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Results for “RNA, Small Interfering”

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At least 793 records · Page 44Linked to original sources

PubMed2005

Estrogen-related receptor-gamma and peroxisome proliferator-activated receptor-gamma coactivator-1alpha regulate estrogen-related receptor-alpha gene expression via a conserved multi-hormone response element.

The expression of estrogen-related receptor-alpha (ERRalpha) is stimulated by estrogen in selective tissues. Recently, a correlation between ERRalpha expression and the induction of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) in the liver of fasting animals and in cold-stressed brown-fat tissues and skeletal muscle was shown. To explore the molecular mechanisms of ERRalpha regulation by diverse signals, the promoter of the human ERRalpha gene was cloned and characterized. Mutation and deletion analyses revealed that a 53 bp region containing repeated core element AGGTCA motifs of the ERRalpha gene serves as a multi-hormone response element (MHRE) for several nuclear receptors in transient co-transfection studies of human endometrial carcinoma (HEC-1B) cells. Among the nuclear receptors tested, ERRgamma bound to and robustly stimulated the transcription of reporters containing at least two AGGTCA motifs. Ectopic expression of PGC-1alpha in HEC-1B cells strongly activated the reporter containing the MHRE, presumably via the endogenous nuclear receptor binding to the element. Reducing the endogenous level of ERRgamma by small interfering RNA, and increasing the ERRgamma level by ectopic expression, substantially decreased and increased respectively the transactivation capability of PGC-1alpha. The activation function 2 domain of the ERRgamma and the L2 and L3 motifs of PGC-1alpha were essential to transactivate the MHRE. Additionally, PGC-1alpha increases the amount of endogenous ERRgamma bound to the MHRE region as determined by a chromatin immunoprecipitation assay. The present study demonstrates that the MHRE of the ERRalpha gene is a target for ERRgamma transactivation, which is enhanced by PGC-1alpha.

Animals↗

PubMed2004

Spermidine/spermine N1-acetyltransferase specifically binds to the integrin alpha9 subunit cytoplasmic domain and enhances cell migration.

The integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation. alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the alpha9 cytoplasmic domain. alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent. Using yeast two-hybrid screening, we identified the polyamine catabolizing enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as a specific binding partner of the alpha9 cytoplasmic domain. Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains. The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration. We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.

Acetyltransferases↗

PubMed2005

17Beta-estradiol induced Ca2+ influx via L-type calcium channels activates the Src/ERK/cyclic-AMP response element binding protein signal pathway and BCL-2 expression in rat hippocampal neurons: a potential initiation mechanism for estrogen-induced neuroprotection.

Our group and others have demonstrated that 17beta-estradiol (E2) induces neurotrophic and neuroprotective responses in hippocampal and cortical neurons which are dependent upon the Src/extracellular signal-regulated kinase (ERK) signaling pathways. The purpose of this study was to determine the upstream mechanism(s) that initiates the signaling cascade leading to E2-inducible neuroprotection. We tested the hypothesis that E2 activates rapid Ca(2+) influx in hippocampal neurons, which would lead to activation of the Src/ERK signaling cascade and up-regulation of Bcl-2 protein expression. Using fura-2 ratiometric Ca(2+) imaging, we demonstrated that E2 induced a rapid rise of intracellular Ca(2+) concentration ([Ca(2+)](i)) within minutes of exposure which was blocked by an L-type Ca(2+) channel antagonist. Inhibition of L-type Ca(2+) channels resulted in a loss of E2 activation of the Src/ERK cascade, activation of cyclic-AMP response element binding protein (CREB) and subsequent increase in Bcl-2. Real-time intracellular Ca(2+) imaging combined with pERK immunofluorescence, demonstrated that E2 induced [Ca(2+)](i) was coincident with ERK activation in the same neuron. Small interfering RNA knockdown of CREB resulted in a loss of E2 activation of CREB and subsequent E2-induced increase of Bcl-2 expression. We further demonstrated the presence of specific membrane E2 binding sites in hippocampal neurons. Together, these data indicate that E2-induced Ca(2+) influx via the L-type Ca(2+) channel is required for E2 activation of the Src/ERK/CREB/Bcl-2 signaling. Implications of these data for understanding estrogen action in brain and use of estrogen therapy for prevention of neurodegenerative disease are discussed.

Animals↗

PubMed2006

ATR-dependent checkpoint modulates XPA nuclear import in response to UV irradiation.

In response to DNA damage, mammalian cells activate various DNA repair pathways to remove DNA lesions and, meanwhile, halt cell cycle progressions to allow sufficient time for repair. The nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint activation are two major cellular responses to DNA damage induced by UV irradiation. However, how these two processes are coordinated in the response is poorly understood. Here we showed that the essential NER factor XPA (xeroderma pigmentosum group A) underwent nuclear accumulation upon UV irradiation, and strikingly, such an event occurred in an ATR (Ataxia-Telangiectasia mutated and RAD3-related)-dependent manner. Either treatment of cells with ATR kinase inhibitors or transfection of cells with small interfering RNA targeting ATR compromised the UV-induced XPA nuclear translocation. Consistently, the ATR-deficient cells displayed no substantial XPA nuclear translocation while the translocation remained intact in ATM (Ataxia-Telangiectasia mutated)-deficient cells in response to UV irradiation. Moreover, we found that ATR is required for the UV-induced nuclear focus formation of XPA. Taken together, our results suggested that the ATR checkpoint pathway may modulate NER activity through the regulation of XPA redistribution in human cells upon UV irradiation.

Active Transport, Cell Nucleus↗

PubMed2004

Neuron-restrictive silencer factor (NRSF) functions as a repressor in neuronal cells to regulate the mu opioid receptor gene.

The mu opioid receptor (MOR) is expressed in the central nervous system and specific cell lines with varying expression levels perhaps playing important roles. One of the neuronal-specific transcription regulators, neuron-restrictive silencer factor (NRSF), has been shown to repress the expression of neuron-specific genes in non-neuronal cells. However, we showed here that the neuron-restrictive silencer element (NRSE) of MOR functions as a critical regulator to repress the MOR gene expression in specific neuronal cells depending on NRSF expression level. Using co-transfection studies, we showed that the NRSE of the MOR promoter is functional in NRSF-positive cells (NS20Y and HeLa) but not in NRSF-negative cells (PC12). NRSF binds to the NRSE of the MOR gene in a sequence-specific manner confirmed by supershift and chromatin immunoprecipitation assays, respectively. The suppression of NRSF activity with either trichostatin A or a dominant-negative NRSF induced MOR promoter activity and transcription of the MOR gene. When the NRSF was disrupted in NS20Y and HeLa cells using small interfering RNA, the transcription of the endogenous target MOR gene increased significantly. This provides direct evidence the role of NRSF in the cells and also indicates that NRSF expression is regulated by post-translational modification in neuronal NMB cells. Our data suggested that NRSF can function as a repressor of MOR transcription in specific cells, via a mechanism dependent on the MOR NRSE.

Animals↗

PubMed2006

Curcumin induces pro-apoptotic endoplasmic reticulum stress in human leukemia HL-60 cells.

Curcumin has been shown to induce apoptosis in many cancer cells. However, the molecular mechanism(s) responsible for curcumin-induced apoptosis is not well understood and most probably involves several pathways. In HL-60 cells, curcumin induced apoptosis and endoplasmic reticulum (ER) stress as evidenced by the survival molecules such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2alpha, glucose-regulated protein-78, and the apoptotic molecules such as caspase-4 and CAAT/enhancer binding protein homologous protein (CHOP). Inhibition of caspase-4 activity by z-LEVD-FMK, blockage of CHOP expression by small interfering RNA, and treatment with salubrinal, an ER inhibitor, significantly reduced curcumin-induced apoptosis. Removing two double bonds in curcumin, which was speculated to form Michael adducts with thiols in secretory proteins, resulted in a loss of the ability of curcumin to induce apoptosis as well as ER stress. Thus, the present study shows that curcumin-induced apoptosis is associated with its ability to cause ER stress.

Antineoplastic Agents↗

PubMed2005

Role of type I receptors for anti-Müllerian hormone in the SMAT-1 Sertoli cell line.

Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-beta family responsible for regression of Müllerian ducts during male sexual differentiation and for regulation of gonadal steroidogenesis. AMH is also a gonadal tumor suppressor which mediates its effects through a specific type II receptor and the bone morphogenetic protein (BMP)-specific Smad proteins, suggesting that AMH and BMPs could also share type I receptors, namely activin-like kinases (ALKs)2, 3 or 6. However, attempts to identify a unique AMH type I receptor among them were unsuccessful. Here, using kinase-deficient type I receptors and small interfering RNA technology, we demonstrate that, in an AMH Sertoli target cell line, ALK3 mediates AMH effects on both Smad1 activation and P450 side-chain cleavage enzyme. In addition, transfecting a combination of normal and kinase-deficient receptors, we show that ALK2 can compensate for the absence of ALK3 and probably acts in synergy with ALK3 at high concentrations of AMH to activate Smad1, whereas ALK6 has a competitive inhibitory effect. These results are a first step in understanding how AMH transduces its effects in immature Sertoli cells.

Animals↗

PubMed2005

Involvement of {beta}-catenin and unusual behavior of CBP and p300 in glucocorticosteroid signaling in Schwann cells.

In the nervous system, glucocorticosteroid hormones play a major role during development and adult life. Myelin-forming cells are among the targets of glucocorticosteroids, which have been shown to promote myelination both in the central and peripheral nervous system. Glucocorticosteroid-stimulated gene transcription is mediated by the glucocorticosteroid receptor (GR) that recruits coactivators of the p160 family, forming a docking platform for secondary coactivators, such as cAMP-response element binding protein (CREB)-binding protein (CBP) or its close homologue, p300. Here, we investigated the role of CBP and p300 in mouse Schwann cells (MSC80). We show that, although the CBP/p300 binding domain of steroid receptor coactivator-1 is crucial for GR transactivation, neither CBP nor p300 enhanced GR transcriptional activation, as shown by overexpression and small interfering RNA (siRNA) knocking-down experiments. Unexpectedly, overexpression of p300, considered as a coactivator of the GR, resulted in inhibition of GR transcriptional activity. Studies with p300 deletion mutants demonstrated that p300-dependent repression is related to its acetyltransferase activity. Functional and pull-down assays showed that beta-catenin may be the coactivator replacing CBP in the GR transcriptional complex. Our results suggest the formation of a GR-coactivator complex within Schwann cells, indicating that glucocorticosteroids may act by means of unusual partners in the nervous system, and we show a repressive effect of p300 on nuclear receptors.

Animals↗

PubMed2004

A fatty acid synthase blockade induces tumor cell-cycle arrest by down-regulating Skp2.

In eukaryotes, fatty acid synthase (FAS) is the enzyme responsible for synthesis of palmitate, the precursor of long-chain nonessential fatty acids. FAS is up-regulated in a wide range of cancers and has been suggested as a relevant drug target. Here, two independent approaches are taken toward knocking down FAS and then probing its connection to tumor cell proliferation. In one approach, Orlistat, a drug approved for treating obesity, is used as a potent inhibitor of the thioesterase function of FAS. In a separate strategy, the expression of FAS is suppressed by targeted knock-down with small interfering RNA. In both circumstances, the ablation of FAS activity causes a dramatic down-regulation of Skp2, a component of the E3 ubiquitin ligase that controls the turnover of p27Kip1. These effects ultimately tie into the retinoblastoma protein pathway and lead to a cell-cycle arrest at the G1/S boundary. Altogether, the findings of the study reveal unappreciated links between fatty acid synthase and ubiquitin-dependent proteolysis of cell-cycle regulatory proteins.

Avidin↗

PubMed2005

Krüppel-like factor-6 promotes preadipocyte differentiation through histone deacetylase 3-dependent repression of DLK1.

Preadipocyte differentiation occurs during distinct periods of human development and is a key determinant of body mass. Transcriptional events underlying adipogenesis continue to emerge, but the link between chromatin remodeling of specific target loci and preadipocyte differentiation remains elusive. We have identified Krüppel-like factor-6 (KLF6), a recently described tumor suppressor gene, as a repressor of the proto-oncogene Delta-like 1 (Dlk1), a gene encoding a transmembrane protein that inhibits adipocyte differentiation. Forced expression of KLF6 strongly inhibits Dlk1 expression in preadipocytes and NIH 3T3 cells in vivo, whereas down-regulation of KLF6 in 3T3-L1 cells by small interfering RNA prevents adipogenesis. Repression of Dlk1 requires HDAC3 deacetylase activity, which is recruited to the endogenous Dlk1 promoter where it interacts with KLF6. Our studies identify the interaction between HDAC3 and KLF6 as a potential mechanism underlying human adipogenesis, and highlight the role of KLF6 as a multifunctional transcriptional regulator capable of mediating adipocyte differentiation through gene repression.

3T3 Cells↗

PubMed2006

Orphan nuclear receptor Nur77 induces zinc finger protein GIOT-1 gene expression, and GIOT-1 acts as a novel corepressor of orphan nuclear receptor SF-1 via recruitment of HDAC2.

Kruppel-associated box (KRAB) domain-containing proteins consist of potential transcriptional repression modules. Previously, gonadotropin-inducible ovarian transcription factor-1 (GIOT-1) was identified as a novel KRAB-containing zinc finger protein and shown to have transcriptional repression activity. Here, we demonstrate that orphan nuclear receptor Nur77 regulates GIOT-1 gene expression in testicular Leydig cell lines and that GIOT-1 acts as a novel corepressor of the orphan nuclear receptor steroidogenic factor 1 (SF-1). Mutation analysis of the GIOT-1 promoter and overexpression analysis of dominant-negative Nur77 revealed that luteinizing hormone activates GIOT-1 gene expression through Nur77. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that Nur77 directly binds to the GIOT-1 promoter. GIOT-1 represses the SF-1 transactivation, and specific interaction between GIOT-1 and SF-1 was observed. We also demonstrate an interaction between GIOT-1 and histone deacetylase 2 (HDAC2). GIOT-1-mediated transrepression was recovered by down-regulation of HDAC2 expression with small interfering RNA of HDAC2. Knock down of the endogenous GIOT-1 results in significant enhancement of CYP17 expression in Leydig cells. In conclusion, this study of cross-talk between GIOT-1 and orphan nuclear receptors will provide new insights into the role of KRAB-containing zinc finger proteins in nuclear receptor action.

Animals↗

PubMed2004

p53 Stabilization and accumulation induced by human vaccinia-related kinase 1.

Variations in intracellular levels of p53 regulate many cellular functions and determine tumor susceptibility. Major mechanisms modulating p53 levels include phosphorylation and interaction of p53 with specific ubiquitin ligases that promote its degradation. N-terminal phosphorylation regulates the interaction of p53 with several regulatory molecules. Vaccinia-related kinase 1 (VRK1) is the prototype of a new Ser-Thr kinase family in the human kinome. VRK1 is located in the nucleus outside the nucleolus. Overexpression of VRK1 increases the stability of p53 by a posttranslational mechanism leading to its accumulation by a mechanism independent of the Chk2 kinase. Catalytically inactive VRK1 protein (a K179E mutant) does not induce p53 accumulation. VRK1 phosphorylates human p53 in Thr18 and disrupts p53-Mdm2 interaction in vitro, although a significant decrease in p53 ubiquitination by Mdm2 in vivo was not detected. VRK1 kinase does not phosphorylate Mdm2. VRK1-mediated p53 stabilization was also detected in Mdm2(-/-) cells. VRK1 also has an additive effect with MdmX or p300 to stabilize p53, and p300 coactivation and acetylation of p53 is enhanced by VRK1. The p53 stabilized by VRK1 is transcriptionally active. Suppression of VRK1 expression by specific small interfering RNA provokes several defects in proliferation, situating the protein in the regulation of this process. VRK1 might function as a switch controlling the proteins that interact with p53 and thus modifying its stability and activity. We propose VRK1 as the first step in a new pathway regulating p53 activity during cell proliferation.

Blotting, Western↗

PubMed2004

Use of siRNA to study the function of MDC1 in DNA damage responses.

Small interfering RNA (siRNA) technology has emerged as a powerful genetic tool to investigate gene function in mammalian cells. Here we use siRNA to study a mediator of DNA damage-checkpoint protein 1 (MDC1), previously known as Kiaa0170 or NFBD1, in DNA damage responses. We show that MDC1 siRNA specifically and efficiently down-regulates MDC1 expression, resulting in defective radiation-induced apoptosis in A549 cells. Transfection with siRNA-resistant MDC1 restores radiation-induced apoptosis. These findings suggest that siRNA can be a very useful tool for the exploration of gene function in mammalian checkpoint responses.

Adaptor Proteins, Signal Transducing↗

PubMed2004

[Telomerase SiRNA inhibits KB cell growth in human oral squamous cell carcinoma].

OBJECTIVE: To test the telomerase SiRNA on telomerase mRNA and on KB cell growth of oral squamous cell carcinoma. METHODS: We synthesized 21-nucleotide SiRNA duplexes with symmetric 2-nucleotide 3' overhangs corresponding to the target sequence (2 657 approximately 2 675 nucleotide downstream of the start codon) of telomerase mRNA. Telomerase activity, cell proliferation, cell cycle and apoptosis were measured after transfection. RESULTS: Twenty one-nucleotide small interfering RNA (SiRNA) duplexes specifically suppressed expression of endogenous telomerase mRNA in human oral squamous carcinoma KB cells. This inhibitory effect lasted only for about 48 h after transfection. Telomerase activity reduction corresponded to the mRNA suppression. Cell proliferation decreased by 30% at 48 h after transfection and lasted for 120 h after treatment. This inhibitory effect resulted from the block of G(1) to S transition. Apoptosis was not involved in this process. CONCLUSIONS: SiRNA is a powerful tool for studying gene function and can be used as gene-specific therapeutics.

Apoptosis↗

PubMed2006

SPIN90/WISH interacts with PSD-95 and regulates dendritic spinogenesis via an N-WASP-independent mechanism.

SPIN90/WISH (SH3 protein interacting with Nck, 90 kDa/Wiskott-Aldrich syndrome protein (WASP) interacting SH3 protein) regulates actin polymerization through its interaction with various actin-regulating proteins. It is highly expressed in the brain, but its role in the nervous system is largely unknown. We report that it is expressed in dendritic spines where it associates with PSD-95. Its overexpression increased the number and length of dendritic filopodia/spines via an N-WASP-independent mechanism, and knock down of its expression with small interfering RNA reduced dendritic spine density. The increase in spinogenesis is accompanied by an increase in synaptogenesis in contacting presynaptic neurons. Interestingly, PSD-95-induced dendritic spinogenesis was completely abolished by knock down of SPIN90/WISH. Finally, in response to chemically induced long-term potentiation, SPIN90/WISH associated with PSD-95 and was redistributed to dendritic spines. Our results suggest that SPIN90/WISH associates with PSD-95, and so becomes localized to dendritic spines where it modulates actin dynamics to control dendritic spinogenesis. They also raise the possibility that SPIN90/WISH is a downstream effector of PSD-95-dependent synaptic remodeling.

Actins↗

PubMed2006

Enhanced SMYD3 expression is essential for the growth of breast cancer cells.

We previously reported that upregulation of SMYD3, a histone H3 lysine-4-specific methyltransferase, plays a key role in the proliferation of colorectal carcinoma (CRC) and hepatocellular carcinoma (HCC). In the present study, we reveal that SMYD3 expression is also elevated in the great majority of breast cancer tissues. Similarly to CRC and HCC, silencing of SMYD3 by small interfering RNA to this gene resulted in the inhibited growth of breast cancer cells, suggesting that increased SMYD3 expression is also essential for the proliferation of breast cancer cells. Moreover, we show here that SMYD3 could promote breast carcinogenesis by directly regulating expression of the proto-oncogene WNT10B. These data imply that augmented SMYD3 expression plays a crucial role in breast carcinogenesis, and that inhibition of SMYD3 should be a novel therapeutic strategy for treatment of breast cancer.

Adult↗

PubMed2002

53BP1, a mediator of the DNA damage checkpoint.

53BP1 binds to the tumor suppressor protein p53 and has a potential role in DNA damage responses. We used small interfering RNA (siRNA) directed against 53BP1 in mammalian cells to demonstrate that 53BP1 is a key transducer of the DNA damage checkpoint signal. 53BP1 was required for p53 accumulation, G2-M checkpoint arrest, and the intra-S-phase checkpoint in response to ionizing radiation. 53BP1 played a partially redundant role in phosphorylation of the downstream checkpoint effector proteins Brca1 and Chk2 but was required for the formation of Brca1 foci in a hierarchical branched pathway for the recruitment of repair and signaling proteins to sites of DNA damage.

BRCA1 Protein↗

PubMed2006

Whole genome microarray analysis of growth hormone-induced gene expression in bone: T-box3, a novel transcription factor, regulates osteoblast proliferation.

Growth hormone (GH) is important in the development and maintenance of bone; however, the IGF-dependent and -independent molecular pathways involved remain to be established. We used microarray analysis to evaluate GH signaling pathways in 4-wk-old GH-deficient mice following a single injection of GH (4 mg/kg body wt) or PBS (n = 6/group) at 6 or 24 h after treatment. Six thousand one hundred sixty genes were differentially expressed at P </= 0.05, and 17% of these genes were identified at both time points. Several of the genes differentially expressed were expressed sequence tags, and the remaining genes fell into 49 Gene Ontology categories. For subsequent studies, we focused on T-box (Tbx)3, a novel transcription factor, which increased more than twofold at both time points. Real-time RT-PCR analysis determined that pretreatment with IGF-binding protein-4 did not block GH-induced Tbx3 expression in vitro. Pretreatment with TNF-alpha blocked GH-induced Tbx3 expression. Tbx3 expression increased during osteoblast differentiation and following BMP-7 and Wnt3a treatment (P </= 0.05). Blocking Tbx3 expression by small interfering RNA decreased cell number and [(3)H]Thymidine incorporation (P < 0.01). In conclusion, 1) GH caused acute changes in several novel genes, suggesting that many GH-induced signaling pathways and target genes remain to be discovered; 2) because Tbx3 expression is regulated in osteoblasts and blockage of Tbx3 expression decreased cell number and DNA synthesis, we propose that Tbx3 is an important determinant of osteoblast cell number.

Animals↗