Cyclic AMP Response Element Research Articles

Evolution of mirror-image pain in temporomandibular joint osteoarthritis mouse model.

Mirror-image pain is a kind of pain that occurs on the contralateral side, but its pathogenesis remains unclear.ObjectiveTo develop an osteoarthritis mouse model for investigating mirror-image pain through observing nocifensive behaviors, histological changes, and nociceptive activity at days 3, 7, 14, 21, and 28 after the chemical induction of unilateral temporomandibular joint (TMJ) osteoarthritis.MethodologyWe randomly divided 6-week-old mice into sham and complete Freund adjuvant groups. To induce nocifensive behaviors, we applied 0.04 g of von Frey filament, 10 psi of air puff, and cold acetone on both sides of whisker pads at different days. The histology of TMJ on both sides was observed by hematoxylin/eosin staining and microcomputed tomography scanning. Furthermore, the nociceptive activity was evaluated using the phosphorylated cyclic AMP response element binding protein (pCREB) and a microglia marker at different days in the trigeminal subnucleus caudalis.ResultsNocifensive behaviors against mechanical and temperature stimuli on the contralateral side became stronger than the baseline on day 28, in agreement with the elevation of the pCREB and the microglia marker in the trigeminal subnucleus caudalis. Thus, hypernociception on the contralateral side occurred at day 28.ConclusionsClearly, the TMJ model with unilateral osteoarthritis exhibited mirror-image pain. Therefore, this model is useful in investigating the pathogenesis of pain and in developing treatments.

ATF3 expression in cardiomyocytes and myofibroblasts following transverse aortic constriction displays distinct phenotypes.

BackgroundActivating transcription 3 (ATF3) is a member of the basic leucine zipper family of transcription factors. ATF3 is an immediate early gene expressed following various cellular stresses. ATF3 acts through binding to cyclic AMP response elements found in the promoters of key regulatory proteins that determine cell fate. In the heart, multiple cardiac stresses result in chronic ATF3 expression. Transgenic mice with ATF3 expression in cardiomyocytes clearly demonstrate that ATF3 serves a leading role in heart hypertrophy, cardiac fibrosis, cardiac dysfunction and death. In contrast, the use of ATF3 whole body knockout mice resulted non-conclusive results. The heart is composed of various cell types such as cardiomyocytes, fibroblasts, endothelial and immune cells. The question that we addressed in this study is whether ablation of ATF3 in unique cell types in the heart results in diverse cardiac phenotypes.MethodsATF3-flox mice were crossed with αMHC and Postn specific promoters directing CRE expression and thus ATF3 ablation in cardiomyocytes and myofibroblast cells. Mice were challenged with transverse aortic constriction (TAC) for eight weeks and heart function, ventricle weight, hypertrophic markers, fibrosis markers and ATF3 expression were assessed by qRT-PCR.ResultsThe results of the study show that ATF3 deletion in cardiomyocytes followed by TAC resulted in reduced heart growth and dampened fibrosis response while ATF3 ablation in myofibroblasts displayed a reduced hypertrophic gene program.ConclusionsTAC-operation results in increased ATF3 expression in both myofibroblasts and cardiomyocytes that promotes a hypertrophic program and fibrotic cardiac growth, respectively.

Attenuation of Anxiety-Like Behavior by Helichrysum stoechas (L.) Moench Methanolic Extract through Up-Regulation of ERK Signaling Pathways in Noradrenergic Neurons.

The long-term use of anxiolytic and antidepressant drugs can cause a plethora of side effects and the use of complementary and alternative medicine, which is generally considered safer than conventional medicine, is consistently increasing. Helichrysum stoechas (L.) Moench methanolic extract (HSE) has shown MAO-A inhibitory properties in previous studies. With the aim of obtaining innovative and safer therapies for mood disorders, this study investigated the potential activity of HSE in the management of anxiety- and depression-related symptoms. HSE showed dose-dependent (30–100 mg/kg p.o.) anxiolytic-like activity in the light dark box and marble burying tests, without any antidepressant-like activity, as shown by the results of the tail suspension test. Additionally, HSE did not have any effect on the modulation of pain, which highlights its selectivity in the control of anxiety-related behavior. At active doses, HSE did not produce any sedative effect or result in impaired motor coordination and memory functions. Western blotting experiments showed the ability of HSE to counteract the reduction in the phosphorylation of ERK44/42, to restore brain-derived neurotrophic factor (BDNF) expression and to return cyclic AMP response element binding (CREB) levels to basal levels in noradrenergic hippocampal neurons of mice exposed to an anxiety-related environment, which indicates a protective role against anxiety behavior. These results suggest that oral administration of HSE might represent an interesting opportunity for the management of anxiety disorders.

Ablation of LGR4 signaling enhances radiation sensitivity of prostate cancer cells

AimOur previous study has shown that leucine-rich repeat containing GPCR-4 (LGR4, or GPR48) LGR4 plays a role in cell migration, invasion, proliferation and apoptosis of prostate cancer (PCa). In this study, we aimed to explore whether LGR4 would affect radiation response in PCa. Materials and methodsLGR4 expression was silenced by shRNA transfection. qRT-PCR was employed to determine mRNA expression of LGR4 and DNA damage repair genes. Western blot was used to evaluate protein expression of LGR4, RSPO1-4, androgen receptor (AR), cyclic AMP response-element binding protein (CREB1), γH2A.X, and H2A.X. Cell proliferation was detected by CCK-8 assay and apoptosis was assayed by flow cytometry. Additionally, a xenograft model was also established to validate the role of LGR4 in PCa cells after radiation. Key findingsLGR4 expression was enhanced in PCa cells by radiation treatment in dose- and time-dependent means. RSPO1-4 were also upregulated post-radiation. Furthermore, LGR4 knockdown exacerbated apoptosis, reduced cell viabilities and strengthened nuclear γH2A.X staining in AR positive PCa cells but not in AR negative cells in the presence of radiation. Likewise, LGR4 ablation diminished AR and CREB1 expression induced by radiation. In contrast, RSPO1 stimulation augmented cell viabilities, promoted AR and CREB1 expression, and upregulated DNA repair gene expression, which could be reversed by enzalutamide, except for AR expression. Additionally, LGR4 knockdown further suppressed tumor growth and AR/CREB1 expression but enhanced γH2A.X expression in xenografts. SignificanceIn all, our study suggested that LGR4 might serve as an important regulator of radiation sensitivity in PCa.

Pp90RSK Isoforms Play Distinct Roles during Hematopoiesis

Ribosomal S6 Kinases (RSKs) include a family of serine/threonine kinases that regulate cell proliferation and survival. In humans, four RSK isoforms (RSK1-4) have been identified. Although they are 73-80% conserved in sequence homology, RSK isoforms are reported to have distinct functions. RSKs phosphorylate cyclic AMP response element binding protein (CREB) which is a regulator of hematopoietic proliferation and differentiation. RSK1 is hyperactivated in AML and is therefore a potential therapeutic target for acute myeloid leukemia (AML). Therefore, inhibition of RSK requires understanding of its role during normal hematopoiesis. Furthermore, the role of RSK isoforms during hematopoiesis and leukemogenesis has not been well characterized. To study the expression of RSK isoforms in hematopoiesis, we cultured human cord blood CD34+ (CBCD34+) hematopoietic stem and progenitor cells (HSPCs) in differentiating media containing SCF, Flt3L, TOP, IL-3, IL-6, GM-CSF, and EPO and performed qPCR with mRNA of the RSK isoforms at different stages of maturation up to 14 days. RPS6KA1 (RSK1) was expressed throughout hematopoiesis, but RPS6KA3 (RSK2) and RPS6KA2 (RSK3) showed higher expression by 2-fold (p<0.05) in the earlier stages of normal hematopoiesis. Moreover, RPS6KA3 is highly expressed in CD38-CD34+ HSCs compared with other progenitor populations (LMPP, MPP, CMP, GMP, and MEP) isolated from human cord blood. RPS6KA2 expression was higher in LMPP by 7-fold (p<0.05) and CMP by 3-fold (p<0.05) compared with HSCs but was barely detectable in MEPs. RPS6KA1 was ubiquitously expressed in all progenitor populations. These results suggest that RSK2 and RSK3 have distinct functions during early myelopoiesis. To study the requirement of RSK isoforms during hematopoiesis, we knocked down RSK expression by transducing lentivirus expressing isoform-specific shRNAs or scramble controls into human CBCD34+ cells. On day 5 following transduction, cells were sorted and plated in methylcellulose media and assessed for the colony-forming activity of RSK knockdown in HSPCs. While RSK3 knockdown increased BFU-E colonies by 1.5-fold (p<0.05) compared with control cells, RSK1 knockdown decreased BFU-E and CFU-E erythroid colonies by 2-fold (p<0.05) but increased CFU-GM colonies by 2-fold (p<0.05) compared with control cells. However, RSK2 knockdown did not show any significant effects on colony-forming activity of HSPCs. We confirmed the isoform-specific effect of RSKs in normal hematopoiesis using liquid culture assays. We collected transduced cells at day 8, 11, and 14 after transduction and analyzed cell populations by flow cytometry. Similar to colony formation assay results, RSK3 knockdown increased the CD71+ erythroid population by 1.5-fold (p<0.001), but suppressed production of the CD14+ monocyte population. RSK1 knockdown enhanced production of the CD11b+/CD14+ myeloid population, but inhibited the CD71+ erythroid production by 1.5-fold (p<0.001) compared with control HSPCs. There was no significant change in blood cell differentiation in RSK2 knockdown cells. These data demonstrate that RSK1 and RSK3 exert opposite functions during erythroid and myeloid differentiation of HSPCs, suggesting a novel role for RSK isoforms as a determinant of early fate decisions of HSCs. Disclosures No relevant conflicts of interest to declare.

Down Syndrome Critical Region 1 Reduces Oxidative Stress-Induced Retinal Ganglion Cells Apoptosis via CREB-Bcl-2 Pathway.

PurposeIrreversible retina ganglion cell (RGC) loss is a key process during glaucoma progression. Down syndrome critical region 1 (DSCR1) has been shown to have protective effects against neuronal death. In this study, we aimed to investigate the neuroprotective mechanisms of DSCR1 on RGCs.MethodsDBA/2J mice and optic nerve crush (ONC) rat model were used for vivo assays. Oxidative stress model of primary RGCs was carried out with in vitro transduction. DSCR1 protein localization was assessed by immunofluorescence. Differential protein expression was validated by Western blot, and gene expression was detected by real-time PCR. TUNEL was used to identify cell apoptosis, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to analyze cell viability.ResultsSignificant upregulation of DSCR1 was observed in DBA/2J mice, ONC rat model, and RGCs treated with H2O2, reaching peaks at the age of 6 months in DBA/2J mice, 5 days after ONC in rats, and 24 hours after H2O2 treatment in RGCs, respectively. DSCR1 was shown to be expressed in the ganglion cell layer. In vitro, overexpressed DSCR1 significantly promoted phosphorylation of cyclic AMP response element binding protein (CREB), B-cell lymphoma 2 (Bcl-2) expression, and RGC survival rate while reducing cleaved caspase 3 expression in H2O2-treated RGCs. On the other hand, the opposite effects were shown after knockdown of DSCR1. In addition, silencing of CREB inhibited expression of DSCR1.ConclusionsOur results suggested that DSCR1 might protect the RGCs against oxidative stress via the CREB–Bcl-2 pathway, which may provide a theoretical basis for future treatments of glaucoma.

Role of cyclic AMP response element binding protein (CREB) in angiotensin II-induced responses in vascular smooth muscle cells.

Cyclic AMP response element (CRE) binding protein (CREB) is a nuclear transcription factor that regulates the transcription of several genes containing the CRE sites on their promoters. CREB is activated by phosphorylation on a key serine residue, Ser311, in response to a wide variety of extracellular stimuli including angiotensin II (Ang II). Ang II is an important vasoactive peptide and mitogen for vascular smooth muscle cells (VSMC) that in addition to regulating the contractile response in VSMC also plays an important role in phenotypic switch of VSMC from contractile to a synthetic state. The synthetic VSMC are known to exhibit proliferative and migratory properties due to hyperactivation of Ang II-induced signaling events. Ang II has been shown to induce CREB phosphorylation/activation and transcription of genes implicated in proliferation, growth, and migration. Here, we have highlighted some key studies that have demonstrated an important role of CREB in Ang II-mediated gene transcription, proliferation, hypertrophy, and migration of VSMC.

Silymarin alleviates docetaxel-induced central and peripheral neurotoxicity by reducing oxidative stress, inflammation and apoptosis in rats.

Docetaxel (DTX) is a chemotherapeutic agent used in the treatment of various malignancies but is often associated with central and peripheral neurotoxicity. The aim of this study was to investigate the neuroprotective effect of silymarin (SLM) against DTX-induced central and peripheral neurotoxicities in rats. Rats received 25 and 50mg/kg body weight SLM orally for seven consecutive days after receiving a single injection of 30mg/kg body weight DTX on the first day. SLM significantly decreased brain lipid peroxidation level and ameliorated brain glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities in DTX-administered rats. SLM attenuated levels of nuclear factor kappa B (NF-κB), tumor necrosis factor-α (TNF-α), glial fibrillary acidic protein (GFAP) and activity of p38α mitogen-activated protein kinase (p38α MAPK) whereas caused an increase in levels of neural cell adhesion molecule (NCAM) in the brain and sciatic nerve of DTX-induced rats. In addition, SLM improved the histological structure of the brain and sciatic nerve tissues and decreased the expression of c-Jun N-terminal kinase (JNK) in the sciatic nerve whereas increased cyclic AMP response element binding protein (CREB) expression in the brain induced by DTX. Additionally, SLM markedly up-regulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and B-cell lymphoma-2 (Bcl-2) and downregulated the expression of Bcl-2 associatedXprotein (Bax) in the brain and sciatic nerve tissues of DTX-induced rats. Our results show that SLM can protect DTX-induced brain and sciatic nerve injuries by enhancing the antioxidant defense system and suppressing apoptosis and inflammation.

Chemotherapy Controls Metastasis Through Stimulatory Effects on GRP78 and Its Transcription Factor CREB3L1.

To achieve a cure for metastatic breast cancer, further understanding of molecular drivers of the metastatic cascade is essential. Currently, chemotherapy regimens include doxorubicin and paclitaxel which act in part by inducing the unfolded protein response (UPR). The master regulator of the UPR, glucose regulated protein 78 (GRP78), localizes on the surface of tumor cells and is associated with metastatic disease. Cyclic AMP responsive element binding protein 3-like 1 (CREB3L1), a member of the UPR, is a breast cancer metastasis suppressor that acts on cyclic AMP to promote the expression of target genes including GRP78. The aim of the present study was to evaluate the effects of chemotherapy on CREB3L1 and cell-surface GRP78 expression and its association with the development of breast cancer metastasis. For this purpose, we use breast cancer cells migration in vitro assays and an in vivo metastatic mouse model. The results showed that chemotherapy activated CREB3L1 and enhanced cell-surface GRP78 expression specifically in triple-negative breast cancer cells (TNBC), reducing their migration and metastatic potential. CREB3L1 knockout (KO) in the triple negative MDAMB231 cell line using CRISPR/Cas9 technology led to inhibition of GRP78 expression and abrogation of the CREB3L1 metastatic suppression function. Inoculation of CREB3L1-KO MDAMB231 cells into a mouse metastatic model induced a massive metastatic profile which chemotherapy failed to prevent. These findings elucidate a potential pathway to the development of a novel treatment strategy for metastatic TNBC based on modulating CREB3L1 and cell-surface GRP78 expression by chemotherapy and GRP78-targeted drugs.

Melatonin Induction of APP Intracellular Domain 50 SUMOylation Alleviates AD through Enhanced Transcriptional Activation and Aβ Degradation

The amyloid precursor protein (APP) intracellular domain (AICD) is implicated in the pathogenesis of Alzheimer’s disease (AD), but post-translational modification of AICD has rarely been studied and its role in AD is unknown. In this study, we examined the role and molecular mechanism of AICD SUMOylation in the pathogenesis of AD. We found that AICD is SUMO-modified by the SUMO E3 ligase protein inhibitor of activated STAT1 (PIAS1) in the hippocampus at Lys-43 predominantly, and that knockdown of PIAS1 decreases endogenous AICD SUMOylation. AICD SUMOylation increases AICD association with its binding protein Fe65 and increases AICD nuclear translocation. Furthermore, AICD SUMOylation increases AICD association with cyclic AMP-responsive element binding protein (CREB) and p65 and their DNA binding for transcriptional activation of neprilysin (NEP) and transthyretin (TTR), two major Aβ-degrading enzymes, respectively. Consequently, AICD SUMOylation decreases the Aβ level, Aβ oligomerization, and amyloid plaque deposits. It also rescues spatial memory deficits in APP/PS1 mice. Conversely, blockade of AICD SUMOylation at Lys-43 produces the opposite effects. Melatonin is identified as an endogenous stimulus that induces AICD SUMOylation. It also decreases the Aβ level and rescues reduction of PIAS1, NEP, and TTR expression in APP/PS1 mice. In this study, we demonstrate that AICD SUMOylation functions as a novel endogenous defense mechanism to combat AD.

Beta amyloid-induced time-dependent learning and memory impairment: involvement of HPA axis dysfunction.

Aβ aggregation is one of the pathological biomarkers of Alzheimer's disease (AD). However, the possible mechanism related to Aβ-induced pathological signaling pathway is still unknown. In the present study, Aβ1-42-induced time-dependent memory impairment and its possible relationship to hypothalamic-pituitary-adrenal (HPA) axis hyperactivity were examined. Aβ1-42-treated mice significantly impaired acquisition activity in the learning curve at 10days, 1 and 4months in the Morris water-maze (MWM) task. This learning activity was back to normal at 8months after Aβ1-42 treatment. In the probe trial test, Aβ1-42-treated mice needed longer latencies to touch the precious platform location and fewer numbers of crossing from 10days to 4months after microinjection. This Aβ1-42 induced memory loss was consistent with the results of the step-down passive avoidance test. The HPA axis related parameters, such as corticosterone (CORT) level in the serum, glucocorticoid receptor (GR) and corticotropin-releasing factor receptor (CRF-R) expression in the frontal cortex and hippocampus increased in Aβ1-42-treated mice from 10days to 4months. While the downstream molecules phosphorylation of cyclic AMP response element binding (pCREB) and brain-derived neurotrophic factor (BDNF) expression decreased during this time. These effects were back to normal 8months after treatment with Aβ1-42. Altogether, our results suggested that Aβ1-42 induced significant learning and memory impairment, which is involved in HPA axis dysfunction.

PTEN participates in airway remodeling of asthma by regulating CD38/Ca2+/CREB signaling.

Both phosphatase and tensin homologue deleted on chromosome ten (PTEN) and cluster of differentiation 38 (CD38) have been suggested to be key regulators of the pathogenesis of asthma. However, the precise role and molecular mechanisms by which PTEN and CD38 are involved in airway remodeling throughout asthma pathogenesis remains poorly understood. This study aimed to elucidate the role of PTEN and CD38 in airway remodeling of asthma. Exposure to tumor necrosis factor-α (TNF-α) in airway smooth muscle (ASM) cells markedly decreased PTEN expression, and increased expression of CD38. Overexpression of PTEN suppressed the expression of CD38 and downregulated proliferation and migration induced by TNF-α stimulation, which was partially reversed by CD38 overexpression. PTEN/CD38 axis regulated Ca2+ levels and cyclic AMP response-element binding protein (CREB) phosphorylation in TNF-α-stimulated ASM cells. The in vitro knockdown of CD38 or overexpression of PTEN remarkably restricted airway remodeling and decreased Ca2+ concentrations and CREB phosphorylation in asthmatic mice. CD38 overexpression abolished the inhibitory effects of PTEN overexpression on airway remodeling. These findings demonstrate that PTEN inhibits airway remodeling of asthma through the downregulation of CD38-mediated Ca2+/CREB signaling, highlighting a key role of PTEN/CD38/Ca2+/CREB signaling in the molecular pathogenesis of asthma.

Selective Fragments for the CREBBP Bromodomain Identified from an Encoded Self-assembly Chemical Library.

DNA-encoded chemical libraries (DECLs) are collections of chemical moieties individually coupled to distinctive DNA barcodes. Compounds can be displayed either at the end of a single DNA strand (i. e., single-pharmacophore libraries) or at the extremities of two complementary DNA strands (i. e., dual-pharmacophore libraries). In this work, we describe the use of a dual-pharmacophore encoded self-assembly chemical (ESAC) library for the affinity maturation of a known 4,5-dihydrobenzodiazepinone ring (THBD) acetyl-lysine (KAc) mimic for the cyclic-AMP response element binding protein (CREB) binding protein (CREBBP or CBP) bromodomain. The new pair of fragments discovered from library selection showed a sub-micromolar affinity for the CREBBP bromodomain in fluorescence polarization and ELISA assays, and selectivity against BRD4(1).

Anti-inflammatory capacity of Apremilast in human chondrocytes is dependent on SOX-9.

Osteoarthritis (OA) impacts the quality of life in middle-aged and elderly people by inducing immobility. The severe inflammation in chondrocytes is reported to be related to the development and process of OA. The present study aims to investigate the protective effects of Apremilast on injured chondrocytes induced by interleukin-1α (IL-1α) and the underlying mechanism. 10ng/mL IL-1α was used to induce the in vitro injured chondrocytes. QRT-PCR was used to evaluate the expression level of Sry-type high-mobility-group box 9 (SOX-9), collagen type II alpha-1 gene (COL2A1), Aggrecan (ACAN) and collagen type X alpha 1 chain (COL10A1). SiRNA technology was utilized to knock down the expression of SOX-9 in the chondrocytes. The expression of SOX-9 was determined by Western Blot assay and/or immunofluorescence assay. Western Blot was used to evaluate the expression level of phosphorylated cyclic AMP response element binding (CREB). SOX9, Col2a1 and Acan were significantly up-regulated and Col10a1 was significantly down-regulated in the chondrocytes by Apremilast in a dose-dependent manner. IL-1α induced the injured chondrocytes by decreasing the expression of SOX9, Col2a1, Acan and increasing the expression of Col10a1, which were greatly reversed by Apremilast. By silencing SOX-9, the effects of Apremilast on SOX9 and marker genes were abolished. Phosphorylated CREB was up-regulated by Apremilast in a time-dependent manner. The up-regulated SOX-9 by Apremilast was reversed by the protein kinase A (PKA)/CREB pathway inhibitor H89. Apremilast may protect chondrocytes from inflammation by up-regulating SOX9.

Abstract 1753: Targeting cancer with selective cbp/p300 bromodomain inhibitors

Abstract Background: The Bromodomain (BRD) and Extra-Terminal domain (BET) family of proteins are key regulators of epigenetic control. Although pan BET Inhibitors show good clinical activity, progressive disease was seen after several months of treatment in clinical responders, likely due to secondary resistance mechanisms. The plausible resistance mechanisms are increased expression of TCF7L2, c-Myc, Survivin and PIM1. Cyclic AMP response element binding protein (CREB)-binding protein (CBP) and E1A interacting protein of 300 kDa (EP300 or p300) are two closely related histone acetyl transferases with oncogenic roles in a variety of cancers. They are known to be co-activators of several key transcription factors that contribute to tumor progression including HIF1a, BRCA-1, p53, c-Myc and androgen receptor (AR). Studies have indicated that p300 is also upregulated in SPOP-mutated prostate cancer. Thus, targeting CBP/p300 represents an attractive approach for developing novel therapies. Methods and Results: Multiple potent and selective CBP/p300 bromodomain inhibitors that are structurally unrelated to known inhibitors were identified by iterative medicinal chemistry and SAR based approaches. The compounds were optimized towards attaining good physicochemical properties and DMPK profile. The anti-proliferative activity of the lead compounds was studied across multiple tumor types in a 3-day assay. The lead compounds potently inhibited viability of a wide range of hematological and solid tumor cell lines including prostate cancer cell lines VCaP and 22Rv1. In H929 cell line the lead compounds showed dose-dependent inhibition of cMYC and increase in cPARP. In a single dose PK-PD study in MV4-11 xenograft model, the compounds showed modulation of cMYC and Survivin. Conclusions: In summary, our studies demonstrate that selective CBP/p300 bromodomain inhibitors are potent in models of hematologic malignancies and solid tumors in-vitro. Profiling of efficacy in xenograft models, and further toxicological evaluation are in progress. Citation Format: Mahaboobi Jaleel, Ramesh S. Senaiar, Chandrasekhar Abbineni, Girish A. Renukappa, Subhendu Mukherjee, Sivapriya Marappan, DS Samiulla, AB Aravind, Naveen R. Kumar, Venkata Siva N. Reddy, Asha Babu, Akhila P. Srinivas, Prasad Yadlapalli, Suraj Tgore, Raghavendra NR, Chandranath D. Naik, Sanjeev Giri, Thomas Antony, Kavitha Nellore, Shekar Chelur, Girish Daginakatte, laura Ravanti, Mikko Myllymäki, Gerd Wohlfahrt, Elina Mattila, Stefan Karlsson, Mari Björkman, Reetta Riikonen, Tarja Ikonen, Laura Leimu, Chira Mälmström, Timo Korjamo, Anu Moilanen, Murali Ramachandra, Susanta Samajdar. Targeting cancer with selective cbp/p300 bromodomain inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1753.

Cognitive Function Improvement in Mouse Model of Alzheimer's Disease Following Transcranial Direct Current Stimulation.

Anodal transcranial direct current stimulation (tDCS) is a painless noninvasive method that reportedly improves cognitive function in Alzheimer’s disease (AD) by stimulating the brain. However, its underlying mechanism remains unclear. Thus, the present study investigates the cognitive effects in a 5xFAD AD mouse model using electrophysiological and pathological methods. We used male 5xFAD C57BL/6J and male C57BL/6J wild-type mice; the dementia model was confirmed through DNA sequencing. The verified AD and wild-type mice were randomly assigned into four groups of five mice each: an induced AD group receiving tDCS treatment (Stim-AD), an induced AD group not receiving tDCS (noStim-AD), a non-induction group receiving tDCS (Stim-WT), and a non-induction group not receiving tDCS (noStim-WT). In the Stim group, mice received tDCS in the frontal bregma areas at an intensity of 200 µA for 20 min. After 2 weeks of treatment, we decapitated the mice, removed the hippocampus from the brain, confirmed its neuronal activation through excitatory postsynaptic potential (EPSP) recording, and performed molecular experiments on the remaining tissue using western blots. EPSP significantly increased in the Stim-AD group compared to that in the noStim-AD, which was comparable to that in the non-induced groups, Stim-WT and noStim-WT. There were no significant differences in cyclic amp-response element binding protein (CREB), phosphorylated CREB (pCREB), and Brain-derived neurotrophic factor (BDNF) levels in the Stim-AD group compared to those in the noStim-AD group. This study demonstrated that a tDCS in both frontal lobes of a transgenic 5xFAD mouse model affects long-term potentiation, indicating possible enhancement of cognitive function.

The adenosine A(2A) receptor agonist CGS 21680 alleviates auditory sensorimotor gating deficits and increases in accumbal CREB in rats neonatally treated with quinpirole.

The adenosine A(2A) receptor forms a mutually inhibitory heteromer with the dopamine D2 receptor, and A(2A) agonists decrease D2 signaling. This study analyzed whether an adenosine A(2A) agonist would alleviate deficits in sensorimotor gating and increases in cyclic-AMP response element binding protein (CREB) in the nucleus accumbens (NAc) in the neonatal quinpirole model of schizophrenia (SZ). Male and female Sprague-Dawley rats were neonatally treated with saline (NS) or quinpirole HCl (NQ; 1mg/kg) from postnatal days (P) 1-21. Animals were raised to P44 and behaviorally tested on auditory sensorimotor gating as measured through prepulse inhibition (PPI) from P44 to P48. Approximately 15min before each session, animals were given an ip administration of saline or the adenosine A(2A) agonist CGS 21680 (0.03 or 0.09mg/kg). One day after PPI was complete on P49, animals were administered a locomotor activity test in the open field after saline or CGS 21680 treatment, respectively. On P50, the nucleus accumbens (NAc) was evaluated for CREB protein. NQ-treated rats demonstrated a deficit in PPI that was alleviated to control levels by either dose of CGS 21680. The 0.03mg/kg dose of CGS 21680 increased startle amplitude in males. The 0.09 mg/kg dose of CGS 21680 resulted in an overall decrease in locomotor activity. NQ treatment significantly increased NAcCREB that was attenuated to control levels by either dose of CGS 21680. This study revealed that an adenosine A(2A) receptor agonist was effective to alleviate PPI deficits in the NQ model of SZ in both male and female rats.

Anti-aging Klotho Protects SH-SY5Y Cells Against Amyloid β1-42 Neurotoxicity: Involvement of Wnt1/pCREB/Nrf2/HO-1 Signaling.

Alzheimer's disease (AD) is considered a prevalent neurological disorder with a neurodegenerative nature in elderly people. Oxidative stress and neuroinflammation due to amyloid β (Aβ) peptides are strongly involved in AD pathogenesis. Klotho is an anti-aging protein with multiple protective effects that its deficiency is involved in development of age-related disorders. In this study, we investigated the beneficial effect of Klotho pretreatment at different concentrations of 0.5, 1, and 2nM against Aβ1-42 toxicity at a concentration of 20μM in human SH-SY5Y neuroblastoma cells. Our findings showed that Klotho could significantly and partially restore cell viability and decrease reactive oxygen species (known as ROS) and improve superoxide dismutase activity (SOD) in addition to reduction of caspase 3 activity and DNA fragmentation following Aβ1-42 challenge. In addition, exogenous Klotho also reduced inflammatory biomarkers consisting of nuclear factor-kB (NF-kB), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in Aβ-exposed cells. Besides, Klotho caused downregulation of Wnt1 level, upregulation of phosphorylated cyclic AMP response element binding (pCREB), and mRNA levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) with no significant alteration of epsilon isoform of protein kinase C (PKCε) after Aβ toxicity. In summary, Klotho could alleviate apoptosis, oxidative stress, and inflammation in human neuroblastoma cells after Aβ challenge and its beneficial effect is partially exerted through appropriate modulation of Wnt1/pCREB/Nrf2/HO-1 signaling.

Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy.

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injuries. The molecular mechanisms are unknown. To clarify these mechanisms, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cyclic AMP (cAMP) response element binding protein 1 (CREB1) was markedly phosphorylated (p-CREB1), and the CREB-binding protein (CBP) was recruited to p-CREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes involved in mitochondrial biogenesis. The results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes, in a manner dependent on cold stimulation frequency and duration. This information will inform further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

Targeting the CBP/β-Catenin Interaction to Suppress Activation of Cancer-Promoting Pancreatic Stellate Cells.

Background: Although cyclic AMP-response element binding protein-binding protein (CBP)/β-catenin signaling is known to promote proliferation and fibrosis in various organ systems, its role in the activation of pancreatic stellate cells (PSCs), the key effector cells of desmoplasia in pancreatic cancer and fibrosis in chronic pancreatitis, is largely unknown. Methods: To investigate the role of the CBP/β-catenin signaling pathway in the activation of PSCs, we have treated mouse and human PSCs with the small molecule specific CBP/β-catenin antagonist ICG-001 and examined the effects of treatment on parameters of activation. Results: We report for the first time that CBP/β-catenin antagonism suppresses activation of PSCs as evidenced by their decreased proliferation, down-regulation of “activation” markers, e.g., α-smooth muscle actin (α-SMA/Acta2), collagen type I alpha 1 (Col1a1), Prolyl 4-hydroxylase, and Survivin, up-regulation of peroxisome proliferator activated receptor gamma (Ppar-γ) which is associated with quiescence, and reduced migration; additionally, CBP/β-catenin antagonism also suppresses PSC-induced migration of cancer cells. Conclusion: CBP/β-catenin antagonism represents a novel therapeutic strategy for suppressing PSC activation and may be effective at countering PSC promotion of pancreatic cancer.