Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), can efficiently increase cAMP levels in pituitary cells and release a number of pituitary hormones, suggesting an important physiological role for this peptide in pituitary function. Exposure of GH3 rat pituitary cells to PACAP results in increases in cellular cAMP levels, PRL promoter activity, and PRL messenger RNA levels. We have employed this system to further characterize PACAP regulation of PRL gene expression. RT-PCR analysis showed that GH3 cells express transcripts for two PACAP receptors, PACAP-R-hop1 and VIP2. As the former can couple PACAP to increases in both cAMP and inositol phosphates, we investigated whether either pathway mediates PACAP action on the PRL promoter. Our observations that TRH, but not PACAP, increases the intracellular Ca2+ concentration in GH3 cell cultures and that the optimal concentrations of TRH and PACAP have additive effects on transient expression of a PRL-CAT construct imply that the inositol trisphosphate-Ca2+ pathway is not significantly involved in PACAP action on the PRL promoter. Four kinase inhibitors exhibited similar profiles of inhibition of the activity on PRL-chloramphenicol acetyltransferase (PRL-CAT) of either the adenylyl cyclase activator forskolin (FSK) or PACAP, suggesting a transcriptional role for protein kinase A (PKA). The observations that coexpression of the dominant PKA inhibitor RAB completely blocked either FSK or PACAP action on PRL-CAT and that these actions of FSK and PACAP were completely nonadditive imply that the cAMP-PKA pathway plays a dominant role in PACAP regulation of PRL gene expression. Coexpression of low levels of KCREB, a cAMP response element (CRE)-binding protein (CREB) dominant inhibitor, partially blocked regulation of PRL-CAT activity by PACAP, but not TRH, implying that PACAP action is mediated at least in part by a CREB family member that can dimerize with CREB. The PRL promoter contains an asymmetric sequence at positions -99/-92 resembling a canonical CRE and termed here the CRE-like element (CLE). Mutation of either the left or right 4 bp of the CLE yielded a strong decrease in the response to either FSK or PACAP, but not to TRH. These data imply that PACAP and TRH employ independent pathways to regulate the PRL promoter, and that PACAP action is exerted virtually entirely via a cAMP/PKA-mediated pathway that is strongly dependent upon an intact CLE sequence and at least partially dependent upon the activity of a CREB-related protein.
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PMID:Pituitary adenylate cyclase-activating polypeptide regulates prolactin promoter activity via a protein kinase A-mediated pathway that is independent of the transcriptional pathway employed by thyrotropin-releasing hormone. 862

The extracellular domain of the PRL receptor (PRL-R) is composed of two subdomains of approximately 100 amino acids, S1 and S2. To explore the functional significance of these subdomains in PRL binding and signal transduction, deletion mutants of S1 or/and S2 subdomains were constructed. We report here the inability of each of these mutant receptor forms to bind PRL after expression in COS-7 cells. We also studied the abilities of these different mutant receptors to respond to hormonal stimulation after transfection of each mutant complementary DNA into CHO-K1 cells along with a chimeric gene containing the promoter of a milk protein gene (beta-lactoglobulin) fused to chloramphenicol acetyltransferase coding sequence. Somewhat unexpectedly, a constitutively (PRL-independent) mutant form of the PRL-R was obtained after deletion of the S2 subdomain. Moreover, we analyzed, in CHO-K1 cells, the biological activity of chimeric receptors constructs in which each subdomain sequence was replaced by an unrelated, but coding, sequence of foreign protein, and we confirmed a specific requirement for the S1 sequence in the constitutive activity. In contrast, the S2 subdomain produced an inhibitory effect on S1 constitutive activity. Cotransfection experiments with the wild-type receptor and the constitutive mutant receptor provided evidence that the wild-type receptor was able to inhibit the constitutive activity of the deleted mutant. Furthermore, in the mouse mammary epithelial cell line HC11, the constitutive PRL-R form was able to induce transcription of the beta-casein gene in the absence of PRL. These results suggest a complex signal transduction process that implicates each extracellular PRL-R subdomain. Possible mechanisms for the constitutive effect are discussed.
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PMID:Development of a constitutively active mutant form of the prolactin receptor, a member of the cytokine receptor family. 883 44

Altered PRL levels are associated with infertility in women. Molecular targets at which PRL elicits these effects have yet to be determined. These studies demonstrate transcriptional regulation by PRL of the gene encoding the final enzymatic step in progesterone biosynthesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD). A 9/9 match with the consensus Stat5 response element was identified at -110 to -118 in the human Type II 3beta-HSD promoter. 3beta-HSD chloramphenicol acetyltransferase (CAT) reporter constructs containing either an intact or mutated Stat5 element were tested for PRL activation. Expression vectors for Stat5 and the PRL receptor were cotransfected with a -300 --> +45 3beta-HSD CAT reporter construct into HeLa cells, which resulted in a 21-fold increase in reporter activity in the presence of PRL. Promoter activity showed an increased response with a stepwise elevation of transfected Stat5 expression or by treatment with increasing concentrations of PRL (max, 250 ng/ml). This effect was dramatically reduced when the putative Stat5 response element was removed by 5'-deletion of the promoter or by the introduction of a 3-bp mutation into critical nucleotides in the element. Furthermore, 32P-labeled promoter fragments containing the Stat5 element were shifted in electrophoretic mobility shift assay experiments using nuclear extracts from cells treated with PRL, and this complex was supershifted with antibodies to Stat5. These results demonstrate that PRL has the ability to regulate expression of a key human enzyme gene (type II 3beta-HSD) in the progesterone biosynthetic pathway, which is essential for maintaining pregnancy.
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PMID:Stat5-mediated regulation of the human type II 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene: activation by prolactin. 1040 60

The lactogen-dependent rat Nb2 lymphoma is a useful model to investigate PRL signaling pathways that lead to regulation of gene transcription. A primary mechanism coupled to PRL receptor (PRLR) activation in Nb2 cells involves phosphorylation by Jak-family tyrosine kinases of one or more signal transducers and activators of transcription (Stat) factors which subsequently bind to gamma-interferon activation sequences (GAS) within promoter regions of target genes. However, it is presently unclear whether this mechanism is operative as a means for regulating PRL-induced gene expression to the exclusion of other signaling pathways. Previously, we reported that PRL directly stimulated rapid expression of the protooncogene, pim-1, at the mRNA and protein levels in lactogen-dependent Nb2-11 cells. In the present study, experiments were conducted to evaluate signaling mechanisms by which PRL regulates transcription of pim-1. Toward this end, a 1,268-bp segment upstream of the transcription initiation site of the 5'-pim-1 promoter and a series of deletion mutants were ligated upstream of the chloramphenicol acetylase transferase (CAT) gene in an expression vector that was introduced into FDC/Nb2 cells, a premyeloid line that stably expresses the intermediate form of the PRLR. Analysis of PRL-treated cultures indicated that two elements [distal (DE), -427 to -336 bp and proximal (PE), - 104 to -1] but not several GAS or GAS-like sequences were required for hormone activation of the pim-1 promoter. Moreover, treatment of Nb2-11 cells with PRL activated protein binding to these elements assessed by gel mobility shift assay. Deoxyribonuclease I (DNase I) protection experiments revealed a motif containing a nuclear factor-1 (NF-1, -224 to -217 bp) half-site that was hydrolyzed when exposed to extracts from PRL-treated cells but protected by proteins from unstimulated cells. Gel mobility shift analysis of this sequence showed decreased protein binding after PRL stimulation. It is concluded that the PRLR initiates pim-1 transcription by a mechanism that involves transcriptional activation by factors that stimulate the DE- and PE-sites and derepress a NF-1-containing element. Moreover, this mechanism appears to be independent of an interaction between Stat transcription factors and GAS-like elements present within the promoter.
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PMID:Prolactin regulation of pim-1 expression: positive and negative promoter elements. 1057 30


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