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C Bancroft

Publications and source records attributed to C Bancroft.

36 records · Page 2Linked to original sources
PubMed1991

Either isoform of the dopamine D2 receptor can mediate dopaminergic repression of the rat prolactin promoter.

Hypophyseal portal dopamine is a major negative regulator of pituitary prolactin (PRL) production. Dopamine has been reported to repress PRL gene transcription in pituitary cells. To facilitate further study of the effect of dopamine on PRL gene activation, we introduced PRL promoter and D2 receptor (D2R) constructs into GH3 cells. Since two D2R isoforms (termed D2S and D2L) have been cloned previously, we first determined which isoform(s) is present in the lactotroph by measuring the level of each mRNA species in rat prolactinoma. mRNA for each D2R isoform was found to be present, with the D2L mRNA in great (c. 6-fold) excess. Because the lactotroph contains both isoforms, the effect of each on the PRL promoter was investigated. The cDNA for each receptor isoform was synthesized by polymerase chain reaction, and cloned into an RSV-based expression vector. GH3 cells were then transiently co-transfected with either of the resulting RSV-D2R constructs plus a PRL-chloramphenicol acetyltransferase (CAT) construct containing the first 1957 base-pairs of PRL gene 5'-flanking DNA. The cells were then incubated 48 h plus or minus the dopamine agonist ergocryptine (ECR). In the presence of either RSV-D2R isoform, ECR yielded a 4-5-fold decrease in CAT activity, an effect not seen in the absence of the RSV-D2R. The promoter specificity of this effect was demonstrated by the inability of ECR to regulate expression of a control RSV-CAT construct. The PRL promoter repression mediated by each receptor isoform had appropriate pharmacology: the specific D2R agonist, quinpirole, yielded results similar to ECR, and the ECR repression was reversed by the dopamine antagonist spiperone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Mediation by calcium of thyrotropin--releasing hormone action on the prolactin promoter via transcription factor pit-1.

Mediation by Ca2+ of TRH action on the PRL promoter was investigated by both additivity and pharmacological studies and by techniques that probe more gene-proximal events. TRH required the presence of Ca2+ in the medium for stimulation of transient expression in GH3 cells of a PRL-chloramphenicol acetyltransferase (PRL-CAT) construct containing proximal PRL promoter sequences [(-187)PRL-CAT]. Chronic 12-O-tetradecanoyl phorbol-13-acetate down-regulation of cellular protein kinase C did not block induction of expression of (-187)PRL-CAT by either Ca2+ or TRH. In studies with Ca2+ blockers, the Ca2+ flux inhibitors cobalt ion and nimodipine blocked induction of (-187)PRL-CAT expression by either Ca2+ or TRH. On the other hand, the Ca2+ immobilizers 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyltetraester and 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate blocked induction of expression of this construct by Ca2+ but not by TRH, suggesting that TRH regulation of the PRL promoter may be dependent on Ca2+ fluxes but insensitive to Ca2+ immobilization. We have shown previously that the PRL promoter pit-1 binding site 1P is a TRH response element. In the present studies, Ca2+ regulation studies with 5'-deletion mutants of (-204)PRL-CAT showed that (-75)PRL-CAT, containing the single pit-1 binding site 1P, also contains a Ca2+ response element. The observation that two copies of a site 1P oligomer transferred a Ca2+ response to either of the two minimal constructs (-39)PRL-CAT or (-39)mouse metallothionein-CAT showed that site 1P is an independent Ca2+ response element. Analysis of site 1P mutants yielded a strong correlation between the ability to bind pit-1 and to transfer a Ca2+ response. In addition, coexpression of a mutant pit-1 possessing reduced trans-activational activity strongly inhibited TRH regulation of (-187)PRL-CAT and partially blocked Ca2+ regulation of this construct. We conclude that Ca2+ mediates TRH action on the PRL promoter, and that pit-1 represents a gene-proximal mediator in this signalling pathway.

Animals↗
PubMed1991

Thyrotropin-releasing hormone action on the prolactin promoter is mediated by the POU protein pit-1.

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)

Animals↗
PubMed1990

Use of a selectable reporter for the isolation of mammalian regulatory genes.

A general method is described for isolating the genes encoding differentiation-specific activators of transcription using genetic selection. Employing regulation of the prolactin encoding gene (PRL) as a model, we have shown that the hamster dihydrofolate reductase-encoding gene (dhfr) is an effective dominant selectable reporter in this methodology. The dhfr coding region was ligated to the rat PRL promoter, and the resultant construct was stably transfected into DHFR- Chinese hamster ovary (CHO) cells, where it had little or no activity. Transfection of these cells with plasmid DNA, containing the coding region of a pituitary-specific transcription factor (Pit-1/GHF-1) in a eukaryotic expression vector, resulted in transfectants in which activation of the chimeric construct, pPRLdhfr, had occurred, enabling these cells to be selected on the basis of their DHFR+ phenotype. Our results suggest that this strategy could be used to isolate unknown genes that regulate a variety of differentiated functions.

Animals↗
PubMed1990

Identification of a growth hormone gene promoter repressor element and its cognate double- and single-stranded DNA-binding proteins.

In previous investigations, cell fusion was found to silence either the endogenous rat growth hormone (GH) gene or a transfected rat GH gene promoter, implying that repression plays a role in regulation of this gene. To search the rat GH gene promoter for repressor sequences, a series of 5'-deleted GH-CAT constructs was analyzed by transient expression in GH3 rat pituitary cells. Deletion of either a distal region between positions -307/-244 or a proximal sequence between -169/-152 increased CAT enzymatic activity by 3-4-fold. Since the action of the proximal repressor element (PRE) at -169/-152 was serum-independent, and the element is located between two strong positive elements, the PRE and its cognate binding proteins were further analyzed. A 5-base pair sequence centered at -163 is critical for PRE repressor activity, since mutation of this sequence in GH-CAT constructs yielded 6-11-fold increases in expression in GH3 cells. Although the PRE is adjacent to the GH thyroid hormone (T3) response region, they are distinct elements, since the PRE mutation has little effect on the T3 response of GH-CAT constructs. Nuclear extracts of 10 cell lines were searched by DNA mobility shift for protein(s) binding specifically to a double-stranded PRE probe. No such protein was detected in any of four rodent pituitary cell lines or three human cell lines. However, three different rodent non-pituitary cell lines yielded a common shifted band, corresponding to a DNA sequence-specific PRE-binding protein (PREB). Similar analysis with the coding strand of the PRE detected no shifted band in any of these cell lines. However, the PRE noncoding strand yielded a common shifted band in all of the cell lines, corresponding to a ubiquitous, strand-specific, single-stranded PRE-binding protein (ssPREB). Mutation of the PRE permitted ssPREB binding to the coding strand, implying that the wild-type coding strand somehow excludes ssPREB binding. That PREB and ssPREB are distinct proteins was confirmed by the inability of their DNA binding sites to cross-compete binding of the proteins.

Animals↗
PubMed1990

Epidermal growth factor and phorbol ester regulate prolactin gene expression via distinct pathways.

Previous studies, involving phosphorylation of cytoplasmic proteins and localization of DNA regulatory elements, have suggested that epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) have similar actions on prolactin (PRL) gene expression by pituitary (GH) cells. However, little is presently known about whether the actions of these two factors involve common gene-distal intermediates. In the present study, we have employed two approaches to examine this question. Chronic exposure of GH3 cells to TPA, which strongly down-regulates protein kinase C activity, completely inhibited acute TPA stimulation of transient expression of a transfected PRL promoter construct ((-187)PRL-CAT), but did not inhibit EGF stimulation of either accumulation of endogenous PRL mRNA or of expression of (-187)PRL-CAT. Furthermore, the acute stimulatory effects of EGF and TPA on expression of (-187)PRL-CAT were additive. Each of these observations implies that EGF and TPA have gene-distal actions on PRL gene expression that are at least partially non-overlapping.

Animals↗
PubMed1989

Calcium induction of the mRNAs for prolactin and c-fos is independent of protein kinase C activity.

Ca2+ is a strong regulator of the expression of a limited number of eukaryotic genes, including prolactin and c-fos. However, little is known about the cellular signal transduction pathways involved in the action of this ion on specific gene expression. The Ca2+-dependent enzyme protein kinase C has been implicated in other transcriptional pathways regulating the prolactin and c-fos genes. We therefore employed down-regulation of protein kinase C in rat pituitary cells to investigate whether this enzyme is involved in Ca2+ regulation of expression of either of these genes. Exposure of Ca2+-deprived GH3 cells to this ion in the presence of the Ca2+ channel modulator Bay K8644 yielded large increases in the mRNAs for both prolactin and c-fos. Incubation of cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) yielded a 7- to 15-fold decrease in the enzymatic activity of C kinase assayed in vitro, but little or no effect on Ca2+ stimulation of cellular levels of either mRNA. The latter observation was apparently not due simply to an inability of TPA to down-regulate the gene-regulatory activity of kinase C in intact GH3 cells, since this phorbol ester blocked the stimulation by platelet-derived growth factor of cellular levels of c-fos mRNA. TPA treatment also yielded no significant effect on the kinetics of the Ca2+-stimulated accumulation of either mRNA, implying that kinase C is not required at any stage of the Ca2+ induction of these two genes.

Animals↗
PubMed1989

Proximal rat prolactin promoter sequences direct optimal, pituitary cell-specific transcription.

Previous studies have shown that transferred hybrid constructs containing the PRL promoter are expressed specifically in rat pituitary (GH) cell lines. However, it is not yet clear which DNA region(s) is primarily responsible for expression directed by this promoter in pituitary cells. In the present studies we have examined the DNA sequences required for cell type-specific transcription of the rat PRL (rPRL) promoter either during transient expression in intact cells or in nuclear chromatin extracts. RNase protection and nuclear run-on transcription assays showed directly that a PRL-chloramphenicol acetyltransferase (CAT) construct containing about two kilobase-pairs of the rPRL promoter region (pPRL-CAT) is transcribed specifically in pituitary (GH3) cells. Analysis by transient expression in GH3 cells of pPRL-CAT and its 5' deletions showed that 1) deletion of sequences between positions -1957 and -958 did not significantly affect CAT activity; 2) the first 187 basepairs (bp) of the rPRL promoter directs full CAT activity; and 3) 98% of this activity is accounted for by rPRL DNA sequences between positions -187 and -113, containing two GH3 chromatin footprinting sites. Analysis in GH3 cell nuclear extracts showed that transcription of PRL-CAT constructs is unaffected by successive 5' deletions from position -1957 to -187, and that further deletion to -75 yielded only a moderate (approximately 2-fold) decrease in transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Calcium channel agonists and antagonists: effects of chronic treatment on pituitary prolactin synthesis and intracellular calcium.

PRL synthesis by GH cells in culture has previously been shown to increase when calcium is added to cultures grown in calcium-depleted medium or when cultures are treated for 18 h or longer with the dihydropyridine calcium channel agonist BAY K8644, whereas the antagonist nimodipine inhibits PRL. The experiments described here were designed to test whether differences in PRL synthesis caused by the dihydropyridines are due to changes in PRL mRNA levels, whether structurally different classes of calcium channel blockers alter PRL production, and whether long term treatment with calcium channel agonists and antagonists alters intracellular free calcium, [Ca2+]i. PRL synthesis and PRL mRNA levels were increased similarly by BAY K8644 and decreased in parallel by the dihydropyridine antagonist nimodipine, while overall protein and RNA synthesis were not changed by either the agonist or antagonist. Two calcium channel blockers which act at different sites on L-type channels than the dihydropyridines also inhibited PRL synthesis without affecting GH; 5 microM verapamil reduced PRL by 64% and 15 microM diltiazem by 89%. Partial depolarization with 5-25 mM KCl increased PRL synthesis up to 2-fold. The intracellular free calcium ion concentration was estimated by Quin 2 and averaged 142 nM for control cultures in normal medium, and 128 and 168 nM for cultures treated 72 h with nimodipine or BAY K8644, respectively. Nimodipine totally prevented the calcium rise obtained upon depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Proximal upstream flanking sequences direct calcium regulation of the rat prolactin gene.

Previous investigations have shown that Ca2+ strongly and specifically stimulates endogenous PRL gene expression by GH3 cells. In this study, addition of Ca2+ to Ca2+-deprived GH3 cells yielded a large (ca. 8-fold) stimulation of transient expression of a transfected PRL-chloramphenical acetyltransferase (CAT) construct containing ca. 1 kilo-base-pair of the PRL promoter region, but only a slight (less than or equal to 2-fold) nonspecific stimulation of CAT activity directed by any of three control promoters: dihydrofolate reductase, Rous sarcoma virus, or thymidine kinase. In GH3 cells never deprived of Ca2+, expression of a PRL-CAT construct was specifically stimulated and inhibited, respectively, by the dihydropyridine voltage-dependent Ca2+ channel modulators Bay K8644 and nimodipine; Ca2+ can thus regulate expression of an exogenous PRL promoter in cells incubated under physiological Ca2+ conditions. By employing a combined protocol, in which Ca2+-deprived cells are exposed to Ca2+ in the presence of Bay K8644, a very large (greater than 35-fold) but still promoter-specific induction of expression of a PRL-CAT construct was obtained. Analysis of 5'-deleted PRL-CAT constructs implied that the PRL gene Ca2+ response element is contained entirely within the first 174 base pairs of upstream flanking DNA sequence.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Identification by cell fusion of gene sequences that interact with positive trans-acting factors.

Cell fusion experiments have implicated positive or negative regulatory factors in the cell type--specific expression of specialized endogenous genes. The inability to readily manipulate such genes has prevented characterization of the cis-acting DNA sequences that interact with these factors. A transfection-fusion technique, which combined stable gene transfer and formation of transient heterokaryons, was used to study this class of factors and their DNA binding sites. Messenger RNA directed by a quiescent, rat prolactin promoter region stably transferred into mouse fibroblasts was detected only after fusion to rat pituitary cells, implying that pituitary cells contain a positive cell type--specific factor or factors. Nuclear run-on assays showed that fusion activation is transcriptional. Fusion did not activate either a stably transferred rat growth hormone gene promoter or expression of the endogenous silent fibroblast prolactin or growth hormone genes. Analysis by 5'-deletion mutation identified a 30-base pair DNA sequence required for cell fusion activation of the rat prolactin promoter region. Comparison with previous results from direct cellular transfer of this region implies that transfection-fusion identifies novel regulatory DNA sequences.

Acetyltransferases↗
PubMed1987

Ca2+/calmodulin regulation of prolactin gene expression.

Despite the extensive literature on the biological actions of Ca2+ and calmodulin, very little is known about their involvement in nuclear functions, e.g., regulation of specific gene expression. To date, the only genes other than prolactin and growth hormone shown to be regulated by perturbations in cell Ca2+ are those coding for two glucose-regulated proteins. However, there is a growing body of indirect evidence for nuclear functions of Ca2+ and calmodulin, and we suspect that other examples of Ca2+-regulated genes will emerge. We have described in this chapter several different experimental approaches which we have employed to examine first whether prolactin gene expression is regulated by changes in cell Ca2+ content, and then to begin searching for the components of the mechanism by which Ca2+ exerts its effects on the prolactin gene. The tentative identification of 56-kDa nuclear matrix protein as both a calmodulin-binding protein and a substrate of a Ca2+-calmodulin-dependent protein kinase suggests that NMP 56 may be a subunit of a multifunctional Ca2+-calmodulin-protein kinase. This enzyme was recently detected in the nuclear matrix fraction of neuronal nuclei, and was shown to phosphorylate a chromatin protein similar to high mobility group protein 17 (HMG 17). Since HMG 17 is associated with actively transcribed chromatin, its phosphorylation in GH3 cells might play a role in the Ca2+-calmodulin-dependent regulation of prolactin gene expression by hormones and growth factors.

Animals↗
PubMed1987

Glucocorticoid stimulation of growth hormone messenger ribonucleic acid levels in GH3 cells is inhibited by calcium but not by somatostatin.

Aside from studies of a possible synergism between T3 and glucocorticoids in regulating GH mRNA, there have been few previous studies of the modulation by hormones and other factors of glucocorticoid hormone regulation of pituitary cell GH gene expression. We have employed a serum-free medium containing no exogenously added Ca2+ to investigate whether Ca2+ or somatostatin influences the stimulation by the synthetic glucocorticoid dexamethasone of GH mRNA levels in GH3 cells. Basal levels were slightly (less than or equal to 2-fold) stimulated by CaCl2 (0.4 mM). The stimulation by dexamethasone (100 nM) of GH mRNA in GH3 cells incubated in serum-free medium with or without Ca2+ was, respectively, 20- and 25-fold. Thus, under these experimental conditions, little effect of Ca2+ on regulation by dexamethasone was observed. To further reduce Ca2+ levels in the incubation medium, EGTA (20 microM) was added to chelate residual Ca2+. In some but not all experiments, EGTA treatment yielded a slight decrease in basal GH mRNA levels. Time-course experiments performed in the presence of EGTA showed that during incubation periods with dexamethasone that yielded a detectable stimulation of GH mRNA (2-4 days), Ca2+ (0.4 mM) inhibited the dexamethasone stimulation by 3- to 5-fold. Investigations of the dose-response relationship of the effect of dexamethasone on GH mRNA performed under the same conditions showed that at dexamethasone concentrations that yielded a significant stimulation of GH mRNA (10 nM or greater), Ca2+ (0.4 mM) inhibited the dexamethasone stimulation by about 2-fold. In contrast to the inhibitory effects of Ca2+ on the stimulation by dexamethasone of GH mRNA in the GH3 cells, somatostatin had no effect at any concentration tested (1-1000 nM) on dexamethasone regulation of this mRNA.

Animals↗
PubMed1985

Regulation by calcium of prolactin and growth hormone mRNA sequences in primary cultures of rat pituitary cells.

Addition of Ca2+ to primary cultures of female pituitary cells incubated in serum-free medium lacking added Ca2+ yielded no effects on levels of prolactin or growth hormone mRNA, assayed by cytoplasmic dot hybridization. However, incubation of the cells in serum-free medium containing sufficient ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to reduce medium Ca2+ levels below the 10-40 microM present as a trace contaminant yielded a decrease in the levels of both mRNAs. The decrease was dose-dependent at extracellular Ca2+ concentrations below 1.0 microM, had an apparent half-maximum at about 0.3 microM, and did not appear to plateau with increasing incubation times. Following 2-3-day incubations of cells in low Ca2+, a reduction of prolactin mRNA (23-70-fold) consistently greater than the reduction of growth hormone mRNA (9-15-fold) was observed. Similar effects of reduced extracellular Ca2+ were obtained with primary cultures of male pituitary cells. The specificity of these effects of lowered extracellular Ca2+ was demonstrated by the following observations. The decreases in these mRNAs were substantially reversible by readdition of Ca2+ to the incubation medium. Reduction of extracellular Ca2+ led to no detectable changes in cellular ribosomal RNA levels or over-all RNA synthesis. In male pituitary cells, the level of another metal-regulated mRNA, that for metallothionein, was not decreased by a reduction of extracellular Ca2+ that caused a 40-fold decrease in levels of prolactin and growth hormone mRNA. Hence, Ca2+ exhibits specificity in its regulation of pituitary prolactin and growth hormone gene expression.

Animals↗
PubMed1985

Detection of two chromatin proteins which bind specifically to the 5'-flanking region of the rat prolactin gene.

We employed a protein gel blotting procedure to search for nuclear proteins from rat pituitary cells that bind preferentially to the 5'-flanking region of the rat prolactin gene. By gel blots of chromatin proteins from GH3 rat pituitary tumor cells with a 32P-labeled prolactin genomic clone, we detected two major binding proteins with molecular weights of approximately 44,000 and 48,000, designated NP44 and NP48, respectively. Both NP44 and NP48 are minor chromatin proteins which are extracted at low salt concentrations (0.4 M NaCl) and exhibit a range of slightly acidic isoelectric variants. NP44 and NP48 were detected at similar levels in chromatin extracts of GH3 cells, the prolactin-negative GC cell variant of the GH3 cells, and normal rat pituitary tissue. Considerably lower levels of these two proteins were found in chromatin extracts from rat liver and rat C6 glial cells. NP44 and NP48 exhibit DNA sequence specificity, as evidenced by their strong binding to the upstream flanking region of the prolactin gene, but only very weak binding to plasmid DNA, rat prolactin or growth hormone cDNAs, or upstream flanking regions of two other rat genes. By analyzing subclones of a rat prolactin genomic clone, we established that NP44 and NP48 bind to at least two sites, which are located between 0.4 and 2.0 kilobases (region I) and between 2.0 and 4.8 kilobases (region II) upstream of the transcription initiation site. These findings are discussed in the context of a possible functional association between the strong binding of NP44 and NP48 to the prolactin 5'-flanking region and pituitary-specific expression of the prolactin gene.

Animals↗
PubMed1984

Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells.

A peptide with high intrinsic activity for specifically stimulating the secretion of immunoreactive growth hormone (GH; somatotropin) has been characterized and reproduced by total synthesis. This peptide, human pancreatic growth hormone-releasing factor, 44-amino-acid form (hpGRF1-44-NH2), was isolated from a tumor localized in the pancreas of a patient with acromegaly. We report here the effect of this growth hormone-releasing factor (GRF) on GH release and the GH mRNA levels in monolayer cultures of rat pituitary. The cytoplasmic dot hybridization technique was used to examine the effect of GRF on GH mRNA levels. Incubation of rat pituitary cultures with GRF for 72 hr resulted in a 2- to 2.5-fold increase in GH mRNA levels, and the maximal levels of stimulation were achieved at GRF concentrations as low as 1 fM. GRF did not stimulate prolactin release, nor did it affect specific prolactin mRNA levels in the pituitary cultures. We conclude that GRF is a potent and specific GH secretagogue that also affects specifically GH mRNA levels in normal pituitary cells.

Animals↗
PubMed1984

Growth hormone (GH)-releasing factor does not regulate GH release or GH mRNA levels in GH3 cells.

Previous studies have shown that GH-releasing factor (GRF) regulates both GH production and GH mRNA levels in primary cultures of rat pituitary cells. Investigations were carried out to ascertain the ability of GRF to regulate GH production or mRNA levels in a clonal strain of rat pituitary tumor (GH3) cells. Incubation of the cells with GRF at 1-1000 nM for 4 h to 10 days did not result in a stimulation of GH or PRL production, nor did it affect the cytoplasmic levels of the corresponding mRNAs. The lack of response to GRF was not affected by dexamethasone, T3, or serum. We conclude that GH3 cells do not provide a useful model system for studies of the mechanism(s) of action of GRF on either GH release or GH gene expression.

Animals↗