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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a human (h) PRL-producing cell line, SKUT-1B-20, which we isolated as a subclone of a uterine sarcoma cell line. Although this cell line is of uterine origin, it does not use the decidual-specific upstream promoter of the hPRL gene, but transcribes the hPRL gene from the downstream pituitary-type transcription start site, as determined by Northern blot, reverse transcriptase-polymerase chain reaction and primer extension analyses. This is particularly intriguing because SKUT-1B-20 cells lack the transcription factor Pit-1. No Pit-1 messenger RNA was detectable by reverse transcriptase-polymerase chain reaction, and endogenous Pit-1 target genes (GH, PRL, and Pit-1) were refractory to transfected Pit-1 expression vector, whereas in cotransfection experiments, Pit-1 efficiently activated reporter gene fusion constructs carrying 5'-flanking sequences of the human and rat PRL or the mouse Pit-1 genes. By transfecting reporter genes containing 8.7 kilobases of DNA flanking the hPRL pituitary-specific start site (hPRL-8700/Luc) and deletions thereof, we located a Pit-1-independent cis-active region more than 7 kilobases upstream of the start site. The most distal 1650 or 880 base pairs of the hPRL genomic fragment (which extends to -8784 base pairs), when placed directly upstream of the homologous hPRL or the heterologous thymidine kinase promoters, conferred transcriptional activation to those promoters. SKUT-1B-20 cell-specific activation of hPRL-8700/Luc could not be suppressed by the introduction of an inhibitor of protein kinase A (PKA), PKI. This is the first demonstration of pituitary-type PRL gene transcription independent of Pit-1 and activation of the PKA pathway. The SKUT-1B-20 cell line was then used in reconstitution experiments to delineate the role of Pit-1 in modulating the transcriptional effects of phorbol ester, PKA, and estrogen receptor (ER) on the hPRL gene. The low response of hPRL/luciferase fusion genes to phorbol ester was greatly enhanced by cotransfected Pit-1 and was mediated by the proximal region between -250 and -38. The catalytic subunit of PKA, C beta, was able to elicit a moderate induction of hPRL-8700/Luc even in the absence of Pit-1. A potential estrogen response element has been located in the hPRL gene sequence at a position similar to that of the estrogen response element of the rat PRL gene immediately adjacent to the distal enhancer.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Endocrinol 1995 Jul
PMID:Pituitary-type transcription of the human prolactin gene in the absence of Pit-1. 747 71

Insulin-like growth factor binding proteins (IGFBPs) comprise a family of secreted proteins that bind insulin-like growth factors-I and -II (IGF-I and -II) with high affinity and potentially modulate their biological effects. We have demonstrated previously that IGFBP-5, the most conserved of the six known IGFBPs, is expressed in muscle cells in the developing embryo and during the terminal differentiation of several myogenic cell lines. In this study we show that an IGF-I analog that binds minimally to IGFBPs potently enhances the differentiation of the stringently controlled inducible C2 myoblast (C2l) cell line and identify IGFBP-5 as the sole IGFBP secreted during C2l differentiation. We find that induction of IGFBP-5 mRNA and protein is coincident with the onset of myogenin gene expression and occurs secondary to the rapid activation of IGFBP-5 gene transcription. By transient gene transfer experiments we demonstrate that a 1004 base pair segment of the IGFBP-5 promoter is very active in directing expression of the reporter gene luciferase in C2l myoblasts. A promoter fragment containing only 156 nucleotides of 5'-flanking DNA retained more than 70% of maximal activity and mediated at least part of the differentiation-dependent rise in IGFBP-5 gene transcription. Within this active segment are several potential binding sites for muscle-enriched transcription factors. Our results show that induction of IGFBP-5 expression is an early event in the myogenic differentiation of the C2l cell line and suggest that one function of this IGFBP is to modulate IGF-induced differentiation. C2l cells are thus an excellent in vitro model for elucidating the developmental factors that control IGFBP-5 gene transcription and action in skeletal muscle.
Mol Endocrinol 1995 Jul
PMID:Rapid activation of insulin-like growth factor binding protein-5 gene transcription during myoblast differentiation. 747 73

We previously identified a codon 351 (Asp-->Tyr) mutant estrogen receptor (ER) in a tamoxifen-stimulated human breast tumor line. To examine its biological activity, we have constructed cell lines from the ER-negative human breast cancer cell line MDA-MB-231 that stably express either the wild type (S30) or mutant ER (BC-2). ER expression was confirmed by Western blot, ligand-binding studies, and ER-enzyme immunoassay. The growth characteristics of the S30 and BC-2 cell lines were compared when treated with estradiol, fixed-ring 4-hydroxytamoxifen [(fr) 4-OH TAM], or ICI 182,780. (fr) 4-OH TAM is a stable, high affinity tamoxifen analog. Many investigators have recognized that growth of ER-negative cell lines stably transfected with ER is inhibited by estradiol. Similarly, both S30 and BC-2 cell lines are inhibited by estradiol in a concentration-dependent manner. (fr) 4-OH TAM has no effect on S30 proliferation but inhibits the growth of BC-2 cells. The pure antiestrogen ICI 182,780 can block the growth-inhibitory effect of estradiol in both cell lines and the growth-inhibitory effect of (fr) 4-OH TAM in the BC-2 cells. In transient transfection analyses using a luciferase reporter plasmid containing two copies of the Xenopus vitellogenin A2 estrogen response element, estradiol stimulated luciferase transcription through both the wild type and mutant estrogen receptors, while (fr) 4-OH TAM stimulated transcription to a greater extent through the mutant receptor. These results demonstrate that the estrogenicity of (fr) 4-OH TAM is increased by binding to the codon 351 mutant ER, and that ER activation and growth inhibition are associated.
Mol Endocrinol 1995 Aug
PMID:A naturally occurring estrogen receptor mutation results in increased estrogenicity of a tamoxifen analog. 747 79

The insulin-like growth factor I (IGF-I) receptor mediates signal transduction by the IGFs and plays a critical role in growth and development. The proximal promoter region of the rat IGF-I receptor gene contains multiple Sp1 consensus-binding sites (GC boxes). Various promoter fragments fused to a luciferase reporter gene were transiently cotransfected together with an Sp1 expression vector into Drosophila Schneider cells, which lack endogenous Sp1. A proximal promoter fragment containing 476 nucleotides of 5'-flanking region and 640 nucleotides of 5'-untranslated region was strongly activated by Sp1 (an average of 116-fold), and progressive 5'-deletions of the promoter that sequentially removed GC boxes reduced Sp1 activation to 15-fold over basal promoter activity. DNase I footprinting studies with purified Sp1 protein revealed four GC boxes in the 5'-flanking region of the promoter and one homopurine/homopyrimidine motif (CT element) in the 5'-untranslated region that bound Sp1. Mutation of the CT element reduced Sp1 activation by 70%. Taken together, these results demonstrate that Sp1 can regulate expression of the IGF-I receptor promoter by acting both on GC boxes in the 5'-flanking region of the promoter and on a CT element in the 5'-untranslated region.
Mol Endocrinol 1995 Sep
PMID:Regulation of insulin-like growth factor I receptor gene expression by Sp1: physical and functional interactions of Sp1 at GC boxes and at a CT element. 749 Nov 7

The estrogenic activity of various 19-norprogestin derivatives has been identified by several laboratories. We have previously hypothesized that the estrogenic activity of these compounds stems from the absence of a methyl group at the 19 position, as various progestins that have a methyl group at this position are not estrogens. To test this hypothesis more directly, we now compare the progestin megestrol acetate against its 19-nor analogue nomegestrol acetate. We also compare these compounds to known estrogens (estradiol, norgestrel, RU486) as well as compounds known to be devoid of estrogenic activity at concentrations as high as 10(-6) M (medroxyprogesterone acetate, R5020, ICI 182780). In growth assays using the MCF-7 and T47D:A18 human breast cancer cell lines, we find that only estradiol, norgestrel and RU486 stimulate proliferation, and this effect can be blocked by the pure antiestrogen ICI 182780. Furthermore, in transient transfection studies using a luciferase reporter construct containing three tandem copies of the Xenopus vitellogenin A2 estrogen response element, estradiol, norgestrel and RU486 can stimulate transcription, while none of the other compounds act as estrogens. Transcriptional stimulation by the estrogenic compounds can be blocked by ICI 182780. Our results demonstrate that the lack of a 19-methyl is not the major determinant for estrogenic activity in 19-norprogestins. We suggest that the 17-hydroxyl group more accurately defines estrogenic action.
J Steroid Biochem Mol Biol 1995 Nov
PMID:Nomegestrol acetate, a clinically useful 19-norprogesterone derivative which lacks estrogenic activity. 749 4

We describe the complete genomic organization of the rat insulin-like growth factor binding protein-2 (rIGFBP-2) gene. This single-copy gene spans over 36 kilobases (kb) and is split into four exons of 475, 224, 141, and 472 nucleotides (nt), and three introns of 32 kb, 686, and 1793 nt, respectively. A single transcription start site (-90) was mapped by S1 protection assay and primer extension. The putative promoter of the rIGFBP-2 gene does not possess TATA or CAAT elements; however, it contains three GC-rich regions located 37, 57, and 81 nt 5' of the cap site. Deletion analysis of the 0.6-kb region of the upstream sequences and transfection of these constructs into BRL-3A and Chinese hamster ovary cells were used to localize possible cis-acting elements. The three GC boxes enhanced chloramphenicol acetyltransferase and luciferase transcription almost to the same level as the XbaI-NsphI (-579 to +1) fragment and displayed synergism and orientation dependence. In addition a similar positive effect on luciferase transcription has been obtained by cotransfecting these fragments with varying amounts of Sp1 expression vector into Drosophila cells that lack endogenous Sp1. In vitro gel mobility shift assays demonstrated that box 1 (GGGCGG), box 2 (GGGAGG), and box 3 (GGGAAGG) bind to SpI with variable affinities and display cooperativity. A protein that gave a similar DNA binding pattern was present in nuclear extracts of BRL-3A cells. To analysis using consensus or aberrant Sp1 elements and a polyclonal Sp1 antiserum to inhibit DNA binding were performed. These in vivo and in vitro data demonstrated that Sp1 plays an important role in the regulation of the expression of rIGFBP-2.
Mol Endocrinol 1993 Sep
PMID:Genomic structure and regulation of the promoter of the rat insulin-like growth factor binding protein-2 gene. 750 79

To define the minimal promoter responsible for expression of CD18 in myeloid and lymphoid cells, we generated 5' and 3' deletion constructs of a segment extending 785 bp upstream and 19 bp downstream of a major transcription start site and determined their effects on driving expression of the luciferase reporter gene in transfected hematopoietic cell lines. A region extending from nucleotides (nt) -302 to +19 was sufficient for cell-restricted and phorbol ester-inducible expression. DNase I footprinting of this region revealed two adjacent protected segments extending from nt -81 to -68 (box A) and -55 to -41 (box B). When a construct of 47 nt in length containing box A and box B and lacking other 3' or 5' elements was cloned into a promoterless vector, it conferred tissue-specific and phorbol ester-inducible expression. Gel retardation revealed that the protein components of two major protein-DNA complexes that form on both box A and box B and are required for transcriptional activation are members of the Ets oncoprotein family; one is related to the GA-binding protein (GABP), and the other is related to PU.1/Spi-1. The minimal CD18 promoter, lacking TATA, CAAT, and initiator elements and consisting primarily of Ets repeats, may exemplify an emerging class of promoters with which the concerted binding of Ets factors is necessary and sufficient to mediate transcriptional activation through direct recruitment of the basal transcription machinery.
Mol Cell Biol 1994 Apr
PMID:The human beta 2 integrin CD18 promoter consists of two inverted Ets cis elements. 751 Dec 9

The human glycoprotein hormone alpha-gene is transcriptionally activated by cAMP in placental cells. We have shown that the novel hypothalamic peptide, pituitary adenylate cyclase activating polypeptide, PACAP-38, significantly stimulates intracellular cAMP levels (12-fold increase; P < 0.001) in JEG-3 choriocarcinoma cells. Regulation of alpha-promoter activity was assessed using both the chloramphenicol acetyl transferase (CAT) and the luciferase (LUC) reporter gene systems. alpha-CAT activity was significantly stimulated by PACAP-38 (4-fold increase; P < 0.05) at 24 h with a similar stimulation being seen with a LUC expression vector. The kinetics of stimulation of the alpha-promoter by PACAP-38 were similar to those seen with 8-Br-cAMP and vasoactive intestinal polypeptide (VIP), a peptide which shares 68% homology with PACAP-38. PACAP-38 also stimulated the production of IL-6 from JEG-3 cells with a time course of response similar to that of alpha-promoter transcription. We conclude that human placental choriocarcinoma cells possess functional receptors for PACAP-38, whose activation enhances cAMP formation, alpha-subunit gene transcription and interleukin-6 (IL-6) production.
Mol Cell Endocrinol 1994 Feb
PMID:PACAP-38 positively regulates glycoprotein hormone alpha-gene expression in placental cells. 751 49

TRH is known to stimulate the transcription of the TSH gene in pituitary cells. To examine TRH-responsive elements of the human TSH alpha-subunit gene, we have used transient transfection of GH3 rat pituitary tumor cells. Using this system, TRH treatment stimulated expression of a reporter gene containing 846 base pairs from the 5'-flanking region of the human glycoprotein hormone alpha-subunit gene linked to luciferase. Analysis of 5'-deletions of the alpha-subunit sequence revealed that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 are important for regulation by TRH. The more proximal region includes a previously defined cAMP-response element (CRE) while the more upstream region contains an element with sequence similarity to the binding site for the pituitary transcription factor, Pit-1. The TRH responsiveness of each individual region was tested by inserting fragments upstream of a thymidine kinase-luciferase reporter gene. The -151 to -100 region had basal enhancer activity and permitted a 3.4-fold response to TRH. The -223 to -168 region did not permit a TRH response, but possessed basal enhancer activity. The combination of both regions resulted in a 5-fold stimulation by TRH. To assess the contributions of different signal transduction pathways, various combinations of treatments were examined. Combined treatment with TRH and forskolin led to an additive activity. Treatment with TRH plus phorbol 12-myristate-13-acetate resulted in the same level of reporter gene activity as with either agent alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1994 Apr
PMID:Involvement of a cAMP-responsive DNA element in mediating TRH responsiveness of the human thyrotropin alpha-subunit gene. 751 24

Insulin-like growth factor-binding protein-1 (IGFBP-1) modulates the action of IGFs on target cells. IGFBP-1 transcription is highly regulated by hormonal and metabolic factors. In rat H4-II-E hepatoma cells, IGFBP-1 messenger RNA is stimulated by dexamethasone, cAMP, and phorbol esters, and dominantly inhibited by insulin. To identify the cis-elements that determine transcriptional regulation by these agents, we have coupled rat IGFBP-1 promoter fragments to a luciferase reporter gene and transfected H4-II-E cells using the cationic liposome procedure. Promoter fragments whose 5'-end was at nucleotide (nt) -925 or -327 (with respect to the transcription initiation site, 1) conferred positive regulation of promoter activity by dexamethasone, cAMP, and phorbol esters. Insulin inhibited promoter activity in the presence of any of the three stimulatory agents. Stimulation by cAMP or phorbol esters was abolished when the region between nt -327 and -235 was deleted. Although this region contains potential activating protein-2 and activating protein-1 sites, the sites responsible for this regulation have not yet been identified. By contrast, stimulation by dexamethasone was retained in deletion constructs whose 5'-end was at nt -92, but was abolished by site mutagenesis of either the left or right half-sites of a potential glucocorticoid response element (GRE) located between nt -91 and -77. Surprisingly, substitution mutations in an up-stream region, -108 to -99 (M4), also decreased dexamethasone-stimulated promoter activity despite the presence of an intact GRE. We postulate that a positive factor that binds to the wild-type M4 region neutralizes factors that inhibit interaction of the glucocorticoid receptor with the GRE. The M4 region also is involved in inhibition by insulin. Insulin inhibition of dexamethasone-stimulated promoter activity was lost after deletion of nt -135 to -92 or mutation of the region between nt -108 and -99. This insulin response element is conserved in the human IGFBP-1 promoter and is homologous to the insulin response element of the phosphoenolpyruvate carboxykinase gene, which also is rapidly inhibited by insulin in H4-II-E cells. The rat IGFBP-1 promoter provides a valuable model system for studying the multihormonal regulation of transcription.
Mol Endocrinol 1994 Jun
PMID:Identification of cis-elements mediating the stimulation of rat insulin-like growth factor-binding protein-1 promoter activity by dexamethasone, cyclic adenosine 3',5'-monophosphate, and phorbol esters, and inhibition by insulin. 752 64


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