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. 2016 Nov 4;158(1):109–120. doi: 10.1210/en.2016-1544

Induction of Tissue Factor Pathway Inhibitor 2 by hCG Regulates Periovulatory Gene Expression and Plasmin Activity

Muraly Puttabyatappa 1, Linah F Al-Alem 1, Farnosh Zakerkish 2, Katherine L Rosewell 1, Mats Brännström 2, Thomas E Curry Jr 1,✉
PMCID: PMC5412983  PMID: 27813674

Abstract

Increased proteolytic activity is a key event that aids in breakdown of the follicular wall to permit oocyte release. How the protease activity is regulated is still unknown. We hypothesize that tissue factor pathway inhibitor 2 (TFPI2), a Kunitz-type serine protease inhibitor, plays a role in regulating periovulatory proteolytic activity as in other tissues. TFPI2 is secreted into the extracellular matrix (ECM) where it is postulated to regulate physiological ECM remodeling. The expression profile of TFPI2 during the periovulatory period was assessed utilizing a well-characterized human menstrual cycle model and a gonadotropin-primed rat model. Administration of an ovulatory dose of human chorionic gonadotropin (hCG) increased TFPI2 expression dramatically in human and rat granulosa and theca cells. This increase in Tfpi2 expression in rat granulosa cells required hCG-mediated epidermal growth factor, protein kinase A, mitogen-activated protein kinase (MAPK) 1/2, p38 MAPK and protease activated receptor 1-dependent cell signaling. A small interferingRNA-mediated knockdown of TFPI2 in rat granulosa cells resulted in increased plasmin activity in the granulosa cell conditioned media. Knockdown of TFPI2 also reduced expression of multiple genes including interleukin 6 (Il6) and amphiregulin (Areg). Overexpression of TFPI2 using an adenoviral vector partially restored the expression of Il6 and Areg in TFPI2 siRNA treated rat granulosa cells. These data support the hypothesis that TFPI2 is important for moderating plasmin activity and regulating granulosa cell gene expression during the periovulatory period. We, therefore, propose that through these actions, TFPI2 aids in the tissue remodeling taking place during follicular rupture and corpus luteum formation.

The ovulatory process is characterized by progressive breakdown of the apical follicular wall that spans over 36 hours in the human and is a prerequisite for the expulsion of the cumulus-oocyte complex (COC). Previous studies have documented that this process involves an increase in follicular proteolytic activity in numerous species, including the human (1, 2). The paramount importance of proteases is highlighted by the demonstration that inhibiting proteases blocks follicular rupture and oocyte release (3–5). The proteases that are mainly induced during the periovulatory period are the plasminogen activator (PA)/plasmin, matrix metalloproteinases (MMPs), and A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family members (1, 2, 6). These proteases degrade the proteinaceous components that form the basement membrane and extracellular matrix (ECM) of the cells in the theca layer and surrounding layers of the follicle, thereby compromising the integrity of the follicular wall to allow expulsion of the COC.

Regulation of this enzymatic activity is essential such that a controlled proteolysis occurs to permit follicular rupture and at the same time protect the cells that remain in the follicle from the adverse effects of uncontrolled enzymatic action. Regulation of these proteases occurs through specific protease inhibitors such as PA inhibitors (PAIs) and tissue inhibitors of MMPs that are induced during the periovulatory period (7, 8). Simultaneous expression of proteases and protease inhibitors by the granulosa and theca cells is one way of regulating local protease action. For example, plasmin initiates activation of the MMP proprotein and the presence of PAIs and tissue inhibitors of MMPs confines the proteolytic activity, resulting in controlled matrix degradation (9). Thus, the balance between the levels of proteases and inhibitors is critical to determine whether proteolysis occurs. This concept is supported in macaques where PA activity levels in the follicular fluid were elevated 36 hours post hCG, whereas PAI1 levels were low, potentially allowing proteolysis to occur at this critical period of oocyte release (8).

Our preliminary studies to identify proteases and protease inhibitors that change following a human chorionic gonadotropin (hCG) stimulus revealed that an ovulatory stimulus increases the expression of a protease inhibitor, tissue factor pathway inhibitor 2 (TFPI2), in human granulosa cells. TFPI2 belongs to a Kunitz family of serine protease inhibitors that contain one or more Kunitz-type domains. This family has over 20 members, including pancreatic trypsin inhibitor (aprotinin) and the homolog of TFPI2, TFPI1, among others (10). TFPI2 is secreted as a glycosylated protein with a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxy-terminal tail (11). Human TFPI2 was first identified in the placenta, where it was reported to inhibit the amidolytic activity of plasmin (12). In the ovary, TFPI2 was first observed in follicular fluid obtained from women undergoing in vitro fertilization (13). Most recently, TFPI2 expression was detected in the human ovary and bovine granulosa cells (14, 15). In patients with polycycstic ovary syndrome (PCOS), TFPI2 is overexpressed in the stroma during the preovulatory phase of the menstrual cycle, although in bovine granulosa cells, TFPI2 expression correlated with oocyte competence. However, these studies did not assess the changes during the periovulatory period.

Unlike TFPI1, which inhibits the tissue factor pathway that forms part of the coagulation cascade, the true function of TFPI2 has not been entirely determined. The current postulate is that TFPI2 plays a crucial role in regulating ECM remodeling (10, 16) by inhibiting protease activity of plasmin directly and MMPs indirectly (17). As noted earlier, the ovary undergoes dynamic changes in the ECM involving proteases that are induced during the periovulatory period (1, 2, 6). Unlike specific inhibitors, TFPI2 can inhibit multiple proteases, making it an ideal candidate to regulate the immense proteolytic process during ovulation.

We report the temporal profile of TFPI2 during the human and rat periovulatory period utilizing well-characterized models of ovulation and its regulation and actions using the rat model of ovulation. Additionally, we demonstrate TFPI2’s ability to regulate granulosa cell gene expression during the periovulatory period.

Methods

Human granulosa and theca cell collection

Human ovarian granulosa and theca cells were collected before and after ovulatory hCG treatment at different times as described previously (18). Briefly, ovarian tissue collection was carried out in women with normal menstrual cycles undergoing tubal sterilization. The Graafian follicle representing the preovulatory stage (0 hours) was collected by laparoscopic surgery when the follicle measured ≥14 mm and ≤17.5 mm. The remainder of the patients received an injection of hCG (s.c., 250 µg recombinant hCG, Ovitrelle, Merck Serono, Oss, Netherlands) to induce ovulatory events. Following hCG injection, follicles were collected by laparoscopic surgery at early ovulatory phase (12 to ≤18 hours), late ovulatory phase (>18 to ≤36 hours), and postovulatory phase (>44 to ≤72 hours). The intact dominant follicle was excised using only scissors and with no diathermy. For granulosa cell isolation, the follicle was bisected to release the loosely attached cells, and the mural granulosa cells were then gently scraped from the follicle wall and pooled together. The theca interna cell layer was collected by mechanically separating this layer from the remnant of the follicle. This harvested cell layer also contains vascular cells, leukocytes, and fibroblasts, and for the sake of clarity, we refer to these as ‘‘theca cells’’ (19). Theca cells were obtained from all four periovulatory phases, but granulosa cells could not be collected from the postovulatory group due to a loss of cells after ovulation. This study was approved by the human ethics committee at the University of Gothenburg. Informed written consent was obtained from all patients before surgery.

Rat ovarian tissue collection

Immature female Sprague-Dawley rats (Harlan, Indianapolis, IN) were maintained at room temperature with food and water provided ad libitum. Rats (24 to 25 days of age) were administered 10 IU of pregnant mare serum gonadotropin (PMSG). Forty-eight hours after PMSG injection, 5 IU of hCG was administered to induce ovulation. Rats were euthanized at varying time points after hCG treatment (0, 4, 8, 12, and 24 hours) for tissue collection as below. Whole, intact ovaries were used as such or used to isolate granulosa cells and residual ovarian tissue as described below. The residual tissue represents the ovarian tissue left behind after granulosa cell collection and is a heterogeneous tissue comprised mostly of theca, interstitial, endothelial, and stromal cells as well as remaining granulosa cells. For collection of granulosa cells and residual ovarian tissue, ovaries collected at defined time points after hCG were punctured with a 26-gauge needle to release the granulosa cells into a petri dish containing media. The remaining residual ovarian tissue was removed, and granulosa cells were partially purified by filtration through a 40-µ pore size nylon filter to remove tissue debris and COCs. The cells were then pelleted by centrifugation at 300 × g. Tissues collected for messenger RNA (mRNA) analysis were snap frozen and stored at –70°C for later analysis. All animal procedures for these experiments were approved by the University of Kentucky Institutional Animal Care and Use Committee.

Rat granulosa cell culture

Preovulatory granulosa cells were isolated by follicular puncture from ovaries collected from immature rats 48 hours after treatment with PMSG and used for cell culture experiments as described before (20). Briefly, isolated granulosa cells were cultured at 5 × 105 cells/well in a 12-well tissue culture plate in OptiMEM media containing insulin, transferrin, selenium (ITS) and gentamycin. These cells were treated with or without hCG for different durations of time (0, 4, 8, 12, and 24 hours), and both the conditioned culture media and cells were collected for hormone assay or RNA analysis, respectively.

We used specific pharmacological agents that attenuate or activate specific signaling mediators to determine the hCG-mediated factors that are required for Tfpi2 expression. These were added to rat granulosa cell cultures in the presence or absence of hCG and cultured as above for 4 or 6 hours depending on the experiment. The specific pharmacological agents and their doses are listed in the results and figure legends. These pharmacological agents when dissolved in dimethyl sulfoxide (DMSO), the final vehicle volume in cultures were 0.1% volume-to-volume ratio (v/v), and control or hCG-alone treatments received equivalent volumes of the vehicle.

TFPI2 knockdown in granulosa cells was brought about utilizing small interfering RNA (siRNA) against TFPI2 from Invitrogen (Carlsbad, CA). Two different siRNAs (Silencer Select siRNAs with catalog numbers141862 and s141863) were used in combination to knockdown Tfpi2 expression. For siRNA transfections, rat granulosa cells were cultured overnight in OptiMEM media containing ITS and gentamycin to allow cells to acclimatize to the culture conditions. The next day, the spent media was removed, and Tfpi2 siRNAs (10 nM each) mixed with Lipofectamine siRNAmax (Invitrogen) in fresh OptiMEM media without antibiotics were added on to the cells as per manufacturer’s recommendation. For control transfections, a scrambled siRNA (Negative Control siRNA; catalog number 12935112; Invitrogen) was used. After 3 hours, cells were treated with the vehicle (ethanol and DMSO; 0.1% v/v) or forskolin (FSK; in ethanol, 10 µM) plus phorbol-12-myristate-13-acetate (PMA; in DMSO, 20 nM) for different times. Following the culture period, media were collected for either hormone assay or measurement of proteolytic activity and cells were processed for RNA isolation.

For TFPI2 overexpression, an adenoviral vector expressing a human recombinant TFPI2 kindly donated by Christina Lupu (Oklahoma Medical Research Foundation, Oklahoma City, OK) (21) was used. Rat granulosa cells were cultured overnight as above and were transduced with either a control adenovirus or human TFPI2-expressing adenovirus at 5 multiplicity of infection for 3 hours. At the end of this time point, cells were treated with the vehicle or FSK+PMA, and cells were processed as above.

Real-time RT-PCR

Total RNA was isolated using Trizol reagent (Invitrogen) from intact rat ovaries or the residual tissue, and an RNAeasy kit (Qiagen, Germantown, MD) was used to isolate RNA from human granulosa and theca cells and rat granulosa cells as per the manufacturer’s guidelines. Total RNA was reverse transcribed into complementary DNA for use in the gene expression analysis. The TFPI2 expression in the rat and human samples was analyzed using the TaqMan real-time reverse transcription (RT) polymerase chain reaction (PCR) assay utilizing Assay-on-Demand primer-probe sets from Applied Biosystems (Carlsbad, CA) on a Stratagene (La Jolla, CA) Mx3000P real-time PCR instrument. The following primer-probe sets were used: human TFPI2 gene expression analysis, Hs00197918_m1; human GAPDH, 4326317E; rat Tfpi2, Rn00597628_m1; and rat Rpl32, Rn00820748_g1. The target gene and the control gene real-time analysis was carried out in the same reaction, and the expression levels were normalized to the control gene (GAPDH for the human and Rpl32 for the rat samples). A SYBRgreen-based real-time RT-PCR assay was used to determine the mRNA levels of rat Il6, Areg, and housekeeping control Rpl32. Oligonucleotide primers for these genes were designed using PRIMER3 software, and the specificity for each primer set was confirmed by both electrophoresis of the PCR products and analyzing the melting (dissociation) curve after each real-time PCR reaction. The sequences for the primers used are: rat Il6 forward, 5′-GAAAAGAGTTGTGCAATGGCAA-3′; reverse, 5′-TTTCAATAGGCAAATTTCCTGGTTA-3′; rat Areg forward, 5′-CGGAAAAGGCAGAAGAAACAGG-3′; reverse, 5′-TGATGACAATGGCAGGTCACC-3′; rat Rpl32 forward, 5′-GAAGCCCAAGATCGTCAAAA-3′; and reverse, 5′-AGGATCTGGCCCTTGAATCT-3′. PCR reactions were performed on an Mx3000P System (Stratagene). The relative amount of each transcript was calculated using the ΔΔCT method and normalized to the endogenous reference gene Rpl32.

Plasmin assays

Plasmin activity was assessed by two different methods, one by using a fluorimetric activity assay kit from Anaspec (San Jose, CA) and the second by utilizing the ability of plasmin to cleave fibrinogen. For both these assays, conditioned media from rat granulosa cell cultures that were concentrated using the Amicon Ultra centrifugal filter units with 10-kDa molecular weight cutoff (Millipore, Billerica, MA) was used. To concentrate the conditioned media, it was loaded in the centrifugal units and centrifuged at 7,500g for 20 minutes at 4°C. The filtrate was discarded, and concentrated conditioned media was then recovered for use in the activity assay. Total protein in the concentrated conditioned media was determined using a BioRad (Hercules, CA) detergent compatible protein assay kit as recommended by the manufacturer. This concentrated media equivalent to 1 µg total protein was then used in the activity assay as described below.

Equivalent volumes of conditioned media corresponding to 1 µg total protein and the fluorogenic peptide substrate were incubated for 60 minutes at room temperature on an orbital shaker. Enzymatic action of the plasmin on the substrate releases a fluorophore (7-amido-4-trifluoromethylcoumarin). Following incubation, the fluorescent signal was measured on a spectrophotometer (Molecular Devices, Sunnyvale, CA; excitation/emission wavelengths were 380 nm/500 nm).

The fibrinogenolytic assay was carried out as reported before (22). Briefly, 10 µg of human fibrinogen was incubated in 10 mM tris(hydroxymethyl)aminomethane HCl in the presence of human plasmin (1 U) or concentrated granulosa cell–conditioned media (1 µg total protein) at 37°C for 2 hours. To assess the specificity of plasmin cleavage of fibrinogen, human plasmin plus fibrinogen was incubated with or without the plasmin inhibitor d-Val-Phe-Lys-CMK (chloromethyl ketone dihydrochloride; 1 µM). After incubation, the reaction was stopped using a denaturing loading buffer containing 2.4 M urea. The reaction mixture was boiled and then separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The reaction products (fibrinogenolytic products) were then visualized by staining the gel with Coomassie blue.

Microarray analysis

Granulosa cells were cultured for siRNA-mediated knockdown of TFPI2 as described above. Total RNA from rat granulosa cells transfected with TFPI2 siRNAs or the negative control siRNA and treated with FSK+PMA for 6 hours was used for the microarray analysis. Five micrograms of total RNA was used as a template for complementary DNA synthesis by the University of Kentucky Microarray Core facility as described previously (23). Biotinylated antisense complementary RNA probes were prepared, and the integrity of the riboprobe was confirmed by gel electrophoresis.

The Affymetrix GeneChip Rat Gene 2.0 ST oligonucleotide array (Affymetrix, Santa Clara, CA) was hybridized, washed, and scanned as per the manufacturer’s protocol. The DNA microarray assays were performed on total RNA pooled from granulosa cells obtained from 3 separate experiments. The changes observed by DNA microarray analysis were confirmed by real-time PCR for a select subset of genes.

Progesterone assay

Progesterone in the conditioned media was assayed using an Immulite kit on an Immulite 1000 (Siemens Healthcare Diagnostics, Los Angeles, CA). Assay sensitivity was 0.2 ng/mL, and the intraassay and interassay coefficients of variation were 6.3% and 9.1%, respectively.

Determination of cell viability

Granulosa cell viability was assessed using the CellTiter Aqueous One Solution MTS assay (Promega, Madison, WI) as per the manufacturer’s instructions. This involved adding the CellTiter 96 Aqueous One reagent into the culture well at the end of the experiment and incubating at 37°C for 4 hours. The absorbance was then measured at 490 nm using an Infinite F200 plate reader (Tecan, Mannedorf, Switzerland).

Statistical analysis

All data were checked for heterogeneity of variance using Bartlett’s χ2 test. Data not normally distributed were log transformed for analysis. Changes in gene expression were analyzed by one-way analysis of variance and followed by Bonferroni posthoc tests. Data for siRNA-mediated knockdown and adenovirus-mediated overexpression were analyzed by two-way analysis of variance and followed by Bonferroni’s multiple comparison tests. All data analysis was performed using GraphPad Prizm (GraphPad Software version 5.00, La Jolla, CA). Changes were considered significantly different if P < 0.05.

Results

hCG induces TFPI2 expression in human granulosa and theca cells

Changes in TFPI2 mRNA were assessed utilizing human granulosa and theca cells obtained from the different stages of the periovulatory period. TFPI2 levels were low in the granulosa cells from the preovulatory stage but increased dramatically (∼2,000-fold) following hCG administration by the early ovulatory stage [Fig. 1(A)]. This expression remained elevated in the late ovulatory stage. Similarly, TFPI2 levels were low in the theca cells collected during the preovulatory stage and with hCG administration increased by 30- to 40-fold [Fig. 1(B)]. This increased TFPI2 expression remained elevated through the late ovulatory phase before declining by the postovulatory phase.

Figure 1.

Figure 1.

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TFPI2 expression in the human granulosa and theca cells. Human follicles were collected from women undergoing elective surgery for tubal sterilization at different stages of the periovulatory period. (A) Granulosa and (B) theca cells isolated from these samples were used to determine the mRNA levels of TFPI2 using real-time RT-PCR. Relative levels of mRNA were normalized to GAPDH in each sample and expressed as a fold change relative to the preovulatory stage. Data are presented as the mean ± standard error of the mean of the fold change in mRNA levels, and different superscripts indicate significant changes in mRNA levels (n = 4/stage; P < 0.05).

hCG induces Tfpi2 expression in the rat ovary in vivo

Changes in Tfpi2 mRNA during the rat periovulatory period were assessed in the intact ovary, isolated granulosa cells, and the residual ovarian tissue. In the intact ovaries, Tfpi2 levels were low prior to hCG administration and increased about 350-fold by 8 hours after hCG [Fig. 2(A)]. By 24 hours post-hCG, Tfpi2 levels declined to levels observed prior to hCG administration. In granulosa cells, Tfpi2 increased by 850-fold 8 hours after hCG and declined to pre-hCG levels by 24 hours in a pattern similar to that observed in intact ovaries [Fig. (2B)]. In the residual ovarian tissue, Tfpi2 levels were low prior to hCG but increased about 180-fold by 8 hours after hCG and declined to pre-hCG levels by 24 hours [Fig. 2(C)].

Figure 2.

Figure 2.

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TFPI2 expression in the rat ovary. The expression profiles of Tfpi2 mRNA in (A) the intact ovary, (B) granulosa cells, and (C) residual ovarian tissue collected from PMSG-primed immature rats at 0, 4, 8, 12, and 24 hours after administration of hCG are shown. Data are presented as the mean ± SEM of the fold change in mRNA over the 0-hour time point (n = 3 to 4/time point). Different superscripts indicate significant changes (P < 0.05).

Regulation of hCG-induced Tfpi2 expression in rat granulosa cells in vitro

Studies were performed to determine if the hCG induction of Tfpi2 mRNA observed in granulosa cells in vivo can be mimicked in vitro. In granulosa cells cultured without hCG, Tfpi2 levels were low and increased after 4 hours in culture. In cells treated with hCG, there was an ∼90-fold increase in Tfpi2 levels by 4 hours after hCG, which declined by 12 hours to control levels and remained low through the 24-hour time point [Fig. 3(A)].

Figure 3.

Figure 3.

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Regulation of Tfpi2 expression in rat granulosa cells in vitro. Granulosa cells isolated from immature rats after 48 hours of PMSG administration were used for in vitro cell culture to examine the regulation of Tfpi2 expression. (A) The induction of Tfpi2 was examined with or without hCG treatment. (B) Regulation of Tfpi2 expression in rat granulosa cells by major pathways activated by luteinizing hormone such as PKA and PKC were examined in vitro using their activators FSK and PMA, respectively, alone or in combination for 4 hours and compared with or without hCG treatment. (C) Cellular signaling pathways required for hCG-mediated induction of Tfpi2 in rat granulosa cells were examined using specific inhibitors against various cell-signaling kinases. Rat granulosa cells were treated with or without hCG in the presence of vehicle (Veh), H89 (PKA inhibitor, 10 μM), GF109203x (GF; PKC inhibitor 1 μM), LY294002 (LY; PI3K inhibitor 25 μM), U0126 (U0; MEK1/2 inhibitor, 10 μM), or SB203580 (SB; p38 MAPK inhibitor 20 μM) for 4 hours, and changes in Tfpi2 mRNA were assessed by real-time RT-PCR. (D) Similarly, rat granulosa cells were treated with or without hCG in the presence of either vehicle, AG1478 (AG; EGFR inhibitor, 1 μM), RU486 (RU; PGR antagonist, 1 μM), or NS398 (NS; PTGS2 inhibitor, 1 μM) for 4h, and changes in Tfpi2 mRNA were measured. (E and F) Thrombin-mediated signaling of hCG-induced Tfpi2 was also examined. Rat granulosa cells were cultured in the presence or absence of hCG with different concentrations of (E) thrombin or (F) the PAR1 antagonist SCH79797 (SCH) for 6 hours, and changes in Tfpi2 were measured. Data are presented as the mean ± standard error of the mean of the fold change in mRNA over the 0-hour time point or control (n = 3 to 4). Different superscripts indicate significant changes (P < 0.05) in mRNA levels.

Because cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and protein kinase C (PKC) are the main intracellular signaling pathways activated by luteinizing hormone/hCG (24), pharmacological activators of PKA and PKC kinases, FSK and PMA, respectively, were used to test if they independently or in combination induced Tfpi2 expression in rat granulosa cells. Treatment with FSK but not PMA for 4 hours induced Tfpi2 expression [Fig. 3(B)]. In contrast, treatment with FSK+PMA in combination resulted in a further increase in Tfpi2 levels beyond that induced by hCG or FSK alone [Fig. (3B)].

Specific pharmacological inhibitors targeting the different cell signaling pathways together with hCG were used to determine the pathways that are required for hCG-mediated induction of Tfpi2 mRNA in the rat granulosa cells [Fig. 3(C)]. Addition of H89 (a PKA inhibitor; 10 µM), U0126 [a mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor; 10 µM], and SB203580 [a p38 mitogen-activated protein kinase (MAPK) inhibitor; 10 µM] reduced the hCG-induced increase in Tfpi2 expression by about 60% to 70%. The PKC inhibitor GF109203x (1 μM) had no effect, although the inhibition of PI3K (LY294002; 25 μM) increased Tfpi2 levels beyond that induced by hCG alone [Fig. 3(C)].

In addition, pharmacological inhibitors or a specific antagonist were also used to ascertain the role of epidermal growth factor (EGF) receptors, progesterone receptors, and prostaglandin synthase in Tfpi2 expression in granulosa cells. The EGF receptor inhibitor AG1478 (1 µM) attenuated by ∼60% the hCG-mediated increase in Tfpi2 expression. However, neither the progesterone receptor antagonist (RU486; 1 µM) nor the prostaglandin synthase 2 inhibitor (NS398; 1 µM) had any effect [Fig. 3(D)].

As thrombin induces Tfpi2 expression in macrophages (25), we explored whether thrombin plays a role in Tfpi2 expression in rat granulosa cells [Fig. 3(E) and 3(F)]. Addition of thrombin in the absence of hCG did not have any effect on Tfpi2 levels, although in the presence of hCG, 1 U/mL thrombin doubled the hCG-induced increase in Tfpi2 [Fig. 3(E)]. Treating rat granulosa cells with a direct thrombin inhibitor, hirudin, in presence of hCG did not affect Tfpi2 expression (data not shown). However, the hCG-mediated increase in Tfpi2 was decreased by ∼80% to 99% following by addition of thrombin receptor protease-activated receptor 1 (PAR1) antagonist (SCH79797) at 2 and 4 μM [Fig. 3(F)].

TFPI2 regulates rat granulosa cell plasmin activity

To determine the functional role of TFPI2 during the periovulatory period, an mRNA knockdown approach was used using siRNAs synthesized against TFPI2. In rat granulosa cells transfected with scrambled siRNA, treatment with FSK+PMA significantly increased Tfpi2 at 24 hours of treatment compared with the vehicle-treated cells [Fig. 4(A)]. In cells transfected with Tfpi2 siRNAs, Tfpi2 was reduced by approximately 75% to 90% in cells treated with FSK+PMA compared with scrambled siRNA-transfected cells treated with FSK+PMA [Fig. 4(A); Supplemental Fig. 1A (207.8KB, TIF) ]. Knockdown of TFPI2 did not affect granulosa cell progesterone synthesis or cell viability [Fig. 4(B) and 4(C)].

Figure 4.

Figure 4.

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Effect of Tfpi2 knockdown on proteolytic activity of rat granulosa cells. Rat granulosa cells in culture were transfected with either a scrambled siRNA (white and black bars) or TFPI2 siRNAs (gray bars) and treated with vehicle (white bars) or FSK+PMA (black and gray bars). After 24 hours in culture, conditioned media and cells were collected. (A) Rat Tfpi2 mRNA in the granulosa cells and (B) progesterone concentrations in the conditioned media are shown. Granulosa cell viability was assessed following mock (white bars), scrambled (black bars), and TFPI2 (gray bars) siRNA with (hatched bars) or without (white bars) FSK+PMA using (C) an MTS assay, and data are presented as the mean ± standard error of the mean of the percent change mock transfected-vehicle treated cells. (D) Conditioned media from rat granulosa cells cultured and treated as above was used to measure the plasmin activity. For the plasmin activity assay, changes in relative fluorescent units are expressed as percent change over that for scrambled siRNA-transfected/vehicle-treated cells. Different superscripts indicate significant changes (P < 0.05; n = 4). (E) A fibrinogenolytic assay was also performed to assess the plasmin activity in the same conditioned media. Conditioned media from sample number 1 is from scrambled siRNA-transfected/vehicle-treated cells. Sample number 2 is from scrambled siRNA-transfected/FSK+PMA-treated cells. Sample 3 is from TFPI2 siRNA-transfected/FSK+PMA-treated cells. Fibrinogen breakdown products following plasmin activity are shown on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels stained with Coomassie blue.MW, molecular weight marker; Sc/Scram, scrambled; Veh, vehicle.

Plasmin activity was measured in conditioned media collected from rat granulosa cells cultured with either a scrambled siRNA treated with vehicle or FSK+PMA or Tfpi2 siRNA treated with FSK+PMA for 24 hours. In cells transfected with scrambled siRNA, treatment with FSK+PMA increased plasmin activity by 30% over vehicle-treated cells [Fig. 4(D)]. In cells with Tfpi2 knockdown, treatment with FSK+PMA increased plasmin activity further by about 115% over vehicle-treated cells [Fig. 4(D)].

A fibrinogenolytic assay was also used to test the effect of TFPI2 knockdown on plasmin activity in the granulosa cell–conditioned media. Incubation of fibrinogen with human plasmin was able to cleave the fibrinogen, and this cleavage was prevented by addition of either a plasmin inhibitor or recombinant TFPI2 [Fig. 4(E)]. Addition of conditioned media from scrambled siRNA-transfected, vehicle-treated cells (sample number 1) did not have any effect on fibrinogen cleavage, but addition of conditioned media from both scrambled siRNA or TFPI2 siRNA-transfected cells treated with FSK+PMA (sample numbers 2 and 3, respectively) increased the cleavage of fibrinogen. Addition of the plasmin inhibitor along with the conditioned media from TFPI2 siRNA-transfected cells treated with FSK+PMA did not prevent fibrinogen cleavage. However, addition of recombinant TFPI2 was able to reduce the cleavage of fibrinogen [Fig. 4(C)].

TFPI2 regulates periovulatory rat granulosa cell gene expression

To test if TFPI2 knockdown had any effect on granulosa cell gene expression, a microarray approach was used. Similar to our real-time PCR analysis results, microarray data showed a reduction in Tfpi2 levels in TFPI2 siRNA-transfected cells treated with FSK+PMA for 6 hour (data not shown). The genes that were highly down-regulated following TFPI2 siRNA knockdown are listed in Table 1. Of these, we examined Il6 and Areg by real-time PCR because of their documented association with the ovulatory process (26–28) and confirmed the microarray findings (Supplemental Fig. 1B and 1C (207.8KB, TIF) ).

Table 1.

Genes Downregulated in Luteinizing Rat Granulosa Cells Following siRNA-Mediated Knockdown of TFPI2

Transcript ID Gene Name Gene Symbol Fold Change Function
17681866 ADAM metallopeptidase with thrombospondin type 1 motif, 4 Adamts4 −2.20 Metalloproteinase
17867261 Adenylate cyclase activating polypeptide 1 Adcyap1 −1.95 Mediates transcription of neuroendocrine stress response genes by increasing cAMP
17693425 Amphiregulin Areg −1.83 Growth factor
17687609 Activating transcription factor 3 Atf3 −2.42 Transcription factor in the CREB family
17684316 B-cell Translocation Gene family, member 2 Btg2 −2.01 Regulates cell cycle G1/S transition
17693465 Chemokine (C-X-C motif) ligand 1 Cxcl1 −1.88 Chemoattractant for neutrophils
17688711 Chemokine (C-X-C motif) ligand 10 Cxcl10 −2.26 Stimulates monocytes, natural killer and T-cell migration
17823500 Deiodinase, iodothyronine, type II Dio2 −2.18 Activates thyroid hormone
17709486 Ephrin B2 Efnb2 −2.30 Stimulates migration, repulsion, and adhesion
17833546 Growth arrest and DNA-damage-inducible, beta Gadd45b −2.04 DNA repair
17749614 Histone cluster 2, H2ac Hist2h2ac −2.31 Basic nuclear protein
17816804 Heat shock protein 2 Hspa2 −2.44 Maintains cellular protein structure under stress
17803153 5-hydroxytryptamine (serotonin) receptor 1D Htr1d −1.82 Receptor for neurotransmitter serotonin
17788345 Interleukin 6 Il6 −2.65 Potent inducer of the acute-phase immune response.
17808683 Jun proto-oncogene Jun −2.12 Transcription factor
17728638 Jun B proto-oncogene Junb −2.25 Transcription factor
17661160 Keratin associated protein 4-3 Krtap4-3 −4.34 Increases stability of keratin
17632926 Protein phosphatase 1, regulatory subunit 15A Ppp1r15a −2.47 Reverses the shutoff of protein synthesis initiated by stress-inducible kinases and facilitates recovery of cells from stress
17725247 Synaptotagmin IV Syt4 −3.60 Calcium sensor
17866930 Thioredoxin domain containing 2 (spermatozoa) Txndc2 −3.76 Regulates sperm fibrous sheath assembly
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Abbreviation: CREB, cAMP response element binding protein.

To test if the increase in plasmin activity following Tfpi2 knockdown is responsible for down-regulation of these genes, we used a pharmacological inhibitor of plasmin. As shown in Figure 5(A), TFPI2 siRNA transfection led to about 80% reduction in Tfpi2 levels, although addition of plasmin inhibitor to these cells did not have any effect. Similarly, addition of the plasmin inhibitor did not have any effect on Tfpi2 siRNA-induced down-regulation of Il6 or Areg expression [Fig. 5(B) and 5(C)]. We then directly tested if TFPI2 is indeed required to increase the expression of these genes using an adenoviral vector that expresses recombinant human TFPI2. Using this approach, we restored human TFPI2 in rat granulosa cells in which rat Tfpi2 was knocked down using siRNA. Transfection of TFPI2 siRNA and infection of adenovirus-expressing human TFPI2 caused a reduction in rat Tfpi2 mRNA levels by about 90%, but human TFPI2 mRNA levels in these cells were markedly elevated [∼4,500-fold over vehicle treated; Fig. 5(D)]. TFPI2 knockdown led to reduction by about 40% to 50% in both Il6 and Areg mRNA [Fig. 5(E) and 5(F)]. The addition back of TFPI2 through overexpression of human TFPI2 increased the expression of Areg, with a trend toward an increase of Il6 [P = 0.09; Fig. 5(E) and 5(F)].

Figure 5.

Figure 5.

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Effect of rat Tfpi2 knockdown with either (A­–C) plasmin inhibition or (D–F) human TFPI2 overexpression on granulosa cell gene expression. Rat granulosa cells were transfected with scrambled siRNA or TFPI2 siRNAs and cultured overnight. To assess the effect of plasmin inhibition, a portion of cells transfected with TFPI2 siRNA was treated with plasmin inhibitor (gray horizontal hatched bars) before being stimulated with FSK+PMA. To determine the effect of human TFPI2 overexpression, a portion of cells transfected with both scrambled and TFPI2 siRNAs was infected with a control adenovirus (white and black bars), and the remaining cells were infected with an adenovirus-expressing recombinant human TFPI2 (gray bars) for 3 hours. At the end of the third hour, additional media containing vehicle (white bars) or FSK+PMA (black and gray bars) were added and cultured for 6 hours. Real-time RT-PCR was used to measure the changes in mRNA levels of rat (r) and human (h; depicted as hatched bars) Tfpi2 (A and D), Il6 (B and E), and Areg (C and F) in these cells. Data are presented as the mean ± SEM fold change in mRNA over scrambled siRNA-transfected and either vehicle-treated or control adenovirus-infected vehicle-treated cells, respectively. Different superscripts indicate significant changes (P < 0.05; n = 3). Ad-CTRL, control adenovirus; Ad-hTFPI2, adenovirus expression human TFPI2; Scram, scrambled.

Discussion

The regulation of the proteolytic activity that directs breakdown of the follicular wall and allows expulsion of the COC during the ovulatory process needs to be exquisitely controlled. This is accomplished by simultaneous expression of proteases and their inhibitors in the ovary. Here, we report that a serine protease inhibitor, TFPI2, is induced during the human and rat periovulatory period. This hCG-mediated induction of Tfpi2 expression in rat granulosa cells requires EGFR, PKA, MEK1/2, p38 MAPK, and thrombin receptor PAR1-dependent cell signaling. Utilizing siRNA, we also provide evidence that TFPI2 is a regulator of ovulatory proteolysis, as knockdown of TFPI2 in rat granulosa cells leads to an increase in plasmin activity. These data support the hypothesis that the moderation of plasmin activity is one of the major roles for TFPI2 in an ovulating follicle. A surprising finding from the current study was that TFPI2 could also regulate the expression of key periovulatory granulosa cell genes such as Il6 and Areg.

TFPI2 has been reported to have a role in ECM remodeling by virtue of being a serine protease inhibitor. TFPI2 has been shown to inhibit plasmin effectively but has no inhibitory effect on PAs (29). Our results show that TFPI2 knockdown led to an increase in specific plasmin activity in rat granulosa cell–conditioned media, suggesting that TFPI2 can regulate plasmin activity in the ovary. As plasmin cleaves fibrinogen (22, 30), the presence of cleaved fibrinogen was used as an indicator for plasmin activity in the conditioned media following TFPI2 knockdown in granulosa cells. We did not observe an increase in cleavage of fibrinogen following TFPI2 knockdown as observed with the activity assay. This may be due to the fact that in the fluorimetric activity assay, plasmin cleaves a peptide containing the plasmin-specific cleavage site, whereas fibrinogen has multiple plasmin cleavage sites as well as other sites sensitive to proteolytic cleavage by enzymes other than plasmin, such as thrombin and MMPs (31). In addition, generation of some fibrinogen degradation peptides has been shown to inhibit both fibrinogenolytic and fibrinolytic activity of the plasmin (32). Nevertheless, this assay provides the proof of principle that addition of recombinant TFPI2 and not a plasmin inhibitor was able to reverse the fibrinogenolytic activity in the conditioned media from granulosa cells with TFPI2 knockdown. This supports the concept that other serine or nonserine proteases activities are also regulated by TFPI2. In fact, TFPI2 is shown to have actions on other protease systems. For example, in other tissues, it has been reported that TFPI2 inhibition of plasmin can also prevent conversions of pro-MMP1, 3, and 13 to their active forms (17). In addition, it has been shown that TFPI2 can directly inhibit MMPs, and it inhibits collagenases more efficiently than gelatinases (11, 33). Our results indicate that TFPI2 is induced during the periovulatory period and has a role in regulating plasmin activity directly that could potentially regulate MMP activity either indirectly or directly. TFPI2 expression is reported to be up-regulated in the ovarian stroma of the women with PCOS (15), which suggests that abnormal expression of TFPI2 could adversely affect the ovarian protease activity, resulting in hyperthecosis, and therefore prevent ovulation as observed in the PCOS ovary. By these actions TFPI2 could have an active role in moderating the ECM remodeling occurring during and after the periovulatory period.

Another potential role of TFPI2 in regulating the dynamic ECM remodeling occurring during the ovulatory process may involve interaction with ADAMTS1. TFPI2 has been identified as a substrate of ADAMTS1, and cleavage by this enzyme alters TFPI2’s extracellular location (34). ADAMTS1 is induced in the granulosa cells during the periovulatory period and could therefore influence where TFPI2 action could localize (35). We attempted to localize TFPI2 with 4 different commercially available antibodies but were unable to determine if there was a spatial localization of the inhibitor. However, the importance of ADAMTS1 in the ovary is underscored by the demonstration that Adamts1 knockout mice have a reduced ovulation rate (36), but the involvement of TFPI2 in this process is unknown. These reports further underpin the importance of TFPI2 in the ovulatory process.

An ovulatory stimulus induces multiple signaling pathways in the granulosa cells, the chief among which are cAMP, PKC, EGFR, progesterone, and prostaglandins that, in turn, activate multiple signaling kinases. These signaling pathways induce or down-regulate expression of various genes that orchestrate the periovulatory process (24). In rat granulosa cells, hCG-induced Tfpi2 expression required cAMP-dependent PKA, EGFR, MEK1/2, p38 MAPK, and PAR1-dependent signaling but not PKC-dependent signaling. However, the specific pathways regulating TFPI2 expression appear to be cell dependent. For example, TFPI2 expression in cultured human endometrial stromal cells was increased by prostaglandin-induced cAMP and PKC signaling (37), which is in contrast to the present observations in the rat granulosa cells where a specific PTGS2 or PKC inhibitor did not affect Tfpi2 expression. Likewise, TFPI2 in human liver myofibroblasts was increased by ERK1/2, independent of the EGFR signaling (38), whereas in rat granulosa cells, both EGFR and ERK1/2 signaling seemed to be required. Because, in the rodent granulosa cells, ERK1/2 activation can occur through both EGF-dependent and -independent mechanisms (39), further studies are required to determine if the Tfpi2 induced in the rat required ERK1/2 and EGFR activation independent of each other.

Recent findings in the mouse ovary have shown that the thrombin signaling pathway is actively involved in periovulatory events (40). In a macrophage cell line, TFPI2 expression was increased by thrombin, and this increase was blocked by addition of the direct thrombin inhibitor hirudin (25). In the current study, thrombin increased Tfpi2 expression in the rat granulosa cells, and a PAR1 antagonist (but not hirudin) was able to abolish hCG-induced Tfpi2 expression. This suggests that thrombin-activated PAR1 signaling is required for the increase in Tfpi2 expression in rat granulosa cells. Thrombin-dependent expression of Tfpi2 required ERK1/2 in macrophages (25), but this remains to be elucidated in granulosa cells. Overall, our results suggest that multiple signaling pathways activated by hCG work in a coordinated manner to increase Tfpi2 mRNA in rat granulosa cells.

Although it has been extensively documented that TFPI2 is a serine protease inhibitor, there are limited data that it may also elicit intracellular changes potentially through an unidentified cell surface receptor (10). In smooth muscle cells, recombinant TFPI2 activated MAPK activity and increased early proto-oncogene c-fos expression, thus stimulating their proliferation (41). Additionally, proteases are known to regulate the release of growth factors and cytokines such as IGFs, EGFs, and tumor necrosis factor α (26, 27, 42–44) that could impact cellular gene expression. Acting as a protease inhibitor, TFPI2 could alter proteolytic processing of growth factor signaling, thereby affecting gene expression. In the current study, TFPI2 knockdown resulted in down-regulation of genes that are known to play important roles in the ovulatory process (43, 45, 46). Because addition of the plasmin inhibitor clearly did not have any effect on Areg or Il6, it appears that these effects may be directed by TFPI2, independent of its actions on plasmin. Thus, either through direct or indirect actions, it appears that TFPI2 has a role in granulosa cell gene expression.

In summary, we have shown that TFPI2 is induced just prior to ovulation in both human and rat ovaries and provide compelling evidence that TFPI2 can regulate granulosa cell plasmin activity. In addition, TFPI2 impacts the expression of multiple key genes that participate in the ovulatory process either dependent or independent of its known function as a protease inhibitor. These data therefore support the hypothesis that the serine protease inhibitor TFPI2 is important for the ECM remodeling as well as regulating granulosa cell gene expression that aids in the normal ovulatory process and formation of the corpus luteum.

Acknowledgments

The authors thank Carole Bryant for assay of progesterone hormone and Rajesh Rajaiah, Misung Jo, Ji Yeon Park, and Birendra Mishra for their valuable input in design of the experiments and critically reading the manuscript.

Acknowledgments

This work was supported in part by National Institutes of Health Grant HD057446 (to T.E.C.) and HD071875 (T.E.C.) and the Swedish Research Council 11607 (to M.B.).

Acknowledgments

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
ADAMTS
A disintegrin and metalloproteinase with thrombospondin motifs
cAMP
cyclic adenosine monophosphate
COC
cumulus-oocyte complex
DMSO
dimethyl sulfoxide
ECM
extracellular matrix
EGF
epidermal growth factor
FSK
forskolin
hCG
human chorionic gonadotropin
ITS
insulin, transferrin, selenium
MAPK
mitogen-activated protein kinase
MEK
mitogen-activated protein kinase kinase
MMP
matrix metalloproteinase
mRNA
messenger RNA
PA
plasminogen activator
PAI
PA inhibitor
PAR1
protease-activated receptor 1
PCOS
polycycstic ovary syndrome
PCR
polymerase chain reaction
PKA
protein kinase A
PMA
phorbol-12-myristate-13-acetate
PMSG
pregnant mare serum gonadotropin
RT
reverse transcription
siRNA
small interfering RNA
TFP12
tissue factor pathway inhibitor 2
v/v
volume-to-volume ratio

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