From the Unité Mixte de Recherche (UMR)
CNRS 6551, IFR47, Université de Caen, Cyceron, Caen Cedex 14074, France, ¶ Department of Psychiatry, Institute of Psychiatric
Research, Indiana University School of Medicine, Indianapolis, Indiana
46202, and
Unité INSERM 422, Vieillissement
Cérébral et Dégénérescence Neuronale,
Lille Cedex 59045, France
Received for publication, January 24, 2003, and in revised form, February 27, 2003
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ABSTRACT |
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Accumulation of the amyloid- The proteolytic cleavage of the amyloid precursor protein
(APP)1 leads to the
production of A Many studies have identified increased levels of a variety of cytokines
in patients developing chronic neurodegenerative disorders such as AD
(3-8). Among these factors, recent reports have associated transforming growth factor- Despite this dual effect of TGF- Semiquantitative Reverse Transcription-PCR (RT-PCR)--
Total
RNAs were prepared using either the RNAxel extraction kit (Eurobio,
Paris, France) or RNAeasy extraction columns (Qiagen, Courtaboeuf,
France). Samples (1 µg) of total mRNA were transcribed into
cDNA. cDNA libraries (1 µl from 20 µl) were amplified by PCR with oligonucleotides for Nuclear Extracts--
Nuclear extracts were prepared from
control and TGF- Electrophoretic Mobility Shift
Assay--
Oligonucleotides were end labeled with
[ Primary Cell Cultures--
Mouse cortical cultures of neurons
were prepared from 14-15-day-old embryos as described previously (15).
After 3 days in vitro, neurons were treated with 10 µM cytosine arabinoside to inhibit the proliferation of
astrocytes. Experiments were realized on pure neuronal cultures (> 98% of microtubule-associated protein-2-positive cells) after 12-14
days in vitro.
Murine cortical cultures of astrocytes were prepared from 1-3 day
postnatal mice (15). Experiments were performed on confluent cultured
cortical astrocytes after 10 days in vitro. Primary cultures of cortical astrocytes were washed three times in PBS each 3 days in vitro to prevent the adhesion of microglial cells onto
the monolayer of astrocytes until use (10-12 days in
vitro). This protocol leads to fewer than 1% of CD11b-positive cells.
Primary cultures of human neurons and astrocytes were established from
brain tissues of therapeutically aborted human brain fetuses after 10 weeks of gestation. The protocol for tissues so obtained complied with
institutional and national guidelines.
Transgenic Animals--
All transgenic mice were of BALB/c × SJL background and heterozygous for the respective transgene.
Nontransgenic littermates served as controls. Glial fibrillary acidic
protein (GFAP)-TGF- Western Blotting Experiments--
Cells were harvested in a
lysis solution containing 50 mM Tris-HCl (pH 7.6), 1%
Nonidet P-40 (Sigma), 150 mM NaCl, 2 mM EDTA, with 100 µM phenylmethylsulfonyl fluoride in the presence
of a protease inhibitor mixture (Sigma). Equally conditioned media were
harvested in the presence of 100 µM phenylmethylsulfonyl fluoride and protease inhibitor mixture prior vacuum concentration (10-fold). Electrophoreses were done on 8% SDS-polyacrylamide Tris-glycine gels or 16.5% Tris-Tricine gels containing 8 M urea. Gels were transferred to a polyvinylidene
difluoride membrane (PolyScreen®, PerkinElmer Life
Sciences), membranes were blocked in nonfat milk and probed with
appropriate antiserum. Blots were finally developed with an enhanced
chemiluminescence Western blotting detection system (PerkinElmer Life Sciences).
Antibodies--
The monoclonal mouse antibody 22C11 (1:1,000)
(Roche Applied Science) to residues 66-81 of APP695 was
used to identify either membrane-bound protein or soluble derivatives.
The following primary antibodies were used: R7 (1:1,000) against
KPI-APP proteins (17), R1736 (1:1,000) against sAPP- Double Fluorescent Immunocytochemistry--
Cultured murine
cortical astrocytes were gently washed in PBS and fixed with ice-cold
4% paraformaldehyde. Cells were washed, and nonantigenic sites were
blocked in PBS plus 4% bovine serum albumin and 0.1% Tween 20 (Sigma)
and incubated overnight at 4 °C with the primary antibody raised
against GFAP in PBS, 1% BSA, and 0.1% Tween 20. Cells were then
washed and incubated for 1 h with the appropriate secondary Alexa
Fluor® 488-conjugated antibody (Molecular Probes).
Thereafter, astrocytes were incubated with the OX42 monoclonal antibody
as described above. The appropriate secondary biotin-conjugated
antibody was used, and antibody-antigen complexes were revealed with
streptavidin Alexa Fluor® 555-conjugate (Molecular
Probes). Cells were finally counterstained with DAPI-containing PBS and
0.1% Tween 20.
A mRNA Decay Experiments--
Mouse cortical astrocytes were
exposed to 10 µg/ml actinomycin D for 0-16 h after 24-h treatment
with recombinant human TGF- Transfection Protocols--
Cells were transiently transfected
with the constructs indicated using the Transfast®
Transfection Reagent (Promega) as described by the manufacturer. For
each transfection experiment, sister culture dishes were used to
control the efficiency of transfection using an enhanced green fluorescent protein (EGFP)-containing plasmid driven by cytomegalovirus promoter (pEGFP-C1 vector) (BD
Biosciences-Clontech). All transfections were
performed with the empty vector (pcDNA3.1, Invitrogen)
corresponding to the experimental plasmid used
(pcDNA3.1-APPtre-luc).
Transfection efficiency was determined by counting total cells and
EGFP-positive astrocytes. In our hands, we reached the ratio of 71 ± 8% of transfected astrocytes. Potential toxicity was estimated by
examination of the cultures under phase microscopy and quantified by
measurement of the activity of the cytosolic enzyme lactate
dehydrogenase released by damaged cells into the bathing medium as
described previously (21). No differences were observed between
untransfected and transfected cells (data not shown). Moreover, the
pRL-TK values provided by the Dual-Luciferase® Reporter
Assay System did not differ from controls (data not shown).
Reporter Gene Assay--
Two days after transfection, cells were
treated and luciferase activities (firefly luciferase and
Renilla luciferase) were evaluated after 1 day using the
Dual Luciferase® Reporter Assay System (Promega). Values
were normalized to the Renilla luciferase activity
(Promega). The Dual-Luciferase® Reporter Assay System
refers to the simultaneous expression and measurement of two individual
reporter enzymes within a single system. Typically, the
"experimental" reporter (firefly luciferase) is correlated with the
effect of specific experimental conditions (e.g. TGF- APP Promoter Constructs--
pGL3-APPluc constructs
were prepared into the pGL3-basic vector (Promega)
containing a major late promoter sequence (pGL3-MLP) (14).
The Densitometric Analyses--
Agarose gels or blots from three
independent experiments were acquired by a CCD camera and saved as a
resolution of 600 dpi for software analysis. PCR products and Western
blot signals were quantified by two-dimension densitometric analysis
using the OptiQuant® software (Packard Instruments
Inc.).
Statistical Analysis--
Results are expressed as the mean ± S.D. Statistical analyses were performed with StatView (Abacus,
Berkeley, CA) by one-way variance analysis (ANOVA) followed by the
Bonferroni-Dunn test or Student's t test.
Endogenous Increase of APP and A TGF-
Because it could be advanced that the up-regulation of APP
observed after TGF-
TGF-
Expression of either Alk-5 or Alk-4 led to an enhanced transcription of
all isoforms of APP, whereas Alks linked to other members of the
TGF- A TGF-
Finally, we mutated the AGAC sequence required for Smad binding to the
DNA into an ACAT sequence, previously reported to abolish Smad binding
to the DNA (14), within the
To characterize further the involvement of TGF-
To determine whether astrocytes and neurons were TGF- A TGF- A Although neurons are known to be the major source of A However, an astrocytic gliosis is always observed in brains of patients
with AD (43). These data led us to reconsider the participation of
astrocytes in the amyloidogenic process. Indeed, recent work performed
from transgenic mice (Tg2576) exhibiting amyloid plaques evidenced that
astrocyte-derived A Our data are in agreement with previous findings demonstrating that the
expression of TGF- In vitro, an overexpression of APP mRNAs induced by
TGF- Overall, these data reveal a molecular mechanism through which TGF- peptide (A
) in
the brain is crucial for development of Alzheimer's disease.
Expression of transforming growth factor-
1 (TGF-
1), an
immunosuppressive cytokine, has been correlated in vivo
with A
accumulation in transgenic mice and recently with A
clearance by activated microglia. Here, we demonstrate that TGF-
1
drives the production of A
40/42 by astrocytes leading to A
production in TGF-
1 transgenic mice. First, TGF-
1 induces the
overexpression of the amyloid precursor protein (APP) in astrocytes but
not in neurons, involving a highly conserved TGF-
1-responsive
element in the 5'-untranslated region (+54/+74) of the APP promoter.
Second, we demonstrated an increased release of soluble APP-
which
led to TGF-
1-induced A
generation in both murine and human
astrocytes. These results demonstrate that TGF-
1 potentiates A
production in human astrocytes and may enhance the formation of plaques
burden in the brain of Alzheimer's disease patients.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
with a large amount of the 40-amino acid variant,
A
40, and to a lesser extent of the 42-amino acid variant, A
42. In
Alzheimer's disease, this amyloidogenesis can lead to the formation of
amyloid deposits within the cerebral parenchyma and vascular walls (1,
2).
1 (TGF-
1), a potent immunosuppressive cytokine, with AD. First, recent data suggest that a genetic
polymorphism of the TGF-
1 gene may be associated with a higher risk
to develop AD (9). Second, post-mortem brain tissue analyses of AD
patients show an increased expression of TGF-
1 correlated with the
degree of cerebral amyloid angiopathy (8). Third, 16-month-old
transgenic mice overexpressing TGF-
1 in astrocytes elicit A
deposition (8). In the same study, these authors generated biogenic
mice expressing both human APP and TGF-
1. In these hAPP/TGF-
1
mice, A
deposits were observed after 3 months of age compared with TGF-
1 mice, suggesting that TGF-
1 would be able to influence APP
metabolism or processing. In addition to its amyloidogenic effects (8),
TGF-
1 was recently associated with A
clearance from the brain
parenchyma to the cerebral blood vasculature in aged hAPP/TGF-
1 mice
by activated microglia (10).
1 in the amyloid plaque metabolism,
the mechanism(s) by which TGF-
1 promotes the production of A
remained to be elucidated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin, APP770,
APP751, and APP695 (respectively 539, 242, 222, and 401 bp of PCR products). All PCR were established in the linear
range of amplification by performing multicycle amplification to reach
half of the saturation curve. The
-actin oligonucleotides were:
sense, 5'-GTGGGCCGCTCTAGGCACAA-3', 25-45 bp; and antisense,
5'-CTCTTTGATGTCACGCACGATTTC-3', 564-540 bp. The APP695
oligonucleotides were: sense,
5'-GCACTAACTTGCACGACTATGGCATGCTGCTGCCCTG-3', 500-536 bp; and
antisense, 5'-GCTGGCTGCCGTCGTGGGAACTCGGACTACCTCCTCCACA-3', 861-1104 bp. The APP751 primers were: sense,
5'-CTACCACTGAGTCTGTGGAG-3', 848-868 bp; and antisense,
5'-GCTGGCTGCCGTCGTGGGAAACACGCTGCCACACACCGCC-3', 1028-1104 bp.
APP770 primers were: sense, 5'-CTACCACTGAGTCTGTGGAG-3', 848-868 bp; and antisense,
5'-CTTGAGTAAACTTTGGGATGACACGCTGCCACACACCGCC-3', 1028-1068 bp (11-13).
Human APP primers were: hAPP751 antisense, 5'-CTTCCCTTCAGTCACATCAAAGT-3', 1070-1092 bp; hAPP770
antisense, 5'-CTTGAGTAAACTTTGGGACATGGCGCTGC-3', 1171-1200 bp; and hAPP
sense, 5'-GAACCCTACGAAGAAGCC-3', 925-942 bp. TGF-
transgene
sense was 5'-GGCTGTATTTAAGGA CAT CG-3' (corresponding to the 1689-1708
porcine TGF-
sequence); TGF-
transgene antisense was
5'-GCAGCTTATAATGGTTAC-3' (corresponding to SV40 sequence). The
conditions of amplification were 30 s at 95 °C, 30 s at
55 °C, and 1 min at 72 °C for 30 cycles, corresponding to the
50% of the saturation curve of the PCR product. Based on the PCR
primers used, TGF-
expression described in Fig. 1A
correspond to the transgene encoded TGF-
. PCR primers used for
IL-1
were: sense, 5'-TGACCTGGGCTGTCCAGATG-3'; antisense, 5'-CTGTCCATTGAGGTGGAGAG-3', 340 bp. The conditions of amplification were 30 s at 95 °C, 30 s at 58 °C, and 1 min at
72 °C for 30 cycles, corresponding to the 50% of the saturation
curve of the PCR product.
1-treated astrocytes and neurons. Cells were
harvested 1 h after treatment and processed as described
previously (14).
-32P]dCTP using the Klenow fragment of DNA
polymerase. Binding reactions, containing 10 µg of nuclear extracts
and 2 ng of labeled oligonucleotides, were performed for 20 min at
37 °C in appropriate binding buffer (14). The sequence of the double
stranded oligonucleotide used as probe was: 5'-CGGGAGACGGCGGCGG-3'.
Protein-DNA complexes were resolved in 5% polyacrylamide gels
containing 0.5 × TBE.
1 line T65 has been generated similarly to lines
64 and 115, which have been described previously (16). Briefly, a
1.35-kb porcine TGF-
1 cDNA was inserted into the first exon of a
modified mouse GFAP gene. This cDNA had been mutated to allow
secreted TGF-
1 to be functional once released into the extracellular
space. Homozygous TGF-
1 transgenic mice develop communicating
hydrocephalus (16); however, for this study we used heterozygous
TGF-
1 mice, which do not develop this complication. Identification
of transgenic mice was performed by analysis of tail genomic DNA and
from cerebral total RNAs.
(18), 192wt
(1:500) against sAPP-
(19), FCA3340 (1:1,000) against human A
40
(20), FCA3542 (1:1,000) against human A
42, sc-146 (1:200) against
human and murine TGF-
1 (Santa Cruz Biotechnology Inc.), OX42 (1:100)
against CD11b (kindly provided by Dr. Anna-Maria Planas), and anti-GFAP
antibody (1:100) and M4403 (1:100) against actin (Sigma). R1742 and
R600 were generated to synthetic A
37-42 and
A
1-10 and tested against synthetic
A
1-40 and A
1-42 peptides (Sigma).
ELISAs--
A
peptides were captured with a monoclonal
antibody raised against the N-terminal domain of A
(8-17) and
revealed using a secondary antibody against the C-terminal extremity
(40 or 42 end) of A
(BioSource Europe, Nivelles, Belgium). Either
recombinant rodent or human A
s (Calbiochem) were used as controls.
1 (R&D Systems Europe) before
semiquantitative RT-PCR analysis. Gels were scanned and quantified by densitometry.
1
treatment), whereas the activity of the cotransfected "control"
(Renilla luciferase) reporter provides an internal control, which serves as the base line of the response. Indeed, the pRL-TK control vector contains the herpes simplex virus thymidine kinase promoter region upstream of Renilla luciferase. It
provides low level and constitutive expression in transfected cells.
Normalizing the activity of the experimental reporter to the activity
of the internal control minimizes experimental variability caused by differences in cell toxicity, transfection efficiency and proliferation.
1104/+104 fragment of the Rhesus monkey APP promoter (GenBank
accession number AF 067971) was provided by Dr. Lahiri (22). The
309/+104 and the
201/+104 fragments were PCR amplified, digested by
KpnI and BglII restriction enzymes, and ligated
into pGL3-MLP. The
309/+104 luciferase vector was
digested by XhoI and BglII restriction enzymes to
obtain the
75/+104 fragment. The +54/+74 luciferase reporter vector
was obtained by inserting into pGL3-MLP a double stranded
oligonucleotide corresponding to the +54/+74 region of the human APP
promoter and flanked by two XhoI restriction sites.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Expression in TGF-
1
Transgenic Mice--
To understand the role of TGF-
1 in A
deposition, the influence of TGF-
1 on APP and A
expression was
investigated in 6-month-old transgenic mice overexpressing TGF-
1
(line T65) through the use of semiquantitative RT-PCR, Western blotting
analysis, and A
ELISAs. Transgenic mice were generated by Prof.
Lennart Mucke's laboratory similarly to the previously described low
expressing GFAP-TGF-
1 transgenic lines, T64 and T115 (16). These
mice express large amounts of the transgene associated with enhanced expression of endogenous TGF-
1 as determined either by RT-PCR (Fig.
1A) or by immunoblotting (Fig.
1B). In cortices of TGF-
1 mice, we observed a significant
up-regulation of the mRNAs encoding APP isoforms compared with
wild-type mice (Fig. 1, C and D). These data were
confirmed after immunoblotting performed from the same brain extracts
(Fig. 1E). When revealed with 22C11 antibody, membrane-bound APP proteins were significantly overexpressed in TGF-
1 mice compared with wild-type mice (Fig. 1E). Because TGF-
1 possesses an
amyloidogenic role (8), we determined A
production by ELISAs in our
T65 transgenic line and observed a significant increase of soluble A
42 (7.41 ± 0.48 in wild-type versus 13.07 ± 1.88 pg/mg in transgenic; p = 0.04) and A
40
(32.91 ± 1.36 in wild-type versus 59.12 ± 3.57 pg/mg in transgenic; p = 0.01) without modifying the
A
42:A
40 ratio (0.225 ± 0.06 in wild-type versus
0.221 ± 0.11 in transgenic) (Fig. 1F).
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Fig. 1.
TGF- 1 transgenic
mice display enhanced A
generation.
A, expression of the GFAP-TGF-
1 transgene, endogenous
TGF-
1 mRNA in the brain of 6-month-old mice overexpressing
TGF-
1 in astrocytes (T65, TGF-
1) was determined by
semiquantitative RT-PCR analysis. Experiments were performed in
triplicate.
-Actin was used as a housekeeping gene. B,
Western blotting analysis of endogenous TGF-
1 in the brain of
6-month-old mice overexpressing TGF-
1 in astrocytes (T65, TGF-
1)
revealed using the sc-146 antibody (n = 3). The
membrane was reprobed to determine the expression of actin to estimate
the homogeneity of proteins loaded. C, expression of APP
mRNAs in the brain of 6-month-old mice overexpressing TGF-
1 in
astrocytes (T65, TGF-
1) was determined by semiquantitative RT-PCR
analysis. Experiments were performed in triplicate.
-Actin was used
as a housekeeping gene. D, densitometric quantification of
brain relative expression of APP mRNA isoforms. Results are the
mean ± S.D. of experiments performed in triplicate.
Asterisk, p < 0.01, Student's t
test. E, Western blotting analysis of total APP proteins in
the brain of 6-month-old mice overexpressing TGF-
1 in astrocytes
(T65, TGF-
1) revealed using the 22C11 antibody (n = 3). The membrane was reprobed to determine the expression of actin to
estimate the homogeneity of proteins loaded. F, A
ELISAs
in the brain of 6-month-old mice overexpressing TGF-
1 in astrocytes
(T65, TGF-
1). Amounts of respective A
species are indicated in
the left panel, and rationalized expressions are shown in
the right panel. Results are the mean ± S.E. of six
independent experiments. Asterisk, p < 0.05, Student's t test).
1-dependent Transcription of APP in
Astrocytes--
To determine which cell type was involved in the
increased expression of APP in TGF-
1 mice, we exposed primary
cultures of mouse cortical neurons or astrocytes to exogenous
recombinant TGF-
1. As reported previously (23), the three APP
isoforms are expressed in astrocytes with a predominant expression of
APP770 and APP751, whereas cortical neurons
abundantly express APP695 mRNA but only small amounts
of APP770 and APP751 mRNAs (Fig. 2, A and C). Although
1 ng/ml TGF-
1 exposure failed to modify the expression pattern of
APP mRNAs in cortical neurons (Fig. 2, A and
B), TGF-
1 markedly enhanced the expression of the
mRNAs encoding the three isoforms of APP in astrocytes (Fig. 2,
C and D).
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Fig. 2.
TGF- 1-dependent expression
of APP mRNAs in cultured astrocytes. A, expression
of APP mRNAs in neurons was determined by semiquantitative RT-PCR
analysis at the indicated time after treatment of cultured cortical
neurons in the presence of 1 ng/ml TGF-
1 (24 and 72 h)
(n = 3).
-Actin was used as a housekeeping gene. All
extracts elicited similar levels for
-actin. The letter C
refers to sham wash control cultures, and T refers to
TGF-
1-treated cell cultures. B, densitometric
quantification of the experiments presented in A 24 h
after treatment. White bars represent sham wash control
neurons, and dark bars represent TGF-
1-treated neurons.
Results are the mean of three experiments. Asterisk,
p < 0.01, Student's t test. C,
expression of APP mRNA in cultured astrocytes was determined by
semiquantitative RT-PCR analysis at the indicated time after treatment
in the presence of 1 ng/ml TGF-
1 (24 and 72 h)
(n = 3).
-Actin was used as a housekeeping gene. All
extracts elicited similar levels for
-actin. D,
densitometric quantification of the experiments presented in
C 24 h after treatment. White bars represent
sham wash control astrocytes, and dark bars represent
TGF-
1-treated astrocytes. Results are the mean of three experiments.
Asterisk, p < 0.03, Student's t
test. E, astrocytes were stained with an antibody raised
against the astrocytic marker GFAP (green) and the
microglial marker CD11b (red). Finally, cells were
counterstained with 1 µg/ml DAPI (blue). Overlaid images
are presented in the bottom right panel.
1 exposure could result from the secondary activation of astrocytes by proinflammatory cytokines such as interleukin-1
released by microglia cells, we have estimated the
amount of microglia in our cultures of astrocytes by performing double
immunocytochemical labeling against the GFAP, an astrocytic marker, and
CD11b, a specific marker of microglial cells. Less than 1% of
microglial contamination in our primary cultures of cortical astrocytes
was evidenced (Fig. 2E).
1 elicits its biological effects through a heteromeric complex
of transmembrane serine/threonine kinase receptors, cloned as type I
and type II receptors. After receptor-dependent
phosphorylation, Smad2 or Smad3 interacts with the common
mediator Smad4 to form a heteromeric complex that translocates to the
nucleus to initiate TGF-
-dependent transcriptional
activity. This complex binds DNA sequences termed Smad-binding elements
that contain a minimal four-nucleotide domain AGAC also called "CAGA
box" (24). Accordingly, we have transiently transfected astrocytes
with expression vectors encoding constitutively activated versions of
the TGF-
type I receptor (Alks) (25) or with an expression vector
encoding the transcription factor Smad3 (Fig.
3). To validate the efficiency of
transfection in our system, murine cultured astrocytes were transfected
with the EGFP-C1 vector encoding EGFP using cationic lipids. 24 h
later, astrocytes were immunolabeled for GFAP and counterstained with
DAPI before determination of transfection efficiency (~70%) (Fig.
3A). Cell viability did not differ from untransfected
astrocytes as estimated by phase microscopic observation and lactate
dehydrogenase activity (data not shown).
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Fig. 3.
Regulation of APP transcription by the
Smad-dependent TGF- 1 signaling
pathway in astrocytes. A, efficiency of transient
transfection of primary cultured astrocytes. The expression vector
pEGFP-C1 encoding EGFP was transiently transfected in astrocytes before
immunostaining with an antibody against the GFAP (red) and
counterstaining with 1 µg/ml DAPI (blue). Overlaid images
are presented in the bottom right panel. B,
transient transfections of cultured murine astrocytes, with expression
vectors encoding constitutively activated versions of the TGF-
family type I receptors, were performed before RT-PCR analyses as
described in Fig. 2. C, relative expression of APP mRNA
isoforms compared with
-actin in transiently transfected astrocytes
presented in B. Dark, dark gray, and
light gray bars represent the ratio obtained for
APP770, APP751, and APP695
isoforms, respectively. Results are the mean ± S.D. of
experiments performed in triplicate. Asterisk,
p < 0.003, Sharp (different from controls or empty
vector), p < 0.01, Student's t
test. D, primary cultures of mouse cortical astrocytes were
transfected either with expression vector encoding the transcription
factors Smad3 or Smad7 or with empty vector (pcDNA3)
before RT-PCR analyses as described in Fig. 2 (n = 3).
Astrocytes transfected with the empty vector were exposed to TGF-
1
at 1 ng/ml for 24 h after transfection. The letter C
refers to sham wash control cultures, and T refers to
TGF-
1-treated cell cultures. E, densitometric
quantification of the experiments presented in D 24 h
after treatment. White bars represent control astrocytes
transfected with the empty vector (pcDNA3), dark
bars represent TGF-
1-treated astrocytes transfected with
the empty vector (pcDNA3), white hatched
bars represent control astrocytes transfected with the expression
vector encoding Smad3, and dark hatched bars represent
control astrocytes transfected with the expression vector encoding
Smad7. Results are the mean of three experiments. Asterisk,
p < 0.01, Sharp (different from TGF-
-treated
cells), p < 0.02, Student's t test.
superfamily did not enhance APP transcription (Fig. 3,
B and C). Indeed, transfecting astrocytes with
Alk-3 unexpectedly lowered APP mRNA expression. Accordingly, Smad3
overexpression potentiated APP mRNA expression (Fig. 3,
D and E). As expected, overexpression of Smad7, a
physiological dominant negative Smad, prevented TGF-
1-induced
expression of APP in cultured astrocytes (Fig. 3, D and
E). Thus, TGF-
1 signaling machinery controls APP transcription in cultured astrocytes by a Smad3-dependent mechanism.
-responsive Element Mediates
TGF-
1-dependent Expression of APP--
We determined
whether the induction of the APP mRNAs by TGF-
1 in astrocytes
was the result of a stabilization of the transcripts. In the presence
of actinomycin D, the relative amount of APP and
-actin mRNAs
was reduced in a time-dependent manner. The administration of TGF-
1 failed to modify the stability of APP transcripts (Fig. 4A). Considering the
TGF-
1-dependent overexpression of the three APP isoforms
mRNAs, we investigated whether TGF-
1 modulates the activity of
the APP promoter. Thus, different regions (
309/+104,
201/+104, and
75/+104) of the Rhesus monkey APP promoter were subcloned into the
pGL3-basic luciferase reporter vector containing a
MLP. When transfected in the mink lung epithelial cell line Mv1Lu, previously characterized as a TGF-
-responsive cell line (26,
27), these entire promoter constructs were activated by TGF-
1
treatment or Smad3 transfection (Fig. 4B). By performing sequence alignments (Fig. 4C), we noticed that a conserved
sequence, located between +56 and +71 within the 5'-untranslated
region, was retrieved in the cloned APP promoter from mouse, rat,
Rhesus monkey, and man (5'-CGGGAGACGGCGGCGG-3'). Although the empty
vector was nonresponsive to TGF-
1 treatment or Smad3 transfection,
it became sensitive to TGF-
1 and Smad3 when five copies of the
+54/+74 sequence of the human APP promoter (named APP
TGF-
-responsive element or APPtre) were inserted (Fig. 5,
A and B). Results
were similar both in Mv1Lu cells (Fig. 5A) and in primary
cultures of murine astrocytes (Fig. 5B). Transfection of
cultured astrocytes with expression vectors encoding Alk-4 and Alk-5
resulted in a marked increase in luciferase activities confirming the
necessity of TGF-
receptor activation to induce its response (Fig.
5C). Moreover, overexpression of Smad7 led to 90%
inhibition of TGF-
1-induced activation of the APPtre reporter
construct (Fig. 5D). In MDA-MB468 cells deficient for
endogenous Smad4 expression (28, 29), TGF-
1 or Smad3 had no effect,
whereas transfection of Smad4 rescued TGF-
1-induced activation of
the APPtre reporter construct (Fig. 5E). These data
demonstrates that a Smad3·Smad4 complex is necessary to mediate the
TGF-
1-induced transcription of APP.
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Fig. 4.
Identification of a
TGF- 1-responsive element within the APP
promoter. A, APP mRNA decay experiments were
performed in cultured astrocytes treated with 10 µg/ml actinomycin D
for the indicated times after a 24 h-treatment in the presence or not
of 1 ng/ml TGF-
1. Open diamonds (
),
-actin for
TGF-
1-treated astrocytes; black diamonds (
),
-actin
for untreated astrocytes; open squares (
),
APP770 for TGF-
1-treated astrocytes; black
squares (
), APP770 for untreated astrocytes;
open circles (
), APP751 for TGF-
1-treated
astrocytes; black circles (
), APP751 for
untreated astrocytes; open triangles (
),
APP695 for TGF-
1-treated astrocytes; black
triangles (
), APP695 for untreated astrocytes.
Results are the mean of three independent experiments.
Asterisk, p < 0.01, Student's t
test. B, mean ± S.D. of the luciferase activity of
Mv1Lu cell line transiently transfected with luciferase reporter
constructs containing the regions
309/+104,
201/+104, or
75/+104
of the Rhesus monkey APP promoter, respectively. For each condition
transfected cells were treated (black bars) or not
(white bars) in the presence of 1 ng/ml TGF-
1 for 24 h or cotransfected with the expression vector encoding Smad3
(hatched bars) (n = 12).
Asterisk, p < 0.01, ANOVA followed by the
Bonferroni-Dunn test. C, alignment of DNA sequences of the
5'-untranslated regions of human, Rhesus monkey, mouse, and rat APP
genes. Letters in bold differ from the human DNA
sequence. Underlined letters indicate the minimal binding
DNA sequence for Smad3. The dark square elicits the
conserved sequences among the different species analyzed.
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Fig. 5.
Smads proteins mediate
TGF- 1-induced activation of the APP
promoter. A, mean ± S.D. of the luciferase
activity of Mv1Lu cell line transiently transfected with a luciferase
reporter construct containing five copies of the +54/+74 sequence of
the human APP promoter inserted 5' of the MLP luciferase reporter gene.
For each condition, transfected cells were treated or not in the
presence of TGF-
1 or cotransfected with Smad3 (n = 12). For each condition transfected cells were treated (black
bars) or not (white bars) in the presence of 1 ng/ml
TGF-
1 for 24 h or cotransfected with the expression vector
encoding Smad3 (hatched bars). Asterisk,
p < 0.01, ANOVA followed by the Bonferroni-Dunn test.
B, the same experiments as described in A were
performed in primary cultures of murine cortical astrocytes
(n = 12). Asterisk, p < 0.01, ANOVA followed by the Bonferroni-Dunn test. C,
mean ± S.D. of the luciferase activity of astrocytes transiently
cotransfected with a luciferase reporter construct containing five
copies of the +54/+74 sequence of the human APP promoter inserted 5' of
the MLP luciferase reporter gene and with expression vectors encoding
autoactivated type I TGF-
receptors (Alks). Black bars
represent astrocytes expressing autoactivated Alks, and white
bars represent astrocytes transfected with the empty vector
(n = 12). Asterisk, p < 0.01, Sharp (different from untreated cells), p < 0.02, ANOVA followed by the Bonferroni-Dunn test. D,
mean ± S.D. of the luciferase activity of Mv1Lu cell line
transiently transfected with a luciferase reporter construct containing
five copies of the +54/+74 sequence of the human APP promoter inserted
5' of the MLP luciferase reporter gene. For each condition transfected
cells were treated (black bars) or not (white
bars) in the presence of TGF-
1 or cotransfected with Smad7
(hatched bars) (n = 8). Asterisk,
p < 0.01, Sharp (different from TGF-
-treated
cells), different from TGF-
1, p < 0.01, ANOVA
followed by the Bonferroni-Dunn test. E, mean ± S.D.
of the luciferase activity of Smad4-deficient MDA cell line transiently
transfected with a luciferase reporter construct containing five copies
of the +54/+74 sequence of the human APP promoter inserted 5' of the
MLP luciferase reporter gene. For each condition transfected cells were
treated (black bars) or not (white bars) in the
presence of TGF-
1 or cotransfected with either Smad3 (dark
hatched bars) or Smad4 (gray bars) or Smad3 and Smad4
(gray hatched bars) (n = 8).
Asterisk, p < 0.03, ANOVA followed by the
Bonferroni-Dunn test. F, mean ± S.D. of the luciferase
activity of astrocytes transiently transfected with a luciferase
reporter construct containing the wild-type (AGAC) or mutated (ACAT)
region
201/+104 of the Rhesus monkey APP promoter inserted 5' of the
MLP luciferase reporter gene. For each condition transfected cells were
treated (black bars) or not (white bars) in the
presence of TGF-
1 (n = 12). Asterisk,
p < 0.01, ANOVA followed by the Bonferroni-Dunn test.
G, immunoblot analyses of full-length APP proteins
associated with the cell monolayer of cultured murine cortical
astrocytes treated (T) or not (C) in the presence
of TGF-
1 for 24 h were performed using the 22C11 antibody. To
determine the involvement of endogenous TGF-
1 in controlling APP
expression, parallel experiments were done in the presence of soluble
TGF-
type II receptor (sT
RII) as a TGF-
antagonist
(n = 2).
204/+104 gene promoter construct (Fig.
5F). This construct also provides evidences of the influence
of this element in a similar context to the full-length APP promoter.
Results obtained by Dual-Luciferase® Reporter Assay System
clearly indicate that this double site mutation abolishes the ability
of this construct to respond to TGF-
1 treatment and to Smad3
transfection. Thus, our findings demonstrate the absolute necessity of
this sequence for TGF-
1-induced transcriptional activity of the APP promoter.
1 in the control of
APP expression in cultured astrocytes, we have performed experiments
using a soluble TGF-
type II receptor as a TGF-
antagonist (30,
31). Addition of the this receptor in the media of cultured astrocytes
prevented TGF-
1-induced APP overexpression. Moreover, it is
interesting to note that treatment in the presence of the soluble
TGF-
type II receptor also decreased the basal expression of APP,
confirming the presence of endogenous TGF-
1 in our model of cultured
astrocytes as demonstrated previously (32). Thus, endogenous
TGF-
1 regulates APP basal expression and can enhance APP transcription.
1-responsive,
we performed an electrophoretic mobility shift assay using astrocytic
and neuronal nuclear extracts in an attempt to characterize the DNA
binding activity on the TGF-
-responsive APP sequence containing the previously characterized TGF-
-responsive AGAC sequence (14). Although TGF-
1 failed to induce
formation of complexes in cultured
neurons, increased binding complexes were observed in TGF-
1-treated
astrocytes (Fig. 6, center lanes). To confirm the
specificity of the assay, a 50-fold excess of unlabeled probe was added
to nuclear extracts from TGF-
1-treated cells, which totally
prevented the formation of complexes (Fig. 6, right lanes).
These data provide evidence that primary cultured mature cortical
neurons (14 days in vitro) are not capable of mediating Smad-dependent TGF-
1 signaling in the APP promoter.
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Fig. 6.
Direct binding of
TGF- 1-induced transcription factors to the
APPtre sequence containing the AGAC. An electrophoretic mobility
shift assay was performed using a 32P-labeled probe
containing the AGAC sequence and nuclear extract from neurons or
astrocytes induced for 1h by TGF-
1 or not induced. Bands
corresponding to specific TGF-
1-induced complexes are indicated. 50 molar excesses of nonradiolabeled oligonucleotide were added as
competitor (n = 3).
Production Is Enhanced by TGF-
1 Signaling--
As
observed at the mRNA level, TGF-
1 failed to modify the
expression of either full-length or derivatives of APP in cortical neurons (Fig. 7). In contrast,
immunoblotting performed from cell extracts of murine astrocytes showed
that membrane-bound APP proteins were increased markedly in the
presence of TGF-
1 over 24-72 h, whereas actin remained unchanged
(Fig. 7, A and B). Using the R7 antiserum
targeted against the KPI domain of KPI-APPs, i.e. APP770 and APP751, we confirmed the
overexpression of APP770 and APP751 at the
membrane of astrocytes stimulated by TGF-
1 (Fig. 7, A and
B). Furthermore, we evidenced that the release of sAPP derivatives was increased in the media of TGF-
1-treated astrocytes (Fig. 7C). Similarly, we showed that sAPP-
was increased
in the conditioned media of astrocytes treated by TGF-
1 (for 24 or
72 h) (Fig. 7C). Because
-secretase activity is
necessary for A
production, incubation with TGF-
1 for 24 or
72 h induced a marked secretion of the 4-kDa A
species in the
bathing medium of cultured astrocytes (Fig. 7D). Moreover,
our results revealed that the overexpression of Smad3 led to an
accumulation of A
as obtained previously after TGF-
1 treatment
(Fig. 7E). In contrast, overexpression of Smad7 prevented
the TGF-
-induced accumulation of A
(Fig. 7E). Using
FCA3340 and FCA3542 antisera raised against A
40 or A
42, we
observed an accumulation of A
40 and A
42 under TGF-
1 treatment,
whereas A
was not be detected in control conditions (Fig.
7F). Because we were unable to detect basal secretion of astrocytic A
using immunoblotting (Fig. 7, D-F), we have
finally estimated A
release by quantitative ELISAs and confirmed a
~2-fold elevation of A
loads (Fig. 7G). This 2-fold
increase in A
secretion (×1.9 for A
42 and ×1.8 for A
40) can
be directly associated with the 2-fold up-regulation of astrocytic APP
transcription. Nevertheless, we cannot exclude the possibility of
indirect effects of TGF-
1 on either the APP processing or the A
degradation. Taken overall, these experiments evidence that TGF-
1
promotes A
generation in astrocytes.
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Fig. 7.
TGF- 1 promotes
sAPP-
and A
accumulation in the conditioned media of astrocytes.
A, immunoblot analyses of full-length APP proteins
associated with the cell monolayer of cultured murine cortical neurons
or astrocytes treated (T) or not (C) in the
presence of TGF-
1 for 24 h were performed using either the
22C11 antibody or the R7 antiserum. Note that only small amounts of
KPI-APPs are detected in neurons. The same immunoblot was reprobed with
actin antibody as control. Experiments were performed in triplicate.
B, densitometric quantification of the experiments presented
in A 24 h after treatment. White bars
represent sham wash control cells, and dark bars represent
TGF-
1-treated cells. Results are the mean of three experiments.
Asterisk, p < 0.01, Student's t
test. N represents neurons, and A, astrocytes.
C, immunoblot analyses of total sAPP and sAPP-
from
conditioned media of cultured murine cortical neurons or astrocytes
treated (T) or not (C) in the presence of
TGF-
1 for 24 h were performed using either the 22C11 antibody
or the 192wt antiserum. The membrane was stained by naphthol blue to
confirm the homogeneity of loaded amounts of proteins (data not shown).
Experiments were performed in triplicate. D, conditioned
media of astrocytes treated in the presence of 1 ng/ml TGF-
1 for
72 h were concentrated as described under "Experimental
Procedures," and Western blot analyses were performed using the R600
polyclonal antiserum raised against the N terminus (residues 1-10) of
the A
peptide. Experiments were performed in triplicate.
E, Western analysis of A
were performed using the R600
antiserum from concentrated conditioned media of primary cultures of
cortical astrocytes previously transfected either with the expression
vector encoding Smad3 or the expression vector encoding Smad7 or with
the empty vector in the presence or not of TGF-
1 for 72 h.
Asterisks indicate nonspecific labeling. F, to
discriminate A
species, Western analyses of A
were performed from
concentrated conditioned media of astrocytes incubated with 1 ng/ml
TGF-
1 for 72 h and probed with a polyclonal antiserum raised
against A
1-40, FCA3340, or A
1-42,
FCA3542. Asterisks indicate nonspecific labeling. The
experiments were performed in triplicate. G, in parallel,
A
ELISAs were realized from the same extracts used for
immunoblotting experiments. White bars correspond to
untreated astrocytes and dark bars to TGF-
1 treated
astrocytes. Asterisk, p < 0.001, Student's
t test.
1 Promotes A
in Cultured Human Astrocytes--
Because
rodents do not display amyloid deposits, we performed a set of
experiments in primary cultures of human astrocytes and neurons. As
observed previously in murine cultures, TGF-
1 treatment failed to
influence either transcription or accumulation of APP derivatives in
cultured human neurons (Fig. 8). However, the addition of exogenous TGF-
1 to human astrocytes induced an increased expression of APP (Fig. 8A) as well as the
activation of the APPtre luciferase reporter vector (Fig.
8B). These transcriptional data were confirmed at the
protein level. Although TGF-
1 failed to influence APP expression in
neurons, it increased the amounts of full-length APP and sAPP-
associated with either the plasma membrane or the conditioned media of
cultured human astrocytes (Fig. 8, C and D).
Moreover, TGF-
1 (Fig. 8E) or transfection with the Smad3
vector (Fig. 8F), respectively, led to a 1.5-2.5 increase
in A
content in the conditioned media of human astrocytes. Finally,
A
ELISAs (n = 8) confirmed a significant enhanced
generation of A
42 (7.32 ± 0.59 in controls versus
10.01 ± 0.97 pg/mg in treated cells; p = 0.02)
and A
40 (43.26 ± 3.77 in controls versus 74.13 ± 5.31 pg/mg in treated cells; p = 0.001) in human
astrocytes after TGF-
1 treatment. Interestingly, the A
42:A
40
ratio decreased (0.169 ± 0.045 in controls versus
0.135 ± 0.018 pg/mg in treated cells; p = 0.04)
in human treated cells (Fig. 6G), whereas mice cultured
astrocytes did not display any difference, suggesting slightly
different mechanisms in A
generation.
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Fig. 8.
TGF- 1 promotes
A
generation in human cultured
astrocytes. A, expression of APP mRNAs in primary
cultures of human astrocytes was determined by RT-PCR after treatment
in the presence of 1 ng/ml TGF-
1 for 24 h (n = 3).
-Actin was used as a housekeeping gene. All extracts elicited
similar levels of
-actin. B, mean ± S.D. of the
luciferase activity of cultured human astrocytes transiently
transfected with a luciferase reporter construct containing five copies
of the +54/+74 sequence of the human APP promoter inserted 5' of the
MLP luciferase reporter gene. For each condition, transfected cells
were treated (T) or not (C) in the presence of
TGF-
1 (n = 12). Asterisk,
p < 0.01, ANOVA followed by the Bonferroni-Dunn test. C,
immunoblot analysis of full-length APP proteins associated with the
cell monolayer of cultured human cortical neurons or astrocytes treated
(T) or not (C) in the presence of TGF-
1 for
24 h using the R7 antibody. The same immunoblot was reprobed with
actin antibody as control. Experiments were performed in triplicate.
The histogram illustrates densitometric quantification of
the experiments presented in C 24 h after treatment.
White bars represent sham wash control cells, and dark
bars represent TGF-
1-treated cells. Results are the mean of
three experiments. Asterisk, p < 0.01, Student's t test. D, immunoblot analysis of
sAPP-
was performed from the conditioned media of cultured human
cortical neurons or astrocytes treated (T) or not
(C) in the presence of TGF-
1 for 24 h using the
192wt antiserum. The histogram illustrates densitometric
quantification of the experiments presented in D 24 h
after treatment. White bars represent sham wash control
cells, and dark bars represent TGF-
1-treated cells.
Results are the mean of three experiments. Asterisk,
p < 0.01, Student's t test. E,
immunoblot analyses of A
were performed using the R600 antiserum
from the conditioned media of cultured human cortical neurons or
astrocytes treated (T) or not (C) in the presence
of TGF-
1 for 24 h. Experiments were performed in triplicate.
Densitometry analysis of A
production in the media of cultured human
cortical neurons or astrocytes treated (black bars) or not
(white bars) in the presence of TGF-
1 for 72 h was
performed. Results are the mean of three independent experiments.
Asterisk, p < 0.03, Student's t
test. F, Western analyses of A
were performed using the
R600 antiserum from concentrated conditioned media of primary cultures
of cortical astrocytes previously transfected with the expression
vector encoding Smad3 or with the empty vector in the presence or not
of TGF-
1 for 72 h. G, A
ELISAs were performed
from human astrocytes treated with TGF-
1 for 24 h. The amounts
of respective A
species are indicated in the left panel,
and rationalized expressions are shown in the right panel.
Results are the mean ± S.E. of eight independent experiments.
Asterisk, p < 0.05, Student's t
test.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
accumulation into amyloid plaques in the brain is one of the
two histopathological hallmarks of AD. The process that regulates the
deposition of A
in the brain is still under investigation. A better
understanding of the mechanism leading to A
production would
facilitate the development of treatments for AD. We document here that
the overexpression of the anti-inflammatory cytokine TGF-
1 in
transgenic mice induces higher expression of endogenous APP isoforms
and increased A
generation in cerebral tissues. Furthermore, we
demonstrate that exogenous TGF-
1 enhances APP synthesis in
astrocytes and leads to A
generation in vitro.
in AD
(33-35), the contribution of astrocytes to amyloidogenic processes has
never been clearly established. Studies have suggested that cultured
astrocytes could generate modest amounts of A
compared with neurons
(36-39). Although TGF-
1 induced the overexpression of APP in
astrocytes by involving a TGF-
-responsive element, we observed no
effect of TGF-
1 in primary cultures of mature neurons (14 days
in vitro). This absence of a TGF-
1 response is puzzling
but has been described previously (40). In addition, contradictory data
report the neuronal expression of type II TGF-
receptor (32, 41),
which binds the ligand and then activates the TGF-
signaling
intracellular pathway. Finally, these data are in agreement with a
previous study (42), demonstrating that although TGF-
induced
transcription of plasminogen activator inhibitor-1 in cultured
astrocytes, it failed to mediate this response in cultured neurons. To
understand better why TGF-
1 did not activate APP transcription in
mature cultured neurons (14 days in vitro), we have
performed an electrophoretic mobility shift assay and observed that
TGF-
1 is unable to activate the Smad pathway in mature neurons.
participate in plaque formation and maturation
at later stages than neuronal A
(44). Here, we demonstrate that
TGF-
1 potentiates A
production in astrocytes.
1 in the brain parenchyma induces cerebrovascular
and meningeal A
deposition at 12-18 months of age in TGF-
1
transgenic mice (line T64 or T115) or at 2-3 months of age in
hAPP/TGF-
1 biogenic mice expressing the human APP and TGF-
1 (line
T64 or T115) (8). In addition to its proamyloidogenic effect, TGF-
1
may exert a more complex role because 12-15-month-old hAPP/TGF-
1
double transgenic mice (10) displayed plaque burden reduction
associated with increased microglia activation and increased clearance
of A
compared with hAPP mice. In the present study, we report that
6-month-old transgenic mice overexpressing TGF-
1 (line T65) display
increased endogenous APP expression and A
production in
vivo. Moreover, we postulate that this effect is sustained by a
transcriptional activation of APP in both murine and human astrocytes.
Although AD cannot be equated with a simple unique gene deregulation,
this is, to our knowledge, the only transgenic animal overexpressing a
brain-derived cytokine that promotes A
deposition in the brain.
Nevertheless, we cannot exclude the possibility of indirect effects of
TGF-
1 on either the APP processing or the A
degradation. These
data are strengthened by several publications. First, TGF-
immunoreactivity has been found within the plaques of AD (5). Second,
increased TGF-
2 levels in reactive astrocytes are associated with AD
(7). Third, aged transgenic mice containing the Swedish double mutation
of APP695 display TGF-
1 immunoreactive astrocytes found
in close proximity to A
deposits (44). We demonstrate that
TGF-
1-induced APP overexpression leads to an enhanced A
production. Similarly, the increase in APP expression because of
duplication of the 21 chromosome in Down's syndrome results in an
overproduction of A
peptides leading to the appearance of AD-type
brain lesions with associated microgliosis and astrogliosis
(45).
1 has been reported previously in astrocytes or in astrocytoma
cell line (46, 47). Amara et al. (47) have described an
increased half-life of the APP transcripts induced by TGF-
1. By
performing mRNA decay experiments after 24 h of TGF-
1
treatment, we demonstrated that in our hands the half-life of APP
mRNA was not stabilized by a TGF-
1 treatment. In contrast, we
evidenced that TGF-
-induced up-regulation of APP mRNA expression
involved the activation of a TGF-
=responsive element within the
+54/+74 region of the APP promoter. Additional data provided evidences
that the 5'-untranslated region of the APP promoter is crucial for
driving APP expression. Indeed, the
75/+104 region has been shown to
mediate up to 40% of the promoter activity compared with the
full-length promoter-driven activity (
7900/+104) (48).
1
promotes A
generation and underline the critical role that
astrocytes could hold in AD pathogenesis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Anna-Maria Planas, Dr. Peter
Seubert, Prof. Nikolaos Robakis, and Prof. Frédéric
Checler, who generously provided the OX42, 192wt, R7 and FCA3340, and
FCA3542 antibodies, respectively. We also thank Dr. Tony Wyss-Coray for
providing brain tissues from TGF-1 transgenic line T65. Alks
plasmids and MDA-MB468 cells were provided by Dr. Joan Massagué
and Dr. Peter ten Dijke.
![]() |
FOOTNOTES |
---|
* This work was supported to grants from the Regional Council of Lower Normandy (to S. L.), the French Ministry of Research and Technology (to G. L.), the National Center for Scientific Research CNRS (to C. G.), and the National Institutes of Health (to D. K. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: CNS Inflammation Group, Centre for Neuroscience at Southampton, University of Southampton, Biomedical Sciences Bldg., Southampton SO16 7PX, UK.
** These authors contributed equally to this work.
To whom correspondence should be addressed: UMR CNRS 6551, Université de Caen, Centre Cyceron, Boulevard Henri Becquerel, BP
5229, Caen Cedex 14074, France. Tel.: 33-2-3156-6039; Fax: 33-2-3156-6199; E-mail: d.vivien@neuro.unicaen.fr.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M300819200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
APP, amyloid
precursor protein;
A, amyloid-
peptide;
AD, Alzheimer's disease;
ANOVA, analysis of variance;
DAPI, 4,6-diamidino-2-phenylindole;
EGFP, enhanced green fluorescent protein;
ELISA, enzyme-linked immunosorbent
assay;
GFAP, glial fibrillary acidic protein;
KPI, Kunitz protease
inhibitor;
MLP, major late promoter;
PBS, phosphate-buffered saline;
RT, reverse transcription;
sAPP, soluble APP;
TGF-
1, transforming
growth factor-
1;
TK, thymidine kinase;
tre, TGF-
-responsive
element;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
Smad, Small Mothers against Decapentaplegie.
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REFERENCES |
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