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.

Ca2+/Calmodulin-Dependent Protein Kinase II and Androgen Signaling Pathways Modulate MEF2 Activity in Testosterone-Induced Cardiac Myocyte Hypertrophy

“…Accordingly, NFAT-controlled gene transcription was found to involve additional transcription factors, including MEF2 ( Cortes et al, 2012 ). Recently, we determined that testosterone stimulates cardiac myocyte hypertrophy through NFAT activation and GSK-3β inhibition, which supports a cooperative mechanism involving cytoplasmic and nuclear signaling ( Duran et al, 2016 ).…”

“…Cardiac myocytes were cultured for 24 h and then incubated with [ 3 H]-leucine (2.5 μCi⋅mL -1 ) for an additional 48 h in the presence or absence of testosterone ( Duran et al, 2016 ). Next, the cells were washed four times with ice-cold PBS and treated with 10% trichloroacetic acid at 4°C for 60 min.…”

Effect of DHT-Induced Hyperandrogenism on the Pro-Inflammatory Cytokines in a Rat Model of Polycystic Ovary Morphology

“…These transcription factors are dephosphorylated through calcineurin, a calcium-dependent phosphate that facilitates the nuclear translocation of NFAT [51]. In this regard, it is pertinent to mention a study that showed the increase in NFAT activity by testosterone and modulation of its transcriptional activity [52].…”

Glycogen synthase kinase 3β (GSK3β) regulates the expression of MyHC2a in goat skeletal muscle satellite cells (SMSCs)

“…As an important regulator of myogenesis in mammal, GSK3β inhibition promotes myocyte differentiation manifested in the enhancement of myogenic regulatory factors such as myocyte enhancer factor 2 (MEF2) and MyoG (Dionyssiou et al, ), targeting insulin‐mediated myogenesis and activation of NFAT (Duran et al, ; Jl, Schols, Willems, Kelders, & Langen, ; Litwiniuk et al, ). Treatment of IGF‐1 or GSK3β inhibitor in differentiated C2C12 cells accelerates myogenesis shown in increased myotube formation, muscle creatine kinase (MCK) activity, and transactivation of troponin I (TnI) promoter, as well as increases the expression of MyoD, Myf5, myogenin, TnI‐slow, TnI‐fast, MCK, and myoglobin (Jl et al, ).…”