cAMP Response Element Sites Research Articles

IL-1β induction of MUC5AC gene expression is mediated by CREB and NF-κB and repressed by dexamethasone.

Chronic airway diseases are characterized by inflammation and mucus overproduction. The MUC5AC mucin gene is upregulated by the proinflammatory cytokine interleukin-1 β (IL-1β) via activation of cAMP response element-binding protein (CREB) in the NCI-H292 cancer cell line and nuclear factor-κB (NF-κB) in the HBE1 transformed cell line, with each transcription factor binding to a cognate cis site in the proximal or distal region, respectively, of the MUC5AC promoter. We utilized primary differentiated human bronchial epithelial (HBE) and A549 lung adenocarcinoma cells to further investigate the contributions of CREB and NF-κB subunits to the IL-1β-induced upregulation of MUC5AC. Data show that ligand binding of IL-1β to the IL-1β receptor is required to increase MUC5AC mRNA abundance. Chromatin immunoprecipitation analyses show direct binding of CREB to the previously identified cAMP response element site and binding of p65 and p50 subunits to a novel NF-κB site in a mucin-regulatory domain in the proximal promoter and to a previously identified NF-κB site in the distal promoter. P50 binds to both NF-κB sites at 1 h following IL-1β exposure, but is replaced at 2 h by p65 in A549 cells and by a p50/p65 heterodimer in HBE cells. Thus IL-1β activates multiple domains in the MUC5AC promoter but exhibits some cell-specific responses, highlighting the complexity of MUC5AC transcriptional regulation. Data show that dexamethasone, a glucocorticoid that transcriptionally represses MUC5AC gene expression under constitutive conditions, also represses IL-1β-mediated upregulation of MUC5AC gene expression. A further understanding of mechanisms mediating MUC5AC regulation should lead to a honing of therapeutic approaches for the treatment of mucus overproduction in inflammatory lung diseases.

Demethylation of the Human Eotaxin-3 Gene Promoter Leads to the Elevated Expression of Eotaxin-3

DNA demethylation has been primarily studied in the context of development biology, cell fate, and cancer, with less attention on inflammation. In this article, we investigate the association between DNA methylation and production of the chemoattractant cytokine eotaxin-3 in the tissue of patients with allergic disease. Regions of the human eotaxin-3 promoter were found to be hypomethylated in primary epithelial cells obtained from allergic tissue compared with normal control tissue. The demethylation of a specific CpG site (designated CpG 2), which is juxtaposed to a key cAMP-responsive element site, was significantly demethylated in patient-derived compared with normal control tissue-derived epithelial cells. Levels of methylation at CpG 2 inversely correlated with basal and IL-13-induced eotaxin-3 gene expression. Conversely, global inhibition of methylation with 5-azacytidine promoted eotaxin-3 production in association with decreasing CpG 2 methylation. In addition, the basal and IL-13-induced eotaxin-3 transcriptional activity was suppressed by promoter methylation using a methylation-free in vitro system. Furthermore, EMSAs demonstrated that the attachment of CREB binding protein and activating transcription factor 2 (ATF-2) to the cAMP-responsive element site was methylation dependent. Taken together, these data identify a contributory role for DNA methylation in regulating eotaxin-3 production in human allergic inflammation.

Transcriptional machinery of TNF-α-inducible YTH domain containing 2 (YTHDC2) gene

We previously demonstrated that a cellular factor, cyclosporin A (CsA) associated helicase-like protein (CAHL) that is identical to YTH domain containing 2 (YTHDC2), forms trimer complex with cyclophilin B and NS5B of hepatitis C virus (HCV) and facilitates HCV genome replication. Gene expression of YTHDC2 was shown in tumor cell lines and tumor necrosis factor (TNF)-α-treated hepatocytes, but not in untreated. However, the function of YTHDC2 in the tumor cells and the mechanism by which the YTHDC2 gene is transcribed in these cells is largely unknown. We first evaluated that the role of YTHDC2 in the proliferation of hepatocellular carcinoma (HCC) cell line Huh7 using RNA interference and found that YTHDC2-downregulated Huh7 were significantly decreased cell growth as compared to control. We next demonstrated that the cAMP response element (CRE) site in the promoter region of the YTHDC2 gene is critical for YTHDC2 transcription. To further investigate the transcription factors bound to the CRE site, we performed chromatin immunoprecipitation assays. Our findings demonstrate that c-Jun and ATF-2 bind to the CRE site in Huh7, and that TNF-α induces the biological activity of these transcription factors in hepatocytes as well as Huh7. Moreover, treatment with the HDAC inhibitor, trichostatin A (TSA), reduces YTHDC2 expression in Huh7 and in TNF-α-stimulated hepatocytes. Collectively, these data show that YTHDC2 plays an important role in tumor cells growth and activation/recruitment of c-Jun and ATF-2 to the YTHDC2 promoter is necessary for the transcription of YTHDC2, and that HDAC activity is required for the efficient expression of YTHDC2 in both of hepatocyte and HCC cells.

Human endogenous retrovirus W family envelope gene activates the small conductance Ca2+-activated K+ channel in human neuroblastoma cells through CREB

Numerous studies have shown that human endogenous retrovirus W family (HERV-W) envelope gene (env) is related to various diseases but the underlying mechanism has remained poorly understood. Our previous study showed that there was abnormal expression of HERV-W env in sera of patients with schizophrenia. In this paper, we reported that overexpression of the HERV-W env elevated the levels of small conductance Ca2+-activated K+ channel protein 3 (SK3) in human neuroblastoma cells. Using a luciferase reporter system and RNA interference method, we found that functional cAMP response element site was required for the expression of SK3 triggered by HERV-W env. In addition, it was also found that the SK3 channel was activated by HERV-W env. Further study indicated that cAMP response element-binding protein (CREB) was required for the activation of the SK3 channel. Thus, a novel signaling mechanism of how HERV-W env influences neuronal activity and contributes to mental illnesses such as schizophrenia was proposed.

Protamine stimulates bone sialoprotein gene expression

Protamine is a small, arginine-rich, nuclear protein that replaces histone late in the haploid phase of spermatogenesis and is believed to be essential for sperm head condensation and DNA stabilization. Protamine has many biological activities and has roles in hematopoiesis, immune responses, the nervous system and bone metabolism. Bone sialoprotein (BSP) is a mineralized connective tissue-specific protein expressed in differentiated osteoblasts that appears to function in the initial mineralization of bone. Protamine (71.35ng/ml) increased BSP mRNA levels by 6h in osteoblast-like ROS 17/2.8 cells. In a transient transfection assay, protamine (71.35ng/ml) increased luciferase activity of the construct (−116 to +60) in ROS 17/2.8 cells and rat bone marrow stromal cells. Luciferase activities induced by protamine were blocked by protein kinase A, tyrosine kinase and ERK1/2 inhibitors. Introduction of 2bp mutations to the luciferase constructs showed that the effects of protamine were mediated by a cAMP response element (CRE), a fibroblast growth factor 2 response element (FRE) and a homeodomain protein-binding site (HOX). Gel shift analyses showed that protamine (71.35ng/ml) increased the nuclear protein binding to CRE, FRE and HOX. CREB, phospho-CREB, c-Fos, c-Jun, JunD and Fra2 antibodies disrupted the formation of CRE-protein complexes. Dlx5, Msx2, Runx2 and Smad1 antibodies disrupted FRE– and HOX–protein complex formations. These studies demonstrate that protamine induces BSP transcription by targeting CRE, FRE and HOX sites in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB, c-Fos, c-Jun, JunD, Fra2, Dlx5, Msx2, Runx2 and Smadl transcription factors appear to be key regulators of protamine effects on BSP transcription.

Transcriptional regulation of mouse neuroglobin gene by cyclic AMP responsive element binding protein (CREB) in N2a cells

Neuroglobin (Ngb) has been demonstrated to be a novel neuroprotective protein that protects against hypoxia/ischemia and oxidative stress-induced injury in the nervous system. However, the regulation mechanisms of Ngb gene expression under both normal resting and stress conditions have not been fully elucidated. The cyclic AMP response element binding protein (CREB) is a key transcription factor that regulates a variety of pro-survival genes, but its role in regulating the neuroprotective gene Ngb has not been studied. In this study we investigated the transcriptional regulation of mouse Ngb gene by CREB in mouse neuroblastoma cell line N2a. Our results showed that CREB knockdown decreased Ngb gene expression, and overexpression of the wild-type CREB, but not the mutant CREB, significantly increased Ngb gene expression in N2a cells. Moreover, a cAMP response element (CRE) site located at −854 in the promoter region of mouse Ngb gene was found to be responsible for both basal and CREB-induced Ngb promoter activity. Using chromatin immunopreciptation (ChIP) assays, we found that CREB could bind to the Ngb promoter region spanning from −1016 to −793 that harbors the CRE site. Taken together, our results suggested that transcription factor CREB participates in the transcriptional regulation of mouse Ngb gene.

TLR 2 Induces Vascular Smooth Muscle Cell Migration Through cAMP Response Element−Binding Protein−Mediated Interleukin-6 Production

Migration of vascular smooth muscle cells (VSMCs) from the media into intima contributes to the development of atherosclerosis. Gene deletion experiments implicate a role for toll-like receptor 2 (TLR2) in atherogenesis. However, the underlying mechanisms remain unclear. We postulate that TLR2 promotes VSMC migration by enhancing interleukin (IL)-6 production. Migration assays revealed that TLR2 agonists promoted VSMC migration but not cell proliferation or viability. TLR2 deficiency or inhibition of TLR2 signaling with anti-TLR2 antibody suppressed TLR2 agonist-induced VSMC migration and IL-6 production, which was mediated via p38 mitogen-associated protein kinase and extracellular signal-regulated kinase 1/2 signaling pathways. Neutralizing anti-IL-6 antibodies impaired TLR2-mediated VSMC migration and formation of filamentous actin fiber and lamellipodia. Blockade of p38 mitogen-associated protein kinase or extracellular signal-regulated kinase 1/2 activation inhibited TLR2 agonist pam3CSK4-induced phosphorylation of cAMP response element-binding protein, which regulates IL-6 promoter activity through the cAMP response element site. Moreover, cAMP response element-binding protein small interfering RNA inhibited pam3CSK4-induced IL-6 production and VSMC migration. Additionally, Rac1 small interfering RNA inhibited pam3CSK4-induced VSMC migration but not IL-6 production. Our results suggest that on ligand binding, TLR2 activates p38 mitogen-associated protein kinase and extracellular signal-regulated kinase 1/2 signaling in VSMCs. These signaling pathways act in concert to activate cAMP response element-binding protein and subsequent IL-6 production, which in turn promotes VSMC migration via Rac1-mediated actin cytoskeletal reorganization.

Effects of ATF4 on PGC1α expression in brown adipose tissue and metabolic responses to cold stress

ObjectiveWe have shown previously that the expression of peroxisome proliferator activated receptor gamma coactivator (PGC1α) increases significantly in the white and brown adipose tissue of activating transcription factor 4 (ATF4) global knockout mice, which suggests that ATF4 is involved in the regulation of PGC1α expression. The goal of the current study is to investigate this possibility and elucidate the underlying cellular mechanisms. Material/methodsThe effects of ATF4 on PGC1α expression and on PGC1α promoter activity were analyzed in vivo and in vitro using mice, HIB-1B, and 293T cell line. The physiological functions of ATF4 in the regulation of PGC1α expression were confirmed by analysis of body temperature of Atf4−/− and Atf4+/+ mice in response to cold stress as well as expression of Complex I, II, III, V in BAT. ResultsIn this study, we showed ATF4 to be a negative regulator of PGC1α expression through competitive binding with cAMP response element binding protein (CREB) at a cAMP response element (CRE) site in the PGC1α promoter. ATF4 was also found to influence the expression of mitochondria-related proteins, including Complex I, II, III, and IV through regulation of PGC1α. Finally, we showed that Atf4−/− mice have higher core body temperatures in reduced-temperature environments than control mice. ConclusionThis study describes the mechanisms underlying ATF4 regulating PGC1α expression. We demonstrate a novel function of ATF4 in the regulation of thermogenesis. Taken together, these observations provide new insight into the physiological functions of ATF4, especially the regulation of thermogenesis and the response to cold stress.

Phytoestrogen Genistein Up-Regulates Endothelial Nitric Oxide Synthase Expression Via Activation of cAMP Response Element-Binding Protein in Human Aortic Endothelial Cells

We previously reported that genistein, a phytoestrogen, up-regulates endothelial nitric oxide synthase (eNOS) and prevents hypertension in rats that are independent of estrogen signaling machinery. However, how genistein regulates eNOS expression is unknown. In the present study, we show that genistein enhanced eNOS expression and NO synthesis in primary human aortic endothelial cells. Inhibition of extracellular signal regulated kinase, phosphoinositol-3 kinase, or protein kinase C did not affect genistein-enhanced eNOS expression and NO synthesis. However, chemical inhibition of protein kinase A (PKA) or adenoviral transfer of the specific endogenous PKA inhibitor gene completely abolished PKA activity and genistein-stimulated eNOS expression and NO production. Accordingly, genistein induced PKA activity and subsequent phosphorylation of cAMP response element (CRE)-binding protein (CREB) at Ser133. Suppression of CREB by small interfering RNA transfection abolished genistein-enhanced eNOS expression and NO production. Consistently, deletion of the CRE site within human eNOS promoter eliminated genistein-stimulated eNOS promoter activity. These findings provide the first evidence to our knowledge that genistein may play a beneficial role in vascular function through targeting the PKA/CREB/eNOS/NO signaling pathway.

Involvement of PGE2 and the cAMP signalling pathway in the up-regulation of COX-2 and mPGES-1 expression in LPS-activated macrophages

PG (prostaglandin) E2 plays an important role in the modulation of the immune response and the inflammatory process. In the present study, we describe a PGE2 positive feedback for COX (cyclo-oxygenase)-2 and mPGES-1 [microsomal PGES (PGE synthase)-1] expression in the macrophage cell line RAW 264.7. Our results show that PGE2 induces COX-2 and mPGES-1 expression, an effect mimicked by dbcAMP (dibutyryl-cAMP) or forskolin. Furthermore, the cAMP signalling pathway co-operates with LPS (lipopolysaccharide) in the induction of COX-2 and mPGES-1 transcriptional activation. Analysis of the involvement of PGE receptors [EPs (E-prostanoids)] showed that incubation with EP2 agonists up-regulated both COX2 and mPGES-1 mRNA levels. Moreover, EP2 receptor overexpression enhanced the transcriptional activation of COX2 and mPGES-1 promoters. This induction was repressed by the PKA (protein kinase A) inhibitor H89. Activation of the PGE2/EP2/PKA signalling pathway induced the phosphorylation of CREB [CRE (cAMP-response element)-binding protein] in macrophages and stimulated the specific binding of this transcription factor to COX2 and mPGES-1 promoters. Deletion or mutation of potential CRE sites in both promoters diminished their transcriptional activity. In summary, the results of the present study demonstrate that activation of PKA/CREB signalling through the EP2 receptor by PGE2 plays a key role in the expression of COX-2 and mPGES-1 in activated macrophages.

PKA/CREB Signaling Triggers Initiation of Endothelial and Hematopoietic Cell Differentiation via Etv2 Induction

Ets family protein Etv2 (also called ER71 or Etsrp) is a key factor for initiation of vascular and blood development from mesodermal cells. However, regulatory mechanisms and inducing signals for Etv2 expression have been largely unknown. Previously, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling enhanced differentiation of vascular progenitors into endothelial cells (ECs) and hematopoietic cells (HPCs) using an embryonic stem cell (ESC) differentiation system. Here, we show that PKA activation in an earlier differentiation stage can trigger EC/HPC differentiation through Etv2 induction. We found Etv2 was markedly upregulated by PKA activation preceding EC and HPC differentiation. We identified two cAMP response element (CRE) sequences in the Etv2 promoter and 5'-untranslated region and confirmed that CRE-binding protein (CREB) directly binds to the CRE sites and activates Etv2 transcription. Expression of a dominant negative form of CREB completely inhibited PKA-elicited Etv2 expression and induction of EC/HPCs from ESCs. Furthermore, blockade of PKA significantly inhibited Etv2 expression in ex vivo whole-embryo culture using Etv2-Venus knockin mice. These data indicated that PKA/CREB pathway is a critical regulator for the initiation of EC/HPC differentiation via Etv2 transcription. This early-stage molecular linkage between a triggering signal and transcriptional cascades for differentiation would provide novel insights in vascular and blood development and cell fate determination.

Molecular mechanisms of early growth response protein‐1 (EGR‐1) expression by quercetin in INS‐1 beta‐cells

Early growth response-1 (EGR-1), one of immediate early response genes, is involved in diverse cellular response. We recently reported that quercetin increased catalytic subunit of γ-glutamylcysteine ligase (GCLC) via the interaction of EGR-1 to GCLC promoter in INS-1 beta-cells. Therefore, this study investigated molecular mechanisms of quercetin-induced EGR-1 expression in INS-1 cells. Quercetin significantly induced EGR-1 protein and its mRNA expressions. This induction of EGR-1 was completely blocked by pretreatment with a PKA inhibitor, H89 and partially blocked by a p38 inhibitor, SB203580. Additionally, the siRNA-mediated inhibition of PKAα and p38 resulted in significant reduction of quercetin-induced EGR-1 promoter activity. Also, quercetin-induced EGR-1 protein expression was significantly decreased in the cells transfected with PKAα siRNA. Study using truncated EGR-1 promoter constructs showed that serum response element (SRE) sites, not cAMP response element site, were essential for EGR-1 transcription. However, electrophoretic mobility shift assay showed that quercetin did not affect the band intensity of DNA-protein complex on SRE site of EGR-1 promoter. Also, immune-shift assay using serum response factor (SRF) and phospho-SRF antibodies showed no difference between control and quercetin-treated groups. Collectively, quercetin-induced EGR-1 expression is largely dependent on PKA and partly on p38 MAPK pathway, and SRE sites of EGR-1 promoter are involved in quercetin-induced EGR-1 transcriptional activity.

CREMα suppresses spleen tyrosine kinase expression in normal but not systemic lupus erythematosus T cells

T cells from patients with systemic lupus erythematosus (SLE) display increased amounts of spleen tyrosine kinase (Syk), which is involved in the aberrant CD3/T cell receptor-mediated signaling process, and increased amounts of CREMα, which suppresses the production of interleukin-2. Syk expression can be suppressed by CREMα. This study was undertaken to investigate why CREMα fails to suppress Syk expression in SLE T cells. CREMα was overexpressed in healthy T cells by transfection with CREMα expression vector, and Syk expression and phosphorylation were measured. A newly identified cAMP response element (CRE) site on the SYK promoter was characterized by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay. The CREMα-mediated repression of Syk expression was further evaluated by analyzing SYK promoter activity. T cells from SLE patients and healthy individuals were subjected to ChIP to evaluate CREMα binding and histone H3 acetylation. Increased CREMα levels suppressed Syk expression by direct binding to a CRE site of the SYK promoter in T cells from healthy individuals but failed to do so in T cells from SLE patients. The failure of CREMα to suppress Syk expression in SLE T cells was due to weaker binding to the CRE site of the SYK promoter compared to healthy T cells because the promoter site is hypoacetylated in SLE T cells and therefore of limited access to transcription factors. Our findings indicate that epigenetic alteration of the SYK promoter in SLE T cells results in the inability of the transcriptional repressor CREMα to bind and suppress the expression of Syk, resulting in aberrant T cell signaling.

Serotonin-Mediated Synapsin Expression Is Necessary for Long-Term Facilitation of theAplysiaSensorimotor Synapse

Serotonin (5-HT)-induced long-term facilitation (LTF) of the Aplysia sensorimotor synapse depends on enhanced gene expression and protein synthesis, but identification of the genes whose expression and regulation are necessary for LTF remains incomplete. In this study, we found that one such gene is synapsin, which encodes a synaptic vesicle-associated protein known to regulate short-term synaptic plasticity. Both synapsin mRNA and protein levels were increased by 5-HT. Upregulation of synapsin protein occurred in presynaptic sensory neurons at neurotransmitter release sites. To investigate the molecular mechanisms underlying synapsin regulation, we cloned the promoter region of Aplysia synapsin, and found that the synapsin promoter contained a cAMP response element (CRE), raising the possibility that the transcriptional activator CRE-binding protein 1 (CREB1) mediates 5-HT-induced regulation of synapsin. Indeed, binding of CREB1 to the synapsin promoter was increased following treatment with 5-HT. Furthermore, increased acetylation of histones H3 and H4 and decreased association of histone deacetylase 5 near the CRE site are consistent with transcriptional activation by CREB1. RNA interference (RNAi) targeting synapsin mRNA blocked the 5-HT-induced increase in synapsin protein levels and LTF; in the absence of 5-HT treatment, basal synapsin levels were unaffected. These results indicate that the 5-HT-induced regulation of synapsin levels is necessary for LTF and that this regulation is part of the cascade of synaptic events involved in the consolidation of memory.

Abstract 9665: PKA / CREB Pathway Initiates Endothelial And Hematopoietic Cell Differentiation Via Etv2 Induction

Ets-family protein Etv2 (also called ER71 or Etsrp) is a key factor for vascular and blood development from mesodermal cells. However, regulatory mechanisms and a triggering signal for Etv2 expression have been largely unknown. We previously established an ES cell differentiation system that reproduces early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors. Recently, we revealed that cAMP/protein kinase A (PKA) signaling enhanced differentiation of vascular progenitors into endothelial cells (ECs) and hematopoietic cells (HPCs) using this system. Here we show that PKA activation induces Etv2 expression, which can trigger EC/HPC differentiation. We found that Etv2 was markedly upregulated by PKA activation preceding EC and HPC differentiation. Inhibition of Etv2 using siRNA completely abolished PKA-elicited EC and HPC differentiation from ES cells. We identified two cAMP response element (CRE) sequences in Etv2 promoter region, and confirmed that CRE-binding protein (CREB) directly binds to the CRE sites. Furthermore, PKA activated Etv2 promoter activity in luciferase assay, which was disappeared in CRE-mutant Etv2 promoters. Expression of dominant negative form of CREB completely inhibited PKA-elicited Etv2 expression and induction of EC/HPCs from ES cells. PKA/CREB pathway is, thus, a critical regulator for the initiation of EC/HPC differentiation through Etv2 transcription. This early stage molecular linkage between a triggering signal and transcriptional cascades for differentiation would provide novel insights in vascular and blood development and novel molecular targets for vascular and blood regeneration medicine.

The Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase/Ribosomal S6 Protein Kinase 1 Cascade Phosphorylates cAMP Response Element-binding Protein to Induce MUC5B Gene Expression via d-Prostanoid Receptor Signaling

Mucus hypersecretion is a prominent feature of respiratory diseases, and MUC5B is a major airway mucin. Mucin gene expression can be affected by inflammatory mediators, including prostaglandin (PG) D(2,) an inflammatory mediator synthesized by hematopoietic PGD synthase (H-PGDS). PGD(2) binds to either D-prostanoid receptor (DP1) or chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2). We investigated the mechanisms by which PGD(2) induces MUC5B gene expression in airway epithelial cells. Western blot analysis showed that H-PGDS was highly expressed in nasal polyps. Similar results were obtained for PGD(2) expression. In addition, we could clearly detect the expressions of both H-PGDS and DP1 in nasal epithelial cells but not CRTH2. We demonstrated that PGD(2) increased MUC5B gene expression in normal human nasal epithelial cells as well as in NCI-H292 cells in vitro. S5751, a DP1 antagonist, inhibited PGD(2)-induced MUC5B expression, whereas a CRTH2 antagonist (OC0459) did not. These data suggest that PGD(2) induced MUC5B expression via DP1. Pretreatment with extracellular signal-regulated kinase (ERK) inhibitor (PD98059) blocked both PGD(2)-induced ERK mitogen-activated protein kinase (MAPK) activation and MUC5B expression. Proximity ligation assays showed direct interaction between RSK1 and cAMP response element-binding protein (CREB). Stimulation with PGD(2) caused an increase in intracellular cAMP levels, whereas intracellular Ca(2+) did not have such an effect. PGD(2)-induced MUC5B mRNA levels were regulated by CREB via direct interaction with two cAMP-response element sites (-921/-914 and -900/-893). Finally, we demonstrated that PGD(2) can induce MUC5B overproduction via ERK MAPK/RSK1/CREB signaling and that DP1 receptor may have suppressive effects in controlling MUC5B overproduction in the airway.

Developmental regulation of TAC1 in peptidergic-induced human mesenchymal stem cells: implication for spinal cord injury in zebrafish.

Human mesenchymal stem cells (MSCs) are easy to expand, are relatively safe, and can be transplanted in allogeneic recipients as off-the-shelf cells. MSCs can be induced to form functional peptidergic neurons and express the neurotransmitter gene, TAC1. Expression of TAC1 requires that the repressor gene, RE-1 silencing transcription factor (REST), is decreased. This study investigated the molecular pathway in TAC1 induction as MSCs differentiated into neurons and then applied the findings in a model of spinal cord injury (SCI) in zebrafish. We studied the developmental roles of the 2 cAMP response element (CRE) sites: CRE1 and CRE2. Activator protein-1 (AP-1) binding site overlaps with CRE2 (CRE2/AP-1). Reporter gene studies with the 5' regulatory region of TAC1 containing wild-type or mutant CRE sites and, parallel studies with ectopically expressed inhibitor of cAMP proteins (inducible cAMP early repressor) indicated that CRE1 and CRE2/AP-1 are activated at days 6 and 12, respectively. Studies with protein kinase-A (PKA) and Jun N-terminal kinase (JNK) inhibitors in the reporter gene studies, chromatin immunoprecipation assay, and ectopic expression of REST indicated the following pathways: Decrease of REST activated upstream c-Jun N-terminal kinase (JNK). In turn, JNK activated ATF-2 and AP-1 for interaction with CRE1 and CRE2/AP-1, respectively. To apply the finding to SCI, we transplanted 6-day-induced MSCs in transgenic HB9-GFP zebrafish larvae with SCI, in the presence or absence of JNK inhibitors. Imaging and functional studies showed significant improvement in the fish. The repair mechanism involved the activation of JNK. The findings have long-term implications for SCI repair with MSCs.

DNA cytosine methylation in the Bovine Leukemia Virus promoter is associated with latency in a Lymphoma-derived B-cell line : potential involvement of direct inhibition of CREB/CREM/ATF binding

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

Activation of the protein kinase A pathway down regulates spleen tyrosine kinase in human T cells (63.30)

Abstract Cyclic adenosine monophosphate (cAMP)-mediated activation of the protein kinase A (PKA) pathway has an overall negative impact on T cell proliferation and down regulates or neutralizes signaling pathways of many genes. Spleen tyrosine kinase (SYK) is an important gene expressed differentially during T cell development and functions by phosphorylating downstream effector molecules in both T and B lymphocytes. We report here that PKA inhibitors promoted, whereas cAMP analogues suppressed SYK expression. Overexpression of a catalytic subunit of PKA significantly abrogated SYK protein and mRNA expression as well as the levels of phosphorylated SYK. A complete cAMP response element (CRE) site on SYK promoter was found functional and capable of binding CREMα and CREB as detected by EMSA and chromatin immunoprecipitation. CREMα overexpression reduced, while disruption of the CRE site enhanced the basal promoter activity of SYK. Collectively, the cAMP-mediated PKA pathway functions as the negative regulator of SYK expression by promoting direct binding of a repressor element to the SYK promoter.

Epigenetic Regulation of the IL-13-induced Human Eotaxin-3 Gene by CREB-binding Protein-mediated Histone 3 Acetylation

The etiology of a variety of chronic inflammatory disorders has been attributed to the interaction of genetic and environmental factors. Herein, we identified a link between epigenetic regulation and IL-13-driven eotaxin-3 in the pathogenesis of chronic allergic inflammation. We first demonstrated that the cAMP-responsive element (CRE) site in the eotaxin-3 promoter affects IL-13-induced eotaxin-3 promoter activity. Furthermore, the CRE-binding protein-binding protein (CBP), a histone acetyltransferase, induced base-line and IL-13-induced eotaxin-3 promoter activity. Additionally, IL-13 treatment promoted global histone 3 acetylation as well as the formation of a complex containing CBP and STAT6 and the subsequent acetylation of histone 3 at the eotaxin-3 promoter. CBP gene silencing decreased IL-13-induced transcription of eotaxin-3. Conversely, inhibition of histone deacetylation increased IL-13-induced eotaxin-3 production. Clinical studies demonstrated markedly increased global acetylation of histone 3 in the inflamed tissue of patients with allergic inflammation. Collectively, these results identify an epigenetic mechanism involving CBP and chromatin remodeling in regulating IL-13-induced chemokine transcription.