(Received for publication, May 9, 1997, and in revised form, May 29, 1997)
From the § Department of Surgery, The early growth response-1 (EGR-1) protein is an
anti-proliferative signal for certain tumor cells and is required for
apoptosis induced by stimuli that elevate intracellular
Ca2+. We present evidence that EGR-1 transactivates
the promoter of the p53 gene and up-regulates p53 RNA and protein
levels. Inhibition of p53 function with dominant-negative p53 mutants
abrogates EGR-1-dependent apoptosis. These findings
establish a direct functional link between EGR-1 and the p53-mediated
cell death pathway and suggest that mutant forms of p53 in tumor cells
may provide resistance to the anti-proliferative effects of EGR-1.
Apoptosis, or programmed cell death, a genetic process of
coordinated deletion of selective cells, is essential for metazoan development and homeostasis (1-4). The primordial forms of apoptosis in Caenorhabditis elegans and Drosophila have
been recapitulated in mammalian cells, and striking similarities have
been observed in the cell death programs of invertebrates and
vertebrates (5-7). Apoptosis is characterized by cell membrane
blebbing, chromatin condensation, changes in nuclear architecture, and
oligonucleosome-length DNA fragmentation (8, 9). The apoptotic pathways
consist of an early component that includes molecular events that are specific for an inducer or a group of inducers and of downstream effector components that are common to diverse apoptotic signals (10).
The common components include a basal cell death machinery composed of
initiator, amplifier, and effector proteases belonging to the
interleukin-1 converting enzyme subfamily or an interleukin-1 converting enzyme-related family (10-14). Downstream targets of these
proteases include the interleukin-1 converting enzyme subfamily proteases themselves; the nuclear enzymes poly(ADP-ribose) polymerase and DNA-dependent protein kinase, which are involved in DNA
repair; the nuclear protein U1 ribonucleoprotein and nuclear lamins;
and cytoplasmic components such as protein kinase C Intracellular calcium is a key second messenger implicated in the
activation of the apoptotic program in diverse cell types and tissues
(15-18). Intracellular calcium levels become elevated after the
activation of T lymphocytes by anti-CD3 or that of B lymphocytes by
anti-IgM antibodies; after withdrawal of survival factors, such as
testosterone in the prostate gland; or after exposure to certain
exogenous stimuli, such as calcium ionophores or thapsigargin
(TG),1 a potent inhibitor of
the Ca2+-dependent ATPase in the endoplasmic
reticulum (19). Elevation of intracellular calcium causes induction of
immediate-early genes that further trigger a cascade of downstream
events leading ultimately to cell death. Although several
immediate-early genes such as early growth response-1
(Egr-1; also referred to as zif268, NGF-IA, TIS8, and
Krox-24), nur77, and par-4 have been functionally
linked to apoptosis caused by intracellular calcium elevation (20-24), the precise downstream events that are important for successful apoptosis via this pathway have not been delineated.
Egr-1 was first identified as an immediate-early gene
induced by mitogenic stimulation and during membrane depolarization and
seizure (25, 26). Subsequently, EGR-1 was shown to be induced by
diverse exogenous stimuli (27). EGR-1 is a nuclear protein that
contains three zinc finger motifs of the C2H2
subtype that bind to a GC-rich consensus DNA sequence
TGCG(T/g)(G/A)GG(C/a/t)G(G/T) (where lowercase letters indicate bases
of relatively lower binding affinity) or to a (TCC)n motif (28,
29). Structure-function mapping studies suggest that the
NH2 terminus of EGR-1 confers transactivation function to
the protein (27, 30). The Egr-1 gene was localized to human
chromosome 5q31.1, a region known to be often deleted from patients
suffering from therapy-induced acute myeloid leukemia (31). Moreover,
studies using diverse tumor cells suggest that endogenous levels of
EGR-1 act to impede proliferation (32, 33). Consistent with an
anti-tumor role for EGR-1, apoptosis-inducing stimuli, such as TG and
ionizing radiation, up-regulate EGR-1 expression (20, 34). Furthermore, our previous studies, which used antisense oligomers to block EGR-1
expression or a dominant-negative EGR-1 mutant to inhibit EGR-1
function, confirmed that EGR-1 is essential for apoptosis induced by TG
or by ionizing radiation (20, 34).
Because EGR-1 is induced very early in the apoptotic process (20, 34),
it is expected to mediate the activation of downstream genes that play
crucial roles in growth control. EGR-1-binding sites (EBS) that conform
to the GC-rich consensus sequence have been identified in the promoters
of genes such as thymidine kinase, an enzyme integral to DNA
biosynthesis; cell cycle regulators such as cyclin D1 and the
retinoblastoma susceptibility gene Rb; and (TCC)n
motifs have been identified in the promoter regions of genes encoding
growth factors such as platelet-derived growth factor and basic
fibroblast growth factor; growth factor receptors such as epidermal
growth factor-receptor and the insulin-like growth factor-receptor; and
protooncogenes c-Ki-ras and c-myc (cited in Refs.
20 and 27). However, none of these genes has been shown to be directly
involved in apoptosis as a consequence of EGR-1 induction.
The tumor suppressor gene p53 is a central mediator of cell
cycle growth arrest and apoptosis (reviewed in Refs. 35-37). The p53
protein encodes a transcription factor that functions as a transcriptional activator or repressor, depending upon the promoter context (38-41). The p53 protein can up-regulate the expression of a
number of downstream genes, such as p21/waf1, insulin-like growth factor binding protein-3, bax, and
fas/apo1, implicated in growth inhibition and
apoptotic cell death (42-45). These regulatory effects require binding
of p53 protein to consensus binding sites in the promoter region of
target genes (35, 46, 47). The p53-dependent downstream
induction or activation of the Fas/Apo1 pathway leads to the activation
of a cascade of downstream effector proteases resulting in apoptotic
death (12, 45). p53-dependent apoptotic pathways that are
independent of the ability of this protein to function as a
transcriptional activator have been also suggested (48, 49). The
induction of apoptosis requires p53 in most cellular experimental
systems, but there are multiple examples of apoptosis in the absence of
functional p53 protein, suggesting the presence of p53-independent
pathway(s) for apoptosis (50-53). The precise molecular components of
the p53-independent pathway, however, are not known.
In a number of different types of cancers, p53 is the most
commonly mutated tumor suppressor gene (54). Lack of p53
expression or function is associated with an increased risk of tumor
development (55-58). Most tumor types either contain no p53 protein
owing to loss of the p53 alleles or of chromosome 17 where
p53 is located or contain point mutations in the
p53 gene that result in mutant protein products that are
functionally inactive (54). Most of the commonly occurring mutations in
p53 are located in the DNA-binding or transactivation
domains of the protein and render p53 inactive in the transcription of
downstream proapoptotic genes such as bax (39, 40, 54, 59).
Some mutant forms of p53 protein can oligomerize with wild-type p53
protein and abrogate its growth-inhibitory functions and thus act as
dominant-negative inhibitors of wild-type p53 (46).
Wild-type but not mutant p53 protein can cause apoptosis in response to
a wide range of exogenous stimuli such as adenovirus E1A protein,
anti-cancer agents, or ionizing radiation (62-68). Moreover, hypoxia
has been shown to induce apoptosis that is dependent on p53, and
hypoxic conditions are suggested to provide a selective pressure for
p53 mutations (69). Several immediate-early genes encoding
transcription factors such as c-Fos, c-Jun, EGR-1, Nur77, and c-MYC
have been implicated in apoptosis (20, 22-24, 70-73), and c-Fos and
c-MYC have been shown to require wild-type p53 for apoptosis (70, 72,
73). It is not known, however, whether the apoptotic action of the
other immediate-early genes is dependent on wild-type p53 function.
Studies directed at identifying whether p53 is required or not for the
transduction of an apoptotic signal from immediate-early proapoptotic
genes encoding transcription factors, which couple the early events in
the plasma membrane to long term cellular phenotypic changes in the
cell, should provide valuable insights into the downstream targets of
the transcription factors and help identify a link with components of
the cell death pathways. In the course of our studies aimed at
understanding the mechanism of EGR-1-mediated apoptosis, we have
identified p53 as a direct transcriptional target of EGR-1. Our data
suggest that the proapoptotic action of EGR-1 is mediated via wild-type p53.
Human melanoma cells
A375-C6 and transfected cell lines that expressed pCMV-mEGR1 or vector
were cultured as described previously (20, 34). Pools of approximately
200 transfected clones were maintained as cell lines. Expression
constructs m143 and m175 that encode mutant p53 proteins (46) were
kindly provided by Bert Vogelstein (Johns Hopkins University School of
Medicine, Baltimore, MD). The constructs pCMV- Whole-cell protein extracts
were prepared and analyzed by using Western blot analysis as described
previously (20, 34). EGR-1 (sc-110) and p53 (DO-1) antibodies were
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the
actin antibody was purchased from Sigma.
Cells were quantified for
apoptosis by terminal transferase-mediated dUTP-nucleotide-end labeling
(TUNEL) as described previously (20, 34).
Transfections were performed
by the calcium phosphate coprecipitation method, and stable
transfectants were selected in 300 µg/ml G418-sulfate as described
previously (20). Pools of about 200 stably transfected clones were
maintained as cell lines. CAT assays were performed by using thin layer
chromatography as described previously (76, 77).
Total RNA extraction and Northern blot
analysis were performed as described previously (77). The cDNA
probe for Egr-1 has been described previously (77). The
cDNA for human p53 was purchased from the American Type Culture
Collection (Rockville, MD).
Preparation of nuclear extracts from transfected cells and
electrophoretic mobility shift assay were preformed as described previously (76). Two complementary primers, 5 To examine ectopically
overexpressed EGR-1 as a cause or enhancer of apoptosis, human A375-C6
cells were stably transfected with pCMV-mEGR1, an expression construct
for mouse EGR-1 (mEGR1), or with vector for a control. For each of the
two constructs, we examined three transfected cell lines for EGR-1
expression by Western blot analysis. Data representative of the
transfected cell lines shown in Fig.
1A confirmed overexpression of
EGR-1 in the pCMV-mEGR1-transfected cell line as compared with the
vector-transfected cell line. These transfected cell lines were then
exposed to 250 nM or 1 µM TG for various
periods of time and examined for apoptosis by TUNEL. As seen in Fig.
1B, vector-transfected cells consistently showed a
significantly lower number of apoptotic cells (p < 0.0001 by the Student's t test) than did the
EGR-1-transfected cells at all the time points ranging from 6 to
36 h of exposure to TG. These findings suggested that ectopic
mEGR1 provided enhanced susceptibility to TG-inducible cell death (Fig.
1B). Thus, consistent with a role for EGR-1 as a positive
regulator of apoptosis, ectopic overexpression of EGR-1 sensitizes
cells to apoptosis.
One logical mechanism by which EGR-1 induces apoptosis may involve the
transcriptional up-regulation of downstream genes. To address this
question, A375-C6 cells were stably transfected with pCMV-mEGR1 or the
following mEGR1 deletion mutant constructs: pCMV-
To identify candidate
EGR-1-responsive genes essential for mediating apoptosis, a GenBankTM
data base search was conducted to identify putative EGR-1-binding sites
in the promoter regions of genes known to be functionally involved in
apoptosis. Interestingly, two putative EGR-1 consensus binding sites,
EBS-1 and EBS-2 (Fig. 3A),
were identified in the promoter region of the p53 gene (75) (GenBankTM accession number X54156). The p53 gene has been
also shown to be essential for apoptosis induced via activation of diverse intracellular signaling pathways (50-53, 62-68). Because regulation of the p53 gene at the transcriptional level
contributes significantly to the control of p53 protein levels and to
phenotypic responses (80), the ability of EGR-1 to induce the
p53 promoter was tested. Reporter constructs,
p53(2.2+1.6)-CAT, which contains about 2.2 kb of sequence upstream of
the p53 cap site and about 1.6 kb of sequence downstream of
the p53 cap site (Fig. 3A and Ref. 75);
p53(0.8)-CAT, which contains the 0.8 kb upstream region of
p53 (Fig. 3A); or the empty CAT vector, pGCAT-C,
were examined in transient transfection and CAT assays for inducibility
with the pCMV-mEGR1 construct. As seen in Fig. 3B
(left panel), cells transfected with pCMV-mEGR1 showed a
4-5-fold higher CAT activity from the p53 reporter construct
p53(2.2+1.6)-CAT than did the empty CAT reporter construct. Also, both
of the p53 reporter constructs p53(2.2+1.6)-CAT and p53(0.8)-CAT showed
induction of CAT activity with pCMV-mEGR1 but not with the
pCB6+ empty vector (Fig. 3B, middle
panel), suggesting that the 0.8-kb region upstream of the cap site
in the p53 promoter was sufficient for transcriptional
activation by EGR-1. To determine whether the transactivation and the
DNA binding functions of EGR-1 were necessary for p53
induction, the cells were transiently cotransfected with
p53(2.2+1.6)-CAT and pCMV-mEGR1, pCMV-
To ascertain that EGR-1 protein from the mEGR-1 overproducer cells
actually bound to EBS-1 of the p53 promoter, electrophoretic mobility shift assays were performed. Nuclear protein extracts prepared
from cells stably transfected with pCMV-mEGR-1 or vector were incubated
with radiolabeled EBS-1. As shown in Fig. 3C (left panel), nuclear extracts from the cells transfected with vector produced two primary bound complexes: cI and cII. On the other hand,
nuclear extracts from cells transfected with mEGR-1 produced three
primary complexes; cI and cII were similar in intensity to those seen
in extracts from vector-transfected cells, whereas the third complex,
cIII, was predominant in extracts from mEGR-1 overproducers (Fig.
3C, left panel). These results suggested that complex cIII most likely contained EGR-1 bound to EBS-1. To ascertain whether complex cIII represented EGR-1, the nuclear extracts were preincubated either with a preimmune antibody or with EGR-1 antibody before incubation with the radiolabeled probe. These experiments showed
that complex cIII was supershifted with the EGR-1 antibody but not with
the preimmune antibody, suggesting that it contained EGR-1 (Fig.
3C, right panel). Together with the results of
CAT assays described above, these findings suggest that the EGR-1 protein can directly bind to and transcriptionally activate the p53 promoter.
To ascertain that the induction of p53 was not merely
restricted to its promoter, we used Northern blot analysis to examine the ability of EGR-1 to induce the endogenous p53 gene at
the RNA level (Fig. 3D) and Western blot analysis to examine
the ability of EGR-1 to induce the endogenous p53 gene at
the protein level (Fig. 3E). Three different pools of stable
transfectants that overexpressed EGR-1 showed 4-5-fold higher levels
of p53 RNA and protein than did transfectants containing the
vector (Fig. 3, D and E). These findings suggest
that ectopic EGR-1 induced expression of the p53 gene,
causing increased production of p53 RNA and protein.
Because A375-C6
cells contain wild-type p53 (34), we used these cells to
examine the functional requirement of wild-type p53 protein in
TG-inducible apoptosis. These studies used dominant-negative mutants of
p53, pCMV-m143 (Val to Ala at amino acid 143), and pCMV-m175 (Arg to
His at amino acid 175) that had been previously shown (46) to
oligomerize with and inhibit the function of wild-type p53 protein.
A375-C6 cells were transfected with expression constructs pCMV-m143 or
pCMV-m175 or with vector for a control, and pools of stably transfected
clones were maintained as cell lines. When whole-cell protein extracts
from these transfected cell lines were subjected to Western blot
analysis, the levels of p53 protein in cells transfected with the p53
mutant constructs were higher than the levels of endogenous wild-type
p53 protein in cells transfected with vector (Fig.
4A), indicating successful
overexpression of the p53 mutants.
Next, the stably transfected cells were tested for their ability to
compete with endogenous wild-type p53 for transactivation of reporter
constructs PG13-CAT (containing intact p53-binding sites)
or MG15-CAT (containing mutations in the p53-binding site) (46). The stably transfected cells expressing m143, m175, or vector
were transiently transfected with PG13-CAT or
MG15-CAT. When cell extracts were tested for CAT activity,
transfectants expressing mutant p53 proteins showed much lower CAT
activity from PG13-CAT than did transfectants containing
the vector (Fig. 4B). As expected, all the three
transfectants produced low level CAT activity from MG15-CAT
(Fig. 4B). These results, in agreement with those obtained
from other cell lines (46), suggest that mutant forms of p53 protein
can inhibit the ability of endogenous wild-type p53 in the A375-C6
cells to cause transcriptional activation.
We then determined the susceptibility of transfected cells expressing
p53 mutant proteins or vector to TG-inducible apoptosis by exposing
them to either 250 nM or 500 nM TG for 48 h. When these cells were assayed for apoptosis with TUNEL, the cell
lines expressing mutant p53 proteins showed a significantly lower
(p < 0.0001 by Student's t test)
percentage of TUNEL-positive cells than did the cell lines expressing
the vector (Fig. 4C). These experiments suggested that the
p53 mutants, which interfere with the transactivation function of
endogenous wild-type p53, inhibited the ability of TG to cause
apoptosis in the transfectants. Thus, wild-type p53 function is
essential for apoptosis induced by TG.
Our previous
studies have shown that EGR-1 protein is induced by TG in A375-C6 cells
(20). To determine whether p53 induction was temporally downstream of
EGR-1-induction, we exposed A375-C6 cells to TG for various periods of
time; whole-cell protein extracts prepared from the cells were then
subjected to Western blot analysis for EGR-1 or p53 protein. As seen in
Fig. 4D, EGR-1 protein levels increased at 1 h of
exposure to TG, and peak levels were reached at 2 h; thereafter,
the levels decreased sharply at 3 and 5 h. The levels of p53
protein were low until 1 h of exposure to TG and then began to
increase at 2 h of exposure, and peak levels were reached at
3 h. These findings suggest that p53 induction is temporally
downstream of EGR-1 induction by TG.
We reasoned that if wild-type p53 functions at a level downstream of
EGR-1 in a linear pathway, the dominant-negative mutants of p53 should
abrogate EGR-1-dependent apoptosis. To address this question, we stably cotransfected A375-C6 cells with pCMV-mEGR-1 plus
vector, pCMV-mEGR-1 plus pCMV-m175, pCMV-m175 plus vector, or with
vector alone. The stably transfected cell lines, thus obtained, were
tested for their responsiveness to TG-inducible apoptosis by exposing
them to 250 nM or 1 µM TG for 24 h, and the apoptotic cells were scored with TUNEL. As seen in Fig.
4E, cell lines coexpressing mEGR1 and m175 showed a
significantly lower (p < 0.0001 by Student's
t test) percentage of apoptotic cells than did cell lines
coexpressing mEGR1 and vector at each of the two different
concentrations of TG, suggesting that mutant p53 proteins can abrogate
the ability of EGR-1 to cause apoptosis. Thus, wild-type p53 function
is essential for EGR-1-dependent apoptosis induced by TG,
and p53 is a downstream effector of EGR-1 function.
The p53 protein is required for apoptosis induced by diverse
exogenous signals (33, 42-45), and the findings of this study have
expanded the range of p53-inducers to EGR-1 and intracellular Ca2+. The demonstration that wild-type p53 function is
essential for apoptosis induced by intracellular Ca2+
elevation is of broad biological significance because intracellular Ca2+ elevation is central to apoptosis in a number of
in vitro and in vivo model systems (15-18, 20).
Consistent with the observations from our previous studies indicating
that inhibition of EGR-1 expression or function abrogates apoptosis
(20), the findings of the present study indicated that ectopic EGR-1
enhances the apoptotic action of TG. Because the proapoptotic action of
EGR-1 requires intact transactivation and DNA binding functions, we examined genes that are central mediators of the apoptotic process for
EGR-1-responsive sites in their promoter. This led to the identification of p53 as a novel downstream target of EGR-1. Two putative EGR-1 consensus binding sites, EBS-1 and EBS-2, were identified in the p53 promoter. EBS-1 conforms to the
GC-rich consensus motif and EBS-2 conforms to the (TCC)n motif. We showed by electrophoretic mobility shift and supershift assays that
EGR-1 binds to EBS-1. These findings showed direct interaction of EGR-1
with the p53 promoter element. Furthermore, we showed that
EGR-1 can cause transcriptional up-regulation of the p53 promoter. The
ability of EGR-1 to induce the p53 promoter required the
transcriptional activation functions of EGR-1, as evident from the
finding that full-length EGR-1 but not mutants that lacked the
transactivation or DNA-binding domain up-regulated the p53 promoter. EGR-1 also caused induction of p53 RNA and
protein. These findings are consistent with the previous observation
that up-regulation of the p53 gene at the transcriptional
level contributes to up-regulation of p53 protein levels (80). Most
importantly, we showed that when transactivation function of p53
protein was blocked with two different dominant-negative mutants, the
ability of EGR-1 to enhance TG-inducible apoptosis was also abrogated. These findings indicate that the transactivation function of p53 protein is required for an apoptotic response to TG and EGR-1. The
proapoptotic gene bax is known to be transcriptionally
induced by wild-type p53 but not by mutant p53 proteins (39, 40, 44), and A375-C6 transfectants that expressed dominant-negative p53 mutants
showed down-regulation of endogenous bax as compared with vector-transfected cells that expressed only endogenous wild-type p53,2 suggesting that
bax induction by p53 may mediate the apoptotic action of TG
and EGR-1. Together, these findings establish the p53 gene
as a direct target of EGR-1 that is functionally important for
apoptosis. Because EGR-1 has been implicated in both positive and
negative regulation of growth, these findings can be extended in future
studies to address the relevance of EGR-1-mediated p53 induction in
other growth control processes.
EGR-1 enhances the apoptotic action of TG or ionizing radiation but
blocks the growth arresting action of interleukin-1 (20, 34, 77). The
mechanisms by which EGR-1 induces these distinct phenotypic effects are
unclear. EGR-1-binding sites have been identified in the promoter
regions of a number of genes, including those encoding growth factors,
growth factor receptors, cell cycle regulators, and protooncogenes
(27), but these genes have not been shown to be transcriptionally
up-regulated or to have biological relevance in the proapoptotic action
of EGR-1. Toward the goal of determining the mechanism of
EGR-1-dependent apoptosis, we used ectopic EGR-1
overexpression and determined its impact on downstream events. A375-C6
transfectants that stably expressed EGR-1 were confirmed by TUNEL
studies to show increased susceptibility to apoptosis induced by TG as
compared with control vector-transfected cells that contained only
endogenous levels of EGR-1, confirming that ectopic overexpression of
EGR-1 sensitizes cells to apoptosis. The successful selection of
transfectants stably expressing high levels of EGR-1 protein suggested
that EGR-1 was not sufficient on its own to cause apoptosis. Because
EGR-1 protein contains several phosphorylation sites and phosphorylated
forms of the protein have been shown to bind the EBS motif (27), we are
investigating the possibility that exposure to TG causes
post-translational phosphorylation of EGR-1 protein that results in
activation of its transregulatory function leading to induction of
downstream genes and apoptosis.
Because EGR-1 protein acts either as a transcriptional up-regulator or
suppressor of promoter constructs depending on the promoter context and
cell background (27), we determined which of these two transregulatory
functions was important for apoptosis in A375-C6 cells. Wild-type EGR-1
or the mutant that lacked the restriction module but contained the
transactivation and DNA-binding domains showed transcriptional
activation of the EBS-CAT reporter construct and an enhanced apoptotic
response to TG. On the other hand, EGR-1 mutants that lacked the
transactivation domain or DNA-binding domain did not transactivate the
reporter construct and did not show an enhanced apoptotic response to
TG. Because these cells contained comparable amounts of the ectopic
proteins, the findings suggested that the DNA-binding and
transactivation domains of EGR-1 are necessary for an enhanced
apoptotic response to TG. In the light of recent reports indicating
that EGR-1 can directly bind to intracellular proteins (81, 82), it
would be reasonable to speculate that EGR-1 may enhance apoptosis by sequestration of key growth/survival promoting proteins by
protein-protein interactions. However, this possibility was ruled out
by the observation that the proapoptotic functions of EGR-1 were absent
in the DNA-binding and transactivation mutants, which together spanned
the entire EGR-1 protein and thus included the domains shown to bind
other proteins. Thus, the proapoptotic function of EGR-1 requires the DNA binding and transactivation functions of EGR-1. By contrast, the
proapoptotic functions of another immediate-early gene product c-Fos
are dependent on transcriptional repressor functions of the protein
that cause negative regulation of survival genes in a
p53-dependent manner (70). Thus, immediate-early genes that encode transcription factors can regulate apoptosis by distinct p53-dependent mechanisms.
The present study has identified p53 as a functionally relevant and
direct target of EGR-1 in the apoptosis pathway. Because p53 is a
central component of apoptotic pathways in diverse model systems (33,
42-45), this observation establishes the importance of EGR-1 in
apoptosis in our model system, i.e. A375-C6 cells exposed to
TG. Because EGR-1 is an indispensable component of the apoptotic or
growth arrest pathways in A375-C6 cells (20, 77), i.e. loss
of EGR-1 function is not compensated by other EGR-1 family members in
these cells, our model system is ideal for studying EGR-1 function and
downstream targets. However, because EGR-1 knock-out mice do not show
any abnormalities related to growth, differentiation, or apoptosis (60,
61), EGR-1 function may be a dispensable component of apoptotic
pathways in other model systems. It is possible that other members of
the EGR-1 family may have compensated for the loss of EGR-1 function
and produced the normal growth, differentiation, or apoptotic features in the knock-out mice. To ascertain that the importance of EGR-1 in
apoptosis is not restricted to our model system, it is necessary to
study EGR-1 function in a number of other apoptosis model systems wherein the results are not influenced by functional redundancy of
other EGR-1 family members. EGR-1 has been shown to function as an
anti-proliferative protein in a number of tumor cell types (32, 33).
Because the presence of functional p53 is required for successful
transduction of the EGR-1-dependent apoptotic signal, tumor
cells that contain mutant forms of p53 or that lack p53 can be expected
to be unresponsive or resistant to the EGR-1-dependent apoptotic pathway. A number of reports have suggested that the process
of tumor progression is linked to the absence of wild-type p53
tumor-suppressor function (54-58). In such tumor cells, transduction of an apoptotic signal by EGR-1 is expected to be unsuccessful. On the
other hand, because p53 protein is wild-type in normal cells, EGR-1
up-regulation may constitute the important cellular event directed at
preventing malignant transformation or tumor progression by
induction of apoptosis via p53 induction. Further studies on a broad
panel of primary cell cultures and tumor models are required to test
the validity of this hypothesis.
The p53(2.2+1.6)-CAT promoter construct was a
gift from Dr. David Reisman (University of South Carolina, Columbia,
SC), and PG13-CAT, MG15-CAT, pCMV-m143, and
pCMV-m175 were gifts from Dr. Bert Vogelstein (Johns Hopkins University
School of Medicine, Baltimore, MD).
Department of Microbiology and Immunology, the
Graduate Center for Toxicology, and the ** Markey Cancer
Center, University of Kentucky, Lexington, Kentucky 40536 and the
¶ Renal Division, the Beth Israel Deaconess Medical Center, and
the Department of Medicine, Harvard Medical School,
Boston, Massachusetts 02215
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and cytoskeleton components such as actin (cited in Ref. 12).
Cell Cultures and Plasmid Constructs
TA, pCMV-
RM,
pCMV-
ZF, and pCMV-
TA/RM that encode mutant EGR-1 proteins have
been previously described (27, 74). Reporter construct
p53(2.2+1.6)-CAT, which contains about 2.2 kb of sequence upstream of
the human p53 cap site and about 1.6 kb of sequence downstream of the
p53 cap site placed downstream of the chloramphenicol acetyltransferase
(CAT) cDNA (75), was kindly provided by David Reisman (University of South Carolina, Columbia, SC). Polymerase chain reaction was employed to synthesize a 0.8-kb p53 promoter fragment from
p53(2.2+1.6)-CAT DNA template. The reactions used a downstream primer
(5
-TTTTTGGCTCTAGACTTTTGAGAA-3
) complementary to the
region +11 to
9 (underlined) and an upstream primer
(5
-TTTTTCTAGAGAAGCGCCTACGCTCCC-3
) corresponding to the region
758 to
774 (underlined) in the p53 promoter. The polymerase chain reaction product was subcloned in the XbaI site
upstream of the CAT sequence in pGCAT-C vector and the construct,
p53(0.8)-CAT, thus obtained was confirmed for orientation.
-GCGCCTACGCTC-3
and
5
-GAGCGTAGGCGC-3
, corresponding to the upper and lower strands of
EBS-1 (
770 to
762) in the upstream region of the p53 promoter were
synthesized at the Macromolecular Synthesis Facility, University of
Kentucky. The primers were annealed, end-labeled with
[
-32P]ATP and used as probe. The reaction mixture
consisting of nuclear extract, 5 × reaction buffer (1 M Tris, pH 7.5, 2.5 M potassium chloride, 0.5 M EDTA, 1 M dithiothreitol, glycerol, bovine
serum albumin), and 1 µM poly(dI-dC)·poly(dI-dC) was
incubated on ice for 15 min. Probe was added, and the mixture was
further incubated at room temperature for 20 min. The reaction mixture
for the supershift assay consisted of all of the reagents described
above for electrophoretic mobility shift assay plus 2 µl of EGR-1
antibody (sc-110X from Santa Cruz Biotechnology, Inc.) or preimmune
antibody. The reaction mixtures were preincubated for 20 min on ice,
then incubated with the probe for 40 min, and subjected to
electrophoresis on polyacrylamide gels to separate the bound complexes
from the free probe.
EGR-1 Sensitizes Cells to Apoptosis
Fig. 1.
Ectopically expressed EGR-1 enhances the
apoptotic response to TG. A375-C6 cells were stably transfected
with vector or pCMV-mEGR1 that encodes full-length mouse EGR-1 protein,
and transfected clones were pooled to obtain cell lines. A,
the cell lines were examined for expression of EGR-1 protein or actin
(for a control) by Western blot analysis. B, the cell lines
were exposed for the indicated time periods to either 250 nM or 1 µM TG, and apoptotic cells were
quantified by TUNEL. A total of 400 cells were scored for
TUNEL-positive cells in each experiment, and the data represent means
of three experiments. The error bars represent standard
deviation.
[View Larger Version of this Image (25K GIF file)]
TA that lacks the
transactivation domain (amino acids 1-240); pCMV-
RM that lacks the
repression module (amino acids 284-330); pCMV-
ZF that lacks the
first two zinc fingers (amino acids 331-374); or pCMV-
TA/RM that
lacks both the transactivation domain and the repression module (amino
acids 1-314) (Fig. 2A and
Ref. 74). Whole-cell protein extracts from each stably transfected cell line were first examined for expression of endogenous or ectopic EGR-1
or mutant proteins by Western blot analysis. As seen in Fig.
2B, EGR-1-transfected cells contained about 12-fold higher EGR-1 levels than did the vector-transfected cells, which contained only endogenous EGR-1. Moreover, the levels of mutant EGR-1 proteins (
TA,
ZF,
RM, or
TA/RM) produced by the mutant constructs
were similar to those produced by full-length EGR-1 (Fig.
2B). Next, the transfectants were transiently transfected
with the EBS-containing reporter construct, EBS-CAT (78, 79), and
analyzed for CAT activity. As seen in Fig. 2C, the cells
expressing pCMV-mEGR1 or pCMV-
RM showed relatively higher CAT
activity than did the cells expressing the vector, pCMV-
TA,
pCMV-
ZF, or pCMV-
TA/RM. When the transfectants were examined for
TG-inducible apoptosis by TUNEL, those expressing pCMV-mEGR1 or
pCMV-
RM showed a higher percentage of TUNEL-positive cells than did
transfectants that expressed vector, pCMV-
TA, pCMV-
ZF, or
pCMV-
TA/RM (Fig. 2D). These findings suggested that
constructs that lacked the transactivation domain or the DNA-binding
zinc finger domain of EGR-1 failed to cause transcriptional activation
or to sensitize the cells to TG-inducible apoptosis. Thus, both the DNA
binding and transactivation functions of EGR-1 are essential for an
enhanced apoptotic response to TG. These findings suggest that EGR-1
induces apoptosis by a mechanism involving transcriptional regulation
of downstream target genes.
Fig. 2.
Both DNA binding and transactivation
functions of EGR-1 are essential for an enhanced apoptotic response.
A, to identify the structural domains of EGR-1 required to
enhance apoptosis, the following expression constructs were used:
pCMV-mEGR1, pCMV-TA, pCMV-
RM, pCMV-
ZF, and pCMV-
TA/RM (see
text for details). B, A375-C6 cells were stably transfected
with the above constructs or vector for a control, and pools of
transfected clones were maintained as cell lines. Whole-cell protein
extracts from each pool of transfectants were subjected to Western blot
analysis with EGR-1 antibody and then with actin antibody. The
intensity of EGR-1 or mutant EGR-1 protein signals was determined by
densitometric scanning and was normalized with respect to the
corresponding signal for actin. The relative protein levels for
endogenous EGR-1 in vector-transfected cells, full-length EGR-1, and
different mutants are shown. C, the stably transfected cell
lines were then transiently transfected with the EBS-CAT reporter
construct that contains three tandem EGR-1-binding sites, and CAT
assays were performed. D, the stably transfected cells were
left unexposed or exposed to either 250 nM or 1 µM TG for 24 h and examined for apoptosis by TUNEL
(see legend for Fig. 1B).
TA, transcription activation-deficient;
RM, repression module-deficient;
ZF, finger 1- and 2-deficient;
TA/RM,
transcription activation- and repression module-deficient.
[View Larger Version of this Image (34K GIF file)]
TA, pCMV-
ZF, or vector. As
seen in Fig. 3B (right panel), pCMV-mEGR1, but
not the vector, pCMV-
TA, or pCMV-
ZF, showed a 3-fold induction of
CAT activity from the p53-promoter construct. Thus, EGR-1 mutants with
deletion of the transactivation domain or DNA-binding domain showed
minimal CAT activity, suggesting that these domains of EGR-1 are
required for induction of the p53 promoter.
Fig. 3.
EGR-1 induces p53 expression. A,
the p53 promoter constructs, p53(2.2+1.6)-CAT and p53(0.8)-CAT, were
used to test the ability of EGR-1 to transactivate the p53 promoter.
Both constructs contain two putative EGR-1-binding sites; EBS-1
(5-CGCCTACGC-3
) located from nucleotide positions
770 to
762 and
EBS-2 ((TCC)n sequence) located from nucleotide positions
139
to
186. B, A375-C6 cells were transiently cotransfected
with pCMV-mEGR1 and either p53(2.2+1.6)-CAT or pGCAT-C (left
panel); with vector or pCMV-mEGR1 and p53(2.2+1.6)-CAT or
p53(0.8)-CAT (middle panel); or with p53(2.2+1.6)-CAT and
vector, pCMV-mEGR1, pCMV-
TA (
TA), or pCMV-
ZF
(
ZF, right panel). Protein extracts were
prepared from the transfected cells and were examined for CAT activity.
C, electrophoretic mobility shift assays were performed by
using radiolabeled EBS-1 and nuclear extracts from cells stably
expressing mEGR1 or vector (left panel). The supershift
assay was performed either with no antibody or with 2 µl of EGR-1
antibody or preimmune rabbit serum as indicated (right
panel) to ascertain the presence of EGR-1 in the bound complex.
The bound complexes cI, cII, cIII, and supershift with EGR-1 antibody
are indicated. D, total RNA was prepared from stably transfected cell lines expressing pCMV-mEGR1 or vector and was subjected to Northern analysis by using p53 cDNA as a probe
(left panel). The corresponding ethidium bromide-stained
agarose gel shows loading of the RNAs (right panel).
E, whole-cell protein extracts were prepared from the stably
transfected cell lines, and 20 µg of each protein extract was
subjected to Western blot analysis for p53 protein by using antibody
DO-1. Data are shown for p53 protein expression in three different
mEGR1-transfected cell lines (mEGR1.L1, mEGR1.L2, and mEGR1.L3) and in
one vector-transfected cell line.
[View Larger Version of this Image (40K GIF file)]
Fig. 4.
p53 is required for TG-inducible and
EGR-1-dependent apoptosis. A, whole-cell protein
extracts from stably transfected cell lines expected to contain either
vector or mutant p53 constructs (pCMV-m143 or pCMV-m175) were examined
for p53 expression or actin as a loading control by Western blot
analysis. B, to determine the effect of ectopically
overexpressed mutant p53 proteins on the transactivation function of
endogenous wild-type p53, mutant-p53 overexpressor cells or
vector-transfected cells were transiently transfected with
PG13-CAT (containing 13 intact p53 binding sites) or with
MG15-CAT (in which the 15 p53-binding sites were mutated). Protein extracts from the transiently transfected cells were tested for
CAT activity. C, A375-C6 cells that stably expressed vector or the mutant p53 proteins m143 or m175 were tested by TUNEL for susceptibility to TG-inducible apoptosis (see legend for Fig. 1B). D, A375-C6 cells were treated with TG (500 nM) for various periods of time; whole-cell protein
extracts from the cells were then subjected to Western blot analysis
for EGR-1, p53, or actin protein levels. The intensity of the signal in
each lane was determined by densitometric scanning and the signals for
EGR-1 or p53 were normalized with respect to the corresponding signal
for actin. The relative normalized expression levels are shown as a
function of time. E, A375-C6 cells were cotransfected with
pCMV-mEGR1 plus vector, with pCMV-mEGR1 plus pCMV-m175, with pCMV-m175
plus vector, or with vector alone, and transfected cell lines stably
expressing the plasmids were tested for apoptosis by TUNEL (see legend
for Fig. 1B).
[View Larger Version of this Image (35K GIF file)]
*
This work was supported by National Institutes of Health
Grants CA52837 and CA60872, by Council for Tobacco Research Grant 3490, and by McDowell Cancer Foundation Gene Therapy Grant 4-32716 (to
V. M. R.).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: Combs Research
Bldg., Rm. 303, University of Kentucky, 800 Rose St., Lexington, KY
40536. Tel.: 606-257-2677; Fax: 606-257-9608.
1
The abbreviations used are: TG, thapsigargin;
EGR-1, early growth response-1; EBS, EGR-1-binding site(s); kb,
kilobase pair(s); CAT, chloramphenicol acetyltransferase; TUNEL,
terminal transferase-mediated dUTP-nucleotide-end labeling; mEGR-1,
mouse EGR-1.
2
P. Nair and V. M. Rangnekar, unpublished
data.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.