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(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-
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). ( 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
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
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-
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
Figure 8:
Induction of phosphotyrosine in Raji and
Raji/CD9f by exposure to fibronectin, laminin, and BSA. 4
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 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 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
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24092-24099
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
1 Integrin-dependent Motility on Fibronectin and Laminin
Substrates and Enhanced Tyrosine Phosphorylation (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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) .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.
Molecular Cloning of CD9
A full-length cDNA
encoding CD9 was selected using the anti-CD9 mAb 50H.19 from an
endothelial cDNA library assembled in the expression vector gt11
(Clontech) and the 1.2-kilobase insert subcloned into PTZ-19,
transformed into XL1-Blue, and single-stranded template DNA isolated
for sequencing. The clone contains the entire coding region for CD9
flanked by 51-base pair 5`- and 450 base pair 3`-untranslated regions
and is identical with the published coding sequence(8) . The
insert was cloned into the eukaryotic Epstein Barr virus episomal
plasmid expression vector pREP4 (Invitrogen), transfected into Raji by
electroporation, and selected by hygromycin resistance, followed by
immunoselection on immobilized mAb 50H.19. Mock-transfected controls
containing the vector alone were selected on the basis of
hygromycin-resistance. A full length cDNA encoding feline CD9 was
obtained using the rapid amplification cDNA ends technique as
described(24) , cloned into the cDNA3 expression vector, and
transfected into the same strain of Raji by electroporation, followed
by immunoselection with anti-CD9 mAb.
Cell Lines, mAb, and FACS Analysis
HOON and Raji
are pre-B cell and B cell lines, respectively. They were obtained from
Dr. Michelle Letarte (University of Toronto) and Dr. B. M. Longenecker
(University of Alberta) and maintained in RPMI 1640, 10% fetal calf
serum. For FACS analysis cells were stained with mAb TS2/7 against
VLA-1 (T Cell Diagnostics, Cambridge, MA), mAb P1E6 against VLA-2,
(Life Technologies, Inc.), mAb P1B5 against VLA-3 (Life Technologies,
Inc.), mAb 44H6 against VLA-4 (kindly provided by Dr. Michelle
Letarte), mAb P1D6 against VLA-5 (Life Technologies, Inc.), and mAb
GoH3 against VLA-6 (AMAC, BioCan, Mississauga, Ontario) using a FACScan
cell sorter (Becton Dickinson) as described previously(18) .
mAb 50H.19 is an anti-CD9 mAb first raised against a melanoma cell
line(25) . mAb 4B4 (Coulter Corp., Miami, FL) and mAb AP-138
recognize the 1 integrin subunit. Isotype-matched controls were
included, and >10,000 events accumulated per sample. Data were
analyzed using the Lysis II program.Immunoprecipitation and Immunoblotting
10
HOON cells were lysed in 0.5 ml of RIPA buffer, precleared with
Protein A-Sepharose, and immunoprecipitated by addition of 5 µg of
the anti-CD9 mAb 50H.19 together with 30 µl of preswollen Protein
A-Sepharose or Protein A-Sepharose alone, and eluted with SDS-PAGE
loading buffer. Following separation on a 5-20% gradient gel, the
proteins were transferred to nitrocellulose and immunoblotted with
horseradish peroxidase-conjugated mAb 50H.19. Positive bands were
detected by enhanced chemiluminescence (Amersham International,
Buckinghamshire, United Kingdom).Adhesion Assay
Cells were grown in RPMI 1640
supplemented with 10% fetal calf serum and washed in RPMI containing
0.5% BSA. 2 
10 cells in a volume of 250 µl of
RPMI 1640, 0.5% BSA were added to a 24-well plate in which wells were
precoated for 2 h with varying concentrations of human plasma
fibronectin (Life Technologies, Inc.) in phosphate-buffered saline at
37° C and blocked for 1 h with 1% BSA(26) . Control wells
were incubated with BSA alone. The effect of inhibitors was determined
by preincubation with the agent for 20 min at 23° C and addition to
the assay without further dilution. Cells were preincubated at 37°
C for 90 min before dislodging the loosely adherent cells by agitation
on a rotary shaker at 110 rpm for 5 min. Detached cells were
quantitated in an automated cell counter (Coulter Electronics, Hialeah,
FL).Motility Assay
Cells were washed and resuspended
in RPMI 1640, 0.5% BSA. 1.5 10
cells in a volume of
100 µl were applied to the upper chamber of 6.5-mm diameter
Transwells (Costar, Sin-Can Inc., Calgary, Alberta), and 600 µl of
RPMI 1640, 0.5% BSA were added to the lower chamber. Polycarbonate
filters (8-µ diameter pores) were precoated with 100 µl of
protein solution for 2 h and blocked with 1% BSA(26) . Cells
were incubated for 18 h at 37° C, and cells migrating to the lower
chamber were quantitated in an automated cell counter (Coulter
Electronics). Examination of the lower surface of the filter confirmed
that transmigrating cells did not adhere to the filter, but accumulated
in the lower chamber.Analysis of Phosphotyrosine
250-µl aliquots of
Raji or Raji/CD9 transfectants at concentrations of 5 10 to 8 10 cells/ml were introduced into the
wells of a 24-well tissue culture plate (Costar) precoated with
fibronectin and blocked with BSA. Cells were incubated for 90 min at
37° C and then lysed in RIPA buffer (pH 8.0) containing 0.02 M Tris-HCl, 0.001 M NaHPO
, 137
mM NaCl, 0.5% Nonidet P-40, 0.25% sodium deoxycholate; 1
µg/ml leupeptin, pepstatin A, antipain, and Trasylol; 1 mM EGTA; 1 mM iodoacetamide; 1 mM sodium
orthovanadate. Proteins were separated by 5-20% SDS-PAGE,
transferred to nitrocellulose, immunoblotted with the horseradish
peroxidase-conjugated anti-phosphotyrosine mAb PY20 (Transduction
Laboratories, Lexington, KY), and developed using enhanced
chemiluminescence.
Ectopic Expression of CD9 in Raji
CD9 was
strongly expressed in lysates of Raji cells transfected with the human
CD9 cDNA insert (Raji/CD9h), but not in Raji transfected with pREP4
alone (Fig. 1, lanes E and C). The level of
expression was very similar to that of the highly motile pre-B cell
line HOON (Fig. 1, lane A). FACS analysis demonstrated
that our strain of Raji cells expressed predominantly VLA-4 and VLA-6
among 1 integrins, and that ectopic expression of CD9 did not
qualitatively affect their VLA profile (Fig. 2). However,
ectopic expression of both human and feline CD9 markedly increased the
expression of VLA-6 (Fig. 2) suggesting that CD9 may
preferentially affect the transport or assembly of this integrin.
CD9 Enhances Motility of Raji on Fibronectin
When
Raji and Raji/CD9 transfectants were compared for their ability to
transmigrate across fibronectin-coated polycarbonate filters, the
transfectants exhibited a dramatically enhanced migratory capacity (Fig. 3A). Migration of the transfectants increased in
proportion to the coating concentration of fibronectin, reaching a
maximum at 3 µg/ml. In contrast, mock-transfected Raji cells barely
migrated except at the highest coating concentration of 10 µg/ml.
Since Raji has the capacity to migrate on fibronectin, but requires a
considerably higher concentration to become motile, it suggests that
CD9 expression may amplify a motorgenic signal induced by fibronectin.
Tyrosine kinases have recently been implicated in cell
motility(27, 28) . We therefore asked whether
preincubation of the cells with the protein tyrosine kinase inhibitor
herbimycin A would affect cell migration. Incubation of cells for 3.5 h
with herbimycin A reduced tyrosine phosphorylation by >70% without
affecting cell viability. Preincubation of Raji/CD9 transfectants with
herbimycin A for this period inhibited migration by almost 80% (Table 1), indicating that tyrosine kinase activity was required
for CD9-enhanced cell migration.
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.CD9 Does Not Confer Enhanced Adhesion to
Fibronectin
B cells exposed to surfaces coated with fibronectin
adhere weakly in comparison to pre-B cells(30) . To investigate
whether CD9 influenced the avidity of cellular adhesion of B
lymphocytes, we quantitated binding of Raji and Raji/CD9 to surfaces
coated with fibronectin. Because B lymphoid cells possess relatively
low avidity for most extracellular matrix proteins, we developed a low
stringency adhesion assay in which the usual method of detaching
loosely adherent cells by washing was replaced by a 10-min period of
mechanical agitation. Using this assay, we found that both Raji and
Raji/CD9 transfectants adhered to fibronectin in a dose-dependent
manner, and that the CD9 transfectants adhered similarly to
mock-transfected controls (Fig. 3B). Since ectopic
expression of CD9 did not significantly affect adhesion of the B cell
line CD9 does not appear to influence motility by increasing the level
of cellular avidity for fibronectin or by inducing a generalized state
of cellular activation. Preincubation of the cells with herbimycin A
did not affect adhesion, indicating that adhesion unlike CD9-enhanced
motility does not require tyrosine phosphorylation (results not shown). CD9-transfected Raji Exhibit Enhanced Migration on
Laminin
Although Raji possesses VLA-6, a laminin receptor, it is
poorly motile on laminin. We asked whether cells transfected with CD9
would show enhanced motility on laminin. Raji/CD9h transfectants
demonstrated a dramatic increase in motility over Raji controls on
polycarbonate filters coated with laminin which increased with the
coating concentration between 1 and 10 µg/ml (Fig. 4A). Raji transfected with feline CD9 also
exhibited a laminin-dependent increase in migration over
mock-transfected controls (Fig. 4B). The ability of CD9
from two different species to confer large increases in cell motility
on laminin substrates strongly suggests that motility enhancement is a
fundamental property of CD9. In a second experiment, Raji/CD9f was
found to show enhanced migration on both fibronectin and laminin, but
not BSA (Fig. 5), confirming that CD9 confers motility on
substrates recognized by the two major Raji VLA-antigens.
CD9 Enhanced Migration Is Inhibitable by Antibodies
against VLA-4 and VLA-6
The ability of anti-1 integrin
subunit mAb to inhibit the migration of Raji/CD9h on extracellular
matrix implicates VLA in the generation of a motorgenic signal. To
investigate which VLA is responsible for the inhibition, cells were
preincubated with the anti-VLA-4 mAb 44H6 and with the anti-VLA-6 mAb
GoH3. Both antibodies strongly inhibited migration of Raji/CD9h on
laminin, and their combination completely prevented cells entering the
lower chamber (Fig. 6A), whereas mAb 44H6, but not mAb
GoH3, significantly inhibited migration on fibronectin-coated filters (Fig. 6B). Since VLA-6 selectively regulates migration
on laminin, but cross-linking VLA-4 affects migration on either laminin
or fibronectin, it suggests that VLA-4 plays an unexpected and
essential role in B cell motility. Cells transfected with feline CD9
were similarly inhibited by both mAbs on laminin substrates, but the
anti-VLA-6 mAb effectively inhibited migration on fibronectin implying
a possible species difference (Fig. 6, C and D). The epitope recognized by the anti-VLA-4 mAb may be
critical since the anti-VLA4 mAb P4G9 had no effect upon migration
(results not shown). mAb against CD9 and the 1 integrin subunit
also effectively inhibited migration of both CD9h and CD9f
transfectants on either substrate (Fig. 6, A, B, C, and D). These results implicate CD9
and the 1 integrin subunit as major determinants of cell motility
on laminin substrates through interactions involving VLA-4 and VLA-6.
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.
The Migratory Subset Is Enriched for CD9
Expression
If CD9 is directly responsible for migratory
activity, we reasoned that CD9 might be preferentially expressed in
cells migrating to the lower chamber of the Transwell apparatus during
the 18-h assay. FACS analysis of this subpopulation produced a mean
fluorescence value for CD9 of 1386.5 compared to 854.3 for the starting
cell population, an enrichment of 162%. In contrast, the mean
fluorescence for 1 integrin expression was essentially unchanged
(1016.47 versus 959.71). Therefore, changes in CD9 expression
rather than changes in 1 expression correlate with the migratory
behavior of the transfected cells. CD9 expression has been associated
with both enhancement and suppression of cell
proliferation(21, 29, 31) . However, in our
hands, the proliferation of cultures of CD9-transfected and
mock-transfected Raji cells in log phase measured over a 4-day interval
were identical (results not shown), indicating that the effects of CD9
expression on motile behavior did not result from differential
proliferative capacity.Raji/CD9 Transfectants Display Increased Tyrosine
Phosphorylation
Adherence to fibronectin or cross-linking of the
1 integrin subunit with mAb is reported to activate protein
tyrosine kinases in B cells and to phosphorylate proteins of
105-110 kda(32) . Since CD9 associates with 1
integrins and enhances migration on fibronectin, we wondered whether
CD9 could affect the ability of cells to phosphorylate proteins on
tyrosine. We therefore investigated whether tyrosine phosphorylation
accompanied contact of Raji or of Raji/CD9 transfectants with
fibronectin substrates (Fig. 7). Tyrosine phosphorylation of
several components was observed in Raji cells adhering to fibronectin,
the major bands migrating at 69 and 130 kDa. Phosphorylation was
observable by 5 min, and the intensity of the signal was observed to
increase progressively over a 90-min period. The pattern of
phosphorylation was identical in both transfected and mock-transfected
cells, indicating that CD9 expression does not influence the
specificity of response. However, CD9 transfectants consistently
displayed enhanced levels of tyrosine phosphorylation. The optical
density of the 130-kDa phosphotyrosine band derived from cells at a
concentration of 4 10 ml was 5.9 greater
for Raji/CD9 cells than for Raji, and the 69-kDa band was 6.2
greater for Raji/CD9 cells than for Raji. Pretreatment of Raji or of
the CD9 transfectants with the tyrosine phosphatase inhibitor sodium
orthovanadate enhanced the level of tyrosine phosphorylation in both
transfected and mock-transfected cells, demonstrating that the
activation of protein tyrosine kinases by immobilized fibronectin is
strongly opposed by tyrosine phosphatase activity (Fig. 7). The
enhanced phosphorylation observed in CD9-positive cells could therefore
reflect a change in tyrosine kinase/phosphatase balance rather than an
increase in overall kinase activity. Whether enhanced phosphorylation
on tyrosine occurred in cells transfected with feline CD9 was
determined by allowing cells to attach to BSA-, laminin-, and
fibronectin-coated surfaces, carefully removing the medium, and lysing
the cells. Cells adherent to all three surfaces strongly phosphorylated
a protein of 72 kDa not seen in mock transfectants, whereas a band of
110 kDa was specifically phosphorylated on the motility-inducing
substrates laminin and fibronectin, but not on cells exposed to BSA (Fig. 8). Consequently, enhanced phosphotyrosine signaling was
observed in CD9-transfected cells adhering to a variety of substrates
and a 110-kDa band specifically observed on targets of the major B cell
integrins. While additional experiments are required to clarify these
issues, the results clearly indicate that CD9-transfected B cells have
a markedly enhanced capacity for tyrosine phosphorylation on
integrin-dependent substrates and may therefore play a role in the
regulation of contact signaling.
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.
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.
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.
v5(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.
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.
)1 integrins; RHAMM, receptor for hyaluronic acid-mediated
motility; mAb, monoclonal antibody; PAGE, polyacrylamide gel
electrophoresis; BSA, bovine serum albumin; FACS,
fluorescence-activated cell sorter.
We are indebted to Helena Marusyk for her expert
assistance with the photography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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