* La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037; Department of Neurobiology, Harvard
Medical School, and Division of Neuroscience, The Children's Hospital, Boston, Massachusetts 02115; and § Department of
Clinical Neuroscience, The Karolinska Hospital, Stockholm, Sweden S-171 76
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Abstract |
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The amyloid- peptide (A
) can mediate
cell attachment by binding to
1 integrins through an
arg-his-asp sequence. We show here that the
5
1 integrin, a fibronectin receptor, is an efficient binder of A
,
and mediates cell attachment to nonfibrillar A
. Cells
engineered to express
5
1 internalized and degraded
more added A
1-40 than did
5
1-negative control
cells. Deposition of an insoluble A
1-40 matrix around
the
5
1-expressing cells was reduced, and the cells
showed less apoptosis than the control cells. Thus, the
5
1 integrin may protect against A
deposition and
toxicity, which is a course of Alzheimer's disease lesions.
![]() |
Introduction |
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INTEGRIN-mediated cell adhesion is necessary for the
survival of many types of cells, and loss of adhesion
causes apoptosis (reviewed in Frisch and Ruoslahti,
1997). The
5
1 integrin may have a particularly prominent antiapoptotic effect because
5
1 is the only integrin
that protects cells from apoptosis in serum-free cultures
(Zhang et al., 1995
; O'Brien et al., 1996
).
5
1-mediated adhesion upregulates the antiapoptosis protein Bcl-2 (Zhang
et al., 1995
), and
5
1 is one of a few integrins that activates the signaling protein Shc (Wary et al., 1996
). These
signaling events may partly explain its antiapoptotic effects.
1 integrins have been shown to mediate cell adhesion
to the amyloid beta (A
)1 protein, and
5
1 has been proposed to be the integrin responsible for the A
binding
(Ghiso et al., 1992
). The amino acid sequence arg-his-asp
(RHD) has been pinpointed as the integrin recognition site in A
(Ghiso et al., 1992
; Sabo et al., 1995
). This sequence resembles the general integrin recognition sequence RGD present in many extracellular matrix proteins (Ruoslahti, 1996a
).
A is a 39-42 amino acid protein derived from proteolytic cleavage of a larger membrane-spanning glycoprotein, the amyloid precursor protein (APP; Kang et al.,
1987
). A
forms fibrillar aggregates that can cause cell
death by apoptosis (Loo et al., 1993
; Pike et al., 1993
;
Lorenzo and Yanker, 1994
). Enhanced deposition of A
matrix within the cortex, hippocampus, and vasculature of the brain correlates with neuronal cell death and ultimately dementia in Alzheimer's disease (AD; reviewed by
Selkoe, 1994
). Two predominant forms of A
(1-40 and
1-42) exist in AD that differ by two amino acid residues at
the hydrophobic COOH terminus, a domain that is required for nucleation-dependent fibril formation (Jarret et al., 1993
). The A
1-40 form has a slower rate of fibril
formation in vitro than the A
1-42 form (Jarret et al.,
1993
).
There is evidence for three mechanisms of A accumulation: overproduction of A
, production of longer forms
of A
(which aggregate more), and impaired clearance of
A
. The clearance pathways for fibrillar and soluble A
are incompletely known. Two cell surface receptors are
known to bind A
. The scavenger receptor present on
glial cells binds specifically to fibrillar A
, and appears to
mediate clearance of small fibrillar A
aggregates in vitro (Paresce et al., 1996
; Khoury et al., 1996
). The receptor for advanced glycation end products binds both the soluble
and fibrillar forms of A
, and may mediate some of the cytotoxic effects of fibrillar A
(Yan et al., 1996
).
Because 5
1 may also be an A
receptor, and because
5
1 and A
have apparently contrasting effects on apoptosis, we sought to determine whether
5
1 is indeed an
A
-binding integrin and, if so, what effect it might have on
the metabolism of A
and on cell survival. We show here
that nonfibrillar A
binds to the
5
1 integrin, and that
this interaction promotes clearance of A
by cultured
cells, reducing the formation of an insoluble A
fibrillar
matrix and counteracting the toxic effects of the A
matrix. These results suggest a new function for
5
1 as a
binder of A
and a regulator of brain cell survival.
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Materials and Methods |
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Cells
The human neuroblastoma cell line (IMR-32) was obtained from the
American Type Culture Collection (Rockville, MD). The CHO-B2 cells
deficient in 5
1 were from Dr. Rudolf Juliano (School of Medicine, University of North Carolina, Chapel Hill, NC; Schreiner et al., 1989
). All
cells were maintained in
-MEM (Sigma Chemical Co., St. Louis, MO)
supplemented with 10% FBS and glutamine/pen-strep (Irvine Scientific,
Santa Ana, CA). G418 (GIBCO BRL, Gaithersburg, MD) was added to
the media of transfected cells at a concentration of 250 µg/ml.
Reagents
Amyloid beta 1-40 peptide (A) was synthesized as previously described
(Nordstedt et al., 1994
). A
was also purchased from a commercial source
(Synthetic Amyloid Beta peptide 1-40; Bachem, Torrance, CA). A
1-40
from both sources was examined for cell adhesion activity. Two out of the
three Bachem lots tested showed adhesive activity (lots zn571 and
wm365), while lot zn327 was not active. For water-free storage to prevent
aggregation of A
into its fibrillar form, the peptide was dissolved and
stored in 1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP; Fluka Chemika, Neu-Ulm, Switerland). Before use, the peptide was lyophilized from HFIP, dissolved in sterile distilled water at 1 mg/ml, and tested immediately. The
control peptide, A
40-1, was purchased from Bachem, solubilized in water at 1 mg/ml, and tested immediately. Fibronectin was purchased from
Chemicon International, Inc. (Temecula, CA), and vitronectin was purified as described (Yatohgo et al., 1988
). Purified anti-human
5 integrin
monoclonal antibody (P1D6; Calbiochem-Novabiochem Corp., La Jolla,
CA; Wayner et al., 1988
) and purified mouse IgG (Sigma Chemical Co.)
were used at a concentration of 50 µg/ml.
Transfection
The CHO-B2/5
1+, CHO-B2/
v
1+, and IMR-32/
5
1+ cells were generated by introducing cDNAs coding for the
5 and
v integrin subunits
into
5
1-deficient CHO-B2 and IMR-32 cells (Schreiner et al., 1989
;
Bauer et al., 1992
; Zhang et al., 1993
, 1995
). Transfectants expressing the
integrin were cloned and expanded (Zhang et al., 1993
; Zhang et al.,
1995
). CHO-B2 and IMR-32 control cells received the empty vector.
Integrin Analysis
Integrin expression of IMR-32 and CHO transfectants was analyzed by
FACS using monoclonal antibodies against human 5 (P1D6),
v (L230),
and
1 (P4C10). FITC-conjugated goat anti-mouse antibody (Sigma
Chemical Co.) was used as the secondary antibody. The same integrin antibodies were used to block integrin function in other experiments.
Cell Adhesion to Nonfibrillar A1-40
The cell attachment assay and the use of antibodies and peptides as inhibitors of adhesion have been described previously (Zhang et al., 1993; Matter and Laurie, 1994
). Microtiter plates coated overnight at room temperature with nonfibrillar A
1-40 peptide, control A
40-1 peptide, or
fibronectin were blocked with 1% BSA for 30 min at room temperature,
the wells were rinsed once with PBS (pH 7.4), and cells were subsequently
added (2 × 10 5 cells/well) in serum-free media and incubated for 60 min
(37°C). Inhibition studies were performed by preincubating cells with antibody for 30 min (37°C; gentle agitation every 10 min), and then cells including antibodies were added to the coated wells. After a 60-min incubation at 37°C, plates were gently washed four times with PBS, fixed with
1% glutaraldehyde (Sigma Chemical Co.), PBS-washed once, stained with
0.5% crystal violet, 20% MEOH, washed under running distilled water,
solubilized in 0.1 N sodium citrate, 50% ETOH, and read on an ELISA
plate reader (Molecular Devices Corp., Sunnyvale, CA) using the 590-nm
filter.
Adhesion assays with fibrillar A1-40 were performed as above. Before the adhesion assay, soluble A
1-40 was incubated at 4°C for 96 h to
allow self-aggregation of A
1-40 into its fibrillar form (Jarret et al., 1993
).
Coating efficiency was measured by coating microtiter wells with either
soluble [125I]A
1-40 or preaggregated [125I]A
1-40 at room temperature
overnight. Nonbound peptide solution was removed, and the well and the
nonbound peptide solution were counted. Both forms of A
1-40 bound to
the wells with an efficiency of ~70%.
Immunostaining of A Fibrillar Matrix
Cells were plated on four-well PermanoxTM plastic slides (Nunc Inc., Naperville, IL) at 50,000 cells/well. 6 h after plating, the media was replaced
with media containing A1-40 peptide (100 µg/ml) and incubated for 72 h
at 37°C. The cultures were washed with PBS and fixed in PBS containing
3.7% paraformaldehyde and 10 mM sucrose, pH 7.4, for 30 min at room
temperature. The cultures were then blocked with 1% BSA/PBS and
stained with a polyclonal rabbit anti-human A
1-40 peptide antibody
(Chemicon International, Inc.) for 2 h, followed by goat anti-rabbit FITC-labeled IgG (Sigma Chemical Co.) secondary antibody. After antibody
treatment, coverslips were mounted with Vectashield mounting medium
(Vector Labs., Inc., Burlingame, CA) and analyzed under a fluorescent
confocal microscope.
Analysis of A in Matrix Deposition with
Radiolabeled [125I]A
125I-labeled A1-40 peptide was purchased as a lyophilized powder (25 µCi) from Nycomed Amersham, Inc. (Princeton, NJ). The powder was
solubilized in sterile water and immediately added to 24-well culture
dishes at a concentration of 2 ng/well. The specific activity of the 125I-labeled
A
1-40 peptide was 2 × 106 µCi/mmol.
Insolubilization of A was analyzed using 125I-labeled A
1-40 peptide
as described previously for fibronectin matrix assembly (McKeown-Longo and Mosher, 1985
; Morla and Ruoslahti, 1992
). Cells were plated at
105 cells/ml (IMR variants) or 0.5 × 105 cells/ml (CHO variants) into 24-well tissue culture plates in media containing 10% serum. Media was replaced 6 h after plating with media containing [125I]A
1-40 and 10% serum. Cells were cultured for 72 h at 37°C. The media was then removed,
the wells were washed three times with PBS, and 5× SDS sample buffer
(0.5M Tris pH 6.8, glycerol, 10% SDS, 0.5% bromophenol blue) was used
to solubilize the [125I]A
matrix in each well.
For antibody inhibition experiments, cells were plated as above. 6 h after plating, the media was replaced with media containing the appropriate
antibody and 10% serum. 125I-labeled A1-40 peptide (2 ng/well) was
added to the antibody-containing media and incubated for 72 h at 37°C.
The cells were then processed as above.
Internalization and Degradation of
[125I]Soluble A1-40
Internalization of A1-40 added to cell layers was measured as described
(Duckworth et al., 1972
; McDermott and Gibson, 1997). Subconfluent
cells were trypsinized and plated onto 24-well plates. Media was replaced
6 h after plating with [125I]A
1-40 (2 ng/ml). The cells were incubated for
1 h with [125I]A
1-40, the media was removed, cells were washed five
times with PBS, and serum-containing media containing no A
1-40 was added. The cells were cultured for 1 to 12 h at 37°C, washed three times
with PBS, detached by EDTA, washed twice with PBS, lysed in 100 µl of
1% NP40 buffer for 10 min at 4°C, and lysate-analyzed for radioactivity.
For TCA precipitations, the cells were cultured for 72 h with [125I]A1-40
at 37°C, washed three times with PBS, detached by EDTA, washed twice
with PBS, and lysed in 100 µl of 1% NP40 buffer for 10 min at 4°C. BSA/
PBS (100 µl, 1%) was added to the samples, the samples were vortexed,
and 1.6 ml of TCA (12.5% wt/vol) was added with vortexing. The samples
were centrifuged at 2,000 rpm for 10 min at 4°C, and the supernatant and
pellet were collected for radioactive counting.
Secretion of 125I-Labeled A1-40
Subconfluent cells were detached with trypsin, washed once with media,
and plated at 105 cells/ml in 24-well plates. 6 h after plating, media was replaced with 2 ng/ml of [125I]A1-40 in serum-containing media and incubated for 1 h at 37°C. The radiolabeled media was removed, and cells were
washed five times in PBS before serum-containing media containing no
A
was added to each well. At designated time points, 100 µl of media was collected, and [125I] was measured.
Apoptosis and Cell Viability Assays
The apoptotic effect of fibrillar A was determined using the Apoptag
Plus In Situ Apoptosis KitTM (Oncor, Inc., Gaithersburg, MD) that detects
the 3'-OH region of cleaved DNA. Cells were plated on eight-chamber
tissue culture glass slides (Miles Scientific Laboratories, Inc., Naperville,
IL), and 6 h after plating the media was replaced with media containing either A
1-40 peptide (50 µg/ml) or A
40-1 control peptide (50 µg/ml) and
10% serum. Cells were cultured for 72 h at 37°C, and were then fixed in a solution containing 3.7% paraformaldehyde, 10 mM sucrose in PBS for 30 min at room temperature. Cells were stained following kit protocol, counterstained with propidium iodide/antifade solution (Oncor, Inc.), mounted,
and viewed under a confocal microscope.
To measure apoptosis by nuclear fragmentation, cells were plated in
wells coated with either 50 µg/ml of fibronectin, vitronectin, or A1-40 for
72 h in serum-free medium. Attached and floating cells were then collected by centrifugation, washed once with PBS, fixed with 3.7%
paraformaldehyde for 10 min at room temperature, and stained with 0.1 µg
of 4', 6-diamidino-2-phenylindole (DAPI) per ml in PBS. The stained cells
were washed three times with PBS and mounted onto slides for analysis
under a fluorescence microscope (Zhang et al., 1995
).
Cell viability was assessed in several assays. The ability of cells to take
up acridine orange/ethidium bromide was measured as described (Cotter
and Martin, 1996). In brief, the assay was performed in 96-well tissue culture plates containing 100 µl media/well. Cells were plated in media containing 10% serum. 6 h after plating, the media was replaced with media
containing various concentrations of the test reagents and 10% serum.
The plates were incubated for 72 h at 37°C. At the 72-h time point, cells
were trypsinized and resuspended in PBS at 0.5 × 106 cells/ml. 1 µl from a
solution of acridine orange (100 µg/ml) and ethidium bromide (100 µg/ml)
was added to a 25-µl cell suspension, incubated for 2 min at room temperature, and examined under 40× magnification using a Zeiss Fluorescence
microscope.
Cells cultured in microtiter wells were pulsed with 25 µl of a 2.5 mg/ml
MTT stock in PBS and incubated for 4 h. Then 100 µl of a solution containing 10% SDS, 0.01 N HCl was added, and the plates were incubated
overnight (Tada et al., 1986). Absorption was read on a Vmax Microplate
ReaderTM (Molecular Devices Corp., Sunnyvale, CA) using a reference
wavelenth of 650 nm and a test wavelength of 590 nm. Test reagents were
added to media alone in order to provide a blank.
To measure lactate dehydrogenase (LDH) release from cells, the colorimetric Cytotox 96-LDH-Release AssayTM (Promega Corp., Madison, WI) was performed according to the instructions of the manufacturer.
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Results |
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The Integrin 5
1 Mediates Cell Adhesion to
Nonfibrillar A
1-40
The RHD sequence in A resembles the integrin recognition sequence RGD, and has been implicated in cell adhesion to A
via one or more of the
1 integrins (Ghiso et al.,
1992
; Sabo et al., 1995
). We set out to determine which of
the RGD-binding integrins bind to A
. A CHO cell line
deficient in
5 integrin subunit expression (CHO-B2) was
transfected with cDNA encoding human
5,
v, or vector alone, and was examined for its ability to adhere to a surface coated with A
1-40. Each of the integrin transfectants adhered to A
in a dose-dependent manner, but cells
that received the vector alone attached to A
within the
BSA background range (Fig. 1 A). CHO-B2/
5
1+ cells
adhered strongly to A
, and CHO-B2/
v
1+ cells were
moderately adhesive, whereas the control cells CHO-B2/c did not adhere above BSA background levels. FACS analysis indicated that CHO-B2/
5
1+ and CHO-B2/
v
1+
cell transfectants were similar in their expression of the
transfected integrin (Fig. 2, D and E). A control peptide in
which the A
sequence is inverted (A
40-1) did not have
adhesive activity with any of the cell types tested (Fig. 1 B).
In addition, integrin transfectants adhered only to soluble
nonfibrillar A
1-40, and not to fibrillar A
1-40 (Fig. 1 B).
Plates were coated with equal amounts of soluble and
fibrillar A
1-40 as measured by [125I]A
1-40. The
5
1-mediated cell adhesion to soluble A
1-40 was inhibitable
by the integrin-binding peptide GRGDSP, and by a function-blocking anti-
5 integrin monoclonal antibody (P1D6;
Fig. 1 C), but not by the control peptide GRGESP or a
monoclonal antibody to
v (Fig. 1 C). The
v
3 integrin,
which also binds to RGD, does not mediate adhesion to A
because
v
3-expressing IMR-90 cells did not adhere to
A
when the
5
1 and
v
1 integrins were blocked with
anti-
5 and anti-
1 monoclonal antibodies (data not shown).
|
|
We also tested the 5-negative human neuroblastoma
cell line IMR-32 (Neill et al., 1994
) for A
attachment
with (IMR-32/
5
1+) and without (IMR-32/c)
5 transfection (Fig. 2 A). Three separate clones were obtained
that expressed human
5
1 on their surface as detected by
FACS analysis (Fig. 2, A-C). Each
5
1-expressing clone
adhered to coated A
1-40 in a dose-dependent manner
(Fig. 3 A), and cell adhesion was inhibitable by an anti-
5
antibody (data not shown). The control-transfected IMR-32
cells (Fig. 2, A-C) attached poorly to this substrate (Fig. 3
A). Both the transfected and control cells attached well to
vitronectin (data not shown), whereas the control peptide
A
40-1 and fibrillar A
1-40 did not promote adhesion
above BSA background levels for any of the IMR-32 cell
lines (Fig. 3 B).
|
5
1 Reduces the Formation of an Insoluble A
Fibrillar Extracellular Matrix
An increase of insoluble A fibrillar matrix is one hallmark of AD (Glenner and Wong, 1984
; Masters et al.,
1985
). As shown above, the
5
1 integrin bound to coated
A
with the highest avidity among the integrins we tested.
Therefore, we asked whether
5
1 would affect the formation of an A
fibrillar matrix. Exogenous A
1-40
added to cell cultures formed a matrix around the cells that was detectable by immunostaining with anti-A
antibodies. There was a substantial decrease in the formation
of matrix from added A
in cultures of the
5
1-expressing IMR-32 cell lines compared with the control lines (Fig.
4, A-D). Moreover, the matrix in the
5
1+ cell cultures
appeared to be cell-associated, whereas in the
5
1
cell
cultures it appeared to be largely independent of the cells.
|
To study quantitatively the formation of the A matrix,
the various IMR-32 lines were incubated with 125I-labeled
A
for 72 h, and the amount of radiolabeled A
that had
become soluble in detergent was measured. The IMR-32
clones expressing
5
1 deposited fivefold less insoluble
A
radioactivity than the control cells. Moreover, the
P1D6 anti-
5 antibody returned A
matrix formation in
the
5
1-expressing IMR-32 cultures to the level in the
parental control cells (Fig. 5 A). A control antibody had
no effect. CHO cells expressing
5
1 also had less A
matrix than their control-transfected counterpart cells as judged from the insolubility of [125I]A
; the difference was
fourfold (Fig. 5 B). Adding the anti-
5 antibody canceled
the
5
1 effect, but a control antibody did not. The insolubility of A
remained the same in the CHO control cell
cultures regardless of the antibody added. These results indicate that cell expression of
5
1 reduces A
matrix deposition threefold relative to the control cells. Because iodinated A
forms fibrils less readily than unlabeled A
1-40
(Bush et al., 1994
), it was not possible to use the [125I]A
to quantitate the proportion of the added A
1-40 that becomes insolubilized.
|
Soluble A1-40 is Taken Up By Cells and Partially
Degraded Via an
5
1-Mediated Pathway
Possible reasons for the 5
1-mediated reduction of A
matrix include internalization of soluble A
1-40, degradation of the peptide, or both. Neuronal cells have been
shown to internalize A
, but the mechanism for this internalization is only incompletely known (Ida et al., 1996
,
Hammad et al., 1997
). To investigate the possibility that
binding
5
1 to soluble A
initiates cellular uptake of A
,
we examined the processing of 125I-labeled A
1-40 by
5
1+ and
5
1
cells. Initially, CHO-B2/c control cells
and transfectants were incubated for 1 h with [125I]A
1-40,
and were then examined for cell-associated radioactivity. The
5
1-expressing CHO-B2 cells contained twofold
more radioactivity at 1 and 12 h than the control CHO-B2/c
cells (Fig. 6 A).
|
Cell cultures were then incubated with 125I-labeled A
over a 72-h period to determine whether the [125I]A
taken up by the cells was degraded.
5
1-expressing IMR-32 cells contained twofold more radioactivity after the 72-h incubation than
5
1-negative IMR-32 cells (Fig. 6 B). Part
of the radioactivity was soluble in TCA, indicating that A
had been degraded. CHO cells internalized and degraded
soluble A
in a similar manner, with
5
1-expressing cells
containing eightfold more TCA-soluble radioactivity than
5
1-negative cells (Fig. 6 C). The CHO cells expressing
5
1 bound 10% of the added A
, whereas the control cells bound only 0.4%. Moreover, 90% of the cell-associated A
was degraded in the CHO-
5
1 expressers. The
higher expression levels of
5
1 on the CHO transfectants
(Fig. 2, D and E) may explain why these cells bound and
internalized more radiolabeled A
than the IMR-32 transfectants.
We next examined whether A was released into the
culture medium. The release of radioactivity into cell culture media was monitored over a 72-h period that followed a 1-h incubation with 125I-labeled A
1-40. The media of
5
1-expressing IMR-32 and CHO cells contained
twofold more radioactivity than the corresponding control
cell media (Fig. 6, D and E). These results point to an
5
1-dependent pathway that internalizes and degrades A
.
5
1 Protects Cells Against A
Induced Apoptosis
Having established an 5
1-dependent mechanism for the
inhibition of A
matrix deposition, we examined whether
the reduction of the A
matrix would promote neuronal
cell survival in cultures treated with A
. IMR-32 cell lines
cultured with exogenous soluble A
1-40 underwent apoptosis in the absence of
5
1 (Fig. 7, A and B), but three
5
1-expressing lines did not (two are shown in Fig. 7, C
and D). The control peptide A
40-1 caused no apoptosis in the control (Fig. 7, E and F) or
5
1-expressing cells
(not shown). Analysis of acridine orange/ethidium bromide uptake revealed three times more apoptosis in the
control cells than in the IMR-32
5
1-expressers (Fig. 8).
|
|
We also assessed the A effect by using the MTT assay,
which measures cell viability by detecting the ability of a
mitochondrial enzyme to reduce its substrate. A
-treated
IMR-32 control cells lost their ability to reduce MTT in a
manner that was dependent on the dose of A
, whereas
A
had almost no effect on the
5
1-expressing cell lines
(Fig. 9 A). The control peptide A
40-1 had no effect on
MTT reduction in any of the cell types, even at the highest test concentration (Fig. 9 B). To examine further the cytotoxicity of A
1-40, we used an assay that measures the release of LDH upon cell lysis (Behl et al., 1994
). A threefold increase in LDH levels relative to controls was seen in
the
5
1
IMR-32 cells cultured in the presence of A
1-40, whereas A
1-40 had no effect on the LDH levels of the
5
1+ cells (Fig. 10 A). These results indicate that
5
1-mediated A
binding protects the IMR-32 cells from the
cytotoxicity of aggregated A
, presumably by inhibiting its
aggregation into fibrils. No apoptosis was caused by A
in
any of the CHO cell lines, as examined by TUNEL staining, the MTT assay, and the LDH assay, indicating that
these cells are resistant to the cytotoxic effects of an A
matrix.
|
|
We previously demonstrated that cell attachment through
5
1 protects CHO cells from apoptosis when cultured in
a serum-free environment (Zhang et al., 1995
). Therefore,
we examined whether ligation of
5
1 to coated A
1-40
would protect
5
1-expressing CHO cells from apoptosis
in serum-free cultures. CHO-B2/
5
1+ cells were plated
on either fibronectin, vitronectin, or A
-coated dishes and
examined for survival 96 h after serum withdrawal. CHO-B2/
5
1+ cells survived on A
and fibronectin, whereas
cells plated on vitronectin underwent apoptosis (Fig. 10
B). These results indicate that
5
1 can also protect cells
from apoptosis by mediating cell attachment to coated A
.
![]() |
Discussion |
---|
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---|
We report that the 5
1 integrin mediates cell adhesion to
A
and promotes internalization and degradation of A
.
This
5
1-A
interaction correlates with both an increase
in the clearance of soluble A
, a reduction in the formation of an insoluble A
fibrillar matrix, and a decrease of
the toxicity of A
to cells. This study provides one mechanism for regulating A
accumulation.
Our data, showing that A binds to
5
1, and to a lesser
extent
v
1, is in agreement with previous reports that A
mediates cell attachment, and that the RHD sequence in it
serves as an integrin-binding site (Ghiso et al., 1992
; Sabo
et al., 1995
). The RHD sequence apparently functions as a
mimic of the RGD sequence in fibronectin, the matrix
ligand of
5
1 (Ruoslahti, 1996a
), because a short peptide
containing the RGD sequence inhibits A
binding.
5
1
binds only to nonfibrillar A
, since we did not see any detectable cell adhesion to aggregated fibrillar A
. Therefore, other receptors presumably mediate cellular interactions with fibrillar A
, and are responsible for the
cytotoxic effects of this form of A
. The
5
1 integrin is
one of the most discriminating of the RGD-directed integrins with regard to its ligand specificity (Ruoslahti,
1996b
). In addition to its main ligand fibronectin, the
5
1
integrin has only been shown to bind to the bacterial protein invasin (Watari et al., 1996
) and the insulin-like
growth factor binding protein IGFBP-X (Jones et al.,
1993
). Our results add A
among its ligands. The binding
site for
5
1 seems to be available only in A
, not in its
precursor protein (APP; B. Bossy, M.L. Matter, and E. Ruoslahti, unpublished results).
The 5
1 integrin may play a role in the rapid clearance
of A
that occurs in the normal brain (Ghersi-Egea et al.,
1996
). We show that expression of the
5
1 integrin is associated with increased cellular uptake and degradation
and decreased matrix deposition of A
in cell cultures.
Moreover, reversal of this effect with a function-blocking
anti-
5 antibody established a causal link between
5
1
activity and increased clearance of A
. Although more
complex explanations of this effect are possible, the binding of A
to
5
1 shown here suggests that A
binds to
5
1 at the cell surface, and is subsequently internalized
into a cellular compartment where it is degraded. This hypothesis is in agreement with previous results showing that
a neuronal cell line internalizes A
from culture medium
in a manner that is dependent on the NH2 terminus of A
where the RHD sequence resides (Ida et al., 1996
). The lipoprotein Apo J can also reduce the formation of fibrillar
A
by causing it to be internalized and degraded (Hammad
et al., 1997
). Thus, it is likely that more than one mechanism plays a role in the regulation of A
accumulation in
vivo. Clearly, a transgenic animal expressing the amyloid
precursor protein with a mutated RHD sequence would
be of great interest in testing the contribution of the RHD
sequence and integrin-binding to the metabolism of A
.
The 5
1 integrin circulates through the endocytic cycle
(Bretscher, 1989
; Bretscher, 1992
). Inhibiting exocytosis
with primaquin causes accumulation of internalized
5
1
in an intracellular pool that returns to the cell surface over
time. Recent studies have shown that internalization of
fibrillar A
promotes accumulation of stable fibrillar A
in the late endosome/secondary lysosome compartment,
whereas internalization of soluble A
leads to degradation of the peptide in the same compartment (Knauer et al.,
1992
; Koo and Squazzo, 1994
; Yang et al., 1995
). This result is in agreement with our data, showing that soluble A
is internalized through an
5
1 integrin-mediated pathway, and is at least partially degraded, presumably within
endosomes. Thus, clearance of soluble A
can be mediated
by the
5
1 integrin, presumably through the receptor-mediated endocytosis pathway that normally internalizes
this integrin.
5
1 may play a protective role in the brain by suppressing A
cytotoxicity. We provide evidence for two
separate mechanisms that could be responsible for such a
protective effect. First, we show that
5
1-mediated adhesion to nonfibrillar A
protects cells from apoptosis in cell
culture. Upregulation of Bcl-2 (Zhang et al., 1995
) and activation of the MAPK pathway (Wary et al., 1997) may be
responsible for this pathway. The second and potentially more important mechanism is suggested by our demonstration that
5
1 suppresses the apoptotic effects of A
by reducing production of toxic A
matrix.
The 5
1 integrin and
v
1 are present in the adult central nervous system (Grooms et al., 1993
). Immunostaining
for
5
1 shows that it is expressed in the vasculature, cortex, and hippocampus of adult rat brain (Bahr et al., 1991
;
Pagani et al., 1992
; Tawil et al., 1994
; for review see Sargent Jones, 1996). Moreover, primary hippocampal neurons express
5
1 (Yamazaki et al., 1997
). Soluble A
1-40
is present in vivo (Seubert et al., 1992
), and is rapidly cleared when injected into normal rats (Ghersi-Egea et al.,
1996
). Our results suggest that
5
1 may mediate the
clearance of A
, and that
5
1 may play a significant role
in protecting the brain from the A
-initiated pathology
that in its extreme form causes AD.
![]() |
Footnotes |
---|
Received for publication 2 December 1997 and in revised form 25 February 1998.
The present address of Z. Zhang is Department of Neurobiology, Harvard Medical School, The Children's Hospital, Enders 260, 300 Longwood Ave., Boston, MA 02115.We thank Drs. Blaise Bossy, Eva Engvall, and Kristiina Vuori for comments on the manuscript, and Dr. Edward Monosov for help with the confocal microscopy. This work was supported by grant CA28896 (E. Ruoslahti) and Cancer Center Support Grant CA 30199 from the National Cancer Institute, Department of Health and Human Services. M.L. Matter is supported by postdoctoral training grant CA09579 from the National Institutes of Health.
![]() |
Abbreviations used in this paper |
---|
A, amyloid
peptide;
AD, Alzheimer's disease, LDH, lactate dehydrogenase;
RHD, arg-his-asp.
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