Glutamine (GLN) plays a key role in cellular protection following injury via enhancement of heat shock protein 70 (HSP70). The pathway by which GLN enhances HSP70 is unknown. GLN is a key substrate for the hexosamine biosynthetic pathway (HBP), which has been shown to induce HSP70. We sought to explore the role of the HBP in GLN-mediated HSP70 expression. Both chemical inhibitors and small interfering (si)RNA knockdown of key HBP enzymes were used in mouse embryonic fibroblast cells to determine the effects of the HBP on HSP70 expression. The O-glycosylation, nuclear translocation, and transcriptional activation of heat shock factor-1 (HSF-1) and Sp1 were evaluated using immunoprecipitation, Western blotting, and luciferase assays. HSP70 expression levels were evaluated via ELISA and Western blotting. GLN augmented HBP activity before and after heat stress (HS). Chemical inhibition of HBP enzymes reduced GLN-mediated HSP70 expression. Specific siRNA targeting of the key HBP enzyme UDP-N-acetylglucosamine (GlcNAc): polypeptide-O-beta-acetylglucosaminyltransferase (OGT) blocked GLN-mediated HSP70 expression and attenuated GLN-mediated cellular protection post-HS. Chemical and siRNA attenuation of the HBP blocked GLN-induced nuclear translocation of Sp1 and HSF-1, which are key to maximal HSP70 expression. Finally, immunoprecipitation revealed HSF-1 was O-glycosylated, and GLN enhanced this effect. These results suggest that metabolism of GLN via the HBP enhances HSP70 expression. This effect appears to be mediated via O-glycosylation, nuclear translocation, and transcriptional activation of Sp1 and HSF-1. This is an important mechanistic description of a pathway that appears responsible for GLN-mediated HSP70 expression.

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

Short interfering RNAs (siRNAs) that mediate specific gene silencing through RNA interference (RNAi) are widely used to study gene function and are also being developed for therapeutic applications. Many nucleic acids, including double-(dsRNA) and single-stranded RNA (ssRNA), can stimulate innate cytokine responses in mammals. Despite this, few studies have questioned whether siRNA may have a similar effect on the immune system. This could significantly influence the in vivo application of siRNA owing to off-target effects and toxicities associated with immune stimulation. Here we report that synthetic siRNAs formulated in nonviral delivery vehicles can be potent inducers of interferons and inflammatory cytokines both in vivo in mice and in vitro in human blood. The immunostimulatory activity of formulated siRNAs and the associated toxicities are dependent on the nucleotide sequence. We have identified putative immunostimulatory motifs that have allowed the design of siRNAs that can mediate RNAi but induce minimal immune activation.

Most paramyxoviruses circumvent the IFN response by blocking IFN signaling and limiting the production of IFN by virus-infected cells. Here we report that the highly conserved cysteine-rich C-terminal domain of the V proteins of a wide variety of paramyxoviruses binds melanoma differentiation-associated gene 5 (mda-5) product. mda-5 is an IFN-inducible host cell DExD/H box helicase that contains a caspase recruitment domain at its N terminus. Overexpression of mda-5 stimulated the basal activity of the IFN-beta promoter in reporter gene assays and significantly enhanced the activation of the IFN-beta promoter by intracellular dsRNA. Both these activities were repressed by coexpression of the V proteins of simian virus 5, human parainfluenza virus 2, mumps virus, Sendai virus, and Hendra virus. Similar results to the reporter assays were obtained by measuring IFN production. Inhibition of mda-5 by RNA interference or by dominant interfering forms of mda-5 significantly inhibited the activation of the IFN-beta promoter by dsRNA. It thus appears that mda-5 plays a central role in an intracellular signal transduction pathway that can lead to the activation of the IFN-beta promoter, and that the V proteins of paramyxoviruses interact with mda-5 to block its activity.

Hypoxia-inducible factor (HIF), a transcriptional complex conserved from Caenorhabditis elegans to vertebrates, plays a pivotal role in cellular adaptation to low oxygen availability. In normoxia, the HIF-alpha subunits are targeted for destruction by prolyl hydroxylation, a specific modification that provides recognition for the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumour suppressor protein (pVHL). Three HIF prolyl-hydroxylases (PHD1, 2 and 3) were identified recently in mammals and shown to hydroxylate HIF-alpha subunits. Here we show that specific 'silencing' of PHD2 with short interfering RNAs is sufficient to stabilize and activate HIF-1alpha in normoxia in all the human cells investigated.'Silencing' of PHD1 and PHD3 has no effect on the stability of HIF-1alpha either in normoxia or upon re-oxygenation of cells briefly exposed to hypoxia. We therefore conclude that, in vivo, PHDs have distinct assigned functions, PHD2 being the critical oxygen sensor setting the low steady-state levels of HIF-1alpha in normoxia. Interestingly, PHD2 is upregulated by hypoxia, providing an HIF-1-dependent auto-regulatory mechanism driven by the oxygen tension.

RNA interference is an evolutionarily conserved process in which expression of a specific gene is post-transcriptionally inhibited by a small interfering RNA (siRNA), which recognizes a complementary mRNA and induces its degradation. Currently, RNA interference is being used extensively to inhibit expression of specific genes for experimental and therapeutic purposes. For applications in mammalian cells, siRNAs are designed to be <approximately 30 base pairs to avoid nonspecific effects that arise from inducing the cellular double-stranded RNA (dsRNA)-dependent protein kinase (PKR) response. Here we perform expression profiling in mammalian tissue-culture cells treated under standard conditions with conventional 21-bp siRNAs and find, unexpectedly, that >1000 genes involved in diverse cellular functions are nonspecifically stimulated or repressed. The effects on gene expression are dependent upon siRNA concentration and are stable throughout the course of siRNA treatment. Our results can be explained by previous studies showing that dsRNAs can affect multiple signaling and transcription pathways in addition to PKR. The potential for this widespread, nonspecific effect on mammalian gene expression must be carefully considered in the design of siRNA experiments and therapeutic applications.

RNA interference is a powerful method for suppressing gene expression in mammalian cells. Stable knock-down can be achieved by continuous expression of synthetic short hairpin RNAs, typically from RNA polymerase III promoters. But primary microRNA transcripts, which are endogenous triggers of RNA interference, are normally synthesized by RNA polymerase II. Here we show that RNA polymerase II promoters expressing rationally designed primary microRNA-based short hairpin RNAs produce potent, stable and regulatable gene knock-down in cultured cells and in animals, even when present at a single copy in the genome. Most notably, by tightly regulating Trp53 knock-down using tetracycline-based systems, we show that cultured mouse fibroblasts can be switched between proliferative and senescent states and that tumors induced by Trp53 suppression and cooperating oncogenes regress upon re-expression of Trp53. In practice, this primary microRNA-based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals.

Small interfering RNAs (siRNAs) were designed to target the bcr-abl oncogene, which causes chronic myeloid leukemia (CML) and bcr-abl-positive acute lymphoblastic leukemia (ALL). Chemically synthesized anti-bcr-abl siRNAs were selected using reporter gene constructs and were found to reduce bcr-abl mRNA up to 87% in bcr-abl-positive cell lines and in primary cells from CML patients. This mRNA reduction was specific for bcr-abl because c-abl and c-bcr mRNA levels remained unaffected. Furthermore, protein expression of BCR-ABL and of laminA/C was reduced by specific siRNAs up to 80% in bcr-abl-positive and normal CD34(+) cells, respectively. Finally, anti-bcr-abl siRNA inhibited BCR-ABL-dependent, but not cytokine-dependent, proliferation in a bcr-abl-positive cell line. These data demonstrate that siRNA can specifically and efficiently interfere with the expression of an oncogenic fusion gene in hematopoietic cells.

Modulation of calcium-sensitive potassium (BK) channels by oxygen is important in several mammalian tissues, and in the carotid body it is crucial to respiratory control. However, the identity of the oxygen sensor remains unknown. We demonstrate that hemoxygenase-2 (HO-2) is part of the BK channel complex and enhances channel activity in normoxia. Knockdown of HO-2 expression reduced channel activity, and carbon monoxide, a product of HO-2 activity, rescued this loss of function. Inhibition of BK channels by hypoxia was dependent on HO-2 expression and was augmented by HO-2 stimulation. Furthermore, carotid body cells demonstrated HO-2-dependent hypoxic BK channel inhibition, which indicates that HO-2 is an oxygen sensor that controls channel activity during oxygen deprivation.

Inappropriate transcriptional repression involving histone deacetylases (HDACs) is a prominent cause for the development of leukemia. We now identify faulty expression of a specific mediator of transcriptional repression in a solid tumor. Loss of the adenomatosis polyposis coli (APC) tumor suppressor induces HDAC2 expression depending on the Wnt pathway and c-Myc. Increased HDAC2 expression is found in the majority of human colon cancer explants, as well as in intestinal mucosa and polyps of APC-deficient mice. HDAC2 is required for, and sufficient on its own to prevent, apoptosis of colonic cancer cells. Interference with HDAC2 by valproic acid largely diminishes adenoma formation in APC(min) mice. These findings point toward HDAC2 as a particularly relevant potential target in cancer therapy.

The SAP family transcription factor myocardin functionally synergizes with serum response factor (SRF) and plays an important role in cardiac development. To determine the function of myocardin in the smooth muscle cell (SMC) lineage, we mapped the pattern of myocardin gene expression and examined the molecular mechanisms underlying transcriptional activity of myocardin in SMCs and embryonic stem (ES) cells. The human and murine myocardin genes were expressed in vascular and visceral SMCs at levels equivalent to or exceeding those observed in the heart. During embryonic development, the myocardin gene was expressed abundantly in a precise, developmentally regulated pattern in SMCs. Forced expression of myocardin transactivated multiple SMC-specific transcriptional regulatory elements in non-SMCs. By contrast, myocardin-induced transactivation was not observed in SRF(-/-) ES cells but could be rescued by forced expression of SRF or the SRF DNA-binding domain. Furthermore, expression of a dominant-negative myocardin mutant protein or small-interfering-RNA-induced myocardin knockdown significantly reduced SM22 alpha promoter activity in SMCs. Most importantly, forced expression of myocardin activated expression of the SM22 alpha, smooth muscle alpha-actin, and calponin-h1 genes in undifferentiated mouse ES cells. Taken together, these data demonstrate that myocardin plays an important role in the SRF-dependent transcriptional program that regulates SMC development and differentiation.