Activin A-induced increase in LOX activity in human granulosa–lutein cells is mediated by CTGF

in Reproduction
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Hsun-Ming ChangDepartment of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada

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Jung-Chien ChengDepartment of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada

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Yingtao LiuDepartment of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada
Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China

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Christian KlausenDepartment of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada

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Congjian XuObstetrics and Gynecology Hospital of Fudan University, Shanghai, China

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Peter C K LeungDepartment of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada

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Lysyl oxidase (LOX) is the key enzyme involved in the crosslinking of collagen and elastin that is essential for the formation of extracellular matrix (ECM). LOX-mediated ECM remodeling plays a critical role in follicle development, oocyte maturation and corpus luteum formation. To date, the regulation of LOX in human ovary has never been elucidated. Activin A and its functional receptors are highly expressed in ovarian follicles from an early developmental stage. They locally regulate follicle progression. The aim of this study was to investigate the effects of activin A on the expression of LOX and its extracellular enzyme activity in primary and immortalized human granulosa–lutein cells obtained from patients undergoing an in vitro fertilization procedure. We demonstrated that activin A significantly upregulated the expression of connective tissue growth factor (CTGF) and LOX via an activin/TGF-β type I receptor mediated-signaling pathway. Using a target depletion small interfering RNA knockdown approach, we further confirmed that the upregulation of CTGF expression resulted in an activin-A-induced increases in LOX expression and activity. These findings may provide insight into the mechanisms by which intrafollicular growth factors regulate the expression of LOX for ECM formation and tissue remodeling in the human ovary.

Abstract

Lysyl oxidase (LOX) is the key enzyme involved in the crosslinking of collagen and elastin that is essential for the formation of extracellular matrix (ECM). LOX-mediated ECM remodeling plays a critical role in follicle development, oocyte maturation and corpus luteum formation. To date, the regulation of LOX in human ovary has never been elucidated. Activin A and its functional receptors are highly expressed in ovarian follicles from an early developmental stage. They locally regulate follicle progression. The aim of this study was to investigate the effects of activin A on the expression of LOX and its extracellular enzyme activity in primary and immortalized human granulosa–lutein cells obtained from patients undergoing an in vitro fertilization procedure. We demonstrated that activin A significantly upregulated the expression of connective tissue growth factor (CTGF) and LOX via an activin/TGF-β type I receptor mediated-signaling pathway. Using a target depletion small interfering RNA knockdown approach, we further confirmed that the upregulation of CTGF expression resulted in an activin-A-induced increases in LOX expression and activity. These findings may provide insight into the mechanisms by which intrafollicular growth factors regulate the expression of LOX for ECM formation and tissue remodeling in the human ovary.

Introduction

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase that plays a central role in the remodeling of extracellular matrix (ECM) (Cox & Erler 2011). This oxidative enzyme catalyzes the crosslinking of collagen and elastin by oxidizing the lysine residues in the ECM structural components, resulting in a condensed form of a mature functional structure (Lucero & Kagan 2006). LOX-driven ECM remodeling and matrix stiffening play essential roles in the regulation of cellular development in many tissues (Cox & Erler 2011). In ovarian follicles, ECM acts in an autocrine and paracrine manner to regulate a variety of cellular processes, including cell proliferation, survival, aggregation, morphology, communication and steroidogenesis (Woodruff & Shea 2007). In an in vivo immature rat model, gene expression profiling revealed a close association between LOX gene expression in mural granulosa cells and the developmental competence of the corresponding oocytes (Jiang et al. 2010), suggesting important roles for LOX and ECM in follicular development and oocyte maturation. LOX mRNA is expressed in the granulosa cells of bovine follicles and late preantral/early antral follicles in rats (Harlow et al. 2003, Kendall et al. 2003). In an in vitro system, FSH suppressed LOX mRNA expression and LOX activity, whereas dihydrotestosterone and several transforming growth factor-β (TGF-β) superfamily members enhanced LOX mRNA and enzyme activity (Harlow et al. 2003). However, the regulation of LOX expression and its enzyme activity in human granulosa cells has still not been elucidated.

Connective tissue growth factor (CTGF), also known as CCN2, is a cysteine-rich extracellular matrix protein that belongs to the CCN family of proteins (CCN1-6), which include CYR61, CTGF, NOV, WISP1, WISP2 and WISP3 (Brigstock 2003). CTGF is an important mitogen encoded by a growth factor-inducible early response gene that has been shown to take part in a variety of cellular functions, including cell proliferation, differentiation, apoptosis, migration adhesion and ECM remodeling (Bradham et al. 1991, Brigstock 1999). Notably, CTGF ovarian conditional knockout mice exhibit multiple reproductive defects and severe subfertility, indicating an essential role for CTGF in the regulation of follicle development and ovulation (Nagashima et al. 2011). Previous studies have shown that CTGF mRNA levels were increased in the theca and granulosa cells of porcine early antral follicles (Wandji et al. 2000). In rats, administration of CTGF enhanced the growth of immature follicles by inducing the expression of several genes related to cell proliferation and cell differentiation (Schindler et al. 2010). CTGF is also expressed in the human corpus luteum and is likely involved in the vascular remodeling that occurs during corpus luteum formation and regression (Duncan et al. 2005). Interestingly, LOX and CTGF mRNA are abundantly co-expressed in the granulosa cells of rat developing follicles, and their expression is inversely correlated with the granulosa cell differentiation (Slee et al. 2001). Taken together, these reports indicate that CTGF along with LOX may function coordinately to regulate ECM formation and tissue remodeling during follicular development and the subsequent ovulation and corpus luteum formation.

Belonging to the TGF-β superfamily, activins are homo- or heterodimers of inhibinβsubunits that play essential roles in reproduction (de Kretser et al. 2002). The mature form of activin proteins and their functional receptors (type I and type II) are expressed in ovarian follicles from the early developmental stage, indicating that activins may regulate follicle progression in an autocrine/paracrine manner (Rabinovici et al. 1991, Drummond et al. 2002). We have recently reported that three activin isoforms (activin A, activin B and activin AB) have comparable effects on ovarian steroid synthesis (Chang et al. 2014a, 2015b). Our studies demonstrated that activins mainly act as potent luteinization inhibitors by suppressing progesterone production and promoting the development and survival of growing follicles (Chang et al. 2015b). Studies in mice preantral follicles have shown that activin A may enhance granulosa cell proliferation and the related follicle growth (Smitz et al. 1998). In addition, in vitro studies have revealed a positive regulatory effect of activin A on the development and maturation of human oocytes (Alak et al. 1998). Furthermore, targeted depletion of the activin type IIB receptor exhibited a reproductive phenotype of arrested follicle development and infertility (Matzuk et al. 1995, Nishimori & Matzuk 1996). Interestingly, the expression of inhibinβsubunits is relatively higher in the granulosa cells of immature antral follicles compared with those in the mature and preovulatory follicles of primate and rodent ovaries (Meunier et al. 1988, Schwall et al. 1990). This expression pattern also led to the hypothesis of an activin-dominant microenvironment in growing follicles, the ‘activin tone’ rather than the ‘inhibin tone’ in an inhibin–follistatin-dominant microenvironment in dominant follicles (Knight & Glister 2001). Taken together, the developmentally regulated expression of three intrafollicular factors (activin A, CTGF and LOX) in the differentiation of growing follicles has led us to propose that CTGF may mediate the effects of activin A to regulate the expression and activity of LOX in human granulosa cells. In the current study, we used primary and immortalized human granulosa–lutein cells as study models to investigate the role of CTGF in activin A-induced regulation of LOX expression and activity.

Materials and methods

Preparation and culture of primary human granulosa–lutein (hGL) cells

Primary hGL cells were obtained from patients who provided informed consent after obtaining approval from the University of British Columbia Research Ethics Board. The controlled ovarian stimulation protocol for in vitro fertilization (IVF) patients consisted of the downregulation of either the luteal phase with nafarelin acetate (Synarel, Pfizer) or the follicular phase with a GnRH antagonist (Ganirelix; Merck, Frosst). Gonadotropin stimulation with human menopausal gonadotropin (hMG; Menopur, Ferring) and recombinant FSH (Puregon, Merck) began on day 2 of the menstrual cycle and was followed by hCG (Pregnyl, Merck) administration 34–36h before oocyte retrieval, which was based on follicle size. Collected hGL cells were purified via density centrifugation from follicular aspirates collected from women undergoing oocyte retrieval as described previously (Chang et al. 2014a, 2016a). Individual primary cell cultures were composed of cells from one individual patient. Purified granulosa cells were seeded (2×105cells/well in 12-well plates) and cultured at 37°C in a humidified atmosphere with 5% CO2 in 95% air in Dulbecco’s Modified Eagle Medium/nutrient mixture F-12 Ham (DMEM/F-12; Sigma-Aldrich) supplemented with 10% charcoal/dextran-treated fetal bovine serum (Hyclone, Logan, UT, USA), 100U/mL penicillin (Life Technologies/BRL), 100μg/mL streptomycin sulfate (Life Technologies/BRL) and 1X GlutaMAX (Invitrogen, Life Technologies). Culture medium was changed every other day in all experiments.

Simian virus 40 large T antigen-immortalized human granulosa cell (SVOG) culture

A nontumorigenic immortalized human granulosa cell line, SVOG, which was previously produced by a Simian virus 40 large T antigen transfection of early luteal phase human granulosa cells obtained from patients undergoing IVF (Lie et al. 1996), was used in this study. Because the immortalized SVOG cells were generated from primary human granulosa–lutein cells, they display biological responses to many different treatments that are similar to the responses of hGL cells (Chang et al. 2014b, 2015a,c,d, 2016b). The SVOG cells were counted with a hemocytometer, and cell viability was assessed by a 0.04% Trypan blue exclusion. The percentage of viable cells before and after culture is over 95%. The cells were seeded (2–4×105cells/mL in 6-well plates) and cultured in DMEM/F-12 medium supplemented with 10% charcoal/dextran-treated fetal bovine serum, 100U/mL penicillin, 100μg/mL streptomycin sulfate and 1X GlutaMAX. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. Culture medium was changed every other day in all experiments. Before being treated with growth factors, SVOG cells were cultured in serum-free medium for 24h.

Antibodies and reagents

Recombinant human activin A was obtained from R&D Systems and was composed of a Chinese hamster ovary cell-derived recombinant human protein that was >95% pure based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. It was supplied lyophilized from a 0.2μM filtered solution of 4mM HCL with 0.1% BSA as a carrier protein. The activin/TGF-β type I receptor inhibitor SB431542 was purchased from Sigma-Aldrich. Polyclonal goat anti-CTGF antibody (sc-34772) (diluted at 1:1000), and monoclonal mouse anti-α tubulin antibody (sc-23948) (diluted at 1:2000) were obtained from Santa Cruz Biotechnology. Polyclonal rabbit anti-LOX (ab31238) antibody (diluted at 1:1000) was obtained from Abcam. A horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse IgGs were obtained from Bio-Rad Laboratories. Horseradish peroxidase-conjugated donkey anti-goat IgG was obtained from Santa Cruz Biotechnology.

Reverse transcription quantitative real-time PCR (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, Life Technologies) according to the manufacturer’s instructions. Reverse transcription into first-strand cDNA was performed with 2μg RNA, random primers and Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega). Each 20μL qPCR contained 1X SYBR Green PCR Master Mix (Applied Biosystems), 20ng of cDNA and 250nM of each specific primer. The following primers were used: CTGF, 5′-GCGTGTGCACCGCCAAAGAT-3′ (sense) and 5′-CAGGGCTGGGCAGACGAACG-3′ (antisense); LOX, 5′-GCCTCAGGCTGCACAATTTC-3′ (sense) and 5′-TCAGAACACCAGGCACTGATTT-3′ (antisense) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GAGTCAACGGATTTGGTCGT-3′ (sense) and 5′-GACAAGCTTCCCGTTCTCAG-3′ (antisense). RT-qPCR was performed using an Applied Biosystems 7300 real-time PCR system equipped with a 96-well optical reaction plate. The specificity of each assay was validated based on a dissociation curve analysis and agarose gel electrophoresis of the PCR products. Three independent experiments were performed on different cultures, and each sample of the RT-qPCR experiment was assayed in triplicate. mRNA levels were determined by taking the mean value of the triplicate measurements. Relative quantification of the mRNA levels was performed using a comparative cycle threshold (Ct) method with the 2−ΔΔCt formula, with GAPDH as the reference gene.

Western blotting analysis

After treatment, cells were lysed in a lysis buffer (20mM Tris, 150mM NaCl, 1mM Na2EDTA, 1mM EGTA, 1% Triton, 2.5mM sodium pyrophosphate, 1mM β-glycerophosphate, 1mM Na3VO4, 1μM aprotinin, 1μM leupeptin and 1mM PMSF) (Cell Signaling Technology) containing a protease inhibitor cocktail (Sigma-Aldrich). Extracts were centrifuged at 20,817g for 15min at 4°C to remove cellular debris. Protein concentrations were quantified using a DC Protein Assay (Bio-Rad Laboratories). Equal amounts (50μg) of protein were separated via 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 for 1h and incubated overnight at 4°C with primary antibodies. After washing with 1X Tris-buffered saline, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1h. Immunoreactive bands were detected via an enhanced chemiluminescent substrate or a SuperSignal West Femto Chemiluminescence Substrate (Pierce). The membranes were stripped with stripping buffer (50mM Tris–HCl (pH 7.6), 10mmol/L β-mercaptoethanol and 1% SDS) at 50°C for 30min and reprobed with mouse anti-α tubulin antibody as a loading control.

Small interfering RNA (siRNA) transfection

To knock down endogenous CTGF expression, cells were transfected with 25 or 50nM ON-TARGET plus SMART pool siRNA targeting specific genes (Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Life Technologies). siCONTROL Non-Targeting pool siRNA (Dharmacon) was used as the transfection control. Knockdown efficiency was confirmed via RT-PCR or Western blotting analysis.

Measurement of LOX activity

Following the specified treatment, the culture medium was assayed immediately or stored at −80°C until it was assayed. LOX activity was measured according to the manufacturer’s instructions using an enzyme fluorescent assay (Abcam, ab112139). The inter- and intra-assay coefficients of variation for this assay were less than 6%, and the detection limit of LOX in solution was 40ng. Each sample was measured in triplicate, and the detected LOX activity was normalized to the total cellular protein content in each sample.

Statistical analysis

The results are presented as the mean±s.e.m. of at least three independent experiments. Multiple comparisons were analyzed via a one-way analysis of variance followed by Tukey’s multiple comparison tests using PRISM software (GraphPad Software). Statistical significance was defined as P<0.05.

Results

Activin A upregulates the expression of CTGF in the SVOG cells

To investigate the effect of activin A on the expression of CTGF in human granulosa cells, we first treated the SVOG cells with a vehicle control or different concentrations (1, 10 or 100ng/mL) of recombinant human activin A (activin A). As shown in Fig. 1A, the treatment with activin A for 3h induced concentration-dependent increases in CTGF mRNA levels. Western blotting analysis of the cell lysates treated with activin A for 6h showed similar increases in CTGF protein (Fig. 1B). Given that the average concentrations of activin A in follicular fluid and serum are 1110–1210ng/mL and 30–40ng/mL, respectively (Harada et al. 1996, Lau et al. 1999), we continued our studies using a treatment concentration of 30ng/mL activin A. To evaluate the time-dependent effects of activin A, we next treated the cells with 30ng/mL activin A for 1, 3, 6, 12 or 24h. The results showed that the CTGF mRNA levels were increased at 3h. This stimulatory effect persisted until 24h after exposure to activin A (Fig. 1C). Likewise, the activin A-induced increase in CTGF protein levels appeared at 3h and persisted until 24h (Fig. 1D).

Figure 1
Figure 1

Activin A (AcA) upregulates CTGF expression in the SVOG cells. (A and B) SVOG cells were treated with a vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 3h (A) or 6h (B), and the levels of CTGF mRNA (A) and CTGF protein (B) were examined using RT-qPCR and Western blotting, respectively. (C and D) SVOG cells were treated with vehicle control or 30ng/mL activin A for 1, 3, 6, 12 or 24h, and the levels of CTGF mRNA (C) and CTGF protein (D) were examined using RT-qPCR and Western blotting, respectively. The results are expressed as the mean±s.e.m. of at least three independent experiments. Values marked by different letters are significantly different (P<0.05). Ctrl, control.

Citation: Reproduction 152, 4; 10.1530/REP-16-0254

Activin A upregulates LOX expression and increases LOX activity in the SVOG cells

To examine the concentration-dependent effects of activin A on the expression of LOX, SVOG cells were treated with a vehicle control or different concentra­tions (1, 10 or 100ng/mL) of activin A. As shown in Fig. 2A and B, the activin A treatment significantly increased the mRNA (at 6h) and protein (at 12h) levels of LOX in a concentration-dependent manner. The time course studies revealed that the LOX mRNA levels were increased after 6h (Fig. 2C), whereas the LOX protein levels were increased after 12h (Fig. 2D). To investigate whether activin A-induced upregulation of LOX expression correlates with an increase in LOX activity, we used an enzyme immunoassay to measure LOX activity in conditioned medium from cells treated with activin A. The results showed that activin A could significantly increase the LOX activity in a concentration-dependent manner (Fig. 2E). Likewise, the time course studies revealed that the LOX activity was increased after 12 and 24h of exposure to 30ng/mL activin A (Fig. 2F).

Figure 2
Figure 2

Activin A (AcA) upregulates LOX expression and increases LOX activity in the SVOG cells. (A and B) SVOG cells were treated with a vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 6h (A) or 12h (B), and the levels of LOX mRNA (A) and LOX protein (B) were examined using RT-qPCR and Western blotting, respectively. (C and D) SVOG cells were treated with vehicle control or 30ng/mL activin A for 1, 3, 6, 12 or 24h, and the levels of LOX mRNA (C) and LOX protein (D) were examined using RT-qPCR and Western blotting, respectively. (E) SVOG cells were treated with vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 24h, and LOX activity in the conditioned medium was determined using an enzyme fluorescent assay. The amount of LOX activity of control group is 44.3±19.8pg/106 cells. (F) SVOG cells were treated with vehicle control or 30ng/mL activin A for 3, 6, 12 or 24h, and LOX activity in the conditioned medium was determined using an enzyme fluorescent assay. The amount of LOX activity of control group is 36.7±27.4pg/106 cells. The results are expressed as the mean±s.e.m. of at least three independent experiments. Values marked by different letters are significantly different (P<0.05). Ctrl, control.

Citation: Reproduction 152, 4; 10.1530/REP-16-0254

Activin/TGF-β type I receptor inhibitor SB431542 abolishes the upregulation of CTGF and LOX by activin A in the SVOG cells

To confirm that the effects of activin A on CTGF and LOX are mediated by conventional activin receptors, we used a potent and specific inhibitor of activin/TGF-β type I receptor kinases, SB431542 (Inman et al. 2002). As shown in Fig. 3A and B, pretreatment of SVOG cells with 5μM of SB431542 abolished the activin A-induced increases of CTGF mRNA and protein. Similarly, pretreatment with SB431542 also abolished the stimulatory effects of activin A on LOX mRNA and protein (Fig. 3C and D).

Figure 3
Figure 3

SB431542 inhibitor abolishes activin A-induced upregulation of CTGF and LOX in the SVOG cells. (A and B) Cells were treated with 30ng/mL activin A for 3h (A) or 6h (B) in the presence of vehicle control (DMSO) or 5μM SB431542. The mRNA (A) and protein (B) levels of CTGF were examined using RT-qPCR and Western blotting, respectively. (C and D) Cells were treated with 30ng/mL activin A for 6h (C) or 12h (D) in the presence of vehicle control (DMSO) or 5μM SB431542. The mRNA (C) and protein (D) levels of LOX were examined using RT-qPCR and Western blotting, respectively. The percentage of viable cells after activin A treatment (3, 6 or 12h) is over 95%. The results are expressed as the mean±s.e.m. of at least three independent experiments. Values marked by different letters are significantly different (P<0.05). Ctrl, control.

Citation: Reproduction 152, 4; 10.1530/REP-16-0254

CTGF mediates the effects of activin A on LOX expression and activity in SVOG cells

It has been shown that CTGF can regulate a variety of cellular functions including the synthesis and formation of ECM in many cell types (Arnott et al. 2011). Small interfering RNA (siRNA; 25 or 50nM) targeting CTGF was used to investigate its involvement in the stimulatory effects of activin A on LOX expression and activity in SVOG cells. As shown in Fig. 4A and B, the transient transfection with siRNA targeting CTGF for 24h could effectively suppress the expression of CTGF mRNA (Fig. 4A) and protein (Fig. 4B). Notably, knockdown of CTGF for 24h abolished the activin A-induced increases in LOX mRNA (Fig. 4C) and protein (Fig. 4D). Most importantly, CTGF knockdown abolished the upregulation of LOX activity by activin A (Fig. 4E). These data suggest that activin A upregulates LOX expression and activity by inducing the production of CTGF.

Figure 4
Figure 4

CTGF mediates the activin A-induced upregulation of LOX and increase in LOX activity in the SVOG cells. (A and B) Cells were transfected with 25 or 50nM control siRNA (siCtrl) or CTGF siRNA (siCTGF) for 24h. Knockdown efficiency of CTGF siRNA was examined using RT-qPCR (A) or Western blotting (B). (C) Cells were transfected for 24h with 25nM control siRNA (siCtrl) or 25nM CTGF siRNA (siCTGF) followed by treatment with vehicle control or 30ng/mL activin A for 6h. The mRNA levels of LOX were measured using RT-qPCR. (D) Cells were transfected for 24h with 25nM control siRNA (siCtrl) or 25nM CTGF siRNA (siCTGF) followed by treatment with vehicle control or 30ng/mL activin A for 12h. The protein levels of LOX were measured using Western blotting. (E) Cells were transfected for 24h with 25nM control siRNA (siCtrl) or 25nM CTGF siRNA (siCTGF) followed by treatment with vehicle control or 30ng/mL activin A for 24h. LOX activity in the conditioned medium was measured using an enzyme fluorescent assay. The amount of LOX activity of siCtrl control group is 32.6±28.8pg/106 cells. The results are expressed as the mean±s.e.m. of at least three independent experiments. Values marked by different letters are significantly different (P<0.05). Ctrl, control.

Citation: Reproduction 152, 4; 10.1530/REP-16-0254

Activin A upregulates the expression of CTGF and LOX in the primary hGL cells

To further confirm our findings in SVOG cells, we next used primary hGL cells obtained from women undergoing IVF to investigate the effects of activin A on CTGF expression. As shown in Fig. 5A and B, treatment with activin A increased the mRNA and protein levels of CTGF in a concentration-dependent manner. Likewise, using the primary hGL cell culture, we further confirmed that activin A can upregulate the expression of LOX at both the mRNA and protein levels (Fig. 5C and D). Finally, we used an enzyme immunoassay to measure LOX activity in conditioned medium from primary hGL cells treated with activin A. The results showed that activin A could significantly increase the LOX activity in a concentration-dependent manner in primary hGL cells (Fig. 5E).

Figure 5
Figure 5

Activin A upregulates the expression of CTGF and LOX in the primary hGL cells. (A and B) Primary hGL cells were treated with a vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 3h (A) or 6h (B), and the levels of CTGF mRNA (3h) (A) and CTGF protein (6h) (B) were examined using RT-qPCR and Western blotting, respectively. (C and D) Primary hGL cells were treated with a vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 6h (C) or 12h (D), and the levels of CTGF mRNA (6h) (C) and CTGF protein (12h) (D) were examined using RT-qPCR and Western blotting, respectively. (E) Primary hGL cells were treated with vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 24h, and LOX activity in the conditioned medium was determined using an enzyme fluorescent assay. The amount of LOX activity of control group is 60.3±33.8pg/106 cells. The results are expressed as the mean±s.e.m. of at least three independent experiments. Values marked by different letters are significantly different (P<0.05). Ctrl, control.

Citation: Reproduction 152, 4; 10.1530/REP-16-0254

Activin A treatment does not affect the cell morphology of hGL cells

To investigate the effects of activin A on the cell morphology of hGL cells, we treated SVOG cells and primary hGL with 30ng/mL activin A for 12h, and the cells were inspected using a digital camera (Leica EC3) mounted on Leica microscope (Leica DM 2000), magnification 100×. As shown in Fig. 6A and B, treatment with activin A for 12h did not affect the cell morphology of both SVOG and primary hGL cells.

Figure 6
Figure 6

Cell morphology of human granulosa–lutein cells. (A and B) The photographs (original magnification 100×) show the cell morphology of SVOG cells (A) and primary human granulosa–lutein cells (B) after treatment with 30ng/mL of activin A (12h) in comparison with the control cells. Scale bar represents 20μm. Ctrl, control.

Citation: Reproduction 152, 4; 10.1530/REP-16-0254

Discussion

LOX-mediated ECM formation determines the mechanical properties and dynamic remodeling of connective tissue and plays a critical role in regulating a variety of biological functions including reproduction (Woodruff & Shea 2007, Karsdal et al. 2013). However, dysregulation of LOX may initiate the onset and progression of certain pathological events, such as fibrosis, tumor progression, cancer metastasis, cardiovascular diseases and neurodegenerative disorders (Maki 2009). In human and animal models, over-expression of LOX has been identified in the lesion of endometriosis and polycystic ovary syndrome (PCOS), two common causes of female infertility (Harlow et al. 2003, Papachroni et al. 2010, Ruiz et al. 2015). Specifically, in a PCOS rat model, administration of DHEA induced the upregulation of LOX, which has been implicated as one of the causes of pathogenesis in PCOS (Harlow et al. 2003). Given its essential role in the development of physiological and pathological conditions, the regulation of LOX expression and activity is considered an important therapeutic target. This study aimed to elucidate the effect of activin A on the regulation of LOX expression and activity using an in vitro cell model. Our experimental data demon­strated that human granulosa–lutein cells exposed to different concentrations of activin A displayed an upregulation of LOX and increase in LOX activity in a concentration- and time-dependent manner. Both immortalized and primary human granulosa–lutein cells displayed similar responses to activin A ligand treatment, although the basal levels (control group) of LOX activity in primary cells are approximately 1.5-fold higher than those of the immortalized cells, which could be due to higher expression of activin receptors in the primary cells. This result is consistent with a previous study showing that three TGF-β superfamily members, TGF-β1, growth differentiation factor (GDF) 9 and activin A, stimulated LOX mRNA and activity at 6h and that the effects persisted until 48h in rat granulosa cells (Harlow et al. 2003), which is the only study that has addressed the regulation of LOX in mammalian ovaries. However, the underlying mechanism of the stimulatory effect of activin A in LOX gene regulation has never been explored.

In this study, we present experimental evidence that activin A induced a rapid upregulation (within 3h) of CTGF expression at both mRNA and protein levels in human granulosa–lutein cells. CTGF has been shown to act as a downstream mediator for the TGF-β superfamily members to modulate cellular biology or influence pathological progression in many tissues (Harlow et al. 2003, Verrecchia & Mauviel 2007, Shi-Wen et al. 2008). We have recently reported that GDF8 suppressed human granulosa cell proliferation by upregulating the expression of CTGF (Chang et al. 2016b). Previous studies have demonstrated that activin A was over-expressed in the hepatocytes of fibrotic liver, and the elevation of activin A induced the upregulation of CTGF, which contributed to the pathogenesis of liver fibrosis (Sugiyama et al. 1998, Gressner et al. 2008). Moreover, a recent study has reported that the activin A-induced synthesis of CTGF in liver progenitor cells was mediated via the canonical SMAD signaling, that is SMAD2/3 and SMAD4 collaboratively contributed to this induction (Ding et al. 2016). Consistent with these results, our data also showed that inhibition of the activin A signaling pathway with the activin/TGF-β type I receptor (ALK4/5/7) inhibitor SB431542 abolished the activin A-induced upregulation of CTGF expression. Interestingly, the inhibition of activin A signaling further abolished the activin A-induced upregulation of LOX expression in our cell model. Given that the activin A-induced stimulatory effect on CTGF expression is 3–6h before that of LOX expression, we speculated that there is a plausible role of CTGF in the mediation of activin A-induced increases in LOX expression and activity. Indeed, our data further confirmed that knockdown of endogenous CTGF using small interfering RNA was able to abolish the effects of activin A on LOX expression and its enzyme activity, indicating that CTGF is the downstream effector of the cellular function exerted by activin A on LOX.

In an in vivo wound repair model, TGF-β and CTGF were abundantly co-expressed in a lesion, indicating that the two growth factors are coordinately involved in the process of wound healing (Igarashi et al. 1993). In a monkey skin fibrosis model, administration of TGF-β alone or CTGF alone produced only a mild transient fibrotic response. However, concomitant treatment with TGF-β and CTGF led to a prominent and persistent fibrotic reaction (Mori et al. 1999). Consistent with our results, inhibition of the expression of CTGF using antisense mRNA or CTGF-binding antibodies prevented the TGF-β-induced increase in collagen synthesis in fibroblasts, indicating that induction of CTGF is mandatory for the TGF-β-induced fibrotic response (Uchio et al. 2004). Notably, our recent study also revealed that knockdown of CTGF reversed the suppressive effect of GDF8 on human granulosa cell proliferation (Chang et al. 2016b). In addition to acting as a mediator of other growth factors, CTGF has been shown to bind to TGF-β to form a complex, which assists the high-affinity binding of TGF-β and its TGF-β type II receptor (Abreu et al. 2002). Taken together, previous studies and our study suggest that CTGF may play a broad role in ECM formation and diverse roles in signal transduction by mediating and interacting with TGF-β superfamily members. Nonetheless, two additional questions are raised by our current results. How does CTGF regulate the expression and activity of LOX? Do the activin A-induced increases in LOX expression and activity contribute to the enhancement of ECM formation and connective tissue remodeling in the follicles? Future studies aimed at addressing these issues using in vivo animal models, such as rodents and nonhuman primates, will be of great interest.

In summary, in vitro experiments using human granulosa–lutein cells were performed to examine the effects of activin A on the expression of CTGF and LOX. Treatment with activin A induced an upregulation of CTGF expression in a concentration- and time-dependent manner. In addition, inhibition of activin A signaling using inhibitor SB431542 abolished this stimulatory effect. Furthermore, the upregulation of CTGF was attributed to the activin A-induced increases in LOX expression and activity. These novel findings may provide insight into the mechanisms by which intrafollicular growth factors regulate the expression of the key enzyme LOX for ECM formation and tissue remodeling. Better understanding of the molecular and cellular basis of the LOX aberration-related diseases, such as endometriosis and PCOS, may help us in developing pharmacological strategies for infertility treatment.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by an operating grant (#143317) from the Canadian Institutes of Health Research to P C K L.

Acknowledgements

We thank Dr Elizabeth L Taylor for coordinating the acquisition of patient follicular fluid samples.

References

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    Activin A (AcA) upregulates CTGF expression in the SVOG cells. (A and B) SVOG cells were treated with a vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 3h (A) or 6h (B), and the levels of CTGF mRNA (A) and CTGF protein (B) were examined using RT-qPCR and Western blotting, respectively. (C and D) SVOG cells were treated with vehicle control or 30ng/mL activin A for 1, 3, 6, 12 or 24h, and the levels of CTGF mRNA (C) and CTGF protein (D) were examined using RT-qPCR and Western blotting, respectively. The results are expressed as the mean±s.e.m. of at least three independent experiments. Values marked by different letters are significantly different (P<0.05). Ctrl, control.

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    Activin A (AcA) upregulates LOX expression and increases LOX activity in the SVOG cells. (A and B) SVOG cells were treated with a vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 6h (A) or 12h (B), and the levels of LOX mRNA (A) and LOX protein (B) were examined using RT-qPCR and Western blotting, respectively. (C and D) SVOG cells were treated with vehicle control or 30ng/mL activin A for 1, 3, 6, 12 or 24h, and the levels of LOX mRNA (C) and LOX protein (D) were examined using RT-qPCR and Western blotting, respectively. (E) SVOG cells were treated with vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 24h, and LOX activity in the conditioned medium was determined using an enzyme fluorescent assay. The amount of LOX activity of control group is 44.3±19.8pg/106 cells. (F) SVOG cells were treated with vehicle control or 30ng/mL activin A for 3, 6, 12 or 24h, and LOX activity in the conditioned medium was determined using an enzyme fluorescent assay. The amount of LOX activity of control group is 36.7±27.4pg/106 cells. The results are expressed as the mean±s.e.m. of at least three independent experiments. Values marked by different letters are significantly different (P<0.05). Ctrl, control.

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    SB431542 inhibitor abolishes activin A-induced upregulation of CTGF and LOX in the SVOG cells. (A and B) Cells were treated with 30ng/mL activin A for 3h (A) or 6h (B) in the presence of vehicle control (DMSO) or 5μM SB431542. The mRNA (A) and protein (B) levels of CTGF were examined using RT-qPCR and Western blotting, respectively. (C and D) Cells were treated with 30ng/mL activin A for 6h (C) or 12h (D) in the presence of vehicle control (DMSO) or 5μM SB431542. The mRNA (C) and protein (D) levels of LOX were examined using RT-qPCR and Western blotting, respectively. The percentage of viable cells after activin A treatment (3, 6 or 12h) is over 95%. The results are expressed as the mean±s.e.m. of at least three independent experiments. Values marked by different letters are significantly different (P<0.05). Ctrl, control.

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    CTGF mediates the activin A-induced upregulation of LOX and increase in LOX activity in the SVOG cells. (A and B) Cells were transfected with 25 or 50nM control siRNA (siCtrl) or CTGF siRNA (siCTGF) for 24h. Knockdown efficiency of CTGF siRNA was examined using RT-qPCR (A) or Western blotting (B). (C) Cells were transfected for 24h with 25nM control siRNA (siCtrl) or 25nM CTGF siRNA (siCTGF) followed by treatment with vehicle control or 30ng/mL activin A for 6h. The mRNA levels of LOX were measured using RT-qPCR. (D) Cells were transfected for 24h with 25nM control siRNA (siCtrl) or 25nM CTGF siRNA (siCTGF) followed by treatment with vehicle control or 30ng/mL activin A for 12h. The protein levels of LOX were measured using Western blotting. (E) Cells were transfected for 24h with 25nM control siRNA (siCtrl) or 25nM CTGF siRNA (siCTGF) followed by treatment with vehicle control or 30ng/mL activin A for 24h. LOX activity in the conditioned medium was measured using an enzyme fluorescent assay. The amount of LOX activity of siCtrl control group is 32.6±28.8pg/106 cells. The results are expressed as the mean±s.e.m. of at least three independent experiments. Values marked by different letters are significantly different (P<0.05). Ctrl, control.

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    Activin A upregulates the expression of CTGF and LOX in the primary hGL cells. (A and B) Primary hGL cells were treated with a vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 3h (A) or 6h (B), and the levels of CTGF mRNA (3h) (A) and CTGF protein (6h) (B) were examined using RT-qPCR and Western blotting, respectively. (C and D) Primary hGL cells were treated with a vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 6h (C) or 12h (D), and the levels of CTGF mRNA (6h) (C) and CTGF protein (12h) (D) were examined using RT-qPCR and Western blotting, respectively. (E) Primary hGL cells were treated with vehicle control or increasing concentrations (1, 10 or 100ng/mL) of activin A for 24h, and LOX activity in the conditioned medium was determined using an enzyme fluorescent assay. The amount of LOX activity of control group is 60.3±33.8pg/106 cells. The results are expressed as the mean±s.e.m. of at least three independent experiments. Values marked by different letters are significantly different (P<0.05). Ctrl, control.

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    Cell morphology of human granulosa–lutein cells. (A and B) The photographs (original magnification 100×) show the cell morphology of SVOG cells (A) and primary human granulosa–lutein cells (B) after treatment with 30ng/mL of activin A (12h) in comparison with the control cells. Scale bar represents 20μm. Ctrl, control.

  • Abreu JG, Ketpura NI, Reversade B & De Robertis EM 2002 Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nature Cell Biology 4 599604. (doi:10.1038/ncb826)

    • Search Google Scholar
    • Export Citation
  • Alak BM, Coskun S, Friedman CI, Kennard EA, Kim MH & Seifer DB 1998 Activin A stimulates meiotic maturation of human oocytes and modulates granulosa cell steroidogenesis in vitro. Fertility and Sterility 70 11261130. (doi:10.1016/S0015-0282(98)00386-0)

    • Search Google Scholar
    • Export Citation
  • Arnott JA, Lambi AG, Mundy C, Hendesi H, Pixley RA, Owen TA, Safadi FF & Popoff SN 2011 The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis. Critical Reviews in Eukaryotic Gene Expression 21 4369. (doi:10.1615/CritRevEukarGeneExpr.v21.i1)

    • Search Google Scholar
    • Export Citation
  • Bradham DM, Igarashi A, Potter RL & Grotendorst GR 1991 Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. Journal of Cell Biology 114 12851294. (doi:10.1083/jcb.114.6.1285)

    • Search Google Scholar
    • Export Citation
  • Brigstock DR 1999 The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocrine Reviews 20 189206. (doi:10.1210/er.20.2.189)

    • Search Google Scholar
    • Export Citation
  • Brigstock DR 2003 The CCN family: a new stimulus package. Journal of Endocrinology 178 169175. (doi:10.1677/joe.0.1780169)

  • Chang HM, Cheng JC, Klausen C, Taylor EL & Leung PC 2014a Effects of recombinant activins on steroidogenesis in human granulosa-lutein cells. Journal of Clinical Endocrinology and Metabolism 99 E1922E1932. (doi:10.1210/jc.2014-1223)

    • Search Google Scholar
    • Export Citation
  • Chang HM, Cheng JC, Taylor E & Leung PC 2014b Oocyte-derived BMP15 but not GDF9 down-regulates connexin43 expression and decreases gap junction intercellular communication activity in immortalized human granulosa cells. Molecular Human Reproduction 20 373383. (doi:10.1093/molehr/gau001)

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