title: Ouabain activates the Na-K-ATPase signalosome to induce autosomal dominant polycystic kidney disease cell proliferation.
abstract: The Na-K-ATPase is part of a cell signaling complex, the Na-K-ATPase signalosome, which upon activation by the hormone ouabain regulates the function of different cell types. We previously showed that ouabain induces proliferation of epithelial cells derived from renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD cells). Here, we investigated the signaling pathways responsible for mediating the effects of ouabain in these cells. Incubation of ADPKD cells with ouabain, in concentrations similar to those found in blood, stimulated phosphorylation of the epidermal growth factor receptor (EGFR) and promoted its association to the Na-K-ATPase. In addition, ouabain activated the kinase Src, but not the related kinase Fyn. Tyrphostin AG1478 and PP2, inhibitors of EGFR and Src, respectively, blocked ouabain-dependent ADPKD cell proliferation. Treatment of ADPKD cells with ouabain also caused phosphorylation of the caveolar protein caveolin-1, and disruption of cell caveolae with methyl-β-cyclodextrin prevented Na-K-ATPase-EGFR interaction and ouabain-induced proliferation of the cells. Downstream effects of ouabain in ADPKD cells included activation of B-Raf and MEK and phosphorylation of the extracellular regulated kinase ERK, which translocated into the ADPKD cell nuclei. Finally, ouabain reduced expression of the cyclin-dependent kinase inhibitors p21 and p27, which are suppressors of cell proliferation. Different from ADPKD cells, ouabain showed no significant effect on B-Raf, p21, and p27 in normal human kidney epithelial cells. Altogether, these results identify intracellular pathways of ouabain-dependent Na-K-ATPase-mediated signaling in ADPKD cells, including EGFR-Src-B-Raf-MEK/ERK, and establish novel mechanisms involved in ADPKD cell proliferation.
authors.affiliation: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA.
authors.forename: Anh-Nguyet T
authors.initials: AN
authors.lastname: Nguyen
authors.forename: Kyle
authors.initials: K
authors.lastname: Jansson
authors.forename: Gladis
authors.initials: G
authors.lastname: Sánchez
authors.forename: Madhulika
authors.initials: M
authors.lastname: Sharma
authors.forename: Gail A
authors.initials: GA
authors.lastname: Reif
authors.forename: Darren P
authors.initials: DP
authors.lastname: Wallace
authors.forename: Gustavo
authors.initials: G
authors.lastname: Blanco
mesh_terms: D051242:Caveolin 1 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology*; D010766:Phosphorylation; D016891:Polycystic Kidney, Autosomal Dominant / Q000139:chemically induced / Q000201:enzymology*; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D011799:Quinazolines; D015398:Signal Transduction / Q000187:drug effects*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C412373:AG 1879; C493956:CDKN1A protein, human; D051242:Caveolin 1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004791:Enzyme Inhibitors; D011743:Pyrimidines; D011799:Quinazolines; D020032:Tyrphostins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D050760:Cyclin-Dependent Kinase Inhibitor p27; C101044:RTKI cpd; D010042:Ouabain; D066246:ErbB Receptors; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020930:MAP Kinase Kinase Kinases; D000254:Sodium-Potassium-Exchanging ATPase