PARP-1 deficiency blocks IL-5 expression through calpain-dependent degradation of STAT-6 in a murine asthma model.

Abstract

We recently showed that poly(ADP-ribose)polymerase-1 (PARP-1) may play a role in allergen (ovalbumin)-induced airway eosinophilia, potentially through a specific effect on IL-5 production. We also reported that while IL-5 replenishment promotes reversal of eosinophilia in lungs of PARP-1(-/-) mice, IL-4 or Immunoglobulin E replenishment do not, suggesting a potentially significant regulatory relationship between PARP-1 and IL-5.
To explore the mechanism by which PARP-1 regulates IL-5 production and to determine how PARP-1 inhibition blocks allergen-induced eosinophilia.
This study was conducted using a murine model of allergic airway inflammation and primary splenocytes.
PARP-1 knockout-associated reduction in IL-5 upon allergen exposure occurs at the mRNA level. Such an effect appears to take place after IL-4 receptor activation as PARP-1 inhibition exerted no effect on JAK1/JAK3 activation. Signal transducer and activator of transcription-6 (STAT-6) protein was severely downregulated in spleens of PARP-1(-/-) mice without any effect on mRNA levels, suggesting an effect on protein integrity rather than gene transcription. Interestingly, the degradation of STAT-6 in PARP-1(-/-) mice required allergen stimulation. Additionally, PARP-1 enzymatic activity appears to be required for STAT-6 integrity. The downregulation of STAT-6 coincided with mRNA and protein reduction of GATA-binding protein-3 and occupancy of its binding site on the IL-5 gene promoter. IL-4 was sufficient to induce STAT-6 downregulation in both PARP-1(-/-) mice and isolated splenocytes. Such degradation may be mediated by calpain, but not by proteasomes.
These results demonstrate a novel function of PARP-1 in regulating IL-5 expression during allergen-induced inflammation and explain the underlying mechanism by which PARP-1 inhibition results in IL-5 reduction.

Full text

Page 1

ORIGINAL ARTICLE
EXPERIMENTAL ALLERGY AND IMMUNOLOGY
PARP-1 deficiency blocks IL-5 expression through
calpain-dependent degradation of STAT-6 in a murine
asthma model
R. Datta1*, A. S. Naura1*, M. Zerfaoui1, Y. Errami1, M. Oumouna1?, H. Kim1, J. Ju1, V. P. Ronchi2,
A. L. Haas2& A. H. Boulares1
1Department of Pharmacology, The Stanley Scott Cancer Center;2Department of Biochemistry and Molecular Biology; Louisiana State
University Health Sciences Center, New Orleans, LA, USA
To cite this article: Datta R, Naura AS, Zerfaoui M, Errami Y, Oumouna M, Kim H, Ju J, Ronchi VP, Haas AL, Boulares AH. PARP-1 deficiency blocks IL-5 expres-
sion through calpain-dependent degradation of STAT-6 in a murine asthma model. Allergy 2011; DOI: 10.1111/j.1398-9995.2011.02549.x.
The control of inflammation has long been one of the major
therapeutic goals of medicine, as inflammatory processes are
involved in the pathogenesis of diseases that affect all physio-
logical systems, including cardiac, pulmonary, and neuro-
logical. Over the last decades, the family of poly (ADP-ribose)
polymerases (PARP) has emerged as an important player in
the development and progression of inflammatory disease. The
involvement of the primary member of the family, PARP-1, in
the inflammatory process is not entirely clear. This enzyme is
believed to mediate inflammation through the promotion of
cell death via ATP depletion as well as the transcription of
Keywords
allergen-induced eosinophilia; IL-4; lung;
transgenic/knockout mice.
Correspondence
A. Hamid Boulares, PhD, 533 Bolivar St
#509, New Orleans, LA 70112, USA.
Tel.: +(504) 568-2304
Fax: +(504) 568-2361
E-mail: hboulr@lsuhsc.edu
?Current address: Biotechnology Laboratory
of Animal Reproduction, Department of
Veterinary Sciences, Faculty of Agronomy
and Veterinary Sciences, Blida University,
BP 270 Blida 09000, Algeria.
*These two authors contributed equally to
the work.
Accepted for publication 3 January 2011
DOI:10.1111/j.1398-9995.2011.02549.x
Edited by: Angela Haczku
Abstract
Background: We recently showed that poly(ADP-ribose)polymerase-1 (PARP-1)
may play a role in allergen (ovalbumin)-induced airway eosinophilia, potentially
through a specific effect on IL-5 production. We also reported that while IL-5
replenishment promotes reversal of eosinophilia in lungs of PARP-1)/)mice, IL-4
or Immunoglobulin E replenishment do not, suggesting a potentially significant
regulatory relationship between PARP-1 and IL-5.
Objective: To explore the mechanism by which PARP-1 regulates IL-5 production
and to determine how PARP-1 inhibition blocks allergen-induced eosinophilia.
Methods: This study was conducted using a murine model of allergic airway
inflammation and primary splenocytes.
Results: PARP-1 knockout-associated reduction in IL-5 upon allergen exposure
occurs at the mRNA level. Such an effect appears to take place after IL-4 recep-
tor activation as PARP-1 inhibition exerted no effect on JAK1/JAK3 activation.
Signal transducer and activator of transcription-6 (STAT-6) protein was severely
downregulated in spleens of PARP-1)/)mice without any effect on mRNA levels,
suggesting an effect on protein integrity rather than gene transcription. Interest-
ingly, the degradation of STAT-6 in PARP-1)/)mice required allergen stimula-
tion. Additionally, PARP-1 enzymatic activity appears to be required for STAT-6
integrity. The downregulation of STAT-6 coincided with mRNA and protein
reduction of GATA-binding protein-3 and occupancy of its binding site on the
IL-5 gene promoter. IL-4 was sufficient to induce STAT-6 downregulation in
both PARP-1)/)mice and isolated splenocytes. Such degradation may be medi-
ated by calpain, but not by proteasomes.
Conclusion: These results demonstrate a novel function of PARP-1 in regulating
IL-5 expression during allergen-induced inflammation and explain the underlying
mechanism by which PARP-1 inhibition results in IL-5 reduction.
Abbreviations
PARP-1, poly(ADP-ribose)polymerase-1; STAT-6, signal transducer
and activator of transcription-6; OVA, ovalbumin; JAK, Janus
kinase; BAL, bronchoalveolar lavage; ALLN, Acetyl-L-Leucyl-L-
Leucyl-L-Norleucinal; GATA-3, GATA-binding protein-3; IL-4R, IL-4
receptor.
Allergy
ª 2011 John Wiley & Sons A/S

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inflammatory factors [for review (1–3)]. We have demonstrated
the involvement of PARP-1 in the pathogenesis of allergen-
induced inflammation (4–6) and airway hyperresponsiveness
(AHR) (6) upon allergen (ovalbumin, OVA) exposure in a
mouse model of asthma. PARP-1 activity appears to be critical
for allergen-induced inflammation, and airway AHR as poly
(ADP-ribosyl)ation is evident in lungs of OVA-exposed ani-
mals (4, 7). Furthermore, inhibition of PARP-1 pharmacologi-
cally with old generation as well as novel drugs confers a
marked protection against the manifestation of airway inflam-
mation and AHR upon allergen exposure (4, 7, 8). PARP-1
inhibition was also shown to reduce the severity of cough and
the occurrence of dyspnea in a guinea pig asthma model (7). In
a number of studies, our laboratory has shown that inhibition
of PARP-1, either pharmacologically or genetically, markedly
attenuated OVA-induced eosinophilic infiltration as well as
reduced the expression of Th2 cytokines, particularly those
downstream of IL-4. A primary target of PARP-1 inhibition is
cytokine IL-5, which is known to be crucial for eosinophilia
(9). In a phenotype-reversal experiment, we were able to rees-
tablish eosinophilia in OVA-challenged PARP-1)/)mice.
However, intranasal administration of either IL-4 or Immuno-
globulin E (IgE) completely failed to reverse eosinophilia in
OVA-challenged PARP-1)/)mice (5). These results clearly
establish a role for PARP-1 in the pathogenesis of OVA-
induced lung inflammation in our murine model of allergic air-
way inflammation and also describe a potentially important
regulatory relationship between PARP-1 and IL-5. More
importantly, these results suggest that the role PARP-1 may be
upstream of IL-5, but downstream of IL-4.
Upon ligand binding, IL-4 receptor (IL-4R) heterodimer-
ization promotes the activation of members of the Janus
family of protein kinases (JAK1 and JAK3) [reviewed (10,
11)]. While JAK1 is constitutively associated with an a chain
of the IL-4R (IL-4Ra), JAK3 is constitutively associated with
the c chain of the receptor. The two Janus kinase (JAK) pro-
teins are subsequently activated by trans-phosphorylation of
the specific and conserved tyrosine residues located in their
activation loops. The activated JAK kinases initiate several
intracellular signaling cascades by phosphorylating multiple
tyrosine residues in the cytoplasmic domain of IL-4Ra, which
facilitates the recruitment of the IL-4Ra-specific transcription
factor signal transducer and activator of transcription-6
(STAT-6) via its SH2 domain. STAT-6 then homodimerizes
upon phosphorylation by JAK kinases, which ultimately pro-
motes its translocation to nuclei of stimulated cells. Nuclear
STAT-6 then binds to the promoters of target genes such as
GATA-binding protein-3 (GATA-3) thus, participating in
their expression (10, 11). The goal of this study was to explore
the mechanism by which PARP-1 regulates IL-5 production
and to determine how PARP-1 inhibition blocks allergen-
induced eosinophilia by focusing primarily on the IL-4 signal-
ing pathway. Given that stimulation of immune cells with
IL-4 results in IL-5 production (9), we examined the various
steps of the signaling cascade to determine whether there are
interruptions in the pathway. Interference with any of the
latter processes would explain an attenuation of Th2 inflam-
mation, which can be bypassed using IL-5 but not IL-4.
Materials and methods
Animals
C57BL/6J wild-type and C57BL/6J PARP-1)/)mice were
bred in a specific pathogen-free facility at LSUHSC and
allowed unlimited access to sterilized chow and water. Main-
tenance and experimental protocols were approved by the
LSUHSC Animal Care and Use Committee. The generation
of PARP-1)/)mice on a C57BL/6J genetic background was
described (5).
Protocol for OVA-challenged model, tissue processing, and
cytokine assessment
Six- to 8-week-old male mice were sensitized and challenged
essentially as described (5). Briefly, mice received i.p. injec-
tions of 100 lg of grade V chicken OVA (Sigma-Aldrich,
St Louis, MO, USA) mixed with 2 mg of aluminum hydrox-
ide in saline once a week for two consecutive weeks, followed
by a challenge with aerosolized OVA 1 week after the second
sensitization. The OVA aerosol was generated by a Bennett
nebulizer (DeVilbiss, Somerset, PA, USA). Control groups
were not sensitized or challenged. The mice used in each
experiment were of the same litter or the same family. Mice
were then left to recover and killed 24 h later for bronchoal-
veolar lavage (BAL) for cytokine determination. Spleens were
also removed to be subjected to homogenization followed by
protein or RNA extraction.
Bronchoalveolar lavage fluids were assessed for IL-5 using
a single-plex assay kit (Bio-Rad Laboratories, Hercules, CA,
USA) as per instructions of the manufacturer and as
described (6). In some experiments, naı¨ve mice received a
single i.p. injection of 100 ng recombinant mouse IL-4 (cat#
404-ML; R&D Systems, Minneapolis, MN, USA) in 120 ll
saline. Mice were killed 6 or 12 h after injection. Spleens
were then collected and processed for protein or RNA
extraction.
Isolation of splenocytes, immunoblot analysis, nuclear protein
extraction, electrophoretic mobility shift assay (EMSA), and
conventional and real-time reverse transcriptase polymerase
chain reaction (RT-PCR)
Splenocytes were isolated essentially as described (5). The
cells were counted and cultured in complete medium and
treated with 10 ng/ml recombinant mouse IL-4 for the time
intervals indicated in the figures. In some experiments,
splenocytes were treated with IL-4 in the presence or absence
of the proteasome inhibitor MG132 (cat# c2211; Sigma
Aldrich) or the calpain and proteasome inhibitor Acetyl-l-
Leucyl-l-Leucyl-l-Norleucinal (ALLN) (sc-29119; Santa Cruz
Biotech, Santa Cruz, CA, USA).
Whole protein extracts were subjected to immunoblot
analysis with antibodies to STAT-6 (sc-621), JAK1 (sc-1677),
JAK3 (sc-513), actin (sc-1615), or GATA-3 (sc-22206), all of
which were purchased from Santa Cruz Biotechnology or
to antibodies to phospho-JAK1 (ab5493), phospho-JAK3
(ab61102), phospho-STAT-6 (ab54461), or ubiquitin (ab7780)
Regulation of STAT-6 by PARP-1
Datta et al.
ª 2011 John Wiley & Sons A/S

Page 3

which were purchased from Abcam Inc (Cambridge, MA,
USA).
RNA was extracted from either whole spleens or isolated
splenocytes using standard methods, and cDNA was generated
using reverse transcriptase III (Invitrogen, Carlsbad, CA,
USA). The primers (Integrated DNA technologies, Coralville,
IA, USA) used for the PCRs were as follows: STAT-6: forward,
5¢-CTTCAACGACAACAGCTT-3¢; reversed, 5¢-CTGGCTC-
ATTGAGGAGAAGG-3¢; GATA3: forward: 5¢-AGCCG-
CGCTGGGTGAGCCA-3¢; reversed: 5¢-CGTGGTGGATG-
GACGTCTTG-3¢; IL-5: forward, 5¢-GAAAGAGACCTTGA-
CACAGCTG-3¢; reversed, 5¢-GAACTCTTGCAGGTAATC-
CAGG-3¢; and b-actin: forward, 5¢-ACC GTG AAA AGA
TGA CCC AGA TC-3¢; reverse, 5¢-TAG TTT CAT GGA
TGC CAC AGG-3¢. The resulting PCR products were sub-
jected to agarose electrophoresis. For real-time PCR, the
specific primers were as follows: IL-5: forward, 5¢-GGG CTT
CCT GCT CCT ATC TA-3¢; reverse, 5¢-CAG TCA TGG CAC
AGT CTG AT-3¢; STAT-6: forward, 5¢-CTC TGT GGG GCC
TAA TTT CCA-3¢; reverse, 5¢-CAT CTG AAC CGA CCA
GGA ACT-3¢; GATA-3: forward, 5¢-CTC GGC CAT TCG
TAC ATG GAA-3¢; reverse, 5¢-GGA TAC CTC TGC ACC
GTA GC-3¢; and b-actin: forward primer, 5¢-TACAGCTTCA-
CCACCACAGC-3¢, reverse primer, 5¢-TCTCCAGGG AGG-
AAGAGGAT-3¢. Quantitative determination of gene expres-
sion levels using a two-step cycling protocol was performed on
a MyIQ Cycler (Bio-Rad). Relative expression levels were cal-
culated using the 2[-Delta Delta C(T)] method (12). Quantities
of all targets from the test samples were normalized to the
mouse b-actin housekeeping gene.
Nuclear extracts were prepared from spleens collected
from the different experimental groups using an extraction
kit (Active Motif, Carlsbad, CA, USA) according to the
manufacturer’s instructions. Nuclear extracts were then sub-
jected to EMSA as described (13) using a radiolabeled
oligonucleotide containing the STAT-6 consensus sequence
5¢-GTA TTT CCC AGA AAA GGA AC-3¢ (Santa Cruz
Biotech).
Chromatin immunoprecipitation assay (ChIP)
Splenocytes were treated with 10 ng/ml IL-4 for 14 h and
fixed with formaldehyde to cross-link the chromatin and
nuclear proteins. The ChIP assay was conducted using a kit
from Active Motif following to the manufacturer’s instruc-
tions. Briefly, after enzymatic shearing, antibodies against
mouse GATA-3 were added to precipitate the sheared chro-
matin. After cross-linking reversal, DNA was isolated and
one-tenth of the final precipitated DNA was used in each
reaction. The following primers were used to target the
GATA-3-binding site on the mouse IL-5 promoter: 5¢-TCGC
CTTTATTAGGTGTCCTC-3¢ and 5¢-GGCCTTCAGCAAA
GGAAGAG-3¢ to target the )70 to )59 region.
Data analysis
All data are expressed as means ± SEM of values from at
least six mice per group or triplicate cell preparations. prism
software (GraphPad, San Diego, CA, USA) was used to ana-
lyze the differences between experimental groups by two-way
anova followed by Bonferroni posttests to compare replicate
means by row.
Results and discussion
PARP-1 gene knockout reduces allergen-induced IL-5
production at the mRNA level without affecting expression
or activation of IL-4 receptor-associated kinases
The mechanism by which PARP-1 inhibition results in a reduc-
tion in eosinophilic infiltration in a mouse model of asthma
remains unclear. The ability of IL-5 replenishment, but not of
either IL-4 or IgE, to restore eosinophilia in OVA-treated
PARP-1)/)mice suggested initially that PARP-1 may play a
role upstream of IL-5 but downstream of IL-4. We postulated
that PARP-1 gene deletion may be associated with a discon-
nect between IL-4 and IL-5, potentially through dysfunctional
IL-4 receptor (IL-4R)-associated signal transduction. Fig-
ure 1A confirms the significant reduction in IL-5 production in
BAL fluids of OVA-challenged PARP-1)/)mice compared
with their wild-type (WT) counterparts (5, 6, 14). An important
question to address was whether the effect of PARP-1 gene
deletion on IL-5 occurred at the transcriptional level.
Figure 1B (upper panels) shows that PARP-1 gene knockout
was associated with a severe reduction in IL-5 mRNA upon
OVA challenge compared with their WT counterparts as
assessed by RT-PCR analysis of RNA isolated from spleens of
the different experimental groups and confirmed by real-time
PCR (Fig. 1B, bottom panel). These results suggest that
PARP-1 may regulate IL-5 at the mRNA level. We and others
have shown the involvement of PARP-1 in the transcription of
many inflammatory genes, namely those dependent on NF-jB
(15, 16), through a number of processes including direct tran-
scription factor binding [reviewed (17)].
Signaltransduction through
complex and a crucial pathway that promotes the effects
of the T cell-mediated pathogenesis of asthma (18). To
determine whether the decrease in IL-5 mRNA expression
is linked to a defect in IL-4R-associated signal transduc-
tion, we examined the expression levels and activation
states of JAK1 or JAK3 upon OVA challenge. Figure 1C
shows that PARP-1 gene deletion affected neither the
integrity of JAK1 and JAK3 expression nor their activa-
tion as assessed by phosphorylation on tyrosines 1034 and
785, respectively. These results clearly suggest that the
effect of PARP-1 gene deletion on IL-5 mRNA expression
may occur after receptor activation through JAK1 and
JAK3 phosphorylation.
theIL-4 receptorisa
PARP-1 inhibition is associated with STAT-6 degradation in
spleens in an allergen-dependent manner and linked to a
severe reduction in GATA-3 expression
The phosphorylated residues on JAK1 and JAK3 serve as
docking sites for STAT-6 (10, 11, 18, 19). Subsequently,
STAT-6 binds to the phosphorylated cytoplasmic sequences,
Datta et al.
Regulation of STAT-6 by PARP-1
ª 2011 John Wiley & Sons A/S

Page 4

becomes phosphorylated, and disengages from the receptor.
Phosphorylated STAT-6 then homodimerizes and translo-
cates into the nucleus, where it serves as a transcription
factor for various genes including GATA-3, which in turn
drives the expression of IL-5 (10, 11). Accordingly, we next
examined the fate of STAT-6 in WT or PARP-1)/)mice
upon OVA challenge. The expression levels of STAT-6
appeared to be comparable between naı¨ve WT and PARP-1)/
)mice (Fig. 2A). It is important to note that OVA challenge
culminated in STAT-6 phosphorylation in spleens of WT
mice and that such event was largely absent in OVA-
challenged PARP-1)/)mice (Fig. 2B). Surprisingly, while
the levels of STAT-6 remained largely unchanged in OVA-
challenged WT mice, its levels were severely reduced in
spleens of PARP-1)/)mice upon OVA challenge (Fig. 2A),
suggesting involvement of an allergen-induced phenomenon.
Figure 2C shows that STAT-6 DNA binding activity, as
assessed by EMSA, was almost completely absent in nuclear
extracts isolated from spleens of OVA-challenged PARP-1)/)
mice when compared to their WT counterparts confirming
the results attained using immunoblot analysis.
The involvement of PARP-1 in the fate of STAT-6 upon
OVA challenge was confirmed using the specific and potent
inhibitor of the enzyme TIQ-A (Fig. 2D). The latter results
also indicate that the actual activity of the PARP-1 is neces-
sary for STAT-6 protein integrity upon allergen exposure.
Interestingly, STAT-6 mRNA levels were not altered in
spleens of either WT or PARP-1)/)mice subjected to OVA
challenge or left untreated as assessed by conventional and
confirmed by real-time PCR (Fig. 2E); a slight increase in
STAT-6 message was observed in spleens of both WT and
PARP-1)/)upon OVA exposure. These results suggest that
the effect of PARP-1 gene knockout in reducing STAT-6
levels was not because of a change in transcription. Rather,
PARP-1 gene knockout may promote conditions conducive
to the degradation of the signal transducer. As stated ear-
lier, GATA-3 is a key transcription factor necessary for
driving IL-5 expression and directly dependent on STAT-6
activation (20). GATA-3 gene expression requires binding of
STAT-6 to its promoter. Accordingly, we surmised that the
loss of STAT-6 would lead to a reduction in GATA-3
expression. Indeed, STAT-6-reduced expression in spleens of
A
B
C
OVA:
WT
*
*#
#
––++
PARP-1–/–
OVA:
p1034-JAK1 relative density
p785-JAK3 relative density
WT
––++
PARP-1–/–
OVA:
WT
––++
PARP-1–/–
OVA:
WT
––++
PARP-1–/–
OVA:
p1034-JAK1
Actin
JAK1
WT
1.6
1.4
1.2
0.8
0.6
0.4
0.2
0
1
0.8
0.6
0.4
0.2
0
1
*
*
*
*
*
*
––++
PARP-1–/–
OVA:
IL-5
3.0
Relative IL-5 mRNA
(fold change)
2.0
1.0
0.0
β-Actin
WT
––++
PARP-1–/–
OVA:
p785-JAK3
Actin
JAK3
WT
––++
PARP-1–/–
IL-5 (pg/ml)
30
25
15
5
0
20
10
*
Figure 1 Poly(ADP-ribose)polymerase-1 (PARP-1)
associated inhibition of IL-5 occurs at the mRNA level without an
effect on IL-4 receptor activation. (A) Wild-type (WT) and PARP-1)/)
mice were sensitized to and challenged with ovalbumin (OVA).
Mice were then killed; lungs were subjected to bronchoalveolar
lavage (BAL), and spleens were collected for RNA or protein extrac-
tion. (A) BAL fluids were assessed for IL-5 using a BioRad single-
plex system. Data are given as means ± SEM of values obtained
from at least six mice per group. *, difference from unchallenged
mice, P < 0.01; #, difference from WT mice subjected to the OVA
challenge, P < 0.01. (B) Total RNA, extracted from portions of the
collected spleens, was subjected to cDNA generation followed by
genedeletion-
conventional (upper panels) or real-time (bottom panel) polymerase
chain reaction with primers specific to murine IL-5 or b-actin. (C)
Protein extracts were prepared from the remaining portions of the
collected spleens and subjected to immunoblot analysis with anti-
bodies to JAK1, JAK3, the phosphorylated form of JAK1 at tyrosine
residue 1034 (p1034-JAK1), the phosphorylated form of JAK3 at
tyrosine residue 785 (p785-JAK3), or actin. Note that JAK1 and
JAK3 blots (C, bottom panels) are of the same samples used for
p1034-JAK1 and p785-JAK3, respectively, but were generated
using a different gel. The immunoblots were quantified using
Adobe Photoshop CS, and data are expressed as relative density;
*Difference from untreated WT control, P < 0.01.
Regulation of STAT-6 by PARP-1
Datta et al.
ª 2011 John Wiley & Sons A/S

Page 5

OVA-challenged PARP-1)/)mice coincided with a severe
reduction in GATA-3 mRNA as assessed by conventional
and confirmed by real-time PCR (Fig. 2F), which culmi-
nated in a drastic reduction in protein levels as assessed
by immunoblot analysis (Fig. 2G). Together, these results
suggest that PARP-1 may regulate IL-5 production by influ-
encing the integrity of STAT-6, affecting, as a result,
the downstream expression levels of GATA-3 mRNA and
protein.
The reduction of STAT-6 levels associated with PARP-1 gene
knockout upon allergen exposure may occur in response to
IL-4 stimulation
Given that PARP-1 gene deletion may be associated with a
disconnect between IL-4-mediated signal transduction and
IL-5 expression, we wished to determine whether OVA-
induced STAT-6 degradation in PARP-1)/)mice may be
achieved by exposure to IL-4 alone. Intraperitoneal adminis-
OVA:
WTPARP-1–/–
–+–+
OVA:
STAT-6 relative density
WTPARP-1–/–
–+–+
TIQ-A:
WTPARP-1–/–
–+–
*#
*
*
*
STAT-6
2.5
2
1.5
1
0.5
0
STAT-6 relative density
Relative STAT-6 mRNA (fold change)
Relative GATA-3 mRNA (fold change)
GATA-3 relative density
0.25
3.0
14
1.4
1.2
1
0.8
0.6
0.4
0.2
0
12
10
8
6
4
2
0
2.5
2.0
1.5
1.0
0.5
0.0
OVA:
0.2
0.15
0.1
0.05
0
OVA:
STAT-6 relative density
p641-STAT-6 relative density
WTPARP-1–/–
–+–+
OVA:
WT PARP-1–/–
–+–+
*#
*#
*#
*
*
*#
*
*
2
1.5
1
0.5
0
1.5
1
0.5
0
Actin
OVA:
WTPARP-1–/–
––++
WTPARP-1–/–
–+–+
OVA:
WT PARP-1–/–
–+–+
OVA:
WT PARP-1–/–
–+–+
STAT-6
β β-Actin
OVA:
WTPARP-1–/–
––++
GATA-3
β β-Actin
OVA:
WTPARP-1–/–
––++
GATA-3
Actin
OVA:
WT
+(+TIQ-A)
PARP-1–/–
++
STAT-6
Actin
OVA:
WTPARP-1–/–
––++
OVA:
STAT-6/DNA
Free probe
WTPARP-1–/–
––++
STAT-6
Actin
p641-STAT-6
A
DEFG
B
C
Figure 2 Poly(ADP-ribose)polymerase-1 (PARP-1)
promotes signal transducer and activator of transcription-6 (STAT-6)
protein degradation in spleens in an allergen-dependent manner
with a consequent reduction in GATA-binding protein-3 (GATA-3)
expression. Spleens from the different experimental groups were
subjected to total or nuclear protein or RNA extraction. Total protein
extracts were then subjected to immunoblot analysis with antibod-
ies to STAT-6 or actin (A) or to its phosphorylated form on tyrosine
residue 641 (p641-STAT-6) (B); the immunoblots were quantified
using Adobe Photoshop CS, and data are expressed as relative
density; *, difference from untreated control, P < 0.05; #, differ-
ence from ovalbumin (OVA)-treated wild-type (WT) mice, P < 0.05.
(C) Nuclear extracts were subjected to electrophoretic mobility shift
assay using a radiolabeled oligonucleotide containing the STAT-6
genedeletion
consensus sequence. (D) Mice received an i.p. injection of TIQ-A
(6 mg/kg) or vehicle alone 1 h prior OVA challenge. Mice were killed
24 h later. Spleens were removed for protein extraction after which
proteins were subjected to immunoblot analysis with antibodies to
STAT-6 or actin; extracts from spleens of OVA-challenged PARP-1)/)
mice were used as a control for STAT-6 degradation. The immuno-
blots were quantified as aforementioned, and data are expressed as
relative density; *, difference from OVA-treated WT mice, P < 0.05.
Total RNA was subjected to cDNA generation followed by conven-
tional (upper panels) or real-time (lower panels) polymerase chain
reaction with primers specific to STAT-6 (E) or GATA-3 (F); b-actin
was used as an internal control. *, difference from OVA-treated WT
mice, P < 0.01. (G) Protein extracts were subjected to immunoblot
analysis with antibodies to murine GATA-3 or actin.
Datta et al.
Regulation of STAT-6 by PARP-1
ª 2011 John Wiley & Sons A/S