine and fine-tuning of body water homeostasis. AVP-mediated PKA activation also causes an increase in the AQP2 protein abundance through a mechanism that involves dephosphorylation of AQP2 at serine 261 and a decrease in its poly-ubiquitination. However, the signaling downstream of PKA that controls the localization and abundance of AQP2 is incompletely understood. We carried out an siRNA screen targeting 719 kinase-related genes, representing the majority of the kinases of the human genome and analyzed the effect of the knockdown on AQP2 by high-content imaging and biochemical approaches. The screening identified 13 hits whose knockdown inhibited the AQP2 accumulation in the plasma membrane. Amongst the candidates was the so far hardly characterized cyclin-dependent kinase 18 (CDK18). Our further analysis revealed a hitherto unrecognized signalosome comprising CDK18, an E3 ubiquitin ligase, STUB1 (CHIP), PKA and AQP2 that controls the localization and abundance of AQP2. CDK18 controls AQP2 through phosphorylation at serine 261 and STUB1-mediated ubiquitination. STUB1 functions as an A-kinase anchoring protein (AKAP) tethering PKA to the protein complex and bridging AQP2 and CDK18. The modulation of the protein complex may lead to novel concepts for the treatment of disorders which are caused or are associated with dysregulated AQP2 and for which a satisfactory treatment is not available, e.g., hyponatremia, liver cirrhosis, diabetes insipidus, ADPKD or heart failure.

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“…9 Some of these interventions indeed result in increased membrane AQP2 expression, and they increase urinary-concentrating ability in animal models, including vasopressin-deficient Brattleboro rats and lithium-treated rats. 2 In the current issue of JASN, Bogum et al 10 use a smallmolecule screening approach to identify chemical compounds that inhibit cAMP-induced AQP2 membrane accumulation. Such compounds would function as aquaretics and counteract the effects of elevated vasopressin/cAMP or increased AQP2 levels in conditions that might lead to excessive fluid reabsorption, hypertension, and hyponatremia.…”

mentioning

confidence: 99%

Aquaporin-2 Inhibitors

Brown

2013

Journal of the American Society of Nephrology

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The critical contribution of aquaporin-2 (AQP) to water homeostasis in mammals is highlighted by the severe concentrating defects seen in humans with mutations in the AQP2 gene and in mice that are AQP2 null or have loss-of-function mutations in AQP2. 1,2 The role of the antidiuretic hormone vasopressin and its receptor, V2R, in the signaling cascade that leads to membrane accumulation of AQP2 in collecting-duct principal cells, and to an increase in renal water reabsorption, is well established. 3 The involvement of VP-induced cAMP elevation by adenylyl cyclase activation, and subsequent protein kinase A-induced phosphorylation of AQP2, is also well known. Defects in any of the major components of this signaling cascade have the potential to lead to aberrant AQP2 trafficking and function, with an associated loss of urine-concentrating ability or an inappropriate increase in collecting-duct water retention.Decreased water reabsorption leads to diabetes insipidus, either central (loss of vasopressin) or nephrogenic (loss of V2R or AQP2 function). Excessive AQP2 expression and plasma membrane localization are associated with such conditions as congestive heart failure, cirrhosis of the liver, and the syndrome of inappropriate antidiuretic hormone secretion. In such cases, inappropriate water re-absorption can lead to moderate or severe hyponatremia and its subsequent complications, sometimes fatal. Furthermore, many defects in water homeostasis result from acquired conditions, such as lithium-induced nephrogenic diabetes insipidus, a common consequence of the use of this compound as therapy for bipolar disorder.Considerable effort has therefore been expended over the past several years to identify components of the AQP2 trafficking pathway that could be targeted by drugs or other therapeutic strategies in order to alleviate the consequences of these diseases. On the basis of current knowledge of the signaling and trafficking pathways that have been elucidated by several groups, different strategies have been explored. These include bypassing the V2R-mediated signaling pathway using alternative cyclic nucleotide-generating agents, such as the cyclic guanosine monophopshate phosphodiesterase inhibitor sildenafil; 4,5 activating alternative pathways, including prostaglandin signaling; 6 and modulating trafficking with drugs, including statins, that affect the actin cytoskeleton, 7,8 a key factor in endo-and exocytotic events, as well as inhibiting endocytosis directly. 9 Some of these interventions indeed result in increased membrane AQP2 expression, and they increase urinary-concentrating ability in animal models, including vasopressin-deficient Brattleboro rats and lithium-treated rats. 2 In the current issue of JASN, Bogum et al. 10 use a smallmolecule screening approach to identify chemical compounds that inhibit cAMP-induced AQP2 membrane accumulation. Such compounds would function as aquaretics and counteract the effects of elevated vasopressin/cAMP or increased AQP2 levels in conditions that might lead to exce...

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Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking

Cited by 25 publications

References 56 publications

G protein-coupled estrogen receptor 1 mediates relaxation of coronary arteries via cAMP/PKA-dependent activation of MLCP

G protein-coupled estrogen receptor 1 mediates relaxation of coronary arteries via cAMP/PKA-dependent activation of MLCP

Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP

Aquaporin-2 Inhibitors

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