Proteomic Analysis of Ischemia-Reperfusion Injury upon Human Liver Transplantation Reveals the Protective Role of IQGAP1

Ischemia-reperfusion injury (IRI) represents a major determinant of liver transplantation. IRI-induced graft dysfunction is related to biliary damage, partly due to a loss of bile canaliculi (BC) integrity associated with a dramatic remodeling of actin cytoskeleton. However, the molecular mechanisms associated with these events remain poorly characterized. Using liver biopsies collected during the early phases of organ procurement (ischemia) and transplantation (reperfusion), we characterized the global patterns of expression and phosphorylation of cytoskeleton-related proteins during hepatic IRI. This targeted functional proteomic approach, which combined protein expression pattern profiling and phosphoprotein enrichment followed by mass spectrometry analysis, allowed us to identify IQGAP1, a Cdc42/Rac1 effector, as a potential regulator of actin cytoskeleton remodeling and maintenance of BC integrity. Cell fractionation and immunohistochemistry revealed that IQGAP1 expression and localization were affected upon IRI and related to actin reorganization. Furthermore using an IRI model in human hepatoma cells, we demonstrated that IQGAP1 silencing decreased the basal level of actin polymerization at BC periphery, reflecting a defect in BC structure coincident with reduced cellular resistance to IRI. In summary, this study uncovered new mechanistic insights into the global regulation of IRI-induced cytoskeleton remodeling and led to the identification of IQGAP1 as a regulator of BC structure. IQGAP1 therefore represents a potential target for the design of new organ preservation strategies to improve transplantation outcome. Ischemia-reperfusion injury (IRI) represents a major determinant of liver transplantation. IRI-induced graft dysfunction is related to biliary damage, partly due to a loss of bile canaliculi (BC) integrity associated with a dramatic remodeling of actin cytoskeleton. However, the molecular mechanisms associated with these events remain poorly characterized. Using liver biopsies collected during the early phases of organ procurement (ischemia) and transplantation (reperfusion), we characterized the global patterns of expression and phosphorylation of cytoskeleton-related proteins during hepatic IRI. This targeted functional proteomic approach, which combined protein expression pattern profiling and phosphoprotein enrichment followed by mass spectrometry analysis, allowed us to identify IQGAP1, a Cdc42/Rac1 effector, as a potential regulator of actin cytoskeleton remodeling and maintenance of BC integrity. Cell fractionation and immunohistochemistry revealed that IQGAP1 expression and localization were affected upon IRI and related to actin reorganization. Furthermore using an IRI model in human hepatoma cells, we demonstrated that IQGAP1 silencing decreased the basal level of actin polymerization at BC periphery, reflecting a defect in BC structure coincident with reduced cellular resistance to IRI. In summary, this study uncovered new mechanistic insights into the global regulation of IRI-induced cytoskeleton remodeling and led to the identification of IQGAP1 as a regulator of BC structure. IQGAP1 therefore represents a potential target for the design of new organ preservation strategies to improve transplantation outcome. Orthotopic liver transplantation is the only viable therapeutic option for the treatment of end stage liver diseases. All donor organs experience some degree of preservation damage related to cold ischemia-reperfusion injury (IRI). 1The abbreviations used are: IRI, ischemia-reperfusion injury; AJ, adherens junctions; BC, bile canaliculi; F-actin, filamentous actin; shRNA, short hairpin RNA; TRITC, tetramethylrhodamine isothiocyanate; PKA, cAMP-dependent protein kinase; MAPK, mitogen-activated protein kinase. IRI has been linked to graft primary dysfunction (1Washington K. Update on post-liver transplantation infections, malignancies, and surgical complications.Adv. Anat. Pathol. 2005; 12: 221-226Crossref PubMed Scopus (39) Google Scholar) and can lead to vascular and biliary complications (2Ben-Ari Z. Pappo O. Mor E. Intrahepatic cholestasis after liver transplantation.Liver Transplant. 2003; 9: 1005-1018Crossref PubMed Scopus (69) Google Scholar) such as intrahepatic cholestasis, a pathologic state of reduced bile formation or flow, and posthepatic jaundice (3Ericzon B.G. Eusufzai S. Kubota K. Einarsson K. Angelin B. Characteristics of biliary lipid metabolism after liver transplantation.Hepatology. 1990; 12: 1222-1228Crossref PubMed Scopus (40) Google Scholar). These conditions involve damage to the biliary tree at the level of either cholangiocyte membrane integrity (4Doctor R.B. Dahl R.H. Salter K.D. Fitz J.G. Reorganization of cholangiocyte membrane domains represents an early event in rat liver ischemia.Hepatology. 1999; 29: 1364-1374Crossref PubMed Scopus (34) Google Scholar) or the bile canaliculi (BC) (5Cutrin J.C. Cantino D. Biasi F. Chiarpotto E. Salizzoni M. Andorno E. Massano G. Lanfranco G. Rizzetto M. Boveris A. Poli G. Reperfusion damage to the bile canaliculi in transplanted human liver.Hepatology. 1996; 24: 1053-1057Crossref PubMed Google Scholar). BC are structural tubules that form the most proximal intrahepatic secretory channels and carry bile secreted by the hepatocytes toward the bile duct to the gall bladder. The BC lumen, delimited by the apical membrane of two or more adjacent hepatocytes, is separated from the basolateral membranes of the hepatocytes by the presence of tight junctions supported by adherens junctions (AJ) (6Farquhar M.G. Palade G.E. Junctional complexes in various epithelia.J. Cell Biol. 1963; 17: 375-412Crossref PubMed Scopus (2132) Google Scholar). The cytoplasmic face of the BC membrane is associated with a dense cytoskeletal network that mostly consists of actin microfilaments and cytokeratin intermediate filaments (7Ishii M. Washioka H. Tonosaki A. Toyota T. Regional orientation of actin filaments in the pericanalicular cytoplasm of rat hepatocytes.Gastroenterology. 1991; 101: 1663-1672Abstract Full Text PDF PubMed Scopus (20) Google Scholar). Treatment of rat liver with compounds that alter actin polymerization, such as cytochalasin B and phalloidin, leads to the alteration of BC structure (canaliculi dilatation and loss of microvilli) as well as contractile properties (8Cooper J.A. Effects of cytochalasin and phalloidin on actin.J. Cell Biol. 1987; 105: 1473-1478Crossref PubMed Scopus (1634) Google Scholar, 9Watanabe N. Tsukada N. Smith C.R. Phillips M.J. Motility of bile canaliculi in the living animal: implications for bile flow.J. Cell Biol. 1991; 113: 1069-1080Crossref PubMed Scopus (111) Google Scholar). In addition, a major actin cytoskeleton alteration around the hepatocyte cell membrane has been reported in human liver in response to IRI and has also been related to an impairment of BC contraction, tight junction permeability, and subsequent maintenance of a normal bile flow during the postoperative period (10Yasui H. Yoshimura N. Kobayashi Y. Ochiai S. Matsuda T. Takamatsu T. Oka T. Microstructural changes of bile canaliculi in canine liver: the effect of cold ischemia-reperfusion in orthotopic liver transplantation.Transplant. Proc. 1998; 30: 3754-3757Crossref PubMed Scopus (10) Google Scholar). An intact actin network is therefore required for BC integrity to ensure proper bile secretion, i.e. both bile polarized transport out of the hepatocytes and maintenance of the blood-biliary barrier. However, although the observed actin cytoskeleton remodeling and alteration of BC structure were reported as primary targets of IRI, critical for graft recovery (5Cutrin J.C. Cantino D. Biasi F. Chiarpotto E. Salizzoni M. Andorno E. Massano G. Lanfranco G. Rizzetto M. Boveris A. Poli G. Reperfusion damage to the bile canaliculi in transplanted human liver.Hepatology. 1996; 24: 1053-1057Crossref PubMed Google Scholar), the underlying molecular mechanisms integrating these events remain largely unknown. In this work, we combined a targeted functional proteomic analyses of human liver biopsies collected during different phases of the transplantation with functional validation both in situ and in cellular models to identify and characterize proteins involved in both IRI-mediated actin cytoskeleton remodeling and maintenance of BC structural integrity. We demonstrated that the protein IQGAP1 represents a potential regulator of both mechanisms and as a consequence mediates cellular IRI resistance. Biopsies were collected from human livers during cold ischemia and/or reperfusion phases (34 livers) as described in Fig. 1A according to McGill University Health Centre ethics regulation (ERB 05-003). 0.5-cm3 biopsies were taken before the donor liver clamping (I0), and 10 and 60 min after clamping (I10 and I60), during reimplantation in the recipient patient just before clamp removal (R0), and 10 and 60 min after blood flow re-establishment in the hepatic portal vein (R10 and R60), respectively. Proteins were extracted from tissue or cultured after using as described membrane lipid and Biol. 1999; PubMed Scopus Google Scholar, A. E. are during human liver Pathol. 2005; PubMed Scopus Google Scholar, S. A. S. A. E. of and regulation of cell during Biol. PubMed Scopus Google Scholar). This leads to of proteins membrane lipid and Biol. 1999; PubMed Scopus Google Scholar). were as described S. A. S. A. E. of and regulation of cell during Biol. PubMed Scopus Google Scholar). protein from ischemia or reperfusion liver biopsies were for to were out in on protein of liver were for as described E. G. K. S. and of an protein that in 1999; PubMed Google Scholar). used in this study are described in were by were used in the or as were using the proteins both ischemia and reperfusion The of the for protein expression and as were either by on with either or as by the and as described A. S. enrichment of intact proteins by 2005; PubMed Scopus Google Scholar). proteins were using and by of the proteins by and to a with and by two of 10 min in before the of an of The were for min with 10 to and for min with to effect an of and the were extracted with at were with in for at and the were extracted in followed by two with of All were using a All from a were combined in the well of This to the of a for mass spectrometry on a with of of at a flow of with to a The with on a min of the the from and B the toward the with 10 at a flow of and to for the with a and B The from B to in from to in from to in at for and to in 10 The on a that the the of the mass on the to a during of were in a from to The most and were to in from to The were by the on the and of the the or to a of The were and to a for the for with a level of or and with an or the were and for All to from and were and using the Z. A. D. M. of the in and Scholar, F. A. F. M. S. of the in and Scholar) to and a of proteins the proteins for which at in in at two of the were proteins with properties from both and were from the were using the IQGAP1 and were from of as by the were by and and were the and as described K. of protein phosphorylation and protein on using a phosphorylation 2003; PubMed Scopus Google Scholar). by the IQGAP1 using the and for into and subsequent in according to the were from the were by and were at a of and for in were using and Z. A. IQGAP1 cell and Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) or and the using according to the were for with University of preservation with 10 Reperfusion by the cold preservation and Fig. Cell on using of were for are as of were using the tissue on or and on were in of filamentous actin using phalloidin as described S. A. M. S. M. E. of membrane by a protein in Biol. 2005; PubMed Google Scholar). tissue were using the or according to the were using and using the of this work, we on human liver during the early phases of transplantation. We ischemia from reperfusion to the molecular events during both Biopsies were collected as described characterize cytoskeleton changes in human liver during in and liver tissue using basal mostly at hepatocyte junctions well BC the of the a polymerization of actin observed the of hepatocytes (I10 and i.e. just before reperfusion (R0), with the junctions more at with an dilatation of BC The alteration of cell junction integrity during the reperfusion coincident with the of at the cell (R10 and of only at the hepatocyte BC, which and These are in with remodeling in human transplanted liver during the of IRI (5Cutrin J.C. Cantino D. Biasi F. Chiarpotto E. Salizzoni M. Andorno E. Massano G. Lanfranco G. Rizzetto M. Boveris A. Poli G. Reperfusion damage to the bile canaliculi in transplanted human liver.Hepatology. 1996; 24: 1053-1057Crossref PubMed Google Scholar, F. D. D. A. of ischemia-reperfusion on bile microfilaments in hepatocytes of human liver by PubMed Scopus Google Scholar). these the alteration of structure with the loss of hepatocyte junctions and BC integrity and hepatocyte bile and (5Cutrin J.C. Cantino D. Biasi F. Chiarpotto E. Salizzoni M. Andorno E. Massano G. Lanfranco G. Rizzetto M. Boveris A. Poli G. Reperfusion damage to the bile canaliculi in transplanted human liver.Hepatology. 1996; 24: 1053-1057Crossref PubMed Google Scholar). characterize the molecular targets involved in the IRI-induced described the expression pattern of cytoskeleton-related proteins during ischemia and reperfusion using an The expression of proteins related to this functional were The proteins were on the of in the These proteins were into functional and and and and protein which been reported to either or involved in cytoskeleton of two on of protein are in Fig. The were by and as described of the proteins and during both ischemia and reperfusion These proteins were therefore from The of the used to the expression patterns for the proteins a upon ischemia or reperfusion an of protein a a of protein The proteins expression expression patterns with a decreased expression during the and an expression during the reperfusion These that the major events during both phases of the transplantation involve the expression of cytoskeleton-related these changes in early phases of ischemia or the of major to both changes and expression The expression of a of proteins to in cytoskeleton and affected upon IRI of these are protein which are of the of protein We therefore that the molecular events in the dramatic observed in Fig. also major of liver at different of the transplantation. These in functional IRI-induced cytoskeletal and the related defect in BC structure and bile phosphoprotein on or and for mass spectrometry as described to the for both ischemia and reperfusion were phosphoprotein the most at these as by or in phosphorylation We proteins at and proteins at that were into functional according to and and of proteins as to the two conditions The most the two proteins involved in response and protein with a from ischemia to In the of proteins involved in and metabolism in reperfusion in The of the functional affected and the of the proteins for both conditions as by the of proteins in both This approach, although a enrichment of A. S. enrichment of intact proteins by 2005; PubMed Scopus Google Scholar), a in by mass spectrometry This more of the for proteins Y. T. of proteins as a for the PubMed Scopus Google Scholar, by mass spectrometry and to PubMed Scopus Google Scholar, H. A. the and Biol. 2003; PubMed Scopus Google Scholar). the functional related to liver and bile were proteins related to the cytoskeleton of the major functional of This to proteins at the ischemia and proteins at the reperfusion proteins were to both conditions and in and the of different mechanisms during the two phases of the transplantation. The proteins structural of the cytoskeleton and proteins involved in and of the proteins involved in cytoskeleton to the membrane and the related to BC S. M. K. Y. T. A. S. H. D. S. with loss of from bile PubMed Scopus Google and of actin cytoskeleton and some of these proteins are involved in the and maintenance of hepatocyte AJ, such as K. M. F. M. The protein is a S. A. 1991; PubMed Scopus Google Scholar) and the Cdc42/Rac1 IQGAP1 S. M. M. K. T. T. T. N. H. Y. S. K. of IQGAP1, a target of the and in regulation of 1998; PubMed Scopus Google Scholar, Z. IQGAP1 and Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). These proteins therefore critical targets IRI-induced actin cytoskeleton remodeling and alteration of the BC structure BC integrity on the maintenance of junctions (6Farquhar M.G. Palade G.E. Junctional complexes in various epithelia.J. Cell Biol. 1963; 17: 375-412Crossref PubMed Scopus (2132) Google cytoskeleton-related of phosphorylation and protein cytoplasmic protein protein protein protein membrane and in a new cytoskeleton-related of phosphorylation and cytoplasmic cytoplasmic protein protein protein cytoskeletal cytoskeletal cytoskeletal protein protein and in a new the targets using the proteomic approach, we the Cdc42/Rac1 IQGAP1 and proteins are to critical of hepatocyte response to IRI. IQGAP1 is a protein that actin and junction proteins are of cytoskeletal 2003; PubMed Scopus Google Scholar). of these has been reported to affected upon IRI M. H. K. M. Ischemia-reperfusion injury of the liver with to PubMed Scopus Google Scholar, and functional of apical membrane during 1999; PubMed Scopus Google Scholar, structure and of the tight 1998; PubMed Google Scholar, of the actin cytoskeleton in cell injury and PubMed Scopus Google Scholar) and that the maintenance of adherens junctions is critical for BC integrity in hepatocytes (6Farquhar M.G. Palade G.E. Junctional complexes in various epithelia.J. Cell Biol. 1963; 17: 375-412Crossref PubMed Scopus (2132) Google Scholar) that IQGAP1 a the IRI-induced actin remodeling and the related alteration of BC structure. the of has been this protein and an of with IQGAP1 and of the protein domains in IQGAP1 proteins are of cytoskeletal 2003; PubMed Scopus Google Scholar), that a to that of therefore to and characterize the of IQGAP1 and in IRI-induced actin remodeling and the related of BC structure. to IQGAP1 during the to as well as to IQGAP1 and were after 60 min of reperfusion The to a to both is in Fig. The of of IQGAP1 and at both ischemia and reperfusion by of proteins followed by and the of to the of proteins as using in phosphorylation and However, we IQGAP1 and expression in liver we that both during ischemia and decreased during reperfusion The of IQGAP1 and expression with the of the expression for the proteins in Fig. a for IQGAP1 and IQGAP1 and expression therefore a and for these two proteins in IRI-induced cytoskeletal reorganization. we the expression level of reported from the and we observed the pattern for during ischemia and a during Fig. and the of two or proteins has been with functional and IQGAP1 and been reported to this the potential of a and during IRI. In we in actin level during IRI Fig. actin a of that that the IRI-induced changes actin are most related to demonstrated in Fig. characterize proteins during IRI, the in situ liver of proteins upon IRI. biopsies were in the of with and were separated by IQGAP1 and a decreased with during this pattern upon reperfusion This with the of of these two proteins in upon ischemia followed by a upon reperfusion These that IQGAP1 and are to localization changes in the liver during on liver upon IRI revealed that IQGAP1 followed a to that of in hepatocytes, at the cell after of ischemia This upon reperfusion This with the fractionation that the of IQGAP1 with hepatocyte membrane observed by immunohistochemistry at of as is with used in fractionation In basal conditions and after of reperfusion IQGAP1 most associated with a to although as a of IQGAP1 S. S. A. The human protein a potential actin and with and Biol. 1996; PubMed Scopus Google Scholar), the protein in hepatocytes conditions The localization of to to in which IQGAP1 also In although fractionation upon IRI only IQGAP1 in hepatocytes to identify potential of actin remodeling in hepatocytes in with of BC, we therefore on IQGAP1 a in the alteration of BC we used both and human hepatoma These cell been used to study structural and functional properties of BC The formation of bile canaliculi in human hepatoma cell 1990; PubMed Scopus Google Scholar, D. membrane and bile membrane in PubMed Scopus Google Scholar). In these cells, intact BC are characterized by an actin at the IQGAP1 expression by two different and Z. A. IQGAP1 cell and Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), the of an actin at the apical the formation of BC, decreased and with the of IQGAP1 silencing observed IQGAP1 B and due to basal expression level Fig. These that IQGAP1 is involved in the formation of actin in both and cells, that IQGAP1 in an of BC in these hepatoma cell the functional of of IQGAP1 expression in the of the to IRI, and were to IRI as in Fig. cell IRI to IQGAP1 expression and with BC structure integrity IQGAP1 expression level the of intact a critical determinant in the of both and to IRI, that IQGAP1 a in hepatocyte resistance to IRI the maintenance of BC integrity. cytoskeleton remodeling and hepatocyte BC integrity were reported as primary targets of IRI and been to functional implications on graft recovery in of bile (5Cutrin J.C. Cantino D. Biasi F. Chiarpotto E. Salizzoni M. Andorno E. Massano G. Lanfranco G. Rizzetto M. Boveris A. Poli G. Reperfusion damage to the bile canaliculi in transplanted human liver.Hepatology. 1996; 24: 1053-1057Crossref PubMed Google Scholar, H. Yoshimura N. Kobayashi Y. Ochiai S. Matsuda T. Takamatsu T. Oka T. Microstructural changes of bile canaliculi in canine liver: the effect of cold ischemia-reperfusion in orthotopic liver transplantation.Transplant. Proc. 1998; 30: 3754-3757Crossref PubMed Scopus (10) Google Scholar, F. D. D. A. of ischemia-reperfusion on bile microfilaments in hepatocytes of human liver by PubMed Scopus Google Scholar). However, the molecular mechanisms underlying these remain characterized. improve the of these we an that to identify potential molecular targets of IRI using human liver to characterize the targets in liver tissue to in IRI-induced actin remodeling and to the in situ characterized targets in cell We demonstrated that IRI an cytoskeleton remodeling a expression pattern for a of and events are linked to protein we to characterize more the during both the and reperfusion We therefore intact using or followed by mass spectrometry to identify of the IRI-induced actin remodeling and the related loss of BC integrity. The by mass spectrometry of two to two conditions and by the of this However, we extracted from two we the patterns with the of proteins involved in response and protein during reperfusion can to the presence of blood in the biopsies and to the of protein the of reperfusion that we reported A. E. are during human liver Pathol. 2005; PubMed Scopus Google Scholar). the of proteins involved in and metabolism during ischemia of to and lipid due to upon blood flow D. and the of PubMed Scopus Google Scholar). we also cytoskeleton-related targets reported to involved in IRI. proteins involved in cytoskeleton to the membrane were at and been reported as targets of in models of IRI G. Y. F. in rat by PubMed Scopus Google Scholar, M. M. K. M. K. H. K. S. M. ischemia-reperfusion injury of the rat liver by and Full Text Full Text PDF PubMed Scopus Google Scholar). we the protein as a upon ischemia has been demonstrated to in liver upon IRI S. T. mediates of the in Full Text Full Text PDF PubMed Scopus Google Scholar). approach, of protein for identification as with to the of the identification of intact complexes A. S. enrichment of intact proteins by 2005; PubMed Scopus Google Scholar). at we PKA, and IQGAP1, of a that has been reported to involved in M. S. of with the 2005; PubMed Scopus Google Scholar). IQGAP1 and were by mass spectrometry after enrichment of protein complexes in the ischemia and the reperfusion liver respectively. of IQGAP1 and of the of However, we were to that both IQGAP1 and were at and as were by IQGAP1 and enrichment on or is most due to the that these two proteins are of However, although we demonstrated at both and a of both IQGAP1 and we the of the phosphorylation The of IQGAP1 phosphorylation is supported by that IQGAP1 is and in a A. S. enrichment of intact proteins by 2005; PubMed Scopus Google Scholar). has been reported that IQGAP1 phosphorylation on to to the loss of K. D. D. M. of IQGAP1 to a new of Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), an event that related to the IRI-induced alteration of BC structure. In addition, revealed an expression for IQGAP1 and and as well as for Fig. with the expression pattern for cytoskeleton-related proteins This a for these most of the in the us to the pattern of IQGAP1 and and during IRI. This due to the that of liver were and complexes been affected by this in situ in liver biopsies and in cell models allowed us to target potential to the IRI-induced actin remodeling and to maintenance of BC structure in led us to expression in and hepatocytes models to the of target proteins toward an of BC structure these mechanisms are well in these cell The formation of bile canaliculi in human hepatoma cell 1990; PubMed Scopus Google Scholar, S. formation in cultured Google Scholar). Using this functional validation we were to that IQGAP1 for the integrity of actin around using a of with a model of IRI ischemia in of and PubMed Google Scholar) combined with we that the integrity of these actin also critical for cell resistance to IRI in IQGAP1 in maintenance of BC can by the demonstrated of IQGAP1 for and proteins are of cytoskeletal 2003; PubMed Scopus Google Scholar). and been to the of and IQGAP1 G. T. T. S. K. Y. of by and proteins IQGAP1 and actin Cell Biol. PubMed Scopus Google Scholar, M. K. M. T. N. S. Y. K. of IQGAP1, an of in formation at of Biol. PubMed Scopus Google Scholar). we can in in IQGAP1 silencing the of and to required for the formation of BC This also to the of IQGAP1 with the and we observed in human and E. this only before ischemia and this with fractionation the of IQGAP1 with in presence in the with membrane during ischemia by a The biliary complications observed in a of BC during the of therefore due to an of IQGAP1 with the The maintenance of the hepatocyte bile properties on to and BC structure upon In summary, in this work, we were to identify and IQGAP1 as a potential target expression and on maintenance of BC structure. the potential of targeted functional proteomic using human to identify critical of conditions to human The of IQGAP1 in hepatocyte resistance to IRI the design of therapeutic in the preservation target IQGAP1 and graft recovery and transplantation We G. and S. as well as D. and for critical of the M. for at the and G. and for with with

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