Regulated expression of prostaglandin E(2) receptors EP2 and EP4 in human ovarian granulosa-luteal cells.

Prostaglandins (PGs) have been implicated in regulation of ovarian function. We have previously shown that the expression of cyclooxygenase-2 and the receptor for PGF(2 alpha) are expressed in periovulatory human granulosa cells and upregulated by gonadotropins and cytokines in cultured human ovarian granulosa-luteal (GL) cells. We now show that transcripts for PGE(2) receptor subtypes EP2 and EP4 are expressed in freshly isolated human granulosa cells and in mouse ovaries as detected by Northern blot analysis. However, EP2 and EP4 receptor mRNA levels were low or nondetectable in cultured human GL cells suggesting that these transcripts may be under hormonal and/or cytokine regulation in the ovaries in vivo. Indeed, the proinflammatory cytokine interleukin-1 beta (IL-1 beta) stimulated expression of EP2 and EP4 transcripts in concentration- and time-dependent manner in the GL cells. Furthermore, the transcript for EP2 receptor was localized in the corpus luteum of the mouse ovary by in situ hybridization, and EP2 protein was expressed in human corpus luteum as detected by immunohistochemistry. Our data suggest that IL-1 beta induces expression of EP2 and EP4 receptors in human GL cells, and that EP2 receptor is expressed in both human and murine luteal glands.

Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice.

The cyclooxygenase (COX)-2 gene encodes an inducible prostaglandin synthase enzyme that is overexpressed in adenocarcinomas and other tumors. Deletion of the murine Cox-2 gene in Min mice reduced the incidence of intestinal tumors, suggesting that it is required for tumorigenesis. However, it is not known if overexpression of Cox-2 is sufficient to induce tumorigenic transformation. We have derived transgenic mice that overexpress the human COX-2 gene in the mammary glands using the murine mammary tumor virus promoter. The human Cox-2 mRNA and protein are expressed in mammary glands of female transgenic mice and were strongly induced during pregnancy and lactation. Female virgin Cox-2 transgenic mice showed precocious lobuloalveolar differentiation and enhanced expression of the beta-casein gene, which was inhibited by the Cox inhibitor indomethacin. Mammary gland involution was delayed in Cox-2 transgenic mice with a decrease in apoptotic index of mammary epithelial cells. Multiparous but not virgin females exhibited a greatly exaggerated incidence of focal mammary gland hyperplasia, dysplasia, and transformation into metastatic tumors. Cox-2-induced tumor tissue expressed reduced levels of the proapoptotic proteins Bax and Bcl-x(L) and an increase in the anti-apoptotic protein Bcl-2, suggesting that decreased apoptosis of mammary epithelial cells contributes to tumorigenesis. These data indicate that enhanced Cox-2 expression is sufficient to induce mammary gland tumorigenesis. Therefore, inhibition of Cox-2 may represent a mechanism-based chemopreventive approach for carcinogenesis.

Expression of cyclooxygenase-2 and prostanoid receptors by human myometrium.

Prostanoids play an important role in the regulation of parturition. All reproductive tissues, including fetal membranes, decidua, and myometrium, have the capacity to synthesize prostanoids, and fetal membranes have been shown to express elevated levels of cyclooxygenase-2 (Cox-2) at the onset of labor. We have now investigated the expression of Cox-2 in human myometrium. Myometrial samples collected from women in labor during lower segment cesarean section expressed 15-fold higher levels of Cox-2 messenger ribonucleic acid (mRNA) compared to myometrial specimens collected from women not in labor, as detected by Northern blot analysis. Immunohistochemical detection of Cox-2 protein showed cytoplasmic staining in the smooth muscle cells of the myometrium. Cultured myometrial cells expressed low levels of Cox-2 mRNA under baseline conditions, but interleukin-1beta (IL-1beta) caused a 17-fold induction of expression of the Cox-2 transcript after incubation for 6 h. IL-1beta also induced expression of biologically active Cox-2 protein, as detected by immunofluorescence, Western blot analysis, and measuring the conversion of arachidonic acid to prostanoids in the presence and absence of a Cox-2-selective inhibitor, NS-398. PGE2 receptor subtype EP2 mRNA was expressed in cultured myometrial smooth muscle cells, whereas transcripts for EP1, EP3, EP4, FP, and IP were low or below the detection limit as measured by Northern blot analysis. However, IL-1beta stimulated expression of EP4 receptor mRNA. Our data suggest that expression of Cox-2 transcript is elevated at the onset of labor in myometrial smooth muscle cells, which may depend on induction by cytokines. As, in addition to Cox-2, the expression of prostanoid receptors is regulated, not only the production of prostanoids, but also responsiveness to them, may be modulated.

Proinflammatory cytokines regulate expression of the lymphatic endothelial mitogen vascular endothelial growth factor-C.

Vascular endothelial growth factor (VEGF) is a prime regulator of normal and pathological angiogenesis. Three related endothelial cell growth factors, VEGF-B, VEGF-C, and VEGF-D were recently cloned. We have here studied the regulation of VEGF-C, a lymphatic endothelial growth factor, by angiogenic proinflammatory cytokines. Interleukin (IL)-1beta induced a concentration- and a time-dependent increase in VEGF-C, but not in VEGF-B, mRNA steady-state levels in human lung fibroblasts. The increase in VEGF-C mRNA levels was mainly due to increased transcription rather than elevated mRNA stability as detected by the nuclear run-on method and by following mRNA decay in the presence of an inhibitor of transcription, respectively. In contrast, angiopoietin-1 mRNA, encoding the ligand for the endothelial-specific Tek/Tie-2 receptor, was down-regulated by IL-1beta. Tumor necrosis factor-alpha and IL-1alpha also elevated VEGF-C mRNA steady-state levels, whereas the IL-1 receptor antagonist and dexamethasone inhibited the effect of IL-1beta. Experiments with cycloheximide indicated that the effect of IL-1beta was independent of protein synthesis. Hypoxia, which is an important inducer of VEGF expression, had no effect on VEGF-B or VEGF-C mRNA levels. IL-1beta and tumor necrosis factor-alpha also stimulated the production of VEGF-C protein by the fibroblasts. Cytokines and growth factors have previously been shown to down-regulate VEGF receptors in vascular endothelial cells. We found that the mRNA for the VEGF- and VEGF-C-binding VEGFR-2 (KDR/Flk-1) was stimulated by IL-1beta in human umbilical vein endothelial cells, whereas the mRNA levels of VEGFR-1 (Flt-1) and VEGFR-3 (Flt-4) were not altered. Our data suggest that in addition to VEGF, VEGF-C may also serve as an endothelial stimulus at sites of cytokine activation. In particular, these results raise the possibility that certain proinflammatory cytokines regulate the lymphatic vessels indirectly via VEGF-C.

Differential hormonal regulation of vascular endothelial growth factors VEGF, VEGF-B, and VEGF-C messenger ribonucleic acid levels in cultured human granulosa-luteal cells.

The development of ovarian follicles and subsequent corpus luteum formation is accompanied by very active angiogenesis. Ovarian granulosa cells produce vascular endothelial growth factor (VEGF), which is a potent endothelial cell mitogen and an angiogenic agent. The complementary DNAs of two other factors structurally related to VEGF, namely VEGF-B and VEGF-C, were recently cloned, but little is known of their regulation in the ovary. We first studied the expression of the messenger RNAs (mRNAs) of the three VEGF isotypes in freshly isolated human granulosa-luteal (GL) cells obtained at oocyte retrieval for in vitro fertilization. The hormonal regulation of these mRNAs was subsequently studied in primary cultures of human GL cells. Analysis of cultured GL cell RNA by reverse transcription-PCR revealed that these cells express the alternatively spliced transcripts representing 121-, 145-, and 165-amino acid VEGF isoforms. Northern blot hybridization analyses indicated that transcripts of 4.5 and 3.7 kilobases for VEGF, and 1.4 and 2.4 kilobases for VEGF-B and VEGF-C, respectively, are expressed in human GL cells. The basal VEGF mRNA levels declined steadily, whereas VEGF-B mRNA levels were rather invariant over a 10-day culture period of GL cells. In contrast, VEGF-C mRNA levels increased toward the end of culture. For studying the hormonal regulation of VEGF isotype mRNAs, GL cells were treated with hCG, recombinant human FSH, PGE2, as well as 8-bromo-cAMP and 12-O-tetradecanoylphorbol 13-acetate, which activate protein kinase A- and protein kinase C-dependent signaling pathways, respectively. All test agents stimulated the expression of VEGF mRNA levels in a concentration-dependent manner. Time-course studies indicated that all treatments induced VEGF mRNA levels as early as incubation for 2 h, and the effect was sustained up to 48 h. VEGF-B mRNA levels were not regulated by any of the test agents. However, we found that hCG and 8-bromo-cAMP decreased VEGF-C mRNA levels with a maximal response observed at 24 and 48 h after cellular treatment. We conclude that the mRNAs of VEGF, VEGF-B, and VEGF-C are expressed in human GL cells and that their mRNA steady state levels are regulated in cultured human GL cells in an isotype-specific manner. The differential regulation of VEGF, VEGF-B, and VEGF-C in human GL cells suggests that distinct VEGF isotypes may play different roles during the vascularization of the human ovarian follicle and corpus luteum.

Induction of cyclooxygenase-2 and prostaglandin F2alpha receptor expression by interleukin-1beta in cultured human granulosa-luteal cells.

Prostanoids are important regulators of ovarian function, especially during ovulation and luteolysis. Cyclooxygenase (Cox) is the rate-limiting enzyme in conversion of arachidonic acid to prostanoids. We have examined the expression and regulation of the inducible Cox isoform (Cox-2) and of the receptor for PGF2alpha (FP) in human granulosa cells obtained from women undergoing oocyte retrieval for in vitro fertilization. Freshly isolated granulosa cells express Cox-2 and FP receptor messenger RNAs (mRNAs). FP receptor mRNA is also expressed in cultured human granulosa-luteal (GL) cells, but Cox-2 transcripts are expressed only upon induction. Interleukin-1beta (IL-1beta) elevated Cox-2 mRNA steady state levels in a concentration-dependent manner, and kinetic studies showed that Cox-2 mRNA levels were already induced at the 2 h point and returned to the basal level after incubation for 24 h. The protein synthesis inhibitor, cycloheximide, induced Cox-2 mRNA expression and potentiated the effect of IL-1beta. Degradation of Cox-2 mRNA was inhibited by IL-1beta, which suggests regulation at the posttranscriptional level. IL-1beta also induced the expression of Cox-2 protein, as detected by immunofluorescence staining using Cox-2-specific polyclonal antibodies. Further, IL-1beta-induced synthesis of prostanoids was blocked by a Cox-2-specific inhibitor, NS-398. In addition, hCG induced Cox-2 mRNA expression and potentiated the effect of IL-1beta. However, in contrast to the rapid and transient effect of IL-1beta on Cox-2 mRNA, the effect of hCG followed slower kinetics. We have previously shown that hCG induces expression of human FP receptor mRNA in cultured human GL cells. We now show that IL-1beta induces FP receptor mRNA in a time- and concentration-dependent manner. Our data suggest that Cox-2 and FP receptor are coexpressed in freshly isolated human granulosa cells and that their expression is up-regulated by IL-1beta and hCG in cultured human GL cells.

Tumorigenic transformation of immortalized ECV endothelial cells by cyclooxygenase-1 overexpression.

The cyclooxygenase (Cox) enzyme catalyzes the rate-limiting oxidative and peroxidative enzymatic steps in the biosynthesis of prostanoids. Both Cox-1 and -2 genes encode the two isoenzymes that carry out similar enzymatic steps. Enhanced Cox activity is associated with proliferative diseases such as colon cancer. To determine if a cause and effect relationship exists between Cox isoenzyme overexpression and tumorigenesis, the human Cox-1 and Cox-2 isoenzymes were transfected into ECV immortalized endothelial cells. Although numerous clones of Cox-1 expressing cells were obtained, Cox-2 overexpression resulted in growth disadvantage and increased cell death. In contrast, Cox-1 overexpressing cells expressed high levels of the functional Cox-1 polypeptide in the endoplasmic reticulum and the nucleus. In vitro proliferation of these cells was reduced compared with vector-transfected ECV cells. Cox-1 overexpression also enhanced the tumor necrosis factor-alpha-induced apoptosis of ECV cells 2-fold. In contrast to the in vitro behavior, ECV-Cox-1 cells proliferated aggressively and formed tumors in athymic "nude" mice, whereas the vector-transfected counterparts did not. The growth of Cox-1-induced tumors was not inhibited by indomethacin, suggesting a nonprostanoid function of Cox-1. ECV-Cox-1-derived tumors were angiosarcoma-like and contained numerous host-derived neovessels. These data suggest that Cox-1 overexpression in immortalized ECV endothelial cells results in nuclear localization of the polypeptide and tumorigenesis.

Down-regulation of cytokine-induced cyclo-oxygenase-2 transcript isoforms by dexamethasone: evidence for post-transcriptional regulation.

The immediate-early cyclo-oxygenase-2 (Cox-2) gene encodes an inducible prostaglandin synthase enzyme that has been implicated in inflammatory and proliferative diseases. We have shown that the inflammatory cytokine interleukin-1 (IL-1) induces the Cox-2 gene in a sustained manner and that post-transcriptional mRNA stabilization is an important even [Ristimäki, Garfinkel, Wessendorf, Maciag and Hla (1994) J. Biol. Chem. 269, 11769-11775]. The anti-inflammatory glucocorticoid dexamethasone potently down-regulates IL-1-induced Cox-2 mRNA expression. Kinetic studies suggest that antagonism of IL-1-induced mRNA stabilization is, at least in part, responsible for the suppression of Cox-2 mRNA. The Cox-2 gene produces two major transcript isoforms, namely Cox-2(4.6) (4.6 kb) and Cox-2(2.8) (2.8 kb), which are derived by alternative polyadenylation in the 3'-untranslated region (UTR). In response to dexamethasone, the short Cox-2(2.8) transcript isoform, which lacks a highly conserved AU-rich region, decays with a longer half-life than the Cox-2(4.6) isoform. Furthermore, heterologous expression of the hybrid Cox-1 open reading frame and the Cox-2 3'-UTR results in the accumulation of high levels of the short isoform and lower levels of the long isoform. These data suggest that multiple elements in the 3'-UTR of the Cox-2 gene are involved in the determination of the differential mRNA stabilities of Cox-2 transcript isoforms. Because dexamethasone destabilizes the Cox-2 transcript, and because the decay of Cox-2 transcript isoforms induced by dexamethasone occurs with different half-lives, post-transcriptional mRNA destabilization may be an important mechanism in the action of anti-inflammatory glucocorticoids.

A rare neutral polymorphism in 21-hydroxylase genes as HLA haplotype marker. Evidence for strong founder effect in the Finnish population.

The usefulness of rare neutral gene polymorphisms as an HLA haplotype marker and as a probe for founder effect in small populations was tested by determining the frequency and MHC associations of an NcoI polymorphism in the P450c21 genes in the Finnish population. In the general population, 13% (9 of 70) of samples had the NcoI site. A very strong association with the HLA-B62, Bf*F, C4A*3, C4B*Q0, DRB1*13, DQA1*0103, DQB1*0603 alleles was observed. P450c21A and P450c21B gene-specific amplifications mapped the polymorphic site to both P450c21A pseudogene and P450c21B functional gene of this haplotype in all cases. The majority of haplotypes with the NcoI cutting site found in this population may thus have derived from a single ancestral haplotype. The HLA homozygous cell lines with the NcoI site showed heterogeneous HLA associations. Our results suggest that in small populations the variety of MHC haplotypes may be surprisingly low and rare polymorphisms can serve as informative markers.

Structure of the human cyclo-oxygenase-2 gene.

Cyclo-oxygenase (Cox), a rate-limiting enzyme in the synthesis of prostanoids, is encoded by two genes, Cox-1 and Cox-2, which are differentially expressed and regulated. Human Cox-1 and -2 polypeptides share 61% primary sequence identity. While the expression of Cox-1 is maximal in quiescent cells. Cox-2 expression is induced by growth factors and cytokines. We have screened a human genomic library with a probe from the 5'-untranslated region (UTR) of the human Cox-2 (hCox-2) cDNA and isolated two overlapping genomic clones. We have determined the DNA sequence of 0.8 kb upstream of the transcription start site, 6 kb of protein coding region, which includes 10 exons and 9 introns, as well as 2.5 kb of the 3'-UTR. The structures of the hCox-1 and hCox-2 and the murine TIS10 (Cox-2) genes are highly conserved, with a few exceptions. The 3'-UTRs of the Cox-1 and -2 genes are distinct; for example, the largest exon in the Cox-2 gene encodes the entire 3'-UTR, containing 22 copies of the 'AUUUA' RNA instability element. Sequence analysis of the 5'-flanking region has shown several potential transcription regulatory sequences, including a TATA box, a C/EBP motif, two AP-2 sites, three SP1 sites, two NF-kappa B sites, a CRE motif and an Ets-1 site. These efforts serve as a basis for future studies on transcriptional and post-transcriptional mechanisms of Cox-2 gene regulation.