TRIB3 regulates Cx43 expression in human granulosa cells via the Wnt/β-catenin signaling pathway: an in vitro study.

A total of 28 women undergoing in vitro fertilization for tubal pathology were recruited from the Center for Reproductive Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. The exclusion criteria were a diagnosis of polycystic ovarian syndrome (according to the Rotterdam criteria), endometriosis, abnormal glucose and lipid metabolism, or other abnormalities that could affect folliculogenesis. This study was approved by the ethics committee of Ruijin Hospital (2020104a). Written informed consent was obtained from all patients. All patients underwent standard GnRH antagonist treatment with recombinant human FSH, which was started on the second day of the menstrual cycle. All viable embryos were frozen, and none of the patients underwent fresh embryo transfer. Over the next 3 months, the patients underwent frozen embryo transfer, with 1–2 blastomeres being transferred. After 36 h of trigger with Human Chorionic Gonadotropin, the follicular fluid was collected from follicles with diameters of ≥ 18 mm, from which oocytes were retrieved. Follicular fluid samples from 28 patients were centrifuged at 250 × g for 10 min. The pellets were resuspended in phosphate-buffered saline (PBS), layered over 40% Percoll (Solarbio, Beijing, China), and separated from red blood cells via centrifugation. GCs were collected from the interface between the Percoll layer and PBS. Finally, the GCs were preserved in DMEM/F12 (Gibco, Waltham, MA, USA) medium and stored at -80 °C until further analysis. Human granulosa-like tumor (KGN) cell line (Feiya Biotechnology Co., Ltd. Jiangsu, China) was used for in vitro experiments. KGN cells were cultured in DMEM/F12 (Gibco) medium, supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Hyclone, Logan, UT, USA), and incubated at 37 °C with 5% CO 2 . The following reagents were used: MG132 (S2619, Selleck Chemicals, Houston, TX, USA), cycloheximide (C7418, Selleck Chemicals), ICG-001 (HY-14428, MedChemExpress) and Palmitic acid (PA; Sigma-Aldrich, St. Louis, MO, USA). The expression lentivirus for TRIB3 , siRNA for TRIB3 , and the corresponding control vector were purchased from Genechem Biotechnology Co., Ltd. (Shanghai, China). A non-targeting scrambled siRNA sequence was used as the negative control for TRIB3 knockdown experiments. For overexpression experiments, an empty vector without the TRIB3 insert was employed as the control. KGN cells were transfected with the lentiviruses (multiplicity of infection: 50) for 16 h. Then, the transfected cells were selected using 1 µg/mL puromycin (Beyotime, Jiangsu, China) to generate stable cell lines. Serum anti-Mullerian hormone levels were measured using an ELISA kit (Biotra, Guangzhou, China). The levels of LH, FSH, estradiol, and progesterone were measured using a chemiluminescence immunoassay (ECLIA) kit (Beckman Coulter, Brea, CA, USA) according to the manufacturer’s instructions. Serum free fatty acid, glucose (GLU), insulin (INS), triglycerides, and total cholesterol were measured using an automated biochemical analyzer (Roche Cobas 8000). Cell viability was measured using the Cell Counting Kit-8 (CCK-8; Yeason Biotech, Shanghai, China) according to the manufacturer’s protocol. KGN cells were seeded in 96-well plates and treated with PA (100 µM) for 24 h, with or without TRIB3 overexpression or knockdown. Medium containing 10% CCK8 reagent replaced the cell culture medium. The plate was incubated for 1 h at 37 °C. Absorbance was measured at 450 nm. Cell proliferation was assessed using an EdU incorporation assay with flow cytometry detection (Beyotime, China). Cells were incubated with EdU (10 µM) for 2 h, fixed, permeabilized, and stained according to the manufacturer’s protocol. The proportion of EdU-positive cells was determined using a flow cytometer (Becton Dickinson, USA). Cell apoptosis was analyzed with a Annexin V-FITC/PI Apoptosis Detection Kit (Yeason Biotech, Shanghai, China). The cells were collected by centrifugation at 300 × g at 4℃ for 5 min. The cells were washed twice with PBS. After centrifugation, 100 µl binding buffer was added, followed by 5 µl Annexin V-FITC and 10 µl PI. After avoiding light and reacting for 10 min, 400 µl binding buffer was added and mixed well. The level of intracellular apoptosis was detected and analyzed using flow cytometry (Becton Dickinson, USA). Total RNA was isolated using the Total RNA Isolation Kit (Vazyme, Nanjing, China). cDNA was synthesized via reverse transcription using an RT reaction kit (Vazyme) according to the manufacturer’s instructions. qRT-PCR was performed to determine the mRNA expression of TRIB3 , Cx43 , and β-catenin using Applied Biosystems 7500 (Thermo Fisher Scientific, Waltham, MA, USA) and the SYBR Green qPCR Mix (Vazyme). The cycling conditions were as follows: denaturation at 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. The 2 −ΔΔCt method was used to calculate relative expression levels (defined as fold-change). The primers were synthesized by Beijing TsingkeBiotech Co., Ltd., and their sequences were list on Supplementary Table (1). GAPDH was used as the housekeeping gene, according to the NormFinder software (MOMA, Aarthus, Denmark). Results of NormFinder were listed on Supplementary Table (2). To ensure the specificity of SYBR Green-based qRT-PCR assays, we implemented multiple quality control steps. All primers were checked for specificity using NCBI Primer-BLAST. After each qRT-PCR run, melting curve analysis was conducted to confirm the presence of a single specific amplification product. No-template controls were included in all reactions to monitor for potential contamination or non-specific amplification. These steps ensured the specificity and reliability of our qRT-PCR data. All qRT-PCR experiments were performed using three independent biological replicates, each with three technical replicates. Immunofluorescence staining was performed as described previously [ 4 ]. KGN cells were incubated with 100µM palmitate for 24 h and fixed in 4% paraformaldehyde for 30 min. After fixation, the cells were blocked with 5% bovine serum albumin (BSA, Servicebio, Wuhan, China) for 30 min. Then, the cells were incubated overnight with the following primary antibodies: TRIB3 (cat. 3868, 1:200, Proteintech, China), Cx43 (cat. 3512, 1:100, Cell Signaling Technology [CST], USA), β-catenin (cat. 8480, 1:200, CST). The samples were washed thrice with PBS and incubated with the secondary antibody AlexaFluro647 (cat. GB25303, 1:200; Servicebio) for 1 h in the dark. Finally, after washing the slides with PBS thrice, the coverslips were mounted on them with an antifade mounting medium containing DAPI (Servicebio). Coimmunoprecipitation was performed as described previously [ 11 ]. Briefly, the cells were cultured in 10-cm dishes, washed with cold PBS, and lysed using lysis buffer. After centrifuging at 12,000 × g and 4 °C for 10 min, 20 µL Protein A/G Magnetic Beads (Biolinkedin, Shanghai, China) per 100 µL of the supernatant was added and incubated with the appropriate antibody overnight at 4 °C. The following day, the immunoprecipitates were washed with wash buffer thrice and boiled in 5× SDS loading buffer. The cells were harvested and lysed with RIPA lysis buffer (EpiZyme, Shanghai, China) containing 1% protease inhibitor cocktail (EpiZyme). Protein concentration was measured using a BCA Protein Assay Kit (EpiZyme), and 20 µg of protein was loaded onto a 10% SDS-PAGE gel and separated via electrophoresis. The resolved proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked with a protein-free rapid blocking buffer (EpiZyme) for 20 min at room temperature, and then incubated with the corresponding primary antibodies overnight at 4 °C. The primary antibodies included those against TRIB3 (1:1000, Abcam, Cambridge, UK), Cx43 (1:1000, Cell Signaling Technology [CST], Danvers, MA, USA), β-catenin (1:1000, CST), ubiquitin (1:1000, CST), β-actin (1:1000, CST) and β-tubulin (1:4000, Proteintech). After washing thrice with Tris-buffered saline containing 0.1% Tween-20 for 10 min, the polyvinylidene difluoride membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000, CST) or anti-mouse secondary antibody (1:2000, Proteintech) for 1 h. Target proteins were detected using the Western Chemiluminescent HRP Substrate Kit (Vazyme) according to the manufacturer’s instructions. Western blotting experiments were performed three times, each using independent biological samples to confirm reproducibility. The scrape loading/dye transfer assay was used to observe functional gap junctions, which were traced using the Lucifer yellow (LY) (L0259, Sigma) dye that was transferred between cells. The assay was performed as described previously [ 12 ]. Briefly, cells were incubated in 3-cm dishes with 100µM palmitate. After 24 h, the cells were washed with PBS, and scrape loading was applied with a surgical blade in PBS solution containing LY (1 mg/ml). After loading for 5 min, the cells were washed with PBS thoroughly and fixed with 4% paraformaldehyde. Fluorescence images were captured using CLSM (A1R MP+, Nikon, Tokyo, Japan). Gap junctions were quantified by continuously evaluating the fluorescence distance over time. All experiments were performed in triplicate, and statistical analyses were performed using SPSS version 26.0 (SPSS Inc., Chicago, IL, USA). Normally distributed continuous variables are represented as mean ± SEM, while non-normally distributed continuous variables are expressed as the median. Normally and non-normally distributed variables were compared between groups using Student’s t-test and Mann-Whitney U-test, respectively. Statistical significance was set at P-values of < 0.05.

The women with infertility were divided into two groups based on their BMI (group I, BMI<24.0 kg/m 2 ; group II, BMI ≥ 24.0 kg/m 2 ) (Table  1 ). There was no significant difference between the two groups in terms of age or basal hormone profiles. The duration of stimulation, total dose of FSH, serum estradiol levels on the trigger day, and total number of retrieved oocytes were similar in both the groups ( P  > 0.05). Meanwhile, serum free fatty acid was higher in group II ( P  < 0.05), but other metabolic biomarkers including glucose, insulin, triglycerides, and total cholesterol showed no differences between the two groups. The fertilization rate and number of top-quality embryos was significantly lower in group II than in group I ( P  < 0.05). However, the average number of embryos transferred, and clinical pregnancy rates did not differ. Table 1 Characteristics of the study participants Parameter Group I ( n  = 17) Group II ( n  = 11) P -value Age (years) 32.18 ± 3.47 33.18 ± 4.71 0.521 BMI (kg/m 2 ) 21.55 ± 1.27 27.08 ± 2.15 <0.001 AMH (ng/mL) 4.09 ± 1.70 4.10 ± 2.18 0.990 Sex hormone levels FSH (mIU/mL) 7.17 ± 2.71 6.64 ± 1.50 0.554 LH (mIU/mL) 4.73 ± 1.57 4.62 ± 1.42 0.853 E2 (pg/mL) 28.84 (19.50, 38.50) 26.00 (22.00, 30.34) 0.962 P (ng/mL) 0.67 (0.34, 0.92) 0.33 (0.31, 0.63) 0.165 Metabolic markers GLU (mmol/L) 5.42 ± 0.57 5.38 ± 0.62 0.862 INS (µIU/ml) 19.26 ± 4.28 20.81 ± 5.62 0.415 Triglyceride (mmol/L) 1.25 ± 0.63 1.31 ± 0.57 0.801 Total cholesterol (mmol/L) 3.24 ± 1.49 3.47 ± 1.63 0.704 Serum free fatty acid (mmol/L) 0.37 ± 0.11 0.44 ± 0.18 0.021 Ovarian stimulation cycle characteristics Total FSH dose (IU) 2908.53 ± 1048.18 2868.18 ± 1062.55 0.922 Duration of stimulation (day) 10.12 ± 1.50 10.09 ± 1.45 0.963 E2 level on hCG day (pg/ml) 2837.0 (2137.0, 5882.0) 2354.0 (2001.0, 2689.0) 0.151 No. of retrieved oocytes 13.53 ± 3.71 11.27 ± 4.52 0.161 Fertilization rate (%) 92.1 (210/228) 77.9 (95/122) <0.001 No. of top-quality embryos 3.22 ± 2.29 1.53 ± 2.88 0.042 Average number of embryos transferred 1.18 ± 0.39 1.18 ± 0.40 0.973 Clinical pregnancy rate (%) 58.8 (10/17) 36.4 (4/11) 0.246 Values are shown as mean ± SEM or median (IQR). Abbreviations: BMI, body mass index; AMH, anti-Müllerian hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; P, progesterone; GLU, glucose; INS, insulin; hCG, human chorionic gonadotropin. The fertilization rate was calculated as the number of oosperms divided by the number of oocytes used for fertilization. The top-quality embryos were blastocysts graded as AA, AB, BA, and BB Characteristics of the study participants Values are shown as mean ± SEM or median (IQR). Abbreviations: BMI, body mass index; AMH, anti-Müllerian hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; P, progesterone; GLU, glucose; INS, insulin; hCG, human chorionic gonadotropin. The fertilization rate was calculated as the number of oosperms divided by the number of oocytes used for fertilization. The top-quality embryos were blastocysts graded as AA, AB, BA, and BB We measured the expression of TRIB3 and Cx43 in all samples. Compared with those in group I, the expression levels of TRIB3 ( P  = 0.014) and Cx43 ( P  = 0.046) were increased in group II (Table  2 ; Fig.  1 ). Table 2 Comparison of mRNA expression in GCs from 28 participants mRNA expression in GCs Group I ( n  = 17) Group II ( n  = 11) P -value Cx43/GAPDH 1.09 ± 0.04 1.13 ± 0.07 0.046 TRIB3/GAPDH 1.21 ± 0.14 1.34 ± 0.13 0.014 Values are shown as mean ± SEM. Abbreviations: Cx43, Connexin43; TRIB3, tribble pseudokinase 3 Comparison of mRNA expression in GCs from 28 participants Values are shown as mean ± SEM. Abbreviations: Cx43, Connexin43; TRIB3, tribble pseudokinase 3 Fig. 1 mRNA expression in granulosa cells (GCs) from 28 women with infertility a Expression of Cx43 was increased in group II. b Expression of TRIB3 was increased in group II. Data are presented as mean ± SEM of values from three independent experiments. * P  < 0.05, compared with that in group I mRNA expression in granulosa cells (GCs) from 28 women with infertility a Expression of Cx43 was increased in group II. b Expression of TRIB3 was increased in group II. Data are presented as mean ± SEM of values from three independent experiments. * P  < 0.05, compared with that in group I KGN cells were treated with palmitate at concentration of 50, 100, and 200 µM for 24 h. We observed that palmitate treatment increased the percentage of apoptotic cells, as determined by flow cytometry following Annexin V-FITC and PI staining. Meanwhile, under the same dose of PA, TRIB3 overexpression significantly increased apoptotic cells, while its knockdown reduced apoptotic cells (Fig.  2 a, b). Following a 24 h exposure to 100 µM palmitate, KGN cells showed markedly decreased viability and proliferative potential. Overexpression of TRIB3 decreased proliferation and viability. On the other hand, these characteristics were restored by TRIB3 knockdown (Fig.  2 c-e). Fig. 2 Palmitate (PA) affects apoptosis, viability and proliferation in KGN cells. a Annexin V and propidium iodide staining, as detected by flow cytometry, is shown for cells treated with BSA, 50, 100 and 200 µM PA. The numbers shown in the lower or upper right indicate the percentage of early or late apoptotic cells, respectively. b All data are presented as mean ± SEM of values from three independent experiments. c Cell viability was assessed using the CCK-8 assay. d Quantification of EdU-positive cell percentage. e EdU incorporation assay with flow cytometry detection. * P  < 0.05 compared with that in control group; ** P  < 0.01 compared with that in control group; *** P  < 0.001 compared with that in the control group; **** P  < 0.0001 compared with that in control group Palmitate (PA) affects apoptosis, viability and proliferation in KGN cells. a Annexin V and propidium iodide staining, as detected by flow cytometry, is shown for cells treated with BSA, 50, 100 and 200 µM PA. The numbers shown in the lower or upper right indicate the percentage of early or late apoptotic cells, respectively. b All data are presented as mean ± SEM of values from three independent experiments. c Cell viability was assessed using the CCK-8 assay. d Quantification of EdU-positive cell percentage. e EdU incorporation assay with flow cytometry detection. * P  < 0.05 compared with that in control group; ** P  < 0.01 compared with that in control group; *** P  < 0.001 compared with that in the control group; **** P  < 0.0001 compared with that in control group We incubated KGN cells with palmitate at a gradient concentration of 0, 50, and 100 µM for 24 h and then measured their TRIB3 and Cx43 protein levels. Western blotting analyses showed that TRIB3 and Cx43 protein expression levels were significantly increased in the PA-treated group and exhibited concentration-dependent promotional effects (Fig.  3 a–c). To observe the association between the two proteins, we overexpressed and knocked down TRIB3 in KGN cells (Fig.  3 d–f) to determine whether it interferes with Cx43 expression. Under the same dose of PA, TRIB3 overexpression enhanced Cx43 expression, while its knockdown reduced Cx43 expression (Fig.  3 g, h). Immunofluorescence analysis revealed the same trend (Fig.  3 i, j). Cx43 was upregulated in TRIB3 -overexpressing cells and downregulated in TRIB3 -knockdown cells compared to that in control group. Fig. 3 Western blotting analysis and immunofluorescence revealed that palmitate (PA) induces TRIB3 and Cx43 expression in KGN cells. a Western blotting analysis was performed in KGN cells treated with PA. b PA treatment increased TRIB3 levels in a concentration-dependent manner. * P  < 0.05 compared with that in the control group; ** P  < 0.01 compared with that in the control group. c PA treatment increased Cx43 levels in a concentration-dependent manner. * P  < 0.05 compared with that in the control group; *** P  < 0.001 compared with that in the control group. d TRIB3 protein levels were determined using western blotting analysis in KGN cells with stable TRIB3 overexpression and knockdown of TRIB3 . e Quantification of TRIB3 protein levels in (d). * P  < 0.05 compared with that in the control group; ** P  < 0.01 compared with that in the control group. f TRIB3 mRNA levels were determined using qRT-PCR in KGN cells with stable TRIB3 overexpression and knockdown of TRIB3 . * P  < 0.05 compared with that in control group; *** P  < 0.001 compared with that in the control group. g Western blotting analysis was performed in TRIB3 -overexpressing KGN cells, TRIB3 -knockdown KGN cells, and control cells treated with PA. h TRIB3 overexpression increased the expression of Cx43, whereas TRIB3 knockdown reduced Cx43 expression levels. **** P  < 0.0001 compared with that in the control group. Data represent the mean ± SEM of values derived from three independent experiments. i Protein expression of TRIB3 in TRIB3 -overexpressing KGN cells, TRIB3 -knockdown KGN cells, and the control group in the presence or absence of 100 µM palmitate (PA). j Protein expression of Cx43 in TRIB3 -overexpressing KGN cells, TRIB3 -knockdown KGN cells, and the control group in the presence or absence of 100 µM PA. The cell membrane was stained with AlexaFluro488 (green), and cell nuclei were stained with DAPI (blue). All images were acquired at 630× magnification. Scale bar: 20 μm Western blotting analysis and immunofluorescence revealed that palmitate (PA) induces TRIB3 and Cx43 expression in KGN cells. a Western blotting analysis was performed in KGN cells treated with PA. b PA treatment increased TRIB3 levels in a concentration-dependent manner. * P  < 0.05 compared with that in the control group; ** P  < 0.01 compared with that in the control group. c PA treatment increased Cx43 levels in a concentration-dependent manner. * P  < 0.05 compared with that in the control group; *** P  < 0.001 compared with that in the control group. d TRIB3 protein levels were determined using western blotting analysis in KGN cells with stable TRIB3 overexpression and knockdown of TRIB3 . e Quantification of TRIB3 protein levels in (d). * P  < 0.05 compared with that in the control group; ** P  < 0.01 compared with that in the control group. f TRIB3 mRNA levels were determined using qRT-PCR in KGN cells with stable TRIB3 overexpression and knockdown of TRIB3 . * P  < 0.05 compared with that in control group; *** P  < 0.001 compared with that in the control group. g Western blotting analysis was performed in TRIB3 -overexpressing KGN cells, TRIB3 -knockdown KGN cells, and control cells treated with PA. h TRIB3 overexpression increased the expression of Cx43, whereas TRIB3 knockdown reduced Cx43 expression levels. **** P  < 0.0001 compared with that in the control group. Data represent the mean ± SEM of values derived from three independent experiments. i Protein expression of TRIB3 in TRIB3 -overexpressing KGN cells, TRIB3 -knockdown KGN cells, and the control group in the presence or absence of 100 µM palmitate (PA). j Protein expression of Cx43 in TRIB3 -overexpressing KGN cells, TRIB3 -knockdown KGN cells, and the control group in the presence or absence of 100 µM PA. The cell membrane was stained with AlexaFluro488 (green), and cell nuclei were stained with DAPI (blue). All images were acquired at 630× magnification. Scale bar: 20 μm To elucidate the effect of TRIB3 on gap junction formation, an LY (green) transfer experiment was performed. PA stimulation enhanced LY transfer, leading to an increased transfer distance of the small-molecule LY (Fig.  4 a–c). Moreover, TRIB3 -overexpressing cells showed a significant increase in LY transfer to adjacent cells, whereas TRIB3 -knockdown cells exhibited a reduced LY transfer extent. Fig. 4 Palmitate (PA) treatment increased the TRIB3-induced promotion of GJIC activity in KGN cells. a Fluorescence micrograph of Lucifer yellow (LY, green) staining. KGN cells were cultured for 24 h in the presence or absence of 100 µM PA. (Original magnification 20×). b The graph shows the distance of LY transfer from the cutting edge to the farthest cells during a 5 min incubation with 1 mg/ml LY. c The transmission distance was calculated by measuring the distance from the cut edge to the most distant cell layers with LY uptake. Bars represent the mean ± SEM of values from three independent experiments. * P  < 0.05, compared with the control Palmitate (PA) treatment increased the TRIB3-induced promotion of GJIC activity in KGN cells. a Fluorescence micrograph of Lucifer yellow (LY, green) staining. KGN cells were cultured for 24 h in the presence or absence of 100 µM PA. (Original magnification 20×). b The graph shows the distance of LY transfer from the cutting edge to the farthest cells during a 5 min incubation with 1 mg/ml LY. c The transmission distance was calculated by measuring the distance from the cut edge to the most distant cell layers with LY uptake. Bars represent the mean ± SEM of values from three independent experiments. * P  < 0.05, compared with the control To explore whether TRIB3 interferes with Cx43 expression via Wnt/β-catenin signaling, we measured β-catenin protein expression levels in KGN cells with stable TRIB3 overexpression and knockdown. In all cell groups, PA treatment led to an increase in β-catenin protein levels. Meanwhile, TRIB3 overexpression enhanced the protein expression of β-catenin, whereas TRIB3 knockdown reduced its protein expression, with or without PA treatment (Fig.  5 a, b). To determine whether the changes in β-catenin expression occurred at the protein or mRNA level, we performed qRT-PCR analysis. We found no change in mRNA expression, although knockdown and overexpression of TRIB3 resulted in a significant decrease and increase in the protein expression of β-catenin, respectively (Fig.  5 c). Fig. 5 Analysis of the Wnt/β-catenin signaling pathway following TRIB3 knockdown and overexpression in KGN cells. a Western blotting was performed on TRIB3 -overexpressing KGN cells, TRIB3 -knockdown KGN cells, and the control cells treated with PA. b TRIB3 overexpression increased the expression of β-catenin, whereas TRIB3 knockdown reduced β-catenin expression levels. * P  < 0.05 compared with that in the control group; ** P  < 0.01 compared with that in the control group; *** P  < 0.001 compared with that in the control group; **** P  < 0.0001 compared with that in the control group. c β-catenin mRNA levels were determined using qRT-PCR in KGN cells with stable TRIB3 overexpression and TRIB3 knockdown. Data represent the mean ± SEM of values from three independent experiments Analysis of the Wnt/β-catenin signaling pathway following TRIB3 knockdown and overexpression in KGN cells. a Western blotting was performed on TRIB3 -overexpressing KGN cells, TRIB3 -knockdown KGN cells, and the control cells treated with PA. b TRIB3 overexpression increased the expression of β-catenin, whereas TRIB3 knockdown reduced β-catenin expression levels. * P  < 0.05 compared with that in the control group; ** P  < 0.01 compared with that in the control group; *** P  < 0.001 compared with that in the control group; **** P  < 0.0001 compared with that in the control group. c β-catenin mRNA levels were determined using qRT-PCR in KGN cells with stable TRIB3 overexpression and TRIB3 knockdown. Data represent the mean ± SEM of values from three independent experiments Considering that the overexpression of TRIB3 altered β-catenin protein levels but not mRNA expression (Fig.  5 b, c), we speculated that TRIB3 may control β-catenin levels by regulating the stability of the protein through the ubiquitination/degradation system. Accordingly, the KGN cells stably overexpressing and those with low expression of TRIB3 were treated with cycloheximide to inhibit protein synthesis, and β-catenin protein levels were analyzed over time (Fig.  6 a). Compared with that in the control group, the degradation of β-catenin was considerably increased in TRIB3 -knockdown cells and significantly reduced in KGN cells stably overexpressing TRIB3 (Fig.  6 b). Next, the TRIB3 -knockdown cells were treated with a proteasome inhibitor (MG132). The suppression of β-catenin expression caused by TRIB3 knockdown was relieved via blockage of the proteasomal degradation pathway (Fig.  6 c, d). Overall, these results indicated that TRIB3 increased the protein stability of β-catenin by inhibiting protein degradation mediated by the ubiquitin/proteasomal pathway. Fig. 6 TRIB3 regulated β-catenin protein stability by affecting β-catenin ubiquitination levels. a KGN cells stably overexpressing TRIB3 were treated with cycloheximide (CHX, 20 umol/L) to inhibit protein synthesis, and β-catenin protein turnover was analyzed over time. Similarly, KGN cells with stable knockdown of TRIB3 were treated with CHX. Representative results from three independent experiments are shown. b Quantification of β-catenin protein levels in ( a ) (mean ± SEM of values from three independent experiments). c KGN cells with stable TRIB3 knockdown were treated with proteasome inhibitor (400 µM MG132) for 24 h. Western blotting was performed to detect the levels of β-catenin. Representative results from three independent experiments are shown. d Quantification of β-catenin protein levels in ( c ) (mean ± SEM of values from three independent experiments). * P  < 0.05 compared with that in the control group. e β-catenin-related immunoprecipitation was used to assess the levels of ubiquitinated β-catenin in KGN cells with stable knockdown of TRIB3 TRIB3 regulated β-catenin protein stability by affecting β-catenin ubiquitination levels. a KGN cells stably overexpressing TRIB3 were treated with cycloheximide (CHX, 20 umol/L) to inhibit protein synthesis, and β-catenin protein turnover was analyzed over time. Similarly, KGN cells with stable knockdown of TRIB3 were treated with CHX. Representative results from three independent experiments are shown. b Quantification of β-catenin protein levels in ( a ) (mean ± SEM of values from three independent experiments). c KGN cells with stable TRIB3 knockdown were treated with proteasome inhibitor (400 µM MG132) for 24 h. Western blotting was performed to detect the levels of β-catenin. Representative results from three independent experiments are shown. d Quantification of β-catenin protein levels in ( c ) (mean ± SEM of values from three independent experiments). * P  < 0.05 compared with that in the control group. e β-catenin-related immunoprecipitation was used to assess the levels of ubiquitinated β-catenin in KGN cells with stable knockdown of TRIB3 Ubiquitination-related immunoprecipitation was performed to assess ubiquitinated β-catenin levels. Results showed that the levels of ubiquitinated β-catenin were significantly increased in TRIB3 -knockdown cells compared to those in the control cells (Fig.  6 e). Collectively, these results suggested that TRIB3 regulated β-catenin protein stability by affecting β-catenin ubiquitination levels. To further verify whether TRIB3 regulates Cx43 expression via β-catenin signaling, we treated KGN cells overexpressing TRIB3 with the β-catenin inhibitor ICG-001. The results showed that ICG-001 treatment significantly reduced PA-induced upregulation of Cx43 protein levels. In TRIB3 -overexpression cells, PA further enhanced β-catenin and Cx43 levels, which were reversed by ICG‑001 (Fig.  7 a-c). This confirmed that TRIB3-mediated Cx43 upregulation is dependent on β-catenin signaling pathways. Fig. 7 ICG-001 blocks TRIB3 and PA-induced Wnt/β-catenin activation and Cx43 upregulation. a Western blotting was performed on TRIB3 -overexpressing KGN cells and the control cells treated with PA and with or without ICG-001. b TRIB3 overexpression or PA treatment increases β-catenin protein levels. ICG-001 did not significantly affect β-catenin expression (mean ± SEM of values from three independent experiments). c TRIB3 overexpression and PA treatment markedly increase Cx43 protein levels. This upregulation is effectively reversed by ICG-001. ns = not significant; ** P  < 0.01; *** P  < 0.001; **** P  < 0.0001 compared with corresponding group without ICG-001 ICG-001 blocks TRIB3 and PA-induced Wnt/β-catenin activation and Cx43 upregulation. a Western blotting was performed on TRIB3 -overexpressing KGN cells and the control cells treated with PA and with or without ICG-001. b TRIB3 overexpression or PA treatment increases β-catenin protein levels. ICG-001 did not significantly affect β-catenin expression (mean ± SEM of values from three independent experiments). c TRIB3 overexpression and PA treatment markedly increase Cx43 protein levels. This upregulation is effectively reversed by ICG-001. ns = not significant; ** P  < 0.01; *** P  < 0.001; **** P  < 0.0001 compared with corresponding group without ICG-001

Obesity is a major global public health concern; it affects both embryo developmental competence and the uterine microenvironment, resulting in reduced pregnancy and live birth rates in patients receiving artificial reproductive technologies [ 1 ]. Although previous in vitro and in vivo studies have suggested that the ovarian follicles exposed to high levels of free fatty acids have decreased oocyte developmental competence [ 2 , 3 ], the mechanism underlying free fatty acid-induced cytotoxicity in GCs, resulting in oocyte maturation failure, remains unclear. Our previous studies demonstrated that increased free fatty acid levels in the follicular fluid of women with obesity are associated with elevated tribbles pseudokinase 3 (TRIB3) expression and decreased oocyte developmental competence [ 4 ]. TRIB3 is a member of the mammalian tribble homologs, which coordinate cell stress in a context- and microenvironment-dependent manner, such as obesity and diabetes. It has been reported that increased expression of TRIB3 resulted in a significant reduction in the oocyte maturation rate in bovine cumulus cells [ 5 ]. Gap junctions of granulosa cells formed by connexins are crucial for oocyte development, as they facilitate the exchange of ions and molecules between the oocyte and the surrounding cumulus or granulosa cells (GCs). Connexin 43 (Cx43) is the most well-studied connexin in human oocyte cumulus complexes. During folliculogenesis, follicle-stimulating hormone (FSH) upregulates Cx43 expression, resulting in increased gap-junctional communication. Before ovulation, meiotic resumption is induced by the interruption of gap junctions, leading to a decrease in intra-oocyte cAMP concentrations. Consequently, the maturation-promoting factor is activated, and the oocyte completes the maturation process [ 6 ]. Accordingly, a decrease in the levels of Cx43 following a surge in luteinizing hormone (LH) levels is key in the initiation of oocyte meiotic resumption. It is worth noting that the GJA1 mRNA (encoding Cx43 protein) expression might be regulated by protein kinase A through the wingless-related integration site (Wnt)/ β-catenin pathway [ 7 , 8 ]. While, the regulation of WNT activity through the competitive binding patterns of TRIB homologs and β-catenin against COP1 E3 ligase has been reported [ 9 , 10 ]. As far as we know, the studies on the regulation of Cx43 expression by TRIB3 in the ovarian GCs are lacking. In this study, we aimed to investigate the role of elevated expression of TRIB3 in human GCs, which being supposed to regulate Cx43 expression through WNT pathway in follicle fluid with excessive fatty acid.

In the present study, we provide preliminary evidence of a correlation between TRIB3 and Cx43 expression mediated by Wnt/β-catenin signaling activity in human GCs. PA stimulation upregulated TRIB3 expression in KGN cells, accompanied by increased β-catenin protein and Cx43 levels. Overexpression of TRIB3 resulted in competitive inhibition of β-catenin degradation, further leading to an increase in β-catenin protein level, which may upregulate Cx43 expression. These findings suggest a regulatory axis involving TRIB3, β-catenin, and Cx43 in response to PA stimulation. Oocyte maturation is naturally triggered by the LH surge [ 7 ], which disrupts GC communication in the cumulus-oocyte complex by downregulating the expression of Cx43. This reduction in Cx43 decreases intra-oocyte cAMP concentrations, thereby contributing to the initiation of meiotic resumption [ 13 , 14 ]. Moreover, during embryonic development, downregulation of Cx43 is strongly correlated with embryo quality [ 15 ]. Consistently, it was reported oocyte maturation is associated with decreased expression of Cx43 in the surrounding cumulus cells of humans [ 16 , 17 ]. Obesity in women is frequently associated with ovarian dysfunction, such as polycystic ovary syndrome (PCOS). Studies have shown that Cx43 expression in oocytes and GCs of PCOS patients decreases, contributing to ovarian dysfunction by disrupting normal hormone secretion and follicle development [ 12 , 18 ]. However, a direct relationship between obesity and Cx43 expression in GCs has not yet been demonstrated. In this study, given the critical role of Cx43 in oocyte maturation, its dysregulation may contribute to the reduced fertilization rates and numbers of top-quality embryos observed in overweight or obese women. The apoptosis of GCs is a critical factor in follicular atresia and ovarian aging. TRIB3 plays a role in endoplasmic reticulum stress response, which can lead to apoptosis when excessive. Supporting this, He et al. [ 19 ] demonstrated that perfluorooctanoic acid (PFOA) promotes mouse pancreatic β-cell apoptosis by upregulating TRIB3 expression. In endometrial cancer cells, TRIB3 inhibits the Akt signaling pathway, enhancing cell apoptosis as demonstrated in TRIB3 overexpression and TRIB3 inhibition experiments [ 20 ]. Consistently, we observed PA-induced apoptosis in KGN cells. Notably, TRIB3 overexpression alone was sufficient to significantly increase the apoptotic rate even in the absence of PA, indicating a pro-apoptotic role of TRIB3 in granulosa cells. Furthermore, under PA stimulation, TRIB3 overexpression amplified the apoptotic effect. Conversely, TRIB3 knockdown reduced apoptosis under both basal and PA-treated conditions. Furthermore, our results demonstrated that TRIB3 not only affects apoptosis but also significantly impacts granulosa cell viability and proliferation. TRIB3 knockdown restored both viability and proliferation capacity under the same PA conditions. By inhibiting cell growth and promoting cell death, elevated TRIB3 may contribute to the impaired follicular development and oocyte competence observed in obesity-related infertility. This indicates that lower expression of TRIB3 has a protective effect on GCs against apoptosis under PA stimulation and it can be used as a potential target for preserving GC viability and improving ovarian function in obese women. Since the percentage of cell apoptosis increased dramatically at the dose of 200 µM PA, this concentration was excluded from subsequent experiments. Our findings also demonstrated that TRIB3 overexpression alone is sufficient to increase Cx43 mRNA and protein levels in KGN cells, indicating a direct regulatory role. Moreover, PA treatment also upregulated Cx43. Notably, when combined, PA and TRIB3 overexpression resulted in a greater increase in Cx43 expression and gap junction intercellular communication. Wnt/β-catenin signaling plays a crucial role in regulating Cx43 expression. Xia et al. [ 21 ] and Zhao et al. [ 8 ] reported that β-catenin binds to the Cx43 promoter directly, stimulating Cx43 expression and functional gap junctions in osteocytes or corpus cavernosum smooth muscle cells. In mouse GCs, knockdown of WNT2 or β-catenin significantly results in a considerable reduction in the expression of Cx43 and gap junction intercellular communication activity [ 22 ]. However, the specific regulatory effects and pathway of TRIB3 on Cx43 have not been previously reported. TRIB3 regulates cell proliferation, differentiation, and metabolism through various signaling pathways [ 23 ] including ubiquitination and proteasomal degradation of some transcription factors through its COP1 domain [ 24 ]. Based on previous studies, we hypothesized that TRIB3 functions as an upstream regulator of β-catenin to regulate the expression of Cx43 in human GCs [ 23 , 24 ]. COP1 is an evolutionarily conserved E3 ligase in the ubiquitin-proteasome system that negatively regulates β-catenin stability by promoting its ubiquitination and degradation. TRIB homologs have a consensus COP1-binding motif at their carboxyl terminus [ 25 ] and bind to the COP1 E3 ligase through their D/E/AQXVPD/E motifs, blocking the binding site for β-catenin attachment. Competitive binding between TRIB proteins and β-catenin with COP1 can regulate proteasomal degradation and protein half-life in the Wnt pathway [ 24 ]. In the present study, TRIB3 overexpression or knockdown in KGN cells resulted in significant changes in β-catenin protein levels, while β-catenin mRNA expression remained unchanged. This suggests that TRIB3 regulates β-catenin post-translationally rather than at the transcriptional level. Furthermore, we observed significantly increased ubiquitinated β-catenin levels in TRIB3 -knockdown cells, confirming that TRIB3 influences β-catenin stability through ubiquitination regulation. Chen Li et al. previously demonstrated direct TRIB3-β-catenin interactions using Co-IP and GST pull-down assays, supporting the biochemical basis of this regulatory mechanism. Furthermore, our additional experiments using the β-catenin inhibitor ICG-001 confirmed that the upregulation of Cx43 by TRIB3 is β-catenin-dependent. This functional verification strengthens the conclusion that TRIB3 regulates Cx43 expression through the Wnt/β-catenin signaling pathway. Our study is the first to report that TRIB3 plays a crucial role in mediating PA-induced upregulation of Cx43 through the Wnt/β-catenin signaling pathway in human GCs. Collectively, these results demonstrate that PA treatment increases TRIB3 expression, leading to reduced β-catenin degradation and Cx43 upregulation. These results suggest that controlling stress-induced elevation of TRIB3 expression under PA stimulation could be a novel approach to improving oocyte quality in overweight or obese women. However, this study has some limitations. One is the small patient cohort, which may not fully capture variability in metabolic or hormonal parameters. Additionally, our study was not powered to detect long-term fertility outcomes. Future studies should incorporate larger populations with longitudinal reproductive follow-up to comprehensively evaluate the clinical impact of TRIB3-Cx43 modulation. Additionally, as we conducted only in vitro experiments using KGN cells, further in vivo studies are necessary to validate our findings. Future research will focus on using a conditional TRIB3 knockout mouse model to examine the effects of TRIB3 on GC function and oocyte maturation. Additionally, we plan to explore pharmacological modulation of TRIB3 to assess its potential clinical implications. The therapeutic implications of TRIB3 modulation have been reported. Several studies have highlighted the role of TRIB3 modulation in cancer therapy. Notably, ABTL0812, a drug targeting TRIB3, has shown therapeutic potential in various cancers [ 26 – 28 ]. A first-in-human phase I/Ib clinical trial of ABTL0812 in patients with advanced solid tumors confirmed its safety and potential therapeutic benefit [ 29 ]. These findings provide substantial support for TRIB3 modulation as a promising therapeutic strategy, enhancing the relevance of our study.

In conclusion, we demonstrated that conditions of overweight and obesity may be associated with increased TRIB3 and Cx43 expression levels in female luteinized GCs. Excessive concentration of PA can disrupt the decrease in Cx43 expression following the surge in LH levels in the KGN cell line via the TRIB3/Wnt/β-catenin pathway. To the best of our knowledge, this is the first study to investigate the association between TRIB3 and Cx43 expression in GCs. This study introduces a novel concept for improving the quality of female oocytes and providing infertility therapy in the clinic.

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 1 Supplementary Material 2 Supplementary Material 2