Prolactin Gene Promoter Research Articles

PIT-1 Protein Localization at Different Optical Sections in a Single Living Cell Using FRET Microscopy and Green Fluorescent Proteins

Abstract The pituitary specific transcription factor Pit-1 is required for transcriptional activity of the prolactin (PRL) gene. The Pit-1 protein is a member of the POU homeodomain transcription factors that is expressed in several different anterior pituitary cell types, where it functions as an important determinant of pituitary-specific gene expression. The Pit-1 protein generally interacts with DNA elements in the PRL gene promoter as a dimer, and has been demonstrated to associate with other transcription factors. The objective of our research is to define the critical molecular events involved in transcriptional regulation of the PRL gene in living cells. Methods that allow monitoring of the intimate interactions between protein partners in living cells provide an unparalleled perspective on these biological processes. Using the jellyfish green fluorescent protein (GFP) as a tag, we applied the fluorescence resonance energy transfer (FRET) technique to visualize where and when the Pit-1 protein interacts in the living cell. FRET is a quantum mechanical effect that occurs between donor (D) and acceptor (A) fluorophores provided: (i) the emission energy of D is coincident with the energy required to excite A, and (ii) the distance that separating the two fluorophores is 10-100 Å. Mutant forms of GFP that fluoresce either green or blue (BFP) have excitation and emission spectra that are suitable for FRET imaging.

Thyroid Hormone Inhibits the Human Prolactin Gene Promoter by Interfering with Activating Protein-1 and Estrogen Stimulations

Transcription of the human PRL (hPRL) gene in the pituitary is subject to tissue-specific and multihormonal regulation involving two main regulatory regions, a proximal promoter and a distal enhancer. In this report we show that thyroid hormone inhibits the expression of the hPRL gene in rat pituitary cells. Transient expression experiments show that thyroid hormone regulation involves a strong inhibitory element, located in the proximal (-164/-35) promoter, which is modulated by a more distal stimulatory response control region. Gel retardation experiments reveal that the thyroid hormone receptor does not bind to the proximal negative element. We show the existence of an activating protein-1 (AP-1) response element located at positions -61 to -54 of the proximal promoter, conferring AP-1 stimulation to the hPRL promoter. This AP-1 induction is abolished when hormone-bound thyroid hormone receptor is present, indicating that there is an interference between the thyroid hormone receptor and AP-1 regulatory pathways. Furthermore, using the complete hPRL upstream region, we show that estrogen induction is abolished by simultaneous thyroid hormone treatment.

Studies on the mechanism of glucocorticoid-mediated repression from a negative glucocorticoid response element from the bovine prolactin gene.

Several models for repression of transcription by glucocorticoid hormone, some of which involve so-called negative glucocorticoid response elements (nGRE), have been suggested. In the cases where nGREs are required, the glucocorticoid receptor (GR) is thought to bind to the nGRE and interfere with transcriptional activation by positively acting transactivating factors. We have studied an nGRE from the bovine prolactin gene promoter (PRL3), which increases basal expression from a heterologous promoter in rat pituitary cells (GH3) and is repressed by glucocorticoids. Two proteins in addition to the GR were identified in pituitary cells to bind specifically to the PRL3 nGRE, one of which was the pituitary-specific transcription factor Pit-1/GHF-1. A mutation in the PRL3 nGRE, which destroyed Pit-1/GHF-1 binding, totally abolished the increased basal expression as well as glucocorticoid repression in transfected GH3 cells. A mutation in the binding site for the second protein, termed XTF, partially impaired basal activity but totally abrogated glucocorticoid repression. The same mutation had no effect on GR binding to the PRL3 nGRE. Mixing experiments with whole-cell extracts containing overexpressed GR from COS cells decreased the binding of both Pit-1/GHF-1 and XTF to the PRL3 element. However, Pit-1/GHF-1 displacement from the PRL3 element by the GR required XTF binding. Furthermore, GR binding to the PRL3 nGRE was required for glucocorticoid repression to occur, because a mutation of the GR binding site abolished the glucocorticoid effect. Moreover, the PRL3 nGRE was found to contain only half a palindromic GRE, allowing only one GR moiety to contact the DNA. These data demonstrate that the PRL3 nGRE is composite in nature and that the ability of the GR to repress transactivation by displacement requires an intermediary factor, XTF.

Hydrogen peroxide-induced expression of the proto-oncogenes, c-jun, c-fos and c-myc in rabbit lens epithelial cells.

The involvement of H2O2 in cataract development has been established in both human patients and animal models. At the molecular level H2O2 has been observed to cause damage to DNA, protein and lipid. To explore the oxidative stress response of the lens system at the gene expression level, we have examined the effects of H2O2 on the mRNA change of the proto-oncogenes, c-jun, c-fos and c-myc in a rabbit lens cell line, N/N1003A. H2O2 treatment of the rabbit lens epithelial cells for 60 min induces quick up-regulation of both c-jun and c-fos mRNAs. The maximal induction is 38 fold for c-jun at 150 microM and 72 fold for c-fos at 250 microM H2O2. Treatment of N/N1003A cells with 50-250 microM H2O2 for 60 min leads to a 2-5 fold increase of the c-myc mRNA level. H2O2 also induces an up-regulation in transactivity of the activating protein-1 (AP-1) as shown with a reporter gene driven by a prolactin gene promoter with 4 copies of AP-1 binding sites inserted in the upstream of the promoter. Maximal induction occurs with 150 microM H2O2. In the same system, the antioxidants, N-acetyl-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) at concentrations shown to up-regulate the mRNAs of both c-jun and c-fos, also enhance the transactivity of AP-1. NAC and PDTC have different effects in modulating the induction of AP-1 activity by H2O2 and TPA. These results reveal that oxidative stress regulates expression of various regulatory genes in lens systems, which likely affects cell proliferation, differentiation and viability and thus affect normal lens functions.

A Dopamine-responsive Domain in the N-terminal Sequence of Pit-1: TRANSCRIPTIONAL INHIBITION IN ENDOCRINE CELL TYPES

The POU transcription factor Pit-1 activates the prolactin gene in pituitary lactotrophs and may integrate responses of the gene to external signals. To study the role of Pit-1 in dopaminergic inhibition of the prolactin gene, we transiently transfected Pit-1 and dopamine D2 receptor vectors into a series of heterologous cell lines and examined dopamine regulation of the prolactin gene promoter. Regulation was Pit-1-dependent in all cell lines tested. Moreover, dopamine responsiveness was cell type-specific: stimulatory in fibroblasts (COS-7) and muscle-type cells (P19/Me2SO-induced) and inhibitory in pancreatic endocrine (RIN, InR1-G9) and neural-like (P19/retinoic acid-induced) cells. Because dopaminergic responses in Pit-1-transfected RIN cells paralleled those in pituitary GH4 cells, the islet cell line was used to test for sequences in Pit-1 that mediate negative hormone signals. Dopamine responsiveness of the Pit-1 transactivation domain (residues 8-80) was examined using a chimeric LexA construct. LxPit-1, LxSp1, and Lx-glucocorticoid receptor fusions all activated basal transcription, but only LxPit-1 was regulated by dopamine. Regulatory responses of LxPit-1 and full-length Pit-1 were quantitatively similar. In addition, gain-of-function G alpha mutants that inhibit Pit-1-dependent promoters in GH4 cells also suppressed selectively Pit-1- or LxPit-1-dependent promoters in RIN cells. This demonstrates that Pit-1 can function as a specific target for distinct inhibitory G protein signals. Interestingly, Pit-1 sequences N-terminal to the DNA-binding POU domain appear to be sufficient in mediating regulation by these pathways.

The insulin and cAMP response elements of the prolactin gene are overlapping sequences.

Prolactin gene expression is affected by numerous signals, but many of the promoter elements required for these responses have not been clearly identified. This report identifies sequences within the prolactin gene promoter that are required for the responses to cAMP and insulin. The cAMP response element, -101 to -92 shares a 6 of 8 base pair homology with previously identified cAMP response elements. Mutation of this element also results in a > 100-fold decrease in basal prolactin gene expression. This is characteristic of cAMP response elements, but the importance of this element to basal prolactin gene transcription was previously unrecognized. The insulin response element, -97 to -67, is not homologous to previously reported insulin response elements and mediates the 10-fold increases in prolactin gene expression due to insulin observed in GH cells. These elements also function to mediate insulin and cAMP responses from the heterologous delta MTV-CAT reporter plasmid. Together, insulin and cAMP increase prolactin gene expression additively. The clustering of these elements may provide clues to the independent and possible coordinate regulation by these effectors.

G(i) alpha 2- and G(o) alpha-mediated signaling in the Pit-1-dependent inhibition of the prolactin gene promoter. Control of transcription by dopamine D2 receptors.

Dopaminergic signaling in pituitary lactotrophs is dependent on coupling of D2 receptors to several inhibitory G-protein subtypes, resulting in the activation of multiple signaling pathways. In prolactin-secreting GH4 cells that express cloned D2 receptors, dopamine selectively inhibits the activity of the prolactin gene promoter, a response mediated in part by the pituitary transcription factor Pit-1. Transfected gain-of-function mutants of the G alpha subtypes, Gi alpha 2 (Q205L) and G(o) alpha (Q205L), mimic the promoter-specific and Pit-1-dependent inhibition by dopamine. Whereas the activated Gi alpha 2 subtype suppresses cAMP levels, the G(o) alpha mutant does not, demonstrating a cAMP-independent pathway in the inhibition of the prolactin gene. This alternate pathway could involve other regulators, possibly calcium. Interestingly, in Ltk- cells in which cloned D2 receptors modestly suppress cAMP, but elevate [Ca2+]i, the activity of the prolactin promoter is enhanced rather than inhibited by dopamine. The response is promoter-specific, dependent on Pit-1, and completely blocked by low concentrations of EGTA, consistent with a calcium-regulated pathway. Last, in GH4 cells, the absence of additivity between Gi alpha 2 and G(o) alpha mutants suggests a convergent mechanism in the reduction of prolactin promoter activity, in which either signaling pathway may be sufficient for maximum inhibition. This apparent redundancy in inhibitory control mechanisms may be of physiological importance for maintaining efficient tonic suppression of prolactin synthesis.

Expression of Pit-1 and related proteins in diverse human pituitary adenomas.

Pit-1, a member of the POU family of homeo-domain transcription factors, activates prolactin and GH gene expression but also has a role in pituitary cell differentiation and proliferation. Expression of Pit-1 may therefore be of central importance in the function and phenotype of human pituitary adenomas. We have found evidence that, in addition to Pit-1 mRNA, Pit-1-like immunoreactivity and DNA-binding activity are readily detectable in a series of human pituitary adenomas. Gel mobility shift assays using adenoma protein extracts with two Pit-1-binding sites from the human prolactin gene promoter demonstrated the formation of several DNA sequence-specific protein-DNA complexes; some of these could be accounted for by Oct-1-binding activity. Pit-1 activity was anticipated in prolactin- and GH-secreting adenomas, but was also detected in a proportion of endocrine-inactive (non-secreting) adenomas that did not express Pit-1 target genes. The data demonstrate the presence of Pit-1 in a range of pituitary adenomas. Different adenomas generated slightly differing patterns of DNA-binding activity, though Pit-1 mRNA and protein size appeared normal in all tumours so far examined.

Interactions between Rat Prolactin Gene Promoter and Enhancer Regions in Mammosomatotrope and Lactotrope Cell Lines

Although both promoter and enhancer sequences of the PRL gene 5'-flanking DNA are required for cell-specific, high level expression in transgenic animals, reports of the relative contributions of these elements determined in transient transfection experiments have varied. In this study we examined the transcriptional activities of proximal promoter (-422/+33) and distal enhancer (-1956/-1530) sequences of the rat (r) PRL gene by transient transfection of hybrid genes containing these sequences into two rat pituitary cell lines, GC and 235-1. These cell lines exhibit characteristics either of mammosomatotropes, which express both PRL and the evolutionarily related GH gene (GC), or lactotropes, which express only PRL (235-1). As lactotropes are thought to differentiate from a mammosomatotrope precursor cell, comparisons between these cell lines provide the opportunity to examine the mechanisms that activate PRL and GH genes during development. We show that the relative contributions of promoter and enhancer elements differ between GC and 235-1 cells. Although maximal enhancer-driven activity was similar between these cell lines, promoter sequences were more active in GC (5-10% maximal) than 235-1 cells (1-2% maximal). However, no apparent differences in factor binding to the rPRL promoter region could be correlated with differences in activity, suggesting that differential factor modification, rather than different factors, is involved. As the rGH promoter exhibited a similar pattern of activity in these cell lines, these observations suggest that promoter as well as enhancer elements contribute to the cell specificity of PRL and GH gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction and characterisation of 3′-deletion mutants of the rat prolactin gene promoter

Construction and characterisation of 3′-deletion mutants of the rat prolactin gene promoter

Characterisation of tissue‐specific trans‐acting factor binding to a proximal element in the rat growth hormone gene promoter

Using an exonuclease III protection assay, tissue-specific binding of rat pituitary tumour cell (GH3 cell) nuclear factors to a proximal region (-68 to -138) of the rat growth hormone gene promoter has been detected. The binding is particularly strong between the borders -68 to -102. The binding is eliminated in the presence of excess unlabelled rat growth hormone gene promoter sequences but also by proximal (-423 to +38) or distal (-1960 to -1260) rat prolactin gene promoter sequences and simian virus 40 enhancer/promoter sequences. Extracts of rat pituitaries showed identical binding characteristics. Methylation interference analysis indicated that the contact points between the pituitary-specific factor and the proximal rat growth hormone gene promoter-binding element (-65 to -95) are over a conserved sequence which occurs twice in the rat growth hormone gene promoter and at least eight times in the rat prolactin gene 5'-flanking sequences. This sequence has previously been proposed to constitute the binding site for the somatotroph/lactotroph tissue-specific transcription factor. Gel-retardation and exonuclease III competition analysis showed that three of the rat prolactin gene promoter elements (-46 to -71, -156 to -180 and -174 to -204) share the ability to bind the pituitary-specific factor. The binding to the most proximal rat prolactin gene promoter element (-46 to -71) was clearly more avid than to the rat growth hormone gene promoter (-65 to -95) proximal element. However, both these elements displayed the formation of two gel-retarded complexes while the more distal rat prolactin gene binding elements (-156 to -180 and -174 to -204) formed only the smaller of the two complexes. Finally, we demonstrated by co-transfection competition analysis that plasmids containing the most proximal rat prolactin gene promoter binding element completely inhibited transcription from the rat growth hormone gene promoter while rat growth hormone gene promoter sequences only partially inhibited transcription from the rat prolactin gene promoter. This suggests that the higher affinity for factor binding displayed by the proximal rat prolactin gene promoter binding site in vitro is reflected in factor binding activity in vivo.

Dual functions of a cis-acting element within the rat prolactin gene promoter.

Within the promoter region of the rat prolactin gene lies a TA-rich imperfect palindrome. The possible functions of the 18-base-pair symmetrical sequence were investigated by using an in vitro transcription system. Prolactin templates with and without the palindrome were transcriptionally assayed in both pituitary and nonpituitary extracts. Our results indicated that the palindromic sequence has at least two functions in the regulation of prolactin transcription.

Dual functions of a cis-acting element within the rat prolactin gene promoter.

Within the promoter region of the rat prolactin gene lies a TA-rich imperfect palindrome. The possible functions of the 18-base-pair symmetrical sequence were investigated by using an in vitro transcription system. Prolactin templates with and without the palindrome were transcriptionally assayed in both pituitary and nonpituitary extracts. Our results indicated that the palindromic sequence has at least two functions in the regulation of prolactin transcription.

Tissue specific trans-acting factor interaction with proximal rat prolactin gene promoter sequences.

Using an exonuclease III protection assay, strong, reversible and tissue-specific binding of GH3 cell nuclear factors to proximal regions of the rat prolactin (rPrl) promoter (-31 to -77) has been detected. A second less prominent region of factor binding, that may have a correlate in HeLa cell extracts, was detected in the region (-155 to -180). The binding is eliminated in the presence of excess unlabelled rPrl promoter sequences (-423 to +38), excess unlabelled distal rPrl 5'-flanking sequences (-1960 to -1260) and SV40-enhancer/promoter sequences; it is largely unaffected by growth hormone (rGH) promoter and RSV-LTR sequences. A plasmid containing the proximal rPrl promoter sequences (-75 to +38) was also shown to be an avid inhibitor, at low concentrations, of rPrl promoter driven chloramphenicol acetyl transferase (CAT) gene expression in transient cotransfection competition studies; under these assay conditions distal rPrl 5'-flanking sequences and RSV and rGH promoter plasmids do not compete. The results emphasize the critical importance of proximal rPrl promoter sequences for prolactin gene expression in GH3 cells but recognize the related functional potential of more distal sequences.