Accumulation of Mutant α1-Antitrypsin Z in the Endoplasmic Reticulum Activates Caspases-4 and -12, NFκB, and BAP31 but Not the Unfolded Protein Response

In α1-antitrypsin (α1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein α1AT, α1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells. It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease. One possible explanation is that the “susceptible” subgroup is unable to mount the appropriate protective cellular responses. Here we examined the effect of mutant α1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant α1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant α1AT. The results show that ER retention of polymerogenic mutant α1ATZ does not result in an unfolded protein response (UPR). The UPR can be induced in the presence of α1ATZ by tunicamycin excluding the possibility that the pathway has been disabled. In striking contrast, ER retention of nonpolymerogenic α1AT mutants does induce the UPR. These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures. Accumulation of mutant α1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFκB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic α1AT mutants. In α1-antitrypsin (α1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein α1AT, α1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells. It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease. One possible explanation is that the “susceptible” subgroup is unable to mount the appropriate protective cellular responses. Here we examined the effect of mutant α1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant α1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant α1AT. The results show that ER retention of polymerogenic mutant α1ATZ does not result in an unfolded protein response (UPR). The UPR can be induced in the presence of α1ATZ by tunicamycin excluding the possibility that the pathway has been disabled. In striking contrast, ER retention of nonpolymerogenic α1AT mutants does induce the UPR. These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures. Accumulation of mutant α1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFκB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic α1AT mutants. In the classical form of α1AT 2The abbreviations used are:α1ATα1-antitrypsinERendoplasmic reticulumUPRunfolded protein responseHTOHeLa Tet-Off cellsdoxdoxycyclineRTreverse transcriptionICintracellularECextracellularPIZZhomozygous for protease inhibitor type Z mutant 2The abbreviations used are:α1ATα1-antitrypsinERendoplasmic reticulumUPRunfolded protein responseHTOHeLa Tet-Off cellsdoxdoxycyclineRTreverse transcriptionICintracellularECextracellularPIZZhomozygous for protease inhibitor type Z mutant deficiency, the mutant α1ATZ molecule is retained in the endoplasmic reticulum (ER) of liver cells rather than secreted. There is an 85-90% reduction in α1AT levels in the blood and body fluids. This deficiency affects ∼1 in 1800 live births and results in the premature development of pulmonary emphysema in adult life. Chronic liver disease develops in a subgroup of homozygotes, usually becoming evident during childhood. There is an of in life. is by a of α1AT in the of the In contrast, liver injury to a the of mutant α1ATZ in the ER liver cells α1-antitrypsin endoplasmic reticulum unfolded protein response Tet-Off cells for protease inhibitor type Z mutant α1-antitrypsin endoplasmic reticulum unfolded protein response Tet-Off cells for protease inhibitor type Z mutant relatively is known the that the “susceptible” of individuals to liver disease the remainder of the from liver disease. using cell lines from individuals with liver disease for expression of α1ATZ, we that is a in ER of mutant α1ATZ in cells from individuals with liver disease These results that the response of cells to the of this mutant protein in the the a in the to liver disease that of mutant α1ATZ is by several pathways, including the as well as In the of protein of misfolded proteins in the ER the several ER response pathways can be the unfolded protein response is a pathway a of in response to the of proteins in the In to of ER and that the of the and for of ER to the protein an in the of proteins that the ER the and cellular There is a in of in such a way that specific can be The of the UPR is the ER activation results in in levels in the ER to as a protective that and the of the UPR of to the of proteins is responsible for in an ER is to to misfolded this results in and activation of the in the of in such a way that a and protein with is can activate the of a of UPR it to to the it is The of has for UPR including the and ER such as and of results in and activation of a that protein by of to ER retention of misfolded proteins been the activation of in cells with and in cells in the protein is retained in the This signaling to ER has been the ER pathway it to be distinct from the UPR. that of cells with that ER including and results in the activation of an that caspase-4 is activated in cells ER is an protein of the ER that to be in the retention of several proteins in the ER of can from the ER to is that ER of be to the that develops in α1AT deficiency with of mutant α1ATZ in the ER that has been to be with In to how cells respond to the retention of mutant α1ATZ in the we cell lines and transgenic mice with inducible expression of In to using cell lines transgenic mice with expression of α1ATZ in it is possible that cells that to retention of α1ATZ in the ER the the inducible to the response of cells to ER retention of α1ATZ the to and to the as it is possible to inducible expression in cell this type of is to that be by in and the cell with Here we used cell lines and mice to pathways activated by of mutant α1ATZ in the to α1AT was from to and from to caspase-12 from and by to was by to was from to caspase-4 and from liver from liver of a liver and from the liver of with α1AT deficiency liver to cell lines with expression of α1AT, we used the cell it does not the α1AT and the Tet-Off inducible it expression and of expression of Tet-Off cells from type α1AT, mutant α1ATZ, mutant α1AT and mutant α1AT of the in the from These used to the cells from for expression of α1AT by of cell and by of α1ATZ in cells of we used the was for expression of using the from of the from used to a with the and for expression of the This was The α1ATZ, α1AT α1AT and α1AT type used to the from the of in the way as the cell lines cell lines that type α1AT and the α1AT mutants been the of ER α1ATZ in we transgenic mice with liver-specific inducible expression of α1AT. used the of with expression of a This is for levels of expression and levels of of expression of the in we transgenic mice with type α1AT, mutant α1ATZ, mutant α1AT and mutant α1AT of the in as mice used an well and in by of that for the to mice from The The mice transgenic for a that the the of the that liver-specific with inducible liver-specific expression of the type α1AT and with inducible liver-specific expression of mutant α1ATZ Z and mice in the mice the and and cells to in with protease inhibitor and as cell of by a and the was on of a in in a for and from the The of was in to and was proteins with from the on and to for to α1AT in the cell cells in and α1AT in the cell cells in with protease inhibitor BAP31, cells in and of and of we used the of for of we the of In cells with tunicamycin used as a of protein as by the with and with and with the using in the and and with that the using the from with by used to the was by an was from the liver of Z and mice that was with from been to the was from mice that with tunicamycin by using the of tunicamycin in a was in several to mice with The mice and for with of to is inducible expression of α1ATZ in the cell for expression of the the is from the cell we examined of α1ATZ in this cell cells that been in the of for in with several of and of the α1AT was by in a with a of cells in with for several of α1ATZ in a with by There was of the of the that the effect on α1ATZ was cells that been in with for several The results show of α1ATZ from to and and results indicate that expression of α1ATZ in the cell is and In to the cell the that α1AT deficiency, it was with the cell in a In cells type α1AT is as a It is to a by of the with the of the in the the and and of the as the In the has retained with by of the and a of the that α1ATZ with the ER protein not These results that the cell is an appropriate for α1AT of α1ATZ on the to the UPR is activated the α1ATZ is induced in cells examined was from the The results show that the α1ATZ is from the is in of is in is in the from the One from is the presence of a that is with is from the This with α1ATZ, and the result that α1ATZ to expression is induced in cells. The was in of cell lines was from cells. of by was by The of the is the UPR is activated and and is the that is with mutant α1AT by The is not a a of the as by not including be to the of the of this The cells of the UPR as in by the of and the in the cells to a known for the lines with inducible expression of α1AT the and cell with and cell in the presence of for and for of from and cells in the of cells and cell to to for α1AT. results in the with of in the of in cell lines with inducible expression of α1AT mutants. and cell lines for in the presence of the cells for in the presence of The cells for and for of in the cell in several of The cell was for in of as the of The cells for and for α1AT and cells examined for of In of α1ATZ is of of but is in of that of not In is in of of in levels in cells of not In to the possibility that levels of expression of α1ATZ to activation of the we several of Tet-Off cells with inducible expression of This we used a of during and to expression of The with the of expression was In cells for α1AT. The results show that of α1ATZ is well in than for in levels of the is activation of with of the that to α1ATZ is in than cells and is α1ATZ expression is induced by of not of of was and α1ATZ the cell not In to the possibility that expression of α1ATZ in a cell to activation of the we a cell with inducible expression of In we this cell to for α1AT and The results show that expression of α1ATZ is is but is in levels of of in the of of α1AT and with α1AT the possibility that the UPR is induced in the liver in we levels of in transgenic of α1AT One is the that has expression of α1ATZ in the liver and The has been the was with The is the Z with inducible liver-specific expression of It was in the using this we the possibility that the UPR been in liver a In of liver from mice that α1AT expression in the Z and is by in the to levels in a The α1AT in the Z and has an to that in the α1AT was not in the that the does not the α1AT for in that is in levels of in the liver of mice as with and of in the the Z liver of from the we examined levels of in from with α1AT deficiency Here was for levels of of on the UPR in we examined the possibility that ER retention of α1ATZ in the inducible cell lines has a effect on the UPR. the and cell lines with a known of the UPR The results show that α1AT is induced is from the in The of α1AT is not type α1AT and mutant α1ATZ in the presence of in to a by the of of has effect on of but tunicamycin an in of the cell lines The in of induced by tunicamycin is in the of the in an in the cell The in is in the presence of that ER of α1ATZ does not a effect on the UPR and that it does not on of the UPR. cells in the presence of for to tunicamycin a in of in the presence of α1ATZ expression not The of UPR the was examined using the of was not induced by the of but was induced by tunicamycin in of by tunicamycin was in the presence of in cell that ER of α1ATZ does not activate the does not a effect on activation of the and does not on the activation of the UPR. One possible with is that by the tunicamycin an in the of α1ATZ in the ER retention In to this potential we examined the effect of tunicamycin and of the on the of α1ATZ in we the of that was for activation of the UPR in cells The results show that of is of we examined the effect of tunicamycin and on the of mutant α1ATZ in cells In the α1ATZ and with a of the by of the In the presence of the α1ATZ is retained of the and of the and of the α1ATZ is secreted. In the presence of is of the α1ATZ from of α1ATZ in the in the presence of as with indicate that tunicamycin and not an in of α1ATZ the ER α1ATZ retention of Accumulation of α1ATZ in the ER on to the possibility that is a relatively of UPR we examined the effect of ER α1ATZ on of is of the in the signaling pathway that the activation of used liver from the and Z and a for we examined the liver of mice that tunicamycin as a of was of we examined the effect of from the of the Z There was of of There was of of in the with expression of on the cells and on liver from the Z not show in expression of of the known of the UPR. and The results of that the UPR is not activated by ER α1ATZ of of α1AT on the we examined the possibility that the UPR is not activated by ER retention of mutant α1ATZ α1ATZ and In to we cells with inducible expression of α1AT and α1AT α1AT mutants that retained in the ER but not α1AT is a the of α1AT, and α1AT is a mutant with the that α1ATZ and the that α1AT In the and cell lines that we The results show that expression of α1AT and α1AT is is The α1AT and α1AT than type α1AT and mutant α1ATZ, of the In using and we that the α1AT and α1AT from α1ATZ in that not form retained in cells. In to that α1AT and α1AT not we cell from the cell lines to a of from from of and cells. The results for α1ATZ show a from to and and in the that the to α1ATZ in with including and and that the and in the α1ATZ of α1AT is a and of in the to of the to of the mutant α1AT in with and in a to to α1ATZ from and for type α1AT and mutant α1AT to results in cells not that α1AT and α1AT retained in the ER not form and that nonpolymerogenic mutants. These results that the cell is a for α1AT deficiency in that α1ATZ in results indicate that for the of α1ATZ, this mutant has a and with to that of the nonpolymerogenic α1AT mutants. we used cell lines to the possibility that ER retention of the nonpolymerogenic α1AT mutants the UPR The results show that is an in of the that in the cells that accumulate α1AT The in is in the of the The effect of α1AT on of is to that of tunicamycin in cells in the presence of The in of was on the of α1AT that in the ER cells in of for of was induced in an of from cells not These show that the UPR is not activated by α1ATZ but is activated by nonpolymerogenic α1AT mutants that retained in the that a with in the cells been from the that α1ATZ to a with in the cells has been and expression This is in to the α1AT molecule of from cells and cells with is to the with in This that the of the is with α1AT is used in and that a of this is with α1ATZ and α1AT These results the possibility that the in activation of the UPR by α1AT as with α1ATZ to of in but not the to of in but not the of ER of α1ATZ on and of cells with that including and has been to result in activation of an in cells examined the possibility that ER retention of α1ATZ results in activation of caspase-12 by using cell lines with inducible expression of α1ATZ as well as in the of Z was used to the that to activated The results show that caspase-12 is activated in cells but not in cells it a relatively for caspase-12 to was of but be in by of This was in the liver of Z mice was but was in in the liver of Z mice on the possibility that does not α1ATZ expression in the liver of the Z we of α1AT in the presence of but is of in the of in not was not in the on The for levels of results that levels not and the of the caspase-12 that activation of caspase-12 in response to ER of α1ATZ was on the of α1ATZ cells for in of and to for The results show that activation of caspase-12 is with the of to and and not that caspase-4 is the ER in we examined the possibility that of α1ATZ in the ER of cells and cells to for caspase-4 The results in and cells but to an in cells. The was evident in in cells that been in the possibility that is a of expression of α1ATZ the of in the is an in the of and from to of ER of α1ATZ on we examined the possibility that ER retention of α1ATZ activated the ER pathway by using cell lines and classical for The results show of for in cells by of The was with that of the It was not in cells The in was by to by but not by we examined the possibility that was activated in the liver of the Z and of α1AT The results show that is an in in the liver of the Z of is not in the as with the liver of the Z of is and the liver of the in the presence of activation in the Z liver and of in the of This to of α1ATZ in the liver not There is in the liver of the as with the In the in the liver of the Z of and in the liver of the to be specific in that by but not in that the is with in These results that ER retention of α1ATZ distinct cellular response ER retention of α1ATZ does not activate the results for the that the is distinct from the UPR. of ER of α1ATZ on of is an protein of the ER that to be in the ER retention of several proteins of can the ER and we that ER retention of α1ATZ is with we examined the possibility that it the of this we used cell lines and for of The results show an of the in and but the was in cells. The effect was of than that of the activation of the pathway by in the presence of and a relatively to It was by but be in by of of was on the of α1ATZ that in the This is in cells for in several of The is as the of is to and is the results show that activation of is a specific effect of the ER α1ATZ retention of ER of on of ER NFκB, and to the nonpolymerogenic mutants in the effect on ER and NFκB, we cell lines with inducible expression of α1AT and α1AT In the cell was in the presence of for The results show that of α1AT is induced and that this is with an in the of with activation of the UPR. In a of α1AT be in the cell in the presence of but with a in with that in the of not we used this cell to of α1AT to activation of caspase-12 The results show for the of caspase-12 in the cell in the presence of the that with in the cell we examined the cell for activation of There was for of BAP31, the in the of is in cells. we used the to is activated in In is in the cell in the of the of the cell in the of of but not caspase-12 was in the liver of the from to of not with results indicate that of a nonpolymerogenic α1AT mutant the UPR and the ER pathway but not ER BAP31, a profile of signaling pathways distinct from that activated by the polymerogenic mutant α1ATZ that does not activate the UPR but does activate the ER the ER and of disease and in the that cellular to mutant proteins that accumulate in the ER can the of of the cellular response pathways that activated by of mutant α1ATZ in the ER is to be liver injury and in α1AT deficiency to a and in the liver disease individuals by this disease the possibility that specific cellular response pathways cellular for to liver disease. that the response is activated and in this is by injury as well In activation of the UPR was not in the by levels of in cells by levels of and activation of the of is for expression of α1ATZ, it is possible that results be by that cell transgenic mice to the of a mutant protein in the In this we used cell lines and transgenic mice with inducible expression of mutant α1AT in to that the the response of cells that not to and to how cells respond to this mutant protein and in the transgenic mice in it is that the mutant protein is in the disease in we that expression is by and ER retention of mutant α1ATZ can be activated specific and in the inducible the to the of that to the of the liver disease. using inducible it was possible to that of mutant α1ATZ in the ER does not activate the UPR. This was by of and of the in a cell with inducible expression of In to the possibility that the UPR be activated by levels of expression of mutant α1ATZ, we a cell with levels of inducible and we that ER of this protein does not activate the UPR. we examined the possibility that the UPR be activated in cells of There was for levels of in a cell for inducible expression of levels of not α1ATZ expression was induced in the liver of a with liver-specific inducible expression of In to the possibility that levels of an of the we the effect of ER retention of α1ATZ on of in the of activation of the ER during the UPR. There was for of in the liver of the Z α1ATZ was induced in the liver of the examined the possibility that of α1ATZ in the ER has a effect on the UPR. There was in induced by tunicamycin in cells in the presence of α1ATZ with the of of results it that of α1ATZ is by the machinery of the but the is not of a in the of the response One possibility that levels of not a of the cellular of was not it was in with α1ATZ was by several as of in of in of expression profile of UPR of of and of of with α1ATZ in In to the possibility that it is the polymerogenic of α1ATZ that for the of activation of the we examined the effect of nonpolymerogenic mutants of α1AT that retained in the In we that the α1AT mutant and the α1AT the that α1ATZ as well as α1AT retained in the ER for as than α1ATZ not of mutants in cells by using a relatively and and in we not of mutants by in the cell lines as well as in cell of the expression of of mutants was with activation of the UPR. These indicate that the of activation of the UPR by ER retention of α1ATZ has to with the of α1ATZ to form in the of UPR activation α1ATZ in the ER be in several possible that the UPR is is from and by misfolded proteins It be that the of the UPR not activated by polymeric proteins is not by we that to a that with α1ATZ α1ATZ expression is and this to α1AT was in the using and and of we that to α1ATZ in cell lines in the way that it to α1AT and α1AT and can be in with that cell lines that α1ATZ, α1AT α1AT These results the possibility that α1ATZ to activate the UPR it does not from the of the UPR. explanation is that mutant α1ATZ in polymeric form does not as as the misfolded α1AT and α1AT of this using several of of the not this to this it is possible that of α1ATZ in specific of the ER with the of and in the remainder of the ER on by It is possible that to and it is in with α1ATZ but not in with α1AT explanation is that activation of the UPR of specific such as that for not accumulate in the this explanation it is with the that from the and ER retention of α1ATZ and α1AT signaling the in the of α1ATZ and α1AT is the presence in the ER of in the of activation proteins accumulate in the the ER pathway we the possibility that it was activated in response to the of α1ATZ in the The results that was activated in cells α1ATZ expression is The that ER of α1ATZ but not the UPR for the that the ER pathway is distinct from the UPR The ER pathway has for injury in NFκB, of α1ATZ in the ER of liver cells and cells the that in the liver and of by the In a we that of α1ATZ in the ER was with injury and and that activation of be in the liver of mice and The that α1ATZ in the ER of a possible for the effect on the that ER of α1ATZ activate caspase-12 in the and caspase-4 in the human, we that activated in the liver in α1AT deficiency by the ER and This that the can be activated in the of the UPR that activation of the pathway is distinct from activation of the UPR. that the UPR can be activated caspase-4 caspase-12 of α1ATZ in the ER to activation of ER is in the liver of of and with α1AT deficiency This that the cellular response to ER of α1ATZ the expression of that of as for the inhibitor protein that several of the protein in cells from and that proteins can by a of It is that of by the ER has been in the activation of by the ER pathway and in the effect of ER on including the of on it is that a distinct profile of signaling pathways is activated in response to of the polymerogenic mutant α1ATZ as with of the nonpolymerogenic mutants α1AT and Accumulation of the Z mutant to activation of ER NFκB, and but not the of the and mutants to activation of the UPR and but not ER the show that the by cells respond to the of proteins in the ER be we we not the of response several of the profile with the Z mutant with on in the of α1AT deficiency These show that that accumulate α1ATZ, relatively in but not of cell the in the of the liver of the is to the of that the that accumulate α1ATZ but not and responsible for in that by with of α1ATZ, that a In we that this is the of the for that is in α1AT deficiency activation of the UPR to cell the of activation in response to of α1ATZ be of the of to cell and in the of in a in the of as it has been with and induced with