Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
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Abstract |
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The 6
4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH
(PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz,
H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91:
949-960). We demonstrate here using MDA-MB-435
breast carcinoma cells that
6
4 stimulates chemotactic
migration, a key component of invasion, but that it has
no influence on haptotaxis. Stimulation of chemotaxis
by
6
4 expression was observed in response to either
lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of
lamellae in these cells is dependent upon
6
4 expression. Both lamellae formation and chemotactic migration are inhibited or "gated" by cAMP and our results
reveal that a critical function of
6
4 is to suppress the
intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for
lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are
both required to promote lamellae formation and
chemotactic migration, our data indicate that they are
components of distinct signaling pathways. The essence
of our findings is that
6
4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion.
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Introduction |
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CARCINOMA invasion is a complex process that involves directed migration and localized proteolysis (24). Although the mechanistic basis of invasion had been elusive, recent advances in molecular cell biology have facilitated a much more rigorous analysis of this important and critical component of cancer progression. In particular, insight into the function and regulation of cell adhesion receptors, as well as proteases, has fueled significant progress in our understanding of the invasive process. Studies aimed at defining specific signal transduction pathways that determine the behavior of invasive carcinoma cells are also contributing to an uncovering of the molecular basis of invasion.
Recent work by our group and others has implicated a
key role for the 6
4 integrin in carcinoma invasion (3, 10,
32, 35, 40, 47). This integrin, which is a receptor for the laminins, is essential for the organization and maintenance
of epithelial structure. In many epithelia,
6
4 mediates
the formation of stable adhesive structures termed hemidesmosomes that link the intermediate filament cytoskeleton with the extracellular matrix (2, 12). The importance
of this integrin in epithelial structure has been reinforced
by the generation of
4-nullizygous mice that exhibit gross
alterations in epithelial morphology and anchorage to the
basement membrane (9, 46). In contrast to its function in
normal epithelia,
6
4 can stimulate carcinoma migration
and invasion through its ability to interact with the actin
cytoskeleton and mediate the formation and stabilization of lamellae (32). This dynamic function of
6
4 in enhancing the migration of invasive carcinoma cells is quite distinct from its role in maintaining stable adhesive contacts
in normal epithelia by associating with intermediate filaments. In fact, we have established that the ability of
6
4
to stimulate carcinoma migration and invasion depends
upon its preferential activation of a phosphoinositide 3-OH
kinase (PI3-K)1/Rac signaling pathway that we (40) and
others (18) have shown is necessary for invasion. In essence, our studies have defined an integrin-mediated
mechanism of carcinoma invasion that involves the stimulation of carcinoma migration by the dynamic association of
6
4 with F-actin and the activation of a specific signaling pathway by this integrin.
Although we have established the involvement of 6
4
in the migration of invasive carcinoma cells, the nature of
this migration has not been well defined. Moreover, signaling pathways distinct from PI3-K/Rac that are also regulated by
6
4 are likely to contribute to carcinoma migration. For these reasons, we sought to examine the
migration mediated by
6
4 in more detail and to identify other signaling pathways regulated by this integrin that
contribute to migration. The results obtained indicate that
6
4 stimulates the chemotactic migration of invasive carcinoma cells but that it has no influence on their haptotactic migration. Importantly, we demonstrate that the ability
of
6
4 to suppress the intracellular cAMP concentration
([cAMP]i) by activating a cAMP-specific phosphodiesterase (PDE) is essential for its enhancement of lamellae formation and chemotactic migration. Although PI3-K and
cAMP-specific PDE activities are required for lamellae formation and chemotactic migration, we conclude that they
are components of distinct signaling pathways.
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Materials and Methods |
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Cell Culture and Antibodies
We used stable subclones of MDA-MB-435 human breast carcinoma cells
that had been transfected with either the expression vector alone (mock
transfectants), a full-length 4 cDNA (MDA/
4 transfectants), or a mutated
4 cDNA that lacked the entire cytoplasmic domain with the exception of four amino acids distal to the transmembrane sequence (MDA/
4-
CYT). The characterization of these transfectants has been described
previously (38, 40). Both the
4 transfectants and the
4-
CYT transfectants expressed the
6
4 heterodimer on the cell surface as assessed by
FACS® analysis and immunoprecipitation of surface-labeled extracts (40).
The surface expression of
6
4 in these transfectants was comparable
to the expression seen in other breast carcinoma cell lines that express
this integrin endogenously such as MDA-MB-231 cells (Shaw, L.M., unpublished observation). All MDA-MB-435 cells were cultured in Dulbecco's modified Eagle's medium (DME) with 10% fetal calf serum plus
1% L-glutamine, 1% penicillin, and 1% streptomycin (GIBCO BRL,
Gaithersburg, MD). Clone A cells, originally isolated from a human,
poorly differentiated colon adenocarcinoma (7) and were cultured in
RPMI 1640 medium containing 10% fetal calf serum plus 1% L-glutamine,
1% penicillin, and 1% streptomycin.
NIH-3T3 cells were cultured in DME containing 10% newborn calf serum plus 1% L-glutamine, 1% penicillin, and 1% streptomycin. NIH-3T3 conditioned medium was prepared from normal culture medium incubated with cells for 2 d before harvest with cellular debris removed by centrifugation.
The following function blocking, integrin-specific monoclonal antibodies (mAb) were used: mAb 13 (mouse anti-1; S. Akiyama, National Institutes of Health, Research Triangle Park, NC), G0H3 (rat anti-
6; Immunotech, Westbrook, ME) and 2B7 (mouse anti-
6, prepared by our
laboratory [39]). Non-specific mouse IgG was purchased from Sigma
Chemical Co. (St. Louis, MO).
Migration and Invasion Assays
Cells were harvested using trypsin, rinsed three times with serum-free DME containing 250 µg/ml heat-inactivated BSA (DME/BSA), and then resuspended in DME/BSA. For migration assays, the lower surface of the membrane in each Transwell chamber (6.5-mm-diam, 8 µm pore size; Costar, Cambridge, MA) was coated for 30 min with either 15 µg/ml laminin-1 purified from Englebreth-Holm-Swarm tumor (19), 15 µg/ml collagen I (Vitrogen®; Collagen Biomaterials, Palo Alto, CA), or NIH-3T3 conditioned medium. For chemotaxis assays, either NIH-3T3 conditioned medium or lysophosphatidic acid (LPA) was added to the lower chamber. For haptotaxis assays, DME/BSA was added to the lower chamber. Cells (5 × 104) suspended in DME/BSA were added to the upper chamber. After incubating for 4 h at 37°C, nonmigrating cells were removed from the upper chamber with a cotton swab and cells that had migrated to the lower surface of the membrane were fixed with 100% methanol and stained with 0.2% (wt/vol) crystal violet in 2% ethanol. Migration was quantified by counting cells per square millimeter using bright-field optics. For antibody inhibition experiments, cells were incubated with 20 µg/ml of antibody for 30 min and then added to the Transwell chambers. The effects of pertussis toxin, IBMX (Calbiochem-Novabiochem, La Jolla, CA), forskolin, and rolipram (Sigma Chemical Co.) on migration were assessed by preincubating the cells with these reagents for 30 min before assay and including them in the assay medium at the concentrations noted in the figure legends.
Invasion assays were performed as described previously (40). In brief, 10 µg of Matrigel (Collaborative Research, Bedford, MA) was diluted with cold water and dried onto each Transwell filter. The Matrigel was reconstituted with DME for 1 h before its use in the assays. Cells were prepared as above and then added to the upper chamber of each well. NIH-3T3 conditioned medium was added to the lower chamber. Cells were allowed to invade for 4 h and then cells that had invaded were stained and quantified as described above.
cAMP Assays
Culture dishes (35-mm) were coated overnight with 20 µg/ml of collagen I in PBS and then blocked with serum-free RPMI containing 250 µg/ml BSA (RPMI/BSA). Cells (1.5 × 106) were then plated for 2 h and harvested by quickly removing the medium and extracting them directly with 80% (vol/vol) ethanol. Cell extracts were collected, cleared by centrifugation in a microcentrifuge for 10 min, dried in a SpeedVac (Savant Instruments, Farmingdale, NY) for 1.5 h, and then resuspended in 50 mM phosphate buffer, pH 6.2. The intracellular cAMP concentration was quantified using a cAMP enzyme-linked immunoabsorption assay (cAMP EIA; Cayman Biochemicals, Ann Arbor, MI) following the manufacturer's recommendation using nonacetylated cAMP as a standard and acetylcholine esterase-linked cAMP as a competitor. Values were corrected for cell number as determined from replicate plates. In some experiments, either 50 µM forskolin alone or forskolin plus 1 mM IBMX was added to cells 15 min before harvesting. To determine the cAMP content of cells under normal culture conditions, cells were plated in 35-mm dishes in DME plus 10% FCS, incubated for 18 h, and then processed as described above.
Phosphodiesterase Assays
cAMP PDE assays were performed according to the protocol of Sette et
al. (37). In brief, cells were plated onto collagen I coated dishes as described for cAMP assays. Cells were then scraped from the dishes in a hypotonic lysis buffer (PDE lysis buffer: 20 mM Tris-HCl, pH 8.0, 1 mM
EDTA, 0.2 mM EGTA, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, 4 µg/ml aprotinin,
and 2 mM PMSF) and then sonicated. Cellular debris was removed by
centrifugation and the supernatants were assayed immediately for PDE
activity. PDE activity of cell extracts (2-4 µg protein) was assayed in
cAMP PDE assay buffer (20 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1.25 mM -mercaptoethanol, 0.14 mg/ml BSA, 1 µM cAMP, and 0.2 µCi [3H]
cAMP) for 10 min at 34°C. The reaction was stopped by adding 40 mM
Tris, pH 7.5, containing 10 mM EDTA and heating for 2 min at 100°C.
The reaction products were then digested with 50 µg Crotalus atrox snake
venom (Sigma Chemical Co.) for 30 min at 34°C, separated from substrate
using SpinZyme acidic alumina devices (Pierce Chemical Co., Rockford,
IL), and quantified using a scintillation counter (Wallace, Gaithersburg,
MD). Values were corrected for protein content and are reported as pmol
cAMP hydrolyzed/min per milligram of protein. Protein content of cell
extracts was determined using the Bio-Rad protein reagent (Hercules,
CA) with BSA as a protein standard. Where noted, cells were incubated
either for 30 min with 50 µM forskolin or 100 nM wortmannin or for 5 min with 100 nM LPA before harvest. For rolipram inhibition of PDE activity, 100 nM rolipram was added to cell extracts for 5 min before assaying
for PDE activity.
Assessment of Lamellae Formation
Glass coverslips were coated overnight at 4°C with 20 µg/ml collagen I or laminin-1 and then blocked with BSA (0.25% in RPMI). The MDA-MB-435 transfectants were trypsinized and rinsed as described above, and then plated onto the coverslips for 2 h. As noted, cells were then treated with either 1 mM IBMX or 0.2% DMSO for 30 min. Subsequently, the cells were either treated with 100 nM LPA for 5 min or left untreated and then fixed for 10 min with 4% paraformaldehyde containing 10 mM Pipes, pH 6.8, 2 mM EGTA, 2 mM MgCl2, 7% sucrose, and 100mM KCl. The coverslips were rinsed three times with PBS and mounted in glycerol. For the analysis of clone A cells, the cells were treated with IBMX or DMSO for 30 min, plated on laminin-1-coated coverslips, incubated for 45 min at 37°C, and then fixed. Clone A cells were then rinsed three times with PBS and incubated with blocking solution containing 1% BSA/5% normal donkey serum for 30 min. Cells were incubated with 20 µg/ml TRITC-labeled phalloidin in blocking solution for 30 min. Cells were rinsed four times with PBS over 30 min and then mounted in glycerol containing 1× PBS, pH 8.5, and 0.1% propylgallate. All cells were imaged with a Nikon Diaphot 300 inverted microscope (Tokyo, Japan) using either Nomarski differential-interference contrast (DIC) or phase-contrast optics. Images were captured with a charge-coupled device camera (Dage-MTI, Michigan City, IN), a frame grabber (Scion, Frederick, MD) and a 7600 Power Macintosh computer (Apple Computer, Cupertino, CA). Images were analyzed and lamellar area quantified using IPLab Spectrum image analysis software (Signal Analytics, Vienna, VA) using the criteria for defining lamellae used previously by our group (32). Lamellae were defined as broad, flat cellular protrusions rich in F-actin and devoid of membrane-bound vesicles. The lamellar area of each cell was determined using both phase contrast optics and FITC-phalloidin staining.
Analysis of PDE Expression
To determine the relative expression of PDE in the cells used in this study, cell extracts (40 µg of protein) were resolved by SDS-PAGE (8%), transferred to nitrocellulose, and then immunoblotted with PDE4-specific antibodies provided by M. Conti (Stanford University, Stanford, CA) (15). Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies and visualized using SuperSignal chemiluminescent substrate (Pierce Chemical Co.).
Analysis of PI3-K Activation
The activation of PI3-K by the integrin 6
4 was assessed as described
previously (40). In brief, cells were trypsinized and rinsed as above, resuspended in RPMI/BSA at a concentration of 2 × 106 cells/ml and incubated
for 30 min with integrin-specific antibodies or in buffer alone. Either
IBMX (1 mM), forskolin (50 µM) or DMSO (0.2%) was added for 10 min
before plating the cells onto tissue culture dishes coated with goat anti-rat
IgG Ab. After incubation for 30 min at 37°C in the presence of IBMX, forskolin, or DMSO, the cells were washed twice with cold PBS and solubilized at 4°C for 10 min with 20 mM Tris, pH 7.4, 137 mM NaCl, 1% NP-40,
10% glycerol, 1 mM sodium orthovanadate, 2 mM PMSF, and 5 µg/ml of aprotinin, pepstatin, and leupeptin. Equivalent amounts of protein from
each extract were incubated for 3 h at 4°C with the antiphosphotyrosine mAb, 4G10 (Upstate Biotechnology, Lake Placid, NY) and protein A-Sepharose (Pharmacia Biotech, Piscataway, NJ). The Sepharose beads were
washed twice with lysis buffer then twice with 10 mM Hepes, pH 7.0, and
0.1 mM EGTA. Beads were then resuspended with kinase buffer plus 100 µM ATP, 25 µM MgCl2, 10 µCi [
-32P]ATP, and 10 µl sonicated brain lipids and incubated for 10 min at room temperature. The reaction was
stopped using 60 µl 2N HCl and 160 µl chloroform/methanol (1:1). Lipids
were resolved using potassium oxalate-coated thin layer chromatography
plates.
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Results |
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Expression of the 6
4 Integrin in MDA-MB-435 Cells
Enhances Their Chemotactic Migration
The possibility that expression of the 6
4 integrin influenced the rate of either haptotactic or chemotactic migration was assessed. For this purpose, stable transfectants of
MDA-MB-435 cells were used that expressed either the
6
4 integrin (MDA/
4) or a deletion mutant of
6
4
(MDA/
4-
CYT) that retains only four amino acids of
the
4 cytoplasmic domain, immediately proximal to the
transmembrane domain (40). As shown in Fig. 1 A, subclones of the MDA/
4 transfectants (5B3 and 3A7) exhibited a rate of haptotactic migration toward laminin-1 that
was slightly lower than the rate observed for subclones of
the mock transfectants (6D7 and 6D2). In marked contrast, expression of
6
4 induced a substantial increase in
the rate of chemotaxis of these cells towards conditioned
medium from NIH-3T3 cells (Fig. 1 B). The rate of chemotaxis of the MDA/
4 transfectants (5B3 and 3A7) was 15-
20-fold greater than that of the mock transfectants (6D7
and 6D2) over a 4-h time period. These data indicate that
expression of
6
4 potentiates chemotactic migration of
MDA-MB-435 cells without substantially altering their
rate of haptotaxis.
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To identify specific factors that could cooperate with
6
4 to promote chemotaxis of MDA-MB-435 cells, we
tested several growth factors known to have chemotactic
potential including epidermal growth factor, basic fibroblast growth factor, hepatocyte growth factor/scatter factor, insulin-like growth factor type I, transforming growth
factor
and
, platelet-derived growth factor (AA and BB), somatostatin, thrombin, and LPA. Of these factors,
only LPA was able to mimic the chemotactic effects of
NIH-3T3 cell conditioned medium on the MDA-MB-435
transfectants (Fig. 1 C and data not shown). LPA stimulated the chemotaxis of MDA-MB-435 cells in a dose dependent manner with maximal stimulation observed at
100 nM. Of note, LPA stimulation of chemotaxis was five-
to sevenfold greater in the MDA/
4 transfectants than in
the mock transfectants. Subclones of the MDA/
4-
CYT
transfectants (
3C12 and
1E10) exhibited a rate of chemotaxis that was similar to the mock transfectants (Fig. 1
D), indicating that the
4 cytoplasmic domain is critical for
mediating the increased chemotaxis seen in the MDA/
4
transfectants.
The increased chemotaxis observed for the MDA/4
transfectants in response to LPA was evident on both collagen I (Fig. 1, C and D) and laminin-1 (data not shown),
indicating that
6
4-enhanced migration is independent
of the matrix protein used for traction. This possibility was
examined further by preincubating the MDA/
4 transfectants with function-blocking mAbs before their use in the
chemotaxis assays. As shown in Fig. 2 A, inhibition of
6 integrin function with the mAb 2B7 did not block the
chemotaxis of the MDA/
4 transfectants on collagen I towards LPA. However, this mAb inhibited the haptotaxis
of MDA-MB-435 cells toward a laminin-1 gradient (Fig. 2
B), a process that is dependent on the
6
1 integrin (38).
Chemotaxis toward LPA was inhibited completely, however, by preincubating the cells with the
1 integrin-specific mAb 13 (Fig. 2 A). Collectively, these data indicate
that the stimulation of chemotaxis by expression of
6
4
can be independent of the adhesive functions of
6
4, and
that the adhesive interactions required for
6
4-enhanced
chemotaxis on collagen I are mediated through
1 integrins.
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Expression of the 6
4 Integrin Is Required for the
Formation of Lamellae in Response to LPA
Chemotactic migration frequently involves the formation
of broad sheets of polymerized actin at the leading edge of
the cell termed lamellae (27). To determine if expression
of the 6
4 integrin influenced the formation of such motile structures, we analyzed the morphology of the MDA-MB-435 transfectants plated on collagen I (Fig. 3). Prominent lamellae were not evident in the mock transfectants
and treatment with 100 nM LPA did not stimulate a significant increase in lamellar area (Fig. 3, C and D). The MDA/
4 transfectants exhibited a similar morphology to
that of the mock transfectants when plated on collagen I
(Fig. 3, compare A with C) or laminin-1 (data not shown).
Within minutes after LPA treatment, however, the MDA/
4 transfectants formed large, ruffling lamellae (Fig. 3 C).
Quantification of these cells by digital image analysis indicated that LPA stimulated a dramatic increase in the
lamellar area of the two subclones of the MDA/
4 transfectants (Fig. 3 D). In contrast, no increase in the lamellar area of the mock transfectants in response to LPA was detected by this analysis (Fig. 3 D).
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Pharmacological Evidence for the Involvement of cAMP in Chemotaxis
LPA is a bioactive phospholipid that can mediate its effects on cells through a receptor linked to heterotrimeric
G proteins, including inhibitory type G (Gi) proteins (29).
To assess the possible involvement of a Gi protein in
6
4-enhanced chemotaxis, we used pertussis toxin, which
inactivates heterotrimeric Gi-proteins by ADP ribosylation (31). The LPA-stimulated chemotaxis of both the
MDA/
4 and mock transfectants was inhibited by pertussis toxin with maximal inhibition observed at 100 ng/ml
(data not shown). These data suggested that the
6
4 integrin enhances chemotaxis that is mediated through pertussis toxin-sensitive, Gi-linked receptors. Gi proteins are
known to inhibit certain classes of adenyl cyclases and thus
limit cAMP production (45). For this reason, we analyzed
the impact of stimulating cAMP production on chemotaxis
using forskolin. Although forskolin inhibited LPA-stimulated chemotaxis, the MDA/
4 and mock transfectants
differed significantly in their response to this activator of
adenyl cyclases. LPA-stimulated chemotaxis of the mock
transfectants was inhibited to basal levels by 50 µM forskolin (Fig. 4 A). At this concentration of forskolin, the inhibition of chemotaxis of the MDA/
4 transfectants was
only 50% and higher concentrations of forskolin (100 µM)
did not abrogate chemotaxis of these cells (Fig. 4 A). Interestingly, treatment of the MDA/
4 or mock transfectants with forskolin did not inhibit haptotactic migration
on laminin-1 (Fig. 4 B). These data indicate that a cAMP-sensitive pathway plays a key role in LPA-stimulated chemotaxis of MDA-MB-435 cells and they suggest that
the
6
4 integrin may regulate this pathway.
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Expression of the 6
4 Integrin in MDA-MB-435 Cells
Influences cAMP Metabolism
To determine if 6
4 expression influences the [cAMP]i,
the [cAMP]i was determined in extracts obtained from
subconfluent cultures of MDA/mock,
4, and
4-
CYT
transfectants using a cAMP enzyme-linked immunoabsorption assay. As shown in Fig. 5 A, the MDA/
4 transfectants had a 30% lower [cAMP]i (2.7 pmol cAMP per 106 cells) than either the mock (3.7 pmol cAMP per 106
cells) or
4-
CYT transfectants (3.8 pmol cAMP per 106
cells). This difference was statistically significant (P < 0.001). Of note, neither clustering of
6
4 using the 2B7
mAb and an appropriate secondary Ab nor LPA treatment reduced cAMP levels further (data not shown).
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The observation that the MDA/4 transfectants were
more resistant to forskolin inhibition of chemotaxis than
the mock transfectants (Fig. 4) suggested that these two
populations of cells differ in their ability to metabolize the
cAMP generated in response to forskolin stimulation. This
possibility was examined by determining the [cAMP]i in
forskolin-treated cells. As shown in Fig. 5 B, the MDA/
4
transfectants exhibited a 30% lower [cAMP]i than the mock
transfectants when plated on collagen I. With forskolin
stimulation, a 2.5-fold greater accumulation of cAMP was
observed in the mock transfectants (6.6 pmol per 106 cells)
compared with the
4 transfectants (2.6 pmol per 106
cells). When the forskolin-treated cells were also treated
with the PDE inhibitor, IBMX, to prevent breakdown of
cAMP, the MDA/
4 transfectants exhibited a [cAMP]i
comparable to the mock transfectants (120 ± 11 versus
104 ± 18 pmol per 106 cells, respectively; Fig. 5 C). Together, these data suggest that expression of
6
4 integrin
suppresses the [cAMP]i by increasing PDE activity.
To establish more directly that expression of the 6
4
integrin can regulate cAMP-dependent PDE activity, the
activity of this enzyme was assayed in cell extracts obtained from the MDA/mock and
4 transfectants. As
shown in Fig. 6 A, the MDA/
4 transfectants exhibited a
significantly higher rate of PDE activity than the mock
transfected cells. Moreover, the PDE activity of the MDA/
4 transfectants was markedly increased (51% for 5B3
and 45% for 3A7) in response to forskolin stimulation
compared with the mock transfectants (29% for 6D7; Fig.
6 A). The difference in PDE activity between the MDA/
4
and mock transfectants was eliminated by rolipram, a type
IV PDE-specific (PDE 4) inhibitor (Fig. 6 B). These data indicate that a cAMP-dependent PDE 4 activity is influenced by
6
4 expression in MDA-MB-435 cells. Also,
this activity is likely responsible for the observed decrease
in [cAMP]i and the resistance to forskolin-mediated inhibition of LPA chemotaxis observed in the MDA/
4 transfectants.
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To examine the possibility that the MDA/4 and mock
transfectants differed in their level of PDE expression, we
assessed PDE 4 expression in these cells using antibodies
specific for the various PDE 4 variants (15). The predominant PDE 4 variant expressed in MDA-MB-435 cells is
PDE 4B based on results obtained with antibodies specific
for PDE 4A, 4B and 4D (data not shown). Importantly, the expression of PDE 4B did not differ significantly between the MDA/
4 and mock transfectants (Fig. 6 C).
These data indicate that the increased PDE activity observed in the MDA/
4 transfectants is not the result of increased PDE expression.
PDE Activity Is Necessary for Chemotaxis, Invasion, and Lamellae Formation
The importance of PDE for chemotactic migration was examined by treating the MDA/mock and 4 transfectants
with IBMX before their use in the chemotaxis assay. As
shown in Fig. 7 A, IBMX inhibited LPA-stimulated chemotaxis with maximal inhibition observed at 1 mM. Similar
results were obtained with the cAMP-specific PDE inhibitor, rolipram (data not shown). We also examined the involvement of PDE in carcinoma invasion by treating cells
with IBMX before their use in a standard Matrigel invasion assay. A substantial inhibition of invasion was observed in the presence of IBMX in comparison to the solvent control (Fig. 7 B).
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The necessity of cAMP-specific PDE activity in the formation of lamellae was also assessed. IBMX had no effect
on the morphology of the MDA/4 transfectants in the absence of LPA (Fig. 8, compare A with B). However,
IBMX-treated cells were unable to form the large, ruffling
lamellae in response to LPA stimulation in comparison to
untreated cells (Fig. 8, compare C with D). Quantitative
analysis of these cell populations revealed that inhibition
of PDE activity resulted in an approximate fourfold reduction in the lamellar area of LPA-stimulated MDA/
4
transfectants (Fig. 8 E).
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Recently, we reported that 6
4 is necessary for the formation and stabilization of lamellae in clone A colon carcinoma cells plated on laminin-1 (32). If PDE activity is
needed for lamellae formation as indicated by the above
results, IBMX should inhibit the formation of lamellae in
clone A cells. To test this possibility, clone A cells were
treated with IBMX or a solvent control and then plated
onto laminin-1 for 45 min. The control cells formed large
fan-shaped lamellae enriched in F-actin when plated on
laminin-1 (Fig. 9, A and B) as we reported previously (32).
In contrast, IBMX-treated cells formed small, immature
lamellae with a marked reduction in F-actin content (Fig.
9, C and D). Quantitative analysis of these images revealed that IBMX reduced the total lamellar area of clone
A cells on laminin-1 by ~75% (629 ± 74 µm2 for control
versus 164 ± 24µm2 with IBMX). Interestingly, inhibition
of PDE activity had no effect on the attachment or spreading of clone A cells on laminin-1 (Fig. 9).
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cAMP Metabolism and PI3-K Signaling Are Not Directly Linked in MDA-MB-435 Cells
A possible relationship between cAMP metabolism and
PI3-K signaling is of interest given our recent finding that
6
4 stimulates the preferential activation of PI3-K and
that this activity is required for invasion and the formation
of lamellae (40). To determine if PI3-K activity is required
for the cAMP-specific PDE activity we observed in the
MDA/
4 transfectants, these cells were incubated in the
presence of wortmannin, a specific inhibitor of PI3-K, before extraction and assay of PDE activity. As shown in Fig.
10 A, wortmannin had no effect on PDE activity in these cells and it did not inhibit the marked induction of PDE
activity that we had observed in response to forskolin
stimulation. The possibility also existed that cAMP influences the
6
4-mediated activation of PI3-K. To address
this issue, we used the
6-specific mAb G0H3 to cluster
6
integrins on the MDA/
4 transfectants in the presence of
the PDE inhibitor IBMX and forskolin. As shown in Fig.
10 B, mAb-mediated clustering of
6
4 in MDA/
4 transfectants activated PI3-K markedly compared with cells
maintained in suspension, in agreement with our previous
results (40). However, treatment of MDA/
4 transfectants
with either IBMX or forskolin did not inhibit
6
4-mediated activation of PI3-K (Fig. 10 B). In fact, no inhibition
of PI3-K was observed when both of these inhibitors were
used in combination, a treatment that increases the [cAMP]i from 4 to 120 pmoles per 106 cells (Fig. 5).
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Discussion |
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Recently, we established that the 6
4 integrin promotes
carcinoma invasion (3, 32, 40). In the current study, we extend this observation by demonstrating that a major function of
6
4 is to stimulate the chemotactic migration of
carcinoma cells, a function that is essential for invasion.
This function is consistent with our previous finding that
6
4 is involved in the formation and stabilization of
lamellae and filopodia (32). Importantly, the data presented here also provide evidence that
6
4 stimulates chemotaxis and lamellae formation by regulating the
[cAMP]i by a mechanism that involves activation of a rolipram-sensitive, cAMP-specific PDE. Our finding that elevated [cAMP]i inhibits the formation of lamellae, chemotactic migration, and invasion is in agreement with recent
studies indicating that cAMP can function to inhibit or
"gate" specific signaling pathways (14, 16, 23). Furthermore, we show that the cAMP-mediated gate does not influence haptotaxis thus providing additional evidence that
the signaling events governing chemotaxis and haptotaxis
differ (1, 20). Collectively, our results strengthen the hypothesis that
6
4 promotes carcinoma invasion through
its ability to regulate signaling pathways required for migration. They also indicate that cAMP metabolism is likely
to be an important factor in the regulation of carcinoma
invasion and progression.
Although integrins can regulate a number of signaling
pathways (4), their ability to influence cAMP metabolism
has not been studied extensively. An earlier study, however, did provide evidence that the simultaneous engagement of 2 integrins and tumor necrosis factor (TNF)
receptors decreases the [cAMP]i in neutrophils (30). Interestingly, the reduction in [cAMP]i observed in response to
2 integrin and TNF receptor engagement in neutrophils is similar to the level of [cAMP]i suppression that we observed in response to
6
4 expression (~30%). This level
of suppression of the total [cAMP]i is quite impressive
given that localized gradients of [cAMP]i are probably required to facilitate chemotactic migration, as well as for
other cell functions that are gated by cAMP. For example,
localized gradients of cAMP have been implicated in regulating the direction of growth cone turning (41). It is also
important to note that we observed an inverse correlation between the [cAMP]i and the rate of chemotaxis (compare
values in Fig. 4 A with Fig. 5 B). This observation reinforces the functional significance of
6
4 suppression of
the [cAMP]i.
A novel aspect of our study is the finding that integrins,
and 6
4 in particular, can regulate the activity of a rolipram-sensitive, cAMP-specific PDE. This family of PDEs
is defined as type IV PDE (PDE 4) and consists of a number of structural variants (6). Because all of these variants
hydrolyze cAMP with a Km comparable to the [cAMP]i, it
is thought that tissue-specific expression and the state of
activity of these variants are the major determinants of
their responsiveness to extracellular stimuli (6). Indeed, the major focus of work in this area has been hormonal
regulation of PDE activity. Regulation of PDE activity can
occur rapidly in response to hormone stimulation through
a mechanism that involves PKA-dependent phosphorylation of the enzyme (36, 37). In addition, long-term, hormonal stimulation can actually increase de novo synthesis
of the cAMP-specific PDEs (6, 43). The data we obtained
suggest that expression of
6
4 does not increase the expression of PDE 4B, a predominant PDE variant expressed by MDA-MB-435 cells. For this reason, regulation
of PDE 4 activity by
6
4 expression may occur through a
mechanism that involves PDE phosphorylation. Another
possibility that has been proposed recently is that the subcellular localization of the cAMP-specific PDEs influences their function and activation (17). The possibility that
6
4 increases the association of PDE 4 with either the
plasma membrane or cytoskeleton is certainly attractive
and could account, at least in part, for its ability to influence cAMP metabolism. Interestingly, LPA stimulation
by itself had no effect on either PDE activity or the
[cAMP]i in MDA-MB-435 cells. This observation reinforces our hypothesis that a major function of
6
4 is to
release cAMP gating of LPA-stimulated chemotaxis.
In previous studies, we established that an important
function of 6
4 in invasive carcinoma cells is its ability to
stimulate the formation of lamellae (32). This function of
6
4 is highlighted by the observation in the present study
that LPA was able to induce significant lamellae formation only in MDA-MB-435 cells that expressed
6
4 (Fig.
3). Importantly, our finding that PDE activity is necessary for lamellae formation promoted by
6
4 expression implies that a localized suppression of the [cAMP]i plays an
important role in controlling the signaling and cytoskeletal
events that are required for lamellae formation. This hypothesis agrees with studies that have shown an inhibitory
effect of cAMP on the organization of the actin cytoskeleton (11, 13, 21, 22). Moreover, the formation of lamellae is
a dynamic process that is linked to the mechanism of cell
migration. Therefore, it is likely that temporal fluxes in the
[cAMP]i regulated by
6
4 contribute to the chemotactic
migration of carcinoma cells.
Of particular relevance to our work, Butcher and colleagues (23) reported that cAMP is a negative regulator of
leukocyte migration signaled through the classical chemoattractants. In this model, cAMP impedes or gates RhoA-mediated leukocyte integrin activation and adhesion.
Our results support their conclusion that cAMP inhibits
chemotactic migration. Importantly, we also provide evidence for an integrin-mediated mechanism of regulating the [cAMP]i to facilitate migration. Our findings support
the work of the Butcher group because LPA is a potent activator of Rho (29) and, in fact, we have observed that the
expression of a dominant-negative Rho can inhibit both
LPA-stimulated chemotactic migration and invasion of
MDA-MB-435 cells (O'Connor, K.L., and A.M. Mercurio,
unpublished observation). Thus, a possibility worth investigating is that LPA-mediated activation of Rho is gated by cAMP and that 6
4 releases cAMP gating by increasing cAMP-specific PDE activity and thereby enhances
Rho activation. A likely target of Rho activation is the actin cytoskeleton (44), which is consistent with the reported
effects of cAMP on the cytoskeleton (11, 13, 21, 22), as
well as our demonstration that cAMP inhibits lamellae formation. Although Rho has been linked to stress fiber
formation and not lamellae formation in fibroblasts (25),
much less is known about Rho function in epithelial-derived cells. In fact, our preliminary data suggest that
LPA-induction of lamellae formation in the MDA/
4 transfectants is inhibited by expression of a dominant-negative Rho. Other integrins, especially the
1 integrins that mediate the adhesive interactions required for chemotactic
migration, are another potential target of Rho (25, 34). It
is worth noting in this context that expression of
6
4 has
been shown to alter the function of collagen I-binding integrins in breast carcinoma cells (42).
The ability of 6
4 to promote lamellae formation and
carcinoma invasion is dependent upon its preferential activation of a PI3-K and Rac signaling pathway (40). Our
current finding that the release of cAMP gating by
6
4 is
also required for these events raised the issue of a possible
link between cAMP and the PI3-K/Rac pathway. Such a
link was suggested, for example, by the finding that the interleukin-2 dependent activation of PI3-K is inhibited by
cAMP (28). In our experiments, however, pharmacological stimulation of cAMP levels had no effect on the ability
of
6
4 to activate PI3-K even under conditions in which
the [cAMP]i increased 30-fold over basal levels. Our data
also indicate that PI3-K probably does not function upstream of cAMP-specific PDE because wortmannin did
not inhibit the activity of this enzyme. We conclude from these findings that PI3-K and cAMP-specific phosphodiesterase function in tandem to promote lamellae formation and chemotactic migration but they are components
of distinct signaling pathways.
An interesting finding in the present study is that the
ability of 6
4 to stimulate chemotactic migration and
suppress the [cAMP]i can be independent of the adhesive
function of this integrin. Although the laminins are the
only known matrix ligands for
6
4 (26), expression of
this integrin also stimulated chemotactic migration on a
collagen I matrix and this migration was not inhibited by
an
6-function blocking mAb. The possibility that the effects of
6
4 expression on migration result from a decrease of
6
1 expression is discounted by the fact that expression of the
6
4
CYT integrin had no effect on
either chemotaxis, cAMP levels or PDE activity even
though expression of this mutant integrin eliminates
6
1
expression in these cells (38). The observation that the
ability of
6
4 to promote chemotactic migration can be
independent of its adhesive function is in agreement with
several recent studies by our group and others that have
revealed `ligand-independent' functions for the
6 integrins in carcinoma cells (3, 5, 8). Insight into the possible
mechanism of this phenomenon was provided by a recent
study that demonstrated self-association of the
4 cytoplasmic domains, a process that could initiate intracellular signaling events independently of ligand binding (33). One
important implication of these findings is that the ability of
6
4 to influence cAMP metabolism and stimulate the
chemotactic migration of carcinoma cells need not be limited to sites of contact with laminin-containing matrices.
This possibility is supported by the numerous studies that
have implicated
6
4 as a major determinant of carcinoma invasion and progression (10, 35, 47).
In summary, we have demonstrated that the 6
4 integrin can stimulate lamellae formation and chemotactic
migration of invasive carcinoma cells by increasing the activity of a rolipram-sensitive cAMP specific-PDE and lowering the [cAMP]i. This cAMP specific-PDE functions in
tandem with a PI3-K/Rac pathway, that is also regulated by
6
4, and is required for carcinoma invasion and lamellae formation. The essence of our findings is that the
6
4
integrin stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling
events that underlie this critical component of carcinoma
invasion.
![]() |
Footnotes |
---|
Address correspondence to Arthur M. Mercurio, Beth Israel Deaconess Medical Center-Dana 601, 330 Brookline Ave., Boston, MA 02215. Tel.: (617) 667-7714. Fax: (617) 975-5531. E-mail: amercuri{at}bidmc.harvard.edu
Received for publication 24 September 1998 and in revised form 27 October 1998.
We would also like to thank M. Conti and S. Iona (Stanford University,
Stanford, CA) for their generous contribution of the PDE 4 antibodies, as
well as S. Akiyama for the 1 integrin antibody. We also thank R. Falcioni, (Regina Elena Cancer Institute, Rome, Italy), R. Bachelder, I. Rabinovitz, and D. Senger (all three from Beth Israel Deaconess Medical
Center, Boston, MA) for their comments and discussions regarding the
manuscript.
This work was supported by grants from the United States Army Medical Research and Material Command to K. O'Connor (DAMD17-98-1-8033), L.M. Shaw (DAMD17-97-1-7313), and A.M. Mercurio (DAMD17- 96-1-6199) and a grant from the National Institutes of Health to A.M. Mercurio (CA-44704).
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Abbreviations used in this paper |
---|
[cAMP]i, intracellular cyclic AMP concentration; DIC, differential-interference contrast; Gi, inhibitory type G protein; IBMX, isobutylmethylxanthine; LPA, lysophosphatidic acid; PDE, phosphodiesterase; PI3-K, phosphoinositide 3-OH kinase.
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