Die Wang

A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells

Nonspecific effects triggered by small interfering RNAs (siRNAs) complicate the use of RNA interference (RNAi) to specifically downregulate gene expression. To uncover the basis of these nonspecific activities, we analyzed the effect of chemically synthesized siRNAs on mammalian double-stranded RNA (dsRNA)-activated signaling pathways. siRNAs ranging from 21 to 27 nucleotides (nt) in length activated the interferon system when they lacked 2-nt 3′ overhangs, a characteristic of Dicer products. We show that the recognition of siRNAs is mediated by the RNA helicase RIG-I and that the presence of 3′ overhangs impairs its ability to unwind the dsRNA substrate and activate downstream signaling to the transcription factor IRF-3. These results suggest a structural basis for discrimination between microRNAs that are endogenous Dicer products, and nonself dsRNAs such as by-products of viral replication. These findings will enable the rational design of siRNAs that avoid nonspecific effects or, alternatively, that induce bystander effects to potentially increase the efficacy of siRNA-based treatments of viral infections or cancer.

Analysis of microRNA turnover in mammalian cells following Dicer1 ablation

Although microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to the full appreciation of their regulatory roles. Accordingly, we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we establish a mathematical model of miRNA turnover and determine an average miRNA half-life of 119 h (i.e. ∼5 days). In addition, we demonstrate that select miRNAs turnover more rapidly than others. This study constitutes, to our knowledge, the first in-depth characterization of miRNA decay in mammalian cells. Our findings indicate that miRNAs are up to 10× more stable than messenger RNA and support the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function.

A miR-19 regulon that controls NF-κB signaling

Fine-tuning of inflammatory responses by microRNAs (miRNAs) is complex, as they can both enhance and repress expression of pro-inflammatory mediators. In this study, we investigate inflammatory responses following global miRNA depletion, to better define the overall contribution of miRNAs to inflammation. We demonstrate that miRNAs positively regulate Toll-like receptor signaling using inducible Dicer1 deletion and global miRNA depletion. We establish an important contribution of miR-19b in this effect, which potentiates nuclear factor-κB (NF-κB) activity in human and mouse cells. Positive regulation of NF-κB signaling by miR-19b involves the coordinated suppression of a regulon of negative regulators of NF-κB signaling (including A20/Tnfaip3, Rnf11, Fbxl11/Kdm2a and Zbtb16). Transfection of miR-19b mimics exacerbated the inflammatory activation of rheumatoid arthritis primary fibroblast-like synoviocytes, demonstrating its physiological importance in the pathology of this disease. This study constitutes, to our knowledge, the first description of a miR-19 regulon that controls NF-κB signaling, and suggests that targeting this miRNA and linked family members could regulate the activity of NF-κB signaling in inflammation.

ATF3 suppresses metastasis of bladder cancer by regulating gelsolin-mediated remodeling of the actin cytoskeleton

Bladder cancer is associated with high recurrence and mortality rates due to metastasis. The elucidation of metastasis suppressors may offer therapeutic opportunities if their mechanisms of action can be elucidated and tractably exploited. In this study, we investigated the clinical and functional significance of the transcription factor activating transcription factor 3 (ATF3) in bladder cancer metastasis. Gene expression analysis revealed that decreased ATF3 was associated with bladder cancer progression and reduced survival of patients with bladder cancer. Correspondingly, ATF3 overexpression in highly metastatic bladder cancer cells decreased migration in vitro and experimental metastasis in vivo. Conversely, ATF3 silencing increased the migration of bladder cancer cells with limited metastatic capability in the absence of any effect on proliferation. In keeping with their increased motility, metastatic bladder cancer cells had increased numbers of actin filaments. Moreover, ATF3 expression correlated with expression of the actin filament severing protein gelsolin (GSN). Mechanistic studies revealed that ATF3 upregulated GSN, whereas ATF3 silencing reduced GSN levels, concomitant with alterations in the actin cytoskeleton. We identified six ATF3 regulatory elements in the first intron of the GSN gene confirmed by chromatin immunoprecipitation analysis. Critically, GSN expression reversed the metastatic capacity of bladder cancer cells with diminished levels of ATF3. Taken together, our results indicate that ATF3 suppresses metastasis of bladder cancer cells, at least in part through the upregulation of GSN-mediated actin remodeling. These findings suggest ATF3 coupled with GSN as prognostic markers for bladder cancer metastasis.

Increased CD45RA+ FoxP3(low) regulatory T cells with impaired suppressive function in patients with systemic lupus erythematosus.

Background The role of naturally occurring regulatory T cells (Treg) in the control of the development of systemic lupus erythematosus (SLE) has not been well defined. Therefore, we dissect the phenotypically heterogeneous CD4+FoxP3+ T cells into subpopulations during the dynamic SLE development. Methodlogy/Principal Findings To evaluate the proliferative and suppressive capacities of different CD4+ T cell subgroups between active SLE patients and healthy donors, we employed CD45RA and CD25 as surface markers and carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay. In addition, multiplex cytokines expression in active SLE patients was assessed using Luminex assay. Here, we showed a significant increase in the frequency of CD45RA+FoxP3low naive Treg cells (nTreg cells) and CD45RA−FoxP3low (non-Treg) cells in patients with active SLE. In active SLE patients, the increased proportions of CD45RA+FoxP3low nTreg cells were positively correlated with the disease based on SLE disease activity index (SLEDAI) and the status of serum anti-dsDNA antibodies. We found that the surface marker combination of CD25+CD45RA+ can be used to defined CD45RA+FoxP3low nTreg cells for functional assays, wherein nTreg cells from active SLE patients demonstrated defective suppression function. A significant correlation was observed between inflammatory cytokines, such as IL-6, IL-12 and TNFα, and the frequency of nTreg cells. Furthermore, the CD45RA+FoxP3low nTreg cell subset increased when cultured with SLE serum compared to healthy donor serum, suggesting that the elevated inflammatory cytokines of SLE serum may promote nTreg cell proliferation/expansion. Conclusions/Significance Our results indicate that impaired numbers of functional CD45RA+FoxP3low naive Treg cell and CD45RA−FoxP3low non-suppressive T cell subsets in inflammatory conditions may contribute to SLE development. Therefore, analysis of subsets of FoxP3+ T cells, using a combination of FoxP3, CD25 and CD45RA, rather than whole FoxP3+ T cells, will help us to better understand the pathogenesis of SLE and may lead to the development of new therapeutic strategies.

BTB-ZF transcriptional regulator PLZF modifies chromatin to restrain inflammatory signaling programs

Significance Maintaining physiological balance is vital in the primary response to infectious and other stress stimuli to avert damaging inflammation. Delineation of the cell regulatory processes that control inflammatory processes better enable the development of informed strategies to treat associated pathologies. Toward this end, we identify that the promyelocytic leukemia zinc finger (PLZF) transcription factor limits pathogen-induced inflammation. PLZF stabilizes a repressor complex that encompasses histone deacetylase activity, which modifies the state of chromatin. This activity maintains homeostasis by decreasing the scale of induction of select immune response genes. In the absence of PLZF, the chromatin structure is altered, enabling active transcriptional complexes to immediately assemble on gene promoters, resulting in inordinate production of inflammatory cytokines. Inflammation is critical for host defense, but without appropriate control, it can cause chronic disease or even provoke fatal responses. Here we identify a mechanism that limits the inflammatory response. Probing the responses of macrophages to the key sensory Toll-like receptors, we identify that the Broad-complex, Tramtrack and Bric-a-brac/poxvirus and zinc finger (BTB/POZ), transcriptional regulator promyelocytic leukemia zinc finger (PLZF) limits the expression of inflammatory gene products. In accord with this finding, PLZF-deficient animals express higher levels of potent inflammatory cytokines and mount exaggerated inflammatory responses to infectious stimuli. Temporal quantitation of inflammatory gene transcripts shows increased gene induction in the absence of PLZF. Genome-wide analysis of histone modifications distinguish that PLZF establishes basal activity states of early response genes to maintain immune homeostasis and limit damaging inflammation. We show that PLZF stabilizes a corepressor complex that encompasses histone deacetylase activity to control chromatin. Together with our previous demonstration that PLZF promotes the antiviral response, these results suggest a strategy that could realize one of the major goals of immune therapy to retain immune resistance to pathogens while curbing damaging inflammation.

Regulation of Actin Dynamics by Protein Kinase R Control of Gelsolin Enforces Basal Innate Immune Defense

Primary resistance to pathogens is reliant on both basal and inducible immune defenses. To date, research has focused upon inducible innate immune responses. In contrast to resistance via cytokine induction, basal defense mechanisms are less evident. Here we showed that the antiviral protein kinase R (PKR) inhibited the key actin-modifying protein gelsolin to regulate actin dynamics and control cytoskeletal cellular functions under homeostatic conditions. Through this mechanism, PKR controlled fundamental innate immune, actin-dependent processes that included membrane ruffling and particle engulfment. Accordingly, PKR counteracted viral entry into the cell. These findings identify a layer of host resistance, showing that the regulation of actin-modifying proteins during the innate immune response bolsters first-line defense against intracellular pathogens and has a sustained effect on virus production. Moreover, these data provide proof of principle for a concept in which the cell cytoskeleton could be targeted to elicit broad antiviral protection.

Salicylates Trigger Protein Synthesis Inhibition in a Protein Kinase R-like Endoplasmic Reticulum Kinase-dependent Manner

The non-steroidal anti-inflammatory drug aspirin and its metabolite, sodium salicylate, have profound effects on cellular protein synthesis and cell physiology. However, the underlying mechanism by which they cause these responses remains unclear. We show here that salicylates induce phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in the inhibition of mRNA translation in cells. Exposure of cells to acetyl salicylic acid resulted in strong activation of eIF2α stress-activated protein kinase R-like endoplasmic reticulum kinase (PERK). Analysis of fibroblasts with a targeted deletion of the perk gene revealed that PERK is indispensable for triggering the phosphorylation of eIF2α as well as the inhibition of protein synthesis induced by salicylates. Although salicylate treatment did not trigger activation of inositol-requiring enzyme 1, there was an increased expression of the pro-apoptotic transcription factor CHOP-(gadd153), a downstream event to eIF2α phosphorylation known to mediate endoplasmic reticulum stress-mediated responses. Thus, salicylates selectively trigger an endoplasmic reticulum stress-responsive signaling pathway initiated through activation of PERK to induce their cellular effects.

Conformational rearrangements of RIG-I receptor on formation of a multiprotein:dsRNA assembly

The retinoic acid inducible gene-I (RIG-I)-like family of receptors is positioned at the front line of our innate cellular defence system. RIG-I detects and binds to foreign duplex RNA in the cytoplasm of both immune and non-immune cells, and initiates the induction of type I interferons and pro-inflammatory cytokines. The mechanism of RIG-I activation by double-stranded RNA (dsRNA) involves a molecular rearrangement proposed to expose the N-terminal pair of caspase activation recruitment domains, enabling an interaction with interferon-beta promoter stimulator 1 (IPS-1) and thereby initiating downstream signalling. dsRNA is particularly stimulatory when longer than 20 bp, potentially through allowing binding of more than one RIG-I molecule. Here, we characterize full-length RIG-I and RIG-I subdomains combined with a stimulatory 29mer dsRNA using multi-angle light scattering and size-exclusion chromatography–coupled small-angle X-ray scattering, to build up a molecular model of RIG-I before and after the formation of a 2:1 protein:dsRNA assembly. We report the small-angle X-ray scattering–derived solution structure of the human apo-RIG-I and observe that on binding of RIG-I to dsRNA in a 2:1 ratio, the complex becomes highly extended and flexible. Hence, here we present the first model of the fully activated oligomeric RIG-I.

The acetyltransferase HAT1 moderates the NF-κB response by regulating the transcription factor PLZF

To date, the activities of protein kinases have formed the core of our understanding of cell signal transduction. Comprehension of the extent of protein acetylation has raised expectations that this alternate post-transcriptional modification will be shown to rival phosphorylation in its importance in mediating cellular responses. However, limited instances have been identified. Here we show that signalling from Toll-like or TNF-α receptors triggers the calcium/calmodulin-dependent protein kinase (CaMK2) to activate histone acetyltransferase-1 (HAT1), which then acetylates the transcriptional regulator PLZF. Acetylation of PLZF promotes the assembly of a repressor complex incorporating HDAC3 and the NF-κB p50 subunit that limits the NF-κB response. Accordingly, diminishing the activity of CaMK2, the expression levels of PLZF or HAT1, or mutating key residues that are covalently modified in PLZF and HAT1, curtails control of the production of inflammatory cytokines. These results identify a central role for acetylation in controlling the inflammatory NF-κB transcriptional programme.