
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12775-2
10.1016/j.heliyon.2024.e36744
e36744
Research Article
TRIM59 is required for mouse GC-1 cell maintenance through modulating the ubiquitination of AXIN1
Wu Tiantian ab1
Zhou Hui c1
Wang Lulu d1
Tan Jianxin d
Gao Wenxin b
Wu Yibo c
Zhao Dan e
Shen Cong a
Zheng Bo bozheng@njmu.edu.cn
a⁎⁎⁎
Huang Xiaoyan bbhxy@njmu.edu.cn
b⁎⁎
Shao Binbin shaobinbin1988@163.com
d⁎
a State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproduction and Genetics, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School of Nanjing Medical University, Suzhou, 215002, China
b Department of Histology and Embryology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, 211166, China
c Human Reproductive and Genetic Center, Affiliated Hospital of Jiangnan University, Wuxi, 214122, China
d Department of Prenatal Diagnosis, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, 210004, China
e Reproductive Medicine Center, The Fourth Affiliated Hospital of Jiangsu University, Zhenjiang, 212001, China
⁎ Corresponding author. shaobinbin1988@163.com
⁎⁎ Corresponding author. bbhxy@njmu.edu.cn
⁎⁎⁎ Corresponding author. bozheng@njmu.edu.cn
1 These authors contributed equally to this work and share first authorship.

23 8 2024
15 9 2024
23 8 2024
10 17 e3674424 6 2024
20 8 2024
21 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Tripartite motif-containing protein 59 (TRIM59) is a biomarker for multiple tumors with crucial roles. However, the specific role of TRIM59 in germ cells remains largely unknown. Here, we investigated the effects and underlying regulatory mechanisms of TRIM59 on germ cells using the mouse spermatogonial cell line GC-1. Our results demonstrated that TRIM59 promoted proliferation and inhibited apoptosis of GC-1 cells. Mechanistically, TRIM59 maintained GC-1 cell behaviors through ubiquitination of AXIN1 to activate β-catenin signaling. Furthermore, activation of β-catenin signaling reversed the effects mediated by Trim59 knockdown in GC-1 cells. Collectively, our study revealed a major role and regulatory mechanism of TRIM59 in GC-1 cells, which sheds new light on the molecular pathogenesis of defects in spermatogenesis and may provide therapeutic targets for treatment of male infertility.

Keywords

TRIM59
GC-1 cells
AXIN1
Ubiquitination
β-catenin signaling
==== Body
pmc1 Introduction

Male infertility which accounts for nearly half cases of infertility, affects approximately 50 million couples worldwide according to the World Health Organization (WTO) [1]. Azoospermia, which is defined as the complete absence of spermatozoa in the ejaculate, occurs in 10∼15 % of male infertility patients and 1 % of normal male population [2]. Azoospermia is classified as obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). NOA accounts for approximately 60 % of azoospermia patients due to spermatogenesis deficit including primary testicular dysfunction or secondary testicular dysfunction [3]. Spermatogenesis is a complex process including the proliferation and differentiation of spermatogonia, meiosis division of spermatocytes and the release of mature spermatozoa [4]. The maintenance of spermatogenesis needs to be precisely regulated through a complex multi-layered gene-protein network; however, the specific mechanisms of the regulation for spermatogenesis remain unclear.

Protein ubiquitination is a crucial post-translational modification process in eukaryotic cells, involving the covalent attachment of ubiquitin molecules to the target proteins, marking them for proteasomal degradation or influencing their localization, activity, or interaction with other molecules [5]. The ubiquitination process consists of three main steps: activation, conjugation, and ligation, mediated by a cascade of enzymes including E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase [6]. This highly regulated process plays pivotal roles in maintaining protein homeostasis, cell cycle progression, signal transduction, DNA repair, and cancer progression, highlighting its significance in cellular physiology and pathology [[7], [8], [9], [10]].

The Wnt/β-catenin signaling pathway is a fundamental and highly conserved signaling cascade that plays critical roles in various biological processes, including embryonic development, cell proliferation, differentiation, and tissue homeostasis [11,12]. The pathway is initiated when Wnt ligands bind to the Frizzled (Fz) family of seven-pass transmembrane receptors and their co-receptors, such as LRP5/6 (Low density lipoprotein receptor-related proteins 5 and 6), leading to the disassembly of the destruction complex. Consequently, β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it interacts with TCF/LEF (T-cell factor/lymphoid enhancer-binding factor) transcription factors to activate target gene expression [[13], [14], [15]]. AXIN1 is a key scaffold protein in the β-catenin destruction complex. In an inactive state of Wnt/β-catenin signaling, AXIN1 binds to β-catenin, APC (Adenomatous polyposis coli), GSK-3β (Glycogen synthase kinase 3 beta), and CK1 (Casein kinase 1), facilitating the phosphorylation and degradation of β-catenin [16,17]. However, the association between Wnt/β-catenin signaling and male infertility remains unclear.

The tripartite motif (TRIM) protein family includes nearly 80 members and is characterized by the highly conserved Trim motif, including a RING domain, B-box motifs, and a coiled-coil region [18]. The TRIM family has been implicated in various biological processes, such as cell proliferation, cell cycle progression, apoptosis, and innate immunity [19]. Tripartite motif-containing protein 59 (TRIM59), which is a member of the TRIM family, contains a RING finger domain and has an E3 ubiquitin ligase activity, which modulates protein ubiquitination, leading to degradation, activation, or functional modification of target proteins [20]. For instance, in gastric cancer, TRIM59 promotes tumor growth by accelerating ubiquitination and degradation of p53 [21]. In glioblastoma, TRIM59 enhances STAT3 (Signal transducer and activator of transcription 3) signaling activation and tumorigenicity by promoting ubiquitination and degradation of the tumor suppressive histone variant macroH2A1 [22]. Additionally, TRIM59 promotes steatosis and ferroptosis in non-alcoholic fatty liver disease by enhancing GPX4 (Glutathione peroxidase 4) ubiquitination [23]. Studies have shown that many TRIM family members trigger β-catenin signaling via ubiquitination of AXIN1 [24,25]. Moreover, AXIN1 is a regulatory factor of the Wnt/β-catenin signaling pathway in spermatogonial stem cells [26]. However, to our knowledge, no studies have suggested an association between TRIM59 and germ cell development.

In this study, we predominantly focused on the role and regulatory mechanism of TRIM59 in mouse spermatogonial cells. We found that TRIM59 is required to maintain mouse spermatogonial cell behaviors through ubiquitination of AXIN1 to activate β-catenin signaling, which may provide new therapeutic targets for treatment of male infertility.

2 Materials and methods

2.1 Cell culture

The mouse spermatogonial cell line GC-1 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA; ATCC number: CRL2053) and authenticated by short tandem repeat profiling. GC-1 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (Gibco, Utah, CA, USA) with 10 % fetal bovine serum (FBS; Thermo Scientific, Waltham, MA, USA) at 37 °C with 5 % CO2. To passage GC-1 cells, the medium was removed, cells rinsed with PBS, and treated with 0.25 % trypsin-EDTA at 37 °C for 2–3 min. After detachment, trypsinization was halted with medium, and cells were centrifuged at 600 g for 5 min. The pellet was resuspended, counted, and seeded into new flasks. Transient transfection of small interfering RNA (siRNA) was performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. siRNAs targeting Trim59 (si-Trim59 1#: 5′-GGAACAACAACUUGAAUUA-3′; si-Trim59 2#: 5′-GGAGCUAAUUGAUACAUUA-3′) and negative control (NC) siRNA (si-NC; 5′-ACGUGACACGUUCGGAGAA-3′) were provided by GenePharma (Suzhou, China). For β-catenin activation and inhibition assays, the same number of GC-1 cells was treated with 10 μmol/L β-catenin activator (Selleck, SKL2001) or β-catenin inhibitor (Selleck, XAV-939).

2.2 RNA extraction and quantitative real-time PCR

Total RNA was extracted from GC-1 cells using Trizol reagent (Invitrogen) and then reverse transcribed into cDNA with a HiScript III 1st Strand cDNA Synthesis Kit (Vazyme) in accordance with the manufacturers’ protocols. Quantitative Real-time PCR (RT-PCR) was performed using AceQ qPCR SYBR Green Master Mix (Vazyme, Q141-02) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Gene expression was calculated using the 2−ΔΔCt method. The primer sequences used for quantitative real-time PCR are listed in Table S1.

2.3 Western blotting

Western blotting was performed using a previously described protocol with minor modifications [27]. Briefly, total cell lysates were prepared by incubation in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Nantong, China). Nuclear protein was obtained using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer's instructions. Equal amounts of extracted proteins were separated by 4–20 % gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5 % (w/v) dry non-fat milk and then incubated overnight with the indicated primary antibody (Table S2). After washing, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (1:1000, Thermo Scientific) for 1 h at room temperature. Protein signals were visualized using SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific). Band intensities were analyzed by Image-Pro Plus Software (Media Cybernetics, San Diego, CA, USA).

2.4 Immunofluorescence

GC-1 cells were cultured on a Millicell EZ slide (Merck Millipore, Darmstadt, Germany), fixed in 4 % (w/v) paraformaldehyde (PFA), and then blocked in 1 % (w/v) bovine serum albumin (Sigma, St. Louis, MO, USA). Subsequently, the samples were incubated with primary antibodies (Table S2) overnight at 4 °C. After washing with phosphate-buffered saline (PBS), the samples were incubated with Alexa Fluor secondary antibodies (1:1000, Thermo Scientific). The cell nuclei were stained with 5 μg/ml DAPI (Beyotime) for 1 min. All samples were imaged under a confocal laser microscope (Zeiss LSM800, Carl Zeiss, Oberkochen, Germany).

2.5 Cell viability, proliferation, and colony formation assay

Cell Counting Kit-8 (CCK-8; Beyotime) was used to evaluate cell viability as described previously [28,29]. After transfection with si-NC or si-Trim59 for 48 h, GC-1 cells were treated with CCK-8 solution (1:100 dilution) for 2 h. Then, absorbance at 450 nm was measured. For the colony formation assay, GC-1 cells were seeded at 1000 cells/well in six-well plates. After cultivation for 2 weeks, cells were fixed using methanol and stained with a crystal violet solution (Beyotime).

A 5-ethynyl-2′-deoxyuridine (EdU) assay was performed to assess cell proliferation using an EdU assay kit as described previously [30,31]. Transfected GC-1 cells were incubated with 20 μM EdU at 37 °C for 1 h and then viewed under the LSM800 confocal microscope after the nuclei were counterstained with DAPI.

2.6 TUNEL assay

Apoptotic cells were detected with a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) BrightRed Apoptosis Detection kit (Vazyme, Nanjing, China) in accordance with the manufacturer's instructions. Briefly, the cells were fixed in 4 % (w/v) PFA, permeabilized with 0.1 % Triton X-100, and then treated with BrightRed Labeling Buffer for 1 h at 37 °C. After washing with PBS, the samples were stained with DAPI. Images were captured under the LSM800 confocal microscope.

2.7 Cell migration assay

A Transwell assay was used to assess the cell migratory ability as described previously [32]. Briefly, 1 × 105 cells in serum-free medium (200 μL) were seeded into the upper culture chamber of a 24-well Transwell plate (Merck Millipore). Medium (600 μL) that was supplemented with 10 % fetal bovine serum (FBS) was added to the lower chamber. After incubation for 24 h, the cells on the surface of the lower membrane were fixed with 4 % polyformaldehyde and then stained with 0.1 % (w/v) crystal violet (Beyotime). Images were captured under a brightfield microscope (Carl Zeiss, Germany).

2.8 Protein half-life assay

To determine the half-life of AXIN1, GC-1 cells were transfected with si-Trim59 or si-NC using Lipofectamine 2000. After 48 h, cells were treated with 100 μg/mL cycloheximide for 0, 3, and 6 h. Cells were lysed in cold RIPA buffer with protease inhibitors and centrifuged at 12,000 g. Protein concentration was measured using the BCA assay, and equal amounts of protein were subjected to SDS-PAGE and probed with primary antibodies against AXIN1 and tubulin, followed by HRP-conjugated secondary antibodies. Protein bands were visualized using ECL and quantified with Image-Pro Plus, normalizing AXIN1 levels to tubulin. The experiment was repeated three times for reproducibility.

2.9 TOP/FOP luciferase assays

A TOP/FOP Flash luciferase assay was performed to investigate the effect of Trim59 knockdown on β-catenin activity [33,34]. GC-1 cells treated with si-NC, si-Trim59 1#, or si-Trim59 2# were co-transfected with TOP/Flash, FOP/Flash, and Renilla luciferase plasmids (Addgene, Cambridge, USA) using Lipofectamine 2000. After 48 h of transfection, the cells were washed with PBS and lysed in lysis buffer. The lysates were then centrifuged at 12,000 g for 1 min to remove cell debris, and the supernatants were transferred to new tubes. Luciferase activities were detected using a Luciferase Reporter Gene Assay Kit (Beyotime), and the luminescence was quantified using a microplate luminometer (BioTek). The TOP/Flash and FOP/Flash values were normalized to the Renilla luciferase activity to account for variations in transfection efficiency. The ratio of TOP/Flash to FOP/Flash luminescence was calculated to determine the β-catenin activity.

2.10 Three-dimensional reconstruction of AXIN1/TRIM59 complexes

The process began with the generation of individual 3D AXIN1 and TRIM59 protein models using Robetta (https://robetta.bakerlab.org/), which predicts the structure based on sequence information and homology modeling. These models were then uploaded to HawkDock (http://cadd.zju.edu.cn/hawkdock/), where protein-protein docking simulations were performed to identify potential interaction sites and binding conformations. The MM/GBSA algorithm provided further refinement by optimizing the interface of the docked complex. This step ensured that the predicted interactions were energetically favorable and structurally feasible. The final complex structures were visualized using PyMOL (https://pymol.org/2/), a molecular visualization system. PyMOL was used to annotate the interacting residues and present a clear visual representation of the AXIN1/TRIM59 complex, highlighting key interaction sites and the overall molecular interface.

2.11 Immunoprecipitation

For the immunoprecipitation (IP) assay, 5 μg/mL anti-TRIM59, anti-β-catenin, or anti-AXIN1 antibody was incubated with equal amounts of proteins for 14 h at 4 °C. Then, the antibody–protein complex was incubated with 20 μl washed protein A/G agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C. The beads were pelleted and washed three times with RIPA lysis buffer. After resuspending with 2 × SDS-PAGE buffer, the bound protein was boiled for 5 min at 95 °C and then centrifuged for 30 min at 12,000 g at 4 °C. The presence of each protein in the IP products was detected using the indicated antibodies. Inputs represented approximately 1/10 of the extract volume used for the IP assay.

2.12 Statistical analysis

Experiments were repeated at least three times. Data are presented as means ± standard deviation (SD). Statistical significance was calculated using Student's t-test or one-way ANOVA by GraphPad Prism 8 (https://www.graph pad.com/) with * P < 0.05, **P < 0.01 and ***P < 0.001.

3 Results

3.1 Trim59 is required to maintain mouse spermatogonial cells

To determine the roles of Trim59 in maintaining spermatogonial cells, we established a GC-1 cell culture system in vitro and used siRNAs targeting Trim59 to knockdown its expression. We used RT-PCR to confirm Trim59 knockdown efficiency. Trim59 expression levels in GC-1 cells treated with si-Trim59 1# and si-Trim59 2# are shown in Fig. 1a. We next used a CCK-8 assay to assess the effect of Trim59 knockdown on cell viability. The growth of GC-1 cells was significantly inhibited after Trim59 knockdown (Fig. 1b). Moreover, the number of colonies was obviously reduced in Trim59 knockdown GC-1 cell cultures (Fig. 1c and d). Then, we used an EdU assay to measure cell proliferation and found that GC-1 cells transfected with si-Trim59 1# and si-Trim59 2# had reduced cell proliferation compared with cells transfected with si-NC (Fig. 1e and f). We used TUNEL staining to determine the extent of apoptosis and observed a significant increase in GC-1 cell apoptosis after Trim59 knockdown (Fig. 1g and h). To determine whether Trim59 knockdown was associated with spermatogonial cell migration, we conducted a Transwell assay to estimate the migratory ability of GC-1 cells following Trim59 knockdown. As expected, the number of migrated GC-1 cells was significantly reduced after transfection with si-Trim59 (Fig. 1i and j). In addition to self-renewal, another critical feature of spermatogonia is their capacity for differentiation, which involves two stages of meiotic divisions, ultimately producing mature spermatozoa. We therefore evaluated two markers for meiosis initiation (Spo11 and Sycp1), and the results revealed a significant increase in their expressions after Trim59 knockdown in GC-1 cells (Fig. 1k). These results suggest that Trim59 is required for mouse spermatogonial cell maintenance.Fig. 1 Effect of Trim59 knockdown in GC-1 cells (a) Real-time PCR showed high knockdown efficiency of Trim59 knockdown in GC-1 cells. si-NC was negative control. N = 3 for each group. (b) CCK-8 assay for assessing cell viability of GC-1 cells after Trim59 knockdown. N = 4 for each group. (c) Colony formation assay for colony formation ability of GC-1 cells after Trim59 knockdown. (d) Quantification for number of colonies (c). N = 3 for each group. (e) EdU assay for measuring cell proliferation of GC-1 cells after Trim59 knockdown. GC-1 cells after Trim59 knockdown were incubated with 20 μM EdU at 37 °C for 1 h, and the nuclei were counterstained with DAPI. Scale bar, 50 μm. (f) Quantification of (e). N = 3 for each group. (g) TUNEL staining for detecting cell apoptosis of GC-1 cells after Trim59 knockdown. Scale bar, 50 μm. (h) Quantification of (g). N = 3 for each group. (i) Transwell assay for detecting cell migratory ability of GC-1 cells after Trim59 knockdown. Scale bar, 100 μm. (j) Quantification of (i). N = 3 for each group. Experiments were repeated at least three times. (k) Real-time PCR analysis of Spo11 and Sycp1 expressions after Trim59 knockdown. N = 3 for each group. Statistical analysis was calculated using one-way ANOVA with Dunnett post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 1

3.2 Trim59 knockdown up-regulates AXIN1 expression and down-regulates β-catenin expression

To further investigate the mechanism of TRIM59 in maintaining mouse spermatogonial cells, we examined changes in AXIN1 and its direct target, β-catenin, expression levels after Trim59 knockdown, and found that AXIN1 expression was up-regulated while β-catenin expression was markedly down-regulated in Trim59 knockdown GC-1 cells (Fig. 2a and b). Reciprocal IP experiments confirmed the interactions between endogenous AXIN1 and β-catenin in GC-1 cells (Fig. 2c). Next, we quantified AXIN1 expression by fluorescence. GC-1 cells transfected with si-Trim59 had an increased fluorescence intensity of AXIN1 (Fig. 2d and e), which was highly consistent with the Western blot data. Nuclear translocation of β-catenin was significantly reduced, indicating that β-catenin signaling was inactivated in Trim59 knockdown GC-1 cells (Fig. 2f–i). Furthermore, knockdown of Trim59 resulted in a remarkable reduction in TOP/FOP luciferase activity (Fig. 2j). These results indicated that Trim59 knockdown up-regulated AXIN1 expression and down-regulated β-catenin expression.Fig. 2 The expression of AXIN1 and β-catenin of GC-1 cells after Trim59 knockdown (a) Western blotting analysis of the expression of TRIM59, AXIN1 and β-catenin of GC-1 cells after Trim59 knockdown. si-NC was negetive control. (b) Quantification of (a). N = 3 for each group. (c) Reciprocal IP assays confirming the interactions between endogenous AXIN1 and β-catenin in GC-1 cells. (d) Immunofluorescence analysis for AXIN1 expression in GC-1 cells after Trim59 knockdown. The cell nuclei were stained with 5 μg/mL DAPI for 1 min. Scale bar, 20 μm. (e) Quantification of AXIN1 relative fluorescene intensity in (d). (f) Immunofluorescence analysis for nuclear translocation of β-catenin in GC-1 cells after Trim59 knockdown. Scale bar, 20 μm. (g) Percentage of nuclear β-catenin-expressing cells in GC-1 cells after Trim59 knockdown. (h) Western blotting analysis of the nuclear β-catenin expression in GC-1 cells after Trim59 knockdown. Histone H3 was used as an internal control. (i) Quantification of (h). (j) TOP/FOP flash luciferase assays performed in GC-1 cells after Trim59 knockdown. Experiments were repeated at least three times. Statistical analysis was calculated using one-way ANOVA with Dunnett post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2

3.3 TRIM59 interacts with AXIN1 and influences its ubiquitination in GC-1 cells

We measured protein stability by the AXIN1 half-life in Trim59 knockdown GC-1 cells and found that it was significantly more stable in cells treated with si-Trim59 than si-NC (Fig. 3a and b). Then, we characterized the interactions between AXIN1 and TRIM59 proteins by 3D modeling. The predicted 3D structural model to dock AXIN1/TRIM59 complexes is visualized in Fig. 3c and d. The critical residues for AXIN1/TRIM59 interactions are listed in Table S3. Moreover, reciprocal IP assays confirmed the interactions between endogenous AXIN1 and TRIM59 (Fig. 3e), suggesting that TRIM59 affects the maintenance of GC-1 cells potentially through interacting with AXIN1.Fig. 3 The interactions between TRIM59 and AXIN1 (a) Half-time assay for the expression stability of AXIN1 in Trim59 knockdown GC-1 cells. (b) Quantification of (a). (c) Visualization of the docking of the AXIN1/TRIM59 complexes. The front and back views of the docked AXIN1 (blue)/TRIM59 (golden) protein complex. The interacting residues are colored purple and red for AXIN1 and TRIM59, respectively. The cartoon mode displays the backbone and the secondary structures of the corresponding proteins. The surface mode represents the solvent-accessible surface area. The merged mode combines the cartoon and transparent surface views. (d) Visualization of the binding interface of the AXIN1/TRIM59 complexes. The interface view zooms and labels the residues (in stick mode) involved in the binding between AXIN1 and TRIM59. The interacting residues are colored in purple and red for AXIN1 and TRIM59, respectively. The molecular contacts between interacting residues are connected with dashed lines. (e) Reciprocal IP of endogenous TRIM59 and AXIN1 from GC-1 cells. IgG was used as the negative control. (f) Ubiquitination analysis of endogenous AXIN1 in GC-1 cells after Trim59 knockdown. For a, e, and f, experiments were repeated at least three times. Statistical analysis was calculated using Student's t-test. *P < 0.05; **P < 0.01.

Fig. 3

TRIM59 has an ubiquitin ligase activity and AXIN1 has ubiquitination modification sites, and therefore we explored whether TRIM59 induces AXIN1 ubiquitination in mouse spermatogonial cells by measuring the ubiquitination level of AXIN1. As expected, the ubiquitination level of endogenous AXIN1 was significantly decreased after Trim59 knockdown (Fig. 3f). Combined with the high expression level of AXIN1 after Trim59 knockdown, we concluded that TRIM59 destabilizes and ubiquitinates AXIN1 protein in GC-1 cells.

3.4 β-catenin signaling is required for GC-1 cell growth

β-Catenin expression was decreased in Trim59 knockdown GC-1 cells (Fig. 2a and b). Therefore, we hypothesized that Trim59 knockdown induced cell proliferation and apoptosis by suppressing the β-catenin signaling. To confirm whether the β-catenin signaling is essential for GC-1 cell growth, we used the specific β-catenin signaling activator SKL2001 and inhibitor XAV-939 to promote or inhibit β-catenin expression in GC-1 cells.

To confirm the activation and inhibition efficiencies of β-catenin, we measured protein expression levels of β-catenin in GC-1 cells treated with SKL2001 or XAV-939. Western blot analysis showed that β-catenin expression in GC-1 cells was significantly increased by SKL2001 and decreased by XAV-939 compared with NC (Fig. 4a and b). Next, the CCK-8, colony formation, and EdU assays were used to measure GC-1 cell proliferative ability. The results indicated that GC-1 cell proliferative ability was significantly increased by SKL2001 and inhibited by XAV-939 (Fig. 4c–g). Moreover, Transwell assays showed that the cell migratory ability was increased by SKL2001 and decreased by XAV-939 (Fig. 4h and i). Taken together, activation of β-catenin signaling plays an important role in promoting GC-1 cell growth.Fig. 4 Effects of activated or depressed β-catenin signaling on GC-1 cells (a) Western blotting analysis of GC-1 cells treated with β-catenin activitor SKL2001 or inhibitor XAV-939, respectively. NC was negative control. (b) Quantification of (a). N = 3 for each group. (c) CCK-8 assay assessing cell viability of GC-1 cells treated with β-catenin activitor SKL2001 or inhibitor XAV-939, respectively. N = 4 for each group. (d) Colony formation assay for colony formation ability of GC-1 cells treated with β-catenin activitor SKL2001 or inhibitor XAV-939, respectively. (e) Quantification for number of colonies in (d). N = 3 for each group. (f) EdU assay for measuring cell proliferation of GC-1 cells treated with β-catenin activitor SKL2001 or inhibitor XAV-939, respectively. Scale bar, 50 μm. (g) Quantification of (f). N = 3 for each group. (h) Transwell assay detecting cell migratory ability of GC-1 cells treated with β-catenin activitor SKL2001 or inhibitor XAV-939. Scale bar, 100 μm. (i) Quantification of (h). N = 3 for each group. Experiments were repeated at least three times. Statistical analysis was calculated using one-way ANOVA with Dunnett post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4

3.5 Activation of β-catenin signaling reverses the effects mediated by Trim59 knockdown

To further clarify the TRIM59/AXIN1/β-catenin axis in GC-1 cells, we carried out rescue experiments using SKL2001 or si-Axin1 to activate β-catenin signaling in Trim59-deficient GC-1 cells. β-catenin protein levels in GC-1 cells treated with si-Trim59 + SKL2001 or si-Trim59 + si-Axin1 were higher than those in cells treated with si-Trim59 alone (Fig. 5a and b). CCK-8, colony formation, and EdU assays showed that GC-1 cells treated with si-Trim59 + SKL2001 or si-Trim59 + si-Axin1 had higher cell proliferative ability than those treated with si-Trim59 (Fig. 5c–g). Moreover, TUNEL staining revealed that cells treated with si-Trim59 + SKL2001 or si-Trim59 + si-Axin1 showed lower apoptosis than cells treated with si-Trim59 (Fig. 5h and i). Transwell assays showed that the migratory ability of cells treated with si-Trim59 + SKL2001 or si-Trim59 + si-Axin1 was increased compared with si-Trim59 (Fig. 5j and k). These results indicated that the phenotypes of Trim59 knockdown cells were rescued by si-Axin1 or a β-catenin signaling activator such as SKL2001. Thus, activation of β-catenin signaling is an effective approach to reverse Trim59 knockdown effects in GC-1 cells. Combined with all findings, we demonstrated that TRIM59 ubiquitinates AXIN1 to activate β-catenin signaling, thereby promoting GC-1 cell growth (Fig. 6).Fig. 5 Activation of β-catenin rescues the altered phenotypes caused by Trim59 knockdown in GC-1 cells (a) Western blotting analysis measuring the expression of β-catenin in GC-1 cells transfected with si-Trim59 alone, si-Trim59 + SKL2001, or si-Trim59 + si-Axin1, respectively. NC was negative control. (b) Quantification of (a). N = 3 for each group. (c) CCK-8 assay assessing cell viability of GC-1 cells transfected with si-Trim59 alone, or si-Trim59 + SKL2001, or si-Trim59 + si-Axin1, respectively. N = 4 for each group. (d) Colony formation assay for colony formation ability of GC-1 cells transfected with si-Trim59 alone, si-Trim59 + SKL2001, or si-Trim59 + si-Axin1, respectively. (e) Quantification for number of colonies in (d). N = 3 for each group. (f) EdU assay measuring cell proliferation of GC-1 cells transfected with si-Trim59 alone, si-Trim59 + SKL2001, or si-Trim59 + si-Axin1, respectively. Scale bar, 50 μm. (g) Quantification of (f). N = 3 for each group. (h) TUNEL staining detecting cell apoptosis of GC-1 cells transfected with si-Trim59 alone, si-Trim59 + SKL2001, or si-Trim59 + si-Axin1, respectively. Scale bar, 50 μm. (i) Quantification of (h). N = 3 for each group. (j) Transwell assay detecting cell migratory ability of GC-1 cells transfected with si-Trim59 alone, si-Trim59 + SKL2001, or si-Trim59 + si-Axin1, respectively. Scale bar, 100 μm. (k) Quantification of (j). N = 3 for each group. Experiments were repeated at least three times. Statistical analysis was calculated using one-way ANOVA with Dunnett post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5

Fig. 6 Schematic illustration of the working model for the role of TRIM59 in GC-1 cells TRIM59 is required for maintaining GC-1 cell behaviors by ubiquitinating of AXIN1 to activate the β-catenin signaling.

Fig. 6

4 Discussion

We investigated the effect of β-catenin on GC-1 cells treated with SKL2001 or XAV-939. The results indicated that activation of the β-catenin signaling pathway promotes maintenance of GC-1 cells, whereas inhibition of β-catenin leads to the opposite trend. Furthermore, we transfected siRNA targeting Trim59 to explore the role of Trim59 in GC-1 cells. Trim59-deficient GC-1 cells exhibited compromised viability, migration, proliferation, and increased rates of apoptosis. Mechanistically, we found that TRIM59 ubiquitinates and degrades AXIN1 to promote β-catenin expression. Furthermore, we performed rescue experiments, which indicated that silencing Axin1 is an effective approach to reverse Trim59 knockdown effects in GC-1 cells.

Trim59 is involved in many pathophysiological processes, such as tumorigenesis, cytotoxicity, and inflammatory responses [35,36]. Trim59 knockdown in cancer cells induces apoptosis and inhibits cell proliferation and migration. In retinoblastoma, Trim59 plays an oncogenic role in its progression and serves as a promising prognostic marker [37]. In lung adenocarcinoma, Trim59 promotes gefitinib resistance in EGFR-mutant lung adenocarcinoma cells [38]. However, little is known about its role in the reproductive system. Other members of the TRIM family have been reported to participate in spermatogenesis. For example, Trim36 is upregulated in mouse spermatogonia in response to a reduction of collagen type I, and Trim36-deficient mouse spermatozoa show lower motility and less complex motility patterns [39]. Trim27, a transcriptional regulatory protein, interacts with Slx2 to regulate the formation of XY bodies and germ cell proliferation during spermatogenesis [40]. In this study, we found that Trim59 knockdown inhibited cell proliferation and migration capacities, and induced apoptosis of GC-1 cells in vitro, which is similar to its roles in carcinomas [41,42]. Trim59 regulates ovarian cancer via the MKP3/ERK pathway [43], neuroblastoma via the Wnt/β-catenin pathway [44], and retinoblastoma via the p38-MARK pathway [37]. Our study showed that Trim59 regulated GC-1 cell behaviors via the β-catenin signaling. We found that expression and nuclear translocation of β-catenin were significantly reduced in Trim59 knockdown GC-1 cells. Similar to the regulatory mechanism of Trim59 in retinoblastoma [44], Trim59 knockdown inhibited cell proliferation by down-regulating the β-catenin signaling in GC-1 cells.

Wnt/β-catenin signaling is an important regulator of spermatogonial fate. However, the specific regulatory mechanism of Wnt/β-catenin signaling in affecting spermatogenesis is controversial. Some studies suggest that activation of Wnt/β-catenin signaling promotes spermatogenesis. Canonical Wnt signaling is active in post-meiotic germ cells of the testis and particularly present at higher levels in mouse testes at day 28 [45]. Liang et al. demonstrated that the Wnt/β-catenin signaling pathway is positively regulated by KIF3A during spermatogenesis [46]. In spermatogonial cells, periostin promotes cell proliferation by activating the Wnt/β-catenin signaling pathway [47]. In human Sertoli cells, miR-202-3p inhibits cell proliferation and the synthesis function by inhibiting activation of Wnt/β-catenin signaling [48]. In peritubular myoid cells (PMCs), activation of Wnt/β-catenin signaling promotes spermatogenesis by mediating the R-spondin receptor Lgr4 [49]. Additionally, the number of PMCs with nuclear β-catenin decreases dramatically in Lgr4 mutant testes. Reactivation of Wnt/β-catenin signaling partially rescues the Lgr4 deficiency phenotypes. Conversely, some studies suggest that activation of Wnt/β-catenin signaling is harmful to spermatogenesis. In Sertoli cells, constitutive activation of the Wnt/β-catenin pathway causes spermatogenic defects, whereas deletion of β-catenin causes no phenotypic defects [50]. In spermatogonia stem cells (SSCs), Wnt5a promotes SSC activity by blocking β-catenin-dependent signaling [51]. Wnt3a activates β-catenin signaling in a subset of cluster cells, which subsequently reduces SSCs activity in vitro [52]. By epigenetically repressing Wnt10b/β-catenin signaling, BMI1 promotes SSC maintenance [53]. In testes, cellular nucleic acid-binding protein maintains testicular development by inhibiting the Wnt/β-catenin pathway [54]. Wnt ligands signal via canonical and non-canonical pathways, and they have opposite effects on spermatogenesis [51]. The effects of Wnt/β-catenin signaling on spermatogenesis may be complex because of the interaction of these two pathways, which may explain the above-mentioned controversial roles of Wnt/β-catenin signaling in spermatogenesis. More studies about the detailed regulatory mechanism of Wnt/β-catenin signaling in affecting spermatogenesis are warranted. Using the activator and inhibitor of the β-catenin signaling pathway in vitro, we found that activated β-catenin signaling promoted GC-1 cell growth.

In our study, we found that TRIM59 ubiquitinated AXIN1 to activate β-catenin signaling, thereby promoting GC-1 cell growth. Moreover, silencing Axin1 significantly attenuated the negative effect of Trim59 knockdown on GC-1 cells. We believe that AXIN1 is required for the function of TRIM59 in GC-1 cells, but we do not exclude other targets of TRIM59 in this intricate regulatory process. Our study indicates that TRIM59 interacts with AXIN1 and enhances ubiquitination and degradation of AXIN1. However, the specific ubiquitination sites and forms will be interesting to explore in the future.

The present study had several limitations. We were unable to determine the cellular localization of TRIM59 in seminiferous tubules or the specific location of the TRIM59 and AXIN1 complex because of the poor quality of antibodies used in immunolocalization. Our study showed that TRIM59 is required to maintain mouse spermatogenic cells via activating β-catenin signaling by ubiquitinating AXIN1. However, because of differences in the cell line in vitro and the internal environment in vivo, whether TRIM59 plays a similar role in the testes of mice and humans as that observed in GC-1 cells remains uncertain. Because of embryonic lethality in mice lacking Trim59 [55], in a future study, we will employ Trim59-CKO (conditional knockout) mice to further investigate its regulatory mechanism in vivo.

This study revealed the major role of TRIM59 in GC-1 cells. We found that TRIM59 maintains GC-1 cells behaviors through ubiquitination of AXIN1 to activate β-catenin signaling. Our findings shed new light on the molecular pathogenesis of spermatogenesis defects and may provide therapeutic targets for treatment of male infertility.

Data availability

The data associated with this study have not been deposited into a publicly accessible database, but have been included in this article and are available from the corresponding author upon reasonable request.

Funding

This work was supported by the 10.13039/501100001809 National Natural Science Foundation of China [Grant number 82001612 ], the Key Research Foundation of Zhenjiang Social Development (SH2022029 ) and the Top Talent Support Program for Young and Middle-aged people of Wuxi Health Committee [Grant number BJ2020047 ].

CRediT authorship contribution statement

Tiantian Wu: Writing – original draft, Methodology, Investigation, Formal analysis. Hui Zhou: Writing – original draft, Methodology, Formal analysis. Lulu Wang: Writing – original draft, Methodology, Formal analysis. Jianxin Tan: Resources, Investigation. Wenxin Gao: Investigation. Yibo Wu: Investigation, Funding acquisition. Dan Zhao: Investigation, Funding acquisition, Formal analysis. Cong Shen: Methodology, Data curation. Bo Zheng: Project administration, Conceptualization. Xiaoyan Huang: Writing – review & editing, Supervision, Conceptualization. Binbin Shao: Writing – review & editing, Funding acquisition, Conceptualization.

Declaration of competing interest

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.

Appendix A Supplementary data

The following is the supplementary data to this article:Multimedia component 1

Multimedia component 1

Acknowledgements

We would like to thank Walgenron Bio-Pharm Co., Ltd (Shenzhen, China) for language editing.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e36744.
==== Refs
References

1 Bose R. Ubiquitin-proteasome system in spermatogenesis Adv. Exp. Med. Biol. 759 2014 181 213 25030765
2 Cocuzza M. Alvarenga C. Pagani R. The epidemiology and etiology of azoospermia Clinics 68 Suppl 1 2013 15 26 Suppl 1 23503951
3 Maor-Sagie E. Deleterious mutation in SYCE1 is associated with non-obstructive azoospermia J. Assist. Reprod. Genet. 32 6 2015 887 891 25899990
4 Neto F.T. Spermatogenesis in humans and its affecting factors Semin. Cell Dev. Biol. 59 2016 10 26 27143445
5 Swatek K.N. Komander D. Ubiquitin modifications Cell Res. 26 4 2016 399 422 27012465
6 Cruz Walma D.A. Ubiquitin ligases: guardians of mammalian development Nat. Rev. Mol. Cell Biol. 23 5 2022 350 367 35079164
7 Yau R. Rape M. The increasing complexity of the ubiquitin code Nat. Cell Biol. 18 6 2016 579 586 27230526
8 Swatek K.N. Insights into ubiquitin chain architecture using Ub-clipping Nature 572 7770 2019 533 537 31413367
9 Sampson C. The roles of E3 ubiquitin ligases in cancer progression and targeted therapy Clin. Transl. Med. 13 3 2023 e1204 36881608
10 Sheng X. The ubiquitin codes in cellular stress responses Protein Cell 15 3 2024 157 190 37470788
11 Liu J. Wnt/beta-catenin signalling: function, biological mechanisms, and therapeutic opportunities Signal Transduct. Targeted Ther. 7 1 2022 3
12 Perugorria M.J. Wnt-beta-catenin signalling in liver development, health and disease Nat. Rev. Gastroenterol. Hepatol. 16 2 2019 121 136 30451972
13 Yu F. Wnt/beta-catenin signaling in cancers and targeted therapies Signal Transduct. Targeted Ther. 6 1 2021 307
14 Clevers H. Nusse R. Wnt/beta-catenin signaling and disease Cell 149 6 2012 1192 1205 22682243
15 Xu C. beta-Catenin signaling in hepatocellular carcinoma J. Clin. Invest. 132 4 2022
16 Qiu L. The scaffold protein AXIN1: gene ontology, signal network, and physiological function Cell Commun. Signal. 22 1 2024 77 38291457
17 Albrecht L.V. Tejeda-Munoz N. De Robertis E.M. Cell biology of canonical Wnt signaling Annu. Rev. Cell Dev. Biol. 37 2021 369 389 34196570
18 Hatakeyama S. TRIM proteins and cancer Nat. Rev. Cancer 11 11 2011 792 804 21979307
19 Hatakeyama S. TRIM family proteins: roles in autophagy, immunity, and carcinogenesis Trends Biochem. Sci. 42 4 2017 297 311 28118948
20 Han T. TRIM59 regulates autophagy through modulating both the transcription and the ubiquitination of BECN1 Autophagy 14 12 2018 2035 2048 30231667
21 Zhou Z. TRIM59 is up-regulated in gastric tumors, promoting ubiquitination and degradation of p53 Gastroenterology 147 5 2014 1043 1054 25046164
22 Sang Y. CDK5-dependent phosphorylation and nuclear translocation of TRIM59 promotes macroH2A1 ubiquitination and tumorigenicity Nat. Commun. 10 1 2019
23 Zhang J. TRIM59 promotes steatosis and ferroptosis in non-alcoholic fatty liver disease via enhancing GPX4 ubiquitination Hum. Cell 36 1 2022 209 222 36417114
24 Hou Y. TRIM11 promotes lymphomas by activating the β-catenin signaling and Axin1 ubiquitination degradation Exp. Cell Res. 387 2 2020
25 Yang, Y.A.-O., et al., TRIM65 Triggers β-catenin Signaling via Ubiquitylation of Axin1 to Promote Hepatocellular Carcinoma. (1477-9137 (Electronic)).
26 Yao L. The effect of regulating the Wnt signaling pathway on the proliferation and differentiation of spermatogonial stem cells Ann. Transl. Med. 8 16 2020 1003 1003 32953803
27 Zhang R. Testis-enriched Asb12 is not required for spermatogenesis and fertility in mice Transl. Androl. Urol. 11 2 2022 168 178 35280661
28 Zhou J. LINC00624/TEX10/NF-κB axis promotes proliferation and migration of human prostate cancer cells Biochem. Biophys. Res. Commun. 601 2022 1 8 35219000
29 Wang Q. BMI1 promotes osteosarcoma proliferation and metastasis by repressing the transcription of SIK1 Cancer Cell Int. 22 1 2022 136 35346195
30 Chen X. SAT2 regulates Sertoli cell-germline interactions via STIM1-mediated ROS/WNT/β-catenin signaling pathway Cell Biol. Int. 46 10 2022 1704 1713 35819096
31 Yu X. E3 ubiquitin ligase RNF187 promotes growth of spermatogonia via lysine 48-linked polyubiquitination-mediated degradation of KRT36/KRT84 Faseb. J. 37 10 2023 e23217
32 Xu B.Y. RNF187 governs the maintenance of mouse GC-2 cell development by facilitating histone H3 ubiquitination at K57/80 Asian J. Androl. 26 3 2023 272 281 38156805
33 Wu Y.-B. Long non-coding RNA NRSN2-AS1 promotes ovarian cancer progression through targeting PTK2/β-catenin pathway Cell Death Dis. 14 10 2023
34 Zheng A. Long non-coding RNA LUCAT1/miR-5582-3p/TCF7L2 axis regulates breast cancer stemness via Wnt/β-catenin pathway J. Exp. Clin. Cancer Res. 38 1 2019
35 Tan P. He L. Zhou Y. TRIM59 deficiency curtails breast cancer metastasis through SQSTM1-selective autophagic degradation of PDCD10 Autophagy 15 4 2019 747 749 30653426
36 An Y. TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages through JNK signaling pathway Cell. Signal. 67 2020
37 Wu C. TRIM59 promotes retinoblastoma progression by activating the p38–MAPK signaling pathway Investigative Opthalmology & Visual Science 61 10 2020
38 Cui Z. TRIM59 promotes gefitinib resistance in EGFR mutant lung adenocarcinoma cells Life Sci. 224 2019 23 32 30902544
39 Mascaro M. Lages I. Meroni G. Microtubular TRIM36 E3 ubiquitin ligase in embryonic development and spermatogenesis Cells 11 2 2022
40 Zhuang X.-J. Trim27 interacts with Slx2, is associated with meiotic processes during spermatogenesis Cell Cycle 15 19 2016 2576 2584 27612028
41 Tian Y. TRIM59: a membrane protein expressed on Bacillus Calmette-Guérin-activated macrophages that induces apoptosis of fibrosarcoma cells by direct contact Exp. Cell Res. 384 1 2019
42 Shen H. Knockdown of tripartite motif 59 (TRIM59) inhibits proliferation in cholangiocarcinoma via the PI3K/AKT/mTOR signalling pathway Gene 698 2019 50 60 30822475
43 Tong X. TRIM59, amplified in ovarian cancer, promotes tumorigenesis through the MKP3/ERK pathway J. Cell. Physiol. 235 11 2020 8236 8245 31951023
44 Chen G. TRIM59 knockdown inhibits cell proliferation by down-regulating the Wnt/β-catenin signaling pathway in neuroblastoma Biosci. Rep. 39 1 2019
45 Kerr G.E. Regulated wnt/beta-catenin signaling sustains adult spermatogenesis in Mice1 Biol. Reprod. 90 1 2014
46 Liang Y.-J. KIF3A regulates the Wnt/β-catenin pathway via transporting β-catenin during spermatogenesis in Eriocheir sinensis Cell Tissue Res. 381 3 2020 527 541 32458081
47 Li C. POSTN promotes the proliferation of spermatogonial cells by activating the wnt/β-catenin signaling pathway Reprod. Sci. 28 10 2021 2906 2915 33959891
48 Yang C. miR-202-3p regulates Sertoli cell proliferation, synthesis function, and apoptosis by targeting LRP6 and cyclin D1 of wnt/β-catenin signaling Mol. Ther. Nucleic Acids 14 2019 1 19 30513418
49 Qian Y. Lgr4-mediated Wnt/β-catenin signaling in peritubular myoid cells is essential for spermatogenesis Development 140 8 2013 1751 1761 23533175
50 Chang H. Wt1negatively regulates β-catenin signaling during testis development Development 135 10 2008 1875 1885 18403409
51 Yeh J.R. Zhang X. Nagano M.C. Wnt5a is a cell-extrinsic factor that supports self-renewal of mouse spermatogonial stem cells J. Cell Sci. 124 14 2011 2357 2366 21693582
52 Singh S.R. Indirect effects of wnt3a/β-catenin signalling Support mouse spermatogonial stem cells in vitro PLoS One 7 6 2012
53 Yu J. BMI1 promotes spermatogonial stem cell maintenance by epigenetically repressing Wnt10b/β-catenin signaling Int. J. Biol. Sci. 18 7 2022 2807 2820 35541907
54 Cheng H. Genetic analysis and intracytoplasmic sperm injection outcomes of Chinese patients with congenital bilateral absence of vas deferens J. Assist. Reprod. Genet. 39 3 2022 719 728 35119551
55 Su X. Embryonic lethality in mice lacking Trim59 due to impaired gastrulation development Cell Death Dis. 9 3 2018
