Cyclic AMP Response Element Research Articles

Coupling of binding and differential subdomain folding of the intrinsically disordered transcription factor CREB.

The cyclic AMP response element binding protein (CREB) contains a basic leucine zipper motif (bZIP) that forms a coiled coil structure upon dimerization and specific DNA binding. Although this state is well characterized, key features of CREB bZIP binding and folding are not well understood. We used single-molecule Förster resonance energy transfer (smFRET) to probe conformations of CREB bZIP subdomains. We found differential folding of the basic region and leucine zipper in response to different binding partners; a strong and previously unreported DNA-independent dimerization affinity; folding upon binding to nonspecific DNA; and evidence of long-range interdomain interactions in full-length CREB that modulate DNA binding. These studies provide new insights into DNA binding and dimerization and have implications for CREB function.

Dietary carbon loaded with nano-ZnO alters the gut microbiota community to mediate bile acid metabolism and potentiate intestinal immune function in fattening beef cattle

BackgroundTo our knowledge, carbon loaded with nano-ZnO (NZnOC) represents a new nutritional additive for the animal husbandry industry. However, the mechanism by which NZnOC mediates beef cattle growth and intestinal health is not fully understood. This study aimed to investigate the effects of carbon loaded with nano-ZnO (NZnOC) supplementation on growth performance, gut microbiota, bile acid (BAs) metabolism and intestinal immunity in fattening cattle. Twenty cattle (16 ± 0.95 months) were randomly assigned to two dietary groups: CON (control, without feed additive) and NZnOC (diet supplemented with 80 mg NZnOC/kg diet dry matter basic) for 60 d. The colon digesta microbiota composition and BAs concentration were determined by microbiota metagenomics and gas chromatography methods, respectively.ResultsThe results showed that the NZnOC-supplemented cattle had greater final weight, average daily gain and gain-to-feed ratio than those in the CON group. Cattle fed the NZnOC diet had a higher relative abundance of the secondary BAs synthesizing phyla Firmicutes, Tenericutes and Actinobacteria than those fed the CON diet. Dietary supplementation with NZnOC increased the relative abundance of the secondary BAs synthesis microbiota genera Clostridium, Ruminococcus, Eubacterium, and Brevibacillus in colon digesta. Cattle fed the NZnOC diet had increased activities of 3α-hydroxysteroid dehydrogenase (EC: 1.1.1.52) and bile acid-CoA ligase BaiB (EC: 6.2.1.7) in the colon digesta compared with those fed the CON diet. The primary BAs taurocholic acid, taurochenodeoxycholic acid and taurodeoxycholate acid were significantly decreased by dietary NZnOC supplementation, while the secondary BAs deoxycholic acid, taurolithocholic acid, beta-muricholic acid, 12-ketolithocholic acid and ursodeoxycholic acid were significantly increased. Dietary supplementation with NZnOC increased the mRNA abundance of G protein-coupled bile acid receptor 1, protein kinase cAMP-activated catalytic subunit alpha, cyclic-AMP response element binding protein 1 and interleukin (IL)-10 in the colon mucosa of cattle, while the mRNA abundance of tumor necrosis factor and IL-1β were significantly decreased.ConclusionsIn summary, dietary supplementation with NZnOC can facilitate the growth performance and intestinal immune function of cattle by improving BAs metabolism. NZnOC can be supplemented in the diet as a safe regulator of gut microbiota and as a feed additive in the ruminants industry.

AKAP13 Enhances CREB1 Activation by FSH in Granulosa Cells.

Granulosa cells (GCs) must respond appropriately to follicle-stimulating hormone (FSH) for proper follicle maturation. FSH activates protein kinase A (PKA) leading to phosphorylation of the cyclic AMP response element binding protein-1 (CREB1). We identified a unique A-kinase anchoring protein (AKAP13) containing a Rho guanine nucleotide exchange factor (RhoGEF) region that was induced in GCs during folliculogenesis. AKAPs are known to coordinate signaling cascades, and we sought to evaluate the role of AKAP13 in GCs in response to FSH. Aromatase reporter activity was increased in COV434 human GCs overexpressing AKAP13. Addition of FSH, or the PKA activator forskolin, significantly enhanced this activity by 1.5- to 2.5-fold, respectively (p < 0.001). Treatment with the PKA inhibitor H89 significantly reduced AKAP13-dependent activation of an aromatase reporter (p = 0.0067). AKAP13 physically interacted with CREB1 in co-immunoprecipitation experiments and increased the phosphorylation of CREB1. CREB1 phosphorylation increased after FSH treatment in a time-specific manner, and this effect was reduced by siRNA directed against AKAP13 (p = 0.05). CREB1 activation increased by 18.5-fold with co-expression of AKAP13 in the presence of FSH (p < 0.001). Aromatase reporter activity was reduced by inhibitors of the RhoGEF region, C3 transferase and A13, and greatly enhanced by the RhoGEF activator, A02. In primary murine and COV43 GCs, siRNA knockdown of Akap13/AKAP13 decreased aromatase and luteinizing hormone receptor transcripts in cells treated with FSH, compared with controls. Collectively, these findings suggest that AKAP13 may function as a scaffolding protein in FSH signal transduction via an interaction with CREB, resulting in phosphorylation of CREB.

Abstract C040: CREB-LIF axis drives immune suppression by promoting macrophage polarization in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains an aggressive malignancy, and its unique genetic makeup and immunosuppressive tumor microenvironment (TME) produce a lack of response to current therapies. Previously, we have identified Cyclic AMP Response Element Binding protein 1 (CREB) as an oncogenic transcriptional factor that promotes disease aggressiveness, poor survival, and immune suppression. Based on these, we sought to determine the role of tumor intrinsic CREB in promoting immunosuppressive TME in PDAC. Methods: We have generated a genetically engineered mouse model (GEMM) of pancreas-specific CREB deletion (CREBfl/fl) in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice that phenocopy human PDAC disease. CRISPR/CAS9-based genomic editing was utilized to ablate CREB (CREBKO) in KPC tumor cells. RNA-sequencing analysis was performed in KPC CREB wild type (CREBWT) vs. CREBKO tumor cells to identify CREB-mediated transcriptomic changes. Chromatin immunoprecipitation (ChIP-qPCR) analysis was performed in KPC tumor cells. Syngeneic orthotopic tumor implantation of these cells was performed in the pancreata of mice. Immunophenotyping was accomplished to assess changes in the immune subsets with CREB deletion invivo. Additionally, these tissues were also processed for single-cell RNA (scRNA) transcriptomics analysis to evaluate changes on different cellular constituents. Results: Pancreas-specific CREB deletion in the KPC GEMM led to a significant reduction in the primary tumor burden, liver metastases, and improved overall survival compared to wild-type KPC. In assessing the immune repercussions of CREB deletion in pancreatic tumors, we observed a decreased infiltration of tumor-promoting CD11b+ F4/80+ CD206+ [M2-like tumor-associated macrophages (TAMs)] and a concomitant increase in the antigen-presenting M1-like macrophages (F4/80+MHC-IIhighCD86high). Additionally, scRNA sequencing analysis within the macrophage compartment in CREBKO tumors revealed significant enrichment of M1 hallmark signaling pathways. Also, CREB ablation in these tumors further facilitated increased infiltration of activated effector memory CD8+ T cells and resulted in enhanced adaptive immune response within the PDAC TME. Mechanistically, RNA transcriptomic-based analysis of CREBKO tumor cells revealed, Leukemia inhibitory factor (LIF) as one of the downstream targets of CREB. ChIP qPCR analysis after CREB1 pulldown confirmed its occupancy on LIF promoter regulatory region. Incubation of macrophages with CREBWT conditioned media in the presence of LIF neutralizing antibody or blocking its receptor expression using EC359 pushed these macrophages towards an M1-like phenotype, confirming its role as a mediator of tumor cell macrophage crosstalk. Conclusion: These findings broaden our understanding of the tumor cell-intrinsic role of CREB and provide new insights into its molecular underpinnings in fostering immunosuppressive profile by promoting skewness of TAMs towards M2 phenotype in PDAC. Citation Format: Siddharth Mehra, Vanessa T. Garrido, Samara Singh, Iago De Castro Silva, Anna Bianchi, Luis A. Nivelo, Nilesh U. Deshpande, Austin R. Dosch, Zhou Zhiqun, Supriya Srinivasan, Christine I. Rafie, Ifeanyichukwu C. Ogobuiro, Xi Chen, Alejandro Villarino, Jashodeep Datta, Nipun B. Merchant, Nagaraj Nagathihalli. CREB-LIF axis drives immune suppression by promoting macrophage polarization in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C040.

Overcoming high level adenosine-mediated immunosuppression by DZD2269, a potent and selective A2aR antagonist

BackgroundAdenosine is a potent immunosuppressant whose levels in the tumor microenvironment (TME) are often much higher than those in normal tissues. Binding of adenosine to its receptor A2aR activates a cascade of genes and leads to immunosuppression. In addition, immune checkpoint blockage markedly increases A2aR expression in T cells, which could dampen their anti-tumor response. Several A2aR antagonists are under clinical development, but with limited clinical benefit reported so far. These A2aR antagonists showed much diminished activity at high adenosine levels found in TME, which may explain their clinical underperformance. We report the discovery and early clinical development of DZD2269, a novel A2aR antagonist which can fully block A2aR mediated immunosuppression commonly found in TME. Adenosine stimulates phosphorylation of cyclic AMP response element binding protein (CREB) in T cells and inhibits anti-tumor cytokine secretion in PBMCs in a dose-dependent manner. DZD2269 was able to reverse the immunosuppression induced by high concentrations of adenosine, as demonstrated by inhibiting CREB phosphorylation in T cells, restoring Th1 cytokine secretion in PBMCs, and stimulating dendritic cells (DCs) maturation. As a single agent, DZD2269 showed anti-tumor growth in multiple syngeneic mouse tumor models, and more profound anti-tumor effects were observed when DZD2269 was in combination with immune checkpoint inhibitors, radiotherapy, or chemotherapy. A good PK/PD relationship was observed in these animal models. In the phase 1 clinical study, downregulation of pCREB was detected in human T cells, consistent with preclinical prediction. Our data support further clinical development of DZD2269 in patients with cancer.MethodsThe selectivity of DZD2269 for adenosine receptors was tested in engineered cell lines, and its efficacy in blocking A2aR signaling and reversing adenosine-mediated immunosuppression was assessed in human T cells and peripheral blood mononuclear cells (PBMCs). The anti-tumor effects of DZD2269 were evaluated in multiple syngeneic mouse models as a single agent as well as in combination with chemotherapy, radiotherapy, or immune checkpoint inhibitors. A phase 1 study in healthy volunteers (NCT04932005) has been initiated to assess safety, pharmacokinetics (PK) and pharmacodynamics (PD) of DZD2269.ResultsAdenosine stimulates phosphorylation of cyclic AMP response element binding protein (CREB) in T cells and inhibits anti-tumor cytokine secretion in PBMCs in a dose-dependent manner. DZD2269 was able to reverse the immunosuppression induced by high concentrations of adenosine, as demonstrated by inhibiting CREB phosphorylation in T cells, restoring Th1 cytokine secretion in PBMCs, and stimulating dendritic cells (DCs) maturation. As a single agent, DZD2269 showed anti-tumor growth in multiple syngeneic mouse tumor models, and more profound anti-tumor effects were observed when DZD2269 was in combination with immune checkpoint inhibitors, radiotherapy, or chemotherapy. A good PK/PD relationship was observed in these animal models. In the phase 1 clinical study, downregulation of pCREB was detected in human T cells, consistent with preclinical prediction.ConclusionDZD2269 is a novel A2aR antagonist which can fully block A2aR mediated immunosuppression commonly found in TME. Clinical development of DZD2269 in patients with cancer is warranted (NCT04634344).

Impact of Long Non-Coding RNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 on the Susceptibility of High Glucose-Treated Cardiomyocytes to Hypoxia/Reoxygenation

To estimate the effect of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on susceptibility of high glucose (HG)-treated cardiomyocytes (CMs) to hypoxia/reoxygenation (H/R). Forty healthy rats were assigned to the control, diabetes mellitus (DM), DM + ischemia/reperfusion injury (IRI) control, and DM IRI groups (n = 10 for each group). Rat CMs (H9C2) were subjected to HG and H/R treatments. LncRNA MALAT1 and cyclic-AMP responsive element modulator (CREM) mRNA levels were measured using quantitative polymerase chain reaction, and protein levels of CREM, myeloid differentiation primary response protein 88 (MyD88), interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor associated factor 6 (TRAF6), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were quantified using western blotting. Flow cytometry was applied for detection of apoptosis, and a cell viability analyzer determined the number of living cells. IRI induced myocardial apoptosis and elevated lncRNA MALAT1, CREM, and MyD88/IRAK1/TRAF6 levels in DM group mice. In H9C2 cells, HG treatment downregulated CREM expression, which resulted in the downregulation of lncRNA MALAT1 expression, reducing the susceptibility to H/R, increasing cell viability and apoptosis, decreasing the activity of the MyD88/IRAK1/TRAF6 signaling pathway, and suppressing TNF-α and IL-6 expression. LncRNA MALAT1 regulates the susceptibility of HG-treated CMs to H/R through the MyD88 signaling pathway.

Urolithin A attenuates severity of chronic pancreatitis associated with continued alcohol intake by inhibiting PI3K/AKT/mTOR signaling.

Heavy alcohol consumption is the dominant risk factor for chronic pancreatitis (CP); however, treatment and prevention strategies for alcoholic chronic pancreatitis (ACP) remains limited. The present study demonstrates that ACP induction in C57BL/6 mice causes significant acinar cell injury, pancreatic stellate cell (PSC) activation, exocrine function insufficiency, and an increased fibroinflammatory response when compared with alcohol or CP alone. Although the withdrawal of alcohol during ACP recovery led to reversion of pancreatic damage, continued alcohol consumption with established ACP perpetuated pancreatic injury. In addition, phosphokinase array and Western blot analysis of ACP-induced mice pancreata revealed activation of the phosphatidylinositol 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and cyclic AMP response element binding protein (CREB) signaling pathways possibly orchestrating the fibroinflammatory program of ACP pathogenesis. Mice treated with urolithin A (Uro A, a gut-derived microbial metabolite) in the setting of ACP with continued alcohol intake (during the recovery period) showed suppression of AKT and P70S6K activation, and acinar damage was significantly reduced with a parallel reduction in pancreas-infiltrating macrophages and proinflammatory cytokine accumulation. These results collectively provide mechanistic insight into the impact of Uro A on attenuation of ACP severity through suppression of PI3K/AKT/mTOR signaling pathways and can be a useful therapeutic approach in patients with ACP with continuous alcohol intake.NEW & NOTEWORTHY Our novel findings presented here demonstrate the utility of Uro A as an effective therapeutic agent in attenuating alcoholic chronic pancreatitis (ACP) severity with alcohol continuation after established disease, through suppression of the PI3K/AKT/mTOR signaling pathway.

CaMKIV/CREB/BDNF signaling pathway expression in prefrontal cortex, amygdala, hippocampus and hypothalamus in streptozotocin-induced diabetic mice with anxious-like behavior.

Individuals with diabetes mellitus (DM) tend to manifest anxiety and depression, which could be related to changes in the expression of calcium/calmodulin-dependent protein kinase IV (CaMKIV), transcription factor cyclic AMP-responsive element binding protein (CREB), phosphorylated CREB (pCREB) and brain-derived neurotrophic factor (BDNF) in different brain regions. The objective of this study was to determine whether mice with type 1 diabetes (T1DM) induced with streptozotocin show a profile of anxious-type behaviors and alterations in the expression/activity of CaMKIV, CREB, pCREB and BDNF in different regions of the brain (prefrontal cortex, amygdala, hippocampus and hypothalamus) in comparison to non-diabetic mice (NDB). Mice with 3months of chronic DM showed an anxious-like behavioral profile in two anxiety tests (Open Field and Elevated Plus Maze), when compared to NDB. There were significant differences in the expression of cell signaling proteins: diabetic mice had a lower expression of CaMKIV in the hippocampus, a greater expression of CREB in the amygdala and hypothalamus, as well as a lower pCREB/CREB in hypothalamus than NDB mice (P < 0.05). This is the first study evaluating the expression of CaMKIV in the brain of animals with DM, who presented lower expression of this protein in the hippocampus. In addition, it is the first time that CREB was evaluated in amygdala and hypothalamus of animals with DM, who presented a higher expression. Further research is necessary to determine the possible link between expression of CaMKIV and CREB, and the behavioral profile of anxiety in diabetic animals.

GDNF Promotes Astrocyte Abnormal Proliferation and Migration Through the GFRα1/RET/MAPK/pCREB/LOXL2 Signaling Axis.

Glial cell-line derived neurotrophic factor (GDNF) is a powerful astroglioma (AG) proliferation and migration factor that is highly expressed in AG cells derived from astrocytes. However, it is still unclear whether high levels of GDNF promote AG occurrence or if they are secondary to AG formation. We previously reported that high concentrations of GDNF (200 and 500ng/mL) can inhibit DNA damage-induced rat primary astrocytes (RA) apoptosis, suggesting that high concentrations of GDNF may be involved in the malignant transformation of astrocytes to AG cells. Here we show that 200ng/mL GDNF significantly increased the proliferation and migration ability of RA cells and human primary astrocytes (HA). This treatment also induced RA cells to highly express Pgf, Itgb2, Ibsp, Loxl2, Lif, Cxcl10, Serpine1, and other genes that enhance AG proliferation and migration. LOXL2 is an important AG occurrence and development promotion factor and was highly expressed in AG tissues and cells. High concentrations of GDNF promote LOXL2 expression and secretion in RA cells through GDNF family receptor alpha-1(GFRα1)/rearranged during transfection proto-oncogene (RET)/mitogen-activated protein kinase (MAPK)/phosphorylated cyclic AMP response element binding protein (pCREB) signaling. GDNF-induced LOXL2 significantly promotes RA and HA cell proliferation and migration, and increases the expression of Ccl2, Gbp5, MMP11, TNN, and other genes that regulate the extracellular microenvironment in RA cells. Our results demonstrate that high concentrations of GDNF activate LOXL2 expression and secretion via the GFRα1/RET/MAPK/pCREB signal axis, which leads to remodeling of the astrocyte extracellular microenvironment through molecules such as Ccl2, Gbp5, MMP11, TNN. This ultimately results in abnormal astrocyte proliferation and migration. Collectively, these findings suggest that high GDNF concentrations may promote the malignant transformation of astrocytes to AG cells.

Antidepressant effect of Jujuboside A on corticosterone-induced depression in mice

ObjectiveThe aim of this study was to investigate the antidepressant effect of Jujuboside A (JuA) on corticosterone (CORT)-induced depression in mice and explore the underlying mechanisms. MethodsThe mice models were submitted to CORT and treated with JuA (10 and 30 mg/kg) for three weeks. Experiments were also performed on mice with brain-derived neurotrophic factor knockdown (BDNF (±)) as control subjects. Behavioral tests, including the open field test (OFT), tail suspension test (TST), forced swimming test (FST) and Morris water maze (MWM), were then performed to evaluate the antidepressant effect of JuA. The expression levels of BDNF, tyrosine kinase receptor B (TrkB), and cyclic AMP response element binding protein (CREB) in the hippocampi of mice were examined by immunohistochemistry (IHC) and Western blot. The effect of JuA on the viability of mouse hippocampal cells (HT22) was also assessed by CCK-8 assay. ResultsJuA significantly decreased the OFT and TST immobility time of the mice, the total distance travelled and the time spent in the central area also effectively increased in the OFT. In the MWM, the escape latencies of the mice decreased remarkably, while the number of times the mice crossed the platform and the target quadrant increased significantly after treatment with JuA. In addition, the BDNF, TrkB, and CREB expression levels were significantly increased in the hippocampi of the mice treated with JuA. Furthermore, JuA clearly attenuated CORT-induced cell injury, as evidenced by the increased viability of the HT22 cells. ConclusionThese findings demonstrated that JuA may exhibit potential antidepressant effect in mice by increasing protein expression levels of BDNF, TrkB, CREB, and improving the viability of the hippocampal cells.

Abstract 1328: Deciphering the role of CREB1 in shaping the tumor immune landscape of pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains a major therapeutic challenge with hallmarks including oncogenic mutations, desmoplastic architecture, and an immunosuppressive tumor microenvironment (TME). Previously, we have identified Cyclic AMP Response Element Binding protein 1 (CREB) as an oncogenic transcriptional factor downstream of KRAS that promotes disease aggressiveness, poor survival, and immune exclusion. Based on these, we sought to determine the impact of tumor intrinsic CREB deletion in shaping the tumor immune microenvironment. Methods: We have generated a novel genetically engineered mouse model (GEMM) of pancreas-specific CREB deletion (CREBfl/fl) in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice that phenocopy human PDAC disease. CRISPR/CAS9-based genomic editing was utilized to ablate CREB (CREBKO) in KPC tumor cells. RNA-sequencing analysis was performed in KPC CREB wild type (CREBEV) vs. CREBKO tumor cells to identify CREB-mediated transcriptomic changes. Syngeneic orthotopic tumor implantation of these cells was performed into the pancreata of mice. High dimensional immunophenotyping was accomplished to assess changes in the immune subsets with CREB deletion in murine tumors. Additionally, these tissues were processed for immunohistochemical and qPCR-based analysis to assess changes in fibroinflammatory and immune mediators. Results: Pancreas-specific CREB deletion in the KPC GEMM led to a significant reduction in the primary tumor burden, liver metastases and improved overall survival compared to wild-type KPC. Additionally, CREB deletion significantly remodeled the tumor stroma, as evidenced by the reduction in the expression of fibroinflammatory and immunosuppressive markers. In assessing the immune repercussions of CREB deletion in pancreatic tumors, we observed a decreased infiltration of CD11b+ myeloid-derived suppressor cells (MDSCs) and granulocytic-PMN MDSCs (CD11b+ Ly6Ghigh Ly6Clow F4/80-), with a concomitant increase in the antigen-presenting M1-like macrophages (F4/80+ MHC-II high CD86 high). Also, CREB ablation in these tumors further facilitated increased infiltration of activated effector CD8+ T cells resulted in enhanced anti-tumor immune response within the PDAC TME. Mechanistically, RNA transcriptomic and secretome analysis in CREBKO Vs. the wild type KPC tumor cells identified several differentially expressed immunomodulatory soluble mediators responsible for shaping CREB dependent immunogenic landscape in PDAC. Conclusion: Overall, depleting CREB reshapes the tumor immune landscape to reduce innate immunosuppressive myeloid infiltration and reinvigorate the antitumor T cell immune responses to improve overall survival in PDAC. Citation Format: Siddharth Mehra, Vanessa Garrido, Samara Singh, Iago D Silva, Luis Alberto Nivelo, Anna Bianchi, Shrey Modi, Zhiqun Zhou, Austin Dosch, Nilesh Deshpande, Supriya Srinivasan, Christine Rafie, Ifeanyichukwu Ogobuiro, Xi Chen, Alejandro Villarino, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. Deciphering the role of CREB1 in shaping the tumor immune landscape of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1328.

Psychoactive Effects of Lactobacillus johnsonii BS15 on Preventing Memory Dysfunction Induced by Acute Ethanol Exposure Through Modulating Intestinal Microenvironment and Improving Alcohol Metabolic Level.

The negative effects of ethanol (EtOH) abuse on the body have been widely reported in recent years. Building on the microbiota-gut-brain axis hypothesis, our study aimed to demonstrate the potential psychobiotic role of Lactobacillus johnsonii BS15 in the preventive effects of acute EtOH intake on memory impairment. We also determined whether L. johnsonii BS15 intake could effectively improve resistance to acute drinking and alleviate the adverse effects of EtOH. Male mice were fed L. johnsonii BS15 orally with (Probiotic group) or without (Control and Alcohol groups) daily dose of 0.2 × 109 CFU/ml per mouse for 28 days. Gavage with L. johnsonii BS15 significantly modified the ileal microbial ecosystem (assessed by 16S rRNA gene sequencing) in favor of Firmicutes and Lactobacillus, indicating the ability of BS15 to restore the gut microbiota. The acute EtOH exposure model (7 g/kg EtOH per mice) was established by gavage, which was administered to the alcohol and probiotic groups on day 28 of the experiment. The L. johnsonii BS15 intake effectively reduced alcohol unconsciousness time, blood alcohol concentration, and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Meanwhile, the improvement of ethanol resistance time and the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the liver were shown by BS15 in acute alcohol-induced mice. We found that acute EtOH exposure reduced the exploration ratio (assessed by the novel object recognition test), escape latency, number of errors (assessed by passive avoidance test), and spontaneous exploration (assessed by T-maze test) in mice, which were obviously improved by L. johnsonii BS15. In the hippocampus, L. johnsonii BS15 significantly reversed the decrease in antioxidant capacity of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) and mRNA expression of memory-related functional proteins of brain-derived neurotrophic factor (BDNF) and cyclic ampresponse element binding protein (CREB) in the hippocampal tissue after acute EtOH exposure. In conclusion, L. johnsonii BS15 intake appears as a promising psychoactive therapy to ameliorate alcohol-mediated memory impairment by increasing EtOH metabolic levels.

Genetic and epigenetic alterations of cyclic AMP response element modulator in rheumatoid arthritis.

Genetic and epigenetic factors are strongly associated with the autoimmune disease rheumatoid arthritis (RA). Cyclic AMP response element modulator (CREM), a gene related to immune system regulation, has been implicated in various immune-mediated inflammatory processes, although it remains unknown whether CREM is involved in RA. This study enrolled 278 RA patients and 262 controls. Three variants [rs12765063, rs17499247, rs1213386] were identified through linkage disequilibrium and expression quantitative trait locus analysis, and CREM transcript abundance was determined by quantitative real-time polymerase chain reaction. The identified variants were genotyped using the TaqMan Allelic Discrimination assay, and CREM promoter methylation was assessed by bisulphite sequencing. Differences between groups and correlations between variables were assessed with Student's t-tests and Pearson's correlation coefficients. Associations between phenotypes and genotypes were evaluated with logistic regression. Rheumatoid arthritis patients exhibited increased CREM expression (p<.0001), which was decreased by methotrexate (p=.0223) and biologics (p=.0001), but could not be attributed to CREM variants. Interestingly, rs17499247 displayed a significant association with serositis (p=.0377), and rs1213386 increased the risk of lymphadenopathy (p=.0398). Furthermore, seven CpG sites showed decreased methylation in RA (p=.0477~ p<.0001). Collectively, our results indicate that CREM hypomethylation and CREM upregulation occur in RA and that CREM variants are involved in the development of serositis and lymphadenopathy in RA. This study highlights the novel roles of CREM in RA pathophysiology.

Effects of inhibiting astrocytes and BET/BRD4 chromatin reader on spatial memory and synaptic proteins in rats with Alzheimer's disease.

Communication between astrocytes and neurons has a profound effect on the pathophysiology of Alzheimer's disease (AD). Astrocytes regulate homeostasis and increase synaptic plasticity in physiological situations, however, they become activated during the progression of AD. Whether or not these reactions are supportive or detrimental for the central nervous system have not been understood yet. Considering epigenetic regulation of neuroinflammatory genes by chromatin readers, particularly bromodomain and extraterminal domain (BET) family, here we examined the effect of chronic co-inhibition of astrocytes metabolism (with fluorocitrate) and also BRD4 (with JQ1) on cognition deficit at early stages of AD. Forty adult male Wistar rats underwent stereotaxic cannulation for inducing AD by intrahippocampal injection of Aβ1-42 (4μg/8μl/rat). Then animals were divided into five groups of Saline+DMSO, Aβ + saline+DMSO, Aβ + JQ1, Aβ + FC (fluorocitrate), and Aβ + JQ1 + FC and received the related treatments. Two weeks later, spatial memory was recorded by Morris Water Maze (MWM), and the levels of phosphorylated cyclic-AMP response element binding protein (CREB), postsynaptic density 95 (PSD95), synaptophysin (SYP), and tumor necrosis factor-alpha (TNF-α) were measured in the hippocampus by western blotting and RT-qPCR. Administration of JQ1 significantly improved both acquisition and retrieval of spatial memory, which were evident by decreased escape latency and increased total time spent (TTS) in target quadrant, and significant rise in p-CREB, PSD95, and synaptophysin compared with Aβ + saline+DMSO group. In contrast, both groups receiving FC demonstrated memory decline, and reduction in p-CREB, PSD95 and synaptophysin in parallel with increase in TNF-α. Our data indicate that chronic inhibition of BRD4 significantly restores memory impaired by amyloid β partly via CREB signaling and upregulating synaptic proteins of PSD95 and synaptophysin. However, inhibition of astrocytes nullifies the memory-boosting effects of JQ1 and reduces CREB/PSD95/synaptophysin levels in hippocampus.

Multivalency enables unidirectional switch-like competition between intrinsically disordered proteins

Intrinsically disordered proteins must compete for binding to common regulatory targets to carry out their biological functions. Previously, we showed that the activation domains of two disordered proteins, the transcription factor HIF-1α and its negative regulator CITED2, function as a unidirectional, allosteric molecular switch to control transcription of critical adaptive genes under conditions of oxygen deprivation. These proteins achieve transcriptional control by competing for binding to the TAZ1 domain of the transcriptional coactivators CREB-binding protein (CBP) and p300 (CREB: cyclic-AMP response element binding protein). To characterize the mechanistic details behind this molecular switch, we used solution NMR spectroscopy and complementary biophysical methods to determine the contributions of individual binding motifs in CITED2 to the overall competition process. An N-terminal region of the CITED2 activation domain, which forms a helix when bound to TAZ1, plays a critical role in initiating competition with HIF-1α by enabling formation of a ternary complex in a process that is highly dependent on the dynamics and disorder of the competing partners. Two other conserved binding motifs in CITED2, the LPEL motif and an aromatic/hydrophobic motif that we term ϕC, function synergistically to enhance binding of CITED2 and inhibit rebinding of HIF-1α. The apparent unidirectionality of competition between HIF-1α and CITED2 is lost when one or more of these binding regions is altered by truncation or mutation of the CITED2 peptide. Our findings illustrate the complexity of molecular interactions involving disordered proteins containing multivalent interaction motifs and provide insight into the unique mechanisms by which disordered proteins compete for occupancy of common molecular targets within the cell.

Maternal omega-3 fatty acid supplementation against prenatal lead exposure induced cognitive impairment in offspring mice.

Maternal lead exposure is associated with poor outcomes in fetal brain development such as cognitive dysfunction. Here, we aimed to reveal the effect and mechanism of omega-3 fatty acids in ameliorating maternal lead exposure-induced cognitive impairment in mouse offspring. The activity levels of locomotor and anxiety, memory and learning capacity, spatial working memory, and cognitive behavioral function were determined using the open field test, Morris water maze, Y-maze, and nest-building test, respectively. The protein levels of brain-derived neurotrophic factor (BDNF), nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were measured using enzyme-linked immunosorbent assay or Western blot. The mRNA levels of BDNF, tyrosine kinase B (TrkB) and cyclic AMP response element binding protein (CREB) were measured by real-time qPCR. Malondialdehyde (MDA) and anti-oxidants, including SOD, GSH and CAT, were measured using bioassay kits. We found that supplementing omega-3 significantly improved cognitive behavioral function in offspring after prenatal lead exposure. The protein and mRNA levels of BDNF, TrkB and CREB in the prenatal lead exposure group were significantly upregulated by omega-3 supplementation. The MDA level in the prenatal lead exposure group was markedly elevated compared with the control group, which was significantly reduced by omega-3. Omega-3 restored anti-oxidants SOD, GSH and CAT to control levels after prenatal lead exposure. Omega-3 significantly upregulated Nrf2 nuclear expression and HO-1 expression after prenatal lead exposure. Overall, omega-3 supplementation significantly elevated the BDNF/TrkB/CREB pathway and restores anti-oxidants by upregulating the Nrf2/HO-1, thereby improving cognitive function in offspring after prenatal lead exposure.

Design, Synthesis, and Biological Evaluation of 1-(Indolizin-3-yl)ethan-1-ones as CBP Bromodomain Inhibitors for the Treatment of Prostate Cancer

CREB (cyclic-AMP responsive element binding protein) binding protein (CBP) is a potential target for prostate cancer treatment. Herein, we report the structural optimization of a series of 1-(indolizin-3-yl)ethan-1-one compounds as new selective CBP bromodomain inhibitors, aiming to improve cellular potency and metabolic stability. This process led to compound 9g (Y08284), which possesses good liver microsomal stability and pharmacokinetic properties (F = 25.9%). Furthermore, the compound is able to inhibit CBP bromodomain as well as the proliferation, colony formation, and migration of prostate cancer cells. Additionally, the new inhibitor shows promising antitumor efficacy in a 22Rv1 xenograft model (TGI = 88%). This study provides new lead compounds for further development of drugs for the treatment of prostate cancer.

CN470 is a BET/CBP/p300 multi-bromodomain inhibitor and has an anti-tumor activity against MLL-rearranged acute lymphoblastic leukemia

Acute lymphoblastic leukemia with chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene (MLL-r ALL) remains an incurable disease. Thus, development of a safe and effective therapeutic agent to treat this disease is crucial to address this unmet medical need. BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, and cyclic AMP response element binding protein binding protein (CBP) and p300, two paralogous histone acetyltransferases, are all considered cancer drug targets and simultaneous targeting of these proteins may have therapeutic advantages. Here, we demonstrate that a BET/CBP/p300 multi-bromodomain inhibitor, CN470, has anti-tumor activity against MLL-r ALL in vitro and in vivo. CN470, potently inhibited ligand binding to the bromodomains of BRD4, CBP, and p300 and suppressed the growth of MLL-r ALL cell lines and patient-derived cells with MLL rearrangements. CN470 suppressed mRNA and protein expression of MYC and induced apoptosis in MLL-r ALL cells, following a cell cycle arrest in the G1 phase. Moreover, CN470 reduced BRD4 binding to acetylated histone H3. The in vivo effects of CN470 were investigated using SEMLuc/GFP cells expressing luminescent markers in an orthotopic mouse model. Mice administered CN470 daily had prolonged survival compared to the vehicle group. Further, CN470 also showed anti-tumor effects against an MLL-r ALL patient-derived xenograft model. These findings suggest that inhibition of BET/CBP/p300 by the multi-bromodomain inhibitor, CN470, represents a promising therapeutic approach against MLL-r ALL.

Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells.

Skin barrier damage is present in the patients with hereditary disorders of the magnesium channel, but the molecular mechanism has not been fully understood. We found that the expressions of hyaluronan synthase (HAS), HAS2 and HAS3 are influenced by MgCl2 concentration in human keratinocyte-derived HaCaT cells. The exposure of cells to a high concentration (5.8 mM) of MgCl2 induced the elevation of HAS2/3 expression, which was inhibited by mRNA knockdown of nonimprinted in Prader-Willi/Angelman syndrome-like domain containing 4 (NIPAL4). Similarly, the content of hyaluronic acid (HA) was changed according to MgCl2 concentration and the expression of NIPAL4. The MgCl2 supplementation increased the reporter activities of HAS2/3, which were inhibited by NIPAL4 knockdown, indicating that the expressions of HAS2/3 are up-regulated at the transcriptional level. The reporter activities and mRNA levels of HAS2/3, and the production of HA were inhibited by CHIR-99021, a glycogen synthase kinase-3 (GSK3) inhibitor, and naphthol AS-E, a cyclic AMP-response element binding protein (CREB) inhibitor. Furthermore, the mutation in putative CREB-binding sites of promoter region in HAS2/3 genes inhibited the MgCl2 supplementation-induced elevation of promoter activity. Our results indicate that the expressions of HAS2/3 are up-regulated by MgCl2 supplementation in HaCaT cells mediated through the activation of GSK3 and CREB. Magnesium may play a pivotal role in maintaining the skin barrier function and magnesium supplementation may be useful to enhance moisturization and wound repair in the skin.

Skeletal muscle-targeted delivery of Fgf6 protects mice from diet-induced obesity and insulin resistance.

Obesity, a major health care issue, is characterized by metabolic abnormalities in multiple tissues, including the skeletal muscle. Although dysregulation of skeletal muscle metabolism can strongly influence the homeostasis of systemic energy, the underlying mechanism remains unclear. We found promoter hypermethylation and decreased gene expression of fibroblast growth factor 6 (FGF6) in the skeletal muscle of individuals with obesity using high-throughput sequencing. Reduced binding of the cyclic AMP responsive element binding protein-1 (CREB1) to the hypermethylated cyclic AMP response element, which is a regulatory element upstream of the transcription initiation site, partially contributed to the downregulation of FGF6 in patients with obesity. Overexpression of Fgf6 in mouse skeletal muscle stimulated protein synthesis, activating the mammalian target of rapamycin pathway, and prevented the increase in weight and the development of insulin resistance in high-fat diet–fed mice. Thus, our findings highlight the role played by Fgf6 in regulating skeletal muscle hypertrophy and whole-body metabolism, indicating its potential in strategies aimed at preventing and treating metabolic diseases.