Vesicle-Associated Membrane Protein-Associated Protein a Is Involved in Androgen Receptor Trafficking in Mouse Sertoli Cells

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Hindawi International Journal of Endocrinology Volume 2018, Article ID 4537214, 6 pages https://doi.org/10.1155/2018/4537214

Research Article Vesicle-Associated Membrane Protein-Associated Protein A Is Involved in Androgen Receptor Trafficking in Mouse Sertoli Cells

Qiong Deng, Jianwen Zhang, Zhu Wang, Shengping Zhang, Fan Zhi, and Hui Liang

Department of Urology, The People’s Hospital of Longhua, Shenzhen, China

Correspondence should be addressed to Hui Liang; [email protected]

Received 10 November 2017; Revised 18 January 2018; Accepted 5 February 2018; Published 4 March 2018

Academic Editor: Rosaria Meccariello

Copyright © 2018 Qiong Deng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Androgen and its receptor (AR) play an important role in maintaining spermatogenesis and male fertility. The nonclassical androgen signaling pathway is proposed to be mediated by an AR in plasma membrane in Sertoli cells. Our previous studies showed that testosterone induces cytoplasmic AR translocation to plasma membrane by binding with caveolin-1. This study was conducted to the underlying molecular mechanism mediating AR trafficking. Data from mass spectrometry using membrane coimmunoprecipitation sample by anti-AR antibody indicated VAPA is a candidate protein. Knockdown of VAPA by shRNA decreased the amount of AR localized to membrane and nuclear fraction and prevented AR trafficking after being exposed to testosterone. Further studies indicated AR trafficking in Sertoli cells might be mediated by VAPA via association with vesicle transport protein OSBP. This study can enrich the mechanism of the androgen actions and will be helpful for further clarifying the nonclassical signaling pathway of androgens in Sertoli cells.

1. Introduction this fast action is mediated by a membrane receptor of androgens. Our previous data showed AR localized in the Androgen and its receptor (AR) play an important role in membrane of murine testicular TM4 cells, and testosterone maintaining spermatogenesis and male fertility. Evidences enhanced the membrane association, AR localizes to plasma showed that the actions of androgen in Sertoli cells were essen- membrane by binding to Caveolin-1 [5, 6]. However, the pre- ffi tial for the proceeding of spermatogenesis. Sertoli cell-selected cised mechanism mediating AR tra cking to the membrane AR knockout mice exhibit spermatogenesis arrest at the dip- remains unclear. lotene stage [1–3], similar to the phenotype of AR knockout In this study, we used coimmunoprecipitation (co-IP) ff and employed mass spectrograph (MS) to find the candidate mice. The selected AR knockout in germ cells does not a ect ffi the process of the spermatogenesis [4]. These data suggested protein involved in AR tra cking. The screened candidate proteins will be knock down using shRNA constructs to that the AR in Sertoli cells is critical of spermatogenesis. ffi AR associates with testosterone, and the complex translo- test the role in AR tra cking. We will also conduct experiments to study the molecular mechanism mediating AR cates to nucleus, binds with the androgen response element trafficking by the candidate protein. This study will enrich (ARE) in the chromatin, and then induces gene transcrip- the mechanism of the action of androgens and provide tional repression or activation. This is the classical pathway new sights into testosterone signaling pathway in Sertoli of androgen, taking several hours since it requires new pro- cells mediating spermatogenesis. tein synthesis and secretion. Compared with the classical pathway, a faster action of androgen called nonclassical 2. Materials and Methods signaling pathway was found. It only takes several seconds to minutes to response since it does not relate to DNA 2.1. Reagents and Constructs. Unless otherwise indicated, all amplification or protein synthesis. And evidences suggested the chemical reagents were obtained from Sigma-Aldrich 2 International Journal of Endocrinology

(USA), all of the cell culture reagents were bought from Life Table 1: Antibodies used in this study. technologies (USA). The shRNA construct was provided by Shanghai Genechem Co. Ltd (China). Antibody Specie Catalog Dilution Source ∗ AR (N-20) Rabbit sc-816 1 : 1000 SCBT 2.2. Cell Culture and Transfection. Murine Sertoli cell line Pan Cadherin Mouse ab22744 1 : 5000 Abcam ∗ TM4 was purchased from the American Type Culture GAPDH Goat sc-23057 1 : 1000 SCBT Collection (ATCC, Manassas, VA). Cells were cultured in DMEM containing 10% FBS and 100 μg/ml penicillin and VAPA Rabbit ab96584 1 : 1000 Abcam ° 100 μg/ml streptomycin at 37 C. Cells plated in 10 cm culture OSBP Rabbit GTX114602 1 : 1000 GeneTex dishes were transfected with shRNA or negative control ∗SCBT: Santa Cruz Biotechnology. using Lipofectamine 3000 (Invitrogen). Cells were changed to medium supplemented with stripped serum when reach- 3. Results ing 80%–85% confluence. Eighteen hours later, the medium was changed to serum-free medium containing 0.1% BSA 3.1. VAPA Is Identified as a Candidate Protein by MS. and incubated for 2 hours before exposure to 10 nM tes- Agreeing with our previous studies [5, 6], Western blot data tosterone (Melone Pharma, China) for 30 minutes unless and statistical analysis showed testosterone induces cytoplas- otherwise indicated. mic AR translocation to membrane fraction (Figure 1(a)). In order to clarify the molecular mechanism of AR transloca- 2.3. Coimmunoprecipitation and Mass Spectrometry. Protein tion to membrane fraction, we used anti-AR antibody to subcellular fractionation isolation was following the instruc- immunoprecipitate the molecules in the membrane fraction tion provided in the kit as previously described [7]. 500– that associated with AR. The co-IP experiment was verified 1000 μg of protein was subjected to immunoprecipitation by Western blot. The co-IP sample was running on a SDS using 2.5 μg of rabbit polyclonal antibody, anti-AR N-20 gel, and silver staining showed multiple different bands. (Santa Cruz Biotechnology) and 2.5 μg of rabbit polyclonal More co-IP samples were preloaded on the gels, and the sites antibody, anti-AR (ab74272, Abcam). After separation of with different bands were cut down, and sent to FitGene Bio- immunoprecipitated proteins using magnetic protein A/G technology Co. Ltd. (Guangzhou, China) for MS detection. Dynabeads (Invitrogen), pellets were washed by lysis buffer As shown in Figure 1(b), in this site, five proteins (VAPA, fi (containing 150 mM NaCl, 5 mM MgCl2, 2 mM EGTA, ACTB, P5CR2, AXN1, and KRT) were identi ed. ACTB 50 mM Tris-HCl, and 1% Triton X-100) three times, 5 min (Actin beta) and KRT (Keratin) were suggested as contami- each. Proteins were disassociated from the magnetic beads nated protein, since they were detected in most of the by incubating with 0.1 M glycine (pH 2.2–2.7) for 10 min. A samples. P5CR2 (Pyrroline-5-carboxylate reductase 2) and part of co-IP samples was sent to FitGene Biotechnology AXN1 (Axin-1) were verified by co-IP and Western blot, Co. Ltd. (Guangzhou, China) for mass spectrometry (MS) and no positive data was observed (data not shown). detection and bioinformatic analysis, and the other was for Vesicle-associated membrane protein-associated protein Western blot analysis. A (VAPA) presents in the plasma membrane and intracellular vesicles. This protein is reported in function in vesicle 2.4. Western Blot. Levels of proteins were measured using trafficking, membrane fusion, protein complex assembly, 20 μg of protein or equal volume of co-IP samples. The sam- and cell motility. Membrane and cytoplasmic fractions were ples were subjected to SDS-PAGE on 10% tricine-glycine isolated from TM4 cell with or without testosterone admin- gels and transferred onto polyvinyl difluoride membranes istration. As shown in Figure 1(c), the expression of VAPA (Millipore). After blocking nonspecific protein-binding sites was unaltered in the input group after testosterone expo- by incubation for 1 h at room temperature in TBST (Tris- sure. The binding of the protein to AR was decreased in buffered saline with 0.2% Tween 20) containing 5% nonfat the membrane fraction (P <001) and increased in the cyto- dry milk, membranes were incubated overnight with primary plasmic fraction under testosterone treatment (P <001). antibody (antibodies information see Table 1). The mem- These data indicated that VAPA may be involved in the AR branes were washed with TBST for 5 min, repeated 3 times. membrane trafficking. Subsequently, the membranes were incubated with the sec- ondary antibody in 5% nonfat dry milk at room tempera- 3.2. VAPA Is Involved in AR Trafficking in Mouse Sertoli ture for 1 h. The specific complexes were detected using Cells. shRNA of VAPA was designed and synthesized to the enhanced chemiluminescence system from GE Health- decrease the expression of VAPA in TM4 cell. Western blot care. The densitometry of the bands was measured using and RT-PCR results in Figure 2(a) showed an effective software Image J. knockdown of VAPA by the shRNA (P <001). Cells transfected with negative control or VAPA shRNA were exposed 2.5. Statistical Analysis. Data are represented as mean ± SEM. to 10 nM testosterone for 30 min. Compared with the neg- The statistical significance of the differences between groups ative control, blots and data analysis showed a decrease in was determined by one- or two-way ANOVA followed the membrane (1.00 ± 0.03 versus 0.55 ± 0.04, P <005) and by Fisher protected least-significant difference posthoc test nuclear (1.00 ± 0.05 versus 0.69 ± 0.06, P <005) localiza- using software SigmaPlot. Statistical significance was set tion at basal condition (Figures 2(b) and 2(c)). After expo- at P <005. sure to testosterone, there were significant increases in International Journal of Endocrinology 3

IgG Basal Testosterone Cytoplasm Membrane CTCT 170 AR 130 100 GAPDH 70 Pan cadherin 55

IP-AR 40 1.5 35

1.0 25 ⁎ ⁎⁎ AR 0.5

15 0 Cytoplasm Membrane Basal Testosterone (a) (b) Membrane Cytoplasm Input IP (AR) Input IP (AR) CCT IgG TCCT IgG T AR AR

VAPA VAPA

PPanan cadcadherinherin GGAPDHAPDH

Input Co-IP 3 ⁎⁎ 1.5 2 1.0 1 VAPA 0.5 VAPA /AR VAPA ⁎⁎ 0 0 Cytoplasm Membrane Cytoplasm Membrane Basal Basal Testosterone Testosterone (c)

Figure 1: VAPA is identified as a candidate protein by MS. (a) Representative Western blots showed the cytoplasmic and membrane AR translocation in input samples and co-IP samples in TM4 cells. Data analysis from collected experiments (n =5) showed a decrease of AR expression in cytoplasmic fraction and an increase in membrane. (b) Silver staining of the co-IP sample of the membrane proteins. The bands indicated by the dotted rectangle were subjected to mass spectrograph. (c) The film images showed the representative blots for AR and VAPA in membrane and cytoplasmic fractions, before (input) or after immunoprecipitation (co-IP) by the anti-AR antibody in TM4 cells treated with 10 nM testosterone for 30 min. For the input samples, data analysis from repeated experiment (n =3) showed that VAPA expression was unchanged after exposure to testosterone. For the co-IP samples, the data indicated the association of VAPA and AR, which was showed as VAPA expression divided by AR expression. This experiment was repeated for three times. C: basal. T: 10 nM testosterone for 30 min. ∗P <005; ∗∗P <001. membrane and nuclear proteins of both NC and shRNA molecular mechanism of AR trafficking mediated by VAPA. group. However, AR trafficking to membrane in shRNA It was reported that VAPA interacts with OSBP (Oxysterol- group (1.38 ± 0.08) was decreased, compared with the NC binding protein) [8]. The expression of OSBP was unaltered (0.67 ± 0.07, P <001); and the nuclear trafficking was also in membrane and cytoplasmic fractions under testosterone inhibited (3.28 ± 0.34 versus 1.56 ± 0.18, P <001). treatment (Figures 3(a) and 3(c)). As shown in Figures 3(b) and 3(d), the binding of OSBP with AR was decreased in 3.3. VAPA-Mediated AR Trafficking via Vesicle Transport membrane fraction (1.00 ± 0.04 versus 0.42 ± 0.09, P <001) Protein OSBP. Experiments were conducted to study the but increased in the cytoplasmic fraction (1.00 ± 0.03 versus 4 International Journal of Endocrinology

NC shRNA VAPA

GAPDH NC shRNA 1.5 CTCT AR (membrane)

1.0 Pan Cadherin ⁎⁎ 0.5 AR (nucleus)

HDAC 0

VAPA mRNA (Fold Change) (Fold mRNA VAPA NC shRNA (a) (b) ⁎⁎ ⁎ ⁎⁎ ⁎⁎ ⁎ 1.5 4 ⁎⁎ ⁎⁎ 1.0 3

2 0.5 AR (nucleus) AR (membrane) 1 0 0 NC shRNA NC shRNA Basal Basal Testosterone Testosterone (c)

Figure 2: VAPA is involved in AR traﬃcking. (a) Representative Western blot analysis and real-time PCR showed VAPA was knocked down by shRNA transfection. ∗∗P <001, n =3. (b) Representative Western blot analysis indicated that AR localization and traﬃcking in membrane and nuclear fractions were decreased in VAPA shRNA group. (c) Statistical analysis of collected data was from three repeated experiments. The left panel was for membrane fraction, and right for nucleus. The data was expressed as the fold change of AR expression level at basal condition in cells transfected with NC, and the data was showed as mean ± SEM. NC: cells transfected with the negative control vector. ∗P <005; ∗∗P <001.

2.21 ± 0.14, P <001). The data analysis also showed that In the nonclassical pathway, stimulation of Sertoli cells the association of VAPA and OSBP was increased in cyto- with levels of testosterone (10–250 nM) that are similar to plasmic fraction (1.00 ± 0.05 versus 1.35 ± 0.09, P <005) but or lower than that found in the testis causes a population of unchanged in membrane samples. AR to localize near the plasma membrane. Studies using Western blot and immunofluorescence confirmed the localization of AR in membrane [10]. Binding of testosterone to 4. Discussion AR allows the receptor to interact with and activate Src tyrosine kinase. Once activated, Src causes the phosphory- Spermatogenesis is a complex and highly ordered process lation of the epidermal growth factor receptor (EGFR) via regulated by a series of gene products. Lots of clinical and an intracellular pathway, as well as kinase Ras. The stimu- experimental studies suggested that androgen and its recep- lation of EGFR results in the activation of the MAP kinase tor are critical for spermatogenesis and male fertility. Andro- cascade including the kinases RAF, MEK, and Erk [9]. In gen controls spermatogenesis mainly by associating with a previous study [6], we detected the membrane localiza- the AR localized in Sertoli cells, and the role of androgen tion of AR, and testosterone induces AR translocation to in Sertoli cells includes maintaining blood-testis barrier, membrane within 5 min (1.20 ± 0.02 fold change of basal meiosis, Sertoli-spermatid adhesion, and sperm release. value), the translocation was increased up to 60 minutes, AR is capable of transmitting testosterone signals by at and at 30-minute point, the fold change of AR translocation least 2 mechanisms, the classical and nonclassical pathways. was 2.56 ± 0.02, then we choose 30 minutes treatment for In the classical signaling pathway, testosterone that diffuses further study. through the cell membrane interacts with AR in the cyto- In order to figure out the molecular mechanism of AR plasm and then translocates to nucleus, binds AREs in gene trafficking to membrane fraction, we identified the candidate regulatory regions, and recruits coactivator or corepressor proteins involved in this action using MS and bioinformatic proteins to regulate gene expression [9]. analysis. 10 candidate proteins were identified; we conducted International Journal of Endocrinology 5

Input Cytoplasm Membrane CCTT Co-IP

AR Cytoplasm Membrane IgG CCTTIgG

OSBP AR AR Ab VAPA OSBP

GAPDH VAPA VAPA Ab Pan cadherin OSBP

(a) (b) Co-IP by AR Ab 1.5 Input 3 Co-IP by VAPA Ab ⁎⁎ 2 ⁎ 1.0 2

1 OSBP OSBP 1 0.5 OSBP/AR ⁎⁎ OSBP/VAPA

0 0 0 Cytoplasm Membrane Cytoplasm Membrane Cytoplasm Membrane

Basal Basal Basal Testosterone Testosterone Testosterone

Figure 3: VAPA-mediated AR trafficking via vesicle transport protein OSBP. The film images showed the representative blots for AR, VAPA, and OSBP in membrane and cytoplasmic fractions, before (input, (a)) or after immunoprecipitation (co-IP, (b)) by the anti-AR antibody or anti-VAPA antibody in TM4 cells treated with 10 nM testosterone for 30 min. (c) The data analysis from three repeated experiments of panel (a). The data was expressed as the fold change of the OSBP expression in cytoplasmic fraction at basal condition, and the data was showed as mean ± SEM. (d) Collected data analysis showed the testosterone enhance AR and OSBP association, as well as the binding of VAPA and OSBP in cytoplasmic fraction, n =4. The left panel was for the ratio of OSBP/AR (OSBP expression divided by AR expression) and the right for OSBP/VAPA (OSBP expression divided by VAPA expression). The data was expressed as the fold change of the values at basal condition, and the data was showed as mean ± SEM. ∗P <005; ∗∗P <001. experiments to confirm the data and study the molecular testosterone treatment. These data indicated that VAPA is mechanism mediating AR trafficking by these proteins. involved AR intracellular trafficking. VAPA is a vesicle membrane-associated protein, evolu- VAPA has been shown to interact with the OSBP to tion conserved in species and participates the formation of modify export from the endoplasmic reticulum [8]. It is lipid bilayer of the tight junction between endoplasmic retic- reported that this association is mediated by the FFAT ulum and of other organelles. VAPA is a type IV membrane motif in OSBP [16]. It is also reported that endosome protein, localizes in the plasma membrane and intracellular endoplasmic reticulum contacts control actin nucleation vesicles [11]. It may also be associated with the cytoskeleton. and retromer function through VAPA-dependent regulation ffi This protein may function in vesicle tra cking, membrane of phosphatidylinositol 4-phosphate (PI4P) [17]. The blots fusion, protein complex assembly, and cell motility [12, 13]. showed that the interaction of OSBP with AR or VAPA It was reported that VAPA is involved in the regulation of decreased in membrane fraction and increased in cytoplas- several biological process by activation of the MAPK signal- mic after exposure to testosterone. This suggested that VAPA ing pathway, including the activation of oncogenes, and the may mediate AR trafficking by interacting with OSBP. development of tumors [14]. VAPA may also facilitate the target protein to the plasma membrane [15]. Our data 5. Conclusions indicated that the association of AR to VAPA was decreased in membrane fraction and increased in cytoplasmic after In this study, we identified proteins involved in cytoplasmic exposure to testosterone. Knock down of VAPA decreases AR translocation to plasma membrane under the treatment AR membrane and nuclear localization at basal condition of testosterone at physiological concentration, and the prevented AR translocation to membrane or nucleus after data showed VAPA plays an important role in this process. 6 International Journal of Endocrinology

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