MMP-9 in B-cell chronic lymphocytic leukemia is ...

Blood First Edition Paper, prepublished online July 13, 2006; DOI 10.1182/blood-2006-03-007294

MMP-9 in B-cell chronic lymphocytic leukemia is upregulated by α4β1 integrin or CXCR4 engagement via distinct signaling pathways, localizes to podosomes, and is involved in cell invasion and migration Javier Redondo-Muñoz1, Elizabeth Escobar-Díaz1, Rafael Samaniego2, Ma José Terol 3, José A. García-Marco4, and Ángeles García-Pardo1

1

Departamento de Inmunología, Centro de Investigaciones Biológicas, CSIC,

Madrid, Spain; 2Servicio de Inmunología, Hospital Gregorio Marañón, Madrid; 3

Departamento de Hematología y Medicina Oncológica, Hospital Clínico,

Valencia, Spain; 4Servicio de Hematología, Hospital Puerta de Hierro, Madrid.

Running title: α4β1 integrin and CXCR4 regulate MMP-9 in B-CLL Scientific heading: Neoplasia

Supported by grant SAF2003-00824 from the Ministerio de Educación y Ciencia (MEC) and by the MMA Foundation (FMMA). JRM was supported by a fellowship from FMMA; EED was supported by a fellowship from MEC.

Correspondence: Ángeles García-Pardo, Departamento de Inmunología, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain. Tel: 34 91 837 3112, FAX: 34 91 536 0432; e-mail: [email protected]

Word count: Abstract: 196; Text: 4899.

Copyright © 2006 American Society of Hematology

Editorial note: JRM: Performed research and designed some experiments EED: Performed research RS: Performed the confocal microscopy analyses MJT: Contributed with patient samples and data JAGM: Contributed with patient samples and data AGP: Designed and supervised research and wrote the manuscript

2

Abstract B-cell chronic lymphocytic leukemia (B-CLL) progression is determined by malignant cell extravasation and lymphoid tissue infiltration. We have studied the role and regulation of matrix metalloproteinase-9 (MMP-9) in B-CLL cell migration and invasion. Adhesion of B-CLL cells to the fibronectin fragment FNH89, VCAM-1, or TNF-α-activated HUVEC, upregulated MMP-9 production, measured by gelatin zymography. This effect was mediated by α4β1 integrin and required PI3-K/Akt signaling. The chemokine CXCL12 also upregulated MMP-9, independently of α4β1 and involving ERK1/2 but not Akt activity. Accordingly, α4β1 engagement activated the PI3-K/Akt/NF-κB pathway, while CXCL12/CXCR4 interaction activated ERK1/2/c-Fos signaling. Anti-MMP-9 antibodies, the MMP-9 inhibitor TIMP-1, or transfection with three different MMP-9 siRNAs significantly blocked migration through Matrigel or HUVEC. Cell-associated MMP-9 was mainly at the membrane and contained the proactive and mature forms. Moreover, B-CLL cells formed podosomes upon adhesion to FN-H89, VCAM-1 or fibronectin; MMP-9 localized to podosomes in a PI3-K-dependent manner and degraded a fibronectin/gelatin matrix. Our results are the first to show that MMP-9 is physiologically regulated by α4β1 integrin and CXCL12 and plays a key role in cell invasion and transendothelial migration, thus contributing to B-CLL progression. MMP-9 could therefore constitute a target for treatment of this malignancy.

3

Introduction B-CLL is characterized by the accumulation of monoclonal, slow-dividing CD5+ B lymphocytes in the peripheral blood1-3. In most cases, these cells progressively infiltrate the bone marrow and secondary lymphoid tissue, resulting in poor prognosis1-3. Extravasation of B-CLL cells and migration through endothelium are mainly directed by three chemokines: CCL21, which is expresed in high endothelial venules (HEV), CCL19 and CXCL12, which are produced by stromal cells of lymph nodes and bone marrow respectively4,5. The corresponding receptors for these chemokines, CCR7 (for CCL21 and CCL19) and CXCR4 (for CXCL12), are highly expressed in B-CLL with widespread involvement of lymph nodes5,6. Other molecules like vascular endothelial growth factor (VEGF) and αLβ2/α4β1 integrins were also recently shown to be involved in B-CLL transendothelial migration in response to chemokines7. Moreover, high expression of α4β1 (but not αLβ2) correlated with the presence of lymphadenopathy7, suggesting an important role of this integrin in B-CLL progression. Transendothelial migration and organ invasion of malignant cells also requires proteolytic degradation of the vascular basement membrane and the extracellular matrix of lymphoid tissues. This can be accomplished by matrix metalloproteinases (MMP)8-10, in particular the gelatinases MMP-2 and MMP-9. MMPs also release matrix-bound growth factors that stimulate malignant cell expansion and angiogenesis11. Indeed angiogenesis is increased in the bone marrow of B-CLL patients12-14 and high levels of the angiogenic factors VEGF

4

and basic fibroblast growth factor (bFGF) have been detected in the urine and serum of these patients12,15,16. Previous studies have shown that early stage B-CLL produce and secrete MMP-9 which can be detected in the serum of these patients and in BCLL cell culture supernatants17. It was later demonstrated that B-CLL cells constitutively produce MMP-9 in various molecular forms and that elevated levels of intracellular MMP-9 correlate with advanced stage and poor patient survival18. Moreover, MMP-9 was highly expressed by B-CLL cells present in the bone marrow (with a diffused pattern) and in lymph nodes, and contributed to B-CLL migration through artificial basement membranes or endothelial cells18. The presence of other MMPs in B-CLL cells has not been reported. These previous reports suggest that MMP-9 contributes to B-CLL progression by facilitating malignant cell migration and tissue invasion, and could constitute a target for therapeutic intervention. It is therefore important to understand the mechanisms that regulate MMP-9 production in B-CLL. To address this, we have studied the role of α4β1 integrin and CXCR4, two molecules involved in B-CLL cell migration, in regulating MMP-9. We show that engagement of either receptor upregulates MMP-9 and we have identified the signaling pathways involved in this effect. We also show that MMP-9 localizes to podosomes upon adhesion to α4β1 integrin ligands, and plays a crucial role in B-CLL cell transendothelial migration and invasion through basement membranes.

5

Materials and Methods Patients, cell purification and cell lines Pheripheral blood samples were obtained after informed consent from twelve BCLL patients, diagnosed according to established clinical and laboratory criteria. None of them had received treatment at the time of this study, and eight patients presented lymphadenopathy. CD5+ B-lymphocytes were purified by Ficoll-Hypaque (Nycomed, Oslo, Norway) centrifugation. B-lymphocytes from healthy donors were purified from buffy coat cells by Ficoll-Hypaque centrifugation and anti-CD19-conjugated Dynabeads (Dynal Biotech ASA, Oslo, Norway). The EBV-transformed CO43, HUT112, and BRO168 cell lines, established from normal B-lymphocytes, have been previously reported19. Human umbilical vein endothelial cells (HUVEC) were kindly provided by Drs. S. Lamas and M. L. Botella (Centro de Investigaciones Biológicas, Madrid, Spain) and cultured in Medium 199 Modified Earle`s Salts (Gibco, Auckland NY USA), containing 15% FCS and 50 µgr/ml endothelial cell grow factor. HUVEC were used up to the fourth passage. Cell viability was assessed by flow cytometry using FITC-Annexin V (Bender Medsystems, Vienna, Austria) and propidium iodide as described20.

Antibodies, reagents and proteins Monoclonal antibodies (mAbs) HP2/1 (anti-α4 integrin subunit), HU5/3 (antiintercellular adhesion molecule-1, ICAM1) and W6/32 (anti-HLA) were obtained from Dr. F. Sánchez-Madrid (Hospital de la Princesa, Madrid, Spain); P1D6 (anti-α5 integrin subunit) has been previously described21; anti-vascular cell

6

adhesion molecule-1 (VCAM-1, sc-8304), anti-IκB-α (sc-1643), and anti-Akt1 (sc-5298) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); TS1/11 (anti-αL integrin subunit) and TS1/18 (anti-β2 integrin subunit) were obtained from Drs. A. Corbí and C. Bernabeu (Centro de Investigaciones Biológicas); LEM2/15 (anti-membrane type 1- MMP, MT1-MMP) was obtained from Dr. A. G. Arroyo (Centro Nacional de Investigaciones Cardiovasculares, Madrid); antiMMP-9 and anti-MMP-2 were from NeoMarkers (Labvision, Freemont, CA); anti-vinculin was from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal antibodies to phospho-Akt (Ser 473), phospho-p44/p42 MAPK, and p44/p42 MAPK were from Cell Signaling (Beverly, MA); rabbit antibodies to MMP-9 were from Chemicon (Temecula, CA). TIMP-1, UO126 (MEK inhibitor), PP2 (Src inhibitor), SB203580 (p38 MAPK inhibitor), Triciribine/API-2 (Akt inhibitor), Wortmannin and LY294002 (PI3K inhibitors), BisI (PKC inhibitor) and pertussis toxin were from Calbiochem (Darmstadt, Germany). The chemokine CXCL12 was from R&D Systems (Minneapolis, MN). MatrigelTM was from BD Biosciences (Erembodegem, Belgium). FITC-gelatin was from Molecular Probes (Leiden, Netherlands). Poly-lysine (p-Lys) was from Sigma. Plasma fibronectin was purified as described21. The fibronectin fragment H89 (FN-H89), containing the CS-1 ligand for α4β1 integrin21, and VCAM-1 were prepared as reported22,23. ICAM-1 was obtained from Dr. C. Cabañas (Instituto de Farmacología y Toxicología, CSIC, Madrid).

7

Results Constitutive expression of MMPs in B-CLL cells To establish the constitutive expression of MMPs in B-CLL, we first analyzed lysates of freshly isolated cells by gelatin zymography. All twelve samples studied contained MMP-9 in the 92 kDa proform (shown in Figure 1A for five representative samples). MMP-9 expression was significantly higher in B-CLL cells than in normal peripheral blood B cells, or in EBV-transformed B-cell lines (Figure 1A). The presence of MMP-2 was also analyzed by gelatin zymography (six samples), flow cytometry after cell permeabilization (three samples), and RT-PCR (five samples) (data not shown). These studies established the absence of MMP-2 in B-CLL cells, confirming previous observations18. We next examined the presence of MT1-MMP in these cells using flow cytometry. As shown in Figure 1B for patient 2 (and not shown for patients 3 and 4), MT1-MMP was not detected at the B-CLL cell surface and was not induced after treatment with PMA. In the same experiment, PMA induced MT1MMP expression on control NCI-H929 myeloma cells (Figure 1B). We then analyzed the intracellular presence of this MMP. As shown in Figure 1C, MT1MMP mRNA was detected in all five samples studied, with heterogeneous levels of expression. At the protein level, western blotting analyses of cell lysates from four different samples revealed the reported 64 kDa form of MT1MMP25 as well as other unidentified bands (Figure 1D). From these results we concluded that although MT1-MMP is found intracellularly in B-CLL cells, it probably plays a minor role in these cells. Accordingly, subsequent studies were focused on MMP-9.

8

α4β1

integrin-mediated

B-CLL

cell

adhesion

upregulates

MMP-9

production Adhesion to fibronectin mediated by α4β1, α5β1 and αVβ1 integrins or VCAM-1 via α4β1, was previously shown to upregulate MMP-9 and MMP-2 in T cells26,27. B-CLL cells encounter fibronectin and VCAM-1 during extravasation and migration within tissues and express α4β1 as a major integrin28,29. We therefore studied whether MMP-9 was upregulated in B-CLL cells by interaction with FNH89 or VCAM-1. Equal number of cells from six different patients was incubated on VCAM-1, FN-H89, or BSA-coated plates and after 24 h the conditioned media was collected and analyzed by gelatin zymography. As shown in Figure 2A for four representative samples, and quantitated for all six samples studied, MMP-9 was significantly upregulated by adhesion of B-CLL cells to VCAM-1 or FN-H89, compared to cells cultured on BSA. This effect was completely blocked by anti-α4 integrin subunit mAbs (Figure 2A). It was also blocked by cell preincubation with cycloheximide (not shown), suggesting that MMP-9 upregulation involved de novo protein synthesis. Enhanced MMP-9 secretion was not due to different viability of cells cultured on FN-H89 or BSA, as confirmed by flow cytometry (Supplemental Figure 1). Moreover, as shown in Figure 2B for patient 7, upregulation of MMP-9 by FN-H89 or VCAM-1 was dose-dependent, further establishing the specificity of the cell adhesion effect. We also studied whether MMP-9 was upregulated by adhesion of B-CLL cells to HUVEC. Unstimulated or TNF-α-activated HUVEC secreted only minimal levels of MMP-9 (Figure 2C). Adhesion of B-CLL cells to unstimulated HUVEC, did not increase MMP-9 production above the basal levels of cells on

9

BSA (Figure 2C). However adhesion to TNF-α-activated HUVEC, significantly increased MMP-9 secretion and this was blocked by anti-α4 mAbs (Figure 2C). In contrast, blocking αL or β2 integrin subunits, had a minor effect (Figure 2C). MMP-9 upregulation was also inhibited by anti-VCAM-1 but not anti-ICAM-1 mAbs (Figure 2C), further supporting the role of α4β1-mediated interactions in upregulating MMP-9 production.

α4β1 integrin upregulates MMP-9 in B-CLL cells via activation of the PI3K/Akt/NF-κB signaling pathway To determine the mechanism involved in the α4β1-mediated upregulation of MMP-9, B-CLL cells from three different patients, were incubated on FN-H89 in the presence of various protein kinase inhibitors. Gelatin zymographic analysis of the conditioned media revealed that inhibition of PKC, p38 MAPK, Src-family kinases, or ERK1/2 signaling, did not affect the enhanced MMP-9 secretion (Figure 3A). In contrast, inhibition of PI3-K by wortmannin, reduced this secretion to basal levels (Figure 3A). Wortmannin (or the other inhibitors, not shown) had no effect on the MMP-9 basal production of B-CLL cells on BSA (Figure 3A). To confirm these results we used two different doses of wortmannin and LY294002, another inhibitor of PI3-K; we also tested the effect of API-2, a specific inhibitor of the PI3-K effector Akt. As shown in Figure 3B for patient 4, wortmannin and LY294002 produced a dose-dependent inhibitory effect, and API-2 reduced MMP-9 production to basal levels. At the concentration used, none of the inhibitors induced B-CLL apoptosis, as confirmed by Annexin-V uptake (Supplemental Figure 2).

10

Having established that upregulation of MMP-9 by α4β1 integrin involved the PI3-K/Akt signaling pathway, we next studied if α4β1-mediated cell adhesion activated Akt. B-CLL cells from two representative samples were incubated on FN-H89 or BSA. After 24 h, cells were lysed and lysates analyzed by western blotting. As shown in Figure 3C, B-CLL cell adhesion to FN-H89 (but not to BSA) induced Akt phosphorylation and this was completely blocked by wortmannin or API-2. α4β1 integrin-mediated adhesion did not activate ERK1/2 (Figure 3C), indicating that the effect was specific for Akt. Moreover, the Akt downstream effector NF-κB was also activated at this time, as determined by measuring the levels of the NF-κB-associated protein IκB-α, which is released from the complex and degraded upon activation30. As shown in Figure 3D, IκBα decreased upon cell adhesion to FN-H89, and this response was reverted by PI3-K or Akt inhibitors. We also studied the kinetics of Akt phosphorylation induced by B-CLL adhesion to FN-H89. Figure 3E shows that Akt was phosphorylated after 30 min and remained activated for the 24 h of the assay. Sustained Akt signaling was further confirmed by kinetic studies of IκB-α levels, which remained low throughout the assay (Supplemental Figure 3). No ERK1/2 activation was observed at any time (Figure 3E).

CXCL12 upregulates MMP-9 in B-CLL cells via the ERK1/2/c-Fos signaling pathway and independently of PI3-K B-CLL cells express functional receptors for CXCL12 (CXCR4), which are involved in the migration of these cells particularly in the bone marrow31,32. To establish whether the interaction CXCL12/CXCR4 could modulate MMP-9

11

production, B-CLL cells from six different patients were incubated for 24 h with or without CXCL12, and the conditioned media analyzed by gelatin zymography. As shown in Figure 4A for four representative samples and quantitated for all six patients studied, CXCL12 significantly enhanced MMP-9 secretion with respect to untreated cells. This effect was completely blocked by inhibiting CXCR4 signaling with pertussis toxin (Figure 4A). To determine whether this CXCL12 regulatory effect involved the same or different signaling pathways as α4β1 integrin, we performed the experiments described above in the presence of various protein kinase inhibitors. As shown in Figure 4B for four representative samples, inhibition of ERK1/2 by UO126 significantly reduced MMP-9 to basal levels, while all other inhibitors, including wortmannin and LY294002 (not shown) had no effect. To determine if CXCL12 induced ERK1/2 phosphorylation, B-CLL cells from the same two samples used in Figure 3C were incubated in the presence or absence of CXCL12. After 24 h, cells were lysed and lysates analyzed by western blotting. Figure 4C shows that CXCL12 phosphorylated ERK1/2 in B-CLL cells and this was inhibited by UO126 and pertussis toxin. CXCL12 did not activate Akt (Figure 4C). We next studied the activation of c-Fos, a well known effector ERK1/233. c-Fos was not detected after 24 h of CXCL12 stimulation in the two samples studied (not shown), and thus we measured c-Fos activation at earlier times. Figure 4D shows that CXCL12 rapidly increased (2-5 min) the basal expression of c-Fos, after which c-Fos levels progressively declined. In correlation with this peak of c-Fos activation, the levels of phospho-ERK1/2 were also maximal after 5 min of CXCL12 stimulation (Figure 4E). Although these levels also decreased with

12

time, ERK1/2 remained activated for at least 24 h, as previously observed (Figures 4C and E). CXCL12 did not activate Akt at any time (Figure 4E).

α4β1 integrin and CXCR4 independently upregulate MMP-9 The preceding results indicated that CXCR4- and α4β1 integrin-dependent upregulation of MMP-9 had different signaling requirements. We next studied if the effect of both receptors was additive. B-CLL cells from four different patients were incubated for 24 h on BSA or FN-H89 and with or without CXCL12, and the conditioned media analyzed by gelatin zymography. Interestingly, α4β1 integrin and CXCR4 co-stimulation did not increase MMP-9 secretion above the levels induced by either stimulus, but resembled the effect of CXCL12 alone (Figure 5A). To confirm this, we studied the signaling pathways involved in MMP-9 upregulation by co-stimulation via α4β1 and CXCL12. As shown in Figure 5B for two representative samples, inhibition of ERK1/2 reduced the enhanced MMP-9 secretion by 80% while inhibition of PI3-K resulted in 48% reduction. The combination of both inhibitors decreased MMP-9 to basal levels (94% inhibition, Figure 5B). Altogether these results established that CXCL12 and α4β1 enhanced MMP-9 production via different signaling pathways, and that the combination of both stimuli was not cooperative.

MMP-9 is involved in B-CLL invasion through basement membranes and transendothelial migration, in a PI3-K/Akt and ERK1/2-dependent manner It was recently shown that MMP-9, when activated by plasmin, contributes to BCLL migration through mixtures of collagen IV/laminin or HUVEC18. We

13

analyzed if the enhanced MMP-9 production observed in the present study, had a functional role in B-CLL invasion and transendothelial migration. For invasion assays we used Matrigel, which is mainly composed of laminin and type IV collagen and resembles vascular basement membranes. Although few cells migrated through Matrigel in the absence of chemokine, CXCL12 increased this number 2.5-fold (Figure 6A). Invasion through Matrigel was significantly inhibited by anti-MMP-9 Abs (86%) or the MMP-9 inhibitor TIMP-1 (80%), while an anti-MT1-MMP mAb had no effect (Figure 6A). These results were further confirmed by MMP-9 gene silencing experiments. B-CLL cells from three patients were transfected with three different siRNA for MMP-9 or a control siRNA, and their invasion through Matrigel was measured. As shown in Figure 6A, all three MMP-9 siRNA significantly reduced B-CLL invasion while the control siRNA had no effect (Figure 6A). Transfection with these siRNA did not significantly reduced B-CLL viability with respect to controls (shown for one patient in Supplemental Figure 4). CXCL12-triggered transendothelial migration was also nearly completely inhibited (95%) by anti-MMP-9 but not anti-MT1-MMP Abs (Figure 6B). As in the case of Matrigel invasion, transfection of B-CLL cells with MMP-9 siRNAs significantly blocked transendothelial migration, while the control siRNA had no effect (Figure 6B). Blocking α4β1 integrin also inhibited B-CLL transendothelial migration (not shown), indicating that α4β1 is also involved in this process, as recently documented7. To confirm that the siRNAs were functional, the conditioned medium of transfected B-CLL cells cultured for 24 h was analyzed by gelatin zymography. As shown in Figure 6C for two representative samples, all three siRNAs

14

significantly diminished MMP-9 secretion, while the control siRNA had no effect. HUVEC-enhanced MMP-9 production was also reduced by 66% upon transfection with MMP-9 siRNAs (not shown). Altogether these results clearly established that MMP-9 plays a key role in B-CLL cell invasion across basement membranes and in transendothelial migration. We

also

studied

the

signaling

pathways

required

for

B-CLL

transendothelial migration. As shown in Figure 6D for two representative patients, inhibition of PI3-K/Akt or ERK1/2 activities, blocked B-CLL cell transendotehlial migration, while inhibition of p38 MAPK (used as control) had no effect. These results indicated that signals provided by α4β1 integrin (upon binding to VCAM-1 in endothelium), and CXCL12 were required for B-CLL cell transendothelial migration, and further supported the role of MMP-9, which is upregulated in response to these signals, in this process.

Cell-associated MMP-9 localizes to the membrane as pro-active and active forms, and to α4β1-induced podosomes, where degrades extracellular matrix MMP-9 is detected at the B-CLL cell surface by flow cytometry and its able to degrade collagen18. We studied the localization of cellular MMP-9 and its possible modulation by α4β1 integrin. B-CLL cells were incubated on FN-H89 for 24 h and after lysing, the membrane and cytosolic fractions analyzed by gelatin zymography. Figure 7A shows that MMP-9 was mainly located at the cell membrane and that the mature form of 85 kDa was present in this fraction. The mature form was also constitutively present at the cell membrane (Figure 7A),

15

confirming that α4β1 integrin regulates MMP-9 by increasing production of the proform, rather than affecting MMP-9 activation at the cell surface. It was previously shown that B-CLL cells, but not normal B cells, form podosomes34,35, which are cytoskeletal structures in close contact with the substratum and associated with cell migration/invasion36. We then studied whether α4β1 engagement induced podosome formation and whether MMP-9 localized to these structures. Confocal microscopy analyses showed that B-CLL cells incubated on FN-H89 or VCAM-1, but not p-Lys, formed the punctuate pattern of an actin-rich core surrounded by a vinculin-containing ring characteristic of podosomes36 (Figure 7B). Aproximately 15-25% of cells formed podosomes, depending on the sample (six samples studied). CXCL12 did not induce podosomes and did not affect induction of podosomes by α4β1 integrin (not shown), confirming that integrin-mediated adhesion was a prerequisite for podosome

formation.

Importantly,

α4β1

engagement

induced

MMP-9

colocalization with actin at the podosome core, as documented in Figure 7C using dot-plot analyses. Some MMP-9 also colocalized with cortical actin (Figure 7C and D). Induction of podosomes by α4β1 integrin did not require PI3-K or ERK1/2 activity, as actin still presented a punctuate pattern in the presence of wortmannin or UO126 (Figure 7D, left panels). However, inhibition of PI3-K completely prevented MMP-9 localization in podosomes, demonstrated by confocal dot-plot analyses (Figure 7D). In contrast, inhibition of ERK1/2 did not affect this MMP-9 localization (Figure 7D).

16

We next studied whether MMP-9 was active in podosomes by examining the degradation of a gelatin/fibronectin matrix. In initial experiments with unlabeled-gelatin and fibronectin, we established that adhesion of B-CLL cells to this matrix was completely dependent on α4β1 integrin (not shown), and resulted in formation of podosomes where MMP-9 co-localized with actin (Figure 7E). Subsequently, B-CLL cells were incubated on FITC-labeled gelatin/fibronectin for 24 h and degradation was visualized by the local loss of matrix immunofluorescence. Figure 7F shows that B-CLL cells containing podosomes, but not those lacking them, degraded the gelatin/fibronectin matrix. This effect was completely inhibited by TIMP-1 (Figure 7F) or anti-MMP-9 Abs (not shown), confirming that MMP-9 was active in podosomes and responsible for the observed matrix degradation. Moreover, inhibition of PI3-K activity also blocked matrix degradation while inhibition of ERK1/2 had no effect (Figure 7F). Altogether these results established that α4β1 integrin in B-CLL cells regulates not only MMP-9 production, but also its localization and activity in podosomes, and both processes involve PI3-K signaling.

Discussion In this report we have studied the role and regulation of MMP-9 in B-CLL cells. We show for the first time that: 1) MMP-9 is upregulated in response to distinct signals elicited by α4β1 integrin or CXCR4 ligand engagement; 2) MMP-9 plays a major role in transendothelial migration and basement membrane invasion; 3) MMP-9 localizes to podosomes in an α4β1-dependent manner and degrades extracellular matrix.

17

Previous studies have shown that MMP-9 is constitutively present in lysates and culture medium of B-CLL cells17,18. We have confirmed and extended these results by showing that MMP-9 expression in B-CLL cells was observed in all samples studied and was significantly higher than in normal Bcells. Our study also establishes that MMP-9 is the major MMP in B-CLL, since at difference with other B-cell malignancies37,38, MMP-2 was absent, confirming a previous report18 and MT1-MMP was not detected at the B-CLL cell surface even after stimulation. Increasing evidence indicates that α4β1 integrin plays an important role in B-CLL progression. Elevated expression of α4β1 correlates with the presence of lymphadenopathy5 and α4β1 is required, together with VEGF, for chemokinedirected B-CLL transendothelial migration5,7. Besides its role in cell migration, we have previously reported that α4β1 protects B-CLL cells from spontaneous and

drug-induced

expression

apoptosis29,39.

profiling,

have

Moreover,

recently

gene

established

and that

surface-antigen the

correlated

overexpression of α4 integrin subunit and CD38 is characteristic of bad B-CLL prognosis40,41. We now show in the present study that α4β1 ligand engagement upregulates MMP-9 production and induces podosomes and the localization of active MMP-9 in these structures, thus affecting the migratory and invasive properties of B-CLL cells. Our results also establish that PI3-K/Akt are the kinases involved in α4β1-induced upregulation of MMP-9. Previous reports have shown that the PI3-K/Akt/NF-κB pathway is constitutively activated in fresh B-CLL cells and that Akt phosphorylation decreases upon cell culture42. We show here that

18

α4β1/ligand engagement increases the constitutive levels of phospho-Akt and induces sustained Akt/NF-κB activation. Ringshausen et al43., have shown that active p38 MAPK is required for spontaneous secretion of MMP-9 by B-CLL cells. We did not observe this requirement for the basal secretion of MMP-9 by cells on BSA, and this may be due to the different experimental conditions used in both studies. Interestingly, upregulation of MMP-9 by fibronectin in T cells was increased by inhibiting PI3-K and the MAPKs MEK1/2 and p38, and was dependent on Src activity26. These observations suggest that MMP-9 regulation, even when triggered by cell adhesion via integrins, involves different signaling pathways depending of the cell type. In support of these differences, we found that both ligands of α4β1, FN-H89 and VCAM-1, upregulated MMP-9 while in T cells VCAM-1 had no effect27. It is well known that CXCL12 enhances MMP-9 expression in myeloma, osteoclasts, and other cell types38,44,45. Our present results constitute the first evidence that CXCL12 upregulates MMP-9 in B-CLL cells. Interestingly, CXCL12 upregulation of MMP-9 was independent of the effect of α4β1 integrin and involved ERK1/2 but not Akt activation. ERK1/2 activation by CXCL12 has been previously observed in several cell systems46,47, but we further show that in B-CLL cells, ERK1/2 phosphorylation was sustained and produced rapid cFos activation. MMP-9 gene promoter contains one NF-κB and two API-1 (the complex of Fos and Jun) elements9 and thus, although this study focused on MMP-9 regulation at the protein level, our results strongly suggest that α4β  integrin and CXCL12 may transcriptionally regulate MMP-9. Nevertheless, modulation of MMP-9 at other levels cannot be disregarded. On the other hand,

19

independent regulation of MMP-9 by α4β1 integrin and CXCL12 may provide the basis for a continuous stimulation of MMP-9 in physiological situations where only α4β1 ligands or chemokine are present. Our results provide strong evidence for a functionally active MMP-9 in BCLL cells. First, mature MMP-9 was present at the cell membrane. Second, anti-MMP-9 Abs, TIMP-1, or MMP-9 siRNAs significantly inhibited B-CLL invasion through basement membranes and transendothelial migration. Third, MMP-9 localized to α4β1-induced podosomes and efficiently degraded extracellular matrix. The fact that matrix degradation was also completely blocked by TIMP-1 or anti-MMP-9 Abs further supports a major role for MMP-9 in B-CLL invasion. MMP-9 has been found in podosomes in other cell types36, but its localization in B-CLL cell podosomes specifically required PI3-K signaling. This requirement emphasizes the role of α4β1 integrin (which activates PI3-K/Akt) not only in upregulating MMP-9 secretion, but also in focalizing its activity in podosomes. This property appears to be unique for α4β1 since CXCL12, which also upregulates MMP-9 secretion, did not induce podosomes. Although these functional assays clearly established that MMP-9 is active in B-CLL cells, in conditioned media we did not observe the mature 85 kDa form. Many previous studies on different cell types have reported similar observations48 and the reason for this remains unclear. One possibility is that soluble mature MMP-9 is produced at undetectable levels, yet sufficient for focalized activity. MMP-9 could also become active by alternative, nonproteolytic mechanisms, involving oxidative modifications or conformational

20

changes48. It is not known if these mechanisms operate in B-CLL cells. On the other hand, proteolytic activation of pro-MMP-9 is accomplished by a variety of proteases including plasmin, and is thought to be more efficient at the cell surface than in solution48,49. Indeed we have observed active MMP-9 in B-CLL cell membranes and podosomes, suggesting that a fraction of secreted MMP-9 binds to the cell surface. MMP-9 can associate with several “docking” molecules (CD44, Ku protein, ICAM-1, integrins) at the cell surface, thereby facilitating its activation and proteolytic activity48-50. In this context, enhanced release of the proform by α4β1 integrin or CXCL12 in B-CLL cells may provide a continuous pool of zymogen, susceptible of activation as needed. The present report therefore identifies novel physiological mechanisms contributing to B-CLL progression and provides evidence for a key role of MMP9 in this malignancy. MMP-9 degrades extracellular matrix and mediates B-CLL migration and invasion, as we show here, but may also release matrix-bound angiogenic factors, such as VEGF and bFGF, and contribute to angiogenesis11. Although still not proven in B-CLL, these angiogenic factors in turn upregulate MMP-9 in other cell systems11, thus establishing a feed-back mechanisim for tumor expansion. It is interesting that VEGF51,52 and the two molecules, α4β1 integrin29,39 and CXCL1253, identified in the present report as physiological regulators of MMP-9 production, provide survival signals in B-CLL, and MMP-9 may also be part of this survival mechanism43. Interferons can suppress MMP-9 production17 and it was recently shown that hyperforin induces apoptosis of BCLL cells by inhibiting the secretion of MMP-9 and VEGF54, suggesting that MMP-9 could be a target for therapeutic intervention in B-CLL. Therapies aimed to neutralize several MMPs are already in progress for other pathologies10,11.

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Since we have also identified the kinase signaling pathways involved in MMP-9 upregulation, targeting these pathways, alone or in combination with other protocols may also prove to be useful for B-CLL treatment.

Acknowledgements We thank the B-CLL patients who donated samples for this research; all mentioned investigators for antibodies or reagents; Drs. Joaquín Teixidó and Alicia G. Arroyo for critical reading of the manuscript; Dr. Paloma SánchezMateos (Hospital Gregorio Marañón, Madrid) for valuable advice with confocal microscopy analyses; Dr. Pedro Lastres for help with flow cytometry; and Mercedes Hernández del Cerro for excellent technical assistance.

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Figure legends Figure 1. Constitutive expression of MMPs in B-CLL. (A) Lysates from 2x106 fresh B–CLL cells from five different patients (P1-P5), peripheral blood B lymphocytes (PB-BL), or EBV-transformed normal B cells (Bro168, CO43, HUT112), were analyzed by gelatin zymography on 10% gels. MMP-9 was identified as the 92 kDa pro-active form. Actin was analyzed in identical aliquots from the same lysates by western blotting, and used as control for protein loading. Quantitative values represent the average of 2 different samples for PB-BL and 12 different samples for B-CLL. ***P < 0.001. (B) Flow cytometric analysis of MT1-MMP surface expression in B-CLL cells from one patient, or NCI-H929 myeloma cells, without (Control), or with PMA (50 ng/ml, 24 h). (C) RT-PCR analysis of constitutive MT1-MMP mRNA expression in B-CLL cells from patients 1-5. BLM melanoma cells were included as positive control. (D) Lysates from 4 different B-CLL samples or BLM cells were analyzed by western blotting and MT1-MMP was identified using the LEM2/15 mAb.

Figure 2. α4β1 integrin engagement upregulates MMP-9 in B-CLL cells. (A) 2x106 B-CLL cells from 4 different patients were suspended in RPMI, 0.1% FCS and incubated on wells coated with 0.5% BSA, 10 µg/ml FN-H89 or 10 µg/ml VCAM-1. Some cells were pre-treated for 30 min with anti-α4 or α5 mAbs (10 µg/ml) and then incubated in the presence of the Abs. After 24 h, the conditioned media was concentrated 50 x and analyzed by gelatin zymography. Values for MMP-9 production (arbitrary units) represent the average of all 6 samples studied; basal levels of MMP-9 on BSA were normalized to 100. (B) B-

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CLL cells from one patient were incubated on wells coated with the indicated concentrations of FN-H89 or VCAM-1, and after 24 h analyzed as explained. (C) B-CLL cells from 2 patients (5 x 105/100 µl) were incubated on the upper chamber of Transwell filters coated with resting or TNF-α activated HUVEC, and in the presence or absence of the indicated mAbs. After 24 h, the conditioned media was concentrated 5 x and analyzed by gelatin zymography. Values for MMP-9 production (arbitrary units) represent the average of the two samples studied. *P < 0.05, **P < 0.01.

Figure 3. α4β1 integrin-induced MMP-9 upregulation involves the PI3K/Akt signaling pathway. (A) B-CLL cells from 3 different patients were incubated or not with either 30 nM wortmannin (Wmn), or 5 µM of the following: Bis I, SB203580 (SB), PP2, and UO126 for 1 h at 370 C, and added to FN-H89coated wells. Cells untreated or treated with 30 nM Wmn were also added to BSA-coated wells. After 24h the conditioned media was concentrated, analyzed by gelatin zymography, and MMP-9 secretion was quantitated. Basal levels of MMP-9 on BSA were normalized to 100, and values represent arbitrary units. (B) B-CLL cells from one patient were preincubated for 1 h with the indicated concentrations of wortmannin, LY294002 (LY), or API-2, and added to FN-H89coated wells. After 24 h, the conditioned media was analyzed by gelatin zymography. Values represent the average of duplicate determinations. (C) 2 x 106 B-CLL cells from 2 patients were incubated on 0.5% BSA or 10 µg/ml FNH89 in the absence or presence of wortmannin or API-2. Cells were lysed after 24 h and phosphorylated and total Akt and ERK were analyzed by western

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blotting using specific Abs. (D) The same lysates used in (C) were also analyzed by western blotting using specific Abs to IκB-α. Relative IκB-α levels were quantitated and values represent the average of the 2 samples studied. IκB-α levels of cells on BSA were normalized to 1. (E) 2 x 106 B-CLL cells from one patient were incubated on 0.5% BSA or 10 µg/ml FN-H89, and at the indicated times phospho-Akt and phospho-ERK were analyzed as explained. Relative P-Akt and P-ERK levels were quantitated and constitutive levels were normalized to 1. * P < 0.05, **P < 0.01.

Figure 4.

CXCL12 upregulates MMP-9 in B-CLL cells via the ERK1/2

signaling pathway. (A) B-CLL cells (2x 106) from 4 different patients were suspended in RPMI, 0.1% FCS and incubated on wells coated with 0.5% BSA and with or without 150 ng/ml CXCL12. Pertussis toxin (PT, 200 ng/ml) was added to some cells. After 24 h, the conditioned media was concentrated and analyzed by gelatin zymography. Values represent the average of the 6 samples studied, and basal levels of MMP-9 without CXCL12 were normalized to 100. (B) B-CLL cells from 4 patients were incubated for 1 h with or without (None) the indicated inhibitors (same concentrations as in Figure 3), and added to BSA-coated wells in the presence or absence of 150 ng/ml CXCL12. After 24 h, the conditioned media was concentrated and MMP-9 analyzed and quantitated. (C) Cells from 2 patients were incubated on 0.5% BSA in the presence of 150 ng/ml CXCL12, and in the presence or absence of pertussis toxin (PT) or UO126. Control cells were incubated in the absence of CXCL12. Phospho- and total Akt and ERK were analyzed by western blotting. (D) B-CLL cells from the same patients used in (C) were incubated with or without 150

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ng/ml CXCL12; at the indicated times, RNA was extracted and expression of cFos mRNA was analyzed by RT-PCR using specific primers. GAPDH was also amplified as an internal control for sample loading. Values represent the average of the two samples studied. (E) Cells (2 x 106) from the same patient used in Figure 3E were incubated with or without 150 ng/ml CXCL12; at the indicated times, cells were lysed and lysates analyzed by western blotting using specific Abs to phospho-Akt and phospho-ERK. Relative P-Akt and P-ERK levels were quantitated and normalized with respect to constitutive levels, which were considered 1. **P < 0.01.

Figure 5. α4β1 integrin and CXCL12 independently enhance MMP-9 production in B-CLL cells. (A) B-CLL cells from 4 different patients were added to BSA- or FN-H89-coated wells and in the presence or absence of 150 ng/ml CXCL12. After 24 h, MMP-9 secretion was analyzed by gelatin zymography and quantitated. Basal MMP-9 levels of cells on BSA and without CXCL12 were normalized to 100. (B) B-CLL cells from 2 patients were incubated on FN-H89 in the presence or absence (None) of the indicated kinase inhibitors. 150 ng/ml CXCL12 was also added to the cells. Control cells (Contr.) were incubated in 0.5% BSA and without CXCL12. The conditioned media was collected after 24 h and MMP-9 was quantitated. Basal values were normalized to 100. *P < 0.05, **P < 0.01.

Figure 6. Role of MMP-9 in B-CLL migration through Matrigel and HUVEC. (A) B-CLL cells (5x105) from 3 different patients, with or without previous incubation with the indicated Abs (10 µg/ml), TIMP-1 (1.5 nM), or transfected

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with control siRNA or 3 different MMP-9 siRNAs (MMP9.1, MMP9.2, and MMP9.3), were added to the upper chamber of Transwell filters coated with Matrigel. CXCL12 (150 ng/ml) was added to the medium in the bottom chamber, except for the control. After 24 h invasive cells were counted by flow cytometry. Values represent the average of the 3 samples studied and are expressed as the percentage of total cells added. (B) B-CLL cells from 3 patients, with or without previous incubation with the indicated Abs, or transfected with the same siRNAs shown in (A), were added to Transwell filters previously coated with inactivated (control) or TNF-α-activated HUVEC. CXCL12 was added to the bottom chamber except for the control. After 24 h transmigrated cells in the bottom chamber, were counted by flow cytometry. Values represent the average of the 3 samples studied. (C) Transfected B-CLL cells from 2 patients were incubated for 24 h, and the conditioned medium concentrated and analyzed by gelatin zymography. Values are the average of the 2 samples studied. (D) B-CLL cells (5 x 105) from 2 different patients were incubated for 1 h with the indicated inhibitor or with medium (None), and added to Transwell filters coated with TNF-α activated HUVEC. Control cells were added to inactivated HUVEC. After 24 h, cells in the bottom chamber were counted by flow cytometry. Values represent the average of the 2 samples studied. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7. Analysis of cell-associated MMP-9 in B-CLL cells. (A) 3x106 BCLL cells from a representative sample were incubated on FN-H89 for 24 h. Cells were removed and membrane and cytosolic fractions separated and subjected to gelatin zymography. Identical aliquots from the same lysate were

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analyzed by western blotting using anti-RhoGDI specific Abs. (B) B-CLL cells in medium containing 80 nM PMA, were added to glass coverslips coated with 5 µg/ml p-Lys, 10 µg/ml FN-H89 or 10 µg/ml VCAM-1. After 1 h at 370C, podosomes were analyzed by confocal microscopy after staining F-actin (red) with Alexa 568-phalloidin, and vinculin (green) with specific primary Abs/FITClabelled secondary Abs. Inserts are x 20 magnifications. Bar, 4 µm. (C) B-CLL cells were added to FN-H89- or VCAM-coated glass coverslips and after 1 h, MMP-9 (green) was visualized with specific primary Abs and Alexa 488-labelled secondary Abs. F-actin (red) was stained as explained in (B) and the merged images are shown. Colocalization (yellow) of MMP-9 and F-actin in podosomes was further demonstrated using dot-plot analyses as explained in Materials and Methods. (D) B-CLL cells were preincubated or not for 1 h with 30 nM wortmannin (Wmn) or 10 µM UO126 and added to glass coverslips coated with 10 µg/ml FN-H89. F-actin and MMP-9 were visualized as explained and the merged images (yellow) and dot-plot analyses are shown. (E) B-CLL cells were added to coverslips coated with gelatin/fibronectin; after 24 h, colocalization of actin and MMP-9 in podosomes was analyzed as explained. (F) B-CLL cells, with or without the indicated inhibitors, were added to coverslips coated with FITC-gelatin/fibronectin; after 24 h, F-actin was stained with Alexa 568phalloidin and sites of matrix degradation were visualized by the loss of green fluorescence. White arrows indicate cells containing podosomes. Bar, 12 µm.

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