Creb activation assay

Rows in the heatmap represent gene expression levels and columns represent each sample. Hierarchical clustering of the differentially expressed phosphatases using Morpheus revealed the sustained reduction in the expression of PP2A regulatory subunits Ppp2r2c and Ppp2r3a, especially for Ppp2r3a whose expression pattern was in accordance with the sustained CREB phosphorylation in the development of MCT-induced PH Fig.

In addition, intracellular zinc was reported to regulate CREB-mediated transcriptional activity [ 38 ]. Role of zinc in regulation of CREB-mediated transcription and cell proliferation. CREB was originally identified, due to its response to cAMP signaling, while prostacyclin and its analogues, the widely used drug for treatment of patients with PH, increases intracellular cAMP formation via adenylyl cyclase stimulation. CREB have three functional isoforms alpha, beta and delta, produced by alternative splicing.

Hypoxia selectively activates the CREB family of transcription factors in the in vivo lung, contributing to pulmonary vascular remodeling and PH [ 7 ]. Unexpectedly, the same group revealed that deletion of alpha and delta isoforms of CREB in mice contributed to elevated pulmonary vascular resistance both in normoxia and following exposure to hypoxic conditions for three weeks [ 41 ].

They concluded that alpha and delta isoforms of CREB regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance and to maintain the normal alveolar structure [ 41 ]. In fact, this paradoxical role of CREB was also appeared in the other conditions [ 4 , 42 ].

Similarly, we were able to identify the CRE motif within the promoters of The exact mechanisms underlying these divergent results remained unclear. However, the divergent results may be explained by the feature of bidirectional promoters. The subsequent study revealed a strong preference for CREB binding at these bidirectional promoters, when CREB was bound at the bidirectional promoters, promoter activity in one direction was upregulated, whereas promoter activity in the other direction was repressed [ 45 ].

Although the concentration of the protein kinase inhibitors used in this study were in accordance to with the instructions provided by the manufacturers and proved to be effective, pretreatment of PASMCs with various protein kinase inhibitors alone failed to significantly inhibit sustained phosphorylation of CREB, thus coincided with previous study showing failure to determine the exact upstream protein kinases in hypoxia-treated PC12 cells [ 46 ].

However, the identification of downregulated multiple protein kinases indicated limited effect of protein kinases on sustained CREB phosphorylation. Most studies on CREB pathway focused on the protein kinases. The regulatory B subunit dictates subcellular localization and substrate specificity, and 20 different regulatory subunits have been identified.

The Ppp2r2c and Ppp2r3a determine the substrate selectivity and catalytic activity[ 48 ], The Ppp2r3a expression was persistently downregulated with a pattern explained the sustained CREB phosphorylation. Therefore, it is likely that the reduced expression of PP2A regulatory subunits, in particular Ppp2r3a, may account for the impaired PP2A activity observed by the previous study in PASMCs from patients with pulmonary arterial hypertension [ 49 ].

Multiple regulatory subunits of PP1 were identified as differential expression in this study, including upregulated and downregulated subunits. In the present study, it was showed that hypoxia enhanced and prolonged serum-induced CREB phosphorylation. It was possible that the prolonged and enhanced serum-induced CREB phosphorylation induced by hypoxia may be due to the reduced phosphatases that negatively regulate CREB phosphorylation. The labile zinc was reported to regulate CREB activation [ 38 , 52 ] and inhibit protein phosphatases [ 32 , 35 , 36 ].

The reduced expression of multiple protein phosphatases were identified and may be associated with elevated intracellular zinc in MCT-induced PH. As an intracellular signaling mediator, zinc is involved in a variety of biological processes. Interestingly, a role for labile zinc in regulating hypoxic pulmonary vasoconstriction has been reported [ 53 , 54 ].

The ZIP12 is localized to the plasma membrane and capable of mediating the influx of extracellular zinc into the cytosol. In addition, intracellular zinc derived from ZIP12 has been reported to regulate neurulation and neurite extension through CREB activation [ 38 ]. A proposed model for the role and regulation of CREB in the proliferation of pulmonary artery smooth muscle cells in pulmonary hypertension.

Other data generated or analysed during this study are included in this article and its supplementary information files. Rabinovitch M. Molecular pathogenesis of pulmonary arterial hypertension. J Clin Investig. J Am Coll Cardiol. Regulation of gene expression by cyclic GMP. Circ Res. Ichiki T. Role of cAMP response element binding protein in cardiovascular remodeling: Good, bad, or both?

Arterioscler Thromb Vasc Biol. Apoptosis induced by inhibition of cyclic AMP response element-binding protein in vascular smooth muscle cells. Hypoxia selectively activates the CREB family of transcription factors in the in vivo lung.

Redox Biol. Mol Med Rep. CREB depletion in smooth muscle cells promotes medial thickening, adventitial fibrosis and elicits pulmonary hypertension.

Pulm Circ. A novel secreted-cAMP pathway inhibits pulmonary hypertension via a feed-forward mechanism. Cardiovasc Res. CPT1 regulates the proliferation of pulmonary artery smooth muscle cells through the AMPK-pp21 pathway in pulmonary arterial hypertension. Mol Cell Biochem. RNA sequencing analysis of monocrotaline-induced PAH reveals dysregulated chemokine and neuroactive ligand receptor pathways. Aging Albany NY.

Transcriptomic analysis identifies Toll-like and Nod-like pathways and necroptosis in pulmonary arterial hypertension. J Cell Mol Med. The zinc transporter ZIP12 regulates the pulmonary vascular response to chronic hypoxia.

Tyrosine hydroxylase transcription depends primarily on cAMP response element activity, regardless of the type of inducing stimulus. Mol Cell Neurosci. Genomic assessment of a multikinase inhibitor, sorafenib, in a rodent model of pulmonary hypertension.

Physiol Genomics. Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues. Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension. Respir Res. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu Rev Biochem. Kinetic studies and molecular bases for the resistance of protein kinase CK2. Eur J Biochem.

Naunyn Schmiedebergs Arch Pharmacol. Regulation of human insulin gene transcription by the immunosuppressive drugs cyclosporin A and tacrolimus at concentrations that inhibit calcineurin activity and involving the transcription factor CREB. Inhibition of cAMP-responsive element-mediated gene transcription by cyclosporin A and FK after membrane depolarization. J Biol Chem. Mol Pharmacol. Transcription factor cAMP response element modulator Crem restrains Pdgf-dependent proliferation of vascular smooth muscle cells in mice.

Pflugers Arch. Complex formation lower arrow could be supershifted with ATF2 antibody upper arrow. D The same mutation from the CREB II mut oligonucleotide, resulting in a lack of specific CREB complex formation, was introduced into the luciferase reporter constructs, and luciferase activity was measured after transfection in Huh7 cells, as described above.

Either the mutation construct 1B mut or the deletion as in construct 2 of the upstream CREB II site resulted in a tremendous decrease in luciferase activity compared with constructs 1 and 1B. C Relative luciferase activity was assessed in transfection experiments.

The reporter plasmids were transfected into Huh7 cells and luciferase activity was determined and compared with controls comprising either both CREB sites construct 1 , one construct 1B , or no CREB sites construct 2.

Thus we next investigated whether cAMP signalling pathways also mediate inducible S promoter activation, as indicated by the cotransfection experiment of HBV wild-type and the PKA expression plasmid fig 1. Constructs 2—5 had low basal luciferase expression but luciferase activity was strongly induced by CREB and even further enhanced by PKA in constructs 2 and 3.

Constructs 4 and 5 also displayed some inducible activity but their relative response to CREB or PKA was remarkably reduced compared with constructs 2 or 3.

Construct 1 conferred the highest basal activity as expected from figs 3—5. However, the highest relative induction was seen in constructs 2 and 3 whereas this inducible activity was remarkably lower in constructs 4 and 5. Constructs 1—3 contain a putative Ets-1 binding motif, which was not present in constructs 4 and 5. Stimulation with forskolin resulted in maximal activation of relative luciferase expression.

Stimulation with forskolin resulted in maximal activation of relative luciferase expression that could not be further stimulated by cotransfecting the PKA expressing construct fig 6B. These experiments indicated that the putative Ets binding motif might be involved in mediating PKA dependent promoter activation. We thus mutated the putative Ets sequences in this part of the promoter region constructs 3-mut-I and 3-mut-II, fig 7A.

The mutant Ets constructs were then cotransfected with the catalytic subunit of PKA. These experiments showed that the intact Ets motif was necessary to confer full PKA mediated gene activation of the S promoter fig 7B. The constructs used for transfection experiments are shown. B Luciferase activity was determined in transfection experiments.

Mutations of the Ets motif resulted in reduced inducibility by PKA, similar to the complete deletion of the sequence constructs 4 and 5. C In electrophoretic mobility shift assay EMSA experiments, nuclear extracts from either unstimulated Huh7 cells lane 2 or forskolin stimulated Huh7 cells lanes 3—10 were incubated with a radioactive labelled double stranded oligonucleotide representing the Ets motif.

In order to reduce an unspecific signal top arrow , nuclear extracts were first preincubated with unlabelled mutated Ets oligonucleotide.

We next attempted to further characterise possible transcription factors involved in binding to the Ets motif. The family of Ets transcription factors consists of more than 40 different proteins, and protein-protein interactions for example, between AP1 and Ets are common. Nuclear extracts from either unstimulated or forskolin stimulated Huh7 cells were incubated with radioactive labelled double stranded Ets or mutated Ets oligonucleotides in EMSA experiments.

Only for the Ets wild-type oligo did forskolin stimulation result in formation of a novel complex fig 7C, bottom arrow, and data not shown. These experiments showed a reduction in complex formation of the slower migrating forskolin inducible signal fig 7C, bottom arrow when extracts were incubated with antibodies directed against CREB1 and ATF4 fig 7C.

Transcriptional regulation via adenylate cyclase signalling pathways is mediated by a family of cyclic AMP responsive nuclear factors, including CREB. The activation function of CRE binding proteins is modulated by phosphorylation by several kinases for example, PKA and other cellular cofactors. Regulation of HBV-S gene expression is crucial for viral pathogenesis, as the envelope proteins are essential for virus formation, infectivity, and immunogenicity.

Interestingly, these regulatory regions are located within the transcribed pre-S2 region of the HBV genome. Similar promoter organisations with regulatory elements located in intronic regions have been shown for a variety of promoters, such as CD95 or caspase 8.

Additional observations indicate the functional relevance of these CREB sites. This would argue that the downstream CREB site may functionally compensate for loss of the upstream site in HBV deletion constructs, although this was not observed in our experiments applying the luciferase reporter system.

Our reporter gene experiments showed that a sequence in the pre-S2 region comprising an Ets motif was significantly involved in mediating cAMP dependent activation. However, we cannot exclude the fact that more than the identified factors are involved in this process. You will be able to get a quick price and instant permission to reuse the content in many different ways.

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Log in via Institution. Email alerts. Article Text. Article menu. Abstract Background and aims: CREB cAMP response element binding protein transcription factors are key regulators of homeostatic functions in the liver, and CRE binding is increased in hepatic inflammation. Statistics from Altmetric. Cloning of luciferase expression plasmids, luciferase, and beta-galactosidase reporter assays A computer based search for potential transcription factor binding sites was performed using MatInspector software.

View this table: View inline View popup. Exp Cell Res ; : — Nature ; : —3. Nature ; : — Hepatology ; 39 : — Mayr B , Montminy M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol ; 2 : — Papavassiliou AG. Anticancer Res ; 14 : —5. This product detects total CREB in human, mouse, rat and monkey samples. It also detects phosphorylated CREB1 in human, mouse, rat and hamster samples.

The members of this family, named bZIP, share a dimerization domain with a leucine zipper motif and a DNA-binding domain rich in basic residues lysines and arginines.

The CREB proteins activate transcription of target genes in response to a diverse array of stimuli, such as peptide hormones, growth factors and neuronal activity.

Upon cell stimulation with forskolin, an activator of adenylyl cyclase, the kinases are activated by cAMP production and translocate to the nucleus where they phosphorylate CREB at Ser Therefore, CREB is almost exclusively nuclear in both unstimulated and stimulated cells. These results are provided for demonstration purposes only.

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