1

ERK 1/2 and JNKs-dependent synthesis of IL-6 and IL-8 by fibroblast-like synoviocytes

stimulated with protein I/II, a modulin from oral streptococci, requires FAK

Laurence Neff1#, Mirjam Zeisel1#, Vanessa Druet1, Ken Takeda3, Jean-Paul Klein1, Jean

Sibilia2 and Dominique Wachsmann1.

1 Laboratoire d’Immunologie et de Biochimie Bactérienne, Inserm U392, Université Louis

Pasteur de Strasbourg, Faculté de Pharmacie, 74 route du Rhin, 67400 Illkirch, France.

2 Département de Rhumatologie, Hôpitaux Universitaires de Strasbourg, France.

3 Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires-UMR CNRS

7034, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, 74 route du Rhin,

67400 Illkirch, France.

running title: Role of FAK in cytokine synthesis

keywords: FAK, MAPKs, oral streptococci, IL-6, IL-8, α5β1 integrins

Corresponding author: Pr Dominique Wachsmann, Laboratoire d’Immunologie et de

Biochimie Bactérienne, Inserm U392, Faculté de Pharmacie, 74 route du Rhin, F-67400

Illkirch, France.

Tel : 33 3 90 24 41 52 ; Fax : 33 3 90 24 43 08 ; e.mail : wachs@pharma.u-strasbg.fr

# Laurence Neff and Mirjam Zeisel are equivalent authors

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 20, 2003 as Manuscript M212065200 by guest on A

pril 5, 2018http://w

ww

.jbc.org/D

ownloaded from

2

SUMMARY

Protein I/II, a PAMP from oral streptococci, is a potent inducer of IL-6 and IL-8

synthesis and release from fibroblast-like synoviocytes (FLSs), cells which are critically

involved in joint inflammation. This synthesis implicates ERK 1/2 and JNKs as well as AP-1-

binding activity and nuclear translocation of NF-κB. The mechanisms by which protein I/II

activates MAPKs remain however elusive. As FAK was proposed to play a role in signaling

to MAPKs, we examined its ability to contribute to the MAPKs-dependent synthesis of IL-6

and IL-8 in response to protein I/II. We used FAK-/- fibroblasts as well as FAK+/+ fibroblasts

and FLSs transfected with FRNK, a dominant negative form of FAK. The results demonstrate

that IL-6 and IL-8 release in response to protein I/II was strongly inhibited in both protein

I/II-stimulated FAK-/- and FRNK-transfected cells. Cytochalasin D, which inhibits protein

I/II-induced phosphorylation of FAK (Tyr397), had no effect either on activation of ERK 1/2

and JNKs or on IL-6 and IL-8 release. Taken together, these results indicate that IL-6 and IL-

8 release by protein I/II-activated FLSs is regulated by FAK independently of Tyr397

phosphorylation.

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

3

INTRODUCTION

Integrins are a family of heterodimeric transmembrane proteins that bind a variety of

ligands including proteins of the extracellular matrix, intercellular adhesion molecules,

plasma proteins and complement factors (1). Furthermore, integrins act as receptors for many

pathogenic bacteria or viruses and are presently referred to as pattern recognition receptors

(PRRs) as they recognize molecular structures called pathogen-associated molecular patterns

(PAMPs) on pathogens. For example, α5ß1 integrin is the known receptor of several bacterial

PAMPs such as invasin of Yersinia pseudotuberculosis (2), fimbrillin A of Porphyromonas

gingivalis (3), ipa proteins of Shigella spp (4), filamentous hemagglutinin of Bordetella

pertussis (5) and protein I/II of Streptococcus mutans (6). In addition, several other

observations have demonstrated that integrin recognition of fibronectin promotes adhesion

and internalization of bacteria expressing fibronectin-binding proteins, for example, protein

M1 of Streptococcus pyogenes (7) or fibronectin binding protein A of Staphylococcus aureus

(8).

Since integrins are known to control cellular processes as diverse as proliferation,

differentiation, apoptosis and cell migration, it is likely that their interactions with pathogens

will probably have an important impact on host cell responses as well as on microbial

pathogenesis. There are many outside-in signaling pathways that have been identified

downstream from integrins, notably, the MAPKs pathway which converts extracellular

stimuli to intracellular signals and which is central to many cellular functions. MAPKs belong

to one of the major pathways transmitting signals to early genes implicated in the regulation

of cytokine responses. Numerous data demonstrate that proinflammatory cytokine synthesis

in response to bacteria or bacterial components (e.g. lipopolysaccharide, polyosides,

lipoteichoic acids and proteins), after binding to their cognate receptors on different

eukaryotic cells, is controlled by the MAPKs pathway and that this synthesis may play an

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

4

important role in innate immunity as well as in various inflammatory disorders (9, 10). Using

protein I/II, a PAMP from oral streptococci, we reported previously that interaction of this

cell wall component with fibroblast-like synoviocytes (FLSs), cells which are critically

involved in rhumatoid arthritis-associated joint inflammation, triggers the production and

release of inflammatory mediators such as IL-6 and IL-8 (11, 12). This cytokine synthesis

involves ERK 1/2 and JNKs as well as AP-1-binding activity and nuclear translocation of NF-

κB (13).

However, the mechanisms by which integrins initiate the MAPKs pathway are

generally not fully understood. There is increasing evidence that FAK is critical in linking

integrins to this pathway insofar as FAK, which colocalizes with integrins in focal adhesions,

is associated with different signaling, adaptator or structural proteins including Src family

protein tyrosine kinases, PLC-γ, PI3-kinase, p130Cas, Shc, Grb2, and paxillin. Several

mechanisms can be used by FAK to activate ERK 1/2. For example, autophosphorylation of

FAK at Tyr397 generates a binding site for Src family protein tyrosine kinases (14), and Src-

mediated phosphorylation of FAK Tyr925 allows the binding of the SH2 domain of Grb2 and

the formation of a Grb2/SoS complex which activates the Ras/MAPKs cascade. In addition,

interaction with Src leads to the phosphorylation of FAK Tyr576 and 577 and full kinase

activity. The Ras/MAPKs pathway can also be activated by recruitment and phosphorylation

of p130Cas which promotes the binding of the adaptator proteins Crk, Nck and SoS (15-19).

Many integrins use more than one mechanism to activate the ERK pathway and some

of them seem to be independent of FAK and cell-specific. One group has provided evidence

that caveolin-1 and the adaptator protein Shc play a role in relaying signals from integrins to

ERK in primary cells but FAK/Src complexes might control the temporal response of ERK

initiated by Shc, in B-Raf-expressing cells (20-22). In addition, FAK, independently of

tyrosine phosphorylation and kinase activity, was proposed to regulate integrin-dependent

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

5

activation of JNKs by a mechanism involving paxillin and the small GTP-binding proteins of

the Rho family (23). Another linkage was suggested, occurring through association of FAK

with Src and p130Cas and the recruitment of Crk and Dock 180 (24). Recent findings have

also demonstrated that FAK coordinates MAPKs signaling, following costimulation of

integrins and growth factors receptors for EGF or PDGF, through interactions mediated by

FAK-C-terminal and –N-terminal domain connections to the respective transmembrane

receptors (25, 26).

Based on these observations, it can be hypothesized that FAK may participate in

various biochemical routes linking integrins to MAPKs cascades. This work was thus

undertaken to examine the ability of FAK to contribute to signaling events leading to ERK

1/2- and JNKs-dependent synthesis of proinflammatory cytokines, in response to protein I/II.

Our results indicate that FAK is critical for IL-6 and IL-8 release by protein I/II-activated

FLSs, but that Tyr397, the major site of autophosphorylation which promotes the assembly of

a number of signaling complexes, is not essential for this process.

EXPERIMENTAL PROCEDURES

Material - Cell culture media (RPMI 1640 and M199), foetal calf serum (FCS), penicillin,

streptomycin, amphotericin B, Taq DNA polymerase, dNTPs, M-MULV, RNase inhibitor,

IL-6 and β actin primers were from Life Technologies (Cergy-Pontoise, France). Cell culture

media had an endotoxin content that never exceeded 0.04 ng/mL, as tested by the Limulus

chromogenic assay. Polymyxin B, Tri-reagent, type XI collagenase, wortmannin, cytochalasin

D and geneticin were obtained from Sigma (Saint-Quentin-Fallavier, France). Protease

inhibitor cocktail was from Roche (Meylan, France). Hexanucleotide mix was from

Boehringer-Mannheim (Mannheim, Germany). The enzyme immunoassay kits for human IL-

8 and IL-6 detection were from Endogen (Interchim, Montluçon, France) and for mouse IL-6

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

6

detection from R & D (Minneapolis, USA). Purified α5β1 integrins were obtained from

Valbiotech (Paris, France). Rabbit anti-FAK [pY397], anti-Shc [pY317] polyclonal antibodies

were from Biosource (Cliniscience, Montrouge, France), rat anti-integrin ß1 chain, clone

9EG7 monoclonal antibodies and rabbit anti-FAK polyclonal antibodies were from

Pharmingen (Cliniscience, Montrouge, France). Rabbit anti-active ERK 1/2 and anti-active

JNKs polyclonal antibodies were from Promega (Charbonnières, France), rabbit anti-ERK

1/2, anti-JNKs and anti-Shc polyclonal antibodies were from Upstate Biotechnology

(Euromedex, Souffelweyersheim, France). Horseradish peroxydase (HRP)-conjugated goat

anti-mouse IgG polyclonal antibodies, anti-rabbit IgG polyclonal antibodies and the

chemiluminescence kit were from Amersham Pharmacia Biotech (Saclay, France). Universal

tyrosine kinase assay kit was from Takara Biomedicals (Genevilliers, France). The FAK-

Related Non Kinase plasmid (FRNK-YCam) has been previously described (27), the pRk5-

ShcY317F plasmid was a generous gift from Dr. Filippo G. Giancotti (Memorial Sloane-

Kettering Cancer Center, New-York). The NucleofectorTM KIT was from Amaxa (Köln,

Germany). Throughout this study, buffers were prepared with apyrogenic water obtained from

Fandre (Ludres, France).

Cell culture and transfections - Human FLSs were isolated from RA synovial tissues from

three different patients, at the time of knee joint arthroscopic synovectomy as described

previously (28). The diagnoses conformed to the revised criteria of the American College of

Rheumatology (29). Briefly, tissues were minced, digested with 1 mg/mL collagenase in

serum-free RPMI 1640 for 3 h at 37°C, centrifuged (130 x g, 10 min, 4°C) and resuspended in

M199-RPMI 1640 (1:1) containing 2 mM L-glutamine, penicillin (100 IU/mL), streptomycin

(100 µg/mL), amphotericin B (0.25 µg/mL) and 20% heat-inactivated FCS (complete

medium). After overnight culture, non-adherent cells were removed and adherent cells were

cultured in complete medium. At confluence, cells were trypsinized and passaged in 75 cm2

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

7

culture flasks in complete medium containing 10% heat-inactivated FCS. Between the 3rd and

the 10th passage, during which time cultures were a homogeneous population of fibroblastic

cells, negative for CD16 as determined by FACS analysis, cells (5.103 cells per well) were

grown to confluence in 96-well plates (7-10 days). Cells were deprived of serum for 24 h,

before addition of the appropriate stimuli, diluted in serum-free RPMI 1640 with antibiotics.

Cell number and cell viability were examined by the MTT test (3-(4,5 dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide test) as described elsewhere (30).

FAK+/+ and FAK-/- primary mouse embryo fibroblasts were a generous gift from Dr

Dusko Ilic (Department of Medicine, University of California, San Francisco). Cells were

cultured in DMEM containing penicillin (100 IU/mL), streptomycin (100 µg/mL),

amphotericin B (0.25 µg/mL), β-mercaptoethanol (0.1 mM), non essential aminoacids (1%),

sodium pyruvate (1%) and 10% heat-inactivated FCS. FAK+/+ cells were transfected with

FRNK-YCam (27) by the calcium phosphate method. Briefly, 15 µg of plasmidic DNA in

1 mL BES–buffered saline (BBS pH 6.95, containing 2.5 mM of CaCl2) was added to 5.105

cells for 20 h at 37°C (3% CO2). Following transfection, cells were rinsed and then cultured in

complete DMEM medium containing geneticin (1.5 mg/mL) for 2 weeks. The antibiotic

resistant cells were then pooled and used for further analysis. Green fluorescent protein (GFP)

was used to determine the transfection efficiency. Transient transfection of FLSs was

performed using the NucleofectorTM kit. 2 µg of plasmidic DNA were added to 5. 105 FLSs

suspended in 100 µL of human dermal fibroblast Nucleofector™ solution. The program U-23

was selected for a high density of transfection according to the manufacturer’s instructions.

Cells were then plated in 96-well plates (5.104 cells per well) and serum-starved for 24 h

before activation experiments.

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8

Purification of protein I/II - Recombinant protein I/II of S. mutans OMZ 175 was purified

from pHBsr-1 transformed Escherichia coli cell extract by gel filtration and immunoaffinity

chromatography as previously described (31). The purity of the protein was checked by SDS-

PAGE after staining with Coomassie blue. Protein I/II migrated as a single band having an

apparent molecular mass of 195 kDa.

Activation of cells - FLSs were preincubated with 100 µl of various inhibitors diluted in

serum-free RPMI 1640 with antibiotics: for 40 min at 37°C with cytochalasin D (0.5, 1 and 2

µM), for 1 h at 37°C with anti-integrin β1 chain mAbs (5, 20, 40 µg/mL), with wortmannin

(50, 100, 200 nM) and then incubated with 100 µL of serum-free RPMI 1640 containing

protein I/II (125 pM final concentration). Protein I/II (125 pM, 200 µL) was also preincubated

for 2 hours at 4°C with purified α5β1 integrins (1, 5, 10 µg/mL) and then used to stimulate

FLSs. After a 20 h incubation-period, culture supernatants were harvested and used to

estimate IL-6 and IL-8 release by a heterologous two-site sandwich ELISA as previously

described (32). To confirm that the observed effects were not due to possible LPS

contamination, all the experiments were performed in presence of polymyxin B (2 µg/mL).

Detection of IL-6–mRNA - Total RNA was extracted from 106 FAK+/+ or FAK-/- cells

activated with 125 pM of protein I/II for 1 h, using 1 mL of Tri-Reagent and reverse

transcribed for 45 min at 42°C. 100 ng of total RNA was mixed with 5 U of M-MuLV, 10 U

of RNAse inhibitor, 10 nM of dNTP, 50 pM of hexanucleotide mix, 100 nM DTT, 2.5 mM

MgCl2, 500 mM KCl and 100 mM Tris-HCl pH 8.3. PCR was performed in a volume of 50

µL containing 1 µL of the reverse transcription mixture, 100 mM Tris-HCl pH 8.3, 500 mM

KCl, 1.25 mM MgCl2 , 20 pM of each primer, 20 mM of dNTPs and 2.5 U of Taq DNA

polymerase, in a 9600 Perkin-Elmer cycler set for 30 cycles. The PCR temperatures used

were 94°C for 1 min (denaturing), 60°C for 1 min (annealing) and 72°C for 1 min

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

9

(polymerization) followed by an extension of 10 min at 72°C. PCR fragments were then

separated on 1.5% agarose gels and visualized with ethidium bromide. The specific primers

for IL-6 and β actin were selected based on published mouse IL-6 and β actin cDNA

sequences. The oligonucleotide primers used were for IL-6: 5’-

TTCCTCTCTGCAAGAGACT-3’ and 5’-TCAGGAAGTCTCTCTATGT-3’, and for β actin:

5’-ATGGATGACGATATCGCT-3’ and 5’-TGGACTGTCTGATGGAGTA-3’.

Western blot detection of tyrosine phosphorylation - 106 cells were incubated for various

times in 100 µl of serum-free RPMI 1640 supplemented with antibiotics with or without

125 pM of protein I/II, in presence or in absence of cytochalasin D (1 µM). After stimulation,

cells were centrifuged (130 x g for 10 min at 4°C), and the pellets were suspended for 20 min

in 100 µL of ice-cold lysis buffer (1% Triton X-100, 20 mM Tris-HCl pH 8.0, 137 mM NaCl,

10% glycerol, 1 mM sodium orthovanadate, 2 mM EDTA, 1 mM phenylmethylsulfonyl

fluoride and protease inhibitors) (33). The Triton X-100-soluble proteins were separated by

centrifugation (14,000 x g for 10 min at 4°C) and the supernatant was subjected to SDS-

PAGE and transferred electrophoretically to nitrocellulose membranes. Membranes were

blocked using 1% bovine serum albumin in TBS (20 mM Tris, pH 7.5, 150 mM NaCl) for 1 h

at 25°C. The blots were then incubated with various antibodies: anti-FAK [pY397], anti-Shc

[pY317], anti-active ERK 1/2 and anti-active JNKs in TBS-Tween (0.1% Tween 20) for 2 h

at 25°C, followed by horseradish peroxydase-conjugated goat anti-rabbit IgG polyclonal

antibodies (1 h at 25°C) and detected by enhanced chemiluminescence according to the

manufacturer’s instructions. To confirm the presence of equal amounts of FAK, ERK 1/2,

JNKs and Shc proteins, bound antibodies were removed from the membrane by incubation in

62.5 mM Tris pH 6.7, 100 mM β-mercaptoethanol, 2% SDS for 30 min at 50°C and probed

again with either anti-FAK, anti-ERK 1/2, anti-JNKs and anti-Shc polyclonal antibodies.

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

10

FAK tyrosine kinase assay – 4 .106 cells were incubated for 15 min in serum-free RPMI 1640

supplemented with antibiotics with or without 125 pM protein I/II, in the presence or absence

of cytochalasin D (1 µM). Cells activated for 15 min with fibronectin (10 µg/mL) were used

as control. Cells were lysed as described above and FAK was immunoprecipitated with anti-

FAK pAbs. Protein A Sepharose CL-4B (50%, 100 µL) was then added for 90 min at 4°C.

After centrifugation (10,000 g, 1 min, 4°C), the pellet was used for a non-radioactive tyrosine

kinase assay according to the manufacturer’s instructions. FAK kinase activity was assessed

using protein tyrosine kinase standards included in the assay kit.

RESULTS

Exposure of FLSs to protein I/II causes phosphorylation of FAK - As previous observations

from our laboratory indicated that in endothelial cells, protein I/II interactions with α5β1

integrins increased the tyrosine phosphorylation of FAK and lead to IL-8 secretion (6), we

first investigated the role of α5β1 integrins in the FLSs activation process. The results show

that addition of either increasing amounts of purified α5β1 integrins or anti-integrin β1 chain

antibodies induced inhibition of protein I/II-stimulated IL-6 and IL-8 release (Fig. 1). An

inhibition of cytokine release of about 80% was obtained with 10 µg/mL of α5β1 integrins

and 45% with 40 µg/mL of anti-integrin β1 chain antibodies (p < 0.05). As observed in

endothelial cells, this suggests that α5β1 integrins are implicated in the activation process

leading to IL-6 and IL-8 release from protein I/II-activated FLSs.

We next examined the capacity of protein I/II to stimulate phosphorylation of FAK in

FLSs. Cell lysates were analyzed directly by blotting with specific anti-FAK (pY397)

antibodies. Stimulation of FLSs with protein I/II for various times (1, 5, 15, 30 and 60 min),

resulted in an increased amount of phosphorylated FAK (Fig. 2) which was detectable within

5 min and remained elevated for at least 30 min. Fibronectin was used as a positive control.

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

11

These results demonstrate that interaction of protein I/II with FLSs induces phosphorylation

of FAK at Tyr397.

Protein I/II-induced signaling to MAPKs and IL-6 and IL-8 release occurs via a FAK-

dependent pathway - Recently, Neff et al. (13) studied the eventual role of bacterial

components such as protein I/II in promoting joint inflammation and reported that NF-κB and

the MAPKs/AP-1 pathways are both involved in IL-6 and IL-8 release from FLSs stimulated

with protein I/II. Thus, we next asked whether FAK could participate in the signaling events

leading to IL-6 and IL-8 release from activated cells via the MAPKs pathway. In one set of

studies and as preliminary experiments, we used FAK+/+ and FAK-/- primary mouse embryo

fibroblasts. FAK+/+ and FAK-/- cells were incubated with protein I/II (125 pM) for 30 min

and then immunoblotting experiments were performed. As shown in Fig. 3A, stimulation of

FAK+/+ fibroblasts with protein I/II increased FAK as well as ERK 1/2 tyrosine

phosphorylation, however protein I/II failed to stimulate tyrosine phosphorylation of ERK 1/2

in FAK-/- cells. These results raise the possibility that FAK participates in MAPKs activation

in protein I/II-stimulated cells. We thus explored the cytokine response of FAK-/- fibroblasts

stimulated with protein I/II, using FAK+/+ fibroblasts as positive controls. As illustrated in

Fig. 3B and 3C, protein I/II increased IL-6 mRNA production and IL-6 release from FAK+/+

fibroblasts, but protein I/II-induced IL-6 production was completely inhibited in FAK-/-

fibroblasts (p < 0.01). This suggests that FAK is involved in protein I/II-induced release of

IL-6. To rule out the possibility that the observed inhibition could be due to a non specific

inhibition of other cellular functions, FAK+/+ fibroblasts were transfected with FRNK, the C-

terminal region of FAK which localizes to focal adhesions but does not contain the FAK

kinase domain and which is known to block activation of FAK when overexpressed. As

shown in Fig. 3A, protein I/II treatment did not increase tyrosine phosphorylation of FAK and

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

12

ERK 1/2 in FRNK-transfected FAK+/+ fibroblasts. Moreover, overexpression of FRNK

inhibits IL-6 release from activated cells as compared to wild-type cells (p < 0.01; Fig 3C).

These observations correlate with the results obtained with FAK-/- fibroblasts.

To further demonstrate the requirement of FAK in cytokine release from protein I/II-

activated fibroblasts, the cytokine response of FLSs transiently transfected with FRNK was

examined. FLSs transfected with a GFP-expressing vector were used as controls. Wild-type

FLSs and transfected FLSs were then incubated with 125 pM of protein I/II for 20 h at 37°C.

As seen in Fig. 4, overexpression of FRNK inhibited significantly IL-6 and IL-8 release from

protein I/II-activated FLSs.

In a complementary approach, we have also evaluated the contribution of the adaptor

protein Shc to this pathway. It is known that a subset of integrins, including α5β1, activates

the Ras/ERK pathway by a mechanism implicating the membrane protein caveolin and

tyrosine phosphorylation of Shc by the tyrosine kinase Fyn (20). This last event is necessary

and sufficient to activate MAPKs as demonstrated by results from dominant negative studies

and from mouse embryos deficient in Shc (18). Two tyrosine phosphorylation sites have been

identified on Shc (34): Y239/240, which has been linked to c-Myc activation and Y317,

which appears to be critical for MAPKs activation in response to integrins and growth factor

receptors. Western blotting analysis using specific anti-Shc (pY317) antibodies showed that

two isoforms of Shc, p46 and p52, were constitutively phosphorylated in control FLSs and

that protein I/II did not increase the level of Shc phosphorylation (Fig. 5A). EGF was used as

a positive control. To further demonstrate that Shc is not involved in cytokine synthesis, FLSs

were transiently transfected with a dominant negative version of Shc (pRk5-Shc-Y317F), in

which the tyrosine residue that is phosphorylated and binds Grb2 is replaced by a

phenylalanine. FLSs transfected with a GFP-expressing vector were used to control

transfection. Wild-type and transfected cells were then incubated with 125 pM of protein I/II

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

13

for 20 h at 37°C. As shown in Fig. 5B, overexpression of a dominant negative version of Shc

had no effect on IL-6 and IL-8 release from protein I/II-activated FLSs, as compared to

activated wild-type FLSs, indicating that IL-6 and IL-8 release from protein I/II-activated

FLSs does not require integrin-mediated Shc (Y317) signaling. Taken together, these results

indicate that FAK plays a predominant role in protein I/II-induced IL-6 and IL-8 release from

activated FLSs.

FAK Tyr397 phosphorylation is not implicated in either signaling to MAPKs or release of IL-

6 and IL-8 – To further study the mechanisms by which FAK is required for protein I/II-

induced IL-6 and IL-8 release, we used cytochalasin D which has been shown to prevent FAK

Tyr397 phosphorylation by disrupting the actin cytoskeleton (35-37). A number of FAK-

signaling events are dependent on FAK phosphorylation at Tyr397. Immunoblotting with

anti-FAK (pY397) polyclonal antibodies revealed that 1 µM cytochalasin D, a concentration

that neither affects cell viability nor basal phosphorylation levels (data not shown), suppresses

protein I/II-induced phosphorylation of FAK at Tyr397 (Fig. 6A). By contrast, at this

concentration, cytochalasin D had no effect on protein I/II-stimulated phosphorylation of

ERK 1/2 and JNKs (Fig. 6B, 6C). These results indicate that cytochalasin D does not suppress

signaling to ERK 1/2 and JNKs in protein I/II-activated FLSs, suggesting that

phosphorylation of FAK at Tyr397 is not required for MAPKs signaling in protein I/II-

stimulated FLSs. In order to confirm that IL-6 and IL-8 release which is known to be an ERK

1/2- and JNKs-mediated event (13), is not dependent on phosphorylation of FAK at Tyr397,

FLSs were first incubated for 40 min with cytochalasin D at various concentrations (0.5, 1

and 2 µM), followed by addition of 125 pM of protein I/II for 20 h at 37°C. As shown in Fig.

6D, protein I/II-induced secretion of IL-6 and IL-8 was not affected by cytochalasin D. From

these findings, we conclude that IL-6 and IL-8 release by protein I/II-activated FLSs proceeds

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

14

independently of FAK Tyr397 phosphorylation. Furthermore, these observations suggest that

this integrin-mediated IL-6 and IL-8 synthesis does not require an intact actin cytoskeleton.

PI-3kinase is not involved in protein I/II-induced IL-6 and IL-8 release.- Interestingly, we

also found that PI-3kinase, which plays a role in the signaling pathway connecting FAK to

ERK 1/2 and JNKs after binding to phosphorylated Tyr397, is not involved in protein I/II-

induced IL-6 and IL-8 production. As shown in Fig. 7, pretreatment of cells with various

concentrations of wortmannin had no effect on the ability of protein I/II to induce IL-6 and

IL-8 synthesis.

Protein I/I induces FAK kinase activity independently of Tyr397 - As caution needs to be

taken in making a correlation between FAK tyrosine phosphorylation and FAK kinase

activity, we then evaluated FAK kinase activity in response to protein I/II. As shown in Fig. 8,

protein I/II induces a 10-fold increase in FAK kinase activity as compared to non activated

control cells (p<0.05). Pretreatment of cells with 1 µM cytochalasin D, a concentration that

totally inhibits FAK Tyr397 phosphorylation, did not affect significantly protein I/II-induced

FAK kinase activity (Fig.8). Based on these results, we suggest that protein I/II activates FAK

by a mechanism that does not involve phosphorylation at Tyr397.

DISCUSSION

Most of the data published to date suggest that FAK is implicated in physiological

pathways that regulate motility and migration and possibly also cell growth and/or survival in

response to engagement of integrins and/or stimulation of growth factor receptors. In addition,

FAK has been shown to be involved in adhesion and internalization of pathogenic bacteria for

example, the invasin-mediated uptake of Y. pseudotuberculosis (38) and the invasion of brain

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

15

endothelial cells by E. coli (39). Presently, little is known on the eventual participation of

FAK in proinflammatory responses induced by PAMPs-PRRs interactions. We previously

demonstrated that interaction of protein I/II with α5β1 integrins activates the MAPKs

cascades and that activation of this pathway (6) accounts for secretion of IL-6 and IL-8. We

thus chose to probe the role of FAK in cytokine synthesis in response to integrin engagement.

In agreement with our previous results showing that protein I/II enhanced tyrosine

phosphorylation of FAK in endothelial cells (6), we found that in FLSs, protein I/II caused

tyrosine phosphorylation of FAK at Tyr397, the major phosphorylation site of FAK.

Moreover, using FAK-/- fibroblasts, we found that protein I/II did not stimulate either

tyrosine phosphorylation of ERK 1/2 or IL-6 synthesis in these cells. FAK-/- fibroblasts are

considered as an excellent tool to test the role of FAK despite the fact that they express

elevated levels of Pyk 2 which may function in a compensatory manner in the absence of

FAK (40). The inability of FAK-/- cells to respond to protein I/II suggests that FAK is critical

to IL-6 synthesis. Moreover, cytokine release is not rescued by the presence of Pyk 2 in this

model. These findings are fully consistent with studies from several groups showing that FAK

plays a major role in mediating signal to MAPKs and to downstream targets in response to

various stimuli. For example, FAK is involved in the MAPKs-dependent production of NO in

chondrocytes stimulated with an aminoterminal fragment of fibronectin (41). In another

study, FAK contributes to the subsequent inflammatory response in response to adenovirus

type 19 infection of human corneal fibroblasts (42). Finally, in ovarian carcinoma cells, FAK

and ERK 1/2 are involved in integrin-stimulated MMP-9 secretion (26).

Consistent with experiments using FAK-/- cells, we also found that overexpression of

FRNK, the C-terminal non catalytic domain of FAK which includes the FAT domain as well

as the p130Cas, CAP and Graf proteins-SH3 binding domains, inhibited MAPKs

phosphorylation and subsequent cytokine synthesis in protein I/II-stimulated FAK+/+ cells.

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

16

This was also demonstrated in FLSs transiently transfected with FRNK. FRNK is known to

function as a negative regulator of FAK activity. In most cells, overexpression of FRNK

inhibits FAK-dependent cell spreading, cell migration as well as growth-factor-mediated

signals to MAPKs (43). Previous observations indicated that the FAT domain is the principal

region by which FRNK inhibits FAK but the proposal that FRNK displaces FAK from focal

adhesion sites or diverts some critical partners from FAK remains to be proven (44). In the

experiments reported here, FRNK is unlikely to function by displacing FAK from focal

adhesion sites since actin cytoskeleton disruption induced by cytochalasin D did not suppress

signaling to MAPKs and subsequent cytokine synthesis. One hypothesis could be that FRNK

might interfere as a competitive inhibitor for a yet unidentified binding partner which

regulates FAK activation.

The fact that FAK is critical for cytokine synthesis in this model, is not fully consistent

with studies from Barberis et al. (22) who proposed that in primary fibroblasts, signaling to

ERK 1/2 can proceed in absence of activation of FAK. There is some controversy in the

literature concerning the connections linking integrins to the activation of MAPKs and it is by

no means obvious that the same mechanisms might take place in all cell types in response to

various stimuli. In some primary cells, integrin-mediated activation of ERK 1/2 has been

proposed to be dependent on the phosphorylation of the adaptor protein Shc at Tyr239 or 317.

Shc is an important intermediate of MAPKs activation by integrins. Shc-/- fibroblasts exhibit

a decrease of ERK 1/2 activation in response to extracellular matrix proteins (45). However,

in our model, even though Shc appears to be slightly phosphorylated in unstimulated FLSs,

protein I/II did not increase tyrosine phosphorylation of Shc. Moreover, IL-6 and IL-8

synthesis was not blocked by overexpression of a dominant negative form of Shc, indicating

that Shc is not required for protein I/II-dependent cytokine synthesis.

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

17

Our initial expectation was that phosphorylation of FAK at Tyr397 would be

implicated in MAPKs activation in response to protein I/II. This residue is essential for the

biological and biochemical functions of FAK as a number of FAK-dependent signaling

events, involving Src family kinases, the p85 subunit of PI3-kinase, Shc, Grb7 and PLC-γ are

dependent on FAK phosphorylation at Tyr397 (18). Here, we demonstrated that cytochalasin

D, at a concentration that profoundly suppresses FAK phosphorylation at Tyr397, does not

impair ERK 1/2 and JNKs tyrosine phosphorylation induced by protein I/II. Moreover,

preincubation of cells with concentrations of cytochalasin D that inhibit FAK

phosphorylation, had no effect on IL-6 and IL-8 synthesis, suggesting that FAK

phosphorylation at Tyr397 is not essential for MAPKs-dependent cytokine synthesis in

response to protein I/II. In accordance with this hypothesis, it is interesting to note that PI3-

kinase signaling activity which is regulated by FAK phosphorylation at Tyr397, is not

involved in protein I/II-induced cytokine synthesis.

Cytochalasin D is probably not the most suitable agent to address the role of FAK, as

it has been reported previously in NIH 3T3 cells that cytochalasin D, which has no effect on

the level of Src, may increase its intrinsic kinase activity and thus could mask the potential

contribution of FAK to the activation of MAPKs and subsequent cytokine synthesis (46).

However, this was not found to be the case here, as in control experiments, increasing

amounts of cytochalasin D failed to induce IL-6 and IL-8 synthesis from unstimulated FLSs

(data not shown).

Our findings that FAK phosphorylation at Tyr397 may not be essential for the

signaling pathway induced in response to protein I/II, are consistent with observations from

several authors suggesting that the phosphorylation state of Tyr397 is not always correlated

with FAK activity. First, Izaguirre et al. (47) restored alpha-actinin phosphorylation normally

induced by FAK by transfecting a Phe-397 mutant of FAK in cells lacking FAK. Second,

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

18

Hamawy et al. (48) demonstrated that transfection of a variant (3B6) of the RBL-2H3 mast

cell line with the FAK-Y397F mutant restored mast cell secretion. Third, Tachibana et al (49)

showed that a FAK-Y397F mutant was still able to phosphorylate Cas proteins. Moreover, by

transfecting cells with a kinase negative mutant of FAK or a FAK COOH-terminal domain

that does not contain the kinase domain, Tachibana et al. (49) demonstrated that the kinase

domain of FAK was required for Cas phosphorylation. As cytochalasin D which prevents

Tyr397 phosphorylation, does not inhibit either protein I/II-induced FAK kinase activity or

cytokine synthesis, it is tempting to speculate that FAK may function in response to protein

I/II, by initiating by itself, integrin-mediated tyrosine phosphorylation of a binding partner yet

unidentified. However we cannot exclude that FAK may also function as an adaptor or linker

protein in the protein I/II-induced signaling pathway. Given that integrins collaborate with

growth factor receptors to signal to MAPKs and that this pathway is cytochalasine D

insensitive, it is also possible that growth factor receptors may function as binding partners in

protein I/II-induced activation of FAK and subsequent cytokine synthesis (43). This remains

to be proven and further studies will be now necessary to define more precisely how FAK is

activated and which FAK substrates are involved in cytokine synthesis in response to protein

I/II-α5β1 integrin interaction.

Acknowledgments – We are grateful to Dr. Fatiha Sadallah for excellent technical assistance

with transfections using NucleofectorTM kit. We would like to thank Dr Dusko Ilic for

critically reading the manuscript. This work was supported in part by grants from Pharmacia,

Wyeth Lederle and Région Alsace.

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

19

REFERENCES

1 Van der Flier, A., and Sonnenberg, A. (2001) Cell Tissue Res. 305, 285-298

2 Gustavsson, A., Armulik, A., Brakebusch, C., Fassler, R., Johansson, S., and Fallman, M.

(2002) J. Cell Sci. 115, 2669-2678

3 Yilmaz, O., Watanabe, K., and Lamont, R. J. (2002) Cell. Microbiol. 4, 305-314

4 Watarai, M., Funato, S., and Sasakawa, C. (1996) J. Exp. Med. 183, 991-999

5 Ishibashi, Y., Relman, D. A., Nishikawa, A. (2001) Microb. Pathogenesis 30, 279-288

6 Al-Okla, S., Chatenay-Rivauday, C., Klein, J. P., and Wachsmann, D. (1999) Cell.

Microbiol. 1, 157-168

7 Cue, D., Southern, S. O., Southern, P. O., Prabhakar, J., Lorelli, W., Smalheer, J. M.,

Mousa, S. A., and Cleary, P. P. (2000) Proc. Natl. Acad. Sci. USA 14, 2858-2863

8 Sinha, B., Francois, P., Que, Y. A., Hussain, M., Heilmann, C., Moreillon, P., Lew, D.,

Krause, K. H., Peters, G., and Ferrmann, M. (2000) Infect. Immun. 68, 6871-6878

9 Rawadi, G., Ramez, V., Lemercier, B., Roman-Roman, S., (1998) J. Immunol. 160, 1330-

1339

10 Scherle, P. A., Jones, E. A., Favata, M. F., Daulerio, A. J., Covington, M. B., Nurnberg,

S.A., Mafolda, R. L., and Trzaskos, J. M. (1998) J. Immunol. 161, 5681-5686

11 Müller-Ladner, U., Gay, R. E., and Gay, S. (2000) Curr. Opin. Rheumatol. 12, 186-194

12 Gourieux, B., AL-Okla, S., Schöller, M., Klein, J. P., Sibilia, J., and Wachsmann, D.

(2000) FEMS Immunol. Med. Microbiol. 1280, 1-7

13 Neff, L., Zeisel, M., Sibilia, J., Schöller-Guinard, M., Klein, J. P., and Wachsmann, D.

(2001) Cell. Microbiol. 3, 703-712

14 Calalb, M., Polte, T., Hanks, S. K. (1995) Mol. Cell. Biol. 15, 954-963

15 Schlaepfer, D. D., Hanks, S. K., Hunter, T., and van der Geer, P. (1995) Nature 372, 786-

791

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

20

16 Polte, T. R., and Hanks, S. K., (1997) J. Biol. Chem. 272, 5501-5509

17 Ilic, D., Damsky, C., and Yamamoto, T. (1997) J. Cell. Sci. 110, 401-407

18 Schlaepfer, D. D., Hauck, C. R., and Sieg, D. J., (1999) Prog. Biophys. Mol. Biol. 71, 435-

478

19 Schaller, M. D., (2001) Biochem. Biophys. Acta 1540, 1-2

20 Wary, K. K., Mariotti, A., Zurzolo, C., and Giancotti, F. G. (1998) Cell 94, 625-6

21 Giancotti, F. G., and Ruoslahti, E. (1999) Science 285, 1028-1032

22 Barberis, L., Wary, K. K., Fiucci, G., Liu, F., Hirsch, E., Brancaccio, M., Altruda, F.,

Tarone, G., and Giancotti, F.G. (2000) J. Biol. Chem. 275, 36532-36540

23 Igishi, T., Fukuhara, S., Patel, V. Katz, B. Z., Yamada, K., and Gutkind, J. S. (1999) J.

Biol. Chem. 274, 30738-30746

24 Oktay, M., Wary K. K., Dans, M., Bireg, R. B., and Giancotti, F. (1999) J. Cell Biol. 145,

1461-1469

25 Sieg, D. J., Hauck, C. R., Ilic, D., Klingbeil, C. K., Damsky, C. H., and Schlaepfer, D.

(2000) Nature Cell Biol. 2, 249-256

26 Hauck, C. R., Sieg, D. J., Hsia, D. J., Loftus, J.C., Gaarde, W. A., Monia, B. P., and

Schlaepfer, D. (2001) Cancer Res. 61, 7079-7090

27 Giannone, G., Rondé, P., Guire, M., Haiech, J., and Takeda K (2002) J. Biol. Chem. 277,

26364-26371

28 Dechanet, J., Taupin, J. L., Chomarat, M. C., Moreau, J. F., Banchereau, J., and Miossec,

P. (1994) Eur. J. Immunol. 24, 3222-3228

29 Arnett, F., Edworthy, S. M., Bloch, D. A., Mc Shane D. J., Fires, J. F., Cooper, N. S. et al.

(1988) Arthritis Rheum. 3, 315-324

30 Mosmann, T. (1983) J. Immunol. Methods 65, 55-63

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

21

31 Chatenay-Rivauday, C., Yamodo, I., Sciotti, M., Ogier, J. A., and Klein, J. P. (1998) Mol.

Microbiol. 29, 39-48

32 Vernier, A., Diab, M., Soell, M., Haan-Archipoff, G., Beretz, A., Wachsmann, D., and

Klein, J. P. (1996) Infect. Immun. 64, 3016-3022

33 Tachado, S. D., Gerold, P., McConvill, M. J., Baldwin, T., Quilici, D., Schwarz, R. T., and

Schofield, L. (1996) J. Immunol. 156, 1897-1907

34 Ravichandran, K. S. Oncogene 20, 6322-6330

35 Chen, Q., Kinch, M., Lin, T., Burridge, K., and Juliano, R. (1994) J. Biol. Chem. 269,

26602-26605

36 Morino, M., Nimjura, T., Hamasaki, K., Tobe, K., Ueki, K., Kikuchi, K., Takehara, K.,

Kadowaki, T., Yazaki, Y., and Nojima, Y. (1995) J. Biol. Chem. 270, 269-273

37 Zhu, X., and Assoian, R. (1995) Mol. Biol. Cell 6, 273-282

38 Alrutz, M. A., Isberg, R. R. (1998) Proc. Natl. Acad. Sci. U S A. 95, 13658-13663

39 Reddy, M. A., Prasadarao, N. V., Wass, C. A., Kim, K. S. (2000) J. Biol. Chem. 275,

36769-36774

40 Sieg, D. J., Ilic, D., Jones, K. C., Damsky, C. H., Hunter, D., and Schlaepfer, D. D. (1998)

EMBO J. 17, 5933-5947

41 Gemba, T., Valbracht, J., Alsalameh, S., Lotz, M. (2002). J. Biol. Chem. 277, 907-911

42 Natarajan, K., Ghalayini, A. J., Sterling, R. S., Holbrook, R. M., Kennedy, R. C., Chodosh,

J. (2002) Invest. Ophthalmol. Vis. Sci. 43, 2685-2690

43 Parsons, J., T. (2003) J. Cell Sci. 116, 1409-1416

44 Mortier, E., Cornelissen, F., van Hove, C., Dillen, L., Richardson, A. (2001) Cell. Signal.

13, 901-909

45 Lai, K. M., Pawson, T. (2000) Genes Dev. 14, 1132-1145

46 Lock, P., Abram, C., Gibson, T., and Courtneidge, S. (1998) EMBO J. 17, 4346-4357

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

22

47 Izaguirre, G., Aguirre, L., Hu, Y. P., Lee, H. Y., Schlaepfer, D. D., Aneskievich, B. J., and

Haimovich, B. (2001) J. Biol. Chem. 276, 28676-28685

48 Hamawy, M. M., Swieter, M. K., Mergenhagen, S. E., and Siraganian, R. P. (1997) J. Biol.

Chem. 272, 30498-30503

49 Tachibana, K., Urano, T., Fujita, H., Ohashi, Y., Kamiguchi, K., Iwata, S., Hirai, H., and

Morimoto, C. (1997) J. Biol. Chem. 272, 29083-29087

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

23

Footnote

Abbreviations:

MAPKs: mitogen-activated protein kinases

ERK 1/2: extracellular-regulated kinases

JNKs: receptor Jun N-terminal kinases

FAK: focal adhesion kinase

PI-3K: phosphatidyl inositol 3-kinase

FRNK: FAK-related non-kinase

FAT: focal adhesion targeting

GFP: green fluorescent protein

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

24

Figure Legends

Figure 1: α5β1 integrins or anti-integrin β1 chain mAbs inhibited protein I/II-induced IL-6

and IL-8 release by FLSs. FLSs were stimulated for 20 h at 37°C with protein I/II

(125 pM) either A, preincubated with purified α5β1integrins (1, 5 and 10 µg/mL)

for 2 h at 4°C or B, added simultaneously with anti-integrin β1 chain mAbs (5, 20

and 40 µg/mL). IL-6 and IL-8 levels in the cell supernatants were determined by a

heterologous two-site sandwich ELISA. Results are expressed as percentage

inhibition of IL-6 (£) and IL-8 (¢) release and are mean values ± SD for

triplicate determinations from three identical experiments.

Figure 2: FAK phosphorylation in protein I/II-stimulated FLSs. FLSs were either incubated

in medium alone (control) or treated with protein I/II (125 pM) or fibronectin

(10 µg/ml) for the indicated times. Cell lysates were analyzed by Western-blotting

with phosphospecific antibodies to FAK [FAK pY397]. The same blot was

stripped and reprobed with anti-FAK antibodies to confirm the presence of equal

levels of FAK protein between samples. The results shown are representative of

three identical experiments.

Figure 3: Involvement of FAK in MAPKs activation and IL-6 and IL-8 synthesis by protein

I/II-stimulated mice fibroblasts. A, FAK +/+, FAK -/- and FRNK transfected FAK

+/+ mice fibroblasts were stimulated with protein I/II (125 pM) for 30 min at

37°C before lysis. Cells lysates were then analysed by Western-blotting with

phosphospecific antibodies to FAK (pFAKY397) or ERK 1/2 (pERK 1/2).

Control cells were incubated for the same time without protein I/II. The same

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

25

membranes were stripped and reprobed with antibodies to total FAK or ERK 1/2.

The results shown are representative of three identical experiments. B, PCR

analysis of mRNA using primers specific for IL-6 and β−actin in FAK +/+ and

FAK -/- mice fibroblasts stimulated with protein I/II (125 pM). The results shown

are representative of three identical experiments. C, FAK +/+, FAK -/- and

FRNK-transfected FAK +/+ mice fibroblasts were incubated in medium alone (£)

or stimulated with protein I/II (125 pM) (¢) for 20 h at 37°C. IL-6 levels in the

cell supernatants were determined by a heterologous two-site sandwich ELISA.

Data are expressed as mean values ± SD for triplicate determinations from three

identical experiments.

Figure 4: Involvement of FAK in protein I/II-induced IL-6 and IL-8 release by human

FLSs. FLSs transiently transfected with FRNK were incubated with (¢) or

without (£) protein I/II (125 pM) for 20 h at 37°C. Non transfected FLSs were

used as controls. IL-6 and IL-8 levels in cell supernatants were determined by a

heterologous two-site sandwich ELISA. Results are expressed as concentrations

of IL-6 and IL-8. Data are expressed as mean values ± SD for triplicate

determinations from three identical experiments.

Figure 5: Shc is not involved in protein I/II-induced IL-6 and IL-8 release. A, FLSs were

either incubated in medium alone (control) or treated with protein I/II (125 pM) or

EGF (100 ng/mL) for the indicated times. Cell lysates were analyzed by Western-

blotting with phosphospecific antibodies to Shc [Shc pY317]. The same blot was

stripped and reprobed with anti-Shc antibodies to confirm the presence of equal

levels of Shc protein between samples. The results shown are representative of

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

26

three identical experiments. B, FLSs were transiently transfected with Shc-Y317F

and then incubated in medium alone (£) or stimulated with protein I/II (125 pM)

(¢) for 20 h at 37°C. Non transfected FLSs were used as controls. IL-6 and IL-8

levels in cell supernatants were determined by a heterologous two-site sandwich

ELISA. Results are expressed as concentrations of IL-6 and IL-8. Data are

expressed as mean values ± SD for triplicate determinations from three identical

experiments.

Figure 6: Cytochalasin D has no effect on protein I/II-induced FAK, ERK 1/2 and JNKs

phosphorylation and IL-6 and IL-8 release. FLSs pretreated (40 min at 37°C) with

cytochalasin D (1 µM) were incubated in medium alone (C) or stimulated with

protein I/II (125 pM) for 15 and 30 min. Cell lysates were then analyzed by

Western-blotting with phosphospecific antibodies to A, FAK (pFAKY397), B,

ERK 1/2 (pERK 1/2), or C, JNKs (pJNKs). The same blot was stripped and

reprobed with anti-FAK, -ERK 1/2 or -JNKs antibodies to confirm the presence of

equal levels of FAK, ERK 1/2 or JNKs proteins between samples. Results shown

are representative of three identical experiments. D, FLSs preincubated (40 min at

37°C) with 0.5, 1 and 2 µM of cytochalasin D, were treated with protein I/II (125

pM) for 20 h at 37°C. IL-6 (£) and IL-8 (¢) levels in the cell supernatants were

determined by a heterologous two-site sandwich ELISA. Results are expressed as

concentrations of IL-6 and IL-8. Data are expressed as mean values ± SD for

triplicate determinations from three identical experiments.

Figure 7: Wortmannin has no effect on protein I/II-induced IL-6 and IL-8 release. FLSs

were preincubated (1 h at 37°C) with increasing amounts of wortmannin at the

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

27

indicated concentrations and then stimulated with protein I/II (125 pM) for 20 h at

37°C. IL-6 (£) and IL-8 (¢) levels in the cell supernatants were determined by a

heterologous two-site sandwich ELISA. Results are expressed as IL-6 and IL-8

concentrations and are mean values ± SD for triplicate determinations from three

identical experiments.

Figure 8: Cytochalasin D has no effect on protein I/II-induced FAK kinase activity. FLSs

preincubated (40 min at 37°C) with 1 µM of cytochalasin D, were treated with

protein I/II (125 pM) for 15 min at 37°C. After immunoprecipitation of FAK, its

tyrosine kinase activity was determined by an in vitro assay. Results are expressed

as protein tyrosine kinase (PTK) activity and are mean values ± SD for triplicate

determinations from three identical experiments.

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Figure 1

0

20

40

60

80

100

1 5 10

% inhibition

0

20

40

60

80

100

5 20 40

% inhibition

A

B

Soluble α5β1 integrins (µg/mL)

Anti-integrin β1 (µg/mL)

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Figure 2

pFAK Y397

Time (min) 1 5 15 30 60Con

trol (

15 m

in)

Fibr

onec

tin

FAK

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Figure 3

A

control I/II control I/II

FAK+/+ FAK-/-

pFAK Y397

FAK

pERK

ERK

control I/II control I/II

FAK+/+ FRNK

050

100150200250300

FAK +/+ FAK -/-

pg/mL

Bcontrol I/II control I/II

FAK+/+ FAK-/-

IL-6

actin

050

100150200250300

FAK+/+ FRNK

pg/mLC

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Figure 4

IL-6 IL-8

0

1

2

3

4

control FRNK

ng/mL ng/mL

0

1

2

3

4

control FRNK

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Figure 5

pShc Y317

Shc

Time (min) 1 5 15 30 60 EGFContro

l

A

B

0

1

2

3

4

control Shc-Y317F

IL-6 IL-8ng/mL ng/mL

0

1

2

3

4

control Shc-Y317F

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Figure 6

pFAK Y397

FAK

Cytochalasin D (1 µM)

Time (min) 15 30C 15 30

+ +- --

pERK1/2

ERK1/2

pJNKs

JNKs

A

B

C

D

0

1

2

3

4

0 0.5 1 2

IL-6

IL-8

ng/mL

Cytochalasin D (µM)

+ + + +Protein I/II (125 pM)

Protein I/II (125 pM) + + + +-

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Figure 7

0

1

2

3

4

5

6

7

0 50 100 200

wortmannin (nM)

IL-6

IL-8

ng/ml

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Figure 8

Cytochalasin D (1 µM)

Protein I/II (125 pM) + +

+ +- -

--

0

1

2

3

4

5

6

PT

K a

cti

vit

y (

x1

0-5

un

its

/µL

)

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Jean Sibilia and Dominique WachsmannLaurence A. Neff, Mirjam B. Zeisel, Vanessa A. Druet, Ken Takeda, Jean-Paul Klein,

requires FAKsynoviocytes stimulated with protein I/II, a modulin from oral streptococci, ERK 1/2 and JNKs-dependent synthesis of IL-6 and IL-8 by fibroblast-like

published online May 20, 2003J. Biol. Chem. 

  10.1074/jbc.M212065200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from