title: GSK-3β/NFAT Signaling Is Involved in Testosterone-Induced Cardiac Myocyte Hypertrophy.
abstract: Testosterone induces cardiac hypertrophy through a mechanism that involves a concerted crosstalk between cytosolic and nuclear signaling pathways. Nuclear factor of activated T-cells (NFAT) is associated with the promotion of cardiac hypertrophy, glycogen synthase kinase-3β (GSK-3β) is considered to function as a negative regulator, mainly by modulating NFAT activity. However, the role played by calcineurin-NFAT and GSK-3β signaling in testosterone-induced cardiac hypertrophy has remained unknown. Here, we determined that testosterone stimulates cardiac myocyte hypertrophy through NFAT activation and GSK-3β inhibition. Testosterone increased the activity of NFAT-luciferase (NFAT-Luc) in a time- and dose-dependent manner, with the activity peaking after 24 h of stimulation with 100 nM testosterone. NFAT-Luc activity induced by testosterone was blocked by the calcineurin inhibitors FK506 and cyclosporine A and by 11R-VIVIT, a specific peptide inhibitor of NFAT. Conversely, testosterone inhibited GSK-3β activity as determined by increased GSK-3β phosphorylation at Ser9 and β-catenin protein accumulation, and also by reduction in β-catenin phosphorylation at residues Ser33, Ser37, and Thr41. GSK-3β inhibition with 1-azakenpaullone or a GSK-3β-targeting siRNA increased NFAT-Luc activity, whereas overexpression of a constitutively active GSK-3β mutant (GSK-3βS9A) inhibited NFAT-Luc activation mediated by testosterone. Testosterone-induced cardiac myocyte hypertrophy was established by increased cardiac myocyte size and [3H]-leucine incorporation (as a measurement of cellular protein synthesis). Calcineurin-NFAT inhibition abolished and GSK-3β inhibition promoted the hypertrophy stimulated by testosterone. GSK-3β activation by GSK-3βS9A blocked the increase of hypertrophic markers induced by testosterone. Moreover, inhibition of intracellular androgen receptor prevented testosterone-induced NFAT-Luc activation. Collectively, these results suggest that cardiac myocyte hypertrophy induced by testosterone involves a cooperative mechanism that links androgen signaling with the recruitment of NFAT through calcineurin activation and GSK-3β inhibition.
authors.affiliation: Laboratorio de Endocrinología Celular, Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Javier
authors.initials: J
authors.lastname: Duran
authors.affiliation: Laboratorio de Endocrinología Celular, Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Cesar
authors.initials: C
authors.lastname: Oyarce
authors.affiliation: Laboratorio de Endocrinología Celular, Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Mario
authors.initials: M
authors.lastname: Pavez
authors.affiliation: Laboratorio de Endocrinología Celular, Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Denisse
authors.initials: D
authors.lastname: Valladares
authors.affiliation: Programa de Anatomía y Biología del Desarrollo, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Carla
authors.initials: C
authors.lastname: Basualto-Alarcon
authors.affiliation: Laboratorio de Endocrinología Celular, Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Daniel
authors.initials: D
authors.lastname: Lagos
authors.affiliation: Laboratorio de Endocrinología Celular, Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Genaro
authors.initials: G
authors.lastname: Barrientos
authors.affiliation: Laboratorio de Endocrinología Celular, Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Mayarling Francisca
authors.initials: MF
authors.lastname: Troncoso
authors.affiliation: Heart Failure Bioscience Department, Cardiovascular and Metabolic Diseases (CVMD), Innovative Medicines & Early Development iMED Biotech unit, AstraZeneca R&D, Mölndal, Sweden.
authors.forename: Cristian
authors.initials: C
authors.lastname: Ibarra
authors.affiliation: Laboratorio de Endocrinología Celular, Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Manuel
authors.identifier: http://orcid.org/0000-0001-7877-8160
authors.initials: M
authors.lastname: Estrada
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D006332:Cardiomegaly / Q000139:chemically induced* / Q000235:genetics; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects; D000071679:Glycogen Synthase Kinase 3 beta / Q000235:genetics / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000473:pathology*; D050778:NFATC Transcription Factors / Q000235:genetics / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013739:Testosterone / Q000009:adverse effects*
chemical_list: D050778:NFATC Transcription Factors; D013739:Testosterone; D000071679:Glycogen Synthase Kinase 3 beta