title: Cardiotonic steroids trigger non-classical testosterone signaling in Sertoli cells via the α4 isoform of the sodium pump.
abstract: The α4 isoform of the Na(+),K(+)-ATPase (sodium pump) is known to be expressed in spermatozoa and to be critical for their motility. In the investigation presented here, we find that the rat-derived Sertoli cell line 93RS2 also expresses considerable amounts of the α4 isoform in addition to the α1 isoform. Since Sertoli cells are not motile, one can assume that the function of the α4 isoform in these cells must differ from that in spermatozoa. Thus, we assessed a potential involvement of this isoform in signaling pathways that are activated by the cardiotonic steroid (CTS) ouabain, a highly specific sodium pump ligand. Treatment of 93RS2 cells with ouabain leads to activation of the c-Src/c-Raf/Erk1/2 signaling cascade. Furthermore, we show for the first time that the activation of this cascade by ouabain results in phosphorylation and activation of the transcription factor CREB. This signaling cascade is induced at low nanomolar concentrations of ouabain, consistent with the involvement of the α4 isoform. This is further supported by experiments involving siRNA: silencing of α4 expression entirely blocks ouabain-induced activation of Erk1/2 whereas silencing of α1 has no effect. The findings of this study unveil new aspects in CTS/sodium pump interactions by demonstrating for the first time ouabain-induced signaling through the α4 isoform. The c-Src/c-Raf/Erk1/2/CREB cascade activated by ouabain is identical to the so-called non-classical signaling cascade that is normally triggered in Sertoli cells by testosterone. Taking into consideration that CTS are produced endogenously, our results may help to gain new insights into the physiological mechanisms associated with male fertility and reproduction.
authors.affiliation: Zentrum f. Frauenheilkunde und Geburtshilfe, Fachbereich Medizin, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Konrad
authors.forename: Raimund
authors.initials: R
authors.lastname: Dietze
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kirch
authors.forename: Herbert
authors.initials: H
authors.lastname: Kirch
authors.forename: Alexander
authors.initials: A
authors.lastname: Eva
authors.forename: Georgios
authors.initials: G
authors.lastname: Scheiner-Bobis
mesh_terms: D000818:Animals; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D002301:Cardiac Glycosides / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D017136:Ion Transport / Q000187:drug effects; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D010042:Ouabain / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012708:Sertoli Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013739:Testosterone / Q000494:pharmacology; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D002301:Cardiac Glycosides; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013739:Testosterone; D010042:Ouabain; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D019908:Proto-Oncogene Proteins c-raf; D000254:Sodium-Potassium-Exchanging ATPase