Putative cAMP Response Element Research Articles

The Hallucinogenic Serotonin2A Receptor Agonist, 2,5-Dimethoxy-4-Iodoamphetamine, Promotes cAMP Response Element Binding Protein-Dependent Gene Expression of Specific Plasticity-Associated Genes in the Rodent Neocortex.

Psychedelic compounds that target the 5-HT2A receptor are reported to evoke psychoplastogenic effects, including enhanced dendritic arborization and synaptogenesis. Transcriptional regulation of neuronal plasticity-associated genes is implicated in the cytoarchitectural effects of serotonergic psychedelics, however, the transcription factors that drive this regulation are poorly elucidated. Here, we addressed the contribution of the transcription factor cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) in the regulation of neuronal plasticity-associated genes by the hallucinogenic 5-HT2A receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI). In vitro studies with rat cortical neurons indicated that DOI enhances the phosphorylation of CREB (pCREB) through mitogen-activated protein (MAP) kinase and calcium/calmodulin dependent kinase II (CaMKII) pathways, with both cascades contributing to the DOI-evoked upregulation of Arc, Bdnf1, Cebpb, and Egr2 expression, whilst the upregulation of Egr1 and cFos mRNA involved the MAP kinase and CaMKII pathway respectively. We observed a robust DOI-evoked increase in the expression of several neuronal plasticity-associated genes in the rat neocortex in vivo. This DOI-evoked upregulation of neuronal plasticity-associated genes was completely blocked by the 5-HT2A receptor antagonist MDL100,907 in vitro and was also abrogated in the neocortex of 5-HT2A receptor deficient mice. Further, 5-HT2A receptor stimulation enhanced pCREB enrichment at putative cAMP response element (CRE) binding sites in the Arc, Bdnf1, Cebpb, cFos, but not Egr1 and Egr2, promoters in the rodent neocortex. The DOI-mediated transcriptional induction of Arc, cFos and Cebpb was significantly attenuated in the neocortex of CREB deficient/knockout (CREBαδ KO) mice. Collectively, these results indicate that the hallucinogenic 5-HT2A receptor agonist DOI leads to a rapid transcriptional upregulation of several neuronal plasticity-associated genes, with a subset of them exhibiting a CREB-dependent regulation. Our findings raise the intriguing possibility that similar to slow-acting classical antidepressants, rapid-action serotonergic psychedelics that target the 5-HT2A receptor may also recruit the transcription factor CREB to enhance the expression of neuronal plasticity-associated genes in the neocortex, which could in turn contribute to the rapid psychoplastogenic changes evoked by these compounds.

Characterization of the G protein-coupled receptor kinase 6 promoter reveals a functional CREB binding site.

BackgroundG protein-coupled receptor kinase 6 (GRK6) is part of the G protein-coupled receptor kinase family, whose members act as key regulators of seven-transmembrane receptor signalling. GRK6 seems to play a role in regulation of inflammatory processes, but mechanisms of transcriptional regulation of GRK6 expression in inflammatory cell lines have not been characterized. Protein kinase C (PKC) signalling is also involved in inflammatory regulation and an impact of PKC activation on GRK6 protein expression was described previously. Thus, the aim of this study was to 1) characterize the GRK6 promoter, and 2) investigate a potential influence of PKC on GRK6 expression.MethodsFive deletion constructs of the GRK6 promoter were cloned. After transient transfection into a human T cell line, promoter activity was assessed using luciferase reporter gene assays. Putative transcription factor binding sites were identified, mutated, and binding was investigated using electrophoretic mobility shift assays (EMSA). Following stimulation with a PKC activator, GRK6 expression on mRNA and protein levels was assessed by reverse transcriptase qPCR and Western blots.ResultsInvestigation of the GRK6 promoter revealed a putative cAMP responsive element (CRE), whose mutation led to decreased promoter activity (p = 0.0006). Functionality of the CRE binding protein (CREB) binding site was verified in EMSA blots. Stimulation with a PKC activator resulted in decreased GRK6 promoter activity (p = 0.0027), mRNA (p = 0.04) and protein expression.ConclusionWe characterized the human GRK6 promoter and identified promoter activity to be influenced by a CREB binding site. PKC might be one determinant contributing to altered GRK6 expression.

Transcriptional Regulation of ZNF638 in Thermogenic Cells by the cAMP Response Element Binding Protein in Male Mice.

Zinc finger factors are implicated in a variety of cellular processes, including adipose tissue differentiation and thermogenesis. We have previously demonstrated that zinc finger protein 638 (ZNF638) is a transcriptional coactivator acting as an early regulator of adipogenesis in vitro. In this study, we show, to our knowledge for the first time, that, in vivo, ZNF638 abounds selectively in mature brown and subcutaneous fat tissues and in fully differentiated thermogenic adipocytes. Furthermore, gene expression studies revealed that ZNF638 is upregulated by cAMP modulators in vitro and by cold exposure and by pharmacological stimulation of β-adrenergic signaling in vivo. In silico analysis of the upstream regulatory region of the ZNF638 gene identified two putative cAMP response elements within 500 bp of the ZNF638 transcription start site. Detailed molecular analysis involving EMSA and chromatin immunoprecipitation assays demonstrated that cAMP response element binding protein (CREB) binds to these cAMP response element regions of the ZNF638 promoter, and functional studies revealed that CREB is necessary and sufficient to regulate the levels of ZNF638 transcripts. Taken together, these results demonstrate that ZNF638 is selectively expressed in mature thermogenic adipocytes and tissues and that its induction in response to classic stimuli that promote heat generation is mediated via CREB signaling, pointing to a possible novel role of ZNF638 in brown and beige fat tissues.

Sympathetic Regulation of Slc2a4 Gene Expression: Participation of a Putative cAMP Responsive Element (CRE) Site in the Slc2a4 Promoter.

Studies have indicated that sympathetic activity enhances GLUT4 expression (Slc2a4 gene) by activating beta-adrenergic receptors. This could be mediated by a direct enhancer effect of cyclic AMP-responsive element binding protein (CREB) and family members upon Slc2a4 gene. However, a cAMP responsive element (CRE) in Slc2a4 promoter has never been demonstrated. Slc2a4 CRE-site was searched by in silico analysis. In skeletal muscles from rats displaying high sympathetic activity (SHR), Slc2a4 CRE-site was investigated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay; and Slc2a4 expression was analyzed by RT-qPCR. Functional activity of the CRE-site was investigated by luciferase assay, 2 hours after 8-br-cAMP stimulation, in 3T3L1 adipocytes transientely transfected with native and mutated CRE-sites. In silico analysis indicated the -480/-473 segment as a putative CRE-site, with 62.5% of identity to CRE consensus sequence, and highly preserved in mouse, rat and human. CREB/CREM binding in this CRE-site was confirmed to occur in vitro (EMSA) and in vivo (ChIP assay). Enhancer activity of this segment in Slc2a4 transcription was confirmed in 3T3-L1 cells. Finally, in extensor digitorum longus muscle from SHR, 80% increase in Slc2a4 mRNA expression was observed to be accompanied by increased CREB/CREM binding into the CRE-site both in vitro and in vivo. This study demonstrates the presence of a functional CRE-site at -480/-473 sequence of the Slc2a4 gene. This CRE-site has an enhancing activity on Slc2a4 expression and participates in the Slc2a4 increased expression observed in glycolytic muscles of rats displaying high sympathetic activity.

NR6A1 couples with cAMP response element binding protein and regulates vascular smooth muscle cell migration

Vascular smooth muscle cell (VSMC) migration is implicated in atherosclerosis and restenosis. Nuclear receptor subfamily 6, group A, member 1 (NR6A1) is involved in regulating embryonic stem cell differentiation, reproduction, neuronal differentiation. Functional cooperation between cAMP response element modulator tau (CREMtau) and NR6A1 can direct gene expression in cells. cAMP response element binding protein (CREB) plays a key role in VSMC migration. In this study, we sought to determine whether CREB involved in NR6A1-modulated VSMC migration. VSMCs treated with platelet-derived growth factor-BB (PDGF-BB) displayed reduced mRNA and protein levels of NR6A1. Adenovirus-mediated expression of NR6A1 (Ad-NR6A1) could inhibit PDGF-BB- and serum-induced VSMC migration. The mRNA and protein expressions of secreted phosphoprotein 1 (SPP1) were down-regulated by NR6A1 overexpression. SPP1 promoter reporter activity was repressed by NR6A1. NR6A1 was found to physically couple with nuclear actin and the large subunit of RNA polymerase II. Furthermore, we showed that CREB interacted with NR6A1 in VSMCs. NR6A1 overexpression repressed cAMP response element (CRE) activity. ChIP assay revealed that NR6A1 bind to SPP1 promoter. Luciferase reporter assay showed that NR6A1 regulated SPP1 promoter activity via a putative CRE site. Adenovirus mediated local NR6A1 gene transfer attenuated stenosis after balloon-induced arterial injury in Sprague–Dawley rats. Taken together, this study provided experimental evidence that NR6A1 modulated SPP1 expression via its binding with CREB protein in VSMCs. We also revealed a NR6A1-CREB-SPP1 axis that serves as a regulatory mechanism for atherosclerosis and restenosis.

Functional cyclic AMP response element in the breast cancer resistance protein (BCRP/ABCG2) promoter modulates epidermal growth factor receptor pathway- or androgen withdrawal-mediated BCRP/ABCG2 transcription in human cancer cells

Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways.

Characterization of a far upstream located promoter expressing the acetyl-CoA carboxylase-alpha in the brain of cattle

The expression of the bovine acetyl-CoA carboxylase-alpha-encoding gene (ACACA) was known to be controlled by three promoters. Here, we characterized a fourth promoter (PI) located 41kb upstream of the adjacent nutritionally-regulated promoter PIA on bovine chromosome 19. Our results showed that PI is an intergenic promoter driving expression of ACACA and conceivably, by analogy with the homologous genomic arrangement in sheep a component of the chromatin-modifying complex gene (TADA2L). 5′-RACE experiments defined the 3′ boundary of the promoter and a novel exon 1 comprising 263bp. It features at position +226 an ORF encoding an N-terminally extended ACC-α enzyme. The PI sequence is GC-rich, has no TATA box and CAAT box, similar to the homologous promoters in sheep, mouse and human. Expression profiles showed that PI is the promoter driving expression of the dominant ACACA-transcript in brain. Reporter gene assays in HC-11 cells indicated that deletion of extended promoter segments harboring putative cAMP response elements (CRE) clustered in the distal promoter region and specificity protein 1 (Sp1) attachment sites lowered PI activity.

Transcriptional Regulation of BMP2 Expression by the PTH-CREB Signaling Pathway in Osteoblasts

Intermittent application of parathyroid hormone (PTH) has well established anabolic effects on bone mass in rodents and humans. Although transcriptional mechanisms responsible for these effects are not fully understood, it is recognized that transcriptional factor cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts, and that there is a communication between the PTH-CREB pathway and the BMP2 signaling pathway, which is important for osteoblast differentiation and bone formations. These findings, in conjunction with putative cAMP response elements (CREs) in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts. To test this hypothesis, we first demonstrated that PTH signaling activated CREB by phosphorylation in osteoblasts, and that both PTH and CREB were capable of promoting osteoblastic differentiation of primary mouse osteoblast cells and multiple rodent osteoblast cell lines. Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells. Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA), we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts. Together, these results demonstrate that the anabolic function of PTH signaling in bone is mediated, at least in part, by CREB transactivation of BMP2 expression in osteoblasts.

Transcriptional regulation of the chemokine co-receptor CCR5 by the cAMP/PKA/CREB pathway

The cyclic adenosine monophosphate (cAMP)-dependent signaling pathway directs the expression of several genes involved in diverse neuroendocrine, immune, metabolic, and developmental pathways. The primary effectors of this pathway are members of the cAMP response element binding (CREB) family of transcription factors, in particular the CREB-1 and cAMP response element modulator (CREM). Both these genes encode alternative splice variants that serve as activators or repressors in a context- and position-specific manner. Although the β-chemokine receptor CC chemokine receptor 5 (CCR5) has been identified on progenitor cells in the bone marrow, the regulatory mechanisms orchestrating its expression are not fully understood. Previous reports have identified putative cAMP response elements in the CCR5 promoter and have described a suppressive role of cAMP in CCR5 expression. In this study, the CD34+CD4+CCR5+ human bone marrow progenitor cell line TF-1 was used to investigate the detailed kinetics of CCR5 transcription in response to the elevation of intracellular cAMP levels and the underlying molecular events. We hypothesize that CCR5 transcription follows an asymmetrical sinusoidal pattern in TF-1 cells that parallels a protein kinase A-dependent alternating change in the ratio of activator pCREB-1-α,Δ to repressor pCREM-α,β isoforms. However, elevated CCR5 mRNA levels do not correlate with enhancement in infectivity with respect to the R5 human immunodeficiency virus type 1 (HIV-1) strain. Our results lend critical insight into the precise mechanism governing the cAMP-CCR5 axis in progenitor cells and pose interesting questions regarding its functional role in HIV-1 infection.

Altered adrenergic receptor signaling following traumatic brain injury contributes to working memory dysfunction

The prefrontal cortex is highly vulnerable to traumatic brain injury (TBI) and its structural and/or functional alterations as a result of TBI can give rise to persistent working memory (WM) dysfunction. Using a rodent model of TBI, we have described profound WM deficits following TBI that are associated with increases in prefrontal catecholamine (both dopamine and norepinephrine) content. In this study, we examined if enhanced norepinephrine signaling contributes to TBI-associated WM dysfunction. We demonstrate that administration of α1 adrenoceptor antagonists, but not α2A agonist, at 14 days post-injury significantly improved WM performance. mRNA analysis revealed increased levels of α1A, but not α1B or α1D, adrenoceptor in the medial prefrontal cortex (mPFC) of brain-injured rats. As α1A and 1B adrenoceptor promoters contain putative cAMP response element (CRE) sequences, we therefore examined if CRE-binding protein (CREB) actively engages these sequences in order to increase receptor gene transcription following TBI. Our results show that the phosphorylation of CREB is enhanced in the mPFC at time points during which increased α1A mRNA expression was observed. Chromatin immunoprecipitation (ChIP) assays using mPFC tissue from injured animals indicated increased phospho-CREB binding to the CRE sites of α1A, but not α1B, promoter compared to that observed in uninjured controls. To address the translatability of our findings, we tested the efficacy of the FDA-approved α1 antagonist Prazosin and observed that this drug improves WM in injured animals. Taken together, these studies suggest that enhanced CREB-mediated expression of α1 adrenoceptor contributes to TBI-associated WM dysfunction, and therapies aimed at reducing α1 signaling may be useful in the treatment of TBI-associated WM deficits in humans.

VEGF stimulates PKD‐mediated CREB‐dependent orphan nuclear receptor Nurr1 expression: Role in VEGF‐induced angiogenesis

New vessel formation is critical for solid tumor growth and it is primarily stimulated by the most potent angiogenic factor vascular endothelial growth factor (VEGF or VEGF-A165). VEGF promotes endothelial cell proliferation by initiating signaling cascades to increase gene transcription. Recent works showed that VEGF potently and rapidly induces expression of orphan nuclear receptor Nurr1 in endothelial cells. However, the signaling pathway for VEGF-induced Nurr1 expression and its role in VEGF-induced endothelial cell proliferation and angiogenic response have not been examined. In our study, we first show that VEGF significantly induces expression of Nurr1 mRNA, protein and its promoter activity in cultured endothelial cells. Furthermore, the promoter analysis shows that deletion of the putative cAMP-responsive element binding protein (CREB) site in the proximal region of the promoter markedly reduces VEGF-induced promoter activity whereas deletion of the upstream NF-κB site has moderate effect. Transfection of a dominant negative CREB mutant (K-CREB) or mutation of this putative CREB site in the Nurr1 promoter attenuates VEGF-induced Nurr1 expression. VEGF also stimulates the binding of nuclear CREB protein to its site in the Nurr1 promoter in vitro and in vivo. Moreover, using pharmacological inhibitors and molecular approaches, we show that VEGF-induced CREB activation is largely mediated by protein kinase C-dependent protein kinase D activation. Finally, our data indicate that knockdown of endogenous Nurr1 expression attenuates VEGF-induced endothelial cell proliferation, migration and in vivo matrigel angiogenesis, suggesting its potential importance in mediating VEGF-induced tumor angiogenesis.

Repression of Cellular Retinoic Acid-binding Protein II during Adipocyte Differentiation

In preadipocytes, retinoic acid (RA) regulates gene expression by activating the nuclear RA receptor (RAR) and its cognate intracellular lipid-binding protein CRABP-II. It was previously reported that RA inhibits adipocyte differentiation but only when administered early during the differentiation program. The data presented here indicate that the diminished ability of RA to activate RAR following induction of differentiation stems from down-regulation of CRABP-II. The observations show that expression of CRABP-II in preadipocytes is repressed by all three components of the classical hormonal mixture that induces adipocyte differentiation, i.e. isobutylmethylxanthine, insulin, and dexamethasone. Isobutylmethylxanthine-dependent activation of protein kinase A triggered the phosphorylation of the transcription factor cAMP-response element-binding protein, which induced the expression of the cAMP-response element-binding protein family repressor cAMP-response element modulator. In turn, cAMP-response element modulator was found to associate with a cognate response element in the CRABP-II promoter and to repress CRABP-II expression. The data further show that CRABP-II is a direct target gene for the glucocorticoid receptor and that it is subjected to dexamethasone-induced glucocorticoid receptor-mediated repression during adipogenesis. Finally, the observations demonstrate that permanent repression of CRABP-II in mature adipocytes is exerted by the master regulator of adipocyte differentiation CCAAT/enhancer-binding protein alpha and is directly mediated through CCAAT/enhancer-binding protein alpha-response elements in the CRABP-II promoter. Taken together, the observations emphasize the important role of CRABP-II in regulating the transcriptional activity of RA through RAR, and they demonstrate that repression of this gene is critical for allowing adipogenesis to proceed.

Increased cAMP Levels Modulate Transforming Growth Factor-β/Smad-induced Expression of Extracellular Matrix Components and Other Key Fibroblast Effector Functions

cAMP is a key messenger of many hormones and neuropeptides, some of which modulate the composition of extracellular matrix. Treatment of human dermal fibroblasts with dibutyryl cyclic AMP and forskolin antagonized the inductive effects of transforming growth factor-beta (TGF-beta) on the expression of collagen, connective tissue growth factor, tissue inhibitor of matrix metalloproteinase-1, and plasminogen activator inhibitor type I, four prototypical TGF-beta-responsive genes. Increased intracellular cAMP prevented TGF-beta-induced Smad-specific gene transactivation, although TGF-beta-mediated Smad phosphorylation and nuclear translocation remained unaffected. However, increased cAMP levels abolished TGF-beta-induced interaction of Smad3 with its transcriptional co-activator cAMP-response element-binding protein (CREB)-binding protein (CBP)/p300. Overexpression of the transcriptional co-activator CBP/p300 rescued Smad-specific gene transcription in the presence of cAMP suggesting that sequestration of limited amounts of CBP/p300 by the activated cAMP/CREB pathway is the molecular basis of this inhibitory effect. These findings were extended by two functional assays. Increased intracellular cAMP levels suppressed the inductive activity of TGF-beta to contract mechanically unloaded collagen lattices and resulted in an attenuation of fibroblast migration of mechanically induced cell layer wounds. Of note, cAMP and TGF-beta synergistically induced hyaluronan synthase 2 (HAS2) expression and hyaluronan secretion, presumably via putative CREB-binding sites adjacent to Smad-binding sites within the HAS2 promoter. Our findings identify the cAMP pathway as a potent but differential and promoter-specific regulator of TGF-beta-mediated effects involved in extracellular matrix homeostasis.

CAMP-response element binding protein (CREB) positively regulates mouse adiponectin gene expression in 3T3-L1 adipocytes

Adiponectin is expressed in adipose tissue by adipogenic transcription factors including PPARγ, C/EBPα, and ADD1/SREBP1c. Because cAMP-response element binding protein (CREB) is also a central transcriptional activator of adipocyte differentiation, we evaluated CREB to determine if it stimulates adiponectin gene expression. To accomplish this, we evaluated the effects of activated CREB on the promoter activity of the mouse adiponectin gene, and identified the cAMP-response element (CRE) in the promoter. The constitutively active form of CREB increased the promoter activity of the mouse adiponectin gene. In addition, transfection studies using 5′ serial deleted promoters revealed the presence of a putative CRE located between the −1250 and −1000bp region. Furthermore, an electrophoresis mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis demonstrated that CREB bound to the region between −1022 and −995 in the adiponectin promoter. Insulin-like growth factor (IGF-1), which activate CREB, increased the adiponectin promoter activity. However, this stimulation was prevented by the dominant negative form of CREB (ACREB) and pretreatment with PD098059, indicating that IGF-1 stimulate adiponectin expression through CREB phosphorylation via the ERK pathway. Importantly, the transactivation of adiponectin expression by CREB was inhibited by ATF3. Coimmunoprecipitation and GST pull-down assay revealed that ATF3 bound to CREB and prevented CREB phosphorylation induced during differentiation of 3T3-L1 adipocytes. Collectively, these findings demonstrate that CREB is a positive regulator of mouse adiponectin gene expression in adipocytes, which play an important role in the regulation of adiponectin expression in response to growth factor.

Identification and characterization of an alternative promoter of the human PGC-1α gene

The transcriptional regulator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1α expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1α transcript (designated PGC-1α-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1α-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca2+- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1α-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1α expression in contracting muscle.

Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

Cyclin D1 expression is elevated and Wnt10b is repressed by cAMP during the first few hours of adipogenesis. cAMP-responsive element-binding protein (CREB) is a primary target for cAMP signaling, and we have shown that activation of CREB promotes adipogenesis and adipocyte survival. Here we tested the impact of CREB on expression of cyclin D1 and wingless-related mouse mammary tumor virus integration site 10b (Wnt10b) in 3T3-L1 cells. Forced depletion of CREB blocked Bt(2)cAMP-stimulated cyclin D1 expression and basal Wnt10b gene expression. Two CREB-binding sites were identified in the Wnt10b promoter region. Ablation of either site partially blocked promoter activity, while mutation of both sites completely suppressed promoter activity. These results suggest that CREB activates transcription from both the cyclin D1 and Wnt10b gene promoters. What accounts for the differential regulation of cyclin D1 and Wnt10b genes by cAMP? Chromatin immunoprecipitation revealed CREB bound to the Wnt10b promoter in untreated preadipocytes but not following treatment with Bt(2)cAMP. CREB binding to the cyclin D1 promoter was detected in untreated cells and post-Bt(2)cAMP. Differences between CREB binding to the two genes correlated with increasing methylation of the Wnt10b promoter following Bt(2)cAMP treatment, whereas no methylation of the cyclin D1 promoter was observed. Treatment of cells with the methylase inhibitor 5-azacytidine restored CREB binding to the Wnt10b gene promoter and prevented the inhibition of Wnt10b RNA expression by Bt(2)cAMP. We conclude that cAMP stimulates phosphorylation and binding of CREB to the cyclin D1 gene promoter. Simultaneously, hypermethylation of the Wnt10b gene promoter suppresses binding of CREB, allowing adipogenesis to proceed.

PGE2 potentiates tonicity-induced COX-2 expression in renal medullary cells in a positive feedback loop involving EP2-cAMP-PKA signaling

Cyooxygenase-2 (COX-2)-derived PGE2 is critical for the integrity and function of renal medullary cells during antidiuresis. The present study extended our previous finding that tonicity-induced COX-2 expression is further stimulated by the major COX-2 product PGE2 and investigated the underlying signaling pathways and the functional relevance of this phenomenon. Hyperosmolality stimulated COX-2 expression and activity in Madin-Darby canine kidney (MDCK) cells, a response that was further increased by PGE2-cAMP signaling, suggesting the existence of a positive feedback loop. This effect was diminished by AH-6809, an EP2 antagonist, and by the PKA inhibitor H-89, but not by AH-23848, an EP4 antagonist. The effect of PGE2 was mimicked by forskolin and dibutyryl-cAMP, suggesting that the stimulatory effect of PGE2 on COX-2 is mediated by a cAMP-PKA-dependent mechanism. Accordingly, cAMP-responsive element (CRE)-driven reporter activity paralleled the effects of PGE2, AH-6809, AH-23848, H-89, forskolin, and dibutyryl-cAMP on COX-2 expression. In addition, the stimulatory effect of PGE2 on tonicity-induced COX-2 expression was blunted in cells transfected with dominant-negative CRE binding (CREB) protein, as was the case in a COX-2 promoter reporter construct in which a putative CRE was deleted. Furthermore, PGE2 resulted in PKA-dependent phosphorylation of the pro-apoptotic protein Bad at Ser155, a mechanism that is known to inactivate Bad, which coincided with reduced caspase-3 activity during osmotic stress. Conversely, pharmacological interruption of the PGE2-EP2-cAMP-PKA pathway abolished Ser155 phosphorylation of Bad and blunted the protective effect of PGE2 on cell survival during osmotic stress. These observations indicate the existence of a positive feedback loop of PGE2 on COX-2 expression during osmotic stress, an effect that apparently is mediated by EP2-cAMP-PKA signaling, and that contributes to cell survival under hypertonic conditions.

The Biologic Meaning of the Structural Components of Defibrotide (DF) in the Setting of HIV Disease with Refractory Herpes, Candida, Cryptosporidium Diarrhea and Multi-Organ Failure Against the Background of Down-Regulation of Cellular Immunity, Autocrine Upregulation of Inflammatory Cytokines of TNF-Alpha, IL2, IL1, IL6 and Dysregulation of cAMP/cGMP Protein Kinase A/C Signalling.

Production of DF from native mammalian DNA involves heat controlled depolimerization of polymerized nucleic acids as a function of hyperchromicity measured at wavelengths of 260–280 nm as the ratio of Optical Density (OD) in (disorderly phase)/(OD) in (orderly phase). In double helix DNA this rearrangement can be of the order of 100%. DF is defined to represent a hyperchromicity ratio around 15%. Its structural formula is static: P1-5, (dAp)12–24, (dGp) 10–20, (dTp)13–26, (dCp)10–20. ASH Abstract #4086, 2004 reported sequence identities of the fragments at MW </=560 Da (the calculated MW of alkali Na-salt of ATP), using Reversed Phase- HPLC and (MALDI-TOF) (Oral, Lewis),to represent the capability of DF for 15% molecular rearrangement to include but not limited to: dC, dA, G, dGMP, AMP, dTTP, CTP, ATP, dGTP, CMP, cGMP, dAMP. 4 aptamers were sequenced from the PCR product of the double-stranded Defibrotide precursor (Schroer). We have analyzed Aptamer #4 of the sequence 5′ggtggtggttgtggt against the gag/pol region of HIV, and found 3 homology sites. Translation of the aptamer region in gag is a peptide “PEPTA”, and a pol gene fragment translates the same DNA sequence into TRANS. There is a conserved hairpin in the gag/pol region, just before the aptamer #4 homology. Other homologies of Aptamer #4 include but are not limited to the self-replicating portion of mitochondrial DNA (G.Gorilla, H.Sapiens), Candida tropicalis, but not Herpes, etc. T he reproducibility of these sequences in different batches of DF are still under study, and to be reported at the time of the ASH meeting. DF's mechanism of efficacy as a dose dependent modulator of 2nd messengers, however, are based on a more universally reported data. Highlighted in the literature are the HIV induced loss of bi-directional phosphorilation between Gs GTP/Gs GDP in favor of unidirectional continuous activation of adenylate cyclase (AC) by Gs GTP and secondary uninhibited upregulation of cAMP production. This leads to a known intracellular imbalance between cAMP/cGMP favoring viral replication. Also known are Beta Adrenoreceptor coupling to AC by Gs GTP, and secondary upregulation of autocrine production of inflammatory cytokines of TNF-Alpha, IL6, IL2, IL1, as well as micelle and spore formation of Candida Albicans (Sabie). Similarly, HSV-1 Alpha0 Promoter isknown to contain a putative cAMP responsive element (CRE) via activating transcriptor protein ATP/cAMP CRE binding protein CREB, NF-kB via CA ((Danaher). Reported are upregulation of cAMP, ATP, NADH, gluthathione, 2–3 DPG, NADP /NADPH, and AC by DF, albeit as static events. We will report on an in-vivo dose system aiming at resetting the broken feedback loops of bi-directional phosphorilation reactions as a common denominator for DF's mechanism of efficacy. The patient data dose-stratified from 40mg/kg/day to 300mg/kg/day using either approach are to be detailed at the time of the upcoming ASH meetings.

Identification of a novel human glucagon receptor promoter: Regulation by cAMP and PGC-1α

Previously we have demonstrated that glucagon receptor mRNA expression in cultured rat hepatocytes and pancreatic islets can be regulated by various factors, including cAMP; however, the regulation of the human glucagon receptor gene has not been well-defined. Here we have characterized the promoter regions of the human glucagon receptor gene. Primer extension studies yielded multiple products in both liver and pancreas, corresponding to transcription start sites situated at − 166 and − 477 relative to the start of translation, indicating two putative promoters. Both transcription start sites were found to be active, when sequence immediately upstream of the start sites were cloned into luciferase reporter constructs. The transcriptional activity of the proximal promoter, but not the distal promoter, could be inhibited approximately 50% by cAMP, indicating that the previously observed inhibitory effects of cAMP on glucagon receptor mRNA expression is mediated at the level of gene transcription. The cAMP-mediated downregulation of the proximal promoter was examined by deletion analysis in the human hepatoma cell line HepG2 and the cAMP responsiveness was found to be located in a region between 1051 and 1016 base pairs upstream of the transcription start site, which contains several putative cAMP responsive elements. Expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), known to be upregulated in the liver by fasting, was found to abolish the cAMP-dependent downregulation of glucagon receptor mRNA expression in vitro, whereas overexpression of PGC-1β had no effect.

Structural and Functional Conservation of Vertebrate Corticotropin-Releasing Factor Genes: Evidence for a Critical Role for a Conserved Cyclic AMP Response Element

Corticotropin-releasing factor (CRF) plays a central role in neuroendocrine, autonomic, immune, and behavioral responses to stressors. We analyzed the proximal promoters of two Xenopus laevis CRF genes and found them to be remarkably conserved with mammalian CRF genes. We found several conserved cis elements in the frog CRF genes including a cAMP response element (CRE), activator protein 1 binding sites, and glucocorticoid response elements. Exposure to a physical stressor caused a rapid elevation in phosphorylated CRE binding protein (CREB; 20 min) and CRF (1 h) in the anterior preoptic area of juvenile frogs. CREB bound to the putative frog CREs in vitro, which was disrupted by point mutations introduced into the CRE. The frog proximal CRF promoters supported basal transcription in transfection assays, and forskolin caused robust transcriptional activation. Mutagenesis of the CRE or overexpression of a dominant-negative CREB reduced forskolin-induced promoter activation. Using electroporation-mediated gene transfer in tadpole brain, we show that the proximal CRF promoters support cAMP or stressor-dependent transcription in vivo, which was abolished by mutation of the CRE. Using chromatin immunoprecipitation, we found that CREB associated with the proximal frog CRF promoter in vivo in a stressor-dependent manner. These data provide strong support for the hypothesis that stressor-induced CRF gene activation in vivo depends on CREB binding to the CRE in the promoter. Our findings show that the basic regulatory elements of the CRF gene responsible for stressor-induced activation arose early in vertebrate evolution and have been maintained by strong positive selection.