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Department of Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI. Ligand-binding affinity of VLA-5 was also augmented by
receptor tyrosine kinases when the phospholipase C
-1/protein kinase
C pathway was inhibited. Wortmannin suppressed induction of the high
affinity state VLA-5 in either case. Conversely, introduction of a
constitutively active p110 subunit of phosphatidylinositol 3-kinase (PI
3-kinase) increased the binding affinity for fibronectin. Failure of a
constitutively active Akt to stimulate adhesion suggested that the
affinity modulation mechanisms mediated by PI 3-kinase are distinct
from the mechanisms to control growth and apoptosis by PI 3-kinase.
Taken together, our findings demonstrated that the increase of affinity
of VLA-5 was induced by physiologically relevant stimuli and PI
3-kinase was a critical affinity modulator of
VLA-5. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Avidity modulation of integrins is thought to be regulated by the
spatial distribution or ligand-binding affinity of the integrin
9, 10, 11, 12 . Affinity modulation in integrins detected with soluble ligands
or Abs recognizing the high affinity state was reported for
4ß1, 5ß1,
Lß2, and
IIbß3 integrins in cells stimulated with
activating Abs, manganese ions, or cross-linking of the TCR 13, 14, 15, 16, 17, 18, 19 .
Cross-linking of the TCR increased the affinity of
VLA-44 for fibronectin
(FN) in a T lymphoid cell line 13 , but it was reported that
affinity of VLA-4 for FN and VCAM-1 did not change in primary T cells
18, 20 . Several studies showed that PMA enhanced adhesion without
detectable change in ligand-binding affinity of integrins 13, 17, 21 ,
suggesting that affinity change does not account for all avidity
modulation processes. In fact, the physiologic relevance of affinity
modulation of integrins has been questioned, since artificial agonists
are often used and it has not been clearly demonstrated that a natural
agonist can modulate the affinity of ß1 integrins 22 .
To gain a clearer understanding of avidity regulations of integrins, we
have examined adhesion mediated by VLA-5 in bone marrow-derived mast
cells. Mast cells are distributed exclusively in peripheral tissues and
play critical roles in allergy and inflammation. The tissue
distribution of mast cells, therefore, influences the magnitude of
these inflammatory events. We and others previously reported that steel
factor (SLF) transiently stimulated mast cells to adhere to FN via
VLA-5 at concentrations 100-fold lower than those required for growth
stimulation 23, 24 . Furthermore, avidity of VLA-5 was regulated
independently by the PI 3-kinase and PLC-1/protein kinase C (PKC)
pathways of c-kit and the PDGF receptor 25, 26, 27 .
Cross-linking of FcRI also stimulated mast cells to bind to FN 28 .
However, it remains unclear whether these agonists affect
ligand-binding affinity of VLA-5 and how ligand-binding affinity is
regulated upon stimulation. In this study, we reported that the
affinity modulation of VLA-5 by physiologically relevant agonists
caused adhesion of mast cells to FN. We further showed that the
ligand-binding affinity of VLA-5 was increased by PI 3-kinase
stimulated by receptor tyrosine kinases and FcRI.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary bone marrow-derived mast cell cultures were conducted as described 23 . Primary mast cells were used from 4–10 wk of culture after establishment. Retrovirus-mediated transfection was employed to introduce the wild-type, mutant PDGF receptors 25 , or constitutively active PI 3-kinase, p110-CAAX 29 into mast cells, as described 25 .
The anti-mouse VLA-5 mAb MFR-5 (5H10) (rat IgG2a) 30 was purified by affinity chromatography on protein G-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ). Anti-DNP IgE was a culture supernatant of a hybridoma, IGEL a2 (American Type Culture Collection, Manassas, VA). Recombinant murine SLF (Genzyme, Boston, MA), PMA (Sigma, St. Louis, MO), and manganese chloride tetrahydrate (Sigma) were purchased.
Purification of FN and production of the 80-kDa fragment
FN was purified as described 31 from frozen human plasma obtained from the Japanese Red Cross Central Blood Center. The 80-kDa tryptic fragment that contains the RGD-binding motif for VLA-5, but lacks VLA-4-binding domains, was produced by a modified procedure 32 . Briefly, purified FN was digested with 1 or 2 µg/ml of trypsin (Wako Pure Chemical Industries, Osaka, Japan) for 90 min at 37°C in digestion buffer containing 25 mM Tris-HCl buffer (pH 7.6), 50 mM NaCl, and 0.5 mM EDTA. The 80-kDa fragment was purified by affinity chromatography on heparin-Sepharose CL-6B (Pharmacia Biotech, Uppsala, Sweden). After extensive washing with digestion buffer, a fraction containing a 80-kDa FN fragment was obtained with elution buffer containing 25 mM Tris-Cl buffer, pH 7.6, 100 mM NaCl, and 0.5 mM EDTA, and further purified by HPLC on Superdex 200 HR 10/30 (Pharmacia Biotech). The purity of the 80-kDa fragment was more than 95%, as assessed by SDS-PAGE, and its function was confirmed by adhesion assays.
Adhesion assays
Assays of adhesion to FN were performed as described 23
Briefly, mast cells labeled with 2',7'-bis-(2-carboxyethyl)-5 (and
-6)-carboxyfluorescein (BCECF) in 96-well plates precoated with FN (1
µg/well) were incubated in triplicate at 37°C for 30 min in the
presence of PMA (10 ng/ml; Sigma), SLF (1 U/ml), DNP (1 µg/ml), or
medium alone (RPMI 1640 supplemented with 0.02% BSA and 10 mM HEPES,
pH 7.4), as indicated. For manganese stimulation, HBSS (Life
Technologies, Grand Island, NY) was used instead of RPMI 1640, and BSA
was dialyzed against PBS. The amounts of these factors were chosen to
give the maximum response. For cross-linking of FcRI, labeled mast
cells were sensitized with a culture supernatant containing
anti-DNP IgE for 30 min at room temperature before stimulation with
DNP-conjugated BSA (1 µg/ml). After washing the plate four times,
bound fluorescence was measured with a fluorescence concentration
analyzer (IDEXX Laboratories, Westbrook, ME). The level of adhesion was
calculated by dividing bound fluorescence by input fluorescence. Mast
cells did not adhere to wells precoated with BSA with or without
stimulation (data not shown). For the assay with Abs, labeled mast
cells were preincubated at room temperature with 20 µg/ml of Abs, as
indicated, and the adhesion assay was performed in the presence of Abs.
For pretreatment with PMA, mast cells were incubated with 100 ng/ml of
PMA for 40 h. The pretreatment had no effect on viability of mast
cells. PMA was washed away before the adhesion assay. For the
experiment with wortmannin (Wako Pure Chemical, Tokyo, Japan), cells
were treated with wortmannin at room temperature for 10 min, as
described 33 .
For the experiment with the soluble 80-kDa FN fragment, the plates were coated with the 80-kDa FN fragment (1 µg/well), then blocked with 3% BSA for 1 h at 37°C. Mast cells labeled with BCECF mixed with varying amounts of the soluble 80-kDa FN fragment were subjected to the adhesion assay described above.
125I labeling of the 80-kDa FN fragment
The 80-kDa FN fragment was radioiodinated with a modified method using chloramine T 34 . Briefly, carrier-free Na125I (1 mCi) (iodine-125, NEZ-033A; DuPont NEN, Boston, MA) was mixed with 250 µg of the 80-kDa FN fragment in labeling buffer containing 10 mM sodium phosphate, pH 7, 150 mM NaCl, and 30 µg/ml of chloramine T (Katayama Chemical, Osaka, Japan). After incubation for 4 min at room temperature, the iodination was stopped by adding sodium pyrosulfite (Na2S2O5; Katayama Chemical) at 60 µg/ml and 0.1% NaI (Wako Pure Chemical Industries). The labeled protein was collected with the PD-10 column (Pharmacia Biotech, Piscataway, NJ). Radioactivity of the protein was measured with a gamma counter. The typical sp. act. of the labeled 80-kDa fragment used in our experiments was about 3.7 x 108 dpm/nmol.
Ligand-binding assay
The ligand-binding assay was performed basically as previously described 31 . Mast cells were washed once with binding buffer containing RPMI 1640 (Sigma), 0.1% BSA (Life Technologies), and 10 mM HEPES, pH 7.4 (Sigma), and suspended with the same buffer at 1 x 107 cells/ml. In a typical binding assay, performed in a 1.5-ml microcentrifuge tube, 100 µl of cells (1 x 106 cells/tube) was mixed with 50 µl of the radiolabeled 80-kDa fragment and 50 µl of stimulant (e.g., 1 mM manganese, 10 ng/ml PMA, 10 U/ml SLF, or 1 µg/ml DNP), with or without inhibitors (unlabeled 80-kDa FN fragment, wortmannin). For inhibition with Abs (20 µg/ml), mast cells were preincubated with Abs for 20 min at 25°C. In the case of manganese stimulation, HBSS was used instead of RPMI 1640 and BSA was dialyzed against PBS. After incubation for 30 min at 37°C, samples were layered onto 100 µl of separation oil (80% Di-n-butyl phthalate (Wako Pure Chemical Industries) and 20% olive oil (Katayama Chemical)) in 0.5-ml tubes, and centrifuged at 8000 rpm for 1 min. The tip of tubes was amputated from the body with a blade and applied to a gamma counter to measure radioactivity of the bound (the tip) and the unbound (the body). The nonspecific binding was determined at each data point in the presence of a 50-fold excess of the unlabeled 80-kDa fragment. The specific binding was calculated by subtracting the nonspecific binding from the total binding.
In vitro PI 3-kinase assay
PI 3-kinase assays were performed basically as described 35 .
Mast cells (107 cells) were stimulated for 5 min at 37°C
with PMA (100 ng/ml) and SLF (50 U/ml). For stimulation with PDGF (50
ng/ml), mast cells expressing the PDGF receptor were used. Cell lysates
prepared as described above were immunoprecipitated for 2 h at
4°C with an anti-phosphotyrosine (PY) mAb (4G10; Upstate
Biotechnology, Lake Placid, NY), followed by incubation for 40 min at
4°C with protein G-Sepharose (Pharmacia Biotech) to collect the
immune complex. The beads were washed three times with lysis buffer,
and three times with PI 3-kinase buffer (10 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 10 mM MgCl2, and 200 µM adenosine). PI 3-kinase
activity was measured in a volume of 100 µl of PI 3-kinase buffer.
The kinase reaction was initiated by adding 10 µg sonicated
phosphatidylinositol (Sigma), 10 µM ATP, and 10 µCi
[-32P]ATP (Amersham, Arlington Heights, IL). Reactions
are conducted for 15 min at room temperature and stopped by the
addition of 20 µl of 6 N HCl. Lipids were extracted by
chloroform:methanol (1:1, v/v) and were separated on oxalate-treated
TLC plates using a solvent of chloroform:methanol:water:28% ammonia
(45:35:7.5:2.5, v/v/v/v). Labeled phosphatidylinositol was detected and
quantified by a phosphor imager (BAS1000; Fujifilm, Tokyo, Japan).
Analysis of expression and phosphorylation of Akt and cell survival
Mast cells were prepared for cell lysates as described 25 . Equal amounts of protein were subjected to SDS-PAGE. Following SDS-PAGE, the separated proteins were electrophoretically transferred to a PVDF membrane. After blocking with 5% BSA, the membrane was incubated with a 1/1000 dilution of anti-phospho-Akt Ab (New England Biolabs, Beverly, MA) and then with a 1/2000 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit Ab. The bands were visualized using enhanced chemoluminescence (ECL; Amersham). The same membrane was stripped and reprobed with a 1/1000 dilution of anti-Akt Ab (New England Biolabs) and a 1/2000 HRP-conjugated anti-rabbit Ab.
To detect HA-tagged c-Akt and gag-Akt in transfected mast cells, cell lysates (5 x 106 cells) were immunoprecipitated with 5 µg of an anti-HA mAb (12CA5; Boehringer Mannheim, Indianapolis, IN) and protein G-Sepharose (Pharmacia Biotech). After blocking with 5% BSA, the membrane was incubated with a 1/500 dilution of anti-Akt-1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h, then with a 1/4000 dilution of HRP-conjugated anti-goat Ab (Santa Cruz Biotechnology).
To measure cell survival of transfected mast cells, cells were washed three times with PBS to remove IL-3, and then they were cultured in RPMI 1640 containing 10% FCS and 50 µM 2 ME with or without IL-3 (100 U/ml). Viable cells were counted with the trypan blue exclusion assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse bone marrow-derived mast cells did not adhere to FN without
stimulation. Upon stimulation with PMA, SLF, and cross-linking of
FcRI for 30 min, mast cells adhere to FN at comparable levels (Fig. 1
A). In all cases, adhesion to
FN was blocked by an anti-VLA-5 Ab, MFR-5, indicating that adhesion
was mediated by VLA-5. The maximal adhesion responses were not changed
significantly in concentrations examined (1–100 ng/ml for PMA, 1–100
U/ml for SLF, and 0.1–100 µg/ml for DNP-BSA). Induction of
adhesion accompanied little change of VLA-5 expressions before and
after stimulation 23 (data not shown), indicating avidity modulation
of VLA-5 occurred in mast cells. These stimuli induced adhesion very
quickly with almost identical kinetics, and a maximum level of adhesion
was reached at 30 min 23 (unpublished data).
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B). Stimulation with PMA or SLF did not
augment the binding of the FN80 fragment, although they had a potent
effect on adhesion (Fig. 1A). In contrast,
FcRI-stimulated mast cells showed significant increase of specific
ligand binding. The increased binding was mediated by VLA-5, because
the binding was inhibited with the anti-VLA-5 Ab. The effect of
manganese on the binding was also measured, as manganese is known to
act directly on integrins and activate them 36, 37, 38 . As expected,
manganese also increased the specific binding, which was also inhibited
with the anti-VLA-5 Ab.
High affinity ligand binding of VLA-5 by FcRI cross-linking
Although unstimulated, PMA-, or SLF-stimulated mast cells showed
low binding affinity for the FN80 fragment as above, we were unable to
obtain the specific saturation curve that was reproducible (data not
shown). In contrast, mast cells stimulated with cross-linking of
FcRI showed specific saturation curves with increased amounts of
labeled FN80 fragment, and Scatchard analysis indicated a
Kd of 37.7 ± 3.8 nM (n =
3) (Fig. 1C). The total number of VLA-5 was calculated as
3.85 ± 0.20 x 104 molecules/cell, which was
similar to the number obtained with 125I-labeled
anti-VLA-5 Ab (data not shown). As a comparison, we also measured
ligand-binding affinity of VLA-5 in mast cells stimulated with
manganese. Manganese also induced dose-dependent bindings of the FN80
fragment, and Scatchard analysis showed a Kd of
about 10 nM (data not shown). The affinity of VLA-5 obtained with
FcRI cross-linking and manganese was in agreement with result of a
previous report using ß1 integrin-activating Abs 15 .
Inhibition of adhesion with soluble FN
To examine whether the increase in ligand-binding affinity of
VLA-5 accounts for adhesion to immobilized FN, various doses of soluble
FN80 fragment were added in adhesion assays. While there was little
inhibition of adhesion of PMA-stimulated mast cells with the soluble
FN80 fragment, FcRI or manganese-induced adhesion was inhibited
almost completely (Fig. 1D). This result indicates that an
excess amount of FN80 competes with immobilized FN for the binding of
the high affinity state VLA-5 and that the ligand-binding affinity of
VLA-5 on PMA-stimulated mast cells could be too low to be inhibited for
adhesion by the amounts of soluble FN examined. SLF-induced adhesion
was inhibited slightly with soluble FN80, suggesting increases of
ligand-binding affinity below the detectable level in our
ligand-binding assay.
Association of PI 3-kinase activity with anti-PY immunoprecipitates in stimulated mast cells
Previously, we showed that avidity of VLA-5 was up-regulated by PI
3-kinase of c-kit and the PDGF receptor 25 . To assess the
relative ability of stimuli used in adhesion assays to activate PI
3-kinase in mast cells, we measured activities of PI 3-kinase
that were immunoprecipitated with an anti-PY Ab in mast cells
stimulated with PMA, SLF, and FcRI. We also measured anti-PY
immunoprecipitable PI 3-kinase in PDGF-stimulated mast cells expressing
the PDGF receptor. As shown in Fig. 2,
stimulation with SLF, FcRI, and PDGF, but not PMA, caused a
substantial increase in the amount of PI 3-kinase activity that was
immunoprecipitated with anti-PY, which is consistent with the
earlier reports 33, 35, 39 . The doses of stimulants used in this
assay gave the equivalent levels of adhesion to FN as seen in Fig. 1.
The activity of the anti-PY-precipitable PI 3-kinase in mast cells
stimulated by SLF was increased about 20-fold and comparable with that
by PDGF. The activity of anti-PY-precipitable PI 3-kinase in mast
cells stimulated with FcRI cross-linking was about 60% to that by
SLF.
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To explore the possibility that PI 3-kinase is involved in the
affinity modulation of VLA-5, we used wortmannin, a specific PI
3-kinase inhibitor 40 . Wortmannin reduced FcRI-induced
ligand-binding affinity for FN by about half at as low as 10 nM (Fig. 3). Further reduction was barely observed
even at 500 nM. On the other hand, manganese-induced ligand-binding
affinity was not affected at all by wortmannin (Fig. 3), demonstrating
the specific inhibition of FcRI-induced ligand-binding affinity of
VLA-5 by wortmannin. These results suggest that PI 3-kinase is involved
at least in part in the affinity modulation of VLA-5 by FcRI
cross-linking.
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To investigate the relationship between adhesiveness and
ligand-binding affinity for FN in more details, we examined adhesion
and ligand-binding affinity for FN of receptor tyrosine kinases. As
shown in our previous study 25 , the PDGF receptor, closely related
c-kit can induce the adhesion of mast cells to FN through
two pathways, in which PI 3-kinase and PLC-1 independently send
signals to increase adhesiveness to FN. The wild-type or mutant PDGF
receptors that were defective in the binding of PI 3-kinase or PLC-1
supported PDGF-induced adhesion, whereas the mutants that were
defective in both binding sites eliminated PDGF-induced adhesion 25 .
Upon stimulation with PDGF, ligand-binding affinity for FN was modestly
increased in mast cells expressing the wild-type receptor, and was
markedly increased in cells expressing the mutant receptor that lacks
the PLC-1 binding site (Fig. 4). In
contrast, there was little change in ligand-binding affinity
for FN in mast cells expressing the mutant receptor that was defective
in binding to PI 3-kinase, although they adhered to FN at levels
comparable with that of cells expressing the wild-type and
PLC-1-defective mutant receptors (data not shown). The mutation of
both the PI 3-kinase and PLC-1 binding sites abolished the ligand
bindings. These results suggest a critical role for PI 3-kinase in
ligand-binding affinity for FN.
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-1 binding site
induced higher ligand-binding affinity than wild-type suggests that
activation of the PLC-1/PKC pathway inhibits affinity modulation for
FN. To examine the possibility that the PLC-1/PKC pathway inhibits
affinity modulation of VLA-5 by SLF/c-kit, mast cells were
pretreated with PMA to down-regulate PKC. Although PMA-pretreated mast
cells did not respond to PMA for adhesion, they still adhered to FN
when stimulated with SLF (Fig. 5A). The adhesion of
PMA-pretreated mast cells was almost completely inhibited with
wortmannin, showing the PI 3-kinase-dependent adhesion to FN (Fig. 5A). In contrast to untreated mast cells (Fig. 1B), PMA-pretreated mast cells showed increased ligand
bindings to FN upon SLF stimulation (Fig. 5B). The increase
in ligand-binding affinity for FN was completely abolished by
wortmannin. Taken together, these results support the notion that the
PLC/PKC pathway counteracts the affinity modulation of PI 3-kinase
through receptor tyrosine kinases.
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To further confirm modulation of ligand-binding affinity by PI
3-kinase, we introduced a constitutively active p110 subunit of PI
3-kinase (p110-CAAX) 29, 41 into mast cells. Two independent mast
cell lines expressing p110-CAAX adhered to FN without stimulation (Fig. 6A). Moreover, the
ligand-binding affinity for FN was also increased (Fig. 6B),
indicating that PI 3-kinase can modulate ligand-binding affinity for
FN. Taken together with the inhibitory effect of wortmannin on ligand
binding to FN, these results indicate that PI 3-kinase modulates
ligand-binding affinity for FN.
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RI cross-linking
induced phosphorylation of Akt, which was abolished by the treatment of
wortmannin, indicating the PI 3-kinase-dependent activation of Akt
(Fig. 7A). The degree of
phosphorylation of Akt by SLF was higher than that by FcRI
cross-linking, consistent with the relative ability of activation of PI
3-kinase (Fig. 2). To examine whether Akt is capable of stimulating
adhesion, a constitutively active Akt, gag-Akt, or c-Akt
43 was introduced into mast cells. Mast cells expressing
gag-Akt or c-Akt failed to stimulate adhesion to FN (Fig. 7B). However, gag-Akt, but not c-Akt, prolonged
the survival of mast cells deprived of IL-3 (Fig. 7C), which
is consistent with the reports that gag-Akt prevents
apoptosis induced by IL-3 withdrawal 44 . Taken together, these
results suggest that the affinity modulation mechanism by PI 3-kinase
is distinct from the mechanisms to control growth and apoptosis by PI
3-kinase.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI with Ag and IgE.
Through our study of adhesiveness and ligand-binding affinity for FN,
we demonstrated that cross-linking FcRI induced high affinity state
VLA-5 that mediated adhesion to FN, indicating that a distinct
physiologic agonist can increase ligand-binding affinity of
ß1 integrin. We also showed that PI 3-kinase was a
critical affinity modulator of VLA-5 by using the specific PI 3-kinase
inhibitor, the mutant PDGF receptor that lacked the binding site of PI
3-kinase, and the constitutively active PI 3-kinase. The affinity
modulation of PI 3-kinase through receptor tyrosine kinase was
augmented when the PLC-1/PKC pathway was inhibited, suggesting a
fine tuning of a balance between these two pathways determines the
ligand-binding affinity of VLA-5.
Cross-linking of FcRI stimulated mast cells to adhere to FN as
reported 28 , through VLA-5 in a rapid and transient fashion as SLF
(unpublished data). The different effects of FcRI cross-linking and
SLF on ligand-binding affinity of VLA-5 could result in distinct
adhesion behaviors under physiologic and pathologic circumstances.
Since FcRI cross-linking enhanced mast cell chemotaxis though
FN-coated matrix by chemokines 45 , induction of the high affinity
state VLA-5 by FcRI could facilitate migration and accumulation
toward Ag. Our result that soluble FN competitively inhibited the
FcRI-induced adhesive interaction suggests that the high affinity
state of VLA-5 rather weakens firm attachment when soluble FN
concentrations are augmented due to plasma protein extravasation at
allergic and inflammatory sites and influences tissue localization of
mast cells. Further studies are required to establish the specific role
of the high affinity state VLA-5 in mast cells.
Although integrin adhesiveness is thought to be regulated through alterations of integrin affinity for ligand or cell surface distribution by diffusion/clustering 9, 10, 11, 12 , the physiologic relevance of affinity modulation of integrins, in particular ß1 integrin, has been questioned 11 , because artificial agonists were used to induce the high affinity state integrins. Our study clearly showed affinity modulation of ß1 integrin in a physiologically relevant system. PI 3-kinase was shown to be an important intracellular mediator in the inside-out signaling pathway by using mutant receptors that were defective in the binding sites of PI 3-kinase or by using a dominant-negative form of PI 3-kinase 25, 46, 47 . Our study using a constitutively active PI 3-kinase ruled out the possibility that other molecules that bind to the same sites of receptors stimulate integrin adhesiveness, and confirmed PI 3-kinase as a critical affinity modulator of VLA-5 and probably other integrin subfamilies as well.
It is unknown how PI 3-kinase modulates the affinity of integrins. Our
result with a constitutively active Akt suggests distinct mechanisms to
control ligand-binding affinity of integrins from those to regulate
growth and apoptosis. We also confirmed that Btk, another downstream
target of PI 3-kinase, which played a critical role in Ca2+
influx, was not involved in FcRI-induced adhesion 48 . Although
downstream targets of PI 3-kinase are unknown at present, affinity
modulation of integrins probably occurs through the cytoplasmic region
of integrin - and/or ß-chains since mutations or deletions of the
cytoplasmic regions resulted in high affinity state integrins 49, 50 .
Interactions of integrin cytoplasmic regions with other molecules such
as cytoskeletal proteins may control the ligand-binding affinity of
integrins.
Apparently, other adhesive mechanisms exist besides affinity modulation
of integrins, because ligand-binding affinity for FN was not changed
significantly by PMA and SLF in mast cells. Recent studies have shown
that an increase in lateral diffusion of integrins by PMA or
cytochalasin D at low doses facilitates adhesion, suggesting that
release of integrins from cytoskeletal constraints is an important step
in activation of adhesion 12, 51 . We observed that PMA or SLF, but
not cross-linking of FcRI, induced surface redistribution of VLA-5
in mast cells (unpublished data). Therefore, regulation of spatial
distribution of integrin molecules by clustering and diffusion may be
an alternative regulatory mechanism to control integrin adhesiveness
probably through PKC, because PMA is a direct activator of PKC 52 .
Our result that suppression of the PLC-1/PKC pathway either by
mutation of binding sites of PLC-1 in the PDGF receptor or
down-regulation of PKC augmented ligand-binding affinity upon
stimulation suggests that a balance between the PI 3-kinase and
PLC-1/PKC pathways determines diffusibility and ligand-binding
affinity of integrins and controls cell adhesion behavior.
Interestingly, down-regulation of PKC did not enhance ligand-binding
affinity of VLA-5 by FcRI (unpublished data), although cross-linking
of FcRI leads to Ca2+ influx and PKC activation. This
result implies differences in activation patterns of PKC among FcRI
and receptor tyrosine kinases. It is currently under investigation
which isotypes of PKC are activated and responsible for stimulation of
adhesion and inhibition in affinity modulation of integrins in mast
cells.
Regulation of the adhesive interactions with extracellular matrix underlies complex mechanisms regulating activation and inactivation of integrin adhesiveness. Elucidation of the mechanistic basis to the control of these processes requires a better understanding of the specific signaling pathways. Our study has yielded important insights into, as well as a useful experimental system with which to examine, inside-out signaling in physiologic conditions.
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Acknowledgments |
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Footnotes |
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3 Address correspondence and reprint requests to Drs. Tatsuo Kinashi or Kiyoshi Takatsu, Department of Immunology, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. E-mail address:
4 Abbreviations used in this paper: VLA, very late Ag; BCECF, 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein; FN, fibronectin; HA, hemagglutinin; HRP, horseradish peroxidase; PDGF, platelet-derived growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; PY, phosphotyrosine; SLF, steel factor.
Received for publication July 20, 1998. Accepted for publication November 12, 1998.
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6ß1 integrin. J. Leukocyte Biol. 61:397.[Abstract]
5ß1: regulation of the functional response by soluble fibronectin. J. Cell Biol. 121:155.4ß1 integrin-dependent cell adhesion is regulated by a low affinity receptor pool that is conformationally responsive to ligand. J. Immunol. 270:28740.
1 pathways. Blood 86:2086.RI cross-linking-induced mast cell activation and adhesion to fibronectin. J. Physiol. Pharmacol. 45:501.[Medline]
5ß1-fibronectin interactions by divalent cations. J. Immunol. 270:26270.
IIbß3 (GPIIb-IIIa) by the cytoplasmic domain IIb. Science 254:845. subunit cytoplasmic domain regulates the assembly and adhesiveness of integrin lymphocyte function-associated antigen-1. J. Immunol. 159:268.[Abstract]
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 T. E. Secott, T. L. Lin, and C. C. Wu Mycobacterium avium subsp. paratuberculosis Fibronectin Attachment Protein Facilitates M-Cell Targeting and Invasion through a Fibronectin Bridge with Host Integrins Infect. Immun., July 1, 2004; 72(7): 3724 - 3732. [Abstract] [Full Text] [PDF]
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 V. Lam, J. Kalesnikoff, C. W. K. Lee, V. Hernandez-Hansen, B. S. Wilson, J. M. Oliver, and G. Krystal IgE alone stimulates mast cell adhesion to fibronectin via pathways similar to those used by IgE + antigen but distinct from those used by Steel factor Blood, August 15, 2003; 102(4): 1405 - 1413. [Abstract] [Full Text] [PDF]
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X. Zhu, B. Jacobs, E. Boetticher, S. Myou, A. Meliton, H. Sano, A. T. Lambertino, N. M. Munoz, and A. R. Leff IL-5-induced integrin adhesion of human eosinophils caused by ERK1/2-mediated activation of cPLA2 J. Leukoc. Biol., November 1, 2002; 72(5): 1046 - 1053. [Abstract] [Full Text] [PDF]
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A. Lorentz, D. Schuppan, A. Gebert, M. P. Manns, and S. C. Bischoff Regulatory effects of stem cell factor and interleukin-4 on adhesion of human mast cells to extracellular matrix proteins Blood, February 1, 2002; 99(3): 966 - 972. [Abstract] [Full Text] [PDF]
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M. L. Lupher Jr., E. A. S. Harris, C. R. Beals, L. Sui, R. C. Liddington, and D. E. Staunton Cellular Activation of Leukocyte Function-Associated Antigen-1 and Its Affinity Are Regulated at the I Domain Allosteric Site J. Immunol., August 1, 2001; 167(3): 1431 - 1439. [Abstract] [Full Text] [PDF]
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M. L. Woods and Y. Shimizu Signaling networks regulating {beta}1 integrin-mediated adhesion of T lymphocytes to extracellular matrix J. Leukoc. Biol., June 1, 2001; 69(6): 874 - 880. [Abstract] [Full Text] [PDF]
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 N. Prasad, R. S. Topping, and S. J. Decker SH2-Containing Inositol 5'-Phosphatase SHIP2 Associates with the p130Cas Adapter Protein and Regulates Cellular Adhesion and Spreading Mol. Cell. Biol., February 15, 2001; 21(4): 1416 - 1428. [Abstract] [Full Text] [PDF]
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R. Houtman, R. Ten Broeke, J. E. Blalock, M. Villain, A. S. Koster, and F. P. Nijkamp Attenuation of Very Late Antigen-5-Mediated Adhesion of Bone Marrow-Derived Mast Cells to Fibronectin by Peptides with Inverted Hydropathy to EF-Hands J. Immunol., January 15, 2001; 166(2): 861 - 867. [Abstract] [Full Text] [PDF]
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J. A. Epler, R. Liu, H. Chung, N. C. Ottoson, and Y. Shimizu Regulation of {beta}1 Integrin-Mediated Adhesion by T Cell Receptor Signaling Involves ZAP-70 but Differs from Signaling Events That Regulate Transcriptional Activity J. Immunol., November 1, 2000; 165(9): 4941 - 4949. [Abstract] [Full Text] [PDF]
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 J. K. Ivins, P. D. Yurchenco, and A. D. Lander Regulation of Neurite Outgrowth by Integrin Activation J. Neurosci., September 1, 2000; 20(17): 6551 - 6560. [Abstract] [Full Text] [PDF]
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K. Katagiri, M. Hattori, N. Minato, S.-k. Irie, K. Takatsu, and T. Kinashi Rap1 Is a Potent Activation Signal for Leukocyte Function-Associated Antigen 1 Distinct from Protein Kinase C and Phosphatidylinositol-3-OH Kinase Mol. Cell. Biol., March 15, 2000; 20(6): 1956 - 1969. [Abstract] [Full Text] [PDF]
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 J. M. Lu-Kuo, D. A. Fruman, D. M. Joyal, L. C. Cantley, and H. R. Katz Impaired Kit- but Not Fcepsilon RI-initiated Mast Cell Activation in the Absence of Phosphoinositide 3-Kinase p85alpha Gene Products J. Biol. Chem., February 25, 2000; 275(8): 6022 - 6029. [Abstract] [Full Text] [PDF]
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T. Kinashi, K. Katagiri, S.-i. Watanabe, B. Vanhaesebroeck, J. Downward, and K. Takatsu Distinct Mechanisms of alpha 5beta 1 Integrin Activation by Ha-Ras and R-Ras J. Biol. Chem., July 14, 2000; 275(29): 22590 - 22596. [Abstract] [Full Text] [PDF]
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