Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
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

We have investigated the ability of a constitutively active Gq-alpha mutant, Q209L-alpha q, to regulate target gene expression. Transient expression in GH3 pituitary cells of a rat proximal prolactin promoter-chloramphenicol acetyltransferase construct (-187)PRL-CAT, was stimulated by co-expression of Q209L alpha q, but not by wild-type alpha q. Q209L-alpha q stimulated expression of constructs driven by promoters for either rat prolactin or growth hormone, but not of a control construct driven by the thymidine kinase promoter. Thus, transcriptional effects of alpha q are specific both for the activated state of this G-alpha subunit and the promoter examined. Since both the prolactin and growth hormone promoters are activated by the pituitary cell-specific transcription factor Pit-1, we examined whether a Pit-1 binding site could direct a response to Q209L-alpha q. Two copies of prolactin promoter Pit-1 binding site 1P conferred upon a heterologous metallothionein promoter a response to Q209L-alpha q, implying an involvement of this site in the transcriptional action of Q209L-alpha q on the prolactin promoter. The phorbol ester activator of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate, stimulated (-187)PRL-CAT activity, but opposed the action of Q209L-alpha q on activity of this PRL-CAT construct. Q209L-alpha q stimulation of (-187)PRL-CAT activity was inhibited by co-expression of a dominant negative Raf mutant, Raf-C4, but not by a point mutant of Raf-C4 with reduced inhibitory properties. These results imply that activated alpha q subunits can stimulate prolactin promoter activity via a pathway that involves a Pit-1 DNA binding site(s), is opposed by protein kinase C, and is mediated by a pathway in which Raf-1 kinase plays a role.
Mol Cell Endocrinol 1995 Aug 11
PMID:Constitutively active Gq-alpha stimulates prolactin promoter activity via a pathway involving Raf activity. 748 29

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

In the present report, we have investigated the role of DNA methylation on the binding and trans-acting properties of transcription factors involved in the regulation of the rat prolactin (rPRL) gene, specifically Pit-1. To this aim we took advantage of a model system composed of three GH3 rat pituitary tumor cell lines that greatly differed in the extent of rPRL gene methylation and in the level of rPRL gene expression. Northern blot analyses indicated that identical species of Pit-1 mRNA were present to similar extent in the three GH3 cell lines. Electrophoretic mobility shift assays further demonstrated that Pit-1 was present in nuclear extracts and displayed equal affinities to bind the 1P responsive element encompassing the -65 to -38 region of the rPRL promoter, whatever the GH3 cell line tested. These data suggested that differential expression of the rPRL gene among cell lines did not result from variable amounts of Pit-1. By combining in vitro methylation and transient transfection experiments with a rPRL promoter-driven CAT construct, we showed that extensive methylation at CpG sites abolished the expression of the reporter gene. Furthermore, in vivo competition assays demonstrated that CpG methylation inhibited gene expression by preventing the binding of transcription factors We propose that related mechanisms linked to DNA methylation might alter the activity of the endogenous PRL gene in the low expressing cell line.
Mol Cell Endocrinol 1995 Feb 27
PMID:CpG methylation represses the activity of the rat prolactin promoter in rat GH3 pituitary cell lines. 753 57

Although the GH3 line of somatolactotropic rat pituitary cells has proven useful for many regulation studies, the absence of functional D2 receptors on these cells long prevented their use in studies of dopaminergic action. However, it is now possible to employ GH3 cells expressing recombinant D2 receptors for such investigations. We have investigated both the level at which expression of functional D2 receptors in GH3 cells is blocked, and the cellular pathways employed by the major pituitary D2 receptor isoform, D2A, to inhibit prolactin (PRL) gene transcription. In run-off transcription assays with nuclei from either parental GH3 cells or a GH3 cell line stably expressing a D2A expression vector, Pit-1 gene transcription was detectable in either cell line, but only the latter cell line yielded detectable D2 receptor transcription, implying that the block in D2 receptor expression by GH3 cells is transcriptional. Further investigations employed GH3 cells transiently co-transfected with a D2A expression vector plus a rat PRL promoter construct (-1957)PRL-CAT. Pertussis toxin blocked repression by quinpirole, a D2 agonist, of PRL-CAT activity, demonstrating that this action is mediated by a pertussis toxin-sensitive G protein. The observations that neither of two agents expected to raise intracellular Ca2+, Bay K8644 or thyrotropin-releasing hormone, prevented quinpirole repression of PRL-CAT activity, and that the repressive effects on this construct of quinpirole and the Ca2+ channel antagonist were independent, suggested that regulation of intracellular Ca2+ levels does not play a major role in D2A-mediated repression of the PRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1995 Jun
PMID:The D2 receptor: blocked transcription in GH3 cells and cellular pathways employed by D2A to regulate prolactin promoter activity. 755 74

Expression of the growth hormone gene is due to the presence of the pituitary-specific transcription factor GHF-1/Pit-1. The action of the thyroid hormone T3 is mediated by nuclear receptors that regulate transcription by interaction with DNA elements located near promoters of the regulated genes. In this study, we show that T3 inhibits expression of the GHF-1/Pit-1 gene in rat pituitary GH4C1 cells by a novel mechanism that involves transcriptional interference with other regulatory elements of the promoter. Sequences between bp -90 and -200 of the rat GHF-1/Pit-1 gene which do not contain a hormone response element but contain two cyclic AMP-responsive elements mediate most of the repressive effect of T3. The hormone reduces basal levels of GHF-1/Pit-1 promoter activity and antagonizes its response to cyclic AMP and the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate). A similar repression is found with a heterologous promoter that contains four copies of the cyclic AMP-responsive element motif. This regulation provides a novel example of the cross-talk between the thyroid hormone receptor and the signal transduction pathways used by different hormones and growth factors. Additionally, T3 interferes with in vitro binding of GHF-1/Pit-1 to a positive autoregulatory element located at bp -45 to -63 and has a detectable inhibitory effect on the activity of a promoter construct which extends to bp -90 of 5'-flanking DNA. The regulation of the transcription factor provides a novel example of negative transcriptional regulation by thyroid hormones.
Mol Cell Biol 1995 Nov
PMID:Negative regulation of expression of the pituitary-specific transcription factor GHF-1/Pit-1 by thyroid hormones through interference with promoter enhancer elements. 756 85

Expression of the rat PRL (rPRL) gene is highly restricted to pituitary lactotroph cells and is induced by the cAMP-dependent protein kinase A (PKA) pathway. Current data indicate that this PKA effect requires at least one of the redundant pituitary-specific elements of the proximal rPRL promoter, suggesting the involvement of the pituitary-specific transcription factor, GHF-1/Pit-1. To directly determine whether GHF-1 is necessary and sufficient to mediate the PKA activation of the rPRL promoter, we established a cotransfection reconstitution assay whereby the activity of an intact and site-specific mutants of the (-425 to +73) rPRL promoter-luciferase reporter gene was reconstituted by cotransfecting expression vectors encoding for either the PKA beta catalytic subunit, GHF-1, or both, into HeLa nonpituitary cells. Cotransfection of PKA beta alone significantly stimulated rPRL promoter activity in HeLa cells in a GHF-1-independent manner, and this PKA beta effect was mapped to the most proximal GHF-1 site [footprint (FP) I; -67/-36]. Site-specific alterations of either FP II (-130/-120), or of the basal transcription element (BTE; -112/-80), did not significantly affect the PKA beta response. As expected, the transactivation effect of cotransfected GHF-1 mapped to the GHF-1/Pit-1 binding sites, FP I and/or FP III, of the rPRL promoter. Finally, cotransfection of PKA beta and GHF-1 resulted in a marked synergistic response of the rPRL promoter, and this response also localized to the FP I site. These data confirm not only that GHF-1/Pit-1 and the FP I site are involved in mediating the PKA response, but also imply that a distinct and possibly ubiquitous factor is involved by binding to FP I and functionally interacting with GHF-1 to modulate PKA beta regulation of the rPRL promoter.
Mol Endocrinol 1995 Apr
PMID:Reconstitution of protein kinase A regulation of the rat prolactin promoter in HeLa nonpituitary cells: identification of both GHF-1/Pit-1-dependent and -independent mechanisms. 765 93

Our previous studies demonstrated that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 of the human TSH gene are important for transcriptional regulation by TRH in GH3 rat pituitary cells. The proximal region (-151 to -135 bp) including the cAMP-responsive element (CRE) was required for the induction of the TSH gene by TRH, while the distal region (-223 to -190 bp) containing an element similar to the binding site for the pituitary-specific transcription factor, Pit-1, was necessary to amplify the effects of TRH. To determine whether a pituitary-specific nuclear protein, in addition to the CRE-binding protein, is involved in the molecular mechanism of TRH regulation, a gel retardation assay and Southwestern blot analysis were performed on the distal region with GH3 cell nuclear extracts. GH3 extracts generated a distinct DNA-protein complex that was effectively eliminated in the presence of excess unlabelled DNA fragment, and TRH treatment increased the affinity of protein binding remarkably. Excess Pit-1 DNA-binding sequence from the rat prolactin gene inhibited formation of the complex, but mutation of the Pit-1 consensus sequence in the distal region did not eliminate the complex. In addition, Southwestern experiments showed that a 33 kDa nuclear protein present in GH3 cells bound to this region and its binding affinity was increased slightly 2 h after TRH treatment, with the maximal increase (fivefold) at 3 h, which was similar to the results when using gel retardation. Phosphatase treatment of nuclear protein also resulted in a loss of binding affinity. Taken together, these data indicate that the interaction of a pituitary-specific nuclear protein, identical or closely related to Pit-1, with the distal region may be involved in the TRH stimulation of human TSH gene expression.
J Mol Endocrinol 1995 Jun
PMID:A 33 kDa Pit-1-like protein binds to the distal region of the human thyrotrophin alpha-subunit gene. 766 23

Previous studies have shown that estrogen responsiveness of the rat PRL gene requires the presence of both the estrogen receptor and the tissue-specific transcription factor, Pit-1. To examine the contribution of individual Pit-1-binding sites in permitting an estrogen response, we mutated specific sites in both the proximal and distal regions of the rat PRL gene. The studies reveal that mutation of Pit-1-binding sites in either the proximal or the distal region can have an effect on estrogen responsiveness. The most important Pit-1-binding site appears to be the site in the distal enhancer, which is adjacent to the estrogen receptor-binding site. However, mutation of combinations of other Pit-1-binding sites reveals that these sites also contribute to the estrogen response of the PRL gene. The binding sequences for another transcription factor cannot substitute for Pit-1 sites in bringing about a wild-type estrogen response, as shown by replacement of Pit-1-binding sites with a consensus cAMP-responsive element. Conversion of the imperfect palindromic estrogen response element of the PRL gene to a perfect palindrome eliminated the positive effects of an intact 1D Pit-1-binding site. To examine potential physical interactions between the estrogen receptor and Pit-1, a protein interaction assay was performed. The results demonstrate that labeled estrogen receptor can bind to Pit-1 immobilized on glutathione agarose beads. However, most of the interaction between Pit-1 and the estrogen receptor appears to be DNA dependent.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1994 Dec
PMID:Multiple Pit-1-binding sites facilitate estrogen responsiveness of the prolactin gene. 770 61

The mechanism by which activation of common signal transduction pathways can elicit cell-specific responses remains an important question in biology. To elucidate the molecular mechanism by which the Ras signaling pathway activates a cell-type-specific gene, we have used the pituitary-specific rat prolactin (rPRL) promoter as a target of oncogenic Ras and Raf in GH4 rat pituitary cells. Here we show that expression of either c-Ets-1 or the POU homeo-domain transcription factor GHF-1/Pit-1 enhance the Ras/Raf activation of the rPRL promoter and that coexpression of the two transcription factors results in an even greater synergistic Ras response. By contrast, the related GHF-1-dependent rat growth hormone promoter fails to respond to Ras or Raf, indicating that GHF-1 alone is insufficient to mediate the Ras/Raf effect. Using amino-terminal truncations of c-Ets-1, we have mapped the c-Ets-1 region required to mediate the optimal Ras response to a 40-amino-acid segment which contains a putative mitogen-activated protein kinase site. Finally, dominant-negative Ets and GHF constructs block Ras activation of the rPRL promoter, and each blocks the synergistic activation mediated by the other partner protein, further corroborating that a functional interaction between c-Ets-1 and GHF-1 is required for an optimal Ras response. Thus, the functional interaction of a pituitary-specific transcription factor, GHF-1, with a widely expressed nuclear proto-oncogene product, c-Ets-1, provides one important molecular mechanism by which the general Ras signaling cascade can be interpreted in a cell-type-specific manner.
Mol Cell Biol 1995 May
PMID:Functional interaction of c-Ets-1 and GHF-1/Pit-1 mediates Ras activation of pituitary-specific gene expression: mapping of the essential c-Ets-1 domain. 773 65


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>