Lidocaine inhibits vascular endothelial growth factor-A-induced angiogenesis.

PURPOSE: Angiogenesis is closely related to the pathophysiology of diseases such as cancer or ischemia. Here, we investigated the effect of lidocaine at clinically effective blood concentrations on vascular endothelial growth factor A (VEGF-A)-induced angiogenesis. In addition, we aimed to clarify the mechanisms by which lidocaine could inhibit angiogenesis. METHODS: Angiogenesis was analyzed using commercially available in vitro assay kits in human umbilical vein endothelial cells (HUVECs)/normal human dermal fibroblast co-culture systems. The effects of lidocaine on cytotoxicity, VEGF-induced cell migration, and VEGF-induced cell proliferation were examined in HUVECs using lactate dehydrogenase cytotoxic, Boyden chamber, and WST-8 assays, respectively. The VEGF signaling pathway via VEGF receptor 2 (VEGFR-2) was analyzed by western blotting. RESULTS: Lidocaine elicited a significant dose-dependent, angiogenesis-inhibitory effect at a concentration range of 1-10 µg/ml. At this concentration range, cell death was not observed. Lidocaine, at a concentration of 10 µg/ml, significantly inhibited cell proliferation but not cell migration, induced by VEGF-A in HUVECs. Furthermore, lidocaine, in a dose-dependent manner, significantly inhibited the VEGF-A-induced phosphorylation of VEGFR-2 at 3 and 10 µg/ml. CONCLUSION: We demonstrated that lidocaine has an anti-angiogenesis effect on clinically effective blood concentrations without causing cell death. This finding could represent a new avenue for future research into anesthesia, cancer-related analgesia, and revascularization therapy.

Characterization of the membrane-targeting C1 domain in Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some forms of pasteurellosis. The toxin activates G(q)- and G(12/13)-dependent pathways through the deamidation of a glutamine residue in the alpha-subunit of heterotrimeric GTPases. We recently reported the crystal structure of the C terminus (residues 575-1285) of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine protease-like catalytic triad. In this study, we analyzed the membrane-targeting function of the C1 domain in detail. The C1 domain consists of seven helices of which the first four (residues 590-670), showing structural similarity to the N terminus of Clostridium difficile toxin B, were found to be involved in the recruitment of C-PMT to the plasma membrane. C-PMT lacking these helices (C-PMT DeltaC1(4H)) neither localized to the plasma membrane nor stimulated the G(q/12/13)-dependent signaling pathways. When the membrane-targeting property was complemented by a peptide tag with an N-myristoylation motif, C-PMT DeltaC1(4H) recovered the PMT activity. Direct binding between the C1 domain and liposomes containing phospholipids was evidenced by surface plasmon resonance analyses. These results indicate that the C1 domain of C-PMT functions as a targeting signal for the plasma membrane.

Clostridium perfringens enterotoxin interacts with claudins via electrostatic attraction.

Clostridium perfringens enterotoxin (CPE), a causative agent of food poisoning, is a pore-forming toxin disrupting the selective permeability of the plasma membrane of target cells, resulting in cell death. We previously identified claudin as the cell surface receptor for CPE. Claudin, a component of tight junctions, is a tetratransmembrane protein and constitutes a large family of more than 20 members, not all of which serve as the receptor for CPE. The mechanism by which the toxin distinguishes the sensitive claudins is unknown. In this study, we localized the region of claudin responsible for interaction with CPE to the C-terminal part of the second extracellular loop and found that the isoelectric point of this region in sensitive claudins was higher than insensitive claudins. Amino acid substitutions to lower the pI resulted in reduced sensitivity to CPE among sensitive claudins, whereas substitutions to raise the pI endowed CPE-insensitive claudins with sensitivity. The steric structure of the claudin-binding domain of CPE reveals an acidic cleft surrounded by Tyr(306), Tyr(310), Tyr(312), and Leu(315), which were reported to be essential for interaction with the sensitive claudins. These results imply that an electrostatic attraction between the basic claudin region and the acidic CPE cleft is involved in their interaction.

A colorimetric assay for studying effector secretion through the bacterial type III secretion system.

We have devised a colorimetric method that monitors secretion of effector proteins into host cytoplasm through the bacterial type III secretion machinery. Here we used constructs of effectors fused with Bordetella adenylate cyclase as a reporter, but evaluated the effector translocation by quantifying cell viability, rather than by measuring the intracellular cAMP concentration. This is based on our findings that cells infected by a secretion-competent bacterium expressing the fusion protein lost their viability under our experimental conditions. Cell death was quantified using commercially available reagents and basic research equipment. An observation that cell death was potentiated when the infected cells were treated with 2-deoxyglucose and sodium azide suggests that the depletion of intracellular ATP is partly involved in the process. Using enteropathogenic Escherichia coli, we demonstrated that the method was applicable to at least three effectors of bacteria, Tir, EspF, and Map, and was useful for studying a secretion signal sequence for Tir. This technically simple and inexpensive method is a good alternative to the existing procedure for studying the mechanism by which effectors are secreted through the type III secretion system in a high-throughput format.

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT), one of the virulence factors produced by the bacteria, exerts its toxicity by up-regulating various signaling cascades downstream of the heterotrimeric GTPases Gq and G12/13 in an unknown fashion. Here, we present the crystal structure of the C-terminal region (residues 575-1,285) of PMT, which carries an intracellularly active moiety. The overall structure of C-terminal region of PMT displays a Trojan horse-like shape, composed of three domains with a "feet"-,"body"-, and "head"-type arrangement, which were designated C1, C2, and C3 from the N to the C terminus, respectively. The C1 domain, showing marked similarity in steric structure to the N-terminal domain of Clostridium difficile toxin B, was found to lead the toxin molecule to the plasma membrane. The C3 domain possesses the Cys-His-Asp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying Cys-His-Asp. PMT toxicities on target cells were completely abrogated when one of the amino acids constituting the triad was mutated. Our results indicate that PMT is an enzyme toxin carrying the cysteine protease-like catalytic triad dependent on the redox state and functions on the cytoplasmic face of the plasma membrane of target cells.

Enteropathogenic Escherichia coli, Shigella flexneri, and Listeria monocytogenes recruit a junctional protein, zonula occludens-1, to actin tails and pedestals.

Enteropathogenic Escherichia coli, Shigella flexneri, and Listeria monocytogenes induce localized actin polymerization at the cytoplasmic face of the plasma membrane or within the host cytoplasm, creating unique actin-rich structures termed pedestals or actin tails. The process is known to be mediated by the actin-related protein 2 and 3 (Arp2/3) complex, which in these cases acts downstream of neural Wiskott-Aldrich syndrome protein (N-WASP) or of a listerial functional homolog of WASP family proteins. Here, we show that zonula occludens-1 (ZO-1), a protein in the tight junctions of polarized epithelial cells, is recruited to actin tails and pedestals. Immunocytochemical analysis revealed that ZO-1 was stained most in the distal part of the actin-rich structures, and the incorporation was mediated by the proline-rich region of the ZO-1 molecule. The direct clustering of membrane-targeted Nck, which is known to activate the N-WASP-Arp2/3 pathway, triggered the formation of the ZO-1-associated actin tails. The results suggest that the activation of the Arp2/3 complex downstream of N-WASP or a WASP-related molecule is a key to the formation of the particular actin-rich structures that bind with ZO-1. We propose that an analysis of the recruitment on a molecular basis will lead to an understanding of how ZO-1 recognizes a distinctive actin-rich structure under pathophysiological conditions.

Fish soluble Toll-like receptor 5 (TLR5S) is an acute-phase protein with integral flagellin-recognition activity.

From an EST fragment of the rainbow trout that was predicted to contain leucine-rich repeats (LRR), we cloned the whole cDNA and identified a soluble form of TLR5 ortholog (rtTLR5S), which does not exist in the mouse and human. rtTLR5S was about 38% homologous to the extracellular domains of human (hu) and mouse (mo)TLR5, while rtTLR5S showed <25% homologous to those of other human or mouse TLRs. A chimera constructed of rtTLR5S and the intra-cellular TIR of huTLR5 expressed on HeLa cells signaled the presence of flagellin A and C from V. anguillarum, resulting in NF-kappaB activation. The mRNA of rtTLR5S was predominantly detected in the liver. The hepatoma cell line of the rainbow trout RTH149 that responded to flagellin, allowed to up-regulate rtTLR5S in response to V. anguillarum or its purified flagellin within 8 h. rtTLR5S, when co-expressed with membrane huTLR5 in HeLa cells, augmented huTLR5-mediated NF-kappaB activation in response to flagellin. These results, together with the genome information of the pufferfish Fugu (Fugu rubripes), suggest that in fish the soluble TLR5 is an acute-phase protein sensing bacterial infection via recognition of a variety of bacterial flagellins to augment NF-kappaB activation, and may be important for fish to survive from bacterial infection in the water.

Bordetella dermonecrotic toxin exerting toxicity through activation of the small GTPase Rho.

Bordetella dermonecrotic toxin (DNT) is a virulence factor produced by bacteria belonging to the genus Bordetella. The toxin possesses novel transglutaminase activity that catalyzes polyamination or deamidation of the small GTPases of the Rho family. The modified GTPases loose their GTP hydrolyzing activity, function as a constitutive active molecule, and continuously transduce signals to downstream effectors, which mediate the consequent phenotypes of cells intoxicated by DNT. A dynamin-dependent endocytosis is required for the toxin to be internalized into cells although it is unlikely transported to deep organelles such as the Golgi apparatus or the ER. Several lines of evidence show that the toxin undergoes proteolytic cleavage by furin or furin-like protease probably in the early endosome, and then escapes into the cytoplasm to reach the GTPase.

A short consensus repeat-containing complement regulatory protein of lamprey that participates in cleavage of lamprey complement 3.

The prototype of the short consensus repeat (SCR)-containing C regulatory protein is of interest in view of its evolutionary significance with regard to the origin of the C regulatory system. Lamprey is an agnathan fish that belongs to the lowest class of vertebrates. Because it does not possess lymphocytes, it lacks Ig and consequently the classical C pathway. We identified an SCR-containing C regulatory protein from the lamprey. The primary structure predicted from the cDNA sequence showed that this is a secretary protein consisting of eight SCRs. This framework is similar to the alpha-chain of C4b-binding protein (C4bp). SCR2 and -3 of human C4bp are essential for C4b inactivation, and this region is fairly well conserved in the lamprey protein. However, the other SCRs of this protein are similar to those of other human C regulatory proteins. The lamprey protein binds to the previously reported lamprey C3b/C3bi deposited on yeast and cleaves lamprey C3b-like C3 together with a putative serum protease. The scheme resembles the C regulatory system of mammals, where factor I and its cofactor inactivate C3b. Unlike human cofactors, the lamprey protein requires divalent cations for C3b-like C3 cleavage. Its artificial membrane-anchored form protects host cells from lamprey C attack via the lectin pathway. Thus, the target of this protein appears to be C3b and/or its family. We named this protein Lacrep, the lamprey C regulatory protein. Lacrep is a member of SCR-containing C regulators, the first of its kind identified in the lowest vertebrates.

Bordetella dermonecrotic toxin undergoes proteolytic processing to be translocated from a dynamin-related endosome into the cytoplasm in an acidification-independent manner.

Bordetella pertussis dermonecrotic toxin (DNT), which activates intracellular Rho GTPases, is a single chain polypeptide composed of an N-terminal receptor-binding domain and a C-terminal enzymatic domain. We found that DNT was cleaved by furin, a mammalian endoprotease, on the C-terminal side of Arg(44), which generates an N-terminal fragment almost corresponding to the receptor-binding domain and a C-terminal remainder (deltaB) containing the enzymatic domain. These two fragments remained associated even after the cleavage and made a nicked form. DNT mutants insensitive to furin had no cellular effect, whereas the nicked toxin was much more potent than the intact form, indicating that the nicking by furin was a prerequisite for action. DeltaB, but not the nicked toxin, associated with artificial liposomes and activated Rho in cells resistant to DNT because of a lack of surface receptor. These results imply that deltaB, dissociated from the binding domain, fully possesses the ability to enter the cytoplasm across the lipid bilayer membrane. The translocation ability of deltaB was found to be attributable to the N-terminal region encompassing amino acids 45-166, including a putative transmembrane domain. Pharmacological analyses with various reagents disturbing vesicular trafficking revealed that the translocation requires neither the acidification of the endosomes nor retrograde vesicular transport to deeper organelles, although DNT appeared to be internalized via a dynamin-dependent endocytosis. We conclude that DNT binds to its receptor and is internalized into endosomes where the proteolytic processing occurs. DeltaB, liberated from the binding domain after the processing, begins to translocate the enzymatic domain into the cytoplasm.

Mapping of the sites responsible for factor I-cofactor activity for cleavage of C3b and C4b on human C4b-binding protein (C4bp) by deletion mutagenesis.

Human C4b-binding protein (C4bp) facilitates the factor I-mediated proteolytic cleavage of the active forms of complement effectors C3b and C4b into their inactive forms. C4bp comprises a disulfide-linked heptamer of alpha-chains with complement (C) regulatory activity and a beta-chain. Each alpha-chain contains 8 short consensus repeat (SCR) domains. Using SCR-deletion mutants of recombinant multimeric C4bp, we identified the domains responsible for the C3b/C4b-binding and C3b/C4b-inactivating cofactor activity. The C4bp mutant with deletion of SCR2 lost the C4b-binding ability, as judged on C3b/C4b-Sepharose binding assaying and ELISA. In contrast, the essential domains for C3b-binding extended more to the C-terminus, exceeding SCR4. Using fluid phase cofactor assaying and deletion mutants of C4bp, SCR2 and 3 were found to be indispensable for C4b cleavage by factor I, and SCR1 contributed to full expression of the factor I-mediated C4b cleaving activity. On the other hand, SCR1, 2, 3, 4, and 5 participated in the factor I-cofactor activity for C3b cleavage, and SCR2, 3, and 4 were absolutely required for C3b inactivation. Thus, different sets of SCRs participate in C3b and C4b inactivation, and the domain repertoire supporting C3b cofactor activity is broader than that supporting C4b inactivation by C4bp and factor I. Furthermore, the domains participating in C3b/C4b binding are not always identical to those responsible for cofactor activity. The necessity of the wide range of SCRs in C3b inactivation compared to C4b inactivation by C4bp and factor I may reflect the physiological properties of C4bp, which is mainly directed to C4b rather than C3b.

Molecular assembly of CD46 with CD9, alpha3-beta1 integrin and protein tyrosine phosphatase SHP-1 in human macrophages through differentiation by GM-CSF.

Human CD46, formerly membrane cofactor protein (MCP), binds and inactivates complement C3b and serves as a receptor for measles virus (MV), thereby protecting cells from homologous complement and sustaining systemic viral infection. CD46 on activated macrophages (Mphi) but not intact monocytes is presumed to be the factor responsible for virus-mediated immune modulation including down-regulation of IL-12 production. As CD46 is expressed on both Mphi and monocytes, the molecular mechanisms responsible for these distinct immune responses remain largely unknown. Here, we found that peripheral blood monocytes treated for 5--8 days with GM-CSF (i.e. mature Mphi) acquired the capacity to assemble CD9, alpha3-beta1 integrin and the tyrosine phosphatase SHP-1 with their CD46. Prior to this maturation stage, Mphi expressed sufficient amounts of CD9 and CD46 but showed no such complex formation, and as in intact monocytes MV replication was markedly suppressed. By flow cytometry and confocal microscopy, the complex was found to assemble on the surface in cells treated with approximately 6 days with GM-CSF but not for approximately 2 days. Notably, an alternative MV receptor SLAM CDw150 was neither expressed nor recruited to this complex throughout GM-CSF-mediated Mphi differentiation. These responses and molecular links were not reproduced in the hamster cell line CHO expressing human CD46 although these cells acquired high susceptibility to MV. Based on these observations, MV susceptibility in human myeloid lineages appears not to be as simple as that observed in human CD46-transfected non-myeloid cells. The molecular complex involving CD46 may confer high MV permissiveness leading to immune modulation in Mphi.