Testosterone regulates tight junction proteins and influences prostatic autoimmune responses

Abstract

Testosterone and inflammation have been linked to the development of common age-associated diseases affecting the prostate gland including prostate cancer, prostatitis, and benign prostatic hypertrophy. We hypothesized that testosterone regulates components of prostate tight junctions which serve as a barrier to inflammation, thus providing a connection between age- and treatment-associated testosterone declines and prostatic pathology. We examined the expression and distribution of tight junction proteins in prostate biospecimens from mouse models and a clinical study of chemical castration, using transcript profiling, immunohistochemistry, and electron microscopy. We determined that low serum testosterone is associated with reduced transcript and protein levels of Claudin 4 and Claudin 8, resulting in defective tight junction ultrastructure in benign prostate glands. Expression of Claudin 4 and Claudin 8 was negatively correlated with the mononuclear inflammatory infiltrate caused by testosterone deprivation. Testosterone suppression also induced an autoimmune humoral response directed toward prostatic proteins. Testosterone supplementation in castrate mice resulted in re-expression of tight junction components in prostate epithelium and significantly reduced prostate inflammatory cell numbers. These data demonstrate that tight junction architecture in the prostate is related to changes in serum testosterone levels, and identify an androgen-regulated mechanism that potentially contributes to the development of prostate inflammation and consequent pathology.

Trial registration: ClinicalTrials.gov NCT00799617.

© Springer Science+Business Media, LLC 2011

Figures

Figure 1. Testosterone suppression is associated with the loss of Claudin 4 and Claudin 8 expression in prostate epithelium

Quantitative RT-PCR analysis of Cldn4 expression (A) and Cldn8 expression (C) in the prostate epithelium of castrated and control mice. The relative abundance of Cldn4 and Cldn8 normalized to Hprt, was significantly reduced compared to normal controls, representing as mean ± SE (mean difference of the cycle threshold numbers is 3.266 ± 0.6151; p=0.0002 for Cldn4) and mean difference of the Ct numbers is 0.9158 ± 0.3793; p=0.0326 for Cldn8) (B) Immunofluorescence detection of Cldn4 on prostate sections from control and castrated mice. Cldn4 proteins were distributed within the epithelium of the mouse prostates, and expression was substantially decreased in the prostates of castrated mice; (D) Immunofluorescence detection of Cldn8 on prostate sections from control and castrated mice, Cldn8 proteins were localized at the intercellular sites in the epithelium of control mouse prostates, some of the proteins concentrated at the edge of the luminal compartment. Cldn8 became undetectable in the prostates of 4 week post castrated mice. Scale bar =20 μm, applied to all images.

Figure 2. Ultrastructure of prostate epithelium in normal and castrated mice

Normal epithelial tight junctions comprise a series of kissing points (A) (arrows) that juxtapose the lipid bilayers on opposing epithelial cells into intimate contact. In castrated mice, the tight junctions in the prostate epithelium lack obvious contact points (B,C), and the intercellular membranous contact regions are separated (arrows).

Figure 3. Androgen suppression decreases the expression of CLDN4 and CLDN8 in the human prostate

Transcript levels of CLDN4 (mean difference of Ct numbers is 3.603 ± 0.6654; p(A) and CLDN8 (mean difference of Ct number is 6.532 ± 1.492; p=0.0003) (B) were significantly decreased in prostate epithelium following testosterone suppression. (C) Immunofluorescence detection of CLDN4 on prostate sections from normal control and castrated men, CLDN4 proteins are localized at the intercelluar tight junction sites within the epithelium of eugonadal prostates, but only detectable at very low levels in the prostates from castrated men. Cytokeratin was stained as a marker for epithelial cells; (D) immunofluorescence detection of CLDN8 on prostate sections from normal control and castrated men. Within the epithelium of eugonadal prostates, CLDN8 proteins are localized at the intercellular tight junction sites of the basal compartment, and strong signals of CLDN8 staining was detected along the edge of luminal compartment of the epithelium. CLDN8 staining was decreased in the prostates from castrated men. Scale bar =20 μm, applied to all images. The mean scores of claudin protein expression levels from prostates following androgen suppression were significantly lower than the control group (the difference of mean scores for CLDN4 is 2.830 ± 0.4101, (P<0.0001) (E); the difference of mean scores for CLDN8 is 2.698 ± 0.6413 (p= 0.0012)(F))

Figure 4. Testosterone suppression is associated with enhanced prostate inflammation

A substantial CD3 positive lymphocyte infiltrate (brown immunoreactivity) was detected in the prostate glands of castrated mice compared to controls (A). Two weeks after testosterone supplementation, the T cell numbers were substantially reduced relative to castrated mice before testosterone treatment: (10 random fields under 20× lens were examined for each animal, 5 animals in each treatment group; student t-test p=0.0005) (B). Significantly more F4/80 positive macrophages (brown immunoreactivity) were detected in the prostates of castrated mice relative to normal controls. After testosterone replacement treatment, the number of macrophages was significantly reduced in the castrated mice (10 random fields under 20× lens were examined for each animal, 5 animals in each treatment group; student t-test: P=0.0068) (C). A CD3 positive lymphocyte cell infiltrate and CD68 positive macrophage infiltrate (D) was detectable in the prostates of castrated men compared to eugonadal controls. Ten random fields under the 20× lens were examined for each patient. Lymphocyte numbers increased from 6.158 ± 1.226/field, n=5 in non-treated patients to 25.72 ± 5.600/field, n=11 in men with suppressed testosterone (p=0.0373), and macrophage numbers increased from 7.614 ± 2.545/field, n=5 in non-treated patients to 24.22 ± 4.990/field, n=11 in men with suppressed testosterone (p= 0.0494) Scale bar =50 μm, applied to all images.

Figure 5. Testosterone suppression induces autoimmune responses in the prostate

(A) Detection of prostatic proteins with antisera from intact or castrate mice. Auto-antigens are evident as distinct bands using sera from 4 week and 12 week castrated mice. (B) Coomassie blue stained gel of prostatic proteins immunoprecipitated from mouse prostate using sera from intact or castrate mice. (C) Immunofluorescence staining shows the autoantigens were distributed at the luminal epithelium of the prostate. Scale bar =20 μm, applied to all images.