Autophagy and Crohn's disease: at the crossroads of infection, inflammation, immunity, and cancer.

Inflammatory bowel diseases (IBD) are common inflammatory disorders of the gastrointestinal tract that include ulcerative colitis (UC) and Crohn's disease (CD). The incidences of IBD are high in North America and Europe, affecting as many as one in 500 people. These diseases are associated with high morbidity and mortality. Colorectal cancer risk is also increased in IBD, correlating with inflammation severity and duration. IBD are now recognized as complex multigenetic disorders involving at least 32 different risk loci. In 2007, two different autophagy-related genes, ATG16L1 (autophagy-related gene 16-like 1) and IRGM (immunity-related GTPase M) were shown to be specifically involved in CD susceptibility by three independent genome-wide association studies. Soon afterwards, more than forty studies confirmed the involvement of ATG16L1 and IRGM variants in CD susceptibility and gave new information on the importance of macroautophagy (hereafter referred to as autophagy) in the control of infection, inflammation, immunity and cancer. In this review, we discuss how such findings have undoubtedly changed our understanding of CD pathogenesis. A unifying autophagy model then emerges that may help in understanding the development of CD from bacterial infection, to inflammation and finally cancer. The Pandora's box is now open, releasing a wave of hope for new therapeutic strategies in treating Crohn's disease.

Modulation of Rho GTPase activity in endothelial cells by selective proteinase-activated receptor (PAR) agonists.

The proteinase-activated receptors (PAR) PAR1 and PAR2 mediate responses to thrombin and trypsin-like proteases, respectively. Both receptors are expressed on endothelial cells where they have been reported to transduce a similar set of intracellular responses. In cultured human umbilical vein endothelial cells (HUVEC), we observed a marked difference in shape changes induced by PAR-activating peptides (PAR-APs); unlike PAR1-AP, PAR2-AP failed to stimulate cell rounding. Objectives were to shed light on the mechanisms underlying PAR-mediated cytoskeletal responses. We examined the activation of the Rho family GTPases in HUVEC using highly selective PAR1- and PAR2-APs to do this. Both peptides induced a robust and transient activation of RhoA, with the time course of activation being more sustained for the PAR1-AP. Interestingly, divergent effects on Rac activity were observed. Addition of PAR1-AP inhibited basal Rac activity as well as the phosphorylation of the Rac effector, p21-activated kinase (PAK). In contrast, PAR2-AP induced a modest activation of Rac, phosphorylation of PAK and translocation of cortactin from the cytosol to membrane ruffles, a Rac-dependent event. In vivo, only PAR1-AP rapidly enhanced vascular permeability in a mouse skin assay. We conclude that the differential regulation of the Rac/PAK pathway by PAR1 and PAR2 agonists in endothelial cells points toward distinct roles for these receptors in the control of vascular permeability and blood vessel remodeling.

Angiogenesis and G-protein-coupled receptors: signals that bridge the gap.

Angiogenesis is a mechanism that has repercussions in a number of physiological and pathological situations. Vascular endothelial growth factor and basic fibroblast growth factor have understandably received enormous research coverage for being the major mediators of new blood vessel growth, often overshadowing other agonist that also have strong angiogenic potential. We wish to put the spotlight on GPCR agonists that undoubtedly have their word to say on the subject of angiogenesis. In this short review, we will discuss our findings along with the work from other groups on the mechanisms by which GPCR agonists, like thrombin and angiotensin II, control a number of angiogenic signals. A complete understanding of these mechanisms could, by the design of new therapeutic strategies, have a strong impact in clinical oncology.

Effects of cytotoxic necrotizing factor 1 and lethal toxin on actin cytoskeleton and VE-cadherin localization in human endothelial cell monolayers.

Integrity of the vascular endothelium is largely dependent on endothelial cell shape and establishment of intercellular junctions. Certain pathogenic bacterial toxins alter the cytoskeletal architecture of intoxicated cells by modulating the GTPase activity of p21 Rho family proteins. In the present study we have analyzed the effect of Rho-directed toxins on the actin cytoskeleton and monolayer integrity of endothelial cells. We report here that Escherichia coli cytotoxic necrotizing factor 1 (CNF1) activates Rho in human umbilical vein endothelial cells (HUVEC). In confluent monolayers, CNF1 treatment induces prominent stress fiber formation without significantly modifying peripheral localization of VE-cadherin, a specific marker of vascular endothelial cell adherens junctions. Further, Rho activation with CNF1 blocks thrombin-induced redistribution of VE-cadherin staining and gap formation in HUVEC monolayers. Inhibition of Rho by prolonged treatment of cells with C3 exoenzyme (Clostridium botulinum) eliminates actin stress fibers without disrupting the continuity of VE-cadherin staining, indicating that Rho-dependent stress fibers are not required for maintaining this adhesion receptor at sites of intercellular contact. Lethal toxin (Clostridium sordellii), an inhibitor of Rac as well as Ras and Rap, potently disrupts the actin microfilament system and monolayer integrity in HUVEC cultures.

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.

Expression of a long pentraxin, PTX3, by monocytes exposed to the mycobacterial cell wall component lipoarabinomannan.

PTX3 is a prototypic long pentraxin composed of a C-terminal domain similar to those of classical pentraxins (e.g., C reactive protein) and an unrelated N-terminal portion. PTX3 is expressed in a variety of cell types, notably mononuclear phagocytes and endothelial cells, after exposure to the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha). The present study was designed to assess whether mycobacterial components were able to induce expression and production of PTX3. Mycobacterial lipoarabinomannan (LAM) induced expression of PTX3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside. Among mononuclear cells, monocytes were responsible for LAM-induced PTX3 mRNA expression. Whole mycobacteria (Mycobacterium bovis BCG) strongly induced PTX3 expression. Pretreatment with actinomycin D abolished LAM-induced PTX3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced PTX3 expression was associated with the production of immunoreactive PTX3. IL-10 and IL-13 did not inhibit the induction of PTX3 by LAM. Under the same conditions, these anti-inflammatory cytokines inhibited MCP-1 expression. In contrast, gamma interferon inhibited LAM-induced PTX3 expression. Thus, in addition to IL-1, TNF, and lipopolysaccharide, mycobacterial cell wall components also induce expression and production of the long pentraxin PTX3. The significance of PTX3 in the immunobiology of mycobacterial infection and its relevance in relation to clinical involvement remain to be determined.

Multimer formation and ligand recognition by the long pentraxin PTX3. Similarities and differences with the short pentraxins C-reactive protein and serum amyloid P component.

PTX3 is a prototypic long pentraxin consisting of a C-terminal 203-amino acid pentraxin-like domain coupled with an N-terminal 178-amino acid unrelated portion. The present study was designed to characterize the structure and ligand binding properties of human PTX3, in comparison with the classical pentraxins C-reactive protein and serum amyloid P component. Sequencing of Chinese hamster ovary cell-expressed PTX3 revealed that the mature secreted protein starts at residue 18 (Glu). Lectin binding and treatment with N-glycosidase F showed that PTX3 is N-glycosylated, sugars accounting for 5 kDa of the monomer mass (45 kDa). Circular dichroism analysis indicated that the protein consists predominantly of beta-sheets with a minor alpha-helical component. While in gel filtration the protein is eluted with a molecular mass of congruent with900 kDa, gel electrophoresis using nondenaturing, nonreducing conditions revealed that PTX3 forms multimers predominantly of 440 kDa apparent molecular mass, corresponding to decamers, and that disulfide bonds are required for multimer formation. The ligand binding properties of PTX3 were then examined. As predicted based on modeling, inductive coupled plasma/atomic emission spectroscopy showed that PTX3 does not have coordinated Ca2+. Unlike the classical pentraxins CRP and SAP, PTX3 did not bind phosphoethanolamine, phosphocholine, or high pyruvate agarose. PTX3 in solution, bound to immobilized C1q, but not C1s, and, reciprocally, C1q bound to immobilized PTX3. Binding of PTX3 to C1q is specific and saturable with a Kd 7.4 x 10(-8) M as determined by solid phase binding assay. The Chinese hamster ovary cell-expressed pentraxin domain bound C1q when multimerized. Thus, as predicted on the basis of computer modeling, the prototypic long pentraxin PTX3 forms multimers, which differ from those formed by classical pentraxins in terms of protomer composition and requirement for disulfide bonds, and does not recognize CRP/SAP ligands. The capacity to bind C1q, mediated by the pentraxin domain, is consistent with the view that PTX3, produced in tissues by endothelial cells or macrophages in response to interleukin-1 and tumor necrosis factor, may act as a local regulator of innate immunity.

Expression of monocyte chemotactic protein-3 in human monocytes exposed to the mycobacterial cell wall component lipoarabinomannan.

Monocyte chemotactic protein-3 (MCP-3) is a C-C chemokine which interacts with the CCR1, CCR2 (MCP-1) and CCR3 receptors and has a distinct spectrum of action. The present study was designed to assess whether mycobacterial components were able to induce expression and production of MCP-3 in human monocytes. Mycobacterial lipoarabinomannan (LAM) induced expression of MCP-3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside (PLM). Among mononuclear cells, monocytes were responsible for LAM-induced MCP-3 mRNA expression. Whole mycobacteria (Mycobacterium bovis BCG) strongly induced MCP-3 expression. Pretreatment with actinomycin D abolished LAM-induced MCP-3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced MCP-3 expression was associated with the production of immunoreactive PTX3. Interleukin 10 (IL-10) and IL-13 inhibited the induction of MCP-3 by LAM. Thus mycobacterial cell wall components induced expression of MCP-3 in human monocytes. MCP-3, a chemokine active on mononuclear phagocytes, NK cells, T cells and dendritic cells, may be relevant to the induction and expression of immunity against mycobacteria.

Post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor.

The purpose of the present study was to analyze the post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor. A human receptor cDNA was engineered to encode an epitope tag derived from the vesicular stomatitis virus glycoprotein at the COOH terminus of the receptor and expressed in human embryonic kidney 293 cells. We show here that the mature receptor is a glycosylated protein with an apparent molecular mass ranging from 68 to 80 kDa by SDS-polyacrylamide gel electrophoresis. Removal of asparagine-linked oligosaccharides with N-glycosidase F leads to the appearance of a 36-40-kDa receptor species. The current model for receptor activation by thrombin involves specific hydrolysis of the arginine-41/serine-42 (Arg-41/Ser-42) peptide bond. Cleavage of the receptor by thrombin was demonstrated directly by Western analyses performed on membranes and glycoprotein-enriched lysates from transfected cells. Whereas thrombin treatment of cells results in increased mobility of the receptor in SDS-polyacrylamide gel electrophoresis, we found that their treatment with the thrombin receptor agonist peptide leads to a decrease in thrombin receptor mobility due, in part, to phosphorylation. The serine proteases trypsin and plasmin also cleave and activate the receptor similar to thrombin, whereas chymotrypsin cleaves the receptor at a site distal to Arg-41, thus rendering it unresponsive to thrombin while still responsive to thrombin receptor agonist peptide.

Distinct mechanisms regulate 5-HT2 and thrombin receptor desensitization.

We have compared the desensitization of two receptors, the thrombin receptor which displays dual coupling to both pertussis toxin-sensitive (Gi) and -insensitive (Gq) proteins and the serotonin type 2 (5-HT2) receptor which selectively couples to Gq. In the case of the thrombin receptor, cleavage induces activation and irreversible receptor modification followed by rapid (T1/2 = 3 min) and extensive desensitization of the receptor's ability to modulate phospholipase C (Gq). 5-HT-induced desensitization of its receptor is markedly slower (T1/2 = 10 min) and by 60 min only 50% of the phospholipase C response is lost. This effect occurs with a parallel disappearance of 5-HT receptors from the cell surface. Whole cell phosphorylation studies showed that the thrombin receptor is rapidly phosphorylated upon activation. In contrast, the 5-HT2 receptor displays a low basal level of phosphorylation which is not increased upon agonist treatment. The cytoplasmic tail of the 5-HT2 receptor which contains several protein kinase consensus sequences was found not to be involved in receptor activation or desensitization. However, a chimeric receptor having the core of the 5-HT2 receptor and the cytoplasmic tail of the thrombin receptor was able to undergo 5-HT-induced desensitization and phosphorylation. These results indicate that (i) both 5-HT2 and thrombin receptors have unique shut-off mechanisms, and (ii) that sequences in the carboxyl terminus of the thrombin receptor are sufficient to trigger rapid uncoupling of the receptor from its G protein(s) and downstream effector(s).

Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first, second or third N-terminal residue leads to a loss of activity, suggesting that a defined spacing of more than one structural component may be important for ligand-receptor interaction. Finally, we did not observe an antagonistic effect of the inactive peptides on phospholipase C activation or DNA synthesis induced by alpha-thrombin (1 nM) or SFLLRNP (3 microM).

Differential activation of p44mapk (ERK1) by alpha-thrombin and thrombin-receptor peptide agonist.

alpha-Thrombin (thrombin), a potent mitogen for CCL39 hamster lung fibroblasts, stimulates phosphoinositide-specific phospholipase C (PI-PLC) and inhibits adenylate cyclase via cleavage of a specific G-protein-coupled receptor (TH-R), recently cloned from human and hamster cells. This action can be entirely mimicked by the synthetic peptide SFFLRNP, referred to here as TMP (thrombin-mimicking peptide). TMP corresponds to the first seven amino acids of the new N-terminus generated by thrombin cleavage of the hamster TH-R. Although thrombin and TMP apparently generate identical early transmembrane signals, only thrombin is mitogenic on its own. TMP needs to be associated with fibroblast growth factor (FGF), a tyrosine kinase-activating growth factor, to induce cell-cycle re-entry. Here, we have examined the early and late phase of p44 MAP kinase (p44mapk) activation in G0-arrested CCL39 cells after stimulation by thrombin, TMP, FGF or TMP+FGF. We found that: (i) both thrombin and TMP rapidly activate p44mapk in a dose-dependent manner with maximum activation at around 5 min, (ii) after the initial burst of activation, a second and long-lasting wave of activation is observed in response to thrombin (10-100 nM) but not to TMP (up to 300 microM), (iii) FGF alone (25 ng/ml), like thrombin, rapidly and persistently activates p44mapk (20-fold at 5 min and about 3-fold after 2 h), (iv) TMP added together with FGF strongly potentiates the second and sustained phase of p44mapk activation. From these results we propose that: (1) thrombin-induced mitogenesis is mediated only in part by the TH-R recently cloned and (2) activation of p44mapk, in particular the long-lasting phase that correlates with DNA synthesis, is an obligatory event for cell-cycle re-entry.

Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis.

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.

cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2+ mobilization.

The serine protease alpha-thrombin (thrombin) potently stimulates G-protein-coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G-protein-coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C-coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N-terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N-terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion-binding exosite of thrombin.

Cloning, functional expression and role in cell growth regulation of a hamster 5-HT2 receptor subtype.

We have isolated a hamster fibroblast cDNA clone that encodes a serotoninergic receptor whose deduced amino acid sequence displays 94% identity with the rat brain serotonin (5-HT) type 2 receptor. When expressed in Xenopus oocytes, the hamster receptor efficiently couples to the phosphoinositide second messenger system and leads to intracellular Ca2+ mobilization in response to 5-HT. To determine the pharmacological properties of this receptor, and to evaluate the role of phospholipase C (PLC) activation in growth modulation by 5-HT, we have expressed it in hamster fibroblasts. Transfected cells that express 5-HT receptors were selected using a novel method based on coexpression of the Na+/H+ antiporter gene as a selectable marker. After co-transfection of the 5-HT receptor and Na+/H+ antiporter cDNAs in fibroblasts lacking antiporter activity (variants of the CCL39 line), 50% of the clones resistant to an acute acid load express functional receptors. The pharmacological profile of the transfected receptor is consistent with it being of the 5-HT2 subtype, and the extent of 5-HT-stimulated PLC activation in independent clones correlates with their relative level of cRNA expression. In cells in where addition of 5-HT leads to strong activation of PLC, and inhibition of adenylate cyclase via endogenous 5-HT1b receptors, 5-HT alone has little effect on DNA synthesis stimulation. Thus we conclude that activation of the PLC signalling pathway in these cells is not sufficient to trigger G0/G1 to S phase transition. Strong activation of PLC via 5-HT2 receptors does however contribute to the synergy observed between 5-HT (Gi-coupled pathway) and fibroblast growth factor (tyrosine kinase-activated pathway) on DNA synthesis reinitiation in transfected cells.