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

In order to identify the mechanism by which cyclic AMP stimulates expression of the human renin gene (REN), the effect of forskolin was tested in transient expression analyses of REN 5'-flanking DNA-chloramphenicol acetyltransferase (CAT) reporter gene constructs in secondary cultures of human chorio-decidual cells, a major site of renin synthesis. Forskolin induced a mean 5-fold stimulation which was localized to DNA in the region -249 to -162 with respect to the transcription start site (+1). Such DNA also mediated a response to forskolin in heterologous (HSV thymidine kinase) promoter constructs. Strong cAMP-response element (CRE) homology at -222 to -218 resembled the target for members of the CRE binding protein (CREB) family. Gel shift assays demonstrated similarly migrating nucleoprotein complexes for oligonucleotides containing the putative REN CRE as for a canonical CRE, in chorio-decidual, JEG-3 and HeLa nuclear extracts. Mutation of residues critical for CREB attachment reduced binding. In conclusion, a CRE was identified at -222 to -218 that appears critical for cAMP-induced human renin gene transcription.
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PMID:Identification of cyclic AMP response element in the human renin gene. 816

Progesterone has been implicated to play a critical role in mediating LH induction of ovulation and possibly luteinization. The present study was undertaken to determine the effects of various agonists (LH, FSH, forskolin, and GnRH) known to stimulate ovulation on their abilities to induce progesterone receptor (PR) mRNA and protein in rat preovulatory follicles and in cultured rat granulosa cells exhibiting a preovulatory phenotype. In cultured granulosa cells, PR mRNA was induced by LH in a dose- and time-dependent manner. Transcripts of approximately 11.0, 7.2, 6.8, 6.2, 3.4, and 3.1 kilobases in size were induced by ovulatory (500 ng/ml), but not low (50 ng/ml), concentrations of LH and FSH as well as by forskolin (10 microM), GnRH (1 microM), and phorbol 12-myristate 13-acetate (200 nM). Two forms (A and B) of PR protein were also induced in an agonist- and time-dependent manner, with the shorter form A (mol wt, 83,000-85,000) appearing in greater abundance than the longer form B (mol wt, 115,000). Indirect immunofluorescent analyses verified nuclear localization of the induced receptor. Forskolin and progesterone, but not progesterone alone, were able to activate a glucocorticoid (progesterone) response element-E1b-chloramphenicol acetyltransferase reporter construct (12-fold) after transfection into cultured granulosa cells. The antiprogestins RU486 and ZK98299 did not inhibit induction of PR mRNA and protein, but effectively blocked agonist activation of glucocorticoid response element2-E1b-chloramphenicol acetyltransferase, as well as LH stimulation of luteinization in vitro. These results provide direct evidence that agonists stimulating diverse intracellular pathways can induce PR in granulosa cells, that progesterone plays a functional role in the luteinization process triggered by the LH surge, and that the effects are mediated at least in part by induction of PR.
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PMID:Hormonal regulation, localization, and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles. 834 15

We examined the effect of PTH on the activity of alpha 1(I) collagen promoter fusion genes in cultured calvariae from transgenic mice. The parent construct, ColCAT 3.6, contains 3520 basepairs of 5' rat alpha 1(I) collagen DNA, 115 basepairs of untranslated alpha 1(I) collagen-coding DNA, and the bacterial chloramphenicol acetyltransferase reporter gene, while the 5'-deletion ColCAT 2.3 contains 2296 kilobases of rat alpha 1(I) collagen promoter sequence. Transgenic mouse lines harboring these collagen promoter fusion genes were developed using the oocyte microinjection technique, and for each construct, three different lines of mice were tested. Calvariae from 6- to 8-day-old transgenic mice were cultured for 48 h with or without bovine PTH-(1-34). ColCAT 3.6 and ColCAT 2.3 were expressed at comparable levels in calvariae and were inhibited by PTH. There were parallel decreases in the incorporation of [3H]proline into collagen and levels of the endogenous alpha 1(I) collagen mRNA and transgene mRNA. Forskolin at 10 microM mimicked the inhibitory effect of PTH on promoter activity in ColCAT 3.6 and ColCAT 2.3 calvariae. A RNase protection assay showed that the transgene was initiated correctly from the transgene promoter. These data show that PTH and cAMP can repress collagen promoter activity in calvariae from transgenic mice, suggesting that the alpha 1(I) collagen promoter may contain cis elements down-stream of -2.3 kilobases that mediate PTH and cAMP repression of collagen gene expression in bone. Cultured bone explants from transgenic mice can be used as a model to study hormonal regulation of alpha 1(I) collagen promoter constructs.
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PMID:Parathyroid hormone represses alpha 1(I) collagen promoter activity in cultured calvariae from neonatal transgenic mice. 848 79

Prostaglandin endoperoxide synthase isoform 2 (PGS-2) mRNA and protein are transiently induced by gonadotropins in granulosa cells of preovulatory follicles prior to ovulation. To better understand the hormonal regulation of the rat PGS-2 (rPGS-2) gene in these cells, genomic clones containing rPGS-2 as well as up to 6 kilobases of 5'-flanking DNA were isolated by screening a rat liver genomic library with a labeled 5'-fragment of the mouse PGS-2 cDNA. Primer extension analysis using ovarian follicular mRNA identified the presence of a single rPGS-2 transcription initiation site located 144 nucleotides upstream of the ATG translation initiation codon. To test for promoter activity within the 5'-flanking region of the rPGS-2 gene, a genomic fragment, -2698/32 (1 = cap site), as well as a series of 5'-deletion mutants, were fused upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into primary cultures of granulosa cells. Forskolin (7.5 microM), follicle-stimulating hormone (500 ng/ml) and luteinizing hormone (500 ng/ml) induced CAT activity following transfection with the -2698/32PGS.CAT, whereas gonadotropin-releasing hormone (10(-6) M) and interleukin-1 beta (30 ng/ml) had no effect. Deletion mutants delineated the region spanning from -192 to -54 of the transcription start site to be essential for both basal and forskolin-regulated expression of the reporter gene. The same DNA fragment (-192/-54) exhibited specific binding to granulosa cell nuclear extract proteins as analyzed by electrophoretic mobility shift assays. Additional specific bands were observed in extracts prepared from granulosa cells exposed to an ovulatory dose of gonadotropin. Collectively, these results provide the first structural and functional evidence that the transcriptional regulation of the rat PGS-2 gene by gonadotropins and forskolin in granulosa cells involves 5'-flanking DNA sequences, specifically a region between -192 and -54 of the transcription initiation site.
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PMID:Characterization and hormonal regulation of the promoter of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Identification of functional and protein-binding regions. 850 40

This study examined the role of the protein kinase C (PKC) signalling pathway in the regulation of expression of human complement factor I (CFI) gene. The production of CFI by Hep G2 cells was enhanced in a dose- and time-dependent fashion by 12-O-tetradecanoyl-1,2-phorbol 13-acetate (TPA), a potent PKC activator. 4Alpha-phorbol didecanoate, an inactive phorbol ester, had no effect on CFI synthesis. The TPA-dependent increase in CFI secretion was correlated with an increase in CFI mRNA levels. Forskolin, a cAMP-inducing agent, augmented the TPA response. W7, an inhibitor of protein kinase A and genistein, an inhibitor of protein tyrosine kinase(s) both did not prevent the increase in CFI expression mediated by TPA. However, calphostin C, a specific inhibitor of PKC, abolished the TPA-induced increase in CFI mRNA levels. Down regulation of intracellular PKC levels by prior exposure of Hep G2 cells to a high concentration of TPA also blocked the increase in CFI mRNA levels induced by TPA suggesting that the TPA effects were mediated via activation of PKC. mRNA decay studies indicated that the half-life of CFI mRNA in TPA-induced cells was not significantly different from control. Nuclear run-on transcriptional assays on the other hand demonstrated that whereas the CFI gene is transcribed under basal conditions in Hep G2 cells, TPA induced a 3-4 fold increase in the transcription rate of CFI gene in 24 h. The transcription rate of GAPDH gene did not change, indicating that the effects were not general on gene transcription. Transient transfections of Hep G2 cells with chloramphenicol acetyltransferase reporter gene (CAT) constructs containing a series of sequential 5' deletions of the CFI promoter and CAT assays showed that the sequence between -136 and -130, containing an AP-1 consensus sequence (TGAGTCA) was required for the TPA response. This observation was substantiated by the finding that mutation of this AP-1 site to TttaTCA or TtAtcCA abolished the TPA responsiveness. The enhancement of the activity of transfected chimeric CAT constructs by TPA was abrogated by calphostin C and by pyrrolidine dithiocarbamate (an inhibitor of NF-kappaB and AP-1 transactivation). These results indicate that TPA regulation of CFI gene requires PKC signalling and is mediated by via a TPA response element (TRE) in the CFI promoter region located at -136/-130 and involves the transactivation of AP-1 and NF-kappaB transcription factors. We suggest that PKC may be one of the intracellular pathways that control CFI gene expression and that cellular processes (involving growth factors, hormones, cytokines etc.) that activate PKC may upregulate the expression of the CFI gene.
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PMID:Transcriptional modulation of the human complement factor I gene in Hep G2 cells by protein kinase C activation. 1063 Jun 30

Major depression is frequently associated with hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. Clinically effective therapy with antidepressant drugs normalizes the disturbed activity of HPA axis, in part, by decreasing corticotropin-releasing hormone (CRH) synthesis, but the mechanism of this action is poorly recognized. In order to find out whether antidepressants directly affect CRH gene promoter activity, we studied their effect on undifferentiated and differentiated Neuro-2A cells, and for comparison the effect of the selected antidepressants on AtT-20 cells was also determined. The cells were stably transfected with a human CRH promoter fragment (-663 to +124 bp) linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The regulation of CRH gene promoter activity is similar in Neuro-2A cells, both intact and differentiated, and in AtT-20 cell line, and cAMP/PKA-dependent pathway plays an important role in the stimulation of CRH gene. It was found that imipramine, amitryptyline, desipramine, fluoxetine, and mianserin, present in the culture medium for 5 days, in a concentration-dependent manner inhibited basal hCRH gene promoter activity in undifferentiated Neuro-2A cells, while other drugs under study (citalopram, tianeptine, moclobemide, venlafaxine, reboxetine, mirtazapine, and milnacipram) were inactive. In the differentiated cells, all examined antidepressants, except moclobemide (no effect) and tianeptine (increase), inhibited hCRH gene transcription. Moreover, in differentiated cells, the drugs acted stronger and were effective at lower concentrations. Forskolin-induced CAT activity was attenuated by imipramine and fluoxetine and to a lesser degree by amitriptyline and desipramine in differentiated cells, whereas other drugs were inactive. Moreover, imipramine and fluoxetine, but not tianeptine, showed moderate inhibitory effect on CRH gene promoter activity also in AtT-20 cell line, commonly used in CRH gene regulation studies. These results indicate that neuron-like differentiated Neuro-2A cells are a better model than pituitary and intact neuroblastoma to investigate the mechanism of psychotropic drug action. Inhibition of CRH gene promoter activity by antidepressant drugs may be a molecular mechanism by which these drugs inhibit the activity of HPA axis.
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PMID:Regulation of the human corticotropin-releasing-hormone gene promoter activity by antidepressant drugs in Neuro-2A and AtT-20 cells. 1473 30


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