Earlygrowthresponse-1gene (
Egr-1
) promoter induction
by ionizingradiationinU87malignantglioma cells
in vitro
Ralph G. Meyer
1,2
, Jan-Heiner KuÈ pper
2
, Reinhard Kandolf
2
and H. Peter Rodemann
1
1
Section of Radiobiology and Molecular Environmental Research, Department of Radiotherapy, and
2
Department of Molecular
Pathology, University of Tu
È
bingen, Germany
The promoter of the earlygrowth response gene (Egr-1)
has been described to b e activated byionizing radiation,
and it seems to be c lear that this process involves dierent
mitogen activated protein (MAP) kinases, dependent on
the speci®c cell type examined. However, early steps
leading to activation of the corresponding pathways and
thus to overexpression of Egr-1 are not well understood.
In this study, deletion m utants of the 5¢ upstream region
of the Egr-1 gene were generated which allowed us t o
correlate the radiation±induction of the Egr-1 promoter in
U87 g lioma cells to ®ve serum response elements. Based on
the d ata shown, a possible role o f two cAMP responsive
elements for radiation-dependent promoter regulation
could be ruled out. O n t he basis of a ctivator/inhibitor
studies applying fetal bovine serum, EGF, PD98059,
anisomycin, SB203580, forskolin a nd w ortmannin, it
couldbedemonstratedthatinU87cellstheERK1/2
and potentially SAPK/JNK, but not the p 38MAPK/
SAPK2, pathway contribute to the radiation-induction of
Egr-1 promoter. In addition, it was observed that irradi-
ated cells secrete a diusible factor into the culture
media which accounts for the radiation-induced promoter
upregulation. By blocking growth factor receptor activa-
tion with suramin, this eect could be completely
abolished.
Keywords: Egr-1 promoter; growth factor receptor; glio-
blastoma cells; ionizing radiation.
The immediate e arly gene Egr-1 (synonyms are NG FI-A,
zif268, TIS8 and kro x24) encodes a transcription factor
involved in cell growth and differentiation. The DNA
binding domain of this protein with its three zinc-®nger
motifs allows speci®c binding to GC rich recognition
sequences in the promoters of many downstream genes
and thus r egulation of their expression. Target genes of the
Egr-1 transcription factor are numerous and include growth
factors such as platelet derived growth factor A chain
(PDGF-A [1]), PDGF-B chain [ 2], b asic ®broblast growth
factor (bFGF) [3], cytokines such as TGF-b [4] and other
proteins that can be affected.
Expression of Egr-1 i tself is t ransiently induced by a
variety of extracellular stimuli such as cytokines, growth
factors s uch as FGF-2 [5], hypoxia [6], shear s tress on
vascular cells caused by blood current [7], tissue injury and
physical stress in¯icted byionizing radiation. Publication of
the latter ®nding [8] initiated investigations on how the Egr-1
promoter may be employed in cancer gene therapy
approaches, controlling ectopic expression of therapeutic
genes with the application of X-rays t o target tissues [9±13].
However, initial s teps of the underlying mechanism of the
observed radio-induction of Egr-1 expression are not
completely understood, although further signal transduc-
tion pathways involved in this gene activation are, at least in
part, well characterized. Functional dissection of the Egr-1
promoter sequence [14±16] r evealed in previous studies t hat
it contains at least ®ve copies [17] of a characteristic
transcription factor binding site designated the serum
response element (SRE), which is described to be respon-
sible for radioinducibility of the ge ne. Due to its consensus
sequence CC(A/T)
6
GG this motif is also known as t he
ÔCArGÕ element, and represents a combined recognition site
for Elk-1, a member of the Ets family which acts a s a ternary
complex f actor (TCF) in concert with other transcription
factors, mainly p68/SRF s erum response factor [18]. The
promoter is strongly activated upon binding of Elk-1/SRF
transcription factor complexes to CArG elements [19].
Complex assembly requires phosphorylation of b oth SRF
and Elk-1 by speci®c kinases which are, at least in the case of
Elk-1, in turn dependent on prior activation of mitogen
activated p rotein kinases (MAPK) downstream of different
signal transduction pathways. At least three differently
regulated but partly overlapping kinase cascade pathways
Correspondence to H. Peter Rodemann, Section of Radiobiology and
Molecular Environmental Research, Department of Radiation
Oncology, University of Tu
È
bingen, Ro
È
ntgenweg 11, D-72076
Tu
È
bingen, Germany. Fax: + 49 7071 29 5900,
Tel.: + 49 7071 29 8596 2,
E-mail: hans-peter.rodemann@uni-tuebingen.de
Abbreviations: Egr-1, earlygrowthresponse-1 gene; PDGF, platelet
derived growth factor; bFGF, basic ®broblast growth factor; SRE,
serum response element; MAPK, mitogen activated protein kinases;
TCF, ternary complex factor; SAPK, stress activated protein kinases;
Raf, ras-activated factor; E RK, extracellularly regulated kinase; PLC,
phospholipase C; FGF, ®broblast growth factor; AP-1, activated
protein-1; TRE, thiophorbolester responsive element; EgrBS, Egr-1
binding site; CRE, cAMP responsive element; MCS, multiple cloning
site; RSV, Rous sarcoma virus; rhEGF, recombinant human epider-
mal growth factor; PtdIns3-kinase, phosphatidylinositol 3-kinase;
b-Gal, b-galactosidase; PVDF, poly(vinylidene di¯uoride); ECL,
enhanced chemoluminescence; IL, interleukin.
De®nition: 1 gray (Gy) 100 rads.
(Received 18 July 2001, revised 26 October 2001, accepted 6 November
2001)
Eur. J. Biochem. 269, 337±346 (2002) Ó FEBS 2002
converge in their activity on the phosphorylation of E lk-1,
including the c ascade leading to the activation of stress
activated protein kinases (JNK/SAPK), the ras-activated
factor/extracellularly regulated kinase (Raf/ERK) pathway
and the activation of the p38 MAPK/SAPK2 pathway.
Independently of these pathways, protein kinase C (PKC) is
able to phosphorylate E lk-1 directly. PKC is activated by
diacylglycerol, which is formed by phospholipase C (PLC)
mediated phosphatidylinositol 4,5-diphosphate cleavage.
PLC in turn is activated by binding to intracellular d omains
of activated g rowth facto r receptors, e.g. the ®broblast
growth factor (FGF) receptor.
In addition to SREs the 700-bp full length Egr-1
promoter comprises several kinds of other binding
sites including a JNK/SAPK dependent activated
protein-1/thiophorbolester responsive element (AP-1/TRE)
site, binding sites f or Egr-1 itself (EgrBS) and two cAMP
responsive elements (CRE). For g ene t herapy purposes a
core promoter of 490 bp (nucleotides )42 5 t o + 65 rela tive
to the putative transcription start [9,10], w as described to be
suf®cient for radioactivation. This includes only the SREs,
Sp1 sites a nd the t wo CREs (see Fig. 1). Whether or not
CRE sites contribute to radioinducibility of the Egr-1
promoter in normal cells was not clearly demonstrated to
date, although t here is evidence that this is the case i n ras-
mutated J urkat cells which exhibit impaired MAP kinase
pathways [20].
In order to i nvestigate the phenomenon of radiation
induction of the human Egr-1 promoter, glioblastoma
cells U87 were transiently transfected with expression
plasmids containing a reporter gene under the control of
wild-type and recombinant versions of the human Egr-1
promoter. This system was used to investigate the Egr-1
promoter regulation under different experimental condi-
tions. We show that Egr-1 promoter can be induced by a
single dose of 4 Gy. The data presented indicate that
radiation induction of the Egr-1 promoter is at least in
part mediated by protein factors secreted byU87 cells
in response t o radiation exposure. The data are discussed
in the context o f the potential use o f Egr-1 pro moter for
radiation-induced gene therapy strategies in radiation
oncology.
MATERIALS AND METHODS
Cloning of Egr-1 promoter variants
A 780-b p fragment of the human Egr-1 promoter was
obtained by double restriction digestion of plasmid
pGL/TiS8 [15] with SmaI/HindIII, with overhanging
5¢ ends ®lled up by using T4 DNA polymerase (Roche,
Mannheim, Germany). T he promoter fragm ent w as then
ligated blunt into pCR-S cript SK+ (Stratagene). The
resulting p lasmid pEgr was used for sequencing a nd as a
source of all following promoter variants which were
generated by conventional plasmid construction methods
using t he restriction enzymes depicted in Fig. 1. As an
exception pD7egr was generated by site directed mutagenesis
of pD5egr (see below). Plasmid pD6egr is similar to pD5egr
but contains an additional cluster of SRE/Ets sites in a
fragment which was generated by cutting pD3egr with
Eco47III/AccIII and polishing the ends of the 207 bp
fragment with T4 DNA polymerase. All promoter versions
were excised from their host plasmids and cloned into
pGFL cut with Sm aI. The resulting plasmids were desig-
nated pwtegrGFL, pD1egrGFL, pD2egrGFL, etc.
Expression plasmid pGFL is based on pGL3basic
(Promega) but the luciferase gene was replaced by an
in-frame ®re¯y luciferase/EGFP fusion with the original
multiple cloning site (MCS) and the synthetic upstream
5¢ polyadenylation signal which blocks unspeci®c transcrip-
tion activation. The presence of EGFP as part of the
luciferase gene allows additional comparison of transfection
ef®ciencies in parallel cultures. Physical properties of the
novel GFL protein were investigated in several sets of
plasmid t ransfection experiments which s howed, that in the
fusion protein luciferase a ctivity was constantly lowered to
70% of the unfused luciferase gene. Also, EGFP ¯uores-
cence was decreased to an estimated 65±70% of free
EGFP. Extensive tests, including complete sets of experi-
ments described in this study were performed in order to
ensure that there was no alteration of promoter behaviour
in corresponding lucifer ase/luciferase±EGFP vectors ( data
not shown). All cloning steps were controlled and veri®ed by
restriction analysis.
Fig. 1. The human Egr-1 promoter and its regulatory e lements as cloned into p wtegrGFL. As a key region the s equence f rom nucleotides )136 to
)56 is shown in detail, comp rising two serum response elemen ts (SRE) ¯anked by two cAMP responsive elements (CRE). Oth er bi nding sites are
located upstream of SRE 1±3, n amely three Sp1 binding s ites, two reco gnition sequences fo r the Egr-1 gene product itself (EgrBS) and an AP1
binding site.
338 R. G. Meyer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Site-directed mutagenesis
Plasmid pD7egrGFL was generated by exchange of ®ve
nucleotides in CRE2 (nucleotides )71 to )58) from
ACGTC t o CTCAT by site directed mutagenesis u sing a
kit (Stratagene). The primer sequence (5 ¢)3¢) w as C CCA
TATATGCCATGTCTCATCACGACGGAGGCGG.
Successful mutagenesis was con®rmed by sequence analysis.
The de®ciency of this altered sequence to bind CREB was
previously published [18].
As a positive control a well characterized Rous Sarcoma
Virus (RSV) promoter, excised from pAdRSVbgal, was
inserted into pGFL, resulting i n pRSVGFL and sequenced.
This promoter stems from rous sarcoma virus, strain
Schmidt±Ruppin (EMBL database accession no. L29198).
Cell culture and transfection procedures/irradiation
of transfected cells
Human U87gliomacells were kept in DMEM (Gibco),
supplemented with 10% fetal bovine serum (Gibco), under
standard cell culture conditions (5% CO
2
,37°C).
FuGene (Boehringer Mannheim) transfections were
performed a ccording t o i nstructions of the m anufacturer.
Transfection ef®ciencies were determined by transfection of
plasmid pCMVb (Clontech) into U87 cells; 48 h after
transfection cells were stained for b-galactosidase (b-Gal)
activity using the b-Gal staining kit purchased from
Invitrogen.
Routine luciferase activity measurement procedures were
performed according t o the following procedure. Cells were
plated at a density of 10
5
cells per 35 mm dish and allowed
to attach for 24 h. FuGene transfections were performed by
using 900 ng pDxEgrGFL per cell culture dish. pCMVb
(100 ng; Clontech) was added to each culture dish for
internal control of transfection ef®ciency. N egative controls
were performed by transfecting p romoterless p GFL a s w ell
as pRSVGFL constructs in parallel. As tested by pilot
experiments, cotransfection of pCMVb did not in¯uence
basal Egr-1 or RSV promoter activity s igni®cantly. Twenty-
four hours after transfection, cells were exposed to a single
dose of 4 Gy of ionizingradiation generated by a linear
accelerator (Mevatron 6MeV, LINAC) as described else-
where [20,21].
Cells were harvested 48 h aft er irradiation and assayed
for luciferase activity. In experiments using speci®c effectors
of Egr-1 promoter activity these factors/substances were
added 6 h prior to cell harvest (i.e. after 42 h).
Inhibitor and activator studies
Speci®c activators and inhibitors to signal transduction
pathways, except for fetal bovine serum, were obtained
from Calbiochem and dissolved to the following working
solutions.
Fetal bovine serum. fetal bovine serum (Gibco Life
Technologies) was added to a ®nal concentration of 30%
in the culture medium serving as a positive control for
ERK1/2 activation.
Recombinant human epidermal growth factor (rhE-
GF). Stock solutions at a concentration of 100 lgámL
)1
in an aqueous solution of 0.3% BSA with 10 m
M
acetic acid
were added to t he culture m edia at a ® nal c oncentration of
100 ngámL
)1
. r hEGF was used as a positive control for the
immediate early activation of the Egr-1 promoter by
activated Ras-dependent pathways.
PD98059. This was added as a 50-m
M
solution in
dimethylsulfoxide t o the media making a ®nal co ncentration
of 100 l
M
. This c ompound is a potent inhibitor of MEK1
(IC
50
5±10 l
M
) a nd to a lesser extent of MEK2 [ 22,23],
two upstream protein kinases which activate ERK1/2.
Anisomycin. This was dissolved in dimethylsulfoxide
(10 mgámL
)1
) and immediately added to the culture media
at a ®nal concentration of 50 l
M
. Anisomycin (from
Streptomyces griseolus) activates p38MAPK/SAPK2 and
SAPK/JNK and served as a positive control in this study. It
is also an inhibitor of protein expression at the translational
level.
SB203580. This was added as a 1-m
M
working solution in
dimethylsulfoxide to the cells to a ®nal concentration of
10 l
M
. It was used to inhibit p38MAPK/SAPK2 activity.
Forskolin. This was prepared a s a 10-m
M
stock solution
in dimethylsulfoxide and directly added to the culture
medium to m ake a ®nal concentration of 10 l
M
.Beinga
strong and speci®c activator of a denylate cyclase, forsko-
lin induces increased levels of cAMP in treated cells. This
in turn activates cAMP-dependent protein kinase A
(PKA) which phosphorylates CREB and other target
proteins.
Wortmannin. A1m
M
stock solution in dimethylsulfoxide
was further diluted 1 : 100 in NaCl/P
i
and ®nally added
as a 10-l
M
working solution with a ®nal c oncentration in
the media of 100 n
M
. As a speci®c inhibitor o f phosphat-
idylinositol 3-kinase (PtdIns3-kinase, IC
50
5n
M
)wort-
mannin a llows us to investigate d o wnstream signalling of
the P tdIns3 pathway and dependent activation e vents.
Suramin. Suramin (Sigma, St Louis, MO, USA) was added
as an aqueous 3 0 m
M
stock to the media t o make a ®nal
concentration o f 300 l
M
. This substance interferes w ith t he
recognition of several growth factors by their membrane
receptors and, in addition, disrupts the interaction of growth
factor receptors with corresponding adenylate cyclase
activating G-proteins.
For control conditions, cells were treated with equal
amounts of the corresponding solvent (e.g. dimethylsulf-
oxide).
Cell lysis and quanti®cation of reporter gene activities
Cell monolayers were scraped off with a rubber policeman
in 100 lL of r eporter lysis buffer ( Promega), transferred t o
1.5 mL reaction t ubes, centrifuged (13 000 g for 60 s) in a
conventional bench-top centrifuge and kept on ice until
measurements of luciferase activity, b-Gal activity and
protein content which were performed f rom the same cell
aliquots.
Protein content was estimated using Bradford's reagent
(Biorad), b-Gal act ivity was measured by employing a
Ó FEBS 2002 Egr-1 promoterinductionbyionizingradiation (Eur. J. Biochem. 269) 339
commercial kit (Invitrogen). Luciferase activity was deter-
mined with a kit from Promega and measured in a
luminometer (Berthold); m easured values were normalized
to speci®c b-Gal activity. Egr-1 promoter stimulation by
ionizing radiation or chemical compounds was performed
only for the three major promoter mutant plasmids
pD1egrGFL, pD5egrGFL and pD7EgrGFL. All transfec-
tion experiments were performed in triplicates an d repro-
duced at least three times.
Immunoblotting and detection of activated protein
factors with phospho-speci®c antibodies
For Western blot analyses cells were cultured in 60 mm
dishes under n ormal cell culture conditions. T reatment with
speci®c inhibitors or exposure to a single dose of 4 Gy o f
c-irradiation were performed as described above. Thirty
minutes after treatment with activators or inhibitors or
radiation exposure, cells were washed twice with NaCl/P
i
,
SDS sample buffer was added and cells scraped off the plate
and subjected t o denaturing SDS/PAGE. Proteins were
subsequently blotted to a poly(vinylidene di¯uoride)
(PVDF) membrane and detected using phosphospeci®c or
phosphorylation-state-independent antibodies according to
the recommendations of th e manufacturer ( Cell Signaling
Technology, New England Biolabs). As s econdary antibod-
ies, monoclonal horseradish coupled anti-(rabbit I g) Ig or
anti-(mouse Ig) Ig was used allowing identi®cation of
protein bands using enhanced chemoluminescence (ECL)
detection. For detection of site-speci®c phosphorylated
target proteins, i.e. ERK1/2 (T202/Y204), SAPK/JNK
(T183/Y185), p38MAPK/SAPK2 (T180/Y182), ATF-2
(T71), c-Jun (S73) and CREB (S133) phosphospeci®c rabbit
polyclonal Ig w as applied. Phosphorylation-state-indepen-
dent polyclonal antibodies were used to detect total Egr-1,
ATF-2 and c-Jun protein.
RESULTS
Basal activity of Egr-1 promoter variants
Basal activities of cloned variants o f the Egr-1 promoter
were analyzed by measuring the luciferase reporter gene
activity. Transfection experiments with pwtegrGFL showed
that basal activity of t he wild-type Egr-1 promoter construct
was roughly 10±12% of the activity displayed b y a control
RSV promoter.
As demonstrated in Fig. 2, the relative activity of
promoter variant p D1egrGFL ( nucleotides )47 4 t o +12)
did not differ signi®cantly from that of the wild-type
promoter construct pwtegrGFL (nucleotides )720 to
+12). These data indicate that regulatory promoter ele-
ments upstream of nucleotide )474 do not contribute
signi®cantly to basal promoter activity under the conditions
applied. In contrast however, deletion o f nucleotides )259 to
)126 which results inpromoter construct pD3egrGFL led to
a s mall but signi®cant increase in relative basal luciferase
activity (110.5 2.3% of control activity, P < 0.05). A
similar, but more pronounced, effect was observed with
promoter variant pD5egrGFL also lacking nucleotides )259
to )126 but, as compared to pD3egrGFL, additionally
missing the sequence )474 to +12. This promoter variant
presented a signi®cantly( p < 0 .005) enhanced relative basal
activity of 137.1 5.7%. These data indicated, that dele-
tion of the CRE1 site and of the putative Sp1 recognition site
results in an upre gulation of basal Eg r-1 p romoter a ctivity.
Insertion of an additional cluster of two SRE/Ets binding
sites ( nucleotides )474 to )265) into pD5egrGFL resulting
Fig. 2. Basal activity o f Egr-1 promoter variants inU87glioma cells. Deletion mutants of the human Egr-1 promoter were generated i n order t o
con®ne the r adio-inductio n ee ct of the promoter to speci®c tran scription f actor b inding sites. For comparison of promo ter ac tivity i n a no nind uced
state, resul ting p ro moter v ariant s D1egr, D2egr, D3egr, D4egr, D5egr, D6egr and D7egr were cloned into pGFL, carrying an in-frame fusion of EGFP
and ®re¯y luciferase (termed GFL) and transfected in to U87 c ells. Luciferase activities under n ormal cell culture conditions were considered as basal
promoter activities, w hich were compared t o wild-type Egr-1 p romoter activity.
340 R. G. Meyer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
in the variant pD6egrGFL, led to a further but, as compared
to pD5egrGFL, nonsigni®cant increase i n b asal promoter
activity (158 9 .1%).
Removal of this cluster of SRE/Ets bindings sites, as
performed i n p D2egrGFL a nd p D4egrGFL, alway s led to a
dramatic decrease of basal promoter performance to
11.1 0.5% (pD2egrGFL) and 10.5 0.3% (pD4egr-
GFL) of wild-type promoter activity.
Site directed mutagenesis of CRE2 in pD5Egr resulted
in promoter variant pD7Egr, which contains only the SRE
and Ets binding sites of the wild-type promoter. p D7egr-
GFL presented a signi®cantly (P < 0.005) reduced pro-
moter activity which was 61.5 2.8% of the wild-type
promoter.
Immunoblotting of U87 cell lysates followed by detec-
tion of activated ERK1/2 with phosphospeci®c ERK1/2
(T202/Y204) a ntibodies demonstrated detectable amounts
of activated ERK1/2 already formed under normal cell
culture conditions. Relatively high c oncentrations of phos-
phorylated CREB (S133) and ATF-1 were detected,
whereas there appeared to be no detectable amounts of
activated ATF-2 nor SAPK/JNK.
Effect of serum, rhEGF and ionizing radiation
on promoter activity
To analyse the effect of fetal bovine serum and human
recombinant EGF on Egr-1 promoter activity the promoter
variants D1egr, D5egr and D7egr were used. Increasing the
concentration of fetal bovine serum in the culture medium
to 30% resulted in an ef®cient induction of all three
promoter plasmids tested (pD1egrGFL pD5egrGFL and
pD7egrGFL) within a 6-h interval of application. As shown
in Fig. 3 t he corresponding luciferase activity increased to
157.3 12.6% (pD1egrGFL), 149.2 13.4% (pD5egr-
GFL), or 206.6 33.1% (pD7egrGFL), respectively, as
compared to wild-type activity.
Treatment of U87cells carrying these promoter con-
structs with 1 0 ngámL
)1
rhEGF resulted i n a pronounced
stimulation of promoter activities to 205.5 18.8% in
pD1egrGFL transfectants, to 151.9 11.4% in pD5egr-
GFL transfectants and to 2 11.5 22.4% to pD7egr GFL
transfectants as compared to cells transfected with the wild-
type promoter construct (Fig. 3).
As indicated by time kinetic experiments, increased l evels
of ®re¯y luciferase activity due to the exposure of
pwtegrGFL-, pD1egrGFL-, pD5egrGFL-, and pD7eg-
rGFL-transfected U87cells to a single dose of 4Gy of
ionizing irradiation could be observed to be maximal
after 40±48 h post IR (data not shown). As compared
to sham-irradiated controls luciferase activities measured
48 h post IR were increased signi®cantly to 133.7 4.3%
for pD1egrGFL-, 119.1 5.0% for pD5egrGFL and
138.7 4.2% for pD7egrGFL-transfected U87cells (all
P <<0.005).
Protein kinase inhibitor/activator studies
As shown in Fig. 3, a 6-h treatment of U87 cells
transfected with the three Egr-1 promoter mutants with
PD98059 (100 l
M
), a speci®c inhibitor of MEK1,
decreased relative luciferase activities by about 20±30%
as compared to wild-type control levels (pD1egrGFL:
82.6 3.5%; pD5egrGFL: 67.6 4.8%; pD7egrGFL:
72.0 4.7%).
Under the same treatment conditions anisomycin, a
speci®c activator of p38MAPK/SAPK and SAPK/JNK,
did not alter the luciferase activity of pD1egrGFL (relative
activity: 103.3 6.8%) and pD7egrGFL (relative activity:
106.5 19.8%) as compared to wild-type controls. How-
ever, anisomycin resulte d in a signi®cant (P < 0.005)
inhibition of the pD5egrGFL luciferase a ctivity by a bout
26% (relative activity: 74 2.1%). Treatment with
SB203580, which i s a potent inhibitor of p38MAPK/SAPK,
resulted in a pronounced promoter activation of pD1eg-
rGFL (131.7 9.5%, P<0.005) and pD7egrGFL
(124.2 12.3%). In contrast to these results a signi®cant
decrease in luciferase activity by about 27% could be
Fig. 3. D1egr, D5egr and D7egr promoter activities as a function of inhibitors/activators of speci®c signal tra nsduction pathways. U87cells were
transfected w ith reporter gen e constructs containing an i n-frame EGFP±luciferase gene under the control of modi®ed Egr-1 promoters (see F ig. 1)
using FuGene transfection reagent (Roche, Mannheim, Germany). Forty-two hours after the start o f t ransfection cells were treated with chemical
eectors as indicated. Cells were further incubated for 6 h and assayed for luciferase activity 48 h post transfection. Irradiated cells were exposed to
a single dose of 4 Gy of ionizingradiation 16 h after the start o f transfection and luciferase activity was deter mined 48 h postirradiation.
Cotransfected plasmid pCMVbGal (Clontech) was used as an internal control of transfection eciency in all samples. Data represent the mean
SE from th ree to six exp eriments performed in triplicates.
Ó FEBS 2002 Egr-1 promoterinductionbyionizingradiation (Eur. J. Biochem. 269) 341
observedinpD5egrGFL-transfected cells ( relative activity:
73.1 6.6%; P < 0.005) (Fig. 3).
Treatment w ith 1 0 l
M
forskolin, a speci®c activ ator o f
adenylate c yclase, did not signi®cantly alter the promoter
activity incells transfected with the luciferase reporter
constructs pD1egrGFL and pD7egrGFL, but l ed to a slight,
but signi®cant decrease to 84.2 8.9% (P <0.05) of
relative activity incells transfected with pD5egrGFL
(Fig. 3 ).
For all three promoter variants tested, treatment of the
transfected cells with wortmannin, a speci®c inhibitor of
PtdIns3 k inase, resulted in a m easurable downregulation,
which ranged between 10% and 17% (pD1egrGFL:
84.0 6.8%; pD5egrGFL: 82.9 15.3%; pD7egrGFL:
90.8% 4.1%) within a 6-h i nterval.
In order to a nalyse, whether factors produced and
secreted in response to radiation exposure may alter the
activity levels of the promoter variant construct pD7egrGFL
in transfected cells, a cross feeding exp eriment as described
in Materials and methods was performed. When culture
media from irradiated was f ed to nonirradiated cells
transfected with promoter variant pD7egrGFL the luciferase
activity was increased to approximately the same level
(130.6 4.8%) as in irradiated cells (132 10.2%).
Levels of activity in the irradiated cells did not decrease
signi®cantly after a ddition of control medium f or up to 6 h,
indicating a l onger lasting s ecretion of activator molecules
or growth factors. Adding suramin, a potent inhibitor of
growth factor receptor±ligand binding, to the culture
medium of irradiated cells or nonirradiated cells fed with
medium from irradiated cells the stimulatory effect on
luciferase activity of promoter variant plasmid pD7egrGFL
could c ompletely b e abolished (87.49 10.15%, 0 Gy a nd
84.89 11.87%, 4 Gy, Fig. 4).
Western-blot analyses
For the interpretation of the regulatory function of the
different Egr-1 promoter elements and t he role of speci®c
signal transduction pathways in activating the Egr-1 pro-
moter with a nd without radiation exposure W estern blot
analyses of untransfected cells for different target proteins
and their phosporylation status were performed. Therefore,
protein extracts from the same set of cells used for the
analyses of luciferase activity under different treatment
conditions with serum, rhEGF and ionizingradiation as
well as inhibitors or activators of speci®c protein kinases
were used.
Fetal bovine serum treatment. After treatment with 30%
fetal bovine s erum increased ERK1/2 phosphorylation w as
shown by immunoblot analysis (Fig. 5). This could account
for the promoterinduction shown in Fig. 3. While there was
slight activation of c-Jun ( S73), ATF-2 phosphorylation did
not seem to be affected by elevated fetal bovine serum
concentration (Fig. 5).
RhEGF-treatment. Treatment with 10 ngámL
)1
rhEGF
resulted in the strongest phosphorylation a nd activation of
ERK1/2 (T202/Y204) (Fig. 5 ), re¯ecting t he highest Egr-1
promoter induction as shown for all mutants tested (Fig. 3).
Immunoblots incubated with phospho-speci®c antibodies
revealed increased levels of phosphorylated CREB and
ATF-1 (S133), S APK/JNK ( T183/Y185) as well as upre-
gulation at the translational level and activation of ATF-2
(T71) and c-Jun (S73). An increase in p38MAPK/SAPK2
(T180/Y182) activation was not observed.
Radiation exposure. After exposure to ionizing radiation,
phosphorylated SAPK/JNK was detectable, but to a much
lesser degree t han it was observed a fter rhEGF treatment
(Fig. 5). Activation of p38MAPK/SAPK2 could not be
detected and ERK1/2 phospho rylation was slightly lower
than in control samples. A s a result, there were only low
amounts of phosphorylated CREB (S133); however, the
level of phosphorylated ATF-1 remained unchanged as
compared to controls. Furthermore, radiation exposure
resulted in high amounts of activated ATF-2 and c-Jun. As
it could be demonstrated using speci®c phosphorylation-
independent antibodies as controls for ATF-2 (T71) and
c-Jun (S73) levels the expression of both proteins was
strongly upregulated.
PD98059. As illustrated in Fig. 5, PD98059 as an inhibitor
of MEK1, clearly prevented activation of MEK1
(IC
50
5±10 l
M
). As ERK1 and ERK2 are activated
by MEK1, treatment of U87cells with 100 l
M
PD98059
speci®cally abolished the phosphorylation of ERK1/2
without affecting t he activity status of ATF-1, ATF-2,
SAPK/JNK, and c-Jun.
Anisomycin. Exposure of U87cells to 10 l
M
anisomycin
did not result in marked changes in phosphorylation
status of ERK1/2. The amount of phospho-SAPK/JNK
(T183/Y185) and especially o f phospho-p38MAPK/SAPK2
(T180/Y182) were strongly increased ( Fig. 5). T his activa-
tion coincided with high l evels of phosphorylated CREB,
ATF-1 and ATF-2; however, phosphorylation or activation
Fig. 4. Suramin inhibits radiation-induced Egr-1 p romoter activation.
After transient transfection with plasmid pD7egr, U87cells were irra-
diated (4 Gy) and in cu bated without subsequent exchange of c ulture
media (Control). After 42 h, media were removed from irradiated cells
and replaced by media from parallel, unirradiated, transfected cells
(A2). The un irradiated cultures, in turn, re ceived media from corre-
sponding irradiated cultures (A1). The addition of culture media from
irradiatedcellsledtoanincreaseinluciferaseactivityintheunirradi-
ated cells (A1) up to the l ev el reached by irradiated cells (control cells
and A2). Addition of 300 l
M
suramin abolished the eect (B1, B2).
Cells were harvested after a t otal time interval of 48 h and luciferase
activities were determined. Data represent the mean SE from three
independent experiments performed at least in triplicates.
342 R. G. Meyer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
of c-Jun (S73) was only marginal. By using a pan-c-Jun
antibody, which recognizes c-Jun independently of its
phosphorylation status, a partial phosphorylation of the
c-Jun protein, presumably in position S63, could be
demonstrated.
SB203580. As control cells, grown under standard
conditions, presented basically no phosphorylated
p38MAPK/SAPK2, no alterations of the phosphorylation
pro®le was to be expected by treating the cells with the
p38MAP-kinase inhibitor SB203580. However, potentially
as side-effects which have also been described i n the recent
literature, SB203580 caused st rong activation of E RK1/2
(T202/Y204) via activation of Raf1 [24±26] and inhibition of
CREB/ATF1 activation as shown in immunoblots at a
concentration of 10 l
M
.
In this context it remains to be addressed in more detail
whether the profoun d promoter a ctivation of p D1egrGFL
and pD7egrGFL shown in Fig. 3 is related to t he Western
blotting results.
Forskolin. While forskolin had no effect on the phospho-
rylation of SAPK/JNK, p38MAPK/SAPK2, ATF-2 or
c-Jun, a marked increase in phospho-CREB (S133) levels
could be observed ( Fig. 5). A s CREB phosphorylation i s a
cAMP-dependent reaction [27], this observation proves
functionality of the drug. A s shown in Fig. 5 in agreement
with results from o thers [28] forskolin abolis hed ERK1/2
phosphorylation inU87 cells. The extent of inhibition was
comparable to that shown above for PD98059.
Wortmannin. As a consequence of w ortmannin treatment
no activation of SAPK/JNK or p38MAPK/SAPK2 was
observable and the amounts of phosphorylated ERK1/2
were slightly diminished. Additionally no effect on ATF-2
phoshorylation or expression could be observed; however,
reduced levels of activated CREB, ATF-1 and c-Jun were
present after treatment with wortmannin, whereas no
phosphorylated c-Jun could be detected.
DISCUSSION
One of the main aims of this work was to investigate
mechanisms of radiationinduction of the human Egr-1
promoter and its potential use for radiation induced gene
therapy as recently reported [9,29]. While it is ®rmly
established, that the Egr-1 promoter is effectively and
quickly activated bygrowth factors, such as EGF, bFGF
and other serum compounds [5,30,31], mechanisms leading
to inductionbyradiation are by far less well understood.
Most likely both, growth factor and radiation stimuli
ultimately lead to the binding of SRF and TCF/Elk-1 to
SREs which are recognized as overlapping CArG/Ets
binding sites forming the core promoter [14±17]. By the
Fig. 5. Immunoblot analyses of activated protein factors inU87cells using polyclonal phosphospeci®c antibodies. U87cells were subjected to various
treatments with chemical compounds for a time interval of 30 min and then lysed. Cells exposed to 4 Gy of ionizingradiation were incubated for
30 min prior to harvest. Lysates were r esolved by SDS/PAGE and subsequent immunoblotting with polyclonal rabb it antisera directed speci®cally
against the phosphorylated proteins i ndicated (phosphorylated amino-acid residues are given in b racket s). Phosphorylation-state-independent
rabbit polyclonal antib odies recognized total E gr-1, ATF-2 and c-Jun protein.
Ó FEBS 2002 Egr-1 promoterinductionbyionizingradiation (Eur. J. Biochem. 269) 343
stepwise removal all other known regulatory e lements from
the 5¢ upstream region of the wild-type Egr-1- promoter,
such as CREs as well as AP1-, Egr-1- and Sp1- binding sites,
we were able to provide additional evidence s upporting the
prevailing view that these binding sites are suf®cient to
maintain responsiveness of the Egr-1 promoter t o EGF and
ionizing radiation.
In general, the radiation-dependent Egr-1 promoter
upregulation in U 87 gliomacells observed in t he presen t
study was weak but signi®cant. A single dose of 4 Gy
upregulated the promoter variant pD7egrGFL by a factor of
1.4 (p < 0 .05). Similar data of induction of the Egr-1
promoter byionizingradiation h ave been reported recently
[29]. Using synthetic promoter constructs consisting merely
of r epetitive SRE consensus sequences Marples et al.[29]
described an upregulation of Egr-1 by a factor of 1.5±2.5
after radiation exposure of U87 cells.
Based on literature data [32] it could be expected that
stimulation w ith EGF results in t he phosphorylation and
thus binding of TCF/Elk-1 to SRE through activation of the
MAP kinases ERK1/2 or SAPK/JNK or both. However, in
our hands strong activation of p38MAPK/SAPK2 was not
observed inU87cells except after anisomycin treatme nt.
Immunoblot analyses showed activation of ERK1/2 after
stimulation of U87 c ells with fe tal bovine serum, E GF and
SB203580 but not after e xposure to i onizing radiation. This
data indicate that ERK1/2 and SAPK/JNK pathways may
be activated apart from each other. Both, SAPK/JNK and
ERK1/2, are activated upon growth factors binding to
their receptors. However, signal tran sduction leading to
JNK/SAPK activation i s known to b e mainly triggered by
ultraviolet light (UV-C) [34±36], ionizing radiation
(reviewed in [ 37]), p roin¯ammatory c ytokines [30,31] a nd
DNA damaging agents [37], whereas the Raf/ERK pathway
is preferentially activated upo n binding of growth factors t o
receptor tyrosine kinases ([18] and many others).
While there i s some overlap of both pathways our data
based on EGF treatment and protein kinase inhibitor
studies suggest a clear preference of the ERK1/2 pathway
after binding of EGF to its receptor, whereas ionizing
radiation seems t o favour SAPK/JNK activation as d em-
onstrated by the pronounced c-Jun activation observed by
Western blot a nalyses. As a clear immediate early reaction
after exposure to ionizingradiation a strong increase in
c-Jun activation and expr ession was apparent. A lthough in
our study no evidence has been obtained that Egr-1
immediate earlygeneinduction could be observed 30 min
after exposure t o i onizing radiation, Liu et al. [ 38] reported
that exposure of serum-depleted, quiescent U87 cells
presented a slight induction of Egr-1 2 h after exposure to
UVC.
Forskolin inhibited ERK1/2 phosphorylation under
normal cell c ulture conditions, but had no signi®cant effect
on Egr-1 promoter activity i n our experimental setup. As it
has also been reported by others [28] as a speci®c side-effect
of forskolin this compound ab olished ERK1/2 phosphory-
lation in the U87cells used in the present study. Therefore,
this ®nding supports the view, that Egr-1 promoter activity
is not strictly dependent on ERK1/2 activity, but may in
part be regulated b y the SAPK/JNK [39,40] or the PKC
pathway. Furthermore, in the present study we were able to
show that binding of EGF to its receptor led to strong Egr-1
expression U87cells transfected with Egr-1 reporter
constructs. On t he other hand, expression of ATF-2 and
c-Jun was not signi®cantly affected after EGF treatment o f
these cells, but strongly induced after exposure to i onizing
radiation. The fact that ATF-2 and c-Jun were phospho-
rylated to a great extent due to the radiation exposure,
provides good evidence for the activation of an intact
SAPK/JNK pathway, as this is the major kinase activating
the two transcription factors [35,41 ].
Secretion of bFGF and interleukin-1a (IL-1a) has been
reported after U V-irradiation of H eLa or normal ®broblast
cells [42]. This phenomenon may help to transmit a
radiation-induced signal to nonirradiated cells. Moreover
it may establish an obligatory growth factor l oop on the
producer cell, both leading to expression of immediate early
genes, e.g. c-Jun, via the activation of S APK/JNK [42,43].
Growth factor-mediated early step in radiation-induced cell
signaling could be inhibited either by speci®c antibodies
directed against b FGF or IL-1a as well as by preincubation
with suramin [42,43]. These results although obtained after
irradiating cells with UV light are in perfect agreement with
the immunoblot analyses and suramin inhibition experi-
ments presented in our study indicating an auto- and/or
paracrine growth factor dependency of Egr-1 promoter
activation byionizing radiation. However, in contrast to the
immediate-early expression observed for c-Jun, r adiation-
induction of Egr-1 seems to be a rather slow, long-lasting
and potentially SAPK/JNK independent phenomenon
which may involve additional, so far unknown, mecha-
nisms. Based on data r eported by Woloschak et al.[44]itis
very likely, that activation of protein kinase C (PKC) a dds
to the phosphorylation of Elk-1 and therefore to the
induction of Egr-1 expression in response to radiation
exposure. With respect to t hese results and our own data it
can be assumed, however, that growth factor r elease from
irradiated cells unequivocally contributes to radiation-
mediated Egr-1 expression via the ERK1/2 or the PKC
pathway. However, how the secretion into the media is
triggered still remains an open question a nd needs to b e
investigated further.
As a second aspect of Egr-1 gene regulation, our data
indicate a strong impact of CRE sites on basal promoter
activity. T his suggests, that the w ild-type promoterin U87
glioma cells is in part also dependent on the phosphoryla-
tion state o f CREB, ATF-1, ATF-2 and c-Jun p roteins. As
reported by Van Dam et al. [ 39], activation o f ATF-2 upon
genotoxic stress is mediated preferentially by SAPK/JNK,
leading to the induction of c-Jun expression. According t o
our data, the SAPK/JNK pathway transmits at least part of
Egr-1 radiation-induction. The CRE sites m ay thus be
involved in the radiation-dependent Egr-1 expression.
Indeed, stress induced activation o f Egr-1 gene expression
via promoter-CRE sites has b een described for ras-mutated
Jurkat cells which present a n impaired Ras/Raf/ERK
pathway [20]. However, under the experimental conditions
applied in t his investigation, a speci®c radiation-dependent
regulation of CRE sites was not observed.
As indicated by the deletion of CRE2 in the pD7egrGFL
mutant, this element positively regulates Egr-1 promoter
activity (see Fig. 2). Inhibition of p38MAPK/SAPK2
dependent activation of these proteins may therefore
account for the low pD5egrGFL promoter activity after
SB203580 incubation. This promoter construct contains
only one CRE2 element. Deletion of CRE1 as performed in
344 R. G. Meyer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
pD5egrGFL revealed its role as a negative r egulatory
element, which is in line with previously published w ork
[15,46]. The p resence of both, an activating CRE2 and an
inhibitory CRE1 in pD1egrGFL m ay therefore neutralize
the effects of S B203580 on CREB/ATF1 phosphorylation.
Thus, in pD1egrGFL and pD7egrGFL transfected U87 cells
SB203580 induces increased luciferase activity merely due to
ERK1/2 activation.
Taken together in the present s tudy we provide evidence
for the ra diation-induction of the Egr-1 promoter and the
regulatory signal transduction pathways involved in this
activation. While no evidence for activation of the
p38MAPK/SAPK2 pathway exist from our data, other
pathways involving ERK1/2 and potentially SAPK/JNK
seem to be primarily involved in the initial signal from the
receptor protein to the Egr-1 p romoter. Most interestingly,
however, a bFGF-like factor released by irradiated cells
contributes markedly to the radiation-induction of the Egr-
1 promoter via upstream S RE elements. Closer insight into
the very early events of Egr-1 geneinduction and identi®-
cation of the secreted factor will be useful for further
successful utilization of the Egr-1 promoterin combined
radiation-inducible gen e therapy approaches as well as for
the understand ing of stress-dependent regulation of cell
growth in general.
ACKNOWLEDGEMENTS
Vector pAdRSVbgal was a kind gift from Dr Ju
È
rgen Kleinschmidt,
Deutsches Krebsforschungszentrum Heidelbe rg. Thanks also to
Kathleen M. Sakamoto, University o f California, Lo s Angeles, fo r
permission to use plasmid pGL/TiS8. T his work was sup ported by a
grant from t he Deutsche Forsc hungsgemeinschaft (Ro527/3-1,2) and
the Dr M ildred Scheel Stiftung (10-1 503-Ku
È
I).
REFERENCES
1. Silverman, E.S., Khachigian, L.M., Lindner, V., Williams, A.J. &
Collins, T. (1997) Inducible PDGF A-chain transcription in
smooth muscle cells is mediated by Egr-1 displacement of Sp1 and
Sp3. Am . J. Physiol. 273, 1415±1426.
2. Khachigian, L., Lindner, L., Williams, A. & Collins, T. (1996)
Egr-1induced endothelial gene expression: a common theme in
vascular i njury. Science 271, 1427±1431.
3. Biesiada, E., Razandi, M. & Levin, E.R. (1996) Egr-1 activates
basic ®broblast growth factor transcription. Mechanistic i mp li-
cations for astrocyte p roliferation. J. Biol . C hem. 271 , 1 8576±
18581.
4. Kim, S J., Jean g, K T., Glick, A.B., S porn , M.B . & Roberts,
A.B. (1989) Promoter sequences of the human transforming
growth f actor-b1 gene respo nsive to transforming growth facto r-
b1 autoinduction. J. Biol. Chem. 264, 7041±7045.
5. Santiago, F.S., Lowe, H.C., Day, F.L., Chesterman, C.N. &
Khachigian, L .M. (1999) Earlygrowth response factor-1 induc-
tion by injury is triggered by release and p aracrine activation by
®broblast g rowth factor-2. Am. J. Pathol. 154, 937±944.
6. Yan, S.F., Lu, J., Zou, Y.S., Soh-Won, J., Coh en, D.M., Buttrick,
P.M., Cooper, D.R., Ste inbe rg, S.F., Mackman, N., P insk y, D.J.
& Stern, D.M. (1999) Hypoxia-associated induction of early
growth response-1gene e xpression. J . Biol. Che m. 274, 15030±
15040.
7. Schwachtgen, J.L., Houston, P., Campbell, C., Sukhatme, V. &
Braddock, M. (1998) Fluid shear stress activation of Egr-1
transcription in cultured human endothelial a nd epithelial cells
is mediated via the extracellular signal-related kinase 1/2 mitogen-
activated protein kinase pathway. J. Clin. Invest. 101, 2540±
2459.
8. Keyse, S.M. (1993) The induc tion of gene expression in mam-
malian cellsby radiation. Sem. Cancer Biol. 4, 119±128.
9. Weichselbaum, R.R., Hallahan, D.E., Beckett, M.A., Mauceri,
H.J., Lee, H., Sukhatme, V.P. & Kufe, D.W. (1994) Gene therapy
targeted byradiation p referentially radiosensitizes tumor cells.
Cancer Res. 54, 4266±4269.
10. Hallahan, D.E., Mauceri, H.J., Se ung, L.P., Dun phy, E.J.,
Wayne, J.D., Hanna, N .N., Toledano, A ., Hellman, S., Kufe,
D.W. & Weichselbaum, R.R. (1995) Spatial and temporal control
of gene therapy using ionizing radiation. Nat. M ed. 1, 786±791.
11. Joki, T., Nakamura, M. & O hno, T. (1 995) Activation o f the
radiosensitive EGR-1 promoter induces expression of the herpes
simplex virus thymidine k inase g ene a nd sensitivity of human
glioma cells to ganciclovir. Hum. Ge ne Ther. 6, 1507±1513.
12. Manome,Y.,Kunieda,T.,Wen,P.Y.,Koga,T.,Kufe,D.W.&
Ohno, T. (1998) Transgene expression inmalignantglioma using a
replication-defective adenoviral vector containing the Egr-1 pro-
moter: activation by ion izing radiation or up take of radioactive
iododeoxyuridine. Hum. Gene Ther. 9, 1409±1417.
13. Kim, S.H., Kim, J.H., Kolozsvary, A., Brown, S.L. & Freytag,
S.O. (1997) Preferential radiosensitization of 9L glioma cells
transduced with HSV-tk geneby acyclovir. J. Neurooncol. 33, 189±
194.
14. Sakamoto, K.M., B ardeleben, C., Y ates, K .E., Rai nes, M .A.,
Golde, D.W. & Gasson, J.C. (1991) 5¢ upstream sequenc e and
genomic structure of the primary response g ene EGR-1 /TIS8.
Oncogene 6, 867±875.
15. Aicher, W.K., Dinkel, A., Grimbacher, B., Haas, C.V.,
Seydlitz-Kurzbach, E., Peter, H.H. & Eibel, H. (1999) S erum
response elements activate and cAMP responsive elements inhibit
expression of tran scription factor E gr-1 in syn ovial ®broblasts of
rheumatoid arthritis p atients. Int. Imm unol. 11, 47±61.
16. Christy, B. & Nathans, D. (1989) Functional serum r esponse
elements up stream of the growth factor-inducible gene zif68. Mol.
Cell Bi ol. 11, 4889±4895.
17. Schwachtgen, J.L., Campbell, C.J. & Braddock, M. (2000) Full
promoter sequence of human earlygrowth response factor-1 (Egr-1):
demonstration of a ®fth functional serum response element. DNA
Seq. 10 , 429±432.
18. Rolli, M., Kotlyarov, A., Sakamoto, K.M., Gaestel, M. & Nein-
inger, A. (1999) Stress-induced stimulation of early growth
response ge ne-1 by p38/stress-activated protein k inase 2 is medi-
ated by a cAMP-responsive promoter e lement in a MAPKAP
kinase 2-indepe ndent manne r. J. Biol. Chem. 274, 1 9559±19564.
19. Marais, R., Wynne, J. & Treisman, R. (1993) The SRF accessory
protein E lk-1 contains a growth f actor-regulated transcriptional
activation d omain. Cell 73, 381±393.
20. Burger, A., L o
È
er, H., B amberg, M. & Rodemann, H.P. (1998)
Molecular and cellular basis of radiation ®brosis. Int. J. Rad. Biol.
73, 401± 408.
21. Hakenjos, L., B amberg, M. & Rodemann, H.P. ( 2000) TG F-
b1-mediated alterations of rat lung ®broblast dierentiation re-
sulting in the radiation-induced ®brotic response. Int. J. Rad. Biol.
76, 503± 509.
22. Alessi, D.R., Cuenda, A., Cohen, P., Dudley, D.T. & Saltiel, A.R.
(1995) PD098059 is a speci®c inhibitor of the activation of
mitogen-activated protein kinase kinase in vit ro and in vivo . J. Biol.
Chem. 270, 27489±27494.
23. Dudley,D.T.,Pang,L.,Decker,S.J.,Bridges,A.J.&Saltiel,A.R.
(1995) A synthetic inhibitor of the mitogen-activated protein
kinase cascade. Proc. Natl Acad. Sci. USA 92, 7686±7689.
24. Hall-Jackson, C.A., Eyers, P.A., Cohen, P., Goedert, M.,
Boyle, F.T., Hewitt, N., Plant, H. & Hedge, P. (1999) Paradoxi-
cal activation of Raf by a novel Raf inhibitor. Chem. Biol. 6,
559±568.
Ó FEBS 2002 Egr-1 promoterinductionbyionizingradiation (Eur. J. Biochem. 269) 345
25. Hall-Jackson, C.A., Goedert, M., Hedge, P. & Cohen, P. (1999)
Eect of S B203580 on the a ctivity of c-Raf invitro and in vivo .
Oncogene 18, 2047±2054.
26. Kalmes,A.,Deou,J.,Clowes,A.W.&Daum,G.(1999)Raf-1is
activated by the mitogen-activated protein kinase inhibitor,
SB203580. FE BS Lett. 444, 71±74.
27. Gonzalez, G.A. & Montminy, M.R. ( 1989) Cyclic AMP stimu-
lates somatostatin gene transcription by phosphorylation of
CREB at serine 133. Cell 59, 675 ±680.
28. David, M., Petricoin, E . & Larner, A .C. (1996) Activation o f
protein kinase A inhibits inte rferon ind uction of t he Jak/Stat
pathway in U266 cells. J. Biol. Chem. 27 1, 4585±4588.
29. Marples, B., Scott, S.D., Hendry, J.H., Embleton, M.J., Lashford,
L.S. & Margison, G.P. (2000 ) Development of synthetic pro-
moters for radiation-mediated gene t herapy. Gene Ther. 7,511±
517.
30. Cao, X.M., Guy, G.R., Sukhatme, V.P. & Tan, Y.H. (1992)
Regulation of the Egr-1 genebytumornecrosisfactorandinter-
ferons in primary h uman ®broblasts. J. Biol. Chem. 267, 1345±
1349.
31. Groo, M., Boxer, L.M. & Thiel, G . (2000) Nerve growth factor-
and epidermal growth factor-regulated gene transcriptio n in PC12
pheochromocyt oma a nd INS-1 insulinoma ce lls. Eur. J. Cell Biol.
79, 924±935.
32. Whitmarsh, A.J., Shore, P., Sharrocks, A.D. & Davis, R.J. (1995)
Integration of MAP kinase signal transduction pathways at the
serum r esponse e lement. Science 269, 403±407.
33. Hibi, M., Lin, A., Smeal, T., Minden, A. & Karin, M. (1993)
Identi®cation of an oncoprotein- and UV-responsive protein
kinase that binds and potentiates the c-Jun activation domain.
Genes Dev. 7, 2135±2148.
34. Derijard, B., Hibi, M., Wu, I.H., Barrett, T., S u, B., Deng, T.,
Karin, M. & Davis, R.J. (1994) JNK1: a protein kinase stimulated
by UV light and Ha-Ras that b inds an d phosp horylates the c-Jun
activation domain. Cell 76, 1025±1037.
35. Wilhelm, D., van Dam, H., Herr, I., Baumann, B., Herrlich, P. &
Angel, P. (1995) Both ATF-2 and c-Jun are phosphorylated by
stress-activated protein kinases in response to UV irradiation.
Immunobiology 193, 143±148.
36. Verheij, M., Ruiter, G.A., Zerp, S.F., van Blitterswijk, W .J., Fuks,
Z., Haimovitz-Friedman, A. & Bartelink, H. (1998) The role of the
stress-activated protein kinase (SAPK/JNK) signaling pathway in
radiation-induced a poptosis. Rad iother. Oncol. 47, 225±232.
37. VanDam,H.,Wilhelm,D.,Herr,I.,Steen,A.,Herrlich,P.&
Angel, P. (1995) ATF-2 is preferentially activated by stress-
activated protein kinases to mediate c-Jun inductionin response to
genotoxic agents. EMBO J. 14 , 1798±1811.
38. Liu, C., Yao, J., M ercola, D. & Adamson, E. (2000) The tran-
scription factor Egr-1 directly transactivates the ®bronectin gene
and enhances attachment of human glioblastoma cell line U 251.
J. Biol. Chem. 275, 2 0315±20323.
39. Cavigelli, M., Dol®, F., Claret, F.X. & Karin, M. (1995) Induction
of c-fos e xpression through JNK-mediated T CF/Elk-1 p ho spho-
rylation. EMBO J. 14, 5957±5964.
40. Gille, H., Strahl, T. & Shaw, P.E. (1995) Activation of ternary
complex factor Elk-1 by stress-activated p rotein kinases. Curr.
Biol. 5, 1191±1200.
41.Gupta,S.,Campbell,D.,Derijard,B.&Davis,R.J.(1995)
Transcription factor ATF-2 regulation by the JNK signal trans-
duction pathway. Science 267 , 389±393.
42. Kra
È
mer, M., Sachsenmaier, C., H errlich, P. & Rahmsdorf, H.J.
(1993) UV irradiation-induced Interleukin-1 and basic ®broblast
growth factor synthesis and release mediate part of the UV
response. J. Bi ol. Chem. 268, 6734±6741.
43. Sachsenmaier, C., Radler-P ohl, A., Zinck, R., Nordheim, A.,
Herrlich, P. & Rahmsdorf, H.J. (1994) Involvement of growth
factor receptors in the mammalian UVC response. Cell 78,963±
972.
44. Woloschak, G .E., C hang-Liu, C.M. & S hearin-Jones, P. (1990)
Regulation of protein kinase C byionizing radiation. Cancer Res.
50, 3963±3967.
45. Aicher, W., Sakamoto, K., Hack, A . & Eibel, H . (1999) Analysis
of fu nctional elements in the hum an Egr-1 gene promoter.
Rheumatol. Int. 18 , 207±214.
346 R. G. Meyer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
. Early growth response-1 gene (
Egr-1
) promoter induction
by ionizing radiation in U87 malignant glioma cells
in vitro
Ralph G. Meyer
1,2
, Jan-Heiner. Molecular
Pathology, University of Tu
È
bingen, Germany
The promoter of the early growth response gene (Egr- 1)
has been described to b e activated by ionizing radiation,
and