Astroglial Control of the Antidepressant-Like Effects of Prefrontal Cortex Deep Brain Stimulation.

Although deep brain stimulation (DBS) shows promising efficacy as a therapy for intractable depression, the neurobiological bases underlying its therapeutic action remain largely unknown. The present study was aimed at characterizing the effects of infralimbic prefrontal cortex (IL-PFC) DBS on several pre-clinical markers of the antidepressant-like response and at investigating putative non-neuronal mechanism underlying DBS action. We found that DBS induced an antidepressant-like response that was prevented by IL-PFC neuronal lesion and by adenosine A1 receptor antagonists including caffeine. Moreover, high frequency DBS induced a rapid increase of hippocampal mitosis and reversed the effects of stress on hippocampal synaptic metaplasticity. In addition, DBS increased spontaneous IL-PFC low-frequency oscillations and both raphe 5-HT firing activity and synaptogenesis. Unambiguously, a local glial lesion counteracted all these neurobiological effects of DBS. Further in vivo electrophysiological results revealed that this astrocytic modulation of DBS involved adenosine A1 receptors and K(+) buffering system. Finally, a glial lesion within the site of stimulation failed to counteract the beneficial effects of low frequency (30 Hz) DBS. It is proposed that an unaltered neuronal-glial system constitutes a major prerequisite to optimize antidepressant DBS efficacy. It is also suggested that decreasing frequency could heighten antidepressant response of partial responders.

In vitro and in vivo regulation of synaptogenesis by the novel antidepressant spadin.

BACKGROUND AND PURPOSE: We have described a novel antidepressant peptide, spadin, that acts by blocking the TWIK-related-potassium channel, type 1 (TREK-1). Here, we examined possible mechanisms of action of spadin at both molecular and cellular levels. EXPERIMENTAL APPROACHES: Effects of spadin were measured in primary cultures of neurons or tissues from mice injected i.v. with spadin. Western blots, qPCR, histochemical and electrophysiological techniques were used. KEY RESULTS: In vitro, spadin increased neuronal membrane potential and activated both the MAPK and PI3K signalling pathways, in a time- and concentration-dependent manner. The latter pathway was involved in the protective effect of spadin against staurosporine-induced apoptosis. Also, spadin enhanced both mRNA expression and protein of two markers of synaptogenesis, the post-synaptic density protein of 95 kDalton (PSD-95) and synapsin. We confirmed these effects on synaptogenesis by the observation that spadin treatment significantly increased the proportion of mature spines in cortical neurons. Finally, in vivo injections of spadin led to a rapid increase in both mRNA expression and protein level of brain-derived neurotrophic factor (BDNF) in the hippocampus, confirming the antidepressant action of the peptide. We argue for a new role of spadin in synaptogenesis as both PSD-95 and synapsin mRNA expression and protein levels were further enhanced in the hippocampus, following treatment in vivo with the peptide. CONCLUSIONS AND IMPLICATIONS: These findings provide new mechanisms of action for the rapidly acting antidepressant peptide spadin by stimulating expression of BDNF and synaptic proteins, both in vitro and in vivo.

Targeting two-pore domain K(+) channels TREK-1 and TASK-3 for the treatment of depression: a new therapeutic concept.

Depression is a disease that is particularly frequent, affecting up to 20% of the population in Western countries. The origins of this pathology involve multiple genes as well as environmental and developmental factors leading to a disorder that remains difficult to treat. Several therapies for depression have been developed and these mainly target monoamine neurotransmitters. However, these treatments are not only associated with numerous adverse effects, but they are also ineffective for more than one-third of patients. Therefore, the need to develop new concepts to treat depression is crucial. Recently, studies using knockout mouse models have provided evidence for a crucial role of two members of the two-pore domain potassium channel (K2P ) family, tandem P-domain weak inward rectifying K(+) (TWIK)-related K(+) channel 1 (TREK-1) and TWIK-related acid-sensitive K(+) channel 3 (TASK-3) in the pathophysiology of depression. It is believed that TREK-1 and TASK-3 antagonists could lead to the development of new antidepressants. Herein, we describe the discovery of spadin, a natural peptide released from the maturation of the neurotensin receptor-3 (also known as sortilin), which specifically blocks the activity of the TREK-1 channel and displays particular antidepressant properties, with a rapid onset of action and the absence of adverse effects. The development of such molecules may open a new era in the field of psychiatry.

Spadin as a new antidepressant: absence of TREK-1-related side effects.

Despite several decades of research, current antidepressant (AD) treatments remain of a limited efficacy justifying the need to find new drugs. These drugs have to be more efficacious, more rapid and display lesser side effects. Using rodent models, we recently identified spadin as a new antidepressant molecule that acts more quickly than classical ADs, working within 4 days to get same effects obtained with other ADs after 21 days. Spadin blocks TREK-1 K(2P) potassium channels that are considered as new targets for ADs. Deletion of the TREK-1 channel is known to increase sensitivity to pain, seizures and ischemia. Thus blocking these channels could result in deleterious side effects. In this study we showed that spadin did not interfere with other TREK-1 controlled functions such as pain, epilepsy and ischemia. We also demonstrated that spadin was unable to inhibit currents generated by TREK-2, TRAAK, TASK and TRESK four other K2P channels. More importantly, spadin did not induce cardiac dysfunctions, did not block I(Kr) and I(Ks) and did not modify the systolic pressure or cardiac pulses. After a three week treatment spadin remained an efficacious AD and did not modify the infarct size in brain following focal ischemia. Finally, we showed that kainate induced seizures and glycemia were not modified by spadin treatments. These data, together with those previously published reinforce the idea that spadin represents a good candidate for a new generation of ADs. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Expression of Delta-like protein 4 in the human endometrium.

Activation of Delta-Notch signaling pathway promotes the development of the vascular system in embryo, normal adult tissues, and cancerous lesions. Delta and Notch genes are known to be expressed in endothelial cells, and little is known of their expression beyond the vascular system. The purpose of this study was to investigate whether Delta gene would be expressed in cells of the uterine endometrium. In this study, we found that the human endometrial cells expressed one of the Delta ligands, Delta-like 4 protein (Dll4). Dll4 was expressed in human endometrium in a spatiotemporal fashion. Immunohistochemistry studies showed the cytoplasm as well as membrane staining with apical localization both in the luminal and glandular epithelium and moderate diffuse staining in the cytoplasm of the stromal cells. Western blot analysis showed that the size of the endometrial Dll4 was identical to that in the human umbilical endothelial cells. The expression of Dll4 mRNA in human endometrial cells was quantitatively determined by real-time PCR. Dll4 mRNA expressed in the glandular epithelium showed large variations, and it was significantly elevated in the mid and late proliferative and early secretory endometrium. Endometrial stromal cells contained less Dll4 mRNA and had no clear correlation with the menstrual cycle. The effect of hormones was studied in the primary culture of isolated glandular epithelial and stromal cells. In glandular cells, estradiol had little effect, and medroxyprogesterone acetate significantly reduced the mRNAs compared with that of control. Relaxin induced the Dll4 mRNA. In stromal cells, both estradiol and medroxyprogesterone acetate reduced the Dll4 mRNA. To our knowledge, this is the first report of the expression of Dll4 in the endometrium. We propose that endometrial Dll4 may enhance the development of the endometrial microvascular system and facilitate the implantation of blastocyst in a fertile cycle.

Disparate effects of relaxin and TGFbeta1: relaxin increases, but TGFbeta1 inhibits, the relaxin receptor and the production of IGFBP-1 in human endometrial stromal/decidual cells.

BACKGROUND: The purpose of this study was to determine the effect of progestin, relaxin (RLX) and transforming growth factor beta1 (TGFbeta1) on the content of relaxin receptor (LGR7) mRNA. The effect of RLX on insulin-like growth factor binding protein-1 (IGFBP-1) production was determined to evaluate the biological function of RLX/receptor in human endometrial cells. METHODS AND RESULTS: The levels of LGR7 mRNA and the effect of hormones were determined by real-time PCR in endometrial cells. LGR7 mRNA was found to be relatively abundant in endometrial glands and decidual cells and much less in endometrial stromal cells. In stromal cells, medroxyprogesterone acetate (MPA), or MPA plus RLX, significantly increased the LGR7 mRNA and RLX alone had little effect. In decidual cells, RLX increased LGR7 mRNA in a dose- and time-dependent fashion. TGFbeta1 reduced the LGR7 mRNA. In stromal cells, MPA alone caused a slight increase (2-4-fold) of the production rate of IGFBP-1 whereas MPA plus RLX synergistically increased (>40-fold) the IGFBP-1 production. In decidual cells in which the basal production rate was already approximately 50-fold higher than in stromal cells, RLX alone caused an additional increase (>30-fold) on the production rate. TGFbeta1 inhibited the IGFBP-1 production. CONCLUSION: The present study showed that in undifferentiated endometrial stromal cells, progestin increases the RLX receptor content to enhance the effect of RLX on the target gene (IGFBP-1). In decidual cells, RLX alone up-regulates its receptor, resulting in a large scale induction of IGFBP-1. TGFbeta1 has an inhibitory effect on LGR7 and IGFBP-1.

Ligand activated hPR modulates the glycodelin promoter activity through the Sp1 sites in human endometrial adenocarcinoma cells.

Human endometrium produces glycodelin-A (GdA). The GdA mRNA is highly expressed in progestin-sensitized human endometrial glandular epithelial cells. The mechanism of GdA gene expression, however, is not clear. To understand the cell specific GdA gene transcription, our first approach was to identify the cis-element in the GdA promoter using transfection assay in a human endometrial adenocarcinoma cell line (HEC-1B, a cell line originally derived from the glandular component of the endometrium). The GdA promoter (-1900 to +20 bp) was linked to the luciferase reporter gene to construct p1900Luc, along with two shorter promoter constructs, p1100Luc and p304Luc. Deletion analysis showed that the basal promoter activity was derived from the region between -304 to +20 bp. This region contains three putative Sp1 binding sites (Sp1-1, -243 to -238 bp; Sp1-2, -207 to -202 bp; and Sp1-3, -56 to -49 bp). Mutation analysis at the Sp1 sites showed that p304Spm2Luc and p304Spm3Luc reduced the activity by 80%, while p304Spm1-2-3Luc reduced the activity by 95%. Sp1-1 mutation, however, had no effect. These results showed that two of the three Sp1 cis-elements mediate the basal promoter activity of the GdA gene. Electrophoretic gel mobility shift showed that at least two specific binding proteins in the nuclear extracts of HEC-1B cells bound to the oligo containing Sp1-2 or Sp1-3 cis-element. Sp1 antibody reduced the specific binding complex by 70% suggesting that Sp1 transcription factor regulates GdA gene expression. In addition, over expression of Sp1 increased the promoter activity. To determine whether progestin would modulate the promoter activity, HEC-1B cells were transfected with p304Luc and with progesterone receptor (either hPR-A or hPR-B) expression vector. Medroxyprogesterone acetate increased the promoter activity (3-fold) derived from p304Luc but not from the mutant, p304Spm1-2-3Luc. In contrast, the promoter activity was slightly reduced in cells treated with estradiol and co-transfected with estrogen receptor expression vector. These data indicate that ligand-activated PR stimulates GdA gene expression mediated through the functional Sp1 sites.

Involvement of the neurotensin receptor subtype NTR3 in the growth effect of neurotensin on cancer cell lines.

The expression of the 3 currently known neurotensin receptors was studied in human cancer cells of prostatic, colonic or pancreatic origin by means of RT-PCR analysis and binding experiments. All the cells selected for this work have been shown to exhibit a growth response to neurotensin. We found that the 7 transmembrane domain, levocabastine insensitive receptor (NTR1) is expressed in most but not all of the cells studied whereas the 7 transmembrane domain, levocabastine sensitive receptor (NTR2) is present in none of these cells. The 100 kDa-type I neurotensin receptor (NTR3) is expressed in all the cells assayed. Moreover, we demonstrated that neurotensin can stimulate the growth of CHO cells stably transfected with the NTR3. Taken together, our results strongly suggest that the NTR3 subtype could be involved in the growth response of human cancer cells to neurotensin.

Pharmacological properties of the mouse neurotensin receptor 3. Maintenance of cell surface receptor during internalization of neurotensin.

We recently reported the molecular identification of a new type of receptor for the neuropeptide neurotensin (NT), the neurotensin receptor 3 (NTR3), identical to sortilin, which binds receptor-associated protein. Here, we demonstrate that the cloned mouse NTR3 is expressed on the plasma membrane of transfected COS-7 cells. The mouse NTR3 is detectable by photoaffinity labeling and immunoblotting at the cell surface as a 100 kDa N-glycosylated protein. Biochemical analysis and confocal microscopic imaging clearly indicate that NT is efficiently internalized after binding to NTR3, and that despite this internalization, the amount of receptor present on the cell surface is maintained.

Sortilin/neurotensin receptor-3: a new tool to investigate neurotensin signaling and cellular trafficking?

The identification of gp95sortilin, a sorting protein, as being the 100 kDa neurotensin (NT) receptor, a non-G-protein coupled receptor, constitutes a new and interesting but intriguing step in the neuropeptide signaling as well as in cellular trafficking. The isolation of the same protein by three different experimental approaches sum up the complexity for researchers involved in the functional significance of the so-called sortilin/neurotensin receptor 3 (NTR3). This review will concentrate on the putative physiological and cellular roles of sortilin/NTR3 as most results so far have proposed hypothetical conclusions rather than concrete evidence.

Ligand-activated progesterone receptor isoform hPR-A is a stronger transactivator than hPR-B for the expression of IGFBP-1 (insulin-like growth factor binding protein-1) in human endometrial stromal cells.

In human endometrium, the levels of progesterone receptor (PR) isoforms hPR-A and hPR-B are differentially regulated during the reproductive cycle. Progesterone significantly increases the content of hPR-A, the predominant isoform in decidualized stromal cells (1). The purpose of this study was to determine the capacity of hPR-A and hPR-B to transactivate the progestin-dependent target gene in human endometrial stromal cells. We examined the effect of cotransfection of hPR-A or hPR-B on the expression of the human insulin-like growth factor binding protein-1 (IGFBP-1) in endometrial stromal cells. The primary culture of human endometrial stromal cells was transfected with the hPR-A or hPR-B expression vector and the IGFBP-1 promoter construct p275CAT, which contains two functional progesterone response elements (PRE1 and PRE2) in decidualized stromal cells. Medroxyprogesterone acetate (MPA) increased the promoter activities ranging from 1.2- to 27-fold in cells cotransfected with hPR-A or hPR-B in eight endometrial specimens. The promoter activity increased by the hPR-A was significantly higher than hPR-B (15 +/- 8 vs. 4 +/- 2, mean +/- SD; n = 8, P < 0.005). Site-specific mutation showed that the induced activity by hPR-A was mediated through the PRE1 and PRE2 sites. Addition of hPR-B reduced the effect of hPR-A. The high transactivation capacity of hPR-A was also activated by other ligands, progesterone, Org 2058, and norethindrone. These observations indicate that hPR-A is a stronger transactivator than hPR-B for the IGFBP-1 promoter in endometrial stromal cells. Previous studies have shown the progestin-dependent production of IGFBP-1 correlates with its mRNA levels and transcription rate. Thus, we have determined the effect of hPR-A and hPR-B on the production of IGFBP-1 in stromal cells treated with MPA. The production rate in cells uniformly infected with AdPRA (recombinant Ad5-directed PR expression system) was significantly higher (P < 0.001) than the rate in uninfected cells and in cells infected with AdPRB or AdCMV (the Ad5 viral expression vector). This result, in concert with the promoter analysis, provides evidence that hPR-A is a strong inducer for the chromosomal IGFBP-1 gene in endometrial stromal cells.

Dopamine and morphine stimulate nitric oxide release in human endometrial glandular epithelial cells.

OBJECTIVE: Previous studies have shown that human endometrial glandular epithelial cells contain endothelial nitric oxide synthase indicating that the endometrium might produce nitric oxide (NO). We conducted this study to identify stimuli that can activate a transient NO release from endometrial glandular epithelial cells because NO is an important intracellular and intercellular signal transduction pathway in reproductive cycle. METHODS: Endometrial glandular epithelial cells, free of endothelial cells, were isolated from human endometrial specimens and maintained viable in RPMI 1640 medium with 2% fetal bovine serum for 2-4 days. Nitric oxide release from the glandular cells in response to stimuli was monitored continuously amperometrically. RESULTS: Among the substances examined, we found that dopamine and morphine stimulated a transient surge of NO production that was dose-dependent, whereas estrogen, progesterone, or relaxin (RLX) had no short-term effect on NO release. Cells treated with RLX or dopamine for 4 days enhanced the dopamine-induced NO release fourfold to sixfold, with the peak of the NO surge shifting from 35 to 15 seconds. CONCLUSION: Endometrial glandular cells were capable of producing NO. Dopamine and morphine were potent stimuli for a transient surge of NO release from endometrial glandular cells. Furthermore, prolonged exposure to dopamine or RLX enhanced the sensitivity of NO release in endometrial glands.

Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor.

The neuropeptide neurotensin (NT) is known to be internalized in a receptor-mediated fashion into its target cells. To gain insight into the mechanisms underlying this process, we monitored in parallel the migration of the NT1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalization was prevented by hypertonic sucrose, potassium depletion and cytosol acidification, demonstrating that it proceeded via clathrin-coated pits. Within 0-30 minutes, fluo-NT accumulated together with its receptor in Acridine Orange-positive, acidic organelles. These organelles concentrated transferrin and immunostained positively for rab 5A, therefore they were early endosomes. After 30-45 minutes, the ligand and its receptor no longer colocalized. Fluo-NT was first found in rab 7-positive late endosomes and later in a nonacidic juxtanuclear compartment identified as the Trans-Golgi Network (TGN) by virtue of its staining for syntaxin 6. This juxtanuclear compartment also stained positively for rab 7 and for the TGN/pericentriolar recycling endosome marker rab 11, suggesting that the ligand could have been recruited to the TGN from either late or recycling endosomes. By that time, internalized receptors were detected in Lamp-1-immunoreactive lysosomes. These results demonstrate that neurotensin/NT1 receptor complexes follow a recycling cycle that is unique among the G protein-coupled receptors studied to date, and provide the first evidence for the targeting of a nonendogenous protein from endosomes to the TGN.

Somatostatin-induced regulation of SST(2A) receptor expression and cellsurface availability in central neurons: role of receptor internalization.

To investigate the effects of somatostatin (somatotropin release-inhibiting factor, SRIF) on the regulation of SST(2A) receptors in mammalian brain, we examined how blockade of SRIF release or stimulation by the SRIF analog [d-Trp(8)]-SRIF would affect the expression and cell surface availability of SST(2A) receptors in rat brain slices. First, we measured the intensity of SST(2A) immunoreactivity, using quantitative light microscopic immunocytochemistry, and levels of SST(2A) mRNA, using semiquantitative RT-PCR, under conditions of acute SRIF release blockade. Incubation of slices from the claustrum or basolateral amygdala, two regions previously shown to contain high concentrations of SST(2A) receptors, in Ca(2+)-free Ringer's for 40 min induced a decrease in the intensity of SST(2A) receptor immunoreactivity and concentration of SST(2A) mRNA as compared with control values obtained in Ca(2+)-supplemented Ringer's. These effects were counteracted in a dose-dependent manner by the addition of 10-100 nm [d-Trp(8)]-SRIF to the Ca(2+)-free medium. Furthermore, both of these effects were abolished in the presence of the endocytosis inhibitors phenylarsine oxide or hyperosmolar sucrose, suggesting that they were dependent on receptor internalization. Electron microscopic immunogold labeling confirmed the existence of an agonist-induced internalization of SST(2A) receptors in central neurons. At a high (10 microm), but not at a low (10 nm), concentration of agonist this internalization resulted in a significant decrease in cell surface receptor density, irrespective of the presence of Ca(2+) in the medium. Taken together, these results suggest that ligand-induced endocytosis is responsible for rapid transcriptional (increase in SST(2A) expression) and trafficking (loss of cell surface receptors) events involved in the control of the somatostatinergic signal.

Partial characterization of the CCAAT box in the promoter of the hLGFBP-1 gene: interaction with negatively acting transcription factors in decidualized human endometrial stromal cells.

The CCAAT cis-element and its adjacent DNA sequence (-82 to -52 bp) in the human insulin-like growth factor binding protein-1 gene (IGFBP-1) promoter are active in both decidualized human endometrial stromal cells and HepG2 cells. In HepG2 cells, CCAAT activity is mediated by interacting with hepatocyte nuclear factor, HNF-1. In endometrial cells, this region is protected by the nuclear extracts of endometrial decidual cells, however, the transactivator which interacts with the region has not been identified. This study was carried out to characterize and identify the stromal/decidual nuclear proteins that interact with the IGFBP-1 CCAAT motif. Gel shift analysis showed that the CCAAT motif (-82 to -52 bp) formed three specific complexes (CI, CII, and CIII) by extracts from human endometrial decidual or stromal cells. The intensity of CIII formed by the nuclear extracts of decidual cells was less compared to that formed by stromal cells whereas CI/CII was found to be opposite. To evaluate the transcription factors that bind to this region, a number of known CCAAT binding proteins were tested. Among them, the CCAAT binding proteins NF-Y (alpha2(1) collagen promoter CCAAT binding protein) and CBF (hsp70 promoter CCAAT binding protein), were characterized by the gel shift assay. The NF-Y consensus binding sequence (the alpha2(1) collagen promoter) and NF-YA,B antibody abolished or shifted CIII. Although the CBF consensus binding sequence (the hsp70 promoter) eliminated all three complexes, the antibody to CBF had no effect on all three complexes. The nuclear extracts of the endometrial stromal/decidual cells did not form a band corresponding to the HNF-1/CCAAT complex. These results indicate that the CCAAT motif binds to NF-Y and the CI/CII binding protein (remains to be identified) but not HNF-1 in endometrium. Systematic mutation in the CCAAT motif showed that NF-Y(CIII binding protein) bound to the 12 bp sequence GGCGCTGCCAAT(-79 to -68 bp) and the CI/CII binding protein bound to 9 bp, TGCCAATCA(-74 to -66 bp). These findings indicate that the CCAAT motif is a composite element. The CCAAT mediated function was analyzed in decidualized endometrial stromal cells. Mutations in the CCAAT motif increased the promoter activity. The maximum activity was found in mutants which abolished the NF-Y complex. The CCAAT core sequence mutants in which both CIII and CI/CII were abolished, also increased the promoter activity. Results indicated that NF-Y and the CI/CII binding protein, yet to be identified, interact with the composite CCAAT element in the IGFBP-1 promoter to repress the promoter activity in endometrial decidual cells.

Intracellular dynamics of sst5 receptors in transfected COS-7 cells: maintenance of cell surface receptors during ligand-induced endocytosis.

Internalization of G protein-coupled receptors is crucial for resensitization of phosphorylation-desensitized receptors, but also for their long term desensitization through sequestration. To elucidate the mechanisms regulating cell surface availability of the somatostatin (SRIF) receptor subtype sst5, we characterized its internalization properties in transfected COS-7 cells using biochemical, confocal microscopic, and electron microscopic techniques. Our results demonstrated rapid and efficient sequestration of specifically bound [125I]Tyr0-D-Trp8-SRIF (up to 45% of bound radioactivity). Combined immunocytochemical detection of sst5 and visualization of a fluorescent SRIF analog by confocal microscopy revealed that whereas the internalized ligand progressively clustered toward the cell center with time, immunoreactive receptors remained predominantly associated with the plasma membrane. The preservation of cell surface receptors was confirmed by binding experiments on whole cells revealing a lack of saturability of [125I]Tyr0-D-Trp8-SRIF binding at 37 C. Binding was rendered saturable by the drug monensin, showing that receptor recycling played a key role in the preservation of cell surface receptors. Electron microscopy demonstrated that in addition to receptor recycling, internalization of receptor-ligand complexes triggered a massive recruitment of sst5 receptor molecules from intracellular stores to the membrane. This combination of recycling and recruitment of spare receptors may protect sst5 from long term down-regulation through sequestration and, therefore, facilitate extended SRIF signaling.

Activation of the insulin-like growth factor binding protein-1 promoter by progesterone receptor in decidualized human endometrial stromal cells.

Insulin-like growth factor binding protein-1 (IGFBP-1) is induced extensively when human endometrial stromal cells are decidualized by progestin and relaxin in a long-term primary culture system. The purpose of this study is to investigate whether progesterone receptor (PR) directly activates the IGFBP-1 gene promoter. In decidualized stromal cells, activity of the IGFBP-1 promoter (from -1.2 kb to +68 bp) containing putative progesterone-response elements (PREs) was increased 80-fold. Mutation of either 5' or 3' half-site of the putative PRE1 site (from -193 to -179 bp) reduced the promoter activity. Mutations that converted PRE1 closer to consensus PRE increased the promoter activity. In undifferentiated stromal cells, mutations of PRE sites had no effect on the promoter activity. When a PR expression vector (hPR1) was cotransfected, progestin increased promoter activity derived from p275CAT but not from p1.2CAT, suggesting that the function of PRE1 was repressed by the region from -1.2 kb to -275 bp in the promoter. Progestin did not increase promoter activity derived from p275CAT without cotransfection of hPR1, suggesting that endogenous PR alone is insufficient to activate PRE1. In summary, results indicate that the PRE1 site of the IGFBP-1 promoter mediates a direct activation of PR on transcription specifically in decidualized stromal cells.

Prolactin and its receptor in human endometrium.

Synthesis of prolactin (PRL) in human endometrium extends from the late luteal phase of the menstrual cycle throughout the pregnancy. We have studied the hormonal requirements for the sustained production of PRL and its receptor (PRL-R) in a long-term primary cell culture system. Progestin stimulates the production PRL and its receptor when stromal cells transform into decidual cells. The rise in PRL production rate correlates with an increase in steady-state PRL mRNA levels which are caused by increased transcription rate gene. Replacing progestin by the antiprogestin, RU 486, causes a transient superinduction of PRL production followed by reduction to basal level of expression. On the other hand, RU 486 exerts immediate inhibition of PRL-receptor mRNA expression. In addition, relaxin (RLX) enhances PRL synthesis. The transcription of the PRL gene in endometrium is dependent upon the promotor 6-kb upstream of the transcription start site in the pituitary. That biological functions of PRL and its receptor are critical to implantation and the maintenance of pregnancy is suggested by the impaired fertility of PRL and PRL-R knockout mice. PRL enhances endometrial cell growth at low concentrations and inhibits it at high concentrations. This dual action indicates an autocrine action of PRL-R-mediated signaling transduction pathways during reproductive cycles and pregnancy. During gestation, decidual-derived prolactin regulates the volume of amniotic and fetal extracellular fluid and electrolytes.

Neurotensin and neurotensin receptors.

Neurotensin is a brain and gastrointestinal peptide that fulfils many central and peripheral functions through its interaction with specific receptors. Three subtypes of neurotensin receptors have been cloned. Two of them belong to the family of G protein-coupled receptors, whereas the third one is an entirely new type of neuropeptide receptor and is identical to gp95/sortilin, a 100 kDa-protein with a single transmembrane domain. In this review, the present knowledge regarding the molecular and pharmacological properties of the three cloned neurotensin receptors is summarized and the relationship between these receptors and the known pharmacological effects of neurotensin is discussed.

Receptor-mediated internalization is critical for the inhibition of the expression of growth hormone by somatostatin in the pituitary cell line AtT-20.

The inhibitory effect of the neuropeptide somatostatin on the expression of growth hormone was measured by quantitative polymerase chain reaction in the pituitary cell line AtT-20. We demonstrate that this effect is dependent on the internalization of somatostatin-receptor complexes and that it is totally independent from the peptide-induced inhibition of adenylate cyclase. Indeed, the inhibitory effect of the peptide on growth hormone mRNA levels was totally insensitive to pertussis toxin treatment but was totally abolished under conditions which block somatostatin receptor internalization. Comparative confocal microscopic imaging of fluorescent somatostatin sequestration and fluorescence immunolabeling of sst1, sst2A, and sst5 receptors suggests that sst2A is most probably responsible of the inhibitory effect of somatostatin on growth hormone expression.