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

---

Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The dihydropyridine Ca2+ channel modulators (-) Bay K 8644 (R5417) and nimodipine were used to study the role of voltage-gated Ca2+ channels in the regulation of PRL gene transcription in GH3 cells. Fusion constructs containing 5'-flanking sequences from the rat PRL gene linked to either the bacterial chloramphenicol acetyltransferase (CAT) gene or the firefly luciferase gene were transiently expressed in GH3 cells and the transcriptional response to Ca2+ channel modulators was assessed. The Ca2+ channel agonist R5417 enhanced the transcription of a PRL-CAT fusion gene containing 2.5 kilobase (kb) pairs of the 5'-flanking sequence. This response was completely blocked by the Ca2+ channel blocker nimodipine demonstrating that sequences in the PRL 5'-flanking region confer response to Ca2+. Transfection with PRL-CAT constructs containing 2.5 kb to 0.6 kb pairs of 5'-flanking sequence were responsive to Ca2+, although those which contained the distal enhancer region (positions-1765 to -1495) had much higher basal expression. The possibility that the distal enhancer might contain Ca2(+)-responsive elements was tested by comparing the response to R5417 and TRH for both the proximal enhancer region (approximately first 300 base pair of the 5'-flanking sequence) and distal enhancer regions linked to the thymidine kinase promoter and CAT. The results demonstrate that these two regions contribute to the overall transcriptional response to Ca2+ and TRH. The distal region does not confer a response to phorbol ester, while the proximal region is responsive to that treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Pituitary calcium channel modulation and regulation of prolactin gene expression. 170 75

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.
...
PMID:Mediation by calcium of thyrotropin--releasing hormone action on the prolactin promoter via transcription factor pit-1. 177 32

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)
...
PMID:Thyrotropin-releasing hormone action on the prolactin promoter is mediated by the POU protein pit-1. 192 85

Our previous studies demonstrated TRH stimulation of TSH beta gene transcription in rat pituitary cell cultures and in transient expression assays, with the TRH-sensitive region located between -1.3 kilobases and -204 basepairs (bp) relative to the major transcriptional start site. Using nuclear runoff and transient expression assays, we have analyzed the interactions among TRH, the phorbol ester 12-myristate 13-acetate (PMA), and the adenylate cyclase activator forskolin on TSH beta gene transcription. In cultured pituitary cells, TSH beta gene transcription was stimulated by 2 h of 10(-9) M TRH (2- to 4-fold), 100 nM PMA (2- to 6-fold), or 2 microM forskolin (1.5- to 2.5-fold) treatment, with additive interactions among all three effectors. Chimeric plasmids containing various 5'-flanking portions of the TSH beta gene and both transcriptional start sites, fused to the chloramphenicol acetyltransferase (CAT) gene, were transfected into the clonal pituitary GH3 cell line to delineate DNA sequences conferring this regulation. Transfected TSH beta CAT constructs containing TSH beta gene sequences from -2100/+27I150, -1295/+27I150, and -520/+27I150 expressed CAT enzyme activity which was stimulated by 24 h of TRH (2- to 3-fold), PMA (3- to 6-fold), or forskolin (1.5- to 3-fold) treatment, similar to observations in normal pituitary cells. In addition, a CAT expression vector construct containing only upstream TSH beta gene sequences from -703 to -85 bp, fused to the heterologous thymidine kinase promoter (tkCAT), exhibited similarly stimulated transcription in a transfection assay in response to TRH, PMA, and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Interactions of thyrotropin-releasing hormone, phorbol ester, and forskolin-sensitive regions of the rat thyrotropin-beta gene. 217 92

In order to investigate the molecular mechanism(s) by which TRH regulates the biosynthesis of TSH, we are studying the effects of TRH on the expression of the TSH subunit genes (alpha and TSH beta). To study the structure-function relation of TRH stimulation of the activity of the single rat TSH beta gene, chimaeric plasmids were constructed. The 5'-flanking region of the rat TSH beta gene including exon 1 (5'-untranslated region) was inserted into a promoterless, modified pBR, chloramphenicol acetyltransferase (CAT) expression vector. After transfection, specific TSH beta promoter activity was evident in both TRH-responsive pituitary-derived GH3 and primary pituitary cell cultures. To determine potential regulation of TSH beta promoter-directed activity in these cells by TRH, cells were incubated with media containing TRH (10(-7) to 10(-11) M) for 1 to 48 h. TRH stimulated a 1.5- to 3-fold increase in TSH beta promoter activity. Concomitant with an increase in CAT activity was an anticipated increase in PRL synthesis in the GH3 cells in response to TRH. The TRH effect on the TSH beta gene was specific; no increase in CAT activity was detected for TKCAT (thymidine kinase of herpes simplex virus promoter), pBRCAT (no promoter), or TSH beta CAT (3'-5'-orientation). Similar results were obtained using primary pituitary cell cultures. Deletion mutation analysis indicated that TRH sensitivity was detected in a 1.1 kilobase, but not in a 0.38 kilobase TSH beta gene fragment suggesting that the TRH responsive element(s) resides at least in part within the 700 base pairs of the 5'-flanking sequence.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Thyrotropin-releasing hormone stimulates the activity of the rat thyrotropin beta-subunit gene promoter transfected into pituitary cells. 249 52

The ability of an upstream element of the rat PRL gene to permit transcriptional regulation in response to several different hormones has been examined. To test the ability of specific DNA sequences to mediate hormone responsiveness, DNA fragments were subcloned upstream of a thymidine kinase-chloramphenicol acetyltransferase fusion gene and transferred into GH3 pituitary tumor cells. Initially, fragments representing a distal enhancer element (positions -1713 to -1495) and a more proximal element (positions -292 to -38) were tested. The results demonstrate that the distal enhancer permits cAMP, TRH, epidermal growth factor (EGF), and estradiol to stimulate expression of the thymidine kinase-chloramphenicol acetyltransferase gene. The proximal element permitted fusion gene regulation in response to cAMP, TRH, EGF, and phorbol esters. For the cAMP, TRH, and EGF responses, the distal element permitted responses approximately equal to or greater than responses conferred by the proximal PRL gene fragment. The response of the distal element to cAMP and TRH was more than additive with the response to estradiol, suggesting that the estrogen response element is distinct but may interact cooperatively with the other hormone response elements. Mutation of the estrogen-responsive element abolished both the response to estrogen and the cooperative response with cAMP, but not the response to cAMP itself. Mutation of a sequence involved in basal enhancer activity of the distal element reduced both basal transcription and the response to cAMP. These results suggest that the distal enhancer sequence of the PRL gene contains, in addition to an estrogen response element, elements that confer responsiveness to cAMP, TRH, and EGF.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:The distal enhancer region of the rat prolactin gene contains elements conferring response to multiple hormones. 253 91

We are interested in the mechanisms by which endocrine and developmental factors regulate TSH synthesis at both pre-translational and post-translational levels. Thyroid hormone profoundly decreases transcription of the TSH-beta gene, while TRH and agents modifying cyclic AMP increase transcription. To elucidate the molecular mechanisms underlying these effects, human embryonal kidney cells were transfected with constructs of the human TSH-beta gene fused to the chloramphenicol acetyltransferase gene. The first exon of human TSH-beta, contains an element that increases basal expression and mediates T3-induced gene repression, probably through a direct interaction with c-erbA beta. This transcriptional repression by T3 appears aberrant in thyrotropic tumors. In contrast, TRH and agents modifying cyclic AMP mediate increased transcription of TSH-beta through interacting with upstream regulatory elements. Thyroid hormone, TRH and developmental factors also regulate the branching pattern and relative sialylation of TSH carbohydrate chains, which may affect TSH action in vitro and in vivo. Certain thyrotropic tumors produce TSH with more complex carbohydrate branching patterns, which may increase its biologic activity.
...
PMID:Pre-translational and post-translational regulation of TSH synthesis in normal and neoplastic thyrotrophs. 269 12

The precursor peptide of TRH (prepro-TRH) contains five copes of pro-TRH linked by other peptide sequences. These peptides are coprocessed with TRH in the median eminence of the hypothalamus and released into the portal circulation, rendering this family of peptides available to act at the level of the anterior pituitary. Therefore, we tested the potential bioactivity of one cryptic peptide, prepro-TRH amino acids 160-169 [prepro-TRH-(160-169)], in a TRH-responsive pituitary cell line (GH3). In a heterologous TSH expression assay, we found that prepro-TRH-(160-169) stimulated TSH beta gene promoter activity in a time- and dose-dependent manner; moreover, the effect of prepro-TRH-(160-169) was more rapid and of greater magnitude than that of TRH on TSH beta-directed chloramphenicol acetyltransferase synthesis. In the same cells, we found that prepro-TRH-(160-169) stimulated PRL synthesis and secretion, but the effect was similar in magnitude and duration to that of TRH. The effect of prepro-TRH-(160-169) appears to be additive to that of TRH, suggesting that prepro-TRH-(160-169) may act through a mechanism separate from that of TRH. Thus, prepro-TRH-(160-169) has potent endocrinological effects at the level of the genome.
...
PMID:A cryptic peptide from the preprothyrotropin-releasing hormone precursor stimulates thyrotropin gene expression. 834 17

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.
...
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

---