Tetrapeptides Modelled to the Androgen Binding Site of ZIP9 Stimulate Expression of Tight Junction Proteins and Tight Junction Formation in Sertoli Cells

Androgens stimulate the expression of tight junction (TJ) proteins and the formation of the blood–testis barrier (BTB). Interactions of testosterone with the zinc transporter ZIP9 stimulate the expression of TJ-forming proteins and promote TJ formation in Sertoli cells. In order to investigate androgenic effects mediated by ZIP9 but not by the nuclear androgen receptor (AR), the effects of three tetrapeptides fitting the androgen binding site of ZIP9 were compared with those induced by testosterone in a Sertoli cell line expressing ZIP9 but not the AR. Three tetrapeptides and testosterone displaced testosterone-BSA-FITC from the surface of 93RS2 cells and stimulated the non-classical testosterone signaling pathway that includes the activation of Erk1/2 kinases and transcription factors CREB and ATF-1. The expression of the TJ-associated proteins ZO-1 and claudin-5 was triggered as was the re-distribution of claudin-1 from the cytosol to the membrane and nucleus. Furthermore, TJ formation was stimulated, indicated by increased transepithelial electrical resistance. Silencing ZIP9 expression by siRNA prevented all of these responses. These results are consistent with an alternative pathway for testosterone action at the BTB that does not involve the nuclear AR and highlight the significant role of ZIP9 as a cell-surface androgen receptor that stimulates TJ formation.

The Effects of Tetrapeptides Designed to Fit the Androgen Binding Site of ZIP9 on Myogenic and Osteogenic Cells

ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance that may mediate certain physiological responses to androgens. Using in silico methods, six tetrapeptides with the best docking properties at the testosterone binding site of ZIP9 were synthesized and further investigated. All tetrapeptides displaced T-BSA-FITC, a membrane-impermeable testosterone analog, from the surface of mouse myogenic L6 cells that express ZIP9 but not the classical androgen receptor (AR). Silencing the expression of ZIP9 with siRNA prevented this labeling. All tetrapeptides were found to be pro-androgenic; in L6 cells they stimulated the expression of myogenin, triggered activation of focal adhesion kinase, and prompted the fusion of L6 myocytes to syncytial myotubes. In human osteoblastic SAOS-2 cells that express AR and ZIP9, they reduced the expression of alkaline phosphatase and stimulated mineralization. These latter effects were prevented by silencing ZIP9 expression, indicating that the osteoblast/osteocyte conversion is exclusively mediated through ZIP9. Our results demonstrate that the synthetic tetrapeptides, by acting as ZIP9-specific androgens, have the potential to replace testosterone or testosterone analogs in the treatment of bone- or muscle-related disorders by circumventing the undesirable effects mediated through the classical AR.

Dietary Intake of 17α-Ethinylestradiol Promotes HCC Progression in Humanized Male Mice Expressing Sex Hormone-Binding Globulin

Hepatocellular carcinoma (HCC) is a male-oriented malignancy; its progression is affected by sex hormones. 17α-ethinylestradiol (EE2) is a synthetic estrogen widely used as an oral contraceptive; however, it is unknown whether EE2 regulates sex hormone action in HCC. We investigated whether EE2 influences HCC risk in male androgenic environments, using mice expressing human sex hormone-binding globulin (SHBG). Two-week-old male mice were injected with diethyl-nitrosamine (DEN, 25 mg/kg) and fed an EE2 diet for 10 weeks from 30 weeks of age. Development and characteristics of liver cancer were evaluated in 40-week-old mice via molecular and histological analyses. Although EE2 did not increase HCC progression in wild-type mice, SHBG mice exhibited remarkably higher HCC risk when fed EE2. The livers of EE2-treated SHBG mice exhibited substantially increased pro-inflammatory necrosis with high plasma levels of ALT and HMGB1, and intrahepatic injury and fibers. Additionally, increased androgen response and androgen-mediated proliferation in the livers of EE2-treated SHBG mice and EE2-exposed hepatocytes under SHBG conditions were observed. As a competitor of SHBG-androgen binding, EE2 could bind with SHBG and increase the bioavailability of androgen. Our results revealed that EE2 is a novel risk factor in androgen-dominant men, predisposing them to HCC risk.

Effects of cholesterol and Lactobacillus acidophilus on testicular function

Objective: In this study, the effects of Lactobacillus acidophilus on testosterone (TES), follicle-stimulating hormone (FSH), luteinizing hormone (LH), androgen-binding protein (ABP), factor-associated apoptosis (FAS), and total cholesterol (TC), as well as histopathological changes, were investigated in male rats fed a high-cholesterol diet. Methods: The study included three groups. The control (C) group was fed standard-diet for 8 weeks. The hypercholesterolemia (HC) group was fed a 2% cholesterol-diet for 8 weeks. The therapeutic group (HCL) was fed a 2% cholesterol-diet for 8 weeks and administered L. acidophilus for the last 4 weeks. FSH, TES, and FAS levels in testicular tissue were determined using an enzyme-linked immunosorbent assay (ELISA), while another sample was examined histopathologically. LH and ABP levels were determined using ELISA, and serum TC levels were assessed via an autoanalyzer. Results: In the HC group, the TC levels were significantly higher and the LH levels were lower (p<0.05) than in the C group. The ABP levels were lower (p>0.05). In the HCL group, the LH and ABP levels were higher (p>0.05) and the TC level significantly lower (p<0.05) than in the HC group. The TES and FSH levels were lower, and the FAS levels were higher, in the HC than in the C group (p<0.05). In the HCL group, levels of all 3 resembled control levels. Histologically, in the testicular tissue of the HC group, the cells in the tubular wall exhibited atrophy, vacuolization, and reduced wall structure integrity. However, in the HCL group, these deteriorations were largely reversed.Conclusion: Supplementary dietary administration of an L. acidophilus to hypercholesterolemic male rats positively impacted testicular tissue and male fertility hormone levels.

Development of an Androgen Receptor Inhibitor Targeting the N-Terminal Domain of Androgen Receptor for Treatment of Castration Resistant Prostate Cancer

Prostate cancer patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer. Resistance can occur when mutations in the androgen receptor (AR) render anti-androgen drugs ineffective or through the expression of constitutively active splice variants lacking the androgen binding domain entirely (e.g., ARV7). In this study, we are reporting the discovery of a novel AR-NTD covalent inhibitor 1-chloro-3-[(5-([(2S)-3-chloro-2-hydroxypropyl]amino)naphthalen-1-yl)amino]propan-2-ol (VPC-220010) targeting the AR-N-terminal Domain (AR-NTD). VPC-220010 inhibits AR-mediated transcription of full length and truncated variant ARV7, downregulates AR response genes, and selectively reduces the growth of both full-length AR- and truncated AR-dependent prostate cancer cell lines. We show that VPC-220010 disrupts interactions between AR and known coactivators and coregulatory proteins, such as CHD4, FOXA1, ZMIZ1, and several SWI/SNF complex proteins. Taken together, our data suggest that VPC-220010 is a promising small molecule that can be further optimized into effective AR-NTD inhibitor for the treatment of CRPC.

SAT-748 Advantages and Limitations of an Integrative Measurement for All Serum Androgens and Anti-Androgens

Background: Analytic measurements of a hormone in bodily fluids are a cornerstone of clinical evaluation. However, clinical presentation may be affected also by currently unmeasured modulators of hormonal function. An alternative could be to measure the cumulative effect of all factors present in a bodily fluid on the function of a hormone receptor. Prior studies showed an androgen receptor (AR) BioAssay to accurately measure urine androgens in males undergoing testosterone (T) supplementation (1). The factors integrated by the AR BioAssay include androgens, anti-androgens and, in serum, androgen binding proteins affecting ligand availability. Methods: The AR BioAssay was exposed to male and female serum samples obtained from the CDC’s Hormone Standardization (HoSt) Program, and to female serum samples from the UCSF PCOS Tissue Bank registry. AR activity was quantified against a testosterone standard curve and recorded as ‘T-equivalent’ (‘T-eq’) androgen activity units. Results: In 40 CDC HoSt sera added directly (no extraction) to AR BioAssay cells, androgen activities ranged from 2.57 to 298 ng ‘T-eq’/dl. In the 20 ‘male’ CDC HoSt sera (T&gt;150 ng/dl), the androgen activity measurements were uniformly less (0.35 ± 0.10) that of the T-measurement. By contrast, in the 20 ‘female’ CDC HoSt sera, in which T concentrations are typically lower than the affinity of T for sex hormone binding globulins such that more of the T is available to the AR BioAssay, the measured androgen levels were on average 1.45-fold higher than the T concentration. This androgen to T comparison showed high variability in females with 5 of 20 CDC HoSt samples having androgen concentrations more than double that of T (maximum, 6.1-fold). In female serum samples from the PCOS registry, androgens were higher in patients with a PCOS diagnosis (57.7 +/-17.6 ng ‘T-eq’/dl; n = 23) compared to women not meeting formal PCOS criteria (38.1 +/-10.8 ng ‘T-eq’/dl; n = 4); androgen values again averaged 1.40-fold (maximum, 4.9-fold) that of the T measurements, regardless of PCOS diagnosis. Conclusions: Androgen-binding globulins appear to most influence androgen activity levels in male serum. In females, the lower T concentrations may minimize the impact of androgen-binding proteins and permit the impact of non-T androgens to be more pronounced with possible clinical consequence. Further investigations are needed to determine whether functional androgen measurements may improve clinical diagnosis of certain conditions. Reference: (1) Bailey et al (2016) PLoS One11(3):e0151860

Protein pengikat hormon seks: sex hormone binding globulin (SHBG) dan aksi steroid seks

Jumlah gen pada manusia sekitar 30 000 gen, salah satunya yaitu gen SHBG (sex hormone binding globulin). Telah terbukti bahwa protein merupakan produk gen. Gen yang diekspresikan berarti mengkode sintesis protein. Pada studi ini mempelajari tentang protein sex hormone binding globulin (SHBG) yang merupakan produk gen SHBG. Gen SHBG terletak pada kromosom 17 p 3.1 di setiap sel tubuh kita. Gen SHBG pada hepatosit mengkode protein SHBG, protein tersebut selanjutnya disekresikan ke sistem sirkulasi. Gen SHBG di dalam hepatosit memiliki kesamaan dengan gen androgen binding protein (ABP) di sel Sertoli dalam testis. Jumlah gen pada manusia sekitar 30 000 gen, salah satunya yaitu gen SHBG (sex hormone binding globulin). Telah terbukti bahwa protein merupakan produk gen. Gen yang diekspresikan berarti mengkode sintesis protein. Pada studi ini mempelajari tentang protein sex hormone binding globulin (SHBG) yang merupakan produk gen SHBG. Gen SHBG terletak pada kromosom 17 p 3.1 di setiap sel tubuh kita. Gen SHBG pada hepatosit mengkode protein SHBG, protein tersebut selanjutnya disekresikan ke sistem sirkulasi. Gen SHBG di dalam hepatosit memiliki kesamaan dengan gen androgen binding protein (ABP) di sel Sertoli dalam testis. Dalam sisntesis protein SHBG maupun ABP ada 2 tahap yaitu tahap sintesis prekursor protein dan tahap selanjutnya pematangan prekursor protein di badan Golgi dengan proses glikosilasi. Protein SHBG maupun ABP memiliki funsgi sama yaitu memperantarai aksi hormon steroid seks ke sel sasaran. Ikatan antara SHBG dengan steroid tersebut bersifat reversibel dan berafinitas tinggi untuk mengikat androgen (dehidrotestosteron/DHT, testosteron, 3α-androstenediol), sedangkan ikatan terhadap estrogen aktif maupun estradiol dengan afinitas yang lebih rendah. Aksi steroid seks ke sel sasaran telah terbukti dengan 2 cara yaitu cara pertama dengan berdifusi melewati membran sel sasaran dan cara kedua dengan sistem transduksi sinyal yang diperantarai oleh reseptor SHBG (R-SHBG) pada permukaan sel sasaran. Protein SHBG di dalam sistem sirkulasi memiliki fungsi untuk mengikat hormon steroid seks dan memperantarai aksi hormon tersebut ke sel sasaran di luar testis, sedangkan ABP berfungsi memperantarai aksi hormon steroid seks di dalam testis.