Tumor necrosis factor-a-induced caspase-1 gene expression
Role of p73
Nishant Jain, Ch Sudhakar and Ghanshyam Swarup
Centre for Cellular and Molecular Biology, Hyderabad, India
Tumor necrosis factor-a (TNF-a) is a multifunctional
cytokine that plays an important role in the immune
response, inflammation, control of cell death and cell
proliferation. The biological effects of TNF-a are med-
iated mostly through tumor necrosis factor receptor-1
(TNF-R1), a cell-surface receptor. TNF-R1 is a type 1
transmembrane protein that contains four cysteine-rich
repeats in the extracellular domain. The distal cysteine-
rich domain mediates homophilic interaction of the
receptor molecules, thereby keeping the receptors in a
silent, homomultimerized state [1]. Binding of the tri-
meric TNF-a ligand results in the re-organization of
pre-assembled TNF-R1 complexes. These events signal
the recruitment of tumor necrosis factor-a receptor
associated death domain to the intracellular death
domain of TNF-R1. TNF-R1-bound tumor necrosis
factor-a receptor associated death domain serves as
platform for the binding of TNF receptor-associated
Keywords
caspase-1; caspase-5; IRF-1; p73; TNF-a
Correspondence
G. Swarup, Centre for Cellular and
Molecular Biology, Uppal Road,
Hyderabad)500 007, India
Fax: +91 40 27160591 ⁄ +91 40 27160311
Tel: +91 40 27192616 ⁄ +91 40 27160222
E-mail: [email protected]

sequence 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN, interferon; IRF-1, interferon regulatory factor-1; NF-jB, nuclear
factor-jB; shRNA, short hairpin RNA; TNF-a, tumor necrosis factor-a; TNF-R1, tumor necrosis factor receptor-1.
4396 FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS
factor and the serine threonine kinase receptor inter-
acting protein 1. These proteins recruit key enzymes to
TNF-R1 that orchestrate the inducible expression of
genes for diverse biological processes, including cell
death, cell growth, stress response and inflammation
[2,3]. One of the major signaling pathways induced by
TNF-a leads to the activation of transcription factor
nuclear factor-jB (NF-jB), which directly mediates the
induction of several genes, including interferon regula-
tory factor-1 (IRF-1) [4,5].
Caspase-1 is a cysteine protease that catalyses the
proteolytic processing of the pro-inflammatory cyto-
kine, interleukin-1b. Caspase-1 plays a pivotal role in
inflammation and apoptosis. Caspase-1 knockout
mice are resistant to bacterial lipopolysaccharide-
induced septic shock and are also defective in the
production of the active cytokines interleukin-1b,
interleukin-18 and interleukin-33 [6–9]. Involvement
of caspase-1 in TNF-a-induced cytotoxicity has
been determined by employing inhibitors of caspase-1
[10–12]. Caspase-1 gene expression is induced by
interferon (IFN)-a, IFN-c and TNF-a [13–17]. In
addition, treatment of tumor cell lines with doxorubi-
cin, cisplatin and UV radiation also induces caspase-1
mRNA [18–20]. However, the mechanism of activa-
tion of caspase-1 gene expression by TNF-a is
unknown, although signaling by TNF-a has been

moter [18,38]. An Ets-1-binding site has also been
identified in the caspase-1 promoter upstream of the
minimal promoter [36]. Endogenous, as well as exoge-
nous, p73 activates caspase-1 promoter primarily
through the p53 ⁄ p73-binding site. Optimal activation
of the caspase-1 promoter by IFN-c requires p73 [19].
However, the transcription factors involved in the acti-
vation of the caspase-1 promoter by TNF-a are not
known. In the present study we analyzed the role of
p73 and IRF-1 in mediating TNF-a-induced caspase-1
promoter activation. Our results showed that p73 plays
an important role in TNF-a-induced caspase-1 gene
expression from endogenous, as well as exogenous,
promoters. In addition, our results revealed that
TNF-a induces p73 gene expression.
Results
TNF-a activates caspase-1 promoter
The human lung carcinoma cell line A549 was treated
with TNF-a and RNA was isolated from TNF-a-trea-
ted and -untreated cells at the time-points indicated.
The level of caspase-1 mRNA was determined by semi-
quantitative RT-PCR. There was a time-dependent
increase in caspase-1 mRNA levels upon treatment of
the cells with TNF-a (Fig. 1A). There was no change in
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
mRNA levels, which was used as a control. Caspase-1
mRNA levels reached maximum levels after 9 h of
treatment with TNF-a and remained high up to 24 h.
The caspase-1 protein level also increased upon treat-
ment of cells with TNF-a, as shown by western blot

mal activation of the caspase-1 promoter by TNF-a.
Role of p73 in TNF-a-induced activation
of the caspase-1 promoter
We used dominant negative mutants of p53 and p73 to
assess the requirement of these proteins for TNF-a-
induced caspase-1 promoter activity. Previously, it has
been shown that p73DD, a deletion mutant of p73a,
inhibits p73 function without affecting p53-dependent
transcriptional activation [39,40]. We observed that
TNF-a-induced caspase-1 promoter activity was inhib-
ited by p73DD (60% inhibition, P < 0.05) but not by
the p53-specific inhibitor, p53DD (Fig. 3A).
To provide further evidence for the requirement of
p73 in TNF-a-induced activation of the caspase-1 pro-
moter, we used a p73-directed short hairpin RNA
(shRNA). This shRNA has been shown to reduce p73
levels and was presumed to be specific for p73 because
it did not affect the level of C3G or other endogenous
proteins tested [19]. The mutation of two nucleotides
inactivated this shRNA, which was used as a control.
The p73-directed shRNA strongly reduced p73-induced
caspase-1 promoter activity (Fig. 3B). TNF-a-induced
caspase-1 promoter activity was inhibited by p73-direc-
ted shRNA (67% inhibition; P < 0.05) (Fig. 3C).
Basal caspase-1 promoter activity was also inhibited
by this shRNA. These results suggest that p73 plays
an important role in the TNF-a-induced activation of
caspase-1 promoter.
Fig. 1. Induction of caspase-1 gene expression and promoter activation by TNF-a. (A) A549 cells were treated with 10 ngÆmL
)1

western blot analysis. The p73 protein level was
knocked down by Adp73shRNA virus but not by con-
trol virus (Fig. 4A). C3G protein levels or endogenous
Cdk-2 levels were not affected significantly by
Adp73shRNA. To determine the effect of knockdown
of endogenous p73 on caspase-1 gene expression, A549
cells were infected with adenoviruses for 24 h; subse-
quently, the cells were treated with TNF-a for 6 or
9 h. RNA was then isolated and subjected to semi-
quantitative RT-PCR analysis. As expected, adenoviral
p73shRNA abrogated endogenous p73 mRNA levels
as compared with the control shRNA-infected cells
(Fig. 4B). The level of TNF-a-induced p73 mRNA
was also reduced by p73shRNA. Next, we determined
caspase-1 mRNA levels in the TNF-a-treated shRNA-
infected cells. There was a significant decrease of
TNF-a-induced caspase-1 mRNA levels in the
Adp73shRNA-infected cells as compared with the con-
trol adenovirus-infected cells (Fig. 4B).
We also investigated whether knockdown of p73
would affect caspase-1 protein expression. A549 cells
C
B
A
D
Fig. 2. Effect of mutation of the p73-respon-
sive and IRF-1-responsive sites on TNF-a
induced caspase-1 promoter activity. (A,B)
Schematic representations of wild-type and
mutated caspase-1 promoter-reporter

) for 24 h. CAT activities relative to
the untreated control are shown (n ¼ 3). (B) shRNA for p73 inhibits
p73-induced caspase-1 promoter activity. A549 cells were transfect-
ed with pC-WT reporter plasmid (100 ng) and p73b (5 ng), along
with 200 ng of p73 shRNA (shRNA) or 200 ng of a control shRNA
(control). After 28 h of transfection, cell lysates were made for
reporter assays. CAT activities relative to the control without p73
are shown. (C) Effect of p73-directed shRNA on caspase-1 pro-
moter activity induced by TNF-a. A549 cells were cotransfected
with pC-WT reporter plasmid (100 ng) along with shRNA for p73 or
control shRNA-expressing plasmids (200 ng). After 6 h of transfec-
tion, cells were treated with TNF-a or left untreated for 24 h. CAT
activities relative to the untreated control are shown (n ¼ 3).
B
C
A
Fig. 4. TNF-a-induced caspase-1 gene expression is inhibited by
p73 shRNA. (A) Efficacy of adenovirus expressing p73-directed
shRNA. HeLa cells were transfected with p73a and C3G expres-
sion plasmids; after 4 h the cells were infected with control or
p73shRNA-expressing adenovirus. After another 24 h, the cells
were harvested and extracts were subjected to western blot analy-
sis using specific antibodies for p73 (anti-HA), C3G and tubulin.
C3G served as a transfection control and tubulin as a loading con-
trol. (B) A549 cells were infected with adenoviruses expressing
control shRNA (Ad con) or p73shRNA (Ad shRNA). After 24 h of
infection, the cells were treated with TNF-a for the indicated time-
periods. Total RNA was isolated and semiquantitative RT-PCR anal-
ysis for p73, caspase-1 and GAPDH was performed. (C) A549 cells
were infected with adenoviruses expressing control shRNA (Ad

induction of caspase-5 mRNA by TNF-a was reduced
in cells infected with Adp73shRNA compared with
control virus-infected cells (Fig. 6B), although the
basal level of caspase-5 mRNA was not reduced. Cas-
pase-5 gene expression was induced by the overexpres-
sion of p73a and also by p73b (Fig. 6C). These results
suggest that caspase-5 gene expression is induced by
p73 and that TNF-a-induced caspase-5 gene expression
is mediated, in part, by p73.
Effect of TNF-a on p73 promoter
The treatment of cells with TNF-a has been shown to
increase the p73 protein level [32,34]. The promoter of
p73 has E2F1-binding sites and the TNF-a treatment
of cells has been shown to recruit E2F1 to these sites
in the p73 promoter that are occupied by E2F3 in
unstimulated cells [34]. However, activation of p73
promoter activity by TNF-a has not been demon-
strated. We found that the p73 promoter reporter was
not activated by TNF-a (Fig. 7A). We have previously
found that IFN-c-induced caspase-1 promoter activa-
tion requires p73 and that p73 protein accumulates in
A
C
B
Fig. 5. Adenovirus-mediated expression of
p73 induces caspase-1 mRNA and protein.
(A) A549 cells were infected with adeno-
viruses Ad Con, Ad p73a or Ad p73b. After
24 or 48 h of infection, cell lysates were
prepared for western blotting with antibod-

induced increase in p73 mRNA are not present in this
promoter and may be present upstream or downstream
of this promoter.
Discussion
The results presented here show that stimulation of the
human lung carcinoma cell line, A549, with TNF-a
increases the expression of caspase-1 mRNA and pro-
tein. The increase in caspase-1 gene expression is prob-
ably caused by activation of the promoter because the
caspase-1 promoter is activated in response to TNF-a.
A
B
C
Fig. 6. TNF-a enhances caspase-5 mRNA levels. (A) Total RNA was
isolated from A549 cells treated with TNF-a at the indicated time-
points and subjected to semiquantitative RT-PCR analysis for cas-
pase-5 and GAPDH. (B) TNF-a-induced caspase-5 gene expression
is inhibited by p73 shRNA. A549 cells were infected with adenovi-
ruses expressing control shRNA (Ad con) or p73shRNA (Ad
shRNA). After 24 h of infection, the cells were treated with TNF-a
for the indicated time. Total RNA was isolated and semiquantitative
RT-PCR analysis for caspase-5 and GAPDH was performed. Num-
bers at the top indicate the relative amount of caspase-5 PCR prod-
uct. (C) Adenovirus-mediated expression of p73 induces caspase-5
mRNA. A549 cells were infected with the adenoviruses Ad con, Ad
p73a or Ad p73b. Total RNA was isolated 24 h postinfection and
caspase-5 mRNA levels were analyzed by RT-PCR. GAPDH was
used as a control.
A
B

ment of p73 for TNF-a-induced signaling to caspase-1,
namely (i) mutation of the p73-responsive site compro-
mises TNF-a-induced caspase-1 promoter activity,
(ii) knockdown of p73 by shRNA (or a dominant nega-
tive mutant) reduces the activation of the caspase-1 pro-
moter in response to TNF-a and (iii) knockdown of p73
by shRNA reduces the expression of caspase-1 mRNA
and protein in response to TNF-a. Further support for
a role of p73 in TNF-a-induced caspase-1 gene expres-
sion is provided by the observation that p73 mRNA and
protein are up-regulated by TNF-a, which precedes the
maximal induction of caspase-1 mRNA.
IRF-1, p53, Ets-1 and p73 have been reported to be
direct transcriptional activators of caspase-1 [18,19,36,
38]. We evaluated their ability to affect the activation
of caspase-1 promoter by TNF-a. Our experiments
revealed that the optimal activation of caspase-1 pro-
moter by TNF-a requires p73 but not p53. These
results are consistent with previous reports that TNF-
a-induced apoptosis requires p73 and not p53 [32]. An
Ets-1-binding site has been identified in the upstream
region of the caspase-1 promoter, which is not present
in the promoter constructs used in this study. As the
caspase-1 promoter-reporter construct does not have
an Ets-1-binding site but is activated by TNF-a to the
same extent as that with an Ets site (data not shown),
a role of Ets-1 in caspase-1 promoter activation by
TNF-a is very unlikely.
A composite GAS ⁄ jB promoter element present in
the IRF-1 promoter mediates the induction of IRF-1

known. It has been speculated that p73 contributes to
a mitochondria-dependent apoptotic mechanism in the
TNF-a-induced pathway [32]. In the present study we
have shown that p73 contributes to TNF-a-induced
caspase-1 and -5 gene expression. Although the
primary role of caspase-1 and -5 is believed to be in
the production of cytokines, we speculate that they
may also contribute, to some extent, to TNF-a-
induced apoptosis in some cells.
In conclusion, our results show that TNF-a-induced
caspase-1 gene expression is mediated by IRF-1 and
p73, which activate the promoter through their respec-
tive binding sites. TNF-a induces p73 and IRF-1 gene
expression, which precede caspase-1 gene expression.
TNF-a induces caspase-5 gene expression, which is
also mediated, in part, by p73. These observations pro-
vide support to the suggestion that p73 is an important
component of the TNF-a-induced signaling pathway
leading to gene expression.
Experimental procedures
Cell culture and transfections
A549, HeLa and 293T cells were maintained at 37 °Cina
CO
2
incubator in Dulbecco’s modified Eagle’s medium sup-
plemented with 10% fetal bovine serum. The transfections
were carried out using Lipofectamine Plus
TM
reagent (Invi-
trogen, San Diego, CA, USA) according to the manufac-

reaction mixture for p73 contained 10% dimethylsulfoxide.
Expression vectors and antibodies
The expression vectors of p73a and p73b , cloned in-frame
with the hemagglutinin tag into pcDNA3-HA, were a kind
gift from Gerry Melino (Department of experimental medi-
cine and biochemical sciences, University of Rome, Italy)
[23]. pcDNA3-p73DD and pcDNA3-p53DD were gifts of
William Kaelin (DFCI, Harvard Medical School, Boston,
MA, USA) [39]. Cdk-2, IRF-1, C3G, tubulin and caspase-1
antibodies were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA, USA); mouse monoclonal anti-hemagglu-
tinin (HA) was from Roche Molecular Biochemicals (India-
napolis, IN, USA); p73 monoclonal antibody (IMG 259)
was from Imgenex (San Diego, CA, USA) and Cy-3-conju-
gated anti-mouse immunoglobulin was from Amersham
Pharmacia Biotech (Piscataway, NJ, USA).
Construction of adenoviral vectors
All adenoviral vectors were generated using the AdEasy
System [46] kindly provided by B. Vogelstein (Howard
Hughes Medical Institute and The Sidney Kimmel Compre-
hensive Cancer Center, The Johns Hopkins Medical Institu-
tions, Baltimore, MD, USA). Adp73a or Adp73b,
expressing the p73a or -b isoform, was constructed as fol-
lows: the p73a or -b cDNA was isolated from the
pcDNA3.1-p73 plasmid by KpnI ⁄ XhoI digestion and cloned
into the pAdtrack-cytomegalovirus (CMV) plasmid under
the control of the CMV promoter terminated by the simian
virus 40 (SV40) polyadenylation signal, resulting in pAd-
track-CMV-p73a or -p73b. The pAdtrack-CMV plasmid
was utilized as a control vector. The adenovirus-based

The primers used were: forward, 5¢-CGCTCGAGGATCC
AGAGCCCGAGCCCACA-3¢ and reverse, 5¢-CGAAGCT
TCCGTCGCAGCCCCGGGCA-3¢ [48]. The amplified pro-
moter fragment of 930 bp was cloned into the pMOSBlue
vector (Amersham) and sequenced. The p73 promoter frag-
ment was then excised by digestion with HindIII and
XhoI, subcloned into the pGL3-BASIC vector (Promega,
Madison, WI, USA) and named p73Pr-Luc.
Vector expressing p73-directed shRNA
The shRNA expression vector targeting p73 was constructed
using the U6 promotor-based vector and has been described
previously [19,47]. The p73 sequence targeted by this shRNA
was from nucleotides 638–656 (Gene Bank
TM
accession num-
ber: NM_005427). A mutant of this shRNA was made by
substituting two bases in the middle of the target sequence
TNF-a-induced caspase-1 expression requires p73 N. Jain et al.
4404 FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS
and was found to be functionally inactive. This mutant
shRNA expression plasmid was used as a control.
Western blot analysis
Cells were washed twice with PBS and lysed in 1 · SDS
sample buffer. Proteins were separated on 10% SDS-poly-
acrylamide gels and blotted onto nitrocellulose membranes.
The blot was washed twice with Tween-Tris-buffered saline
before blocking nonspecific binding with 5% nonfat dry
milk (BLOTTO; Santa Cruz Biotechnology). The caspase-1,
C3G, Cdk-2 and other antibodies were used at 1 : 1000
dilutions, and the blot was incubated for 1 h at room tem-

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