The cAMP signaling system regulates LHbeta gene expression: roles of early growth response protein-1, SP1 and steroidogenic factor-1.

Expression of the gonadotropin genes has been shown to be modulated by pharmacological or physiological activators of both the protein kinase C (PKC) and the cAMP second messenger signaling pathways. Over the past few years, a substantial amount of progress has been made in the identification and characterization of the transcription factors and cognate cis-elements which mediate the PKC response in the LH beta-subunit (LHbeta) gene. In contrast, little is known regarding the molecular mechanisms which mediate cAMP-mediated regulation of this gene. Using pituitary cell lines, we now demonstrate that rat LHbeta gene promoter activity is stimulated following activation of the cAMP system by the adenylate cyclase activating agent, forskolin, or by the peptide, pituitary adenylate cyclase-activating peptide. The forskolin response was eliminated with mutation of a previously identified 3' cis-acting element for the early growth response protein-1 (Egr-1) when evaluated in the context of region -207/+5 of the LHbeta gene. Activation of the cAMP system increased Egr-1 gene promoter activity, Egr-1 protein levels and Egr-1 binding to the LHbeta gene promoter, supporting the role of this transcription factor in mediating the cAMP response. Analysis of a longer LHbeta promoter construct (-797/+5) revealed additional contribution by upstream Sp1 DNA-regulatory regions. Of interest, forskolin-induced stimulation of LHbeta gene promoter activity was observed to increase synergistically with introduction of the transcription factor, steroidogenic factor-1 (SF-1). Although SF-1 is a critical mediator of the cAMP response in other genes, mutation of the SF-1 DNA-binding sites in the rat LHbeta gene did not alter the forskolin response nor did forskolin increase SF-1 protein levels in a gonadotrope cell line. In a further set of experiments, it was determined that forskolin-responsiveness was maintained following mutation of the previously defined homeobox-binding element at position -100. We conclude that both Egr-1 and Sp1 contribute to cAMP-dependent transcription of the rat LHbeta gene promoter. While SF-1 does not act independently to mediate the cAMP/PKA response, SF-1 is important for magnification of this response.

Sp1, steroidogenic factor 1 (SF-1), and early growth response protein 1 (egr-1) binding sites form a tripartite gonadotropin-releasing hormone response element in the rat luteinizing hormone-beta gene promoter: an integral role for SF-1.

Recently, several cis-regulatory elements that play roles in LHbeta gene expression, and their cognate DNA-binding transcription factors, have been identified. These factors include Sp1, steroidogenic factor-1 (SF-1), and early growth response protein 1 (Egr-1). Using the GH3 pituitary cell line (which lacks SF-1) as a model, we demonstrate that expression of SF-1 or Egr-1 increases rat LHbeta gene promoter activity but has little effect on the fold response to GnRH. However, expression of both SF-1 and Egr-1 synergistically enhances LHbeta gene promoter activity and prevents further stimulation of activity by GnRH. Mutations in the Sp1 binding sites of the rat LHbeta gene promoter decrease GnRH responsiveness, whereas mutations in the SF-1 and/or Egr-1 binding sites alone have little effect on the GnRH response. Combinatorial mutations in both the Sp1 and Egr-1 binding elements result in almost complete loss of the GnRH response. In contrast, in GH3 cells cotransfected with SF-1, mutations in the Sp1, SF-1, or Egr-1 binding elements independently decrease GnRH responsiveness. In LbetaT2 cells, a gonadotrope-derived cell line that expresses SF-1 endogenously, mutations in either the Sp1 or Egr-1 binding elements decrease GnRH responsiveness. These data suggest that the Sp1, SF-1, and Egr-1 binding sites form a tripartite GnRH response element in the rat LHbeta gene promoter. Changes in the spacing between the upstream Sp1 binding sites and the downstream SF-1/Egr-1 binding elements reduce the response to GnRH. SF-1, while having little direct effect on GnRH responsiveness, has a critical role in integrating the effects of Sp1 and Egr-1.

The protein kinase C system acts through the early growth response protein 1 to increase LHbeta gene expression in synergy with steroidogenic factor-1.

Expression of the LHbeta gene has been shown to be modulated by both the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response protein 1, Egr-1. It is also well known that LHbeta mRNA levels are increased after hormonal activation of the protein kinase C (PKC) signaling system, for example by GnRH; however, the mechanisms by which the PKC system exerts this effect has not been fully characterized. By transient transfection of the GH3 cell line, we demonstrate that activation of the PKC system with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), increases activity of region -207/+5 of the rat LHbeta gene promoter (approximately 2-fold) and markedly augments SF-1-induced stimulation (95-fold in the presence of both factors vs. 13-fold for SF-1 alone). Mutation of the two previously identified Egr-1 sites not only prevents Egr-1 effects on the LHbeta gene promoter, but also eliminates the synergistic response to PMA and SF-1 together, findings that were confirmed in a longer construct spanning region -797/+5. In the gonadotrope-derived cell line, alphaT3-1, these mutations eliminate the GnRH responsiveness of the -207/+5 LHbeta promoter construct. We next show that PMA treatment (GH3 and alphaT3-1 cells) or GnRH treatment (alphaT3-1 cells) induces expression of Egr-1, as detected by Egr-1 interaction with Egr-1 DNA-binding sites in the rat LHbeta gene promoter sequence. Furthermore, we demonstrate that PMA increases steady-state Egr-1 mRNA levels via increased Egr-1 transcription. We conclude that PMA-induced stimulation of LHbeta gene expression is achieved, at least in part, by induction of Egr-1 expression.

Steroidogenic factor-1 and early growth response protein 1 act through two composite DNA binding sites to regulate luteinizing hormone beta-subunit gene expression.

Recent in vivo and in vitro studies have implicated the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response protein 1 (Egr-1) in the transcriptional regulation of the luteinizing hormone beta-subunit (LHbeta) gene. We have previously demonstrated the ability of SF-1 to bind to and transactivate the rat LHbeta gene promoter acting at a consensus gonadotrope-specific element (GSE) located at position -127. We have now identified a second functional GSE site at position -59. In addition, based on electrophoretic mobility shift assay, in vitro translated Egr-1 is shown to bind to two putative Egr-1 binding sites (positions -112 and -50), which appear to be paired with the identified GSE sites. By transient transfection assay in pituitary-derived GH3 cells, it was seen that Egr-1 increases promoter activity of region -207/+5 of the rat LHbeta gene promoter through action at both Egr-1 sites. Furthermore, LHbeta gene promoter activity is markedly augmented in the presence of both factors together relative to activity in the presence of SF-1 or Egr-1 alone (150-fold versus 14-fold and 12-fold, respectively). These data define two composite SF-1-Egr-1 response-elements in the proximal LHbeta gene promoter and suggest that SF-1 and Egr-1 act synergistically to increase expression of the LHbeta gene in the gonadotrope.

Stimulation of luteinizing hormone beta gene promoter activity by the orphan nuclear receptor, steroidogenic factor-1.

The orphan nuclear receptor, steroidogenic factor-1 (SF-1), is expressed in the pituitary and in the gonadotrope precursor cell line, alphaT3-1, where it is believed to enhance expression of the common gonadotropin alpha-subunit gene through transactivation of the gonadotrope-specific element (GSE). Sequence analysis of the rat luteinizing hormone beta-subunit (LH beta) gene promoter revealed the presence of a consensus GSE at -127 to -119 (TGACCTTGT). We have demonstrated the ability of SF-1 to bind specifically to this putative GSE sequence by electrophoretic mobility shift assay, utilizing both alphaT3-1 nuclear extracts and in vitro translated SF-1. In addition, mutation of the putative LHbeta-GSE (TGAAATTGT) eliminated specific DNA binding. To examine the ability of SF-1 to enhance LHbeta promoter activity, CV-1 cells, which lack endogenous SF-1, were cotransfected with an SF-1-containing expression vector and an LHbeta-luciferase reporter construct. When cotransfected with -209/+5 of the LHbeta promoter, SF-1 increased luciferase activity by 56-fold. SF-1 responsiveness was markedly diminished with loss of the putative GSE region in deletion constructs and in the presence of a two base pair mutation, analogous to the mutation which eliminated DNA binding. Finally, the LHbeta-GSE was able to confer SF-1 responsiveness on a heterologous minimal growth hormone promoter, GH50 (57-fold). We conclude that SF-1 both binds to and transactivates the rat LHbeta promoter. These data suggest that SF-1 may participate in the expression of the LHbeta gene by the gonadotrope.

Inhibin, activin, and follistatin in reproductive medicine.

OBJECTIVE: To review the available information regarding the polypeptide factors inhibin, activin, and follistatin in reproductive physiology. DESIGN: The protein structure, tissue expression, regulation, and effects of these factors are outlined, with an emphasis on the reproductive tissues in both females and males. Although some information is only available in animal model systems, human data has been selected whenever possible. CONCLUSIONS: Inhibin and activin are closely related peptides with opposing actions, whereas follistatin is a structurally unrelated peptide that may act indirectly through modulation of inhibin-activin effects. These three peptides are secreted in highest levels by the adult gonads; however, they are also present in a wide variety of reproductive and nonreproductive tissues where they are believed to exert local, tissue-specific effects. Within the reproductive system, these peptides play a role in the regulation of gonadotropin biosynthesis and secretion, ovarian and placental steroidogenesis, and oocyte and spermatogonial maturation.

Transcriptional activation of the follicle-stimulating hormone beta-subunit gene by activin.

Activin markedly stimulates FSH beta messenger RNA (mRNA) levels in rat pituitary cells. Nonetheless, the molecular mechanisms through which activin enhances FSH beta gene expression are not clear. To assess the role of transcriptional activation in activin stimulation, we first transfected two -2300FSH beta Luc constructs into primary pituitary cells and treated them with activin in plated culture and perifusion. Basal expression of the constructs was low, and no activin response was observed. These results suggested that additional FSH beta sequences are required for basal expression or that the effects of activin are not transcriptional. An alternative approach for measuring transcriptional responses was developed based upon changes in the levels of FSH beta primary transcripts (FSH beta-PT; newly transcribed mRNAs that still contain the first intron) after activin (3 ng/ml) stimulation of perifused rat pituitary cells. An increase in FSH beta-PT was observed after 30 min of activin stimulation, preceding the first observable rise in mature FSH beta mRNA. Levels of FSH beta-PT peaked between 1-2 h, then fell to a lower level (28% of maximal) at 4 h, which was maintained through 10 h of activin stimulation (36% of maximal at 10 h). Mature FSH beta mRNA levels peaked between 2-4 h, which is after the increase in FSH beta-PT, and fell more gradually between 4-10 h of stimulation (56% of maximal at 10 h). Unstimulated levels of mature mRNA and FSH beta-PT did not vary significantly over the course of the experiments. Cotreatment with the transcriptional inhibitor actinomycin-D (2 microM) blocked activin stimulation of both FSH beta-PT and FSH beta mRNA, confirming the transcriptional basis for these events. In summary, we have documented rapid and sequential increases in FSH beta-PT and mature FSH beta mRNAs after activin stimulation, which are prevented by transcriptional blockade. These data provide evidence that the increase in FSH beta mRNA levels after activin treatment are at least partly due to transcriptional activation of the FSH beta gene.

Dynamic regulation of pituitary follistatin messenger ribonucleic acids during the rat estrous cycle.

FSH synthesis and secretion are regulated by a complex interplay of hypothalamic, gonadal, and pituitary factors. Recent evidence suggests that inhibin, activin, and follistatin, although originally identified as gonadal peptides, are also expressed in the pituitary, where they may be secreted and play an autocrine/paracrine role in the control of FSH beta gene expression. Attempts to study pituitary regulation of the genes encoding these proteins have been hampered by low levels of mRNA expression. Consequently, we developed quantitative reverse transcription-polymerase chain reaction assays for follistatin (FS) and the three subunits (alpha, beta A, and beta B) that comprise activin and inhibin. Expression of activin type II receptor (actRII) mRNA was also analyzed. Reasoning that mRNA levels of these FSH-regulating pituitary peptides might be modulated at times of increased FSH gene expression, two in vivo models were chosen for further investigation. 1) Two weeks after ovariectomy, rat pituitary FS mRNA levels increased substantially (4.09-fold vs. intact) with a modest increase in beta B-inhibin mRNA levels (1.88-fold vs. intact). No changes in alpha-inhibin, beta A-inhibin, or actRII mRNA levels were observed. 2) During the estrous cycle, pituitary FS gene expression varied strikingly, with a peak at 1800 h on proestrus (13.69-fold vs. 0900 h on proestrus), followed by a rapid decline at 2400 h on proestrus (2.10-fold vs. 0900 h on proestrus). A more detailed analysis of expression during proestrus revealed that peak FS mRNA levels preceded peak FSH beta gene expression by 6 h. Levels of the inhibin subunit and actRII transcripts varied minimally across the estrous cycle. We conclude that during the estrous cycle in rats, pituitary FS mRNA levels are regulated dynamically, whereas levels of inhibin/activin subunits and the activin receptor, actRII, very minimally. The observation that FS mRNA levels peak before maximal expression of FSH beta mRNA raises the possibility that FS facilitates, rather than inhibits, FSH biosynthesis in vivo.

Perifusion of rat pituitary cells with gonadotropin-releasing hormone, activin, and inhibin reveals distinct effects on gonadotropin gene expression and secretion.

Gonadotropin biosynthesis and secretion are influenced by pulsatile GnRH derived from the hypothalamus as well as by paracrine factors. In the current studies, we compared the effects of inhibin, activin, and GnRH, alone and in combination, on gonadotropin subunit messenger RNA (mRNA) levels and gonadotropin secretion. A pituitary perifusion system was used to allow GnRH to be administered as pulses and to minimize paracrine effects. FSH beta mRNA levels were increased 25-fold by a maximal concentration of activin (3 ng/ml) and suppressed 83% by a maximal concentration of inhibin (30 ng/ml). When activin and inhibin were perifused together, inhibin attenuated the effects of maximal activin stimulation in a concentration-dependent manner, with a 10-fold excess of inhibin required to block the effects of activin entirely. Whole cell receptor assays using 125I-labeled activin confirmed that the inhibin used in the perifusion experiments competed for activin binding sites, although with a lower affinity. Direct competition at the activin receptor may thus account for part of the activin/inhibin antagonism observed at the level of FSH beta mRNA. Neither activin nor inhibin had a significant effect on levels of LH beta or alpha mRNAs. Hourly pulses of 10 nM GnRH elicited a 2- to 5-fold increase in FSH beta mRNA. This increment was maintained in the presence of activin and inhibin, suggesting separate, but dependent, mechanisms of action for GnRH vs. inhibin and activin. In studies of secretion, continuous activin stimulation (3 ng/ml) elicited only a small (approximately 30%) increase in basal FSH secretion. However, the response of FSH to pulses of GnRH was amplified 3-fold in the presence of activin. A similar enhancement of GnRH-induced, but not basal, LH release was also observed. Inhibin, in contrast, elicited no changes in basal or GnRH-stimulated release of FSH or LH. We conclude that activin and inhibin are the primary regulators of FSH beta mRNA levels, whereas GnRH appears to be the major effector for gonadotropin secretion. There is significant functional overlap, however, and the combined actions of activin, inhibin, and GnRH determine the final level of FSH beta mRNA and the pattern of gonadotropin secretion.

Spontaneous uterine rupture after hysteroscopic metroplasty with uterine perforation. A case report.

This is the first documented case of spontaneous uterine rupture during pregnancy following hysteroscopic metroplasty complicated by uterine perforation. This case emphasizes the importance of treating these patients as high risk throughout pregnancy.

Ovarian angiogenesis in rabbits: endotheliotrophic chemoattractant activity from isolated follicles and dispersed granulosa cells.

To test the hypothesis that angiogenesis is an important variable in ovarian folliculogenesis, we measured endothelial cell migration (chemotaxis) in media conditioned by rabbit ovarian cells. Endothelial cell migration, a reliable predictor of angiogenesis in vivo, was stimulated by media conditioned by isolated intact follicles (0.4-2.2 mm in diameter) from either unstimulated or hCG-stimulated (pseudopregnant) rabbits. In separate experiments, endothelial cell migration was also stimulated by granulosa cell-conditioned media. Follicular chemoattractant activity was associated with a molecular weight greater than 30,000 but was not correlated with follicular size or steroid concentrations in the media, although there was no evidence to suggest that the biological activity detected in media conditioned by either intact follicles or dispersed granulosa cells was the same. Demonstration of nonsteroidal chemoattractant activity in media conditioned by intact follicles or by dispersed granulosa cells provides evidence that follicles secrete a vascular chemotactic factor, and is consistent with a role for angiogenesis in follicle growth.

Dynamic regulation of follicle-stimulating hormone-beta messenger ribonucleic acid levels by activin and gonadotropin-releasing hormone in perifused rat pituitary cells.

Maintenance of FSH biosynthesis requires ongoing exposure to pulsatile GnRH. Recent data demonstrate that activin also stimulates FSH biosynthesis. We used a perifused pituitary system to examine regulation of FSH beta mRNA levels by pulsatile GnRH and activin. Hourly pulses of 10 nM GnRH increased FSH beta mRNA levels by 3-fold. In the same experiment, continuous infusion of 50 ng/ml activin elicited a 50-fold increase in FSH beta mRNA. This magnitude of response to activin in perifusion was unexpected, as only a 2.7-fold increase in FSH beta mRNA was observed when activin was administered to pituitary cells that were cultured in dishes. Since perifusion columns, unlike culture dishes, are exposed to a continuous supply of fresh medium, we examined the possibility that endogenous factors produced by pituitary cells cultured in dishes were stimulating the cells in a paracrine fashion, thereby precluding the full response to exogenously added activin. The kinetics of FSH beta mRNA expression were examined immediately after pituitary dispersion and at different times after culturing the cells in plates. FSH beta mRNA levels fell rapidly after dispersion to 8% of initial levels and remained low over 8 h. Thereafter, FSH beta mRNA levels increased slowly and exceeded initial levels by the second day of culture. In a parallel set of experiments, when medium conditioned by exposure to plated cells was applied to the perifusion system, FSH beta mRNA levels were selectively stimulated (6-fold). These data suggest the removal during dispersion and subsequent accumulation in culture of pituitary-derived factors that are important for the maintenance of FSH beta mRNA levels. We conclude that activin plays a greater role in the regulation of FSH beta mRNA levels than was suggested by previous experiments employing static culture systems in which autocrine or paracrine stimulation may have obscured the effects of exogenously added activin.