Stimulation of macrophages with lipopolysaccharide (LPS) leads to the production of cytokines that elicit massive liver apoptosis. We investigated the in vivo role of stress-responsive transcription factors (SRTFs) in this process focusing on the precipitating events that are sensitive to a cell-permeant peptide inhibitor of SRTF nuclear import (cSN50). In the absence of cSN50, mice challenged with LPS displayed very early bursts of inflammatory cytokines/chemokines, tumor necrosis factor ␣ (1 h), interleukin 6 (2 h), interleukin 1 ␤ (2 h), and monocyte chemoattractant protein 1 (2 h). Activation of both initiator caspases 8 and 9 and effector caspase 3 was noted 4 h later when full-blown DNA fragmentation and chromatin condensation were first observed (6 h). At this time an increase of pro-apoptotic Bax gene expression was observed. It was preceded by a decrease of anti-apoptotic Bcl2 and BclX L gene transcripts. Massive apoptosis was accompanied by microvascular injury manifested by hemorrhagic necrosis and a precipitous drop in blood platelets observed at 6 h. An increase in fibrinogen/fibrin degradation products and a rise in plasminogen activator inhibitor 1 occurred between 4 and 6 h. Inhibition of SRTFs nuclear import with the cSN50 peptide abrogated all these changes and increased survival from 7 to 71%. Thus, the nuclear import of SRTFs induced by LPS is a prerequisite for activation of the genetic program that governs cytokines/ chemokines production, liver apoptosis, microvascular injury, and death. These results should facilitate the rational design of drugs that protect the liver from inflammation-driven apoptosis.Programmed cell death (apoptosis) is the major mechanism of embryonic development and remodeling of tissues and organs, homeostatic control of immune cells that recognize self and non-self antigens, and removal of virally infected cells (1). Apoptosis of hepatocytes may occur in fulminant hepatitis, an inflammatory process that is caused by viral and non-viral agents (2). For example, recent gene therapy approaches to correct an inborn error of metabolism led to fulminant liver failure (3). This inflammation-related complication of gene therapy impedes broader application of viral vectors (4, 5). The sequence of intracellular signaling events that underlie inflammation-driven development of ultimately fatal liver apoptosis remains incompletely understood.Fulminant liver apoptosis has been studied in several animal models. These studies indicate that activation of T cells with concanavalin A (6) or with agonists that interact with T cell receptor such as staphylococcal enterotoxin B can lead to massive apoptosis (7,8). Staphylococcal enterotoxin B-induced apoptosis occurs under conditions of metabolic stress imposed by 2-amino-2-deoxy-D-galactosamine (D-Gal).1 Similarly, activation of macrophages with their Toll-like receptors (TLR) agonists, such as lipopolysaccharide (LPS, endotoxin), induces massive liver apoptosis when animals are treated with ethanol or D-Gal (9, 10). By reversibly dep...

Targeting of peptides, proteins, and other functional cargo into living cells is contingent upon efficient transport across the plasma membrane barrier. We have harnessed the signal sequence hydrophobic region (SSHR) to deliver functional cargoes to cultured cells and to experimental animals. We now report evidence that two chirally distinct forms of SSHR composed of all L or all D amino acids showed similar membrane-translocating activity as assessed by confocal microscopy, flow cytometry, and direct fluorescence measurement. An attached nuclear localization sequence ferried by the SSHR enantiomers displayed similar intracellular function by inhibiting inducible nuclear import of transcription factor nuclear factor B and suppressing nuclear factor B-dependent gene expression of cytokines. A nuclear localization sequence comprised of a positively charged cluster of amino acids was rapidly translocated by SSHR enantiomers to the interior of unilamellar phospholipid vesicles. These findings indicate that the SSHR translocates functional peptides directly through the plasma membrane phospholipid bilayer without involving chirally specific receptor/transporter mechanisms. This mechanism of SSHR translocation is suitable for facile delivery of biologically active peptides for cell-based and animal-based functional proteomic studies.The plasma membrane imposes tight control on the access of extracellular peptides and proteins to the cell interior. Through its mosaic structure of proteins and glycolipids embedded into the phospholipid bilayer, the plasma membrane provides a boundary for the 10,000 -15,000 proteins expressed in a typical mammalian cell (1, 2). Despite this barrier, transfer of information across the membrane is essential for cell development, function, and survival. Membrane receptors and transporters sense the extracellular environment of growth-promoting or -inhibiting ligands, short peptides, ions, and nutrients. This recognition allows their cellular uptake through specific receptor-mediated endocytosis or transporter-based translocation. To bypass these inherent mainstays of the plasma membrane functional integrity, we harnessed a signal sequence-derived hydrophobic region to deliver functional cargoes composed of peptides and proteins to probe and modulate intracellular signaling (3, 4). However, the mechanism of SSHR 1 -directed translocation of functional cargo across the plasma membrane remains unexplained.The overall structure of signal peptides is conserved in evolution between prokaryotes and eukaryotes, although the sequences of signal peptides are highly diverse (5). Their tripartite structure comprises an NH 2 -terminal region (n region), and a hydrophobic h region of variable length; this is followed by a cleavage site (c region) for signal peptidase. The hydrophobic region, which usually forms a helix, is endowed with a membrane-translocating activity (6). Its primary function is to guide a nascent polypeptide chain from the ribosomal tunnel through a translocon pore that is open latera...

Staphylococcal enterotoxin B and related toxins that target T cells have the capacity to elicit systemic inflammation, tissue injury, and death. Genes that encode mediators of inflammation can be globally inhibited by blocking the nuclear import of stress-responsive transcription factors. Here we show that cell-permeant peptides targeting Rch1/importin ␣/karyopherin ␣ 2, a nuclear import adaptor protein, are delivered to T cells where they inhibit the staphylococcal enterotoxin B-induced production of inflammatory cytokines ex vivo in cultured primary spleen cells and in vivo. The systemic production of tumor necrosis factor ␣, interferon ␥, and interleukin-6 was attenuated in mice either by a cell-permeant cyclized form of SN50 peptide or by a transgene whose product suppresses the nuclear import of transcription factor nuclear factor B in T cells. The extent of liver apoptosis and hemorrhagic necrosis was also reduced, which correlated with significantly decreased mortality rates. These findings highlight nuclear import inhibitors as a potentially useful countermeasure for staphylococcal enterotoxin B and other toxins that trigger harmful systemic inflammatory responses. Staphylococcal enterotoxin B (SEB)1 causes a spectrum of human diseases, including food poisoning and non-menstrual toxic shock syndrome (NMTSS) (1, 2). SEB is one of the major virulence factors regulated by a quorum-sensing mechanism in the setting of staphylococcal infections caused by antibioticresistant strains. These high-risk community-acquired infections, which may lead to NMTSS, occur with increasing frequency as compared with the greater than 2 million hospitalacquired infections recorded annually in the United States (3, 4). Strikingly, SEB induces a fatal respiratory distress syndrome in non-human primates, suggesting its potential use as a bioweapon on the battlefield or in mass civilian settings (5, 6). Potential air-borne, water-borne, and food-borne use of SEB led to its designation by the United States Centers for Disease Control as a category B agent.In terms of its mechanism of action, SEB is avidly bound by the T cell receptor V␤ chain and by major histocompatibility complex class II proteins on dendritic cells or macrophages (7-9). The resulting intercellular "synapse" generated by SEB engagement leads to excessive production of the inflammatory cytokines tumor necrosis factor ␣ (TNF␣), interferon ␥ (IFN␥), interleukin (IL)-1␤, IL-2, and IL-6. T cell-produced inflammatory cytokines contribute to massive vascular injury, organ failure, and depending on the mode of exposure potentially lethal respiratory distress syndrome or toxic shock (1, 2, 5, 6). Active immunization prior to SEB exposure and passive immunization immediately after exposure are not readily available (6). We have designed an alternative approach to antibodymediated neutralization of SEB and related toxins by targeting a common step in their intracellular signaling to the nucleus required for inflammatory cytokine gene expression.The genes that encode inflamm...

Shiga toxins (Stx) are the virulence factors of enterohemorrhagic Escherichia coli O157:H7, a worldwide emerging diarrheal pathogen, which precipitates postdiarrheal hemolytic uremic syndrome, the leading cause of acute renal failure in children. In this study, we show that Stx2 triggered expression of fractalkine (FKN), a CX3C transmembrane chemokine, acting as both adhesion counterreceptor on endothelial cells and soluble chemoattractant. Stx2 caused in HUVEC expression of FKN mRNA and protein, which promoted leukocyte capture, ablated by Abs to either endothelial FKN or leukocyte CX3CR1 receptor. Exposure of human glomerular endothelial cells to Stx2 recapitulated its FKN-inducing activity and FKN-mediated leukocyte adhesion. Both processes required phosphorylation of Src-family protein tyrosine kinase and p38 MAPK in endothelial cells. Furthermore, they depended on nuclear import of NF-κB and other stress-responsive transcription factors. Inhibition of their nuclear import with the cell-penetrating SN50 peptide reduced FKN mRNA levels and FKN-mediated leukocyte capture by endothelial cells. Adenoviral overexpression of IκBα inhibited FKN mRNA up-regulation. The FKN-mediated responses to Stx2 were also dependent on AP-1. In mice, both virulence factors of Stx-producing E. coli, Stx and LPS, are required to elicit hemolytic uremic syndrome. In this study, FKN was detected within glomeruli of C57BL/6 mice injected with Stx2, and further increased after Stx2 plus LPS coadministration. This was associated with recruitment of CX3CR1-positive cells. Thus, in response to Stx2, FKN is induced playing an essential role in the promotion of leukocyte-endothelial cell interaction thereby potentially contributing to the renal microvascular dysfunction and thrombotic microangiopathy that underlie hemolytic uremic syndrome due to enterohemorrhagic E. coli O157:H7 infection.

Signal-dependent nuclear translocation of transcription factor nuclear factor kappaB (NF-kappaB) is required for the activation of downstream target genes encoding the mediators of immune and inflammatory responses. To inhibit this inducible signaling to the nucleus, we designed a cyclic peptide (cSN50) containing a cell-permeable motif and a cyclized form of the nuclear localization sequence for the p50-NF-kappaB1 subunit of NF-kappaB. When delivered into cultured macrophages treated with the pro-inflammatory agonist lipopolysaccharide, cSN50 was a more efficient inhibitor of NF-kappaB nuclear import than its linear analog. When delivered into mice challenged with lipopolysaccharide, cSN50 potently blocked the production of proinflammatory cytokines (tumor necrosis factor alpha and interferon gamma) and significantly reduced the lethality associated with ensuing endotoxic shock. Based on specificity studies conducted with a mutated form of cSN50, a functional nuclear localization motif is required for this protective effect. Taken together, our findings demonstrate effective targeting of a cell-permeable peptide that attenuates cytokine signaling in vivo. This new class of biological response modifiers may be applicable to the control of systemic inflammatory reactions.