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Sequence Motifs in the Integrin ₄ Cytoplasmic Tail Required for Regulation of In Vivo Expansion of Murine Lymphoma Cells¹

Michaela Bittner^*, Uwe Gosslar^*, Arne Luz and Bernhard Holzmann^2,*,

^* Institute of Medical Microbiology, Immunology, and Hygiene, Technische Universität, Munich, Germany; Gesellschaft für Strahlung und Umweltforschung-Institut für Pathologie Neuherberg, Oberschleissheim, Germany; and Department of Surgery, Klinikum rechts der Isar, Technische Universität, Munich, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The binding of integrins to cognate ligands is tightly controlled by intracellular signals. Conversely, integrin occupancy generates biochemical signals inside the cell. The present study examined whether concepts of integrin function established by in vitro analysis apply to regulation of receptor function in complex biologic settings in vivo using a mouse model of tumor metastasis. Integrin ₄ subunits were truncated at amino acid Gln¹⁰¹⁴ (A4-1014), preserving the conserved GFFKR motif, and at position Glu¹⁰²¹ (A4-1021). In vitro adhesion assays revealed that cytoplasmic tail truncations did not affect constitutive ligand binding of ₄ integrins, while agonist-induced adhesion was abolished by the A4-1014, but not by the A4-1021, mutation. Inducible ligand binding of ₄ integrins was dependent on cytoskeletal function, whereas constitutive adhesion was not. In vivo metastasis formation assays demonstrated that expansion of murine T lymphoma cells in spleen is strongly inhibited by the wild-type ₄ subunit and the A4-1021 mutant. In contrast, the in vivo phenotype of ₄ integrin expression in lymphoma cells was completely abrogated by the A4-1014 mutation. Cross-linking of ₄ integrins in vitro inhibited proliferation and induced apoptosis of LB cells expressing wild-type ₄ subunits or the A4-1021 mutant, but not of LB-A4-1014 cells. In summary, these results demonstrate that sequence motifs regulating cytoskeleton-dependent ₄ integrin activation in vitro are essential for the control of LB lymphoma cell expansion both in vitro and in vivo.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Integrins are a family of heterodimeric transmembrane receptors that mediate cell-cell and cell-matrix adhesion, but also fulfill important functions in signal transduction (1, 2, 3). Integrins of the ₄ subfamily are expressed on most leukocyte cell types and function as receptors for VCAM-1 and the alternatively spliced CS-1 region of fibronectin (4, 5, 6). Mucosal addressin cell adhesion molecule-1 (MAdCAM-1)³ is preferentially recognized by integrin ₄ß₇ and directs recirculating lymphocyte to mucosal sites (7). In contrast to other members of the integrin family, ₄ integrins have the capacity to mediate cell adhesion both under static conditions and in the presence of shear stress (8, 9, 10, 11, 12). Cell adhesion mediated by ₄ß₁ and ₄ß₇ integrins appears to play an important role in the differentiation of lymphocytes, the development of lymphoid organs, the induction of protective immune responses, immunopathology, muscle differentiation, and metastasis formation of malignant tumors (6, 13, 14, 15, 16).

Regulation of ligand binding activity by cellular signaling processes is considered an important aspect of integrin function. During the transition from virgin T cells to memory or effector T cells not only is expression of ß₁ integrins increased (17), but ß₁ and ß₂ integrins also undergo coordinated activation (18). Besides modulation of integrin activity associated with cellular differentiation, numerous environmental stimuli may cause transient and reversible activation of integrin ligand binding functions. Engagement of the TCR/CD3 complex or binding of the chemoattractant formyl-methionyl-leucyl-phenylalanine to its serpentine receptor induces transient ₄ integrin activity in lymphocytes (17, 19). The chemokines macrophage inflammatory protein-1, macrophage inflammatory protein-1ß, RANTES, and IFN-inducible protein-10 were also shown to enhance transient adhesion of resting and activated T cells to recombinant VCAM-1 (20). Recent results revealed that binding of _Vß₃ to its ligand CD31 resulted in increased locomotion of lymphocytes on VCAM-1 (21), suggesting regulation of integrin function by cross-talk between different integrins.

Integrin activation, as evidenced by increased ligand binding, may result from two distinct, but not mutually exclusive, mechanisms (22, 23, 24). First, a conformational change in the extracellular domain may enhance integrin affinity for ligand. Second, cytoskeletal reorganization may induce aggregation of integrin receptors, thereby increasing avidity and ligand binding capacity. Regulation of integrin function by affinity modulation and receptor clustering appears to variably apply to different members of the integrin family. Integrin _IIbß₃ binding to fibrinogen was shown to be increased by conformational changes in the extracellular integrin domain leading to a high affinity state (25). Chemoattractants such as RANTES or macrophage chemotactic protein-5 increased the affinity of integrin _Mß₂, whereas activation of ₄ß₁ was dependent on cytoskeleton-regulated integrin clustering (26). Moreover, chemoattractant-induced ₄ integrin activation is mediated by the small GTPase Rho (27) that is involved in modulation of the actin cytoskeleton and organization of focal adhesions (28, 29).

Ligand binding and aggregation of ₄ integrins were shown to induce multiple signals inside the cell. Enhanced phosphorylation of proteins such as focal adhesion kinase, paxilin, Fyn, Lck, and mitogen-activated protein kinase was observed after stimulating a human T lymphoblastic cell line with Abs against very late antigen-4 (VLA-4) or with the CS-1 region of fibronectin (30). Adhesion by ₄ß₁ integrins also provides a costimulatory signal for activation of resting T lymphocytes. When exposed to immobilized VCAM-1 in conjunction with anti-TCR/CD3 mAb or superantigens, resting T lymphocytes respond by cell proliferation; secretion of cytokines including IL-2, TNF-, IFN-, and granulocyte-macrophage CSF; up-regulation of the IL-2R; and induction of the transcription factors nuclear factor of activated T cells, activating protein-1, and NF-B (31, 32, 33, 34). In contrast to resting or short term activated T cells, coligation of the TCR and ₄ integrins on chronically stimulated T cells results in activation-dependent death (35). Additional studies have shown that adhesion of T cells to VCAM-1 augments the expression of 72-kDa gelatinase (36). In monocytes, engagement of ₄ integrins causes induction of TNF-, IL-1ß, IL-1R antagonist, monocyte adherence-derived inflammatory gene 6, and tissue factor production (37, 38, 39).

The cytoplasmic domains of integrin and ß subunits are critical for regulation of integrin transmembrane responses. Besides a highly conserved membrane proximal GFFKR motif, the cytoplasmic domains contain highly divergent amino acid sequences, whereas the ß-chains exhibit partial sequence conservation (40, 41). The importance of the ₄ cytoplasmic domain for integrin function was elucidated by domain swapping and deletion experiments. Exchange of the cytoplasmic ₂ domain with the ₄ domain enhanced cell migration of rhabdomyosarcoma cells (42). In another study the effects of ₄, ₂, and ₅ cytoplasmic domains on functions of integrins carrying ₄ and ß₁ extracellular domains were investigated. The results indicated that the ₄ cytoplasmic tail enhanced VLA-4-dependent chemotactic cell migration, but cell spreading and localization in focal contacts were greater with the ₂ or ₅ cytoplasmic tails (43, 44). Deletions of cytoplasmic tail sequences revealed that the -chain is critically involved in the stimulation of adhesion by cellular agonists such as PMA. Truncation of the ₄ chain C-terminal of the conserved GFFKR motif abolished the stimulatory effect of PMA and greatly diminished the adhesive capacity of the mutant receptor (43). It therefore appears that sequences on the C-terminal side of the GFFKR motif may mediate integrin activation.

Previously, we have shown that expression of the murine integrin ₄ subunit on the spontaneous, ₄ integrin-deficient T cell lymphoma LB strongly inhibits expansion of disseminated lymphoma cells in spleen and other organs (45). In the present report, experimental metastasis formation of LB cells was used as a model to study the role of amino acid motifs in the cytoplasmic tail of the ₄ subunit for in vivo integrin function as opposed to the regulation of in vitro ligand binding. The results demonstrate that for in vivo ₄ integrin function cytoplasmic tail sequences are required that mediate cytoskeleton-dependent receptor activation by cellular agonists, but do not affect constitutive integrin activity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abs and reagents

The following Abs were used: rat anti-murine integrin ₄, R1/2, PS/2, and 9C10; hamster anti-rat ß₁, HA2/5; rat anti-murine ß₇, Fib504 (46); rat anti-murine _E, M290; rat anti-murine ₆, EA-1 (47); rat anti-murine LFA-1 -chain, M17/4.3; rat anti-murine CD44, IM781 (48); and rat anti-murine ICAM-1, BE29G1 (American Type Culture Collection, Manassas, VA). Rat anti-murine TCR V_ß7, TR310, and rat anti-murine MAdCAM-1, R3/3 (49), neither of which binds LB lymphoma cells, were used as negative controls. Unless otherwise indicated, mAb were obtained from PharMingen (Hamburg, Germany). Recombinant soluble human VCAM-1 (domains 1–6) and VCAM-Ig consisting of domains 1 and 2 of human VCAM-1 fused to the human IgG1 constant region were provided by Dr. John Clements, British Biotechnology (Oxford, U.K.). Fluorescein-conjugated annexin V was obtained from R & D Systems (Minneapolis, MN).

Flow cytometric analysis

For flow cytometric analysis, lymphoma cells were incubated with saturating amounts of mAbs, and Ab binding was detected by FITC-conjugated mouse F(ab')₂ reacting with rat Ig (Dianova, Hamburg, Germany). Streptavidin coupled with FITC (Dianova) was used as a second-stage reagent to examine the reactivity of biotinylated mAb HA2/5. The samples were analyzed on a Coulter EPICS XL cytometer (Hialeah, FL).

Generation of ₄ cytoplasmic tail mutants in LB lymphoma cells

Deletions in the murine ₄ integrin subunit cytoplasmic tail were created by introduction of termination codons at amino acid position Q1014 or E1021 by double-stranded, site-directed mutagenesis using the Chameleon mutagenesis kit (Stratagene, Heidelberg, Germany) as recommended by the manufacturer. Mutations were confirmed by DNA sequencing. The cDNA constructs encoding mutant ₄ subunits were subcloned into the retroviral vector pNTK (50). Recombinant retroviruses were prepared from the mouse ecotropic, helper virus-free producer line GP+E-86 (51). The BALB/c T cell lymphoma LB was infected with recombinant retroviruses as previously described (45). Transduced cells were selected with G418, and clonal cell lines were obtained by limiting dilution. From each construct at least two independent clones derived from separate infections were used for the experiments reported. Generation of LB cell clones expressing wild-type ₄ subunits (LB-A4-wt) and of control LB-NTK cells has been described previously (45).

Cell adhesion assays

Flat-bottom 96-well plates were coated overnight at 4°C with recombinant human VCAM-1 at 0.25 µg/well in 100 µl of PBS. Alternatively, plates were coated overnight at 4°C with 5 µg/well of glutathione-S-transferase (GST) linked to the fibronectin CS-1 segment (amino acid sequence: FLPHPNLHGPEILDVPST). For controls, plates were coated with 5 µg/well of GST in 100 µl of PBS. The plates were washed and blocked for 1 h at 37°C with TBS buffer (14 mM Tris-HCl (pH 7.4), 137 mM NaCl, 2.7 mM KCl, 1% BSA, and 2 mM glucose). Lymphoma cells were labeled in cell culture medium for 30 min at 37°C with 12 µg/ml H33342 (Calbiochem, Bad Soden, Germany). The cells were washed with PBS followed by PBS containing 1 mM EDTA. For some experiments, cells were treated for 20 min at 37°C with 10 µM cytochalasin D (Sigma, Deisenhofen, Germany) in TBS. Thereafter, divalent cations at the concentrations indicated or 20 ng/ml PMA were added, and cells were incubated for 10 min at 37°C. Cells were plated at 5 x 10⁴/well and centrifuged for 10 min at 10 x g. Cells were allowed to adhere for 30 min at 37°C, and unbound cells were removed by inverse centrifugation of the plate at 50 x g for 10 min. Adhesion was quantified by fluorometry using a Cytofluor 2300 (Millipore, Bedford, MA).

Experimental metastasis formation

Syngeneic BALB/c mice were injected into the tail vein with 1 x 10⁶ lymphoma cells suspended in 100 µl of PBS. After 7 days, single-cell suspensions from spleen were prepared. Spleen cells were cultured in 96-well plates for 24 h at 2 x 10⁵ cells/well in RPMI 1640 containing 7% FCS. Subsequently, the cells were labeled for 20 h with 1 µCi/well of [³H]thymidine (Amersham, Braunschweig, Germany). Incorporation of [³H]thymidine was quantified in a beta counter. For histopathologic examination, syngeneic BALB/c mice were injected i.v. with LB-NTK, LB-A4-wt, LB-A4-1014, or LB-A4-1021 cells at 1 x 10⁶ cells/mouse. Spleens were removed on day 7 after injection, fixed in 10% buffered formalin, and embedded in paraffin. Sections of 5 µm were stained with hematoxylin and eosin.

In vivo migration assay

Control LB-NTK cells were labeled with 1 µM of the green fluorochrome 5-chloromethylfluorescein diacetate (CMFDA) (Molecular Probes, Leiden, The Netherlands), while lymphoma cells expressing wild-type integrin ₄ subunits or ₄-cytoplasmic deletion mutants were traced by incubation with 5 µM of the red fluorescent dye 5-(and-6)-4-chloromethylbenzoylaminotetramethyl-rhodamine (CMTMR) (Molecular Probes). Cell labeling was performed according to the manufacturer’s protocol. Equal numbers of LB-NTK cells and lymphoma cells expressing various ₄ constructs (2.5 x 10⁷ each) were mixed and injected i.v. into syngeneic BALB/c mice. Spleens were removed after 2 or 24 h, and single-cell suspensions were fixed in PBS containing 1% paraformaldehyde. The ratios of LB-A4-wt cells or ₄-cytoplasmic deletion mutants to LB-NTK cells in the injection mixture (R_I) and in spleen (R_SPL) were determined by flow cytometry, and the relative localization ratios (RLR = R_SPL/R_I) were calculated for each sample.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of cytoplasmic domain mutants of the integrin ₄ subunit and expression in LB lymphoma cells

To evaluate the function of sequence motifs in the ₄ cytoplasmic domain for integrin regulation in vitro and receptor function in vivo, two deletion mutants were constructed. Using in vitro mutagenesis a stop codon was inserted at amino acid position 1014 of the murine ₄ subunit (A4-1014), thus preserving only the GFFKR motif that is common to all integrin -chains. In the second construct (A4-1021) seven amino acids C-terminal to the GFFKR motif were retained in addition (Table I). Truncated ₄ subunits were transduced into LB lymphoma cells in the expression vector pNTK using retrovirus-mediated gene transfer. LB lymphoma cells lack ₄ integrins, but synthesize ß₁ and ß₇ subunits (45). Generation of LB cells expressing wild-type ₄ subunits (LB-A4-wt) and of control LB-NTK cells has been reported previously (45). For each ₄ construct at least two independent LB cell clones derived from independent retroviral infections were used in subsequent experiments.

View this table: [in this window] [in a new window]   Table I. Structure of 4 integrin cytoplasmic tail mutants

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Expression of adhesion molecules on transduced LB cell lines. Expression of surface receptors (filled histograms) on selected LB cell clones was determined by immunofluorescence staining and flow cytometric analysis. Negative control staining was performed using mAb TR310 against the Vß7 segment of the murine TCR (open histograms). Mean fluorescence intensities are indicated in each histogram. Values obtained with negative control staining using mAb TR310 have been subtracted.

Differential regulation of constitutive and inducible cell adhesion mediated by ₄ integrin mutants in vitro

To determine the basal activity state of mutant ₄ integrins, adhesion of LB cell clones to recombinant soluble VCAM-1 was analyzed over a wide range of Ca²⁺ concentrations, and the concentrations resulting in half-maximal adhesion (ED₅₀ values) were determined. It has been demonstrated previously that distinct activity states of ₄ integrins can be distinguished by titration of various divalent cations and that ED₅₀ values are independent of integrin expression levels (52, 53). The results presented in Fig. 2 indicate that the ED₅₀ values for Ca²⁺ did not significantly differ between LB cell clones expressing full-length or mutant ₄ subunits. Moreover, when adhesion assays were performed at 1 mM Ca²⁺ unstimulated LB-A4-wt, LB-A4-1014, and LB-A4-1021 clones, but not LB-NTK cells, bound to VCAM-1 with comparable and high efficiency (Fig. 3 and data not shown). In the presence of 1 mM EDTA, VCAM-1 adhesion of LB cell clones expressing wild-type or mutant ₄ constructs was completely abrogated (Fig. 3). These results therefore indicate that the constitutive activity of wild-type or mutant ₄ integrins was sufficient to mediate VCAM-1 adhesion at divalent cation concentrations that are in a physiologic range. In addition, these findings document the functional integrity of the extracellular domains of the various ₄ integrin constructs and demonstrate that differences in expression levels between wild-type or mutant ₄ subunits did not affect constitutive ligand binding capacity.

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Constitutive binding of 4 integrins to VCAM-1 is not affected by truncations of the subunit cytoplasmic tail. Adhesion of clonal LB-A4-wt (filled circles), LB-A4-1021 (open squares), and LB-A4-1014 (gray diamonds) cell lines to recombinant soluble VCAM-1 was analyzed over a wide range of Ca2+ concentrations. The results of a representative experiment are presented. The ED50 values representing half-maximal adhesion were 86 ± 6 µM for LB-A4-wt, 91 ± 30 for LB-A4-1021, and 77 ± 30 for LB-A4-1014 (mean ± SD of at least three independent experiments for each mutant).

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Constitutive binding of 4 integrins to VCAM-1 is independent of cytoskeletal function. Constitutive adhesion to recombinant soluble VCAM-1 was measured in the presence of 1 mM Ca2+ and 1 mM Mg2+. Adhesion was blocked in the presence of 1 mM EDTA. To inhibit cytoskeletal functions, cells were preincubated with 10 µM cytochalasin D for 20 min. Results are expressed as the percentage of adherent cells and represent the mean ± SD from at least three independent experiments.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Inducible adhesion to CS-1 is dependent on 4 cytoplasmic tail sequences and cytoskeletal function. GST and GST-CS-1 were produced in Escherichia coli, and purified recombinant proteins were adsorbed onto 96-well plates. Constitutive binding of LB cell clones expressing wild-type or mutant 4 subunits was measured in the presence of 1 mM Ca2+ and 1 mM Mg2+. To stimulate ligand binding cells were incubated with 20 ng/ml PMA or 1 mM Mn2+. Cytochalasin D treatment was performed as described in Fig. 3. The data represent the mean percentage of adherent cells ± SD and were obtained from at least three separate experiments. Filled bars, GST; gray bars, GST-CS-1.

In vivo expansion of LB lymphoma cells in spleen is regulated by sequence motifs in the ₄ subunit cytoplasmic tail

To address the question of whether concepts of inside-out activation of integrin function established by in vitro experiments also apply to regulation of receptor function in complex biologic settings in vivo, spleen metastasis formation of LB lymphoma cells was examined. The LB lymphoma model was selected based on previous results showing that de novo expression of the wild-type ₄ integrin subunit inhibits expansion of metastatic LB cells in spleen and other organs (45). In the present study, syngeneic BALB/c mice were injected i.v. with LB-NTK, LB-A4-wt, LB-A4-1014, or LB-A4-1021 cells, and metastatic growth of lymphoma cells was evaluated after 7 days. The results summarized in Figs. 5 and 6 directly demonstrate that in contrast to wild-type ₄ subunits the mutant A4-1014, which lacks the cytoplasmic domain except for the GFFKR motif, completely lost the ability to suppress metastasis formation of LB cells. Both [³H]thymidine incorporation assays and histopathologic analyses indicated that expansion of disseminated LB-A4-1014 cells in spleen was comparable to that of LB-NTK control cells (Figs. 5 and 6). Interestingly, addition of seven amino acids C-terminal of the GFFKR motif (LB-A4-1021 cells) was sufficient to reconstitute ₄ integrin function in vivo and to inhibit metastasis formation with the same efficiency as wild-type ₄ subunits (Figs. 5 and 6). To exclude that the high metastatic potency of the LB-A4-1014 cells was due to selection of variants lacking mutant ₄ integrin subunits, lymphoma cells derived from day 7 spleen tumors were analyzed upon short term in vitro culture for expression of the A4-1014 construct. Immunofluorescence staining and flow cytometric analysis of LB-A4-1014 cells revealed homogeneous expression of mutant ₄ subunits (data not shown). In summary, these results emphasize that sequence motifs regulating inside-out activation of ₄ integrins in vitro are also critical for the control of ₄ integrin function in vivo.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. Spleen metastasis formation of LB lymphoma cells is regulated by amino acid motifs in the cytoplasmic tail of the integrin 4 subunit. Independent LB cell clones (1 x 106 cells/mouse) were injected i.v. into syngeneic BALB/c mice. After 7 days spleens were removed from untreated BALB/c mice or from mice injected with LB-NTK, LB-A4-wt, LB-A4-1014, or LB-A4-1021 clones. The lymphoma cell load in spleen was determined by [3H]thymidine incorporation assay. Data are presented as the mean ± SE from six independent mice for each cell line.

View larger version (109K): [in this window] [in a new window]   FIGURE 6. Histopathologic analysis of spleen metastases. Syngeneic BALB/c mice were injected i.v. with 1 x 106 clonal LB-NTK, LB-A4-wt, LB-A4-1014, or LB-A4-1021 cells. Spleens were removed after 7 days, and tissue sections were stained with hematoxylin and eosin. For each cell line, three independent mice were examined, yielding similar results (original magnification, x10).

LB lymphoma cell migration to spleen is not affected by ₄ integrins

To test whether in vivo trafficking of lymphoma cells may be altered by expression of wild-type or mutant integrin ₄ subunits, short term migration of LB cell clones to the spleen was examined. Control LB-NTK cells were labeled with the green fluorescent dye CMFDA, while LB-A4-wt, LB-A4-1014, or LB-A4-1021 cells were traced with the red fluorochrome CMTMR. For each experiment, equivalent numbers of LB-NTK cells and LB clones expressing truncated or wild-type ₄ subunits were mixed and injected into the tail vein of syngeneic BALB/c mice. After 2 h lymphoma cell numbers in spleen were determined by flow cytometry. As shown in Fig. 7A the ratios of LB-A4-wt, LB-A4-1021, or LB-A4-1014 cells to LB-NTK cells in spleen were nearly equivalent, suggesting that LB lymphoma cells expressing wild-type or mutant ₄ integrin receptors do not differ in their capacity to invade spleen.

View larger version (26K): [in this window] [in a new window]   FIGURE 7. Short term accumulation of lymphoma cells in spleen. To determine the short term accumulation of control lymphoma LB-NTK as well as LB-A4-wt, LB-A4-1014, and LB-A4-1021 in spleen, LB-NTK cells were labeled with a green fluorochrome and used as internal standard, while LB-A4-wt, LB-A4-1014, and LB-A4-1021 cells were stained with a red fluorochrome. For each experiment, 2.5 x 107 LB-NTK and 2.5 x 107 LB-A4-wt, LB-A4-1014, or LB-A4-1021 lymphoma cells were mixed and injected into the tail vein of syngeneic BALB/c mice. Spleens were removed 2 h (A) and 24 h (B) after injection of lymphoma cells. Absolute numbers of green and red fluorescent cells in spleen were determined by flow cytometric analysis. Results represent the mean relative localization ratios ± SD from at least three independent experiments.

Effects of ₄ integrins on lymphoma cell growth and apoptosis in vitro are dependent on cytoplasmic tail motifs

To directly address the question of whether the effects of ₄ cytoplasmic tail mutants on metastasis formation by lymphoma cells are caused by post-trafficking events, LB cell proliferation and apoptosis were analyzed following ₄ integrin cross-linking by solid phase Ab or natural ligand. The results presented in Fig. 8A demonstrate that proliferation of LB-A4-wt cells was inhibited by ₄ integrin ligation using specific mAb or VCAM-Ig. In contrast, proliferation of LB-A4-1014 mutants and LB-NTK control cells was not affected by ₄ integrin cross-linking, whereas LB-A4-1021 cells were growth inhibited (Fig. 8A). Proliferation of LB-NTK cells or LB cells expressing various ₄ constructs was not affected by cross-linking of ICAM-1 that is also expressed by LB cells (Fig. 8A and data not shown). In additional experiments, induction of apoptosis was investigated using annexin V, which binds to phosphatidylserine relocated to the outer plasma membrane of apoptotic cells. As is shown in Fig. 8B, the fraction of apoptotic LB-A4-wt cells increased about twofold after ₄ integrin cross-linking, whereas apoptosis was not induced in LB-A4-1014 cells. Thus, ₄ integrin-induced growth inhibition and apoptosis of LB lymphoma cells are dependent on sequence motifs in the ₄ cytoplasmic tail.

View larger version (48K): [in this window] [in a new window]   FIGURE 8. Inhibition of lymphoma cell growth and induction of apoptosis in vitro by cross-linking of 4 integrins are dependent on cytoplasmic tail motifs. A, Microtiter wells were coated with 0.5 µg of mAbs R3/3 (anti-MAdCAM-1), BE29G1 (anti-ICAM-1), PS/2, or 9C10 (both directed against the integrin 4 subunit) or with 1.0 µg of VCAM-Ig. To each well, 5 x 103 LB-NTK, LB-A4-wt, LB-A4-1014, or LB-A4-1021 cells were added; incubated for 3 days in cell culture medium; and pulsed with 1 µCi/well [3H]thymidine for 20 h. Incorporation of [3H]thymidine obtained in the presence of mAb R3/3, which does not bind LB lymphoma cells, was defined as 100%. Results are presented as the mean ± SD from two to five independent experiments. Filled bars, LB-NTK; gray bars, LB-A4-wt; hatched bars (coarse), LB-A4-1021; hatched bars (fine), LB-A4-1014. B, LB-A4-wt (gray bars) or LB-A4-1014 cells (hatched bars) were incubated with mAb as described in A, stained with fluorescein-conjugated annexin V, and analyzed by flow cytometry. Results are presented as the mean ± SD from three or four independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The results of this study demonstrate that in vivo and in vitro expansion of LB cells was inhibited by the A4-wt and A4-1021 constructs with similar efficiency, suggesting a minor role for the 18 C-terminal amino acids. Together with the findings for the A4-1014 mutant, these data also suggest that the seven amino acids C-terminal of the GFFKR motif are sufficient for regulation of LB cell growth and apoptosis by ₄ integrins. Consistent with previous findings we noted that the length of the cytoplasmic ₄-chain influences receptor sensitivity to activation by the phorbol ester PMA (43, 56). While ligand binding of the A4-1014 mutant could not be stimulated by PMA, addition of seven amino acids C-terminal (A4-1021) was sufficient to completely restore cytoskeleton-dependent induction of adhesion, suggesting an important role of this sequence in avidity regulation. Consistent with these data, a previous study observed that addition of six amino acids immediately following the GFFKR motif was sufficient to restore maximal adhesive activity of ₄ integrins in CHO cells (54). Taken together, these observations reveal a striking correlation between the structural requirements for integrin activation by intracellular signals and those for suppression of metastasis formation. It is therefore conceivable that signals induced by some factor(s) in the spleen stimulate or increase LB cell binding to natural ligand, leading to ₄ integrin signals that promote growth arrest and/or apoptosis. Although the nature of the integrin-activating signals remains to be identified, the results presented suggest that they may act through protein kinase C.

In vitro adhesion assays revealed that the constitutive ligand binding activity of integrins may vary according to the cellular background (43, 57). Thus, ₄ integrins expressed in naive or memory T cells show different constitutive ligand binding capacity (18). Our present results demonstrate that in LB cells basal ₄ integrin activity is sufficient to mediate binding to VCAM-1, but not to the CS-1 splice fragment of fibronectin, and are consistent with results showing that CS-1 binding requires a higher integrin activation state (52, 53). In previous studies the GFFKR motif of integrin subunits was identified as a regulatory element acting in a dominant negative manner. Thus, deletion of ₄ at a homologous amino acid position abolished ligand binding in carcinoma and CHO cells (54). Recent evidence indicates that the reduced adhesive capacity of ₄ integrin mutants truncated C-terminal of the GFFKR sequence results from deficient lateral mobility and clustering of integrin receptors (56). In the present study using divalent cation titration assays it was demonstrated that in contrast to phorbol ester stimulation, constitutive ligand binding by ₄ integrins was not affected by -chain cytoplasmic tail truncations. Moreover, inhibition of cytoskeletal function abolished PMA-inducible ligand binding, but did not alter constitutive adhesion to VCAM-1. These results therefore indicate that in LB cells basal ₄ integrin activity is independent of a putative negative regulatory role of the GFFKR motif and suggest that basal ₄ integrin activity is mainly determined by receptor affinity, whereas integrin activation by PMA appears to be regulated by receptor aggregation.

Signal transduction through ₄ integrins may be triggered by Ab-mediated cross-linking or by adhesion to cognate ligands (58), resulting in the formation of focal adhesion sites (59). In focal contacts, integrin cytoplasmic domains colocalize with cytoskeletal components such as talin, vinculin, -actinin, and filamin, as well as with signal transducing proteins, including focal adhesion kinase, c-Src, RhoA, Rac1, Ras, Raf, and c-Jun kinase (60, 61). The accumulation of structural and regulatory proteins in focal contacts is blocked by protein kinase inhibitors or disruption of the cytoskeleton using cytochalasin D (61). Notably, previous studies have demonstrated that integrin clustering and receptor occupancy by ligand synergize to induce integrin transmembrane responses and that both events are required for efficient redistribution of cytoskeletal and signaling proteins to adhesion sites (61, 62). The results presented here indicate that functions of ₄ integrins in vivo correlate with integrin responsiveness to cytoskeleton-dependent receptor activation. It is therefore conceivable that deficient aggregation of A4-1014 mutants severely impairs ₄ integrin-mediated signaling in LB cells and thereby abolishes the inhibitory effects of ₄ integrin ligation on metastasis formation.

	Acknowledgments

 
We thank Anja Schuster for expert technical assistance, Dr. Eugene C. Butcher for providing mAb Fib504, Dr. Beat Imhof for providing mAb EA-1, and Dr. John Clements for providing VCAM-1 protein.

	Footnotes

 
¹ This work was supported by a grant from the Deutsche Krebshilfe (Dr. Mildred Scheel Stiftung).

² Address correspondence and reprint requests to Prof. Dr. Bernhard Holzmann, Department of Surgery, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Str. 22, D-81675 Munich, Germany. E-mail address:

³ Abbreviations used in this paper: MAdCAM-1, mucosal addressin cell adhesion molecule-1; VLA, very late antigen; GST, glutathione-S-transferase.

Received for publication March 27, 1998. Accepted for publication August 6, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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