posttranslational modifications play a key role in the regulation of  protein activity , localization , and stability , as well as in the control of protein - protein interactions . among them , ubiquitylation counts as one of the most prominent and best - characterized modifications .
ubiquitin is a small protein that can be covalently attached to lysine residues of other proteins through the interplay of different ubiquitin - activating ( e1 ) and -conjugating ( e2 ) enzymes and ubiquitin ligases ( e3 ) ( hershko and ciechanover , 1998 ) .
the analysis of mice lacking various components of the ubiquitylation machinery highlighted the importance of ubiquitylation in several cellular and physiological processes , spanning from autophagy , transcription factor regulation , and dna repair to the control of cell cycle and cell death ( bergink and jentsch , 2009 , bernassola et  al . , 2010 , hershko and ciechanover , 1998 , mukhopadhyay and riezman , 2007 ) , which in turn can affect complex processes such as the immune response ( bhoj and chen , 2009 , corn and vucic , 2014 ) , the maintenance of tissue homeostasis ( kumari et  al .
, 2014 ) , and tumorigenesis ( hoeller and dikic , 2009 , paolino et  al . , 2014 ) .
life or death cell fate decisions are critical for development , against infectious diseases , or cancer formation .
apoptosis was considered the sole form of programmed cell death during  development or disease ( elmore , 2007 ) . however , besides apoptosis , alternative programmed necrosis or necroptosis can be induced by death receptors ( vandenabeele et  al . ,
survival , apoptosis , and necroptosis can all be triggered by the same cell - surface receptors .
there has been intense interest in how these fate decisions are made by identifying the gatekeepers of apoptotic versus necroptotic cell fate and the signaling cascades involved ( christofferson and yuan , 2010 ) .
one of the most paradigmatic signaling pathways that regulates cellular fate is triggered by the tumor necrosis factor receptor 1 ( tnfr1 ) .
tnfr1 engagement elicits cell survival pathways via nf-b induction ( beg and baltimore , 1996 , van antwerp et  al .
when nf-b activation is inhibited , tnf stimulation triggers caspase-8-mediated apoptosis via a  defined death effector complex ( micheau and tschopp , 2003 , wang et  al . , 1996 ) .
in specific conditions , blockade of both nf-b and apoptosis may result in programmed necrotic cell death ( cho et  al . , 2009 , he et  al . , 2009 ,
necroptosis is mediated via the rip1/rip3/mlkl kinase pathway and recently has been implicated in normal development and in pathogenesis of diseases , including ischemic injury , neurodegeneration , and intestinal inflammation ( duprez et  al .
, 2011 , kaiser et  al . , 2011 , kang et  al . , 2013 ,
2011 ) . hect domain and ankyrin repeat - containing e3 ligase 1 ( hace1 ) is an e3 ligase initially identified in the context of sporadic wilms tumor ( anglesio et  al . , 2004 ) .
a number of reports showed that hace1 is downregulated in various types of tumors ( anglesio et  al .
, 2004 , hibi et  al . , 2008 , kk et  al . , 2013 , zhang et  al . , 2007 ) , and
a previous study from our group highlighted the important function of hace1 as a tumor suppressor : mice lacking hace1 showed an increased propensity to develop spontaneous tumors as well as a reduced survival in different cancer models ( zhang et  al . , 2007 ) .
in addition to this function , hace1 has been implicated in golgi membrane dynamics ( tang et  al . , 2011 ) ,
2014 ) , and heart failure following cardiac injury ( zhang et  al . , 2014 ) . however , the complexity of the molecular and cellular processes potentially regulated by hace1 is still largely unexplored . here
genetic inactivation of hace1 impairs nf-b  activation and apoptosis downstream of tnfr1 , whereas rip3-regulated necroptotic cell fate is not affected .
importantly , we found that hace1 mutant mice exhibit enhanced intestinal inflammation and colon cancer following epithelial injury , which can be rescued by simultaneous genetic inactivation of either rip3 or tnfr1 .
to identify pathways relevant to hace1 function , we scanned the signaling pathways downstream of multiple ligand / receptor pairs , using hace1 mutant ( hace1 ) mouse embryonic fibroblasts ( mefs ) .
intriguingly , hace1 mefs stimulated with tnf showed compromised phosphorylation and incomplete degradation of the nf-b inhibitor ib. as a consequence , tnf - induced phosphorylation and activation of the nf-b subunit p65 was strongly reduced in hace1 mefs ( figure  1a ) . impaired nf-b activation
was confirmed using an  electrophoretic mobility shift assay ( emsa ) filter plate assay ( figure  1b ) .
re - expression of wild - type ( wt ) hace1 in hace1 mefs , but not that of the e3 ligase inactive hace1 c876s mutant ( anglesio et  al . ,
2004 ) , restored tnf - induced p65 nf-b activation ( figure  1b ; figure  s1a ) . moreover , tnf - induced nf-b - dependent expression of interleukin-6 ( il-6 ) ( bollrath and greten , 2009 ) also was markedly inhibited in hace1-deficient mefs ( figure  1c ) .
in addition to defective nf-b induction , tnfr1-induced jnk and consequent c - jun phosphorylation were markedly reduced in hace1 mefs , a phenomenon that was accompanied by increased and prolonged erk1/2 and p38-mapk phosphorylation ( figure  1a ) .
tnfr1 and nf-b are essential for the in  vivo clearance of the intracellular pathogen listeria monocytogenes ( pfeffer et  al . , 1993 ,
1995 ) . considering the observed in  vitro defects in tnfr1/nf-b activation , we tested whether hace1 also controls the response to l.  monocytogenes in  vivo .
importantly , we observed impaired clearance of l.  monocytogenes ( figure  1d ) , accompanied by enhanced lethality of the hace1 mice as compared to their wt littermates ( figure  1e ) , a phenotype that is compatible with and might be dependent on defective tnfr1/nf-b activation .
thus , hace1 e3 ligase activity is essential for tnf - induced activation of both nf-b and jnk downstream signaling in  vitro , and hace1 controls in  vivo immunity to l.  monocytogenes .
when nf-b activation is blocked , tnf stimulation results in apoptotic cell death ( beg and baltimore , 1996 , van antwerp et  al .
, 1996 , wang et  al . , 1996 ) . given that tnf - mediated nf-b activation
is impaired in hace1 cells , we reasoned that loss of hace1 might sensitize cells to tnfr1-induced apoptosis .
surprisingly , tnf stimulation alone did not induce significant cell death in hace1 cells ( figures 2a and 2b ) , even when we monitored these cells for 72  hr ( figure  s1b ) .
moreover , whereas , as expected , control wt mefs underwent apoptosis in response to treatment with tnf and actinomycin - d for 8  hr ( actd ; used to block nf-b anti - apoptotic transcriptional activity ; lasek et  al .
, 1996 ) , hace1 mefs remained resistant to apoptotic death ( figures 2a and 2b ) and cells remained viable , albeit only partially , even at later time points ( figure  s1c ) .
treatment with actd alone did not induce cell death in control nor hace1 mefs ( figure  s1d ) .
consistent with a critical role for hace1 in mediating tnf / actd - induced apoptosis , activation of caspase-8 and its downstream death effector caspase-3 as well as cleavage of rip1 kinase ( rip1 ) were markedly impaired in hace1 mefs ( figure  2c ) .
we also failed to detect caspase-3 activation in hace1 mefs using a devdase activity assay ( figure  s1e ) .
similarly , apoptotic death and caspase-3 activation were absent in hace1 mefs challenged with tnf plus cycloheximide ( chx ) ( figure  2d ; figure  s1f ) , an inhibitor of the nf-b - mediated survival pathway ( tsuchida et  al . , 1995 ) .
importantly , re - expression of wt hace1 , but not e3 ligase - dead c876s hace1 , restored tnf / actd- as well as tnf / chx - induced cell death in hace1 mefs ( figures 2e and 2f ) .
smac mimetic drugs such as bv6 induce intrinsic apoptosis by blocking the inhibitor of apoptosis ( iap ) proteins downstream of tnfr1 ( vercammen et  al .
we therefore used this model as an additional  trigger of tnf - induced apoptotic cell death .
baseline ciap1 and xiap expression levels before and after tnf stimulation were comparable in control and hace1 mefs ( figure  s1
as expected , we observed tnf / bv6-induced cell death in control mefs ; by contrast , hace1 mefs again were resistant to apoptosis induced by tnf and bv6 ( figure  s1h ) .
these results show that tnfr1-induced apoptosis depends on hace1 . to test whether hace1 also regulates tnfr1-mediated apoptosis in  vivo , we first challenged control and hace1 mice with recombinant murine tnf .
hace1 mice were completely resistant to tnf - induced death and systemic inflammation , as determined by the liver injury markers alt and ast ( figures 3a3c ) . to extend these data to additional in  vivo models , we next challenged mice with lipopolysaccharide ( lps)/d - gal , which induces liver damage and death in wt mice , but not in tnfr1 mutant mice ( pfeffer et  al . , 1993 ) .
hace1 knockout mice were , to a large extent albeit not completely , resistant to lps / d - gal - induced liver damage and apoptotic death ( figures 3d3f ) .
these in  vivo data indicate that hace1 deficiency renders mice resistant to apoptotic liver damage and death downstream of tnfr1 . in the condition where both nf-b and caspase activation are  either genetically or pharmacologically blocked , tnf stimulation may result in a non - apoptotic form of cell  death termed necroptosis ( cho et  al . , 2009 , christofferson and yuan , 2010 , declercq et  al . , 2009 ,
to determine whether loss of hace1 also interferes with tnf - induced necroptosis , wt and hace1 mefs were pre - treated with the pan - caspase inhibitor z - val - ala - asp - fluoromethyl ketone ( z - vad ) and then stimulated with tnf / actd to induce cell death .
we observed that both wt and hace1 mefs are comparably sensitive to necroptosis triggered by tnf / actd / z - vad ( figure  4a ) .
these findings also were confirmed using tnf / chx / z - vad as an alternative necroptotic drug combination ( figure  4a ) . of note ,
treatment with z - vad alone or in combination with either actd or tnf did not induce cell death in control or hace1 mefs ( figures s1d and s2a ) .
two kinases , rip1 and rip3 , mediate necroptotic cell fate downstream of tnfr1 ( christofferson and yuan , 2010 , declercq et  al . , 2009 ) .
pre - treatment with the specific rip1 kinase inhibitor necrostatin 1 ( nec-1 ) ( degterev et  al . ,
2008 ) reverted necroptotic cell death in both control and hace1 mefs ( figure  4a ) .
we next generated hace1 ripk3 double - knockout ( hace1 
 ripk3 ) mefs to genetically determine the contribution of rip3 on cell death in hace1 mefs .
these double - mutant cells exhibited growth curves comparable to control mefs ( figure  s2b ) .
importantly , whereas hace1 mefs were sensitive to necroptosis , hace1 
 ripk3 mefs were resistant to necroptotic death ( figure  4b ) .
thus , hace1 is required for tnf - induced nf-b activation and apoptosis induction , but it is dispensable for the necroptotic response following tnfr1 stimulation . to identify molecular targets of hace1 that could explain impaired nf-b activation and defective apoptosis downstream of tnfr1 , we established a hace1-dependent in  vitro ubiquitylation assay using hemagglutinin ( ha)-ubiquitin , the ubiquitin - activating e1 enzyme , and multiple e2 enzymes .
the most efficient e2 for hace1 e3 ligase activity was found to be ubc13 ( figure  s3a ) . using recombinant hace1 , ubc13 , the e1 enzyme , and ha - ubiquitin
, we then performed in  vitro ubiquitylation assays on microchips spotted with > 9,000 natively folded human proteins .
intriguingly , our microchip array data revealed multiple hace1 ubiquitylation targets , many of which had been annotated previously to the tnfr1/nf-b - signaling pathway ( figures s3b and s3c ) . using in  vitro ubiquitylation assays
previous reports indicated that traf2 plays a pivotal role in driving nf-b and jnk activation and inhibiting necroptosis after stimulation with tnf .
this function is modulated by k63-linked ubiquitylation of traf2 ( karl et  al . , 2014 , li et  al .
, 2009 , petersen et  al . , 2015 , yeh et  al . , 1997 ) .
to identify the nature of traf2 ubiquitylation mediated by hace1 , we performed an in  vitro ubiquitylation assay .
we found that hace1 directly catalyzes k63-linked ubiquitylation of traf2 ( figure  s4a ) .
importantly , hace1 is essential for this particular modification : lack of hace1 led to the abrogation of tnf - driven k63-linked ubiquitylation of traf2 ( figure  s4b ) .
we failed to detect tnf - induced k48-specific ubiquitylation of traf2 in control and hace1 mefs ( figure  s4c ) as well as in in  vitro ubiquitylation assays ( not shown ) .
defective ubiquitylation was restored in hace1 mefs reconstituted with wt hace1 , but not in the e3 ligase - defective hace1 c876s mutant ( figure  s4d ) .
similar to hace1 mefs , rnai - mediated downregulation of hace1 in wt mefs impaired ubiquitylation of traf2 following tnf stimulation ( figure  s4e ) .
activation of tnfr1 induces rapid formation of a traf2-containing plasma membrane - bound signaling complex ( complex i ) ( micheau and tschopp , 2003 ) . within complex
i , k63 ubiquitylation of traf2 has been previously associated with the ability to activate nf-b and to induce jnk signaling ( habelhah et  al . , 2004 , li et  al . , 2009 , liang et  al . , 2010 , xia and chen , 2005 ) .
endogenous hace1 associated with traf2 , an interaction that was markedly enhanced upon tnf stimulation ( figures s5a and s5b ) .
moreover , endogenous hace1 co - immunoprecipitated with tnfr1 in wt mefs , an interaction that appears to be constitutive ( figures s5c and s5d ) .
cell surface expression of tnfr1 in hace1 mefs was comparable to that in control cells ( figure  s5e ) .
immunoprecipitation of complex i using flag - tagged human tnf ( micheau and tschopp , 2003 ) showed that all the essential components of complex i , such as ikk , ikk , traf2 , as well as rip1 , were recruited to the activated tnfr1 in both control and hace1 mefs ( figure  5a ; figure  s5c ) .
rip1 kinase appeared to be normally phosphorylated and ubiquitylated within complex i ( figure  5a ; figures s5f and s5 g ) .
serial immunoprecipitation experiments , carried out to pull down traf2 from the tnf - activated complex i ( lu et  al . , 2013 ) , showed a marked defect in k63 ubiquitylation of traf2 in hace1 versus control mefs ( figure  5b ; figure  s5h ) .
thus , loss of hace1 has no apparent effect on the assembly of complex i ; however , hace1 is essential for the ubiquitylation of complex i - associated traf2 . following initial formation of complex i , tnfr1 stimulation results in the assembly of a secondary cytoplasmic complex , termed complex ii ( micheau and tschopp , 2003 ) , which drives caspase-8-dependent apoptotic cell death ( declercq et  al . , 2009 , kaiser et  al . , 2011 ,
2011 ) . to investigate whether the observed resistance of hace1 mefs to tnf - induced apoptosis originates from defective complex ii formation , we analyzed the assembly of the apoptotic complex ii following tnf / actd stimulation . in hace1 mefs , the association of traf2 with fadd ( figure  5c ) , as well as its tnf stimulation - dependent association with rip1 kinase ( figure  5d ) , was severely impaired .
further in  vitro assays using recombinant proteins showed that traf2 and fadd interaction depends on ubiquitylation of traf2 ( figure  s6a ) .
moreover , the recruitment of rip1 kinase and caspase-8 to fadd also was severely affected ( figure  5e ) , demonstrating that loss of hace1 expression results in defective assembly of the caspase-8-activating complex .
loss of hace1 does not impair necroptosis downstream of tnfr1 stimulation ( figure  4a ) .
necroptosis is mediated via the formation of complex ii containing , among other proteins , fadd , caspase-8 , and both rip1 and rip3 kinases .
when caspase-8 is inactivated , rip1 and rip3 kinases together induce programmed necrosis ( cho et  al . , 2009 , christofferson and yuan , 2010 , declercq et  al . , 2009 ,
galluzzi and kroemer , 2008 , zhang et  al . , 2011 ) . as reported previously ( wang et  al . ,
2008 ) , z - vad inhibition of caspase-8 in tnf / actd - treated wt mefs was associated with enhanced stability of the fadd / caspase-8/rip1 kinase complex ( figure  5e ; figure  s6b ) .
intriguingly , the association of rip1 kinase and caspase-8 with fadd was found to be virtually absent in hace1 mefs undergoing tnf / actd / z - vad - triggered necroptosis ( figure  5e ; figure  s6b ) . in both control and hace1 mefs ,
this interaction is mirrored by the phosphorylation of the necroptotic downstream mediator mlkl ( figure  5 g ) ( vanden berghe et  al . , 2014 ) , indicating that induction of rip1/rip3-dependent necroptosis is not affected by the absence of hace1 .
interestingly , rip1/rip3 interactions can be detected , albeit at reduced levels , in hace1 mefs even under apoptotic conditions ( figure  5f ) .
however , we failed to detect any association between the rip1/rip3 kinases and caspase-8 under necroptotic conditions in hace1 mefs treated with either tnf / actd / z - vad or with tnf / bv6/z - vad ( figures s6b and s6c ) ( he et  al . , 2009 ) .
importantly , we still observed rip1 phosphorylation , a marker of rip1 kinase activity and necroptotic pre - disposition ( declercq et  al . , 2009 ) ,
in both control and hace1 mefs treated with tnf / actd / z - vad ( figure  5e ; figure  s6b ) or tnf / bv6/z - vad ( figure  s6c ) .
thus , loss of hace1 expression impairs the assembly of the canonical complex ii without any apparent effect on the formation and activity of the necrosome formed by rip1 and rip3 kinases and phosphorylation of mlkl .
our data so far showed that loss of hace1 protects cells from  tnf - induced apoptosis in  vitro while leaving tnf - mediated rip1/rip3 kinase - dependent necroptosis intact . since tnfr1 , nf-b , and rip3 kinase
recently have been implicated in intestinal homeostasis ( gnther et  al . , 2011 , pasparakis , 2009 , welz et  al .
, 2011 ) , we tested whether hace1 might play a role in gut inflammation .
naive hace1 mice showed normal body weight ( figure  s7a ) , and morphological and flow cytometric analyses of the intestine of naive hace1 mice did not reveal any overt alteration compared to wt littermates ( figures s7b and s7c ) .
however , when we challenged mice with dextran sodium sulfate ( dss ) , a chemical irritant that disrupts the intestinal epithelial barrier and induces colitis ( de robertis et  al . , 2011 ) , we observed markedly increased intestinal inflammation in hace1 versus control hace1 mice .
hace1 mice suffered a more pronounced weight loss than wt mice when treated with dss ( figure  6a ) , and histopathological analysis revealed markedly more severe inflammation , tissue destruction , and epithelial cell death in hace1 mice compared to wt littermate controls ( figure  6b ; figure  s7d ) .
in addition , hace1 mice showed more severe diarrhea and increased intestinal bleeding ( hemoccult test ) compared to wt littermates ( figures 6c and 6d ) .
flow cytometric analysis of the immune system in the lamina propria revealed markedly increased infiltration of cd11b gr-1 ly6 g inflammatory monocytes in the intestines of hace1-deficient mice after treatment with dss ( figure  6e ) , confirming the increased severity of intestinal inflammation in hace1 compared to control wt mice .
nf-b acts downstream of surface receptors recognizing pro - inflammatory cytokines , including tnf itself , as well as pathogen - associated pattern receptors , which in turn leads to the expression of further inflammatory mediators ( bonizzi and karin , 2004 ) . on the other hand , nf-b expression in epithelia such as
the intestine plays a fundamental role in ensuring epithelial cell survival and in maintaining the structural integrity of mucosal surfaces ( pasparakis , 2009 ) .
hace1 mrna is expressed at comparable levels in a variety of immune cells as well as in intestinal epithelial cells ( figure  s7f ) . to discern in which cells hace1 is acting to prevent severe colitis , we generated bone marrow chimeric mice lacking hace1 exclusively in the immune system by transferring hace1-deficient bone marrow cells in lethally irradiated hace1-sufficient host mice .
as t  cells in the intestine are largely radioresistant , we used lymphopenic rag2 mice as hosts ( korag2 ) to prevent host - derived wt t  cells to persist in the intestine , thus affecting the validity of the system . as a control group , we reconstituted irradiated rag2 mice with wt bone marrow ( wtrag2 ) .
treatment of these chimeric mice with dss revealed that the absence or presence of hace1 in the immune system had no apparent effect on the development of colitis : both groups showed comparable weight loss ( figure  s7 g ) and similar recruitment of inflammatory cells to the intestinal lamina propria ( figure  s7h ) .
these results indicate that the loss of hace1 in radiosensitive immune cells is not responsible for the increased susceptibility of hace1 mice to colitis .
importantly , genetic inactivation of rip3 kinase in hace1 
 ripk3 double - knockout mice and inactivation of tnfr1 in hace1 
 tnfr1 double - knockout mice completely rescued all parameters of the severe inflammatory phenotype in hace1 mice to levels observed in wt littermates , including weight loss , tissue damage , death of epithelial cells , recruitment of inflammatory cells , as well as diarrhea and intestinal bleeding ( figures 6a6e ; figure  s7d ) .
also , the expression of several pro - inflammatory mediators was upregulated in the colon of hace1 mice ; in hace1 
 tnfr1 and hace1 
 ripk3 mice the levels of these pro - inflammatory factors were again restored to levels detected in wt controls ( figure  s7e ) .
the increased death of intestinal epithelial cells in dss - treated hace1 mice is probably the consequence of exaggerated immune activation and inflammation resulting from the preferential induction of tnf - driven necroptosis instead of apoptosis in the absence of hace1 ( pasparakis and vandenabeele , 2015 ) . of note , while others reported increased severity of colitis in ripk3 ( moriwaki et  al . , 2014 ) and
, to a lesser extent , in tnfr1 mice ( stillie and stadnyk , 2009 , wang et  al . , 2012 )
, in our hands single - knockout mice showed no change in weight loss compared to wt littermates ( figure  6f ) .
the difference in ripk3 mice might be due to the time point of analysis : moriwaki et  al .
( 2014 ) reported a defect of ripk3 animals in the recovery from dss , while we focused on the earlier phase of tissue destruction during which enhanced necroptosis is known to be detrimental ( gnther et  al . , 2011 ) .
together , our data show that the increased susceptibility of hace1-deficient mice to dss - induced colitis depends on deregulated tnfr1 signaling and rip3 kinase .
we initially identified hace1 as a tumor suppressor gene downregulated in multiple cancers ( anglesio et  al .
since we observed increased intestinal inflammation , we next explored whether hace1 mice are prone to develop colon cancer upon challenge with dss and the genotoxic drug azoxymethane ( aom ) ( tanaka et  al . , 2003 ) .
since hace1 mice are particularly sensitive to dss , we reduced the dss treatment regimen to a level not sufficient to induce colon cancer in wt mice ( de robertis et  al .
as expected using this low dss dose , we failed to detect any tumors in control hace1-expressing mice ( figure  7a ) .
however , in hace1 mice , we observed a markedly increased incidence of colon cancer as determined by colonoscopy or histopathological examination ( figures 7a and 7b ) .
colonic tumors in hace1 mice show features consistent with adenomatous polyps as well as nuclear atypia , multiple mitotic figures , areas of carcinoma in  situ , and focal invasion ( figure  7b ) .
most importantly , hace1 
 ripk3 as well as hace1 
 tnfr1 double - knockout mice were similar to wt mice and did not show aom / dss - induced colonic tumors ( figures 7a and 7b ) , indicating that simultaneous genetic inactivation of tnfr1 and rip3 kinase considerably reduced colon cancer formation secondary to hace1 deficiency . in conclusion , hace1 deficiency predisposes to colonic inflammation and carcinogenesis , which is markedly alleviated by genetic inactivation of tnfr1 and rip3 kinase .
loss of hace1 impairs tnfr1-mediated activation of nf-b signaling and apoptosis in  vitro and in  vivo , but it does not inhibit tnfr1-dependent rip1/rip3-induced necroptosis . moreover ,
our data show that enhanced inflammation and colon cancer in hace1 mutant mice can be reverted by genetic inactivation of rip3 kinase and tnfr1 .
loss of hace1 does not impair formation of complex i at tnfr1 ; however , nf-b and jnk activation are markedly impaired in hace1 mefs , a function dependent on its e3 ligase activity .
our results indicate that hace1 expression is essential for k63 ubiquitylation of traf2 within complex i upon tnfr1 stimulation , which previously has been associated with the ability to activate the canonical nf-b pathway and to induce jnk signaling ( habelhah et  al . , 2004 , liang et  al . , 2010 , xia and chen , 2005 ) .
although nf-b signaling is impaired , hace1 mefs do not undergo apoptosis in response to tnfr1 stimulation , which can be biochemically explained by the fact that hace1 cells fail to form pro - apoptotic complex ii .
moreover , our results show that traf2 ubiquitylation is required for traf2 to associate with the key death molecule fadd , which in turn nucleates the caspase-8 apoptosis effector complex .
however , besides traf2 , we found multiple additional  hace1 ubiquitylation targets that previously have been linked to cell death and signaling pathways downstream of tnfr1 , suggesting that hace1 could regulate tnfr1-regulated cell fate at various interception points .
our in  vitro and in  vivo data indicate that necroptosis in hace1 mutant cells is mediated via the rip1/rip3/mlkl pathway .
since we failed to detect formation of an intact necrosome , such death signals might progress independently of the canonical molecular necroptotic complex .
necroptosis in the context of hace1 deficiency appears to occur without direct caspase-8 interaction with rip1/rip3 kinases , raising the possibility that alternative rip1/rip3 kinase complexes might exist when hace1 is inactivated .
of note , in our experimental system , induction of necroptosis in hace1 knockout mefs still requires caspase inhibition via z - vad .
whether regulation of cflip ( oberst et  al . , 2011 ) could explain this remaining caspase dependency requires further exploration .
moreover , a feedback inhibition loop via rip3 kinase activity has been reported recently in the control of caspase-8-mediated apoptosis ( newton et  al . , 2014 ) , suggesting complex interplays between the apoptotic and necroptotic death effector machineries .
we initially identified hace1 as a candidate tumor suppressor gene in a child with wilms tumor ( anglesio et  al .
in accord with its putative tumor suppressor function , we found hace1 to be downregulated in multiple cancers ( hibi et  al .
, 2008 , sakata et  al . , 2009 ) . that hace1 can indeed suppress cancer
was shown in hace1 knockout mice that develop tumors of multiple tissues ( zhang et  al . , 2007 ) .
since the tnfr1 and rip1/rip3 kinase pathway has been reported to induce oxidative stress and possibly alter cellular metabolism ( christofferson and yuan , 2010 , declercq et  al .
, 2009 , galluzzi and kroemer , 2008 ) , our results suggest that this activation pathway downstream of tnfr1 might not only trigger necroptosis but also could be involved in oncogenesis .
moreover , rip1/rip3 kinases downstream of tnfr1 have been implicated in a pro - inflammatory cascade in the intestine unleashed by smac mimetics or caspase-8 deficiency ( kang et  al . , 2013 , vince et  al . , 2012 ) , driven by the release of necroptosis - mediated damage - associated molecular pattern molecules ( damps ) ( duprez et  al .
mutant mice indeed develop increased intestinal inflammation following epithelial injury and increased incidence of colon cancer .
importantly , genetic deletion of either rip3 kinase or tnfr1 not only reversed the enhanced inflammation but also strongly reduced the development of colon cancer in hace1 single - mutant mice .
these data demonstrate that deregulation of the tnfr1-mediated rip3 kinase signaling is a key driving principle of inflammation and subsequent colon cancer development controlled by hace1 .
the mice , hace1 , hace1 , hace1 
 tnfr1 , and hace1 
 ripk3 on a c57bl/6 background , were bred and maintained under specific pathogen - free conditions at the institute of molecular biotechnology of the austrian academy of sciences ( imba ) animal facility . for experiments , age- and sex - matched mice ( 812  weeks of age )
all animal experiments were approved by austrian authorities , and mice were kept following the guidelines of austrian animal laboratory law .
mefs derived from hace1 and hace1 mice were prepared as described previously ( zhang et  al . , 2007 ) .
mefs were seeded at 2   10 cells per 60-cm petri dish and cultured in dmem supplemented with 5% fetal calf serum ( fcs ) and 1% pen - strep ( sigma ) in a 5% co2 incubator .
the next day , cells were treated with 10  ng / ml mouse tnf ( 3014 , immunotools ) , 1  g / ml actd - mannitol ( a-5156 , sigma ) , 5  g / ml z - vad ( bachem ) , and 50  nm nec-1 ( n-9037 , sigma ) .
in addition , cells were treated with 10  ng / ml mouse tnf ( 3014 , immunotools ) and 10  g / ml chx ( c1988 , sigma ) . following the treatments ,
the cells were lysed in 1  ml ice - cold np-40 buffer ( 10  mm tris [ ph  8.0 ] , 150  mm nacl , and 1% nonidet p-40 ) supplemented with protease and phosphatase inhibitor tablets ( 11873580001 and 04906837001 , roche ) .
the lysates were then centrifuged at 14,000  rpm for 10  min at 4c .
immunoprecipitations were performed according to the manufacturer s protocol . for high - dose lethal tnf challenge
, animals were injected via the tail vein with 450  g / kg of purified endotoxin - free tnf ( immunotools ) in 200  l pbs ( ph 6.8 ) .
for induction of hyper - acute shock , tnf was used at a dose of 5  g / kg .
lps ( l2880 , sigma ) was administrated intraperitoneally ( i.p . ) at a concentration of 10  g / kg .
d - gal ( g0500 , sigma ) was given to mice together with lps at a concentration of 1 g / kg .
liver enzymes were assayed using the cobas c111 system ( roche ) . for histology , livers were embedded in paraffin , sectioned , and stained with h&e .
listeria monocytogenes ( strain lo28 ) was prepared for infection as described previously ( stockinger et  al . , 2009 ) .
the bacterial suspension ( 500  l ) was injected into the peritoneum of 8- to 10-week - old male hace1 and hace1 littermate mice .
survival of infected mice was monitored for 10  days . for determination of the bacterial load
, mice were euthanized 96  hr after infection and  spleens were homogenized in pbs .
serial dilutions of homogenates were plated on oxford agar plates and colonies were counted after growth at 37c for 24  hr . if not otherwise stated , all data are expressed as the means  sd .
significance was assessed by student s two - sided t test unless otherwise stated in the figure legends . for multiple comparisons
, we used a two - way anova test with a bonferroni post - test . differences with p values
< 0.05 were considered as significant ( p  < 0.05 , p  < 0.01 , and p  < 0.001 ) .
g.s .- f . , f.i . , g.k . , p.v . , h.k . , a.p . , and
 summarythe hect domain e3 ligase hace1 has been identified as a tumor suppressor in multiple cancers . here , we report that hace1 is a central gatekeeper of tnfr1-induced cell fate . 
 genetic inactivation of  hace1 inhibits tnf - stimulated nf-b activation and tnfr1-nf-b - dependent pathogen clearance in  vivo . 
 moreover , tnf - induced apoptosis was impaired in hace1 mutant cells and knockout mice in  vivo . 
 mechanistically , hace1 is essential for the ubiquitylation of the adaptor protein traf2 and formation of the apoptotic caspase-8 effector complex . 
 intriguingly , loss of hace1 does not impair tnfr1-mediated necroptotic cell fate via rip1 and rip3 kinases . 
 loss of hace1 predisposes animals to colonic inflammation and carcinogenesis in  vivo , which is markedly alleviated by genetic inactivation of rip3 kinase and tnfr1 . 
 thus , hace1 controls tnf - elicited cell fate decisions and exerts tumor suppressor and anti - inflammatory activities via a tnfr1-rip3 kinase - necroptosis pathway . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions