| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Department of Medicine, Division of Hematology, and
Department of Surgery, University of Washington, Seattle, WA 98104
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4
1-dependent
adhesion of Jurkat cells at 30 min. Similarly, Y-27632 treatment
increased stimulated 2 integrin-dependent neutrophil
adhesion at 30 min but not at 5 min. Because reduced de-adhesion could
mimic augmentation of adhesion at later time points, we developed an
assay to measure de-adhesion specifically. Treatment of phorbol
ester—or bacterial chemoattractant peptide—but not
Mn2+-stimulated neutrophils adherent to serum-coated
plastic or endothelial cells with Y-27632 or C3 exoenzyme markedly
reduced the rate of de-adhesion, while markedly increasing their
spreading. RhoA kinase inhibitor effects on de-adhesion and spreading
were reversed by treatment with the cytoskeletal-disrupting agent
cytochalasin D. Treatment with Y-27632 influenced neither integrin
activation epitope nor integrin clustering. We conclude that activation
of RhoA kinase promotes leukocyte de-adhesion by inhibiting
cytoskeletal-dependent spreading, and that these effects of RhoA kinase
constitute a new mechanism for regulation of integrin receptor
avidity. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2 integrin-dependent stimulated neutrophil
adhesion (6). However, in other studies treatment with C3
exoenzyme did not inhibit 2 integrin-dependent
adhesion of neutrophils (7) or JY lymphocytic cells
(8) or 1 integrin-dependent
adhesion of peripheral blood T cells (9) or U937 monocytic
cells (10). Cultured human T lymphocytic cells adhering
via 1 integrin and freshly prepared human
polymorphonuclear leukocytes (neutrophils), which undergo rapid
2 integrin-dependent adhesion and de-adhesion
when stimulated, were used to study the intracellular mechanisms
regulating these events. We focused on RhoA and RhoA kinase, the
downstream kinase of RhoA, which has been shown to mediate many RhoA
functions (11, 12, 13, 14). We were able to distinguish between
effects on adhesion and de-adhesion by designing an assay to
specifically measure de-adhesion of adherent leukocytes using a rapidly
penetrating and fast-acting reagent that could be added after removal
of stimulus and nonadherent cells. |
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human T lymphocytic Jurkat cells (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 (BioWhittaker, Walkersville, MD) with 10% FBS (HyClone Laboratories, Logan, UT). HUVEC were isolated and cultured as previously described (15). They were grown in RPMI 1640 with the addition of 2 mM glutamine, sodium pyruvate, nonessential amino acids, 10 mM HEPES, 100 U/ml penicillin, 100 U/ml streptomycin, 250 ng/ml fungizone (BioWhittaker), 90 µg/ml heparin (Sigma-Aldrich, St. Louis, MO), bovine hypothalamic extract (gift of E. Raines, University of Washington, Seattle, WA), 10% bovine calf serum and 10% bovine calf serum supplemented with iron (HyClone Laboratories). For experiments, they were cultured in cell culture microwell plates (Nalge Nunc International, Naperville, IL).
Preparation of neutrophils from peripheral blood
Peripheral blood was obtained from normal donors with informed consent according to procedures approved by the Human Subjects Review Committee of the University of Washington. Blood was drawn into heparinized syringes and neutrophils were isolated using Ficoll-Hypaque Plus (Amersham Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation, followed by lysis of RBC (0.155 M NH4Cl, 0.1 M KHCO3, 1 mM EDTA, pH 7.4, at 4°C) and washing in RPMI containing 2% FBS.
Adhesion and de-adhesion assays
Jurkat cells were incubated overnight with C3 exoenzyme from Clostridium botulinum (Calbiochem, La Jolla, CA) at 37°C. Leukocytes were labeled with 5 µM calcein-AM (Molecular Probes, Eugene, OR) at room temperature for 45 min and washed. Neutrophils were treated with C3 exoenzyme for 4 h, with calcein-AM added for the last 15 min. For inhibition of RhoA kinase, calcein-labeled leukocytes were treated with indicated concentrations of RhoA kinase inhibitor Y-27632 (Ref. 16 ; a gift of Welfide Corporation, Osaka, Japan) for 10 min. Jurkat cells (5 x104/well) were stimulated with PMA (Sigma-Aldrich) or with mAb 8A2 (17, 18) to adhere to fibronectin (1 µg/ml; Life Technologies, Rockville, MD) for 30 min at 37°C. Neutrophils (1 x 105/well) were stimulated with phorbol 12,13-dibutyrate (PDBu)4 (Calbiochem), fMLP (Sigma-Aldrich), or Mn2+ (MnCl2; Sigma-Aldrich) to adhere to serum-blocked cell culture microwell plates or to confluent HUVEC at 37°C. One-tenth volume of 10% methanol-free formaldehyde (Polysciences, Warrington, PA) was added to fix the cells at times indicated. Percent adherence was calculated by measuring the fluorescence in each well of the total cells, and of the adherent cells after two washes, in a Cytofluor 4000 fluorescence plate reader (PerSeptive Biosystems, Framingham, MA). To measure de-adhesion, neutrophils were stimulated to adhere to serum-blocked plastic or to HUVEC, after which nonadherent cells were decanted and the plates were washed twice. Medium with or without 10 µM Y-27632 was then added and the plates were incubated at 37°C. Cytochalasin D (Sigma-Aldrich) was added 10 min after the Y-27632. At the indicated times, the wells were fixed, and percent neutrophils remaining adherent was determined at the end by measuring fluorescence before and after additional washes. MnCl2 quenched calcein fluorescence, even as it induced strong adhesion. Consequently, staining with crystal violet was performed at the end to confirm relative numbers of cells. After fluorescence readings on neutrophil adherence to confirm the even loading of cells, washed plates were read again and stained with 0.5% crystal violet (Sigma-Aldrich) in 20% methanol, extensively washed in water, and stain solubilized with 0.1% sodium citrate in 50% ethanol. The absorbance at 570 nm (reference 650 nm) was read on a Titertek Multiscan MCC/340 plate reader (Flow Laboratories, McLean, VA).
ADP-ribosylation assay of RhoA
Jurkat cells (2 x 106) were treated with 10 µg/ml C3 exoenzyme as indicated. After overnight incubation, the cells were washed with cold PBS, harvested in 250 µl of cold lysis buffer, 50 mM HEPES, pH 7.4, containing CØmplete Mini EDTA-free protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN) and sonicated. Aliquots (100 µg of protein) of the lysate were incubated with [32P]NAD (American Radiolabeled Chemicals, St. Louis, MO) and 10 µg of C3 exoenzyme at 37°C for 30 min before Laemmli buffer was added. The samples were heated for 3 min at 95°C and run on 12% SDS-PAGE. [32P]ADP-ribosylated proteins were detected by autoradiography with a PhosphorImager from Molecular Dynamics (Krefeld, Germany).
Confocal microscopy
Neutrophils in RPMI 1640 plus 5% heat-inactivated adult bovine serum (HyClone) were stimulated with 1 µM fMLP for 10–15 min at 37°C for cells to adhere on serum-blocked eight-well Lab-Tek chamber slides. fMLP and nonadherent cells were removed by three washes with medium. The adherent cells were either fixed immediately or incubated at 37°C for 15 min in the presence or absence of 15 µM Y-27632 and then fixed. Integrin CD11b was stained with mAb OKM1 (ATCC CRL 8026) purified from mouse ascites followed by FITC-goat anti-mouse F(ab')2, human-adsorbed second Ab (Caltag Laboratories, So. San Francisco, CA). Photomicrographs were made with a Bio-Rad Radiance 2000 confocal microscope (Hercules, CA) equipped with a krypton-argon laser and a Nikon inverted microscope (Tokyo, Japan).
Neutrophil spreading
Neutrophils in RPMI with 2% FBS were incubated with or without 10 µM Y-27632 for 30 min and then adhered to serum-blocked eight-well Lab-Tek chamber slides (Nalge Nunc International) with or without 10 µM fMLP in the presence or absence of 0.25 µg/ml cytochalasin D. Cells were incubated at room temperature for 30 min before fixing with 0.5% formaldehyde. After three washes to remove nonadherent cells, actin was stained with rhodamine-phalloidin and nuclei with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes). Photomicrographs were made on a Nikon Eclipse E800 Fluorescent microscope, using a 60 x 0.95 Nikon dry objective lens and filters to detect DAPI and rhodamine fluorescence above 590 nm. Areas were determined on a Nikon Diaphot 200 microscope using NIH Image Scion 1.6 software. At least 150 cells were measured per sample.
Flow cytometry
Neutrophils were suspended in RPMI containing 5%
heat-inactivated adult bovine serum and were incubated with or without
30 µM Y-27632 before activation at 37°C for 15 min with fMLP in the
presence of Alexa-488 mAb 327C, which recognizes a 2
integrin activation epitope (Ref. 17 ; the generous gift of
D. Staunton, ICOS Corporation, Bothell, WA). To measure the effect of
Y-27632 on disappearance of the active epitope, neutrophils that had
bound mAb 327C were washed to remove stimulant and unbound mAb and
incubated with or without Y-27632 at 37°C for 30 min. Samples were
fixed with 1% formaldehyde, data were acquired on a BD Biosciences
FACScan flow cytometer (Mountain View, CA) and were analyzed using
WinMDI (J. Trotter, Scripps Institute, La Jolla, CA). Data were
normalized by dividing mean fluorescences in each experiment by those
obtained with mAb 5D1, which recognizes the 1
integrin subunit, not expressed by neutrophils under normal
circumstances.
Significance was determined with the two-tailed Student’s t test with unequal variance, using Microsoft Excel (Redmond, WA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 integrin-dependent Jurkat cell adhesion
Overnight treatment of Jurkat cells with C3 exoenzyme
significantly reduced the amount of RhoA protein that could be labeled
with 32P in vitro, demonstrating that the C3
exoenzyme had penetrated the intact cells and ADP-ribosylated RhoA in
vivo (Fig. 1
A, lanes
1 and 2; compare to lane 4). In the absence
of C3 exoenzyme in vitro there was no RhoA ADP-ribosylation
(Fig. 1A, lane 3). However, treatment with a
concentration of C3 exoenzyme which ADP-ribosylated most of the RhoA
protein in the cells did not inhibit 1
integrin-dependent PMA-stimulated adhesion to fibronectin (Fig. 1B). This PMA-stimulated adhesion was inhibited by mAbs to
4 and 1 integrin
subunits (data not shown). Furthermore, the specific RhoA kinase
inhibitor Y-27632 did not inhibit Jurkat cell adherence to
fibronectin (Fig. 1B). In fact, inhibition of the RhoA/RhoA
kinase pathway appeared to increase PMA-stimulated Jurkat cell
adhesion, but had no effect on adhesion stimulated by mAb 8A2, which
directly increases 1 integrin affinity (Refs.
18 and 19 ; data not shown).
|
2
integrin-dependent adherent neutrophil de-adhesion
Neutrophils pretreated with C3 exoenzyme and stimulated to adhere
to serum-blocked plastic evidenced adhesion similar to control (Fig. 1
C), but less de-adhesion than control (Fig. 1D).
Similarly, in the presence of Y-27632, neutrophils adhered more and
de-adhered less than controls (Fig. 1, C and D).
Stimulation of neutrophils with the chemoattractant fMLP or the protein
kinase C activator PDBu produced a marked, time-dependent increase in
adhesion to serum-coated plastic, and this adhesion was totally
inhibited by the blocking CD18 mAb 60.3 (data not shown). Inhibition of
RhoA kinase with Y-27632 enhanced both fMLP- and PDBu-stimulated
neutrophil adhesion at 30 min (Fig. 2).
However, further investigation of the time course of adhesion showed
that Y-27632 had no effect on fMLP- or PDBu-stimulated neutrophil
adhesion at 5 min, despite a 30-min pretreatment, but significantly
enhanced adhesion at 20 min (Fig. 3, A and B). The lack of effect on early adhesion,
despite preincubation with the inhibitor, but apparent augmentation of
adhesion at later time points suggested that the inhibitor might
actually be reducing de-adhesion of the adherent cells. In support of
this, the effect of C3 exoenzyme implicated RhoA specifically in
neutrophil de-adhesion. To separate the process of de-adhesion from
that of adhesion, it was necessary first to stimulate neutrophils to
adhere using reagents that could be removed, and second to administer
to the adherent cell reagent(s) that penetrate easily and act quickly.
fMLP and PDBu met the first requirement, and the RhoA kinase inhibitor,
Y-27632, met the second. Therefore, we examined the participation of
RhoA in neutrophil de-adhesion using stimulated neutrophils and
Y-27632.
|
|
, C and D). Y-27632 had no effect on
neutrophils that had been stimulated to adhere using
Mn2+ to augment 2
integrin affinity directly (Fig. 3E). The induction and
decay of an activation epitope of 2 integrin
was investigated using mAb 327C (17). Y-27632 treatment
had no effect on either induction (Fig. 4A) or disappearance (Fig. 4B) of activation epitope. Staining for integrin with
anti-CD11b mAb did not demonstrate clear changes in integrin
clustering (Fig. 4C). Adherent neutrophils incubated with
Y-27632 after removal of stimulus and nonadherent cells exhibited a
more spread morphology than those incubated in its absence. CD11b
integrin was distributed in a much larger area in the presence of
Y-27632 (Fig. 4C).
|
A). This effect was not
caused by carryover of a trace amount of inhibitor from neutrophil to
HUVEC, because fMLP-stimulated neutrophils adhered less well to HUVEC
pretreated with Y-27632 (Fig. 5B), consistent with the
proposed role of endothelial RhoA in integrin-ligand clustering
(20). Treatment with Y-27632 did reduce the rate of
neutrophil de-adhesion from HUVEC (Fig. 5, C and
D). Y-27632 also slowed de-adhesion of MO7E from Chinese
hamster ovary cells transfected with VCAM-1, a
1 integrin-dependent process (data not
shown).
|
Previous studies had shown that inhibition of RhoA function by
treatment with C3 exoenzyme promoted monocyte spreading
(21). Consistent with this result, we found that blockade
of RhoA kinase by Y-27632 dramatically increased neutrophil spreading
on serum-coated plastic (Fig. 6; Table I). The average area of Y-27632-treated,
fMLP-stimulated cells was 1.8 times that of control neutrophils.
Because spreading requires a reorganization of the actin
cytoskeleton, we tested the effect of cytochalasin D, which
disrupts actin filament reorganization and thereby prevents cell
spreading. Cytochalasin D completely blocked the increased neutrophil
spreading induced by Y-27632 (Fig. 6; Table I), and neutrophils
co-incubated with cytochalasin D and Y-27632 de-adhered at a rate
similar to untreated cells (Fig. 7).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 and 2
integrin-dependent leukocyte adhesion and de-adhesion. We found that
blockade of RhoA did not inhibit
41 integrin-mediated
adhesion of Jurkat T lymphocytes to fibronectin. When assayed at 30
min, PMA-stimulated, but not mAb 8A2-stimulated, adhesion of Jurkat
cells treated overnight with C3 exoenzyme or treated with the specific
RhoA kinase inhibitor Y-27632 was apparently enhanced. Similarly,
neutrophils treated with Y-27632 evidenced greater
2 integrin-dependent adhesion. A careful
analysis of the time course of stimulated neutrophil adhesion showed
that the Rho kinase inhibitor had no effect on early adhesion at 5 min,
but significantly augmented adhesion at 20 and 30 min. We considered
three possible explanations for the effect on late but not early
adhesion: 1) drug penetration dynamically controls the process; 2) the
inhibitor is a slow-acting drug; 3) the effect is on the process of
de-adhesion rather than on adhesion. Regardless of whether cells were
preincubated with the inhibitor for 30 min, there was no effect on
early adhesion. Therefore, it seemed most likely that the effect of the
inhibitor was to reduce de-adhesion. The short life span of neutrophils
ex vivo limits the use of approaches such as overexpression of
constitutive active or dominant negative constructs, which after all do
not distinguish between effects on initial adhesion and de-adhesion. We
designed an assay to study de-adhesion of already adherent cells,
taking advantage of the ability of Y-27632 to penetrate cells rapidly
and act quickly (22). Using this assay system, we observed
that treatment with C3 exoenzyme or Y-27632 had more significant
effects on the fraction of cells that de-adhered than they had had on
the fraction of cells that had adhered in the same time frame (Fig. 1, C and D). This further suggested that RhoA and
RhoA kinase were involved in leukocyte de-adhesion signal pathways.
Treatment of adherent neutrophils with Y-27632 significantly increased
the fraction of cells remaining adherent (Fig. 3D).
Differences could be observed within 5 min, confirming that the
apparent increase in adhesion seen with Y-27632 was actually inhibition
of de-adhesion and was not due to slow penetration of or slow activity
by Y-27632 (Fig. 3B). The de-adhesion studies reported in
this study focused on stimulated neutrophils, which adhere to
serum-blocked plastic and to HUVEC entirely via
2 integrins (data not shown). However, we
obtained identical results with Y-27362 on 1
integrin-dependent MO7E adhesion to and de-adhesion from
VCAM-1-transfected Chinese hamster ovary cells (data not shown), which
argues that the role of Rho kinase in de-adhesion is not leukocyte type
or integrin subunit-specific.
Mn2+ stimulates neutrophil adherence via affinity
modulation (23), and de-adhesion of
Mn2+-induced adherent neutrophils was insensitive
to the Rho kinase inhibitor (Fig. 3E). Y-27632 also did not
induce spreading of Mn2+-stimulated neutrophils
(data not shown). Similarly, mAb 8A2-induced adherence of Jurkat cells
was not enhanced by Y-27632 (data not shown). These results likely
reflect differences in cytoskeletal involvement between affinity- and
avidity-dependent mechanisms of cell adhesion (19, 24).
Phorbol esters and fMLP, but not 8A2 and Mn2+,
increase integrin mobility and clustering, and induce cell adhesion
predominantly by modulation of avidity involving cytoskeletal
reorganization (37). Thus, the
effect of Y-27632 on phorbol ester- or fMLP-stimulated, but not on mAb
8A2- or Mn2+-stimulated, adherence is consistent
with the involvement of the cytoskeleton in cell spreading. In further
support of this is the failure of Y-27632 to influence either induction
of the 2 integrin activation epitope by fMLP
(Fig. 4A) or its disappearance (Fig. 4B). There were no evident changes in clustering
of integrin CD11b, but adherent cells washed and then incubated with
Y-27632 were much more spread than those incubated without it (Fig. 4C). This, in turn, increased the number of integrin-ligand
interactions, which would strengthen cell adhesion.
Aepfelbacher et al. (21) reported that blockade of RhoA by
C3 exoenzyme caused monocyte spreading, suggesting that the RhoA
signaling pathway promoted cell rounding. Similarly, we found that the
RhoA kinase inhibitor Y-27632 markedly increased neutrophil spreading
by 80% (Fig. 6; Table I). Although high doses of cytochalasin D
inhibit adhesion, low doses increase adhesion by promoting integrin
receptor diffusion and ligand-induced clustering (24, 25).
Cytochalasin D, at a dose that did not inhibit adhesion of either
control or Y-27632-treated fMLP-stimulated neutrophils (data not
shown), increased de-adhesion of Y-27632-treated neutrophils (Fig. 7).
When actin filament reorganization was prevented by cytochalasin D,
Y-27632-treated cells remained round (Fig. 6). Even though cytochalasin
D has been reported to activate RhoA in adherent cells
(26), it is unlikely RhoA activation was responsible for
the effect of cytochalasin D observed in this study because RhoA kinase
is downstream of RhoA. It is more likely that cytochalasin D
accelerated Y-27632-treated cell de-adhesion and abrogated cell
spreading by interrupting the cytoskeleton network (24).
It has been reported that Y-27632 can suppress myosin L chain
phosphorylation (27). The effect of RhoA may result from
RhoA kinase-mediated phosphorylation of myosin L chain
(28) and the myosin-binding subunit of myosin L chain, as
has been reported for neutrophils and multiple other cell types
(27, 28, 29, 30, 31, 32, 33). Phosphorylation of myosin by RhoA kinase
stimulates actin-activated ATPase activity of myosin II, whereas
phosphorylation of myosin L chain phosphatase by RhoA kinase inhibits
its activity, leading indirectly to increased myosin L chain
phosphorylation (11). Both of these activities of RhoA
kinase would thus promote actomyosin assembly and cell contraction or
rounding. The RhoA kinase-mediated rounding would decrease cell
spreading and thereby reduce interactions between neutrophil
2 integrin receptors and cellular or
extracellular ligands, promoting de-adhesion (Fig. 7). Conversely,
increased spreading and a consequent increase in
2 integrin-dependent avidity produced by
blockade of RhoA kinase would impair neutrophil migration, as has been
reported (27).
Anderson et al. (7) reported slower migration of neutrophils pretreated with C3 exoenzyme in a flow chamber model. Several recent studies have used time-lapse video microscopy to observe the effects of pretreatment with RhoA and RhoA kinase reagents on leukocyte migration. Worthylake et al. (34) reported that blockade of RhoA and RhoA kinase prevented monocyte tail retraction and transendothelial migration. Alblas et al. (35) found that RhoA and RhoA kinases were involved in eosinophil rear release in migration assays. Lymphocyte chemotaxis was abolished by pretreatment with C3 exoenzyme or Y-27632 (36). Our investigation of de-adhesion separately from adhesion extends the conclusions of these migration studies and confirms that the inability of cells pretreated with blockers of RhoA and RhoA kinase activity to migrate results specifically from impairment of de-adhesion.
In summary, blockade of RhoA kinase promoted cell spreading and
impaired de-adhesion. By preventing spreading and promoting
de-adhesion, activation of the RhoA/RhoA kinase pathway constitutes
another mechanism to regulate integrin avidity (Fig. 8).
|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 L.L. and B.R.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Li Liu, Division of Hematology, Harborview Medical Center, Box 359756, 325 9th Avenue, Seattle, WA 98104-2499. E-mail address: liliu{at}u.washington.edu
4 Abbreviations used in this paper: PDBu, phorbol 12,13-dibutyrate; DAPI, 4',6-diamidino-2-phenylindole.
Received for publication January 25, 2002. Accepted for publication June 26, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
51 integrin receptor. FEBS Lett. 363:78.[Medline]
1 integrin (CD29) stimulates VLA-dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components. J. Cell Biol. 116:499.51: regulation of the functional response by soluble fibronectin. J. Cell Biol. 121:155.1-integrin-dependent leukocyte adhesion. J. Biol.
Chem. In press.This article has been cited by other articles:
|
|
 |
|
  P. Y. Mong and Q. Wang Activation of Rho Kinase Isoforms in Lung Endothelial Cells during Inflammation J. Immunol., February 15, 2009; 182(4): 2385 - 2394. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. M. Maher, T. N. Dhonnchu, J. P. Burke, A. Soo, A. E. Wood, and R. W. G. Watson Statins alter neutrophil migration by modulating cellular Rho activity--a potential mechanism for statins-mediated pleotropic effects? J. Leukoc. Biol., January 1, 2009; 85(1): 186 - 193. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-H. Lee and Z.-F. Chang Regulation of RhoA-dependent ROCKII activation by Shp2 J. Cell Biol., October 21, 2008; 181(6): 999 - 1012. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Weinmeister, R. Lukowski, S. Linder, C. Traidl-Hoffmann, L. Hengst, F. Hofmann, and R. Feil Cyclic Guanosine Monophosphate-dependent Protein Kinase I Promotes Adhesion of Primary Vascular Smooth Muscle Cells Mol. Biol. Cell, October 1, 2008; 19(10): 4434 - 4441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nagamatsu, Y. Rikitake, M. Takahashi, Y. Deki, W. Ikeda, K.-i. Hirata, and Y. Takai Roles of Necl-5/Poliovirus Receptor and Rho-associated Kinase (ROCK) in the Regulation of Transformation of Integrin {alpha}V{beta}3-based Focal Complexes into Focal Adhesions J. Biol. Chem., May 23, 2008; 283(21): 14532 - 14541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Chilcoat, Y. Sharief, and S. L. Jones Tonic protein kinase A activity maintains inactive {beta}2 integrins in unstimulated neutrophils by reducing myosin light-chain phosphorylation: role of myosin light-chain kinase and Rho kinase J. Leukoc. Biol., April 1, 2008; 83(4): 964 - 971. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Rubtsov, C. L. Swanson, S. Troy, P. Strauch, R. Pelanda, and R. M. Torres TLR Agonists Promote Marginal Zone B Cell Activation and Facilitate T-Dependent IgM Responses J. Immunol., March 15, 2008; 180(6): 3882 - 3888. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hidaka, Y. Suzuki, M. Yamashita, T. Shibata, Y. Tanaka, S. Horikoshi, and Y. Tomino Amelioration of Crescentic Glomerulonephritis by RhoA Kinase Inhibitor, Fasudil, through Podocyte Protection and Prevention of Leukocyte Migration Am. J. Pathol., March 1, 2008; 172(3): 603 - 614. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Hopkins, A. A. Pineda, L. M. Winfree, G. T. Brown, M. G. Laukoetter, and A. Nusrat Organized migration of epithelial cells requires control of adhesion and protrusion through Rho kinase effectors Am J Physiol Gastrointest Liver Physiol, March 1, 2007; 292(3): G806 - G817. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Francis, X. Shen, J. B. Young, P. Kaul, and D. J. Lerner Rho GEF Lsc is required for normal polarization, migration, and adhesion of formyl-peptide-stimulated neutrophils Blood, February 15, 2006; 107(4): 1627 - 1635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Wettschureck and S. Offermanns Mammalian G Proteins and Their Cell Type Specific Functions Physiol Rev, October 1, 2005; 85(4): 1159 - 1204. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Vielkind, M. Gallagher-Gambarelli, M. Gomez, H. J. Hinton, and D. A. Cantrell Integrin Regulation by RhoA in Thymocytes J. Immunol., July 1, 2005; 175(1): 350 - 357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Tasaka, H. Koh, W. Yamada, M. Shimizu, Y. Ogawa, N. Hasegawa, K. Yamaguchi, Y. Ishii, S. E. Richer, C. M. Doerschuk, et al. Attenuation of Endotoxin-Induced Acute Lung Injury by the Rho-Associated Kinase Inhibitor, Y-27632 Am. J. Respir. Cell Mol. Biol., June 1, 2005; 32(6): 504 - 510. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. van Buul and P. L. Hordijk Signaling in Leukocyte Transendothelial Migration Arterioscler Thromb Vasc Biol, May 1, 2004; 24(5): 824 - 833. [Abstract] [Full Text]
|
|
|
|
|
|
H. Honing, T. K. van den Berg, S. M. A. van der Pol, C. D. Dijkstra, R. A. van der Kammen, J. G. Collard, and H. E. de Vries RhoA activation promotes transendothelial migration of monocytes via ROCK J. Leukoc. Biol., March 1, 2004; 75(3): 523 - 528. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Suriano, M. J. Oliveira, D. Huntsman, A. R. Mateus, P. Ferreira, F. Casares, C. Oliveira, F. Carneiro, J. C. Machado, M. Mareel, et al. E-cadherin germline missense mutations and cell phenotype: evidence for the independence of cell invasion on the motile capabilities of the cells Hum. Mol. Genet., November 15, 2003; 12(22): 3007 - 3016. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. D. Coghill, S. Brown, D. L. Cottle, M. J. McGrath, P. A. Robinson, H. H. Nandurkar, J. M. Dyson, and C. A. Mitchell FHL3 Is an Actin-binding Protein That Regulates {alpha}-Actinin-mediated Actin Bundling: FHL3 LOCALIZES TO ACTIN STRESS FIBERS AND ENHANCES CELL SPREADING AND STRESS FIBER DISASSEMBLY J. Biol. Chem., June 20, 2003; 278(26): 24139 - 24152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Worthylake and K. Burridge RhoA and ROCK Promote Migration by Limiting Membrane Protrusions J. Biol. Chem., April 4, 2003; 278(15): 13578 - 13584. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Liu, B. R. Schwartz, J. Tupper, N. Lin, R. K. Winn, and J. M. Harlan The GTPase Rap1 Regulates Phorbol 12-Myristate 13-Acetate-stimulated but Not Ligand-induced beta 1 Integrin-dependent Leukocyte Adhesion J. Biol. Chem., October 18, 2002; 277(43): 40893 - 40900. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, control; 
, Y-27632. Data
are the means ± SEM of 327C binding from four experiments (NS).
B, Neutrophils that had been stimulated with fMLP in the
presence of Alexa-mAb 327C were washed and incubated at 37°C for 30
min in the presence or absence of Y-27632 to measure effect of the RhoA
kinase inhibitor on decay of expression. C, Neutrophils
were allowed to adhere (control) or were stimulated with fMLP to adhere
to serum-blocked chamber coverslips for 15 min at 37°C. fMLP and
nonadherent cells were removed by three washes with medium. The
adherent cells were either fixed immediately (no decay) or incubated at
37°C for 15 min in the presence (fMLP, decay + Y27632) or absence
(fMLP, decay in medium) of Y-27632. Neutrophils were stained
with mAb OKM1 for CD11b. Photomicrographs were made with a confocal
microscope (Bio-Rad Radiance 2000).
