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Biology of Reproduction 65, 1565-1572 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Transcriptional Regulation of Cyclooxygenase-2 Gene in Ovine Large Luteal Cells1

Yuh-Lin Wua, and Milo C. Wiltbank,a,b

a Endocrinology-Reproductive Physiology Program and b Department of Dairy Science, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT

There is positive feedback pathway in the ovine large luteal cell, such that prostaglandin (PG) F2{alpha} stimulation induces intraluteal PGF2{alpha} production as the result of induction of one of the rate-limiting enzymes in PG production, cyclooxygenase-2 (Cox-2). The objective of the present study was to evaluate the intracellular effector systems and important DNA transcriptional element(s) involved in regulating the Cox-2 gene in ovine large luteal cells. In transient transfection assays, Cox-2 promoter was rapidly induced (4 h) by phorbol didecanoate (a protein kinase [PK] C activator), ionomycin, and cloprostenol (PGF2{alpha} analogue), with a peak induction at 12 h. Cloprostenol-mediated promoter activation was not blocked by inhibition of various second messenger systems, including PKA, calcium calmodulin kinase II, or mitogen-activated protein kinases. However, myristoylated PKC pseudosubstrate peptide inhibited cloprostenol stimulation of Cox-2 promoter, indicating the critical role of PKC in this stimulation. The Cox-2 promoter could be reduced to 282 base pairs (bp) of the 5' flanking sequence with retention of full inducibility by cloprostenol. Mutation of three critical cis-responsive elements within this 282-bp region (C/EBP, cAMP responsive element [CRE], and E-box) indicated that E-box was critical in both basal and cloprostenol-induced promoter activity. However, there was also significant but less dramatic inhibition of cloprostenol stimulation by mutation of C/EBP and CRE in the Cox-2 promoter, and mutation of all three elements eliminated cloprostenol induction of this promoter. Electrophoretic mobility shift assays of nuclear extracts from large luteal cells revealed that upstream stimulatory factor (USF)-1 and USF-2 bound to the E-box in Cox-2. Thus, PKC directly regulates transcription of the Cox-2 gene in large luteal cells by acting through DNA elements close to the putative transcriptional start point, particularly an E-box region at -50 bp.

corpus luteum, gene regulation, kinases, signal transduction

INTRODUCTION

An essential, rate-limiting step in the biosynthesis of prostaglandins (PGs) is the conversion of arachidonic acid to PGs of the G/H series by cyclooxygenase (Cox), also known as PG G/H synthase. Two different isoforms of Cox have been identified. The Cox-1 isoform is expressed in many tissues under basal conditions and generally is not inducible [1, 2]. In contrast, Cox-2 is usually absent and is induced in many different cell types after diverse stimuli, including mitogens, growth factors, cytokines, and tumor promoters [1, 2]. The key role of Cox-2 in female reproduction is highlighted by the reduction in efficiency of many reproductive processes in Cox-2 but not Cox-1 knockout mice, including ovulation, fertilization, implantation, and decidualization [3, 4]. There is a dramatic induction of Cox-2 but not Cox-1 expression in follicular granulosa cells by the LH surge (hCG) or protein kinase (PK) A activation in rats [5, 6], horses [7], and cows [8]. There is a substantial delay between hCG treatment of granulosa cells and detection of Cox-2 protein. For example, in cattle, Cox-2 is first detected about 18 h after hCG or about 10 h prior to expected ovulation [7]. Sirois and Dore [7] suggested that Cox-2 may be a determinant of the timing of ovulation because of the surprisingly consistent interval from induction of Cox-2 until ovulation in species that vary widely in time from the LH surge until ovulation.

Prostaglandins are also involved in initiation of regression of the corpus luteum (CL) in many species because of secretion of PGF2{alpha} from the uterus. Production of PGF2{alpha} by luteal cells has also been documented in cows [9, 10], pigs [11, 12], rhesus monkeys [13], sheep [14, 15], and pseudopregnant rats [16]. Cyclooxygenase-2 mRNA was induced in the CL [17] or in large luteal cells following treatment with PGF2{alpha} [12, 15]. Thus, a small amount of PGF2{alpha} from the uterus or another source could induce intraluteal production of PGF2{alpha} in an autoamplification cascade that may be critical for complete luteolysis [15]. Induction of Cox-2 by PGF2{alpha} in large luteal cells is very rapid [12, 15], similar to the immediate early gene designation of Cox-2 in other cells but different from the delayed expression pattern in granulosa cells. Thus, although large luteal cells are generally regarded as having differentiated from granulosa cells, it appears that regulation of Cox-2 changes from a delayed PKA-dependent regulation in granulosa cells to a rapid PKC-dependent regulation in large luteal cells [18].

The Cox-2 promoter sequence has been analyzed in rats [19, 20], mice [21], humans [22–24], and cows [25]. A variety of cis-responsive elements, e.g., NF-{kappa}B, interferon-response elements, C/EBP (NF-interleukin 6), cAMP response element (CRE), and E-box (-CACGTG-), have been proposed as key regulatory elements that mediate Cox-2 transcription in various cell types. In rat granulosa cells [19, 20], either C/EBP or E-box are essential elements, but E-box is the critical regulatory element in bovine granulosa cells [25]. In luteal cells, the critical regulatory elements of the Cox-2 promoter have not yet been determined.

Binding of PGF2{alpha} to the PGF2{alpha} receptor activates a cascade of intracellular effector systems [26, 27], including PKC, free intracellular calcium, and mitogen-activated protein kinase (MAPK). Pharmacologic activation of PKC increases Cox-2 mRNA, but whether this pathway or other pathways directly alter luteal Cox-2 gene transcription has not been addressed. In the present study, we used the 5' upstream region of the ovine Cox-2 gene combined with a luciferase expression plasmid to investigate the intracellular effector systems and critical DNA regulatory elements involved in Cox-2 gene transcription in primary cultures of ovine large luteal cells.

MATERIALS AND METHODS

Chemicals and Reagents

Fetal bovine serum (FBS) was obtained from Gibco BRL (Rockville, MD). The luciferase assay kit, pGL2B plasmid, pGEM-T easy vector, T4 polynucleotide kinase, and restriction enzymes were purchased from Promega (Madison, WI). The MicroSpin G-50 column and Poly(dI-dC) oligonucleotides were purchased from Amersham Pharmacia (Piscataway, NJ). SuperFect transfection reagent and endotoxin-free plasmid purification columns were from Qiagen (Valencia, CA). Intravaginal progesterone implants (EAZI-BREED CIDR for sheep and goats) were from InterAg (Hamilton, New Zealand). Pfu DNA polymerase was from Stratagene (La Jolla, CA). ABI Prism BigDye terminator cycle sequencing kits were from Applied Biosystems (Foster City, CA). Polyclonal antibodies against upstream stimulatory factor (USF)-1 and USF-2, donkey anti-rabbit IgG-horseradish peroxidase (HRP), and Jurkat whole cell lysates were from Santa Cruz Biotechnology (Santa Cruz, CA). Cloprostenol was purchased from Cayman Chemical Company (Ann Arbor, MI). The 18 different kinase inhibitors (KN-93, K-252a, U0126, Lavendustin C, PD 98059, SB 202190, KT5720, H-89, Staurosporine, H-8, 4-cyano-3-methylisoquinoline, myristoylated PKA pseudosubstrate peptide, calphostin C, chelerythrine chloride, Go 6976, RO-32-0432, bisindolylmaleimide I, myristoylated PKC pseudosubstrate peptide) and the 3-isobutyl-1-methyl-xanthine were all purchased from Calbiochem (San Diego, CA). Western blot transfer membrane immobilon-P was from Millipore (Bedford, MA). Western blot chemiluminescence reagent was purchased from NEN Life Science Products (Boston, MA). Unless otherwise specified, other chemicals and reagents used in these studies were purchased from Sigma (St. Louis, MO).

Animals and Cell Preparation

Western range or polypay ewes from Equity Livestock (Baraboo, WI) were used in this study. To obtain large luteal cells, mature ewes were synchronized by insertion of a controlled internal drug device (CIDR) for 5 days. Superovulation was stimulated with i.m. injection of 1500 IU eCG 2 days before removal of the CIDR. Prostaglandin F2{alpha} (Lutalyse, Kalamazoo, MI) injection was given i.m. when the CIDR was removed. Nine to 11 days later, the CL were removed by midventral laparotomy, decapsulated, sliced, and dissociated into cell suspensions by incubating at 39°C in a shaking water bath in the presence of collagenase (92.5 mg/ml) and DNAase (50 µg/ml). Large and small luteal cells were separated by centrifugal elutriation as previously described [15]. The purity and concentration of cells were determined by hemacytometer. Cells with diameters larger than 22 µm were designated large luteal cells. The purity of large luteal cells in this project averaged 86.9% ± 4.3% (range, 79%–91%). Large luteal cells were plated down overnight (50 000/well) in 24-well tissue culture plates in M199 medium with 0.1% BSA (fraction V powder), 1 µg/ml insulin, and 1% FBS.

Isolation and Mutation of the 5' Flanking Sequence of the Ovine Cox-2 Gene

To obtain the 5' flanking sequence of the ovine Cox-2 gene, primers were designed that corresponded to -1553 base pairs (bp) and +61 bp of the bovine Cox-2 genomic DNA sequence [25] (5'-TGACTAGAGGAGAAAGGCTTCC-3', 5'-GAGGAGGGCGGTGCGGAGTT-3'). Ovine genomic DNA (100 ng) was amplified by polymerase chain reaction (PCR) using these primers with Taq DNA polymerase. PCR was done by preheating to 95°C for 1 min followed by 35 cycles of 1 min at 95°C, 1 min at 57°C, and 1.5 min at 72°C, followed by 10 min at 72°C. The amplified product was separated on a 0.7% agarose gel and visualized after staining with ethidium bromide. The PCR product was treated twice with phenol/chloroform, precipitated with 100% ethanol, and resuspended in 1x Tris-EDTA buffer. The pure PCR product was ligated into a pGEM-T easy vector through a TA cloning site. The ligation mixture was used to transform competent Escherichia coli (DH5{alpha}). Plasmid DNA, isolated from a positive colony, was subjected to DNA sequencing. Plasmid DNA (500 ng) was amplified with BSF primer (5'-TTTTCCCAGTCACGACGTTG-3') or BSR primer (5'-TGTGGAATTGTGAGCGGATAA-3') using an ABI Prism BigDye terminator cycle sequencing kit. The product was purified through a MicroSpin G-50 column, lyophilized, and sequenced by the Biotech Center at the University of Wisconsin-Madison. The sequence was compared to the bovine sequence from GenBank (AF031699). Based on the comparison, a second pair of sheep-specific primers was designed (5'-CTTTTGTCTCACGTAGAATCAC-3'; 5'-AAACAACTGGAAGCTAACGGAG-3') with two restriction enzyme sites, KpnI (5'-GGTACC-3') and XhoI (5'-CTCGAG-3'). These primers were used to amplify the ovine sequence from sheep genomic DNA by PCR using Pfu DNA polymerase. The product was digested with restriction enzymes KpnI and XhoI and subcloned into a luciferase reporter vector pGL2B through the KpnI and XhoI sites. The entire insert within the pGL2B vector was subjected to DNA sequencing as described earlier, and the sequence was submitted to GenBank (AF369795). Deletion mutants of the ovine Cox-2 promoter sequence were generated with different combinations of restriction enzyme digestion. Combinations of KpnI with one of the following enzymes, PstI, SnaBI, SacI, or EagI, were used to delete a DNA region between the restriction sites for each pair of enzymes. The digested plasmid DNA was eluted from the agarose gel to remove the deleted fragment. The digested plasmids were precipitated with ethanol and ligated back to form intact plasmids. The ligated plasmids contained different sizes of the 5' flanking sequence with deletions from the 5' end: 1046 bp, 770 bp, 461 bp, and 282 bp. Specific mutations were introduced into the C/EBP, CRE, and E-box regions of the 282-bp sequence using the Quick-change kit (Stratagene) (Fig. 1). Two random mutations were also introduced at -192 and -90 bp. Plasmids containing Cox-2 upstream sequences (mutant or different lengths of native) were amplified in bacteria and column purified for transfection.



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  		FIG. 1. Schematic display of the 5' flanking sequence for the ovine Cox-2 gene showing key cis-elements and sites chosen for site-directed mutagenesis. A) Potential cis-elements within the ovine Cox-2 promoter. The CRE and E-box have an overlapping sequence of two nucleotides. The random mutation sites are located upstream of C/EBP (random mutant 1) or between C/EBP and CRE/E-box (random mutant 2). B) The primer sequences (sense strand) used for site-directed mutagenesis of C/EBP, CRE, and E-box elements. The wild-type sequences for each of the elements are underlined; lowercase letters indicate the mutated nucleotides

Experiment I

To examine the intracellular effector systems leading to activation of Cox-2 transcription, large luteal cells were transfected with the Cox-2 promoter construct according to previously described methods used to optimize transfection efficiency in bovine granulosa cells [28]. A luciferase-reporter plasmid (1 µg) containing the full-length Cox-2 5' flanking sequence was mixed with transfection reagent SuperFect (2 µl) to form a DNA-SuperFect complex. Before transfection, large luteal cells that were attached to 24-well plates were washed three times with M199 + 1% BSA. The DNA-SuperFect mixture was added directly to cells. After 2 h of incubation at 39°C, the cells were washed three times with M199. Cells were then incubated with 8-bromo-cAMP (1 mM; a PKA activator), phorbol didecanoate (PDD, 10 nM; a PKC activator), ionomycin (1 µM; increases free intracellular Ca2+), or cloprostenol (100 nM; a PGF2{alpha} analogue). The dose of 8-bromo-cAMP was previously found to stimulate Cox-2 transcription in bovine granulosa cells (data not shown). The doses of PDD and cloprostenol were based on results of previous studies on induction of Cox-2 in ovine large luteal cells [15, 18]. To measure luciferase activity in the cell lysates, medium was removed and cells were washed once with 1x PBS and lysed with luciferase assay lysis reagent. Cell lysates were centrifuged at 15 000 rpm for 5 sec, and the luciferase activity in the supernatant was measured using a commercial luciferase assay kit (Promega). Transfection efficiency was determined by direct measurement of the number of transfected plasmids per cell using a PCR method previously described [28]. Luciferase activity of samples was normalized by transfection efficiencies and expressed as n-fold increases over promoterless (pGL2B) control.

Experiment II

To determine the intracellular pathways involved in PGF2{alpha} stimulation of Cox-2 transcription, the expression plasmid containing the full-length Cox-2 promoter construct was analyzed in the presence of various PK inhibitors. After transfection, inhibitors were added to large luteal cells, and 1 h later some cells were treated with cloprostenol (100 nM) for another 12 h. All inhibitors except 3-isobutyl-1-methyl-xanthine (IBMX) were evaluated in preliminary dose-response experiments to determine doses that had no detectable cellular toxicity. All doses that were utilized were greater than reported inhibitory constants (Ki) and most were fivefold or greater than the reported Ki for the inhibitors. The inhibitors that were evaluated and their respective final concentrations in the cell cultures were calcium calmodulin kinase II inhibitors KN-93 (2.5 µM), K-252a (0.5 µM), and Lavendustin C (2.5 µM); MAPK inhibitors PD 98059 (10 µM), SB 202190 (5 µM), and U0126 (1 µM); PKA inhibitors KT5720 (250 nM), H-89 (0.5 µM), H-8 (2.5 µM), 4-cyano-3-methylisoquinoline (50 nM), and myristoylated PKA pseudosubstrate peptide (25 µM); and PKC inhibitors Saturosporine (100 nM), Calphostin C (0.5 µM), chelerythrine chloride (1 µM), Go 6976 (1 µM), RO-32-0432 (0.5 µM), bisindolylmaleimide I (0.5 µM), and myristoylated PKC pseudosubstrate peptide (25 µM). An inhibitor of cAMP phosphodiesterase, IBMX (1 µM), was also used as a control. A dose-response evaluation for PGF2{alpha} was also performed in the presence or absence of myristoylated PKA or PKC pseudosubstrate peptide (25 µM). Some of the large luteal cells transfected with the full-length Cox-2 promoter plasmid were treated for 1 h with inhibitor and then challenged with different concentrations of cloprostenol (1, 3, 10, 30, or 100 nM) for another 12 h.

Experiment IIIa

To localize essential regions of the 5' flanking sequence mediating Cox-2 transcription in large luteal cells, progressive deletion mutants of the Cox-2 promoter construct were transfected into large luteal cells. The cells were then treated with 8-bromo-cAMP (1 mM), PDD (10 nM), or cloprostenol (100 nM) for 12 h. To further identify critical elements within the Cox-2 promoter region, expression plasmids were prepared with mutations in the C/EBP, CRE, E-box, and two random sites (Fig. 1) and were transfected into large luteal cells. Following transfection, the cells were treated with PDD (10 nM) for 12 h.

Experiment IIIb

An electrophoretic mobility shift assay (EMSA) was used to define potential transcription factors capable of binding to the E-box element in the Cox-2 promoter region. Nuclear extracts were prepared according to the method of Schreiber et al. [29]. A double-strand oligonucleotide containing the CRE/E-box (5'-CAGTCATGCCGTCACGTGGGCTATTT-3') was end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase and purified in a MicroSpin G-50 column. The 32P-labeled probe was mixed with nuclear extracts (5 µg) in 10 mM Hepes (pH 7.9) containing 50 mM KCl, 2.5 mM MgCl2, 10% glycerol (v/v), 0.1 µg/µl BSA, 1 mM dithiothreitol, and 0.25 µg/µl poly(dI-dC) and incubated in the presence or absence of unlabeled competitor oligonucleotide for 20 min at room temperature. The unlabeled competitor sequences were either wild type (same as probe) or had an E-box mutation (5'-CAGTCATGCCGTCAatgaGGCTATTT-3') or a CRE mutation (CAGTCATGCaagCACGTGGGCTATTT). For supershift assays, nuclear extracts were incubated with USF-1 or USF-2 antibodies for 10 min at room temperature prior to addition of the labeled oligonucleotide. The mixtures were analyzed on a 4% acrylamide gel in 22.5 mM Tris-borate containing 0.5 mM EDTA. The gels were dried and analyzed with the Cyclone Storage Phosphor System (Packard Instrument Company, Meriden, CT).

To examine USF-1 and USF-2 in nuclear extracts, nuclear proteins (10 µg) were mixed in 20 µl 2x SDS-loading buffer, and samples were steamed for 10 min. Samples were then loaded on a 10% polyacrylamide gel and separated at a constant current of 30 mA for 2 h. Proteins were transferred from gels to Immobilon-P membranes (Millipore) using the miniprotean II gel transfer system (Bio-Rad, Hercules, CA). Following transfer, blots were incubated in blocking buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween-20, and 5% nonfat dry milk) at 4°C overnight. The blots were incubated with anti-USF-1 or anti-USF-2 antibodies (Santa Cruz Biotechnology) at 1:5000 dilution for 2 h at room temperature, followed by three washes (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween-20). Anti-rabbit HRP (Santa Cruz Biotechnology) was added at 1:10 000 dilution for 1 h at room temperature, followed by three washes. The intensity of the bands was increased with enhanced chemiluminescence reagent (NEN Life Science Products). The developed blots were exposed to x-ray film for 1 min.

Statistical Analyses

Results were analyzed by one-way ANOVA using the general linear model of the Statistical Analysis System (SAS, Cary, NC) to test for treatment effects. If a significant F-test was detected by ANOVA, a Fisher least significant difference (LSD) test was used to compare different treatments, using an {alpha}-value of 0.05. In experiment II, means were compared for different inhibitors in the absence (control) or presence of cloprostenol. In the experiment using different doses of cloprostenol, treatments (no inhibitor, PKC pseudosubstrate peptide, or PKA pseudosubstrate peptide) were compared only within each dose of cloprostenol (0, 1, 3, 10, 30, and 100 nM) by ANOVA and LSD. In experiment IIIa, luciferase activity from different deletion mutants was compared with that of the full-length promoter within untreated (control) and 8-bromo-cAMP, PDD, and cloprostenol treatment groups. The luciferase activity for each group of different site-directed mutants was compared with that of the full-length promoter within control or PDD treatment groups.

RESULTS

Experiment I

In large luteal cells, there was a significant induction of luciferase activity by PDD, ionomycin, and cloprostenol but not by 8-bromo-cAMP (Fig. 2). The increase in promoter activity induced by PDD, ionomycin, and cloprostenol was more dramatic at 12 h (PDD: 30x; ionomycin: 15x; cloprostenol: 40x) than at 4 h (PDD: 9x; ionomycin: 7x; cloprostenol: 9x).



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  		FIG. 2. Functional analysis of ovine Cox-2 promoter in ovine large luteal cells. Cells were transfected with the luciferase expression vector driven by the Cox-2 promoter sequence (1500 bp) and treated with 8-bromo cAMP (1 mM), PDD (10 nM), ionomycin (1 µM), or cloprostenol (100 nM) for 4 or 12 h. Data are normalized for transfection efficiency and represent the mean ± SEM for three different experiments, with each treatment evaluated in triplicate wells in each experiment. Differences (P < 0.05) between treatments and control within a time point are indicated by an asterisk

Experiment II

In large luteal cells transfected with the full-length Cox-2 promoter construct, the calcium calmodulin kinase II inhibitors (KN-93, K-252a, and Lavendustin C), MAPK inhibitors (PD 98059, SB 202190, and U0126), PKA inhibitors (KT5720, H-89, H-8, 4-cyano-3methylisoquinoline, and PKA pseudosubstrate peptide), and cAMP phosphodiesterase inhibitor did not have any effect on either untreated or cloprostenol-treated cells (Fig. 3). The PKC inhibitors chelerythrine chloride, RO-32-0432, and PKC pseudosubstrate peptide significantly decreased the cloprostenol-mediated promoter activation but not the basal promoter activity (Fig. 4). However, the PKC inhibitors Staurosporine, bisindolylmaleimide I, Calphostin C, and Go 6976 did not alter either basal or cloprostenol-stimulated luciferase activity (Fig. 4). In the presence of different concentrations of cloprostenol, there was a dose-dependent induction of Cox-2 promoter activity by cloprostenol from 1 nM to 100 nM at both 4 h (Fig. 5A) and 12 h (Fig. 5B). The PKC pseudosubstrate peptide, but not the PKA pseudosubstrate peptide, dramatically reduced cloprostenol-mediated luciferase activity by 64%–68% at 4 h (Fig. 5A) and by 74%–84% at 12 h (Fig. 5B).



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  		FIG. 3. Luciferase activity in lysates of ovine large luteal cells transfected with luciferase expression vector driven by the ovine Cox-2 promoter (1500 bp) and treated with or without cloprostenol (100 nM) for 12 h in the presence or absence of inhibitors of calcium calmodulin kinase II, MAPKs, and PKA. Data are normalized for transfection efficiency and represent the mean ± SEM for four experiments, with each treatment evaluated in triplicate wells in each experiment. There were no differences (P < 0.05) between cells treated with inhibitors and untreated cells in the control or cloprostenol-treated groups



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  		FIG. 4. Luciferase activity in lysates of ovine large luteal cells transfected with luciferase expression vector driven by the ovine Cox-2 promoter (1500 bp) and treated with or without cloprostenol (100 nM) in the presence of PKC inhibitors for 12 h. Data are normalized for transfection efficiency and represent the mean ± SEM for four experiments, with each treatment evaluated in triplicate wells in each experiment. Differences (P < 0.05) due to inhibitors in cloprostenol-treated cells are indicated by an asterisk



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  		FIG. 5. The PKC pseudosubstrate peptide blocks PGF2{alpha}-mediated Cox-2 promoter activity in ovine large luteal cells at 4 (A) or 12 (B) h after cloprostenol treatment. Ovine large luteal cells were transfected with the luciferase expression vector driven by the ovine Cox-2 promoter (1500 bp). These cells were treated with or without the myristolated PKC pseudosubstrate peptide (25 µM) or the myristolated PKA pseudosubstrate peptide (25 µM); 1 h later increasing doses of cloprostenol were added for an additional 4 or 12 h. Data are normalized for transfection efficiency and represent the mean ± SEM for three experiments, with each treatment evaluated in triplicate wells in each experiment. Differences (P < 0.05) between inhibitory peptide treatments within each dose of cloprostenol are indicated by an asterisk at 4 or 12 h

Experiment IIIa

Progressive deletion of the ovine Cox-2 promoter region revealed that deletion from full-length 1500 bp to 282 bp did not result in any change in basal or PDD- and cloprostenol-induced luciferase activity (data not shown). Cyclic AMP did not alter luciferase activity from any deletion mutants (data not shown). In untreated large luteal cells, single or combined mutations of C/EBP and CRE did not cause any change in basal luciferase activity (Fig. 6). Mutation of the E-box element reduced the basal luciferase expression (P < 0.05), and this reduction was similar whether it was caused by the E-box mutation alone or by the E-box mutation combined with mutations of the C/EBP and/or CRE. In the presence of the PKC activator PDD, the scenario was more complicated. The PDD-stimulated luciferase activity was reduced (P < 0.05) by single mutations in C/EBP (55%), CRE (32%), or E-box (68%). Combined mutations produced more dramatic inhibition of PDD-stimulated luciferase activity: C/EBP plus CRE (60%), C/EBP plus E-box (78%), CRE plus E-box (83%), and C/EBP plus CRE plus E-box (95%). The two random mutants did not alter basal or PDD-stimulated luciferase activity (Fig. 6).



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  		FIG. 6. Identification of critical element(s) in ovine Cox-2 promoter mediating PDD-regulated luciferase activity in ovine large luteal cells. Large luteal cells were transfected with the full-length construct (Cox-2/1500 bp), the 282-bp wild-type construct (Cox-2/282 bp), and various mutations of the 282-bp construct, including mutations of C/EBP, CRE, and E-box and combinations of these mutations and two random mutations (see Fig. 1). Following transfection, cells were treated with or without PDD (10 nM) for 12 h. Data are normalized for transfection efficiency and represent the mean ± SEM for four experiments, with each treatment evaluated in triplicate wells in each experiment. Differences (P < 0.05) between mutants and full-length promoter construct in the presence or absence of PDD are indicated by an asterisk

Experiment IIIb

There were four distinct bands that could be identified after incubation of nuclear extracts from untreated or PGF2{alpha}-treated large luteal cells with the 32P-labeled oligonucleotide containing the CRE and E-box element (Fig. 7, lane 2). Addition of a 50-fold excess of unlabeled wild-type oligonucleotide to the incubation mixture completely eliminated all of the bands (Fig. 7, lane 5). Addition of a 50-fold excess of unlabeled oligonucleotide that contained an E-box mutation eliminated all bands except the uppermost band (Fig. 7, lane 6). Addition of a 50-fold excess of unlabeled oligonucleotide containing a CRE mutation but not an E-box mutation eliminated all bands (data not shown). Use of a 50-fold excess of unrelated oligonucleotide as a competitor eliminated all bands except the uppermost band (data not shown). Incubation of the nuclear extract with antibodies against USF-1 or USF-2 eliminated only the uppermost band (Fig. 7, lanes 3 and 4). The banding pattern was similar whether nuclear proteins were from untreated or PGF2{alpha}-treated large luteal cells (Fig. 7, A vs. B). Western blotting analysis revealed no detectable differences in amounts of USF-1 or USF-2 proteins in nuclear extracts from control or PGF2{alpha}-treated large luteal cells (Fig. 8, A and B).



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  		FIG. 7. EMSAs using a 32P-labeled CRE/E-box oligonucleotide probe incubated with nuclear extracts from ovine large luteal cells that were untreated (A) or treated for 1 h with 100 nM cloprostenol (B). Lane 1 is probe only without nuclear extract, and lanes 2–6 contain probe incubated with nuclear extracts. Lanes 3 and 4 had nuclear extract preincubated with antibodies against USF 1 (lane 3) or USF-2 (lane 4). Lane 5 contains labeled probe and nuclear extract incubated with a 50-fold excess of wild-type unlabeled probe, and lane 6 contains a 50-fold excess of unlabeled probe with mutation in the E-box element. The uppermost band that appears to be specific for E-box and USF proteins is indicated by an arrow



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  		FIG. 8. Western Blotting of USF-1 and USF-2 proteins in nuclear extracts from ovine large luteal cells untreated (control) or treated with PGF2{alpha} (100 nM) for 1 h. The blot was analyzed with anti-USF-1 antibody (A) or anti-USF-2 antibody (B). Whole cell lysates from Jurkat cells are used as a positive control for USF-2. Molecular masses of USF-1 (43 kDa) and USF-2 (44 kDa) are indicated with an arrow.

DISCUSSION

Prostaglandin F2{alpha} is the initiator of luteolysis in sheep. An autoamplification pathway has been reported in which PGF2{alpha} treatment will induce Cox-2 and PGF2{alpha} production within large luteal cells, although no previous study has defined the potential transcriptional mechanisms involved in this pathway. In the present study, we established that induction of Cox-2 mRNA by PGF2{alpha} in large luteal cells is mediated by direct control of gene transcription induced by a short (less than 300 bp) region of DNA immediately 5' to the Cox-2 gene. The action of PGF2{alpha} on Cox-2 transcription was mediated by three critical elements within this Cox-2 promoter, with regulation through the PKC intracellular effector system.

A number of intracellular effectors are activated by PGF2{alpha} in large luteal cells, including free intracellular calcium, PKC, and MAPKs [26, 27]. Treatment of ovine large luteal cells with a PKC activator increased steady state concentrations of Cox-2 mRNA [15]. However, no previous investigators have utilized PKC inhibitors to evaluate whether the PGF2{alpha}-induced increase in Cox-2 mRNA is mediated through this pathway. We utilized a number of different inhibitors in an attempt to dissect the pathways involved in PGF2{alpha} induction of Cox-2 gene transcription. Three different calcium calmodulin kinase inhibitors (KN-93, K252a, and Lavendustin C) did not alter induction of Cox-2 by PGF2{alpha}. Likewise, inhibition of MAPK kinase (MEK inhibited by PD 98059), P38 MAPK (by SB 202190), or P42/44 MAPK (by U0126) did not alter PGF2{alpha} induction of Cox-2 gene transcription in spite of the elegant studies that clearly show that PGF2{alpha} activates the Raf/MEK and P42/44 MAPK pathways [27]. In contrast, PKC inhibitors inhibit Cox-2 induction by PGF2{alpha}, with the most dramatic inhibition by the highly specific myristolated PKC pseudosubstrate peptide. This pseudosubstrate peptide was designed based on the autoinhibitory domains present in the PKC{alpha} and PKCß isoforms [30]. This autoinhibitory domain has a peptide sequence that resembles a PKC phosphorylation site (a set of basic amino acids), except an alanine is substituted for the serine or threonine residue that would normally be the phosphate acceptor. Thus, inhibition of PGF2{alpha} action by this highly specific PKC inhibitor is taken as strong evidence that PKC is involved in Cox-2 activation in large luteal cells. We did not clearly delineate which of the 12 identified PKC isoforms [31] are involved in this action of PGF2{alpha}. However, this pseudosubstrate should inhibit PKC{alpha} and PKCß, because the sequence exactly matches their pseudosubstrate sequences, and should inhibit the PKC{gamma} isoform, which contains a pseudosubstrate with only a single amino acid substitution [30]. Other subtypes of PKC show considerable variability in their pseudosubstrate sequences and may not be inhibited by this peptide [30].

Expression of the Cox-2 gene is regulated in a myriad of different cell types by numerous regulators during many physiologic and pathophysiologic processes. Many different DNA response elements (see Fig. 1) are utilized in a remarkably cell-specific and activator-specific manner. For example, interleukin-1ß and dexamethasone acted through the NF-{kappa}B pathway in human amnion-derived cells [32], whereas the actions of platelet-derived growth factors and dexamethasone did not utilize the NF-{kappa}B site in renal mesangial cells [33]. Neither basal nor PGF2{alpha}-induced expression of Cox-2 utilized the NF-{kappa}B site in ovine large luteal cells because this site was eliminated from the shorter promoter sequence (282 bp), yet the promoter remained fully functional. Other more distal response elements such as the interferon response element [34] were critical for Cox-2 regulation in other cells but were not essential for basal or PGF2{alpha}-induced Cox-2 expression in large luteal cells. Similar to the effect in large luteal cells, a short region of 5' DNA was sufficient for complete Cox-2 promoter activity in granulosa cells [19, 20, 25], hepatocytes [35], microvascular endothelial cells [36], and osteoblastic cells [37]. There are a number of potential DNA regulatory elements in the 282-bp promoter sequence. We focused on the E-box, an adjacent CRE, and a C/EBP element because previous research indicated the regulatory importance of these elements in other cell types.

The E-box is critical for basal and forskolin-induced expression of Cox-2 in bovine granulosa cells [25] and for basal and LH- or GnRH-stimulated expression of Cox-2 in rat granulosa cells [20]. Our results showed that E-box is critical for basal and PGF2{alpha}-stimulated expression of the Cox-2 gene in ovine large luteal cells. In the EMSA, nuclear proteins from large luteal cells specifically bound to an oligonucleotide that spanned the E-box sequence. This binding was eliminated by incubation with an unlabeled E-box oligonucleotide but not with a similar oligonucleotide with a mutated E-box. Disruption of the E-box band by antibodies to either USF-1 or USF-2 indicates that both USF-1 and USF-2 are involved in the complex formation between E-box and large luteal cell nuclear extracts. The USF proteins are basic-helix-loop-helix-leucine zipper transcription factors that have polypeptide structures similar to those of Myc oncoproteins, and both Myc and USF bind to E-box. Overexpression of c-Myc often contributes to rapid proliferation of tumor cells, whereas overexpression of USF is known to inhibit cellular proliferation in a number of cancer cell lines [38, 39]. There appears to be ubiquitous expression of USF-1 and USF-2 in most tissues, with perhaps some differences among tissues in the ratio of USF-1 to USF-2 [40]. There was no detectable difference in USF proteins between untreated and PGF2{alpha}-treated large luteal cells using Western blotting techniques. In addition, there were no differences in banding patterns between untreated and PGF2{alpha}-treated large luteal cells in EMSA using labeled oligonucleotides spanning the E-box region. Although similar binding of USF to E-box in transcriptionally active and inactive cells seems paradoxical, it is consistent with the results of the elegant studies of Qyang et al. [41] on USF/E-box transcriptional activation. They showed that USF proteins (USF-1 or USF-2) activated transcription in HeLa cells but not in Saos-2 cells. The inactivity of USF proteins in Saos-2 cells was not due to a lack of proper localization of USF to the nucleus or lack of USF binding to the DNA promoter. Their results are most consistent with mediation of the transcriptional activity of USF by interaction with a not yet identified coactivator that was inactivated or lacking in Saos-2 cells [41]. Although the E-box region appears to be crucial for either basal or PGF2{alpha}-induced Cox-2 gene transcription in large luteal cells, factors other than binding of USF proteins to E-box may be crucial for explaining E-box-mediated transcriptional activation.

Mutations of the CRE and C/EBP elements did not reduce basal Cox-2 gene activity but did reduce PKC-mediated transcriptional activation. Mutation of all three critical elements, E-box, CRE, and C/EBP, eliminated PGF2{alpha} induction of the Cox-2 gene. Thus, there was a combinatorial action of all three cis-acting elements in regulating transcriptional activation of the Cox-2 gene in large luteal cells. Combinatorial involvement of more than one DNA element in Cox-2 transcription has been reported in other cell types, such as AP-2, C/EBP, and CRE in human microvascular endothelial cells [36], CRE and AP-1 in osteoblastic cells [37], CRE and C/EBP in macrophage cells [42], and C/EBP and E-box in skin carcinoma cells [43].

This study provides clear evidence that PGF2{alpha} activates Cox-2 gene transcription through the PKC signaling pathway. An E-box DNA element is critical for both basal and PGF2{alpha}-induced Cox-2 gene transcription. However, CRE and C/EBP DNA elements are also involved in PGF2{alpha}-induced Cox-2 gene transcription, acting in combination with E-box. Further studies are needed on the proteins involved in regulating Cox-2 transcription, particularly the proteins that interact with USF/E-box, to precisely characterize the transcriptional mechanisms involved in PGF2{alpha} induction of Cox-2 in large luteal cells.

FOOTNOTES

First decision: 29 May 2001.

1 Supported by NIH grant HD-32623. Back

2 Correspondence: Milo C. Wiltbank, University of Wisconsin-Madison, 1675 Observatory Drive, Madison, WI 53706. FAX: 608 263 9412;wiltbank{at}calshp.cals.wisc.edu Back

Accepted: June 28, 2001.

Received: April 30, 2001.

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