Abstract
The molecular regulation of Sertoli cells and their crosstalk with germ cells has not been fully characterized. SUMO proteins are essential for normal development and are expressed in mouse and human Sertoli cells; However, the cell-specific role of sumoylation in those cells has only started to be elucidated. In other cell types, including granulosa cells, sumoylation is regulated by a SUMO ligase KAP1/Trim28. Deletion of KAP1 in Sertoli cells causes testicular degeneration; However, the role of Kap1 in those cells has not been identified. Here we show that both mouse and human Sertoli undergo apoptosis upon inhibition of sumoylation with a chemical inhibitor or via a siRNA technology. We have additionally detected changes in the Sertoli cell proteome upon the inhibition of sumoylation, and our data suggest that among others, the expression of ER/stress-related proteins is highly affected by this inhibition. Sumoylation may also regulate the NOTCH signaling which is important for the maintenance of the developing germ cells. Furthermore, we show that a siRNA-down-regulation of KAP1 in a Sertoli-derived cell line causes an almost complete inactivation of sumoylation. In conclusion, sumoylation regulates important survival and signaling pathways in Sertoli cells, and KAP1 can be a major regulator of sumoylation in these cells.
Keywords: sumoylation, Sertoli cells, KAP1, SUMO ligases, ER stress, NOTCH signaling
Introduction.
Spermatogenesis consists of mitosis of spermatogonia, meiosis of spermatocytes and maturation of spermatids into mature sperm. This process is tightly regulated by hormones and growth factors secreted by testicular somatic cells such as Sertoli, Leydig and myoepithelial cells. Sertoli cells subdivide the seminiferous epithelium into basal and adluminal compartments, forming a blood–testes barrier supported by the tight junctions between the adjacent cells [1, 2]. Sertoli cells secrete growth factors that bind to specific receptors on the surface of germ cells, thus providing the proper environment for the developing cells. Sertoli cells proliferate during pubertal testicular development, but their number in the mature testis is constant.
Spermatogenesis is supported by various posttranslational modifications. Sumoylation is a modification of other proteins by a covalent addition of SUMO peptides through the serial modification by specific enzymes such as SUMO-activation enzyme (E1), SUMO-conjugating enzyme (E2) and cell-specific SUMO ligases (E3s) [3–5]. We have localized SUMO proteins to all testicular cells in mouse and human testes and obtained evidence suggesting a role of sumoylation in the regulation of proliferation of spermatogonia, meiosis spermatocytes and sperm functions [6–11]. In Sertoli cells, SUMO is localized to the nucleus of the cells [7, 9]. However, the role of sumoylation in Sertoli cell functions has not been well-understood. In other cell types, including granulosa cells (the female counterparts of the Sertoli cells), sumoylation is regulated by KAP1/Trim28/ TIF1β. This protein has a role in various cellular activities, but its SUMO-ligase activity has only recently been characterized [12, 13]. Abolishment of the KAP-1 mediated sumoylation in granulosa cells mimics the KAP1 knockout and causes developmental defects in vivo, suggesting that the regulation of sumoylation can be the major role of KAP1 in those cells [12]. KAP1 is expressed in mouse and human Sertoli cells, and previous studies have shown that inactivation of KAP1 in those cells causes testicular degeneration [14]. We have also shown that KAP1 interacts and co-localizes with SUMO in both mouse and human Sertoli cells [9]. However, the role of KAP1 in Sertoli cells has not been identified.
To better understand the processes regulated by SUMO in Sertoli cells, in this study, we have shown, that inhibition of sumoylation with either a sumoylation inhibitor (Gingkolic acid) or UB9 (SUMO-conjugating enzyme) si-RNAs caused a decrease in the cell viability and an increase in apoptosis of mouse and human Sertoli cells. We have also detected changes in the Sertoli cell proteome upon the inhibition of sumoylation, and our data suggest that among others, the level of ER/stress-related proteins is highly affected by this inhibition. Sumoylation may regulate the Notch signaling which is important for the maintenance of the developing germ cells.
Furthermore, our data strongly suggest that KAP1 is a major regulator of sumoylaion in Sertoli cells.
Materials and Methods.
Cell lines, primary mouse and human Sertoli.
The mouse Sertoli cell line 15P-1 (ATCC®, CRL-2618) was purchased from ATCC (Manassas, VA) and grown in DMEM media with 5% fetal bovine serum (FBS, Life Technologies, 16140–071), 5% bovine growth serum (Fisher Scientific, SH30541.03), 1% penicillin/streptomycin (Life Technologies, 15140–122) and 0.5% Fungizone (Life Technologies, 15290–018) at 32°C with 5% CO2. According to the ATCC and references within, “the 15P-1 cell line was established from testicular cells of transgenic mice that express the large T protein of polyoma virus (PyLT) in the seminiferous epithelium. This line exhibits characteristics of Sertoli cells, including transcription of the Wilms’ tumor (WT1) and Steel genes. 15P-1 cultures express phagocytic activity evidenced by the internalization of latex beads and the disposal of dead cells. Interaction with germ cells enhances the phagocytic activity of 15P-1 cells. The cells support the meiotic and postmeiotic differentiation in cocultures of diploid premeiotic germ cells into haploid spermatids expressing the protamine (Prm-1) gene. When cocultured with 15P-1 cells, testicular cells explanted from immature 9-day-old animals, before the onset of the first meiosis, generate tetrads of haploid cells with the morphology of round spermatids and initiate protamine transcription. The 15P-1 cell line releases protease-sensitive material with broad-spectrum”.
The primary human Sertoli cell comprehensive kit was purchased from ScienCell Research Laboratories (Carlsbad, CA), and the cells were cultured at 32°C with 5% CO2. The human Sertoli were isolated from human testis and characterized by immunofluorescence with antibody specific to GATA-4 and Sox-9.
C57BL/6NCrl mice were purchased from Charles River (Kingston, NY). The Animal Committee of Albert Einstein College of Medicine approved all animal protocols that followed the National Institutes of Health guide for the care and use of laboratory animals. A differentiating plating procedure was used to separate primary mouse Sertoli from germ cells as described in our recent publication with some modifications [9, 11]. Briefly, Petri dishes were covered with FBS and left open to dry at the laminar floor hood. Testicular suspension was prepared from the testes of pubertal mice (11–15 dpp) using a two-step enzymatic digestion as previously described [11]. The cells were cultured on the FBS-coated dishes at the concentration of 1–5×106 cells/100 mm dish for a duration of 2–3 hours. Sertoli cells attached to the dishes, but floating spermatogonia and spermatocytes were removed with the medium. Sertoli cells continued to grow for several days and were used for the experiments at 70–80% confluency.
Gingkolic acid and Si-RNA treatments.
Sumoylation inhibitor Gingkolic acid (GA) was diluted at DMSO and used at the concentration of 25 −150uM for 2 hours. The concentration for each cell type was chosen based on our previous studies[15] and at the range that would not cause massive cell death and detachment. For the proteomic experiments, FSH (F4021, sigma Aldrich) was added at the concentration of 20 IU/ml 4 hours before an addition of the GA (50uM for 2 hours) to some FSH and control wells. Eighty pmols of UBC9 (the only conjugating enzyme for all SUMO isoforms), KAP1 or control siRNAs (Santa Cruz Biotechnology; sc-36773, sc-38551 and sc-36869, respectively) were used for the transfection of the Sertoli-derived cell line using siRNA transfection reagent (sc-29528) and siRNA transfection medium (sc-36868). The cells were subjected to 6 h of transfection followed by a 48h recovery period prior to analysis. The dose of the siRNAs and the transfection time were determined in pilot experiments, and the downregulation was assessed using western blot analysis.
Viability and apoptotic assays; statistical analysis.
WST-1 cell proliferation assay (Cayman Chemical, Ann Arbor, MI) and The Caspase-Glo® 3/7 assay (Pomega, WI, USA) were performed according to the manufacture instructions. The measurements were collected using a Promega Glomax microplate reader. Each experiment was repeated three times in triplicates. To calculate the difference between the samples, Student’s paired t-test was used. P< 0.05 was considered statistically significant.
Whole-cell protein lysate preparation.
Whole-cell protein lysates were prepared as previously described [9], using the whole-cell extraction kit and protease inhibitor from Millipore (2910, Sigma-Aldrich) complemented with 2.5 mg ml−1 of NEM (a de-sumoylation inhibitor; E3876–100G, Sigma-Aldrich) according to the manufacturer’s instructions.
Protein concentrations were determined via bicinchoninic acid (BCA) protein assay (23225, Thermo Fisher) using bovine serum albumin (BSA) as the standard.
Gel electrophoresis and western blotting.
Gel electrophoresis and western blotting was performed as previously described [9, 15] under reducing conditions using NuPAGE 4%–12% gradient bis-tris polyacrylamide gels (Thermo Fisher) and MOPS running buffer (Thermo Fisher). The membrane was then incubated with primary antibodies in PBS containing 2% BSA and 0.1% sodium azide for either 2 h at room temperature or overnight at 4°C”. Rabbit polyclonal anti-SUMO1 antibody (Abcam, ab32058) was used at a 1:500 dilution; a rabbit polyclonal antibody against KAP1 (Bethyl, Montgomery, TX, A300–274A) was used at a 1:1000 dilution; a mouse anti- ubiquitin antibody from Millipore (MAB1510) was used at a 1:100 dilution. Equal loading was ensured with monoclonal anti-β-actin (sc-1615, Santa Cruz) at a 1:1000 dilution Following three washes with PBS-T, the membrane was further incubated with secondary antibodies that were diluted to 1:5000 in PBS-T for 1 h at room temperature. The secondary antibodies used in this study included the following: anti-rabbit IgG horseradish peroxidase (HRP) linked (NA934V, GE Healthcare UK Limited) and goat anti-mouse IgG (H + L) HRP (AP308P, EMD Millipore Corporation, Sigma Aldrich). Western blot detection was performed using Luminata™ Forte (Sigma-Aldrich), in accordance with the manufacturer’s instructions. All antibodies have been validated by the companies and in previous studies at our laboratory using the molecular weight markers, localization studies and siRNA experiments. Each experiment was repeated at least three times.
Two-dimensional gel electrophoresis, profiling and protein identification.
Two-dimensional gel electrophoresis (2-D DIGE) profiling and protein identification was performed by Applied Biomics, Inc (Hayward, CA) according to the following protocol:
Preparation of samples.
Protein sample buffer was exchanged into 2D lysis buffer (30 mM Tris-HCl, pH 8.8, containing 7 M urea, 2 M thiourea and 4% CHAPS). Protein concentration was measured using Bio-Rad Protein Assay Kit II #500–0002 according to manufacturer’s protocol.
CyDye labeling.
For each sample, 30ug of protein was mixed with 1.0 ul of diluted CyDye, and kept in the dark on ice for 30 min. The labeling reaction was stopped by adding 1.0 ul of 10 mM Lysine to each sample and incubating in the dark on ice for an additional 15 min. The labeled samples were then mixed. The 2X 2-D Sample buffer (8 M urea, 4% CHAPS, 20 mg/ml DTT, 2% pharmalytes and trace amount of bromophenol blue), 100 ul destreak solution and Rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mg/ml DTT, 1% pharmalytes and trace amount of bromophenol blue) were added to the labeling mix to make the total volume of 250 ul. The samples were mixed and spun before being loaded into the strip holder IEF and SDS-PAGE. After loading the labeled samples, IEF (pH3–10 Linear) was run following the protocol provided by Amersham BioSciences. Upon finishing the IEF, the IPG strips were incubated in the freshly made equilibration buffer-1 (50 mM Tris-HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, trace amount of bromophenol blue and 10 mg/ml DTT) for 15 minutes with gentle shaking. Then the strips were rinsed in the freshly made equilibration buffer-2 (50 mM Tris-HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, trace amount of bromophenol blue and 45 mg/ml DTT) for 10 minutes with gentle shaking. Next the IPG strips were rinsed in the SDS-gel running buffer before transferring into 12% SDS-gels. The SDS-gels were run at 15 °C until the dye front ran out of the gels
Image scan and data analysis.
Gel images were scanned immediately following the SDS-PAGE using Typhoon TRIO (GE Healthcare). The protein ratio analysis was performed using DeCyder software version 6.0 (GE Healthcare).
Results.
Inhibition of sumoylation with Gingkolic acid and siRNA decreases the viability and induces apoptosis in mouse and human Sertoli cells.
To test whether sumoylation is important for the Sertoli cell survival and functioning we performed a treatment of a mouse Sertoli-derived cell line and human primary Sertoli cells with an increasing concentration of the sumoylation inhibitor Gingkcolic acid. The inhibition of sumoylation caused a significant decrease in the cell viability and an increase in apoptosis (Fig. 1, A, D). While in mouse cells the percentage of cell death and apoptosis was always proportional to the GA concentration, in human cells, the apoptosis did not increase between the 25 and 50 uM of the GA, suggesting that other types of cell death could be activated at the higher concentrations (Fig. 1, A, D).
Figure 1. Inhibition of sumoylation in mouse and human Sertoli cells causes a decrease in the cell viability and initiation of apoptosis.
A. Measurement of the cell viability (WST-1 assay) and activation of caspases (Caspase Glow-assay) in mouse Sertoli-derived cell line following a treatment with the increasing concentrations of the Gingkcolic acid (GA).
B. Measurement of the cell viability and activation of caspases in mouse Sertoli-derived cell line following the treatment of the cells with anti UBC-9 si RNA (+) or a scramble control (−).
C. A PARP cleavage (about 90 kDa; blue arrows) was observed following a treatment of the cells with the increasing concentrations of the GA. The uncleaved isoform (about 116 kDa) is indicated by the black arrows. Molecular weight markers (MW) as well as the control lysate (C) from the company (prepared using a different sample buffer) are shown. The higher molecular weight bands are most likely sumoylated PARP isoforms.
D. Measurement of the cell viability and activation of caspases in human primary Sertoli cells following a treatment of the cells with the increasing concentrations of the GA.
E. Immunoblot with anti-SUMO antibody to confirm the decrease in sumoylation after the inhibition of sumoylation by the GA (human cells) or UBC-9 si RNA in triplicates (mouse cells). The same mouse membrane was incubated with anti-ubiquitin antibody.
In a similar manner, the use of anti UBC-9 (the only SUMO-conjugating enzyme) siRNAs caused a consistent and statistically significant decrease in the cell viability and increase in the cell apoptosis (Fig. 1, B).
A PARP cleavage (one of the main caspase substates; blue arrows) was also observed (Fig. 1, C). Interestingly, the additional bands above the uncleaved PARP isoform (black arrow) are likely sumoylated PARP isoforms; their presence is consistent with the previously reported PARP sumoylation and the use of desumoylation inhibitor in our lysate buffer. Interestingly, it looks like the sumoylated isoforms are being cleaved first. The control lysate from the company with both cleaved and uncleavd isoform of PARP (C) was sent in a different lysate buffer which is less compatible with our gel buffer system.
Immunoblot with anti-SUMO antibody (Fig. 1, E) was used to confirm the decrease in sumoylation after its inhibition by the GA (the dose-response effect is seen) and by UBC-9 si RNAs. Notably, the global ubiquitination pattern of the same samples has not been affected significantly suggesting that the inhibition specifically affected sumoylation pathways (Fig. 1, E);
Inhibition of sumoylation causes significant changes in the expression level of ER proteins and a Notch signaling regulator.
To better understand the mechanism behind the cell death /apoptosis of the Sertoli cells, we analyzed the changes in the proteome of the Sertoli cells before and after their treatment with the sumoylation inhibitor GA. We have performed this experiment with and without an addition of FSH to the Sertoli cells to better understand whether a specific pathway is FSH-regulated. For each pair of the conditions, the two-dimensional gel electrophoresis (2-D DIGE) profiling was performed using two fluorescent dies as described at the Materials and Methods. A representative 2-D gel image is shown at Fig. 2A.
Figure 2. Analysis of changes in the proteome of the primary mouse Sertoli cells upon inhibition of sumoylation.
A. A representative 2-D gel image used for the analysis.
B. Summary of proteins differently expressed upon an inhibition of sumoylation in Sertoli cells. Proteins are grouped based on their functions (* - regulated by FSH).
We found changes in the expression of different groups of the proteins, but the most significant changes were observed in the level of ER proteins (Fig. 2B); the full proteomic data and the fold changes for each target are available at the supplementary Table1). These results suggest that ER stress can be responsible for the decreased viability and increased apoptosis of the Sertoli cells. Furthermore, a significant change was observed in the expression level of the Recombination signal binding protein for immunoglobulin kappa J region (Rbpj), an important regulator of the Notch signaling; this pathway in Sertoli cells regulates germ cell fate and numbers [16]. Interestingly, the Rbpj expression is significantly upregulated by FSH but downregulated by the sumoylation inhibitor at both basal and FSH -activated conditions (supplementary Table1). The same supplementary table summarizes all the proteins affected by the FSH treatment. These data can be used in other studies of Sertoli cells and their regulation by FSH.
KAP1 regulates sumoylation in Sertoli-derived cell line.
Our data suggest that sumoylation may regulate ER stress and Notch signaling in Sertoli cells. However, what proteins regulate sumoylation in Sertoli cells is not currently known. A recent study in granulosa cells (female counterparts of the Sertoli) has identified KAP1 as a major SUMO ligase. KAP-1 is highly expressed in the testes including Sertoli cells. Notably, a Sertoli-specific inactivation of KAP1 causes testicular degeneration. Our previously published mass spectrometry and co-IP studies have shown that Kap-1 interacts and colocalizes with SUMO in mouse spermatocytes, spermatids, mouse and Human Sertoli while being sumoylated itself [9]. We, therefore, were interested to test whether KAP1 can regulate sumoylation in Sertoli-derived cell line. Indeed, upon downregulation of KAP1 using siRNAs, a dramatic decrease in the overall sumoylation could be observed (Figure 3). Almost all high molecular weight (HMW) bands disappeared while the level of the free SUMO was significantly increased; Those results support the possible specific role of KAP1 as SUMO ligase.
Figure 3. Kap1 regulates sumoylation in Sertoli-derived cell line.
Downregulation of Kap1 in Sertoli-derived cell line using si RNA, causes an almost complete block in sumoylation. Immunoblots with anti-KAP1 and SUMO antibodies are shown on the control (−) and si-RNA (+) samples in triplicates. Anti-actin antibody was used as a loading control.
Discussion.
SUMO proteins are highly expressed in all testicular cells where they were implicated in cell-specific functions during spermatogenesis. Sertoli cells provide support and regulation of developing germ cell; However, the molecular regulation of Sertoli cells and their crosstalk with germ cells has not been fully characterized. In this study we have shown that both mouse and human Sertoli cells undergo apoptosis upon inhibition of sumoylation with a chemical inhibitor or using si-RNA technology. We have additionally detected changes in the Sertoli cell proteome upon the inhibition of sumoylation, and our data suggest that among others, ER/stress-related proteins are highly affected by this inhibition. ER stress in testicular cells, including Sertoli cells, has been identified as a possible cause of male infertility [17]. Many substances (bisphenol, sodium fluoride, nonylphenol, dibutyl phthalate) induce apoptosis in Sertoli cells through an activation of an ER stress[18–20]. Therefore, ER stress can be one of the underlying mechanisms in initiation of apoptosis in Sertoli cells upon inhibition of sumoylation.
Our data suggest that sumoylation may also regulate the NOTCH signaling which is important for the maintenance of developing germ cells [21]. Specifically, we see a decrease in the level of Rbpj which is required for the proper regulation of the NOTCH signaling [22]. A knockout of Rbpj in mouse Sertoli cells in vivo caused a significant increase in the number of atrophic seminiferous tubules, increase in testes size, increase in the expression of niche factors, such as Gdnf and Cyp26b1 and the number of germ cells, including spermatogonia stem cells (SSCs) [16]. However, the regulation of RBPJ in Sertoli cells has not been well-characterized. Our data suggest that sumoylation can directly or indirectly regulate the transcription or translation level of Rbpj since the inhibition of sumoylation caused a significant decrease in its expression level. Furthermore, our data suggest that Rbpj is also regulated by FSH.
All cells studied so far, share the same and the only E1 (SUMO-activating) and E2 (SUMO-conjugating) enzymes; however, E3s (SUMO-ligases) show a tissue- and cell-specific distribution. In other cell types, including granulosa cells, sumoylation is regulated by a newly identified SUMO ligase KAP1/Trim28 [12]. Kap1 is expressed in mouse and human Sertoli cells, and previous studies have shown that inactivation of KAP1 in Sertoli cells causes testicular degeneration [14]. We have shown that KAP1 interacts and co-localizes with SUMO in mouse and human Sertoli [9]. Remarkably, the results herein show that a down-regulation of KAP1 in Sertoli-derived cell line causes an almost complete inactivation of sumoylation in these cells. Therefore, KAP1 may be a major regulator of sumoylation in Sertoli cells. These results should be confirmed by in-vivo experiments since the transfection procedure affected the viability of the primary Sertoli cells. Nevertheless, the Sertoli-derived cell line exhibit many characteristics of Sertoli cells, including transcription of the Wilms’ tumor (WT1) and Steel genes, ability to phagocytize beads and support differentiation of the meiotic and postmeiotic cells in cocultures (Materials and Methods). It is also possible that the function of KAP1 is not identical in mitotic pubertal and postmitotic adult Sertoli, although co-localization and interaction with SUMO in all three models (the cell line, pubertal mouse and adult human Sertoli [9]) suggest some overlapping functions.
These studies lay a foundation for the future studies in vivo that would include a generation of transgenic animals with inactivated sumoylation machinery and a mutation abolishing the SUMO-ligase activity of KAP1 specifically in Sertoli cells. Analysis and comparison of these phenotypes among themselves and to the one reported for the KAP1 Sertoli knockout will be important in dissecting the molecular events regulated by sumoylation and its corresponding KAP-1-regulating activity in Sertoli cells.
Supplementary Material
Highlights.
Downregulation of sumoylation causes apoptosis in mouse and human Sertoli cells
Inhibition of sumoylation activates ER stress response in primary Sertoli cells
Sumoylation regulates the level of RBPJ 1, an important NOTCH signaling regulator
KAP1 may be a major regulator of sumoylation in Sertoli cells
Acknowledgement:
This study was supported by the NIH, NICHD, and Academic Research Enhancement Award 1R15HD067944-01A1 (MV, PI). Undergraduate student research was supported by Appelbaum Foundation and by Stern College for Women, Yeshiva University.
We would like to thank the Laboratory for Macromolecular Analysis and Proteomics (LMAP) at Albert Einstein College of Medicine for their assistance in proteomic analyses.
Footnotes
Conflict of interests: The authors declare no conflict of interest
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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