Báo cáo khoa học: Tumor necrosis factor-a-induced caspase-1 gene expression pptx

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

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:
(Received 2 May 2007, revised 15 June
2007, accepted 2 July 2007)
doi:10.1111/j.1742-4658.2007.05969.x

Tumour necrosis factor-a (TNF-a) is a cytokine that is involved in many
functions, including the inflammatory response, immunity and apoptosis.
Some of the responses of TNF-a are mediated by caspase-1, which is
involved in the production of the pro-inflammatory cytokines interleukin1b, interleukin-18 and interleukin-33. The molecular mechanisms involved
in TNF-a-induced caspase-1 gene expression remain poorly defined, despite
the fact that signaling by TNF-a has been well studied. The present study
was undertaken to investigate the mechanisms involved in the induction of
caspase-1 gene expression by TNF-a. Treatment of A549 cells with TNF-a
resulted in an increase in caspase-1 mRNA and protein expression, which
was preceded by an increase in interferon regulatory factor-1 and p73 protein levels. Caspase-1 promoter reporter was activated by the treatment of
cells with TNF-a. Mutation of the interferon regulatory factor-1 binding
site resulted in the almost complete loss of basal as well as of TNF-ainduced caspase-1 promoter activity. Mutation of the p53 ⁄ p73 responsive
site resulted in reduced TNF-a-induced promoter activity. Blocking of p73

function by a dominant negative mutant or by a p73-directed small hairpin
RNA reduced basal as well as TNF-a-induced caspase-1 promoter activity.
TNF-a-induced caspase-1 mRNA and protein levels were reduced when
p73 mRNA was down-regulated by small hairpin RNA. Caspase-5 gene
expression was induced by TNF-a, which was inhibited by the small hairpin RNA-mediated down-regulation of p73. Our results show that TNF-a
induces p73 gene expression, which, together with interferon regulatory
factor-1, plays an important role in mediating caspase-1 promoter
activation by TNF-a.

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 mediated 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 cysteinerich domain mediates homophilic interaction of the

receptor molecules, thereby keeping the receptors in a
silent, homomultimerized state [1]. Binding of the trimeric 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

Abbreviations
CAT, chloramphenicol acetyltransferase; Cdk-2, cyclin dependent kinase 2; CMV, cytomegalovirus; Ets-1, E26 transformation-specific
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.

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N. Jain et al.

factor and the serine threonine kinase receptor interacting 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 regulatory factor-1 (IRF-1) [4,5].
Caspase-1 is a cysteine protease that catalyses the
proteolytic processing of the pro-inflammatory cytokine, interleukin-1b. Caspase-1 plays a pivotal role in
inflammation and apoptosis. Caspase-1 knockout
mice are resistant to bacterial lipopolysaccharideinduced 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 doxorubicin, cisplatin and UV radiation also induces caspase-1
mRNA [18–20]. However, the mechanism of activation of caspase-1 gene expression by TNF-a is
unknown, although signaling by TNF-a has been
studied extensively.

The p73 protein belongs to the p53 family of transcription factors. Unlike the p53 gene, which shows
only little alternative splicing, the p73 gene encodes
multiple protein isoforms, which arise as a result of
alternative promoter usage and differential mRNA
splicing [21–26]. Exposure to chemotherapeutic agents,
such as cisplatin, camptothecin and doxorubicin,
causes the stabilization and activation of the p73 protein [27–29]. When overexpressed, p73 binds to p53
DNA target sites, transactivates p53-responsive genes
and is capable of inducing cell cycle arrest and apoptosis in a p53-like manner. Clues to the physiological
roles of p53 and p73 came from the respective knockout mice. The main phenotype of the p53-deficient
mouse is the high incidence of spontaneous tumours
[30]. In contrast, p73-deficient mice exhibit chronic
infections, inflammation and neural defects [31]. Previous reports have shown that p73 contributes to
TNF-a-induced apoptosis in mouse thymocytes and
vascular smooth muscle cells [32,33]. These findings
are consistent with a recent study in a human B-cell
lymphoblastoid cell line (Ramos cells) in which TNF-a
increased p73 protein levels [34].
Activation of caspase-1 gene expression can be
mediated by IRF-1, signal transducer and activator of

TNF-a-induced caspase-1 expression requires p73

transcription 1, p53, p73 and E26 transformation-specific sequence 1 (Ets-1) [13,18,19,35–37]. Analysis of
the human caspase-1 promoter has shown functional
binding sites for IRF-1 and p53 in the minimal promoter [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 exogenous, 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 activation 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-treated and -untreated cells at the time-points indicated.
The level of caspase-1 mRNA was determined by semiquantitative 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 treatment of cells with TNF-a, as shown by western blot
analysis (Fig. 1B). The level of IRF-1 mRNA and protein also increased upon TNF-a treatment of these cells
and this increase was transient (Fig. 1A,C). There was
a decrease in IRF-1 mRNA as well as in protein levels
when cells were treated for longer than 3 h with TNF-a
(Fig. 1A,C). The levels of p73 mRNA and protein
increased upon treatment of cells with TNF-a
(Fig. 1A,C). By employing specific primers, we detected
that the alpha-isoform of p73 was induced in A549
cells. These results raised the possibility that IRF-1 and

p73 may be involved in regulating or maintaining caspase-1 gene expression in cells treated with TNF-a.
A caspase-1 promoter reporter plasmid was transfected into A549 cells and, 6 h after transfection,
the cells were treated with TNF-a for 24 h. TNF-a
treatment of cells resulted in an increase in caspase-1

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Fig. 1. Induction of caspase-1 gene expression and promoter activation by TNF-a. (A) A549 cells were treated with 10 ngỈmL)1 of TNF-a for
3, 6, 9, 12 or 24 h. After total RNA isolation, caspase-1, IRF-1, p73 and GAPDH mRNA levels were analyzed by semiquantitative RT-PCR. C,
untreated control cells. Numbers at the top of the lower panel indicate the relative amount of the p73 PCR product. (B,C) Immunoblotting
was performed with total proteins isolated from A549 cells treated with TNF-a for the indicated time. The immunoblot was performed with
antibodies against caspase-1, IRF-1, p73 and cyclin dependent kinase 2 (Cdk-2). Cdk-2 was used as a loading control. Numbers at the top of
(C) indicate the relative amount of the p73 protein. (D) TNF-a activates caspase-1 promoter. A549 cells were transfected with pC-WT
(100 ng), and, after 6 h, were treated with the indicated concentrations of TNF-a for 24 h. Chloramphenicol acetyltransferase (CAT) activities
relative to the untreated control are shown.

promoter activity in a dose-dependent manner
(Fig. 1D). Functional binding sites for IRF-1 and
p53 ⁄ p73 have been identified in the human caspase-1
promoter [18,19,38]. Mutation of the IRF-1-binding
site resulted in a near-complete loss of basal, as well as
of TNF-a-induced, promoter activity (Fig. 2A,C).
Mutation of the p53 ⁄ p73 responsive site resulted in a

reduction of TNF-a-induced caspase-1 promoter activity from 4.7-fold to 2.3-fold (Fig. 2B,D); however, the
basal activity was not affected, as reported previously
[19]. These results suggested that, in addition to
IRF-1, a p53 family member is also required for optimal 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-ainduced caspase-1 promoter activity. Previously, it has
been shown that p73DD, a deletion mutant of p73a,
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inhibits p73 function without affecting p53-dependent
transcriptional activation [39,40]. We observed that
TNF-a-induced caspase-1 promoter activity was inhibited 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 promoter, 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-directed 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.

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TNF-a-induced caspase-1 expression requires p73

A

B

C

D

Fig. 2. Effect of mutation of the p73-responsive 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
constructs. (C,D) pC-WT, pC-MT-IRF-1 or
pC-MT-p53 (100 ng) were transfected into
A549 cells, and, after 6 h, were treated with
TNF-a (10 ngỈmL)1) for 24 h. CAT activities
relative to the untreated control are shown
(n ¼ 3).

Knockdown of endogenous p73 inhibits
TNF-a-induced caspase-1 gene expression
We hypothesized that p73 is required for the optimal
activation of caspase-1 gene expression by TNF-a, as
evident from our dominant negative and shRNA-based

promoter assay experiments. To test this assumption,
we generated an adenovirus- based vector, which
expressed shRNA, to knock down the expression of
p73. We derived recombinant adenoviruses encoding
control shRNA or p73shRNA under the control of the
U6 promoter (Ad control shRNA or Adp73shRNA),
as described in the Experimental procedures. The control virus expresses the mutated shRNA. These adenoviruses co-expressed green fluorescent protein as a
reporter for infection efficiency. To determine the
knockdown efficacy of this virus, HeLa cells were
transfected with p73a and C3G expression plasmids
and, 4 h later, the cells were infected with control or
Adp73shRNA viruses. After another 24 h, the cells
were harvested and the cell lysates were subjected to

western blot analysis. The p73 protein level was
knocked down by Adp73shRNA virus but not by control 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; subsequently, the cells were treated with TNF-a for 6 or
9 h. RNA was then isolated and subjected to semiquantitative 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 shRNAinfected cells. There was a significant decrease of
TNF-a-induced caspase-1 mRNA levels in the
Adp73shRNA-infected cells as compared with the control adenovirus-infected cells (Fig. 4B).
We also investigated whether knockdown of p73
would affect caspase-1 protein expression. A549 cells

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A

B

B
C

Fig. 3. Role of p73 in TNF-a-induced caspase-1 promoter activity.
(A) pC-WT reporter plasmid was transfected along with p53DD or
p73DD (100 ng of each) or control plasmid. After 6 h the cells were
treated with TNF-a (10 ngỈmL)1) 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 transfected 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 promoter 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 transfection, cells were treated with TNF-a or left untreated for 24 h. CAT
activities relative to the untreated control are shown (n ¼ 3).

were infected with control or Adp73shRNA viruses
and then treated with TNF-a. In the Adp73shRNAinfected cells, the TNF-a-induced caspase-1 protein
level was also markedly lower than that of the control
virus-infected cells (Fig. 4C). Overall, these results suggest that p73 plays an important role in TNF-ainduced caspase-1 gene and protein expression.
p73 induces caspase-1 gene and protein
expression
To determine the effect of p73 on caspase-1 protein
expression, adenoviruses were constructed that express
the a and b isoforms of p73. A549 cells were infected
with adenoviruses expressing p73 proteins or with
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C

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 expression 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 analysis using specific antibodies for p73 (anti-HA), C3G and tubulin.
C3G served as a transfection control and tubulin as a loading control. (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 timeperiods. Total RNA was isolated and semiquantitative RT-PCR analysis for p73, caspase-1 and GAPDH was performed. (C) A549 cells
were infected with adenoviruses expressing control shRNA (Ad
con) or p73shRNA (Ad shRNA) for 24 h, followed by treatment
with TNF-a for 12 or 18 h. Western blot analysis for caspase-1 and
Cdk-2 is shown.

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TNF-a-induced caspase-1 expression requires p73

A

B

C
Fig. 5. Adenovirus-mediated expression of
p73 induces caspase-1 mRNA and protein.
(A) A549 cells were infected with adenoviruses Ad Con, Ad p73a or Ad p73b. After
24 or 48 h of infection, cell lysates were
prepared for western blotting with antibodies for caspase-1, p73 and Cdk-2. (B) A549
cells were infected with the indicated adenoviruses. RNA was isolated 24 and 48 h
postinfection and caspase-1 mRNA levels
were analyzed by RT-PCR. GAPDH
was used as a control. (C) Activation of
caspase-1 promoter by p73a, p73b and
IRF-1. A549 cells were transfected with
100 ng of pC-WT and the indicated amounts
of p73a, p73b or IRF-1 expression plasmids.
CAT activities relative to the control are
shown.

control adenovirus, and, after 24 or 48 h of infection,
cell lysates were prepared for western blotting. Expression of p73a and p73b in A549 cells resulted in the

induction of caspase-1 protein expression, as determined by western blotting (Fig. 5A). Infection with control virus did not induce caspase-1. Caspase-1 mRNA
levels were also increased upon the expression of p73a
or p73b (Fig. 5B). Caspase-1 promoter was strongly
activated by p73a and p73b in A549 cells (Fig. 5C).
TNF-a-induced caspase-5 gene expression:
role of p73
The treatment of murine osteoblastic cells with TNF-a
has been shown to induce caspase-11 gene expression,
in addition to the induction of caspase-1 and -7 [41].
Caspase-5 is believed to be a human counterpart of
murine caspase-11 [42,43]. Caspase-11 is an upstream
regulator of caspase-1 activation [44]. Therefore, we
explored the possibility of regulation of caspase-5 by
TNF-a and p73. We found that caspase-5 mRNA levels increased in TNF-a-treated A549 cells, reaching
maximum levels after 9 h of treatment, and remained
high up to 24 h (Fig. 6A). To determine the effect of
knockdown of endogenous p73 on caspase-5 gene

expression, A549 cells were infected with adenovirus
(Adp73shRNA) and then treated with TNF-a. The
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. Caspase-5 gene expression was induced by the overexpression 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 demonstrated. 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 activation requires p73 and that p73 protein accumulates in

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A

A

B

C

B

Fig. 6. TNF-a enhances caspase-5 mRNA levels. (A) Total RNA was
isolated from A549 cells treated with TNF-a at the indicated timepoints and subjected to semiquantitative RT-PCR analysis for caspase-5 and GAPDH. (B) TNF-a-induced caspase-5 gene expression
is inhibited by p73 shRNA. 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. Total RNA was isolated and semiquantitative
RT-PCR analysis for caspase-5 and GAPDH was performed. Numbers at the top indicate the relative amount of caspase-5 PCR product. (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.

response to treatment with IFN-c [19]. We explored
the possibility of regulation of p73 gene expression by
IFN-c. To achieve this, we treated A549 cells with
IFN-c for various periods of time; the p73 mRNA
level was enhanced by IFN-c treatment of cells but to
a much lesser extent than that induced by TNF-a
(Fig. 7B). In contrast to TNF-a, the IFN-c treatment
of A549 cells resulted in a small, but significant
(P < 0.01), increase in p73 promoter activity
(Fig. 7A), which is consistent with a small increase in
the p73 mRNA level observed upon IFN-c treatment
of cells. These observations indicate that the TNF-ainduced increase in p73 mRNA level may not be a
result of promoter activation but may involve a posttranscriptional mechanism. Alternatively, it is possible
that the DNA elements which mediate the TNF-a4402

Fig. 7. Effect of TNF-a on p73 promoter activity. (A) A549 cells
were transfected with 100 ng of p73 promoter-reporter plasmid
(p73Pr-Luc) treated with TNF-a (10 ngỈmL)1) and interferon-c (IFN-c)
(100 ngỈmL)1) for 24 h. Luciferase activities relative to the
untreated control are shown (n ¼ 3) after normalizing with b-galactosidase activities. (B) A549 cells were treated with IFN-c for the
indicated periods of time; subsequently, total RNA was isolated
and subjected to semiquantitative RT-PCR analysis for p73,

GAPDH, caspase-1 and IRF-1. Cells treated with TNF-a for 6 h
were used for comparison.

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 protein. The increase in caspase-1 gene expression is probably caused by activation of the promoter because the
caspase-1 promoter is activated in response to TNF-a.

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Mutation of the IRF-1-binding site abolished TNF-ainduced caspase-1 promoter activity. Optimal activation of the caspase-1 promoter by TNF-a required
the p73 ⁄ p53 responsive site. Moreover, blocking the
function of p73 by employing specific inhibitors significantly compromised the activation of the caspase-1
promoter. However, blocking the function of p53 had
no significant effect on TNF-a-induced promoter activity. TNF-a also enhances the gene expression of the
full-length isoform of p73, p73a. Taken together, these
results are consistent with a pathway in which TNF-ainduced p73 and IRF-1 contribute to caspase-1 promoter activation and gene expression.
Various lines of evidence have established a requirement of p73 for TNF-a-induced signaling to caspase-1,
namely (i) mutation of the p73-responsive site compromises TNF-a-induced caspase-1 promoter activity,
(ii) knockdown of p73 by shRNA (or a dominant negative mutant) reduces the activation of the caspase-1 promoter 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 expression 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 promoter by TNF-a requires p73 but not p53. These
results are consistent with previous reports that TNFa-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
transcription in response to TNF-a. The jB motif has
been demonstrated to be occupied by the p50 ⁄ p65
subunits of NF-jB [4,5]. Blocking of NF-jB by super
repressor inhibitor of NF-jB (I-jB) strongly inhibited
activation of the caspase-1 promoter by TNF-a but
not by overexpressed IRF-1 (data not shown). Taken
together, our results are consistent with the suggestion
that NF-jB-mediated IRF-1 expression is required for
TNF-a-induced caspase-1 promoter activation.

TNF-a-induced caspase-1 expression requires p73

In murine cells, caspase-1 activation requires caspase-11 [44]. Caspase-5 is believed to be the human
ortholog of caspase-11 because both are expressed at
a low level in most tissues and are induced by IFN-c
and lipopolysaccharide in responsive cells. Expression
of caspase-11 mRNA is induced by TNF-a in murine
osteoblastic cells [41]. We found that caspase-5 gene
expression is induced by TNF-a in A549 cells and
also by the overexpression of p73. Induction of caspase-5 by TNF-a provides further support to the suggestion that in human cells caspase-5 serves a
function similar to that of caspase-11 in murine cells.
TNF-a-induced caspase-5 gene expression, like that
of caspase-1, was partly inhibited by p73-directed
shRNA. Thus, it is probable that the role of p73 in
TNF-a-induced gene expression is not restricted to
caspase-1 and that p73 may be involved in the regulation of other genes.
Although the requirement of p73 for TNF-a-induced
apoptosis has been demonstrated in various cells
[32,33], the precise role of p73 in this pathway is not
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-ainduced 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 respective 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 provide 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 °C in a
CO2 incubator in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The transfections
were carried out using Lipofectamine PlusTM reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s instructions. All the plasmids for transfection were
prepared by using Qiagen columns (Hilden, Germany).
Human TNF-a (Sigma, St Louis, MO, USA) was added

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wherever indicated at a final concentration of 10–
20 ngỈmL)1.

RT-PCR
Total RNA was isolated using the TRIzol reagent (Invitrogen). Semiquantitative RT-PCR was carried out
essentially as described previously [18,45]. RNA was
reverse transcribed using reagents from the first-strand
cDNA synthesis kit (Invitrogen). Primers for amplification

of caspase-1 and GAPDH have been described previously
[18]. Primers IRF-2 (5¢-CGGAATTCTACGGTGCA
CAGGGAATGGCC-3¢) and IRF-3 (5¢-TACAACAGA
TGAGGATGAGGAAGGG-3¢) were used for the amplification of human IRF-1 mRNA. Primers C5F2 (5¢-CCT
GCAAGGAATGGGGCTCACTAT-3¢)
and
RCASP
(5¢-CTCTGCAGGCCTGGACAATGATGAC-3¢)
were
used for the amplification of human caspase-5 mRNA.
The primers used for p73 amplification – p73P1 (5¢-ACT
TTGAGATCCTGATGAAGCTG-3¢) and p73P2 (5¢-CA
GATGGTCATGCGGTACTG-3¢) – were designed in a
region common to various TA isoforms (a, b, c and d) of
p73. The PCR conditions for p73 were: 1 cycle of 3 min
at 95 °C; 37 cycles of 1 min at 95 °C, 1 min at 60 °C and
1 min at 72 °C; and 1 cycle of 7 min at 72 °C. The PCR
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 medicine 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-hemagglutinin (HA) was from Roche Molecular Biochemicals (Indianapolis, IN, USA); p73 monoclonal antibody (IMG 259)
was from Imgenex (San Diego, CA, USA) and Cy-3-conjugated 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 Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, Baltimore, MD, USA). Adp73a or Adp73b,
expressing the p73a or -b isoform, was constructed as follows: the p73a or -b cDNA was isolated from the
pcDNA3.1-p73 plasmid by KpnI ⁄ XhoI digestion and cloned

4404

into the pAdtrack-cytomegalovirus (CMV) plasmid under
the control of the CMV promoter terminated by the simian
virus 40 (SV40) polyadenylation signal, resulting in pAdtrack-CMV-p73a or -p73b. The pAdtrack-CMV plasmid
was utilized as a control vector. The adenovirus-based
shRNA vector was generated by subcloning the transcriptional unit of p73 shRNA (0.4 kb) from the pmu6 vector
described previously [19,47]. The U6-SH cassette was
cloned into the pAdTrack plasmid upstream of the CMVgreen fluorescent protein cassette (1.6 kb). Recombinant
plasmids were generated by homologous recombination in
AdEasier cells. The 293T cells were transfected with the
recombinant adenoviral plasmids using Lipofectamine 2000
(Invitrogen), and adenoviruses were collected.

Reporter plasmids and reporter assays
The reporter plasmid pC-WT, which contains the human
caspase-1 promoter from positions )182 to +42, relative to
the transcriptional start site, cloned upstream of the CAT
reporter gene, has been described previously [38]. The
reporter plasmid pC-MT-p53 and pC-MT-IRF-1, were
derived from pC-WT by mutating the p53 and the IRF-1responsive sites, respectively, and have been described
previously [18,19]. Cells grown in 24-well plates were

transfected with 100 ng of pC-WT (or pC-MT-p53 or pCMT-IRF-1), 50 ng of pCMV.SPORT-b-gal (Invitrogen) and
with the required amount of the other plasmids. The total
amount of plasmid in each transfection was kept constant
(400 ng for each well of a 24-well plate) by adding control
plasmid. Lysates were generally made 30 h post-transfection. Preparation of lysates and CAT assays were carried
out as described previously [18]. Relative CAT activities
were calculated after normalizing with b-galactosidase
enzyme activities.
The p73 promoter was cloned from human genomic
DNA by utilizing the PCR as described previously [48].
The primers used were: forward, 5¢-CGCTCGAGGATCC
AGAGCCCGAGCCCACA-3¢ and reverse, 5¢-CGAAGCT
TCCGTCGCAGCCCCGGGCA-3¢ [48]. The amplified promoter fragment of 930 bp was cloned into the pMOSBlue
vector (Amersham) and sequenced. The p73 promoter fragment 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 BankTM accession number: NM_005427). A mutant of this shRNA was made by
substituting two bases in the middle of the target sequence

FEBS Journal 274 (2007) 4396–4407 ª 2007 The Authors Journal compilation ª 2007 FEBS


N. Jain et al.

TNF-a-induced caspase-1 expression requires p73

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-polyacrylamide 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 temperature. The blots were washed three times, and detection
was performed by using horseradish peroxidase-conjugated
secondary antibody or alkaline phosphatase-conjugated
secondary antibody, as described previously [19]. The
immunoblotting procedure for the p73 blot has been
described previously [19].

Acknowledgements
We thank Drs Gerry Melino, William Kaelin and Bert
Vogelstein for providing reagents, and Dr V. Radha
for critical reading of the manuscript. This work was
supported by a grant from the Indian Council of Medical Research to GS.

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