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GSK-3β/NFAT Signaling Is Involved in Testosterone-Induced Cardiac Myocyte Hypertrophy
Javier Duran1, Cesar Oyarce1, Mario Pavez1
1Laboratorio de Endocrinología Celular, Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
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.
Insights
Testosterone promotes cardiac hypertrophy by activating Nuclear Factor of Activated T-cells (NFAT) and inhibiting Glycogen Synthase Kinase-3β (GSK-3β). This study elucidates the signaling pathways involved in testosterone-induced heart muscle growth.
Area of Science:
- Cardiovascular Biology
- Molecular Endocrinology
- Cell Signaling
Background:
- Testosterone is known to induce cardiac hypertrophy, a process involving complex intracellular signaling.
- Nuclear Factor of Activated T-cells (NFAT) promotes cardiac hypertrophy, while Glycogen Synthase Kinase-3β (GSK-3β) acts as a negative regulator, primarily by modulating NFAT.
- The specific roles of calcineurin-NFAT and GSK-3β signaling in testosterone-induced cardiac hypertrophy were previously undefined.
Purpose of the Study:
- To investigate the molecular mechanisms by which testosterone stimulates cardiac myocyte hypertrophy.
- To determine the involvement of calcineurin-NFAT and GSK-3β signaling pathways in testosterone-mediated cardiac hypertrophy.
- To elucidate the crosstalk between androgen signaling and these key intracellular pathways.
Main Methods:
- Utilized NFAT-luciferase reporter assays to measure NFAT activity under testosterone stimulation.
- Employed calcineurin inhibitors (FK506, cyclosporine A) and a specific NFAT inhibitor (11R-VIVIT).
- Assessed GSK-3β activity via phosphorylation status and β-catenin levels, using inhibitors (1-azakenpaullone) and siRNA, and a constitutively active mutant (GSK-3βS9A).
- Quantified cardiac myocyte hypertrophy by measuring cell size and protein synthesis ([3H]-leucine incorporation).
- Investigated the role of the intracellular androgen receptor.
Main Results:
- Testosterone dose- and time-dependently increased NFAT activity, peaking at 24 hours with 100 nM testosterone.
- Testosterone inhibited GSK-3β activity, evidenced by increased GSK-3β phosphorylation at Ser9 and β-catenin accumulation.
- Calcineurin-NFAT inhibition abolished testosterone-induced hypertrophy, while GSK-3β inhibition promoted it.
- Inhibition of the intracellular androgen receptor blocked testosterone-induced NFAT-Luc activation.
- Testosterone-induced cardiac myocyte hypertrophy was confirmed by increased cell size and protein synthesis.
Conclusions:
- Testosterone stimulates cardiac myocyte hypertrophy through NFAT activation and GSK-3β inhibition.
- The findings reveal a cooperative mechanism linking androgen signaling to NFAT recruitment via calcineurin activation and GSK-3β inhibition.
- These pathways are critical mediators of testosterone's hypertrophic effects on cardiac myocytes.
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