Diversity of bone matrix adhesion proteins modulates osteoblast

Morphologie (2014) 98, 53—64
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ORIGINAL ARTICLE
Diversity of bone matrix adhesion proteins
modulates osteoblast attachment and
organization of actin cytoskeleton
Les différents types de molécules d’adhésion modulent
l’ancrage des ostéoblastes et l’organisation des fibres
d’actine du cytosquelette
V. Demais a, C. Audrain a,b, G. Mabilleau a,b, D. Chappard a,∗,b,
M.F. Baslé a,b
a
Groupe études remodelage osseux et biomatériaux (GEROM), LHEA, IRIS-IBS (institut de biologie en
santé), LUNAM université, CHU d’Angers, 49933 Angers cedex, France
b
Service commun d’imageries et d’analyses microscopiques (SCIAM), IRIS-IBS (institut de biologie en
santé), LUNAM université, CHU d’Angers, 49933 Angers cedex, France
Available online 13 April 2014
KEYWORDS
Matrix protein;
Osteoblast;
Cytoskeleton;
Scanning electron
microscopy;
Immunogold labeling;
Fractal analysis;
Vitronectin;
Integrin;
Fibronectin
∗
Summary Interaction of cells with extracellular matrix is an essential event for differentiation, proliferation and activity of osteoblasts. In bone, binding of osteoblasts to bone matrix
is required to determine specific activities of the cells and to synthesize matrix bone proteins.
Integrins are the major cell receptors involved in the cell linkage to matrix proteins such as
fibronectin, type I collagen and vitronectin, via the RGD-sequences. In this study, cultures of
osteoblast-like cells (Saos-2) were done on coated glass coverslips in various culture conditions:
DMEM alone or DMEM supplemented with poly-L-lysine (PL), fetal calf serum (FCS), fibronectin
(FN), vitronectin (VN) and type I collagen (Col-I). The aim of the study was to determine the
specific effect of these bone matrix proteins on cell adherence and morphology and on the
cytoskeleton status. Morphological characteristics of cultured cells were studied using scanning
electron microscopy and image analysis. The heterogeneity of cytoskeleton was studied using
fractal analysis (skyscrapers and blanket algorithms) after specific preparation of cells to expose
the cytoskeleton. FAK and MAPK signaling pathways were studied by western blotting in these
various culture conditions. Results demonstrated that cell adhesion was reduced with PL and
VN after 240 min. After 60 min of adhesion, cytoskeleton organization was enhanced with FN,
VN and Col-I. No difference in FAK phosphorylation was observed but MAPK phosphorylation
was modulated by specific adhesion on extracellular proteins. These results indicate that
Corresponding author.
E-mail address: [email protected] (D. Chappard).
1286-0115/$ – see front matter © 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.morpho.2014.02.003
54
V. Demais et al.
culture conditions modulate cell adhesion, cytoskeleton organization and intracellular protein
pathways according to extracellular proteins present for adhesion.
© 2014 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS
Protéines
matricielles ;
Ostéoblaste ;
Cytosquelette ;
Microscopie
électronique à
balayage ;
Immunomarquage à
l’or ;
Analyse fractale ;
Vitronectine ;
Intégrine ;
Fibronectine
Résumé L’interaction des cellules avec la matrice extracellulaire est un événement essentiel
pour la différenciation, la prolifération et l’activité des ostéoblastes. Dans l’os, l’adhérence
des ostéoblastes à la matrice osseuse est nécessaire pour déterminer les activités spécifiques de
ces cellules et pour qu’elles synthétisent les protéines matricielles. Les intégrines sont les principaux récepteurs cellulaires impliqués dans la liaison des cellules aux protéines matricielles
telles que la fibronectine, le collagène de type I et la vitronectine (via la séquence RGD).
Dans cette étude, des cultures de cellules de type ostéoblastique (Saos-2) ont été réalisées
sur des lamelles de verre recouvertes de différents supports de culture : DMEM seul ou DMEM
supplémenté avec de la poly-L-lysine (PL), sérum de veau fœtal (FCS), fibronectine (FN), vitronectine (VN) et collagène de type I (Col-I). Le but de l’étude était de déterminer l’effet
spécifique de ces protéines matricielles sur l’adhérence des cellules, leur morphologie et sur
leur cytosquelette. Les caractéristiques morphologiques des cellules en culture ont été étudiées
en microscopie électronique à balayage et analyse d’images. L’hétérogénéité du cytosquelette
a été étudiée par analyse fractale (algorithmes des gratte-ciel et des couvertures) après une
préparation spécifique des cellules pour exposer le cytosquelette. Les voies de signalisation
FAK et MAPK ont été étudiées par western blot dans ces différentes conditions de culture. Les
résultats ont montré que l’adhésion cellulaire a été réduite avec PL et VN après 240 minutes.
Après 60 minutes, l’organisation du cytosquelette a été optimisée sur les lamelles recouvertes
de FN, VN et Col-I. Aucune différence dans la phosphorylation de FAK n’a été observée mais
la phosphorylation MAPK était modulée par l’adhérence spécifique aux protéines extracellulaires. Ces résultats indiquent que les conditions de culture modulent l’adhésion cellulaire,
l’organisation du cytosquelette et les voies de signalisation intracellulaire en fonction des
protéines extracellulaires présentes qui permettent l’adhérence des cellules ostéoblastiques.
© 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction
Osteoblasts are the cells responsible for bone formation.
They synthesize numerous proteins of the extracellular bone
matrix and are involved in the process of mineralization.
Osteoblasts are generated from bone marrow osteoprogenitors and several hormones and growth factors contribute
to their differentiation and activity. However, interaction of
cells with the extracellular matrix is an essential event for
differentiation, proliferation and activity of osteoblasts [1].
Osteoblasts are linked to the bone matrix via cell surface
receptors (mainly integrin) that bind the specific arg-gly-asp
amino-acid sequence (RGD) shared by some glycoproteins
of the matrix (thrombospondin, fibronectin, vitronectin,
type I collagen, fibrilline and osteopontin, bone sialoprotein [belonging to the SIBLING family: small integrin-binding
ligand, N-linked glycoproteins]) [2]. Integrins are ∝␤ heterodimers with an extracellular domain that binds the RGD
sequence, a transmembranous part and a short intracellular
sequence linked to cytoskeleton proteins. At least 16 ∝ subunits and 8 ␤ subunits have been identified leading to more
than 20 different types of integrins. Each type of integrin is
able to bind a specific RGD-containing protein but several
integrins may also bind the same protein [3]. The major cell
vitronectin receptor is ∝V ␤3 that can also bind fibronectin
(FN); conversely, vitronectin (VN) may also link other integrins. Numerous integrins mediate cell attachment to FN,
among them ∝3 ␤1 , ∝4 ␤1 and ∝V ␤3 [4]. All these integrins
are expressed at the osteoblast surface [5]. However, in all
human osteosarcoma cells some of them may be absent. For
example, Saos-2 cells express ∝1 ␤1 but not ∝2 ␤1 [6] contrary
to MG-63 where only ∝2 ␤1 (and not ∝1 ␤1 ) is present as type
I collagen receptor [7].
When bound to their ligands, integrins transmit signals
by recruiting cytoskeleton and signaling proteins to sites
known as focal adhesions. Formation of focal adhesion provides the physical contact sites of attachment between
cells and extracellular matrix. Clustering of integrins in
focal adhesion site induces the recruitment and the phosphorylation of tensin and focal adhesion kinase (FAK) that
subsequently induces the recruitment of talin, vinculin and
∝-actinin [8,9]. Talin, vinculin and ∝-actinin link the F-actin
fibers to the plasma membrane and interact with several
proteins such as vaso-dilatator phosphoprotein (VASP) and
Arp2/3 proteins which are implicated in the regulation of
actin polymerization [10,11]. Rearrangement of F-actin bundles induced by their liaison to the integrin ␤ subunit can
induce changes in the cell shape [12,13]. This can produce
finger-like or sheet-like protrusions (respectively filopodia
and lamellipodia) [14].
Synthesis and distribution of cytoskeleton proteins have
been involved as a cause or a consequence, in osteoblast
morphology, growth, migration, attachment, signaling and
functions [15]. The growth of bone cells is associated with
changes in organization and synthesis of cytoskeleton proteins and migration implicates dynamic reorganization of the
cytoskeleton. Rearrangement and polymerization of actin
filaments are a consequence of the linkage between cell
surface receptor and bone matrix adhesion protein. As a
consequence of the involvement of osteoblast attachment
via the integrin-cytoskeleton system, signal transduction
events are activated allowing control of gene expression
Effects of matrix proteins on osteoblasts
in osteoblasts [15]. Orientation of matrix collagen fibers
deposited by osteoblasts is controlled by cell orientation, which depends of the cytoskeleton organization. In
osteoblasts, actin filaments are abundant and mainly distributed at the periphery of the cytoplasm and in the core of
the cell processes [15]. Two other cytoskeleton elements are
also present in osteoblasts, microtubules of tubulin (which
cross the cytoplasm as continuous structure for long distance between perinuclear space and plasma membrane)
and intermediate filaments involved in the nuclear anchorage [16]. Actin is a major cytoskeleton protein that plays
a central role in cell shape determination. Changes in the
pattern of the actin fibers distribution induce cell morphological changes, particularly in term of cell spreading. It may
also determine or reflect functional modifications. A growing consensus of experimental data now suggests that the
changes within cytoarchitecture of the cell and activation
of specific signal transduction pathways are linked [17].
In this study, osteoblast-like cells from human osteosarcoma (Saos-2) were cultured on FN, VN and collagen type I
(Col-I) coated coverslips. Kinetic of cell attachment, morphology and proliferation were studied and the organization
of the actin cytoskeleton was determined using light and
electron microscopy.
55
Cell culture
Saos-2 were routinely grown on 250 mm plastic dishes in
DMEM supplemented with 10% FCS, 4 mm L-glutamine and
50 U/ml penicillin/streptomycin, at 37 ◦ C in humidified and
5% CO2 atmosphere. At confluence and before experiments,
cells were FCS-deprived overnight at 37 ◦ C with 5% CO2
in a humidified atmosphere and then passaged with 0.1%
trypsin/EDTA. For experiments, the supports of culture (coverslips or plastic dishes) were coated according to the
followed conditions:
•
•
•
•
•
•
DMEM
DMEM
DMEM
DMEM
DMEM
DMEM
medium culture alone;
with 0.001% PL as unspecific substrate;
supplemented with 10% FCS;
supplemented with 2 ␮g/ml FN;
supplemented with 1 ␮g/ml VN;
supplemented with 10 ␮g/ml type I collagen.
For each condition, 100 mm plastic dishes and 25 mm
glass coverslips were sterilized by ultraviolet irradiation for
2 h and then coated by pre-incubation at 4 ◦ C, overnight,
just before experiment. The coating medium was removed
and cells were plated on coated glass coverslips at 2.5 × 105
cells per coverslip and at 2 × 105 per cm2 in 100 mm plastic dishes. For all the following experiments, cultures were
done in DMEM alone without any supplementation.
Materials and methods
Kinetic of cell adherence
Materials and reagents
Human osteoblast-like cells, Saos-2, derived from
osteogenic osteosarcoma (HTB 85) were purchased
from ATCC (Manassas, USA). Dulbecco’s Modified Eagle’s
Medium (DMEM), L-glutamine, penicillin/streptomycin and
trypsin/EDTA were all from Biomedia (Boussens, France) and
fetal calf serum (FCS) from Polylabo (Strasbourg, France).
Poly-L-lysine (PL), human fibronectin (F 0895), calf collagen
type I (C9791), hexamethyldisilazane (HMDS), 3,3 dithio bis
(propionic acid N. hydroxysuccinimide ester) (DTSP), Triton
X-100, EGTA, Piperazine-N, N -bis 2-ethanesulfonic acid
(Pipes), Dnase I (D 7291), Rnase A (R 6513), Phalloidin-TRITC
(P1951), paraformaldehyde, sodium borohydride, rabbit
polyclonal antibody against-actin (A 2066), rabbit polyclonal
antibody against — focal adhesion kinase (F-2918) were purchased from Aldrich-Sigma (Saint Quentin Fallavier, France).
The mouse monoclonal antibody against phospho-p44/42
MAPK was from Ozyme (Saint-Quentin-en-Yvelines, France).
Chloroform was from Aldrich. Human VN (12165064) and
6-well plates came from Life Technology (Eragny, France).
Bovine serum albumin (BSA) was from Euromedex (Souffelweyershein, France), PEG 6000 and glutaraldehyde
from Merck (Fontenay-sous-bois, France). Glass coverslips
were from CML (Nemours, France), and 100 and 250 mm
plastic dishes from Fisher Scientific (Elancourt, France).
DNA quantification was done on a Shimatzu spectrometer
(Kyoto, Japan). Carbon-sputtering was done with a MED 020,
Bal-Tec (Chatillon sur Cher, France). The scanning electron
microscopy (SEM) was done using a JEOL JSM-6301 F from
JEOL (Paris, France) and the fluorescent microscopy with
an Olympus Fluoview confocal microscope (Paris, France).
For each cultured condition, cells were seeded and cultivated in triplicate onto coated glass coverslips (2.5 × 105
cells/coverslip) for 5, 30, 60, 120 and 240 min at 37 ◦ C
in humidified and 5% CO2 atmosphere. Before harvesting,
cultures were rinsed in PBS and then treated with 0.1%
trypsin/EDTA. The number of cells was counted using a
Malassez cell and results were expressed as percentage of
adherent cells vs. seeded cells
For DNA content measurements, cell cultures were rinsed
in PBS and lysed in potassium hydroxide 0.6 N by 3 cycles
+37 ◦ C/−196 ◦ C. Suspensions were incubated 1 h at 37 ◦ C to
hydrolyze RNA. Hydrogen perchloride was then added at the
final concentration of 0.6 N and incubated 1 h at 37 ◦ C to precipitate total DNA and proteins. After centrifugation 10 min
at 30,000 g, pellets were solubilized in hydrogen perchloride
0.6 N and incubated 10 min at 80 ◦ C for DNA hydrolyze and
then 1 h at 4 ◦ C. After centrifugation, proteins are pelleted
and the DNA in the supernatants was quantified by dosage
at 260 nm using a spectrometer. Results were expressed in
␮g of DNA per coverslip.
Actin visualization with TRITC-labelled phalloidin
Cells were cultured onto coated coverslips (2.5 × 105
cells/coverslip) for 5, 30, 60, 120, 240 min and 24 h. After
washing in PBS, cells were fixed with 4% paraformaldehyde
in PBS for 20 min, at room temperature. Following extensive rinsing with PBS, cells were treated in the dark with
12.7 ␮M TRITC-labeled phalloidin in PBS, for 30 min. The
actin cytoskeleton was identified under confocal microscope
with a highpass BA610IF filter.
56
Scanning electron microscopy (SEM)
For morphological study and quantitative measurements,
cells were plated onto coated glass coverslips (2.5 × 105
cells/coverslip) and cultured for 5, 30, 60, 120, 240 min and
24 h. Coverslips were fixed in 2.5% glutaraldehyde in PBS,
dehydrated in graded ethanol series, desiccated in hexamethyldisilazane and air dried. Samples were carbon-coated
prior SEM examination at 5 kV. Analysis and measurements
of cell surface area were performed on at least 10 cells, from
each culture condition and time, by image analysis using SEM
photographs at the original magnification of ×1500 and NIH
Scion image for Windows.
For cytoskeleton examination, cells were plated onto
coated glass coverslips (2.5 × 105 cells/coverslip), cultured
for 5, 30, 60, 120, 240 min and 24 h and prepared according to the method of Bell et al. [18—20]. Briefly, cells were
pre-fixed during 5 min at 37 ◦ C in 0.02% DTSP in PBS. The
cytoskeleton was then prepared by extracting cells with a
non-ionic detergent, which solubilized the membrane and
revealed the cytoskeleton. Coverslips were treated for 5 min
at 37 ◦ C, with 1% Triton X-100 and 0.02% DTSP in Micro Tubule
Stabilizing Buffer, MTSB (MTSB: EGTA 1 mM, PEG 6000 4%
and Pipes 100 mM, at pH 6.9 in deionised water), and then
treated twice with 1% Triton X-100 in MTSB, without DTSP, for
5 min each, at room temperature. Cells were then incubated
in 100U/ml Dnase I and 100 ␮g/ml Rnase A in MTSB, 30 min
at 37 ◦ C. Coverslips were post-fixed in 2% paraformaldehyde
with 0.1% glutaraldehyde in PBS for 5 min, rinsed 3 times in
PBS and treated by sodium borohydride for 5 min at room
temperature to block aldehyde sites. For SEM examination
of the cytoskeleton, cells were then treated as for morphological study.
Proteins of the cytoskeleton (actin, tubulin and vimentin)
were identified using immunogold labeling. After sodium
borohydride incubation, cells were treated with 5% BSA in
PBS to block non-specific antigenic sites and rinsed in PBS
with 0.1% BSA. Cells were incubated for 2 h at 37 ◦ C with
the following first antibodies diluted at 1/100 in 0.1% BSA
in PBS: goat anti human actin, rabbit anti human tubulin or
goat anti human vimentin. Protein A conjugated with 20 nm
colloidal gold beads, diluted at 1/100 in PBS with 0.1% BSA,
was applied overnight at 4 ◦ C. The cultured coverslips were
then treated as for SEM examination.
V. Demais et al.
Dblank was computed by plotting the logarithm of the volume
against the logarithm of ␧ and searching the slope coefficient
by the least-squares method.
The ‘‘skyscrapers’’ fractal analysis [23]
Pixels that constituted an image were considered as
skyscrapers whose height was represented by the grey level.
The roof of the skyscrapers was a square of side ␧. The surface area of the image was obtained by measuring the sum
of top surfaces and sum of the exposed lateral sides of the
skyscrapers. Gray levels of adjacent pixels were then averaged in squares of ␧: 2, 4, 8, 16, and 32 pixels to produce new
images; the surface area of each image was calculated for
each ␧. The fractal dimension of the surface was determined
by plotting the logarithm of the surface against the logarithm log ␧. A linear regression line was computed only on
the aligned points by the least-squares method. The fractal
dimension was obtained as Dsky = 2 + slope [22].
Immunoprecipitation and western blot analysis
Cells were plated onto 100-mm coated plastic Petri dishes
(2 × 105 per cm2 ) for 5, 30, 60 and 120 min at 37 ◦ C in
Image analysis
Two-texture analysis were developed in VisualBasic
(Microsoft) and used sequentially on the SEM images of the
cytoskeleton.
The ‘‘blanket’’ fractal analysis [21]
We calculated the fractal dimension by using dilatation and
erosion of an image, the mathematical details have been
presented elsewhere [22]. The structural element used was
a cross rod-shaped element. Given this structuring element
of size ␧, a dilation and an erosion of the image provided two
new covering images: respectively, the upper and the lower
ones. The volume of the blanket, i.e., the volume enclosed
between the dilatation and erosion images, was measured.
The number of dilations and erosions ranged from 1 to 10 and
the volume as measured each time. The fractal dimension
Figure 1 Effects of matrix proteins on DNA contents. Cells
were grown during 5, 30, 60, 120 and 240 min in DMEM alone
(DMEM) and in presence of poly-L-lysine (PL), fetal calf serum
(FCS), fibronectin (FN), vitronectin (VN) and type I collagen
(Col-I). * P < 0.05 vs. 5 min, § vs. 30 min, vs. 60 min,
vs.
120 min.
Effet des protéines matricielles sur le contenu en ADN. Les cellules ont été cultivées pendant 5, 30, 60, 120 et 240 minutes
dans du milieu DMEM seul (DMEM) et en présence de polyL-lysine (PL), sérum de veau fœtal (FCS), fibronectine (FN),
vitronectine (VN) et collagène de type I (Col-I). Différences significatives à * p < 0,05 vs à 5 minutes, § vs 30 minutes, vs 60
minutes, vs 120 minutes.
Effects of matrix proteins on osteoblasts
57
Figure 2 Scanning electron microscopy of the effects of matrix proteins on the morphological aspect of cells cultured during 5,
30, 60 min and 24 h. Coverslips coated with poly-L-lysine (PL), DMEM alone, fetal calf serum (FCS), fibronectin (FN), vitronectin (VN)
and type I collagen (Col-I). Cell spreading increased with time, excepted on PL.
Microscopie électronique à balayage des effets des protéines matricielles sur l’aspect morphologique des cellules cultivées pendant
5, 30, 60 minutes et 24 heures. Lamelles couvre-objet revêtues de poly-L-lysine (PL), DMEM seul, sérum de veau fœtal (FCS),
fibronectine (FN), vitronectine (VN) et collagène de type I (Col-I). L’étalement cellulaire a augmenté avec le temps, à l’exception
de la PL.
serum-free medium. Cells were then solubilized in 1 ml
of lysis buffer (50 mM Tris pH 7, 100 mM NaCl, 50 mM
NaF, 3 mM Na3 PO4 , 2 ␮g/ml leupeptin, 5 ␮g/ml aprotinin,
1 ␮g/␮l pepstatin, 1 mM PMSF, 1% NP40) for 1 h at 4 ◦ C. The
lysate was centrifuged for 30 seconds at 4 ◦ C at 12,000 rpm
and the lysate protein concentrations were determined
by a colorimetric assay (Bio-Rad Laboratories, Hercules,
California) and standardized prior to further analysis.
For MAPK analysis, 15 ␮l of sample buffer (1 M Tris,
0.025% bromophenol blue, 10% SDS, 10% (v/v) glycerol, 5%
mercaptopropanediol) was added to 25 ␮l of lysate. After
boiling 10 min at 100 ◦ C, cell lysates were ready to western
blot analysis. For FAK analysis, cell lysates must previously
undergo immunoprecipitation. For immunoprecipitation,
cell lysates were incubated with primary antibody against
focal adhesion kinase overnight at 4 ◦ C with rocking,
followed by incubation with protein A-sepharose for an
additional 2 h at 4 ◦ C. Protein A-sepharose complexes were
pelleted and washed 2 times with lysis buffer, one time
with lysis buffer without detergent and one time with Tris
50 mM. Bound proteins were eluted in 40 ␮l sample buffer
(1 M Tris, 0.025% bromophenol blue 10% SDS, 10% (v/v)
glycerol, 5% mercaptopropanediol) by boiling 10 min at
100 ◦ C.
Proteins in the sample buffer were subjected to electrophoresis on a 7.5% SDS-polyacrylamide gels for FAK
analysis and on a 10% SDS-polyacrylamide gels for MAPK
analysis. Proteins were electrophoretically transferred onto
polyvinylidene fluoride membranes (Immobilon P, Millipore
Corp., Bedford, MA). Blots were blocked with 6% BSA for FAK
and 5% milk for MAPK in 200 mM NaCl and 50 mM Tris pH 7.6
overnight at 4 ◦ C, and subsequently incubated with primary
antibody (0.5 ␮g/ml) in 1% BSA/200 mM NaCl/50 mM Tris pH
7.6/0.05% Tween 20, for 5 h at room temperature. Membranes were then washed briefly and incubated with goat
anti-mouse IgG or goat anti-rabbit IgG peroxidase conjugates
58
V. Demais et al.
Figure 3 Effects of matrix proteins on cell surface area determined on cells cultured during 5, 30, 60 min and 24 h. DMEM alone
(DMEM), and in presence of poly-L-lysine (PL), fetal calf serum (FCS), fibronectin (FN), vitronectin (VN) and type I collagen (Col-I).
Cell surface significantly increased with time excepted on PL (* P < 0.05 vs. all time).
Effets des protéines matricielles sur la surface des cellules en culture pendant 5, 30, 60 minutes et 24 heures. DMEM seul (DMEM),
et en présence de poly-L-lysine (PL), sérum de veau fœtal (FCS), fibronectine (FN), vitronectine (VN) et collagène de type I (Col-I).
La surface cellulaire a considérablement augmenté avec le temps sauf avec la PL (différences significatives à * p < 0,05 vs tous les
temps).
for 1 h. Following extensive washing, immunoreactivity was
detected on film using chemiluminescent immunodetection
(ECL kit, Amersham Corp. Arlington Heights, IL). Images
from ECL autoradiograms were captured using ImageJ (NIH
image software).
Statistical analysis
Statistical analysis was done using Systat software, release
6.0.1 (SPSS, Chicago, Ltd) with a nonparametric analysis of
variance (ANOVA) with Bonferroni’s probability least significant difference post-hoc test. All data were expressed as
mean and standard deviation. Differences were considered
significant when P < 0.05.
Results
Cell attachment assays
The number of adherent cells onto the different coated coverslips was evaluated by counting with the Malassez cell
(result no shown) and the dosage of DNA content (Fig. 1).
Results were similar with the two methods. For each type
of coated coverslips, cell adherence was time dependent.
The comparison at each time did not show any important
differences until 60 min. After 60 min, cell adherence was
significantly decreased on VN and PL as compared with other
conditions. At 240 min, adherence on VN was decreased
compared to FCS, FN and Col-I and adherence on PL was
diminished versus on FN or Col-I. There were no significant
differences for other coatings.
Morphological aspects
Numerous adherent cells were observed in SEM as soon as
from 5 min onto coated supports. However on FCS-coated
coverslips scarcely distributed cells were observed (Fig. 2).
Cells were small and globulous until 30 min on DMEM, FCS,
FN, VN and Col-I but larger on PL. After 1 h, very few cells
were observed on FCS coatings. Cells appeared rounded but
some flat cells were presents on FN, VN and Col-I. On PL,
cells were numerous, large and very flat. At 4 h (results not
shown) and 24 h of adhesion, cells cultured on PL showed a
globular appearance and on the other coatings, large cells
were in majority (Fig. 2).
Cell surfaces were measured by image analysis (Fig. 3).
After 30 min, cells cultivated on the PL coating were significantly larger than on all other supports. However, their
surface significantly decreased with time while the surface
of cells cultivated on all others coated supports significantly
increased with time. After 24 h of culture, the cell surface area was significantly increased on FN vs. all other
supports.
After treatment with TRITC-phalloidin, the actin network was identified in confocal microscopy as red filaments
(Fig. 4). After 5 min of adhesion, cells were round in all
culture conditions but no organized cytoskeleton network
was observed. Actin filaments were observed first in cells
cultivated on VN, FN and Col-I from 1 h of culture. Cells
on VN showed actin filaments mainly distributed in periphery while the actin filaments were observed throughout the
cytoplasm on FN and Col-I. At 24 h, flat cells were present
on FCS, FN, VN and Col-I with an important network of actin
fibers. On PL and DMEM, cells are smaller, rounder and no
actin fibers were observed (Fig. 4).
Effects of matrix proteins on osteoblasts
59
After extraction of the plasma membrane and immunogold labeling, elements of the cytoskeleton were observed:
actin microfilaments, microtubules of ␣- and ␤-tubulin and
vimentin intermediate filaments (Fig. 5). On FCS, cells
appeared very small and round until 240 min so no cytoskeleton could be analyzed on such samples. Two regions in the
cell cytoskeleton were observed: one perinuclear and one
at the cell periphery. The perinuclear cytoskeleton did not
show any differences whatever time of culture and coated
support and was mainly represented by vimentin as identified by immunogold (Fig. 5a). After 1 h, the cytoskeleton at
the periphery of the cell showed different aspects of organization according to the coating used. Immunogold labeling
identified actin (Fig. 5b), vimentin (Fig. 5c), and tubulin
(Fig. 5d) but it was not possible to characterize each structure of the cytoskeleton so it was not possible to determine
the degree of organization of each element of the cytoskeleton (Figs. 5 and 6). On PL and FCS, cytoskeleton appeared as
a compact mesh with no individualized filament, no filipodia
or lamellipodia. On DMEM, the cytoskeleton was not really
developed, but cells send long filipodia towards other cells.
These filipodia contained a single and very long filament.
On VN, the filaments seemed to be much more polymerized
and oriented parallel to the cell profile. On FN and Col-I,
the cytoskeleton seemed to be much more oriented; individuals filaments were packaged together in large bundles
extending throughout the cell (Figs. 5 and 6).
Texture analysis provided different results according to
the algorithm used. Dblank showed few differences between
coatings and times. The heterogeneity of the cytoskeleton
was significantly different between 5 min and 24 h on PL
and Col-I (P < 0.05). At 24 h VN heterogeneity was significantly lower than on cells grown on PL or FCS (P < 0.05). Dsky
analysis revealed much more differences. The Dsky dimension, reflecting cytoskeleton heterogeneity was significantly
higher at 24 h than at 5 min for PL, DMEM, FN and Col-I
(P < 0.05). No differences were observed with FCS and VN
at any time. Dsky measured on VN was lower than on DMEM
and FCS at 240 min and 24 h and also lower than on PL, FN
and Col-I at 24 h (P < 0.05).
Western blot analysis
Figure 4 Effects of matrix proteins on actin cytoskeleton
analyzed by confocal microscopy. Cells were grown during 5,
60 min and 24 h. DMEM alone, and onto coverslips coated with
poly-L-lysine (PL), fetal calf serum (FCS), fibronectin (FN), vitronectin (VN) and type I collagen (Col-I). Clearly organized actin
cytoskeleton was detected with FCS at 24 h, FN, VN and Col-I
from 60 min.
Effets des protéines matricielles sur les fibres d’actine du
cytosquelette ; analyse par microscopie confocale. Les cellules ont été cultivées pendant 5, 60 minutes et 24 heures.
DMEM seul, et sur des lamelles recouvertes de poly-L-lysine
(PL), sérum de veau fœtal (FCS), fibronectine (FN), vitronectine (VN) et collagène de type I (Col-I). Les fibres d’actine
du cytosquelette sont clairement détectées avec le FCS à
24 heures, FN, VN et Col-I à partir de 60 minutes.
FAK phosphorylation (Fig. 7) showed no significant difference
between culture conditions at all times. However, a weaker
expression was observed on PL and DMEM until 60 min. For
each cultured condition, FAK phosphorylation seemed to
grow up with time.
MAPK phosphorylation (Fig. 8) was lower at each times in
cells plated on PL until 60 min. In cells cultured with DMEM,
FCS and Col-I, MAPK phosphorylation showed no significant
difference with time. MAPK phosphorylation decreased with
time in cells plated on FN and VN.
Discussion
In this study, the influence of extracellular proteins on
osteoblastic adhesion and shape was evaluated. Osteoblasts
synthesize numerous extracellular proteins including type I
collagen, the principal organic component of bone matrix
and a wide variety of non-collagen proteins such as
60
V. Demais et al.
Figure 5 Immunogold labeling of cytoskeleton proteins for (A) perinuclear vimentin, (B) actin, (C) vimentin intermediate filaments, (D) tubulin microtubules, (E) negative control for vimentin and (F): negative control for actin. White arrows indicate some
of 20 nm gold nanoparticles and arrowhead points out to the nucleus.
Immunomarquage des protéines du cytosquelette (A) vimentine périnucléaire, (B) l’actine, (C) filaments intermédiaires vimentine,
(D) microtubules de tubuline, (E) contrôle négatif pour la vimentine et (F) contrôle négatif pour l’actine. Les flèches blanches
indiquent des nanoparticules d’or de 20 nm et les têtes de flèche pointent vers le noyau.
fibronectin, osteopontin, and bone sialoprotein. Adhesive
interactions between cells and their substrata, mediated
by cell surface integrin and extracellular matrix proteins,
appear to result in massive rearrangement of the cell
cytoskeleton. These early events promote several structural
changes such as cell spreading and initiate signaling cascades, which modulate genes expression [24,25].
This study strongly supported the hypothesis that the
organization of the cytoskeleton was mainly related to the
extracellular matrix protein on which osteoblast adhered.
The kinetics of osteoblast-cell adherence and the aspects
of the cell shape were dependent of the nature of the substrate without any influence of cell density which was similar
after long time culture whatever matrix protein used was
[26]. This is in agreement with reports showing different
adhesive and spreading kinetics of cell cultured on FN, Col-I
or serum-free medium [27—29]. Quantification of the SEM
images of the cytoskeleton was only possible by using techniques developed to evaluate the complexity of an image.
Fractal methods for measuring the texture of an image are
useful and can provide more information than those based
on Euclidean geometry. It is also well known that the fractal
algorithms should be adapted to the texture to measure. In
the present study, the skyscraper algorithm provided more
information than the blanket because there was no preferential distribution of the cytoskeleton fibers [30,31].
Cells cultured on purified adhesion proteins that contain
RGD-sequences had the highest surface area and only cells
on PL showed a decrease with time in cell surface area. The
cell rounding on PL might be related to disassociation of cell
receptor-substratum complexes, which causes a dissipation
of isotonic tension at cell surface and retraction of cortical
microfilaments. This might lead to a growth arrest.
The cellular attachment in serum-free medium indicates
that osteoblast binding can occur in the absence of serum
or exogenously added cell adhesion proteins. When cells
adhere to glass, they use presumably other mechanisms
or non-integrin receptors to mediate cell attachment. Our
Effects of matrix proteins on osteoblasts
61
Figure 6 Effects of matrix proteins on ultrastructural cytoskeleton aspect. Cells were cultivated in DMEM alone for 5, 60 min
and 24 h onto coverslips coated with poly-L-lysine (PL), DMEM, fetal calf serum (FCS), fibronectin (FN), vitronectin (VN) and type I
collagen (Col-I).
Effets des protéines matricielles sur l’aspect ultrastructural du cytosquelette. Les cellules ont été cultivées dans du milieu DMEM
seul pendant 5, 60 minutes et 24 heures sur des lamelles recouvertes de poly-L-lysine (PL), DMEM, sérum de veau fœtal (FCS),
fibronectine (FN), vitronectine (VN) et collagène de type I (Col-I).
results confirm previous observations, which did not report
marked changes in attachment in presence or absence of
a specific or preferred ligand such as fibronectin [32]. For
other matrix proteins (FN, VN or Col-I), the osteoblastcell had a relatively flat shape. These findings demonstrate
the specific role of extra cellular matrix molecules to
the cell binding, the actin polymerization triggering, the
contact adhesion formation and the stabilization of cell
shape [16,33].
Previous authors suggested that fibronectin promoted
both osteoblast attachment and spreading [34] and that
plating on matrices such as type I collagen and fibronectin
accelerated osteoblastic cell maturation [35—37]. Moreover,
FN functions as a survival factor for mature osteoblasts [38].
62
Figure 7 Effects of matrix proteins on FAK phosphorylation.
Cells were grown in DMEM alone during 5, 30, 60 and 120 min
onto coverslips coated with poly-L-lysine (PL), DMEM, fetal calf
serum (FCS), fibronectin (FN), vitronectin (VN) and type I collagen (Col-I) and FAK phosphorylation study was performed by
western blot.
Effets des protéines matricielles sur la phosphorylation de FAK.
Les cellules ont été cultivées dans du DMEM seul pendant 5,
30, 60 et 120 minutes sur des lamelles recouvertes de polyL-lysine (PL), DMEM, sérum de veau fœtal (FCS), fibronectine
(FN), vitronectine (VN) et collagène de type I (Col-I) et l’étude
de la phosphorylation de FAK a été réalisée par western blot.
Osteoblastic cells cultured on a collagen substratum adhered
and spread over it. Col-I, like FN anchorage is important
for osteogenesis but may function at different stages of
the osteoblast differentiation program [39]. The attachment of osteoblasts to the collagenous matrix had important
effects on the expression of the osteoblast phenotype and
osteoblast marker genes such as alkaline phosphatase and
osteocalcin [40]. In cell cultivated on VN, polymerized actin
fibers are observed but they are mainly at the periphery of
the cell. Some authors supposed that, in epithelial cells,
this circumferential band of actin filaments provided the
structural support for cell-cell junctions [41].
Results reported in this study seem to confirm that
integrins convey information from the extracellular environment by linkage between the extracellular matrix and
actin cytoskeleton. They transmit signals by organizing the
cytoskeleton and regulating cell shape [42,43]. Such changes
in cell shape and cytoskeleton organization might regulate
gene expression and cell biosynthesis [44,45] and thereby
control the differentiation and growth of cells [46]. Current
speculation postulates that the formation of large aggregates of signal transduction molecules on a cytoskeleton
framework provides high local concentrations of receptors,
enzymes, substrates, and structural molecules. This protein clustering facilitates activation of signaling pathways
by stimulating tyrosine phosphorylation cascades linked to
V. Demais et al.
Figure 8 Effects of matrix proteins on MAPK phosphorylation.
Cells were grown in DMEM alone during 5, 30, 60 and 120 min
onto coverslips coated with poly-L-lysine (PL), DMEM, fetal calf
serum (FCS), fibronectin (FN), vitronectin (VN) and type I collagen (Col-I) and MAPK phosphorylation study was performed by
western blot.
Effets des protéines matricielles sur phosphorylation de MAPK.
Les cellules ont été cultivées dans du DMEM seul pendant 5,
30, 60 et 120 minutes sur des lamelles recouvertes de polyL-lysine (PL), DMEM, sérum de veau fœtal (FCS), fibronectine
(FN), vitronectine (VN) et collagène de type I (Col-I) et l’étude
de la phosphorylation de MAPK a été réalisée par western blot.
MAPK and other pathways [47] and organization of adhesive
sites. Adhesion on various matrix proteins induces structural
changes in cells, and may alter the conformation and interactions of integrin receptor thereby leading to activation of
different signal transduction pathways, than would be activated by cell adhesion or receptor occupancy. Finally it may
be necessary that the integrin receptors could be engaged
with an extracellular ligand in order to mediate signal transduction. Such a result is clearly consistent with the unique
dependency of mechanotransduction on the maintenance of
a intact cytoskeleton [32].
Binding of integrin and the subsequent cell adherence stimulate the formation of membrane-associated focal
adhesion complexes [32]. Some authors have reported that
FAK might play a role in the regulation of MAPK in inducing tyrosine phosphorylation of p44MAPK in NIH 3T3. So,
MAPK activity may be related to cell shape and cytoskeleton
organization [48]. In this study FAK and MAPK are phosphorylated after cell adhesion. If no significant differences were
observed for FAK phosphorylation, the MAPK phosphorylation
differs with time and type of matrices. These data suggested
that FAK and MAPK phosphorylations are part of regulation in
cytoskeleton organization but other cell signaling pathways
might be involved. That is in agreement with findings showing that Rho pathway was implicated in integrin-mediated
focal adhesion formation and may also enhance actin polymerization [49].
In this study the influence of matrix proteins on
cell attachment and cytoskeleton organization was investigated. These results show that binding to different adhesion
Effects of matrix proteins on osteoblasts
proteins induces changes in cytoskeleton organization. This
suggests that cells are able to distinguish specific integrinligand binding, to induce different intracellular pathways
in response and in consequence to modulate cytoskeleton
organization.
Disclosure of interest
The authors declare that they have no conflicts of interest
concerning this article.
Acknowledgments
This work was made possible by grants from Contrat Region
Pays de la Loire: Bioregos2 program.
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