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. 2009 Apr 2;150(7):3291–3300. doi: 10.1210/en.2008-1527

Runt-Related Transcription Factor 1 Regulates Luteinized Hormone-Induced Prostaglandin-Endoperoxide Synthase 2 Expression in Rat Periovulatory Granulosa Cells

Jing Liu 1, Eun-Sil Park 1, Misung Jo 1
PMCID: PMC2703554  PMID: 19342459

Abstract

Runt-related transcription factor 1 (RUNX1), a transcription factor, is transiently induced by the LH surge and regulates gene expression in periovulatory granulosa cells. Potential binding sites for RUNX are present in the 5′-flanking region of the Ptgs2 (prostaglandin-endoperoxide synthase 2) gene. Periovulatory Ptgs2 expression is essential for ovulation. In the present study, we investigated the role of RUNX1 in mediating the LH-induced expression of Ptgs2 in periovulatory granulosa cells. We first determined whether the suppression of Runx1 expression or activity affects Ptgs2 expression using cultured preovulatory granulosa cells isolated from immature rat ovaries primed with pregnant mare serum gonadotropin for 48 h. Knockdown of human chorionic gonadotropin-induced Runx1 expression by small interfering RNA or inhibition of endogenous RUNX activities by dominant-negative RUNX decreased human chorionic gonadotropin or agonist-stimulated Ptgs2 expression and transcriptional activity of Ptgs2 promoter reporter constructs. Results from chromatin immunoprecipitation assays revealed in vivo binding of endogenous RUNX1 to the Ptgs2 promoter region in rat periovulatory granulosa cells. Direct binding of RUNX1 to two RUNX-binding motifs in the Ptgs2 promoter region was confirmed by EMSA. The mutation of these two binding motifs resulted in decreased transcriptional activity of Ptgs2 promoter reporter constructs in preovulatory granulosa cells. Taken together, these findings provide experimental evidence that the LH-dependent induction of Ptgs2 expression results, in part, from RUNX1-mediated transactivation of the Ptgs2 promoter. The results of the present study assign potential significance for LH-induced RUNX1 in the ovulatory process via regulating Ptgs2 gene expression.

These findings provide experimental evidence that the LH-dependent induction of Ptgs2 expression results, in part, from RUNX1-mediated transactivation of the Ptgs2 promoter.

The preovulatory gonadotropin surge initiates the ovulatory process by reprogramming the transcriptional profile of a myriad of genes in periovulatory follicles (1,2,3,4). Specific transcription factors rapidly induced by the ovulatory LH stimulus in periovulatory follicular cells play a key role in successful ovulation and/or luteinization by directly controlling the transcription of diverse effector genes (5,6). Such transcription factors identified include progesterone receptor, CAAT enhancer binding protein-β (C/EBPβ) and early growth response 1 (7,8,9). Recently our laboratory and others documented the dramatic and transient induction of runt-related transcription factor 1 (RUNX1), in rat and mice periovulatory follicles by the LH surge in naturally cycling animals and by human chorionic gonadotropin (hCG) in immature animals (1,2,10). Runx1 expression was rapidly increased within 3 h after hCG treatment and declined by 24 h after hCG (2,10). Previously we demonstrated that knockdown of Runx1 expression decreased the expression of a cohort of periovulatory genes (e.g. Cyp11a1, Mt1a, Hapln1, and Rgc32) and progesterone production in vitro (10,11), suggesting the involvement of RUNX1 in the periovulatory process. However, the functional significance in the ovulatory process and mechanisms of RUNX1 action in follicular cells remain to be determined.

RUNX1, also known as acute myeloid leukemia 1/core binding factor (CBF)-α2/polyomavirus enhancer binding protein 2αB, belongs to the mammalian RUNX family of transcription factors. The other two members identified are RUNX2 (AML3/Cbfα1/PEBP2αA) and RUNX3 (AML2/Cbfα3/PEBP2αC) (12,13,14). The RUNX proteins bind DNA through a conserved runt-homology domain (RHD), which is essentially identical and recognizes the same consensus sequences (14,15,16). To be fully functional as transcriptional regulators, the RUNX proteins must dimerize with CBFβ, a small non-DNA-binding protein that is expressed in virtually all cells (15,16). Runx1 knockout mice are embryonic lethal due to a complete block in fetal liver hematopoiesis (17). RUNX1 also has an important role in development of the myeloid and lymphoid lineages, neuronal differentiation, osteoblast growth, and the inflammatory process (12,18,19).

The ovulatory process has been viewed as a self-controlled inflammatory reaction involving an increase in the production of prostaglandins (20,21,22). Prostaglandin-endoperoxide synthase 2 (PTGS2), also called cyclooxygenase-2, is a rate-limiting enzyme in the biosynthesis of prostaglandins. Previous studies have shown that the increase in follicular prostaglandin synthesis before ovulation is caused by a rapid and transient LH-dependent induction of Ptgs2 expression in periovulatory granulosa cells (23,24,25,26,27). Studies in rats revealed that Ptgs2 mRNA was dramatically increased at 4–5 h after hCG treatment, and the PTGS2 protein remained present at 11 h but disappeared in the newly formed corpus luteum (24 h after hCG) (28). The obligatory role of PTGS2 in ovulation was underscored by genetic studies in which mice deficient in the Ptgs2 gene proved to be infertile because of compromised ovulation (29,30). The LH-induced expression of Ptgs2 in granulosa cells of the periovulatory follicle is mediated by actions of transcription factors, such as C/EBPβ, E-box elements, and upstream stimulatory factor (USF)-1 and -2 (9,31,32,33). In mouse osteoblasts, a consensus RUNX binding sequences in the 5′-flanking region of the Ptgs2 gene is required for bone morphogenetic protein-2 (BMP-2)-induced Ptgs2 expression (34,35). This finding suggests the possibility of Ptgs2 regulation by RUNX transcription factors in ovarian cells.

Given the temporal and spatial expression pattern between Runx1 and Ptgs2 after the LH surge and the presence of RUNX binding sites in the promoter region of the Ptgs2 gene, we hypothesized that LH-induced RUNX1 plays a key role in regulating the transcription of the Ptgs2 gene in periovulatory granulosa cells. The present study tested this hypothesis by: 1) determining the effect of suppressing LH-induced Runx1 expression or inhibiting RUNX activity on Ptgs2 expression and 2) examining the significance of RUNX binding sites in the transcriptional activity of Ptgs2 promoter constructs in periovulatory granulosa cells. The direct interaction of RUNX1 with the Ptgs2 gene in periovulatory granulosa cells was analyzed using both in vivo and in vitro models.

Materials and Methods

Animals

All animal procedures were approved by the University of Kentucky Animal Care and Use Committee. Sprague Dawley rats were obtained from Harlan, Inc. (Indianapolis, IN) and provided with water and chow ad libitum. Immature female rats (22 or 23 d old) were injected with pregnant mare serum gonadotropin (PMSG, 10 IU) sc to stimulate follicular development. Forty-eight hours later, the animals were injected with hCG (10 IU) sc to induce ovulation. Animals were killed at 0 h (48 h after PMSG) or defined times after hCG administration.

Isolation and culture of rat granulosa cells

Ovaries were collected from immature rats 48 h after PMSG administration and punctured to isolate granulosa cells as previously described (2,10). The cells were pooled, filtered, pelleted by centrifugation at 200× g for 5 min, and resuspended in Opti-MEM (Life Technologies, Inc., Grand Island, NY) media supplemented with 0.05 mg/ml gentamicin and 1× insulin, transferrin, and selenium. The cells were cultured at 37 C in a humidified atmosphere of 5% CO2. Granulosa cells cultured under these conditions acquire a periovulatory phenotype (36) and respond to an ovulatory dose of hCG (1 IU/ml) as demonstrated by the induction of specific genes, including Runx1 and Ptgs2 within 3–6 h.

Knockdown of RUNX1 by small interfering RNA (siRNA) in granulosa cells in vitro

Granulosa cells were collected from immature rats 48 h after PMSG administration. A specific siRNA against Runx1 (5′-CAAACCUGAGGUCGUUGAAUCUCGC-3′; Runx1 stealth select RNA interference; Invitrogen, Carlsbad, CA), Runx2 (5′-GCACGCUAUUAAAUCCAAAtt-3′; 5′-UUUGGAUUUAAUAGCGUGCtg-3′; Ambion, Inc., Austin, TX) or scrambled siRNA (stealth RNA interference negative control; Invitrogen) was transfected into granulosa cells using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Four hours later, transfection media were replaced with fresh culture media containing hCG (1 IU/ml) and the cells were further cultured for 6 h. The cells were collected and snap frozen for later isolation of total RNA for real-time PCR or processed to prepare cell lysates for Western blot analyses.

Quantification of mRNA

The levels of mRNA for Runx1, Ptgs2, Runx2, 20α-HSD, p21, and TGF-β1 were measured by real-time PCR. Briefly, total RNA was isolated from granulosa cells using Trizol (Invitrogen). Synthesis of first-strand cDNA was performed by reverse transcription of 1.0 μg total RNA using SuperScript III with Oligo(dT)20 primer (Invitrogen) according to the manufacturer’s protocol. Oligonucleotide primers corresponding to cDNA for rat L32 (forward 5′-GAAGCCCAAGATCGTCAAA A-3′, reverse 5′-AGGATCTGGCCCTGGCCCTTGAATCT-3′); Runx1 (forward 5′-AACCCTCAGCCTCAAAGTCA-3′, reverse 5′-GGGTGCACAGAAGAGGTGAT-3′); Ptgs2 (forward 5′-GATCACATTTGATTGACAGC-3′, Reverse 5′-TCCTTATTTCCTTTCACACC-3′); Runx2 (forward 5′-CTCACTACCACACGTACCTGC-3′, reverse 5′-ATAGGACGCTGACGAAGTACC-3′); 20α-HSD (forward 5′-G ATAGGTCAGGCCATTGTAAGC-3′, reverse 5′-CGGGAAATGAATGAGATAGAGG-3′); p21 (forward 5′-ACCCCTGTTTCTGTAACACC-3′, reverse 5′-GAAGTATTTATTGAGCACCAGC-3′); and TGF-β1 (forward 5′-AATTCCTGGCGTTACCTTGG-3′, reverse 5′-AAGCGAAAGCCCTGTATTCC-3′) were designed using Primer3(Whitehead Institute for Biomedical Research, Cambridge, MA; http://fokker.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi; Ref. 63) software. The specificity for each primer set was confirmed by both electrophoresis of the PCR products on a 2.0% agarose gel and analyzing the melting (dissociation) curve using a MxPro real-time PCR analysis program (Stratagene, La Jolla, CA) after each real-time PCR. The real-time PCRs were carried out as previously described (11). The relative amount of transcripts was calculated using the 2−ΔΔCT method (37) and normalized to the endogenous reference gene L32.

Western blot analysis

Granulosa cell lysate was prepared using radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology, Santa Cruz, CA). The protein concentration in the lysate was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). Thirty micrograms of protein were separated on a 7.5% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were incubated with antibodies against RUNX1 (1:200; Active Motif, Carlsbad, CA), PTGS2 (1:400; Cayman Chemical, Ann Arbor, MI), or β-actin (1:1000; Cell Signaling Technology, Danvers, MA), overnight at 4 C. Western blotting was analyzed using an enhanced chemiluminescence detection system (Pierce, Rockford, IL) and exposed to x-ray film.

Chromatin immunoprecipitation (ChIP) analysis

ChIP assay was performed on RUNX1 in the Ptgs2 promoter region using a ChIP kit (Upstate Biotechnology, Inc., Lake Placid, NY) as described previously (11). Briefly, the periovulatory granulosa cells were isolated from PMSG-primed immature rat ovaries at 8 h after hCG injection. The cells were treated with 1% formaldehyde and lysed in lysis buffer to release nuclei. The nuclei were sonicated with a Fisher sonic dismembrator model 550 (Thermo Fisher Scientific, Inc., Waltham, MA) to obtain DNA fragments of an average length of approximately 100–500 bp. Chromatin was immunoprecipitated overnight at 4 C with anti-RUNX1 (5 μg/reaction; Calbiochem) or normal rabbit IgG (5 μg/reaction; Santa Cruz Biotechnology) as a negative control. The immunoprecipitated chromatin and 1:10 dilution of input chromatin were analyzed by PCR using the primers designed to amplify fragments spanning the RUNX motif in the Ptgs2 promoter (see Fig. 2B, distal forward, 5′-CTT AAA GCA ATG CGG TGG AC-3′, distal reverse, 5′-GAA ATG AGA GGG CTG CTG TC-3′, and proximal forward, 5′-GGG GAA GCT GTG ACA TTC TC T-3′, proximal reverse, 5′-CCA TAG GGG CAG GCT TTA CT-3′). After 25–30 cycles of amplification, PCR products were run on a 2% agarose gel, stained with ethidium bromide, and visualized under UV light.

Figure 2.

Figure 2

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Endogenous RUNX1 binds to the Ptgs2 promoter region in periovulatory granulosa cells. A, The rat Ptgs2 promoter nucleotide sequence was analyzed using a genomic library. Nucleotide sequences were numbered from transcription start site at +1. Putative transcription factor binding sites (boxed sequences) were predicted by a TFSEARCH program. B, ChIP detection of RUNX1 transcription factor binding to the rat Ptgs2 promoter region in periovulatory granulosa cells. Ptgs2 proximal (227 bp) and Ptgs2 distal (159 bp) DNA fragments containing RUNX binding sites were enriched in chromatin samples immunoprecipitated with RUNX1 antibody as well as input DNA. C, EMSA of the proximal and distal probe containing RUNX binding sites. Lane 1, nuclear extract alone for negative control; lanes 2 and 5, labeled proximal and distal probe, respectively; lanes 3 and 6, 200-fold molar excess of unlabeled wild-type of the proximal and distal probe (wt), respectively; lanes 4 and 7, 200-fold molar excess of unlabeled mutant of the proximal and distal probe (mt), respectively; lane 8, labeled proximal probe as a negative control for supershift analysis; lane 9, labeled proximal probe (p) plus anti-RUNX1 antibody; lane 10, labeled distal probe (d) plus anti-RUNX1 antibody. Experiments were repeated at least five times, each with different granulosa cell samples.

EMSA

Annealed wild-type probes of the proximal RUNX binding motif (5′-AGCAGGCACAGCGAACCACAGGGCGCCTGGAAGGA-3′) and distal binding motif (5′-ATACCTTAAAGCAATGCGGTGGACACTTAGCATTC-3′) were labeled with biotin-11-deoxyuridine 5-triphosphate using a biotin 3′ end DNA labeling kit (Pierce). GATATC replaced the consensus RUNX binding sequence ACCACA or TGCGGT in mutant unlabeled probes. Nuclear protein was isolated from periovulatory granulosa cells using a nuclear extract kit (Active Motif). EMSA was performed using the LightShift chemiluminescent EMSA kit (Pierce). Briefly, 5 μg of nuclear extracts were incubated with different unlabeled competitor probes (10 pmol) and biotin end-labeled wild-type probe (50 fmol) in binding reaction buffer for 20 min. For supershift assays, nuclear extract and labeled wild-type probes were incubated in binding reaction buffer for 20 min, and 1 μl RUNX1 antibody (Active Motif) was added for another 30 min. Samples were run on a 6% nondenaturing polyacrylamide gel in 0.5M Tris borate EDTA buffer at 100 V for 1–2 h. Probes were transferred to a nylon membrane, baked at 80 C in the vacuum oven for 1 h for cross-linking, and developed using a chemiluminescence nucleic acid detection system (Pierce).

Cloning of the rat Ptgs2 promoter and generation of Ptgs2 promoter-reporter plasmid constructs

Genomic DNA was isolated from tail samples from rats using an easy-DNA kit (Invitrogen). A 693-bp (−673/+20) fragment and 220-bp (−200/+20) fragment of the Ptgs2 gene was amplified using the primers attached with restriction enzyme sites (KpnI and BglII) and cloned into the pCRII-TOPO vector (Invitrogen) as described previously (2). Cloned fragments were digested with KpnI and BglII enzyme and subcloned into a multiple cloning site of the pGL3 basic vector (Promega, Madison, WI). Site-directed point mutations of the Ptgs2 promoter were generated using a QuickChange II site-directed mutagenesis kit according to the manufacturer’s protocol (Stratagene). The sequences of the oligonucleotide primers used to generate respective Ptgs2 promoters containing mutations (shown in lowercase) are following: mutant A (5′-AGGTACCACCTTAAAGCAATGattTGGACACTTAGCATTC CGACG-3′), mutant B (5′-AGTCTTGGAGCAGGCACAGCGActtACAGGGCGCCTG-3′), mutant D (5′-GAGTAAAGCCTGCCCCTATGGGTAgacTagtATTGGAAGCGGAGATGGGGGAAAG-3′), mutant E (5′-GAAAGACACAGTCACGAAGTCACtTtGAGTCCACTTTACTAAGATTTAA-3′). All constructs cloned into the expression vector were sequenced commercially to verify their authenticity (MWG Biotech, Inc., High Point, NC).

Transient transfection and luciferase reporter assay

Granulosa cells were isolated from immature rats (48 h after PMSG) as described above. The cells were transfected with respective firefly luciferase reporter plasmids (pGL3-basic vector or pGL3-Ptgs2 promoter constructs) and Renilla luciferase vector (pRL-TK vector) using a Lipofectamine 2000 reagent (Invitrogen). Fresh culture medium was added 4 h after transfection. The next day, cells were treated with forskolin (FSK; 10 μm), phorbol 12-myristate 13-acetate (PMA; 20 nm), or FSK + PMA for 6 h. The cells were harvested to measure Firefly and Renilla luciferase activities using a dual-luciferase reporter assay system (Promega), and each reaction was monitored for 10 sec by luminescence system in the Tecan Infinite 200 microplate reader (Tecan U.S., Durham, NC). Firefly luciferase activities were normalized by Renilla luciferase activities and each experiment was performed in triplicate at least three times.

Construction of RUNX1 and dominant-negative RUNX (DNRUNX) recombinant adenovirus

The RUNX1 and DNRUNX recombinant adenovirus were constructed using an AdEasy XL adenoviral vector system kit (Stratagene). The processes for generating and propagating recombinant adenoviruses were performed according to the manufacturer’s instructions. Briefly, a full length of rat Runx1 gene and 411-bp fragment of RHD coding region were cloned into a multiple cloning site of the pShuttle-CMV vector. The pShuttle-CMV-RUNX1 and pShuttle-CMV-RHD plasmids were linearized with PmeI enzyme and then transformed into BJ5183-AD-1 cells. The recombination event that took place in the BJ5183-AD-1 cells resulted in the production of recombinant AdEasy-RUNX1 and AdEasy-RHD plasmid DNA. After selecting colonies by restriction of recombinant DNA with PacI enzyme, the recombinant AdEasy-RHD plasmid was amplified in XL10-Gold cells. Ten micrograms of linearized AdEasy-RUNX1 and AdEasy-RHD plasmid were transfected into human AD-293 cells by the calcium phosphate precipitation method. After transfection the cells were cultured in DMEM media (Life Technologies) with 10% fetal bovine serum for 7–10 d at which time the cells exhibited viral cytopathic effect. Adenoviruses were collected and the titer of virus stocks was determined using the AdEasy viral titer kit (Stratagene).

Overexpression of RUNX1 and DNRUNX after adenoviral infection in vitro

Granulosa cells were isolated from immature rats (48 h after PMSG) as described above. The cells were plated and subsequently incubated with adenoviral RUNX1 (AdEasy-RUNX1), DNRUNX (AdEasy-RHD), or green fluorescent protein (GFP; AdEasy-GFP) at 10 multiplicity of infection (MOI) for 24 h. The next day, cells were treated with FSK plus PMA for 6 h. The cells were collected for isolation of total RNA for real-time PCR or protein for Western blot analysis. To assess the functional relevance of RUNX1 on Ptgs2 promoter activity, cells were transfected with pGL3-Ptgs2 promoter constructs and pRL-TK vector, and fresh medium containing adenoviral RUNX1, DNRUNX, or GFP was added 4 h after transfection. Twenty-four hours later, adenoviral GFP-, RUNX1-, or DNRUNX-infected cells were incubated for 6 h with FSK plus PMA, and luciferase reporter assay was performed as described above.

Statistical analyses

All data are presented as means ± sem. One-way ANOVA was used to test differences in luciferase activities of respective Ptgs2 promoter constructs among treatments and in levels of Runx1 and Ptgs2 mRNA and protein among treatments. If ANOVA revealed significant effects of treatments, the means were compared by Tukey’s test, with P < 0.05 considered significant.

Results

Knockdown of RUNX1 expression reduced the expression of Ptgs2 in preovulatory granulosa cell cultures

To determine whether LH-induced RUNX1 regulates the expression of the Ptgs2 gene, the rat preovulatory granulosa cells were transfected with specific siRNA for Runx1 and cultured in the presence of hCG for 6 h. Real-time PCR data showed that Runx1 siRNA effectively attenuated hCG-induced expression of Runx1 mRNA compared with that of hCG plus scrambled siRNA-treated cells (Fig. 1A). As shown in Fig. 1B, Western blotting results confirmed the knockdown of RUNX1 protein. It has previously been shown that Ptgs2 is rapidly and transiently induced by hCG in preovulatory granulosa cells in vitro (24,25,28). Real-time PCR and Western blot data revealed that the hCG-stimulated expression of Ptgs2 was dramatically decreased in Runx1 siRNA-treated cells (Fig. 1, A and B). These data indicated that the Ptgs2 gene may be a direct target of LH-induced RUNX1. In the cells treated with Runx1 siRNA, levels of Hapln1 mRNA were also significantly decreased as comparable with that of a previous study (10). However, the expression of Runx2 as well as other LH-induced genes including 20α-HSD, p21, and TGF-β1 was not affected by Runx1 siRNA (Fig. 1C), confirming the specific effect of RUNX1 silencing on Ptgs2 expression.

Figure 1.

Figure 1

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Knockdown of Runx1 expression reduced the expression of Ptgs2 in preovulatory granulosa cell cultures. Granulosa cells obtained from rat preovulatory ovaries (48 h after PMSG) were transfected with scrambled siRNA or Runx1 siRNA, treated with hCG (1 IU/ml), and cultured for 6 h. A, Levels of Runx1 and Ptgs2 in granulosa cell cultures were measured by real-time PCR. Levels of mRNA for Runx1 and Ptgs2 were normalized to the L32 in each sample (mean ± sem; n = 5 independent experiments). B, RUNX1 and PTGS2 protein in granulosa cell cultures were assessed by Western blotting analysis. C, Levels of Runx2, 20α-HSD, p21, and TGF-β1 mRNA in granulosa cell cultures were measured by real-time PCR. Levels of mRNA for Runx2, 20α-HSD, p21, and TGF-β1 were normalized to the L32 in each sample (mean ± sem; n = 5 independent experiments). Experiments were repeated at least five times, each with different granulosa cell samples. Bars with no common superscripts are significantly different (P < 0.05).

Endogenous RUNX1 binds to the Ptgs2 promoter region in periovulatory granulosa cells

The putative promoter region (1.0 kb upstream of transcription start site) of the rat Ptgs2 gene was analyzed using a TFSEARCH program (http://www.cbrc.jp/research/db/TFSEARCH. html). The analysis revealed several putative RUNX binding sites, with two consensus binding sequences (Fig. 2A).

To investigate whether RUNX1 specifically binds to these candidate sites in the Ptgs2 promoter in vivo, we performed ChIP assays on chromatin samples extracted from periovulatory granulosa cells. PCR analysis revealed that immunoprecipitation of endogenous RUNX1 enriches chromatin fragments containing the RUNX binding sequence in both the proximal and distal promoter region compared with that of normal rabbit IgG immunoprecipitation (Fig. 2B). This result demonstrated that endogenous RUNX1 associates with the Ptgs2 promoter in periovulatory granulosa cells.

To further determine the interaction between RUNX1 protein and RUNX binding motifs in the Ptgs2 promoter, the 35-bp probes including the proximal motif (ACCACA) or distal motif (TGCGGT) were incubated with periovulatory granulosa cell nuclear extracts and subjected to EMSA. The hybridization reactions resulted in a shift of both proximal and distal probes, demonstrating an interaction of a nuclear transcription factor(s) in periovulatory granulosa cells with RUNX binding motifs (Fig. 2C). The binding was competed by an excess of unlabeled wild-type probe but not by an unlabeled probe carrying a mutation of the ACCACA or TGCGGT motif to GATATC, indicating the interaction is specific for the RUNX binding motif. Addition of RUNX1 antibody to probe/extract mixtures led to the formation of supershift bands, confirming that RUNX1 is indeed able to interact with these motifs on the Ptgs2 promoter region (Fig. 2C).

Transactivation of Ptgs2 promoter reporter constructs was reduced by mutation of RUNX binding sequences in preovulatory granulosa cell cultures

Two rat Ptgs2 promoter reporter constructs (−673/+20 and −200/+20 bp) were transfected to preovulatory granulosa cells and stimulated with FSK, PMA, or FSK plus PMA for 6 h. These respective protein kinase A and protein kinase C activators were used to induce Ptgs2 and Runx1 expression in preovulatory granulosa cells by mimicking the action of an ovulatory dose of LH/hCG (10,11,38). Both FSK and PMA treatments increased the luciferase activities of −200/+20 and −673/+20 bp reporter constructs compared with that of control cultures, and FSK plus PMA had synergistic effects on transactivation of the Ptgs2 promoter (Fig. 3A). Noticeably, FSK- plus PMA-stimulated transactivation of the full-length Ptgs2 promoter construct (−673/+20 bp) was significantly higher than that of the truncated promoter reporter construct (−200/+20 bp), suggesting that the distal region (−661/−212 bp) containing multiple RUNX binding sites enhanced the activity of the Ptgs2 promoter (Fig. 3A).

Figure 3.

Figure 3

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Transcriptional activity of Ptgs2 promoter reporter constructs was reduced by mutation in RUNX binding sequences in preovulatory granulosa cell cultures. A, Granulosa cells isolated from gonadotropin-primed immature (48 h after PMSG) were transiently transfected with empty luciferase reporter vector (LUC), −200/+20 bp, or −673/+20 bp Ptgs2-luciferase reporter constructs, treated with FSK (10 μm), PMA (20 nm), or FSK+PMA and cultured for 6 h. Firefly luciferase activities were normalized by Renilla luciferase activities. B, Granulosa cells isolated from gonadotropin-primed immature rats (48 h after PMSG) were transiently transfected with empty luciferase reporter vector (LUC), wild-type, mutant A, mutant B, mutant C, mutant D, or mutant E Ptgs2-luciferase reporter constructs, treated with FSK (10 μm)+PMA (20 nm) and cultured for 6 h. Fold change of FSK+PMA-induced luciferase activities were normalized by basal levels. Each experiment was performed in triplicate and the experiment was repeated at least three times. Bars with no common superscripts are significantly different (P < 0.05).

To determine the roles of consensus RUNX binding sites within the −661/−212 bp region of Ptgs2 promoter, site-directed mutants of the distal binding site (−656/−651 bp, mutant A) and proximal binding site (−260/−255 bp, mutant B), double mutation of these two sites (mutant C) as well as additional mutation of C/EBP (mutant D) or USF binding site (mutant E) were generated and transfected into cultured preovulatory granulosa cells in the presence of FSK plus PMA (Fig. 3B). The results indicated that mutation of either the distal or proximal RUNX binding site markedly decreased FSK+PMA-induced activity of Ptgs2 promoter from 5.6- to 3.5-fold (Fig. 3B). The double mutation of both regions of consensus RUNX binding sites had no significantly additive effect on the agonist-induced fold change of the Ptgs2 promoter activity (Fig. 3B). However, the additional mutation of either C/EBP or USF binding site resulted in a significantly additive decrease in the fold induction by FSK+PMA vs. control compared with that of wild-type control plasmid (Fig. 3B). These data suggest that coordinate action of multiple transcription factors including RUNX1, C/EBPβ, or USF is required for full transactivation of the Ptgs2 promoter.

DNRUNX reduced Ptgs2 expression in preovulatory granulosa cell cultures

It has been well documented that truncated RUNX containing only RHD acts as a DNRUNX by competing with RUNX transcriptional factors for DNA binding and inhibiting their transcriptional activities (39,40,41,42). To further clarify the role of RUNX proteins in LH-induced Ptgs2 expression, we developed adenoviral DNRUNX (Ad DNRUNX). The preovulatory granulosa cells were infected with Ad DNRUNX or viral control (Ad GFP) at 10 MOI. The efficiency of infection, as examined by observing granulosa cells infected with Ad GFP for 24 h under a fluorescence microscope, was more than 80% (data not shown). The expression of DNRUNX construct was confirmed by real-time PCR: a 300-fold higher RHD fragment was detected in Ad DNRUNX-infected cells than controls (Fig. 4, A and B). As expected, levels of endogenous RUNX1 expression were not changed in Ad DNRUNX-infected cells compare with that of control cells (Fig. 4, B and D).

Figure 4.

Figure 4

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DNRUNX reduced Ptgs2 expression in preovulatory granulosa cell cultures. A, A schematic representation of the RUNX1 protein structure. The numbers indicate amino acid numbers in each polypeptide. The RHD is boxed. The primers used for the real-time PCR analysis are designated by arrows. B, Real-time PCR data showing mRNA expression of DNRUNX in rat granulosa cells infected with Ad DNRUNX at 10 MOI for 24 h. The Ad GFP was used as a negative control for the viral infection effect on cells. RHD primers were designed to recognize both Runx genes and DNRUNX, whereas Runx1 primers recognize only Runx1 mRNA. C, Real-time PCR analysis was used to measure the levels of Ptgs2 mRNA in the rat granulosa cells. The cells were isolated from gonadotropin-primed immature rats (48 h after PMSG), infected with Ad GFP or Ad DNRUNX at 10 MOI, treated with FSK (10 μm) + PMA (20 nm), and cultured for 6 h. Levels of mRNA for Ptgs2 were normalized to the L32 in each sample (mean ± sem; n = 5 independent experiments). Bars with no common superscripts are significantly different (P < 0.05). D, The PTSG2, RUNX1, and RUNX2 protein in granulosa cell cultures was detected by Western blotting analysis. Experiments were repeated at least five times, each with different granulosa cell samples.

The mRNA and protein levels of Ptgs2 were assessed in cultured granulosa cells infected with Ad DNRUNX and Ad GFP. FSK+PMA treatment induced a dramatic increase in Ptgs2 expression in both Ad GFP-infected and no virus-infected control cells. The overexpression of DNRUNX significantly attenuated the FSK+PMA-stimulated expression of Ptgs2 (Fig. 4, C and D), indicating that RUNX protein(s) is actively involved in LH-induced Ptgs2 expression.

Transcriptional activity of the Ptgs2 promoter was reduced by RUNX1 knockdown or DNRUNX overexpression and increased by RUNX1 overexpression in preovulatory granulosa cell cultures

To further determine whether knockdown of Runx1 expression affects the transcriptional activity of the Ptgs2 promoter, the −673/+20 bp construct and Runx1-specific siRNA were cotransfected into cultured preovulatory granulosa cells stimulated with FSK+PMA. The luciferase activity assay revealed that the FSK+PMA-stimulated transcriptional activity of the Ptgs2 promoter reporter construct was significantly reduced by 40% in Runx1 siRNA-treated granulosa cells compared with cells with scrambled siRNA (Fig. 5A). Likewise, infection of granulosa cells with the adenoviral DNRUNX at a MOI of 10 significantly decreased FSK+PMA-induced Ptgs2 promoter activity (Fig. 5B). Next, to determine whether the overexpression of RUNX1 affects Ptgs2 promoter activity, we established adenoviral RUNX1 (Ad RUNX1) to overexpress wild-type RUNX1 protein. The luciferase reporter assay showed that infection of granulosa cells with the Ad RUNX1 significantly increased the fold-induction of FSK+PMA-stimulated Ptgs2 promoter activity compared with that of Ad GFP-infected cells (Fig. 5C).

Figure 5.

Figure 5

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Transcriptional activity of the Ptgs2 promoter was reduced by RUNX1 knockdown or DNRUNX overexpression and increased by RUNX1 overexpression in preovulatory granulosa cell cultures. A, Granulosa cells isolated from gonadotropin-primed immature rats (48 h after PMSG) were transiently transfected with −673/+20 bp Ptgs2-luciferase reporter constructs in the presence of scrambled siRNA or Runx1 siRNA, treated with FSK (10 μm) + PMA (20 nm), and cultured for 6 h. B, Granulosa cells isolated from gonadotropin-primed immature rats (48 h after PMSG) were transiently transfected with −673/+20 bp Ptgs2-luciferase reporter constructs and infected with Ad GFP or Ad DNRUNX at 10 MOI, treated with FSK (10 μm) + PMA (20 nm), and cultured for 6 h. Firefly luciferase activities were normalized by Renilla luciferase activities. Each experiment was performed in triplicate and the experiment was repeated at least three times. Bars with no common superscripts are significantly different (P < 0.05). C, Granulosa cells isolated from gonadotropin-primed immature rats (48 h after PMSG) were transiently transfected with −673/+20 bp Ptgs2-luciferase reporter constructs and infected with Ad GFP or Ad RUNX1 at 10 MOI, treated with or without FSK (10 μm) + PMA (20 nm), and cultured for 6 h. Fold change of FSK+PMA-induced luciferase activities were normalized by basal levels. Western blotting analysis shows overexpression of RUNX1 protein in rat granulosa cells infected with Ad RUNX1 at 10 MOI for 24 h. The experiments were repeated at least three times and analyzed by paired t tests. *, P < 0.05.

Discussion

Successful ovulation requires the induction of specific transcription factors that drive the expression of pertinent downstream genes in periovulatory follicular cells. The expression of Ptgs2 induced in periovulatory follicles is responsible for the production of prostaglandins that play an essential role in ovulation (22,23,26,27,28,30). In this study, we demonstrated a functional link between LH-induced transcription factor RUNX1 and Ptgs2 gene expression in periovulatory granulosa cells by showing: 1) suppression of Runx1 expression resulted in reduced Ptgs2 expression, 2) RUNX1 responsiveness of Ptgs2 promoter activity, and 3) RUNX1 binding to the endogenous Ptgs2 gene.

RUNX1 is a member of the RUNX family of transcription regulators that also includes RUNX2 and RUNX3 (12,13). RUNX proteins contain a highly conserved region of 128 amino acids in their N-terminal portion referred to as a RHD domain that is responsible for both DNA binding and heterodimerization with its binding partner, CBFβ (14). The unique feature of the RHD allowed us to generate a dominant-negative inhibitor for RUNX proteins. Infection of an adenovirus carrying DNRUNX into preovulatory granulosa cells resulted in reduced Ptgs2 gene expression and promoter activity comparable with those of Runx1 knockdown approaches, providing strong support for the role of RUNX1 in the up-regulation of Ptgs2 gene expression.

In an effort to delineate the molecular mechanism by which RUNX1 regulates Ptgs2 gene expression, we studied the rat Ptgs2 gene and identified two consensus RUNX binding motifs in the Ptgs2 promoter region. The direct binding of RUNX1 to the Ptgs2 promoter was observed in vivo by ChIP analysis, and the ability of RUNX1 binding to these consensus sequences in vitro was verified by EMSA. In addition, mutation of RUNX consensus sequence significantly reduced the agonist-stimulated luciferase activity of Ptgs2 promoter reporter constructs. Taken together, these data provided experimental evidence that the Ptgs2 gene is a direct downstream target of RUNX1 in periovulatory granulosa cells.

Interestingly, the mutation of RUNX consensus sequence did not completely obliterate the agonist-induced Ptgs2 promoter reporter activity, whereas the overexpression of RUNX1 protein increased the agonist-stimulated Ptgs2 promoter activity but not for basal promoter activity, indicating the involvement of other functional transcriptional regulators in LH-induced Ptgs2 expression. The finding that the additional mutation of either C/EBP or USF binding site resulted in a significantly additive decrease in agonist-stimulated transactivation of the Ptgs2 promoter indicated C/EBPβ and USF transcription factors as cotranscriptional regulators of the Ptgs2 gene. This observation agrees with those of others (9,31,32,33) that additional elements located within the −200/+20 bp region, such as C/EBP (−143/−130 bp) and USF (−56/−49 bp), are involved in the transcriptional regulation of the Ptgs2 gene. C/EBP has been proposed to act as an auxiliary factor in the transcription of the Ptgs2 gene in various cells (43,44,45). In granulosa cells, C/EBPβ is induced in response to an LH stimulus and the consensus C/EBP binding sequence is functionally relevant to the transcriptional activity of the Ptgs2 promoter (9). RUNX1 and RUNX2 have been shown to interact with C/EBP factors (α, β and δ) in a synergistic manner to enhance the transcription of specific genes in nonovarian cells (46,47). Thus, it is possible that RUNX1 may interact with C/EBP or USF to synergistically enhance Ptgs2 promoter activity in periovulatory granulosa cells.

The expression of Runx1 was induced in not only mural granulosa cells but also cumulus cells of periovulatory follicles (1,10,48). Runx1 expression was highly induced in mouse cumulus-oocyte complexes (COCs) at 8–12 h and maintained in ovulated COCs at 16 h after hCG administration in vivo (1). In the in vitro studies, the expression of Runx1 was induced by FSH and ampiregulin, an epidermal growth factor-related peptide, both of which are known to alter gene expression profiles in cumulus cells leading to COC expansion (1,48). Our observations confirmed the similar increase in Runx1 expression in rat COCs both in vivo and in vitro (data not shown). Ptgs2 expression was also increased in cumulus cells of periovulatory follicles by hCG in vivo (49) and stimulation with FSH or ampiregulin in vitro (49,50). Importantly, cumulus expression of Ptgs2 is crucial for COC expansion and meiotic maturation in the oocyte (29,30,51). Taken together, it is conceivable that RUNX1 is involved in cumulus expression of the Ptgs2 gene in periovulatory follicles, which is critical for COC expansion and oocyte maturation.

A recent study showed that the ectopic expression of Runx1, Runx2, or Runx3 genes in the common cell background induced an indistinguishable phenotype and a strongly overlapping change in global transcription patterns, suggesting a high degree of functional redundancy in the RUNX family (52). In mouse osteoblasts, RUNX2 mediates bone morphogenetic protein 2 (BMP2)-induced Ptgs2 expression by enhancing the transcriptional activity of the Ptgs2 promoter (34,35). In the ovary, up-regulation of Runx2 expression was documented in granulosa cells and COCs of periovulatory follicles after hCG injection in mice and rats (1,53). Therefore, we tested whether RUNX2 is involved in periovulatory Ptgs2 expression by suppressing Runx2 expression by siRNA in vitro and found no effect on Ptgs2 mRNA expression (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). These data suggest that RUNX1, not RUNX2, is involved in Ptgs2 expression in rat granulosa cells. However, it is noteworthy that unlike Runx1, the expression of which was transient and peaked at 6–8 h after hCG (2,10), Runx2 mRNA levels began to increase at 4 h after hCG, continued to increase at 12 h and remained high in newly formed corpora lutea (24 h after hCG) (supplemental Fig. 2). We also confirmed corresponding increases in RUNX2 protein; the levels were highest at 24 h after hCG in vivo and at 48 h after hCG treatment in vitro (our unpublished data). These observations suggested differential contribution of RUNX1 vs. RUNX2 during the early vs. late/postovulatory period, respectively, in the rat ovary. Support of this notion came from our recent findings that RUNX1 was involved in the preovulatory granulosa cell expression of Rgc32 (10,11), whereas luteal expression of Rgc32 was regulated by RUNX2 (our unpublished data). The Rgc32 gene was also shown to be a common target of RUNX1, RUNX2, and RUNX3 transcription factors in nonovarian cells (52).

It is well established that RUNX1 plays an important role in the immune system and the inflammatory response (12,54,55,56,57,58,59,60,61). The expression of Runx1 is required for T and B lymphocyte development (57,58,59). Misregulation of Runx1 expression is associated with human inflammatory and autoimmune diseases (54,55,56,60,61). Notably, some of the immune-related genes regulated by RUNX1 in nonovarian cells, such as Alcam (52), Pdcd1 (55), and Cd97a (59), were also found to be expressed in periovulatory follicular cells (1). The Ptgs2 gene is often referred to as an inducible effector involved in inflammatory and immune processes (62). Therefore, it is likely that RUNX1 mediates the transcription of a subset of immune-related gene expression in periovulatory follicular cells, contributing to the immune/ inflammation-associated function during the ovulatory process.

In summary, this is the first study to provide direct experimental evidence that LH-induced RUNX1 regulates the expression of Ptgs2 gene by enhancing the transactivation of the Ptgs2 promoter in periovulatory granulosa cells before ovulation. Given the obligatory role of the LH-induced Ptgs2 in ovulation and COC expansion, results of the present study provide new insight into our understanding of the molecular mechanisms responsible for this key physiological process.

Supplementary Material

[Supplemental Data]
en.2008-1527_index.html (2.2KB, html)

Acknowledgments

We thank Dr. Feixue Li for helpful consultation on adenovirus constructs and Drs. Phillip Bridges, Carolyn Komar, and Thomas E. Curry Jr. for critical reading of the manuscript.

Footnotes

This work was supported by National Institutes of Health Grants P20 RR 15592 and HD051727.

Disclosure Summary: The authors have nothing to disclose.

First Published Online April 2, 2009

Abbreviations: Ad DNRUNX, Adenoviral DNRUNX; Ad GFP, viral control; Ad RUNX1, adenoviral RUNX1; CBF, core binding factor; C/EBP, CAAT enhancer binding protein; ChIP, chromatin immunoprecipitation; COC, cumulus-oocyte complex; DNRUNX, dominant-negative RUNX; FSK, forskolin; GFP, green fluorescent protein; hCG, human chorionic gonadotropin; MOI, multiplicity of infection; PMA, phorbol 12-myristate 13-acetate; PMSG, pregnant mare serum gonadotropin; PTGS2, prostaglandin-endoperoxide synthase 2; RHD, runt-homology domain; RUNX1, runt-related transcription factor 1; siRNA, small interfering RNA; USF, upstream stimulatory factor.

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