Short Inhibitory RNA Research Articles

Epidermal Growth Factor Receptor and Protein Kinase C Signaling to ERK2: SPATIOTEMPORAL REGULATION OF ERK2 BY DUAL SPECIFICITY PHOSPHATASES

Spatiotemporal aspects of ERK activation are stimulus-specific and dictate cellular consequences. They are dependent upon dual specificity phosphatases (DUSPs) that bind ERK via docking domains and can both inactivate and anchor ERK in cellular compartments. Using high throughput fluorescence microscopy in combination with a system where endogenous ERKs are removed and replaced with wild-type or mutated ERK2-green fluorescent protein (GFP), we show that ERK2 activation responses to epidermal growth factor (EGF) and protein kinase C (PKC) are transient and sustained, respectively. PKC-mediated ERK2 activation is associated with prolonged nuclear localization in the dephosphorylated form, whereas EGF-stimulated ERK2 activation mediates only transient nuclear accumulation. By using short inhibitory RNAs to nuclear inducible DUSP1, -2, or -4 (alone or in combination), we demonstrate that all three of these enzymes contribute to the dephosphorylation of PKC (but not EGF)-activated ERK2 in the nucleus but that they have opposing effects on localization. DUSP2 and -4 inactivate and anchor ERK2, whereas DUSP1 dephosphorylates ERK in the nucleus but allows its traffic back to the cytoplasm. Overexpression of DUSP1, -2, or -4 prevented ERK2 activation, but only DUSP2 and -4 caused ERK2-GFP nuclear accumulation or could be immunoprecipitated with ERK2. Furthermore, protein synthesis inhibition or replacement of wild-type ERK2-GFP with docking domain mutants selectively increased PKC effects on ERK activity and altered ERK2-GFP localization. These mutations also impaired the ability of ERK2-GFP to bind DUSP2 and -4. Together, our data reveal a novel, stimulus-specific, and phosphatase-specific mechanism of ERK2 regulation in the nucleus by DUSP1, -2, and -4.

Protocols for gene silencing in schistosomes

Schistosomes are parasitic platyhelminths that infect over 200 million people globally. In recent years there have been many advances in schistosome genomics and proteomics and in the development of molecular tools for use with these parasites. Among the more promising methodologies is RNA interference (RNAi) which is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. We aim to develop effective protocols utilizing RNAi for use in the intra-mammalian life stages of Schistosoma mansoni. In this work, the gene encoding alkaline phosphatase (SmAP) was targeted by exposing the parasites to dsRNA encoding part of the SmAP coding region. SmAP is known to be expressed in a variety of parasite tissues. We report that both long dsRNAs as well as synthetic short inhibitory RNAs (siRNAs) are effective at eliciting SmAP gene suppression in cultured schistosomula and in adult males and females. Electroporation as a mode of dsRNA delivery is more efficient than simply soaking the parasites in an equivalent dose. Relative SmAP RNA levels >90% lower than controls were routinely detected, when measured 2 days after treatment by electroporation, using quantitative real-time PCR. Commensurate with this decline in SmAP RNA, relative alkaline phosphatase enzyme activity levels >70% lower than controls were detected, 5 days after treatment. Protocols described here that result in the robust suppression of target genes in intravascular schistosomes may have wide applicability and promote functional schistosome genomics.

Optimizing gene suppression in schistosomes using RNA interference

Schistosomes are parasitic platyhelminths that constitute an important public health problem globally. Here we describe optimized protocols for effectively suppressing gene expression in the intra-mammalian life stages of Schistosoma mansoni using RNA interference (RNAi). RNAi is a mechanism by which gene-specific double stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. The gene encoding the cysteine protease cathepsin B (SmCB1 or Sm31) was targeted by exposing the parasites to dsRNA encoding part of the cathepsin B coding region. Suppression was measured using quantitative real time PCR. Electroporation as a mode of dsRNA delivery was substantially more efficient (100–1000-fold) than simply soaking the parasites in an equivalent dose. Soaking the parasites with dsRNA in the presence of different proprietary liposome preparations did not enhance gene suppression. In fact, all three reagents tested were variably toxic to the cultured schistosomes. Both long dsRNAs as well as synthetic short inhibitory RNAs (siRNAs) were effective at eliciting gene suppression. Different siRNAs exhibited variable efficiencies of suppression, perhaps reflecting differences in siRNA accessibility to the cathepsin B mRNA. Parasites cultured in vitro for 7 days or more following their emergence from the intermediate snail host were more susceptible to RNAi than those treated with dsRNA on the day of emergence (during the process of cercarial transformation into schistosomula). In addition, adult male and female parasites (49 days old) were susceptible to RNAi. Using fluorescein-labeled dsRNA to monitor the process, it was seen that in schistosomula (cultured for 7 days), electroporated dsRNA entered primarily through the mouth into the caecum while in young parasites (freshly emerged from snails) dsRNA appeared to enter primarily into the pre- and post-acetabular glands. The cathepsin B gene was significantly suppressed for up to 40 days after treatment suggesting that, as in some other organisms, the RNAi process can be amplified in schistosomes.

Experimental research of effectiveness of siRNA-Her-2/neu on drug sensitivity of Her-2/neu-over-expressing lung adenocarcinoma cell line

Objective: Her-2/neu protein overexpression has been demonstrated in lung adenocarcinoma. Its overexpression often indicates a poor prognosis and resistance to chemotherapeutic agents. The objectives of this paper is to explore the effectiveness of double-stranded short inhibitory RNAs (siRNA) targeting Her-2/neu oncogene on the drug sensitivity of Her-2/neu-overexpressing lung adenocarcinoma cells. Methods: Lung adenocarcinoma cell line calu-3 was transfected with siRNAs formulated LipofectAMINE 2000, and Her-2/neu protein and P-gp were determined by flow cytometry (FCM). The chemosensitivity of transfected cells to cisplatin (CDDP) was measured by MTT. Cell apoptosis detection kit (Annexin V method) was used to examine the drug induced apoptosis rate. Results: siRNA targeting Her-2/neu greatly reduced the cell surface expression of Her-2/neu protein and had no effect on P-gp. Consequently the inhibitory rate of CDDP in combination with siRNA targeting Her-2/neu was (67.1±2.3)%, while the inhibitory rates were (48.1±3.5)%, (46.3±5.9)% and (50.2±2.9)% in untreated control, empty vector and unrelated siRNA groups, respectively. The FCM results showed that the apoptosis rate of cells treated with CDDP combined with siRNAs-Her-2/neu was elevated when compared with unrelated siRNA group and empty vector group. Conclusion: Sequence specific siRNA targeting Her-2/neu was capable of enhancing the chemosensitivity of calu-3 cell to cisplatin.

The Healing Effects of TGF-β

Proper wound healing requires orderly migration of multiple cell types. In humans, epidermal keratinocytes (HKs) move across the wound to form a new continuous epithelial layer. Dermal cells like dermal fibroblasts (DFs) and human dermal microvascular endothelial cells (HDMECs) move in to restore the extracellular matrix and help build new blood vessels. But how do these cells know when and where to do their work? New work from Bandyopadhyay et al. suggests that one important factor is that cells at the wound are first exposed to plasma (blood liquid without the cells), then serum (the cell-free liquid that remains after blood clotting), and then again to plasma. The authors observed the effects of serum and plasma on the various cell types in an assay in which cells left visible tracks on a surface of extracellular matrix coated with colloidal gold or in an in vitro wound-healing assay. Plasma stimulated migration of dermal cells (DFs and HDMECs) but had little effect on epidermal HKs. Conversely, serum stimulated migration of HKs but had little effect on DFs and HDMECs. The authors suspected transforming growth factor-β (TGF-β) as a factor that could account for these differential responses because it is relatively abundant in serum and scarce or undetectable in plasma. Cells responded to serum from which the TGF-β3 isoform had been removed with antibodies as though they were responding to plasma. The authors also found that type II TGF-β receptors were abundant in dermal cells but less prevalent in epidermal cells. Exogenous expression of TGF-βRII in HKs caused the cell to respond to serum with reduced, rather than enhanced, motility. When short inhibitory RNA was used to decrease expression of TGF-βRII in dermal cells, their migratory responses to plasma and serum were reversed. The authors propose that the sequential exposure of cells at a wound to plasma, then serum, and then plasma again coordinates the movement of the various cell types to allow tissue remodeling and healing. Wounds that fail to heal are a problem in certain disease states and in the elderly, and better understanding of the orchestration of the normal healing process may permit more effective use of growth-factor therapies to enhance healing of refractory wounds in the clinic. B. Bandyopadhyay, J. Fan, S. Guan, Y. Li, M. Chen, D. T. Woodley, W. Li, A "traffic control" role for TGFβ3: Orchestrating dermal and epidermal cell motility during wound healing. J. Cell Biol. 172 , 1093-1105 (2006). [Abstract] [Full Text]

Activation Function-1 Domain of Estrogen Receptor Regulates the Agonistic and Antagonistic Actions of Tamoxifen

The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.

Melanophilin and myosin Va track the microtubule plus end on EB1

In mouse melanocytes, myosin Va is recruited onto the surface of melanosomes by a receptor complex containing Rab27a that is present in the melanosome membrane and melanophilin (Mlp), which links myosin Va to Rab27a. In this study, we show that Mlp is also a microtubule plus end–tracking protein or +TIP. Moreover, myosin Va tracks the plus end in a Mlp-dependent manner. Data showing that overexpression and short inhibitory RNA knockdown of the +TIP EB1 have opposite effects on Mlp–microtubule interaction, that Mlp interacts directly with EB1, and that deletion from Mlp of a region similar to one in the adenomatous polyposis coli protein involved in EB1 binding blocks Mlp's ability to plus end track argue that Mlp tracks the plus end directly by hitchhiking on EB1. These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p–Kar9p–Bim1p complex in yeast. We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.

Stable Plasmid-based siRNA Silencing of Gene Expression in Human Embryonic Stem Cells

RNA interference (RNAi) using short inhibitory RNAs (siRNAs) has been widely explored for the suppression of cellular mRNA levels to investigate the function of specific genes, including gene function in differentiation and development. The establishment of human embryonic stem cell (hESC) models for differentiation of selected lineages is an area of intense interest and activity. On the basis of our previous work with stable overexpression of enhanced green fluorescent protein (EGFP) in hESC, we used plasmid vector-based siRNA expression to silence EGFP expression in stably-transfected hESC. After hygromycin selection, we derived several cell lines in which EGFP expression was significantly reduced. At the genomic DNA level, there was no difference between the two cell lines and the parental H1EGFP cell line when analyzed with quantitative PCR; however, there were significant differences among the three cell lines at the RNA and protein levels as analyzed with real-time RT-PCR and Western blotting. From these data, we conclude that the decrease in EGFP expression was caused by RNAi, not by genomic DNA loss. Down-regulation of EGFP expression was sustained through multiple passages of both siEGFP cell lines. This simple silencing system will allow novel investigations of target gene function in hESC self-renewal or differentiation, as well as differentiated function in other cell types.

The p38 mitogen-activated protein kinase pathway links the DNA mismatch repair system to the G2 checkpoint and to resistance to chemotherapeutic DNA-methylating agents.

Although human cells exposed to DNA-methylating agents undergo mismatch repair (MMR)-dependent G(2) arrest, the basis for the linkage between MMR and the G(2) checkpoint is unclear. We noted that mitogen-activated protein kinase p38alpha was activated in MMR-proficient human glioma cells exposed to the chemotherapeutic methylating agent temozolomide (TMZ) but not in paired cells made MMR deficient by expression of a short inhibitory RNA (siRNA) targeted to the MMR protein Mlh1. Furthermore, activation of p38alpha in MMR-proficient cells was associated with nuclear inactivation of the cell cycle regulator Cdc25C phosphatase and its downstream target Cdc2 and with activation of the G(2) checkpoint, actions which were suppressed by the p38alpha/beta inhibitors SB203580 and SB202590 or by expression of a p38alpha siRNA. Finally, pharmacologic or genetic inhibition of p38alpha increased the sensitivity of MMR-proficient cells to the cytotoxic actions of TMZ by increasing the percentage of cells that underwent mitotic catastrophe as a consequence of G(2) checkpoint bypass. These results suggest that p38alpha links DNA MMR to the G(2) checkpoint and to resistance to chemotherapeutic DNA-methylating agents. The p38 pathway may therefore represent a new target for the development of agents to sensitize tumor cells to chemotherapeutic methylating agents.

The Effectiveness of Double-Stranded Short Inhibitory RNAs (siRNAs) May Depend on the Method of Transfection

RNA interference (RNAi) is a recently described powerful experimental tool that can cause sequence-specific gene silencing, thereby facilitating functional analysis of gene function. Consequently, we became interested in using RNAi to determine the function of aberrantly expressed ErbB3 in the KAS-6/1 human myeloma cell line. Despite the wealth of information available on the use of RNAi, dsRNA target design, and the transfection of dsRNA in vitro, little information is available for transfecting dsRNA into nonadherent cells from any species. In the present study, we report that gene silencing of ErbB3 was not observed in myeloma cells when dsRNA targeting ErbB3 was introduced using conventional transfection agents and protocols that have proved successful for several adherent cell lines. Silencing of ErbB3, however, was observed in T47D cells, an adherent breast carcinoma cell line, using the same transfection methods, indicating that our target sequence was functional for gene silencing of ErbB3. Interestingly, ErbB3 was silenced in myeloma cells when the dsRNA target was introduced by electroporation. Thus, our studies illustrate the striking dependence of dsRNA-mediated gene silencing in some cells on the methods of dsRNA transfection.

Deregulated human Cdc14A phosphatase disrupts centrosome separation and chromosome segregation.

We show that human Cdc14A phosphatase interacts with interphase centrosomes, and that this interaction is independent of microtubules and Cdc14A phosphatase activity, but requires active nuclear export. Disrupting the nuclear export signal (NES) led to Cdc14A being localized in nucleoli, which in unperturbed cells selectively contain Cdc14B (ref. 1). Conditional overproduction of Cdc14A, but not its phosphatase-dead or NES-deficient mutants, or Cdc14B, resulted in premature centrosome splitting and formation of supernumerary mitotic spindles. In contrast, downregulation of endogenous Cdc14A by short inhibitory RNA duplexes (siRNA) induced mitotic defects including impaired centrosome separation and failure to undergo productive cytokinesis. Consequently, both overexpression and downregulation of Cdc14A caused aberrant chromosome partitioning into daughter cells. These results indicate that Cdc14A is a physiological regulator of the centrosome duplication cycle, which, when disrupted, can lead to genomic instability in mammalian cells.