Regulation Of CREB Research Articles

PABPN1 functions as a downstream gene of CREB to inhibit the proliferation of preadipocytes.

This study was designed to reveal the role of nuclear poly(A) binding protein 1 (PABPN1) in the proliferation of preadipocytes, and to reveal the relationship between PABPN1 and cAMP response element (CRE)-binding protein (CREB) in the regulation of preadipocyte proliferation. Vectors overexpressing and siRNAs against PABPN1/CREB were transiently transfected into both porcine preadipocytes and mouse 3T3-L1 cells. Preadipocyte proliferation was measured with CCK-8, EdU, real-time quantitative PCR, Western blotting, and flow cytometry analyses. Additionally, the transcriptional regulation of CREB on PABPN1 were analyzed with dual-luciferase reporter gene and EMSA assays. Overexpression of PABPN1 inhibits, and knockdown of PABPN1 promotes, the proliferation of both porcine preadipocytes and 3T3-L1 cell lines. PABPN1 overexpression increased, while knockdown decreased, the cell population in the G0/G1 phase. These indicates that PABPN1 repressed preadipocyte proliferation by inhibiting cell cycle progress. Additionally, it was revealed that CREB regulated the expression of PABPN1 through binding to the promoter and that CREB inhibited preadipocyte proliferation by repressed cell cycle progress. Furthermore, we showed that PABPN1 functions as a downstream gene of CREB to regulate the proliferation of preadipocytes. PABPN1 inhibits preadipocyte proliferation by suppressing the cell cycle. We also found that CREB could promote PABPN1 expression by binding to a motif in the promoter. Further analysis confirmed that PABPN1 functions as a downstream gene of CREB to regulate the proliferation of preadipocytes. These results suggest that the CREB/PABPN1 axis plays a role in the regulation of preadipocyte proliferation, which will contribute to further revealing the mechanism of fat accumulation.

CBP Expression Contributes to Neuropathic Pain via CREB and MeCP2 Regulation in the Spared Nerve Injury Rat Model.

Background and Objectives: This study aimed to investigate the relationship between neuropathic pain and CREB-binding protein (CBP) and methyl-CpG-binding protein 2 (MeCP2) expression levels in a rat model with spared nerve injury (SNI). Materials and Methods: Rat (male Sprague-Dawley white rats) models with surgical SNI (n = 6) were prepared, and naive rats (n = 5) were used as controls. The expression levels of CBP and MeCP2 in the spinal cord and dorsal root ganglion (DRG) were compared through immunohistochemistry at 7 and 14 days after surgery. The relationship between neuropathic pain and CBP/MeCP2 was also analyzed through intrathecal siRNA administration. Results: SNI induced a significant increase in the number of CBPs in L4 compared with contralateral DRG as well as with naive rats. The number of MeCP2 cells in the dorsal horn on the ipsilateral side decreased significantly compared with the contralateral dorsal horn and the control group. SNI induced a significant decrease in the number of MeCP2 neurons in the L4 ipsilateral DRG compared with the contralateral DRG and naive rats. The intrathecal injection of CBP siRNA significantly inhibited mechanical allodynia induced by SNI compared with non-targeting siRNA treatment. MeCP2 siRNA injection showed no significant effect on mechanical allodynia. Conclusions: The results suggest that CBP and MeCP2 may play an important role in the generation of neuropathic pain following peripheral nerve injury.

Abstract 4277: CREB-ROCK driven extracellular matrix remodeling exasperates pancreatic cancer progression

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by a heightened oncogenic mutational burden, an immunosuppressive tumor microenvironment, and a dense desmoplastic stroma. During the tumor progression from in situ carcinoma to intraductal neoplasia, cancer cells manage to evade the stromal compartment and infiltrate into the ducts. This invasive tumor progression is often associated with the overexpression of extracellular matrix (ECM) modulating genes. Despite this correlation, there are currently no effective and specific inhibitors of ECM-altering pathways. By understanding the signaling mechanism, the ECM remodeling pathways may be intercepted to prevent invasive tumor progression. This study aims to elucidate the role of cAMP-response element binding protein 1 (CREB1) and its downstream effectors in regulating PDAC ECM remodeling. Methods: The Cancer Genome Atlas (TCGA) was quired for CREB1 expression and other ECM modulators in PDAC patients. In vitro, CRISPR/CAS9-based genomic editing was utilized to knockout CREB1 in the murine KPC cell line, on which RNA-seq analysis was performed. To investigate the effects of CREB in PDAC progression, a novel conditional CREB1 knockout (CREBfl/fl) was created in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice (KPCC-/-). Tumors were extracted and histologically evaluated. Immunoblotting and staining were conducted on both in vitro and in vivo samples. Chromatin immunoprecipitation sequencing (ChIP-seq) and qPCR were performed on the KPC CREBKO. Eukaryotic Promoter Database (EPD) and ENCODE databases were explored to evaluate the transcriptional role of CREB in regulating ECM genes. Results: Patient PDAC samples data from TCGA revealed elevated levels of CREB1, ROCK1, and ROCK2 compared to normal pancreatic tissues. When comparing high CREB expression to low CREB expression, upregulation of several ECM genes was noted, including ROCK1 and ROCK2 in patients with high CREB expression. In CREBKO, RNA-seq analysis revealed the downregulation of key ECM genes, including Mmp3, Mmp10, Lama3, and Fn1 compared to CREB wildtype. In our novel murine conditional knockout model, a substantial reduction in fibrosis was observed. In both in vitro and in vivo, immunoblot and staining demonstrated a marked reduction in the expression of ECM remodeling proteins upon CREB deletion. Through ChIP-seq, the direct involvement of CREB1 in the transcriptional regulation of Rock1 and Rock2 was established. EPD and ENCODE were utilized to confirm and validate our CREB regulation experimental findings. Conclusion: These findings demonstrate the role of CREB in PDAC progression, which may provide a potential target to intercept ECM remodeling via inhibition of the CREB-ROCK axis to suppress PDAC tumor progression and invasion. Citation Format: Sudhakar Jinka, Siddharth Mehra, Varunkumar Krishnamoorthy, Anna Bianchi, Karthik Rajkumar, Andrew Adams, Haleh Amirian, Samara Singh, Edmond W. Box, Erin Dickey, Nivelo L. Alberto, Ban Yuguang, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. CREB-ROCK driven extracellular matrix remodeling exasperates pancreatic cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4277.

Oleuropein confers neuroprotection against rotenone-induced model of Parkinson’s disease via BDNF/CREB/Akt pathway

Major pathological features of Parkinson’s disease (PD) include increase in oxidative stress leading to the aggregation of α-synuclein, mitochondrial dysfunction and apoptosis of dopaminergic neurons. In addition, downregulation of the expression of neurotrophic factors like-Brain Derived Neurotrophic Factor (BDNF) is also involved in PD progression. There has been a lot of interest in trophic factor-based neuroprotective medicines over the past few decades to treat PD symptoms. Rotenone, an insecticide, inhibits the mitochondrial complex I causing overproduction of ROS, oxidative stress, and aggregation of α-synuclein. It has been shown that BDNF and Tropomyosin receptor kinase B (TrkB) interaction initiates the regulation of neuronal cell development and differentiation by the serine/threonine protein kinases like Akt and GSK-3β. Additionally, Transcription factor CREB (cAMP Response Element-binding protein) also determines the gene expression of BDNF. The homeostasis of these signalling cascades is compromised with the progression of PD. Therefore, maintaining the equilibrium of these signalling cascades will delay the onset of PD. Oleuropein (OLE), a polyphenolic compound present in olive leaves has been documented to cross blood brain barrier and shows potent antioxidative property. In the present study, the dose of 8, 16 and 32 mg/kg body weight (bwt) OLE was taken for dose standardisation. The optimised doses of 16 and 32 mg/kg bwt was found to be neuroprotective in Rotenone induced PD mouse model. OLE improves motor impairment and upregulate CREB regulation along with phosphorylation of Akt and GSK-3β in PD mouse. In addition, OLE also reduces the mitochondrial dysfunction by activation of enzyme complexes and downregulates the proapoptotic markers in Rotenone intoxicated mouse model. Overall, our study suggests that OLE may be used as a therapeutic agent for treatment of PD by regulating BDNF/CREB/Akt signalling pathway.

Irisin regulates thermogenesis and lipolysis in 3T3-L1 adipocytes

BackgroundAdipose tissue plays a pivotal role in the development and progression of the metabolic syndrome which along with its complications is an epidemic of the 21st century. Irisin is an adipo-myokine secreted mainly by skeletal muscle and targeting, among others, adipose tissue. In brown adipose tissue it upregulates uncoupling protein-1 (UCP1) which is responsible for mitochondrial non-shivering thermogenesis. MethodsHere we analyzed the effects of irisin on the metabolic activity of 3T3-L1 derived adipocytes through a mitochondrial flux assay. We also assessed the effects of irisin on the intracellular signaling through Western Blot. Finally, the gene expression of ucp1 and lipolytic genes was examined through RT-qPCR. ResultsIrisin affects mitochondrial respiration and lipolysis in a time-dependent manner through the regulation of PI3K-AKT pathway. Irisin also induces the expression of UCP1 and the regulation of NF-κB, and CREB and ERK pathways. ConclusionOur data supports the role of irisin in the induction of non-shivering thermogenesis, the regulation of energy expenditure and lipolysis in adipocytes. General significanceIrisin may be an attractive therapeutic target in the treatment of obesity and related metabolic disorders.

Zinc-mediated activation of CREB pathway in proliferation of pulmonary artery smooth muscle cells in pulmonary hypertension

BackgroundTranscription factor CREB is involved in the development of pulmonary hypertension (PH). However, little is known about the role and regulatory signaling of CREB in PH.MethodsA series of techniques, including bioinformatics methods, western blot, cell proliferation and luciferase reporter assay were used to perform a comprehensive analysis of the role and regulation of CREB in proliferation of pulmonary artery smooth muscle cells (PASMCs) in PH.ResultsUsing bioinformatic analysis of the differentially expressed genes (DEGs) identified in the development of monocrotaline (MCT)- and hypoxia-induced PH, we found the overrepresentation of CRE-containing DEGs. Western blot analysis revealed a sustained increase in total- and phosphorylated-CREB in PASMCs isolated from rats treated with MCT. Similarly, an enhanced and prolonged serum-induced CREB phosphorylation was observed in hypoxia-pretreated PASMCs. The sustained CREB phosphorylation in PASMCs may be associated with multiple protein kinases phosphorylated CREB. Additionally, hierarchical clustering analysis showed reduced expression of the majority of CREB phosphatases in PH, including regulatory subunits of PP2A, Ppp2r2c and Ppp2r3a. Cell proliferation analysis showed increased PASMCs proliferation in MCT-induced PH, an effect relied on CREB-mediated transcriptional activity. Further analysis revealed the raised intracellular labile zinc possibly from ZIP12 was associated with reduced phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation.ConclusionsCREB pathway was overactivated in the development of PH and contributed to PASMCs proliferation, which was associated with multiple protein kinases and/or reduced CREB phosphatases and raised intracellular zinc. Thus, this study may provide a novel insight into the CREB pathway in the pathogenesis of PH.B6vttFt3ef-LGEoxvha7NyVideo abstract

Dehydropeptidase 1 Promoted Survival and Cell Cycle Progression through Regulation of CREB and P53 Pathways in B-Cell ALL

Background: Outcome of adults with B-cell acute lymphoblastic leukemia (ALL) remains poor and relapse is the major cause of treatment-failure. Identifying new biomarkers in B-cell ALL and studying their clinical significance and biological function will be helpful for risk-stratification, treatment decision and targeted therapy. Dehydropeptidase 1 (DPEP1) is a zinc-dependent metalloproteinase that is expressed aberrantly in several cancers. The role of DPEP1 in cancer remains controversial. In our previously conducted microarray analysis, DPEP1 was identified as one of most differentially expressed genes which were up-regulated in B-cell ALL more than 20-fold compared with normal bone marrow. However, the expression and biological function of DPEP1 in B-cell ALL remains unknown.Aims: To investigate the expression levels of DPEP1 in adults with B-cell ALL and to explore the biological function of DPEP1 in the cell line derived from B-cell ALL.Methods: A real-time quantitative RT-PCR was used to examine DPEP1 expression in 11 malignant hematological disorders cell lines and bone marrow samples from 146 adult acute leukemia and 26 health donors. We constructed 2 B-cell lines: DPEP1 over-expressing (OE) and DPEP1 knocked-down (KD) BV173 cell line to study the biological role of DPEP1 in B-cell ALL. The cell proliferation and colony formation were detected by CCK-8 and colony formation assay. The cell apoptosis and cell cycle were analysed by flow cytometry. The cell migration ability was assayed by the transwell assay. Western blot was used to determine the protein level of signaling pathway.Results: These results showed that the relative levels of DPEP1 gene expression in marrow from the 126 newly diagnosed B-cell ALL(median 136%; range 0-1066%) were significantly higher than those of marrow from the 26 healthy donors(8.4%; 0-20.8%; p <0.01), whereas no significant difference was detected in newly diagnosed AML patients compared with health donors (Figure A). DPEP1 gene expression was higher in BV-173 cells derived from B-cell ALL, but nearly undetectable in cell lines derived from AML(KG-1, NB4, HL60, MEG-01), CML(K562), MM(MOLP2, SKO-007), lymphoma(U937, Raji, Ramos) and the normal B cell line IM-9. Proliferation was significantly decreased in DPEP1-KD cells compared with cells transfected with control lentivirus (OD value: 1.90±0.05 vs 1.68±0.06, p < 0.05). In contrast, DPEP1 over-expression markedly increased proliferation of transfected cells (OD value: 1.32±0.06 vs 1.72±0.03, p < 0.05) (Figure B). Silencing of DPEP1 expression significantly decreased numbers of colony-forming units compared with controls (65.7±10.4 vs 109.7±8.3, p <0.01), whereas DPEP1 over-expression had a converse effect (116.7±15.5 vs 106.7±6.6, p <0.05). Data from flow cytometry analyses showed DPEP1 expression promoted cell-cycle progression: DPEP1-OE BV173 had a higher percentage cells in S phases (43.19±0.35% vs 38.58±0.29%, p <0.01), whereas DPEP1-KD BV173 cells significantly prone to accumulate in G0/G1 phases compared with control cells (57.75±1.66% vs 50.78±0.39%, p <0.01) (Figure C). Migration and invasion activity were significantly enhanced in DPEP1-OE BV173 cells but attenuated in DPEP-KD BV173 cells as detected by transwell assay. However, DPEP1-KD significantly increased the apoptosis in BV173 cells (11.68±0.18% vs 1.72±0.12%, p <0.001) (Figure C). Then we sought to identify the pathway and molecular mechanisms that mediated the effect of DPEP1.The p-CREB were significantly up-regulated in DPEP1-OE cells but down-regulated in DPEP1-KD cells. And expression of p53 was increased in DPEP1-KD cells (Figure D).Conclusion: These findings suggest that abnormal expression of DPEP1 in leukemia may be involved in the pathomechanism of B-cell ALL. DPEP1 improves the proliferation, migration, invasion and promote the survival in B-cell ALL cells, which might be mediated via CREB and p53 pathways. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Roles of constitutive and signal-dependent protein phosphatase 2A docking motifs in burst attenuation of the cyclic AMP response element-binding protein

The cAMP response element-binding protein (CREB) is an important regulator of cell growth, metabolism, and synaptic plasticity. CREB is activated through phosphorylation of an evolutionarily conserved Ser residue (S133) within its intrinsically disordered kinase-inducible domain (KID). Phosphorylation of S133 in response to cAMP, Ca2+, and other stimuli triggers an association of the KID with the KID-interacting (KIX) domain of the CREB-binding protein (CBP), a histone acetyl transferase (HAT) that promotes transcriptional activation. Here we addressed the mechanisms of CREB attenuation following bursts in CREB phosphorylation. We show that phosphorylation of S133 is reversed by protein phosphatase 2A (PP2A), which is recruited to CREB through its B56 regulatory subunits. We found that a B56-binding site located at the carboxyl-terminal boundary of the KID (BS2) mediates high-affinity B56 binding, while a second binding site (BS1) located near the amino terminus of the KID mediates low affinity binding enhanced by phosphorylation of adjacent casein kinase (CK) phosphosites. Mutations that diminished B56 binding to BS2 elevated both basal and stimulus-induced phosphorylation of S133, increased CBP interaction with CREB, and potentiated the expression of CREB-dependent reporter genes. Cells from mice harboring a homozygous CrebE153D mutation that disrupts BS2 exhibited increased S133 phosphorylation stoichiometry and elevated transcriptional bursts to cAMP. These findings provide insights into substrate targeting by PP2A holoenzymes and establish a new mechanism of CREB attenuation that has implications for understanding CREB signaling in cell growth, metabolism, synaptic plasticity, and other physiologic contexts.

The Critical Role and Regulation of CREB Pathway in Pulmonary Hypertension

Background: Transcription factor CREB is involved in the development of pulmonary hypertension (PH). However, the regulatory mechanisms of CREB activation in PH remain unknown. Methods: A series of techniques, including bioinformatics methods, western blot and luciferase reporter assay were used to perform a comprehensive analysis of expression and regulation of CREB in experimental PH. Findings: Using bioinformatic analysis of the differentially expressed genes (DEGs) identified in the development of monocrotaline (MCT)- and hypoxia-induced PH, we found the overrepresentation of CRE-containing DEGs. Western blot analysis revealed a sustained increase in total and phosphorylated CREB in pulmonary artery smooth muscle cells (PASMCs) from rats treated with MCT. Similarly, an enhanced and prolonged serum-induced CREB phosphorylation was observed in hypoxia-pretreated PASMCs. The sustained CREB phosphorylation in PASMCs may be associated with multiple protein kinases phosphorylated CREB. Additionally, hierarchical clustering analysis showed reduced expression of the majority of CREB phosphatases in MCT-induced PH, including regulatory subunits of PP2A, Ppp2r2c and Ppp2r3a. Cell proliferation assay showed increased proliferation of PASMCs derived from rats with PH, an effect relied on CREB-mediated transcriptional activity. Further analysis revealed the raised intracellular labile zinc possibly from ZIP12 was associated with reduced phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation. Interpretation: CREB pathway was overactivated in the development of PH and contributed to PASMCs proliferation, which may be associated with multiple protein kinases, reduced CREB phosphatases and raised intracellular labile zinc. Funding: This work was supported by a grant from the National Natural Science Foundation of China (Grant No. 81873537). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The procedures were approved by the Laboratory Animal Welfare and Ethics Committee of Fujian Medical University (Approval No. 2017–070, Fuzhou, China)

Remote Ischemic Postconditioning vs. Physical Exercise After Stroke: an Alternative Rehabilitation Strategy?

There remain debates on neuroprotection and rehabilitation techniques for acute ischemic stroke patients. Therapeutic physical exercise following stroke has shown promise but is challenging to apply clinically. Ischemic conditioning, which has several clinical advantages, is a potential neuroprotective method for stroke rehabilitation that is less understood. In the present study, the rehabilitative properties and mechanisms of physical exercise and remote ischemic postconditioning (RIPostC) after stroke were compared and determined. A total of 248 adult male Sprague-Dawley rats were divided into five groups: (1) sham, (2) stroke, (3) stroke with intense treadmill exercise, (4) stroke with mild treadmill exercise, and (5) stroke with RIPostC. Focal ischemia was evaluated by infarct volume and neurological deficit. Long-term functional outcomes were represented through neurobehavioral function tests: adhesive removal, beam balance, forelimb placing, grid walk, rota-rod, and Morris water maze. To further understand the mechanisms underlying neurorehabilitation and verify the presence thereof, we measured mRNA and protein levels of neuroplasticity factors, synaptic proteins, angiogenesis factors, and regulation molecules, including HIF-1α, BDNF, TrkB, and CREB. The key role of HIF-1α was elucidated by using the inhibitor, YC-1. Both exercise intensities and RIPostC significantly decreased infarct volumes and neurological deficits and outperformed the stroke group in the neurobehavioral function tests. All treatment groups showed significant increases in mRNA and protein expression levels of the target molecules for neurogenesis, synaptogenesis, and angiogenesis, with intermittent further increases in the RIPostC group. HIF-1α inhibition nullified most beneficial effects and indicative molecule expressions, including HIF-1α, BDNF, TrkB, and CREB, in both procedures. RIPostC is equally, or superiorly, effective in inducing neuroprotection and rehabilitation compared to exercise in ischemic rats. HIF-1α likely plays an important role in the efficacy of neuroplasticity conditioning, possibly through HIF-1α/BDNF/TrkB/CREB regulation.

Long non-coding RNA MALAT1 targeting STING transcription promotes bronchopulmonary dysplasia through regulation of CREB.

Bronchopulmonary dysplasia (BPD) is a severe complication of preterm infants characterized by increased alveolarization and inflammation. Premature exposure to hyperoxia is believed to be a key contributor to the pathogenesis of BPD. No effective preventive or therapeutic agents have been created. Stimulator of interferon gene (STING) is associated with inflammation and apoptosis in various lung diseases. Long non‐coding RNA MALAT1 has been reported to be involved in BPD. However, how MALAT1 regulates STING expression remains unknown. In this study, we assessed that STING and MALAT1 were up‐regulated in the lung tissue from BPD neonates, hyperoxia‐based rat models and lung epithelial cell lines. Then, using the flow cytometry and cell proliferation assay, we found that down‐regulating of STING or MALAT1 inhibited the apoptosis and promoted the proliferation of hyperoxia‐treated cells. Subsequently, qRT‐PCR, Western blotting and dual‐luciferase reporter assays showed that suppressing MALAT1 decreased the expression and promoter activity of STING. Moreover, transcription factor CREB showed its regulatory role in the transcription of STING via a chromatin immunoprecipitation. In conclusion, MALAT1 interacts with CREB to regulate STING transcription in BPD neonates. STING, CREB and MALAT1 may be promising therapeutic targets in the prevention and treatment of BPD.

New Synthesized Galloyl‐RGD Inhibits Melanogenesis by Regulating the CREB and ERK Signaling Pathway in B16F10 Melanoma Cells

Gallic acid (3, 4, 5-trihydroxybenzoic acid) is a phytochemical derived from diverse herbs. It has been reported to have effective antifungal, antiviral and antioxidant activity. However, gallic acid exhibits low solubility and instability at high temperatures. In a previous study, in order to overcome these limitations, we synthesized galloyl-RGD by combining gallic acid with arginine, glycine and asparaginic acid (RGD peptide). This compound showed better thermal stability than gallic acid. In this study, we investigated the antimelanogenic effect of galloyl-RGD and the underlying mechanism for this effect. Galloyl-RGD markedly inhibited melanin content and tyrosinase activity in a concentration-dependent manner. We also found that galloyl-RGD decreased the levels of melanogenesis-related gene and protein. In addition, galloyl-RGD reduces intracellular cyclic adenosine monophosphate (cAMP) levels that leads to inhibition of cAMP-responsive element binding protein (CREB) phosphorylation and activates extracellular signal-regulated kinase (ERK) expression. These results indicate that CREB and ERK regulation by galloyl-RGD contributes to reduced melanin synthesis via degradation of microphthalmia-associated transcription factor. Therefore, galloyl-RGD can be potential candidate for application in cosmetic or pharmaceutical industry.

The role of hepatic transcription factor cAMP response element-binding protein (CREB) during the development of experimental nonalcoholic fatty liver: a biochemical and histomorphometric study

BackgroundSeveral molecular mechanisms contribute to the initiation and progression of nonalcoholic fatty liver disease (NAFLD); however, the exact mechanism is not completely understood. Cyclic adenosine monophosphate (cAMP) is one of the most promising pathways that regulates various cellular functions including lipid and carbohydrate metabolism. cAMP induces gene transcription through phosphorylation of the transcription factor, cAMP response element-binding protein (CREB). The action of cAMP is tightly regulated by its level and repression. Among the repressors, Inducible cAMP Early Repressor (ICER) is the only inducible CRE-binding protein. The present study aimed to evaluate the role of hepatic CREB level in the development of experimental NAFLD model to clarify the pathogenesis of the disease. NAFLD 35 male Wistar rats fed a high fat diet for a period of 14 weeks were studied compared with 35 control rats fed a standard diet. Five fasting rats were sacrificed each 2 weeks intervals for a period of 14 weeks.ResultsNAFLD group revealed a remarkable duration—dependent elevation in cAMP and CREB levels in the liver tissue compared to control group (P value < 0.004, P value < 0.006, respectively). In contrast, ICER gene expression, as a dominant-negative regulator of CREB, was downregulated in the liver of NAFLD group compared to control group. We also demonstrated that CREB levels were positively correlated with liver function tests, and glucose homeostasis parameters.ConclusionsOur results indicate that cAMP/CREB pathway provides an early signal in the progression to NAFLD representing a noninvasive biomarker that can early detect NAFLD and a promising therapeutic target for the treatment of the disease as well.

Manganese induced nervous injury by α-synuclein accumulation via ATP-sensitive K(+) channels and GABA receptors

Manganese (Mn) is an environmental pollutant having a toxic effect on Parkinson's disease, with significant damage seen in the neurons of basal ganglia. Hence, Mn pollution is a public health concern. A Sprague–Dawley rat model was used to determine the damage to basal nuclei, and the effect of Mn intake was detected using the Morris water maze test and transmission electron microscopy. The SH-SY5Y cell line was exposed to Mn, and downstream signaling was assessed to determine the mechanism of toxicity. Mn exposure injured neurons, repressing GABAAR receptors and inducing GABABR receptors. The synergistic effect of the GABABR receptor and Kir6.1-SUR1 or Kir6.2-SUR1 was found to be one of the potential factors for the secretion of α-synuclein. The accumulation of α-synuclein regulated downstream factors calmodulin (CAM) cAMP response element–binding protein (CREB), thereby impairing learning and memory. Other genes downstream of CREB, rather than the feedback regulation of CREB, and brain-derived neurotrophic factor might also be involved.

Circulating microRNAs Profile in Patients With Transthyretin Variant Amyloidosis.

Transthyretin variant amyloidosis (ATTRv) is a rare autosomal dominant disease characterized by the accumulation of amyloid in many organs, mostly causing a sensory-motor neuropathy, cardiomyopathy, and dysautonomia. The aim of the study was to report microRNAs (miRNAs) expression profile identified in the blood of ATTRv patients. Ten ATTRv patients, 10 asymptomatic carriers of transthyretin variant (TTRv), 10 patients with Charcot-Marie-Tooth (CMT) disease, and 10 healthy controls were studied. Human Schwann cells cultures were used to study the regulatory effects of miR-150-5p on the expression of cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF). ATTRv patients had 33 miRNAs up-regulated and 48 down-regulated versus healthy controls; 9 miRNAs were up-regulated and 30 down-regulated versus CMT patients; 19 miRNAs were up-regulated and 38 down-regulated versus asymptomatic TTRv carriers. Twelve out of the 19 upregulated miRNAs had a fold increase higher than 100. The validation experiment indicated miR-150-5p as a valuable biomarker to differentiate ATTRv patients from asymptomatic TTRv carriers (AUC: 0.9728; p < 0.0001). Schwann cells culture model demonstrated that miR-150-5p is a powerful negative regulator of CREB, BDNF, and NGF genes. Identification of deregulated miRNAs can help in understanding the complex pathomechamism underlying the development of ATTRv and related multisystemic pathology. Further investigations are needed on the role of circulating miR-150-5p to predict the shift of TTRv carriers from an asymptomatic status to symptoms appearance.

S.21.02 Epigenetic regulation of cognitive decline in Alzheimer's disease

Manganese (Mn) is an environmental pollutant having a toxic effect on Parkinson's disease, with significant damage seen in the neurons of basal ganglia. Hence, Mn pollution is a public health concern. A Sprague–Dawley rat model was used to determine the damage to basal nuclei, and the effect of Mn intake was detected using the Morris water maze test and transmission electron microscopy. The SH-SY5Y cell line was exposed to Mn, and downstream signaling was assessed to determine the mechanism of toxicity. Mn exposure injured neurons, repressing GABAAR receptors and inducing GABABR receptors. The synergistic effect of the GABABR receptor and Kir6.1-SUR1 or Kir6.2-SUR1 was found to be one of the potential factors for the secretion of α-synuclein. The accumulation of α-synuclein regulated downstream factors calmodulin (CAM) cAMP response element–binding protein (CREB), thereby impairing learning and memory. Other genes downstream of CREB, rather than the feedback regulation of CREB, and brain-derived neurotrophic factor might also be involved.

Novel role of CREB in regulating maturation of alveolar macrophages and thereby in maintaining lung‐fluid balance

Alveolar macrophages (AM) represent a specialized subpopulation of lung resident macrophages responsible for coordinating both the initiation and resolution of inflammatory lung injury. Analysis of the dynamics of lung resident macrophage populations using LyzM‐GFP mice (green myeloid cells) over the course of five days post‐injury by nebulized LPS showed a gradual decline in AM during injury phase but AM expanded and returned to baseline levels by 5th day post injury. While evidence suggests that recruited bone marrow monocytes can adopt AM phenotype following their differentiation into interstitial macrophage mechanisms reconstituting AM remains elusive. CREB, a cAMP‐Response Element Binding protein, is known to transcribe genes which regulate several cellular processes including cell differentiation, angiogenesis and anti‐inflammatory signaling. Mice lacking CREB rapidly die after birth primarily due to impairment of lung function. Thus, we generated mice lacking CREB in myeloid cells (CREBΔMye) by crossing CREBfl/fl mice with LyzM‐Cre mice to test the hypothesis that CREB regulates AM generation and thereby maintains lung‐fluid homeostasis. We show that in contrast to CREBfl/fl mice, CREBΔMye lungs developed lung edema basally. LPS induced significantly more edema formation at 4h in CREBΔMye than control mice. Whereas the edema resolved fully in control mice at 24h CREBΔMye lungs remained edematous. FACS analysis showed ~50% reduction in AM in CREBΔMye lungs (SiglecF+/CD11b‐/CD11c+ cells) basally and at 24 h following LPS‐induced lung injury. Intriguingly, we found increase in MHCII+/Siglec+/CD11clo lung cell population in CREBΔMye lungs suggesting loss of CREB in myeloid cells led to generation of incompetent pre‐AM/interstitial phenotype. Bone marrow derived macrophages from floxed and CREB lacking mice indicated dysregulation of several glycolytic enzymes, including hexokinase and pyruvate dehydrogenase. Further studies are being carried out to fully characterize the transcriptome of CREB lacking lung macrophages and whether CREB regulation of metabolic reprogramming is required for full differentiation of Pre‐AM/interstitial macrophages into AMs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Design principles of pleiotropic G‐protein signaling through guanine nucleotide exchange modulators (GEMs)

Heterotrimeric (henceforth trimeric) G‐proteins are molecular switches that control signal transduction. Dysregulation of the G‐protein pathway can lead to aberrant signal transduction and herald oncogenesis. Canonical G‐protein signaling, which is triggered exclusively by G protein‐coupled receptors (GPCRs), is spatially and temporally restricted, i.e. triggered exclusively at the plasma membrane (PM) via a process that completes within a few hundred milliseconds. Recently, a new paradigm has emerged, in which tyrosine‐based signals initiated by growth factor receptor tyrosine kinases (RTKs; e.g., EGFR) are relayed via non‐canonical activation of trimeric G proteins; such signaling has unique spatiotemporal features and is orchestrated by a non‐receptor guanine exchange modulator (GEM), GIV (a.k.a Girdin). GIV‐GEM acts pleiotropically as a guanine exchange factor (GEF) for Gαi and a guanine dissociation inhibitor (GDI) for Gαs; such modulation of two opposing Gα‐proteins integrates downstream cAMP signals depending on the abundance of functional copies in cells, fueling sinister properties such as invasiveness, stemness, survival, chemoresistance and angiogenesis. Here we developed the first network‐based compartmental model for signal transduction between RTKs and G proteins; two pathways of paramount importance in biology. The primary goal of this model was to identify the mechanistic regulation of cAMP, PKA, and CREB in different cellular compartments over time and to predict how network crosstalk affects cAMP dynamics. The model was constrained by experimental data and was robust to changes in kinetic parameters but sensitive to changes in initial concentrations of signaling species. Our model not only captured the unique temporal aspects of RTK→GIV→Gαi/s→cAMP signaling, but also predicted previously unforeseen impacts of EGF signaling [receptor endocytosis, copy number variations, activating mutations, etc.] and PDE activity on GIV‐dependent cAMP modulation. Predictions of the model appear to hold true in various disease states, most prominently in cancers.

Soluble cpg15 from Astrocytes Ameliorates Neurite Outgrowth Recovery of Hippocampal Neurons after Mouse Cerebral Ischemia.

The present study focuses on the function of cpg15, a neurotrophic factor, in ischemic neuronal recovery using transient global cerebral ischemic (TGI) mouse model and oxygen-glucose deprivation (OGD)-treated primary cultured cells. The results showed that expression of cpg15 proteins in astrocytes, predominantly the soluble form, was significantly increased in mouse hippocampus after TGI and in the cultured astrocytes after OGD. Addition of the medium from the cpg15-overexpressed astrocytic culture into the OGD-treated hippocampal neuronal cultures reduces the neuronal injury, whereas the recovery of neurite outgrowths of OGD-injured neurons was prevented when cpg15 in the OGD-treated astrocytes was knocked down, or the OGD-treated-astrocytic medium was immunoadsorbed by cpg15 antibody. Furthermore, lentivirus-delivered knockdown of cpg15 expression in mouse hippocampal astrocytes diminishes the dendritic branches and exacerbates injury of neurons in CA1 region after TGI. In addition, treatment with inhibitors of MEK1/2, PI3K, and TrkA decreases, whereas overexpression of p-CREB, but not dp-CREB, increases the expression of cpg15 in U118 or primary cultured astrocytes. Also, it is observed that the Flag-tagged soluble cpg15 from the astrocytes transfected with Flag-tagged cpg15-expressing plasmids adheres to the surface of neuronal bodies and the neurites. In conclusion, our results suggest that the soluble cpg15 from astrocytes induced by ischemia could ameliorate the recovery of the ischemic-injured hippocampal neurons via adhering to the surface of neurons. The upregulated expression of cpg15 in astrocytes may be activated via MAPK and PI3K signal pathways, and regulation of CREB phosphorylation.SIGNIFICANCE STATEMENT Neuronal plasticity plays a crucial role in the amelioration of neurological recovery of ischemic injured brain, which remains a challenge for clinic treatment of cerebral ischemia. cpg15 as a synaptic plasticity-related factor may participate in promoting the recovery process; however, the underlying mechanisms are still largely unknown. The objective of this study is to reveal the function and mechanism of neuronal-specific cpg15 expressed in astrocytes after ischemia induction, in promoting the recovery of injured neurons. Our findings provided new mechanistic insight into the neurological recovery, which might help develop novel therapeutic options for cerebral ischemia via astrocytic-targeting interference of gene expression.

P.1.g.041 - Inhibition of GSK-3beta signalling improves cognitive deficits and glutamate-excitotoxicity in diabetes through transcription regulation of CREB

P.1.g.041 - Inhibition of GSK-3beta signalling improves cognitive deficits and glutamate-excitotoxicity in diabetes through transcription regulation of CREB