Rapid upregulation of α4 integrin-mediated leukocyte adhesion by transforming growth factor-ß1

Rubén A. Bartolomé*, Francisco Sanz-Rodríguez*, Mar M. Robledo, Andrés Hidalgo and Joaquin Teixidó From the Centro de Investigaciones Biológicas, Department of Immunology, Madrid, Spain Running title: TGF-ß1 modulates α4 integrin adhesion Correspondence: Joaquin Teixidó. Centro de Investigaciones Biológicas, Department of Immunology, Velázquez 144, 28006 Madrid, Spain. Phone 34-91-5611800; Fax 34-91-5627518; e-mail joaquint@cib.csic.es Keywords: α4 integrins, cell adhesion, TGF-ß1, SDF-1α *These authors equally contributed to this work. This work was supported by grant SAF99-0057 from Ministerio de Ciencia y Tecnología. Rubén A. Bartolomé is a recipient of a pre-doctoral fellowship from CSIC-Glaxo Wellcome. Francisco Sanz-Rodríguez and Andrés Hidalgo were recipients of pre-doctoral fellowships from Fundación Ramón Areces and the Comunidad de Madrid, respectively. Mar M. Robledo was a recipient of a post-doctoral fellowship from the Comunidad de Madrid. Andrés Hidalgo, present address: Department of Hematology, Mount Sinai School of Medicine, One Gustave L. Levy Place. New York, NY 10029.

MBC in Press, published on October 16, 2002 as 10.1091/mbc.E02-05-0275

The α4 integrins (α4ß1 and α4ß7) are cell surface heterodimers expressed mostly on

leukocytes that mediate cell-cell and cell-extracellular matrix adhesion. A characteristic feature

of α4 integrins is that their adhesive activity can be subjected to rapid modulation during the

process of cell migration. Here we show that transforming growth factor-ß1 (TGF-ß1) rapidly

(0.5-5 min) and transiently upregulated α4 integrin-dependent adhesion of different human

leukocyte cell lines and human peripheral blood lymphocytes (PBL) to their ligands VCAM-1

and CS-1/fibronectin. In addition, TGF-ß1 enhanced the α4 integrin-mediated adhesion of PBL to

TNF-α-treated human umbilical vein endothelial cells, indicating the stimulation of

α4ß1/VCAM-1 interaction. Although TGF-ß1 rapidly activated the small GTP-ase RhoA and the

p38 MAP kinase, enhanced adhesion did not require activation of both signalling molecules.

Instead, polymerization of actin cytoskeleton triggered by TGF-ß1 was necessary for α4 integrin-

dependent upregulated adhesion, and elevation of intracellular cAMP opposed this upregulation.

Moreover, TGF-ß1 further increased cell adhesion mediated by α4 integrins in response to the

chemokine SDF-1α. These data suggest that TGF-ß1 can potentially contribute to cell migration

by dinamically regulating cell adhesion mediated by α4 integrins.

3

INTRODUCTION

The α4 integrins (α4ß1 and α4ß7) are heterodimer cell adhesion receptors mainly

expressed on cells of hematopoietic origin that mediate cell-cell and cell-extracellular matrix

(ECM) interactions (reviewed in Lobb and Hemler, 1994; Hynes, 1992). VCAM-1 and the

alternatively-spliced CS-1 region of fibronectin constitute ligands for both integrins, whereas

α4ß7 can additionally interact with MAdCAM-1 (Lobb and Hemler, 1994). α4ß1 and α4ß7 play

key roles in leukocyte recruitment to inflammatory sites and in lymphocyte recirculation, and

α4ß1 function is required during hematopoiesis in the bone marrow (Springer, 1995; Butcher and

Picker, 1996; Arroyo et al., 1996; Butcher et al., 1999).

A characteristic feature of α4 integrins on most leukocytes is that its adhesive activity can

be upregulated by external stimuli leading to firm attachment. Several chemokines binding to

their G protein-coupled receptors, as well as cytokines whose receptors have tyrosine kinase

activity, have been previously demonstrated to rapidly and transiently increase α4 integrin-

dependent cell adhesion (Tanaka et al., 1993; Woldemar Carr et al., 1996; Grabovsky et al.,

2000; Lévesque et al., 1995). For instance, the chemokine SDF-1α upregulates α4 integrin-

mediated lymphocyte, hematopoietic progenitor and myeloma cell adhesion (Grabovsky et al.,

2000; Wright et al., 2002; Peled et al., 1999; Hidalgo et al., 2001; Sanz-Rodríguez et al., 2001).

The enhancement in adhesion was shown to be independent of changes in α4 surface expression,

and was suggested to be the result of variations in the avidity and/or affinity of these integrins for

their ligands.

Three mammalians isoforms of transforming growth factor-beta1 (TGF-ß1) have been

described, TGF-ß1, ß2 and ß3, encoded by different genes (Massagué, 1998). TGF-ß1 is a

multifunctional cytokine which regulates cell proliferation, differentiation and migration, and has

4

an important role in tissue recycling and repair (Massagué, 1998; Piek et al., 1999). In addition,

TGF-ß1 plays a pivotal role in tumor development, eliciting both positive and negative effects in

carcinogenesis (Massagué et al., 2000; Derynck et al., 2001; Wakefield and Roberts, 2002). TGF-

ß1 is a potent immunosuppresor (Letterio and Roberts, 1998), and its absence causes massive

leukocyte infiltration due to increased expression of inflammatory mediators and adhesion

molecules whose expression is negatively regulated by TGF-ß1 (Shull et al., 1992; Kulkarni et

al., 1993). TGF-ß1 exerts its biological functions through binding to a heteromeric cell surface

complex formed by types I and II TGF-ß receptors, which are transmembrane serine/threonine

kinases which are required for TGF-ß signalling (Derynck and Feng, 1997; Massagué, 1998).

Members of the Smad group of proteins mediate signalling from the receptors to the nuclei,

acting as intermediates for transcriptional regulation and cell cycle arrest (Derynck et al., 1998;

Piek et al., 1999; Massagué and Wotton, 2000; Miyazono et al., 2000).

TGF-ß1 controls the synthesis of several integrins (Ignotz and Massagué, 1988; Heino et

al., 1989; Parker et al., 1992; Wahl et al., 1993), which is detected after several hours of exposure

to the cytokine. On the other hand, TGF-ß1 can rapidly (minutes) and independently of Smads,

activate several signaling pathways including mitogen-activated protein (MAP) kinases

(Hartsough and Mulder, 1995; Hannigan et al., 1998; Sano et al., 1999; Hanafusa et al., 1999)

and small GTP-ases of the Rho subfamily (Mucsi et al., 1996; Bhowmick et al., 2001; Edlund et

al., 2002). The latter comprises a group of several proteins which are key regulators of the

organization of actin cytoskeleton and play important roles in cell migration (Hall, 1998).

In the present study we have investigated whether short exposure to TGF-ß1 could

influence α4-integrin dependent cell adhesion. A potential modulation of this adhesion by TGF-

ß1 could contribute to lymphocyte extravasation at sites of inflammation, as well as in the

trafficking of precursor cells in hematopoietic organs and in secondary lymphoid tissue.

5

MATERIALS AND METHODS

Cells and antibodies. Human peripheral blood lymphocytes (PBL) were isolated from buffy

coats by ficoll density gradient centrifugation (Biochrom KG, Berlin, Germany), followed by two

steps of cell adherence at 37ºC onto plastic flasks. Peripheral blood T lymphocytes (PBL-T) were

purified from PBL by immunomagnetic negative selection using a mixture of anti-CD19, anti-

CD16, and anti-CD14 mAb, following the method already described (Sancho et al., 1999). The

negatively-selected cell population was always more than 97% positive for CD3 expression, as

analyzed by flow cytometry. Isolated cells were washed and resuspended in adhesion medium

(RPMI/BSA 0.5%) for the assays. Human umbilical vein endothelial cells (HUVEC) were

isolated and cultured as previously described (Dejana et al., 1987). Cells were seeded on tissue

culture dishes coated with 0.5% gelatin and grown in Medium 199 (Biowhitaker, Verviers,

Belgium) supplemented with 20% fetal bovine serum (FBS) (Biowhittaker), 50 µg/ml endothelial

cell growth supplement (prepared from bovine brain), 100 µg/ml heparin (Sigma, St. Louis, MO)

and antibiotics, and used up to the second passage. The erythroleukemia K562, myeloid U937,

and lymphoid JY, RPMI 8866 and Jurkat human cell lines, were cultured in RPMI 1640 media

(Biowhitaker) supplemented with 10% FBS and antibiotics (complete medium). K562 α4

transfectants (Muñoz et al., 1996) were maintained in the same medium containing 1 mg/ml of

G418 (Calbiochem). The megakaryocytic leukemia-derived human cell line Mo7e was mantained

in complete medium and 5 ng/ml rhGM-CSF (R&D Systems, Abingdon, UK). The integrin anti-

α4 HP1/2, anti-CD19 (Bu12) and the control P3X63 mAb were gifts of Dr. Francisco Sánchez-

Madrid (Hospital de la Princesa, Madrid, Spain). Anti-CD16 and anti-CD14 were from

Pharmigen Transduction Laboratories (San Diego, CA).

α4 integrin ligands and cell adhesion assays. The recombinant FN-H89 fragment of fibronectin

which contains the CS-1 site and lacks the RGD central binding domain, was generated as

6

previously described (Mould et al., 1994). For adhesion to VCAM-1, we used the soluble 7-

extracellular domain recombinant human VCAM-1 (sVCAM-1) (R&D Systems). Before the

adhesion assays, cell lines were starved for 4 h by incubation in adhesion medium, without

detectable loss of viability as determined by trypan blue exclusion. PBL and PBL-T were used

directly after their isolation. Cells were labelled for 20 min at 37ºC with BCECF-AM (Molecular

Probes, Leiden, The Netherlands), washed and resuspended in adhesion medium, followed by

treatment with or without inhibitors or antibodies, and finally incubated with rhTGF-β1 (R&D

Systems). Cells (5-10x104 in 100 µl) were added in triplicates to 96-wells dishes (High-binding,

Costar, Cambridge, MA) coated with 2.5 µg/ml FN-H89 or 1-2 µg/ml of sVCAM-1. After

incubation for 10 min at 37ºC, or 2 min after a 15-sec centrifugation of plates, unbound cells

were removed by 3 washes with RPMI medium, and adhered cells quantified using a

fluorescence analyzer (POLARstar Galaxy, BMG Labtechnologies, Offenburg, Germany). For

cell adhesion to HUVEC, monolayers were treated for 10 h with or without 10 ng/ml of

recombinant human TNF-α (Peprotech, London, UK.). Cell adhesion to HUVEC was carried out

for 10 min at 37ºC, and unbound cells were processed and analyzed as indicated above. In

experiments using rhSDF-1α (R&D Systems), the chemokine was incubated with cells in

adhesion medium in the presence or in the absence of TGF-ß1. Inhibitors used in this study

included cytochalasin D, SB 203580, phenylarsine oxide, forskolin and 8-Br-cAMP (Sigma),

PD98059 (New England Biolabs, Beverly, MA), and okadaic acid (Calbiochem). Recombinant

C3 transferase was expressed and purified as previously described (Nobes and Hall, 1995). The

concentrations used for the different inhibitors were not cytotoxic, as measured in cell cycle

analysis by flow cytometry (not shown).

Actin polymerization assays. To determine the content of polymerized actin (F-actin) induced

by TGF-β1 treatment, 105 cells per condition were permeabilized, fixed and stained in a single

7

step by the addition of a 2x solution containing 0.5 mg/ml L-α-lysophosphatidylcholine (Sigma),

8% formaldehyde and 4 U/ml FITC-phalloidin (Molecular-Probes, Eugene, OR). Cells were

incubated at 22ºC for 10 min, washed twice with PBS and subjected to flow cytometry.

GTP-ase activity assays and MAP kinase activation. For GTP-ase assays we followed

essentially the method already reported (Robledo et al., 2001). The GST-C21 and GST-PAK-CD

fusion proteins were generated as described (Sander et al., 1998). To determine the effect of

TGF-ß1 on RhoA and Rac1 activation, cells (107 per condition) were first starved in adhesion

medium, followed by incubation at 37ºC in the same medium in the abscence or in the presence

of TGF-β1. Cells were washed in ice-cold PBS and incubated for 15 min at 4ºC in 200 µl of lysis

buffer (Robledo et al., 2001). Lysates were centrifuged, 15 µl of supernatant were kept for total

lysate samples, and the remaining 185 µl were mixed with fusion proteins precoupled to

glutathione-agarose beads. The beads and proteins bound to the fusion protein were washed in an

excess of lysis buffer, eluted in Laemmli sample buffer, and analyzed for bound Rac1 or RhoA

by Western blotting using monoclonal antibodies against human Rac1 (Pharmingen/Transduction

Laboratories, San Diego, CA) or RhoA (Santa Cruz Biotechnology, Santa Cruz, CA). For p38

MAP kinase activation, 107 cells were starved and subsequently incubated in the presence or in

the absence of TGF-ß1, followed by solubilization in 1 ml of RIPA lysis buffer (Sanz-Rodríguez

et al., 2001). After SDS-PAGE, immunoblots were blocked, incubated with anti-phospho-p38

antibodies (New England Biolabs), washed in T-PBS (0.05% Tween 20 in PBS), and further

incubated with HRP-conjugated secondary antibodies (DAKO A/S, Copenhagen, Denmark).

Blots were developped by a chemiluminiscent reaction and exposed to radiographic films. After

stripping and saturation of nonspecific protein binding sites, the same blots were reprobed with

anti-p38 antibodies (New England Biolabs) to test for total p38 protein content.

Statistical analysis. The results are expressed as the mean ±SD of data obtained from two or

8

more experiments each performed in triplicate. Statistical significance was determined using the

two-tailed Student's t-test.

9

RESULTS

TGF-ß1 rapidly upregulates α4 integrin-mediated cell adhesion. To investigate whether

TGF-ß1 was capable of influencing α4 integrin-dependent cell adhesion we first used the Mo7e

and JY human cell lines as models for studies on α4ß1- and α4ß7-mediated adhesion,

respectively, as they exhibit low adhesive efficiencies to α4 integrin ligands (Lévesque et al.,

1995; Hidalgo et al., 2001; Chan et al., 1992). We pre-incubated these cells at 37ºC for short

periods of time with TGF-ß1, followed by their addition for 10 min to wells coated with α4

integrin ligands. TGF-ß1 rapidly (2.5-5 min) and transiently upregulated the attachment of Mo7e

cells to sVCAM-1, a soluble form of VCAM-1, and to FN-H89, a CS-1-containing fragment of

fibronectin (Fig. 1A, left). The anti-α4 HP1/2 mAb blocked the increased adhesion, indicating

that α4ß1 was mediating the enhancement in cell adhesion. Dose-response experiments showed

that concentrations of TGF-ß1 between 0.5-2 ng/ml were optimal for stimulation of adhesion,

whereas higher concentrations were without effect or slightly inhibitory (not shown). JY cells

displayed a low capability to attach to sVCAM-1, but TGF-ß1 rapidly (1 min) enhanced their

adhesion, as well as the attachment to FN-H89, whereas HP1/2 mAb blocked this adhesion (Fig.

1A, right) indicating the involvement of α4ß7. Additionally, TGF-ß1 upregulated the adhesion to

FN-H89 of the α4ß7+/α4ß1- RPMI 8866 cell line (not shown).

In cell lines which express α4ß1 and display high basal adhesion levels to α4 integrin

ligands (over 40% of the input), such as monocytic U937 cells, K562 cells expressing transfected

α4 (4M7 cells; Muñoz et al., 1996) (Fig. 1B) and Jurkat T cells (not shown), TGF-ß1 had a small

or no effect in their α4ß1-dependent adhesion. Parental K562 cells did not attach to FN-H89 or

sVCAM-1, and addition of TGF-ß1 did not induce their attachment to these ligands (not shown).

When we shortened the time of adhesion to 2 min after a short spin to place cells faster in

contact with α4 integrin ligands, we still detected a notable upregulation in Mo7e cell adhesion to

10

FN-H89 and sVCAM-1 after 2.5-5 min pre-incubation with TGF-ß1 (Fig. 2A), suggesting the

participation of early signalling events in the increased adhesion. To analyze if TGF-ß1 was

selectively targetting a cell population during its modulation of α4 integrin function, we pre-

incubated Mo7e cells with TGF-ß1 before attachment for 5 min to FN-H89, and subsequently,

non-bound cells were recovered and further pre-incubated with the cytokine before new adhesion

to FN-H89. Analysis of extent of cell adhesion in both cases revealed that TGF-ß1 upregulated

the attachment of both the initial and the non-bound cell population (Fig. 2B), suggesting that

TGF-ß1 did not exclusively targetted a determined cell population. When we examined whether

FN-H89-attached Mo7e cells which had not been previously in the presence of TGF-ß1, were still

capable of responding to TGF-ß1, we observed no significative changes in the levels of adhesion

compared with adhered cells incubated in medium alone (not shown).

Peripheral blood lymphocytes (PBL) express cell surface α4 integrins (α4ß1 and α4ß7),

whose adhesive activity has been shown to be the target of modulation by several chemokines

(Tanaka et al., 1993; Woldemar Carr et al. 1996; Pachynski et al., 1998; Grabovsky et al., 2000;

Wright et al., 2002). Therefore we used PBL, as well as T lymphocytes purified from PBL (PBL-

T), to study if TGF-ß1 could modulate their attachment to α4 integrin ligands. TGF-ß1 rapidly

and transiently augmented the attachment of PBL and PBL-T to sVCAM-1, which was blocked

by HP1/2 mAb, indicating the involvement of α4 integrins (Fig. 3, left). TGF-ß1 also increased

the adhesion of PBL and PBL-T to FN-H89 but to a lower extent compared to attachment to

sVCAM-1 (Fig. 3, right).

Control flow cytometry experiments addressed to study whether cell incubation with

TGF-ß1 altered α4 integrin surface expression showed that incubations up to 25 min with TGF-

ß1 did not influence the expression of α4 integrins on PBL and Mo7e cells (not shown).

As pro-inflammatory cytokines induce VCAM-1 expression in HUVEC (Osborn et al.,

11

1989), we incubated them with TNF-α and analyzed whether TGF-ß1 was capable of modulating

the subsequent adhesion of PBL and PBL-T. TGF-ß1 did not influence PBL (Fig. 4) or PBL-T

(not shown) adhesion to resting HUVEC. PBL and PBL-T adhered to higher levels upon TNF-α

treatment of HUVEC, and TGF-ß1 rapidly upregulated their α4ß1-dependent adhesion, as HP1/2

inhibited the increased adhesion. These results suggest that TGF-ß1-triggered upregulation of

PBL and PBL-T adhesion to HUVEC involves at least an increase in α4ß1/VCAM-1 interaction.

Activation of Rho GTP-ases and F-actin polymerization by TGF-ß1. Role in upregulation

of α4 integrin-dependent cell adhesion. To identify TGF-ß1-activated signalling routes which

could be involved in the increase in α4 integrin-mediated adhesion, we first tested on Mo7e cells

the effect of TGF-ß1 on the activation of the small GTPases RhoA and Rac1, key regulators of

the organization of actin cytoskeleton (Van Aelst and D'Souza-Schorey, 1997; Hall, 1998), as

they are activated by this cytokine in epithelial and fibroblastic cells (Mucsi et al., 1996; Atfi et

al., 1997; Bhowmick et al., 2001; Edlund et al., 2002 ). Mo7e cells were treated for different

times with TGF-ß1, following by cell solubilization and incubation of lysates with GST-fusion

proteins containing domains derived from Rho GTPase targets. For the detection of active RhoA

we used GST-C21, which contains the Rho binding domain of the Rho efector Rhotekin, and for

active Rac1 the CRIB (Cdc42/Rac interacting binding) domain of the Rac/Cdc42 effector

molecule PAK (GST-PAK-CD) was used. SDS-PAGE followed by Western blot using anti-

RhoA or anti-Rac1 antibodies showed that TGF-ß1 rapidly and transiently activated RhoA and

Rac1 (i.e.,GTP-loaded Rho A and Rac1) (Fig. 5A). The C3 transferase, an enzyme that

specifically ADP-ribosylates and blocks RhoA activation (Aktories et al., 1989), inhibited the

activation of Rho A triggered by TGF-ß1. Therefore, activation of RhoA and Rac1 by TGF-ß1 in

Mo7e cells takes place at times when cells become stimulated for increased α4 integrin-

dependent adhesion, raising the posibility that downstream signalling activated by these Rho

12

GTP-ases could participate in the upregulated adhesion.

We used C3 transferase to obtain some insight into a potential participation of RhoA

activation in TGF-ß1-triggered increase in Mo7e adhesion. C3 did not significantly alter the

enhancement in Mo7e adhesion to sVCAM-1 and FN-H89 in response to TGF-ß1 (Fig. 5B),

whereas it substantially inhibited the upregulation of α4 integrin-mediated cell adhesion in

response to the chemokine SDF-1α (Wright et al., 2002; Parmo-Cabañas et al. manuscript in

preparation). Moreover, Mo7e treatment with Y-27632, a specific inhibitor of the Rho

downstream effector ROCK, did not alter the increase in adhesion to FN-H89 in response to

TGF-ß1 (not shown). These results suggest that rapid activation of RhoA by TGF-ß1 in Mo7e

cells is not required for the increase in α4ß1-dependent adhesion.

In addition of activating RhoA and Rac1, TGF-ß1 rapidly (1-5 min) and transiently

triggered F-actin polymerization on Mo7e and JY cells (Fig. 6A, Left). Cell pre-incubation with

cytochalasin D, an agent that disrupts actin filaments, blocked the enhancement of F-actin

polymerization induced by TGF-ß1, whereas treatment with C3 resulted in partial inhibition (Fig.

6A, Right). Pre-incubation of PBL and Mo7e cells with cytochalasin D before adding TGF-ß1

substantially inhibited the subsequent TGF-ß1-stimulated upregulation of α4 integrin-dependent

cell adhesion (Fig. 6B), suggesting that a reorganization of the actin cytoskeleton was implicated

in the increased adhesion.

p38 MAP kinase activation by TGF-ß1 is not necessary for enhancement of α4 integrin-

dependent cell adhesion. TGF-ß1 triggered a rapid (1 min) phosphorylation of p38 MAP kinase

in Mo7e cells (Fig. 7A), which was sustained after 15 min incubation with the cytokine. Pre-

incubation of Mo7e cells with SB 203580, an inhibitor of p38 MAP kinase activation abrogated

the phosphorylation of this kinase induced by TGF-ß1, but it did not affect the increase in cell

adhesion to FN-H89 in response to TGF-ß1 was detected (Fig. 7B), indicating that activation of

13

p38 MAP kinases is not necessary for TGF-ß1-induced increase in adhesion. Moreover,

enhancement of α4ß1-dependent adhesion of Mo7e cells to FN-H89 and sVCAM-1 by TGF-ß1

was not influenced by pre-incubation with the MEK1 inhibitor PD98058, nor by the protein

phosphatase inhibitors okadaic acid and phenylarsine oxide (not shown).

Alterations in intracellular cAMP oppose the upregulation of α4 integrin-dependent cell

adhesion in response to TGF-ß1. We used forskolin, a direct activator of adenylate cyclase,

and 8-Br-cAMP, a cell-permeable cAMP derivative, to test whether alterations in the levels of

cAMP could affect the increase in TGF-ß1-triggered adhesion mediated by α4 integrins. Pre-

treatment of Mo7e cells with these two agents substantially inhibited the subsequent upregulation

of cell adhesion to sVCAM-1 and FN-H89 in response to TGF-ß1 (Fig. 8A), suggesting that

elevation of cAMP levels functionally opposed the signalling mechanisms involved in the α4

integrin-dependent enhanced adhesion.

As Cyt D inhibits both TGF-ß1-triggered F-actin polymerization and the increase in α4

integrin-dependent cell adhesion, together with the reported data showing that alterations in

cAMP levels affected the organization of actin cytoskeleton (Valitutti et al., 1993; Busca et al.,

1998; Dong et al., 1998), we tested the effect of 8-Br-cAMP in the induction of F-actin

polymerization by TGF-ß1. The results showed that 8-Br-cAMP substantially inhibited early (up

to 2.5 min) but not late (5 min or more) TGF-ß1-activated F-actin polymerization in Mo7e cells

(Fig. 8B). These data raise the possibility that interference with TGF-ß1-stimulated F-actin

polymerization by agents that affect intracellular cAMP levels could mediate the inhibitory

effects of these agents in upregulation of α4 integrin-dependent adhesion by TGF-ß1.

TGF-ß1 further increases SDF-1α-stimulated α4 integrin-dependent cell adhesion. We

previously reported that SDF-1α rapidly and transiently upregulated α4ß1-mediated adhesion of

Mo7e cells to sVCAM-1 and FN-H89 (Hidalgo et al., 2001). To study whether TGF-ß1 could

14

influence the increase in these adhesions, we incubated Mo7e cells with TGF-ß1 in the presence

or in the absence of SDF-1α, before subjecting them to adhesion to sVCAM-1 or FN-H89. As

expected, cell adhesion was notably upregulated by SDF-1α, and the presence of TGF-ß1 resulted

in a substantial further increase in the adhesion, that was totally blocked by HP1/2, indicating

involvement of α4ß1 in the stimulated adhesion (Fig. 9A). However, the additive effect of TGF-

ß1 in cell attachment was not observed when α4 integrin-mediated upregulation of adhesion by

SDF-1α reached 30% or more of the total cellular input in the assay, and was barely detected

after the effect of SDF-1α on stimulation of cell adhesion finished. It has also been demonstrated

that SDF-1α enhances the adhesion of T lymphocytes to VCAM-1 (Grabovsky et al., 2000).

Accordingly, SDF-1α triggered a large upregulation of T lymphocyte adhesion to sVCAM-1, and

TGF-ß1 induced a modest but statistically significant further increase in adhesion, whereas

HP1/2 blocked this adhesion (Fig. 9B). Furthermore, TGF-ß1 upregulated to a greater extent the

enhancement of T lymphocyte adhesion to FN-H89 in response to SDF-1α. Together these data

indicate that TGF-ß1 has an additive effect on the SDF-1α-induced increase in α4 integrin-

dependent cell adhesion.

15

DISCUSSION

A characteristic feature of α4 integrins is that their adhesive activity on leukocytes can be

rapidly modulated by external stimuli during the process of cell migration. Several chemokines

and cytokines binding to G protein-coupled receptors and tyrosine-kinase receptors, respectively,

have been previously demonstrated to trigger rapid and transient upregulation of α4 integrin-

dependent cell adhesion (Woldemar Carr et al., 1996; Grabovsky et al., 2000; Lévesque et al.,

1995; Peled et al., 1999; Hidalgo et al., 2001; Sanz-Rodríguez et al., 2001). In the present study

we show that TGF-ß1, a cytokine whose receptors are serine-threonine kinases, is also capable of

rapidly and transiently increase α4 integrin-mediated cell adhesion. Incubation with TGF-ß1 for

short periods of time (0.5-5 min) resulted in an enhancement in the subsequent adhesion of Mo7e

and JY cell lines, which express α4ß1 and α4ß7, respectively, to sVCAM-1 and the CS-1-

containing FN-H89 fragment of fibronectin. The upregulation of α4 integrin-mediated adhesion

was detected in adhesion assays as short as 2 min and was independent of changes in α4 cell

surface expression, suggesting that rapid TGF-ß1-triggered changes in α4 integrin avidity and/or

affinity for its ligands were responsible for the augmented adhesion. Cell exposure to TGF-ß1 for

longer than 30 min, or to concentrations of the cytokine of 4 ng/ml or higher, resulted in the

absence of adhesion-triggering effects or even produced a slight inhibition in cell attachment to

α4 integrin ligands.

Upregulation of cell adhesion by TGF-ß1 was detected in cell lines, such as the

mentioned Mo7e and JY, and cells displaying low levels of α4 integrin-dependent adhesion,

including whole PBL as well as PBL-T cells. Instead, cell lines showing high adhesion to α4

integrin ligands were refractory to TGF-ß1 adhesion modulatory effects. TGF-ß1 transiently

enhanced the adhesion of both PBL and PBL-T to sVCAM-1, and increased their attachment to

TNF-α-treated HUVEC, indicating the activation of the α4ß1/VCAM-1 adhesion pathway. TGF-

16

ß1 also augmented the adhesion of these cells to FN-H89, but to a lesser extent compared with

sVCAM-1.

Enhancement of adhesion by TGF-ß1 could either exclusively target a specific cell

population expressing α4 integrins activated enough to mediate a basal level of cell attachment

and TGF-ß1 producing a further activation, or it could target cells expressing non-active α4

integrins unable to mediate adhesion and TGF-ß1 activating them, or else a mixture of both

effects. The capability of unbound Mo7e cells recovered from TGF-ß1-stimulated adhesions, to

respond to this cytokine in a subsequent adhesion suggest that TGF-ß1 is not targetting a specific

cell population for its adhesion activating properties. However, whereas close to 30% of Mo7e

and JY cells in adhesion assays were cabable of enhancing their α4 integrin-dependent adhesion

in response to TGF-ß1, this percentage was reduced to less than 10% when using PBL or PBL-T.

At this point we do not know whether a particular lymphocyte subset or most lymphocytes are

potentially capable to respond to TGF-ß1 by stimulating their α4 integrin-mediated adhesion.

Several important signalling molecules are rapidly activated by TGF-ß1, including small

GTP-ases of the Rho family (Mucsi et al., 1996; Bhowmick et al., 2001; Edlund et al., 2002;

Hartsough and Mulder, 1995). We show here that TGF-ß1 rapidly and transiently activated

RhoA and Rac1 in Mo7e cells, coincident with the times of increased cell adhesion in response to

the cytokine. However, activation of RhoA by TGF-ß1 was found not to be required for α4

integrin-dependent upregulation of cell adhesion, as inhibition of RhoA activation by C3

transferase did not influence the enhanced adhesion. In addition to activate RhoA and Rac1, key

regulatory molecules of the actin cytoskeleton (Hall, 1998), TGF-ß1 triggered a transient increase

in cell F-actin polymerization, which was blocked by cytochalasin D and partially inhibited by

C3. Moreover, cytochalasin D substantially inhibited TGF-ß1-induced increase in α4 integrin-

mediated adhesion in PBL and Mo7e cells. These data suggest that a reorganization of actin

17

cytoskeleton in response to TGF-ß1, that is independent of RhoA activity, might result in an

enhancement in the avidity and/or affinity of α4 integrins for their ligands. As it has been

reported that Rac1 activation results in α4ß1 clustering in T lymphocytes, leading to increased

avidity and upregulation of T cell adhesion to fibronectin (D'Souza-Schorey et al., 1998), we can

not exclude that activation of Rac1 could represent a potential mediator response in the enhanced

α4 integrin-dependent adhesion by TGF-ß1.

The p38 MAP kinase represents an additional signalling molecule rapidly activated by

TGF-ß1 (Sano et al., 1999; Hanafusa et al., 1999). TGF-ß1 triggered a rapid phosphorylation of

p38 MAP-kinase in Mo7e cells, but inhibition of activation of this kinase, as well as that of ERK

MAP-kinase, did not influence the upregulation in α4 integrin-mediated adhesion, suggesting that

activation of MAP kinases was not necessary for the TGF-ß1-triggered increase in adhesion.

, , .. It has been earlier demonstrated that cell treatment with agents that increase

intracellular cAMP levels can inhibit TGF-ß1 actions (Daniel et al., 1987; Grainger et al., 1994;

Steiner et al., 1994). We show here that elevation of intracellular cAMP by incubating cells with

the cAMP analog 8-Br-cAMP, or with forskolin, notably reduced the upregulation of Mo7e cell

adhesion to sVCAM-1 and FN-H89 in response to TGF-ß1, and inhibited TGF-ß1-induced

increase in F-actin polymerization, indicating that these agents were altering at some point the

signalling leading to actin-regulated enhanced adhesion. Altogether, these results suggest that

TGF-ß1 might promote a rapid and actin-controlled spatial reorganization of α4 integrins in the

plasma membrane, potentially leading to the clustering of these integrins, and increased cell

adhesion as a result of an enhancement in the avidity for their ligands. It is possible that in the

absence of stimulation, actin fibers might hold α4 integrins in an inactive form, and

reorganization of actin by TGF-ß1 could contribute to a transient release towards an active

integrin conformation, which could reflect the mentioned clustering, but also to a conformation

18

displaying higher affinity for their ligands.

Several chemokines are potent activators of lymphocyte adhesion involving α4 integrin

adhesive activity (Tanaka et al., 1993; Woldemar Carr et al., 1996; Pachynski et al., 1998;

Grabovsky et al., 2000; Wright et al., 2002). For instance, SDF-1α bound to the surface of

endothelial cells can interact with CXCR4-bearing lymphocytes in the blood stream and trigger

their α4ß1-dependent firm adhesion which enables them to resist blood shear flow, before

initiating transendothelial migration (Grabovsky et al., 2000). TGF-ß1 is expressed by endothelial

cells and, similarly to SDF-1α, it can bind to proteoglycans and ECM proteins (Fava and

McClure, 1987; Murphy-Ullrich et al., 1992; Butzow et al., 1993; Hannan et al., 1988; Antonelli-

Orlidge et al., 1989) and therefore could also be displayed by these molecules on endothelial cells

in the lumen of blood vessels, as well as in the underlying extracellular matrices. Consequently,

lymphocyte extravasation could represent a process potentially modulated by TGF-ß1 by its

transient upregulation of α4 integrin-dependent cell adhesion. Additionally, we show here that

TGF-ß1 modestly, but significantly further augmented the number of PBL-T cells adhered to α4

integrin ligands during rapid SDF-1α activation of this adhesion. The additional enhancement in

adhesion to both sVCAM-1 and FN-H89 was much larger when we used Mo7e cells, indicating

the likelihood of TGF-ß1 additive effects to SDF-1α in the modulation of α4 integrin-dependent

cell adhesion. It is clear from these results that, compared to SDF-1α and possibly to other

chemokines, TGF-ß1 contribution to increase in lymphocyte adhesion mediated by α4 integrins is

low. However, the results presented here on time kinetics of activation of this adhesion by SDF-

1α and TGF-ß1 raises the possibility that this cytokine might prolong SDF-1α-activated adhesion

and hence contribute to cell migration.

In addition to its potential contribution to lymphocyte extravasation at sites of

inflammation, rapid activation of α4ß1-dependent adhesion by TGF-ß1 might also have a role in

19

the trafficking of precursor cells in hematopoietic organs, where cell motility between different

niches are thought to occur, allowing controlled maturation responses. Moreover, modulation by

TGF-ß1 of lymphocyte α4ß7-dependent adhesion within secondary lymphoid organs could also

contribute to their motility during lymphocyte immune surveillance functions. All the above

processes require a rapid and dynamic modulation of α4 integrin-dependent adhesion, and hence,

TGF-ß1 together with chemokines could mediate controlled adhesion/detachment cycles

allowing cell migration.

A previous work reported that TGF-ß1 was capable of enhancing T lymphocyte

attachment to whole fibronectin and that TGF-ß1 downmodulated SDF-1α-triggered increase in

adhesion (Brill et al., 2001). A possible explanation for the discrepancy with our results is that we

used much shorter times (maximum 7 min) of cell exposure to SDF-1α and TGF-ß1, whereas in

the other case T lymphocyte adhesion was measured after 1 h of incubation with both stimuli. As

mentioned above, we have observed that long incubations with TGF-ß1 results in a subsequent

reduction in α4 integrin-mediated cell adhesion, that could also explain these different results.

The present data indicate that different extracellular stimuli acting through different types

of membrane receptors, such as TGF-ß1 receptors and CXCR4, are capable of triggering the

activity of the same adhesion molecules, although with different kinetics and potencies. In

addition, the signalling pathways activated by these different stimuli have common molecular

points, including Rho GTP-ases and MAP kinases, as well as common cell responses, such as

polymerization of the actin cytoskeleton. Interfering with the induction of actin polymerization

triggered by TGF-ß1 and SDF-1α results in both cases in the inhibition of upregulation of α4

integrin-mediated cell adhesion, indicating that the actin reorganization response might represent

a common mechanism for enhanced avidity/affinity of α4 integrins for their ligands. Collectively,

the results presented here suggest that rapid modulation of α4 integrin-dependent cell adhesion by

20

TGF-ß1 could represent an additional stimulus contributing to the process of cell migration.

21

ACKNOWLEDGMENTS

We thank Drs. Francisco Sánchez-Madrid, Santiago Lamas and Angel Corbí for

providing reagents. Natalia Wright is acknowledged for preparation of fusion proteins.

FOOTNOTES

Abreviations.

CS-1: Connecting segment-1 of fibronectin

VCAM-1: Vascular cell adhesion molecule-1

HUVEC: Human umbilical vein endothelial cells

SDF-1α: Stromal cell-derived factor-1α

ECM: Extracellular matrix

22

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Figure Legends

Figure 1. Modulation of α4 integrin-mediated adhesion by TGF-ß1 in human cell lines. BCECF-

AM-labelled cells were pre-incubated for the indicated times (A), or 2.5 min (B), in adhesion

medium with or without TGF-ß1 (1 ng/ml). Cells were then allowed to adhere for 10 min to

sVCAM-1- or FN-H89-coated wells. Some samples were pre-incubated with the anti-α4 HP1/2

mAb prior to exposure to TGF-ß1. Adhesions were quantified in a fluorescence analyzer and data

represent the mean ± SD of triplicate samples from representative results of at least three

independent experiments. **Adhesion was significantly stimulated, P<0.005, or * P<0.05,

according to the Student's two-tailed t test.

Figure 2. Characterization of α4ß1-mediated upregulation of cell adhesion by TGF-ß1. (A)

Mo7e cells were labelled with BCECF-AM, pre-incubated for the indicated times in adhesion

medium with or without TGF-ß1 (1 ng/ml), and allowed to adhere to FN-H89- or sVCAM-1-

coated wells for 2 min after a 15-sec plate centrifugation. (B) BCECF-labelled Mo7e cells were

pre-incubated for 5 min in adhesion medium in the presence or in the absence of TGF-ß1, and

allowed to adhere to FN-H89-coated wells for 5 min. Non-bound cells were recovered, again pre-

incubated for 5 min with or without TGF-ß1, and allowed to adhere to new FN-H89-coated wells

for 10 min. All adhesions were quantified in a fluorescence analyzer and data represent the mean

± SD of triplicate samples from representative results of at least two experiments. ***Adhesion

was significantly stimulated, P<0.001, or ** P<0.005, according to the Student's two-tailed t test.

Figure 3. Modulation of α4 integrin-mediated adhesion by TGF-ß1 in PBL and PBL-T.

BCECF-AM-labelled cells were pre-incubated for the indicated times in adhesion medium in the

presence or in the absence of TGF-ß1 (1 ng/ml). Cells were then allowed to adhere to sVCAM-1-

or FN-H89-coated wells for 10 min. Some samples were pre-incubated with the anti-α4 HP1/2

mAb prior to exposure to TGF-ß1. Adhesions were quantified in a fluorescence analyzer and data

31

represent the mean ± SD of triplicate samples from representative results of at least three

independent experiments. .

**Adhesion was significantly stimulated, P<0.005, or * P<0.05, according to the Student's two-

tailed t test.

Figure 4. Modulation by TGF-ß1 of α4 integrin-mediated PBL and PBL-T adhesion to HUVEC.

BCECF-AM-labelled cells were pre-incubated in adhesion medium for the indicated times with

or without TGF-ß1 (2 ng/ml), and allowed to adhere for 10 min to HUVEC monolayers, which

had been pre-treated for 10 h in the presence or in the absence of TNF-α. Some samples were

pre-incubated with the anti-α4 HP1/2 mAb prior to exposure to TGF-ß1. Adhesions were

quantified in a fluorescence analyzer and data represent the mean ± SD of triplicate samples from

representative results out of three independent experiments. **Adhesion was significantly

stimulated, P<0.005, or * P<0.05, according to the Student's two-tailed t test.

32

Figure 5. Activation of RhoA by TGF-ß1 is not required for upregulation of α4 integrin-

dependent cell adhesion. (A) TGF-ß1 activates RhoA and Rac1. Mo7e cells were pre-incubated

in adhesion medium in the presence or in the absence of C3 exozyme (50 µg/ml, 16 h) and

subsequently treated with TGF-ß1 (0.5 ng/ml) or with LPA (5 µM) for the stated times. Cells

were solubilized and lysates incubated with glutathione agarose-bound fusion proteins to detect

active RhoA and Rac1. Beads and proteins bound to fusion proteins were eluted in Laemli

sample buffer and analyzed for bound RhoA and Rac1 by Western Blot using anti-RhoA and

anti-Rac1 antibodies. Aliquots of the respective lysates served as controls for analyzing total

amounts of RhoA and Rac1. (B) Effect of C3 on the upregulation of α4 integrin-dependent cell

adhesion by TGF-ß1. Mo7e cells were exposed to C3 or adhesion medium alone (Control). After

labelling with BCECF-AM, cells were pre-incubated for 2.5 min in adhesion medium with or

without TGF-ß1 (1 ng/ml), and allowed to adhere to sVCAM-1- or FN-H89-coated wells for 10

min. Adhesions were quantified in a fluorescence analyzer and data represent the mean ± SD of

triplicate samples from representative results of at least two independent experiments.

Figure 6. Role of actin cytoskeleton in upregulation of α4 integrin-mediated cell adhesion by

TGF-ß1. (A) Left. Cells were incubated for the indicated times with TGF-ß1 (1 ng/ml) or medium

alone, stained with FITC-phalloidin and subjected to flow cytometry. Right. Cells were pre-

incubated with cytochalasin D (0.5 µg/ml, 30 min; Cyt D), with C3, or in adhesion medium alone

(Control), followed by exposure to TGF-ß1 for the stated times and processing for flow

cytometry as above. (B) BCECF-AM-labelled cells were treated for 30 min at 37ºC in adhesion

medium alone (Control) or with cytochalasin D (Cyt D; 0.5 µg/ml), pre-incubated for the stated

times in adhesion medium with or without TGF-ß1, and allowed to adhere to FN-H89-coated

wells for 10 min. Adhesions were quantified in a fluorescence analyzer and data represent the

mean ± SD of triplicate samples from representative results of three (PBL) or five (Mo7e)

33

independent experiments. **Adhesion was significantly inhibited, P<0.005, or * P<0.05 according

to the Student's two-tailed t test.

Figure 7. p38 MAP kinase activation by TGF-ß1 is not required for enhancement of α4 integrin-

dependent cell adhesion. (A) Mo7e cells were pre-incubated in adhesion medium for 45 min

with or without SB 203580 (13 µM), followed by treatment for the indicated times at 37ºC with

TGF-ß1 (1 ng/ml). Cells were solubilized and extracts subjected to immunoblotting using anti-

phospho-p38 MAP kinase antibodies. After stripping and saturating non-specific protein binding

sites, the same blots were reprobed with anti-p38 antibodies to check for total protein content.

Blots were developped by a chemiluminiscent reaction and exposed to a radiographic film. (B)

Mo7e cells were labelled with BCECF-AM, incubated for 45 min in adhesion medium with or

witout SB 203580 followed by incubation with TGF-ß1 (1 ng/ml). Cells were subjected to

adhesion to FN-H89 for 10 min at 37ºC. Adhesions were quantified in a fluorescence analyzer

and data represent the mean ± SD of triplicate samples from a representative result of three

experiments.

Figure 8. Alterations in intracellular cAMP oppose the upregulation of α4 integrin-dependent

cell adhesion in response to TGF-ß1. (A) Mo7e cells were labelled with BCECF-AM, incubated

for 1 h in adhesion medium without (Control) or with forskolin (35 µM) or 8-Br-cAMP (500

µM), following by incubation for 5 min with TGF-ß1 (1 ng/ml). Cells were added to wells coated

with sVCAM-1 or FN-H89 and subjected to adhesion for 2 min at 37ºC after a short spin.

Adhesions were quantified in a fluorescence analyzer and data represent the mean ± SD of

triplicate samples from a representative result of three independent experiments. **Adhesion was

significantly inhibited, P<0.005, or * P<0.05, according to the Student's two-tailed t test. (B)

Mo7e cells were treated with or without 8-Br-cAMP as above followed by incubation with TGF-

ß1 for the indicated times and staining with FITC-phalloidin and analysis by flow cytometry.

34

Figure 9. Role of TGF-ß1 in SDF-1α-stimulated α4 integrin-dependent cell adhesion. Mo7e (A)

or PBL-T (B) cells were labelled with BCECF-AM, pre-incubated for the indicated times with or

without TGF-ß1 (1 ng/ml), in the presence of SDF-1α (150 ng/ml; +SDF-1α) or in adhesion

medium alone (-SDF-1α) within the 2.5 or 5 min incubation with TGF-ß1. When SDF-1α

incubation was longer (5 min) compared to incubation with TGF-ß1 (2.5 min), then SDF-1α was

added before the cytokine. Cells were allowed to adhere for 2 min at 37ºC to sVCAM-1- or FN-

H89-coated wells after the plate was spun for 15 seconds. Some samples were pre-incubated with

the anti-α4 HP1/2 mAb before cytokine treatments. Non-bound cells were washed and extent of

adhesion measured in a fluorescence analyzer. Data represent the mean ± SD of triplicate samples

from a representative result of three experiments for each cell type. ***Adhesion was significantly

stimulated, P<0.001, or * P<0.05, according to the Student's two-tailed t test.

A

sVC

AM

-1

Tim

e (m

in)

Cells bound/mm2

100

200

300

400

500 0

510

1520

Tim

e (m

in)

510

1520

FN-H

89

TGF-

β βββ1M

ediu

m

TGF-

β βββ1+H

P1/2

Mo7

eJY

510

1520

FN-H

89

05

1015

20

Tim

e (m

in)

Tim

e (m

in)

sVC

AM

-1

**

**

***

*

Fig

1 A

U93

7B

Cells bound/mm2

250

500

750

1000 0

Medium TGF-ß1

TGF-ß1+HP1/2

FN-H

89

500

1000

1500

0

Medium TGF-ß1

TGF-ß1+HP1/2

sVC

AM

-120

00

K56

2-α ααα4 25

0

500

750

1000

0

FN-H

89

MediumTGF-ß1

TGF-ß1+HP1/2

500

1000

1500 0

sVC

AM

-1

MediumTGF-ß1

TGF-ß1+HP1/2

2000

Fig

1 B

Cells bound/mm2

AB

2 m

in a

dhes

ion

Medium

TGF-ββββ1-2.5 min

TGF-ββββ1-5 min

0

100

200

300

400

500

600

MediumTGF-ββββ1

MediumTGF-ββββ1

5 m

in

adhe

sion

Non

-bou

nd c

ells

,ne

w a

dhes

ion

***

**

0

100

200

300

400

500

600

***

***

0

100

200

300

***

FN-H

89sV

CA

M-1

TGF-ββββ1-5 min+HP1/2

TGF-ββββ1-5 min+HP1/2

TGF-ββββ1-5 min

Medium

FN-H

89

Fig

2 A

B

TGF-ββββ1-2.5 min+HP1/2

TGF-ββββ1-2.5 min

PBL-

T20

0 04080120

160 Medium TGF-ββββ1-5 min

sVC

AM

-1

0Ti

me

(min

)

Cells bound/mm2

50100

150

200

250

PBL

510

15

TGF-

β βββ1M

ediu

m

HP1

/2TG

F-β βββ1

+

20

PBL

0

100

200

300

400

500

515

20

Tim

e (m

in)

10

PBL-

T

050100

150

200

250

FN-H

89

TGF-

β βββ1M

ediu

m

HP1

/2TG

F-β βββ1

+

TGF-ββββ1-2.5 min

Medium TGF-ββββ1-5 min

TGF-ββββ1-2.5 min+HP1/2

***

**

**

Fig

3

0

50100

150

200

PBL

PBL-

T

0

100

200

300

400

500

Cells bound/mm2

Medium

TGF-ββββ1-2.5 min

TGF-ββββ1-5 min

TGF-ββββ1-5 min+HP1/2

Medium

Medium

-TN

F-α ααα

+TN

F-α ααα

TGF-ββββ1-2.5 min

TGF-ββββ1-5 min+HP1/2

TGF-ββββ1-5 min

TGF-ββββ1-2.5 min

TGF-ββββ1-5 min+HP1/2

TGF-ββββ1-5 min

*

**

**

**

Fig

4

0 1 2.5 5 15Time (min)

Active Rac1

Total Rac1

TGF-ββββ1

0 1 2.5 5 15

LPA

TGF-ββββ1

Active RhoA

Total RhoA

Time (min)

A

Cel

ls bo

und/

mm

²

Med

ium

Med

ium

TGF-ß1

TGF-ß1

0

50

100

150

200

250FN-H89

Control C3

0

150

300

450

600sVCAM-1

Med

ium

Med

ium

TGF-ß1

TGF-ß1

Control C3

B

Fig 5 A B

C3

1 2.5 5 15

Control

0

50

100

150

200

250

0

100

200

300

400

500

600

Med

iumBSA

Med

ium

TGF-ββββ1-2

.5 min

TGF-ββββ1-2

.5 min

TGF-β1β1β1β1-5

min

TGF-β1β1β1β1-5

min

Control Cyt D

Med

iumTGF-β

1β1β1β1-5

min

TGF-β1β1β1β1-5

min

Control Cyt D

BSA

Med

ium

Cel

ls bo

und/

mm

2

PBL Mo7e

B

AF-

actin

con

tent

(% o

f con

trol

)

Time (min) 50

80

100

120

140

160

10 15 20 25 5 10 15 20

Mo7e JY

* **

**

Fig 6 A B

TGF-ββββ1Medium

80

100

120

140

160 Mo7e

0 0.5 2.51 0 0.5 2.51 0 0.5 2.51 minControl Cyt D C3

TGF-ββββ1

BA

050100

150

200

250

Cells bound/mm²

TGF-β1β1β1β1-5 min

TGF-β1β1β1β1-5 min

BSA

Medium

Medium

Con

trol

SB20

3580

TG

F-ß1

(min

) 0

1

5

15

phos

pho-

p38

p38

0

1

5

15

SB20

3580

Med

ium

Fig

7

04080120

160

200

Cells bound/mm²

sVC

AM

-1

Med

ium

TG

F-ß1*

**

0

100

200

300

FN-H

89

Med

ium

TG

F-ß1**

**

Con

trol

8-B

r-cA

MP

Fors

kolin

75100

125

150

A

F-actin content (% of control)

Con

trol

8-B

r-cA

MP

B

0

1

2.

5

5

TG

F-ß1

(min

)

Fig

8 A

B

Mo7

eCells bound/mm²

-SD

F-1α ααα

+SD

F-1α ααα

-SD

F-1α ααα

+SD

F-1α ααα

sVC

AM

-1FN

-H89

Med

ium

TGF-ß1-5

min

TGF-ß1-5 m

in+HP1/2

TGF-ß1-2.5

min

TGF-ß1-5 m

in

TGF-ß1-2.5

min0

100

200

300

400

500

Med

ium0

100

200

300

400

500 Med

ium

TGF-ß1-5 m

in

Med

ium

TGF-ß1-5 m

in

TGF-ß1-5 m

in+HP1/2

***

Med

ium

TGF-ß1-5 m

in

Med

ium

TGF-ß1-5 m

in

***

***

1 m

in2.

5 m

in5

min

Med

ium TGF-ß1-5 m

in

TGF-ß1-2.5

min

Med

ium TGF-ß1-5 m

in

TGF-ß1-2.5

min

1 m

in2.

5 m

in5

min

A

***

Fig

9 A

0

200

400

600

800

0

100

200

300

400

500

sVC

AM

-1FN

-H89

PBL-

T

Med

ium

Med

ium

TGF-ß1 5 m

in

TGF-ß1-5 m

in+HP1/2

Med

ium

Med

ium

TGF-ß1-5 m

in+HP1/2

TGF-ß1 5 m

in

+SD

F-1α ααα

+SD

F-1α ααα

***

*

B

Fig

9 B