1 Departamento de Bioquímica Médica, Universidade Federal do Rio
de Janeiro, Rio de Janeiro, 21941-590, Brazil
2 Departamento de Anatomia, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, 21941-590, Brazil
3 Departamento de Histologia e Embriologia, Instituto de Ciências
Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
21941-590, Brazil
4 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio
de Janeiro, Rio de Janeiro, 21941-590, Brazil
* Author for correspondence (e-mail: tcsampaio{at}bioqmed.ufrj.br)
Accepted 15 September 2002
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Summary |
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Key words: Laminin, Neuritogenesis, Protein kinase A
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Introduction |
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In vitro studies of the effects of laminin on cell function have been
achieved by using artificial matrices, prepared by adsorbing the protein on
the surfaces of cell-culture plastic or glassware. It is usually assumed that
adsorbed laminin will form a molecular network retaining the overall
properties of laminin matrices in vivo. This assumption, however, is not
necessarily correct. First, laminin polymerization does not correspond to
random protein aggregation, but, instead, it is a well defined assembly
process, involving interactions of specific domains of the protein
(Schittny and Yurchenco, 1990;
Yurchenco and Cheng, 1993
).
Second, there is evidence that laminin matrices assembled on either cultured
astrocytes or Schwann cells can vary their morphologies, which has been
considered of biological relevance
(Garcia-Abreu et al., 1995a
;
Garcia-Abreu et al., 1995b
;
Farwell and Dubord-Tomasetti,
1999
; Tsiper and Yurchenco,
2002
). In addition, analysis of laminin distribution in developing
rat brain revealed the occurrence of polymers of distinct morphologies also in
vivo (Zhou, 1990
).
Laminin had previously been shown to self-assemble in solution at a minimal
protein concentration of approximately 60 nM
(Yurchenco et al., 1985). More
recently, we showed that solution polymerization could alternatively be
triggered at low laminin concentrations by acidification of bulk pH
(Freire and Coelho-Sampaio,
2000
). We hypothesized that acidic polymerization may occur in
vivo in regions of the plasma membrane where circumstantially the negatively
charged groups provided by glycoproteins, glycolipids and proteoglycans reduce
the local pH (Wettreich et al.,
1999
; Freire and
Coelho-Sampaio, 2000
). In this work, we investigated whether
manipulation of solution pH generates morphologically distinct polymers from
purified laminin and whether such polymers would induce distinct phenotypes on
overlaying cells. We showed that laminin polymerized at neutral or acidic
condition self-assembled into structurally distinct matrices and that such
matrices favored either neuritogenesis or cell division through activation of
distinct signaling pathways.
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Materials and Methods |
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Light scattering and fluorescence measurements
Light scattering and intrinsic fluorescence were both measured on an
ISSPC-PCI spectrofluorometer (ISS Inc., Champaign, IL) at 35°C. In the
first case the wavelength of the incident light was fixed at 400 nm and
scattered light was collected at 90° between 350 and 450 nm. In
fluorescence measurements excitation was at 295 nm and emission between 300
and 400 nm. Laminin self-polymerization was initiated by diluting the protein
from a stock solution to a final concentration of 50 µg/ml in either 20 mM
sodium acetate, pH 4 or 20 mM Tris-HCl, pH 7 (final volume of 600 µl).
Laminin stock solutions (1 mg/ml) were kept at 4°C until they were diluted
into assay media previously warmed to 35°C in the sample compartment of
the instrument. Under these conditions, i.e. at 4°C, aggregates are not
formed in the stock solutions until the exact time when dilution in pre-warmed
buffer takes place (Yurchenco et al.,
1985).
After addition of laminin, samples were gently mixed and the measurements were taken within approximately 1 minute. Data were corrected by subtracting appropriate blanks containing buffer only. Prior to use, cuvettes were pre-treated with Repel silane overnight to avoid adherence of laminin to the quartz surface.
Preparation of laminin matrices
Laminin was diluted to a final concentration of 50 µg/ml in either 20 mM
sodium acetate, pH 4 or 20 mM Tris-HCl, pH 7, both containing 1 mM
CaCl2. Aliquots of 200 µl were immediately placed on 5.5 mm
diameter glass coverslips and incubated at 37°C for 12 hours. Coverslips
were then washed three times with PBS, pH 7 and used either directly for
immunocytochemical analysis or as a substrate for cell plating. The percentage
of laminin adsorbed on coverslips was calculated by measuring the
radioactivity remaining in solution after 12 hours of incubation of the
iodinated protein with glass coverslips. Laminin labeling was carried out
using the Iodo beads iodination reagent as described by the manufacturer.
Retinal whole-mounts
Whole mounted retinas were prepared as previously described
(Linden and Perry, 1982).
Briefly, newborn Lister hooded rat pups were instantaneously killed by
decapitation with a single cut of sharp scissors, and their retinae were
dissected out of the eyeballs with fine forceps and whole-mounted onto
gelatinized glass slides with the vitreal side up. A few radial cuts were made
with a sharp scalpel blade to flatten the retina onto the slides. Care was
taken to avoid touching the vitreal surface of the retinae to prevent
disruption of the extracellular matrix. Following fixation with 4%
paraformaldehyde in phosphate buffer pH 7.2, the retinae were processed for
laminin immunostaining as described below but omitting the permeabilization
step.
Neuron primary cultures
Primary cultures of neurons were prepared from cerebral cortex of Wistar
rats (UFRJ, Rio de Janeiro, Brazil) at embryonic day 14, as previously
described (Gomes et al., 1999;
Fróes et al., 1999
).
Briefly, single cell suspensions were obtained by dissociating cells of
cerebral cortex in DMEM/F12 medium supplemented with glucose (33 mM),
glutamine (2 mM) and sodium bicarbonate (3 mM). 100,000 cells were plated on
each laminin-coated coverslip previously placed on a 24-well plate. Neuron
cultures were kept in DMEM/F12 medium without serum or supplements for up to
24 hours at 37°C in humidified, 5% CO and 95% air atmosphere. In some
experiments, instead of dissociated cells, cortex tissue was cut into small
pieces with sharpened forceps and placed on culture wells as explants.
Explants of postnatal day 2 rat pups were also prepared.
Immunocytochemistry
For immunocytochemistry, cultured cells were fixed with 4% paraformaldehyde
for 20 minutes, washed three times with PBS and permeabilized with 0.2% triton
X-100 for 5 minutes at room temperature. After permeabilization, cells were
washed again twice with PBS. Immunocytochemistry was performed as previously
described (Garcia-Abreu, 1995a; Gomes et
al., 1999). Cells were blocked with 5% BSA in PBS for 1 hour and
subsequently incubated with the specific primary antibodies diluted in
blocking solution, overnight, at room temperature. Cells were then washed
three times with blocking solution and incubated with secondary antibodies for
2 hours, at room temperature. In the case of biotin-conjugated antibodies,
development of secondary antibodies was performed by incubating the cells with
the Texas-Red-streptavidin conjugate according to the specifications of the
manufacturer. Nuclei were labeled with DAPI. Negative controls were performed
by omitting primary antibodies. In all cases no reactivity was observed when
the primary antibody was absent. Cell preparations were mounted directly on
N-propyl gallate. The coverslips were visualized using a Zeiss Axioplan
microscope.
Trypan blue viability assay
Cell viability was assayed at 24 hours on neuronal cultures by replacing
culture medium with 0.4% trypan blue solution in PBS for 1 minute. At least 5
fields of attached cells were counted per well.
Bromodeoxyuridil incorporation and detection
Neuronal cultures were incubated for 24 hours in the presence of 1 µg/ml
of BrdU. In order to prevent BrdU interfering with cell adhesion, cortical
cells were allowed to settle for 2 hours before addition of BrdU. After 24
hours of incubation, cells were fixed with 4% paraformaldehyde for 20 minutes.
Cultures were then washed twice with distilled water and incubated in 2 N HCl
at 50°C for 15 minutes. Subsequently, neuronal cultures were washed twice
with 0.1 M borate buffer for 10 minutes at room temperature. After washing
with PBS, cells were incubated with anti-BrdU antibody as described above and
visualized using a Zeiss Axioplan microscope. The percentage of proliferating
cells was quantified by counting the percentage of labeled cells in at least
10 different fields per coverslip.
Morphometry and statistical analyses
Neurons stained for either Tau or ß tubulin were photographed in a
Zeiss Axioplan microscope. Photos were scanned and the number of neurites and
the total neurite length were analyzed using the Sigma Scan Pro Software
(Jandel Scientific). Two forms of quantitative analyses are presented. In the
first one, 100 neurons in six or seven fields chosen randomly were considered
independently of their size. In the second one, only the 30 longest neurites
were averaged. Each experiment was repeated at least three times producing
very similar results. Statistical analyses were performed using the Microsoft
Excel version 7.0. Error bars in histograms represent s.e.m.
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Results |
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In order to compare the morphologies of the polymers formed at each pH,
laminin deposited onto coverslips at either pH 7 or 4 were immunostained with
anti-laminin antibodies. Matrices formed at either pH presented clearly
distinct morphologies, as seen by comparing panels A and B with panels C and D
of Fig. 1. At pH 7, the laminin
matrix consisted mainly of large aggregates, protruding from the surface of
the coverslip, and could not be homogeneously focused on a single optical
plane (Fig. 1A,B; see also
Fig. 6A). At pH 4, laminin
produced a flat network, where regular polygons could be distinguished
(Fig. 1C,D). Laminin
organization in polygonal flat arrays had previously been observed on the
surfaces of cultured myotubes, Schwann cells and on embryoid bodies
(Colognato et al., 1999;
Lohikangas et al., 2001
;
Tsiper and Yurchenco, 2002
),
which suggests that the artificial matrix obtained at low pH is structurally
similar to some cell-assembled laminin polymers.
To investigate whether the polygonal structure of the artificial laminin
matrix produced at low pH is also found in vivo, we carried out an
immunohistochemical analysis of the surface of whole-mounted retinae. In most
tissues laminin can be assessed only in sections and therefore cannot be
visualized as a naturally deposited layer. The inner (vitreal) surface of the
developing retina expresses laminin (e.g.
Linden et al., 1999), promotes
neuritogenesis (Halfter et al.,
1987
) and can be directly accessed in the whole-mount preparation.
As seen in Fig. 1, panels E and
F, laminin at the periphery of the immature retina is also organized in a
polygonal pattern, visualized in a single optical plane. Interestingly,
laminin deposited at the center of the whole mount was not similarly organized
and presented protruding laminin aggregates, as seen on the artificial laminin
matrix produced at neutral pH. Because retinal maturation progresses from the
center to the periphery (Rapaport and Stone, 1984), it is conceivable that the
organized laminin matrix occurring at the periphery of the retina of newborn
rats is involved in early events of axonal extension from ganglion cells.
Because previous studies have implicated calcium ions in the modulation of
laminin polymerization (Schittny and
Yurchenco, 1990; Lallier and
Bronner-Fraser, 1991
), we compared the morphologies of acidic
matrices obtained in the presence of 2 mM CaCl2
(Fig. 1) with matrices obtained
in the presence of contaminating calcium only and in the presence of 2 mM
EDTA. In the absence of added calcium (contaminating Ca2+) matrices
presented an intermediate morphology, that is, flat, organized, but disrupted
polymers alternated with rare large aggregates (data not shown). In the
complete absence of calcium ions (added EDTA), matrices were not formed (data
not shown).
Neurite outgrowth on different laminin matrices
Artificial laminin matrices were compared with respect to their abilities
to promote neurite outgrowth of embryonic neurons. Dissociated cells from the
cerebral cortex of E14 rat embryos were added to coverslips previously coated
with either neutral or acidic laminin. After 24 hours, immunocytochemistry for
the neuron marker Tau revealed a clear difference between cells plated on the
two surfaces (Fig. 2A,B). On
the neutral matrix, we observed poor neuritogenesis and a clear tendency for
cells to clump together. Measurement of only the 30 longest neurites (which is
the usual procedure detailed in the literature) showed that the average
neurite length was of 104.1±6.0 and 244.9±14.9 µm for neurons
plated onto neutral and acidic matrices, respectively
(Fig. 2C). When 100 neurites
from randomly chosen cells were averaged, the respective values were
46.7±4.6 and 141.9±9.4 µm
(Fig. 2D-E). The discrepancy
between values obtained using each approach was mainly due to the fact that
there were many more cells without neurites on the neutral than on the acidic
matrix (Fig. 2F). Laminin
matrices obtained at intermediate pH values between 7 and 4 induced
neuritogenesis with increasing efficiencies, for example, matrix assembled at
pH 6 promoted significantly higher neurite outgrowth than matrix obtained at
pH 7 (data not shown). In another control we observed that neurites did not
develop on substrates coated with laminin in the presence of EDTA. Finally, we
evaluated the behavior of cortical cells plated directly onto plastic, glass
and on poly-ornithine-coated glass using DMEM/F12 in the absence of serum or
other supplements. In such conditions, cells attached very poorly to the
substrate, and the few attached cells were not viable within 24 hours (data
not shown). It is noteworthy that cortical cells cultured in Neurobasal medium
plus B-27 supplement (Invitrogen) attach and develop neurites after 48 hours
on either untreated or on poly-ornithine or laminin-coated substrates (data
not shown).
Further, we compared neuritogenesis induced by the two laminin matrices using brain explants. In this case, overall neurite outgrowth was again more pronounced at pH 4 than at pH 7 (Fig. 3). As also seen in Fig. 2, neuronal cell bodies clumped on the neutral (Fig. 3B) but not on the acidic matrix. The few isolated cells seen on the neutral matrix had short neurites (Fig. 3A, inset). It is interesting to note that both laminin matrices were capable of promoting cell migration out of the explant, although migration on the acidic matrix seemed slightly more effective.
Closer inspection of isolated neurons revealed that neurites appearing in
the acidic matrix after 6 hours of culture already have multiple fine
filopodia along their axes, whereas neurites formed on the neutral matrix
remained unbranched even after 24 hours. Furthermore, growth cones in the
acidic matrix exhibited lamellipodia and filopodia extending for large areas,
whereas growth cones from neurons on the neutral matrix were reduced, usually
without filopodia (Fig. 4). The
increase in the lamellar and filopodial form of neurons on the acidic matrix
could be due to superior adhesive properties of this matrix. To test this
hypothesis we measured cell attachment to each matrix at different times
either using 35S-labeled neurons or by directly counting cells
remaining in the culture supernatant
(Lallier and Bronner-Fraser,
1991). Twenty minutes after plating, cell attachment to either
neutral or acidic matrix was about 60%. Two hours later, attachment increased
to 80% in both cases. These results indicate that differential effects of
laminin matrices on cell morphology were not dictated by selectivity of cell
attachment.
Neuritogenesis was additionally investigated at increasing incubation times ranging from 2 to 24 hours (Fig. 5). The kinetics of neurite outgrowth on each substrate were significantly different: on the acidic matrix neurites grew linearly with time up to 24 hours, on laminin assembled at neutral pH neurite extension tended to stabilize at an average length of approximately 100 µm at around 16 hours.
|
Cell growth on distinct matrices
Comparison of panels A and B on Fig.
2 shows that, besides a lower neuritogenic potential, the
neutral-assembled laminin matrix induced clumping of cell bodies. The
appearance of clumped cells could be attributed to several effects. First, it
could be due to cell death. To examine this possibility, we estimated cell
viability using trypan blue in both conditions and found that after 24 hours
more than 90% of the cells were viable in both cases (data not shown). As
second possibility is that cell bodies may preferentially attach to the large
laminin aggregates present on neutral matrices. This could be the case because
clumps appearing 24 hours after plating cells over the neutral matrix indeed
colocalize with large laminin aggregates
(Fig. 6). Nevertheless, after 2
hours, a time frame sufficient to guarantee maximal cell attachment (see
above), clumps were still not present, ruling out this possibility
(Fig. 5, inset). We thus
hypothesized that clumps could be due to proliferating cells. To test this
hypothesis we treated cells with the thymidine analogue, BrdU, which labels
cells in S phase. Visual inspection revealed that the majority of cells in
contact with the neutral matrix showed overlapping labeling for both the
nuclear marker DAPI and for BrdU, whereas only a fraction of cell nuclei
stained with DAPI was co-stained for BrdU on the acidic matrix
(Fig. 7). Quantitative analysis
showed that the fractions of proliferative cells in culture after 24 hours
were 60 and 30% for cultures on neutral and acidic matrices, respectively
(Fig. 7G). The observation that
the neutral matrix supported a larger fraction of proliferating cells was
confirmed by directly counting cell number at increasing times. On the neutral
matrix, cells doubled after 2 hours and then increased three-fold in the
following 22 hours, leading to a total increase of six-fold in 24 hours. In
the same time interval cells plated on the acidic matrix did not increase in
number. Taken as a whole these results indicate that, although the two laminin
matrices promote cell adhesion with similar efficiency, the acidic matrix
favors neuritogenesis while the neutral matrix favors cell proliferation.
Differential effects of kinase inhibitors upon neurite outgrowth on
distinct laminin matrices
To investigate whether neuritogenesis on the two laminin matrices involves
similar signal transduction pathways, we tested the effects of selective
inhibitors of protein kinases upon neuritogenesis of embryonic cortical cells
plated on each matrix. Staurosporine, a wide spectrum inhibitor of protein
kinases, with particular effects upon protein kinase C (PKC) and myosin light
chain kinase (MLCK), reduced the average neurite length by 40% when added to
cultures on the neutral laminin matrix
(Fig. 8). On the other hand,
neuritogenesis on the acidic matrix was not significantly affected by this
inhibitor. Conversely, H-89, which inhibits protein kinase A, did not affect
neuritogenesis on the neutral matrix but decreased the average neurite length
from a mean value of 244.9 to 126.2 µm when tested in cultures plated on
the acidic matrix. These results are in agreement both with previous studies
using artificial matrices assembled in neutral buffer, which reported that
modulation of neuritogenesis by laminin involved activation of protein kinase
C (Bixby, 1989;
Ary-Pires and Linden, 2000
), as
well as with evidence for the involvement of MLCK in neurite growth
(Jian et al., 1996
; Ramaker et
al., 2001). Notwithstanding, our data raise the possibility of alternative
activation of protein kinase A in the transduction of neuritogenic signals.
Based on the emerging concept that increased intracellular levels of cyclic
AMP can prompt neurons to neuroplasticity events
(Bailey et al., 1996
;
Cai et al., 2001
), we
investigated whether contact with the acidic laminin matrix would promote
neuritogenesis in cells that are otherwise non-responsive to laminin. In this
investigation, we used cortical explants of newborn rats, which do not respond
to neutral laminin, when plated in total absence of serum or growth factors
(see below).
Migration and neurite outgrowth of postnatal neurons
Explants of cerebral cortex of postnatal day 2 rats were plated either on
the neutral or on the acidic laminin matrix and examined after 24 hours. As
seen in Fig. 9, explants did
attach to the neutral matrix but cells were virtually unable to migrate or
develop neurites. On the other hand, cells from explants plated on the acidic
matrix migrated outwards and developed long neurites. Since virtually all
migrating cells were double-labeled for the nuclear marker DAPI and for the
neuronal cytoskeletal protein Tau, it is likely that they are committed to
neuronal differentiation
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Discussion |
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The morphologically distinct laminin matrices described here presented
different functional properties. Laminin polymers obtained at neutral pH had
already been shown to promote neuritogenesis of neurons from both the
peripheral and central nervous system
(Rogers et al., 1983,
Hammarback et al., 1988
;
Chamak and Prochiantz, 1989
).
Here we confirmed that neurite outgrowth is promoted by the neutral laminin
matrix, but we observed that neritogenesis was further increased on the acidic
matrix. A correlation between the morphology of a cell-associated laminin
matrix and its neuritogenic potential was described in cultivated astrocytes,
isolated from different regions of rat brain
(Garcia-Abreu et al., 1995a
).
Interestingly, a similar structure/function correlation occurred in brain
cells isolated at different developmental stages (E.F. and T.C.-S.,
unpublished). These observations support the notion that, in the nervous
system, glial cells can use differential laminin assembly modes to control
their biological properties in vivo. Our finding that laminin is organized in
vivo in either permissive or non-permissive morphologies, respectively, at the
periphery and center of newborn retina is consistent with this view.
Comparison of the distributions of cell bodies on the two matrices revealed
that the neutral matrix led to cell clumping. Such effect had previously been
observed in primary cultures of rat embryonic neurons either plated on laminin
(Ivins et al., 1998) or in
co-cultures with astroglial cells
(Garcia-Abreu et al., 1995a
),
and in both cases cell clumping correlated with poor neuritogenesis. Here we
showed that the clumps of cell bodies observed after 24 hours of culture
resulted from an increase in cell proliferation. It can be speculated that the
distinct functional properties of the two matrices, that is, their capacities
for favoring either neuritogenesis or cell division, are related to
differential exposure of laminin domains in the polymers. In this regard, it
has previously been proposed that distinct laminin fragments, namely E8 and P1
control neurite outgrowth and proliferation, respectively, in cultured retinal
neuroepithelial cells (Frade et al.,
1996
).
A consistent body of evidence has defined a crucial role for the isoform 1
of laminin (LN-1) as a positive cue during development of the nervous system.
Besides inducing neuronal migration and differentiation in vitro, LN-1 has
been implicated in neuritogenesis in vivo (reviewed by
Luckenbill-Edds, 1997;
Colognato and Yurchenco,
2000
). For instance, LN-1 has been shown to localize along the
routes of migrating neuroblasts and growing fiber tracts in the embryonic
brain (Liesi, 1985
;
Letourneau et al., 1988
;
Liesi and Silver, 1988
;
Hunter et al., 1992
).
Furthermore, mice lacking the laminin-specific
6 integrin chain present
abnormal laminar organization in the developing cerebral cortex and retina
(Georges-Labouesse et al.,
1998
). Late in development, neurons lose their ability to respond
to LN-1, which contributes to the impairment of regeneration in the adult
central nervous system. Lack of response to LN-1 has been ascribed to either
downregulation of integrin receptors or a decrease in their activation state
(Cohen et al., 1986
;
Cohen et al., 1987
;
Hall et al., 1987
;
de Curtis et al., 1991
;
de Curtis and Reichardt, 1993
;
Ivins et al., 2000
).
Nevertheless, adult neurons can still respond to the isolated long arm
(fragment E8) of LN-1 or to `activated' LN-1, that is, LN-1 blocked with
antibodies directed to the short arms
(Calof et al., 1994
;
Ivins et al., 1998
). In this
study we report that acidic polymerization can reverse the lack of response to
full-length LN-1 in explants of newborn rat cortex. This suggests that, upon
acidic polymerization, laminin can hide its short arms and predominantly
expose the distal region of the long arm to recognition by cellular receptors.
This hypothesis is compatible with our experimental data
(Fig. 1), showing that the
acidic matrix presents a sheet-like arrangement, which indicates prevalence of
the interactions occurring in a single spatial plane (between short arms). By
contrast, the neutral laminin matrix assembles into 3D aggregates, indicative
of higher contribution of interactions involving domains located at different
planes (between short and long arms). Several laminin receptors have been
implicated in neuritogenesis, namely integrins
1ß1,
6ß1 and
3ß1, dystroglycan, the amyloid precursor
protein (reviewed in Luckenbill-Edds,
1997
; Powell and Kleinman,
1997
) and the cellular prion protein
(Graner et al., 2000
). Except
for
1ß1, all these receptors bind to laminin sequences located at
the distal region of the long arm, which is probably available for cell
recognition in both neutral and acid matrices. Conversely, the N-terminal
globular domain of laminin
1 recognized by the
1ß1 integrin
(Colognato-Pyke et al., 1995
)
is located in a short arm and expected to be committed to laminin-laminin
interactions in the acidic polymer. It is thus conceivable that the
proliferative response of cortical cells observed here on neutral laminin
involves recognition by
1ß1 integrins.
Laminin-induced neuritogenesis involves activation of PKC
(Bixby, 1989;
Ary-Pires and Linden, 2000
).
Here we have shown that staurosporin, which inhibits both PKC and MLCK
activation, selectively inhibits neurite extension on the neutral matrix. This
result is not surprising since activation of these two kinases can be
connected to the Ras/MAP kinase signaling cascade, which mediates integrin
signal transduction (Hood and Cheresh,
2002
). In addition, ligation of dystroglycan has also been
connected to the same signaling pathway, precisely through activation of the
adaptor protein Grb-2 (Yang et al.,
1995
). Interestingly, neuritogenesis on acidic laminin was not
affected by staurosporin but, instead, by the specific inhibitor of PKA, H-89.
Increased levels of endogenous cAMP are associated with enhancement of
neuritogenesis induced by neurotrophic factor (e.g.
Meyer-Franke et al., 1995
),
whereas decreased levels have recently been identified as the cause for the
developmentally regulated loss of neuronal response to myelin
(Cai et al., 2001
). Despite the
identification of a connection between PKA and neuritogenic potential
downstream from cAMP, no correlation between activation of PKA and occupancy
of laminin receptors has been established so far. One possibility is that cell
attachment to the acidic, flat, matrix would permit better cell spreading and,
consequently, transduction of mechanochemical signals through simultaneous
engagement of integrin receptors. Such a mechanism has recently been described
in endothelial cells attached to a fibronectin substrate
(Meyer et al., 2000
).
The question of how acidification controls laminin self-assembly in vivo
remains to be answered. Despite the morphological similarities between
artificial matrices assembled at acidic pH and the natural matrix in the
retinal inner limiting membrane, it cannot be concluded that acidification
plays a role in vivo. In vitro acidification could simply mimic a putative
physiological event, such as a conformational change, thereby privileging
interactions between laminin short arms (necessary for organization into a
sheet-like matrix). Alternatively, it is possible that acidification of bulk
pH actually simulates an in vivo situation, because the accumulation of
negative charges at the outer surface of cell membranes leads to a net
decrease in local pH. If this is the case, then laminin binding to cell
receptors [in particular to integrins containing the ß1 subunit, reported
to localize to lipid rafts along with gangliosides
(Claas et al., 2001)], would
trigger acid-induced conformational changes and the corresponding
self-assembly process. In this regard, we have observed that laminin matrices
formed over films of gangliosides in neutral buffer presented the same
morphological and functional properties as the acidic matrix described here
(E.F., unpublished).
In conclusion, the alternative modes of assembly of laminin matrices lead
to differential growth of neurites, with the acidic matrix, which shows a
morphology similar to that found in vivo on a permissive surface for axonal
growth, favoring neuritogenesis over cell proliferation. These results,
together with recent findings demonstrating the pivotal role of laminin for in
vivo regeneration in the central nervous system
(Grimpe at al., 2002), point
to the possibility of using acidic laminin matrices to stimulate axonal
regeneration.
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Acknowledgments |
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