Ligand Binding Prolongs Androgen Receptor Protein Half-Life by Reducing its Degradation

Abstract

Androgens are important in female reproduction, but the molecular actions of androgens in female reproductive tissues are not fully understood. We investigated the androgen-responsive transcriptome in human and mouse granulosa cells (GCs) and surprisingly found that the gene-regulation activity of androgen receptor (AR) in these cells is negligible. We then investigated extranuclear actions of AR and found that in human and mouse GCs, as well as in prostate cancer cells, dihydrotestosterone (DHT) dramatically increases the half-life of its own receptor protein. Using the human granulosa-like KGN cells, we show that this effect is not the result of increased AR gene transcription or protein synthesis, nor is it fully abrogated by proteasome inhibition. Knockdown of PTEN, which contributes to degradation of cytoplasmic AR, did not diminish AR accumulation in the presence of DHT. Using immunofluorescence cellular localization studies, we show that nuclear AR is selectively protected from degradation in the presence of DHT. Knockdown of importin 7 expression, a potential regulator of AR nuclear import, does not affect DHT-mediated nuclear accumulation of AR, suggesting importin 7-independent nuclear import of AR in GCs. Further, DNA binding is not required for this protective mechanism. In summary, we show that ligand binding sequesters AR in the nucleus through enhanced nuclear localization independent of DNA binding, thereby protecting it from proteasome degradation in the cytoplasm. This phenomenon distinguishes AR from other sex steroid receptors and may have physiological significance through a positive feedback loop in which androgen induces its own activity in male and female reproductive tissues.

Keywords: androgen; granulosa; ovary; prostate; proteasome; transcriptome.

Figure 1.

A, RNA sequencing heat map of differentially expressed genes from KGN cells after 12 hours of 25-nM dihydrotestosterone (DHT) treatment (n = 5 per group, individual samples indicated). B, Gene expression measured by quantitative polymerase chain reaction of the same samples as in A (n = 5 per group). Data are relative to control and analyzed as paired samples. C, β-Galactoside–adjusted luciferase activity in cultured cells (n = 3) transfected with MMTV-luciferase and β-galactosidase, and treated as indicated (BAY: nuclear factor κB inhibitor Bay-11-7082, 5 μM). Data are means ± SEM relative to control within each group. *P less than .05. **P less than .01. ****P less than 10^-4.

Figure 2.

A, Western blot of androgen receptor (AR) expression in KGN cells treated with indicated concentrations of dihydrotestosterone (DHT) for 24 hours (top) and quantification of band density (bottom) fitted to a one-site binding model (n = 4). B, Western blot and quantification of AR expression in KGN cells treated with 25-nM DHT for indicated times. Experiment was repeated 3 times with similar results. C, Western blot of AR expression in mouse primary granulosa cells cultured with or without 25-nM DHT, as indicated, for 24 hours. D, Western blot and quantification of AR expression in C4-2 cells treated with vehicle (veh) or 25-nM DHT for 24 hours.

Figure 3.

A, Androgen receptor (AR) messenger RNA (mRNA) expression measured by quantitative polymerase chain reaction (qPCR) in KGN cells after treatment with indicated doses of dihydrotestosterone (DHT) for 24 hours. B, AR mRNA expression measured by qPCR in KGN cells treated with 25-nM DHT and/or 5-μM actinomycin D (ActD) as indicated for 6 to 24 hours. Data are relative to time 0 and not adjusted to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) because of GAPDH degradation with actinomycin D treatment. C, Western blot of AR protein expression in KGN cells treated as indicated for up to 12 hours (left) and quantification of band density (right, n = 3) with best-fit curves analyzed by analysis of variance. *P less than .05. **P less than .01.

Figure 4.

A, Western blot of androgen receptor (AR) expression in KGN cells treated with 25-nM dihydrotestosterone (DHT) and/or 50-μM cycloheximide (CHX) as indicated for 6 to 24 hours. B, Quantification of band density from A (n = 3) fitted to a one-phase decay model and analyzed by analysis of variance. Protein half-life was estimated based on the curves as shown by the dotted lines. **P less than .01.

Figure 5.

A, Western blot of androgen receptor (AR) expression in KGN cells treated with 50-μM cycloheximide (CHX—all samples), 10-μM bortezomib (BTZ), and/or 25-nM dihydrotestosterone (DHT) as indicated for up to 48 hours. B, Quantification of band density from A (n = 3) fitted to a one-phase decay model and analyzed by analysis of variance. Protein half-life was estimated based on the curves as shown by the dotted lines. ****P less than 10^-4.

Figure 6.

Top: Western blots of androgen receptor (AR) and PTEN expression in KGN cells treated with 50-μM cycloheximide (CHX—all samples), with or without 25-nM dihydrotestosterone (DHT) as indicated for up to 24 hours, after transfection with small interfering RNA (siRNA) targeting PTEN or nonspecific (NSP) siRNA. Bottom: quantification of band density (n = 3) fitted to best-fit curves (VEH: vehicle).

Figure 7.

Immunofluorescence of androgen receptor (AR) expression in KGN cells treated with 25-nM dihydrotestosterone (DHT) and/or 50μM cycloheximide (CHX) for 12 to 24 hours. Primary antibody was diluted 1:200 and secondary 1:500. Exposure time was 500 ms for all samples under 40× magnification.

Figure 8.

A, Immunofluorescence of androgen receptor (AR) expression in KGN cells treated with 25-nM dihydrotestosterone (DHT) and/or 50-μM cycloheximide (CHX) for 24 hours after transfection with small interfering RNA (siRNA) targeting importin 7 (IPO7) or nonspecific (NSP) siRNA. B, Western blots of AR and IPO7 expression and quantification of AR in KGN cells treated as indicated. Data are representative of 2 experiments. For Westerns, all conditions included treatment with cycloheximide.

Figure 9.

A, Luciferase reporter assay of DNA-binding mutant androgen receptor (AR) transcriptional activity. HEK-293 cells were transfected with the empty vector control, wild-type AR, or mutant AR plasmids, then treated for 18 hours with 25-nM dihydrotestosterone (DHT). B, Protein degradation study of the DNA-binding AR mutant. HEK-293 cells were transfected with wild-type (WT) or DNA-binding mutant (MUT) AR and treated with 25-nM DHT and/or 50-μM cycloheximide (CHX) for 24 hours, before Western blotting for AR and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Data are representative of 2 experiments.