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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4700-4711
Functional Cooperation of Cyclin C and c-Myc in Mediating Homotypic
Cell Adhesion Via Very Late Antigen-4 Activation and Vascular Cell
Adhesion Molecule-1 Induction
By
Zhao-Jun Liu,
Yoshiya Tanaka,
Shinichiro Mine,
Akio Morinobu,
Hideo Yagita,
Ko Okumura,
Tadatsugu Taniguchi,
Hirohei Yamamura, and
Yasuhiro Minami
From the Department of Biochemistry and of Laboratory Medicine, Kobe
University, School of Medicine, Kobe, Japan; the First Department of
Internal Medicine, University of Occupational and Environmental Health,
School of Medicine, Kitakyushu, Japan; the Department of Immunology,
Juntendo University, School of Medicine, Tokyo, Japan; CREST (Core
Research for Evolutional Science and Technology) of Japan Science and
Technology Corporation (JST), Tokyo, Japan; and the Department of
Immunology, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
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ABSTRACT |
Very late antigen-4 (VLA-4)/vascular cell adhesion molecule-1
(VCAM-1) are a pair of adhesion molecules mediating cell-cell interaction. The binding activity of each depends on its surface expression, yet integrin activity can also be modulated through inside-out signaling. However, the specific intracellular molecules involved in modulating integrin VLA-4 activation via inside-out signaling or in regulating VCAM-1 expression are poorly understood. We
show here that constitutive coexpression of cyclin C and c-Myc in
hematopoietic BAF-B03 cells induces homotypic cell adhesion, which
results from enhanced VLA-4 ligand-binding activity and induced
expression of VCAM-1. Furthermore, regulation of cell adhesion appears
to be a feature unique to cyclin C, but not other G1 cyclins, E and D3,
and its regulatory function is independent of CDK8 kinase activity. Our
results provide a novel role for cyclin C and c-Myc in the regulation
of cell adhesion through distinct mechanisms.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CELL-CELL INTERACTION via very late
antigen-4 (VLA-4;  4 1) and its
counterreceptor vascular cell adhesion molecule-1 (VCAM-1) has been
implicated in numerous physiologic and pathologic processes.1 Although adhesion of VCAM-1 to its ligand
depends fundamentally on the molecule numbers expressed on the cell
surface, integrin-mediated cell adhesion can be regulated either
through altering the repertoire of integrin expression, by clustering on the cell surface, or by modulating the affinity of the integrins for
their ligands without changes in surface expression through an
inside-out signaling mechanism.2,3
The precise molecular mechanism of integrin activation modulated by
inside-out signaling remains largely unknown. It has been proposed that
conformational changes in integrins are required for their activation.
Studies using several unique monoclonal antibodies (MoAbs) demonstrate
that these MoAbs recognize integrins in their conformation in which the
expression of neoepitopes or ligand-induced binding sites are induced
on the integrin.4-6 The cytoplasmic domains of both and
subunits have been shown to be essential for the regulation of
inside-out signaling.7,8 For instance, a membrane-proximal
GFFKR sequence within the subunit may possess helical structure
and has been proposed to function as a component of a hinge that
connects the cytoplasmic and the transmembrane domains. Deletions in
GFFKR result in a default high-affinity integrin that may be caused by
locking the hinge in an irreversible high-affinity state. Several
intracellular signaling molecule(s), such as
calreticulin,9,10 ILK (Integrin-Linked Kinase),11 and cytohesin-1,12 have
been identified as associating with the cytoplasmic domain of
integrins. Such interactions may induce changes in the spatial
relationships or conformations of the cytoplasmic tails of the integrin
and subnuits and may participate in the regulation of both
inside-out and outside-in signalings.
The activities of both protein kinases and phosphatases have been
implicated in the inside-out signaling regulation.13-16 For instance, it has been shown that modulation of the ligand-binding affinity of 1 and 2 by phorbol ester
(PMA) stimulation depends on protein kinase C (PKC) activation, and the
cytoplasmic domain of the 6A subunit has been proven to
be a substrate for PKC.17 Participation of oncogenic
proteins in integrin activation has also been reported. Bcr/Abl
exhibits positive effects on the function of VLA-4 and VLA-5
integrins,18 whereas pp60v-src was suggested to
function as a negative regulator to reduce the binding capacity of
1 for its ligands via tyrosine
phosphorylation.19,20 Intriguingly, recent studies
demonstrate that members of the small GTPase family such as R-Ras and
H-Ras are involved in this regulation, albeit these two members exhibit
opposing effects.21,22 In addition, it has been shown that
cytokine stimuli as well as elevation of intracellular Ca2+
or cyclic AMP (cAMP) levels activate various integrin
molecules.14,23,24
VCAM-1 is an Ig-superfamily protein and is primarily distributed on
activated endothelium. Although it can also be expressed on many other
cells, its expression on hematopoietic cells has not been reported. The
ligand-binding activity of VCAM-1 is generally regulated at the level
of its expression. Cytokines such as tumor necrosis factor-
(TNF-), interleukin-1 (IL-1), IL-4, and lipopolysaccharide are
known to be able to induce VCAM-1 expression.25,26 Two cell-type-specific NF- B sites in the promoter region of VCAM-1 have
been shown to be responsible for cytokine-induced expression of
VCAM-1.27,28 In addition, two GATA elements28
and an element 3 to the TATA-box29 may also be
important for the regulation of VCAM-1 expression.
During our study aimed at understanding the function of cyclin C in the
regulation of cell cycle, we made an interesting observation that
constitutive coexpression of cyclin C and c-Myc in hematopoietic progenitor BAF-B03 cells, which normally exists as a suspension of
single cells, induces a homotypic cell adhesion. We show here that this
homotypic aggregation is mediated by the interaction between activated
4 integrin and inducibly expressed VCAM-1. Cyclin C,
which possesses an important function in the promotion of cell cycle
progression,30 also plays a critical role in the regulation
of cell adhesion in cooperation with c-Myc. Such a cell adhesive
regulatory function is unique to cyclin C and is not seen with cyclin E
and D3, the other two G1 cyclins. The involvement of both cyclin C and
c-Myc in either the activation of integrin or regulation of VCAM-1
expression has not previously been defined. Our results provide not
only a novel function for cyclin C and c-Myc, but also an interesting
notion that a set of intracellular molecules is capable of regulating
each partner of a ligand-receptor pair in a hematopoietic cell line
through distinct mechanisms.
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MATERIALS AND METHODS |
Antibodies.
MoAbs used in this study are as follows: PS/2
(anti-4),31 HM5-1
(anti-5),32 KBA
(anti-L),33 Mac-1
(anti-M),34 RMV-7
(anti-v),35 HM1-1
(anti-1),36 M18/2
(anti-2),34 HM3-1
(anti-3),37 M293
(anti-7),38 KAT-1 (anti-intercellular adhesion molecule-1 [ICAM-1]),39 and M/K-2
(anti-VCAM-1).40 Anti-Thy-1 MoAb was provided by Dr E. Shevach (NIH, Bethesda, MD). Fluorescein isothiocyanate
(FITC)-conjugated goat-antirat and goat-antihamster IgG
were purchased from Cappel Laboratories (Malvern, PA).
Cells and cell culture.
BAF-B03, a subclone of the Ba/F3 cell line, is a bone marrow-derived
murine IL-3-dependent pro-B-cell line.41 BER2 cells were
obtained by transfecting human epidermal growth factor (EGF) receptor
gene into BAF-B03 cells42; BC and BEC cells were
established by transfecting a human cyclin C expression plasmid
(Rc-cycC43; kindly provided by R.A. Weinberg, MIT,
Cambridge, MA) into BAF-B03 or BER2 cells, respectively; BM and BEM
cells were obtained by transfection of the human c-myc
expression plasmid, pN-LTR-myc44 into BAF-B03 or BER2
cells, respectively; and BMC and BEMC clones were established by
transfecting a human cyclin C expression plasmid into pooled BM
or BEM cells, respectively. BMC-K8AMG cells were obtained
by transfection of a catalytically inactive mutant of CDK8, in which
the Asp (D) residue in the DMG motif within kinase subdomain VII was
replaced by Ala (A). BMC, BMC-K8AMG, and
BMC-K8WT (for BMC-K8AMG and
BMC-K8WT, drug selection was performed in the presence of
IL-3) were maintained in RPMI 1640 (Nissui, Tokyo, Japan) medium
supplemented with 10% (vol/vol) fetal calf serum (FCS; JRH
Biosciences, Lexena, KS); other cells were cultured in the same medium
containing 10% (vol/vol) WEHI-3B culture supernatant as a source of
IL-3.
DNA transfection.
Plasmid DNAs were transfected into cells by an electroporation
procedure as described previously.45 Selection was
initiated 24 hours after DNA transfection, using 2 mg/mL G418 for BC
and BMC; 1 mg/mL hygromycin for BM, BEM, and BEC cells; or 0.75 µg/mL puromycin for BEMC, BEME, BEMD3, BMC-K8WT, and
BMC-K8AMG cells. Drug-resistant clones were either pooled
or subsequently cloned by limiting dilution, as described
previously.46
Flow cytometry.
Cell surface expression of adhesion molecules was analyzed by
immunofluorescence using MoAbs against the respective molecules as
described previously.47 For each sample, a total of 1 × 106 cells were treated with respective MoAb for 30 minutes at 4°C. After washing, cells were stained with
FITC-conjugated goat antirat or antihamster antibodies. The stained
cells were analyzed by a Coulter Epics XL-MCL Flow Cytometer (Coulter
K.K., Miami, FL).
Conjugation assay.
Formation of conjugates was measured using a two-color flow cytometer
essentially as described.48 Briefly, cells for conjugate formations assay were divided into two portions. The one portion was
labeled with sulfofluorescein diacetate to be fluorescent green and
another portion was labeled with hydroethidine to be fluorescent red;
these portions were mixed at a 4:1 ratio and allowed to settle for 1 hour at 4°C. The cells were then incubated for 6 minutes at
37°C, vortexed, and transferred into medium at 4°C. Conjugates
were enumerated as a percentage of red cells in the conjugates. Because
5,000 events were analyzed, the counting errors are less than 2% and
differences between samples of 2% to 3% conjugates are generally
reproducible. In all experiments, the background due to coincident
detection of red and green cells in a cell mix lacking conjugate (ie, a
green to red mix analyzed immediately after mixing in suspension) was
determined, and this background was subtracted from experimental
values, which normally range from 2% to 5%. In assays of MoAb
inhibition, saturating concentration of MoAb and human intact globulin
to block Fc-binding sites were added before setting and were present
continuously thereafter.
Antibody-blocking assay.
Aggregated BMC cells were mechanically separated. For each sample, 5 × 105 cells were replated in a 24-well plate (1 mL/well) along with various MoAbs at a saturating concentration of 10 µg/mL, which was shown in previous studies to produce a maximum
inhibition of the relevant adhesive interaction.49
Efficiency of MoAb on cell aggregation was evaluated by observation of
photomicrographs after 2 to 8 hours of incubation.
Cell adhesion assay.
Adhesion assay of BAF-B03, BM, BC, and BMC cells to fibronectin (Fn)
was performed essentially as previously described.50 Fn (5 µg/well; Seikagaku, Tokyo, Japan) or control 3% human serum albumin
(HSA; Green-Cross, Osaka, Japan) was applied to a 48-well plate in
phosphate-buffered saline (PBS) at 4°C overnight. Wells were
subsequently blocked with Ca2+/Mg2+-free
PBS/3% HSA for 2 hours at 37°C to reduce nonspecific attachment. The plates were washed three times with PBS before the addition of
BAF-B03 or different transfectants. BAF-B03 (2 × 105)
or transfectants were labeled with 51Cr (Dupont NEN,
Wilmington, DE) in RPMI 1640 with 1% HSA and were added to the wells
with or without blocking MoAb (10 µg/mL) in the presence or absence
of PMA (10 ng/mL; Sigma). After settling phase for 30 minutes at
4°C, which also allowed MoAb binding, the plates were rapidly
warmed to 37°C for 30 minutes, then gently washed twice with
RPMI-1640 at room temperature to completely remove nonadherent cells.
The contents of each well containing adherent cells were lysed with 250 µL of 1% Triton X-100 (Sigma), and the 51Cr
radioactivity was measured using a  -counter. Data were expressed as
the mean percentage of the binding of indicated cells from a
representative experiment.
Cell growth assay.
For the cell growth assay, factor-independent cells were cultured at a
density of 5 × 105 cells/mL in RPMI 1640 supplemented
with 10% FCS. Culture medium was changed every other day. Viable cell
numbers were determined by trypan blue exclusion assay as
described.51
| |
RESULTS |
Constitutive coexpression of cyclin C and c-Myc induces a homotypic
cell adhesion.
As an attempt to elucidate the role of cyclin C in the regulation of
cell cycle, we investigated whether cyclin C by itself or in
cooperation with cytokine-responsive immediately early gene products
such as c-Myc was able to promote cell cycle progression of
cytokine-dependent hematopoietic cells via substitution for growth
factor-induced proliferative signals and therefore to be able to render
these cells able to proliferate in a cytokine-independent manner. To
this end, a human cyclin C gene (Rc-cycC), either singly or
combined with a human c-myc expression plasmid (pN-LTR-myc), was transfected into BAF-B03 cell, an IL-3-dependent murine
hematopoietic progenitor cell line and the BAF-B03-derived BER2 cell
line42 (abbreviated here as BE cells), which expresses
human epidermal growth factor receptor (EGFR), respectively. It was
found that, although both BAF-B03 cells and BER2 cells ectopically
expressing cyclin C (termed BC and BEC cells) or c-Myc (named BM and
BEM cells) alone were unable to proliferate in the absence of growth factor, cells coexpressing cyclin C and c-Myc become able to
proliferate in a growth factor-independent manner
(Fig 1, termed BMC and BEMC cells).30 Interestingly, we also found that, like cyclin C, the other two G1 cyclins, cyclin E and cyclin D3, were also capable of
inducing cytokine-independence in BAF-B03-derived cells in cooperation
with c-Myc (Fig 1), despite the fact that expression of these cyclins
by themselves failed to do so. The transfectants that coexpress cyclin
E (Rc-cycE) with c-Myc or cyclin D3 (Rc-cycD3) with c-Myc in BER2 cells
were named BEME or BEMD3 (Liu et al, manuscript in
preparation), respectively. These results indicated that
cyclin C plays a critical role in the promotion of cell cycle progression and that cyclin C may share a similar function(s) with
cyclins E and D3 in the regulation of cell cycle progression.
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| Fig 1.
Proliferation profiles for BAF-B03-derived cells. For
growth assay, IL-3-dependent BAF-B03 and BM cells as well as
IL-3-independent BMC, BEMC, BEME, and BEMD3 cells (pooled
transfectants) were plated at 5 × 105 cells/mL in the
absence of IL-3 after washing with PBS. The concentration of viable
cells was counted at various times after plating and represented on a
logarithmic scale.
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We noted that both BAF-B03 and BER2 cells, which grow as isolated cells
in suspension (Fig 2A and B),
form cell aggregates when cyclin C and c-Myc were ectopically
coexpressed (Fig 2E and F). Cells expressing cyclin C (BC cells; Fig
2C) or c-Myc (BM cells, Fig 2D) alone did not form such aggregates (BEC
and BEM, data not shown), indicating that cooperation between cyclin C and c-Myc is required for homotypic cell adhesion. Interestingly, unlike BMC and BEMC cells, neither BEME (Fig 2G) nor BEMD3 cells (Fig
2H) formed aggregates, suggesting that, among the G1 cyclins examined,
cyclin C is required selectively for this observed cell adhesion.
Consistent with the above-noted results, F-actin polymerization was
apparent only in BMC and BEMC cells but not in BAF-B03, BER2, BEME,
BEMD3, BC, BEC, BM, or BEM cells (data not shown), even in the presence
of IL-3. Furthermore, it was found that homotypic cell adhesion of BMC
cells requires divalent cations, because cell aggregation was blocked
by the addition of EDTA (data not shown). Taken together, these results
suggested that the cyclin C- and c-Myc-induced homotypic cell
adhesion may be mediated by cell surface adhesion molecules, such as
members of the integrin family.
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| Fig 2.
Morphological properties of BAF-B03-derived cells.
BAF-B03 (A), BER2 (B), BC (C), and BM (D) cells were cultured in the
presence of IL-3, whereas BMC (E), BEMC (F) , BEME (G), and BEMD3 (H)
cells were cultured in the absence of IL-3. The addition of IL-3 does
not alter the cell cluster-forming properties of BMC, BEMC, BEME, and
BEMD3 cells.
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The homotypic cell adhesion observed in both BMC and BEMC cells could
occur even in the absence of IL-3, and restimulation with IL-3 did not
alter their adhesive properties. To determine whether BMC cell adhesion
results from stimulation by soluble factors that may be secreted as a
result of ectopic coexpression of cyclin C and c-Myc, we harvested the
supernants from growing BMC and BEMC cells; used these to culture
either parental BAF-B03, BM, BC, BEME, or BEMD3 cells; and found that
these cells do not form clusters, indicating that homotypic cell
adhesion is not due to an indirect cytokine stimulation.
It should be noted that the exogenous human EGFR responds only to human
EGF rather than any other factor that existed in the medium. In fact,
ectopically expressed EGFR was not phosphorylated under our culture
conditions, and BMC and BEMC exhibited almost similar degrees of both
cytokine-independent growth and homotypic cell adhesion (data not
shown). Thus, the difference between BMC or BEMC and BEME or BEMD3
cells primarily reflects a unique function of cyclin C; therefore, we
used BMC cells in subsequent experiments.
Function of cyclin C in regulating cell adhesion is independent of
CDK8 kinase activity.
Because regulation of cell adhesion appears to be a unique
characteristic of cyclin C and a recent work has demonstrated that cyclin C can associate with a novel cyclin-dependent kinase,
CDK8,52 it is important to address whether the role of
cyclin C in the regulation of cell adhesion is dependent on CDK8. To
this end, we transfected a catalytically inactive mutant of CDK8
(kindly provided by Dr E. Nigg, Swiss Inst. Exp. Cancer Research,
ISREL, Switzerland), in which an Asp (D) residue in the DMG motif
within the kinase subdomain VII was mutated to Ala (A), into BMC cells and the resultant clones were designated BMC-k8AMG. It is
noteworthy that this mutant form of CDK8 is still able to form a
complex with cyclin C in vitro.30 As a control, wild-type CDK8 was also used in our transfection experiments and the resultant cells were termed BMC-k8WT. To avoid the possibility that
expression of a catalytically inactive mutant of CDK8 may affect the
role of cyclin C in cell proliferation and may induce cell death in BMC
cells, drug selection was performed in the presence of IL-3. For each
kind of transfectants, at least three independent clones were obtained
and the results from a representative clone are presented. As shown in
Fig 3 (inset panels), expression levels of
exogenous CDK8 were about sixfold higher than those of endogenous CDK8.
It was found that BMC cell aggregation was not affected by ectopic
expression of either the catalytically inactive mutant or wild-type
CDK8 (Fig 3), suggesting that the function of cyclin C in the
regulation of cell adhesion is independent of CDK8 kinase activity.
Furthermore, the activation and expression of the adhesion molecules,
which are involved in homotypic adhesion of BMC cells (see below), were
not altered by ectopic expression of either the catalytically inactive
mutant or wild-type CDK8 in BMC cells, confirming that CDK8 kinase
activity is not required for the function of cyclin C in the regulation of cell adhesion.
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| Fig 3.
Effect of enforced expression of a catalytically inactive
mutant of CDK8 on the homotypic cell adhesion of BMC cells.
Morphological properties of BMC-mock, BMC-k8AMG, and
BMC-k8WT are shown. Expression of CDK8 was detected by
anti-CDK8 antibody (inset panels). Upper and lower bands indicate
Myc-tagged exogenous CDK8 and endogenous CDK8.
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Expression of VCAM-1 is induced in BMC cells.
To investigate whether BMC cell adhesion is induced through increasing
the cell surface expression of adhesion molecules such as integrins or
Ig-superfamily proteins, flow cytometric analysis was performed using
MoAbs against integrin 4, 5,
L, M, v, 1,
2, 3, 7 chain, VCAM-1, and
ICAM-1. We found that the integrin 4
(Fig 4A), 5, and
1 chains as well as ICAM-1 (Fig 4B) are expressed on BMC
cells, but at levels comparable with those on the parental BAF-B03
cells. The L, M, v,
2, 3, and 7 chains were
not detectable on either BAF-B03 or BMC cells (Fig 4B). Noticeably, VCAM-1 is not expressed on parental BAF-B03 cells but is expressed substantially on the surface of BMC cells (Fig 4A). Induced VCAM-1 expression was not detectable on either BC or BM cells (data not shown). Furthermore, expression of VCAM-1 was not detectable on the
surface of BEME and BEMD3 cells, indicating that, among the G1 cyclins
examined, cyclin C is unique for the induced expression of VCAM-1 in
conjunction with c-Myc (Fig 4A). Some other integrin subunits, such as
1 and 2, which are not generally involved in homotypic cell adhesion, were also examined and were undetectable on
either BAF-B03 cells or transfectants (data not shown). It should be
noted that expression of 4 integrin, the counterreceptor for VCAM-1, is not affected by ectopic coexpression of cyclin C and
c-Myc (Fig 4A). Taken together, these results suggested that the
cooperative effect of cyclin C and c-Myc on homotypic cell adhesion of
BMC cells can be at least partly mediated by the induced expression of
VCAM-1.
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| Fig 4.
(A) Flow cytometric analysis of 4 integrin
and VCAM-1 expression in BAF-B03 cells and transfectants. The various
cells were unstained (dotted line) or stained with
anti-4 integrin and anti-VCAM-1 MoAbs (solid line),
followed by staining with FITC-labeled secondary antibodies and a flow
cytometric analysis, as described in Materials and Methods. (B) Flow
cytometric analysis of expression of various integrin subunits and
ICAM-1 on BAF-B03 cells ( ) and BMC cells ( ). The mean
fluorescence intensity values are shown.
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VLA-4 and VCAM-1 are involved in homotypic aggregation of BMC cells.
To characterize further the nature of BMC cell aggregation, we used
several function-blocking MoAbs to identify the candidate adhesion
molecules involved in mediating homotypic aggregation of BMC cells. It
was found that homotypic aggregation of BMC cells was almost completely
inhibited by anti-4 MoAb (PS/2), whereas anti-L (KBA), anti-ICAM-1(KAT-1),
anti-v (RMV-7), or a control nonblocking
anti-2 (M18) MoAb failed to affect homotypic cell adhesion of BMC cells (Fig 5). Thus, the
results suggested that 4 integrin is involved in
homotypic cell adhesion of BMC cells and may be activated in these
cells. On the other hand, anti-VCAM-1 MoAb, M/K-2, which has been
demonstrated to inhibit VCAM-1-mediated cell adhesion,53
exhibited an incompletely blocking effect on homotypic adhesion of BMC
cells. This partial blocking effect of anti-VCAM-1 MoAb (M/K-2) might
be due to the fact that this MoAb can bind to only one of the
4 integrin binding sites of VCAM-1, the D1 domain, but
not to the other 4 integrin binding site, D4
domain.54 The D4 domain, which remains unoccupied by M/K-2,
may still be able to partially mediate the association with 4
integrin. Alternatively, the homotypic aggregation of BMC cells may be
partly mediated by the bridge of serum Fn in the medium.
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| Fig 5.
(a) Antibody-blocking analysis of homotypic cell adhesion
of BMC cells. Morphological properties of BMC after treatment with a
control nonblocking anti-2 MoAb (A),
anti-L (B), anti-v (C),
anti-4 (D), as well as anti-ICAM-1 (E) and
anti-VCAM-1 MoAb (F) are shown. (b) Effects of anti-4
integrin and anti-VCAM-1 MoAbs on early phase of homotypic aggregation
of BMC cells. The cells are pretreated by 10 µg/mL of human Ig,
divided into effector cells (labeled with hydroethidine) and target
cells (labeled with sulfofluorescein diacetate) at a 4:1 ratio, added
to 1 µg/106 cells of indicated MoAbs, and enumerated
using a flow cytometric analysis. Data are reported as the percentage
of total targets presented as conjugates. The conjugated percentage is
shown in the top, right-hand corner of each panel.
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We also tested the abilities of anti-4 integrin and
anti-VCAM-1 MoAbs to inhibit homotypic aggregation of BMC cells at the very early phase of the conjugate formation. Homotypic conjugates were
observed on BMC cells but not parental BAF-B03 cells within only 6 minutes after the culture at 37°C. As shown in Fig 5b, inhibition
of the BMC cell conjugates was consistently observed with either
antibodies against 4 integrin (PS/2) or VCAM-1 (M/K-2). Combination of these two MoAbs did not further increase their blocking
efficiency. Taken together, these data indicated that 4
integrin and VCAM-1 are involved in mediating homotypic aggregation of
BMC cells.
The 4 subunit is capable of associating with either 1
(41, also termed VLA-4) or an alternative
subunit, 7.55 Because 7
subunit is not detectable on both BAF-B03 and BMC cells, it is
indicated that 4 associates with 1 to
form heterodimer (VLA-4) in these cells. Noticeably, an
anti-1 MoAb, Hm1, which has been demonstrated to be able to block the binding of
41 to Fn,36 failed to inhibit
homotypic aggregation of BMC cells in our hand (up to 50 µg/mL; data
not shown). Perhaps this is simply due to the fact that
Hm1 does not recogize the epitope on the
1, which is required for interaction with VCAM-1,
because it was known that the site(s) involved in the ligation of
VLA-4/VCAM-1 and VLA-4/Fn is different.56 To test whether
1 is indeed involved in mediating cell aggregation of
BMC cells, we used another antimouse 1 MoAb,
9EG7,57 which can recognize either activated or
ligand-binding related epitopes of integrin 1, to
perform a flow cytometric analysis. The analysis showed that expression
of activated or ligand-binding related epitopes of integrin
1 is induced on BMC at a higher level compared with
BAF-B03 cells (data not shown). Hence, VLA-4 is primarily responsible
for the homotypic aggregation of BMC cells.
VLA-4 is activated on BMC cells.
VLA-4 is known to be able to bind to either the cellular
counter-receptor VCAM-1 or extracellular matrix Fn. Because the
activities of the integrins can be modulated via inside-out signaling,
it is of importance to determine whether VLA-4 expressed on BMC cells is indeed activated as a result of the functional cooperation between
cyclin C and c-Myc. For this purpose, we have examined the abilities of
BAF-B03, BMC, and other BAF-B03-derived transfectants to attach to Fn
coated on the plates. It was found that BMC cells bind efficiently to
Fn-coated plates, but not to control HSA-coated plates
(Fig 6A). About a fourfold increase in
specific attachment to Fn was observed for BMC cells over that seen in
BAF-B03, BM, BC, BEME, and BEMD3 cells (Fig 6A and data not shown).
Furthermore, adhesion of BMC cells to Fn was selectively inhibited by
anti-4 MoAb, PS/2, but not by an irrelevant anti-Thy-1
MoAb. However, inhibition by PS/2 was incomplete and its blocking
efficiency is approximately 50%. Probably another integrin(s), such as
5 1, may also be activated on BMC cells. Phorbol ester
PMA is able to activate the integrins through a PKC-mediated inside-out
signaling mechanism.58 Treatment with PMA drastically
increased attachment of BAF-B03, BM, and BC as well as BEME and BEMD3
cells to Fn, whereas it had a marginal effect on BMC cells. Increased
adhesion of BAF-B03, BM, and BC as well as on BEME and BEMD3 cells to
Fn after PMA treatment was also inhibited by anti-4
MoAb, PS/2 (Fig 6B). Collectively, these results suggest that the VLA-4
expressed on the surface of BMC cells has already been activated by the cooperative effect of cyclin C and c-Myc before PMA stimulation.
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| Fig 6.
(A) Adhesion of BAF-B03 cells and transfectants to
Fn-coated or HSA-coated plates. Mean percentage of indicated cells
binding to Fn () or HSA () is shown. (B) Effect of
anti-4 blocking MoAb (PS/2) and PMA treatment on the
adhesion of BAF-B03 cells and transfectants to Fn. The cells indicated
were untreated or treated with anti-4 integrin MoAb in
the presence or absence of PMA. The mean percentage of binding is
shown.
|
|
| |
DISCUSSION |
A salient feature of the function of certain integrins is that their
activities can be modulated through intracellular signaling without
changing levels of expression. In contrast, the function of VCAM-1
depends primarily on its surface expression. It was originally supposed
that these two processes are regulated by distinct mechanisms in
different cells, because the cellular and tissue distribution of VLA-4
and VCAM-1 is quite different and VLA-4/VCAM-1 generally mediates
heterotypic cell-cell interactions, such as those between leukocytes
and endothelial cells. Also, it remains unclear whether the
VLA-4/VCAM-1 pair can be regulated by the same intracellular
molecule(s). In this study, we investigated the cooperative effect of
cyclin C and c-Myc on hematopoietic cellular behavior and found that
interaction of VLA-4 and VCAM-1 occurred on the same cells with VCAM-1
being induced, whereas VLA-4 is activated without alteration of its
expression. Our studies provide the first example in which these two
partners are regulated by a set of intracellular molecules but through
distinct mechanisms in a given hematopoietic cell line.
Concomitant activation of VLA-4 and VCAM-1 in BMC cells appears not to
be a result of mutual stimulation between VLA-4 and VCAM-1. Activation
of VLA-4 by PMA in BAF-B03, BM, or BC cells does not induce homotypic
aggregation of these cells (data not shown), suggesting that VCAM-1 is
not simultanously induced. Thus, activation of VLA-4 by itself is not
likely to be responsible for the induction of VCAM-1 expression.
Likewise, VCAM-1 expression seems not to be responsible for the
activation of VLA-4. The observation that coculture of BAF-B03, BM, or
BC cells with COS cells expressing VCAM-1 does not result in apparent
heterotypic cell-cell interactions (data not shown) supports this
possibility. Further study will be required to confirm such a
possibility.
Regulation of VLA-4/VCAM-1-mediated homotypic aggregation seems to be
a unique character of cyclin C, because neither cyclin E nor cyclin D3
can induce cell adhesion or enhance cell attachment to Fn in
cooperation with c-Myc, albeit the respective G1 cyclins in cooperation
with c-Myc are sufficient to render BAF-B03 cells able to proliferate
in a cytokine-independent fashion. Cyclin C was originally isolated
through its ability to complement a Saccharomyces cerevisiae
strain lacking the G1 cyclin CLN1-3.59-61 Our very
recent study demonstrated a critical role for cyclin C in the
regulation of cell cycle transition by cooperation with c-Myc.30 Moreover, it has recently been reported that
cyclin C is capable of associating with a unique cyclin-dependent
protein kinase, CDK8, and that the cyclin C/CDK8 complex may regulate transcription via phoshorylation of RNA polymerase II.62
Thus, it appears that cyclin C may play different roles in the
regulation of cell cycle progression, transcription, and cell adhesion.
However, the relationship between cyclin C-regulated cell cycle
progression, transcription, and cell adhesion is not yet well
understood. First, it remains unclear whether homotypic cell adhesion
induced by cyclin C and c-Myc is a prerequisite for cell cycle
progression mediated by these molecules. It seems that
cytokine-independent BMC cell proliferation driven by the cooperative
effect of cyclin C and c-Myc is not tightly dependent on homotypic cell
aggregation, because BMC cells exhibit drastically decreased cell
adhesion properties after treatment with anti-4 integrin
blocking MoAb, PS/2, but do not undergo apoptosis and can still
proliferate well in the absence of cytokine (data not shown). In
addition, PS/2 MoAb itself failed to inhibit apoptosis or promote
proliferation of BAF-B03 cells (date not shown). These data suggest
that cyclin C may function directly to promote cell cycle progression
rather than via regulation of cell adhesion, which can supply survival signals to cooperate with c-Myc. This is likely because BAF-B03 cells
are not anchorage-dependent and both BEME and BEMD3 cells can
proliferate without cell adhesion. Thus, these observations suggest
that cytokine-independent BMC cell proliferation is at least partly
independent of cell-cell interaction that may provide adhesion-mediated
proliferative signals. Second, kinase activity of CDK8, which has been
implicated in transcription control, is not required for the cyclin C
and c-Myc-mediated cell proliferation and cell adhesion. The
observation that amounts of CDK8 remain constant throughout the cell
cycle62 and that CDK8 kinase activity was not upregulated
in BMC cells30 suggest that the function of exogenous
cyclin C in promoting cell proliferation is not mediated by CDK8.
Furthermore, it was found that enforced expression of a catalytically
inactive mutant of CDK8 fails to reverse or alleviate homotypic
aggregation of BMC cells or their attachment to Fn, indicating that
CDK8 kinase activity is not required for cyclin C-dependent
VLA-4/VCAM-1 activation. Collectively, these data implicate that cyclin
C is a multifunctional molecule.
On the other hand, c-Myc is also required for the regulation of both
VLA-4 activation and VCAM-1 expression. Involvement of c-Myc in the
regulation of both integrin and VCAM-1 is poorly understood, except for
the finding that c-Myc expression can downregulate LFA-1.63
Our observation of the participation of c-Myc in the activation of
VLA-4 integrin and induction of VCAM-1 is the first example of a novel
role for c-Myc in regulating cell adhesion. However, how c-Myc
cooperates with cyclin C is unclear. At least ectopic expression of
c-Myc by itself does not upregulate cyclin C expression and vice versa
(data not shown). It is unlikely that c-Myc can directly regulate
VCAM-1 expression through its activity as a transcription factor,
because c-Myc binding sequences have not been reported within the
promoter region of VCAM-1. One possible mechanism for the induction of
VCAM-1 is that c-Myc and cyclin C may induce the expression of
cytokine(s), such as TNF-, IL-1, and IL-4, that are known to be
able to induce VCAM-1 expression.25,26,64 However, our
finding that the culture supernatants from growing BMC cells fail to
induce cell cluster formation in PMA-stimulated BAF-B03, BM, or BC
cells, on which VLA-4 is activated, suggests that VCAM-1 expression is
not induced on these cells under these conditions (data not shown).
Likewise, supernatants from growing BMC cells do not enhance the
attachment of BAF-B03, BM, or BC cells to Fn (data not shown),
excluding the possibility that activation of VLA-4 is mediated by such
indirect cytokine stimulation, which is known to be able to activate
certain integrins.14 It will be of interest to examine
whether the signals of those cytokines in regulating cell adhesion are
transmitted through activation of cyclin C and c-Myc. Because both
cyclin C and c-myc can be induced in hematopoietic
cells by IL-3 stimulation,30,42 our findings may represent
a novel mechanism responsible for cytokine-induced hematopoietic cell
adhesiveness14 in which activation of VLA-4 is involved.
Interestingly, dose-dependent and transient cell adhesiveness of
hematopoietic cells, including BAF-B03 cells, is induced upon cytokine
stimulation of factor-deprived cells, whereas activation of VLA-4 and
induction of VCAM-1 observed in our experimental system are somehow
constitutive. This may simply reflect a difference in the magnitude and
duration of expression of these genes, because induction of these genes
by cytokine stimulation is normally of a transient nature, whereas
cyclin C and c-Myc are constitutively overexpressed in BMC cells.
Adhesion-induced outside-in signals, in particular in certain
anchorage-dependent cells, have been known to integrate into the cell
cycle machinery.65 Conversely, little is known about whether and how the cell cycle machinery is involved in the regulation of cell adhesion. It has been assumed that the intrinsic cell cycle
machinery may play an important role(s) in the regulation of cell
adhesion. For example, many types of cells become rounded and appear to
lose adhesion during mitosis. In addition, it was found that ectopic
expression of cyclin A in anchorage-dependent NRK fibroblasts renders
cells able to proliferate in suspension,66 suggesting that
cyclin A is involved in the control of cell adhesion. Our findings
provide direct evidence of a critical role for cyclin C in the
regulation of cell adhesion in cooperation with c-Myc. However, the
exact mechanism of the cooperative effect of cyclin C and c-Myc on
integrin activation remains unclear. Because neither c-Myc nor cyclin C
possess any enzymatic activity, it is likely that any modulation of the
cytoplasmic tails of integrins is brought about through an indirect
regulatory mechanism. The cooperative effect of cyclin C and c-Myc on
integrin activation may represent a novel signaling pathway, yet it
will also be of interest to examine whether the function of cyclin C
and c-Myc is mediated via regulation of several previously identified
signaling pathways, such as R-Ras, PKC, protein tyrosine kinases, and
intracellular Ca2+ or cAMP, etc. Alternatively, it may be
also interesting to test whether cyclin C and c-Myc are targets of
those signaling pathways. Treatment of BMC cells with either wortmannin
(100 ng/mL) or rapamycin (10 ng/mL) exhibits no apparent effect on
homotypic aggregation of BMC cells (data not shown), suggesting that
the cooperative effect of cyclin C and c-Myc on VLA-4 activation may
not be mediated by activation of phosphoinositide 3-OH kinase or the S6
kinase pathway. Further study will be required to elucidate the precise mechanism by which cyclin C and c-Myc cooperatively activate intergin and induce VCAM-1 expression.
We demonstrate here that the enhancement of BMC cell binding to VCAM-1
or Fn induced by the cooperative effects of cyclin C and c-Myc is a
result of functional modulation of VLA-4 already present on the cell
surface, because increased VCAM-1 ligation and Fn attachment occurred
without alteration in the surface expression of this integrin. Hence,
we conclude that functional cooperation of cyclin C and c-Myc is able
to activate integrin, at least partly, by increasing integrin
ligand-binding affinity via an inside-out signaling. However, our
results do not exclude possible avidity changes that might result from
integrin clustering, a postligand binding event that can also enhance
ligand-binding ability. It should also be noted that we have examined
only a limited selection of adhesion molecules in this study. It will
be of interest to investigate whether cadherins, or other members of
the integrin family, such as VLA-5, are also modulated. In addition,
our results indicate that the VLA-4/VCAM-1 pair is primarily
responsible for mediating homotypic aggregation of BMC cells; however,
we do not exclude the possibility that BMC cell aggregation can be
partly mediated by a VLA-4-VLA-4 interaction. In fact, it has been
reported that the 4 integrin is a ligand for
41 and
47.67 It is also possible
that serum Fn may also contribute to this cell adhesion through
VLA-4-Fn-VLA-4 interactions.
The physiological and pathological significance of regulating
functional properties of VLA-4 and VCAM-1 on hematopoietic cells remains to be determined. Thus far, the only example of a VLA-4/VCAM-1 interaction occuring between the same type of cells is an observation made by Rosen et al,68 in which they found that VCAM-1 and
VLA-4 were expressed concomitantly on myoblasts. This VLA-4/VCAM-1
interaction has been suggested to be important for myogenesis.
Activation of a VLA-4/VCAM-1 pair on the hematopoietic progenitors may
be of importance for the regulation of hematopoiesis. Adhesive
interactions of VLA-4 with VCAM-1 on stromal cells or with
extracellular matrix retain hematopoietic progenitor cells in close
proximity to the components of bone marrow microenvironment that is
essential for the regulation of normal hematopoiesis. The importance of
VLA-4 in hematopoiesis has been demonstrated by Miyake et
al,31 who reported that the addition of anti-VLA-4 MoAb to
long-term born marrow cultures abrogated lymphopoiesis and retarded
myelopoiesis. VLA-4-specific antibodies have also been shown to
abrogate stroma-dependent erythropoiesis.32 VCAM-1 may also
help to promote lymphopoiesis and myelopoiesis.53,69,70 In
addition, VLA-4/VCAM-1-mediated cell adhesion has been suggested to be
involved in the migration of leukocytes71,72 and
circulating malignant cells,73,74 which is a critical step
in the process of inflammation and metastasis.
In summary, we show in this study that (1) cooperation of cyclin C and
c-Myc induces homotypic aggregation of a hematopoietic progenitor,
BAF-B03 cells, via activation of VLA-4 and induction of VCAM-1; (2)
regulation of cell adhesion represents a novel function for cyclin C,
and CDK8 kinase activity is not required for this function; (3)
activation of VLA-4 by cyclin C and c-Myc is at least partly due to an
increase in its ligand-binding activity through inside-out signaling;
and (4) VCAM-1, which is not normally expressed on hematopoietic cells,
can be induced on the BAF-B03 cells. These results clearly demonstrate
a novel role for cyclin C and c-Myc in the regulation of cell adhesion.
The elucidation of cyclin C and c-Myc-mediated signal transduction
pathways governing cell adhesion will enable us to gain further
important insights into mechanisms regulating multiple biological
processes in which the VLA-4/VCAM-1 pair plays essential roles.
| |
ACKNOWLEDGMENT |
The authors are grateful to Dr R.A. Weinberg for human cyclin C, E, and
D3 expression plasmids (Rc-cycC, Rc-cycE, and Rc-cycD3) and to Dr E.A.
Nigg for pCMV-cdk8WT and pCMV-cdk8AMG plasmids.
We also thank Dr A. Kukula for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted April 30, 1998;
accepted August 7, 1998.
Supported by a Grant-in-Aid for Scientific Research on Priority Areas
provided by the Ministry of Education, Science, Sports and Culture,
Japan; by Nippon Boehringer Ingelheim Co, Ltd, Kawanishi Pharma
Research Institute; by the Kato Memorial Bioscience Foundation; by The
Naito Foundation; and by the Kanae Foundation For Life & Socio-Medical
Science. Z.-J.L. was supported by a Grant-in-Aid for Japan Society for
the Promotion of Science Fellows.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Yasuhiro Minami, MD,
Department of Biochemistry, Kobe University, School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan; e-mail:
minami{at}kobe-u.ac.jp.
| |
REFERENCES |
1.
Springer TA:
Adhesion receptors of the immune system.
Nature
346:425, 1990[Medline]
[Order article via Infotrieve]
2.
Ginsberg MH, Du X, Plow EF:
Inside-out integrin signalling.
Curr Opin Cell Biol
4:766, 1992[Medline]
[Order article via Infotrieve]
3.
Hynes RO:
Integrins: Versatility, modulation, and signalling in cell adhesion.
Cell
69:11, 1992[Medline]
[Order article via Infotrieve]
4.
Van Kooyk Y, Weder P, Hogervorst F, Verhoeven AJ, Van Seventer G, Te Velde AA, Borst J, Keizer GD, Figdor CG:
Activation of LFA-1 through a Ca2(+)-dependent epitope stimulates lymphocyte adhesion.
J Cell Biol
112:345, 1991[Abstract/Free Full Text]
5.
Dransfield I, Cabanas C, Craig A, Hogg N:
Divalent cation regulation of the function of the leukocyte integrin LFA-1.
J Cell Biol
116:219, 1992[Abstract/Free Full Text]
6.
Dransfield I, Cabanas C, Barrett J, Hogg N:
Interaction of leukocyte integrins with ligand is necessary but not sufficient for function.
J Cell Biol
116:1527, 1992[Abstract/Free Full Text]
7.
O'Toole TE, Katagiri Y, Faull RJ, Peter K, Tamura R, Quaranta V, Loftus JC, Shattil SJ, Ginsberg MH:
Integrin cytoplasmic domains mediate inside-out signal transduction.
J Cell Biol
124:1047, 1994[Abstract/Free Full Text]
8.
O'Toole TE, Ylanne J, Culley BM:
Regulation of integrin affinity states through an NPXY motif in the subunit cytoplasmic domain.
J Biol Chem
270:8553, 1995[Abstract/Free Full Text]
9.
Rojiani M, Finlay BB, Gray V, Dedhar S:
In vitro interaction of a polypeptide homologous to human Ro/SSA antigen (calreticulin) with a highly conserved amino acid sequence in the cytoplasmic domain of integrin subunits.
Biochemistry
30:9859, 1991[Medline]
[Order article via Infotrieve]
10.
Coppolino M, Leung-Hagesteijn C, Dedhar S, Wilkins S:
Inducible interaction of integrin 21 with calreticulin: Dependence on the activation-state of the integrin.
J Biol Chem
270:23132, 1995[Abstract/Free Full Text]
11.
Hannigan GE, Leung-Hagesteijn C, Fitz-Gibbon L, Coppolino MG, Radeva G, Filmus J, Bell JC, Dedhar S:
Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase.
Nature
379:91, 1996[Medline]
[Order article via Infotrieve]
12.
Kolanus W, Nagel W, Schiller B, Zeitlmann L, Stockinger S, Seed B:
Alpha L beta 2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule.
Cell
86:233, 1996[Medline]
[Order article via Infotrieve]
13.
Dustin ML, Springer TA:
T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1.
Nature
341:619, 1989[Medline]
[Order article via Infotrieve]
14.
Levesque J-P, Leavesley DI, Niutta S, Vadas M, Simmons PJ:
Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA-4 and VLA-5 integrins).
J Exp Med
181:1805, 1995[Abstract/Free Full Text]
15.
Valmu L, Gahmberg CG:
Treatment with okadaic acid reveals strong threonine phosphorylation of CD18 after activation of CD11/CD18 leukocyte integrin with phorbol esters or CD3 antibodies.
J Immunol
155:1175, 1995[Abstract]
16.
Dedhar S, Hannigan GE:
Integrin cytoplasmic interactions and bidirectional transmembrane signalling.
Curr Opin Cell Biol
8:657, 1996[Medline]
[Order article via Infotrieve]
17.
Gimond C, De Melker A, Aumailley M, Sonnenberg A:
The cytoplasmic domain of 6A integrin subunit is an in vitro substrate for protein kinase C.
Exp Cell Res
216:232, 1995[Medline]
[Order article via Infotrieve]
18.
Bazzoni G, Carlesso N, Griffin JD, Hemler ME:
Bcr/Abl expression stimulates integrin function in hematopoietic cell lines.
J Clin Invest
98:521, 1996[Medline]
[Order article via Infotrieve]
19.
Tapley P, Horwitz A, Buck C, Burridge K, Duggan K, Rohrschneider L:
Analysis of the avian fibronectin receptor (integrin) as a direct substrate for pp60v-src.
Oncogene
4:325, 1989[Medline]
[Order article via Infotrieve]
20.
Horvath AR, Elmore MA, Kellie S:
Differential tyrosine-specific phosphorylation of integrin in Rous sarcoma virus transformed cells with differing transformed phenotypes.
Oncogene
5:1349, 1990[Medline]
[Order article via Infotrieve]
21.
Zhang Z, Vuori K, Wang H-G, Reed JC, Ruoslahti E:
Integrin activation by R-ras.
Cell
85:61, 1996[Medline]
[Order article via Infotrieve]
22.
Hughes PE, Renshaw MW, Pfaff M, Forsyth J, Keivens VM, Schwartz MA, Ginsberg MH:
Suppression of integrin activation: A novel function of a Ras/Raf-initiated MAP kinase pathway.
Cell
88:521, 1997[Medline]
[Order article via Infotrieve]
23.
Haverstick DM, Gray LS:
Lymphocyte adhesion mediated by lymphocyte function-associated antigen-1. I. Long term augmentation by transient increases in intracellular cAMP.
J Immunol
149:389, 1992[Abstract]
24.
Haverstick DM, Gray LS:
Lymphocyte adhesion mediated by lymphocyte function-associated antigen-1. II. Interaction between phorbol ester- and cAMP-sensitive pathways.
J Immunol
149:397, 1992[Abstract]
25.
Osborn L, Hession C, Tizard R, Vassallo C, Luhowskyj S, Chi-Rosso G, Lobb R:
Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes.
Cell
59:1203, 1989[Medline]
[Order article via Infotrieve]
26.
Rice GE, Bevilacqua MP:
An inducable endothelial cell surface glycoprotein mediates melanoma adhesion.
Science
246:1303, 1989[Abstract/Free Full Text]
27.
Iademarco MF, McQuillan JJ, Resen GD, Dean DC:
Characterization of the promoter for vascular cell adhesion molecule-1 (VCAM-1).
J Biol Chem
267:16323, 1992[Abstract/Free Full Text]
28.
Neish AS, Willians AJ, Palmer HJ, Whitley MZ, Collins T:
Functional analysis of the human vascular cell adhesion molecule-1 promoter.
J Exp Med
176:1583, 1992[Abstract/Free Full Text]
29.
Iademarco MF, McQuillan JJ, Dean DC:
Vascular cell adhesion molecule-1: Contrasting transcriptional control mechanisms in muscle and endothelium.
Proc Natl Acad Sci USA
90:3943, 1993[Abstract/Free Full Text]
30.
Liu Z-J, Ueda T, Miyazaki T, Tanaka N, Mine S, Tanaka Y, Taniguchi T, Yamamura H, Minami Y:
A critical role for cyclin C in promotion of the hematopoietic cell cycle by cooperation with c-Myc.
Mol Cell Biol
18:3445, 1998[Abstract/Free Full Text]
31.
Miyake K, Weissman IL, Greenberger JS, Kincade PW:
Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis.
J Exp Med
173:599, 1991[Abstract/Free Full Text]
32.
Yanai N, Sekine C, Yagita H, Obinata M:
Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis.
Blood
83:2844, 1994[Abstract/Free Full Text]
33.
Nishimura T, Yagi H, Yagita H, Uchiyama Y, Hashimoto Y:
Lymphokine-activated cell-associated antigen involved in broad-reactive killer cell-mediated cytotoxicity.
Cell Immunol
94:122, 1985[Medline]
[Order article via Infotrieve]
34.
Sanchez-Madrid F, Simon P, Thompson S, Springer TA:
Mapping of antigenic and functional epitopes on the - and -subnuits of two related mouse glycoproteins involved in cell interations, LFA-1 and MAC-1.
J Exp Med
158:586, 1983[Abstract/Free Full Text]
35.
Takahashi K, Nakamura T, Koyanagi M, Kato K, Hashimoto Y, Yagita H, Okumura K:
A murine very late activation antigen-like extracellular matrix receptor involved in CD2- and lymphocyte function-associated antigen-1-independent killer-target cell interaction.
J Immunol
145:4371, 1990[Abstract]
36.
Noto K, Kato K, Okumura K, Yagita H:
Identification and functional characterization of mouse CD29 with a mAb.
Int Immunol
7:835, 1995[Abstract/Free Full Text]
37.
Yasuda M, Hasunuma Y, Adachi H, Sekine C, Sakanishi T, Hashimoto H, Ra C, Yagita H, Okumura K:
Expression and function of fibronectin binding integrins on rat mast cells.
Int Immunol
7:251, 1995[Abstract/Free Full Text]
38.
Ni J, Porter AG, Hollander D:
Beta 7 integrins and other cell adhesion molecules are differentially expressed and modulated by TNF beta in different lymphocyte populations.
Cell Immunol
161:166, 1995[Medline]
[Order article via Infotrieve]
39.
Seko Y, Matsuda H, Kato K, Hashimoto Y, Yagita H, Okumura K, Yazaki Y:
Expression of intercellular adhesion molecule-1 in murine hearts with acute myocarditis caused by coxsackievirus B3.
J Clin Invest
91:1327, 1993
40.
Hession C, Moy P, Tizard R, Chisholm P, Williams C, Wysk M, Burkly L, Miyake K, Kincade P, Lobb R:
Cloning of murine and rat vascular cell adhesion molecule-1.
Biochem Biophys Res Commun
183:163, 1992[Medline]
[Order article via Infotrieve]
41.
Palacios R, Steinmetz M:
IL-3-dependent mouse clones that express B-220 surface antigen, contain Ig genes in germ-line configuration, and generate B lymphocytes in vivo.
Cell
41:727, 1985[Medline]
[Order article via Infotrieve]
42.
Shibuya H, Yoneyama M, Ninomiya-Tsuji J, Matsumoto K, Tanighchi T:
IL-2 and EGF receptors stimulate the hematopoietic cell cycle via different signaling pathways: Demonstration of a novel role for c-myc.
Cell
70:57, 1992[Medline]
[Order article via Infotrieve]
43.
Dowdy SF, Hinds PW, Louie K, Reed SI, Arnold A, Weinberg RA:
Physical interaction of the retinoblastoma protein with human D cyclins.
Cell
73:499, 1993[Medline]
[Order article via Infotrieve]
44.
Battey J, Moulding C, Taub R, Murphy W, Stewart T, Potter H, Lenoir G, Leder P:
The human c-myc oncogene: Structural consequences of translocation into the IgH locus in Burkitt lymphoma.
Cell
34:779, 1983[Medline]
[Order article via Infotrieve]
45.
Doi T, Hatakeyama M, Minamoto S, Kono T, Mori H, Taniguchi T:
Human interleukin 2 (IL-2) receptor chain allows transduction of IL-2-induced proliferation signal(s) in a murine cell line.
Eur J Immunol
19:2375, 1989[Medline]
[Order article via Infotrieve]
46.
Miyazaki T, Liu Z-J, Kawahara A, Minami Y, Yamada K, Tsujimoto Y, Barsoumian EL, Perlmutter RM, Taniguchi T:
Three distinct IL-2 signaling pathways mediated by bcl-2, c-myc, and lck cooperate in hematopoietic cell proliferation.
Cell
81:223, 1995[Medline]
[Order article via Infotrieve]
47.
Minami Y, Nakagawa Y, Kawahara A, Miyazaki T, Sada K, Yamamura H, Taniguchi T:
Protein tyrosine kinase Syk is associated with and activated by the IL-2 receptor: Possible link with the c-myc induction pathway.
Immunity
2:89, 1995[Medline]
[Order article via Infotrieve]
48.
Wake A, Tanaka Y, Nakatsuka K, Misago M, Oda S, Morimoto I, Eto S:
Calcium dependent homotypic adhesion through leukocyte function-associated antigen 1/intracellular adhesion molecule-1 induces interleukin-1 and parathyroid hormone-related protein production on adult T-cell leukemia cells in vitro.
Blood
86:2257, 1995[Abstract/Free Full Text]
49.
Tanaka Y, Adams DH, Hubscher S, Hirano H, Siebenlist U, Shaw S:
T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1.
Nature
361:79, 1993[Medline]
[Order article via Infotrieve]
50.
Tanaka Y, Kimata K, Wake A, Mine S, Morimoto I, Yamakawa N, Habuchi H, Ashikari S, Yamamoto H, Sakurai K, Yoshida K, Suzuki S, Eto S:
Heparan sulfate proteoglycan on leukemic cells is primarily involved in integrin triggering and its mediated adhesion to endothelial cells.
J Exp Med
184:1987, 1996[Abstract/Free Full Text]
51.
Miyazaki T, Liu Z-J, Taniguchi T:
Selective cooperation of HTLV-1-encoded p40tax-1 with cellular oncoproteins in the induction of hematopoietic cell proliferation.
Oncogene
12:2403, 1996[Medline]
[Order article via Infotrieve]
52.
Tassan J-P, Jaquenoud M, Leopold P, Schultz SJ, Nigg EA:
Identification of human cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C.
Proc Natl Acad Sci USA
92:8871, 1995[Abstract/Free Full Text]
53.
Miyake K, Medina K, Ishihara K, Kimoto M, Auerbach R, Kincade PW:
A VCAM-like adhesion molecule on murine bone marrow stroma cells mediates binding of lymphocyte precursors in culture.
J Cell Biol
114:557, 1991[Abstract/Free Full Text]
54.
Terry RW, Kwee L, Levine JF, Labow MA:
Cytokine induction of an alternatively spliced murine vascular cell adhesion molecule (VCAM) mRNA encoding a glycosylphosphatidylinositol-anchored VCAM protein.
Proc Natl Acad Sci USA
90:5919, 1993[Abstract/Free Full Text]
55.
Erle DJ, Ruegg C, Sheppard D, Pytela D:
Complete amino acid sequence of an integrin beta subunit (7) identified in leukocytes.
J Biol Chem
266:11009, 1991[Abstract/Free Full Text]
56.
Elices MJ, Osborn L, Takada Y, Crouse C, Luhowskyi S, Hemler ME, Lobb RR:
VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site.
Cell
60:577, 1990[Medline]
[Order article via Infotrieve]
57.
Lenter M, Uhlig H, Hamann A, Jeno P, Imhof B, Vestweber D:
A monoclonal antibody against an activation epitope on mouse integrin chain 1 blocks adhesion of lymphocytes to the endothelial integrin 61.
Proc Natl Acad Sci USA
90:9051, 1993[Abstract/Free Full Text]
58.
Boudignon-Proudhon C, Patel PM, Parise LV:
Phorbol ester enhances integrin alpha IIb beta 3-dependent adhesion of human erythroleukemic cells to activation dependent monoclonal antibodies.
Blood
87:968, 1996[Abstract/Free Full Text]
59.
Lahue EE, Smith AV, Orr-Weaver TL:
A novel cyclin gene from Drosophila complements CLN function in yeast.
Genes Dev
5:2166, 1991[Abstract/Free Full Text]
60.
Leopold P, O'Farrell PH:
An evolutionarily conserved cyclin homolog from Drosophila rescues yeast deficient in G1 cyclins.
Cell
66:1207, 1991[Medline]
[Order article via Infotrieve]
61.
Lew DJ, Dulic V, Reed SI:
Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast.
Cell
66:1197, 1991[Medline]
[Order article via Infotrieve]
62.
Rickert P, Seghezzi W, Shanahan F, Cho H, Lees E:
Cyclin C/CDK8 is a novel CTD kinase associated with RNA polymerase II.
Oncogene
12:2631, 1996[Medline]
[Order article via Infotrieve]
63.
Inghirami G, Grignani F, Sternas L, Lombardi L, Knowles DM, Dalla-Favera R:
Down-regulation of LFA-1 adhesion receptors by C-myc oncogene in human B lymphoblastoid cells.
Science
250:682, 1990[Abstract/Free Full Text]
64.
Schleimer RP, Sterbinsky SA, Kaiser J, Bickel A, Klunk DA, Tomioka K, Newman W, Luscinskas FW, Gimbrone MA, McIntyre BW, Bochner BS:
IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium: Association with expression of VCAM-1.
J Immunol
148:1086, 1992[Abstract]
65.
Bottazzi ME, Assoian RK:
The extracellular matrix and mitogenic growth factors control G1 phase cyclins and cyclin-dependent kinase inhibitors.
Trends Cell Biol
7:348, 1997[Medline]
[Order article via Infotrieve]
66.
Guadagno TM, Ohtsubo M, Roberts JM, Assoian RK:
A link between cyclin A expression and adhesion-dependent cell cycle progression.
Science
262:1572, 1993[Abstract/Free Full Text]
67.
Altevogt P, Hubbe M, Ruppert M, Lohr J, von Hoegen P, Sammar M, Andrew DP, McEvoy L, Humphries MJ, Butcher EC:
The 4 integrin chain is a ligand for 47 and 41.
J Exp Med
182:345, 1995[Abstract/Free Full Text]
68.
Rosen GD, Sanes JR, LaChance R, Cunningham JM, Roman J, Dean DC:
Role of the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis.
Cell
69:1107, 1992[Medline]
[Order article via Infotrieve]
69.
Ryan DH, Nuccie BL, Abboud CN, Winslow JM:
Vascular cell adhesion molecule-1 and the integrin VLA-4 mediate adhesion of human B cell precursors to cultured bone marrow adherent cells.
J Clin Invest
88:995, 1991
70.
Teixido J, Hemler ME, Greenberger JS, Anklesaria P:
Role of 1 and 2 integrins in the adhesion of human CD34hi stem cells to bone marrow stroma.
J Clin Invest
90:358, 1992
71.
Morales-ducret J, Wayner E, Elices MJ, Alvaro-Gracia JM, Zvaifler NJ, Firestein GS:
41 integrin (VLA-4) ligands in arthritis. Vascular cell adhesion molecule-1 expression in synovium and on fibroblast-like synoviocytes.
J Immunol
149:1424, 1992[Abstract]
72.
Weg VB, Willians TJ, Lobb RR, Nourshargh S:
A monoclonal antibody recognizing very late activation antigen-4 inhibits eosinophil accumulation in vivo.
J Exp Med
177:561, 1993[Abstract/Free Full Text]
73.
Taichman DB, Cybulsky MI, Djaffar I, Longenecker BM, Teixido J, Rice GE, Aruffo A, Bevilacqua MP:
Tumor cell surface 41 integrin mediates adhesion to vascular endothelium: Demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-1.
Cell Regul
2:347, 1991[Medline]
[Order article via Infotrieve]
74.
Garofalo A, Chirivi RGS, Foglieni C, Pigott R, Mortarini R, Martin-Padura I, Anichini A, Gearing AJ, Sanchez-Madrid F, Dejana E, Giavazzi R:
Involvement of the very late antigen 4 integrin on melanoma in interleukin 1- augmented experimental metastases.
Cancer Res
55:414, 1995[Abstract/Free Full Text]
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Abstract
Introduction