(Received for publication, April 21, 1995; and in revised form, June 14, 1995)
From the
Few molecules have been shown to confer cell motility. Although
the motility-arresting properties of anti-CD9 monoclonal antibody (mAb)
suggest the transmembrane 4 superfamily (TM4SF) member CD9 can induce a
motorgenic signal, gene transfection studies have failed to confirm
this hypothesis. We report here that ectopic expression of human CD9
(CD9h) and feline CD9 (CD9f) in the CD9-negative, poorly motile, human
B cell line Raji dramatically enhances migration across fibronectin-
and laminin-coated polycarbonate filters. Migration of Raji/CD9h and
Raji/CD9f on either substrate was inhibited by the anti-CD9 mAb 50H.19
and by the anti-1 integrin mAb AP-138. Migration of Raji/CD9h on
laminin was potently inhibited by the anti-VLA-6 integrin mAb GoH3 and
by the anti-VLA-4 integrin mAb 44H6, whereas migration of Raji/CD9h on
fibronectin was inhibited only by mAb 44H6. Since CD9h-transfected Raji
cells adhered to fibronectin as effectively as mock transfectants,
expression of CD9 enhanced motility, but not adhesion. CD9-enhanced
migration was inhibited by the protein tyrosine kinase inhibitor
herbimycin A suggesting that tyrosine phosphorylation played a role in
the generation of a motorgenic signal. Raji/CD9h transfectants adherent
to fibronectin expressed 6-fold higher levels of phosphotyrosine than
Raji. Raji/CD9f transfectants also phosphorylated proteins on tyrosine
more effectively than Raji including a protein of 110 kDa which was
phosphorylated on the motility-inducing substrates laminin and
fibronectin, but not on bovine serum albumin. Our results support a
role for CD9 in the amplification of a motorgenic signal in B cells
involving
1 integrins and the activation of protein tyrosine
kinases.
CD9, a 22-24-kDa cell surface glycoprotein, highly
expressed in developing B cells, blood platelets, neuroblastoma cell
lines, normal and transformed epithelia, peripheral glia, and neurones (1, 2, 3, 4) is a member of the
transmembrane 4 protein superfamily (TM4SF). ()TM4SF
proteins possess two external loops and four hydrophobic domains of
membrane-spanning length (5, 6, 7, 8) . The putative
transmembrane regions and certain residues within the external loops
are highly conserved suggesting the proteins perform closely related
functions. However, the nature of those functions is not well
understood(6) . There is evidence that TM4SF proteins play a
role in the initiation of signals controlling cell proliferation. For
example, CD53 is found exclusively on subsets of proliferating
thymocytes(9) , CD81 exerts both positive and negative effects
on the proliferation of T and B lymphoid cell lines(10) , and
an anti-CD9 mAb was recently shown to induce the proliferation of
Schwann cells(11) . Some family members may also regulate
adhesive and morphogenetic functions. CD9 is expressed at high density
on peripheral blood platelets(12) , and anti-CD9 mAb are
exceptional platelet agonists (2, 13) when Fc-receptor
interactions are not impeded(14) . Immobilized, but not
soluble, F(ab`)
fragments of anti-CD9 mAb activate
platelets(15) , and immobilized, but not soluble, antibody
induces proliferation in nerve cells(11) . These findings
suggest that CD9 may transduce signals involving immobilized ligands
such as extracellular matrix proteins. Integrins are heterodimeric
adhesion molecules linking extracellular matrix proteins to an active
cytoskeleton(16) . We have reported that anti-CD9 mAb promote
physical association between CD9 and the
3 integrin
GPIIb-IIIa(17) , and that anti-CD9 mAb induce homotypic and
heterotypic adhesive interactions in pre-B cells through pathways which
may involve
1 integrins(18, 19) . Very recently,
these observations have been extended by a report that CD9 physically
associates with
1 integrins(20) . CD9 may therefore
interact with integrins to participate in the transduction of
integrin-dependent signals across the plasma membrane (17, 20) .
Antibodies which uniquely prevent cell
motility in a variety of tumor cell lines were found to recognize a
protein subsequently identified as CD9 suggesting that CD9 is a
positive regulator of cell motility(14) . However, ectopic
expression of CD9 in heterologous tumor cell lines either failed to
confer motility (20, 21) or in some cases suppressed
migratory and metastatic activity(21) . We chose to study CD9
function in cells of the B lymphocyte lineage in which CD9 expression
is modulated in a stage-specific manner. Pre-B lymphocytes express high
levels of CD9, and we have observed that transformed pre-B cell lines
readily penetrate fibronectin-coated polycarbonate filters. B cell
lines on the other hand lack CD9 and are poorly migratory in Transwell
assays(21) . To investigate the possibility that CD9 plays a
role in integrin-dependent motility, we transfected cDNA encoding human
and feline CD9 into the immature B cell Raji which lacks CD9. Raji
expresses the 1 integrin VLA-4, a fibronectin receptor found in
highly motile cells (22, 23, 24, 25, 26) implicated
in invasiveness, and metastasis(22, 23) , as well as
the laminin receptor VLA-6. In our experiments, Raji cells penetrated
laminin or fibronectin-coated filters poorly, suggesting they might
lack an accessory molecule required for migration on integrin-dependent
substrates. We report here that ectopic expression of CD9 in Raji cells
exposed to laminin and fibronectin dramatically enhanced both their
ability to penetrate polycarbonate filters and their ability to
phosphorylate protein targets on tyrosine.
Figure 1: Ectopic expression of CD9 in Raji. Cell lysates from the CD9+ pre-B cell line HOON (lanes A and B), the CD9- B cell line Raji (lanes C and D), and Raji transfected with a full-length cDNA encoding human CD9 (lanes E and F) were immunoprecipitated with the anti-CD9 mAb 50H.19/Protein A-Sepharose (lanes A, C, and E) or with Protein A-Sepharose alone (lanes B, D, and F). Following SDS-PAGE, the proteins were immunoblotted with horseradish peroxidase-conjugated mAb 50H.19. Molecular size markers: 116 kDa, 66 kDa, 45 kDa, and 31 kDa.
Figure 2: FACS analysis of VLA antigen and CD9 expression on Raji, Raji transfected with human CD9 (Raji/CD9h), and Raji transfected with feline CD9 (Raji/CD9f). Cells were stained with mAb against VLA antigens and CD9 as described under ``Experimental Procedures'' followed by a fluorescein isothiocyanate-conjugated second antibody and analyzed on a FACScan. The fluorescence profiles of stained cells (black) are compared to isotype controls (white).
Figure 3: A, motility of Raji and of Raji/CD9 transfectants on fibronectin-coated filters. Motility was assessed by counting the number of cells penetrating the lower chamber of a Transwell apparatus through a perforated polycarbonate filter (8-µ diameter pore). The abscissa shows the coating concentration of plasma fibronectin. Error bars = 1 S.D. The result is representative of six experiments. B, adhesion of Raji and Raji/CD9 to fibronectin-coated plates. Cells were plated on wells coated with varying amounts of plasma fibronectin, and adhesion-quantitated after 90 min by mechanically agitating the plate and counting the nonadherent cells. The abscissa shows the coating concentration of fibronectin. The result is representative of five experiments. Error bars = 1 S.D.
Since CD9 is reported to associate
with 1 integrins(20) , we investigated whether
1
integrins were involved in cell migration on fibronectin by
preincubating the cells with mAb AP-138, an antibody which recognizes
the
1 integrin subunit. mAb AP-138 inhibited cell motility by 59%
indicating that
1 integrins play a regulatory role in the
migratory behavior (Table 1). In keeping with reports that
anti-CD9 mAb block cell movement in a variety of transformed
cells(20, 21, 29) , we observed that the
anti-CD9 mAb 50H.19 inhibited migration of the transfectants by 70.4% (Table 1). mAb 50H.19 and mAb AP-138 also blocked motility of the
highly motile pre-B cell line HOON by 71.2 and 72.4%, respectively.
Transfection of CD9 therefore confers motility upon a poorly motile B
cell line which is inhibitable by anti-CD9 mAb in the manner of pre-B
cells which constitutively express the protein.
Figure 4: A, migration of Raji and Raji/CD9h through laminin-coated polycarbonate filters. Polycarbonate filters were coated with between 1 and 10 µg/ml of laminin, and cells were applied to the upper chamber of a Transwell apparatus. Cells penetrating to the lower chamber over 18 h were quantitated by electronic cell counting. B, migration of Raji and Raji/CD9f through laminin-coated polycarbonate filters. Polycarbonate filters were coated with between 1 and 10 µg/ml of laminin, and the number of cells penetrating to the lower chamber of a Transwell apparatus was determined over 18 h by electronic cell counting.
Figure 5: Migration of Raji/CD9f through polycarbonate filters. Polycarbonate filters were coated with 10 µg/ml plasma fibronectin, or laminin, and the percentage of cells penetrating to the lower chamber of a Transwell apparatus was determined over 18 h by electronic cell counting. Error bars represent 1 S.D. about the mean.
Figure 6:
Effect of antibodies to VLA- antigens and
CD9 on the migration of Raji/CD9h and Raji/CD9f through laminin- or
fibronectin-coated polycarbonate filters. Cells were preincubated with
the anti-CD9 mAb 50H.19, the anti-1 subunit mAb AP-138, the
anti-VLA-4 mAb 44H6, the anti-VLA-6 mAb GoH3, or a combination of mAb
44H6 and GoH3 at 10 µg/ml for 30 min at 37° C before
introduction into the upper chamber of a Transwell apparatus. Cells
penetrating to the lower chamber were quantitated over 18 h. A, Raji/CD9 on laminin-coated filters. B, Raji/CD9h
on fibronectin-coated filters. C, Raji/CD9f on laminin-coated
filters. D, Raji/CD9f on fibronectin-coated filters. Laminin
and fibronectin were coated at a concentration of 10
µg/ml.
Figure 7:
Fibronectin-stimulated tyrosine
phosphorylation of Raji and Raji/CD9. Graded numbers of cells were
allowed to attach to fibronectin-coated surfaces (5 µg/ml plasma
fibronectin) for 90 min in the presence or absence of sodium
orthovanadate (OV), lysed in RIPA buffer, and the proteins
were separated by SDS-PAGE and immunoblotted with horseradish
peroxidase-conjugated anti-phosphotyrosine antibody. Densitometric
comparison of phosphotyrosine bands obtained from 4 10
cells gave a ratio of Raji:Raji/CD9 of 1:5.9 (130 kDa) and 1:6.2
(69 kDa), respectively. The result is representative of five
experiments.
Figure 8:
Induction of phosphotyrosine in Raji and
Raji/CD9f by exposure to fibronectin, laminin, and BSA. 4
10
cells were allowed to adhere to plastic surfaces coated
with BSA, laminin (LN), and fibronectin (FN) at 10
µg/ml for 15 h before lysis in RIPA buffer, analysis by SDS-PAGE,
and immunoblotting with horseradish peroxidase-conjugated
anti-phosphotyrosine mAb.
Cell motility is a complex process involving extracellular matrix proteins, adhesion receptors, cytoskeletal components, and signaling molecules. A number of agents have been identified which modulate motility including fibronectin and certain growth factors, but few transmembrane molecules other than integrins have been shown to play key roles(33) . CD9 (29) and the hyaluronan receptor RHAMM (34) are two non-integrin cell surface proteins demonstrated to affect motility. However, although CD9 was first identified through exhaustive selection of mAbs which block motile function, experimental investigation of CD9 function by gene transfection has not supported a role for CD9 as a motorgenic molecule. For example, transfection of CD9 into the CD9-positive human lung adenocarcinoma cell line MAC10 led to a reduction rather than gain in cellular motility(21) . This might be explained by a gene dosage effect in which overexpression of CD9 is inhibitory. However, overexpression cannot explain why transfection of CD9 into the CD9-negative human myeloma cell line ARH77 also suppressed motility(21) , or why transfection of CD9 into the T cell line CEM failed to influence motility at all(20) . Furthermore, the conclusion that CD9 is a suppressor of cell motility and metastasis (21) seems at odds with its widespread expression in carcinoma cell lines and biopsy specimens(29) . The evidence to date therefore suggests a complex role for CD9 which may be critically affected by the cellular environment in which CD9 is expressed. Since CD9 is expressed at early and late stages of B cell differentiation, but not in nonactivated B cells, we chose to express CD9 in Raji cells which possess a phenotype typical of an early B lymphocyte.
Our
findings partially redress the paradox of CD9 function by demonstrating
that Raji cells expressing physiological levels of CD9 and 1
integrins provide a permissive environment for CD9-dependent motility
on extracellular matrix proteins which can be blocked by antibodies
against CD9 and the two major integrins. The finding that both human
and feline CD9 effect very similar increases in motility involving
1 integrins confirms that CD9 modulates migratory responses to
extracellular matrix proteins and is consonant with the 95.1% homology
in amino acid sequence between the two proteins(24) . However,
the effectiveness of anti-VLA-6 mAb in inhibiting the motile response
of Raji/CD9f, but not of Raji/CD9h, on fibronectin substrates suggests
a regulatory role for those regions of CD9 which are differentially
expressed including amino acids 169-180 of the second external
loop and a potential N-linked glycosylation site within the
first external loop(24) . Our finding that an anti-VLA-4 mAb
inhibited migration of the transfectants on laminin to which VLA-4 is
not known to bind was unexpected. Although VLA-4 is recognized to play
a major role in the migratory activity of several cells, VLA-6 has been
not been linked to cell motility, but rather to the regulation of
differentiated events(35, 36) . However, a recent
report that an anti-VLA-6 mAb could partially inhibit pre-B cell
transmigration under human bone marrow stroma suggests that VLA-6 may
also function in cooperation with other adhesion molecules to modulate
motile behavior(37) . Possibly in our experiment, VLA-4 affects
VLA-6-dependent motility through lateral interactions between VLA-6 and
sites of VLA-4 cytoskeletal association.
The presence of enhanced
levels of tyrosine-phosphorylated proteins in CD9-transfected cells and
the sensitivity of CD9-enhanced motility to inhibitors of tyrosine
kinases suggest a connection between tyrosine phosphorylation and
motility. Platelet-derived growth factor(38) , epidermal growth
factor(27, 39) , and hepatocyte growth factor (40) have all recently been shown to induce cell motility
through stimulation of their respective receptor protein tyrosine
kinases. Integrin-dependent motility may be targeted by growth factors
since it was recently reported that epidermal growth factor could
induce motility through the selective modulation of events under the
control of the integrin vitronectin receptor v
5(28) .
In that study, the effect of epidermal growth factor was specific for
motility, but had no effect upon cellular adhesion. Similarly, in the
present study, CD9 modulated motility, but not adhesion, to
fibronectin. In order to migrate, cells need to re-organize their actin
networks through local assembly and deassembly of actin. Actin is
assembled at points of substrate adherence known as focal contacts
which also serve as sites of integrin localization. Recently it was
reported that the hyaluronan receptor RHAMM promotes motility at least
in part through tyrosine phosphorylation of the
1 integrin target,
the focal adhesion kinase pp125
(41) . CD9,
through its ability to physically associate with
1 integrins,
could therefore participate in either of these mechanisms. There is
evidence that lymphocytes may transduce integrin-dependent signals
through phosphorylation of proteins other than the focal adhesion
kinase pp125
. For example, stimulation of B lineage cells
by fibronectin or by specifically cross-linking VLA-4 is reported to
phosphorylate a protein of 105-110 kDa on tyrosine(32) .
The specific phosphorylation of a protein of this size in feline CD9
transfectants exposed to the motorgenic substrates laminin and
fibronectin, but not to BSA, could possibly implicate CD9 as an
accessory component of VLA-4 signaling.
B cell development is
accompanied by pronounced stage-specific changes in fibronectin
receptor expression and cellular avidity, suggesting that interactions
with fibronectin are of prime importance to B cell
maturation(30, 42) . We provide evidence that CD9, a
putative 1 integrin accessory molecule expressed during early, but
not late, B cell development, dramatically amplifies tyrosine
phosphorylation and motility on fibronectin and laminin on
reconstitution in a B cell. Such interactions are likely to be relevant
to pre-B cell exploitation of inductive microenvironments within the
bone marrow stroma and to the ability of leukemic cells and solid tumor
cells to gain the motility required to disseminate to distant sites.
Our results extend previous work indicating a physical association
between CD9 and members of the
1 integrin family (17, 20) by suggesting that such associations may
serve to amplify the ability of
1 integrins to activate protein
tyrosine kinase-dependent signal pathways regulating motile, but not
adhesive, behavior.