| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Pathology, University of Connecticut Health Center, Farmington, CT 06030; and
Center for Biochemistry/Institute of Biochemistry, Medical School of Hannover, Hanover, Germany.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(3), chemotactic
factors (4, 5, 6), and various stresses (2, 7).
It has roles in many cellular processes, including inflammatory
responses (8), mRNA stabilization (9), cell
division (10), apoptosis (11), and cancer
cell invasiveness (12). There are four known isoforms of
p38 MAPK termed p38, p38
, p38
, and p38
(13, 15). The p38 MAPKs are activated by the upstream kinases through
threonine and tyrosine phosphorylation (16, 17, 18). The
downstream substrates of p38 MAPK include a number of transcription
factors such as activating transcription factor 2 (19),
CHOP/GADD153 (20), muscle-specific transcription factor 2
(21), and p53 (22), as well as a number of
protein kinases such as MAPK-activated protein kinase (MK) 2
(23), MK3 (24), MAPK interacting kinase 2
(25), p38 regulated/activated protein kinase
(26), and mitogen- and stress-activated protein kinase
(MSK) 1 (27). Among these protein kinases, MK2 is the first identified and the best-studied substrate for p38 MAPK (28). Fully activated MK2 requires multiple sites of phosphorylation (29, 30). Within cells, MK2 specifically associates with p38 MAPK (31, 32). Two MK2 isoforms of 54 kDa and 46 kDa are known that vary in their C terminus (33). The N terminus of MK2 contains a proline-rich domain that may interact with src homology (SH)3 domain-containing proteins (34). The sequence of the C-terminal region of the p54 isoform of MK2 contains a nuclear localization signal and nuclear export signal (35). MK2 phosphorylates small heat shock protein (HSP) 27 (3), and induces dissociation of HSP27 multimers, thereby regulating the chaperone function of HSP27 and cellular resistance to oxidative stress (36, 37). MK2 also is involved in cytokine-induced mRNA stabilization by an AU-rich region-targeted mechanism (9). In addition to HSP27, other known substrates of MK2 include SRF (38), E47 (39), CREB (40), 5-lipoxygenase (41), vimentin (42), myosin L chain (43), and leukocyte-specific protein (LSP) 1 (44).
Neutrophils play a vital role in defense against infection and in a
number of allergic and nonallergic tissue-damaging inflammatory
reactions (45, 46). Rapid and transient activation of p38
MAPK and MK2 has been observed in neutrophils treated with chemotactic
factors fMLP (4, 5, 6), platelet-activating factor
(4), and TNF- (6). Treatment of
neutrophils with the p38 MAPK and inhibitor SB203580 or its
analogs inhibits fMLP-induced MK2 activation (4, 5, 6),
chemotaxis (4, 5, 6), superoxide production (47, 48), IL-8 expression (6), TNF--induced adhesion
(47), cell death (49), and inflammatory
responses in vivo (50, 51). Recent work with
MK2-/- mice revealed that MK2 is
essential for LPS-induced TNF- biosynthesis (52).
In this paper, we studied the migration of wild-type (WT) and MK2-/- neutrophils in Zigmond chambers containing fMLP gradients. Confocal images of polarized WT neutrophils showed an intracellular gradient of phospho-MK2 from the anterior to the posterior regions of the neutrophils. Compared with WT neutrophils, the MK2-/- neutrophils showed impaired directionality but higher migration speed. These results suggest that MK2 plays an important role in neutrophil migration.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
MK2-/- mice were generated at Martin Luther University (Halle, Germany; Ref. 52). Breeding pairs of MK2-/- mice were transferred to the University of Connecticut Health Center and maintained in sterile microisolator cages on standard mouse chow in a 12-h light-dark cycle. C57BL6/J WT control mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in similar conditions. In each experiment, age- and sex-matched experimental and control mice were used. All experiments involving the mice were approved by the University of Connecticut Health Center Animal Care Committee.
The following materials were purchased: NIM2 reagents from Cardinal
Associates (Santa Fe, NM); crystalline BSA, lysolecithin, normal goat
serum, PMA, fMLP, fibrinogen, vitronectin, and thioglycollate from
Sigma (St. Louis, MO); rhodamine-phalloidin, FITC-phalloidin, and
Slowfade reagent from Molecular Probes (Eugene, OR); peroxidase and
FITC-conjugated goat anti-rabbit immunoglobulin from Jackson
Immunoresearch (West Grove, PA); rat monoclonal anti-GR1 from
Accurate Chemical and Scientific (Westbury, NY); Percoll and
[-32P]ATP from Amersham Pharmacia Biotech
(Piscataway NJ); paraformaldehyde from Polysciences (Warrington, PA);
Abs to p38, phospho-p38, ERK 1/2, and phospho-ERK 1/2 from New England
Biolabs, (Beverly, MA); and HSP25 from Stessgen, (Victoria, British
Columbia, Canada).
Isolation of neutrophils
Murine bone marrow neutrophils were prepared with Percoll gradients as described previously (53). In some experiments, mouse peripheral blood neutrophils were purified by using NIM 2 reagents from anticoagulant (0.05 M EDTA)-treated blood obtained by cardiac exsanguination. In both cases, after isolation, neutrophils were suspended in Hanks buffer solution (0.14 M NaCl, 5.4 mM KCl, 1 mM Tris, 1.1 mM CaCl2, 0.4 mM MgSO4, and 1 mM HEPES, pH 7.2) containing 1 mg/ml BSA. Granulocyte differentiation Ag 1 staining as analyzed by flow cytometry and detection of mRNA by RT-PCR for myeloperoxidase, elastase, cathepsin, lysozyme, lactoferrin, and gelatinase showed similar levels in WT and MK2-/- neutrophils (data not shown). There was no significant difference between the WT and MK2-/- mice in their neutrophil counts with either peripheral blood or bone marrow (data not shown).
Videomicroscopy of migrating neutrophils
Time-lapsed videomicroscopy was used to examine neutrophil movements in Zigmond chambers. In these chambers, neutrophils purified from peripheral blood were recorded crawling in the absence or presence of fMLP gradients. The microscope was equipped with differential interference contrast optics and a x20 objective. Images were captured at 5-s intervals with a PXL-EEV37 CCD camera (Photometrics, Waterloo, Ontario, Canada) and Metamorph Imaging software (West Chester, PA). Individual cell tracings were made from the captured images. From these tracings, the final position of a neutrophil relative to its starting position was graphed. On these graphs, a positive X distance reflects travel up the gradient. Absolute Y values represents lateral deviation along the gradient.
Confocal microscopy of migrating neutrophils
For indirect immunofluorescence, neutrophils were fixed in Zigmond chambers by the careful removal of chemotaxis buffers and replacement with 2.4% paraformaldehyde in PBS for 15 min at 37°C (54). This was followed by incubation with a solution of 0.1% lysolecithin in PBS containing 1% normal goat serum and Abs to the protein of interest (1/100 dilution). After 30 min at room temperature, the cells were washed and incubated in lysolecithin/PBS containing FITC-conjugated goat anti-rabbit immunoglobulin and rhodamine-phalloidin. After this incubation, cells were washed and coverslips mounted with a drop of Slowfade reagent. These samples were then examined using a Zeiss (Oberkochen, Germany) confocal imaging system with excitation wavelengths of 488 and 568 nm and emission filters to detect FITC (515–520 nm) and rhodamine (590 nm). The pinhole of the confocal microscope was set at 25 µm to minimize staining differences attributable to cell thickness (55) Measurements of staining intensity were obtained from original images using NIH Image software (http://rsb.info.nih.gov/nih-image).
F-actin polymerization assay
WT or MK2-/- neutrophils were stimulated with fMLP (10-5 M) for the indicated times and stained with FITC-phalloidin for flow cytometric analysis to detect F-actin content as described (56). Analysis on a FACScan cytometer (BD Biosciences, Mountain View, CA) was performed with a linear scale fluorescence channel (FL1). The level of F-actin polymerization stimulated by fMLP was determined by the increase of mean fluorescence over time zero.
Adhesion assay
Determination of neutrophil adhesiveness with or without fMLP (10-5 M) or PMA (2.5 ng/ml) treatments was performed essentially as described (57). Briefly, bone marrow neutrophils (3 x 105 in 100 µl) were incubated (37°C in 5% CO2 atmosphere) in wells of a microtiter plate previously coated with fibrinogen (100 µg/ml overnight at 4°C). At the indicated times, samples were removed and the plates inverted and centrifuged on Whatman (Clifton, NJ) #3 filter paper. The remaining cells were quantified by measuring membrane acid phosphatase. The 100% standard was determined by measuring the value of a sample (100 µl) of the original cell suspension.
In vitro MK2 kinase activity assay
Bone marrow-derived neutrophils from WT and
MK2-/- mice were suspended in Hanks
buffer. Cells were preincubated (5 min at 37°C) and then treated with
buffer, 5 x 10-6 M fMLP, or 0.1 µg/ml
PMA for the indicated times. Stimulation was stopped by the addition of
an equal volume of ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM
EDTA, 1% NP40, 150 mM NaCl, and 2 mM diisopropyl fluorophosphate)
followed by incubation on ice for 20 min. Cell lysates were centrifuged
(13,000 x g, 10 min) and 20 µl of the supernatants
added to a kinase assay mixture (20 µl, 20 mM HEPES, pH 7.3, 10 mM
MgCl2, 1 mM EGTA, 5 µM sodium orthovanadate, 5
µM okadaic acid, 2 mM DTT, 0.2 mM
[-32P]ATP (105
cpm/pmol), and 1 µg HSP25). The phosphorylation reaction was allowed
to proceed for 30 min at 37°C. Samples were analyzed by SDS-PAGE
(10%), and phosphorylation of HSP25 was detected by
autoradiography.
Peritoneal neutrophil influxes assay
Mice were injected i.p. (1 ml) with sterile solutions of thioglycollate (2.4%), fMLP (105 M), or PBS. Four hours after the injections, the mice were sacrificed. The peritonea of WT and MK2-/- mice were rinsed (3 times), and total neutrophils recovered were counted.
Immunoblots
Neutrophils (2 x 105) were lysed with sample buffer, boiled, and resolved in 10% PAGE. Samples then were analyzed as immunoblots with Abs to the proteins of interest (1:1000) followed by peroxidase-conjugated goat anti-rabbit immunoglobulin (1:5000). Bound Abs were visualized with the ECL detection system. In some experiments, immunoreactivity of the protein bands was determined by densitometric scanning with NIH Image software.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It recently has been found that signaling molecules involved in
chemotaxis can be asymmetrically distributed in migrating cells
(58, 59). Because p38 MAPK pathway may be involved in
neutrophil chemotaxis (5, 6), we wondered whether an
intracellular gradient of phospho-MK2 may also exist in migrating
neutrophils. Thr317 of the p54 isoform of MK2
(Thr334 of the p46; Ref. 60) has
been shown as the major phosphorylation site of p38 MAPK, and its
phosphorylation is required for activation and nuclear export of MK2
(29, 34). We have previously prepared
anti-phosphopeptide Abs that recognize the
Thr317-phosphorylated form of MK2 in neutrophils
stimulated by fMLP (56 , 60). We used these
antiphospho-MK2 Abs to examine the intracellular staining patterns of
phospho-MK2 in polarized WT mouse neutrophils. Phospho-MK2 was found to
be colocalized with F-actin in the leading front of the polarized
neutrophils (Fig. 1
A,
middle). When measured from anterior to posterior (Fig. 1B, top, a and b), the intensity of
phospho-MK2 staining decreased rapidly toward the posterior region,
showing a pattern of intracellular gradient. When examined laterally
(Fig. 1B, top, c and d), no internal gradient
of phospho-MK2 staining was observed. The results indicate that
phospho-MK2 is primarily found in the lamelipodia regions of the
neutrophil. Intracellular F-actin staining of polarized WT neutrophils
showed a decrease of staining from the anterior to posterior regions,
although not as dramatic as the phospho-MK2 (Fig. 1B,
bottom). No phospho-MK2 staining was observed in the confocal
images of MK2-/- neutrophils (Fig. 1A, bottom).
|
The antiphospho-Thr317 MK2 Abs and an in
vitro kinase assay were used to demonstrate that stimulation with fMLP
activates MK2 in murine neutrophils. The Abs to
phospho-Thr317 MK2 were used in immunoblots and
showed that stimulation of murine bone marrow neutrophils with fMLP
caused a time-dependent increase in the phosphorylation of the p46 and
p54 protein bands (Table I and
Fig. 1
C). These two protein bands correspond to the two
isoforms (Mr 46,000 and
Mr 54,000) (p46/54) of MK2 in mouse
(34, 52). Stimulation with PMA (10 min) also induced
increased phosphorylation of p46 and p54 (Table I and Fig. 1C). No
phospho-MK2 immunoreactivity was observed in
MK2-/- neutrophils (Fig. 1C).
The in vitro MK2 kinase assay with mouse HSP25 as a substrate
demonstrated a large increase in MK2 activity in the cell lysates of
fMLP- or PMA-stimulated WT but not
MK2-/- neutrophils
(p < 0.005, n = 3; Fig. 1D). These results demonstrate that fMLP induces the
phosphorylation and activation of MK2 and suggest that MK2 is the major
kinase for HSP25 in mouse neutrophils.
|
It has been known that fMLP and PMA stimulate both p38 MAPK
(4, 5, 6) and ERK (57) pathways in human
neutrophils. We examined both kinases in WT and
MK2-/- mouse neutrophils on fMLP and PMA
stimulation. Phosphorylation of ERK (p42/44) was observed in WT
neutrophils stimulated with fMLP or PMA (Fig. 2A, left). Compared with WT
neutrophils, the phosphorylation of ERK was greatly reduced when
stimulated by fMLP and partially reduced when stimulated by PMA (Fig. 2A). This is not attributable to a low protein level of ERK
in the MK2-/- neutrophils, as the
protein levels of ERK present in both WT and
MK2-/- neutrophils were similar (Fig. 2A, right). This suggests that MK2 is required for
activation of upstream signaling molecules involved in ERK
phosphorylation stimulated by fMLP.
|
B, left).
Compared with WT neutrophils, a lower level of p38 MAPK phosphorylation
increase was observed in fMLP- or PMA-stimulated
MK2-/- neutrophils (Fig. 2B
left). Immunoblots with Abs to the nonphospho-p38 MAPK showed that
the lower level of phospho-p38 MAPK may be attributable to the lower
protein level of p38 MAPK in the MK2-/-
neutrophils (69 ± 5.6% of WT; Fig. 2B, right). These
results suggest that MK2 may regulate the intracellular protein level
of p38 MAPK. Migration of MK2-/- neutrophils
The intracellular staining pattern of MK2 in neutrophils in fMLP
gradients (Fig. 1A) suggests that MK2 may be involved in
regulating neutrophil chemotaxis. To examine chemotaxis, in vitro,
time-lapsed video microscopy was used to analyze the migration of WT
and MK2-/- neutrophils on BSA-coated
coverslips in fMLP gradients as described by Zigmond (61).
MK2-/- neutrophils migrated faster (49
µm/min ± 12.7, n = 86 vs WT 29 µm/min ±
5.4, n = 55) but much less directionally toward fMLP
than WT neutrophils (54 vs 100% WT, Fig. 3A). Similar results were
obtained when the mouse chemokine KC was used as the
chemoattractant (not shown). Without a fMLP gradient, the random
migration speed of MK2-/- neutrophils is
also higher than WT (8.0 ± 3.7 vs 3.6 ± 1.4 µm/min).
|
A). However, when
using fibrinogen or vitronectin coated coverslips, the
MK2-/- neutrophils still showed higher
migration speeds (Fig. 3B) and less directionality toward
fMLP than did WT neutrophils (data not shown), similar to what was
observed with BSA-coated coverslips (Fig. 3A). These results
suggest the abnormal migration phenotype of
MK2-/- neutrophils is not dependent on
the substrates used in the assays. Morphological examination of migrating WT and MK2-/- neutrophils
We examined the morphology of WT and
MK2-/- neutrophils to better understand
the nature of the chemotactic defects observed in vitro. Tracings of
the boundaries of individual migrating neutrophils indicated that
MK2-/- neutrophils, in fMLP gradients,
extended their leading fronts further than WT at each time interval (50
s; Fig. 4A). In addition, the
MK2-/- neutrophils had twice as many
membrane protrusions as WT and showed lateral and rearward protrusions
that were not detected in WT (Table II).
|
|
B, left). The
MK2-/- neutrophils showed polarized cell
bodies but had multiple protrusions, especially side protrusions and
dramatic misalignments of their heads and tails (Fig. 4B,
right). These results suggest that MK2 regulates cell morphology
during chemotaxis. Adhesion of WT and MK2-/- neutrophils to fibrinogen
Adhesion strength can affect migration speed, so WT and
MK2-/- neutrophil adhesiveness was
quantitated with fibrinogen as a substrate. Compared with WT,
MK2-/- neutrophils showed slightly
reduced fMLP-stimulated adherence (25.6 ± 6.4%
MK2-/- vs 33.1 ± 7.7% WT at 60
min; Fig. 5A). Adherence
induced by PMA was not reduced in MK2-/-
neutrophils (66.0 ± 6.4% MK2-/-
vs 63.1 ± 10.0% WT at 60 min; Fig. 5B). These results
suggest that the abnormal migration phenotype of
MK2-/- neutrophils described above is
not attributable to a large change of adhesiveness induced by
fMLP.
|
To ensure that the fMLP receptor was functional on
MK2-/- neutrophils, the F-actin
polymerization response to fMLP was examined. Both the rate and extent
of F-actin polymerization in fMLP-stimulated WT and
MK2-/- neutrophils are similar when
assayed by FACS (Fig. 6).
|
To evaluate the migration phenotype in vivo, mice were injected
with chemotactic stimuli. Injections of fMLP but not thioglycollate
into the peritoneal cavities of MK2-/-
mice resulted in the influxes of higher numbers of peritoneal
neutrophils (4 h after injection) when compared with WT mice (5.94
± 0.86 x 106
MK2-/- (n = 4) vs
1.86 ± 0.25 x 106 WT
(n = 4) with fMLP injection; Fig. 7).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(59)
and the plekstrin homology domain of kinase AKT/protein kinase B
(58) shows that these signaling molecules are localized to
regions of the cell receiving the strongest chemotactic signals. These
regions also contain psuedopods and are the sites of actin
polymerization. Therefore, it has been suggested that these molecules
are part of the compass that directs neutrophil movement
(64). In this study, we observed that in WT neutrophils,
phospho-MK2 colocalized with F-actin and formed an intracellular
gradient during neutrophil chemotaxis in fMLP gradients. Additionally,
the loss of MK2 resulted in the loss of directionality during
migration. Therefore, phospho-MK2 may be part of the compass directing
the neutrophil during chemotaxis. In addition to a loss of directionality, MK2-/- neutrophils showed a higher migration speed than WT neutrophils. Similar to what we observed, microinjection of a dominant-negative Cdc42 mutant into Bacl.2F5 macrophages enhances migration speed but eliminates polarization during chemotaxis toward colony stimulating factor-1 (65). Additionally, mice lacking the nonreceptor tyrosine kinases Abl and Arg showed that these kinases negatively control cell migration through the regulation of Crk and CAS adapter protein complexes (66).
The control of directionality and migration speed may be related. MK2
may regulate speed and directionality through its unique primary
structure and the phosphorylation of its cytoskeletal substrates. The N
terminus of MK2 contains a proline-rich domain that may interact with
SH3 domain-containing proteins localized to the actin cytoskeleton.
Previously, we identified LSP1, an F-actin cross-linking protein, as a
major substrate for MK2 in neutrophils (44). A recent
study showed that the expression levels of LSP1 could modulate the
migration speed of monocyte-differentiated U937 cells
(67). We speculate that MK2 may regulate both
directionality and migration speed by controlling the phosphorylation
of an F-actin cross-linking protein such as LSP1. A weaker cross-linked
F-actin cytoskeleton may have a less stiff membrane, which will produce
more membrane protrusions during cell migration, as we observed in
MK2-/- neutrophils (Table I). This could
also explain the appearance of MK2-/-
neutrophils in confocal images, characterized by multiple protrusions
and the dramatic misalignment of fronts and tails (Fig. 4B).
Recently we reported that LSP1-/-
neutrophils have reduced directionality and multiple membrane
protrusions in chemokine KC gradients (68). However, the
LSP1-/- neutrophils have a reduced
migration speed. This suggests that other potential cytoskeletal
substrates for MK2 such as myosin L chain (43), vimentin
(42), and HSP25 (37, 38) also may be
important.
The protein level of p38 MAPK was reduced in the MK2-/- neutrophils. This suggests that MK2 may help maintain p38 MAPK protein level in the cell, perhaps through their binding interactions (33). MK2 also may regulate the stability or expression of p38 MAPK mRNA. In contrast to the finding of a reduced level of p38 MAPK, a reduction of ERK protein level was not observed in MK2-/- neutrophils. However, the phosphorylation of ERK induced by fMLP was greatly reduced in the MK2-/- neutrophils. Recent studies indicate that ERK can be activated by RAS/RAF/mitogen-activated protein/extracellular signal-related kinase kinase (MEK) pathway as well as AKT/PAK/MEK pathway (69, 70). mitogen-activated protein/extracellular signal-related kinase kinase In human neutrophils, it has been shown that MEK2 is the major upstream kinase of ERK (71). Further work is required to study the effects of MK2 knockout on the low protein level of p38 MAPK and the loss of fMLP-induced ERK phosphorylation.
Treatment of neutrophils with SB203580 or its analogs has been shown to
inhibit neutrophil fMLP-induced MK2 activation and chemotaxis as
assayed by Boyden chambers (4, 5, 6). The downstream
targets of p38 MAPK include MK2 (23), MK3
(24), MNK2 (25), PRAK (26), and
MSK1 (27). If MK2 is the major downstream target of p38
MAPK for regulating neutrophil migration, one would expect a similar
phenotype between MK2-/- and
SB203580-treated WT neutrophils. In this study, we observed that the
phenotype of MK2-/- neutrophils
undergoing chemotaxis in Zigmond chambers containing fMLP gradients
showed a partial loss of directionality but enhanced migration speeds.
Several possibilities need to be considered. First, in
MK2-/- neutrophils a loss of
fMLP-induced ERK phosphorylation was observed. In contrast, SB203580
does not inhibit fMLP-induced ERK phosphorylation (72).
The MEK inhibitor PD098059 does not inhibit neutrophil chemotaxis
(73), suggesting that ERK activation is not required for
chemotaxis. Second, in addition to MK2, other kinase substrates (MK3,
MNK2, PRAK and MSK1) of p38 MAPK may also be involved in chemotaxis.
One of them, MK3, is known to also phosphorylate HSP25
(74). However, the near-complete loss of HSP25 kinase
activity in MK2-/- neutrophils (Fig. 1C) suggests that MK3 is not a major HSP25 kinase in mouse
neutrophils. Third, the structure of MK2 contains a proline-rich domain
that may bind to SH3 domain-containing molecules. A complete loss of
MK2 may have results different from simply inhibiting the activation of
MK2. Finally, a partial loss of p38 MAPK protein was observed in
MK2-/- neutrophils. We cannot rule out
that this may alter the phenotypes of
MK2-/- neutrophils. The development of
MK2-/- neutrophils appears to be normal
as shown by adhesion assay (Fig. 5), fMLP-induced F-actin
polymerization (Fig. 7), and FACs analysis of GR1 expression (data not
shown).
We saw no difference in the rate or extent of actin polymerization in neutrophils stimulated with fMLP and analyzed by FACS. However, we did observe an abnormal migration phenotype of MK2-/- neutrophils in Zigmond chambers containing fMLP gradients. This suggests that migration speed and direction are not determined solely by actin polymerization. In fact, fibroblasts from gelsolin-/- mice have reduced motility despite increased levels of F-actin (76). It is likely that cross-linking of F-actin filaments and intracellular calcium levels (77) also play roles in controlling neutrophil speed.
Compared with WT mice, MK2-/- mice showed higher influxes of neutrophils into their peritoneal cavities after fMLP injection. In Zigmond chambers containing fMLP gradients, MK2-/- neutrophils migrated with higher speed but less direction. The discrepancy between these in vitro and in vivo results could be attributable to 1) in vivo the peritoneal cavity is a large target area and the increase in migration speed compensates for the lack of directionality; and 2) neutrophil chemotaxis in two-dimensional Zigmond chambers in vitro may be different from the in vivo transmigration across endothelial cells followed by migration through the three-dimensional intercellular matrixes.
Originally, MK2 was found to be involved in the cellular responses to
stress (7, 2), bacterial LPS (2), and the
proinflammatory cytokines IL-1 and TNF- (3). Our
results indicate that MK2 plays a regulatory role in fMLP-induced
neutrophil migration and may also affect other signaling molecules.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Chi-Kuang Huang, Department of Pathology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3105. E-mail address: HUANGCHI{at}sun.uch.edu
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ERK, extracellular regulated kinase; MK, MAPK-activated protein kinase; MSK, mitogen- and stress-activated kinase; HSP, heat shock protein; LSP, leukocyte-specific gene protein; WT; wild type; MEK, mitogen-activated protein/extracellular signal-related kinase kinase; SH, src homology.
Received for publication April 24, 2001. Accepted for publication July 25, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
or FMLP stimulation. J. Immunol. 160:1982.2-integrin-dependent neutrophil adhesion and the adhesion-dependent oxidative burst. J. Immunol. 161:1921. biosynthesis. Nature Cell Biol. 1:94.[Medline]
1-induced signal transduction in human neutrophils. Biochem. Biophys. Res. Commun. 246:55.[Medline]
complex in living cells during chemotaxis. Science 287:1034. and its lipid products. Trends Cell Biol. 10:466.[Medline]
This article has been cited by other articles:
|
|
 |
|
  P. D. A. Issuree, P. N. Pushparaj, S. Pervaiz, and A. J. Melendez Resveratrol attenuates C5a-induced inflammatory responses in vitro and in vivo by inhibiting phospholipase D and sphingosine kinase activities FASEB J, August 1, 2009; 23(8): 2412 - 2424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Katsube, T. Kato, M. Kitagawa, H. Noma, H. Fujita, and S. Kitagawa Calpain-mediated regulation of the distinct signaling pathways and cell migration in human neutrophils J. Leukoc. Biol., July 1, 2008; 84(1): 255 - 263. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-K. Park, N. Ronkina, A. Hoft, C. Prohl, J. Menne, M. Gaestel, H. Haller, and M. Meier Deletion of MK2 signalling in vivo inhibits small Hsp phosphorylation but not diabetic nephropathy Nephrol. Dial. Transplant., June 1, 2008; 23(6): 1844 - 1853. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Liu, R. R. Warburton, O. E. Guevara, N. S. Hill, B. L. Fanburg, M. Gaestel, and U. S. Kayyali Lack of MK2 Inhibits Myofibroblast Formation and Exacerbates Pulmonary Fibrosis Am. J. Respir. Cell Mol. Biol., November 1, 2007; 37(5): 507 - 517. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. R. Jog, V. R. Jala, R. A. Ward, M. J. Rane, B. Haribabu, and K. R. McLeish Heat Shock Protein 27 Regulates Neutrophil Chemotaxis and Exocytosis through Two Independent Mechanisms J. Immunol., February 15, 2007; 178(4): 2421 - 2428. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Ronkina, A. Kotlyarov, O. Dittrich-Breiholz, M. Kracht, E. Hitti, K. Milarski, R. Askew, S. Marusic, L.-L. Lin, M. Gaestel, et al. The Mitogen-Activated Protein Kinase (MAPK)-Activated Protein Kinases MK2 and MK3 Cooperate in Stimulation of Tumor Necrosis Factor Biosynthesis and Stabilization of p38 MAPK Mol. Cell. Biol., January 1, 2007; 27(1): 170 - 181. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Zheng, Z. Lin, Z. J. Zhao, Y. Yang, H. Niu, and X. Shen MAPK-activated Protein Kinase-2 (MK2)-mediated Formation and Phosphorylation-regulated Dissociation of the Signal Complex Consisting of p38, MK2, Akt, and Hsp27 J. Biol. Chem., December 1, 2006; 281(48): 37215 - 37226. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Aberg, M. Perander, B. Johansen, C. Julien, S. Meloche, S. M. Keyse, and O.-M. Seternes Regulation of MAPK-activated Protein Kinase 5 Activity and Subcellular Localization by the Atypical MAPK ERK4/MAPK4 J. Biol. Chem., November 17, 2006; 281(46): 35499 - 35510. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hegen, M. Gaestel, C. L. Nickerson-Nutter, L.-L. Lin, and J.-B. Telliez MAPKAP Kinase 2-Deficient Mice Are Resistant to Collagen-Induced Arthritis J. Immunol., August 1, 2006; 177(3): 1913 - 1917. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. T. Kneass and R. B. Marchase Protein O-GlcNAc Modulates Motility-associated Signaling Intermediates in Neutrophils J. Biol. Chem., April 15, 2005; 280(15): 14579 - 14585. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Liu, D. C. Cara, J. Kaur, E. Raharjo, S. C. Mullaly, J. Jongstra-Bilen, J. Jongstra, and P. Kubes LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration J. Exp. Med., February 7, 2005; 201(3): 409 - 418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. C. Tsai, M. L. Rodriguez, K. S. Young, J. C. Deng, V. J. Thannickal, K. Tateda, M. B. Hershenson, and T. J. Standiford Azithromycin Blocks Neutrophil Recruitment in Pseudomonas Endobronchial Infection Am. J. Respir. Crit. Care Med., December 15, 2004; 170(12): 1331 - 1339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Yu, D. Bian, C. Mahanivong, R. K. Cheng, W. Zhou, and S. Huang p38 Mitogen-activated Protein Kinase Regulation of Endothelial Cell Migration Depends on Urokinase Plasminogen Activator Expression J. Biol. Chem., November 26, 2004; 279(48): 50446 - 50454. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ueda, H. Kosako, Y. Fukui, and S. Hattori Proteomic Identification of Bcl2-associated Athanogene 2 as a Novel MAPK-activated Protein Kinase 2 Substrate J. Biol. Chem., October 1, 2004; 279(40): 41815 - 41821. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Huang, K. Jacobson, and M. D. Schaller MAP kinases and cell migration J. Cell Sci., September 15, 2004; 117(20): 4619 - 4628. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. P. Roux and J. Blenis ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions Microbiol. Mol. Biol. Rev., June 1, 2004; 68(2): 320 - 344. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Shi, A. Kotlyarov, K. Laass, A. D. Gruber, E. Butt, K. Marcus, H. E. Meyer, A. Friedrich, H.-D. Volk, and M. Gaestel Elimination of Protein Kinase MK5/PRAK Activity by Targeted Homologous Recombination Mol. Cell. Biol., November 1, 2003; 23(21): 7732 - 7741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Singh, D. W. Powell, M. J. Rane, T. H. Millard, J. O. Trent, W. M. Pierce, J. B. Klein, L. M. Machesky, and K. R. McLeish Identification of the p16-Arc Subunit of the Arp 2/3 Complex as a Substrate of MAPK-activated Protein Kinase 2 by Proteomic Analysis J. Biol. Chem., September 19, 2003; 278(38): 36410 - 36417. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. W. Powell, M. J. Rane, B. A. Joughin, R. Kalmukova, J.-H. Hong, B. Tidor, W. L. Dean, W. M. Pierce, J. B. Klein, M. B. Yaffe, et al. Proteomic Identification of 14-3-3{zeta} as a Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Substrate: Role in Dimer Formation and Ligand Binding Mol. Cell. Biol., August 1, 2003; 23(15): 5376 - 5387. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Nick, S. K. Young, P. G. Arndt, J. G. Lieber, B. T. Suratt, K. R. Poch, N. J. Avdi, K. C. Malcolm, C. Taube, P. M. Henson, et al. Selective Suppression of Neutrophil Accumulation in Ongoing Pulmonary Inflammation by Systemic Inhibition of p38 Mitogen-Activated Protein Kinase J. Immunol., November 1, 2002; 169(9): 5260 - 5269. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). Scale bar, 10
µm. B, Analysis of phospho-MK2 and F-actin
distributions in a migrating neutrophil. The green channel
(top) and red channel (bottom)
intensities obtained from images of a neutrophil stained with FITC
anti-MK2 and rhodamine phalloidin were measured with NIH Image
software. Measurements were taken along lines drawn from anterior to
posterior (a and b, left)
or laterally (c and d,
right). Scale bar, 10 µm. C, Immunoblot
of phospho-MK2 in WT and MK2-/-
neutrophils. Neutrophils treated with buffer or fMLP for the indicated
times were used in immunoblot analysis to detect phospho-MK2.
D, In vitro MK2 kinase activity. WT or
MK2-/- bone marrow cells were
treated with buffer, fMLP, or PMA for the indicated times, and
32P incorporation into HSP25 was examined using
autoradiography.
