Abstract
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.
Highlights
Ischemia-reperfusion injury (IRI) represents a major determinant of liver transplantation
Actin Cytoskeleton Is Subjected to Dramatic Changes during IRI in Hepatocytes—For the purpose of this work, we focused on human liver during the early phases of transplantation
The alteration of filamentous actin (F-actin) structure correlates with the loss of hepatocyte junctions and bile canaliculi (BC) integrity and significantly impacts hepatocyte bile secretion and function [5]
Summary
Ischemia-reperfusion injury (IRI) represents a major determinant of liver transplantation. 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 cytoskeletonrelated 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/Rac effector, as a potential regulator of actin cytoskeleton remodeling and maintenance of BC integrity. A major actin cytoskeleton alteration around the hepatocyte cell membrane has been reported in human liver in response to IRI and has been related to an impairment of BC contraction, tight junction permeability, and subsequent maintenance of a normal bile flow during the postoperative period [10, 11]. We combined a targeted functional proteomic analyses of human liver biopsies collected during different phases of the transplantation with functional validation both in
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