Non-Genomic Action of Androgens is Mediated by Rapid Phosphorylation and Regulation of Androgen Receptor Trafficking

Abstract

Background: Testosterone is critical for maintaining spermatogenesis and male fertility. The accomplishment of these processes requires the synergistic actions of the classical and non-classical signaling pathways of androgens. Methods: A murine testicular Sertoli cell line, TM4 cell was used to examine androgen actions in Sertoli cells. Western blot analysis and immunofluorescence assay were employed to study the testosterone-induced Androgen receptor (AR) translocation. Protein phosphorylation antibody array was applied to identify the phosphorylation sites under testosterone treatment, and these findings were verified by Western blot analysis. Results: We found that a physiological dose of testosterone induced fast membrane association of AR. By using a phosphorylation antibody array, several phosphorylation sites, such as MEK1/2 (Ser217/221), Akt (Ser473), and Erk1/2 (Thr202/Tyr204) were rapidly phosphorylated within 5 min of testosterone treatment. Inhibition of the MEK and Akt signaling pathways prevented AR trafficking. Blocking of AR by flutamide eliminated the stimulation effect of testosterone on kinase phosphorylation. Testosterone induced kinase Src phosphorylation, and inhibition of Src restricted AR translocation to the membrane and the nucleus. Conclusion: Findings suggested that the membrane association of AR was mediated by the MEK and Akt phosphorylation signaling pathways, which resulted in Src activation and was initiated by testosterone binding to the membrane-localized AR. This study provides new insights into the testosterone signaling pathway in Sertoli cells, which mediate spermatogenesis. In addition, the study can be used in the diagnosis and treatment of male infertility caused by disorders in spermatogenesis.

Membrane-associated androgen receptor (AR) potentiates its transcriptional activities by activating heat shock protein 27 (HSP27)

“…previous report has shown membrane-associated AR mediates rapid AKT activation stimulated by androgen (9,25). Consistent with this study, we found androgen-induced AKT phosphorylation at 30 min was blocked in the presence of the KIF5Btail, but not the KIFC3-tail ( Fig.…”

CSN6 and Rab34 Are Involved in Androgen Receptor Trafficking in Mouse Testicular Sertoli Cells

“…Consistent with the previous studies [6,14,17], Western blot analysis using TM4 cells revealed the rapid increase in the phosphorylation of kinase protein kinase B (Akt) (Ser473), Erk1/2 (Thr202/Tyr204), and mitogen activated protein kinase kinase (MEK1/2) (Ser217/221) within 5 min. Knockdown of CSN6 by shRNA inhibited the increase in the phosphorylation caused by testosterone stimulation (Fig.…”

“…As described in a previous study, testosterone induces AR translocation to membrane in primary Sertoli cells, with translocation becoming significantly evident within 5 min [14].…”

“…Rapid increases in kinase Akt (Ser473), Erk1/2 (Thr202/Tyr204), and MEK1/2 (Ser217/221) were induced by testosterone within 5 min, and knockdown of CSN6 prevented the stimulation. Our previous studies showed that inhibition of the kinase (Akt, Erk and MEK) by inhibitors block AR trafficking caused by testosterone administration [14]. These data suggested that CSN6 regulates AR trafficking via the phosphorylation signaling pathway.…”

Haloperidol ameliorates androgen-induced behavioral deficits in developing male rats

“…Testosterone is known to mediate its effects by two different pathways of action, classical pathway of cytosolic/nuclear androgen receptors and non-classical pathway of membrane-associated androgen receptors (Deng et al 2017). The classical effects of testosterone occur slowly because of the translation of newly synthesized proteins and the non-classical effects of testosterone occur fast due to the rapid phosphorylation of signal molecules (Deng et al 2017). In Experiment 3 of the present study, we did not find the differences of motor behavior and grooming behavior between the rats receiving Hal treatment and the rats receiving Hal concurrent with TP treatment.…”