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• W2045704709 abstract "Integrins are important mammalian receptors involved in normal cellular functions and the pathogenesis of inflammation and disease. Entamoeba histolytica is a protozoan parasite that colonizes the gut, and in 10% of infected individuals, causes amebic colitis and liver abscess resulting in 105 deaths/year. E. histolytica-induced host inflammatory responses are critical in the pathogenesis of the disease, yet the host and parasite factors involved in disease are poorly defined. Here we show that pro-mature cysteine proteinase 5 (PCP5), a major virulent factor that is abundantly secreted and/or present on the surface of ameba, binds via its RGD motif to αVβ3 integrin on Caco-2 colonic cells and stimulates NFκB-mediated pro-inflammatory responses. PCP5 RGD binding to αVβ3 integrin triggered integrin-linked kinase(ILK)-mediated phosphorylation of Akt-473 that bound and induced the ubiquitination of NF-κB essential modulator (NEMO). As NEMO is required for activation of the IKKα-IKKβ complex and NFκB signaling, these events markedly up-regulated pro-inflammatory mediator expressions in vitro in Caco-2 cells and in vivo in colonic loop studies in wild-type and Muc2−/− mice lacking an intact protective mucus barrier. These results have revealed that EhPCP5 RGD motif is a ligand for αVβ3 integrin-mediated adhesion on colonic cells and represents a novel mechanism that E. histolytica trophozoites use to trigger an inflammatory response in the pathogenesis of intestinal amebiasis. Integrins are important mammalian receptors involved in normal cellular functions and the pathogenesis of inflammation and disease. Entamoeba histolytica is a protozoan parasite that colonizes the gut, and in 10% of infected individuals, causes amebic colitis and liver abscess resulting in 105 deaths/year. E. histolytica-induced host inflammatory responses are critical in the pathogenesis of the disease, yet the host and parasite factors involved in disease are poorly defined. Here we show that pro-mature cysteine proteinase 5 (PCP5), a major virulent factor that is abundantly secreted and/or present on the surface of ameba, binds via its RGD motif to αVβ3 integrin on Caco-2 colonic cells and stimulates NFκB-mediated pro-inflammatory responses. PCP5 RGD binding to αVβ3 integrin triggered integrin-linked kinase(ILK)-mediated phosphorylation of Akt-473 that bound and induced the ubiquitination of NF-κB essential modulator (NEMO). As NEMO is required for activation of the IKKα-IKKβ complex and NFκB signaling, these events markedly up-regulated pro-inflammatory mediator expressions in vitro in Caco-2 cells and in vivo in colonic loop studies in wild-type and Muc2−/− mice lacking an intact protective mucus barrier. These results have revealed that EhPCP5 RGD motif is a ligand for αVβ3 integrin-mediated adhesion on colonic cells and represents a novel mechanism that E. histolytica trophozoites use to trigger an inflammatory response in the pathogenesis of intestinal amebiasis. The colonic protozoan parasite Entamoeba histolytica (Eh) 2The abbreviations used are: EhE. histolyticaCPcysteine proteinaseSPsecreted proteinasesPCP5pro-mature cysteine proteinase 5ILKintegrin-linked kinasNEMONF-κB essential modulatorIKKIκB kinaseLYLY294002WMwortmanninZbenzyloxycarbonylpNAp-nitroanilidepphosphorylated. infects 10% of the world population and causes 100,000 deaths/year (1World Health OrganizationThe World Health Report. 1998: 51-54Google Scholar). Intestinal amebiasis is characterized by severe colitis and bloody dysentery. Unfortunately, we know little about the host and parasite factors involved in the sequence of events leading to the pathogenesis of intestinal amebiasis. Most evidence to date suggests that a host response to E. histolytica components (e.g. Gal/GalNAc adherence lectin) following adherence to the epithelium or to inflammatory cells triggers an acute inflammatory response as an initiating factor central to the genesis of disease. Among the potential virulent factors that can play a role in disease are the parasite cysteine proteinases (CPs). Cysteine proteinases are major virulent molecules produced by ameba of which EhCP1, EhCP2, and EhCP5 account for ∼90% of all EhCP transcripts (2Bruchhaus I. Loftus B.J. Hall N. Tannich E. Eukaryot Cell. 2003; 2: 501-509Crossref PubMed Scopus (146) Google Scholar). Of these, EhCP5 is the major protease that is secreted and/or present on the ameba surface (3Jacobs T. Bruchhaus I. Dandekar T. Tannich E. Leippe M. Mol. Microbiol. 1998; 27: 269-276Crossref PubMed Scopus (104) Google Scholar). EhCP5 is directly involved in disrupting the protective mucin barrier of the colon and in tissue invasion through degradation of extracellular matrix proteins and plays a key role in immune evasion by degrading host antibodies and complement (4Moncada D. Keller K. Ankri S. Mirelman D. Chadee K. Gastroenterology. 2006; 130: 721-730Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 5Reed S.L. Ember J.A. Herdman D.S. DiScipio R.G. Hugli T.E. Gigli I. J. Immunol. 1995; 155: 266-274PubMed Google Scholar, 6Tran V.Q. Herdman D.S. Torian B.E. Reed S.L. J. Infect. Dis. 1998; 177: 508-511Crossref PubMed Scopus (62) Google Scholar). E. histolytica cysteine proteinase secreted proteinases pro-mature cysteine proteinase 5 integrin-linked kinas NF-κB essential modulator IκB kinase LY294002 wortmannin benzyloxycarbonyl p-nitroanilide phosphorylated. Integrins are αβ heterodimeric trans-membrane proteins located on the surface of mammalian cells that convey an intracellular signaling network controlling cellular processes such as cell-cell adhesion, differentiation, migration, and survival (7Scatena M. Giachelli C. Trends Cardiovasc. Med. 2002; 12: 83-88Crossref PubMed Scopus (69) Google Scholar, 8Humphries J.D. Byron A. Humphries M.J. J. Cell Sci. 2006; 119: 3901-3903Crossref PubMed Scopus (1194) Google Scholar, 9Dupuy A.G. Caron E. J. Cell Sci. 2008; 121: 1773-1783Crossref PubMed Scopus (178) Google Scholar). Integrins on the cell surface can also act as cellular receptors for soluble plasma proteins and a variety of microbial pathogens such as Borrelia burgdorferi, Yersinia spp., Bordetella pertussis, adenovirus, and rotavirus (reviewed in Ref. 10Isberg R.R. Tran Van Nhieu G. Trends Microbiol. 1994; 2: 10-14Abstract Full Text PDF PubMed Scopus (166) Google Scholar). Pathogen recognition is mediated by the 3-amino acid RGD sequence arginine-glycine-aspartate (11Wickham T.J. Mathias P. Cheresh D.A. Nemerow G.R. Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1959) Google Scholar). The expression of integrins is cell type-dependent, and those that are expressed on the epithelium include several αvβ integrins, which are present on the apical and around the lateral surfaces (8Humphries J.D. Byron A. Humphries M.J. J. Cell Sci. 2006; 119: 3901-3903Crossref PubMed Scopus (1194) Google Scholar, 12Gilcrease M.Z. Cancer Lett. 2007; 247: 1-25Crossref PubMed Scopus (151) Google Scholar). As proteases can interfere with cell adhesion and/or migration in a non-proteolytic fashion by integrin receptors (13Lechner A.M. Assfalg-Machleidt I. Zahler S. Stoeckelhuber M. Machleidt W. Jochum M. Nägler D.K. J. Biol. Chem. 2006; 281: 39588-39597Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 14Papaconstantinou M.E. Carrell C.J. Pineda A.O. Bobofchak K.M. Mathews F.S. Flordellis C.S. Maragoudakis M.E. Tsopanoglou N.E. Di Cera E. J. Biol. Chem. 2005; 280: 29393-29396Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 15Brooks P.C. Strömblad S. Sanders L.C. von Schalscha T.L. Aimes R.T. Stetler-Stevenson W.G. Quigley J.P. Cheresh D.A. Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1435) Google Scholar), we explored whether EhCP5 can interact with colonic epithelial cell integrins to trigger host inflammatory responses, which are critical in the pathogenesis of intestinal amebiasis. Caco-2 human colonic cells were obtained from the ATCC (Manassas, VA). Secreted proteinases (SPs) were prepared as described previously (4Moncada D. Keller K. Ankri S. Mirelman D. Chadee K. Gastroenterology. 2006; 130: 721-730Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). E. histolytica cultures deficient in CP5 were a gift from Dr. David Mirelman (Weizmann Institute of Science, Rehovot, Israel). NEMO (sc-8330), integrin-linked kinase (ILK), αVβ3, ubiquitin (P4D1), antibody, ILK siRNA (sc-35666, Santa Cruz Biotechnology), ILK shRNA (RHS4430-101029656, Open Biosystems), human fibronectin, E64, PD98029, LY294002, and wortmannin were obtained from Sigma. αVβ3 blocking antibody LM609 (16Stockbauer K.E. Grigsby D. Pan X. Fu Y.X. Mejia L.M. Cravioto A. Musser J.M. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 3128-3133Crossref PubMed Scopus (52) Google Scholar) and αVβ3 integrin protein were from Millipore; p-Akt, p-IκBα, His antibody, and GSK3β fusion protein were from Cell Signaling. GRGDSP and GRADSP peptide were purchased from Calbiochem. EhCP5 was expressed in Escherichia coli strain BL21(DE3) (pAPlacIQ) using the expression vector pJC45. Recombinant RAD and RGA were mutated by QuikChange site-directed mutagenesis kit (Stratagene). The recombinant protein was expressed as an insoluble histidine-tagged pro-enzyme and was solubilized, purified, and refolded as described elsewhere (17Hellberg A. Nickel R. Lotter H. Tannich E. Bruchhaus I. Cell. Microbiol. 2001; 3: 13-20Crossref PubMed Scopus (81) Google Scholar). Protein purity was >95% as revealed by SDS-PAGE. C57BL/6 wild-type mice were purchased from Charles River, and Muc2−/− of the same background were obtained from Dr. Anna Velcich (Montefiore Medical Center) and bred at the University of Calgary Animal Facilities. Mice were anesthetized with ketamine/xylazine, and colonic loops were injected with log-phase virulent wild-type trophozoites (106) and pro-mature CP5 (PCP5) or PCP5-RAD (0.7 μg/μl). Sham-challenged mice were injected with 200 μl of the vehicle PBS, and animals were kept on a 37 °C warm plate for 3 h. The colons were excised, and pro-inflammatory gene expression was analyzed by using quantitative real time PCR. All studies were carried out with the approval of the University of Calgary Animal Care Committee. After treatment with various additions, cells were washed with ice-cold phosphate-buffered saline and lysed in lysis buffer (20 mm Tris-HCl, pH 7.6, 120 mm NaCl, 1 mm EDTA, 50 mm NaF, 1% Nonidet P-40, 1 mm Na3VO4, protease inhibitor mixture (Sigma)). Lysates were immunoprecipitated for 4 h at 4 °C with the appropriate primary antibody, and protein A/G (Santa Cruz Biotechnology) was added overnight. Western blot analysis was performed as described previously (18Hou Y. Gao F. Wang Q. Zhao J. Flagg T. Zhang Y. Deng X. J. Biol. Chem. 2007; 282: 9279-9287Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In vitro binding of purified αVβ3 (10 nm) integrin protein to PCP5 (1 μm) and mutants (1 μm) lacking the RGD motif was also assessed by co-immunoprecipitation as described previously (13Lechner A.M. Assfalg-Machleidt I. Zahler S. Stoeckelhuber M. Machleidt W. Jochum M. Nägler D.K. J. Biol. Chem. 2006; 281: 39588-39597Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Cells were fixed for 15 min with 3.7% paraformaldehyde, incubated with primary antibodies, and subsequently incubated with secondary antibodies. After the final washing, cells were covered with Permafluor mounting medium. Immunostained cells were viewed on a confocal microscope (Olympus FV 1000 Confocal). Caco-2 cells were co-transfected with NFκB-luc plasmid and pTK-Renilla luciferase plasmid as an internal control (ratio: 1:100) by using LipofectamineTM 2000 (Invitrogen); 24 h after transfection, cells were treated as indicated. Cell lysates were measured using a Dual-Luciferase reporter assay system (Promega), and all data were normalized using pTK-Renilla luciferase. Results are expressed as -fold increase over matched controls. Cell attachment assay was performed as described previously (13Lechner A.M. Assfalg-Machleidt I. Zahler S. Stoeckelhuber M. Machleidt W. Jochum M. Nägler D.K. J. Biol. Chem. 2006; 281: 39588-39597Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Briefly, 96-microwell plates were coated with PCP5, RGD, RAD, fibronectin, and BSA as a control (all at 5 μg/ml) in 100 μl of PBS/well for 18 h at 4 °C. The wells were blocked with 10 mg/ml BSA for 30 min. Subconfluent Caco-2 were detached using trypsin/EDTA and resuspended at 2 × 105 cells/ml in DMEM/F12 containing 1% BSA, and 100 μl of cells were added to each well. Inhibition studies were performed by preincubating Caco-2 cells with αVβ3 antibody (5 μg/ml), GRGDSP (50 μg/ml), and GRADSP (50 μg/ml) for 30 min before addition to the wells. Control cells were treated with BSA (5 μg/ml). The plates were incubated for 90 min at 37 °C in 5% CO2. Cells were washed with PBS, fixed with 4% formaldehyde in PBS, stained with 0.1% crystal violet for 20 min, and washed and dissolved, and cell numbers were quantified by measurement of the optical density at 595 nm in a microplate reader. Total RNA from colon was isolated using an RNeasy mini kit (Qiagen) and analyzed by real time PCR with SYBR Green (Qiagen). mRNA expression was normalized against GAPDH. -Fold change over control was determined according to the cycle threshold (Ct) method (19Bas A. Forsberg G. Hammarström S. Hammarström M.L. Scand. J. Immunol. 2004; 59: 566-573Crossref PubMed Scopus (347) Google Scholar). ILK assay was described previously (20Nho R.S. Xia H. Kahm J. Kleidon J. Diebold D. Henke C.A. J. Biol. Chem. 2005; 280: 26630-26639Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Caco-2 cells were treated as indicated. Cells were lysed and immunoprecipitated with 4 μg of anti-ILK antibody. 1 μg of GSK3β fusion protein (Cell Signaling) was then added to the immunoprecipitated lysate buffer followed by incubation for 30 min at 30 °C. After reaction, 20 μl of SDS sample buffer were added to terminate the reaction. Samples were analyzed by Western analysis with phospho-GSK3β and total GSK3β antibodies. The values for each parameter within a group are expressed as the mean ± S.E. Statistical significance was determined by Student's t test (when comparing two groups) or analysis of variance with a post hoc test (when comparing more than two groups). Significance was defined as p < 0.05. We have previously shown (4Moncada D. Keller K. Ankri S. Mirelman D. Chadee K. Gastroenterology. 2006; 130: 721-730Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) that the specific cysteine protease inhibitor E64 markedly inhibited mucin degradation by E. histolytica SPs. To determine whether ameba SPs can directly activate colonic cells, we performed a time-dependent study to quantify the activation of NFκB. As shown in Fig. 1A, ameba SPs rapidly induced the ubiquitination of NEMO and the phosphorylation of IκBα (Fig. 1B) even in the presence of the cysteine proteinase inhibitor E64, suggesting that cysteine proteinase enzyme activity was not required for activating NFκB. Phosphorylation of IκBα is catalyzed by IκB kinase (IKK), a complex composed of three subunits, IKKα/IKK1, IKKβ/IKK2, and NEMO/IKKγ. IKK1 and IKK2 are the catalytic subunits, whereas NEMO serves a non-enzymatic, regulatory function (21Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1595) Google Scholar, 22Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar). IKKα and IKKβ are serine/threonine protein kinases, whereas NEMO contains several protein interaction motifs but no apparent catalytic domains (23Miller B.S. Zandi E. J. Biol. Chem. 2001; 276: 36320-36326Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Genetic studies suggest that NEMO is absolutely required for the activation of IKK and NFκB in response to diverse stimuli (24Nenci A. Becker C. Wullaert A. Gareus R. van Loo G. Danese S. Huth M. Nikolaev A. Neufert C. Madison B. Gumucio D. Neurath M.F. Pasparakis M. Nature. 2007; 446: 557-561Crossref PubMed Scopus (856) Google Scholar, 25Tokunaga F. Sakata S. Saeki Y. Satomi Y. Kirisako T. Kamei K. Nakagawa T. Kato M. Murata S. Yamaoka S. Yamamoto M. Akira S. Takao T. Tanaka K. Iwai K. Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (758) Google Scholar), and ubiquitination of NEMO is required for the activation IKK (26Zhou H. Wertz I. O'Rourke K. Ultsch M. Seshagiri S. Eby M. Xiao W. Dixit V.M. Nature. 2004; 427: 167-171Crossref PubMed Scopus (453) Google Scholar). E. histolytica intrinsic and secreted proteinases contain both PCP5 and mature CP5 (27Nowak N. Lotter H. Tannich E. Bruchhaus I. J. Biol. Chem. 2004; 279: 38260-38266Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Interestingly, when ameba was grown in the presence of E64, PCP5 levels were significantly increased (Fig. 1C) as compared with untreated controls. This finding is consistent with a previous report that showed ameba secretes both PCP5 and mature CP5 (27Nowak N. Lotter H. Tannich E. Bruchhaus I. J. Biol. Chem. 2004; 279: 38260-38266Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Thus, to elucidate a specific functional role for EhCP5, colonic cells were exposed to secreted proteinases derived from wild-type (WT), amebapore A vector control (AP−/−) or E. histolytica genetically silenced for CP5 (AP/CP−/−) expression. Under these conditions, the WT or vector control (AP−/−) significantly induced the ubiquitination of NEMO, but CP5−/− did not (Fig. 1D). These data clearly show that PCP5 was the critical proteinase involved in activating NFκB and inducing the ubiquitination of NEMO. As inhibition of CP activity did not prevent CP-induced ubiquitination of NEMO and phosphorylation of IκB, we were led to inquire whether CP5 was directly binding and interacting with an epithelial cell surface receptor to activate NFκB. Amino acid sequence analysis revealed that PCP5 encodes an RGD motif (92–94 amino acids, Fig. 2A), the peptide motif recognized by integrin receptors, in the pro-domain region upstream toward the carboxyl-terminal end. The RGD motif is also found on EhCP18 and EhCP112 within the catalytic domain; however, these enzymes are not highly expressed or secreted by ameba (3Jacobs T. Bruchhaus I. Dandekar T. Tannich E. Leippe M. Mol. Microbiol. 1998; 27: 269-276Crossref PubMed Scopus (104) Google Scholar). Based on this finding, we postulated that PCP5 might function as a ligand with integrin to induce pro-inflammatory responses. αVβ3 integrin is abundantly expressed on Caco-2 cells in culture and colonic epithelial cells and binds RGD motifs (28Croyle M.A. Walter E. Janich S. Roessler B.J. Amidon G.L. Hum. Gene Ther. 1998; 9: 561-573Crossref PubMed Scopus (53) Google Scholar). Thus, to determine binding of PCP5 to αVβ3, co-immunoprecipitation studies were done that demonstrated that PCP5 bound αVβ3 integrin on Caco-2 cells (Fig. 2B). However, a single amino acid substitution in the RGD motif where either the aspartate or the glycine residue was substituted by an alanine residue to generate the mutants RAD or RGA, respectively (Fig. 2A), abrogated binding (Fig. 2B). Similarly, Caco-2 cells were pretreated with the GRGDSP peptide that specifically binds integrin (but not to the nonspecific GRADSP peptide), as well as neutralizing antibodies against αVβ3, which markedly inhibited PCP5 binding to αVβ3 integrin. Moreover, co-immunoprecipitation of purified recombinant PCP5 alone demonstrated that PCP5 bound to the αVβ3 receptor, whereas the mutants PCP5 RAD and RGA did not (Fig. 2C). As integrin αVβ3 is also expressed on the apical surface of many human cells types including colonic epithelial cells (12Gilcrease M.Z. Cancer Lett. 2007; 247: 1-25Crossref PubMed Scopus (151) Google Scholar), we determined the binging of PCP5 to Caco-2 monolayer. As shown in Fig. 2D, cell attachment analysis revealed that PCP5 bound αVβ3 integrin, whereas the mutants RAD and RGD did not. However, cells pretreated with GRGDSP and αVβ3 antibody inhibited PCP5 binding to αVβ3 integrin. To determine cellular localization of the αVβ3 integrin, Caco-2 cells were exposed with PCP5 and analyzed by confocal fluorescence microscopy. As shown in the merged photomicrograph (Fig. 2E, arrows), PCP5 co-localized with αVβ3 on the surface of the colonic cells. Taken together, these findings demonstrate that PCP5 bound to αVβ3 integrin receptor on colonic cells via the RGD motif. ILK is implicated in the direct phosphorylation of serine 473 of Akt (29Attwell S. Mills J. Troussard A. Wu C. Dedhar S. Mol. Biol. Cell. 2003; 14: 4813-4825Crossref PubMed Scopus (123) Google Scholar). ILK functions downstream of PI3-kinase and promotes the phosphorylation of serine 473 of Akt (30Zhang Y. Guo L. Chen K. Wu C.A. J. Biol. Chem. 2002; 277: 318-326Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We have recently shown that E. histolytica can activate the PI3-kinase/Akt pathway, leading to NFκB-mediated pro-inflammatory MCP-1 expression and secretion by inducing the phosphorylation of Akt (31Kammanadiminti S.J. Dey I. Chadee K. Infect Immun. 2007; 75: 1765-1770Crossref PubMed Scopus (17) Google Scholar). Here we show that PCP5 can induce the phosphorylation of Akt-473 rather than Akt-308 in a dose- and time-dependent manner (supplemental Fig. S1, A and B). In particular, PCP5 significantly induced the phosphorylation of Akt-473, whereas the PCP5 mutants RAD and RGA had no effect (Fig. 3A), clearly implicating that the RGD motif is required and essential for PCP5-induced Akt phosphorylation. As predicted, as compared with the control peptide GRADSP, GRGDSP significantly inhibited PCP5-mediated phosphorylation of Akt (Fig. 3B). Moreover, the PI3K inhibitors LY294002 (LY) and wortmannin (WM) significantly abolished PCP5-mediated phosphorylation of Akt (Fig. 3C). As activated ILK can phosphorylate the substrate GSK3β in vitro (20Nho R.S. Xia H. Kahm J. Kleidon J. Diebold D. Henke C.A. J. Biol. Chem. 2005; 280: 26630-26639Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), our results show that PCP5 induced ILK activation, whereas the mutants RAD and RGD did not. Moreover, the peptide GRGDSP and neutralizing antibodies against αVβ3 inhibited ILK activation in response to PCP5 (supplemental Fig. S1C). It is well known that ligand coupling through the αVβ3 integrin dependently induces the activation of NFκB (32Urbinati C. Bugatti A. Giacca M. Schlaepfer D. Presta M. Rusnati M. J. Cell Sci. 2005; 118: 3949-3958Crossref PubMed Scopus (42) Google Scholar). Although PCP5 can significantly induce NFκB activity (supplemental Fig. S2, A and B, Fig. 3D), the RAD and RGA mutants did not (supplemental Fig. S3A). As the GRGDSP peptide can also inhibit PCP5-induced phosphorylation of IκBα (Fig. 3E, supplemental Fig. S3B), this suggests that the RGD motif of PCP5 is essential for PCP5-induced NFκB activity. In support of this, inhibiting the phosphorylation of Akt with LY and WM markedly decreased PCP5-induced phosphorylation of IκBα (Fig. 3F, supplemental Fig. S3C). These results were corroborated by NFκB luciferase activity analysis (Fig. 3, G and H) where PCP5 (or PCP5 treated with polymycin B to remove any potential LPS contaminant) stimulated high NFκB activity. Fibronectin, as a nonspecific ligand for αVβ3 integrin (8Humphries J.D. Byron A. Humphries M.J. J. Cell Sci. 2006; 119: 3901-3903Crossref PubMed Scopus (1194) Google Scholar, 33Qwarnström E.E. Ostberg C.O. Turk G.L. Richardson C.A. Bomsztyk K. J. Biol. Chem. 1994; 269: 30765-30768Abstract Full Text PDF PubMed Google Scholar), also induced robust NFκB activation in Caco-2 cells (supplemental Fig. S4B). Considering the nonspecific effects of siRNA on ILK (34Verreault M. Bally M.B. Oligonucleotides. 2009; 19: 129-140Crossref PubMed Scopus (8) Google Scholar), we silenced ILK using siRNA or shRNA. As shown in supplemental Fig. S4A, silencing ILK reduced PCP5 ILK-mediated phosphorylation of IκBα and the phosphorylation of Akt. These findings clearly show that PCP5 binding to ανβ3 led to the activation of the ILK/Akt/NFκB pathways. Ubiquitination of NEMO is required for NFκB activation (24Nenci A. Becker C. Wullaert A. Gareus R. van Loo G. Danese S. Huth M. Nikolaev A. Neufert C. Madison B. Gumucio D. Neurath M.F. Pasparakis M. Nature. 2007; 446: 557-561Crossref PubMed Scopus (856) Google Scholar, 25Tokunaga F. Sakata S. Saeki Y. Satomi Y. Kirisako T. Kamei K. Nakagawa T. Kato M. Murata S. Yamaoka S. Yamamoto M. Akira S. Takao T. Tanaka K. Iwai K. Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (758) Google Scholar, 26Zhou H. Wertz I. O'Rourke K. Ultsch M. Seshagiri S. Eby M. Xiao W. Dixit V.M. Nature. 2004; 427: 167-171Crossref PubMed Scopus (453) Google Scholar). The Akt/IKK pathway mediates cell survival (35Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 36Madrid L.V. Wang C.Y. Guttridge D.C. Schottelius A.J. Baldwin Jr., A.S. Mayo M.W. Mol. Cell. Biol. 2000; 20: 1626-1638Crossref PubMed Scopus (589) Google Scholar) and pro-inflammatory responses (31Kammanadiminti S.J. Dey I. Chadee K. Infect Immun. 2007; 75: 1765-1770Crossref PubMed Scopus (17) Google Scholar). Here we found that PCP5 but not the mutants RAD and RGA can rapidly induce the ubiquitination of NEMO (Fig. 4, A and B, supplemental Fig. S5). Akt induces NFκB activity by activating IKK (35Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 36Madrid L.V. Wang C.Y. Guttridge D.C. Schottelius A.J. Baldwin Jr., A.S. Mayo M.W. Mol. Cell. Biol. 2000; 20: 1626-1638Crossref PubMed Scopus (589) Google Scholar), and as expected, we show that inhibitors of Akt (LY and WM) abrogated PCP5-induced ubiquitination of NEMO (Fig. 4C). As shown in Fig. 4D, PCP5-induced Akt activation resulted in Akt binding to NEMO, whereas the mutants RAD, RGA, and PCP5, with the addition of the competitor peptide GRGDSP, did not. Under these conditions, the PI3K inhibitors LY and WM completely inhibited PCP5-induced interaction between Akt and NEMO (Fig. 4E). As ILK functions downstream of PI3-kinase and promotes the phosphorylation of Akt (29Attwell S. Mills J. Troussard A. Wu C. Dedhar S. Mol. Biol. Cell. 2003; 14: 4813-4825Crossref PubMed Scopus (123) Google Scholar, 30Zhang Y. Guo L. Chen K. Wu C.A. J. Biol. Chem. 2002; 277: 318-326Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) as predicted, silencing ILK by siRNA or shRNA abolished PCP5-induced ubiquitination of NEMO, as well as Akt binding to NEMO (Fig. 4, F and G). Taken together, these findings clearly implicate PCP5 binding to αvβ3 integrin, which in turn triggers ILK/Akt/NEMO signaling in colonic epithelial cells. As ameba SPs can induce Akt and NFκB-mediated pro-inflammatory responses (31Kammanadiminti S.J. Dey I. Chadee K. Infect Immun. 2007; 75: 1765-1770Crossref PubMed Scopus (17) Google Scholar), we determined whether PCP5 coupling through integrin could activate the Akt/NFκB pathway, resulting in pro-inflammatory responses. As shown in Fig. 5A and B, PCP5 induced the secretion of the pro-inflammatory cytokines TNF-α and IL-1β by Caco-2 cells; however, cells pretreated with αvβ3 antibody, GRGDSP, or Akt inhibitor abolished PCP5-induced pro-inflammatory responses. To determine whether a similar response occurred in vivo, we used an animal model of the disease and demonstrated that wild-type mice colons infected with live highly virulent E. histolytica trophozoites or challenged with purified PCP5 stimulated robust TNF-α, IL-1β, IL-6, and Cox-2 gene expression (Fig. 6, A–D). In parallel, mice infected with CP5−/− ameba or inoculated with the RAD-PCP5 mutant could not mimic the effect of virulent E. histolytica or PCP5 (Fig. 6, A–D). As ameba CPs have high enzyme activity against Muc2 mucin substrates (37Lidell M.E. Moncada D.M. Chadee K. Hansson G.C. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9298-9303Crossref PubMed Scopus (214) Google Scholar), which can substantiality affect PCP5 binding to ανβ3 integrin receptor on colonic cells, studies were done in Muc2−/− mice, which are deficient for the major structural component of colonic mucus, to facilitate direct binding of PCP5 to the surface of the epithelium. Surprisingly, in Muc2−/− mice, pro-inflammatory cytokine gene expressions were significantly up-regulated in response to PCP5 but not to the mutant RAD as compared with WT mice with an intact mucus barrier (Fig. 6, E–H). These results suggest that loss of Muc2 mucin mucosal barrier protective function led to enhanced interaction of PCP5 with the integrin receptor on colonic cells (Fig. 5). Moreover, Muc2−/− mice colons pretreated with blocking αvβ3 integrin antibody significantly inhibited E. histolytica-induced pro-inflammatory gene expression (supplemental Fig. S6, A–D), suggesting that the αvβ3 integrin pathway is critically involved in E. histolytica-mediated pro-inflammatory responses.FIGURE 6PCP5 induces pro-inflammatory responses in mouse colon. A–D, WT and Muc2−/− mice colons were infected with virulent E. histolytica trophozoites and AP−/−CP5−/− deficient E. histolytica trophozoites for 3 h as described under “Experimental Procedures.” TNF-α, IL-1β, IL-6, and Cox-2 gene expression was detected by real-time PCR. Asterisk, p < 0.01; double asterisk, p < 0.05. Error bars indicate S.E. E–H, WT and Muc2−/− mice colons were treated with PCP5 and PCP5-RAD for 3 h, and TNF-α, IL-1β, IL-6, and Cox-2 gene expression was detected by real-time PCR. Asterisk, p < 0.01; double asterisk, p < 0.05. Error bars indicate S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The pathogenesis of intestinal amebiasis is a multifaceted event involving both host and parasite factors. Host inflammatory responses are critical in the pathogenesis of intestinal amebiasis (38Lejeune M. Rybicka J.M. Chadee K. Future Microbiol. 2009; 4: 105-118Crossref PubMed Scopus (36) Google Scholar). In this study, we have revealed a novel role for integrins in the pathogenesis of E. histolytica, making ανβ3 integrin the first human receptor to be exploited by a colonic mucosal parasite. Our findings support an intriguing mechanism by which the PCP5 RGD motif can bind ανβ3 integrin to trigger integrin/NFκB signaling that elicits pro-inflammatory cytokine production. E. histolytica cysteine proteinases are key virulence factors involved in pathogenesis (3Jacobs T. Bruchhaus I. Dandekar T. Tannich E. Leippe M. Mol. Microbiol. 1998; 27: 269-276Crossref PubMed Scopus (104) Google Scholar, 4Moncada D. Keller K. Ankri S. Mirelman D. Chadee K. Gastroenterology. 2006; 130: 721-730Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Trophozoites secrete both pro-mature CP5 and mature CP5 (27Nowak N. Lotter H. Tannich E. Bruchhaus I. J. Biol. Chem. 2004; 279: 38260-38266Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). CP5-deficient trophozoites are known to inflict less gut inflammation and damage to the intestinal barrier and are incapable of inducing amebic liver abscesses (39Ankri S. Stolarsky T. Mirelman D. Mol. Microbiol. 1998; 28: 777-785Crossref PubMed Scopus (106) Google Scholar). Thus, it was not surprising to find that E. histolytica genetically deficient in CP5 production elicited significantly less NFκB activation and pro-inflammatory responses, reinforcing the notion that CP5 is an important virulence factor. Importantly, as both pro-mature CP5 and mature CP5 are secreted, it was critical to determine which form of the enzyme was essential for activating NFκB-mediated pro-inflammatory responses. With the use of the specific CP5 enzyme inhibitor E64 to abolish CP5 enzyme activity, we were intrigued that it had no effect on PCP5-mediated NFκB activation, suggesting that PCP5 may activate NFκB. Although previous studies have shown that inhibiting cysteine proteinase activity decreases E. histolytica-induced liver abscess formation, it is important to note that only E64-resistant ameba cultures, rather than wild-type ameba cultured with E64, had a significant effect on liver abscess formation (27Nowak N. Lotter H. Tannich E. Bruchhaus I. J. Biol. Chem. 2004; 279: 38260-38266Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). As E64-resistant E. histolytica cultures have significantly decreased CP5 gene expression (27Nowak N. Lotter H. Tannich E. Bruchhaus I. J. Biol. Chem. 2004; 279: 38260-38266Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), this finding demonstrates precedence of CP5 as a virulent factor for E. histolytica. Interestingly, our results show that the pro-mature form, PCP5, is also an important virulent factor, and in this regard, this is the first report to ascribe a functional role for PCP5. These findings also explain why E. histolytica cultured with E64 cannot inhibit liver abscess formation (27Nowak N. Lotter H. Tannich E. Bruchhaus I. J. Biol. Chem. 2004; 279: 38260-38266Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) as CP5 enzyme activity is not required for the NFκB-mediated pro-inflammatory responses that potentiate amebic liver abscess formation (reviewed in 38Lejeune M. Rybicka J.M. Chadee K. Future Microbiol. 2009; 4: 105-118Crossref PubMed Scopus (36) Google Scholar). Amino acid sequence analysis revealed that PCP5 encodes an RGD motif, which is known to interact with ανβ3 integrin to activate NFκB (31Kammanadiminti S.J. Dey I. Chadee K. Infect Immun. 2007; 75: 1765-1770Crossref PubMed Scopus (17) Google Scholar). Other integrins including α3β1, α5β1, α8β1, αVβ3, and αVβ1 can recognize ligands containing an RGD tripeptide active site (8Humphries J.D. Byron A. Humphries M.J. J. Cell Sci. 2006; 119: 3901-3903Crossref PubMed Scopus (1194) Google Scholar). Although αVβ3 integrins are abundantly expressed on Caco-2 and colonic epithelial cells (28Croyle M.A. Walter E. Janich S. Roessler B.J. Amidon G.L. Hum. Gene Ther. 1998; 9: 561-573Crossref PubMed Scopus (53) Google Scholar), we cannot exclude the possibility that PCP5 may bind other integrins and have a functional role. Our results show that the RGD motif was required for PCP5-mediated NFκB activation by inducing ubiquitination of NEMO. NEMO is the regulatory subunit of the IKKα-IKKβ complex, which, predominantly acting through IKKβ, phosphorylates IκBs that sequester NFκB in the cytoplasm. This phosphorylation event signals IκB ubiquitin-mediated degradation, and thereby, the release of NFκB into the nucleus, where it activates pro-inflammatory gene expression. This pathway of NFκB activation is dependent on NEMO as pro-inflammatory signal-induced NFκB activity is completely blocked in NEMO-deficient cells but only partially affected in IKK2/β-deficient cells (40Schmidt C. Peng B. Li Z. Sclabas G.M. Fujioka S. Niu J. Schmidt-Supprian M. Evans D.B. Abbruzzese J.L. Chiao P.J. Mol. Cell. 2003; 12: 1287-1300Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 41Li Q. Estepa G. Memet S. Israel A. Verma I.M. Genes Dev. 2000; 14: 1729-1733Crossref PubMed Google Scholar). ILK has been implicated in direct phosphorylation of Akt serine 473 (29Attwell S. Mills J. Troussard A. Wu C. Dedhar S. Mol. Biol. Cell. 2003; 14: 4813-4825Crossref PubMed Scopus (123) Google Scholar) and functions downstream of PI3-kinase in promoting phosphorylation of Akt serine 473 (30Zhang Y. Guo L. Chen K. Wu C.A. J. Biol. Chem. 2002; 277: 318-326Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We have previously shown that proteins secreted by virulent E. histolytica activate the PI3-kinase/Akt pathway, leading to NFκB-mediated pro-inflammatory MCP-1 expression, by inducing Akt phosphorylation at serine 308 (30Zhang Y. Guo L. Chen K. Wu C.A. J. Biol. Chem. 2002; 277: 318-326Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Here we found that PCP5 had no effect on the phosphorylation of Akt308. Unexpectedly, high concentrations of PCP5 (>500 μg/ml) decreased the phosphorylation of Akt473 and IκBα in a time-dependent fashion, suggesting that PCP5 may function within a narrow range, but this is only speculation. Silencing ILK abolished ILK-mediated phosphorylation of Akt 473. These data suggest that PCP5 integrin binding activated the ILK/Akt pathway and activated NFκB. The Akt/IKK pathway is known to mediate cell survival (35Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 36Madrid L.V. Wang C.Y. Guttridge D.C. Schottelius A.J. Baldwin Jr., A.S. Mayo M.W. Mol. Cell. Biol. 2000; 20: 1626-1638Crossref PubMed Scopus (589) Google Scholar) and pro-inflammatory responses (31Kammanadiminti S.J. Dey I. Chadee K. Infect Immun. 2007; 75: 1765-1770Crossref PubMed Scopus (17) Google Scholar); however, it is still unclear how Akt induces the activation of NEMO and activates downstream NFκB signaling. Our results show that Akt bound to NEMO and caused NEMO to be ubiquitinated and that NEMO ubiquitination in turn led to the activation of IKK and subsequent activation of NFκB. These results not only clarify the mechanism whereby PCP5 activates NFκB but also provide a novel mechanism of Akt interaction with NEMO. Activation of the NFκB family mediates inflammation (42Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3380) Google Scholar, 43Ben-Neriah Y. Schmidt-Supprian M. Nat. Immunol. 2007; 8: 479-481Crossref PubMed Scopus (31) Google Scholar). Thus, it was not surprising that PCP5 significantly induced pro-inflammatory cytokine secretion (TNF-α and IL-1β) in human colonic cells. More importantly, mice colons challenged with wild-type virulent E. histolytica trophozoites and recombinant PCP5 induced robust pro-inflammatory cytokine gene expression (TNF-α, IL-1β, Cox-2, and IL-6), whereas CP5−/− deficient trophozoites and a RAD-PCP5 mutant elicited a greatly reduced pro-inflammatory response. We have previously shown that CP5 has high enzyme activity against MUC2 colonic mucin (37Lidell M.E. Moncada D.M. Chadee K. Hansson G.C. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9298-9303Crossref PubMed Scopus (214) Google Scholar), which can substantially prevent CP5 from engaging epithelial cell integrin in the colon. Accordingly, studies done in Muc2−/− mice showed a significant increase in pro-inflammatory cytokine gene expression as compared with wild-type mice, clearly implicating that loss of the protective mucus barrier led to enhanced interaction of PCP5 with the epithelial integrin receptor. This study substantiates our hypothesis that secreted CP5/PCP5 can exert multiple functions in the gut lumen by first exhibiting enzyme activity against mucin and that only when the luminal barrier is compromised can PCP5 bind integrin on colonic cells via the RGD motif. This stepwise mode of action for PCP5 in the pathogenesis of intestinal amebiasis seems the most likely scenario. Taken together, initiation of host pro-inflammatory responses through PCP5 binding to αvβ3 integrin on colonic cells via the activation of the ILK/Akt/NEMO pathway provides novel insights into the molecular basis of E. histolytica-induced intestinal inflammation, the central event involved in instigating amebic colitis. We thank Dr. Iris Bruchhaus for generously providing the CP5 plasmid and CP5 antibody. Download .zip (.31 MB) Help with zip files" @default.
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• W2045704709 title "Entamoeba histolytica Cysteine Proteinase 5 Binds Integrin on Colonic Cells and Stimulates NFκB-mediated Pro-inflammatory Responses" @default.
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