Download extracellular matrix remodeling and integrin

The matrix reorganized: extracellular matrix remodeling
and integrin signaling
Melinda Larsen1, Vira V Artym1,2, J Angelo Green1 and
Kenneth M Yamada1
Via integrins, cells can sense dimensionality and other physical
and biochemical properties of the extracellular matrix (ECM).
Cells respond differently to two-dimensional substrates and
three-dimensional environments, activating distinct signaling
pathways for each. Direct integrin signaling and indirect integrin
modulation of growth factor and other intracellular signaling
pathways regulate ECM remodeling and control subsequent
cell behavior and tissue organization. ECM remodeling is
critical for many developmental processes, and remodeled
ECM contributes to tumorigenesis. These recent advances in
the field provide new insights and raise new questions about
the mechanisms of ECM synthesis and proteolytic degradation,
as well as the roles of integrins and tension in ECM remodeling.
Addresses
1
Craniofacial Developmental Biology and Regeneration Branch,
National Institute of Dental and Craniofacial Research, National Institutes
of Health, 30 Convent Drive, MSC 4370, Bethesda, MD 20892-4370,
USA
2
Department of Oncology, Lombardi Comprehensive Cancer Center,
Georgetown University Medical School, Washington, DC 20057-1469,
USA
(2D) surfaces and complex, malleable three-dimensional
(3D) environments can therefore be sensed by integrins
that respond to these surfaces with altered signaling
(reviewed in [3–7]).
In this review, we discuss recent findings regarding ECM
remodeling, with emphasis on collagen, fibronectin and
associated integrin signaling. We examine how ECM
physical properties and higher-order organization influence cell behavior and integrin signaling pathways. ECM
remodeling is required in vivo for proper development,
but ECM alterations can also create an environment
conducive to tumorigenesis. In this review, we will consider some examples of these processes. We apologize for
the omissions imposed by the need for brevity.
Fibroblast-mediated collagen matrix
remodeling in vitro
Introduction
Increasing numbers of studies show that the morphology,
cytoskeletal structure and signaling of cells grown on 2D
surfaces differ from those of cells grown within 3D
environments, where collagen fibers contact both ventral
(lower) and dorsal (upper) surfaces of the cells. In fact,
upon engagement of receptors on their dorsal surface,
well-spread fibroblasts in 2D culture quickly convert to a
bipolar or stellate morphology characteristic of fibroblasts
in 3D environments [8]. A recent study [9] of cell
interactions with collagen reveals that a2b1 integrinmediated transport of collagen fibers and subsequent
contraction of in vitro 3D collagen matrices requires
non-muscle myosin II-B. Although this myosin is important for fibroblast cell motility in 3D collagen matrices, it
is not required for migration on 2D surfaces. Another
difference is that myosin II-B only localizes to cellular
extensions when cells are plated within 3D matrices
rather than on 2D surfaces [9].
Extracellular matrix (ECM) remodeling is involved in
development, fibrosis, tissue repair and tumor-associated
desmoplasia (stromatogenesis). The ECM can be remodeled by many processes, including synthesis, contraction
and proteolytic degradation. Integrins are the primary
ECM receptors mediating ECM remodeling (reviewed
in [1,2]). In response to changes in the ECM, integrin
signaling also regulates many other interrelated cellular
processes: proliferation, survival, cell migration and invasion (Figure 1). Integrins function as mechanotransducers
and can transform mechanical forces created by the ECM
or the cytoskeleton into chemical signals. Differences
between simple, rigid and non-pliable two-dimensional
Specific serum components can facilitate integrin-dependent 3D collagen gel contraction through different signaling pathways. PDGF-stimulated contraction of
floating collagen matrices utilizes phosphatidylinositol
3-kinase (PI3K) and myosin II, whereas LPA-stimulated
contraction depends on signaling by the monomeric Gprotein GaI, but does not require myosin II [10,11].
Using siRNA knockdown and inhibitors, it was demonstrated that signaling through PDGF and LPA converge
on p21-activated kinase-1 (PAK1) to regulate collagen
matrix contraction through cofilin-1 [11]. PAK1 therefore links these two distinct matrix remodeling pathways.
Corresponding author: Yamada, Kenneth M ([email protected])
Current Opinion in Cell Biology 2006, 18:463–471
This review comes from a themed issue on
Cell-to-cell contact and extracellular matrix
Edited by Martin Schwartz and Alpha Yap
Available online 17th August 2006
0955-0674/$ – see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.ceb.2006.08.009
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Current Opinion in Cell Biology 2006, 18:463–471
464 Cell-to-cell contact and ECM
Figure 1
Generalized schematic diagram of integrin signaling, focusing on pathways specifically covered in this review. Integrins signal through
recruitment of FAK, recruitment and activation of SFKs, and activation of PI3K. Src phosphorylates p130CAS and recruits Crk to activate Rac.
Rac is also activated by FAK via stimulation from PIX/GIT/paxillin complexes. FAK activates ERK signaling that, together with Rac downstream
signaling, exerts a regulatory effect on cell proliferation and survival. Signaling downstream of PI3K affects activation of Akt and the small
GTPases Rac, Cdc42, and Rho to induce changes in the cytoskeleton, cell contractility, cell migration, invasion and gene expression. Crosstalk
between integrin and GFR signaling pathways ensures proper integration of integrin- and GFR-mediated signaling required for optimal cell
function. LPA, acting through a seven-transmembrane G-protein-coupled receptor, signals through PAK and cofilin, and cooperates with the
ROCK/MLCP/myosin II pathway to promote collagen matrix contraction. Abbreviations: guanine nucleotide-exchange factors, GEFs; growth
factor, GF; LIM kinase, LIMK; mammalian diaphanous, mDIA; myosin light chain phosphatase, MLCP; phosphatidylinositol-3,4,5-trisphosphate,
PIP3; protein kinase C, PKC; Src-family kinases, SFKs; Wiskott-Aldrich syndrome protein, WASP.
ROCK was specifically implicated in PDGF-induced and
mDia1 in LPA-induced ECM contraction. These findings
suggest that Rho effectors act parallel to and/or cooperatively with PAK1 to regulate contraction of floating collagen matrices.
Fibronectin matrix remodeling by fibroblasts
in vitro
The major receptor for fibronectin, a5b1 integrin, can be
found in different adhesion structures, such as focal
complexes, focal adhesions, fibrillar adhesions and 3Dmatrix adhesions [3]. Fibronectin fibrillogenesis is
mediated by actin-dependent, directed translocation of
a5b1 out of focal adhesions into fibrillar adhesions [12],
and a recent study indicates that this translocation
depends upon a specific integrin conformation [13]. It
Current Opinion in Cell Biology 2006, 18:463–471
is well known that conformation directly affects integrin
activation state and ligand-binding activity; in 2D substrates, exogenous activating antibodies or manganese can
activate integrins and induce matrix formation [1].
Recent studies implicate the urokinase-type plasminogen
activator receptor (uPAR) in regulating integrin a5b1 activity. Addition of a uPAR ligand, the P-25 peptide, stimulated fibronectin fibril assembly [14] by activating integrin
a5b1 through the EGF receptor and Src [15]. Interestingly,
fibronectin fibril assembly by cells expressing activationdependent integrins can be stimulated specifically by a 3D
fibronectin matrix without exogenous activators [16].
These data support the idea that a 3D matrix can activate
integrins to induce fibronectin matrix assembly (Figure 2),
although the mechanism remains to be elucidated. One
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Extracellular matrix remodeling and integrin signaling Larsen, Artym, Green and Yamada 465
Figure 2
Extracellular factors promote ECM remodeling by stimulating integrin
activation. On a 2D substrate in vitro, fibroblast cells are polarized such
that only the ventral surface contacts the substrate. In this state, cells
are typically well-spread and only a subset of integrins is activated: bent
conformation, inactive integrin; extended conformation, activated
integrin. Upon plating within a 3D matrix or following treatment with
exogenous stimulators (e.g. Mn2+, activating antibodies or uPAR ligand),
integrins undergo a conformational change and are activated, leading to
enhanced integrin–ECM interactions and increased ECM production. As
the matrix assumes a more 3D character, cell morphology becomes
more bipolar or stellate, at least partly as a result of increased
engagement of receptors on the dorsal cell surface.
experimental approach to this question would be to
use conformation-specific antibodies to detect activated
integrins within different 3D environments.
Cells in vivo exist in complex environments, where they
constantly interact with multiple ECM molecules rather
than a single component. In vitro studies show that
specific cell–ECM interactions affect the dynamics of
other ECM components and the physical state of the
ECM. For example, cell-dependent fibronectin polymerization increases the tensile strength of a 3D collagen
biogel without affecting rigidity [17]. Additionally, LPA
induces both fibronectin and collagen assembly concurrently in smooth muscle cells. Inhibition of LPA-induced
fibronectin assembly with an anti-integrin a5b1 antibody
prevents collagen type I fibril assembly [18]. Other
studies also show that fibrillar collagen deposition is
dependent on fibronectin [19,20].
ECM remodeling in epithelial morphogenesis
ex vivo
Organ culture model systems provide powerful tools to
study developmental events using intact epithelial
embryonic tissues ex vivo. Branching organs, such as lung,
kidney and salivary gland, can be studied as organ
explants, since internally driven morphogenetic programs
continue ex vivo and recapitulate in vivo processes.
Branching morphogenesis involves a number of repetitive
steps, starting with formation of a cleft or indentation in
the basement membrane, which is a specialized ECM
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located at the basal surface of differentiated epithelial
cells. ECM remodeling and integrin signaling are clearly
important for branching, especially in the salivary gland,
where fibronectin and its major receptor, integrin a5b1,
are required (Figure 3a) [21]. Fibronectin accumulation in
cleft sites is associated with a decrease in E-cadherin, the
prototypic epithelial cadherin that mediates cell–cell
adhesion. In this study [21], exogenous cellular fibronectin also locally decreased cadherin levels in a human
salivary gland cell line, indicating that a function of
fibronectin is negative regulation of E-cadherin, although
the mechanism of this inhibition is not known.
Many classical studies point to the importance of basement membrane remodeling during branching morphogenesis. Recently, cytoskeletal tension was found to be
important for branching: inhibitors of ROCK, myosin
light chain kinase and myosin ATPase inhibited branching of lung rudiments (Figure 3b) [22]. Interestingly,
ROCK stimulated basement membrane thinning at the
lung bud distal tips. This thinning may facilitate cell
proliferation and promote bud elongation through
decreased synthesis or increased protease-mediated
degradation of basement membrane components.
Fibronectin matrix remodeling in vivo
Important new insights into the role of fibronectin matrix
remodeling during development have emerged recently
from work in model organisms. Somite formation is a major
event during vertebrate development, whereby the unsegmented paraxial mesoderm undergoes a mesenchymal-toepithelial transition to form epithelial segments. Although
fibronectin is known to be critical for somitogenesis
because fibronectin-null mouse embryos lack somites,
recent work has provided insight into the mechanism of
this morphological segmentation (Figure 3b). Mutation of
integrin a5 in zebrafish prevents fibronectin accumulation,
resulting in a defect in somite boundaries and epithelialization [23]. Another recent study reports that fibronectin is
required for somite maintenance but not initiation [24].
The mechanism by which fibronectin induces somite
epithelialization involves focal adhesion kinase (FAK).
In zebrafish integrin a5 mutants, no active FAK (phosphorylated at Y397) was detected [24]. Inhibition of FAK
signaling by the dominant-negative form of FAK, FAKrelated nonkinase (FRNK), results in defective fibronectin matrix deposition and disrupted somite boundaries in
Xenopus embryos [25]. It is interesting that FNRKinjected Xenopus embryos display a phenotype more
severe than that of FAK-null mouse embryos. FRNKinjected Xenopus embryos also show defective localization
of Xenopus Ena, an Ena/VASP family protein linking
integrins with the actin cytoskeleton. Neutralization of
Ena/VASP activity results in defective fibronectin accumulation around somites and impaired FAK activation
[25]. Together, these data suggest the existence of a
Current Opinion in Cell Biology 2006, 18:463–471
466 Cell-to-cell contact and ECM
Figure 3
Current Opinion in Cell Biology 2006, 18:463–471
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Extracellular matrix remodeling and integrin signaling Larsen, Artym, Green and Yamada 467
bidirectional signaling pathway between FAK and Ena/
VASP protein. These studies imply that Ena/VASP proteins and FAK activate integrin a5 to stimulate fibronectin fibrillogenesis and induce downstream signaling
leading to epithelialization. This provocative finding
indicates that fibronectin can promote the transition of
certain mesenchymal cells to an epithelial phenotype.
It is not clear why fibronectin can decrease E-cadherin in
some contexts [21], whereas in other contexts, such as
somite formation, it induces epithelialization [24] and
increased cadherin expression. However, integrin signaling often forms part of a signaling network. In the complex process of somitogenesis, fibronectin-integrin a5
signaling acts cooperatively with other pathways: Eph–
Ephrin signaling cooperates with fibronectin–integrin
signaling to maintain somite boundaries [24], and
Notch/Delta and integrin a5 interdependently regulate
somite epithelialization and fibronectin assembly [23].
The interaction between these molecules is complex, and
it changes along the anterior–posterior axis. The cellular
response to fibronectin can therefore depend on the local
signaling context.
ECM rigidity and cytoskeletal tension in
tumor progression
Stromatogenesis is a desmoplastic alteration in tumorassociated stroma, occurring in parallel with neoplasia,
which is characterized by many changes, including
increased expression of organized fibronectin and type
I collagen by adjacent stromal fibroblasts [26,27]. A consequence of stromatogenesis is that the tumor-associated
stroma becomes more rigid. Measurements of the rigidity
of normal mammary tissue, malignant breast tissue and
tumor-associated stroma revealed that malignancy is
accompanied by substantial increases in ECM rigidity
in both the tumor and stroma [28]. Cells sense elevated
ECM rigidity through integrins and respond with modified signaling, according to many studies. Increased
ECM rigidity stimulates integrin expression [29] and
induces conformational changes to activate avb3 integrin
[30]. Inappropriate increases in ECM rigidity perturb
normal tissue architecture, activate Rho, induce Rhogenerated cytoskeletal tension and activate ERK-dependent growth [31,32], whereas subsequent increases in
cytoskeletal tension promote growth and focal adhesion
assembly [33,34]. Pharmacological inhibition of ROCK or
myosin II can reverse the malignant phenotype, indicating a dependence of the transformed phenotype on Rhodependent tension [28]. ERK inhibition can also
reverse the malignant phenotype, underscoring the
cross-talk between mitogenic growth factor and mechanotransducing integrin pathways [28]. As a result of the
functional link between ECM rigidity, Rho, cell contractility and cell behavior, a mechanosensitive positive feedback loop appears to amplify cell proliferation,
transformation and ECM rigidity in tumors (Figure 4).
Proteolytic remodeling of ECM in tumor
progression
Many proteases can cleave ECM molecules involved in
tumor progression (reviewed in [35]), but recent studies
have identified MT1-MMP as a major protease supporting the invasive phenotype. MT1-MMP confers an
advantage on tumor cells in vitro and in vivo by enabling
them to escape growth suppression by fibrillar type I
collagen [36]. Membrane-bound MT1-MMP cleaves collagen and activates proMMP-2 in the immediate vicinity
of cancer cells, creating a proliferation-promoting microenvironment of cleaved collagen. Interestingly, in MCF-7
cells expressing MT1-MMP, type I collagen increases
cell-surface MT1-MMP activity by a novel mechanism:
impaired clathrin-mediated internalization of MT1MMP [37]. MT1-MMP activity is essential for fibroblast and tumor cell invasion through 3D collagen gels,
independent of plasminogen or secreted MMP activity
[38]. However, it should be noted that because epithelial cells rarely express MT1-MMP [39], even though
tumor cell lines and the surrounding stroma express
it, there are conflicting views on the significance of
MT1-MMP in human epithelial tumors.
The heterogeneity of ECM organization (reviewed in
[40]), from loose connective tissue to dense basement
membranes, requires cancer cells to employ a range of
migration modes. Two types of cancer cell migration
through ECM have been identified: mesenchymal migration that utilizes integrins and proteases for adhesive and
proteolytic interactions with ECM proteins, and amoeboid
migration that is integrin-independent and non-proteolytic (reviewed in [41]). Experimental models predict that
cells can switch from mesenchymal to amoeboid migration
(Figure Legend 3) ECM remodeling during organogenesis. (a) ECM remodeling in branching morphogenesis. Branching morphogenesis in the
salivary gland and other organs requires fibronectin. Acting through integrin a5b1, fibronectin locally decreases E-cadherin to stimulate cleft
formation. In the lung, cytoskeletal contraction mediated through ROCK signaling is critical for branching morphogenesis. Cytoskeletal
contraction leads to basement membrane remodeling, which may facilitate localized proliferation and subsequent bud elongation. (b) Integrin
signaling during somite morphogenesis. Somite formation depends upon fibronectin assembly at the intersomitic boundary. Activation of integrin
a5b1 induces fibronectin fibrillogenesis. Notch signaling stimulates integrin a5b1 signaling either directly, through Eph signaling, or through
cytoskeletal alterations. This was concluded since expression of active notch induced high-affinity integrin b1. Eph signaling induces cytoskeletal
changes and integrin a5b1 activation. FAK phosphorylation is required for somite formation, fibronectin fibrillogenesis, and colocalization of
Ena/VASP with integrin a5b1. FAK localization depends upon integrin ligation, and Ena/VASP is required for FAK activation by phosphorylation (P).
Activated integrin a5b1 signals downstream via FAK to induce epithelialization.
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Current Opinion in Cell Biology 2006, 18:463–471
468 Cell-to-cell contact and ECM
Figure 4
Effect of ECM rigidity on integrin signaling and tumorigenesis. Increased ECM rigidity activates integrins to promote focal adhesion formation, leading
to stimulation of the Rho/ROCK pathway and increased cell contractility, cell migration and invasion. ERK is activated either directly by integrins
or indirectly by growth factors to increase cell proliferation and possibly cell contractility. As transformation proceeds, increased cell contractility
contributes to further ECM stiffening, leading to increased integrin expression, focal adhesion formation and amplified signaling, creating a
self-sustained positive feedback loop (long red arrow) that may promote cell transformation. The phenotypic consequence of ECM stiffening
associated with cellular transformation is disruption of tissue homeostasis and conversion from a differentiated to a malignant phenotype.
Malignancy is associated with high Rho activity, lumen obstruction and loss of tissue polarity and adherens junctions in glandular tissues.
if pericellular proteolysis is inhibited by protease inhibitors, if the Rho/ROCK signaling pathway is activated, or if
integrin–ECM interactions are blocked [41].
Focal pericellular proteolysis of ECM molecules is a hallmark of mesenchymal migration by tumor cells [41].
Studies of malignant cells on 2D fluorescent matrices
indicate that pericellular proteolysis and invasion are
mediated by specialized cell membrane protrusions
termed invadopodia (reviewed in [42]). MT1-MMP was
identified as a key invadopodial protease responsible for
local ECM degradation by carcinoma cells. The dynamics
of the sequential accumulation of cortactin plus actin at
Current Opinion in Cell Biology 2006, 18:463–471
invadopodia followed by MT1-MMP recruitment, and the
subsequent onset of ECM degradation, have been
revealed by live-cell imaging [43]. Although intravital
imaging has detected invadopodium-like protrusions in
extravasating carcinoma cells [44], further studies are
required to detect invadopodial markers and test the
ECM-degrading ability of these protrusions to verify the
physiological importance of invadopodia in vivo.
Remodeled ECM as a carcinogen and
inducer of metastasis
Although, in normal tissue, one function of fibroblasts is
to maintain tissue homeostasis, the fibroblasts and ECM
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Extracellular matrix remodeling and integrin signaling Larsen, Artym, Green and Yamada 469
associated with tumors (the tumor microenvironment)
can function as a carcinogen. The tumor microenvironment can send erroneous signals to tumors that induce
accumulation of MMPs, activate soluble growth factors
and facilitate paracrine signaling. The epithelium often
responds by undergoing an epithelial–mesenchymal transition (EMT), where epithelial cells lose epithelial characteristics, acquire mesenchymal characteristics and
become invasive. Exposure of epithelial cells to MMP3 stimulates expression of Rac1b, an alternatively spliced
and highly active form of Rac1, which induces cellular
reactive oxygen species (ROS). ROS both stimulate
EMT and cause oxidative damage to DNA, resulting
in genomic instability [45]. Tumor microenvironments
are often hypoxic. An interesting but puzzling recent
study reports that lysyl oxidase, the well-known enzyme
that crosslinks collagen in vivo, is induced under hypoxic
conditions [46]. Although not required for primary tumor
growth, lysyl oxidase is required for metastasis, and it
induces FAK phosphorylation to stimulate cell motility.
The role of this matrix-modifying enzyme in metastasis
will be intriguing to resolve.
Conclusions and future directions
Great strides have been made in our understanding of
ECM remodeling through the increasing use of 3D culture systems in recent years. Although it is now clear that
integrin signaling and cytoskeletal organization are different in 2D and 3D environments, the factors responsible for these differences remain to be determined.
Understanding the mechanistic differences between
ECM remodeling on 2D surfaces and in 3D systems is
critical for developing valid model systems characteristic
of in vivo biology. Which factors are responsible for the
differences: the complex linkages of ECM proteins, the
physical forces associated with ECM presentation to the
cell, or a combination of these and other factors? Clarification is needed concerning how ECM components
structurally regulate each other to influence cell morphology and signaling. It may be possible to answer many
questions regarding the impact of structure on cell behavior using techniques that can sense local ECM rigidity.
Future improvements in atomic force microscopy (AFM)
or alternative approaches are needed to measure, apply
and evaluate the effects of mechanical stresses on morphology and signaling of live cells in 3D environments.
Since ECM remodeling is now known to be a dynamic
process involving cytoskeletal molecules and proteases,
live imaging in vivo and in 3D culture systems should
provide many new insights into mechanisms of ECM
remodeling. One issue with studying matrix assembly is
distinguishing new ECM from old. A recent report
applied the Timer reporter, a DsRed derivative that
changes from green to red over time, to overcome this
problem in studies of elastic fiber formation [47]. Multicolor in vivo time-lapse imaging of fluorescently labeled
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ECM and cell adhesion proteins should illuminate the
mechanisms of ECM assembly that control developmental processes, whereas time-lapse imaging of tumor cell
proteases and specialized cell membrane structures (e.g.
invadopodia) will help elucidate their role in ECM degradation. Studies employing electron microscopy will also
be useful to validate the significance of invadopodia in
vivo. Although the ECM and microenvironment are now
known to be involved in tumor initiation and progression
(e.g. through ECM rigidity), the mechanisms need elucidation. This gap could be addressed by development of
in vitro 3D models in which matrix rigidity and biochemical content can be carefully controlled. A difficulty with
studying tumor-associated fibroblasts has been maintaining their phenotype in culture, but a recent report showed
that their phenotype can be maintained within a 3D
matrix [48]. Developing a deeper understanding of how
ECM remodeling occurs and how it modifies cell behavior should lead to better clinical interventions for pathologies that develop when ECM remodeling goes awry.
Update
A new study proposes that local translocation of a component of the 3D matrix guides branching morphogenesis. Live confocal imaging of glands labeled with twocolor fluorescently labeled fibronectin was used to establish that translocating wedges of fibronectin move steadily
inward through a population of surprisingly highly motile
epithelial cells. This 3D matrix translocation separates
the cells to form clefts during embryonic mouse salivary
gland development [49].
Acknowledgements
The authors thank A. Doyle and S. Even-Ram for valuable comments and
suggestions and H. Grant for excellent proofreading. Research support
was provided by the Intramural Research Program of the NIH, NIDCR,
to K.M.Y.
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