Heat shock inhibits caspase‐1 activity while also preventing its inflammasome‐mediated activation by anthrax lethal toxin

Anthrax lethal toxin (LT) rapidly kills macrophages from certain mouse strains in a mechanism dependent on the breakdown of unknown protein(s) by the proteasome, formation of the Nalp1b (NLRP1b) inflammasome and subsequent activation of caspase‐1. We report that heat‐shocking LT‐sensitive macrophages rapidly protects them against cytolysis by inhibiting caspase‐1 activation without upstream effects on LT endocytosis or cleavage of the toxin's known cytosolic substrates (mitogen‐activated protein kinases). Heat shock protection against LT occurred through a mechanism independent of de novo protein synthesis, HSP90 activity, p38 activation or proteasome inhibition and was downstream of mitogen‐activated protein kinase cleavage and degradation of an unknown substrate by the proteasome. The heat shock inhibition of LT‐mediated caspase‐1 activation was not specific to the Nalp1b (NLRP1b) inflammasome, as heat shock also inhibited Nalp3 (NLRP3) inflammasome‐mediated caspase‐1 activation in macrophages. We found that heat shock induced pro‐caspase‐1 association with a large cellular complex that could prevent its activation. Additionally, while heat‐shocking recombinant caspase‐1 did not affect its activity in vitro, lysates from heat‐shocked cells completely inhibited recombinant active caspase‐1 activity. Our results suggest that heat shock inhibition of active caspase‐1 can occur independently of an inflammasome platform, through a titratable factor present within intact, functioning heat‐shocked cells.     

Sodium fluoride induces podosome formation in endothelial cells

Background information. Fluoride is a well‐known G‐protein activator. Exposure of cultured cells to its derivatives results in actin cytoskeleton remodelling. Podosomes are actin‐based structures endowed with adhesion and matrix‐degradation functions. This study investigates actin cytoskeleton reorganization induced by fluoride in endothelial cells. Results. Treatment of cultured endothelial cells with sodium fluoride (NaF) results in a rapid and potent stimulation of podosome formation. Furthermore, we show that Cdc42 (cell‐division cycle 42), Rac1 and RhoA activities are stimulated in NaF‐treated cells. However, podosome assembly is dependent on Cdc42 and Rac1, but not RhoA. Although the sole activation of Cdc42 is sufficient to induce individual podosomes, a balance between RhoGTPase activities regulates podosome formation in response to NaF, which in this case are often found in groups or rosettes. As in other models, podosome formation in endothelial cells exposed to NaF also involves Src. Finally, we demonstrate that NaF‐induced podosomes are fully competent for matrix protein degradation. Conclusions. Taken together, our findings establish NaF as a novel inducer of podosomes in endothelial cells in vitro.     

Arabidopsis Formin3 Directs the Formation of Actin Cables and Polarized Growth in Pollen Tubes

Cytoplasmic actin cables are the most prominent actin structures in plant cells, but the molecular mechanism underlying their formation is unknown. The function of these actin cables, which are proposed to modulate cytoplasmic streaming and intracellular movement of many organelles in plants, has not been studied by genetic means. Here, we show that Arabidopsis thaliana formin3 (AFH3) is an actin nucleation factor responsible for the formation of longitudinal actin cables in pollen tubes. The Arabidopsis AFH3 gene encodes a 785–amino acid polypeptide, which contains a formin homology 1 (FH1) and a FH2 domain. In vitro analysis revealed that the AFH3 FH1FH2 domains interact with the barbed end of actin filaments and have actin nucleation activity in the presence of G-actin or G actin-profilin. Overexpression of AFH3 in tobacco (Nicotiana tabacum) pollen tubes induced excessive actin cables, which extended into the tubes'' apices. Specific downregulation of AFH3 eliminated actin cables in Arabidopsis pollen tubes and reduced the level of actin polymers in pollen grains. This led to the disruption of the reverse fountain streaming pattern in pollen tubes, confirming a role for actin cables in the regulation of cytoplasmic streaming. Furthermore, these tubes became wide and short and swelled at their tips, suggesting that actin cables may regulate growth polarity in pollen tubes. Thus, AFH3 regulates the formation of actin cables, which are important for cytoplasmic streaming and polarized growth in pollen tubes.     

Activation of CXCR7 promotes endothelial repair and reduces the carotid atherosclerotic lesions through inhibition of pyroptosis signaling pathways

Endothelial injuries, including cell pyroptosis, are ongoing inflammatory processes with key roles in atherosclerosis development. Our previous report showed that the chemokine CXCL12 and its receptor CXCR7 are associated with the proliferation and angiogenesis of endothelial cells. Nevertheless, the mechanism underlying these effects on atherosclerotic lesions, especially on endothelial dysfunction, remains unknown. Here, we demonstrated that CXCR7 was upregulated in human carotid atherosclerotic plaques, apolipoprotein E knockout (ApoE^?/?) mice fed with a high‐fat diet (HFD), and oxidized lipopolysaccharide‐treated (ox‐LDL) human umbilical vein endothelial cells (HUVECs). Further, the activation of CXCR7 reversed ox‐LDL‐induced HUVEC dysfunction, such as migration, tube formation, and cell pyroptosis; all of these protective effects were alleviated by inhibition of CXCR7. The NOD‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasomes were also elevated in human carotid atherosclerotic plaques, ApoE^?/? mice fed with HFD, and ox‐LDL‐injured HUVECs by regulation of caspase‐1 and interleukin (IL)‐1β expression. The activation of CXCR7 by TC14012 led to a decrease in atherosclerotic lesions in ApoE^?/? mice fed with HFD. TC14012 also inhibited the expression of the NLRP3 inflammasome signaling pathway in vivo. In conclusion, our study suggests that CXCR7 plays an important role in regulating NLRP3 inflammasome‐modulated pyroptosis in HUVECs, providing a potential novel therapy for atherosclerosis.     

A Gly65Val substitution in an actin,GhACT_LI1, disrupts cell polarity and F‐actin organization resulting in dwarf,lintless cotton plants

Actin polymerizes to form part of the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hirsutum) actin gene in the organization of actin filaments in lobed cotyledon pavement cells and the highly elongated single‐celled trichomes that comprise cotton lint fibers. Using mapping‐by‐sequencing, virus‐induced gene silencing, and molecular modeling, we identified the causative mutation of the dominant dwarf Ligon lintless Li₁ short fiber mutant as a single Gly65Val amino acid substitution in a polymerization domain of an actin gene, GhACT_LI1 (Gh_D04G0865). We observed altered cell morphology and disrupted organization of F‐actin in Li₁ plant cells by confocal microscopy. Mutant leaf cells lacked interdigitation of lobes and F‐actin did not uniformly decorate the nuclear envelope. While wild‐type lint fiber trichome cells contained long longitudinal actin cables, the short Li₁ fiber cells accumulated disoriented transverse cables. The polymerization‐defective Gly65Val allele in Li₁ plants likely disrupts processive elongation of F‐actin, resulting in a disorganized cytoskeleton and reduced cell polarity, which likely accounts for the dominant gene action and diverse pleiotropic effects associated with the Li₁ mutation. Lastly, we propose a model to account for these effects, and underscore the roles of actin organization in determining plant cell polarity, shape and plant growth.     

Nitric oxide promotes MPK6‐mediated caspase‐3‐like activation in cadmium‐induced Arabidopsis thaliana programmed cell death

Nitric oxide (NO), a vital cell‐signalling molecule, has been reported to regulate toxic metal responses in plants. This work investigated the effects of NO and the relationship between NO and mitogen‐activated protein kinase (MAPK) in Arabidopsis (Arabidopsis thaliana) programmed cell death (PCD) induced by cadmium (Cd²⁺) exposure. With fluorescence resonance energy transfer (FRET) analysis, caspase‐3‐like protease activation was detected after Cd²⁺ treatment. This was further confirmed with a caspase‐3 substrate assay. Cd²⁺‐induced caspase‐3‐like activity was inhibited in the presence of the NO‐specific scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), suggesting that NO mediated caspase‐3‐like protease activation under Cd²⁺ stress conditions. Pretreatment with cPTIO effectively inhibited Cd²⁺‐induced MAPK activation, indicating that NO also affected the MAPK pathway. Interestingly, Cd²⁺‐induced caspase‐3‐like activity was significantly suppressed in the mpk6 mutant, suggesting that MPK6 was required for caspase‐3‐like protease activation. To our knowledge, this is the first demonstration that NO promotes Cd²⁺‐induced Arabidopsis PCD by promoting MPK6‐mediated caspase‐3‐like activation.     

Rsk-mediated phosphorylation and 14-3-3ɛ binding of Apaf-1 suppresses cytochrome c-induced apoptosis

Many pro‐apoptotic signals trigger mitochondrial cytochrome c release, leading to caspase activation and ultimate cellular breakdown. Cell survival pathways, including the mitogen‐activated protein kinase (MAPK) cascade, promote cell viability by impeding mitochondrial cytochrome c release and by inhibiting subsequent caspase activation. Here, we describe a mechanism for the inhibition of cytochrome c‐induced caspase activation by MAPK signalling, identifying a novel mode of apoptotic regulation exerted through Apaf‐1 phosphorylation by the 90‐kDa ribosomal S6 kinase (Rsk). Recruitment of 14‐3‐3ε to phosphorylated Ser268 impedes the ability of cytochrome c to nucleate apoptosome formation and activate downstream caspases. High endogenous levels of Rsk in PC3 prostate cancer cells or Rsk activation in other cell types promoted 14‐3‐3ε binding to Apaf‐1 and rendered the cells insensitive to cytochrome c, suggesting a potential role for Rsk signalling in apoptotic resistance of prostate cancers and other cancers with elevated Rsk activity. Collectively, these results identify a novel locus of apoptosomal regulation wherein MAPK signalling promotes Rsk‐catalysed Apaf‐1 phosphorylation and consequent binding of 14‐3‐3ε, resulting in decreased cellular responsiveness to cytochrome c.     

Induction of caspase 3 activation by multiple Legionella pneumophila Dot/Icm substrates

The intracellular pathogen Legionella pneumophila is able to strike a balance between the death and survival of the host cell during infection. Despite the presence of high level of active caspase 3, the executioner caspase of apoptotic cell death, infected permissive macrophages are markedly resistant to exogenous apoptotic stimuli. Several bacterial molecules capable of promoting the cell survival pathways have been identified, but proteins involved in the activation of caspase 3 remain unknown. To study the mechanism of L. pneumophila‐mediated caspase 3 activation, we tested all known Dot/Icm substrates for their ability to activate caspase 3. Five effectors capable of causing caspase 3 activation upon transient expression were identified. Among these, by using its ability to activate caspase 3 by inducing the release of cytochrome c from the mitochondria, we demonstrated that VipD is a phospholipase A2, which hydrolyses phosphatidylethanolamine (PE) and phosphocholine (PC) on the mitochondrial membrane in a manner that appears to require host cofactor(s). The lipase activity leads to the production of free fatty acids and 2‐lysophospholipids, which destabilize the mitochondrial membrane and may contribute to the release of cytochrome c and the subsequent caspase 3 activation. Furthermore, we found that whereas it is not detectably defectively in caspase 3 activation in permissive cells, amutant lacking all of these five genes is less potent in inducing apoptosis in dendritic cells. Our results reveal that activation of host cell death pathways by L. pneumophila is a result of the effects of multiple bacterial proteins with diverse biochemical functions.     

Central involvement of Rho family GTPases in TNF-alpha-mediated bovine pulmonary endothelial cell apoptosis

In our recent studies, we defined a critical role for increased levels of myosin light chain (MLC) phosphorylation, a regulatory event in the interaction between actin and myosin in TNF-alpha-induced pulmonary endothelial cell actomyosin rearrangement and apoptosis. The Rho GTPase effector, Rho kinase is an important signaling effector governing levels of MLC phosphorylation which contributes to plasma membrane blebbing in several models of apoptosis. In this study, we directly assessed the role of Rho kinase in TNF-alpha-induced endothelial cell microfilament rearrangement and apoptosis. Inhibition of RhoA GTPase activity by the overexpression of dominant negative RhoA attenuates TNF-alpha-triggered stress fiber formation, consistent with Rho activation as a key event in TNF-alpha-induced cytoskeletal rearrangement. Furthermore, pharmacologic inhibition of Rho kinase as well as dominant negative RhoA overexpression dramatically reduced TNF-alpha-induced bovine endothelial apoptosis reflected by nucleosomal fragmentation as well as caspase 7, 3, and 8 activation. These results indicate that Rho kinase-dependent cytoskeletal rearrangement is critical for early apoptotic events, possibly in the assembly of the death-inducing signaling complex leading to initiator and effector caspase activation, and suggest a novel role for Rho GTPases in endothelial cell apoptosis.     

Madecassoside,a triterpenoid saponin isolated from Centella asiatica herbs,protects endothelial cells against oxidative stress

This study aimed to investigate the effect of madecassoside against oxidative stress‐induced injury of endothelial cells. Hydrogen peroxide (H₂O₂, 500 µmol/L) was employed as an inducer of oxidative stress in human umbilical vein endothelial cells (HUVECs). Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry. Caspase‐3 activity and mitochondria membrane potential were further examined. As a result, madecassoside (10, 30, 100 µmol/L) could reverse morphological changes, elevate cell viability, increase glutathione levels, and decrease lactate dehydrogenase and malondialdehyde levels caused by H₂O₂ in a concentration‐dependent manner. It attenuated apoptosis, preventing the activation of caspase‐3 and the loss of mitochondria membrane potential, as well as the phosphorylation of p38 mitogen‐activated protein kinase (MAPK) in HUVECs. These data suggested that madecassoside could protect HUVECs from oxidative injury, which was probably achieved by inhibiting cell apoptosis via protection of mitochondria membranes and downregulation of the activation of caspase‐3 and p38 MAPK. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:399–406, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21434     

Propofol inhibits proliferation and induces neuroapoptosis of hippocampal neurons in vitro via downregulation of NF‐κB p65 and Bcl‐2 and upregulation of caspase‐3

Propofol is widely used in paediatric anaesthesia and intensive care unit because of its essentially short‐acting anaesthetic effect. Recent data have shown that propofol induced neurotoxicity in developing brain. However, the mechanisms are not extremely clear. To gain a better insight into the toxic effects of propofol on hippocampal neurons, we treated cells at the days in vitro 7 (DIV 7), which were prepared from Sprague–Dawley embryos at the 18th day of gestation, with propofol (0.1–1000 μM) for 3 h. A significant decrease in neuronal proliferation and a remarkable increase in neuroapoptosis were observed in DIV 7 hippocampal neurons as measured by 3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and apoptosis assay respectively. Moreover, propofol treatment decreased the nuclear factor kappaB (NF‐κB) p65 expression, which was accompanied by a reduction in B‐cell lymphoma 2 (Bcl‐2) mRNA and protein levels, increased caspase‐3 mRNA and activation of caspase‐3 protein. These results indicated that downregulation of NF‐κB p65 and Bcl‐2 were involved in the potential mechanisms of propofol‐induced neurotoxicity. This likely led to the caspase‐3 activation, triggered apoptosis and inhibited the neuronal growth and proliferation that we have observed in our in vitro systems. Copyright © 2014 John Wiley & Sons, Ltd.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter‐endothelial junction disruption: role of HMGB1

Recent studies have indicated that the inflammasome plays a critical role in the pathogenesis of vascular diseases. However, the pathological relevance of this inflammasome activation, particularly in vascular cells, remains largely unknown. Here, we investigated the role of endothelial (Nucleotide‐binding Oligomerization Domain) NOD‐like receptor family pyrin domain containing three (Nlrp3) inflammasomes in modulating inter‐endothelial junction proteins, which are associated with endothelial barrier dysfunction, an early onset of obesity‐associated endothelial injury. Our findings demonstrate that the activation of Nlrp3 inflammasome by visfatin markedly decreased the expression of inter‐endothelial junction proteins including tight junction proteins ZO‐1, ZO‐2 and occludin, and adherens junction protein VE‐cadherin in cultured mouse vascular endothelial (VE) cell monolayers. Such visfatin‐induced down‐regulation of junction proteins in endothelial cells was attributed to high mobility group box protein 1 (HMGB1) release derived from endothelial inflammasome‐dependent caspase‐1 activity. Similarly, in the coronary arteries of wild‐type mice, high‐fat diet (HFD) treatment caused a down‐regulation of inter‐endothelial junction proteins ZO‐1, ZO‐2, occludin and VE‐cadherin, which was accompanied with enhanced inflammasome activation and HMGB1 expression in the endothelium as well as transmigration of CD43⁺ T cells into the coronary arterial wall. In contrast, all these HFD‐induced alterations in coronary arteries were prevented in mice with Nlrp3 gene deletion. Taken together, these data strongly suggest that the activation of endothelial Nlrp3 inflammasomes as a result of the increased actions of injurious adipokines such as visfatin produces HMGB1, which act in paracrine or autocrine fashion to disrupt inter‐endothelial junctions and increase paracellular permeability of the endothelium contributing to the early onset of endothelial injury during metabolic disorders such as obesity or high‐fat/cholesterol diet.     

Alternation of extracellular matrix remodeling and apoptosis by activation of the aryl hydrocarbon receptor pathway in human periodontal ligament cells

It is well known that the aryl hydrocarbon receptor (AhR) is involved in the toxicity of halogenated aromatic hydrocarbons (HAH) and polycyclic aromatic hydrocarbons (PAH). Recent experiments have shown the induction of impaired tooth and hard‐tissue formation by AhR pathway activation, however, the effect on periodontal ligament (PDL) tissue remains unclear. Here, we investigated the effects of benzo(a)pyrene (BaP), a member of PAH, on the extracellular matrix (ECM) remodeling‐related molecules, collagen type I (COL‐I), matrix metalloproteinase‐1 (MMP‐1), alpha‐smooth muscle actin (α‐SMA) expression, and apoptosis in two different human periodontal ligament cells (HPDLCs). The transduction of AhR from the cytoplasm to the nucleus and the increase of AhR‐responsive genes; that is, cytochrome P450 1A1 (CYP1A1), cytochrome P450 1B1 (CYP1B1), and aryl‐hydrocarbon receptor repressor (AhRR), expression was induced by BaP exposure in both HPDLCs. BaP treatment significantly enhanced MMP‐1 mRNA expression and MMP‐1 protein production, while markedly suppressing COL‐I and a‐SMA mRNA expression in both HPDLCs. Furthermore, these BaP‐treated HPDLCs fell into apoptotic cell death as evidenced by induction in annexin V and caspase‐3/7 staining and reduction of total cell number and Bcl‐2 mRNA expression. Thus, BaP exposure altered the expression of ECM‐related molecules and induced apoptosis in HPDLCs through activation of the AhR pathway. Overactivity of the AhR pathway may induce an inappropriate turnover of PDL tissue via disordered ECM remodeling and apoptosis in PDL cells. J. Cell. Biochem. 113: 3093–3103, 2012. © 2012 Wiley Periodicals, Inc.     

Significance of the caspase family in Helicobacter pylori induced gastric epithelial apoptosis

Background and Aims. H. pylori infection results in an increased epithelial apoptosis in gastritis and duodenal ulcer patients. We investigated the role and type of activation of caspases in H. pylori‐induced apoptosis in gastric epithelial cells. Methods. Differentiated human gastric cancer cells (AGS) and human gastric mucous cell primary cultures were incubated with H. pylori for 0.5–24 hours in RPMI 1640 medium, and the effects on cell viability, epithelial apoptosis, and activity of caspases were monitored. Apoptosis was analyzed by detection of DNA‐fragments by Hoechst stain®, DNA‐laddering, and Histone‐ELISA. Activities of caspases were determined in fluorogenic assays and by Western blotting. Cleavage of BID and release of cytochrome c were analyzed by Western blot. Significance of caspase activation was investigated by preincubation of gastric epithelial cells with cell permeable specific caspase inhibitors. Results. Incubation of gastric epithelial cells with H. pylori caused a time and concentration dependent induction of DNA fragmentation (3‐fold increase), cleavage of BID, release of cytochrome c and a concomittant sequential activation of caspase‐9 (4‐fold), caspase‐8 (2‐fold), caspase‐6 (2‐fold), and caspase‐3 (6‐fold). No effects on caspase‐1 and ‐7 were observed. Activation of caspases preceded the induction of DNA fragmentation. Apoptosis could be inhibited by prior incubation with the inhibitors of caspase‐3, ‐8, and ‐9, but not with that of caspase‐1. Conclusions. Activation of certain caspases and activation of the mitochondrial apoptotic pathway are essential for H. pylori induced apoptosis in gastric epithelial cells.     

1,4‐butanediyl‐bismethanethiosulfonate (BMTS) induces apoptosis through reactive oxygen species‐mediated mechanism

Although methane sulfonate compounds are widely used for the protein modification for their selectivity of thiol groups in proteins, their intracellular signaling events have not yet been clearly documented. This study demonstrated the methane sulfonate chemical 1,4‐butanediyl‐bismethanethiosulfonate (BMTS)‐induced cascades of signals that ultimately led to apoptosis of Jurkat cells. BMTS induced apoptosis through fragmentation of DNA, activation of caspase‐9 and caspase‐3, and downregulation of Bcl‐2 protein with reduction of mitochondrial membrane potential. Moreover, BMTS intensely and transiently induced intracellular reactive oxygen species (ROS) production and ROS produced by BMTS was mediated through mitochondria. We also found that a reducing agent dithiothreitol (DTT) and an anti‐oxidant N‐acetyl cysteine (NAC) inhibited BMTS‐mediated caspase‐9 and ‐3 activation, ROS production and induction of Annexin V/propidium iodide double positive cells, suggesting the involvement of ROS in the apoptosis process. Therefore, this study further extends our understanding on the basic mechanism of redox‐linked apoptosis induced by sulfhydryl‐reactive chemicals. J. Cell. Biochem. 108: 1059–1065, 2009. © 2009 Wiley‐Liss, Inc.     

Early sequence of events triggered by the interaction of Neisseria meningitidis with endothelial cells

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus‐mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high‐resolution quantitative live‐cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili‐associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl‐β‐cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria‐induced plasma membrane reorganization is a rapid event driven by a direct cross‐talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.     

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.