Peter Rodemann1 1 Section of Radiobiology and Molecular Environmental Research, Department of Radiotherapy, and 2 Department of Molecular Pathology, University of TuÈbingen, Germany The
Trang 1Early growth response-1 gene ( Egr-1 ) promoter induction
Ralph G Meyer1,2, Jan-Heiner KuÈpper2, Reinhard Kandolf2and H Peter Rodemann1
1 Section of Radiobiology and Molecular Environmental Research, Department of Radiotherapy, and 2 Department of Molecular Pathology, University of TuÈbingen, Germany
The promoter of the early growth response gene (Egr-1)
has been described to be activated by ionizing radiation,
and it seems to be clear 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 mutants of the 5¢ upstream region
of the Egr-1 gene were generated which allowed us to
correlate the radiation±induction of the Egr-1 promoter in
U87 glioma cells to ®ve serum response elements Based on
the data shown, a possible role of two cAMP responsive
elements for radiation-dependent promoter regulation
could be ruled out On the basis of activator/inhibitor
studies applying fetal bovine serum, EGF, PD98059, anisomycin, SB203580, forskolin and wortmannin, it could be demonstrated that in U87 cells the ERK1/2 and potentially SAPK/JNK, but not the p38MAPK/ 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 early gene Egr-1 (synonyms are NGFI-A,
zif268, TIS8 and krox24) 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 regulation 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], basic ®broblast growth
factor (bFGF) [3], cytokines such as TGF-b [4] and other proteins that can be affected
Expression of Egr-1 itself is transiently induced by a variety of extracellular stimuli such as cytokines, growth factors such as FGF-2 [5], hypoxia [6], shear stress on vascular cells caused by blood current [7], tissue injury and physical stress in¯icted by ionizing 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 to target tissues [9±13] However, initial steps 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 activatransduc-tion are, at least in part, well characterized Functional dissection of the Egr-1 promoter sequence [14±16] revealed in previous studies that
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 gene Due to its consensus sequence CC(A/T)6GG this motif is also known as the ÔCArGÕ element, and represents a combined recognition site for Elk-1, a member of the Ets family which acts as a ternary complex factor (TCF) in concert with other transcription factors, mainly p68/SRF serum 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 both 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 protein 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 TuÈbingen, RoÈntgenweg 11, D-72076
TuÈbingen, Germany Fax: + 49 7071 29 5900,
Tel.: + 49 7071 29 85962,
E-mail: hans-peter.rodemann@uni-tuebingen.de
Abbreviations: Egr-1, early growth response-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; ERK, 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)
Trang 2converge in their activity on the phosphorylation of Elk-1,
including the cascade 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 Elk-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 domains
of activated growth factor 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 for Egr-1 itself (EgrBS) and two cAMP
responsive elements (CRE) For gene therapy purposes a
core promoter of 490 bp (nucleotides )425 to +65 relative
to the putative transcription start [9,10], was described to be
suf®cient for radioactivation This includes only the SREs,
Sp1 sites and the two 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 there is evidence that this is the case in
ras-mutated Jurkat cells which exhibit impaired MAP kinase
pathways [20]
In order to investigate 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 by U87 cells
in response to radiation exposure The data are discussed
in the context of the potential use of Egr-1 promoter for
radiation-induced gene therapy strategies in radiation
oncology
M A T E R I A L S A N D M E T H O D S
Cloning of Egr-1 promoter variants
A 780-bp 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) The promoter fragment was then ligated blunt into pCR-Script SK+ (Stratagene) The resulting plasmid pEgr was used for sequencing and as a source of all following promoter variants which were generated by conventional plasmid construction methods using the 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 SmaI 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 activatranscrip-tion 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 transfection experiments which showed, that in the fusion protein luciferase activity 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 luciferase/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 elements as cloned into pwtegrGFL As a key region the sequence from nucleotides )136 to )56 is shown in detail, comprising two serum response elements (SRE) ¯anked by two cAMP responsive elements (CRE) Other binding sites are located upstream of SRE 1±3, namely three Sp1 binding sites, two recognition sequences for the Egr-1 gene product itself (EgrBS) and an AP1 binding site.
Trang 3Site-directed mutagenesis
Plasmid pD7egrGFL was generated by exchange of ®ve
nucleotides in CRE2 (nucleotides )71 to )58) from
ACGTC to CTCAT by site directed mutagenesis using a
kit (Stratagene) The primer sequence (5¢)3¢) was CCCA
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 in 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 U87 glioma cells were kept in DMEM (Gibco),
supplemented with 10% fetal bovine serum (Gibco), under
standard cell culture conditions (5% CO2, 37 °C)
FuGene (Boehringer Mannheim) transfections were
performed according to instructions of the manufacturer
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 to the following procedure Cells were
plated at a density of 105cells 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 Negative controls
were performed by transfecting promoterless pGFL as well
as pRSVGFL constructs in parallel As tested by pilot
experiments, cotransfection of pCMVb did not in¯uence
basal Egr-1 or RSV promoter activity signi®cantly
Twenty-four hours after transfection, cells were exposed to a single
dose of 4 Gy of ionizing radiation generated by a linear
accelerator (Mevatron 6MeV, LINAC) as described
else-where [20,21]
Cells were harvested 48 h after 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 mMacetic acid were added to the culture media at a ®nal concentration of
100 ngámL)1 rhEGF 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-mM solution in dimethylsulfoxide to the media making a ®nal concentration
of 100 lM This compound is a potent inhibitor of MEK1 (IC50 5±10 lM) and 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 lM 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-mMworking solution in dimethylsulfoxide to the cells to a ®nal concentration of
10 lM It was used to inhibit p38MAPK/SAPK2 activity Forskolin This was prepared as a 10-mM stock solution
in dimethylsulfoxide and directly added to the culture medium to make a ®nal concentration of 10 lM Being a strong and speci®c activator of adenylate 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 A 1 mMstock solution in dimethylsulfoxide was further diluted 1 : 100 in NaCl/Pi and ®nally added
as a 10-lMworking solution with a ®nal concentration in the media of 100 nM As a speci®c inhibitor of phosphat-idylinositol 3-kinase (PtdIns3-kinase, IC50 5 nM) wort-mannin allows us to investigate downstream signalling of the PtdIns3 pathway and dependent activation events Suramin Suramin (Sigma, St Louis, MO, USA) was added
as an aqueous 30 mMstock to the media to make a ®nal concentration of 300 lM This substance interferes with the 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 reporter lysis buffer (Promega), transferred to 1.5 mL reaction tubes, 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 from the same cell aliquots
Protein content was estimated using Bradford's reagent (Biorad), b-Gal activity was measured by employing a
Trang 4commercial kit (Invitrogen) Luciferase activity was
deter-mined with a kit from Promega and measured in a
luminometer (Berthold); measured 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 and
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 normal cell culture conditions Treatment with
speci®c inhibitors or exposure to a single dose of 4 Gy of
c-irradiation were performed as described above Thirty
minutes after treatment with activators or inhibitors or
radiation exposure, cells were washed twice with NaCl/Pi,
SDS sample buffer was added and cells scraped off the plate
and subjected to 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 the manufacturer (Cell Signaling
Technology, New England Biolabs) As secondary
antibod-ies, monoclonal horseradish coupled anti-(rabbit Ig) 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 was applied
Phosphorylation-state-indepen-dent polyclonal antibodies were used to detect total Egr-1, ATF-2 and c-Jun protein
R E S U L T S
Basal activity of Egr-1 promoter variants Basal activities of cloned variants of the Egr-1 promoter were analyzed by measuring the luciferase reporter gene activity Transfection experiments with pwtegrGFL showed that basal activity of the wild-type Egr-1 promoter construct was roughly 10±12% of the activity displayed by a control RSV promoter
As demonstrated in Fig 2, the relative activity of promoter variant pD1egrGFL (nucleotides )474 to +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 of nucleotides )259 to )126 which results in promoter construct pD3egrGFL led to
a small 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 recognidele-tion site results in an upregulation of basal Egr-1 promoter activity Insertion of an additional cluster of two SRE/Ets binding sites (nucleotides )474 to )265) into pD5egrGFL resulting
Fig 2 Basal activity of Egr-1 promoter variants in U87 glioma cells Deletion mutants of the human Egr-1 promoter were generated in order to con®ne the radio-induction eect of the promoter to speci®c transcription factor binding sites For comparison of promoter activity in a noninduced state, resulting promoter variants 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 into U87 cells Luciferase activities under normal cell culture conditions were considered as basal promoter activities, which were compared to wild-type Egr-1 promoter activity.
Trang 5in the variant pD6egrGFL, led to a further but, as compared
to pD5egrGFL, nonsigni®cant increase in basal promoter
activity (158 9.1%)
Removal of this cluster of SRE/Ets bindings sites, as
performed in pD2egrGFL and pD4egrGFL, always 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
pD7egr-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) antibodies demonstrated detectable amounts
of activated ERK1/2 already formed under normal cell
culture conditions Relatively high concentrations 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 the corresponding luciferase activity increased to
157.3 12.6% (pD1egrGFL), 149.2 13.4%
(pD5egr-GFL), or 206.6 33.1% (pD7egr(pD5egr-GFL), respectively, as
compared to wild-type activity
Treatment of U87 cells carrying these promoter
con-structs with 10 ngámL)1rhEGF resulted in 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 211.5 22.4% to pD7egrpD5egr-GFL transfectants as compared to cells transfected with the wild-type promoter construct (Fig 3)
As indicated by time kinetic experiments, increased levels
of ®re¯y luciferase activity due to the exposure of pwtegrGFL-, pD1egrGFL-, pD5egrGFL-, and pD7eg-rGFL-transfected U87 cells 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 U87 cells (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 lM), 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 resulted in a signi®cant (P < 0.005) inhibition of the pD5egrGFL luciferase activity by about 26% (relative activity: 74 2.1%) Treatment with SB203580, which is a potent inhibitor of p38MAPK/SAPK, resulted in a pronounced promoter activation of pD1eg-rGFL (131.7 9.5%, P < 0.005) and pD7egpD1eg-rGFL (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 transduction pathways U87 cells were transfected with reporter gene constructs containing an in-frame EGFP±luciferase gene under the control of modi®ed Egr-1 promoters (see Fig 1) using FuGene transfection reagent (Roche, Mannheim, Germany) Forty-two hours after the start of transfection 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 ionizing radiation 16 h after the start of transfection and luciferase activity was determined 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 three to six experiments performed in triplicates.
Trang 6observed in pD5egrGFL-transfected cells (relative activity:
73.1 6.6%; P < 0.005) (Fig 3)
Treatment with 10 lM forskolin, a speci®c activator of
adenylate cyclase, did not signi®cantly alter the promoter
activity in cells transfected with the luciferase reporter
constructs pD1egrGFL and pD7egrGFL, but led to a slight,
but signi®cant decrease to 84.2 8.9% (P < 0.05) of
relative activity in cells transfected with pD5egrGFL
(Fig 3)
For all three promoter variants tested, treatment of the
transfected cells with wortmannin, a speci®c inhibitor of
PtdIns3 kinase, resulted in a measurable 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 interval
In order to analyse, 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 experiment as described
in Materials and methods was performed When culture
media from irradiated was fed 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 addition of control medium for up to 6 h,
indicating a longer lasting secretion 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 completely be abolished (87.49 10.15%, 0 Gy and
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 the role of speci®c signal transduction pathways in activating the Egr-1 pro-moter with and without radiation exposure Western 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 ionizing radiation as well as inhibitors or activators of speci®c protein kinases were used
Fetal bovine serum treatment After treatment with 30% fetal bovine serum increased ERK1/2 phosphorylation was shown by immunoblot analysis (Fig 5) This could account for the promoter induction 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 and activation of ERK1/2 (T202/Y204) (Fig 5), re¯ecting the 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), SAPK/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 than it was observed after rhEGF treatment (Fig 5) Activation of p38MAPK/SAPK2 could not be detected and ERK1/2 phosphorylation was slightly lower than in control samples As 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 (IC50 5±10 lM) As ERK1 and ERK2 are activated
by MEK1, treatment of U87 cells with 100 lM PD98059 speci®cally abolished the phosphorylation of ERK1/2 without affecting the activity status of ATF-1, ATF-2, SAPK/JNK, and c-Jun
Anisomycin Exposure of U87 cells to 10 lM anisomycin did not result in marked changes in phosphorylation status of ERK1/2 The amount of phospho-SAPK/JNK (T183/Y185) and especially of phospho-p38MAPK/SAPK2 (T180/Y182) were strongly increased (Fig 5) This activa-tion coincided with high levels of phosphorylated CREB, ATF-1 and ATF-2; however, phosphorylation or activation
Fig 4 Suramin inhibits radiation-induced Egr-1 promoter activation.
After transient transfection with plasmid pD7egr, U87 cells were
irra-diated (4 Gy) and incubated without subsequent exchange of culture
media (Control) After 42 h, media were removed from irradiated cells
and replaced by media from parallel, unirradiated, transfected cells
(A2) The unirradiated cultures, in turn, received media from
corre-sponding irradiated cultures (A1) The addition of culture media from
irradiated cells led to an increase in luciferase activity in the
unirradi-ated cells (A1) up to the level reached by irradiunirradi-ated cells (control cells
and A2) Addition of 300 l M suramin abolished the eect (B1, B2).
Cells were harvested after a total time interval of 48 h and luciferase
activities were determined Data represent the mean SE from three
independent experiments performed at least in triplicates.
Trang 7of 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 in the recent
literature, SB203580 caused strong activation of ERK1/2
(T202/Y204) via activation of Raf1 [24±26] and inhibition of
CREB/ATF1 activation as shown in immunoblots at a
concentration of 10 lM
In this context it remains to be addressed in more detail
whether the profound promoter activation of pD1egrGFL
and pD7egrGFL shown in Fig 3 is related to the 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) As CREB phosphorylation is a
cAMP-dependent reaction [27], this observation proves
functionality of the drug As shown in Fig 5 in agreement
with results from others [28] forskolin abolished ERK1/2 phosphorylation in U87 cells The extent of inhibition was comparable to that shown above for PD98059
Wortmannin As a consequence of wortmannin 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
D I S C U S S I O N
One of the main aims of this work was to investigate mechanisms of radiation induction 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 by growth factors, such as EGF, bFGF and other serum compounds [5,30,31], mechanisms leading
to induction by radiation 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 in U87 cells using polyclonal phosphospeci®c antibodies U87 cells were subjected to various treatments with chemical compounds for a time interval of 30 min and then lysed Cells exposed to 4 Gy of ionizing radiation were incubated for
30 min prior to harvest Lysates were resolved by SDS/PAGE and subsequent immunoblotting with polyclonal rabbit antisera directed speci®cally against the phosphorylated proteins indicated (phosphorylated amino-acid residues are given in brackets) Phosphorylation-state-independent rabbit polyclonal antibodies recognized total Egr-1, ATF-2 and c-Jun protein.
Trang 8stepwise removal all other known regulatory elements 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 supporting the
prevailing view that these binding sites are suf®cient to
maintain responsiveness of the Egr-1 promoter to EGF and
ionizing radiation
In general, the radiation-dependent Egr-1 promoter
upregulation in U87 glioma cells observed in the present
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 by ionizing radiation have been reported recently
[29] Using synthetic promoter constructs consisting merely
of repetitive 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 with EGF results in the 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 in U87 cells except after anisomycin treatment
Immunoblot analyses showed activation of ERK1/2 after
stimulation of U87 cells with fetal bovine serum, EGF and
SB203580 but not after exposure to ionizing 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 transduction leading to
JNK/SAPK activation is known to be mainly triggered by
ultraviolet light (UV-C) [34±36], ionizing radiation
(reviewed in [37]), proin¯ammatory cytokines [30,31] and
DNA damaging agents [37], whereas the Raf/ERK pathway
is preferentially activated upon binding of growth factors to
receptor tyrosine kinases ([18] and many others)
While there is 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 to favour SAPK/JNK activation as
dem-onstrated by the pronounced c-Jun activation observed by
Western blot analyses As a clear immediate early reaction
after exposure to ionizing radiation a strong increase in
c-Jun activation and expression was apparent Although in
our study no evidence has been obtained that Egr-1
immediate early gene induction could be observed 30 min
after exposure to ionizing 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 culture conditions, but had no signi®cant effect
on Egr-1 promoter activity in our experimental setup As it
has also been reported by others [28] as a speci®c side-effect
of forskolin this compound abolished ERK1/2
phosphory-lation in the U87 cells 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 by 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 U87 cells transfected with Egr-1 reporter
constructs On the other hand, expression of ATF-2 and c-Jun was not signi®cantly affected after EGF treatment of these cells, but strongly induced after exposure to ionizing 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 UV-irradiation of HeLa 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 loop on the producer cell, both leading to expression of immediate early genes, e.g c-Jun, via the activation of SAPK/JNK [42,43] Growth factor-mediated early step in radiation-induced cell signaling could be inhibited either by speci®c antibodies directed against bFGF 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 by ionizing radiation However, in contrast to the immediate-early expression observed for c-Jun, radiation-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 reported by Woloschak et al [44] it is very likely, that activation of protein kinase C (PKC) adds
to the phosphorylation of Elk-1 and therefore to the induction of Egr-1 expression in response to radiation exposure With respect to these results and our own data it can be assumed, however, that growth factor release 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 and needs to be investigated further
As a second aspect of Egr-1 gene regulation, our data indicate a strong impact of CRE sites on basal promoter activity This suggests, that the wild-type promoter in U87 glioma cells is in part also dependent on the phosphoryla-tion state of CREB, ATF-1, ATF-2 and c-Jun proteins As reported by Van Dam et al [39], activation of ATF-2 upon genotoxic stress is mediated preferentially by SAPK/JNK, leading to the induction of c-Jun expression According to our data, the SAPK/JNK pathway transmits at least part of Egr-1 radiation-induction The CRE sites may thus be involved in the radiation-dependent Egr-1 expression Indeed, stress induced activation of Egr-1 gene expression via promoter-CRE sites has been described for ras-mutated Jurkat cells which present an impaired Ras/Raf/ERK pathway [20] However, under the experimental conditions applied in this 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
Trang 9pD5egrGFL revealed its role as a negative regulatory
element, which is in line with previously published work
[15,46] The presence of both, an activating CRE2 and an
inhibitory CRE1 in pD1egrGFL may therefore neutralize
the effects of SB203580 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 study we provide evidence
for the radiation-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 promoter Most interestingly,
however, a bFGF-like factor released by irradiated cells
contributes markedly to the radiation-induction of the
Egr-1 promoter via upstream SRE elements Closer insight into
the very early events of Egr-1 gene induction and
identi®-cation of the secreted factor will be useful for further
successful utilization of the Egr-1 promoter in combined
radiation-inducible gene therapy approaches as well as for
the understanding of stress-dependent regulation of cell
growth in general
A C K N O W L E D G E M E N T S
Vector pAdRSVbgal was a kind gift from Dr JuÈrgen Kleinschmidt,
Deutsches Krebsforschungszentrum Heidelberg Thanks also to
Kathleen M Sakamoto, University of California, Los Angeles, for
permission to use plasmid pGL/TiS8 This work was supported by a
grant from the Deutsche Forschungsgemeinschaft (Ro527/3-1,2) and
the Dr Mildred Scheel Stiftung (10-1503-KuÈ I).
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