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Lysosomal Accumulation of Gliadin

Lysosomal Accumulation of Gliadin

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Lysosomal accumulation of gliadin p31
e
43 peptideinduces oxidative stress and tissuetransglutaminase-mediated PPAR
g
downregulationin intestinal epithelial cells and coeliac mucosa
Luigi Maiuri,
1
Alessandro Luciani,
1,2
Valeria Rachela Villella,
3
Angela Vasaturo,
3
Ida Giardino,
4
Massimo Pettoello-Mantovani,
1
Stefano Guido,
2,3
Olivier N Cexus,
5
Nick Peake,
5
Marco Londei,
6
Sonia Quaratino
5
ABSTRACTBackground
An unresolved question in coeliac disease isto understand how some toxic gliadin peptides, inparticular p31
e
43, can initiate an innate response andlead to tissue transglutaminase (TG2) upregulation incoeliac intestine and gliadin sensitive epithelial cell lines.
Aim
We addressed whether the epithelial uptake ofp31
e
43 induces an intracellular pro-oxidativeenvoronment favouring TG2 activation and leading to theinnate immune response.
Methods
The time course of intracellular delivery tolysosomes of p31
e
43, p
a
-2 or p
a
-9 gliadin peptides wasanalysed in T84 and Caco-2 epithelial cells. The effects ofpeptide challenge on oxidative stress, TG2 andperoxisome proliferator-activated receptor (PPAR)
g
ubiquitination and p42/44
e
mitogen activated protein(MAP) kinase or tyrosine phosphorylation wereinvestigated in cell lines and cultured coeliac diseasebiopsies with/without anti-oxidant treatment or TG2 genesilencing by immunoprecipitation, western blot, confocalmicroscopy and Fluorenscence Transfer ResonanceEnergy (FRET) analysis.
Results
After 24 h of challenge p31
e
43, but not p
a
-2 orp
a
-9, is still retained within LAMP1-positive perinuclearvesicles and leads to increased levels of reactive oxygenspecies (ROS) that inhibit TG2 ubiquitination and lead toincreases of TG2 protein levels and activation. TG2induces cross-linking, ubiquitination and proteasomedegradation of PPAR
g
. Treatment with the antioxidantEUK-134 as well as TG2 gene silencing restored PPAR
g
levels and reversed all monitored signs of innateactivation, as indicated by the dramatic reduction oftyrosine and p42/p44 phosphorylation.
Conclusion
p31
e
43 accumulation in lysosomes leads toepithelial activation via the ROS
e
TG2 axis. TG2 works asa rheostat of ubiquitination and proteasome degradationand drives inflammation via PPAR
g
downregulation.
INTRODUCTION
Coeliac disease is a relatively common pathology that affects 1% of the population, and is precipi-tated by the ingestion of gluten proteins.
1
e
3
Inpredisposed individuals, gliadin acts as a stimulatorof innate responses, in turn setting the type andintensity of the adaptive immune response
4
leadingto a powerful activation of lamina propria gliadin-speci
c Tcells.
5
e
7
Several fragments of gliadin havebeen found to be
toxic
for predisposed individualsandithasalsobeensuggestedthatdifferentportion(s)of gliadin, ostensibly not those recognised by Tcells,modulates this innate activation of coeliac smallintestine.
7 8
 Among these, p31
e
43 induces theexpression of early markers of epithelial activationand leads to enterocyte apoptosis
4
both in coeliacintestine and in sensitive intestinal epithelial celllines.
4
The mechanisms by which p31
e
43 initiates aninnate immune response in coeliac duodenum are,however, still elusive. It has been reported thatp31
e
43 can delay epidermal growth factor receptor(EGFR) inactivation through interference with theendocytic pathway,
9
this suggesting that gliadinfragments could amplify the effects of traceamounts of EGF. Moreover, p31
e
43 induces early tissue transglutaminase (TG2) upregulationcompared to immunodominant gliadin peptides.
10
This indicates that TG2 is not only central to theadaptive response to gliadin as the main deami-dating enzyme of the immunodominant epitopes,but is also a key player in the initiation of in
am-mation in coeliac disease.
10
However, it is stillunknown how p31
e
43 induces early TG2 activa-tion in both coeliac intestine and
gliadin-sensitive
intestinal epithelial cells.
9 10
TG2 expression is regulated by retinoids, steroidhormones, peptide growth factors and cytokines,which also lead to a time-dependent decrease inTG2 ubiquitination,
11
indicating that post-trans-lational control mechanisms also play a role in TG2regulation. We have demonstrated that in cystic
brosis (CF) airway epithelia,
12
sustained TG2activation is driven by the increased levels of reac-tive oxygen species (ROS) caused by the CFTgenetic defect.
12
 We therefore investigated whetherp31
e
43 was able to drive an intracellular pro-oxidative environment and demonstrate thatp31
e
43 leads to an increase of ROS levels in coeliacduodenum as well as T84 and Caco-2 intestinalepithelial cells.To understand how p31
e
43 challenge inducesoxidative stress, we analysed the delivery of thispeptidetotheintracellular degradationsystems. Ithasbeen reported that the epithelial translocation of thegliadin immunostimulatory peptide 33-mer occurs by transcytosis after partial degradation through a rab5endocytic compartment.
13
Increased transepithelial
See Commentary, p 286
<
Supplementary methods and figures are published online onlyat http://gut.bmj.com/content/ vol59/issue3
1
Institute of Pediatrics,University of Foggia, Foggia, Italy
2
Dynamic Imaging Microscopy,CEINGE, Naples, Italy
3
Department of ChemicalEngineering, University FedericoII of Naples, Naples, Italy
4
Department of BiomedicalScience, University of Foggia,Foggia, Italy
5
Cancer Research UK OncologyUnit, University of Southampton,Southampton, UK
6
Novartis Pharma AGTranslational Medicine, Basel,Switzerland
Correspondence to
Professor Luigi Maiuri, Instituteof Pediatrics, University ofFoggia, viale Pinto 1, Foggia71100, Italy; maiuri@unina.itRevised 13 July 2009Accepted 1 September 2009Published Online First1 December 2009
Gut 
2010;
59
:311
e
319. doi:10.1136/gut.2009.183608 311
Coeliac disease
 
translocation of the 33-mer was demonstrated in active coeliacdisease compared to healthy controls. Moreover, incomplete degra-dation of the 33-mer and protected transport of the peptide 31
e
49occurs in patients with untreated coeliac disease, thus favouringtheir respective immunostimulatory and toxic effects.
14
Here weshow that p31
e
43 is retained within the lysosomes as compared tothe more rapid disappearance of the immunodominant peptidesp
a
-2orp
a
-9fromtheintracellularcompartments.Blockingp31
e
43endocytosis prevents the increase of ROS. We have previously reported that in CF airways sustainedTG2 activation drives in
ammation by inducing cross-linking,ubiquitination and proteasome degradation, of the anti-in
am-matory peroxisome proliferator-activated receptor (PPAR)
g
,
12
a negative regulator of in
ammatory gene expression.
15 1
Blocking TG2 through speci
c gene silencing
12
or the speci
cTG2 inhibitors restores a physiological control of in
ammationin CF airways.
12
 We therefore addressed whether the p31
e
43-induced innateresponse was mediated by the ROS
e
TG2 axis via PPAR 
g
downregulation.
METHODSPeptide preparation
Biotinylated gliadin peptides p31
e
43, p
a
-9 (57
e
68) or p
a
-2(62
e
75), control human thyroid peroxidase pTPO (535
e
551)and pTPO (536 
e
547), peptide LGQQQPFAAVQPY (referred tobelow as pZ), and scrambled peptides with amino acid compo-sition similar to p31
e
43 (QQGQPFPQPQLQY (referred to belowas pX), GLQQFQPPPPQQY (referred to below as pY)) weresynthesised by Primm (Milan, Italy) and the purity was deter-mined by reverse-phase HPLC.
Cell lines
Human colon adenocarcinoma T84 or human colorectal carci-noma Caco-2 cell lines were cultured as recommended by  American Type Culture Collection (ATCC).
Cell cultures
The cells were challenged with p31
e
43 for 24 h in the presenceor absence of the ROS scavenger EUK-134 (50
m
g/ml; AlexisBiochemicals, Florence, Italy), glutathione (GSH) (10 mmol/l;Sigma, Milan, Italy)
17
or the TG inhibitor cystamine (400
m
mol/l;Sigma). To study internalisation of peptides, cells were chal-lenged for 15, 30, 60 or 90 min, 3 or 24 h at 37
8
C with thebiotinylated peptides (20
m
g/ml). To inhibit endocytosis, the cellswere pre-treated with the cholesterol-binding agent methyl-
b
-cyclo-dextrin (M-
b
-CD, 10 mmol/l; Sigma) or
lipin (5
m
g/ml;Sigma) and then challenged with biotinylated peptides. Cell lineswere also incubated for 6 h with the PPAR 
g
agonist rosiglitazone(10
m
mol/l; Alexis Biochemicals) and then challenged withp31
e
43. The PPAR 
g
antagonist GW9662 (1
m
mol/l; AlexisBiochemicals) was added for 24 h to p31
e
43 in the presence orabsence of TG2 siRNA oligos. The proteosome inhibitor MG132(50
m
mol/l; Calbiochem, Milan, Italy) was added 6 h beforep31
e
43 in the presence or absence of TG2 siRNA oligos orEUK134.
Patients
Duodenal multiple endoscopic biopsies were performed fordiagnostic purposes in seven treated patients with coeliac disease(mean age, 25 years; range, 16 
e
41 years), and
ve non-coeliaccontrols affected by oesophagitis (22.4 years; range,18
e
29 years). Informed consent was obtained from all individ-uals, and the study was performed according to the EthicsCommittee of Regione Campania Health Authority. One spec-imen from each patient was used for diagnosis; the other sampleswere cultured in vitro as described.
4
In vitro organ culture of biopsy specimens from patients withcoeliac disease and from controls
Duodenal biopsy specimens were cultured as previously reported
4
for 3 or 24 h with medium alone, p31
e
43, p
a
-9, p
a
-2,pTPO (20
m
g/ml) in the presence or absence of cystamine(400
m
mol/l).Duodenal biopsyspecimenswere alsoincubatedfor6 h with the PPAR 
g
agonist rosiglitazone (10
m
mol/l; AlexisBiochemicals) and then re-challenged for 3 h with p31
e
43.
RNA interference
The cells were transfected with 50 nmol/l human TG2 andscrambled small interfering RNAs (siRNAs) duplex usingHiperfect Transfection Reagent (Qiagen, Milan, Italy) at 37
8
C for72 h as previously described.
12
Adenoviral vector
Human manganese superoxide dismutase (MnSOD) cDNA wascloned into the shuttle vector pAd5CMVK-NpA.
18
MnSODadenovirus was a gift from Michael Brownlee (Albert EinsteinCollege ofMedicine, New York). T84 cell lines were infected withMnSOD or the control adenovirus for 2 h, as previously described.
18
Immunolocalisations
Tissue sections were individually incubated with the antibodiesanti-phospho-tyrosine (PY99 mAb, 1:80, mouse IgG2b; SantaCruz Biotechnology, Santa Cruz, California, USA), anti-PPAR 
g
(clone E8, sc-7273, 1:100, mouse IgG1; clone H100, sc-7196,1:100, rabbit polyclonal IgG; Santa Cruz Biotechnology) andcontrol antibodies.
4 12
Cell lines were incubated with the anti-bodies against phospho-tyrosine, Early Endosome Antigen-1(EEA-1; 1:100, rabbit polyclonal IgG; Abcam, Cambridge, UK),Rab7 (1:200, rabbit polyclonal IgG; Abcam), ARF-1 (1:100, rabbitpolyclonal IgG; Abcam), LAMP-1 (1:100, rabbit polyclonal IgG; Abcam). Two-colour and indirect immuno
uorescence were usedfor the detection of the tested markers by using an LSM510 Zeissconfocal laser scanning unit (Zeiss, Jena, Germany).
12
Thecorrelation coef 
cient analysis was used as value of co-local-isation between peptides and EEA-1 or LAMP-1 as described.
19
In situ detection of TG2 enzymatic activity 
The detection of in situ TG2 activity was performed by detec-tion of biotinylated mono-dansyl-cadaverine (MDC) incorpora-tion by Alexa-546 streptavidin as previously described.
4 12
FRET microscopy 
For acceptor photobleaching 4-
m
m frozen tissue sections of biopsy samples were
xed with buffered 2% paraformaldehyde andpermeabilised with 0.5% Triton X-100. Upon
xation, tissues wereimmunostained with Alexa 546/anti-PPAR 
g
(Santa CruzBiotechnology, Santa Cruz, California, USA) and Cy5/anti-
 N 
e
(
g
-
L
-glutamyl)-
L
-lysineisopeptide(Covolab,Cambridge,UK).Alexa546 
uorescence was detected before and after Cy5 photobleaching.
Immunoblot and immunoprecipitation
Blots were incubated with anti-phospho-tyrosine (PY99 mAb,1:500), anti-phospho-p42/p44 MAP kinases (Cell SignalingTechnology, Danvers, Massachusetts, USA), PPAR 
g
(clone E8 sc-7273; Santa Cruz Biotechnology, Santa Cruz, California, USA),
312
Gut 
2010;
59
:311
e
319. doi:10.1136/gut.2009.183608
Coeliac disease
 
TG2 (clone CUB7402, 1:100; NeoMarkers, Fremont, California,USA),
e
(
g
-
L
-glutamyl)-
L
-lysine isopeptide (clone 81DIC2;Covalab), ubiquitin (1:100 clone FL-76, rabbit polyclonal IgG;Santa Cruz Biotechnology), as previously described.
12
Immunoprecipitations with anti-TG2 CUB 7402 mAb, anti-phospho-tyrosine and anti-PPAR 
g
were carried out as previously described.
12
ROS detection
Cell lines were pulsed with 10
m
mol/l 5-(and-6)-chloromethyl-2
9
,7
9
-dichlorodihydro
uorescein diacetate acetyl ester (CM-H2DCFDA) (Molecular Probes, Invitrogen, Milan, Italy) andanalysed as previously described.
12
Statistical analysis
Cells challenged with peptides were compared with thosecultured in medium alone, and with those cultured in thepresence of the tested inhibitors. All experiments wereperformed at least in triplicate. Data distribution was analysed,and statistical differences were evaluated using ANOVTukey 
e
Kramer test and SPSS 12 software. A p value of 
<
0.05was considered signi
cant.Detailed methods are described in the online supplementary information section.
RESULTSp31
e
43 is retained within the lysosomes in T84 epithelial cells
T84 cells were incubated with biotinylated p31
e
43, p-
a
2, p-
a
9(20
m
g/ml) for 15, 30, 60, 90 min, 3 h and 24 h at 37
8
C. Thepeptides were rapidly detected (upon 15 min) in intracellularvesicles (
gure 1A). Prolonged incubation (after 90 min to 24 h)increased the amount of p31
e
43 (
gure 1A), but not of p-
a
9(
gure1A)orp-
a
2(datanotshown),whichresidedinperinuclearvesicles. Soon after 15 min of challenge, peptides co-localisedwith EEA-1 (
gure 1B), a marker of early endosomes,
13
whereasafter 60 min they co-localised with Rab7-positive late endosomes(data not shown).
20
These data are in agreement with thosedescribed for the gliadin peptide 33-mer.
13
Co-staining with anti-body against Arf1 (data not shown) did not reveal translocationofthepeptidestotheGolgi complex.After 90 minthe internalisedpeptides co-localised with Lysosomal Associated MembraneProtein-1 (LAMP-1) (
gure 1C), a marker of lysosomes.
20
Thepeptide/EEA-1 or peptide/LAMP-1 correlation coef 
cients of thespatial intensity distributions yielded a fairly high correlationcoef 
cient (
gure 1D), indicating that gliadin peptides areinternalised and transported through early and late endocyticendosomes to lysosomes. No internalisation of peptides or co-localisation with LAMP-1 were observed when T84 cells werepre-incubated with M-
b
-CD, which inhibits clathrin-mediatedendocytosis,
20
or
lipin, a speci
c inhibitor of lipid raft- orcaveolae-dependent endocytosis
20
(data not shown). Thissuggests that endocytosis is essential for the delivery of gliadinpeptides to the lysosomes for degradation.Increasing appearance of the internalised p31
e
43 in LAMP-1-positive vesicles, mainly limited to perinucelar localisation, wasobserved after 3
e
24 h of challenge (
gure 1E,F). At these latetime points p-
a
9(
gure1E,F) orp
a
-2(datanot shown) wereonlfaintly detected in LAMP-1-positive structures. The coef 
cientsof p-
a
2 or p-
a
9 co-localisation with LAMP-1 were signi
cantly lower compared to p31
e
43 (
gure 1G), thus indicating thatp31
e
43, but not p-
a
9 or p-
a
2 is retained within the lysosomes.Caco-2 cells showed the same behaviour as T84 after peptidechallenge (data not shown). The control pTPO (535
e
551) (datanot shown), pTPO (536 
e
547) and pZ, as well as scrambled pXand pY peptides, behaved like p
a
-9 and p
a
-2 in both T84(supplementary 
gure 1) and Caco-2 cell lines (data not shown).
p31
e
43 induces increase of ROS in T84 epithelial cells
T84 cells were pulsed with 10
m
mol/l CM-H2DCFDA in thepresence of the tested peptides and the levels of ROS weremonitored after 90 min, 3 h and 24 h of challenge. The intracel-lular transport of gliadin peptides through early and late endo-cytic endosomes did not induce ROS generation (data notshown). Moreover, after 90 min of challenge neither p31
e
43 norp
a
-9, both detected in LAMP-1 positive vesicles (
gure 1C),induced signi
cant increase of ROS (
gure 2A). At the later timepoints (3
e
24 h of challenge), p31
e
43, but not p
a
-9 (
gure 2A),p
a
-2, pTPO (535
e
551) (data not shown) or peptides pTPO(536 
e
547), pX, pY and pZ (supplementary 
gure 1) induceda time-dependent signi
cant increase of ROS levels, suggestingthat the prolonged persistence of p31
e
43 in LAMP-1 positivevesicles (
gure 1E,F) generates a pro-oxidative environment. Noincrease of ROS was observed after challenge with p31
e
43 uponM-
b
-CD or
lipin treatment (
gure 2B).The generation of a pro-oxidative environment induces acti-vation of different stress sensitive signalling pathways.
9 12 21
Since the challenge with p31
e
43 induces phosphorylation of theextracellular signal-regulated kinases 1/2 (Erk1/2, p42/44mitogen-activated protein kinases, MAPK),
9
we investigatedwhether p31
e
43-induced ROS-mediated p42/44 MAPK phos-phorylation in T84 cells. We demonstrated that p42
e
44 MAPK phosphorylation induced by the challenge with p31
e
43 wasprevented by the incubation with the catalase
e
superoxidedismutase (SOD) mimetic EUK-134
12
(
gure 2C), as well as by GSH
17
or by MnSOD over-expression
18
(supplementary 
gure 2).This further indicates that the generation of a pro-oxidativeenvironment is critical to p31
e
43 biological activity in T84 cells.
p31
e
43 induces ROS-dependent inhibition of TG2 ubiquitination
To investigate whether the increased levels of ROS may accountfor the p31
e
43-induced increase of TG2 levels,
12
we incubatedp31
e
43-challenged T84 cells with EUK-134. We demonstratedthat EUK-134 was effective in controlling the increase of TG2protein levels (
gure 3A) and TG2 activity (data not shown)induced by p31
e
43. The same effects as EUK-134 were observedafter incubation with GSH or upon MnSOD over-expression(supplementary 
gure 2). No increase of TG2 was observed afterchallenge with p31
e
43 upon M-
b
-CD or
lipin treatment (datanot shown). This indicates that p31
e
43 internalisation is criticalfor the induction of ROS-mediated TG2 activation.Since post-translational modi
cations of TG2, such as ubiq-uitination, play a role in regulating the levels of TG2 protein
11
we investigated whether TG2 ubiquitination was in
uenced by ROS generation induced byp31
e
43. Weincubated T84 cellswithp31
e
43 in the presence or absence of EUK-134 upon inhibition of proteasome function by MG132,
12
then immunoprecipitatedTG2 protein and detected ubiquitin co-reactivity. The ubiquitinimmunoreactivity on TG2 immunoprecipitates was enhancedupon treatment with EUK-134 (
gure 3B). No effects of p31
e
43on TG2 ubiquitination were observed when T84 cells werepre-treated with M-
b
-CD or
lipin (data not shown).
p31
e
43-induced TG2 drives PPAR
g
cross-linking andproteasome degradation in T84 cells
 We investigated whether the p31
e
43-induced TG2 activationwas effective in inducing PPAR 
g
cross-linking and proteasomedegradation as we have reported in CF airways.
12
 We immunoprecipitated PPAR 
g
species from T84 cell lysatesafter challenge with p31
e
43 and detected the immunoreactivity 
Gut 
2010;
59
:311
e
319. doi:10.1136/gut.2009.183608 313
Coeliac disease

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