title: The ubiquitous isoform of Na/K-ATPase (ATP1A1) regulates junctional proteins, connexin 43 and claudin 11 via Src-EGFR-ERK1/2-CREB pathway in rat Sertoli cells.
abstract: Interaction of Na/K-ATPase with its ligand ouabain has been implicated in the regulation of various biological processes. The objective was to investigate roles of Na/K-ATPase isoforms in formation and function of junctional complexes in Sertoli cells. Primary cultures of Sertoli cells were obtained by enzymatic digestion of 20-day-old rat testes and grown on Matrigel-coated dishes for 7 days. Sertoli cells predominantly expressed the ubiquitous isoform of Na/K-ATPase (ATP1A1), confirmed by immunoblotting, PCR, immunofluorescence, and mass spectrometry. Treatment of Sertoli cells with 50 nM ouabain increased transepithelial electrical resistance (TER) and expression of claudin 11 (tight junctions) and connexin 43 (gap junctions), whereas 1 mM ouabain had opposite effects. Involvement of Src-EGFR-ERK1/2-CREB pathway in ouabain-mediated expression of claudin 11 and connexin 43 was evaluated. Incubation of Sertoli cells with 50 nM ouabain increased content of p-Src, p-EGFR, p-ERK1/2, and p-CREB; in contrast, 1 mM ouabain decreased phosphorylation of these signaling molecules. Preincubation of Sertoli cells with inhibitors of Src and MAPK pathways inhibited ouabain-induced effects on these signaling molecules, TER, and expression of claudin 11 and connexin 43. In conclusion, we inferred that ATP1A1 regulated Sertoli cell tight junctions and gap junctions through the Src-EGFR-ERK1/2-CREB pathway. Ouabain is an endogenous steroid; therefore, its interaction with ATP1A1 may be a critical signaling mechanism for the regulation of Sertoli cell function and male fertility.
authors.affiliation: Department of Production Animal Health, Faculty of Veterinary Medicine, Heritage Medical Research Building RM 400, 3330 Hospital Drive NW, University of Calgary, Calgary, AB, Canada.
authors.forename: Gayathri D
authors.initials: GD
authors.lastname: Rajamanickam
authors.affiliation: Department of Production Animal Health, Faculty of Veterinary Medicine, Heritage Medical Research Building RM 400, 3330 Hospital Drive NW, University of Calgary, Calgary, AB, Canada.
authors.forename: John P
authors.initials: JP
authors.lastname: Kastelic
authors.affiliation: Department of Production Animal Health, Faculty of Veterinary Medicine, Heritage Medical Research Building RM 400, 3330 Hospital Drive NW, University of Calgary, Calgary, AB, Canada.
authors.forename: Jacob C
authors.initials: JC
authors.lastname: Thundathil
mesh_terms: D000818:Animals; D057167:Claudins / Q000235:genetics / Q000378:metabolism*; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012708:Sertoli Cells / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D057167:Claudins; C492353:Cldn11 protein, rat; D018031:Connexin 43; D017362:Cyclic AMP Response Element-Binding Protein; C580592:Gja1 protein, rat; D066246:ErbB Receptors; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; C499075:Atp1a1 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Sertoli cell; Na/K-ATPase; tight junction; gap junction