From the Division of Hematology, Department of
Medicine, Brigham and Women's Hospital, Harvard Medical School,
Boston, Massachusetts 02115, the ** Diabetes Unit, Department of
Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, and the
Department of Biochemistry and
Molecular Biology and Suncoast Gerontology Center, University of South
Florida, Tampa, Florida 33612
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ABSTRACT |
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The amyloid precursor
protein (APP) has been associated with Alzheimer's disease
(AD) because APP is processed into the The amyloid precursor protein
(APP)1 is a 110-130-kDa type
I membrane-spanning glycoprotein expressed ubiquitously in mammalian tissues (1, 2). A portion of APP is processed constitutively into
40-42-amino acid In addition to altered A IL-1 is the first proinflammatory cytokine secreted after the
activation of macrophage/microglial cells (28). IL-1 is expressed abundantly in microglia around developing amyloid plaques in brain cells, particularly in those brain regions that are prone to develop the mature amyloid plaques enriched in Microglial IL-1, which is known as a stimulator of astroglial
proliferation (31), is increased in the rat brain after injury (32). A
protein kinase C-dependent pathway (9) has linked IL-1 to a 3-fold
increase in APP gene expression in human endothelial cells at the level
of enhanced transcription. IL-1 greatly increased the transcriptional
regulation of the amyloid plaque-associated protein ACT in human
primary astrocytes (17) and a human astrocytoma cell line (33).
However, a smaller induction of APP gene transcription by IL-1 in rat
neuronal cells was not matched by an increase in the steady-state
levels of APP mRNA in glial cells, including astrocytes (2, 34).
In this report we have identified and characterized a novel mechanism
of IL-1-dependent regulation of APP gene expression at the
level of increased APP mRNA translation in astrocytes. Here,
increased synthesis of APP by IL-1 was found to be mediated through a
translational regulatory sequence in the 5'-untranslated region
(5'-UTR) of APP mRNAs. We showed previously that IL-1 specifically enhances translation of the mRNAs encoding the light (L) and heavy (H) subunits of ferritin, the central iron storage protein present in
all cells (35). The APP mRNA 5'-UTR sequence can fold into a single
RNA stem-loop and is related to hepatic RNA enhancers, the acute box
sequences, in the 5'-UTR of L- and H-ferritin mRNAs (36, 37).
Cell Culture and Immunocytochemistry
Primary human astrocytes were prepared by trypsinization of
human fetal brain tissue as described previously (17), treatment with 5 mM H-Leu-O-methyl ester to eliminate microglia
(38), seeding onto coated plates, and growing to 70% confluence in
DMEM (low glucose) supplemented with 10% fetal bovine serum. For
immunohistochemistry the cells were grown on 10 µg/ml
poly-L-lysine-coated microscope slides, washed briefly in
1 × PBS, and then fixed in 4% paraformaldehyde and 0.1% Triton
X-100 in 1 × PBS, pH 7.4, for 30 min on ice. The cells were then
incubated in 10% normal goat serum (Life Technologies, Inc.) and 0.4%
Triton X-100 in 1 × PBS for 30 min at 37 °C to block
nonspecific binding. The primary antibody (monoclonal anti-glial fibrillary acidic protein (GFAP), clone G-A-5, Sigma, dilution 1:400),
was applied in 1% normal goat serum and 0.4% Triton X-100 in 1 × PBS for 1 h at 37 °C. The astrocytes were washed for 5 min
in 1 × PBS and incubated with the secondary antibody
(affinity-purified polyclonal Cy3-labeled goat anti-mouse IgG (H+L),
Jackson ImmunoResearch, dilution 1:400) in 10% normal goat serum and
0.4% Triton X-100 in 1 × PBS for 30 min at room temperature. The
cells were again washed in 1 × PBS for 5 min, counterstained with
4 ng/ml 4,6-diamidino-2-phenylindole, mounted in 50% glycerol, and
examined with a light microscope (Axioskop, Zeiss).
The U373MG astrocytoma cell line was obtained from Dr. H. Fine (DFCI,
Boston, MA) and cultured on uncoated dishes to 60-80% confluence in
DMEM (high glucose) supplemented with 10% fetal bovine serum.
Plasmid Constructs
The eukaryote expression vector pSV2CAT contains a
unique StuI site 45 base pairs (bp) downstream from the SV40
early T-antigen promoter. A unique HindIII site is present
17 bp further downstream from the StuI site (62 bp from the
CAT gene 5'-cap site (36)). The pSV2(APP)CATconstruct was
prepared by inserting 90 bp of the APP gene 5'-UTR (between the
SmaI and the NruI sites, respectively +55 and
+144 nucleotides from the 5'-cap site) into the StuI and HindIII sites in the 5'-UTR of the CAT gene in
pSV2CAT (see Fig. 6). This pSV2(APP)CAT
construct was prepared by two steps of subcloning because the APP gene
5'-UTR is GC-rich and was refractory to accurate amplification by
polymerase chain reaction. First, a 3-kb
SmaI-HindIII fragment containing the APP gene was
subcloned into compatible StuI and HindIII sites
unique to the 5'-UTR of the CAT gene in the pSV2CAT
expression vector. The fragment between the NruI and HindIII sites in the APP gene was then removed from the
construct. The restriction sites were then blunt ended and religated.
The pBS2CAT construct contained a 254-bp
HindIII-EcoRI fragment from the CAT gene coding
for 218 bp of coding sequences from CAT gene ligated immediately
downstream from 36-bp 5'-UTR sequences from the SV40 early T-antigen
promoter region. These sequences from the CAT gene were ligated into
the unique polylinker site in the pBS vector (Stratagene). The
pGEM3zF±-hACT, pGEM3zF±-hAPP, and
pGEM3zF±-hGAPDHconstructs, respectively, contained a
407-bp PstI-SacI fragment (amino acids 175-311
in the human ACT gene (39)), a 1,056-bp
EcoRI-EcoRI fragment (bp 1795-2856 in the human
APP gene (40)), and a 548-bp HindIII-XbaI
fragment (amino acids 66-248 in the human GAPDH gene (41)). These
inserts were subcloned into the pGEM3zf+vector
(Stratagene). Restriction-digested DNA from these constructs was used
as a template to synthesize antisense cRNAs.
APP Synthesis
Primary Astrocytes--
GFAP-positive astrocytes (1 × 105 cells/well) and astrocytoma cells (70% confluent) were
measured for intracellular APP synthesis and ferritin synthesis after
stimulation with 0.5 ng/ml recombinant IL-1 Astrocytoma Cells--
U373MG astrocytoma cells (80% confluent)
were stimulated with IL-1 Immunoprecipitations--
In all labeling experiments,
antigen-antibody complex was collected with protein A-Sepharose beads
(Pierce) and the immunoprecipitates applied to 10-20% Tris-Tricine
gels (Novex) and fractionated by electrophoresis in a buffer containing
0.1 M Tris-Tricine buffer and 0.1% SDS, pH 8.3. The gels
were fixed with 25% methanol, 7% (v/v) acetic acid for 1 h,
incubated with a fluorographic reagent (Amplify; Amersham Pharmacia
Biotech) for 30 min, dried, and exposed to Kodak X-Omat film overnight
at RNA Purification and Northern Blot Hybridization
Equal numbers of cells were pelleted by centrifugation at 1,000 rpm and the pellets lysed using 1 ml of modified guanidinium/phenol reagent according to the manufacturer's instructions (Tri-reagent, MRC
Research Inc., Cincinnati, OH). The RNA pellet was resuspended in TES
buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 0.5%
SDS, pH 7.6), and the A260/280
nM was measured as an estimate of purity and
RNA concentration. A cesium chloride procedure for the purification of
RNA from DNA was followed for all experiments involving the use of
transfected DNA (36). Total RNA samples from both transfected and
untransfected cells were denatured in 50% formamide, 2.2 M formaldehyde, 20 mM MOPS, 5 mM sodium acetate,
0.5 mM EDTA, pH 7.4, at 60 °C for 10 min. RNA samples
were separated by electrophoresis on 1.5% agarose-formaldehyde gels,
blotted onto Hybond-N filters, and immobilized by UV cross-linking (2 min) and heating of filters to 80 °C for 1 h. Filters were
prehybridized overnight to reduce background hybridization signal and
then hybridized overnight in 50% formamide, 50 mg/ml denatured salmon
sperm DNA, 5 × SSC, 0.1% SDS, and 5 × Denhardt's
solution. After hybridization, filters were washed twice for 1 h
in 2 × SSC and 0.2% SDS at room temperature. Filters were washed
further through four changes of 0.5 × SSC and 0.1% SDS at
55 °C. In all experiments, equal loading of Northern gels was
verified by ethidium bromide staining of 18 S and 28 S rRNAs and
GAPDH hybridization as an internal standard (41). Specific RNAs were
detected either by hybridization of Northern blot filters with randomly
primed cDNA probes (35) or with copy RNA (cRNA) probes (37).
Antisense cDNA hybridization probes were labeled by random primed
labeling (specific activity = 2 × 108 cpm/µg),
and cRNA probes were transcribed in vitro with
T7 RNA polymerase (Promega, Madison, WI) in the presence of
[32P]CTP (3,000 Ci/mmol, NEN Life Science Products).
Transient Transfections and CAT Assays
Transient Transfections--
Astrocytoma cells (1 × 107 cells/10-cm2 plate) were transfected with
20 µg of DNA, each purified from the pSV2CAT or
pSV2(APP)CAT constructs. Inclusion of RSV2-GAL
(5 µg) in each transfection was used to standardize for differences
in the transfection efficiency of the pSV2CAT and
pSV2(APP)CAT constructs. Briefly, LipofectAMINE reagent
(Boehringer Mannheim) was added to DMEM (without serum) and incubated
for 45 min at room temperature. Plasmids (20 µg), in an equal volume
of DMEM, were incubated for 45 min at room temperature with the
LipofectAMINE reaction mixture. LipofectAMINE-DNA complex was then
applied for 4 h to U373MG astrocytoma cells grown to 60%
confluence. The cells were washed twice in DMEM (without serum), and
subsequently cells were incubated for 24-48 h with DMEM supplemented
with 10% fetal calf serum in the presence or absence of cytokine
before harvesting and CAT assay. Transfected cells were stimulated with
cytokine by the administration of 0.5 ng/ml IL-1 CAT Assays--
After harvesting, equal numbers of cells from
10-cm2 tissue culture plates were resuspended in 1 ml of
0.25 M Tris, pH 7.8, and subjected to three cycles of
freezing and thawing (liquid nitrogen, 37 °C) to lyse the cells.
Lysates were collected after centrifugation at 10,000 rpm for 5 min.
The protein concentration was measured using the manufacturer's
conditions (Bio-Rad assay). Lysates (20 µg of protein) were added to
50 µl of a CAT reaction mix containing 1 M Tris, pH 7.8, 20 µl of acetyl-CoA (3.5 mg/ml), and 5 µl of
14C-labeled chloramphenicol (25 µCi/ml). After incubation
for 1 h at 37 °C, reaction products were extracted with 1 ml of
ethyl acetate, and the samples were resolved by thin layer
chromatography as described previously (37). Marked areas on the TLC
plate were excised and quantified by scintillation counting
(Econofluor, NEN Life Science Products) using a Wallac 1409 counter
(Amersham Pharmacia Biotech). The average CAT activity from a number of separate transfection experiments (see Fig. 7B) includes CAT
activity as estimated by use of an assay directly counting the amount
of CAT reaction product partitioning into liquid scintillation fluid (37).
Galactosidase Assay--
Lysates were diluted at equal protein
concentration in a CAT lysis buffer (Promega). Extracts were then
incubated in the presence of ONPG color dye, and IL-1 Stimulates APP Gene Expression at the Translational Level in
Primary Human Astrocytes--
APP synthesis was measured in primary
human fetal brain astrocytes (95-100% GFAP-positive cells in culture
(Fig. 1)) after treatment with both
IL-1
Further immunoprecipitations from two additional time course
experiments (2, 6, and 16 h) showed that APP synthesis increased most sharply after 6 h of stimulus of primary astrocytes with the
IL-1
IL-1 also induced a 1.7-fold increase in the secretion of APP
(Protease-Nexin II or APP(s)) from primary human astrocytes as measured
by direct scintillation counting of labeled immunoprecipitates (Fig.
2D). Densitometry of autoradiographs from an additional experiment revealed that IL-1
Northern blot analysis was used to compare the possible action of
IL-1 IL-1 Enhances APP mRNA Translation in Astrocytoma
Cells--
The effect of IL-1
Maximal IL-1 induction of APP synthesis in U373MG cells was observed in
dose-response experiments using concentrations of IL-1
Additional time course experiments (2 h and 16 h) with astrocytoma
cells reflected closely the pattern of translational regulation of APP
gene expression by IL-1 observed in primary astrocytes (as shown in
Fig. 2C). In U373MG cells, IL-1 An IL-1-dependent Translational Enhancer in the APP
mRNA 5'-UTR--
A consistent feature of our cell culture labeling
experiments was that IL-1 and iron chelation with desferrioxamine
generated a similar profile for APP and ferritin synthesis (Fig. 2).
Previously, the 5'-UTRs of the L-ferritin and H-ferritin genes (+74 bp
to +142 bp from the L-ferritin gene cap site and +139 bp to +199 bp
from the H-ferritin gene cap site) have been shown to confer both
base-line and IL-1
Multiple transfection experiments with the pSV2(APP)CAT
construct showed that both IL-1 Enhanced Base-line Translation by the APP Gene 5'-UTR
mRNA--
Standardized transfections also revealed that base-line
CAT gene expression was increased when APP gene 5'-UTR sequences were inserted between the unique HindIII and StuI
sites in the 5'-UTR of the CAT gene in pSV2CAT (Fig.
8). This effect accounts for the
consistent difference in the base-line CAT gene expression derived from
pSV2(APP)CAT compared with pSV2CAT in
astrocytoma cells, as observed in Fig. 7C. In addition to
mediating an IL-1-dependent increase in translation, the
APP 5'-UTR conferred a consistent 3-4-fold increase in basal CAT
activity in pSV2(APP)CAT-transfected U373MG cells compared
with the parental pSV2CAT vector transfectants (Fig. 8,
upper panel). The amount of CAT gene expression was
standardized with the RSV2GAL plasmid (5 µg/transfection)
to account for differences in transfection efficiency. In a separate
study we have been examining the translational regulatory action of the
APP gene 5'-UTR in human neuroblastoma cells (SKN SH neuroblastoma
cells). In addition to basal translational regulation in
astrocyte-derived cells, multiple transfections of the
pSV2(APP)CAT construct confirmed that the APP mRNA
5'-UTR is also a basal translational regulatory element in
neuroblastoma cells (n = 6) (Fig. 8, lower
panel). Increased base-line translational efficiency conferred by
the APP mRNA 5'-UTR was measured at 3.6-fold in astrocytoma cells (4.2 ± 0.06-fold in neuroblastoma cells, n = 6).
This report provides the first evidence that IL-1 substantially
induces APP synthesis in primary human astrocytes and astrocytoma cells
by a mechanism of enhanced message translation. The translational efficiency of astrocytic APP mRNA was specifically and selectively enhanced by IL-1, while the translational efficiencies of the astrocytic mRNAs for Both cytokine isoforms, IL-1 We identified a novel IL-1-responsive and basal translational enhancer
in the 5'-UTR of the APP gene, consistent with computer alignment with
similar 5'-UTR sequences in the ferritin genes. Ferritin gene
expression has been well characterized and is known to be regulated at
the level of message translation in hepatoma cells (37). Transfection
studies with a hybrid APP/CAT mRNA construct confirmed that the
action of this sequence, mapping from + 55 to + 144 nucleotides from
the APP mRNA 5'-cap site, was sufficient to mediate the
translational regulation of APP mRNA by IL-1 in U373MG cells, as
measured by CAT reporter activity. In contrast, the steady-state levels
of transfected hybrid APP/CAT mRNA was unchanged, similar to
findings from parallel CAT reporter studies with the ferritin mRNA
acute box elements (36). The most straightforward interpretation of our
results is that IL-1 elevates APP mRNA translation through the
action of an IL-1-responsive stem-loop structure in APP mRNAs.
Computer alignment showed that sequences in the 5'-UTR of APP mRNAs
are homologous, but not identical, to the acute box sequence of
L-ferritin mRNA 5'-UTR which confers IL-1-dependent
translation specifically in hepatoma cells. The L-ferritin mRNA
sequence differs from the APP mRNA sequence, likely explaining the
lack of L-ferritin gene translational regulation by IL-1 in astrocytoma
cells. The APP mRNA 5'-UTR sequence is highly GC-rich (80%) and is
predicted to fold into a single stable RNA stem-loop structure
( There are striking overlaps in the regulation of the APP gene and the
L- and H-ferritin genes, each of which encodes the subunits for the
central iron storage protein shown in Fig. 2. First, ferritin is an
acute phase reactant, and increased ferritin synthesis and concomitant
iron sequestration are consistent with the anemia associated with the
inflammation of chronic diseases (35, 45). Ferritin gene expression is
regulated at the translational level in hepatoma cells (35). Here, the
APP gene is also shown to be an acute phase reactant, regulated at the
translational levels in astrocytes. Second, APP mRNA 5'-UTR
sequences confer significant IL-1-dependent and basal
translational enhancement to activate CAT reporter gene expression in
pSV2(APP)CAT- transfected astrocytoma cells. Similar
hepatic translational regulation is conferred by the IL-1-responsive
acute box RNA sequences in the L-and H-ferritin mRNA 5'-UTRs (37).
Like the ferritin genes, the APP gene 5'-UTR maintains efficient
translation of APP in both astrocyte-derived and neuroblastoma cells.
The L- and H-ferritin gene 5'-UTRs are organized into two regulatory
sequences: an iron-responsive element at the 5'-cap site, which is
responsive to iron (48), oxidative stress (49), phorbol esters (50) and
thyroid hormone receptor (51); and a downstream acute box sequence that
is both a base-line and an IL-1-dependent translational
regulatory element that works in an iron-dependent fashion
(Fig. 4B) (35). The presence of similar translational
regulatory sequences in the 5'-UTRs of both APP mRNA and ferritin
mRNA is consistent with the known role for metal binding, including
copper and likely iron, as a part of the normal function of APP in
cells (52). APP mRNA 3'-UTR sequences regulate APP gene expression
by modulating message stability in human peripheral blood mononuclear
cells (53) and regulating message translation in Chinese hamster ovary
cells (54). In addition, other studies have indirectly shown
translational regulation of APP gene expression. The steady-state
levels of APP protein in the rat cerebral cortex, meninges, and in
primary astroglial, microglial, and neuronal cultures have been
reported not to reflect APP mRNA levels (55). Furthermore, the
relative levels of APP-695 (KPI Several reports suggest a direct connection between increased APP
levels and the development of AD pathogenesis. This increase might be
linked to inflammatory mechanisms. 1) Down's syndrome brains and
trisomy 16 mice show increased APP levels beyond the 0.5-fold increase
that would be expected by gene dosage (12). 2) Overexpression of APP in
transgenic mice is necessary, even in the presence of FAD mutations,
for sufficient A Overexpression of IL-1 by centrally located microglia has been shown to
be associated even with early forms of amyloid plaques, the
non-neuritic diffuse plaques, as well as being increased strikingly during plaque development (17, 18, 62). IL-1 has been suggested as a
driving force for amyloid plaque maturation (62), perhaps mediated by
signaling by the cytokine to astrocytes surrounding the plaque
structures and subsequent induction of APP and ACT protein synthesis
(64). However, published findings are not consistent about whether
sporadic AD is associated with increased APP gene transcription, with
reports of both increased (65) as well as decreased levels of APP
mRNA (66). Our data provide substantial in vitro
evidence for increased APP synthesis by enhanced message translation in
response to IL-1 in astrocytes. IL-1 enhancement of APP synthesis in
astrocytes suggests that the accumulation of A-peptide that accumulates
in amyloid plaques, and APP gene mutations can cause early onset AD.
Inflammation is also associated with AD as exemplified by increased
expression of interleukin-1 (IL-1) in microglia in affected areas of
the AD brain. Here we demonstrate that IL-1
and IL-1
increase APP
synthesis by up to 6-fold in primary human astrocytes and by 15-fold in
human astrocytoma cells without changing the steady-state levels of APP
mRNA. A 90-nucleotide sequence in the APP gene 5'-untranslated
region (5'-UTR) conferred translational regulation by IL-1
and
IL-1
to a chloramphenicol acetyltransferase (CAT) reporter gene.
Steady-state levels of transfected APP(5'-UTR)/CAT mRNAs were
unchanged, whereas both base-line and IL-1-dependent CAT
protein synthesis were increased. This APP mRNA translational
enhancer maps from +55 to +144 nucleotides from the 5'-cap site and is
homologous to related translational control elements in the 5'-UTR of
the light and and heavy ferritin genes. Enhanced translation of APP
mRNA provides a mechanism by which IL-1 influences the
pathogenesis of AD.
INTRODUCTION
Top
Abstract
Introduction
References
-amyloid (A
) peptides, which then polymerize and deposit as the amyloid filaments as one of the pathological hallmarks of Alzheimer's disease (AD) and Down syndrome (3-7). The
regulation of APP gene expression as a pathogenic factor for AD has
received considerable attention. Several putative physiological activators of APP gene transcription have been defined (8-10). Overexpression of APP evidently can cause AD because all individuals with Down syndrome have an extra copy of the APP gene on chromosome 21 and invariably develop AD pathology by the age of 40-50 years (11,
12).
cleavage, secretion, and deposition (5),
accumulating evidence has revealed that local inflammation at the site
of developing extracellular plaques in the brain is important to AD
pathogenesis (13-15). For example,
1-antichymotrypsin (ACT) is present in amyloid plaques, and its production by adjacent astrocytes suggests the occurrence of an inherent inflammation in the
AD brain, similar to the hepatic acute phase response (16, 17). Other
significant markers, such as interleukin-1 (IL-1)-positive microglia
and complement protein, confirm the presence of local inflammatory
events during AD progression (18, 19). Epidemiological studies
identifying traumatic head injury as a risk factor for AD strengthen
the hypothesis that inflammatory mechanisms contribute to the disease
pathogenesis (20). Hippocampal lesion has been shown to increase APP
immunoreactivity in neighboring astrocytes (21). In vitro
studies, and recently an in vivo study, have shown that
certain proteins, e.g. ACT and apolipoprotein E (apoE), which are expressed during traumatic injury and inflammation of the
brain parenchyma, might regulate the polymerization of A
peptides
into amyloid filaments (22-27).
-sheet protein structure (17,
18, 29). IL-1 action is mediated by two separate cytokines, IL-1
and
IL-1
, which share low sequence homology (30%) and are encoded by
two separate genes derived from a common ancestor gene (30). IL-1
and IL-1
target the same signaling receptor and exert overlapping
proinflammatory effects, although the processing and site of action of
these cytokines differ (28).
EXPERIMENTAL PROCEDURES
(Genzyme), 0.5 ng/ml
IL-1
(Genzyme), 10 µM ferrotransferrin (Boehringer
Mannheim), 100 µM desferrioxamine (Ciba Geigy), or left
untreated as controls. Cell numbers from individual wells were counted
to ensure that 1 × 105 cells were present in each
well at the beginning of each labeling experiment. Astrocytes were
preincubated for 15 min in methionine-free medium (RPMI 1640; Life
Technologies, Inc.) and pulse labeled for 30 min with 300 µCi/ml
[35S]methionine. Each microtiter plate was washed twice
in cold PBS at 4 °C before lysis of astrocytes in 25 µl of STEN
buffer (0.2% Nonidet P-40, 2 mM EDTA, 50 mM
Tris, pH 7.6) using a sterile glass rod.
L-Phenylmethylsulfonyl fluoride (100 µg/ml) and leupeptin (2 µg/ml) were added to the STEN lysis buffer to prevent proteolysis. Half of the pooled lysates (i.e. 300-µl total volume from
each row of 12 wells) were immunoprecipitated with a 1:500 dilution of
a COOH-terminal directed APP antibody (C-8, 1:500 dilution, against
amino acids 676-695 of APP-695; gift from D. Selkoe (42)). The other
half of the lysates were immunoprecipitated with human ferritin
antiserum (1:500 dilution, Boehringer Mannheim). Labeling of secreted
APP (APP(s); Protease-Nexin-2) was measured after 2 h of pulse
labeling astrocytes with 300 µCi/ml
[35S]methionine, after which 2 ml of medium was
precleared by centrifugation in Eppendorf tubes (10,000 rpm for 10 min), and the supernatant was immunoprecipitated (1:1,500 dilution,
against amino acids 595-611 of APP, R1736, gift from D. Selkoe). ApoE
was immunoprecipitated from both the lysate and the medium using a
1:200 dilution of a polyclonal antiserum (Chemicon).
and IL-1
at concentrations between 0.05 and 5 ng/ml. After stimulation, equal numbers of cells were labeled for
30 min with 300 µCi/ml [35S]methionine in DMEM lacking
methionine, washed twice with PBS, and the cell pellets were lysed in
200 µl of cold STEN buffer containing 100 µg/ml
phenylmethylsulfonyl fluoride. There were 2 × 107
cells/10-cm2 plate at the beginning of each pulse labeling
(see Fig. 4), and 2 × 106 cells were present in each
well of six-well plates in the experiment described in Fig. 5. The
protein concentrations of each lysate were measured to confirm that
equal numbers of cells had been pulse labeled. Total protein synthesis
was measured by the amount of [35S]methionine
incorporation using 10% trichloroacetic acid and 2% casamino acids to
precipitate labeled proteins present in each lysate (4 °C).
Triplicate samples (10 µl) were assayed after hydrolysis of
methionine charged tRNAs with 250 µl of 1 M NaOH and
1.5% H2O2 at 65 °C for 30 min. APP and
ferritin were immunoprecipitated from U373MG astrocytoma lysates by
adding 2 µl of anti-APP antibody (C-8 antibody) (42) as described for
primary astrocytes.
80 °C.
, 0.5 ng/ml IL-1
,
or left as unstimulated controls. After defined times of cytokine
stimulus (20 h in Fig. 7A), cells were harvested in PBS and
assayed immediately for CAT activity or CAT mRNA levels (Northern blotting).
-galactosidase
enzyme activity was determined according to the manufacturer's conditions.
RESULTS
and IL-1
(0.5 ng/ml) for 16 h. Fig. 2A (left panel)
shows the results of a representative experiment in which 30 min of
metabolic labeling with [35S]methionine was followed by
APP immunoprecipitation and gel electrophoresis (n = 3). In the labeling shown, a 4-fold increase in the synthesis of
intracellular APP was observed in response to a 16-h stimulus with
IL-1
and a 3-fold increase in response to a 16-h stimulus with
IL-1
(maximal 5.9-fold induction of APP synthesis by IL-1
for
primary astrocytes (Table I)). The C-8
antibody immunoprecipitated two proteins, of 110 and 130 kDa,
corresponding to the mature and immature glycosylated APP holoprotein
(43). We used the rate of synthesis of other proteins as positive and
negative controls to confirm that IL-1
and IL-1
specifically
increase APP mRNA translation. IL-1
and IL-1
induced a 4-fold
increase of H-ferritin synthesis in primary astrocytes (Fig.
2A, right panel), whereas the rate of astrocytic
L-ferritin synthesis was unchanged in response to both IL-1
and
IL-1
(Fig. 2A, right panel). The level of apoE protein synthesis was also determined and found to be unchanged after
IL-1
or IL-1
stimulus, thus serving as an additional internal loading control to the L-ferritin for measuring specific increases in
the relative rate of APP synthesis in astrocytes (n = 3) (Fig. 2B). IL-1
increased the rate of total protein
synthesis by 60% (maximal increase) in primary astrocyte cultures, as
measured by trichloroacetic acid precipitation of labeled proteins from triplicate lysates (Table I). The action of iron chelation with 100 µM desferrioxamine generated a similar reduction of
synthesis of both L-ferritin and H-ferritin in addition to APP (Fig.
2A). Iron as ferrotransferrin had no effect on the rate of
either APP or ferritin protein synthesis in primary astrocyte cultures.
The apparent coordinate regulation of the APP and ferritin genes is discussed below in terms of the presence of homologous translational regulatory sequences in their 5'-UTRs (see Fig. 6).
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Fig. 1.
GFAP-positive astrocytes. Human primary
astrocytes displayed immunofluorescent GFAP staining using a monclonal
antibody (clone G-A-5) and a Cy3-labeled goat anti-mouse secondary
antibody (panel A) but very faint background staining
following omission of primary antibody (panel B).
Panels C and D, 4,6-diamidino-2-phenylindole
staining of the same cells.
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Fig. 2.
IL-1 and
IL-1
increased APP and H-ferritin subunit
protein synthesis and APP(s) secretion in human primary astrocyte
cells, whereas protein synthesis for apoE,
-actin, and L-ferritin subunit remained
unchanged. Panel A. Left group, cells were
treated for 16 h, pulse labeled for 30 min, and derivative lysates
were immunoprecipitated with an antibody specific to the COOH terminus
of APP (C-8). From left, first lane, unstimulated
cells; second lane, 0.5 ng/ml IL-1
;
third lane, 0.5 ng/ml IL-1
; fourth
lane, 10 µM Fe2Tf;
fifth lane, 100 µM desferrioxamine
(Df). Right group, these lysates were
immunoprecipitated with a ferritin antibody. Panel B, the
culture medium was treated and pulse labeled as in panel A
and immunoprecipitated with apoE antibody. Panel C, cells
and matched controls were treated with 0.5 ng/ml IL-1
for 2, 6, and
16 h, pulsed labeled for 30 min, and derivative lysates were
immunoprecipitated with C-8 antibody and a
-actin antibody.
Panel D, culture medium from cells were treated for 16 h, pulse labeled for 120 min, and immunoprecipitated with an
NH2-terminal APP antibody (R1736). First
lane, unstimulated; second lane, 0.5 ng/ml IL-1
; third lane, 0.5 ng/ml
IL-1
.
Time course of IL-1-dependent increase of APP synthesis
for comparison with total protein synthesis in primary human astrocytes
(2 h) = 113 cpm ± 21 cpm;
IL-1
(2 h) = 89 cpm ± 12.5 cpm. Second
row, +IL-1
(6 h) = 272 cpm ± 17 cpm;
IL-1
(6 h) = 167 cpm ± 33 cpm. Third row, +IL-1
(16 h) = 179 cpm ± 62 cpm;
IL-1
(16 h) = 175 cpm ± 39 cpm.
(Fig. 2C). Our data showed that a 6-h stimulation
with IL-1
induced a maximal 5.9-fold increase in APP synthesis
(Table I). A 3.8-fold increase of APP synthesis was observed after a 16-h IL-1
stimulation.
-Actin protein synthesis was increased by
70% (maximum) relative to unstimulated astrocytes after IL-1
stimulation (Fig. 2C). 2 h of stimulation with IL-1
increased APP synthesis by only 40% relative to untreated cells,
whereas
-actin synthesis changed by 0.65-fold under the same
conditions. We concluded that APP synthesis peaked at a time point
between 6 and 16 h after the IL-1
stimulation of primary
astrocytes. Total protein synthesis increased by 60% in response to
IL-1
, as measured by trichloroacetic acid precipitation of labeled
proteins from each lysate (Table I). Evidently, IL-1
increased APP
synthesis by a margin 3.7-fold greater than the induction of total
protein synthesis in primary astrocytes.
induced APP(s) synthesis by 2.6-fold. In these experiments the medium was collected after a 2-h pulse labeling with [35S]methionine and immunoprecipitated
using an NH2-terminal antibody (against amino acids
595-611 of the APP). IL-1
also enhanced secretion of APP(s) into
the medium, causing a smaller 25% accumulation of APP(s). Thus, the
levels of both cell-associated and secreted APP (APP(s)) were increased
by exposure of primary astrocytes to IL-1.
to increase the steady-state levels of APP mRNA (3 kb) in
primary astrocytes over the same time course for IL-1
induction of
APP synthesis (Fig. 3). We measured no
increase in APP mRNA levels in primary astrocytes after 2, 6, or
16 h of IL-1
stimulation (n = 4). As a positive
control for effective IL-1
signal transduction, exposure of
astrocytes to the cytokine caused a pronounced increase in the
steady-state levels of ACT mRNA (1.5 kb of ACT mRNA), as has
been demonstrated previously (>10-fold; n = 4) (17).
As Northern blot loading controls, steady-state levels of astrocytic
GAPDH mRNA and 28 S rRNA were unchanged after IL-1
stimulation
(Fig. 3). Our data also demonstrated that not only IL-1
stimulation,
but also IL-1
stimulation leaves APP mRNA levels unchanged in
primary astrocytes (n = 4) (data not shown). Therefore,
astrocytic APP gene expression by IL-1 is mediated by translational
mechanisms.
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Fig. 3.
Time course showing that APP mRNA
expression was unchanged in response to 0.5 ng/ml
IL-1 stimulation of human primary
astrocytes. Sequential Northern blot hybridizations were performed
with cRNA probes against ACT mRNA (upper), APP mRNA
(middle), and GAPDH mRNA (lower). The
molecular weights of each RNA are: APP mRNA, 3,500; ACT mRNA,
1,500; and GAPDH mRNA, 1,000.
and IL-1
stimulation on APP and
ferritin protein synthesis was also measured in human astrocytoma
(U373MG) cells. Fig. 4A shows
a representative experiment in which APP synthesis was, respectively,
increased 3- and 5-fold after 16 h of IL-1
or IL-1
stimulation. As for primary astrocytes, protein synthesis of
L-ferritin subunit was unchanged, thereby serving as an
internal control for the specificity of induced APP synthesis (Fig.
4B). Both IL-1
and IL-1
increased the rate of
H-ferritin synthesis by 4-5-fold in U373MG astrocytoma cells (Fig.
4B). At the same time, IL-1
and IL-1
stimulation for
16 h caused a marked increase in the protein synthesis of mature
and immature (glycosylated form) of ACT, as measured by a 30-min pulse
labeling and immunoprecipitation (Fig. 4C). Northern blot
experiments (Fig. 4D) showed consistently that IL-1
and
IL-1
stimulation did not increase the steady-state levels of APP
mRNA when standardized for RNA loading by GAPDH mRNA expression
(n = 4). As in primary astrocytes, IL-1 induction of
APP synthesis in U373MG cells did not reflect the steady-state levels
of APP mRNA, clearly indicating that IL-1 regulates APP gene
expression at the translational level in astrocytoma cells. To confirm
that IL-1
and IL-1
signaling invokes a well characterized transcriptional activation in astrocytoma cells (33), we demonstrated that both cytokines specifically increased the steady-state levels of
ACT mRNA (Fig. 4D). Therefore, IL-1 signal transduction
pathways in astrocytoma cells operate to increase ACT gene expression
(ACT protein synthesis) at the level of ACT gene transcription but enhance APP gene expression at a level of enhanced APP mRNA
translation.
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Fig. 4.
IL-1 and
IL-1
increase APP synthesis but have no effect
on APP mRNA levels in U373MG astrocytoma cells. Panel
A, cells were treated for 16 h, pulse labeled for 30 min, and
derivative lysates were immunoprecipitated with COOH-terminal directed
APP antibody (C-8). From left, first
lane, unstimulated cells; second lane,
0.5 ng/ml IL-1
; third lane, 0.5 ng/ml IL-1
.
Panel B, the same lysates immunoprecipitated with a ferritin
antibody. Panel C, ACT protein synthesis after 16 h of
IL-1 stimulation of astrocytoma cells. First lane,
unstimulated cells; second lane, 0.5 ng/ml IL-1
;
third lane, 0.5 ng/ml IL-1
. Panel D, Northern
blot hybridizations with cRNA probes complementary to APP mRNA,
GAPDH mRNA, and ACT mRNA sequences. First lane,
unstimulated cells; second lane, 0.5 ng/ml IL-1
;
third lane, unstimulated cells; fourth lane, 0.5 ng/ml IL-1
. U373MG cells were stimulated with IL-1 for 16 h.
and IL-1
varying by 2 orders of magnitude (0.05, 0.5, and 5 ng/ml IL-1
and
IL-1
). For example, a 15-fold increase in APP synthesis was
quantitated from immunoprecipitations of astrocytoma cells stimulated
with 5 ng/ml IL-1
for 16 h (Fig.
5A). Two dose-response experiments showed that all three concentrations of IL-1
and IL-1
generated an average 12-fold increase of APP synthesis in U373MG cells
(Table II, n = 2). In the
same experiments, IL-1 elevated total protein synthesis by only
2-3-fold in U373MG cells, as measured by [35S]methionine
incorporation into trichloroacetic acid-insoluble counts (Table II). To
confirm translational regulation, IL-1 stimulation did not increase APP
mRNA levels over the same 0.05-5 ng/ml concentration range of
IL-1
and IL-1
used to generate a 12-fold increase APP synthesis
(Fig. 5B).
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Fig. 5.
APP synthesis in U373MG cells is induced in a
dose- and time-dependent fashion by IL-1
and IL-1
stimulation in the absence of
change in the steady-state levels of APP mRNA. Panel
A, dose-response experiment measuring APP synthesis. Cells were
treated and lysates harvested and immunoprecipitated with COOH-terminal
directed APP antibody (C-8) as described in Fig. 4. Left
group: from left, first lane, unstimulated
cells; second lane, 0.05 ng/ml IL-1
;
third lane, 0.5 ng/ml IL-1
; fourth
lane, 5 ng/ml IL-1
. Right group: from
left, first lane, unstimulated cells;
second lane, 0.05 ng/ml IL-1
; third lane, 0.5 ng/ml IL-1
; fourth lane, 5 ng/ml IL-1
. Panel
B, dose-response experiment measuring the steady-state levels of
APP mRNA. Northern blot hybridization was performed with cDNA
probe complementary to APP mRNA sequences. Lane 1,
unstimulated cells; lane 2, 0.05 ng/ml IL-1
; lane
3, 5 ng/ml IL-1
; lane 4, 0.05 ng/ml IL-1
;
lane 5, 5 ng/ml IL-1
. Panel C, left
group, time course experiment measuring APP synthesis. Cells were
treated, and lysates were harvested and immunoprecipitated with
COOH-terminal directed APP antibody (C-8) as described in Fig.
4A. From left, first lane,
unstimulated cells; second lane, 0.5 ng/ml IL-1
stimulation for 2 h; third lane, 0.5 ng/ml IL-1
stimulation for 6 h. Right group, time course
experiment measuring the steady-state levels of APP mRNA. Northern
blot hybridization was performed using a labeled cDNA probe against
APP mRNA. First lane, in vitro translated APP
mRNA marker (3.5 kb); second lane, unstimulated cells;
third and fourth lanes, 0.5 ng/ml IL-1
, 2 and
6 h, respectively.
Dose-responsive increase of APP synthesis for comparison with total
protein synthesis in U373MG cells (16-h stimulation with IL-1 and
IL-1
)
for 16 h). Total protein synthesis was measured as trichloroacetic
acid-precipitable counts (cpm × 105 (n = 6)). The relative increase in protein synthesis was calculated after
quantitation of trichloroacetic acid-insoluble counts as follows: 1, no
stimulation = 247 cpm ± 29 cpm; 2, IL-1
(0.05 ng/ml) = 524 cpm ± 79 cpm; 3, IL-1
(0.5 ng/ml) = 597 cpm ± 76 cpm; 4, IL-1
(5 ng/ml) = 519 cpm ± 75 cpm; 5, no stimulus = 190 cpm ± 75 cpm; 6, IL-1
(0.05 ng/ml) = 560 cpm ± 18 cpm; 7, IL-1
(0.5 ng/ml) = 602 cpm ± 18 cpm; 8, IL-1
(5 ng/ml) = 412 cpm ± 29 cpm.
(0.5 ng/ml) increased APP
synthesis starting 6 h after cytokine stimulation (Fig.
5C, left panel). APP levels were unchanged after
2 h stimulation. By contrast, the steady-state levels of APP
mRNA were unchanged at all time points after IL-1
stimulation
(Fig. 5C, right panel). Multiple
immunoprecipitation experiments demonstrated that IL-1
and IL-1
each generated an overall average 5-fold and 9-fold induction of APP
synthesis in astrocytoma cells (n = 7). These data
confirm that 1) IL-1 regulates APP synthesis at the level of message
translation in astrocytoma cells; that 2) IL-1-dependent translational regulation of astrocytoma APP mRNA begins after 6 h of cytokine stimulation, reflecting the induction profile of
APP mRNA translation in primary astrocytes; and that 3) IL-1 induction of APP synthesis is 2-5-fold greater than the induction of
total protein synthesis in astrocytoma cells.
-dependent translation to a CAT
reporter gene transfected in human hepatoma cells (37). Therefore we aligned L- and H-ferritin gene 5'-UTR sequences with the APP gene. Fig.
6 shows the presence of an unexpectedly
high 51% sequence homology sequence alignment between the L-ferritin
and APP mRNA 5'-UTRs (Gap program, Genetics Software from
University of Wisconsin, Madison). Because the APP mRNA 5'-UTR
contained a significant sequence homology to IL-1-responsive 5'-UTR
translational regulatory sequences in both L- and H-ferritin mRNAs
(+85 bp to + 146 bp from the 5'-cap site of the APP gene; Fig. 6), we
tested whether these APP mRNA 5'-UTR sequences could confer
IL-1-dependent translation enhancement. A 90-nucleotide DNA
fragment, encoding sequences from positions + 55 to +144 between the
SmaI to NruI sites of the APP gene 5'-UTR, was
inserted immediately upstream of a hybrid CAT reporter gene. The
resulting reporter construct was designated as pSV2(APP)CAT
because it was a derivative of the pSV2CAT expression vector.
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Fig. 6.
Hybrid CAT constructs expressing the APP
mRNA 5'-UTR. Upper panel, the
pSV2(APP)CAT construct was prepared by inserting a 90-bp
SmaI-NruI fragment of the APP gene 5'-UTR
immediately upstream of the CAT mRNA start codon. Computer
alignment between the 5'-UTR of the APP gene and the IL-1-responsive
5'-UTR translational enhancer in L-ferritin mRNA revealed 51%
sequence homology (bold lettering). The acute box homology
motif is underlined (36). Lower panel, comparison
of the predicted folding of RNA by computer analysis of the APP
mRNA 5'-UTR and the IL-1-responsive L-ferritin mRNA
5'-UTR translational enhancer (37). The APP mRNA acute box sequence
is predicted to fold into a stable RNA stem-loop structure (47). This
RNA stem-loop is identical to a larger stem-loop folded from the
complete APP mRNA 5'-UTR ( G =
54 kCal/mol).
The corresponding L-ferritin mRNA 5'-UTR stem-loop, specific to the
acute box consensus, folds into a less stable RNA structure
(
G =
16 kCal/mol).
and IL-1
, respectively, conferred an average 3-fold and 4-fold translational enhancement to CAT reporter
mRNAs in U373MG astrocytoma cells (n = 6) (Fig.
7B). Panel A shows
a duplicate experiment where IL-1
increased CAT gene expression by
6-fold, and IL-1
increased CAT gene expression by 9-fold in
pSV2(APP)CAT-transfected astrocytoma cells. This induction
was sufficient to account for a significant proportion of the
IL-1-enhanced APP synthesis in astrocytoma cells. As a negative
control, IL-1
stimulation of pSV2CAT-transfected
astrocytoma cells did not increase CAT activity, confirming that the
APP mRNA 5'-UTR is a translational regulatory element (36). In the
representative experiment shown in Fig. 7C no sequences in
the parental vector pSV2CAT conferred
IL-1-dependent translational regulation. At the same time
CAT activity was enhanced 3-fold in pSV2(APP)CAT transfectants. Northern blot analysis confirmed that a 16-h exposure to
both IL-1
and IL-1
(0.5 ng/ml) did not significantly change the
steady-state levels of the transfected APP/CAT hybrid mRNA in
pSV2(APP)CAT-transfected astrocytoma cells (Fig.
7D). Purified RNA from either pSV2(APP)CAT or
pSV2CAT (negative control) transfectants was hybridized to
labeled antisense RNA sequences homologous to the 5'-end of the CAT
gene. The pSV2CAT transfectants expressed a
1,527-nucleotide CAT mRNA as expected (Fig. 7D,
lanes 1 and 2, shows two separate loadings, 10 and 2 µg, respectively). The pSV2(APP)CAT-transfected
cells expressed a closely migrating APP/CAT mRNA (1,617 nucleotides), larger by the presence of the 90-nucleotide insert coding
for the APP gene 5'-UTR, but also transcribed another larger (1,640 nucleotides) APP/CAT transcript (Fig. 7D, lane
3). This APP/CAT mRNA was likely the result of using a second
poly(A) addition site downstream from the CAT gene stop codon in
pSV2CAT. IL-1
and IL-1
stimulated the reappearance of
only the single 1,617-nucleotide APP/CAT mRNA transcript using the
upstream poly(A) addition site. Densitometric quantitation of
autoradiographs showed that IL-1
or IL-1
only modestly (30%)
increased the total quantity of APP/CAT mRNA transcribed in
pSV2(APP)CAT transfectants relative to standardizing GAPDH
mRNA levels (Fig. 7D, lanes 3-5). Slot-blot analysis has convincingly confirmed that neither IL-1
nor IL-1
altered the steady-state levels of APP/CAT mRNA in
pSV2(APP)CAT-transfected astrocytoma (U373MG) cells (data
not shown). These measurements confirm previous reports characterizing
IL-1-dependent translational regulation mediated by
equivalent L- and H-ferritin 5'-UTR acute box sequences (36,
37).
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Fig. 7.
A 90-nucleotide SmaI to
NruI sequence element from the 5'-UTR of APP mRNA
confers IL-1-dependent CAT gene expression in U373MG
cells. IL-1 did not change the steady-state levels of APP/CAT
mRNA, and IL-1 did not change CAT gene expression in
pSV2CAT transfected astrocytoma cells. Panel A,
representative duplicate assay for CAT activity in lysates of
pSV2(APP)CAT-transfected U373MG astrocytoma cells. From
left, first and second lanes,
unstimulated; third and fourth lanes,
IL-1 -stimulated; fifth and sixth lanes,
IL-1
-stimulated for 16 h. Panel B, average fold CAT
activity conferred by the APP mRNA 5'-UTR in response to IL-1
and IL-1
stimulation of astrocytoma cells from multiple separate
transfections (mean ± S.E.; n = 6). Panel
C, left group, representative experiment showing CAT
activity in lysates of U373MG astrocytoma cells transfected with the
pSV2CAT parental vector and treated for 16 h
(n = 6). First lane, unstimulated cells;
second lane, 0.5 ng/ml IL-1
. Right group,
astrocytoma cells transfected with the pSV2(APP)CAT vector
and treated for 16 h. First lane, unstimulated cells;
second lane, 0.5 ng/ml IL-1
. The lysates were
normalized for transfection efficiency using 5 µg of a reference
RSV2GAL plasmid. Panel D, Northern blot
hybridizations of RNA purified from pSV2CAT and
pSV2(APP)CAT transfected astrocytoma cells (control and IL-1-stimulated) with
a labeled cRNA probe complementary to the 5'-end coding sequences and
5'-UTR of the CAT gene from the pSBCAT subclone (36). Lanes
1 and 2, 10 and 2 µg of RNA from astrocytoma cells
transfected with the parental vector (pSV2CAT); lanes
3-5, 10 µg of RNA from astrocytoma cells transfected with the
pSV2(APP)CAT vector and treated for 16 h as follows:
lane 3, unstimulated cells; lane 4, 0.5 ng/ml
IL-1
; lane 5, 0.5 ng/ml IL-1
. The Northern blot shown
was standardized for loading by use of a GAPDH gene probe. The ratio of
IL-1 induction of APP/CAT to GAPDH mRNA established that IL-1 only
increased the overall expression of transfected APP/CAT mRNA by a
30% margin.
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Fig. 8.
Top histogram, representative
transfection experiment showing that the APP gene 5'-UTR sequences
conferred a 3.8-fold increased basal CAT gene expression in U373MG
cells transfected with pSV2(APP)CAT compared with the
parental pSV2CAT vector (ratio of 1.7 to 6.4% acetylation
(n = 2). Lower histogram, quantitation of
multiple transfection experiments showing that the APP gene 5'-UTR
sequences conferred 4.3-fold increased basal CAT gene expression in
SKN-SH neuroblastoma cells transfected with pSV2(APP)CAT
compared with the parental pSV2CAT vector
(n = 6). Differences in transfection efficiency were
normalized using a reference RSV2GAL plasmid (mean ± S.E. n = 6).
DISCUSSION
-actin, L-ferritin, and apoE were
unaffected (H-ferritin synthesis is increased in astrocytes (Figs.
2A and 4B)). Induced APP synthesis was not
observable after 2 h, but required 6 h of IL-1
stimulation
in both primary astrocytes and in U373MG astrocytoma cells. IL-1 was
shown to increase total astrocytoma protein synthesis by 2-3-fold,
similar to insulin signaling of protein synthesis in HEK293 cells (44).
The cytokine specifically induced a more substantial level of APP
synthesis (Tables I and II). A similar time course of increased L- and H-ferritin mRNA translation during inflammation has been
demonstrated in rat liver cells (45). The expression ratio of APP
isoforms (APP-695:APP-751:APP-770) in astrocytes is 1:4:2
(i.e. the Kunitz-containing APP isoforms are the major APP
isoforms in this cell type as reported previously), whereas APP-695
predominates in neuronal cells (2, 42, 43).
and IL-1
, increased APP synthesis,
although IL-1
enhanced the secretion of APP(s) to a greater extent
than IL-1
. Differences in the magnitude of cytokine-stimulated secretion of [35S]methionine-labeled APP(s) and nascent
APP are likely the result of additional actions by IL-1 to alter the
APP processing (1, 46). It has been shown previously that the effect of
IL-1
on APP processing in human umbilical vein endothelial cells is
mediated by the IL-1 receptor (46). The two cytokines are known to
differ in a number of biological responses that they illicit (28). IL-1
is a cell-associated cytokine expressed as a fully active 31-kDa precursor protein (pro-L-1
) that is cleaved into a mature 17-Da IL-1
product. In contrast, IL-1
operates at the systemic level, where only the cleaved 17-Da cytokine is active. Additionally, only IL-1
preferentially binds to the IL-1 receptor II, perhaps also
modifying signal transduction though the IL-1 receptor I (28). IL-1
does not bind at a high affinity to IL-1 receptor II.
G =
54 kCal/mol in Fig. 6, lower)
(47).
) and APP-751 (KPI+) mRNA and
their proteins have been found to be discordant in human brain. Each
transcript was approximately equally abundant, whereas KPI+ proteins
predominated (>82%) and at elevated levels in the Alzheimer's brain
(56, 57).
peptide production to lead to development of
amyloid filament deposits and an Alzheimer's-like pathology (59, 60).
Furthermore, APP synthesis correlates with A
peptide production
in vivo (61). 3) Traumatic brain injury, a known risk factor
for AD, increases IL-1 as well as APP immunoreactivity in rat brain
(20, 32). 4) IL-1 injected into the rat cerebral cortex increases the
steady-state levels of APP protein at the site of the lesion (62), and
primary astrocytes have been shown to be a source of secreted A
peptides (63).
peptides into plaques
during AD may be accelerated by a pattern of local protein synthesis in
glial cells. This model of elevated local APP synthesis by a
cytokine-mediated mechanism is consistent with increasing experimental
epidemiological evidence linking the use of nonsteroidal
anti-inflammatory drugs to the risk for AD pathology (58, 67).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. D. Selkoe for helpful discussions and the use of the C-8 antiserum specific to the carboxyl-terminal of APP and the anti-APP(s) antibody (R1736). We also are grateful to Dr. K. Bridges, Dr. F. Bunn, and R. Handin of the Hematology Division, Department of Medicine, Brigham and Women's Hospital for their support. J. T. R. also acknowledges Dr. John Growdon, Alzheimer's Disease Research Center Massachusetts, and Dr. L. Thal of the Neurosciences and Education Research Foundation at the University of California, San Diego.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant AR29I32717, American Federation for Aging Research Grant 96022, Alzheimer's Association Grant PRG-94-146, a pilot grant from the Alzheimer's Disease Research Center, Boston (to J. T. R.), and a National Institutes of Health Grant AG09665 (to H. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: 604 LMRC, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-278-0370; Fax: 617-739-3324; E-mail: Rogers{at}calvin.bwh.harvard.edu.
¶ Recipient of Postdoctoral Fellowship 6872302 from the NCI, National Institutes of Health.
Present address: Dept. of Genetics, Harvard Medical School,
Boston, MA 02115.
§§ Present address: Schepens Eye Institute, 20 Staniford St., Boston, MA 02114.
¶¶ Recipient of postdoctoral fellowships from the Swedish Foundation for International Cooperation in Research and Higher Education and Riksbankens Jubileimsfond (following a donation from Erik Rönnberg).
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ABBREVIATIONS |
---|
The abbreviations used are:
APP, amyloid
precursor protein;
A,
-amyloid;
AD, Alzheimer's disease;
ACT,
1-antichymotrypsin;
IL-1, interleukin-1;
apoE, apolipoprotein E;
UTR, untranslated region;
L, light;
H, heavy;
DMEM, Dulbecco's modified Eagle's medium;
PBS, phosphate-buffered saline;
GFAP, glial fibrillary acidic protein;
bp, base pair(s);
kb, kilobase(s);
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
APP(s), secreted APP;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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