Androgen receptor transcriptionally regulates μ-opioid receptor expression in rat trigeminal ganglia

Abstract

The involvement of testosterone in pain, inflammation, and analgesia has been reported, but the role of androgen receptor (AR), a steroid receptor for testosterone, is not well understood. We have previously shown that peripheral inflammation upregulates μ-opioid receptor (MOR) in rat trigeminal ganglia (TG) in a testosterone-dependent manner. In this study, we hypothesized that testosterone regulates MOR expression via transcriptional activities of AR in TG. We first examined whether AR is co-expressed with MOR in TG neurons. Our immunohistochemical experiment revealed that AR staining is detected in neurons of all sizes in TG and that a subset of AR is expressed in MOR as well as in TRPV1-positive neurons. We identified the promoter region of the rat MOR gene contains putative AR binding sites. Using chromatin immunoprecipitation assay, we demonstrated that AR directly binds to these sites in TG extracts. We confirmed with luciferase reporter assay that AR activated the MOR promoter in response to androgens in a human neuroblastoma cell line (5H-5YSY). These data demonstrated that AR functions as a transcriptional regulator of the MOR gene activity. Finally, we showed that flutamide, a specific AR antagonist, prevents complete Freund's adjuvant (CFA)-induced upregulation of MOR mRNA in TG, and that flutamide dose-dependently blocks the efficacy of DAMGO, a specific MOR agonist, on CFA-induced mechanical hypersensitivity. Our results expand the knowledge regarding the role of androgens and their receptor in pain and analgesia and have important clinical implications, particularly for inflammatory pain patients with low or compromised plasma testosterone levels.

Keywords: flutamide; inflammation; peripheral; sensory neurons; testosterone.

Fig. 1

AR is co-expressed with MOR in TG. Photomicrographs show immunohistochemical staining of AR in TRPV1 (A–C) and MOR (D–F)- positive neurons in TG. MOR in TRPV1-positive neurons in TG is also shown (G–I). Arrows indicate examples of co-expression. Scale bar=30 μm.

Fig. 2

Soma size distribution of AR-positive neurons in TG based on cell body area (μm²). The histograms were constructed from area measurements of 237 AR-positive neurons from two TG.

Fig. 3

AR directly binds to the MOR promoter in rat TG. (A) A schematic diagram of the cloned 2.3-kb rat MOR promoter showing two putative AR-binding sites located at −982 bp to −789 bp and −635 bp to −404 bp. Luc, firefly luciferase (B) The rat TG were subjected to ChIP assay using anti-AR antibody and PCR primers specific for the two putative AR binding sites in the MOR promoter region. IgG was used as a negative control for IP. The PCR products were analyzed on 1% agarose gel and stained with ethidium bromide for visualization. The input represents PCR product amplified from 10% ChIP assay input material. M, male TG. F, female TG.

Fig. 4

AR transcriptionally activates MOR expression. The SH-SY5Y cells were co-transfected with pFLAG-AR and 2.3-kb MOR promoter-luciferase construct (pGL3-MOR-1) as shown in Fig. 2A and then treated with 100 nM dihydrotestosterone (DHT) or testosterone (TS) for 24 h. Cell lysates were then analyzed for luciferase activity. The means±SEM are presented. ^*p<0.05 in Student’s t-test.

Fig. 5

Flutamide blocks CFA-induced MOR expression and function. (A). MOR mRNA levels in TG were compared between vehicle (n=5) and flutamide (n=6)-treated rats under CFA-induced inflammatory condition. The relative RNA levels in both groups were normalized to the mean RNA contents of the flutamide-treated group. (B) Effects of peripheral DAMGO (10 μg) on mechanical hyperalgesia before and after CFA injection in vehicle (n=6) and flutamide (1, 5, and 10 mg/kg, i.p.; n=6 per group)-treated rats. For both RT-PCR and behavioral experiments, flutamide was administered daily for 3 days. The data are presented as means±SEM. ^*p<0.05.