Hepatocyte-specific c-Met Deletion Disrupts Redox Homeostasis and Sensitizes to Fas-mediated Apoptosis

Abstract

The hepatocyte growth factor and its receptor c-Met direct a pleiotropic signal transduction pathway that controls cell survival. We previously demonstrated that mice lacking c-Met (Met-KO) in hepatocytes were hypersensitive to Fas-induced liver injury. In this study, we used primary hepatocytes isolated from Met-KO and control (Cre-Ctrl) mice to address more directly the protective effects of c-Met signaling. Loss of c-Met function increased sensitivity to Fas-mediated apoptosis. Hepatocyte growth factor suppressed apoptosis in Cre-Ctrl but not Met-KO hepatocytes concurrently with up-regulation of NF-κB and major antiapoptotic proteins Bcl-2 and Bcl-xL. Intriguingly, Met-KO hepatocytes exhibited intrinsic activation of NF-κBas well as Bcl-2 and Bcl-xL. Furthermore, unchallenged Met-KO cells displayed oxidative stress as evidenced by overproduction of reactive oxygen species, which was associated with greater NADPH and Rac1 activities, was blocked by the known NADPH oxidase inhibitors, and was paralleled by increased lipid peroxidation and reduced glutathione (GSH) content. N-Acetylcysteine, an antioxidant and GSH reduced cell the the protective effects of in Met-KO In of c-Met in hepatocytes in which the increased sensitivity to Fas-induced apoptosis and up-regulation of NF-κB signaling. that c-Met a factor in the of reactive oxygen The hepatocyte growth factor and its receptor c-Met direct a pleiotropic signal transduction pathway that controls cell survival. We previously demonstrated that mice lacking c-Met (Met-KO) in hepatocytes were hypersensitive to Fas-induced liver injury. In this study, we used primary hepatocytes isolated from Met-KO and control (Cre-Ctrl) mice to address more directly the protective effects of c-Met signaling. Loss of c-Met function increased sensitivity to Fas-mediated apoptosis. Hepatocyte growth factor suppressed apoptosis in Cre-Ctrl but not Met-KO hepatocytes concurrently with up-regulation of NF-κB and major antiapoptotic proteins Bcl-2 and Bcl-xL. Intriguingly, Met-KO hepatocytes exhibited intrinsic activation of NF-κBas well as Bcl-2 and Bcl-xL. Furthermore, unchallenged Met-KO cells displayed oxidative stress as evidenced by overproduction of reactive oxygen species, which was associated with greater NADPH and Rac1 activities, was blocked by the known NADPH oxidase inhibitors, and was paralleled by increased lipid peroxidation and reduced glutathione (GSH) content. N-Acetylcysteine, an antioxidant and GSH reduced cell the the protective effects of in Met-KO In of c-Met in hepatocytes in which the increased sensitivity to Fas-induced apoptosis and up-regulation of NF-κB signaling. that c-Met a factor in the of reactive oxygen known as a of the receptor that of a by activation of in the and to to hepatocyte with growth factor used hepatocyte growth factor factor reactive oxygen used hepatocyte growth factor factor reactive oxygen a and that was as a effects of by a receptor c-Met activation of The the pathway that the that hepatocyte survival. of of apoptosis of hepatocytes of that the of oxidative stress a in Fas-mediated apoptosis to that antioxidant the of reactive oxygen was to the and hepatocyte apoptosis by that function as an antioxidant factor to oxidative cell of of a major of and Furthermore, a of cell in and to in the control of oxidative stress The to the activation of the factor which in of antioxidant and antiapoptotic with we demonstrated that hepatocytes increased of a of in the c-Met to control of previously demonstrated that mice lacking the c-Met receptor were hypersensitive to Fas-induced liver and from with of that not the of mice was to that Fas-induced apoptosis by primary hepatocyte from c-Met The that c-Met hepatocytes to Fas-mediated apoptosis increased and GSH was from and were from was from was from and were from were from Hepatocyte and and control (Cre-Ctrl) mice were previously were in and in with the the and of were isolated from to mice by a by in a as previously and were as by were cells in in the hepatocyte growth with a the was to a hepatocyte growth The cells were to and as cells were with with of and NF-κB and of cells were by with in as previously cells with were in from cells were and the of apoptosis was as a of the cell of was by the to were with and with with and with The of the was The cell was as the to and proteins were isolated from cells with and to the of of were to and with and were with the of the primary were with was demonstrated by the with were with in and were in were and the was and The was by NF-κB was a as a was with and and The in of of and The were In of was in the of the were with of the of by oxygen were the in were with in were with and the were a by and was by as by and with cells were in of and of the in was to and and the was to and were in the by the of of in were the of the and a were by peroxidation was by the of as by and of NADPH Cre-Ctrl and Met-KO cells were by and in NADPH oxidase was as by were with NADPH was by the in NADPH oxidase was by the of of NADPH by that was the of cells with was used to content. were as of NADPH of of Rac1 was Rac1 activation to the The used were used to Rac1 by Rac1 as were were of c-Met of to with in hepatocytes isolated from c-Met mice (Met-KO) were more to cell as by and and in Met-KO but cell in Cre-Ctrl hepatocytes The of was of and with a in activation and of in hepatocytes cell NF-κB and in the the increased of apoptosis in we the activation of known to in of and was in control hepatocytes and was in Met-KO primary hepatocytes activation of NF-κB and antiapoptotic and in Cre-Ctrl but not Met-KO cell from Cre-Ctrl and Met-KO with and were with and and The of was by to The of in was as the of NF-κB in Cre-Ctrl and Met-KO hepatocytes with the The of with the of cell from Cre-Ctrl and Met-KO hepatocytes with and were with of antiapoptotic proteins in Cre-Ctrl and Met-KO Cre-Ctrl and Met-KO hepatocytes were and with the cell were with and of in and we which of a major in the protective of in this we used and of and and we the activation of factor that NF-κB hepatocytes apoptosis of proteins In control increased NF-κB that in with a and was and In with the activation of NF-κB was associated with up-regulation of the Bcl-2 The of Bcl-2 and were increased in cell from Cre-Ctrl hepatocytes of and a The of which to the of hepatocytes was and increased to a the of of the pathway by NF-κB of Bcl-2 and and apoptosis in Cre-Ctrl hepatocytes The of were by in Met-KO cells that activation of the pathway and not of proteins with the NF-κB was in Bcl-2 and and apoptosis in cell and of and NF-κB activation cell not activation the antiapoptotic effects of in Cre-Ctrl of NF-κB Cre-Ctrl hepatocytes were with and by were in of antiapoptotic proteins in Cre-Ctrl were with and by of of and NF-κB of apoptosis. of cells were with by with and the in was by cells the were from from of antiapoptotic proteins in Met-KO were with and by of in and GSH in Met-KO hepatocytes sensitivity to apoptosis. Cre-Ctrl and Met-KO hepatocytes were with with by was by cells the were from from Cre-Ctrl cells with Met-KO with of NF-κB was from Cre-Ctrl cells and Met-KO cells and Met-KO cells with cell in Cre-Ctrl and Met-KO hepatocytes with as by of Cre-Ctrl of unchallenged hepatocytes exhibited greater NF-κB in with control cells of hepatocytes of Bcl-2 and with NF-κB and of activation of NF-κB and was to hepatocytes well known that NF-κB oxidative stress we of in the oxidative stress in primary hepatocytes We the of c-Met function to the We that was by as by the In the of a of oxidative were increased in the were reduced as with the control and the proteins of the and were in c-Met as an to the increased oxidative stress hepatocytes to oxidative Cre-Ctrl and Met-KO cells were with the Met-KO cells were with and and with lipid peroxidation was by in Cre-Ctrl and Met-KO hepatocytes with was used as the of of reduced glutathione (GSH) to glutathione by in Cre-Ctrl and Met-KO Cre-Ctrl hepatocytes were with as the of of antioxidant cell from Cre-Ctrl and Met-KO hepatocytes with and were with and of NADPH oxidase of cell and as were with Rac1 activation was used to the from Cre-Ctrl and Met-KO cell was Rac1 to the of in to by the that signal was blocked by known NADPH oxidase as and with oxidase as well as with not to the of the The of increased in Met-KO cells was associated with a greater NADPH oxidase as well as up-regulation of a of the NADPH oxidase in hepatocytes and In with Met-KO primary hepatocytes exhibited a in the of Rac1 in the and activation of NADPH oxidase In hepatocytes displayed increased of not to of a of the NADPH oxidase that of c-Met in hepatocytes of the increased of the of GSH in in we the of GSH in the of Met-KO cells to apoptosis. this hepatocytes were in the of an antioxidant and GSH an of the in the glutathione The that a protective cell The of Met-KO cells apoptosis was reduced as with the with and apoptosis in Met-KO cells and Cre-Ctrl a GSH in Fas-induced of NF-κB Met-KO that NF-κB activation in Met-KO cells of increased oxidative we hepatocytes with the antioxidant with to an in with blocked and the of NF-κB to the in controls that GSH reduced we cells with a NF-κB that NF-κB to reduced of Met-KO hepatocytes with a in the of NF-κB and In of Cre-Ctrl cell that NF-κB activation was to Met-KO hepatocytes this study, we used and to address the of cell survival. primary hepatocytes from mice to the apoptosis by c-Met receptor with the c-Met the of hepatocytes to Fas-induced a the in the of the receptor of the effects of a c-Met and receptor in c-Met that blocked the with an of activation of in and hepatocytes well and activation of as well as and In with we the pathway as a of which NF-κB activation and of the Bcl-2 proteins as Bcl-2 and Bcl-xL. of pathway by the NF-κB up-regulation of Bcl-2 and as well as of cell of and and with NF-κB activation cell and NF-κB activation with up-regulation of Bcl-2 and of a in of hepatocytes of Bcl-2 and in a and a NF-κB in in cells NF-κB activation as an hepatocytes more to apoptosis the in the of NF-κB activation and up-regulation of antiapoptotic well known that in and in NF-κB activation as a the cell a of oxidative which the sensitivity to apoptosis with hepatocytes overproduction of lipid and up-regulation of antioxidant proteins and In the were in Met-KO cells In the GSH and the GSH by a in GSH that NF-κB directly of of in the liver we a in GSH in Cre-Ctrl cells with the of GSH in of we used the of the and a in the of the GSH reduced cell in the and apoptosis in Met-KO and Cre-Ctrl In the of NF-κB in Met-KO cells to the in Cre-Ctrl cells oxidative stress in the NF-κB Furthermore, NF-κB with a reduced of Met-KO not the in Cre-Ctrl the that NF-κB in cells was and to the effects of but not the with increased oxidative In with we and up-regulation of factor factor and a of of of c-Met of apoptosis in cells the antioxidant function of in a of oxidative by in a of cell with that in primary hepatocytes a by the GSH and the of a of the NADPH oxidase in hepatocytes The NADPH oxidase as a major of with Furthermore, a of a as a direct of in cells with this to of Rac1 in a of oxidative stress and apoptosis In with Met-KO hepatocytes exhibited in with Rac1 and NADPH to the that c-Met and the that activation of and Rac1 in the oxidative stress by c-Met of c-Met in primary hepatocytes that in a of of The a the of cells to Fas-mediated apoptosis and that c-Met a factor in the of known as a of the receptor that of a by activation of in the and to to hepatocyte with Hepatocyte growth factor used hepatocyte growth factor factor reactive oxygen used hepatocyte growth factor factor reactive oxygen a and that was as a effects of by a receptor c-Met activation of The the pathway that the that hepatocyte survival. of of apoptosis of hepatocytes of that the of oxidative stress a in Fas-mediated apoptosis to that antioxidant the of reactive oxygen was to the and hepatocyte apoptosis by that function as an antioxidant factor to oxidative cell of of a major of and Furthermore, a of cell in and to in the control of oxidative stress The to the activation of the factor which in of antioxidant and antiapoptotic with we demonstrated that hepatocytes increased of a of in the c-Met to control of We previously demonstrated that mice lacking the c-Met receptor were hypersensitive to Fas-induced liver and from with of that not the of mice was to that Fas-induced apoptosis by primary hepatocyte from c-Met The that c-Met hepatocytes to Fas-mediated apoptosis increased and GSH was from and were from was from was from and were from were from Hepatocyte and and control (Cre-Ctrl) mice were previously were in and in with the the and of were isolated from to mice by a by in a as previously and were as by were cells in in the hepatocyte growth with a the was to a hepatocyte growth The cells were to and as cells were with with of and NF-κB and of cells were by with in as previously cells with were in from cells were and the of apoptosis was as a of the cell of was by the to were with and with with and with The of the was The cell was as the to and proteins were isolated from cells with and to the of of were to and with and were with the of the primary were with was demonstrated by the with were with in and were in were and the was and The was by NF-κB was a as a was with and and The in of of and The were In of was in the of the were with of the of by oxygen were the in were with in were with and the were a by and was by as by and with cells were in of and of the in was to and and the was to and were in the by the of of in were the of the and a were by peroxidation was by the of as by and of NADPH Cre-Ctrl and Met-KO cells were by and in NADPH oxidase was as by were with NADPH was by the in NADPH oxidase was by the of of NADPH by that was the of cells with was used to content. were as of NADPH of of Rac1 was Rac1 activation to the The used were used to Rac1 by Rac1 as were were was from and were from was from was from and were from were from Hepatocyte and and control (Cre-Ctrl) mice were previously were in and in with the the and of were isolated from to mice by a by in a as previously and were as by were cells in in the hepatocyte growth with a the was to a hepatocyte growth The cells were to and as cells were with with of and NF-κB and of cells were by with in as previously cells with were in from cells were and the of apoptosis was as a of the cell of was by the to were with and with with and with The of the was The cell was as the to and proteins were isolated from cells with and to the of of were to and with and were with the of the primary were with was demonstrated by the with were with in and were in were and the was and The was by NF-κB was a as a was with and and The in of of and The were In of was in the of the were with of the of by oxygen were the in were with in were with and the were a GSH by and was by as by and with cells were in of and of the in was to and and the was to and were in the by the of of in were the of the and a were by peroxidation was by the of as by and of NADPH Cre-Ctrl and Met-KO cells were by and in NADPH oxidase was as by were with NADPH was by the in NADPH oxidase was by the of of NADPH by that was the of cells with was used to content. were as of NADPH of Rac1 of Rac1 was Rac1 activation to the The used were used to Rac1 by Rac1 as were were of c-Met of to with in hepatocytes isolated from c-Met mice (Met-KO) were more to cell as by and and in Met-KO but cell in Cre-Ctrl hepatocytes The of was of and with a in activation and of in hepatocytes cell NF-κB and in the the increased of apoptosis in we the activation of known to in of and was in control hepatocytes and was in Met-KO we which of a major in the protective of in this we used and of and and we the activation of factor that NF-κB hepatocytes apoptosis of proteins In control increased NF-κB that in with a and was and In with the activation of NF-κB was associated with up-regulation of the Bcl-2 The of Bcl-2 and were increased in cell from Cre-Ctrl hepatocytes of and a The of which to the of hepatocytes was and increased to a the of of the pathway by NF-κB of Bcl-2 and and apoptosis in Cre-Ctrl hepatocytes The of were by in Met-KO cells that activation of the pathway and not of proteins with the NF-κB was in Bcl-2 and and apoptosis in cell and of and NF-κB activation cell not activation the antiapoptotic effects of in Cre-Ctrl of NF-κB Cre-Ctrl hepatocytes were with and by were in of antiapoptotic proteins in Cre-Ctrl were with and by of of and NF-κB of apoptosis. of cells were with by with and the in was by cells the were from from of antiapoptotic proteins in Met-KO were with and by of in and GSH in Met-KO hepatocytes sensitivity to apoptosis. Cre-Ctrl and Met-KO hepatocytes were with with by was by cells the were from from Cre-Ctrl cells with Met-KO with of NF-κB was from Cre-Ctrl cells and Met-KO cells and Met-KO cells with cell in Cre-Ctrl and Met-KO hepatocytes with as by of Cre-Ctrl of unchallenged hepatocytes exhibited greater NF-κB in with control cells of hepatocytes of Bcl-2 and with NF-κB and of activation of NF-κB and was to hepatocytes well known that NF-κB oxidative stress we of in the oxidative stress in primary hepatocytes We the of c-Met function to the We that was by as by the In the of a of oxidative were increased in the were reduced as with the control and the proteins of the and were in c-Met as an to the increased oxidative stress hepatocytes to oxidative Cre-Ctrl and Met-KO cells were with the Met-KO cells were with and and with lipid peroxidation was by in Cre-Ctrl and Met-KO hepatocytes with was used as the of of reduced glutathione (GSH) to glutathione by in Cre-Ctrl and Met-KO Cre-Ctrl hepatocytes were with as the of of antioxidant cell from Cre-Ctrl and Met-KO hepatocytes with and were with and of NADPH oxidase of cell and as were with Rac1 activation was used to the from Cre-Ctrl and Met-KO cell was Rac1 to the of in to by the that signal was blocked by known NADPH oxidase as and with oxidase as well as with not to the of the The of increased in Met-KO cells was associated with a greater NADPH oxidase as well as up-regulation of a of the NADPH oxidase in hepatocytes and In with Met-KO primary hepatocytes exhibited a in the of Rac1 in the and activation of NADPH oxidase In hepatocytes displayed increased of not to of a of the NADPH oxidase that of c-Met in hepatocytes of the increased of the of GSH in in we the of GSH in the of Met-KO cells to apoptosis. this hepatocytes were in the of an antioxidant and GSH an of the in the glutathione The that a protective cell The of Met-KO cells apoptosis was reduced as with the with and apoptosis in Met-KO cells and Cre-Ctrl a GSH in Fas-induced of NF-κB Met-KO that NF-κB activation in Met-KO cells of increased oxidative we hepatocytes with the antioxidant with to an in with blocked and the of NF-κB to the in controls that GSH reduced we cells with a NF-κB that NF-κB to reduced of Met-KO hepatocytes with a in the of NF-κB and In of Cre-Ctrl cell that NF-κB activation was to Met-KO hepatocytes of c-Met of to with in hepatocytes isolated from c-Met mice (Met-KO) were more to cell as by and and in Met-KO but cell in Cre-Ctrl hepatocytes The of was of and with a in activation and of in hepatocytes cell NF-κB and in the the increased of apoptosis in we the activation of known to in of and was in control hepatocytes and was in Met-KO we which of a major in the protective of in this we used and of and and we the activation of factor that NF-κB hepatocytes apoptosis of proteins In control increased NF-κB that in with a and was and In with the activation of NF-κB was associated with up-regulation of the Bcl-2 The of Bcl-2 and were increased in cell from Cre-Ctrl hepatocytes of and a The of which to the of hepatocytes was and increased to a the of of the pathway by NF-κB of Bcl-2 and and apoptosis in Cre-Ctrl hepatocytes The of were by in Met-KO cells that activation of the pathway and not of proteins with the NF-κB was in Bcl-2 and and apoptosis in cell and of and NF-κB activation cell not Met-KO of unchallenged hepatocytes exhibited greater NF-κB in with control cells of hepatocytes of Bcl-2 and with NF-κB and of activation of NF-κB and was to hepatocytes apoptosis. well known that NF-κB oxidative stress we of in the oxidative stress in primary hepatocytes We the of c-Met function to the We that was by as by the In the of a of oxidative were increased in the were reduced as with the control and the proteins of the and were in c-Met as an to the increased oxidative stress in to by the that signal was blocked by known NADPH oxidase as and with oxidase as well as with not to the of the The of increased in Met-KO cells was associated with a greater NADPH oxidase as well as up-regulation of a of the NADPH oxidase in hepatocytes and In with Met-KO primary hepatocytes exhibited a in the of Rac1 in the and activation of NADPH oxidase In hepatocytes displayed increased of not to of a of the NADPH oxidase that of c-Met in hepatocytes of the increased of GSH the of GSH in in we the of GSH in the of Met-KO cells to apoptosis. this hepatocytes were in the of an antioxidant and GSH an of the in the glutathione The that a protective cell The of Met-KO cells apoptosis was reduced as with the with and apoptosis in Met-KO cells and Cre-Ctrl a GSH in Fas-induced apoptosis. of NF-κB Met-KO that NF-κB activation in Met-KO cells of increased oxidative we hepatocytes with the antioxidant with to an in with blocked and the of NF-κB to the in controls that GSH reduced we cells with a NF-κB that NF-κB to reduced of Met-KO hepatocytes with a in the of NF-κB and In of Cre-Ctrl cell that NF-κB activation was to Met-KO hepatocytes this study, we used and to address the of cell survival. primary hepatocytes from mice to the apoptosis by c-Met receptor with the c-Met the of hepatocytes to Fas-induced a the in the of the receptor of the effects of a c-Met and receptor in c-Met that blocked the with an of activation of in and hepatocytes well and activation of as well as and In with we the pathway as a of which NF-κB activation and of the Bcl-2 proteins as Bcl-2 and Bcl-xL. of pathway by the NF-κB up-regulation of Bcl-2 and as well as of cell of and and with NF-κB activation cell and NF-κB activation with up-regulation of Bcl-2 and of a in of hepatocytes of Bcl-2 and in a and a NF-κB in in cells NF-κB activation as an hepatocytes more to apoptosis the in the of NF-κB activation and up-regulation of antiapoptotic well known that in and in NF-κB activation as a the cell a of oxidative which the sensitivity to apoptosis with hepatocytes overproduction of lipid and up-regulation of antioxidant proteins and In the were in Met-KO cells In the GSH and the GSH by a in GSH that NF-κB directly of of in the liver we a in GSH in Cre-Ctrl cells with the of GSH in of we used the of the and a in the of the GSH reduced cell in the and apoptosis in Met-KO and Cre-Ctrl In the of NF-κB in Met-KO cells to the in Cre-Ctrl cells oxidative stress in the NF-κB Furthermore, NF-κB with a reduced of Met-KO not the in Cre-Ctrl the that NF-κB in cells was and to the effects of but not the with increased oxidative In with we and up-regulation of factor factor and a of of of c-Met of apoptosis in cells the antioxidant function of in a of oxidative by in a of cell with that in primary hepatocytes a by the GSH and the of a of the NADPH oxidase in hepatocytes The NADPH oxidase as a major of with Furthermore, a of a as a direct of in cells with this to of Rac1 in a of oxidative stress and apoptosis In with Met-KO hepatocytes exhibited in with Rac1 and NADPH to the that c-Met and the that activation of and Rac1 in the oxidative stress by c-Met of c-Met in primary hepatocytes that in a of of The a the of cells to Fas-mediated apoptosis and that c-Met a factor in the of In this study, we used and to address the of cell survival. primary hepatocytes from mice to the apoptosis by c-Met receptor with the c-Met the of hepatocytes to Fas-induced a the in the of the receptor of the effects of a c-Met and receptor in c-Met that blocked the with an of activation The of in and hepatocytes well and activation of as well as and In with we the pathway as a of which NF-κB activation and of the Bcl-2 proteins as Bcl-2 and Bcl-xL. of pathway by the NF-κB up-regulation of Bcl-2 and as well as of cell of and and with NF-κB activation cell and NF-κB activation with up-regulation of Bcl-2 and of a in of hepatocytes of Bcl-2 and in a and a NF-κB in in cells NF-κB activation as an Intriguingly, hepatocytes more to apoptosis the in the of NF-κB activation and up-regulation of antiapoptotic well known that in and in NF-κB activation as a the cell a of oxidative which the sensitivity to apoptosis with hepatocytes overproduction of lipid and up-regulation of antioxidant proteins and In the were in Met-KO cells In the GSH and the GSH by a in GSH that NF-κB directly of of in the liver we a in GSH in Cre-Ctrl cells with the of GSH in of we used the of the and a in the of the GSH reduced cell in the and apoptosis in Met-KO and Cre-Ctrl In the of NF-κB in Met-KO cells to the in Cre-Ctrl cells oxidative stress in the NF-κB Furthermore, NF-κB with a reduced of Met-KO not the in Cre-Ctrl the that NF-κB in cells was and to the effects of but not the with increased oxidative In with we and up-regulation of factor factor and a of of of c-Met of apoptosis in cells the antioxidant function of in a of oxidative by in a of cell with that in primary hepatocytes a by the GSH and the of a of the NADPH oxidase in hepatocytes The NADPH oxidase as a major of with Furthermore, a of a as a direct of in cells with this to of Rac1 in a of oxidative stress and apoptosis In with Met-KO hepatocytes exhibited in with Rac1 and NADPH to the that c-Met and the that activation of and Rac1 in the oxidative stress by c-Met In of c-Met in primary hepatocytes that in a of of The a the of cells to Fas-mediated apoptosis and that c-Met a factor in the of We and with and