Proopiomelanocortin Gene Transcription Research Articles

Glucocorticoid receptor binding to a specific DNA sequence is required for hormone-dependent repression of pro-opiomelanocortin gene transcription.

Glucocorticoids rapidly and specifically inhibit transcription of the pro-opiomelanocortin (POMC) gene in the anterior pituitary, thus offering a model for studying negative control of transcription in mammals. We have defined an element within the rat POMC gene 5'-flanking region that is required for glucocorticoid inhibition of POMC gene transcription in POMC-expressing pituitary tumor cells (AtT-20). This element contains an in vitro binding site for purified glucocorticoid receptor. Site-directed mutagenesis revealed that binding of the receptor to this site located at position base pair -63 is essential for glucocorticoid repression of transcription. Although related to the well-defined glucocorticoid response element (GRE) found in glucocorticoid-inducible genes, the DNA sequence of the POMC negative glucocorticoid response element (nGRE) differs significantly from the GRE consensus; this sequence divergence may result in different receptor-DNA interactions and may account at least in part for the opposite transcriptional properties of these elements. Hormone-dependent repression of POMC gene transcription may be due to binding of the receptor over a positive regulatory element of the promoter. Thus, repression may result from mutually exclusive binding of two DNA-binding proteins to overlapping DNA sequences.

Glucocorticoid receptor binding to a specific DNA sequence is required for hormone-dependent repression of pro-opiomelanocortin gene transcription.

Glucocorticoids rapidly and specifically inhibit transcription of the pro-opiomelanocortin (POMC) gene in the anterior pituitary, thus offering a model for studying negative control of transcription in mammals. We have defined an element within the rat POMC gene 5'-flanking region that is required for glucocorticoid inhibition of POMC gene transcription in POMC-expressing pituitary tumor cells (AtT-20). This element contains an in vitro binding site for purified glucocorticoid receptor. Site-directed mutagenesis revealed that binding of the receptor to this site located at position base pair -63 is essential for glucocorticoid repression of transcription. Although related to the well-defined glucocorticoid response element (GRE) found in glucocorticoid-inducible genes, the DNA sequence of the POMC negative glucocorticoid response element (nGRE) differs significantly from the GRE consensus; this sequence divergence may result in different receptor-DNA interactions and may account at least in part for the opposite transcriptional properties of these elements. Hormone-dependent repression of POMC gene transcription may be due to binding of the receptor over a positive regulatory element of the promoter. Thus, repression may result from mutually exclusive binding of two DNA-binding proteins to overlapping DNA sequences.

Effect of ethane dimethane sulphonate on proopiomelanocortin (POMC) mRNA and POMC-derived peptides in the rat testis

Recent studies have shown that both proopiomelanocortin (POMC)-derived peptides and a range of POMC gene transcripts are present in the testis. Previous immunocytochemical studies have reported immunoreactive (ir)-β-endorphin (EP) and ir-ACTH to be localized in the Leydig cells, and ir-NacEP in spermatogonia and primary spermatocytes. In the present study, we have further examined the hypothesis that testicular Leydig cells are the principal site of synthesis of these peptides, by determining the effects of the administration of the cytotoxic drug ethane dimethane sulphonate (EDS) which selectively destroys the Leydig cells of the testis. As expected, serum testosterone levels fell and serum FSH/LH levels increased within 3 days of EDS administration, returning to normal levels 4–8 weeks later. In contrast, the testicular content of POMC-derived peptides and POMC mRNA levels in these animals was not significantly altered throughout the experimental period. In addition, POMC mRNA was not detected in a purified Leydig cell preparation derived from adult male rats, and POMC-derived peptides were also undetectable in the media of a similar preparation following cell culture. These data suggest that in the adult the predominant site of rat POMC gene expression is in testicular interstitial cells other than Leydig cells.

Pituitary-specific expression and glucocorticoid regulation of a proopiomelanocortin fusion gene in transgenic mice.

The product of a single gene encoding proopiomelanocortin (POMC) is differentially processed to produce corticotropin and alpha-melanotropin in anterior and intermediate pituitary cells, respectively. Hormonal control of POMC gene transcription and of corticotropin or alpha-melanotropin release is also tissue-specific; for example, glucocorticoids specifically inhibit anterior but not intermediate pituitary POMC transcription. Outside the pituitary gland, very low levels of POMC mRNAs are present in brain, testes, ovaries, and placenta. We have used transgenic mice to identify POMC 5' flanking sequences that are sufficient for tissue-specific expression and glucocorticoid regulation in anterior and intermediate pituitary cells. Three lines of transgenic mice were established, each carrying 50-75 copies (per cell) of a chimeric rPOMCneo gene constituted of rat POMC promoter sequences and of bacterial neomycin-resistance coding sequence. High levels of rPOMCneo transcripts were detected in pituitaries of mice from all three lineages. In situ hybridization revealed that the ratio of intermediate to anterior pituitary transcripts was similar for the transgene and endogenous POMC mRNA. rPOMCneo transcripts were not detected in any other tissue except at very low levels in the testes in two transgenic lines. Endogenous mouse POMC mRNA increased in response to depletion of plasma glucocorticoids (adrenalectomy) and decreased after glucocorticoid treatment; rPOMCneo transcripts were altered to the same extent by these treatments in all three lines. Intermediate pituitary and testicular rPOMCneo transgene expression was not altered by these treatments. Thus, no more than 769 base pairs of the rat POMC promoter are required for pituitary-specific expression and for specific glucocorticoid inhibition of the POMC gene in the anterior pituitary.

Human lymphocytes produce pro-opiomelanocortin gene-related transcripts. Effects of lymphotropic viruses.

Expression of the pro-opiomelanocortin (POMC) gene was examined in normal human lymphocytes and lymphocyte cell lines infected by lymphotropic viruses. POMC gene transcripts were detected in human lymphocytes using stringent RNA-RNA hybridizations. Low transcript levels were found in normal phytohemagglutinin-stimulated peripheral blood mononuclear cells and in tonsillar T and B cells. The highest levels were found in cells infected with Epstein-Barr virus (EBV). However, T cell lines infected with human T lymphotropic viruses did not have increased levels of transcripts. The transcript levels in an EBV-transformed B lymphocyte line were not affected by dexamethasone or corticotropin-releasing hormone, known regulators of anterior pituitary POMC gene expression. Therefore, it is possible that EBV infections could result in abnormal POMC expression.

Complex transcriptional regulation by glucocorticoids and corticotropin-releasing hormone of proopiomelanocortin gene expression in rat pituitary cultures.

Proopiomelanocortin (POMC) peptide secretion from rat anterior pituitary corticotrophs and intermediate pituitary melanotrophs is stimulated by corticotropin-releasing hormone (CRH). CRH-stimulated secretion in the corticotrophs is inhibited by glucocorticoids in a complex fashion, involving both a fast, direct blockade of POMC secretion (minutes to hours) and a longer inhibitory action (hours to days) that decreases the amount of POMC peptide available for release. The current studies tested the ability of CRH to stimulate beta-endorphin (a peptide derived from POMC) secretion and POMC gene transcription in cultured anterior and neurointermediate lobe pituitary cells, and examined interactions between CRH and glucocorticoids in regulating POMC gene expression using an in vitro nuclear transcription run-on assay. In both tissues, CRH elicited a time-dependent stimulation of POMC gene transcription that was maximal at 60 min and remained elevated for at least 18 hr. Glucocorticoids rapidly inhibited POMC gene transcription fourfold in the anterior lobe with maximal effects within 20 min. Glucocorticoids also blocked CRH-stimulated POMC gene transcription in anterior pituitary cultures in a temporal manner paralleling their inhibitory effects on CRH-stimulated beta-endorphin secretion. In neurointermediate lobe cultures, the effects of glucocorticoids and CRH on POMC gene transcription were qualitatively similar to, but of lesser magnitude than those observed in the anterior lobe. These studies indicate that the regulation of POMC gene transcription by glucocorticoids and CRH is complex and that the two modulators do not function independently.

Tissue-specific regulation of pituitary proopiomelanocortin gene transcription by corticotropin-releasing hormone, 3',5'-cyclic adenosine monophosphate, and glucocorticoids.

The release of pituitary hormones derived from POMC is under multihormonal and tissue-specific control in the anterior and intermediate lobes of the pituitary where the same single-copy POMC gene is expressed. In order to assess the tissue-specificity of POMC regulation at the gene level, we have previously shown that glucocorticoids inhibit POMC gene transcription in the anterior but not in the intermediate pituitary. In the present work, we have investigated the role of corticotropin-releasing hormone (CRH) and cAMP in the differential regulation of anterior and intermediate pituitary POMC gene transcription. Using pituitary cells in primary culture and nuclear run-on transcription assays, we found that cAMP increases POMC gene transcription rate to the same extent in both anterior and intermediate pituitary cells while CRH only increases anterior pituitary POMC transcription rate. This observation contrasts with the stimulation of ACTH and alpha MSH release from anterior and intermediate pituitary cells, respectively, by both CRH and cAMP. In the anterior pituitary, both CRH and cAMP stimulated as well as basal POMC transcription rates are inhibited by glucocorticoids. In the anterior pituitary, both CRH stimulation and glucocorticoid inhibition of POMC transcription are rapid and do not require de novo protein synthesis. Thus, we report that transcription of the POMC gene is differentially regulated by CRH, cAMP, and glucocorticoids in anterior and intermediate pituitary tissues, in much the same way as the control of POMC processing and release.

Regulation of pro-opiomelanocortin gene transcription in individual cell nuclei.

A nonrepetitive complementary RNA probe specific for an intervening sequence of the rat pro-opiomelanocortin (POMC) gene primary transcript was used to analyze the hormonal regulation of POMC gene transcription in individual cell nuclei in the rat pituitary by in situ hybridization. This probe recognized only full-length POMC heterogeneous nuclear RNA, as verified by Northern blots of pituitary RNA. When pituitary sections were hybridized with this 3H-labeled POMC intron A probe, silver grains were predominantly localized over the nuclei of cells that expressed POMC in the anterior and intermediate lobes. Adrenalectomy increased both the average grain density over corticotroph nuclei and the number of cells in the anterior pituitary with significant numbers of silver grains over their nucleus. Dexamethasone administration to intact or adrenalectomized rats results in the rapid (within 30 minutes) disappearance of silver grains over the nuclei of corticotrophs in the anterior lobe, suggesting that POMC gene transcription had been inhibited. However, adrenalectomy or dexamethasone administration did not alter the silver grain density over nuclei of intermediate lobe melanotrophs. Thus, this in situ hybridization assay utilizing an intervening sequence-specific POMC probe can measure rapid physiological changes in POMC heterogeneous nuclear RNA in individual cell nuclei.

A Diurnal Rhythm in Proopiomelanocortin Messenger Ribonucleic Acid that Varies Concomitantly with the Content and Secretion ofβ-Endorphin in the Intermediate Lobe of the Rat Pituitary*

The present study investigated whether diurnal variations in the secretion of alpha MSH and beta-endorphin from the intermediate lobe (IL) of the pituitary are associated with parallel changes in the synthesis of mRNA specifically encoding proopiomelanocortin (POMC). The results demonstrate that concomitant diurnal variations occur in both plasma and IL concentrations of immunoreactive beta-endorphin and alpha MSH. Plasma and IL peptide levels were relatively constant during daylight hours (0600-1800 h), but increased after the onset of darkness and reached maximal concentrations at 0200 h. To examine the possibility that this diurnal rhythm in the content and secretion of POMC-derived peptides resulted from diurnal changes in the biosynthesis of POMC, the concentration and rate of synthesis of POMC mRNA were examined. POMC mRNA levels were elevated during the dark period, reaching a maximum level at 0200 h that was 2-fold higher than that occurring during the light period. POMC mRNA synthesis, determined by measuring the number of RNA polymerase II complexes transcribing the POMC gene in isolated cell nuclei, also varied diurnally, with maximum transcription rates occurring at 1800 h, thus preceding maximal increases in POMC mRNA content and POMC peptide secretion by 8 h. Together, these data indicate that diurnal variations in the content and secretion of POMC-derived peptides are associated with parallel changes in POMC mRNA concentrations and are preceded by similar changes in POMC gene transcription.

DNA sequence element responsible for inhibition of pro-opiomelanocortin gene transcription by glucocorticoids

DNA sequence element responsible for inhibition of pro-opiomelanocortin gene transcription by glucocorticoids

Opposite regulation of pro-opiomelanocortin gene transcription by glucocorticoids and CRH

Pro-opiomelanocortin (POMC) is the pituitary precursor for adrenocorticotropin (ACTH), β-endorphin, β-lipotropin and the melanotropins. The level of ACTH secretion from the anterior pituitary is largely determined by the competing action of the stimulatory hypothalamic hormone, corticoliberin (corticotropin-releasing hormone, CRC), and the inhibitory effect of glucocorticoids. We now demonstrate that these two hormones, glucocorticoids and CRH, also inhibit and stimulate, respectively, the transcription rate of the POMC gene as measured by nuclear run-on transcription assays. Indeed, we show both by in vivo treatment and with rat anterior pituitary cells in primary culture that glucocorticoids inhibit within 30 min transcription of the POMC gene. Similarly, we find that CRH stimulates POMC gene transcription within 15 min. CRH and glucocorticoids can compete with each other to set the rate of POMC transcription. Our results indicate that CRH and glucocorticoids regulate anterior pituitary POMC gene transcription in addition to their well-documented role in the control of POMC peptide release.

Glucocorticoid regulation of pro-opiomelanocortin gene transcription in the rat pituitary.

The effect of glucocorticoids on pro-opiomelanocortin (POMC) gene transcription in the rat pituitary has been investigated by an in vitro nuclear runoff transcription assay. Both dexamethasone and corticosterone had rapid inhibitory effects on POMC transcription in the anterior lobe but not in the intermediate lobe of the pituitary. These effects were maximal during the first hour after injection of the steroid. Bilateral adrenalectomy had a time-dependent stimulatory effect on anterior lobe POMC transcription, presumably because of the removal of endogenous glucocorticoids. This study shows that the well documented inhibitory effects of glucocorticoids on POMC mRNA in the pituitary anterior lobe are due at least in part to an inhibition in POMC mRNA synthesis.

Corticotropin-releasing factor regulates proopiomelanocortin messenger ribonucleic acid levels in vivo.

Ovine corticotropin-releasing factor (oCRF) was administered to male rats for 3-15 days. Continuous intravenous infusion, repeated subcutaneous, and intracerebroventricular injections of oCRF resulted in a 50-100% increase in messenger ribonucleic acid (mRNA) coding for proopiomelanocortin (POMC) in the anterior pituitaries (AP) of these animals. The synthetic glucocorticoid, dexamethasone, reversed the actions of oCRF. None of the above treatments altered POMC mRNA content in the neurointermediate lobe. Bilateral adrenalectomy enhanced POMC mRNA levels 4.8-fold. To determine whether this effect was dependent on increased hypothalamic production and release of CRF, a bilateral ablation of the paraventricular nucleus (PVN), known to contain CRF neurons directly involved in ACTH secretion, was performed. 6 days after the PVN destruction a parallel 75% reduction of both stalk-median eminence CRF-like immunoreactivity and plasma ACTH had occurred. The AP content of POMC mRNA decreased by 53%, while the ACTH content increased by 56%. These observations suggest that CRF regulates the biosynthesis of POMC-derived polypeptide products in the AP, while the peptide does not seem to be directly involved in intermediate lobe POMC biosynthesis. The reduction of POMC mRNA levels following PVN destruction demonstrates that CRF and other substances located in the nucleus may cause the increased POMC gene transcription following adrenalectomy.