Paracrine regulation of matrix metalloproteinase expression in the normal human endometrium.

Endometrial expression of matrix metalloproteinase (MMP)-3, MMP-7 and MMP-11 occurs during menstrual breakdown and subsequent estrogen-mediated growth, but not during the secretory phase. These enzymes are suppressed by progesterone treatment. Paracrine factors, including transforming growth factor-beta (TGF-beta) and retinoic acid, are also critical for MMP regulation in the endometrium. In contrast, inflammatory cytokines such as interleukin-1alpha may block or interfere with steroid-mediated MMP regulation at ectopic sites of growth. Using in vitro models, our laboratory has investigated the complex interactions between progesterone and locally produced cytokines that may affect MMP expression during the development of endometriosis. Our results indicate that targeting the regulation of MMPs may represent an appropriate therapeutic strategy for the treatment of endometriosis. Copyrightz1999S. KargerAG,Basel

Stat5-mediated regulation of the human type II 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene: activation by prolactin.

Altered PRL levels are associated with infertility in women. Molecular targets at which PRL elicits these effects have yet to be determined. These studies demonstrate transcriptional regulation by PRL of the gene encoding the final enzymatic step in progesterone biosynthesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD). A 9/9 match with the consensus Stat5 response element was identified at -110 to -118 in the human Type II 3beta-HSD promoter. 3beta-HSD chloramphenicol acetyltransferase (CAT) reporter constructs containing either an intact or mutated Stat5 element were tested for PRL activation. Expression vectors for Stat5 and the PRL receptor were cotransfected with a -300 --> +45 3beta-HSD CAT reporter construct into HeLa cells, which resulted in a 21-fold increase in reporter activity in the presence of PRL. Promoter activity showed an increased response with a stepwise elevation of transfected Stat5 expression or by treatment with increasing concentrations of PRL (max, 250 ng/ml). This effect was dramatically reduced when the putative Stat5 response element was removed by 5'-deletion of the promoter or by the introduction of a 3-bp mutation into critical nucleotides in the element. Furthermore, 32P-labeled promoter fragments containing the Stat5 element were shifted in electrophoretic mobility shift assay experiments using nuclear extracts from cells treated with PRL, and this complex was supershifted with antibodies to Stat5. These results demonstrate that PRL has the ability to regulate expression of a key human enzyme gene (type II 3beta-HSD) in the progesterone biosynthetic pathway, which is essential for maintaining pregnancy.

Synthesis of retinoic acid by rat ovarian cells that express cellular retinoic acid-binding protein-II.

The induction of pseudopregnancy by the injection of eCG in rats results in the appearance of cellular retinoic acid-binding protein type II (CRABP[II]) in the granulosa cells of the ovary and the lining epithelium of the uterus within 48 h. This expression pattern is also seen in the normal mature female rat, in which CRABP(II) is expressed in the uterine epithelium during estrus (but not diestrus) and in the granulosa and luteal cells of the ovary. We have previously demonstrated that the uterine epithelial cells from the pseudopregnant rat have gained the ability to synthesize retinoic acid from retinol, in correlation with the induced expression of CRABP(II). If this is true for other sites of CRABP(II) expression, then local production of retinoic acid is intimately connected with various stages of reproduction in the female. Here we report that granulosa cells from the ovary of the eCG-treated immature rat and luteal cells from the ovary of the eCG/hCG-treated immature rat (both of which express CRABP[II]) synthesized markedly higher amounts of retinoic acid when cultured, compared to granulosa cells cultured from the ovary of the prepubertal rat treated with control vehicle. Culturing the granulosa cells from either control or eCG-treated animals had no effect on the expression of CRABP(II) cells. These data are consistent with our hypothesis that CRABP(II) expression is associated with retinoic acid synthesis and strengthen the case that local generation of retinoic acid plays an important role in reproduction.

Steady-state concentrations of mRNA encoding two inhibitors of protein kinase C in ovine luteal tissue.

Prostaglandin F2 alpha (PGF2 alpha) decreases secretion of progesterone from the corpus luteum in domestic ruminants. However, it is less effective during the early part of the oestrous cycle (Louis et al., 1973) and at the time of maternal recognition of pregnancy (Silvia and Niswender, 1984; Lacroix and Kann, 1986). Decreased luteal responsiveness may be due to failure of PGF2 alpha to activate fully its normal second messenger system, protein kinase C (PKC). Alternatively, increased resistance of the corpus luteum to PGF2 alpha might be attributable to greater concentrations of recently identified biological inhibitors of PKC. These possibilities were addressed by measuring steady-state concentrations of mRNA encoding PGF2 alpha receptor and two inhibitors of PKC, protein kinase C inhibitor-1 (PKCI-1) and kinase C inhibitor protein-1 (KCIP-1, brain 14-3-3 protein), in corpora lutea collected from ewes on days 4, 10 and 15 of the oestrous cycle (n = 5 per day) and day 15 of pregnancy (n = 7). There were no differences in mean concentrations of mRNA encoding PGF2 alpha receptor among the groups. However, concentrations of mRNA encoding both inhibitors of PKC were higher (P < 0.01) on day 4 of the oestrous cycle compared with the other groups. Treatment of ewes with a luteolytic dose of PGF2 alpha, which activates PKC, did not change concentrations of mRNA encoding either PKCI-1 or KCIP-I up to 24 h later. Luteal expression of mRNA encoding the PKC inhibitors and PGF2 alpha receptor was also examined in ewes treated with oestradiol in vivo for 16 h in the midluteal phase. High concentrations of oestradiol in serum (20 and 70 pg ml-1) did not influence quantities of any of the mRNAs examined. Therefore, an increase in PKC inhibitors may be involved in resistance of the corpus luteum to PGF2 alpha during the early part of the oestrous cycle but does not appear to mediate the increased resistance of the corpus luteum to PGF2 alpha during maternal recognition of pregnancy. Neither PGF2 alpha nor oestradiol affected steady-state concentrations of mRNAs encoding PKCI-1 or KCIP-I.

DAX-1 blocks steroid production at multiple levels.

DAX-1 is an unusual member of the nuclear hormone receptor superfamily whose expression is mainly, but not uniquely, restricted to steroidogenic tissues. We have recently shown that DAX-1 can block the first and rate-limiting step in steroid biosynthesis by repressing StAR (steroidogenic acute regulatory protein) expression. Here we show that DAX-1 blocks steroid production at multiple levels in the Y-1 mouse adrenocortical tumor cell line. Expression of DAX-1 in Y-1 cells significantly impairs both basal and cAMP-stimulated steroid production, without affecting the functionality of the cAMP-responsive PKA pathway. Experiments using an hydroxylated cholesterol derivative show that biochemical steps in steroidogenesis subsequent to cholesterol delivery to mitochondria are also impaired in Y-1 cells expressing DAX-1. This is explained by the repression of P450scc and 3beta-HSD expression, in addition to StAR. DAX-1 expression in Y-1 cells results in the inhibition of the activity of the StAR, P450scc and 3beta-HSD promoters. An inappropriate steroidogenic block in the male fetus might have an important role in the pathogenesis of sex reversal syndromes caused by a duplication of the genomic region of the X chromosome containing the DAX-1 gene.

Targeting of the domain-specific integral membrane protein PM52 to the periacrosomal plasma membrane during guinea pig spermiogenesis.

The sperm plasma membrane is segregated into functionally, biochemically, and structurally distinct domains yet the protein sorting pathways and assembly mechanisms that assemble these domains during spermiogenesis are incompletely understood. We previously characterized two structurally related size-variant, integral membrane proteins of 52 kDa (PM52) and 35 kDa localized to the periacrosomal plasma membrane of guinea pig cauda epididymal spermatozoa (Westbrook-Case et al., 1994). In this study we used light and electron microscopic immunocytochemistry to define the expression pattern and sorting pathway that establishes the domain-specific distribution of PM52 during spermiogenesis. The PM52 is first expressed in acrosome-phase spermatids and it localizes exclusively to the cytoplasmic lobe. Immunoelectron microscopy revealed that both cytoplasmic vesicles and the plasma membrane of the cytoplasmic lobe labeled with anti-PM52. During early stages of expression, PM52 appeared to be absent from the head region, but significant PM52 accumulation over the spermatid head was noted in late acrosomal phase spermatids. Throughout spermiogenesis PM52 extended posteriorly to the annulus, which represents a barrier preventing PM52 diffusion into the posterior tail. Following the migration of the annulus to the midpiece-principal piece junction, PM52 began to disappear from the flagellar region and at the completion of spermiogenesis most of the PM52 was restricted to the acrosomal segment. Spermatids and epididymal sperm PM52 exhibited identical sizes by SDS-PAGE and immunoblotting, indicating that they are not proteolytically modified during epididymal maturation. The PM52 antibodies were also used to screen a guinea pig testis cDNA library, and sequence determination of full-length PM52 clones demonstrated identity of a sperm membrane protein recently termed "sperad" (Quill and Garbers, 1996). Membrane barriers and potential mechanisms establishing the domain-specific residence of PM52 are discussed.

Synergistic activation of the human type II 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase promoter by the transcription factor steroidogenic factor-1/adrenal 4-binding protein and phorbol ester.

Steroidogenic factor-1/adrenal 4-binding protein (SF-1/Ad4BP) is an orphan nuclear receptor/transcription factor known to regulate the P450 steroid hydroxylases; however, mechanisms that regulate the activity of SF-1/Ad4BP are not well defined. In addition, little is known about the mechanisms that regulate the human steroidogenic enzyme, type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD II), the major gonadal and adrenal isoform. Regulation of the 3beta-HSD II promoter was examined using human adrenal cortical (H295R; steroidogenic) and cervical (HeLa; non-steroidogenic) carcinoma cells. H295R cells were transfected with a series of 5' deletions of 1251 base pairs (bp) of the 3beta-HSD II 5'-flanking region fused to a chloramphenicol acetyltransferase (CAT) reporter gene followed by treatment with or without phorbol ester (phorbol 12-myristate 13-acetate; PMA). CAT assay data indicated that the region from -101 to -52 bp of the promoter was required for PMA-induced expression. A putative SF-1/Ad4BP regulatory element, TCAAGGTAA, was identified by sequence homology at -64 to -56 bp of the promoter. Cotransfection of HeLa cells with the -101 3beta-HSD-CAT construct and an expression vector for SF-1/Ad4BP increased CAT activity 49-fold. Subsequent treatment with PMA induced an unexpected synergistic increase in transcriptional activity 540-fold over basal. Mutation of the putative response element (TCAAGGTAA to TCAATTTAA) abolished SF-1-induced CAT activity and the synergistic response to PMA. Gel mobility shift assays confirmed that SF-1/Ad4BP interacts with the putative element and transcripts for SF-1/Ad4BP were detected in H295R cells by Northern analysis. These data are the first to demonstrate 1) regulation of a non-cytochrome P450 steroidogenic enzyme promoter by SF-1/Ad4BP, 2) a powerful synergistic effect of PMA on SF-1/Ad4BP-induced transcription, and 3) the importance of the SF-1/Ad4BP regulatory element in the regulation of the 3beta-HSD II promoter.

The regulation of 3 beta-hydroxysteroid dehydrogenase expression.

3 beta-Hydroxysteroid dehydrogenasel delta 5-->4-isomerase (3 beta-HSD) catalyzes the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids, an obligate step in the biosynthesis not only of androgens and estrogens but also of mineralocorticoids and glucocorticoids. The enzyme is expressed in the adrenal cortex and in steroidogenic cells of the gonads, consistent with this role. However, 3 beta-HSD is also expressed in many other tissues, such as the liver and kidney, where its function is not entirely clear. It is established that a family of closely related genes encode for 3 beta-HSD. The various 3 beta-HSD isoforms are expressed in a tissue-specific manner involving separate mechanisms of regulation. The human type I 3 beta-HSD is expressed at high levels in syncytial trophoblast and in sebaceous glands, and the type II isoform is almost exclusively expressed in the adrenal cortex and gonads. An important feature in liver and kidney (at least of hamster, mouse, rabbit, and rat) is the sexual dimorphic nature of 3 beta-HSD expression. We briefly review studies on the regulation of the human 3 beta-HSD I and II genes in human trophoblast and adrenal cortex and extend this to discuss the rat 3 beta-HSD I gene expressed in adrenals and gonads. The complexity of 3 beta-HSD expression through multiple signaling pathways acting on a multigene family of enzymes may contribute importantly to the diverse patterns and locations of steroid hormone biosynthesis.

Specific expression of haptoglobin mRNA in implantation-stage rabbit uterine epithelium.

A glycoprotein, termed GP42, was previously identified in uterine fluid obtained from peri-implantation-stage rabbits. N-terminus amino acid sequencing of purified GP42 demonstrated identity through the first 13 amino acids with the beta subunit of liver haptoglobin. The present study was undertaken to determine if GP42 is indeed identical to haptoglobin and, if so, to determine whether it is expressed in the uterus as opposed to being present as a transudate from plasma. Reverse transcription-PCR amplification of poly(A)+ RNA prepared from implantation-stage rabbit endometrium with GP42- and haptoglobin-specific primers yielded a predicted 667 bp cDNA product. Sequence analysis of the cloned cDNA confirmed the identity of GP42 with beta-haptoglobin. Northern blot analysis demonstrated the specific expression of haptoglobin mRNA in the peri-implantation-stage endometrium and the absence of its expression in the estrous or day 4 pseudopregnant endometrium. Non-isotopic in situ hybridization revealed that the haptoglobin mRNA was restricted to the epithelium lining the luminal surface and mucosal folds of day 6(3/4) pregnant or pseudo-pregnant uteri and that no haptoglobin mRNA was detectable in the epithelium of the deep glands or cells of the stroma or myometrium. Similarly, in situ hybridization revealed no expression of haptoglobin mRNA in any cell types of the estrous uterus. These data establish the identity of GP42 with beta-haptoglobin and demonstrate that it is expressed in a stage-specific manner just prior to implantation, correlating with uterine receptivity to blastocyst implantation. Endometrial GP42 mRNA expression is not dependent on the presence of blastocysts.

Induction and activation of Stat 5 in the ovaries of pseudopregnant rats.

PRL acts in the ovary to promote the maintenance and function of the corpus luteum. However, the cellular signals that induce these responses have not been clearly defined. In the present report we demonstrate that Stat 5, previously identified as a transcription factor activated by PRL in the mammary gland, is also activated by PRL in the ovaries of pseudopregnant rats. Intraperitoneal injection of PRL into pseudopregnant rats results in the tyrosine phosphorylation and nuclear translocation of Stat 5. This activated Stat 5 possesses DNA-binding activity for a sequence containing the PRL-inducible element. In addition, we report that luteinization of the PMSG-primed ovary by the administration of hCG is accompanied by an induction of Stat 5 protein.

Regulation and localization of cellular retinol-binding protein, retinol-binding protein, cellular retinoic acid-binding protein (CRABP), and CRABP II in the uterus of the pseudopregnant rat.

Three members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP), and cellular retinoic acid-binding protein II (CRABP II). Retinol-binding protein (RBP) is a secreted protein that binds and solubilizes vitamin A for transport. Here we report the coordinate regulation of RBP, CRBP, retinol, and CRABP II in the uterus of the pseudopregnant rat. In the proliferative stage of the uterus, which was induced by PMSG, the messenger RNA (mRNA) and protein levels of RBP and CRBP as well as retinol levels significantly decreased. This pattern of regulation was duplicated by estrogen treatment of prepubertal rats. In addition, CRBP and RBP were found to be colocalized to the stromal cells of the rat uterus by immunohistochemistry and [35S]methionine-labeled affinity chromatography, respectively, and were not detected in other cell populations. CRABP II mRNA and protein expression were up-regulated in the proliferative phase of the uterus brought about by PMSG injection or, alternatively, by estrogen treatment of prepubertal rats. CRABP II was localized to the surface epithelium, but was not seen elsewhere, including glandular epithelium. Immunolocalization of CRABP showed staining of the smooth muscle and stromal cells of the uterus. The appearance of CRABP in the stroma of the uterus also correlated with PMSG injection as well as estrogen treatment. Although estrogen induced the appearance of both binding proteins, CRABP mRNA levels peaked between 4-24 h postestrogen treatment, whereas CRABP II mRNA levels continued to rise 48 h postestrogen treatment. These data demonstrate an important role for vitamin A and retinoid-binding proteins in rat uterine physiology.

Physiological inhibitors of protein kinase C.

Increasing numbers of proteins that have the capacity of interacting with protein kinase C isozymes in vitro and inhibiting their enzymatic activity in a noncompetitive manner have been purified. While these proteins can be hypothesized to be part of a tight regulatory system for protein kinase C enzymatic activity, critical examinations of the roles of these proteins in the context of whole cells have not yet been performed. Interesting new data suggest that some of the classes of protein kinase C inhibitors may have a much broader role of interacting with multiple types of kinases and proto-oncogene products. cDNAs encoding a number of these inhibitor proteins have been isolated, which will allow the design and implementation of experiments on their cell biology and help address their function outside of the context of their operational definitions.

Inducible expression of cellular retinoic acid-binding protein II in rat ovary: gonadotropin regulation during luteal development.

Two members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular retinoic acid-binding protein and cellular retinoic acid-binding protein II [CRABP(II)]. CRABP is found in many adult tissues, whereas CRABP(II) is more restricted and is reported as abundant primarily in skin. Here we report a much greater expression of CRABP(II) in rat corpus luteum than in any other organ/tissue examined, including skin. A rat complementary DNA clone encoding CRABP(II) was isolated and the ovarian expression followed during gonadotropin induction of follicular development in the pseudopregnant rat. The pattern of rat CRABP(II) messenger RNA and protein expression correlated with the appearance of corpora lutea and the rise in progesterone production as the corpora lutea developed, and was similar to the induction of 3 beta-hydroxysteroid dehydrogenase. Immunohistochemical localization revealed that CRABP(II) appeared in luteal cells and was dramatically restricted to their cytoplasmic compartment, with no apparent presence in the nucleus. This suggests that CRABP(II) may be expressed to restrict retinoic acid from occupying nuclear retinoic acid receptors, implying that the differentiation and maintenance of the rat corpus luteum may involve in part a release of certain pathways from retinoid suppression.

Widespread tissue distribution of steroid sulfatase, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD), 17 beta-HSD 5 alpha-reductase and aromatase activities in the rhesus monkey.

Dehydroepiandrosterone-sulfate (DHEA-S), the main secretory product of the human adrenal, requires the presence of steroid sulfatase, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD), 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), 5 alpha-reductase, and aromatase to form the active androgen dihydrotestosterone (DHT) and the estrogens 17 beta-estradiol (E2) and 5-androst-ene-3 beta,17 beta-diol (delta 5-diol) in peripheral target tissues. Because humans, along with non-human primates are unique in having adrenals that secrete large amounts of DHEA-S, the present study investigated the tissue distribution of the enzymatic activity of the above-mentioned steroidogenic enzymes required for the formation of active sex steroids in the male and female rhesus monkey. Estrone and DHEA sulfatase activities were measured in all 25 tissues examined, and with the exception of the salivary glands, estrogenic and androgenic 17 beta-HSDs were present in all the tissues examined. The adrenal, small and large intestine, kidney, liver, lung, fat, testis, prostate, seminal vesicle, ovary, myometrium, and endometrium all possess the above-mentioned enzymatic activities, thus suggesting that these tissues could possibly form the biologically active steroids E2 and DHT from the adrenal precursor DHEA-S. On the other hand, the oviduct, cervix, mammary gland, heart, and skeletal muscle possess all the enzymatic activities required to synthesize E2 from DHEA-S. The present study describes the widespread tissue distribution of steroid sulfatase, 3 beta-HSD, 17 beta-HSD, 5 alpha-reductase, and aromatase activities in rhesus monkey peripheral tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone receptor, but not estradiol receptor, messenger ribonucleic acid is expressed in luteinizing granulosa cells and the corpus luteum in rhesus monkeys.

Estrogens (i.e. estradiol) and progestins (i.e. progesterone) may act as local regulators of ovarian function in various species. This study tested the hypothesis that if progesterone and estradiol act via receptor-mediated pathways in the primate ovary, then receptor messenger RNAs (mRNAs) should be detectable in ovarian cells. The reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect progesterone and estradiol receptor (PR and ER, respectively) mRNAs in the rhesus monkey ovary. Total RNA was isolated from macaque uterine myometrium (positive control), spleen (negative control), whole ovary, germinal (surface) epithelium-enriched cortical and medullary compartments of the ovary, granulosa cells in preovulatory follicles before and after an ovulatory stimulus, and corpora lutea from early (days 3-5), mid (days 7 and 8)-, and late (days 14 and 15) luteal phase of the menstrual cycle. Using primers to the hormone-binding region encoded by the receptor mRNAs, RT-PCR products of the expected sizes were detected for PR and ER from 1 microgram myometrial RNA, whereas products were not obtained from spleen. PR mRNA product was detected in all ovaries, germinal epithelium-enriched cortical and medullary compartments, and corpora lutea from all three stages of the luteal phase (n = 3/stage). PR mRNA product was detected as a strong band in one of three preparations obtained from granulosa cells before an ovulatory stimulus. In contrast, PR mRNA was detected in granulosa cells from all animals after an ovulatory dose of hCG. ER mRNA was detected in whole ovary and in germinal epithelium-enriched cortical compartments, with a barely visible product occasionally observed in medullary compartments of the ovary. In contrast to PR mRNA, ER mRNA was not detected in any corpora lutea throughout the luteal phase or in granulosa cells obtained before or after an ovulatory stimulus. To confirm the specificity of the RT-PCR products, restriction enzymes cleaved the PR product from myometrium, germinal epithelium-enriched cortical compartment, and corpus luteum into the predicted size fragments. Similarly, the ER product from the myometrium and the germinal epithelium-enriched compartment was cleaved into the expected size fragments. Sequence analysis of the PR and ER RT-PCR products revealed 99% homology to the complementary DNA for the hormone-binding region of human PR and ER, respectively. Thus, PR mRNA detection supports the hypothesis of progesterone action via classical receptor-mediated pathways in the luteinizing follicle and corpus luteum of the primate ovary.(ABSTRACT TRUNCATED AT 400 WORDS)

Novel interactions between human T-cell leukemia virus type I Tax and activating transcription factor 3 at a cyclic AMP-responsive element.

Human proenkephalin gene transcription is transactivated by human T-cell leukemia virus type I (HTLV-I) Tax in human Jurkat T lymphocytes. This transactivation was further enhanced in Jurkat cells treated with concanavalin A, cyclic AMP, or 12-O-tetradecanoylphorbol-13-acetate. Deletion and cis-element transfer analyses of the human proenkephalin promoter identified a cyclic AMP-responsive AP-1 element (-92 to -86) as both necessary and sufficient to confer Tax-dependent transactivation. Different AP-1 or cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) proteins which bind this element were expressed in murine teratocarcinoma F9 cells to identify those capable of mediating Tax-dependent transactivation of human proenkephalin gene transcription. Although CREB, c-Fos, c-Jun, and JunD did not have significant effects, JunB inhibited the Tax-dependent transactivation. In contrast, ATF3 dramatically induced Tax-dependent transactivation, which was further enhanced by protein kinase A. Electrophoretic mobility shift assays with recombinant fusion proteins expressed and purified from bacteria indicate that the DNA-binding activity of ATF3 is also dramatically enhanced by Tax. Chimeric fusion proteins consisting of the DNA-binding domain of the yeast transcription factor Gal4 and the amino-terminal domain (residues 1 to 66) of ATF3 were able to mediate Tax-dependent transactivation of a Gal4-responsive promoter, which suggests a direct involvement of this region of ATF3. Recombinant fusion proteins of glutathione S-transferase with either the amino- or carboxy-terminal (residues 139 to 181) domain of ATF3 were able to specifically interact with Tax. Furthermore, specific antisera directed against Tax coimmunoprecipitated ATF3 only in the presence of Tax.

Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity.

Examination of the regulation of prodynorphin (pro-DYN) promoter activity is limited by the absence of a good cell model. The discovery of pro-DYN mRNA and derived peptides in the reproductive tract led us to examine the cellular localization and hormonal regulation of ovarian pro-DYN expression and to evaluate normal granulosa cells as a model for studying pro-DYN gene regulation. Ovarian pro-DYN mRNA levels were significantly elevated in PMSG-primed immature rats 12-24 h after receiving an ovulatory dose of hCG. Levels peaked 2 days after hCG, remained elevated throughout the ensuing pseudopregnancy, and rose again at the end of pseudopregnancy. In situ hybridization localized pro-DYN expression predominantly to granulosa and luteal cells. Transfection of primary cultures of granulosa cells revealed that the activity of the rat pro-DYN promoter [-1858 to 133 base pairs (bp)] was increased 18- to 19-fold by hCG and human FSH treatments and 7-fold by cAMP analog treatment. Deletion analysis identified a 358 bp fragment as the primary hormone-responsive sequence (-1858 to -1500 bp; containing three potential cAMP-responsive elements); its deletion resulted in severely reduced FSH responsiveness, and its ligation to hormone-unresponsive basal promoter sequences completely restored FSH responsiveness. This is an unusually distal position for cAMP-responsive elements compared to other cAMP-regulated genes. These data demonstrate specific expression of pro-DYN in granulosa and luteal cells, which is under sensitive gonadotropin regulation, and identify a distal hormone-responsive sequence within the promoter.