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The Journal of Neuroscience, December 1, 1998, 18(23):9703-9715
Neuronal Receptors Mediating Responses to AntibodyActivated
Laminin-1
Jonathan K.
Ivins1,
Holly
Colognato2,
Jordan A.
Kreidberg3,
Peter D.
Yurchenco2, and
Arthur D.
Lander1
1 Department of Developmental and Cell Biology and the
Developmental Biology Center, University of California at Irvine,
Irvine, California 92697, 2 Department of Pathology and
Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway,
New Jersey 08854, and 3 Divisions of Nephrology and
Developmental and Newborn Biology, Children's Hospital and Department
of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
Embryonic retinal neurons lose the ability to extend neurites on
laminin-1 (LN-1) with increasing developmental age yet still do so on
other laminin isoforms. However, after treatment of LN-1 with
antibodies to "short-arm" regions or removal of the short arms
proteolytically, LN-1 supports attachment and extension of neurites
even by late embryonic retinal neurons. We have mapped a domain for
antibody-mediated "activation" of LN-1 to the N-terminal end of the
1 chain. Furthermore, we show that the primary receptors used in the
retinal neuron response to "activated" LN-1 are integrins 3
1
and 61; these are the same receptors used by these neurons for
outgrowth on other LN isoforms. Interestingly, 31 is
preferentially involved in neurite outgrowth, whereas 61
preferentially mediates attachment and spreading. However, in cultures
from 3 integrin-deficient mice, 61 mediates retinal ganglion
cell neurite outgrowth and compensates for the absence of 31.
Finally, we show that key features of the retinal neuron response to
LN-1 also characterize neurons of the hippocampus, thalamus, and
cerebral cortex; these include poor response to untreated LN-1,
extensive neurite outgrowth on antibody-activated LN-1 or on fragment
E8, and dependence of this response on integrin 61 and at
least one other long arm-binding 1 integrin. These data suggest that
regulation of LN-1 function via the process of activation could have
important consequences for axonal regeneration. Curiously, the data
also imply that the mechanism of laminin activation involves enhanced
function at sites that cannot be considered cryptic.
Key words:
integrin; retina; neurite outgrowth; extracellular
matrix; laminin; antibody activation; knock-out; VLA-3; VLA-6; retinal ganglion cell
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INTRODUCTION |
Signals from the extracellular
matrix (ECM) control numerous aspects of cell behavior, including
proliferation, survival, gene expression, and morphological
differentiation. Regulation of cell-matrix interactions can occur at
the level of how cells recognize and respond to ECM molecules, as well
as at the level of how ECM molecules are organized in space and
presented to cells. Studies of retinal neurons have provided examples
of both types of regulation; during embryonic development, retinal
neurons lose the ability to attach and extend neurites in response to
substrata containing the ECM protein laminin-1 (LN-1), a change that is thought to reflect decreases in the number and/or activation state of
integrin receptors (Cohen et al., 1986
, 1987; Hall et al., 1987; de
Curtis et al., 1991; de Curtis and Reichardt, 1993). Despite this
change, however, any of several manipulations of the LN-1 molecule,
including the binding of antibodies to short-arm domains or proteolytic
cleavages that isolate the long-arm domain, restore the ability of LN-1
to promote neurite outgrowth by late embryonic retinal neurons, a
phenomenon we refer to as laminin "activation" (Calof et al.,
1994).
Because LNs are thought to play roles both in axonal development and in
regeneration, it is important to understand the mechanisms of laminin
activation and the possible physiological significance of such
activation. In the present study, we sought to address the following
questions. What are the minimal manipulations of the LN-1 molecule
required for its activation? What receptors do late embryonic retinal
neurons use to respond to activated LN-1, and how do these receptors
compare with the receptors that other types of neurons use to respond
to native LN-1? Finally, given that induction of LN expression is a
common injury response in many parts of the central and peripheral
nervous systems (Zak et al., 1987; Brodkey et al., 1993; Frisen, 1997;
Fu and Gordon, 1997), we sought to determine whether activation of LN-1
increases its ability to promote neurite outgrowth from CNS neurons
other than those of the retina.
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MATERIALS AND METHODS |
Anti-integrin antibodies. Function-blocking
monoclonal hamster anti-rat 1 [clone Ha31/8 (Mendrick et al.,
1995)], 2 [clone Ha1/29 (Mendrick and Kelly, 1993)], and 1
[clone Ha2/5 (Mendrick and Kelly, 1993)] integrins were obtained from
PharMingen (San Diego, CA) and were used in cell culture
experiments at a final concentration of 10 µg/ml. The mouse anti-rat
1 integrin antibody 3A3 (Turner et al., 1989) was a generous gift
from Dr. Sal Carbonetto (Montreal, Canada) and was provided as an
ascites fluid. Results with 3A3 were identical to those obtained with
Ha31/8. The function-blocking rabbit anti-rodent 1 integrin
antiserum designated "lenny" was a generous gift of Dr. Clayton
Buck (The Wistar Institute, Philadelphia, PA) and was typically used at
a 1:200 dilution. The function-blocking mouse anti-chicken 2
integrin monoclonal antibody mep-17 (McNagny et al., 1992; Bradshaw et
al., 1995) was the generous gift of Dr. Kelly McNagny (Heidelberg,
Germany) and Dr. Amy Bradshaw (University of California at Santa
Barbara). A rabbit polyclonal antiserum directed against the
cytoplasmic domain of the 3 integrin subunit was the generous gift
of Drs. Mike DiPersio and Richard Hynes (Massachusetts Institute of
Technology, Cambridge, MA). Hybridoma cells secreting Ralph 3.1, a
function-blocking mouse anti-rat 3 integrin antibody (DeFreitas et
al., 1995), were the generous gift of Dr. Louis Reichardt (University
of California at San Francisco) and were grown in DMEM containing 10%
FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and glutamine
(2 mM). The Ralph 3.1 antibody was isolated from spent
culture supernatants by ammonium sulfate precipitation (50%) followed
by passage over anti-mouse IgG-agarose (Sigma, St. Louis, MO). Antibody
was eluted with 100 mM glycine, pH 2.5, neutralized with 1 M Tris, pH 9.0, concentrated by centrifugation in a
Centricon 10 (Millipore, Bedford, MA), dialyzed into F12 culture media,
sterile filtered, and used at a final concentration of 50 µg/ml. The
function-blocking rat monoclonal anti-mouse 6 integrin antibody GoH3
was obtained from AMAC (Westbrook, ME) and was used at a
concentration of 2 µg/ml. All antibody reagents were tested for toxic
effects using either cultures of retinal neurons derived from
noncross-reacting species, cultures of primary chick sensory neurons,
or PC12 pheochromocytoma cells grown on various substrata. Normal mouse
IgG, rabbit IgG, or nonimmune rabbit sera were also typically included
as controls at the same concentrations.
Anti-laminin antibodies. Antibodies to laminin fragments
E1', P1', recombinant-1(VI-IVb),
recombinant-1(VI), E8, and E4 were prepared and used as described
(Sung et al., 1993; Calof et al., 1994; Colognato-Pyke et al., 1995).
Antibodies were applied at a concentration of 10 µg/ml; anti-E4 was
an exception and was used as a whole serum diluted 1:100. Nonspecific
rabbit IgG or nonimmune rabbit sera were included as controls at the
same concentrations. For cross-absorption, antibodies were diluted to
their final working concentration in HBSS containing 0.2% BSA and the
indicated laminin fragment at 50 µg/ml and were incubated overnight
at 4°C before their use.
Preparation of substrata. Murine LN-1 was purified from the
Engelbreth-Holm-Swarm tumor as described (Calof et al., 1994). Human merosin (a mixture of LN-2 and LN-4) was purchased from Life
Technologies (Gaithersburg, MD). LN-1 fragments E8 and E1' were
prepared as described (Sung et al., 1993; Colognato-Pyke et al., 1995).
Proteins were diluted into Ca2+- and
Mg2+-free HBSS to a final concentration of 40 µg/ml for LN-1 and LN-2/4 or 20 µg/ml for E8 and E1' and were
applied to 96-well tissue culture plates (#3596; Costar, Cambridge,
MA). Typically, proteins were allowed to adsorb overnight at 4°C. Rat
LN-5 (Baker et al., 1996) was adsorbed directly onto tissue culture
plastic from culture supernatants conditioned by the rat bladder cell
line 804G, which were the generous gift of Mark Fitchmun (Desmos, San
Diego, CA). After adsorption, substrata were washed a minimum of three
times and blocked with HBSS containing 0.2% heat-inactivated BSA
(#7030; Sigma). When used, antibodies to substrate-absorbed
molecules (see below) were applied in this blocking solution for a
minimum of 1 hr at 37°C before the plating of cells.
Cell culture. Cultures of retinal neurons were derived from
embryos of timed pregnant Sprague Dawley rats (Charles River
Laboratories, Wilmington, MA; Bantin-Kingman, Freemont, CA; or Taconic
Farms), CD-1 mice, or chick embryos (Spafas Farms) as described
(Calof et al., 1994). Typically, rats at embryonic day 18, mice
at embryonic day 16, and chicks at embryonic day 7 or 11 were used.
Briefly, the neuroretina was dissected free of surrounding tissues,
minced with sharpened forceps, trypsinized briefly, and triturated
through a fire-polished Pasteur pipette to obtain a suspension of
single cells and/or small clusters of cells. Retinal neurons were
plated in DMEM/F12 (1:1) containing N2 supplements (Bottenstein et al., 1979) and 0.5% ultrapure crystalline BSA (ICN Biochemicals, Costa Mesa, CA) on substrata prepared as described above and were cultured overnight at 37°C in a humidified atmosphere containing 5%
CO2. For some experiments, retinal tissue was cut into
small pieces with sharpened forceps and placed into culture as
explants. Cultures were also established from embryonic day 18 rat
cortex and hippocampus as well as embryonic day 14 and 15 mouse cortex
and thalamus. These cultures were grown in DMEM supplemented with 0.2%
BSA, pen/strep, and B27 (Life Technologies). Cultures were fixed by underlay with warm PBS containing 10% formalin and 5% sucrose and
were observed on a Zeiss Axiovert microscope equipped for phase
contrast and 35 mm photography. Typically, cultures were scored for the
presence of cells or clumps of cells with neurites greater than two
cell body diameters in length. A minimum of 100 cells per well were
counted from duplicate wells for each condition scored. In some
instances, measurements of neurite length were made with
Image1/AT (Universal Imaging Corporation, West Chester, PA)
using a Dage-MTI CCD camera housed on a Zeiss Axiovert microscope.
Cell surface biotinylation and immunoprecipitation. After
dissociation, cells were allowed to recover from trypsinization overnight in culture media on Petri plastic at a density of 5 × 106 cells/ml. Cells were collected by
centrifugation, washed into HBSS containing 1 mM
biotin-LC-NHS (Pierce, Rockford, IL), and incubated with gentle
rocking at 4°C for 1 hr. The reaction was terminated by the addition
of 5 ml of Leibovitz L15 culture media. Cells were then spun through a
cushion of L15 containing 4% BSA and lysed with 2 ml of lysis buffer
containing 100 mM -octyl-glucoside, 150 mM
NaCl, 50 mM Tris-Cl, pH 8.0, 1 mM
MgCl2, 100 µM PMSF, 10 µg/ml
N-ethylmaleimide, and 1 µg/ml pepstatin A. This material was precleared by incubation for 15 min at 4°C with 25 µl each of a
slurry of 50 mg/ml protein A-Sepharose (Sigma) and 50 mg/ml anti-mouse
IgG-agarose, followed by centrifugation. Primary antibodies (5 µg of
purified antibody or 2 µl of antiserum) were added to aliquots of
this supernatant and incubated overnight at 4°C. Immune complexes
were isolated by incubation with 50 µl of either anti-mouse IgG-agarose or protein A-Sepharose for 2 hr at 4°C followed by centrifugation. Beads were washed once with lysis buffer containing 750 mM NaCl and again with lysis buffer. Beads were eluted with 95°C double-strength SDS sample buffer, separated by SDS-PAGE under
nonreducing conditions, and transferred to Immobilon P using a semidry
blotter (Owl Scientific). Filters were blocked by incubation in TBS
containing 0.2% Tween-20, 2% BSA, and 3% goat serum and then were
incubated with avidin-HRP (ABC; Vector Laboratories, Burlingame, CA).
Biotinylated proteins were visualized by enhanced chemiluminescence
(Amersham, Arlington Heights, IL).
Immunohistochemistry. Immunohistochemistry was performed as
described (Ivins et al., 1997) on horizontal sections of embryonic day
18-19 rat embryos and embryonic day 16-17 mouse embryos. Cryostat sections were cut at 20 µm from fresh frozen tissue and post-fixed either with acetone at 
20°C for 10 min or with 4% paraformaldehyde in PBS for 10 min at room temperature. In some cases, tissue was fixed
overnight in PBS containing 4% paraformaldehyde at 4°C and cryoprotected in PBS containing 20% sucrose before cryosectioning. Sections were blocked for 30 min in TBST (50 mM Tris, pH
8.0, 150 mM NaCl, and 0.2% Tween-20) containing 3% normal
goat serum and 2% BSA and then were incubated overnight at room
temperature in a humidified chamber in primary antibody diluted in
blocking solution. Sections were washed five times with TBST and then
incubated in Cy3-conjugated secondary antibodies (Jackson
ImmunoResearch, West Grove, PA) also diluted in block for 1-2 hr at
room temperature. Sections were washed five times with TBST and
coverslipped with Aquamount (Biomeda) containing ProLong
antifade reagent (Molecular Probes, Eugene, OR). Sections were observed
and photographed on a Zeiss Axiophot photomicroscope equipped for
bright field, differential interference contrast, and epifluorescence.
31 integrin "knock-out" mice. Mice harboring a
targeted deletion of the 3 integrin gene (Kreidberg et al., 1996)
were maintained as heterozygotes through outcrosses with CD-1 mice.
Mice were genotyped by PCR performed on DNA isolated from tail
biopsies; a PCR protocol generously provided by Dr. H. Gardner
(Scripps Research Institute, La Jolla, CA) was used as described
(DiPersio et al., 1997). For tissue culture experiments, embryos were
harvested from timed pregnancies from heterozygote crosses, and tissue
from each embryo was cultured separately. Tissue was also collected from each embryo for DNA isolation and subsequent genotyping. Because
the time required to generate dissociated cell suspensions from the
retinas of many embryos in parallel was too long to ensure adequate
neuronal viability, these experiments were performed with retinal
explants rather than with dissociated cells.
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RESULTS |
A domain for antibody activation maps to the N terminal of the 1
chain of LN-1
Retinal neurons lose the ability to attach and extend neurites on
LN-1 substrata with increasing developmental age (Cohen et al., 1986),
but decorating substratum-bound LN-1 with antibodies to its short-arm
domains allows even late embryonic retinal neurons to extend neurites
(Calof et al., 1994). Because neurite outgrowth on activated LN-1 is
blocked by antibodies to long-arm domains and because isolated long-arm
fragments of LN-1 (fragments E8, C8-9) have the same effects on retinal
neurons as does LN-1 that has been antibody-activated, it has been
argued that activation of LN-1 is the result of blockade or removal of
some type of inhibitory activity that resides in the short-arm domains
(Calof et al., 1994).
To date, the antibodies that have been shown to activate LN-1 have been
directed against either the E1' or the slightly smaller P1' fragments
of LN (Fig. 1A). Both
of these fragments contain parts of all three LN-1 chains (1, 1,
and 
1). We suspected that the site responsible for antibody
activation of LN-1 might reside on the 1 or possibly the 1 chain
because late embryonic retinal neurons respond to preparations of
merosin, a mixture of LN isoforms 211 (LN-2) and 221
(LN-4), with vigorous neurite outgrowth (Calof et al., 1994). The
simplest explanation for this difference between merosin and LN-1 is
that the critical site for activation of LN-1 resides on a chain that
is absent in merosin.
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Figure 1.
Mapping a domain for antibody activation of LN-1.
A, The domain structure of LN-1 and the domain-specific
antibodies used in this study are shown. B,
C, Dissociated retinal neurons from embryonic day 18 rat
retinas were grown on LN-1 treated with anti-E4
(B) or with anti-VI-IVb
(C) antibodies. D, Retinal neurons
were grown either on untreated LN-1 substrata or on LN-1 that had been
decorated with an anti-E1' antiserum. The anti-E1' antiserum was
further cross-absorbed with LN-1 short-arm fragments VI-IVb, E1X, or
E35 as indicated. After 18 hr, cultures were fixed and scored for
the percentage of cells or clumps of cells with neurites. Scale bars:
B, C, 50 µm.
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To test this hypothesis, we treated substratum-bound LN-1 with
affinity-purified polyclonal antibodies specific for an N-terminal 75 kDa fragment of the 1 chain (fragment E4) or with antibodies to
either of two overlapping domains near the N-terminal end of the 1
chain. As shown in Figure 1, antibodies against fragment E4 failed to
activate LN-1 (Fig. 1B), but antibodies against
recombinant 1 domains VI-IVb [r1(VI-IVb)] rendered LN-1
substrata as effective in promoting late embryonic retinal neurite
outgrowth as did anti-E1' or anti-P1' antibodies (Fig. 1C).
Domains VI-IVb represent the N-terminal 120 kDa portion of the 1
chain and are entirely contained within the E1' fragment and overlap
significantly with the P1' fragment. An antiserum specific for the
isolated 1 domain VI (Colognato-Pyke et al., 1995) had some
activating ability, but it was very weak at all concentrations tested
(up to 20 µg/ml; data not shown). As with LN-1 that has been
activated with anti-E1' or anti-P1' antibodies, the effects of
anti-1(VI-IVb)-treated LN-1 on retinal neurons could be blocked by
antibodies to the LN-1 long-arm domain E8 (Calof et al., 1994) (data
not shown).
To test whether 1 chain sites that are sufficient for antibody
activation are also necessary for antibody activation, we cross-absorbed an activating anti-E1' antiserum with preparations of
either recombinant 1(VI-IVb), fragment E1X (1 domain
IVb), or fragment E35 (1 domain VI). As shown in Figure
1D, each of these fragments strongly inhibited
activation of LN-1 by the anti-E1' antibodies. Together, these data
suggest that regions both necessary and sufficient for activation of
LN-1 reside on the short arm of the 1 chain of LN-1, but these
regions may span more than a single domain.
Identification of candidate receptors for retinal neurite outgrowth
on activated LN-1
Integrin receptors of the 1 family are required for the
responses of early embryonic retinal neurons to LNs (Hall et al., 1987;
Cohen and Johnson, 1991; Neugebauer and Reichardt, 1991; Calof et al.,
1994), and we have reported previously that the response of late
embryonic retinal neurons to activated LN-1 is also 1
integrin-dependent (Calof et al., 1994). The 1 integrins that are
known to be able to bind LN-1 include 11 and 21, which
interact near the N terminal of the chains of both LN-1 and LN-2
(Colognato-Pyke et al., 1995; Colognato et al., 1997), and 31,
61, and 71, which interact at sites on the LN-1 long arm
(Hall et al., 1990; Sonnenberg et al., 1990; Tomaselli et al., 1990;
Kramer et al., 1991; Gehlsen et al., 1992; Pattaramalai et al., 1996).
Three of these integrins have been reported to be expressed in the
neural retina during at least some stages of development: 11
(Duband et al., 1992), 21 (Bradshaw et al., 1995; Cann et al.,
1996), and 61 (de Curtis et al., 1991; Cann et al., 1996).
Expression of 31 and 71 in the retina has apparently not
been studied.
To identify candidate integrin receptors mediating the response of late
embryonic retinal neurons to activated LN-1, we performed immunoprecipitations from -octyl-glucoside extracts of cell
surface-biotinylated retinal neurons as well as immunohistochemical
localization using integrin-specific antibodies. The results are
shown in Figure 2. Immunoprecipitations
with the 1-specific antiserum lenny yielded two major bands with
Mr of ~110 and 130 kDa when analyzed under nonreducing conditions by SDS-PAGE and Western blotting; these bands
presumably correspond to the 1 chain and one or more associated chains, respectively (Fig. 2A). Immunoprecipitation
with the 1-specific monoclonal antibody (mAb) 3A3 (Turner et
al., 1989) yielded a single band with Mr of
~185 kDa (Fig. 2A) consistent with the 1
integrin subunit. Immunoprecipitation with an antiserum directed
against the cytoplasmic domain of the 3A integrin
subunit yielded a single predominant band with
Mr of 130 kDa (Fig. 2A). Immunoprecipitation with an anti-7 antibody failed to produce any
bands, and in situ hybridization, using a partial 7 cDNA derived by reverse transcription-PCR from skeletal muscle RNA, also failed to provide any evidence of expression of this integrin in
the retina (data not shown).
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Figure 2.
Integrin expression in the rodent retina.
A, Immunoprecipitations from cell surface-biotinylated
retinal neurons performed using the rabbit anti-1 antibody lenny,
the anti-1 mAb 3A3, and a rabbit antiserum raised against the 3A
cytoplasmic domain. Immunoprecipitates were analyzed as described to
confirm the cell surface expression of 1 (110 kDa and associated integrin chains of 130-150 kDa), 1 (185 kDa), and 3A integrin
(130 kDa) subunits. B, Immunofluorescent localization of
31 in a cryosection of an embryonic day 18 rat retina stained
with conditioned medium from the Ralph 3.1 hybridoma. Strongest
expression is seen throughout the retinal ganglion cell layer and
within the optic nerve, although diffuse staining is evident throughout
the neuroretina. C, Immunofluorescent localization of
61 in a cryosection of an equivalent stage (embryonic day 16)
mouse retina stained with the GoH3 antibody (5 µg/ml). 61
immunoreactivity is present throughout all retinal layers.
D, Cryosection of embryonic day 18 rat retina stained
with nonconditioned medium. The exposure is matched for that shown in
B. Scale bars: B-D, 100 µm.
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Immunoprecipitations with the anti-mouse 6-specific mAb GoH3 also
failed to produce any bands (data not shown), but this most likely
reflects the known poor ability of this monoclonal antibody to perform
in immunoprecipitations. Integrin 6 has been shown previously to be
present in the chick neuroretina (de Curtis et al., 1991; de Curtis and
Reichardt, 1993). Furthermore, we confirmed the presence of both
31 and 61 in the retina immunohistochemically. Using the
function-blocking anti-rat 3 mAb Ralph 3.1 (DeFreitas et al., 1995)
to stain horizontal sections through embryonic day 18 rat retina, we
found immunoreactivity in all retinal layers, but with the highest
levels of expression associated with retinal ganglion cells and their
axons (Fig. 2B). An identical staining pattern was
observed with an antibody specific for the cytoplasmic domain of the A
isoform of the 3 integrin (data not shown). In contrast to this
pattern, the anti-mouse 6 antibody GoH3 stained all retinal layers
with similar intensity (Fig. 2C).
Functional analysis of laminin-binding integrins in
retinal neurons
To test whether 11 or 21 integrins are involved in the
response of retinal neurons to LNs, including antibody-activated LN-1,
cultures of embryonic day 11 chick and embryonic day 18 rat retinal
neurons were plated on LN-1, LN-2/4 (merosin), LN-5, E8, and LN-1
activated by treatment with anti-E1' antibodies in the presence or
absence of monoclonal antibodies that block the functions of rat 1
[3A3 (Turner et al., 1989)], rat 2 [Ha1/29 (Mendrick et al.,
1995)], or chick 2 [mep-17 (McNagny et al., 1992)]. None of these
antibodies inhibited the ability of retinal neurons to extend neurites
on antibody-activated LN-1, E8, LN-2/4, or LN-5 (data not shown).
Furthermore, these antibodies did not alter the inability of retinal
neurons to respond to untreated LN-1. These data suggest that 11
and 21 do not mediate neurite outgrowth on LNs, nor are they
responsible for inhibition of neurite outgrowth on LN-1 that has not
been activated.
To test for a role for the 31 integrin in retinal neurite
outgrowth on antibody-activated LN-1, we plated dissociated cultures of
embryonic retinal neurons from embryonic day 18 rats on untreated and
activated LN-1 substrata in the presence or absence of
function-blocking anti-3 antibodies (Fig.
3). As expected, retinal neurons failed to attach and extend neurites on untreated LN-1 (Fig. 3A)
but attached, spread, and extended long neurites on LN-1 substrata treated with anti-P1' antibodies (Fig. 3B). The
function-blocking anti-31 mAb Ralph 3.1 (50 µg/ml)
substantively inhibited the ability of retinal neurons to extend
neurites on antibody-activated LN-1 (Fig. 3C, see also Figs.
5, 6). Interestingly, however, the spreading response of the neurons
remained unaffected (Fig. 3C). Incubation with the
function-blocking anti-1 antibodies lenny (Fig. 3D) or
Ha2/5 (data not shown) completely inhibited both the attachment and
spreading responses as well as all neurite outgrowth. Incubation with
nonimmune mouse IgG was without effect (data not shown).
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Figure 3.
Effect of 31 integrin blockade on neurite
outgrowth on antibody-activated LN-1. The effect of integrin blockade
on the morphology of embryonic day 18 rat retinal neurons growing on an
LN-1 substrata in the absence (A) or presence
(B-D) of anti-P1' antibodies. A,
Retinal neurons growing on untreated LN-1 attach poorly (remaining
phase-bright and rounded) and do not extend neurites. B,
Decoration of the short arms of LN-1 with anti-P1' antibodies permits
embryonic day 18 rat retinal neurons to attach and extend neurites.
Note the phase-dark appearance of most cells, indicating spreading.
C, Blockade of 31 integrin function by Ralph 3.1 (50 µg/ml) inhibits neurite outgrowth while significantly sparing
cell attachment and spreading. D, Blockade of all 1
integrins with lenny (1:200) inhibits both neurite outgrowth and cell
attachment. Scale bar, 75 µm.
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Because 31 function seemed to be important for retinal neurite
outgrowth on antibody-activated LN-1, we also asked whether 31
was required for outgrowth on LN-1 that was activated by proteolytic
removal of short-arm domains, as well as on other LN isoforms that do
not require activation to promote neurite outgrowth from late embryonic
retinal neurons (Fig. 4). As described previously (Calof et al., 1994), LN-1 long-arm fragment E8 (Fig. 4A) and LN-2/4 (Fig. 4C) both
promote neurite outgrowth from late embryonic retinal neurons as does
thrombospondin-1, an ECM protein known to interact with 31 (Fig.
4G). Additionally, these cells extend neurites in response
to LN-5 (Fig. 4E), a LN isoform that shares no chains
in common with LN-1 (Marinkovich et al., 1992). However, when retinal
neurons were cultured on any of these substrata in the presence of
Ralph 3.1 (50 µg/ml), neurite outgrowth was greatly reduced (Figs.
4B,D,F,H, 5,
6). This effect can be seen quantitatively both in terms of the number of neurite-bearing cells in
the culture (Fig. 5) and in the lengths of neurite-bearing cells (Fig.
6).
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Figure 4.
Effect of 31 integrin blockade on neurite
outgrowth on LN-1 fragment E8 and on other LN isoforms. Embryonic day
18 rat retinal neurons were grown on substrata as indicated in the
absence (A, C, E,
G) or in the presence (B,
D, F, H) of the
anti-3 function-blocking antibody Ralph 3.1 (50 µg/ml). Ralph 3.1 strongly inhibits neurite outgrowth on the E8 long-arm fragment of LN-1
(A, B), on LN-5 (E,
F), and on thrombospondin-1 (G,
H) but only has a minor effect on LN-2/4
(C, D). Scale bar, 50 µm.
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Figure 5.
Effect of 31 integrin blockade on numbers of
neurite-bearing cells. Cultures of embryonic day 18 rat retinal neurons
were grown on the indicated substrata in the presence or absence of
antibodies as described. Cultures were fixed after 24 hr and scored for
the number of cells or clumps of cells bearing neurites greater than
two cell body diameters in length. A, Ralph 3.1 (50 µg/ml; hatched bars) blocks neurite outgrowth to the
same extent relative to controls (open bars) on both
anti-P1'-treated LN-1 (LN-1 + @P1') and the E8
fragment of LN-1. In each case, cell attachment and neurite outgrowth
are completely blocked by anti-1 integrin antibodies (solid
bars). B, Ralph 3.1 (50 µg/ml; hatched
bars) blocks neurite outgrowth on LN-2 and LN-5. The effect of
31 blockade is minor on LN-2 compared with that seen on the other
LN substrates. Counts were performed on duplicate wells from a total of
six separate experiments, except that for anti-P1'-treated LN-1, two
separate experiments were performed. All differences are significant at
p < 0.01 by Student's t
test.
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Figure 6.
Effect of 31 integrin blockade on neurite
lengths. Cultures of embryonic day 18 rat retinal neurons were grown
overnight on antibody-activated LN-1 (A) or on
long-arm fragment E8 (B) in the absence
(thin line) or presence of Ralph 3.1 (50 µg/ml;
thick line) and then fixed; neurite lengths were
measured. Only neurites greater than two cell body diameters in length
were included. Neurites that do grow in the presence of
31-blocking antibodies are shorter than controls. All differences
are significant (p < 0.01) by Student's
t test.
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These results suggest that 31 is a major, although possibly not
the only, retinal neuron receptor for antibody-activated LN-1 and
further support the idea that antibody activation of LN-1 and
activation by proteolytic removal of short-arm fragments have similar
functional consequences for LN-1. The data also support the idea that
activation of LN-1 imparts functionality that is "constitutively"
present in other LN isoforms.
The role that the 61 integrin plays in the responses of retinal
neuron to LN-1 substrata was also tested with blocking antibodies, this
time using embryonic day 16 mouse retinal neurons and the rat
monoclonal anti-6 integrin antibody GoH3. Neurons were cultured on a
variety of substrata in the presence (Fig.
7B,D,F) or
absence (Fig. 7A,C,E) of the antibody. In contrast to the
effects seen with 31 blockade on neurite outgrowth, only a slight
effect on neurite outgrowth was seen with 61 blockade with GoH3
(2 µg/ml). However, 61 blockade resulted in a marked inhibition of cell attachment and spreading on E8 (Fig.
7A,B), on LN-2/4 (Fig.
7C,D), and on LN-5 (Fig.
7E,F). These results suggest
that the 61 integrin is important for the attachment and
spreading of retinal neurons on LN substrata but is much less so for
neurite outgrowth. The data also demonstrate, however, that activation of LN-1 is characterized not only by an increase in the ability of LN-1
to promote neurite outgrowth but also by an increase in the ability to
promote, via a separate mechanism that uses a different receptor, cell
spreading.
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Figure 7.
Effect of 61 blockade on the spreading
response of retinal neurons. Embryonic day 16 mouse retinal neurons
were grown on E8 (A, B), on LN-2/4
(C, D), or on LN-5 (E,
F) in the presence (B,
D, F) or absence
(A, C, E) of the
anti-61 antibody GoH3 (2 µg/ml). Cultures were fixed after 24 hr and photographed. In each case, 61 blockade inhibited the
ability of cells to spread on these LN substrates but still allowed
considerable neurite growth. In many cases, dissociated retinal neurons
aggregated to form large clumps of cells rather than remaining as
single cells or small clumps. Because of this tendency of cells to form
large aggregates, the effects of 61 blockade on neurite outgrowth
were not measured quantitatively. The effect of GoH3 on
antibody-activated LN-1 (data not shown) was indistinguishable from
that seen here with E8. Scale bar, 50 µm.
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31 is required by retinal ganglion cell axons for outgrowth
on the long arm of LN-1
The immunohistochemical localization of the 31 integrin
suggests that it is predominantly expressed by retinal ganglion cells
(Fig. 2B). To assess the role of 31
specifically in retinal ganglion cell outgrowth on activated LN-1, we
cultured retinas from embryonic day 18 rats as explants. Under these
conditions, a number of investigators have shown that the only axons
that extend away from the explant are those of retinal ganglion cells (e.g., Bates and Meyer, 1993). We observed that, as was the case with
dissociated retinal cultures, retinal ganglion cell axons from such
explants were unable to extend axons on substrata of LN-1 (Fig.
8A). However, these
explants exhibited robust axon growth when plated on the long-arm
fragment E8 (Fig. 8B) or on anti-E1'-treated LN-1
(data not shown). Furthermore, in both cases this outgrowth was
strongly inhibited by 50 µg/ml Ralph 3.1 (Fig. 8C;
data not shown). Interestingly, in the presence of the anti-3 antibody, retinal ganglion cell axons did manage to grow but mainly in
highly fasciculated bundles that wrapped many times around the margin
of the explant without extending out onto the substratum. These results
confirm that 31 is an important receptor for retinal ganglion
cell axon outgrowth on activated LN-1.
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Figure 8.
Effect of 31 integrin blockade on retinal
ganglion cell outgrowth. Explants of embryonic day 18 rat retina were
plated on LN-1 (A) or on E8 (B,
C) in the absence (B) or presence
(C) of Ralph 3.1 and were allowed to grow
overnight. In B, the edge of the explant is visible in
the lower left corner of the photograph. Scale bar, 50 µm.
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61 can support neurite growth when 31 is
genetically disrupted
One criticism of the above experiments with blocking antibodies to
31 and 61 is that, because of limitations in the types of
antibodies available, it was only possible to block 31 in rat
neurons and 61 in mouse neurons. Because each reagent inhibited some of the neuronal response (e.g., attachment and/or neurite outgrowth) to activated LN-1, it is tempting to speculate that the two
receptors together account for all of the response, but demonstrating
this requires blocking both integrins in the same cells.
To circumvent the lack of appropriate antibodies, we turned to a mouse
model in which the 3 integrin subunit has been deleted by homologous
recombination (Kreidberg et al., 1996). Cultures established from the
retinas of littermates derived from 3+/
× 3+/ matings were then grown on the LN-1
long-arm fragment E8 in the presence or absence of the anti-61
antibody GoH3. Explants established from wild-type, heterozygous, and
homozygous null retinas all exhibited robust axon growth on E8 (Fig.
9). In agreement with the data
presented above, neurite outgrowth produced by wild-type (data not
shown) and heterozygote cultures was only partly inhibited by GoH3, but
outgrowth by homozygous null retinas was almost completely blocked
(Fig. 9). These data support the view that, together, 31 and
61 mediate the retinal neuron response to activated LN-1.
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Figure 9.
Effect of disruption of the 3 integrin gene on
retinal ganglion cell outgrowth on the LN-1 long arm. Explants of
retina from embryonic day 16 mice derived from crosses of heterozygote
3 integrin null mice were plated on E8. Explants were grown in the
absence (A, C) or presence
(B, D) of GoH3 (2 µg/ml) to inhibit
61. Explants in A and B were
derived from a heterozygote, whereas explants in C and
D were derived from a null embryo. Insets
(A, C), Embryos were genotyped by PCR.
ko, Knock-out; wt, wild type.
Scale bar, 50 µm.
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A surprising feature of the data is that, despite the absence of
functional 31, the magnitude of the neurite outgrowth response was not noticeably lower in the homozygous null cells than in the
wild-type neurons (Fig. 9). This strongly suggests that the mutant
cells compensate for the loss of 31 by increasing the part of
their response that is mediated by 61. Whether this is
attributable to a change in 61 expression or function remains to
be determined. However, such compensation may explain why DiI labeling
of the visual system of 3-/- mice demonstrates an
apparently normal pattern of retinal innervation of the superior
colliculus (J. K. Ivins, J. A. Kreidberg, and A. D. Lander, unpublished observations).
Activated LN-1 exhibits enhanced neurite outgrowth-promoting
activity for many types of CNS neurons
Integrin 61, which was shown above to be one of two
integrins used by late embryonic retinal neurons to respond to
activated LN-1, is widely expressed in the CNS. This raised the
possibility that other types of CNS neurons might also exhibit an
enhanced response to LN-1 that has been activated. To test this, we
cultured neurons from several CNS regions on LN-1 substrates in the
presence or absence of activating antibodies. The results with cultures of embryonic day 18 rat hippocampal neurons are shown in Figure 10. When cultured overnight on
untreated LN-1 substrata, dissociated hippocampal neurons attach but
tend to form large cell aggregates that exhibit sparse neurite
outgrowth (Fig. 10A). Treatment of the substratum
with anti-E1' antibodies, however, allowed these neurons to attach
individually and to extend extensive neurites (Fig.
10B), comparable with that seen on LN-2/4
(Fig. 10C). Similar extensive outgrowth was also seen on
LN-1 fragment E8 (data not shown). As was the case with retinal
neurons, neurite outgrowth on activated LN-1 was completely blocked by
function-blocking anti-1 integrin antibodies (Fig.
10D). Similar results were also obtained with
cultures of embryonic day 18 rat cerebral cortical neurons (data not
shown).
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Figure 10.
Effect of antibody activation of LN-1 on the
neurite outgrowth of other CNS neurons. A-D,
Hippocampal neurons from embryonic day 18 rats were plated on LN-1 in
the absence (A) or presence (B,
D) of anti-E1' antibodies or on LN-2/4
(C). The culture in D was
additionally treated with anti-1 function-blocking antibody Ha2/5
(10 µg/ml). Like late embryonic retinal neurons, hippocampal neurons
respond to activated LN-1 with vigorous neurite outgrowth.
E, F, Embryonic day 14 mouse cortical
neurons were grown overnight on E8 in the absence
(E) or presence (F) of the
anti-6 mAb GoH3 (2 µg/ml). GoH3 treatment resulted in a
significant reduction both in the number of cells with neurites (78 vs
45%) and in neurite lengths (67.2 ± 6.3 vs 28.0 ± 3.3 µm, mean ± SEM; Student's t test,
p < 0.0001). No outgrowth was seen on untreated
LN-1 (data not shown). Outgrowth was completely blocked by anti-1
antibodies (data not shown). Scale bar, 75 µm.
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To test whether these responses are mediated by integrin 61, we
repeated the above experiments with mouse cortical neurons, culturing
them on E8 in the absence (Fig. 10E) or presence
(Fig. 10F) of mAb GoH3. Treatment with mAb GoH3
resulted in a significant reduction in the number of cells with
neurites (78% for control vs 45% for GoH3-treated) as well as a
significant reduction in neurite lengths (67.2 ± 6.3 µm for
control vs 28.0 ± 3.3 µm for GoH3-treated, mean ± SEM;
Student's t test, p < 0.0001). No
outgrowth was observed on untreated LN-1 (data not shown). Neurite
outgrowth was completely blocked by anti-1 antibodies on all
substrates tested (data not shown). Similar results were obtained with
cultures of mouse thalamic neurons (data not shown).
In cultures of central neurons, therefore, as in cultures of retinal
neurons, neurite outgrowth on activated LN-1 is only partially mediated
by the 61 integrin. However, 31 is unlikely to mediate
outgrowth in these cultures for several reasons. First, immunohistochemical localization studies by us (data not shown) and
others (DeFreitas et al., 1995) have failed to detect the 3 integrin
subunit in the rodent cerebral cortex, hippocampus, or thalamus.
Furthermore, treatment of rat hippocampal or cortical neuronal cultures
grown on activated LN-1 with mAb Ralph 3.1 (anti-rat integrin 3) had
no effect on neurite outgrowth (data not shown). Together, these data
show that not only do CNS neurons other than retinal neurons respond to
the activation of LN-1 but suggest that at least one 1 integrin
other than 61 and 31 may be involved.
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DISCUSSION |
In previous studies, treatment of substratum-bound LN-1 with
antibodies to its E1' or P1' domains rendered LN-1 a potent promoter of
neurite outgrowth by late embryonic retinal neurons (Calof et al.,
1994). Proteolytic fragments containing the long arm of LN-1 had
effects similar to those of antibody-activated LN-1, and antibodies to
distal long-arm fragments of LN-1, or to 1 integrins, blocked the
neurite outgrowth activity of both antibody-activated and
proteolytically activated LN-1 (Calof et al., 1994). These observations
suggest the existence of two discrete activities in LN-1: one located
in the short-arm domains, the blockade or removal of which leads to
activation, and one located at the end of the long arm, which mediates
integrin-dependent neurite outgrowth in the activated LN-1 molecule but
fails to do so in native LN-1. In the present study, we have shown that
a major site for LN-1 activation resides in domains VI-IVb of the 1
chain (Fig. 1). In addition, via the study of retinal neurons (Figs.
2-9) and other CNS neurons (Fig. 10), we have shown that multiple
integrins can mediate neuronal responses to activated LN-1, including
integrins that, on other cell types, mediate robust responses to
unmanipulated LN-1.
Multiple integrins mediate the neuronal response to
activated LN-1
In late embryonic retinal neurons, the response to activated LN-1
is mediated by two integrins, 31 and 61 (Figs. 2-9). Both of these interact with the distal long arm of LN-1 (Sonnenberg et al.,
1990; Gehlsen et al., 1992; Pattaramalai et al., 1996), consistent with
the observation that effects of activated LN-1 are blocked by
antibodies to distal long-arm fragments (Calof et al., 1994).
It is noteworthy that, although isolated 31 has been shown to
bind LN-1 (DeFreitas et al., 1995) and LN-1-derived peptides (Gehlsen
et al., 1992) in biochemical assays, there is little evidence that
cells use 31 as a receptor for interactions with LN-1, and cells
transfected with 3 do not acquire the ability to attach to LN-1
(Delwel et al., 1994; Shaw et al., 1996). In contrast, there are
numerous cases in which 31 is used by cells, often including
neurons, as a receptor for LN-2/4 (Tomaselli et al., 1993), LN-5 (Smith
et al., 1996), and LN preparations likely to contain isoforms other
than LN-1 (Gehlsen et al., 1989; Delwel et al., 1994), as well as for
other extracellular matrix proteins such as thrombospondin-1 (DeFreitas
et al., 1995). These data are consistent with the view that there is an
31 binding site on LN-1 but that it is normally not accessible to
cells unless LN-1 is altered by antibody or proteolytic activation.
Interestingly, whereas the inability of retinal neurons to use 31
to respond to unaltered LN-1 could potentially be ascribed to
inaccessibility of the receptor binding site, the same cannot be said
for 61, which late embryonic retinal neurons also seem to use to
respond only to activated, and not untreated, LN-1. This is because
61 is known to be used by many cell types, including neurons, as
a receptor for (unmanipulated) LN-1 (Hall et al., 1990; Sonnenberg et
al., 1990; Calof et al., 1994). Indeed, 61 has been identified as
the major LN-1 receptor used by early embryonic retinal neurons before
they lose LN-1 responsiveness (de Curtis et al., 1991; de Curtis and
Reichardt, 1993). Accordingly, there is little reason to believe that
the 61 binding site on LN-1 is inaccessible to cells in
unmanipulated LN-1 molecules. Thus, activation of LN-1 seems to affect
the interaction of cells with receptor binding sites that cannot
normally be considered cryptic. It is interesting that only late
embryonic, and not early embryonic, retinal neurons require LN-1 to be
activated before 61-mediated responses can be elicited (de Curtis
et al., 1991; de Curtis and Reichardt, 1993). This difference might
reflect developmental changes in molecules that control the cell
surface exposure or activation state of integrins, such that some
threshold for signaling is no longer reached by unactivated LN-1.
Structurally, the 3 and 6 integrin subunits are more closely
related to each other than to other integrin subunits (Schwartz et
al., 1995), yet on late embryonic retinal neurons, 31 is concerned mainly with the promotion of neurite growth, whereas 61
preferentially mediates attachment and spreading (Figs. 2-7). This may
represent an intrinsic functional difference between 3 and 6, or
it may reflect differential usage in neurons of the alternative "A"
and "B" type cytoplasmic domains that either of these integrins can
possess (Tamura et al., 1991; de Curtis and Reichardt, 1993; Schwartz
et al., 1995). Interestingly, despite the functional difference
observed between 3 and 6 in normal retinal neurons, the
experiments with 3-deficient mice (Fig. 9) or with neurons derived
from other CNS regions (Fig. 10) suggest that, at least under some
circumstances, 61 can mediate neurite outgrowth as effectively as
31. It will be interesting to see whether this effect reflects
differences in the level of expression or splicing of 6.
Potential mechanisms underlying LN activation
It has been suggested previously that activation of LN-1 reflects
the blockade or removal of an inhibitory, or suppressive, domain
residing in the short arms of LN-1 (Calof et al., 1994). The mapping of
a domain involved in LN-1 activation within the N-terminal portion of
the 1 chain (Fig. 1) is consistent with the observation that LN
isoforms that lack the 1 chain [e.g., merosin (21/21) and
LN-5 (332)] do not require any activating manipulations to
promote neurite outgrowth by late embryonic retinal neurons (Figs. 3,
4, 6).
If the activation domain of LN-1 does have an inhibitory function, the
mechanism of action must involve either an effect of this domain on
cells (altering the way cells respond to the rest of the LN-1 molecule)
or an effect of this domain on the structure or accessibility of other
parts of LN-1. If this domain acts on cells, it apparently does not do
so via signaling mediated by integrins 11 and 21 (the only
known integrin receptors for this part of the LN-1 molecule), because
blocking antibodies to these integrins did not render late embryonic
retinal neurons capable of extending neurites on LN-1.
If the activation domain acts via an influence on LN-1 structure, it
must alter the activity of integrin binding sites located 50-75 nm
away, at the distal end of the long arm (Sonnenberg et al., 1990;
Gehlsen et al., 1992; Pattaramalai et al., 1996), affecting the
activities of binding sites for 31 (Figs. 3-6), 61 (Fig. 7), and probably at least one more 1 integrin (Fig. 10). One
large-scale structural change that LN-1 is known to undergo is
polymerization, a process that involves end-to-end interactions of the
short arms (Schittny and Yurchenco, 1990; Colognato-Pyke et al., 1995).
However, LN activation is unlikely to involve changes in the
polymerization state of LN-1, because antibodies to short-arm domains
of LN-1 (including those that activate LN-1) generally have little
effect on polymerization (H. Colognato and P. D. Yurchenco,
unpublished observations). Furthermore, chemically treated LN-1
that cannot polymerize also fails to promote neurite outgrowth from
late embryonic retinal neurons but is activated by anti-E1' antibodies
(J. K. Ivins, H. Colognato, P. D. Yurchenco, and A. D. Lander, unpublished observations).
Recently, a phenomenon strikingly similar to LN-1 activation was
described for LN-5. Cleavage of LN-5 at a site near the
short-arm-long-arm junction of the 2 chain changed LN-5 from a
molecule that promotes only epithelial cell attachment to one that
promotes 31-integrin-dependent epithelial cell migration
(Giannelli et al., 1997). Intriguingly, this new activity mapped to an
epitope that has since been shown to be located at the distal end of
the long arm (V. Quaranta, personal communication). It will
be interesting to determine whether the mechanism of activation of LN-5
shows similarity to that underlying activation of LN-1.
Does LN activation occur in vivo?
Whether endogenous, physiological activators of LN-1 exist is not
known. Although antibodies to the short arms of LN-1 are unlikely to be
present in vivo, at least one extracellular matrix protein,
fibulin-2, is thought to interact with LN-1 via domain IVb of the 1
chain (Utani et al., 1997), within the region defined here for antibody
activation of LN-1. Thus LN-1 activation may be a function of the ECM
molecules with which LN-1 is complexed. Alternatively, activation may
occur as the result of endogenous proteolytic cleavage. Indeed, the
LN-5 activation phenomenon referred to above has been shown to result
from an matrix metalloproteinase-2-mediated cleavage event that
can be detected in LN-5 extracted from multiple tissues (Giannelli et
al., 1997).
It is noteworthy that parenchymal LN-1 is primarily present in the CNS
only during development [in locations that include the optic pathways
followed by retinal ganglion cell axons (Cohen et al., 1986; Halfter
and Fua, 1987)], but it frequently reappears after neural injury (Zak
et al., 1987; Brodkey et al., 1993; Frisen, 1997; Fu and Gordon, 1997).
If the CNS neurons that encounter LN-1 at injury sites behave like the
CNS neurons that were tested here (retinal, thalamic, cortical, and
hippocampal), then the state of LN-1 activation could have considerable
influence over whether the ECM at those sites succeeds or fails to
promote the regeneration of axons. Likewise, if LN-1 is not normally
activated at sites of neural injury, then the possibility of activating it with exogenous agents (e.g., specific anti-short-arm antibodies) might have potential therapeutic value.
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FOOTNOTES |
Received July 30, 1998; revised Sept. 17, 1998; accepted Sept. 18, 1998.
This work was supported by National Institutes of Health Grants EY10324
and NS36049 and American Paralysis Association Grant LA2-9603 to
A.D.L. and by National Institutes of Health Grant DK48045 to P.D.Y. We
wish to thank the following for their generous gifts of antibodies:
Mike DiPer
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Abstract
Introduction
