Direct Involvement of Androgen Receptor in Oxytocin Gene Expression: Possible Relevance for Mood Disorders

Abstract

Oxytocin (OXT), synthesized in the hypothalamic paraventricular nucleus (PVN) and then released into different brain areas, may play a crucial role in various behaviors and neuropsychiatric disorders, including depression. Testosterone has been proposed by clinical studies to have the opposite effect of oxytocin in these disorders. We began by studying, in the postmortem hypothalamus of fifteen patients with mood disorders and fifteen matched controls, the expression of OXT in the PVN by means of immunocytochemistry (ICC) and the co-localization of OXT and androgen receptor (AR) by means of double labeling ICC. Subsequently, the regulatory effect of AR on OXT gene expression was studied in vitro. We found a higher expression of PVN OXT in the mood disorder patients than in the control subjects, and observed a clear co-localization of AR in OXT-expressing neurons, both in the cytoplasm and in the nucleus. In addition, a significant decrease in OXT-mRNA levels was observed after pre-incubation of the SK-N-SH cells with testosterone. A further potential androgen-responsive element in the human OXT gene promotor was revealed by electrophoretic mobility shift assays and co-transfections in neuroblastoma cells. Finally, in vitro studies demonstrated that AR mediated the down-regulation of OXT gene expression. These results suggest that the fact that OXT and testosterone appear to have opposite effects in neuropsychiatric disorders might be based upon a direct inhibition of AR on OXT transcription, which may provide a novel target for therapeutic strategies in depression.

Figure 1

Comparison of oxytocin (OXT)-immunoreactivity in hypothalamic paraventricular nucleus (PVN) between mood disorder and controls. Data are presented as mean±SEM. BD: bipolar disorder; IOD: integrated optical density; MDD: major depressive disorder. *p=0.027.

Figure 2

(a–c) Co-localization of androgen receptor (AR) and oxytocin (OXT) in the hypothalamic paraventricular nucleus (PVN). Asterisks indicate co-localization of AR (red) and OXT (blue), arrows indicate single AR-stained cells, and arrowheads indicate single OXT-stained cells. Bar=25 μm. (a1–c1) the mask of AR (red) and OXT (blue) signals which had optical density values more than two times the background. The double-stained cells are indicated by asterisks.

Figure 3

Endogenous expression of androgen receptor (AR) and oxytocin (OXT) in SK-N-SH cells, and the comparison of OXT-mRNA levels between testosterone treated cells and controls. (a) Immunofluorescence staining for the endogenous expression of AR (a, left) and OXT (a, right) in SK-N-SH cells. Bar=50 μm. (b) Comparison of OXT-mRNA levels in SK-N-SH cells with adding in the control group the estrogen receptor antagonist ICI 182 780 to the medium and adding ICI 182 780 followed by testosterone administration for 24 h to the medium of the experimental group. Data are presented as mean±SEM from three independent experiments. *p=0.05. T treatment: Testosterone treatment.

Figure 4

Electrophoretic mobility shift assay (EMSA) of two predicted androgen responsive elements (AREs). (a) Comparison of ARE sites A and site B in the human oxytocin (OXT) gene promoter with a consensus ARE. Site A is located at −346 to −332, while site B is located at +204 to +218, relative to the start site ATG. Nucleotides of site A and site B are shown respectively. N=any nucleotide. (b) EMSA performed with androgen receptor (AR)-containing nuclear extracts and potential ARE sequences site A and site B. Site A strongly bound to nuclear extracts from PSV-AR-transfected 293T cells, as is evident from the band shift indicated by arrows. Bands completely disappeared when nuclear extracts were incubated with a 200-fold excess of non-labeled probe. The same holds true when nuclear extracts were omitted from the reaction mixture. (c) Western blot detection of AR variants. 293T cells were transfected with human AR expressing plasmid PSV-AR and detected by western blot. AR variants were indicated by two arrows between 70 KD and 130 KD, which were consistent with the literature (Saatcioglu, 2011; Wilson and McPhaul, 1996).

Figure 5

Inhibitory effect of testosterone on oxytocin (OXT) gene expression via androgen responsive element (ARE). (a) Diagram of the two fragments from the human OXT promoter sequence used in construction of the reporter plasmids. Full-length OXT promoter (OXTA), spanning both ARE sites, A and B, from −533 bp to 235 bp, was cloned into the PGL3 luciferase reporter basic vector. The OXTAm fragment is shorter and lacks site A. (b) An interaction assay was performed on 293FT cells co-transfected with PSV-androgen receptor (AR) and one of the OXT promoter plasmids (POXTA or POXTAm). Panel I: Western blot analyses were performed to determine the presence of AR after co-transfection, compared with the untransfected 293FT cells. Panel II: PCR amplification of ARE site A and site B in nucleus sonication products by Primer A (PA) and Primer B (PB). Panel III: Immunoprecipitated complexes were detected by PCR analysis. Only 293 FT cells co-transfected with POXTA and incubated with anti-AR antibody demonstrated PCR product when Primer A was used. However, cells co-transfected with POXTAm and incubated with anti-AR antibody did not show the PCR products when Primer B was used. In addition, neither untransfected cells treated with anti-AR antibody nor cells co-transfected with POXTA and incubated with IgG demonstrated PCR products when Primer A was used. (c) Luciferase activity in 293FT cells, which were co-transfected with human OXT promoter plasmids POXTA with or without plasmid contained AR (PSV-AR), followed by testosterone treatment. Note the significantly decreased luciferase activity in 293FT cells transfected with PSV-AR, pointing to an inhibitory role of testosterone via AR on OXT gene expression. Results are expressed as mean luciferase activity±SEM from three independent experiments. *p=0.0126.