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siRNA
Kinds of siRNA Terms modified by siRNA Selected AbstractsRegulation of PSA secretion and survival signaling by calcium-independent phopholipase A2, in prostate cancer cellsTHE PROSTATE, Issue 12 2009Thomas M. Nicotera Abstract BACKGROUND Serum prostate specific antigen (PSA) levels in prostate cancer patients serve as a useful biomarker for diagnosing and monitoring prostate cancer. Recently, secreted PSA has been characterized as an autocrine survival factor through activation of Akt and induction of AR. In the normal prostate, PSA is secreted in the lumen of prostatic ducts to lyse proteins in the seminal coagulum. METHODS However, the mechanism for constitutive PSA secretion from benign prostate and its transport across the prostate-blood barrier into serum are unknown. Regulation of peptide secretion by iPLA2 -, has been reported in non-prostatic tissue and in prostate tissue iPLA2 -, is reported to be under androgen regulation. We investigated whether iPLA2 plays a role for in PSA secretion by comparing iPLA2 activity and expression in normal prostate epithelial RWPE-1 cells and in LNCaP prostate cancer cells. Expression of the two active iPLA2 -, mRNA splice variants, LH-iPLA2 and SH-iPLA2, were increased and the inhibitory ankyrin-iPLA2 isoform was markedly reduced in LNCaP cells as compared to normal prostate epithelial RWPE-1 cells. RESULTS These changes are consistent with a higher enzymatic activity in LNCaP cells. The iPLA2 -,-specific inhibitor BEL inhibited PSA secretion and induced apoptosis in LNCaP cells. iPLA2 knockdown using SiRNA inhibited PSA secretion, downregulated AR and induced apoptosis. Exogenous PSA suppressed BEL-induced apoptosis and neutralizing anti-PSA antibody blocked the survival effect of PSA. CONCLUSIONS These data indicate that iPLA2 -, participates in regulating PSA secretion and supports the concept that secreted PSA provides an autocrine survival function in LNCaP cells. Prostate 69:1270,1280, 2009. © 2009 Wiley-Liss, Inc. [source] TRAF6 knockdown promotes survival and inhibits inflammatory response to lipopolysaccharides in rat primary renal proximal tubule cellsACTA PHYSIOLOGICA, Issue 3 2010S. Liu Abstract Aim:, TRAF6 is a unique adaptor protein of the tumour necrosis factor receptor-associated factor family that mediates both tumour necrosis factor receptor (TNFR) and interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) signalling. Activation of IL-1R/TLR and TNFR pathways in renal tubular cells contributes to renal injury. This study aimed to investigate if blockade of lipopolysaccharide (LPS)-triggered TLR4 signalling by small interfering RNA (siRNA) targeting TRAF6 protects survival and inhibits inflammatory response in isolated rat renal proximal tubular cells (PTCs). Methods:, PTCs isolated from F344 rat kidneys were transfected with chemically synthesized siRNA targeting TRAF6 mRNA. Real-time quantitative PCR was applied to measure mRNA level of TRAF6, TNF-,, IL-6 and monocyte chemoattractant protein-1 (MCP-1). Protein levels of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase, caspase 3 and cleaved caspase 3 were evaluated by Western blotting. Cell viability was analysed with XTT reagents. Results:, We found that the TRAF6 gene was effectively silenced in PTCs using siRNA. TRAF6 knockdown resulted in reduced TNF-, and IL-6 mRNA expression upon LPS challenge. LPS-induced phosphorylation of JNK and p38 was attenuated in TRAF6 siRNA-transfected cells while the change in the phosphorylation of ERK was not remarkable. TRAF6 knockdown was associated with increased cell viability and reduced protein level of cleaved caspase-3, both, in the absence and presence of LPS. Conclusion:, Our studies suggest that TRAF6 knockdown may inhibit inflammatory response and promote cell survival upon LPS challenge in primary rat proximal renal tubular cells. [source] Normoxic destabilization of ATF-4 depends on proteasomal degradationACTA PHYSIOLOGICA, Issue 4 2010M. Wottawa Abstract Aim:, Hypoxia-inducible gene expression is an important physiological adaptive mechanism in response to a decreased oxygen supply. We have recently described an oxygen- and prolyl-4-hydroxylase (PHD)3-dependent stabilization of the activating transcription factor 4 (ATF-4). The aim of the present study was to examine if the normoxic destabilization of ATF-4 is regulated by oxygen-dependent proteasomal degradation. Methods:, We determined poly-ubiquitination of ATF-4 in normoxia compared to hypoxia by immunoprecipitation and immunoblots. Furthermore, we analysed the expression of the ATF-4 target gene GADD153 as a function of oxygen concentration. Results:, ATF-4 protein levels were not detectable in normoxia. Normoxic degradation correlated with an oxygen-dependent poly-ubiquitination of ATF-4, which was hindered by hypoxic incubation of the cells. As a result of hypoxia, GADD153 was expressed. The hypoxic GADD153 expression was attenuated or increased by transfecting the cells with ATF-4 siRNA or PHD3 siRNA respectively. Conclusion:, Our results demonstrate the involvement of oxygen-dependent proteasomal degradation of ATF-4 in the hypoxia-induced expression of GADD153. Taken together, hypoxia/PHD3-regulated stabilization of ATF-4 by hindering oxygen-dependent degradation may play a critical role in linking cell fate decisions to oxygen availability. [source] TOGp regulates microtubule assembly and density during mitosis and contributes to chromosome directional instabilityCYTOSKELETON, Issue 8 2009Lynne Cassimeris Abstract TOGp, a member of the XMAP215 MAP family, is required for bipolar mitotic spindle assembly. To understand how TOGp contributes to spindle assembly, we examined microtubule dynamics after depleting TOGp by siRNA. Fluorescence recovery after photobleaching of GFP-tubulin demonstrated that spindle microtubule turnover is slowed two-fold in the absence of TOGp. Consistent with photobleaching results, microtubule regrowth after washout of the microtubule depolymerizing drug nocodazole was slower at the centrosomes and in the vicinity of mitotic chromatin in cells depleted of TOGp. The slower microtubule turnover is likely due to either nucleation or the transitions of dynamic instability because TOGp depletion did not effect the rate of plus end growth, measured by tracking EB1-GFP at microtubule ends. In contrast, microtubule regrowth after nocodazole washout was unaffected by prior depletion of TACC3, a centrosomal protein that interacts with TOGp. Kinetochore fibers in both untreated and TOGp-depleted cells were stable to incubation at 4°C or lysis in buffer containing calcium indicating that stable kinetochore-microtubule attachments are formed in the absence of TOGp. Depletion of TOGp, but not TACC3, reduced kinetochore oscillations during prometaphase/metaphase. Defects in oscillations are not due simply to multipolarity or loss of centrosome focus in the TOGp-depleted cells, since kinetochore oscillations appear normal in cells treated with the proteosome inhibitor MG132, which also results in multipolar spindles and centrosome fragmentation. We hypothesize that TOGp is required for chromosome motility as a downstream consequence of reduced microtubule dynamics and/or density. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Inflammatory cytokines augments TGF-,1-induced epithelial-mesenchymal transition in A549 cells by up-regulating T,R-ICYTOSKELETON, Issue 12 2008Xiangde Liu Abstract Epithelial-mesenchymal transition (EMT) is believed to play an important role in fibrosis and tumor invasion. EMT can be induced in vitro cell culture by various stimuli including growth factors and matrix metalloproteinases. In this study, we report that cytomix (a mixture of IL-1,, TNF-, and IFN-,) significantly enhances TGF-,1-induced EMT in A549 cells as evidenced by acquisition of fibroblast-like cell shape, loss of E-cadherin, and reorganization of F-actin. IL-1, or TNF-, alone can also augment TGF-,1-induced EMT. However, a combination of IL-1, and TNF-, or the cytomix is more potent to induce EMT. Cytomix, but not individual cytokine of IL-1,, TNF-, or IFN-,, significantly up-regulates expression of TGF-, receptor type I (T,R-I). Suppression of T,R-I, Smad2 or Smad3 by siRNA partially blocks EMT induction by cytomix plus TGF-,1, indicating cytomix augments TGF-,1-induced EMT through enhancing T,R-I and Smad signaling. These results indicate that inflammatory cytokines together with TGF-,1 may play an important role in the development of fibrosis and tumor progress via the mechanism of epithelial-mesenchymal transition. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Alpha-smooth muscle actin expression enhances cell traction forceCYTOSKELETON, Issue 4 2007Jianxin Chen Abstract Using an established corneal stromal cell differentiation model, we manipulated ,-smooth muscle actin (,-SMA) protein expression levels in fibroblasts by treating them with TGF-,1, bFGF, TGF-, type I receptor inhibitor (SB-431542), and siRNA against ,-SMA. The corresponding cell traction forces (CTFs) were determined by cell traction force microscopy. With all these treatments, we found that ,-SMA is not required for CTF induction, but its expression upregulates CTF. This upregulation involves the modification of stress fibers but does not appear to relate to non-muscle myosin II expression or ,-actin expression. Moreover, there exists a linear relationship between ,-SMA protein expression level and CTF magnitude. Finally, CTFs were found to vary among a population of myofibroblasts, suggesting that ,-SMA protein expression levels of individual cells also vary. Cell Motil. Cytoskeleton 2007. © 2006 Wiley-Liss, Inc. [source] RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisationCYTOSKELETON, Issue 3 2005Elen Griffith Abstract TES was originally identified as a candidate tumour suppressor gene and has subsequently been found to encode a novel focal adhesion protein. As well as localising to cell-matrix adhesions, TES localises to cell-cell contacts and to actin stress fibres. TES interacts with a variety of cytoskeletal proteins including zyxin, mena, VASP, talin and actin. There is evidence that TES may function in actin-dependent processes as overexpression of TES results in increased cell spreading and decreased cell motility. Together with TES's interacting partners, these data suggest that TES might be involved in regulation of the actin cytoskeleton. Here, for the first time, we have used RNAi to successfully knockdown TES in HeLa cells and we demonstrate that loss of TES from focal adhesions results in loss of actin stress fibres. Similarly, and as previously reported, RNAi-mediated knockdown of zyxin results in loss of actin stress fibres. TES siRNA treated cells show reduced RhoA activity, suggesting that the Rho GTPase pathway may be involved in the TES RNAi-induced loss of stress fibres. We have also used RNAi to examine the requirement of TES and zyxin for each other's localisation at focal adhesions, and we propose a hierarchy of recruitment, with zyxin being first, followed by VASP and then TES. Cell Motil. Cytoskeleton 60:140,152, 2005. © 2005 Wiley-Liss, Inc. [source] Delivery of small interfering RNA with a synthetic collagen poly(Pro-Hyp-Gly) for gene silencing in vitro and in vivoDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 8 2010Taro Adachi Silencing gene expression by small interfering RNAs (siRNAs) has become a powerful tool for the genetic analysis of many animals. However, the rapid degradation of siRNA and the limited duration of its action in vivo have called for an efficient delivery technology. Here, we describe that siRNA complexed with a synthetic collagen poly(Pro-Hyp-Gly) (SYCOL) is resistant to nucleases and is efficiently transferred into cells in vitro and in vivo, thereby allowing long-term gene silencing in vivo. We found that the SYCOL-mediated local application of siRNA targeting myostatin, coding a negative regulator of skeletal muscle growth, in mouse skeletal muscles, caused a marked increase in the muscle mass within a few weeks after application. Furthermore, in vivo administration of an anti-luciferase siRNA/SYCOL complex partially reduced luciferase expression in xenografted tumors in vivo. These results indicate a SYCOL-based non-viral delivery method could be a reliable simple approach to knockdown gene expression by RNAi in vivo as well as in vitro. [source] The role of twist during palate development,DEVELOPMENTAL DYNAMICS, Issue 10 2008Wenli Yu Abstract In palatogenesis, the MEE (Medial Edge Epithelium) cells disappear when palates fuse. We hypothesize that the MEE cells undergo EMT (Epithelial-Mesenchymal Transition) to achieve mesenchyme confluence. Twist has an important role in EMT for tumor metastasis. The purpose of this study was to analyze Twist function during palatal fusion. Twist protein was expressed in palatal shelves and MEE both in vivo and in vitro just prior to fusion. Twist mRNA increased in chicken palates 3 and 6 hr after TGF,3 treatment. Palatal fusion was decreased when cultured palatal shelves were treated with 200 nM Twist siRNA and the subcellular localization of ,-catenin was altered. Twist mRNA decreased in palatal shelves treated with TGF,3 neutralizing antibody or LY294002, a specific phosphatidylinositol-3 kinase (PI-3K) inhibitor. In summary, Twist is downstream of TGF,3 and PI-3K pathways during palatal fusion. However, decreasing Twist with siRNA did not completely block palate fusion, indicating that the function of Twist may be duplicated by other transcription factors. Developmental Dynamics 237:2716,2725, 2008. © 2008 Wiley-Liss, Inc. [source] Divergent roles of the DEAD-box protein BS-PL10, the urochordate homologue of human DDX3 and DDX3Y proteins, in colony astogeny and ontogenyDEVELOPMENTAL DYNAMICS, Issue 6 2006Amalia Rosner Abstract Proteins of the highly conserved PL-10 (Ded1P) subfamily of DEAD-box family, participate in a wide variety of biological functions. However, the entire spectrum of their functions in both vertebrates and invertebrates is still unknown. Here, we isolated the Botryllus schlosseri (Urochordata) homologue, BS-PL10, revealing its distributions and functions in ontogeny and colony astogeny. In botryllid ascidians, the colony grows by increasing the number of modular units (each called a zooid) through a whole colony synchronized and weekly cyclical astogenic budding process (blastogenesis). At the level of the colony, both BS-PL10 mRNA and its protein (78 kDa) fluctuate in a weekly pattern that corresponds with the animal's blastogenic cycle, increasing from blastogenic stage A to blastogenic stage D. At the organ/module level, a sharp decline is revealed. Primary and secondary developing buds express high levels of BS-PL10 mRNA and protein at all blastogeneic stages. These levels are reduced four to nine times in the new set of functional zooids. This portrait of colony astogeny differed from its ontogeny. Oocytes and sperm cells express high levels of BS-PL10 protein only at early stages of development. Young embryos reveal background levels with increased expressions in some organs at more developed stages. Results reveal that higher levels of BS-PL10 mRNA and protein are characteristic to multipotent soma and germ cells, but patterns deviate between two populations of differentiating stem cells, the stem cells involved in weekly blastogenesis and stem cells involved in embryogenesis. Two types of experimental manipulations, zooidectomy and siRNA assays, have confirmed the importance of BS-PL10 for cell differentiation and organogenesis. BS-PL10 (phylogenetically matching the animal's position in the evolutionary tree), is the only member of this subfamily in B. schlosseri, featuring a wide range of biological activities, some of which represent pivotal roles. The surprising weekly cyclical expression and the participation in cell differentiation posit this molecule as a model system for studying PL10 protein subfamily. Developmental Dynamics 235:1508,1521, 2006. © 2006 Wiley-Liss, Inc. [source] LAR protein tyrosine phosphatase receptor associates with TrkB and modulates neurotrophic signaling pathwaysDEVELOPMENTAL NEUROBIOLOGY, Issue 13 2006Tao Yang Abstract The identities of receptor protein tyrosine phosphatases (PTPs) that associate with Trk protein tyrosine kinase (PTK) receptors and modulate neurotrophic signaling are unknown. The leukocyte common antigen-related (LAR) receptor PTP is present in neurons expressing TrkB, and like TrkB is associated with caveolae and regulates survival and neurite outgrowth. We tested the hypothesis that LAR associates with TrkB and regulates neurotrophic signaling in embryonic hippocampal neurons. Coimmunoprecipitation and coimmunostaining demonstrated LAR interaction with TrkB that is increased by BDNF exposure. BDNF neurotrophic activity was reduced in LAR,/, and LAR siRNA-treated LAR+/+ neurons and was augmented in LAR-transfected neurons. In LAR,/, neurons, BDNF-induced activation of TrkB, Shc, AKT, ERK, and CREB was significantly decreased; while in LAR-transfected neurons, BDNF-induced CREB activation was augmented. Similarly, LAR+/+ neurons treated with LAR siRNA demonstrated decreased activation of Trk and AKT. LAR is known to activate the Src PTK by dephosphorylation of its negative regulatory domain and Src transactivates Trk. In LAR,/, neurons, or neurons treated with LAR siRNA, phosphorylation of the Src regulatory domain was increased (indicating Src inactivation), consistent with a role for Src in mediating LAR's ability to up-regulate neurotrophic signaling. Interactions between LAR, TrkB, and Src were further confirmed by the findings that Src coimmunoprecipitated with LAR, that the Src inhibitor PP2 blocked the ability of LAR to augment TrkB signaling, and that siRNA-induced depletion of Src decreased LAR interaction with TrkB. These studies demonstrate that receptor PTPs can associate with Trk complexes and promote neurotrophic signaling and point to receptor PTP-based strategies as a novel approach for modulating neurotrophin function. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] A possible role for dihydrodiol dehydrogenase in the formation of benzo[a]pyrene-DNA adducts in lung cancer cells and tumor tissuesENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2007Ya-Wen Cheng Abstract Epidemiological studies indicate that there is a gender difference in the susceptibility to tobacco and environmental carcinogens, and this gender difference is suspected to result in a higher risk for lung cancer among women. However, the molecular mechanisms underlying this sexual dimorphism remain unclear. In the present study, we have evaluated the roles of CYP1A1 and dihydrodiol dehydrogenase (DDH) in the formation of benzo[a]pyrene (BaP) DNA adducts in various lung cancer cell lines. Among six lung cancer cell lines tested, higher adduct levels were observed in CL-3 and CL1-1 cells, which had relatively high expression of both CYP1A1 and DDH isoform 1 (DHH1). To determine whether a reduction in DDH expression changed the adduct levels, an siRNA was used to knock down DDH1 expression in CL-3 cells. The BaP adduct levels in siDDH-CL-3 cells increased 1.4,2.2-fold relative to that of the parental CL-3 cells. We also examined BaP-like DNA adducts, and CYP1A1 and DDH1 expression by immunohistochemistry in 120 lung tumors. Detection of DNA adducts correlated with CYP1A1-positive tumors (P = 0.023), but not with DDH1-positive tumors. In addition, 28 of 33 tumors (85%) that were CYP1A1-positive and DDH1-negative contained detectable levels of DNA adducts, a proportion that was higher than for tumors from the other three categories of CYP1A1 and DDH1 expression (P = 0.012). Finally, a greater proportion of adduct-positive tumors from females were CYP1A1-positive/DDH1-negative (45.3%) than were tumors from males (27.3%). These results suggest that the reduction of DDH expression in lung tumors may contribute to an increase in DNA adduct levels, which may be partly responsible for the higher susceptibility of female lung cancer patients to DNA damage. Environ. Mol. Mutagen., 2007. © 2006 Wiley-Liss, Inc. [source] Regulation of GADD153 induced by mechanical stress in cardiomyocytesEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2009W. P. Cheng Abstract Background, Growth arrest and DNA damage-inducible gene 153 (GADD153), an apoptosis regulated gene, increased during endoplasmic reticulum stress. However, the expression of GADD153 in cardiomyocytes under mechanical stress is little known. We aimed to investigate the regulation mechanism of GADD153 expression and apoptosis induced by mechanical stress in cardiomyocytes. Materials and methods, Aorta-caval shunt was performed in adult Sprague,Dawley rats to induce volume overload. Rat neonatal cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles min,1. Results, The increased ventricular dimension measured using echocardiography in the shunt group (n = 8) was reversed to normal by treatment with chaperon 4-phenylbutyric acid (PBA) (n = 8) at 500 mg kg,1 day,1 orally for 3 days. GADD153 protein and mRNA were up-regulated in the shunt group when compared with sham group (n = 8). Treatment with PBA reversed the protein of GADD153 to the baseline values. The TUNEL assay showed that PBA reduced the apoptosis induced by volume overload. Cyclic stretch significantly increased GADD153 protein and mRNA expression after 14 h of stretch. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA and tumour necrosis factor-, (TNF-,) antibody 30 min before stretch, reduced the induction of GADD153 protein. Stretch increased, while GADD153-Mut plasmid, SP600125 and TNF-, antibody abolished the GADD153 promoter activity induced by stretch. GADD153 mediated apoptosis induced by stretch was reversed by GADD153 siRNA, GADD153-Mut plasmid and PBA. Conclusions, Mechanical stress enhanced apoptosis and GADD153 expression in cardiomyocytes. Treatment with PBA reversed both GADD153 expression and apoptosis induced by mechanical stress in cardiomyocytes. [source] Involvement of hypoxia-inducible factor-1 HiF(1,) in IgE-mediated primary human basophil responsesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2009Vadim V. Sumbayev Abstract Basophils play a pivotal role in regulating chronic allergic inflammation as well as angiogenesis. Here, we show for the first time that IgE-mediated activation of primary human basophils results in protein accumulation of the ,-subunit of hypoxia-inducible factor 1, (HIF-1,), which is differentially regulated compared with signals controlling histamine release. HIF-1 facilitates cellular adaptation to hypoxic conditions such as inflammation and tumour growth by controlling glycolysis, angiogenesis and cell adhesion. ERK and p38 MAPK, but not reactive oxygen species (ROS), ASK1 or PI 3-kinase, were critical for IgE-mediated accumulation of HIF-1,, although the latter crucially affected degranulation. Abrogating HIF-1, expression in basophils using siRNA demonstrated that this protein is essential for vascular endothelial growth factor (VEGF) mRNA expression and, consequently, release of VEGF protein. In addition, HIF-1, protein alters IgE-induced ATP depletion in basophils, thus also supporting the production of the pro-allergic cytokine IL-4. [source] PI3K limits TNF- , production in CD16-activated monocytesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2009Phillip R. Kramer Abstract IgG complexes bind to Fc receptor family members Fc,RI (CD64), Fc,RII (CD32) and Fc,RIII (CD16), activating cell MAPK and PI3K resulting in increased cytokine production from particular leukocytes. The signaling molecules involved in cytokine production after cross-linking CD16 have not been determined in monocytes. To address this question, TNF-,, IL-1, and IL-6 were measured in activated monocytes after inhibiting MEK1/2, PI3K and glycogen synthase kinase-, (GSK-3,). The roles of GSK-3, and NF-,B were then determined using reporter assays and siRNA treatment. The data suggested that an MAPK pathway stimulated TNF-, release but that active PI3K limited TNF-,, IL-1, and IL-6 cytokine production after cross-linking CD16. PI3K was also shown to limit nuclear translocation of NF-,B. The limiting effect of PI3K on TNF-, production from activated monocytes depended on the decrease of GSK-3, activity, which significantly reduced the transactivation of NF-,B. Moreover, the TNF-, production induced by CD16 cross-linking was reduced in monocytes after treatment with siRNA against NF-,B, implying that this transcription factor functioned in TNF-, production. The results suggest that CD16 cross-linking activated PI3K and that active PI3K limited TNF-, production by inhibiting GSK-3, activity, that blocked the action of NF-,B. [source] Requirement of the tumour suppressor APC for the clustering of PSD-95 and AMPA receptors in hippocampal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2007Atsushi Shimomura Abstract Mutations in the adenomatous polyposis coli (APC) gene are associated with familial adenomatous polyposis and sporadic colorectal tumours. The APC gene is expressed ubiquitously in various tissues, especially throughout the large intestine and central nervous system (CNS). In the CNS, the expression of the APC protein is highest during embryonic and early postnatal development. APC associates through its C-terminal region with postsynaptic density (PSD)-95, a neuronal protein that participates in synapse development. Here, we examined the involvement of APC in synaptogenesis. In cultured hippocampal neurons, both overexpression of a dominant-negative construct that disrupts the APC,PSD-95 interaction and knockdown of APC expression using small interfering RNA (siRNA) inhibited the clustering of PSD-95 and a glutamate receptor subunit, and reduced alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)-induced activity of AMPA receptors; however, the clustering of an N -methyl- d -aspartate (NMDA) receptor subunit was unaffected. These results are suggestive of APC involvement in the development of glutamatergic synapses. [source] Impact of basic FGF expression in astrocytes on dopamine neuron synaptic function and developmentEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2006Caroline Forget Abstract Behavioural sensitization to amphetamine (AMPH) requires action of the drug in the ventral midbrain where dopamine (DA) neurons are located. In vivo studies suggest that AMPH sensitization requires enhanced expression of basic fibroblast growth factor (bFGF) in the nucleus of midbrain astrocytes. One idea is that the AMPH-induced increase in bFGF expression in astrocytes leads to enhanced secretion of this peptide and to long-term plasticity in DA neurons. To study directly the effects of astrocytic expression of bFGF on DA neurons, we established a cell-culture model of mesencephalic astrocytes and DA neurons. Immunolabelling showed that even in the absence of a pharmacological stimulus, the majority of mesencephalic astrocytes in culture express bFGF at a nuclear level. Arguing against the idea that bFGF was secreted, bFGF was undetectable in the extracellular medium (below 10 pg/mL). However, supplementing culture medium with exogenous bFGF at standard concentrations (20 ng/mL) led to a dramatic change in the morphology of astrocytes, increased spontaneous DA release, and inhibited synapse formation by individual DA neurons. RNA interference (siRNA) against bFGF mRNA, caused a reduction in DA release but produced no change in synaptic development. Together these data demonstrate that under basal conditions (in the absence of a pharmacological stimulus such as amphetamine) bFGF is not secreted even though there is abundant nuclear expression in astrocytes. The effects of bFGF seen here on DA neurons are thus likely to be mediated through more indirect glial,neuronal interactions, leading to enhanced DA release without a necessary change in synapse number. [source] MyD88 expression in the rat dental follicle: implications for osteoclastogenesis and tooth eruptionEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 4 2010Dawen Liu Liu D, Yao S, Wise GE. MyD88 expression in the rat dental follicle: implications for osteoclastogenesis and tooth eruption. Eur J Oral Sci 2010; 118: 333,341. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Myeloid differentiation factor 88 (MyD88) is a key adaptor molecule in the interleukin (IL)-1 and IL-18 toll-like receptor signaling pathways. Because MyD88 is present in dental follicle (DF) cells in vitro, the purpose of this study was to determine its chronological expression in vivo, as well as its possible role in osteoclastogenesis and tooth eruption. An oligo DNA microarray was used to determine expression of the Myd88 gene in vivo in the DFs from the first mandibular molars of postnatal rats from days 1 to 11. The results showed that MyD88 was expressed maximally on day 3. Using small interfering RNA (siRNA) to knock down MyD88 expression in the DF cells also reduced the expression of the nuclear factor-kappa B-1 (NFKB1) and monocyte chemoattractant protein 1 (MCP-1) genes. Interleukin-1, up-regulated the expression of NFKB1, MCP-1, and receptor activator of nuclear factor kappa B ligand (RANKL), but knockdown of MyD88 nullified this IL-1, effect. Conditioned medium from DF cells with MyD88 knocked down had reduced chemotactic activity for mononuclear cells and reduced osteoclastogenesis, as opposed to controls. In conclusion, the maximal expression of MyD88 in the DF of postnatal day 3 rats may contribute to the major burst of osteoclastogenesis needed for eruption by up-regulating MCP-1 and RANKL expression. [source] Effect of siRNA terminal mismatches on TRBP and Dicer binding and silencing efficacyFEBS JOURNAL, Issue 22 2009Hemant K. Kini To enhance silencing and avoid off-target effects, siRNAs are often designed with an intentional bias to ensure that the end of the siRNA that contains the guide strand 5, end is less stably hybridized relative to the end containing the passenger strand 5, end. One means by which this is accomplished is to introduce a terminal mismatch, typically by changing the passenger strand sequence to impair its hybridization with the guide strand 5, end. However, there are conflicting reports about the influence of terminal mismatches on the silencing efficacy of siRNAs. Here, the silencing efficiency of siRNAs with a terminal mismatch generated either by altering the guide strand (at the 5, end, nucleotide 1) or the passenger strand (nucleotide 19 from the 5, end) was examined. Subsequently, we studied the relationship between the silencing efficiency of the siRNAs and their binding to the RNA-induced silencing complex loading complex proteins HIV transactivating response RNA-binding protein and Dicer in H1299 cytoplasmic extracts. Binding of siRNA and the transactivating response RNA-binding protein was significantly reduced by terminal mismatches, which largely agrees with the reduction in eventual silencing efficacy of the siRNAs. Single terminal mismatches led to a small increase in Dicer binding, as expected, but this did not lead to an improvement in silencing activity. These results demonstrate that introduction of mismatches to control siRNA asymmetry may not always improve target silencing, and that care should be taken when designing siRNAs using this technique. [source] Inhibition of PI3K/Akt partially leads to the inhibition of PrPC -induced drug resistance in gastric cancer cellsFEBS JOURNAL, Issue 3 2009Jie Liang Cellular prion protein (PrPC), a glycosyl-phosphatidylinositol-anchored membrane protein with unclear physiological function, was previous found to be upregulated in adriamycin (ADR)-resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901. Overexpression of PrPC in gastric cancer has certain effects on drug accumulation through upregulation of P-glycoprotein (P-gp), which is suggested to play an important role in determining the sensitivity of tumor cells to chemotherapy and is linked to activation of the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway. In the present study, we further investigate the role of the PI3K/Akt pathway in PrPC -induced multidrug-resistance (MDR) in gastric cancer. Immunohistochemistry and confocal microscope detection suggest a positive correlation between PrPC and phosphorylated Akt (p-Akt) expression in gastric cancer. Using established stable PrPC transfectant cell lines, we demonstrated that the level of p-Akt was increased in PrPC -transfected cells. Inhibition of PrPC expression by RNA interference resulted in decreased p-Akt expression. Inhibition of the PI3K/Akt pathway by one of its specific inhibitors, LY294002, or by Akt small interfering RNA (siRNA) resulted in decreased multidrug resistance of SGC7901 cells, partly through downregulation of P-gp induced by PrPC. Taken together, our results suggest that PrPC -induced MDR in gastric cancer is associated with activation of the PI3K/Akt pathway. Inhibition of PI3K/Akt by LY2940002 or Akt siRNA leads to inhibition of PrPC -induced drug resistance and P-gp upregulation in gastric cancer cells, indicating a possible novel mechanism by which PrPC regulates gastric cancer cell survival. [source] Down-regulation of heme oxygenase-2 is associated with the increased expression of heme oxygenase-1 in human cell linesFEBS JOURNAL, Issue 23 2006Yuanying Ding Intracellular heme concentrations are maintained in part by heme degradation, which is catalyzed by heme oxygenase. Heme oxygenase consists of two structurally related isozymes, HO-1 and HO-2. Recent studies have identified HO-2 as a potential oxygen sensor. To gain further insights into the regulatory role of HO-2 in heme homeostasis, we analyzed the expression profiles of HO-2 and the biochemical consequences of HO-2 knockdown with specific short interfering RNA (siRNA) in human cells. Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and HepG2 hepatoma. Down-regulation of HO-2 expression with siRNA against HO-2 (siHO-2) caused induction of HO-1 expression at both mRNA and protein levels in HeLa and HepG2 cells. In contrast, knockdown of HO-1 expression did not noticeably influence HO-2 expression. HO-2 knockdown prolonged the half-life of HO-1 mRNA twofold in HeLa cells. Transient transfection assays in HeLa cells revealed that the 4.5-kb human HO-1 gene promoter was activated with selective knockdown of HO-2 in a sequence-dependent manner. Moreover, HO-2 knockdown caused heme accumulation in HeLa and HepG2 cells only when exposed to exogenous hemin. HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellular heme homeostasis. These results suggest that HO-2 may down-regulate the expression of HO-1, thereby directing the co-ordinated expression of HO-1 and HO-2. [source] Aly/,REF, a factor for mRNA transport, activates RH gene promoter functionFEBS JOURNAL, Issue 11 2005Hiroshi Suganuma The rhesus (Rh) blood group antigens are of considerable importance in transfusion medicine as well as in newborn or autoimmune hemolytic diseases due to their high antigenicity. We identified a major DNaseI hypersensitive site at the 5, flanking regions of both RHD and RHCE exon 1. A 34 bp fragment located at ,191 to ,158 from a translation start position, and containing the TCCCCTCCC sequence, was involved in enhancing promoter activity, which was assessed by luciferase reporter gene assay. A biotin-labelled 34 bp probe isolated an mRNA transporter protein, Aly/REF. The specific binding of Aly/REF to RH promoter in erythroid was confirmed by chromatin immunoprecipitation assay. The silencing of Aly/REF by siRNA reduced not only the RH promoter activity of the reporter gene but also transcription from the native genome. These facts provide second proof of Aly/REF as a transcription coactivator, initially identified as a coactivator for the TCR, enhancer function. Aly/REF might be a novel transcription cofactor for erythroid-specific genes. [source] Delivery of Nucleic Acids through the Controlled Disassembly of Multifunctional NanocomplexesADVANCED FUNCTIONAL MATERIALS, Issue 24 2009Mahmoud Elsabahy Abstract In this study, novel pH-responsive polyion complex micelles (PICMs) were developed for the efficient delivery of nucleic acid drugs, such as antisense oligonucleotide (AON) and short interfering RNA (siRNA). The PICMs consisted of a poly(amidoamine) (PAMAM) dendrimer,nucleic acid core and a detachable poly(ethylene glycol)- block -poly(propyl methacrylate- co -methacrylic acid) (PEG- b -P(PrMA- co -MAA)) shell. The micelles displayed a mean hydrodynamic diameter ranging from 50 to 70,nm, a narrow size distribution, and a nearly neutral surface charge. They could be lyophilized without any additives and stored in dried form. Upon redispersion in water, no change in complexation efficiency or colloidal properties was observed. Entry of the micelles into cancers cells was mediated by a monoclonal antibody fragment positioned at the extremity of the PEG segment via a disulfide linkage. Upon cellular uptake and protonation of the MAA units in the acidic endosomal environment, the micelles lost their corona, thereby exposing their positively charged endosomolytic PAMAM/nucleic acid core. When these pH-responsive targeted PICMs were loaded with AON or siRNAs that targeted the oncoprotein Bcl-2, they exhibited a greater transfection activity than nontargeted PICMs or commercial PAMAM dendrimers. Moreover, their nonspecific cytotoxicity was lower than that of PAMAM. The pH-responsive PICMs reported here appear as promising carriers for the delivery of nucleic acids. [source] Lipid-Like Nanoparticles for Small Interfering RNA Delivery to Endothelial CellsADVANCED FUNCTIONAL MATERIALS, Issue 19 2009Seung-Woo Cho Abstract Here, nanoparticles composed of lipid-like materials (lipidoids) to facilitate non-viral delivery of small interfering RNA (siRNA) to endothelial cells (ECs) are developed. Nanoparticles composed of siRNA and lipidoids with small size (,200,nm) and positive charge (,34,mV) are formed by self-assembly of lipidoids and siRNA. Ten lipidoids are synthesized and screened for their ability to facilitate the delivery of siRNA into ECs. Particles composed of leading lipidoids show significantly better delivery to ECs than a leading commercially available transfection reagent, Lipofectamine 2000. As a model of potential therapeutic application, nanoparticles composed of the top performing lipidoid, NA114, are studied for their ability to deliver siRNA targeting anti-angiogenic factor (SHP-1) to human ECs. Silencing of SHP-1 expression significantly enhances EC proliferation and decreases EC apoptosis under a simulated ischemic condition. [source] Anti-apoptotic effect of the basic helix-loop-helix (bHLH) transcription factor DEC2 in human breast cancer cellsGENES TO CELLS, Issue 4 2010Yang 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 PRMT5GENES TO CELLS, Issue 1 2009Takanori 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] CANu1, a novel nucleolar protein, accumulated on centromere in response to DNA damageGENES TO CELLS, Issue 8 2008Choong-Ryoul Sihn Single nucleotide polymorphism is known to be an ideal marker to detect human diseases. We isolated a novel human gene, to be called as CANu1, by the large-scale genome-wide association analysis to screen specific Single nucleotide polymorphisms in colon cancer. It is mapped to chromosome 14q11.2 and its transcript contains a 948-nt open reading frame encoding a protein of 315 aa. Here, we observed that green fluorescence protein (GFP)-fused CANu1 protein was localized to nucleoli and the C-termini of CANu1 protein were essential for its localization. Moreover, the silencing of the CANu1 gene by siRNA caused ribosomal stress leading to G1 cell cycle arrest, the induction of p53 protein, and the translocation of B23 protein. In addition, CANu1 protein was translocated from nucleolus to nuclear foci in response to UV damage. Interestingly, the mobility of a GFP-CANu1 protein in the UV damaged cells was two times faster than non-irradiated cells. Taken together, we report that a novel nucleolar protein, CANu1, is essential to maintain ribosomal structure and responsive upon UV damage. [source] Sin1 binds to both ATF-2 and p38 and enhances ATF-2-dependent transcription in an SAPK signaling pathwayGENES TO CELLS, Issue 11 2006Chieko Makino Yeast Sin1 binds to the Sty1 kinase, a member of the stress-activated kinases (SAPKs), and is required for stress-induced phosphorylation and activation of the transcription factor Atf1, a homolog of the vertebrate-activating transcription factor-2 (ATF-2). Here we report that mammalian Sin1 plays an important role in the SAPK signaling pathway by binding to both ATF-2 and p38. In response to stress, ATF-2, a member of the ATF/cAMP response element-binding protein family, is phosphorylated by p38/Jun NH2 -terminal protein kinase and activates the transcription of apoptosis-related genes. In contrast, in response to serum stimulation, ATF-2 is phosphorylated via the Ras effector pathway and leads to the induction of growth-related genes. We found that Sin1 binds directly to both ATF-2 and p38. Sin1 over-expression enhanced osmotic stress-induced phosphorylation of ATF-2 and ATF-2-mediated transcription, whereas knockdown of Sin1 expression by siRNA suppressed these responses. Moreover, a reduction in Sin1 expression suppressed osmotic stress-induced apoptosis and the expression of Gadd45,, one of the ATF-2 target genes that is correlated with apoptosis. Decreased Sin1 expression, however, did not affect the serum stimulation-induced phosphorylation of ATF-2. Sin1 may contribute to ATF-2 signaling specificity by acting as a nuclear scaffold. [source] Regulation of mitotic function of Chk1 through phosphorylation at novel sites by cyclin-dependent kinase 1 (Cdk1)GENES TO CELLS, Issue 5 2006Takashi Shiromizu Chk1 is phosphorylated at Ser317 and Ser345 by ATR in response to stalled replication and genotoxic stresses. This Chk1 activation is thought to play critical roles in the prevention of premature mitosis. However, the behavior of Chk1 in mitosis remains largely unknown. Here we reported that Chk1 was phosphorylated in mitosis. The reduction of this phosphorylation was observed at the metaphase-anaphase transition. Two-dimensional phosphopeptide mapping revealed that Chk1 phosphorylation sites in vivo were completely overlapped with the in vitro sites by cyclin-dependent protein kinase (Cdk) 1 or by p38 MAP kinase. Ser286 and Ser301 were identified as novel phosphorylation sites on Chk1. Treatment with Cdk inhibitor butyrolactone I induced the reduction of Chk1-S301 phosphorylation, although treatment with p38-specific inhibitor SB203580 or siRNA did not. In addition, ionizing radiation (IR) or ultraviolet (UV) light did not induce Chk1 phosphorylation at Ser317 and Ser345 in nocodazole-arrested mitotic cells. These observations imply the regulation of mitotic Chk1 function through Chk1 phosphorylation at novel sites by Cdk1. [source] Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA DeliveryADVANCED FUNCTIONAL MATERIALS, Issue 9 2009Koen Raemdonck Abstract The successful therapeutic application of small interfering RNA (siRNA) largely relies on the development of safe and effective delivery systems that are able to guide the siRNA therapeutics to the cytoplasm of the target cell. In this report, biodegradable cationic dextran nanogels are engineered by inverse emulsion photopolymerization and their potential as siRNA carriers is evaluated. The nanogels are able to entrap siRNA with a high loading capacity, based on electrostatic interaction. Confocal microscopy and flow cytometry analysis reveal that large amounts of siRNA-loaded nanogels can be internalized by HuH-7 human hepatoma cells without significant cytotoxicity. Following their cellular uptake, it is found that the nanogels are mainly trafficked towards the endolysosomes. The influence of two different strategies to enhance endosomal escape on the extent of gene silencing is investigated. It is found that both the application of photochemical internalization (PCI) and the use of an influenza-derived fusogenic peptide (diINF-7) can significantly improve the silencing efficiency of siRNA-loaded nanogels. Furthermore, it is shown that an efficient gene silencing requires the degradation of the nanogels. As the degradation kinetics of the nanogels can easily be tailored, these particles show potential for intracellular controlled release of short interfering RNA. [source] | ||||||||||||||||||||||||||||||||||||||||