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J Biol Chem, Vol. 273, Issue 40, 25533-25536, October 2, 1998
andFrom the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
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ABSTRACT |
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Abstract
Introduction
Procedures
Results & Discussion
References
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Developmental patterning and differentiation,
maintenance of parenchymal cell function, and the size, shape, and
invasiveness of tumors are all orchestrated by cell interactions with
the extracellular matrix. Here we show that the fibrillar structure of
fibronectin (FN) matrix encodes essential regulatory cues and controls
cell proliferation and signaling through changes in matrix
architecture. A matrix assembled from native FN stimulated cell growth.
In contrast, a mutant FN (FN
III1-7) that contains
all known cell binding motifs but forms a structurally distinct matrix
inhibited progression from G0/G1 into S phase.
Furthermore, FNIII1-7 suppressed the stimulatory
capacity of native FN and induced different levels of tyrosine
phosphorylation of pp125FAK. The differential effects on
cell growth were ablated by blocking formation of matrix fibrils. Thus,
modification of matrix architecture provides a novel approach to
control cell proliferation.
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INTRODUCTION |
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Top
Abstract
Introduction
Procedures
Results & Discussion
References
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In tissues and tumors, cells live within a multidimensional
fibrillar extracellular matrix. The surrounding matrix fibrils thus are
uniquely poised to supply environmental signals to cells, suggesting
that the structural organization of the matrix itself contributes to
the control of cell behavior. Extracellular matrix regulation of cell
adhesion, migration, and gene expression occurs through binding to
integrins and other cell surface receptors (1-3). As an integral
component of extracellular matrices,
FN1 matrix fibrils interact
with integrin receptors through well characterized binding sites (4,
5). Multiple FN domains are required to maintain the integrity of the
matrix and deletion or mutation of specific sites in FN can affect
matrix structure and assembly (6-11). In particular, a recombinant FN,
FNIII1-7, that contains all known cell binding sites
but lacks the first seven type III repeats (Fig. 1A)
exhibits an altered rate of matrix assembly with unique intermediates
as compared with native FN (11). FNIII1-7 and FN
matrices also differ in their capacities to re-organize the actin
cytoskeleton (12), suggesting that the structurally distinct
architecture of native and altered FN matrices may have significantly
different intracellular consequences.
Matrix engagement of specific receptors represents an important control point for determining cell shape, cytoskeletal geometry, and the organization of intracellular components (13, 14) as well as initiating the signaling events that lead to cell cycle progression (15-17). We now show that the architecture of the FN matrix can also regulate this process. FN matrices with distinct morphologies had opposite effects on cell growth by specifically altering the rate of G0/G1 to S phase progression. These effects required FN fibril formation demonstrating that the structure of the matrix plays an active role in modulating cell signaling.
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EXPERIMENTAL PROCEDURES |
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Abstract
Introduction
Procedures
Results & Discussion
References
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Proteins and Cells--
SVT2 (SV40-transformed 3T3) cells were
grown in Dulbecco's modified Eagle's medium plus 10% calf serum.
Growth conditions for CHO
5 cells and expression and purification of
pFN and recombinant FNs were as described previously (11).
FNIII1-2 is a baculovirus-expressed full-length
recombinant FN lacking the first two type III repeats.
Cell Synchronization--
SVT2 and CHO5 cells were seeded at
a density of 2 × 105 cells in either four-well (Nunc)
plastic chamber slides (for SVT2 cells) or 24-well dishes with glass
coverslips (CHO5), allowed to attach and spread for 16-24 h, then
incubated in serum-free medium for 16-22 h to obtain a population of
cells in G0. As is the case for most transformed cell
lines, serum starvation was not sufficient to totally synchronize SVT2
or CHO5 cells but did enrich a G0 population as
determined by propidium iodide staining and FACS analysis. For
synchronization at G1/S, CHO5 and SVT2 cells were seeded
in 24-well dishes at a concentration of 2.5 × 105
cells/well. After a 16-h incubation in complete medium, SVT2 cells were
cultured with 0.5 mM hydroxyurea for 14 h and CHO5 cells in 1 mM hydroxyurea for 18 h to synchronize
cells at late G1/S phase. Cells were released from
hydroxyurea, washed, and refed with complete medium containing
FN-depleted serum and 50 µg/ml pFN or FNIII1-7. Cells
were stained with propidium iodide (Cycle Test Plus DNA Reagent Kit,
Becton Dickinson), and 3 × 104 cells were analyzed by
FACS 10 h after release from hydroxyurea to monitor progression
through S phase and G2/M. A set of nonsynchronized cells
and cells after hydroxyurea incubation were also stained and analyzed
by FACS using Cell Quest software (Becton Dickinson).
Matrix Assembly and Cell Adhesion--
After synchronization,
cells were transferred into complete medium containing FN-depleted
serum plus 50 µg/ml pFN, 50 µg/ml FNIII1-7, or
other recombinant FNs or without added FN. For the mixture of
FNIII1-7 with pFN, 50 µg/ml of each protein were
added. For 70-kDa inhibition of fibril formation, 50 µg/ml pFN or
FNIII1-7 was mixed with 250 µg/ml 70-kDa fragment.
Cells were allowed to assemble FN matrix for the indicated time
periods. For immobilized proteins, 96-well microtiter plates were
coated overnight at 4 °C with 10 µg/ml pFN or
FNIII1-7 for BrdUrd incorporation or 5, 10, and 15 µg/ml for adhesion. Serum-starved CHO5 cells were trypsinized and
plated onto coated wells at a concentration of 2 × 104 cells/well and then cultured in medium containing
FN-depleted serum for a 10-20 h time course.
BrdUrd Labeling-- BrdUrd was added to a final concentration of 10 µM and incubated for 30 min. BrdUrd-positive cells were detected with an anti-BrdUrd antibody followed by an alkaline phosphatase-conjugated secondary antibody and developed with nitro blue tetrazolium and X-phosphate substrate solution (Boehringer Mannheim). The numbers of total and BrdUrd-positive cells were counted for several fields (750-1000 total cells) and the percentage of positive cells calculated. Alternatively, cells were labeled with 10 µM BrdUrd and processed for ELISA using BrdU Detection Kit III and ABTS substrate (Boehringer Mannheim). Plates were read on a microtiter plate reader at 405 nm with a reference wavelength of 490 nm.
Immunoblots--
For adhesion, serum-starved CHO5 cells were
trypsinized and plated at a concentration of 2 × 105
cells onto a 48-well dish coated with either 10 µg/ml pFN or 10 µg/ml FNIII1-7. Cells were lysed (50 mM
Tris-HCl, pH 7.5, 250 mM NaCl, 2 mM EDTA, 1%
Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 50 mM sodium fluoride, and 0.1 mM sodium orthovanadate) 0.5, 1, 3, 6, and 9 h after plating. For matrix assembly, CHO5 cells were
seeded, serum-starved, and incubated with FNs. Cells were lysed as
described 3, 9, 16 and 24 h after release from serum-free medium.
The concentration of total protein in each lysate was determined by BCA
assay (Pierce). 30 µg/lane of total protein was separated by 6%
SDS-PAGE, transferred to nitrocellulose, blocked overnight in 5%
bovine serum albumin, Buffer A, and probed with anti-phosphotyrosine
antibody PT66 (Sigma) at a concentration of 1:3000. For adhesion, blots
were developed by ECL (Pierce) and exposed to x-ray film. For matrix
assembly, blots were incubated with 125I-protein A then
analyzed and quantitated using a Molecular Dynamics PhosphorImager as
described (11). The level of phosphorylation after serum starvation
(time 0) was set to 1.0, and all other values are expressed relative to
that value. p130 was detected by immunoprecipitation of 150 µg of total cell lysates with 20 µg of polyclonal
anti-p130 antibody (Santa Cruz). Immunoprecipitated proteins
were separated by 6% SDS-PAGE and the level of phosphorylation detected with anti-phosphotyrosine antibody PT66 and developed by
ECL.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results & Discussion
References
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Native and Mutant FN Matrices Differentially Regulate Cell
Proliferation--
Native FN and FNIII1-7 matrices
have markedly different effects on cell proliferation as shown in Fig.
1B. CHO5 cells, which fail
to synthesize a FN matrix (11), were serum-starved to obtain an
enriched population of quiescent cells in G0. Cells were
then released from serum-free conditions and incubated with medium
containing native pFN, baculovirus-expressed mutant
FNIII1-7, or no exogenous FN. During the incubation,
CHO5 cells bind and assemble exogenous FN into a fibrillar matrix at
the cell surface (11). Labeling of newly synthesized DNA by
incorporation of BrdUrd was then used to monitor cell cycle
progression. Compared with cells without FN matrix, pFN stimulated and
FNIII1-7 inhibited entry into S phase (Fig.
1B). Twice as many cells with a native FN matrix were
BrdUrd-positive 16 h after release from serum-free conditions as
compared with cells with FNIII1-7 matrix (Fig.
1C). With native FN matrix, cells entered S phase at least
8 h earlier than cells with FNIII1-7 matrix. Cells with no FN matrix progressed into S phase at a rate intermediate to
native FN and FNIII1-7. BrdUrd incorporation by cells with a full-length recombinant FN matrix was the same as cells with
pFN, indicating that differences in growth were not due to the source
of the baculovirus-expressed recombinant protein.
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III1-7 matrix inhibit cell growth, it
also suppresses the stimulatory effects of native FN. A matrix composed
of equal proportions of pFN and FNIII1-7 reduced BrdUrd
incorporation by 30% relative to cells in the presence of native FN
matrix (Fig. 1D). This result shows that the
FNIII1-7 matrix has a dominant-negative effect on
growth stimulation by native FN.
FNIII1-7 matrix also slowed the proliferation of SVT2
cells that assemble a matrix using endogenously produced FN (Fig. 2A). Incubation of cells with
exogenous pFN or FNIII1-7 results in co-assembly with
endogenous SVT2 FN as well as a significant increase in the overall
level of matrix-associated FN (9). The G0 to S phase
interval of SVT2 cells with FNIII1-7-containing matrix
was 4 h longer than that of cells assembling a pFN matrix, suggesting a dominant inhibitory effect of the FNIII1-7 matrix on cell growth. Morphologically, SVT2 cell matrices containing pFN and FNIII1-7 are distinct. Immunofluorescence
staining of cells with exogenous pFN shows an ordered fibrillar matrix (Fig. 2B). In contrast, FNIII1-7 matrix
appears less uniform and is characterized by fibrils of varying length
and thickness (Fig. 2, C and D). Together, these
results demonstrate that co-assembly of FNIII1-7 with
SVT2 FN has a dominant-negative effect on cell growth and, as with
CHO5 cells, this effect correlates with differences in matrix fibril
organization.
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5 and SVT2
cells synchronized at G1/S with hydroxyurea were released
into complete medium containing pFN, FNIII1-7, or no FN
and stained with propidium iodide to monitor DNA synthesis and cell
cycle progression by FACS analysis. Neither native FN or
FNIII1-7 matrix altered the progression of cells
through S or G2/M as the percentages of cells traveling
through these phases and back to G1 were identical (not
shown).
Growth Responses Are Abolished by Inhibition of FN Fibril
Formation--
pFN and FNIII1-7 both contain all known
cell binding sites and both use 5
1 integrin to initiate matrix
assembly (11, 18, 19), yet the two types of matrices have opposite
effects on cell growth. Cell proliferation would be affected if there are different levels of integrin-mediated binding and adhesion to these
two FNs (16, 17, 20). To address this possibility, attachment and
growth of CHO5 cells on immobilized pFN or FNIII1-7 protein were measured. Equal numbers of cells attached to pFN and
FNIII1-7 substrates in 30 min (not shown). Attached cells showed identical levels of BrdUrd incorporation on the two proteins (Fig. 3A). Therefore,
cell adhesive interactions with native and mutant FNs are
indistinguishable. An alternative explanation for the opposite growth
responses is that the distinct architectures of the two matrices may
influence cell proliferation. If fibrillar matrix structure controls
the rate of growth, then inhibition of fibril formation should
eliminate the differences between native FN and
FNIII1-7 matrices. Inclusion of excess 70-kDa
amino-terminal fragment of FN during matrix assembly blocks FN-FN
interactions via the assembly domain (see Fig. 1A) thus
preventing fibril formation by pFN (6, 7) and FNIII1-7
(11). 70-kDa fragment does not interfere with FN-integrin interactions.
Inhibition of fibril formation by addition of 70-kDa fragment reversed
the growth stimulatory effects of native FN matrix and the inhibitory
effects of FNIII1-7 matrix (Fig. 3B). In
both cases, a block in fibril assembly resulted in BrdUrd incorporation
comparable with that of cells with no matrix. Further evidence for the
role of fibrillar matrix structure in regulating cell growth was
provided by experiments with two other mutant recombinant FNs,
FNIII1-2 and FN(syn-). FNIII1-2 is
defective in FN binding and
polymerization,2 while
FN(syn-) does not bind well to 51 integrin (21). As a result of
these defects, each can only form short fibrils but cannot assemble
into an extensive matrix of the type shown in Fig. 2. Cells grown in
the presence of either of these two mutant FNs progressed into S phase
at a rate similar to cells with no FN matrix (Fig. 3B).
These results demonstrate that the differential effects of native FN
and FNIII1-7 on cell growth occur only when each is
interacting with cells from within a fibrillar matrix. More
importantly, the opposite effects of these FNs on cell proliferation
are directly attributable to the structural differences between
native FN and FNIII1-7 matrix fibrils.
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Induction of Intracellular Signaling Responses--
Cell
interactions with FN activate a number of intracellular signal
transduction cascades (2, 22, 23). In particular, phosphorylation of
focal adhesion kinase (pp125FAK) is an early biochemical
response to integrin-mediated adhesion to FN substrates (24-26). If
FNIII1-7 matrix structure can perturb cell cycle
progression, then intracellular signaling in response to matrix should
be affected. Phosphorylation levels of pp125FAK were
compared in cells assembling different fibrillar matrices. Cells
assembling native FN matrix exhibited a 1.5-2-fold increase in
pp125FAK phosphorylation over cells with either no FN or
FNIII1-7 matrix (Fig.
4A). The differential effects
of these matrices were maintained even at the earliest time point where
a transient increase in pp125FAK phosphorylation was
observed under all three conditions, probably due to the addition of
serum (2). The high levels of phosphorylation in response to native FN
matrix were sustained throughout the entire time course. However,
pp125FAK phosphorylation in cells with
FNIII1-7 matrix remained low until the 24-h time point
when it gradually increased to within 1.5-fold of levels with pFN (Fig.
4A). In contrast to the effects of fibrillar matrix
assembly, cell adhesion on immobilized pFN or FNIII1-7
protein resulted in a rapid phosphorylation of pp125FAK to
equivalent levels (Fig. 4B). Moreover, on both substrates, pp125FAK phosphorylation was transient with obvious
decreases after 3 h. A second focal adhesion protein,
p130 (27, 28), did not exhibit differential phosphorylation
(Fig. 4C). p130 phosphorylation was transient,
and comparable levels were observed with both pFN and
FNIII1-7 during matrix assembly and cell adhesion. Therefore, cell interactions with fibrillar matrix elicit specific responses that are not observed with cell binding to immobilized protein substrates. Similar to the stimulatory effect on cell proliferation, native FN matrix induced pp125FAK
phosphorylation. FNIII1-7 matrix, on the other hand,
inhibited both pp125FAK phosphorylation and cell cycle
progression. Clearly, the structure of the FN matrix can regulate
receptor signaling and downstream pathways.
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III1-7
matrix structures (12). Extracellular matrix rigidity can modify the
strength of connections between integrins and the cytoskeleton (32) and
may constitute another link between matrix architecture and cell growth
control. Interestingly, our results also show that altering the
structure of the FN matrix can be more detrimental than having no
matrix at all. pFN polymers have been shown to have antimetastatic
activity (33), and they may be acting through effects on matrix
organization. Thus the design of reagents that can modify existing
matrix architecture through dominant-negative effects could be a novel
strategy for controlling cell and tumor growth and spread.
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ACKNOWLEDGEMENTS |
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We thank Drs. Mark Ginsberg, Arnold Levine, Bonnie Bassler, and Ruth Steward for critical reading of the manuscript and Drs. Siobhan Corbett and Stuart Emanuel for helpful discussions. We are grateful to Andrew Beavis (Flow Cytometry Laboratory) and to Darren Hasara (Animal Facility) for their help on various aspects of this project.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA 44627 (to J. E. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Fellow of the New Jersey Commission on Cancer Research.
§ To whom correspondence should be addressed. Tel.: 609-258-2893; Fax: 609-258-1035; E-mail: jschwarzbauer{at}molbio.princeton.edu.
The abbreviations used are:
FN, fibronectin; pFN, plasma FN; FNIII1-7, recombinant FN lacking
repeats III1-7SVT2, SV40-transformed 3T3 cellsFACS, fluorescence-activated cell sortingBrdUrd, bromodeoxyuridineELISA, enzyme-linked immunosorbent assayPAGE, polyacrylamide gel
electrophoresis.
2 J. L. Sechler and J. E. Schwarzbauer, unpublished observations.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results & Discussion
References
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G Davey, M Buzzai, and R. Assoian Reduced expression of (a |
