Fos mRNA Expression Research Articles

Increase of c-FOS promoter transcriptional activity by the dual leucine zipper kinase

The dual leucine zipper kinase (DLK) and the ubiquitously expressed transcription factor c-FOS have important roles in beta-cell proliferation and function. Some studies in neuronal cells suggest that DLK can influence c-FOS expression. Given that c-FOS is mainly regulated at the transcriptional level, the effect of DLK on c-FOS promoter activity was investigated in the beta-cell line HIT. The methods used in this study are the following: Luciferase reporter gene assays, immunoblot analysis, CRISPR-Cas9-mediated genome editing, and real-time quantitative PCR. In the beta-cell line HIT, overexpressed DLK increased c-FOS promoter activity twofold. Using 5′-,3′-promoter deletions, the promoter regions from − 348 to − 339 base pairs (bp) and from a − 284 to − 53 bp conferred basal activity, whereas the promoter region from − 711 to − 348 bp and from − 53 to + 48 bp mediated DLK responsiveness. Mutation of the cAMP response element within the promoter prevented the stimulatory effect of DLK. Treatment of HIT cells with KCl and the adenylate cyclase activator forskolin increased c-FOS promoter transcriptional activity ninefold. Since the transcriptional activity of those promoter fragments activated by KCl and forskolin was decreased by DLK, DLK might interfere with KCl/forskolin-induced signaling. In a newly generated, genome-edited HIT cell line lacking catalytically active DLK, c-Fos mRNA levels were reduced by 80% compared to the wild-type cell line. DLK increased c-FOS promoter activity but decreased stimulated transcriptional activity, suggesting that DLK fine-tunes c-FOS promoter-dependent gene transcription. Moreover, at least in HIT cells, DLK is required for FOS mRNA expression.

P2X7-dependent constitutive Interleukin-1β release from pyramidal neurons of the normal mouse hippocampus: Evidence for a role in maintenance of the innate seizure threshold

Disruption of Interleukin-1β (IL-1β) signaling sensitized mice to convulsant stimuli, suggesting that this quintessential cytokine of the innate immune system contributes to maintenance of the innate seizure threshold (ST). However, much remains unknown about where and how IL-1β secretion occurs in the normal brain. This study examined the possibility that neurons of the hippocampus are key sources of constitutive IL-1β secretion and that the release from these cells is dependent on the purinoceptor, P2X7. It was posited that treatment with the P2X7 antagonist, JNJ-47965567 (JNJ), would cause IL-1β to accumulate in cells that produce it, and consequently, lower the ST. No IL-1β immunoreactivity was detected in any region of the hippocampal formation of mice treated with the JNJ vehicle, Sulfobutylether-β-cyclodextrin. In contrast, prominent immunoreactivity was discovered in the pyramidal neurons of the CA3 region 60 min after treatment with the P2X7 antagonist. Lower levels were found in CA1 neurons, and no immunoreactivity was detected in granule cells of the dentate gyrus. JNJ also increased IL-1β immunoreactivity in the cell bodies of hippocampal neurons in culture. Interestingly, JNJ potentiated bicuculline-induced Fos and COX-2 mRNA expression in the cultures and this was blocked by an NMDA receptor antagonist. Moreover, pentylenetetrazole-induced seizure severity and incidence of convulsions were increased in mice treated with JNJ and this resembled that observed with IL-1 signaling-deficient mice. Overall, the results from this study support the notion that constitutive P2X7-dependent IL-1β release from hippocampal pyramidal neurons contributes to maintenance of the ST in the normal brain, perhaps by modulating neuronal excitability. These findings may have implications for epilepsy, a brain disorder in which the ST is compromised.

Prognostic role of EGR1 in breast cancer: a systematic review

EGR1 (early growth response 1) is dysregulated in many cancers and exhibits both tumor suppressor and promoter activities, making it an appealing target for cancer therapy. Here, we used a systematic multiomics analysis to review the expression of EGR1 and its role in regulating clinical outcomes in breast cancer (BC). EGR1 expression, its promoter methylation, and protein expression pattern were assessed using various publicly available tools. COSMIC-based somatic mutations and cBioPortal-based copy number alterations were analyzed, and the prognostic roles of EGR1 in BC were determined using Prognoscan and Kaplan-Meier Plotter. We also used bc-GenEx-Miner to investigate the EGR1 co-expression profile. EGR1 was more often downregulated in BC tissues than in normal breast tissue, and its knockdown was positively correlated with poor survival. Low EGR1 expression levels were also associated with increased risk of ER+, PR+, and HER2-BCs. High positive correlations were observed among EGR1, DUSP1, FOS, FOSB, CYR61, and JUN mRNA expression in BC tissue. This systematic review suggested that EGR1 expression may serve as a prognostic marker for BC patients and that clinicopathological parameters influence its prognostic utility. In addition to EGR1, DUSP1, FOS, FOSB, CYR61, and JUN can jointly be considered prognostic indicators for BC.

A high-androgen microenvironment inhibits granulosa cell proliferation and alters cell identity

A naturally occurring bovine model with excess follicular fluid androstenedione (High A4), reduced fertility, and polycystic ovary syndrome (PCOS)-like characteristics has been identified. We hypothesized High A4 granulosa cells (GCs) would exhibit altered cell proliferation and/or steroidogenesis. Microarrays of Control and High A4 GCs combined with Ingenuity Pathway Analysis indicated that High A4 GCs had cell cycle inhibition and increased expression of microRNAs that inhibit cell cycle genes. Granulosa cell culture confirmed that A4 treatment decreased GC proliferation, increased anti-Müllerian hormone, and increased mRNA for CTNNBIP1. Increased CTNNBIP1 prevents CTNNB1 from interacting with members of the WNT signaling pathway thereby inhibiting the cell cycle. Expression of CYP17A1 was upregulated in High A4 GCs presumably due to reduced FOS mRNA expression compared to Control granulosa cells. Furthermore, comparisons of High A4 GC with thecal and luteal cell transcriptomes indicated an altered cellular identity and function contributing to a PCOS-like phenotype.

A Novel lncRNA Regulates the Toll-Like Receptor Signaling Pathway and Related Immune Function by Stabilizing FOS mRNA as a Competitive Endogenous RNA.

Long non-coding RNAs (lncRNAs) have recently emerged as new regulatory molecules with diverse functions in regulating gene expression and significant roles in the immune response. However, the function of many unknown lncRNAs is still unclear. By studying the regulatory effect of daidzein (DA) on immunity, we identified a novel lncRNA with an immune regulatory function: lncRNA- XLOC_098131. In vivo, DA treatment upregulated the expression of lncRNA- XLOC_098131, FOS, and JUN in chickens and affected the expression of activator protein 1 (AP-1) to regulate MAPK signaling, Toll-like receptor signaling, and related mRNA expression. It also enhanced macrophage activity and increased the numbers of blood neutrophils and mononuclear cells, which can improve the body's ability to respond to stress and bacterial and viral infections. Furthermore, DA treatment also reduced B lymphocyte apoptosis and promoted the differentiation of B lymphocytes into plasma cells, which in turn resulted in the production of more immunoglobulins and the promotion of antigen presentation. In vitro, using HEK293FT cells, we demonstrated that mir-548s could bind to and decrease the expression of both FOS and lncRNA- XLOC_098131. LncRNA- XLOC_098131 served as a competitive endogenous RNA to stabilize FOS by competitively binding to miR-548s and thereby reducing its inhibitory effect of FOS expression. Therefore, we concluded that the novel lncRNA XLOC_098131 acts as a key regulatory molecule that can regulate the Toll-like receptor signaling pathway and related immune function by serving as a competitive endogenous RNA to stabilize FOS mRNA expression.

Dysregulated gene expressions of MEX3D, FOS and BCL2 in human induced-neuronal (iN) cells from NF1 patients: a pilot study

Direct conversion technique to produce induced-neuronal (iN) cells from human fibroblasts within 2 weeks is expected to discover unknown neuronal phenotypes of neuropsychiatric disorders. Here, we present unique gene expression profiles in iN cells from patients with neurofibromatosis type 1 (NF1), a single-gene multifaceted disorder with comparatively high co-occurrence of autism spectrum disorder (ASD). Microarray-based transcriptomic analysis on iN cells from male healthy controls and male NF1 patients (NF1-iN cells) revealed that 149 genes expressions were significantly different (110 upregulated and 39 downregulated). We validated that mRNA of MEX3D (mex-3 RNA binding family member D) was lower in NF1-iN cells by real-time PCR with 12 sex-mixed samples. In NF1-iN cells on day 14, higher expression of FOS mRNA was observed with lower expression of MEX3D mRNA. Interestingly, BCL2 mRNA was higher in NF1-iN cells on day 5 (early-period) but not on day 14. Our data suggest that aberrant molecular signals due to NF1 mutations may disturb gene expressions, a subset of which defines continuum of the neuronal phenotypes of NF1 with ASD. Further translational studies using induced pluripotent stem (iPS) cell-derived neuronal cells are needed to validate our preliminary findings especially confirming meanings of analysis using early-period iN cells.

Early emergence of altered 5-HT2A receptor-evoked behavior, neural activation and gene expression following maternal separation

The early stress of Maternal Separation (MS) contributes to the establishment of adult psychopathology. The serotonergic (5-HT) system is implicated during this temporal window in mediating the development of mood-related behaviors. MS is reported to evoke altered 5-HT2A receptor function in adulthood. However, the ontogeny of altered 5-HT2A receptor responsivity following MS remains unknown. Here, we examined 5-HT2A receptor agonist, DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) (2mg/kg) evoked responses, namely stereotypical head-twitch behaviors in control and MS Sprague-Dawley rat pups at postnatal day 21 (P21). MS involved a separation of pups from the dam for 3h daily from postnatal day 2–14. MS pups at P21 exhibited significantly enhanced head-twitch behaviors compared to controls. Using c-Fos cell counting we examined neural activation in control and MS pups following DOI treatment. MS pups exhibited altered DOI-evoked c-Fos expression within all mPFC subdivisions, but not in the hippocampus, lateral septum and hypothalamus, suggesting differential prefrontal neural activation upon 5-HT2A receptor stimulation following early stress. Gene profiling of 5-HT2A receptor-regulated immediate early genes (IEGs) indicated a decline in the expression of Fos, Fra1 and Egr1 mRNA under baseline conditions in the mPFC of MS pups. MS pups also showed an altered pattern in the regulation of several 5-HT2A receptor-regulated IEGs (Fos, Fra1, Bdnf, Egr1, Egr3) following DOI treatment. Collectively, these results highlight an early emergence of altered 5-HT2A receptor-evoked behavioral responses and neural activation patterns in multiple brain regions in animals with a history of MS.

Gene expression analysis of Arc mRNA as a neuronal cell activity marker in the hippocampus and amygdala in two-way active avoidance test in rats

IntroductionImmediate early genes are widely used as neuronal cell activity markers in neuroscience. The present study investigated the relationship between their expression and abnormality in context fear conditioning. MethodsThe learning test (two-way active avoidance test) was conducted in male rats administered with nonselective muscarinic antagonist scopolamine or selective dopamine D1-like receptor antagonist SCH 23390 at a dose level of 2.0 or 0.1mg/kg, respectively, for 4days. Expression levels of Arc and Fos mRNA in the hippocampus and amygdala were also evaluated on the second day of dosing by fluorescent in situ hybridization (FISH) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). ResultsScopolamine had no effect on avoidance rate, but decreased freezing in the two-way active avoidance test. SCH 23390 decreased avoidance rate and increased freezing. In FISH and RT-qPCR assays, scopolamine decreased Arc mRNA in the hippocampus and amygdala, whereas SCH 23390 increased Arc mRNA in the hippocampus. By contrast, scopolamine and SCH 23390 did not change Fos mRNA expression compared to Arc mRNA expression. DiscussionThe results of the learning test indicated that scopolamine or SCH 23390 respectively inhibited fear or context conditioning in rats. Furthermore, alteration of the expression of Arc mRNA but not of Fos mRNA in the hippocampus and amygdala of the brain was suggested to be a sensitive neuronal cell activity marker to detect behavioral abnormality in the two-way active avoidance test.

Binding and inhibition of the ternary complex factor Elk-4/Sap1 by the adapter protein Dok-4.

The adapter protein Dok-4 (downstream of kinase-4) has been reported as both an activator and inhibitor of Erk and Elk-1, but lack of knowledge about the identity of its partner molecules has precluded any mechanistic insight into these seemingly conflicting properties. We report that Dok-4 interacts with the transactivation domain of Elk-4 through an atypical phosphotyrosine-binding domain-mediated interaction. Dok-4 possesses a nuclear export signal and can relocalize Elk-4 from nucleus to cytosol, whereas Elk-4 possesses two nuclear localization signals that restrict interaction with Dok-4. The Elk-4 protein, unlike Elk-1, is highly unstable in the presence of Dok-4, through both an interaction-dependent mechanism and a pleckstrin homology domain-dependent but interaction-independent mechanism. This is reversed by proteasome inhibition, depletion of endogenous Dok-4 or lysine-to-arginine mutation of putative Elk-4 ubiquitination sites. Finally, Elk-4 transactivation is potently inhibited by Dok-4 overexpression but enhanced by Dok-4 knockdown in MDCK renal tubular cells, which correlates with increased basal and EGF-induced expression of Egr-1, Fos and cylcinD1 mRNA, and cell proliferation despite reduced Erk activation. Thus, Dok-4 can target Elk-4 activity through multiple mechanisms, including binding of the transactivation domain, nuclear exclusion and protein destabilization, without a requirement for inhibition of Erk.

Brain-wide maps of Fos expression during fear learning and recall.

Fos induction during learning labels neuronal ensembles in the hippocampus that encode a specific physical environment, revealing a memory trace. In the cortex and other regions, the extent to which Fos induction during learning reveals specific sensory representations is unknown. Here we generate high-quality brain-wide maps of Fos mRNA expression during auditory fear conditioning and recall in the setting of the home cage. These maps reveal a brain-wide pattern of Fos induction that is remarkably similar among fear conditioning, shock-only, tone-only, and fear recall conditions, casting doubt on the idea that Fos reveals auditory-specific sensory representations. Indeed, novel auditory tones lead to as much gene induction in visual as in auditory cortex, while familiar (nonconditioned) tones do not appreciably induce Fos anywhere in the brain. Fos expression levels do not correlate with physical activity, suggesting that they are not determined by behavioral activity-driven alterations in sensory experience. In the thalamus, Fos is induced more prominently in limbic than in sensory relay nuclei, suggesting that Fos may be most sensitive to emotional state. Thus, our data suggest that Fos expression during simple associative learning labels ensembles activated generally by arousal rather than specifically by a particular sensory cue.

TGF-β Effects on Prostate Cancer Cell Migration and Invasion Require FosB.

Activator Protein-1 (AP-1) family (cJun, JunB, JunD, cFos, FosB, Fra1, and Fra2) plays a central role in the transcriptional regulation of many genes that are associated with cell proliferation, differentiation, migration, metastasis, and survival. Many oncogenic signaling pathways converge at the AP-1 transcription complex. Transforming growth factor beta (TGF-β) is a multifunctional regulatory cytokine that regulates many aspects of cellular function, including cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance, and survival. This study investigated, the role of FOS proteins in TGF-β signaling in prostate cancer cell proliferation, migration, and invasion. Steady state expression levels of FOS mRNA and proteins were determined using RT-PCR and western blotting analyses. DU145 and PC3 prostate cancer cells were exposed to TGF-β1 at varying time and dosage, RT-PCR, western blot, and immunofluorescence analyses were used to determine TGF-β1 effect on FOS mRNA and protein expression levels as well as FosB subcellular localization. Transient silencing of FosB protein was used to determine its role in cell proliferation, migration, and invasion. Our data show that FOS mRNA and proteins were differentially expressed in human prostate epithelial (RWPE-1) and prostate cancer cell lines (LNCaP, DU145, and PC3). TGF-β1 induced the expression of FosB at both the mRNA and protein levels in DU145 and PC3 cells, whereas cFos and Fra1 were unaffected. Immunofluorescence analysis showed an increase in the accumulation of FosB protein in the nucleus of PC3 cells after treatment with exogenous TGF-β1. Selective knockdown of endogenous FosB by specific siRNA did not have any effect on cell proliferation in PC3 and DU145 cells. However, basal and TGF-β1- and EGF-induced cell migration was significantly reduced in DU145 and PC3 cells lacking endogenous FosB. TGF-β1- and EGF-induced cell invasion were also significantly decreased after FosB knockdown in PC3 cells. Our data suggest that FosB is required for migration and invasion in prostate cancer cells. We also conclude that TGF-β1 effect on prostate cancer cell migration and invasion may be mediated through the induction of FosB. Prostate 77:72-81, 2017. © 2016 Wiley Periodicals, Inc.

Oocyte-Specific Expression of Mouse MEX3C652AA in the Ovary and Its Potential Role in Regulating Maternal Fos mRNA.

Currently, the human MEX3C gene is known to encode an RNA-binding protein of 659 amino acid residues. Here we show that the MEX3C gene has alternative splicing forms giving rise to multiple MEX3C variants, and some cells express MEX3C transcripts coding for short MEX3C isoforms but not transcripts for MEX3C(659AA) MEX3C(659AA) functions as an adaptor protein for Exportin 1 (XPO1)-mediated nuclear export since it increases the cytoplasmic distribution of poly(A)(+) RNA and since addition of the nuclear export signal (NES) sequence to a short MEX3C isoform MEX3C(464AA) confers similar cytoplasmic poly(A)(+) RNA accumulation activity as MEX3C(659AA) FOS mRNA is a potential MEX3C target mRNA. One mechanism by which MEX3C(659AA) could regulate FOS mRNA is by promoting its nuclear export. Overexpressing MEX3C(659AA) significantly increased FOS mRNA expression, whereas mutating the NES of MEX3C(659AA) and treating cells with leptomycin B to inhibit XPO1-mediated nuclear export attenuated FOS upregulation. FOS mRNA is unstable in somatic cells but less so in oocytes; how it is stabilized in the oocytes is unknown. Transcripts for the mouse counterpart of human MEX3C(659AA) (MEX3C(652AA)) are specifically expressed in developing oocytes in the ovary, although total Mex3c transcripts are expressed in both granulosa cells and oocytes. The specific expression of this long MEX3C isoform in oocytes and its ability to enhance FOS mRNA nuclear export and stability all suggest that MEX3C(659AA) is an RNA-binding protein that preserves maternal FOS mRNA in oocytes.

말의 운동 스트레스에 관한 새로운 생물지표

(Received: October 27, 2015; Revised: December 16, 2015; Accepted: December 24, 2015)Abstract : Exercise is one of the most common stressors in horses. Although various physiological parameters suchas cortisol respond to exercise, there is no reliable parameter for the measurement of exercise-induced stress insport horses. This study was performed to discover a new biomarker with high sensitivity for exercise-induced stress.The expression of fos mRNA was increased more than 10-fold in horse blood samples collected after an hour ofexercise, as compared with before the exercise. The plasma cortisol levels were also increased after the exercise,but only by about two-fold. The fos mRNA levels were well-correlated with plasma cortisol concentrations. Thesefindings suggest that fos mRNA expression in blood may be useful for the measurement of exercise-induced stressin horses.Keywords : biomarker, exercise, fos, horse, stress

Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Expression of factors associated with apoptosis in the porcine corpus luteum throughout the luteal phase of the estrous cycle and early pregnancy: Their possible involvement in acquisition of luteolytic sensitivity

The studies on the acquisition of luteolytic sensitivity have been focused mainly on molecular changes induced in the luteal tissue after treatment with exogenous PGF2α or on physiological changes occurring during the estrous cycle. The comparison of changes leading to the acquisition of luteolytic sensitivity after Day 12 of the estrous cycle and corresponding days of pregnancy has not been investigated in the pig. The present study was undertaken to evaluate (1) apoptosis measured as the proportions of early apoptotic, late apoptotic, and viable cells; (2) expression of factors involved in the extrinsic (TNFA/TNFα, TNFRSF1A/TNFR1, TNFRSF1B/TNFR2, FAS/Fas, and FASLG/FasL) and intrinsic (CASP3/Casp3, TP53/p-53, BAX/Bax, and BCL2/Bcl-2) apoptotic pathways, with two components of the activating protein-1 complex, i.e., FOS/Fos and JUN/Jun and IFNG/IFNγ; and (3) concentrations of luteal and blood plasma progesterone (P4) throughout the luteal phase of the estrous cycle and early pregnancy. Corpora lutea (CL) were collected postmortem on Days 8, 10, 12, and 14 of the estrous cycle and the corresponding days of pregnancy. The luteal tissue was subjected to RNA and/or protein isolation and disaggregation of CL cells followed by flow cytometry analysis aimed to determine apoptotic changes. Luteal and blood plasma P4 concentrations decreased on Day 14 of the estrous cycle versus pregnancy (P < 0.05 and P < 0.001, respectively). A significant increase in the number of early apoptotic cells and a decrease in the number of viable cells were observed on Day 14 of the estrous cycle (P < 0.001 and P < 0.05, respectively). Increase (P < 0.05) of TNFA messenger RNA (mRNA) level coincided with that of IFNG on Day 12 of the estrous cycle but not on the corresponding day of pregnancy. The content of FAS mRNA and protein increased on Day 14 of the estrous cycle versus pregnancy (P < 0.05). The mRNA expression of CASP3, BCL-2 and BAX was unchanged in cyclic and pregnant CL, while level of TP53 increased (P < 0.05) on Day 12 of the estrous cycle versus Day 8. The level of FOS and JUN mRNA increased (P < 0.05) on Day 14 of the estrous cycle versus the remaining days. The level of FOS and JUN mRNA was significantly higher (P < 0.001 and P < 0.05, respectively) on Day 14 of the estrous cycle than that on the corresponding day of pregnancy. In summary, the simultaneous increase of TNFA and IFNG transcript in cyclic CL suggests the crucial role of both cytokines in sensitization of porcine CL to further luteolytic action of PGF2α. The upregulated expression of FAS, FOS, and JUN mRNA in the late luteal phase in cyclic CL can indicate their involvement in structural luteolysis. The increased viability of luteal cells and elevated P4 concentrations in pregnant CL confirm the protective role of luteal P4 against apoptosis.

Buckwheat Rutin Inhibits AngII-induced Cardiomyocyte Hypertrophy via Blockade of CaN-dependent Signal Pathway.

Buckwheat rutin has been found to be able to inhibit angiotensin II (AngII) - induced hypertrophy in cultured neonatal rat cardiomyocytes, but the mechanism remains uncertain. In this study, myocardial hypertrophy model was made by adding AngII to the medium of cardiac myocytes of neonatal rats; meanwhile, different concentrations of buckwheat rutin were applied to observe their effects. Intracellular Ca(2+) level was detected by Hitachi - 850 fluorospectrophotometer, calcineurin (CaN) activity was measured by colorimetric method, the expression of CaN protein was observed with immunocytochemistry, and the proto - oncogene c - fos mRNA expression was assessed with reverse transcription polymerase chain reaction (RT - PCR). Compared with control group, AngII could greatly stimulate the increase of intracellular Ca(2+) level, the activities and protein expression of cardiomyocytes CaN, and the expression of proto - oncogene c - fos mRNA in cultured neonatal rat cardiomyocytes, which could be effectively decreased by buckwheat rutin. Our results demonstrated that buckwheat rutin exhibited inhibitory effect on AngII - induced hypertrophy in cultured neonatal rat cardiomyocytes via Ca(2+) antagonism action thus block the CaN - dependent signal pathway.

Toll-like receptor 2 modulates the expression of T cell transcription factors following alcohol and burn injury

s / Alcohol 47 (2013) 567–576 572 17. Toll-like receptor 2 modulates the expression of T cell transcription factors following alcohol and burn injury X. Li, J.L. Rendon, M.A. Choudhry, Alcohol Research Program, Burn & Shock Trauma Research Institute, Department of Surgery, Loyola University Chicago Health Sciences Division, Maywood, IL 60153, USA Previous studies from our laboratory have shown that acute alcohol (ethanol) intoxication combined with burn injury suppresses T cell proliferation, IL-2 and IFN-g production. We further found that T cell stimulation with Toll-like receptor 2 (TLR2) agonist (HKLM) prevents the decrease in T cell IFN-g release following ethanol and burn injury. This study examined the effect of ethanol and burn injury on T cell transcription factors (e.g., NFAT, Tbx21, Jun and Fos) and determined whether TLR2 agonist modulates the expression of these factors following ethanol and burn injury. To test this, male C57/BL6 mice (w25 g) were gavaged with ethanol (w2.9 mg/kg) to achieve a blood ethanol level of w100 mg/dl prior to receiving w12.5% total body surface area sham or burn injury. One day after injury, mice were sacrificed and splenic T cells were isolated. T cells were cultured with platebound anti-CD3 antibody in the presence or absence of TLR2 agonist (HKLM 108 cells/ml) for 48 h. Supernatants were collected for the measurement of IFN-g by ELISA and cells were lysed. The cells lysates were analyzed for NFAT, Tbx21, Jun and Fos mRNA expression by RT-PCR. As reported earlier, there was a significant decrease in IFN-g release following ethanol and burn injury compared to shams. T cells stimulated with anti-CD3 combined with TLR2 agonist did not show a decrease in IFN-g release following ethanol and burn injury. This was accompanied with a significant decrease in NFAT, Tbx21, Jun and Fos expression in T cells stimulated with anti-CD3 following ethanol and burn injury. However T cells stimulated with anti-CD3 combined with TLR2 agonist did not exhibit a decrease in expression of these transcription factors. Together, these findings suggest that TLR2 stimulation of T cells normalizes IFN-g by modulating NFAT, Tbx21, Jun and Fos expression following ethanol intoxication combined with burn injury (Supported by NIH R01 AA015731 [MAC], F30 AA020167 [JR] and the Dr. Ralph and Marian C. Falk Medical

Immediate early genes expression in the cerebellar cortex correlates with LTP and LTD induction

The consolidation of changes following activity-dependent neural plasticity are believed to involve specific patterns of gene expression. In the hippocampus, immediate early genes are thought to contribute to long-term synaptic plasticity (LTP and LTD); this phenomenon may occur also in the cerebellum, in which the transcription factors c-Fos and P-CREB have been identified. The cerebellum granular layer (GL) can manifest both LTP and LTD following a Theta Burst Stimulus (TBS) delivered to the mossy fibers. We have employed VSD imaging in rat cerebellar slices (P18-24) in order to map the spatial distribution of LTP and LTD in the cerebellum GL. Fluorescence changes were correlated to LTP or LTD in two different post-TBS time ranges (15 and 120 min). Slices were then fixed and processed for immunohistochemistry in order to identify levels of c-Fos and P-CREB expression. The induction of long-term plasticity increased the average level of P-CREB both at 15 min (+39±4.9, p<0.01%) and 120 min (+24±7.2, p<0.05%) after TBS. The level of c-Fos was unaltered at 15 min, while it significantly increased at 120 min (+37±8.9, p<0.05%). By spatially correlating longterm synaptic plasticity with the corresponding variation of P-CREB and c-Fos, we observed that regions showing LTP well correlated (p<0.05) with positive variations of P-CREB and c-Fos. Conversely, areas showing LTD correlated exclusively (p<0.05) with negative variations of P-CREB. Slices were also evaluated by in situ hybridization and a similar analysis was performed. The levels of fos and CREB mRNA expression and their spatial correlation with the sign of long-term synaptic plasticity corresponded with the immunohistochemical results. As a further test, VSD recordings showed that the induction of granular layer LTP and LTD could be prevented by applying 50 mM D-APV, a selective NMDA receptor blocker. Moreover, in situ hybridization and immunohistochemistry analysis evidenced that in these conditions both mRNA and protein expression levels of c-fos and CREB were unchanged, confirming the involvement of these two transcription factors in cerebellar granular layer plasticity.

Brain activation following peripheral administration of the GLP-1 receptor agonist exendin-4

The aim of our study was to investigate the anorectic and brain stimulatory effects of various doses of exendin-4 (Ex-4) and to investigate the role of the vagus nerve in Ex-4-induced brain activation. A dose-related increase in c-fos mRNA expression was observed following Ex-4 administration (0.155-15.5 μg/kg). Doses of Ex-4 that caused anorexia without aversive effects (0.155, 0.775 μg/kg) induced c-fos expression in the hypothalamic arcuate and paraventricular (PVH; parvocellular) nuclei as well as in the limbic and brainstem structures. Doses of Ex-4 that caused aversion (1.55, 15.5 μg/kg) stimulated the same regions (in a more intense way) and additionally activated the magnocellular hypothalamic structures (supraoptic nucleus and PVH magnocellular). The brain c-fos pattern induced by Ex-4 showed both similarities and differences with that induced by refeeding. Subdiaphragmatic vagotomy significantly blunted the stimulation of c-fos mRNA expression induced by Ex-4 in the nodose ganglion, the medial part of nucleus of the solitary tract, and the parvocellular division of the PVH. Pretreatment with Ex-9-39 (330 μg/kg ip) impaired the neuronal activation evoked by Ex-4 in all brain regions and in the nodose ganglion. Effects of Ex-4 on hypothalamic-pituitary-adrenal axis activity were not altered by vagotomy. Results of this study demonstrate and relate the anorectic and brain stimulatory effects of aversive and nonaversive doses of Ex-4 and indicate that the activation of specific central regions induced by the peripheral administration of Ex-4 is, at least in part, dependent on the integrity of the vagus nerve.

Serotonin transporter, sex, and hypoxia: microarray analysis in the pulmonary arteries of mice identifies genes with relevance to human PAH

Pulmonary arterial hypertension (PAH) is up to threefold more prevalent in women than men. Female mice overexpressing the serotonin transporter (SERT; SERT+ mice) exhibit PAH and exaggerated hypoxia-induced PAH, whereas male SERT+ mice remain unaffected. To further investigate these sex differences, microarray analysis was performed in the pulmonary arteries of normoxic and chronically hypoxic female and male SERT+ mice. Quantitative RT-PCR analysis was employed for validation of the microarray data. In relevant groups, immunoblotting was performed for genes of interest (CEBPβ, CYP1B1, and FOS). To translate clinical relevance to our findings, CEBPβ, CYP1B1, and FOS mRNA and protein expression was assessed in pulmonary artery smooth muscle cells (PASMCs) derived from idiopathic PAH (IPAH) patients and controls. In female SERT+ mice, multiple pathways with relevance to PAH were altered. This was also observed in chronically hypoxic female SERT+ mice. We selected 10 genes of interest for qRT-PCR analysis (FOS, CEBPβ, CYP1B1, MYL3, HAMP2, LTF, PLN, NPPA, UCP1, and C1S), and 100% concordance was reported. Protein expression of three selected genes, CEBPβ, CYP1B1, FOS, was also upregulated in female SERT+ mice. Serotonin and 17β-estradiol increased CEBPβ, CYP1B1, and FOS protein expression in PASMCs. In addition, CEBPβ, CYP1B1, and FOS mRNA and protein expression was also increased in PASMCs derived from IPAH patients. Here, we have identified a number of genes that may predispose female SERT+ mice to PAH, and these findings may also be relevant to human PAH.