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PubMed2005

siRNA produced by recombinant dicer mediates efficient gene silencing in islet cells.

RNA interference (RNAi) is emerging as a powerful and convenient tool for studying gene function and genetic variation. RNAi is mediated by 21- to 23-nucleotide-long, small interfering RNAs (siRNA) produced from larger double-stranded RNAs in vivo by the RNase III family enzyme Dicer. To overcome the problems associated with the use of predesigned synthetic siRNA molecules, a novel method utilizing the in vitro activity of recombinant Dicer has been developed recently. In nonislet cells, it has been demonstrated that a pool of siRNA, generated by Dicer from in vitro transcribed dsRNA (d-siRNA), mediates convenient, efficient, and reproducible gene silencing in various cell types. The aim of this study was to evaluate the ability of d-siRNA to silence endogenous gene expression in pancreatic islet cells. We observed that liposomal transfection mediates efficient transport of siRNA in up to 90% of dispersed islet cells and that d-siRNA mediates almost complete and nontoxic silencing of an endogenous mRNA, the messenger coding for the nonreceptor tyrosine kinase c-Abl. The approach described here using d-siRNA provides an important tool for elucidating gene function in further studies of pancreatic islets and diabetes pathophysiology.

Animals↗
PubMed2004

Control of siRNA expression using the Cre-loxP recombination system.

Gene silencing mediated by RNA interference (RNAi) was first discovered in Caenorhabditis elegans, and was subsequently recognized in various other organisms. In mammalian cells, RNAi can be induced by small interfering RNAs (siRNAs). In earlier studies, our group developed a vector-based system for expression of siRNA under control of a polymerase III promoter, the U6 promoter, which can induce RNAi in living cells. We here describe a system for controlling the U6 promoter-driven expression of siRNA using the Cre-loxP recombination system. We constructed a 'Cre-On' siRNA expression vector which could be switched on upon excision catalyzed by Cre recombinase, which was delivered to cells directly from the medium as a fusion protein. An examination of the effectiveness of RNAi against a reporter gene revealed that addition of TAT-NLS-Cre (where NLS is a nuclear localization signal and TAT is a peptide of 11 amino acids derived from HIV) to the medium resulted in plasmid recombination, generation of siRNA and suppression of reporter activity. This system should allow us to induce RNAi in a spatially, temporally, cell type-specifically or tissue-specifically controlled manner and potentiate the improved application of RNAi in both an experimental and a therapeutic context.

Amino Acid Sequence↗
PubMed2003

Short interfering RNAs (siRNAs) for reducing dopaminergic phenotypic markers.

Short interfering RNAs (siRNAs) are double-stranded RNAs of approximately 19-29 nucleotides designed to suppress the expression of homologous genes by a process known as RNA interference (RNAi). In this study we have characterized several short interfering RNAs to reduce (knockdown) the expression of genes related to the dopaminergic system of the ventral mesencephalon. We report here effective suppression of all targeted genes, tyrosine hydroxylase (TH), the transcription factor Nr4a2 (Nurr1) and the GDNF receptor moiety cRet, by co-transfection of plasmids expressing gene specific siRNAs under control of the human U6 promoter with a reporter plasmid coding for firefly luciferase and fused in tandem to the cDNA for the gene of interest. The effective suppression of Nr4a2, cRet and tyrosine hydroxylase suggest that the U6 expression cassette could be used to deliver siRNA to mammalian cells in vivo and actively suppress the expression of dopamine related mammalian genes. The characterization of highly effective siRNAs for DA phenotypic markers may open new avenues for loss-of-function phenotypic analysis, and may lead to new approaches for gene therapy.

Analysis of Variance↗
PubMed2005

Loss of phosphatase and tensin homologue increases transforming growth factor beta-mediated invasion with enhanced SMAD3 transcriptional activity.

In normal epithelial tissues, the multifunctional cytokine transforming growth factor-beta (TGF-beta) acts as a tumor suppressor through growth inhibition and induction of differentiation whereas in advanced cancers, TGF-beta promotes tumor progression through induction of tumor invasion, neoangiogenesis, and immunosuppression. The molecular mechanisms through which TGF-beta shifts from a tumor suppressor to a tumor enhancer are poorly understood. We now show a role for the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in repressing the protumorigenic effects of TGF-beta. The TGF-beta effector SMAD3 inducibly interacts with PTEN on TGF-beta treatment under endogenous conditions. RNA interference (RNAi) suppression of PTEN expression enhances SMAD3 transcriptional activity and TGF-beta-mediated induction of SMAD3 target genes whereas reconstitution of PTEN in a null cancer cell line represses the expression of TGF-beta-regulated target genes. Targeting PTEN expression through RNAi in a PTEN wild-type cell line increases TGF-beta-mediated invasion but does not affect TGF-beta-mediated growth inhibition. Reconstitution of PTEN expression in a PTEN-null cell line blocks TGF-beta-induced invasion but does not modulate TGF-beta-mediated growth regulation. These effects are distinct from Akt and Forkhead family members that also interact with SMAD3 to regulate apoptosis or proliferation, respectively. Pharmacologic inhibitors targeting TGF-beta receptors and phosphatidylinositol 3-kinase signaling downstream from PTEN cooperate to block TGF-beta-mediated invasion. Thus, the loss of PTEN expression in human cancers may contribute to a role for TGF-beta as a tumor enhancer with specific effects on cellular motility and invasion.

Cell Movement↗
PubMed2005

LAR receptor protein tyrosine phosphatases in the development and maintenance of excitatory synapses.

Leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-alpha (SYD2) and are implicated in axon guidance. We report that LAR-RPTP is concentrated in mature synapses in cultured rat hippocampal neurons, and is important for the development and maintenance of excitatory synapses in hippocampal neurons. RNA interference (RNAi) knockdown of LAR or dominant-negative disruption of LAR function results in loss of excitatory synapses and dendritic spines, reduction of surface AMPA receptors, impairment of dendritic targeting of the cadherin-beta-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, beta-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-alpha and GRIP from rat brain extracts. We propose that the cadherin-beta-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-alpha to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Induction of hyaluronic acid synthase 2 (HAS2) in human vascular smooth muscle cells by vasodilatory prostaglandins.

Hyaluronic acid (HA) is a prominent constituent of the extracellular matrix of atherosclerotic vascular lesions in humans known to modulate vascular smooth muscle phenotype. The regulation of HA synthesis by vasodilatory prostaglandins was analyzed in human arterial smooth muscle cells (SMCs). The prostacyclin analogue, iloprost (100 nmol/L), markedly increased pericellular formation of HA coats and HA secretion into the cell culture medium in human arterial SMCs (8.7+/-1.6-fold). Expression of HA synthase 2 (HAS2) was determined by semiquantitative RT-PCR and found to be strongly upregulated at concentrations of iloprost between 1 and 100 nmol/L after 3 hours. Furthermore, endogenous cyclooxygenase-2 (COX2) activity was required for basal expression of HAS2 mRNA in SMCs in vitro. Total HA secretion in response to iloprost was markedly decreased by RNA interference (RNAi), specific for HAS2. In addition, siRNA targeting HAS2 strongly increased the spreading of human SMCs compared with mock-transfected cells. HAS2 mRNA levels were also stimulated by a selective prostacyclin receptor (IP) agonist, cicaprost (10 nmol/L), prostaglandin E(2) (10 nmol/L), and the EP(2) receptor agonist, butaprost (1 micromol/L). Induction of HAS2 mRNA and HA synthesis by prostaglandins was mimicked by stable cAMP analogues and forskolin. In human atherectomy specimens from the internal carotid artery, HA deposits and COX2 expression colocalized frequently. In addition, strong EP(2) receptor expression was detected in SMCs in HA-rich areas. Therefore, upregulation of HAS2 expression via EP(2) and IP receptors might contribute to the accumulation of HA during human atherosclerosis, thereby mediating proatherosclerotic functions of COX2.

6-Ketoprostaglandin F1 alpha↗
PubMed2000

Distinct and redundant functions of mu1 medium chains of the AP-1 clathrin-associated protein complex in the nematode Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, there exist two micro1 medium chains of the AP-1 clathrin-associated protein complex. Mutations of unc-101, the gene that encodes one of the micro1 chains, cause pleiotropic effects (). In this report, we identified and analyzed the second mu1 chain gene, apm-1. Unlike the mammalian homologs, the two medium chains are expressed ubiquitously throughout development. RNA interference (RNAi) experiments with apm-1 showed that apm-1 and unc-101 were redundant in embryogenesis and in vulval development. Consistent with this, a hybrid protein containing APM-1, when overexpressed, rescued the phenotype of an unc-101 mutant. However, single disruptions of apm-1 or unc-101 have distinct phenotypes, indicating that the two medium chains may have distinct functions. RNAi of any one of the small or large chains of AP-1 complex (sigma1, beta1, or gamma) showed a phenotype identical to that caused by the simultaneous disruption of unc-101 and apm-1, but not that by single disruption of either gene. This suggests that the two medium chains may share large and small chains in the AP-1 complexes. Thus, apm-1 and unc-101 encode two highly related micro1 chains that share redundant and distinct functions within AP-1 clathrin-associated protein complexes of the same tissue.

Adaptor Protein Complex 1↗
PubMed2003

A gene encoding an RNase D exonuclease-like protein is required for post-transcriptional silencing in Arabidopsis.

Post-transcriptional gene silencing (PTGS) and the closely related phenomenon RNA interference (RNAi) result from the initial endonucleolytic cleavage of target mRNAs, which are then presumed to be completely hydrolyzed by exoribonucleases. To date, no plant genes required for PTGS are known to encode exoribonucleases. The Arabidopsis Werner Syndrome-like exonuclease (WEX) gene encodes an RNase D domain most similar to that in human Werner Syndrome protein (WRN), but lacks the RecQ helicase domain. It is also related to Caenorhabditis elegans mut-7, which is essential for RNAi, PTGS, and transposon activity. We isolated a loss-of-function mutant, wex-1, that showed greatly reduced expression of WEX mRNA and early flowering. Although wex-1 did not affect expression of a robust marker for transcriptional gene silencing (TGS), PTGS of a green-fluorescent-protein (GFP) reporter gene was blocked in wex-1 and restored by ectopic expression of WEX, indicating that WEX is required for PTGS but not TGS. Thus, members of the RNase D protein family are required for PTGS in both plants and animals. Interestingly, WEX has been shown to interact with an Arabidopsis RecQ helicase, suggesting that these proteins might comprise a functional equivalent of WRN.

Arabidopsis↗
PubMed2005

Quantum dot-conjugated hybridization probes for preliminary screening of siRNA sequences.

In the present study, we describe the design and fabrication of quantum dot-conjugated hybridization probes and their application to the development of a comparatively simple and rapid procedure for the selection of highly effective small-interfering RNA (siRNA) sequences for RNA interference (RNAi) in mammalian cells, for example, siRNAs with high accessibility and affinity to the respective mRNA target. A single-stranded siRNA was conjugated with a quantum dot and used as a hybridization probe. The target mRNA was amplified in the presence of Cy5-labeled nucleotides, and Cy5-mRNA served as a hybridization sample. The formation of siRNA/mRNA duplexes during a comparatively short hybridization time (1 h) was used as a criterion for the selection of highly effective, target-specific siRNA sequences. The accessibility and affinity of the siRNA sequence for the target mRNA site were determined by fluorescence resonance energy transfer (FRET) between a quantum dot (donor) and a fluorescent dye molecule (Cy5, acceptor) localized at an appropriate distance from each other when hybridization occurred. The FRET signal was observed only when there was high accessibility between an antisense siRNA and a sense mRNA and did not appear in the case of mismatch siRNAs. Moreover, the amplitude of the FRET signal significantly correlated with the specific effect of siRNA on the expression of the target mRNA and protein, determined in native cells by RT-PCR and immunoblot analysis, respectively.

Base Sequence↗
PubMed2006

[Specific inhibition of HER-2 expression in ovarian carcinoma cells by siRNA targeting HER-2].

OBJECTIVE: To investigate the effects of RNA interference (RNAi) targeting HER-2 gene on the biological traits of human ovarian carcinoma. METHODS: siRNA specific to HER-2 gene was synthesized according to the sequence in the GenBank. Human ovarian carcinoma cells of the line SKOV-3 were cultured and divided into 3 groups: control group; non-specific group, transfected with non-specific siRNA; and specific group, transfected with specific HER-2 siRNA. On the 5th day after transfection cisplatin was added into the culture fluid. The expression of HER-2 mRNA and the expression of protein both before and after transfection were detected by RT-PCR and Western blotting. The cell apoptosis was assessed by flow cytometry. The chemosensitivity of transfected cells to cisplatin was determined by MTT method. RESULTS: Since the 3rd day after transfection the expression of HER-2 mRNA of the HER-2 siRNA group was suppressed till 10 days later. On the 7th day after transfection the expression rate of HER-2 protein of the HER-2 siRNA group was (25.5 +/- 0.8)%, significantly lower than those of the nonspecific siRNA group and control group, (95.7 +/- 0.8)% and (96.6 +/- 1.2)% respectively (both P < 0.001). On the 9th day after transfection no expression of HER-2 protein was found in the HER-2 siRNA group. The apoptosis rate of SKOV-3 cells increased time-dependently after transfection in the HER-2 siRNA group and reached the peak, (53.2 +/- 1.0)% on the 6th day, significantly higher than those of the non-specific siRNA group and control group, (4.1 +/- 0.3)% and (4.1 +/- 0.3)% respectively (both P < 0.001). After exposure to cisplatin for 24 hours, the survival rate of the HER-2 siRNA group was (58.4 +/- 0.8)%, significantly higher than those of the nonspecific siRNA group and control group, (68.0 +/- 0.6)% and (67.0 +/- 0.3)% respectively (both P < 0.001). CONCLUSION: siRNA targeting HER-2 synthesized in vitro and transfected into human ovarian carcinoma cells effectively suppresses the HER-2 expression, induces cell apoptosis, and increases the sensitivity to cisplatin of the cells. The successful application of HER-2 siRNA extends the list of available therapeutic modalities in the treatment of human ovarian cancer.

Antineoplastic Agents↗
PubMed2003

Variations of the 3' protruding ends in synthetic short interfering RNA (siRNA) tested by microinjection in Drosophila embryos.

Short interfering RNAs (siRNAs) are the processing product originating from long double-stranded RNAs (dsRNAs) that are cleaved by the RNase III-like ribonuclease Dicer. As siRNAs mediate cleavage of specific single-stranded target RNAs, they are essential intermediates of RNA interference (RNAi). When applied in synthetic form, siRNAs likewise can induce the silencing process in the absence of long dsRNAs. Here, we tested variations of a conventional synthetic siRNA that had been used successfully to silence the Drosophila notch gene. The variants had two 3 ' -terminal deoxynucleotides in their protruding single-stranded ends. In one case, the deoxynulceotides would match to the notch mRNA, whereas the other variant had nonmatching deoxy-T residues, representing a widely used siRNA design. siRNAs with different combinations of sense and antisense strands were injected into Drosophila embryos at two different concentrations. We found that the all-ribonucleotide siRNA gave the best inhibition of notch expression. The combination of two modified strands with 3 ' -terminal deoxynucleotides was effective, but if combined with a sense or antisense ribostrand, the efficacy dropped. The siRNAs with nonmatching 3 ' -terminal TT residues showed a reduced silencing potential, which became evident at low concentration. An siRNA with a nonmatching 3 ' -terminal ribonucleotide in the antisense strand retained most of its silencing potential in accordance with the hypothesis that primer extension for generation of ssRNA from single-stranded mRNA does not operate in Drosophila.

3' Untranslated Regions↗
PubMed2006

Loss of the VHR dual-specific phosphatase causes cell-cycle arrest and senescence.

Protein tyrosine phosphatases regulate important processes in eukaryotic cells and have critical functions in many human diseases including diabetes to cancer. Here, we report that the human Vaccinia H1-related (VHR) dual-specific protein tyrosine phosphatase regulates cell-cycle progression and is itself modulated during the cell cycle. Using RNA interference (RNAi), we demonstrate that cells lacking VHR arrest at the G1-S and G2-M transitions of the cell cycle and show the initial signs of senescence, such as flattening, spreading, appearance of autophagosomes, beta-galactosidase staining and decreased telomerase activity. In agreement with this notion, cells lacking VHR were found to upregulate p21(Cip-Waf1), whereas they downregulated the expression of genes for cell-cycle regulators, DNA replication, transcription and mRNA processing. Loss of VHR also caused a several-fold increase in serum-induced activation of its substrates, the mitogen-activated protein (MAP) kinases Jnk and Erk. VHR-induced cell-cycle arrest was dependent on this hyperactivation of Jnk and Erk, and was reversed by Jnk and Erk inhibition or knock-down. We conclude that VHR is required for cell-cycle progression as it modulates MAP kinase activation in a cell-cycle phase-dependent manner.

Animals↗
PubMed2006

Design and cloning strategies for constructing shRNA expression vectors.

BACKGROUND: Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). RESULTS: We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20-40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. CONCLUSION: In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process.

Base Sequence↗
PubMed2001

Efficient and heritable functional knock-out of an adult phenotype in Drosophila using a GAL4-driven hairpin RNA incorporating a heterologous spacer.

We have developed a modified RNA interference (RNAi) method for generating gene knock-outs in Drosophila melanogaster. We used the sequence of the yellow (y) locus to construct an inverted repeat that will form a double-stranded hairpin structure (y-IR) that is under the control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. Hairpins are extremely difficult to manipulate in Escherichia coli, so our method makes use of a heterologous 330 bp spacer encoding sequences from green fluorescent protein to facilitate the cloning steps. When the UAS-y-IR hairpin is expressed under the control of different promoter-GAL4 fusions, a high frequency of y pigment phenocopies is obtained in adults. Consequently this method for producing gene knock-outs has several advantages over previous methods in that it is applicable to any gene within the fly genome, greatly facilitates cloning of the hairpin, can be used if required with GAL4 drivers to avoid lethality or to induce RNAi in a specific developmental stage and/or tissue, is useful for generating knock-outs of adult phenotypes as reported here and, finally, the system can be manipulated to investigate the trans-acting factors that are involved in the RNAi mechanism.

Animals↗
PubMed2006

Interleukin-1alpha stimulates non-amyloidogenic pathway by alpha-secretase (ADAM-10 and ADAM-17) cleavage of APP in human astrocytic cells involving p38 MAP kinase.

Interleukin-1alpha (IL-1alpha) stimulates a disintegrin and metalloproteinase, ADAM-17 synthesis, consistent with activation of the soluble fragment of Amyloid Precursor Protein, APP, (sAPPalpha) in human primary astrocytes. To characterize the mechanism by which IL-1alpha promotes the non-amyloidogenic pathway of APP metabolism, we used U373 MG astrocytoma cells. IL-1alpha significantly increased levels of ADAM-10 and ADAM-17 mRNA in 16 hr. Upregulation of ADAM-17 mRNA by IL-1alpha was more pronounced despite higher basal levels of ADAM-10 mRNA. This pattern was also observed at the protein level with the upregulation of alpha-secretase. RNA interference (RNAi) of ADAM-10 and ADAM-17 inhibited IL-1alpha-stimulated sAPPalpha release and the effect was more pronounced with ADAM-17 RNAi. Concomitantly, the level of sAPPalpha was significantly increased by IL-1alpha in 48 hr; however, IL-1alpha stimulated cell-associated APP levels maximally at 6 h but the induction declined at 48 hr. IL-1alpha treatment of cells for 48 h reduced both intracellular and secreted levels of amyloid-beta, Abeta-40, and Abeta-42 peptides. Multiple MAP kinases (MAPK), including MEK/ERK, p38 kinase, PI3 kinase (PI3K) but not JNK were involved in the regulation of IL-1alpha-stimulated alpha-secretase activity and sAPPalpha release. p38 MAPK seems to be the most proximal of these MAPKs, as it was the earliest to be activated by IL-1alpha and blocking this pathway attenuated activation of IL-1alpha-induced MEK and PI3K pathways. Our data show a complex mechanism of sAPPalpha regulation by IL-1alpha that involves ADAM-10, ADAM-17 and p38 MAPK upstream of MEK and PI3K.

ADAM Proteins↗
PubMed2003

ago1 and dcr1, two core components of the RNA interference pathway, functionally diverge from rdp1 in regulating cell cycle events in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, three genes that function in the RNA interference (RNAi) pathway, ago1+, dcr1+, and rdp1+, have recently been shown to be important for timely formation of heterochromatin and accurate chromosome segregation. In the present study, we present evidence that null mutants for ago1+ and dcr1+ but not rdp1+, exhibit abnormal cytokinesis, cell cycle arrest deficiencies, and mating defects. Subsequent analyses showed that ago1+ and dcr1+ are required for regulated hyperphosphorylation of Cdc2 when encountering genotoxic insults. Because rdp1+ is dispensable for this process, the functions of ago1+ and dcr1+ in this pathway are presumably independent of their roles in RNAi-mediated heterochromatin formation and chromosome segregation. This was further supported by the finding that ago1+ is a multicopy suppressor of the S-M checkpoint deficiency and cytokinesis defects associated with loss of Dcr1 function, but not for the chromosome segregation defects of this mutant. Accordingly, we conclude that Dcr1-dependent production of small interfering RNAs is not required for enactment and/or maintenance of certain cell cycle checkpoints and that Ago1 and Dcr1 functionally diverge from Rdp1 to control cell cycle events in fission yeast. Finally, exogenous expression of hGERp95/EIF2C2/hAgo2, a human Ago1 homolog implicated in posttranscriptional gene silencing, compensated for the loss of ago1+ function in S. pombe. This suggests that PPD proteins may also be important for regulation of cell cycle events in higher eukaryotes.

Adenosine Triphosphatases↗
PubMed2005

Cdc42 downregulates MMP-1 expression by inhibiting the ERK1/2 pathway.

The small GTPases of the Rho family are key intermediates in cellular signalling triggered by activated cell-adhesion receptors. In this study, we took advantage of RNA interference (RNAi) using small interfering RNAs (siRNAs) to define the roles of the best-characterized members of the RhoGTPase family, RhoA, Rac1 and Cdc42, in the control of MMP-1, MMP-2 and type-I-collagen expression in normal human skin fibroblasts (HSFs). A specific and long-lasting repression, up to 7 days after transfection, of the three GTPases was achieved by transient transfection of specific siRNA. The silencing of Cdc42, but not that of RhoA or Rac1, induced a 15-fold increase in MMP-1 secretion. This upregulation was confirmed at the mRNA level and observed with two different siRNAs targeting Cdc42. Such a regulation was also observed in various human cell lines and was rescued by re-expressing wild-type Cdc42 encoded by a construct bearing silent mutations impeding its recognition by the siRNA. By contrast, MMP-2 and type-I-collagen expression was not affected by the individual silencing of each Rho GTPase. Cytokine protein array, enzyme-linked immunosorbent assays and reverse-transcription PCR measurements revealed that ablation of Cdc42 induced an overexpression of interleukin 8 and MCP-1. Although these cytokines are known to induce the expression of MMP-1, we showed that they were not involved in the Cdc42-mediated upregulation of MMP-1. Silencing of Cdc42 also induced an increased phosphorylation of ERK1/2 and p38 MAP kinase. The use of chemical inhibitors on Cdc42-ablated cells revealed that the upregulation of MMP-1 is dependent on the ERK1/2 pathways, whereas the p38 MAP kinase pathway displayed an inhibitory role. Simultaneous knock-down of two or three Rho GTPases allowed us to demonstrate that the RhoA-ROCK pathway was not involved in this regulation but that the silencing of Rac1 reduced the effect of Cdc42 suppression. These data suggest that, in vivo, when cell/extracellular-matrix interactions via integrins induce cytoskeleton organization, MMP-1 expression is maintained at a low level by Cdc42 via a repression of the Rac1 and ERK1/2 pathways. Therefore, Cdc42 contributes to ECM homeostasis and connective tissue integrity.

Blotting, Western↗
PubMed2003

Potent suppression of HIV type 1 infection by a short hairpin anti-CXCR4 siRNA.

The phenomenon of RNA interference (RNAi) sparked a new surge in the area of posttranscriptional gene silencing methodologies and their potential application for HIV-1 gene therapy. A potentially promising strategy is to exploit siRNAs to prevent viral entry at the cell surface by down-regulating essential cell surface HIV-1 coreceptors. In the present studies we targeted the CXCR4 coreceptor for disruption with siRNA to inhibit HIV-1 entry as a first step toward the ultimate goal of translating this to gene therapy for AIDS. A stem-loop hairpin structured anti-CXCR4 siRNA was designed and synthesized in vitro by transcription with T7 polymerase. Down-regulation of the coreceptor was assayed in U373-Magi-CXCR4 cells. FACS analysis showed marked down-regulation of CXCR4 on the cell surface and Western blot analysis confirmed the reduced levels of intracellular synthesis. When challenged with X4-tropic HIV-1 NL4-3, the siRNA-transfected cells exhibited marked viral resistance. Consistent with these results, siRNA-transfected primary lymphocytes also exhibited significant resistance to HIV-1 entry. These proof-of-concept studies demonstrated the efficacy of an siRNA targeted to an essential cellular coreceptor CXCR4 in protecting from HIV-1 infection. Delivery of this siRNA into hematopoietic stem cells via lentiviral vectors may have potential gene therapeutic applications.

Blotting, Western↗