Experimental Cell Research 274, 169 –177 (2002)
doi:10.1006/excr.2001.5463, available online at http://www.idealibrary.com on
Effects of Collagen IV on Neuroblastoma Cell Matrix-Related Functions
Athina K. Tzinia,* ,1 Paraskevi V. Kitsiou,* Argiris A. Talamagas,* Angelique Georgopoulos,†
and Effie C. Tsilibary*
*Institute of Biology, National Center for Scientific Research “Demokritos,” 15310 Agia Paraskevi, Athens, Greece; and
†Veterans Administration Medical Center, Minneapolis, Minnesota 55455
Integrin-mediated interactions with collagen IV and
its domains were examined in a human neuroblastoma
cell line (SK-N-SH). By adhesion assays we demon-
strated that neuroblastoma cells bound to solid-phase
intact collagen IV and synthetic cell-binding peptide
HEP-III, derived from the collagenous part of the mol-
ecule, but not to the main noncollagenous NC1 domain
or to the synthetic cell-binding peptide HEP-I, derived
from this domain. Monoclonal antibodies against ␤1,
␣3, and ␣v␤3 integrins resulted in inhibition of cell
binding to collagenous substrates by 95, 30, and 35%,
respectively. By flow cytometry and immunoblotting it
was shown that culture of SK-N-SH cells on collagen IV
resulted in alteration in the expression of major neu-
roblastoma cell integrins. Binding to collagen IV in-
duced the expression and activation of matrix metallo-
proteinases A and B (MMP-2, MMP-9), with a
concomitant increase at the protein level of tissue-spe-
cific inhibitors of metalloproteinases (TIMP-1, TIMP-2).
Finally, the expression of MMP-2 was significantly up-
regulated by anti-␣3␤1 antibodies, whereas ligation of
anti-␣v␤3 antibodies resulted in a modest down-regula-
tion of MMP-2. Our results indicate that the presence of
collagen IV modulates the expression of integrins, which
are used for binding to this glycoprotein, and MMP-2
secreted by SK-N-SH cells. © 2002 Elsevier Science (USA)
Key Words: collagen type IV; adhesion; neuroblas-
toma cells; integrins; MMP-2.
INTRODUCTION
Extracellular matrix (ECM), 2 including basement
membranes, is a complex network of interacting mole-
cules such as collagens, fibronectin, and laminin. This
matrix, in addition to acting as a scaffold that stabi-
lizes the physical structure of tissues, regulates vital
cell processes, including cell migration, growth, and
1
To whom correspondence and reprint requests should be ad-
dressed. Fax: 01-6511767. E-mail: atzin@mail.demokritos.gr.
2
Abbreviations used: MMPs, matrix metalloproteinases; TIMPs,
tissue inhibitors of metalloproteinases; ECM, extracellular matrix;
PBS, phosphate-buffered saline; FACS, fluorescence-activated cell
sorting.
169
differentiation [1, 2]. Collagen IV (Col IV) not only
forms the main structural framework of most base-
ment membranes, but also has the ability to promote
cell adhesion of various cell types [3, 4], and it is
important for axonal regeneration [5]. Col IV is a trim-
mer composed of three monomeric chains with the
usual composition (␣1) 2(␣2) [6], and four isoform
chains have been described [7]. Intact Col IV, its main
noncollagenous NC1 domain, and synthetic peptides
representing sequences from the (␣1) NC1 and (␣1)
collagenous domains (HEP-I and HEP-III, respec-
tively) have been shown to interact with several cell
types mainly via integrin receptors [3, 8, 9]. Integrins
are the major family of cell-surface receptors, ex-
pressed by all cell types. These transmembrane pro-
teins consist of ␣ and ␤ heterodimers. It is considered
that each integrin has distinct ligand binding capacity
and most known integrins recognize various extracel-
lular matrix proteins, such as fibronectin, laminin, and
collagens [10 –12]. In the nervous system, integrins are
the major family of adhesion receptors, which mediate
the differentiating effects of ECM components of neu-
ronal cells during development [13]. These integrin-
mediated interactions are intimately involved in the
regulation of cell behavior and gene expression, for
example the expression of matrix metalloproteinases
(MMPs) [14, 15].
MMPs are a family of zinc-dependant proteolytic
enzymes involved in the remodeling of the ECM in a
variety of physiological and pathological processes, in-
creasingly being implicated in the pathogenesis of sev-
eral diseases of the central nervous system (CNS) such
as multiple sclerosis and Alzheimer’s disease [16]. Ac-
cording to their substrate specificity they have been
classified to gelatinases, stromelysines, and collag-
enases [17, 18]. The 92-kDa gelatinase B (MMP-9) and
the 72-kDa gelatinase A (MMP-2) efficiently degrade
native collagens such as types IV and V, fibronectin,
entactin, and elastin [19, 20]. The proteolytic activity of
MMP-9 and MMP-2 is in part regulated by the action of
tissue inhibitors of metalloproteinases TIMP-1 and
TIMP-2, respectively [21]. One of the mechanisms in-
volved in the regulation of the expression of MMPs is
0014-4827/02 $35.00
© 2002 Elsevier Science (USA)
All rights reserved.
170 TZINIA ET AL.
via integrin-mediated signaling [22, 23]. Regulation of
the expression of MMPs, and the expression of their
specific inhibitors (TIMPs), represents a mechanism
for controlling ECM turnover, in addition to the ex-
pression and secretion of ECM components by the cells.
The aim of the present study was to examine the
effect of Col IV, an important component for neuronal
cell differentiation and axonal regeneration, on the
expression of specific integrin subunits, which are
present on the surface of SK-N-SH cells, and on the
expression of proteins involved in the degradative
pathways of ECM.
MATERIALS AND METHODS
Cell cultures and treatments. The human neuroblastoma cell line
SK-N-SH was cultured in minimum essential medium Eagle with 2
mM L-glutamine adjusted to contain 0.1 mM nonessential amino
acids, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, 10% fetal
bovine serum, and antibiotics.
To estimate the expression of MMPs and TIMPs in the presence of
Col IV, 2 ⫻ 10 6 cells were cultured in 58-cm 2 tissue culture dishes
precoated with 50 ␮g/ml Col IV (⬃1 ␮g/cm 2) for 48 h. The medium
was then removed, the dishes were washed three times with serum-
free medium, and cells were incubated for 24 h in 10 ml serum-free
medium. At the end of the 24-h incubation period, the conditioned
media were transferred to 15-ml conical tubes, centrifuged to remove
cells and cell fragments, supplemented to 1 mM Na 2EDTA and 0.02%
sodium azide, and stored at ⫺20°C for further use.
Antibodies and substrates. The following antibodies were used in
this study: Rabbit anti-human polyclonal antibodies to the integrin
subunits ␣2, ␣3, ␣5, ␣v, ␤1, and ␤3 were purchased from Chemicon
International. Monoclonal antibodies against ␣3 (P1B5), ␤1 (P5D2),
␣2 (P1E6), and ␣v␤3 (LM609) integrins were also purchased from
Chemicon International. Well-characterized polyclonal antibodies
Ab45 and Ab110 [24, 25] against the collagenases MMP-2 and
MMP-9, respectively, and antibodies against their inhibitors TIMP-1
and TIMP-2 were kindly provided by Dr. Stetler-Stevenson (NCI,
National Institute of Health, Bethesda, MD).
Intact Col IV was isolated from the Engelbreth–Holm–Swarm
tumor system using previously described techniques [26]. The car-
boxyl terminal noncollagenous domain 1 (NC1) was prepared by
digestion with bacterial collagenase as previously described [27].
Peptides derived from the sequence of either NC1 (HEP-I) or triple
helix-rich domains (HEP-III) were synthesized according to the
method of Barany and Merrifield and purified through an HPLC
reverse-phase column as described previously [26].
Total protein extraction. Cells grown on culture dishes either
precoated or not with 50 ␮g/ml Col IV (⬃1 ␮g/cm 2) were collected by
treatment with trypsin/EDTA and lysed in PBS, pH 7.4, containing
1 mM phenylmethylsulfonyl fluoride, 1 mM N-ethylmalemide, 1%
Triton X-100, 1 mM CaCl 2, and a cocktail of protease inhibitors
(Sigma P8340) for 30 min at 4°C. Insoluble material was removed by
centrifugation and the supernatant was stored at ⫺20°C. Protein
estimation was done by the method of Bradford (Pierce).
Immunoblotting. Electrophoresis in the presence of SDS was per-
formed according to the method of Laemmli [28] on either 7.5 or 10%
polyacrylamide gels, under reducing or nonreducing conditions, as
indicated in the figure legends. The resolved proteins were subse-
quently transferred to Hybond–ECL nitrocellulose membrane (Am-
ersham) as described by Towbin et al. [29]. Blots were saturated for
2 h at room temperature with 5% nonfat milk in Tris-buffered saline,
0.1% Tween 20 and incubated overnight at 4°C with the appropriate
dilutions of polyclonal antibodies, in the same buffer without Tween
20. Incubations with peroxidase-conjugated anti-rabbit immuno-
globulins and detection of bound peroxidase activity were carried out
as described in the ECL-blotting detection system (Amersham). Im-
ages of Western blots were analyzed using image-processing soft-
ware (Bioprofil Vilber Loumart).
Flow cytometry. Expression of integrin subunits was evaluated
by indirect immunofluorescence staining and flow cytometry. Briefly,
cells were released from confluent monolayer cultures (either in the
presence or in the absence of Col IV) with trypsin, washed, and
resuspended in FACS buffer (2% fetal calf serum, 0.02% sodium
azide in PBS); 1 ⫻ 10 6 cells were incubated with the primary anti-
body for 45 min on ice and washed twice with FACS buffer. Anti-
mouse IgG conjugated with fluorescein isothiocyanate was then
added in a total volume of 0.1 ml FACS buffer, and cells were
incubated for 45 min on ice. After washing twice with FACS buffer
the cells were fixed with 1% formaldehyde in PBS. Analysis was
performed using CELL QUEST software on a FACScan (Becton–
Dickinson). Positive fluorescence was determined on a four-decade
log scale and fluorescence intensity (log F1) was expressed as the
mean channel number of 10,000 cells.
Cell adhesion assays. Ninety-six-well plates were coated with 50
␮l/well of increasing concentrations of various substrates (0.3–100
␮g/ml) in PBS for 24 h at 29°C to complete dryness, followed by a 2-h
incubation at 37°C in 0.5 mg/ml bovine serum albumin in PBS to
cover uncoated sites on plastic. Neuroblastoma cells were cultured
until they reached ⬃80% confluency. The cells were metabolically
labeled overnight with 0.15 mCi of [ 35S]methionine per T-25-cm 2
flask. Cells were released with trypsin/EDTA, washed, resuspended
in binding buffer (Dulbecco’s modified Eagle’s medium, 2 mg/ml
bovine serum albumin, 20 mM Hepes, pH 7.5), and plated at a
density of 5000 cells/well. Adhesion was allowed to occur for 30 – 60
min at 37°C. At the end of the incubation period, nonadherent cells
were removed by washing with binding buffer, the adhering cells
were lysed and transferred to scintillation vials, and radioactivity
was measured with a ␤-counter. Percentage adhesion of cells was
calculated as (cpm of cells adhered per well/cpm of 5000 cells) ⫻ 100.
For competition assays, cells were preincubated in suspension
with appropriate concentrations of monoclonal antibodies against
human integrins for 30 min at 4°C and then plated to wells and
allowed to adhere for 30 min. Inhibition of adhesion was calculated
as a percentage of the cell adhesion in the absence of antibodies. Cell
adhesion and competition experiments were done in six replicates
and repeated a minimum of three times.
Zymography. Gelatin zymography was performed as previously
described [30]. Briefly, aliquots of each sample of conditioned me-
dium were subjected to SDS–PAGE under nonreducing conditions in
10% polyacrylamide gels containing 0.1% gelatin. The volume of
conditioned medium loaded per lane was adjusted according to the
cell number obtained at harvest. After electrophoresis, SDS was
removed from the gel by washing in 50 mM Tris–HCl, pH 7.5, 5 mM
CaCl 2, 1 ␮M ZnCl 2, 2.5% Triton X-100, and 0.02% NaN 3 for 3⫻ 30
min at room temperature. The gel was then incubated in the same
buffer excluding Triton X-100 for 48 h at 37°C. After staining with
Coomassie brilliant blue R-250 for 3 h and destaining in water,
gelatin-degrading enzymes were identified by their ability to clear
the gelatinous substrate at their respective molecular weights.
Incubation of cells with antibodies. Cells were detached from
confluent monolayer cultures by treatment with trypsin/EDTA, col-
lected by centrifugation, resuspended in culture medium (3 ⫻ 10 6
cells/ml), preincubated with anti-␣3, -␣2, and -␣v␤3 monoclonal an-
tibodies at a concentration of 40 ␮g/ml each and anti-␤1 (0.5 ␮g/ml)
for 30 min at 37°C, and then diluted 10-fold and cultured for 24 h on
24-well plates precoated with 50 ␮g/ml Col IV. Control cells were
cultured in the absence of antibodies. The medium was then re-
moved, the plates were washed three times with serum-free medium,
and cells were incubated for 24 h in serum-free medium in the
absence or presence of 4 ␮g/ml of each anti-␣3, anti-␣2, and anti-
 NEUROBLASTOMA CELLS–COLLAGEN IV INTERACTIONS
FIG. 1. Adhesion of neuroblastoma cells to substrates. (A)
[ 35S]methionine-labeled cells were seeded in 96-well plates (5000
cells/well) coated with 20 ␮g/ml of either solid phase collagen IV or
NC1 or 50 ␮g/ml peptides HEP-I and HEP-III. Cells were allowed to
adhere for 30 min to 1 h at 37°C. Adherent cells were lysed, and
radioactivity was quantitated as stated under Materials and Meth-
ods. Percentage adhesion of cells was calculated as (cpm of cells
adhered per well/cpm of 5000 cells) ⫻ 100. Nonspecific adhesion of
cells to bovine serum albumin, which was less than 1%, was sub-
tracted from adhesion to substrates. (B) In dose–response cell adhe-
sion, cells were allowed to adhere to wells coated with increasing
concentrations of substrates for 30 min. Results are expressed as
mean ⫾ SD of six replicates, from three experiments. Differences
were significant at P ⬍ 0.05.
␣v␤3 monoclonal antibody (mAb) and 0.05 ␮g/ml anti-␤1 mAb. At the
end of the 24-h incubation period, the conditioned media were col-
lected for zymography.
Densitometric analysis. Densitometric analysis of images of
Western blots and zymograms was performed using image-process-
ing software (Bioprofil Vilber Loumart).
Statistical analysis. Mean values were derived from experiments
performed in triplicate. These values were compared using the Stu-
dent t test. A P value of ⬍0.05 was considered statistically signifi-
cant.
RESULTS
Neuroblastoma Cell Adhesion to Substrates
We examined neuroblastoma cell binding to intact
Col IV, its noncollagenous domain NC1, and two syn-
thetic peptides, HEP-I and HEP-III, which were previ-
ously reported to promote adhesion of several cell
types. Figure 1A shows that neuroblastoma cells ad-
hered to intact Col IV up to 45% after 1 h and up to 30%
to the synthetic peptide HEP-III, derived from a cell-
171
binding amino acid sequence which represents an in-
terruption of the Gly-X-Y sequence of the ␣1 chain of
Col IV from the collagenous part of the ␣1 (IV). In
contrast, within the same time interval the percentage
adhesion to the major noncollagenous NC1 domain and
to the synthetic peptide HEP-I, the sequence of which
was derived from the ␣1 (NC1-IV) domain, was ob-
served to be only 5 and 3%, respectively. This indicates
a preference of SK-N-SH neuroblastoma cells to bind to
the collagenous domain of the molecule rather than to
the noncollagenous NC1 domain. NC1 and HEP-I have
been reported to promote adhesion to other cell types,
such as human mesangial cells, sympathetic neurons,
and aortic endothelial cells [8, 9, 31]. In dose–response
cell adhesion experiments (Fig. 1B), the highest per-
centage adhesion (⬃25% over 30 min) was achieved
when intact Col IV was plated at a concentration of 20
␮g/ml and did not increase further. Results obtained
using the synthetic peptide HEP-III as substrate indi-
cated that, for concentrations of HEP-III ranging from
0.3 to 100 ␮g/ml, maximum adhesion (⬃20%) was ob-
served at 50 ␮g/ml of peptide, following an incubation
of 30 min.
Integrins Mediating the Adhesion of Neuroblastoma
Cells to Col IV and Peptide HEP-III
To identify the integrin receptors of neuroblastoma
cells, which are involved in the interaction of these
cells with Col IV and HEP-III, we performed adhesion
experiments in the presence of well-characterized
monoclonal antibodies, directed against various inte-
grin subunits expressed by this cell line. A panel of
monoclonal antibodies was used, all of which were pre-
viously reported to inhibit the adhesion of different
cells types to various substrates [9, 32, 33]. Antibodies
were used at increasing concentrations, ranging from
0.3 to 10 ␮g/ml, whereas the selected substrate concen-
trations promoted half-maximal binding of cells. These
concentrations were 10 ␮g/ml for Col IV and 25 ␮g/ml
for peptide HEP-III. Cells were preincubated with
monoclonal antibodies at 4°C for 30 min before plating.
Inhibition of adhesion was determined compared to
adhesion in the absence of antibodies considered as
100%. From the examined panel of ␣2, ␣3, ␤1, and
␣v␤3 anti-integrin antibodies used, as shown in Fig. 2,
the maximal observed inhibition on both substrates
was obtained by the use of anti-␤1 integrin antibody.
Almost up to 100% inhibition was observed at concen-
trations of antibody higher than 1.25 ␮g/ml. At the
same concentration, anti-␣3 antibody resulted in 30
and 35% inhibition of adhesion on Col IV and HEP-III,
respectively. Anti-␣v␤3 antibody resulted in inhibition
of adhesion on both substrates similar to that of
anti-␣3 (⬃30%), but at eightfold higher concentrations.
172 TZINIA ET AL.
FIG. 2. mAb inhibition of neuroblastoma cell adhesion to sub-
strates. [ 35S]methionine-labeled cells (5000 cells/well) were coincu-
bated in suspension with the indicated concentrations of integrin
mAbs anti-␣3 (P1B5), anti-␣v␤3 (Lm609), and anti-␤1 (P5D2) at 4°C
for 30 min before plating. The cells were then allowed to adhere on
wells precoated with Col IV (10 ␮g/ml) or HEP-III (25 ␮g/ml) for 30
min at 37°C. At the end of the incubation period, adherent cells were
lysed, and radioactivity was quantitated. Bound counts were ex-
pressed as a percentage of the counts bound in the absence of mAb,
to give percentage adhesion. Results are expressed as mean ⫾ SD of
six replicates, from three experiments. Differences were significant
at P ⬍ 0.05.
Anti-␣2 antibodies had no effect on the adhesion on
either substrate in all concentrations used.
Integrins Expressed in the Presence of Collagen IV
Integrin expression in neuroblastoma cells, in the
presence or absence of Col IV, was examined by flow
cytometry and immunoblotting. Initially, fluorescence-
activated cell sorting was performed in nonpermeabi-
lized cell populations to verify the cell surface avail-
ability of specific integrin subunits expressed by SK-
N-SH neuroblastoma cells in the absence or presence of
Col IV, by the use of specific anti-integrin monoclonal
antibodies. Cells were allowed to grow for 48 h on
either plastic or culture dishes precoated with 50 ␮g/ml
Col IV. From the panel of integrin subunits examined
(␣2, ␣3, ␣5, ␣v, ␤1, and ␤3), 96% of cells grown on
plastic expressed ␤1 integrin subunit at high density
on cell surface. Forty-five and thirty percent of cells
expressed the ␣3 and ␣2 subunits, respectively, while
the percentage of ␣v␤3-positive cells was about 20%
(Fig. 3A). In the presence of Col IV as substrate, the
levels of ␣3 and ␤1 integrin were decreased by 29 and
55%, respectively, whereas the levels of ␣2 and ␣v␤3
were not significantly changed (Fig. 3B). Under both
experimental conditions SK-N-SH cells did not express
␣5 integrin subunits.
To confirm the above results at the protein level,
immunoblotting was performed on lysates from cells
cultured either on plastic (Fig. 4A, lanes 1, 3, 5, 7, and
9) or on 50 ␮g/ml Col IV (Fig. 4A, lanes 2, 4, 6, 8, and
10) and probed with the use of specific anti-integrin
polyclonal antibodies. Densitometric analysis of the
blots (Fig. 4B) confirmed the results obtained by
FACscan analysis.
Induction of the Expression of MMPs and TIMPs
To determine whether the interaction of neuroblas-
toma cells with Col IV induced the expression of pro-
teins involved in the degradative pathway, we per-
formed gelatin zymography and Western blotting on
conditioned media from cultures of cells grown either
on plastic or on immobilized Col IV substrate. Gelatin
zymography is a sensitive technique for the direct de-
tection of any MMPs that can degrade gelatin. Cells
cultured on plastic synthesized and secreted high lev-
els of 72/68-kDa MMP-2 (Fig. 5, lane 1), but very low
levels of MMP-9, detected only after loading 10⫻ con-
centrated amounts of conditioned medium for zymog-
raphy (data not shown). When cells were allowed to
grow on Col IV for 48 h, the levels of 72/68-kDa MMP-2
increased by 2-fold, and the 92/88-kDa MMP-9 was
also detected in both the pro-MMP and the active
forms, albeit in smaller amounts compared to MMP-2
(Fig. 5, lane 2). To confirm the above results, 10-fold-
concentrated conditioned media were blotted with spe-
cific antibodies against MMP-2 and MMP-9 proteins.
Media assessed by Western blotting showed that
MMP-9 was secreted by cells grown on plastic but at
very low levels compared to the levels of MMP-2 (Fig.
6A, lanes 1 and 3, respectively). In addition, the acti-
vated form of both proteins was present when the cells
were grown on Col IV (Fig. 6A, lanes 2 and 4).
Since TIMPs regulate in part the biological activity
of MMPs, we studied whether the expression of
TIMP-1 and TIMP-2 was modulated by Col IV. The
results obtained from Western analysis demonstrated
an increase in the protein level of TIMP-1 and TIMP-2
by 25 and 35%, respectively, expressed by cells grown
on Col IV (Fig. 6A, lanes 6 and 8), compared to the
protein levels expressed by cells grown on plastic (Fig.
6A, lanes 5 and 7). Increased expression of MMPs in
parallel with an increase in the protein levels of TIMPs
could represent a regulatory mechanism of the cells to
maintain homeostasis of the ECM environment.
Effect of Anti-integrin Antibodies on MMP-2
Expression
Since ␣3␤1 and ␣v␤3 integrins were shown to medi-
ate the adhesion of neuroblastoma cells to Col IV, and
integrins were reported to be involved in signal trans-
duction regulating MMP transcription and secretion
[14, 15, 34], we examined the possible roles of ␣2, ␣3␤1,
and ␣v␤3 integrins in the secretion of MMP-2, the
predominant collagenase expressed by these cells. For
this purpose, cells were cultured on Col IV for 48 h at
37°C, in the absence or presence of 4 ␮g/ml of each
anti-␣3, -␣v␤3, and -␣2 monoclonal antibodies, which
 NEUROBLASTOMA CELLS–COLLAGEN IV INTERACTIONS 173

FIG. 3. Fluorescence intensity distribution of integrin subunits on SK-N-SH cells. (A) Integrin expression on neuroblastoma cells grown
on plastic was analyzed using anti-␤1, -␣3, -␣2, and -␣v␤3 integrin monoclonal antibodies at saturating concentrations (10 ␮g/ml).
Fluorescein activity was examined using fluorescein isothiocyanate-anti-human IgG as second antibody. (B) Alterations in integrin expres-
sion of neuroblastoma cells, grown on Col IV (50 ␮g/ml), expressed as mean of fluorescence. Results are expressed as mean ⫾ SD from three
experiments.

were used as integrin ligands [22, 35]. Anti-␤1 mAb
was used at a concentration of 0.05 ␮g/ml, since at this
antibody concentration cell adhesion to Col IV was not
significantly affected. Conditioned media were col-
lected for zymography. As illustrated in Fig. 7, treat-
ment of cells with a combination of mAbs against ␣3
and ␤1 integrin subunits enhanced the secretion of
MMP-2 by ⬃300% (Fig. 7, lane 2), whereas treatment
of cells with anti-␣v␤3 mAb down-regulated the secre-
tion of MMP-2 by 30% (Fig. 7, lane 3) in comparison to
untreated cells (Fig. 7, lane 1). Treatment of cells with
mAb against ␣2 subunit failed to induce any significant
changes in MMP-2 secretion (Fig. 7, lane 4).
DISCUSSION
ECM influences cellular functions including adhe-
sion, migration, differentiation, and gene expression.
Collagen IV has a pivotal role in ECM interaction with
cells, in addition to providing mechanical stability by
network formation and incorporation of other matrix
components. In neuronal cells, Col IV plays an impor-
174 TZINIA ET AL.
FIG. 4. Alterations in integrin protein levels due to Col IV. (A)
Western blot analysis of integrin subunits in neuroblastoma cells
cultured either on plastic or on 50 ␮g/ml Col IV. Total protein (20 ␮g)
extracted from cells cultured on both conditions was analyzed on
7.5% SDS–PAGE under nonreducing conditions and immunoblotted
with the appropriate dilution of polyclonal antibodies against inte-
grin subunits. (B) Quantification of the protein content of each inte-
grin subunit by scanning densitometry. Each bar represents the
mean ⫾ SD of three independent experiments. Differences were
significant at P ⬍ 0.05.
tant role for axonal regeneration [5]. In the present
study, we addressed additional effects of Col IV on
neuroblastoma cell functions. The aim was to deter-
mine the domains of Col IV involved in neuroblastoma
cell binding, the surface receptor(s) participating in the
binding, and the modulation of gene expression follow-
ing interactions of SK-N-SH cells with Col IV. Col IV is
an ECM component essential during the differentia-
tion of neuronal cells [13, 36]. To unravel the role of Col
IV, adhesion experiments were performed using as
substrates intact Col IV, the main noncollagenous NC1
domain of Col IV, and the synthetic peptides HEP-I
and HEP-III derived from the ␣1 (NC1) and ␣1 (IV),
respectively. Other neuroblastoma cell lines were pre-
viously shown to bind to intact Col IV [37]. The NC1
domain of Col IV and peptides HEP-I and HEP-III
were additionally reported to promote adhesion to dif-
ferent cell types [8, 9, 38]. Peptide HEP-I originates
from the ␣1 (noncollagenous NC1) chain of Col IV and
represents a major recognition site, which binds to
adjacent Col IV molecules during the process of poly-
merization to a network. In addition, this peptide pro-
motes cell adhesion [8]. Peptide HEP-III contains a
cell-binding sequence from an interruption of the Gly-
X-Y motif in the ␣1 chain of Col IV [9]. We documented
herein that SK-N-SH neuroblastoma cells used intact
Col IV and HEP-III for binding, in a concentration- and
time-dependent manner. Larger peptide concentra-
tions were required for adhesion compared to Col IV,
apparently because of lack of conformation of this pep-
tide and because intact Col IV contains more than one
cell-binding site [9].
Domain NC1 and peptide HEP-I did not significantly
sustain adhesion of SK-N-SH cells. Thus, these cells
apparently do not use this domain for binding to Col
IV, in contrast to other neuronal cells [31]. We conclude
that SK-N-SH cell–Col IV interactions are mediated by
the major collagenous part of this glycoprotein.
We next examined which integrin subunits of neuro-
blastoma cells mediate the binding to Col IV and HEP-
III. Competition experiments were performed, in which
adhesion to Col IV was examined in the presence or
absence of monoclonal antibodies to different integrin
subunits. These experiments demonstrated that bind-
ing to both Col IV and HEP-III was mainly mediated by
the ␤1 integrin subunit (␤1 is apparently the key sub-
unit interacting with different ␣ subunits). Even small
concentrations of anti-␤1 antibody (0.3 ␮g/ml) were
able to inhibit the adhesion of neuroblastoma cells on
Col IV by almost 50%. Based on inhibition experi-
ments, the ␣3␤1 integrin is a major integrin mediating
binding to Col IV, whereas the ␣2 integrin subunit was
not apparently involved in this binding. Additionally,
the ␣v␤3 receptor mediates neuroblastoma–Col IV in-
teractions, albeit to a lesser extent, since the maximal
observed inhibition in the presence of anti-␣v␤3 anti-
body was ⬃30%. These results suggest that both ␣3␤1
and ␣v␤3 represent two of the integrin receptors me-
diating SK-N-SH–Col IV interactions. Furthermore,
the obtained results indicate that the sequence con-
tained in the synthetic peptide HEP-III represents a
major recognition site for ␣3␤1 binding of neuroblas-
toma cells to Col IV. The ␣v␤3 integrin recognized this
sequence minimally, based on inhibition experiments,
since the concentration of antibody used to inhibit cell
FIG. 5. Alterations in gelatinolytic activity of MMP-2 and
MMP-9 secreted by neuroblastoma cells, in the presence of Col IV.
Aliquots of serum-free media conditioned by cells cultured on plastic
(lane 1) or on 50 ␮g/ml Col IV (lane 2) were analyzed on 10%
polyacrylamide gel containing 1 mg/ml gelatin under nonreducing
conditions. The volume of conditioned medium loaded per lane was
adjusted according to the cell number obtained upon harvest. Gela-
tinolytic activity was detected as clear bands following incubation in
enzyme buffer and staining with Coomassie brilliant blue.
 NEUROBLASTOMA CELLS–COLLAGEN IV INTERACTIONS 175

FIG. 6. Induction of the expression of MMPs and TIMPs in the presence of Col IV. (A) Serum-free media conditioned by neuroblastoma
cells previously cultured either on plastic or on 50 ␮g/ml Col IV were collected. Aliquots of each sample were concentrated and subjected to
10% SDS–PAGE under reducing conditions. The volume of conditioned medium loaded per lane was adjusted according to the cell number
obtained upon harvest. Electrophoreticaly transferred proteins were immunoblotted using polyclonal antibodies against MMP-2, MMP-9,
TIMP-1, and TIMP-2 (B–C). Western blots were analyzed by densitometry. Each bar represents the mean ⫾ SD of three independent
experiments. Differences were significant at P ⬍ 0.05.

adhesion was eightfold higher than the concentrations
of anti-␣3 and anti-␤1 antibodies used. The ␣3␤1 inte-
grin from melanoma cells was previously shown to
recognize this peptide [38], whereas in mesangial cells
the same sequence was recognized by ␣2␤1 integrin [9].
Apparently then, the sequence represented in HEP-III
peptide is a major recognition site for more than one
integrin complex in different cell types. In the case of
SK-N-SH cells, peptide HEP-III primarily interacted
via the ␣3␤1 integrin. Our experiments cannot exclude,
however, the possibility that additional sites in the
major, triple-helical domain of Col IV may be used for
the binding of SK-N-SH cells via ␣3␤1, ␣v␤3, or addi-
tional integrin complexes. The major recognition site
for ␣v␤3 on the triple-helical domain of Col IV remains
to be elucidated.
In our experiments, the presence of Col IV resulted
in down-regulation of ␣3␤1 integrin, a major ligand for
this matrix protein. It was previously reported that
integrin receptors are regulated by matrix compo-
nents. More specifically, in the presence of high con-
centrations of laminin, the expression of laminin-bind-
ing ␣6␤1 integrin was decreased on the surface of
sensory neurons [39].
Previous studies have shown the ability of ECM pro-
teins, mainly fibronectin, laminin, and Col I, to induce
their own degradation by promoting the production
and release of several matrix metalloproteinases by
different adherent cell types. For instance, human
rhabdomyosarcoma cells cultured on matrigel, largely
composed of laminin, induced MMP-2 activation [35],
fibrosarcoma cells promoted MMP-2 production cul-
tured on fibronectin [40], the adhesion of macrophages
to fibronectin induced MMP-9 expression [41], and en-
dothelial cells cultured on three-dimensional Col I pro-
duced increased amounts of MMP-2 protein [42]. In the
present study we demonstrated that culturing of neu-
roblastoma cells on intact Col IV, an essential matrix
component for the differentiation of neuronal cells [13,
36], not only induced the expression of both MMP-2
and, to a lesser extent, MMP-9, but also resulted in the
secretion of the activated forms of these enzymes.
It has been suggested that various integrins can
signal increased transcription of MMPs depending on
the cell type [15, 34, 43]. Based on experiments in
which cells were incubated in the presence of antibod-
ies against the ␣3 and ␤1 integrin subunits combined,
used as ligands for integrins, we propose that SK-N-SH
neuroblastoma cells mainly use the ␣3␤1 integrin re-
ceptor to up-regulate, at least in part, the expression of
MMP-2. Culturing neuroblastoma cells in the presence
of anti-␣v␤3 antibodies resulted in moderate down-
regulation of MMP-2. Thus, the two major integrins
that are used for the binding of these cells to Col IV
apparently have opposite effects on the expression of
MMP-2. This could be the result of two different sig-
naling mechanisms mediated by two different sites of
cell binding on Col IV, one of which up-regulates the
176 TZINIA ET AL.
FIG. 7. Effect of anti-integrin antibodies on MMP-2 expression
and secretion. (A) Cells in suspension were preincubated with anti-
integrin monoclonal antibodies for 30 min at 37°C as described under
Materials and Methods (lanes 2– 4) or without antibody addition
(lane 1) and then diluted 10-fold and cultured for 24 h on 24-well
plates precoated with 50 ␮g/ml Col IV. The medium was removed,
the plates were washed with serum-free medium, and cells were
incubated for 24 h in serum-free medium containing or not anti-
integrin mAbs. Aliquots of each sample of conditioned media were
analyzed by gelatin zymography (the volume of conditioned medium
loaded per lane was adjusted according to the cell number obtained
upon harvest). (B) Quantification of the 72/68-kDa form of MMP-2
using scanning densitometry.
expression of MMP-2 and the other of which has the
opposite effect. We conclude that interactions of SK-
N-SH neuroblastoma cells with Col IV lead to regula-
tion of MMP-2 expression, depending on the site of
binding and the integrins that become engaged in each
case. The binding event(s) and the ensuing result of Col
IV–neuroblastoma cell interactions would then be de-
termined, based on the needs of these cells to degrade
their immediate environment. Furthermore, Col IV-
induced increase of protein levels of TIMP-1 and
TIMP-2, which are the specific inhibitors for MMP-2
and MMP-9, respectively, suggested that this increase
could be, in part, a balancing effect to the increased
expression of MMPs.
In summary, the data presented in this report sug-
gest that neuroblastoma cell binding to Col IV is me-
diated by the main collagenous domain of this glyco-
protein and does not involve the main noncollagenous
NC1 domain. A recognition site for the ␣3␤1 integrin
was primarily the sequence of peptide Hep-III in the ␣1
(IV) chain, although additional sites should exist on
Col IV. Col IV–SK-N-SH cell interactions, apparently
mediated by ␣3␤1 and to a lesser extent by ␣v␤3 inte-
grins, modulate the expression of MMP-2 and MMP-9
and, moreover, promote the activation of both MMPs.
This could be important for pathological conditions of
the central nervous system, in which MMPs are impli-
cated, including Alzheimer’s disease.
The authors are indebted to Drs. Christina Zioudrou and George
Konidakis from the Medical School of the University of Crete, for
solid-phase synthesis of peptide Hep-III, and to K. Economou and P.
Karamessinis, for expert assistance with image processing and anal-
ysis.
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