 |
INTRODUCTION |
Circulating leukocytes respond to infection or injury by
dramatically altering cell shape and adhesive properties to facilitate migration from the bloodstream to the affected tissue (1, 2). Leukocytes follow biochemical cues to guide the timing and location of
activation (3, 4). These cues include soluble inflammatory cytokines
and chemokines as well as stationary adhesion molecules. Both types of
signals initiate immune system responses from resting leukocytes and
provide directional information for appropriate migration.
The process of cell migration can be described as the result of
coordination between membrane protrusive activity at a leading edge,
movement of the cell body, and retraction of the rear of the cell (5,
6). New membrane extensions at the leading edge are stabilized by
adhesive interactions between integrins and stationary adhesion
molecules located on another cell or deposited as an extracellular
matrix. Conversely, the retraction of membrane at the rear of the cell
requires the disruption of the adhesive interaction either by
disengagement between the ligand and receptor or dissociation of the
links between the integrin and the internal cytoskeleton (7).
The actin cytoskeleton plays an important role in regulating both cell
shape and adhesive properties. Actin polymerization at the leading edge
can physically drive membrane protrusion (8). Interactions between the
actin cytoskeleton and integrins regulate integrin activity (9, 10).
The links between the actin cytoskeleton and integrins can either
promote or restrain integrin adhesiveness depending on the cell type
and environmental context. Early investigations of integrin-actin
linkages in stationary fibroblasts demonstrated that
actomyosin-dependent integrin clustering was required for strong integrin adhesions (11). More recently, a study of highly motile
leukocytes and lymphocytes have revealed that when these cells are
circulating, interactions between integrins and cortical actin can
restrain integrin activity until the appropriate signals are received
(12-15). Upon activation, the restraining integrin-actin linkage is
broken, resulting in increased integrin mobility to allow clustering
and the formation of new integrin-actin interactions that promote
adhesion and signaling (16, 17).
The Rho family of small GTPases regulates many facets of cytoskeletal
dynamics that underlie changes in cell shape and adhesion during
migration (18-24). These signaling proteins act as molecular switches
that are responsive to a range of extracellular signals that influence
cell migration including those transduced by tyrosine kinase receptors,
G-protein coupled receptors, and integrin adhesion molecule receptors
(25-27). The targets of Rho GTPase signaling pathways modulate
actomyosin contractility through regulation of myosin
phosphorylation and actin dynamics by either promoting polymerization
or by stimulating depolymerization and severing of existing
F-actin filaments (28-31).
Previously, we have investigated the role of RhoA in regulating cell
shape and migration properties. In fibroblasts, we have shown that
down-regulation of RhoA by p190RhoGAP is necessary for cell spreading
and directed migration into a wound (32). In leukocytes, we have shown
that RhoA signaling to a serine/threonine kinase,
ROCK,1 regulates the
retraction of the tail of migrating monocytes and negatively influences
integrin adhesion at the cell rear (33, 34). This role for RhoA and
ROCK in negative regulation of integrin adhesion was initially
unexpected based on previous findings that RhoA promotes the formation
of large integrin-based focal adhesions in fibroblasts. However, these
large focal adhesions are a characteristic of stationary cells and are
absent from leukocytes. Instead, leukocytes form small focal complexes
of tethering and signaling molecules surrounding ligand-engaged
integrins. These focal complexes coordinate integrin signaling during
migration and are present both in leukocytes and the leading edge of
migrating fibroblasts. In both cases, membrane protrusions and their
accompanying focal complexes have been shown to be dependent on Rac or
Cdc42 and negatively regulated by RhoA and ROCK activity (33-38).
In our current study, we have further investigated the RhoA/ROCK
signaling pathway in regulating integrin activity. We find that ROCK
negatively regulates two integrin-mediated functions: phosphytyrosine
signaling and membrane protrusion. We present evidence suggesting that
ROCK regulates integrin adhesion and membrane activity by suppressing
cytoskeletal remodeling. Finally, we show that a biological consequence
of RhoA/ROCK signaling is to promote migration by limiting membrane
protrusions to the leading edge.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
THP-1 monocytes were routinely cultured
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and antibiotics. Transfections were carried out with 15 µl of LipofectAMINE 2000 (Invitrogen) and 1 µg of DNA in 100 µl of Dulbecco's modified Eagle's medium in 24-well plates
containing 3 × 105 cells for 24 h. Primary
monocytes were isolated and electroporated as described previously
(24).
Plasmids, Proteins, Pharmacological Reagents, and
Antibodies--
Myc-wild type cofilin, and Myc-S3A cofilin were
generous gifts from Drs. Audrey Minden and Ora Bernard. The cofilin
sequences were amplified by PCR and subcloned into a PET 28 vector for the generation of His-tagged fusion proteins. Fusion
proteins were isolated using a His-Bind kit (Novagen) according to
manufacturer's instructions. N17 RAP1a and E63 RAP1a were a
kind gift from Dr. Lawrence Quillam and were subsequently subcloned
into an enhanced green fluorescent protein N1 vector by Dr. William
Arthur. GST and GST-C3 fusion proteins were prepared as described
previously (34). Cytochalasin D, latrunculin A, jasplakinolide, and
Y-27632 were purchased from Calbiochem. Concentrations of cytochalasin D and latrunculin A are indicated in the text. Jasplakinolide was used
either at 1 or 10 µM, each concentration giving
equivalent results. Y-27632 was used at 10 µM.
Recombinant human ICAM-1, VCAM, and MCP-1 were purchased from R&D
Systems. Human plasma fibronectin was purchased from Invitrogen.
Pyk-2, paxillin, and PY-20 antibodies from Transduction
Laboratories were used according to manufacturer's instructions.
Cofilin antibody was purchased from Cytoskeleton Inc., and the
phosphospecific cofilin antibody was a generous gift from Dr. Jim Bamburg.
Cell Adhesion, Morphology, and Migration Assays--
Adhesion to
10 µg/ml ICAM-1 or VCAM was performed as described previously (34).
Cell morphology was assessed by F-actin staining with Alexa
594-phalloidin (Molecular Probes) or by phase-contrast microscopy using
a Zeiss Axiophot microscope with either a ×100 or ×63 objective.
Images were obtained with a cooled CCD camera and processed in
Metamorph Imaging software (Universal Imaging) and Adobe PhotoShop.
Scion Image software (NIH) was used to quantitate cell area. For cells
plated on fibronectin, ICAM+VCAM or poly-L-lysine, a
minimum of 10 cells from three separate experiments was analyzed. Cells
transfected with Myc-S3A cofilin were detected by immunostaining, and
those determined to be expressing low levels of S3A cofilin were
selected for the measurement of cell area. Data were collected from
three separate transfection experiments. Time-lapse videos were
recorded with Scion Image software. Images were taken every 20 s
for 15 min to record cell behavior at a magnification of ×40. Still
images were further processed in Adobe PhotoShop. Transwell assays were
performed by coating the 8.0-µm pore membrane with a solution of 10 µg/ml ICAM-1 and 3 µg/ml VCAM (using concentrations previously
shown to promote monocyte migration) (39), placing 105
monocytes in the upper chamber and 10 ng/ml MCP-1 in the lower chamber
to induce migration. Primary monocytes were allowed to migrate 1 h, whereas THP-1 cell migration was assessed after 3 h. Cells
adherent to the top of the Transwell filter were removed with a cotton
swab, and the number of cells attached to the underside of the filter
was counted by microscopy.
 |
RESULTS |
ROCK Limits Membrane Protrusions--
Previous work has shown that
ROCK negatively influences integrin-mediated adhesion in primary
monocytes, eosinophils, and neutrophils (33, 34, 38). To better
understand the molecular mechanism underlying this observation, we used
the THP-1 monocytic cell line to study the function of ROCK on integrin
adhesion, membrane activity, and leukocyte migration. THP-1 cells grow
in suspension, but placing them in wells coated with the integrin substrates, ICAM-1 or VCAM, induces adhesion that is up-regulated >2-fold in the presence of the ROCK inhibitor Y-27632 (Fig.
1). This indicates that integrin adhesion
of the THP-1 cell line is regulated in the same manner as primary
cells.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
Inhibition of ROCK promotes integrin
adhesion. THP-1 cells were allowed to adhere to wells coated with
ICAM-1 or VCAM for 30 min in the presence or absence of Y-27632. The
number of adherent cells was determined by crystal violet staining as
described previously. Data shown represent results from triplicate
wells from three separate experiments normalized to the maximal binding
achieved with the addition of 100 ng/ml phorbol 12-myristate
13-acetate.
|
|
Because Y-27632-induced adhesion was correlated with a dramatic
increase in cell spreading in primary monocytes, we examined the
morphology of THP-1 cells treated with Y-27632. For these experiments,
we used F-actin staining to observe the shape of THP-1 cells adherent
to different integrin substrates. Fibronectin represents an
extracellular matrix material present in wounds that engages several
1 integrins, whereas ICAM-1 and VCAM are cell adhesion
molecules expressed by activated endothelial cells to recruit
leukocytes through
L/M
2 and
4
1, respectively. THP-1 cells adherent to
fibronectin or ICAM + VCAM displayed a rounded appearance, although the
cells plated on ICAM + VCAM had more fine filopodial extensions and
more distinct punctate structures characteristic of podosomes (Fig.
2A). To investigate the role of ROCK on monocyte morphology, we allowed the cells to engage the
substrates in the presence of Y-27632. The area occupied by THP-1 cells
plated under various conditions was measured, and the average results
are depicted graphically in Fig. 2B. The inhibition of ROCK
augmented cell spreading in response to integrin engagement, particularly when plated on fibronectin. In addition to a simple increase in cell spreading, the F-actin staining in Fig. 2A
shows that inhibition of ROCK promoted excessive membrane activity as indicated by F-actin content and numerous lamellipodia around the
perimeter of the cell. The up-regulated protrusions were dependent on
the integrin substrates, because cells adherent to a nonspecific charged surface (poly-L-lysine) were round and flat with no
active lamellipodia or membrane ruffling as indicated by the smooth
appearance and reduced levels of F-actin content.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 2.
Inhibition of ROCK leads to increased
integrin-induced membrane protrusions. A, THP-1
cells were plated on coverslips coated with the indicated integrin
substrates or poly-L-lysine for 40 min prior to fixation,
permeabilization, and staining for F-actin. Images shown are
representative of the cell morphology observed in a minimum of three
separate experiments. B, quantitation of cell spreading
using Scion Image software is shown as average cell area from at least
10 cells in three separate experiments. Application of the Student's
t test yields p = 0.1 and p = 0.3 for cells spread on fibronectin (FN) and
ICAM+VCAM (I/V),
respectively. Cells plated on PLL did not occupy significantly
different areas.
|
|
These findings prompted us to determine whether integrin signaling was
similarly misregulated by the inhibition of ROCK with Y-27632. Because
tyrosine kinases are potent mediators of integrin signaling,
we examined the pattern of tyrosine kinase signaling in THP-1 cells
adherent to integrin substrates. Fig.
3A shows localization of
phosphotyrosine in punctate structures concentrated toward the leading
edge of cells bound to fibronectin or ICAM + VCAM. THP-1 cells adherent
to integrin substrates in the presence of Y-27632 displayed multiple
membrane extensions on the cell, each with a concentration of
phosphotryosine signal. The phosphotyrosine content of cells bound to
poly-L-lysine was significantly reduced, indicating that
the integrin substrates initiated the phosphotyrosine signaling. Thus,
the increased membrane protrusions were accompanied by phosphotyrosine
signaling triggered by integrin substrates.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 3.
Inhibition of ROCK leads to increased
adhesion-induced phosphotyrosine signaling. A, THP-1
cells were plated on coverslips coated with the indicated integrin
substrates or poly-L-lysine for 40 min prior to fixation,
permeabilization, and staining for phosphotyrosine. Images shown are
representative of the cell morphology observed in a minimum of three
separate experiments. B, THP-1 cells were kept in a
suspension or plated onto fibronectin-coated dishes for 20 min in the
presence or absence of Y-27632. Total phosphotyrosine content was
assessed by Western blot. C, THP-1 cells were plated as in
B, and then Pyk-2 or paxillin were immunoprecipitated and
the tyrosine phosphorylated status was measured by Western blot.
|
|
To further explore the effect of inhibiting ROCK on tyrosine
phosphorylation signaling, we analyzed cell lysates for the pattern of
tyrosine phosphorylation by Western blot. Fig. 3B shows that when THP-1 cells adhere to fibronectin, there is an increase in tyrosine-phosphorylated proteins in the region of ~100 and ~64-70 kDa. This adhesion-induced phosphotyrosine signaling is further enhanced when the cells are allowed to adhere in the presence of
Y-2732. We next sought to determine the identity of specific proteins
that may display increased tyrosine phosphorylation when ROCK is
inhibited with Y-27632. Based on the molecular mass of the
observed increase in phosphotyrosine-containing proteins, we evaluated
the phosphorylation status of the tyrosine kinase Pyk-2 (CADTK,
Cak
, and RAFT-TK) and paxillin, which have molecular masses of 110 and 68 kDa, respectively. Again, THP-1 cells were allowed to adhere to
fibronectin for 20 min followed by immunoprecipitation of Pyk-2
or paxillin and subjected to analysis of phosphotyrosine content by
Western blot. In suspension, Y-27632 decreased Pyk-2 tyrosine phosphorylation, suggesting that integrin-independent signaling to Pyk-2 requires ROCK signaling. We found that the highest level of tyrosine phosphorylation for both Pyk-2 and paxillin occurred in cells adherent to fibronectin in the presence of Y-27632 (Fig. 3C).
Actin Remodeling Is Required for Adhesion and Membrane
Protrusions--
Having found that inhibition of ROCK with Y-27632
promoted increased integrin adhesion that is accompanied by increases
in membrane protrusions and phosphotyrosine signaling, we then
investigated the molecular mechanism responsible for increased integrin
adhesion. Regulation of integrin avidity is an important mechanism
controlling of integrin adhesion in leukocytes (40). It is thought that the round circulating cells maintain integrins inactive by tethering them to the band of cortical actin until appropriate signals are received. Upon activation, integrins are freed from their cytoskeletal constraints and allowed to cluster, forming qualitatively different connections with the actin cytoskeleton for signaling and migration. Evidence both for and against the model of cytoskeletal constraint has
been reported, so we performed experiments to test this model with our
cell system using two different drugs that destabilize the
cytoskeleton, cytochalasin D, and latrunculin A. Fig.
4A shows dose-dependent adhesion of THP-1 cells to either ICAM-1- or
VCAM-coated surfaces when the cells are plated in the presence of
cytochalasin D or latrunculin A. Our results support the cytoskeletal
constraint model because both drugs stimulate adhesion to integrin
substrates at low doses. Thus, limited cytoskeletal destabilization
promotes adhesion, whereas completely abolishing F-actin does not allow the formation of the subsequent cytoskeletal linkages thought to be
required for productive integrin clustering and signaling. This is
further supported by the observation that cells plated in the presence
of 0.1 µM cytochalasin D displayed a well spread morphology, whereas those plated in 1 µM cytochalasin D
were very small and rounded (Fig. 4B).

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 4.
Actin remodeling is required for integrin
adhesion induced by ROCK inhibition. A, THP-1 cells
were allowed to adhere to ICAM-1- or VCAM-coated surfaces for 30 min in
the presence of the indicated doses of either cytochalasin D or
latrinculin A. Adhesion was measured by crystal violet staining as
described previously. Results shown are the average of triplicate wells
in two separate experiments. B, the morphology of THP-1
cells treated with either an adhesion-inducing (0.1 µM)
or adhesion-blocking (1 µM) dose of cytochalasin is shown
by F-actin staining. C, THP-1 cells were adherent to
fibronectin-coated coverslips in the presence of Y-27632 ± jasplakinolide for 30 min. Because jasplakinolide competes with
phalloidin for binding to F-actin, phase-contrast images are
shown.
|
|
We then asked whether the role of Y-27632 in promoting integrin
functions could be attributed to a destabilization of the cortical
actin network. To address this question, we utilized another
pharmacological reagent, jasplakinolide, which stabilizes F-actin
structures, thereby preventing actin remodeling. Fig. 4C
shows that jasplakinolide counteracts the effects of Y-27632 on THP-1
cell morphology as the cells treated with both jasplakinolide and
Y-27632 are more rounded than those treated with only Y-27632. In
addition, jasplakinolide and Y-27632 have opposite and ameliorating effects on THP-1 cells adhesion. Jasplakinolide abolishes adhesion, Y-27632 promotes adhesion, and cells plated in the presence of both
drugs show an intermediate level of adhesion (data not shown). These
results are consistent with a model in which ROCK negatively regulates
integrin function by maintaining the cortical actin structure that
constrains integrins.
ROCK Signaling Pathways That Regulate Integrin Function--
Our
next objective was to determine signaling pathways downstream from ROCK
that influenced integrin function in a manner dependent upon
cytoskeletal remodeling. The small GTPase RAP1 plays an important role
in up-regulating integrin adhesion, especially in leukocytes (41-44).
Cross-talk between members of the small GTPase superfamily is a common
regulatory mechanism, so we asked whether ROCK might exert its effect
through RAP1 by examining the effect of RAP1 mutants on THP-1
cell membrane protrusions. If Y-27632 is activating RAP-1 to stimulate
integrin function, transfection with constitutively active RAP1 should
mimic the Y-27632 phenotype, whereas a dominant negative RAP1 should
block the Y-27632 effect on morphology. Fig.
5 shows that these RAP1 mutants have
little effect on the cell morphology with or without Y-27632. In
addition, we found that Y-27632 did not increase the levels of GTP-RAP1
when cells were plated on fibronectin (data not shown). These results
indicate that RAP1 is not a component of ROCK signaling that
modulates integrin function.

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 5.
RAP1 does not act downstream
of ROCK inhibition to promote membrane protrusions. THP-1 cells
were transfected with either GFP-N17 Rap1 or GFP-E63 RAP1 and plated
±Y-27632 onto fibronectin-coated coverslips. Cell morphology was
assessed by F-actin staining, whereas transfected cells were visualized
by GFP expression.
|
|
Another potentially interesting candidate downstream from ROCK is the
LIM kinase to cofilin signaling pathway. ROCK phosphorylates and
activates LIM kinase, which phosphorylates and inactivates cofilin.
Cofilin both depolymerizes and severs F-actin filaments, thereby
facilitating actin remodeling (45). In this model, ROCK activity would
lead to phosphorylated inactive cofilin that would tend to stabilize
the existing F-actin networks, whereas Y-27632 would lead to
de-phosphorylated active cofilin that would promote actin remodeling.
However, LIM kinase and cofilin are regulated by additional pathways
besides ROCK. To determine the contribution of ROCK signaling in
cofilin regulation in our cell system, we first examined the effect of
Y-27632 on cofilin phosphorylation status. We used an antibody that
specifically recognizes cofilin that is phosphorylated on serine 3 to
show that in THP-1 cells plated on fibronectin, there is a significant
level of phosphorylated inactive cofilin. When cells are allowed to
adhere to fibronectin in the presence of Y-27632, the levels of cofilin
phosphorylation are nearly abolished. This indicates that ROCK plays an
important role in regulating the phosphorylation and thus activation,
the status of cofilin in response to THP-1 cell adhesion.
We next used an S3A mutant of cofilin that cannot be phosphorylated and
inactivated to examine the importance of cofilin activity in cell shape
and membrane protrusions. In THP-1 cells transfected with S3A cofilin,
we found that low level expression of activated cofilin led to a
~40% increase in cell spreading (Fig.
6B). In cells expressing high
levels of activated cofilin, F-actin structures are completely
abolished, leading to cells with a small rounded morphology (data not
shown). This finding is consistent with our previous data using
cytochalasin D and latrunculin A that showed strong
dose-dependent differences on integrin function. We also examined the effect of activated cofilin on the morphology of primary
monocytes. In these experiments, cells were loaded with either wild
type or S3A cofilin proteins by electroporation as described previously
(34). Previously, we have demonstrated that inhibiting ROCK in primary
monocytes increased membrane protrusive activity, and because the cells
are highly migratory, ROCK inhibition results in cells with
unretracted tails (34). Fig. 6C shows that overexpression of
wild type cofilin leads to a small increase in the percentage of cells
with unretracted tails (22% in controls, 34% with wild type cofilin),
whereas loading the cells with active cofilin results in a greater
percentage of cells with unretracted tails (65%). The effect of
loading primary monocytes with active cofilin is very similar to the
phenotype observed with Y-27632 treatment (80% unretracted tails),
albeit less potent. Together, these results indicate that cofilin is
inactivated downstream from ROCK, which contributes to the promotion of
membrane protrusions and inhibition of tail retraction.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 6.
Cofilin activation contributes to
Y-27632-induced lamellipodial protrusions. A, THP-1
cells were allowed to adhere to fibronectin-coated plates for 20 and 40 min in the presence or absence of Y-27632. Cell lysates were analyzed
by Western blot for the levels of serine 3-phosphorylated cofilin and
total cofilin. B, THP-1 cells were transfected with
Myc-tagged S3A cofilin or empty vector followed by analysis of cell
morphology by F-actin staining. Low expressing transfectants were
identified by Myc staining. Data shown are representative of one of
three separate experiments. C, Primary monocytes were loaded
with wild type, S3A cofilin, or GFP (control) as described
previously. Loaded cells were plated on coverslips for 40 min prior to
fixation and staining for F-actin. The number of cells with tails was
counted in 20 individual fields in 3-5 separate experiments.
Representative data are shown.
|
|
ROCK Limits Membrane Protrusions and Promotes Directed
Migration--
This study has examined the molecular mechanisms
underlying of membrane protrusions induced by inhibition of ROCK. Our
next goal was to determine the biological consequence of
RhoA/ROCK-regulated membrane protrusions. To study monocyte migration,
we used our primary monocyte system in which cells were loaded with C3
protein, a potent inhibitor of RhoA, and subsequently ROCK activity.
The loaded monocytes were plated onto coverslips and their migration was monitored by time-lapse video microscopy. Control cells loaded with
GST displayed a typical migration pattern in which the membrane at the
leading edge protrudes, the cell body moves forward, and the rear of
the cell retracts. This results in a cell with a relatively round
morphology, reflecting the balance between protrusions at the front of
the cells and retraction of membrane at the rear of the cell. To change
direction, the monocyte sends out a lamellipodial extension in an
alternate direction and the former leading edge stops protruding and
retracts (Fig. 7A). In
contrast, monocytes loaded with C3 display competing membrane lamellae,
pulling the cell in different directions at the same time (Fig.
7B). Membrane activity is not limited to a single leading
edge, resulting in an elongated cell with unregulated protrusions along
the length of the cell. Thus, RhoA activity is required to suppress
membrane protrusions at the rear of a migrating monocyte.

View larger version (123K):
[in this window]
[in a new window]
|
Fig. 7.
RhoA signaling is required to suppress
protrusions away from the leading edge. A, primary
monocytes were loaded with either GST (control) or C3 and
plated on a glass slide, and time-lapse images were taken every 20 s for 20 min. Selected frames show the cell behavior of representative
cells from a total of 35 cells in three separate experiments.
Arrows indicate the direction of migration. Note the abrupt
change in the direction of the control (GST) cell in
panels 5-7.
|
|
Monocytes in which RhoA or ROCK is inhibited have robust lamellipodial
protrusions, a behavior typically thought to promote migration. Yet
over time, the C3-loaded monocytes do not appear to be able to migrate
any significant distance, despite vigorous short term movements. To
directly test whether monocytes require ROCK signaling to undergo
productive migration, we used a Transwell migration assay. Primary
monocytes (Fig. 8) or THP-1 cells (data not shown) were placed in the upper chamber of a Transwell and allowed
to migrate toward the chemokine MCP-1 in the lower chamber. The
dividing membrane was coated with the ICAM + VCAM combination to
simulate integrin substrates normally used by monocytes for migration.
The number of cells that migrated to the lower chamber was counted by
microscopy, and the results are presented in Fig. 8. As expected, MCP-1
induced >10-fold induction of monocyte migration for control cells.
When the migration assay was performed in the presence of the ROCK
inhibitor Y-27632, migration was reduced to ~3-fold. Together, these
results demonstrate that RhoA/ROCK signaling is required to suppress
protrusions at the rear of migrating monocytes to allow productive
migration.

View larger version (7K):
[in this window]
[in a new window]
|
Fig. 8.
ROCK signaling is required for MCP-1-induced
migration. Primary monocytes were added to the upper chamber of a
Transwell and allowed to migrate for 1 h toward 10 ng/ml
MCP-1 in the lower chamber ±Y-27632 (see "Experimental
Procedures" for details). Data are plotted as fold increase in the
number of cells that migrated to the underside of the Transwell filter
in the absence of MCP-1 or Y-27632. Data show the average from
duplicate wells in two separate experiments.
|
|
 |
DISCUSSION |
In this study, we have extended results from previous work
examining the function of RhoA and ROCK signaling in leukocyte migration. We report that RhoA and ROCK signaling are required to limit
membrane protrusions to a single leading edge. When ROCK signaling was
inhibited with Y-27632, we found increased integrin adhesion
accompanied by increased membrane activity and up-regulated phosphotyrosine signaling. We described an important role for cytoskeletal remodeling in regulating integrin adhesion in experiments utilizing either actin depolymerizing or stabilizing agents. An investigation into the signaling pathways that influence cytoskeletal remodeling to regulate integrin activity revealed that both ROCK and
cofilin contribute to regulating membrane protrusions. Finally, we
showed that in the absence of RhoA/ROCK signaling, monocytes send out
competing lamellipodia that interfere with productive migration.
We observed a strong correlation among increased integrin adhesion,
increased integrin-dependent phosphotyrosine signaling, and
inappropriate lamellipodial protrusions. We propose that the misregulated integrin engagement initiates phosphotyrosine signaling, which promotes membrane protrusion. Evidence for a direct link between
integrin signaling and lamellipodia formation comes from our
identification of Pyk-2 and paxillin as two proteins whose adhesion-induced tyrosine phosphorylation levels are influenced by ROCK
activity. Both Pyk-2 and paxillin have been implicated in promoting
protrusive activity in leukocytes and fibroblasts (46-50). This is
further supported by studies from the Cabanas laboratory showing that
increased Pyk-2 signaling in neutrophils treated with C3 was downstream
from integrin clustering (51). Our data highlight a previously
unappreciated role for ROCK signaling in suppressing integrin adhesion,
signaling, and membrane protrusions away from the leading edge.
Circulating leukocytes must maintain tight regulation over the state of
integrin adhesiveness. Leukocytes inhibit integrin function in the
resting state to prevent unwarranted inflammatory responses but quickly
activate integrin adhesion in response to infection or injury. A rigid
band of cortical actin maintains the round shape of circulating
leukocytes, and it has been proposed that this cortical actin
constrains integrin mobility, rendering them unable to cluster with
other integrins (14, 40). Our data support the model for cytoskeletal
remodeling as a molecular mechanism that regulates integrin adhesion in
leukocytes. We found that low doses of two actin destabilizing agents,
cytochalasin D and latrunculin A, promote integrin adhesion, whereas
the jasplakinolide, a F-actin stabilizing agent, prevents adhesion.
Despite the pharmacological evidence for the role of actin remodeling
in integrin activation, the in vivo signaling pathways that
would regulate actin dynamics have not been well characterized. Here,
we implicate ROCK in governing actin remodeling, because inhibition of
ROCK has the same effect as low doses of actin destabilizing agents on
integrin adhesion. In addition, the combination of the ROCK inhibitor
with an actin stabilizer had counteracting effects on adhesion and
membrane protrusion. Further investigation of signaling pathways
downstream from ROCK showed that the actin remodeling protein, cofilin,
is involved in regulating membrane protrusions in THP-1 cells and tail
retraction in primary monocytes. Cofilin is known to promote actin
remodeling by virtue of its actin severing and depolymerizing activity
(45). Furthermore, cofilin activity has been implicated in promoting
lamellipodial protrusions in response to growth factors (52, 53). The
effects of expressing non-phosphorylatable, activated cofilin were not as striking as those observed with inhibition of ROCK, which indicates a role for additional signaling molecules. These results indicate that
regulation of integrin function by actin remodeling was regulated by
ROCK signaling through a pathway involving inactivation of cofilin.
To evaluate the biological consequence of RhoA and ROCK regulation of
integrin functions, we analyzed the behavior of primary monocytes in
addition to THP-1 cells. Using time-lapse video microscopy, we observed
that inhibition of RhoA signaling leads to the formation of multiple
competing lamellae. These monocytes appear to be unable to choose a
leading edge and do not appear to migrate any appreciable distance,
despite robust membrane protrusive activity. We measured the capability
of the monocytes to migrate in a Transwell assay, which demonstrated
the ROCK dependence of MCP-1-induced migration. This finding is
consistent with results reported by Ashida et al. (54) that
show that Y-27632 inhibits migration of THP-1 cells. Our results
confirm those findings and build upon them by implicating two
integrin-dependent functions, phosphotyrosine signaling and
membrane protrusions, as molecular mechanisms underlying the block in
directed migration.
Combining data from the present studies with that from many different
investigators and systems, we propose a model in which spatial
regulation of RhoA activity is required for productive migration. In
this model, RhoA must be actively inhibited at the leading edge to
allow lamellipodial protrusions. This is supported by other studies in
fibroblasts that show that the inhibition of RhoA/ROCK signaling with
C3, Y-27632, or overexpression of p190RhoGAP promotes cell spreading,
protrusion, and polarity (32, 36). Furthermore, p190RhoGAP is localized
to sites of membrane protrusions and ruffling (55, 56). Although RhoA
must be inhibited at the leading edge, our model also proposes that
RhoA must be activated at the rear of the cell to promote migration.
Myosin-based contractility has previously been implicated in the tail
retraction of migrating neutrophils (57), and the RhoA/ROCK signaling
pathway is known to regulate myosin phosphorylation. However, our
previous studies in migrating monocytes did not reveal a major role for myosin contractility in RhoA-dependent tail retraction. Our
current data suggest an additional mechanism that RhoA and ROCK are
required to suppress lamellipodia formation away from the leading edge and that failure to limit membrane activity inhibits productive migration.