ACCELERATED PUBLICATION
Direct Transcriptional Activation of Human Caspase-1 by Tumor
Suppressor p53*
Sanjeev
Gupta
,
Vegesna
Radha
,
Yusuke
Furukawa§, and
Ghanshyam
Swarup
¶
From the
Centre for Cellular and Molecular Biology,
Hyderabad 500 007, India and § Division of Molecular
Hemopoiesis, Centre for Molecular Medicine and Department of
Hematology, Jichi Medical School, Tochigi 329-0498, Japan
Received for publication, January 18, 2001, and in revised form, February 9, 2001
 |
ABSTRACT |
The tumor suppressor protein p53 is a
sequence-specific DNA-binding protein, and its biological
responses are very often mediated by transcriptional activation of
various target genes. Here we show that caspase-1 (interleukin-1
converting enzyme), which plays a role in the production of
proinflammatory cytokines and in apoptosis, is a transcriptional target
of p53. Caspase-1 mRNA levels increased upon overexpression of p53
by transfection in MCF-7 cells. Human caspase-1 promoter showed
a sequence homologous to the consensus p53-binding site. This sequence
bound to p53 in gel shift assays. A caspase-1 promoter-reporter
construct was activated 6-8-fold by cotransfection with normal p53 but
not by mutant p53 (His273) in HeLa, as well as MCF-7,
cells. Mutation of the p53-binding site in caspase-1 promoter abolished
transactivation by p53. Treatment of p53-positive MCF-7 cells with the
DNA-damaging drug, doxorubicin, which increases p53 levels, enhanced
caspase-1 promoter activity 4-5-fold, but similar treatment of
MCF-7-mp53 (a clone of MCF-7 cells expressing mutant p53) and
p53-negative HeLa cells with doxorubicin did not increase caspase-1
promoter activity. Doxorubicin treatment increased caspase-1 mRNA
levels in MCF-7 cells but not in MCF-7-mp53 or HeLa cells. These
results show that endogenous p53 can regulate caspase-1 gene expression.
 |
INTRODUCTION |
The tumor suppressor protein p53 plays an important role in
mediating response to stress such as that induced by DNA damage and
hypoxia resulting in either growth arrest or apoptosis (1-3). It
is a sequence-specific DNA-binding protein, and its biological effects
are generally mediated by transcriptional activation of various target
genes (1-3). The p53 gene is mutated in over 50% of human tumors and
in some inflammatory disorders like rheumatoid arthritis (2-4). These
p53 mutations are clustered in the sequence-specific DNA-binding domain
of the molecule leading to inactivation of its sequence-specific
transactivation function (2).
Caspase-1, also known as interleukin-1
converting enzyme, is a
member of the cysteine protease family, which cleaves cellular substrates after aspartic acid (5-7). The primary function of caspase-1 is the proteolytic processing of the precursors of
proinflammatory cytokines such as interleukin-1
into active
cytokines (5-7). In addition caspase-1 is also involved in some forms
of apoptosis (5-7). Caspase-1 knockout mice are developmentally normal
but are defective in the production of mature cytokines
interleukin-1
and interleukin-18. These mice are resistant to septic
shock and show a partial defect in apoptosis (8, 9).
Several p53-responsive genes have been identified by using different
approaches and various cell types (10, 11). These p53-responsive genes
include various functional categories such as those involved in
apoptosis, cell cycle, signal transduction, angiogenesis, etc. (11).
Induction of various genes by p53 is dependent on the type of inducer
used, and even with the same inducer it may be cell
type-dependent (11). However none of the members of the
caspase family have been identified as a transcriptional target of p53.
Here we report that human caspase-1 is a transcriptional target of
exogenous, as well as endogenous, p53. In addition we have identified a
site in the caspase-1 promoter that is required for transcriptional
activation by p53.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Transfections--
The cell lines were
maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum at 37 °C in a CO2
incubator. The transfections were done using LipofectAMINE PLUSTM reagent (Life Technologies, Inc.) according to
manufacturer's instructions. All the plasmids for transfections were
prepared by using Qiagen columns.
Reverse Transcriptase Polymerase Chain Reaction
Analysis--
Total RNA was isolated using
TRIZOLTM reagent (Life Technologies, Inc).
Semiquantitative RT-PCR1 was
carried out essentially as described previously (12). RNA was reverse
transcribed using reagents from an RNA-PCR kit (PerkinElmer Life
Sciences). The GAPDH and caspase-1 mRNAs were amplified for 23 and 40 cycles, respectively, in the same reactions. The PCR products
were analyzed on a 1.2% agarose gel containing ethidium bromide
followed by Southern blot analysis for caspase-1. Primers for
amplification of GAPDH mRNA have been described (12). Primers C1P2,
5'-CGAATTCAATGTCCTGGGAAGAGGTAGAA-3', and C1P3,
5'-CGAATTCAAGGACAAACCGAAGGTGATC-3', were used for amplification of
human caspase-1 mRNA. Primers C1P4, 5'-AAGGAGAAGAGAAAGCTGTTTATC-3',
and C1P5, 5'-ATTATTGGATAAATCTCTGCCGAC-3', were used to distinguish
among
-,
-, and
- or
-isoforms of caspase-1.
CAT Assay--
Cells grown in 35-mm dishes were transfected with
250 ng of pCAT-ICE, 150 ng of pCMV·SPORT-
GAL (Promega), and
500 ng of wild-type p53, mutant p53 (His273), or control
plasmids. Lysates were prepared 30 h post-transfection from HeLa
cells and 48 h post-transfection from MCF-7 cells using reporter
lysis buffer from Promega according to the manufacturer's instructions. For CAT assay 40 µl of lysate was mixed with 2 µl of
14C-labeled chloramphenicol (25 µCi ml
1; 54 mCi mmol
1) and 10 µl of acetyl coenzyme A (3.5 mg
ml
1) in a total volume of 60 µl and incubated at
37 °C for 3 h. Relative CAT activities were calculated after
normalizing with
-galactosidase enzyme activities.
Electrophoretic Mobility Shift Assay--
Double-stranded
oligonucleotide corresponding to the putative p53-binding site in
caspase-1 promoter (Casp-1; see Fig. 3A) was end-labeled
with polynucleotide kinase using [
-32P]ATP. Nuclear
extracts were prepared by high salt extraction of nuclei (13). Binding
reactions with labeled oligonucleotide and nuclear extracts were
performed essentially as described (14) in 10 mM Tris-HCl,
pH 7.5, 0.1% Triton X-100, 4.5% glycerol, 1 mM EDTA, 0.05 mM dithiothreitol, 1 µg of poly(dI-dC), 100 mM sodium chloride. Nuclear extract (4 µg of protein) was
then added followed by addition of 2 ng of labeled probe (50,000 cpm).
The reaction mix was incubated at 25 °C for 45 min followed by
incubation at 4 °C for 15 min. A polyclonal antibody (1 µg) from
Roche Molecular Biochemicals was included where indicated in the
binding reaction.
Reporter Plasmids--
The promoter region of human caspase-1
gene from nucleotide position
182 to +42 relative to the
transcription start site was cloned in pCAT-Basic vector (Promega) and
designated as pCAT-ICE-wt (15). Mutated promoter-reporter
plasmid named as pCAT-ICE-mt was constructed using primers
mut-1, 5'-GGGAAAAGAAATAAAGAAATTCATATGAATTCACAGTGAGTATTTCC-3', and
mut-2, 5'-GGAAATACTCACTGTGAATTCATATGAATTTCTTTATTTCTTTTCCC-3', by
PCR-based site-directed mutagenesis using overlap extension PCR (16).
The nucleotide sequence of the mutant, as well as wild-type promoter,
in these constructs was confirmed by automated sequencing.
 |
RESULTS AND DISCUSSION |
The level of caspase-1 mRNA was determined by RT-PCR analysis
in response to transient overexpression of human wild-type p53 in MCF-7
cells. Caspase-1 mRNA level increased severalfold by overexpression
of p53 as compared with the control-transfected cells or untransfected
cells (Fig. 1A). This increase
in the caspase-1 mRNA level was not the result of induction of
apoptosis by p53, because treatment of MCF-7 cells with some
apoptosis-inducing agents, staurosporine, and cycloheximide did not
increase the caspase-1 mRNA level (Fig. 1B). Treatment
with staurosporine in fact decreased the level of caspase-1 mRNA.
There are five isoforms of caspase-1 mRNA (17). Using another set
of primers, we found that the
form, which is proapoptotic, was
induced by p53 (Fig. 1, C and D), and
,
,
and
forms were not induced. By using appropriate primers we found
that the
-isoform was also not induced (data not shown).

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Fig. 1.
p53-dependent expression of
caspase-1. Panel A, RT-PCR analysis of total RNA from MCF-7
cells transfected with wild-type human p53 (Wp53), control
plasmid (C), and untransfected (U) cells using
primers for human caspase-1 and GADPH. Upper panel shows an
ethidium bromide-stained agarose gel of PCR products as indicated, and
lower panel shows a Southern blot for caspase-1. Panel
B, effect of cycloheximide (Chx) and staurosporine
(Sta) on caspase-1 mRNA levels in MCF-7 cells. The cells
were treated with 100 µg ml 1 of cycloheximide for
24 h or 0.1 µM staurosporine for 8 h or was
untreated (C). Panel C, schematic representation
of splice variants of human caspase-1. The positions of primers used
for amplifying caspase-1 are indicated by arrows.
Panel D, RT-PCR analysis for caspase-1 expression using
primers C1P4 and C1P5. A PCR product of 451 bp is produced from
-isoform. -Isoform would give a PCR product of 388 bp whereas
- and -isoforms would give a PCR product of 172 bp.
M, molecular weight markers; C, control;
Wp53, wild-type p53.
|
|
Examination of the nucleotide sequence of human caspase-1 promoter (18)
showed a sequence homologous to the consensus p53-binding site at
nucleotide position
85 to
66 relative to the transcriptional start
site (Fig. 2A). A caspase-1
promoter-reporter construct (pCAT-ICE-wt) containing this region
(nucleotide position
182 to +42) was activated over 6-8-fold by
cotransfection with normal p53 in MCF-7, as well as HeLa, cells (Fig.
2, B and C). In these experiments the ratio of
p53 to reporter plasmid was 1:1 with HeLa cells (Fig. 2C)
and 2:1 with MCF-7 cells (Fig. 2B). At a higher ratio (2:1)
of p53 to reporter plasmid in HeLa cells there was an over 12-fold
increase in activation of transcription from this promoter (Fig.
2D). Mutant p53 (His273) did not activate this
transcription in p53-negative HeLa cells, but in MCF-7 cells, which are
p53-positive, it gave a small (less than 2-fold) increase in activity
(Fig. 2, B and C). The control plasmid
(pCAT-Basic) gave much lower activity and did not show any activation
by p53 (data not shown). Mutation of the putative p53-binding site in
caspase-1 promoter completely abolished transactivation by p53 (Fig.
2D). These observations suggest that there is only one
functional p53-responsive site in this region (
182 to +42) of
caspase-1 promoter.

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Fig. 2.
Activation of transcription from caspase-1
promoter by wild-type p53. Panel A, schematic representation
of caspase-1 promoter-reporter constructs, pCAT-ICE-wt and pCAT-ICE-mt.
The nucleotide sequence of human caspase-1 promoter from position 85
to 66 relative to the transcription start site is shown. Panels
B and C, activation of caspase-1 promoter by wild-type
p53. pCAT-ICE-wt and pCMV·SPORT- GAL were cotransfected, along with
wild-type p53 (Wp53) or mutant p53 (Mp53) or
control plasmids (C) in MCF-7 (B) and HeLa
(C) cells. CAT activities relative to control are shown
(n = 4). The ratio of p53 to reporter plasmid was 2:1
in panel B and 1:1 in panel C. Panel
D, effect of mutation of the p53-binding site in caspase-1
promoter on transactivation by wild-type p53. The ratio of p53 to
reporter plasmid was 2:1 (n = 3).
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|
To determine whether p53 binds to the putative p53-binding site in
human caspase-1 promoter, we carried out electrophoretic mobility shift
assays using a synthetic oligonucleotide corresponding to this site
(Fig. 3A). Binding to this
oligonucleotide was seen with nuclear extract prepared from MCF-7 cells
treated with doxorubicin, which is known to increase the p53 protein
level (Fig. 3B, lane 2). This binding was
competed out with a 50-fold excess of unlabeled self-oligonucleotide
and also with a consensus p53-binding oligonucleotide but not with a
mutated oligonucleotide in which the p53-binding core sequence CATG was
mutated to AATT (Fig. 3, A and B, lanes 3-5). A polyclonal antibody to p53 immunodepleted the shifted band (Fig. 3B, lane 6). These results suggest
that the binding to this oligonucleotide corresponding to the putative
p53-binding site in caspase-1 promoter is specific and dependent on
p53.

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Fig. 3.
Electrophoretic mobility shift assay using
the putative p53-binding site sequence of caspase-1 promoter.
Panel A, sequences of oligonucleotides corresponding to
nucleotide positions 89 to 65 relative to the transcription start
site in Casp-1. Sequences of mutant oligonucleotide
(mt-Casp-1) and p53-binding consensus oligonucleotide
(Consensus) are also shown. Panel B,
electrophoretic mobility shift assays were done using radiolabeled
Casp-1 oligonucleotide with nuclear extracts from MCF-7 cells treated
with 500 ng ml 1 of doxorubicin. Lane 1 is
binding without nuclear extract. The arrow shows the
p53-specific band that was competed out by a 50-fold excess of
unlabeled Casp-1 oligonucleotide (lane 3) and consensus
oligonucleotide (lane 4) but not by mt-Casp-1
oligonucleotide (lane 5). The addition of p53 polyclonal
antibody (p53 Ab; 1 µg) immunodepleted the shifted band
(lane 6).
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|
To address the role of endogenous p53 in regulating endogenous
caspase-1 gene expression, MCF-7 cells were treated with doxorubicin, which increases the level of p53 protein. Treatment of MCF-7 cells with
doxorubicin enhanced the caspase-1 mRNA level 4-5-fold (Fig. 4). Similar treatment of MCF-7-mp53, a
clone of MCF-7 cells expressing mutant p53 (His273) or
p53-negative HeLa cells, did not increase the caspase-1 mRNA level
(Fig. 4). The basal level of caspase-1 mRNA was higher in MCF-7-mp53 that decreased upon treatment with doxorubicin.The MCF-7-mp53 cell line was obtained by transfection of MCF-7 cells with
the His273 mutant of p53 followed by selection with G418.
This mutant of p53 is known to function as a dominant inhibitor of
wild-type p53 function (19). Treatment of A549 cells (which have
normal p53) with doxorubicin also resulted in an increase in the
caspase-1 mRNA level (Fig. 4). These results showed that endogenous
p53 can regulate expression of the endogenous caspase-1 gene.

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Fig. 4.
Regulation of caspase-1 gene expression by
endogenous p53. Indicated cells were treated with 500 ng
ml 1 doxorubicin for 24 and 48 h. After RNA isolation
caspase-1 mRNA levels were analyzed by
RT-PCR.U, untreated.
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To determine the role of endogenous p53 in regulating caspase-1
promoter, MCF-7, MCF-7-mp53, and HeLa cells were transfected with
caspase-1 promoter-reporter plasmid, and after 24 h they were
treated with doxorubicin for 40 or 48 h. Doxorubicin treatment resulted in a 4-5-fold increase in caspase-1 promoter activity in
MCF-7 cells but not in MCF-7-mp53 or HeLa cells (Fig.
5). These results showed that endogenous
wild-type p53 can also activate transcription from the caspase-1
promoter, which is inhibited by the His273 mutant of
p53.

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Fig. 5.
Transactivation of caspase-1 promoter by
up-regulating endogenous p53. The indicated cells were
cotransfected with pCAT-ICE-wt and pCMV·SPORT- GAL, and 24 h
post-transfection they were treated with 500 ng ml 1
doxorubicin for 40 and 48 h. CAT activities relative to untreated
control are shown (n = 3).
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Ectopic expression of caspase-1 is known to induce apoptosis (17, 20).
The wild-type p53-induced apoptosis in MCF-7 cells was partially
inhibited (50% inhibition) by YVAD-cmk (which preferentially inhibits caspase-1) but not by the caspase-3 family inhibitor DEVD-cmk
(data not shown). Doxorubicin-induced apoptosis in MCF-7 cells was also
partially inhibited (45% inhibition) by YVAD-cmk and not by DEVD-cmk
(data not shown). These observations suggest that caspase-1 contributes
in part to p53-mediated apoptosis. Apoptotic pathways are cell type-
and stimulus-specific, and it is likely that caspase-1, along with
other transcriptional targets, may play a role in p53-mediated
apoptosis at least in some cells.
The primary role of caspase-1 is in the production of proinflammatory
cytokines interleukin-1
, interleukin-16, and interleukin-18 (5-7).
Wild-type p53 is overexpressed in several inflammatory diseases
(reviewed in Ref. 4), but its potential role in inflammation is not
understood. Our results, showing that caspase-1 is transcriptionally activated by p53, suggest that p53 has a role in inflammation. Mutational inactivation of p53 in human tumors would, therefore, lead
to reduced inflammatory response, in addition to resistance to apoptosis.
 |
ACKNOWLEDGEMENT |
S. G. gratefully acknowledges the Council of
Scientific and Industrial Research, Government of India, for a senior
research fellowship.
 |
FOOTNOTES |
*
This work was supported by a research grant from the
Department of Biotechnology, Government of India (to G. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
91-40-7172241; Fax: 91-40-7171195; E-mail:
gshyam@ccmb.ap.nic.in.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.C100025200
 |
ABBREVIATIONS |
The abbreviations used are:
RT-PCR, reverse transcriptase polymerase chain reaction;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
CAT, chloramphenicol
acetyltransferase;
CMV, cytomegalovirus;
GAL,
-galactosidase;
Casp-1, caspase-1;
wt, wild-type;
mt, mutated;
bp, base pairs;
cmk, chloromethylketone.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.