Activation of p38 mitogen-activated protein kinase promotes epidermal growth factor receptor internalization

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions.     

Interaction of variable bacterial outer membrane lipoproteins with brain endothelium

Background

Previously we reported that the variable outer membrane lipoprotein Vsp1 from the relapsing fever spirochete Borrelia turicatae disseminates from blood to brain better than the closely related Vsp2 [1]. Here we studied the interaction between Vsp1 and Vsp2 with brain endothelium in more detail.

Methodology/Principal Findings

We compared Vsp1 to Vsp2 using human brain microvascular endothelial cell (HBMEC) association assays with aminoacid radiolabeled Vsp-expressing clones of recombinant Borrelia burgdorferi and lanthanide-labeled purified lipidated Vsp1 (LVsp1) and Vsp2 (LVsp2) and inoculations of the lanthanide-labeled proteins into mice. The results showed that heterologous expression of LVsp1 or LVsp2 in B. burgdorferi increased its association with HBMEC to a similar degree. Purified lanthanide-labeled lipidated Vsp1 (LVsp1) and LVsp2 by themselves were capable of associating with HBMEC. The association of LVsp1 with brain endothelium was time-dependent, saturable, and required the lipidation. The association of Vsp1 with HBMEC was inhibited by incubation at lower temperature or with excess unlabeled LVsp1 or LVsp2 but not with excess rVsp1 or mouse albumin or an anti Vsp1 monoclonal antibody. The association of LVsp2 with HBMEC and its movement from blood to brain parenchyma significantly increased in the presence of LVsp1.

Conclusions/Significance

Variable bacterial outer membrane lipoproteins interact with brain endothelium differently; the lipidation and variable features at the protein dome region are key modulators of this interaction.     

Activation of RAW264.7 macrophages by bacterial DNA and lipopolysaccharide increases cell surface DNA binding and internalization

Bacterial DNA containing unmethylated CpG motifs is a pathogen-associated molecular pattern (PAMP) that interacts with host immune cells via a toll-like receptor (TLR) to induce immune responses. DNA binding and internalization into cells is independent of TLR expression, receptor-mediated, and required for cell activation. The objective of this study was to determine whether exposure of immune cells to bacterial DNA affects DNA binding and internalization. Treatment of RAW264.7 cells with CpG oligodeoxynucleotide (ODN) for both 18 and 42 h resulted in a significant increase in DNA binding, whereas non-CpG ODN had no effect on DNA binding. Enhanced DNA binding was non-sequence-specific, inhibited by unlabeled DNA, showed saturation, was consistent with increased cell surface DNA receptors, and resulted in enhanced internalization of DNA. Treatment with Escherichia coli DNA or lipopolysaccharide (LPS) also resulted in a significant increase in DNA binding, but treatment with interleukin-1alpha, tumor necrosis factor-alpha, or phorbol 12-myristate 13-acetate had no effect on DNA binding. Soluble factors produced in response to treatment with CpG ODN or LPS did not affect DNA binding. These studies demonstrate that one consequence of activating the host innate immune response by bacterial infection is enhanced binding and internalization of DNA. During this period of increased DNA internalization, RAW264.7 cells were hypo-responsive to continued stimulation by CpG ODN, as assessed by tumor necrosis factor-alpha activity. We speculate the biological significance of increasing DNA binding and internalization following interaction with bacterial PAMPs may provide a mechanism to limit an ongoing immune inflammatory response by enhancing clearance of bacterial DNA from the extracellular environment.     

Activation of the alternative pathway by pneumococcal cell walls.

The present studies were performed in order to identify the pneumococcal subcellular component responsible for activating the alternative pathway. Purified pneumococcal cell walls were able to activate the alternative pathway at a concentration as low as 5 mug/ml and were more active than crude cell walls, which in turn were more active than the whole organism. Purified pneumococcal cell membranes also were able to activiate the alternative pathway but had less than 10% of the activity of the purified walls. Thus, the cell wall appears to play a major role in pneumococcal activation of the alternative pathway. Pneumococcal cell walls containing ethanolamine were as effective as cell walls containing choline in activating the alternative pathway. Since C-reactive protein binds specifically to the phosphorylcholine residue of pneumococcal C-polysaccharide, it is unlikely that pneumococcal cell walls must combine with C-reactive protein in order to activate the alternative pathway.     

Activation of bacterial ribonuclease P by macrolides

The effect of macrolide antibiotic spiramycin on RNase P holoenzyme and M1 RNA from Escherichia coli was investigated. Ribonuclease P (RNase P) is a ribozyme that is responsible for the maturation of 5' termini of tRNA molecules. Spiramycin revealed a dose-dependent activation on pre-tRNA cleavage by E. coli RNase P holoenzyme and M1 RNA. The K s and V max, as well as the K s(app) and V max(app) values of RNase P holoenzyme and M1 RNA in the presence or absence of spiramycin, were calculated from primary and secondary kinetic plots. It was found that the activity status of RNase P holoenzyme and M1 RNA is improved by the presence of spiramycin 18- and 12-fold, respectively. Primer extension analysis revealed that spiramycin induces a conformational change of the P10/11 structural element of M1 RNA, which is involved in substrate recognition.     

Cloning and expression of the pneumococcal neuraminidase gene in Escherichia coli

A gene bank of Sau3AI-generated Streptococcus pneumoniae DNA fragments was constructed in Escherichia coli K-12 by cloning into the BamHI site of the cosmid vector pHC79. One clone capable of cleaving the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid was isolated. This activity was inhibited by treatment with a mouse antiserum raised against purified pneumococcal neuraminidase. The recombinant plasmid purified from this clone (designated pJCP301) contained approximately 3.0 kb of pneumococcal DNA. Western-blot analysis indicated that E. coli K-12[pJCP301] produced a 98-kDa polypeptide which reacted with antineuraminidase serum.     

IAP antagonization promotes inflammatory destruction of vascular endothelium

In this study, we show for the first time that the therapeutic antagonization of inhibitor of apoptosis proteins (IAPs) inhibits B16 melanoma growth by disrupting tumor vasculature. Specifically, the treatment of mice bearing B16 melanoma with an IAP antagonist compound A (Comp A) inhibits tumor growth not by inducing direct cytotoxicity against B16 cells but rather by a hitherto unrecognized antiangiogenic activity against tumor vessels. Our detailed analysis showed that Comp A treatment induces NF-κB activity in B16 tumor cells and facilitates the production of TNF. In the presence of Comp A, endothelial cells (ECs) become highly susceptible to TNF and undergo apoptotic cell death. Accordingly, the antiangiogenic and growth-attenuating effects of Comp A treatment were completely abolished in TNF-R knockout mice. This novel targeting approach could be of clinical value in controlling pathological neoangiogenesis under inflammatory condition while sparing blood vessels under normal condition.     

Role of lipid-mediated signal transduction in bacterial internalization

Receptor-mediated phagocytosis normally represents an important first line of immune defence. Invading microbes are internalized into phagosomes and are typically killed by exposure to a battery of microbicidal agents. To some intracellular pathogens, however, receptor-mediated phagocytosis represents an opportunity to access a protected niche within the host cell. Another type of intracellular pathogen, including Salmonella enterica serovar Typhimurium and Shigella flexneri, invade host cells in a more direct manner. These pathogens deliver effectors into the host cell via a type III secretion apparatus, initiating a ruffling response that leads to their uptake into intracellular vacuoles. Recent studies have demonstrated the importance of lipid signal transduction events in the uptake of pathogenic bacteria by both receptor-mediated phagocytosis and type III secretion-mediated invasion. In this review we highlight some of these discoveries, with a focus on phospholipid-dependent signalling events.     

The role of clathrin-dependent endocytosis in bacterial internalization

Internalization of bacteria into mammalian host cells has been studied extensively in the past two decades. These studies have highlighted the amazingly diverse strategies used by bacterial pathogens to induce their entry in non-phagocytic cells. The roles of actin and of the whole cytoskeletal machinery have been investigated in great detail for several invasive organisms, such as Salmonella, Shigella, Yersinia and Listeria. Recent results using Listeria highlight a role for the endocytosis machinery in bacterial entry, suggesting that clathrin-dependent endocytic mechanisms are also involved in internalization of large particles. This contrasts with the generally accepted dogma but agrees with previous studies of bacterial and viral infections and also of phagocytosis.     

Activation of macrophages and neutrophils by an endothelium growth suppressing factor.

An endothelial cell growth-suppressing factor (EGSF) was purified from the serum-free conditioned medium of the mouse P388D1 culture in the presence of carboxymethylated curdlan. The purified EGSF showed two bands corresponding to the molecular masses of 55 and 63 kDa by silver staining on a SDS-polyacrylamide gel under reducing conditions. This factor strongly suppressed the proliferation of endothelial cells from bovine artery, human umbilical vein, and human dermal vas capillare and this suppression was observed to be reversible. We found that EGSF was a potent chemoattractant for macrophages and neutrophils. EGSF mediated the adhesion of neutrophils to BAEs and transendothelial migration of neutrophils. Macrophages stimulated by EGSF produced nitrite in a dose-dependent manner. EGSF did not affect the proliferation of T lymphocytes. These findings suggest that EGSF acts not only as a potent inhibitor for the growth of endothelial cells but also an activator for macrophages and neutrophils. Thus EGSF plays a role in an inflammatory response in the endothelium.     

Lipoprotein-derived lysophosphatidic acid promotes atherosclerosis by releasing CXCL1 from the endothelium

Oxidatively modified low-density lipoprotein (oxLDL) plays a key role in the initiation of atherosclerosis by increasing monocyte adhesion. The mechanism that is responsible for the oxLDL-induced atherogenic monocyte recruitment in vivo, however, still remains unknown. Oxidation of LDL generates lysophosphatidylcholine, which is the main substrate for the lysophosphatidic acid (LPA) generating enzyme autotaxin. We show that oxLDL requires endothelial LPA receptors and autotaxin to elicit CXCL1-dependent arterial monocyte adhesion. Unsaturated LPA releases endothelial CXCL1, which is subsequently immobilized on the cell surface and mediates LPA-induced monocyte adhesion. Local and systemic application of LPA accelerates the progression of atherosclerosis in mice. Blocking the LPA receptors LPA(1) and LPA(3) reduced hyperlipidemia-induced arterial leukocyte arrest and atherosclerosis in the presence of functional CXCL1. Thus, atherogenic monocyte recruitment mediated by hyperlipidemia and modified LDL crucially depends on LPA, which triggers endothelial deposition of CXCL1, revealing LPA signaling as a target for cardiovascular disease treatments.     

Activation of ERK signaling upon alternative protease nexin-1 internalization mediated by syndecan-1

Protease nexin-1 (PN-1), an inhibitor of serine proteases, contributes to tissue homeostasis and influences the behavior of some tumor cells. The internalization of PN-1 protease complexes is considered to be mediated by the low-density lipoprotein receptor related protein 1 (LRP1). In this study, both wild-type and LRP1-/- mouse embryonic fibroblasts (MEF) were shown to internalize PN-1. Receptor associated protein (RAP) interfered with PN-1 uptake only in wild-type MEF cells, indicating that another receptor mediates PN-1 uptake in the absence of LRP1. In LRP1-/- MEF cells, inhibitor sensitivity and kinetic values (t(1/2) at 45 min) of PN-1 uptake showed a similarity to syndecan-1-mediated endocytosis. In these cells, PN-1 uptake was increased by overexpression of full-length syndecan-1 and decreased by RNA interference targeting this proteoglycan. Most important, in contrast to PKA activation known to be triggered by LRP1-mediated internalization, our study shows that syndecan-1-mediated internalization of PN-1 stimulated the Ras-ERK signaling pathway.     

Shigella deliver an effector protein to trigger host microtubule destabilization,which promotes Rac1 activity and efficient bacterial internalization

Shigella deliver a subset of effectors into the host cell via the type III secretion system, that stimulate host cell signal pathways to modulate the actin dynamics required for invasion of epithelial cells. Here we show that one of the Shigella effectors, called VirA, can interact with tubulin to promote microtubule (MT) destabilization, and elicit protrusions of membrane ruffling. Under in vitro conditions, VirA inhibited polymerization of tubulin and stimulated MT destabilization. Upon microinjection of VirA into HeLa cells, a localized membrane ruffling was induced rapidly. Overexpression of VirA in host cells caused MT destruction and protruding membrane ruffles which were absent when VirA was co-expressed with a dominant-negative Rac1 mutant. Indeed, Shigella but not the virA mutant stimulated Rac1, including the formation of membrane ruffles in infected cells. Importantly, the MT structure beneath the protruding ruffling was destroyed. Furthermore, drug-induced MT growth in HeLa cells greatly enhanced the Shigella entry. These results indicate that VirA is a novel type of bacterial effector capable of inducing membrane ruffling through the stimulation of MT destabilization.     

Epithelial properties of brain capillary endothelium

The specialized endothelial cells (ECs) in brain capillaries provide a blood-brain barrier to some solutes while facilitating transcapillary exchange of other solutes. In addition, brain capillaries may contribute to the secretion of spinal fluid, a process that is typically mediated by epithelial cells. This proposal is supported by the many epithelial properties of brain capillary ECs including the presence of 1) continuous tight junctions, 2) low transcellular permeability, 3) transcellular concentration gradients, 4) a transcellular potential difference, 5) a high transcellular resistance, and 6) an asymmetrical distribution of transport systems between the luminal and antiluminal plasma membranes. Thus, the brain capillary contains ECs that are structurally and perhaps functionally related to an epithelial cell. These unique features of brain ECs undoubtedly play an important role in regulating the formation and composition of the brain's interstitial fluid.     

Activation of human brain galactosylceramidase by phosphatidylserine.

Assays of sphingolipid hydrolases in vitro generally require bile salts or other detergents. A few 'activator proteins' have been reported that can partially replace the detergents in the assay mixture. We report here that phosphatidylserine from bovine brain is a relatively specific activator of human brain galactosylceramidase in the absence of sodium taurocholate (phosphatidylserine system). Activity similar to that obtained with the conventional assay system containing taurocholate and oleic acid (taurocholate system) could be obtained. Other lipids tested generally gave less than 10% of the taurocholate system activity, but sulfatide could activate human brain galactosylceramidase to 20--30% of the taurocholate system. The properties of the reaction in the phosphatidylserine system were examined with human brain whole homogenate, crude soluble post-concanavalin A preparations, and partially purified preparations as the enzyme source and compared with those obtained with the taurocholate system. The pH optimum shifted from 4.2 in the taurocholate system to 4.7 in the phosphatidylserine system. The phosphatidylserine system was superior in the linearity of the reaction with respect to the enzyme protein. Reasonably linear Lineweaver-Burk plots could be obtained. The Km values for the phosphatidylserine system were greater than those for the taurocholate system. The effect of phosphatidylserine was not additive to that of taurocholate. Additional phosphatidylserine to the taurocholate system was either without effect at lower concentrations or inhibitory at higher concentrations. The assays of galactosylceramidase with phosphatidylserine and without taurocholate do not necessarily provide pragmatic advantages but offer a potentially useful system with which to study the mechanism of in vivo degradation of the membrane-bound glycosphingolipid.     

Activation of urocortin transport into brain by leptin

There are several transport systems for peptides and polypeptides at the blood-brain barrier (BBB) which facilitate the passage of bioactive substances from blood to brain or from brain to blood. Nonetheless, it would be a novel concept for one peptide or polypeptide to activate the transport of another peptide with a similar function but unrelated structure. In this study, we report the first observation of such a phenomenon: activation of a urocortin transport system at the BBB by leptin. Urocortin, a corticotropin-releasing factor (CRF)-related neuropeptide, is a more potent suppressor of food intake than leptin or CRF when injected peripherally. Radiolabeled urocortin (¹²⁵I-urocortin) was used for these in vivo studies in mice; it remained stable and intact during the experimental period. Unlike CRF, urocortin was not saturably transported out of the brain. There was no substantial entry of ¹²⁵I-urocortin into brain as determined by sensitive multiple-time regression analysis after iv bolus injection. Addition of leptin, however, caused a dose-related increase in the influx of ¹²⁵I-urocortin and greatly facilitated its entry into brain parenchyma; this effect disappeared at higher doses of leptin. Moreover, in the presence of an activating dose of leptin, the entry of ¹²⁵I-urocortin into brain was saturable. The results indicate that the presence of leptin contributes to the potent satiety effects of urocortin after peripheral administration. Thus, the action of leptin in the periphery extends beyond its direct passage across the BBB and involves acute modulation of an inert transport system. We believe that these findings have broad physiological implications and indicate a unique function of the BBB as a regulatory interface.