Identification of a network involved in thapsigargin-induced apoptosis using a library of small interfering RNA expression vectors

We describe here the construction of a library of small interfering RNA expression vectors targeted to a few hundred apoptosis-related genes and the application of this library to an investigation of thapsigargin (TG)-induced apoptosis. Thapsigargin triggers endoplasmic reticulum stress, with subsequent apoptosis, but the molecular mechanisms underlying this process are incompletely understood. Using our library, we identified three anti-apoptotic genes, namely, NOXA, E2F1, and MAPK1, in addition to already characterized genes in the apoptotic pathway. In contrast to proposals by others, our data revealed (i) that TG-induced apoptosis is associated with Apaf1 in a caspase-3- and caspase-9-independent manner; (ii) that the E2F1-PUMA pathway might be involved; and (iii) that the ERK pathway, via MAP3K8 (mitogen-activated protein kinase kinase 8), is required for the induction by TG of apoptosis. Our study demonstrates clearly that unexpected and novel genes can be identified effectively by our method, and it provides evidence for the efficacy and utility of the comprehensive analysis of signaling networks and pathways using a library of small interfering RNA expression vectors.     

Strategies for generation of an siRNA expression library directed against the human genome

RNA interference (RNAi) is a phenomenon whereby expression of an individual gene is specifically silenced by the introduction of a double-stranded RNA (dsRNA) whose sequence is homologous to that of the gene in question. The generation of a small interfering RNA (siRNA) expression library directed against the entire human genome is a project that requires solutions to many difficult technical problems. We present here some strategies for solving some of these problems, including the development of genetically stable and highly active siRNA expression vectors, a procedure for selection of favorable target sites, and an efficient and inexpensive procedure for constructing an siRNA expression library.     

Protein kinase C-dependent protein kinase D activation modulates ERK signal pathway and endothelial cell proliferation by vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal conditions and in pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are not well defined. Protein kinase D (PKD), a newly described serine/threonine protein kinase, has been implicated in many signal transduction pathways and in cell proliferation. We hypothesized that PKD would mediate VEGF signaling and function in endothelial cells. Here we found that VEGF rapidly and strongly stimulated PKD phosphorylation and activation in endothelial cells via VEGF receptor 2 (VEGFR2). The pharmacological inhibitors for phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) significantly inhibited VEGF-induced PKD activation, suggesting the involvement of the PLCgamma/PKC pathway. In particular, PKCalpha was critical for VEGF-induced PKD activation since both overexpression of adenovirus PKCalpha dominant negative mutant and reduction of PKCalpha expression by small interfering RNA markedly inhibited VEGF-induced PKD activation. Importantly, we found that small interfering RNA knockdown of PKD and PKCalpha expression significantly attenuated ERK activation and DNA synthesis in endothelial cells by VEGF. Taken together, our results demonstrated for the first time that VEGF activates PKD via the VEGFR2/PLCgamma/PKCalpha pathway and revealed a critical role of PKD in VEGF-induced ERK signaling and endothelial cell proliferation.     

Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis

Resistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2-related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)-induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system.     

WNK1 activates ERK5 by an MEKK2/3-dependent mechanism

WNK1 belongs to a unique protein kinase family that lacks the catalytic lysine in its normal position. Mutations in human WNK1 and WNK4 have been implicated in causing a familial form of hypertension. Here we report that overexpression of WNK1 led to increased activity of cotransfected ERK5 in HEK293 cells. ERK5 activation was blocked by the MEK5 inhibitor U0126 and expression of a dominant negative MEK5 mutant. Expression of dominant negative mutants of MEKK2 and MEKK3 also blocked activation of ERK5 by WNK1. Moreover, both MEKK2 and MEKK3 coimmunoprecipitated with endogenous WNK1 from cell lysates. WNK1 phosphorylated both MEKK2 and -3 in vitro, and MEKK3 was activated by WNK1 in 293 cells. Finally, ERK5 activation by epidermal growth factor was attenuated by suppression of WNK1 expression using small interfering RNA. Taken together, these results place WNK1 in the ERK5 MAP kinase pathway upstream of MEKK2/3.     

Determination of the Role of DDX3 a Factor Involved in Mammalian RNAi Pathway Using an shRNA-Expression Library

RNA interference (RNAi) is an endogenous RNA-destruction phenomenon induced by certain double-stranded RNAs (dsRNAs). In RNAi, dsRNAs are processed into small interfering RNAs (siRNAs) which in turn trigger the cleavage of the target mRNA. Here, using a short hairpin RNA-expression library, we identified a DEAD-box helicase 3, DDX3, as an essential factor involved in RNAi pathway and revealed that DDX3 is colocalized with Ago2, an essential factor in RNAi pathway that cleaves target mRNA. Results of experiments with a dominant negative mutant of DDX3 further confirmed that this factor affects the RNAi activity. Together, DDX3 functions to assure mammalian RNAi pathway. Together, our results indicate that DDX3 is a new key molecule to understand the molecular mechanism underlying RNAi pathway in mammals.     

Regulation of docetaxel-induced apoptosis of human melanoma cells by different isoforms of protein kinase C

Our previous studies showed that docetaxel-induced apoptosis of human melanoma cells was dependent on the activation of the c-jun NH(2)-terminal kinase (JNK) signaling pathway but was inhibited by the extracellular signal-regulated kinase (ERK)-1/2 pathway. However, the mechanisms by which these pathways were modulated by docetaxel were not clear. We report here that docetaxel induces activation of protein kinase C (PKC) signaling differentially through PKCepsilon and PKCdelta isoforms. Activation of PKCepsilon was most marked in docetaxel-resistant cells and paralleled the activation of the ERK1/2 pathway. Inhibition of PKCepsilon by small interfering RNA molecules resulted in down-regulation of phosphorylated ERK1/2 and sensitization of cells to docetaxel-induced apoptosis. Experiments also showed that beta-tubulin class III, a molecular target of docetaxel, coimmunoprecipitated with PKCepsilon and colocalized in confocal microscopic studies. In contrast to PKCepsilon, high levels of activated PKCdelta were associated with activation of the JNK pathway and sensitivity to docetaxel. Activation of PKCdelta seemed to be upstream of JNK because inhibition of PKCdelta by small interfering RNA abrogated activation of the JNK pathway. Although PKCdelta could be activated in resistant cells, downstream activation of JNK and c-Jun did not occur. In summary, these results suggest that the outcome of docetaxel-induced apoptotic events in human melanoma cells depends on their PKC isoform content and signaling responses. PKCepsilon was associated with prosurvival signaling through ERK, whereas PKCdelta was associated with proapoptotic responses through JNK activation.     

Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and -2

Protease-activated receptor-2 (PAR-2) is activated by trypsin-like serine proteases and can promote cell migration through an ERK1/2-dependent pathway, involving formation of a scaffolding complex at the leading edge of the cell. Previous studies also showed that expression of a dominant negative fragment of beta-arrestin-1 reduces PAR-2-stimulated internalization, ERK1/2 activation, and cell migration; however, this reagent may block association of many proteins, including beta-arrestin-2 with clathrin-coated pits. Here we investigate the role of PAR-2 in the constitutive migration of a metastatic breast cancer cell line, MDA MB-231, and use small interfering RNA to determine the contribution of each beta-arrestin to this process. We demonstrate that a trypsin-like protease secreted from MDA MB-231 cells can promote cell migration through autocrine activation of PAR-2 and this correlates with constitutive localization of PAR-2, beta-arrestin-2, and activated ERK1/2 to pseudopodia. Addition of MEK-1 inhibitors, trypsin inhibitors, a scrambled PAR-2 peptide, and silencing of beta-arrestins with small interfering RNA also reduce base-line migration of MDA MB-231 cells. In contrast, a less metastatic PAR-2 expressing breast cancer cell line does not exhibit constitutive migration, pseudopodia formation, or trypsin secretion; in these cells PAR-2 is more uniformly distributed around the cell periphery. These data demonstrate a requirement for both beta-arrestins in PAR-2-mediated motility and suggest that autocrine activation of PAR-2 by secreted proteases may contribute to the migration of metastatic tumor cells through beta-arrestin-dependent ERK1/2 activation.     

Eukaryotic translation initiation factor 3, subunit a, regulates the extracellular signal-regulated kinase pathway

The extracellular signal-regulated kinase (ERK) pathway participates in the control of numerous cellular processes, including cell proliferation. Since its activation kinetics are critical for to its biological effects, they are tightly regulated. We report that the protein translation factor, eukaryotic translation initiation factor 3, subunit a (eIF3a), binds to SHC and Raf-1, two components of the ERK pathway. The interaction of eIF3a with Raf-1 is increased by β-arrestin2 expression and transiently decreased by epidermal growth factor (EGF) stimulation in a concentration-dependent manner. The EGF-induced decrease in Raf-1-eIF3a association kinetically correlates with the time course of ERK activation. eIF3a interferes with Raf-1 activation and eIF3a downregulation by small interfering RNA enhances ERK activation, early gene expression, DNA synthesis, expression of neuronal differentiation markers in PC12 cells, and Ras-induced focus formation in NIH 3T3 cells. Thus, eIF3a is a negative modulator of ERK pathway activation and its biological effects.     

OxLDL up-regulates Niemann-Pick type C1 expression through ERK1/2/COX-2/PPARα-signaling pathway in macrophages

The Niemann-Pick type C1 (NPC1) is located mainly in the membranes of the late endosome/lysosome and controls the intracellular cholesterol trafficking from the late endosome/lysosome to the plasma membrane. It has been reported that oxidized low-density lipoprotein (oxLDL) can up-regulate NPC1 expression. However, the detailed mechanisms are not fully understood. In this study, we investigated the effect of oxLDL stimulation on NPC1 expression in THP-1 macrophages. Our results showed that oxLDL up-regulated NPC1 expression at both mRNA and protein levels in a dose-dependent and time-dependent manner. In addition, oxLDL also induced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Treatment with oxLDL significantly increased cyclooxygenase-2 (COX-2) mRNA and protein expression in the macrophages, and these increases were suppressed by the ERK1/2 inhibitor PD98059 or ERK1/2 small interfering RNA (siRNA) treatment. OxLDL up-regulated the expression of peroxisome proliferator-activated receptor α (PPARα) at the mRNA and protein levels, which could be abolished by COX-2 siRNA or COX-2 inhibitor NS398 treatment in these macrophages. OxLDL dramatically elevated cellular cholesterol efflux, which was abrogated by inhibiting ERK1/2 and/or COX-2. In addition, oxLDL-induced NPC1 expression and cellular cholesterol efflux were reversed by PPARα siRNA or GW6471, an antagonist of PPARα. Taken together, these results provide the evidence that oxLDL can up-regulate the expression of the NPC1 through ERK1/2/COX-2/PPARα-signaling pathway in macrophages.     

B-RAF regulation of Rnd3 participates in actin cytoskeletal and focal adhesion organization

The actin cytoskeleton controls multiple cellular functions, including cell morphology, movement, and growth. Accumulating evidence indicates that oncogenic activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) pathway is accompanied by actin cytoskeletal reorganization. However, the signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation are largely unknown. Mutant B-RAF is found in a variety of cancers, including melanoma, and it enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with small interfering RNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/Rho kinase (ROCK)/LIM kinase-2 signaling pathway, cumulating in the inactivation of the actin depolymerizing/severing protein cofilin. The expression of Rnd3, a Rho antagonist, was attenuated after B-RAF knockdown or MEK inhibition, but it was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Together, our results identify Rnd3 as a regulator of cross talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions.     

Long double-stranded RNA-mediated RNA interference and immunostimulation: long interfering double-stranded RNA as a potent anticancer therapeutics

In most applications, small interfering RNAs are designed to execute specific gene silencing via RNA interference (RNAi) without triggering nonspecific responses such as immunostimulation. However, in anticancer therapeutics, immunostimulation combined with specific oncogene silencing could be beneficial, resulting in the synergistic inhibition of cancer cell growth. In this study, we report an immunostimulatory long double-stranded RNA (dsRNA) structure with the ability to trigger RNAi-mediated specific target gene silencing, termed as long interfering dsRNA (liRNA). liRNA targeting Survivin mRNA not only efficiently and specifically triggered target gene silencing via RNAi, but also stimulated the protein kinase R pathway to induce the expression of interferon β. As a result, the ability of Survivin-targeting liRNA to inhibit cancer cell growth was superior over conventional small interfering RNA or nontargeting dsRNA structures. Our results thus provide a simple yet efficient dual function immunostimulatory RNAi-triggering structure, which is potentially applicable for the development of anticancer therapeutics.     

细胞MEK1/2蛋白及其相关通路在单纯疱疹病毒Ⅱ型（HSV-2）复制中的作用

基于细胞Raf/MEK/ERK信号通路与病毒复制的关系,应用Western印迹检测 p-ERK1/2蛋白的表达、用终点滴定法测定病毒增殖量（TCID_５０）,以及观察感染细胞的细胞病变效应（CPE）等,揭示单纯疱疹病毒Ⅱ型(HSV-2)复制与 ERK通路的关系. 结果表明,HSV-2的复制可引起细胞ERK通路的活化;用U0126预先抑制ERK通路的活化,或用特异性siRNA敲减MEK1/2基因的表达可显著地抑制病毒复制.提示ERK信号通路以及MEK1/2蛋白对HSV-2的复制具有重要的作用.该研究对进一步阐明细胞ERK通路各激酶蛋白在病毒复制中的作用机制、寻找抗病毒作用靶标等奠定了良好的基础.     

Hepatitis C virus infection induces the beta interferon signaling pathway in immortalized human hepatocytes

Beta interferon (IFN-beta) expression is triggered by double-stranded RNA, a common intermediate in the replication of many viruses including hepatitis C virus (HCV). The recent development of cell culture-grown HCV allowed us to analyze the IFN signaling pathway following virus infection. In this study, we have examined the IFN-beta signaling pathway following infection of immortalized human hepatocytes (IHH) with HCV genotype 1a (clone H77) or 2a (clone JFH1). We observed that IHH possesses a functional Toll-like receptor 3 pathway. HCV infection in IHH enhanced IFN-beta and IFN-stimulated gene 56 (ISG56) promoter activities; however, poly(I-C)-induced IFN-beta and ISG56 expression levels were modestly inhibited upon HCV infection. IHH infected with HCV (genotype 1a or 2a) exhibited various levels of translocation of IRF-3 into the nucleus. The upregulation of endogenous IFN-beta and 2',5'-oligoadenylate synthetase 1 mRNA expression was also observed in HCV-infected IHH. Subsequent studies suggested that HCV infection in IHH enhanced STAT1 and ISG56 protein expression. A functional antiviral response of HCV-infected IHH was observed by the growth-inhibitory role in vesicular stomatitis virus. Together, our results suggested that HCV infection in IHH induces the IFN signaling pathway, which corroborates observations from natural HCV infection in humans.