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Luteinizing Hormone-Induced RUNX1 Regulates the Expression of Genes in Granulosa Cells of Rat Periovulatory Follicles

Department of Obstetrics and Gynecology, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0298

Address all correspondence and requests for reprints to: Misung Jo, Department of Obstetrics and Gynecology, Chandler Medical Center, 800 Rose Street, Room MS 335, University of Kentucky, Lexington, Kentucky 40536-0298. E-mail: mjo2{at}uky.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The LH surge induces specific transcription factors that regulate the expression of a myriad of genes in periovulatory follicles to bring about ovulation and luteinization. The present study determined 1) the localization of RUNX1, a nuclear transcription factor, 2) regulation of Runx1 mRNA expression, and 3) its potential function in rat ovaries. Up-regulation of mRNA and protein for RUNX1 is detected in preovulatory follicles after human chorionic gonadotropin (hCG) injection in gonadotropin-treated immature rats as well as after the LH surge in cycling animals by in situ hybridization and immunohistochemical and Western blot analyses. The regulation of Runx1 mRNA expression was investigated in vitro using granulosa cells from rat preovulatory ovaries. Treatments with hCG, forskolin, or phorbol 12 myristate 13-acetate stimulated Runx1 mRNA expression. The effects of hCG were reduced by inhibitors of protein kinase A, MAPK kinase, or p38 kinase, indicating that Runx1 expression is regulated by the LH-initiated activation of these signaling mediators. In addition, hCG-induced Runx1 mRNA expression was inhibited by a progesterone receptor antagonist and an epidermal growth factor receptor tyrosine kinase inhibitor, whereas amphiregulin stimulated Runx1 mRNA expression, demonstrating that the expression is mediated by the activation of the progesterone receptor and epidermal growth factor receptor. Finally, knockdown of Runx1 mRNA by small interfering RNA decreased progesterone secretion and reduced levels of mRNA for Cyp11a1, Hapln1, Mt1a, and Rgc32. The hormonally regulated expression of Runx1 in periovulatory follicles, its involvement in progesterone production, and regulation of preovulatory gene expression suggest important roles of RUNX1 in the periovulatory process.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
THE PREOVULATORY GONADOTROPIN surge induces profound changes in the preovulatory follicle, including breakdown of the extracellular matrix at the apex of the follicle to allow for release of the cumulus-oocyte complex, differentiation of follicular cells to luteal cells, and the follicular/luteal shift in steroidogenesis from estradiol to progesterone. These LH-induced preovulatory changes are mediated by the expression of specific genes, such as genes for inflammation-related proteins, matrix-remodeling enzymes, cell cycle inhibitors, and specific steroidogenic enzymes (reviewed in Refs. 1, 2, 3). Furthermore, transient induction of specific transcription factors by the LH surge [e.g. progesterone receptor (PGR) (4) and CCAAT-enhancer binding protein ß (C/EBPß) (5)] has been shown to be crucial for ovulation and/or luteinization. Recently, we have found that the expression of mRNA for the transcription factor, Runx1, is transiently increased in periovulatory ovaries of gonadotropin-stimulated immature rats after human chorionic gonadotropin (hCG) injection (6), suggesting that RUNX1 may be a key transcriptional modulator in periovulatory follicular changes. However, nothing is known about the regulatory mechanism of Runx1 expression or its function in the ovary.

Runx1 mRNA is highly expressed in thymus, bone marrow, and various hematopoietic cell lines (7). RUNX1 has been shown to play an essential role in the differentiation of various cell types. For instance, RUNX1 is required for differentiation of myeloid progenitor cells into granulocytes (8, 9), maturation of megakaryocytes (10), and differentiation of T and B cells (10, 11). RUNX1 has been shown to be involved in nerve cell innervation (12), fibroblast transformation (13), and angiogenesis (14). In addition, the Runx1 gene is the most frequent target of chromosomal translocation associated with leukemia, and the resultant mutant fusion proteins inhibit the normal transcriptional activity of RUNX1 in various hematopoietic cells (15). All of this evidence points to the involvement of RUNX1 in cell differentiation, maturation, and/or transformation.

RUNX1, also called AML1 (acute myeloid leukemia 1)/core binding factor A2/ polyomavirus enhancer binding protein 2B, belongs to a family of runt-related transcription factors (RUNX/CBF/PEBP2). RUNX1 binds to a consensus recognition sequence, designated as a PEPB2 site [5'-PyGPyGGTPy-3' (16)], in the promoter or enhancer binding regions of target genes, thus regulating their transcriptional activity. Several genes have been characterized as downstream targets of RUNX1 in various hematopoietic cells, although the transcriptional regulation of RUNX1 on these genes was found to be cell-type specific (17). Among these genes, a few have been found to be up-regulated in periovulatory ovaries, such as cyclin-dependent kinase inhibitor 1A (cdkn1a) (18, 19), tissue inhibitor of metalloproteinase-1 (Timp1) (20, 21), and matrix metalloproteinase 13 (Mmp13) (22, 23). Studies with promoter regions of RUNX1 target genes have demonstrated the importance of the cooperation of RUNX1 with other transcription factors in regulating gene expression (19). Such transcription factors include activator protein-1 factors (23) and C/EBP (24), both of which are increased in preovulatory follicles after the LH surge (25, 26). Based on these findings, we hypothesized that the LH-induced RUNX1 may play an important role in the differentiation of follicular cells to luteal cells via regulating the transcription of specific genes in the ovary. The present study tested this hypothesis by characterizing the expression of both mRNA and protein for RUNX1 during the periovulatory period using ovaries from PMSG/hCG-primed immature mice and rats, as well as ovaries from cycling rats. In addition, in vitro experimental models were used to determine the regulatory mechanisms by which the LH surge induces Runx1 expression and identify genes downstream of RUNX1 action in granulosa cells of periovulatory follicles.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Expression of Runx1 mRNA and Protein during the Periovulatory Period in Rat Ovaries
We have previously demonstrated the transient induction of Runx1 mRNA in granulosa cells of preovulatory follicles and theca cells of all antral follicles after hCG injection in PMSG-treated immature rat ovaries using in situ hybridization and Northern blot analyses (6). In the present study, to further determine whether the expression of Runx1 mRNA is followed by its translation into protein, immunohistochemical analyses were performed using an antibody for RUNX1 (AML1: N-20) in ovarian tissue sections from gonadotropin-treated immature rats. No staining for RUNX1 was detected in granulosa cells of preovulatory follicles in ovaries obtained before hCG injection (0 h post-hCG; Fig. 1A, panels a and b). After hCG injection, positive staining was evident in the nuclei of granulosa cells in preovulatory follicles, whereas granulosa cells of adjacent small follicles were not stained (Fig. 1A, panels c–f). After ovulation, positive staining was also observed in cells of corpora lutea (Fig. 1A, panels g and h). In all the ovarian sections examined, positive staining for RUNX1 was localized to the nuclei of theca cells of the majority of antral follicles (Fig. 1A, a–f). RUNX1 was up-regulated in periovulatory ovaries after hCG injection as demonstrated by Western blot analyses using a different anti-RUNX1 antibody (ab11903) and nuclear extracts from granulosa cells, residual ovarian tissues, or whole ovaries (Fig. 1B; two representative blots, a and b, are depicted). Two predominant sizes of RUNX1 ( 53 kDa and 57 kDa) were detected in granulosa cells only at 6 and 12 h after hCG injection, whereas in residual ovarian tissues the proteins were detected at all time points examined and in ovaries containing newly forming corpora lutea (24 h post-hCG). RUNX1 level was highest in granulosa cells obtained at 12 h post-hCG. We initially speculated that the approximately 57-kDa RUNX1 may be a phosphorylated form of RUNX1. However, when the nuclear extracts from 12 h post-hCG granulosa cells were incubated with alkaline phosphatase (1 unit; Promega Corp., Madison, WI), there was no shift in size in Western blot analyses (data not shown).

View larger version (84K): [in this window] [in a new window]   Fig. 1. Expression Profile of RUNX1 in Gonadotropin-Primed Immature Rat Ovaries A, Immunohistochemical localization for RUNX1 protein during the periovulatory period in gonadotropin-primed immature rat ovaries. Sections of rat ovaries obtained at 0 h (48 h post-PMSG, a and b), 6 h (c, d, i, and j), 12 h (e and f), or 24 h (g and h) after hCG injection were immunostained with the antibody for RUNX1 (AML-1: N-20). The boxed areas in panels a, c, e, g, and i are magnified in panel b, d, f, h, and j, respectively. The tissue section depicted in i and j was incubated with the primary antibody preadsorbed with the blocking peptide for AML1 as a negative control. F, Follicle; Gc, granulosa cell layer; Th, theca cell layer; PF, periovulatory follicles; nCL, newly forming corpus luteum. Arrowheads indicate positive stained nuclei of granulosa cells. Arrows indicate positive stained nuclei of theca cells. Wavy arrows indicate positively stained nuclei of luteal cells. Stars indicate small antral follicles lacking RUNX1 immunoreactivity in the granulosa cells. Original magnification of a, c, e, g, and i is x150. Original magnification of b, d, f, h, and j panels is x400. B, Western blot analysis of RUNX1 protein in granulosa cells, residual ovarian tissue, or whole ovaries of immature rats obtained at indicated times after hCG injection. Arrows to the right of panel B) indicate different forms of RUNX1 detected. Each lane was loaded with 25 µl of nuclear fraction (30 µg) extracted from ovaries of each animal. The membrane was stained with Ponceau-S to show the relative loading of sample protein. The blots (a and b in panel B) are representatives of four separate experiments (n = 4 animals/time point).

View larger version (102K): [in this window] [in a new window]   Fig. 2. Expression Profile of RUNX1 in Naturally Cycling Rat Ovaries A, In situ hybridization analysis of Runx1 mRNA during the periovulatory period in ovaries from naturally cycling rats. Representative bright-field (a, c, and e) and corresponding dark-field (b, d, and f) photomicrographs are depicted. Ovaries were collected at 1600 h (a and b; at the peak of the LH surge) and 2400 h (c and d) on proestrus, and 400 h on estrus (e and f). Arrows in d indicate a small antral follicle expressing Runx1 mRNA in the theca layer. Arrowheads in d indicate Runx1 mRNA expression in periovulatory follicles. Original magnification of all slides is x40. B, Immunohistochemical localization for RUNX1 during the periovulatory period in ovaries from naturally cycling rats. Sections of rat ovaries obtained at 1600 h (a and b; at the peak of the LH surge) and 2400 h (c and d) on proestrus, and 400 h on estrus (e and f) were immunostained with the RUNX1 antibody (AML-1: N-20). Arrows, arrowheads, and wavy arrows indicate theca cells, granulosa cells, and luteal cells, respectively, that show positive staining for Runx1 protein. Stars indicate small follicles lacking RUNX1 immunoreactivity in the granulosa cells. The boxed areas in panels a, c, and e were magnified in panels b, d, and f, respectively. Original magnification of a, c, and e panels is x150. Original magnification of b, d, and f panels is x400. Gc, Granulosa cell layer; Th, theca cell layer; PF, periovulatory follicles; nCL, newly forming corpus luteum; pCL, corpus luteum from previous estrous cycles; F, follicle.

We also examined the expression pattern of mRNA and protein for RUNX1 in mouse ovaries during the periovulatory period. Northern blot, in situ hybridization, and immunohistochemical and Western blot analyses of ovaries obtained from PMSG/hCG-stimulated immature mice revealed virtually identical expression patterns of mRNA and protein for RUNX1 in periovulatory ovaries compared with that in the rat ovary (data not shown).

Effects of LH on the Expression of Runx1 mRNA in Granulosa Cell Cultures
To determine whether the transient increase in levels of Runx1 mRNA after hCG injection in vivo can be mimicked in vitro and whether the induction of Runx1 mRNA is directly mediated by the action of LH, granulosa cells isolated from PMSG-primed immature rat ovaries (48 h post-PMSG) were cultured in the absence or presence of a luteinizing dose of hCG (1 IU/ml). Northern blot analysis revealed expression of multiple transcripts (6.3, 3.8, and 3.6 kb) of the Runx1 gene in cultured rat granulosa cells (Fig. 3A). hCG treatment induced a dramatic, transient increase in levels of Runx1 mRNA. The levels were highest at 3 and 6 h of culture and then declined to basal levels by 24 h (Fig. 3A). This transient expression pattern is similar to that of Runx1 mRNA in rat granulosa cells of preovulatory follicles in vivo (6). To confirm the induction of RUNX1 protein by hCG, Western blot analysis was conducted with nuclear fractions extracted from the cultured granulosa cells. As expected, hCG induced a transient increase in RUNX1 protein (Fig. 3B). RUNX1 was detected in the granulosa cells collected at 6 h, further increased by 12 h, and then began to decline at 24 h after culture, indicating the time delay ( 3 h) in RUNX1 protein accumulation compared with the profile of Runx1 mRNA expression. The highest level of RUNX1 was detected by 9–12 h of culture (data not shown). This profile of RUNX1 protein in vitro mimics that in vivo (Fig. 1B).

View larger version (62K): [in this window] [in a new window]   Fig. 3. Transient Up-Regulation of mRNA and Protein for RUNX1 in Granulosa Cells by hCG in Vitro A, Autoradiograph of a representative Northern blot analysis shows multiple transcripts for the Runx1 gene and ribosomal protein L32 mRNA in granulosa cells from rat preovulatory ovaries (48 h post-PMSG) cultured in medium alone (Control) or with hCG (1 IU/ml) for 0, 3, 6, 12, or 24 h. The levels of Runx1 mRNA were calculated by combining the intensity of all three different transcripts of the Runx1 gene detected in the Northern blot. Relative levels of Runx1 mRNA were normalized to the L32 band in each sample (mean ± SEM; n = 3 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05). B, Western blot analysis of RUNX1 protein in preovulatory granulosa cells cultured in medium alone (Control) or with hCG for 3, 6, 12, or 24 h. Arrows to the right of panel B indicate the different forms of RUNX1 detected. Protein concentrations were controlled by plating an equal number of cells per well for each treatment and then loading an equal volume (30 µl) of nuclear extracts to each lane. The membrane was stained with Ponceau-S to show the relative loading of sample protein. Panel B is a representative of four separate culture experiments. C, Control.

View larger version (47K): [in this window] [in a new window]   Fig. 4. Regulation of Runx1 mRNA Expression by Agonists or Inhibitors of Various Intracellular Signaling Modulators in Granulosa Cells in Vitro A, Autoradiograph of a representative Northern blot analysis shows mRNA for Runx1 and ribosomal protein L32 in granulosa cells from rat preovulatory ovaries (48 h post-PMSG) cultured in medium alone (Cont) or with hCG (1 IU/ml), forskolin (FSK, 10 µM), PMA (20 nM), or FSK + PMA for 6 h. The levels of Runx1 mRNA were measured as described in Fig. 3. Relative levels of Runx1 mRNA were normalized to the L32 band in each sample (mean ± SEM; n = 7 independent culture experiments). B, Granulosa cells from rat preovulatory ovaries (48 h post-PMSG) were cultured with medium alone (Cont), inhibitors of various signaling molecules [an inhibitor of PKA (H89, 10 µM), PKC (GF109203X[GF], 1 µM), phosphatidylinositol 3-kinase (LY294002[LY], 25 µM), MEK (PD98059[PD], 20 µM), and p38 kinase (SB2035850[SB], 20 µM)], hCG, or hCG + inhibitors of various signaling molecules for 6 h. Levels of Runx1 mRNA were measured by Northern blot analyses. Relative levels of Runx1 mRNA were normalized to the L32 band in each sample (mean ± SEM; n = 4 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05). C, The experimental results are summarized in the diagram. The LH surge or an ovulatory dose of hCG stimulates Runx1 mRNA expression via activating PKA, MEK, and p38 kinase. PI3K, Phosphatidylinositol 3-kinase; PLC, phospholipase C.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Complete Inhibition of the hCG-Induced Runx1 mRNA Accumulation by Cyclohexamide Granulosa cells from rat preovulatory ovaries (48 h post-PMSG) were cultured in medium alone (Cont) or with cyclohexamide (1 µg/ml, CHX), hCG (1 IU/ml), or hCG + CHX for 6 h. Relative levels of Runx1 mRNA were normalized to the L32 band in each sample in Northern blots (mean ± SEM; n = 4). Bars with no common superscripts in each panel are significantly different (P < 0.01).

View larger version (40K): [in this window] [in a new window]   Fig. 6. Regulation of Runx1 mRNA Expression by Hormones A, A representative Northern blot and RT-PCR show a transient increase in levels of mRNA for Areg and Pgr, respectively. L32 and L19 were used as an internal control for each assay. Granulosa cells from rat preovulatory ovaries (48 h post-PMSG) were cultured in medium alone (C) or with hCG for 0, 3, 6, 12, or 24 h. B, hCG stimulates progesterone production in cultured granulosa cells. Concentrations of progesterone were measured in preovulatory granulosa cell conditioned culture media collected at 3, 6, or 24 h. Bars with no common superscripts are significantly different (P < 0.05). C, hCG-induced Runx1 mRNA expression was reduced by a PGR antagonist (ZK98299) and an EGF receptor tyrosine kinase-selective inhibitor (AG1478), as determined by Northern blot analyses. Granulosa cells from rat preovulatory ovaries (48 h post-PMSG) were cultured in medium alone (Cont) or with NS-398 (1 µM; NS, a specific inhibitor of prostaglandin-endoperoxide synthase 2), ZK98299 (1 µM; ZK, a progesterone receptor antagonist), AG1478 (1 µM; AG, an EGF receptor tyrosine kinase-selective inhibitor), ZK + AG, hCG (1 IU/ml), hCG + NS, hCG + ZK, hCG + AG, hCG + ZK + AG for 6 h. Bars with no common superscripts in each panel are significantly different (P < 0.05).

View larger version (25K): [in this window] [in a new window]   Fig. 7. Induction of Runx1 mRNA by AREG Was Partially Mediated by the Activation and Induction of PGR Granulosa cells from rat preovulatory ovaries (48 h post-PMSG) were cultured in medium alone (Cont) or with agonists and/or inhibitors for 6 h to detect Runx1 mRNA and 4 h to measure the levels of Pgr mRNA. Concentrations of agonists and inhibitors are as follows: hCG (1 IU/ml), AREG (0.1 mg/ml), AG1478 (AG1, 1 µM; AG3, 3 µM), ZK98299 (1 µM, ZK), and cyclohexamide (1 µg/ml, CHX). The levels of Runx1 and Pgr mRNA were determined by Northern blot analyses. L32 was used as an internal control. Relative levels of mRNA for the gene were normalized to the L32 band in each sample (mean ± SEM; n = 3 or more independent culture experiments). A, The activation of EGF-receptor by AREG stimulated Runx1 mRNA expression, and cyclohexamide blocked AREG-induced Runx1 mRNA expression. B, The stimulatory effect of AREG on Runx1 mRNA expression was inhibited by ZK98299. C, AREG stimulated Pgr mRNA expression. D, The stimulatory effect of hCG on Pgr mRNA expression was reduced by AG1478. Bars with no common superscripts in each panel are significantly different (P < 0.05). E, Hypothetical model of Runx1 mRNA regulation by LH. Cont, Control; EGF-R, EGF receptor.

View larger version (45K): [in this window] [in a new window]   Fig. 8. Effects of Reduction in RUNX1 by Runx1 siRNA in Cultured Granulosa Cells Granulosa cells obtained from rat preovulatory ovaries (48 h post-PMSG) were cultured with hCG (1 IU/ml) + control siRNA (scrambled siRNA) or hCG + Runx1 siRNA. A, Autoradiograph of a representative Northern blot shows mRNA for Runx1 and L32 in the granulosa cells cultured for 4 and 24 h. Relative levels of Runx1 mRNA were normalized to the L32 band in each sample (mean ± SEM; n = 7 independent culture experiments). B, Western blot analysis of RUNX1 protein in preovulatory granulosa cells cultured with hCG (1 IU/ml) + control siRNA (scrambled siRNA) or hCG + Runx1 siRNA for 10 h. Arrows to the right of panel B indicate the different forms of Runx1 detected. Protein concentrations were controlled by plating an equal number of cells per well in each treatment and then loading an equal volume (30 µl) of nuclear extract in each lane. The membrane was stained with Ponceau-S to show the relative loading of sample protein. Panel B is a representative of four separate culture experiments. C, Concentrations of progesterone in granulosa cell culture media collected at 24 h after hCG treatment (mean ± SEM; n = 6 independent culture experiments). D, Autoradiograph of a representative Northern blot shows mRNA for Cyp11a1, Timp1, and L32 in granulosa cells cultured for 4 and 24 h. Relative levels of Cyp11a1 mRNA were normalized to the L32 band in each sample (mean ± SEM; n = 7 independent culture experiments). *, P < 0.001; **, P < 0.01. Cont, Control.

View larger version (52K): [in this window] [in a new window]   Fig. 9. Regulation of Periovulatory Gene Expression by Suppression of RUNX1 via siRNA in Cultured Granulosa Cells Granulosa cells obtained from rat preovulatory ovaries (48 h post-PMSG) were cultured with hCG (1 IU/ml) + control siRNA (scrambled siRNA) or hCG + Runx1 siRNA. A, Autoradiograph of a representative Northern blot shows mRNA for Runx1 and L32 in the granulosa cells cultured with hCG + siRNA for 4, 10, and 24 h. B, Autoradiograph of a representative Northern blot shows mRNA for Runx1, Cdkn1a, Mt1a Hapln1, Rgc32, and L32 (internal control) in the granulosa cells cultured with hCG + siRNA for 10 and 24 h. Relative levels of Mt1a (C), Hapln1 (D), and Rgc32 (E) mRNA were normalized to the L32 band in each sample (mean ± SEM; n = 4 independent culture experiments). *, P < 0.001; **, P < 0.01. Cont, Control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In response to the LH surge, preovulatory follicles undergo rapid and dramatic changes in the expression patterns of a myriad of genes (40). For instance, many genes that are highly expressed in growing preovulatory follicles are turned off by the LH surge, whereas the expression of numerous genes that are involved in ovulation and luteinization increases dramatically during the periovulatory period (reviewed in Refs. 1 and 32). Therefore, it is conceivable that transcriptional regulators induced in periovulatory follicles after the LH surge play a crucial role in controlling the transcriptional activation/repression of periovulatory genes. Such transcriptional modulators previously identified in periovulatory ovaries include PGR (31), C/EBPß (5), and early growth response 1 (41). Herein, we report RUNX1 as a transcriptional regulator involved in periovulatory gene expression; this is based on experimental evidence of LH surge-induced, hormone-regulated expression of Runx1 in periovulatory follicles and alteration of periovulatory gene expression by suppression of hCG-induced RUNX1 expression.

In the present study, we demonstrated the rapid induction and transient expression of RUNX1 protein during the periovulatory period in rodent ovaries of gonadotropin-stimulated immature mice and rats. Using cycling adult rats, we confirmed the induction of RUNX1 in periovulatory follicles and newly forming corpora lutea. In addition, RUNX1 protein was detected in theca cells of antral follicles even before the LH surge in cycling rats and before hCG injection in the gonadotropin-induced model. Previously, we reported a minor and transient increase in Runx1 mRNA levels at 12 h after PMSG injection (6). This increase is due to the expression of Runx1 mRNA in the theca layer of antral follicles as determined by in situ hybridization studies (Jo, M., unpublished data). Thus, we speculated that the thecal expression of RUNX1 protein is initiated before hCG injection or the LH surge, presumably during the follicular growth stimulated by the LH-like activity of PMSG in gonadotropin-treated immature rats or LH in cycling animals.

Using primary granulosa cell cultures, we demonstrated that hCG induces a rapid and transient expression of Runx1 mRNA, which was followed by that of RUNX1 protein, as determined by immunoblotting. These expression patterns of both mRNA and protein for RUNX1 in vitro are strikingly similar to those seen in vivo, demonstrating that the up-regulation of RUNX1 expression results from the direct action of LH on its cognate receptor in preovulatory granulosa cells. Importantly, the induction of Runx1 mRNA in cultured granulosa cells afforded us an experimental model in which the LH-initiated signaling pathway involved in Runx1 expression was further investigated. It is well recognized that the binding of LH to its receptor activates adenylate cyclase and leads to cAMP-mediated events, predominantly through the PKA-signaling pathway (32). In addition, the involvement of other kinase-signaling pathways has been implicated for mediating the complex series of LH-induced periovulatory processes, including PKC (27), phosphatidylinositol 3-kinase (42), and receptor tyrosine kinase (43). A previous study by Salvador et al. (30) has shown that the activation of LH receptor by administration of hCG to rat preovulatory granulosa cells in vitro leads to the activation of MEK and p42/44 MAPK through the PKA-dependent pathway and also stimulates p38 kinase phosphorylation by a PKA- and PKC-independent pathway. In this study, we demonstrated that Runx1 mRNA expression in cultured granulosa cells is dependent on the LH-induced activation of PKA, MEK, and p38 kinase, implicating the involvement of multiple signaling pathways, presumably the PKA-dependent pathway (cAMP/PKA/MEK) as well as the PKA- and PKC-independent pathway (p38 kinase) (depicted in Fig. 4C).

In addition to the involvement of multiple signaling pathways, the LH-induced Runx1 expression appears to require the action of autocrine and/or paracrine factors, which are synthesized in preovulatory granulosa cells in response to the LH surge. For instance, the present data show that the hCG-induced Runx1 mRNA accumulation was completely abolished by cyclohexamide, demonstrating that the induction of Runx1 mRNA requires the synthesis of a new protein(s). We hypothesized that the newly synthesized protein(s) could be PGR, EGF-related peptides, or PTGS2, all of which have been shown to be rapidly and transiently induced in granulosa cells of preovulatory follicles in response to the ovulatory level of LH in vivo and in vitro (28, 34, 36). This hypothesis was supported in part by the present finding that blocking PGR by a specific antagonist (ZK98299) and inhibiting the activation of the EGF receptor (AG1478) by an EGF receptor tyrosine kinase-selective inhibitor reduced the hCG-induced Runx1 mRNA expression in cultured granulosa cells. Furthermore, the present data demonstrating the stimulatory effect of AREG on Runx1 mRNA expression and its complete abolishment by AG1478 confirmed the involvement of EGF signaling on Runx1 mRNA expression. Interestingly, this stimulatory effect of AREG was completely repressed when the de novo protein synthesis was blocked by cyclohexamide, indicating that a newly synthesized protein(s) is required for the up-regulation of Runx1 mRNA. We explored the possibility that PGR may be one of the synthesized proteins induced by AREG and required for Runx1 mRNA expression. This speculation was derived from our finding that the stimulatory effect of AREG on Runx1 mRNA expression was reduced by a PGR antagonist. Our novel finding demonstrating the ability of AREG to stimulate Pgr mRNA expression and AG1478 to inhibit the hCG-induced Pgr mRNA expression provides support for this hypothesis. The fact that the levels of Runx1 and Pgr mRNA were partially reduced by each inhibitor used in the present study implicates the presence of multiple pathways involved in the LH-induced gene expression. Taken together, these data suggest that the up-regulation of Runx1 mRNA expression in periovulatory granulosa cells is mediated by the coordinate actions of multiple autocrine/paracrine factors that are induced by the LH surge, including actions of progesterone/PGR and EGF-related peptides/EGF receptor (depicted in Fig. 7E).

RUNX1 has been shown to play an essential role in differentiation of various cell types (8, 10, 11, 13). In the ovary, the LH surge promotes differentiation of follicular cells to luteal cells that predominantly produce and secrete progesterone [reviewed in (44)]. In addition, luteinizing granulosa cells have been shown to exit the cell cycle and subsequently stop dividing (18, 45). Robker and Richards (18) have demonstrated that during the LH-induced terminal differentiation of granulosa cells (luteinization), the expression of a cell cycle promoter, cyclin D2, was rapidly down-regulated, whereas cell cycle inhibitors (e.g. Cdkn1a and Cdknlb) were dramatically increased in rat periovulatory granulosa cells. Previously, the expression of Cdkn1a has been shown to be regulated by RUNX1 in hematopoietic cells (19). Based on our finding of the rapid induction of RUNX1 expression by the LH surge and a previous report of the up-regulation of Cdkn1a mRNA expression in periovulatory granulosa cells (18), we speculated that RUNX1 may be involved in differentiation of luteinizing granulosa cells, perhaps by regulating the expression of Cdkn1a. However, we found that the levels of Cdkn1a mRNA were not affected by the reduction of RUNX1 expression in siRNA-treated granulosa cell cultures. Considering that RUNX1 expression was not completely blocked by siRNA treatment in the present study, it is possible that only complete abolishment of RUNX1 would exert an effect on Cdkn1a gene expression or it may be that RUNX1 regulates the transcription of Cdkn1a gene in a cell type-specific manner.

Another potential role of RUNX1 in the ovary may be the follicular/luteal shift in steroidogenesis toward progesterone production. For instance, we found that the suppression of RUNX1 by siRNA resulted in decreased progesterone accumulation as well as a reduction of the level of Cyp11a1 mRNA, a key steroidogenic enzyme that is involved in converting cholesterol to progesterone. In the preovulatory ovary, LH/hCG has been shown to stimulate the expression of Cyp11a1 mRNA and protein in vivo and in vitro (46, 47) in luteinizing granulosa cells, which produce predominantly progesterone. Therefore, our novel findings suggest that RUNX1 plays a role in the follicular/luteal shift in steroidogenesis in luteinizing granulosa cells, possibly through regulating the expression of genes for steroidogenic enzymes such as Cyp11a1.

One of the important findings in this study was the identification of candidate genes that are regulated by RUNX1 in periovulatory granulosa cells. Herein, we reported three genes, Mt1a, Hapln1, and Rgc32, as potential downstream targets of RUNX1. Metallothionein is a zinc-binding protein that acts to regulate the availability of zinc and other heavy metal ions in cells, thus known to play an important role in cellular metabolism, redox balance, and various cellular processes such as proliferation, inflammation, and apoptosis (reviewed in Ref. 48). A recent study by Espey et al. (38) has documented that Mt1a mRNA is induced in periovulatory follicles and highly expressed in corpora lutea of gonadotropin-stimulated immature rat ovaries. These authors suggested potential roles for this protein in regulating the activity of metalloproteinases, steroidogenesis, and inflammatory processes during the periovulatory period (38). HAPLN1 is a glycoprotein that interacts with both hyaluronic acid and proteoglycan (e.g. versicans) to form aggregates in a hyaluronan-rich extracellular matrix. In the ovary, HAPLN1 was localized exclusively to the extracellular matrix and cytoplasm of cumulus granulosa cells of periovulatory follicles in rodent ovaries (39, 49), indicating the involvement of this protein in cumulus-oocyte complex expansion. Rgc32 was first identified as a gene that is induced by sublytic complement activation in oligodendrocytes (50). A recent study by Badea et al. (50) showed that RGC32 increased the activity of p34^cdc2 kinase and the overexpression of this protein enhanced S-phase entry in aortic smooth muscle cells, suggesting the role of RGC32 as a cell cycle regulator. Rat Ovarian Gene Expression database (6) and our Northern blot data (Park, E., unpublished data) showed that Rgc32 mRNA expression is increased in ovulating follicles of rodent ovaries. Therefore, it is of interest to further determine whether RGC32 is involved in changes in the cell cycle of luteinizing granulosa cells. Although the results from the present study suggest the involvement of RUNX1 in the gene expression of Mt1a, Hap1n1, and Rgc32, there has been no report for the binding site of RUNX1 in the known promoter/enhancer regions of these genes. A computational search (TFSEARCH, http://www.cbrc.jp/research/db/TFSEARCH.html) revealed the presence of potential binding sites for RUNX1 within 1000-bp genomic sequences 5' of the first transcription start site of Hapln1 and Mt1a gene of rats. As for the Rgc32 gene, rat genomic sequence of this gene is not presently available, although in the mouse Rgc32 gene, three possible binding sites for RUNX1 were found. However, further experimental analyses (e.g. promoter analysis and chromatin immunoprecipitation assay) are needed to determine whether these genes are direct targets of RUNX1 in luteinizing granulosa cells.

In summary, the present study demonstrated the rapid and transient induction of Runx1 mRNA by the LH surge in periovulatory ovaries and the concomitant increase in RUNX1 protein in granulosa cells of periovulatory follicles and luteal cells in newly forming corpora lutea. We provide experimental evidence that the hCG-induced Runx1 mRNA expression is dependent on the activation of the EGF receptor by EGF-related peptides and PGR activation, revealing coordinate regulation of Runx1 mRNA expression by these two key mediators of the periovulatory process. Finally, the findings that reduction of hCG-induced RUNX1 expression by siRNA resulted in decreased progesterone accumulation as well as a reduction in levels of mRNA for Cyp11a1, Mt1a, Hapln1, and the novel gene, Rgc32, provide strong support for the role of RUNX1 in the follicular/luteal shift in steroidogenesis and periovulatory gene expression in luteinizing granulosa cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Materials
Unless otherwise noted, all chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO), or Calbiochem (La Jolla, CA). Molecular biological enzymes, BenchMark Prestained Protein ladder, oligonucleotides, and Trizol were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA).

Animals
All animal procedures were approved by the University of Kentucky Animal Care and Use Committee. In the present study, gonadotropin-treated immature female mice and rats, as well as sexually mature adult female rats exhibiting regular 4-d estrous cycles were used.

Immature female Sprague Dawley rats and C57BL/6 mice were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and provided with water and chow ad libitum. Rats were maintained on a 14-h light, 10-h dark cycle, and mice were maintained on a 12-h light, 12-h dark cycle. The animals (22 d old) were injected with PMSG (10 IU to rats, 5 IU to mice) s.c. to stimulate follicular development and 48 h later with hCG (5 IU to rat, 2.5 IU to mice) s.c. to induce ovulation and subsequent formation of corpora lutea. In these models (51), ovulation occurs approximately 12–16 h post-hCG administration. Animals were killed at 0 h (at the time of hCG administration) and defined times after hCG administration (n = 3–4 animals/time point). Ovaries were collected and stored at –70 C for later isolation of total RNA, or placed in Tissue-Tek OCT compound (VWR Scientific, Atlanta, GA), snap frozen, and stored at –70 C until sectioned and processed for in situ hybridization and immunohistochemical analyses. For western immnuoblot analyses, fresh ovaries were used to extract nuclear fractions from granulosa cells or residual ovarian tissues.

Adult female Sprague Dawley rats (150–180 g body weight, 2 months old) were purchased from Harlan Sprague Dawley, Inc., and housed as described above. Stages of the estrous cycle were determined by daily examination of vaginal cytology, and only animals showing at least two consecutive 4-d cycles were used for the experiment. Rats were killed at 1600 h, 2000 h, and 2400 h on proestrus, and 0400 h on estrus. In this colony of rats, the peak of the LH surge was at 1600 h on proestrus. Ovaries were collected, placed in Tissue-Tek OCT compound, and stored at –70 C until sectioned and processed for in situ hybridization and immunohistochemistry.

Isolation and Culture of Granulosa Cells
To isolate granulosa cells, ovaries were collected from immature rats 48 h after PMSG administration and processed as described previously (21). Briefly, granulosa cells were isolated by the method of follicular puncture. The cells were pooled, filtered, pelleted by centrifugation at 200 x g for 5 min, and resuspended in defined medium consisting of DMEM-Ham’s F-12 medium supplemented with 1% BSA, 0.01% pyruvic acid, 0.22% bicarbonate, gentamycin (0.05 mg/ml), and ITS (insulin, transferrin, and selenium). The cells were cultured in the absence or presence of various reagents for 0, 3, 6, 12, or 24 h at 37 C in a humidified atmosphere of 5% CO₂. When reagents were dissolved in dimethylsulfoxide (DMSO) and added to the culture medium, the same concentration of DMSO was added to medium for the control cells. The final concentration of DMSO in cultures was less than 0.05%. At the end of each culture period, cells were collected and processed for extraction of nuclear fractions or snap-frozen for later isolation of total RNA.

siRNA Knockdown of Runx1 mRNA in Granulosa Cells in Vitro
Granulosa cells were collected from immature rats 48 h after PMSG administration described above and resuspended in siPORT siRNA electroporation buffer (Ambion, Inc., Austin, TX). The cells were mixed with siRNA specific for Runx1 (sense, 5'-GGCUCCUACCAAUUCUCCAtt-3'; antisense, 5'-UGGAGAUUGGUAGGAGCCtg-3'; Ambion, Inc.) or Silencer negative control siRNA (scrambled siRNA; Ambion Inc.) and then electrophorated at 400 V, 100 µsec, and one pulse using a Multiporator (Eppendorf, Hamburg, Germany). These conditions were chosen based on the results of preliminary optimization experiments. The electroporated cells (5 x 10⁵ cells and 100 nM of siRNA per well) were cultured for 1 h in the defined medium described above before hCG (1 IU/ml) treatment, and further cultured for 4, 10, or 24 h at 37 C in a humidified atmosphere of 5% CO₂. At the end of each culture period, cells were collected and snap frozen for isolation of total RNA for Northern blot analyses or processed to isolate nuclear extracts for Western blot analyses. Conditioned culture media were collected and stored at –20 C and measured for progesterone by RIA.

DNA Microarray
Total RNA was extracted from the granulosa cells treated with Runx1 siRNA or scrambled siRNA and cultured for 10 or 24 h using a RNeasy Mini kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer’s instructions and quantified by spectrophotometry. The integrity of total RNA was verified by visualizing the intact and distinct 28S rRNA and 18S rRNA stained with ethidium bromide in 1.5% agarose-formaldehyde gel. The total RNA was transferred to a nitrocellulose membrane. The reduction of Runx1 mRNA in Runx1 siRNA-treated granulosa cells was confirmed by Northern blot analyses. Total RNA (5µg) pooled from two independent experiments was used as a template for cDNA synthesis, and biotinylated antisense cRNA probe was prepared as described by the manufacturers of the SuperScript System Kit (Invitrogen) and the ENZO BioArray HighYield RNA labeling Kit (Enzo Diagnostics, Farmingdale, NY). Unincorporated nucleotides were removed from the riboprobe preparation using the RNeasy Mini Kit. The integrity of the riboprobe was checked by gel electrophoresis. The Affymetrix Rat 230 2.0 genechip array was hybridized, washed, and scanned using Affymetrix equipment and protocols (Affymetrix; DNA Microarray Core Facility, University of Kentucky, Lexington, KY).

Generation of the Plasmid-Containing cDNA for Rat Cdkn1a, Hapln1, Mt1a, Pgr, and Rgc32
Partial cDNAs corresponding to each of the reference sequences listed in Table 1 were generated by RT-PCR and cloned into the pCRII-TOPO Vector (Invitrogen) as described previously (6). Oligonucleotide primer pairs were designed based on published sequence data (the primer sequence for each gene is listed in Table 1). DNA sequences of cloned rat partial cDNAs for the selected genes were verified commercially (MWG Biotech, Inc., High Point, NC).

In Situ Hybridization of Runx1 mRNA
Ovaries collected from PMSG-primed immature mouse ovaries or naturally cycling adult rats were sectioned at 10 µm and mounted on Probe On Plus slides (Fisher Scientific, Pittsburgh, PA). In situ hybridization was carried out as described previously (54). Briefly, plasmids containing cDNA for rat Runx1 were linearized with SaI I and BamHI to generate sense and antisense riboprobes, respectively. Linearized plasmids were labeled with [-³⁵S]UTP (10 mCi/ml; MP Biomedicals, Inc., Costa Mesa, CA) and appropriate RNA polymerases. The sections were hybridized overnight with 1 x 10⁶ cpm ³⁵S-labeled riboprobe/slide in a humidified chamber at 55 C. The next day, slides were washed and treated with RNase A (0.1 mg/ml) for 30 min at 45 C. Tissue sections were washed again at high stringency, dried, dipped in Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY), and exposed at 4 C for 4 wk. To visualize the hybridized riboprobes, slides were developed with Kodak D19 and counterstained with hematoxylin solution. Tissues were examined with an Eclipse E800 Nikon microscope (Nikon Corp., Melville, NY) under bright- and dark-field optics. One ovary from each of three animals was used for in situ hybridization. At least 12 sections per ovary were analyzed for each antisense probe, making a total of at least 36 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.

Immunohistochemistry
Frozen ovaries collected from untreated or gonadotropin-treated immature mice and rats were sectioned at 8 µm, thaw-mounted on Probe On Plus slides, and air dried. The sections were fixed in acetone for 10 min. The endogenous peroxidase activity was quenched by immersing the sections in methanol containing 0.3% H₂O₂. Immunohistostaining was performed using the Vectorstain Elite ABC (avidin-biotin complex) kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. Briefly, after a PBS rinse, the sections were incubated with normal rabbit serum for 30 min and then treated with the primary RUNX1 antibody (AML1; N-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted to 2 µg/ml in PBS for 16 h at 4 C. For the negative control, the sections were incubated with the primary antibody preadsorbed with a blocking peptide for AML1 (Santa Cruz Biotechnology, Inc.) or without primary antibody. The sections were washed with PBS and then incubated with antigoat biotin-conjugated secondary antibody for 30 min. The biotinylated antibody complex was incubated with a peroxidase-labeled ABC for 1 h at room temperature by use of the ABC Elite reagent and then visualized with substrate-chromagen solution (3,3'-diaminobenzidine). One ovary from each of three animals was used for immunohistochemistry. At least eight sections per ovary were analyzed by immunohistochemistry, making a total of at least 24 tissue sections analyzed for each time point.

Western Blot Analysis
Nuclear extracts were isolated from granulosa cells, the residual ovarian tissue (ovary after expelling granulosa cells), or whole ovaries obtained from gonadotropin-stimulated immature rats or cultured rat granulosa cells using a Nuclear Extraction Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instruction. Briefly, the cells were collected in ice-cold PBS/phosphatase inhibitors (Active Motif), pelleted by centrifugation at 200 x g for 5 min, and resuspended in hypotonic buffer to swell the cell membrane for 15 min. Then, 25 µl of detergent was added to release cytoplasmic proteins into the supernatant. To collect nuclear pellet, the cell suspensions were pelleted by centrifugation at 14,000 x g for 30 sec. The supernatant was removed, and the nuclei were lysed with 50 µl Complete Lysis buffer/protease inhibitor cocktail for 30 min on ice. After centrifugation for 10 min at 14,000 x g, supernatants (nuclear fraction) were aliquoted and stored at –80 C until use. For the ovarian tissues, they were homogenized in hypotonic buffer/dithiothreitol + detergent with prechilled Dounce homogenizers. Homogenized tissues were pelleted by centrifugation at 850 x g for 10 min. The pellets were resuspended in hypotonic buffer and processed as described above. Nuclear extracts were denatured by boiling for 5 min and separated by SDS-PAGE on an 8% polyacrylamide gel and then transferred onto nitrocellulose membrane (Schleicher & Schuell Inc., Keene, NH). Immunoblotting of RUNX1 protein was performed using Vectorstain Elite ABC kit (Vector Laboratories, Inc.). Primary antibody [rabbit polyclonal antibody to RUNX1 (ab11903, 1:1000 dilution; Abcam, Cambridge, MA) was added in 1% casein solution and incubated overnight at 4 C. Peroxidase activity was visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL). Jurkat nuclear extracts (Active Motif) were included as a positive control sample for RUNX1 protein and prestained protein size marker (Invitrogen) in most Western blots.

RIA
Concentrations of progesterone in 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.

Statistical Analyses
All data are presented as means ± SEM. One-way ANOVA was used to test differences in levels of Runx1 mRNA across time of tissue collection, time of culture, or among treatments in vitro. If ANOVA revealed significant effects of time of tissue collection, time of culture, or treatment, the means were compared by Duncan’s test, with P < 0.05 considered significant. The experiments examining the effect of Runx1 siRNA in granulosa cell cultures were analyzed by paired t tests.

	ACKNOWLEDGMENTS

 
We thank Dr. O.K. Park-Sarge for providing plasmids containing cDNAs for L32 and ZK98299 and Dr. J. S. Richards for providing plasmids containing cDNAs for Cyp11a1.

	FOOTNOTES

 
This work was supported by National Institutes of Health Grants NCRR P20 RR 15592 (to T.E.C. and M.J.) and RO3 HD051727-01 (to M.J.).

M.J. and T.E.C. have nothing to declare.

First Published Online May 4, 2006

Abbreviations: AML1, Acute myeloid leukemia 1; AREG, amphiregulin; cdkn, cyclin-dependent kinase inhibitor; C/EBPß, CCAAT-enhancer binding protein ß; CG, chorionic gonadotropin; DMSO, dimethylsulfoxide; EGF, epidermal growth factor; Hapln1, hyaluronan and proteoglycan link protein 1; MEK, MAPK kinase; Mt1a, metallothionein 1a; PGR, progesterone receptor; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12 myristate 13-acetate; PMSG, pregnant mare serum gonadotropin; Rgc32, response gene to complement 32; siRNA, small interfering RNA; Timp1, tissue inhibitor of metalloproteinase-1.

Received for publication December 14, 2005. Accepted for publication April 25, 2006.

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