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. 2010 Mar 2;24(4):846–858. doi: 10.1210/me.2009-0392

RUNX2 Transcription Factor Regulates Gene Expression in Luteinizing Granulosa Cells of Rat Ovaries

Eun-Sil Park 1, Anna-Karin Lind 1, Pernilla Dahm-Kähler 1, Mats Brännström 1, Martha Z Carletti 1, Lane K Christenson 1, Thomas E Curry Jr 1, Misung Jo 1
PMCID: PMC2852356  PMID: 20197312

Abstract

The LH surge promotes terminal differentiation of follicular cells to become luteal cells. RUNX2 has been shown to play an important role in cell differentiation, but the regulation of Runx2 expression and its function in the ovary remain to be determined. The present study examined 1) the expression profile of Runx2 and its partner CBFβ during the periovulatory period, 2) regulatory mechanisms of Runx2 expression, and 3) its potential function in the ovary. Runx2 expression was induced in periovulatory granulosa cells of human and rodent ovaries. RUNX2 and core binding factor-β (CBFβ) proteins in nuclear extracts and RUNX2 binding to a consensus binding sequence increased after human chorionic gonadotropin (hCG) administration. This in vivo up-regulation of Runx2 expression was recapitulated in vitro in preovulatory granulosa cells by stimulation with hCG. The hCG-induced Runx2 expression was reduced by antiprogestin (RU486) and EGF-receptor tyrosine kinase inhibitor (AG1478), indicating the involvement of EGF-signaling and progesterone-mediated pathways. We also found that in the C/EBPβ knockout mouse ovary, Runx2 expression was reduced, indicating C/EBPβ-mediated expression. Next, the function of RUNX2 was investigated by suppressing Runx2 expression by small interfering RNA in vitro. Runx2 knockdown resulted in reduced levels of mRNA for Rgc32, Ptgds, Fabp6, Mmp13, and Abcb1a genes. Chromatin immunoprecipitation analysis demonstrated the binding of RUNX2 in the promoter region of these genes, suggesting that these genes are direct downstream targets of RUNX2. Collectively, the present data indicate that the LH surge-induced RUNX2 is involved in various aspects of luteal function by directly regulating the expression of diverse luteal genes.

The LH-induced RUNX2 regulates luteal gene expression in luteinizing rat granulosa cells.

In response to the LH surge, a preovulatory follicle undergoes morphological and physiological changes that culminate in ovulation and luteinization. These changes are accomplished by the expression of a distinct group of genes, such as matrix-remodeling proteins, cell cycle inhibitors, inflammation/immune-related proteins, and steroidogenic factors (1,2,3). Specific transcriptional regulators induced by the LH surge ensure the timely expression of these periovulatory genes, thus playing a vital role in the periovulatory process (4). RUNX2 transcription factor was recently documented to be highly expressed in cumulus-oocyte complexes (COCs) and granulosa cells of periovulatory ovaries after human chorionic gonadotropin (hCG) injection in mice (5). This finding suggested that RUNX2 plays a role in regulating periovulatory gene expression. However, little is known about the regulatory mechanism of Runx2 expression and the specific function of this protein in the ovary.

RUNX2 is a member of the core binding factor (CBF)/poliomavirus enhancer binding protein (PEBP) family (6). CBF is a heterodimeric transcription factor: the α-subunit is encoded by one of three Runx genes (Runx1, Runx2, and Runx3), and the β-subunit is encoded by a single gene, CBFβ. RUNX proteins share the conserved DNA-binding domain referred to as a runt-homology domain. The runt-homology domain interacts with the dimeric partner, CBFβ, which does not bind DNA itself but stimulates DNA binding of RUNX proteins (7,8).

RUNX2 plays an essential role in osteoblast differentiation, as demonstrated by the absence of bone formation and neonatal death in Runx2 knockout mice (9). A growing number of genes have been identified as being regulated by RUNX2 (10,11). Some of these genes are highly expressed in periovulatory ovaries, such as matrix metalloproteinase 9 (Mmp9) (12), Mmp13 (13), secreted phosphoprotein 1 (Spp1) (14), and response gene to complement 32 (Rgc32) (15). For example, induction of matrix-related proteins [e.g. matrix components (Spp1) and proteolytic enzymes (Mmp9 and Mmp13)] by the LH surge is important for matrix remodeling during follicular rupture and luteal formation (12,13,14,16,17). Likewise, our previous study documented the up-regulation of Rgc32 expression by the LH surge in periovulatory follicles and corpora lutea (CL) (15). Because response gene to complement 32 (RGC32) has a role in cell cycle suppression (18), it was suggested that RGC32 may play a similar role in the ovary (15).

Based on these studies, we hypothesized that 1) the preovulatory gonadotropin surge increases Runx2 expression in periovulatory ovaries and 2) RUNX2 regulates gene expression in periovulatory follicular cells, thus playing a role in ovulation and/or luteal development. The present study tested this hypothesis by characterizing the expression of Runx2 and its partner, CBFβ during the periovulatory period using ovaries from pregnant mare serum gonadotropin (PMSG)/hCG-primed immature rats as well as ovaries from cycling rats. We also determined the expression profile of RUNX2 in human periovulatory follicles. Next, the regulatory mechanisms by which the LH surge induces Runx2 expression were determined using in vivo and in vitro models. Last, we identified downstream target genes of RUNX2 in luteinizing granulosa cells.

Results

Cellular localization of Runx2 mRNA in rat ovaries

This is the first report of in situ localization of Runx2 mRNA in the ovary. In PMSG/hCG-simulated immature rat ovaries, Runx2 mRNA was localized to periovulatory follicles and newly forming CL (nCL) (Fig. 1A, f, g, and h), whereas little expression was detected in ovaries obtained before hCG stimulation (Fig. 1Ae). We also performed in situ hybridization analysis in PMSG/hCG-stimulated immature mouse ovaries and found the identical localization pattern of Runx2 mRNA (data not shown).

Figure 1.

Figure 1

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In situ localization of Runx2 mRNA in rat ovaries obtained from gonadotropin-primed immature rats (A) and naturally cycling rats (B). Representative bright-field (A, a–d; B, a–d; and C, a and b) and corresponding dark-field (A, e–h; B, e–h; and C, c and d) photomicrographs are depicted. Ovaries were collected at the indicated time points before or after hCG injection (A or C) or the LH surge (B or C). Arrows indicate Runx2 mRNA expression in periovulatory follicles (PF). Arrowheads indicate nCL expressing Runx2 mRNA. Asterisks indicate CL generated during previous estrous cycles (pCL). Wavy arrows indicate Runx2 expression in cumulus cells in C. F, Follicle. Original magnification of all slides in A and B is ×40. Magnification of all slides in C is ×100.

To further confirm whether the induction of Runx2 mRNA observed in PMSG/hCG-stimulated immature animals occurs in the natural setting, in situ localization analyses were performed in naturally cycling rat ovaries collected throughout the periovulatory period. Similar to results from the immature rat model, Runx2 mRNA was localized to granulosa cells of periovulatory follicles and nCL (Fig. 1B, f–h). Interestingly, Runx2 mRNA was also localized to the CL from previous cycles, although the expression appeared to be low compared with that observed in the adjacent nCL (Fig. 1Bh).

The expression of Runx2 mRNA was also localized to cumulus cells of periovulatory follicles as evident in the ovary from both immature and cycling adult rats (Fig. 1C).

Runx2 and CBFβ expression in rat periovulatory ovaries

Ovarian levels of Runx2 mRNA began to increase at 4 h and continued to increase after ovulation (24 h after hCG, Fig. 2A). To be fully functional, RUNX2 needs to be dimerized with CBFβ. Northern blot analysis revealed the steady-state levels of CBFβ mRNA throughout the periovulatory period (Fig. 2B).

Figure 2.

Figure 2

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Ovarian expression of Runx2 and Cbfβ during the periovulatory period. Ovaries were collected before or at indicated hours (h) after hCG injection from PMSG-primed immature rats (n = 4 animals per time point). A and B, Whole ovarian levels of Runx2 (A) and Cbfβ (B) mRNA in rats. C, RUNX2 and CBFβ proteins in whole-cell extracts or nuclear extracts of rat ovaries obtained at indicated time points after hCG injection. D, The DNA binding activity of RUNX2 in nuclear extracts collected at indicated time points in rats using a TransAM kit. The levels of Runx2 mRNA and Cbfβ mRNA were measured by real-time PCR and Northern blot analysis, respectively. The levels of target genes were normalized to the L32 value in each sample. For the Western blot analysis, each lane was loaded with 50 μg protein extracts from ovaries of each animal. The membrane was reprobed with a monoclonal antibody against TATA binding protein (TBP) as a nuclear loading control and β-actin as a whole cell loading control. Bars with no common superscripts are significantly different (P < 0.05).

RUNX2 protein levels were increased in both whole-cell extracts and nuclear extracts of periovulatory ovaries; the high expression of RUNX2 protein was detected at 24 and 48 h after hCG (Fig. 2C). Similar to the mRNA expression profile, CBFβ protein was readily detected, and the levels were constant in whole ovarian extracts throughout the periovulatory period (Fig. 2C). But in nuclear extracts of periovulatory ovaries, CBFβ protein levels began to increase at 8 h and continued to increase by 48 h after hCG (Fig. 2C).

Next, we measured the DNA binding activity of RUNX2 in periovulatory ovaries using an ELISA-based TransAM RUNX2 kit. The increase in RUNX2 binding to DNA consensus sequences was detected at 12 and 24 h after hCG injection (Fig. 2D).

Up-regulation of Runx1 and Runx2 mRNA in granulosa cells or COCs of rat or human ovaries during the periovulatory period

To determine the relative expression of Runx2 mRNA in the granulosa cell compartment, granulosa cells were isolated from ovaries obtained before or at various times after hCG administration. The levels of Runx2 mRNA began to increase at 8 h and continued to increase at 12 h after hCG (Fig. 3A). Because Runx2 mRNA was localized to cumulus cells by in situ hybridization analyses, the levels of Runx2 mRNA in periovulatory COCs were also determined. Cumulus cell expression of Runx2 mRNA was increased at 6 h and continued to increase at 12 and 24 h after hCG (Fig. 3B).

Figure 3.

Figure 3

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Granulosa cells (GC) or COC expression of Runx1 or Runx2 in rats or humans during the periovulatory period. A and B, Granulosa cells or COCs were collected from periovulatory ovaries obtained before or at indicated hours (h) after hCG injection from PMSG-primed immature rats (n = 4 animals per time point). COCs were also collected from the oviduct at 24 h after hCG. C and D, Granulosa cells were isolated from periovulatory follicles collected before (PEO, preovulatory phase) or indicated times (EO, early ovulatory phase; LO, late ovulatory phase) after rhCG injection from women (n = 5 patients per time point). The levels of Runx1 and Runx2 mRNA were measured by real-time PCR and normalized to the L32 (rat) and GAPDH (human) value in each sample. Bars with no common superscripts are significantly different (P < 0.05).

Virtually nothing is known about RUNX2 expression in human ovaries. To relate our findings from animal studies to humans, it is critical to determine whether RUNX2 is expressed and also regulated in human granulosa cells. To determine the periovulatory expression pattern of RUNX2 mRNA, granulosa cells were isolated from preovulatory or periovulatory follicles surgically removed from ovaries at different stages of the periovulatory period. Real-time PCR data revealed the dramatic up-regulation of RUNX2 expression within 12 h after recombinant hCG (rhCG) injection (early ovulatory period), and this increase persisted during the late ovulatory phase (Fig. 3C). Because RUNX1 expression was not reported in human ovaries, we also measured the levels of RUNX1 mRNA in corresponding granulosa cell samples. The expression of RUNX1 mRNA was transient; the levels increased during the early ovulatory period but declined during the late ovulatory period (Fig. 3D). The expression pattern of RUNX1 and RUNX2 mRNA in granulosa cells of periovulatory follicles in humans was similar to that observed in rats.

The effect of hCG on Runx2 and CBFβ expression in preovulatory granulosa cell cultures

To determine whether the in vivo induction of Runx2 expression can be mimicked in vitro, granulosa cells isolated from PMSG-primed immature rat ovaries were treated with hCG to activate LH receptors. hCG stimulated Runx2 mRNA expression; the levels were highest at 48 h of culture (Fig. 4A). A transient increase in Runx2 mRNA level was also observed in control cells at 6 h of culture, but this elevation returned to the basal level by 24 h. Nuclear accumulation of RUNX2 protein was evident in hCG-treated granulosa cells collected at 24 and 48 h (Fig. 4C). We also detected RUNX2 protein in granulosa cells treated with hCG for 8 h when the x-ray film was exposed for a longer period (Supplemental Fig. 1 published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). To compare the expression profile of Runx2 with that of Runx1, another member of the RUNX family, we also measured Runx1 mRNA and protein in corresponding cultured granulosa cell samples. hCG stimulated a transient increase in Runx1 mRNA (Fig. 4B) and protein (Fig. 4C). hCG also increased levels of CBFβ protein in nuclear extracts of cultured granulosa cells (Fig. 4C). But in whole-cell extracts, CBFβ was readily detected and unaffected by hCG (Fig. 4C). To further determine whether RUNX2 binds to CBFβ, nuclear extracts from cultured granulosa cells were immunoprecipitated with RUNX2 antibody. CBFβ protein was detected in nuclear extracts of hCG-treated granulosa cells (Fig. 4D).

Figure 4.

Figure 4

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Regulation of Runx2 and Cbfβ expression in periovulatory granulosa cells. A and B, Granulosa cells obtained from rat preovulatory ovaries (48 h after PMSG) were cultured for 0, 6, 12, 24, or 48 h in medium alone (control) or with hCG (1 IU/ml). The levels of Runx2 mRNA (A) and Runx1 mRNA (B) were measured using real-time PCR (mean ± sem; n = 3 independent experiments). C, Granulosa cells isolated from rat ovaries were cultured in medium alone (C) or with hCG (1 IU/ml) for 0, 8, 24, or 48 h. RUNX1, RUNX2, and CBFβ proteins in nuclear extracts and CBFβ protein in whole-cell extracts were detected by Western blot analyses. Each lane was loaded with 40 μg protein extracts. The membranes were reprobed with a monoclonal antibody against TATA binding protein (TBP) for nuclear loading control or β-actin for whole cell loading control (n = 4 independent experiments). D, Granulosa cells were cultured with hCG or without (Cont) for 48 h. The direct interaction of RUNX2 and CBFβ was determined by immunoprecipitation (IP) assays. Cytoplasmic and nuclear fractions (100 μg) were immunoprecipitated with RUNX2 antibody. The resulting precipitates were analyzed for CBFβ protein by Western blot (WB) analysis. E, Preovulatory granulosa cells were cultured for 48 h in medium alone (Cont) or with hCG (1 IU/ml), FSK (10 μm), or PMA (20 nm). The levels of Runx2 mRNA were measured using real-time PCR (mean ± sem; n = 4 independent culture experiments). F, Preovulatory granulosa cells were cultured for 24 h in medium alone (Cont) or with AG1479 (AG, 1 μm), NS-398 (NS, 1 μm), RU486 (RU, 10 μm), hCG (1 IU/ml), or hCG plus inhibitor. The levels of Runx2 mRNA were measured using real-time PCR (mean ± sem; n = 3 independent culture experiments). In A, B, E, and F, bars with no common superscripts are significantly different (P < 0.05). G and H, Granulosa cells were isolated from ovaries collected before or at indicated hours after hCG injection from PMSG-primed immature wild-type (WT) and C/EBPβ knockout (KO) mice. Levels of mRNA for Runx2 (G) and Runx1 mRNA (H) were measured by real-time PCR. For mean comparison between KO vs. WT: *, P < 0.05.

Regulation of Runx2 expression in preovulatory granulosa cells in vitro

To determine which signaling pathway is involved in hCG-induced Runx2 expression, we cultured preovulatory granulosa cells in the absence or presence of forskolin (FSK), an activator of adenylate cyclase, and/or phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. These agonists have been frequently used to mimic the action of an ovulatory LH stimulus in preovulatory granulosa cell cultures (19). The stimulatory effect of hCG on Runx2 mRNA was mimicked by FSK, but not by PMA, suggesting that the induction of Runx2 expression is primarily mediated by LH-activated adenylate cyclase-mediated signaling pathway (Fig. 4E).

Next, we tested whether the up-regulation of Runx2 expression is regulated by LH-induced mediators such as progesterone receptors (20), EGF-related peptides (21), and prostaglandin synthase 2 (PTGS2) (19). These mediators are rapidly, yet transiently induced by LH/hCG in periovulatory granulosa cells in vivo and in vitro and are known to be essential for successful ovulation (reviewed in Ref. 4). Preovulatory granulosa cells were cultured in medium alone or with RU486 (1 μm, antiprogestin), AG1478 (1 μm, EGF-receptor tyrosine kinase inhibitor), NS-398 (1 μm, PTGS2 inhibitor), hCG (1 IU), or hCG plus inhibitor for 24 h. AG1479 and RU486 reduced hCG-stimulated Runx2 mRNA expression, whereas NS-398 had no effect. In line with this finding, treatment with amphiregulin (one of the EGF-like peptides induced by hCG/LH) (21) stimulated Runx2 expression, although the stimulatory effect is lower than that of hCG (40%) (Supplemental Fig. 2). These data indicate that LH-dependent activation of progesterone receptors as well as EGF-receptor tyrosine kinase contributes Runx2 expression in cultured granulosa cells (Fig. 4F).

The CCAAT/enhancer-binding protein beta(C/EBPβ) transcription factor is highly induced by the LH surge in periovulatory follicles and found to regulate the expression of periovulatory genes, such as Ptgs2 (22), StAR (23), and Inha (24). In addition, C/EBP has been shown to regulate Runx2 expression in nonovarian cells (25). To determine whether C/EBPβ is involved in the LH-induced expression of Runx1 and Runx2, the levels of Runx1 and Runx2 mRNA were compared between granulosa cells isolated from ovaries of C/EBPβ knockout mice and those from wild-type mice collected before and at various hours after hCG administration. The levels of Runx2 mRNA in C/EBPβ-null mouse ovaries were lower compared with those in wild-type mice (Fig. 4G). But Runx1 mRNA levels were reduced only in granulosa cell samples collected at 2 h after hCG injection (Fig. 4H). These data indicate that hCG-induced C/EBPβ is important for full up-regulation of Runx2 expression but not for Runx1 expression.

Knockdown of hCG-induced Runx2 expression by small interfering RNA (siRNA) in luteinizing granulosa cells

To determine the role of RUNX2 in periovulatory granulosa cells, we used a siRNA knockdown approach. Preovulatory granulosa cells were preincubated overnight in OptiMEM media supplemented with gentamycin. This is to acclimatize the cells before transfection and also to prevent bacterial contamination during the culture period. In this culture condition, we found the most efficient knockdown of Runx2 expression. Next morning, the cells were changed with fresh OptiMEM media and then transfected with Runx2 siRNA or negative siRNA before stimulating with FSK for 48 h. Because of the down-regulation of LH receptors during overnight preincubation (26,27), FSK was used in preovulatory granulosa cell cultures to mimic the action of an ovulatory dose of LH/hCG in inducing Runx2 expression. Runx2 siRNA effectively suppressed agonist-stimulated Runx2 expression (Fig. 5, A and B). Knockdown of Runx2 expression resulted in a reduction of nuclear levels of CBFβ and higher accumulation of CBFβ in the cytoplasmic fraction (Fig. 5B), suggesting that the induction and subsequent dimerization with RUNX2 promotes the translocation of CBFβ to the nucleus.

Figure 5.

Figure 5

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Reduction in Runx2 expression by Runx2 siRNA in cultured granulosa cells. Granulosa cells obtained from rat preovulatory ovaries (48 h after PMSG) were transfected without (vehicle) or with negative control scrambled siRNA (NC siRNA) and Runx2 siRNA, and then treated with FSK for 48 h. The levels of mRNA for Runx2 (A) and Cyp11a1 (C) were measured by real-time PCR and normalized to the L32 in each sample (mean ± sem; n = 4 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05). Western blot (B) shows RUNX2 and CBFβ proteins in nuclear and cytoplasmic fractions isolated from siRNA-transfected cells. Each lane was loaded with 50 μg protein extracts. The membrane was reprobed with a monoclonal antibody against TATA binding protein (TBP) and β-actin for the nuclear and cytoplasmic loading control, respectively. The blots are representatives of three separate experiments. Concentrations of progesterone (D) were measured in granulosa cell culture media collected at 48 h after FSK treatment (mean ± sem; n = 6 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05).

To determine whether the knockdown of Runx2 expression affects the steroidogenic capacity of luteinizing granulosa cells, levels of Cyp11a1 mRNA and the concentration of progesterone in culture media were measured. FSK increased the levels of Cyp11a1 mRNA and progesterone production, but knockdown of Runx2 expression had no effect (Fig. 5, C and D).

Identification of RUNX2-regulated genes in luteinizing granulosa cells

To screen the potential targets of RUNX2, DNA microarray analysis was performed using total RNA isolated from FSK plus Runx2 siRNA-treated granulosa cells and FSK plus negative siRNA-treated cells. These cells were cultured for 48 h, the time point of maximal Runx2 expression. Consistent with the real-time PCR results (Fig. 5A), the microarray data confirmed the reduction of Runx2 mRNA levels in Runx2 siRNA-treated cells (data not shown). Initially, we selected the top 12 genes that were down-regulated 50% or more compared with scrambled siRNA-treated cells from the microarray data. Among these genes, the expression of only six genes was confirmed to be stimulated by FSK treatment compared with control cultures (vehicle) and down-regulated by Runx2 siRNA treatment by real-time PCR (Fig. 6). These genes included Rgc32, Spp1, Mmp13, Ptgds, Fabp6, and Abcb1a.

Figure 6.

Figure 6

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RUNX2 regulation of Rgc32, Spp1, Mmp13, Ptgds, Fabp6, and Abcb1a expression in luteinizing granulosa cells in vitro. Granulosa cells obtained from rat preovulatory ovaries (48 h after PMSG) were transfected without (vehicle) or with FSK plus negative control scrambled siRNA (NC siRNA) and FSK plus Runx2 siRNA for 48 h. The levels of mRNA for Rgc32 (A), Spp1 (B), Mmp13 (C), Ptgds (D), Fabp6 (E), and Abcb1a (F) were measured by real-time PCR and normalized to the L32 value in each sample (mean ± sem; n = 4 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05).

To make it more relevant to physiological conditions, we determined whether these genes were up-regulated in response to hCG stimulation. In preovulatory granulosa cells cultured with hCG, the levels of Rgc32, Mmp13, Ptgds, Fabp6, and Abcb1a mRNA were increased (Fig. 7, A–E). The hCG-stimulated expression of these genes was highest at 48 h culture, similar to that of Runx2 expression. On the contrary, Spp1 expression was not stimulated by hCG and decreased over the culture period (Fig. 7F).

Figure 7.

Figure 7

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hCG stimulation of Rgc32, Mmp13, Ptgds, Fabp6, and Abcb1a expression in luteinizing granulosa cells in vitro. Granulosa cells obtained from rat preovulatory ovaries (48 h after PMSG) were cultured for 0, 6, 24, or 48 h in medium alone (control) or with hCG (1 IU/ml). Levels of Rgc32 (A), Mmp13 (B), Ptgds (C), Fabp6 (D), Abcb1a (E), and Spp1 (F) mRNA were measured using real-time PCR and normalized to the L32 value in each sample (mean ± sem; n = 3 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05).

Next, to assess whether these genes are direct transcriptional targets of RUNX2, we first screened for potential RUNX binding sites in the 5′-flanking region of each gene using a TFSEARCH program (http://www.cbrc.jp/research/db/TRSEARCH.html) (Fig. 8). Once potential RUNX binding sites were identified, ChIP analyses were performed using preovulatory granulosa cells cultured with hCG for 48 h. PCR analysis revealed the enrichment of chromatin fragments of Rgc32, Mmp13, Ptgds, Fabp6, and Abcb1a promoter regions, demonstrating the binding of RUNX2 in the promoter region of these genes (Fig. 8, A–E).

Figure 8.

Figure 8

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ChIP analysis for RUNX2 transcription factor binding to the Rgc32, Mmp13, Ptgds, Fabp6, and Abcb1a promoter regions in luteinizing granulosa cells. RUNX binding sites were predicted by a TFSEARCH program and numbered from transcription start site at +1 in A–E. ChIP assays were performed using granulosa cells cultured with hCG (1 IU/ml) for 48 h. DNAs were analyzed by PCR using primers listed in Supplemental Table 2 and represented as arrows in A–E. Amplified DNA fragments containing RUNX transcription factor binding sites are represented as black boxes with the indicated PCR product size. Experiments were repeated at least three times, each with different cultured luteinizing granulosa cell samples.

Effects of PGD2 on granulosa cell viability

To determine the functional impact of the RUNX2 downstream gene, Ptgds, an enzyme that converts prostaglandin H2 (PGH2) to PGD2 (28) in the rat ovary, we tested the effect of PGD2 on granulosa cell viability. Preovulatory granulosa cells were cultured with hCG or various concentrations of PGD2 for 48 h. At the end of culture, the cells were subjected to 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, which measures the reductase activity of mitochondria and is widely used as a measure of cell viability. PGD2 treatment increased cell viability in a dose-dependent manner, mimicking the effect of hCG on granulosa cell viability (Fig. 9).

Figure 9.

Figure 9

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Effects of PGD2 on granulosa cell viability. Granulosa cells obtained from rat preovulatory ovaries (48 h after PMSG) were cultured without (control) or with DMSO (0.025%), hCG (1 IU/ml), or synthetic PGD2 (0.1, 0.5, 1, 2.5, or 5 μm) for 24 h. Granulosa cell viability was measured using a MTS kit (mean ± sem; n = 3 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05).

Discussion

Transcriptional factors induced in periovulatory follicles by the LH surge play an important role in ovulation and CL formation by directly controlling gene expression. The present study demonstrated the increase in Runx2 expression in periovulatory granulosa cells of rodent ovaries and, importantly, human ovaries. Runx2 expression is also highly expressed in the forming CL, suggesting that RUNX2 may play a role in ovulation as well as luteal development. We provided evidence that the LH-induced expression of Runx2 is important for the up-regulation of specific luteal genes: three of them (Ptgds, Fabp6, and Abcb1a) are newly identified as RUNX2-regulated genes; the other two (Rgc32 and Mmp13) are previously identified in nonovarian cells but are now verified in luteinizing granulosa cells. These findings suggest that RUNX2 is functionally involved in luteal formation and/or function.

The data from the present study showed that RUNX2 protein and its activity followed a pattern similar to that of Runx2 mRNA in periovulatory granulosa cells, although nuclear accumulation of RUNX2 protein was delayed a few hours, possibly due to the time required for translation of Runx2 mRNA and transfer of this protein to the nucleus. In addition, we found that CBFβ, the dimeric partner of RUNX proteins, was constitutively expressed, but nuclear accumulation of CBFβ was stimulated by hCG. Together with evidence of RUNX2 binding to CBFβ, these data indicated that the LH surge-induced RUNX2 forms a dimeric complex with CBFβ and exerts its function in the nucleus of luteinizing granulosa cells.

Another member of the RUNX family, Runx1, was documented to be rapidly, yet transiently induced in LH/hCG-stimulated granulosa cells of rodent ovaries (5,29,30). Herein, we reported the similar pattern of RUNX1 expression in human periovulatory granulosa cells. Previous studies demonstrated that RUNX1 is involved in regulating the expression of several key periovulatory genes, such as Ptgs2, Cyp11a1, Mt1a, Hapln1, and Rgc32 (30,31). Interestingly, Rgc32 mRNA was highly expressed in luteal cells (15) where Runx1 expression is low (30). The present findings of RUNX2 binding to the Rgc32 promoter and reduction of Rgc32 mRNA levels by Runx2 siRNA suggested that luteal expression of Rgc32 is mediated by RUNX2. Our result is consistent with a recent finding by Wotton et al. (32) showing Rgc32 as a common target of RUNX1, RUNX2, and RUNX3 transcription factors.

The overlapping expression of Runx1 and Runx2 and their redundant function in periovulatory granulosa cells presented a challenge in determining RUNX2-specific target genes. However, the in vitro analysis of Runx1 and Runx2 expression revealed that the hCG-stimulatory effect on Runx1 expression was transient, whereas Runx2 expression gradually increased and reached a maximum at 48 h of cultures. This observation suggested differential roles for each RUNX protein during the early vs. late ovulatory period. Moreover, the temporal deviation of Runx1 and Runx2 expression pattern allowed us to identify RUNX2-specific target genes using luteinizing granulosa cell cultures (48 h of culture).

In the present study, we focused on identifying the genes that are up-regulated by LH and mediated by RUNX2. Herein, we report Rgc32, Mmp13, Ptgds, Fabp6, and Abcb1a as potential direct downstream target genes of RUNX2 in LH-stimulated luteinizing granulosa cells. This is based on evidence that 1) suppression of Runx2 expression by siRNA reduced levels of mRNA for these genes, 2) RUNX2 binds to their promoter region, and 3) hCG stimulated the expression of these genes, similar to that of Runx2. Additional studies will be needed to verify the transcriptional activation of these genes by RUNX2 in ovarian cells. Nonetheless, the genes identified as being regulated by RUNX2 in luteinizing granulosa cells appear to have a role in luteal formation or function. For examples, RGC32 is known to be involved in controlling the cell cycle (18,33). Rgc32 expression is up-regulated by the LH surge in periovulatory granulosa cells and luteal cells (15), which exit the cell cycle, suggesting that RGC32 may act as a suppressor of the cell cycle in these cells. Matrix metalloproteinase 13 (MMP13) has potent collagenolytic and gelatinolytic enzyme activity that is important for connective tissue turnover (34). Mmp13 expression was found to be up-regulated during follicular development (35), by LH stimulation (13) and luteal regression (36), suggesting involvement of this enzyme during various stages of the reproductive cycle in the ovary. ABCB1 functions as an efflux pump that expels various xenobiotic compounds out of the cells (37) and is expressed in cells with secretory/excretory function (38,39). Abcb1 expression was highly up-regulated in preovulatory granulosa cells and luteal cells by hCG (40). Given the production of abundant secretory factors by luteinizing granulosa cells, the LH-induced ABCB1 likely contributes to the rapid transport of these products. FABP6 is involved in fatty acid transport and metabolism (41,42,43). The expression of Fabp6 mRNA and protein was detected in luteal cells of the rat ovary (44), suggesting a role of FABP6 in uptake, intracellular transport, and/or metabolism of certain steroid hormones. Lastly, PGD synthase (Ptgds) catalyzes the conversion of PGH2 to PGD2 (28). PGD synthase was found to be highly up-regulated in human CL from the early to middle luteal phase. PGD2 treatment increased cAMP content and progesterone production in pieces of human CL (45,46). We also found that PGD2 treatment increased the viability of cultured luteinizing granulosa cells, suggesting a luteotropic function of this prostaglandin in developing CL.

In terms of upstream regulators of Runx2 expression, we investigated whether Runx2 expression is regulated by LH/hCG-induced key mediators, such as progesterone receptors, EGF-like peptides, PTGS2, and C/EBPβ (19,20,21,22). These factors are rapidly and transiently induced in periovulatory granulosa cells and are critical for successful ovulation and/or luteal formation (19,20,21,22). We found that inhibition of progesterone receptors and EGF receptors reduced hCG-stimulated Runx2 mRNA levels in cultured granulosa cells, indicating that Runx2 expression is regulated downstream of progesterone/progesterone receptors and EGF-signaling pathways. We also observed a reduction of Runx2 mRNA levels in periovulatory granulosa cells of C/EBPβ knockout mice, indicating that Runx2 is a downstream target of C/EBPβ. Considering the crucial contribution of EGF signaling, progesterone receptors, and C/EBPβ in ovulation and/or luteal formation (19,20,21,22), the full up-regulation of Runx2 expression induced by these mediators may be important for the successful periovulatory process.

In summary, this study documented for the first time that the LH surge-induced Runx1 and Runx2 expression in periovulatory follicles is conserved between humans and rodents. Their partner, CBFβ, is constitutively expressed but accumulates in the nucleus and dimerizes with RUNX proteins, indicating the presence of functional CBF complex (RUNXs plus CBFβ) that can regulate periovulatory gene expression. The present study suggest that the up-regulation of RUNX2 is functionally linked to various aspects of luteal development by controlling the expression of the genes involved the cell cycle, matrix turnover, cell signaling, and cellular transport. Additional studies will be needed to determine the specific function of RUNX2 downstream genes, thus delineating physiological impacts of Runx2 expression in periovulatory ovaries.

Materials and Methods

Animals

Sprague Dawley rats were obtained from Harlan, Inc. (Indianapolis, IN) and maintained at the University of Kentucky Laboratory Animal Resources. Animal protocols for the rat study were approved by University of Kentucky Animal Care and Use Committees. For the induced model, rats (25 or 26 d old) were injected with PMSG (10 IU) to stimulate follicular development. Forty-eight hours later, the animals were injected with hCG (10 IU) to induce ovulation and formation of CL. Animals were killed at 0 h (at the time of hCG administration) and defined times after hCG. Ovaries were collected and processed for in situ hybridization, Northern blot, RT-PCR, or Western blot analyses.

Sexually mature female rats exhibiting regular 4-d estrous cycles (150–180 g body weight, 2 months old) were killed at 1600, 2000, and 2400 h on proestrus and 0400 h on estrus. In this colony of rats, the LH surge occurred at 1600 h on proestrus (30). Ovaries were collected and processed for in situ hybridization analyses.

Female C/EBPβ knockout mice (n = 15, C57BL/6 × C129 strain, 30–40 d old) and littermate wild-type controls (n = 15) were propagated at the University of Kansas Medical Center Laboratory Animal Resources. Animal protocols for the mouse study were approved by University of Kansas Medical Center Animal Care and Use Committees. Mice were primed with PMSG (5 IU) and 44–48 h later stimulated with hCG (5 IU). The mice were killed at 0, 2, 4, or 8 h after hCG. The granulosa cells from two to three mice at each time point were pooled and processed for RNA isolation.

Culture of rat granulosa cells

To isolate granulosa cells, ovaries were collected from immature rats at 48 h after PMSG. Granulosa cells were isolated by the method of follicular puncture (15). The cells were pooled, filtered, pelleted, and resuspended in defined medium consisting of Opti-MEM I reduced serum medium supplemented with 0.05 mg/ml gentamycin, and 1× ITS (insulin, transferrin, and selenium). The cells were cultured in the absence or presence of various reagents at 37 C in a humidified atmosphere of 5% CO2.

Collection of human granulosa cells

This study has been approved by the ethics committee of Sahlgrenska Academy at Göthenburg University. Informed written consent was obtained from all patients. Human ovarian tissues were obtained before, during, and after ovulation as previously described (47). Briefly, when the dominant follicle reaches a diameter of 15–17 mm on transvaginal ultrasound, 250 μg rhCG (Ovitrelle; Serono International S.A., Geneva, Switzerland) was injected to mimic the natural LH surge. To obtain periovulatory follicles at different stages of the ovulatory process, patients were divided into three groups: preovulatory phase, early ovulatory phase, and late ovulatory phase. The preovulatory phase is defined as the stage when the dominant follicle reaches a diameter of at least 14 mm and no more than 17.5 mm before an LH surge. For a group of women, surgery was performed at this preovulatory stage without giving rhCG. Blood was collected, and serum levels of FSH, LH, estradiol, and progesterone were determined to ensure that ovulation had not been initiated. The rest of the women underwent surgery during one of two different time intervals after rhCG injection. The early ovulatory phase is defined as 12–18 h after rhCG and late ovulatory phase is more than 18 h to 34 h after rhCG. The whole preovulatory or periovulatory follicles with adjacent ovarian stroma was excised from the ovary. Total RNA was isolated from granulosa cells of these follicles (n = 5 individuals per stage) as described previously (47) and was used to synthesize cDNA using the one-cycle target labeling protocol (Invitrogen Corp., Carlsbad, CA).

Quantification of Runx2, Runx1, Spp1, Rgc32, Mmp13, Ptgds, Fabp6, and Abcb1a mRNA

Total RNA was isolated from rat ovaries and cultured granulosa cells using a Trizol reagent (Invitrogen) and a RNeasy mini kit (QIAGEN, Inc., Valencia, CA), respectively. To measure levels of rat Runx2, Runx1, Spp1, Rgc32, Ptgds, Fabp6, Mmp13, and Abcb1a mRNA, we used real-time PCR as described previously (15). Oligonucleotide primers corresponding to each gene were designed using PRIMER3 software and listed in Supplemental Table 1. The specificity for each primer set was confirmed by both running the PCR products on a 2.0% agarose gel and analyzing the melting (dissociation) curve using the MxPro real-time PCR analysis program (Stratagene, La Jolla, CA) after each real-time PCR.

To measure the levels of Runx1 and Runx2 mRNA in mouse granulosa cells, primers for mouse Runx1 and Runx2 were designed using the Primer Express 2.0 software, whereas the GAPDH primers and probe were purchased from Applied Biosystems (Foster City, CA). Real-time PCR was performed using either SYBR Green (Runx1 and Runx2) or TaqMan (GAPDH) (48).

To measure the levels of RUNX1 and RUNX2 mRNA in human granulosa cells, TaqMan primers and probes for RUNX1, RUNX2, and GAPDH were purchased from Applied Biosystems. Real-time PCR was performed according to the manufacturer’s protocol.

All samples were measured in duplicate or triplicate, and the amplification efficiency of each transcript primer set was determined by running a standard curve. The relative abundance of the target transcripts was normalized to the endogenous reference gene GAPDH (for mouse and human granulosa cells) or L32 (for rats) and calculated according to the Pfaffl method (49).

In situ localization of Runx2 mRNA

Ovaries were sectioned at 10 μm and mounted on Probe On Plus slides (Fisher Scientific, Pittsburgh, PA). In situ hybridization analysis was carried out as described previously (12). Briefly, a 582-bp DNA fragment corresponding to partial rat Runx2 cDNA was generated by RT-PCR and cloned into the pCRII-TOPO Vector (Invitrogen) as described previously (29). Oligonucleotide primer pairs were designed based on published sequence data (XM_346016, 5′-GCC GGG AAT GAT GAG AAC TA-3′, 5′-GAG GCA GAA GTC AGA GGT GG-3′). DNA sequences of cloned rat partial cDNA were verified commercially (Eurofins MWG Operon, Huntsville, AL). Plasmids containing cDNA for Runx2 were linearized with BamHI and EcoRV to generate sense and antisense riboprobes, respectively. Linearized plasmids were labeled with [α-35S]UTP (10 mCi/ml; MP Biomedicals, Inc., Costa Mesa, CA) and T7 and SP6 RNA polymerases, as appropriate. One ovary from each of three animals was used for in situ hybridization. At least four sections per ovary were analyzed for each antisense probe, making a total of at least 12 tissue sections analyzed for each time point. A sense riboprobe, used as a control for nonspecific binding, was included for each ovary and each time point.

Western blot analysis

Nuclear extracts, cytoplasmic fractions, or whole-cell extracts were isolated from ovaries or cultured granulosa cells using a nuclear extraction kit (Active Motif, Carlsbad, CA) as previously described (29). All lysates were denatured by boiling for 5 min and separated by SDS-PAGE on a 9% polyacrylamide gel and then transferred onto a nitrocellulose membrane. The membrane was incubated overnight at 4 C in 1% casein solution containing primary antibody against RUNX2 (Calbiochem, La Jolla, CA), RUNX1 (Calbiochem), or CBFβ (Abcam, Cambridge, MA). Primary antibodies against TATA binding protein (TBP; Abcam) and β-actin (Cell Signaling Technology Inc., Danvers, MA) were used as a loading control for nuclear and cytoplasmic/whole-cell extracts, respectively. The blots were incubated with the respective secondary horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. Peroxidase activity was visualized using the SuperSignal West Pico chemiluminescent substrate (Pierce Chemical Co., Rockford, IL).

Immunoprecipitation assay

Nuclear and cytoplasmic fractions were isolated from cultured granulosa cells using a nuclear extraction kit (Active Motif). All lysates were precleared by adding protein A-agarose (Upstate Biotechnology, Inc., Lake Placid, NY) for 30 min at 4 C on a rocker. An aliquot of cytoplasmic and nuclear fraction (100 μg/reaction) was immunoprecipitated with 5 μg anti-RUNX2 antibody (Santa Cruz Biotechnology) overnight at 4 C. The complexes were then collected by the addition of protein A-agarose and overnight incubation at 4 C. Immunoprecipitates were washed three times with ice-cold PBS, resuspended in RIPA buffer, and separated by SDS-PAGE for Western blot analysis.

Knockdown of Runx2 mRNA by siRNA in granulosa cell cultures

Granulosa cells were isolated from ovaries collected at 48 h after PMSG and cultured overnight to acclimatize the cells. On the next day, the medium was changed to Opti-MEM I reduced serum medium. Runx2 siRNA (sense, GCA CGC UAU UAA AUC CAA Att; antisense, UUU GGA UUU AAU AGC GUG Ctg; Ambion, Inc., Austin, TX) or negative control siRNA (Stealth RNAi Negative Control Med GC; Invitrogen) were transfected to the cells using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instruction. Transfected cells were incubated for 1 h before FSK treatment and further cultured for 48 h. At the end of culture, cells were used to isolate total RNA or extract nuclear fractions for Western blot analyses. Culture media were collected and used to measure progesterone.

Concentrations of progesterone in the culture media were measured using an Immulite kit (Diagnostic Products, Los Angeles, CA). Assay sensitivity was 0.02 ng/ml. The intraassay and interassay coefficients of variation were 9.6 and 10%, respectively.

DNA microarray of rat granulosa cell cultures

Total RNA was extracted from cultured granulosa cells treated with siRNA using a RNeasy Mini kit. The reduction of Runx2 mRNA in Runx2 siRNA-treated granulosa cells was confirmed by real-time PCR. DNA microarray was performed as described previously (30). Briefly, two independent culture experiments were performed, and 5 μg total RNA isolated from each treatment was used as a template for cDNA synthesis (n = 2 experiments per treatment; four chips). The Affymetrix Rat 230 2.0 genechip array was hybridized, washed, and scanned using Affymetrix (Santa Clara, CA) equipment (DNA Microarray Core Facility, University of Kentucky, Lexington, KY). The microarray data were organized using a GEPRO software program (http://www.mc.uky.edu/cls/ko/gepro2.html).

Chromatin immunoprecipitation (ChIP) analysis

ChIP assay was performed on RUNX2 binding sites in Rgc32, Mmp13, Ptgds, Fabp6, and Abcb1a promoter regions using a ChIP kit (Upstate Biotechnology) as described previously (15). Briefly, chromatin isolated from cultured granulosa cells was immunoprecipitated overnight at 4 C with anti-RUNX2 antibody (5 μg/reaction; Santa Cruz Biotechnology) or rabbit IgG (5 μg/reaction). 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 Rgc32, Spp1, Mmp13, Ptgds, Fabp6, and Abcb1a genes. The primer sequences and location of RUNX binding sites on the promoter region of each gene were listed in Supplemental Table 2. After 25–30 cycles amplification, PCR products were run on a 2% agarose gel, stained with ethidium bromide, and visualized under UV light.

DNA binding activity assay

The DNA binding activity of RUNX2 was determined by using a TransAM AML3/RUNX2 transcription factor assay kit (Active Motif) according to the manufacturer’s protocol. Briefly, nuclear fraction was extracted from PMSG/hCG-treated immature rat ovaries as described above. Protein concentrations were measured by using a DC protein assay kit (Bio-Rad, Richmond, CA). Nuclear extracts (5 μg) were added to the immobilized oligonucleotides containing the RUNX2 consensus binding sequences (5′-AACCACA-3′) on a 96-well plate. After 1 h incubation and a series of washes, primary antibody for RUNX2 was added and incubated for 1 h. After incubation with a secondary horseradish peroxidase-conjugated antibody, specific binding was detected by spectrophotometer at 450 nm in a plate reader (Infinite F200; TECAN USA, Inc., Durham, NC).

MTS cell viability assay

Granulosa cells were collected from immature rat ovaries (48 h after PMSG) and seeded in 96-well plates (10,000 cells per well). Cells were incubated without (control) or with hCG (1 IU/ml) or synthetic PGD2 (0. 1, 0.5, 1, 2.5, or 5 μm; Cayman Chemical, Ann Arbor, MI) for 24 h. Because PGD2 was dissolved in dimethylsulfoxide (DMSO), DMSO control (0.025%) was included for each experiment. The final concentration of DMSO was 0.025% for 2.5 μm PGD2. Cell viability was measured using CellTiter 96 Aqueous One Solution cell proliferation assay (MTS) according to the manufacturer’s protocol (Promega, Madison, WI). Briefly, at the end of culture, 20 μl reagent was pipetted into each well containing the cells in 100 μl culture medium, and the cells were then returned to the incubator for an additional 3 h. The absorbance was measured at 492 nm in the Infinite F200 plate reader (Tecan USA) to determine the formazan concentration, which is proportional to the number of live cells (50).

Statistical analyses

Results are expressed as mean ± sem. Data were tested for homogeneity of variance by Levene test, and log transformations were performed on data set that had heterogeneous variance. All data were analyzed by ANOVA (one-way ANOVA or two-way ANOVA for C/EBPβ knockout mouse study) to determine the significant difference across time of culture, tissue collection, or among treatments in vitro. If ANOVA revealed significant effects, the means were compared by Tukey’s test, with P < 0.05 considered significant.

Supplementary Material

[Supplemental Data]
me.2009-0392_index.html (733B, html)

Footnotes

This work was supported by National Institutes of Health Grants NCRR P20 RR 15592 (to T.E.C. and M.J.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online March 2, 2010

Abbreviations: CBF, Core binding factor; C/EBPβ, CCAAT/enhancer-binding protein beta; ChIP, chromatin immunoprecipitation; CL, corpora lutea; COC, cumulus-oocyte complex; DMSO, dimethylsulfoxide; FSK, forskolin; hCG, human chorionic gonadotropin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; nCL, newly forming CL; PGH2, prostaglandin H2; PMA, phorbol 12-myristate 13-acetate; PMSG, pregnant mare serum gonadotropin; PTGS2, prostaglandin synthase 2; RGC32, response gene to complement 32; rhCG, recombinant hCG; siRNA, small interfering RNA.

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Supplementary Materials

[Supplemental Data]
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