Transcriptional Regulation of Pituitary POMC Is Conserved at the Vertebrate Extremes Despite Great Promoter Sequence Divergence (original) (raw)
2007, Molecular Endocrinology
The stress response involves complex physiological mechanisms that maximize behavioral efficacy during attack or defense and is highly conserved in all vertebrates. Key mediators of the stress response are pituitary hormones encoded by the proopiomelanocortin gene (POMC). Despite conservation of physiological function and expression pattern of POMC in all vertebrates, phylogenetic footprinting analyses at the POMC locus across vertebrates failed to detect conserved noncoding sequences with potential regulatory function. To investigate whether ortholog POMC promoters from extremely distant vertebrates are functionally conserved, we used 5-flanking sequences of the teleost fish Tetraodon nigroviridis POMC␣ gene to produce transgenic mice. Tetraodon POMC␣ promoter targeted reporter gene expression exclusively to mouse pituitary cells that normally express Pomc. Importantly, transgenic expression in mouse corticotrophs was increased after adrenalectomy. To understand how conservation of precise gene expression mechanisms coexists with great sequence divergence, we investigated whether very short elements are still conserved in all vertebrate POMC promoters. Multiple local sequence alignments that consider phylogenetic relationships of ortholog regions identified a unique 10-bp motif GTGCTAA(T/G)CC that is usually present in two copies in POMC 5-flanking sequences of all vertebrates. Underlined nucleotides represent totally conserved sequences. Deletion of these paired motifs from Tetraodon POMC␣ promoter markedly reduced its transcriptional activity in a mouse corticotropic cell line and in pituitary POMC cells of transgenic mice. In mammals, the conserved motifs correspond to reported binding sites for pituitary-specific nuclear proteins that participate in POMC transcriptional regulation. Together, these results demonstrate that mechanisms that participate in pituitary-specific and hormonally regulated expression of POMC have been preserved since mammals and teleosts diverged from a common ancestor 450 million years ago despite great promoter sequence divergence. (Molecular Endocrinology 21: [2738][2739][2740][2741][2742][2743][2744][2745][2746][2747][2748][2749] 2007) T HE PROOPIOMELANOCORTIN GENE (POMC) encodes a prohormone that gives rise to several bioactive peptides including ACTH, the melanocortins ␣-, -, and ␥-MSH, and the opioid peptide -endor-phin. POMC is mainly transcribed in discrete populations of brain neurons and in the pituitary gland, where it is expressed in melanotrophs of the intermediate lobe and corticotrophs of the anterior lobe. ACTH is a principal component of the hypothalamic-pituitary-adrenal (HPA) axis that mediates the stress response in all vertebrates (1-3). Stress-induced release of the hypothalamic peptide CRH stimulates the release of ACTH from pituitary corticotrophs, which in turn promotes glucocorticoid release from the adrenal gland cortex. As part of a negative feedback loop, glucocorticoids decrease the expression of CRH in the hypothalamus and POMC in pituitary corticotrophs. The crucial importance of ACTH in the stress response can be appreciated in humans carrying mutations that inactivate the POMC gene, who die from adrenal insufficiency unless permanently supplemented with glu-
Sign up to get access to over 50M papers
Sign up for access to the world's latest research
Related papers
DNA, 1987
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 ßendorphin (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 CRHstimulated j-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.
Proceedings of the National Academy of Sciences, 1988
The product of a single gene encoding proopiomelanocortin (POMC) is differentially processed to produce corticotropin and a-melanotropin in anterior and intermediate pituitary cells, respectively. Hormonal control of POMC gene transcription and of corticotropin or amelanotropin 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 tissuespecific 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. The gene encoding proopiomelanocortin (POMC) is expressed predominantly in the pituitary gland. In addition, POMC transcripts are detected in the hypothalamus, brain, testes, ovaries, and placenta (1-9). Comparison of POMC genomic sequences with the structure of pituitary and extrapituitary POMC transcripts reveals a differential pattern of transcription. Pituitary POMC mRNA is produced by splicing of three exons, whereas testicular transcripts are shorter, do not contain exon 1 and 2 sequences, and appear to be transcribed from a site within the third exon (8, 9). In the testes and in other tissues where these shorter transcripts are found they are at least 100 times less abundant than POMC mRNA in the pituitary. Both pituitary and extrapituitary transcripts are likely transcribed from the same genomic sequences, since there is no evidence for multiple POMC genes (10). Within the pituitary itself, control of POMC
Molecular Endocrinology, 1988
We have verified the possibility that the POMC gene of the rat hypothalamus might be subject to regulation by glucocorticoids. Adrenalectomy increased the concentration of POMC mRNA in anterior pituitary and in hypothalamus, but not in the neurointermediate lobe of the pituitary gland. Dexamethasone and, to a slightly lesser extent, corticosterone treatments reversed the adrenalectomy-induced increase in POMC mRNA concentrations in both anterior pituitary and hypothalamus. Dexamethasone caused a slight decrease of POMC mRNA levels in the neurointermediate lobe of the pituitary gland, while corticosterone had no effect. These results indicate that the POMC gene of the rat brain hypothalamus is also under negative control by glucocorticoids. (Molecular Endocrinology 2: 727-731, 1988) of the hypothalamus by ACTH and /3-end-containing terminals (14) and of the stimulatory role of opioid peptides in the control of the secretion of CRF (15). Recent immunohistochemical studies have also demonstrated the presence of glucocorticoid receptor immunoreactivity in numerous monoaminergic neurons including those of the arcuate nucleus (16). Finally, it is of primordial importance to gain a better understanding of the limbic-hypothalamic mechanisms regulating ACTH secretion, particularly in view of the ACTH and CRF hypersecretion observed in patients suffering from endogenous depressive illness (17-19) and of the abnormal hypothalamic-pituitary-adrenal function in anorexia nervosa (20). Our results demonstrate a negative regulation of hypothalamic POMC gene expression by glucocorticoids.
Regulation of Hypothalamic Corticotropin-Releasing Hormone Transcription by Elevated Glucocorticoids
Molecular Endocrinology, 2013
Negative glucocorticoid feedback is essential for preventing the deleterious effects of excessive hypothalamic pituitary adrenal axis axis activation, with an important target being CRH transcription in the hypothalamic paraventricular nucleus. The aim of these studies was to determine whether glucocorticoids repress CRH transcription directly in CRH neurons, by examining glucocorticoid effects on glucocorticoid receptor (GR)-CRH promoter interaction and the activation of proteins required for CRH transcription. Immunoprecipitation of hypothalamic chromatin from intact or adrenalectomized rats subjected to either stress or corticosterone injections showed minor association of the proximal CRH promoter with the GR compared with that with phospho-CREB (pCREB). In contrast, the Period-1 (Per1, a glucocorticoid-responsive gene) promoter markedly recruited GR. Stress increased pCREB recruitment by the CRH but not the Per1 promoter, irrespective of circulating glucocorticoids. In vitro, corticosterone pretreatment (30 minutes or 18 hours) only slightly inhibited basal and forskolin-stimulated CRH heteronuclear RNA in primary hypothalamic neuronal cultures and CRH promoter activity in hypothalamic 4B cells. In 4B cells, 30 minutes or 18 hours of corticosterone exposure had no effect on forskolin-induced nuclear accumulation of the recognized CRH transcriptional regulators, pCREB and transducer of regulated CREB activity 2. The data show that inhibition of CRH transcription by physiological glucocorticoids in vitro is minor and that direct interaction of GR with DNA in the proximal CRH promoter may not be a major mechanism of CRH gene repression. Although GR interaction with distal promoter elements may have a role, the data suggest that transcriptional repression of CRH by glucocorticoids involves protein-protein interactions and/or modulation of afferent inputs to the hypothalamic paraventricular nucleus. (Molecular Endocrinology 27: 1796 -1807, 2013) N ormal activity of the hypothalamic-pituitary-adrenal (HPA) axis leading to adrenal glucocorticoid production is essential for homeostasis and for survival during severe stress situations. Activation of the HPA axis is initiated by release of CRH produced in the hypothalamic paraventricular nucleus (PVN) into the pituitary portal circulation (1, 2). Episodes of CRH release during stress are usually associated with increases in CRH transcription, as evidenced by rapid and transient increases in primary transcript or heteronuclear RNA (hnRNA). Inap-propriate transcriptional regulation with consequent deficient or excessive CRH expression can lead to HPA axis dysregulation and pathological conditions, such as depression and immune and metabolic disorders (3-5). A major mechanism for limiting HPA axis activation is negative feedback by glucocorticoids at the pituitary corticotroph and several sites in the brain, including hypothalamic CRH neurons in .
Journal of Clinical Investigation, 1998
Corticotropin-releasing hormone (CRH) is the primary hypothalamic releasing factor that mediates the mammalian stress response. The CRH-binding protein (CRH-BP) is secreted from corticotropes, the pituitary CRH target cells, suggesting that the CRH-BP may modulate hypothalamicpituitary-adrenal (HPA) axis activity by preventing CRH receptor stimulation. Transgenic mice were generated that constitutively express elevated levels of CRH-BP in the anterior pituitary gland. RNA and protein analyses confirmed the elevation of pituitary CRH-BP. Basal plasma concentrations of corticosterone and adrenocorticotropin hormone (ACTH) are unchanged, and a normal pattern of increased corticosterone and ACTH was observed after restraint stress. However, CRH and vasopressin (AVP) mRNA levels in the transgenic mice are increased by 82 and 35%, respectively, to compensate for the excess CRH-BP, consistent with the idea that CRH-BP levels are important for homeostasis. The transgenic mice exhibit increased activity in standard behavioral tests, and an altered circadian pattern of food intake which may be due to transgene expression in the brain. Alterations in CRH and AVP in response to elevated pituitary CRH-BP clearly demonstrate that regulation of CRH-BP is important in the function of the HPA axis. ( J.
Loading Preview
Sorry, preview is currently unavailable. You can download the paper by clicking the button above.