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Sample records for down-regulates nr2b expression

  1. Inhibiting effects of rhynchophylline on zebrafish methamphetamine dependence are associated with amelioration of neurotransmitters content and down-regulation of TH and NR2B expression.

    PubMed

    Jiang, Mingjin; Chen, Yifei; Li, Chan; Peng, Qiuxian; Fang, Miao; Liu, Wei; Kang, Qunzhao; Lin, Yingbo; Yung, Ken Kin Lam; Mo, Zhixian

    2016-07-01

    Others and we have reported that rhynchophylline reverses amphetamine-induced conditioned place preference (CPP) effect which may be partly mediated by amelioration of central neurotransmitters and N-methyl-d-aspartate receptor 2B (NR2B) levels in the rat brains. The current study investigated the inhibiting effects of rhynchophylline on methamphetamine-induced (METH-induced) CPP in adult zebrafish and METH-induced locomotor activity in tyrosine hydroxylase-green fluorescent protein (TH-GFP) transgenic zebrafish larvae and attempted to confirm the hypothesis that these effects were mediated via regulation of neurotransmitters and dopaminergic and glutamatergic systems. After baseline preference test (on days 1-3), zebrafish were injected intraperitoneally METH (on days 4, 6 and 8) or the same volume of fish physiological saline (on days 5 and 7) and were immediately conditioned. Rhynchophylline was administered at 12h after injection of METH. On day 9, zebrafish were tested for METH-induced CPP. Results revealed that rhynchophylline (100mg/kg) significantly inhibited the acquisition of METH-induced CPP, reduced the content of dopamine and glutamate and down-regulated the expression of TH and NR2B in the CPP zebrafish brains. Furthermore, the influence of rhynchophylline on METH-induced locomotor activity was also observed in TH-GFP transgenic zebrafish larvae. Results showed that rhynchophylline (50mg/L) treatment led to a significant reduction on the locomotor activity and TH expression in TH-GFP transgenic zebrafish larvae. Taken together, these data indicate that the inhibition of the formation of METH dependence by rhynchophylline in zebrafish is associated with amelioration of the neurotransmitters dopamine and glutamate content and down-regulation of TH and NR2B expression. PMID:27009763

  2. Cloning, expression, and purification of a recombinant Tat-HA-NR2B9c peptide.

    PubMed

    Zhou, Hai-Hui; Zhang, Ai-Xia; Zhang, Yu; Zhu, Dong-Ya

    2012-10-01

    To design a peptide disrupting the interaction between N-methyl-d-aspartate receptors-2B (NR2B) and postsynaptic density protein-95 (PSD-95), a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide contained a fragment of the cell membrane transduction domain of the human immunodeficiency virus type1 (HIV-1) Tat, a influenza virus hemagglutinin (HA) epitope-tag, and the C-terminal 9 amino acids of NR2B (NR2B9c). We named the chimeric peptide Tat-HA-NR2B9c. The expression plasmid contained a gene fragment encoding the Tat-HA-NR2B9c was ligated to the C-terminal fragment of l-asparaginase (AnsB-C) via a unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in inclusion body in Escherichia coli under isopropyl β-d-1-thiogalactopyranoside (IPTG) and purified by washing with 2M urea, solubilizing in 4M urea, and then ethanol precipitation. The target chimeric peptide Tat-HA-NR2B9c was released from the fusion partner following acid hydrolysis and purified by isoelectric point precipitation and ultrafiltration. SDS-PAGE analysis and MALDI-TOF-MS analysis showed that the purified Tat-HA-NR2B9c was highly homogeneous. Furthermore, we investigated the effects of Tat-HA-NR2B9c on ischemia-induced cerebral injury in the rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion, and found that the peptide reduced infarct size and improved neurological functions. PMID:22944204

  3. SRC Inhibition Reduces NR2B Surface Expression and Synaptic Plasticity in the Amygdala

    ERIC Educational Resources Information Center

    Sinai, Laleh; Duffy, Steven; Roder, John C.

    2010-01-01

    The Src protein tyrosine kinase plays a central role in the regulation of N-methyl-d-aspartate receptor (NMDAR) activity by regulating NMDAR subunit 2B (NR2B) surface expression. In the amygdala, NMDA-dependent synaptic plasticity resulting from convergent somatosensory and auditory inputs contributes to emotional memory; however, the role of Src…

  4. Setdb1 histone methyltransferase regulates mood-related behaviors and expression of the NMDA receptor subunit NR2B.

    PubMed

    Jiang, Yan; Jakovcevski, Mira; Bharadwaj, Rahul; Connor, Caroline; Schroeder, Frederick A; Lin, Cong L; Straubhaar, Juerg; Martin, Gilles; Akbarian, Schahram

    2010-05-26

    Histone methyltransferases specific for the histone H3-lysine 9 residue, including Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e are associated with repressive chromatin remodeling and expressed in adult brain, but potential effects on neuronal function and behavior remain unexplored. Here, we report that transgenic mice with increased Setdb1 expression in adult forebrain neurons show antidepressant-like phenotypes in behavioral paradigms for anhedonia, despair, and learned helplessness. Chromatin immunoprecipitation in conjunction with DNA tiling arrays (ChIP-chip) revealed that genomic occupancies of neuronal Setdb1 are limited to <1% of annotated genes, which include the NMDA receptor subunit NR2B/Grin2B and other ionotropic glutamate receptor genes. Chromatin conformation capture and Setdb1-ChIP revealed a loop formation tethering the NR2B/Grin2b promoter to the Setdb1 target site positioned 30 kb downstream of the transcription start site. In hippocampus and ventral striatum, two key structures in the neuronal circuitry regulating mood-related behaviors, Setdb1-mediated repressive histone methylation at NR2B/Grin2b was associated with decreased NR2B expression and EPSP insensitivity to pharmacological blockade of NR2B, and accelerated NMDA receptor desensitization consistent with a shift in NR2A/B subunit ratios. In wild-type mice, systemic treatment with the NR2B antagonist, Ro25-6981 [R-(R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol], and hippocampal small interfering RNA-mediated NR2B/Grin2b knockdown resulted in behavioral changes similar to those elicited by the Setdb1 transgene. Together, these findings point to a role for neuronal Setdb1 in the regulation of affective and motivational behaviors through repressive chromatin remodeling at a select set of target genes, resulting in altered NMDA receptor subunit composition and other molecular adaptations. PMID:20505083

  5. Effects of diazoxide on Aβ1-42-induced expression of the NR2B subunit in cultured cholinergic neurons.

    PubMed

    Zhu, Jin; Fu, Qingxi; Xia, Chunfeng; Ma, Guozhao

    2015-12-01

    The accumulation of amyloid-β protein (Aβ) is significant in the pathogenesis of Alzheimer's disease. Several previous studies indicate that the NR2B‑containing N‑methyl‑D‑aspartate receptors are critically involved in the Aβ mediated disruption of neuronal function. Diazoxide (DZ), a highly selective drug capable of opening mitochondrial ATP‑sensitive potassium channels, has neuroprotective effects against neuronal cell death. However, the mechanism by which DZ protects cholinergic neurons against Aβ‑induced cytotoxicity remains to be elucidated. The present study was designed to investigate the effects of DZ pretreatment against Aβ1‑42‑induced expression of NR2B in order to gain novel insights into the neuroprotective mechanisms. Following exposure to Aβ1‑42 for 24 h, the expression of the NR2B subunit remained unchanged compared with the control group. However, a significant increase in the expression of the NR2B subunit was observed following treatment with Aβ1‑42 for 72 h (P<0.05); and the upregulation of the expression of the NR2B subunit was reversed by pretreatment with DZ (P<0.05). These results suggested that DZ may counteract Aβ1‑42‑mediated cytotoxicity by alleviating the expression of NR2B. PMID:26496862

  6. Senegenin Attenuates Hepatic Ischemia-Reperfusion Induced Cognitive Dysfunction by Increasing Hippocampal NR2B Expression in Rats

    PubMed Central

    Gu, Xiaoping; Zheng, Yaguo; Sun, Yu-e; Liang, Ying; Bo, Jinhua; Ma, Zhengliang

    2012-01-01

    Background The root of Polygala tenuifolia, a traditional Chinese medicine, has been used to improve memory and intelligence, while the underlying mechanisms remain largely unknown. In this study, we investigated the protective effects of senegenin, an component of Polygala tenuifolia root extracts, on cognitive dysfunction induced by hepatic ischemia-reperfusion. Methodology/Principal Findings Initially, we constructed a rat model of hepatic ischemia-reperfusion (HIR) and found that the memory retention ability of rats in the step-down and Y maze test was impaired after HIR, paralleled by a decrease of N-methyl-D-aspartate (NMDA) receptor NR2B subunit mRNA and protein expressions in hippocampus. Furthermore, we found that administration of senegenin by gavage attenuated HIR-induced cognitive impairment in a dose and time dependent manner, and its mechanisms might partly due to the increasing expression of NR2B in rat hippocampus. Conclusions/Significance Cognitive dysfunction induced by HIR is associated with reduction of NR2B expression. Senegenin plays a neuroprotective role in HIR via increasing NR2B expression in rat hippocampus. These findings suggest that senegenin might be a potential agent for prevention and treatment of postoperative cognitive dysfunction (POCD) or other neurodegenerative diseases. PMID:23029109

  7. Early auditory enrichment with music enhances auditory discrimination learning and alters NR2B protein expression in rat auditory cortex.

    PubMed

    Xu, Jinghong; Yu, Liping; Cai, Rui; Zhang, Jiping; Sun, Xinde

    2009-01-01

    Previous studies have shown that the functional development of auditory system is substantially influenced by the structure of environmental acoustic inputs in early life. In our present study, we investigated the effects of early auditory enrichment with music on rat auditory discrimination learning. We found that early auditory enrichment with music from postnatal day (PND) 14 enhanced learning ability in auditory signal-detection task and in sound duration-discrimination task. In parallel, a significant increase was noted in NMDA receptor subunit NR2B protein expression in the auditory cortex. Furthermore, we found that auditory enrichment with music starting from PND 28 or 56 did not influence NR2B expression in the auditory cortex. No difference was found in the NR2B expression in the inferior colliculus (IC) between music-exposed and normal rats, regardless of when the auditory enrichment with music was initiated. Our findings suggest that early auditory enrichment with music influences NMDA-mediated neural plasticity, which results in enhanced auditory discrimination learning. PMID:18706452

  8. Vorinostat, a histone deacetylase inhibitor, facilitates fear extinction and enhances expression of the hippocampal NR2B-containing NMDA receptor gene.

    PubMed

    Fujita, Yosuke; Morinobu, Shigeru; Takei, Shiro; Fuchikami, Manabu; Matsumoto, Tomoya; Yamamoto, Shigeto; Yamawaki, Shigeto

    2012-05-01

    Histone acetylation, which alters the compact chromatin structure and changes the accessibility of DNA to regulatory proteins, is emerging as a fundamental mechanism for regulating gene expression. Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance fear extinction. In this study, we examined whether vorinostat, an HDAC inhibitor, facilitates fear extinction, using a contextual fear conditioning (FC) paradigm, in Sprague-Dawley rats. We found that vorinostat facilitated fear extinction. Next, the levels of global acetylated histone H3 and H4 were measured by Western blotting. We also assessed the effect of vorinostat on the hippocampal levels of NMDA receptor mRNA by real-time quantitative PCR (RT-PCR) and protein by Western blotting. 2 h after vorinostat administration, the levels acetylated histones and NR2B mRNA, but not NR1 or NR2A mRNA, were elevated in the hippocampus. The NR2B protein level was elevated 4 h after vorinostat administration. Last, we investigated the levels of acetylated histones and phospho-CREB (p-CREB) binding at the promoter of the NR2B gene using the chromatin immunoprecipitation (ChIP) assay followed by RT-PCR. The ChIP assay revealed increases in the levels of acetylated histones and they were accompanied by enhanced binding of p-CREB to its binding site at the promoter of the NR2B gene 2 h after vorinostat administration. These findings suggest that vorinostat increases the expression of NR2B in the hippocampus by enhancing histone acetylation, and this process may be implicated in fear extinction. PMID:22364833

  9. Effects of L-3-n-butylphthalide on cognitive dysfunction and NR2B expression in hippocampus of streptozotocin (STZ)-induced diabetic rats.

    PubMed

    Li, Jie; Zhang, Songyun; Zhang, Lihui; Wang, Ruiying; Wang, Mian

    2015-01-01

    Diabetes mellitus is associated with rapid cognitive decline. Currently, there is no effective treatment for cognitive dysfunction induced by diabetes. L-3-n-Butylphthalide (L-NBP) is a nerve protective drug extracted from seeds of celery, which has been proved to improve learning and memory in vascular dementia animal models by improving microcirculation, protecting mitochondria and increasing long-term potentiation (LTP). NR2B, one of the subunits of N-methyl-D-aspartate receptor, has been proved to be an important factor for the formation of LTP. The study aimed to investigate the role of NR2B in cognitive dysfunction in the rats with type 1 diabetes and define the protective effects of L-NBP on cognition. A rat model of type 1 diabetes was established by a single intraperitoneal injection of streptozotocin at 60 mg/kg. Animals were randomly allocated to four groups: normal control (NC); diabetic control (DC); diabetic + low L-NBP (DL, administered L-NBP 60 mg/kg per day for 12 weeks); and diabetic + high L-NBP (DH, administered L-NBP 120 mg/kg per day, for 12 weeks). After 12 weeks, cognitive and memory changes were investigated in the Morris water maze. The expression of NR2B was assessed by real-time polymerase chain reaction, Western blotting, and immunohistochemistry. Our results indicated that the escape latency was significantly increased and the number of crossing platform was significantly decreased in DC group compared to NC group. Also, the expression of NR2B was significantly declined in DC group. However, compared to DC group, the expression of NR2B of the L-NBP-treated groups was significantly increased and the escape latency was shortened with the DH group being the most obvious. Therefore, L-NBP can improve the cognitive function by up-regulating the expression of NR2B in STZ-diabetic rats, which may provide the direction for future diabetic encephalopathy therapy. PMID:25149651

  10. Mas-Related Gene (Mrg) C Activation Attenuates Bone Cancer Pain via Modulating Gi and NR2B

    PubMed Central

    Lu, Cui’e; Lei, Yishan; Ma, Zhengliang; Gu, Xiaoping

    2016-01-01

    Objective This study is to investigate the role of Mas-related gene (Mrg) C in the pathogenesis and treatment of bone cancer pain (BCP). Methods BCP mouse model was established by osteosarcoma cell inoculation. Pain-related behaviors were assessed with the spontaneous lifting behavior test and mechanical allodynia test. Expression levels of MrgC, Gi, and NR2B in the spinal cord were detected with Western blot analysis and immunohistochemistry. Results Pain-related behavior tests showed significantly increased spontaneous flinches (NSF) and decreased paw withdrawal mechanical threshold (PWMT) in mouse models of BCP. Western blot analysis showed that, compared with the control group and before modeling, all the expression levels of MrgC, Gi, and NR2B in the spinal cord of BCP mice were dramatically elevated, which were especially increased at day 7 after operation and thereafter, in a time-dependent manner. Moreover, the treatment of MrgC agonist BAM8-22 significantly up-regulated Gi and down-regulated NR2B expression levels, in the spinal cord of BCP mice, in a time-dependent manner. On the other hand, anti-MrgC significantly down-regulated Gi expression, while dramatically up-regulated NR2B expression, in the BCP mice. Similar results were obtained from the immunohistochemical detection. Importantly, BAM8-22 significantly attenuated the nociceptive behaviors in the BCP mice. Conclusion Our results indicated the MrgC-mediated Gi and NR2B expression alterations in the BCP mice, which might contribute to the pain hypersensitivity. These findings may provide a novel strategy for the treatment of BCP in clinic. PMID:27152740

  11. Distinct roles of NR2A and NR2B cytoplasmic tails in long term potentiation

    PubMed Central

    Foster, Kelly A.; McLaughlin, Nathan; Edbauer, Dieter; Phillips, Marnie; Bolton, Andrew; Constantine-Paton, Martha; Sheng, Morgan

    2010-01-01

    NMDA receptors (NMDARs) are critical mediators of activity-dependent synaptic plasticity, but the differential roles of NR2A- versus NR2B-containing NMDARs have been controversial. Here, we investigate the roles of NR2A and NR2B in LTP in organotypic hippocampal slice cultures using RNAi and overexpression, to complement pharmacological approaches. In young slices, when NR2B is the predominant subunit expressed, LTP is blocked by the NR2B-selective antagonist Ro25-6981. As slices mature, and NR2A expression rises, activation of NR2B receptors became no longer necessary for LTP induction. LTP was blocked, however, by RNAi knockdown of NR2B, and this was rescued by coexpression of an RNAi-resistant NR2B (NR2B*) cDNA. Interestingly, a chimeric NR2B subunit in which the C-terminal cytoplasmic tail was replaced by that of NR2A failed to rescue LTP while the reverse chimera, NR2A channel with NR2B tail, was able to restore LTP. Thus expression of NR2B with its intact cytoplasmic tail is required for LTP induction, at an age when channel activity of NR2B-NMDARs is not required for LTP. Overexpression of wildtype NR2A failed to rescue LTP in neurons transfected with NR2B-RNAi construct, despite restoring NMDA-EPSC amplitude to a similar level as NR2B*. Surprisingly, an NR2A construct lacking its entire C-terminal cytoplasmic tail regained its ability to restore LTP. Together these data suggest that the NR2B subunit plays a critical role for LTP, presumably by recruiting relevant molecules important for LTP via its cytoplasmic tail. By contrast, NR2A is not essential for LTP and its cytoplasmic tail seems to carry inhibitory factors for LTP. PMID:20164351

  12. Amygdala Infusions of an NR2B-Selective or an NR2A-Preferring NMDA Receptor Antagonist Differentially Influence Fear Conditioning and Expression in the Fear-Potentiated Startle Test

    ERIC Educational Resources Information Center

    Walker, David L.; Davis, Michael

    2008-01-01

    Within the amygdala, most N-methyl-D-aspartic acid (NMDA) receptors consist of NR1 subunits in combination with either NR2A or NR2B subunits. Because the particular subunit composition greatly influences the receptors' properties, we investigated the contribution of both subtypes to fear conditioning and expression. To do so, we infused the…

  13. Intrahippocampal Administration of Ibotenic Acid Induced Cholinergic Dysfunction via NR2A/NR2B Expression: Implications of Resveratrol against Alzheimer Disease Pathophysiology

    PubMed Central

    Karthick, Chennakesavan; Periyasamy, Sabapathy; Jayachandran, Kesavan S.; Anusuyadevi, Muthuswamy

    2016-01-01

    Although several drugs revealed moderate amelioration of symptoms, none of them have sufficient potency to prevent or reverse the progression toward Alzheimer's disease (AD) pathology. Resveratrol (RSV), a polyphenolic compound has shown an outstanding therapeutic effect on a broad spectrum of diseases like age-associated neurodegeneration, inflammation etc. The present study was thus conducted to assess the therapeutic efficacy of RSV in ameliorating the deleterious effects of Ibotenic acid (IBO) in male Wistar rats. Stereotactic intrahippocampal administration of IBO (5 μg/μl) lesioned rats impairs cholinergic transmission, learning and memory performance that is rather related to AD and thus chosen as a suitable model to understand the drug efficacy in preventing AD pathophysiology. Since IBO is an agonist of glutamate, it is expected to exhibit an excitotoxic effect by altering glutamatergic receptors like NMDA receptor. The current study displayed significant alterations in the mRNA expression of NR2A and NR2B subunits of NMDA receptors, and further it is surprising to note that cholinergic receptors decreased in expression particularly α7-nAChR with increased m1AChR. RSV administration (20 mg/kg body weight, i.p.) significantly reduced these changes in IBO induced rats. Glutamatergic and cholinergic receptor alterations were associated with significant changes in the behavioral parameters of rats induced by IBO. While RSV improved spatial learning performance, attenuated immobility, and improvised open field activity in IBO induced rats. NR2B activation in the present study might mediate cell death through oxidative stress that form the basis of abnormal behavioral pattern in IBO induced rats. Interestingly, RSV that could efficiently encounter oxidative stress have significantly decreased stress markers viz., nitrite, PCO, and MDA levels by enhancing antioxidant status. Histopathological analysis displayed significant reduction in the hippocampal

  14. Intrahippocampal Administration of Ibotenic Acid Induced Cholinergic Dysfunction via NR2A/NR2B Expression: Implications of Resveratrol against Alzheimer Disease Pathophysiology.

    PubMed

    Karthick, Chennakesavan; Periyasamy, Sabapathy; Jayachandran, Kesavan S; Anusuyadevi, Muthuswamy

    2016-01-01

    Although several drugs revealed moderate amelioration of symptoms, none of them have sufficient potency to prevent or reverse the progression toward Alzheimer's disease (AD) pathology. Resveratrol (RSV), a polyphenolic compound has shown an outstanding therapeutic effect on a broad spectrum of diseases like age-associated neurodegeneration, inflammation etc. The present study was thus conducted to assess the therapeutic efficacy of RSV in ameliorating the deleterious effects of Ibotenic acid (IBO) in male Wistar rats. Stereotactic intrahippocampal administration of IBO (5 μg/μl) lesioned rats impairs cholinergic transmission, learning and memory performance that is rather related to AD and thus chosen as a suitable model to understand the drug efficacy in preventing AD pathophysiology. Since IBO is an agonist of glutamate, it is expected to exhibit an excitotoxic effect by altering glutamatergic receptors like NMDA receptor. The current study displayed significant alterations in the mRNA expression of NR2A and NR2B subunits of NMDA receptors, and further it is surprising to note that cholinergic receptors decreased in expression particularly α7-nAChR with increased m1AChR. RSV administration (20 mg/kg body weight, i.p.) significantly reduced these changes in IBO induced rats. Glutamatergic and cholinergic receptor alterations were associated with significant changes in the behavioral parameters of rats induced by IBO. While RSV improved spatial learning performance, attenuated immobility, and improvised open field activity in IBO induced rats. NR2B activation in the present study might mediate cell death through oxidative stress that form the basis of abnormal behavioral pattern in IBO induced rats. Interestingly, RSV that could efficiently encounter oxidative stress have significantly decreased stress markers viz., nitrite, PCO, and MDA levels by enhancing antioxidant status. Histopathological analysis displayed significant reduction in the hippocampal

  15. Improving solubility of NR2B amino-terminal domain of N-methyl-d-aspartate receptor expressed in Escherichia coli.

    PubMed

    Ng, Fui-Mee; Soh, Wanqin; Geballe, Matthew T; Low, Chian-Ming

    2007-10-12

    The amino-terminal domains (ATDs) of N-methyl-d-aspartate (NMDA) receptors contain binding sites for modulators and may serve as potential drug targets in neurological diseases. Here, three fusion tags (6xHis-, GST-, and MBP-) were fused to the ATD of NMDA receptor NR2B subunit (ATD2B) and expressed in Escherichia coli. Each tag's ability to confer enhanced solubility to ATD2B was assessed. Soluble ATD2B was successfully obtained as a MBP fusion protein. Dynamic light scattering revealed the protein (1mg/ml) exists as monodispersed species at 25 degrees C. Functional studies using circular dichroism showed that the soluble MBP-ATD2B bound ifenprodil in a dose-dependent manner. The dissociation constants obtained for ifenprodil were similar in the absence (64nM) and presence (116nM) of saturating concentration of maltose. Moreover, the yield of soluble MBP-ATD2B is 18 times higher than the refolded 6xHis-ATD2B. We have reported a systematic comparison of three different affinity tagging strategies and identified a rapid and efficient method to obtain large amount of ATD2B recombinant protein for biochemical and structural studies. PMID:17706601

  16. Distinct expression of synaptic NR2A and NR2B in the central nervous system and impaired morphine tolerance and physical dependence in mice deficient in postsynaptic density-93 protein

    PubMed Central

    Liaw, Wen-Jinn; Zhu, Xu-Guang; Yaster, Myron; Johns, Roger A; Gauda, Estelle B; Tao, Yuan-Xiang

    2008-01-01

    Postsynaptic density (PSD)-93, a neuronal scaffolding protein, binds to and clusters N-methyl-D-aspartate receptor (NMDAR) subunits NR2A and NR2B at cellular membranes in vitro. However, the roles of PSD-93 in synaptic NR2A and NR2B targeting in the central nervous system and NMDAR-dependent physiologic and pathologic processes are still unclear. We report here that PSD-93 deficiency significantly decreased the amount of NR2A and NR2B in the synaptosomal membrane fractions derived from spinal cord dorsal horn and forebrain cortex but did not change their levels in the total soluble fraction from either region. However, PSD-93 deficiency did not markedly change the amounts of NR2A and NR2B in either synaptosomal or total soluble fractions from cerebellum. In mice deficient in PSD-93, morphine dose-dependent curve failed to shift significantly rightward as it did in wild type (WT) mice after acute and chronic morphine challenge. Unlike WT mice, PSD-93 knockout mice also showed marked losses of NMDAR-dependent morphine analgesic tolerance and associated abnormal sensitivity in response to mechanical, noxious thermal, and formalin-induced inflammatory stimuli after repeated morphine injection. In addition, PSD-93 knockout mice displayed dramatic loss of jumping activity, a typical NMDAR-mediated morphine withdrawal abstinence behavior. These findings indicate that impaired NMDAR-dependent neuronal plasticity following repeated morphine injection in PSD-93 knockout mice is attributed to PSD-93 deletion-induced alterations of synaptic NR2A and NR2B expression in dorsal horn and forebrain cortex neurons. The selective effect of PSD-93 deletion on synaptic NMDAR expression in these two major pain-related regions might provide the better strategies for the prevention and treatment of opioid tolerance and physical dependence. PMID:18851757

  17. Chronic Administration of Benzo(a)pyrene Induces Memory Impairment and Anxiety-Like Behavior and Increases of NR2B DNA Methylation

    PubMed Central

    Zhang, Wenping; Tian, Fengjie; Zheng, Jinping; Li, Senlin; Qiang, Mei

    2016-01-01

    Background Recently, an increasing number of human and animal studies have reported that exposure to benzo(a)pyrene (BaP) induces neurological abnormalities and is also associated with adverse effects, such as tumor formation, immunosuppression, teratogenicity, and hormonal disorders. However, the exact mechanisms underlying BaP-induced impairment of neurological function remain unclear. The aim of this study was to examine the regulating mechanisms underlying the impact of chronic BaP exposure on neurobehavioral performance. Methods C57BL mice received either BaP in different doses (1.0, 2.5, 6.25 mg/kg) or olive oil twice a week for 90 days. Memory and emotional behaviors were evaluated using Y-maze and open-field tests, respectively. Furthermore, levels of mRNA expression were measured by using qPCR, and DNA methylation of NMDA receptor 2B subunit (NR2B) was examined using bisulfate pyrosequencing in the prefrontal cortex and hippocampus. Results Compared to controls, mice that received BaP (2.5, 6.25 mg/kg) showed deficits in short-term memory and an anxiety-like behavior. These behavioral alterations were associated with a down-regulation of the NR2B gene and a concomitant increase in the level of DNA methylation in the NR2B promoter in the two brain regions. Conclusions Chronic BaP exposure induces an increase in DNA methylation in the NR2B gene promoter and down-regulates NR2B expression, which may contribute to its neurotoxic effects on behavioral performance. The results suggest that NR2B vulnerability represents a target for environmental toxicants in the brain. PMID:26901155

  18. Dopamine receptor D5 deficiency results in a selective reduction of hippocampal NMDA receptor subunit NR2B expression and impaired memory.

    PubMed

    Moraga-Amaro, Rodrigo; González, Hugo; Ugalde, Valentina; Donoso-Ramos, Juan Pablo; Quintana-Donoso, Daisy; Lara, Marcelo; Morales, Bernardo; Rojas, Patricio; Pacheco, Rodrigo; Stehberg, Jimmy

    2016-04-01

    Pharmacological evidence associates type I dopamine receptors, including subtypes D1 and D5, with learning and memory. Analyses using genetic approaches have determined the relative contribution of dopamine receptor D1 (D1R) in cognitive tasks. However, the lack of drugs that can discriminate between D1R and D5R has made the pharmacological distinction between the two receptors difficult. Here, we aimed to determine the role of D5R in learning and memory. In this study we tested D5R knockout mice and wild-type littermates in a battery of behavioral tests, including memory, attention, locomotion, anxiety and motivational evaluations. Our results show that genetic deficiency of D5R significantly impairs performance in the Morris water maze paradigm, object location and object recognition memory, indicating a relevant role for D5R in spatial memory and recognition memory. Moreover, the lack of D5R resulted in decreased exploration and locomotion. In contrast, D5R deficiency had no impact on working memory, anxiety and depressive-like behavior, measured using the spontaneous alternation, open-field, tail suspension test, and forced swimming test. Electrophysiological analyses performed on hippocampal slices showed impairment in long-term-potentiation in mice lacking D5R. Further analyses at the molecular level showed that genetic deficiency of D5R results in a strong and selective reduction in the expression of the NMDA receptor subunit NR2B in the hippocampus. These findings demonstrate the relevant contribution of D5R in memory and suggest a functional interaction of D5R with hippocampal glutamatergic pathways. PMID:26714288

  19. Postsynaptic density protein 95-regulated NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in levodopa-induced dyskinesia rat models

    PubMed Central

    Ba, Maowen; Kong, Min; Ma, Guozhao

    2015-01-01

    Context Abnormality in interactions between N-methyl-d-aspartate (NMDA) receptor and its signaling molecules occurs in the lesioned striatum in Parkinson’s disease (PD) and levodopa-induced dyskinesia (LID). It was reported that Fyn-mediated NR2B tyrosine phosphorylation, can enhance NMDA receptor function. Postsynaptic density protein 95 (PSD-95), one of the synapse-associated proteins, regulates interactions between receptor and downstream-signaling molecules. In light of the relationship between PSD-95, NR2B, and Fyn kinases, does PSD-95 contribute to the overactivity of NMDA receptor function induced by dopaminergic treatment? To further prove the possibility, the effects of regulating the PSD-95 expression on the augmented NR2B tyrosine phosphorylation and on the interactions of Fyn and NR2B in LID rat models were evaluated. Methods In the present study, parkinsonian rat models were established by injecting 6-hydroxydopamine. Subsequently, valid PD rats were treated with levodopa (50 mg/kg/day with benserazide 12.5 mg/kg/day, twice daily) intraperitoneally for 22 days to create LID rat models. Then, the effect of pretreatment with an intrastriatal injection of the PSD-95mRNA antisense oligonucleotides (PSD-95 ASO) on the rotational response to levodopa challenge was assessed. The effects of pretreatment with an intrastriatal injection of PSD-95 ASO on the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in the LID rat models were detected by immunoblotting and immunoprecipitation. Results Levodopa administration twice daily for 22 days to parkinsonian rats shortened the rotational duration and increased the peak turning responses. The altered rotational responses were attenuated by PSD-95 ASO pretreatment. Meanwhile, PSD-95 ASO pretreatment decreased the level of PSD-95 protein expression and reduced both the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B triggered during the levodopa administration in the

  20. Instant and Lasting Down-Regulation of NR1 Expression in the Hippocampus is Associated Temporally with Antidepressant Activity After Acute Yueju.

    PubMed

    Xia, Baomei; Zhang, Hailou; Xue, Wenda; Tao, Weiwei; Chen, Chang; Wu, Ruyan; Ren, Li; Tang, Juanjuan; Wu, Haoxin; Cai, Baochang; Doronc, Ravid; Chen, Gang

    2016-10-01

    Accumulating evidence indicated that N-methyl-D-aspartate (NMDA) receptors are involved in the pathophysiology of depression and implicated in therapeutic targets. NMDA antagonists, such as ketamine, displayed fast-onset and long-lasting antidepressant activity in preclinical and clinical studies. Previous studies showed that Yueju pill exerts antidepressant effects similar to ketamine. Here, we focused on investigating the association of acute and lasting antidepressant responses of Yueju with time course changes of NMDA receptor subunits NR1, NR2A, and NR2B expressions in the hippocampus, a key region regulating depression response. As a result, Yueju reduced immobility time in the forced swimming test from 30 min to 5 days post a single administration. Yueju acutely decreased NR1 and NR2B protein expression in the hippocampus, with NR2A expression unaltered. NR1 expression remained down-regulated 5 days post Yueju administration, whereas NR2B returned to normal level in 24 h. Yueju and ketamine similarly ameliorated the depression-like symptoms at least for 72 h in learned helplessness test. They both reversed the up-regulated expression of NR1 in the learned helpless mice 1 or 3 days post administration. Different from ketamine, the antidepressant effects of Yueju were not influenced by blockade of amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor. These findings served as preclinical evidence that Yueju may confer acute and long-lasting antidepressant effects by favorably modulating NMDA function in the hippocampus. PMID:26825573

  1. Activation of spinal MrgC-Gi-NR2B-nNOS signaling pathway by Mas oncogene-related gene C receptor agonist bovine adrenal medulla 8-22 attenuates bone cancer pain in mice

    PubMed Central

    Sun, Yu’e; Zhang, Juan; Lei, Yishan; Lu, Cui’e; Hou, Bailing; Ma, Zhengliang; Gu, Xiaoping

    2016-01-01

    Objectives: In the present study, we investigate the effects of Mas oncogene-related gene (Mrg) C receptors (MrgC) on the expression and activation of spinal Gi protein, N-methyl-D-aspartate receptor subunit 2B (NR2B), and neuronal nitric oxide synthase (nNOS) in mouse model of bone cancer pain. Methods: The number of spontaneous foot lift (NSF) and paw withdrawal mechanical threshold (PWMT) were measured after inoculation of tumor cells and intrathecal injection of MrgC agonist bovine adrenal medulla 8-22 (BAM8-22) or MrgC antagonist anti-MrgC for 14 days after operation. Expression of spinal MrgC, Gi protein, NR2B and nNOS and their phosphorylated forms after inoculation was examined by immunohistochemistry and Western blotting. Double labeling was used to identify the co-localization of NR2B or nNOS with MrgC in spinal cord dorsal horn (SCDH) neurons. The effects of intrathecal injection of BAM8-22 or anti-MrgC on nociceptive behaviors and the corresponding expression of spinal MrgC, Gi protein, NR2B and nNOS were also investigated. Results: The expression of spinal MrgC, Gi protein, NR2B, and nNOS was higher in tumor-bearing mice in comparison to sham mice or normal mice. Intrathecal injection of MrgC agonist BAM8-22 significantly alleviated bone cancer pain, up-regulated MrgC and Gi protein expression, and down-regulated the expression of spinal p-NR2B, t-nNOS and p-nNOS in SCDH on day 14 after operation, whereas administration of anti-MrgC produced the opposite effect. Meanwhile, MrgC-like immunoreactivity (IR) co-localizes with NR2B-IR or nNOS-IR in SCDH neurons. Conclusions: The present study demonstrates that MrgC-activated spinal Gi-NR2B-nNOS signaling pathway plays important roles in the development of bone cancer pain. These findings may provide a novel strategy for the treatment of bone cancer pain. PMID:27158400

  2. Repeated treatment with electroconvulsive seizures induces HDAC2 expression and down-regulation of NMDA receptor-related genes through histone deacetylation in the rat frontal cortex.

    PubMed

    Park, Hong Geun; Yu, Hyun Sook; Park, Soyoung; Ahn, Yong Min; Kim, Yong Sik; Kim, Se Hyun

    2014-09-01

    The enzymatic activity of histone deacetylases (HDACs) leads to a histone deacetylation-mediated condensed chromatic structure, resulting in transcriptional repression, which has been implicated in the modifications of neural circuits and behaviors. Repeated treatment with electroconvulsive seizure (ECS) induces changes in histone acetylation, expression of various genes, and intrabrain cellular changes, including neurogenesis. In this study, we examined the effects of repeated ECS on the expression of class I HDACs and related changes in histone modifications and gene expression in the rat frontal cortex. Ten days of repeated ECS treatments (E10X) up-regulated HDAC2 expression at the mRNA and protein levels in the rat frontal cortex compared with sham-treated controls; this was evident in the nuclei of neuronal cells in the prefrontal, cingulate, orbital, and insular cortices. Among the known HDAC2 target genes, mRNA expression of N-methyl-d-aspartate (NMDA) receptor signaling-related genes, including early growth response-1 (Egr1), c-Fos, glutamate receptor, ionotropic, N-methyl d-aspartate 2A (Nr2a), Nr2b, neuritin1 (Nrn1), and calcium/calmodulin-dependent protein kinase II alpha (Camk2α), were decreased, and the histone acetylation of H3 and/or H4 proteins was also reduced by E10X. Chromatin immunoprecipitation analysis revealed that HDAC2 occupancy in the promoters of down-regulated genes was increased significantly. Moreover, administration of sodium butyrate, a HDAC inhibitor, during the course of E10X ameliorated the ECS-induced down-regulation of genes in the rat frontal cortex. These findings suggest that induction of HDAC2 by repeated ECS treatment could play an important role in the down-regulation of NMDA receptor signaling-related genes in the rat frontal cortex through histone modification. PMID:24606669

  3. NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

    SciTech Connect

    Li, Mei; Zhang, Dong-Qing; Wang, Xiang-Zhen; Xu, Tie-Jun

    2011-08-12

    Highlights: {yields} The NR2B component of the NMDARs is important for the NSPC proliferation. {yields} pCaMKIV and pCREB exist in NSPCs. {yields} The CaMKIV/CREB pathway mediates NSPC proliferation. -- Abstract: Accumulating evidence indicates the involvement of N-methyl-D-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Ro 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.

  4. Novel approach to probe subunit-specific contributions to N-methyl-D-aspartate (NMDA) receptor trafficking reveals a dominant role for NR2B in receptor recycling.

    PubMed

    Tang, Tina Tze-Tsang; Badger, John D; Roche, Paul A; Roche, Katherine W

    2010-07-01

    N-Methyl-d-aspartate (NMDA) receptors are expressed at excitatory synapses throughout the brain and are essential for neuronal development and synaptic plasticity. Functional NMDA receptors are tetramers, typically composed of NR1 and NR2 subunits (NR2A-D). NR2A and NR2B are expressed in the forebrain and are thought to assemble as diheteromers (NR1/NR2A, NR1/NR2B) and triheteromers (NR1/NR2A/NR2B). NR2A and NR2B contain cytosolic domains that regulate distinct postendocytic sorting events, with NR2A sorting predominantly into the degradation pathway, and NR2B preferentially trafficking through the recycling pathway. However, the interplay between these two subunits remains an open question. We have now developed a novel approach based on the dimeric feature of the alpha- and beta-chains of the human major histocompatibility complex class II molecule. We created chimeras of alpha- and beta-chains with the NR2A and NR2B C termini and evaluated endocytosis of dimers. Like chimeric proteins containing only a single NR2A or NR2B C-terminal domain, major histocompatibility complex class II-NR2A homodimers sort predominantly to late endosomes, whereas NR2B homodimers traffic to recycling endosomes. Interestingly, NR2A/NR2B heterodimers traffic preferentially through the recycling pathway, and NR2B is dominant in regulating dimer trafficking in both heterologous cells and neurons. In addition, the recycling of NR2B-containing NMDARs in wild-type neurons is not significantly different from NR2A(-/-) neurons. These data support a dominant role for NR2B in regulating the trafficking of triheteromeric NMDARs in vivo. Furthermore, our molecular approach allows for the direct and selective evaluation of dimeric assemblies and can be used to define dominant trafficking domains in other multisubunit protein complexes. PMID:20427279

  5. Down-regulation of EZH2 expression in myelodysplastic syndromes.

    PubMed

    Cabrero, Monica; Wei, Yue; Yang, Hui; Ganan-Gomez, Irene; Bohannan, Zach; Colla, Simona; Marchesini, Matteo; Bravo, Guillermo Montalban; Takahashi, Koichi; Bueso-Ramos, Carlos; Garcia-Manero, Guillermo

    2016-05-01

    EZH2 genetic mutations are common in myelodysplastic syndrome (MDS), which implies that this gene has a pathophysiological role in the disease. To further characterize molecular alterations of EZH2, and their potential prognostic impact in MDS, we assessed EZH2 RNA expression in primary bone marrow CD34+ cells from 78 patients. We found that 47% of patients have reduced EZH2 expression compared to normal controls. Further analyses revealed that EZH2 is significantly underexpressed in patients bearing chromosome 7 or 7q deletions (7-alt) when compared to controls, diploid patients, and patients with other cytogenetic alterations (p<0.05). In survival analysis, we found a non-significant trend toward overall survival (OS) being better among patients with EZH2 underexpression (median OS 55 vs. 36 months; p=0.71). Importantly, this trend became significant when the analysis was restricted to the subset of cases without alterations in chromosome 7 (62 vs. 36 months; p=0.033). Furthermore, our previous work has identified a spectrum of innate immune genes in MDS CD34+ cells that are deregulated via abnormal promoter histone methylation. Because EZH2 is a key regulator of histone methylation, we assessed the relationship between deregulation of these genes and EZH2 underexpression. We observed that the mRNA levels of 11 immune genes were higher in the EZH2 underexpression group and that immune gene expression was significantly higher in patients with concomitant EZH2 underexpression and KDM6B (also known as JMJD3, an H3K27 demethylase) overexpression. Taken together, these data indicate that EZH2 underexpression may have unique impact on the molecular pathogenesis and prognosis in MDS and be an important marker for patients without chromosome 7 alteration. PMID:26970171

  6. CDK14 expression is down-regulated by cigarette smoke in vivo and in vitro

    PubMed Central

    Pollack, Daniel; Xiao, Yuxuan; Shrivasatava, Vibha; Levy, Avi; Andrusier, Miriam; D’Armiento, Jeanine; Holz, Marina K.; Vigodner, Margarita

    2016-01-01

    In this study, DNA arrays have been employed to monitor gene expression patterns in testis of mice exposed to tobacco smoke for 24 weeks and compared to control animals. The results of the analysis revealed significant changes in expression of several genes that may have a role in spermatogenesis. Cdk14 was chosen for further characterization because of a suggested role in the testis and in regulation of Wnt signaling. RT-PCR analysis confirmed down regulation of Cdk14 in mice exposed to cigarette smoke (CS). Cdk14 is expressed in all testicular cells; spermatogonia- and Sertoli-derived cell lines treated with cigarette smoke extract (CSE) in vitro showed down-regulation of CDK14 mRNA and protein levels as well as down-regulation of β-catenin levels. CS-induced down-regulation of CDK14 mRNA and protein levels was also observed in several lung epithelium-derived cell lines including primary normal human bronchial epithelial cells (NHBE), suggesting that the effect is not restricted to the testis. Similar to testicular cells, CS-induced down-regulation of CDK14 in lung cells correlated with decreased levels of β-catenin, a finding suggesting impaired Wnt signaling. In the lungs, CDK14 was localized to the alveolar and bronchial epithelium. PMID:25680692

  7. Protective immunity of rAd5/NR2B vaccine against concomitant aversiveness of spontaneous neuropathic pain following spinal nerve ligation injury

    PubMed Central

    Wang, Gong-Ming; Wang, Xiao-Yan; Liu, Guang-Jie; Cheng, Kun; Wang, Hua; Guo, Shou-Gang

    2015-01-01

    Objective: Peripheral nerve injury elicits an aversive state of spontaneous neuropathic pain, and up to now, the modulation of this concomitant aversive state remains a major therapeutic challenge. NMDA receptor subunits NR2B in the rACC are critically involved in the processing of this aversive state and then a strategy targeted at the NR2B subunit might be promising for modulation of the aversive state. Thus, in the present study, using negative reinforcement animal model to reveal spontaneous pain, we investigated the effect of oral immunization with recombinant adenovirus serotype 5-mediated NR2B gene transfer (rAd5/NR2B) on the modulation of the tonic pain. Material and methods: Following oral administration of the rAd5/NR2B vaccine, NR2B-specific antibodies were induced in serum. And the humoral response was involved in the decreased expression of NR2B protein in the rACC. Results: The present study demonstrated that CPP achieved by spinal administration of clonidine in spinal nerve ligation (SNL) rats revealed the presence of aversive state of spontaneous neuropathic pain. Notably, the humoral autoimmune response blocked the CPP by spinal clonidine, suggesting the relief of the concomitant aversive of spontaneous neuropathic pain in the SNL rats. Conclusion: These data proved the feasibility of oral immunization with rAd5/NR2B for modulation of concomitant aversive of spontaneous neuropathic pain due to peripheral nerve injury. PMID:26309509

  8. Co-activation of NR2A and NR2B subunits induces resistance to fear extinction.

    PubMed

    Leaderbrand, Katherine; Corcoran, Kevin A; Radulovic, Jelena

    2014-09-01

    Unpredictable stress is known to profoundly enhance susceptibility to fear and anxiety while reducing the ability to extinguish fear when threat is no longer present. Accordingly, partial aversive reinforcement, via random exposure to footshocks, induces fear that is resistant to extinction. Here we sought to determine the hippocampal mechanisms underlying susceptibility versus resistance to context fear extinction as a result of continuous (CR) and partial (PR) reinforcement, respectively. We focused on N-methyl-D-aspartate receptor (NMDAR) subunits 2A and B (NR2A and NR2B) as well as their downstream signaling effector, extracellular signal-regulated kinase (ERK), based on their critical role in the acquisition and extinction of fear. Pharmacological inactivation of NR2A, but not NR2B, blocked extinction after CR, whereas inactivation of NR2A, NR2B, or both subunits facilitated extinction after PR. The latter finding suggests that co-activation of NR2A and NR2B contributes to persistent fear following PR. In contrast to CR, PR increased membrane levels of ERK and NR2 subunits after the conditioning and extinction sessions, respectively. In parallel, nuclear activation of ERK was significantly reduced after the extinction session. Thus, co-activation and increased surface expression of NR2A and NR2B, possibly mediated by ERK, may cause persistent fear. These findings suggest that patients with post-traumatic stress disorder (PTSD) may benefit from antagonism of specific NR2 subunits. PMID:24055686

  9. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    SciTech Connect

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa; Shin, Incheol

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  10. Density enhanced phosphatase-1 down-regulates urokinase receptor surface expression in confluent endothelial cells

    PubMed Central

    Brunner, Patrick M.; Heier, Patricia C.; Mihaly-Bison, Judit; Priglinger, Ute; Binder, Bernd R.

    2011-01-01

    VEGF165, the major angiogenic growth factor, is known to activate various steps in proangiogenic endothelial cell behavior, such as endothelial cell migration and invasion, or endothelial cell survival. Thereby, the urokinase-type plasminogen activator (uPA) system has been shown to play an essential role not only by its proteolytic capacities, but also by induction of intracellular signal transduction. Therefore, expression of its cell surface receptor uPAR is thought to be an essential regulatory mechanism in angiogenesis. We found that uPAR expression on the surface of confluent endothelial cells was down-regulated compared with subconfluent proliferating endothelial cells. Regulation of uPAR expression was most probably affected by extracellular signal-regulated kinase 1/2 (ERK1/2) activation, a downstream signaling event of the VEGF/VEGF-receptor system. Consistently, the receptor-like protein tyrosine phosphatase DEP-1 (density enhanced phosphatase-1/CD148), which is abundantly expressed in confluent endothelial cells, inhibited the VEGF-dependent activation of ERK1/2, leading to down-regulation of uPAR expression. Overexpression of active ERK1 rescued the DEP-1 effect on uPAR. That DEP-1 plays a biologic role in angiogenic endothelial cell behavior was demonstrated in endothelial cell migration, proliferation, and capillary-like tube formation assays in vitro. PMID:21304107

  11. Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein.

    PubMed

    Werner, Ernst R; Bahrami, Soheyl; Heller, Regine; Werner-Felmayer, Gabriele

    2002-03-22

    GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine. PMID:11799107

  12. The selectivity of conantokin-G for ion channel inhibition of NR2B subunit-containing NMDA receptors is regulated by amino acid residues in the S2 region of NR2B.

    PubMed

    Sheng, Zhenyu; Liang, Zhong; Geiger, James H; Prorok, Mary; Castellino, Francis J

    2009-08-01

    The conantokins are short, naturally occurring peptides that inhibit ion flow through N-methyl-d-aspartate receptor (NMDAR) channels. One member of this peptide family, conantokin-G (con-G), shows high selectivity for antagonism of NR2B-containing NMDAR channels, whereas other known conantokins are less selective inhibitors with regard to the nature of the NR2 subunit of the NMDAR complex. In order to define the molecular determinants of NR2B that govern con-G selectivity, we evaluated the ability of con-G to inhibit NMDAR ion channels expressed in human embryonic kidney (HEK)293 cells transfected with NR1, in combination with various NR2A/2B chimeras and point mutants, by electrophysiology using cells voltage-clamped in the whole-cell configuration. We found that a variant of the con-G-insensitive subunit, NR2A, in which the 158 residues comprising the S2 peptide segment (E(657)-I(814)) were replaced by the corresponding S2 region of NR2B (E(658)-I(815)), results in receptors that are highly sensitive to inhibition by con-G. Of the 22 amino acids that are different between the NR2A-S2 and the NR2B-S2 regions, exchange of one of these, M(739) of NR2B for the equivalent K(738) of NR2A, was sufficient to completely import the inhibitory activity of con-G into NR1b/NR2A-containing NMDARs. Some reinforcement of this effect was found by substitution of a second amino acid, K(755) of NR2B for Y(754) of NR2A. The discovery of the molecular determinants of NR2B selectivity with con-G has implications for the design of subunit-selective neurobiological probes and drug therapies, in addition to advancing our understanding of NR2B- versus NR2A-mediated neurological processes. PMID:19427876

  13. Cell surface modulation of gene expression in brain cells by down regulation of glucocorticoid receptors

    SciTech Connect

    McGinnis, J.F.; de Vellis, J.

    1981-02-01

    The concentration of glycerol-3-phosphate dehydrogenase (GPDH; sn-glycerol-3-phosphate:NAD/sup +/ 2-oxidoreductase, EC 1.1.1.8) had previously been determined to be regulated by glucocorticoids in rat brain cells in vivo and in cell culture. We now demonstrate that concanavalin A (Con A) can inhibit the induction of GPDH in a dose-dependent manner in C6 rat glioma cells and in primary cultures of rat brain oligodendrocytes. The inhibition specifically prevents the appearance of new molecules of GPDH, although Con A does not significantly inhibit protein synthesis in these cells, nor does it affect the activity of another solube enzyme, lactate dehydrogenase. The ability to block enzyme induction is not limited to Con A, because other lectins also inhibit induction. The molecular mechanism by which Con A inhibits GPDH induction appears to be by the down regulation of the cytoplasmic glucocorticoid receptors, because exposure to Con A results in the loss of more than 90% of the receptor activity. Con A does not inhibit the receptor assay and no direct interaction between the receptor and Con A could be demonstrated. This down regulation is not tumor cell specific and appears to be a general phenomenon, because it occurs in normal oligodendrocytes and even in normal astrocytes (a cell type in which the gene for GPDH is not expressed). The down regulation of glucocorticoid receptors in normal brain cells suggests two important corollaries. First, it demonstrates the existence of a rate-limiting step controlling the glucocorticoid-dependent gene expression in brain cells and possibly represents a regulatory site common to all glucocorticoid target cells. Second, it suggests that the response to glucocorticoids of oligodendrocytes and astrocytes can be regulated in vivo by cell surface contact with endogenous lectins, neighboring cells, or both.

  14. Down-Regulation of Brush Border Efflux Transporter Expression in the Kidneys of Pregnant Mice

    PubMed Central

    Yacovino, Lindsay L.; Gibson, Christopher J.

    2013-01-01

    Pregnancy increases the urinary excretion of chemicals in women and rodents. It is unknown whether the enhanced clearance of drugs during pregnancy involves changes in the expression of transporters that mediate chemical secretion and reabsorption. The purpose of this study was to quantify the mRNA and protein expression of efflux transporters in kidneys from virgin and pregnant mice on gestational days 7, 11, 14, and 17 and postnatal days 1, 15, and 30 with use of quantitative polymerase chain reaction, Western blot, and immunofluorescence. Multidrug resistance protein (Mdr) 1b mRNA, multidrug resistance-associated protein (Mrp) 4 mRNA, and protein levels decreased significantly by 25–75% throughout pregnancy and lactation. Similarly, Mrp2 and multidrug and toxin extrusion transporter (Mate) 1 mRNA expression were down-regulated 20–40% during mid to late gestation but returned to control levels by postnatal day 15. In contrast, Mrp3 mRNA and protein increased 225% and 31%, respectively, at gestational day 14. Coordinated down-regulation of brush border transporters Mate1, Mrp2, and Mrp4 and up-regulation of the basolateral Mrp3 transporter would reduce chemical secretion into urine. PMID:22896729

  15. Arctiin induces cell growth inhibition through the down-regulation of cyclin D1 expression.

    PubMed

    Matsuzaki, Youichirou; Koyama, Makoto; Hitomi, Toshiaki; Yokota, Tomoya; Kawanaka, Mayumi; Nishikawa, Akiyoshi; Germain, Doris; Sakai, Toshiyuki

    2008-03-01

    Arctiin is a major lignan constituent of Arctium lappa and has anti-cancer properties in animal models. It was recently reported that arctiin induces growth inhibition in human prostate cancer PC-3 cells. However, the growth inhibitory mechanism of arctiin remains unknown. Herein we report that arctiin induces growth inhibition and dephosphorylates the tumor-suppressor retinoblastoma protein in human immortalized keratinocyte HaCaT cells. We also show that the growth inhibition caused by arctiin is associated with the down-regulation of cyclin D1 protein expression. Furthermore, the arctiin-induced suppression of cyclin D1 protein expression occurs in various types of human tumor cells, including osteosarcoma, lung, colorectal, cervical and breast cancer, melanoma, transformed renal cells and prostate cancer. Depletion of the cyclin D1 protein using small interfering RNA-rendered human breast cancer MCF-7 cells insensitive to the growth inhibitory effects of arctiin, implicates cyclin D1 as an important target of arctiin. Taken together, these results suggest that arctiin down-regulates cyclin D1 protein expression and that this at least partially contributes to the anti-proliferative effect of arctiin. PMID:18288407

  16. Down-Regulation of Nogo-B Expression as a Newly Identified Feature of Intrahepatic Cholangiocarcinoma.

    PubMed

    Nanashima, Atsushi; Hatachi, Go; Tominaga, Tetsurou; Murakami, Goushi; Takagi, Katsunori; Arai, Junichi; Wada, Hideo; Nagayasu, Takeshi; Sumida, Yorihisa

    2016-01-01

    Nogo-B, located in the endoplasmic reticulum, is an isoform belonging to the reticulon protein family, which is expressed specifically in cholangiocytes and non-parenchymal cells in the liver. Nogo-B expression is down-regulated with the progression of liver fibrosis, but its distinct function in liver malignancies has not been fully clarified. We have hypothesized that Nogo-B expression may be altered in intrahepatic cholangiocarcinoma (ICC), a relatively rare type of primary liver cancer with highly malignant behavior. The present study aimed to investigate the relationship between Nogo-B expression, assessed by immunohistochemical staining, and clinicopathological factors and prognosis in 34 ICC patients. Positive expression was observed in 19 (56%) of 34 ICC specimens: 6 patients (18%) with positivity levels of 1+ (positive cells in 10-50% of cancer cells) and 13 patients (38%) with 2+ (positive cells over 50%). Importantly, the remaining 15 patients (44%) were categorized as negative expression (Nogo-B-positive cells, less than 10%). Conversely, the mass-forming type of ICC tended to express Nogo-B with the degree of 2+ positivity, compared to the periductal infiltration type (p = 0.064), and the mass-forming type showed a better 5-year survival rate (66% vs. 5%) after hepatectomy (p < 0.05). However, the degree of positivity was not associated with tumor relapse rate, disease-free and overall survival, although each of the periductal infiltration type, intrahepatic metastasis, larger tumor size, and lower microvessel counts was associated with lower survival rates. We propose that Nogo-B expression is down-regulated in ICC, the implication of which, however, remains to be investigated. PMID:26656426

  17. Curcumin down-regulates AR gene expression and activation in prostate cancer cell lines.

    PubMed

    Nakamura, Keiichiro; Yasunaga, Yutaka; Segawa, Takehiko; Ko, Daejin; Moul, Judd W; Srivastava, Shiv; Rhim, Johng S

    2002-10-01

    Curcumin, traditionally used as a seasoning spice in Indian cuisine, has been reported to decrease the proliferation potential of prostate cancer cells, by a mechanism that is not fully understood. In the current study, we have evaluated the effects of curcumin in cell growth, activation of signal transduction, and transforming activities of both androgen-dependent and independent cell lines. Prostate cancer cell lines, LNCaP and PC-3, were treated with curcumin and its effects were further analyzed on signal transduction and expression of androgen receptor (AR) and AR-related cofactors using transient transfection assay and Western blotting. Our results show that curcumin down-regulates transactivation and expression of AR, activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), and CREB (cAMP response element-binding protein)-binding protein (CBP). Curcumin also inhibited the transforming activities of both cell lines as evidenced by the reduced colony forming ability in soft agar. The results obtained here demonstrate that curcumin has a potential therapeutic effect on prostate cancer cells through down-regulation of AR and AR-related cofactors (AP-1, NF-kappaB and CBP). PMID:12239622

  18. Down-Regulated CK8 Expression in Human Intervertebral Disc Degeneration

    PubMed Central

    Sun, Zhen; Wang, Hai-Qiang; Liu, Zhi-Heng; Chang, Le; Chen, Yu-Fei; Zhang, Yong-Zhao; Zhang, Wei-Lin; Gao, Yang; Wan, Zhong-Yuan; Che, Lu; Liu, Xu; Samartzis, Dino; Luo, Zhuo-Jing

    2013-01-01

    As an intermediate filament protein, cytokeratin 8 (CK8) exerts multiple cellular functions. Moreover, it has been identified as a marker of notochord cells, which play essential roles in human nucleus pulposus (NP). However, the distribution of CK8 positive cells in human NP and their relationship with intervertebral disc degeneration (IDD) have not been clarified until now. Here, we found the percentage of CK8 positive cells in IDD (25.7±4.14%) was significantly lower than that in normal and scoliosis NP (51.9±9.73% and 47.8±5.51%, respectively, p<0.05). Western blotting and qRT-PCR results confirmed the down-regulation of CK8 expression in IDD on both of protein and mRNA levels. Furthermore, approximately 37.4% of cell clusters were CK8 positive in IDD. Taken together, this is the first study to show a down-regulated CK8 expression and the percentage of CK8 positive cell clusters in IDD based upon multiple lines of evidence. Consequently, CK8 positive cells might be considered as a potential option in the development of cellular treatment strategies for NP repair. PMID:23801880

  19. Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways.

    PubMed

    Zhang, Jian; Lazarenko, Oxana P; Blackburn, Michael L; Badger, Thomas M; Ronis, Martin J J; Chen, Jin-Ran

    2014-07-01

    It has been suggested that the beneficial effects of soy protein isolate (SPI) on bone quality are due to either stimulation of estrogenic signaling via isoflavones or through a novel and as yet uncharacterized nonestrogenic pathway. In our study, SPI-fed rat serum inhibited the osteoblastic cell senescence pathway. This effect was accompanied by stimulation of cell differentiation, proliferation, and significant restoration of replicative senescent bone marrow mesenchymal ST2 cells (passaged 30 times). These effects were reproduced in bone from 5-wk-old intact and 10-wk-old ovariectomized female rats fed SPI diets. Caveolin-1 and p53 expression was decreased in bone in SPI-fed, but not in 17β-estradiol (E2)-treated rats. In cell culture studies, membranous caveolin-1 and nuclear p53 expression was greater in replicative senescent ST2 cell cultures than in earlier passaged cells. SPI-fed rat serum significantly down-regulated both caveolin-1 and p53 in senescent and nonsenescent cells. Replicative senescent ST2 cells exhibited a strong association among caveolin-1, p53, and mouse double minute 2 homologue (mdm2), which was inhibited by SPI-fed rat serum. Overexpression of caveolin-1 in ST2 cells resulted in increased expression of p53 and p21, whereas, knockdown of caveolin-1 using shRNA led to increases in mdm2 and eliminated SPI-fed rat serum's effects on p53 and p21 expression. In contrast, manipulation of caveolin-1 expression did not affect the actions of E2 or isoflavones on p53 expression in either ST2 or OB6 cells. These results suggest that caveolin-1 is a mediator of nonestrogenic SPI effects on bone cells.-Zhang, J., Lazarenko, O. P., Blackburn, M. L., Badger, T. M., Ronis, M. J. J., Chen, J.-R. Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways. PMID:24719353

  20. RASSF6 expression in adipocytes is down-regulated by interaction with macrophages.

    PubMed

    Sanada, Yohei; Kumoto, Takahiro; Suehiro, Haruna; Nishimura, Fusanori; Kato, Norihisa; Hata, Yutaka; Sorisky, Alexander; Yanaka, Noriyuki

    2013-01-01

    Macrophage infiltration into adipose tissue is associated with obesity and the crosstalk between adipocytes and infiltrated macrophages has been investigated as an important pathological phenomenon during adipose tissue inflammation. Here, we sought to identify adipocyte mRNAs that are regulated by interaction with infiltrated macrophages in vivo. An anti-inflammatory vitamin, vitamin B6, suppressed macrophage infiltration into white adipose tissue and altered mRNA expression. We identified >3500 genes whose expression is significantly altered during the development of obesity in db/db mice, and compared them to the adipose tissue mRNA expression profile of mice supplemented with vitamin B6. We identified PTX3 and MMP3 as candidate genes regulated by macrophage infiltration. PTX3 and MMP3 mRNA expression in 3T3-L1 adipocytes was up-regulated by activated RAW264.7 cells and these mRNA levels were positively correlated with macrophage number in adipose tissue in vivo. Next, we screened adipose genes down-regulated by the interaction with macrophages, and isolated RASSF6 (Ras association domain family 6). RASSF6 mRNA in adipocytes was decreased by culture medium conditioned by activated RAW264.7 cells, and RASSF6 mRNA level was negatively correlated with macrophage number in adipose tissue, suggesting that adipocyte RASSF6 mRNA expression is down-regulated by infiltrated macrophages in vivo. Finally, this study also showed that decreased RASSF6 expression up-regulates mRNA expression of several genes, such as CD44 and high mobility group protein HMGA2. These data provide novel insights into the biological significance of interactions between adipocytes and macrophages in adipose tissue during the development of obesity. PMID:23626755

  1. Paeoniflorin ameliorates symptoms of experimental Sjogren's syndrome associated with down-regulating Cyr61 expression.

    PubMed

    Li, Huidan; Sun, Xiaoxuan; Zhang, Jie; Sun, Yue; Huo, Rongfen; Li, Haichuan; Zhai, Tianhang; Shen, Baihua; Zhang, Miaojia; Li, Ningli

    2016-01-01

    Paeoniflorin (PF), an active compound extracted from Paeony root, has been used in therapy of autoimmune diseases with effective clinical efficiency and higher safety. Sjogren's syndrome (SS) is a chronic, systemic, immune-mediated inflammatory disease. In this study, we demonstrated that novel pro-inflammatory factor Cyr61/CCN1 was up-regulated in epithelial cells of salivary glands of primary SS patients and submandibular gland autoantigen-induced experimental SS mice. Blocking Cyr61 expression with special monoclonal antibody improved saliva secretion by ameliorating inflammatory infiltration and cytokines production in vivo. Furthermore, we showed that PF could alleviate inflammation by down-regulating Cyr61 expression in experimental SS mice. In conclusion, our new findings revealed for the first time that Cyr61 involves the pathogenesis of primary SS and PF alleviates SS-like symptoms associated with inhibiting Cyr61 expression, providing new insights into the potential molecular mechanism of PF in primary SS treatment. PMID:26630293

  2. A Novel Protein Complex in Membrane Rafts Linking the NR2B Glutamate Receptor and Autophagy Is Disrupted following Traumatic Brain Injury

    PubMed Central

    Bigford, Gregory E.; Alonso, Ofelia F.; Dietrich, W. Dalton

    2009-01-01

    Abstract Hyperactivation of N-methyl-d-aspartate receptors (NRs) is associated with neuronal cell death induced by traumatic brain injury (TBI) and many neurodegenerative conditions. NR signaling efficiency is dependent on receptor localization in membrane raft microdomains. Recently, excitotoxicity has been linked to autophagy, but mechanisms governing signal transduction remain unclear. Here we have identified protein interactions between NR2B signaling intermediates and the autophagic protein Beclin-1 in membrane rafts of the normal rat cerebral cortex. Moderate TBI induced rapid recruitment and association of NR2B and pCaMKII to membrane rafts, and translocation of Beclin-1 out of membrane microdomains. Furthermore, TBI caused significant increases in expression of key autophagic proteins and morphological hallmarks of autophagy that were significantly attenuated by treatment with the NR2B antagonist Ro 25-6981. Thus, stimulation of autophagy by NR2B signaling may be regulated by redistribution of Beclin-1 in membrane rafts after TBI. PMID:19335206

  3. Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation through activating the NR2B subunits of NMDA receptors

    SciTech Connect

    Shi, Wen-Zhu; Miao, Yu-Liang; Guo, Wen-Zhi; Wu, Wei; Li, Bao-Wei; An, Li-Na; Fang, Wei-Wu; Mi, Wei-Dong

    2014-04-25

    Highlights: • Leptin promotes the proliferation of neural stem cells isolated from embryonic mouse hippocampus. • Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation. • The effects of leptin are partially mediated by upregulating NR2B subunits. - Abstract: Corticosterone inhibits the proliferation of hippocampal neural stem cells (NSCs). The removal of corticosterone-induced inhibition of NSCs proliferation has been reported to contribute to neural regeneration. Leptin has been shown to regulate brain development, improve angiogenesis, and promote neural regeneration; however, its effects on corticosterone-induced inhibition of NSCs proliferation remain unclear. Here we reported that leptin significantly promoted the proliferation of hippocampal NSCs in a concentration-dependent pattern. Also, leptin efficiently reversed the inhibition of NSCs proliferation induced by corticosterone. Interestingly, pre-treatment with non-specific NMDA antagonist MK-801, specific NR2B antagonist Ro 25-6981, or small interfering RNA (siRNA) targeting NR2B, significantly blocked the effect of leptin on corticosterone-induced inhibition of NSCs proliferation. Furthermore, corticosterone significantly reduced the protein expression of NR2B, whereas pre-treatment with leptin greatly reversed the attenuation of NR2B expression caused by corticosterone in cultured hippocampal NSCs. Our findings demonstrate that leptin reverses the corticosterone-induced inhibition of NSCs proliferation. This process is, at least partially mediated by increased expression of NR2B subunits of NMDA receptors.

  4. Hyperinsulinemia Down-Regulates TLR4 Expression in the Mammalian Heart

    PubMed Central

    de Laat, Melody A.; Gruntmeir, Kaylynn J.; Pollitt, Christopher C.; McGowan, Catherine M.; Sillence, Martin N.; Lacombe, Véronique A.

    2014-01-01

    Toll-like receptors (TLR) are key regulators of innate immune and inflammatory responses and their activation is linked to impaired glucose metabolism during metabolic disease. Determination of whether TLR4 signaling can be activated in the heart by insulin may shed light on the pathogenesis of diabetic cardiomyopathy, a process that is often complicated by obesity and insulin resistance. The aim of the current study was to determine if supraphysiological insulin concentrations alter the expression of TLR4, markers of TLR4 signaling and glucose transporters (GLUTs) in the heart. Firstly, the effect of insulin on TLR4 protein expression was investigated in vitro in isolated rat cardiac myocytes. Secondly, protein expression of TLR4, the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) suppressor of cytokine signaling 3 (SOCS3) and GLUTs (1, 4, 8, 12) were examined in the equine ventricular myocardium following a prolonged, euglycemic, hyperinsulinemic clamp. Down-regulation of TLR4 protein content in rat cardiac myocytes was observed after incubation with a supraphysiologic concentration of insulin as well as in the equine myocardium after prolonged insulin infusion. Further, cardiac TLR4 expression was negatively correlated with serum insulin concentration. Markers of cardiac TLR4 signaling and GLUT expression were not affected by hyperinsulinemia and concomitant TLR4 down-regulation. Since TLRs are major determinants of the inflammatory response, our findings suggest that insulin infusion exerts an anti-inflammatory effect in the hearts of non-obese individuals. Understanding the regulation of cardiac TLR4 signaling during metabolic dysfunction will facilitate improved management of cardiac sequela to metabolic syndrome and diabetes. PMID:25101057

  5. p65 down-regulates DEPTOR expression in response to LPS stimulation in hepatocytes.

    PubMed

    Yu, Xiaoling; Jin, Dan; Yu, An; Sun, Jun; Chen, Xiaodong; Yang, Zaiqing

    2016-09-01

    DEPTOR, a novel endogenous inhibitor of mTOR, plays an important role in regulating the inflammatory response in vascular endothelial cells (ECs) and in mouse skeletal muscle. However, the regulatory mechanism of DEPTOR transcription and its effects on liver inflammation are unknown presently. Here we reported the role of DEPTOR in regulating inflammatory response in mouse liver-derived Hepa1-6 cells and in a mouse model with LPS-induced hepatic inflammation. The results revealed that DEPTOR over-expression in Hepa1-6 liver cells increased the mRNA levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1). Contrasting results were observed in Hepa1-6 cells with DEPTOR interference. Treatment Hepa1-6 cells with rapamycin, a specific inhibitor of mTORC1, increased MCP-1 mRNA, but have no significant effect on IL-6 mRNA. DEPTOR expression was down-regulated in Hepa1-6 cells with the treatment of inflammatory stimuli LPS or the over-expression of p65/NF-κB, a key inflammatory transcription factor. NF-κB antagonist (PDTC) and inhibitor (IκBα) blocked the effect of LPS on DEPTOR expression. The study in vivo showed that DEPTOR mRNA and protein were significantly reduced in a mouse model with LPS-induced hepatic inflammation, which was accompanied by a concurrent activation of the mTOR signaling pathway. Further, the transcriptional regulation of DEPTOR was explored, which revealed that DEPTOR promoter activity was significantly down-regulated by NF-κB. The progressive deletions and mutations demonstrated that the NF-κB binding motif situated at -145/-127 region is an essential component required for the DEPTOR promoter activity. Chromatin immunoprecipitation (ChIP) assays determined that p65 can directly interact with the DEPTOR promoter DNA. Those results indicate DEPTOR regulates liver inflammation at least partially via mTORC1 pathway, and is down-regulated by LPS through p65. PMID:27179948

  6. Down-regulation of osteoprotegerin expression as a novel biomarker for colorectal carcinoma

    PubMed Central

    Kim, Hyun-Soo; Yoon, Gun; Do, Sung-Im; Kim, Sung-Joo; Kim, Youn-Wha

    2016-01-01

    A better understanding of tumor biology is important in the identification of molecules that are down-regulated in malignancy and in determining their role in tumor suppression. The aim of this study was to analyze osteoprotegerin (OPG) expression in colorectal carcinoma (CRC) and to investigate the underlying mechanism for changes in the expression of OPG. OPG expression was assessed in CRC tissue samples and cell lines. The methylation status of the OPG promoter region was determined, and the effects of demethylation on OPG expression were analyzed. The effects of recombinant OPG (rOPG) administration on cellular functions were also investigated. Clinical and prognostic implications of OPG protein expression in CRC patients were analyzed. The CRC tissues and cells showed significantly lower OPG expression. Pyrosequencing of OPG-silenced CRC cells revealed that the OPG gene promoter was highly methylated. Treatment with demethylating agent significantly elevated OPG mRNA and protein expression. rOPG significantly decreased cell viability and MMP-2 and VEGF-A production in CRC cells. Reduced OPG immunoreactivity was associated with aggressive oncogenic behavior in CRC. Also, OPG expression was found to be an independent predictor of recurrent hepatic metastasis and independent prognostic factor for worse survival rates. We demonstrated that OPG silencing in CRC occurs through epigenetic repression, and is involved in the development and progression of CRC. Our data suggest that OPG is a novel prognostic biomarker and a new therapeutic target for the treatment of patients with CRC. PMID:26942563

  7. Silkworm hemolymph down-regulates the expression of endoplasmic reticulum chaperones under radiation-irradiation.

    PubMed

    Lee, Kyeong Ryong; Kim, Seung-Whan; Kim, Young Kook; Kwon, Kisang; Choi, Jong-Soon; Yu, Kweon; Kwon, O-Yu

    2011-01-01

    We demonstrated that up-regulation of gene expression of endoplasmic reticulum (ER) chaperones (BiP, calnexin, calreticulin, ERp29) and ER membrane kinases (IRE1, PERK, ATF6) was induced by radiation in neuronal PC12 cells. However, addition of silkworm, Bombyx mori, hemolymph to irradiated cells resulted in an obvious decrease in expression of these genes, compared with a single radiation treatment. In contrast, one of the ER chaperones, "ischemia-responsive protein 94 kDa" (irp94), was up-regulated by radiation. However, addition of silkworm hemolymph resulted in no change in the expression of irp94, with an expression pattern that differed from that of ER chaperones. Based on these results, we propose that silkworm hemolymph contains factors that regulate a decrease in the expression of ER chaperones under radiation-irradiation conditions, with the exception of irp94, which is not down-regulated. We suggest that this difference in the molecular character of irp94 may provide a clue to the biological functions associated with ER stress pathways, particularly the effects of radiation. PMID:21845089

  8. Silkworm Hemolymph Down-Regulates the Expression of Endoplasmic Reticulum Chaperones under Radiation-Irradiation

    PubMed Central

    Lee, Kyeong Ryong; Kim, Seung-Whan; Kim, Young Kook; Kwon, Kisang; Choi, Jong-Soon; Yu, Kweon; Kwon, O-Yu

    2011-01-01

    We demonstrated that up-regulation of gene expression of endoplasmic reticulum (ER) chaperones (BiP, calnexin, calreticulin, ERp29) and ER membrane kinases (IRE1, PERK, ATF6) was induced by radiation in neuronal PC12 cells. However, addition of silkworm, Bombyx mori, hemolymph to irradiated cells resulted in an obvious decrease in expression of these genes, compared with a single radiation treatment. In contrast, one of the ER chaperones, “ischemia-responsive protein 94 kDa” (irp94), was up-regulated by radiation. However, addition of silkworm hemolymph resulted in no change in the expression of irp94, with an expression pattern that differed from that of ER chaperones. Based on these results, we propose that silkworm hemolymph contains factors that regulate a decrease in the expression of ER chaperones under radiation-irradiation conditions, with the exception of irp94, which is not down-regulated. We suggest that this difference in the molecular character of irp94 may provide a clue to the biological functions associated with ER stress pathways, particularly the effects of radiation. PMID:21845089

  9. Down-Regulation of CD9 Expression and its Correlation to Tumor Progression in B Lymphomas

    PubMed Central

    Yoon, Sun-Ok; Zhang, Xin; Freedman, Arnold S.; Zahrieh, David; Lossos, Izidore S.; Li, Li; Choi, Yong Sung

    2010-01-01

    Histological transformation, a pivotal event in the natural history of cancers including lymphomas, is typically associated with more aggressive clinical behavior. L3055, a B lymphoma cell line of germinal center (GC) origin, is dependent on follicular dendritic cells (FDCs) for survival and proliferation, similar to GC-B cells. However, L3055 cells become less FDC-dependent after prolonged culture, which is analogous to transformation in vivo. Comparison of two L3055 subclones (i.e., the FDC-dependent indolent clone 12 and the FDC-independent aggressive clone 33) by DNA microarray revealed that CD9 was the most differentially expressed gene (P = 0.05). L3055-12 expresses high levels of CD9 while L3055-33 does not. Reduced levels or loss of CD9 expression is also observed in other CD9-positive B lymphoma cell lines. The resultant CD9-negative cells grow faster than CD9-positive cells due to their greater resistance to apoptosis. Furthermore, CD9-negative cells are less dependent on FDCs for their survival and growth compared with CD9-positive cells. CD9 down-regulation in B lymphomas appears to be controlled epigenetically, mainly through histone modifications. These findings imply that CD9 is inversely correlated with B lymphoma progression, and CD9 inactivation may play an important role in B lymphoma transformation. PMID:20566742

  10. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    SciTech Connect

    Walsh, Erica M.; Niu, MengMeng; Bergholz, Johann; Jim Xiao, Zhi-Xiong

    2015-05-29

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

  11. Delta-Like Ligand 4 Modulates Liver Damage by Down-Regulating Chemokine Expression.

    PubMed

    Shen, Zhe; Liu, Yan; Dewidar, Bedair; Hu, Junhao; Park, Ogyi; Feng, Teng; Xu, Chengfu; Yu, Chaohui; Li, Qi; Meyer, Christoph; Ilkavets, Iryna; Müller, Alexandra; Stump-Guthier, Carolin; Munker, Stefan; Liebe, Roman; Zimmer, Vincent; Lammert, Frank; Mertens, Peter R; Li, Hai; Ten Dijke, Peter; Augustin, Hellmut G; Li, Jun; Gao, Bin; Ebert, Matthias P; Dooley, Steven; Li, Youming; Weng, Hong-Lei

    2016-07-01

    Disrupting Notch signaling ameliorates experimental liver fibrosis. However, the role of individual Notch ligands in liver damage is unknown. We investigated the effects of Delta-like ligand 4 (Dll4) in liver disease. DLL4 expression was measured in 31 human liver tissues by immunohistochemistry. Dll4 function was examined in carbon tetrachloride- and bile duct ligation-challenged mouse models in vivo and evaluated in hepatic stellate cells, hepatocytes, and Kupffer cells in vitro. DLL4 was expressed in patients' Kupffer and liver sinusoidal endothelial cells. Recombinant Dll4 protein (rDll4) ameliorated hepatocyte apoptosis, inflammation, and fibrosis in mice after carbon tetrachloride challenge. In vitro, rDll4 significantly decreased lipopolysaccharide-dependent chemokine expression in both Kupffer and hepatic stellate cells. In bile duct ligation mice, rDll4 induced massive hepatic necrosis, resulting in the death of all animals within 1 week. Inflammatory cell infiltration and chemokine ligand 2 (Ccl2) expression were significantly reduced in rDll4-receiving bile duct ligation mice. Recombinant Ccl2 rescued bile duct ligation mice from rDll4-mediated death. In patients with acute-on-chronic liver failure, DLL4 expression was inversely associated with CCL2 abundance. Mechanistically, Dll4 regulated Ccl2 expression via NF-κB. Taken together, Dll4 modulates liver inflammatory response by down-regulating chemokine expression. rDll4 application results in opposing outcomes in two models of liver damage. Loss of DLL4 may be associated with CCL2-mediated cytokine storm in patients with acute-on-chronic liver failure. PMID:27171900

  12. MicroRNA-21 Down-regulates Rb1 Expression by Targeting PDCD4 in Retinoblastoma

    PubMed Central

    Shen, Fengmei; Mo, Meng-Hsuan; Chen, Liang; An, Shejuan; Tan, Xiaohui; Fu, Yebo; Rezaei, Katayoon; Wang, Zuoren; Zhang, Lin; Fu, Sidney W.

    2014-01-01

    Retinoblastoma (RB) is a children's ocular cancer caused by mutated retinoblastoma 1 (Rb1) gene on both alleles. Rb1 and other related genes could be regulated by microRNAs (miRNA) via complementarily pairing with their target sites. MicroRNA-21 (miR-21) possesses the oncogenic potential to target several tumor suppressor genes, including PDCD4, and regulates tumor progression and metastasis. However, the mechanism of how miR-21 regulates PDCD4 is poorly understood in RB. We investigated the expression of miRNAs in RB cell lines and identified that miR-21 is one of the most deregulated miRNAs in RB. Using qRT-PCR, we verified the expression level of several miRNAs identified by independent microarray assays, and analyzed miRNA expression patterns in three RB cell lines, including Weri-Rb1, Y79 and RB355. We found that miR-19b, -21, -26a, -195 and -222 were highly expressed in all three cell lines, suggesting their potential role in RB tumorigenesis. Using the TargetScan program, we identified a list of potential target genes of these miRNAs, of which PDCD4 is one the targets of miR-21. In this study, we focused on the regulatory mechanism of miR-21 on PDCD4 in RB. We demonstrated an inverse correlation between miR-21 and PDCD4 expression in Weri-Rb1 and Y79 cells. These data suggest that miR-21 down-regulates Rb1 by targeting PDCD4 tumor suppressor. Therefore, miR-21 could serve as a therapeutic target for retinoblastoma. PMID:25520758

  13. Dissociable Roles for the Ventromedial Prefrontal Cortex and Amygdala in Fear Extinction: NR2B Contribution

    PubMed Central

    Diaz-Mataix, Llorenç; Bush, David E.A.; LeDoux, Joseph E.

    2009-01-01

    Fear extinction, which involves learning to suppress the expression of previously learned fear, requires N-methyl-D-aspartate receptors (NMDARs) and is mediated by the amygdala and ventromedial prefrontal cortex (vmPFC). Like other types of learning, extinction involves acquisition and consolidation phases. We recently demonstrated that NR2B-containing NMDARs (NR2Bs) in the lateral amygdala (LA) are required for extinction acquisition, but whether they are involved in consolidation is not known. Further, although it has been shown that NMDARs in the vmPFC are required for extinction consolidation, whether NR2Bs in vmPFC are involved in consolidation is not known. In this report, we investigated the possible role of LA and vmPFC NR2Bs in the consolidation of fear extinction using the NR2B-selective antagonist ifenprodil. We show that systemic treatment with ifenprodil immediately after extinction training disrupts extinction consolidation. Ifenprodil infusion into vmPFC, but not the LA, immediately after extinction training also disrupts extinction consolidation. In contrast, we also show pre-extinction training infusions into vmPFC has no effect. These results, together with our previous findings showing that LA NR2Bs are required during the acquisition phase in extinction, indicate a double dissociation for the phase-dependent role of NR2Bs in the LA (acquisition, not consolidation) and vmPFC (consolidation, not acquisition). PMID:18562331

  14. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    NASA Astrophysics Data System (ADS)

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  15. Galectin-3 down-regulates IL-5 gene expression on different cell types.

    PubMed

    Cortegano, I; del Pozo, V; Cárdaba, B; de Andrés, B; Gallardo, S; del Amo, A; Arrieta, I; Jurado, A; Palomino, P; Liu, F T; Lahoz, C

    1998-07-01

    Galectin-3 is an animal lectin, formerly named epsilon-binding protein or Mac-2, which has been described to play an important role in some inflammatory processes by the implication of different cells and the increase in cell adhesion functions through laminin binding activity. In this work we analyzed the role of galectin-3 in the modulation of Th2 cytokines that have an important role in the development of the inflammatory response. We have found that the addition of galectin-3 to human eosinophils, the eosinophilic cell line EoL-3, PBMC, and an Ag-specific T cell line (CD4+) produced a selective inhibition of IL-5 transcription. No inhibitory effect was found on the IL-4 mRNA transcription rate. The inhibitory effect on IL-5 transcription was reversed by incubation with lactose and using specific Ab against galectin-3. Galectin-3 is able to induce inhibition of the IL-5 released in the supernatants from PBMC stimulated with phorbol 12,13-dibutyrate and anti-CD3. Similar results were obtained when a T-specific cell line was stimulated with Ag. Also, EoL-3 stimulated with anti-CD32 produced IL-5 protein, the synthesis of which was partially inhibited by galectin-3. The present results demonstrate that galectin-3 induces a selective down-regulation of IL-5 expression in different cell types, opening important new possibilities in the regulation of the allergic reactions. PMID:9647247

  16. Functional contributions of synaptically localized NR2B subunits of the NMDA receptor to synaptic transmission and long-term potentiation in the adult mouse CNS

    PubMed Central

    Miwa, Hideki; Fukaya, Masahiro; Watabe, Ayako M; Watanabe, Masahiko; Manabe, Toshiya

    2008-01-01

    The NMDA-type glutamate receptor is a heteromeric complex composed of the NR1 and at least one of the NR2 subunits. Switching from the NR2B to the NR2A subunit is thought to underlie functional alteration of the NMDA receptor during synaptic maturation, and it is generally believed that it results in preferential localization of NR2A subunits on the synaptic site and that of NR2B subunits on the extracellular site in the mature brain. It has also been proposed that activation of the NR2A and NR2B subunits results in long-term potentiation (LTP) and long-term depression (LTD), respectively. Furthermore, recent reports suggest that synaptic and extrasynaptic receptors may have distinct roles in synaptic plasticity as well as in gene expression associated with neuronal death. Here, we have investigated whether NR2B subunit-containing receptors are present and functional at mature synapses in the lateral nucleus of the amygdala (LA) and the CA1 region of the hippocampus, comparing their properties between the two brain regions. We have found, in contrast to the above hypotheses, that the NR2B subunit significantly contributes to synaptic transmission as well as LTP induction. Furthermore, its contribution is greater in the LA than in the CA1 region, and biophysical properties of NMDA receptors and the NR2B/NR2A ratio are different between the two brain regions. These results indicate that NR2B subunit-containing NMDA receptors accumulate on the synaptic site and are responsible for the unique properties of synaptic function and plasticity in the amygdala. PMID:18372311

  17. Age- and Hormone-Regulation of N-Methyl-d-Aspartate Receptor Subunit NR2b in the Anteroventral Periventricular Nucleus of the Female Rat

    PubMed Central

    Maffucci, J. A.; Noel, M. L.; Gillette, R.; Wu, D.; Gore, A. C.

    2009-01-01

    Glutamate, acting through its N-methyl-d-aspartate (NMDA) and non-NMDA receptors in the hypothalamus, regulates reproductive neuroendocrine functions via direct and indirect actions upon gonadotrophin-releasing hormone (GnRH) neurones. Previous studies indicate that the NMDA receptor subunit NR2b undergoes changes in protein and gene expression in the hypothalamus in general, and on GnRH neurones in particular, during reproductive ageing. In the present study, we examined whether the NR2b-expressing cell population, both alone and in association with the NR1 subunit (i.e. the latter subunit is necessary for a functional NMDA receptor), is altered as a function of age and/or steroid hormone treatment. Studies focused on the anteroventral periventricular (AVPV) nucleus of the hypothalamus, a region critically involved in the control of reproduction. Young (3-5 months), middle-aged (9-12 months), and aged (approximately 22 months) female rats were ovariectomised and, 1 month later, they were treated sequentially with oestradiol plus progesterone, oestradiol plus vehicle, or vehicle plus vehicle, then perfused. Quantitative stereologic analysis of NR2b-immunoreactive cell numbers in the AVPV showed an age-associated decrease in the density of NR2b-immunoreactive cells, but no effect of hormone treatment. In a second study, immunofluorescent double labelling of NR2b and NR1 was analysed by confocal microscopy of fraction volume, a semi-quantitative measure of fluorescence intensity. No effect of ageing was detected for immunofluorescent NR1 or NR2b alone, whereas the NR2b fraction volume increased in the oestradiol plus vehicle group. With ageing, the fraction volume of the NR2b/NR1-colocalised subunits increased. Together with the stereology results, this suggests that, although fewer cells express the NR2b subunit in the ageing AVPV, a greater percentage of these subunits are co-expressed with NR1. Our results suggest that the subunit composition of NMDA receptors in

  18. FOXL2 down-regulates vitellogenin expression at mature stage in Eriocheir sinensis

    PubMed Central

    Li, Qing; Xie, Jing; He, Lin; Wang, Yuanli; Yang, Hongdan; Duan, Zelin; Wang, Qun

    2015-01-01

    Ovarian development in crustaceans is characterized by rapid production of egg yolk protein in a process called vitellogenesis. In the present study, we investigated the involvement of a DEAD (Asp-Glu-Ala-Asp) box RNA helicase 20 (DDX20), forkhead transcription factor (FOXL)2 and fushi tarazu factor (FTZ-F)1 in the regulation of vitellogenesis. Based on ESTs from the testis and accessory gland of Eriocheir sinensis, we cloned the full-length cDNAs of foxl2 and fushitarazu factor 1 (ftz-f1), which include the conserved structural features of the forkhead family and nuclear receptor 5A (NR5A) family respectively. The expression of foxl2 mRNA surged at the mature stage of the ovary, when vtg mRNA swooped, suggesting that foxl2 negatively affects the vitellogenin (VTG) synthesis at this developmental stage. Etoposide (inducing germ cell apoptosis) treatment up-regulated FOXL2 and DDX20 at both the mRNA and the protein levels, primarily in the follicular cells as shown by immunofluorescence analysis. Furthermore, foxl2, ddx20 and ftz-f1 mRNA levels increased significantly with right-eyestalk ablation. Interactions between FOXL2 and DDX20 or FTZ-F1 were confirmed by co-immunoprecipitation and the forkhead domain of FOXL2 was identified as the specific structure interacting with FTZ-F1. In conclusion, FOXL2 down-regulates VTG expression by binding with DDX20 in regulation of follicular cell apoptosis and with FTZ-F1 to repress the synthesis of VTG at the mature stage. This report is the first to describe the molecular mechanism of VTG synthesis in E. sinensis and may shed new light on the regulation of cytochrome P450 enzyme by FOXL2 and FTZ-F1 in vitellogenesis. PMID:26430246

  19. FOXL2 down-regulates vitellogenin expression at mature stage in Eriocheir sinensis.

    PubMed

    Li, Qing; Xie, Jing; He, Lin; Wang, Yuanli; Yang, Hongdan; Duan, Zelin; Wang, Qun

    2015-01-01

    Ovarian development in crustaceans is characterized by rapid production of egg yolk protein in a process called vitellogenesis. In the present study, we investigated the involvement of a DEAD (Asp-Glu-Ala-Asp) box RNA helicase 20 (DDX20), forkhead transcription factor (FOXL)2 and fushi tarazu factor (FTZ-F)1 in the regulation of vitellogenesis. Based on ESTs from the testis and accessory gland of Eriocheir sinensis, we cloned the full-length cDNAs of foxl2 and fushitarazu factor 1 (ftz-f1), which include the conserved structural features of the forkhead family and nuclear receptor 5A (NR5A) family respectively. The expression of foxl2 mRNA surged at the mature stage of the ovary, when vtg mRNA swooped, suggesting that foxl2 negatively affects the vitellogenin (VTG) synthesis at this developmental stage. Etoposide (inducing germ cell apoptosis) treatment up-regulated FOXL2 and DDX20 at both the mRNA and the protein levels, primarily in the follicular cells as shown by immunofluorescence analysis. Furthermore, foxl2, ddx20 and ftz-f1 mRNA levels increased significantly with right-eyestalk ablation. Interactions between FOXL2 and DDX20 or FTZ-F1 were confirmed by co-immunoprecipitation and the forkhead domain of FOXL2 was identified as the specific structure interacting with FTZ-F1. In conclusion, FOXL2 down-regulates VTG expression by binding with DDX20 in regulation of follicular cell apoptosis and with FTZ-F1 to repress the synthesis of VTG at the mature stage. This report is the first to describe the molecular mechanism of VTG synthesis in E. sinensis and may shed new light on the regulation of cytochrome P450 enzyme by FOXL2 and FTZ-F1 in vitellogenesis. PMID:26430246

  20. Prostaglandin E2 mediates growth arrest in NFS-60 cells by down-regulating interleukin-6 receptor expression.

    PubMed Central

    de Silva, Kumudika I; Daud, Asif N; Deng, JiangPing; Jones, Stephen B; Gamelli, Richard L; Shankar, Ravi

    2003-01-01

    Interleukin-6 (IL-6), a potent myeloid mitogen, and the immunosuppressive prostanoid prostaglandin E2 (PGE2) are elevated following thermal injury and sepsis. We have previously demonstrated that bone marrow myeloid commitment shifts toward monocytopoiesis and away from granulocytopoiesis during thermal injury and sepsis and that PGE2 plays a central role in this alteration. Here we investigated whether PGE2 can modulate IL-6-stimulated growth in the promyelocytic cell line, NFS-60, by down-regulating IL-6 receptor (IL-6r) expression. Exposure of NFS-60 cells to PGE2 suppressed IL-6-stimulated proliferation as well as IL-6r expression. Receptor down-regulation is functionally significant since IL-6-induced signal transduction through activators of transcription (STAT)-3 is also decreased. Down-regulation of IL-6r correlated with the ability of PGE2 to arrest cells in the G0/G1 phase of the cell cycle. PGE2 appears to signal through EP2 receptors. Butaprost (EP2 agonist) but not sulprostone (EP3 agonist) inhibited IL-6-stimulated proliferation. In addition, an EP2 antagonist (AH6809) alleviated the anti-proliferative effects of PGE2. NFS-60 cells express predominantly EP2 and EP4 receptors. While PGE2 down-regulated both the IL-6r protein and mRNA expression, it had no influence on EP2 or EP4 mRNA expression. The present study demonstrates that PGE2 is a potent down-regulator of IL-6r expression and thus may provide a mechanistic explanation for the granulocytopenia seen in thermal injury and sepsis. PMID:12429018

  1. Phosphorylation of NR2B NMDA subunits by protein kinase C in arcuate nucleus contributes to inflammatory pain in rats

    PubMed Central

    Bu, Fan; Tian, Huiyu; Gong, Shan; Zhu, Qi; Xu, Guang-Yin; Tao, Jin; Jiang, Xinghong

    2015-01-01

    The arcuate nucleus (ARC) of the hypothalamus plays a key role in pain processing. Although it is well known that inhibition of NMDA receptor (NMDAR) in ARC attenuates hyperalgesia induced by peripheral inflammation, the underlying mechanism of NMDAR activation in ARC remains unclear. Protein kinase C (PKC) is involved in several signalling cascades activated in physiological and pathological conditions. Therefore, we hypothesised that upregulation of PKC activates NMDARs in the ARC, thus contributing to inflammatory hyperalgesia. Intra-ARC injection of chelerythrine (CC), a specific PKC inhibitor, attenuated complete Freund’s adjuvant (CFA) induced thermal and mechanical hyperalgesia in a dose-dependent manner. In vivo extracellular recordings showed that microelectrophoresis of CC or MK-801 (a NMDAR antagonist) significantly reduced the enhancement of spontaneous discharges and pain-evoked discharges of ARC neurons. In addition, CFA injection greatly enhanced the expression of total and phosphorylated PKCγ in the ARC. Interestingly, CFA injection also remarkably elevated the level of phosphorylated NR2B (Tyr1472) without affecting the expression of total NR2B. Importantly, intra-ARC injection of CC reversed the upregulation of phosphorylated NR2B subunits in the ARC. Taken together, peripheral inflammation leads to an activation of NMDARs mediated by PKC activation in the ARC, thus producing thermal and mechanical hyperalgesia. PMID:26515544

  2. Dexamethasone down-regulates the expression of microRNA-155 in the livers of septic mice.

    PubMed

    Wang, Zhong-hua; Liang, Yan-bing; Tang, Hao; Chen, Zhi-bin; Li, Zhen-yu; Hu, Xu-chu; Ma, Zhong-fu

    2013-01-01

    To investigate the expression of microRNA-155 (miRNA-155) in the livers of mice with lipopolysaccharide (LPS)-induced sepsis and to determine the role of dexamethasone (DXM) in the regulation of miRNA-155 expression, we pretreated mice with or without DXM prior to LPS exposure. Our study demonstrated that the expression of miRNA-155 and inflammatory factors increased in the liver tissues of mice with LPS-induced sepsis and that DXM down-regulated their expression in a dose-dependent manner. Moreover, DXM alone inhibited the expression of miRNA-155 to below the baseline level, but did not impact the expression of inflammatory factors, suggesting that the down-regulation of miRNA-155 by DXM may partially, but not completely, depend on the suppression of pro-inflammatory cytokines by DXM. Our data indicate that the overexpression of miRNA-155 in the livers of mice with LPS-induced sepsis may play an important role in the pathological processes of sepsis and that the down-regulation of miRNA-155 by DXM may be a novel mechanism regulating inflammation and immunity. PMID:24244697

  3. Sesamin, a lignan of sesame, down-regulates cyclin D1 protein expression in human tumor cells.

    PubMed

    Yokota, Tomoya; Matsuzaki, Youichirou; Koyama, Makoto; Hitomi, Toshiaki; Kawanaka, Mayumi; Enoki-Konishi, Masako; Okuyama, Yusuke; Takayasu, Junko; Nishino, Hoyoku; Nishikawa, Akiyoshi; Osawa, Toshihiko; Sakai, Toshiyuki

    2007-09-01

    Sesamin is a major lignan constituent of sesame and possesses multiple functions such as antihypertensive, cholesterol-lowering, lipid-lowering and anticancer activities. Several groups have previously reported that sesamin induces growth inhibition in human cancer cells. However, the nature of this growth inhibitory mechanism remains unknown. The authors here report that sesamin induces growth arrest at the G1 phase in cell cycle progression in the human breast cancer cell line MCF-7. Furthermore, sesamin dephosphorylates tumor-suppressor retinoblastoma protein (RB). It is also shown that inhibition of MCF-7 cell proliferation by sesamin is correlated with down-regulated cyclin D1 protein expression, a proto-oncogene that is overexpressed in many human cancer cells. It was found that sesamin-induced down-regulation of cyclin D1 was inhibited by proteasome inhibitors, suggesting that sesamin suppresses cyclin D1 protein expression by promoting proteasome degradation of cyclin D1 protein. Sesamin down-regulates cyclin D1 protein expression in various kinds of human tumor cells, including lung cancer, transformed renal cells, immortalized keratinocyte, melanoma and osteosarcoma. Furthermore, depletion of cyclin D1 protein using small interfering RNA rendered MCF-7 cells insensitive to the growth inhibitory effects of sesamin, implicating that cyclin D1 is at least partially related to the antiproliferative effects of sesamin. Taken together, these results suggest that the ability of sesamin to down-regulate cyclin D1 protein expression through the activation of proteasome degradation could be one of the mechanisms of the antiproliferative activity of this agent. PMID:17640297

  4. HMGA1 down-regulation is crucial for chromatin composition and a gene expression profile permitting myogenic differentiation

    PubMed Central

    2010-01-01

    Background High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation. Results We found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable over-expression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1, Igf2, Igfbp1-3 or up-regulation of the transcriptional repressor Msx1. Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells. Conclusions Our data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin

  5. Cryptotanshinone targets tumor-initiating cells through down-regulation of stemness genes expression

    PubMed Central

    ZHANG, YING; CABARCAS, STEPHANIE M.; ZHENG, JI; SUN, LEI; MATHEWS, LESLEY A.; ZHANG, XIAOHU; LIN, HONGSHENG; FARRAR, WILLIAM L.

    2016-01-01

    Recent evidence indicates that tumor-initiating cells (TICs), also called cancer stem cells (CSCs), are responsible for tumor initiation and progression, therefore representing an important cell population that may be used as a target for the development of future anticancer therapies. In the present study, Cryptotanshinone (CT), a traditional Chinese herbal medicine, was demonstrated to regulate the behaviors of LNCaP prostate cells and prostate LNCaP TICs. The results demonstrate that treatment with CT alters cellular proliferation, cell cycle status, migration, viability, colony formation and notably, sphere formation and down-regulation of stemness genes (Nanog, OCT4, SOX2, β-catenin, CXCR4) in TICs. The present study demonstrates that CT targets the LNCaP CD44+CD24- population that is representative of prostate TICs and also affects total LNCaP cells as well via down-regulation of stemness genes. The strong effect with which CT has on prostate TICs suggests that CT may potentially function as a novel natural anticancer agent that specifically targets TICs. PMID:27313698

  6. Kynurenic acid amides as novel NR2B selective NMDA receptor antagonists.

    PubMed

    Borza, István; Kolok, Sándor; Galgóczy, Kornél; Gere, Anikó; Horváth, Csilla; Farkas, Sándor; Greiner, István; Domány, György

    2007-01-15

    A novel series of kynurenic acid amides, ring-enlarged derivatives of indole-2-carboxamides, was prepared and identified as in vivo active NR2B subtype selective NMDA receptor antagonists. The synthesis and SAR studies are discussed. PMID:17074483

  7. Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression.

    PubMed

    Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong; Khanal, Tilak; Choi, Jae Ho; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-10-01

    Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 and CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. PMID:25110054

  8. DAPK1 Interaction with NMDA Receptor NR2B Subunits Mediates Brain Damage in Stroke

    PubMed Central

    Tu, Weihong; Xu, Xin; Peng, Lisheng; Zhong, Xiaofen; Zhang, Wenfeng; Soundarapandian, Mangala M.; Balel, Cherine; Wang, Manqi; Jia, Nali; Zhang, Wen; Lew, Frank; Chan, Sic Lung; Chen, Yanfang; Lu, Youming

    2010-01-01

    SUMMARY N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extra-synaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292–1304 (NR2BCT). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2BCT that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca2+ influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extra-synaptic sites and this interaction acts as a central mediator for stroke damage. PMID:20141836

  9. Glucocorticoids down-regulate beta 1-adrenergic-receptor expression by suppressing transcription of the receptor gene.

    PubMed Central

    Kiely, J; Hadcock, J R; Bahouth, S W; Malbon, C C

    1994-01-01

    The expression of beta 2-adrenergic receptors is up-regulated by glucocorticoids. In contrast, beta 1-adrenergic receptors display glucocorticoid-induced down-regulation. In rat C6 glioma cells, which express both of these subtypes of beta-adrenergic receptors, the synthetic glucocorticoid dexamethasone stimulates no change in the total beta-adrenergic receptor content, but rather shifts the beta 1:beta 2 ratio from 80:20 to 50:50. Radioligand binding and immunoblotting demonstrate a sharp decline in beta 1-adrenergic receptor expression. Metabolic labelling of cells with [35S]-methionine in tandem with immunoprecipitation by beta 1-adrenergic-receptor-specific antibodies reveals a sharp decline in the synthesis of the receptor within 48 h for cells challenged with glucocorticoid. Steady-state levels of beta 1-adrenergic-receptor mRNA declined from 0.47 to 0.26 amol/microgram of total cellular RNA within 2 h of dexamethasone challenge, as measured by DNA-excess solution hybridization. The stability of receptor mRNA was not influenced by glucocorticoid; the half-lives of the beta 1- and beta 2-subtype mRNAs were 1.7 and 1.5 h respectively. Nuclear run-on assays revealed the basis for the down-regulation of receptor expression, i.e. a sharp decline in the relative rate of transcription for the beta 1-adrenergic-receptor gene in nuclei from dexamethasone-treated as compared with vehicle-treated cells. These data demonstrate transcriptional suppression as a molecular explanation for glucocorticoid-induced down-regulation of beta 1-adrenergic receptors. Images Figure 1 Figure 2 Figure 6 PMID:8092990

  10. Endogenous ephrinB2 mediates colon-urethra cross-organ sensitization via Src kinase-dependent tyrosine phosphorylation of NR2B.

    PubMed

    Peng, Hsien-Yu; Chen, Gin-Den; Lai, Cheng-Hung; Tung, Kwong-Chung; Chang, Junn-Liang; Lin, Tzer-Bin

    2010-01-01

    Recently, the role of EphB receptor (EphBR) tyrosine kinase and their ephrinB ligands in spinal pain-related neural plasticity has been identified. To test whether Src-family non-receptor tyrosine kinase-dependent glutamatergic N-methyl-d-aspartate receptor (NMDAR) NR2B subunit phosphorylation underlies lumbosacral spinal EphBR activation to mediate cross-organ sensitization between the colon and the urethra, external urethra sphincter electromyogram activity evoked by pelvic nerve stimulation and protein expression in the lumbosacral (L6-S2) dorsal horn were studied before and after intracolonic mustard oil (MO) instillation. We found MO instillation produced colon-urethra reflex sensitization along with an upregulation of endogenous ephrinB2 expression as well as phosphorylation of EphB 1/2, Src-family kinase, and NR2B tyrosine residues. Intrathecal immunoglobulin fusion protein of EphB1 and EphB2 as well as PP2 reversed the reflex sensitization and NR2B phosphorylation caused by MO. All these results suggest that EphBR-ephrinB interactions, which provoke Src-family kinase-dependent NMDAR NR2B phosphorylation at the lumbosacral spinal cord level, are involved in cross-organ sensitization, contributing to the development of viscero-visceral referred pain between the bowel and the urethra. PMID:19864302

  11. Acute hypoxia differentially affects the NMDA receptor NR1, NR2A and NR2B subunit mRNA levels in the developing chick optic tectum: stage-dependent plasticity in the 2B-2A ratio.

    PubMed

    Vacotto, Marina; Rapacioli, Melina; Flores, Vladimir; de Plazas, Sara Fiszer

    2010-10-01

    It is known that the NMDA-R NR1 subunit is needed for the receptor activity and that under hypoxia the evolution toward apoptosis or neuronal survival depends on the balance NR2A/NR2B subunits. This paper analyzes the effect of acute hypoxia on the above mentioned subunits mRNAs during development. The mean percentage of NR1+ neurons displayed the higher plasticity during development while the NR2A+ neurons the higher stability. Acute hypoxia increased the mean percentage of NR1+ and NR2B+ neurons at ED12 but only that of NR1+ neurons at ED18. Acute hypoxia increased the levels of expression of NR1 and NR2B mRNAs at ED12 without changes in the NR2A mRNA. During early stages there is a higher sensitivity to change the subunits mRNA levels under a hypoxic treatment. At ED12 acute hypoxia increased the probability of co-expression of the NR1-NR2A and NR1-NR2B subunits combinations, the level of NR1 and NR2B and the ratio NR2B/NR2A. These conditions facilitate the evolution towards apoptosis. PMID:20596770

  12. Mifepristone Suppresses Basal Triple-Negative Breast Cancer Stem Cells by Down-regulating KLF5 Expression

    PubMed Central

    Liu, Rong; Shi, Peiguo; Nie, Zhi; Liang, Huichun; Zhou, Zhongmei; Chen, Wenlin; Chen, Haijun; Dong, Chao; Yang, Runxiang; Liu, Suling; Chen, Ceshi

    2016-01-01

    Triple-negative breast cancer (TNBC) is currently the most malignant subtype of breast cancers without effective targeted therapies. Mifepristone (MIF), a drug regularly used for abortion, has been reported to have anti-tumor activity in multiple hormone-dependent cancers, including luminal type breast cancers. In this study, we showed that MIF suppressed tumor growth of the TNBC cell lines and patient-derived xenografts in NOD-SCID mice. Furthermore, MIF reduced the TNBC cancer stem cell (CSC) population through down-regulating KLF5 expression, a stem cell transcription factor over-expressed in basal type TNBC and promoting cell proliferation, survival and stemness. Interestingly, MIF suppresses the expression of KLF5 through inducing the expression of miR-153. Consistently, miR-153 decreases CSC and miR-153 inhibitor rescued MIF-induced down-regulation of the KLF5 protein level and CSC ratio. Taken together, our findings suggest that MIF inhibits basal TNBC via the miR-153/KLF5 axis and MIF may be used for the treatment of TNBC. PMID:26941846

  13. Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

    SciTech Connect

    Xu Xiaohong Ye Yinping; Li Tao; Chen Lei; Tian Dong; Luo Qingqing; Lu Mei

    2010-12-01

    Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) {beta} and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs. The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ER{beta} were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17{beta}-estradiol (17{beta}-E{sub 2}) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17{beta}-E{sub 2}. The present results suggest that BPA, like 17{beta}-E{sub 2}, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17{beta}-E{sub 2} when it coexists with 17{beta}-E{sub 2}. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.

  14. Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

    SciTech Connect

    Kim, Sung Young; Ryu, Sung Jin; Ahn, Hong Ju; Choi, Hae Ri; Kang, Hyun Tae; Park, Sang Chul

    2010-01-01

    One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin {alpha}, karyopherin {beta}, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.

  15. Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression

    SciTech Connect

    Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong; Khanal, Tilak; Choi, Jae Ho; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-10-01

    Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 and CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO expression.

  16. Induction of somatic embryos in Arabidopsis requires local YUCCA expression mediated by the down-regulation of ethylene biosynthesis.

    PubMed

    Bai, Bo; Su, Ying Hua; Yuan, Jia; Zhang, Xian Sheng

    2013-07-01

    Somatic embryogenesis is an important experimental model for studying cellular and molecular mechanisms of early embryo development. Although it has long been known that removal of exogenous auxin from medium results in somatic embryogenesis, the mechanisms underlying the initiation of somatic embryos (SEs) are poorly understood. In this study, we showed that YUCCAs (YUCs) encoding key enzymes in auxin biosynthesis are required for SE induction in Arabidopsis. To identify other factors mediating SE initiation, we performed transcriptional profiling and gene expression analysis. The results showed that genes involved in ethylene biosynthesis and its responses were down-regulated during SE initiation. Ethylene level decreased progressively during SE initiation, whereas treatment with the metabolic precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), or mutation of ETHYLENE-OVERPRODUCTION1 (ETO1) disrupted SE induction, suggesting that ethylene plays a role in this process. Suppression of SE induction was also observed in the constitutive triple response 1 (ctr1) mutant, in which ethylene signaling was enhanced. These results indicate that down-regulation of not only ethylene biosynthesis, but also ethylene response is critical for SE induction. We further showed that ethylene disturbed SE initiation through inhibiting YUC expression that might be involved in local auxin biosynthesis and subsequent auxin distribution. Our results provide new information on the mechanisms of hormone-regulated SE initiation. PMID:23271028

  17. Expression of SPARC like protein 1 (SPARCL1), extracellular matrix-associated protein is down regulated in gastric adenocarcinoma

    PubMed Central

    Jakharia, Aniruddha; Borkakoty, Biswajyoti

    2016-01-01

    Background SPARC-like protein 1 (SPARCL1/Hevin), a member of the SPARC family is defined by the presence of a highly acidic domain-I, a follistatin-like domain, and an extracellular calcium (EC) binding domain. SPARCL1 has been shown to be down-regulated in many types of cancer and may serve as a negative regulator of cell growth and proliferation. Methods Both tumor and adjacent normal tissue were collected from patients with gastric adenocarcinoma. Monoclonal antibody developed against recombinant SPARCL1 was used to analyze the expression of SPARCL1 by immunohisto chemical and western blotting (WB) analysis. Results The expression of SPARCL1 was found to be significantly lower or negligible in gastric adenocarcinoma tissues in nearly all of the cases in comparison with adjacent normal tissue. This comparison was found to be independent of the patient’s age, sex, and stage of cancer. Conclusions We postulate that down regulation of SPARCL1 may be related to inactivation of its tumor suppressor functions and might play an important role in the development of gastric adenocarcinoma. PMID:27034797

  18. Pamidronate Down-regulates Tumor Necrosis Factor-alpha Induced Matrix Metalloproteinases Expression in Human Intervertebral Disc Cells

    PubMed Central

    Kang, Young-Mi; Hong, Seong-Hwan; Yang, Jae-Ho; Oh, Jin-Cheol; Park, Jin-Oh; Lee, Byung Ho; Lee, Sang-Yoon; Kim, Hak-Sun; Lee, Hwan-Mo

    2016-01-01

    Background N-containing bisphosphonates (BPs), such as pamidronate and risedronate, can inhibit osteoclastic function and reduce osteoclast number by inducing apoptotic cell death in osteoclasts. The aim of this study is to demonstrate the effect of pamidronate, second generation nitrogen-containing BPs and to elucidate matrix metallo-proteinases (MMPs) mRNA expression under serum starvation and/or tumor necrosis factor alpha (TNF-α) stimulation on metabolism of intervertebral disc (IVD) cells in vitro. Methods Firstly, to test the effect of pamidronate on IVD cells in vitro, various concentrations (10-12, 10-10, 10-8, and 10-6 M) of pamidronate were administered to IVD cells. Then DNA and proteoglycan synthesis were measured and messenger RNA (mRNA) expressions of type I collagen, type II collagen, and aggrecan were analyzed. Secondly, to elucidate the expression of MMPs mRNA in human IVD cells under the lower serum status, IVD cells were cultivated in full serum or 1% serum. Thirdly, to elucidate the expression of MMPs mRNA in IVD cells under the stimulation of 1% serum and TNF-α (10 ng/mL) In this study, IVD cells were cultivated in three dimensional alginate bead. Results Under the lower serum culture, IVD cells in alginate beads showed upregulation of MMP 2, 3, 9, 13 mRNA. The cells in lower serum and TNF-α also demonstrated upregulation of MMP-2, 3, 9, and 13 mRNA. The cells with various doses of pamidronate and lower serum and TNF-α were reveled partial down-regulation of MMPs. Conclusions Pamidronate, N-containing second generation BPs, was safe in metabolism of IVD in vitro maintaining chondrogenic phenotype and matrix synthesis, and down-regulated TNF-α induced MMPs expression. PMID:27622181

  19. Triptolide inhibits the proliferation of prostate cancer cells and down-regulates SUMO-specific protease 1 expression.

    PubMed

    Huang, Weiwei; He, Tiantian; Chai, Chengsen; Yang, Yuan; Zheng, Yahong; Zhou, Pei; Qiao, Xiaoxia; Zhang, Bin; Liu, Zengzhen; Wang, Junru; Shi, Changhong; Lei, Liping; Gao, Kun; Li, Hewei; Zhong, Sue; Yao, Libo; Huang, Meng-Er; Lei, Ming

    2012-01-01

    Recently, traditional Chinese medicine and medicinal herbs have attracted more attentions worldwide for its anti-tumor efficacy. Celastrol and Triptolide, two active components extracted from the Chinese herb Tripterygium wilfordii Hook F (known as Lei Gong Teng or Thunder of God Vine), have shown anti-tumor effects. Celastrol was identified as a natural 26 s proteasome inhibitor which promotes cell apoptosis and inhibits tumor growth. The effect and mechanism of Triptolide on prostate cancer (PCa) is not well studied. Here we demonstrated that Triptolide, more potent than Celastrol, inhibited cell growth and induced cell death in LNCaP and PC-3 cell lines. Triptolide also significantly inhibited the xenografted PC-3 tumor growth in nude mice. Moreover, Triptolide induced PCa cell apoptosis through caspases activation and PARP cleavage. Unbalance between SUMOylation and deSUMOylation was reported to play an important role in PCa progression. SUMO-specific protease 1 (SENP1) was thought to be a potential marker and therapeutical target of PCa. Importantly, we observed that Triptolide down-regulated SENP1 expression in both mRNA and protein levels in dose-dependent and time-dependent manners, resulting in an enhanced cellular SUMOylation in PCa cells. Meanwhile, Triptolide decreased AR and c-Jun expression at similar manners, and suppressed AR and c-Jun transcription activity. Furthermore, knockdown or ectopic SENP1, c-Jun and AR expression in PCa cells inhibited the Triptolide anti-PCa effects. Taken together, our data suggest that Triptolide is a natural compound with potential therapeutic value for PCa. Its anti-tumor activity may be attributed to mechanisms involving down-regulation of SENP1 that restores SUMOylation and deSUMOyaltion balance and negative regulation of AR and c-Jun expression that inhibits the AR and c-Jun mediated transcription in PCa. PMID:22666381

  20. STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide.

    PubMed

    Starzynski, Rafal Radoslaw; Gonçalves, Ana Sofia; Muzeau, Françoise; Tyrolczyk, Zofia; Smuda, Ewa; Drapier, Jean-Claude; Beaumont, Carole; Lipinski, Pawel

    2006-12-01

    RNA-binding activity of IRP1 (iron regulatory protein 1) is regulated by the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule. NO (nitic oxide), whose ability to activate IRP1 by removing its [4Fe-4S] cluster is well known, has also been shown to down-regulate expression of the IRP1 gene. In the present study, we examine whether this regulation occurs at the transcriptional level. Analysis of the mouse IRP1 promoter sequence revealed two conserved putative binding sites for transcription factor(s) regulated by NO and/or changes in intracellular iron level: Sp1 (promoter-selective transcription factor 1) and MTF1 (metal transcription factor 1), plus GAS (interferon-gamma-activated sequence), a binding site for STAT (signal transducer and activator of transcription) proteins. In order to define the functional activity of these sequences, reporter constructs were generated through the insertion of overlapping fragments of the mouse IRP1 promoter upstream of the luciferase gene. Transient expression assays following transfection of HuH7 cells with these plasmids revealed that while both the Sp1 and GAS sequences are involved in basal transcriptional activity of the IRP1 promoter, the role of the latter is predominant. Analysis of protein binding to these sequences in EMSAs (electrophoretic mobility-shift assays) using nuclear extracts from mouse RAW 264.7 macrophages stimulated to synthesize NO showed a significant decrease in the formation of Sp1-DNA and STAT-DNA complexes, compared with controls. We have also demonstrated that the GAS sequence is involved in NO-dependent down-regulation of IRP1 transcription. Further analysis revealed that levels of STAT5a and STAT5b in the nucleus and cytosol of NO-producing macrophages are substantially lower than in control cells. These findings provide evidence that STAT5 proteins play a role in NO-mediated down-regulation of IRP1 gene expression. PMID:16886906

  1. STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide

    PubMed Central

    Starzynski, Rafal Radoslaw; Gonçalves, Ana Sofia; Muzeau, Françoise; Tyrolczyk, Zofia; Smuda, Ewa; Drapier, Jean-Claude; Beaumont, Carole; Lipinski, Pawel

    2006-01-01

    RNA-binding activity of IRP1 (iron regulatory protein 1) is regulated by the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule. NO (nitic oxide), whose ability to activate IRP1 by removing its [4Fe-4S] cluster is well known, has also been shown to down-regulate expression of the IRP1 gene. In the present study, we examine whether this regulation occurs at the transcriptional level. Analysis of the mouse IRP1 promoter sequence revealed two conserved putative binding sites for transcription factor(s) regulated by NO and/or changes in intracellular iron level: Sp1 (promoter-selective transcription factor 1) and MTF1 (metal transcription factor 1), plus GAS (interferon-γ-activated sequence), a binding site for STAT (signal transducer and activator of transcription) proteins. In order to define the functional activity of these sequences, reporter constructs were generated through the insertion of overlapping fragments of the mouse IRP1 promoter upstream of the luciferase gene. Transient expression assays following transfection of HuH7 cells with these plasmids revealed that while both the Sp1 and GAS sequences are involved in basal transcriptional activity of the IRP1 promoter, the role of the latter is predominant. Analysis of protein binding to these sequences in EMSAs (electrophoretic mobility-shift assays) using nuclear extracts from mouse RAW 264.7 macrophages stimulated to synthesize NO showed a significant decrease in the formation of Sp1–DNA and STAT–DNA complexes, compared with controls. We have also demonstrated that the GAS sequence is involved in NO-dependent down-regulation of IRP1 transcription. Further analysis revealed that levels of STAT5a and STAT5b in the nucleus and cytosol of NO-producing macrophages are substantially lower than in control cells. These findings provide evidence that STAT5 proteins play a role in NO-mediated down-regulation of IRP1 gene expression. PMID:16886906

  2. Hsa-mir-182 suppresses lung tumorigenesis through down regulation of RGS17 expression in vitro

    SciTech Connect

    Sun, Yihua; Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai 200031 ; Fang, Rong; Li, Chenguang; Li, Li; Li, Fei; Ye, Xiaolei; Chen, Haiquan

    2010-05-28

    Lung cancer is one of the most devastating diseases worldwide. RGS17 is previously shown to be over-expressed in human lung adenocarcinomas and plays an important role in lung tumor growth. Here we have identified a miRNA, has-mir-182, involved in the regulation of RGS17 expression through two conserved sites located in its 3' UTR region. Consistently, endogenous RGS17 expression level is regulated by hsa-mir-182 in human lung cancer cell lines. Similar to the knockdown of RGS17, ectopic expression of hsa-mir-182 significantly inhibits lung cancer cell proliferation and anchorage-independent cell growth, which can be rescued by re-expression of RGS17. Taken together, these data have provided the first evidence of miRNA regulation of RGS17 expression in lung cancer.

  3. SNARE Protein Syntaxin-1 Colocalizes Closely with NMDA Receptor Subunit NR2B in Postsynaptic Spines in the Hippocampus

    PubMed Central

    Hussain, Suleman; Ringsevjen, Håvard; Egbenya, Daniel L.; Skjervold, Torstein L.; Davanger, Svend

    2016-01-01

    Syntaxins are a family of membrane-integrated proteins that are instrumental in exocytosis of vesicles. Syntaxin-1 is an essential component of the presynaptic exocytotic fusion machinery in the brain and interacts with several other proteins. Syntaxin-1 forms a four-helical bundle complex with proteins SNAP-25 and VAMP2 that drives fusion of vesicles with the plasma membrane in the active zone (AZ). Little is known, however, about the ultrastructural localization of syntaxin-1 at the synapse. We have analyzed the intrasynaptic expression of syntaxin-1 in glutamatergic hippocampal synapses in detail by using quantitative postembedding immunogold labeling. Syntaxin-1 was present in highest concentrations at the presynaptic AZ, supporting its role in transmitter release. Presynaptic plasma membrane lateral to the AZ, as well as presynaptic cytoplasmic (PreCy) vesicles were also labeled. However, syntaxin-1 was also significantly expressed in postsynaptic spines, where it was localized at the postsynaptic density (PSD), at postsynaptic lateral membranes and in postsynaptic cytoplasm. Postsynaptically, syntaxin-1 colocalized in the nanometer range with the N-methyl-D-aspartate (NMDA) receptor subunit NR2B, but only weakly with the AMPA receptor subunits GluA2/3. This observation points to the possibility that syntaxin-1 may be involved with NR2B vesicular trafficking from cytoplasmic stores to the postsynaptic plasma membrane, thus facilitating synaptic plasticity. Confocal immunofluorescence double labeling with PSD-95 and ultrastructural fractionation of synaptosomes also confirm localization of syntaxin-1 at the PSD. PMID:26903802

  4. Labisia pumila extract down-regulates hydroxysteroid (11-beta) dehydrogenase 1 expression and corticosterone levels in ovariectomized rats.

    PubMed

    Fazliana, Mansor; Gu, Harvest F; Östenson, Claes-Göran; Yusoff, Mashitah Mohd; Wan Nazaimoon, W M

    2012-04-01

    We evaluated the effects of a standardized Labisia pumila var. alata (LPva) extract on body weight change, hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) expressions and corticosterone (CORT) level in ovariectomized (OVX) rats. The decoction of LPva has been used for generations among Malay women in Malaysia to maintain a healthy reproductive system.Thirty-six Sprague-Dawley OVX rats were treated orally with LPva extract (10, 20 or 50 mg/kg/day) or estrogen replacement (ERT) for 30 days. Sham operated rats were used as controls. Compared to untreated OVX rats, LPva-treated rats showed less weight gain and had significantly down-regulated HSD11B1 mRNA in liver tissues. HSD11B1 mRNA in adipose tissues increased by 55% (p < 0.05) in OVX rats but normalized in rats treated with LPva. Similarly, there was significant down-regulation (p < 0.05) of protein levels of HSD11B1 in both liver and adipose tissue of LPva and ERT groups, and CORT levels were significantly reduced in both groups of rats. This is the first study ever conducted to evaluate the beneficial effects of LPva in relation to weight gain caused by estrogen insufficiency. Results implied that the bioactive components in LPva extract affect not only HSD11B1 expressions in both adipose and liver tissues but also decrease circulating CORT. The extract should be explored for its potential use as a natural remedy for weight management. PMID:21833773

  5. Histone Deacetylase 1 (HDAC1) Participates in the Down-Regulation of Corticotropin Releasing Hormone Gene (crh) Expression

    PubMed Central

    Miller, Lydia; Foradori, Chad D.; Lalmansingh, Avin S.; Sharma, Dharmendra; Handa, Robert J.; Uht, Rosalie M.

    2011-01-01

    The paraventricular nucleus of the hypothalamus (PVH) plays a central role in regulating the hypothalamic-pituitary-adrenal (HPA) axis. Medial parvocellular neurons of the PVH (mpPVH) integrate sensory and humoral inputs to maintain homeostasis. Humeral inputs include glucocorticoids secreted by the adrenals, which down-regulate HPA activation. A primary glucocorticoid target is the population of mpPVH neurons that synthesize and secrete corticotropin-releasing factors, the most potent of which is corticotropin-releasing hormone (CRH). Although CRH gene (crh) expression is known to be down-regulated by glucocorticoids, the mechanisms by which this process occurs are still poorly understood. To begin this study we postulated that glucocorticoid repression of crh involves HDAC recruitment to the region of the crh proximal promoter. To evaluate this hypothesis, we treated hypothalamic cells that express CRH with the HDAC inhibitor trichostatin A (TSA). As predicted, treatment with TSA led to increased CRH mRNA levels and crh promoter activity. Although co-treatment with Dex (10−7 M) reduced the TSA effect on mRNA levels, it failed to reduce promoter activity; however co-transfection of HDAC1 but not 3 restored Dex inhibition. A distinction between HDAC1 and 3 was also apparent with respect to crh promoter occupancy. Dex led to increased HDAC1 but not HDAC3 occupancy. In vivo studies revealed that CRH-immunoreactive (-ir) neurons contained HDAC1- and HDAC3-ir. Collectively, these data point to a role for HDAC1 in the physiologic regulation of crh. PMID:21463644

  6. Histone deacetylase 1 (HDAC1) participates in the down-regulation of corticotropin releasing hormone gene (crh) expression.

    PubMed

    Miller, Lydia; Foradori, Chad D; Lalmansingh, Avin S; Sharma, Dharmendra; Handa, Robert J; Uht, Rosalie M

    2011-08-01

    The paraventricular nucleus of the hypothalamus (PVH) plays a central role in regulating the hypothalamic-pituitary-adrenal (HPA) axis. Medial parvocellular neurons of the PVH (mpPVH) integrate sensory and humoral inputs to maintain homeostasis. Humoral inputs include glucocorticoids secreted by the adrenals, which down-regulate HPA activation. A primary glucocorticoid target is the population of mpPVH neurons that synthesize and secrete corticotropin-releasing factors, the most potent of which is corticotropin-releasing hormone (CRH). Although CRH gene (crh) expression is known to be down-regulated by glucocorticoids, the mechanisms by which this process occurs are still poorly understood. To begin this study we postulated that glucocorticoid repression of crh involves HDAC recruitment to the region of the crh proximal promoter. To evaluate this hypothesis, we treated hypothalamic cells that express CRH with the HDAC inhibitor trichostatin A (TSA). As predicted, treatment with TSA led to increased CRH mRNA levels and crh promoter activity. Although co-treatment with Dex (10(-7)M) reduced the TSA effect on mRNA levels, it failed to reduce promoter activity; however co-transfection of HDAC1 but not 3 restored Dex inhibition. A distinction between HDAC1 and 3 was also apparent with respect to crh promoter occupancy. Dex led to increased HDAC1 but not HDAC3 occupancy. In vivo studies revealed that CRH-immunoreactive (-ir) neurons contained HDAC1- and HDAC3-ir. Collectively, these data point to a role for HDAC1 in the physiologic regulation of crh. PMID:21463644

  7. Pioglitazone reverses down-regulation of cardiac PPAR{gamma} expression in Zucker diabetic fatty rats

    SciTech Connect

    Pelzer, Theo . E-mail: pelzer_t@klinik.uni-wuerzburg.de; Jazbutyte, Virginija; Arias-Loza, Paula Anahi; Segerer, Stephan; Lichtenwald, Margit; Law, Marilyn P.; Schaefers, Michael; Ertl, Georg; Neyses, Ludwig

    2005-04-08

    Peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) plays a critical role in peripheral glucose homeostasis and energy metabolism, and inhibits cardiac hypertrophy in non-diabetic animal models. The functional role of PPAR{gamma} in the diabetic heart, however, is not fully understood. Therefore, we analyzed cardiac gene expression, metabolic control, and cardiac glucose uptake in male Zucker diabetic fatty rats (ZDF fa/fa) and lean ZDF rats (+/+) treated with the high affinity PPAR{gamma} agonist pioglitazone or placebo from 12 to 24 weeks of age. Hyperglycemia, hyperinsulinemia, and hypertriglyceridemia as well as lower cardiac PPAR{gamma}, glucose transporter-4 and {alpha}-myosin heavy chain expression levels were detected in diabetic ZDF rats compared to lean animals. Pioglitazone increased body weight and improved metabolic control, cardiac PPAR{gamma}, glut-4, and {alpha}-MHC expression levels in diabetic ZDF rats. Cardiac [{sup 18}F]fluorodeoxyglucose uptake was not detectable by micro-PET studies in untreated and pioglitazone treated ZDF fa/fa rats but was observed after administration of insulin to pioglitazone treated ZDF fa/fa rats. PPAR{gamma} agonists favorably affect cardiac gene expression in type-2 diabetic rats via activation and up-regulation of cardiac PPAR{gamma} expression whereas improvement of impaired cardiac glucose uptake in advanced type-2 diabetes requires co-administration of insulin.

  8. [Ox-LDL down-regulates expression of pigment epithelium-derived factor in human umbilical vein endothelial cells].

    PubMed

    Liu, Jie; Yao, Shu-Tong; Zhai, Lei; Feng, Yue-Long; Song, Guo-Hua; Yu, Yang; Zhu, Ping; Qin, Shu-Cun

    2014-08-25

    Pigment epithelium-derived factor (PEDF) is a multifunctional protein with anti-inflammatory, antioxidant and antithrombotic properties and plays a protective role against atherosclerosis (AS). The purpose of the present study is to explore the effects of oxidized low density lipoprotein (ox-LDL) on the expression of PEDF in cultured human umbilical vein endothelial cells (HUVECs). HUVECs were cultured and incubated with ox-LDL at different concentrations (6.25, 12.5, 25, 50, 100 and 150 mg/L) for 24 h. Apoptosis of endothelial cells were assayed by morphological staining and flow cytometry. The intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. Cell viability was assayed by MTT assay. PEDF protein and mRNA expressions in HUVECs were analyzed by Western blot and quantitative real-time PCR, respectively. The results showed that ox-LDL significantly induced apoptosis, reduced cell viability, increased intracellular ROS levels and decreased the PEDF expression in HUVECs in a concentration-dependent manner. Ox-LDL at 50 mg/L obviously decreased the PEDF protein expression compared with control group (P < 0.05), whereas 25 mg/L ox-LDL already markedly reduced the PEDF mRNA expression (P < 0.05). In conclusion, the results suggest that ox-LDL down-regulates the PEDF expression through an increased ox-LDL-induced intracellular production of ROS. PMID:25131792

  9. Galectin-4 expression is down-regulated in response to autophagy during differentiation of rat trophoblast cells.

    PubMed

    Arikawa, Tomohiro; Liao, Shengjun; Shimada, Hiroki; Inoue, Tomoki; Sakata-Haga, Hiromi; Nakamura, Takanori; Hatta, Toshihisa; Shoji, Hiroki

    2016-01-01

    Placental development and trophoblast invasion of the maternal endometrium establish the maternal-fetal interface, which is critical for the developing embryo and fetus. Herein we show that overexpression of Galectin-4 (Gal-4) during trophoblast differentiation inhibited the enlargement of Rcho-1 cells (a model for rat trophoblast differentiation) and promoted cell-cell adhesion, whereas trophoblast specific markers and MMP-9 activity were not affected. In the rat placenta, microtubule associated protein 1 light chain 3 alpha (LC3) protein, an autophagy marker, is highly expressed on the maternal side of the decidua where Gal-4 expression is weak. In vitro assays showed that the expression of trophoblast-specific differentiation markers was reduced by 3-Methyladenine (3-MA) and Bafilomycin A1, known as autophagy inhibitors, compared to control cells. Furthermore, Gal-4 expression in Rcho-1 cells, which is normally down-regulated during differentiation, was not attenuated in the presence of autophagy inhibitors, suggesting that autophagy is upstream of Gal-4 expression. We herein describe a possible mechanism by which autophagy regulates trophoblast differentiation via regulation of Gal-4 expression in order to establish the maternal-fetal interface. PMID:27572741

  10. Galectin-4 expression is down-regulated in response to autophagy during differentiation of rat trophoblast cells

    PubMed Central

    Arikawa, Tomohiro; Liao, Shengjun; Shimada, Hiroki; Inoue, Tomoki; Sakata-Haga, Hiromi; Nakamura, Takanori; Hatta, Toshihisa; Shoji, Hiroki

    2016-01-01

    Placental development and trophoblast invasion of the maternal endometrium establish the maternal-fetal interface, which is critical for the developing embryo and fetus. Herein we show that overexpression of Galectin-4 (Gal-4) during trophoblast differentiation inhibited the enlargement of Rcho-1 cells (a model for rat trophoblast differentiation) and promoted cell-cell adhesion, whereas trophoblast specific markers and MMP-9 activity were not affected. In the rat placenta, microtubule associated protein 1 light chain 3 alpha (LC3) protein, an autophagy marker, is highly expressed on the maternal side of the decidua where Gal-4 expression is weak. In vitro assays showed that the expression of trophoblast-specific differentiation markers was reduced by 3-Methyladenine (3-MA) and Bafilomycin A1, known as autophagy inhibitors, compared to control cells. Furthermore, Gal-4 expression in Rcho-1 cells, which is normally down-regulated during differentiation, was not attenuated in the presence of autophagy inhibitors, suggesting that autophagy is upstream of Gal-4 expression. We herein describe a possible mechanism by which autophagy regulates trophoblast differentiation via regulation of Gal-4 expression in order to establish the maternal-fetal interface. PMID:27572741

  11. Rapamycin down-regulates LDL-receptor expression independently of SREBP-2

    SciTech Connect

    Sharpe, Laura J.; Brown, Andrew J.

    2008-09-05

    As a key regulator of cholesterol homeostasis, sterol-regulatory element binding protein-2 (SREBP-2) up-regulates expression of genes involved in cholesterol synthesis (e.g., 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) Reductase) and uptake (the low density lipoprotein (LDL)-receptor). Previously, we showed that Akt, a critical kinase in cell growth and proliferation, contributes to SREBP-2 activation. However, the specific Akt target involved is unknown. A potential candidate is the mammalian target of rapamycin, mTOR. Rapamycin can cause hyperlipidaemia clinically, and we hypothesised that this may be mediated via an effect of mTOR on SREBP-2. Herein, we found that SREBP-2 activation and HMG-CoA Reductase gene expression were unaffected by rapamycin treatment. However, LDL-receptor gene expression was decreased by rapamycin, suggesting that this may contribute to the hyperlipidaemia observed in rapamycin-treated patients. Rapamycin did not affect mRNA stability, so the decrease in LDL-receptor gene expression is likely to be occurring at the transcriptional level, although independently of SREBP-2.

  12. Cuprous oxide nanoparticles inhibit angiogenesis via down regulation of VEGFR2 expression

    NASA Astrophysics Data System (ADS)

    Song, Hongyuan; Wang, Wenbo; Zhao, Ping; Qi, Zhongtian; Zhao, Shihong

    2014-02-01

    Angiogenesis is a process that forms new blood capillaries from existing vessels, which is of great physiological and pathological significance. Although recent studies provide evidence that cuprous oxide nanoparticles (CO-NPs) may have biomedical potential, the mechanisms of CO-NPs in angiogenesis have not been investigated to date. We have studied the anti-angiogenic properties of CO-NPs on primary human umbilical vein endothelial cells (HUVECs). We found that CO-NPs were able to induce cell morphology changes and suppress cell proliferation, migration and tube formation in vitro and in vivo dose dependently. Furthermore, CO-NPs could induce cell apoptosis both at the early and late apoptotic stage and induce cell cycle arrest at S phase in a dose dependent manner. As signalling via the vascular endothelial growth factor receptor-2 (VEGFR2) is critical for angiogenic responses, we further explored the expression of VEGFR2 after the treatment of CO-NPs. They were found to inhibit VEGFR2 expression dose and time dependently both at the protein and mRNA level while had no effect on VEGF and VEGFR1 expression. Together, we report for the first time that CO-NPs can act as an anti-angiogenic agent by suppressing VEGFR2 expression, which may be a potential nanomedicine for angiogenesis therapy.Angiogenesis is a process that forms new blood capillaries from existing vessels, which is of great physiological and pathological significance. Although recent studies provide evidence that cuprous oxide nanoparticles (CO-NPs) may have biomedical potential, the mechanisms of CO-NPs in angiogenesis have not been investigated to date. We have studied the anti-angiogenic properties of CO-NPs on primary human umbilical vein endothelial cells (HUVECs). We found that CO-NPs were able to induce cell morphology changes and suppress cell proliferation, migration and tube formation in vitro and in vivo dose dependently. Furthermore, CO-NPs could induce cell apoptosis both at the early and

  13. Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma.

    PubMed Central

    Perlino, E.; Loverro, G.; Maiorano, E.; Giannini, T.; Cazzolla, A.; Napoli, A.; Fiore, M. G.; Ricco, R.; Marra, E.; Selvaggi, L.

    1998-01-01

    Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma. Images Figure 1 Figure 5 Figure 6 Figure 8 PMID:9579831

  14. Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma.

    PubMed

    Perlino, E; Loverro, G; Maiorano, E; Giannini, T; Cazzolla, A; Napoli, A; Fiore, M G; Ricco, R; Marra, E; Selvaggi, L

    1998-04-01

    Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma. PMID:9579831

  15. Piperlongumine and its analogs down-regulate expression of c-Met in renal cell carcinoma.

    PubMed

    Golovine, Konstantin; Makhov, Peter; Naito, Sei; Raiyani, Henish; Tomaszewski, Jeffrey; Mehrazin, Reza; Tulin, Alexei; Kutikov, Alexander; Uzzo, Robert G; Kolenko, Vladimir M

    2015-01-01

    The c-Met protein, a transmembrane receptor tyrosine kinase, is the product of a proto-oncogene. Its only known ligand, hepatocyte growth factor (HGF), regulates cell growth, motility, migration, invasion, proliferation, and angiogenesis. The aberrant expression of c-Met is often associated with poor prognosis in multiple cancers, including renal cell carcinoma (RCC). Silencing or inactivation of c-Met leads to decreased viability of cancer cells, thereby making ablation of c-Met signaling an attractive concept for developing novel strategies for the treatment of renal tumors. Naturally-occurring products or substances are the most consistent source of drug development. As such, we investigated the functional impact of piperlongumine (PL), a naturally occurring alkaloid present in the Long pepper (Piper longum) on c-Met expression in RCC cells and demonstrated that PL and its analogs rapidly reduce c-Met protein and RNA levels in RCC cells via ROS-dependent mechanism. PL-mediated c-Met depletion coincided with the inhibition of downstream c-Met signaling; namely Erk/MAPK, STAT3, NF-κB and Akt/mTOR. As such, PL and PL analogs hold promise as potential therapeutic agents for the treatment of metastatic RCC and the prevention of postoperative RCC recurrence. PMID:25801713

  16. Piperlongumine and its analogs down-regulate expression of c-Met in renal cell carcinoma

    PubMed Central

    Golovine, Konstantin; Makhov, Peter; Naito, Sei; Raiyani, Henish; Tomaszewski, Jeffrey; Mehrazin, Reza; Tulin, Alexei; Kutikov, Alexander; Uzzo, Robert G; Kolenko, Vladimir M

    2015-01-01

    The c-Met protein, a transmembrane receptor tyrosine kinase, is the product of a proto-oncogene. Its only known ligand, hepatocyte growth factor (HGF), regulates cell growth, motility, migration, invasion, proliferation, and angiogenesis. The aberrant expression of c-Met is often associated with poor prognosis in multiple cancers, including renal cell carcinoma (RCC). Silencing or inactivation of c-Met leads to decreased viability of cancer cells, thereby making ablation of c-Met signaling an attractive concept for developing novel strategies for the treatment of renal tumors. Naturally-occurring products or substances are the most consistent source of drug development. As such, we investigated the functional impact of piperlongumine (PL), a naturally occurring alkaloid present in the Long pepper (Piper longum) on c-Met expression in RCC cells and demonstrated that PL and its analogs rapidly reduce c-Met protein and RNA levels in RCC cells via ROS-dependent mechanism. PL-mediated c-Met depletion coincided with the inhibition of downstream c-Met signaling; namely Erk/MAPK, STAT3, NF-κB and Akt/mTOR. As such, PL and PL analogs hold promise as potential therapeutic agents for the treatment of metastatic RCC and the prevention of postoperative RCC recurrence. PMID:25801713

  17. A Molecular Toolbox for Rapid Generation of Viral Vectors to Up- or Down-Regulate Neuronal Gene Expression in vivo

    PubMed Central

    White, Melanie D.; Milne, Ruth V. J.; Nolan, Matthew F.

    2011-01-01

    We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins, and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV) or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1, and Kir3.2) and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miRNA). We show that AAV assembled to express HCN1 miRNA produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miRNA with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience. PMID:21772812

  18. Induction and down-regulation of PLK, a human serine/threonine kinase expressed in proliferating cells and tumors.

    PubMed Central

    Holtrich, U; Wolf, G; Bräuninger, A; Karn, T; Böhme, B; Rübsamen-Waigmann, H; Strebhardt, K

    1994-01-01

    We have identified the nucleotide sequence of the cDNA encoding the human counterpart of the mouse gene Plk (polo-like kinase). The sequence of the human gene, PLK, predicts a serine/threonine kinase of 603 aa. Expression of PLK mRNA appeared to be strongly correlated with the mitotic activity of cells. Resting peripheral lymphocytes did not express the gene at all. When primary T cells were activated by phytohemagglutinin, a high level of PLK transcripts resulted within 2-3 days. In some cases, addition of interleukin 2 to these cells increased the expression of PLK mRNA further. In contrast, primary cultures of human peripheral macrophages, which were not dividing under the culture conditions applied, showed very little or no PLK mRNA. Stimulation of these cells by bacterial lipopolysaccharide, an inducer of several cytokines in macrophages, totally abrogated the expression of PLK mRNA. In line with a function of PLK mRNA expression in mitotically active cells is our finding that six immortalized cell lines examined expressed the gene. In A-431 epidermoid carcinoma cells this expression was down-regulated by serum starvation and enhanced after serum was added again. Tumors of various origin (lung, colon, stomach, smooth muscle, and esophagus as well as non-Hodgkin lymphomas) expressed high levels of PLK transcripts in about 80% of the samples studied, whereas PLK mRNA was absent in surrounding tissue, except for colon. The only normal tissues where PLK mRNA expression was observed were colon and placenta, both known to be mitotically active. No PLK transcripts were found in normal adult lung, brain, heart, liver, kidney, skeletal muscle, and pancreas. In Northern blot experiments with RNA from lymphocytes which were treated with phytohemagglutinin and cycloheximide, PLK transcripts were not detectable, suggesting that PLK is not an early growth-response gene. Images PMID:8127874

  19. Glucose transporter 2 expression is down regulated following P2X7 activation in enterocytes.

    PubMed

    Bourzac, Jean-François; L'Ériger, Karine; Larrivée, Jean-François; Arguin, Guillaume; Bilodeau, Maude S; Stankova, Jana; Gendron, Fernand-Pierre

    2013-01-01

    With the diabetes epidemic affecting the world population, there is an increasing demand for means to regulate glycemia. Dietary glucose is first absorbed by the intestine before entering the blood stream. Thus, the regulation of glucose absorption by intestinal epithelial cells (IECs) could represent a way to regulate glycemia. Among the molecules involved in glycemia homeostasis, extracellular ATP, a paracrine signaling molecule, was reported to induce insulin secretion from pancreatic β cells by activating P2Y and P2X receptors. In rat's jejunum, P2X7 expression was previously immunolocalized to the apex of villi, where it has been suspected to play a role in apoptosis. However, using an antibody recognizing the receptor extracellular domain and thus most of the P2X7 isoforms, we showed that expression of this receptor is apparent in the top two-thirds of villi. These data suggest a different role for this receptor in IECs. Using the non-cancerous IEC-6 cells and differentiated Caco-2 cells, glucose transport was reduced by more than 30% following P2X7 stimulation. This effect on glucose transport was not due to P2X7-induced cell apoptosis, but rather was the consequence of glucose transporter 2 (Glut2)'s internalization. The signaling pathway leading to P2X7-dependent Glut2 internalization involved the calcium-independent activation of phospholipase Cγ1 (PLCγ1), PKCδ, and PKD1. Although the complete mechanism regulating Glut2 internalization following P2X7 activation is not fully understood, modulation of P2X7 receptor activation could represent an interesting approach to regulate intestinal glucose absorption. PMID:22566162

  20. New benzoyl urea derivatives as novel NR2B selective NMDA receptor antagonists.

    PubMed

    Borza, I; Greiner, I; Kolok, S; Galgóczy, K; Ignácz-Szendrei, Gy; Horváth, Cs; Farkas, S; Gáti, T; Háda, V; Domány, Gy

    2006-09-01

    A novel series of benzoyl urea derivatives was prepared and identified as NR2B selective NMDA receptor antagonists. The influence of the substitution of the piperidine ring on the biological activity of the compounds was studied. Compound 9 was active in the formalin test in mice. PMID:17020160

  1. Down-regulation of CD53 expression in Epinephelus coioides under LPS, poly (I:C), and cytokine stimulation.

    PubMed

    Hou, Chia-Yi; Lin, John Han-You; Lin, Shih-Jie; Kuo, Wan-Ching; Lin, Han-Tso

    2016-04-01

    Tetraspanins are a group of cell surface molecules involved in cell adhesion, motility, metastasis, signal transduction, and immune cell activation. Members of the tetraspanin family include CD9, CD37, CD63, CD53, and others. However, few tetraspanins have been investigated in teleosts. In this study, we obtained the open reading frame of CD53 cDNA from orange spotted grouper (Epinephelus coioices), an economically important fish. The predicted amino acid structure contains four membrane-spanning domains and a conserved CCG motif. The amino acid identity between human and grouper CD53 was only 38%; however, both CD53 proteins share the same structure. Quantitative real-time PCR revealed that mRNA is abundant in immune organs, including the head and trunk kidneys, spleen, thymus, gill, and blood. Immunochemistry and immunofluorescence analyses further revealed that CD53 was majorly expressed in the leukocytes of various organs. Finally, mRNA and protein expression for CD53 was down-regulated in fish treated with immune stimulators, including LPS, Poly (I:C), Vibrio, recombinant grouper IL-6, and CCL4. Our results indicate that the expression of CD53 may play important roles in pathogen invasion and inflammation reaction. PMID:26631805

  2. Identification of a Novel Rat NR2B Subunit Gene Promoter Region Variant and Its Association with Microwave-Induced Neuron Impairment.

    PubMed

    Wang, Li-Feng; Tian, Da-Wei; Li, Hai-Juan; Gao, Ya-Bing; Wang, Chang-Zhen; Zhao, Li; Zuo, Hong-Yan; Dong, Ji; Qiao, Si-Mo; Zou, Yong; Xiong, Lu; Zhou, Hong-Mei; Yang, Yue-Feng; Peng, Rui-Yun; Hu, Xiang-Jun

    2016-05-01

    Microwave radiation has been implicated in cognitive dysfunction and neuronal injury in animal models and in human investigations; however, the mechanism of these effects is unclear. In this study, single nucleotide polymorphism (SNP) sites in the rat GRIN2B promoter region were screened. The associations of these SNPs with microwave-induced rat brain dysfunction and with rat pheochromocytoma-12 (PC12) cell function were investigated. Wistar rats (n = 160) were exposed to microwave radiation (30 mW/cm(2) for 5 min/day, 5 days/week, over a period of 2 months). Screening of the GRIN2B promoter region revealed a stable C-to-T variant at nucleotide position -217 that was not induced by microwave exposure. The learning and memory ability, amino acid contents in the hippocampus and cerebrospinal fluid, and NR2B expression were then investigated in the different genotypes. Following microwave exposure, NR2B protein expression decreased, while the Glu contents in the hippocampus and CSF increased, and memory impairment was observed in the TT genotype but not the CC and CT genotypes. In PC12 cells, the effects of the T allele were more pronounced than those of the C allele on transcription factor binding ability, transcriptional activity, NR2B mRNA, and protein expression. These effects may be related to the detrimental role of the T allele and the protective role of the C allele in rat brain function and PC12 cells exposed to microwave radiation. PMID:25917873

  3. Hepatitis C Virus Increases Free Fatty Acids Absorption and Promotes its Replication Via Down-Regulating GADD45α Expression

    PubMed Central

    Chen, Wei; Li, Xiao-ming; Li, An-ling; Yang, Gui; Hu, Han-ning

    2016-01-01

    Background Hepatitis C virus (HCV) infection, as a major cause of chronic hepatic diseases, is always accompanied with an abnormality of lipid metabolism. The aim of this study was to investigate the pathogenic role of free fatty acids (FFA) in human HCV infection. Material/Methods Peripheral blood lipid indexes among HCV patients with different viral loads (199 samples) and healthy donors (80 samples) were detected by clinical biochemistry tests. HCV replication and the expression of growth arrest and DNA-damage-inducible gene 45-α (GADD45α) in Huh7 cells and clinical samples were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Lipid accumulation in Huh7 cells was detected by immunofluorescence. Results In this study, we found that FFA showed a significant positive correlation with viral load in peripheral blood of HCV patients, but not total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), or low-density lipoprotein cholesterol (LDL-C). GADD45α expression in HCV patients dramatically decreased with the increase of viral load. In Huh7 cells, FFA treatment significantly enhanced HCV replication. HCV infection inhibited GADD45α expression, and this effect was further enhanced with the presence of FFA treatment. Ectopic expression of GADD45α in HCV-infected Huh7 cells markedly inhibited the absorption of FFA and HCV replication. However, FFA significantly elevated GADD45α expression without HCV infection. Conclusions These results demonstrated that HCV down-regulates GADD45α expression to enhance FFA absorption and thus facilitate its replication. GADD45α is an essential mediator for the pathogenesis of HCV infection. Thus, our study provides potential clues in the search for novel therapeutics and fatty lipid control options for HCV patients. PMID:27381636

  4. Sinomenine down-regulates TLR4/TRAF6 expression and attenuates lipopolysaccharide-induced osteoclastogenesis and osteolysis.

    PubMed

    He, Longgang; Duan, Heng; Li, Xianglian; Wang, Song; Zhang, Yueyang; Lei, Linsheng; Xu, Jiake; Liu, Shuwen; Li, Xiaojuan

    2016-05-15

    Sinomenine (SIN) is an anti-inflammatory and anti-arthritic alkaloid derived from Sinomenioum acutum. Effects of SIN on lipopolysaccharide (LPS)-induced osteolysis have not been reported. Here, we found that SIN reduced LPS-induced erosion of skull bones in C57BL/6 mice significantly. LPS can induce bone-absorbing osteoclast formation independent of RANKL in pre-osteoclastic RAW264.7 cells in vitro. Here, SIN suppressed LPS-induced osteoclast formation and osteoclast survival in RAW264.7 cells. Expression of osteoclastic-specific marker genes was also inhibited by SIN during osteoclast differentiation and osteoclast survival stimulated with LPS. SIN showed much stronger inhibitory effects on expression of Fra-1 and MMP-9 mRNA in osteoclast differentiation rather than osteoclast survival. SIN dramatically inhibited LPS-induced TNF-α production in vitro and in vivo. Further signaling studies revealed that SIN suppressed the activation and relative gene expression of three notable nuclear factors (NF-κB, AP-1, NFAT), reduced intracellular levels of Ca(2+), and down-regulated phosphorylation of MAPK p38 (but not JNK) in LPS-induced osteoclastogenesis. Focusing on upstream signals after LPS stimulation, SIN decreased expression of TLR4 and TRAF6 during osteoclast differentiation, and reduced expression of TLR4 (but not TRAF6) in osteoclast survival. These data suggest that SIN might be a potential agent for the treatment of osteolysis caused by Gram-negative bacteria infection or inflammation due to its inhibition of osteoclastogenesis through reduction of TLR4/TRAF6 expression and downstream signal transduction. PMID:26965104

  5. Metformin reduces the endotoxin-induced down-regulation of apolipoprotein E gene expression in macrophages

    SciTech Connect

    Stavri, Simona; Trusca, Violeta G.; Simionescu, Maya; Gafencu, Anca V.

    2015-05-29

    The atheroprotective role of macrophage-derived apolipoprotein E (apoE) is well known. Our previous reports demonstrated that inflammatory stress down-regulates apoE expression in macrophages, aggravating atherogenesis. Metformin, extensively used as an anti-diabetic drug, has also anti-inflammatory properties, and thus confers vascular protection. In this study, we questioned whether metformin could have an effect on apoE expression in macrophages in normal conditions or under lipopolysaccharide (LPS)-induced stress. The results showed that metformin slightly increases the apoE expression only at high doses (5–10 mM). Low doses of metformin (1–3 mM) significantly reduce the LPS down-regulatory effect on apoE expression in macrophages. Our experiments demonstrated that LPS-induced NF-κB binds to the macrophage-specific distal regulatory element of apoE gene, namely to the multienhancer 2 (ME.2) and its 5′-deletion fragments. The NF-κB binding on ME.2 and apoE promoter has a down-regulatory effect. In addition, data revealed that metformin impairs NF-κB nuclear translocation, and thus, improves the apoE levels in macrophages under inflammatory stress. The positive effect of metformin in the inflammatory states, its clinical safety and low cost, make this drug a potential adjuvant in the therapeutic strategies for atherosclerosis. - Highlights: • High doses of metformin slightly increase apoE expression in macrophages. • Low doses of metformin up-regulate apoE gene in endotoxin-stressed macrophages. • Metformin reduces the negative effect of LPS on apoE expression by NF-κB inhibition.

  6. Resistin impairs glucose permeability in EA.hy926 cells by down-regulating GLUT1 expression.

    PubMed

    Li, Qiang; Cai, Yuxi; Huang, Jing; Yu, Xiaolan; Sun, Jun; Yang, Zaiqing; Zhou, Lei

    2016-10-15

    Type 2 diabetes mellitus (T2DM) is a chronic disease which is now affecting the health of more and more people in the world. Resistin, discovered in 2001, is considered to be closely related to metabolic dysfunction and obesity. Previous study showed that hyperglycemia is always accompanied by a high serum resistin concentration. We therefore investigated whether resistin can mediate glucose transfer across the blood-tissue barrier. Here, we employed a transwell system to analyze glucose permeability in EA.hy926 human endothelial cells treated without or with human resistin. In EA.hy926 cells treated with resistin, the permeability to glucose was heavily impaired. This was due to the down-regulation of GLUT1 expression as a result of the treatment, rather than regulation of tight junctions. In addition, overexpression of GLUT1 in EA.hy926 cells was able to recover the blocking effect of resistin on glucose permeability. We further found that resistin could inhibit the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and consequently impede the transcription of GLUT1. The results of the present study suggested that resistin could cause glucose retention in serum and thus result in hyperglycemia. This provides a novel explanation for hyperglycemia and a potential new way of treating type 2 diabetes mellitus. PMID:27353463

  7. Down-Regulation of miR-146a Expression Induces Allergic Conjunctivitis in Mice by Increasing TSLP Level

    PubMed Central

    Sun, Wen; Sheng, Yan; Chen, Jie; Xu, Dong; Gu, Yangshun

    2015-01-01

    Background Pollen is the most common aeroallergen to cause conjunctivitis. In this study, we established a short ragweed (SRW)-induced mouse model of allergic conjunctivitis (AC) and aimed to explore the potential role of miR-146a and its downstream molecules in the development of ocular allergic inflammation. Material/Methods The mouse model of challenge pollen was used for in vivo study. The culture model of primary human limbal epithelium (HLE) exposed to lipopolysaccharide (LPS) was performed for in vitro research. The numbers of eosinophils and total inflammatory cells were examined using Giemsa staining. The expression of mRNA and miR-146a was determined by quantitative RT-PCR, and protein production was evaluated by Western blotting. Results In vivo of mice, pollen challenge induced conjunctiva inflammatory response indicated by increased number of eosinophils and total inflammatory cells. Interestingly, pollen significantly attenuated miR-146a expression while it enhanced expression of thymic stromal lymphopoietin (TSLP) and its downstream molecules, including TSLP receptor (TSLPR)/ OX40 ligand (OX40L)/CD11C. In vitro of HCE, downregulation effect of miR-146a expression induced by LPS was reversed by Bay treatment, an inhibitor for nuclear factor kappa B (NF-κB), and LPS-induced cell inflammation is mediated by miR-146a-TSLP/TSLPR/OX40L/CD11C signaling pathway. This was further demonstrated by overexpression of miR-146a in mouse abrogated pollen-triggered conjunctiva inflammatory reaction as well as pollen-induced activity of TSLP/TSLPR/OX40L/CD11C signaling. Conclusions Down-regulation of miR-146a expression induces allergic conjunctivitis in mice by increasing TSLP level. PMID:26166175

  8. Melittin induces PTCH1 expression by down-regulating MeCP2 in human hepatocellular carcinoma SMMC-7721 cells.

    PubMed

    Wu, Xiaoqin; Zhao, Bin; Cheng, Yahui; Yang, Yang; Huang, Cheng; Meng, Xiaoming; Wu, Baoming; Zhang, Lei; Lv, Xiongwen; Li, Jun

    2015-10-01

    Hepatocellular carcinoma (HCC) has a high mortality rate worldwide and still remains to be a noticeable public health problem. Therefore, new remedies are urgently needed. Melittin, a major component of bee venom, is known to suppress cell growth in various cancers including HCC. However, the mechanism of the anticancer effect of melittin on HCC has not been fully elucidated. It has been reported that Methyl-CpG binding protein 2 (MeCP2) plays a key role in tumor proliferation, apoptosis, migration and invasion. In the present study, we found the high expression of MeCP2 in human HCC tissues and in the SMMC-7721 cell line. MeCP2 silencing inhibited cell proliferation, while over-expression of MeCP2 promoted cell growth in SMMC-7721 cells. It indicates that MeCP2 may be an attractive target for human HCC. We further found that melittin could inhibit cell proliferation by reducing MeCP2 expression in vitro. Interestingly, the inhibitory effect of melittin on cell proliferation was due to a delay in G0/G1 cell cycle progression, without influencing cell apoptosis. Next, we investigated the potential molecular mechanisms and found that MeCP2 could modulate Shh signaling in SMMC-7721 cells. Further study indicates that melittin may induce the demethylation of PTCH1 promoter, resulting in the increased expression of PTCH1. Furthermore, the expression of Shh and GLI1 was significantly lowered upon treatment of melittin. These results suggest that melittin can block Shh signaling in vitro. In short, these results indicate that melittin inhibits cell proliferation by down-regulating MeCP2 through Shh signaling in SMMC-7721 cells. PMID:26189965

  9. NDRG2 Expression Decreases Tumor-Induced Osteoclast Differentiation by Down-regulating ICAM1 in Breast Cancer Cells.

    PubMed

    Kim, Bomi; Nam, Sorim; Lim, Ji Hyun; Lim, Jong-Seok

    2016-01-01

    Bone matrix is properly maintained by osteoclasts and osteoblasts. In the tumor microenvironment, osteoclasts are increasingly differentiated by the various ligands and cytokines secreted from the metastasized cancer cells at the bone metastasis niche. The activated osteoclasts generate osteolytic lesions. For this reason, studies focusing on the differentiation of osteoclasts are important to reduce bone destruction by tumor metastasis. The N-myc downstream-regulated gene 2 (NDRG2) has been known to contribute to the suppression of tumor growth and metastasis, but the precise role of NDRG2 in osteoclast differentiation induced by cancer cells has not been elucidated. In this study, we demonstrate that NDRG2 expression in breast cancer cells has an inhibitory effect on osteoclast differentiation. RAW 264.7 cells, which are monocytic preosteoclast cells, treated with the conditioned media (CM) of murine breast cancer cells (4T1) expressing NDRG2 are less differentiated into the multinucleated osteoclast-like cells than those treated with the CM of 4T1-WT or 4T1-mock cells. Interestingly, 4T1 cells stably expressing NDRG2 showed a decreased mRNA and protein level of intercellular adhesion molecule 1 (ICAM1), which is known to enhance osteoclast maturation. Osteoclast differentiation was also reduced by ICAM1 knockdown in 4T1 cells. In addition, blocking the interaction between soluble ICAM1 and ICAM1 receptors significantly decreased osteoclastogenesis of RAW 264.7 cells in the tumor environment. Collectively, these results suggest that the reduction of ICAM1 expression by NDRG2 in breast cancer cells decreases osteoclast differentiation, and demonstrate that excessive bone resorption could be inhibited via ICAM1 down-regulation by NDRG2 expression. PMID:26759696

  10. NDRG2 Expression Decreases Tumor-Induced Osteoclast Differentiation by Down-regulating ICAM1 in Breast Cancer Cells

    PubMed Central

    Kim, Bomi; Nam, Sorim; Lim, Ji Hyun; Lim, Jong-Seok

    2016-01-01

    Bone matrix is properly maintained by osteoclasts and osteoblasts. In the tumor microenvironment, osteoclasts are increasingly differentiated by the various ligands and cytokines secreted from the metastasized cancer cells at the bone metastasis niche. The activated osteoclasts generate osteolytic lesions. For this reason, studies focusing on the differentiation of osteoclasts are important to reduce bone destruction by tumor metastasis. The N-myc downstream-regulated gene 2 (NDRG2) has been known to contribute to the suppression of tumor growth and metastasis, but the precise role of NDRG2 in osteoclast differentiation induced by cancer cells has not been elucidated. In this study, we demonstrate that NDRG2 expression in breast cancer cells has an inhibitory effect on osteoclast differentiation. RAW 264.7 cells, which are monocytic preosteoclast cells, treated with the conditioned media (CM) of murine breast cancer cells (4T1) expressing NDRG2 are less differentiated into the multinucleated osteoclast-like cells than those treated with the CM of 4T1-WT or 4T1-mock cells. Interestingly, 4T1 cells stably expressing NDRG2 showed a decreased mRNA and protein level of intercellular adhesion molecule 1 (ICAM1), which is known to enhance osteoclast maturation. Osteoclast differentiation was also reduced by ICAM1 knockdown in 4T1 cells. In addition, blocking the interaction between soluble ICAM1 and ICAM1 receptors significantly decreased osteoclastogenesis of RAW 264.7 cells in the tumor environment. Collectively, these results suggest that the reduction of ICAM1 expression by NDRG2 in breast cancer cells decreases osteoclast differentiation, and demonstrate that excessive bone resorption could be inhibited via ICAM1 down-regulation by NDRG2 expression. PMID:26759696

  11. Reduced levels of NR2A and NR2B subunits of NMDA receptor and PSD-95 in the prefrontal cortex in major depression

    PubMed Central

    Feyissa, Anteneh M.; Zyga, Agata; Stockmeier, Craig A.; Karolewicz, Beata

    2009-01-01

    Recent neuroimaging and postmortem studies have demonstrated abnormalities in glutamatergic transmission in major depression. Glutamate NMDA (N-methyl-D-aspartate) receptors are one of the major mediators of excitatory neurotransmission in the central nervous system. At synaptic sites, NMDA receptors are linked with postsynaptic density protein-95 (PSD-95) that plays a key role in mediating trafficking, clustering, and downstream signaling events, following receptor activation. In this study, we examined the expression of NMDA receptor subunits NR1, NR2A, and NR2B as well as PSD-95 in the anterior prefrontal cortex (PFC) using Western blot method. Cortical samples were obtained from age, gender and postmortem interval matched depressed and psychiatrically healthy controls. The results revealed that there was a reduced expression of the NMDA receptor subunits NR2A (−54%) and NR2B (−48%), and PSD-95 protein level (−40%) in the PFC of depressed subjects relative to controls, with no change in the NR1 subunit. The alterations in NMDA receptor subunits, especially the NR2A and NR2B, as well as PSD-95 suggest an abnormality in the NMDA receptor signaling in the PFC in major depression. Our findings in conjunction with recent clinical, cellular, and neuroimaging studies further implicate the involvement of glutamate neurotransmission in the pathophysiology of depression. This study provides additional evidence that NMDA receptor complex is a target for discovery of novel antidepressants. PMID:18992785

  12. Arthrophytum scoparium inhibits melanogenesis through the down-regulation of tyrosinase and melanogenic gene expressions in B16 melanoma cells.

    PubMed

    Chao, Hui-Chia; Najjaa, Hanen; Villareal, Myra O; Ksouri, Riadh; Han, Junkyu; Neffati, Mohamed; Isoda, Hiroko

    2013-02-01

    Melanin performs a crucial role in protecting the skin against harmful ultraviolet light. However, hyperpigmentation may lead to aesthetic problems and disorders such as solar lentigines (SL), melasma, postinflammatory hyperpigmentation and even melanoma. Arthrophytum scoparium grows in the desert in the North African region, and given this type of environment, A. scoparium exhibits adaptations for storing water and produces useful bioactive factors. In this study, the effect of A. scoparium ethanol extract (ASEE) on melanogenesis regulation in B16 murine melanoma cells was investigated. Cells treated with 0.017% (w/v) ASEE showed a significant inhibition of melanin biosynthesis in a time-dependent manner without cytotoxicity. To clarify the mechanism behind the ASEE-treated melanogenesis regulation, the expressions of tyrosinase enzyme and melanogenesis-related genes were determined. Results showed that the expression of tyrosinase enzyme was significantly decreased and Tyr, Trp-1, Mitf and Mc1R mRNA expressions were significantly down-regulated. LC-ESI-TOF-MS analysis of the extract identified the presence of six phenolic compounds: coumaric acid, cinnamic acid, chrysoeriol, cyanidin, catechol and caffeoylquinic acid. The melanogenesis inhibitory effect of ASEE may therefore be attributed to its catechol and tetrahydroisoquinoline derivative content. We report here that ASEE can inhibit melanogenesis in a time-dependent manner by decreasing the tyrosinase protein and Tyr, Trp-1, Mitf and Mc1R mRNA expressions. This is the first report on the antimelanogenesis effect of A. scoparium and on its potential as a whitening agent. PMID:23362872

  13. DDX6 post-transcriptionally down-regulates miR-143/145 expression through host gene NCR143/145 in cancer cells.

    PubMed

    Iio, Akio; Takagi, Takeshi; Miki, Kohei; Naoe, Tomoki; Nakayama, Atsuo; Akao, Yukihiro

    2013-10-01

    In various human malignancies, widespread dysregulation of microRNA (miRNA) expression is reported to occur and affects various cell growth programs. Recent studies suggest that the expression levels of miRNAs that act as tumor suppressors are frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription, and disturbances in miRNA processing. MiR-143 and -145 are well-recognized miRNAs that are highly expressed in several tissues, but down-regulated in most types of cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that DEAD-box RNA helicase 6, DDX6 (p54/RCK), post-transcriptionally down-regulated miR-143/145 expression by prompting the degradation of its host gene product, NCR143/145 RNA. In human gastric cancer cell line MKN45, DDX6 protein was abundantly expressed and accumulated in processing bodies (P-bodies). DDX6 preferentially increased the instability of non-coding RNA, NCR143/145, which encompasses the miR-143/145 cluster, and down-regulated the expression of mature miR-143/145. In human monocytic cell line THP-1, lipopolysaccharide treatment promoted the assembly of P-bodies and down-regulated the expression of NCR143/145 and its miR-143/145 rapidly. In these cells, cycloheximide treatment led to a loss of P-bodies and to an increase in NCR143/145 RNA stability, thus resulting in up-regulation of miR-143/145 expression. These data demonstrate that DDX6 contributed to the control of NCR143/145 RNA stability in P-bodies and post-transcriptionally regulated miR-143/145 expression in cancer cells. PMID:23932921

  14. Acacetin inhibits in vitro and in vivo angiogenesis and down-regulates Stat signaling and VEGF expression

    PubMed Central

    Bhat, Tariq A.; Nambiar, Dhanya; Tailor, Dhanir; Pal, Arttatrana; Agarwal, Rajesh; Singh, Rana P.

    2013-01-01

    Angiogenesis is an effective target in cancer control. The anti-angiogenic efficacy and associated mechanisms of acacetin, a plant flavone, is poorly known. In the present study, acacetin inhibited growth and survival (upto 92%, p<0.001), and capillary-like tube formation on matrigel (upto 98%, p<0.001) by human umbilical vein endothelial cells (HUVEC) in regular condition, as well as VEGF-induced and tumor cells conditioned medium-stimulated growth conditions. It caused retraction and disintegration of preformed capillary networks (upto 91%, p<0.001). HUVEC migration and invasion were suppressed by 68-100% (p<0.001). Acacetin inhibited Stat-1 (Tyr701) and Stat-3 (Tyr705) phosphorylation, and down-regulated pro-angiogenic factors including VEGF, eNOS, iNOS, MMP-2 and bFGF in HUVEC. It also suppressed nuclear localization of pStat-3 (Tyr705). Acacetin strongly inhibited capillary sprouting and networking from rat aortic rings and fertilized chicken egg chorioallantoic membrane (CAM) (~71%, p<0.001). Furthermore, it suppressed angiogenesis in matrigel plugs implanted in Swiss albino mice. Acacetin also inhibited tyrosine phosphorylation of Stat-1 and Stat-3, and expression of VEGF in cancer cells. Overall, acacetin inhibits Stat signaling and suppresses angiogenesis in vitro, ex vivo and in vivo, and therefore, it could be a potential agent to inhibit tumor angiogenesis and growth. PMID:23943785

  15. Niemann-Pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine

    SciTech Connect

    Duval, Caroline; Touche, Veronique; Tailleux, Anne; Fruchart, Jean-Charles; Fievet, Catherine; Clavey, Veronique; Staels, Bart . E-mail: Bart.Staels@pasteur-lille.fr; Lestavel, Sophie

    2006-02-24

    Niemann-Pick C1 like 1 (NPC1L1) is a protein critical for intestinal cholesterol absorption. The nuclear receptors peroxisome proliferator-activated receptor alpha (PPAR{alpha}) and liver X receptors (LXR{alpha} and LXR{beta}) are major regulators of cholesterol homeostasis and their activation results in a reduced absorption of intestinal cholesterol. The goal of this study was to define the role of PPAR{alpha} and LXR nuclear receptors in the regulation of NPC1L1 gene expression. We show that LXR activators down-regulate NPC1L1 mRNA levels in the human enterocyte cell line Caco-2/TC7, whereas PPAR{alpha} ligands have no effect. Furthermore, NPC1L1 mRNA levels are decreased in vivo, in duodenum of mice treated with the LXR agonist T0901317. In conclusion, the present study identifies NPC1L1 as a novel LXR target gene further supporting a crucial role of LXR in intestinal cholesterol homeostasis.

  16. Pyrroloquinoline Quinone Induces Cancer Cell Apoptosis via Mitochondrial-Dependent Pathway and Down-Regulating Cellular Bcl-2 Protein Expression.

    PubMed

    Min, Zhihui; Wang, Lingyan; Jin, Jianjun; Wang, Xiangdong; Zhu, Bijun; Chen, Hao; Cheng, Yunfeng

    2014-01-01

    Pyrroloquinoline quinone (PQQ) has been reported as a promising agent that might contribute to tumor cell apoptosis and death, yet little is known on its mechanisms. In current study, the effect of PQQ on cell proliferation and mitochondrial-dependent apoptosis were examined in 3 solid tumor cell lines (A549, Neuro-2A and HCC-LM3). PQQ treatment at low to medium dosage exhibited potent anti-tumor activity on A549 and Neuro-2A cells, while had comparably minimal impact on the viabilities of 2 human normal cell lines (HRPTEpiC and HUVEC). The apoptosis of the 3 tumor cell lines induced by PQQ were increased in a concentration-dependent manner, which might be attributed to the accumulation of intracellular reactive oxygen species (ROS), decline in ATP levels and dissipation of mitochondrial membrane potential (MMP), in conjunction with down-regulation of Bcl-2 protein expression, up-regulation of activated caspase-3, and disturbed phosphorylated MAPK protein levels. PQQ induced tumor cells apoptosis was significantly alleviated by pan-caspase inhibitor Z-VAD-FMK. The present work highlights the potential capability of PQQ as an anti-tumor agent with low toxicity towards normal cells through activating mitochondrial-dependent apoptosis pathways, and warrants its development for cancer therapy. PMID:25161699

  17. Mxi1 inhibits the proliferation of U87 glioma cells through down-regulation of cyclin B1 gene expression

    PubMed Central

    Manni, I; Tunici, P; Cirenei, N; Albarosa, R; Colombo, B M; Roz, L; Sacchi, A; Piaggio, G; Finocchiaro, G

    2002-01-01

    Mxi1 is a Mad family member that plays a role in cell proliferation and differentiation. To test the role of Mxi1 on tumorigenesis of glioma cells we transfected a CMV-driven MXI1 cDNA in U87 human glioblastoma cells. Two clones were isolated expressing MXI1 levels 18- and 3.5-fold higher than wild-type U87 cells (clone U87.Mxi1.14 and U87.Mxi1.22, respectively). In vivo, U87.Mxi1.14 cells were not tumorigenic in nude mice and delayed development of tumours was observed with U87.Mxi1.22 cells. In vitro, the proliferation rate was partially and strongly inhibited in U87.Mxi1.22 and U87.Mxi1.14 cells respectively. The cell cycle analysis revealed a relevant accumulation of U87.Mxi1.14 cells in the G2/M phase. Interestingly, the expression of cyclin B1 was inhibited to about 60% in U87.Mxi1.14 cells. This inhibition occurs at the transcriptional level and depends, at least in part, on the E-box present on the cyclin B1 promoter. Consistent with this, the endogenous Mxi1 binds this E-box in vitro. Thus, our findings indicate that Mxi1 can act as a tumour suppressor in human glioblastomas through a molecular mechanism involving the transcriptional down-regulation of cyclin B1 gene expression. British Journal of Cancer (2002) 86, 477–484. DOI: 10.1038/sj/bjc/6600065 www.bjcancer.com © 2002 The Cancer Research Campaign PMID:11875718

  18. The non-structural protein Nsp2TF of porcine reproductive and respiratory syndrome virus down-regulates the expression of Swine Leukocyte Antigen class I.

    PubMed

    Cao, Qian M; Subramaniam, Sakthivel; Ni, Yan-Yan; Cao, Dianjun; Meng, Xiang-Jin

    2016-04-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically-important global swine pathogen. Here we demonstrated that PRRSV down-regulates Swine Leukocyte Antigen class I (SLA-I) expression in porcine alveolar macrophages, PK15-CD163 cells and monocyte-derived dendritic cells. To identify the viral protein(s) involved in SLA-I down-regulation, we tested all 22 PRRSV structural and non-structural proteins and identified that Nsp1α and Nsp2TF, and GP3 significantly down-regulated SLA-I expression with Nsp2TF showing the greatest effect. We further generated a panel of mutant viruses in which the Nsp2TF protein synthesis was abolished, and found that the two mutants with disrupted -2 ribosomal frameshifting elements and additional stop codons in the TF domain were unable to down-regulate SLA-I expression. Additionally we demonstrated that the last 68 amino acids of TF domain in Nsp2TF are critical for this function. Collectively, the results indicate a novel function of Nsp2TF in negative modulation of SLA-I expression. PMID:26895249

  19. Estrogen down-regulates nicotine-induced adhesion molecule expression via nongenomic signal pathway in endothelial cells.

    PubMed

    Wang, Yajing; Wang, Zhaoxia; Wang, Lianyun; Zhou, Ying; Zhao, Yangxing; Liu, Liming; Yao, Chenjiang; Qiao, Zhongdong

    2006-06-01

    Although gonadal hormone mostly causes genotropic actions through the members of nuclear receptor family, it also can regulate these actions via membrane receptor. To explore the possibility of plasma membrane estrogen receptors (mER) mediating genotropic events, we have investigated estrogen's effect on nicotine-stimulated adhesion molecule expression and evaluated whether this effect depends on calcium, MAPK signal pathway. Fluorescence Spectroscopy analysis of Ca2+ from human umbilical vein endothelial cells (HUVECs) showed through mER, estrogen induced a rapid rise of intracellular free Ca2+ concentration and this rise could not be inhibited by tamoxifen (classic ER inhibitor). In the context of nicotine stimulating, however, estrogen attenuated phosphorylation of mitogen-activated protein kinase (MAPK) family members, extracellular signal regulated kinase 1/2 (ERK1/2), p38 but not c-Jun-N-terminal kinase (JNK) in HUVECs and this effect could not still be prevented by tamoxifen. In the meantime, estrogen also down-regulated surface/soluble vascular cell adhesion molecule (VCAM-1, sVCAM-1) and endothelial selectin (E-selectin, sE-selectin) levels, which was not abolished by tamoxifen either. Moreover, calcium chelator BAPTA, ERK1/2 inhibitor PD98059, p38 inhibitor SB203580 significantly reduced the production of nicotine-activated surface/soluble VCAM-1 and E-selectin and both of the remained levels were no longer regulated by estrogen. Our study here provides the information of decrease effect of mER-mediated estrogen through Ca2+ and ERK1/2, p38 MAPK signaling pathway on nicotine-stimulated expression of surface/soluble VCAM-1 and E-selectin in HUVECs. PMID:16644474

  20. Benzimidazole-2-carboxamides as novel NR2B selective NMDA receptor antagonists.

    PubMed

    Borza, István; Kolok, Sándor; Gere, Anikó; Nagy, József; Fodor, László; Galgóczy, Kornél; Fetter, József; Bertha, Ferenc; Agai, Béla; Horváth, Csilla; Farkas, Sándor; Domány, György

    2006-09-01

    A novel series of benzimidazole-2-carboxamide derivatives was prepared and identified as NR2B selective NMDA receptor antagonists. The influence of some structural elements, like H-bond donor groups placed on the benzimidazole skeleton and the substitution pattern of the piperidine ring, on the biological activity was studied. Compound 6a showed excellent analgetic activity in the mouse formalin test following po administration. PMID:16782335

  1. Allosteric modulators of NR2B-containing NMDA receptors: molecular mechanisms and therapeutic potential.

    PubMed

    Mony, Laetitia; Kew, James N C; Gunthorpe, Martin J; Paoletti, Pierre

    2009-08-01

    N-methyl-D-aspartate receptors (NMDARs) are ion channels gated by glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS). They are widespread in the CNS and are involved in numerous physiological and pathological processes including synaptic plasticity, chronic pain and psychosis. Aberrant NMDAR activity also plays an important role in the neuronal loss associated with ischaemic insults and major degenerative disorders including Parkinson's and Alzheimer's disease. Agents that target and alter NMDAR function may, thus, have therapeutic benefit. Interestingly, NMDARs are endowed with multiple extracellular regulatory sites that recognize ions or small molecule ligands, some of which are likely to regulate receptor function in vivo. These allosteric sites, which differ from agonist-binding and channel-permeation sites, provide means to modulate, either positively or negatively, NMDAR activity. The present review focuses on allosteric modulation of NMDARs containing the NR2B subunit. Indeed, the NR2B subunit confers a particularly rich pharmacology with distinct recognition sites for exogenous and endogenous allosteric ligands. Moreover, NR2B-containing receptors, compared with other NMDAR subtypes, appear to contribute preferentially to pathological processes linked to overexcitation of glutamatergic pathways. The actions of extracellular H+, Mg2+, Zn2+, of polyamines and neurosteroids, and of the synthetic compounds ifenprodil and derivatives ('prodils') are presented. Particular emphasis is put upon the structural determinants and molecular mechanisms that underlie the effects exerted by these agents. A better understanding of how NR2B-containing NMDARs (and NMDARs in general) operate and how they can be modulated should help define new strategies to counteract the deleterious effects of dysregulated NMDAR activity. PMID:19594762

  2. Down-regulation of cytochrome P450 2C family members and positive acute-phase response gene expression by peroxisome proliferator chemicals.

    PubMed

    Corton, J C; Fan, L Q; Brown, S; Anderson, S P; Bocos, C; Cattley, R C; Mode, A; Gustafsson, J A

    1998-09-01

    In this study, we show that peroxisome proliferator chemical (PPC) exposure leads to alterations in the expression of genes in rat liver regulated by the sex-specific growth hormone secretory pattern and induced during inflammation. Expression of the male-specific cytochrome P450 (P450) 2C11 and alpha2 urinary globulin (alpha2u) genes and the female-specific P450 2C12 gene was down-regulated by some PPC. Expression of P450 2C13, also under control by the sex-specific growth hormone secretory pattern, was not altered by PPC treatment, indicating that regulation of CYP2C family members does not involve perturbation of the growth hormone secretory pattern. In contrast to the increases in expression observed when inflammation was induced in male rats, two positive acute-phase response genes, alpha1-acid glycoprotein and beta-fibrinogen, were decreased by PPC exposure. The down-regulation of the P450 2C11 by WY-14,643 could be reproduced in cultured rat hepatocytes, indicating the down-regulation is a direct effect. Experiments in wild-type mice and mice that lacked a functional peroxisome proliferator-activated receptor-alpha gene showed that down-regulation by WY of alpha1-acid glycoprotein, beta-fibrinogen, and a mouse homologue of alpha2u was dependent on peroxisome proliferator-activated receptor-alpha expression. Our results demonstrate that PPC exposure leads to down-regulation of diverse liver-specific genes, including CYP2C family members important in hormonal homeostasis and acute-phase response genes important in inflammatory responses. PMID:9730905

  3. Exosomes Derived from Mesenchymal Stem Cells Suppress Angiogenesis by Down-Regulating VEGF Expression in Breast Cancer Cells

    PubMed Central

    Lee, Jong-Kuen; Park, Sae-Ra; Jung, Bong-Kwang; Jeon, Yoon-Kyung; Lee, Yeong-Shin; Kim, Min-Kyoung; Kim, Yong-Goo; Jang, Ji-Young; Kim, Chul-Woo

    2013-01-01

    Exosomes are small membrane vesicles released by a variety of cell types. Exosomes contain genetic materials, such as mRNAs and microRNAs (miRNAs), implying that they may play a pivotal role in cell-to-cell communication. Mesenchymal stem cells (MSCs), which potentially differentiate into multiple cell types, can migrate to the tumor sites and have been reported to exert complex effects on tumor progression. To elucidate the role of MSCs within the tumor microenvironment, previous studies have suggested various mechanisms such as immune modulation and secreted factors of MSCs. However, the paracrine effects of MSC-derived exosomes on the tumor microenvironment remain to be explored. The hypothesis of this study was that MSC-derived exosomes might reprogram tumor behavior by transferring their molecular contents. To test this hypothesis, exosomes from MSCs were isolated and characterized. MSC-derived exosomes exhibited different protein and RNA profiles compared with their donor cells and these vesicles could be internalized by breast cancer cells. The results demonstrated that MSC-derived exosomes significantly down-regulated the expression of vascular endothelial growth factor (VEGF) in tumor cells, which lead to inhibition of angiogenesis in vitro and in vivo. Additionally, miR-16, a miRNA known to target VEGF, was enriched in MSC-derived exosomes and it was partially responsible for the anti-angiogenic effect of MSC-derived exosomes. The collective results suggest that MSC-derived exosomes may serve as a significant mediator of cell-to-cell communication within the tumor microenvironment and suppress angiogenesis by transferring anti-angiogenic molecules. PMID:24391924

  4. Apigenin suppresses migration and invasion of transformed cells through down-regulation of C-X-C chemokine receptor 4 expression

    SciTech Connect

    Wang, Lei; Kuang, Lisha; Hitron, John Andrew; Son, Young-Ok; Wang, Xin; Budhraja, Amit; Lee, Jeong-Chae; Pratheeshkumar, Poyil; Chen, Gang; Zhang, Zhuo; Luo, Jia; Shi, Xianglin

    2013-10-01

    Environmental exposure to arsenic is known to cause various cancers. There are some potential relationships between cell malignant transformation and C-X-C chemokine receptor type 4 (CXCR4) expressions. Metastasis, one of the major characteristics of malignantly transformed cells, contributes to the high mortality of cells. CXCR4 and its natural chemokine ligand C-X-C motif ligand 12 (CXCL12) play a critical role in metastasis. Therefore, identification of nutritional factors which are able to inhibit CXCR4 is important for protection from environmental arsenic-induced carcinogenesis and for abolishing metastasis of malignantly transformed cells. The present study demonstrates that apigenin (4′,5,7-trihydroxyflavone), a natural dietary flavonoid, suppressed CXCR4 expression in arsenic-transformed Beas-2B cells (B-AsT) and several other types of transformed/cancer cells in a dose- and time-dependent manner. Neither proteasome nor lysosome inhibitor had any effect in reducing the apigenin-induced down-regulation of CXCR4, indicating that apigenin-induced down-regulation of CXCR4 is not due to proteolytic degradation. The down-regulation of CXCR4 is mainly due to the inhibition of nuclear factor κB (NF-κB) transcriptional activity. Apigenin also abolished migration and invasion of transformed cells induced by CXCL12. In a xenograft mouse model, apigenin down-regulated CXCR4 expression and suppressed tumor growth. Taken together, our results show that apigenin is a novel inhibitor of CXCR4 expression. This dietary flavonoid has the potential to suppress migration and invasion of transformed cells and prevent environmental arsenic-induced carcinogenesis. - Highlights: • Apigenin has a potential in preventing environmental arsenic induced carcinogenesis. • Apigenin suppresses CXCR4 in malignant transformed cells in vitro and in vivo. • The down-regulation of CXCR4 is mainly due to inhibition of NF-κB activity.

  5. Down Regulation of Gene Expression Between the Diapause Initiation and Maintenance Phases of the Colorado Potato Beetle, Leptinotarsa Decemlineata

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The diapause initiation and maintenance phases of the Colorado potato beetle, Leptinotarsa decemlineata were screened. Eight transcripts were found to be down regulated as the beetles enter the diapause maintenance phase of diapause development after day 15 postemergence. These transcripts were also...

  6. Cyclic mechanical stretch down-regulates cathelicidin antimicrobial peptide expression and activates a pro-inflammatory response in human bronchial epithelial cells

    PubMed Central

    Gudjonsson, Thorarinn; Karason, Sigurbergur

    2015-01-01

    Mechanical ventilation (MV) of patients can cause damage to bronchoalveolar epithelium, leading to a sterile inflammatory response, infection and in severe cases sepsis. Limited knowledge is available on the effects of MV on the innate immune defense system in the human lung. In this study, we demonstrate that cyclic stretch of the human bronchial epithelial cell lines VA10 and BCi NS 1.1 leads to down-regulation of cathelicidin antimicrobial peptide (CAMP) gene expression. We show that treatment of VA10 cells with vitamin D3 and/or 4-phenyl butyric acid counteracted cyclic stretch mediated down-regulation of CAMP mRNA and protein expression (LL-37). Further, we observed an increase in pro-inflammatory responses in the VA10 cell line subjected to cyclic stretch. The mRNA expression of the genes encoding pro-inflammatory cytokines IL-8 and IL-1β was increased after cyclic stretching, where as a decrease in gene expression of chemokines IP-10 and RANTES was observed. Cyclic stretch enhanced oxidative stress in the VA10 cells. The mRNA expression of toll-like receptor (TLR) 3, TLR5 and TLR8 was reduced, while the gene expression of TLR2 was increased in VA10 cells after cyclic stretch. In conclusion, our in vitro results indicate that cyclic stretch may differentially modulate innate immunity by down-regulation of antimicrobial peptide expression and increase in pro-inflammatory responses. PMID:26664810

  7. Insulin down-regulates the expression of ubiquitin E3 ligases partially by inhibiting the activity and expression of AMP-activated protein kinase in L6 myotubes

    PubMed Central

    Deng, Hu-Ping; Chai, Jia-Ke; Shen, Chuan-An; Zhang, Xi-Bo; Ma, Li; Sun, Tian-Jun; Hu, Qing-Gang; Chi, Yun-Fei; Dong, Ning

    2015-01-01

    While insulin is an anabolic hormone, AMP-activated protein kinase (AMPK) is not only a key energy regulator, but it can also control substrate metabolism directly by inducing skeletal muscle protein degradation. The hypothesis of the present study was that insulin inhibits AMPK and thus down-regulates the expression of the ubiquitin E3 ligases, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) in skeletal muscle cells. Differentiated L6 myotubes were treated with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and/or compound C to stimulate and/or block AMPK respectively. These treatments were also conducted in the presence or absence of insulin and the cells were analysed by western blot and quantitative real-time PCR. In addition, nuleotide levels were determined using HPLC. The activation of AMPK with AICAR enhanced the mRNA levels of MAFbx and MuRF1. Insulin reduced the phosphorylation and activity AMPK, which was accompanied by reduced MAFbx and MuRF1 mRNA levels. Using a protein kinase B (PKB/Akt) inhibitor, we found that insulin regulates AMPK through the activation of Akt. Furthermore, insulin down-regulated AMPK α2 mRNA. We conclude that insulin inhibits AMPK through Akt phosphorylation in L6 myotubes, which may serve as a possible signalling pathway for the down-regulation of protein degradation. In addition, decreased expression of AMPK α2 may partially participate in inhibiting the activity of AMPK. PMID:26193886

  8. Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

    PubMed Central

    2013-01-01

    Introduction Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration. Methods Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age. Results Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice

  9. Down-regulated expression of monocyte/macrophage major histocompatibility complex receptors in human and mouse monocytes by expression of their ligands

    PubMed Central

    Yamana, H; Tashiro-Yamaji, J; Hayashi, M; Maeda, S; Shimizu, T; Tanigawa, N; Uchiyama, K; Kubota, T; Yoshida, R

    2014-01-01

    Mouse monocyte/macrophage major histocompatibility complex (MHC) receptor 1 (MMR1; or MMR2) specific for H-2Dd (or H-2Kd) molecules is expressed on monocytes from non-H-2Dd (or non-H-2Kd), but not those from H-2Dd (or H-2Kd), inbred mice. The MMR1 and/or MMR2 is essential for the rejection of H-2Dd- and/or H-2Kd-transgenic mouse skin onto C57BL/6 (H-2DbKb) mice. Recently, we found that human leucocyte antigen (HLA)-B44 was the sole ligand of human MMR1 using microbeads that had been conjugated with 80 types of HLA class I molecules covering 94·2% (or 99·4%) and 92·4% (or 96·2%) of HLA-A and B molecules of Native Americans (or Japanese), respectively. In the present study, we also explored the ligand specificity of human MMR2 using microbeads. Microbeads coated with HLA-A32, HLA-B13 or HLA-B62 antigens bound specifically to human embryonic kidney (HEK)293T or EL-4 cells expressing human MMR2 and to the solubilized MMR2-green fluorescent protein (GFP) fusion protein; and MMR2+ monocytes from a volunteer bound HLA-B62 molecules with a Kd of 8·7 × 10−9 M, implying a three times down-regulation of MMR2 expression by the ligand expression. H-2Kd (or H-2Dd) transgene into C57BL/6 mice down-regulated not only MMR2 (or MMR1) but also MMR1 (or MMR2) expression, leading to further down-regulation of MMR expression. In fact, monocytes from two (i.e. MMR1+/MMR2+ and MMR1–/MMR2–) volunteers bound seven to nine types of microbeads among 80, indicating ≤ 10 types of MMR expression on monocytes. The physiological role of constitutive MMRs on monocytes possibly towards allogeneic (e.g. fetal) cells in the blood appears to be distinct from that of inducible MMRs on macrophages toward allografts in tissue. PMID:24842626

  10. ER stress upregulated PGE2/IFNγ-induced IL-6 expression and down-regulated iNOS expression in glial cells

    NASA Astrophysics Data System (ADS)

    Hosoi, Toru; Honda, Miya; Oba, Tatsuya; Ozawa, Koichiro

    2013-12-01

    The disruption of endoplasmic reticulum (ER) function can lead to neurodegenerative disorders, in which inflammation has also been implicated. We investigated the possible correlation between ER stress and immune function using glial cells. We demonstrated that ER stress synergistically enhanced prostaglandin (PG) E2 + interferon (IFN) γ-induced interleukin (IL)-6 production. This effect was mediated through cAMP. Immune-activated glial cells produced inducible nitric oxide synthase (iNOS). Interestingly, ER stress inhibited PGE2 + IFNγ-induced iNOS expression. Similar results were obtained when cells were treated with dbcAMP + IFNγ. Thus, cAMP has a dual effect on immune reactions; cAMP up-regulated IL-6 expression, but down-regulated iNOS expression under ER stress. Therefore, our results suggest a link between ER stress and immune reactions in neurodegenerative diseases.

  11. Role for the NR2B Subunit of the NMDA Receptor in Mediating Light Input to the Circadian System

    PubMed Central

    Wang, LM; Schroeder, A; Loh, D; Smith, D; Lin, K; Han, JH; Michel, S; Hummer, DL; Ehlen, JC; Albers, HE; Colwell, CS

    2008-01-01

    Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells that utilize glutamate as a neurotransmitter. A variety of evidence suggests that the release of glutamate then activates N-methyl-Daspartate (NMDA) receptors within the SCN and triggers a signaling cascade that ultimately leads to phase shifts in the circadian system. In this study, we first sought to explore the role of the NR2B subunit in mediating the effects of light on the circadian system. We found that localized microinjection of the NR2B subunit antagonist ifenprodil into the SCN region inhibits the magnitude of light-induced phase shifts of the circadian rhythm in wheel-running activity. Next, we found that the NR2B message and levels of phospho-NR2B levels vary with time of day in SCN tissue using semi-quantitative real-time PCR and Western blot analysis, respectively. Functionally, we found that blocking the NR2B subunit with ifenprodil significantly reduced the magnitude of NMDA currents recorded in SCN neurons. Ifenprodil also significantly reduced the magnitude of NMDA-induced calcium changes in SCN cells. Together, these results demonstrate that the NR2B subunit is an important component of NMDA receptor mediated responses within SCN neurons and that this subunit contributes to light-induced phase shifts of the mammalian circadian system. PMID:18380671

  12. DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

    SciTech Connect

    Li, C.-C.; Lii, C.-K.; Liu, K.-L.; Yang, J.-J.; Chen, H.-W.

    2007-12-15

    The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 {mu}M arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.

  13. CREB1-driven expression of miR-320a promotes mitophagy by down-regulating VDAC1 expression during serum starvation in cervical cancer cells

    PubMed Central

    Liu, Min; Li, Xin; Tang, Hua

    2015-01-01

    The altered expression of miRNAs in response to stresses contributes to cancer pathogenesis. However, little is known regarding the mechanism by which cellular stresses drive alterations in miRNA expression. Here, we found that serum starvation enhanced mitophagy by downregulating the mitophagy-associated protein voltage-dependent anion channel 1 (VDAC1) and by inducing the expression of miR-320a and the transcription factor cAMP responsive element binding protein 1(CREB1). Furthermore, we cloned the promoter of miR-320a and identified the core promoter of miR-320a in the upstream −16 to −130 region of pre-miR-320a. Moreover, CREB1 was found to bind to the promoter of miR-320a to activate its expression and to induce mitophagy during serum starvation. Collectively, our results reveal a new mechanism underlying serum starvation-induced mitophagy in which serum starvation induces CREB1 expression, in turn activating miR-320a expression, which then down-regulates VDAC1 expression to facilitate mitophagy. These findings may provide new insights into cancer cell survival in response to environmental stresses. PMID:26472185

  14. Aquaporin 9 is down-regulated in hepatocellular carcinoma and its over-expression suppresses hepatoma cell invasion through inhibiting epithelial-to-mesenchymal transition.

    PubMed

    Zhang, Wen-Guang; Li, Chuan-Fei; Liu, Min; Chen, Xiao-Feng; Shuai, Kai; Kong, Xin; Lv, Lin; Mei, Zhe-Chuan

    2016-08-10

    Aquaporin 9 (AQP9) is the main aquaglyceroporin in the liver. Few studies have been performed regarding the role of AQP9 in hepatocellular carcinoma (HCC). Here, we report the expression and function of AQP9 in HCC tissues and cell lines. We found that AQP9 mRNA and protein levels were down-regulated in HCC tissues and human hepatoma cell lines compared to the para-cancer normal liver tissues and normal hepatocyte line, respectively. In a human HCC SMMC-7721 cell line, over-expression of AQP9 suppressed cell invasion in vitro and xenograft tumor growth in vivo. AQP9 over-expression increased the expression of E-cadherin and decreased the expression of N-cadherin in SMMC-7721 cells and xenografted tumors, which was correlated with decreased levels of phosphoinositide 3-kinase (PI3K) and p-Akt. Conversely, using siRNA to knock down AQP9 over-expression could reverse the phenotype caused by AQP9 over-expression. Our findings suggest that AQP9 is down-regulated in hepatocellular carcinoma and its over-expression suppresses hepatoma cell invasion through inhibiting epithelial-to-mesenchymal transition. PMID:27216981

  15. PI3K/Akt pathway restricts epithelial adhesion of Dr+ Escherichia coli by down-regulating the expression of Decay Accelerating Factor (DAF)

    PubMed Central

    Banadakoppa, Manu; Goluszko, Pawel; Liebenthal, Daniel; Nowicki, Bogdan J.; Nowicki, Stella; Yallampalli, Chandra

    2014-01-01

    The urogenital microbial infection in pregnancy is an important cause of maternal and neonatal morbidity and mortality. Uropathogenic Escherichia coli strains which express Dr fimbriae (Dr+) are associated with unique gestational virulence and they utilize cell surface decay accelerating factor (DAF or CD55) as one of the cellular receptor before invading the epithelial cells. Previous studies in our laboratory established that nitric oxide reduces the rate of E. coli invasion by delocalizing the DAF protein from cell surface lipid rafts and down-regulating its expression. The phosphoinositide 3-kinase/ protein kinase B (PI3K/Akt) cell signal pathway plays an important role in host-microbe interaction because many bacteria including E. coli activate this pathway in order to establish infection. In the present study we showed that the PI3K/Akt pathway negatively regulates the expression of DAF on the epithelial cell surface and thus inhibits the adhesion of Dr+ E. coli to epithelial cells. Initially, using two human cell lines Ishikawa and HeLa which differ in constitutive activity of PI3K/Akt we showed that DAF levels were associated with the PI3K/Akt pathway. We then showed that the DAF gene expression was up-regulated and the Dr+ E. coli adhesion increased after the suppression of PI3K/Akt pathway in Ishikawa cells using inhibitor LY-294002, and a plasmid which allowed the expression of PI3K/Akt regulatory protein PTEN. The down-regulation of PTEN protein using PTEN-specific siRNA activated the PI3K/Akt pathway, down-regulated the DAF and decreased the adhesion of Dr+ E. coli. We conclude that the PI3K/Akt pathway regulated the DAF expression in a nitric oxide independent manner. PMID:24599886

  16. Glucocorticoid mediates water avoidance stress-sensitized colon-bladder cross-talk via RSK2/PSD-95/NR2B in rats.

    PubMed

    Peng, Hsien-Yu; Hsieh, Ming-Chun; Lai, Cheng-Yuan; Chen, Gin-Den; Huang, Yi-Ping; Lin, Tzer-Bin

    2012-11-01

    Unexpected environmental and social stimuli could trigger stress. Although coping with stress is essential for survival, long-term stress impacts visceral functions, and therefore, it plays a role in the development and exacerbation of symptoms of gastrointestinal/urogenital disorders. The aim of this study is to characterize the role of corticosterone in stress-sensitized colon-bladder cross-talk, a phenomenon presumed to underlie the comorbidity of functional bowel and bladder disorders. Cystometry and protein/mRNA expression in the lumbosacral dorsal horn (L6-S1) in response to intracolonic mustard oil (MO) instillation were analyzed in female Wistar-Kyoto rats subjected to water avoidance stress (WAS; 1 h/day for 10 days) or sham stress (WAsham). Whereas it had no effect on baseline-voiding function, chronic stress upregulated plasma corticosterone concentration and dorsal horn spinal p90 ribosomal S6 kinase 2 (RSK2) protein/mRNA levels, and RSK2 immunoreactivity colocalized with NeuN-positive neurons. Intracolonic MO dose-dependently decreased intrercontraction intervals and threshold pressure, provoked spinal RSK2 and NR2B phosphorylation, and enhanced PSD-95-RSK2 and PSD-95-NR2B coupling. Intrathecal kaempferol (a RSK2 activation antagonist; 30 min before MO instillation), bilateral adrenalectomy (7 days prior the stress paradigm), and subcutaneous RU-38486 (a glucocorticoid receptor antagonist; 30 min daily before stress sessions), but not RU-28318 (a mineralocorticoid receptor antagonist), attenuated MO-induced bladder hyperactivity, protein phosphorylation, and protein-protein interactions in the WAS group. Our results suggest that stress-associated glucocorticoid release mediates WAS-dependent sensitization of colon-bladder cross-talk via the spinal RSK2/PSD-95/NR2B cascade and offer a possibility for developing pharmacological strategies for the treatment of stress-related pelvic pain. PMID:23125098

  17. Cortical effect of oxaliplatin associated with sustained neuropathic pain: exacerbation of cortical activity and down-regulation of potassium channel expression in somatosensory cortex.

    PubMed

    Thibault, Karine; Calvino, Bernard; Dubacq, Sophie; Roualle-de-Rouville, Marie; Sordoillet, Vallier; Rivals, Isabelle; Pezet, Sophie

    2012-08-01

    Oxaliplatin is a third-generation platinum-based chemotherapy drug that has gained importance in the treatment of advanced metastatic colorectal cancer. Its dose-limiting side effect is the production of chronic peripheral neuropathy. Using a modified model of oxaliplatin-induced sensory neuropathy, we investigated plastic changes at the cortical level as possible mechanisms underlying the chronicity of pain sensation in this model. Changes in gene expression were studied using DNA microarray which revealed that when oxaliplatin-treated animals displayed clinical neuropathic pain symptoms, including mechanical and thermal hypersensitivity, approximately 900 were down-regulated in the somatosensory cortex. Because of the known role of potassium channels in neuronal excitability, the study further focussed on the down-regulation of these channels as the possible molecular origin of cortical hyperexcitability. Quantification of the magnitude of neuronal extracellular signal-regulated kinase (ERK) phosphorylation in cortical neurons as a marker of neuronal activity revealed a 10-fold increase induced by oxaliplatin treatment, suggesting that neurons of cortical areas involved in transmission of painful stimuli undergo a chronic cortical excitability. We further demonstrated, using cortical injection of lentiviral vector shRNA against Kv2.2, that down-regulation of this potassium channel in naive animals induced a sustained thermal and mechanical hypersensitivity. In conclusion, although the detailed mechanisms leading to this cortical excitability are still unknown, our study demonstrated that a cortical down regulation of potassium channels could underlie pain chronicity in this model of chemotherapy-induced neuropathic pain. PMID:22652385

  18. Down-regulation of IKKβ expression in glioma-infiltrating microglia/macrophages is associated with defective inflammatory/immune gene responses in glioblastoma

    PubMed Central

    Nauman, Pawel; Gabrusiewicz, Konrad; Sielska, Małgorzata; Przanowski, Piotr; Maleszewska, Marta; Rajan, Wenson D.; Pszczolkowska, Dominika; Tykocki, Tomasz; Grajkowska, Wieslawa; Kotulska, Katarzyna; Roszkowski, Marcin; Kostkiewicz, Boguslaw; Kaminska, Bozena

    2015-01-01

    Glioblastoma (GBM) is an aggressive malignancy associated with profound host immunosuppression. Microglia and macrophages infiltrating GBM acquire the pro-tumorigenic, M2 phenotype and support tumor invasion, proliferation, survival, angiogenesis and block immune responses both locally and systematically. Mechanisms responsible for immunological deficits in GBM patients are poorly understood. We analyzed immune/inflammatory gene expression in five datasets of low and high grade gliomas, and performed Gene Ontology and signaling pathway analyses to identify defective transcriptional responses. The expression of many immune/inflammatory response and TLR signaling pathway genes was reduced in high grade gliomas compared to low grade gliomas. In particular, we found the reduced expression of the IKBKB, a gene coding for IKKβ, which phosphorylates IκB proteins and represents a convergence point for most signal transduction pathways leading to NFκB activation. The reduced IKBKB expression and IKKβ levels in GBM tissues were demonstrated by qPCR, Western blotting and immunohistochemistry. The IKKβ expression was down-regulated in microglia/macrophages infiltrating glioblastoma. NFκB activation, prominent in microglia/macrophages infiltrating low grade gliomas, was reduced in microglia/macrophages in glioblastoma tissues. Down-regulation of IKBKB expression and NFκB signaling in microglia/macrophages infiltrating glioblastoma correlates with defective expression of immune/inflammatory genes and M2 polarization that may result in the global impairment of anti-tumor immune responses in glioblastoma. PMID:26427514

  19. Down-Regulation of EBV-LMP1 Radio-Sensitizes Nasal Pharyngeal Carcinoma Cells via NF-κB Regulated ATM Expression

    PubMed Central

    Xiao, Lanbo; Tang, Min; Liu, Liyu; Li, Zijian; Deng, Mengyao; Sun, Lunquan; Cao, Ya

    2011-01-01

    Background The latent membrane protein 1 (LMP1) encoded by EBV is expressed in the majority of EBV-associated human malignancies and has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. In previous studies we experimentally demonstrated that down-regulation of LMP1 expression by DNAzymes could increase radiosensitivity both in cells and in a xenograft NPC model in mice. Results In this study we explored the molecular mechanisms underlying the radiosensitization caused by the down-regulation of LMP1 in nasopharyngeal carcinoma. It was confirmed that LMP1 could up-regulate ATM expression in NPCs. Bioinformatic analysis of the ATM ptomoter region revealed three tentative binding sites for NF-κB. By using a specific inhibitor of NF-κB signaling and the dominant negative mutant of IkappaB, it was shown that the ATM expression in CNE1-LMP1 cells could be efficiently suppressed. Inhibition of LMP1 expression by the DNAzyme led to attenuation of the NF-κB DNA binding activity. We further showed that the silence of ATM expression by ATM-targeted siRNA could enhance the radiosensitivity in LMP1 positive NPC cells. Conclusions Together, our results indicate that ATM expression can be regulated by LMP1 via the NF-κB pathways through direct promoter binding, which resulted in the change of radiosensitivity in NPCs. PMID:22096476

  20. Negative Allosteric Modulators Selective for The NR2B Subtype of The NMDA Receptor Impair Cognition in Multiple Domains.

    PubMed

    Weed, Michael R; Bookbinder, Mark; Polino, Joseph; Keavy, Deborah; Cardinal, Rudolf N; Simmermacher-Mayer, Jean; Cometa, Fu-ni L; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E; Bristow, Linda J

    2016-01-01

    Antidepressant activity of N-methyl-D-aspartate (NMDA) receptor antagonists and negative allosteric modulators (NAMs) has led to increased investigation of their behavioral pharmacology. NMDA antagonists, such as ketamine, impair cognition in multiple species and in multiple cognitive domains. However, studies with NR2B subtype-selective NAMs have reported mixed results in rodents including increased impulsivity, no effect on cognition, impairment or even improvement of some cognitive tasks. To date, the effects of NR2B-selective NAMs on cognitive tests have not been reported in nonhuman primates. The current study evaluated two selective NR2B NAMs, CP101,606 and BMT-108908, along with the nonselective NMDA antagonists, ketamine and AZD6765, in the nonhuman primate Cambridge Neuropsychological Test Automated Battery (CANTAB) list-based delayed match to sample (list-DMS) task. Ketamine and the two NMDA NR2B NAMs produced selective impairments in memory in the list-DMS task. AZD6765 impaired performance in a non-specific manner. In a separate cohort, CP101,606 impaired performance of the nonhuman primate CANTAB visuo-spatial Paired Associates Learning (vsPAL) task with a selective impairment at more difficult conditions. The results of these studies clearly show that systemic administration of a selective NR2B NAM can cause transient cognitive impairment in multiple cognitive domains. PMID:26105137

  1. The candidate tumor suppressor CST6 alters the gene expression profile of human breast carcinoma cells: Down-regulation of the potent mitogenic, motogenic, and angiogenic factor autotaxin

    SciTech Connect

    Song Jin; Jie Chunfa; Polk, Paula; Shridhar, Ravi; Clair, Timothy; Zhang, Jun; Yin, Lijia; Keppler, Daniel . E-mail: dkeppl@lsuhsc.edu

    2006-02-03

    We recently coined CST6 as a novel candidate tumor suppressor gene for breast cancer. CST6 indeed is expressed in the normal human breast epithelium, but little or not at all in breast carcinomas and breast cancer cell lines. Moreover, ectopic expression of CST6 in human breast cancer cells suppressed cell proliferation, migration, invasion, and orthotopic tumor growth. To obtain insights into the molecular mechanism by which CST6 exhibits its pleiotropic effects on tumor cells, we compared global gene expression profiles in mock- and CST6-transfected human MDA-MB-435S cells. Out of 12,625 transcript species, 61 showed altered expression. These included genes for extracellular matrix components, cytokines, kinases, and phosphatases, as well as several key transcription factors. TaqMan PCR assays were used to confirm the microarray data for 7 out of 11 genes. One down-regulated gene product, secreted autotaxin/lyso-phospholipase D, was of particular interest because its down-regulation by CST6 could explain most of CST6's effect on the breast cancer cells. This study thus provides First evidence that CST6 plays a role in the modulation of genes, particularly, genes that are highly relevant to breast cancer progression.

  2. NR2B subunit in the prefrontal cortex: A double-edged sword for working memory function and psychiatric disorders.

    PubMed

    Monaco, Sarah A; Gulchina, Yelena; Gao, Wen-Jun

    2015-09-01

    The prefrontal cortex (PFC) is a brain region featured with working memory function. The exact mechanism of how working memory operates within the PFC circuitry is unknown, but persistent neuronal firing recorded from prefrontal neurons during a working memory task is proposed to be the neural correlate of this mnemonic encoding. The PFC appears to be specialized for sustaining persistent firing, with N-methyl-D-aspartate (NMDA) receptors, especially slow-decay NR2B subunits, playing an essential role in the maintenance of sustained activity and normal working memory function. However, the NR2B subunit serves as a double-edged sword for PFC function. Because of its slow kinetics, NR2B endows the PFC with not only "neural psychic" properties, but also susceptibilities for neuroexcitotoxicity and psychiatric disorders. This review aims to clarify the interplay among working memory, the PFC, and NMDA receptors; demonstrate the importance of NR2B in the maintenance of persistent activity; understand the risks and vulnerabilities of how NR2B is related to the development of neuropsychiatric disorders; identify gaps that currently exist in our understanding of these processes; and provide insights regarding future directions that may clarify these issues. We conclude that the PFC is a specialized brain region with distinct delayed maturation, unique neuronal circuitry, and characteristic NMDA receptor function. The unique properties and development of NMDA receptors, especially enrichment of NR2B subunits, endow the PFC with not only the capability to generate sustained activity for working memory, but also serves as a major vulnerability to environmental insults and risk factors for psychiatric disorders. PMID:26143512

  3. Down-regulation of CBP80 gene expression as a strategy to engineer a drought-tolerant potato.

    PubMed

    Pieczynski, Marcin; Marczewski, Waldemar; Hennig, Jacek; Dolata, Jakub; Bielewicz, Dawid; Piontek, Paulina; Wyrzykowska, Anna; Krusiewicz, Dominika; Strzelczyk-Zyta, Danuta; Konopka-Postupolska, Dorota; Krzeslowska, Magdalena; Jarmolowski, Artur; Szweykowska-Kulinska, Zofia

    2013-05-01

    Developing new strategies for crop plants to respond to drought is crucial for their innovative breeding. The down-regulation of nuclear cap-binding proteins in Arabidopsis renders plants drought tolerant. The CBP80 gene in the potato cultivar Desiree was silenced using artificial microRNAs. Transgenic plants displayed a higher tolerance to drought, ABA-hypersensitive stomatal closing, an increase in leaf stomata and trichome density, and compact cuticle structures with a lower number of microchannels. These findings were correlated with a higher tolerance to water stress. The level of miR159 was decreased, and the levels of its target mRNAs MYB33 and MYB101 increased in the transgenic plants subjected to drought. Similar trends were observed in an Arabidopsis cbp80 mutant. The evolutionary conservation of CBP80, a gene that plays a role in the response to drought, suggests that it is a candidate for genetic manipulations that aim to obtain improved water-deficit tolerance of crop plants. PMID:23231480

  4. The Liver X Receptor Ligand T0901317 Down-regulates APOA5 GeneExpression through Activation of SREBP-1c

    SciTech Connect

    Jakel, Heidelinde; Nowak, Maxime; Moitrot, Emanuelle; Dehondt, Helene; Hum, Dean W.; Pennacchio, Len A.; Fruchart-Najib, Jamila; Fruchart,Jean-Charles

    2004-07-23

    Alterations in the expression of the recently discovered apolipoprotein A5 gene strongly affect plasma triglyceride levels. In this study, we investigated the contribution of APOA5 to the liver X-receptor (LXR) ligand mediated effect on plasma triglyceride levels.Following treatment with the LXR ligand T0901317, we found that APOA5mRNA levels were decreased in hepatoma cell lines. The observation that no down-regulation of APOA5 promoter activity was obtained by LXR-retinoid X receptor (RXR) co-transfection prompted us to explore the possible involvement of the known LXR target gene SREBP-1c (sterol regulatory element-binding protein 1c). In fact, we found that co-transfection with the active form of SREBP-1c down-regulated APOA5promoter activity in a dose-dependent manner. We then scanned the human APOA5 promoter sequence and identified two putative E-box elements that were able to bind specifically SREBP-1c in gel-shift assays and were shown to be functional by mutation analysis. Subsequent suppression of SREBP-1 mRNA through small interfering RNA interference abolished the decrease of APOA5 mRNA in response to T0901317. Finally, administration of T0901317 to hAPOA5 transgenic mice revealed a significant decrease OF APOA5 mRNA in liver tissue and circulating apolipoprotein AV protein in plasma, confirming that the described down-regulation also occurs in vivo. Taken together, our results demonstrate that APOA5 gene expression is regulated by the LXR ligand T0901317 in a negative manner through SREBP-1c. These findings may provide a new mechanism responsible for the elevation of plasma triglyceride levels by LXR ligands and support the development of selective LXR agonists, not affecting SREBP-1c, as beneficial modulators of lipid metabolism.

  5. Escin Ia suppresses the metastasis of triple-negative breast cancer by inhibiting epithelial-mesenchymal transition via down-regulating LOXL2 expression.

    PubMed

    Wang, Yuhui; Xu, Xiaotian; Zhao, Peng; Tong, Bei; Wei, Zhifeng; Dai, Yue

    2016-04-26

    The saponin fraction of Aesculus chinensis Bunge fruits (SFAC) could inhibit the invasion and migration of MDA-MB-231 cells. Among which, escin Ia showed more potent inhibition of the invasion than other five main saponin constituents. It selectively reduced the expression of LOXL2 mRNA and promoted the expression of E-cadherin mRNA, and prevented the EMT process of MDA-MB-231 cells and TNF-α/TGF-β-stimulated MCF-7 cells. Moreover, it reduced the LOXL2 level in MDA-MB-231 cells but not in MCF-7 cells. When MCF-7 cells were stimulated with TNF-α/TGF-β, transfected with LOXL2 or treated with hypoxia, escin Ia down-regulated the level of LOXL2 in MCF-7 cells. Meanwhile, escin Ia suppressed the EMT process in LOXL2-transfected or hypoxia-treated MCF-7 cells. Of interest, escin Ia did not alter the level of HIF-1α in hypoxia-induced MCF-7 cells. In TNBC xenograft mice, the metastasis and EMT of MDA-MB-231 cells were suppressed by escin Ia. In conclusion, escin Ia was the main active ingredient of SFAC for the anti-TNBC metastasis activity, and its action mechanisms involved inhibition of EMT process by down-regulating LOXL2 expression. PMID:27008697

  6. Hyperosmotic Stimulus Down-regulates 1α, 25-dihydroxyvitamin D3-induced Osteoclastogenesis by Suppressing the RANKL Expression in a Co-culture System

    PubMed Central

    Tian, Yu Shun; Jeong, Hyun Joo; Lee, Sang-Do; Kong, Seok Heui; Ohk, Seung-Ho; Yoo, Yun-Jung; Seo, Jeong-Taeg; Shin, Dong Min; Sohn, Byung-Wha

    2010-01-01

    The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects 1α, 25-dihydroxyvitamin D3 (1α,25(OH)2D3)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on 1α,25(OH)2D3-induced osteoclastogenesis was investigated in an osteoblast-preosteoclast co-culture system. Serial doses of sucrose were applied as a mechanical force. These hyperosmotic stimuli significantly evoked a reduced number of 1α,25(OH)2D3-induced tartrate-resistant acid phosphatase-positive multinucleated cells and 1α,25(OH)2D3-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor κB ligand (RANKL) and Runx2 expressions were down-regulated in response to 1α,25(OH)2D3. Knockdown of Runx2 inhibited 1α,25(OH)2D3-induced RANKL expression in osteoblastic cells. Finally, the hyperosmotic stimulus induced the overexpression of TonEBP in osteoblastic cells. These results suggest that hyperosmolarity leads to the down-regulation of 1α,25(OH)2D3-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells. PMID:20631890

  7. MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

    SciTech Connect

    Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang; Sun, Ming; Stolz, Donna B.; He, Fengtian; Fan, Jie; Xie, Wen; Li, Song

    2014-04-18

    Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl{sub 4}-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.

  8. Isolation of up- or down-regulated genes in PPARgamma-expressing NIH-3T3 cells during differentiation into adipocytes.

    PubMed

    Okuno, Masaaki; Arimoto, Emi; Nishizuka, Makoto; Nishihara, Tsutomu; Imagawa, Masayoshi

    2002-05-22

    Adipocyte differentiation is a complex process in which the expression of many transcription factors and adipocyte-specific genes is regulated under a strict program. The peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the steroid/thyroid nuclear hormone receptor superfamily of ligand-activated transcription factors, is an important regulator of adipocyte gene expression and differentiation. In this study, we tried to identify downstream target genes of PPARgamma, by using PPARgamma-expressing cells and a subtractive cloning strategy, and isolated cDNA clones which were up-regulated or down-regulated by PPARgamma. Northern blot analyses revealed that the expression levels of the aldehyde dehydrogenase-2-like, type VI collagen alpha 3 subunit, cellular retinoic acid binding protein 1 and thrombospondin 1 are changed during the differentiation of mouse 3T3-L1 preadipocyte cells, indicating that these genes might be downstream targets of PPARgamma in adipocyte differentiation. PMID:12023027

  9. The Tau/A152T mutation, a risk factor for frontotemporal-spectrum disorders, leads to NR2B receptor-mediated excitotoxicity.

    PubMed

    Decker, Jochen Martin; Krüger, Lars; Sydow, Astrid; Dennissen, Frank Ja; Siskova, Zuzana; Mandelkow, Eckhard; Mandelkow, Eva-Maria

    2016-04-01

    We report on a novel transgenic mouse model expressing human full-length Tau with the Tau mutation A152T (hTau(AT)), a risk factor for FTD-spectrum disorders including PSP and CBD Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis-sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short- or long-term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage-gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau(AT) causes excitotoxicity mediated by NR2B-containing NMDA receptors due to enhanced extracellular glutamate. PMID:26931569

  10. Increased NR2A:NR2B ratio compresses long-term depression range and constrains long-term memory.

    PubMed

    Cui, Zhenzhong; Feng, Ruiben; Jacobs, Stephanie; Duan, Yanhong; Wang, Huimin; Cao, Xiaohua; Tsien, Joe Z

    2013-01-01

    The NR2A:NR2B subunit ratio of the NMDA receptors is widely known to increase in the brain from postnatal development to sexual maturity and to aging, yet its impact on memory function remains speculative. We have generated forebrain-specific NR2A overexpression transgenic mice and show that these mice had normal basic behaviors and short-term memory, but exhibited broad long-term memory deficits as revealed by several behavioral paradigms. Surprisingly, increased NR2A expression did not affect 1-Hz-induced long-term depression (LTD) or 100 Hz-induced long-term potentiation (LTP) in the CA1 region of the hippocampus, but selectively abolished LTD responses in the 3-5 Hz frequency range. Our results demonstrate that the increased NR2A:NR2B ratio is a critical genetic factor in constraining long-term memory in the adult brain. We postulate that LTD-like process underlies post-learning information sculpting, a novel and essential consolidation step in transforming new information into long-term memory. PMID:23301157

  11. Harmine combined with paclitaxel inhibits tumor proliferation and induces apoptosis through down-regulation of cyclooxygenase-2 expression in gastric cancer

    PubMed Central

    Yu, Xiao-Juan; Sun, Kun; Tang, Xiao-He; Zhou, Cun-Jin; Sun, Hui; Yan, Zhe; Fang, Ling; Wu, Hong-Wen; Xie, Yi-Kui; Gu, Bin

    2016-01-01

    Cyclooxygenase-2 (COX-2) serves an important role in the carcinogenesis and progression of gastric cancer. Harmine (HM) and paclitaxel (PTX) are reported as promising drug candidates for cancer therapy, but whether a synergistic anti-tumor effect of HM combined with PTX exists in human gastric cancer remains unknown. The present study evaluated the effects of HM and/or PTX on cell proliferation and apoptosis in a gastric cancer cell line, SGC-7901. HM and PTX inhibited cell proliferation in a dose-dependent manner. Both HM and PTX alone induced apoptosis in gastric cancer cells. The combination of HM and PTX exerted synergistic effects on proliferation inhibition and apoptosis induction in SGC-7901 cells, with down-regulation of COX-2, PCNA and Bcl-2 and up-regulation of Bax expression. The results indicated that combination chemotherapy using HM with PTX exerts an anti-tumor effect for treating gastric cancer. The combination of the two drugs inhibits gastric cancer development more effectively than each drug alone through down-regulation of COX-2 expression.

  12. Down-regulation of FoxO-dependent c-FLIP expression mediates TRAIL-induced apoptosis in activated hepatic stellate cells.

    PubMed

    Park, Soo-Jung; Sohn, Hee-Young; Yoon, Jeongsook; Park, Sang Ick

    2009-10-01

    Activated hepatic stellate cells which contribute to liver fibrosis have represented an important target for antifibrotic therapy. In this study, we found that TRAIL inhibited PI3K/Akt-dependent FoxO phosphorylation and relocated FoxO proteins into the nucleus from the cytosol in activated human hepatic stellate LX-2 cells. The accumulated FoxO proteins in the nucleus led to down-regulation of c-FLIP(L/S) expression, resulting in the activation of apoptosis-related signaling molecules including the activation of caspase-8, -3, and Bid, as well as mitochondrial cytochrome c release. These results were supported by showing that siRNA-mediated knockdown of FoxO led to restoration of c-FLIP(L/S) expression and resistance to TRAIL-induced apoptosis after treatment of LX-2 cells with TRAIL. Furthermore, c-FLIP(L/S)-transfected LX-2 cells showed the decreased sensitivity to TRAIL-induced apoptosis. Collectively, our data suggest that sequential activation of FoxO proteins under conditions of suppressed PI3K/Akt signaling by TRAIL can down-regulate c-FLIP(L/S), consequently promoting TRAIL-induced apoptosis in LX-2 cells. Therefore, the present study suggests TRAIL may be an effective strategy for antifibrotic therapy in liver fibrosis. PMID:19470406

  13. Thrombomodulin reduces tumorigenic and metastatic potential of lung cancer cells by up-regulation of E-cadherin and down-regulation of N-cadherin expression.

    PubMed

    Zheng, Nana; Huo, Zihe; Zhang, Bin; Meng, Mei; Cao, Zhifei; Wang, Zhiwei; Zhou, Quansheng

    2016-08-01

    Thrombomodulin (TM) is an endothelial cell membrane protein and plays critical roles in anti-thrombosis, anti-inflammation, vascular endothelial protection, and is traditionally regarded as a "vascular protection god". In recent years, although TM has been reported to be down-regulated in a variety of malignant tumors including lung cancer, the role and mechanism of TM in lung cancer are enigmatic. In this study, we found that induction of TM overexpression by cholesterol-reducing drug atorvastatin significantly diminished the tumorigenic capability of the lung cancer cells. Moreover, we demonstrated that TM overexpression caused G0/G1 phase arrest and markedly reduced the colony forming capability of the cells. Furthermore, overexpression of TM inhibited cell migration and invasion. Consistently, depletion of TM promoted cell growth, reduced the cell population at the G0/G1 phase, and enhanced cell migratory ability. Mechanistic study revealed that TM up-regulated E-cadherin but down-regulated N-cadherin expression, resulting in reversal of epithelial-mesenchymal transition (EMT) in the lung cancer cells. Moreover, silencing TM expression led to decreased E-cadherin and increased N-cadherin. Taken together, our study suggests that TM functions as a tumor suppressive protein, providing a conceptual framework for inducing TM overexpression as a sensible strategy and approach for novel anti-lung cancer drug discovery. PMID:27223053

  14. The physiological response of Populus tremula x alba leaves to the down-regulation of PIP1 aquaporin gene expression under no water stress

    PubMed Central

    Secchi, Francesca; Zwieniecki, Maciej A.

    2013-01-01

    In order to study the role of PIP1 aquaporins in leaf water and CO2 transport, several lines of PIP1-deficient transgenic Populus tremula x alba were generated using a reverse genetic approach. These transgenic lines displayed no visible developmental or morphological phenotypes when grown under conditions of no water stress. Major photosynthetic parameters were also not affected by PIP1 down regulation. However, low levels of PIP1 expression resulted in greater leaf hydraulic resistance (an increase of 27%), which effectively implicated PIP1 role in water transport. Additionally, the expression level of PIP1 genes in the various transgenic lines was correlated with reductions in mesophyll conductance to CO2 (gm), suggesting that in poplar, these aquaporins influenced membrane permeability to CO2. Overall, although analysis showed that PIP1 genes contributed to the mass transfer of water and CO2 in poplar leaves, their down-regulation did not dramatically impair the physiological needs of this fast growing tree when cultivated under conditions of no stress. PMID:24379822

  15. In vitro mechanism for down-regulation of ERalpha expression by epigallocatechin gallate in ER+/PR+ human breast cancer cells

    PubMed Central

    De Amicis, Francesca; Russo, Alessandra; Avena, Paola; Santoro, Marta; Vivacqua, Adele; Bonofiglio, Daniela; Mauro, Loredana; Aquila, Saveria; Tramontano, Donatella; Fuqua, Suzanne AW; Andò, Sebastiano

    2015-01-01

    Scope Exposure of the breast to estrogens and other sex hormones is an important cancer risk factor and estrogen receptor down-regulators are attracting significant clinical interest. Epigallocatechin gallate (EGCG), a polyphenolic compound found in green tea, has gained considerable attention for its antitumor properties. Here we aimed to investigate the molecular mechanisms through which EGCG regulates ERα expression in ER+ PR+ breast cancer cells. Material and Methods Western blotting analysis, real time PCR and transient transfections of deletion fragments of the ERα gene promoter show that EGCG down-regulates ERα protein, mRNA and gene promoter activity with a concomitant reduction of ERα genomic and non genomic signal. These events occur through p38MAPK/CK2 activation, causing the release from Hsp90 of PR-B and its consequent nuclear translocation as evidenced by immunofluorescence studies. EMSA and ChIP assay reveal that, upon EGCG treatment, PR-B is recruited at the half PRE site on ERα promoter. This is concomitant with the formation of a corepressor complex containing NCoR and HDAC1 while RNA polymerase II is displaced. The events are crucially mediated by PR-B isoform, since they are abrogated with PR-B siRNA. Conclusions Our data provide evidence for a mechanism by which EGCG down-regulates ERα and explain the inhibitory action of EGCG on the proliferation of ER+ PR+ cancer cells tested. We suggest that the EGCG/PR-B signaling should be further exploited for clinical approach. PMID:23322423

  16. Protective effect of rutin on LPS-induced acute lung injury via down-regulation of MIP-2 expression and MMP-9 activation through inhibition of Akt phosphorylation.

    PubMed

    Chen, Wen-Ying; Huang, Yi-Chun; Yang, Ming-Ling; Lee, Chien-Ying; Chen, Chun-Jung; Yeh, Chung-Hsin; Pan, Pin-Ho; Horng, Chi-Ting; Kuo, Wu-Hsien; Kuan, Yu-Hsiang

    2014-10-01

    Lipopolysaccharide (LPS), also called endotoxin, is the important pathogen of acute lung injury (ALI), which is a clinical syndrome that still lacks effective therapeutic medicine. Rutin belongs to vitamin P and possesses various beneficial effects. In this study, we investigate the potential protective effects and the mechanisms of rutin on LPS-induced ALI. Pre-administration with rutin inhibited LPS-induced arterial blood gas exchange and neutrophils infiltration in the lungs. LPS-induced expression of macrophage inflammatory protein (MIP)-2 and activation of matrix metalloproteinase (MMP)-9 were suppressed by rutin. In addition, the inhibitory concentration of rutin on phosphorylation of Akt was similar as MIP-2 expression and MMP-9 activation. In conclusion, rutin is a potential protective agent for ALI via suppressing the blood gas exchange and neutrophil infiltration. The mechanism of rutin is down-regulation of MIP-2 expression and MMP-9 activation through inhibition of Akt phosphorylation. PMID:25091621

  17. Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes

    PubMed Central

    Lee, Eon-Seok; Won, Yeo Jin; Kim, Byoung-Chul; Park, Daeui; Bae, Jin-Han; Park, Seong-Joon; Noh, Sung Jin; Kang, Yeong-Rok; Choi, Si Ho; Yoon, Je-Hyun; Heo, Kyu; Yang, Kwangmo; Son, Tae Gen

    2016-01-01

    Current evidence indicates that there is a relationship between microRNA (miRNA)-mediated gene silencing and low-dose irradiation (LDIR) responses. Here, alterations of miRNA expression in response to LDIR exposure in male BALB/c mice and three different types of hepatocytes were investigated. The miRNome of the LDIR-exposed mouse spleens (0.01 Gy, 6.5 mGy/h) was analyzed, and the expression of miRNA and mRNA was validated by qRT-PCR. Western blotting, chromatin immunoprecipitation (ChIP), and luciferase assays were also performed to evaluate the interaction between miRNAs and their target genes and to gain insight into the regulation of miRNA expression. The expression of miRNA-193b-3p was down-regulated in the mouse spleen and liver and in various hepatocytes (NCTC, Hepa, and HepG2 cell lines) in response to LDIR. The down-regulation of miR-193b-3p expression was caused by histone deacetylation on the miR-193b-3p promoter in the HepG2 cells irradiated with 0.01 Gy. However, the alteration of histone deacetylation and miR-193b-3p and Rad51 expression in response to LDIR was restored by pretreatment with N-acetyl-cyctein. In conclusion, we provide evidence that miRNA responses to LDIR include the modulation of cellular stress responses and repair mechanisms. PMID:27225532

  18. Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes

    NASA Astrophysics Data System (ADS)

    Lee, Eon-Seok; Won, Yeo Jin; Kim, Byoung-Chul; Park, Daeui; Bae, Jin-Han; Park, Seong-Joon; Noh, Sung Jin; Kang, Yeong-Rok; Choi, Si Ho; Yoon, Je-Hyun; Heo, Kyu; Yang, Kwangmo; Son, Tae Gen

    2016-05-01

    Current evidence indicates that there is a relationship between microRNA (miRNA)-mediated gene silencing and low-dose irradiation (LDIR) responses. Here, alterations of miRNA expression in response to LDIR exposure in male BALB/c mice and three different types of hepatocytes were investigated. The miRNome of the LDIR-exposed mouse spleens (0.01 Gy, 6.5 mGy/h) was analyzed, and the expression of miRNA and mRNA was validated by qRT-PCR. Western blotting, chromatin immunoprecipitation (ChIP), and luciferase assays were also performed to evaluate the interaction between miRNAs and their target genes and to gain insight into the regulation of miRNA expression. The expression of miRNA-193b-3p was down-regulated in the mouse spleen and liver and in various hepatocytes (NCTC, Hepa, and HepG2 cell lines) in response to LDIR. The down-regulation of miR-193b-3p expression was caused by histone deacetylation on the miR-193b-3p promoter in the HepG2 cells irradiated with 0.01 Gy. However, the alteration of histone deacetylation and miR-193b-3p and Rad51 expression in response to LDIR was restored by pretreatment with N-acetyl-cyctein. In conclusion, we provide evidence that miRNA responses to LDIR include the modulation of cellular stress responses and repair mechanisms.

  19. Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes.

    PubMed

    Lee, Eon-Seok; Won, Yeo Jin; Kim, Byoung-Chul; Park, Daeui; Bae, Jin-Han; Park, Seong-Joon; Noh, Sung Jin; Kang, Yeong-Rok; Choi, Si Ho; Yoon, Je-Hyun; Heo, Kyu; Yang, Kwangmo; Son, Tae Gen

    2016-01-01

    Current evidence indicates that there is a relationship between microRNA (miRNA)-mediated gene silencing and low-dose irradiation (LDIR) responses. Here, alterations of miRNA expression in response to LDIR exposure in male BALB/c mice and three different types of hepatocytes were investigated. The miRNome of the LDIR-exposed mouse spleens (0.01 Gy, 6.5 mGy/h) was analyzed, and the expression of miRNA and mRNA was validated by qRT-PCR. Western blotting, chromatin immunoprecipitation (ChIP), and luciferase assays were also performed to evaluate the interaction between miRNAs and their target genes and to gain insight into the regulation of miRNA expression. The expression of miRNA-193b-3p was down-regulated in the mouse spleen and liver and in various hepatocytes (NCTC, Hepa, and HepG2 cell lines) in response to LDIR. The down-regulation of miR-193b-3p expression was caused by histone deacetylation on the miR-193b-3p promoter in the HepG2 cells irradiated with 0.01 Gy. However, the alteration of histone deacetylation and miR-193b-3p and Rad51 expression in response to LDIR was restored by pretreatment with N-acetyl-cyctein. In conclusion, we provide evidence that miRNA responses to LDIR include the modulation of cellular stress responses and repair mechanisms. PMID:27225532

  20. Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region

    PubMed Central

    Ortmann, Christina A.; Burchert, Andreas; Hölzle, Katharina; Nitsche, Andreas; Wittig, Burghardt; Neubauer, Andreas; Schmidt, Manuel

    2005-01-01

    Although the bcr-abl translocation has been shown to be the causative genetic aberration in chronic myeloid leukemia (CML), there is mounting evidence that the deregulation of other genes, such as the transcription factor interferon regulatory factor 4 (IRF-4), is also implicated in the pathogenesis of CML. Promoter methylation of CpG target sites or direct deletions/insertions of genes are mechanisms of a reversible or permanent silencing of gene expression, respectively. Therefore, we investigated whether IRF-4 promoter methylation or mutation may be involved in the regulation of IRF-4 expression in leukemia cells. Whereas promoter mutations or structural rearrangements could be excluded as a cause of altered IRF-4 expression in hematopoietic cells, the IRF-4 promoter methylation status was found to significantly influence IRF-4 transcription. First, treatment of IRF-4-negative lymphoid, myeloid and monocytic cell lines with the methylation-inhibitor 5-aza-2-deoxycytidine resulted in a time- and concentration-dependent increase of IRF-4 mRNA and protein levels. Second, using a restriction-PCR-assay and bisulfite-sequencing we identified specifically methylated CpG sites in IRF-4-negative but not in IRF-4-positive cells. Third, we clearly determined promoter methylation as a mechanism for IRF-4 down-regulation via reporter gene assays, but did not detect an association of methylational status and mRNA expression of DNA methyltransferases or methyl-CpG-binding proteins. Together, these data suggest CpG site-specific IRF-4 promoter methylation as a putative mechanism of down-regulated IRF-4 expression in leukemia. PMID:16396836

  1. Microarray Analysis of Gene Expression Reveals that Cyclo-oxygenase-2 Gene Therapy Up-regulates Hematopoiesis and Down-regulates Inflammation During Endochondral Bone Fracture Healing

    PubMed Central

    Lau, K.-H. William; Popa, Nicoleta L.

    2014-01-01

    Background Cyclo-oxygenase-2 (Cox-2) is an inflammatory mediator that is necessary for the tissue repair, including bone fracture healing. Although the application of Cox-2 gene therapy to a murine closed femoral fracture has accelerated bony union, but the beneficial effect was not observed until the endochondral stage of bone repair that is well after the inflammatory stage normally subsides. Methods To identify the molecular pathways through which Cox-2 regulates fracture healing, we examined gene expression profile in fracture tissues in response to Cox-2 gene therapy during the endochondral bone repair phase. Cox-2 gene therapy was applied to the closed murine femur fracture model. Microarray analysis was performed at 10 days post-fracture to examine global gene expression profile in the fracture tissues during the endochondral bone repair phase. The entire repertoire of significantly expressed genes was examined by gene set enrichment analysis, and the most up-regulated individual genes were evaluated further. Results The genes that normally promote inflammation were under-represented in the microarray analysis, and the expression of several inflammatory chemokines was significantly down-regulated. There was an up-regulation of two key transcription factor genes that regulate hematopoiesis and erythropoiesis. More surprisingly, there was no significant up-regulation in the genes that are normally involved in angiogenesis or bone formation. However, the expression of two tissue remodeling genes was up-regulated. Conclusions The down-regulation of the inflammatory genes in response to Cox-2 gene therapy was unexpected, given the pro-inflammatory role of prostaglandins. Cox-2 gene therapy could promote bony union through hematopoietic precursor proliferation during endochondral bone repair and thereby enhances subsequently fracture callus remodeling that leads to bony union of the fracture gap. PMID:25247155

  2. Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression.

    PubMed

    Yuan, Wei-Qi; Zhang, Rong-Rong; Wang, Jun; Ma, Yan; Li, Wen-Xue; Jiang, Ren-Wang; Cai, Shao-Hui

    2016-05-24

    Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is a major cause of cancer therapy failure. In this study, we identified a novel C21 steroidal glycoside, asclepiasterol, capable of reversing P-gp-mediated MDR. Asclepiasterol (2.5 and 5.0μM) enhanced the cytotoxity of P-gp substrate anticancer drugs in MCF-7/ADR and HepG-2/ADM cells. MDR cells were more responsive to paclitaxel in the presence of asclepiasterol, and colony formation of MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin. Consistent with these findings, asclepiasterol treatment increased the intracellular accumulation of doxorubicin and rhodamine 123 (Rh123) in MDR cells. Asclepiasterol decreased expression of P-gp protein without stimulating or suppressing MDR1 mRNA levels. Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types, and EGF, an activator of the MAPK/ERK pathway, reversed the P-gp down-regulation, implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation. These results suggest that asclepiasterol could be developed as a modulator for reversing P-gp-mediated MDR in P-gp-overexpressing cancer variants. PMID:27129170

  3. SIRT1 attenuates PAF-induced MMP-2 production via down-regulation of PAF receptor expression in vascular smooth muscle cells.

    PubMed

    Kim, Yun H; Bae, Jin U; Lee, Seung J; Park, So Y; Kim, Chi D

    2015-09-01

    Silent mating type information regulation 2 homolog 1 (SIRT1) is known as a key regulator in the protection of various vascular disorders, however, no direct evidences have been reported in the progression of atherosclerosis. Considering the pivotal role of matrix metalloproteinase-2 (MMP-2) in plaque destabilization, this study investigated the role of SIRT1 on MMP-2 production in vascular smooth muscle cells (VSMCs) induced by platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). In VSMCs stimulated with resveratrol, SIRT1 activator, PAF receptor (PAFR) was internalized and then its protein levels were diminished. It was attenuated in cells pretreated with proteasome or lysosome inhibitor. Also, the degradation of PAFR in SIRT1-stimulated cells was significantly attenuated by β-arrestin2 depletion. In cells treated with nicotinamide, SIRT1 deacetylase inhibitor, PAFR internalization by resveratrol or reSIRT1 was inhibited, demonstrating that deacetylation of SIRT1 is an important step in SIRT1-induced PAFR down-regulation. Moreover, PAF-induced MMP-2 production in VSMCs and aorta was attenuated by resveratrol. In the aorta of SIRT1 transgenic mice, the PAF-induced MMP-2 expression was prominently attenuated compared to that in wild type mice. Taken together, it was suggested that SIRT1 down-regulated PAFR in VSMCs via β-arrestin2-mediated internalization and degradation, leading to an inhibition of PAF-induced MMP-2 production. PMID:25967595

  4. A peptide fraction from germinated soybean protein down-regulates PTTG1 and TOP2A mRNA expression, inducing apoptosis in cervical cancer cells.

    PubMed

    Robles-Ramírez, María del Carmen; Ramón-Gallegos, Eva; Mora-Escobedo, Rosalva; Torres-Torres, Nimbe

    2012-01-01

    The aim of this study was to evaluate the effect of a peptide fraction, obtained from a germinated soybean protein hydrolysate, on the viability, apoptosis and cancer related gene expression in HeLa cells. Soybean was germinated for 0-6 days and proteins were isolated from the seeds. Protein isolates, without ethanol-soluble phytochemicals, were hydrolyzed with digestive enzymes and their effect on growth in HeLa cells was evaluated. The most active hydrolysate was separated by ultrafiltration into five peptide fractions. A >10 kDa fraction was the most active against cancer cells. This fraction down-regulated PTTG1 and TOP2A mRNA expression (two genes considered as therapeutic targets) and induced apoptosis in cancer cells activating the caspase cascade and causing DNA fragmentation. Germinated soy protein isolates could be a bioactive ingredient of functional food. PMID:22545419

  5. Shu-Gan-Liang-Xue Decoction Simultaneously Down-regulates Expressions of Aromatase and Steroid Sulfatase in Estrogen Receptor Positive Breast Cancer Cells

    PubMed Central

    Fu, Xue-song; Li, Ping-ping

    2011-01-01

    Objective Estradiol (E2) plays an important role in the development of breast cancer. In postmenopausal women, the estrogen can be synthesized via aromatase (CYP19) pathway and steroid-sulfatase (STS) pathway in peripheral tissues, when the production in ovary has ceased. The objective of our study was to explore the effects of Shu-Gan-Liang-Xue Decoction (SGLXD) on the expressions of CYP19 and STS in estrogen receptor positive breast cancer MCF-7 and T47D cells. Methods The effects of SGLXD on the cell viability of MCF-7 and T47D were analyzed by MTT assay. By quantitative real-time RT-PCR and Western blot, we evaluated the mRNA and protein expressions of CYP19 and STS in MCF-7 and T47D cells after SGLXD treatment. Results By MTT assay, the cell viability rates of MCF-7 and T47D were significantly inhibited by SGLXD in a dose-dependent manner, the IC50 values were 40.07 mg/ml for MCF-7 cells and 25.62 mg/ml for T47D cells, respectively. As evidenced by real-time PCR and Western blot, the high concentrations of SGLXD significantly down-regulated the expressions of CYP19 and STS both in the transcript level and the protein level. Conclusion The results suggest that SGLXD is a potential dual aromatase-sulfatase inhibitor by simultaneously down-regulating the expressions of CYP19 and STS in MCF-7 and T47D cells. PMID:23467843

  6. BAFF induces spleen CD4{sup +} T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

    SciTech Connect

    Ji, Fang; Chen, Rongjing; Liu, Baojun; Zhang, Xiaoping; Han, Junli; Wang, Haining; Shen, Gang; Tao, Jiang

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4{sup +} T cells. Black-Right-Pointing-Pointer Carrying out siRNA technology to study FOXO3A protein function. Black-Right-Pointing-Pointer Helpful to understand the T cell especially CD4{sup +} T cell's role in immunological reaction. -- Abstract: The TNF ligand family member 'B cell-activating factor belonging to the TNF family' (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4{sup +} spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4{sup +} T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4{sup +} spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4{sup +} T cell proliferation.

  7. A Common Variant of PROK1 (V67I) Acts as a Genetic Modifier in Early Human Pregnancy through Down-Regulation of Gene Expression

    PubMed Central

    Su, Mei-Tsz; Huang, Jyun-Yuan; Tsai, Hui-Ling; Chen, Yi-Chi; Kuo, Pao-Lin

    2016-01-01

    PROK1-V67I has been shown to play a role as a modifier gene in the PROK1-PROKR system of human early pregnancy. To explore the related modifier mechanism of PROK1-V67I, we carried out a comparison study at the gene expression level and the cell function alternation of V67I, and its wild-type (WT), in transiently-transfected cells. We, respectively, performed quantitative RT-PCR and ELISA assays to evaluate the protein and/or transcript level of V67I and WT in HTR-8/SV neo, JAR, Ishikawa, and HEK293 cells. Transiently V67I- or WT-transfected HTR-8/SV neo and HEK293 cells were used to investigate cell function alternations. The transcript and protein expressions were down-regulated in all cell lines, ranging from 20% to 70%, compared with WT. There were no significant differences in the ligand activities of V67I and WT with regard to cell proliferation, cell invasion, calcium influx, and tubal formation. Both PROK1 alleles promoted cell invasion and intracellular calcium mobilization, whereas they had no significant effects on cell proliferation and tubal formation. In conclusion, the biological effects of PROK1-V67I on cell functions are similar to those of WT, and the common variant of V67I may act as a modifier in the PROK1-PROKR system through down-regulation of PROK1 expression. This study may provide a general mechanism that the common variant of V67I, modifying the disease severity of PROK1-related pathophysiologies. PMID:26828479

  8. Targeting HCCR expression resensitizes gastric cancer cells to chemotherapy via down-regulating the activation of STAT3

    PubMed Central

    Zhang, Jun-Ling; Liu, Xiang-Zheng; Wang, Peng-Yuan; Chen, Guo-Wei; Jiang, Yong; Qiao, Shu-Kai; Zhu, Jing; Wang, Xin; Pan, Yi-Sheng; Liu, Yu-Cun

    2016-01-01

    The human cervical cancer oncogene (HCCR) has been found to be overexpressed in a variety of human cancers. However, the level of expression of HCCR and its biological function in gastric cancer are largely unknown. In this study, we evaluated HCCR expression in several gastric cancer cell lines and in one normal gastric mucosal cell line. We established a 5-FU-resistant gastric cancer cell subline, and we evaluated its HCCR expression. HCCR expression levels were high in gastric cancer lines, and expression was significantly increased in the 5-FU-resistant cancer cell subline. HCCR expression affected cell growth by regulating apoptosis in the cancer cells, and it had a positive correlation with p-STAT3 expression. Western blot and luciferase reporter assays showed that the activation of STAT3 upregulated HCCR expression in a positive feedback loop model. In vivo and in vitro studies showed that HCCR plays an important role in the apoptosis induced by 5-FU. Our data demonstrate that HCCR is probably involved in apoptosis and cancer growth and that it functions as a p-STAT3 stimulator in a positive feedback loop model. In gastric cancer cells, HCCR confers a more aggressive phenotype and resistance to 5-FU-based chemotherapy. PMID:27052330

  9. The cervical malignant cells display a down regulation of ER-α but retain the ER-β expression

    PubMed Central

    López-Romero, Ricardo; Garrido-Guerrero, Efraín; Rangel-López, Angélica; Manuel-Apolinar, Leticia; Piña-Sánchez, Patricia; Lazos-Ochoa, Minerva; Mantilla-Morales, Alejandra; Bandala, Cindy; Salcedo, Mauricio

    2013-01-01

    The human cervix is a tissue target of sex steroid hormones as estradiol (E2) which exerts its action through of the estrogen receptors alpha and beta (ER-α and ER-β). In this study we investigated the expression of ER-α and ER-β in human invasive cervical carcinomas using immunohistochemistry and RT-PCR analyses and compared with that observed in the corresponding normal tissue. The results show nuclear expression of ER-α mainly in the first third of normal cervical epithelium, however, decreased or absent expression were present in invasive cervical carcinoma, indicating that expression of ER-α is lost in cervical cancer. Nevertheless, by RT-PCR we were able to demonstrate mRNA expression of ER-α in invasive cervical tissues. These results suggest that loss of ER-α could be due to a mechanism of post-transcriptional and/or post-translational regulation of its gene during the progression to invasive carcinoma. On the other hand, ER-β was expressed in normal cervix with an expression pattern similar to ER-α. In addition to its nuclear localization, cytoplasmic immunoreaction of ER-β was present in the epithelium of invasive cervical carcinomas, suggesting an association between cytoplasmic ER-β expression and invasive phenotype in the cervical tumors. In summary, the results show that the cervical malignant cells tend to loss the ER-α but maintain the ER-β actively expressed. Loss of expression of ER-α in neoplastic tissue suggests that the estrogenic effects could be conducted through the ER-β in human neoplastic cervical tissue. More detailed studies are needed to confirm this suggestion and to determine the role of ER-β in cervical cancer. PMID:23923078

  10. CB1 Blockade Potentiates Down-Regulation of Lipogenic Gene Expression in Perirenal Adipose Tissue in High Carbohydrate Diet-Induced Obesity

    PubMed Central

    Gavito, Ana Luisa; Suárez, Juan; Pavón, Francisco Javier; Arrabal, Sergio; Romero-Cuevas, Miguel; Bautista, Dolores; Martínez, Ana; de Fonseca, Fernando Rodríguez; Serrano, Antonia; Baixeras, Elena

    2014-01-01

    De novo lipogenesis and hypercaloric diets are thought to contribute to increased fat mass, particularly in abdominal fat depots. CB1 is highly expressed in adipose tissue, and CB1-mediated signalling is associated with stimulation of lipogenesis and diet-induced obesity, though its contribution to increasing fat deposition in adipose tissue is controversial. Lipogenesis is regulated by transcription factors such as liver X receptor (LXR), sterol-response element binding protein (SREBP) and carbohydrate-responsive-element-binding protein (ChREBP). We evaluated the role of CB1 in the gene expression of these factors and their target genes in relation to lipogenesis in the perirenal adipose tissue (PrAT) of rats fed a high-carbohydrate diet (HCHD) or a high-fat diet (HFD). Both obesity models showed an up-regulated gene expression of CB1 and Lxrα in this adipose pad. The Srebf-1 and ChREBP gene expressions were down-regulated in HFD but not in HCHD. The expression of their target genes encoding for lipogenic enzymes showed a decrease in diet-induced obesity and was particularly dramatic in HFD. In HCHD, CB1 blockade by AM251 reduced the Srebf-1 and ChREBP expression and totally abrogated the remnant gene expression of their target lipogenic enzymes. The phosphorylated form of the extracellular signal-regulated kinase (ERK-p), which participates in the CB1-mediated signalling pathway, was markedly present in the PrAT of obese rats. ERK-p was drastically repressed by AM251 indicating that CB1 is actually functional in PrAT of obese animals, though its activation loses the ability to stimulate lipogenesis in PrAT of obese rats. Even so, the remnant expression levels of lipogenic transcription factors found in HCHD-fed rats are still dependent on CB1 activity. Hence, in HCHD-induced obesity, CB1 blockade may help to further potentiate the reduction of lipogenesis in PrAT by means of inducing down-regulation of the ChREBP and Srebf-1 gene expression, and consequently in

  11. Depletion of mitochondrial DNA by down-regulation of deoxyguanosine kinase expression in non-proliferating HeLa cells

    SciTech Connect

    Franco, Maribel; Johansson, Magnus . E-mail: magnus.johansson@ki.se; Karlsson, Anna

    2007-07-15

    Purine deoxyribonucleotides required for mitochondrial DNA replication are either imported from the cytosol or derived from phosphorylation of deoxyadenosine or deoxyguanosine catalyzed by mitochondrial deoxyguanosine kinase (DGUOK). DGUOK deficiency has been linked to mitochondrial DNA depletion syndromes suggesting an important role for this enzyme in dNTP supply. We have generated HeLa cell lines with 20-30% decreased levels of DGUOK mRNA by the expression of small interfering RNAs directed towards the DGUOK mRNA. The cells with decreased expression of the enzyme showed similar levels of mtDNA as control cells when grown exponentially in culture. However, mtDNA levels rapidly decreased in the cells when cell cycle arrest was induced by serum starvation. DNA incorporation of 9-{beta}-D-arabino-furanosylguanine (araG) was lower in the cells with decreased deoxyguanosine kinase expression, but the total rate of araG phosphorylation was increased in the cells. The increase in araG phosphorylation was shown to be due to increased expression of deoxycytidine kinase. In summary, our findings show that DGUOK is required for mitochondrial DNA replication in resting cells and that small changes in expression of this enzyme may cause mitochondrial DNA depletion. Our data also suggest that alterations in the expression level of DGUOK may induce compensatory changes in the expression of other nucleoside kinases.

  12. Oxidative stress and ROS metabolism via down-regulation of sirtuin 3 expression in Cmah-null mice affect hearing loss

    PubMed Central

    Choi, Yun-Jung; Gurunathan, Sangiliyandi; Kim, Jin-Hoi

    2015-01-01

    CMP-Neu5Ac hydroxylase (Cmah) disruption caused several abnormalities and diseases including hearing loss in old age. However, underling molecular mechanisms that give rise to age-related hearing loss (AHL) in Cmah-null mouse are still obscure. In this study, Cmah-null mice showed age-related decline of hearing associated with loss of sensory hair cells, spiral ganglion neurons, and/or stria vascularis degeneration in the cochlea. To identify differential gene expression profiles and pathway associated with AHL, we performed microarray analysis using Illumina MouseRef-8 v2 Expression BeadChip and pathway-focused PCR array in the cochlear tissues of Cmah-null mouse. Pathway and molecular mechanism analysis using differentially expressed genes provided evidences that altered biological pathway due to oxidative damage by low expressed antioxidants and dysregulated reactive oxygen species (ROS) metabolism. Especially, low sirtuin 3 (Sirt3) gene expressions in Cmah-null mice decreased both of downstream regulator (Foxo1 and MnSod) and regulatory transcription factor (Hif1α and Foxo3a) gene expression. Taken together, we suggest that down-regulation of Sirt3 expression leads to oxidative stress and mitochondrial dysfunction by regulation of ROS and that it could alter various signaling pathways in Cmah-null mice with AHL. PMID:26319214

  13. Lentiviral vector platform for improved erythropoietin expression concomitant with shRNA mediated host cell elastase down regulation.

    PubMed

    Dhamne, Hemant; Chande, Ajit G; Mukhopadhyaya, Robin

    2014-01-01

    Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through an in silico approach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance. PMID:24325878

  14. 6-Methoxyluteolin from Chrysanthemum zawadskii var. latilobum suppresses histamine release and calcium influx via down-regulation of FcεRI α chain expression.

    PubMed

    Shim, Sun-Yup; Park, Jeong-Ro; Byun, Dae-Seok

    2012-05-01

    Mast cells and basophils are important effector cells in immunoglobulin-E (IgE)-mediated allergic reactions. Using the human basophilic KU812F cells, we assessed the inhibitory effects of 6-methoxyluteolin, isolated from Chrysanthemum zawadskii, in the FcεRI-mediated allergic reaction. We determined that 6-methoxyluteolin inhibited anti-FcεRI α chain antibody (CRA-1)-induced histamine release, as well as elevation of intracellular calcium concentration [Ca2+]i in a dose-dependent manner. Moreover, the inhibitory effects of 6-methoxyluteolin on the cell surface expression and the mRNA level of the FcεRI α chain were determined by flow cytometric analysis and reverse transcription-polymerase chain reaction (RTPCR), respectively. Therefore, these results show that 6- methoxyluteolin is a potent inhibitor of histamine release and calcium influx via down-regulation of the FcεRI α chain. PMID:22561855

  15. microRNA-106b-mediated down-regulation of C1orf24 expression induces apoptosis and suppresses invasion of thyroid cancer

    PubMed Central

    Cerutti, Janete Maria

    2015-01-01

    We previously showed that C1orf24 expression is increased in thyroid carcinomas. Nonetheless, the mechanism underlying C1orf24 deregulation is not fully understood. It has been widely demonstrated that microRNAs are involved in post-transcriptional gene regulation in several diseases, including cancer. Using in silico prediction approach, five microRNAs that bind to the 3′-untranslated region (3′-UTR) of C1orf24 were identified. The expression of two selected microRNAs (miR-17-5p, miR-106b) and the expression of C1orf24 were tested in 48 benign and malignant thyroid lesions and in five thyroid carcinoma cell lines. miR-106b was down-regulated in thyroid cancer specimens and thyroid carcinoma cell lines, while C1orf24 expression was markedly increased. To demonstrate that miR-106b reduces C1orf24 expression, follicular (WRO) and papillary (TPC1) thyroid carcinoma cell lines were transiently transfected with miR-106b mimic. Ectopic expression of the miR-106b mimic significantly inhibits C1orf24 mRNA and protein expression in both WRO and TPC1 cells. Dual-luciferase report assays demonstrated that miR-106b directly targets C1orf24 by binding its 3′- UTR. Moreover, miR-106b-mediated down-regulation of C1orf24 expression increased apoptosis and inhibited migration. We additionally demonstrated that siRNA against C1orf24 significantly decreased its expression, inhibited cell migration and cell cycle progression while induced apoptosis. In summary, our findings not only provide new insights into molecular mechanism associated with C1orf24 overexpression in thyroid carcinomas but also show that C1orf24 might increase proliferation and cell migration. Thus, decreasing C1orf24 levels, by restoring miR-106b function, may have therapeutic implications. PMID:26317551

  16. Induced ICER I{gamma} down-regulates cyclin A expression and cell proliferation in insulin-producing {beta} cells

    SciTech Connect

    Inada, Akari; Weir, Gordon C.; Bonner-Weir, Susan . E-mail: susan.bonner-weir@joslin.harvard.edu

    2005-04-15

    We have previously found that cyclin A expression is markedly reduced in pancreatic {beta}-cells by cell-specific overexpression of repressor inducible cyclic AMP early repressor (ICER I{gamma}) in transgenic mice. Here we further examined regulatory effects of ICER I{gamma} on cyclin A gene expression using Min6 cells, an insulin-producing cell line. The cyclin A promoter luciferase assay showed that ICER I{gamma} directly repressed cyclin A gene transcription. In addition, upon ICER I{gamma} overexpression, cyclin A mRNA levels markedly decreased, thereby confirming an inhibitory effect of ICER I{gamma} on cyclin A expression. Suppression of cyclin A results in inhibition of BrdU incorporation. Under normal culture conditions endogenous cyclin A is abundant in these cells, whereas ICER is hardly detectable. However, serum starvation of Min6 cells induces ICER I{gamma} expression with a concomitant very low expression level of cyclin A. Cyclin A protein is not expressed unless the cells are in active DNA replication. These results indicate a potentially important anti-proliferative effect of ICER I{gamma} in pancreatic {beta} cells. Since ICER I{gamma} is greatly increased in diabetes as well as in FFA- or high glucose-treated islets, this effect may in part exacerbate diabetes by limiting {beta}-cell proliferation.

  17. Expression of the Bacterial Type III Effector DspA/E in Saccharomyces cerevisiae Down-regulates the Sphingolipid Biosynthetic Pathway Leading to Growth Arrest*

    PubMed Central

    Siamer, Sabrina; Guillas, Isabelle; Shimobayashi, Mitsugu; Kunz, Caroline; Hall, Michael N.; Barny, Marie-Anne

    2014-01-01

    Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest. PMID:24828506

  18. The Dietary Bioflavonoid, Quercetin, Selectively Induces Apoptosis of Prostate Cancer Cells by Down-Regulating the Expression of Heat Shock Protein 90

    PubMed Central

    Aalinkeel, Ravikumar; Bindukumar, B.; Reynolds, Jessica L.; Sykes, Donald E.; Mahajan, Supriya D.; Chadha, Kailash C.; Schwartz, Stanley A.

    2010-01-01

    BACKGROUND Human and animal studies have suggested that diet-derived flavonoids, in particular quercetin may play a beneficial role by preventing or inhibiting oncogenesis, but the underlying mechanism remains unclear. The aim of this study is to evaluate the effect(s) of quercetin on normal and malignant prostate cells and to identify the target(s) of quercetin’s action. METHODOLOGY We addressed this question using cells in culture and investigated whether quercetin affects key biological processes responsible for tumor cell properties such as cell proliferation and apoptosis and also studied the effect of quercetin on the proteome of prostate cancer cells using difference gel electrophoresis (DIGE) to assess changes in the expression of relevant proteins. RESULTS Our findings demonstrate that quercetin treatment of prostate cancer cells results in decreased cell proliferation and viability. Furthermore, we demonstrate that quercetin promotes cancer cell apoptosis by down-regulating the levels of heat shock protein (Hsp) 90. Depletion of Hsp90 by quercetin results in decreased cell viability, levels of surrogate markers of Hsp90 inhibition (intracellular and secreted), induced apoptosis and activation of caspases in cancer cells but not in normal prostate epithelial cells. Knockdown of Hsp90 by short interfering RNA also resulted in induction apoptosis similar to quercetin in cancer cells as indicated by annexin V staining. CONCLUSION Our results demonstrate that quercetin down-regulates the expression of Hsp90 which, in turn, induces inhibition of growth and cell death in prostate cancer cells while exerting no quantifiable effect on normal prostate epithelial cells. PMID:18726985

  19. Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae down-regulates the sphingolipid biosynthetic pathway leading to growth arrest.

    PubMed

    Siamer, Sabrina; Guillas, Isabelle; Shimobayashi, Mitsugu; Kunz, Caroline; Hall, Michael N; Barny, Marie-Anne

    2014-06-27

    Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest. PMID:24828506

  20. The Ayurvedic drug Ksheerabala (101) ameliorates alcohol-induced neurotoxicity by down-regulating the expression of transcription factor (NFkB) in rat brain

    PubMed Central

    Rejitha, S.; Prathibha, P.; Madambath, Indira

    2015-01-01

    Introduction: Most of the pharmaceutical effects of alcohol are due to its accumulation in the brain. Ksheerabala (101) an Ayurvedic formulation mainly used against central nervous system disorders. Aim: To determine the antioxidant and neuroprotective effect of Ksheerabala (101) on alcohol-induced oxidative stress in rats. Materials and Methods: Male Albino rats of Sprague-Dawley strain were grouped into four; control, alcohol (4 g/kg), Ksheerabala (15 μL/1 ml milk/100 g) and Ksheerabala (15 μL/1 ml milk/100 g) + alcohol (4 g/kg). After the experimental period (90 days), the animals were sacrificed and the effect of Ksheerabala (101) was studied on oxidative stress, inflammatory markers, and induction of transcription factor in brain. Results were statistically analyzed by one-way ANOVA. Results: The activities of antioxidant enzymes and reduced glutathione which were decreased in alcohol-treated rats, increased significantly in co-administered groups. The lipid peroxidation products and protein carbonyls which were increased significantly in alcohol-treated rats decreased significantly in co-administered groups. The expression of gamma-glutamyl cysteine synthase decreased significantly in alcohol-treated rats and increased significantly in co-administered groups. The transcription factor nuclear factor-κB (NFκB) which was up-regulated in alcohol-treated rats was down-regulated in co-administered rats. The histopathology reinforced these results. Conclusion: Ksheerabala (101) attenuates alcohol-induced oxidative stress and down-regulates the expression of NFκB in rat brain. PMID:27313421

  1. Melittin Restores PTEN Expression by Down-Regulating HDAC2 in Human Hepatocelluar Carcinoma HepG2 Cells

    PubMed Central

    Huang, Cheng; Meng, Xiao-Ming; Bian, Er-Bao; Li, Jun

    2014-01-01

    Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation in vitro and further mechanisms was investigated. We found melittin could inhibit cell proliferation in vitro using Flow cytometry and MTT method. Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4. Results of western Blot and Real-time PCR analysis indicated that melittin was capable to upregulate the expression of PTEN and attenuate histone deacetylase 2 (HDAC2) expression. Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression. Conversely, it was that the potential utility of melittin on PTEN expression was reversed in cells treated with a recombinant pEGFP-C2-HDAC2 plasmid. In addition, treatment with melittin caused a downregulation of Akt phosphorylation, while overexpression of HDAC2 promoted Akt phosphorylation. These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways. PMID:24788349

  2. Gene expression down-regulation in CD90+ prostate tumor-associated stromal cells involves potential organ-specific genes

    PubMed Central

    2009-01-01

    Background The prostate stroma is a key mediator of epithelial differentiation and development, and potentially plays a role in the initiation and progression of prostate cancer. The tumor-associated stroma is marked by increased expression of CD90/THY1. Isolation and characterization of these stromal cells could provide valuable insight into the biology of the tumor microenvironment. Methods Prostate CD90+ stromal fibromuscular cells from tumor specimens were isolated by cell-sorting and analyzed by DNA microarray. Dataset analysis was used to compare gene expression between histologically normal and tumor-associated stromal cells. For comparison, stromal cells were also isolated and analyzed from the urinary bladder. Results The tumor-associated stromal cells were found to have decreased expression of genes involved in smooth muscle differentiation, and those detected in prostate but not bladder. Other differential expression between the stromal cell types included that of the CXC-chemokine genes. Conclusion CD90+ prostate tumor-associated stromal cells differed from their normal counterpart in expression of multiple genes, some of which are potentially involved in organ development. PMID:19737398

  3. Down-regulated expression of miR-134 contributes to paclitaxel resistance in human ovarian cancer cells.

    PubMed

    Shuang, Ting; Wang, Min; Shi, Cong; Zhou, Yingying; Wang, Dandan

    2015-10-01

    MiR-134 has been reported to have a role in the development and progression of various cancers. In this study, we found that miR-134 expression was significantly decreased in chemo-resistant serous epithelial ovarian cancer (EOC) patients. Over-expression of miR-134 enhanced the sensitivity of SKOV3-TR30 cells to paclitaxel, and increased paclitaxel-induced apoptosis. Further, Pak2 was identified as a direct target of miR-134, and Pak2-specific siRNA increased cell inhibition rate and promoted paclitaxal-induced apoptosis. By regulating Pak2 expression, miR-134 could mediate Bad phosphorylation at Ser112 and Ser136, which affected cell survival and apoptosis. In conclusion, our findings indicate that repression of miR-134 and consequent up-regulation of Pak2 might contribute to paclitaxel resistance. PMID:26363097

  4. Role of PPARα in down-regulating AGE-induced TGF-β and MMP-9 expressions in chondrocytes.

    PubMed

    Wang, J; Wang, G; Sun, G W

    2016-01-01

    Peroxisome proliferator-activated receptor is closely associated with the pathogenesis of osteoarthritis. The level of exogenous advanced glycation end-products (AGEs) in articular cartilage is highly associated with the severity of osteoarthritic lesions. However, their interactions and role in promoting osteoarthritisprogression remain unclear. Here, we investigated the effect of AGEs on transforming growth factor (TGF)-β and matrix metalloproteinase (MMP)-9 expression, and discussed the correlation between AGEs and osteoarthritis, possible signaling pathways and mechanism in rabbit chondrocytes. TGF-β and MMP-9 mRNA and protein expression, catalase (CAT) and superoxide dismutase (SOD) activity, and malondialdehyde (MDA) and reactive oxygen species (ROS) levels were analyzed in chondrocytes treated with different concentrations of AGEs using RT-PCR and/or western blot; we detected NF-κB nuclear translocation by immunofluorescence. AGE treatment significantly increased TGF-β and MMP-9 mRNA and protein expression compared to controls (P < 0.01) in a dose-dependent manner (highest at 100 μg/mL). AGE-induced TGF-β and MMP-9 expressions in chondrocytes were significantly inhibited by anti-RAGE and PDTC (0.1 mM) treatment (P < 0.01). Furthermore, AGE-treatment significantly decreased CAT and SOD activity and increased MDA levels in a concentration-dependent manner compared to controls (P < 0.05), significantly promoting NF-κB nuclear translocation. AGE significantly inhibited the increased expression of TGF-β and MMP- 9, and induced chondrocyte damage. Its mechanism is associated with RAGE activation, increased ROS expression, and activation of the NF- κB signaling pathways. PMID:27173350

  5. Urban particulate matter down-regulates filaggrin via COX2 expression/PGE2 production leading to skin barrier dysfunction

    PubMed Central

    Lee, Chiang-Wen; Lin, Zih-Chan; Hu, Stephen Chu-Sung; Chiang, Yao-Chang; Hsu, Lee-Fen; Lin, Yu-Ching; Lee, I-Ta; Tsai, Ming-Horng; Fang, Jia-You

    2016-01-01

    We explored the regulation of filaggrin, cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) expression induced by urban particulate matter (PM) in human keratinocytes. In addition, we investigated the signaling pathways involved in PM-induced effects on COX2/PGE2 and filaggrin. PMs induced increases in COX2 expression and PGE2 production, and decreased filaggrin expression. These effects were attenuated by pretreatment with COX2 inhibitor and PGE2 receptor antagonist, or after transfection with siRNAs of the aryl hydrocarbon receptor (AhR), gp91phox and p47phox. Furthermore, PM-induced generation of reactive oxygen species (ROS) and NADPH oxidase activity was attenuated by pretreatment with an AhR antagonist (AhRI) or antioxidants. Moreover, Nox-dependent ROS generation led to phosphorylation of ERK1/2, p38, and JNK, which then activated the downstream molecules NF-κB and AP-1, respectively. In vivo studies in PMs-treated mice showed that AhRI and apocynin (a Nox2 inhibitor) had anti-inflammatory effects by decreasing COX2 and increasing filaggrin expression. Our results reveal for the first time that PMs-induced ROS generation is mediated through the AhR/p47 phox/NADPH oxidase pathway, which in turn activates ERK1/2, p38/NF-κB and JNK/AP-1, and which ultimately induces COX2 expression and filaggrin downregulation. Up-regulated expression of COX2 and production of PGE2 may lead to impairment of skin barrier function. PMID:27313009

  6. Down-regulation of miR-503 expression predicate advanced mythological features and poor prognosis in patients with NSCLC

    PubMed Central

    Liu, Lei; Qu, Weiqing; Zhong, Zhaokun

    2015-01-01

    Objective: We aimed to explore what impact miR-503 has on the prognosis of patients with non-small cell lung cancer (NSCLC). Methods: Cancer and matched non-malignant lung tissue specimens were collected from 109 patients who underwent surgery in Tanisha Hospital from Jun 2006 to July 2013. Overall survival (OS) curves were analyzed using the Lapland-Meier method, and the differences were examined using log-rank tests. Cox proportional- hazards regression analysis was applied in order to estimate univariate and multivariate hazard ratios for OS. Results: The relative expression of miR-503 in NSCLC tissues (0.366 ± 0.130) was significantly lower than that in matched noncancerous lung tissues (1.667 ± 1.047, P < 0.01). Statistically significant association was observed between miR-503 expression and lymphatic invasion (P = 0.005), distant metastasis (P = 0.002), TNM stage (P = 0.008), and tumor grade (P = 0.043). Lapland Meier analysis clearly illustrated that the patients with the lower expression of miR-503 had a worse outcome compared to patients with higher miR-503 expression (P = 0.004). Furthermore, multivariate analysis revealed that miR-503 expression level was an independent prognostic factor for overall survival (HR = 3.992, 95% CI: 2.276-9.872; P = 0.018) in NSCLC. Conclusion: In patients with NSCLC, low miR-503 expression is an independent prognostic factor. PMID:26191272

  7. Urban particulate matter down-regulates filaggrin via COX2 expression/PGE2 production leading to skin barrier dysfunction.

    PubMed

    Lee, Chiang-Wen; Lin, Zih-Chan; Hu, Stephen Chu-Sung; Chiang, Yao-Chang; Hsu, Lee-Fen; Lin, Yu-Ching; Lee, I-Ta; Tsai, Ming-Horng; Fang, Jia-You

    2016-01-01

    We explored the regulation of filaggrin, cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) expression induced by urban particulate matter (PM) in human keratinocytes. In addition, we investigated the signaling pathways involved in PM-induced effects on COX2/PGE2 and filaggrin. PMs induced increases in COX2 expression and PGE2 production, and decreased filaggrin expression. These effects were attenuated by pretreatment with COX2 inhibitor and PGE2 receptor antagonist, or after transfection with siRNAs of the aryl hydrocarbon receptor (AhR), gp91phox and p47phox. Furthermore, PM-induced generation of reactive oxygen species (ROS) and NADPH oxidase activity was attenuated by pretreatment with an AhR antagonist (AhRI) or antioxidants. Moreover, Nox-dependent ROS generation led to phosphorylation of ERK1/2, p38, and JNK, which then activated the downstream molecules NF-κB and AP-1, respectively. In vivo studies in PMs-treated mice showed that AhRI and apocynin (a Nox2 inhibitor) had anti-inflammatory effects by decreasing COX2 and increasing filaggrin expression. Our results reveal for the first time that PMs-induced ROS generation is mediated through the AhR/p47 phox/NADPH oxidase pathway, which in turn activates ERK1/2, p38/NF-κB and JNK/AP-1, and which ultimately induces COX2 expression and filaggrin downregulation. Up-regulated expression of COX2 and production of PGE2 may lead to impairment of skin barrier function. PMID:27313009

  8. Transgenic down-regulation of ARGONAUTE2 expression in Nicotiana benthamiana interferes with several layers of antiviral defenses.

    PubMed

    Odokonyero, Denis; Mendoza, Maria R; Alvarado, Veria Y; Zhang, Jiantao; Wang, Xiaofeng; Scholthof, Herman B

    2015-12-01

    The present study aimed to analyze the contribution of Nicotiana benthamiana ARGONAUTE2 (NbAGO2) to its antiviral response against different viruses. For this purpose, dsRNA hairpin technology was used to reduce NbAGO2 expression in transgenic plants as verified with RT-PCR. This reduction was specific because the expression of other NbAGOs was not affected, and did not cause obvious developmental defects under normal growth conditions. Inoculation of transgenic plants with an otherwise silencing-sensitive GFP-expressing Tomato bushy stunt virus (TBSV) variant resulted in high GFP accumulation because antiviral silencing was compromised. These transgenic plants also exhibited accelerated spread and/or enhanced susceptibility and symptoms for TBSV mutants defective for P19 or coat protein expression, other tombusviruses, Tobacco mosaic virus, and Potato virus X; but not noticeably for Foxtail mosaic virus. These findings support the notion that NbAGO2 in N. benthamiana can contribute to antiviral defense at different levels. PMID:26454664

  9. Peroxisome proliferator-activated receptor {alpha} agonist-induced down-regulation of hepatic glucocorticoid receptor expression in SD rats

    SciTech Connect

    Chen Xiang; Li Ming; Sun Weiping; Bi Yan; Cai Mengyin; Liang Hua; Yu Qiuqiong; He Xiaoying; Weng Jianping

    2008-04-18

    It was reported that glucocorticoid production was inhibited by fenofibrate through suppression of type-1 11{beta}-hydroxysteroid dehydrogenase gene expression in liver. The inhibition might be a negative-feedback regulation of glucocorticoid receptor (GR) activity by peroxisome proliferator-activated receptor alpha (PPAR{alpha}), which is quickly induced by glucocorticoid in the liver. However, it is not clear if GR expression is changed by fenofibrate-induced PPAR{alpha} activation. In this study, we tested this possibility in the liver of Sprague-Dawley rats. GR expression was reduced by fenofibrate in a time- and does-dependent manner. The inhibition was observed in liver, but not in fat and muscle. The corticosterone level in the blood was increased significantly by fenofibrate. These effects of fenofibrate were abolished by PPAR{alpha} inhibitor MK886, suggesting that fenofibrate activated through PPAR{alpha}. In conclusion, inhibition of GR expression may represent a new molecular mechanism for the negative feedback regulation of GR activity by PPAR{alpha}.

  10. Anti-cancer effect of snake venom toxin through down regulation of AP-1 mediated PRDX6 expression

    PubMed Central

    Son, Dong Ju; Song, Ho Sub; Kim, Jung Hyun; Ko, Seong Cheol; Song, Min Jong; Lee, Won Hyoung; Yoon, Joo Hee; Ham, Young Wan; Han, Sang Bae; Hong, Jin Tae

    2015-01-01

    Snake venom toxin (SVT) from Vipera lebetina turanica contains a mixture of different enzymes and proteins. Peroxiredoxin 6 (PRDX6) is known to be a stimulator of lung cancer cell growth. PRDX6 is a member of peroxidases, and has calcium-independent phospholipase A2 (iPLA2) activities. PRDX6 has an AP-1 binding site in its promoter region of the gene. Since AP-1 is implicated in tumor growth and PRDX6 expression, in the present study, we investigated whether SVT inhibits PRDX6, thereby preventing human lung cancer cell growth (A549 and NCI-H460) through inactivation of AP-1. A docking model study and pull down assay showed that SVT completely fits on the basic leucine zipper (bZIP) region of c-Fos of AP-1. SVT (0–10 μg/ml) inhibited lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decreased cIAP and Bcl2 expression via inactivation of AP-1. In an xenograft in vivo model, SVT (0.5 mg/kg and 1 mg/kg) also inhibited tumor growth accompanied with the reduction of PRDX6 expression, but increased expression of proapoptotic proteins. These data indicate that SVT inhibits tumor growth via inhibition of PRDX6 activity through interaction with its transcription factor AP-1. PMID:26061816

  11. Down-regulation of 8-oxoguanine DNA glycosylase 1 expression in the airway epithelium ameliorates allergic lung inflammation.

    PubMed

    Bacsi, Attila; Aguilera-Aguirre, Leopoldo; Szczesny, Bartosz; Radak, Zsolt; Hazra, Tapas K; Sur, Sanjiv; Ba, Xueqing; Boldogh, Istvan

    2013-01-01

    Allergic airway inflammation is characterized by increased expression of pro-inflammatory mediators, inflammatory cell infiltration, mucus hypersecretion, and airway hyperresponsiveness, in parallel with oxidative DNA base and strand damage, whose etiological role is not understood. Our goal was to establish the role of 8-oxoguanine (8-oxoG), a common oxidatively damaged base, and its repair by 8-oxoguanine DNA glycosylase 1 (Ogg1) in allergic airway inflammatory processes. Airway inflammation was induced by intranasally administered ragweed (Ambrosia artemisiifolia) pollen grain extract (RWPE) in sensitized BALB/c mice. We utilized siRNA technology to deplete Ogg1 from airway epithelium; 8-oxoG and DNA strand break levels were quantified by Comet assays. Inflammatory cell infiltration and epithelial methaplasia were determined histologically, mucus and cytokines levels biochemically and enhanced pause was used as the main index of airway hyperresponsiveness. Decreased Ogg1 expression and thereby 8-oxoG repair in the airway epithelium conveyed a lower inflammatory response after RWPE challenge of sensitized mice, as determined by expression of Th2 cytokines, eosinophilia, epithelial methaplasia, and airway hyperresponsiveness. In contrast, 8-oxoG repair in Ogg1-proficient airway epithelium was coupled to an increase in DNA single-strand break (SSB) levels and exacerbation of allergen challenge-dependent inflammation. Decreased expression of the Nei-like glycosylases Neil1 and Neil2 that preferentially excise ring-opened purines and 5-hydroxyuracil, respectively, did not alter the above parameters of allergic immune responses to RWPE. These results show that DNA SSBs formed during Ogg1-mediated repair of 8-oxoG augment antigen-driven allergic immune responses. A transient modulation of OGG1 expression/activity in airway epithelial cells could have clinical benefits. PMID:23127499

  12. Dexmedetomidine protects against learning and memory impairments caused by electroconvulsive shock in depressed rats: Involvement of the NMDA receptor subunit 2B (NR2B)-ERK signaling pathway.

    PubMed

    Gao, Xin; Zhuang, Fu-Zhi; Qin, Shou-Jun; Zhou, Li; Wang, Yun; Shen, Qing-Feng; Li, Mei; Villarreal, Michelle; Benefield, Lauren; Gu, Shu-Ling; Ma, Teng-Fei

    2016-09-30

    Cognitive impairment is a common adverse effect of electroconvulsive therapy (ECT) during treatment for severe depression. Dexmedetomidine (DEX), a sedative-anesthetic drug, is used to treat post-ECT agitation. However, it is not known if DEX can protect against ECT-induced cognitive impairments. To address this, we used chronic unpredictable mild stress (CUMS) to establish a model of depression for ECT treatment. Our Morris water maze and sucrose preference test results suggest that DEX alleviates ECT-induced learning and memory impairments without altering the antidepressant efficacy of ECT. To further investigate the underlying mechanisms of DEX, hippocampal expression of NR2B, p-ERK/ERK, p-CREB/CREB, and BDNF were quantified by western blotting. These results show that DEX suppresses over-activation of NR2B and enhances phosphorylation of ERK1/2 in the hippocampus of ECT-treated depressed rats. Furthermore, DEX had no significant effect on ECT-induced increases in p-CREB and BDNF. Overall, our findings suggest that DEX ameliorates ECT-induced learning and memory impairments in depressed rats via the NR2B-ERK signaling cascade. Moreover, CREB/BDNF seems not appear to participate in the cognitive protective mechanisms of DEX during ECT treatment. PMID:27455425

  13. Down-regulated expression of the protein-tyrosine phosphatase 1B (PTP1B) is associated with aggressive clinicopathologic features and poor prognosis in hepatocellular carcinoma

    SciTech Connect

    Zheng, Long-Yi; Zhou, Dong-Xun; Lu, Jin; Zhang, Wen-Jun; Zou, Da-Jin

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer PTP1B protein showed decreased expression in 67.79% of the HCC patients. Black-Right-Pointing-Pointer Low PTP1B expression predicts poor prognosis of HCC. Black-Right-Pointing-Pointer Low PTP1B expression is correlated with expansion of OV6{sup +} tumor-initiating cells. Black-Right-Pointing-Pointer Down-regulation of PTP1B is associated with activation of Wnt/{beta}-Catenin signaling. -- Abstract: The protein-tyrosine phosphatase 1B (PTP1B) is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and can exert both tumor suppressing and tumor promoting effects in different cancers depending on the substrate involved and the cellular context. However, the expression level and function of PTP1B in hepatocellular carcinoma (HCC) remain unclear. In this study, PTP1B expression was detected by immunohistochemistry in normal liver tissue (n = 16) and hepatocellular carcinoma (n = 169). The correlations between PTP1B expression level and clinicopathologic features and patient survival were also analyzed. One hundred and eleven of 169 HCC patients (65.7%) had negative or low PTP1B expression in tumorous tissues, whereas normal tissues always expressed strong PTP1B. Decreased PTP1B expression was significantly associated with aggressive clinicopathologic features and poor prognosis. Immunohistochemistry also showed that low PTP1B expression level was correlated with high percentage of OV6{sup +} tumor-initiating cells (T-ICs) and high frequency of nuclear {beta}-Catenin expression in HCC specimens. Our findings demonstrate for the first time that the loss of inhibitory effect of PTP1B may contribute to progression and invasion of HCC through activation of Wnt/{beta}-Catenin signaling and expansion of liver T-ICs. PTP1B may serve as a valuable prognostic biomarker and potential therapeutic target in HCC.

  14. Rat peptide methionine sulphoxide reductase: cloning of the cDNA, and down-regulation of gene expression and enzyme activity during aging.

    PubMed Central

    Petropoulos, I; Mary, J; Perichon, M; Friguet, B

    2001-01-01

    Peptide methionine sulphoxide reductase (PMSR, EC 1.8.4.6), the msrA or pmsR gene product, is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins. Decreased expression and/or activity of the PMSR with age could explain, at least in part, the accumulation of oxidized protein observed upon aging. To test this hypothesis, the rat pmsR cDNA was cloned and sequenced. The recombinant protein was expressed, its catalytic activity checked with a synthetic substrate and polyclonal antibodies were raised against recombinant PMSR. The expression of the pmsR gene and protein as well as its catalytic activity were then analysed as a function of age in the rat brain and in two organs that express the most PMSR, liver and kidney. It appears that pmsR gene expression decreases with age in liver and kidney as early as 18 months, whereas protein level and protein activity are reduced in the three organs at the very end of the life of the rat (26 months). These results suggest that the down-regulation of PMSR can contribute to the accumulation of oxidized protein that has been associated with the aging process. PMID:11311146

  15. Glucocorticoid dexamethasone down-regulates basal and vitamin D3 induced cathelicidin expression in human monocytes and bronchial epithelial cell line.

    PubMed

    Kulkarni, Nikhil Nitin; Gunnarsson, Hörður Ingi; Yi, Zhiqian; Gudmundsdottir, Steinunn; Sigurjonsson, Olafur E; Agerberth, Birgitta; Gudmundsson, Gudmundur H

    2016-02-01

    Glucocorticoids (GCs) have been extensively used as the mainstream treatment for chronic inflammatory disorders. The persistent use of steroids in the past decades and the association with secondary infections warrants for detailed investigation into their effects on the innate immune system and the therapeutic outcome. In this study, we analyse the effect of GCs on antimicrobial polypeptide (AMP) expression. We hypothesize that GC related side effects, including secondary infections are a result of compromised innate immune responses. Here, we show that treatment with dexamethasone (Dex) inhibits basal mRNA expression of the following AMPs; human cathelicidin, human beta defensin 1, lysozyme and secretory leukocyte peptidase 1 in the THP-1 monocytic cell-line (THP-1 monocytes). Furthermore, pre-treatment with Dex inhibits vitamin D3 induced cathelicidin expression in THP-1 monocytes, primary monocytes and in the human bronchial epithelial cell line BCi NS 1.1. We also demonstrate that treatment with the glucocorticoid receptor (GR) inhibitor RU486 counteracts Dex mediated down-regulation of basal and vitamin D3 induced cathelicidin expression in THP-1 monocytes. Moreover, we confirmed the anti-inflammatory effect of Dex. Pre-treatment with Dex inhibits dsRNA mimic poly IC induction of the inflammatory chemokine IP10 (CXCL10) and cytokine IL1B mRNA expression in THP-1 monocytes. These results suggest that GCs inhibit innate immune responses, in addition to exerting beneficial anti-inflammatory effects. PMID:26358366

  16. Labdane type diterpenes down-regulate the expression of c-Myc protein, but not of Bcl-2, in human leukemia T-cells undergoing apoptosis.

    PubMed

    Dimas, K; Demetzos, C; Vaos, V; Ioannidis, P; Trangas, T

    2001-06-01

    Sclareol (1) and ent-3beta-hydroxy-13-epi-manoyl oxide (2) belong to the labdane type diterpenes. They were isolated from the leaves and from the fruits of Cistus creticus subsp. creticus, and were found to be active against human leukemic cell lines. Compound 2 was converted to its thiomidazolide derivative (3). Compounds 1 and 3 were found to induce apoptotic cell death in human T-cell leukemia lines and to interfere with their cell cycle, arresting cells at G(0/1) phase. Apoptosis can involve the activation and/or suppression of critical genes such as c-myc whose reduction or its inappropriate expression can be associated with induction of cell death and bcl-2 whose activation prevents apoptosis in the latter case. In order to detect any concomitant effect (1 and 3) upon c-myc and bcl-2 oncogene expression, we performed Western blot analysis to determine the levels of expression of these two genes upon treatment with the above compounds. Western blot analysis showed that of c-myc proto-oncogene levels were markedly reduced before massive apoptosis ensued in H33AJ-JA1 and MOLT3 cells, while bcl-2 expression remained unaffected. Thus, induction of apoptosis due to compounds 1 and 3 in these T-cell leukemic cell lines is preceded by c-myc down regulation and furthermore sustained bcl-2 expression does not rescue cells from apoptosis under the conditions used. PMID:11337016

  17. Prenatal Stress Down-Regulates Reelin Expression by Methylation of Its Promoter and Induces Adult Behavioral Impairments in Rats

    PubMed Central

    Palacios-García, Ismael; Lara-Vásquez, Ariel; Montiel, Juan F.; Díaz-Véliz, Gabriela F.; Sepúlveda, Hugo; Utreras, Elías; Montecino, Martín; González-Billault, Christian; Aboitiz, Francisco

    2015-01-01

    Prenatal stress causes predisposition to cognitive and emotional disturbances and is a risk factor towards the development of neuropsychiatric conditions like depression, bipolar disorders and schizophrenia. The extracellular protein Reelin, expressed by Cajal-Retzius cells during cortical development, plays critical roles on cortical lamination and synaptic maturation, and its deregulation has been associated with maladaptive conditions. In the present study, we address the effect of prenatal restraint stress (PNS) upon Reelin expression and signaling in pregnant rats during the last 10 days of pregnancy. Animals from one group, including control and PNS exposed fetuses, were sacrificed and analyzed using immunohistochemical, biochemical, cell biology and molecular biology approaches. We scored changes in the expression of Reelin, its signaling pathway and in the methylation of its promoter. A second group included control and PNS exposed animals maintained until young adulthood for behavioral studies. Using the optical dissector, we show decreased numbers of Reelin-positive neurons in cortical layer I of PNS exposed animals. In addition, neurons from PNS exposed animals display decreased Reelin expression that is paralleled by changes in components of the Reelin-signaling cascade, both in vivo and in vitro. Furthermore, PNS induced changes in the DNA methylation levels of the Reelin promoter in culture and in histological samples. PNS adult rats display excessive spontaneous locomotor activity, high anxiety levels and problems of learning and memory consolidation. No significant visuo-spatial memory impairment was detected on the Morris water maze. These results highlight the effects of prenatal stress on the Cajal-Retzius neuronal population, and the persistence of behavioral consequences using this treatment in adults, thereby supporting a relevant role of PNS in the genesis of neuropsychiatric diseases. We also propose an in vitro model that can yield new

  18. Glycyrrhizin down-regulates CCL2 and CXCL2 expression in cerulein-stimulated pancreatic acinar cells

    PubMed Central

    Panahi, Yaser; Fakhari, Shohreh; Mohammadi, Mehdi; Rahmani, Mohammad Reza; Hakhamaneshi, Mohammad Saeid; Jalili, Ali

    2015-01-01

    Many inflammatory chemokines release from leukocytes and pancreatic acinar cells which play important roles in pathophysiology of acute pancreatitis (AP). Of interests, CXCL2 and CCL2 have been shown elevated in the plasma of patients with AP. We have recently found that Glycyrrhizin (GZ) attenuates AP in mice model. In this study, we aimed to investigate the direct effect of GZ on expression levels of CCL2 and CXCl2 in isolated pancreatic acinar cells. Isolated acinar cells were isolated from the pancreas of healthy C57BL/6 mice, stimulated with cerulein (10-7 M) and then treated with either PBS or different doses of GZ. The levels of CCL2 and CXCL2 expression at mRNA were assessed by qRT-PCR. Conditioned media from supernatants of each cells culture condition were collected for detection of CCL2 and CXCL2 levels by ELISA. First, we observed that cerulein significantly upregulates both cytokines expression in acinar cells. Moreover, we treated the acinar cells with GZ and found that GZ significantly downregulates CCL2 and CXCL2 expression at mRNA levels in a dose-dependent manner. Consistently, the conditioned media of GZ-treated cells contained a significant lower levels of CCL2 and CXCL2 (p<0.05). In conclusion, our data demonstrate for the first time that GZ directly downregulates CCL2 and CXCL2 levels in cerulein-stimulated acinar cells which may explain the mechanism of therapeutic effects of GZ in cerulein-induced AP in mice. PMID:26155433

  19. Down-regulation of STAT3 expression using vector-based RNA interference promotes apoptosis in Hepatocarcinoma cells.

    PubMed

    Zhang, Junwei; Du, Jiajun; Liu, Qi; Zhang, Yi

    2016-08-01

    In this study, we followed a DNA vector-based RNAi approach to silence the signal transducer and activator of transcription 3 (STAT3) expression in Bel-7402 cells, to explore how the Janus kinase (JAK)/STAT3 signaling pathway influences the apoptosis of hepatocarcinoma cells. According to GenBank's STAT3 cDNA, the plasmid pGCsi.U6/neoRFP STAT3, which was designed for expression of STAT3 small interfering RNA (siRNA), was constructed and synthesized, and then transfected into Bel-7402 cells using Lipofectamine 2000. Cells with or without siRNA transfection were treated in wells. The apoptotic rate was detected by flow cytometry (FCM) and by staining with the Annexin V/propidium iodide (PI) apoptosis detection kit. Simultaneously, the mitochondrial membrane potential (ΔΨm) was visualized by JC-1 fluorescence staining and observed using the inverted fluorescence microscope. Furthermore, the expression of caspase-3 protein was analyzed by Western blotting. The results showed that treatment with STAT3 siRNA displayed effects in the Bel-7402 cells, causing a significantly increased apoptotic ratio (P < 0.05). The mitochondrial membrane potential of the STAT3 siRNA group, observed by the JC-1 fluorescence staining, decreased significantly. The protein expression of active caspase-3 increased with STAT3 siRNA treatment, and was significantly higher than that of the control group (P < 0.05). STAT3 gene-silencing significantly improves the apoptotic effect against Bel-7402 cells. PMID:26134753

  20. PPARgamma inhibits osteogenesis via the down-regulation of the expression of COX-2 and iNOS in rats.

    PubMed

    Lin, Tzu-Hung; Yang, Rong-Sen; Tang, Chih-Hsin; Lin, Chih-Peng; Fu, Wen-Mei

    2007-10-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor, is considered as an anti-osteoblastic factor associated with adiposity and the elderly osteoporosis due to a defect in osteoblastogenesis. We have found that oral administration of PPARgamma activator rosiglitazone decreased tibia BMD and serum ALP but left serum calcium and osteoclast marker C-terminal telopeptide unaffected. In addition, we examined the inhibitory mechanisms of PPARgamma on the bone formation by using PPARgamma activators ciglitazone and 15-deoxy-Delta(12,14)-prostaglandin-J2 (15d-PGJ2). Our data indicated that PPARgamma ligands decreased both mineralized bone nodules and alkaline phosphatase (ALP) activities in cultured primary osteoblasts. Reverse transcription polymerase chain reaction (RT-PCR) showed that the expression of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) was inhibited by ciglitizone and 15d-PGJ2. Furthermore, PPARgamma ligands inhibited NF-kappaB associated downstream COX-2 and iNOS osteogenic signaling. The ultrasound (US)-induced elevation of COX-2 and iNOS expression and nitric oxide (NO) production were attenuated in the presence of PPARgamma ligands. Furthermore, local administration of PPARgamma ligands into the metaphysis of rat tibia decreased the bone volume in secondary spongiosa. These results suggest that the activation of PPARgamma inhibits osteoblastic differentiation and the expression of several anabolic mediators involved in bone formation. These data may reflect osteoporosis and less bone formation in the aging people and patients treated with thiazolidinediones. PMID:17669705

  1. High Glucose Induces Down-Regulated GRIM-19 Expression to Activate STAT3 Signaling and Promote Cell Proliferation in Cell Culture

    PubMed Central

    Li, Yong-Guang; Han, Bei-Bei; Li, Feng; Yu, Jian-Wu; Dong, Zhi-Feng; Niu, Geng-Ming; Qing, Yan-Wei; Li, Jing-Bo; Wei, Meng; Zhu, Wei

    2016-01-01

    Recent studies indicated that Gene Associated with Retinoid-IFN-Induced Mortality 19 (GRIM-19), a newly discovered mitochondria-related protein, can regulate mitochondrial function and modulate cell viability possibly via interacting with STAT3 signal. In the present study we sought to test: 1) whether GRIM-19 is involved in high glucose (HG) induced altered cell metabolism in both cancer and cardiac cells, 2) whether GRIM-19/STAT3 signaling pathway plays a role in HG induced biological effects, especially whether AMPK activity could be involved. Our data showed that HG enhanced cell proliferation of both HeLa and H9C2 cells, which was closely associated with down-regulated GRIM-19 expression and increased phosphorylated STAT3 level. We showed that GRIM-19 knock-down alone in normal glucose cultured cells can also result in an increase in phosphorylated STAT3 level and enhanced proliferation capability, whereas GRIM-19 over-expression can abolished HG induced STAT3 activation and enhanced cell proliferation. Importantly, both down-regulated or over-expression of GRIM-19 increased lactate production in both HeLa and H9C2 cells. The activated STAT3 was responsible for increased cell proliferation as either AG-490, an inhibitor of JAK2, or siRNA targeting STAT3 can attenuate cell proliferation increased by HG. In addition, HG increased lactate acid levels in HeLa cells, which was also observed when GRIM-19 was genetically manipulated. However, HG did not affect the lactate levels in H9C2 cells. Of note, over-expression of GRIM-19 and silencing of STAT3 both increased lactate production in H9C2 cells. As expected, HG resulted in significant decreases in phosphorylated AMPKα levels in H9C2 cells, but not in HeLa cells. Interestingy, activation of AMPKα by metformin was associated with a reversal of the suppressed GRIM-19 expression in H9C2 cells, the fold of changes in GRIM-19 expression by metformin were much less in HeLa cells. Metformin did not affect the

  2. Down regulation by p60v-src of genes specifically expressed and developmentally regulated in postmitotic quail neuroretina cells.

    PubMed Central

    Guermah, M; Gillet, G; Michel, D; Laugier, D; Brun, G; Calothy, G

    1990-01-01

    The avian neuroretina (NR) is composed of photoreceptors and different neurons that are derived from proliferating precursor cells. Neuronal differentiation takes place after terminal mitosis. We have previously shown that differentiating NR cells can be induced to proliferate by infection with Rous sarcoma virus (RSV) and that cell multiplication requires expression of a functional v-src gene. We speculated that the quiescence of NR cells could be determined by specific genes. Cell proliferation could then result from the negative regulation of these genes by the v-src protein. By differential hybridization of a cDNA library, we isolated eight clones corresponding to genes expressed in postmitotic NR cells from 13-day-old quail embryos, transcriptional levels of which are significantly reduced in NR cells induced to proliferate by tsNY68, an RSV mutant with temperature-sensitive mitogenic activity. Partial sequencing analysis indicated that one RNA encoded the calmodulin gene, whereas the other seven showed no similarity to known sequences. By using v-src mutants that induce NR cell proliferation in the absence of transformation, we showed that transcription of six genes was negatively regulated by the v-src protein and that of four genes was correlated with NR cell quiescence. We also report that a subset of genes are specifically transcribed in neural cells and developmentally regulated in the NR. These results indicate that the v-src protein regulates expression of genes likely to play a role in the control of neural cell growth or differentiation. Images PMID:2162475

  3. Thymosin From Bombyx mori Is Down-Regulated in Expression by BmNPV Exhibiting Antiviral Activity

    PubMed Central

    Zhang, Chen; Wang, Yongdi; Fang, Qiang; Xu, Minlin; Lv, Mengyuan; Liao, Jinxu; Li, Si; Nie, Zuoming; Zhang, Wenping

    2016-01-01

    Thymosins have been highly conserved during evolution. These hormones exist in many animal species and play an essential role in many biological events. However, little is known regarding the physiological function of silkworm Bombyx mori thymosin (BmTHY). In this study, we investigated the expression pattern of BmTHY in a Bombyx mori larval ovarian cell line (BmN) challenged with Bombyx mori nuclear polyhydrosis virus (BmNPV) and the antiviral effect of recombinant BmTHY (rBmTHY) for Bombyx mori against BmNPV. Western-blot assay and qRT-PCR analysis revealed that the level of BmTHY protein expression and transcription decreased over time when BmN cells were infected by BmNPV. Treatment with endotoxin-free rBmTHY led to a significant reduction in viral titer in the supernatant of BmN cells challenged with BmNPV. The results from antiviral tests performed in vitro and in vivo showed that endotoxin-free rBmTHY improved the survival rate of Bombyx mori infected with BmNPV. These findings suggest that BmTHY exerts immunomodulatory effects on Bombyx mori, rendering them resistant to viral infection. PMID:27432352

  4. Thymosin From Bombyx mori Is Down-Regulated in Expression by BmNPV Exhibiting Antiviral Activity.

    PubMed

    Zhang, Chen; Wang, Yongdi; Fang, Qiang; Xu, Minlin; Lv, Mengyuan; Liao, Jinxu; Li, Si; Nie, Zuoming; Zhang, Wenping

    2016-01-01

    Thymosins have been highly conserved during evolution. These hormones exist in many animal species and play an essential role in many biological events. However, little is known regarding the physiological function of silkworm Bombyx mori thymosin (BmTHY). In this study, we investigated the expression pattern of BmTHY in a Bombyx mori larval ovarian cell line (BmN) challenged with Bombyx mori nuclear polyhydrosis virus (BmNPV) and the antiviral effect of recombinant BmTHY (rBmTHY) for Bombyx mori against BmNPV. Western-blot assay and qRT-PCR analysis revealed that the level of BmTHY protein expression and transcription decreased over time when BmN cells were infected by BmNPV. Treatment with endotoxin-free rBmTHY led to a significant reduction in viral titer in the supernatant of BmN cells challenged with BmNPV. The results from antiviral tests performed in vitro and in vivo showed that endotoxin-free rBmTHY improved the survival rate of Bombyx mori infected with BmNPV. These findings suggest that BmTHY exerts immunomodulatory effects on Bombyx mori, rendering them resistant to viral infection. PMID:27432352

  5. Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

    PubMed Central

    CAO, Huibi; WANG, Anan; MARTIN, Bernard; KOEHLER, David R.; ZEITLIN, Pamela L.; TANAWELL, A. Keith; HU, Jim

    2015-01-01

    Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation. Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr−/−; C38, Cftr-corrected) stimulated with TNF-α, IL-1β or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels of IκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases. PMID:15740640

  6. Reduction in bile acid pool causes delayed liver regeneration accompanied by down-regulated expression of FXR and c-Jun mRNA in rats.

    PubMed

    Dong, Xiushan; Zhao, Haoliang; Ma, Xiaoming; Wang, Shiming

    2010-02-01

    The present study attempted to examine the effects of bile acid pool size on liver regeneration after hepatectomy. The rats were fed on 0.2% cholic acid (CA) or 2% cholestyramine for 7 days to induce a change in the bile acid size, and then a partial hepatectomy (PH) was performed. Rats fed on the normal diet served as the controls. Measurements were made on the rate of liver regeneration, the labeling indices of PCNA, the plasma total bile acids (TBA), and the mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), farnesoid X receptor (FXR), and transcription factor c-Jun or c-fos. As compared with the normal and CA groups, the rate of liver regeneration was decreased on the day 3, and 7 after PH; the peak of the labeling indices of PCNA was delayed and the labeling indices were significantly reduced on the day 1; the TBA were also decreased on the day 1; the expression of FXR decreased but that of CYP7A1 increased at any given time; at the 1st, and 3rd h, the expression of c-Jun was declined in the cholestyramine group. The reduction in the bile acid pool size was found to delay the liver regeneration, which may be caused by the down-regulation of FXR and c-Jun expression. PMID:20155456

  7. 1,25-hydroxyvitamin D relieves colitis in rats via down-regulation of toll-like receptor 9 expression

    PubMed Central

    Dai, Zhang-han; Tan, Bei; Yang, Hong; Wang, Ou; Qian, Jia-ming; Lv, Hong

    2015-01-01

    Aim To investigate the therapeutic and immunoregulatory effects of 1,25-dihydroxyvitamin D (1,25(OH)D3) on 2,4,6-trinitrobenzenesulfonic acid (TNBS) -induced colitis in rats. Methods Experimental colitis induced by enema administration of TNBS plus ethanol was treated with 5-aminosalicylic acid (5-ASA) and/or 1,25(OH)D3. Disease activity was measured using the disease activation index (DAI), colon macroscopic damage index (CMDI), histological colonic damage score, and myeloperoxidase (MPO) activity. The expression of toll-like receptor 9 (TLR9) in the colon was determined by reverse transcription-polymerase chain reaction and immunohistochemistry. Results Rats with TNBS-induced colitis had significantly elevated DAI, CMDI, histological colonic damage score, and MPO activity (all P < 0.001) compared to rats without colitis. Treatment with 5-ASA or 1,25(OH)D3 ameliorated colitis by lowering CMDI (P = 0.049, P = 0.040, respectively), histological colonic damage score (P = 0.010, P = 0.005, respectively), and MPO activity (P = 0.0003, P = 0.0013, respectively) compared with the TNBS group. Combined treatment with 5-ASA and 1,25(OH)D3 significantly decreased MPO activity (P = 0.003). 1,25(OH)D3 attenuated colitis without causing hypercalcemia or renal insufficiency. TNBS significantly increased the number of TLR9 positive cells compared to control (P < 0.010), while 5-ASA, 1,25(OH)D3, and combined treatment with 5-ASA and 1,25(OH)D3 significantly decreased it compared to TNBS group (all P < 0.010). In TNBS group a moderate correlation was observed between MPO activity and the number of TLR9-positive cells (r = 0.654, P < 0.001). Conclusion TLR9 expression correlates with the extent of inflammation in TNBS-induced colitis. 1,25(OH)D3 relieves this inflammation possibly by decreasing TLR9 expression. PMID:26718757

  8. Difluorinated-Curcumin (CDF) Restores PTEN Expression in Colon Cancer Cells by Down-Regulating miR-21

    PubMed Central

    Roy, Sanchita; Yu, Yingjie; Padhye, Subhash B.; Sarkar, Fazlul H.; Majumdar, Adhip P.N.

    2013-01-01

    Despite recent advancement in medicine, nearly 50% of patients with colorectal cancer show recurrence of the disease. Although the reasons for the high relapse are not fully understood, the presence of chemo- and radiotherapy-resistant cancer stem/stem-like cells, where many oncomirs like microRNA-21 (miR-21) are upregulated, could be one of the underlying causes. miR-21 regulates the processes of invasion and metastasis by downregulating multiple tumor/metastatic suppressor genes including PTEN (phosphatase and tensin homolog). Tumor suppressor protein PTEN controls self-renewal of stem cells. Indeed, our current data demonstrate a marked downregulation of PTEN in SCID mice xenografts of miR-21 over-expressing colon cancer HCT116 cells. Colonospheres that are highly enriched in cancer stem/stem like cells reveal increased miR-21 expression and decreased PTEN. Difluorinated curcumin (CDF), a novel analog of the dietary ingredient curcumin, which has been shown to inhibit the growth of 5-Flurouracil + Oxaliplatin resistant colon cancer cells, downregulated miR-21 in chemo-resistant colon cancer HCT116 and HT-29 cells and restored PTEN levels with subsequent reduction in Akt phosphorylation. Similar results were also observed in metastatic colon cancer SW620 cells. Since PTEN-Akt confers drug resistance to different malignancies including colorectal cancer, our observation of normalization of miR-21-PTEN-Akt pathway by CDF suggests that the compound could be a potential therapeutic agent for chemotherapy-resistant colorectal cancer. PMID:23894315

  9. NR2B-dependent cyclophilin D translocation suppresses the recovery of synaptic transmission after oxygen-glucose deprivation.

    PubMed

    Zhang, Zhihua; Wang, Yongfu; Yan, Shijun; Du, Fang; Yan, Shirley Shidu

    2015-10-01

    N-methyl d-aspartate receptor (NMDA) subunit 2B (NR2B)-containing NMDA receptors and mitochondrial protein cyclophilin D (CypD) are well characterized in mediating neuronal death after ischemia, respectively. However, whether and how NR2B and CypD work together in mediating synaptic injury after ischemia remains elusive. Using an ex vivo ischemia model of oxygen-glucose deprivation (OGD) in hippocampal slices, we identified a NR2B-dependent mechanism for CypD translocation onto the mitochondrial inner membrane. CypD depletion (CypD null mice) prevented OGD-induced impairment in synaptic transmission recovery. Overexpression of neuronal CypD mice (CypD+) exacerbated OGD-induced loss of synaptic transmission. Inhibition of CypD-dependent mitochondrial permeability transition pore (mPTP) opening by cyclosporine A (CSA) attenuated ischemia-induced synaptic perturbation in CypD+ and non-transgenic (non-Tg) mice. The treatment of antioxidant EUK134 to suppress mitochondrial oxidative stress rescued CypD-mediated synaptic dysfunction following OGD in CypD+ slices. Furthermore, OGD provoked the interaction of CypD with P53, which was enhanced in slices overexpressing CypD but was diminished in CypD-null slices. Inhibition of p53 using a specific inhibitor of p53 (pifithrin-μ) attenuated the CypD/p53 interaction following OGD, along with a restored synaptic transmission in both non-Tg and CypD+ hippocampal slices. Our results indicate that OGD-induced CypD translocation potentiates CypD/P53 interaction in a NR2B dependent manner, promoting oxidative stress and loss of synaptic transmission. We also evaluate a new ex vivo chronic OGD-induced ischemia model for studying the effect of oxidative stress on synaptic damage. PMID:26232180

  10. Extreme value theory in analysis of differential expression in microarrays where either only up- or down-regulated genes are relevant or expected

    PubMed Central

    Ivanek, Renata; Gröhn, Yrjö T.; Wells, Martin T.; Raengpradub, Sarita; Kazmierczak, Mark J.; Wiedmann, Martin

    2013-01-01

    Summary We propose an empirical Bayes method based on the extreme value theory (EVT) (BE) for the analysis of data from spotted microarrays where the interest of the investigator (e.g. to identify up-regulated gene markers of a disease) or the design of the experiment (e.g. in certain ‘wild-type versus mutant’ experiments) limits identification of differentially expressed genes to those regulated in a single direction (either up or down). In such experiments, unlike in genome-wide microarrays, analysis is restricted to the tail of the distribution (extremes) of all the genes in the genome. The EVT provides a platform to account for this extreme behaviour, and is therefore a natural candidate for inference about differential expression. We compared the performance of the developed BE method with two other empirical Bayes methods on two real ‘wild-type versus mutant’ datasets where a single direction of regulation was expected due to experimental design, and in a simulation study. The BE method appears to have a better fit to the real data. In the analysis of simulated data, the BE method showed better accuracy and precision while being robust to different characteristics of microarray experiments. The BE method, therefore, seems promising and useful for inference about differential expression in microarrays where either only up- or down-regulated genes are relevant or expected. PMID:18840309

  11. Over-expression of microRNA-223 inhibited the proinflammatory responses in Helicobacter pylori-infection macrophages by down-regulating IRAK-1.

    PubMed

    Wang, Jianjun; Wu, Jianhong; Cheng, Yang; Jiang, Yibiao; Li, Guangxin

    2016-01-01

    MicroRNA-223 plays an important role in the inflammatory response of macrophages. Recent studies have identified that miR-223 was highly expressed in H. pylori infection macrophages, the significance of the elevation, however, has not yet been investigated. In this study, we analyzed the impact of elevated miR-233 to macrophage inflammatory response and possible mechanisms. We found that miR-223 not only could inhibit the expression of inflammatory cytokines including IL-6, IL-8, IL-12 and TNF-α, but also was able to decrease the expression of CD40, CD68, CD80, and CD163. Furthermore, proteins relating to inflammatory signal pathways, such as IRAK-1, NF-κB and MAPK, in H. pylori infected macrophages were down-regulated. Taken together, these results indicated that miR-223 may act as an inflammatory inhibitory factor in H. pylori infected macrophages by IRAK-1, NF-κB or MAPK signal pathways. These findings contribute to the understanding of miR-223 in macrophages inflammatory responses induced by H. pylori. PMID:27158353

  12. Antisense Down-Regulation of 4CL Expression Alters Lignification, Tree Growth, and Saccharification Potential of Field-Grown Poplar1[W][OA

    PubMed Central

    Voelker, Steven L.; Lachenbruch, Barbara; Meinzer, Frederick C.; Jourdes, Michael; Ki, Chanyoung; Patten, Ann M.; Davin, Laurence B.; Lewis, Norman G.; Tuskan, Gerald A.; Gunter, Lee; Decker, Stephen R.; Selig, Michael J.; Sykes, Robert; Himmel, Michael E.; Kitin, Peter; Shevchenko, Olga; Strauss, Steven H.

    2010-01-01

    Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula × Populus alba), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below approximately 50% of the nontransgenic control. Relatively small lignin reductions of approximately 10% were associated with reduced productivity, decreased wood syringyl/guaiacyl lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than approximately 50% 4CL RNA expression were characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees. PMID:20729393

  13. Hoxb-5 down regulation alters Tenascin-C, FGF10 and Hoxb gene expression patterns in pseudoglandular period fetal mouse lung.

    PubMed

    Volpe, MaryAnn V; Ramadurai, Sujatha M; Pham, Lucia D; Nielsen, Heber C

    2007-01-01

    Organ-specific patterning is partly determined by Hox gene regulatory interactions with the extracellular matrix (ECM), cell adhesion and fibroblast growth factor (FGFs) signaling pathways but coordination of these mechanisms in lung development is unknown. We have previously shown that Hoxb-5 affects airway patterning during lung morphogenesis. Hoxb-5 regulation in fetal lung affects ECM expression of tenascin-C and alters FGF10 spatial and cellular expression. To test this hypothesis, gestational day 13.5 (Gd13.5) fetal mouse lung fibroblasts and whole lungs were cultured with Hoxb-5-specific small interfering RNA (siRNA). Western blots showed that siRNA-down regulation of Hoxb-5 led to decreased tenascin-C and FGF10 and was associated with increased Hoxb-4 and decreased Hoxb-6 protein levels. Hoxa-5 protein levels were not affected. Hoxb-5 siRNA-treated whole lung cultures had a significant decrease in total lung and peripheral branching region surface area. Immunostaining showed negligible levels of Hoxb-5 protein and tenascin-C, and loss of FGF10 spatial restriction. We conclude that Hoxb-5 helps regulate lung airway development through modulation of ECM expression of tenascin-C. ECM changes induced by Hoxb-5 may affect mesenchymal-epithelial cell signaling to alter spatial and cellular restriction of FGF10. Hoxb-5 may also affect lung airway branching indirectly by cross regulation of other Hoxb genes. PMID:17127343

  14. All-trans-retinoic Acid Increases SLC26A3 DRA (Down-regulated in Adenoma) Expression in Intestinal Epithelial Cells via HNF-1β*

    PubMed Central

    Priyamvada, Shubha; Anbazhagan, Arivarasu N.; Gujral, Tarunmeet; Borthakur, Alip; Saksena, Seema; Gill, Ravinder K.; Alrefai, Waddah A.; Dudeja, Pradeep K.

    2015-01-01

    All-trans-retinoic acid (ATRA) is an active vitamin A derivative known to modulate a number of physiological processes, including growth and development, differentiation, and gene transcription. The protective effect of ATRA in gut inflammation and diarrheal diseases has been documented. In this regard, down-regulated in adenoma (DRA, a key luminal membrane Cl− transporter involved in NaCl absorption) has been shown to be suppressed in intestinal inflammation. This suppression of DRA is associated with diarrheal phenotype. Therefore, current studies were undertaken to examine the effects of ATRA on DRA expression. DRA mRNA levels were significantly elevated (∼4-fold) in response to ATRA with induction starting as early as 8 h of incubation. Similarly, ATRA increased DRA protein expression by ∼50%. Furthermore, DRA promoter activity was significantly increased in response to ATRA indicating transcriptional activation. ATRA effects on DRA expression appeared to be mediated via the RAR-β receptor subtype, as ATRA remarkably induced RAR-β mRNA levels, whereas RAR-β knockdown substantially attenuated the ability of ATRA to increase DRA expression. Results obtained from agonist (CH-55) and antagonist (LE-135) studies further confirmed that ATRA exerts its effects through RAR-β. Furthermore, ATRA treatment resulted in a significant increase in HNF-1β mRNA levels. The ability of ATRA to induce DRA expression was inhibited in the presence of HNF-1β siRNA indicative of its involvement in ATRA-induced effects on DRA expression. In conclusion, ATRA may act as an antidiarrheal agent by increasing DRA expression via the RAR-β/HNF-1β-dependent pathway. PMID:25887398

  15. Effects of Hypoxia Exposure on Hepatic Cytochrome P450 1A (CYP1A) Expression in Atlantic Croaker: Molecular Mechanisms of CYP1A Down-Regulation

    PubMed Central

    Rahman, Md. Saydur; Thomas, Peter

    2012-01-01

    Hypoxia-inducible factor-α (HIF-α) and cytochrome P450 1A (CYP1A) are biomarkers of environmental exposure to hypoxia and organic xenobiotic chemicals that act through the aryl hydrocarbon receptor, respectively. Many aquatic environments heavily contaminated with organic chemicals, such as harbors, are also hypoxic. Recently, we and other scientists reported HIF-α genes are upregulated by hypoxia exposure in aquatic organisms, but the molecular mechanisms of hypoxia regulation of CYP1A expression have not been investigated in teleost fishes. As a first step in understanding the molecular mechanisms of hypoxia modulation of CYP1A expression in fish, we characterized CYP1A cDNA from croaker liver. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 to 4 weeks) caused significant decreases in hepatic CYP1A mRNA and protein levels compared to CYP1A levels in fish held in normoxic conditions. In vivo studies showed that the nitric oxide (NO)-donor, S-nitroso-N-acetyl-DL-penicillamine, significantly decreased CYP1A expression in croaker livers, whereas the competitive inhibitor of NO synthase (NOS), Nω-nitro-L-arginine methyl ester, restored CYP1A mRNA and protein levels in hypoxia-exposed (1.7 mg DO/L for 4 weeks) fish. In vivo hypoxia exposure also markedly increased interleukin-1β (IL-1β, a cytokine), HIF-2α mRNA and endothelial NOS (eNOS) protein levels in croaker livers. Pharmacological treatment with vitamin E, an antioxidant, lowered the IL-1β, HIF-2α mRNA and eNOS protein levels in hypoxia-exposed fish and completely reversed the down-regulation of hepatic CYP1A mRNA and protein levels in response to hypoxia exposure. These results suggest that hypoxia-induced down-regulation of CYP1A is due to alterations of NO and oxidant status, and cellular IL-1β and HIF-α levels. Moreover, the present study provides the first evidence of a role for antioxidants in hepatic eNOS and IL-1β regulation in aquatic vertebrates during hypoxic stress. PMID:22815834

  16. bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro.

    PubMed

    Chen, Chen; Fan, Yang-yang; Wang, Xin; Song, Fei; Jiang, Tao; Qian, Ping; Tang, Shun-ming; Shen, Xing-jia

    2016-02-01

    Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmo-miR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland (PSG). Firstly, we examined the expression of bmo-miR-0001 and bmo-miR 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of BmFib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pcDNA3.0 and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40]. Finally, the BmN cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected by pcDNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] or pcDNA3.0 with pGL3.0 [A3-luc-Fib-L-3'UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro. PMID:26834013

  17. Ectopic expression of Bcl-XL or Ku70 protects human colon cancer cells (SW480) against curcumin-induced apoptosis while their down-regulation potentiates it.

    PubMed

    Rashmi, Ramachandran; Kumar, Santhosh; Karunagaran, Devarajan

    2004-10-01

    Curcumin, the yellow pigment derived from Curcuma longa, is known to induce apoptosis of several cancer cells. However, many cancer cells protect themselves by over-expressing antiapoptotic proteins such as Bcl-XL or Ku70. To study their role in curcumin-induced apoptosis, human colon cancer cells (SW480) were made to over-express or under-express Bcl-XL (by stable transfection) and Ku70 (by transient transfection) using plasmid constructs that express their genes in sense or antisense orientation, respectively. Stable cells that express Bax [Bax-GFP (green fluorescent protein)], a proapoptotic member of the Bcl-2 family, were also established. Curcumin-induced cell death and nuclear condensation was more in AsBcl-XL and AsKu70 cells that under-express Bcl-XL and Ku70, respectively, compared with the vector-transfected cells. Bcl-XL and Ku70 protected the cells by inhibiting the release of cytochrome c, Smac (second mitochondria derived activator of caspase) and apoptosis inducing factor (AIF), and the activation of caspases 9, 8 and 3 triggered by curcumin. AsBcl-XL and AsKu70 cells were more sensitive to curcumin through enhanced activation of caspases 9 and 3 and release of cytochrome c, Smac and AIF. Curcumin-induced activation of caspase 8 was blocked by Ku70 but not by Bcl-XL. However, caspase 8 activation by curcumin was accelerated in both AsBcl-XL and AsKu70 cells suggesting a possible feedback activation of caspase 8 by caspase 3. Bax-GFP cells were highly sensitized when Ku70 was down-regulated supporting the reported role of Ku70 in the retention of Bax within the cytosol. The study reveals the potential of antisense inhibition of antiapoptotic proteins as an effective strategy to tackle chemoresistant cancers with curcumin. PMID:15205359

  18. bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro*

    PubMed Central

    Chen, Chen; Fan, Yang-yang; Wang, Xin; Song, Fei; Jiang, Tao; Qian, Ping; Tang, Shun-ming; Shen, Xing-jia

    2016-01-01

    Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmo-miR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland (PSG). Firstly, we examined the expression of bmo-miR-0001 and bmo-miR-0015 in 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of BmFib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40]. Finally, the BmN cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected by pcDNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] or pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40] with pGL3.0 [A3-luc-Fib-L-3'UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro. PMID:26834013

  19. Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase.

    PubMed Central

    Sewalt, VJH.; Ni, W.; Blount, J. W.; Jung, H. G.; Masoud, S. A.; Howles, P. A.; Lamb, C.; Dixon, R. A.

    1997-01-01

    We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors. PMID:12223790

  20. Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase.

    PubMed

    Sewalt, VJH.; Ni, W.; Blount, J. W.; Jung, H. G.; Masoud, S. A.; Howles, P. A.; Lamb, C.; Dixon, R. A.

    1997-09-01

    We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors. PMID:12223790

  1. mRNA-Binding Protein TIA-1 Reduces Cytokine Expression in Human Endometrial Stromal Cells and Is Down-Regulated in Ectopic Endometrium

    PubMed Central

    Karalok, Hakan Mete; Aydin, Ebru; Saglam, Ozlen; Torun, Aysenur; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D.; Kristiansson, Helena; Duke, Cindy M. P.; Choe, Gina; Flannery, Clare; Kallen, Caleb B.

    2014-01-01

    Background: Cytokines and growth factors play important roles in endometrial function and the pathogenesis of endometriosis. mRNAs encoding cytokines and growth factors undergo rapid turnover; primarily mediated by adenosine- and uridine-rich elements (AREs) located in their 3′-untranslated regions. T-cell intracellular antigen (TIA-1), an mRNA-binding protein, binds to AREs in target transcripts, leading to decreased gene expression. Objective: The purpose of this article was to determine whether TIA-1 plays a role in the regulation of endometrial cytokine and growth factor expression during the normal menstrual cycle and whether TIA-1 expression is altered in women with endometriosis. Methods: Eutopic endometrial tissue obtained from women without endometriosis (n = 30) and eutopic and ectopic endometrial tissues from women with endometriosis (n = 17) were immunostained for TIA-1. Staining intensities were evaluated by histological scores (HSCOREs). The regulation of endometrial TIA-1 expression by immune factors and steroid hormones was studied by treating primary cultured human endometrial stromal cells (HESCs) with vehicle, lipopolysaccharide, TNF-α, IL-6, estradiol, or progesterone, followed by protein blot analyses. HESCs were engineered to over- or underexpress TIA-1 to test whether TIA-1 regulates IL-6 or TNF-α expression in these cells. Results: We found that TIA-1 is expressed in endometrial stromal and glandular cells throughout the menstrual cycle and that this expression is significantly higher in the perimenstrual phase. In women with endometriosis, TIA-1 expression in eutopic and ectopic endometrium was reduced compared with TIA-1 expression in eutopic endometrium of unaffected control women. Lipopolysaccharide and TNF-α increased TIA-1 expression in HESCs in vitro, whereas IL-6 or steroid hormones had no effect. In HESCs, down-regulation of TIA-1 resulted in elevated IL-6 and TNF-α expression, whereas TIA-1 overexpression resulted in

  2. Nandrolone-pretreatment enhances cardiac beta(2)-adrenoceptor expression and reverses heart contractile down-regulation in the post-stress period of acute-stressed rats.

    PubMed

    Penna, Claudia; Abbadessa, Giuliana; Mancardi, Daniele; Spaccamiglio, Angela; Racca, Silvia; Pagliaro, Pasquale

    2007-10-01

    To investigate whether nandrolone decanoate (ND)-pretreatment can modulate (1) beta-adrenoceptor expression and (2) myocardial contractility in response to beta-adrenoceptors stimulation with isoproterenol (ISO), in hearts of both normal and stressed rats. Rats were treated with 15 mg/(kgday) of Deca-Durabolin (ND, 1 ml i.m.) or with vehicle (oil) for 14 days. The day after the last injection, the dose-response to ISO (1 x 10(-8), 5 x 10(-8) and 10(-7)M), was studied in isolated rat hearts harvested from unstressed animals (unstressed+vehicle (control) or unstressed+ND) or from stressed animals (stressed+vehicle or stressed+ND): acute stress protocol consisted in restrain for 1h immediately before sacrifice. ND-pretreatment increased beta(2)-adrenoceptor expression. In baseline conditions all hearts had a similar left ventricular developed pressure (LVDP) and maximum rate of increase of LVDP (dP/dt(max)). In hearts of unstressed+vehicle or unstressed+ND, ISO caused a similar increase in LVDP (+90-100%) and dP/dt(max) (+120-150%). However, hearts of stressed+vehicle animals showed a marked depression of inotropic response to ISO (i.e. for ISO 1 x 10(-8),-55% in LVDP response versus unstressed). Yet, in hearts of stressed+ND-animals the effect of stress was reversed, showing the highest response to ISO (i.e. for ISO 1 x 10(-7), +30% LVDP response versus unstressed). The ND-induced beta(2)-adrenoceptor overexpression does not affect ISO-response in unstressed animals. However, acute stress induces a down-regulation of ISO-response, which is reversed by ND-pretreatment. Since the physiological post-stress down-regulation of adrenergic-response is absent after nandrolone treatment, the heart may be exposed to a sympathetic over-stimulation. This might represent a risk for cardiovascular incidents in anabolic steroid addicts under stressing conditions. PMID:17611100

  3. IL-6 down-regulates HLA class II expression and IL-12 production of human dendritic cells to impair activation of antigen-specific CD4(+) T cells.

    PubMed

    Ohno, Yosuke; Kitamura, Hidemitsu; Takahashi, Norihiko; Ohtake, Junya; Kaneumi, Shun; Sumida, Kentaro; Homma, Shigenori; Kawamura, Hideki; Minagawa, Nozomi; Shibasaki, Susumu; Taketomi, Akinobu

    2016-02-01

    Immunosuppression in tumor microenvironments critically affects the success of cancer immunotherapy. Here, we focused on the role of interleukin (IL)-6/signal transducer and activator of transcription (STAT3) signaling cascade in immune regulation by human dendritic cells (DCs). IL-6-conditioned monocyte-derived DCs (MoDCs) impaired the presenting ability of cancer-related antigens. Interferon (IFN)-γ production attenuated by CD4(+) T cells co-cultured with IL-6-conditioned MoDCs corresponded with decreased DC IL-12p70 production. Human leukocyte antigen (HLA)-DR and CD86 expression was significantly reduced in CD11b(+)CD11c(+) cells obtained from peripheral blood mononuclear cells (PBMCs) of healthy donors by IL-6 treatment and was STAT3 dependent. Arginase-1 (ARG1), lysosomal protease, cathepsin L (CTSL), and cyclooxygenase-2 (COX2) were involved in the reduction of surface HLA-DR expression. Gene expressions of ARG1, CTSL, COX2, and IL6 were higher in tumor-infiltrating CD11b(+)CD11c(+) cells compared with PBMCs isolated from colorectal cancer patients. Expression of surface HLA-DR and CD86 on CD11b(+)CD11c(+) cells was down-regulated, and T cell-stimulating ability was attenuated compared with PBMCs, suggesting that an immunosuppressive phenotype might be induced by IL-6, ARG1, CTSL, and COX2 in tumor sites of colorectal cancer patients. There was a relationship between HLA-DR expression levels in tumor tissues and the size of CD4(+) T and CD8(+) T cell compartments. Our findings indicate that IL-6 causes a dysfunction in human DCs that activates cancer antigen-specific Th cells, suggesting that blocking the IL-6/STAT3 signaling pathway might be a promising strategy to improve cancer immunotherapy. PMID:26759006

  4. Differential expression of gp200-MR6 molecule in benign hyperplasia and down-regulation in invasive carcinoma of the breast.

    PubMed Central

    al-Tubuly, A. A.; Luqmani, Y. A.; Shousha, S.; Melcher, D.; Ritter, M. A.

    1996-01-01

    In this study, we used immunohistochemical and biochemical analysis to show that gp200-MR6, a 200 kDa molecule that is functionally associated with the human interleukin 4 (IL-4) receptor complex, is expressed at high levels on normal breast epithelial tissues, at lower levels on in situ carcinomas, and that the expression is lost in the invasive carcinoma of the breast. Furthermore, a preliminary study showed that benign epithelial hyperplasia of the breast expresses the gp200-MR6 heterogeneously. Two populations of cells have been observed: MR6 positive and MR6 negative. Interestingly, MR6-positive cells were observed to have different morphology from those that were MR6 negative; the nuclei of the former were larger and rounded in shape, whereas the nuclei of the latter were relatively small and oval in shape. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, monoclonal antibody MR6 detects the same molecular weight molecule in both normal and transformed tissue, indicating that the molecule is not a product of a truncated gene. The intensity of the gp200-MR6 bands correlates with the immunohistochemical data, indicating that the molecule is expressed at high levels in normal tissue and at lower levels in malignant tissue. These results suggest that analysis of gp200-MR6 expression may be useful in tumour grading and prognostic evaluation in breast cancer. Moreover, the molecule may be involved early in the process of tumorigenesis of the breast, in which a loss or a down-regulation of gp200-MR6 could contribute towards tumour development and progression via an effect on cell growth and differentiation. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:8855966

  5. Tetrandrine down-regulates expression of miRNA-155 to inhibit signal-induced NF-κB activation in a rat model of diabetes mellitus

    PubMed Central

    Song, Chunhui; Ji, Yunxi; Zou, Guohui; Wan, Chunxia

    2015-01-01

    Aims: This study is to investigate expression of miRNA-155 and the related signaling pathway in a rat model of diabetes mellitus (DM). Methods: Thirty-six SD rats were divided into control, DM, and tetrandrine groups. A rat model of DM was constructed by tail vein injection with alloxan. Levels of related cytokines in serum samples were detected. The mRNA levels of IκBα and TNF-α in pancreatic islet tissues were detected by real-time PCR. Protein expression of IκBα and TNF-α was detected by western blotting. Expression of miRNA-155 in pancreatic islet tissues and serum samples was detected by real-time PCR. Results: Compared with those in the control and the tetrandrine groups, activities of methane dicarboxylic aldehyde and reactive oxygen species in serum samples and pancreatic islet mitochondria tissues in the DM group were increased (P < 0.05), while activity of superoxide dismutase in the DM group was decreased (P < 0.05). Activities of haemoglobin A1c and glucose in serum samples in the DM group were increased, while insulin in the DM group was decreased (P < 0.05). The mRNA and protein levels of IκBα in pancreatic islet tissues in the DM group were decreased (P < 0.05), while the mRNA and protein levels of TNF-α in the DM group were increased (P < 0.05). Expression of miRNA-155 in pancreatic islet tissues and serum samples in the DM group was increased (P < 0.05). Conclusion: Tetrandrine prevented injury in rat pancreatic islet caused by alloxan, which was related with decreased oxidative stress, down-regulated miRNA-155 and decreased TNF-α in the NF-κB signaling pathway. These results indicate that tetrandrine plays an important role in DM by regulating expression of miRNA-155. PMID:26064305

  6. Cytoplasmic localization of Lrh-1 down-regulates ovarian follicular cyp19a1a expression in a teleost, the orange-spotted grouper Epinephelus coioides.

    PubMed

    Lu, Huijie; Zhang, Shen; Liu, Qiongyou; Zhang, Lihong; Zhang, Weimin

    2014-08-01

    Liver receptor homolog-1 (LRH-1) is a conserved member of the NR5A subfamily in vertebrates and a potential regulator of estrogen synthesis in the ovarian granulosa cells. An Lrh-1 homologue was obtained from the orange-spotted grouper Epinephelus coioides that contains the conserved structural features of NR5A and is phylogenetically closely related to NR5A2. The expression of the orange-spotted grouper Lrh-1 is tissue-specific with relatively higher levels in the liver and ovary. The immunoreactive signals for Lrh-1 and Cyp19a1a were present in the ovarian follicular cells and germ cells. In the ovarian follicular cells, Lrh-1 was present both in the nucleus and cytoplasm, and colocalized with Cyp19a1a. The expression levels of both increased during vitellogenesis whereas only Cyp19a1a dramatically decreased toward maturation when Lrh-1 was localized almost exclusively to the cytoplasm of the follicular cells. The orange-spotted grouper Lrh-1 could up-regulate cyp19a1a transcription in vitro via the two conserved Ftz-f1 sites in cyp19a1a promoter. Chromatin immunoprecipitation analysis showed that the orange-spotted grouper Lrh-1 could bind cyp19a1a promoter in vivo with a higher abundance in the vitellogenic ovary, whereas the binding was dramatically decreased in the mature ovary. Taken together, the results of present study demonstrate that Lrh-1 plays an important role in up-regulating cyp19a1a gene in the ovarian follicular cells during vitellogenesis, and the sequestration of Lrh-1 to the cytoplasm may down-regulate cyp19a1a expression in the mature ovary. This mechanism for modifying transcriptional roles of the orange-spotted grouper Lrh-1 may shed new light on the regulation of Cyp19a1 expression in other vertebrates as well. PMID:24943038

  7. Sodium arsenite down-regulates the expression of X-linked inhibitor of apoptosis protein via translational and post-translational mechanisms in hepatocellular carcinoma

    SciTech Connect

    Chen, Hong; Hao, Yuqing; Wang, Lijing; Jia, Dongwei; Ruan, Yuanyuan; Gu, Jianxin

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Sodium arsenite down-regulates the protein expression level of XIAP in HCC. Black-Right-Pointing-Pointer Sodium arsenite inhibits the de novo XIAP synthesis and its IRES activity. Black-Right-Pointing-Pointer Sodium arsenite decreases XIAP stability and promotes its proteasomal degradation. Black-Right-Pointing-Pointer Overexpression of XIAP attenuates the pro-apoptotic effect of sodium arsenite. -- Abstract: X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitors of apoptosis protein (IAP) family, and has been reported to exhibit elevated expression levels in hepatocellular carcinoma (HCC) and promote cell survival, metastasis and tumor recurrence. Targeting XIAP has proven effective for the inhibition of cancer cell proliferation and restoration of cancer cell chemosensitivity. Arsenic (or sodium arsenite) is a potent anti-tumor agent used to treat patients with acute promyelocytic leukemia (APL). Additionally, arsenic induces cell growth inhibition, cell cycle arrest and apoptosis in human HCC cells. In this study, we identified XIAP as a target for sodium arsenite-induced cytotoxicity in HCC. The exposure of HCC cell lines to sodium arsenite resulted in inhibition of XIAP expression in both a dose- and time-dependent manner. Sodium arsenite blocked the de novo XIAP synthesis and the activity of its internal ribosome entry site (IRES) element. Moreover, treatment with sodium arsenite decreased the protein stability of XIAP and induced its ubiquitin-proteasomal degradation. Overexpression of XIAP attenuated the pro-apoptotic effect of sodium arsenite in HCC. Taken together, our data demonstrate that sodium arsenite suppresses XIAP expression via translational and post-translational mechanisms in HCC.

  8. Deletions of multidrug resistance gene loci in breast cancer leads to the down-regulation of its expression and predict tumor response to neoadjuvant chemotherapy

    PubMed Central

    Litviakov, Nikolai V.; Cherdyntseva, Nadezhda V.; Tsyganov, Matvey M.; Slonimskaya, Elena M.; Ibragimova, Marina K.; Kazantseva, Polina V.; Kzhyshkowska, Julia; Choinzonov, Eugeniy L.

    2016-01-01

    Neoadjuvant chemotherapy (NAC) is intensively used for the treatment of primary breast cancer. In our previous studies, we reported that clinical tumor response to NAC is associated with the change of multidrug resistance (MDR) gene expression in tumors after chemotherapy. In this study we performed a combined analysis of MDR gene locus deletions in tumor DNA, MDR gene expression and clinical response to NAC in 73 BC patients. Copy number variations (CNVs) in biopsy specimens were tested using high-density microarray platform CytoScanTM HD Array (Affymetrix, USA). 75%–100% persons having deletions of MDR gene loci demonstrated the down-regulation of MDR gene expression. Expression of MDR genes was 2–8 times lower in patients with deletion than in patients having no deletion only in post-NAC tumors samples but not in tumor tissue before chemotherapy. All patients with deletions of ABCB1 ABCB 3 ABCC5 gene loci – 7q21.1, 6p21.32, 3q27 correspondingly, and most patients having deletions in ABCC1 (16p13.1), ABCC2 (10q24), ABCG1 (21q22.3), ABCG2 (4q22.1), responded favorably to NAC. The analysis of all CNVs, including both amplification and deletion showed that the frequency of 13q14.2 deletion was 85% among patients bearing tumor with the deletion at least in one MDR gene locus versus 9% in patients with no deletions. Differences in the frequency of 13q14.2 deletions between the two groups were statistically significant (p = 2.03 ×10−11, Fisher test, Bonferroni-adjusted p = 1.73 × 10−8). In conclusion, our study for the first time demonstrates that deletion MDR gene loci can be used as predictive marker for tumor response to NAC. PMID:26799285

  9. Triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes by down-regulating expression of a viral protein LMP1

    SciTech Connect

    Zhou, Heng; Guo, Wei; Long, Cong; Wang, Huan; Wang, Jingchao; Sun, Xiaoping

    2015-01-16

    Highlights: • Triptolide inhibits proliferation of EBV-positive lymphoma cells in vitro and in vivo. • Triptolide reduces expression of LMP1 by decreasing its transcription level. • Triptolide inhibits ED-L1 promoter activity. - Abstract: Epstein–Barr virus (EBV) infects various types of cells and mainly establishes latent infection in B lymphocytes. The viral latent membrane protein 1 (LMP1) plays important roles in transformation and proliferation of B lymphocytes infected with EBV. Triptolide is a compound of Tripterygium extracts, showing anti-inflammatory, immunosuppressive, and anti-cancer activities. In this study, it is determined whether triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes. The CCK-8 assays were performed to examine cell viabilities of EBV-positive B95-8 and P3HR-1 cells treated by triptolide. The mRNA and protein levels of LMP1 were examined by real time-PCR and Western blotting, respectively. The activities of two LMP1 promoters (ED-L1 and TR-L1) were determined by Dual luciferase reportor assay. The results showed that triptolide inhibited the cell viability of EBV-positive B lymphocytes, and the over-expression of LMP1 attenuated this inhibitory effect. Triptolide decreased the LMP1 expression and transcriptional levels in EBV-positive B cells. The activity of LMP1 promoter ED-L1 in type III latent infection was strongly suppressed by triptolide treatment. In addition, triptolide strongly reduced growth of B95-8 induced B lymphoma in BALB/c nude mice. These results suggest that triptolide decreases proliferation of EBV-induced B lymphocytes possibly by a mechanism related to down-regulation of the LMP1 expression.

  10. DZNep, inhibitor of S-adenosylhomocysteine hydrolase, down-regulates expression of SETDB1 H3K9me3 HMTase in human lung cancer cells.

    PubMed

    Lee, Ju-Kyung; Kim, Keun-Cheol

    2013-09-01

    3-Deazaneplanocin A (DZNep), an epigenetic anticancer drug, leads to the indirect suppression of S-adenosyl methionine-dependent cellular methylations by inhibiting S-adenosyl homocystein (AdoHcy) hydrolase. Although it is well known that DZNep targets the degradation of EZH2 protein, H3K27me3 HMTase, there are still uncertainties about the regulation of other types of HMTases during cell death. In this study, we describe that SETDB1 gene expression was regulated by DZNep treatment in human lung cancer cells. We confirm that DZNep induced growth inhibition and increased the dead cell population of lung cancer cells. DZNep treatment affected histone methylations, including H3K27me3 and H3K9me3, but not H3K4me3. Reduced levels of H3K27me3 and H3K9me3 were related with the decreased EZH2 and SETDB1 proteins. Real time PCR analysis showed that SETDB1 gene expression was decreased by DZNep treatment, but no effect was observed for EZH2 gene expression. We cloned the promoter region of SETDB1 and SUV39H1 genes, and performed luciferase assays. The promoter activity of SETDB1 gene was down regulated by DZNep treatment, whereas no effect on SUV39H1 promoter activity was observed. In conclusion, we suggest that DZNep regulates not only on H3K27me3 HMTase EZH2, but also H3K9 HMTase SETDB1 gene expression at the transcription level, implicating that the mechanism of action of DZNep targets multiple HMTases during the death of lung cancer cells. PMID:23933322

  11. Wild-type and mutated presenilins 2 trigger p53-dependent apoptosis and down-regulate presenilin 1 expression in HEK293 human cells and in murine neurons.

    PubMed

    Alves da Costa, Cristine; Paitel, Erwan; Mattson, Mark P; Amson, Robert; Telerman, Adam; Ancolio, Karine; Checler, Frédéric; Mattson, Marc P

    2002-03-19

    Presenilins 1 and 2 are two homologous proteins that, when mutated, account for most early onset Alzheimer's disease. Several lines of evidence suggest that, among various functions, presenilins could modulate cell apoptotic responses. Here we establish that the overexpression of presenilin 2 (PS2) and its mutated form Asn-141-Ile-PS2 alters the viability of human embryonic kidney (HEK)293 cells as established by combined trypan blue exclusion, sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay, and propidium iodide incorporation FACS analyses. The two parent proteins increase the acetyl-DEVD-al-sensitive caspase-3-like activity in both HEK293 cells and Telencephalon specific murine neurons, modulate Bax and bcl-2 expressions, and enhance cytochrome C translocation into the cytosol. We show that overexpression of both wild-type and mutated PS2 increases p53-like immunoreactivity and transcriptional activity. We also establish that wild-type- and mutated PS2-induced caspase activation is reduced by p53 antisense approach and by pifithrin-alpha, a chemical inhibitor of p53. Furthermore, mouse fibroblasts in which the PS2 gene has been knocked out exhibited strongly reduced p53-transcriptional activity. Finally, we establish that the overexpression of both wild-type and mutated PS2 is accompanied by a drastic reduction of endogenous presenilin 1 (PS1) expression. Interestingly, pifithrin-alpha diminished endogenous PS2 immunoreactivity, whereas the inhibitor increases PS1 expression. Altogether, our data demonstrate that wild-type and familial Alzheimer's disease-linked PS2 trigger apoptosis and down-regulate PS1 expression through p53-dependent mechanisms. PMID:11904448

  12. Angiotensin II receptor blockade promotes repair of skeletal muscle through down-regulation of aging-promoting C1q expression

    PubMed Central

    Yabumoto, Chizuru; Akazawa, Hiroshi; Yamamoto, Rie; Yano, Masamichi; Kudo-Sakamoto, Yoko; Sumida, Tomokazu; Kamo, Takehiro; Yagi, Hiroki; Shimizu, Yu; Saga-Kamo, Akiko; Naito, Atsuhiko T.; Oka, Toru; Lee, Jong-Kook; Suzuki, Jun-ichi; Sakata, Yasushi; Uejima, Etsuko; Komuro, Issei

    2015-01-01

    Disruption of angiotensin II type 1 (AT1) receptor prolonged life span in mice. Since aging-related decline in skeletal muscle function was retarded in Atgr1a−/− mice, we examined the role of AT1 receptor in muscle regeneration after injury. Administration of AT1 receptor blocker irbesartan increased the size of regenerating myofibers, decreased fibrosis, and enhanced functional muscle recovery after cryoinjury. We recently reported that complement C1q, secreted by macrophages, activated Wnt/β-catenin signaling and promoted aging-related decline in regenerative capacity of skeletal muscle. Notably, irbesartan induced M2 polarization of macrophages, but reduced C1q expression in cryoinjured muscles and in cultured macrophage cells. Irbesartan inhibited up-regulation of Axin2, a downstream gene of Wnt/β-catenin pathway, in cryoinjured muscles. In addition, topical administration of C1q reversed beneficial effects of irbesartan on skeletal muscle regeneration after injury. These results suggest that AT1 receptor blockade improves muscle repair and regeneration through down-regulation of the aging-promoting C1q-Wnt/β-catenin signaling pathway. PMID:26571361

  13. Down-regulation of tyrosinase, TRP-1, TRP-2 and MITF expressions by citrus press-cakes in murine B16 F10 melanoma

    PubMed Central

    Kim, Sang Suk; Kim, Min-Jin; Choi, Young Hun; Kim, Byung Kok; Kim, Kwang Sik; Park, Kyung Jin; Park, Suk Man; Lee, Nam Ho; Hyun, Chang-Gu

    2013-01-01

    Objective To investigate the suitability of citrus-press cakes, by-products of the juice industry as a source for the whitening agents for cosmetic industry. Methods Ethylacetate extracts of citrus-press cakes (CCE) were examined for their anti-melanogenic potentials in terms of the inhibition of melanin production and mechanisim of melanogenesis by using Western Blot analysis with tyrosinese, tyrosinase-related protein-1 (TRP-1), TRP2, and microphthalmia-associated transcription factor (MITF) proteins. To apply the topical agents, citrus-press cakes was investigated the safety in human skin cell line. Finally flavonoid analysis of CCE was also determined by HPLC analysis. Results Results indicated that CCE were shown to down-regulate melanin content in a dose-dependent pattern. The CCE inhibited tyrosinase, TRP-2, and MITF expressions in a dose-dependent manner. To test the applicability of CCE to human skin, we used MTT assay to assess the cytotoxic effects of CCE on human keratinocyte HaCaT cells. The CCE exhibited low cytotoxicity at 50 µg/mL. Characterization of the citrus-press cakes for flavonoid contents using HPLC showed varied quantity of rutin, narirutin, and hesperidin. Conclusions Considering the anti-melanogenic activity and human safety, CCE is considered as a potential anti-melanogenic agent and may be effective for topical application for treating hyperpigmentation disorders. PMID:23905018

  14. Down Regulation of ackA-pta Pathway in Escherichia coli BL21 (DE3): A Step Toward Optimized Recombinant Protein Expression System

    PubMed Central

    Bakhtiari, Nahid; Mirshahi, Manouchehr; Babaeipour, Valiollah; Maghsoudi, Nader; Tahzibi, Abbas

    2014-01-01

    Background: One of the most important problems in production of recombinant protein is to attain over-expression of the target gene and high cell density. In such conditions, the secondary metabolites of bacteria become toxic for the medium and cause cells to die. One of these aforementioned metabolites is acetate, which enormously accumulated in the medium, so that both cell and protein yields are affected. Objectives: To overcome this problem, several strategies applied. In this research we used antisense RNA strategy, where the transcription of phosphotransacetylase (PTA) and acetate kinase (ACK), two acetate pathway key enzymes, could be controlled, which led to reduced acetate production. Materials and Methods: In order to achieve this, recombinant plasmid harboring antisense sequences targeting both of pta and ackA was assembled, after transfecting to the cells, its effects on the cell growth and acetate accumulation in the minimal media was assessed and compared with the control, the plasmid without antisense cassette, in presence and absence of IPTG in Escherichia coli BL21 (DE3). Results: It was observed that the mentioned strategy partially affect the growth and amount of excreted acetate in comparison with the control. In addition it was found that high down-regulation of the acetate production pathway reduces the growth rate of E. coli BL21 (DE3). Conclusions: The study principally proved the importance of this strategy in acetate excretion control. PMID:25147677

  15. Down-regulation of MSH2 expression by an Hsp90 inhibitor enhances pemetrexed-induced cytotoxicity in human non-small-cell lung cancer cells.

    PubMed

    Tung, Chun-Liang; Chiu, Hsien-Chun; Jian, Yi-Jun; Jian, Yun-Ting; Chen, Chien-Yu; Syu, Jhan-Jhang; Wo, Ting-Yu; Huang, Yi-Jhen; Tseng, Sheng-Chieh; Lin, Yun-Wei

    2014-04-01

    Elevated heat shock protein 90 (Hsp90) expression has been linked to poor prognosis in patients with non-small cell lung cancer (NSCLC). The multitargeted antifolate pemetrexed has demonstrated certain clinical activities against NSCLC. However, the efficacy of the combination of pemtrexed and Hsp90 inhibitor to prolong the survival of patients with NSCLC still remains unclear. Human MutS homolog 2 (MSH2), a crucial element of the highly conserved DNA mismatch repair system, and defects or polymorphisms of MSH2 have been found in lung cancer. In this study, we evaluated the effects of pemetrexed on NSCLC cell lines (H520 and H1703) and found that treatment with this drug at 20-50 µM increased the MSH2 mRNA and protein levels in a MKK3/6-p38 MAPK signal activation-dependent manner. Furthermore, the knockdown of MSH2 expression by transfection with small interfering RNA of MSH2 or the blockage of p38 MAPK activation by SB202190 enhanced the cytotoxicity of pemetrexed. Combining the drug treatment with an Hsp90 inhibitor resulted in an enhanced pemetrexed-induced cytotoxic effect, accompanied with the reduction of MSH2 protein and mRNA levels. The expression of constitutively active MKK6 (MKK6E) or HA-p38 MAPK vectors significantly rescued the decreased p38 MAPK activity, and restored the MSH2 protein levels and cell survival in NSCLC cells co-treated with pemetrexed and Hsp90 inhibitor. In this study, we have demonstrated that down-regulation of the MKK3/6-p38 MAPK signal with the subsequent reduction of MSH2 enhanced the cytotoxic effect of pemetrexed in H520 and H1703 cells. The results suggest a potential future benefit of combining pemetrexed and the Hsp90 inhibitor to treat lung cancer. PMID:24530475

  16. The qEEG Signature of Selective NMDA NR2B Negative Allosteric Modulators; A Potential Translational Biomarker for Drug Development

    PubMed Central

    Keavy, Deborah; Bristow, Linda J.; Sivarao, Digavalli V.; Batchelder, Margaret; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E.; Weed, Michael R.

    2016-01-01

    The antidepressant activity of the N-methyl-D-aspartate (NMDA) receptor channel blocker, ketamine, has led to the investigation of negative allosteric modulators (NAMs) selective for the NR2B receptor subtype. The clinical development of NR2B NAMs would benefit from a translational pharmacodynamic biomarker that demonstrates brain penetration and functional inhibition of NR2B receptors in preclinical species and humans. Quantitative electroencephalography (qEEG) is a translational measure that can be used to demonstrate pharmacodynamic effects across species. NMDA receptor channel blockers, such as ketamine and phencyclidine, increase the EEG gamma power band, which has been used as a pharmacodynamic biomarker in the development of NMDA receptor antagonists. However, detailed qEEG studies with ketamine or NR2B NAMs are lacking in nonhuman primates. The aim of the present study was to determine the effects on the qEEG power spectra of the NR2B NAMs traxoprodil (CP-101,606) and BMT-108908 in nonhuman primates, and to compare them to the NMDA receptor channel blockers, ketamine and lanicemine. Cynomolgus monkeys were surgically implanted with EEG radio-telemetry transmitters, and qEEG was measured after vehicle or drug administration. The relative power for a number of frequency bands was determined. Ketamine and lanicemine increased relative gamma power, whereas the NR2B NAMs traxoprodil and BMT-108908 had no effect. Robust decreases in beta power were elicited by ketamine, traxoprodil and BMT-108908; and these agents also produced decreases in alpha power and increases in delta power at the doses tested. These results suggest that measurement of power spectra in the beta and delta bands may represent a translational pharmacodynamic biomarker to demonstrate functional effects of NR2B NAMs. The results of these studies may help guide the selection of qEEG measures that can be incorporated into early clinical evaluation of NR2B NAMs in healthy humans. PMID:27035340

  17. 6-Shogaol enhances renal carcinoma Caki cells to TRAIL-induced apoptosis through reactive oxygen species-mediated cytochrome c release and down-regulation of c-FLIP(L) expression.

    PubMed

    Han, Min Ae; Woo, Seon Min; Min, Kyoung-jin; Kim, Shin; Park, Jong-Wook; Kim, Dong Eun; Kim, Sang Hyun; Choi, Yung Hyun; Kwon, Taeg Kyu

    2015-02-25

    6-Shogaol, a potent bioactive compound in ginger (Zingiber officinale Roscoe), has been reported for anti-inflammatory and anti-cancer activity. In this study, we investigated the effect of 6-shogaol to enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. The combined treatment with 6-shogaol and TRAIL markedly induces apoptosis in various cancer cells (renal carcinoma Caki cells, breast carcinoma MDA-MB-231 cells and glioma U118MG cells), but not in normal mesangial cells and normal mouse kidney cells. 6-Shogaol reduced the mitochondrial membrane potential (MMP) and released cytochrome c from mitochondria to cytosol via Bax activation. Furthermore, we found that 6-shogaol induced down-regulation of c-FLIP(L) expression at the post-translational levels and the overexpression of c-FLIP(L) markedly inhibited 6-shogaol plus TRAIL-induced apoptosis. Moreover, 6-shogaol increased reactive oxygen species (ROS) production in Caki cells. Pretreatment with ROS scavengers attenuated 6-shogaol plus TRAIL-induced apoptosis through inhibition of MMP reduction and down-regulation of c-FLIP(L) expression. In addition, 6-gingerol, another phenolic alkanone isolated from ginger, did not enhance TRAIL-induced apoptosis and down-regulate c-FLIP(L) expression. Taken together, our results demonstrated that 6-shogaol enhances TRAIL-mediated apoptosis in renal carcinoma Caki cells via ROS-mediated cytochrome c release and down-regulation of c-FLIP(L) expression. PMID:25619640

  18. Inhibition of NR2B-Containing N-methyl-D-Aspartate Receptors (NMDARs) in Experimental Autoimmune Encephalomyelitis, a Model of Multiple Sclerosis

    PubMed Central

    Farjam, Mojtaba; Beigi Zarandi, Faegheh Baha'addini; Farjadian, Shirin; Geramizadeh, Bita; Nikseresht, Ali Reza; Panjehshahin, Mohammad Reza

    2014-01-01

    Neurodegeneration is the pathophysiological basis for permanent neurological disabilities in multiple sclerosis (MS); thus neuroprotection is emerging as a therapeutic approach in MS research. Modulation of excitotoxicity by inhibition of NMDARs has been suggested for neuroprotection, but selective antagonisation of the NR2B subtype of these receptors, a subtype believed to play a more pivotal role in neurodegeneration, has not been tested in MS. In this study inhibition of NR2B-containing NMDAR was evaluated on the animal model of MS, experimental autoimmune encephalomyelitis (EAE). EAE induction was done using MOG in C57BL/6 mice. Therapeutic administration of different doses of highly selective NR2B-containing NMDAR inhibitor (RO25-6981) was compared with memantine (non-selective NMDAR antagonist) and vehicle. Neurological deficits in EAE animals were more efficiently decreased by selective inhibition of NR2B-containing NMDARs. Histological studies of the spinal cords also showed decreased inflammation, myelin degradation and neuro-axonal degeneration when RO25-6981was administered with higher doses. The effects were dose dependent. Regarding the role of NR2B-containing NMDARs in excitotoxicity, selective inhibition of these receptor subtypes seems to modulate the neurological disabilities and pathological changes in EAE. Further elucidation of the exact mechanism of action as well as more experimental studies can suggest NR2B-containing NMDAR inhibition as a potentially effective treatment strategy for slowing down the clinical deterioration of disability in MS. PMID:25237366

  19. Bitter melon (Momordica charantia L.) inhibits adipocyte hypertrophy and down regulates lipogenic gene expression in adipose tissue of diet-induced obese rats.

    PubMed

    Huang, Hui-Ling; Hong, Ya-Wen; Wong, You-Hong; Chen, Ying-Nien; Chyuan, Jong-Ho; Huang, Ching-Jang; Chao, Pei-Min

    2008-02-01

    Bitter melon (Momordica charantia; BM) has been shown to ameliorate diet-induced obesity and insulin resistance. To examine the effect of BM supplementation on cell size and lipid metabolism in adipose tissues, three groups of rats were respectively fed a high-fat diet supplemented without (HF group) or with 5 % lyophilised BM powder (HFB group), or with 0.01 % thiazolidinedione (TZD) (HFT group). A group of rats fed a low-fat diet was also included as a normal control. Hyperinsulinaemia and glucose intolerance were observed in the HF group but not in HFT and HFB groups. Although the number of large adipocytes (>180 microm) of both the HFB and HFT groups was significantly lower than that of the HF group, the adipose tissue mass, TAG content and glycerol-3-phosphate dehydrogenase activity of the HFB group were significantly lower than those of the HFT group, implying that BM might reduce lipogenesis in adipose tissue. Experiment 2 was then conducted to examine the expression of lipogenic genes in adipose tissues of rats fed low-fat, HF or HFB diets. The HFB group showed significantly lower mRNA levels of fatty acid synthase, acetyl-CoA carboxylase-1, lipoprotein lipase and adipocyte fatty acid-binding protein than the HF group (P < 0.05). These results indicate BM can reduce insulin resistance as effective as the anti-diabetic drug TZD. Furthermore, BM can suppress the visceral fat accumulation and inhibit adipocyte hypertrophy, which may be associated with markedly down regulated expressions of lipogenic genes in the adipose. PMID:17651527

  20. Staphylococcus aureus Enterotoxin B Down-Regulates the Expression of Transforming Growth Factor-Beta (TGF-β) Signaling Transducers in Human Glioblastoma

    PubMed Central

    Akbari, Abolfazl; Farahnejad, Zohreh; Akhtari, Javad; Abastabar, Mahdi; Mobini, Gholam Reza; Mehbod, Amir Seied Ali

    2016-01-01

    Background It has been revealed that Staphylococcus aureus enterotoxin B (SEB) may feature anti-cancer and anti-metastatic advantages due to its ability to modify cell immunity processes and signaling pathways. Glioblastoma is one of the most aggressive human cancers; it has a high mortality nature, which makes it an attractive area for the development of novel therapies. Objectives We examined whether the SEB could exert its growth inhibitory effects on glioblastoma cells partially through the manipulation of a key tumor growth factor termed transforming growth factor-beta (TGF-β). Materials and Methods A human primary glioblastoma cell line, U87, was treated with different concentrations of SEB. The cell quantity was measured by the MTT assay at different exposure times. For molecular assessments, total ribonucleic acid (RNA) was extracted from either non-treated or SEB-treated cells. Subsequently, the gene expression of TGF-β transducers, smad2/3, at the messenger RNA (mRNA) level, was analyzed via a quantitative real-time polymerase chain reaction (qPCR) using the SYBR Green method. Significant differences between cell viability and gene expression levels were determined (Prism 5.0 software) using one-way analyses of variance (ANOVA) test. Results We reported that SEB could effectively down-regulate smad2/3 expression in glioblastoma cells at concentrations as quantity as 1 μg/mL and 2 μg/mL (P < 0.05 and P < 0.01, respectively). The SEB concentrations effective at regulating smad2/3 expression were correlated with those used to inhibit the proliferation of glioblastoma cells. Our results also showed that SEB was able to decrease smad2/3 expression at the mRNA level in a concentration- and time-dependent manner. Conclusions We suggested that SEB could represent an agent that can significantly decrease smad2/3 expression in glioblastoma cells, leading to moderate TGF-β growth signaling and the reduction of tumor cell proliferation. PMID:27540448

  1. Expression of Caveolin-1 reduces cellular responses to TGF-{beta}1 through down-regulating the expression of TGF-{beta} type II receptor gene in NIH3T3 fibroblast cells

    SciTech Connect

    Lee, Eun Kyung; Lee, Youn Sook; Han, In-Oc; Park, Seok Hee . E-mail: parks@skku.edu

    2007-07-27

    Transcriptional repression of Transforming Growth Factor-{beta} type II receptor (T{beta}RII) gene has been proposed to be one of the major mechanisms leading to TGF-{beta} resistance. In this study, we demonstrate that expression of Caveolin-1 (Cav-1) gene in NIH3T3 fibroblast cells down-regulates the expression of T{beta}RII gene in the transcriptional level, eventually resulting in the decreased responses to TGF-{beta}. The reduced expression of T{beta}RII gene by Cav-1 appeared to be due to the changes of the sequence-specific DNA binding proteins to either Positive Regulatory Element 1 (PRE1) or PRE2 of the T{beta}RII promoter. In addition, Cav-1 expression inhibited TGF-{beta}-mediated cellular proliferation and Plasminogen Activator Inhibitor (PAI)-1 gene expression as well as TGF-{beta}-induced luciferase activity. Furthermore, the inhibition of endogeneous Cav-1 by small interfering RNA increased the expression of T{beta}RII gene. These findings strongly suggest that expression of Cav-1 leads to the decreased cellular responsiveness to TGF-{beta} through down-regulating T{beta}RII gene expression.

  2. The Neuropeptide Y Y1 Receptor: A Diagnostic Marker? Expression in MCF-7 Breast Cancer Cells Is Down-Regulated by Antiestrogens In Vitro and in Xenografts

    PubMed Central

    Memminger, Martin; Keller, Max; Lopuch, Miroslaw; Pop, Nathalie; Bernhardt, Günther; von Angerer, Erwin; Buschauer, Armin

    2012-01-01

    The neuropeptide Y (NPY) Y1 receptor (Y1R) has been suggested as a tumor marker for in vivo imaging and as a therapeutic target. In view of the assumed link between estrogen receptor (ER) and Y1R in mammary carcinoma and with respect to the development of new diagnostic tools, we investigated the Y1R protein expression in human MCF-7 cell variants differing in ER content and sensitivity against antiestrogens. ER and Y1R expression were quantified by radioligand binding using [3H]-17β-estradiol and the Y1R selective antagonist [3H]-UR-MK114, respectively. The latter was used for cellular binding studies and for autoradiography of MCF-7 xenografts. The fluorescent ligands Cy5-pNPY (universal Y1R, Y2R and Y5R agonist) and UR-MK22 (selective Y1R antagonist), as well as the selective antagonists BIBP3226 (Y1R), BIIE0246 (Y2R) and CGP71683 (Y5R) were used to identify the NPY receptor subtype(s) by confocal microscopy. Y1R functionality was determined by mobilization of intracellular Ca2+. Sensitivity of MCF-7 cells against antiestrogen 4-hydroxytamoxifen correlated directly with the ER content. The exclusive expression of Y1Rs was confirmed by confocal microscopy. The Y1R protein was up-regulated (100%) by 17β-estradiol (EC50 20 pM) and the predominant role of ERα was demonstrated by using the ERα-selective agonist “propylpyrazole triol”. 17β-Estradiol-induced over-expression of functional Y1R protein was reverted by the antiestrogen fulvestrant (IC50 5 nM) in vitro. Furthermore, tamoxifen treatment of nude mice resulted in an almost total loss of Y1Rs in MCF-7 xenografts. In conclusion, the value of the Y1R as a target for therapy and imaging in breast cancer patients may be compromised due to Y1R down-regulation induced by hormonal (antiestrogen) treatment. PMID:23236424

  3. Neuroprotective Effect of Scutellarin on Ischemic Cerebral Injury by Down-Regulating the Expression of Angiotensin-Converting Enzyme and AT1 Receptor

    PubMed Central

    Han, Jichun; Zhou, Mingjie; Ren, Huanhuan; Pan, Qunwen; Zheng, Chunli; Zheng, Qiusheng

    2016-01-01

    Background and Purpose Previous studies have demonstrated that angiotensin-converting enzyme (ACE) is involved in brain ischemic injury. In the present study, we investigated whether Scutellarin (Scu) exerts neuroprotective effects by down-regulating the Expression of Angiotensin-Converting Enzyme and AT1 receptor in a rat model of permanent focal cerebral ischemia. Methods Adult Sprague–Dawley rats were administrated with different dosages of Scu by oral gavage for 7 days and underwent permanent middle cerebral artery occlusion (pMCAO). Blood pressure was measured 7 days after Scu administration and 24 h after pMCAO surgery by using a noninvasive tail cuff method. Cerebral blood flow (CBF) was determined by Laser Doppler perfusion monitor and the neuronal dysfunction was evaluated by analysis of neurological deficits before being sacrificed at 24 h after pMCAO. Histopathological change, cell apoptosis and infarct area were respectively determined by hematoxylin–eosin staining, terminal deoxynucleotidyl transfer-mediated dUTP nick end labeling (TUNEL) analysis and 2,3,5-triphenyltetrazolium chloride staining. Tissue angiotensin II (Ang II) and ACE activity were detected by enzyme-linked immunosorbent assays. The expression levels of ACE, Ang II type 1 receptor (AT1R), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were measured by Western blot and real-time PCR. ACE inhibitory activity of Scu in vitro was detected by the photometric determination. Results Scu treatment dose-dependently decreased neurological deficit score, infarct area, cell apoptosis and morphological changes induced by pMCAO, which were associated with reductions of ACE and AT1R expression and the levels of Ang II, TNF-α, IL-6, and IL-1β in ischemic brains. Scu has a potent ACE inhibiting activity. Conclusion Scu protects brain from acute ischemic injury probably through its inhibitory effect on the ACE/Ang II/AT1 axis, CBF preservation and

  4. Effects of C-phycocyanin and Spirulina on Salicylate-Induced Tinnitus, Expression of NMDA Receptor and Inflammatory Genes

    PubMed Central

    Hwang, Juen-Haur; Chen, Jin-Cherng; Chan, Yin-Ching

    2013-01-01

    Effects of C-phycocyanin (C-PC), the active component of Spirulina platensis water extract on the expressions of N-methyl D-aspartate receptor subunit 2B (NR2B), tumor necrosis factor–α (TNF-α), interleukin-1β (IL-1β), and cyclooxygenase type 2 (COX-2) genes in the cochlea and inferior colliculus (IC) of mice were evaluated after tinnitus was induced by intraperitoneal injection of salicylate. The results showed that 4-day salicylate treatment (unlike 4-day saline treatment) caused a significant increase in NR2B, TNF-α, and IL-1β mRNAs expression in the cochlea and IC. On the other hand, dietary supplementation with C-PC or Spirulina platensis water extract significantly reduced the salicylate-induced tinnitus and down-regulated the mRNAs expression of NR2B, TNF-α, IL-1β mRNAs, and COX-2 genes in the cochlea and IC of mice. The changes of protein expression levels were generally correlated with those of mRNAs expression levels in the IC for above genes. PMID:23533584

  5. Hypoxia in Tumor Angiogenesis and Metastasis: Evaluation of VEGF and MMP Over-expression and Down-Regulation of HIF-1alpha with RNAi in Hypoxic Tumor Cells

    NASA Astrophysics Data System (ADS)

    Shah, Shruti

    Background: As tumor mass grows beyond a few millimeters in diameter, the angiogenic "switch" is turned on leading to recruitment of blood vessels from surrounding artery and veins. However, the tumor mass is poorly perfused and there are pockets of hypoxia or lower oxygen concentrations relative to normal tissue. Hypoxia-inducing factor-1a (HIF-1a), a transcription factor, is activated when the oxygen concentration is low. Upon activation of HIF-1a, a number of other genes also turn on that allows the tumor to become more aggressive and resistant to therapy. Purpose: The main objectives of this study were to evaluate the effect of hypoxia-induced HIF-1a followed by over-expression of angiogenic and metastatic markers in tumor cells and down-regulation of HIF-1a using nanoparticle-delivered RNA interference therapy. Methods: Human ovarian (SKOV3) and breast (MDA-MB-231) adenocarcinoma cells were incubated under normoxic and hypoxic conditions. Following hypoxia treatment of the cells, HIF-1α, vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), and MMP-9 expression was analyzed qualitatively and quantitatively. For intracellular delivery of HIF-1a gene silencing small interfering RNA (siRNA), type B gelatin nanoparticles were fabricated using the solvent displacement method and the surface was modified with poly(ethylene glycol) (PEG, Mol. wt. 2kDa). Cellular uptake and distribution of the nanoparticles was observed with Cy3-siRNA loaded, FITC-conjugated gelatin nanoparticles. Cytotoxicity of the nanoparticle formulations was evaluated in both the cell lines. siRNA was transfected in the gelatin nanoparticles under hypoxic conditions. Total cellular protein and RNA were extracted for analysis of HIF1a, VEGF, MMP-2 and MMP-9 expression. Results: MDA-MB-231 and SKOV3 cells show increased expression of HIF1a under hypoxic conditions compared to baseline levels at normoxic conditions. ELISA and western blots of VEGF, MMP-2 and MMP-9 appear to

  6. Experimental evidence that overexpression of NR2B glutamate receptor subunit is associated with brain vacuolation in adult glutaryl-CoA dehydrogenase deficient mice: A potential role for glutamatergic-induced excitotoxicity in GA I neuropathology.

    PubMed

    Rodrigues, Marília Danyelle Nunes; Seminotti, Bianca; Amaral, Alexandre Umpierrez; Leipnitz, Guilhian; Goodman, Stephen Irwin; Woontner, Michael; de Souza, Diogo Onofre Gomes; Wajner, Moacir

    2015-12-15

    Glutaric aciduria type I (GA I) is biochemically characterized by accumulation of glutaric and 3-hydroxyglutaric acids in body fluids and tissues, particularly in the brain. Affected patients show progressive cortical leukoencephalopathy and chronic degeneration of the basal ganglia whose pathogenesis is still unclear. In the present work we investigated parameters of bioenergetics and redox homeostasis in various cerebral structures (cerebral cortex, striatum and hippocampus) and heart of adult wild type (Gcdh(+/+)) and glutaryl-CoA dehydrogenase deficient knockout (Gcdh(-/-)) mice fed a baseline chow. Oxidative stress parameters were also measured after acute lysine overload. Finally, mRNA expression of NMDA subunits and GLT1 transporter was determined in cerebral cortex and striatum of these animals fed a baseline or high lysine (4.7%) chow. No significant alterations of bioenergetics or redox status were observed in these mice. In contrast, mRNA expression of the NR2B glutamate receptor subunit and of the GLT1 glutamate transporter was higher in cerebral cortex of Gcdh(-/-) mice. Furthermore, NR2B expression was markedly elevated in striatum of Gcdh(-/-) animals receiving chronic Lys overload. These data indicate higher susceptibility of Gcdh(-/-) mice to excitotoxic damage, implying that this pathomechanism may contribute to the cortical and striatum alterations observed in GA I patients. PMID:26671102

  7. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

    SciTech Connect

    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui; Cheng, Tian-Lu; Lin, Shinne-Ren; Chang, Long-Sen

    2015-04-01

    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.

  8. Down-Regulation of TLR and JAK/STAT Pathway Genes Is Associated with Diffuse Cutaneous Leishmaniasis: A Gene Expression Analysis in NK Cells from Patients Infected with Leishmania mexicana

    PubMed Central

    Castillo-Fernández, Juan E.; Miranda-Ortíz, Haydee; Fernández-López, Juan C.; Becker, Ingeborg; Rangel-Escareño, Claudia

    2016-01-01

    An important NK-cell inhibition with reduced TNF-α, IFN-γ and TLR2 expression had previously been identified in patients with diffuse cutaneous leishmaniasis (DCL) infected with Leishmania mexicana. In an attempt to pinpoint alterations in the signaling pathways responsible for the NK-cell dysfunction in patients with DCL, this study aimed at identifying differences in the NK-cell response towards Leishmania mexicana lipophosphoglycan (LPG) between patients with localized and diffuse cutaneous leishmaniasis through gene expression profiling. Our results indicate that important genes involved in the innate immune response to Leishmania are down-regulated in NK cells from DCL patients, particularly TLR and JAK/STAT signaling pathways. This down-regulation showed to be independent of LPG stimulation. The study sheds new light for understanding the mechanisms that undermine the correct effector functions of NK cells in patients with diffuse cutaneous leishmaniasis contributing to a better understanding of the pathobiology of leishmaniasis. PMID:27031998

  9. Fragile X Mental Retardation Protein Interactions with a G quadruplex structure in the 3′-Untranslated Region of NR2B mRNA

    PubMed Central

    Stefanovic, Snezana; DeMarco, Brett A.; Underwood, Ayana; Williams, Kathryn R.; Bassell, Gary J.; Mihailescu, Mihaela Rita

    2015-01-01

    Fragile X syndrome, the most common cause of inherited intellectual disability, is caused by a trinucleotide CGG expansion in the 5′-untranslated region of the FMR1 gene, which leads to the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, an RNA-binding protein that regulates the translation of specific mRNAs, has been shown to bind a subset of its mRNA targets by recognizing G quadruplex structures. It has been suggested that FMRP controls the local protein synthesis of several protein components of the Post Synaptic Density (PSD) in response to specific cellular needs. We have previously shown that the interactions between FMRP and mRNAs of the PSD scaffold proteins PSD-95 and Shank1 are mediated via stable G-quadruplex structures formed within the 3′-untranslated regions of these mRNAs. In this study we used biophysical methods to show that a comparable G quadruplex structure forms in the 3′-untranslated region of the glutamate receptor subunit NR2B mRNA encoding for a subunit of N-methyl-D-aspartate (NMDA) receptors that is recognized specifically by FMRP, suggesting a common theme for FMRP recognition of its dendritic mRNA targets. PMID:26412477

  10. Structure of the Zinc-Bound Amino-Terminal Domain of the NMDA Receptor NR2B Subunit

    SciTech Connect

    Karakas, E.; Simorowski, N; Furukawa, H

    2009-01-01

    N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino-terminal domain (ATD) distinct from the L-glutamate-binding domain. The molecular basis for the ATD-mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc-free and zinc-bound states. The structures reveal the overall clamshell-like architecture distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc-binding site, ion-binding sites, and the architecture of the putative phenylethanolamine-binding site.

  11. Limoniastrum guyonianum aqueous gall extract induces apoptosis in human cervical cancer cells involving p16INK4A re-expression related to UHRF1 and DNMT1 down-regulation

    PubMed Central

    2013-01-01

    Several reports have described the potential effects of natural compounds as anti-cancer agents in vitro as well as in vivo. The aim of this study was to evaluate the anti-cancer effect of Limoniastrum guyonianum aqueous gall extract (G extract) and luteolin in the human cervical cancer HeLa cell line, and, if so, to clarify the underlying mechanism. Our results show that G extract and luteolin inhibited cell proliferation and induced G2/M cell cycle arrest in a concentration and time-dependent manner. Both natural products induced programmed cell death as confirmed by the presence of hypodiploid G0/G1 cells. These effects are associated with an up-regulation of the expression of the tumor suppressor gene p16INK4A and a down-regulation of the expression of the anti-apoptotic actor UHRF1 and its main partner DNMT1. Moreover, G extract- and luteolin-induced UHRF1 and DNMT1 down-regulation is accompanied with a global DNA hypomethylation in HeLa cell line. Altogether our results show that G extract mediates its growth inhibitory effects on human cervical cancer HeLa cell line likely via the activation of a p16INK4A -dependent cell cycle checkpoint signalling pathway orchestrated by UHRF1 and DNMT1 down-regulation. PMID:23688286

  12. Gene expression profiles in rice gametes and zygotes: identification of gamete-enriched genes and up- or down-regulated genes in zygotes after fertilization

    PubMed Central

    Abiko, Mafumi; Maeda, Hiroki; Tamura, Kentaro; Hara-Nishimura, Ikuko; Okamoto, Takashi

    2013-01-01

    In angiosperms, fertilization and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries; therefore, these processes are poorly elucidated. In this study, microarray-based transcriptome analyses were conducted on rice sperm cells, egg cells, and zygotes isolated from flowers to identify candidate genes involved in gametic and/or early zygotic development. Cell type-specific transcriptomes were obtained, and up- or down-regulated genes in zygotes after fertilization were identified, in addition to genes enriched in male and female gametes. A total of 325 putatively up-regulated and 94 putatively down-regulated genes in zygotes were obtained. Interestingly, several genes encoding homeobox proteins or transcription factors were identified as highly up-regulated genes after fertilization, and the gene ontology for up-regulated genes was highly enriched in functions related to chromatin/DNA organization and assembly. Because a gene encoding methyltransferase 1 was identified as a highly up-regulated gene in zygotes after fertilization, the effect of an inhibitor of this enzyme on zygote development was monitored. The inhibitor appeared partially to affect polarity or division asymmetry in rice zygotes, but it did not block normal embryo generation. PMID:23570690

  13. Halofuginone down-regulates Smad3 expression and inhibits the TGFbeta-induced expression of fibrotic markers in human corneal fibroblasts

    PubMed Central

    Nelson, Elizabeth F.; Huang, Craig W.; Ewel, Jillian M.; Chang, Angela A.

    2012-01-01

    Purpose Due to its ability to disrupt transforming growth factor beta (TGF-β) signaling, halofuginone has been successfully used to treat various fibrotic disorders. Here we investigated the antifibrotic potential of halofuginone in human corneal fibroblasts. Methods Human corneal fibroblasts were isolated from human donor corneas for in vitro experiments. TGF-β was used to stimulate pro-fibrotic responses from corneal fibroblasts under halofuginone treatment. The expression of alpha smooth muscle actin (α-SMA) and fibronectin was analyzed by western blots. Phalloidin toxin was used to stain cultures for stress fiber assemblies. Quantitative reverse transcription PCR (qRT–PCR) and immunostaining were used to analyze the expression of type I collagen mRNA and protein, respectively. The expression of Smad2, Smad3, phospho-Smad2, and phospho-Smad3 was determined by western blots. Results Halofuginone was well tolerated by human corneal fibroblasts up to 10 ng/ml as demonstrated by a cell viability assay. At this concentration, TGF-β-induced expression of the fibrotic markers α-SMA, fibronectin, and type I collagen was significantly reduced. Interestingly, under our experimental conditions, halofuginone treatment led to reduced protein expression of Smad3, which was both dose- and time-dependent. Conclusions Our results suggest that halofuginone may exert its antifibrotic effects in the cornea via a novel molecular mechanism and may be used as an antifibrotic agent for corneal fibrosis treatment. PMID:22393274

  14. DIFFERENTIAL ROLE OF NR2A AND NR2B NMDA RECEPTORS IN MEDIATING PHENCYCLIDINE-INDUCED PERINATAL NEURONAL APOPTOSIS AND BEHAVIORAL DEFICITS

    PubMed Central

    Anastasio, Noelle C.; Xia, Yan; O’Connor, Zoe R.; Johnson, Kenneth M.

    2009-01-01

    The mechanism underlying PCP-induced apoptosis in perinatal rats and the development of schizophrenic-like behaviors is incompletely understood. We used antagonists for NR2A- and NR2B-containing NMDARs to test the hypothesis that the behavioral and apoptotic effects of PCP are mediated by blockade of NR1/NR2A-containing receptors, rather than NR1/NR2B-containing receptors. Sprague-Dawley rats were treated on PN7, 9, and 11 with PCP (10 mg/kg), PEAQX (NR2A-preferring antagonist, 10, 20, or 40 mg/kg), or ifenprodil (selective NR2B antagonist, 1, 5, or 10 mg/kg) and sacrificed for measurement of caspase-3 activity (an index of apoptosis) or allowed to age and tested for locomotor sensitization to PCP challenge on PN28-35. PCP or PEAQX on PN7, 9, and 11 markedly elevated caspase-3 activity in the cortex; ifenprodil showed no effect. Striatal apoptosis was evident only after sub-chronic treatment with a high dose of PEAQX (20 mg/kg). Animals treated with PCP or PEAQX on PN7, 9 and 11 showed a sensitized locomotor response to PCP challenge on PN28-35. Ifenprodil treatment had no effect on either measure. Therefore, PCP blockade of cortical NR1/NR2A, rather than NR1/NR2B, appears to be responsible for PCP-induced apoptosis and the development of long-lasting behavioral deficits. PMID:19654040

  15. NR2A- and NR2B-Containing NMDA Receptors in the Prelimbic Medial Prefrontal Cortex Differentially Mediate Trace, Delay, and Contextual Fear Conditioning

    ERIC Educational Resources Information Center

    Gilmartin, Marieke R.; Kwapis, Janine L.; Helmstetter, Fred J.

    2013-01-01

    Activation of "N"-methyl-D-aspartate receptors (NMDAR) in the prelimbic medial prefrontal cortex (PL mPFC) is necessary for the acquisition of both trace and contextual fear memories, but it is not known how specific NR2 subunits support each association. The NR2B subunit confers unique properties to the NMDAR and may differentially…

  16. Activation of the JAK/STAT-1 Signaling Pathway by IFN-γ Can Down-Regulate Functional Expression of the MHC Class I-Related Neonatal Fc Receptor for IgG1

    PubMed Central

    Liu, Xindong; Ye, Lilin; Bai, Yu; Mojidi, Habi; Simister, Neil E.; Zhu, Xiaoping

    2009-01-01

    Expression of many MHC genes is enhanced at the transcriptional or posttranscriptional level following exposure to the cytokine IFN-γ. However, in this study we found that IFN-γ down-regulated the constitutive expression of the neonatal Fc receptor (FcRn), an MHC class I-related molecule that functions to transport maternal IgG and protect IgG and albumin from degradation. Epithelial cell, macrophage-like THP-1 cell, and freshly isolated human PBMC exposure to IFN-γ resulted in a significant decrease of FcRn expression as assessed by real-time RT-PCR and Western blotting. The down-regulation of FcRn was not caused by apoptosis or the instability of FcRn mRNA. Chromatin immunoprecipitation and gel mobility shift assays showed that STAT-1 bound to an IFN-γ activation site in the human FcRn promoter region. Luciferase expression from an FcRn promoter-luciferase reporter gene construct was not altered in JAK1- and STAT-1-deficient cells following exposure to IFN-γ, whereas expression of JAK1 or STAT-1 protein restored the IFN-γ inhibitory effect on luciferase activity. The repressive effect of IFN-γ on the FcRn promoter was selectively reversed or blocked by mutations of the core nucleotides in the IFN-γ activation site sequence and by over-expression of the STAT-1 inhibitor PIAS1 or the dominant negative phospho-STAT-1 mutations at Tyr-701 and/or Ser-727 residues. Furthermore, STAT-1 might down-regulate FcRn transcription through sequestering the transcriptional coactivator CREB binding protein/p300. Functionally, IFN-γ stimulation dampened bidirectional transport of IgG across a polarized Calu-3 lung epithelial monolayer. Taken together, our results indicate that the JAK/STAT-1 signaling pathway was necessary and sufficient to mediate the down-regulation of FcRn gene expression by IFN-γ. PMID:18566411

  17. MiR-26 down-regulates TNF-α/NF-κB signalling and IL-6 expression by silencing HMGA1 and MALT1

    PubMed Central

    Chen, Chyi-Ying A.; Chang, Jeffrey T.; Ho, Yi-Fang; Shyu, Ann-Bin

    2016-01-01

    MiR-26 has emerged as a key tumour suppressor in various cancers. Accumulating evidence supports that miR-26 regulates inflammation and tumourigenicity largely through down-regulating IL-6 production, but the underlying mechanism remains obscure. Here, combining a transcriptome-wide approach with manipulation of cellular miR-26 levels, we showed that instead of directly targeting IL-6 mRNA for gene silencing, miR-26 diminishes IL-6 transcription activated by TNF-α through silencing NF-κB signalling related factors HMGA1 and MALT1. We demonstrated that miR-26 extensively dampens the induction of many inflammation-related cytokine, chemokine and tissue-remodelling genes that are activated via NF-κB signalling pathway. Knocking down both HMGA1 and MALT1 by RNAi had a silencing effect on NF-κB-responsive genes similar to that caused by miR-26. Moreover, we discovered that poor patient prognosis in human lung adenocarcinoma is associated with low miR-26 and high HMGA1 or MALT1 levels and not with levels of any of them individually. These new findings not only unravel a novel mechanism by which miR-26 dampens IL-6 production transcriptionally but also demonstrate a direct role of miR-26 in down-regulating NF-κB signalling pathway, thereby revealing a more critical and broader role of miR-26 in inflammation and cancer than previously realized. PMID:27025651

  18. Overactivation of NR2B-containing NMDA receptors through entorhinal-hippocampal connection initiates accumulation of hyperphosphorylated tau in rat hippocampus after transient middle cerebral artery occlusion.

    PubMed

    Xu, Cheng-Shi; Liu, An-Chun; Chen, Juan; Pan, Zhi-Yong; Wan, Qi; Li, Zhi-Qiang; Wang, Ze-Fen

    2015-08-01

    Middle cerebral artery occlusion (MCAO) induces secondary damages in the hippocampus that is remote from primary ischemic regions. Tau hyperphosphorylation is an important risk for neurodegenerative diseases. Increased tau phosphorylation has been identified in ischemic cortex, but little is known regarding the changes in the hippocampus. We showed that unilateral transient MCAO induced accumulation of hyperphosphorylated tau and concurrent dephosphorylation of glycogen synthase kinase-3β at Ser 9 in the ipsilateral hippocampus. These MCAO-induced changes were not reproduced when glutamatergic inputs from the entorhinal cortex to the hippocampus were transected; however, the changes were mimicked by intrahippocampal N-methyl-d-aspartate (NMDA) administration. Inhibition of NMDA receptor (NMDAR) subunit NR2B, but not NR2A activity in the hippocampus attenuated the accumulation of hyperphosphorylated tau and spatial cognitive impairment in MCAO rats. Together, our data suggest that overactivation of NR2B-containing NMDARs through entorhinal-hippocampal connection plays an important role in the accumulation of hyperphosphorylated tau in the hippocampus following MCAO. Glycogen synthase kinase-3β is an important protein kinase involved in NMDARs-mediated tau hyperphosphorylation. This study indicates that early inhibition of NR2B-containing NMDARs may represent a potential strategy to prevent or delay the occurrence of post-stroke dementia. Middle cerebral artery occlusion induces secondary damage in the hippocampus that is remote from primary ischemic regions. We propose that excessive activation of NR2B-containing NMDA receptors through entorhinal-hippocampal connection initiated the accumulation of hyperphosphorylated tau in the hippocampus, which subsequently induced cognitive deficit. This study provides new insights into the prospects of NR2B inhibition in stoke therapy. PMID:25903928

  19. Dual contribution of NR2B subunit of NMDA receptor and SK3 Ca(2+)-activated K+ channel to genetic predisposition to anorexia nervosa.

    PubMed

    Koronyo-Hamaoui, Maya; Frisch, Amos; Stein, Daniel; Denziger, Yardena; Leor, Shani; Michaelovsky, Elena; Laufer, Neil; Carel, Cynthia; Fennig, Silvana; Mimouni, Mark; Ram, Anca; Zubery, Eynat; Jeczmien, Pablo; Apter, Alan; Weizman, Abraham; Gak, Eva

    2007-01-01

    Since identification of the genetic component in anorexia nervosa (AN), genes that partake in serotonergic and dopaminergic systems and in hormonal and weight regulation have been suggested as potential candidates for AN susceptibility. We propose another set of candidate genes. Those are genes that are involved in the signaling pathway using NMDA-R and SK channels and have been suggested as possible effectors of NMDA-R driven signaling. The role of NMDA-R in the etiology of schizophrenia has already been substantiated on various levels. Several studies based on population and family groups have implicated SK3 in schizophrenia and more recently in AN as well. Our study group consisted of 90 AN family trios. We examined the transmission of two potentially functional polymorphisms, 5073T>G polymorphism in the gene encoding the NR2B subunit of NMDA-R and CAG repeats in the coding region of SK3 channel gene. Using HHRR and TDT approaches, we found that both polymorphisms were preferentially transmitted to AN offspring (TDT yielded chi(2)=5.01, p=0.025 for NR2B 5073G alleles and chi(2)=11.75, p<0.001 for SK3 L alleles including >19 repeats). Distribution analysis of the combined NR2B/SK3 genotypes suggests that the contribution of both polymorphisms to AN risk is independent and cumulative (OR=2.44 for NR2B GG genotype and OR=3.01 for SK3 SL and LL genotypes, and OR=6.8 for the combined NR2B/SK3 genotypes including high-risk alleles). These findings point to the contribution of genes associated with the NMDA-R signaling pathway to predisposition and development of AN. PMID:16157352

  20. Conantokins Derived from the Asprella Clade Impart ConRl-B, an NMDA Receptor Antagonist with a Unique Selectivity Profile for NR2B Subunits

    PubMed Central

    Gowd, Konkallu Hanumae; Han, Tiffany S.; Twede, Vernon; Gajewiak, Joanna; Smith, Misty D.; Watkins, Maren; Platt, Randall J.; Toledo, Gabriela; White, H. Steve; Olivera, Baldomero M.; Bulaj, Grzegorz

    2014-01-01

    Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-Methyl D-Asparate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity to the most studied NR2B-selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogs for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings from Tyr5 substitutions in other conantokins, conRl-B [L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis and pharmacology for discovering new bioactive natural products. PMID:22594498

  1. IL-10-produced by human transitional B-cells down-regulates CD86 expression on B-cells leading to inhibition of CD4+T-cell responses

    PubMed Central

    Nova-Lamperti, Estefania; Fanelli, Giorgia; Becker, Pablo D.; Chana, Prabhjoat; Elgueta, Raul; Dodd, Philippa C.; Lord, Graham M.; Lombardi, Giovanna; Hernandez-Fuentes, Maria P.

    2016-01-01

    A novel subset of human regulatory B-cells has recently been described. They arise from within the transitional B-cell subpopulation and are characterised by the production of IL-10. They appear to be of significant importance in regulating T-cell immunity in vivo. Despite this important function, the molecular mechanisms by which they control T-cell activation are incompletely defined. Here we show that transitional B-cells produced more IL-10 and expressed higher levels of IL-10 receptor after CD40 engagement compared to other B-cell subsets. Furthermore, under this stimulatory condition, CD86 expressed by transitional B-cells was down regulated and T-cell proliferation was reduced. We provide evidence to demonstrate that the down-regulation of CD86 expression by transitional B-cells was due to the autocrine effect of IL-10, which in turn leads to decreased T-cell proliferation and TNF-α production. This analysis was further extended to peripheral B-cells in kidney transplant recipients. We observed that B-cells from patients tolerant to the graft maintained higher IL-10 production after CD40 ligation, which correlates with lower CD86 expression compared to patients with chronic rejection. Hence, the results obtained in this study shed light on a new alternative mechanism by which transitional B-cells inhibit T-cell proliferation and cytokine production. PMID:26795594

  2. Down-regulation of dihydrofolate reductase inhibits the growth of endothelial EA.hy926 cell through induction of G1 cell cycle arrest via up-regulating p53 and p21waf1/cip1 expression

    PubMed Central

    Fei, Zhewei; Gao, Yong; Qiu, Mingke; Qi, Xianqin; Dai, Yuxin; Wang, Shuqing; Quan, Zhiwei; Liu, Yingbin; Ou, Jingmin

    2016-01-01

    Folic acid supplementation may meliorate cardiovascular disease risk by improving vascular endothelial structure and function. However, the underlying mechanisms are still lack of a global understanding. To be used, folic acid must be converted to 7,8-dihydrofolate by dihydrofolate reductase to generate one-carbon derivatives serving as important cellular cofactors in the synthesis of nucleotides and amino acids required for cell growth. Therefore, this study explored the effect of dihydrofolate reductase knockdown on endothelial EA.hy926 cell growth and the mechanism involved. We found that down-regulation of dihydrofolate reductase inhibited EA.hy926 cell proliferation, and induced G1 phase arrest. Meanwhile, the expression of regulators necessary for G1/S phase transition, such as cyclin-dependent kinases CDK2, CDK4 and CDK6, were remarkably down-regulated; by contrast, the cell cycle inhibitors p21waf/cip1, p27Kip1 and p53 were significantly up-regulated after dihydrofolate reductase knockdown. Furthermore, supplementation of 5-methyltetrahydrofolate to the dihydrofolate reductase knockdown cells could weaken the inhibitory effect of dihydrofolate reductase knockdown on cell proliferation, simultaneously, inducing the expression of p53 and p21waf/cip1 falling back moderately. Our findings suggest that attenuating dihydrofolate reductase may cause imbalanced expression of cell cycle regulators, especially up-regulation of p53-p21waf/cip1 pathway, leading to G1 cell cycle arrest, thereby inhibiting the growth of endothelial EA.hy926 cells. PMID:27013776

  3. Sida rhomboidea. Roxb Leaf Extract Down-Regulates Expression of PPARγ2 and Leptin Genes in High Fat Diet Fed C57BL/6J Mice and Retards in Vitro 3T3L1 Pre-Adipocyte Differentiation

    PubMed Central

    Thounaojam, Menaka C.; Jadeja, Ravirajsinh N.; Ramani, Umed V.; Devkar, Ranjitsinh V.; Ramachandran, A. V.

    2011-01-01

    Sida rhomboidea. Roxb leaf extract (SRLE) is being used by the populace of North-East India to alleviate symptoms of diabetes and obesity. We have previously reported its hypolipidemic and anti-diabetic properties. In this study, we report the effect of SRLE on (i) in vivo modulation of genes controlling high fat diet (HFD) induced obesity and (ii) in vitro 3T3L1 pre-adipocyte differentiation and leptin release. Supplementation with SRLE significantly prevented HFD induced increment in bodyweight, plasma lipids and leptin, visceral adiposity and adipocyte hypertrophy. Also, SRLE supplementation reduced food intake, down regulated PPARγ2, SREBP1c, FAS and LEP expressions and up-regulated CPT-1 in epididymal adipose tissue compared to obese mice. In vitro adipogenesis of 3T3L1 pre-adipocytes was significantly retarded in the presence of SRLE extract. Also decreased triglyceride accumulation, leptin release and glyceraldehyde-3-Phosphate dehydrogenase activity along with higher glycerol release without significant alteration of viability of 3T3L1 pre-adipocytes, was recorded. Our findings suggest that prevention of HFD induced visceral adiposity is primarily by down regulation of PPARγ2 and leptin gene expression coupled with attenuation of food intake in C57BL/6J mice. SRLE induced prevention of pre-adipocytes differentiation, and leptin release further substantiated these findings and scientifically validates the potential application of SRLE as a therapeutic agent against obesity. PMID:21845103

  4. Interleukin-18 Down-Regulates Multidrug Resistance-Associated Protein 2 Expression through Farnesoid X Receptor Associated with Nuclear Factor Kappa B and Yin Yang 1 in Human Hepatoma HepG2 Cells

    PubMed Central

    Liu, Xiao-cong; Lian, Wei; Zhang, Liang-jun; Feng, Xin-chan; Gao, Yu; Li, Shao-xue; Liu, Chang; Cheng, Ying; Yang, Long; Wang, Xiao-Juan; Chen, Lei; Wang, Rong-quan; Chai, Jin; Chen, Wen-sheng

    2015-01-01

    Multidrug resistance-associated protein 2 (MRP2) plays an important role in bile acid metabolism by transporting toxic organic anion conjugates, including conjugated bilirubin, glutathione, sulfate, and multifarious drugs. MRP2 expression is reduced in cholestatic patients and rodents. However, the molecular mechanism of MRP2 down-regulation remains elusive. In this report, we treated human hepatoma HepG2 cells with interleukin-18 (IL-18) and measured the expression of MRP2, nuclear factor kappa B (NF-κB), farnesoid X receptor (FXR), and the transcription factor Yin Yang 1 (YY1) by quantitative real-time quantitative polymerase chain reaction (PCR) and western blotting. We found that expression of MRP2 was repressed by IL-18 at both the mRNA and protein levels in a dose- and time-dependent manner. Furthermore, the activated NF-κB pathway increased YY1 and reduced FXR. These changes were all attenuated in HepG2 cells with knockdown of the NF-κB subunit, p65. The reduced expression of FXR and MRP2 in HepG2 cells that had been caused by IL-18 treatment was also attenuated by YY1 knockdown. We further observed significantly elevated IL-18, NF-κB, and YY1 expression and decreased FXR and MRP2 expression in bile duct-ligated Sprague Dawley rat livers. Chromatin immunoprecipitation assays also showed that FXR bound to the promoter region in MRP2 was less abundant in liver extracts from bile duct-ligated rats than sham-operated rats. Our findings indicate that IL-18 down-regulates MRP2 expression through the nuclear receptor FXR in HepG2 cells, and may be mediated by NF-κB and YY1. PMID:26292095

  5. miR-27a-3p suppresses tumor metastasis and VM by down-regulating VE-cadherin expression and inhibiting EMT: an essential role for Twist-1 in HCC

    PubMed Central

    Zhao, Nan; Sun, Huizhi; Sun, Baocun; Zhu, Dongwang; Zhao, Xiulan; Wang, Yong; Gu, Qiang; Dong, Xueyi; Liu, Fang; Zhang, Yanhui; Li, Xiao

    2016-01-01

    Twist-1 and miRNAs have been reported to be associated with tumor metastasis and angiogenesis. However, the relationship between Twist-1 and miRNAs and the function of miRNAs remain largely undefined. We aimed to reveal the Twist-1-related miRNA expression profile and to determine whether Twist-1 functions in tumor metastasis and vasculogenic mimicry (VM) by regulating miRNA expression in hepatocellular carcinoma (HCC). Results showed that the expression of miR-27a-3p was consistently down-regulated in HCC cell lines and tissue samples displaying high expression of Twist-1. Both loss- and gain-of-function assays revealed suppressive effects of miR-27a-3p. Low miR-27a-3p expression was significantly associated with early metastasis in HCC. Subsequent investigations revealed that miR-27a-3p mediated the inhibition of epithelial–mesenchymal transition (EMT). Additional experiments showed that VE-cadherin is a direct target of miR-27a-3p and further demonstrated the critical role of miR-27a-3p in suppressing tumor metastasis and VM. Conclusions: Twist-1 up-regulation in HepG2 cells resulted in the differential expression of 18 miRNAs. Among them, miR-27a-3p deregulation contributed to VM and metastasis. The miR-27a-3p-mediated down-regulation of VE-cadherin and inhibition of EMT may be essential for Twist-1 to induce tumor metastasis and VM. Our findings highlight the importance of miR-27a-3p and suggest a promising new strategy for anti-HCC therapy. PMID:26980408

  6. Propofol attenuates LPS-induced tumor necrosis factor-α, interleukin-6 and nitric oxide expression in canine peripheral blood mononuclear cells possibly through down-regulation of nuclear factor (NF)-κB activation.

    PubMed

    Pei, Zengyang; Wang, Jinqiu

    2015-02-01

    Sepsis is a major cause of mortality in intensive care medicine. Propofol, an intravenous general anesthetic, has been suggested to have anti-inflammatory properties and able to prevent sepsis induced by Gram-positive and Gram-negative bacteria by down-regulating the gene expression of pro-inflammatory cytokines. However, propofol's anti-inflammatory effects upon canine peripheral blood mononuclear cells (PBMCs) have not yet been clarified. Here, we isolate canine PBMCs and investigate the effects of propofol on the gene expressions of both lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α and upon the production of nitric oxide (NO). Through real-time quantitative PCR and the Griess reagent system, we found that non-cytotoxic levels of propofol significantly inhibited the release of NO and IL-6 and TNF-α gene expression in LPS-induced canine PBMCs. Western blotting revealed that LPS does significantly increase the expression of inducible NO synthase (iNOS) protein in canine PBMCs, while pretreatment with propofol significantly decreases the LPS-induced iNOS protein expression. Propofol, at concentration of 25 µM and 50 µM, also significantly inhibited the LPS-induced nuclear translocation of nuclear factor (NF)-κB p65 protein in canine PBMCs. This diminished TNF-α, IL-6 and iNOS expression, and NO production was in parallel to the respective decreased NF-κB p65 protein nuclear translocation in the LPS-activated canine PBMCs pretreated with 25 µM and 50 µM propofol. This suggests that non-cytotoxic levels of propofol pretreatment can down-regulate LPS-induced inflammatory responses in canine PBMCs, possibly by inhibiting the nuclear translocation of the NF-κB p65 protein. PMID:25312048

  7. Activated macrophages down-regulate expression of E-cadherin in hepatocellular carcinoma cells via NF-κB/Slug pathway.

    PubMed

    Wang, Xianteng; Wang, Hao; Li, Guosheng; Song, Yonghong; Wang, Shurong; Zhu, Faliang; Guo, Chun; Zhang, Lining; Shi, Yongyu

    2014-09-01

    Hepatocellular carcinomas are an aggressive malignancy mainly due to metastasis or postsurgical recurrence. Expression of E-cadherin is strongly reduced in Hepatocellular carcinoma (HCC) tissues, and its downregulation is connected to invasiveness and metastasis in hepatocellular carcinomas. The previous study showed that the supernatant from activated macrophages can downregulate the expression of E-cadherin in HCC cells. The partial known molecular mechanism is that tyrosine kinases c-Src- and EGFR phosphorylate β-catenin and E-cadherin leading to destabilization of E-cadherin/β-catenin complex. The aim of this study is to clarify other mechanism by which activated macrophages downregulate the expression of E-cadherin. We detect the expression of E-cadherin and macrophage infiltration in hepatocellular carcinoma tissues by double-staining immunohistochemistry and evaluate the relationship between macrophages and E-cadherin expression in hepatocellular carcinoma cells in vitro experiments. We found that reduced expression of E-cadherin was associated with macrophage infiltration along the border between the tumor nest and stroma in hepatocellular carcinoma tissues. Besides, protein expression of E-cadherin was significantly decreased in hepatocellular carcinoma cells co-cultured with macrophages derived from THP-1 cells. Consistently, mRNA expression of E-cadherin was also decreased in cancer cells co-cultured with THP-1-differentiated macrophages. Moreover, the downregulation of E-cadherin expression was companied by upregulation of Slug expression in cancer cells with conditional medium from THP-1-differentiated macrophage culture. The change in expression of E-cadherin and Slug was abrogated when NF-κB signaling pathway was blocked. All the findings suggested that macrophages contributed to the decreased expression of E-cadherin by NF-κB/Slug pathway in hepatocellular carcinomas. PMID:24894673

  8. Epidermal growth factor down-regulates the expression of neutrophil gelatinase-associated lipocalin (NGAL) through E-cadherin in pancreatic cancer cells

    PubMed Central

    Tong, Zhimin; Chakraborty, Subhankar; Sung, Bokyung; Koolwal, Pooja; Kaur, Sukhwinder; Aggarwal, Bharat B.; Mani, Sendurai A.; Bresalier, Robert S.; Batra, Surinder K.; Guha, Sushovan

    2010-01-01

    BACKGROUND Our group previously reported that neutrophil gelatinase-associated lipocalin (NGAL) overexpression significantly blocked invasion and angiogenesis of pancreatic ductal adenocarcinoma (PDAC) and also demonstrated a loss of NGAL expression in the advanced stages of PDAC. However, little is known regarding mechanisms of NGAL regulation in PDAC. As EGF-EGFR axis is significantly upregulated in PDAC, we examined EGF-mediated NGAL regulation in these cells. METHODS NGAL-positive AsPC-1 and BxPC-3 cells were used as model system. Quantitative RT-PCR, western blot analysis, and immunofluorescence studies were used to investigate EGF-mediated effects on NGAL expression. E-cadherin expression was manipulated using lentiviral overexpression or shRNA constructs. NGAL promoter activity was assessed by luciferase-reporter assay and electrophoretic mobility shift assay (EMSA). RESULTS NGAL expression was positively associated with tumor differentiation and was significantly downregulated after EGF treatment along with a concomitant reduction of E-cadherin expression in PDAC cells. E-cadherin downregulation was partly through the EGF receptor (EGFR)-dependent MEK-ERK signaling pathway. In addition, E-cadherin downregulation reduced NGAL expression in PDAC cells, whereas overexpression of E-cadherin led to increased NGAL expression and partly rescued inhibition of NGAL expression by EGF. Furthermore, EGF in part through E-cadherin reduced NGAL promoter activity by blocking NF-κB activation. CONCLUSIONS We demonstrated for the first time that EGF potently blocked NGAL expression in PDAC cells. This effect is partly mediated through activation of the EGFR-MEK-ERK signaling pathway, which in turn downregulated E-cadherin with a subsequent reduction in NF-κB activation. Our findings illustrate a novel mechanism by which EGF regulates NGAL expression in PDAC. PMID:24048788

  9. Programmed cell death 4 (PDCD4) mediates the sensitivity of gastric cancer cells to TRAIL-induced apoptosis by down-regulation of FLIP expression.

    PubMed

    Wang, Weiqiang; Zhao, Jingjing; Wang, Hongbin; Sun, Yonggang; Peng, Zhihong; Zhou, Gang; Fan, Lilin; Wang, Xingwei; Yang, Shiming; Wang, Rongquan; Fang, Dianchun

    2010-09-10

    Tumor necrosis factor-related apoptosis induced ligand (TRAIL) is an important apoptosis inducer in a variety of tumor cells. In the present study, we determined the underlying molecular mechanisms by which certain gastric cancer cells are resistant to TRAIL. We first detected expression of programmed cell death 4 (PDCD4) in three gastric cancer cell lines and identified its association with the sensitivity of gastric cancer cells to TRAIL. We then stably transfected PDCD4 cDNA or shRNA into these gastric cell lines. Our data showed that restoration of PDCD4 expression induced TRAIL sensitivity, whereas knockdown of PDCD4 expression reduced the sensitivity of these tumor cells to TRAIL treatment. PDCD4 was able to suppress expression of FLICE-inhibiting protein (FLIP), a negative regulator of apoptosis. Knockdown of FLIP expression using FLIP shRNA had similar effects as those of restored PDCD4 expression. Furthermore, the proteasome inhibitor MG132 was able to inhibit expression of FLIP mRNA and protein and upregulate the sensitivity of these cells to TRAIL treatment. Taken together, the results from the current study demonstrated that PDCD4 plays an important role in mediating the sensitivity of gastric cancer cells to TRAIL-induced apoptosis through FLIP suppression. Therefore, the proteasome inhibitor MG132 should be further evaluated for combination therapy with TRAIL. PMID:20595005

  10. Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists down-regulate alpha2-macroglobulin expression by a PPARalpha-dependent mechanism.

    PubMed

    González, María del Carmen; Corton, J Christopher; Cattley, Russell C; Herrera, Emilio; Bocos, Carlos

    2009-08-01

    Fibrates are peroxisome proliferator-activated receptor alpha (PPARalpha) ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. The acute-phase response (APR) is an important inflammatory process. One of the most important acute-phase proteins in rats is alpha2-macroglobulin (A2Mg). Whereas normal adult rats present low serum levels, pregnant rats display high amounts. Therefore, we used pregnant rats to detect the effect of fenofibrate on hepatic A2Mg expression by RT-PCR and Northern blot. Virgin rats were used as controls. The expression of other APR genes, a known fibrate-responder gene, gamma-chain fibrinogen (gamma-Fib), and one gene from the same family as A2Mg, complement component 3 (C3), were also measured in liver. In order to determine whether the fibrate-effects were mediated by PPARalpha, wild-type mice and PPARalpha-null mice were also used and treated with WY-14,643 (WY) or di-2-ethylhexyl phthalate (DEHP). Fenofibrate depressed A2Mg expression in virgin rats, but expression was decreased more sharply in pregnant rats. Expression of C3 and gamma-Fib was diminished after treatment only in pregnant rats. On the other hand, WY, but not DEHP, reduced A2Mg and gamma-Fib expression in the livers of wild-type mice, without any effect in PPARalpha-null mice. WY or DEHP did not affect C3 expression. Therefore, A2Mg expression is modified by PPARalpha agonists not only in pregnant rats under augmented APR protein synthesis, but also in virgin rats and mice under basal conditions. Interestingly, our results also identify A2Mg as a novel PPARalpha agonist-regulated gene. PMID:19497347

  11. Squamosamide derivative FLZ inhibits TNF-α-induced ICAM-1 expression via down-regulation of the NF-κB signaling pathway in ARPE-19 cells

    PubMed Central

    Feng, Ting-Ting; Liang, Ze-Yu; Chen, Song

    2015-01-01

    Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). It has been reported that tumor necrosis factor-α (TNF-α) could induce intercellular adhesion molecule-1 (ICAM-1) expression in RPE cells. FLZ, a novel synthetic squamosamide derivative from a Chinese herb, Annona glabra, has displayed significant anti-inflammatory activity. However, the effects of FLZ on TNF-α-induced ICAM-1 expression in RPE cells remain unknown. Therefore, in the present study, we evaluated the effects of FLZ on TNF-α-induced ICAM-1 expression in RPE cells. We found that FLZ prevented TNF-α-induced ICAM-1 expression and the ability of monocytes to adhere to ARPE-19 cells induced by TNF-α. Furthermore, FLZ inhibited TNF-α-induced NF-κB p65 expression, as well as phosphorylation of IκBα in ARPE-19 cells. Taken together, these results suggest that FLZ inhibited TNF-α-induced ICAM-1 expression through blocking NF-κB signaling pathway in ARPE-19 cells. Thus, FLZ could be used for designing novel therapeutic agents against AMD. PMID:26464656

  12. Squamosamide derivative FLZ inhibits TNF-α-induced ICAM-1 expression via down-regulation of the NF-κB signaling pathway in ARPE-19 cells.

    PubMed

    Feng, Ting-Ting; Liang, Ze-Yu; Chen, Song

    2015-01-01

    Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). It has been reported that tumor necrosis factor-α (TNF-α) could induce intercellular adhesion molecule-1 (ICAM-1) expression in RPE cells. FLZ, a novel synthetic squamosamide derivative from a Chinese herb, Annona glabra, has displayed significant anti-inflammatory activity. However, the effects of FLZ on TNF-α-induced ICAM-1 expression in RPE cells remain unknown. Therefore, in the present study, we evaluated the effects of FLZ on TNF-α-induced ICAM-1 expression in RPE cells. We found that FLZ prevented TNF-α-induced ICAM-1 expression and the ability of monocytes to adhere to ARPE-19 cells induced by TNF-α. Furthermore, FLZ inhibited TNF-α-induced NF-κB p65 expression, as well as phosphorylation of IκBα in ARPE-19 cells. Taken together, these results suggest that FLZ inhibited TNF-α-induced ICAM-1 expression through blocking NF-κB signaling pathway in ARPE-19 cells. Thus, FLZ could be used for designing novel therapeutic agents against AMD. PMID:26464656

  13. Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

    2016-04-01

    Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC. PMID:26921637

  14. Down-regulation of Thanatos-associated protein 11 by BCR-ABL promotes CML cell proliferation through c-Myc expression.

    PubMed

    Nakamura, Satoki; Yokota, Daisuke; Tan, Lin; Nagata, Yasuyuki; Takemura, Tomonari; Hirano, Isao; Shigeno, Kazuyuki; Shibata, Kiyoshi; Fujisawa, Shinya; Ohnishi, Kazunori

    2012-03-01

    Bcr-Abl activates various signaling pathways in chronic myelogenous leukemia (CML) cells. The proliferation of Bcr-Abl transformed cells is promoted by c-Myc through the activation of Akt, JAK2 and NF-κB. However, the mechanism by which c-Myc regulates CML cell proliferation is unclear. In our study, we investigated the role of Thanatos-associated protein 11 (THAP11), which inhibits c-Myc transcription, in CML cell lines and in hematopoietic progenitor cells derived from CML patients. The induction of THAP11 expression by Abl kinase inhibitors in CML cell lines and in CML-derived hematopoietic progenitor cells resulted in the suppression of c-Myc. In addition, over-expression of THAP11 inhibited CML cell proliferation. In colony forming cells derived from CML-aldehyde dehydrogenase (ALDH)(hi) /CD34(+) cells, treatment with Abl kinase inhibitors and siRNA depletion of Bcr-Abl induced THAP11 expression and reduced c-Myc expression, resulting in inhibited colony formation. Moreover, overexpression of THAP11 significantly decreased the colony numbers, and also inhibited the expression of c-myc target genes such as Cyclin D1, ODC and induced the expression of p21(Cip1) . The depletion of THAP11 inhibited JAK2 or STAT5 inactivation-mediated c-Myc reduction in ALDH(hi) /CD34(+) CML cells. Thus, the induced THAP11 might be one of transcriptional regulators of c-Myc expression in CML cell. Therefore, the induction of THAP11 has a potential possibility as a target for the inhibition of CML cell proliferation. PMID:21400515

  15. Microtubule-associated protein tau (Mapt) is expressed in terminally differentiated odontoblasts and severely down-regulated in morphologically disturbed odontoblasts of Runx2 transgenic mice.

    PubMed

    Miyazaki, Toshihiro; Baba, Tomomi T; Mori, Masako; Moriishi, Takeshi; Komori, Toshihisa

    2015-08-01

    Runx2 is an essential transcription factor for osteoblast and odontoblast differentiation and the terminal differentiation of chondrocytes. We have previously shown that the terminal differentiation of odontoblasts is inhibited in Runx2 transgenic {Tg(Col1a1-Runx2)} mice under the control of the 2.3-kb Col1a1 promoter, which directs the transgene expression to osteoblasts and odontoblasts. Odontoblasts show severe reductions in Dspp and nestin expression and lose their characteristic polarized morphology, including a long process extending to dentin, in Tg(Col1a1-Runx2) mice. We study the molecular mechanism of odontoblast morphogenesis by comparing gene expression in the molars of wild-type and Tg(Col1a1-Runx2) mice, focusing on cytoskeleton-related genes. Using microarray, we found that the gene expression of microtubule-associated protein tau (Mapt), a neuronal phosphoprotein with important roles in neuronal biology and microtubule dynamics and assembly, was high in wild-type molars but severely reduced in Tg(Col1a1-Runx2) molars. Immunohistochemical analysis revealed that Mapt was specifically expressed in terminally differentiated odontoblasts including their processes in wild-type molars but its expression was barely detectable in Tg(Col1a1-Runx2) molars. Double-staining of Mapt and Runx2 showed their reciprocal expression in odontoblasts. Mapt and tubulin co-localized in odontoblasts in wild-type molars. Immunoelectron microscopic analysis demonstrated Mapt lying around α-tubulin-positive filamentous structures in odontoblast processes. Thus, Mapt is a useful marker for terminally differentiated odontoblasts and might play an important role in odontoblast morphogenesis. PMID:25707508

  16. Feeding a High Concentrate Diet Down-Regulates Expression of ACACA, LPL and SCD and Modifies Milk Composition in Lactating Goats

    PubMed Central

    Tao, Hui; Chang, Guangjun; Xu, Tianle; Zhao, Huajian; Zhang, Kai; Shen, Xiangzhen

    2015-01-01

    High concentrate diets are fed to early and mid-lactation stages dairy ruminants to meet the energy demands for high milk production in modern milk industry. The present study evaluated the effects of a high concentrate diet on milk fat and milk composition, especially, cis-9, trans-11 CLA content in milk and gene expression of lactating goats. Eight mid-lactating goats with rumen fistula were randomly assigned into a high concentrate diet (HCD) group and low concentrate diet (LCD) group. High concentrate diet feeding significantly increased lipopolysaccharides (LPS) in plasma and decreased milk fat content, vaccenic acid (VA) and cis-9, trans-11 CLA in milk of the lactating goats. The mRNA expression levels of sterol regulatory element binding protein B 1c (SREBP1c), lipoprotein lipase (LPL), fatty acid synthetase (FASN) and acetyl-CoA carboxylase α (ACACA, ACCα) involving in lipid metabolism were analyzed, and ACACA and LPL all decreased in their expression level in the mammary glands of goats fed a high concentrate diet. DNA methylation rate of stearoyl-CoA desaturase (SCD) was elevated and decreased, and SCD mRNA and protein expression was reduced significantly in the mammary glands of goats fed a high concentrate diet. In conclusion, feeding a high concentrate diet to lactating goats decreases milk fat and reduced expression of SCD in the mammary gland, which finally induced cis-9, trans-11 CLA content in milk. PMID:26086219

  17. Effects of penta-O-galloyl-β-D-glucose on human neutrophil function: significant down-regulation of L-selectin expression.

    PubMed

    Kiss, Anna K; Filipek, Agnieszka; Zyżyńska-Granica, Barbara; Naruszewicz, Marek

    2013-07-01

    Penta-O-galloyl-β-D-glucose (PGG) occurrs in high concentrations in medicinal herbs such as Rhus chinensis, Paeonia suffruticosa, Acer truncatum and Terminalia chebula, which demonstrate anti-inflammatory activity. We investigated the effect of PGG on stimulated and non-stimulated neutrophils in processes which included reactive oxygen species generation (ROS), metalloproteinase-9 and interleukin-8 secretion (IL-8), β₂ integrin (CD11b) and L-selectin (CD62L) expression and apoptosis. In concentrations of 5 μM-20 μM, PGG demonstrated statistically significant inhibition of ROS generation, IL-8 secretion and β₂ integrin expression in stimulated neutrophils. The inhibition of L-selectin expression by PGG resulted in prevention in neutrophils' endothelial attachment. The result obtained may explain the anti-inflammatory activity of this compound and underline the contribution of PGG in the activity of PGG rich plant extracts. PMID:22899541

  18. Down-Regulation of MRP1 Expression in C6/VP16 Cells by Chitosan-MRP1-siRNA Nanoparticles.

    PubMed

    Xu, Haitao; Nie, Xiaoqi; Wu, Liquan; Zhu, Xiaonan; Yi, Wei; Huang, Shulan

    2015-05-01

    High expression of multidrug resistance-associated protein 1 (MRP1) in tumor cells reduces effectiveness of chemotherapy drugs. In this study, we screened MRP1 interfering RNA (MRP1-siRNA) molecules that are able to reverse etoposide (VP16) resistance in multidrug resistance rat glioma cell line C6/VP16, and identified one siRNA molecule that is able to effectively deplete the expression of MRP1 gene and reverse tumor cells resistance to etoposide. Since siRNA instability limits its application in treatment of diseases, we next tested silencing effect of chitosan-MRP1-siRNA nanoparticles and found that the nanoparticles with N:P ratio 175 are able to effectively inhibit MRP1 mRNA and protein expression. Our data demonstrate that chitosan can be used as siRNA carrier for high efficient gene silencing in tumor cells. PMID:25543328

  19. MiR-302c-3p suppresses invasion and proliferation of glioma cells via down-regulating metadherin (MTDH) expression.

    PubMed

    Wang, Yonghong; Wei, Yujun; Tong, Haibo; Chen, Laizhao; Fan, Yimin; Ji, Yuchen; Jia, Wenqing; Liu, Dongkang; Wang, Guihuai

    2015-01-01

    Glioma is the most common malignant brain tumors with poor prognosis. The molecular events involved in the development and progression of glioma remain unclear. In this study, the expression levels of miR-302c-3p were examined in glioma tissues by qRT-PCR. The in vitro and in vivo functional effects of miR-302c-3p were examined further. Luciferase reporter assays were conducted to confirm the targeting associations. Results showed that the expression level of miR-302c-3p in glioma tissues was significantly lower than those in normal brain tissues (P < 0.001). The decreased expression of mi-302c-3p in glioma was positively associated with WHO grade (P < 0.001). Up-regulation of MTDH was also detected in glioma tumors compared with normal brain tissues (P = 0.0027) and is inversely correlated with miR-302c-3p expression (P = 0.003, R(2) = 0.4065). MTDH mRNA is a direct target of miR-302c-3p, whose ectopic expression decreases MTDH expression through binding to its 3'-untranslated region. Overexpression of miR-302c-3p results in a dramatic inhibition of glioma cells proliferation and invasion in vitro and in vivo. These data suggest that miR-302c-3p play a pivotal role in the progression of glioma by targeting MTDH and is a potential inhibitor in glioma treatment. PMID:26176806

  20. Deletion of Interleukin-6 Signal Transducer gp130 in Small Sensory Neurons Attenuates Mechanonociception and Down-Regulates TRPA1 Expression

    PubMed Central

    Malsch, Philipp; Andratsch, Manfred; Vogl, Christian; Link, Andrea S.; Alzheimer, Christian; Brierley, Stuart M.; Hughes, Patrick A.

    2014-01-01

    Glycoprotein 130 (gp130) is the signal transducing receptor subunit for cytokines of the interleukin-6 (IL-6) family, and it is expressed in a multitude of cell types of the immune and nervous system. IL-6-like cytokines are not only key regulators of innate immunity and inflammation but are also essential factors for the differentiation and development of the somatosensory system. Mice with a null mutation of gp130 in primary nociceptive afferents (SNS-gp130−/−) are largely protected from hypersensitivity to mechanical stimuli in mouse models of pathological pain. Therefore, we set out to investigate how neuronal gp130 regulates mechanonociception. SNS-gp130−/− mice revealed reduced mechanosensitivity to high mechanical forces in the von Frey assay in vivo, and this was associated with a reduced sensitivity of nociceptive primary afferents in vitro. Together with these findings, transient receptor potential ankyrin 1 (TRPA1) mRNA expression was significantly reduced in DRG from SNS-gp130−/− mice. This was also reflected by a reduced number of neurons responding with calcium transients to TRPA1 agonists in primary DRG cultures. Downregulation of Trpa1 expression was predominantly discovered in nonpeptidergic neurons, with the deficit becoming evident during stages of early postnatal development. Regulation of Trpa1 mRNA expression levels downstream of gp130 involved the classical Janus kinase family-signal transducer and activator of transcription pathway. Our results closely link proinflammatory cytokines to the expression of TRPA1, both of which have been shown to contribute to hypersensitive pain states. We suggest that gp130 has an essential role in mechanonociception and in the regulation of TRPA1 expression. PMID:25057188

  1. Fenofibrate down-regulates the expressions of androgen receptor (AR) and AR target genes and induces oxidative stress in the prostate cancer cell line LNCaP

    SciTech Connect

    Zhao, Hu; Zhu, Chen; Qin, Chao; Tao, Tao; Li, Jie; Cheng, Gong; Li, Pu; Cao, Qiang; Meng, Xiaoxin; Ju, Xiaobing; Shao, Pengfei; Hua, Lixin; Gu, Min; Yin, Changjun

    2013-03-08

    Highlights: ► Fenofibrate induces cell cycle arrest in G1 phase and apoptosis in LNCaP cells. ► Fenofibrate reduces the expressions of androgen receptor in LNCaP cells. ► Fenofibrate induces oxidative stress in the prostate cancer cell line LNCaP. -- Abstract: Fenofibrate, a peroxisome proliferator-androgen receptor-alpha agonist, is widely used in treating different forms of hyperlipidemia and hypercholesterolemia. Recent reports have indicated that fenofibrate exerts anti-proliferative and pro-apoptotic properties. This study aims to investigate the effects of fenofibrate on the prostate cancer (PCa) cell line LNCaP. The effects of fenofibrate on LNCaP cells were evaluated by flow cytometry, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assays, Western blot analysis, and dual-luciferase reporter assay. Fenofibrate induces cell cycle arrest in G1 phase and apoptosis in LNCaP cells, reduces the expressions of androgen receptor (AR) and AR target genes (prostate-specific antigen and TMPRSS2), and inhibits Akt phosphorylation. Fenofibrate can induce the accumulation of intracellular reactive oxygen species and malondialdehyde, and decrease the activities of total anti-oxidant and superoxide dismutase in LNCaP cells. Fenofibrate exerts an anti-proliferative property by inhibiting the expression of AR and induces apoptosis by causing oxidative stress. Therefore, our data suggest fenofibrate may have beneficial effects in fenofibrate users by preventing prostate cancer growth through inhibition of androgen activation and expression.

  2. The effects of down-regulating expression of Arabidopsis thaliana membrane-associated acyl-CoA binding protein 2 on acyl-lipid composition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiple classes of acyl-CoA binding proteins are encoded by plant genomes, including a plant-unique class of predicted integral membrane-proteins. Transcript analysis revealed that both of the integral membrane-acyl-CoA binding proteins of Arabidopsis thaliana, ACBP1 and ACBP2, are expressed in al...

  3. DEXAMETHASONE DIFFERENTIALLY DOWN-REGULATES L-SELECTIN (CD62L) EXPRESSION BY BOVINE LYMPHOCYTE SUBSETS IN VIVO AND DEPLETES THE INTESTINAL MUCOSA OF INTRAEPITHELIAL GAMMA DELTA CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A hallmark of immune modulation by dexamethasone (DEX) is the suppression of lymphocyte proliferation. DEX also alters the expression of adhesion molecules on bovine lymphocytes. Although intraepithelial lymphocytes (IEL) mainly are non-proliferating cells, we hypothesized that DEX treatment of ca...

  4. Down-regulation of oxidative phosphorylation in the liver by expression of the ATPase inhibitory factor 1 induces a tumor-promoter metabolic state

    PubMed Central

    Mobasher, Maysa A.; Casas, Estela; Rueda, Carlos B.; Martínez-Reyes, Inmaculada; de Arenas, Cristina Núñez; García-Bermúdez, Javier; Zapata, Juan M.; Sánchez-Aragó, María; Satrústegui, Jorgina; Valverde, Ángela M.; Cuezva, José M.

    2016-01-01

    The ATPase Inhibitory Factor 1 (IF1) is an inhibitor of the mitochondrial H+-ATP synthase that regulates the activity of both oxidative phosphorylation (OXPHOS) and cell death. Here, we have developed transgenic Tet-On and Tet-Off mice that express a mutant active form of hIF1 in the hepatocytes to restrain OXPHOS in the liver to investigate the relevance of mitochondrial activity in hepatocarcinogenesis. The expression of hIF1 promotes the inhibition of OXPHOS in both Tet-On and Tet-Off mouse models and induces a state of metabolic preconditioning guided by the activation of the stress kinases AMPK and p38 MAPK. Expression of the transgene significantly augmented proliferation and apoptotic resistance of carcinoma cells, which contributed to an enhanced diethylnitrosamine-induced liver carcinogenesis. Moreover, the expression of hIF1 also diminished acetaminophen-induced apoptosis, which is unrelated to differences in permeability transition pore opening. Mechanistically, cell survival in hIF1-preconditioned hepatocytes results from a nuclear factor-erythroid 2-related factor (Nrf2)-guided antioxidant response. The results emphasize in vivo that a metabolic phenotype with a restrained OXPHOS in the liver is prone to the development of cancer. PMID:26595676

  5. Heat shock protein-27 attenuates foam cell formation and atherogenesis by down-regulating scavenger receptor-A expression via NF-κB signaling.

    PubMed

    Raizman, Joshua E; Chen, Yong-Xiang; Seibert, Tara; Hibbert, Benjamin; Cuerrier, Charles M; Salari, Samira; Zhao, Xiaoling; Hu, Tieqiang; Shi, Chunhua; Ma, Xiaoli; Simard, Trevor; Caravaggio, Justin; Rayner, Katey; Bowdish, Dawn; Moore, Kathryn; O'Brien, Edward R

    2013-12-01

    Previously, we showed an inverse correlation between HSP27 serum levels and experimental atherogenesis in ApoE(-/-) mice that over-express HSP27 and speculated that the apparent binding of HSP27 to scavenger receptor-A (SR-A) was of mechanistic importance in attenuating foam cell formation. However, the nature and importance of the interplay between HSP27 and SR-A in atheroprotection remained unclear. Treatment of THP-1 macrophages with recombinant HSP27 (rHSP27) inhibited acLDL binding (-34%; p<0.005) and uptake (-38%, p<0.05). rHSP27 reduced SR-A mRNA (-39%, p=0.02), total protein (-56%, p=0.01) and cell surface (-53%, p<0.001) expression. The reduction in SR-A expression by rHSP27 was associated with a 4-fold increase in nuclear factor-kappa B (NF-κB) signaling (p<0.001 versus control), while an inhibitor of NF-κB signaling, BAY11-7082, attenuated the negative effects of rHSP27 on both SR-A expression and lipid uptake. To determine if SR-A is required for HSP27 mediated atheroprotection in vivo, ApoE(-/-) and ApoE(-/-) SR-A(-/-) mice fed with a high fat diet were treated for 3weeks with rHSP25. Compared to controls, rHSP25 therapy reduced aortic en face and aortic sinus atherosclerotic lesion size in ApoE(-/-) mice by 39% and 36% (p<0.05), respectively, but not in ApoE(-/-)SR-A(-/-) mice. In conclusion, rHSP27 diminishes SR-A expression, resulting in attenuated foam cell formation in vitro. Regulation of SR-A by HSP27 may involve the participation of NF-κB signaling. Lastly, SR-A is required for HSP27-mediated atheroprotection in vivo. PMID:23939398

  6. Minocycline enhances mitomycin C-induced cytotoxicity through down-regulating ERK1/2-mediated Rad51 expression in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Wang, Tai-Jing; Chang, Po-Yuan; Syu, Jhan-Jhang; Chen, Jyh-Cheng; Chen, Chien-Yu; Jian, Yun-Ting; Jian, Yi-Jun; Zheng, Hao-Yu; Chen, Wen-Ching; Lin, Yun-Wei

    2015-10-01

    Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC. PMID:26212550

  7. The cyclin-dependent kinase 2 inhibitor down-regulates interleukin-1beta-mediated induction of cyclooxygenase-2 expression in human lung carcinoma cells.

    PubMed

    Mukhopadhyay, Partha; Ali, M Aktar; Nandi, Animesh; Carreon, Peter; Choy, Hak; Saha, Debabrata

    2006-02-01

    Overexpression of cyclooxygenase-2 (COX-2) is frequently observed in several human cancers, including lung, colon, and head and neck. Malignancies are also associated with the dysregulation of cell cycle events and concomitant elevated activity of cyclin-dependent kinases (CDK). CDK2 is a key cell cycle regulatory protein that controls the transition of cells from G(1) to S phase. In this study, we furnish several lines of evidence that show a functional role for the CDK2 in interleukin-1beta (IL-1beta)-induced COX-2 expression in H358 human non-small cell lung carcinoma cell line by blocking CDK2 activity. First, we show that BMS-387032, a potent CDK2 inhibitor, blocks IL-1beta-induced expression as well as steady-state mRNA levels of COX-2. Second, we show that small interfering RNA that abrogates CDK2 expression also blocks IL-1beta-induced COX-2 expression. Third, results from in vitro kinase assays clearly show that IL-1beta induces CDK2 activity in H358 cells and this activity is significantly inhibited by BMS-387032. Moreover, CDK2 inhibition blocks IL-1beta-induced binding to the NF-IL6 element of the COX-2 promoter and inhibits transcription of the COX-2 gene. We also observed that BMS-387032 does not inhibit endogenous expression of COX-2 or prostaglandin synthesis in lung carcinoma cells. Finally, we provide evidence showing that IL-1beta-induced signaling events, such as p38 mitogen-activated protein kinase, phosphorylated stress-activated protein kinase/c-Jun NH(2)-terminal kinase, phosphorylated AKT, and phosphorylated extracellular signal-regulated kinase 1/2, are not inhibited by CDK2 inhibitor. Taken together, the data suggest that CDK2 activity may play an important event in the IL-1beta-induced COX-2 expression and prostaglandin E(2) synthesis and might represent a novel target for BMS-387032. PMID:16452236

  8. Baicalin down regulates the expression of TLR4 and NFkB-p65 in colon tissue in mice with colitis induced by dextran sulfate sodium

    PubMed Central

    Feng, Jinshan; Guo, Cancan; Zhu, Yuzhen; Pang, Liping; Yang, Zheng; Zou, Ying; Zheng, Xuebao

    2014-01-01

    Background: Baicalin is one of flavonoid extracts from Scutellaria baicalensis, which has several functions including anti-inflammation, anti-bacteria, antitumor and et al. However, the mechanisms of anti-inflammatory of baicalin in ulcerative colitis is not clear. Methods: Mice colitis models were established by dextran sodium sulfate, Mice administrated with baicalin (100 mg/kg) and mesalazine (100 mg/kg) twice daily by intragastric injection for 7 days after colitis induced were defined as treated group. Then the mice were sacrificed and the colon samples were collected. Toll-like receptor-2, 4, 9 were detected by immunohistochemistry. Signaling proteins such as TLR4, MyD88, and NF-κB p65 were analyzed by western blotting. Cytokine’s mRNA include TNF-α, IL-6 IL-10 and IL-13 were measured by reverse transcription polymerase chain reaction. Modified disease activity index were used to analyse the severity of the disease by assessed of diarrhea, stool (occult) blood and body weight loss of the mice. Results: Compared with control and model groups, modified disease activity index in baicalin and mesalazine treated, mice decreased gradually. Immunohistochemistry analysis showed the expression of TLR4, but not TLR2 and TLR9, in the mucosa of mice colon were decreased. Western blot analysis showed that in colitis model, the expression of NF-κB p65 and TLR4 decreased (P < 0.05), while the expression of MyD88 increased significantly compared to control group, and MyD88 expression can not be repressed by baicalin (P < 0.05). Baicalin and mesalazine treatment suppressed the expression of TNF-α, IL-6 and IL-13 mRNA (P < 0.05), yet up-regulated the expression of IL-10 mRNA (P < 0.05), compared to the DDS and control groups. Conclusions: Baicalin administration by intragastric injection ameliorates the severity of colon inflammation. The possible mechanism of anti-inflammatory response by baicalin may involve in the blocking of the TLR4/NF-κB-p65/IL-6 signaling pathway

  9. MicroRNA-21 promotes cell proliferation and down-regulates the expression of programmed cell death 4 (PDCD4) in HeLa cervical carcinoma cells

    SciTech Connect

    Yao, Qing; Xu, Hui; Zhang, Qian-Qian; Zhou, Hui; Qu, Liang-Hu

    2009-10-23

    MicroRNAs are involved in cancer-related processes. The microRNA-21(miR-21) has been identified as the only miRNA over-expressed in a wide variety of cancers, including cervical cancer. However, the function of miR-21 is unknown in cervical carcinomas. In this study, we found that the inhibition of miR-21 in HeLa cervical cancer cells caused profound suppression of cell proliferation, and up-regulated the expression of the tumor suppressor gene PDCD4. We also provide direct evidence that PDCD4-3'UTR is a functional target of miR-21 and that the 18 bp putative target site can function as the sole regulatory element in HeLa cells. These results suggest that miR-21 may play an oncogenic role in the cellular processes of cervical cancer and may serve as a target for effective therapies.

  10. BDE-47 causes developmental retardation with down-regulated expression profiles of ecdysteroid signaling pathway-involved nuclear receptor (NR) genes in the copepod Tigriopus japonicus.

    PubMed

    Hwang, Dae-Sik; Han, Jeonghoon; Won, Eun-Ji; Kim, Duck-Hyun; Jeong, Chang-Bum; Hwang, Un-Ki; Zhou, Bingsheng; Choe, Joonho; Lee, Jae-Seong

    2016-08-01

    2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is a persistent organic pollutant (POP) in marine environments. Despite its adverse effects (e.g. developmental retardation) in ecdysozoa, the effects of BDE-47 on transcription of ecdysteroid signaling pathway-involved-nuclear receptor (NR) genes and metamorphosis-related genes have not been examined in copepods. To examine the deleterious effect of BDE-47 on copepod molting and metamorphosis, BDE-47 was exposed to the harpacticoid copepod Tigriopus japonicus, followed by monitoring developmental retardation and transcriptional alteration of NR genes. The developmental rate was significantly inhibited (P<0.05) in response to BDE-47 and the agricultural insecticide gamma-hexachlorocyclohexane. Conversely, the ecdysteroid agonist ponasterone A (PoA) led to decreased molting and metamorphosis time (P<0.05) from the nauplius stage to the adult stage. In particular, expression profiles of all NR genes were the highest at naupliar stages 5-6 except for SVP, FTZ-F1, and HR96 genes. Nuclear receptor USP, HR96, and FTZ-F1 genes also showed significant sex differences (P<0.05) in gene expression levels over different developmental stages, indicating that these genes may be involved in vitellogenesis. NR gene expression patterns showed significant decreases (P<0.05) in response to BDE-47 exposure, implying that molting and metamorphosis retardation is likely associated with NR gene expression. In summary, BDE-47 leads to molting and metamorphosis retardation and suppresses transcription of NR genes. This information will be helpful in understanding the molting and metamorphosis delay mechanism in response to BDE-47 exposure. PMID:27337698

  11. Inhibition of PCSK9 Transcription by Berberine Involves Down-regulation of Hepatic HNF1α Protein Expression through the Ubiquitin-Proteasome Degradation Pathway*

    PubMed Central

    Dong, Bin; Li, Hai; Singh, Amar Bahadur; Cao, Aiqin; Liu, Jingwen

    2015-01-01

    Our previous in vitro studies have identified hepatocyte nuclear factor 1α (HNF1α) as an obligated trans-activator for PCSK9 gene expression and demonstrated its functional involvement in the suppression of PCSK9 expression by berberine (BBR), a natural cholesterol-lowering compound. In this study, we investigated the mechanism underlying the inhibitory effect of BBR on HNF1α-mediated PCSK9 transcription. Administration of BBR to hyperlipidemic mice and hamsters lowered circulating PCSK9 concentrations and hepatic PCSK9 mRNA levels without affecting the gene expression of HNF1α. However, hepatic HNF1α protein levels were markedly reduced in BBR-treated animals as compared with the control. Using HepG2 cells as a model system, we obtained evidence that BBR treatment let to accelerated degradation of HNF1α protein. By applying inhibitors to selectively block the ubiquitin proteasome system (UPS) and autophagy-lysosomal pathway, we show that HNF1α protein content in HepG2 cells was not affected by bafilomycin A1 treatment, but it was dose-dependently increased by UPS inhibitors bortezomib and MG132. Bortezomib treatment elevated HNF1α and PCSK9 cellular levels with concomitant reductions of LDL receptor protein. Moreover, HNF1α protein displayed a multiubiquitination ladder pattern in cells treated with BBR or overexpressing ubiquitin. By expressing GFP-HNF1α fusion protein in cells, we observed that blocking UPS resulted in accumulation of GFP-HNF1α in cytoplasm. Importantly, we show that the BBR reducing effects on HNF1α protein and PCSK9 gene transcription can be eradicated by proteasome inhibitors. Altogether, our studies using BBR as a probe uncovered a new aspect of PCSK9 regulation by ubiquitin-induced proteasomal degradation of HNF1α. PMID:25540198

  12. Lack of estrogen down-regulates CXCR4 expression on Treg cells and reduces Treg cell population in bone marrow in OVX mice.

    PubMed

    Fan, X-L; Duan, X-B; Chen, Z-H; Li, M; Xu, J-S; Ding, G-M

    2015-01-01

    Postmenopausal osteoporosis (PMO) is the most common metabolic bone disease in women after menopausal. Recent works focused on cross—talk between immune regulation and bone metabolism pathways and suggested Treg cells suppressed bone resorption and osteoclasts (OC) differentiation in bone marrow via cell—cell contact interaction and/or secreting of IL—10 and TGF—beta. In this study, we investigated the impact of estrogen on regulatory T cells (Treg cells) trafficking and staying in bone marrow and we found that a significant reduction of Treg cell population in bone marrow in estrogen deficiency ovariectomied (OVX) mice. We then studied the expressions of chemokines CXCL12/CXCR4 axes, which were critical to Treg cells migration and our data show the expression of CXCR4 on Treg cells was relative with oestrogen in vivo, however, the expression of CXCL12 was not. Furthermore, the loss of trafficking ability of Treg cells in OVX mice was recoverable in our system. These findings may mechanistically explain why Treg cells lose their suppressive functions on the regulation of OC cells and demonstrate a previously unappreciated role for estrogen, which may be critical to the novel therapy in clinical practice of PMO patients. PMID:26025396

  13. [Effect of down-regulating mll-af9 gene expression on proliferation of acute monocytic leukemia cell line THP-1].

    PubMed

    Li, Lei; Zhang, Ai-Hua; Liu, Ling-Bo; Bi, Lan; Wang, Li; Zhao, Ya-Jie; Zou, Ping

    2008-04-01

    This study was aimed to investigate the effect of small interfering RNA (siRNA) on the expression of mll-af9 oncogene and the proliferation of human acute monocytic leukemia cell line THP-1. One group of siRNA was designed targeting mll-af9 mRNA and finally obtained by chemosynthesis. Then the obtained siRNA was transfected into cultured human acute monocytic leukemia cell line THP-1 by lipofectamine. Flow cytometry was used to detect siRNA transfection efficiency. The level of mll-af9 mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). The cell proliferation rate was assayed by MTT. The change of cell cycles and apoptosis rate was detected by flow cytometry. The results showed that the siRNA transfection efficiency was 69.1%+/-1.8%. The level of mll-af9 mRNA expression was significantly inhibited in siRNA-transfected cells as compared with the controls. mll-af9-targeted siRNA inhibited the proliferation of THP-1 cells and induced cell apoptosis effectively after transfection. The percentage of G0/G1 phase cells significantly increased in siRNA-transfected cells in comparion with the control cells, but the percentage of S phase cells significantly decreased. It is concluded that the mll-af9-targeted siRNA can effectively inhibit the proliferation of human acute monocytic leukemia cell line THP-1. PMID:18426643

  14. The Shwachman-Bodian-Diamond syndrome associated protein interacts with HsNip7 and its down-regulation affects gene expression at the transcriptional and translational levels

    SciTech Connect

    Hesling, Cedric; Oliveira, Carla C.; Castilho, Beatriz A.; Zanchin, Nilson I.T.

    2007-12-10

    The Shwachman-Bodian-Diamond syndrome (SDS) is an autosomal disorder with pleiotropic phenotypes including pancreatic, skeletal and bone marrow deficiencies and predisposition to hematological dysfunctions. SDS has been associated to mutations in the SBDS gene, encoding a highly conserved protein that was shown to function in ribosome biogenesis in yeast. In this work, we show that SBDS is found in complexes containing the human Nip7 ortholog. Analysis of pre-rRNA processing in a stable SBDS knock-down HEK293-derivative cell line revealed accumulation of a small RNA which is a further indication of SBDS involvement in rRNA biosynthesis. Global transcription and polysome-bound mRNA profiling revealed that SBDS knock-down affects expression of critical genes involved in brain development and function, bone morphogenesis, blood cell proliferation and differentiation, and cell adhesion. Expression of a group of growth and signal transduction factors and of DNA damage response genes is also affected. In SBDS knock-down cells, 34 mRNAs showed decreased and 55 mRNAs showed increased association to polysomes, among which is a group encoding proteins involved in alternative splicing and RNA modification. These results indicate that SBDS is required for accurate expression of genes important for proper brain, skeletal, and blood cell development.

  15. HSI2 Repressor Recruits MED13 and HDA6 to Down-Regulate Seed Maturation Gene Expression Directly During Arabidopsis Early Seedling Growth.

    PubMed

    Chhun, Tory; Chong, Suet Yen; Park, Bong Soo; Wong, Eriko Chi Cheng; Yin, Jun-Lin; Kim, Mijung; Chua, Nam-Hai

    2016-08-01

    Arabidopsis HSI2 (HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE 2) which carries a EAR (ERF-associated amphiphilic repression) motif acts as a repressor of seed maturation genes and lipid biosynthesis, whereas MEDIATOR (MED) is a conserved multiprotein complex linking DNA-bound transcription factors to RNA polymerase II transcription machinery. How HSI2 executes its repressive function through MED is hitherto unknown. Here, we show that HSI2 and its homolog, HSI2-lik (HSL1), are able to form homo- and heterocomplexes. Both factors bind to the TRAP240 domain of MED13, a subunit of the MED CDK8 module. Mutant alleles of the med13 mutant show elevated seed maturation gene expression and increased lipid accumulation in cotyledons; in contrast, HSI2- or MED13-overexpressing plants display the opposite phenotypes. The overexpression phenotypes of HSI2 and MED13 are abolished in med13 and hsi2 hsl1, respectively, indicating that HSI2 and MED13 together are required for these functions. The HSI2 C-terminal region interacts with HDA6, whose overexpression also reduces seed maturation gene expression and lipid accumulation. Moreover, HSI2, MED13 and HDA6 bind to the proximal promoter and 5'-coding regions of seed maturation genes. Taken together, our results suggest that HSI2 recruits MED13 and HDA6 to suppress directly a subset of seed maturation genes post-germination. PMID:27335347

  16. Down-Regulation of ClC-3 Expression Reduces Epidermal Stem Cell Migration by Inhibiting Volume-Activated Chloride Currents.

    PubMed

    Guo, Rui; Pan, Fuqiang; Tian, Yanping; Li, Hongli; Li, Shirong; Cao, Chuan

    2016-06-01

    ClC-3, a member of the ClC chloride (Cl(-)) channel family, has recently been proposed as the primary Cl(-) channel involved in cell volume regulation. Changes in cell volume influence excitability, contraction, migration, pathogen-host interactions, cell proliferation, and cell death processes. In this study, expression and function of ClC-3 channels were investigated during epidermal stem cell (ESC) migration. We observed differential expression of CLC-3 regulates migration of ESCs. Further, whole-cell patch-clamp recordings and image analysis demonstrated ClC-3 expression affected volume-activated Cl(-) current (I Cl,Vol) within ESCs. Live cell imaging systems, designed to observe cellular responses to overexpression and suppression of ClC-3 in real time, indicated ClC-3 may regulate ESC migratory dynamics. We employed IMARIS software to analyze the velocity and distance of ESC migration in vitro to demonstrate the function of ClC-3 channel in ESCs. As our data suggest volume-activated Cl(-) channels play a vital role in migration of ESCs, which contribute to skin repair by migrating from neighboring unwounded epidermis infundibulum, hair follicle or sebaceous glands, ClC-3 may represent a new and valuable target for stem cell therapies. PMID:26769712

  17. Impaired NFKBIE gene function decreases cellular uptake of methotrexate by down-regulating SLC19A1 expression in a human rheumatoid arthritis cell line

    PubMed Central

    Imamura, Hitoshi; Yoshina, Sawako; Ikari, Katsunori; Miyazawa, Keiji; Momohara, Shigeki; Mitani, Shohei

    2016-01-01

    Abstract Objective: A non-synonymous single nucleotide polymorphism (nsSNP, rs2233434, Val194Ala) in the NFKBIE (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon) gene is known to be a rheumatoid arthritis (RA) susceptibility polymorphism in the Japanese RA population and could be closely associated with nuclear factor kappaB (NF-κB) activity. Inflammation caused by RA is sometimes associated with changes in expression levels of MTX (methotrexate) pathway-related genes. It is of interest to examine whether the NFKBIE gene had any influences on the mode of MTX action. Methods: Both knockdown of NFKBIE gene expression and overexpression of wild-type NFKBIE and Val194Ala mutation were performed. A transfected human RA synovial cell line was cultured and then gene expressions in the MTX pathway were measured. In addition, we measured the uptake and efflux of MTX derivatives under the NFKBIE knockdown condition. Results: Knockdown of NFKBIE reduced the mRNA for SLC19A1, a main MTX membrane transporter, and the intracellular accumulations of MTX derivatives. Moreover, our experiments also confirmed that overexpression of Val194Ala mutant NFKBIE decreased the SLC19A1 mRNA when compared to that of wild-type NFKBIE. Conclusions: We suggest that the impairment of NFKBIE gene function can reduce the uptake of MTX into cells, suggesting that the gene is an important factor for the RA outcome. PMID:26587663

  18. Tamoxifen enhances erlotinib-induced cytotoxicity through down-regulating AKT-mediated thymidine phosphorylase expression in human non-small-cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Chiu, Hsien-Chun; Syu, Jhan-Jhang; Jian, Yi-Jun; Chen, Chien-Yu; Jian, Yun-Ting; Huang, Yi-Jhen; Wo, Ting-Yu; Lin, Yun-Wei

    2014-03-01

    Tamoxifen is a triphenylethylene nonsteroidal estrogen receptor (ER) antagonist used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Thymidine phosphorylase (TP) is an enzyme of the pyrimidine salvage pathway which is upregulated in cancers. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels through AKT inactivation. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, combination treatment with PI3K inhibitors (LY294002 or wortmannin) and tamoxifen further decreased the TP expression and cell viability of NSCLC cells. Knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available small molecular inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical treatment of NSCLC. Compared to a single agent alone, tamoxifen combined with erlotinib resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-AKT and phospho-ERK1/2, and reduced TP protein levels. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and erlotinib for the treatment of NSCLC. PMID:24447935

  19. Anthrax lethal toxin down-regulates type-IIA secreted phospholipase A(2) expression through MAPK/NF-kappaB inactivation.

    PubMed

    Raymond, Benoit; Ravaux, Lucas; Mémet, Sylvie; Wu, YongZheng; Sturny-Leclère, Aude; Leduc, Dominique; Denoyelle, Chantal; Goossens, Pierre L; Payá, Miguel; Raymondjean, Michel; Touqui, Lhousseine

    2010-04-15

    Bacillus anthracis, the etiological agent of anthrax, produces lethal toxin (LT) that displays a metallo-proteolytic activity toward the N-terminus of the MAPK-kinases. We have previously shown that secreted type-IIA phospholipase A(2) (sPLA(2)-IIA) exhibits potent anthracidal activity. In vitro expression of sPLA(2)-IIA in guinea pig alveolar macrophages (AMs), the major source of this enzyme in lung tissues, is inhibited by LT. Here, we examined the mechanisms involved in sPLA(2)-IIA inhibition by LT. We first showed that chemical inhibitors of p38 and ERK MAPKs reduced sPLA(2)-IIA expression in AMs indicating that these kinases play a role in sPLA(2)-IIA expression. LT inhibited IL-1beta-induced p38 phosphorylation as well as sPLA(2)-IIA promoter activity in CHO cells. Inhibition of sPLA(2)-IIA promoter activity was mimicked by co-transfection with dominant negative construct of p38 (DN-p38) and reversed by the active form of p38-MAPK (AC-p38). Both LT and DN-p38 decreased IL-1beta-induced NF-kappaB luciferase activity. This contrasted with the effect of AC-p38, which enhanced this activity. However, neither LT nor specific p-38 inhibitor interfered with LPS-induced IkappaBalpha degradation or NF-kappaB nuclear translocation in AMs. Subcutaneous administration of LT to guinea pig before LPS challenge reduced sPLA(2)-IIA levels in broncho-alveolar lavages and ears. We conclude that sPLA(2)-IIA expression is induced via a sequential MAPK-NF-kappaB activation and that LT inhibits this expression likely by interfering with the transactivation of NF-kappaB in the nucleus. This inhibition, which is operating both in vitro and in vivo, may represent a mechanism by which B. anthracis subvert host defense. PMID:19962969

  20. MicroRNA-212 functions as an epigenetic-silenced tumor suppressor involving in tumor metastasis and invasion of gastric cancer through down-regulating PXN expression

    PubMed Central

    Li, Daojiang; Li, Zhengrong; Xiong, Jianbo; Gong, Binbin; Zhang, Guoyang; Cao, Chao; Jie, Zhigang; Liu, Yi; Cao, Yi; Yan, Yufeng; Xiong, Hong; Qiu, Lingyu; Yang, Miantian; Chen, Hongping; Jiang, Shuping; Yang, Xiongwen; Chen, Heping

    2015-01-01

    Altered expression of paxillin (PXN) is closely linked to the pathogenesis progression, metastasis and prognosis of different malignancies including gastric cancer (GC). Epigenetic silencing of tumor-suppressive microRNAs (miRNAs) is a crucial component of the mechanism underlying activation of oncogenes in tumor. To screen for epigenetically silenced miRNAs which target PXN in GC, we performed bioinformatics algorithms and real-time PCR analysis, and identified miR-212 as the optimum candidate gene. A luciferase reporter gene assay validated that miR-212 directly targets the 3’UTR region of PXN. Importantly, miR-212 levels were inversely correlated with PXN expression in GC cell lines and clinical tumor tissues. The use of miR-212 minics decrease PXN mRNA and protein level in GC cell lines. Moreover, low expression of miR-212 and its promoter hypermethylation were causally related and were associated with aggressive tumor phenotype and adverse prognosis in GC. Restoring mir-212 expression by exogenous mirprecursor molecules transfection or reexpression of endogenous miR-212 treated by 5-aza-2’-deoxycytidine (5-aza) can exert similar effect that reduce GC cells invasion and metastasis abilities in vitro by interacting PXN gene. In addition, 5-aza-induced PXN reduction could be partically blocked by miR-212 inhibitor, resulting in a reversal of weankening cell migration and invasion ability of 5-aza. A rescue experiment and a loss-of-function experiment in vitro and vivo showed that PXN restoration rescues migration and invasion phenotype in miR-212 overexpressed GC cell lines and PXN knockdown blocks GC cells migration and invasion in the presence miR-212 inhibitors. Taken together, our results clearly show that overexpression of PXN induced by methylationsuppressed miR-212 promotes tumor metastasis and invasion, and regulation of miR-212 expression may be a novel therapeutic strategy for gastric cancer. PMID:26693054

  1. Hydroxy decenoic acid down regulates gtfB and gtfC expression and prevents Streptococcus mutans adherence to the cell surfaces

    PubMed Central

    2012-01-01

    Background 10-Hydroxy-2-decenoic acid, an unsaturated fatty acid is the most active and unique component to the royal jelly that has antimicrobial properties. Streptococcus mutans is associated with pathogenesis of oral cavity, gingivoperiodontal diseases and bacteremia following dental manipulations. In the oral cavity, S. mutans colonize the soft tissues including tongue, palate, and buccal mucosa. When considering the role of supragingival dental plaque in caries, the proportion of acid producing bacteria (particularly S. mutans), has direct relevance to the pathogenicity of the plaque. The genes that encode glucosyltransferases (gtfs) especially gtfB and gtfC are important in S. mutans colonization and pathogenesis. This study investigated the hydroxy-decenoic acid (HDA) effects on gtfB and gtfC expression and S. mutans adherence to cells surfaces. Methods Streptococcus mutans was treated by different concentrations of HPLC purified HDA supplied by Iran Beekeeping and Veterinary Association. Real time RT-PCR and western blot assays were conducted to evaluate gtfB and gtfC genes transcription and translation before and after HDA treatment. The bacterial attachment to the cell surfaces was evaluated microscopically. Results 500 μg ml-1 of HDA inhibited gtfB and gtfC mRNA transcription and its expression. The same concentration of HDA decreased 60% the adherence of S. mutans to the surface of P19 cells. Conclusion Hydroxy-decenoic acid prevents gtfB and gtfC expression efficiently in the bactericide sub-concentrations and it could effectively reduce S. mutans adherence to the cell surfaces. In the future, therapeutic approaches to affecting S. mutans could be selective and it’s not necessary to put down the oral flora completely. PMID:22839724

  2. Stearoylethanolamide exerts anorexic effects in mice via down-regulation of liver stearoyl-coenzyme A desaturase-1 mRNA expression.

    PubMed

    Terrazzino, Salvatore; Berto, Fiorenzo; Dalle Carbonare, Maurizio; Fabris, Michele; Guiotto, Adriano; Bernardini, Daniele; Leon, Alberta

    2004-10-01

    Given the recent demonstration that oleoylethanolamide (OEA), a cannabinoid receptor-inactive N-acylethanolamine, decreases food intake by activating the nuclear receptor PPARalpha (peroxisome proliferator-activated receptor alpha) in the periphery, we here evaluated the effects of both saturated and unsaturated C18 N-acylethanolamides (C18:0; C18:1; C18:2) in mice feeding behavior after overnight starvation. Our results show stearoylethanolamide (SEA, C18:0) exerts, unlike other unsaturated C18 homologs, a marked dose-dependent anorexic effect evident already at 2 h after its intraperitoneal administration. In addition, oral administration of SEA (25 mg/kg) was also effective in reducing food consumption, an effect ascribed to the molecule itself and not to its catabolites. Moreover, although the anorexic response to oral administered SEA was not associated with changes in the levels of various hematochemical parameters (e.g., glucose, cholesterol, triglycerides, leptin) nor in liver mRNA expression of peroxisome proliferator-activated receptors (PPARs) including PPARalpha, the anorexic effect of SEA was interestingly accompanied by a reduction in liver stearoyl-CoA desaturase-1 (SCD-1) mRNA expression. As SCD-1 has been recently proposed as a molecular target for the treatment of obesity, the novel observation provided here that SEA reduces food intake in mice in a structurally selective manner, in turn, correlated with downregulation of liver SCD-1 mRNA expression, has the potential of providing new insights on a class of lipid mediators with suitable properties for the pharmacological treatment of over-eating dysfunctions. PMID:15289450

  3. Down-Regulating the Expression of 53 Soybean Transcription Factor Genes Uncovers a Role for SPEECHLESS in Initiating Stomatal Cell Lineages during Embryo Development.

    PubMed

    Danzer, John; Mellott, Eric; Bui, Anhthu Q; Le, Brandon H; Martin, Patrick; Hashimoto, Meryl; Perez-Lesher, Jeanett; Chen, Min; Pelletier, Julie M; Somers, David A; Goldberg, Robert B; Harada, John J

    2015-07-01

    We used an RNA interference screen to assay the function of 53 transcription factor messenger RNAs (mRNAs) that accumulate specifically within soybean (Glycine max) seed regions, subregions, and tissues during development. We show that basic helix-loop-helix (bHLH) transcription factor genes represented by Glyma04g41710 and its paralogs are required for the formation of stoma in leaves and stomatal precursor complexes in mature embryo cotyledons. Phylogenetic analysis indicates that these bHLH transcription factor genes are orthologous to Arabidopsis (Arabidopsis thaliana) SPEECHLESS (SPCH) that initiate asymmetric cell divisions in the leaf protoderm layer and establish stomatal cell lineages. Soybean SPCH (GmSPCH) mRNAs accumulate primarily in embryo, seedling, and leaf epidermal layers. Expression of Glyma04g41710 under the control of the SPCH promoter rescues the Arabidopsis spch mutant, indicating that Glyma04g41710 is a functional ortholog of SPCH. Developing soybean embryos do not form mature stoma, and stomatal differentiation is arrested at the guard mother cell stage. We analyzed the accumulation of GmSPCH mRNAs during soybean seed development and mRNAs orthologous to MUTE, FAMA, and inducer of C-repeat/dehydration responsive element-binding factor expression1/scream2 that are required for stoma formation in Arabidopsis. The mRNA accumulation patterns provide a potential explanation for guard mother cell dormancy in soybean embryos. Our results suggest that variation in the timing of bHLH transcription factor gene expression can explain the diversity of stomatal forms observed during plant development. PMID:25963149

  4. Down-Regulating the Expression of 53 Soybean Transcription Factor Genes Uncovers a Role for SPEECHLESS in Initiating Stomatal Cell Lineages during Embryo Development1[OPEN

    PubMed Central

    Danzer, John; Mellott, Eric; Bui, Anhthu Q.; Le, Brandon H.; Martin, Patrick; Hashimoto, Meryl; Perez-Lesher, Jeanett; Chen, Min; Pelletier, Julie M.; Somers, David A.; Goldberg, Robert B.; Harada, John J.

    2015-01-01

    We used an RNA interference screen to assay the function of 53 transcription factor messenger RNAs (mRNAs) that accumulate specifically within soybean (Glycine max) seed regions, subregions, and tissues during development. We show that basic helix-loop-helix (bHLH) transcription factor genes represented by Glyma04g41710 and its paralogs are required for the formation of stoma in leaves and stomatal precursor complexes in mature embryo cotyledons. Phylogenetic analysis indicates that these bHLH transcription factor genes are orthologous to Arabidopsis (Arabidopsis thaliana) SPEECHLESS (SPCH) that initiate asymmetric cell divisions in the leaf protoderm layer and establish stomatal cell lineages. Soybean SPCH (GmSPCH) mRNAs accumulate primarily in embryo, seedling, and leaf epidermal layers. Expression of Glyma04g41710 under the control of the SPCH promoter rescues the Arabidopsis spch mutant, indicating that Glyma04g41710 is a functional ortholog of SPCH. Developing soybean embryos do not form mature stoma, and stomatal differentiation is arrested at the guard mother cell stage. We analyzed the accumulation of GmSPCH mRNAs during soybean seed development and mRNAs orthologous to MUTE, FAMA, and INDUCER OF C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT-BINDING FACTOR EXPRESSION1/SCREAM2 that are required for stoma formation in Arabidopsis. The mRNA accumulation patterns provide a potential explanation for guard mother cell dormancy in soybean embryos. Our results suggest that variation in the timing of bHLH transcription factor gene expression can explain the diversity of stomatal forms observed during plant development. PMID:25963149

  5. Oral and IV dosages of doxorubicin-methotrexate loaded- nanoparticles inhibit progression of oral cancer by down- regulation of matrix Methaloproteinase 2 expression in vivo.

    PubMed

    Abbasi, Mehran Mesgari; Jahanban-Esfahlan, Rana; Monfaredan, Amir; Seidi, Khaled; Hamishehkar, Hamed; Khiavi, Monir Moradzadeh

    2014-01-01

    Oral cancer is one of the most common and lethal cancers in the world. Combination chemotherapy coupled with nanoparticle drug delivery holds substantial promise in cancer therapy. This study aimed to evaluate the efficacy and safety of two dosages of our novel pH and temperature sensitive doxorubicin-methotrexate-loaded nanoparticles (DOX-MTX NPs) with attention to the MMP-2 mRNA profile in a 4-nitroquinoline-1-oxide induced oral squamous cell carcinoma (OSCC) model in the rat. Our results showed that both IV and oral dosages of DOX-MTX NP caused significant decrease in mRNA levels of MMP-2 compared to the untreated group (p<0.003). Surprisingly, MMP-2 mRNA was not affected in DOX treated compared to cancer group (p>0.05). Our results indicated that IV dosage of MTX-DOX is more effective than free DOX (12 fold) in inhibiting the activity of MMP-2 in OSCCs (P<0.001). Furthermore, MMP-2 mRNA expression in the DOX-MTX treated group showed a significant relation with histopathological changes (P=0.011). Compared to the untreated cancer group, we observed no pathological changes and neither a significant alteration in MMP-2 amount in either of healthy controls that were treated with oral and IV dosages of DOX-MTX NPs whilst cancer group showed a high level of MMP-2 expression compared to healthy controls (p<0.001).Taking together our results indicate that DOX- MTX NPs is a safe chemotherapeutic nanodrug that its oral and IV forms possess potent anti-cancer properties on aggressive tumors like OSCC, possibly by affecting the expression of genes that drive tumor invasion and metastasis. PMID:25605162

  6. Down-regulation of ATP-binding cassette C2 protein expression in HepG2 cells after rifampicin treatment is mediated by microRNA-379.

    PubMed

    Haenisch, Sierk; Laechelt, Sandra; Bruckmueller, Henrike; Werk, Anneke; Noack, Andreas; Bruhn, Oliver; Remmler, Cornelia; Cascorbi, Ingolf

    2011-08-01

    microRNAs (miRNAs), which contribute to the post-transcriptional processing through 3'-untranslated region-interference, have been shown to be involved in the regulation of ATP-binding cassette (ABC) membrane transporters. The aim of this study was to investigate whether ABCC2, an important efflux transporter for various endogenous and exogenous compounds at several compartment barriers, is subject to miRNA-mediated post-transcriptional gene regulation. We screened the expression of 377 human miRNAs in HepG2 cells after 48 h of treatment with 5 μM rifampicin [a pregnane X receptor (PXR) ligand] or vehicle using reverse transcription-polymerase chain reaction-based low-density arrays. Specific miRNA, ABCC2 mRNA, and protein expression were monitored in HepG2 cells undergoing rifampicin treatment for 72 h. Loss- and gain-of-function experiments and reporter gene assays were performed for further confirmation. Highly deregulated miRNAs compared with in silico data revealed miRNA (miR) 379 as candidate miRNA targeting ABCC2 mRNA. Under rifampicin treatment, ABCC2 mRNA increased significantly, with a maximal fold change of 1.56 ± 0.43 after 24 h. In addition, miR-379 increased (maximally 4.10 ± 1.33-fold after 48 h), whereas ABCC2 protein decreased with a maximal fold change of 0.47 ± 0.08 after 72 h. In contrast, transfection of miR-379 inhibitor led to an elevation of ABCC2 protein expression after rifampicin incubation for 48 h. We identify a miRNA negatively regulating ABCC2 on the post-transcriptional level and provide evidence that this miRNA impedes overexpression of ABCC2 protein after a PXR-mediated external transcriptional stimulus in HepG2 cells. PMID:21540293

  7. Lactobacillus plantarum CS24.2 prevents Escherichia coli adhesion to HT-29 cells and also down-regulates enteropathogen-induced tumor necrosis factor-α and interleukin-8 expression.

    PubMed

    Dhanani, Akhilesh S; Bagchi, Tamishraha

    2013-04-01

    The aim of the present study was to evaluate the potential of Lactobacillus plantarum CS24.2 to antagonize Escherichia coli adhesion and modulate expression of the responses by HT-29 cells of inflammatory molecules to E. coli adhesion. Experiments were performed under different adhesion conditions and findings compared with the responses of Lactobacillus rhamnosus GG. Tests of competitive adhesion, adhesion inhibition and displacement assays were performed for lactobacilli (L. rhamnosus GG and L. plantarum CS24.2) and E. coli O26:H11 to HT-29 cells. Both the lactobacilli significantly reduced E. coli adhesion to HT-29 cells (P < 0.05). The ability of lactobacilli to modulate tumor necrosis factor-α and interleukin-8 expression was analyzed in HT-29 cells stimulated with E. coli using qRT-PCR. L. plantarum CS24.2 significantly down regulated expression of both the genes induced by E. coli in HT-29 cells at 6 hr as well as 24 hr, which was more significant than the corresponding findings for L. rhamnosus GG. The present findings suggest that L. plantarum CS24.2 inhibits pathogen adhesion to a similar extent as does the established probiotic strain L. rhamnosus GG. It may also attenuate tumor necrosis factor-α and interleukin-8 expression in HT-29 cells stimulated with E. coli. PMID:23586634

  8. Down regulation of BCL11B expression inhibits proliferation and induces apoptosis in malignant T cells by BCL11B-935-siRNA.

    PubMed

    Huang, Xin; Chen, Si; Shen, Qi; Chen, Shaohua; Yang, Lijian; Grabarczyk, Piotr; Przybylski, Grzegorz K; Schmidt, Christian A; Li, Yangqiu

    2011-07-01

    To screen the highly efficient and specific B-cell chronic lymphocytic leukemia/lymphoma 11B (BCL11B) small interfering RNA (siRNA) which are able to downregulate the BCL11B gene expression in human T-cell acute lymphoblastic leukemia, thereby inhibiting the leukemic T-cell proliferation and inducing apoptosis, four BCL11B-siRNAs and the scrambled non-silencing siRNA control (sc) were designed and obtained by chemosynthesis. After nucleofection, BCL11B expression in the mRNA and the protein levels were measured by qRT-PCR and immunoblotting, respectively. The biological consequences based on the highly efficient and specific BCL11B-siRNA were demonstrated by CCK-8 kit, morphological changes (Hoechst 33258 staining), high-resolution imaging, and flow cytometry. Reduction in the BCL11B mRNA level was observed at 24 or 48 hours in molt-4 T cells with BCL11B-935-siRNA, BCL11B-434-siRNA, or BCL11B-748-siRNA, respectively. BCL11B protein expression levels were reduced by 34·77% and 41·73% in the BCL11B-935-siRNA- and BCL11B-434-siRNA-treated cells, compared with the control level at 72 hours. In comparison with BCL11B-434-siRNA treatment group, the Molt-4 cells transfected with the BCL11B-935-siRNA showed significantly inhibited proliferation and effectively induced apoptosis (P<0·05). When highly efficient and specific BCL11B-935-siRNA was used to analyze the inhibition of BCL11B mRNA level in primary T-cell acute lymphoblastic leukemia (T-ALL) cells, similar result was obtained. In conclusion, siRNAs targeting the different exon domains resulted in different silencing effects and biological consequences. Suppression of BCL11B by RNA interference could inhibit the proliferation and induce the apoptosis effectively in leukemic T cells, which might be considered as a new target therapeutic strategy in T-cell malignancies. PMID:21756541

  9. Generation of a Mouse Model with Down-Regulated U50 snoRNA (SNORD50) Expression and Its Organ-Specific Phenotypic Modulation

    PubMed Central

    Soeno, Yuuichi; Fujita, Kazuya; Kudo, Tomoo; Asagiri, Masataka; Kakuta, Shigeru; Taya, Yuji; Shimazu, Yoshihito; Sato, Kaori; Tanaka-Fujita, Ritsuko; Kubo, Sachiko; Iwakura, Yoichiro; Nakamura, Yoshikazu; Mori, Shigeo; Aoba, Takaaki

    2013-01-01

    Box C/D-type small nucleolar RNAs (snoRNAs) are functional RNAs responsible for mediating 2′-O-ribose methylation of ribosomal RNAs (rRNAs) within the nucleolus. In the past years, evidence for the involvement of human U50 snoRNA in tumorigenesis has been accumulating. We previously identified U50HG, a non-protein-coding gene that hosted a box C/D-type U50 snoRNA, in a chromosomal breakpoint in a human B-cell lymphoma. Mouse genome analysis revealed four mouse U50 (mU50) host-genes: three mU50HG-a gene variants that were clustered in the genome and an mU50HG-b gene that we supposed to be the U50HG ortholog. In this study, to investigate the physiological importance of mU50 snoRNA and its involvement in tumorigenesis, we eliminated mU50 snoRNA sequences from the mU50HG-b gene. The established mouse line (ΔmU50(HG-b)) showed a significant reduction of mU50 snoRNA expression without alteration of the host-gene length and exon-intron structure, and the corresponding target rRNA methylation in various organs was reduced. Lifelong phenotypic monitoring showed that the ΔmU50(HG-b) mice looked almost normal without accelerated tumorigenicity; however, a notable difference was the propensity for anomalies in the lymphoid organs. Transcriptome analysis showed that dozens of genes, including heat shock proteins, were differentially expressed in ΔmU50(HG-b) mouse lymphocytes. This unique model of a single snoRNA knockdown with intact host-gene expression revealed further new insights into the discrete transcriptional regulation of multiple mU50 host-genes and the complicated dynamics involved in organ-specific processing and maintenance of snoRNAs. PMID:23991050

  10. NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy

    PubMed Central

    Dib-Hajj, S. D.; Tyrrell, L.; Black, J. A.; Waxman, S. G.

    1998-01-01

    Although physiological and pharmacological evidence suggests the presence of multiple tetrodotoxin-resistant (TTX-R) Na channels in neurons of peripheral nervous system ganglia, only one, SNS/PN3, has been identified in these cells to date. We have identified and sequenced a novel Na channel α-subunit (NaN), predicted to be TTX-R and voltage-gated, that is expressed preferentially in sensory neurons within dorsal root ganglia (DRG) and trigeminal ganglia. The predicted amino acid sequence of NaN can be aligned with the predicted structure of known Na channel α-subunits; all relevant landmark sequences, including positively charged S4 and pore-lining SS1–SS2 segments, and the inactivation tripeptide IFM, are present at predicted positions. However, NaN exhibits only 42–53% similarity to other mammalian Na channels, including SNS/PN3, indicating that it is a novel channel, and suggesting that it may represent a third subfamily of Na channels. NaN transcript levels are reduced significantly 7 days post axotomy in DRG neurons, consistent with previous findings of a reduction in TTX-R Na currents. The preferential expression of NaN in DRG and trigeminal ganglia and the reduction of NaN mRNA levels in DRG after axonal injury suggest that NaN, together with SNS/PN3, may produce TTX-R currents in peripheral sensory neurons and may influence the generation of electrical activity in these cells. PMID:9671787

  11. The Fission Yeast GATA Factor, Gaf1, Modulates Sexual Development via Direct Down-Regulation of ste11+ Expression in Response to Nitrogen Starvation

    PubMed Central

    Yu, Yeong Man; Yeon, Ji-Hyun; Maeng, Pil Jae

    2012-01-01

    Gaf1 is the first GATA family zinc-finger transcription factor identified in Schizosaccharomyces pombe. Here, we report that Gaf1 functions as a negatively acting transcription factor of ste11+, delaying the entrance of cells exposed to transient nitrogen starvation into the meiotic cycle. gaf1Δ strains exhibited accelerated G1-arrest upon nitrogen starvation. Moreover, gaf1Δ mutation caused increased mating and sporulation frequency under both nitrogen-starved and unstarved conditions, while overexpression of gaf1+ led to a significant impairment of sporulation. By microarray analysis, we found that approximately 63% (116 genes) of the 183 genes up-regulated in unstarved gaf1Δ cells were nitrogen starvation-responsive genes, and furthermore that 25 genes among the genes up-regulated by gaf1Δ mutation are Ste11 targets (e.g., gpa1+, ste4+, spk1+, ste11+, and mei2+). The phenotype caused by gaf1Δ mutation was masked by ste11Δ mutation, indicating that ste11+ is epistatic to gaf1+ with respect to sporulation efficiency, and accordingly that gaf1+ functions upstream of ste11+ in the signaling pathway governing sexual development. gaf1Δ strains showed accelerated ste11+ expression under nitrogen starvation and increased ste11+ expression even under normal conditions. Electrophoretic mobility shift assay analysis demonstrated that Gaf1 specifically binds to the canonical GATA motif (5′-HGATAR-3′) spanning from −371 to −366 in ste11+ promoter. Consequently, Gaf1 provides the prime example for negative regulation of ste11+ transcription through direct binding to a cis-acting motif of its promoter. PMID:22900017

  12. AB153. Down-regulated expression of excision of repair cross-complementing gene 1 reduces resistance to hydroxycamptothecine in bladder cancer

    PubMed Central

    Liu, Yili

    2016-01-01

    Objective The purpose of the study was to explore the potential mechanisms that interference of excision of repair cross-complementing gene 1 (ERCC1) mediated by lentiviral vector in bladder cancer T24 Cells. Methods the expression of ERCC1 was observed by immunohistochemical method in 25 cases of primary bladder cancer and recurrent bladder cancer tissues respectively from 25 patients. T24 cells were silenced targeting ERCC1 by lentiviruses .The transfection efficiency for ERCC1 was observed by fluorescence microscope and the interference efficiency was detected by real-time polymerase clain reaction and western blot assay. CCK-8 assay was used to assess the cell proliferation. Effects of cell apoptosis were detected by flow cytometry. Finally, the pathway of apoptosis was studied by using western blot method. Results As a result, we discovered that the expression level of ERCC1 in recurrent bladder cancer tissues (52%) was significantly higher than that in primary bladder cancer tissues (20%) (P<0.05). Compared with the T24 cells that did not silence the ERCC1 gene (control group) at different time periods (29.45%, 36.48%, 38.45%, 40.35%), the proliferation of T24 cells that silenced ERCC1 gene (experimental group) (27.25%, 37.45%, 32.5%, 42.05%) was not significantly changed (P>0.05). Hydroxycamptothecine (HCPT) inhibited the proliferation of T24 cells in dosage and time dependent manner. The inhibitory effect of HCPT on the experimental group was significantly higher than that of the control group (P<0.05).With the HCPT concentration increased, the apoptosis rate of the experimental group was significantly higher than that of the control group (P<0.05). After silencing of ERCC1, the sensitivity of T24 cells was increased to HCPT which could inhibit cell proliferation and induce cell apoptosis. Conclusions Therefore, ERCC1 may be a potential target protein used to guide the postoperative chemotherapy of bladder cancer.

  13. Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling

    PubMed Central

    Corcoran, K A; Leaderbrand, K; Jovasevic, V; Guedea, A L; Kassam, F; Radulovic, J

    2015-01-01

    In patients suffering from post-traumatic stress disorder (PTSD), fear evoked by trauma-related memories lasts long past the traumatic event and it is often complicated by general anxiety and depressed mood. This poses a treatment challenge, as drugs beneficial for some symptoms might exacerbate others. For example, in preclinical studies, antagonists of the NR2B subunit of N-methyl-d-aspartate receptors and activators of cAMP-dependent protein kinase (PKA) act as potent antidepressants and anxiolytics, but they block fear extinction. Using mice, we attempted to overcome this problem by interfering with individual NR2B and PKA signaling complexes organized by scaffolding proteins. We infused cell-permeable Tat peptides that displaced either NR2B from receptor for activated C kinase 1 (RACK1), or PKA from A-kinase anchor proteins (AKAPs) or microtubule-associated proteins (MAPs). The infusions were targeted to the retrosplenial cortex, an area involved in both fear extinction of remotely acquired memories and in mood regulation. Tat-RACK1 and Tat-AKAP enhanced fear extinction, all peptides reduced anxiety and none affected baseline depression-like behavior. However, disruption of PKA complexes distinctively interfered with the rapid antidepressant actions of the N-methyl-D-aspartate receptors antagonist MK-801 in that Tat-MAP2 blocked, whereas Tat-AKAP completely inverted the effect of MK-801 from antidepressant to depressant. These effects were unrelated to the MK-801-induced changes of brain-derived neurotrophic factor messenger RNA levels. Together, the findings suggest that NR2B–RACK1 complexes specifically contribute to fear extinction, and may provide a target for the treatment of PTSD. AKAP-PKA, on the other hand, appears to modulate fear extinction and antidepressant responses in opposite directions. PMID:26460481

  14. Hydrogen sulfide inhibits opioid withdrawal-induced pain sensitization in rats by down-regulation of spinal calcitonin gene-related peptide expression in the spine.

    PubMed

    Yang, Hai-Yu; Wu, Zhi-Yuan; Bian, Jin-Song

    2014-09-01

    Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a μ-opioid receptor agonist) followed by naloxone administration at the fourth hour significantly decreased rat paw nociceptive threshold, indicating the induction of withdrawal hyperalgesia. Application of NaHS (a hydrogen sulfide donor) together with each injection of DAMGO attenuated naloxone-precipitated withdrawal hyperalgesia. RT-PCR and Western blot analysis showed that NaHS significantly reversed the gene and protein expression of up-regulated spinal calcitonin gene-related peptide (CGRP) in naloxone-treated animals. NaHS also inhibited naloxone-induced cAMP rebound and cAMP response element-binding protein (CREB) phosphorylation in rat spinal cord. In SH-SY5Y neuronal cells, NaHS inhibited forskolin-stimulated cAMP production and adenylate cyclase (AC) activity. Moreover, NaHS pre-treatment suppressed naloxone-stimulated activation of protein kinase C (PKC) α, Raf-1, and extracellular signal-regulated kinase (ERK) 1/2 in rat spinal cord. Our data suggest that H2S prevents the development of opioid withdrawal-induced hyperalgesia via suppression of synthesis of CGRP in spine through inhibition of AC/cAMP and PKC/Raf-1/ERK pathways. PMID:24824948

  15. Kaempferol Reduces Matrix Metalloproteinase-2 Expression by Down-Regulating ERK1/2 and the Activator Protein-1 Signaling Pathways in Oral Cancer Cells

    PubMed Central

    Lin, Chiao-Wen; Chen, Pei-Ni; Chen, Mu-Kuan; Yang, Wei-En; Tang, Chih-Hsin; Yang, Shun-Fa; Hsieh, Yih-Shou

    2013-01-01

    Background Kaempferol has been proposed as a potential drug for cancer chemoprevention and treatment because it is a natural polyphenol contained in plant-based foods. Recent studies have demonstrated that kaempferol protects against cardiovascular disease and cancer. Based on this finding, we investigated the mechanisms by which kaempferol produces the anti-metastatic effect in human tongue squamous cell carcinoma SCC4 cells. Methodology/Principal Findings In this study, we provided molecular evidence associated with the anti-metastatic effect of kaempferol by demonstrating a substantial suppression of SCC4 cell migration and invasion. This effect was associated with reduced expressions of MMP-2 and TIMP-2 mRNA and protein levels. Analysis of the transcriptional regulation indicated that kaempferol inhibited MMP-2 transcription by suppressing c-Jun activity. Kaempferol also produced an inhibitory effect on the phosphorylation of ERK1/2. Conclusions These findings provide new insights into the molecular mechanisms involved in the anti-metastatic effect of kaempferol, and are valuable in the prevention of oral cancer metastasis. PMID:24278338

  16. Ethyl pyruvate and ethyl lactate down-regulate the production of pro-inflammatory cytokines and modulate expression of immune receptors.

    PubMed

    Hollenbach, Marcus; Hintersdorf, Anja; Huse, Klaus; Sack, Ulrich; Bigl, Marina; Groth, Marco; Santel, Thore; Buchold, Martin; Lindner, Inge; Otto, Andreas; Sicker, Dieter; Schellenberger, Wolfgang; Almendinger, Johannes; Pustowoit, Barbara; Birkemeyer, Claudia; Platzer, Mathias; Oerlecke, Ilka; Hemdan, Nasr; Birkenmeier, Gerd

    2008-09-01

    Esters of alpha-oxo-carbonic acids such as ethyl pyruvate (EP) have been demonstrated to exert inhibitory effects on the production of anti-inflammatory cytokines. So far, there is no information about effects, if any, of ethyl lactate (EL), an obviously inactive analogue of EP, on inflammatory immune responses. In the present study, we provide evidence that the anti-inflammatory action of alpha-oxo-carbonic acid esters is mediated by inhibition of glyoxalases (Glo), cytosolic enzymes that catalyse the conversion of alpha-oxo-aldehydes such as methylglyoxal (MGO) into the corresponding alpha-hydroxy acids using glutathione as a cofactor. In vitro enzyme activity measurements revealed the inhibition of human Glo1 by alpha-oxo-carbonic acid esters, whilst alpha-hydroxy-carbonic acid esters such as EL were not inhibitory. In contrast, both EP and EL were shown to suppress the Lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and IL-8 from human immunocompetent cells, and modulated the expression of the immune receptors HLA-DR, CD14 and CD91 on human monocytes. Here, we show a crossing link between glyoxalases and the immune system. The results described herein introduce glyoxalases as a possible target for therapeutic approaches of immune suppression. PMID:18625205

  17. Trans-chalcone and quercetin down-regulate fatty acid synthase gene expression and reduce ergosterol content in the human pathogenic dermatophyte Trichophyton rubrum

    PubMed Central

    2013-01-01

    .5 MIC of trans-chalcone and cerulenin drastically reduced protoplast regeneration in the two strains. The FAS1 gene was repressed in the presence of MICs of quercetin, trans-chalcone, fluconazole and cerulenin. The ERG6 gene was induced in the presence of MICs of fluconazole and cerulenin and was repressed in the presence of MICs of trans-chalcone and quercetin. Trans-chalcone and quercetin inhibited the enzymatic activity of FAS, with IC50 values of 68.23 and 17.1 μg/mL, respectively. Conclusion Trans-chalcone and quercetin showed antifungal activity against T. rubrum, reducing ergosterol levels and modulating the expression of FAS1 and ERG6. PMID:24044691

  18. Carnosic acid sensitized TRAIL-mediated apoptosis through down-regulation of c-FLIP and Bcl-2 expression at the post translational levels and CHOP-dependent up-regulation of DR5, Bim, and PUMA expression in human carcinoma caki cells

    PubMed Central

    Bae, Jae Hoon; Kwon, Taeg Kyu

    2015-01-01

    Carnosic acid is a phenolic diterpene from rosmarinus officinalis, and has multiple functions, such as anti-inflammatory, anti-viral, and anti-tumor activity. In this study, we examined whether carnosic acid could sensitize TRAIL-mediated apoptosis in human renal carcinoma Caki cells. We found that carnosic acid markedly induced TRAIL-mediated apoptosis in human renal carcinoma (Caki, ACHN, and A498), and human hepatocellular carcinoma (SK-HEP-1), and human breast carcinoma (MDA-MB-231) cells, but not normal cells (TMCK-1 and HSF). Carnosic acid induced down-regulation of c-FLIP and Bcl-2 expression at the post-translational levels, and the over-expression of c-FLIP and Bcl-2 markedly blocked carnosic acid-induced TRAIL sensitization. Furthermore, carnosic acid induced death receptor (DR)5, Bcl-2 interacting mediator of cell death (Bim), and p53 up-regulated modulator of apoptosis (PUMA) expression at the transcriptional levels via CCAAT/enhancer-binding protein-homologous protein (CHOP). Down-regulation of CHOP expression by siRNA inhibited DR5, Bim, and PUMA expression, and attenuated carnosic acid plus TRAIL-induced apoptosis. Taken together, our study demonstrates that carnosic acid enhances sensitization against TRAIL-mediated apoptosis through the down-regulation of c-FLIP and Bcl-2 expression, and up-regulation of ER stress-mediated DR5, Bim, and PUMA expression at the transcriptional levels. PMID:25596735

  19. Down-regulation of ERK1/2 and AKT-mediated X-ray repair cross-complement group 1 protein (XRCC1) expression by Hsp90 inhibition enhances the gefitinib-induced cytotoxicity in human lung cancer cells

    SciTech Connect

    Tung, Chun-Liang; Jian, Yi-Jun; Syu, Jhan-Jhang; Wang, Tai-Jing; Chang, Po-Yuan; Chen, Chien-Yu; Jian, Yun-Ting; Lin, Yun-Wei

    2015-05-15

    Gefitinib (Iressa{sup R}, ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreased XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells. - Highlights: • Gefitinib treatment decreased XRCC1 mRNA and protein expression in NSCLC cells. • Knocking down XRCC1 expression enhanced the cytotoxic effect of gefitinib. • Gefitinib combined with an Hsp90 inhibitor resulted in synergistically cytotoxicity.

  20. Expression of cell wall related genes in basal and ear internodes of silking brown-midrib-3, caffeic acid O-methyltransferase (COMT) down-regulated, and normal maize plants

    PubMed Central

    Guillaumie, Sabine; Goffner, Deborah; Barbier, Odile; Martinant, Jean-Pierre; Pichon, Magalie; Barrière, Yves

    2008-01-01

    Background Silage maize is a major forage and energy resource for cattle feeding, and several studies have shown that lignin content and structure are the determining factors in forage maize feeding value. In maize, four natural brown-midrib mutants have modified lignin content, lignin structure and cell wall digestibility. The greatest lignin reduction and the highest cell wall digestibility were observed in the brown-midrib-3 (bm3) mutant, which is disrupted in the caffeic acid O-methyltransferase (COMT) gene. Results Expression of cell wall related genes was investigated in basal and ear internodes of normal, COMT antisens (AS225), and bm3 maize plants of the INRA F2 line. A cell wall macro-array was developed with 651 gene specific tags of genes specifically involved in cell wall biogenesis. When comparing basal (older lignifying) and ear (younger lignifying) internodes of the normal line, all genes known to be involved in constitutive monolignol biosynthesis had a higher expression in younger ear internodes. The expression of the COMT gene was heavily reduced, especially in the younger lignifying tissues of the ear internode. Despite the fact that AS225 transgene expression was driven only in sclerenchyma tissues, COMT expression was also heavily reduced in AS225 ear and basal internodes. COMT disruption or down-regulation led to differential expressions of a few lignin pathway genes, which were all over-expressed, except for a phenylalanine ammonia-lyase gene. More unexpectedly, several transcription factor genes, cell signaling genes, transport and detoxification genes, genes involved in cell wall carbohydrate metabolism and genes encoding cell wall proteins, were differentially expressed, and mostly over-expressed, in COMT-deficient plants. Conclusion Differential gene expressions in COMT-deficient plants highlighted a probable disturbance in cell wall assembly. In addition, the gene expressions suggested modified chronology of the different events leading

  1. Pseudomonas syringae Effector Avirulence Protein E Localizes to the Host Plasma Membrane and Down-Regulates the Expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 Gene Required for Antibacterial Immunity in Arabidopsis.

    PubMed

    Xin, Xiu-Fang; Nomura, Kinya; Ding, Xinhua; Chen, Xujun; Wang, Kun; Aung, Kyaw; Uribe, Francisco; Rosa, Bruce; Yao, Jian; Chen, Jin; He, Sheng Yang

    2015-09-01

    Many bacterial pathogens of plants and animals deliver effector proteins into host cells to promote infection. Elucidation of how pathogen effector proteins function not only is critical for understanding bacterial pathogenesis but also provides a useful tool in discovering the functions of host genes. In this study, we characterized the Pseudomonas syringae pv tomato DC3000 effector protein Avirulence Protein E (AvrE), the founding member of a widely distributed, yet functionally enigmatic, bacterial effector family. We show that AvrE is localized in the plasma membrane (PM) and PM-associated vesicle-like structures in the plant cell. AvrE contains two physically interacting domains, and the amino-terminal portion contains a PM-localization signal. Genome-wide microarray analysis indicates that AvrE, as well as the functionally redundant effector Hypersensitive response and pathogenicity-dependent Outer Protein M1, down-regulates the expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13) gene in Arabidopsis (Arabidopsis thaliana). Mutational analysis shows that NHL13 is required for plant immunity, as the nhl13 mutant plant displayed enhanced disease susceptibility. Our results defined the action site of one of the most important bacterial virulence proteins in plants and the antibacterial immunity function of the NHL13 gene. PMID:26206852

  2. Artemisolide is a typical inhibitor of I{kappa}B kinase {beta} targeting cysteine-179 residue and down-regulates NF-{kappa}B-dependent TNF-{alpha} expression in LPS-activated macrophages

    SciTech Connect

    Kim, Byung Hak; Lee, Jun-Young; Seo, Jee Hee; Lee, Hwa Young; Ryu, Shi Yong; Ahn, Byung Woo; Lee, Chong-Kil; Hwang, Bang Yeon; Han, Sang-Bae; Kim, Youngsoo

    2007-09-28

    Nuclear factor (NF)-{kappa}B regulates a central common signaling for immunity and cell survival. Artemisolide (ATM) was previously isolated as a NF-{kappa}B inhibitor from a plant of Artemisia asiatica. However, molecular basis of ATM on NF-{kappa}B activation remains to be defined. Here, we demonstrate that ATM is a typical inhibitor of I{kappa}B kinase {beta} (IKK{beta}), resulting in inhibition of lipopolysaccharide (LPS)-induced NF-{kappa}B activation in RAW 264.7 macrophages. ATM inhibited the kinase activity of highly purified IKK{beta} and also LPS-induced IKK activity in the cells. Moreover, the effect of ATM on IKK{beta} activity was completely abolished by substitution of Cys-179 residue of IKK{beta} to Ala residue, indicating direct targeting site of ATM. ATM could inhibit I{kappa}B{alpha} phosphorylation in LPS-activated RAW 264.7 cells and subsequently prevent NF-{kappa}B activation. Further, we demonstrate that ATM down-regulates NF-{kappa}B-dependent TNF-{alpha} expression. Taken together, this study provides a pharmacological potential of ATM in NF-{kappa}B-dependent inflammatory disorders.

  3. Thrombospondin-1 Silencing Down-Regulates Integrin Expression Levels in Human Anaplastic Thyroid Cancer Cells with BRAFV600E: New Insights in the Host Tissue Adaptation and Homeostasis of Tumor Microenvironment

    PubMed Central

    Duquette, Mark; Sadow, Peter M.; Lawler, Jack; Nucera, Carmelo

    2013-01-01

    Background and Rationale: Anaplastic thyroid cancer (ATC) is characterized by pleomorphic cells, has a poor prognosis, is highly devastating disease, and is not curable. No reliable biomarkers of metastatic potential, helpful for early diagnosis of ATC and therapeutic response have been found yet. Thrombospondin-1 (TSP-1) plays a fundamental role in cancer progression by regulating cell stromal cross-talk in the tumor microenvironment. Goals: Our goal was to understand whether TSP-1 could affect protein levels of its integrin receptors (e.g., ITGα3, α6, and β1) and cell morphology in BRAFV600E-ATC cells in vitro and in vivo. Experimental Design: Anaplastic thyroid cancer-derived cell cultures and western blotting were used to assess integrin protein expression upon TSP-1 silencing. Immunohistochemistry was performed on orthotopic primary human ATC and metastatic ATC in lung tissue to compare TSP-1 and integrin protein expression levels. Results: TSP-1 knock-down down-regulates ITGα3, α6, and β1 in BRAFV600E-human ATC cells. BRAFV600E-ATC cells with TSP-1 knock-down were rounded compared to control cells, which displayed a spread morphology. TSP-1 knock-down also reduced TSP-1, ITGα3, α6, and β1 protein expression levels in vivo in the ATC microenvironment, which is enriched in stromal and inflammatory cells. Conclusion: TSP-1 silencing causes changes in ITG levels and ATC cell morphology. The assessment of TSP-1 and ITG levels might contribute to earlier metastatic potential of BRAFV600E-positive aggressive thyroid cancers, and allow improved patient selection for clinical trials. PMID:24348463

  4. Low Intensity Ultrasound Promotes the Sensitivity of Rat Brain Glioma to Doxorubicin by Down-Regulating the Expressions of P-Glucoprotein and Multidrug Resistance Protein 1 In Vitro and In Vivo

    PubMed Central

    Zhang, Zhen; Xu, Ke; Bi, Yonghua; Yu, Guibo; Wang, Siwei; Qi, Xun; Zhong, Hongshan

    2013-01-01

    The overall prognosis for malignant glioma is extremely poor, and treatment options are limited in part because of multidrug resistant proteins. Our previous findings suggest low intensity ultrasound (LIUS) can induce apoptosis of glioma cells. Given this finding, we were interested in determining if LIUS could help treat glioma by inhibiting multidrug resistant proteins, and if so, which pathways are involved. In this study, the toxicity sensitivity and multidrug resistance proteins of glioma induced by LIUS were investigated using CCK-8, immunohistochemistry, immunofluorency, and RT-PCR in tissue samples and cultured cells. LIUS inhibited increase of C6 cells in an intensity- and time-dependent manner. The toxicity sensitivity of C6 cells increased significantly after LIUS sonication (intensity of 142.0 mW/cm2) or Doxorubicin (DOX) at different concentration, particularly by the combination of LIUS sonication and DOX. The expressions of P-gp and MRP1 decreased significantly post-sonication at intensity of 142.0 mW/cm2 both in vitro and in vivo. The expressions of p110 delta (PI3K), NF-κB-p65, Akt/PKB, and p-Akt/PKB were downregulated by LIUS sonication and DOX treatment separately or in combination at the same parameters in rat glioma. These results indicate that LIUS could increase the toxicity sensitivity of glioma by down-regulating the expressions of P-gp and MRP1, which might be mediated by the PI3K/Akt/NF-κB pathway. PMID:23940624

  5. Down-regulation of ERK1/2 and AKT-mediated X-ray repair cross-complement group 1 protein (XRCC1) expression by Hsp90 inhibition enhances the gefitinib-induced cytotoxicity in human lung cancer cells.

    PubMed

    Tung, Chun-Liang; Jian, Yi-Jun; Syu, Jhan-Jhang; Wang, Tai-Jing; Chang, Po-Yuan; Chen, Chien-Yu; Jian, Yun-Ting; Lin, Yun-Wei

    2015-05-15

    Gefitinib (Iressa(R), ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreased XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells. PMID:25662161

  6. Synthesis, structural activity-relationships, and biological evaluation of novel amide-based allosteric binding site antagonists in NR1A/NR2B N-methyl-D-aspartate receptors☆

    PubMed Central

    Mosley, Cara A.; Myers, Scott J.; Murray, Ernest E.; Santangelo, Rose; Tahirovic, Yesim A.; Kurtkaya, Natalie; Mullasseril, Praseeda; Yuan, Hongjie; Lyuboslavsky, Polina; Le, Phuong; Wilson, Lawrence J.; Yepes, Manuel; Dingledine, Ray; Traynelis, Stephen F.; Liotta, Dennis C.

    2010-01-01

    The synthesis and structure–activity relationship analysis of a novel class of amide-based biaryl NR2B-selective NMDA receptor antagonists are presented. Some of the studied compounds are potent, selective, non-competitive, and voltage-independent antagonists of NR2B-containing NMDA receptors. Like the founding member of this class of antagonists (ifenprodil), several interesting compounds of the series bind to the amino terminal domain of the NR2B subunit to inhibit function. Analogue potency is modu-lated by linker length, flexibility, and hydrogen bonding opportunities. However, unlike previously described classes of NR2B-selective NMDA antagonists that exhibit off-target activity at a variety of monoamine receptors, the compounds described herein show much diminished effects against the hERG channel and α1-adrenergic receptors. Selections of the compounds discussed have acceptable half-lives in vivo and are predicted to permeate the blood–brain barrier. These data together suggest that masking charged atoms on the linker region of NR2B-selective antagonists can decrease undesirable side effects while still maintaining on-target potency. PMID:19648014

  7. The R18 Polyarginine Peptide Is More Effective Than the TAT-NR2B9c (NA-1) Peptide When Administered 60 Minutes after Permanent Middle Cerebral Artery Occlusion in the Rat

    PubMed Central

    Milani, D.; Knuckey, N. W.; Anderton, R. S.; Cross, J. L.; Meloni, B. P.

    2016-01-01

    We examined the dose responsiveness of polyarginine R18 (100, 300, and 1000 nmol/kg) when administered 60 minutes after permanent middle cerebral artery occlusion (MCAO). The TAT-NR2B9c peptide, which is known to be neuroprotective in rodent and nonhuman primate stroke models, served as a positive control. At 24 hours after MCAO, there was reduced total infarct volume in R18 treated animals at all doses, but this reduction only reached statistical significance at doses of 100 and 1000 nmol/kg. The TAT-NR2B9c peptide reduced infarct volume at doses of 300 and 1000 nmol/kg, but not to a statistically significant extent, while the 100 nmol/kg dose was ineffective. The reduction in infarct volume with R18 and TAT-NR2B9c peptide treatments was mirrored by improvements in one or more functional outcomes (namely, neurological score, adhesive tape removal, and rota-rod), but not to a statistically significant extent. These findings further confirm the neuroprotective properties of polyarginine peptides and for R18 extend its therapeutic time window and dose range, as well as demonstrating its greater efficacy compared to TAT-NR2B9c in a severe stroke model. The superior neuroprotective efficacy of R18 over TAT-NR2B9c highlights the potential of this polyarginine peptide as a lead candidate for studies in human stroke. PMID:27247825

  8. Eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kappaB AND AP-1 through inhibition of MAPKS and AKT/IkappaBalpha signaling pathways in macrophages.

    PubMed

    Yeh, J L; Hsu, J H; Hong, Y S; Wu, J R; Liang, J C; Wu, B N; Chen, I J; Liou, S F

    2011-01-01

    Eugenol and isoeugenol, two components of clover oil, have been reported to possess several biomedical properties, such as anti-inflammatory, antimicrobial and antioxidant effects. This study aims to examine the anti-inflammatory effects of eugenol, isoeugenol and four of their derivatives on expression of inducible nitric oxide synthase (iNOS) activated by lipopolysaccharide (LPS) in mouse macrophages (RAW 264.7), and to investigate molecular mechanisms underlying these effects. We found that two derivatives, eugenolol and glyceryl-isoeugenol, had potent inhibitory effects on LPS-induced upregulation of nitrite levels, iNOS protein and iNOS mRNA. In addition, they both suppressed the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) induced by LPS. Moreover, they both attenuated the DNA binding of NF-kB and AP-1, phosphorylation of inhibitory kB-alpha (IkB-alpha), and nuclear translocation of p65 protein induced by LPS. Finally, we demonstrated that glyceryl-isoeugenol suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK, whereas eugenolol suppressed the phosphorylation of ERK1/2 and p38 MAPK. Taken together, these results suggest that that eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kB and AP-1 through inhibition of MAPKs and Akt/IkB-alpha signaling pathways. Thus, this study implies that eugenolol and glyceryl-isoeugenol may provide therapeutic benefits for inflammatory diseases. PMID:21658309

  9. Characteristic and Expression Analysis of a Metallothionein Gene, OsMT2b, Down-Regulated by Cytokinin Suggests Functions in Root Development and Seed Embryo Germination of Rice1[OA

    PubMed Central

    Yuan, Jing; Chen, Dan; Ren, Yujun; Zhang, Xuelian; Zhao, Jie

    2008-01-01

    Metallothioneins (MTs) are low molecular mass and cysteine-rich metal-binding proteins known to be mainly involved in maintaining metal homeostasis and stress responses. But, their functions in higher plant development are scarcely studied. Here, we characterized rice (Oryza sativa) METALLOTHIONEIN2b (OsMT2b) molecularly and found that its expression was down-regulated by cytokinins. OsMT2b was preferentially expressed in rice immature panicles, scutellum of germinating embryos, and primordium of lateral roots. In contrast with wild-type plants, OsMT2b-RNA interference (RNAi) transgenic plants had serious handicap in plant growth and root formation, whereas OsMT2b-overexpressing transformants were dwarfed and presented more adventitious roots and big lateral roots. The increased cytokinin levels in RNAi plants and decreased cytokinin levels in overexpressing plants were confirmed by high-performance liquid chromatography quantitative analysis in the roots of wild-type and transgenic plants. In RNAi plants, localization of isopentenyladenosine, a kind of endogenous cytokinin, in roots and germinating embryos expanded to the whole tissues, whereas in overexpressing plants, the isopentenyladenosine signals were very faint in the vascular tissues of roots and scutellum cells of germinating embryos. In vitro culture of embryos could largely resume the reduced germination frequency in RNAi plants but had no obvious change in overexpressing plants. Taken together, these results indicate a possible feedback regulation mechanism of OsMT2b to the level of endogenous cytokinins that is involved in root development and seed embryo germination of rice. PMID:18258694

  10. Down-Regulation of KCC2 Expression and Phosphorylation in Motoneurons, and Increases the Number of in Primary Afferent Projections to Motoneurons in Mice with Post-Stroke Spasticity

    PubMed Central

    Toda, Takuya; Ishida, Kazuto; Kiyama, Hiroshi; Yamashita, Toshihide; Lee, Sachiko

    2014-01-01

    Spasticity obstructs motor function recovery post-stroke, and has been reported to occur in spinal cord injury and electrophysiological studies. The purpose of the present study was to assess spinal cord circuit spasticity in post-stroke mice. At 3, 7, 21, and 42 d after photothrombotic ischemic cortical injury in C57BL/6J mice, we observed decreased rate-dependent depression (RDD) of the Hoffmann reflex (H reflex) in the affected forelimb of mice compared with the limbs of sham mice and the non-affected forelimb. This finding suggests a hyper-excitable stretch reflex in the affected forelimb. We then performed immunohistochemical and western blot analyses to examine the expression of the potassium-chloride cotransporter 2 (KCC2) and phosphorylation of the KCC2 serine residue, 940 (S940), since this is the main chloride extruder that affects neuronal excitability. We also performed immunohistochemical analyses on the number of vesicular glutamate transporter 1 (vGluT1)-positive boutons to count the number of Ia afferent fibers that connect to motoneurons. Western bolts revealed that, compared with sham mice, experimental mice had significantly reduced KCC2 expression at 7 d post-stroke, and dephosphorylated S940 at 3 and 7 d post-stroke in motoneuron plasma membranes. We also observed a lower density of KCC2-positive areas in the plasma membrane of motoneurons at 3 and 7 d post-stroke. However, western blot and immunohistochemical analyses revealed that there were no differences between groups 21 and 42 d post-stroke, respectively. In addition, at 7 and 42 d post-stroke, experimental mice exhibited a significant increase in vGluT1 boutons compared with sham mice. Our findings suggest that both the down-regulation of KCC2 and increases in Ia afferent fibers are involved in post-stroke spasticity. PMID:25546454

  11. Down-regulation of PROS1 Gene Expression by 17β-Estradiol via Estrogen Receptor α (ERα)-Sp1 Interaction Recruiting Receptor-interacting Protein 140 and the Corepressor-HDAC3 Complex*

    PubMed Central

    Suzuki, Atsuo; Sanda, Naomi; Miyawaki, Yuhri; Fujimori, Yuta; Yamada, Takayuki; Takagi, Akira; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

    2010-01-01

    Pregnant women show a low level of protein S (PS) in plasma, which is known to be a risk for deep venous thrombosis. 17β-Estradiol (E2), an estrogen that increases in concentration in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E2 in HepG2-ERα cells, which stably express ERα, and also the genomic ER signaling pathway, which modulates the ligand-dependent repression of the PSα gene (PROS1). We observed that E2 repressed the production of mRNA and antigen of PS. A luciferase reporter assay revealed that E2 down-regulated PROS1 promoter activity and that this E2-dependent repression disappeared upon the deletion or mutation of two adjacent GC-rich motifs in the promoter. An electrophoretic mobility shift assay and DNA pulldown assay revealed that the GC-rich motifs were associated with Sp1, Sp3, and ERα. In a chromatin immunoprecipitation assay, we found ERα-Sp protein-promoter interaction involved in the E2-dependent repression of PROS1 transcription. Furthermore, we demonstrated that E2 treatment recruited RIP140 and the NCoR-SMRT-HDAC3 complex to the PROS1 promoter, which hypoacetylated chromatin. Taken together, this suggested that E2 might repress PROS1 transcription depending upon ERα-Sp1 recruiting transcriptional repressors in HepG2-ERα cells and, consequently, that high levels of E2 leading to reduced levels of plasma PS would be a risk for deep venous thrombosis in pregnant women. PMID:20200160

  12. Secreted Heat Shock Protein 90α (HSP90α) Induces Nuclear Factor-κB-mediated TCF12 Protein Expression to Down-regulate E-cadherin and to Enhance Colorectal Cancer Cell Migration and Invasion*

    PubMed Central

    Chen, Wei-Shone; Chen, Chia-Chi; Chen, Li-Li; Lee, Chun-Chung; Huang, Tze-Sing

    2013-01-01

    Secreted levels of HSP90α and overexpression of TCF12 have been associated with the enhancement of colorectal cancer (CRC) cell migration and invasion. In this study, we observed that CRC patients with tumor TCF12 overexpression exhibited both a higher rate of metastatic occurrence and a higher average serum HSP90α level compared with patients without TCF12 overexpression. Therefore, we studied the relationship between the actions of secreted HSP90α and TCF12. Like overexpressed TCF12, secreted HSP90α or recombinant HSP90α (rHSP90α) induced fibronectin expression and repressed E-cadherin, connexin-26, connexin-43, and gap junction levels in CRC cells. Consistently, rHSP90α stimulated invasive outgrowths of CRC cells from spherical structures during three-dimensional culture. rHSP90α also induced TCF12 expression in CRC cells. Its effects on CRC cell epithelial-mesenchymal transition, migration, and invasion were drastically prevented when TCF12 was knocked down. This suggests that TCF12 expression is required for secreted HSP90α to enhance CRC cell spreading. Through the cellular receptor CD91, rHSP90α facilitated the complex formation of CD91 with IκB kinases (IKKs) α and β and increased the levels of phosphorylated (active) IKKα/β and NF-κB. Use of an IKKα/β inhibitor or ectopic overexpression of dominant-negative IκBα efficiently repressed rHSP90α-induced TCF12 expression. Moreover, κB motifs were recognized in the gene sequence of the TCF12 promoter, and a physical association between NF-κB and the TCF12 promoter was detected in rHSP90α-treated CRC cells. Together, these results suggest that the CD91/IKK/NF-κB signaling cascade is involved in secreted HSP90α-induced TCF12 expression, leading to E-cadherin down-regulation and enhanced CRC cell migration/invasion. PMID:23386606

  13. Secreted heat shock protein 90α (HSP90α) induces nuclear factor-κB-mediated TCF12 protein expression to down-regulate E-cadherin and to enhance colorectal cancer cell migration and invasion.

    PubMed

    Chen, Wei-Shone; Chen, Chia-Chi; Chen, Li-Li; Lee, Chun-Chung; Huang, Tze-Sing

    2013-03-29

    Secreted levels of HSP90α and overexpression of TCF12 have been associated with the enhancement of colorectal cancer (CRC) cell migration and invasion. In this study, we observed that CRC patients with tumor TCF12 overexpression exhibited both a higher rate of metastatic occurrence and a higher average serum HSP90α level compared with patients without TCF12 overexpression. Therefore, we studied the relationship between the actions of secreted HSP90α and TCF12. Like overexpressed TCF12, secreted HSP90α or recombinant HSP90α (rHSP90α) induced fibronectin expression and repressed E-cadherin, connexin-26, connexin-43, and gap junction levels in CRC cells. Consistently, rHSP90α stimulated invasive outgrowths of CRC cells from spherical structures during three-dimensional culture. rHSP90α also induced TCF12 expression in CRC cells. Its effects on CRC cell epithelial-mesenchymal transition, migration, and invasion were drastically prevented when TCF12 was knocked down. This suggests that TCF12 expression is required for secreted HSP90α to enhance CRC cell spreading. Through the cellular receptor CD91, rHSP90α facilitated the complex formation of CD91 with IκB kinases (IKKs) α and β and increased the levels of phosphorylated (active) IKKα/β and NF-κB. Use of an IKKα/β inhibitor or ectopic overexpression of dominant-negative IκBα efficiently repressed rHSP90α-induced TCF12 expression. Moreover, κB motifs were recognized in the gene sequence of the TCF12 promoter, and a physical association between NF-κB and the TCF12 promoter was detected in rHSP90α-treated CRC cells. Together, these results suggest that the CD91/IKK/NF-κB signaling cascade is involved in secreted HSP90α-induced TCF12 expression, leading to E-cadherin down-regulation and enhanced CRC cell migration/invasion. PMID:23386606

  14. High Brain Ammonia Tolerance and Down-Regulation of Na+:K+:2Cl- Cotransporter 1b mRNA and Protein Expression in the Brain of the Swamp Eel, Monopterus albus, Exposed to Environmental Ammonia or Terrestrial Conditions

    PubMed Central

    Ip, Yuen K.; Hou, Zhisheng; Chen, Xiu L.; Ong, Jasmine L. Y.; Chng, You R.; Ching, Biyun; Hiong, Kum C.; Chew, Shit F.

    2013-01-01

    Na+:K+:2Cl- cotransporter 1 (NKCC1) has been implicated in mediating ischemia-, trauma- or ammonia-induced astrocyte swelling/brain edema in mammals. This study aimed to determine the effects of ammonia or terrestrial exposure on ammonia concentrations in the plasma and brain, and the mRNA expression and protein abundance of nkcc/Nkcc in the brain, of the swamp eel Monopterusalbus. Ammonia exposure led to a greater increase in the ammonia concentration in the brain of M. albus than terrestrial exposure. The brain ammonia concentration of M. albus reached 4.5 µmol g-1 and 2.7 µmol g-1 after 6 days of exposure to 50 mmol l-1 NH4Cl and terrestrial conditions, respectively. The full cDNA coding sequence of nkcc1b from M. albus brain comprised 3276 bp and coded for 1092 amino acids with an estimated molecular mass of 119.6 kDa. A molecular characterization indicated that it could be activated through phosphorylation and/or glycosylation by osmotic and/or oxidative stresses. Ammonia exposure for 1 day or 6 days led to significant decreases in the nkcc1b mRNA expression and Nkcc1b protein abundance in the brain of M. albus. In comparison, a significant decrease in nkcc1b mRNA expression was observed in the brain of M. albus only after 6 days of terrestrial exposure, but both 1 day and 6 days of terrestrial exposure resulted in significant decreases in the protein abundance of Nkcc1b. These results are novel because it has been established in mammals that ammonia up-regulates NKCC1 expression in astrocytes and NKCC1 plays an important role in ammonia-induced astrocyte swelling and brain edema. By contrast, our results indicate for the first time that M. albus is able to down-regulate the mRNA and protein expression of nkcc1b/Nkcc1b in the brain when confronted with ammonia toxicity, which could be one of the contributing factors to its extraordinarily high brain ammonia tolerance. PMID:24069137

  15. Unprecedented Therapeutic Potential with a Combination of A2A/NR2B Receptor Antagonists as Observed in the 6-OHDA Lesioned Rat Model of Parkinson's Disease

    PubMed Central

    Michel, Anne; Downey, Patrick; Nicolas, Jean-Marie; Scheller, Dieter

    2014-01-01

    In Parkinson's disease, the long-term use of dopamine replacing agents is associated with the development of motor complications; therefore, there is a need for non-dopaminergic drugs. This study evaluated the potential therapeutic impact of six different NR2B and A2A receptor antagonists given either alone or in combination in unilateral 6-OHDA-lesioned rats without (monotherapy) or with (add-on therapy) the co-administration of L-Dopa: Sch-58261+ Merck 22; Sch-58261+Co-101244; Preladenant + Merck 22; Preladenant + Radiprodil; Tozadenant + Radiprodil; Istradefylline + Co-101244. Animals given monotherapy were assessed on distance traveled and rearing, whereas those given add-on therapy were assessed on contralateral rotations. Three-way mixed ANOVA were conducted to assess the main effect of each drug separately and to determine whether any interaction between two drugs was additive or synergistic. Additional post hoc analyses were conducted to compare the effect of the combination with the effect of the drugs alone. Motor activity improved significantly and was sustained for longer when the drugs were given in combination than when administered separately at the same dose. Similarly, when tested as add-on treatment to L-Dopa, the combinations resulted in higher levels of contralateral rotation in comparison to the single drugs. Of special interest, the activity observed with some combinations could not be described by a simplistic additive effect and involved more subtle synergistic pharmacological interactions. The combined administration of A2A/NR2B-receptor antagonists improved motor behaviour in 6-OHDA rats. Given the proven translatability of this model such a combination may be expected to be effective in improving motor symptoms in patients. PMID:25513815

  16. Gibberellin application at pre-bloom in grapevines down-regulates the expressions of VvIAA9 and VvARF7, negative regulators of fruit set initiation, during parthenocarpic fruit development.

    PubMed

    Jung, Chan Jin; Hur, Youn Young; Yu, Hee-Ju; Noh, Jung-Ho; Park, Kyo-Sun; Lee, Hee Jae

    2014-01-01

    Fruit set is initiated only after fertilization and is tightly regulated primarily by gibberellins (GAs) and auxins. The application of either of these hormones induces parthenocarpy, fruit set without fertilization, but the molecular mechanism underlying this induction is poorly understood. In the present study, we have shown that the parthenocarpic fruits induced by GA application at pre-bloom result from the interaction of GA with auxin signaling. The transcriptional levels of the putative negative regulators of fruit set initiation, including Vitis auxin/indole-3-acetic acid transcription factor 9 (VvIAA9), Vitis auxin response factor 7 (VvARF7), and VvARF8 were monitored during inflorescence development in seeded diploid 'Tamnara' grapevines with or without GA application. Without GA application, VvIAA9, VvARF7, and VvARF8 were expressed at a relatively high level before full bloom, but decreased thereafter following pollination. After GA application at 14 days before full bloom (DBF); however, the expression levels of VvIAA9 and VvARF7 declined at 5 DBF prior to pollination. The effects of GA application on auxin levels or auxin signaling were also analyzed by monitoring the expression patterns of auxin biosynthesis genes and auxin-responsive genes with or without GA application. Transcription levels of the auxin biosynthesis genes Vitis anthranilate synthase β subunit (VvASB1-like), Vitis YUCCA2 (VvYUC2), and VvYUC6 were not significantly changed by GA application. However, the expressions of Vitis Gretchen Hagen3.2 (VvGH3.2) and VvGH3.3, auxin-responsive genes, were up-regulated from 2 DBF to full bloom with GA application. Furthermore, the Vitis GA signaling gene, VvDELLA was up-regulated by GA application during 12 DBF to 7 DBF, prior to down-regulation of VvIAA9 and VvARF7. These results suggest that VvIAA9 and VvARF7 are negative regulators of fruit set initiation in grapevines, and GA signaling is integrated with auxin signaling via VvDELLA during

  17. Gibberellin Application at Pre-Bloom in Grapevines Down-Regulates the Expressions of VvIAA9 and VvARF7, Negative Regulators of Fruit Set Initiation, during Parthenocarpic Fruit Development

    PubMed Central

    Jung, Chan Jin; Hur, Youn Young; Yu, Hee-Ju; Noh, Jung-Ho; Park, Kyo-Sun; Lee, Hee Jae

    2014-01-01

    Fruit set is initiated only after fertilization and is tightly regulated primarily by gibberellins (GAs) and auxins. The application of either of these hormones induces parthenocarpy, fruit set without fertilization, but the molecular mechanism underlying this induction is poorly understood. In the present study, we have shown that the parthenocarpic fruits induced by GA application at pre-bloom result from the interaction of GA with auxin signaling. The transcriptional levels of the putative negative regulators of fruit set initiation, including Vitis auxin/indole-3-acetic acid transcription factor 9 (VvIAA9), Vitis auxin response factor 7 (VvARF7), and VvARF8 were monitored during inflorescence development in seeded diploid ‘Tamnara’ grapevines with or without GA application. Without GA application, VvIAA9, VvARF7, and VvARF8 were expressed at a relatively high level before full bloom, but decreased thereafter following pollination. After GA application at 14 days before full bloom (DBF); however, the expression levels of VvIAA9 and VvARF7 declined at 5 DBF prior to pollination. The effects of GA application on auxin levels or auxin signaling were also analyzed by monitoring the expression patterns of auxin biosynthesis genes and auxin-responsive genes with or without GA application. Transcription levels of the auxin biosynthesis genes Vitis anthranilate synthase β subunit (VvASB1-like), Vitis YUCCA2 (VvYUC2), and VvYUC6 were not significantly changed by GA application. However, the expressions of Vitis Gretchen Hagen3.2 (VvGH3.2) and VvGH3.3, auxin-responsive genes, were up-regulated from 2 DBF to full bloom with GA application. Furthermore, the Vitis GA signaling gene, VvDELLA was up-regulated by GA application during 12 DBF to 7 DBF, prior to down-regulation of VvIAA9 and VvARF7. These results suggest that VvIAA9 and VvARF7 are negative regulators of fruit set initiation in grapevines, and GA signaling is integrated with auxin signaling via VvDELLA during

  18. Both NR2A and NR2B Subunits of the NMDA Receptor Are Critical for Long-Term Potentiation and Long-Term Depression in the Lateral Amygdala of Horizontal Slices of Adult Mice

    ERIC Educational Resources Information Center

    Muller, Tobias; Albrecht, Doris; Gebhardt, Christine

    2009-01-01

    The lateral nucleus of the amygdala (LA) is implicated in emotional and social behaviors. We recently showed that in horizontal brain slices, activation of NMDA receptors (NMDARs) is a requirement for persistent synaptic alterations in the LA, such as long-term potentiation (LTP) and long-term depression (LTD). In the LA, NR2A- and NR2B-type NMDRs…

  19. Down-regulation of laminin-5 in breast carcinoma cells.

    PubMed Central

    Martin, K. J.; Kwan, C. P.; Nagasaki, K.; Zhang, X.; O'Hare, M. J.; Kaelin, C. M.; Burgeson, R. E.; Pardee, A. B.; Sager, R.

    1998-01-01

    BACKGROUND: Laminin-5 (ln-5), a large heterotrimeric glycoprotein consisting of an alpha 3, beta 3, and gamma 2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the alpha 3 beta 1 and alpha 6 beta 4 integrins to regulate cell adhesion, migration, and morphogenesis. The ln-5 chains show tissue-specific patterns of regulation in tumors derived from different tissues. For example, ln-5 is often up-regulated in gliomas, gastric carcinomas, and squamous carcinomas and down-regulated in prostate and basal cell carcinomas. Ln-5 expression patterns may represent useful tumor markers and help to elucidate the role of ln-5 in tumor progression in different tissue types. MATERIALS AND METHODS: We have studied ln-5 expression patterns in the breast. mRNA levels were examined in tumor and normal breast epithelial cell lines, tissue samples, and immunomagnetically sorted primary cultures using differential display, Northern blotting, and hybridization arrays. Protein levels were examined by immunoprecipitation. Gene integrity was assessed by Southern blotting of representative cell types. RESULTS: Ln-5 alpha 3, beta 3, and gamma 2 mRNA expression was found to be markedly down-regulated in a panel of breast tumor cell lines when compared with normal breast epithelial cells. Ln-5 mRNA was expressed at relatively high levels in MCF-10A immortal normal breast epithelial cells, long-term cultures of normal breast cells, and sorted primary cultures of normal breast luminal epithelial and myoepithelial cells. Reduced, but detectable, levels of ln-5 tended to be expressed in cell lines derived from early-stage breast tumors, whereas expression was generally not detected in cell lines derived from later-stage tumors. In breast tumor tissue specimens, expression of ln alpha 3 and beta 3 mRNAs tended to be reduced relative to levels observed in adjacent nontumor tissue, whereas in gamma 2 levels were elevated in specimens with increased amounts of

  20. Okadaic acid induces epileptic seizures and hyperphosphorylation of the NR2B subunit of the NMDA receptor in rat hippocampus in vivo.

    PubMed

    Arias, Clorinda; Montiel, Teresa; Peña, Fernando; Ferrera, Patricia; Tapia, Ricardo

    2002-09-01

    Overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors is closely related to epilepsy and excitotoxicity, and the phosphorylation of these receptors may facilitate glutamate-mediated synaptic transmission. Here we show that in awake rats the microinjection into the hippocampus of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, induces in about 20 min intense electroencephalographic and behavioral limbic-type seizures, which are suppressed by the systemic administration of the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine hydrogen maleate and by the intrahippocampal administration of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinases. Two hours after okadaic acid, when the EEG seizures were intense, an increased serine phosphorylation of some hippocampal proteins, including an enhancement of the serine phosphorylation of the NMDA receptor subunit NR2B, was detected by immunoblotting. Twenty-four hours after okadaic acid a marked destruction of hippocampal CA1 region was observed, which was not prevented by the receptor antagonists. These findings suggest that hyperphosphorylation of glutamate receptors in vivo may result in an increased sensitivity to the endogenous transmitter and therefore induce neuronal hyperexcitability and epilepsy. PMID:12429230

  1. Identification and characterization of 4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate, an orally bioavailable, brain penetrant NR2B selective N-methyl-D-aspartate receptor antagonist.

    PubMed

    Liverton, Nigel J; Bednar, Rodney A; Bednar, Bohumil; Butcher, John W; Claiborne, Christopher F; Claremon, David A; Cunningham, Michael; DiLella, Anthony G; Gaul, Stanley L; Libby, Brian E; Lyle, Elizabeth A; Lynch, Joseph J; McCauley, John A; Mosser, Scott D; Nguyen, Kevin T; Stump, Gary L; Sun, Hong; Wang, Hao; Yergey, James; Koblan, Kenneth S

    2007-02-22

    The discovery of a novel series of NR2B subtype selective N-methyl-d-aspartate (NMDA) antagonists is reported. Initial optimization of a high-throughput screening lead afforded an aminopyridine derivative 13 with significant NR2B antagonist potency but limited selectivity over hERG-channel and other off-target activities. Further structure-activity studies on the aminoheterocycle moiety and optimization of the carbamate led to the highly potent 2-aminopyrimidine derivative 20j with a significantly improved off-target activity profile and oral bioavailability in multiple species coupled with good brain penetration. Compound 20j demonstrated efficacy in in vivo rodent models of antinociception, allodynia, and Parkinson's disease. PMID:17249648

  2. Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors.

    PubMed

    Chaturvedi, K; Bandari, P; Chinen, N; Howells, R D

    2001-04-13

    This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors. PMID:11152677

  3. 12-O-Tetradecanoyl phorbol-13-acetate (TPA)-induced growth arrest is increased by silibinin by the down-regulation of cyclin B1 and cdc2 and the up-regulation of p21 expression in MDA-MB231 human breast cancer cells.

    PubMed

    Kim, Sangmin; Lee, Hye Sook; Lee, Se-Kyung; Kim, Sung Hoon; Hur, Sung Mo; Kim, Jee Soo; Kim, Jung-Han; Choe, Jun-Ho; Shin, Incheol; Yang, Jung-Hyun; Lee, Jeong Eon; Nam, Seok Jin

    2010-12-01

    TPA is a potent regulator of cell growth, including cell proliferation and differentiation. In this study, we determined the effect of silibinin on TPA-induced growth arrest in breast cancer cells. Silibinin increased growth arrest of the G2/M phase in a dose-dependent fashion. Silibinin decreased the basal level of cyclin B1 and cdc2 expression, which is involved in S phase and G2/M transition. In addition, TPA-induced G2/M phase arrest was increased by silibinin. Under the same conditions, TPA-induced down-regulation of cyclin B1 and cdc2 was decreased by silibinin. In contrast, TPA-induced p21 expression was further increased by silibinin. To determine the regulatory mechanism of TPA-induced growth arrest, we pretreated cells with various inhibitors, such as UO126, SB203580, and LY294002. Interestingly, TPA-induced growth arrest was significantly increased by LY294002, but not by UO126 and SB203580. In addition, TPA-induced down-regulation of cyclin B1 was inhibited by LY294002; however, the basal level of p21 was increased by TPA and TPA-induced p21 expression was further increased by LY294002. Finally, adenoviral constitutively active-Akt (Ad-CA-Akt) overexpression regulated the up-regulation of cyclin B1 and the down-regulation of p21. Therefore, we have demonstrated that silibinin has an additive effect on TPA-induced growth arrest through the PI-3-kinase/Akt-dependent pathway. PMID:20554189

  4. An 8-gene signature, including methylated and down-regulated glutathione peroxidase 3, of gastric cancer.

    PubMed

    Zhang, Xianglan; Yang, Jung-Jin; Kim, Yoon Sun; Kim, Ki-Yeol; Ahn, Woong Shick; Yang, Sanghwa

    2010-02-01

    We have identified an 8-gene signature with significant expression differences between gastric cancer and normal gastric tissues. This 8-gene set can predict the normal and cancer status of gastric tissues with more than 96% accuracy in a totally independent microarray dataset. The 8 genes are composed of down-regulated KLF4, GPX3, SST and LIPF, together with up-regulated SERPINH1, THY1 and INHBA in gastric cancer. To corroborate the differential gene expression pattern, we chose GPX3 and examined its expression pattern in detail. A comparison of GPX3 expression pattern shows a broader down-regulated pattern in multiple types of cancers, including cervical, thyroid, head and neck, lung cancers and melanoma than in healthy controls. An immuno-histostaining analysis in tissue microarrays confirms GPX3 down-regulation in gastric cancer. Mechanism-wise GPX3 down-regulation in gastric cancer is due to promoter hypermethylation. Collectively, these results show a correct identification of 8 genes as gastric cancer biomarkers. PMID:20043075

  5. Behavioural Assessment of the A2a/NR2B Combination in the Unilateral 6-OHDA-Lesioned Rat Model: A New Method to Examine the Therapeutic Potential of Non-Dopaminergic Drugs.

    PubMed

    Michel, Anne; Downey, Patrick; Van Damme, Xavier; De Wolf, Catherine; Schwarting, Rainer; Scheller, Dieter

    2015-01-01

    In Parkinson's disease (PD), dopaminergic therapies are often associated with the development of motor complications. Attention has therefore been focused on the use of non-dopaminergic drugs. This study developed a new behavioural method capable of demonstrating the added value of combining adenosinergic and glutamatergic receptor antagonists in unilateral 6-OHDA lesioned rats. Rats were dosed orally with Tozadenant, a selective A2A receptor antagonist, and three different doses of Radiprodil, an NR2B-selective NMDA receptor antagonist. The drugs were given alone or in combination and rats were placed in an open-field for behavioural monitoring. Video recordings were automatically analysed. Five different behaviours were scored: distance traveled, ipsi- and contraversive turns, body position, and space occupancy. The results show that A2A or NR2B receptor antagonists given alone or in combination did not produce enhanced turning as observed with an active dose of L-Dopa/benserazide. Instead the treated rats maintained a straight body position, were able to shift from one direction to the other and occupied a significantly larger space in the arena. The highest "Tozadenant/Radiprodil" dose combination significantly increased all five behavioural parameters recorded compared to rats treated with vehicle or the same doses of the drugs alone. Our data suggest that the A2A/NR2B antagonist combination may be able to stimulate motor activity to a similar level as that achieved by L-Dopa but in the absence of the side-effects that are associated with dopaminergic hyperstimulation. If these results translate into the clinic, this combination could represent an alternative symptomatic treatment option for PD. PMID:26322641

  6. Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42, and RhoA GTPases and activation of the PI3K/Akt and JNK signaling pathways.

    PubMed

    Lv, Qi; Zhu, Xian-Yang; Xia, Yu-Feng; Dai, Yue; Wei, Zhi-Feng

    2015-11-01

    Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 μmol·L(-1)) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK. PMID:26614458

  7. Tryptanthrin ameliorates atopic dermatitis through down-regulation of TSLP.

    PubMed

    Han, Na-Ra; Moon, Phil-Dong; Kim, Hyung-Min; Jeong, Hyun-Ja

    2014-01-15

    Atopic dermatitis (AD) is a common skin disease that greatly worsens quality of life. Thymic stromal lymphopoietin (TSLP) plays a decisive role in the development of AD. The purpose of this study is to examine whether tryptanthrin (TR) would suppress AD through the regulation of TSLP. We analyzed the effect of TR on the level of TSLP from phorbol myristate acetate/calcium ionophore A23187-activated human mast cell line, HMC-1 cells, in 2,4-dinitrofluorobenzene-induced AD-like skin lesions of NC/Nga mice, and in anti-CD3/anti-CD28-stimulated splenocytes. TR significantly suppressed the level of intracellular calcium and the production and mRNA expression of TSLP through the blockade of receptor-interacting protein 2/caspase-1/nuclear factor-κB pathway in the activated HMC-1 cells. TR also significantly suppressed the levels of histidine decarboxylase and IL-1β. Furthermore, TR ameliorated clinical symptoms in the AD model. TR significantly reduced the levels of TSLP, IL-4, IFN-γ, IL-6, TNF-α, thymus and activation-regulated chemokine, and caspase-1 in AD skin lesions. Also, TR significantly reduced the serum levels of histamine and IL-4 in the AD model. Finally, TR significantly inhibited the production of IL-4, IFN-γ, and TNF-α from the stimulated splenocytes. Taken together, TR exhibits the potential to be a therapeutic agent for AD through down-regulation of TSLP. PMID:24295961

  8. SAMHD1 is down regulated in lung cancer by methylation and inhibits tumor cell proliferation

    SciTech Connect

    Wang, Jia-lei; Lu, Fan-zhen; Shen, Xiao-Yong; Wu, Yun; Zhao, Li-ting

    2014-12-12

    Highlights: • SAMHD1 expression level is down regulated in lung adenocarcinoma. • The promoter of SAMHD1 is methylated in lung adenocarcinoma. • Over expression of SAMHD1 inhibits the proliferation of lung cancer cells. - Abstract: The function of dNTP hydrolase SAMHD1 as a viral restriction factor to inhibit the replication of several viruses in human immune cells was well established. However, its regulation and function in lung cancer have been elusive. Here, we report that SAMHD1 is down regulated both on protein and mRNA levels in lung adenocarcinoma compared to adjacent normal tissue. We also found that SAMHD1 promoter is highly methylated in lung adenocarcinoma, which may inhibit its gene expression. Furthermore, over expression of the SAMHD1 reduces dNTP level and inhibits the proliferation of lung tumor cells. These results reveal the regulation and function of SAMHD1 in lung cancer, which is important for the proliferation of lung tumor cells.

  9. [The down-regulation of miR-125b-5p and up-regulation of Smad4 expression in human umbilical vein endothelial cells treated with hydrogen peroxide].

    PubMed

    Wei, Ming; Gan, Lu; Yang, Xiaomei; Jiang, Xiangyang; Liu, Jiahong; Chen, Lihong

    2016-08-01

    Objective To explore the expressions and functions of miR-155, miR-125b-5p, miR-210, and their target proteins, in vascular endothelial cells in oxidative stress. Methods Human umbilical vein endothelial cells (HUVECs) were isolated and cultured; the expression of endothelial cell factor VIII related antigen was detected by immunofluorescence staining; and the expression of CD31 was detected and identified by flow cytometry. The oxidative stress model of vascular endothelial cells was established by preeclampsia serum and hydrogen peroxide (H2O2) treatment. The treated HUVECs were divided into 4 groups: normal pregnancy serum group, preeclampsia serum group, 0 μmol/L H2O2 group and 100 μmol/L H2O2 group. The levels of miR-155, miR-125b-5p, and miR-210 were tested by real-time quantitative PCR within 24 hours after modeling. The expression of Smad4 protein was detected by Western blotting and immunofluorescence staining. Cell apoptosis was detected by flow cytometry combined with annexin V-FITC/PI staining. Results Compared with 0 μmol/L H2O2 group, miR-125b-5p significantly decreased in 100 μmol/L H2O2 group, while Smad4 protein significantly increased. In addition, 100 μmol/L H2O2 treatment promoted cell apoptosis and necrosis. Compared with normal pregnancy serum group, the apoptosis and necrosis were significantly enhanced in preeclampsia serum group, but there were no significant differences in miR-125b-5p and Smad4 between the groups. Conclusion High level of H2O2 up-regulates Smad4 protein expression and inhibits miR-125b-5p expression. PMID:27412941

  10. A novel cognitive impairment mechanism that astrocytic p-connexin 43 promotes neuronic autophagy via activation of P2X7R and down-regulation of GLT-1 expression in the hippocampus following traumatic brain injury in rats.

    PubMed

    Sun, Liqian; Gao, Junling; Zhao, Manman; Cui, Jianzhong; Li, Youxiang; Yang, Xinjian; Jing, Xiaobin; Wu, Zhongxue

    2015-09-15

    Connexin 43 (Cx43) is one of the major gap junction proteins in astrocytes. Our previous studies reported that astrocytic phosphorylated Cx43 (p-CX43) regulated neuronic autophagy levels in the rat hippocampus after traumatic brain injury (TBI). In this study, we explored the underlying molecular mechanism by which gap junctional intercellular communication influenced neuronic autophagy and therefore initiated cognitive and memory impairments after TBI. The gap junctional blocker carbenoxolone (CBX) or autophagy inhibitor 3-methyladenine (3-MA) reduced latencies, as compared to TBI rats. Similarly, CBX or 3-MA restored long-term potentiation (LTP), relative to TBI hippocampal slices. Immunoblotting analysis showed that the expression of autophagy-related gene Beclin-1 in the hippocampus post-TBI were decreased in response to treatment with CBX, the P2X7 receptor (P2X7R) antagonist Oxidized ATP (OxATP) or ceftriaxone (Cef) which increased the expression and activity of the glutamate transporter (GLT-1) in the central nervous system (CNS). Moreover, CBX or OxATP pretreatment increased GLT-1 level in the rat hippocampus after TBI. However, CBX pretreatment suppressed P2X7R expression whereas maintained P2X7 level post-TBI. Confocal images revealed that p-CX43, P2X7 and GLT-1 strongly colocalized with glial fibrillary acidic protein (GFAP). Taken together, these results implied that Cx43, might induce neuronic autophagy by activation of P2X7R and reduce the expression of GLT-1 in the hippocampus, promoting TBI-induced cognitive deficits repair. Therefore, control of this communication may be serve as therapeutic strategies for intervention against TBI. PMID:26031379

  11. Amelioration of Diabetic Mouse Nephropathy by Catalpol Correlates with Down-Regulation of Grb10 Expression and Activation of Insulin-Like Growth Factor 1 / Insulin-Like Growth Factor 1 Receptor Signaling

    PubMed Central

    Yang, Shasha; Deng, Huacong; Zhang, Qunzhou; Xie, Jing; Zeng, Hui; Jin, Xiaolong; Ling, Zixi; Shan, Qiaoyun; Liu, Momo; Ma, Yuefei; Tang, Juan; Wei, Qianping

    2016-01-01

    Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that can negatively regulate the insulin-like growth factor 1 receptor (IGF-1R). The IGF1-1R pathway is critical for cell growth and apoptosis and has been implicated in kidney diseases; however, it is still unknown whether Grb10 expression is up-regulated and plays a role in diabetic nephropathy. Catalpol, a major active ingredient of a traditional Chinese medicine, Rehmannia, has been reported to possess anti-inflammatory and anti-aging activities and then used to treat diabetes. Herein, we aimed to assess the therapeutic effect of catalpol on a mouse model diabetic nephropathy and the potential role of Grb10 in the pathogenesis of this diabetes-associated complication. Our results showed that catalpol treatment improved diabetes-associated impaired renal functions and ameliorated pathological changes in kidneys of diabetic mice. We also found that Grb10 expression was significantly elevated in kidneys of diabetic mice as compared with that in non-diabetic mice, while treatment with catalpol significantly abrogated the elevated Grb10 expression in diabetic kidneys. On the contrary, IGF-1 mRNA levels and IGF-1R phosphorylation were significantly higher in kidneys of catalpol-treated diabetic mice than those in non-treated diabetic mice. Our results suggest that elevated Grb10 expression may play an important role in the pathogenesis of diabetic nephropathy through suppressing IGF-1/IGF-1R signaling pathway, which might be a potential molecular target of catalpol for the treatment of this diabetic complication. PMID:26986757

  12. 2,3,5,6-Tetramethylpyrazine (TMP) down-regulated arsenic-induced heme oxygenase-1 and ARS2 expression by inhibiting Nrf2, NF-κB, AP-1 and MAPK pathways in human proximal tubular cells.

    PubMed

    Gong, Xuezhong; Ivanov, Vladimir N; Hei, Tom K

    2016-09-01

    Our recent study demonstrated that sodium arsenite at a clinically relevant dose induced nephrotoxicity in human renal proximal tubular epithelial cell line HK-2, which could be inhibited by natural product 2,3,5,6-tetramethylpyrazine (TMP) with antioxidant activity. The present study demonstrated that arsenic exposure resulted in protein and enzymatic induction of heme oxygenase-1 (HO-1) in dose- and time-dependent manners in HK-2 cells. Blocking HO-1 enzymatic activity by zinc protoporphyrin (ZnPP) augmented arsenic-induced apoptosis, ROS production and mitochondrial dysfunction, suggesting a critical role for HO-1 as a renal protectant in this procession. On the other hand, TMP, upstream of HO-1, inhibited arsenic-induced ROS production and ROS-dependent HO-1 expression. TMP also prevented mitochondria dysfunction and suppressed activation of the intrinsic apoptotic pathway in HK-2 cells. Our results revealed that the regulation of arsenic-induced HO-1 expression was performed through multiple ROS-dependent signal pathways and the corresponding transcription factors, including p38 MAPK and JNK (but not ERK), AP-1, Nrf2 and NF-κB. TMP inhibited arsenic-induced activations of JNK, p38 MAPK, ERK, AP-1 and Nrf2 and block HO-1 protein expression. The present study, furthermore, demonstrated arsenic-induced expression of arsenic response protein 2 (ARS2) that was regulated by p38 MAPK, ERK and NF-κB. To our knowledge, this is the first report showing that ARS2 involved in arsenic-induced nephrotoxicity, while TMP pretreatment prevented such an up-regulation of ARS2 in HK-2 cells. Given ARS2 and HO-1 sharing the similar regulation mechanism, we speculated that ARS2 might also mediate cell survival in this procession. In summary, our study highlighted a role of HO-1 in the protection against arsenic-induced cytotoxicity downstream from the primary targets of TMP and further indicated that TMP may be used as a potential therapeutic agent in the treatment of arsenic

  13. Tannic acid down-regulates the angiotensin type 1 receptor through a MAPK-dependent mechanism.

    PubMed

    Yesudas, Rekha; Gumaste, Upendra; Snyder, Russell; Thekkumkara, Thomas

    2012-03-01

    In the present study, we investigated the effects of tannic acid (TA), a hydrolysable polyphenol, on angiotensin type 1 receptor (AT1R) expression in continuously passaged rat liver epithelial cells. Under normal conditions, exposure of cells to TA resulted in the down-regulation of AT1R-specific binding in concentrations ranging from 12.5-100 μg/ml (7.34-58.78 μm) over a time period of 2-24 h with no change in receptor affinity to angiotensin II (AngII). The inhibitory effect of TA on AT1R was specific and reversible. In TA-treated cells, we observed a significant reduction in AngII-mediated intracellular calcium signaling, a finding consistent with receptor down-regulation. Under similar conditions, TA down-regulated AT1R mRNA expression without changing the rate of mRNA degradation, suggesting that TA's effect is mediated through transcriptional inhibition. Cells expressing recombinant AT1R without the native promoter show no change in receptor expression, whereas a pCAT reporter construct possessing the rat AT1R promoter was significantly reduced in activity. Furthermore, TA induced the phosphorylation of MAPK p42/p44. Pretreatment of the cells with a MAPK kinase (MEK)-specific inhibitor PD98059 prevented TA-induced MAPK phosphorylation and down-regulation of the AT1R. Moreover, there was no reduction in AngII-mediated intracellular calcium release upon MEK inhibition, suggesting that TA's observed inhibitory effect is mediated through MEK/MAPK signaling. Our findings demonstrate, for the first time, that TA inhibits AT1R gene expression and cellular response, suggesting the observed protective effects of dietary polyphenols on cardiovascular conditions may be, in part, through inhibition of AT1R expression. PMID:22322600

  14. Ghrelin inhibits oxLDL-induced inflammation in RAW264.7 mouse macrophages through down-regulation of LOX-1 expression via NF-κB signaling pathway.

    PubMed

    Sun, N; Wang, H; Wang, L

    2016-01-01

    Oxidized low-density lipoprotein (oxLDL) is one of the many causes of the initiation and progression of atherosclerosis, which can subsequently promote the uptake of oxLDL by macrophages and lead to inflammation in the blood vessels. In the present study, we evaluated the protective effects of ghrelin on oxLDL-induced RAW264.7 mouse macrophages. Ghrelin was able to inhibit the release of several pro-inflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-6. In addition, ghrelin also inhibited the expression of Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in oxLDL treated macrophages. Furthermore, we demonstrated that ghrelin could inhibit the expression of p-IκBα, and the inhibitory effects could be blocked by BAY 117082. Taken together, ghrelin possesses anti-inflammatory effects on oxLDL-induced inflammation in macrophages, suggesting that it can prevent or treat atherosclerosis, and deserves to be further studied and developed to be potent drug for treating atherosclerosis. PMID:26950452

  15. Down-regulation of PERK enhances resistance to ionizing radiation

    SciTech Connect

    Oommen, Deepu Prise, Kevin M.

    2013-11-08

    Highlights: •PERK enhances the sensitivity of cancer cells to ionizing radiation. •Down-regulation of PERK results in enhanced DNA repair. •Ionizing radiation-induced apoptosis is inhibited in PERK-down regulated cancer cells. -- Abstract: Although, ionizing radiation (IR) has been implicated to cause stress in endoplasmic reticulum (ER), how ER stress signaling and major ER stress sensors modulate cellular response to IR is unclear. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER transmembrane protein which initiates unfolded protein response (UPR) or ER stress signaling when ER homeostasis is disturbed. Here, we report that down-regulation of PERK resulted in increased clonogenic survival, enhanced DNA repair and reduced apoptosis in irradiated cancer cells. Our study demonstrated that PERK has a role in sensitizing cancer cells to IR.

  16. Impaired hippocampal synaptic plasticity and NR2A/2B expression ratio in remifentanil withdrawal rats.

    PubMed

    Wang, Yi-Yi; Liu, Shichang; Zhang, Nan; Yang, Jing; Zhang, Yinguo

    2016-03-01

    Remifentanil is a kind of synthetic opioid which has gained wide clinical acceptance by anesthesiologists. In this study, we attempted to test whether withdrawal effects on learning mechanisms can be triggered by repeated low-dose remifentanil treatment. Male Sprague-Dawley (SD) rats were subjected to remifentanil (50μg/kgs.c.) twice per day at 12h intervals for 15 days. When the animals of remifentanil group were withdrawn from remifentanil at 10h after the last injection, changes in open field test, Morris water maze test (MWM) and synaptic efficacy were examined in each group. We demonstrated that repeated exposure to 50μg/kg remifentanil produced enhanced locomotor activity indicating that a remifentanil addiction animal model in rats was established. MWM results showed that exposure to remifentanil had no influence on the spatial cognition. After withdrawal of remifentanil rats showed impaired spatial cognition. In electrophysiology test, remifentanil group rats showed a trend for a rightward shift of input/output relationship and significant deficits in maintenance of STP and LTP. Immunohistochemistry results demonstrated increased NR2A/NR2B ratio that should be included depression of LTP. In the whole-cell patch-clamp recording, after elimination from remifentanil incubation, mEPSC frequency was down regulated in hippocampal CA1 neurons, indicating that basal synaptic transmission were affected by remifentanil withdrawal. Taken together, the current findings demonstrate that the remifentanil withdrawn rats exhibit obvious impairment of hippocampus-dependent memory and synaptic plasticity. Increased hippocampal NR2A/NR2B expression ratio and the changes of basal synaptic transmission may participate in the impairment of LTP. PMID:26777139

  17. Myxoma virus M141R expresses a viral CD200 (vOX-2) that is responsible for down-regulation of macrophage and T-cell activation in vivo.

    PubMed

    Cameron, Cheryl M; Barrett, John W; Liu, Liying; Lucas, Alexandra R; McFadden, Grant

    2005-05-01

    M141R is a myxoma virus gene that encodes a cell surface protein with significant amino acid similarity to the family of cellular CD200 (OX-2) proteins implicated in the regulation of myeloid lineage cell activation. The creation of an M141R deletion mutant myxoma virus strain (vMyx141KO) and its subsequent infection of European rabbits demonstrated that M141R is required for the full development of a lethal infection in vivo but is not required for efficient virus replication in susceptible cell lines in vitro. Minor secondary sites of infection were detected in the majority of rabbits infected with the M141R deletion mutant, demonstrating that the M141R protein is not required for the dissemination of virus within the host. When compared to wild-type myxoma virus-infected rabbits, vMyx141KO-infected rabbits showed higher activation levels of both monocytes/macrophages and lymphocytes in situ through assessments of inducible nitric oxide synthase-positive and CD25(+) infiltrating cells in infected and lymphoid tissues. Purified peripheral blood mononuclear cells from vMyx141KO-infected rabbits demonstrated an increased ability to express gamma interferon upon activation by phorbol myristate acetate plus ionomycin compared to cells purified from wild-type myxoma virus-infected rabbits. We concluded that the M141R protein is a bona fide CD200-like immunomodulator protein which is required for the full pathogenesis of myxoma virus in the European rabbit and that its loss from the virus results in increased activation levels of macrophages in infected lesions and draining lymph nodes as well as an increased activation level of circulating T lymphocytes during infection. We propose a model whereby M141R transmits inhibitory signals to tissue macrophages, and possibly resident CD200R(+) dendritic cells, that reduce their ability to antigenically prime lymphocytes and possibly provides anergic signals to T cells directly. PMID:15857991

  18. Myxoma Virus M141R Expresses a Viral CD200 (vOX-2) That Is Responsible for Down-Regulation of Macrophage and T-Cell Activation In Vivo

    PubMed Central

    Cameron, Cheryl M.; Barrett, John W.; Liu, Liying; Lucas, Alexandra R.; McFadden, Grant

    2005-01-01

    M141R is a myxoma virus gene that encodes a cell surface protein with significant amino acid similarity to the family of cellular CD200 (OX-2) proteins implicated in the regulation of myeloid lineage cell activation. The creation of an M141R deletion mutant myxoma virus strain (vMyx141KO) and its subsequent infection of European rabbits demonstrated that M141R is required for the full development of a lethal infection in vivo but is not required for efficient virus replication in susceptible cell lines in vitro. Minor secondary sites of infection were detected in the majority of rabbits infected with the M141R deletion mutant, demonstrating that the M141R protein is not required for the dissemination of virus within the host. When compared to wild-type myxoma virus-infected rabbits, vMyx141KO-infected rabbits showed higher activation levels of both monocytes/macrophages and lymphocytes in situ through assessments of inducible nitric oxide synthase-positive and CD25+ infiltrating cells in infected and lymphoid tissues. Purified peripheral blood mononuclear cells from vMyx141KO-infected rabbits demonstrated an increased ability to express gamma interferon upon activation by phorbol myristate acetate plus ionomycin compared to cells purified from wild-type myxoma virus-infected rabbits. We concluded that the M141R protein is a bona fide CD200-like immunomodulator protein which is required for the full pathogenesis of myxoma virus in the European rabbit and that its loss from the virus results in increased activation levels of macrophages in infected lesions and draining lymph nodes as well as an increased activation level of circulating T lymphocytes during infection. We propose a model whereby M141R transmits inhibitory signals to tissue macrophages, and possibly resident CD200R+ dendritic cells, that reduce their ability to antigenically prime lymphocytes and possibly provides anergic signals to T cells directly. PMID:15857991

  19. Biotic Stress Globally Down-Regulates Photosynthesis Genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Upon herbivore and pathogen attacks, plants switch from processes supporting growth and reproduction to defense by inducing a set of defense genes and down-regulating most of the nuclear encoded photosynthetic genes. To determine if this transcriptional response is universal we used transcriptome da...

  20. CHIP mediates down-regulation of nucleobindin-1 in preosteoblast cell line models.

    PubMed

    Xue, Fuying; Wu, Yanping; Zhao, Xinghui; Zhao, Taoran; Meng, Ying; Zhao, Zhanzhong; Guo, Junwei; Chen, Wei

    2016-08-01

    Nucleobindin-1 (NUCB1), also known as Calnuc, is a highly conserved, multifunctional protein widely expressed in tissues and cells. It contains two EF-hand motifs which have been shown to play a crucial role in binding Ca(2+) ions. In this study, we applied comparative two-dimensional gel electrophoresis to characterize differentially expressed proteins in HA-CHIP over-expressed and endogenous CHIP depleted MC3T3-E1 stable cell lines, identifying NUCB1 as a novel CHIP/Stub1 targeted protein. NUCB1 interacts with and is down-regulated by CHIP by both proteasomal dependent and independent pathways, suggesting that CHIP-mediated down-regulation of nucleobindin-1 might play a role in osteoblast differentiation. The chaperone protein Hsp70 was found to be important for CHIP and NUCB1 interaction as well as CHIP-mediated NUCB1 down-regulation. Our findings provide new insights into understanding the stability regulation of NUCB1. PMID:27178152

  1. Down-regulation of SOSTDC1 promotes thyroid cancer cell proliferation via regulating cyclin A2 and cyclin E2

    PubMed Central

    He, Xiaoying; Ke, Weijian; Xu, Lijuan; Liu, Liehua; Xiao, Haipeng; Li, Yanbing

    2015-01-01

    Sclerostin domain containing protein 1 (SOSTDC1) is down-regulated and acts as a tumor suppressor in some kinds of cancers. However, the expression pattern and biological significance of SOSTDC1 in thyroid cancer are largely unknown. We demonstrated that SOSTDC1 was significantly down-regulated in thyroid cancer. Ectopic over-expression of SOSTDC1 inhibited proliferation and induced G1/S arrest in thyroid cancer cells. Moreover, SOSTDC1 over-expression suppressed the growth of tumor xenografts in nude mice. We also found that elevated SOSTDC1 led to inhibition of cyclin A2 and cyclin E2. Together, our results demonstrate that SOSTDC1 is down-regulated in thyroid cancer and might be a potential therapeutic target in the treatment of thyroid cancer. PMID:26378658

  2. DNA methylation down-regulates EGFR expression in chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The epidermal growth factor receptor (EGFR), a growth-factor-receptor tyrosine kinase, was found up-regulated in numerous tumors, which provides a good target for cancer therapy. Although it was documented that oncoviruses are responsible for the activation of EGFR in tumors, the impact of Marek’s d...

  3. Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer

    PubMed Central

    Wendt, Michael K.; Taylor, Molly A.; Schiemann, Barbara J.; Schiemann, William P.

    2011-01-01

    Reduced epithelial cadherin (E-cad) is a hallmark of invasive carcinomas that have acquired epithelial-mesenchymal transition (EMT) phenotypes. Here we show that down-regulated E-cad expression induced by transforming growth factor-β (TGF-β) and EMT preceded breast cancer outgrowth in three-dimensional (3D) organotypic assays and in the lungs of mice. Pharmacological inhibitors against focal adhesion kinase prevented metastatic outgrowth of newly seeded organoids, but not that of their fully established counterparts. Interrogating the D2-HAN (hyperplastic alveolar nodule) model of breast cancer dormancy and metastasis showed that dormant D2.OR cells produced branched organoid morphologies in 3D-cultures, and expressed robust quantities of E-cad that was uncoupled from regulation by TGF-β. In contrast, metastatic D2.A1 organoids were spherical and wholly lacked E-cad expression. Interestingly, D2.A1 cells engineered to re-express E-cad formed branched organoids, down-regulated β1 integrin expression, and failed to undergo metastatic outgrowth. The tumor-suppressing function of E-cad was inactivated by increased microenvironmental rigidity, and was not recapitulated by expression of an E-cad mutant lacking its extracellular domain. Twist expression, but not that of Snail, reinitiated metastatic outgrowth in dormant D2.OR cells. Our findings show that EMT and its down-regulated expression of E-cad circumvent breast cancer dormancy in part by facilitating β1 integrin expression necessary for metastatic outgrowth. PMID:21613543

  4. Down-regulation of lipoprotein lipase increases glucose uptake in L6 muscle cells

    SciTech Connect

    Lopez, Veronica; Saraff, Kumuda; Medh, Jheem D.

    2009-11-06

    Thiazolidinediones (TZDs) are synthetic hypoglycemic agents used to treat type 2 diabetes. TZDs target the peroxisome proliferator activated receptor-gamma (PPAR-{gamma}) and improve systemic insulin sensitivity. The contributions of specific tissues to TZD action, or the downstream effects of PPAR-{gamma} activation, are not very clear. We have used a rat skeletal muscle cell line (L6 cells) to demonstrate that TZDs directly target PPAR-{gamma} in muscle cells. TZD treatment resulted in a significant repression of lipoprotein lipase (LPL) expression in L6 cells. This repression correlated with an increase in glucose uptake. Down-regulation of LPL message and protein levels using siRNA resulted in a similar increase in insulin-dependent glucose uptake. Thus, LPL down-regulation improved insulin sensitivity independent of TZDs. This finding provides a novel method for the management of insulin resistance.

  5. Antigenic modulation and down regulation-their similarities suggest they may have a common origin.

    PubMed

    Cohen, E P

    1980-01-01

    Antigenic modulation, the antibody-induced disappearance of membrane-associated determinants of neoplastic cells, has a fundamental similarity to the down regulation of receptors for hormones and other ligands affecting the physiology of various nonneoplastic cell types. After hormone stimulation, cells previously responsive become refractory to further stimulation. Diminution in responsiveness correlates with a quantitative reduction in the numbers of specific complementary hormone receptors present. Murine leukemia cells, as one example "escape" antibody-mediated destruction through the mechanism of antigenic modulation. Resistance of the cells correlates with a reduction of the numbers of specific antigens present. In both down regulation and modulation, continued cellular metabolism in the absence of ligand leads to a re-expression of the hormone receptor/membrane antigen involved. It is postulated that the two phenomena have evolved from similar underlying cellular mechanisms. PMID:6256059

  6. Down-regulation of the desmosomal cadherin desmocollin 3 in human breast cancer.

    PubMed

    Klus, G T; Rokaeus, N; Bittner, M L; Chen, Y; Korz, D M; Sukumar, S; Schick, A; Szallasi, Z

    2001-07-01

    In previous studies using cDNA microarray analysis, we have identified an expressed sequence tag which is consistently down-regulated in six human breast tumor cell lines. In the current study, we have determined this tag to be part of the mRNA sequence of human desmocollin 3, a member of the cadherin superfamily of proteins and an integral component of desmosomes. Desmosomes are sites of adhesion between adjacent cells in layers of epithelia, as well as in some non-epithelial tissues, and play an important role in the maintenance of tissue structure. Northern analysis, quantitative real-time polymerase chain reaction assay and Western blot analysis showed that desmocollin 3 is present in normal and immortalized human mammary epithelial cells, but consistently exhibits a significant, and often complete, down-regulation in breast cancer cell lines and primary breast tumors, both at the mRNA and protein levels. PMID:11408939

  7. Dipeptides Inhibit Melanin Synthesis in Mel-Ab Cells through Down-Regulation of Tyrosinase

    PubMed Central

    Lee, Hyun-e; Kim, Eun-Hyun; Choi, Hye-Ryung; Sohn, Uy Dong; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Park, Kyoung-Chan

    2012-01-01

    This study investigated the effects of proline-serine (PS) and valine-serine (VS) dipeptides on melanogenesis in Mel-Ab cells. Proline-serine and VS significantly inhibited melanin synthesis in a concentration-dependent manner, though neither dipeptide directly inhibited tyrosinase activity in a cell-free system. Both PS and VS down-regulated the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. In a follow-up study also described here, the effects of these dipeptides on melanogenesis-related signal transduction were quantified. Specifically, PS and VS induced ERK phosphorylation, though they had no effect on phosphorylation of the cAMP response element binding protein (CREB). These data suggest that PS and VS inhibit melanogenesis through ERK phosphorylation and subsequent down-regulation of MITF and tyrosinase. Properties of these dipeptides are compatible with application as skin-whitening agents. PMID:22915995

  8. Prodigiosin down-regulates survivin to facilitate paclitaxel sensitization in human breast carcinoma cell lines

    SciTech Connect

    Ho, T.-F.; Peng, Y.-T.; Chuang, S.-M.; Lin, S.-C.; Feng, B.-L.; Lu, C.-H.; Yu, W.-J.; Chang, J.-S. Chang, C.-C.

    2009-03-01

    Prodigiosin is a bacterial metabolite with potent anticancer activity, which is attributed to its proapoptotic effect selectively active in malignant cells. Still, the molecular mechanisms whereby prodigiosin induces apoptosis remain largely unknown. In particular, the role of survivin, a vital inhibitor of apoptosis, in prodigiosin-induced apoptosis has never been addressed before and hence was the primary goal of this study. Our results showed that prodigiosin dose-dependently induced down-regulation of survivin in multiple breast carcinoma cell lines, including MCF-7, T-47D and MDA-MB-231. This down-regulation is mainly regulated at the level of transcription, as prodigiosin reduced the levels of both survivin mRNA and survivin promoter activity but failed to rescue survivin expression when proteasome-mediated degradation is abolished. Importantly, overexpression of survivin rendered cells more resistant to prodigiosin, indicating an essential role of survivin down-regulation in prodigiosin-induced apoptosis. In addition, we found that prodigiosin synergistically enhanced cell death induced by paclitaxel, a chemotherapy drug known to up-regulate survivin that in turn confers its own resistance. This paclitaxel sensitization effect of prodigiosin is ascribed to the lowering of survivin expression, because prodigiosin was shown to counteract survivin induction by paclitaxel and, notably, the sensitization effect was severely abrogated in cells that overexpress survivin. Taken together, our results argue that down-regulation of survivin is an integral component mediating prodigiosin-induced apoptosis in human breast cancer cells, and further suggest the potential of prodigiosin to sensitize anticancer drugs, including paclitaxel, in the treatment of breast cancer.

  9. Down-regulation of microRNA-144 in air pollution-related lung cancer

    PubMed Central

    Pan, Hong-Li; Wen, Zhe-Sheng; Huang, Yun-Chao; Cheng, Xin; Wang, Gui-Zhen; Zhou, Yong-Chun; Wang, Zai-Yong; Guo, Yong-Qing; Cao, Yi; Zhou, Guang-Biao

    2015-01-01

    Air pollution has been classified as a group 1 carcinogen in humans, but the underlying tumourigenic mechanisms remain unclear. In Xuanwei city of Yunnan Province, the lung cancer incidence is among the highest in China, owing to severe air pollution generated by the combustion of smoky coal, providing a unique opportunity to dissect lung carcinogenesis. To identify abnormal miRNAs critical for air pollution-related tumourigenesis, we performed microRNA microarray analysis in 6 Xuanwei non-small cell lung cancers (NSCLCs) and 4 NSCLCs from control regions where smoky coal was not used. We found 13 down-regulated and 2 up-regulated miRNAs in Xuanwei NSCLCs. Among them, miR-144 was one of the most significantly down-regulated miRNAs. The expanded experiments showed that miR-144 was down-regulated in 45/51 (88.2%) Xuanwei NSCLCs and 34/54 (63%) control region NSCLCs (p = 0.016). MiR-144 interacted with the oncogene Zeb1 at 2 sites in its 3′ untranslated region, and a decrease in miR-144 resulted in increased Zeb1 expression and an epithelial mesenchymal transition phenotype. Ectopic expression of miR-144 suppressed NSCLCs in vitro and in vivo by targeting Zeb1. These results indicate that down-regulation of miR-144 is critical for air pollution-related lung cancer, and the miR-144-Zeb1 signalling pathway could represent a potential therapeutic target. PMID:26395400

  10. Photosynthesis down-regulation precedes carbohydrate accumulation under sink limitation in Citrus.

    PubMed

    Nebauer, Sergio G; Renau-Morata, Begoña; Guardiola, José Luis; Molina, Rosa-Victoria

    2011-02-01

    Photosynthesis down-regulation due to an imbalance between sources and sinks in Citrus leaves could be mediated by excessive accumulation of carbohydrates. However, there is limited understanding of the physiological role of soluble and insoluble carbohydrates in photosynthesis regulation and the elements triggering the down-regulation process. In this work, the role of non-structural carbohydrates in the regulation of photosynthesis under a broad spectrum of source-sink relationships has been investigated in the Salustiana sweet orange. Soluble sugar and starch accumulation in leaves, induced by girdling experiments, did not induce down-regulation of the photosynthetic rate in the presence of sinks (fruits). The leaf-to-fruit ratio did not modulate photosynthesis but allocation of photoassimilates to the fruits. The lack of strong sink activity led to a decrease in the photosynthetic rate and starch accumulation in leaves. However, photosynthesis down-regulation due to an excess of total soluble sugars or starch was discarded because photosynthesis and stomatal conductance reduction occurred prior to any significant accumulation of these carbohydrates. Gas exchange and fluorescence parameters suggested biochemical limitations to photosynthesis. In addition, the expression of carbon metabolism-related genes was altered within 24 h when strong sinks were removed. Sucrose synthesis and export genes were inhibited, whereas the expression of ADP-glucose pyrophosphorylase was increased to cope with the excess of assimilates. In conclusion, changes in starch and soluble sugar turnover, but not sugar content per se, could provide the signal for photosynthesis regulation. In these conditions, non-stomatal limitations strongly inhibited the photosynthetic rate prior to any significant increase in carbohydrate levels. PMID:21367744

  11. Down-regulation of Bcl-2-interacting protein BAG-1 confers resistance to anti-cancer drugs.

    PubMed

    Takahashi, Noriko; Yanagihara, Miyako; Ogawa, Yuzi; Yamanoha, Banri; Andoh, Toshiwo

    2003-02-14

    BAG-1 was originally identified as a binding partner of anti-apoptotic factor Bcl-2 [Takayama et al., Cell 80 (1995) 279-284]. Exogenous expression of BAG-1 was reported to confer cells resistance to several stresses [Chen et al., Oncogene 21 (2002) 7050]. We have obtained human cervical cancer HeLa cells with down-regulated BAG-1 levels by using a highly specific and efficient RNA interference approach. Surprisingly, cells with down-regulated BAG-1 exhibited significantly lower sensitivity against several anti-cancer drugs than parental cells expressing normal levels of the protein. Furthermore, growth rate of the cells was reduced when BAG-1 was down-regulated. Activity of ERK pathway appeared to be decreased in BAG-1 down-regulated cells, as shown by the reduced phosphorylation of ERK1/2 proteins. Taken together resistance against anti-cancer drugs acquired by BAG-1 down-regulated cells may well be accounted for by the retardation of cell cycle progression, implicating the importance of BAG-1 in cell growth regulation. PMID:12565851

  12. Pseudomonas syringae Effector Avirulence Protein E Localizes to the Host Plasma Membrane and Down-Regulates the Expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 Gene Required for Antibacterial Immunity in Arabidopsis1[OPEN

    PubMed Central

    Xin, Xiu-Fang; Nomura, Kinya; Ding, Xinhua; Chen, Xujun; Wang, Kun; Aung, Kyaw; Uribe, Francisco; Rosa, Bruce; Yao, Jian; Chen, Jin; He, Sheng Yang

    2015-01-01

    Many bacterial pathogens of plants and animals deliver effector proteins into host cells to promote infection. Elucidation of how pathogen effector proteins function not only is critical for understanding bacterial pathogenesis but also provides a useful tool in discovering the functions of host genes. In this study, we characterized the Pseudomonas syringae pv tomato DC3000 effector protein Avirulence Protein E (AvrE), the founding member of a widely distributed, yet functionally enigmatic, bacterial effector family. We show that AvrE is localized in the plasma membrane (PM) and PM-associated vesicle-like structures in the plant cell. AvrE contains two physically interacting domains, and the amino-terminal portion contains a PM-localization signal. Genome-wide microarray analysis indicates that AvrE, as well as the functionally redundant effector Hypersensitive response and pathogenicity-dependent Outer Protein M1, down-regulates the expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13) gene in Arabidopsis (Arabidopsis thaliana). Mutational analysis shows that NHL13 is required for plant immunity, as the nhl13 mutant plant displayed enhanced disease susceptibility. Our results defined the action site of one of the most important bacterial virulence proteins in plants and the antibacterial immunity function of the NHL13 gene. PMID:26206852

  13. Down-regulation of SIRT3 promotes ovarian carcinoma metastasis.

    PubMed

    Dong, Xue-Cai; Jing, Li-Min; Wang, Wen-Xiang; Gao, Yu-Xia

    2016-07-01

    Distant metastasis and local recurrence are still the major causes for failure of treatment in patients with ovarian carcinoma (OC), making it urgent to further elicit the molecular mechanisms of OC metastasis. Sirtuin-3 (SIRT3), a member of the NAD(+)-dependent Class III histone deacetylases, may function as different role depending on the cell-type and tumor-type. However, the function and mechanism of SIRT3 has been not explored in OC metastasis. Here, we found that SIRT3 was significantly down-regulated in the metastatic tissues and highly metastatic cell line of ovarian cancer. In addition, knockdown of SIRT3 enhanced the migration and invasion in vitro and the liver metastasis in vivo of ovarian cancer cell. By contrast, ectopic overexpression of SIRT3 dramatically suppressed cancer cell metastatic capability. Mechanistically, SIRT3 inhibits epithelial-to-mesenchymal transition (EMT) by down-regulating Twist in ovarian cancer cells. Furthermore, an interaction between SIRT3 and Twist was detected. In conclusion, our results demonstrated that SIRT3 plays a crucial suppressive role in the metastasis of ovarian cancer by down-regulating Twist, and that this novel SIRT3/Twist axis may be valuable to develop new strategies for treating OC patients with metastasis. PMID:27216459

  14. Down-regulation of telomerase activity by anticancer drugs in human ovarian cancer cells.

    PubMed

    Kunifuji, Yasumasa; Gotoh, Sadao; Abe, Tetsuya; Miura, Masayoshi; Karasaki, Yuji

    2002-07-01

    Maintenance of telomere length is crucial for survival of cells. Telomerase, an enzyme that is responsible for elongation of shortened telomeres, is active in human germ cells as well as most tumor tissues and experimentally immortalized cells. In contrast, most mature somatic cells in human tissues express undetectable or low telomerase activity, implying the existence of a stringent and negative regulatory mechanism. In this study we report the effects of anticancer drugs on telomerase activity in human cancer cells. In assaying for telomerase activity, we basically followed the original TRAP assay system, but with some modifications. A down-regulation of telomerase activity was found when cells of a human ovarian cancer cell line, A2780, were treated with;cis-diamminedichloroplatinum(II) (CDDP; cisplatin). However, down-regulation of telomerase activity was not found in cells of a cisplatin-resistant cell line, A2780CP, treated with cisplatin. On the other hand, telomerase activity in both the cell lines A2780 and A2780CP was reduced when A2780 or A2780CP was treated with adriamycin, an anthracycline antibiotic having a broad spectrum of antineoplastic activity. The different effects on the telomerase activity of the two types of anticancer drugs may be due the distinct chemical functions of these drugs. The present results may indicate a positive relationship between anticancer effects and down-regulation of telomerase activity by anticancer drugs. PMID:12172504

  15. Requirements for Cell Rounding and Surface Protein Down-Regulation by Ebola Virus Glycoprotein

    PubMed Central

    Francica, Joseph R.; Matukonis, Meghan K.; Bates, Paul

    2009-01-01

    Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of β1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects. PMID:19013626

  16. Mechanisms of allele-selective down-regulation of HLA class I in Burkitt's lymphoma.

    PubMed

    Imreh, M P; Zhang, Q J; de Campos-Lima, P O; Imreh, S; Krausa, P; Browning, M; Klein, G; Masucci, M G

    1995-07-01

    Burkitt lymphomas (BL) that arise in HLA-AII-positive individuals are characterized by selective loss/down-regulation of the HLA AII polypeptide. We have investigated the molecular basis of such down-regulation by comparing 5 pairs of BL lines and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) derived from the normal B cells of the same individuals. The presence of apparently intact HLA AII genes was confirmed in all 5 BL/LCL pairs by polymerase chain reaction (PCR) typing and by Southern-blot hybridization with HLA A locus-specific probes. Northern-blot analysis with locus- and allele-specific probes revealed a significantly lower expression or absence of AII-specific mRNA in all 5 BL lines compared to the corresponding LCLs. Up-regulation of AII-specific mRNA was achieved by IFN alpha treatment of 2 BL lines with low HLA AII expression (BL-28 and BL-72) while the treatment had no effect in 3 BL lines (WWI-BL, WW2-BL and BL41) that did not express the endogenous gene. HLA AII expression was restored by transfection of the gene in WWI-BL whereas transfectants of BL-41 remained AII-negative. An HLA-AII-promoter-driven chloramphenicol acetyl transferase reporter gene (pAIICAT) was active in WWI-BL but not in BL-41. HLA-AII was expressed in hybrids of BL-41 with an AII-positive LCL, while expression of the endogenous HLA AII gene could not be restored by fusion of BL-41 with an AII-negative LCL, although an adequate set of transcription factors was present in the hybrid. Our results suggest that genetic defects and lack of transcription factors may contribute to the selective down-regulation of HLA AII in BL cells. PMID:7601573

  17. [Down-regulation of TIPE2 promotes the proliferation and immune activity of T lymphocytes].

    PubMed

    Huang, Lihong; Chen, Jiangyong; Hong, Bin

    2016-07-01

    Objective To utilize specific small interfering RNA (siRNA) to silence the expression of tumor necrosis factor α-induced protein 8 like-2 (TIPE2) gene of T lymphocytes and investigate the effect of TIPE2 targeting siRNA on T lymphocyte proliferation and immune function. Methods Mouse spleen T lymphocytes were sorted by magnetic beads. Western blotting was used to screen and validate an effective siRNA to silence the TIPE2 gene expression of T lymphocytes. Twenty-four hours after transfection with the siRNA into T lymphocytes, the expression of CD69 in each group was detected by flow cytometry. Seventy-two hours after transfection, the proliferation of the T lymphocytes was measured with CCK-8 assay; meanwhile, the secretion levels of interleukin 2 (IL-2) and interferon γ (IFN-γ) in each group were measured by ELISA. Results We obtained TIPE2 targeting siRNA sequences and effectively silenced the expression of TIPE2 gene. After TIPE2 gene expression was down-regulated, the expression of the CD69 on T lymphocytes increased, and the proliferation of T lymphocytes and the secretion of IL-2 and IFN-γ were enhanced. Conclusion Down-regulation of TIPE2 gene expression can promote the T lymphocyte proliferation and immune activity. PMID:27363266

  18. Involvement of the MEK/ERK pathway in EGF-induced E-cadherin down-regulation.

    PubMed

    Tashiro, Etsu; Henmi, Shizuka; Odake, Hiroyuki; Ino, Seitaro; Imoto, Masaya

    2016-09-01

    E-cadherin is a major component of the epithelial adherens junction. However, the regulatory mechanism of E-cadherin expression is still poorly understood. In this study, we found that EGF decreased E-cadherin expression at both mRNA and protein levels in colorectal carcinoma LoVo cells. Since E-cadherin down-regulation is a well-known hallmark of the EMT (Epithelial-Mesenchymal Transition), we investigated whether EGF induced E-cadherin down-regulation during the EMT. EGF was unable to affect the expression of mesenchymal markers (such as N-cadherin, vimentin or fibronectin) or EMT-regulating transcription factors (such as SNAIL, SLUG, ZEB1, ZEB2 or TWIST), suggesting that EGF induced E-cadherin down-regulation via an EMT-independent mechanism. On the other hand, the MEK inhibitor U0126 was found to suppress EGF-induced E-cadherin down-regulation at the transcriptional level, suggesting that the MEK/ERK pathway is involved in EGF-induced E-cadherin down-regulation. Moreover, we also found that EGF disrupted cell-cell contact, stimulated cells to form an elongated shape with filamentous protrusions, and induced cell migration in LoVo cells. These effects were suppressed by U0126. Therefore, EGF is suggested to induce E-cadherin down-regulation at the transcriptional level through the MEK/ERK pathway, which might result in, at least in part, the induction of cellular morphological changes and cell migration in LoVo cells. PMID:27369075

  19. Mechanism of Wnt signaling induced down regulation of mrhl long non-coding RNA in mouse spermatogonial cells

    PubMed Central

    Akhade, Vijay Suresh; Dighe, Shrinivas Nivrutti; Kataruka, Shubhangini; Rao, Manchanahalli R. Satyanarayana

    2016-01-01

    Long non coding RNAs (lncRNAs) have emerged as important regulators of various biological processes. LncRNAs also behave as response elements or targets of signaling pathway(s) mediating cellular function. Wnt signaling is important in regulating mammalian spermatogenesis. Mrhl RNA negatively regulates canonical Wnt pathway and gets down regulated upon Wnt signaling activation in mouse spermatogonial cells. Also, mrhl RNA regulates expression of genes pertaining to Wnt pathway and spermatogenesis by binding to chromatin. In the present study, we delineate the detailed molecular mechanism of Wnt signaling induced mrhl RNA down regulation in mouse spermatogonial cells. Mrhl RNA has an independent transcription unit and our various experiments like Chromatin Immunoprecipitation (in cell line as well as mouse testis) and shRNA mediated down regulation convincingly show that β-catenin and TCF4, which are the key effector proteins of the Wnt signaling pathway are required for down regulation of mrhl RNA. We have identified Ctbp1 as the co-repressor and its occupancy on mrhl RNA promoter depends on both β-catenin and TCF4. Upon Wnt signaling activation, Ctbp1 mediated histone repression marks increase at the mrhl RNA promoter. We also demonstrate that Wnt signaling induced mrhl RNA down regulation results in an up regulation of various meiotic differentiation marker genes. PMID:26446991

  20. Mechanism of Wnt signaling induced down regulation of mrhl long non-coding RNA in mouse spermatogonial cells.

    PubMed

    Akhade, Vijay Suresh; Dighe, Shrinivas Nivrutti; Kataruka, Shubhangini; Rao, Manchanahalli R Satyanarayana

    2016-01-01

    Long non coding RNAs (lncRNAs) have emerged as important regulators of various biological processes. LncRNAs also behave as response elements or targets of signaling pathway(s) mediating cellular function. Wnt signaling is important in regulating mammalian spermatogenesis. Mrhl RNA negatively regulates canonical Wnt pathway and gets down regulated upon Wnt signaling activation in mouse spermatogonial cells. Also, mrhl RNA regulates expression of genes pertaining to Wnt pathway and spermatogenesis by binding to chromatin. In the present study, we delineate the detailed molecular mechanism of Wnt signaling induced mrhl RNA down regulation in mouse spermatogonial cells. Mrhl RNA has an independent transcription unit and our various experiments like Chromatin Immunoprecipitation (in cell line as well as mouse testis) and shRNA mediated down regulation convincingly show that β-catenin and TCF4, which are the key effector proteins of the Wnt signaling pathway are required for down regulation of mrhl RNA. We have identified Ctbp1 as the co-repressor and its occupancy on mrhl RNA promoter depends on both β-catenin and TCF4. Upon Wnt signaling activation, Ctbp1 mediated histone repression marks increase at the mrhl RNA promoter. We also demonstrate that Wnt signaling induced mrhl RNA down regulation results in an up regulation of various meiotic differentiation marker genes. PMID:26446991

  1. CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

    PubMed Central

    He, Jin; Zhao, Long; Wang, Xiaodong; Li, Zhennan; Qian, Jianjun

    2016-01-01

    Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein which participates in inhibiting tumor apoptosis in pancreatic cancer cells. Using immunohistochemical staining, we investigated the expression of CIP2A protein in 72 cases of human pancreatic ductal adenocarcinoma (PDAC) tissue and 27 cases of adjacent normal pancreatic tissue. The positive rate of CIP2A protein expression in pancreatic cancer tissue was70.83 %, which was significantly higher than that in adjacent non- cancerous pancreatic tissue (11.11%). The expression of CIP2A was found to be correlated with TNM stage, but not correlated with age, gender, tumor location, smoking status, alcohol consumption, diabetes, high blood pressure, BMI, tumor size, lymph node metastasis or distant metastases. Kaplan- Meier survival analysis showed that patients with positive CIP2A protein expression had a lower overall survival rate than patients without CIP2A expression. COX regression analysis indicated that expression of CIP2A was an independent prognostic factor for pancreatic ductal adenocarcinoma. In addition, down-regulation of CIP2A inhibited cell proliferation and increased sensitivity to gemcitabine in pancreatic cancer cells by decreasing AKT signaling pathway. Our results indicated that down-regulation of CIP2A could be a novel therapeutic strategy for pancreatic cancer. PMID:26894380

  2. The siRNA-Mediated Down-Regulation of Vascular Endothelial Growth Factor Receptor1

    PubMed Central

    Jafari Sani, Moslem; Yazdi, Foad; Masoomi Karimi, Masoomeh; Alizadeh, Javad; Rahmati, Majid; Zarei Mahmudabadi, Ali

    2016-01-01

    Background Angiogenesis is an important biological process involved in the proliferation of endothelial cells, tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is considered as a prominent regulator of angiogenesis which exerts the aforementioned effect(s) through its respective receptors (VEGFR1 and VEGFR2). VEGF receptors are targeted as a therapeutic candidate for cancer growth inhibition. RNAi as a new and promising strategy has provided a useful means to specifically suppress gene expression in cancer cells. Objectives The current study aimed to down-regulate expression of the VEGFR1 using siRNA. Materials and Methods This experimental study designed specific siRNAs against VEGFR1. Total RNA was extracted from human umbilical vain endothelial cell (HUVEC) and subsequently cDNA was synthetized. PCR was performed using specific primers to amplify the target gene. After double digestion and purification, the gene was cloned into pEFGP-N1 expression vector. Then, AGS cells were transfected with recombinant pEGFP-N1 using lipofectamin. The gene expression and down-regulation were evaluated by fluorescence scanning, reverse transcription PCR (RT-PCR) and Western blot techniques. Results Fluorescent scanning, RT-PCR (27.68%) and western blot analysis (31.06%) showed that the expression of VEGFR1 was suppressed effectively. Conclusions The results of the current study showed that specifically designed siRNA can be considered as an appropriate strategy to suppress gene expression and might be a promising tool to prevent angiogenesis. PMID:27275397

  3. Mild Hypothermia Combined with Hydrogen Sulfide Treatment During Resuscitation Reduces Hippocampal Neuron Apoptosis Via NR2A, NR2B, and PI3K-Akt Signaling in a Rat Model of Cerebral Ischemia-Reperfusion Injury.

    PubMed

    Dai, Hai-Bin; Xu, Miao-Miao; Lv, Jia; Ji, Xiang-Jun; Zhu, Si-Hai; Ma, Ru-Meng; Miao, Xiao-Lei; Duan, Man-Lin

    2016-09-01

    We investigated whether mild hypothermia combined with sodium hydrosulfide treatment during resuscitation improves neuron survival following cerebral ischemia-reperfusion injury beyond that observed for the individual treatments. Male Sprague-Dawley rats were divided into seven groups (n = 20 for each group). All rats underwent Pulsinelli 4-vessel occlusion. Ischemia was induced for 15 min using ligatures around the common carotid arteries, except for the sham group. Immediately after initiating reperfusion, the mild hypothermia (MH), sodium hydrosulfide (NaHS), hydroxylamine (HA), MH + NaHS, MH + HA, and ischemia-reperfusion (I/R) control groups received an intraperitoneal injection of saline, sodium hydrosulfide, hydroxylamine, sodium hydrosulfide, hydroxylamine, and saline, respectively, and mild hypothermia (32 to 33 °C) was induced in the MH, MH + NaHS, and MH + HA groups for 6 h. The levels of NR2A, NR2B, p-Akt, and p-Gsk-3β in the hippocampus of the MH, NaHS, and MH + NaHS groups were higher than those in the I/R control group, with the highest levels observed in the MH + NaHS group (P < 0.05). Treatment with hydroxylamine reduced the levels of these proteins in the HA and MH + HA groups, compared with the I/R control and MH groups, respectively. The apoptotic index of the CA1 region of the hippocampus was 45.2, 66.5, 63.5, and 84.8 % in the MH + NaHS, MH, NaHS, and I/R control groups, respectively (P < 0.05), indicating that the combination treatment shifted the NR2A/NR2B balance in favor of synaptic neuron stimulation and phosphatidylinositol 3'-kinase (PI3K)/Akt signaling. The combination of mild hypothermia and sodium hydrosulfide treatment for resuscitation following ischemia-reperfusion injury was more beneficial for reducing hippocampal apoptosis and pathology than that of mild hypothermia or hydrogen sulfide treatment alone. PMID:26350917

  4. AMPA receptors serum-dependently mediate GABAA receptor alpha1 and alpha6 subunit down-regulation in cultured mouse cerebellar granule cells.

    PubMed

    Uusi-Oukari, Mikko; Kontturi, Leena-Stiina; Kallinen, Sampsa A; Salonen, Virpi

    2010-04-01

    Depolarization of cultured mouse cerebellar granule cells with potassium or kainate results in developmentally arrested state that includes down-regulation of GABA(A) receptor alpha1, alpha6 and beta2 subunit expression. These subunits are normally strongly expressed in cerebellar granule cells from second postnatal week throughout the adulthood. In the present study we demonstrate that selective activation of AMPA subtype of glutamate receptors down-regulates alpha1 and alpha6 subunit mRNA expression. Removal of AMPA agonist from culture medium restores expression of these subunits indicating reversibility of the down-regulation. In serum-free culture medium AMPA receptor activation did not down-regulate alpha1 or alpha6 subunit expression. Furthermore, the down-regulation was strongly attenuated when the cells were cultured in the presence of dialysed fetal calf serum. The results indicate that down-regulation of GABA(A) receptor alpha1 and alpha6 subunits by AMPA receptor activation is dependent on the presence of low molecular weight compounds present in fetal calf serum. In order to study mouse cerebellar granule cell maturation and/or regulation of GABA(A) receptor subunit expression in culture, the experiments should be performed in the absence of fetal calf serum. PMID:20170697

  5. Luteinizing Hormone Receptor mRNA Down-Regulation Is Mediated through ERK-Dependent Induction of RNA Binding Protein

    PubMed Central

    Menon, Bindu; Franzo-Romain, Megan; Damanpour, Shadi

    2011-01-01

    The ligand-induced down-regulation of LH receptor (LHR) expression in the ovaries, at least in part, is regulated by a posttranscriptional process mediated by a specific LH receptor mRNA binding protein (LRBP). The LH-mediated signaling pathways involved in this process were examined in primary cultures of human granulosa cells. Treatment with 10 IU human chorionic gonadotropin (hCG) for 12 h resulted in the down-regulation of LHR mRNA expression while producing an increase in LHR mRNA binding to LRBP as well as a 2-fold increase in LRBP levels. The activation of ERK½ pathway in LH-mediated LHR mRNA down-regulation was also established by demonstrating the translocation of ERK½ from the cytosol to the nucleus using confocal microcopy. Inhibition of protein kinase A using H-89 or ERK½ by U0126 abolished the LH-induced LHR mRNA down-regulation. These treatments also abrogated both the increases in LRBP levels as well as the LHR mRNA binding activity. The abolishment of the hCG-induced increase in LRBP levels and LHR mRNA binding activity was further confirmed by transfecting granulosa cells with ERK½ specific small interfering RNA. This treatment also reversed the hCG-induced down-regulation of LHR mRNA. These data show that LH-regulated ERK½ signaling is required for the LRBP-mediated down-regulation of LHR mRNA. PMID:21147848

  6. Down-Regulation of Rad51 and Decreased Homologous Recombination in Hypoxic Cancer Cells

    PubMed Central

    Bindra, Ranjit S.; Schaffer, Paul J.; Meng, Alice; Woo, Jennifer; Måseide, Kårstein; Roth, Matt E.; Lizardi, Paul; Hedley, David W.; Bristow, Robert G.; Glazer, Peter M.

    2004-01-01

    There is an emerging concept that acquired genetic instability in cancer cells can arise from the dysregulation of critical DNA repair pathways due to cell stresses such as inflammation and hypoxia. Here we report that hypoxia specifically down-regulates the expression of RAD51, a key mediator of homologous recombination in mammalian cells. Decreased levels of Rad51 were observed in multiple cancer cell types during hypoxic exposure and were not associated with the cell cycle profile or with expression of hypoxia-inducible factor. Analyses of RAD51 gene promoter activity, as well as mRNA and protein stability, indicate that the hypoxia-mediated regulation of this gene occurs via transcriptional repression. Decreased expression of Rad51 was also observed to persist in posthypoxic cells for as long as 48 h following reoxygenation. Correspondingly, we found reduced levels of homologous recombination in both hypoxic and posthypoxic cells, suggesting that the hypoxia-associated reduction in Rad51 expression has functional consequences for DNA repair. In addition, hypoxia-mediated down-regulation of Rad51 was confirmed in vivo via immunofluorescent image analysis of experimental tumors in mice. Based on these findings, we propose a novel mechanism of genetic instability in the tumor microenvironment mediated by hypoxia-induced suppression of the homologous recombination pathway in cancer cells. The aberrant regulation of Rad51 expression may also create heterogeneity in the DNA damage response among cells within tumors, with implications for the response to cancer therapies. PMID:15367671

  7. Role of EGF receptor ligands in TCDD-induced EGFR down-regulation and cellular proliferation.

    PubMed

    Campion, Christina M; Leon Carrion, Sandra; Mamidanna, Gayatri; Sutter, Carrie Hayes; Sutter, Thomas R; Cole, Judith A

    2016-06-25

    In cultures of normal human epidermal keratinocytes (NHEKs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces the expression of the epidermal growth factor receptor ligands transforming growth factor-α (TGF-α) and epiregulin (EREG). TCDD also down-regulates EGF receptors (EGFR), suggesting that decreases in signaling contribute to the effects of TCDD. In this study, we treated post-confluent NHEKs with 10 nM TCDD and assessed its effects on EGFR binding, EGFR ligand secretion, basal ERK activity, and proliferation. TCDD caused time-dependent deceases in [(125)I]-EGF binding to levels 78% of basal cell values at 72 h. Amphiregulin (AREG) levels increased with time in culture in basal and TCDD-treated cells, while TGF-α and epiregulin (EREG) secretion were stimulated by TCDD. Inhibiting EGFR ligand release with the metalloproteinase inhibitor batimastat prevented EGFR down-regulation and neutralizing antibodies for AREG and EREG relieved receptor down-regulation. In contrast, neutralizing TGF-α intensified EGFR down-regulation. Treating NHEKs with AREG or TGF-α caused rapid internalization of receptors with TGF-α promoting recycling within 90 min. EREG had limited effects on rapid internalization or recycling. TCDD treatment increased ERK activity, a response reduced by batimastat and the neutralization of all three ligands indicating that the EGFR and its ligands maintain ERK activity. All three EGFR ligands were required for the maintenance of total cell number in basal and TCDD-treated cultures. The EGFR inhibitor PD1530305 blocked basal and TCDD-induced increases in the number of cells labeled by 5-ethynyl-2'-deoxyuridine, identifying an EGFR-dependent pool of proliferating cells that is larger in TCDD-treated cultures. Overall, these data indicate that TCDD-induced EGFR down-regulation in NHEKs is caused by AREG, TGF-α, and EREG, while TGF-α enhances receptor recycling to maintain a pool of EGFR at the cell surface. These receptors are required for

  8. Down-regulation of Wnt10a affects odontogenesis and proliferation in mesenchymal cells

    SciTech Connect

    Liu, Yang Han, Dong Wang, Lei Feng, Hailan

    2013-05-17

    Highlights: •Down-regulation of Wnt10a in dental mesenchymal cells impairs odontogenesis of reassociated tooth germs. •Dspp is down- and up-regulated after Wnt10a-knockdown and overexpression in dental mesenchymal cells. •Down-regulation of Wnt10a inhibits proliferation of dental mesenchymal cells. -- Abstract: The WNT10a mutation has been found in patients with abnormal odontogenesis. In mice, Wnt10a expression is found in the tooth germ, but its role has not yet been elucidated. We aimed to investigate the role of Wnt10a in odontogenesis. Mesenchymal cells of the first mandibular molar germ at the bell stage were isolated, transfected with Wnt10a SiRNA or plasmid, and reassociated with epithelial part of the molar germ. Scrambled SiRNA or empty vector was used in the control group. The reassociated tooth germs were transplanted into mice subrenal capsules. After gene modification, dental mesenchymal cells cultured in vitro were checked for cell proliferation and the expression of Dspp was examined. All 12 reassociated tooth germs in the control group resumed odontogenesis, while only 5 of 12 in the Wnt10a knockdown group developed into teeth. After Wnt10a knockdown, the mesenchymal cells cultured in vitro presented repressed proliferation. Wnt10a knockdown and overexpression led to both down- and up-regulation of Dspp. We conclude that the down-regulation of Wnt10a impairs odontogensis and cell proliferation, and that Wnt10a regulates Dspp expression in mesenchymal cells. These findings help to elucidate the mechanism of abnormal tooth development in patients with the WNT10A mutation.

  9. Serotonin-induced down-regulation of cell surface serotonin transporter

    PubMed Central

    Jørgensen, Trine Nygaard; Christensen, Peter Møller; Gether, Ulrik

    2014-01-01

    The serotonin transporter (SERT) terminates serotonergic signaling and enables refilling of synaptic vesicles by mediating reuptake of serotonin (5-HT) released into the synaptic cleft. The molecular and cellular mechanisms controlling SERT activity and surface expression are not fully understood. Here we demonstrate that the substrate 5-HT itself causes acute down-regulation of SERT cell surface expression. To assess surface SERT expression by ELISA, we used a SERT variant (TacSERT) where the N-terminus of SERT was fused to the intracellular tail of the extracellularly FLAG-tagged single-membrane spanning protein Tac. In stably transfected HEK293 cells, 5-HT caused a dose-dependent reduction in TacSERT surface signal with an EC50 value equivalent to the Km value observed for 5-HT uptake. The 5-HT-induced reduction in surface signal reached maximum within 40-60 min and was blocked by the selective SERT inhibitor S-citalopram. 5-HT-induced reduction in SERT expression was further supported by surface biotinylation experiments showing 5-HT-induced reduction in wild type SERT plasma membrane levels. Moreover, preincubation with 5-HT lowered the Vmax for 5-HT uptake in cultured raphe serotonergic neurons, indicting that endogenous cell-surface resident SERT likewise is down-regulated in the presence of substrate. PMID:24462583

  10. Dysregulation of Ack1 inhibits down-regulation of the EGF receptor

    SciTech Connect

    Grovdal, Lene Melsaether; Johannessen, Lene E.; Rodland, Marianne Skeie; Madshus, Inger Helene; Stang, Espen

    2008-04-01

    The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of {sup 125}I-EGF internalization, whereas internalization of {sup 125}I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized {sup 125}I-EGF was increased, while degradation of {sup 125}I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.

  11. LPS Down-Regulates Specificity Protein 1 Activity by Activating NF-κB Pathway in Endotoxemic Mice

    PubMed Central

    Ye, Xiaobing; Liu, Hong; Gong, Yong-Sheng; Liu, Shu Fang

    2015-01-01

    Background Specificity protein (Sp) 1 mediates the transcription of a large number of constitutive genes encoding physiological mediators. NF-κB mediates the expression of hundreds of inducible genes encoding pathological mediators. Crosstalk between Sp1 and NF-κB pathways could be pathophysiologically significant, but has not been studied. This study examined the crosstalk between the two pathways and defined the role of NF-κB signaling in LPS-induced down-regulation of Sp1 activity. Methods and Main Findings Challenge of wild type mice with samonelia enteritidis LPS (10 mg/kg, i.p.) down-regulated Sp1 binding activity in lungs in a time-dependent manner, which was concomitantly associated with an increased NF-κB activity. LPS down-regulates Sp1 activity by inducing an LPS inducible Sp1-degrading enzyme (LISPDE) activity, which selectively degrades Sp1 protein, resulting in Sp1 down-regulation. Blockade of NF-κB activation in mice deficient in NF-κB p50 gene (NF-κB-KO) suppressed LISPDE activity, prevented Sp1 protein degradation, and reversed the down-regulation of Sp1 DNA binding activity and eNOS expression (an indicator of Sp1 transactivation activity). Inhibition of LISPDE activity using a selective LISPDE inhibitor mimicked the effects of NF-κB blockade. Pretreatment of LPS-challenged WT mice with a selective LISPDE inhibitor increased nuclear Sp1 protein content, restored Sp1 DNA binding activity and reversed eNOS protein down-regulation in lungs. Enhancing tissue level of Sp1 activity by inhibiting NF-κB-mediated Sp1 down-regulation increased tissue level of IL-10 and decreased tissue level of TNF- αin the lungs. Conclusions NF-κB signaling mediates LPS-induced down-regulation of Sp1 activity. Activation of NF-κB pathway suppresses Sp1 activity and Sp1-mediated anti-inflammatory signals. Conversely, Sp1 signaling counter-regulates NF-κB-mediated inflammatory response. Crosstalk between NF-κB and Sp1 pathways regulates the balance between pro

  12. Curcumin and Emodin Down-Regulate TGF-β Signaling Pathway in Human Cervical Cancer Cells

    PubMed Central

    Thacker, Pooja Chandrakant; Karunagaran, Devarajan

    2015-01-01

    Cervical cancer is the major cause of cancer related deaths in women, especially in developing countries and Human Papilloma Virus infection in conjunction with multiple deregulated signaling pathways leads to cervical carcinogenesis. TGF-β signaling in later stages of cancer is known to induce epithelial to mesenchymal transition promoting tumor growth. Phytochemicals, curcumin and emodin, are effective as chemopreventive and chemotherapeutic compounds against several cancers including cervical cancer. The main objective of this work was to study the effect of curcumin and emodin on TGF-β signaling pathway and its functional relevance to growth, migration and invasion in two cervical cancer cell lines, SiHa and HeLa. Since TGF-β and Wnt/β-catenin signaling pathways are known to cross talk having common downstream targets, we analyzed the effect of TGF-β on β-catenin (an important player in Wnt/β-catenin signaling) and also studied whether curcumin and emodin modulate them. We observed that curcumin and emodin effectively down regulate TGF-β signaling pathway by decreasing the expression of TGF-β Receptor II, P-Smad3 and Smad4, and also counterbalance the tumorigenic effects of TGF-β by inhibiting the TGF-β-induced migration and invasion. Expression of downstream effectors of TGF-β signaling pathway, cyclinD1, p21 and Pin1, was inhibited along with the down regulation of key mesenchymal markers (Snail and Slug) upon curcumin and emodin treatment. Curcumin and emodin were also found to synergistically inhibit cell population and migration in SiHa and HeLa cells. Moreover, we found that TGF-β activates Wnt/β-catenin signaling pathway in HeLa cells, and curcumin and emodin down regulate the pathway by inhibiting β-catenin. Taken together our data provide a mechanistic basis for the use of curcumin and emodin in the treatment of cervical cancer. PMID:25786122

  13. Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function

    SciTech Connect

    Yoshihara, Takashi; Collado, Denise; Hamaguchi, Masaaki . E-mail: hamaguchi@fordham.edu

    2007-07-13

    The expression of tumor suppressor gene DBC2 causes certain breast cancer cells to stop growing [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. Recently, DBC2 was found to participate in diverse cellular functions such as protein transport, cytoskeleton regulation, apoptosis, and cell cycle control [V. Siripurapu, J.L. Meth, N. Kobayashi, M. Hamaguchi, DBC2 significantly influences cell cycle, apoptosis, cytoskeleton, and membrane trafficking pathways. J. Mol. Biol. 346 (2005) 83-89]. Its tumor suppression mechanism, however, remains unclear. In this paper, we demonstrate that DBC2 suppresses breast cancer proliferation through down-regulation of Cyclin D1 (CCND1). Additionally, the constitutional overexpression of CCND1 prevented the negative impact of DBC2 expression on their growth. Under a CCND1 promoter, the expression of CCNE1 exhibited the same protective effect. Our results indicate that the down-regulation of CCND1 is an essential step for DBC2's growth suppression of cancer cells. We believe that this discovery contributes to a better understanding of DBC2's tumor suppressor function.

  14. Down-regulation of cyclooxygenase-2 (COX-2) by cannabidiolic acid in human breast cancer cells.

    PubMed

    Takeda, Shuso; Okazaki, Hiroyuki; Ikeda, Eriko; Abe, Satomi; Yoshioka, Yasushi; Watanabe, Kazuhito; Aramaki, Hironori

    2014-01-01

    Metastases are known to be responsible for approximately 90% of breast cancer-related deaths. Cyclooxygenase-2 (COX-2) is involved not only in inflammatory processes, but also in the metastasis of cancer cells; it is expressed in 40% of human invasive breast cancers. To comprehensively analyze the effects of cannabidiolic acid (CBDA), a selective COX-2 inhibitor found in the fiber-type cannabis plant (Takeda et al., 2008), on COX-2 expression and the genes involved in metastasis, we performed a DNA microarray analysis of human breast cancer MDA-MB-231 cells, which are invasive breast cancer cells that express high levels of COX-2, treated with CBDA for 48 hr at 25 µM. The results obtained revealed that COX-2 and Id-1, a positive regulator of breast cancer metastasis, were down-regulated (0.19-fold and 0.52-fold, respectively), while SHARP1 (or BHLHE41), a suppressor of breast cancer metastasis, was up-regulated (1.72-fold) and CHIP (or STUB1) was unaffected (1.03-fold). These changes were confirmed by real-time RT-PCR analyses. Taken together, the results obtained here demonstrated that i) CBDA had dual inhibitory effects on COX-2 through down-regulation and enzyme inhibition, and ii) CBDA may possess the ability to suppress genes that are positively involved in the metastasis of cancer cells in vitro. PMID:25242400

  15. Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

    SciTech Connect

    Silva, Patricio; Soto, Nicolás; Díaz, Jorge; Mendoza, Pablo; Díaz, Natalia; Quest, Andrew F.G.; Torres, Vicente A.

    2015-08-21

    The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation. - Highlights: • Rab5 is important to the maintenance of cell transformation characteristics. • Down-regulation of Rab5 decreases cell proliferation and increases apoptosis in different cancer cells. • Rab5 is required for anchorage-independent growth and tumorigenicity in-vivo.

  16. Transcriptional down regulation of the nov proto-oncogene in fibroblasts transformed by p60v-src.

    PubMed Central

    Scholz, G; Martinerie, C; Perbal, B; Hanafusa, H

    1996-01-01

    We have sought to identify genes whose expression is altered as a consequence of transformation by p60v-src. Using the mRNA differential display method, we have identified the nov proto-oncogene as one gene that is down regulated in chicken embryo fibroblasts (CEFs) transformed by p60v-src. nov transcripts were also found to be present at only very low levels in proliferating CEFs in comparison with quiescent CEFs. Serum stimulation of quiescent CEFs also resulted in a decline in the steady-state level of nov transcripts. Taken together, these findings suggest that the nov gene is expressed only in quiescent fibroblasts and that its down regulation may contribute to cellular transformation by the v-src oncogene. Down regulation of the nov gene appears to occur at both the transcriptional and posttranscriptional levels. Results obtained from experiments with a protein kinase inhibitor suggest that protein kinase C may be a key downstream effector in mediating the down regulation of nov transcripts in response to activation of p60src or serum stimulation. In addition, we found that transcription of an unknown gene is required for the decline in the steady-state level of nov transcripts in response to serum stimulation. PMID:8552074

  17. Down-regulation of TRPS1 stimulates epithelial-mesenchymal transition and metastasis through repression of FOXA1.

    PubMed

    Huang, Jin-Zhou; Chen, Min; Zeng, Ming; Xu, Song-Hui; Zou, Fei-Yan; Chen, De; Yan, Guang-Rong

    2016-06-01

    The tricho-rhino-phalangeal syndrome 1 gene (TRPS1), which was initially found to be associated with tricho-rhino-phalangeal syndrome, is critical for the development and differentiation of bone, hair follicles and kidney. However, its role in cancer progression is largely unknown. In this study, we demonstrated that down-regulation of TRPS1 correlated with distant metastasis, tumour recurrence and poor survival rate in cancer patients. TRPS1 was frequently down-regulated in high-metastatic cancer cell lines from the breast, colon and nasopharynx. Silencing of TRPS1 stimulated epithelial-mesenchymal transition (EMT), migration and invasion in vitro and metastasis in vivo, while TRPS1 over-expression exhibited the opposite effects. Using quantitative proteomics, FOXA1, a negative regulator of epithelial-mesenchymal transition (EMT), was shown to be down-regulated by TRPS1 knockdown. Ectopic expression of FOXA1 blocked the enhancement of EMT, migration and invasion induced by TRPS1 silencing. Mechanistically, TRPS1, acting as a transcription activator, directly induced FOXA1 transcription by binding to the FOXA1 promoter. We further showed that down-regulation of TRPS1 was induced by miR-373 binding to the 3' UTR of TRPS1. Over-expression of TRPS1, but not TRPS1 3' UTR, blocked the enhancement of migration and invasion induced by miR-373. Taken together, we consider that down-regulation of TRPS1 by miR-373, acting as a transcriptional activator, promotes EMT and metastasis by repressing FOXA1 transcription, expanding upon its previously reported role as a transcription repressor. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:26969828

  18. High glucose induces dysfunction of airway epithelial barrier through down-regulation of connexin 43.

    PubMed

    Yu, Hongmei; Yang, Juan; Zhou, Xiangdong; Xiao, Qian; Lü, Yang; Xia, Li

    2016-03-01

    The airway epithelium is a barrier to the inhaled antigens and pathogens. Connexin 43 (Cx43) has been found to play critical role in maintaining the function of airway epithelial barrier and be involved in the pathogenesis of the diabetic retinal vasculature, diabetes nephropathy and diabetes skin. Hyperglycemia has been shown to be an independent risk factor for respiratory infections. We hypothesize that the down-regulation of Cx43 induced by HG alters the expression of tight junctions (zonula occludens-1 (ZO-1) and occludin) and contributes to dysfunction of airway epithelial barrier, and Cx43 plays a critical role in the process in human airway epithelial cells (16 HBE). We show that high glucose (HG) decreased the expression of ZO-1 and occludin, disassociated interaction between Cx43 and tight junctions, and then increased airway epithelial transepithelial electrical resistance (TER) and permeability by down-regulation of Cx43 in human airway epithelial cells. These observations demonstrate an important role for Cx43 in regulating HG-induced dysfunction of airway epithelial barrier. These findings may bring new insights into the molecular pathogenesis of pulmonary infection related to diabetes mellitus and lead to novel therapeutic intervention for the dysfunction of airway epithelial barrier in chronic inflammatory airway diseases. PMID:26902399

  19. Tyrosine Kinase Inhibitors Induce Down-Regulation of c-Kit by Targeting the ATP Pocket

    PubMed Central

    Descarpentries, Clotilde; Frisan, Emilie; Adam, Kevin; Verdier, Frederique; Floquet, Célia; Dubreuil, Patrice; Lacombe, Catherine; Fontenay, Michaela; Mayeux, Patrick; Kosmider, Olivier

    2013-01-01

    The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket. PMID:23637779

  20. Traxoprodil, a selective antagonist of the NR2B subunit of the NMDA receptor, potentiates the antidepressant-like effects of certain antidepressant drugs in the forced swim test in mice.

    PubMed

    Poleszak, Ewa; Stasiuk, Weronika; Szopa, Aleksandra; Wyska, Elżbieta; Serefko, Anna; Oniszczuk, Anna; Wośko, Sylwia; Świąder, Katarzyna; Wlaź, Piotr

    2016-08-01

    One of the newest substances, whose antidepressant activity was shown is traxoprodil, which is a selective antagonist of the NR2B subunit of the NMDA receptor. The main goal of the present study was to evaluate the effect of traxoprodil on animals' behavior using the forced swim test (FST), as well as the effect of traxoprodil (10 mg/kg) on the activity of antidepressants, such as imipramine (15 mg/kg), fluoxetine (5 mg/kg), escitalopram (2 mg/kg) and reboxetine (2.5 mg/kg). Serotonergic lesion and experiment using the selective agonists of serotonin receptors 5-HT1A and 5-HT2 was conducted to evaluate the role of the serotonergic system in the antidepressant action of traxoprodil. Brain concentrations of tested agents were determined using HPLC. The results showed that traxoprodil at a dose of 20 and 40 mg/kg exhibited antidepressant activity in the FST and it was not related to changes in animals' locomotor activity. Co-administration of traxoprodil with imipramine, fluoxetine or escitalopram, each in subtherapeutic doses, significantly affected the animals' behavior in the FST and, what is important, these changes were not due to the severity of locomotor activity. The observed effect of traxoprodil is only partially associated with serotonergic system and is independent of the effect on the 5-HT1A and 5-HT2 serotonin receptors. The results of an attempt to assess the nature of the interaction between traxoprodil and the tested drugs show that in the case of joint administration of traxoprodil and fluoxetine, imipramine or escitalopram, there were interactions in the pharmacokinetic phase. PMID:26924124

  1. Dioscin enhances methotrexate absorption by down-regulating MDR1 in vitro and in vivo.

    PubMed

    Wang, Lijuan; Wang, Changyuan; Peng, Jinyong; Liu, Qi; Meng, Qiang; Sun, Huijun; Huo, Xiaokui; Sun, Pengyuan; Yang, Xiaobo; Zhen, Yuhong; Liu, Kexin

    2014-06-01

    The purpose of this study was to investigate the enhancing effect of dioscin on the absorption of methotrexate (MTX) and clarify the molecular mechanism involved in vivo and in vitro. Dioscin increased MTX chemosensitivity and transepithelial flux in the absorptive direction, significantly inhibiting multidrug resistance 1 (MDR1) mRNA and protein expression and MDR1 promoter and nuclear factor κ-B (NF-κB) activities in Caco-2 cells. Moreover, inhibitor κB-α (IκB-α) degradation was inhibited by dioscin. Dioscin enhanced the intracellular concentration of MTX by down-regulating MDR1 expression through a mechanism that involves NF-κB signaling pathway inhibition in Caco-2 cells. Dioscin strengthened MTX absorption by inhibiting MDR1 expression in rat intestine. In addition, even though MTX is absorbed into the enterocytes, there was no increase in toxicity observed, and that, in fact, decreased toxicity was seen. PMID:24680847

  2. DNA Methylation mediated down-regulating of MicroRNA-33b and its role in gastric cancer

    PubMed Central

    Yin, Haixin; Song, Peng; Su, Rui; Yang, Guihua; Dong, Lei; Luo, Min; Wang, Bin; Gong, Bei; Liu, Changzheng; Song, Wei; Wang, Fang; Ma, Yanni; Zhang, Junwu; Wang, Weibin; Yu, Jia

    2016-01-01

    The discovery of microRNAs (miRNAs) provides a new and powerful tool for studying the mechanism, diagnosis and treatment of human cancers. Currently, down-regulation of tumor suppressive miRNAs by CpG island hypermethylation is emerging as a common hallmark of cancer. Here, we reported that the down-regulation of miR-33b was associated with pM stage of gastric cancer (GC) patients. Ectopic expression of miR-33b in HGC-27 and MGC-803 cells inhibited cell proliferation, migration and invasion, which might be due to miR-33b targeting oncogene c-Myc. Moreover, enhanced methylation level of the CpG island upstream of miR-33b in GC patients with down-regulated miR-33b was confirmed by methylation-specific PCR (MSP) amplification. Furthermore, re-introduction of miR-33b significantly suppressed tumorigenesis of GC cells in the nude mice. In conclusion, miR-33b acts as a tumor suppressor and hypermethylation of the CpG island upstream of miR-33b is responsible for its down-regulation in gastric cancer. PMID:26729612

  3. Mechanism of endogenous myc gene down-regulation in E mu-N-myc tumors.

    PubMed

    Ma, A; Smith, R K; Tesfaye, A; Achacoso, P; Dildrop, R; Rosenberg, N; Alt, F W

    1991-01-01

    Transgenic mouse lines carrying the N-myc oncogene deregulated by the immunoglobulin heavy-chain enhancer spontaneously develop B-lymphoid tumors (R. Dildrop, A. Ma, K. Zimmerman, E. Hsu, A. Tesfaye, R. DePinho, and F. W. Alt, EMBO J. 8:1121-1128, 1989; H. Rosenbaum, E. Webb, J. M. Adams, S. Cory, and A. W. Harris, EMBO J. 8:749-755). Permanent cell lines derived from these tumors (E mu-N-myc cell lines) express extremely high levels of the N-myc transgene but little or no detectable endogenous N-myc or c-myc. We have employed nuclear run-on assays to show that down-regulation of endogenous N- and c-myc expression occurs at the transcriptional level. To determine whether the lack of endogenous myc gene transcription is a direct effect of high-level N-myc transgene expression, we have generated Abelson murine leukemia virus (A-MuLV)-transformed cell lines from prelymphomatous E mu-N-myc mice (A-MuLV/E mu-N-myc cell lines). Although these A-MuLV/E mu-N-myc lines express very high levels of the N-myc transgene, they continue to transcribe the endogenous c-myc gene. These findings demonstrate that high-level N-myc gene expression alone does not necessarily lead to down-regulation of endogenous myc gene expression and suggest that events associated with transformation by N-myc may be critical to this process. PMID:1986238

  4. Fasting induces CART down-regulation in the zebrafish nervous system in a cannabinoid receptor 1-dependent manner.

    PubMed

    Nishio, Shin-Ichi; Gibert, Yann; Berekelya, Liubov; Bernard, Laure; Brunet, Frédéric; Guillot, Etienne; Le Bail, Jean-Christophe; Sánchez, Juan Antonio; Galzin, Anne Marie; Triqueneaux, Gerard; Laudet, Vincent

    2012-08-01

    Central and peripheral mechanisms modulate food intake and energy balance in mammals and the precise role of the type 1 cannabinoid receptor (CB1) in these processes is still being explored. Using the zebrafish, Danio rerio, we show that rimonabant, a CB1-specific antagonist with an EC(50) of 5.15 × 10(-8) m, decreases embryonic yolk sac reserve use. We reveal a developmental overlap between CART genes and CB1 expression in the hypothalamus and medulla oblongata, two brain structures that play crucial roles in appetite regulation in mammals. We show that morpholino knockdown of CB1 or fasting decreases cocaine- and amphetamine-related transcript (CART)-3 expression. Strikingly, this down-regulation occurs only in regions coexpressing CB1 and CART3, reinforcing the link between CB1, CART, and appetite regulation. We show that rimonabant treatment impairs the fasting-induced down-regulation of CART expression in specific brain regions, whereas vehicle alone-treated embryos do not display this rescue of CART expression. Our data reveal that CB1 lies upstream of CART and signals the appetite through the down-regulation of CART expression. Thus, our results establish the zebrafish as a promising system to study appetite regulation. PMID:22700585

  5. In Vitro Ischemia Triggers a Transcriptional Response to Down-Regulate Synaptic Proteins in Hippocampal Neurons

    PubMed Central

    Fernandes, Joana; Vieira, Marta; Carreto, Laura; Santos, Manuel A. S.; Duarte, Carlos B.; Carvalho, Ana Luísa; Santos, Armanda E.

    2014-01-01

    Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells, which is partially associated with the induction or repression of genes that influence the ischemic response. However, the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults, we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD), an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD, total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes, whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD, confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins, such as those encoding for PICK1, GRIP1, TARPγ3, calsyntenin-2/3, SAPAP2 and SNAP-25, were down-regulated after OGD. Additionally, OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors, but increased the mRNA expression of the GluN3A subunit, thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together, our results present the expression profile elicited by in vitro ischemia in hippocampal neurons, and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins, suggesting that the synaptic proteome may change after ischemia. PMID:24960035

  6. Potent anti-prostate cancer agents derived from a novel androgen receptor down-regulating agent.

    PubMed

    Purushottamachar, Puranik; Khandelwal, Aakanksha; Vasaitis, Tadas S; Bruno, Robert D; Gediya, Lalji K; Njar, Vincent C O

    2008-04-01

    The search for novel androgen receptor (AR) down-regulating agents by catalyst HipHop pharmacophore modeling led to the discovery of some lead molecules. Unexpectedly, the effect of these leads on human prostate cancer LNCaP cell viability did not correlate with the ability of the compounds to cause down-regulation of AR protein expression. Through rational synthetic optimization of the lead compound (BTB01434), we have discovered a series of novel substituted diaryl molecules as potent anti-prostate cancer agents. Some compounds (1-6) were shown to be extremely potent inhibitors of LNCaP cell viability with GI(50) values in the nanomolar range (1.45-83 nM). The most potent compound (4-methylphenyl)[(4-methylphenyl)sulfonyl]amine (5) with a GI(50) value of 1.45 nM is 27,000 times more potent than our lead compound BTB01434 (GI(50)=39.8 microM). In addition, some of the compounds exhibited modest anti-androgenic activities and one was also a potent inhibitor (GI(50)=850 nM) of PC-3 (AR-null) cell growth. A clear structure-activity relationship (SAR) has been established for activity against LNCaP cells, where potent molecules possess two substituted/unsubstituted aromatic rings connected through a sulfonamide linker. These novel compounds are strong candidates for development for the treatment of hormone-sensitive and importantly hormone-refractory prostate cancers in humans. PMID:18316193

  7. Effects of p21 Gene Down-Regulation through RNAi on Antler Stem Cells In Vitro

    PubMed Central

    Guo, Qianqian; Wang, Datao; Liu, Zhen; Li, Chunyi

    2015-01-01

    Cell cycle is an integral part of cell proliferation, and consists mainly of four phases, G1, S, G2 and M. The p21 protein, a cyclin dependent kinase inhibitor, plays a key role in regulating cell cyclevia G1 phase control. Cells capable of epimorphic regeneration have G2/M accumulation as their distinctive feature, whilst the majority of somatic cells rest at G1 phase. To investigate the role played byp21 in antler regeneration, we studied the cell cycle distribution of antler stem cells (ASCs), via down-regulation of p21 in vitro using RNAi. The results showed that ASCs had high levels of p21 mRNA expression and rested at G1 phase, which was comparable to the control somatic cells. Down-regulation of p21 did not result in ASC cell cycle re-distribution toward G2/M accumulation, but DNA damage and apoptosis of the ASCs significantly increased and the process of cell aging was slowed. These findings suggest that the ASCs may have evolved to use an alternative, p21-independent cell cycle regulation mechanism. Also a unique p21-dependent inhibitory effect may control DNA damage as a protective mechanism to ensure the fast proliferating ASCs do not become dysplastic/cancerous. Understanding of the mechanism underlying the role played by p21 in the ASCs could give insight into a mammalian system where epimorphic regeneration is initiated whilst the genome stability is effectively maintained. PMID:26308075

  8. Amino acid limitation induces down-regulation of WNT5a at transcriptional level

    SciTech Connect

    Wang Zuguang; Chen Hong

    2009-01-23

    An aberrant WNT signaling contributes to the development and progression of multiple cancers. WNT5a is one of the WNT signaling molecules. This study was designed to test the hypothesis that amino acid deprivation induces changes in the WNT signaling pathway in colon cancer cells. Results showed that targets of the amino acid response pathway, ATF3 and p21, were induced in the human colon cancer cell line SW480 during amino acid limitation. There was a significant decrease in the WNT5a mRNA level following amino acid deprivation. The down-regulation of WNT5a mRNA by amino acid deprivation is not due to mRNA destabilization. There is a reduction of nuclear {beta}-catenin protein level by amino acid limitation. Under amino acid limitation, phosphorylation of ERK1/2 was increased and the blockage of ERK1/2 by the inhibitor U0126 partially restored WNT5a mRNA level. In conclusion, amino acid limitation in colon cancer cells induces phosphorylation of ERK1/2, which then down-regulates WNT5a expression.

  9. Phosphorylation-dependent down-regulation of apolipoprotein A5 by insulin

    SciTech Connect

    Nowak, Maxine; Helleboid-Chapman, Audrey; Jakel, Heidelinde; Rommens, Corinne; Martin, Genevieve; Duran-Sandoval, Daniel; Staels, Bart; Rubin, Edward M.; Pennacchio, Len A.; Taskinen, Marja-Riitta; Fruchart-Najib, Jamila; Fruchart, Jean-Charles

    2004-02-15

    The apolipoprotein A5 (APOA5) gene has been shown to be important in lowering plasma triglyceride levels. Since several studies have shown that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 gene is regulated by insulin. We show here that cell and mouse treatments with insulin down-regulated APOA5 expression in a dose-dependent manner. Furthermore, we determined that insulin decreases APOA5 promoter activity and subsequent deletion analyses revealed an E-box-containing fragment. We showed that Upstream Stimulatory Factors, USF1/USF2, bind to the identified E-box in the APOA5 promoter. Moreover, in cotransfection studies, USF1 stimulates APOA5 promoter activity. The treatment with insulin reduces the binding of USF1/USF2 to APOA5 promoter. The inhibition of PI3K pathway with wortmannin abolished the insulin s effect on APOA5 gene transcription. Using oligoprecipitation method of USF from nuclear extracts, we demonstrated that phosphorylated USF1 failed to bind to APOA5 promoter. This indicates that the APOA5 gene transrepression by insulin involves a phosphorylation of USF through PI3K, that modulate their binding to APOA5 promoter and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in healthy men shows a decrease of the plasma ApoAV level. These data suggest a potential mechanism involving APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.

  10. Terpene down-regulation triggers defense responses in transgenic orange leading to resistance against fungal pathogens.

    PubMed

    Rodríguez, Ana; Shimada, Takehiko; Cervera, Magdalena; Alquézar, Berta; Gadea, José; Gómez-Cadenas, Aurelio; De Ollas, Carlos José; Rodrigo, María Jesús; Zacarías, Lorenzo; Peña, Leandro

    2014-01-01

    Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with d-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a d-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of d-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, d-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in d-limonene content and the decrease in the defense response suggests that d-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores. PMID:24192451

  11. CyclinD1 Down-Regulation and Increased Apoptosis Are Common Features of Cohesinopathies.

    PubMed

    Fazio, Grazia; Gaston-Massuet, Carles; Bettini, Laura Rachele; Graziola, Federica; Scagliotti, Valeria; Cereda, Anna; Ferrari, Luca; Mazzola, Mara; Cazzaniga, Gianni; Giordano, Antonio; Cotelli, Franco; Bellipanni, Gianfranco; Biondi, Andrea; Selicorni, Angelo; Pistocchi, Anna; Massa, Valentina

    2016-03-01

    Genetic variants within components of the cohesin complex (NIPBL, SMC1A, SMC3, RAD21, PDS5, ESCO2, HDAC8) are believed to be responsible for a spectrum of human syndromes known as "cohesinopathies" that includes Cornelia de Lange Syndrome (CdLS). CdLS is a multiple malformation syndrome affecting almost any organ and causing severe developmental delay. Cohesinopathies seem to be caused by dysregulation of specific developmental pathways downstream of mutations in cohesin components. However, it is still unclear how mutations in different components of the cohesin complex affect the output of gene regulation. In this study, zebrafish embryos and SMC1A-mutated patient-derived fibroblasts were used to analyze abnormalities induced by SMC1A loss of function. We show that the knockdown of smc1a in zebrafish impairs neural development, increases apoptosis, and specifically down-regulates Ccnd1 levels. The same down-regulation of cohesin targets is observed in SMC1A-mutated patient fibroblasts. Previously, we have demonstrated that haploinsufficiency of NIPBL produces similar effects in zebrafish and in patients fibroblasts indicating a possible common feature for neurological defects and mental retardation in cohesinopathies. Interestingly, expression analysis of Smc1a and Nipbl in developing mouse embryos reveals a specific pattern in the hindbrain, suggesting a role for cohesins in neural development in vertebrates. PMID:26206533

  12. Mutations and Down-Regulation of CDX1 in Children with Anorectal Malformations

    PubMed Central

    Zhang, Tao; Tang, Xiao Bing; Wang, Li Li; Bai, Yu Zuo; Qiu, Guang Rong; Yuan, Zheng Wei; Wang, Wei Lin

    2013-01-01

    Background: Anorectal malformations (ARMs) represent a variety of congenital disorders that involve abnormal termination of the anorectum. This study was to reveal relation between CDX1 and human ARMs phenotypes. Methods: 108 Chinese patients and 120 Chinese controls were included in this study. We analyzed the relation between two by PCR, qRT-PCR, western blot and immunofluorescence. Results: Four heterozygous mutations in CDX1 gene were identified in ARMs patients (3.7%, 4/108), no found in controls. CDX1 protein expression was significantly decreased in the ARMs compared with the control anorectum. All samples analyzed in ARMs group exhibited down-regulated CDX1 mRNA expression in comparison to matched normal group, demonstrated significant differences statistically. Conclusion: The findings represented the relation between CDX1 mutations and CDX1 genotype. Furthermore, it was suggested that the downregulation of CDX1 might be related to the development of ARMs. PMID:23329892

  13. Nucleolin Down-Regulation Is Involved in ADP-Induced Cell Cycle Arrest in S Phase and Cell Apoptosis in Vascular Endothelial Cells

    PubMed Central

    Wang, Wenmeng; Luo, Junqing; Xiang, Fang; Liu, Xueting; Jiang, Manli; Liao, Lingjuan; Hu, Jinyue

    2014-01-01

    High concentration of extracellular ADP has been reported to induce cell apoptosis, but the molecular mechanisms remain not fully elucidated. In this study, we found by serendipity that ADP treatment of human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) down-regulated the protein level of nucleolin in a dose- and time-dependent manner. ADP treatment did not decrease the transcript level of nucloelin, suggesting that ADP might induce nucleolin protein degradation. HUVEC and HAEC expressed ADP receptor P2Y13 receptor, but did not express P2Y1 or P2Y12 receptors. However, P2Y1, 12, 13 receptor antagonists MRS2179, PSB0739, MRS2211 did not inhibit ADP-induced down-regulation of nucleolin. Moreover, MRS2211 itself down-regulated nucleolin protein level. In addition, 2-MeSADP, an agonist for P2Y1, 12 and 13 receptors, did not down-regulate nucleolin protein. These results suggested that ADP-induced nucleolin down-regulation was not due to the activation of P2Y1, 12, or 13 receptors. We also found that ADP treatment induced cell cycle arrest in S phase, cell apoptosis and cell proliferation inhibition via nucleolin down-regulation. The over-expression of nucleolin by gene transfer partly reversed ADP-induced cell cycle arrest, cell apoptosis and cell proliferation inhibition. Furthermore, ADP sensitized HUVEC to cisplatin-induced cell death by the down-regulation of Bcl-2 expression. Taken together, we found, for the first time to our knowledge, a novel mechanism by which ADP regulates cell proliferation by induction of cell cycle arrest and cell apoptosis via targeting nucelolin. PMID:25290311

  14. Nucleolin down-regulation is involved in ADP-induced cell cycle arrest in S phase and cell apoptosis in vascular endothelial cells.

    PubMed

    Wang, Wenmeng; Luo, Junqing; Xiang, Fang; Liu, Xueting; Jiang, Manli; Liao, Lingjuan; Hu, Jinyue

    2014-01-01

    High concentration of extracellular ADP has been reported to induce cell apoptosis, but the molecular mechanisms remain not fully elucidated. In this study, we found by serendipity that ADP treatment of human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) down-regulated the protein level of nucleolin in a dose- and time-dependent manner. ADP treatment did not decrease the transcript level of nucloelin, suggesting that ADP might induce nucleolin protein degradation. HUVEC and HAEC expressed ADP receptor P2Y13 receptor, but did not express P2Y1 or P2Y12 receptors. However, P2Y1, 12, 13 receptor antagonists MRS2179, PSB0739, MRS2211 did not inhibit ADP-induced down-regulation of nucleolin. Moreover, MRS2211 itself down-regulated nucleolin protein level. In addition, 2-MeSADP, an agonist for P2Y1, 12 and 13 receptors, did not down-regulate nucleolin protein. These results suggested that ADP-induced nucleolin down-regulation was not due to the activation of P2Y1, 12, or 13 receptors. We also found that ADP treatment induced cell cycle arrest in S phase, cell apoptosis and cell proliferation inhibition via nucleolin down-regulation. The over-expression of nucleolin by gene transfer partly reversed ADP-induced cell cycle arrest, cell apoptosis and cell proliferation inhibition. Furthermore, ADP sensitized HUVEC to cisplatin-induced cell death by the down-regulation of Bcl-2 expression. Taken together, we found, for the first time to our knowledge, a novel mechanism by which ADP regulates cell proliferation by induction of cell cycle arrest and cell apoptosis via targeting nucelolin. PMID:25290311

  15. The herpes simplex virus receptor nectin-1 is down-regulated after trans-interaction with glycoprotein D

    SciTech Connect

    Stiles, Katie M.; Milne, Richard S.B.; Cohen, Gary H.; Eisenberg, Roselyn J.; Krummenacher, Claude

    2008-03-30

    During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with nectin-1 expressing cells to investigate the effects of gD on nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, nectin-1 was not down-regulated. Further analysis of B78H1-derived cells showed that nectin-1 down-regulation corresponds to the ability of gD to bind nectin-1 and is achieved by internalization and low-pH-dependent degradation of nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing nectin-1. These data suggest that the determinants of gD-mediated internalization of nectin-1 may direct HSV to an endocytic pathway during entry.

  16. Steatogenesis in adult-onset type II citrullinemia is associated with down-regulation of PPARα.

    PubMed

    Komatsu, Michiharu; Kimura, Takefumi; Yazaki, Masahide; Tanaka, Naoki; Yang, Yang; Nakajima, Takero; Horiuchi, Akira; Fang, Zhong-Ze; Joshita, Satoru; Matsumoto, Akihiro; Umemura, Takeji; Tanaka, Eiji; Gonzalez, Frank J; Ikeda, Shu-Ichi; Aoyama, Toshifumi

    2015-03-01

    SLC25A13 (citrin or aspartate-glutamate carrier 2) is located in the mitochondrial membrane in the liver and its genetic deficiency causes adult-onset type II citrullinemia (CTLN2). CTLN2 is one of the urea cycle disorders characterized by sudden-onset hyperammonemia due to reduced argininosuccinate synthase activity. This disorder is frequently accompanied with hepatosteatosis in the absence of obesity and ethanol consumption. However, the precise mechanism of steatogenesis remains unclear. The expression of genes associated with fatty acid (FA) and triglyceride (TG) metabolism was examined using liver samples obtained from 16 CTLN2 patients and compared with 7 healthy individuals. Although expression of hepatic genes associated with lipogenesis and TG hydrolysis was not changed, the mRNAs encoding enzymes/proteins involved in FA oxidation (carnitine palmitoyl-CoA transferase 1α, medium- and very-long-chain acyl-CoA dehydrogenases, and acyl-CoA oxidase 1), very-low-density lipoprotein secretion (microsomal TG transfer protein), and FA transport (CD36 and FA-binding protein 1), were markedly suppressed in CTLN2 patients. Serum concentrations of ketone bodies were also decreased in these patients, suggesting reduced mitochondrial β-oxidation activity. Consistent with these findings, the expression of peroxisome proliferator-activated receptor α (PPARα), a master regulator of hepatic lipid metabolism, was significantly down-regulated. Hepatic PPARα expression was inversely correlated with severity of steatosis and circulating ammonia and citrulline levels. Additionally, phosphorylation of c-Jun-N-terminal kinase was enhanced in CTLN2 livers, which was likely associated with lower hepatic PPARα. Collectively, down-regulation of PPARα is associated with steatogenesis in CTLN2 patients. These findings provide a novel link between urea cycle disorder, lipid metabolism, and PPARα. PMID:25533124

  17. Let-7c down-regulation in Helicobacter pylori-related gastric carcinogenesis.

    PubMed

    Fassan, Matteo; Saraggi, Deborah; Balsamo, Laura; Cascione, Luciano; Castoro, Carlo; Coati, Irene; De Bernard, Marina; Farinati, Fabio; Guzzardo, Vincenza; Valeri, Nicola; Zambon, Carlo Federico; Rugge, Massimo

    2016-01-26

    Aberrant let-7c microRNA (miRNA) expression has been observed in Helicobacter pylori-related gastric cancer (GC) but fragmentary information is available on the let-7c dysregulation occurring with each phenotypic change involved in gastric carcinogenesis. Let-7c expression was assessed (qRT-PCR) in a series of 175 gastric biopsy samples representative of the whole spectrum of phenotypic changes involved in H. pylori-related gastric oncogenesis including: i) normal gastric mucosa, as obtained from dyspeptic controls (40 biopsy samples); ii) non-atrophic gastritis (40 samples); iii) atrophic-metaplastic gastritis (35 samples); iv) intra-epithelial neoplasia (30 samples); v) GC (30 samples). Let-7c expression was also tested in 20 biopsy samples obtained from 10 patients before and after H. pylori eradication therapy (median follow-up: 10 weeks; range: 7-14). The results obtained were further validated by in situ hybridization on multiple tissue specimens obtained from 5 surgically treated H. pylori-related GCs. The study also included 40 oxyntic biopsy samples obtained from serologically/histologically confirmed autoimmune gastritis (AIG: 20 corpus-restricted, non-atrophic; 20 corpus-restricted, atrophic-metaplastic). Let-7c expression dropped from non-atrophic gastritis to atrophic-metaplastic gastritis, intra-epithelial neoplasia, and invasive GC (p<0.001). It rose again significantly following H. pylori eradication (p=0.009). As in the H. pylori model, AIG also featured a significant let-7c down-regulation (p<0.001). The earliest phases of the two pathways to gastric oncogenesis (H. pylori-environmental and autoimmune host-related) are characterized by similar let-7c dysregulations. In H. pylori infection, let-7c down-regulation regresses after the bacterium's eradication, while it progresses significantly with the increasing severity of the histological lesions. PMID:26701848

  18. Additional Nitrogen Fertilization at Heading Time of Rice Down-Regulates Cellulose Synthesis in Seed Endosperm

    PubMed Central

    Midorikawa, Keiko; Kuroda, Masaharu; Terauchi, Kaede; Hoshi, Masako; Ikenaga, Sachiko; Ishimaru, Yoshiro; Abe, Keiko; Asakura, Tomiko

    2014-01-01

    The balance between carbon and nitrogen is a key determinant of seed storage components, and thus, is of great importance to rice and other seed-based food crops. To clarify the influence of the rhizosphere carbon/nitrogen balance during the maturation stage of several seed components, transcriptome analysis was performed on the seeds from rice plants that were provided additional nitrogen fertilization at heading time. As a result, it was assessed that genes associated with molecular processes such as photosynthesis, trehalose metabolism, carbon fixation, amino acid metabolism, and cell wall metabolism were differentially expressed. Moreover, cellulose and sucrose synthases, which are involved in cellulose synthesis, were down-regulated. Therefore, we compared cellulose content of mature seeds that were treated with additional nitrogen fertilization with those from control plants using calcofluor staining. In these experiments, cellulose content in endosperm from plants receiving additional nitrogen fertilization was less than that in control endosperm. Other starch synthesis-related genes such as starch synthase 1, starch phosphorylase 2, and branching enzyme 3 were also down-regulated, whereas some α-amylase and β-amylase genes were up-regulated. On the other hand, mRNA expression of amino acid biosynthesis-related molecules was up-regulated. Moreover, additional nitrogen fertilization caused accumulation of storage proteins and up-regulated Cys-poor prolamin mRNA expression. These data suggest that additional nitrogen fertilization at heading time changes the expression of some storage substance-related genes and reduces cellulose levels in endosperm. PMID:24905454

  19. Down regulation of hepatic PPARalpha function by AhR ligand.

    PubMed

    Shaban, Zein; El-Shazly, Samir; Abdelhady, Shawky; Fattouh, Ibrahim; Muzandu, Kaampwe; Ishizuka, Mayumi; Kimura, Kazuhiro; Kazusaka, Akio; Fujita, Shoichi

    2004-11-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachloro dibenzo-p-dioxin (TCDD) and related compounds. Peroxisome proliferator activated receptor alpha (PPARalpha) is a nuclear receptor involved in the maintenance of lipid and glucose homeostasis. In this study we hypothesized that one of the possible mechanisms for the effect of TCDD and its related chemicals on fat metabolism could be through down regulation of PPARalpha functions. We treated Wistar rats with an AhR ligand, Sudan III (S.III), and/or PPARalpha ligand, Clofibric Acid (CA), for 3 days. We analysed the expression of one of the PPARalpha-target gene products, CYP4A protein and its mRNA. We also tested HepG2 cells with the afore-mentioned treatments and evaluated their effects on PPARalpha and RXRalpha protein. Treatment of Wistar rats with S.III was found to down regulates CYP4A protein expression and reduced its induction with CA. It also decreased mRNA expressions of CYP4A1, CYP4A2, CYP4A3 and PPARalpha. In HepG2 cells, PPARalpha and RXRalpha protein expression was decreased by S.III treatment in a dose dependent manner. Our results suggest that AhR has an inhibitory effect on PPARalpha function and a new pathway by which AhR ligands could disturb lipid metabolism. PMID:15585952

  20. Simulated weightlessness down-regulated antioxidant defense system in rats

    NASA Astrophysics Data System (ADS)

    Li, Qi; Qu, Lina; Li, Yingxian; Bi, Lei; Huang, Zengming; Wang, Bo

    A variety of experiments suggest that space flight is associated with an increase in oxidative stress in organism The aim of the present study is to investigate whether or not simulated weightlessness by tail-suspension can affect the antioxidant defense system in rats and the possible protection effects of Chinese medicine named Liu Wei Di Huang Wan LWDHW Blood plasma of rats was taken after 21 days - tail-suspension for the assessment of the change of antioxidant defense system The total antioxidant capacity T-AOC was significantly decreased and the content of malondialdehyde MDA was increased after simulated weightlessness Activities of antioxidant enzymes such as superoxide dismutase and catalase were lower than those in the controlled groups However the activity of glutathione peroxidase was increased in comparison with the controlled groups Adequate dosage of LWDHW could inhibit the production of MAD and improve T-AOC in tail-suspension rats These results suggested that tail-suspension might break the oxidative antioxidative balance and down-regulate antioxidant defense system and Chinese medicine LWDHW was shown to protect rats from oxidative damage during simulated weightlessness Key words Simulated weightlessness Tail-suspension Antioxidant defense system Rats

  1. Human cell receptor CD46 is down regulated through recognition of a membrane-proximal region of the cytoplasmic domain in persistent measles virus infection.

    PubMed Central

    Hirano, A; Yant, S; Iwata, K; Korte-Sarfaty, J; Seya, T; Nagasawa, S; Wong, T C

    1996-01-01

    Monkey cells persistently infected by measles virus (MV) Biken strain (Biken-CV-1 cells) showed no cytopathic effects and lacked surface expression of a homolog of human cell receptor, membrane cofactor protein CD46. Transfection of a human CD46 gene into these cells induced extensive cell fusion, indicating that down regulation of the endogenous CD46 homolog was essential for the maintenance of a noncytopathic mode of infection. Surface expression of the exogenously introduced human CD46 was also drastically down regulated in the persistently infected cells compared with uninfected cells. The down regulation was specific for CD46 and did not affect surface expression of exogenously introduced CD4. Exogenous human CD46 was synthesized efficiently in the persistently infected cells, but it did not accumulate on the cell surface. Fusion of Biken-CV-1 cells required the extracellular hemagglutinin (H-protein)-binding domain but not the cytoplasmic domain. Replacing the transmembrane and cytoplasmic domains of CD46 with a glycosylphosphatidylinositol anchor did not prevent cell fusion but completely alleviated down regulation of the glycosylphosphatidylinositol-anchored CD46 in Biken-CV-1 cells. Deletion analyses revealed that the membrane-distal sequences of the CD46 cytoplasmic domain were not only unnecessary but also inhibitory for CD46 down regulation. By contrast, the six amino acid residues proximal to the membrane contained a sequence required for CD46 down regulation in the persistently infected cells. These results indicate that CD46 is down regulated in the persistently infected cells by a mechanism that recognizes a membrane-proximal sequence in the CD46 cytoplasmic domain. PMID:8794336

  2. miR-340 predicts glioblastoma survival and modulates key cancer hallmarks through down-regulation of NRAS.

    PubMed

    Fiore, Danilo; Donnarumma, Elvira; Roscigno, Giuseppina; Iaboni, Margherita; Russo, Valentina; Affinito, Alessandra; Adamo, Assunta; De Martino, Fabio; Quintavalle, Cristina; Romano, Giulia; Greco, Adelaide; Soini, Ylermi; Brunetti, Arturo; Croce, Carlo M; Condorelli, Gerolama

    2016-04-12

    Glioblastoma is the most common primary brain tumor in adults; with a survival rate of 12 months from diagnosis. However, a small subgroup of patients, termed long-term survivors (LTS), has a survival rate longer then 12-14 months. There is thus increasing interest in the identification of molecular signatures predicting glioblastoma prognosis and in how to improve the therapeutic approach. Here, we report miR-340 as prognostic tumor-suppressor microRNA for glioblastoma. We analyzed microRNA expression in > 500 glioblastoma patients and found that although miR-340 is strongly down-regulated in glioblastoma overall, it is up-regulated in LTS patients compared to short-term survivors (STS). Indeed, miR-340 expression predicted better prognosis in glioblastoma patients. Coherently, overexpression of miR-340 in glioblastoma cells was found to produce a tumor-suppressive activity. We identified NRAS mRNA as a critical, direct target of miR-340: in fact, miR-340 negatively influenced multiple aspects of glioblastoma tumorigenesis by down-regulating NRAS and downstream AKT and ERK pathways. Thus, we demonstrate that expression of miR-340 in glioblastoma is responsible for a strong tumor-suppressive effect in LTS patients by down-regulating NRAS. miR-340 may thus represent a novel marker for glioblastoma diagnosis and prognosis, and may be developed into a tool to improve treatment of glioblastoma. PMID:26799668

  3. miR-340 predicts glioblastoma survival and modulates key cancer hallmarks through down-regulation of NRAS

    PubMed Central

    Fiore, Danilo; Donnarumma, Elvira; Roscigno, Giuseppina; Iaboni, Margherita; Russo, Valentina; Affinito, Alessandra; Adamo, Assunta; De Martino, Fabio; Quintavalle, Cristina; Romano, Giulia; Greco, Adelaide; Soini, Ylermi; Brunetti, Arturo; Croce, Carlo M.; Condorelli, Gerolama

    2016-01-01

    Glioblastoma is the most common primary brain tumor in adults; with a survival rate of 12 months from diagnosis. However, a small subgroup of patients, termed long-term survivors (LTS), has a survival rate longer then 12–14 months. There is thus increasing interest in the identification of molecular signatures predicting glioblastoma prognosis and in how to improve the therapeutic approach. Here, we report miR-340 as prognostic tumor-suppressor microRNA for glioblastoma. We analyzed microRNA expression in > 500 glioblastoma patients and found that although miR-340 is strongly down-regulated in glioblastoma overall, it is up-regulated in LTS patients compared to short-term survivors (STS). Indeed, miR-340 expression predicted better prognosis in glioblastoma patients. Coherently, overexpression of miR-340 in glioblastoma cells was found to produce a tumor-suppressive activity. We identified NRAS mRNA as a critical, direct target of miR-340: in fact, miR-340 negatively influenced multiple aspects of glioblastoma tumorigenesis by down-regulating NRAS and downstream AKT and ERK pathways. Thus, we demonstrate that expression of miR-340 in glioblastoma is responsible for a strong tumor-suppressive effect in LTS patients by down-regulating NRAS. miR-340 may thus represent a novel marker for glioblastoma diagnosis and prognosis, and may be developed into a tool to improve treatment of glioblastoma. PMID:26799668

  4. Extracellular 2'5'-oligoadenylate synthetase 2 mediates T-cell receptor CD3-ζ chain down-regulation via caspase-3 activation in oral cancer.

    PubMed

    Dar, Asif A; Pradhan, Trupti N; Kulkarni, Dakshayni P; Shah, Sagar U; Rao, Kanury V; Chaukar, Devendra A; D'Cruz, Anil K; Chiplunkar, Shubhada V

    2016-02-01

    Decreased expression of CD3-ζ chain, an adaptor protein associated with T-cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3-ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour-derived factors promote immune suppression by down-regulating CD3-ζ chain expression. 2'5'-Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3-ζ chain down-regulation and OAS2 stimulation. The surrogate situation was established by over-expressing OAS2 in a HEK293 cell line and cell-free supernatant was collected. These supernatants when incubated with T cells resulted in down-regulation of CD3-ζ chain, which shows that the secreted OAS2 is capable of regulating CD3-ζ chain expression. Incubation of T cells with cell-free supernatants of oral tumours or recombinant human OAS2 (rh-OAS2) induced caspase-3 activation, which resulted in CD3-ζ chain down-regulation. Caspase-3 inhibition/down-regulation using pharmacological inhibitor or small interfering RNA restored down-regulated CD3-ζ chain expression in T cells induced by cell-free tumour supernatant or rh-OAS2. Collectively these results show that OAS2 leads to impairment in CD3-ζ chain expression, so offering an explanation that might be applicable to the CD3-ζ chain deficiency observed in cancer and diverse disease conditions. PMID:26595239

  5. Down-regulation of human leukocyte antigen class I (HLA-I) is associated with poor prognosis in patients with clear cell renal cell carcinoma.

    PubMed

    Yuan, Jinyang; Liu, Sulai; Yu, Qiuxia; Lin, Yang; Bi, Yunke; Wang, Yi; An, Ruihua

    2013-06-01

    Human leukocyte antigen class I (HLA-I) molecules are transmembrane glycoproteins that have been reported to be down-regulated in multiple types of human malignancies, including clear cell renal cell carcinoma (CCRCC). However, only one study has investigated its prognostic value in CCRCC. In the present study, HLA-I protein expression was analyzed in 120 archived, paraffin-embedded CCRCC samples and 10 adjacent normal tissues using immunohistochemistry. The correlation between HLA-I expression and clinicopathological factors was evaluated by the χ(2) test. Patients' overall survival was analyzed by the Kaplan-Meier method. HLA-I down-regulation was observed in 38.3% (46/120) of renal tumor samples, but only in 10% (1/10) of adjacent normal tissues. Statistical analysis showed a significant correlation of HLA-I expression with TNM stage, lymph node metastasis, and Fuhrman grade. Patients with tumors displaying down-regulation of HLA-I showed significantly shorter overall survival (P=0.021, log-rank test). More importantly, multivariate analysis indicated that down-regulation of HLA-I was an independent prognostic factor for CCRCC patients (P=0.033). Overall, our data suggest that