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Originally published In Press as doi:10.1074/jbc.M109068200 on December 20, 2001

J. Biol. Chem., Vol. 277, Issue 10, 7875-7881, March 8, 2002
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Vascular Endothelial Growth Factor-induced Migration of Multiple Myeloma Cells Is Associated with beta 1 Integrin- and Phosphatidylinositol 3-Kinase-dependent PKCalpha Activation*

Klaus Podar, Yu-Tzu Tai, Boris K. Lin, Radha P. Narsimhan, Martin Sattler, Takashi Kijima, Ravi Salgia, Deepak Gupta, Dharminder Chauhan, and Kenneth C. AndersonDagger

From the Jerome Lipper Multiple Myeloma Research Center/Dana-Farber Cancer Institute and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, September 9, 2001, and in revised form, November 27, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

In multiple myeloma (MM), migration is necessary for the homing of tumor cells to bone marrow (BM), for expansion within the BM microenvironment, and for egress into the peripheral blood. In the present study we characterize the role of vascular endothelial growth factor (VEGF) and beta 1 integrin (CD29) in MM cell migration. We show that protein kinase C (PKC) alpha  is translocated to the plasma membrane and activated by adhesion of MM cells to fibronectin and VEGF. We identify beta 1 integrin modulating VEGF-triggered MM cell migration on fibronectin. We show that transient enhancement of MM cell adhesion to fibronectin triggered by VEGF is dependent on the activity of both PKC and beta 1 integrin. Moreover, we demonstrate that PKCalpha is constitutively associated with beta 1 integrin. These data are consistent with PKCalpha -dependent exocytosis of activated beta 1 integrin to the plasma membrane, where its increased surface expression mediates binding to fibronectin; conversely, catalytically active PKCalpha -driven internalization of beta 1 integrin results in MM cell de-adhesion. We show that the regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85) is constitutively associated with FMS-like tyrosine kinase-1 (Flt-1). VEGF stimulates activation of PI 3-kinase, and both MM cell adhesion and migration are PI 3-kinase-dependent. Moreover, both VEGF-induced PI 3-kinase activation and beta 1 integrin-mediated binding to fibronectin are required for the recruitment and activation of PKCalpha . Time-lapse phase contrast video microscopy (TLVM) studies confirm the importance of these signaling components in VEGF-triggered MM cell migration on fibronectin.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

In multiple myeloma (MM)1 migration is necessary for the homing of tumor cells to bone marrow (BM), for expansion of malignant plasma cells within the BM microenvironment, and for the egress into peripheral blood. It has been reported that the extracellular matrix (ECM) proteins laminin, microfibrillar collagen type VI, and fibronectin are strong adhesive components for MM cells and that adhesion to laminin and fibronectin is beta 1 integrin (CD29)-mediated (1). beta 1 integrins are typically expressed on MM cells, specifically alpha  integrins VLA-4 (alpha 4beta 1) and VLA-5 (alpha 5beta 1) (2, 3). beta 1 integrin-mediated adhesion of MM cells to fibronectin confers protection against drug-induced apoptosis and triggers NFkappa B-dependent transcription and secretion of interleukin-6, the major MM growth and survival factor (4, 5). Interestingly, chimeric mice (beta 1-/- right-arrow wild-type chimeras) lack beta 1-null cells in blood and in hematopoietic organs such as spleen, thymus, and BM as a consequence of the inability of beta 1-null cells to invade the fetal liver (6). In addition to up-regulation of cell surface expression and induction of surface-clustering, integrin activity can be triggered by multiple agonists through "inside-out" signaling independent of changes in integrin expression levels (e.g. ligand binding to growth factor receptors is associated with changes in the way in which adhesion receptors on the cell surface engage the ECM). This concept is illustrated in human umbilical vein endothelial cells in which VEGF stimulates beta 1 integrins and leads to markedly enhanced movement (7).

Although VEGF induces migration as a key step in angiogenesis, the interplay between VEGF and integrins is not restricted to angiogenesis. VEGF and VEGFR are expressed by many tumor cell lines; moreover, elevated levels of VEGF are found in cancer patients, and inhibition of VEGF can suppress tumor growth (8). Indeed, clinical studies are underway investigating VEGF as a novel therapeutic target (9). In MM VEGF is expressed and secreted by tumor cells as well as BM stromal cells (10, 11); moreover, binding of MM cells to BM stromal cells enhances both interleukin-6 and VEGF secretion (11). We recently showed that in addition to stimulating angiogenesis, VEGF directly induces MM cell proliferation via a protein kinase C (PKC)-independent mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway and triggers MM cell migration on fibronectin via a PKC-dependent pathway (12). Members of the PKC family mediate multiple physiological functions (13-18) including integrin-mediated cell spreading and migration (19-22). To date, 11 isoenzymes of the serine/threonine kinase PKC have been identified and classified into three subgroups based on structure and cofactor regulation: conventional PKC, novel PKC, and atypical PKC (15, 18). The conventional PKC isoforms participate in the inside-out signaling activation of cell adhesion mediated by beta 1, beta 2, and beta 3 integrins and are also required for cell spreading (23-25). Moreover, PKCalpha associates with beta 1 integrins, thereby regulating cell trafficking (26, 27).

In the present study, we describe the close interrelationship between integrin and growth factor-induced signaling pathways in MM. We identify PKCalpha as the primary PKC isozyme involved in VEGF-induced MM cell migration. By showing that VEGF-mediated MM cell migration is associated with beta 1 integrin- and PI 3-kinase-dependent PKCalpha activation, we further confirm the importance of tumor cell-BM microenvironment interaction as a pivotal process in the pathogenesis of MM. Moreover, our studies identify several potential targets for novel therapies to improve outcome in MM.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Materials-- Recombinant human VEGF165 was purchased from R&D Systems (Minneapolis, MN). Human plasma fibronectin was obtained from Invitrogen. beta 1 integrin-specific mAb was purified from P4C10 ascites (Chemicon, Temecula, CA). PKC isoforms were purchased from Transduction Laboratories. The goat polyclonal Ab raised against the carboxyl terminus of PYK2, the mouse mAb raised against full-length beta 1 integrin (4B7R), and the rabbit polyclonal Ab directed against amino acids within the extracellular domain of Flt-1 were purchased from Santa Cruz. Anti-phosphotyrosine 4G10 antibody was kindly provided by Dr. Tom Roberts (Dana-Farber Cancer Institute, Boston, MA).

Cells and Cell Culture-- The human MM cell line MM.1S (dexamethasone-sensitive) (28) was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 10 µg/ml streptomycin, and 2 mM L-glutamine.

Stimulation of Cells-- Cells were starved for 15-18 h in medium with 3% FBS overnight and then for 3 h without FBS prior to stimulation with indicated VEGF concentrations (recombinant human VEGF, R&D Systems) or 50 nM/300 nM PMA for 20-30 min at 37 °C.

Cell Lysis, Immunoprecipitation, and Western Blot Analysis-- Cells were washed three times with phosphate-buffered saline and lysed with either lysis buffer (10 mM Tris, 50 mM NaCl, Na-pyrophosphate, 1% Triton, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and protein inhibitor mixture; Roche Molecular Biochemicals) or radioimmune precipitation assay lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% v/v Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture). Insoluble material was removed by centrifugation (15,000 rpm for 30 min at 4 °C).

Immunocomplexes were collected following overnight incubation at 4 °C with 10-20 µl of 100% protein A-Sepharose CL-4B beads (Amersham Biosciences, Inc.). For Western blotting, cell lysates (30-100 µg/lane) or immunoprecipitates (500 µg-1.5 mg total proteins) were separated by 8 or 10% SDS-PAGE prior to electrophoretic transfer onto HybondTM-C super nitrocellulose membranes (Amersham Biosciences, Inc.). After blocking with 5% nonfat milk in phosphate-buffered saline-Tween 20 buffer at room temperature for 1 h, membranes were sequentially blotted with the indicated specific primary Abs and then with horseradish peroxidase-conjugated secondary mouse, rabbit, or goat Abs and were developed using chemiluminescence (Amersham Biosciences, Inc.).

Cell Fractionation-- After washing three times with phosphate-buffered saline, cells were transferred into 200 µl of hypotonic lysis buffer (HLB: 10 mM Tris-HCl, 1 mM EDTA, 1 mM sodium vanadate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture) and incubated for 20 min on ice. The cells were then lysed by 80 strokes in a Dounce homogenizer and subjected to centrifugation at 1500 × g to pellet nuclei and unbroken cells followed by centrifugation of the supernatant at 100,000 × g for 20 min. The supernatant was collected (S100 fraction), and the pellet was resuspended in 70 µl of HLB containing 0.1% Triton X (P100 fraction).

Measurement of PKCalpha Activity-- PKCalpha activity was measured with a PKC assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer's instructions. The phosphotransferase activity of PKCalpha was quantitated in a scintillation counter (Beckman LS 6500, multi-purpose scintillation counter) measuring the amount of the gamma -phosphate of [gamma -32P]ATP incorporated in a specific PKC substrate peptide (QKRPSQRSKYL) bound to P81 phosphocellulose paper. Endogenous phosphorylation of proteins in the sample was determined by substituting the assay dilution buffer for the substrate mixture. To assure that equal amounts of PKCalpha were used in the assay, immunoprecipitates were denaturated, eluted, separated by 10% SDS-PAGE, electrophoretically transferred, and immunoblotted with PKCalpha .

Phosphoinositide Kinase Assays-- PI 3-kinase assays were performed as described previously (29) in a total volume of 50 µl. The radioactivity was visualized and quantitated on a PhosphorImager (Amersham Biosciences, Inc.).

Cell Adhesion Assays-- These were performed using the VybrantTM cell adhesion assay kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. Briefly, after an 18-h starvation in RPMI/2% FBS, MM.1S cells (4 × 106 cells/ml) were harvested and labeled with calcein-acetoxymethyl ester (5 µM per cell loading) for 30 min. After washing with prewarmed (37 °C) RPMI 1640 (without serum), the cells were preincubated with or without blocking beta 1 integrin neutralizing mAb, bisindolylmaleimide I (BIM I, Calbiochem), or LY294002 (Calbiochem), respectively, and then stimulated with VEGF. The cell suspensions were immediately added to fibronectin- or polylysine (1 µg/ml)-coated or non-coated wells. After 90-120 min, nonadherent calcein-labeled cells were removed by gently washing twice with RPMI 1640 by inversion of the plates. Adherent cells were quantitated in a fluorescence multi-well plate reader (Molecular Devices, Sunnyvale, CA) and examined microscopically. All experiments were done in triplicate.

Transwell Migration Assay-- Cell migration was assayed as described previously (12, 30, 31). After 2-5 h, cells that had migrated into the lower compartment of a Boyden-modified chamber were counted using a Coulter counter ZBII (Beckman Coulter).

Time-lapse Video Microscopy-- MM.1S cells were starved in RPMI medium containing 2% FBS for 16 h and plated to uncoated or fibronectin-coated tissue culture plates (35 × 10-mm plates, BD PharMingen), respectively, in the presence or absence of VEGF (100 ng/ml). For image capturing, an Olympus IX70 inverted microscope (Olympus, Lake Success, NY) with Hoffman optics (×10/×20/×40) equipped with a temperature- (Therm-Omega-Tech, Warmington, PA) and CO2 (5%)-controlled chamber was connected to an Optronics Engineering DEI-750 3CCD digital video camera (Optronics, Galeta, CA). Animation and export to a Quick Time movie were performed using the QED Camera Standalone 145 software at 2-min intervals. Images were analyzed with the NIH Image 1.62 software. To generate migration tracks, the position of the centroid of individual cells on each image were marked. The migratory speed was calculated based on the sum of distances divided by the time of observation. Migration of at least 15 cells was analyzed for each experimental condition.

Statistical Analysis-- Statistical significance of differences observed in VEGF-treated versus control cultures was determined using an unpaired Student's t test. The minimal level of significance was p < 0.05.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Previously we showed that VEGF-triggered MM cell migration on fibronectin is mediated via a PKC-dependent signaling pathway (12). In the present study we define and characterize the interrelationship of VEGF- and integrin-signaling in activating PKC that ultimately leads to MM cell migration.

PKC Isoform Expression in MM Cells-- Several PKC isoforms, including PKCalpha (32), PKCdelta (33), and PKCtheta (34), have been implicated in a cell migratory phenotype. Additionally, overexpression of PKCalpha was shown to enhance cell motility/invasiveness of breast cancer cells (26). We have shown previously that MM cell migration is PKC-dependent because it can be selectively inhibited by the PKC inhibitor BIM I (12). As a first step to identify the class of PKC mediating VEGF-induced migration in MM cells, we examined the expression of the PKC isoforms in MM cell lines and patient cells (Fig. 1). Immunoblot analysis revealed that PKCalpha , PKCgamma , PKCepsilon , and PKCzeta are significantly expressed in all human MM cell lines and MM patient cells investigated, in contrast to PKCbeta (low expression), PKCdelta (variable expression), and PKCtheta (no expression, data not shown). As in our previous studies we chose the MM.1S human MM cell line (28) as a representative model system for this study.


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  		Fig. 1.   PKC isoform expression in MM cells. Cell lysates were prepared from MM cell lines and MM patient cells as described under "Materials and Methods." Aliquots of cell extracts (80 µg) were separated on 10% polyacrylamide gels by SDS-PAGE and transferred to nitrocellulose; blots were stained with the isozyme-specific Abs indicated. Cell lysates from rat brain were used as a positive control for PKC expression. Molecular mass standards in kDa are shown.

VEGF-induced Signaling Pathways and Adhesion to Fibronectin Mediate PKCalpha Activation-- Because the activation of PKC occurs concomitantly with recruitment to the plasma membrane, we next performed immunoblotting of cell fractions with isoform-specific Abs against PKCalpha , PKCgamma , PKCepsilon , and PKCzeta to delineate the intracellular distribution after VEGF stimulation (Fig. 2). In non-treated MM cells all PKC isoforms were detected primarily in the cytosolic fraction. After VEGF stimulation of cells seeded on fibronectin, PKCalpha translocated into the membrane fraction after 30 min (Fig. 2a), whereas no significant changes in distribution of PKC isoforms as PKCgamma , PKCepsilon , and PKCzeta were observed. The activation of PKCalpha after VEGF treatment of cells attached to fibronectin was further supported by a 2-fold increase in PKCalpha -IP kinase activity; PKCalpha -IP kinase activation by PMA served as a positive control (Fig. 2b). Although a cAMP-dependent protein kinase/calmodulin kinase (PKA/CaMK) inhibitor mixture was used, this assay (Upstate Biotechnology) may not exclude the phosphorylation of a specific substrate peptide (QKRPSQRSKYL) by unknown PKCalpha -coprecipitated kinases. Neither plating of cells on fibronectin alone nor stimulation of suspended cells with VEGF alone significantly changed the subcellular distribution of PKCalpha (Fig. 2, c and d). In contrast, treatment with PMA, a major PKC activator, induced translocation of responsive PKC (PKCalpha , PKCgamma , and PKCepsilon ) into the detergent-soluble membrane fraction as expected (Fig. 2d) (35, 36)


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  		Fig. 2.   Effects of VEGF and adherence to fibronectin induce PKCalpha subcellular localization and activation. Panel a, MM.1S cells were either untreated or treated with 100 ng/ml VEGF for indicated intervals and were placed in suspension (S) or plated on fibronectin (FN). Panel b, PKCalpha activity was determined using a PKC kinase assay as described under "Materials and Methods." Stimulation of MM.1S cells with 300 nM PMA for 20 min was used as a positive control. L, rat brain lysate; C, IP control. Results of a representative experiment are shown. Panel c, MM.1S cells in suspension were either untreated or treated with 100 ng/ml VEGF for indicated time points. Treatment with 300 nM PMA was performed as a positive control for PKC membrane translocation. d, MM.1S cells were either placed in suspension (S) or plated on fibronectin (FN) for the indicated time points. Panels a, c, and d, SP100 cell fractionation was performed as described under "Materials and Methods." Immunoblots of cytosolic (C) and membrane (M) fractions were probed for expression of the indicated PKC isoforms. Molecular mass standards in kDa are shown.

VEGF Enhances MM Cell Adhesion to Fibronectin-- Laminin, the microfibrillar collagen type VI, and fibronectin bind MM cells, and adhesion to laminin and fibronectin is beta 1 integrin (CD29)-mediated (1). beta 1 integrins expressed on MM cells include VLA-4 (alpha 4beta 1) and VLA-5 (alpha 5beta 1) (2-5), which mediate adherence to both the ECM and BM stromal cells. In human umbilical vein endothelial cells, VEGF stimulates beta 1 integrins via inside-out signaling, leading to significantly increased motility (7). Because migration is a dynamic process of cell adhesion formation and release, we next investigated whether VEGF can modulate beta 1 integrin-mediated MM cell adhesion. As shown previously, MM cells spontaneously adhered to fibronectin, and this adhesion was increased upon stimulation with VEGF (Fig. 3a). Maximal increments of VEGF-mediated cell adhesion were observed at fibronectin concentrations of 25-30 µg/ml, whereas adhesion decreased to baseline levels at higher fibronectin concentrations (Fig. 3b). Notably, VEGF-mediated increments in adhesion were time-dependent, with maximal binding observed after 75-90 min of VEGF treatment and decreasing after 120 min (Fig. 3c).


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  		Fig. 3.   VEGF enhances MM cell adhesion to fibronectin. a, MM cell adhesion to fibronectin is enhanced in a dose-dependent manner by VEGF. contr, adhesion of cells in suspension. b, VEGF-mediated MM cell adhesion to various concentrations of fibronectin. c, VEGF-mediated MM cell adhesion is transient. d, dependence of MM cell adhesion on PKC, PMA (50 nM, 300 nM) was used as a positive control. e, dependence of VEGF-mediated MM cell adhesion on beta 1 integrin shown using a beta 1 integrin-neutralizing mAb (dilution: 1:1000, 1:500, 1:100). S, soluble; neg c, cells seeded to fibronectin without stimulation; pos c, VEGF (100 ng/ml); IgG, non-immune IgG. f, dependence of VEGF-mediated MM cell migration on beta 1 integrin shown using beta 1 integrin-neutralizing mAb (anti-beta 1, 1:1000 and 1:100). neg c, migration without chemoattractant; pos c, VEGF (10 ng/ml); IgG, non-immune IgG. The data shown are the mean ± S.D. of three separate experiments; adhesion assays were performed in triplicate.

We next examined whether the increment of adhesion observed at 90 min of VEGF stimulation was PKC- and beta 1 integrin-dependent. As shown in Fig. 3, d-e, incubation with the PKC inhibitor BIM I (as well as with the beta 1-neutralizing mAb) blocked dose-dependent VEGF-induced cell adhesion to fibronectin. This involvement of PKC in MM cell adhesion to fibronectin was further confirmed by a dose-dependent increment of adhesion triggered by PMA stimulation (Fig. 3d) similar to that induced by VEGF. Taken together, our results show that VEGF transiently enhances MM cell adhesion to fibronectin, dependent on both PKC and beta 1 integrin activity.

beta 1 Integrins Modulate VEGF-triggered Migration on Fibronectin-- We next sought to determine whether beta 1 integrin modulates VEGF-mediated MM cell migration on fibronectin. As seen in Fig. 3f, beta 1 integrin neutralizing mAb (but not irrelevant IgG) mediated dose-dependent inhibition of VEGF-triggered MM cell migration in a Boyden-modified microchemotaxis chamber. These data confirm that beta 1 integrin (CD29) is the integrin primarily associated with VEGF-triggered MM cell migration on fibronectin.

PKCalpha Associates with beta 1 Integrin and CADTK-- The control mechanisms leading to the various stages of the integrin receptor life cycle are largely unknown. Propagation of cell movement is thought to be regulated by the distribution and redistribution of integrins through surface diffusion, internalization, clustering at the leading front, and ripping release from the cell rear (37). In mammary epithelial cells Ng et al. (26) recently have found that PKCalpha interacts with activated beta 1 integrin, which regulates its exocytosis to the plasma membrane; moreover, catalytically active PKCalpha is responsible for beta 1 integrin internalization through a Ca2+- and PI 3-kinase-dependent, dynamin I-controlled endocytic pathway. PKCalpha induced up-regulation of the integrin-dependent cell migration of these cells, which was blocked under conditions that prevented the internalization of the receptor complex. We therefore next determined whether PKCalpha and beta 1 integrin are associated in MM cells. Constitutive complex formation between these two proteins was demonstrated by co-immunoprecipitation (Fig. 4a). Upon activation and translocation, conventional PKCs associate with proteins of the transmembrane-4 superfamily (TM4SF or tetraspans) linking PKC to several subsets of integrins (38) including alpha 4beta 1 (39) and alpha 5beta 1 (40). Specificity of binding to TM4SF is dependent on the extracellular domain of the integrin alpha  chain, and the integrin-TM4SF-PKC complex formation results in phosphorylation of the integrin alpha  cytoplasmic tail (41). In ongoing studies, we are investigating the regulatory role of these integrin-associated proteins on VEGF-induced MM cell migration.


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  		Fig. 4.   VEGFR-1/Flt-1 associates with beta 1 integrin and CADTK and enhances CADTK-phosphorylation, dependent on PKC and beta 1 integrin activation. MM.1S cells were serum-starved overnight in 2% FBS (and an additional 2 h without serum), were detached from the culture dish, and were either maintained in suspension (S) or incubated on fibronectin-coated plates (FN) for the indicated intervals. Immunoprecipitation was performed as described under "Materials and Methods." Panel a, co-immunoprecipitation of CADTK and beta 1 integrin with PKCalpha . Panel b, constitutive association of Flt-1 and CADTK. Panel c, activation of CADTK upon treatment with PMA (300 nM), 20 min. Panel d, additional increase of CADTK phosphorylation in the presence of VEGF (100 ng/ml). Panel e, dependence of VEGF-induced CADTK activation on PKC and beta 1 integrin. beta 1, beta 1 integrin; pY, phosphotyrosine; C, IP control

VEGF Stimulates Tyrosine Phosphorylation of CADTK in Adherent MM Cells-- In addition to influencing cell motility via controlling beta 1 integrin trafficking, catalytically active PKC also regulates other components of the focal complexes, including the small GTPases (Cdc42, Rho, Rac) and/or actin cytoskeleton-binding proteins. Calcium-dependent tyrosine kinase (CADTK), also known as proline-rich tyrosine kinase 2 (PYK2), calcium-dependent tyrosine-kinase beta  (CAKbeta ), and related adhesion focal tyrosine kinase (RAFTK), is a cytoplasmic tyrosine kinase homologous to focal adhesion kinase (FAK). Like FAK, CADTK is a platform kinase site for the coalescence of signaling and adaptor molecules, thereby facilitating the transmission of surface signals to the cytoskeleton and signaling pathways associated with cell growth, apoptosis, and migration. A number of studies have demonstrated tyrosine phosphorylation of CADTK in cells of hematopoietic origin (e.g. T and B cells, monocytes, natural killer cells, granulocytes, bone marrow progenitors, mast cells, megakaryocytes, and platelets). Stimuli that activate CADTK in these cells are associated with cell motility or at least cytoskeletal rearrangement (e.g. CADTK activation is required for cytoskeletal reorganization and monocyte motility) (42).

In MM.1S cells, we have shown previously that CADTK is activated upon dexamethasone treatment, suggesting its role in dexamethasone-induced apoptosis (47). Notably, CADTK has been reported to link calcium- and integrin-mediated signaling to the cytoskeleton in brain and hematopoietic cells (43-46). Our results show that VEGF induces the association of CADTK with the beta 1 integrin/PKCalpha protein complex (Fig. 4a). VEGFR immunoprecipitation and immunoblotting studies using anti-Flt-1 and anti-CADTK Abs showed specific constitutive association (Fig. 4b). We next investigated whether the VEGF-induced formation of a membrane complex containing PKCalpha , beta 1 integrin, and Flt-1 changes the activity of the associated CADTK in adherent MM cells. As shown in Fig. 4d, VEGF increased (2-fold) fibronectin-induced CADTK tyrosine phosphorylation in MM.1S cells; equivalent loading of proteins was confirmed by stripping and reprobing the membrane with anti-CADTK Ab. The PKC activator PMA enhanced constitutive phosphorylation of CADTK (Fig. 4c). Finally, VEGF-triggered CADTK activation was blocked by both the PKC inhibitor BIM I and a beta 1 integrin neutralizing mAb (Fig. 4e). These results indicate that the activation of CADTK in adherent MM cells is influenced by both PKC- and beta 1 integrin-mediated signaling pathways. Moreover, a recent report shows that CADTK in hematopoietic cells is also associated with the cytoskeletal protein paxillin (48). We therefore postulate that VEGF may facilitate signal transduction to the cytoskeleton in MM cells and modulate MM cell migration and/or adhesion on fibronectin via CADTK. Ongoing studies are directed toward understanding this role of CADTK in MM cells in more detail.

p85 Interacts with Flt-1-- We next sought to identify the inside-out signaling components mediating regulation of beta 1 integrin function by VEGF in MM cells. PI 3-kinases phosphorylate PI(4,5)P2, and PLCgamma hydrolyzes PI(4,5)P2 to diacylglycerol and inositol 1,4,5-trisphosphate, thereby mediating intracellular calcium release and PKC activation (49). PLCgamma activity is enhanced by 3-phosphoinositides, both indirectly via Btk-related tyrosine kinases and directly through the binding of PI(3,4,5)P3 to the amino-terminal pleckstrin homology (PH) domain and the tandem Src homology (SH) 2 domains of PLCgamma (50-53). We have shown recently that Flt-1 VEGFR is expressed in the human MM cell line and patient MM cells and is tyrosine-phosphorylated upon VEGF stimulation (12). Because Flt-1 and the p85 subunit of PI 3-kinase were associated when overexpressed in yeast cells (54), we next determined whether this interaction is biologically significant in MM cells. Flt-1 was immunoprecipitated from VEGF treated and non-treated MM.1S cells followed by immunoblotting with anti-p85 Ab. As illustrated in Fig. 5a, p85 is constitutively associated with Flt-1, suggesting that PI 3-kinase participates in Flt-1-mediated VEGF signal transduction.


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  		Fig. 5.   VEGF-induced PI 3-kinase activation is required for MM cell migration. Serum-starved cells were stimulated with VEGF for indicated intervals, lysed, and immunoprecipitated with anti-Flt-1 Ab (panel a), anti-p85 (panel b), or anti-pY (panel c). Immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. Panel d, for the PI 3-kinase assay, equal amounts of lysates were immunoprecipitated with anti-phosphotyrosine mAb, and immunocomplexes were assayed for the ability to phosphorylate PIP2. PIP3, phosphatidylinositol (3,4,5)P3. C, control; indicates a PI 3-kinase assay performed on protein A alone.

VEGF Mediates PI 3-Kinase Activation in MM Cells-- To confirm this hypothesis, we next examined whether PI 3-kinase activation is an early event in VEGF-induced signaling in MM cells. VEGF induced tyrosine phosphorylation of the regulatory p85 subunit of PI 3-kinase, evidenced by immunoblotting of anti-p85 immunoprecipitates with an anti-pY mAb (Fig. 5b). Moreover, immunoblotting of anti-pY immunoprecipitates with specific p85 mAb showed an increased association of p85 with tyrosine-phosphorylated proteins, which was maximal after 5 min of VEGF treatment (Fig. 5c). To determine whether VEGF-induced p85 phosphorylation regulates p110, the catalytic counterpart of PI 3-kinase, we performed in vitro kinase assays using PI 4,5-bisphosphate as a substrate. As shown in Fig. 5d, VEGF induced peak activation (6-fold) of PI 3-kinase activity at 5 min. Taken together, these data show that activation of PI 3-kinase is an early event in the VEGF-triggered Flt-1 signaling cascade.

VEGF-induced PI 3-Kinase Activation and Adhesion to Fibronectin Mediate PKCalpha Translocation to the Plasma Membrane-- We next examined whether activation of PI 3-kinase is required for activation of PKCalpha . When PI 3-kinase activation in MM cells seeded on fibronectin was inhibited by LY294002, PKCalpha recruitment to the plasma membrane was also abrogated (Fig. 6). These results indicate that VEGF-induced PI 3-kinase activation along with beta 1 integrin binding to fibronectin activate PKCalpha .


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  		Fig. 6.   Subcellular localization of PKC isoforms and activation is PI 3-kinase-dependent. MM.1S cells placed in suspension (S) or plated on fibronectin (FN) were either pretreated with LY 294002 (LY, 20 µM) or left untreated prior to stimulation with 100 ng/ml VEGF for 30 min. SP100 cell fractionation was performed as described under "Materials and Methods." Immunoblots of cytosolic (C) and membrane (M) fractions were detected with PKCalpha and reprobed with PKCepsilon . Molecular mass standards in kDa are shown.

MM Cell Migration and Cell Adhesion Are PI 3-Kinase-dependent-- To determine whether activation of PI 3-kinase is necessary for MM cell migration, we next used a Boyden-modified chamber to assay the effect of VEGF on the transfilter migration activity of MM.1S cells seeded on membranes precoated with fibronectin or polylysine as a control for ECM specificity. As seen in Fig. 7a, VEGF added to the conditioned medium in the lower chamber induced a dose-dependent migration of growth factor-deprived MM.1S cells seeded on fibronectin in the upper chamber, which was totally inhibited by the PKC inhibitor BIM I. Preincubation with the PI 3-kinase inhibitor LY294002 (20 µM) at 37 °C for 45 min prior to VEGF stimulation similarly inhibited MM cell migration. In contrast, VEGF did not induce migration of MM.1S cells seeded on polylysine in the upper chamber. We next examined whether VEGF-induced increments in adhesion were also PI 3-kinase-dependent. As shown in Fig. 7b, incubation with the PI 3-kinase inhibitor LY294002 (20 µM) blocked VEGF-induced MM cell adhesion to fibronectin. In contrast, VEGF did not effect MM cell adhesion toward polylysine. Taken together, these results demonstrate that PI 3-kinase activation during MM cell migration on fibronectin regulates PKC. These observations are similar to the Ca2+- and PI 3-kinase-dependent PKCalpha -catalyzed endocytosis reported in breast cancer cells (26).


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  		Fig. 7.   VEGF-mediated MM cell adhesion and migration are PI 3-kinase-dependent. a, MM cell migration was performed as described under "Materials and Methods." no inh, VEGF (10 ng/ml); results shown are representative of three independent experiments. b, MM cell adhesion. S, soluble; neg c, cells seeded to fibronectin or polylysine without stimulation; pos c, VEGF (100 ng/ml); LY, LY294002 (5 and 20 µM). The data shown are the mean ± S.D. of three separate experiments; adhesion assays were performed in triplicate.

Time-lapse Phase Contrast Video Microscopy-- Migration is a complex dynamic process of cell adhesion formation and release organized and coordinated both in time and space. To enhance our understanding of the above results and to link them with the dynamic changes in MM cell morphology that ultimately mediate cell migration, we used time-lapse phase contrast video microscopy (TLVM) (Figs. 8 and 9). During migration filopodia and lamellipodia together with new adhesions are formed at the leading edge, whereas detachment of adhesions are concomitantly observed at the trailing cell edge. In time-course experiments, MM.1S cells were seeded on either fibronectin precoated or control tissue culture plates in the presence or absence of VEGF. Representative cells were investigated for 30 min starting at 3 h after plating, and composite time-lapse phase contrast micrographs (magnification, ×40) were acquired at 2-min intervals tracked with the NIH Image 1.62 software (Fig. 8). MM cells adherent to non-coated tissue culture plates failed to polarize, and the random movement was slightly increased by VEGF (Fig. 8, a and b). In contrast, MM cells adherent to fibronectin rapidly polarized and exhibited increased continuous membrane ruffling (Fig. 8c). Although additional stimulation with VEGF did not significantly change membrane ruffling, the migration rate was markedly increased (Fig. 8d). Importantly, in the presence of the PKC inhibitor BIM I, migration rate was again decreased to levels obtained in MM cells adherent to fibronectin alone (Fig. 8e).


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  		Fig. 8.   Morphology and velocity of migrating cells. Top left, average speed of representative cells shown in bars a-e. Panels a-e, top, composite time-lapse series of phase contrast micrographs (magnification ×40) acquired at 2-min intervals were outlined and tracked with the NIH Image 1.62 software. Images show migration of representative cells over 30 min, starting 3 h after plating. Cells were seeded on tissue culture plates precoated (panels c-e) or not precoated (panels a and b) with fibronectin in the presence (panels b, d, and e) or absence (panels a and c) of VEGF (100 ng/ml). Panels a-e, bottom, graphs represent surface area and length as a description of morphological changes. Bar, 20 µm.


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  		Fig. 9.   Quantitative evaluation of MM cell motility. Average speed of MM cells was measured as described under "Materials and Methods." In time-course experiments, MM.1S cells were seeded on fibronectin precoated or not precoated tissue culture plates, in the presence or absence of VEGF.

The average speed of MM cell migration was next measured as described under "Materials and Methods" (Fig. 9). The baseline migration rate of MM cells seeded on tissue culture plates (not precoated with fibronectin) corresponded to the spontaneous motility of resting cells. The average speed remained steady for the whole period of observation and never exceeded 15-20 µm/hr. Similar results were observed using VEGF-treated cells in the absence of fibronectin. Importantly, binding of MM cells to fibronectin induced a marked acceleration of cell motility, peaking 2-3 h after seeding and remaining higher compared with the conditions described above. VEGF induced an additional increase in motility peaking at 3 h at 100 µm/hr and remaining significantly higher compared with non-stimulated cells seeded to fibronectin. This acceleration was inhibited to baseline migration rates in the presence of 2 µM BIM I.

This study therefore demonstrates that VEGF-mediated MM cell migration is associated with beta 1 integrin- and PI 3-kinase-dependent PKCalpha activation. To further verify the role of PKCalpha as a potential new therapeutic target in MM, ongoing studies are investigating the effect of PKCalpha -antisense both in vitro and in murine MM models.

    ACKNOWLEDGEMENTS

We thank Dr. Frank Gesbert (Dana-Farber Cancer Institute), Dr. Werner Lubitz (Vienna Biocenter, Vienna), and Dr. Heinz Ludwig (Wilhelminenspital, Vienna) for helpful discussions.

    FOOTNOTES

* This work was supported by an International Myeloma Foundation/ Brian D. Novis/Benson Klein Research grant award (to K. P.), National Institutes of Health Grant PO-1 78378, and the Doris Duke Distinguished Clinical Research Scientist Award (to K. C. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dana-Farber Cancer Inst., 44 Binney St., Boston, MA 02115. Tel.: 617-632-2144; Fax: 617-632-2140; E-mail: kenneth_anderson@dfci.harvard.edu.

Published, JBC Papers in Press, December 20, 2001, DOI 10.1074/jbc.M109068200

    ABBREVIATIONS

The abbreviations used are: MM, multiple myeloma; BM, bone marrow; VEGF, vascular endothelial growth factor; PI, phosphatidylinositol; VEGFR, VEGF receptor; PKC, protein kinase C; Flt-1, FMS-like tyrosine kinase; VLA, very late antigens; ECM, extracellular matrix; Ab, antibody; mAb, monoclonal Ab; FBS, fetal bovine serum; BIM I, bisindolylmaleimide I; CADTK, Ca-dependent tyrosine kinase; PMA, phorbol-12-myristate-13-acetate; PLCgamma , phospholipase C gamma ; IP, immunoprecipitation.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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