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*
Department of Biochemistry, Kobe University School of Medicine, Kobe, Japan;
First Department of Internal Medicine, University of Occupational and Environmental Health School of Medicine, Kitakyushu, Japan;
School of Allied Health Science, Faculty of Medicine, Osaka University, Osaka, Japan;
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Department of Laboratory Medicine, Kobe University School of Medicine, Kobe, Japan;
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Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan;
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Core Research for Evolutional Science and Technology of Japan, Science and Technology Corporation, Tokyo, Japan; and
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Department of Anatomy, Shimane Medical University, Izumo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1) integrin without changing its
surface expression. Intriguingly, coexpression of H-Rasv12
and c-Myc in these cells results in not only the activation of VLA-4,
but also the induction of expression of VCAM-1, the counterreceptor for
VLA-4, thereby mediating a marked homotypic cell aggregation. In
addition, H-Rasv12-induced VLA-4 activation appears to be
partly down-regulated by coexpression with c-Myc. Our results represent
an unprecedented example demonstrating a novel role for
H-Rasv12 in the regulation of cell adhesion via
c-Myc-independent and -dependent mechanisms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß heterodimeric structure
and have been divided into three major subgroups according to the ß
subunit expression: ß1,
ß2, and ß3. Within the
ß1 subfamily, also termed very late Ag
(VLA)3 integrins,
VLA-4 is unique in that it mediates both cell-to-cell and
cell-to-extracellular matrix interactions by binding to its
counterreceptor VCAM-1 and fibronectin (Fn), respectively
(3). In contrast, VCAM-1 is an Ig-superfamily protein that
is expressed on endothelial cells in response to IL-1, IL-4,
TNF-, or LPS and is also expressed constitutively on a few other
cell types, such as follicular dendritic cells in lymph nodes
(4) and bone marrow stromal cells (5). The binding activity of both integrins and VCAM-1 depends upon their surface expression, yet integrin activity can also be modulated through inside-out signaling (6). Although the precise molecular mechanism of integrin activation modulated by inside-out signaling remains largely unknown, multiple intracellular signaling pathways and proteins, such as protein kinase C (PKC) (7, 8), phosphoinositide 3-kinase (9, 10), and some of the small G proteins (see below), have been implicated to be directly involved in modulating intergrin-mediated cell adhesion.
H-Ras and c-Myc proteins play crucial roles in regulating proliferation and transformation in multiple cell types (11, 12). It has been shown that H-Ras and c-Myc cooperate to induce cellular transformation in vitro and tumorigenesis in vivo (13, 14, 15). However, the roles of H-Ras and c-Myc, and in particular their cooperative roles in regulating cell adhesion, remain largely unknown. Involvement of small GTP-binding proteins in the regulation of cell adhesion has been suggested from an early observation that injection of GTP analogues into Xenopus XTC fibroblasts inhibits ruffling and increases cell spreading (16). Recent studies further demonstrate participation of H-Ras and R-Ras in regulating the activity of integrins via inside-out signaling. It has been reported that ectopic expression of an active form of H-Ras in Chinese hamster ovary (CHO) cells, stably expressing a chimeric integrin, suppressed the function of the chimeric integrin (17). In contrast, expression of an active form of R-Ras, which is related to H-Ras, has been found to enhance cell adhesion to the extracellular matrix via activation of several integrins (18). In contrast, previous studies demonstrate that Rho plays essential roles in regulating cytoskeletal organization and adhesive activity (19, 20, 21, 22). Furthermore, it has been shown that the effects of Ras on cytoskeletal organization, cell adhesiveness, and proliferation are mediated by Rac signaling pathway (23, 24, 25). However, little is known about the function of c-Myc in modulating cell adhesion, except for an observation that c-Myc can down-regulate the LFA-1 adhesion receptor (26).
In this study, we investigated the effects of protooncoproteins H-Ras and c-Myc on hemopoietic cellular behavior using hemopoietic progenitor BAF-B03 cells and found that constitutive expression of an active form of H-Ras (H-RasV12), but not c-Myc alone, enhances cell adhesion to Fn by activating VLA-4 without alteration of its surface expression. Interestingly, coexpression of H-RasV12 and c-Myc induces a homotypic cell adhesion, which is mediated, at least partly, by the interaction between activated VLA-4 integrin and inducibly expressed VCAM-1. Hence, it becomes evident that H-Ras can regulate cell adhesion molecules through c-Myc-dependent and -independent mechanisms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monoclonal Abs used in this study are as follows: HM1
(anti-1; Ref. 27), HM2
(anti-2; Ref. 27), PS/2
(anti-4; Ref. 28), HM5-1
(anti-5; Ref. 29), KBA
(anti-L; Ref. 30), Mac-1
(anti-M; Ref. 31), RMV-7
(anti-v; Ref. 32), HMß1-1
(anti-ß1; Ref. 33), 9EG7
(anti-ß1; Ref. 34), M18/2
(anti-ß2; Ref. 31), HMß3–1
(anti-ß3; Ref. 35), M293
(anti-ß7; Ref. 36), KAT-1
(anti-ICAM-1; Ref. 37), and M/K-2 (anti-VCAM-1;
Ref. 38). Anti-Thy-1 mAb was provided by Dr. E. Shevach
(National Institutes of Health, Bethesda, MD). FITC-conjugated goat
anti-rat and goat anti-hamster IgG were purchased from Cappel
Laboratories (Malvern, PA). Phospho-p44/42 mitogen-activated protein
(MAP) kinase (Thr202/Tyr204) Ab and phospho-specific p38 MAP kinase
(Thr180/Tyr182) Ab were obtained from New England Biolabs (Beverly,
MA). PD98059, a specific inhibitor of MAP/extracellular signal-related
kinase (ERK) kinase (MEK) 1, was obtained from New England Biolabs.
SB202190, a potent inhibitor of p38 MAP kinase, and SB202474, a
negative control compound for p38 MAP kinase inhibition studies, were
obtained from Calbiochem (San Diego, CA).
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 (39). BRV12 cells were established by transfecting an active form of human H-Ras expression plasmid, pEF-BOS-HA-RasV12 (40) into BAF-B03 cells; BM cells were obtained by transfecting a human c-Myc expression plasmid (pN-LTR-myc; Ref. 41) into BAF-B03 cells; BMRV12 cells were established by transfecting pEF-BOS-HA-Rasv12 into BM cells. For all cell lines, at least three independent clones were established, and the results from a representative clone are shown. BMRV12 cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) FCS, and other cells were cultured in the same medium containing 10% (v/v) 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 (42). Selection was initiated 24 h after DNA transfection using 2 mg/ml G418 for BRV12 and BMRV12 and 1 mg/ml hygromycin for BMRV12 cells. Drug-resistant clones were either pooled or subsequently cloned by limiting dilution as described previously (43).
Western blot analysis
Cells (5 x 106) were harvested and solubilized in lysis buffer (50 mM Tris-HCl, pH 7.4, 0.5% (v/v) Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) by sonication. Postnuclear supernatants were prepared by centrifugation at 10,000 x g for 10 min. Protein was quantified using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). For Western blot analysis, samples containing equal amounts of protein were subjected to SDS-PAGE (10% polyacrylamide gel). Separated proteins were transferred onto polyvinylidene difluoride membranes (Immobilon, Millipore, Bedford, MA). After blocking with TBST-milk (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% (v/v) Tween 20, 5% nonfat dry milk), membranes were incubated with anti-hemagglutinin (HA) mAb, 12CA25 (Boehringer Mannheim, Mannheim, Germany) (1:1000 dilution in TBST-0.5% milk) overnight at 4°C. Then, membranes were washed with TBST and incubated with HRP-conjugated secondary Abs (1:3000 dilution in TBST-0.5% milk) for 1 h at room temperature. After three washes in TBST, proteins were detected using the enhanced chemiluminescence kit according to the manufacturer’s instructions (Amersham, Buckinghamshire, U.K.).
Flow cytometry
Cell-surface expression of adhesion molecules was analyzed by immunofluorescence using mAbs against the respective molecules as described previously (44). For each sample, a total of 1 x 106 cells were treated with the respective mAbs for 30 min at 4°C. After washing, cells were stained with FITC-conjugated goat anti-rat or anti-hamster Abs. The stained cells were analyzed by a Coulter Epics XL-MCL flow cytometer (Coulter, Miami, FL).
Ab-blocking assay
Aggregated BMRV12 cells were mechanically separated into a suspension of single cells. For each sample, 5 x 105 cells were replated in a 24-well plate (1 ml/well) along with various mAb at a saturating concentration of 10 µg/ml, which was shown in previous studies to produce a maximum inhibition of the relevant adhesive interaction (45). The effect of mAb on cell aggregation was evaluated by observation of photomicrographs after 1–4 h incubation.
Cell adhesion assay
Adhesion assay of BAF-B03, BM, BRV12, and BMRV12 cells to Fn was performed essentially as previously described (46). Fn (5 µg/well; Seikagaku, Tokyo, Japan) or control 3% human serum albumin (Green-Cross, Osaka, Japan) was applied to a 48-well plate in PBS at 4°C overnight. Wells were subsequently blocked with Ca2+/Mg2+-free PBS/3% human serum albumin for 2 h at 37°C. After the plates were washed three times with PBS, 2 x 105 BAF-B03 or transfectants labeled with 51Cr (DuPont NEN, Wilmington, DE) were added to each well with or without blocking mAb (10 µg/ml) in the presence or absence of PMA (10 ng/ml; Sigma, St. Louis, MO). To examine the effects of MEK1 inhibitor or p38 inhibitor, cells were pretreated with PD98059 (50 µM), SB202190 (50 µM), or SB202474 (50 µM) for 1 h. After settling for 30 min at 4°C, the plates were rapidly warmed to 37°C for 30 min, then gently washed twice with RPMI 1640 at room temperature to completely remove nonadherent cells. The adherent cells contained in each well were lysed with 250 ml of 1% Triton X-100, and the 51Cr radioactivity was measured using a gamma-counter. Data were expressed as mean percentage of the binding of indicated cells from a representative experiment.
Cell growth assay
For cell growth assay, factor-independent BMRV12 cells were cultured at a density of 5 x 105 cells/ml in RPMI 1640 supplemented with 10% FCS, and other cells were cultured in the same RPMI 1640 medium containing 10% WEHI-3B supernatant as a source of IL-3. The culture medium was changed every other day. For the cell viability assay, BAF-B03, BM, and BRV12 cells were washed with PBS to remove cytokines and then cultured at a density of 5 x 105 cells/ml in RPMI 1640 supplemented with 10% FCS. Viable cell numbers were determined with a trypan blue exclusion assay.
Actin polymerization
The presence of F-actin was detected as described previously (47). In brief, cells (106/ml) were fixed on slides and then permeabilized. F-actin was detected by being stained with rhodamine-phalloidin (1 U/slide; Molecular Probes, Eugene, OR) and was analyzed by a confocal laser scan microscope system (LSM410UV, Carl Zeiss, Oberkochen, Germany).
Northern blot analysis
Total RNAs from cells were prepared by using Isogen (Wako,
Osaka, Japan). For RNA blot analysis, 10 µg of total RNA was
electrophoresed on 1% agarose formaldehyde gels and transferred onto
nylon membranes. The probe DNA (
1.4 kb) was prepared from
pCR2.1-TOPO-mouse VCAM-1 (nt 733-2200 of the open reading frame) by
digestion with EcoRI and labeled with
[-32P]dCTP (3000 Ci/mmol; Amersham) using
the Multiprime labeling kit (Amersham) and hybridized as described
previously (44). Specific activity was 1 x
106 cpm/ng for the probe DNA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As an attempt to investigate the effect of protooncoproteins H-Ras
and c-Myc on hemopoietic cellular behavior, an active form of the human
H-ras (H-rasV12) and c-Myc were stably expressed
singly or in combination in an IL-3-dependent mouse pro-B cell line,
BAF-B03, which normally grows as a suspension of single cells. We
noticed that BAF-B03 cells became slightly adhesive to culture plates
after transfection with an expression plasmid encoding an active form
of human H-Rasv12 (termed
BRv12) (data not shown), yet the
BRv12 cells still required IL-3 for their
proliferation. Interestingly, BAF-B03 cells expressing both active
human H-Rasv12 and human c-Myc (termed
BMRv12 cells) were able to proliferate in a
cytokine-independent fashion (Fig. 1
) as
well as to form cell aggregates (Fig. 2A), despite the fact that
cells expressing human c-Myc alone (BM cells) failed to display
analogous behavior (Figs. 1 and 2A). Furthermore, we
examined the actin polymerization in these transfectants as well as
parental BAF-B03 cells. Remarkable actin polymerization was observed in
BRv12 and BMRv12 cells, but
not in BAF-B03 (Fig. 2B) and BM cells (data not shown).
These observations suggested that H-Rasv12, by
itself or in cooperation with c-Myc, may play an important role in the
regulation of certain cell adhesion molecule(s) and cytoskeletal
molecule(s).
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To determine which molecules might be responsible for the observed
adhesive properties of BRv12 and
BMRv12 cells, candidate molecules were sought
using a panel of Abs and flow cytometric analysis (see Materials
and Methods). The expression of 1,
2, L,
M, V,
ß2, ß3, and
ß7 chains was not detectable on either BAF-B03,
BM, or BRv12 and BMRv12
cells (Fig. 3A). We found that
the integrin 5 and ß1
chains as well as ICAM-1 (Fig. 3A) are comparably expressed
on BAF-B03, BRv12, BM, and
BMRv12 cells. The expression of the integrin
4 chain was also detectable on these cells,
although its expression level on BMRv12 cells was
somewhat lower than the others (Fig. 3B, left),
Noticeably, VCAM-1 is not expressed on either the BAF-B03,
BRv12, or BM cells, but is expressed
substantially on the surface of BMRv12 cells
(Fig. 3B, right). Furthermore, consistent with
the result obtained by flow cytometric analysis, Northern blot analysis
revealed that a high level of VCAM-1 transcripts was
detected in BMRv12 cells, although the expression
of VCAM-1 transcripts was hardly detectable in the BAF-B03,
BRv12, or BM cells (Fig. 3C). This
results indicated that expression of H-Rasv12 or
c-Myc alone is insufficient for the induction of VCAM-1 and that a
cooperative effect of H-Rasv12 and c-Myc is
required for the induction of VCAM-1 expression.
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4ß1) and VCAM-1 are
involved in homotypic cell adhesion induced by coexpression of
H-RasV12 and c-Myc
To identify the adhesion molecules mediating the homotypic
aggregation of BMRv12 cells, we examined the
effects of several function-blocking mAbs on the homotypic aggregation
of the cells. It was found that homotypic aggregation of
BMRv12 cells was almost completely inhibited by
either anti-4 (PS/2) or anti-VCAM-1
(M/K2) mAbs, whereas anti-L (KBA),
anti-ICAM-1(KAT-1), anti-v (RMV-7), or
a control nonblocking anti-ß2 (M18) mAbs
failed to inhibit homotypic aggregation of BMRv12
cells (Fig. 4A). Thus, the
results suggested that 4 integrin may be
activated on these cells, and that both 4
integrin and VCAM-1 are primarily involved in the homotypic aggregation
of BMRv12 cells. Although the integrin
4 chain is capable of associating with either
ß1
(4ß1, VLA-4) or
ß7 (48) to mediate adhesion to
VCAM-1 or Fn, the ß1 but not the
ß7 subunit is expressed on BAF-B03 cells and
their derived transfectants (Fig. 2
A), suggesting that
4 associates with ß1
to form the heterodimer (VLA-4) on these cells. Noticeably, an
anti-ß1 mAb, HMß1–1, which has been
shown to block the binding of
4ß1 to Fn
(33), failed to inhibit homotypic aggregation of
BMRv12 cells under our experimental conditions
(up to 50 µg/ml, data not shown), and it is likely that HMß1–1
fails to recognize the epitope on the ß1 that
is required for the interaction with VCAM-1. In fact, it was shown that
the sites within the ß1 integrin involved in
the ligation of VLA-4/VCAM-1 and VLA-4/Fn are different
(3). Importantly, anti-mouse
ß1 mAb, 9EG7 (34), which
recognizes the ligand-binding or activated epitope of the
ß1 integrin, considerably discriminates
BRv12 and BMRv12 cells from
BAF-B03 and BM cells. As shown in Fig. 4B, the
ligand-binding (or activated) epitope of ß1 is
induced on BRv12 and BMRv12
cells at higher levels compared with BAF-B03 or BM cells as assessed by
flow cytometric analyses, suggesting that expression of
H-Rasv12 alone is sufficient to activate
ß1 integrin. Collectively, our results indicate
that VLA-4 and VCAM-1 are primarily responsible for the homotypic
aggregation of BMRv12 cells.
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Because the activated ß1 is detectable on
BRv12 and BMRv12 cells
among cells examined, we assessed the adhesive abilities of integrin
ß1 on BRv12 and
BMRv12 cells to Fn coated on plates. It was found
that BMRv12 and BRv12 cells
can bind to Fn efficiently. Binding of BRv12 and
BMRv12 cells to Fn was augmented about 6- and
4-fold over the control, respectively (Fig. 5A). Furthermore, the addition
of phorbol ester, a potent integrin trigger, resulted in apparently
enhanced adhesion of BAF-B03 cells to Fn, whereas the adhesion of
BRv12 and BMRv12 cells to
Fn was only moderately and weakly augmented, respectively (Fig. 5A), indicating that H-Rasv12 can play
a pivotal role in the activation of integrin. The fact that attachment
of BMRV12 cells to Fn was weaker than that of
BRV12 cells might reflect the down-regulation of
VLA-4 integrin on BMRV12 cells by H-Ras and c-Myc
(see Discussion). In contrast, no enhanced attachment of
BAF-B03 (Fig. 5A), BM, BRS17N (BAF-B03
cells expressing a dominant negative form of H-Ras,
H-RasS17N), and BAF-B03 cells transfected with an
empty vector to Fn was observed (data not shown). Adhesion of
BRv12 and BMRv12 cells to
Fn was selectively inhibited by the anti-4
mAb (PS/2), but not by an irrelevant anti-Thy-1 mAb (Fig. 5A). However, the inhibition by the
anti-4 mAb was incomplete (50%),
suggesting that other integrins such as 5 may
also be activated on these cells. Collectively, these results indicate
that VLA-4 expressed on the surface of both BRv12
and BMRv12 cells are dominantly activated by
H-RasV12, and thus VLA-4 activation is a
c-Myc-independent process.
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B). Furthermore, the addition of PD98059 or SB202190 failed
to inhibit the homotypic aggregation of BMRv12
cells (data not shown). These results suggest that MEK/ERK as well as
p38 MAP kinase are not involved in
H-Rasv12-mediated activation of VLA-4. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B). One plausible explanation
for such a discrepancy is that H-Ras may possess distinct functions in
regulating different types of integrins, although cell type-specific
functions of H-Ras have also to be considered. In contrast, Zhang et
al. have reported that another Ras family member, R-Ras, is able to
activate integrins (18). R-Ras is a GTP-binding protein
highly homologous to H-Ras protein, but has an additional 26 aa at the
amino terminus (49). R-Ras and H-Ras have similar effector
binding domains and bind to many identical effectors, including Raf and
Ral-GDS (50). It is of interest to examine whether
R-Ras-mediated activation of integrins also involve the activation of
Raf/MAPK pathway.
While the activation of VLA-4 on BAF-B03 cells relies solely on
H-Rasv12, induction of VCAM-1 by
H-Rasv12 is a c-Myc-dependent process. In this
respect, it is of importance to note that ectopic expression of
H-Rasv12 itself did not affect the expression of
c-Myc and vice versa (data not shown). At present, the mechanism of
c-Myc-dependent expression of VCAM-1 remains unclear. 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 of the VCAM-1 induction is that H-Ras and c-Myc act
cooperatively to induce some cytokines, such as TNF-, IL-1, and
IL-4, which are known to be able to induce VCAM-1 expression
(51, 52, 53). To test this, we used the supernatants of
growing BMRv12 cells to culture either parental
BAF-B03 or BM and BRv12 cells and found that
expression of VCAM-1 on these cells was not induced under our
experimental conditions and that BRv12 cells did
not form aggregates (data not shown), indicating that induction of
VCAM-1 is not due to indirect cytokine stimulation. Further study will
be required to elucidate the molecular basis of VCAM-1 induction by
H-RasV12 and c-Myc.
Although the c-Myc-dependent mechanism is primarily responsible for the
induction of VCAM-1, the function of c-Myc appears not to be so simple.
c-Myc may also participate in the down-regulation of VLA-4 expression
on BMRv12 cells in collaboration with
H-Rasv12; 1) expression of the
4 integrin on BMRv12
cells is partly (30%) down-regulated compared with that on BAF-B03,
BM, or BRv12 cells (Fig. 3B); 2)
although expression of total ß1 on
BRv12 and BMRv12 cells is
comparable, the amount of activated ß1 on
BMRv12 cells is partly (30%) down-regulated
compared with that on BRv12 cells (Fig. 4B); 3) attachment of BMRv12 cells to
Fn is weaker than that of BRv12 cells (Fig. 5A). Collectively, these results suggest that VLA-4
expression as well as VLA-4 activity on BMRv12
cells may be down-regulated. The evidence of a possible negative
regulatory effect of c-Myc on VLA-4 is reminiscent of the previous
report, showing that c-Myc could down-regulate LFA-1 (26).
In addition, down-regulation of integrins by N-myc has been
demonstrated (54, 55). It has been reported that integrin
function (or expression) is often diminished upon oncogenic
transformation (56, 57). Hence, down-regulation of VLA-4
integrin observed on BMRv12 cells may reflect the
cellular transformation of BMRv12 cells. In fact,
we found that BMRv12 cells proliferate in a
cytokine-independent manner (Fig. 1) and form foci in soft agar in the
absence of IL-3 (data not shown). Thus, our results may also provide a
possible mechanism to explain such down-regulation of integrins upon
cellular transformation.
Simultaneous activation of VLA-4 and expression of VCAM-1 on the same cell is an interesting phenomenon. It was originally supposed that these two processes are regulated by distinct mechanisms in different types of cells, because the cellular and tissue distributions of VLA-4 and VCAM-1 are quite different and VLA-4/VCAM-1 generally mediates heterotypic cell-to-cell interactions, such as those between leukocytes and endothelial cells. An interesting example of a VLA-4/VCAM-1 interaction occurring on the same types of cells is the observation of Rosen et al. (58) that VLA-4 and VCAM-1 are expressed concomitantly on myoblasts. The VLA-4/VCAM-1 interaction has been suggested to be crucial for myogenesis. However, in the above case, activity of VLA-4 is regulated at the expression level rather than modulation of its ligand-binding activity (affinity) by inside-out signaling. In addition, it remains unclear which intracellular molecules are responsible for this regulation.
Importantly, we have recently observed that BAF-B03 cells expressing both cyclin C and c-Myc (termed BMC cells) exhibit essentially identical cellular behaviors with BMRv12 cells (59). It can be assumed that cyclin C may be one of candidate downstream targets of H-Ras, because cyclin C, like H-Rasv12, is able to cooperate with c-Myc to induce cytokine-independent growth and homotypic adhesion of BAF-B03 cells. However, unlike H-Rasv12, ectopic expression of cyclin C alone in BAF-B03 cells fails to activate VLA-4. It is perhaps due to that cyclin C may be just one of multiple H-Rasv12 targets required for VLA-4 activation, or it simply reflects the difference in the functional strength between constitutively active H-Ras (H-Rasv12) and the wild-type cyclin C. By contrast, it seems unlikely that the function of cyclin C is mediated by the activation of endogenous H-Ras, because overexpression of a dominant negative form of H-Ras (H-RasS17N) in BMC cells, where cyclin C as well as c-Myc are expressed ectopically at high levels, fails to affect their cell adhesion properties (data not shown). It is also possible that cyclin C and H-Ras may use distinct mechanisms to mediate activation of VLA-4 and expression of VCAM-1 in cooperation with c-Myc.
The physiological and pathological significance of the regulation of
the functional properties of VLA-4 and VCAM-1 on hemopoietic cells
remains to be determined. Activation of VLA-4/VCAM-1 pair on the
hemopoietic progenitors may be of importance for the regulation of
hemopoiesis. Adhesive interactions of VLA-4 with VCAM-1 on stromal
cells or with extracellular matrix retain hemopoietic progenitor cells
in close vicinity to components of the bone marrow microenvironment
that are required for the regulation of physiological hemopoiesis. The
importance of VLA-4 in hemopoiesis has been proved by the fact that the
addition of anti-4 mAb to long-term bone
marrow cultures abrogated lymphopoiesis and retarded myelopoiesis
(28). VLA-4-specific Abs have also been shown to abrogate
stroma-dependent erythropoiesis (29). VCAM-1 may also
contribute to promote lympho- and myelopoiesis (5, 60, 61). In addition, VLA-4/VCAM-1-mediated cell adhesion has been
assumed to play an important role(s) in the migration of leukocytes
(62, 63) and circulating malignant cells (64, 65), which is a critical step in the process of inflammation and
metastasis. The elucidation of the c-Myc-dependent and -independent
functions of H-Ras in governing cell adhesion warrants further study in
the context of gaining insights into the pathophysiologic mechanisms
regulating multiple biological processes, such as hemopoiesis,
differentiation, inflammation, and metastases, in which VLA-4 integrin
and VCAM-1 play essential roles.
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Acknowledgments |
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4 mAb, respectively. We also thank Drs. L.
E. Samelson and A. Kukula for critical reading of the
manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Yasuhiro Minami, Department of Biochemistry, Kobe University, School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address:
3 Abbreviations used in this paper: VLA, very late Ag; Fn, fibronectin; PKC, protein kinase C; CHO, Chinese hamster ovary; MAP, mitogen-activated protein; ERK, extracellular signal-related kinase; MEK, MAP/ERK kinase; HA, hemagglutinin.
Received for publication March 8, 1999. Accepted for publication August 24, 1999.
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  S. Nakayamada, Y. Okada, K. Saito, M. Tamura, and Y. Tanaka {beta}1 Integrin/Focal Adhesion Kinase-mediated Signaling Induces Intercellular Adhesion Molecule 1 and Receptor Activator of Nuclear Factor {kappa}B Ligand on Osteoblasts and Osteoclast Maturation J. Biol. Chem., November 14, 2003; 278(46): 45368 - 45374. [Abstract] [Full Text] [PDF]
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 K. H. Shain and W. S. Dalton Cell Adhesion Is a Key Determinant in de Novo Multidrug Resistance (MDR): New Targets for the Prevention of Acquired MDR Mol. Cancer Ther., November 1, 2001; 1(1): 69 - 78. [Abstract] [Full Text] [PDF]
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 K. A. Reedquist, E. Ross, E. A. Koop, R. M.F. Wolthuis, F. J.T. Zwartkruis, Y. van Kooyk, M. Salmon, C. D. Buckley, and J. L. Bos The Small Gtpase, Rap1, Mediates Cd31-Induced Integrin Adhesion J. Cell Biol., March 20, 2000; 148(6): 1151 - 1158. [Abstract] [Full Text] [PDF]
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C.-F. Lai, L. Chaudhary, A. Fausto, L. R. Halstead, D. S. Ory, L. V. Avioli, and S.-L. Cheng Erk Is Essential for Growth, Differentiation, Integrin Expression, and Cell Function in Human Osteoblastic Cells J. Biol. Chem., April 20, 2001; 276(17): 14443 - 14450. [Abstract] [Full Text] [PDF]
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