Despite extensive studies on the crucial functions of Ras and c-Myc in cellular proliferation and transformation, their roles in regulating cell adhesion are not yet fully understood. Involvement of Ras in modulating integrin activity by inside-out signaling has been recently reported. However, in contrast to R-Ras, H-Ras was found to exhibit a suppressive effect. Here we show that ectopic expression of a constitutively active H-Rasv12, but not c-Myc alone, in a hemopoietic cell line induces activation of very late Ag-4 (VLA-4, α4β1) 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.

Cell adhesive interactions play crucial roles in directing the migration, proliferation, survival, and differentiation of cells (1). Integrins are the major family of cell-surface receptors that mediate cell adhesion and link extracellular ligands with cytoskeletal proteins (2). Integrins exhibit an αβ heterodimeric structure and have been divided into three major subgroups according to the β subunit expression: β1, β2, and β3. Within the β1 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-1α, IL-4, TNF-α, 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.

Monoclonal Abs used in this study are as follows: HMα1 (anti-α1; Ref. 27), HMα2 (anti-α2; Ref. 27), PS/2 (anti-α4; Ref. 28), HMα5-1 (anti-α5; Ref. 29), KBA (anti-αL; Ref. 30), Mac-1 (anti-αM; Ref. 31), RMV-7 (anti-αv; Ref. 32), HMβ1-1 (anti-β1; Ref. 33), 9EG7 (anti-β1; Ref. 34), M18/2 (anti-β2; Ref. 31), HMβ3–1 (anti-β3; Ref. 35), M293 (anti-β7; 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).

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.

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).

Cells (5 × 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 × 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.).

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 × 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).

Aggregated BMRV12 cells were mechanically separated into a suspension of single cells. For each sample, 5 × 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.

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 × 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.

For cell growth assay, factor-independent BMRV12 cells were cultured at a density of 5 × 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 × 105 cells/ml in RPMI 1640 supplemented with 10% FCS. Viable cell numbers were determined with a trypan blue exclusion assay.

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).

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 [α-32P]dCTP (3000 Ci/mmol; Amersham) using the Multiprime labeling kit (Amersham) and hybridized as described previously (44). Specific activity was ∼1 × 106 cpm/ng for the probe DNA.

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. 2,A), despite the fact that cells expressing human c-Myc alone (BM cells) failed to display analogous behavior (Figs. 1 and 2,A). 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. 2 B) 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).

FIGURE 1.

Proliferation and viability profiles of BAF-B03-derived cells. Cells were plated at 5 × 105 cells/ml in the presence or absence of IL-3. The concentration of viable cells was counted at various time points after plating and are represented on a logarithmic scale. Expression of exogenous H-RasV12 was detected by Western blot analysis using anti-HA mAb (inset). Abbreviations: BM, BAF-B03 cells expressing human c-Myc alone; BRV12, BAF-B03 cells expressing human H-RasV12 alone; BMRV12, BAF-B03 cells expressing both human H-RasV12 and c-Myc.

FIGURE 1.

Proliferation and viability profiles of BAF-B03-derived cells. Cells were plated at 5 × 105 cells/ml in the presence or absence of IL-3. The concentration of viable cells was counted at various time points after plating and are represented on a logarithmic scale. Expression of exogenous H-RasV12 was detected by Western blot analysis using anti-HA mAb (inset). Abbreviations: BM, BAF-B03 cells expressing human c-Myc alone; BRV12, BAF-B03 cells expressing human H-RasV12 alone; BMRV12, BAF-B03 cells expressing both human H-RasV12 and c-Myc.

Close modal
FIGURE 2.

A, Morphological properties of BAF-B03-derived cells. BAF-B03, BM, and BRV12 cells were cultured in the presence of IL-3 while BMRV12 cells were cultured in the absence of IL-3. The addition of IL-3 did not alter the cell adhesion property of BMRV12 cells. B, Induction of actin polymerization in BRV12 and BMRV12 cells. BAF-B03 (+IL-3), BRV12 (+IL-3), and BMRV12 (-IL-3) cells were stained with rhodamine-phalloidin to detect F-actin. Remarkable actin polymerization was observed in BRV12 and BMRV12 cells, while weak staining was detected in BAF-B03 cells. Quantitational analysis revealed that intensities of rhodamine-phalloidin staining of BRV12 and BMRV12 cells were about 2.7- and 2.5-fold higher than those of BAF-B03 cells, respectively. The magnification of each panel is ×3427. The size of BAF-B03 and BRV12 cells is somewhat smaller and larger than that of BMRV12 cells, respectively.

FIGURE 2.

A, Morphological properties of BAF-B03-derived cells. BAF-B03, BM, and BRV12 cells were cultured in the presence of IL-3 while BMRV12 cells were cultured in the absence of IL-3. The addition of IL-3 did not alter the cell adhesion property of BMRV12 cells. B, Induction of actin polymerization in BRV12 and BMRV12 cells. BAF-B03 (+IL-3), BRV12 (+IL-3), and BMRV12 (-IL-3) cells were stained with rhodamine-phalloidin to detect F-actin. Remarkable actin polymerization was observed in BRV12 and BMRV12 cells, while weak staining was detected in BAF-B03 cells. Quantitational analysis revealed that intensities of rhodamine-phalloidin staining of BRV12 and BMRV12 cells were about 2.7- and 2.5-fold higher than those of BAF-B03 cells, respectively. The magnification of each panel is ×3427. The size of BAF-B03 and BRV12 cells is somewhat smaller and larger than that of BMRV12 cells, respectively.

Close modal

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 α1, α2, αL, αM, αV, β2, β3, and β7 chains was not detectable on either BAF-B03, BM, or BRv12 and BMRv12 cells (Fig. 3,A). We found that the integrin α5 and β1 chains as well as ICAM-1 (Fig. 3,A) are comparably expressed on BAF-B03, BRv12, BM, and BMRv12 cells. The expression of the integrin α4 chain was also detectable on these cells, although its expression level on BMRv12 cells was somewhat lower than the others (Fig. 3,B, 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. 3,B, 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. 3 C). 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.

FIGURE 3.

A, Flow cytometric analysis of expression of various integrin subunits and ICAM-1 on BAF-B03 cells (white bar), BRV12 cells (shaded bar), and BMRV12 cells (black bar). Mean fluorescence intensity values are shown. B, Flow cytometric analysis of α4 integrin and VCAM-1 expression on BAF-B03 cells and transfectants. The indicated cells were unstained (dotted line) or stained with anti-α4 integrin or anti-VCAM-1 mAbs (continuous line) followed by staining with FITC-labeled secondary Abs and flow cytometric analysis, as described in Materials and Methods. C, Northern blot analysis of expression of VCAM-1 transcripts in BAF-B03, BM, BRV12, and BMRV12 cells. Northern blot analysis was performed as described in Materials and Methods. The filters were stained with methylene blue to show 18S and 28S ribosomal RNAs (28S rRNA in bottom of each panel).

FIGURE 3.

A, Flow cytometric analysis of expression of various integrin subunits and ICAM-1 on BAF-B03 cells (white bar), BRV12 cells (shaded bar), and BMRV12 cells (black bar). Mean fluorescence intensity values are shown. B, Flow cytometric analysis of α4 integrin and VCAM-1 expression on BAF-B03 cells and transfectants. The indicated cells were unstained (dotted line) or stained with anti-α4 integrin or anti-VCAM-1 mAbs (continuous line) followed by staining with FITC-labeled secondary Abs and flow cytometric analysis, as described in Materials and Methods. C, Northern blot analysis of expression of VCAM-1 transcripts in BAF-B03, BM, BRV12, and BMRV12 cells. Northern blot analysis was performed as described in Materials and Methods. The filters were stained with methylene blue to show 18S and 28S ribosomal RNAs (28S rRNA in bottom of each panel).

Close modal

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-α4 (PS/2) or anti-VCAM-1 (M/K2) mAbs, whereas anti-αL (KBA), anti-ICAM-1(KAT-1), anti-αv (RMV-7), or a control nonblocking anti-β2 (M18) mAbs failed to inhibit homotypic aggregation of BMRv12 cells (Fig. 4,A). Thus, the results suggested that α4 integrin may be activated on these cells, and that both α4 integrin and VCAM-1 are primarily involved in the homotypic aggregation of BMRv12 cells. Although the integrin α4 chain is capable of associating with either β14β1, VLA-4) or β7 (48) to mediate adhesion to VCAM-1 or Fn, the β1 but not the β7 subunit is expressed on BAF-B03 cells and their derived transfectants (Fig. 2,A), suggesting that α4 associates with β1 to form the heterodimer (VLA-4) on these cells. Noticeably, an anti-β1 mAb, HMβ1–1, which has been shown to block the binding of α4β1 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 HMβ1–1 fails to recognize the epitope on the β1 that is required for the interaction with VCAM-1. In fact, it was shown that the sites within the β1 integrin involved in the ligation of VLA-4/VCAM-1 and VLA-4/Fn are different (3). Importantly, anti-mouse β1 mAb, 9EG7 (34), which recognizes the ligand-binding or activated epitope of the β1 integrin, considerably discriminates BRv12 and BMRv12 cells from BAF-B03 and BM cells. As shown in Fig. 4 B, the ligand-binding (or activated) epitope of β1 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 β1 integrin. Collectively, our results indicate that VLA-4 and VCAM-1 are primarily responsible for the homotypic aggregation of BMRv12 cells.

FIGURE 4.

A, Ab-blocking analysis of homotypic cell adhesion of BMRV12 cells. Morphological properties of BMRV12 following treatment with the respective mAbs (control nonblocking anti-β2 mAb, anti-αL, anti-αv, anti-α4, anti-ICAM-1, or anti-VCAM-1 mAb) for 2 h are shown. B, Expression of ligand-binding epitope of β1 integrin on BAF-B03 cells and transfectants. The various cells were unstained (dotted line) or stained with anti-mouse β1 integrin mAb (9EG7), which recognizes ligand-binding epitope or activated epitope of β1 integrin (continuous line), followed by staining with FITC-labeled secondary Abs and flow cytometric analysis, as described in Materials and Methods.

FIGURE 4.

A, Ab-blocking analysis of homotypic cell adhesion of BMRV12 cells. Morphological properties of BMRV12 following treatment with the respective mAbs (control nonblocking anti-β2 mAb, anti-αL, anti-αv, anti-α4, anti-ICAM-1, or anti-VCAM-1 mAb) for 2 h are shown. B, Expression of ligand-binding epitope of β1 integrin on BAF-B03 cells and transfectants. The various cells were unstained (dotted line) or stained with anti-mouse β1 integrin mAb (9EG7), which recognizes ligand-binding epitope or activated epitope of β1 integrin (continuous line), followed by staining with FITC-labeled secondary Abs and flow cytometric analysis, as described in Materials and Methods.

Close modal

Because the activated β1 is detectable on BRv12 and BMRv12 cells among cells examined, we assessed the adhesive abilities of integrin β1 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. 5,A). 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. 5,A), 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. 5,A), 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-α4 mAb (PS/2), but not by an irrelevant anti-Thy-1 mAb (Fig. 5 A). However, the inhibition by the anti-α4 mAb was incomplete (∼50%), suggesting that other integrins such as α5 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.

FIGURE 5.

A, Effect of anti-α4 blocking mAb (PS/2) on the adhesion of BAF-B03 cells and of their derived transfectants to Fn. The indicated cells were untreated or treated with anti-α4 integrin mAb as described in Materials and Methods. Mean percentages of binding are shown. Essentially identical results were obtained in three independent experiments. B, Effect of MEK1 inhibitor (PD98059) or p38 inhibitor (SB202190) on the adhesion of BAF-B03 and BRV12 cells to Fn. The indicated cells were untreated or treated with PD98059 (50 μM), SB202190 (50 μM), or with SB202474 (50 μM) as described in Materials and Methods. Mean percentages of binding are shown. Essentially identical results were obtained in independent experiments. Immunoblot analysis with phospho-ERK and phospho-p38 MAP kinase Abs revealed that PD98059 and SB202190 almost completely inhibit ERK and p38 activities, respectively, under our experimental conditions (data not shown).

FIGURE 5.

A, Effect of anti-α4 blocking mAb (PS/2) on the adhesion of BAF-B03 cells and of their derived transfectants to Fn. The indicated cells were untreated or treated with anti-α4 integrin mAb as described in Materials and Methods. Mean percentages of binding are shown. Essentially identical results were obtained in three independent experiments. B, Effect of MEK1 inhibitor (PD98059) or p38 inhibitor (SB202190) on the adhesion of BAF-B03 and BRV12 cells to Fn. The indicated cells were untreated or treated with PD98059 (50 μM), SB202190 (50 μM), or with SB202474 (50 μM) as described in Materials and Methods. Mean percentages of binding are shown. Essentially identical results were obtained in independent experiments. Immunoblot analysis with phospho-ERK and phospho-p38 MAP kinase Abs revealed that PD98059 and SB202190 almost completely inhibit ERK and p38 activities, respectively, under our experimental conditions (data not shown).

Close modal

It was found that both ERK1 and ERK2 are activated constitutively in BRv12 cells (data not shown). Thus, we examined whether or not MEK/ERK (or p38 MAP kinase) is involved in H-RasV12-mediated activation of VLA-4 in BRv12 cells by using the MEK inhibitor (PD98059) or p38 inhibitor (SB202190). It was found that PD98059, SB202190, and SB202474 (negative control compound) have marginal effects on the binding of BAF-B03 and BRv12 cells to Fn (Fig. 5 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.

Activation of VLA-4 by H-Ras is a particularly surprising observation, because an inhibitory effect of H-Ras on certain integrins has been recently reported (17). However, it was unclear whether some types of integrins, such as VLA-4, were actually activated by H-Ras in their study, because a constitutively active chimeric integrin system was employed to monitor the suppressive effect rather than to detect activated function. The suppression of integrin function by H-Ras has been shown to be mediated through the Ras/Raf/MAPK (ERK) pathway (17). Interestingly, although both ERK1 and ERK2 are activated constitutively in BRV12 cells (data not shown), it was suggested that ERKs are not involved in activation of VLA-4 in the cells (Fig. 5 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-α, IL-1α, 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 α4 integrin on BMRv12 cells is partly (∼30%) down-regulated compared with that on BAF-B03, BM, or BRv12 cells (Fig. 3,B); 2) although expression of total β1 on BRv12 and BMRv12 cells is comparable, the amount of activated β1 on BMRv12 cells is partly (∼30%) down-regulated compared with that on BRv12 cells (Fig. 4,B); 3) attachment of BMRv12 cells to Fn is weaker than that of BRv12 cells (Fig. 5,A). 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-α4 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.

We are grateful to Drs. T. Kataoka and K. Miyake for providing pEF BOS-HA-Rasv12 vector and anti-α4 mAb, respectively. We also thank Drs. L. E. Samelson and A. Kukula for critical reading of the manuscript.

1

This work was 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, Kawanishi Pharma Research Institute; by Daiichi Pharmaceutical; and by the Kanae Foundation For Life and Socio-Medical Science. Z.-J.L was supported by a Grant-in-Aid for Japan Society for the Promotion of Science Fellows.

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.

1
Rosales, C., V. O’Brien, L. Kornberg, R. Juliano.
1995
. Signal transduction by cell adhesion receptors.
Biochim. Biophys. Acta
1242
:
77
2
Hynes, R. O..
1992
. Integrins: versatility, modulation, and signalling in cell adhesion.
Cell
69
:
11
3
Elices, M. J., L. Osborn, Y. Takada, C. Crouse, S. Luhowskyj, M. E. Hemler, R. R. Lobb.
1990
. 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
4
Freedman, A. S., J. M. Munro, G. E. Rice, M. P. Bevilacqua, C. Morimoto, B. W. McIntyre, K. Rhynhart, J. S. Pober, L. M. Nadler.
1990
. Adhesion of human B cells to germinal centers in vitro involves VLA-4 and INCAM-110.
Science
249
:
1030
5
Miyake, K., K. Medina, K. Ishihara, M. Kimoto, R. Auerbach, P. W. Kincade.
1991
. A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture.
J. Cell Biol.
114
:
557
6
Ginsberg, M. H., X. Du, E. F. Plow.
1992
. Inside-out integrin signalling.
Curr. Opin. Cell Biol.
4
:
766
7
Pacifici, R., J. Roman, R. Kimble, R. Civitelli, C. M. Brownfield, C. Bizzarri.
1994
. Ligand binding to monocytes α5β1 integrin activates the α2β1 receptor via the α5 subunit cytoplasmic domain and protein kinase C.
J. Immunol.
153
:
2222
8
Gimond, C. A., A. De Melker, M. Aumailley, A. Sonnenberg.
1995
. The cytoplasmic α6A integrin subunit is an in vitro substrate for protein kinase C.
Exp. Cell Res.
216
:
232
9
Shimizu, Y., J. Y. Mobley, L. D. Finkelstein, A. S. Chan.
1995
. A role for phosphatidylinositol 3-kinase in the regulation of β1 integrin activity by the CD2 antigen.
J. Cell Biol.
131
:
1867
10
Zhang, J., J. Zhang, S. J. Shattil, M. C. Cunningham, S. E. Rittenhouse.
1996
. Phosphoinositide 3-kinase γ and p85/phosphoinositide 3-kinase in platelets: relative activation by thrombin receptor or β-phorbol myristate acetate and roles in promoting the ligand-binding function of αIIbβ3 integrin.
J. Biol. Chem.
271
:
6265
11
Hunter, T..
1997
. Oncoprotein networks.
Cell
88
:
333
12
Weinberg, R. A..
1997
. The cat and mouse games that genes, viruses, and cells play.
Cell
88
:
573
13
Land, H., L. F. Parada, R. A. Weinberg.
1983
. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes.
Nature
304
:
596
14
Lee, W. M. F., M. Schwab, D. Westaway, H. E. Varmus.
1985
. Augmented expression of normal c-myc is sufficient for cotransformation of rat embryo cells with a mutant ras gene.
Mol. Cell Biol.
5
:
3345
15
Sinn, E., W. Muller, P. Pattengale, I. Tepler, R. Wallace, P. Leder.
1987
. Coexpression of MMTV/v-Ha-ras and MMTV/c-myc genes in transgenic mice: synergistic action of oncogenes in vivo.
Cell
49
:
465
16
Symons, M. H., T. J. Mitchison.
1992
. A GTPase controls cell-substrate adhesion in Xenopus XTC fibroblasts.
J. Cell Biol.
118
:
1235
17
Hughes, P. E., M. W. Renshaw, M. Pfaff, J. Forsyth, V. M. Keivens, M. A. Schwartz, M. H. Ginsberg.
1997
. Suppression of integrin activation: a novel function of a Ras/Raf-initiated MAP kinase pathway.
Cell
88
:
521
18
Zhang, Z., K. Vuori, H.-G. Wang, J. C. Reed, E. Ruoslahti.
1996
. Integrin activation by R-ras.
Cell
85
:
61
19
Landanna, C., J. J. Campbell, E. C. Butcher.
1996
. Role of Rho in chemoattractant-activated leukocyte adhesion through integrins.
Science
271
:
981
20
Nobes, C. D., A. Hall.
1995
. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.
Cell
81
:
53
21
Tapon, N., A. Hall.
1997
. Rho, Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton.
Curr. Opin. Cell Biol.
9
:
86
22
Hall, A..
1998
. Rho GTPases and the actin cytoskeleton.
Science
279
:
509
23
Qin, R-G., J. Chen, D. Kirn, F. McCormick, M. Symons.
1995
. An essential role for Rac in Ras transformation.
Nature
374
:
457
24
Joneson, T., M. A. White, M. H. Wigler, D. Bar-Sagi.
1996
. Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of Ras.
Science
271
:
810
25
Nimnual, A. S., B. A. Yatsula, D. Bar-Sagi.
1998
. Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos.
Science
279
:
560
26
Inghirami, G., F. Grignani, L. Sternas, L. Lombardi, D. M. Knowles, R. Dalla-Favera.
1990
. Down-regulation of LFA-1 adhesion receptors by c-myc oncogene in human B lymphoblastoid cells.
Science
250
:
682
27
Miyake, S., T. Sakurai, K. Okumura, H. Yagita.
1994
. Identification of collagen and laminin receptor integrins on murine T lymphocytes.
Eur. J. Immunol.
24
:
2000
28
Miyake, K., I. L. Weissman, J. S. Greenberger, P. W. Kincade.
1991
. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis.
J. Exp. Med.
173
:
599
29
Yanai, N., C. Sekine, H. Yagita, M. Obinata.
1994
. Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis.
Blood
83
:
2844
30
Nishimura, T., H. Yagi, H. Yagita, Y. Uchiyama, Y. Hashimoto.
1985
. Lymphokine-activated cell-associated antigen involved in broad-reactive killer cell-mediated cytotoxicity.
Cell Immunol.
94
:
122
31
Sanchez-Madrid, F., P. Simon, S. Thompson, T. A. Springer.
1983
. Mapping of antigenic and functional epitopes on the α- and β-subunits of two related mouse glycoproteins involved in cell interations, LFA-1 and MAC-1.
J. Exp. Med.
158
:
586
32
Takahashi, K., T. Nakamura, M. Koyanagi, K. Kato, Y. Hashimoto, H. Yagita, K. Okumura.
1990
. 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
33
Noto, K., K. Kato, K. Okumura, H. Yagita.
1995
. Identification and functional characterization of mouse CD29 with a mAb.
Int. Immunol.
7
:
835
34
Lenter, M., H. Uhlig, A. Hamann, P. Jenš, B. Imhof, D. Vestweber.
1993
. A monoclonal antibody against an activation epitope on mouse integrin chain β1 blocks adhesion of lymphocytes to the endothelial integrin α6β1.
Proc. Natl. Acad. Sci. USA
90
:
9051
35
Yasuda, M., Y. Hasunuma, H. Adachi, C. Sekine, T. Sakanishi, H. Hashimoto, C. Ra, H. Yagita, K. Okumura.
1995
. Expression and function of fibronectin binding integrins on rat mast cells.
Int. Immunol.
7
:
251
36
Ni, J., A. G. Porter, D. Hollander.
1995
. β7 Integrins and other cell adhesion molecules are differentially expressed and modulated by TNFβ in different lymphocyte populations.
Cell Immunol.
161
:
166
37
Seko, Y., H. Matsuda, K. Kato, Y. Hashimoto, H. Yagita, K. Okumura, Y. Yazaki.
1993
. Expression of intracellular adhesion molecule-1 in murine hearts with acute myocarditis caused by coxsackievirus B3.
J. Clin. Invest.
91
:
1327
38
Hession, C., P. Moy, R. Tizard, P. Chisholm, C. Williams, M. Wysk, L. Burkly, K. Miyake, P. Kincade, R. Lobb.
1992
. Cloning of murine and rat vascular cell adhesion molecule-1.
Biochem. Biophys. Res. Commun.
183
:
163
39
Palacios, R., M. Steinmetz.
1985
. 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
40
Tamada, M., C.-D. Hu, K.-I. Kariya, T. Okada, T. Kataoka.
1997
. Membrane recruitment of Raf-1 is not the only function of Ras in Raf-1 activation.
Oncogene
15
:
2959
41
Battey, J., C. Moulding, R. Taub, W. Murphy, T. Stewart, H. Potter, G. Lenoir, P. Leder.
1983
. The human c-myc oncogene: structural consequences of translocation into the IgH locus in Burkitt lymphoma.
Cell
34
:
779
42
Doi, T., M. Hatakeyama, S. Minamoto, T. Kono, H. Mori, T. Taniguchi.
1989
. Human interleukin 2 (IL-2) receptor β chain allows transduction of IL-2-induced proliferation signal(s) in a murine cell line.
Eur. J. Immunol.
19
:
2375
43
Miyazaki, T., Z.-J. Liu, A. Kawahara, Y. Minami, K. Yamada, Y. Tsujimoto, E. L. Barsoumian, R. M. Perlmutter, T. Taniguchi.
1995
. Three distinct IL-2 signaling pathways mediated by bcl-2, c-myc, and lck cooperate in hematopoietic cell proliferation.
Cell
81
:
223
44
Minami, Y., Y. Nakagawa, A. Kawahara, T. Miyazaki, K. Sada, H. Yamamura, T. Taniguchi.
1995
. 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
45
Tanaka, Y., D. H. Adams, S. Hubscher, H. Hirano, U. Siebenlist, S. Shaw.
1993
. T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1β.
Nature
361
:
79
46
Tanaka, Y., K. Kimata, A. Wake, S. Mine, I. Morimoto, N. Yamakawa, H. Habuchi, S. Ashikari, H. Yamamoto, K. Sakurai, K. Yoshida, S. Suzuki, S. Eto.
1996
. Heparan sulfate proteoglycan on leukemic cells is primarily involved in integrin triggering and its mediated adhesion to endothelial cells.
J. Exp. Med.
184
:
1987
47
Adams, D. H., L. Harvath, D. P. Bottaro, R. Interrante, G. Catalano, Y. Tanaka, A. Strain, S. G. Hubscher, S. Shaw.
1994
. Hepatocyte growth factor and macrophage inflammatory protein 1β: structurally distinct cytokines that induce rapid cytoskeletal changes and subset-preferential migration in T cells.
Proc. Natl. Acad. Sci. USA
91
:
7144
48
Erle, D. J., C. Ruegg, D. Sheppard, D. Pytela.
1991
. Complete amino acid sequence of an integrin β subunit (β7) identified in leukocytes.
J. Biol. Chem.
266
:
11009
49
Lowe, D. G., D. J. Capon, E. Delwart, A. Y. Sakaguchi, S. L. Naylor, D. V. Goeddel.
1987
. Structure of the human and murine R-ras genes, novel genes closely related to ras proto-oncogenes.
Cell
48
:
137
50
Urano, T., R. Emkey, L. A. Feig.
1996
. Ral-GTPases mediates a distinct downstream signaling pathway from Ras that facilitates cellular transformation.
EMBO J.
15
:
810
51
Osborn, L., C. Hession, R. Tizard, C. Vassallo, S. Luhowskyj, G. Chi-Rosso, R. Lobb.
1989
. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes.
Cell
59
:
1203
52
Rice, G. E., M. P. Bevilacqua.
1989
. An inducible endothelial cell surface glycoprotein mediates melanoma adhesion.
Science
246
:
1303
53
Schleimer, R. P., S. A. Sterbinsky, J. Kaiser, C. A. Bickel, D. A. Klunk, K. Tomioka, W. Newman, F. W. Luscinskas, M. A. Gimbrone, B. W. McIntyre, B. S. Bochner.
1992
. IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium: association with expression of VCAM-1.
J. Immunol.
148
:
1086
54
Judware, R., L. A. Culp.
1995
. Over-expression of transfected N-myc oncogene in human SKNSH neuroblastoma cells down-regulates expression of β1 integrin subunit.
Oncogene
11
:
2599
55
Judware, R., L. A. Culp.
1997
. Concomitant down-regulation of expression of integrin subunits by N-myc in human neuroblastoma cells: differential regulation of α2, α3, and β1.
Oncogene
14
:
1341
56
Hynes, R., I. Ali, A. Destree, V. Maunter, M. Perkins, D. Senger, D. Wagner, K. Smith.
1978
. A large glycoprotein lost from the surfaces of transformed cells.
Ann. NY Acad. Sci.
312
:
317
57
Schreiner, C., M. Fisher, S. Hussein, R. L. Juliano.
1991
. Increased tumorigenicity of fibronectin receptor deficient chinese hamster ovary cell variants.
Cancer Res.
51
:
1738
58
Rosen, G. D., J. R. Sanes, R. LaChance, J. M. Cunningham, J. Roman, D. C. Dean.
1992
. Role of the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis.
Cell
69
:
1107
59
Liu, Z.-J., Y. Tanaka, S. Mine, A. Morinobu, H. Yagita, K. Okumura, T. Taniguchi, H. Yamamura, Y. Minami.
1998
. Functional co-operation of cyclin C and c-Myc in mediating homotypic cell adhesion via VLA-4 activation and VCAM-1 induction.
Blood
92
:
4700
60
Ryan, D. H., B. L. Nuccie, C. N. Abboud, J. M. Winslow.
1991
. 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
61
Teixido, J., M. E. Hemler, J. S. Greenberger, P. Anklesaria.
1992
. Role of β1 and β2 integrins in the adhesion of human CD34hi stem cells to bone marrow stroma.
J. Clin. Invest.
90
:
358
62
Morales-Ducret, J., E. Wayner, M. J. Elices, J. M. Alvaro-Garcia, N. J. Zvaifler, G. S. Firestein.
1992
. α4β1 integrin (VLA-4) ligands in arthritis: vascular cell adhesion molecule-1 expression in synovium and on fibroblast-like synoviocytes.
J. Immunol.
149
:
1424
63
Weg, V. B., T. J. Willians, R. R. Lobb, S. Nourshargh.
1993
. A monoclonal antibody recognizing very late activation antigen-4 inhibits eosinophil accumulation in vivo.
J. Exp. Med.
177
:
561
64
Taichman, D. B., M. I. Cybulsky, I. Djaffar, B. M. Longenecker, J. Teixido, G. E. Rice, A. Aruffo, M. P. Bevilacqua.
1991
. Tumor cell surface α4β1 integrin mediates adhesion to vascular endothelium: demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-1.
Cell Regul.
2
:
347
65
Garofalo, A., R. G. S. Chirivi, C. Foglieni, R. Pigott, R. Mortarini, I. Martin-Padura, A. Anichini, A. J. Gearing, F. Sanchez-Madrid, E. Dejana, R. Giavazzi.
1995
. Involvement of the very late antigen 4 integrin on melanoma in interleukin 1-augmented experimental metastases.
Cancer Res.
55
:
414