Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.23.15 (renin)
 35,795 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have recently identified a human pulmonary carcinoma cell line (Calu-6) that expresses human renin (hREN) mRNA endogenously, and we use it herein as a model to examine the regulation of the hREN gene. Transfection analysis of a deletion series (-2750 to -149) of hREN promoter-luciferase fusion constructs revealed the presence of multiple weak regulatory elements within the first 1,301 bp of the 5'-flanking region and a classic silencer element within the first intron (intron A) of the gene. The 5'-flanking regulatory domain consisted of three closely linked elements, two negative and one positive, each contributing a cell-specific threefold modulation of transcriptional activity. Treating Calu-6 cells with forskolin caused a 100-fold increase in steady-state endogenous hREN mRNA but no increase in hREN promoter activity in transient transfections or in nuclear runoff transcription assays. Nevertheless, de novo transcription and translation were necessary for adenosine 3',5'-cyclic monophosphate (cAMP)-mediated induction. Our results suggest that multiple regulatory elements regulate basal transcriptional activity of the hREN gene and the increase in hREN mRNA by cAMP may be mediated by posttranscriptional mechanisms.
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PMID:Transcriptional and posttranscriptional mechanisms regulate human renin gene expression in Calu-6 cells. 876 Feb 48

We examined the DNA sequence and transcription factor requirements for cAMP-induced transactivation of the human renin promoter using Calu-6 cells that express human renin mRNA endogenously. A series of constructs containing 896 base pairs of human renin 5'-flanking DNA fused to the luciferase gene and containing either the native, a consensus, or a nonfunctional cAMP response element (CRE) were used to assess DNA sequence requirements mediating the cAMP response. Expression vectors encoding the CREB-1 transcription factor, a dominant negative mutant form of CREB-1, and the catalytic subunit of protein kinase A (PKA) were used to assess transcription factor requirements mediating the cAMP response. Forskolin treatment alone only caused a 2-3-fold activation of the HREN promoter in Calu-6 cells, but nearly a 10-fold activation in JEG-3 cells, which do not express renin but are highly responsive to cAMP. Gel shift assays revealed the binding of five specific DNA-protein complexes consisting of the ATF-1 and CREB-1 transcription factors, one of which was an ATF-1.CREB-1 heterodimer suggesting the potential for regulation of CREB-1 activity by ATF-1. However, over-expression of CREB-1 did not significantly enhance forskolin-induced human renin transcriptional activity. Transfection of both Calu-6 and JEG-3 cells with a PKA expression vector resulted in a 10-fold induction of human renin transcriptional activity in constructs containing the native or consensus CRE and 5-fold activation in a construct containing a nonfunctional CRE. We confirmed that the PKA response has both a CREB-dependent and CREB-independent component by demonstrating that the PKA response was abolished by co-transfection of a dominant negative mutant form of CREB-1 into cells containing the native or consensus CRE construct but not in cells containing the nonfunctional CRE construct. We therefore conclude that the human renin promoter can be transcriptionally activated in a renin expressing cell line through the cAMP-PKA pathway and is mediated by both a CREB-dependent and CREB-independent mechanism.
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PMID:Transactivation of the human renin promoter by the cyclic AMP/protein kinase A pathway is mediated by both cAMP-responsive element binding protein-1 (CREB)-dependent and CREB-independent mechanisms in Calu-6 cells. 899 53

Calu-6 cells were characterized for studying the transcriptional regulation of the human renin gene. Analysis of cis-acting elements of the renin promoter showed the highest activity within the first 582 bp in serum-free conditions and of the 892 bp in the presence of serum. cAMP activates renin mRNA synthesis parallel to renin production (20-fold increase) as well renin promoter activity (2-fold). cAMP response element and the (-77 to -67) element are both necessary for activation of the renin promoter but do not act independently. Functional analysis of Intron A revealed the presence of a silencer specific to renin-producing cells.
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PMID:Regulation of human renin secretion and gene transcription in Calu-6 cells. 916 95

Renin catalyzes the rate-limiting step of the renin-angiotensin system, which regulates blood pressure and electrolyte homeostasis. To determine cell-specific human renin gene control elements, the transcriptional activity of promoter regions up to position -8876 was studied in renin-expressing cells. A positive regulatory region conferring approximately 57-fold higher transcriptional activity to the human renin gene promoter in chorionic cells was identified between nucleotides -5777 and -5552. It had the orientation-independent activity typical of classical enhancers. It also conferred approximately 59-fold higher transcriptional levels from the heterologous simian virus 40 (SV40) promoter in chorionic cells and approximately 6-fold higher transcriptional levels in Calu-6 and As4.1 cells, whereas no effect was measured in non-renin-expressing cells. DNase I footprinting showed that this enhancer contains three binding sites for chorionic cell nuclear extracts. Functional analysis suggested that the activity of the enhancer is regulated by differential mechanisms in the three renin-expressing cells involving a complex arrangement of AP-1 motifs binding cell-specific members of the basic leucine zipper family of transcription factors. Thus, our results demonstrate that this enhancer plays a key role in the expression of the human renin gene in the chorion and may also be involved in its regulated expression in other tissues.
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PMID:A novel distal enhancer confers chorionic expression on the human renin gene. 973 95

The human carcinoma-derived cell line Calu-6 has previously been demonstrated to endogenously express human renin (hREN) mRNA and to markedly increase steady-state hREN mRNA levels (100-fold after 24 hours) in response to analogues of cAMP and postreceptor activators of adenylyl cyclase such as forskolin. However, both transfection analysis using hREN promoter-reporter constructs and nuclear run-on experiments suggest that transcriptional activity alone cannot account for this level of induction. We performed primer extension, reverse transcription-polymerase chain reaction, and 3' rapid amplification of cDNA ends to compare hREN mRNA between unstimulated and forskolin-stimulated cells. We demonstrate that hREN mRNA is identical under both conditions with respect to (1) utilization of the appropriate transcription start site, (2) processing of renin mRNA, and (3) utilization of the proper polyadenylation site and length of the poly-A tail. To address the mechanism of induction caused by cAMP, we used transcriptional inhibition and measured decay of hREN mRNA before and after forskolin or phorbol ester treatment. Experiments with both actinomycin D and 5, 6-dichlororibofuranosylbenzimidazole (DRB) showed that forskolin treatment markedly stabilized hREN mRNA in Calu-6 cells. A 2.3-fold increase in hREN mRNA half-life was also observed after treatment of Calu-6 cells with phorbol ester. Experiments with DRB demonstrated a similar robust stabilization of hREN mRNA after forskolin and phorbol ester treatment. These data demonstrate that the induction in hREN mRNA in response to both cAMP and phorbol ester occurs by a mechanism involving a posttranscriptional component.
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PMID:Human renin mRNA stability is increased in response to cAMP in Calu-6 cells. 1008 6

Renin secretion can be stimulated by ATP via purinergic P2Y receptors. ATP is a cotransmitter with norepinephrine and is released from the cytosol during cell damage. Such release could account for the de novo renin expression seen in the proximal tubule in renal disease and in myocardial infarct borders. Whereas most P2Y purinoceptor subtypes utilize phosphoinositide signal-transduction pathways, the effector mechanisms of the subtype P2Y(11) also involve increases in cAMP, a well-known renin secretagogue and stimulus to renin production. The present study tested the effect of ATP on human renin gene (REN) promoter activity and the role of P2Y(11). By means of reverse transcriptase-polymerase chain reaction, we found that renin-expressing Calu-6 cells express P2Y(11) mRNA. Expression was also detected in the brain, kidney, testis, muscle, liver, and spleen. We made a novel cell line (Calu-6/P2Y11) in which P2Y(11) cDNA, under the control of a strong promoter, was stably integrated into genomic DNA. These cells produced P2Y(11) mRNA during culture. Treatment of Calu-6/P2Y11 cells with 1 mmol/L ATP caused a 3-fold increase in renin mRNA and protein over 36 hours. Transient transfection of Calu-6/P2Y11 cells with constructs containing 896 bp of human REN 5'-flanking DNA linked to the luciferase reporter gene led to a 5.8+/-0.6-fold increase (mean+/-SEM) in reporter activity in response to ATP (P=0.0015). In contrast, UTP produced only a 1.4+/-0.1-fold increase (P=0.016). For ADP, it was 1.7+/-0.1-fold (P=0.011). The response profile was ATP>ADP>AMP=adenosine=0, consistent with a P2Y(11) effect. Mutation of the cAMP response element (CRE) located at -222 in the REN promoter DNA abolished the effect of ATP. Furthermore, ATP induced a rapid, time-dependent increase in the phosphorylation of CRE binding protein (CREB) and activating transcription factor-1. These data implicate a cAMP pathway in mediation of the P2Y(11) effect. In conclusion, we have made a novel cell line that overexpresses the P2Y(11) purinoceptor. Stimulation of these cells by ATP activates a cAMP signal-transduction pathway that phosphorylates CREB and stimulates renin promoter activity via the CRE at -222. The data raise the possibility of a contribution of ATP/P2Y(11) effects to sympathetic stimulation of renin, as well as to responses in renin seen after tissue damage, such as in kidney disease and myocardial infarction.
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PMID:Capacity for purinergic control of renin promoter via P2Y(11) receptor and cAMP pathways. 1111 31

-We previously reported that thyroid hormone stimulates renin synthesis in vivo and in vitro. Here, we analyzed the 5'-flanking sequence of the human renin gene for promoter activity responsive to thyroid hormone using Calu-6 cells, which secrete renin endogenously and express thyroid hormone receptor-ss. The luciferase reporter gene was cloned together with 5'-flanking portions of the human renin gene of various lengths into the pGL3-Basic vector. Luciferase activity assays were performed using the Dual Luciferase Reporter Assay System. 3,3',5-Triiodo-L-thyronine stimulated the promoter activity of pGL3-Basic-1111/+12 and pGL3-Basic-1298/+12 by 2.3+/-0.1- and 1.7+/-0.1-fold, respectively. Shorter constructs (pGL3-Basic-144/+12, pGL3-Basic-226/+12, pGL3-Basic-452/+12, and pGL3-Basic-953/+12) were not stimulated by thyroid hormone. These results suggest that there is a possible thyroid hormone response element (5'-AGG TCA GGT CAc aat GTT CCT-3') between nucleotides -1111 and -953. In 3 constructs with site-directed mutations in this sequence, basal promoter activities were significantly increased, whereas promoter activation by thyroid hormone was abolished. Electrophoretic mobility shift assays showed that the -1111/-953 DNA fragment of the intact human renin gene was bound to nuclear proteins of Calu-6 cells; however, none of the 3 mutant probes were bound to any nuclear proteins. These results suggest that thyroid hormone stimulates the promoter activity of the human renin gene through thyroid hormone response element-dependent mechanisms in Calu-6 cells.
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PMID:Thyroid Hormone Stimulates Renin Gene Expression Through the Thyroid Hormone Response Element. 1120 63

Stabilization and correct localization of mRNA are important features of renin synthesis. To elucidate the molecular basis of cAMP-mediated posttranscriptional control via mRNA stabilization, we analyzed the interaction of human preprorenin (hREN) mRNA 3'-untranslated region (3'-UTR) with proteins of renin synthesizing Calu-6 cells and investigated their functional impact on messenger integrity. To identify hREN mRNA binding proteins, electrophoretic mobility shift assays, UV cross-linking and RNA-affinity chromatography with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were performed. The following six proteins were unambiguously identified as hREN mRNA 3'-UTR binding proteins: hnRNP E1 (synonyms alpha-CP or PCBP), hnRNP K, dynamin, nucleolin, YB-1, and MINT-homologous protein. All proteins contain various RNA binding motifs, and most have been described in the context of mRNA binding and mRNA stabilization. Four proteins for which antibodies were available were verified by immunological techniques (dynamin, nucleolin, hnRNP E1, and YB-1). Forskolin, an activator of cAMP synthesis, considerably stimulates renin synthesis via inhibition of REN mRNA decay. Functionally, this cAMP-based mRNA stabilization is accompanied by a 3- to 6-fold upregulation of REN mRNA binding proteins. RNase degradation assays confirm that 3'-UTR binding proteins are able to protect and stabilize REN mRNA in vitro.
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PMID:Posttranscriptional control of renin synthesis: identification of proteins interacting with renin mRNA 3'-untranslated region. 1260 Aug 97

Production of renin is critically dependent on modulation of REN mRNA stability. Here we sought to elucidate the molecular mechanisms involved. Transfections of renin-expressing Calu-6 cells with reporter constructs showed that a cis-acting 34-nucleotide AU-rich "renin stability regulatory element" in the REN 3'-untranslated region (3'-UTR) contributes to basal REN mRNA instability. Yeast three-hybrid screening with the REN 3'-UTR as bait isolated HADHB (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) beta-subunit) as a novel REN mRNA-binding protein. Recombinant HADHB bound specifically to the 3'-UTR of REN mRNA, as did the known mRNA stabilizers HuR and CP1 (poly(C)-binding protein-1). This required the renin stability regulatory element. Forskolin, which augments REN mRNA stability in Calu-6 cells, increased binding of several proteins, including HuR and CP1, to the REN 3'-UTR, whereas 4-bromocrotonic acid, a specific thiolase inhibitor, decreased binding and elevated renin protein levels. Upon decreasing HADHB mRNA with RNA interference, renin protein and mRNA stability increased, whereas RNA interference against HuR caused these to decrease. Immunoprecipitation and reverse transcription-PCR of Calu-6 extracts confirmed that HADHB, HuR, and CP1 each associate with REN mRNA in vivo. Intracellular imaging revealed distinct localization of HADHB to mitochondria, HuR to nuclei, and CP1 throughout the cell. Immunohistochemistry demonstrated enrichment of HADHB in renin-producing renal juxtaglomerular cells. In conclusion, HADHB, HuR, and CP1 are novel REN mRNA-binding proteins that target a cis-element in the 3'-UTR of REN mRNA and regulate renin production. cAMP-mediated increased REN mRNA stability may involve stimulation of HuR and CP1, whereas REN mRNA decay may involve thiolase-dependent pathways.
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PMID:HADHB, HuR, and CP1 bind to the distal 3'-untranslated region of human renin mRNA and differentially modulate renin expression. 1293 94

It is now recognized that post-transcriptional mechanisms are pivotal to renin production. These involve factors that modulate renin mRNA stability. In 2003 new data has emerged from work in Australia and Germany that has identified several of the, as many as, 20 or so proteins involved. These include CP1 (hnRNP E1), HuR, HADHB, dynamin, nucleolin, YP-1, hnRNP K and MINT-homologous protein. Cyclic AMP (cAMP) is a crucial regulator of renin secretion as well as transcriptional and post-transcriptional control of expression. Many of the RNA-binding proteins that were identified responded to forskolin, increasing in amount by two to 10-fold. The cAMP mechanisms that regulate renin mRNA target, at least in large part, other genes that presumably encode some of these proteins. The increase in the expression of these then facilitates, sequentially, renin mRNA stabilization and destabilization. Our data, using a battery of different techniques, confirm that CP1 and HuR stabilize renin mRNA, whereas HADHB causes destabilization. These proteins target cis-acting C-rich sequences (in the case of CP1) and AU-rich sequences (HuR) in the distal region of the 3'-untranslated region of renin mRNA. We found HADHB was enriched in juxtaglomerular cells and that that within Calu-6 cells HADHB, HuR and CP1 all localized in nuclear subregions, as well as cytoplasm (HADHB and CP1) and mitochondria (HADHB) commensurate with the role each plays in control of renin mRNA stability. The specific proteins that bind to human renin mRNA have begun to be revealed. Cyclic AMP upregulates the binding of several of these proteins, which in turn affect renin mRNA stability and thus overall expression of renin.
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PMID:cAMP controls human renin mRNA stability via specific RNA-binding proteins. 1528 47


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