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Query: UNIPROT:P06889 (Mol)
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A combined reverse hemolytic plaque-in situ hybridization assay was developed to allow analysis of the relationship between peptide secretion and gene expression within individual cells. We used the pituitary lactotroph as a model system, but this strategy should be widely applicable. It can be used to test hypotheses regarding if and when peptide secretion and gene expression are coupled in any system in which antibodies to the secreted peptide and probes complementary to the mRNA are available. Using the mRNA hybridization signal to identify certain cell types, this method may also be useful in further studies on the biochemical mechanism of peptide secretion. In addition, questions regarding whether a cell known to secrete a given peptide contains other specific mRNAs and the relationship between these mRNAs and the secretion of the peptide can be studied using this strategy. We found striking heterogeneity among lactotrophs in both gene expression and PRL secretion and a lack of correlation of these parameters within individual lactotrophs under every treatment examined. We also present the first direct visualization and quantitation of the percentage of nonsecreting PRL mRNA-containing cells after estradiol treatment and in the presence or absence of the PRL secretagogue, TRH. Finally, we found that in ovariectomized rats, nonsecreting lactotrophs exhibited significantly higher levels of PRL mRNA than lactotrophs that were actively secreting PRL during the assay.
Mol Endocrinol 1991 Jan
PMID:Measurement of peptide secretion and gene expression in the same cell. 190 53

The effects of the calcium/calmodulin signaling system on expression of the rat PRL gene were studied in rat pituitary GH3 cells using two specific naphthalene sulfonamide calmodulin (CaM) antagonist drugs, W7 and a more potent and more highly specific iodo-derivative, 5-iodo-1-C8. PRL (but not GH) mRNA accumulation was markedly inhibited by W7, which in coincubations abolished the stimulation normally seen with TRH. Transient transfection assays showed that expression of the reporter gene chloramphenicol acetyl transferase (CAT) linked to 5'-flanking sequences from the PRL gene was inhibited by the calcium-channel blocker verapamil and by the two CaM antagonists. The calcium effects showed partial promoter specificity, in that transcription of PRL-CAT constructs was markedly inhibited by verapamil, but the Rous sarcoma virus-CAT construct also showed significant inhibition, whereas the pBL-CAT2 construct was unaffected. Three hundred ninety five base pairs were sufficient to confer the full inhibitory effect of calcium channel blockade or CaM antagonist seen with longer constructs. The data indicate that CaM is important for PRL gene transcription, and that the effects of CaM are exerted on DNA sequences within the proximal 395bp of prolactin 5'-flanking DNA.
Mol Endocrinol 1991 Jan
PMID:Calcium/calmodulin regulation of the rat prolactin gene is conferred by the proximal enhancer region. 190 54

TRH is known to regulate transcription of the PRL gene in pituitary cells, but little is known about the mechanism involved. We have characterized TRH response elements (TRHREs) in the promoter region of the rat PRL gene and the gene-proximal protein that transmits the TRH signal to these elements. Exposure of GH3 rat pituitary cells to TRH yielded a large specific stimulation of transient expression of a PRL-chloramphenicol acetyltransferase (PRL-CAT) construct containing the PRL promoter region [(-204)PRL-CAT]. Analysis of 5' deletions of this construct implied that regions -174/-113 and -75/+38 each contain a TRHRE. GH3 cell nuclear extracts are known to footprint four sites, termed, respectively, 1P-4P, on the PRL promoter region. The TRHRE between positions -75/+38 was identified as element 1P, residing at -63/-39, since two copies of a 1P oligodeoxynucleotide transferred a TRH response to either (-39)PRL-CAT or mouse metallothionein-CAT construct (-39)mMT-CAT. Similarly, the more proximal TRHRE may be element 3P, residing at -167/-144, since two copies of this element also transferred a TRH response to (-39)PRL-CAT. Binding of pit-1 to site 1P is known to be capable of activating pituitary cell-specific PRL gene expression. To investigate whether pit-1 can also transduce a TRH signal to this site, oligodeoxynucleotides were prepared corresponding to mutations in either or both of two consensus sequences in site 1P.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1991 Apr
PMID:Thyrotropin-releasing hormone action on the prolactin promoter is mediated by the POU protein pit-1. 192 85

From a chum salmon genomic DNA library, clones coding for somatolactin (SL), a presumed pituitary hormone belonging to the GH/PRL family, were isolated and analyzed. The salmon SL gene was 16 kilobases (kb) in length, this being the largest among mammalian GH (approximately 2 kb) and PRL (approximately 10 kb) and fish GH (approximately 4 kb) genes. As in the case of mammalian GH/PRL genes, the salmon SL gene was comprised of five exons in contrast to rainbow trout and Atlantic salmon GH genes, which each have six exons. Upstream from the transcriptional initiation site were found one TATA box identical to those of mammalian PRL genes and four consensus sequences corresponding to the Pit-1/GHF-1 binding element which is essential to the expression of mammalian GH and/or PRL genes. The similarity in amino acid sequences and organization of the SL gene with mammalian GH/PRL genes indicates that the SL gene is quite likely produced from an ancestral gene common to GH/PRL genes by gene duplication. Based on these considerations, an evolutionary model for the GH/PRL/SL gene family was made and shows the common ancestral gene to originate not from duplication of a primordial gene but from shuffling of independent exons and a regulatory sequence.
Mol Endocrinol 1991 Jun
PMID:Gene structure of chum salmon somatolactin, a presumed pituitary hormone of the growth hormone/prolactin family. 192 96

Expression of PRL, a member of the GH family of genes, is restricted to the lactotroph cells of the anterior pituitary. The proximal promoter of the rat PRL (rPRL) gene contains four factor-binding sites. Three nonadjacent elements, footprints (FP) I, III, and IV, are separated by an integral number of helical turns and bind a pituitary-specific factor, LSF-1. FP II binds another factor present in pituitary and nonpituitary cells. The mechanisms by which DNA-bound proteins influence RNA polymerase-II activity over large distances are not fully understood, but protein-protein interactions, with looping of intervening DNA, may bring distant sites into close proximity. Here, we demonstrate, using protein titration studies, that LSF-1 binds to the most proximal FP I element with the highest affinity, whereas it binds the more distal elements, FP III and FP IV, with progressively lower affinities. Time-course and salt-sensitivity studies reveal that binding of LSF-1 to all three pituitary-specific rPRL promoter sites occurs rapidly (less than or equal to 1 min) and requires fairly high salt concentrations (greater than or equal to 300 mM KCl) to destabilize protein-DNA interactions. Moreover, once bound, the pituitary nuclear factor(s) induces a conformational change in rPRL DNA structure with greatly delayed kinetics (greater than 15 min) and at a different salt concentration than are required for simply factor binding. Taken together, these data suggest a model in which LSF-1 initially binds fairly rapidly to multiple nonadjacent elements and then interacts with itself or other DNA-bound proteins much more slowly, possibly looping or bending the rPRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1991 Jun
PMID:Structure-function analysis of the rat prolactin promoter: phasing requirements of proximal cell-specific elements. 192 98

Four isoforms of glycosylated prolactin (G-pPRL) were isolated from porcine pituitary glands by affinity chromatography and concanavalin A-Sepharose, based upon differences in their affinity for the lectin. Structural analysis indicated differences in the carbohydrate units of the four G-pPRLs. N-glycanase treatment cleaved the oligosaccharide from the G-pPRLs, establishing N-linked glycosylation. The binding of G-pPRLs to receptors from lactating rabbit mammary glands was only 3-8% that of nonglycosylated pPRL (NG-pPRL). The immunological crossreactivity of the G-pPRLs varied from 36 to 65% that of NG-pPRL. When tested in the pigeon crop sac bioassay, G-pPRLs were only 11-40% as active as NG-pPRL. The metabolic clearance rate of one of the G-pPRLs was slower and another faster than that of NG-pPRL. We conclude that there are several forms of G-PRL of variable immuno- and bio-potencies in the porcine pituitary, and that the current radioimmunoassay for the hormone does not measure the actual bioactivity.
Mol Cell Endocrinol 1991 Sep
PMID:Isolation and biochemical properties of four forms of glycosylated porcine prolactin. 195 78

We have cloned DNA sequences extending up to 6000 bp upstream from the first exon of the human prolactin (hPRL) gene. 5000 bp of these upstream sequences were fused to a CAT reporter gene and shown to provide tissue-specific transient expression in rat pituitary GH3 cells. Multihormonal response was found in this transient expression assay, leading to significant 2- to 5-fold induction by addition of 8-chlorophenylthio-cyclic AMP, thyrotropin-releasing hormone, epidermal growth factor, basic fibroblast growth factor, phorbol myristate acetate, a calcium channel agonist (Bay K-8644) and triiodothyronine. A 3-fold inhibition was observed in the presence of the glucocorticoid agonist dexamethasone. The sequence of the hPRL promoter was determined up to coordinate -3470. Computer similarity search between the rat and human sequences showed two highly conserved regions corresponding to the proximal and distal tissue specific enhancers described in both PRL promoters.
Mol Cell Endocrinol 1991 Sep
PMID:Multihormonal regulation of the human prolactin gene expression from 5000 bp of its upstream sequence. 195 81

In vitro studies have demonstrated that the estrogen receptor (ER) can bind to the rat PRL estrogen response element (ERE) located 1700 basepairs upstream of the transcriptional start site. However, the mechanism by which the receptor-DNA complex influences the activity of RNA polymerase located in the promoter region is not understood. To begin investigating this process, we developed cell lines derived from GH3 cells that contain steroid-responsive bovine papillomavirus minichromosomes. Within these minichromosomes is a hybrid gene composed of the 5' flanking region of the PRL gene, driving the expression of the Tn5 gene. The episomal PRL DNA sequences responded to 17 beta-estradiol (E2) by increasing the rate of Tn5 gene transcription. Nucleosome mapping experiments using micrococcal nuclease demonstrated that nucleosome-like structures were assembled on the minichromosome in an ordered array separated by 150-200 basepairs of DNA. Novel S1 nuclease as well as DNase-I-hypersensitive sites in the chromatin of the promoter and distal regulatory regions of the episomal PRL gene were detected by indirect end-labeling studies. The nuclease hypersensitive sites in the distal region containing the ERE were modified after treatment of the cells with either E2 or the antiestrogen 4-hydroxytamoxifen. However, only E2 treatment of cells resulted in an increase in the nuclease hypersensitivity of the promoter region and induced gene expression, while antiestrogen treatment had no effect on either parameter. This suggests that complex interactions between factors located at the distal and proximal regulatory regions ultimately determine the transcriptional response of the PRL gene to E2.
Mol Endocrinol 1990 Aug
PMID:An interaction between the 5' flanking distal and proximal regulatory domains of the rat prolactin gene is required for transcriptional activation by estrogens. 196 74

Regulation by PRL of aromatase (P450arom) mRNA and protein and estradiol (E) biosynthesis was examined in granulosa cells during early stages of luteinization in vitro and in vivo. PRL caused a dose-dependent (10-1000 ng/ml) decrease in P450arom mRNA and E biosynthesis (greater than 99%) in luteinized rat granulosa cells in vitro, even when the cells were cultured in the presence of insulin and hydrocortisone (hormones known to synergize with PRL to induce proteins in mammary tissue) or in the presence of forskolin (a nonhormonal stimulator of cAMP). PRL also prevented the marked increases in aromatase mRNA and E biosynthesis stimulated by FSH and forskolin in nonluteinized preovulatory granulosa cells in culture. These effects of PRL on granulosa cells in culture were specific for aromatase and were not observed for other proteins, such as cholesterol side-chain cleavage cytochrome P450 (P450scc) and alpha 2-macroglobulin. PRL also decreased P450arom mRNA and protein during the early stages of luteinization in vivo. PRL administered to rats beginning day 1 postovulation to mimic hormone release during pseudopregnancy reduced the progressive increase in P450arom mRNA occurring in corpora lutea on days 3-4 in ovulated rats not treated with PRL. CB 154, a dopamine agonist that inhibits pituitary release of PRL, caused P450arom mRNA and protein to decrease 50% if given to pregnant rats on days 8-10 of gestation, but increased P450arom mRNA and protein if given to pregnant rats on days 10-12 of gestation. These diverse effects of PRL in pregnancy suggest that placental factors may modify the response of luteal cells to PRL during gestation.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1990 Jan
PMID:Regulation of aromatase mRNA and estradiol biosynthesis in rat ovarian granulosa and luteal cells by prolactin. 197 Jan 19

Expression of the TSH beta subunit gene is restricted to the thyrotroph cells of the anterior pituitary. Previously we identified several AT-rich DNA elements within the murine (m) TSH beta 5'-flanking region, denoted as D1 (-253 to -227), P4 (-142 to -131), P3 (-126 to -112), P2 (-106 to -98), and P1 (-76 to -68) which bind thyrotroph-specific factor(s). These sites are related to, but distinct from GHF-1 and LSF-1 binding sites, which restrict GH and PRL gene expression to pituitary somatotrophs and lactotrophs, respectively. To determine whether different pituitary cell types contain related factors capable of activating the mTSH beta promoter, cell-free transcription studies were performed using extracts from GH4 rat pituitary somatomammotroph cells. AI-through the endogenous mTSH beta gene is not expressed in GH4 cells, in vitro transcription of the mTSH beta promoter, normalized to the Rous sarcoma virus internal control, revealed faithful transcription initiation from the authentic mTSH beta CAP sites in GH4 but not in HeLa cell extracts. Cell-free transcription analysis of mTSH beta 5'-deletion mutants revealed consistent promoter activity with deletion to position -46 but complete loss of activity when deleted to position -9. To better define the specific factors in pituitary somatomammotrophs which interact with and activate the mTSH beta promoter, DNase I protection and gel-shift studies were performed using extracts from GC rat pituitary somatomammotroph cells and DNA affinity-purified lactotroph-specific transcription factor, LSF-1, required for rat PRL promoter activity, and purified from GC cells. These cells contain a factor(s) which binds to thyrotroph-specific elements of the mTSH beta promoter. These studies also show that LSF-1 binds the D1 and proximal thyrotroph-specific elements of the mTSH beta promoter and is capable of reconstituting the trans-activation of the mTSH beta promoter in HeLa nonpituitary cell extracts in vitro. Conversely, nuclear factors present in TtT-97 murine thyrotrophs bind the proximal lactotroph-specific elements on the rPRL promoter. This in vitro transcription assay provides a means to biochemically dissect the trans-activation of the mTSH beta promoter and to determine the functional overlap of distinct pituitary cell-specific factors in regulating GH, PRL, and TSH beta gene expression.
Mol Endocrinol 1990 Dec
PMID:Activation of the murine thyrotropin beta-subunit promoter by GH4 rat pituitary cell-free extracts. 208 87


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