(Received for publication, September 5, 1995)
From the
Several observations support the hypothesis that pathogenetic
mechanisms of amyloid formation in Alzheimer's disease may
involve alterations in amyloid precursor protein (APP) gene expression.
In this regard, molecular dissection of the APP gene transcriptional
regulation is of primary importance. We report evidence that members of
the family of transcription factors NF
B/Rel can specifically
recognize two identical sequences located in the 5`-regulatory region
of APP. These sequences, which we refer to as APP
B sites, interact
preferentially with p50-containing members of the family. In
particular, p50 homodimers and p50/p65 and p50/c-Rel heterodimers act
as transcriptional activators at the APP
B site. Finally, the
nuclear complex specifically binding to the APP
B sites proves to
be an integral part of neurons and lymphocytes.
Molecular genetic studies in familial forms of Alzheimer's
disease (AD) ()and in Down's syndrome have clearly
pointed out that brain deposition of
amyloid peptide in senile
plaques and cerebrovasculature plays a central role in the pathogenesis
of AD (see (1) for review). The molecular nature and origin of
the
peptide have also been clarified.
amyloid is a small
polypeptide fragment of 39-42 amino acids which is generated by
proteolytic cleavage of a family of alternatively spliced transmembrane
proteins, the amyloid precursor proteins (APP), whose functional
significance is still controversial. The APP gene, localized on
chromosome 21, is expressed in brain and all major tissues.
In the
past few years, in vitro studies on processing of endogenous
or transfected (wild-type and mutated) APP molecules have emerged as
major experimental endeavors(1) . This kind of approach has
been extremely important for unraveling some of the molecular
mechanisms involved in the production of the amyloid and, likely,
in the disease. Nevertheless, the possibility that pathogenetic
mechanisms of plaque formation in AD may also involve alterations in
APP gene expression as a required step should not be underestimated.
Several observations support this hypothesis. Among them: (i) augmented
expression of the APP gene transcripts in Down's syndrome and in
specific areas of the brain of AD
patients(2, 3, 4, 5) ; (ii) in
vitro degeneration of postmitotic neurons overexpressing
full-length APP(6) ; (iii) marked increase of the APP gene
expression after head trauma, a well-recognized enviromental risk
factor for AD (7) .
Increased and/or dysregulated expression of the APP gene may potentially result from disturbances at any step in the transcriptional regulatory pathway, like altered activation or expression of specific transcription factors and/or mutations in the 5`-regulatory region of the gene. It appears important therefore to elucidate the molecular mechanisms of APP gene regulation by identifying pathophysiologically Relevant cis-elements and transcriptional regulators interacting with these sites. Despite cloning of its promoter region (8) , little is known about the participants in the transcriptional control of the APP gene. In particular, although several potential recognition sequences for transcriptional control proteins have been identified(9, 10, 11, 12, 13, 14) , only for AP-1 has a direct role in the control of the APP gene expression been established(15) .
The present paper reports
evidence that members of the NFB/Rel family of transcriptional
control proteins may represent critical regulators for modulation of
the APP gene expression. Regulatory proteins belonging to this family
have been widely characterized as very pleiotropic factors, able to
respond to a wide variety of signals and to control expression of a
large number of genes mainly implicated in defensive responses, such as
immune and inflammatory reactions(16, 17) . On the
contrary, there has been only modest progress in determining their
contribution to regulated expression of genes whose products are
functionally Relevant in the central nervous system (18, 19, 20) .
The embryonal carcinoma F9 was grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml).
Both strands of each oligonucleotide probe have a 5`-TCGA-3` overhang at their 5` end to facilitate cloning.
We identified a sequence corresponding to 5`-GGGGTTTCAC-3`,
repeated in positions -2250 to -2241 and -1837 to
-1828 in the 5`-regulatory region of the APP gene and remarkably
similar to the consensus sequence for NFB/Rel transcription
factors. The possibility that the sequence is actually a binding site
for
B/Rel proteins and is operative in the central nervous system
was investigated. Double-stranded oligonucleotides comprising the
sequence and designated as APP1 (-2257 to -2234) and APP2
(-1844 to -1821) were tested, in gel shift analysis, for
binding of nuclear proteins in extracts from rat cerebellum. As shown
in Fig. 1, lanes 1 and 2, both
oligonucleotides detected a complex with identical retarded migration.
To verify if the complex interacting with the sequence from the APP
gene was a
B-Related protein, APP1 and APP2 oligonucleotides were
tested for their ability to compete with a characterized
B
sequence for binding of members of the family of transcription factors.
An established model for
B-mediated gene transcription is the
murine CD4
T cell clone A.E7, where IL-2 gene
regulation has been studied extensively(23) . A
B site in
the IL-2 gene enhancer region (IL-2
B site) interacts with at least
two dimeric complexes with different subunit compositions, both
constitutively present in A.E7 nuclear extracts, p50/p65 (NF
B) and
p50/p50 (NF
C) dimers (Fig. 1, lane 9). Interplay
between these two complexes ensures fine control of IL-2 gene
regulation in nontransformed lymphocytes(23) . APP1 and APP2
oligonucleotides displaced both NF
B and NF
C complexes from
binding to the IL-2
B site probe in a concentration-dependent
manner (lanes 3-6). As a control, the same amount of an
oligonucleotide sequence for octamer proteins (OCTA) failed to compete
for interaction between the IL-2
B sequence and the two
B
complexes (lanes 7 and 8). Furthermore, the APP1 and
APP2 oligonucleotide sequences, when used as probes and incubated with
A.E7 extracts, recognized a major complex (lanes 10 and 11) whose binding activity was disrupted by three base changes
in the core
B-like sequence within the APP1 oligonucleotide (lane 12). Nuclear extracts were obtained also from rat cortex
and hippocampus and tested for binding activity to the APP1 (and APP2,
not shown) oligonucleotide. As depicted in Fig. 2, a single
nuclear complex comigrating with the one from cerebellum was
intercepted by the probe. More importantly, the rat brain complex
binding the APP1 sequence comigrated with the complex from A.E7 cell
extracts. To confirm specificity of DNA-protein interaction, binding of
the constitutive nuclear activity from rat brain could be abolished by
competition with the unlabeled APP1 sequence (20-fold excess) and by
the same mutations in the APP
B sequence which abolished binding in
A.E7 extracts. A representative experiment of gel shift analysis with
cerebellum nuclear extracts is shown in Fig. 2, but comparable
results were observed with extracts from cortex and hippocampus (not
shown). The results clearly demonstrated that the sequence from the APP
regulatory region could indeed interact with
B complexes.
Moreover, they strongly suggested that the specific complex recognized
in rat brain extracts from the APP sequence was either identical with
or very similar to p50 homodimers.
Figure 1:
The sequences from the APP gene
5`-regulatory region bind a specific complex constitutively present in
rat cerebellum extracts and Related to members of the NFB/Rel
family of transcription factors. a, lanes 1 and 2, electrophoretic mobility shift assay with 2 µg of
nuclear extracts from rat cerebellum and
-
P
end-labeled oligonucleotide probes APP1 and APP2. b, lanes
3-9, the sequences APP1 and APP2, unlike an oligonucleotide
for octamer binding proteins (OCTA), can compete binding of p50/p65
heterodimers (or NF
B, upper migrating complex) and p50 homodimers
(or NF
C, lower migrating complex) to the
B sequence from the
IL-2 enhancer region. Nuclear extracts from the murine CD4
T cell clone A.E7 were used. c, lanes
10-12, the APP1 and APP2 oligonucleotides incubated with
A.E7 nuclear extracts bind a complex comigrating with NF
C. Binding
specificity was proven using an APP oligonucleotide sequence with three
base changes in the core
B site (mut).
Figure 2:
A constitutive nuclear factor from several
rat brain regions binds specifically to the APP1 sequence and
comigrates with the APP1 binding complex from A.E7 cell extracts.
Electrophoretic mobility shift assay obtained by incubating nuclear
extracts from A.E7, rat hippocampus, cortex, and cerebellum with the
wild type P-labeled APP1 oligonucleotide sequence or a
mutated version, with three base changes in the core
B-like
sequence (mut), in the absence(-) or presence (+)
of a 20-fold excess unlabeled APP1 oligonucleotide as competitor, to
prove specificity of DNA-protein
interaction.
To better characterize binding
specificity of the APP sequence, the APP1 oligonucleotide was incubated
in the presence of 100 ng of recombinant p50 protein (bact.
p50): a specific complex was obtained (Fig. 3A, lane 2) which comigrated with the one identified by the
IL-2B oligonucleotide probe (lane 1). The same results
were obtained using the APP2 oligonucleotide (data not shown). Finally,
the APP
B complex identified in rat cerebellum nuclear extracts (as
well as in extracts from other brain regions, data not shown) could be
recognized by a polyclonal antibody against p50 (Fig. 3A, lane 5) but not by a polyclonal
antibody against p50B (lane 7), a highly Related NF
B/Rel
family member, or the corresponding preimmune sera (lanes 4 and 6).
Figure 3:
The nuclear complex binding the sequence
from the APP gene contains the p50 subunit of the NFB/Rel family. A, lanes 1 and 2, recombinant p50 protein
binds indistinguishably oligonucleotide probes containing
B sites
from the IL-2 gene enhancer region (IL2
B) and the APP
gene (APP1
B). Lanes 3-7, supershift of the
APP
B binding complex from rat cerebellum by 1 µl of anti-p50
antiserum but not by the same amount of anti-p50B or of their
respective preimmune sera. I = immune serum, PI = preimmune serum. B, lanes 8-15,
APP
B binding activity corRelates with APP gene expression in H9
and HeLa cell lines. Electrophoretic mobility shift assay with H9
nuclear extracts incubated with
-
P end-labeled
oligonucleotide probes APP1 (lane 8), APP2 (lane 9),
or an APP sequence with three base changes (lane 10).
Electrophoretic mobility shift assay with nuclear extracts from HeLa
cells either untreated(-) (lanes 11, 13, and 15) or treated with
12-O-tetradecanoylphorbol-13-acetate (TPA) (60 ng/ml
for 12 h) (+) (lanes 12, 14, and 16) in
the presence of
-
P-labeled APP1 (lanes 11 and 12), APP2 (lanes 13 and 14), or
mutated oligonucleotide (lanes 15 and 16). ns indicates a nonspecific band which appeared in all nuclear
extracts of human origin.
CorRelation between APPB binding activity
and gene expression was analyzed in cell lines expressing different
levels of APP (Fig. 3B). Nuclear extracts from H9
cells, a human T cell line which expresses high levels of
APP(26) , displayed extremely high amounts of APP1 and APP2
B binding activity (lanes 8 and 9,
respectively). HeLa cells, which express lower levels of APP mRNA
compared to H9, displayed low amounts of APP1 (lane 11) and
APP2 (lane 13)
B binding activity. Furthermore, treatment
with 12-O-tetradecanoylphorbol-13-acetate (60 ng/ml for 12 h),
which has been proven to augment APP mRNA levels in HeLa cells (27) , increased both APP1 (lane 12) and APP2 (lane 14) binding activity. In both cell lines, specificity of
protein complexes was confirmed using a version of the APP
B
sequence with three base changes (lanes 10, 15, and 16). In conclusion, the nuclear complex specifically binding
to the APP
B sites proved to be an integral component in the
examined cell lines. Its basal activity corRelated with levels of APP
gene expression and was induced in response to signals that augment
gene transcription.
Transcriptional responses at B sites have
been shown to involve complex molecular mechanisms, being the result of
the different combinatorial possibilities among different members of
the regulatory family, to form homo- and
heterodimers(16, 17) . F9, a mouse embryonal carcinoma
in which endogenous
B binding activity is very low(28) ,
appeared to be a suitable model for dissecting functional significance
of the APP
B binding site and contribution of different subunit
members of the
B/Rel-Related family. Expression vectors for three
B-Related proteins, p50, c-Rel, and p65 (24) were
cotransfected in F9 cells with a reporter plasmid obtained by cloning
the APP
B sequence at the SalI site of a PBLCAT2
vector(25) . As shown in Fig. 4, expression of p50, by
itself or in combination with p65 and c-Rel, led to an increase in CAT
activity, while expression of c-Rel, p65, and p65/c-Rel was devoid of
effect on the reporter gene transcription. Comparable results were
obtained with a reporter plasmid obtained by inserting, at the SalI site of PBLCAT2, the APP2 oligonucleotide sequence (not
shown). Since none of the
B/Rel-Related proteins had any effect on
the vector itself, which lacks
B binding sites, we concluded that
the observed effect on CAT gene transcription resulted from specific
binding to the inserted sequence from the APP gene.
Figure 4:
Transcriptional activities of different
members of the NFB/Rel family at the APP
B site.
Undifferentiated F9 cells were transfected with 10 µg of the
reporter plasmid APP
BCAT (obtained by cloning the APP1
oligonucleotide sequence at the SalI site of PBLCAT2) either
in the absence or presence of various combinations of the expression
vectors for
B/Rel proteins: pSG-p50, pSG-p65, pSG-Rel, pSG-p50
plus pSG-Rel, pSG-p65 plus pSG-Rel, pSG-p50 plus pSG-p65. In
cotransfection experiments, 2 µg of expression vector were used and
the total amount of DNA was adjusted to 15 µg with pSG5.
Transfection experiments were repeated three times in duplicate with at
least two independent plasmid preparations. CAT activities are
expressed as % chloramphenicol conversion.
The APP promoter contains numerous potential recognition
sequences for known transcription
factors(9, 10, 11, 12, 13, 14, 15) .
However, except for the AP1 site(15) , none of these regulatory
regions has been implicated directly in the control of the APP gene
expression. We report the identification of two identical sequences in
the 5`-regulatory region of the APP gene which are specific binding
sites for regulatory members of the NFB/Rel family of
transcription factors(16, 17) . The two nuclear factor
binding domains, which we referred to as APP
B sites, show high
affinity for p50-containing members of the family, which appear to be
constitutively expressed in the CNS. Furthermore, the APP
B binding
activity corRelates with APP gene expression in cell lines of various
origin in both resting and stimulated conditions. Analogously to
previously characterized
B sequences, in transfection assays we
observed that
B-Related complexes with different subunit
composition displayed different transcriptional activities at the
APP
B site. Cotransfection experiments showed that p50-containing
complexes, unlike c-Rel/c-Rel, c-Rel/p65, and p65/p65 dimers, behave as
transactivators when interacting with the APP
B sequence.
Interestingly, differences in transcriptional activity were observed
with the different p50-containing complexes. Although the reason for
these differences has not been addressed directly, this is in agreement
with previous elegant demonstrations by other groups(17) . It
appears that whether complexes with different subunit composition serve
as weak or strong activators (or even repressors, in some cases) is
determined by their conformation on DNA or whether their
transcriptional activation domains are accessible to components of the
general transcription machinery.
An interesting observation made by
several groups is that the APP gene is rapidly transcribed in brain in
response to a number of circumstances, ranging from head trauma, focal
ischemia, neurotoxicity, and heat
shock(29, 30, 31, 32) . Although
very different, these situations can be grouped together under the
generic term of stress conditions. The finding that B/Rel-Related
proteins may be implicated in the control of the APP gene
transcriptional control is intriguing. In fact, these regulatory
proteins are utilized in most cell types for genetic interpretation of
cellular events underlying responses to stress, since among their
target genes are those encoding for cytokines, and they are themselves
activated by cytokines(16) . Intracerebral responses to damage
are often mediated by cytokines, like interleukin 1 (IL-1), interleukin
6, tumor necrosis factor
, so that in analogy to what happens in
the liver, the existence of a ``brain acute phase response''
has been suggested(33) . In this regard, it should be
underlined that the levels of one of the best characterized inducers
for
B/Rel proteins, IL-1, is augmented in the brains of patients
with AD and Down's syndrome(34) . Furthermore, it has
been shown that IL-1 induces an increase in APP transcript levels in
endothelial (9) and in neuronal cells(35) . PReliminary
results indicate that the APP
B sites are indeed responsive to
IL-1
in primary neuronal cultures. (
)
We speculate
that the APP gene may be one of a set of B site-containing genes
coordinately modulated in brain in response to situations that require
a defensive response.