Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of Knockdown

  • gene knockdown
  • mediated knockdown
  • rnai knockdown
  • siRNA-mediat knockdown
  • sirna knockdown

  • Terms modified by Knockdown

  • knockdown approach
  • knockdown cell
  • knockdown embryo
  • knockdown experiment
  • knockdown resistance

  • Selected Abstracts

    Actin stress fiber pre-extension in human aortic endothelial cells

    CYTOSKELETON, Issue 4 2008
    Lan Lu
    Abstract Actin stress fibers (SFs) enable cells to sense and respond to mechanical stimuli and affect adhesion, motility and apoptosis. We and others have demonstrated that cultured human aortic endothelial cells (HAECs) are internally stressed so that SFs are pre-extended beyond their unloaded lengths. The present study explores factors affecting SF pre-extension. In HAECs cultured overnight the baseline pre-extension was 1.10 and independent of the amount of cell shortening. Decreasing contractility with 30 mM BDM or 10 ,M blebbistatin decreased pre-extension to 1.05 whereas increasing contractility with 2 nM calyculin A increased pre-extension to 1.26. Knockdown of ,-actinin-1 with an interfering RNA increased pre-extension to 1.28. None of these affected the wavelength of the buckled SFs. Pre-extension was the same in unperturbed cells as in those in which the actin cytoskeleton was disrupted by both chemical and mechanical means and then allowed to reassemble. Finally, disrupting MTs or IFs did not affect pre-extension but increased the wavelength. Taken together, these results suggest that pre-extension of SFs is determined primarily by intrinsic factors, i.e. the level of actin-myosin interaction. This intrinsic control of pre-extension is sufficiently robust that pre-extension is the same even after the actin cytoskeleton has been disrupted and reorganized. Unlike pre-extension, the morphology of the compressed SFs is partially determined by MTs and IFs which appear to support the SFs along their lengths. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source]

    Molecular characterization of the effects of Y-27632

    CYTOSKELETON, Issue 2 2007
    Hassina Darenfed
    Abstract Many key cellular functions, such as cell motility and cellular differentiation are mediated by Rho-associated protein kinases (ROCKs). Numerous studies have been conducted to examine the ROCK signal transduction pathways involved in these motile and contractile events with the aid of pharmacological inhibitors such as Y-27632. However the molecular mechanism of action of Y-27632 has not been fully defined. To assess the relative contribution of these Rho effectors to the effects of Y-27632, we compared the cytoskeletal phenotype, wound healing and neurite outgrowth in cells treated with Y-27632 or subjected to knockdown with ROCK-I, ROCK-II or PRK-2- specific siRNAs. Reduction of ROCK-I enhances the formation of thin actin-rich membrane extensions, a phenotype that closely resembles the effect of Y-27632. Knockdown of ROCK II or PRK-2, leads to the formation of disc-like extenstions and thick actin bundles, respectively. The effect of ROCK-I knockdown also mimicked the effect of Y-27632 on wound closer rates. ROCK-I knockdown and Y-27632 enhanced wound closure rates, while ROCK-II and PRK-2 were not appreciably different from control cells. In neurite outgrowth assays, knockdown of ROCK-I, ROCK-II or PRK-2 enhances neurite lengths, however no individual knockdown stimulated neurite outgrowth as robustly as Y-27632. We conclude that several kinases contribute to the global effect of Y-27632 on cellular responses. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source]

    Novel genes involved in canonical Wnt/, -catenin signaling pathway in early Ciona intestinalis embryos

    Shuichi Wada
    We report here characterization of five genes for novel components of the canonical Wnt/, -catenin signaling pathway. These genes were identified in the ascidian Ciona intestinalis through a loss-of-function screening for genes required for embryogenesis with morpholinos, and four of them have counterparts in vertebrates. The five genes we studied are as follows: Ci-PGAP1, a Ciona orthologue of human PGAP1, which encodes GPI (glycosylphosphatidylinositol) inositol-deacylase, Ci-ZF278, a gene encoding a C2H2 zinc-finger protein, Ci-C10orf11, a Ciona orthologue of human C10orf11 that encodes a protein with leucine-rich repeats, Ci-Spatial/C4orf17, a single counterpart for two human genes Spatial and C4orf17, and Ci-FLJ10634, a Ciona orthologue of human FLJ10634 that encodes a member of the J-protein family. Knockdown of each of the genes mimicked , -catenin knockdown and resulted in suppression of the expression of , -catenin downstream genes (Ci-FoxD, Ci-Lhx3, Ci-Otx and Ci-Fgf9/16/20) and subsequent endoderm formation. For every gene, defects in knockdown embryos were rescued by overexpression of a constitutively active form, but not wild-type, of Ci- , -catenin. Dosage-sensitive interactions were found between Ci-,-catenin and each of the genes. These results suggest that these five genes act upstream of or parallel to Ci- , -catenin in the Wnt/, -catenin signaling pathway in early Ciona embryos. [source]

    Identification and characterization of Xenopus OMP25

    Masafumi Inui
    This study describes the isolation of mitochondrial outer membrane protein 25 (OMP25) from Xenopus laevis and an analysis of its role in early development. X. laevis OMP25 (xOMP25) is a transmembrane protein of the mitochondrial outer membrane with a PDZ domain in the cytoplasmic tail, and an approximate molecular size of 25 kDa. We isolated xOMP25 from a cDNA library of X. laevis tailbud embryos. Amino acid sequence analysis of xOMP25 showed 57% identity to mouse OMP25, with 73% identity in the PDZ domains. XOMP25 mRNA is expressed maternally, and at a constant level throughout early development. The transcript is localized to eye, otic vesicle, branchial arch and neural tube. Mitochondrial targeting of an EGFP-fusion protein of xOMP25 was visualized using a mitochondria-specific fluorescent dye. Overexpression of xOMP25 in embryos caused curved axes, small eyes and disorganized head structures. Knockdown of xOMP25 protein using antisense morpholino oligonucleotides resulted in slightly shortened axes and decreased neural tissue. Although the mechanism remains unclear, our results implicate xOMP25 protein in the formation of the intact neural tube. [source]

    Churchill and Sip1a repress fibroblast growth factor signaling during zebrafish somitogenesis

    Fatma O. Kok
    Abstract Cell-type specific regulation of a small number of growth factor signal transduction pathways generates diverse developmental outcomes. The zinc finger protein Churchill (ChCh) is a key effector of fibroblast growth factor (FGF) signaling during gastrulation. ChCh is largely thought to act by inducing expression of the multifunctional Sip1 (Smad Interacting Protein 1). We investigated the function of ChCh and Sip1a during zebrafish somitogenesis. Knockdown of ChCh or Sip1a results in misshapen somites that are short and narrow. As in wild-type embryos, cycling gene expression occurs in the developing somites in ChCh and Sip1a compromised embryos, but expression of her1 and her7 is maintained in formed somites. In addition, tail bud fgf8 expression is expanded anteriorly in these embryos. Finally, we found that blocking FGF8 restores somite morphology in ChCh and Sip1a compromised embryos. These results demonstrate a novel role for ChCh and Sip1a in repression of FGF activity. Developmental Dynamics 239:548,558, 2010. © 2009 Wiley-Liss, Inc. [source]

    Emx3 is required for the differentiation of dorsal telencephalic neurons

    Gudrun Viktorin
    Abstract emx3 is first expressed in prospective telencephalic cells at the anterior border of the zebrafish neural plate. Knockdown of Emx3 function by morpholino reduces the expression of markers specific to dorsal telencephalon, and impairs axon tract formation. Rescue of both early and late markers requires low-level expression of emx3 at the one- or two-somite stage. Higher emx3 expression levels cause dorsal telencephalic markers to expand ventrally, which points to a possible role of emx3 in specifying dorsal telencephalon and a potential new function for Wnt/beta-catenin pathway activation. In contrast to mice, where Emx2 plays a major role in dorsal telencephalic development, knockdown of zebrafish Emx2 apparently does not affect telencephalic development. Similarly, Emx1 knockdown has little effect. Previously, emx3 was thought to be fish-specific. However, we found all three emx orthologs in Xenopus tropicalis and opossum (Monodelphis domestica) genomes, indicating that emx3 was present in an ancestral tetrapod genome. Developmental Dynamics 238:1984,1998, 2009. © 2009 Wiley-Liss, Inc. [source]

    The embryonic expression patterns and the knockdown phenotypes of zebrafish ADP-ribosylation factor-like 6 interacting protein gene

    Hsing-Yen Huang
    Abstract ADP-ribosylation factor-like 6 (Arl6) mutation is linked to human disease and Arl6 interacts with Arl6 interacting protein (Arl6ip). However, the expression pattern and function of Arl6ip during embryogenesis are unknown. To confirm whether abnormal Arl6ip function might result in embryonic defects in zebrafish, we examined the expression patterns of arl6ip during embryogenesis, and they were maternally expressed and exhibited in the brain, optic primordia, hypochord, spinal cord, myotome, heart, fin-bud, kidney, trunk, and retina. Knockdown of Arl6ip revealed the following phenotypic defects: microphthalmia, disorganized pigment pattern, flat head, defective tectum, deficient pectoral fins, abnormal pneumatic duct, pericardial edema, and deformed trunk. Particularly, histological dissection of the retinae of arl6ip -morphants revealed that neuronal differentiation is severely delayed, resulting in no formation of retinal layers. We further confirmed that opsins of arl6ip -morphants were not transcribed. Based on this evidence, Arl6ip may play important roles in zebrafish ocular, heart, and fin-bud development. Developmental Dynamics 238:232,240, 2009. © 2008 Wiley-Liss, Inc. [source]

    Zebrafish KLF4 is essential for anterior mesendoderm/pre-polster differentiation and hatching

    Melissa R. Gardiner
    Abstract Gene knockout studies of Krüppel-like factors (KLFs) in mice have shown essential roles in organogenesis. A screen for KLF family members in zebrafish identified many KLFs. One of these, zebrafish KLF4 (zKLF4) is the homologue of neptune, a Xenopus laevis KLF. zKLF4 is expressed from approximately 80% epiboly a patch of dorsal/anterior mesendodermal cells called the pre-polster and, subsequently, in the polster and hatching gland. Here we investigate the function of zKLF4 using morpholino-based antisense oligonucleotides. Knockdown of zKLF4 resulted in complete absence of hatching gland formation and subsequent hatching in zebrafish. In addition, there was early knockdown of expression of the pre-polster/anterior mesendoderm markers CatL, cap1, and BMP4. These results indicate zKLF4 is expressed within the pre-polster, an early mesendodermal site, and that it plays a critical role in the differentiation of these cells into hatching gland cells. Developmental Dynamics 234:992,996, 2005. © 2005 Wiley-Liss, Inc. [source]

    Neuronal calcium sensor-1 gene ncs-1a is essential for semicircular canal formation in zebrafish inner ear

    Brian Blasiole
    Abstract We have analyzed the functional role of neuronal calcium sensor-1 (Ncs-1) in zebrafish development. We identified two orthologs of the mammalian NCS-1 gene. Full-length cDNAs encoding zebrafish Ncs-1a and Ncs-1b polypeptides were cloned and characterized. Whole-mount in situ hybridization revealed that ncs-1a mRNA was expressed beginning at early somitogenesis. As development progressed, ncs-1a mRNA was present throughout the embryo with expression detected in ventral hematopoietic mesoderm, pronephric tubules, CNS nuclei, and otic vesicle. By 4.5 days post fertilization (dpf), ncs-1a expression was detected primarily in the brain. Expression of ncs-1b mRNA was first detected at 36 hours post fertilization (hpf) and was restricted to the olfactory bulb. By 4.5 dpf, ncs-1b was expressed at low levels throughout the brain. Knockdown of ncs-1a mRNA translation with antisense morpholinos blocked formation of semicircular canals. These studies identify a novel function for ncs-1a in inner ear development and suggest that this calcium sensor plays an important role in vestibular function. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005 [source]

    Differences in the Rapid Knockdown and Lethal Effects of Aerosol Formulations against German Cockroach (Blattaria, Blattellidae) Strains

    Dong-Kyu LEE
    ABSTRACT The knockdown and lethal efficacies of five aerosol formulations including Combat Speed® (AIs: 0.1 % imiprothrin and 0.3% cyphenothrin), Raid Power® (AIs: 1.0% pyrethrin and 0.2% permethrin), Home Keeper®, (AIs: 0.2% tetramethrin and 0.3% permethrin), Super Killer® (AIs: 0.32% tetramethrin and 0.08% bioresmethrin), and Perma Kill-K® (AIs: 0.3% dichlorvos and 0.1% tetramethrin) against five strains of the German cockroach, Blattella germanica (L.) were assessed. The results show that the mean value of KT50 (5.4 sec.) of Combat Speed® was 4.5 and 3.1-folds lower than those of Perma Kill-K® and Home Keeper®, respectively. The mean value of KT90 (9.0 sec; slope = 10.02) of Combat Speed® was 3.8 to 5.8-folds lower than Perma Kill-K®, Supper Killer® and Home Keeper®. As lethal effects, the mean value of LT50 (17.3 sec.) of Combat Speed® was over 26 folds lower than Supper Killer® and Perma Kill-K®. The mean value of LT90 (32.9 sec.) of Combat Speed® was 37.4 and 15.1-folds lower than those of Supper Killer® and Perma Kill-K®, respectively. In general, Combat Speed® and Raid Power® were considered the insecticide aerosols with faster knockdown and higher lethal effects than Supper Killer®, Perma Kill-K®, and Home Keeper® against five strains of German cockroaches in Korea. Also, the knockdown and lethal effects of Supper Killer®, Perma Kill-K®, and Home Keeper® were highly variable depends on the strains. [source]

    Sodium/bicarbonate cotransporter NBCn1/slc4a7 increases cytotoxicity in magnesium depletion in primary cultures of hippocampal neurons

    Deborah S. Cooper
    Abstract Growing evidence suggests that pharmacological inhibition of Na/H exchange and Na/HCO3 transport provides protection against damage or injury in cardiac ischemia. In this study, we examined the contribution of the sodium/bicarbonate cotransporter NBCn1 (slc4a7) to cytotoxicity in cultured hippocampal neurons of rats. In neurons exposed to extracellular pH (pHo) ranging from 6.2 to 8.3, NBCn1 protein expression increased by fivefold at pH < 6.5 compared to the expression at pHo 7.4. At pHo 6.5, the intracellular pH of neurons was ,1 unit lower than that at pH 7.4. Immunochemistry showed a marked increase in NBCn1 immunofluorescence in plasma membranes and cytosol of the soma as well as in dendrites, at pHo 6.5. NBCn1 expression also increased by 40% in a prolonged Mg2+ -free incubation at normal pHo. Knockdown of NBCn1 in neurons had negligible effect on cell viability. The effect of NBCn1 knockdown on cytotoxicity was then determined by exposing neurons to 0.5 mm glutamate for 10 min and measuring lactate dehydrogenase (LDH) release from neurons. Compared to normal incubation (pHo 7.2 for 6 h) after glutamate exposure, acidic incubation (pHo 6.3 for 6 h) reduced cytotoxicity by 75% for control neurons and 78% for NBCn1-knockdown neurons. Thus, both controls and knockdown neurons showed acidic protection from cytotoxicity. However, in Mg2+ -free incubation after glutamate exposure, NBCn1 knockdown progressively attenuated cytotoxicity. This attenuation was unaffected by acidic preincubation before glutamate exposure. We conclude that NBCn1 has a dynamic upregulation in low pHo and Mg2+ depletion. NBCn1 is not required for acidic protection, but increases cytotoxicity in Mg2+ -free conditions. [source]

    Mixed lineage leukemia histone methylases play critical roles in estrogen-mediated regulation of HOXC13

    FEBS JOURNAL, Issue 24 2009
    Khairul I. Ansari
    HOXC13, a homeobox-containing gene, is involved in hair development and human leukemia. The regulatory mechanism that drives HOXC13 expression is mostly unknown. Our studies have demonstrated that HOXC13 is transcriptionally activated by the steroid hormone estrogen (17,-estradiol; E2). The HOXC13 promoter contains several estrogen-response elements (EREs), including ERE1 and ERE2, which are close to the transcription start site, and are associated with E2-mediated activation of HOXC13. Knockdown of the estrogen receptors (ERs) ER, and ER, suppressed E2-mediated activation of HOXC13. Similarly, knockdown of mixed lineage leukemia histone methylase (MLL)3 suppressed E2-induced activation of HOXC13. MLLs (MLL1,MLL4) were bound to the HOXC13 promoter in an E2-dependent manner. Knockdown of either ER, or ER, affected the E2-dependent binding of MLLs (MLL1,MLL4) into HOXC13 EREs, suggesting critical roles of ERs in recruiting MLLs in the HOXC13 promoter. Overall, our studies have demonstrated that HOXC13 is transcriptionally regulated by E2 and MLLs, which, in coordination with ER, and ER,, play critical roles in this process. Although MLLs are known to regulate HOX genes, the roles of MLLs in hormone-mediated regulation of HOX genes are unknown. Herein, we have demonstrated that MLLs are critical players in E2-dependent regulation of the HOX gene. [source]

    Systemic RNAi of the cockroach vitellogenin receptor results in a phenotype similar to that of the Drosophila yolkless mutant

    FEBS JOURNAL, Issue 2 2006
    Laura Ciudad
    During vitellogenesis, one of the most tightly regulated processes in oviparous reproduction, vitellogenins are incorporated into the oocyte through vitellogenin receptor (VgR)-mediated endocytosis. In this paper, we report the cloning of the VgR cDNA from Blattella germanica, as well as the first functional analysis of VgR following an RNA interference (RNAi) approach. We characterized the VgR, VgR mRNA and protein expression patterns in pre-adult and adult stages of this cockroach, as well as VgR immunolocalization in ovarioles, belonging to the panoistic type. We then specifically disrupted VgR gene function using RNAi techniques. Knockdown of VgR expression led to a phenotype characterized by low yolk content in the ovary and high vitellogenin concentration in the haemolymph. This phenotype is equivalent to that of the yolkless mutant of Drosophila melanogaster, which have the yl (VgR) gene disrupted. The results additionally open the perspective that development genes can be functionally analyzed via systemic RNAi in this basal species. [source]

    Anti-apoptotic effect of the basic helix-loop-helix (bHLH) transcription factor DEC2 in human breast cancer cells

    GENES TO CELLS, Issue 4 2010
    Yang Liu
    DEC1 (BHLHB2/Stra13/Sharp2) and DEC2 (BHLHB3/Sharp1) are basic helix-loop-helix (bHLH) transcription factors that are involved in circadian rhythms, differentiation and the responses to hypoxia. We examined whether DEC1 and DEC2 are involved in apoptosis regulation, in human breast cancer MCF-7 cells. We found that siRNA-mediated knockdown of DEC2 resulted in marked enhancement of apoptosis compared with that in control cells transfected with nonspecific siRNA. However, knockdown of DEC1 by siRNA did not affect cell survival. Knockdown of DEC2 affected the expression of mRNA or proteins related to apoptosis, such as Fas, c-Myc, caspase-8, poly (ADP-ribose) polymerase (PARP) and Bax. We also showed that tumor necrosis factor-, (TNF-,) up-regulates the expression of DEC1 and DEC2. DEC2 over-expression caused by the transfection of an expression vector reduced the amounts of cleaved PARP and caspase-8 induced by TNF-, treatment, whereas DEC1 over-expression increased it. Finally, we revealed that treatment with double knockdown against both DEC1 and DEC2 decreased the amounts of cleaved PARP and caspase-8 induced by DEC2 siRNA with or without TNF-,. These data indicate that DEC2 has an anti-apoptotic effect, whereas DEC1 has a pro-apoptotic effect, which are involved in the balance of survival of human breast cancer MCF-7 cells. [source]

    Ski co-repressor complexes maintain the basal repressed state of the TGF-, target gene, SMAD7, via HDAC3 and PRMT5

    GENES TO CELLS, Issue 1 2009
    Takanori Tabata
    The products encoded by ski and its related gene, sno, (Ski and Sno) act as transcriptional co-repressors and interact with other co-repressors such as N-CoR/SMRT and mSin3A. Ski and Sno mediate transcriptional repression by various repressors, including Mad, Rb and Gli3. Ski/Sno also suppress transcription induced by multiple activators, such as Smads and c-Myb. In particular, the inhibition of TGF-,-induced transcription by binding to Smads is correlated with the oncogenic activity of Ski and Sno. However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown. In this study, we report the purification and characterization of Ski complexes. The Ski complexes purified from HeLa cells contained histone deacetylase 3 (HDAC3) and protein arginine methyltransferase 5 (PRMT5), in addition to multiple Smad proteins (Smad2, Smad3 and Smad4). Chromatin immunoprecipitation assays indicated that these components of the Ski complexes were localized on the SMAD7 gene promoter, which is the TGF-, target gene, in TGF-,-untreated HepG2 cells. Knockdown of these components using siRNA led to up-regulation of SMAD7 mRNA. These results indicate that Ski complexes serve to maintain a TGF-,-responsive promoter at a repressed basal level via the activities of histone deacetylase and histone arginine methyltransferase. [source]

    Transcription factor NF-Y is involved in regulation of the JNK pathway during Drosophila thorax development

    GENES TO CELLS, Issue 2 2008
    Yasuhide Yoshioka
    The CCAAT motif-binding factor, nuclear factor Y (NF-Y) consists of three different subunits, NF-YA, NF-YB and NF-YC. Knockdown of Drosophila NF-YA (dNF-YA) in the notum compartment of wing discs by a pannir -GAL4 and UAS- dNF-YAIR mainly resulted in a thorax disclosed phenotype. Reduction of the Drosophila c-Jun N-terminal kinase (JNK) basket (bsk) gene dose enhanced the knockdown of dNF-YA-induced phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc) -LacZ enhancer trap line revealed reduction in the level of the JNK reporter, puc-LacZ signals, in dNF-YA RNAi clones. In addition, expression of wild-type Bsk effectively suppressed the phenotype induced by knockdown of dNF-YA. The bsk gene promoter contains a CCAAT motif and this motif plays a positive role in the promoter activity. We performed chromatin immunoprecipitation (ChIP) assays in S2 cells with anti-dNF-YA IgG and quantitative real-time PCR. The bsk gene promoter region containing the CCAAT boxes was effectively amplified in the immunoprecipitates by PCR. However, this region was not amplified in the immunoprecipitates from dNF-YA knockdown cells. Furthermore, the level of endogenous bsk mRNA is reduced in the dNF-YA knockdown larvae. These results suggest that dNF-Y is necessary for proper bsk expression and activity of JNK pathway during thorax development. [source]

    RBP2 is an MRG15 complex component and down-regulates intragenic histone H3 lysine 4 methylation

    GENES TO CELLS, Issue 6 2007
    Tomohiro Hayakawa
    MRG15 is a conserved chromodomain protein that associates with histone deacetylases (HDACs) and Tip60-containing histone acetyltransferase (HAT) complexes. Here we further characterize MRG15-containing complexes and show a functional link between MRG15 and histone H3K4 demethylase activity in mammalian cells. MRG15 was predominantly localized to discrete nuclear subdomains enriched for Ser2 -phosphorylated RNA polymerase II, suggesting it is involved specifically with active transcription. Protein analysis of the MRG15-containing complexes led to the identification of RBP2, a JmjC domain-containing protein. Remarkably, over-expression of RBP2 greatly reduced the H3K4 methylation in culture human cells in vivo, and recombinant RBP2 efficiently removed H3K4 methylation of histone tails in vitro. Knockdown of RBP2 resulted in increased H3K4 methylation levels within transcribed regions of active genes. Our findings demonstrate that RBP2 associated with MRG15 complex to maintain reduced H3K4 methylation at transcribed regions, which may ensure the transcriptional elongation state. [source]

    Actin filaments-stabilizing and -bundling activities of cofilin-phosphatase Slingshot-1

    GENES TO CELLS, Issue 5 2007
    Souichi Kurita
    Slingshot-1 (SSH1) is known to regulate actin filament dynamics by dephosphorylating and activating cofilin, an actin-depolymerizing factor. SSH1 binds to filamentous (F-) actin through its multiple F-actin-binding sites and its cofilin-phosphatase activity is enhanced by binding to F-actin. In this study, we demonstrate that SSH1 has F-actin-stabilizing and -bundling activities. In vitro actin depolymerization assays revealed that SSH1 suppressed spontaneous and cofilin-induced actin depolymerization in a dose-dependent manner. SSH1 inhibited F-actin binding and severing activities of cofilin. Low-speed centrifugation assays combined with fluorescence and electron microscopic analysis revealed that SSH1 has F-actin-bundling activity, independently of its cofilin-phosphatase activity. Deletion of N- or C-terminal regions of SSH1 significantly reduced its F-actin-stabilizing and -bundling activities, indicating that both regions are critical for these functions. As SSH1 does not form a homodimer, it probably bundles F-actin through its multiple F-actin-binding sites. Knockdown of SSH1 expression by RNA interference significantly suppressed stress fiber formation in C2C12 myoblast cells, indicating a role for SSH1 in stress fiber formation or stabilization in cells. SSH1 thus has the potential to regulate actin filament dynamics and organization in cells via F-actin-stabilizing and -bundling activities, in addition to its ability to dephosphorylate cofilin. [source]

    Sustained activation of M-Ras induced by nerve growth factor is essential for neuronal differentiation of PC12 cells

    GENES TO CELLS, Issue 9 2006
    Peng Sun
    Neuronal differentiation in PC12 cells induced by nerve growth factor (NGF) requires sustained activation of ERK/MAP kinase pathway (Raf,MEK,ERK cascade). Although classical Ras (H-Ras, K-Ras, and N-Ras) activated by NGF signaling induces activation of ERK pathway, the activation is transient and not sufficient for PC12 cell differentiation. Instead, it has been widely accepted that NGF signaling-mediated Rap1 activation causes sustained activation of ERK pathway. There has been no direct evidence, however, that Rap1 participates in neuronal differentiation. Here we show that NGF signaling induces sustained activation of M-Ras and subsequent sustained activation of ERK pathway and the transcription factor CREB leading to PC12 cell differentiation. Exogenously expressed constitutively active mutant of M-Ras caused neurite outgrowth in PC12 cells and activating phosphorylation of ERK, whereas activated Rap1 did not. Knockdown of endogenous M-Ras by small interfering RNAs as well as the expression of a dominant,negative mutant of M-Ras interfered with NGF-induced neuritogenesis. Since MEK inhibitors prevented M-Ras-induced neurite outgrowth, ERK pathway participates in this differentiation pathway. Furthermore, M-Ras brought about ERK pathway-mediated activating phosphorylation of CREB and the CREB-mediated transcription. In addition, a dominant,negative mutant of CREB inhibited M-Ras-induced neuritogenesis. Taken together, NGF-induced PC12 cell differentiation requires M-Ras,ERK pathway-mediated activation of CREB. M-Ras was predominantly expressed in the hippocampus and cerebellum of mouse brain and in the gray matter of the spinal cord. All these properties of M-Ras were apparently indistinguishable from those of H-Ras. However, NGF stimulation caused transient activation of classical Ras proteins but sustained activation of M-Ras as well as sustained activating phosphorylation of ERK and CREB. Therefore, M-Ras is essential for neuronal differentiation in PC12 cells by inducing sustained activation of ERK pathway. [source]

    Induction of avian musculoaponeurotic fibrosarcoma proteins by toxic bile acid inhibits expression of glutathione synthetic enzymes and contributes to cholestatic liver injury in mice,

    HEPATOLOGY, Issue 4 2010
    Heping Yang
    We previously showed that hepatic expression of glutathione (GSH) synthetic enzymes and GSH levels fell 2 weeks after bile duct ligation (BDL) in mice. This correlated with a switch in nuclear anti-oxidant response element (ARE) binding activity from nuclear factor erythroid 2,related factor 2 (Nrf2) to c,avian musculoaponeurotic fibrosarcoma (c-Maf)/V-maf musculoaponeurotic fibrosarcoma oncogene homolog G (MafG). Our current aims were to examine whether the switch in ARE binding activity from Nrf2 to Mafs is responsible for decreased expression of GSH synthetic enzymes and the outcome of blocking this switch. Huh7 cells treated with lithocholic acid (LCA) exhibited a similar pattern of change in GSH synthetic enzyme expression as BDL mice. Nuclear protein levels of Nrf2 fell at 20 hours after LCA treatment, whereas c-Maf and MafG remained persistently induced. These changes translated to ARE nuclear binding activity. Knockdown of c-Maf or MafG individually blunted the LCA-induced decrease in Nrf2 ARE binding and increased ARE-dependent promoter activity, whereas combined knockdown was more effective. Knockdown of c-Maf or MafG individually increased the expression of GSH synthetic enzymes and raised GSH levels, and combined knockdown exerted an additive effect. Ursodeoxycholic acid (UDCA) or S-adenosylmethionine (SAMe) prevented the LCA-induced decrease in expression of GSH synthetic enzymes and promoter activity and prevented the increase in MafG and c-Maf levels. In vivo knockdown of the Maf genes protected against the decrease in GSH enzyme expression, GSH level, and liver injury after BDL. Conclusion: Toxic bile acid induces a switch from Nrf2 to c-Maf/MafG ARE nuclear binding, which leads to decreased expression of GSH synthetic enzymes and GSH levels and contributes to liver injury during BDL. UDCA and SAMe treatment targets this switch. (HEPATOLOGY 2010.) [source]

    New mechanism of transforming growth factor-, signaling in hepatoma: Dramatic up-regulation of tumor initiating cells and epidermal growth factor receptor expression

    Takeshi Nishimura
    Aim:, Transforming growth factor-, (TGF-,) has dual activity in tumor cells. We studied the effect of TGF-, on tumor-initiating cells (TICs), which are similar in self-renewal and differentiation features to normal adult stem cells. Methods:, We used side population (SP) cells that exclude DNA binding dye Hoechst 33342 to obtain TICs, studied the differences in the kinetics of the SP cell response to TGF-, treatment between hepatic tumor cell lines, and performed gene analysis. Results:, SP cells from all cell lines have higher proliferative ability compared to non-SP cells and they are drug resistant. TGF-, treatment increased the percentage of SP cells (%SP) and the survival rate; chemotherapeutic drug resistance developed only in K-251 SP cells. Gene analysis showed that TGF-, up-regulated epidermal growth factor receptor (EGFR) only in K-251 cells. There were no EGFR mutations in K-251, which had been reported in lung cancer. Knockdown of Smad4 using the small-interfering RNA technique in K-251 cells inhibited EGFR overexpression and significantly decreased the %SP. In contrast, the JNK inhibitor had little effect on EGFR expression or the %SP. Conclusion:, TGF-, treatment of K-251 cells causes tumor progression and the anti-cancer drug resistant phenotype by increasing SP. [source]

    Interferon alpha receptors are important for antiproliferative effect of interferon-, against human hepatocellular carcinoma cells

    Bazarragchaa Damdinsuren
    Aim:, Interferon (IFN)-, is a promising drug for the prevention and treatment of hepatocellular carcinoma (HCC). We reported that responders to IFN-,/5-fluorouracil combination therapy expressed higher IFN alpha receptor (IFNAR)2 in tumor. Herein we studied involvement of IFNARs in response to IFN-, in HCC cells. Methods:, IFN-, sensitivity and expression of IFNARs were studied in six HCC cell lines (HuH7, PLC/PRF/5, HLE, HLF, HepG2, Hep3B) using growth-inhibitory and RT-PCR, Western blot assays. Short interfering RNAs (SiRNAs) against IFNAR1 and 2 were used to analyze the role of the IFNARs in IFN-,'s effect and signal transduction. Results:, The expressions of IFNAR1 and 2c mRNAs were higher in PLC/PRF/5 cells than those in other cell lines, and PLC/PRF/5 cells expressed abundant IFNAR2c on their cell membrane. When we examined the sensitivity of the HCC cell lines to the growth-inhibitory effect of IFN-,, PLC/PRF/5 exhibited a significant response, while the other cells were much more resistant. Knockdown of either IFNAR1 or 2 using siRNAs suppressed the IFN-,'s signal transduction (2.5-fold), and decreased the growth-inhibitory effect (down by 69.9% and 67.3%). Conclusion:, The results suggest that the expression of IFNAR1 and IFNAR2c independently are important for the antiproliferative effect of IFN-, in HCC cells. [source]

    Molecular characterization of two novel milk proteins in the tsetse fly (Glossina morsitans morsitans)

    G. Yang
    Abstract Purpose: Milk proteins are an essential component of viviparous reproduction in the tsetse fly. Milk proteins are synthesized in and secreted from the milk gland tissue and constitute 50% of the secretions from which the intrauterine larva derives its nourishment. To understand milk protein function and regulation during viviparous reproduction, milk proteins need to be identified and characterized. Methods: Two putative unknown secretory proteins (GmmMGP2 and GmmMGP3) were selected by bioinformatic analysis of tissue specific tsetse cDNA libraries. RT-PCR analysis was performed to verify their milk gland/fat body specific expression profile. Detailed characterization of developmental and tissue specific expression of these proteins was performed by northern blot analysis and fluorescent in situ hybridization. Functional analysis of the milk gland proteins during the tsetse gonotrophic cycle was performed using RNA interference (RNAi). Results: The predicted proteins from gmmmgp2 and gmmmgp3 are small ,22 kD and contain a high proportion of hydrophobic amino acids and potential phosphorylation sites. Expression of both genes is tissue specific to the secretory cells of the milk gland. Transcript abundance for both genes increases over the course of intrauterine larval development and parallels that of gmmmgp, a well characterized milk protein gene considered to be the major milk protein. Phenotypic analysis of flies after RNA interference treatment revealed a significant effect upon fecundity in the gmmmgp2 knockdown flies, but not the gmmmgp3 flies. Knockdown of gmmmgp2 resulted in disruption of ovulation and consequent oocyte accumulation and degradation. Gmmmgp2 knockdown also had a significant impact on fly mortality. Conclusions: This work identifies two novel genes, the proteins of which appear to function in response to intrauterine larvigenesis in tsetse. These proteins may be nutritional components of the milk secretions provided to the larva from the mother. Phenotypic data from knockdown of gmmmgp2 suggests that this protein may also have a regulatory function given the defect in ovulation observed in knockdown flies. Further analysis of these genes will be important (in conjunction with other milk proteins) for identification of transcriptional regulation mechanisms that direct milk gland/pregnancy specific gene expression. [source]

    The effect of focal adhesion kinase gene silencing on 5-fluorouracil chemosensitivity involves an Akt/NF-,B signaling pathway in colorectal carcinomas

    Yuying Chen
    Abstract Multicellular resistance (MCR) is produced because multicellular spheroids (MCSs) are formed with a broad cell,cell connection when cultured in three-dimensions, which limits the clinical treatment efficacy in solid tumors. Focal adhesion kinase (FAK) plays an important role in apoptosis, survival and cell adhesion between cells and their extracellular matrix. In this study, we investigated the expressions of FAK, Akt and NF-,B in human colorectal cancer (CRC), and the effects of FAK gene silencing on MCSs formation and 5-fluorouracil (5-FU) chemosensitivity in colon carcinoma MCSs culture cells. In CRC samples, FAK, Akt and NF-,B were overexpressed. The positive expression of FAK correlated notably with lymph node metastasis and cellular differentiation. Positive expressions of Akt and NF-,B were significantly related to cellular differentiation and lymph node metastasis, respectively. Furthermore, positive expression of FAK correlated with that of Akt and NF-,B. The expression of FAK was inhibited significantly by a small hairpin RNA targeting FAK. Knockdown of FAK reversed the formation and aggregation of MCSs, significantly decreased the 50% inhibitory concentration of 5-FU, and markedly increased MCS culture cells apoptosis. These effects were associated with reduced levels of Akt and NF-,B. These results indicate that suppressing FAK expression potentiated 5-FU-induced cytotoxicity and contributed to its chemosensitizing effect by suppressing Akt/NF-,B signaling in colon carcinoma MCS culture cells. These data also imply that FAK mediates MCR of CRC through the survival signaling pathway FAK/Akt/NF-,B. [source]

    Prostaglandin E2 promotes cell proliferation via protein kinase C/extracellular signal regulated kinase pathway-dependent induction of c-Myc expression in human esophageal squamous cell carcinoma cells

    Le Yu
    Abstract Overexpression of cyclooxygenase-2 (COX-2) and elevation of its derivative prostaglandin E2 (PGE2) are implicated in human esophageal squamous cell carcinoma. The expression of c-Myc, an oncogenic transcription factor, is also upregulated in this malignant disease. This study sought to elucidate whether a functional connection exists between COX-2/PGE2 and c-Myc in esophageal squamous cell carcinoma. Results showed that PGE2 substantially increased the proliferation of cultured esophageal squamous cell carcinoma cells. In this regard, PGE2 substantially increased the mRNA and protein expression of c-Myc and its association with the binding partner Max. Knockdown of c-Myc by RNA interference also significantly attenuated PGE2 -induced cell proliferation. Further, mechanistic study revealed that PGE2 increased the protein stability and nuclear accumulation of c-Myc via phosphorylation on serine 62 in an extracellular signal regulated kinase (ERK)-dependent manner. To this end, ERK activation by PGE2 was completely abolished by protein kinase C (PKC) inhibitors. Moreover, the effect of PGE2 on c-Myc expression was mimicked by EP2 receptor agonist. In addition, knockdown of EP2 receptor by EP2 siRNA attenuated PGE2 -induced c-Myc expression. Collectively, our findings suggest that PGE2 upregulates c-Myc via the EP2/PKC/ERK pathway. This study sheds new light on the carcinogenic mechanism of PGE2 in esophageal squamous cell carcinoma. © 2009 UICC [source]

    CXCR7 is inducible by HTLV-1 Tax and promotes growth and survival of HTLV-1-infected T cells

    Zhe Jin
    Abstract Human T-lymphotropic virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia (ATL), encodes the potent transcriptional activator Tax, which is required for HTLV-1-induced immortalization of T cells. CXCR7 is an atypical chemokine receptor frequently expressed by tumor cells and known to promote cell growth and survival. We found that HTLV-1-immortalized T cells expressing Tax consistently expressed CXCR7. Induction of Tax in JPX-9 upregulated CXCR7. Wild-type Tax efficiently activated the CXCR7 promoter via a proximal NF-,B site, while a mutant Tax selectively defective in NF-,B activation did not. CCX754, a synthetic CXCR7 antagonist, inhibited cell growth and increased apoptosis of HTLV-1-immortalized T cells. Knockdown of CXCR7 by small interfering RNA also reduced cell growth. Stable expression of CXCR7 in a CXCR7-negative ATL cell line promoted cell growth and survival. Taken together, CXCR7 is inducible by Tax and may play an important role in HTLV-1-induced immortalization of T cells by promoting growth and survival of HTLV-1-infected T cells. © 2009 UICC [source]

    Pml and TAp73 interacting at nuclear body mediate imatinib-induced p53-independent apoptosis of chronic myeloid leukemia cells

    Jin-Hwang Liu
    Abstract Bcr-abl signals for leukemogenesis of chronic myeloid leukemia (CML) and activates ras. Since the function of promyelocytic leukemia protein (pml) is provoked by ras to promote apoptosis and senescence in untransformed cells, the function is probably masked in CML. Imatinib specifically inhibits bcr-abl and induces apoptosis of CML cells. As reported previously, p53wild CML was more resistant to imatinib than that lacking p53. Here, we searched for an imatinib-induced p53 independent proapoptotic mechanism. We found imatinib up-regulated phosphorylation of p38 mitogen-activated protein kinase (MAPK), checkpoint kinase 2 (chk2) and transactivation-competent (TA) p73; expression of pml and bax; formation of PML-nuclear body (NB); and co-localization of TAp73/PML-NB in p53-nonfunctioning K562 and p53mutant Meg-01 CML cells, but not in BCR-ABL - HL60 cells. In K562 cells, with short interfering RNAs (siRNAs), knockdown of pml led to dephosphorylation of TAp73. Knockdown of either pml or TAp73 abolished the imatinib-induced apoptosis. Inhibition of p38 MAPK with SB203580 led to dephosphorylation of TAp73, abolishment of TAp73/PML-NB co-localization, and the subsequent apoptosis. Conversely, interferon ,-2a (IFN,), which increased phosphrylated TAp73 and TAp73/PML-NB co-localization, increased additively apoptosis with imatinib. The imatinib-induced TAp73/PML-NB co-localization was accompanied by co-immpunoprecipitation of TAp73 with pml. The imatinib-induced co-localization was also found in primary CML cells from 3 of 6 patients, including 2 with p53mutant and one with p53wild. A novel p53-independent proapoptotic mechanism using p38 MAPK /pml/TAp73 axis with a step processing at PML-NB and probably with chk2 and bax being involved is hereby evident in some imatinib-treated CML cells. © 2009 UICC [source]

    Upregulation of miR-23a,27a,24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells

    Shenglin Huang
    Abstract Transforming growth factor-beta (TGF-beta) plays a dual and complex role in human cancer. In this report, we observe a specific set of MicroRNAs (miRNAs) changed in response to TGF-beta in human hepatocellular carcinoma (HCC) cells by miRNA microarray screening. A cluster of miRNA, miR-23a,27a,24, is induced in an early stage by TGF-beta in Huh-7 cells. Knockdown of Smad4, Smad2 or Smad3 expression by RNA interference can attenuate the response of miR-23a,27a,24 to TGF-beta addition, indicating that this induction is dependent on Smad pathway. We also explore that miR-23a,27a,24 can function as an antiapoptotic and proliferation-promoting factor in liver cancer cells. In addition, expression of this miRNA cluster is found to be remarkably upregulated in HCC tissues versus normal liver tissues. These findings suggest a novel, alternative mechanism through which TGF-beta could induce specific miRNA expression to escape from tumor-suppressive response in HCC cells. © 2008 Wiley-Liss, Inc. [source]

    Oxygen Tension Regulates the Expression of ANK (Progressive Ankylosis) in an HIF-1-Dependent Manner in Growth Plate Chondrocytes,,

    Raihana Zaka
    Abstract The proximal promoter region of ANK, a gene that codes for a protein that regulates the transport of inorganic pyrophosphate, contains two hypoxia responsive elements (HREs); therefore, we studied the expression and function of ANK at different oxygen tensions. ATDC5 and N1511 clonal chondrocytic cells were cultured in either hypoxia (2% O2) or normoxia (21% O2). Transcript and protein levels of ANK were depressed in hypoxic conditions, as were levels of extracellular pyrophosphate (ePPi). To determine whether HIF-1 was involved in the oxemic response, Hif-1, knockdown cells were exposed to varying oxygen conditions and ANK expression was assessed. Knockdown of Hif-1, resulted in low levels of expression of ANK in hypoxia and normoxia. Chromatin immunoprecipitation (ChIP) assays explored the binding of Hif-1, to ANK HREs and showed that Hif-1, is able to bind to the HREs of ANK more avidly in normoxia than in hypoxia. Furthermore, functional studies of Hif-1, activity using luciferase reporter assays of wildtype and mutagenized HREs showed that only HRE-1 binds Hif-1, in normoxia. Expression of ANK in growth plate and articular cartilage was low in hypoxic regions of the tissues, and higher levels of ANK expression were observed in the synovium and meniscus in regions that have a normally higher oxygen tension. The data suggest that ANK expression and function in vitro and in vivo are repressed in hypoxic environments and that the effect is regulated by HIF-1. [source]

    Carboxypeptidase Z (CPZ) Links Thyroid Hormone and Wnt Signaling Pathways in Growth Plate Chondrocytes,

    Lai Wang
    Abstract Carboxypeptidase Z (CPZ) removes carboxyl-terminal basic amino acid residues, particularly arginine residues, from proteins. CPZ contains a cysteine-rich domain (CRD) similar to the CRD found in the frizzled family of Wnt receptors. We have previously shown that thyroid hormone regulates terminal differentiation of growth plate chondrocytes through activation of Wnt-4 expression and Wnt/,-catenin signaling. The Wnt-4 protein contains a C-terminal arginine residue and binds to CPZ through the CRD. The objective of this study was to determine whether CPZ modulates Wnt/,-catenin signaling and terminal differentiation of growth plate chondrocytes. Our results show that CPZ and Wnt-4 mRNA are co-expressed throughout growth plate cartilage. In primary pellet cultures of rat growth plate chondrocytes, thyroid hormone increases both Wnt-4 and CPZ expression, as well as CPZ enzymatic activity. Knockdown of either Wnt-4 or CPZ mRNA levels using an RNA interference technique or blocking CPZ enzymatic activity with the carboxypeptidase inhibitor GEMSA reduces the thyroid hormone effect on both alkaline phosphatase activity and Col10a1 mRNA expression. Adenoviral overexpression of CPZ activates Wnt/,-catenin signaling and promotes the terminal differentiation of growth plate cells. Overexpression of CPZ in growth plate chondrocytes also removes the C-terminal arginine residue from a synthetic peptide consisting of the carboxyl-terminal 16 amino acids of the Wnt-4 protein. Removal of the C-terminal arginine residue of Wnt-4 by site-directed mutagenesis enhances the positive effect of Wnt-4 on terminal differentiation. These data indicate that thyroid hormone may regulate terminal differentiation of growth plate chondrocytes in part by modulating Wnt signaling pathways through the induction of CPZ and subsequent CPZ-enhanced activation of Wnt-4. [source]