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. 2021 Jul 1;105(5):1189–1204. doi: 10.1093/biolre/ioab128

The interleukin 6 trans-signaling increases prostaglandin E2 production in human granulosa cells†

Sai-Jiao Li 1,2,3, Hsun-Ming Chang 4, Jiamin Xie 5, Jeremy H Wang 6, Jing Yang 7,8,✉, Peter C K Leung 9,✉
PMCID: PMC8599049  PMID: 34198336

Abstract

As a potent autocrine regulator, the proinflammatory cytokine interleukin 6 (IL6) is expressed in granulosa cells and is involved in the modulation of various follicular functions, including follicular development and ovulation. At present, the detailed molecular mechanisms by which IL6 regulates the event of ovulation remain to be elucidated. In the present study, primary and immortalized (SVOG) human granulosa–lutein (hGL) cells were used to investigate the effects of IL6 on the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and the subsequent synthesis of prostaglandin E2 (PGE2) and to investigate the underlying molecular mechanisms. We found that instead of classic signaling, IL6/soluble form of the IL6 receptor (sIL-6Ralpha) trans-signaling induced the expression of PTGS2 and production of PGE2 in both SVOG cells and primary hGL cells. Moreover, IL6/sIL-6Ralpha activated the phosphorylation of Janus-activated kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), which in turn induced STAT3 nuclear translocation. In addition, these effects were suppressed by the addition of inhibitors (AG490 for JAK2 and C188-9 for STAT3) and by the small interfering RNA-mediated knockdown of STAT3. In addition, suppressor of cytokine signaling 3 (SOCS3) acts as a negative-feedback regulator in IL6/sIL-6Ralpha-induced cellular activities, including the activation and nuclear translocation of STAT3, upregulation of PTGS2 expression, and increase in PGE2 production in SVOG cells. In conclusion, IL6 trans-signaling upregulates the expression of PTGS2 and increases the production of PGE2 via the JAK2/STAT3/SOCS3 signaling pathway in hGL cells. Our findings provide insights into the molecular mechanisms by which IL6 trans-signaling may potentially modulate the event of ovulation in human ovaries.

Keywords: interleukin 6, IL6 trans-signaling, SOCS3, PTGS2, PGE2

This is a detailed study of the underlying molecular mechanisms by which interleukin 6 trans-signaling upregulates the expression of prostaglandin-endoperoxide 2 and increases prostaglandin E2 production in the human ovary.

Graphical Abstract

Graphical Abstract.

Graphical Abstract

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Introduction

In mammals, ovulation is a highly complex process that is controlled by multiple factors [1]. Prostaglandin (PG), especially prostaglandin E2 (PGE2), is a crucial factor for the ovulatory process [2]. The prostaglandin-endoperoxide synthase (PTGS) enzyme is the rate-limiting enzyme involved in the synthesis of PGs [3, 4]. Previous studies have demonstrated the important role of PTGS2 in the regulation of mammalian ovulatory event. For instance, the targeted disruption of the Ptgs2 gene in mice led to multiple female reproductive defects, including failures in ovulation, fertilization, implantation, and decidualization [3]. However, the administration of PGE2 could restore ovulation events in Ptgs2-deficient mice [5]. Given the critical roles of PTGS2 and its major derivative product PGE2 in the process of ovulation, studies regarding how the expression of PTGS2 is regulated have been a specific area of research interest.

Interleukin 6 (IL6) is a proinflammatory cytokine that has many biological effects on physiologic and pathogenic processes. In classical signaling, IL6 signals by binding to membrane-bound IL6R/gp130 complexes. Alternatively, IL6 can bind the soluble form of the IL6 receptor (sIL-6Ralpha); this process has been called IL6 trans-signaling. Indeed, IL6 trans-signaling has been observed in human serum and follicle fluids [6, 7]. Formation of either the IL6 classic or trans-signaling ligand–receptor complexes results in the homodimerization of the gp130 receptor, subsequently leading to the activation of multiple downstream signaling pathways, including the Janus kinase/signal transducer and activator of transcription (JAK/STAT), protein kinase B (also known as AKT), and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascades [7, 8]. Most cytokines exert their cellular activity through the JAK/STAT signaling pathway, and this pathway is negatively regulated by suppressor of cytokine signaling (SOCS) proteins. As classic negative-feedback regulators, SOCS proteins can be induced by cytokine stimulation [9]. At present, the functional roles of IL6 trans-signaling and SOCS proteins in human granulosa–lutein (hGL) cells are largely unknown. In the ovaries of rodents and humans, IL6 is highly expressed in cumulus-oopholus complexes (COCs) during ovulation, which promotes the maturation of oocytes and cumulus cells, leading to ovulation [8, 10, 11]. Moreover, information obtained from clinical studies showed that the concentrations of IL6 in follicular fluid were elevated in patients with polycystic ovary syndrome [12], endometriosis [13], and ovarian hyperstimulation syndrome [6, 14], indicating the involvement of IL6 in the pathogenesis of these ovarian disorders. Taken together, these studies suggest functional roles for granulosa cell-derived IL6 in the pathophysiological processes of follicular development, ovarian function, and female fertility [12, 15].

To date, whether PTGS2 contributes to IL6-mediated ovulation in humans remains unclear. In mouse COCs, IL6-induced COC expansion by upregulating the expression of PTGS2 [16]. In bovine granulosa cells, IL6 could promote the follicle-stimulating hormone (FSH)-induced upregulation of PTGS2 expression via the activation of the JAK/STAT3 signaling pathway [17]. Collectively, these findings prompted us to propose that granulosa cell-derived IL6 may combine with sIL-6Ralpha in the follicular fluid, which activates IL6 trans-signaling and induces the upregulation of PTGS2/PGE2 in an autocrine/paracrine manner during the periovulatory phase, with the aim of promoting ovulation. To test this hypothesis, we sought to examine the effects of IL6/sIL-6Ralpha trans-signaling on the expression of PTGS2 and the production of PGE2 and to examine the related underlying mechanisms in hGL cells.

Materials and methods

Culture of immortalized human granulosa cells

In the present study, both primary and immortalized hGL cells were used. SVOG cells, a nontumorigenic, immortalized hGL cell line, were previously produced by transfecting hGL cells with the SV40 large T antigen [18]; this cell line was generated from primary hGL cells and displays biological responses to many different treatments that are similar to those of primary hGL cells, as shown in our previous studies [19–22]. The SVOG cells were counted with a hemocytometer, and the cell viability was assessed by Trypan blue (0.04%) exclusion. The cells were seeded (4–8 × 105 cells per well in six-well plates) and cultured in a humidified atmosphere containing 5% CO2 and 95% air at 37°C in dulbecco's modified eagle medium/nutrient mixture F-12 (DMEM-F-12) supplemented with 10% charcoal/dextran-treated fetal bovine serum (HyClone), 100 U/ml penicillin (Life Technologies), 100 mg/ml streptomycin sulfate (Life Technologies) and 1× GlutaMAX (Life Technologies). The culture medium was changed every other day in all of the experiments, and the cells were maintained in serum-free medium for 24 h before treatment.

Preparation of primary hGL cells

Primary hGL cells were extracted and purified from in vitro fertilization patients’ follicular fluid, as previously described [23, 24]. All the patients provided informed consent according to the protocol that was approved by the University of British Columbia Research Ethics Board. The luteal-phase nafarelin acetate (Synarel, Pfizer, Kirkland, Quebec, Canada) or follicular phase gonadotropin-releasing hormone (GnRH) antagonist (Ganirelix; Merck, Frosst, Montreal, Canada) upregulation protocols were used for the in vitro fertilization patients. On day 2 of the menstrual cycle, gonadotrophins, including human menopausal gonadotrophin (Menopur, Ferring, Toronto, Ontario, Canada) and recombinant FSH (Puregon, Merck), were used to stimulate follicle growth until the follicular size reached the standard size of mature follicles. Then, human chorionic gonadotrophin (Pregnyl, Merck) was administered, and oocytes were retrieved 34–36 h after human chorionic gonadotropin (hCG) injection. Follicular samples were anonymized immediately after collection and individual primary cultures were comprised of cells from each individual patient (20 patients in total).

Antibodies and reagents

The polyclonal rabbit anti-PTGS2 antibodies [ab52237, Research Resource Identifier (RRID) AB_869240: diluted at 1:1000] were obtained from Abcam (Cambridge, MA). The monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (sc-365062, GAPDH; RRID AB_10847862: diluted at 1:2000) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal rabbit anti-p-STAT3 (9131, RRID AB_331586: diluted at 1:1000), monoclonal rabbit anti-STAT3 (4904, RRID AB_331269: diluted at 1:1000), monoclonal rabbit anti-p-JAK2 (3776, RRID AB_2617123: diluted at 1:1000), monoclonal rabbit anti-JAK2 (3230, RRID AB_2128522: diluted at 1:1000), monoclonal rabbit anti-SOCS3 (52113S, RRID AB_2799408, diluted at 1:500), polyclonal rabbit anti-phospho-AKTSer473 (9271, RRID AB_329825: diluted at 1:1000), polyclonal rabbit anti-AKT (9272, RRID AB_329827: diluted at 1:1000), monoclonal mouse anti-phospho-p44/42 MAPK (ERK1/2) Thr202/Tyr204 (9106, RRID AB_331768: diluted at 1:1000), and polyclonal rabbit anti-p44/42 MAPK (ERK1/2) (9102, RRID AB_330744: diluted at 1:1000) antibodies were obtained from Cell Signaling Technology (Danvers, MA). The horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit immunoglobulin G (IgG) were purchased from Bio-Rad Laboratories (Richmond, CA, USA). The recombinant human IL6 protein and recombinant human IL-6R alpha protein were purchased from R&D Systems (Minneapolis, MN, USA). Tyrphostin AG-490 and C188-9 were purchased from Cell Signaling Technology and Sigma, respectively.

Reverse transcription quantitative real-time polymerase chain reaction

Total RNA was extracted with TRIzol Reagent (Life Technologies) according to the manufacturer’s instructions. RNA (2 μg) was reverse transcribed into first-strand complementary DNA (cDNA) with random primers and moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI, USA). Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was performed on the Applied Biosystems 7300 Real-Time PCR System in 96-well optical reaction plates. Each 20 μl RT-qPCR reaction contained 1× SYBR Green PCR Master Mix (Applied Biosystems), 20 ng of cDNA, and 250 nM of each specific primer. The primers used were as follows: PTGS2:5′-CCCTTGGGTGTCAAAGGTAA-3′ (sense) and 5′-GCCCTCGCTTATGATCTGTC-3′ (antisense); STAT3: 5′-GTGATGCTTCCC TGATTGTG-3′ (sense) and 5′-CAAGGAGTGGGTCTCTAGG-3′ (antisense); SOCS3: 5′-CTCCAAGAGCGAGTACCA-3′ (sense) and 5′-GTTCTTGGTCCCAGACTG-3′ (antisense); and GAPDH: 5′-GAGTCAACGGATTTTGGTCGT-3′ (sense) and 5′-GACAAGCTTCCCGTTCTCAG-3′ (antisense). The specificity of each assay was validated by dissociation curve analysis. The assay performance was validated by evaluating the amplification efficiencies utilizing calibration curves, ensuring that the plot of the log input amount versus the ΔCq (also known as ΔCt) had a slope <|0.1|. The PCR parameters for the reaction were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Three separate experiments were performed on different cultures, and each sample was assayed in triplicate. A mean value was used to determine the messenger RNA (mRNA) levels by the comparative ΔCq (ΔCt) method using the formula 2−ΔΔCq (2−ΔΔCt) with GAPDH as the reference gene.

Western blotting

The cells were lysed in cell lysis buffer (Cell Signaling). Equal amounts (30 μg) of protein were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinyl difluoride membranes. After 1 h of blocking with 5% skim milk in tris-buffered saline, the membranes were incubated overnight at 4°C with the primary antibodies, which were diluted in 5% skim milk/tris-buffered saline. After the primary antibody incubation, the membranes were incubated with the appropriate HRP-conjugated secondary antibody for 1 h. The immunoreactive bands were detected using an enhanced chemiluminescent substrate.

Immunofluorescence staining

The SVOG cells were washed three times with phosphate-buffered saline (PBS) after IL6/sIL-6Ralpha treatment and fixed with 100% methanol for 20 min. The eells were washed for three times with 0.1 M glycine in PBS and incubated with a rabbit anti-p-STAT3 antibody (diluted at 1:200) overnight at 4°C. After the removal of the primary antibody, the cells were washed three times with detergent buffer (0.01% Tween-20 in PBS) and incubated with a goat anti-rabbit IgG-Alexa Fluor 594 antibody (Abcam Inc., Cambridge, MA, USA; diluted at 1:500). Then the membranes were incubated for 1 h at room temperature under light-protected conditions. The cells were washed for three times with PBS, and the cell nuclei were stained with 406-diamidino-2-phenylindole dihydrochloride (DAPI, 0.1 μg/ml in PBS) in a dark room for 5 min. The plates were stored at 4°C until imaging. The images were captured using a fluorescence microscope, and the fluorescence intensity was analyzed by IMAGEJ software (US National Institutes of Health, Bethesda, MD, USA). To confirm the subcellular localization, DAPI staining was used as an approach to stain the nuclei. Overlapping the Alexa Fluor 594 antibody signal with both the nuclei structure and the DAPI signals confirms the p-STAT3 nuclear localization changes after treatment. When portions of images are measured, the value of each pixel being measured was recorded, and the number of pixels that were measured was recorded. When measuring an image, we recorded the area measured, the mean fluorescence value in that area, and the total fluorescence measured. Total fluorescence intensity is the sum of the intensity of every pixel measured. The image measurement is the mean fluorescence intensity per pixel (mean fluorescence intensity = total fluorescence intensity measured/number of pixels measured), as it is the total fluorescence intensity divided by the number of pixels measured. Regarding the calculation of nuclear localization, we calculated the ratio of the nuclear mean fluorescence to the cytoplasmic mean fluorescence in the treatment group versus in the control group.

Small interfering RNA transfection

We performed transient knockdown assays with an ON-TARGET plus nontargeting control small interfering RNA (siRNA) pool or with separate ON-TARGET plus SMART pools targeting STAT3 and SOCS3 (Dharmacon, GE Healthcare Life Sciences). The cells were precultured in antibiotic-free DMEM/F-12 medium containing 10% charcoal/dextran-treated fetal bovine serum until they were 60–70% confluent, and then they were transfected with 25 nM siRNA using Lipofectamine RNAi MAX and Opti-MEM (Life Technologies) for 24 or 48 h.

Measurement of secreted PGE2

The cells were cultured in a six-well plate with 2 ml medium and then incubated with IL6/sIL-6Ralpha at the indicated concentrations for the indicated time. The culture media were collected, and the PGE2 levels in the culture media were measured by a human PGE2 ELISA kit (Cayman Chemical) according to the manufacturer’s instructions. The PGE2 levels were normalized to the protein concentrations in the cell lysates. The normalized PGE2 values were determined by comparing the values from the treatment groups with those from the control group and are presented as relative values.

Statistical analysis

Data are presented as the mean ± standard deviation of at least three independent experiments. The GraphPad Prism statistical software package (version 6) was used to analyze all the measured parameters. Multiple comparisons were analyzed first by one-way analysis of variance followed by Tukey’s multiple-comparison tests. Values are considered significant when P ˂ 0.05.

Results

A combination of IL6 and sIL-6Ralpha (IL6 trans-signaling) induced an increase in PTGS2 expression in hGL cells

To investigate the effect of IL6 on the expression of PTGS2 in human granulosa cells (GCs), we first used SVOG cells as a model to examine the concentration-dependent effect of IL6 on the expression of PTGS2. The SVOG cells were treated with vehicle control or various concentrations (from 0.001 to 50 ng/ml) of IL6 for 12 h. However, the mRNA levels of PTGS2 were not different between the control and treatment groups (Figure 1A, n = 4). We then utilized different combinations of IL6 and sIL-6Ralpha (0/25, 0.1/25, 1/25, 10/25, or 20/25 ng/ml) for 12 h, and the results showed that IL6/sIL-6Ralpha significantly increased the mRNA (Figure 1B, n = 4) and protein (Figure 1D, n = 4) levels of PTGS2 in a concentration-dependent manner. The greatest effect was achieved with concentrations of 10 ng/ml IL6 and 25 ng/ml sIL-6Ralpha (Figure 1B and D). Therefore, we used this combination to perform the following experiments. The time-course experiments showed that treating SVOG cells with 10/25 ng/ml IL6/sIL-6Ralpha significantly increased the mRNA (Figure 1C, n = 4) and protein (Figure 1E, n = 3) levels of PTGS2, and the effects started at 6 h and persisted until 24 h after treatment. To increase the physiological relevance of the results, we next used primary hGL cells to confirm the effects of IL6 on PTGS2 expression. Consistent with the results obtained in SVOG cells, treatment with IL6/sIL-6Ralpha (10/25 or 20/25 ng/ml) for 12 h (Figure 1F, n = 5) and 24 h (Figure 1G, n = 4) significantly increased the mRNA and protein levels of PTGS2 in primary hGL cells. These findings indicate that IL6 trans-signaling, but not classic IL6R signaling, is the principal signaling pathway by which IL6 upregulates the expression of PTGS2 in hGL cells.

Figure 1.

Figure 1

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Effects of combined IL6 and sIL-6Ralpha on PTGS2 expression in human granulosa–lutein cells. (A) SVOG cells (n = 4) were treated with vehicle control or various concentrations (0.001, 0.01, 0.1, 1, 10, or 50 ng/ml) of IL6 for 12 h, and the mRNA levels of PTGS2 were examined using RT-qPCR. (B) SVOG cells (n = 4) were treated with vehicle control or various combinations of IL6 and sIL-6Ralpha (IL6/sIL-6Ralpha: 0/25, 0.1/25, 1/25, 10/25, or 20/25 ng/ml) for 12 h, and the mRNA levels of PTGS2 were examined using RT-qPCR. (C) SVOG cells (n = 4) were treated with vehicle control or a combination of 10 ng/ml IL6 and 25 ng/ml sIL-6Ralpha (IL6/sIL-6Ralpha), and the mRNA levels of PTGS2 were examined at various time points (3, 6, 12, or 24 h) using RT-qPCR. (D) SVOG cells (n = 4) were treated with IL6/sIL-6Ralpha (0/25, 1/25, 10/25, or 20/25 ng/ml) for 24 h, and the protein levels of PTGS2 were examined using western blot analysis. (E) SVOG cells (n = 3) were treated with 10/25 ng/ml IL6/sIL-6Ralpha for 6, 12, or 24 h, and the protein levels of PTGS2 were examined using western blot analysis. (F and G) Primary hGL cells (n = 5) were treated with vehicle control or various concentrations (1/0, 10/0, 20/0, 1/25, 10/25, or 20/25 ng/ml) of IL6/sIL-6Ralpha for 12 or 24 h, and the mRNA (F, n = 5) and protein (G, n = 4) levels of PTGS2 were examined using RT-qPCR and western blot analysis, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked with different letters are significantly different (P < 0.05). Ctrl, control.

IL6/sIL-6Ralpha activated the JAK2 and STAT3 signaling pathways, but not the AKT and ERK1/2 signaling pathways in SVOG cells

To identify the detailed mechanism by which IL6/sIL-6Ralpha induced the expression of PTGS2, we investigated several intracellular signal transduction molecules (including JAK, STAT3, AKT, and ERK1/2) that have been reported to mediate IL6/sIL-6Ralpha-induced cellular activities [25–27]. To this end, SVOG cells were treated with IL6/sIL-6Ralpha (10/25 ng/ml) for 15, 30, or 60 min, and the levels of the phosphorylated AKT, ERK1/2, STAT3, and JAK2 proteins were examined using western blot analysis. The results showed that IL6/sIL-6Ralpha did not increase the levels of the phosphorylated AKT (Figure 2A, n = 3) and ERK1/2 (Figure 2B, n = 3) proteins at any of the examined time points. However, the treatment of SVOG cells with 10/25 ng/ml IL6/sIL-6Ralpha for 15, 30, and 60 min rapidly increased the levels of the phosphorylated STAT3 (Figure 2C, n = 3) and JAK2 (Figure 2D, n = 3) proteins. These results indicate the IL6 trans-signaling can activate the STAT3 and JAK2 signaling pathways in SVOG cells.

Figure 2.

Figure 2

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Effects of IL6/sIL-6Ralpha on AKT, ERK1/2, JAK2, and STAT3 signaling activity in SVOG cells. (A–D) SVOG cells were treated with vehicle control or 10 ng/ml IL6 and 25 ng/ml sIL-6Ralpha for various time points (15, 30, or 60 min), and the levels of the phosphorylated AKT (A, n = 3), ERK1/2 (B, n = 3), JAK2 (C, n = 3), or STAT3 (D, n = 3) proteins were examined using western blot analysis. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked with different letters are significantly different (P < 0.05).

IL6/sIL-6Ralpha induced the phosphorylation of JAK2 and STAT3 and promoted the nuclear translocation of p-STAT3 in SVOG cells

We then used the JAK2-specific inhibitor AG490 [28] and the STAT3 inhibitor C188-9 [29] to determine the relevance of JAK2 and STAT3 signaling pathway and its biological response to extracellular stimuli. Notably, the addition of the JAK2 inhibitor AG490 (50 μM, pretreatment for 1 h) abolished the increase in the levels of the phosphorylated JAK2 and STAT3 proteins induced by IL6/sIL-6Ralpha in SVOG cells (Figure 3A, n = 4). Furthermore, the addition of the STAT3 inhibitor C188-9 (10 μM, pretreatment for 1 h) completely abolished the increase in the levels of the phosphorylated STAT3 protein induced by IL6/sIL-6Ralpha in SVOG cells (Figure 3B, n = 3).

Figure 3.

Figure 3

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Effects of the AG490 and C188-9 inhibitors on the IL6/sIL-6Ralpha-induced increase in phosphorylated JAK2 and STAT3 in SVOG cells. (A) SVOG cells (n = 4) were pretreated for 1 h with DMSO (vehicle control) or the JAK inhibitor AG490 (50 μM) and then treated with 10/25 ng/ml IL6/sIL-6Ralpha for an additional 30 min. The levels of the phosphorylated JAK2 and STAT3 proteins were examined using western blot analysis. (B) SVOG cells (n = 3) were pretreated for 1 h with DMSO or the STAT3 inhibitor C188-9 (10 μM) and then treated with 10/25 ng/ml IL6/sIL-6Ralpha for an additional 30 min. The levels of the phosphorylated STAT3 protein were examined using western blot analysis. (C) SVOG cells were pretreated with DMSO, 50 μM AG490, or 10 μM C188-9 for 1 h and then treated with IL6/sIL-6Ralpha for an additional 30 min. (C) The localization of the phosphorylated STAT3 protein was examined using immunofluorescence microscopy. The scale bar represents 100 μm. The nuclear presence of phosphorylated STAT3 (n = 6) was quantified and analyzed by the amount of the immunofluorescence intensity. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked with different letters are significantly different (P < 0.05). Ctrl, control.

Upon activation and phosphorylation, STAT3 can dimerize and translocate to the nucleus, where it binds to consensus STAT3-binding sequences within the promoter regions of its target genes [30]. To determine the effect of IL6 trans-signaling on the cellular localization of p-STAT3 in SVOG cells, immunofluorescence analysis was carried out. As shown in Figure 3C, treatment of SVOG cells with 10/25 ng/ml IL6/sIL-6Ralpha significantly promoted p-STAT3 nuclear translocation. Similarly, the addition of either the JAK2 inhibitor AG490 (50 μM, pretreatment for 1 h) or the STAT3 inhibitor C188-9 (10 μM, pretreatment for 1 h) attenuated the IL6/sIL-6Ralpha-induced p-STAT3 nuclear translocation in SVOG cells (Figure 3C, n = 6).

The activation of JAK2/STAT3 was required for the IL6/sIL-6Ralpha-induced upregulation of PTGS2 expression in SVOG cells

To investigate the involvement of JAK2/STAT3 signaling in the IL6/sIL-6Ralpha-induced upregulation of PTGS2 expression, SVOG cells were pretreated with AG490 (50 μM) or C188-9 (10 μM) for 1 h prior to IL6/sIL-6Ralpha stimulation for 12 or 24 h. As shown in Figure 4A and B, the increase in the mRNA (Figure 4A, n = 3) and protein (Figure 4B, n = 3) levels of PTGS2 induced by IL6/sIL-6Ralpha was abolished by the addition of AG490. Similarly, the addition of C188-9 abolished the IL6/sIL-6Ralpha-induced increase in the mRNA (Figure 4C, n = 4) and protein (Figure 4D, n = 3) levels of PTGS2. These results indicate that the activation of JAK2/STAT3 signaling is required for the IL6/sIL-6Ralpha-induced upregulation of PTGS2 expression in SVOG cells.

Figure 4.

Figure 4

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Effects of the AG490 and C188-9 inhibitors and the knockdown of STAT3 on the IL6/sIL-6Ralpha-induced increase in PTGS2 expression in SVOG cells. (A–D) SVOG cells were pre-pretreated with DMSO, 50 μM AG490 (A, n = 3, and B, n = 3), or 10 μM C188-9 (C, n = 4, and D, n = 3) for 1 h and then treated with IL6/sIL-6Ralpha for an additional 24 h. The mRNA (A and C) and protein (B and D) levels of PTGS2 were examined using RT-qPCR and western blot analysis, respectively. (E) SVOG cells (n = 3) were transfected with 25 nM siCtrl or 25 nM siSTAT3 for 24 h, and the protein levels of STAT3 were examined using western blot analysis. (F and G) SVOG cells were transfected with 25 nM siCtrl or siSTAT3 for 24 h and then treated with 10/25 ng/ml IL6/sIL-6Ralpha for an additional 24 h. The mRNA (F, n = 3) and protein (G, n = 3) levels of PTGS2 and STAT3 were examined using RT-qPCR and western blot analysis, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked with different letters are significantly different (P < 0.05).

To further investigate the involvement of STAT3 signaling in the IL6/sIL-6Ralpha-induced PTGS2 regulation, siRNAs specifically targeting STAT3 were used to knockdown endogenous STAT3 in SVOG cells. The knockdown efficiency and specificity were examined using western blot analysis, and the results showed that the knockdown of STAT3 significantly decreased the protein levels of STAT3 (Figure 4E, n = 3). Notably, the knockdown of endogenous STAT3 completely abolished the IL6/sIL-6Ralpha-induced increase in the mRNA (Figure 4F, n = 3) and protein (Figure 4G, n = 3) levels of PTGS2 in SVOG cells, indicating that STAT3 signaling is involved in the IL6/sIL-6Ralpha-mediated induction of PTGS2 expression in SVOG cells.

IL6/sIL-6Ralpha increases PGE2 production via the JAK2/STAT3 signaling pathway

Previous studies have shown that PGE2 is the major product of the PTGS2 enzyme in hGL cells [31–33]. To further investigate whether the IL6/sIL-6Ralpha-induced upregulation of PTGS2 expression contributes to the increase in PGE2 production, we treated SVOG cells with vehicle control or 10/25 ng/mL of IL6/sIL-6Ralpha for 24 h, and the accumulated levels of PGE2 in the culture medium were examined using an enzyme immunoassay. As shown in Figure 5A (n = 6), IL6/sIL-6Ralpha significantly increased the accumulated levels of PGE2 in SVOG cells. In addition, pretreatment with AG490 (Figure 5B, n = 4) or C188-9 (Figure 5C, n = 3) entirely abolished the IL6/sIL-6Ralpha-induced increase in the PGE2 levels in SVOG cells. Moreover, the transfection of cells with STAT3 siRNA for 24 h completely abolished the IL6/sIL-6Ralpha-induced increase in the PGE2 levels (Figure 5D, n = 4). Consistent with the results obtained in SVOG cells, the stimulatory effect of IL6/sIL-6Ralpha on PGE2 production was confirmed in primary hGL cells (Figure 5E, n = 4). These results indicate that the IL6/sIL-6Ralpha-induced upregulation of PTGS2 expression and the increase in PGE2 production in hGL cells are mediated via the JAK2/STAT3 signaling pathway.

Figure 5.

Figure 5

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The effect of IL6/sIL-6Ralpha, AG490, and C188-9 on the production of PGE2 in human granulosa–lutein (hGL) cells. (A) SVOG cells (n = 6) were treated with vehicle control or a combination of 10 ng/ml IL6 and 25 ng/ml sIL-6Ralpha (IL6/sIL-6Ralpha) for 24 h, and the accumulated levels of PGE2 in the conditioned medium were examined using an enzyme immunoassay. (B and C) SVOG cells were pretreated for 1 h with DMSO, 50 μM AG490 (B, n = 4), or 10 μM C188-9 (C, n = 3), followed by treatment with vehicle control or a combination of 10 ng/ml IL6 and 25 ng/ml sIL-6Ralpha (IL6/sIL-6Ralpha) for an additional 24 h. The accumulated levels of PGE2 in the conditioned medium were examined using an enzyme immunoassay. (D) SVOG cells (n = 4) were transfected with 25 nM siCtrl or 25 nM siSTAT3 for 24 h, followed by treatment with vehicle control or IL6/sIL-6Ralpha (10/25 ng/ml) for an additional 24 h. The accumulated levels of PGE2 in the conditioned medium were examined using an enzyme immunoassay. (E) Primary hGL cells (n = 4) were treated with vehicle control or a combination of 10 ng/ml IL6 and 25 ng/ml sIL-6Ralpha (IL6/sIL-6Ralpha) for 24 h, and the accumulated levels of PGE2 in the conditioned medium were examined using an enzyme immunoassay. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked with different letters are significantly different (P < 0.05).

IL6/sIL-6Ralpha upregulated the expression of SOCS3 in hGL cells

To further elucidate the regulatory mechanism by which IL6/sIL-6Ralpha upregulates the expression of PTGS2 in hGL cells, we sought to investigate whether IL6/sIL-6Ralpha can induce the expression of SOCS3 in both SVOG cells and primary hGL cells. As shown in Figure 6A and B, treatment with 10/25 and 20/25 ng/ml IL6/sIL-6Ralpha for 12 h significantly increased the mRNA and protein levels of SOCS3 (Figure 6A, n = 3, Figure 6B, n = 4, Figure 6E, n = 5, and Figure 6F, n = 3) in both SVOG cells and primary hGL cells. In addition, the time-course experiments showed that the treatment of SVOG cells with 10/25 ng/ml IL6/sIL-6Ralpha for 3, 6, 12, or 24 h significantly increased the mRNA (Figure 6C, n = 4) and protein (Figure 6D, n = 3) levels of SOCS3.

Figure 6.

Figure 6

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Effect of combined IL6 and sIL-6Ralpha on the expression of SOCS3 in hGL cells. (A) SVOG cells (n = 3) were treated with vehicle control or various combinations of IL6 and sIL-6Ralpha (IL6/sIL-6Ralpha: 0/25, 0.1/25, 1/25, 10/25, or 20/25 ng/ml) for 12 h, and the mRNA levels of SOCS3 were examined using RT-qPCR. (B) SVOG cells (n = 4) were treated with IL6/sIL-6Ralpha (0/25, 1/25, 10/25, or 20/25 ng/ml) for 24 h, and the protein levels of SOCS3 were examined using western blot analysis. (C and D) SVOG cells were treated with vehicle control or 10/25 ng/ml IL6/sIL-6Ralpha for 12 h, and the mRNA (C, n = 4) and protein (D, n = 3) levels of SOCS3 were examined at various time points (3, 6, 12, or 24 h) using RT-qPCR and western blot analysis, respectively. (E and F) Primary hGL cells were treated with vehicle control or various combinations of IL6 and sIL-6Ralpha (IL6/sIL-6Ralpha: 0/25, 1/25, 10/25, or 20/25 ng/ml) for 12 h (E, n = 5) or 24 h (F, n = 3), and the mRNA (E) and protein (F) levels of SOCS3 were examined using RT-qPCR and western blot analysis, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked with different letters are significantly different (P < 0.05).

SOCS3 is the major regulator that negatively regulates IL6/sIL-6Ralpha-induced increase in PTGS2 expression and PGE2 production in SVOG cells

To further ascertain the role that SOCS3 plays in regulating the IL6/sIL-6Ralpha-induced cellular activities in SVOG cells, we transfected SVOG cells with SOCS3 siRNA to knockdown the endogenous SOCS3. RT-qPCR was used to validate the knockdown effect of SOCS3 siRNAs, and the results showed that knockdown of SOCS3 for 24 or 48 h resulted in a significant decrease in the mRNA levels of SOCS3 (Figure 7A, n = 3). In particular, the knockdown of SOCS3 did not affect the phosphorylation of JAK2 in response to IL6/sIL-6Ralpha (Figure 7B, n = 5). However, the knockdown of SOCS3 significantly increased the levels of the phosphorylated STAT3 protein in response to IL6/sIL-6Ralpha (Figure 7C, n = 4). Similarly, the immunofluorescence analysis showed that the knockdown of SOCS3 increased the nuclear translocation of p-STAT3 compared with that observed after transfection with control siRNA (Figure 7D, n = 5). Most importantly, the knockdown of SOCS3 further enhanced the IL6/sIL-6Ralpha-induced increase in PTGS2 expression (Figure 7E, n = 4) and PGE2 production (Figure 7F, n = 4) in SVOG cells. Taken together, these results indicate that the SOCS3 protein acts as a negative-feedback regulator of IL6/sIL-6Ralpha-induced cellular activities, including the activation and nuclear translocation of STAT3, upregulation of PTGS2 expression, and increase in PGE2 production, in immortalized hGL (SVOG) cells (Figure 8).

Figure 7.

Figure 7

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Effects of knocking down SOCS3 on the IL6/sIL-6Ralpha-induced increase in phosphorylated JAK2, phosphorylated STAT3, PTGS2 expression, and PGE2 production in SVOG cells. (A) SVOG cells (n = 3) were transfected with 25 nM siCtrl or 25 nM siSOCS3 for 24 or 48 h, and the mRNA levels of SOCS3 were examined via RT-qPCR. (B and C) SVOG cells were transfected with 25 nM siCtrl or 25 nM siSOCS3 for 24 h and then treated with vehicle control or IL6/sIL-6Ralpha (10/25 ng/ml) for an additional 30 min. The levels of the phosphorylated JAK2 (B, n = 5) and STAT3 (C, n = 4) proteins were examined using western blot analysis. (D) SVOG cells (n = 5) were transfected with 25 nM siCtrl or 25 nM siSOCS3 for 24 h and then treated with 10/25 ng/mL of IL6/sIL-6Ralpha for an additional 30 min. The localization of phosphorylated STAT3 was examined using immunofluorescence microscopy. The scale bar represents 100 μm. The nuclear levels of STAT3 was quantified and analyzed by the immunofluorescence intensity. (E and F) SVOG cells were transfected with 25 nM siCtrl or 25 nM siSOCS3 for 24 h and then treated with 10/25 ng/ml IL6/sIL-6Ralpha for an additional 24 h. The protein levels of PTGS2 (E, n = 4) and the accumulated levels of PGE2 in the conditioned medium (F, n = 4) were examined using western blot analysis and an enzyme immunoassay, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked with different letters are significantly different (P < 0.05).

Figure 8.

Figure 8

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Schematic diagram of the proposed molecular mechanism by which IL6/sIL-6Ralpha upregulates the expression of PTGS2 and the production of PGE2 in immortalized hGL cells. Treatment with IL6 alone does not lead to homodimerization of gp130 and activation of JAK2 tyrosine kinase, whereas the IL6/sIL-6Ralpha complex leads to homodimerization of gp130 on the cell surface, which subsequently transduces a signal that activates intracytoplasmic JAK2. However, the AKT and ERK1/2 signaling pathways are not activated by IL6/sIL-6Ralpha. JAK tyrosine kinase preferentially induces tyrosine phosphorylation of STAT3, which translocates to the nucleus and subsequently leads to the stimulation of PTGS2 expression and an increase in PGE2 production in hGL cells. Furthermore, as the negative feedback inhibitor of the JAK2/STAT3 signaling pathway, SOCS3 is induced by the JAK2/STAT3 signaling pathway, which suppresses the phosphorylation of STAT3 and subsequently affects the expression of PTGS2 and production of PGE2 in hGL cells. gp130, glycoprotein 130; PTGS2, cyclooxygenase enzyme 2.

Discussion

Interleukins and PGs, which play predominant roles in inflammation, have long been recognized as principal mediators of mammalian ovulation [34]. In the current study, we present data that show, for the first time, that IL6/sIL-6Ralpha trans-signaling, but not IL6 classic signaling, induces an increase in the production of PGE2 in both primary and immortalized hGL cells. This biological effect is most likely achieved by the upregulation of PTGS2 expression. Using immortalized hGL (SVOG) cells as the study model, we further demonstrated that IL6 trans-signaling-induced cellular activities most likely occur via the activation of the JAK2/STAT3 signaling pathway. In various mammalian cells, IL6 has been reported to engage in classic and trans-signaling downstream pathways to achieve its biological functions. IL6 can signal by binding to either membrane-bound IL-6R (classic signaling) or sIL-6Ralpha (trans-signaling). The results obtained from our study clearly demonstrated that IL6 trans-signaling, but not classic IL6 signaling, is the most possible signaling pathway that mediates the IL6-induced cellular activities in SVOG cells. During the late preovulatory stage, the levels of IL6 and sIL-6Ralpha steadily increased in the ovaries, especially during the process of ovulation stimulation, in which sIL-6Ralpha acts as a key factor that orchestrates the cascade of molecular events leading to ovarian hyperstimulation syndrome [14]. In addition, evidence obtained from clinical studies showed that the concentrations of IL6 and sIL-6Ralpha were significantly higher in follicular fluid containing mature oocytes than in follicular fluid containing immature oocytes [7], indicating that IL6 and sIL-6Ralpha could potentially be applied as biochemical markers for the evaluation of oocyte maturation. Taken together, previous studies and our results suggest that during the periovulatory stage, IL6 trans-signaling, but not IL6 classic signaling, most likely contributes to the event of ovulation in humans. However, there are limitations in the present study as all the results were obtained from in vitro experiments using cultured cell models. Moreover, the cells that we used were luteinized granulosa cells, and the morphology and gene expression profile of these cells could have been changed because of luteinization. Future in vivo studies performed using animal models or human samples will be needed to help us generate more information regarding to the pathophysiological function of IL6 trans-signaling in the human ovary.

A comprehensive understanding of the underlying molecular mechanism by which IL6 trans-signaling regulates the production of PGE2 may be advantageous for the development of novel therapeutic strategies for ovarian disorders. However, despite the apparent roles of IL6 and PGE2 in mammalian ovulation, the detailed molecular mechanisms responsible for mediating this biological function remain to be defined. To investigate the intracellular signal transduction molecules downstream of IL6/sIL-6Ralpha trans-signaling, we examined the levels of the phosphorylation of several previously reported protein targets, including JAK/STAT3, AKT, and ERK1/2 [8, 27, 35]. Our results showed that the treatment of SVOG cells with IL6/sIL-6Ralpha significantly increased the levels of both phosphorylated JAK2 and STAT3. As an important member of the JAK family, JAK2 is a nonreceptor tyrosine kinase that is subsequently linked to the constitutive phosphorylation of another intracellular signal transducer STAT3 [36]. Upon phosphorylation, STAT3 dimerizes and then translocates to the nucleus, where it binds to consensus STAT3-binding sequences within the promoter regions of its target genes, thereby activating their transcription [36]. Our results showed that IL6/sIL-6Ralpha activated both JAK2 and STAT3 molecules in SVOG cells. In addition, our immunofluorescence assay showed that IL6/sIL-6Ralpha can promote the nuclear translocation of phosphorylated STAT3. Notably, the increase in JAK2/STAT3 phosphorylation and STAT3 nuclear translocation induced by IL6/sIL-6Ralpha were dramatically abolished by the addition of the specific kinase inhibitors AG490 and C188-9, respectively. Considering the possible off target effects and limitations of these kinase inhibitors, we therefore used specific siRNAs to precisely define which endogenous molecules are involved in downstream signaling. In particular, the knockdown of endogenous STAT3 completely abolished the IL6/sIL-6Ralpha-induced increases in PTGS2 expression and PGE2 production. Collectively, these results suggest that JAK2 and STAT3 are most likely the principal signal transduction mediator of IL6 trans-signaling and that STAT3 is the main downstream molecule of JAK2 in SVOG cells. Consistent with our results, a previous study showed that the activation of the JAK2/STAT3 system is required for the IL6-mediated increase in the FSH-induced LHR production in porcine granulosa cells [11]. However, in human granulosa tumor (KGN) cells, the addition of IL6 reduced the secretion of estradiol via the activation of ERK1/2 signaling but not of STAT3 signaling [13]. In this regard, the downstream signaling pathways that mediate IL6 classic or IL6 trans-signaling are cell type- or target gene-specific. There is considerable academic and clinical value in elucidating the key molecules involved in the relevant essential pathophysiological event. Indeed, STAT3 has recently been applied as a therapeutic target for hepatocellular carcinoma [37].

In many mammalian cells, cytokines control various biological responses, and thus multiple temporally distinct inhibitory mechanisms exist to ensure that the responses are transient in nature [38]. As an inducible inhibitor, SOCS3 is essential for limiting the signaling activated by cytokine receptors and can effectively suppress JAK/STAT signaling in many cells [38]. In this study, we observed that after stimulation with IL6/sIL-6Ralpha, the expression levels of the SOCS3 protein were markedly increased in a dose-dependent manner in both SVOG cells and primary hGL cells. In addition, our results showed that the knockdown of SOCS3 significantly enhanced the IL6/sIL-6Ralpha-induced increase in cellular activities, including STAT3 phosphorylation, p-STAT3 nuclear translocation, PTGS2 expression, and subsequent PGE2 production. These results suggest that SOCS3 acts as a negative regulator of the IL6/sIL-6Ralpha-induced JAK2/STAT3 signaling. Similarly, a previous study using an SOCS3 gene-deficient mouse model demonstrated that IL6 can induce an anti-inflammatory response in macrophages, indicating that SOCS3 plays a pivotal role in negatively regulating the responses to IL6 [39]. Based on our findings, future studies will therefore need to examine how to target SOCS3 and control the ovulation event elicited by IL6 trans-signaling and the subsequent PGE2 production.

Conclusion

In summary, we demonstrated that IL6/sIL-6Ralpha upregulates the expression of PTGS2 and increases the production of PGE2 in primary and immortalized hGL cells. IL6/sIL-6Ralpha initiates its cellular activity by activating the JAK2/STAT3 signaling pathways but not the AKT or ERK1/2 signaling pathways. The phosphorylation of JAK2 and STAT3 further induces the nuclear translocation of STAT3 in immortalized hGL cells. In addition, the IL6/sIL-6Ralpha-induced activation of JAK2/STAT3 signaling upregulates the expression of SOCS3, which negatively regulates IL6 trans-signaling (Figure 8). Our findings shed light on the cellular and molecular mechanisms by which IL6 trans-signaling modulates the production of PGE2 in the human ovary, which deepen our understanding of the molecular mechanisms underlying the ovulation process in humans. Consequently, selectively targeting IL6 trans-signaling or SOCS3 represents a promising potential therapeutic strategy for ovulatory dysfunction, such as polycystic ovary syndrome.

Authors’ contributions

S.-J.L. and H.-M. C. conceived and designed the experiments and wrote the manuscript. S.-J.L., J.X., and J.H.W. performed the experiments and analyzed the data. J.Y. and P.C.K.L. participated in critically discussing and revising the manuscript. All authors read and approved the final manuscript.

Conflict of interest: The authors have declared that no conflict of interest exists.

Data availability

All the data underlying this article are available in the article.

Footnotes

† Grant Support: S.-J.L. is the recipient of a scholarship from the China Scholarship Council (# 201806275007). This work was supported by the Canadian Institutes of Health Research (#143317) to P.C.K.L. and was partly supported by operating grants from the National Natural Science Foundation of China (81701412), the Nature Science Foundation of Hubei Province Grant (2018CFB491), and the Hubei Chenguang Talented Youth Development Foundation (Class B project in 2018) to S.-J.L.

Contributor Information

Sai-Jiao Li, Reproductive Medicine Center, Renmin Hospital of Wuhan University, Wuhan, China; Department of Obstetrics and Gynaecology, BC Children’s Hospital Research Institute, University of British Columbia, Vancouver, British Columbia, Canada; Hubei Clinic Research Center for Assisted Reproductive Technology and Embryonic Development, Wuhan, China.

Hsun-Ming Chang, Department of Obstetrics and Gynaecology, BC Children’s Hospital Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

Jiamin Xie, Department of Obstetrics and Gynaecology, BC Children’s Hospital Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

Jeremy H Wang, Department of Obstetrics and Gynaecology, BC Children’s Hospital Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

Jing Yang, Reproductive Medicine Center, Renmin Hospital of Wuhan University, Wuhan, China; Hubei Clinic Research Center for Assisted Reproductive Technology and Embryonic Development, Wuhan, China.

Peter C K Leung, Department of Obstetrics and Gynaecology, BC Children’s Hospital Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

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