SK-N-SH neuronal cells were treated with 0.2 lM TPA for 30 min to 24 h and the level of expression of the endogenous PS1 gene was measured by Northern blot ana-lysis.. A similar increase
Trang 1Activation of transcription of the human presenilin 1 gene
Martine Pastorcic1and Hriday K Das1,2
1
Department of Pharmacology & Neuroscience and2Department of Molecular Biology & Immunology, and Institute of Cancer Research University of North Texas Health Science Center at Fort Worth, Fort Worth, TX, USA
We have recently identified an Ets element controlling over
90% of the basal expression of the human presenilin 1 (PS1)
gene We have also shown that Ets1 and Ets2 act as
trans-activators of the PS1 gene by cotransfection experiments in
SK-N-SH neuronal cells The PS1 gene is widely but
dif-ferentially expressed across tissues and the expression in
brain appears to be restricted to neurons To gain further
insight into the regulation of the gene we have examined the
regulation of PS1 by 12-O-tetradecanoylphorbol 13-acetate
(TPA) SK-N-SH neuronal cells were treated with 0.2 lM
TPA for 30 min to 24 h and the level of expression of the
endogenous PS1 gene was measured by Northern blot
ana-lysis A two- to threefold increase in the level of PS1 mRNA
appeared 4–8 h after the addition of TPA A similar increase
in transcription activity was observed in nuclear run off
experiments, indicating that the increased mRNA level
results from an activation in the initiation of transcription of
PS1 Consistently, TPA also increased the level of PS1 pro-tein No activation of the PS1 endogenous gene by TPA was observed in hepatoma HepG2 cells Next we tested the effect
of TPA on the expression of the PS1 promoter by trans-fecting fusion genes including various fragments of the PS1 promoter linked to a CAT reporter into SK-N-SH cells TPA also stimulated the expression of the PS1CAT con-structs Generally wild type constructs)687/+178, )118/ +178, )22/+178 including the short )35/+6 fragment showed a minor two- to threefold activation by TPA Point mutations eliminating the)10 Ets motif or the )6 CREB/ AP1 motif did not decrease the stimulation by TPA Thus TPA appears to activate the transcription of the PS1 gene by
a mechanism which does not require these elements Keywords: presenilin; transcription; TPA; SK-N-SH; PKC
Mutations in the presenilin 1 (PS1) gene are the cause of a
majority of familial early onset Alzheimer’s disease (FAD)
cases [1,2] PS1 is an integral membrane protein involved in
the regulation of gamma secretase cleavage generating
amyloid beta protein [3] and appears to play a crucial role in
the normal metabolism of beta amyloid precursor protein as
well as in the pathological increase of the Ab42 cleavage
product [4] Furthermore, the global phenotype of PS1
knockout mice indicates that PS1 function is also required
for mammalian embryogenesis, including CNSand skeletal
development [5,6] Hence the identification of the
mecha-nisms controlling the expression of the PS1 gene should
relate directly to understanding further the development
and differentiation pathways and the pathogenesis of FAD
PS1 is differentially expressed in a variety of tissues [2] and
brain expression is restricted to neurons [7–11] We have
previously identified the promoter sequences controlling the
basal expression of the PS1 gene [12] In particular we have identified at position )10 an Ets element which controls over 90% of the basal expression Typically Ets factors act
in conjunction with other transcription factors binding at adjacent sites [13,14] A Ca2+/cAMP response element binding protein (CREB) as well as an AP1 consensus homology are located immediately downstream from the Ets motif Recent data has shown that the )5 CREB homology is required for activation of PS1 by N-methyl-D -aspartate (NMDA) in SK-N-SH cells [15] TPA (12-O-tetradecanoylphorbol 13-acetate) is a known activator of protein kinase C- (PKC) and AP1-dependent transcription Prolonged treatment by TPA induces morphological and functional differentiation in cultured neurons including SH-SY5Y human neuroblastoma cells and the parental cell line SK-N-SH [16–19] We have examined the regulation of PS1 during short (< 24 h) exposure to 0.2 lM TPA in SK-N-SH cells
E X P E R I M E N T A L P R O C E D U R E S
Northern blot analysis SK-N-SH and HepG2 cells were grown to 75% confluency
in MEM Eagle’s culture medium containing 12.5% (v/v) fetal bovine serum The TPA treatment was started by replacing the culture medium with serum-free medium containing 0.2 lM TPA After various incubation times (from 30 min to 48 h) cells were harvested and total RNA was prepared by guanidine thiocyanate extraction [20] RNA samples (15 lg) were resolved on denaturing 1%
Correspondence to H K Das, University of North Texas Health
Science Center at Fort Worth, 3500 Camp Bowie Boulevard,
Fort Worth, Texas 76107, USA.
Fax: + 1 817 735 2091, Tel.: + 1 817 735 5448,
E-mail: hdas@hsc.unt.edu
Abbreviations: EMSA, Electrophoretic mobility shift assays; FAD,
familial early onset Alzheimer’s disease; GAPDH, glyceraldehyde-3
phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; PKC,
protein kinase C; PS1, presenilin 1; TPA, 12-O-tetradecanoylphorbol
13-acetate; wt, wild type.
(Received 9 August 2002, revised 11 October 2002,
accepted 22 October 2002)
Trang 2(w/v) agarose gels containing formaldehyde, blotted onto
MSI nylon filters (Micron Separation Inc., Westboro, MA,
USA), UV cross-linked and hybridized sequentially with
DNA probes Prehybridizations were for 2 h, and
hybrid-izations were for 20 h, in 50% (v/v) formamide, 1MNaCl,
10% (w/v) dextran sulfate, 1· Denhardt’s solution, 2% (w/
v) SDS and 0.1 mgÆmL)1 salmon sperm DNA at 42C
After hybridizations, filters were washed three times with 1·
NaCl/Cit for 10 min at 24C and once for 10 min at 55 C
The DNA probes used were labeled by random priming
with [a-32P]dCTP to specific activity > 2· 109cpmÆlg)1
The PS1 probe was the 1115 bp fragment from 429–1543 of
the human presenilin 1 cDNA sequence clone cc44
(acces-sion number L76517) obtained by PCR amplification of the
cDNA with the forward primer 5¢-GGAGCCTGCAAGT
GACAACAGC-3¢ and the reverse primer 5¢-GCCATCAT
CATTCTCTGCAACAG-3¢ The human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe included the
entire cDNA
Nuclear run off analysis of transcripts initiated
during TPA treatment
At the end of treatment with TPA, SK-N-SH cells were
washed with NaCl/Piand harvested Aliquots of 107cells
were resuspended into 1 mL of 10 mMTris, pH 7.4, 10 mM
NaCl, 3 mMMgCl2and 0.5% Igepal CA-630 (Sigma) The
cells were allowed to lyze on ice for 5 min Nuclei were then
pelleted for 5 min at 500 g, washed once with same buffer
and resuspended into 50 lL of 50 mMTris, pH 8.3, 5 mM
MgCl2, 0.1 mMEDTA and 40% (v/v) glycerol and stored at
)70 C Transcription reactions were started by adding an
equal volume of 10 mM Tris, pH 8, 5 mM MgCl2, 0.3M
KCl, 1 mM ATP, 1 mM CTP, 1 mM GTP and 5 mM
dithiothreitol to the nuclei suspension with 10 lCi
[a-32P]UTP Mixtures were incubated for 30 min at 30C
with agitation at 150 r.p.m Reactions were stopped by
adding 150 lL of buffer containing 0.5M NaCl, 50 mM
MgCl2, 2 mMCaCl2, 10 mMTris, pH 7.4, and 40 lgÆmL)1
of RNase-free DNase I DNase I treatment was stopped by
adding 50 lL of 5% (w/v) S DS , 0.5MTris, pH 7.4, 0.125M
EDTA and 50 lg of proteinase K After 30 min incubation
at 42C, samples were extracted with phenol :
chloro-form : isoamyl alcohol (25 : 24 : 1, v/v/v) RNA was
pre-cipitated by adding 2 mL ice cold H2O containing 100 lg
tRNA and 2.5 mL of 10% (v/v) trichloroacetic acid After
incubation on ice for 40 min the precipitates were collected
by filtration onto 0.45 lm Milllipore HA filters Filters were
washed three times with 10 lL of 5% (v/v) TCA, 30 mM
sodium pyrophosphate and transferred to vials containing
2 mL of 20 mMHepes, pH 7.5, 5 mMMgCl2, 1 mMCaCl2
and 20 lgÆmL)1DNase I After 30 min treatment at 37C
reactions were stopped with 50 lL of 0.5 M EDTA and
70 lL of 20% (w/v) SDS, and heat-treated at 65C for
10 min Samples were then treated with proteinase K for
30 min at 37C and extracted with an equal volume of
phenol RNA was precipitated with 0.3Msodium acetate
RNA pellets were resuspended in 1 mL 10 mMTes, pH 7.4,
0.2% (w/v) SDS and 10 mMEDTA An equal volume of
the same buffer containing 0.6M NaCl was added and
nitrocellulose strips bearing DNA samples to be tested were
added to the vials and incubated at 65C for 48 h
Membranes were washed with 2· NaCl/Cit, 1% (w/v)
SDS at 24C for 30 min and at 65 C for 15 min Filters were exposed for 24 h DNA probes for presenilin 1, GAPDH and 18SRNA were the same DNA fragments used in Northern blotting DNA was denatured in 50 lL of 0.1MNaOH for 30 min at 24C Solutions were neutral-ized by addition of 450 lL 6· NaCl/Cit and applied to nitrocellulose membrane
Transfection assays SK-N-SH cells were transfected with PS1CAT fusion genes containing various fragments of PS1 sequences flanking the transcription initiation site [12] Cells were seeded at a density of 104Æcm)22 days before transfection Transfection
by calcium phosphate precipitation and glycerol shock were
as described previously [12] After glycerol shock cells were treated with 0.2 lMTPA or dimethylsulfoxide for 16–18 h
in serum-free MEM Promoter activity in different samples was compared using the amount of protein present in the cellular extracts as an internal control Each experiment was repeated three times, with a minimum of triplicate tests of each construct and treatment The ()118, +178) m6 PS1CAT construct contains a mutation within the )6 CREB motif from AATGACGA (wt) to AATcgaGA (m6)
It was generated by PCR-based site-directed mutagenesis using the QuickChange kit from Stratagene and the complementary primers 5¢-CAGAGCCGGAAATCGAG ACAACGGTGAG-3¢ and 5¢-CTCACCGTTGTCTCGA TTTCCGGCTCTG-3¢ including the mutant CREB site with PS1CAT ()118, +178) as a template
Electrophoretic mobility shift assays Nuclear extracts from SK-N-SH cells treated with 0.2 lM TPA or dimethylsulfoxide for 5 h in serum–free MEM were prepared as described previously [12] Electrophoretic mobility shift assays (EMSAs) included either a32P-labeled probe containing the wild type sequences () 22, + 6) or a mutation of the)10 Ets motif from GGAAA to ttAAA Reactions were carried out by incubating 0.1–0.2 ng of probe with 2–5 lg of nuclear extracts in the presence of 1–2 lg of poly(dI-dC)Æpoly(dI-dC) in 10 mM Hepes,
pH 7.9, 50 mM NaCl, 0.75 mM MgCl2, 0.1 mM EDTA,
1 mMdithiothreitol, 1% Igepal CA-630 (Sigma) and 10% (v/v) glycerol for 30 min at 4C DNAÆprotein complexes formed were then analyzed by electrophoresis on nondena-turing 6% polyacrylamide gels containing 0.5% Igepal
CA-630 The electrophoresis buffer was 0.25· TBE (89 mM Tris, 89 mM boric acid and 1 mMEDTA) The gels were prerun for 20 min, and sample electrophoresis was for
90 min at 10 V cm)1at 4C
Western blotting SK-N-SH cells were washed twice with NaCl/Pi and harvested in 2· sample buffer [0.1 M Tris/HCl, pH 6.8, 4% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 20% (v/v) glycerol containing 200 lgÆmL)1 aprotinin, 100 lgÆmL)1 pepstatin, 50 lgÆmL)1leupeptin and 10 mM benzamidine] [21] The DNA was sheared with a 22-gauge needle and extracts were centrifuged at 14 000 g for 30 min at 4C The supernatant was stored at )70 C Aliquots (25 lg) were fractionated by electrophoresis on 12% polyacrylamide
Trang 3gels Proteins were transferred to poly(vinylidene difluoride)
(PVDF) membranes (Millipore) Membranes were blocked
with 1% (w/v) BSA for 60 min at 24C, and incubated with
a 1 : 1000 dilution of the primary antibody aPS1-N [21] in
1% (w/v) BSA for 60 min, and with 1 : 2000 dilution of the
secondary antibody for 45 min Blots were stained with
ECL reagent (Amersham) The same blots were stripped in
60 mM Tris, pH 6.8, 2% (w/v) SDS, and 100 mM
b-mercaptoethanol at 75C for 30 min and retested for
the level of actin protein with 1 : 1000 aActin (sc-8432,
Santa Cruz Biotechnology, CA, USA)
R E S U L T S
TPA treatment increases the level of PS1 mRNA
in SK-N-SH cells
SK-N-SH cells were treated with 0.2 lMTPA for increasing
amounts of time from 30 min to 24 h Total cellular RNA
samples (15 lg) from each time point were analyzed by
Northern blotting (Fig 1A) The PS1 cDNA probe revealed
a major transcript at about 3 kb (Fig 1A) and a lesser
amount of a larger mRNA of about 7 kb was also visible
only in the samples with higher expression of PS1 (not
shown) This is consistent with the size of 3 kb reported for
PS1 mRNA and 7 kb for a minor transcript initiating at an
alternative site [22] No significant difference in mRNA level
between control and TPA treated samples was observed
before the 1 h time point as displayed in the histogram
quantification of the Northern data (Fig 1B) By 2 and 4 h
TPA treatment increased PS1 mRNA level by twofold to
threefold Over longer treatment time (24 h) no significant
difference was observed between TPA treated and control
samples The GAPDH mRNA level used as an internal
control showed no difference over time or with TPA
treatment In the same experiment carried out with
hepa-toma HepG2 cells the level of PS1 mRNA remained
unchanged over time or in the presence of TPA (Fig 1C,
Table 1) Therefore treatment of SK-N-SH cells by 0.2 lM
TPA results in a transient increase in the level of PS1
mRNA, showing a maximum at 4–8 h
TPA increases the rate of transcription initiation
of the PS1 gene in SK-N-SH cells
To determine whether the increase in the level of PS1
mRNA results from the activation of the transcription of
the gene we have performed nuclear run-off assays (Fig 2)
We prepared nuclei from SK-N-SH cells treated with
0.2 lMTPA for 5 h The transcripts already initiated within
the nuclei at the time of harvest were allowed to elongate
in vitroin the presence of32P-labeled ribonucleotides The
labeled RNAs were then purified and the level of specific
mRNAs was quantified by hybridization to DNA probes
for the human PS1 cDNA and 18S RNA immobilized onto
nitrocellulose filters The level of 18Stranscription remained
unchanged after TPA treatment and was used as an internal
control to quantify the changes in PS1 transcription The
rate of PS1 transcription appeared to increase by 2.5– to
threefold in the presence of TPA Thus the increase in the
level of PS1 mRNA observed by Northern blotting of total
cellular RNA results from an increase in the rate of
initiation of transcription of PS1
PS1 protein level increases with TPA
To confirm and extend the previous observations we have examined the level of PS1 protein in SK-N-SH cells Cellular proteins were fractionated by electrophoresis on 12% (w/v) polyacrylamide gels and analyzed by Western
Fig 1 TPA increases the level of PS1 mRNA in SK-N-SH cells (A) SK-N-SH cells were incubated in the presence of 0.2 l M TPA (T) or dimethylsulfoxide (C) for increasing amounts of time from 30 min to
24 h as indicated above the lanes RNA (15 lg) was fractionated on denaturing 1.4% (w/v) agarose gels and analyzed by Northern blot-ting Membranes were sequentially hybridized with cDNA probes for the human PS1 and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) genes (B) The level of transcription at each time point was quantified by laser scanning of the autoradiograms The level of the PS1 3 kb transcript in each lane was expressed as its ratio to GAPDH mRNA in the same sample The average level of the normalized PS1 mRNA at each time point was estimated with n ¼ 4 or n ¼ 5 in each
of three experiments The histogram displays the ratio between the average level of PS1 mRNA in the TPA-treated samples and the average level in dimethylsulfoxide control at each time point (C) HepG2 cells were incubated with TPA or dimethylsulfoxide and total RNA was analyzed by Northern blotting as described for SK-N-SH cells in (A) The average level of the normalized PS1 mRNA at each time point was estimated with n ¼ 3 or n ¼ 4 in each of two experiments In the
2 h control lane the PS1 band is partially masked by a gel artefact.
Trang 4blotting using an antibody recognizing specifically the N
terminus of the PS1 protein Three species were detected: the
full length PS1 appearing as a 45 kDa polypeptide, as well
as a larger aggregated form and the 30 kDa N-terminal
fragment (Fig 3) After a 17-h TPA treatment the level of
the full length 45 kDa species and aggregated form
increased by 1.5– twofold No significant increase in the
30 kDa N-terminal fragment protein was observed Thus
TPA treatment increases the level of the PS1 protein The
full length PS1 has a relatively short half-life, and it is
normally cleaved by endoproteolysis into a 30 kDa
N-terminal fragment and 17 kDa C-terminal fragment
which are considerably more stable [23] It is possible that
any increase in newly synthesized PS1 in the presence of
TPA does not appear against the background of the larger
cellular pool of the stable 30 kDa form Hence we observe
an increase in the level of the PS1 protein by TPA treatment
which is consistent with the increased mRNA level
DNA sequences required to confer activation
of transcription of PS1 by TPA
We have recently identified a promoter area required for
efficient expression of the PS1 gene in SK-N-SH cells and
HepG2 cells including DNA sequences from)35 to +178
flanking the transcription initiation site [12] We have transfected SK-N-SH cells with PS1CAT fusion gene constructs containing various fragments of PS1 sequences from )687 to +178 inserted upstream from the CAT reporter gene With constructs including sequences from
Table 1 TPA does not alter the level of the PS1 mRNA in HepG2 cells HepG2 cells were incubated with TPA or DMSO and total RNA was analyzed by Northern blotting as described for SK-N-SH cells in (A) The level of PS1 mRNA was quantified by laser scanning of the auto-radiograms and normalized with the level of GAPDH mRNA in the same samples The average level of PS1 mRNA at each time point was estimated with n ¼ 3 or 4 in each of 2 experiments.
Dimethylsulfoxide 3 ± 0.4 1.4 ± 0.4 2.7 ± 1 3.9 ± 0.8 1.06 ± 0.34 1.3 ± 0.02 TPA 2.5 ± 0.4 1.9 ± 0.6 2.6 ± 0.6 3.3 ± 0.8 0.97 ± 0.06 1.4 ± 0.3
Fig 3 TPA increases the level of PS1 protein in SH cells
SK-N-SH cells were treated with 0.2 l M TPA for 17 h and cell extracts were fractionated by electrophoresis on 12% (w/v) polyacrylamide gels and analyzed by Western blotting as described in Experimental procedures Control extract (C) and TPA-treated extract (T) (25 lg) were loaded in lanes 1 and 2, respectively The size of molecular mass markers is indicated in kDa alongside the gel Arrows mark the position of the full length 45 kDa, the aggregated form and the 30 kDa N-terminal fragment The same blot was stripped and the level of actin protein was analyzed as a control Bands were quantified by laser scanning of the autoradiograms The level of PS1 was normalized to actin and was determined in three distinct experiments Values were analyzed by the paired t-test/ ANOVA method, and a value of P < 0.05 was considered significant The average level of the aggregated form was 1.7 ± 0.36 (P < 0.05) in TPA-treated samples and 0.88 ± 0.15 in control sam-ples The full length PS1 was 1.74 ± 0.2 in TPA samples and the control level was 0.94 ± 0.2 (P < 0.05) The 30 kDa species was 1.2 ± 0.28 in the TPA-treated samples and 0.98 ± 0.4 in the controls All averages were derived from n ¼ 3.
Fig 2 Nuclear run-off analysis of the transcription of PS1 in the
presence of TPA SK-N-SH cells were incubated in the presence of
0.2 l M TPA for 5 h Nuclei were then purified and used in
transcrip-tion run-off analysis to quantify the RNAs being actively transcribed
at the time of harvest as described in Experimental procedures.
Transcription was quantified by laser scanning of the autoradiograms.
The changes in level of PS1 transcripts were quantified after
normal-ization with 18SRNA TPA increased transcription of PS1 by 2.8
(± 0.8) with n ¼ 3 in two independent experiments.
Trang 5)687 to +178, )118 to +178, )22 to +178, )22 to +42 or
the minimal promoter)35 to +6 the activation by TPA
was two- to threefold (Fig 4) Thus the minimal promoter
)35 to +6 is sufficient to confer activation by TPA This
sequence interval contains an Ets element at )10 (Fig 5)
which is crucial for the expression of PS1 It also contains a
sequence element sharing homology with the consensus
CREB/AP1 binding motif immediately adjacent to the Ets
site [24] The effects of TPA on transcription are commonly mediated by AP1 Furthermore, Ets factors are known to act in conjunction with a number of other regulatory proteins including AP1 Thus we have tested the effect of a point mutation eliminating the AP1 homology (m6) as well
as a point mutation abolishing the)10 Ets site (m1) (Fig 5) M6 reduced the activity of the)118 to +178 construct by about twofold; however, the mutant promoter retained
two-to threefold stimulation by TPA, similar two-to the )118 to +178 wild type construct (Fig 4) Thus the)6 CREB/AP1 homology is not required for TPA activation Similarly, the point mutation m1 eliminating the)10 Ets binding site did not abolish induction by TPA This may indicate that neither the)10 Ets element, nor the )6 CREB/AP1 motif are required for stimulation by TPA
Changes in DNAÆprotein interactions over the)22/+6 region of the PS1 promoter in nuclear extracts from SK-N-SH cells treated with TPA
We have used EMSAs to detect changes in the binding activity of the proteins recognizing specifically the )10 region of the PS1 promoter in nuclear extracts of SK-N-SH cells treated with TPA (Fig 6) In dimethylsulfoxide-treated
Fig 4 DNA sequences requiredfor activation of PS1 transcription by
TPA PS1CAT fusion genes containing various fragments of the PS1
promoter linked to the CAT reporter gene were transfected into
SK-N-SH cells The end-points of the promoter fragments used in each of the
constructs are indicated below the graph m1 is a mutation from
CCGGAAATGACGA to CCttAAATGACGA eliminating the )10
Ets site In m6 the mutation to CCGGAAATcgaGA eliminates the
adjacent CREB and AP1 homologies (underlined) [24].
Fig 5 PS1 promoter sequence PS1 promoter sequence from )118 to
+178 The endpoint of the 3¢ and 5¢deletions used in this study are
indicated by arrows The transcription initiation site is shown (+ 1).
The position of the Ets, CREB and Sp1 binding sites are underlined.
Fig 6 Changes in DNAÆprotein interactions over the )22 to +6 region
of the PS1 promoter induced by TPA treatment of SK-N-SH cells (A) Nuclear extracts from SK-N-SH cells were prepared from cells treated with 0.2 l M TPA for 5 h as well as from cultures where the same dilution of dimethylsulfoxide was added (D) DNAÆprotein interactions over the ( )22 to +6) region of the PS1 promoter were examined by EMSAs The positions of the specific complexes are indicated Extracts were preincubated with aEts1/2 (aE lanes), an antibody recognizing specifically Ets1 and Ets2, for 45 min at 24 C in the absence of DNA probe An antibody unrelated to Ets factors (anti-PS1 sc-1245, from Santa Cruz Biotechnology, CA, USA) was included
in control lanes (C) The probe was added and incubation was con-tinued for another 20 min Reactions were analyzed by electrophoresis
on 6% (w/v) native polyacrylamide gels at 4 C Lanes 1–5 include the wild type probe, lanes 6–10 display binding to the probe containing a mutation (GGAA fi ttAA) within the )10 Ets motif (B) Low exposure of the region of the gel including complex B.
Trang 6nuclear extracts (D) the pattern of DNAÆprotein complexes
observed with the PS1 probe produced the specific
com-plexes A, B, C, D, E, F, G and H These specific
proteinÆDNA complexes (A–H) appear to be generated by
proteinÆprotein interaction with Ets factors and other
proteins [12] These complexes (A–H) are found to be
absent in assays with the Ets motif mutant probe similar to
the data described previously [12] TPA treatment appears
to result in the loss of the specific complexes F and H, a
decrease in complex B, as well as an increase in complexes
A, C, D, E and G Complexes A and G are eliminated by
preincubation of the control or TPA treated nuclear extracts
with anti-Ets1/2 Ig, indicating that at least these complexes
involve interactions with Ets1/2 Therefore TPA treatment
generally increased the specific interactions of the )10
region of the PS1 promoter with nuclear factors, including
the amount of complexes involving Ets1/2 factors
D I S C U S S I O N
The loss of PKC is a prognostic element in the severity of
neuronal damage resulting from ischemia in vivo [25] The
activation of PKC by TPA inhibits cell death in vitro
through a complex set of pathways where different PKC
isozymes appear to play opposite roles [26] TPA increases
the level of the expression of the endogenous PS1 gene in
SK-N-SH cells at the level of initiation of transcription
TPA had no effect on the mRNA level in HepG2 cells (data
not shown), thus the regulation pathway implicated here
may be somewhat cell specific Most of the known
biological effects of TPA are attributed to its ability to
activate PKC The effect(s) of TPA observed here are likely
to result from the activation of PKC because the increase in
PS1 mRNA appears to be abolished by
bisindolylmalei-mide, a specific inhibitor of PKC [27,28], in preliminary data
(not shown) Furthermore, the time course of activation of
PS1 indicates that the maximum increase in the level of PS1
mRNA is reached by 4 h and that a longer exposure to TPA
(24 h) no longer activates PS1 expression This is consistent
with the down-regulation of protein kinase C with long
exposure to TPA observed in many cell types [29]
In order to analyze further the mechanism of activation
we have tested the effect of TPA on the activity of the PS1
promoter in transient infection assays in SK-N-SH cells
TPA treatment activated similarly by two- to 2.5-fold the
transcriptional activity of all promoter fragments tested
The minimal promoter including sequences )35/+6
appears to retain TPA activation Mutations eliminating
the)10 Ets binding or the )6 AP1/CREB motifs did not
reduce activation by TPA This suggests that induction
results from the modification of protein(s) of the initiation
complex which do not bind directly to DNA They may
however, interact with factors recognizing specific motifs,
such as Ets, and promote changes in proteinÆDNA
interac-tions within complexes including Ets For example there is a
significant increase in the amount of the larger complexes A
and B (Fig 6) in the TPA treated extracts Complex A is
likely to contain Ets1 or Ets2, as it is eliminated by the
addition of anti-Ets1/2 Ig It is possibly converted into
complex B (which increases from lane 4 to lane 5) This may
indicate that Ets 1/2 is not required for the formation of
complex B The identity of the protein recognizing
specif-ically the PS1 promoter within B is not known However its
ability to interact directly or indirectly with Ets1/2 should enable its identification by the 2-hybrid selection technique Members of the AP1 protein complex have been impli-cated in the onset of apoptosis Induction by c-fos is an early event in apoptosis [30], the overexpression of c-jun domin-ant negative mutdomin-ants protects sympathetic neurons against programmed cell death induced by the withdrawal of nerve growth factor whereas overexpression of wild type c-jun appears to trigger apoptosis [31] Retinoic acid-induced apoptosis in F9 cells also induces c-jun, and the reduction of c-jun levels by antisense reduces apoptosis [32] In contrast, the same F9 cells stably transformed with wild type PS1 show a significantly reduced level of apoptosis after retinoic acid treatment, whereas mutant PS1 suppresses apoptosis only weakly This indicates that PS1 may play a protective role in the development of c-jun-mediated apoptosis Thus the induction of PS1 gene expression after treatment with TPA in the experiments described here is consistent with a role of PS1 in the c-jun cascade leading to apoptosis However, the role of the c-jun N-terminal kinase (JNK)/ c-jun cascade for in vivo apoptosis and particularly in Alzheimer’s disease is still unclear [33] Growing evidence implicates JNK-dependent pathways in Ab-dependent apoptosis [34] A role of PS1 in the development of Ab-induced apoptosis has previously been suggested [35] Overexpression of mutant PS1 increased the susceptibility of PC12 cells to apoptosis induced by Ab or the withdrawal of trophic factors In contrast with this proapoptotic effect of PS1 mutants, the wild type PS1 suppresses apoptosis induced by the activation of p53 [36], which is a target of JNK Therefore, increasing evidence is indicating the importance of the JNK/c-jun pathway in the neuronal death in Alzheimer’s disease, and its differential interaction with mutant and wild type PS1 suggests further its importance in the development of the disease Thus it should be important to understand how the regulation of the genes in both pathways interface It is also important to note that neuron specific activation of PS1 may increase Notch-1 processing which could lead to neurite outgrowth and decrease the risk of Alzheimer’s disease [37,38]
A C K N O W L E D G E M E N T
We wish to thank Dr B Yankner for his very generous gift of the aPS1 N-terminal antibody This research was supported by a grant from the National Institute of Health (AG18452) to H.K.D.
R E F E R E N C E S
1 Tanzi, R.E., Kowacs, D.M., Kim, T.-W., Moir, R., Guenette, S Y.
& Wasco, W (1996) The gene defects responsible for familial Alzheimer’s disease Neurobiol Dis 3, 159–168.
2 Sherrington R., Rogaev, E.I., Liang, Y., Rogaeva, E.A., Levesque, G., Ikeda, M., Chi, H., Lin, G., Holman, K., Tsuda, T.,
et al (1995) Cloning of a gene bearing missense mutations in early-onset familial Alzheimer’s disease Nature 375, 754–760.
3 Wolfe, M.S., Xia, W., Ostaszewski, B.L., Diehl, T.S., Kimberly, W.T & Selkoe, D.J (1999) Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity Nature 398, 513–517.
4 De Strooper, B., Saftig, P., Craessaerts, K., Vanderstichele, H., Guhge, G., Annaert, W., Von Figura, K & Van Leuven, F (1998) Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein Nature 391, 387–390.
Trang 75 Wong, P.C., Zeng, H., Chen, H., Becher, M.W., Sirinathsinghji,
D.J.S., Trumbauer, M.E., Chen, H.Y., Price, D.L., Van der Ploeg,
L.H.T & Sisodia, S.S (1997) Presenilin 1 is required for Notch1
and DII1 expression in the paraxial mesoderm Nature 387,
288–292.
6 Shen, J., Bronson, R.T., Chen, D.F., Xia, W., Selkoe, D.J &
Tonegawa, S (1997) Skeletal and CNS defects in
presenilin-1-deficient mice Cell 89, 629–639.
7 Kovacs, D.M., Fausett, H.J., Page, K.J., Kim, T., Moir, R.D.,
Merriam, D.E., Hollister, R.D., Hallmark, O.G., Mancini, R.,
Felsenstein, K.M., et al (1996) Alzheimer-associated presenilins
1 and 2: neuronal expression in brain and localization to
intracellular membranes in mammalian cells Nat Med 2,
224–229.
8 Cribbs, D.H., Chen, L.S., Bende, S.M & LaFerla, F.M (1996)
Widespread neuronal expression of the presenilin-1 early-onset
Alzheimer’s disease gene in the murine brain Am J Pathol 148,
1797–1806.
9 Lee, M.K., Slunt, H.H., Martin, L.J., Thinakaran, G., Kim, G.,
Gandy, S.E., Seeger, M., Koo, E., Price, D.L & Sisodia, S.S.
(1996) Expression of presenilin 1 and 2 (PS1 and PS2) in human
and murine tissues J Neurosci 16, 7513–7525.
10 Quarteronet, D., Pradier, L., Czech, C., Delalonde, L., Burgevin,
M.C., Doble, A & Petitet, F (1996) Localization of presenilin-1
mRNA in rat brain Neuroreport 7, 2587–2591.
11 Takami, K., Terai, K., Matsuo, A., Walker, D.G & McGeer, P.L.
(1997) Expression of presenilin-1 and -2 mRNAs in rat and
Alzheimer’s disease brains Brain Res 748, 122–130.
12 Pastorcic, M & Das, H.K (1999) An upstream element
contain-ing an ETSbindcontain-ing site is crucial for transcription of the human
presenilin-1 gene J Biol Chem 274, 24297–24307.
13 Wasylyk, B., Hahn, S.L & Giovane, A (1993) The Ets family of
transcription factors Eur J Biochem 211, 7–18.
14 Janknecht, R & Nordheim, A (1993) Gene regulation by Ets
proteins Biochim Biophys Acta 1155, 346–356.
15 Mitsuda, N., Ohkubo, N., Tamatani, M., Lee, Y.D., Taniguchi,
M., Namikawa, K., Kiyama, H., Yamaguchi, A., Sato, N.,
Sakata, K., et al (2001) Activated cAMP-response
element-binding protein regulates neuronal expression of presenilin-1.
J Biol Chem 276, 9688–9698.
16 Pahlman, S., Odelstad, L., Larsson, E., Grotte, G & Nilsson, K.
(1981) Phenotypic changes of human neuroblastoma cells in
cul-ture induced by 12-O-tetradecanoyl-phorbol-13-acetate Int J.
Cancer 28, 583–589.
17 Pahlman, S., Ruusala, A.I., Abrahamsson, L., Odelstad, L &
Nilsson, K (1983) Kinetics and concentration effects of
TPA-induced differentiation of cultured human neuroblastoma cells.
Cell Differ 12, 165–170.
18 Pahlman, S., Ruusala, A.I., Abrahamsson, L., Mattsson, M.E &
Esscher, T (1984) Retinoic acid-induced differentiation of
cul-tured human neuroblastoma cells: a comparison with
phorbo-lester-induced differentiation Cell Differ 14, 135–144.
19 Pahlman, S., Hoehner, J.C., Nanberg, E., Hedborg, F.,
Fagerstrom, S., Gestblom, C., Johansson, I., Larsson, U.,
Lavenius, E., Ortoft, E et al (1995) Differentiation and survival
influences of growth factors in human neuroblastoma Eur J.
Cancer 31A, 453–458.
20 Chomczynski, P & Sacchi, N (1987) Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction Anal Biochem 162, 156–159.
21 Hartmann, H., Busciglio, J., Baumann, K.-H., Staufenbiel, M &
Yankner, B.A (1997) Developmental regulation of presenilin-1
processing in the brain suggests a role in neuronal differentiation.
J Biol Chem 272, 14505–14508.
22 Rogaev, E.I., Sherrington, R., Wu, C., Levesque, G., Liang, Y.,
Rogaeva, E.A., Ikeda, M., Holman, K., Lin, C., Lukiw, W.J.,
et al (1997) Analysis of the 5¢ sequence, genomic structure, and
alternative splicing of the presenilin-1 gene (PSEN1) associated with early onset Alzheimer disease Genomics 40, 415–424.
23 Ratovitski, T., Slunt, H.H., Thinakaran, G., Price, D.L., Sisodia, S.S & Borchelt, D.R (1997) Endoproteolytic processing and stabilization of wild-type and mutant presenilin J Biol Chem.
272, 24536–24541.
24 Wingender, E., Chen, X., Hehl, R., Karas, H., Liebich, I., Matys, V., Meinhardt, T., Pru¨ß, M., Reuter, I & Schacherer, F (2000) TRANSFAC: an integrated system for gene expression regulation Nucleic Acids Res 28, 316–319.
25 Durkin, J.P., Tremblay, R., Chakravarthy, B., Mealing, G., Morley, P., Small, D & Song, D (1997) Evidence that the early loss of membrane protein kinase C is a necessary step in the excitatory amino acid-induced death of primary cortical neurons.
J Neurochem 68, 1400–1412.
26 Maher, P (2001) How protein kinase C activation protects nerve cells from oxidative stress-induced cell death J Neurosci 21, 2929–2938.
27 Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F.
et al (1991) The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C J Biol Chem 266, 15771– 15781.
28 Heikkila, J., Jalava, A & Eriksson, K (1993) The selective protein kinase C inhibitor GF 109203X inhibits phorbol ester-induced morphological and functional differentiation of SH-SY5Y human neuroblastoma cells Biochem Biophys Res Commun 197, 1185– 1193.
29 Parrow, V., Nnberg, E., Heikkila, J., Hammerling, U & Pa˚hlman,
S (1992) Protein kinase C remains functionally active during TPA induced neuronal differentiation of SH-SY5Y human neuro-blastoma cells J Cell Physiol 152, 536–544.
30 Smeyne, R.J., Vendrell, M., Hayward, M., Baker, S.J., Miao, G.G., Schilling, K., Robertson, L.M., Curran, T & Morgan, J.I (1993) Continuous c-fos expression precedes programmed cell death in vivo Nature 363, 166–169.
31 Ham, J., Babij, C., Whitfield, J., Pfarr, C.M., Lallemand, D., Yaniv, M & Rubin, L.L (1995) A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death Neuron 14, 927–939.
32 Imafuku, I., Masaki, T., Waragai, M., Takeuchi, S., Kawabata, M., Hirai, S., Ohno, S., Nee, L.E., Lippa, C.F., Kanazawa, I.,
et al (1999) Presenilin 1 suppresses the function of c-Jun homo-dimers via interaction with QM/Jif-1 J Cell Biol 147, 121–134.
33 Marx, J (2001) Neuroscience New leads on the how of Alzhei-mer’s Science 293, 2192–2194.
34 Morishima, Y., Gotoh, Y., Zieg, J., Barrett, T., Takano, H., Flavell, R., Davis, R.J., Shirasaki, Y & Greenberg, M.E (2001) Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of Fas ligand J Neurosci 21, 7551–7560.
35 Guo, Q., Sopher, B.L., Furukawa, K., Pham, D.G., Robinson, N., George, M., Martin, G.M & Mattson, M.P (1997) Alzheimer’s presenilin mutation sensitizes neural cells to apoptosis induced by trophic factor withdrawal and amyloid beta-peptide: involvement
of calcium and oxyradicals J Neurosci 17, 4212–4222.
36 Roperch, J.P., Alvaro, V., Prieur, S., Tuynder, M., Nemani, M., Lethrosne, F., Piouffre, L., Gendron, M.C., Israeli, D., Dausset, J., Oren, M., Amson, R & Telerman, A (1998) Inhibition of presenilin 1 expression is promoted by p53 and p21WAF-1 and results in apoptosis and tumor suppression Nat Med 4, 835–838.
37 Sestan, N., Artavanis-Tsakonas, S & Rakic, P (1999) Contact-dependent inhibition of cortical neurite growth mediated by Notch signaling Science 286, 741–746.
38 Redmond, L., Oh, S.R., Hicks, C., Weinmaster, G & Ghosh, A (2000) Nuclear Notch1 signaling and the regulation of dendritic development Nature Neurosci 3, 30–40.