Mol Cell Biochem (2012) 369:205–216
DOI 10.1007/s11010-012-1383-y
Selective integrin subunit reduction disrupts fibronectin
extracellular matrix deposition and fibrillin 1 gene expression
Rajeev K. Boregowda • Brooke M. Krovic •
Timothy M. Ritty
Received: 10 April 2012 / Accepted: 20 June 2012 / Published online: 11 July 2012
Ó Springer Science+Business Media, LLC. 2012
Abstract Integrins are transmembrane receptors that can
specifically bind extracellular matrix (ECM) proteins.
Assembly of the ECM protein fibronectin into fibrils has
been shown to be a cell-mediated process that requires
integrins. Like fibronectin, fibrillin 1 is an ECM glyco-
protein that can assemble into fibrils, but the role of inte-
grins in fibril formation is not understood. To investigate
the role of integrins in fibrillin 1 ECM deposition, cells that
normally produce and assemble fibrillin 1 fibers in vitro
were stably transfected with plasmid constructs encoding
short interfering RNAs that target specific integrin sub-
units. Cells that were deficient in a2- and b3-integrin
subunits produced and deposited fibronectin normally, but
cells that were deficient for a5 and aV were unable to
elaborate a fibronectin matrix, although they continued to
produce and secrete the protein. Surprisingly, the cells that
were unable to elaborate a fibronectin matrix also lost
fibrillin 1 gene expression.
Keywords Extracellular matrix  Fibronectin 
Fibrillin 1  Integrins
R. K. Boregowda  B. M. Krovic  T. M. Ritty
Division of Musculoskeletal Sciences, Department
of Orthopaedics, Penn State College of Medicine,
500 University Drive, Hershey, PA 17033, USA
e-mail: tmritty@gmail.com
R. K. Boregowda (&)
Department of Microbiology and Immunology-H107, Penn State
College of Medicine, 500 University Drive, Hershey,
PA 17033-0850, USA
e-mail: rajeev04mys@yahoo.com; rkb15@psu.edu
Introduction
The processes by which extracellular matrix (ECM) proteins
are assembled in the intercellular space are not well under-
stood. It is not known if most ECM proteins are secreted and
assembled spontaneously, or if cells play an active role in
directing the assembly of newly made ECM proteins into the
existing ECM. Assembly of the most abundant class of
matrix protein—collagens—has been widely studied and
they are known to wind into a trimer intracellularly. It has
been demonstrated that collagen can assemble spontane-
ously into fibrils independent of cells after proteolytic pro-
cessing of the C-terminal propeptide [1]. However, an
interesting model of extracellular compartments that medi-
ate collagen assembly has been proposed (for review [2]).
An understanding of the specific role that cells play in col-
lagen matrix deposition remains incomplete.
Similarly, great experimental effort has been directed at
understanding fibronectin assembly. Fibronectin is a large
(circa 270 kDa) ECM glycoprotein that is expressed early in
development and present in abundance in most adult tissues.
The model that has emerged proposes that fibronectin is
secreted from the cell as a covalently linked dimer, held
upon the cell surface and stretched (for review [3, 4]). It is
thought that stretching reveals cryptic sites that mediate the
polymerization of fibronectin fibrils. There is no evidence
that this compelling model is a general paradigm that can be
applied to the assembly of other matrix proteins, especially
because fibronectin is secreted as a dimer and many other
ECM proteins are secreted as monomers.
If the matrix deposition of a given ECM protein is cell
mediated, as in the case of fibronectin, then cell surface
receptors are likely to be involved. Integrins and syndecans are
two families of ECM receptors that may play a role. Integrins
are heterodimeric transmembrane receptors whose cell surface
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domains bind short peptide sequences in a variety of extra-
cellular proteins, including ECM proteins. Integrin binding of
an extracellular ligand results in extensive intracellular sig-
naling that alters gene expression (for review [5–8). Synde-
cans are monomeric heparan sulfated proteoglycans that also
play a role in cell adhesion primarily through their sugar
modifications. Like integrins, syndecans also interact with
cytoskeletal elements, and a variety of intracellular signaling
molecules (for review [9]). We and others have previously
demonstrated that cell surface heparan sulfated proteoglycans
are involved in fibrillin 1 and 2 matrix assembly [10–12].
Like fibronectin, fibrillin 1 is a large (circa 311 kDa
core) glycoprotein that somehow assembles into extensive
fibrillar networks in the ECM, although fibrillin 1 tissue
distribution is much more restricted. A fibrillin network can
serve as a scaffold that nucleates the deposition of tropo-
elastin and the ultimate assembly of the various proteins
that make up the elastic fiber. Fibrillin 1 exon 37 encodes
the only Arg-Gly-Asp (RGD) amino acid triplet in the
protein. This sequence has been demonstrated in vitro to be
a ligand for aVb3, a8b1, aVb6, and a5b1 integrins by a
variety of techniques using isolated fibrillin and recombi-
nant peptides [13–20]. No other site within the fibrillin 1
protein has been shown to be a ligand for integrins.
In this study, we sought to examine the role of integrins
in the deposition of fibronectin and fibrillin 1 into the
ECM. A thorough understanding of the mechanisms of
fibronectin and fibrillin deposition is essential for events
from gastrulation to musculoskeletal development, vascu-
lar and pulmonary morphogenesis, and homeostatic main-
tenance of several adult tissues that rely upon sufficient
elastic fiber function.
We hypothesized that fibrillin-1-producing cells employ
integrins during the deposition of fibrillin 1 into the ECM.
To test this idea, we used MG63 and ARPE cells that make
and deposit both fibronectin and fibrillin 1 as parental lines
to develop daughter lines that have reduced expression of
individual integrin subunits. These reductions had an
impact on cell attachment and spreading. Two of these
lines were unable to assemble a fibronectin matrix even
though they continued to produce and secrete the protein.
This study also yielded a surprising outcome that, although
preventing the initial hypothesis from being tested, pro-
vided new insights into the expression of fibrillin 1.
Experimental procedures
Cell lines, cell culture, and siRNA constructs
Osteoblastic osteosarcoma MG63 cells (ATCC # CRL
1427) and retinal pigmented epithelium ARPE-19 cells
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Mol Cell Biochem (2012) 369:205–216
(ATCC # CRL 2302) were cultured with Dulbecco’s
minimal essential medium (Hyclone) supplemented with
10 % fetal bovine serum (Gibco) in a 37 °C CO2 incubator.
The expression vector, pSilencer 4.1-CMV neo (Ambion,
Inc.), was used to transfect and constitutively express small
interfering RNAs designed to down-regulate specific inte-
grin subunits. Sequence information was purchased (Am-
bion, Inc.) and 55-mer oligonucleotides were synthesized
for the sense and antisense directions (IDT, Inc.). The
following is a list of target genes and the siRNA design
numbers (Ambion, Inc.) used are ITGA2, ID#111111;
ITGA3, ID#111567; ITGA5, ID#111113; ITGAV,
ID#111116; ITGB3, ID#112581 validated sequence.
Sequences were annealed and cloned into pSilencer and
confirmed by DNA sequencing. To make integrin-reduced
cell lines, plasmid DNA encoding short interfering RNA
was transfected into MG63 and ARPE cells.
Stable transfectants were selected by plasmid conferred
resistance to G418 at 500 lg/ml. For each siRNA, six
clonal lines were established through dilution and colony
isolation. All clones were screened and based upon integrin
subunit down-regulation as assessed by flow cytometry,
one clonal line was chosen for use in most subsequent
experiments.
Flow cytometry
Medium was removed and cells were washed with PBS and
lifted non-enzymatically with versene (0.05 M EDTA in
PBS) at 37 °C for 3-5 min. The cells were pelleted in
DMEM(H) without serum. Pellets were resuspended in
100 ll of the primary antibody solution and incubated for
30 min at room temperature. Primary antibodies used were
mouse monoclonal anti-integrin a2b1–FITC conjugate
(1:100, Chemicon CBL477F), mouse monoclonal anti-
integrin a5–FITC conjugate (1:100, Chemicon CBL497F),
mouse monoclonal anti-integrin aV (1:100, Chemicon
MAB1980), rabbit polyclonal anti-integrin b3 (1:50,
Chemicon AB1932). The samples were washed once in
PBS, then the pellets were resuspended in 100 ll of the
secondary antibody solution (goat anti-mouse IgG–FITC
conjugate, 1:500, Santa Cruz, or Alexa Fluor 488 chicken
anti-rabbit IgG, 1:500, Molecular Probes) and incubated for
20 min at room temperature. The samples were washed
once with PBS before being resuspended in 1 ml of PBS, to
be analyzed by flow cytometry using a Becton Dickson
FACScan. 500,000 cells/sample were used for all flow
cytometry experiments and a minimum of 10,000 cells
were counted. Gating was set at \1 % of no primary
antibody negative control cells. Data were evaluated with
BD CellQuest Pro software.
Mol Cell Biochem (2012) 369:205–216
Cell adhesion assay
Non-tissue culture-treated 24-well plates (Becton–Dickin-
son Labware, NJ) that normally support only low or no
adhesion for attachment-dependent cells were used. Wells
were coated with 10 lg/ml human plasma fibronectin in
distilled water (Chemicon International, CA) or 40 lg/ml
collagen in 25 mM Tris, pH 8.8 (PureColTM Inamed Bio-
materials, CA), overnight in the tissue culture hood. Gelatin
(porcine skin, type A, Sigma, St. Louis) coating was per-
formed using 250 lg/well in distilled water for 1 h in a 37 °C
incubator. All proteins were coated in quadruplicate and
wells were washed with PBS before use. The clonal cell lines
were washed with PBS and lifted non-enzymatically with
versene, washed by centrifugation and resuspended in
serum-free medium (SFM4MegaVir, HyClone). After
counting the cells using a hemocytometer, 1 9 105 cells
from each clone were seeded in the ECM protein pre-coated
wells and the same numbers of cells were taken in triplicate
in microcentrifuge tubes for final assay normalization by
DNA assay. The cells were allowed to attach for 2 h in a
37 °C CO2 incubator, whereas the cells in microcentrifuge
tubes were pelleted by centrifugation and stored at -80 °C in
200 ll lysis buffer containing 0.5 % NP-40 (Fluka Bioche-
mika) in double-distilled water. After 2 h, the media were
removed from the 24-well plates and the wells were washed
with PBS to remove unbound cells. The cells remaining in
the wells were lysed with 200 ll lysis buffer and placed in -
80 °C to aid lysis. To quantify cell numbers, 100 ll of cell
lysate from each sample was mixed with 100 ll of 1:2,000
diluted SYBR Safe DNA Gel Stain (Molecular Probes) in TE
buffer and placed in a 96-well plate with a black flat bottom
(BD Falcon, NJ). The DNA and dye complex were read at
kex = 485, kem = 538 nm with a cutoff at 515 nm, using
Spectramax Gemini Spectrofluorimeter (Molecular Devices,
CA). The results were analyzed using one-way ANOVA with
the help of GraphPad Prism 4.
Cell spreading
Cells from the a2, a5, aV, and negative control plasmid clonal
lines were plated at low density on each of three substrates:
tissue culture plastic, plastic coated with 10 lg/ml fibronectin,
or 100 lg/ml collagen I. The cells were allowed to attach and
spread for 18 h in serum-free medium. They were then
washed three times with PBS and fixed with 3 % parafor-
maldehyde in PBS for 45 min. Cells were stained with ‘‘G’’
Coomassie stain 2.5 % (methanol 10 %) for 45 min, de-
stained with distilled water, and allowed to air dry. A Nikon
Optiphot-2 microscope mounted with a Spot RT CCD digital
camera driven by SPOT software v3.5 (Diagnostic Instru-
ments, Inc.) was used for image capture. Random pictures
were taken of each cell type on each growth substrate at 109
207
magnification. ImageJ was used to quantify both the area and
the perimeter of individual cells. Cells at the edge of the frame
or two cells touching were not measured. Between 100 and
300 cells were analyzed per substrate. Using GraphPad Prism
4, the data were analyzed using one-way ANOVA, comparing
the results against the NCP cells.
Immunofluorescence
Cells were plated at confluency on glass coverslips and
grown for 7 days in standard culture conditions. Cells were
washed with PBS and fixed for 30 min in 2 % paraformal-
dehyde in PBS, pH 7.2. Cells were blocked with PBS/BSA
(1 mg/ml), then incubated with the primary antibody for
30 min at room temperature. A polyclonal fibronectin anti-
body (Sigma, St. Louis, MO) was used at a 1:500 dilution
while a polyclonal fibrillin 1 antibody [21] targeting the
C-terminus was used at a 1:200 dilution. Cells were washed
in PBS/BSA before being incubated with a chicken anti-
rabbit IgG Alexa Fluor 594 conjugate (Molecular Probes)
secondary antibody at a dilution of 1:800 for 30 min at room
temperature. Hoechst 33258 dye (Sigma, St. Louis, MO) was
included at a 1:2,000 dilution to stain cell nuclei. Cells were
washed in PBS/BSA before being mounted in 25 % V/V
glycerol in sterile H2O. A Nikon Optiphot-2 fluorescent
microscope mounted with a Spot RT CCD digital camera
driven by SPOT software v3.5 (Diagnostic Instruments, Inc.)
was used for image capture.
ECM deposition and extraction
Cells were plated at confluency and grown for 4-5 days in
serum-containing media. On Day 5, cells were washed
extensively with PBS. Serum-free medium (SFM4Mega-
Vir, HyClone) was added. For the last 2 days, conditioned
medium was removed and saved every 24 h. The collected
media were dialyzed against weak acetic acid at 4 °C with
a 14 K MWCO, and lyophilized. For ECM harvest on Day
7, the cells were washed three times with ice-cold PBS and
lysed with ice-cold water containing 0.5 % Triton X-100,
protease inhibitor cocktail (1:100, Sigma), and DNase
(1:1,000, Promega). Dishes were tilted gently and kept on
ice for 30 min to completely solubilize the cellular proteins
and membranes. The cell lysates were removed and saved
for normalization. Ice-cold water containing 1:100 protease
inhibitors was added to the remaining insoluble ECM and a
rubber policeman was used to scrape up the ECM, which
was pelleted at 14,000 rpm for 5 min at 4 °C.
Immunoblotting
Sample loads were normalized for cell number by relative
DNA content in cell lysates using SYBR Safe DNA Gel
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Stain as above. Samples were electrophoresed in 1.0-mm
6.0 % SDS polyacrylamide gels at a constant current of
20 mA/gel for 60 min. Proteins were transferred to PVDF
membranes in CAPS buffer with 5 % MeOH at a constant
voltage of 75 V for 75 min. The membranes were blocked
for at least 30 min at room temperature in 2.5 % non-fat
milk in PBS containing 0.05 % Tween 20. The primary
antibodies were incubated at room temperature for 1 h, FN
1:1,000, FBN1 1:500 dilutions. The membranes were
washed three times in PBST before being incubated with
goat anti-rabbit IgG–HRP conjugated secondary antibody
at 1:2,000 for 1 h at room temperature. The membranes
were washed three times in PBST before being developed
by ECL treatment.
RT-PCR and rqRT-PCR
RNA was harvested using Tri Reagent (Ambion) following
the manufacturer’s instructions, and 5 lg total RNA was
reverse transcribed using SuperScript III reverse trans-
criptase (Invitrogen). For PCR amplification of human
fibrillin 1 sequences, 10 ng of cDNA was used for each
25 ll reaction containing the following: 1X PCR buffer,
0.2 mM dNTPs, 1.5 mM Mg2?, 0.5 lM of each primer,
and 0.625 U GoTaq DNA Polymerase (Promega). Primer
sequences: fibrillin 1 set 1 forward-GCCCTGGGATTT
ACCGTGCTT, fibrillin 1 set 1 reverse-TTCCATCCAG
GGCAACAGTAA, fibrillin 1 set 2 forward-TCTGCTC
TGTGCCTTCCGAT, fibrillin 1 set 2 reverse-TCCCTCCG
TCACAGCAGCAT, 18S forward-GAGAAACGGCTACC
ACATCC, 18S reverse-CACCAGACTTGCCCTCCA.
Both fibrillin 1 primer sets were exon spanning. PCR was
performed for 35 cycles. PCR products were resolved on a
10 % polyacrylamide gel at 25 mA/gel for 30 min, stained
with SYBR Safe and visualized with UV light. Relative
quantitative real-time PCR was performed using 15 ng of
cDNA per well in triplicate under standard conditions on
an ABI 7900 using the following TaqMan probes (Applied
Biosystems): a2 integrin—Hs00158127, a5 integrin—
Hs00233732, aV integrin—Hs00233790. Samples were nor-
malized to each other using a probe for 18s RNA. Data were
analyzed using ABI SDS2.2.2 software and Excel (Microsoft).
Results
Integrin subunits were down-regulated with siRNA
MG63 cells were stably transfected with short interfering
RNA expression vectors designed to down-regulate spe-
cific integrin subunits. This resulted in daughter lines with
stable reduction of a2, a5, or aV integrins. A cell line,
labeled negative control plasmid (NCP), was produced by
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Mol Cell Biochem (2012) 369:205–216
transfecting a vector that contained an inactive siRNA
sequence. Integrin expression by NCP controls was not
significantly different from untransfected MG63 cells (data
not shown). We were not able to establish a MG63 cell line
in which the b3-integrin subunit was sufficiently reduced to
be experimentally useful. For this reason, we turned to
human ARPE cells, which also produce and deposit
fibrillin 1 in vitro. ARPE cells were stably transfected with
the b3 siRNA expression vector to down-regulate this
integrin subunit. A NCP ARPE cell line was also produced
and used as a positive control for the b3-reduced ARPE
cells. Flow cytometry demonstrated that cell surface inte-
grin subunit protein levels were reduced as compared to the
NCP control cells (Fig. 1). Expression of all four integrin
subunits (a2, a5, aV, and b3) was successfully diminished
within each respective clonal line (Table 1). These lines
were cultured under selection and continued low levels of
integrin subunit expression were confirmed after months in
culture (data not shown).
Integrin-deficient lines display adhesion differences
The NCP control and integrin-reduced MG63 daughter
lines were evaluated for differences in their abilities to
attach to ECM proteins (Fig. 2). The reduction of a5- and
aV-integrin expression altered the ability of the cells to
adhere to tissue culture plastic coated with ECM proteins.
With these two lines, adhesion to gelatin was nearly
ablated, and attachment to fibronectin and type I collagen
was reduced to 45 % of NCP controls. The ability of cells
with lowered a2 expression to attach to gelatin and type I
collagen was not affected. Although the a2-reduced cells
appeared to attach to fibronectin slightly better than control
cells, the difference did not reach statistical significance.
ARPE cells were not evaluated in adhesion studies.
Cell spreading is altered
The integrin-reduced daughter lines displayed morpholo-
gies that differed from the original MG63 cells. By visual
inspection, the a5 and aV lines in particular appeared to
have reduced spreading. These apparent differences in cell
spreading and perimeter were quantified using computer
analysis (Fig. 3). Indeed, after 18 h of attachment in
serum-free conditions, all three experimental cell lines (a2,
a5, and aV) demonstrated reduced spreading compared to
control cells on tissue culture plastic, type I collagen, and
fibronectin.
For all cell lines, the greatest spreading and perimeters
were observed on fibronectin, followed by collagen, with
the least amount of spreading on tissue culture plastic
(Fig. 3a). Overall, the loss of a5 and aV had the greatest
effect on spreading and perimeter, while the loss of a2 was
Mol Cell Biochem (2012) 369:205–216 209

Fig. 1 Generation of clonal cell

lines reduced in the expression
of specific integrin subunits.
Overlay graphs from flow
cytometry analysis of a2-, a5-,
and aV-reduced MG63 cell
lines and b3-reduced ARPE cell
line. Results from negative
control cells (no antibody) are in
black, positive control cells
(negative control plasmid cells)
are in green, positive control
cells for b3 integrins (BJ
fibroblasts) are in blue, and the
siRNA integrin-reduced cell
line is in red. (Color figure
online)

intermediate. The most dramatically impaired spreading
compared to controls was seen in a5- and aV-reduced cells
that were plated on fibronectin.
For each cell line, the percent increase in spreading on
the two ECM proteins as compared to plastic differed
among the integrin-reduced clones (Fig. 3b). When grown
on collagen I, the a2- and aV-reduced cells increased two-
fold, the same proportion as the control cells. The a5-
reduced cells had a smaller increase in size, only increasing
about 50 %. When grown on fibronectin, the a2-reduced

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210 Mol Cell Biochem (2012) 369:205–216

Table 1 Levels of integrin expression in MG63 and ARPE clonal cell lines
Integrin Clone % Positive % Ctrl mean FU

A
a2 MG63 a2.6 0.29 0
MG63 NCP 43.22 100
a5 MG63 a5.2 3.50 7
MG63 aV.4 19.41 3
MG63 NCP 99.78 100
aV MG63 a5.2 93.01 48
MG63 aV.4 98.65 52
MG63 NCP 99.77 100
b3 ARPE b33.1 10.04 21
ARPE NCP 46.49 32
hFibroblast 96.13 100
Integrin Cell line Relative mRNA level

B
a5 MG63 a5.2 0.10
MG63 aV.4 0.06
MG63 NCP 1.00
hFibroblast 4.02
aV MG63 a5.2 0.65
MG63 aV.4 0.26
MG63 NCP 1.00
hFibroblast 6.98

(A) The data obtained from flow cytometry analysis of integrin-reduced MG63 and ARPE cell lines. Results are presented as the percentage of
gated cells, i.e., above no antibody control levels, and the average fluorescent units (arbitrary) of those cells that were gated. (B) Relative levels
of expression for a5- and aV-integrin subunit mRNAs from the indicated cell types as measured by relative quantitative real-time RT-PCR using
the 50 nuclease assay (TaqMan). Inter-sample differences were normalized with 18s RNA, and levels in the table are relative to MG63 NCP
control cells

cells and control cells both doubled in size. However, the
increase in size was about 50 % for the aV-reduced cells,
and only 16 % for the a5-reduced cells.
Fibronectin and fibrillin 1 are not deposited by a5-
and aV-reduced cells
b3-reduced (Fig. 4b) cells were comparable to normal cells
in their ability to deposit fibrillin 1 into the ECM.
Fibrillin 1 expression is undetectable in a5- and aV-
reduced cells, while fibronectin continues to be
produced but partitions to the medium

Parent MG63 and ARPE cells deposit fibrillin 1 and
copious fibronectin in vitro. Immunofluorescent staining
revealed a5- and aV-reduced clones failed to deposit either
fibronectin or fibrillin 1, while the a2- and b3-reduced
clones deposited both ECM proteins normally (Fig. 4a).
The inability of a5- and aV-reduced cells to deposit
fibronectin and fibrillin 1 into the ECM was confirmed by
western blots of the insoluble ECM proteins produced by
these cells (Fig. 4b). Only a small amount of fibronectin
was detected in these samples, as compared to control and
a2-reduced cells (Fig. 4b), in which ample amounts of FN
were detected. In contrast, fibrillin 1 was not detected in
the ECM of either the a5- or the aV-reduced cells. a2- and
The lack of fibrillin 1 and fibronectin in the ECM prompted
us to verify that the proteins were still being produced. To
determine if the a5- and aV-reduced cells were still pro-
ducing fibronectin and fibrillin 1, 48-h conditioned medium
was evaluated by western blot (Fig. 5). All the lines con-
tinued to produce fibronectin, although increased amounts
were seen in conditioned medium from a5- and aV-
reduced cells. This confirms that fibronectin is being pro-
duced but not being deposited into the insoluble ECM, and
is instead partitioning into the medium.
Western blotting for fibrillin 1 revealed the absence of
this protein in conditioned medium from a5- and aV-
reduced lines, suggesting that they have stopped producing

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Fig. 2 The ability of cells to attach to gelatin, fibronectin, and type I
collagen is diminished when a5 and aV integrin expression is
reduced. Cells were allowed to attach to non-tissue culture-treated
plastic coated with gelatin, fibronectin, or type I collagen for 2 h, then
washed to remove unbound cells. The number of attached cells was
determined by DNA assay. Error bars indicate standard error of the
mean, N = 4. *p \ 0.05
fibrillin 1 protein. This unexpected finding prompted us to
evaluate FBN1 gene expression levels in the a5- and aV-
reduced and parent MG63 lines by RT-PCR (Fig. 6). RNA
was harvested from the NCP control cell line and the a2-
reduced cell line, both of which produce and deposit
fibrillin 1 protein into the insoluble ECM in vitro. RNA
was also harvested from three different clones, each of
which is reduced for a5- or aV-integrin subunits. Reverse
transcription of total RNA followed by PCR amplification
of FBN1 message using exon spanning primers was
211
performed. Fibrillin 1 message was detected normally in
the fibrillin positive lines, but was undetectable in the three
a5-reduced and the three aV-reduced clonal cell lines. This
indicates that fibrillin 1 protein was not detected in the
ECM or conditioned medium of a5- or aV-reduced cells
because message from the gene has been greatly down-
regulated.
Discussion
To study the mechanism by which cells deposit fibrillin 1
into the ECM, we developed integrin subunit-reduced cell
lines from a parent osteosarcoma line, MG63, and a retinal
pigmented epithelial line, ARPE-19, that normally produce
and deposit fibrillin 1 into the ECM in vitro. We estab-
lished clonal lines reduced in a2-, a5-, aV-, or b3-integrin
expression and confirmed this down-regulation by both
flow cytometry analysis and cell function assays. Reduced
expression of two specific integrin subunits, a5 and aV,
resulted in the inability of MG63 cells to deposit fibro-
nectin into their ECM although the cells were still pro-
ducing it. Fibrillin 1 was also not detectable in either the
matrix or the conditioned medium from these cells. This
led to the surprising finding that fibrillin 1 gene expression
is turned off in a5- and aV-reduced clones.
Functional effects of integrin down-regulation
The down-regulation of specific integrin subunits was
determined by flow cytometry, and was confirmed by
functional assays. Cell attachment to fibronectin, gelatin,
and type I collagen was impaired in a5- and aV-reduced
cells, but was unaffected in a2-reduced cells. Cells bind to
RGD sequences contained in gelatin (denatured collagen),
as well as the archetypical RGD of fibronectin. There was a
near total loss in the a5- and aV-reduced cells ability to
attach to gelatin, and a 45 % reduction in ability to bind
fibronectin. This difference may be explained by noting
that fibronectin also has other non-RGD cell binding and
heparin-binding sites. The almost total lack of a5- and aV-
reduced cell attachment to gelatin is probably due to the
lack of heparin-binding domains in gelatin by which cells
can mediate attachment through cell surface heparin sulfate
proteoglycans [22]. The effect of reducing a5- and aV-
integrin subunits on the cell’s ability to adhere to type I
collagen was the least dramatic, compared to all substrates
tested. Again, no change in adhesion to type I collagen was
seen in a2-reduced cells, likely due to the presence of other
collagen receptors on the cell surface. When grown on
plastic, significantly impaired spreading was found for a2-,
a5-, and aV-reduced clones, as compared to controls. The
reason for this reduction is unclear, as attachment to plastic
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Fig. 3 Integrin-reduced cell lines exhibit impaired spreading on
plastic, type I collagen, and fibronectin. a Cells were plated at sub-
confluent densities on tissue culture-treated plastic, or plastic coated
with 10 lg/ml fibronectin, or 100 lg/ml collagen I and allowed to
attach and spread for 18 h in serum-free medium. Cells were fixed,
stained, digitally photographed, and cell area and perimeter were
quantified using ImageJ. Number of cells is between 100 and 300 for
each cell type and substrate. Error bars indicate standard error of the
mean. *p \ 0.05. b Cell spreading data were analyzed to determine
is presumably non-specific, but it may be that the loss of
these subunits results in some impairment of the ability of
these cells to organize cytoskeletal elements. All cell lines
exhibited their largest area and perimeter on fibronectin,
followed by collagen, with the least amount of spreading
on plastic. This likely reflects integrin signaling differences
between attachment to a non-specific surface (e.g. plastic)
and a proteinaceous ligand.
When plated on fibronectin, the relative increase in size of
a2-reduced cells was equal to control cells, i.e., about 110 %.
In contrast, aV-reduced cells increase by just 50 % and the
a5 nulls by only 16 %. Previous study has established a5b1
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Mol Cell Biochem (2012) 369:205–216
the relative increase in cell area of each clone on fibronectin and type
I collagen as compared to spreading on plastic. When plated on type I
collagen, a2- and aV-reduced clones increase their area in the same
proportion as control cells, while increased area of a5-reduced cells is
only half of this. On fibronectin, a2-reduced cells increase in the same
proportion as control cells, while the increase in area of aV-reduced
cells is only half of this. a5-Reduced cells experience the greatest
impairment, only increasing their size 16 % when plated on
fibronectin
as the primary fibronectin receptor, and that aV integrins can
act as a secondary receptor in the absence of a5b1 [23]. Our
data are consistent with this previous study in that the most
profound effect is present in the a5-reduced line and a lesser
effect is displayed by the aV-reduced line.
When plated on collagen I, no decrease in spreading
occurs in the a2-reduced cells, which mirrors the cell
adhesion data where a2 adhesion to collagen I was unal-
tered. At least three other integrin receptors are known to
mediate attachment to collagen, and so it is not surprising
that these data suggest the availability of another collagen
receptor on the a2-reduced cells.
Mol Cell Biochem (2012) 369:205–216
Fig. 4 a5- and aV-Reduced lines do not assemble fibronectin or
fibrillin 1 in vitro. a MG63 and ARPE cells were grown at confluent
density for 1 week on glass coverslips and fibronectin and fibrillin 1
were immunofluorescently labeled. All microscopy images were
captured using a 209 objective lens. No difference in staining was
seen in a2- and b3-reduced cell lines compared to controls. However,
neither fibronectin nor fibrillin 1 was detectable in the ECM of a5-
and aV-reduced cell lines. b Western blot of insoluble ECM proteins
extracted from tissue culture dishes in which cells had been grown for
1 week. Lane loads were normalized to cell number. Fibronectin was
Failure of fibronectin matrix assembly
The a5- and aV-reduced MG63 cells produce ample
fibronectin (Fig. 5), but are unable to elaborate a normal
fibronectin matrix (Fig. 4a, b). Cells from a5- and aV-null
mice are able to deposit fibronectin into their ECM, as the
loss of one can be compensated for by the other [24, 25].
However, a5-null cells lose the ability to deposit FN in the
presence of an aV-blocking antibody [23], and a5- and aV-
double null mice are unable to deposit fibronectin during
development [26]. Therefore, the simultaneous loss of both
a5 and aV results in the failure of fibronectin matrix
assembly. Our data indicate that MG63 cells normally
express very low levels of the a5- and aV-integrin subunits
as compared to fibroblasts (Fig. 1; Table 1B). Also, rela-
tive quantitative real-time RT-PCR and flow cytometry
data show that both subunits are reduced in both cell lines
compared to control cells (Table 1A, B). The reason for
213
detected in all integrin-reduced cell lines. However, matrix deposition
of FN was greatly decreased in a5- and aV-reduced cells. DMEM
containing 10 % FBS and an extracted matrix sample from a previous
experiment known to contain fibronectin were included to positively
identify the fibronectin band. Fibrillin 1 was detected in ECM extracts
from negative control plasmid, a2-reduced, and b3-reduced cells. No
fibrillin 1 was detected in the a5- and aV-reduced cell ECM extracts.
A control ECM sample, known to contain FBN1, was included as a
positive control for reactivity with the a-fibrillin 1 antibody
this is not clear, but given that the a5 and aV subunits have
a high degree of sequence identity, it may be that the anti-
aV and anti-a5 siRNAs are having an off target effect as
has been noted elsewhere [27–29]. These data indicate that
our a5- and aV-reduced cell lines are essentially a5/aV-
double knock-downs. Our immunofluorescent and matrix
extraction studies (Fig. 4a, b) show that integrin protein
levels are not sufficient to support efficient fibronectin
assembly by these cells. As such, our findings support the
previous fibronectin matrix deposition data (described
above) obtained from null mice indicating that the loss of
both aV and a5 ablates the ability of cells to assemble
fibronectin into the ECM.
Loss of fibrillin 1 gene expression
Immunofluorescent staining of cultured cells revealed no
detectable fibrillin 1 matrix in a5- and aV-reduced MG63
123
214
Fig. 5 Conditioned media from a5- and aV-reduced cells contains
increased fibronectin, but no fibrillin 1. Serum-free conditioned media
was collected for 48 h from integrin-reduced MG63 and ARPE cells
that had been grown at confluent density for 5 days. Lane loads were
normalized to cell number. Fibronectin was detected in the condi-
tioned media from all integrin-reduced cell lines, but in much greater
amounts in the samples from a5- and aV-reduced cell lines as
compared to negative control plasmid and a2-reduced cells. This
increase indicates that these clones are unable to deposit fibronectin
normally into the ECM, and it is partitioning only to the medium.
DMEM containing 10 % FBS was included as a positive control.
Fibrillin 1 was detected in conditioned media from negative control
plasmid, a2-reduced, and b3-reduced cell lines. However, no FBN1
was detected in conditioned media samples from a5- and aV-reduced
cell lines, suggesting that these cells no longer express FBN1.
Conditioned medium known to contain FBN1 was included as a
positive control
Fig. 6 Fibrillin 1 gene expression is undetectable in a5- and aV-
reduced cell lines. Cells were grown to confluence on tissue culture-
treated plastic. Total RNA was collected, reverse transcribed, and
using the same amount of cDNA for each sample PCR was performed
using two sets of exon-spanning primers specific to human fibrillin 1.
Three clonal lines each for a5- and aV-reduced cells were examined.
No amplification of fibrillin 1 message was observed in any of the a5-
or aV-reduced cell lines after 35 cycles
clones (Fig. 4a). Extraction of the ECM and evaluation of
conditioned medium from these cells confirmed the
absence of fibrillin 1 protein production (Figs. 4b, 5).
Reverse transcription and PCR amplification of FBN1
message demonstrated that expression from the FBN1 gene
had been silenced in these two lines. To insure that fibrillin
down-regulation was not unique to these two clonal lines,
we examined FBN1 expression in two additional a5 and
aV clones and found that FBN1 expression was not
detectable in these as well (Fig. 6). This has led us to the
123
Mol Cell Biochem (2012) 369:205–216
novel finding that fibrillin 1 gene expression is down-reg-
ulated as a result of the reduction in a5- and aV-integrin
expression and/or the absence of a fibronectin matrix.
It has been suggested that fibronectin is needed for the
nucleation of fibrillin 1 and its subsequent deposition [30,
31]. The deposition of type I and III collagen assembly is
impaired in fibronectin null cells, but both proteins are still
produced [32]. Similarly, fibronectin null cells produce but
fail to deposit LTBP-1 [30]. Matrix assembly of fibulin 1
[33, 34], thrombospondin 1 [35], and fibrinogen [36] has
also been shown to depend upon the presence of a fibro-
nectin matrix. These studies did not indicate if the ECM
protein in question was still produced in the absence of a
fibronectin matrix. It may be that expression of some of
these proteins is, like fibrillin 1, also turned off in the
absence of a fibronectin matrix.
We hypothesize that loss of FBN1 gene expression in
these cells is due to either loss of integrin signaling or
differences in the biomechanical environment caused by
the absence of fibronectin. Mechanotransduction, the con-
version of mechanical load into biochemical signal, holds
strong regulatory influence in most cell types. It is rea-
sonable to think that the in vitro biomechanical environ-
ment of cells lacking a fibronectin matrix would be quite
different from cells within a well-elaborated fibronectin
matrix. Our a5- and aV-deficient MG63 cells may down-
regulate FBN1 expression because of the relative instabil-
ity of their matrix due to the loss of fibronectin. Previous
studies have found that epithelial and fibroblastic cells
grown on soft substrates exhibit reduced spreading and cell
area compared to cells grown on a rigid substrate [37].
Considering both the impaired spreading of our a5- and
aV-reduced cell lines and their failure to elaborate an intact
fibronectin matrix, it is reasonable to believe that the
matrix that these cells produce is not as mechanically
stable as that deposited by normal cells.
Biomechanical regulation of the FBN1 gene by some
cell types would not be surprising given the accepted
structural role for the protein and its distribution in
extensible tissues [38, 39]. Prior studies support the idea of
biomechanical regulation of the FBN1 gene. Fibrillin 1
gene expression in lung spikes just after birth when the
lungs are first fully loaded by use [40]. Yin et al. [41] noted
that FBN1 expression in murine endocardium correlated
with the onset of strong and regular contractions and sug-
gested that fibrillin may be expressed at this time to bolster
the mechanical needs of the organ. In mouse aorta, fibrillin
1 expression peaks at birth—a time that corresponds to the
greatest change in pressure seen by the vasculature [42].
Fibrillin 2 expression in these same tissues does not
undergo similar peaks. Indeed, the only elastic tissue that
does not undergo cyclic strain is elastic cartilage, which
contains mostly fibrillin 2 and has little fibrillin 1 [38, 39].
Mol Cell Biochem (2012) 369:205–216
If fibrillin 2 expression is not biomechanically influenced,
then some of the differential expression of the two genes
could be explained.
Signaling from integrins is well known to have wide-
ranging effects on gene expression. In solid tissues, the
ECM is a component of the cellular niche and, acting
through integrin signaling, can strongly influence cellular
behaviors ranging from lineage specification [43], to
tumorigenesis [44, 45], and even the production of other
ECM proteins [46–48]. The question of integrin signaling
versus mechanotransduction is not mutually exclusive;
clearly, overlap exists between these two known influential
regulators of gene expression. Distinguishing to what
extent these two mechanisms influence the expression of
fibrillin 1 is beyond the scope of this manuscript, but it will
be an interesting avenue for further investigation. In sum-
mary, we found that selective down-regulation of integrin
subunits had phenotypic effects on cell spreading, func-
tional effects on adhesion and ECM deposition, and genetic
effects on fibrillin 1 gene expression.
Acknowledgments RKB is grateful to TMR for his excellent
mentorship during this project. TMR gratefully acknowledges support
from NIH NIAMS RO3 AR 049887-02, Pennsylvania, Department of
Health, and the Penn State, Department of Orthopaedic Surgery,
Division of Musculoskeletal Sciences.
References
1. Miyahara M, Njieha FK, Prockop DJ (1982) Formation of col-
lagen fibrils in vitro by cleavage of procollagen with procollagen
proteinases. J Biol Chem 257(14):8442–8448
2. Zhang G, Young BB, Ezura Y, Favata M, Soslowsky LJ, Chak-
ravarti S, Birk DE (2005) Development of tendon structure and
function: regulation of collagen fibrillogenesis. J Musculoskelet
Neuronal Interact 5(1):5–21
3. Mao Y, Schwarzbauer JE (2005) Fibronectin fibrillogenesis, a
cell-mediated matrix assembly process. Matrix Biol 24(6):
389–399. doi:10.1016/j.matbio.2005.06.008
4. Singh P, Carraher C, Schwarzbauer JE (2010) Assembly of
fibronectin extracellular matrix. Annu Rev Cell Dev Biol
26:397–419. doi:10.1146/annurev-cellbio-100109-104020
5. Ginsberg MH, Partridge A, Shattil SJ (2005) Integrin regulation.
Curr Opin Cell Biol 17(5):509–516. doi:10.1016/j.ceb.2005.
08.010
6. DeMali KA, Wennerberg K, Burridge K (2003) Integrin signaling
to the actin cytoskeleton. Curr Opin Cell Biol 15(5):572–582
7. Mariko B, Ghandour Z, Raveaud S, Quentin M, Usson Y,
Verdetti J, Huber P, Kielty C, Faury G (2010) Microfibrils and
fibrillin-1 induce integrin-mediated signaling, proliferation and
migration in human endothelial cells. Am J Physiol Cell Physiol
299(5):C977–C987. doi:10.1152/ajpcell.00377.2009
8. Piha-Gossack A, Sossin W, Reinhardt DP (2012) The evolution
of extracellular fibrillins and their functional domains. PLoS One
7(3):e33560. doi:10.1371/journal.pone.0033560
9. Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML,
Lincecum J, Zako M (1999) Functions of cell surface heparan
sulfate proteoglycans. Annu Rev Biochem 68:729–777. doi:
10.1146/annurev.biochem.68.1.729
215
10. Ritty TM, Broekelmann TJ, Werneck CC, Mecham RP (2003)
Fibrillin-1 and -2 contain heparin-binding sites important for
matrix deposition and that support cell attachment. Biochem J
375(Pt 2):425–432. doi:10.1042/BJ20030649
11. Tiedemann K, Batge B, Muller PK, Reinhardt DP (2001) Inter-
actions of fibrillin-1 with heparin/heparan sulfate, implications
for microfibrillar assembly. J Biol Chem 276(38):36035–36042.
doi:10.1074/jbc.M104985200
12. Parsi MK, Adams JR, Whitelock J, Gibson MA (2010) LTBP-2
has multiple heparin/heparan sulfate binding sites. Matrix Biol
29(5):393–401. doi:10.1016/j.matbio.2010.03.005
13. Pfaff M, Reinhardt DP, Sakai LY, Timpl R (1996) Cell adhesion
and integrin binding to recombinant human fibrillin-1. FEBS Lett
384(3):247–250
14. Sakamoto H, Broekelmann T, Cheresh DA, Ramirez F, Rosen-
bloom J, Mecham RP (1996) Cell-type specific recognition of
RGD- and non-RGD-containing cell binding domains in fibrillin-
1. J Biol Chem 271(9):4916–4922
15. D’Arrigo C, Burl S, Withers AP, Dobson H, Black C, Boxer M
(1998) TGF-beta1 binding protein-like modules of fibrillin-1 and
-2 mediate integrin-dependent cell adhesion. Connect Tissue Res
37(1–2):29–51
16. Bax DV, Bernard SE, Lomas A, Morgan A, Humphries J, Shut-
tleworth CA, Humphries MJ, Kielty CM (2003) Cell adhesion to
fibrillin-1 molecules and microfibrils is mediated by alpha 5
beta 1 and alpha v beta 3 integrins. J Biol Chem 278(36):
34605–34616. doi:10.1074/jbc.M303159200
17. Lee SS, Knott V, Jovanovic J, Harlos K, Grimes JM, Choulier L,
Mardon HJ, Stuart DI, Handford PA (2004) Structure of the
integrin binding fragment from fibrillin-1 gives new insights into
microfibril organization. Structure 12(4):717–729. doi:10.1016/j.str.
2004.02.023
18. Bouzeghrane F, Reinhardt DP, Reudelhuber TL, Thibault G (2005)
Enhanced expression of fibrillin-1, a constituent of the myocardial
extracellular matrix in fibrosis. Am J Physiol Heart Circ Physiol
289(3):H982–H991. doi:10.1152/ajpheart.00151.2005
19. Jovanovic J, Takagi J, Choulier L, Abrescia NG, Stuart DI, van
der Merwe PA, Mardon HJ, Handford PA (2007) alphaVbeta6 is
a novel receptor for human fibrillin-1. Comparative studies of
molecular determinants underlying integrin-rgd affinity and
specificity. J Biol Chem 282(9):6743–6751. doi:10.1074/jbc.
M607008200
20. Tsuruga E, Sato A, Ueki T, Nakashima K, Nakatomi Y, Ishikawa
H, Yajima T, Sawa Y (2009) Integrin alphavbeta3 regulates
microfibril assembly in human periodontal ligament cells. Tissue
Cell 41(2):85–89. doi:10.1016/j.tice.2008.07.005
21. Ritty TM, Broekelmann T, Tisdale C, Milewicz DM, Mecham RP
(1999) Processing of the fibrillin-1 carboxyl-terminal domain.
J Biol Chem 274(13):8933–8940
22. Woods A, McCarthy JB, Furcht LT, Couchman JR (1993) A
synthetic peptide from the COOH-terminal heparin-binding
domain of fibronectin promotes focal adhesion formation. Mol
Biol Cell 4(6):605–613
23. Yang JT, Hynes RO (1996) Fibronectin receptor functions in
embryonic cells deficient in alpha 5 beta 1 integrin can be
replaced by alpha V integrins. Mol Biol Cell 7(11):1737–1748
24. Bader BL, Rayburn H, Crowley D, Hynes RO (1998) Extensive
vasculogenesis, angiogenesis, and organogenesis precede lethal-
ity in mice lacking all alpha v integrins. Cell 95(4):507–519
25. Yang JT, Rayburn H, Hynes RO (1993) Embryonic mesodermal
defects in alpha 5 integrin-deficient mice. Development 119(4):
1093–1105
26. Yang JT, Bader BL, Kreidberg JA, Ullman-Cullere M, Trevithick
JE, Hynes RO (1999) Overlapping and independent functions of
fibronectin receptor integrins in early mesodermal development.
Dev Biol 215(2):264–277. doi:10.1006/dbio.1999.9451
123
216
27. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J,
Mao M, Li B, Cavet G, Linsley PS (2003) Expression profiling
reveals off-target gene regulation by RNAi. Nat Biotechnol
21(6):635–637. doi:10.1038/nbt831nbt831
28. Persengiev SP, Zhu X, Green MR (2004) Nonspecific, concen-
tration-dependent stimulation and repression of mammalian gene
expression by small interfering RNAs (siRNAs). RNA 10(1):
12–18
29. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG,
Umayam L, Lee JC, Hughes CM, Shanmugam KS, Bhattacharjee
A, Meyerson M, Collins FS (2004) Short interfering RNAs can
induce unexpected and divergent changes in the levels of untar-
geted proteins in mammalian cells. Proc Natl Acad Sci USA
101(7):1892–1897. doi:10.1073/pnas.03086981000308698100
30. Dallas SL, Sivakumar P, Jones CJ, Chen Q, Peters DM, Mosher
DF, Humphries MJ, Kielty CM (2005) Fibronectin regulates
latent transforming growth factor-beta (TGF beta) by controlling
matrix assembly of latent TGF beta-binding protein-1. J Biol
Chem 280(19):18871–18880. doi:10.1074/jbc.M410762200
31. Kinsey R, Williamson MR, Chaudhry S, Mellody KT, McGovern
A, Takahashi S, Shuttleworth CA, Kielty CM (2008) Fibrillin-1
microfibril deposition is dependent on fibronectin assembly.
J Cell Sci 121(Pt 16):2696–2704. doi:10.1242/jcs.029819
32. Velling T, Risteli J, Wennerberg K, Mosher DF, Johansson S
(2002) Polymerization of type I and III collagens is dependent on
fibronectin and enhanced by integrins alpha 11beta 1 and alpha
2beta 1. J Biol Chem 277(40):37377–37381. doi:10.1074/jbc.M
206286200M206286200
33. Roman J, McDonald JA (1993) Fibulin’s organization into the
extracellular matrix of fetal lung fibroblasts is dependent on
fibronectin matrix assembly. Am J Respir Cell Mol Biol 8(5):
538–545
34. Godyna S, Mann DM, Argraves WS (1995) A quantitative
analysis of the incorporation of fibulin-1 into extracellular matrix
indicates that fibronectin assembly is required. Matrix Biol
14(6):467–477
35. Sottile J, Hocking DC (2002) Fibronectin polymerization regu-
lates the composition and stability of extracellular matrix fibrils
and cell-matrix adhesions. Mol Biol Cell 13(10):3546–3559. doi:
10.1091/mbc.E02-01-0048
36. Pereira M, Rybarczyk BJ, Odrljin TM, Hocking DC, Sottile J,
Simpson-Haidaris PJ (2002) The incorporation of fibrinogen into
123
Mol Cell Biochem (2012) 369:205–216
extracellular matrix is dependent on active assembly of a fibro-
nectin matrix. J Cell Sci 115(Pt 3):609–617
37. Pelham RJ Jr, Wang Y (1997) Cell locomotion and focal adhe-
sions are regulated by substrate flexibility. Proc Natl Acad Sci
USA 94(25):13661–13665
38. Zhang H, Hu W, Ramirez F (1995) Developmental expression of
fibrillin genes suggests heterogeneity of extracellular microfibrils.
J Cell Biol 129(4):1165–1176
39. Mariencheck MC, Davis EC, Zhang H, Ramirez F, Rosenbloom J,
Gibson MA, Parks WC, Mecham RP (1995) Fibrillin-1 and
fibrillin-2 show temporal and tissue-specific regulation of expres-
sion in developing elastic tissues. Connect Tissue Res 31(2):
87–97
40. Mariani TJ, Reed JJ, Shapiro SD (2002) Expression profiling of
the developing mouse lung: insights into the establishment of the
extracellular matrix. Am J Respir Cell Mol Biol 26(5):541–548
41. Yin W, Smiley E, Germiller J, Sanguineti C, Lawton T, Pereira L,
Ramirez F, Bonadio J (1995) Primary structure and develop-
mental expression of Fbn-1, the mouse fibrillin gene. J Biol Chem
270(4):1798–1806
42. Kelleher CM, McLean SE, Mecham RP (2004) Vascular extra-
cellular matrix and aortic development. Curr Top Dev Biol
62:153–188. doi:10.1016/S0070-2153(04)62006-0
43. Czyz J, Wobus A (2001) Embryonic stem cell differentiation: the
role of extracellular factors. Differentiation 68(4–5):167–174
44. Larsen M, Artym VV, Green JA, Yamada KM (2006) The matrix
reorganized: extracellular matrix remodeling and integrin sig-
naling. Curr Opin Cell Biol 18(5):463–471. doi:10.1016/j.ceb.
2006.08.009
45. Berrier AL, Yamada KM (2007) Cell-matrix adhesion. J Cell
Physiol 213(3):565–573. doi:10.1002/jcp.21237
46. Klees RF, Salasznyk RM, Vandenberg S, Bennett K, Plopper GE
(2007) Laminin-5 activates extracellular matrix production and
osteogenic gene focusing in human mesenchymal stem cells.
Matrix Biol 26(2):106–114. doi:10.1016/j.matbio.2006.10.001
47. Hwang NS, Varghese S, Zhang Z, Elisseeff J (2006) Chondro-
genic differentiation of human embryonic stem cell-derived cells
in arginine-glycine-aspartate-modified hydrogels. Tissue Eng
12(9):2695–2706. doi:10.1089/ten.2006.12.2695
48. Jean C, Gravelle P, Fournie JJ, Laurent G (2011) Influence of
stress on extracellular matrix and integrin biology. Oncogene
30(24):2697–2706. doi:10.1038/onc.2011.27