1 Department of Biomedical Engineering, Johns Hopkins School of Medicine, 720
Rutland Avenue, Baltimore, MD 21205, USA
2 Department of Oncology, Johns Hopkins School of Medicine, 720 Rutland Avenue,
Baltimore, MD 21205, USA
* Author for correspondence (e-mail: cchen{at}bme.jhu.edu)
Accepted 22 May 2003
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Summary |
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Key words: Cell shape, Microfabrication, Intercellular adhesion, Adherens junctions
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Introduction |
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Although growth factors and integrins convey mitogenic signals leading to
proliferation (Assoian and Schwartz,
2001; Danen and Yamada,
2001
), adhesion between cells is thought to inhibit growth
(Castilla et al., 1999
;
Caveda et al., 1996
). As a
result, cadherin engagement has been suggested to be another mechanism,
distinct from decreased cell spreading, for the cessation of growth when cells
reach confluence (Gumbiner,
1996
). Transformed cells lacking a variety of components of the
cell-cell adhesion machinery proliferate at higher rates than when they are
transfected with neural (N)-cadherin
(Levenberg et al., 1999
) or
epithelial (E)-cadherin (Stockinger et
al., 2001
). In endothelial cells, vascular endothelial
(VE)-cadherin has been implicated as the major receptor responsible for this
effect (Castilla et al., 1999
;
Caveda et al., 1996
). Blocking
the function of VE-cadherin increases proliferation, whereas exogenous
expression of VE-cadherin in CHO cells decreases growth rates
(Caveda et al., 1996
). Despite
these findings, little is known about the mechanism by which cadherins inhibit
growth, or the relative contributions of changes in cell spreading versus
cadherin engagement to growth arrest at confluence. Recent work has suggested
that increasing cell-cell adhesion mechanically competes with and hence
decreases cell-substrate adhesion
(Lauffenburger and Griffith,
2001
; Ryan et al.,
2001
). Thus, growth arrest by cell-cell contact and by changes in
cell spreading may be linked; VE-cadherin could inhibit proliferation in part
by altering cytoskeletal structure and decreasing cell spreading.
VE-cadherin can modulate the organization of the cytoskeleton through
multiple mechanisms. The homotypic engagement of cadherins initiates both
soluble signaling cascades as well as the recruitment of scaffolding proteins
to form the adherens junction. Cadherins increase signaling through ERK
(Pece and Gutkind, 2000), PKC
(Lewis et al., 1994
) and Akt
(Carmeliet et al., 1999
), all
of which have significant effects in reorganizing the actin cytoskeleton
(Clark et al., 1998
;
Keenan and Kelleher, 1998
;
Klemke et al., 1997
;
Reif et al., 1996
). Cadherins
also modulate structural changes to microtubules
(Chausovsky et al., 2000
) and
actin through proteins including the Rho family GTPases
(Noren et al., 2001
) and
ß-catenin (Barth et al.,
1997
). The formation of adherens junctions by VE-cadherin directly
recruits and anchors the actin cytoskeleton to intercellular junctions
(Breviario et al., 1995
).
Modulating cadherin-mediated intercellular adhesion also directly alters the
expression of integrins (Zhu and Watt,
1996
). Thus, VE-cadherin engages numerous pathways that can alter
cytoskeletal structure and tension, cell shape and integrin signaling
(Braga, 2000
;
Chen et al., 1997
;
Pawlak and Helfman, 2001
;
Schwartz and Assoian, 2001
) -
all potent regulators of proliferation.
The complex interplay between intercellular and extracellular matrix
adhesion highlights the importance of developing an experimental system to
independently manipulate these signals, and thereby decouple their respective
effects on cell function. Using a new microengineering approach to control
cell-cell contact and cell spreading independently, we recently reported that
allowing physical contact between cells while preventing changes in cell
spreading stimulated proliferation in multiple cell types, and that this
stimulation was masked by an apparently distinct inhibitory signal when cell
spreading was not controlled (Nelson and
Chen, 2002). Now, using our new system to examine the role of cell
spreading in VE-cadherin-mediated regulation of endothelial cell
proliferation, we show that VE-cadherin exerts its inhibitory effects
specifically by actively decreasing cell spreading. Preventing the
cadherin-induced changes in cell morphology, we unexpectedly reveal that
VE-cadherin also elicits a stimulatory signal for growth that depends on
Rho-mediated tension in the actin cytoskeleton. Thus, VE-cadherin appears to
regulate two distinct proliferative signals in endothelial cells, both through
crosstalk with regulatory pathways traditionally associated with cell shape
and cytoskeletal structure.
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Materials and Methods |
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Fabrication of substrates with microwells
Stamps of poly(dimethylsiloxane) (PDMS, Sylgard 184, Essex Brownell, Fort
Wayne, IN) containing a relief of the desired pattern (bowties raised from the
surface) were fabricated as previously described
(Nelson and Chen, 2002).
Briefly, PDMS was cast on a silicon master with 20 µm-thick bowtie-shaped
wells made by photolithography. To aid in release of the cured PDMS, the
master was first silanized overnight with a vapor of
(tridecafluoro-1,1,2,2,tetrahydrooctyl)-1-trichlorosilane (United Chemical
Technologies, Bristol, PA) under vacuum before casting the PDMS. A stamp
oxidized under UV/ozone (UVO Cleaner, Jelight Company, Irvine, CA) was placed
on a SuperFrost slide (Fisher Scientific) such that only the raised
bowtie-shaped regions of the stamp sealed against the glass surface. A
solution of 0.6% agarose (Life Technologies)/40% ethanol in water was heated
(80°C), perfused through the channels formed between the sealed features,
and allowed to cool for 12 minutes under vacuum. The stamps were peeled from
the substrate, leaving behind bowtie-shaped wells with bases of glass and
walls of agarose. The substrates were sterilized in ethanol, washed in PBS and
treated with a 25 µg/mL solution of fibronectin (Collaborative Biomedical
Products) in PBS, which coated the glass bases of the bowtie-shaped wells.
Measurement of proliferation
To obtain changes in cell number, cells were photographed with a Spot CCD
camera (Diagnostic Instruments, Sterling Heights, NY). Cell proliferation was
calculated by counting cells in microscopy images at indicated times, and
normalized to initial cell number. Mean cell area was determined by outlining
cells in phase contrast images with the Spot software. To determine entry into
S phase, the percentage of cells incorporating 5-bromo-2'-deoxyuridine
(BrdU) was quantified using a commercial assay (Amersham). Cells were
G0-synchronized by holding cultures at confluence for 2 days, then
plated onto substrates in full culture media. BrdU was added to the media 2
hours after plating. Cells were fixed and stained according to the
manufacturer's instructions at 24 hours. BrdU-positive fluorescent cells were
visualized and scored using a Nikon epifluorescence microscope (Nikon). The
DNA-binding dye Hoechst 33258 (Molecular Probes) was used as a counterstain (1
µg/ml). To distinguish between cell types in co-culture experiments, A431
cells were labeled with Cell Tracker Orange (Molecular Probes) prior to
seeding with endothelial cells. For all proliferation conditions using random
seeding, at least 200 cells were counted per condition across two independent
experiments. For all proliferation conditions using patterned wells, at least
500 cells were counted per condition across three independent experiments.
Flow cytometry analysis
To determine position in the cell cycle, trypsinized cells were collected
by centrifugation and resuspended in a 0.05 mg/mL solution of propidium iodide
(PI) (Molecular Probes) in 1% sodium citrate, 0.1% Triton-X-100, and 7
units/mL DNAse-free ribonuclease A (Sigma). Analysis was performed on a
FACScan flow cytometer (Becton Dickinson). All data were acquired and analyzed
with the CellQuest software. For cell-cycle analysis, gating was set around
cell populations based on fluorescence intensity and sideward scatter.
Immunofluorescence
Cells were fixed and stained at 24 hours after seeding. For the detection
of cell-cell adhesion molecules, cells were fixed in 4% paraformaldehyde in
PBS, permeabilized in 0.2% Triton-X-100 in PBS, washed in 33% goat serum in
PBS, incubated in primary antibodies diluted in 33% goat serum in PBS, and
visualized with Alexa 488- or 594-conjugated secondary antibodies (Molecular
Probes). For the detection of actin, fixed and permeabilized cells were
stained with 0.1 µg/mL TRITC-conjugated phalloidin (Sigma) in PBS.
Construction of recombinant adenoviruses
Recombinant adenoviruses encoding RhoN19 and GFP were prepared using the
AdEasy XL System (Stratagene) as per kit instructions. Briefly, the cDNA
fragment encoding RhoN19 was mutagenized from pEGFP-WT-RhoA (gift from M.
Philips, New York University). The fragment was PCR amplified and cloned into
the shuttle vector pShuttle-IRES-hrGFP-1. After construction, the shuttle
vector was linearized with Pme I and transformed into
BJ5183-AD-1-competent cells pretransformed with the pAdEasy-1 adenoviral
vector, to generate recombinant adenoviral plasmids, which were purified and
transfected into HEK 293 cells. Adenoviral infection was monitored by GFP
fluorescence, and adenoviral particles were obtained by cell extraction after
7-10 days. The virus was further amplified and purified by centrifugation on a
CsCl gradient. Stocks of 109-1010 infectious
particles/mL were retained and used in subsequent experiments. The virus was
titrated by infecting HEK 293 cells with serially diluted stocks and counting
GFP-expressing cells.
To infect endothelial cells, a solution of recombinant adenovirus was mixed with culture medium, and cells were exposed to the virus with a multiplicity of 10-100 viral particles/cell for 3 hours. Cells were then washed, trypsinized and plated onto substrates. Cells were analyzed 24 hours after plating; under these conditions, >95% of the cells were infected.
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Results |
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To manipulate VE-cadherin engagement on shorter time scales and further
explore its effects on cell spreading and cell-cycle progression, we altered
cell-cell contact by seeding cells at different densities. Cells were
synchronized by holding at confluence for 2 days, which arrests the cells in
G0/G1 of the cell cycle (Davis et al.,
2001), as confirmed by flow cytometry
(Fig. 1E). Cells were released
by plating onto fibronectin-coated substrates. Under these conditions, cells
synchronously enter S phase at
20 hours after plating, and subsequently
enter mitosis 10 hours later at
30 hours
(Fig. 1F). Synchronized cells
were plated at densities ranging from 300 to 30,000 cells/cm2
(Fig. 1G,H), in the presence of
either the function-blocking anti-VE-cadherin antibody or the non-reactive
control. After 24 hours, cells were fixed and analyzed for cell spreading and
cell proliferation. Increasing the density of cells simultaneously decreased
cell spreading and entry into S phase. Blocking VE-cadherin increased cell
spreading and proliferation rate relative to control at the highest plating
densities. Thus, the engagement of VE-cadherin decreases endothelial cell
spreading and proliferation. Because increased cell spreading itself is known
to have a positive effect on the proliferation of endothelial cells
(Chen et al., 1997
), these
findings raised the possibility that VE-cadherin may inhibit cell
proliferation by causing a change in cell spreading.
Cell-cell contact increases proliferation when spreading is
controlled
To investigate whether contact inhibition of proliferation requires the
decrease in cell spreading, we used a method to prevent changes in cell
spreading in cells cultured with or without cell-cell contact
(Fig. 2A). Cells were plated on
substrates that contained bowtie-shaped, micrometer-sized wells with bases
made of fibronectin-coated glass and 20 µm-high walls made of agarose.
Cells plated on these substrates attached only in the fibronectin-coated wells
and not on the agarose. When two cells attached and spread in the well, each
was constrained to spread triangularly to cover half of the area of the well
(Fig. 2B). The cells contacted
each other at the central constriction to form adherens junctions containing
VE-cadherin and ß-catenin identical to those of unpatterned cells
(Fig. 2C). When a single cell
attached in a well, it spread to fill half of the well, leaving the other half
empty. Thus, pairs of cells only differed from single cells by the presence of
a cell-cell contact; cell spreading was identical.
|
Using the patterned substrates, we first examined whether cell-cell contact
would decrease proliferation in the absence of changes in cell spreading.
Endothelial cells were synchronized at confluence, plated on two sizes of
patterned substrates (750 µm2 or 1000 µm2 per
half) such that single cells or pairs of cells populated the wells, and
proliferation assessed by measuring the incorporation of BrdU. Consistent with
previous studies (Chen et al.,
1997), proliferation increased with cell spreading (1000
µm2 as compared to 750 µm2) in both single cells
and pairs of cells (Fig. 2D).
Interestingly, cells grown in pairs proliferated more than single cells of
equal cell spreading. At both degrees of cell spreading, cell-cell contact
increased proliferation by the same absolute magnitude, implying that the
proliferative signals from spreading and intercellular contact are distinct
and additive.
Engagement of VE-cadherin is required for cell-cell-induced
proliferation
To determine whether VE-cadherin engagement was necessary for the
contact-mediated stimulation of growth, we examined the proliferation of cells
in the bowties with and without VE-cadherin function-blocking antibodies.
Treatment with two different VE-cadherin function-blocking antibodies
inhibited the contact-mediated increase in proliferation
(Fig. 2E). At the concentration
used to inhibit cell-cell-induced proliferation, treatment with
function-blocking anti-VE-cadherin diminished the localization of VE-cadherin
and ß-catenin, but did not induce gap formation or retraction between
pairs of cells or within monolayers (Fig.
2F,G), and did not affect the proliferation of cells without
contacts. However, cadherin-blocking studies do not exclude the role of other
junctional proteins, because inhibiting cadherin engagement has been shown to
inhibit the formation of other types of contacts
(Gottardi et al., 2001),
including gap junctions, tight junctions and PECAM-1-containing contacts.
To determine whether VE-cadherin alone was responsible for the contact-mediated increase in proliferation, we co-cultured endothelial cells with A431 carcinoma cell lines that were either cadherin-null (null) or expressed recombinant human VE-cadherin (VE+). In monolayers, endothelial cells formed VE-cadherin-containing contacts with VE+ cells (Fig. 3A) but not with null cells (Fig. 3B). Neither null nor VE+ cells formed gap junctions, tight junctions or PECAM-1 contacts with the endothelial cells as determined by immunofluorescence staining (Fig. 3A,B) and functional gap junction analysis (data not shown). Thus, the cell lines were used to present VE-cadherin to the synchronized endothelial cells. When endothelial cells were co-cultured with null cells in heterotypic pairs on the bowtie-shaped patterns, the proliferation rate of the endothelial cells was similar to that of single endothelial cells; co-culturing endothelial cells with VE+ cells increased proliferation of the endothelial cells to that of pairs of endothelial cells (Fig. 3C). Thus, VE-cadherin alone reproduced the stimulatory signal for proliferation.
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VE-cadherin inhibits proliferation by decreasing cell spreading
Although these findings suggest that engagement of VE-cadherin can either
inhibit (Fig. 1) or stimulate
(Figs 2,
3) proliferation under
different experimental conditions, it remained unclear what caused the switch
in proliferative response. Because the inhibition of proliferation by
VE-cadherin occurred in an experimental system that allowed cells to spread
and form cell-cell contacts freely, whereas stimulation occurred when both
cell spreading and cell-cell contact were constrained, we examined how
constraining cell-cell contact but allowing cells to spread freely would
affect the proliferative response. We controlled cell-cell contact but
increased the ability of cells to spread by culturing endothelial cells in
bowtie-shaped patterns with constant constriction size but increasing area.
Cells plated on these substrates continued to form contacts, but as the sizes
of the wells progressively increased, progressively fewer cells spread to fill
the wells (Fig. 4A). The
percentage of cells that fully spread to fill the wells was significantly
lower in cells cultured in pairs than in single cells; this difference became
more pronounced with the larger bowties. Cell-cell contact stimulated
proliferation on the smaller bowties, but switched to an inhibitory effect on
the largest bowties (Fig. 4B).
Interestingly, when we divided the proliferation data for each condition into
fully spread and partially spread sub-groupings, we revealed that the
cadherin-mediated stimulus for proliferation was still present
(Fig. 4C,D). The inhibition of
proliferation observed for cells in pairs in the largest bowties was therefore
a direct consequence of the high percentage of these cells that failed to
spread fully. Co-culturing endothelial cells with null or VE+ cells
on the larger wells confirmed that VE-cadherin was responsible for the
decrease in cell spreading (Fig.
4A). Taken together, these data suggest that cell-cell contact
emits two opposing signals that regulate growth: one inhibits proliferation by
decreasing cell spreading; the other promotes proliferation via a
spreading-independent pathway. Both signals - the inhibition and stimulation
of proliferation by intercellular contact - are mediated by VE-cadherin and
are operating simultaneously.
|
Cell-cell-induced proliferation is blocked by inhibiting MEK or
PKC
To explore further the contact-mediated stimulation of proliferation, we
examined possible cellular pathways that might be involved. To determine
whether specific signal transduction pathways associated with cadherin
engagement were involved, we pharmacologically inhibited MEK and PKC.
Pharmacological inhibitors were added to cells 2 hours after they were plated
on bowtie-shaped patterns. The cell-cell contact-induced increase in
proliferation was selectively inhibited over single cell controls by the MEK
inhibitor U0126 at concentrations greater than 60 nM
(Fig. 5A). Similarly,
inhibiting PKC with Ro-31-7549 (Fig.
5B) or H-7 (Fig.
5C) abrogated the increase in proliferation of cells grown in
pairs. At higher concentrations, the drugs also affected cell spreading and
equally inhibited proliferation in single cells and pairs of cells, indicating
possible toxicity. Interestingly, treating cells with drugs at the
concentrations that specifically inhibited cell-cell-induced proliferation
also altered the actin cytoskeleton. Untreated cells cultured in pairs
exhibited a characteristic, continuous band of actin fibers across the
cell-cell contact, whereas treatment with U0126, Ro-31-7549 or H-7
dramatically altered or inhibited the formation of these fibers, without
disrupting adherens junctions (Fig.
5D-G). Because adherens junctions are structurally analogous to
focal adhesions, which regulate proliferation through both growth factor- and
cytoskeleton-mediated pathways (Giancotti,
1997), and because both MAPK and PKC signaling dually affect
proliferation and the cytoskeleton, our findings raised the possibility that
the contact-mediated stimulatory signal for growth is mediated directly
through the actin cytoskeleton.
|
Cell-cell-induced proliferation requires actin cytoskeletal structure
and tension
To examine directly whether the structural changes in actin we observed
after drug treatment were involved in the specific inhibition of
cell-cell-induced proliferation, we disrupted actin filament structure. Cells
grown on the patterned substrates were treated with varying concentrations of
cytochalasin D (Fig. 6A,B) or
latrunculin B (Fig. 6C,D). At
0.01 µg/mL - which disrupted stress fibers but not cortical actin -
cytochalasin D inhibited the contact-mediated increase in proliferation of
pairs over single cells, which were unaffected. At 0.1 µg/mL - which
disrupted both stress fibers and cortical actin - cytochalasin D dramatically
reduced the proliferation of single cells. Higher concentrations of the drug
interfered with cell spreading. Similar results were obtained with latrunculin
B. These findings suggested that stress fibers alone were necessary and
specific for the cell-cell-induced proliferative signal. To test directly
whether it is the presence of the actin cytoskeleton or specifically the
generation of cytoskeletal tension that is required for the increase in
proliferation, we inhibited actinmyosin cycling with BDM
(Fig. 6E,F). At low
concentrations, BDM specifically reduced the cell-cell contact-mediated
increase in proliferation without altering the growth of single cells. This
result was confirmed with another myosin light chain kinase inhibitor, ML-7
(Fig. 6G,H). Staining for
ß-catenin and VE-cadherin in drug-treated cells verified that disrupting
cytoskeletal structure and mechanics did not disrupt the formation of adherens
junctions. These data suggest that cadherin-dependent proliferation depends on
the integrity of the cytoskeleton and the generation of tension.
|
One of the major regulators affecting actomyosin tension generation is the
Rho-ROCK pathway (Etienne-Manneville and
Hall, 2002). To determine whether signaling through the Rho-ROCK
pathway was required for the VE-cadherin-induced proliferation, we
pharmacologically inhibited ROCK with Y-27632
(Fig. 7A,B). Treatment with
Y-27632 was sufficient to selectively inhibit cell-cell-induced proliferation.
To confirm that Rho was required for the proliferative effect, we infected
cells with an adenovirus expressing dominant-negative RhoA (Ad-RhoN19)
bicistronic with GFP (Fig.
7C,D). Adenovirus expressing GFP alone was used as a control
(Ad-GFP). Infection with Ad-RhoN19 specifically inhibited the
cell-cell-induced proliferation in pairs of cells without altering the growth
of single cells. Infection with Ad-GFP had no effect on proliferation or cell
morphology. Inhibiting ROCK and Rho disrupted the actin cytoskeleton without
disrupting the formation of adherens junctions, as verified by staining for
actin and ß-catenin. Collectively, these data suggest that the Rho-ROCK
pathway is involved in the cytoskeletally dependent VE-cadherin-stimulated
proliferation of endothelial cells.
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Discussion |
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Culturing pairs of cells on bowtie-shaped patterns revealed a distinct stimulatory signal for proliferation, also initiated by VE-cadherin. The patterned substrates that stimulated growth (Figs 2, 3) differed from the high-density cultures that inhibited growth (Fig. 1) in two important ways - first, the patterned cells could not change cell spreading upon formation of cell-cell adhesion, and second, they had less cell-cell contact. Holding the degree of cell-cell contact constant while freeing the cells to change cell spreading, we found that 'small' contacts are sufficient to suppress proliferation (Fig. 4). Thus, the same amount of VE-cadherin engagement is sufficient for both the increases and decreases in proliferation. Although increasing the sizes or numbers of contacts may enhance either or both of these opposing signals, and may reveal additional layers of proliferative regulation, our data currently support a model in which VE-cadherin inhibits proliferation by decreasing cell spreading, and stimulates proliferation via a spreading-independent signal (Fig. 8).
Even when cell-cell contact inhibited the overall proliferation rate of bulk populations, examining subpopulations of cells of equal spreading demonstrated the existence of the stimulatory cue. Our data thus suggest the ubiquitous presence of both inhibitory and stimulatory signals by VE-cadherin engagement. The inhibitory signal can be attenuated by restricting how cells spread, whereas the stimulatory signal cannot. Which of these two signals then dominates the response of the population depends on how well the surrounding microenvironment supports cell spreading.
These findings may explain why cell-cell contact appears to inhibit
proliferation in vitro (when cells are freely spread and can therefore retract
with cell-cell contact), but is capable of stimulating proliferation in vivo
(when cells are physically restricted from changing cell shape). For example,
it has been demonstrated in vivo that endothelial cells proliferate while in
contact with their neighbors, such as during large vessel morphogenesis or
capillary angiogenesis (Carmeliet and Jain,
2000; Hanahan and Folkman,
1996
), and that these processes can be blocked by knocking out or
pharmacologically inhibiting VE-cadherin
(Carmeliet et al., 1999
;
Liao et al., 2000
).
The newly identified VE-cadherin-dependent increase in proliferation
appears to depend not only on signals previously known to be stimulated by
cadherin engagement, including the MAPK and PKC pathways
(Lewis et al., 1994;
Pece and Gutkind, 2000
), but
also specifically on the cytoskeleton. The dual dependence on signaling and
the cytoskeleton for VE-cadherin-dependent proliferation is analogous to the
regulation of G1 progression by integrins and growth factors
(Huang et al., 1998
),
suggesting a functional comparison between adherens junctions and focal
adhesions. The linkages between extracellular environment, cytoskeleton and
signaling machinery at these types of adhesions
(Gumbiner, 1996
;
Yamada and Geiger, 1997
) now
appears to be a general mechanism for the integration of mechanical and
chemical signaling. However, proliferative signals arising from cell-cell
contacts are distinct, at least initially, from those from cell-ECM adhesions.
The selective inhibition of cell-cell contact-induced proliferation over
single cells with various inhibitors highlights these differences. Even the
dependence of proliferative signaling on the actomyosin system appears to be
selective: contact-mediated proliferation appears to require stress fibers,
whereas integrin-mediated signals only require cortical actin
(Zhu and Assoian, 1995
).
Although it is not yet clear how cytoskeletal tension modulates cadherin
signaling, these findings suggest the possibility that the adherens junction
may indeed act as a distinct mechanosensor.
The decreases in cell spreading and simultaneous tension-dependent
mitogenic signals generated from cadherin engagement each support the
involvement of the Rho family of small GTPases. Inhibiting Rho and ROCK in our
system both disrupted the actin cytoskeleton and blocked the
VE-cadherin-mediated stimulation of proliferation. Members of the Rho family,
including RhoA, Rac1 and Cdc42, are well-established regulatory molecules
responsible for mediating specific changes in the actin cytoskeleton important
to both cell spreading and cytoskeletal tension
(Nobes and Hall, 1995). In
addition to their direct effects on cytoskeletal structure, the GTPases also
regulate mitogenic pathways. For example, Rho is required for sustained ERK
activity and mid-G1 phase production of cyclin D1 for adhesion-dependent
cell-cycle progression (Welsh et al.,
2001
). Our data are consistent with recent evidence from other
groups suggesting that cadherins actively alter the actin cytoskeleton and
focal adhesion components, possibly through Rho GTPases
(Kovacs et al., 2002
;
Lampugnani et al., 2002
).
Thus, such crosstalk between cadherins and integrins may be responsible for
the simultaneous cadherin-mediated changes in cell spreading and
proliferation.
Previous linkages between the integrin and growth factor pathways have suggested a complex inter-relationship between chemical signaling, structural organization and mechanical cues. Our findings would suggest that cadherins and cell-cell contacts are also intricately linked to this web of mechanochemical signaling and highlight the presence of structural cues that coordinate neighboring cells in a multicellular environment. The fact that the same intercellular adhesion molecule, VE-cadherin, exerts both positive and negative growth signals simultaneously through cytoskeleton-dependent pathways emphasizes the organizational complexity of both the signaling and structural networks of the cell. The inextricable relationships between structure and function in the whole cell may point to a general paradigm for how cells are able to coordinate the many distinct cues from their environment, including juxtacrine, growth factor, extracellular matrix and mechanical signals, into a single network to produce functional responses such as proliferation. Such integration of mechanochemical signals may be fundamental to the development and maintenance of the spatial organization of multicellular life.
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Acknowledgments |
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