In vitro efficacy of a first-generation valosin-containing protein inhibitor (CB-5083) against canine lymphoma.

Valosin-containing protein (VCP), through its critical role in the maintenance of protein homeostasis, is a promising target for the treatment of several malignancies, including canine lymphoma. CB-5083, a first-in-class VCP inhibitor, exerts cytotoxicity through the induction of irreversible proteotoxic stress and possesses a broad spectrum of anticancer activity. Here, we determined the cytotoxicity CB-5083 in canine lymphoma cells and its mechanism of action in vitro. Canine lymphoma cell lines were treated with varying concentrations of CB-5083 and assessed for viability by trypan blue exclusion and apoptosis by caspase activity assays. The mechanism of CB-5083 action was determined by immunoblotting and RT-qPCR analyses of Lys48 ubiquitination and markers of ER stress (DDIT3), autophagy (SQSTM1, MAP1LC3A) and DNA damage (γH2AX). Unfolded protein response markers were also evaluated by immunoblotting (eIF2α, P-eIF2α) and RT-qPCR (ATF4). CB-5083 treatment resulted in preferential cytotoxicity in canine lymphoma cell lines over control peripheral blood mononuclear cells. CB-5083 rapidly disrupted the ubiquitin-dependent protein degradation system, inducing sustained ER stress as indicated by a dramatic increase in DDIT3. Activation of the unfolded protein response occurred through the increase eIF2α phosphorylation and increased transcription of ATF4, but did not re-establish protein homeostasis. Cells rapidly underwent apoptosis through activation of the caspase cascade. These results further validate VCP as an attractive target for the treatment of canine lymphoma and identify CB-5083 as a novel therapy with clinical potential for this malignancy.

Nuclear localization and cell cycle regulation of a murine protein tyrosine phosphatase.

MPTP is a murine homolog of the human T-cell protein tyrosine phosphatase (PTPase) and the rat PTP-S enzyme. Enzymatic activity of this ubiquitously expressed protein was demonstrated in immunoprecipitates from NIH 3T3 cells and in recombinant protein overexpressed in bacteria. Expression of beta-galactosidase-MPTP MPTP chimeric proteins in COS1 cells identified a nuclear localization signal at the carboxyl terminus of the MPTP that was sufficient to direct beta-galactosidase as well as a tagged version of the MPTP to the nucleus. Deletion analysis of amino acids within the nuclear targeting signal showed that this sequence does not conform to the bipartite type of nuclear localization signals. Furthermore, it was shown that the steady-state levels of MPTP RNA fluctuate in a cell cycle-specific manner. On the basis of these experiments, we discuss the possible function of MPTP in the cell cycle and other nuclear processes.

Human chorionic gonadotropin-dependent induction of an equine aldo-keto reductase (AKR1C23) with 20alpha-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo.

Aldo-keto reductases (AKRs) are multifunctional enzymes capable of acting on a wide variety of substrates, including sex steroids. AKRs having 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity can reduce progesterone to 20alpha-hydroxy-4-pregnen-3-one (20alpha-DHP), a metabolite with lower affinity for the progesterone receptor. The objective of this study was to investigate the regulation of equine AKR1C23 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine AKR1C23 cDNA was cloned and shown to encode a 322 amino acid protein that is conserved (71-81% identity) when compared with mammalian orthologs. RT-PCR/Southern blotting analyses were performed to study the regulation of AKR1C23 transcripts in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment (ovulation occurring 39-42 h post-hCG). Results showed the presence of low AKR1C23 expression before hCG treatment, but a marked increase was observed in follicles obtained 12 h after hCG (P<0.05). Analyses of isolated preparations of granulosa and theca interna cells identified low mRNA expression in both cell types prior to hCG treatment, with granulosa cells clearly being the predominant site of follicular AKR1C23 mRNA induction. A specific polyclonal antibody was raised against a fragment of the equine protein and immunoblotting analyses showed an increase in AKR1C23 protein in granulosa cell extracts when comparing follicles isolated at 36 h post-hCG vs those collected prior to treatment, in keeping with mRNA results. Immunohistochemical data confirmed the induction of the enzyme in follicular cells after hCG treatment. The enzyme was tested for 20alpha-HSD activity and was shown to exhibit a K(M) of 3.12 microM, and a V(max) of 0.86 pmol/min per 10 microg protein towards progesterone. The levels of 20alpha-DHP measured in follicular fluid reflected this activity. Collectively, these results demonstrate for the first time that the gonadotropin-dependent induction of follicular luteinization is accompanied by an increase in AKR1C23 expression. Considering the 20alpha-HSD activity of AKR1C23, its regulated expression in luteinizing preovulatory follicles may provide a biochemical basis for the increase in ovarian 20alpha-DHP observed during gonadotropin-induced luteinization/ovulation. (The nucleotide sequence reported in this paper has been submitted to GenBank with accession number AY955082.).

Molecular characterization of equine prostaglandin G/H synthase-2 and regulation of its messenger ribonucleic acid in preovulatory follicles.

To increase our understanding of the molecular control of PG synthesis in equine preovulatory follicles, the specific objectives of this study were to clone and determine the primary structure of equine prostaglandin G/H synthase-2 (PGHS-2) and to characterize the regulation of PGHS-2 messenger RNA (mRNA) in follicles before ovulation. A complementary DNA (cDNA) library prepared from follicular mRNA and a genomic library were screened with a mouse PGHS-2 cDNA probe to isolate the equine PGHS-2 cDNA and gene, respectively. The expression library yielded three nearly full-length clones that differed only in their 5'-ends; clones 3, 5, and 6 were 2946, 3138, and 3398 bp in length, respectively. The longest clone was shown to start 9 bp downstream of the transcription initiation site, as determined by primer extension analysis, and to contain 120 bp of 5'-untranslated region (UTR), 1812 bp of open reading frame, and 1466 bp of 3'-UTR. The open reading frame encodes a 604-amino acid protein that is more than 80% identical to PGHS-2 homologs in other species. Numerous repeats (n = 11) of the Shaw-Kamen's sequence (ATTTA) are present in the 3'-UTR, a motif typically indicative of mRNAs with a short half-life. The complete equine PGHS-2 gene was isolated and sequenced from a approximately 17-kilobase clone obtained from the genomic library. The equine PGHS-2 gene structure (10 exons and 9 introns; total length of 6991 bp) is similar to its human homolog except for lacking sequence elements in introns 4, 8, and 9 and in the 3'-UTR region of exon 10. To characterize the regulation of PGHS-2 mRNA in equine follicles before ovulation, preovulatory follicles were isolated during estrus, 0, 12, 24, 30, 33, 36, and 39 h (n = 4-5 follicles/time point) after an ovulatory dose of hCG. Results from Northern blots showed significant changes in steady state levels of PGHS-2 mRNA in preovulatory follicles after hCG treatment (P < 0.05). The transcript remained undetectable between 0-24 h post-hCG, first appeared (approximately 4 kilobases) only at 30 h, and reached maximal levels 33 h post-hCG. PGHS-2 mRNA was selectively induced in granulosa cells and not in theca interna. Thus, this study provides for the first time the primary structure of the equine PGHS-2 gene, transcript, and protein. It also demonstrates that the induction of PGHS-2 gene expression in equine granulosa cells is a long molecular process (30 h post-hCG), thereby providing a model to study the molecular basis for the late transcriptional activation of PGHS-2 in species with a long ovulatory process.

Dual regulation of promoter II- and promoter 1f-derived cytochrome P450 aromatase transcripts in equine granulosa cells during human chorionic gonadotropin-induced ovulation: a novel model for the study of aromatase promoter switching.

Estradiol biosynthesis is a key biochemical trait of developing follicles. To study its regulation in equine follicles, the objectives of this study were to clone and determine the structure of equine cytochrome P450 aromatase (P450AROM), and characterize the regulation of P450AROM and P450 17alpha-hydroxylase/C17-20 lyase (P45017alpha) messenger RNAs (mRNAs) in vivo in equine preovulatory follicles isolated during hCG-induced ovulation. Two distinct P450AROM complementary DNAs (cDNAs) were isolated from an equine preovulatory follicle cDNA library. One clone was 2682 bp in length and included 115 bp of 5'-untranslated region (UTR), 1509 bp of open reading frame encoding a well conserved 503-amino acid protein, and 1058 bp of 3'-UTR. Its 5'-most region represented the equine homolog of exon 1f, previously designated brain specific. The other cDNA clone encoded a truncated protein and contained a distinct 5'-UTR characteristic of transcripts derived from promoter II, previously identified as the predominant ovarian mRNA. Northern blot analyses were performed using preovulatory follicles obtained during estrus between 0-39 h after the administration of hCG and with corpora lutea isolated on day 8 of the estrous cycle (day 0 = day of ovulation). The results showed a biphasic regulation of P450AROM mRNA expression: levels were highest in follicles at 0 h post-hCG, decreased significantly during the ovulatory process at 12 and 24 h (P < 0.05), and increased again between 30-39 h post-hCG and in corpora lutea. When oligonucleotides specific for P450AROM mRNA variants were used as probes, a novel switching phenomenon was observed. Promoter II-derived transcripts accounted for the message present in follicles at 0 h post-hCG and in corpora lutea, whereas promoter 1f-derived mRNA was expressed exclusively during the ovulatory process (30-39 h post-hCG). Levels of P45017alpha mRNA were high in follicles at 0 h, but significantly decreased after hCG treatment (P < 0.05), with lowest levels in follicles at 36 and 39 h post-hCG and in corpora lutea. Northern blots performed on isolated cellular preparations revealed that P450AROM and P45017alpha transcripts were localized exclusively in granulosa cells and theca interna, respectively. Equine aromatase promoters II and 1f were cloned from a genomic library, and putative transcription start sites were characterized by primer extension assays. Sequence analyses identified distinct potential regulatory elements in each promoter. Thus, this study identifies a novel aromatase promoter-switching phenomenon in equine granulosa cells during follicular luteinization and provides a new model in which aromatase promoter switching is induced in vivo.

Human chorionic gonadotropin induces an inverse regulation of steroidogenic acute regulatory protein messenger ribonucleic acid in theca interna and granulosa cells of equine preovulatory follicles.

The time- and gonadotropin-dependent regulation of steroidogenic acute regulatory protein (StAR) has not been characterized in vivo in preovulatory follicles of large monoovulatory species or sexually mature animals. The objectives of this study were to clone equine StAR and describe the regulation of its messenger RNA (mRNA) in equine follicles after the administration of an ovulatory dose of hCG. The screening of an equine follicle complementary DNA (cDNA) library with a mouse StAR cDNA probe revealed two forms of equine StAR that differ only in the length of their 3'-untranslated region (3'-UTR); a long form of 2918 bp and a short form of 1599 bp. The StAR long form cDNA contains a 5'-UTR of 117 bp, an open reading frame (ORF) of 855 bp, and a 3'-UTR of 1946 bp. Primer extension analysis showed that the cDNA clone lacked the first 10 bp of the primary transcript, giving a total of 127 bp for the complete StAR 5'-UTR. The ORF encodes a 285-amino acid protein that is 86-90% identical to StAR of other species characterized to date. The regulation of StAR mRNA in vivo was studied in equine preovulatory follicles isolated during estrus at 0, 12, 24, 30, 33, 36, and 39 h (n = 4-5 follicles/time point) after an ovulatory dose of hCG. Results from Northern blots showed no significant changes in StAR mRNA levels after hCG treatment when analyses were performed on intact follicle wall (theca interna with attached granulosa cells). However, Northern blots performed on isolated follicle cells revealed an unexpected regulation of StAR mRNA. In granulosa cells, StAR transcripts were undetectable at 0 h but were significantly increased at 30 h post-hCG, and this induction was associated with a rise in follicular fluid concentrations of progesterone (P < 0.05). In contrast, StAR mRNA levels were high in theca interna at 0 h, remained unchanged until 33 h post-hCG, and dropped dramatically thereafter (P < 0.05). Thus, this study describes the primary structure of equine StAR, documents the regulation of StAR mRNA in vivo in preovulatory follicles of a large monoovulatory species, and identifies a novel inverse regulation of StAR transcripts in theca interna and granulosa cells of equine follicles before ovulation.

Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process.

Steroidogenic factor-1 (SF-1, NR5A1a) is a member of the NR5A nuclear receptor subfamily and has been implicated as a key transcriptional regulator of all ovarian steroidogenic genes in vitro. To establish links between the expression of SF-1 and that of the steroidogenic genes in vivo, the objectives of this study were to clone equine SF-1 and examine the regulation of its messenger RNA (mRNA) in follicular cells during human CG (hCG)-induced ovulation. The equine SF-1 primary transcript was cloned by a combination of RT-PCR techniques. Results showed that the transcript was composed of a 5'-untranslated region (UTR) of 161 bp, an open reading frame (ORF) of 1386 bp that encodes a highly-conserved 461-amino acid protein, and a 3'-UTR of 518 bp. The cloning of SF-1 also led to the unexpected and serendipitous isolation of the highly-related orphan nuclear receptor NR5A2, which was shown to include a 5'-UTR of 243 bp, an ORF of 1488 bp, and a 3'-UTR of 1358 bp. The NR5A2 ORF encodes a 495-amino acid protein that is 60% identical to SF-1, including 99%-similar DNA-binding domains. Northern blot analysis revealed that SF-1 and NR5A2 were expressed in all major steroidogenic tissues, with the exception that NR5A2 was not present in the adrenal. Interestingly, NR5A2 was found to be, by far, the major NR5A subfamily member expressed in the preovulatory follicle and the corpus luteum. Using a semiquantitative RT-PCR/Southern blotting approach, the regulation of SF-1 and NR5A2 mRNAs in vivo was studied in equine follicular cells obtained from preovulatory follicles isolated between 0 and 39 h post hCG. Results showed that the theca interna was the predominant site of SF-1 mRNA expression in the follicle, and that hCG caused a significant decrease in SF-1 levels between 12-39 h in theca interna and between 24-39 h post hCG in granulosa cells (P < 0.05). In contrast, the granulosa cell layer was the predominant, if not the sole, site of NR5A2 mRNA expression in the follicle. Importantly, NR5A2 was much more highly expressed in granulosa cells than SF-1. The administration of hCG caused a significant decrease in NR5A2 transcripts in granulosa cells at 30, 36, and 39 h post hCG (P < 0.05). Thus, this study is the first to report the concomitant regulation of SF-1 in theca interna and granulosa cells throughout the ovulation/luteinization process, and to demonstrate the novel expression and hormonal regulation of NR5A2 in ovarian cells. Based on the marked expression of NR5A2 in equine granulosa and luteal cells and on mounting evidence of a functional redundancy between SF-1 and NR5A2 in other species, it is proposed that NR5A2 may play a key role in the regulation of gonadal steroidogenic gene expression.

Regulation of transcripts encoding ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs-1) and progesterone receptor by human chorionic gonadotropin in equine preovulatory follicles.

One member of a new family of metalloproteinases, a disintegrin and metalloproteinase with thrombospondin-like motifs-1 (ADAMTS-1), has been found to be expressed and hormonally induced in granulosa cells of ovulating rodent follicles. Furthermore, the targeted disruption of the ADAMTS-1 gene resulted in ovarian defects associated with severely impaired fertility. While these data demonstrate the importance of ADAMTS-1 in rodent ovarian physiology, the potential role of ADAMTS-1 in the ovulatory process of monoovulatory species remains unknown. The objectives of this study were to clone the equine ADAMTS-1 primary transcript and to study its regulation during human chorionic gonadotropin (hCG)-induced ovulation. A 3573 bp follicular cDNA library clone was isolated and found to encode a nearly complete, highly conserved ADAMTS-1 homologue. Real-time RT-PCR analysis detected this transcript in diverse tIssues, including previously unreported sites of ADAMTS-1 expression such as the male reproductive tract, the follicular theca interna and the mature corpus luteum. The tIssue distribution of the progesterone receptor (PR), a known regulator of ADAMTS-1 expression in rodent preovulatory follicles, was found to overlap that of ADAMTS-1 in some tIssues. A study of the regulation of follicular ADAMTS-1 and PR mRNAs during the hCG-induced ovulatory process revealed distinct patterns of regulation in granulosa cells and in theca interna. In granulosa cells, ADAMTS-1 mRNA was found to be induced at 12 h post-hCG (P<0.05), followed by a return to basal levels by 30 h and a re-increase at 33-39 h (P<0.05). A concomitant increase in PR mRNA (P<0.05) was observed at 12 h post-hCG. In theca interna, abundant ADAMTS-1 mRNA was detected at all timepoints, and levels increased transiently at 33 h post-hCG (P<0.05), whereas no significant change was observed in PR mRNA. Together, these data demonstrate for the first time the hormonally regulated ovarian expression of ADAMTS-1 in a monoovulatory species, and identify a novel biphasic regulation of ADAMTS-1 in granulosa cells and a regulated expression in theca interna that were not previously observed in rodents.

Either Kras activation or Pten loss similarly enhance the dominant-stable CTNNB1-induced genetic program to promote granulosa cell tumor development in the ovary and testis.

WNT, RAS or phosphoinositide 3-kinase signaling pathways control specific stages of ovarian follicular development. To analyze the functional interactions of these pathways in granulosa cells during follicular development in vivo, we generated specific mutant mouse models. Stable activation of the WNT signaling effector ß-catenin (CTNNB1) in granulosa cells results in the formation of premalignant lesions that develop into granulosa cell tumors (GCTs) spontaneously later in life or following targeted deletion of the tumor suppressor gene Pten. Conversely, expression of oncogenic KRAS(G12D) dramatically arrests proliferation, differentiation and apoptosis in granulosa cells, and consequently, small abnormal follicle-like structures devoid of oocytes accumulate in the ovary. Because of the potent anti-proliferative effects of KRAS(G12D) in granulosa cells, we sought to determine whether KRAS(G12D) would block precancerous lesion and tumor formation in follicles of the CTNNB1-mutant mice. Unexpectedly, transgenic Ctnnb1;Kras-mutant mice exhibited increased GC proliferation, decreased apoptosis and impaired differentiation and developed early-onset GCTs leading to premature death in a manner similar to the Ctnnb1;Pten-mutant mice. Microarray and reverse transcription-PCR analyses revealed that gene regulatory processes induced by CTNNB1 were mostly enhanced by either KRAS activation or Pten loss in remarkably similar patterns and degree. The concomitant activation of CTNNB1 and KRAS in Sertoli cells also caused testicular granulosa cell tumors that showed gene expression patterns that partially overlapped those observed in GCTs of the ovary. Although the mutations analyzed herein have not yet been linked to adult GCTs in humans, they may be related to juvenile GCTs or to tumors in other tissues where CTNNB1 is mutated. Importantly, the results provide strong evidence that CTNNB1 is the driver in these contexts and that KRAS(G12D) and Pten loss promote the program set in motion by the CTNNB1.

Equine P450 cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase/delta(5)-delta(4) isomerase: molecular cloning and regulation of their messenger ribonucleic acids in equine follicles during the ovulatory process.

The preovulatory LH rise is the physiological trigger of follicular luteinization, a process during which the synthesis of progesterone is markedly increased. To study the control of follicular progesterone biosynthesis in mares, the objectives of this study were to clone and characterize the equine cholesterol side-chain cleavage cytochrome P450 (P450(scc)) and 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta-HSD), and describe the regulation and cellular localization of their transcripts in equine follicles during hCG-induced ovulation. Complementary DNA cloning and primer extension analyses revealed that the equine P450(scc) transcript is composed of a 5'-untranslated region (UTR) of 52 nucleotides, an open reading frame (ORF) of 1560 nucleotides, and a 3'-UTR of 225 nucleotides, whereas the equine 3 beta-HSD mRNA consists of a 5'-UTR of 61 nucleotides, an ORF of 1119 nucleotides, and a 3'-UTR of 432 nucleotides. The equine P450(scc) and 3 beta-HSD ORF encode 520 and 373 amino acid proteins, respectively, that are highly conserved (68-79% identity) when compared to homologs of other mammalian species. Northern blot analyses were performed with preovulatory follicles isolated 0, 12, 24, 30, 33, 36, and 39 h post-hCG, and corpora lutea obtained on day 8 of the cycle. Results showed that levels of P450(scc) mRNA in follicular wall (theca interna with attached granulosa cells) decreased after hCG treatment (30-39 h versus 0 h post-hCG, P: < 0.05), and increased again after ovulation to reach their highest levels in corpora lutea (P: < 0.05). Northern blots on isolated cellular preparations revealed that theca interna was the predominant site of P450(scc) expression in follicles prior to hCG (P: < 0.05). However, transcript levels decreased in theca interna between 30-39 h (P: < 0.05) and increased in granulosa cells at 39 h (P: < 0.05), making the granulosa cell layer the predominant site of P450(scc) expression at the end of the ovulatory process. A different pattern of regulation was observed for 3 beta-HSD, as transcript levels remained constant throughout the luteinization process (P: > 0.05). Also, in contrast to other species, expression of 3 beta-HSD mRNA in equine preovulatory follicles was localized only in granulosa cells and not in theca interna. Thus, this study characterizes for the first time the complete structure of equine P450(scc) and 3 beta-HSD mRNA and identifies novel patterns of expression and regulation of these transcripts in equine follicles prior to ovulation.

Molecular cloning and tissue-specific RNA processing of a murine receptor-type protein tyrosine phosphatase.

The molecular cloning of a murine receptor-type protein tyrosine phosphatase, termed PTP NU-3, with an extracellular cell-adhesion-molecule-like domain is reported. NU-3 was isolated from 11.5-day total mouse embryonic RNA by reverse-transcriptase PCR using degenerate oligonucleotides flanking the conserved protein tyrosine phosphatase catalytic domain. This produced a 280-bp DNA probe which was subsequently employed to screen a mouse embryonic kidney library. Several overlapping cDNA clones were isolated, collectively forming a cDNA of 6.0 kb that encodes a putative 211-kDa protein. Northern-blot analysis of total RNA from adult and embryonic mouse tissues indicates the existence of two major PTP NU-3 transcripts of approximately 6 kb and 7 kb. Both messages are expressed predominantly in brain tissues and neuronal-derived cell lines, although detectable levels of the 7-kb message were found in other non-neuronal tissues. We have identified a unique 132-bp exon segment that is present in the 7-kb message but is completely absent in the 6-kb transcript, suggesting tissue-specific levels of expression and RNA processing. Analysis of the amino acid sequence encoded by the 132-bp segment reveals that it completes a partial fibronectin type-III element resulting in a protein with a total of nine such elements. Bacterial expression of the two catalytic domains demonstrated that only the first domain possesses enzymic activity towards a tyrosine phosphorylated substrate.

The delayed activation of the prostaglandin G/H synthase-2 promoter in bovine granulosa cells is associated with down-regulation of truncated upstream stimulatory factor-2.

To elucidate the molecular mechanisms involved in the delayed induction of PGHS-2 in species with a long ovulatory process, a 1. 6-kilobase fragment of the bovine PGHS-2 promoter was isolated, and its activity was characterized in primary cultures of bovine granulosa cells. Promoter activity assays performed with a series of deletion mutants revealed that the promoter region from -149 to -2 (+1 = transcription start site) confers full-length promoter activity in response to forskolin (10 microM). Four consensus cis-elements were identified within this region, including an E-box, ATF/CRE, C/EBP, and AP2 site. Site-directed mutagenesis showed that the E-box was required for PGHS-2 promoter activity, that disruption of the C/EBP element decreased forskolin inducible activity by 29%, whereas point mutation within the ATF/CRE and AP2 element had no inhibitory effect. Electrophoretic mobility shift assays (EMSAs) performed with the -149/-2 fragment and granulosa cell nuclear extracts obtained before (0 h) and after (18 and 20 h) human chorionic gonadotropin (hCG) revealed the regulation of multiple DNA-protein complexes. The 0-h extract generated four complexes at the E-box, whereas only one complex was produced at this site with the 18-h extract. Supershift EMSAs identified that upstream stimulatory factor-1 and -2 (USF-1 and -2) were part of these complexes. Interestingly, the presence of the amino-terminal truncated USF-2, which lacks the transcription activation domain, was detected in the 0-h extract, but not in extracts prepared post-hCG. Supershift EMSAs also indicated high levels of C/EBPbeta binding to its cis-element in the 0-h extract, which contrasts with results previously reported in rats. Thus, high levels of amino-terminal truncated USF-2 and C/EBPbeta in bovine granulosa cells prior to hCG treatment could repress gene expression, and be involved in the delayed induction of PGHS-2 in species with a long ovulatory process.

Molecular characterization of bovine prostaglandin G/H synthase-2 and regulation in uterine stromal cells.

Prostaglandin G/H synthase (PGHS) is a key rate-limiting enzyme in the prostaglandin biosynthetic pathway, and prostaglandins play a central role in the control of the reproductive cycle. The objectives of this study were to clone and characterize the primary structure of bovine PGHS-2 and to study its regulation in uterine stromal cells in vitro. The bovine PGHS-2 cDNA was cloned by a combination of reverse transcription-polymerase chain reaction and cDNA library screening. Results showed that the complete bovine PGHS-2 cDNA is composed of a 5'-untranslated region of 128 bp, an open reading frame of 1815 bp, and a 3'-untranslated region of 1565 bp containing multiple repeats (n = 11) of the Shaw-Kamen sequence 5'-ATTTA-3'. The open reading frame encodes a 604-amino acid protein that is 86-97% identical to other mammalian PGHS-2 homologs. The regulation of PGHS-2 mRNA and protein was studied in primary cultures of bovine uterine stromal cells stimulated with phorbol 12-myristate 13-acetate (PMA; 100 nM). Northern and Western blot analyses reveal a marked induction in PGHS-2 transcript (4.0 kilobases) and protein (M(r) = 72 000) after 3-12 h of PMA stimulation (P < 0.05). However, this induction was transient in nature as levels of PGHS-2 mRNA and protein returned to basal levels after 24 h of PMA stimulation. In contrast, PMA had no effect on levels of PGHS-1 (P > 0.05). The PMA-dependent induction of PGHS-2 was associated with a significant increase in prostaglandin E2 secretion in the culture media (P < 0.05). To study promoter activity of the 5'-flanking DNA region of the bovine PGHS-2 gene, the genomic fragment -1574/-2 (+1 = transcription start site), as well as a series of 5'-deletion mutants, were fused upstream of the firefly luciferase gene and transiently transfected into primary cultures of bovine uterine stromal cells. Results showed that a first promoter region located between -1574 and -492 and a second region between -88 and -39 appear to play important roles in PMA-dependent regulation of PGHS-2 promoter activity in bovine uterine cells. Thus, this study characterizes for the first time the structure of the bovine PGHS-2 transcript and the deduced amino acid sequence of its encoded protein and establishes an in vitro model to study the regulation of PGHS-2 gene expression in bovine uterine tissue.