GSK-3β/NFAT Signaling Is Involved in Testosterone-Induced Cardiac Myocyte Hypertrophy

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.

睾酮可通过涉及胞浆与核信号通路协同串扰的机制诱导心肌肥厚。活化T细胞核因子（Nuclear factor of activated T-cells, NFAT）与心肌肥厚的发生密切相关，而糖原合成激酶3β（glycogen synthase kinase-3β, GSK-3β）被认为是主要的负调控因子，其功能主要通过调控NFAT活性实现。然而，钙调神经磷酸酶-NFAT与GSK-3β信号通路在睾酮诱导的心肌肥厚中所发挥的作用仍未明确。本研究证实，睾酮可通过激活NFAT并抑制GSK-3β活性，进而刺激心肌细胞肥厚。睾酮以时间和剂量依赖性方式升高NFAT荧光素酶（NFAT-luciferase, NFAT-Luc）报告基因活性，在100 nM睾酮刺激24小时后活性达到峰值。睾酮诱导的NFAT-Luc活性可被钙调神经磷酸酶抑制剂FK506、环孢素A（cyclosporine A）以及特异性NFAT肽抑制剂11R-VIVIT阻断。与之相反，睾酮可抑制GSK-3β活性，具体表现为GSK-3β在丝氨酸9（Ser9）位点的磷酸化水平升高、β-连环蛋白（β-catenin）蛋白积累增加，同时β-连环蛋白在Ser33、Ser37及苏氨酸41（Thr41）位点的磷酸化水平降低。使用1-氮杂肯帕隆（1-azakenpaullone）或靶向GSK-3β的小干扰RNA（siRNA）抑制GSK-3β活性，可升高NFAT-Luc活性；而过表达组成型激活的GSK-3β突变体（GSK-3βS9A）则可抑制睾酮介导的NFAT-Luc激活。睾酮诱导的心肌细胞肥厚可通过心肌细胞体积增大以及[3H]-亮氨酸掺入法（用于检测细胞蛋白质合成）测得的亮氨酸掺入量升高来证实。钙调神经磷酸酶-NFAT通路的抑制可阻断睾酮诱导的心肌肥厚，而GSK-3β的抑制则可促进该过程。过表达GSK-3βS9A以激活GSK-3β，可阻断睾酮诱导的肥厚标志物升高。此外，抑制细胞内雄激素受体可阻止睾酮诱导的NFAT-Luc激活。综上，本研究结果表明，睾酮诱导的心肌细胞肥厚涉及一种协同机制，该机制将雄激素信号通路与通过钙调神经磷酸酶激活及GSK-3β抑制招募NFAT的过程联系起来。