The Extra Domain A of Fibronectin Activates Toll-like Receptor
4*
Yoshinori
Okamura
,
Michiko
Watari
,
Elliot S.
Jerud§,
Donna
W.
Young§,
Sally T.
Ishizaka§,
Jeffrey
Rose§,
Jesse C.
Chow§, and
Jerome F.
Strauss III
¶
From the
Center for Research on Reproduction and
Women's Health, University of Pennsylvania Medical Center,
Philadelphia, Pennsylvania 19104 and § Department of
Molecular Biology and Biochemistry and Signal Transduction, Eisai
Research Institute, Wilmington, Massachusetts 01887
Received for publication, January 5, 2001
 |
ABSTRACT |
Cellular fibronectin, which contains an
alternatively spliced exon encoding type III repeat extra domain A
(EDA), is produced in response to tissue injury. Fragments of
fibronectin have been implicated in physiological and pathological
processes, especially tissue remodeling associated with inflammation.
Because EDA-containing fibronectin fragments produce cellular responses
similar to those provoked by bacterial lipopolysaccharide (LPS), we
examined the ability of recombinant EDA to activate Toll-like receptor
4 (TLR4), the signaling receptor stimulated by LPS. We found that
recombinant EDA, but not other recombinant fibronectin domains,
activates human TLR4 expressed in a cell type (HEK 293 cells) that
normally lacks this Toll-like receptor. EDA stimulation of TLR4 was
dependent upon co-expression of MD-2, a TLR4 accessory protein. Unlike
LPS, the activity of EDA was heat-sensitive and persisted in the
presence of the LPS-binding antibiotic polymyxin B and a potent LPS
antagonist, E5564, which completely suppressed LPS activation of TLR4.
These observations provided a mechanism by which EDA-containing
fibronectin fragments promote expression of genes involved in the
inflammatory response.
 |
INTRODUCTION |
Cellular fibronectins, which contain alternatively spliced exons
encoding type III repeat extra domain A
(EDA)1 and EDB (1), are
produced in response to tissue injury (2-5). Fragments of fibronectin
or specific fibronectin domains are believed to play important roles in
physiological and pathological processes, including tissue remodeling
in response to inflammation (2-8). The responses of cells exposed to
recombinant EDA or EDA-containing fibronectin are similar to those
observed when cells are treated with bacterial lipopolysaccharide
(LPS), including the induction of genes encoding proinflammatory
cytokines and matrix metalloproteinases (2-5). However, the mechanism
by which cells sense the EDA domain is not known. Because of the
similarities between the effects of the EDA domain and LPS, we
hypothesized that these molecules may be recognized by the same receptor.
Constituents of pathogens, including LPS, are detected by a family of
recently discovered receptors related to the Drosophila Toll
protein, which is involved in dorsoventral polarization and the
induction of antimicrobial factors in response to infection (9, 10).
The mammalian Toll-like receptors have an extracellular domain
containing leucine-rich repeats and an intracellular domain, related to
that of the IL-1 signaling receptor, that activates a signal
transduction cascade resulting in nuclear translocation of nuclear
factor
B (NF-
B) (9-15). The endogenous ligand for the
Drosophila Toll receptor, spätzle, is a secreted
protein that requires proteolytic activation (16, 17).
Mammalian TLR4 is the signaling receptor activated by LPS (9, 13-15,
18, 19), although the accessory proteins MD-2 (20) and CD14 (21) are
required for maximal responses to LPS. The endogenous
ligands/activators of the mammalian TLRs have not yet been identified,
but it has been proposed that a proteolytic processing reaction
generates the peptide ligands/activators in a manner analogous to the
Drosophila Toll-spätzle system. Recent observations suggest that heat shock protein 60 (hsp 60) activates TLR4 (22), but it
is not yet known if processing of hsp 60 is required to achieve TLR4
activation. In the present study, we determined that the EDA domain of
fibronectin is capable of activating TLR4, which would account for the
ability of EDA or EDA-containing fibronectin fragments to induce
LPS-like responses.
 |
EXPERIMENTAL PROCEDURES |
Production of Recombinant Fibronectin Type III Repeat
Proteins--
Reverse transcription-polymerase chain reaction was
employed to generate cDNAs encoding the individual fibronectin type
III domains. The primers for EDA were: sense primer (EDA-s),
5'-CGGGATCCAACATTGATCGCCCTAAAGG-3'; and antisense primer
(EDA-a), 5'-TCCCCCGGGTGTGGACTGGGTTCCAATC-3'. The primers
for EDB were: sense primer (EDB-s),
5'-CGGGATCCGAGGTGCCCCAACTCACTGACC-3', and antisense primer
(EDB-a), 5'-TCCCCCGGGCGTTTGTTGTGTCAGTGTAGT-3'. The primers
for III11 were: sense primer,
5'-CGGGATCCGAAATTGACAAACCATCCCCA-3'; and antisense primer,
5'-TCCCCCGGGGGTTACTGCAGTCTGAACC-3'. The BamHI
linker incorporated into the sense primers and the SmaI linker incorporated into the antisense primers are underlined. The
polymerase chain reaction-amplified cDNAs were subcloned into the
pQE-30 vector (Qiagen), which places a His6-tag at the N
terminus. Recombinant proteins were purified on a
nickel-nitrilotriacetic acid resin and then passed through an
Acticlean Etox (Sterogene Bioseparations Inc., Carlsbad, CA) column to
remove LPS.
A 7-kDa recombinant protein representing the C terminus of type III
repeat 1 (III1C), poly-L-histidine, and human
fibroblast cellular fibronectin were purchased from Sigma. Polymyxin B
(10 µg/ml) was added to all proteins to bind residual LPS. LPS in the
recombinant proteins was quantified using the Limulus
amebocyte lysate assay (Sigma), and Western blotting was used to detect bacterial hsp 60 with an antibody recognizing Escherichia
coli hsp 60.
THP-1 Cell Experiments--
THP-1 cells (American Type Culture
Collection, Manassas, VA) were cultured in RPMI 1640 medium
supplemented with 10% fetal bovine serum (FBS) and gentamicin (50 µg/ml). Cells were resuspended in serum-free medium before each
experiment and all studies were carried out in serum-free culture
fluid. Cells were treated with recombinant fibronectin domains or LPS
(serotype 055:B5) (Sigma) in the absence or presence of the LPS
antagonist, E5531 (Eisai). The conditioned medium was collected
for analysis of matrix metalloproteinase 9 (MMP-9) expression by
zymography or ELISA. Cell numbers were counted with a hemocytometer.
Assay of MMP-9--
Conditioned medium was subjected to
zymography as previously described (23). MMP-9 released into the
culture fluid was also measured using an MMP-9 ELISA kit (Oncogene
Research Products, Boston, MA).
Western Blot Analysis--
Cells were lysed in Nonidet P-40
lysis buffer, and nuclear extracts were prepared and protein
quantitated using the Micro BCA protein assay (Pierce). Nuclear
extracts (15 µg/lane) were subjected to Western blotting using
antibodies raised against human NF-
B p65 and p50 (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA).
Activation of TLR4--
For transfection experiments, HEK 293 cells, which do not normally express TLR4, were plated in 48-well
tissue culture plates at a density of 2 × 105
cells/well and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS for 24 h. Cells were then
transfected with TLR4 cDNA or empty vector and/or MD-2 cDNA
plus 50 ng of pELAM-1-luc using a calcium phosphate protocol
(13). All cells were also transfected with pRL-TK plasmid, a
Renilla luciferase control reporter vector (Promega, Madison, WI) to
normalize transfection efficiencies. After transfection, cells were
maintained in DMEM supplemented with 10% FBS for 24 h. The
medium was subsequently removed and replaced with DMEM plus
0.5% FBS. The cells were either left untreated or incubated with 1 µM of the indicated recombinant fibronectin domain or 100 ng/ml LPS plus 10 nM soluble CD14 (sCD14) for 18 h.
Cells were harvested in lysis buffer and assayed for Firefly and
Renilla luciferase activity as described by the manufacturer of the
Dual Luciferase Reporter System (Promega).
HEK 293 cells stably carrying plasmids for TLR4, MD2, and
ELAM-1-luciferase (HEK-TLR4/MD2/ELAM-1-luc) were generated as described (15). Cells were plated in 96-well plates at a density of 50,000 cells
per well and maintained in DMEM plus 10% FBS for 24 h. The medium
was removed and replaced with DMEM plus 0.5% FBS and the cells were
incubated with 1 µM of the indicated fibronectin domain protein or 10 or 100 ng/ml LPS plus sCD14 and, in some cases, in the
presence of 1 µM E5564 or 10 µg/ml polymyxin B for
18 h. Fibronectin domain proteins and LPS were also heat-treated
at 95 °C for 20-60 min prior to addition to the cells. Steady-Glo reagent (Promega) was added to the wells and the amount of luciferase activity was quantified. Data are shown as means ± S.D. from one representative experiment in which each transfection or treatment was
performed in quadruplicate. Each experiment was repeated on at least
three separate occasions.
Mouse Splenocyte Assay--
Spleens from C3H/HeN (control;
Charles River Laboratories) and eC3H/HeJ (LPS-insensitive because of
Tlr4 mutation; Jackson Laboratories) mice were broken apart
with sterile forceps and passed through a sterile 21-gauge needle.
Splenocytes were collected, washed once in serum-free RPMI 1640 medium
and then seeded into 24 well tissue culture plates at a density of
5 × 106 cells/well in 500 µl of RPMI 1640 medium
supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 µM
-mercaptoethanol and 100 units/ml penicillin and streptomycin.
Recombinant proteins (1 µM) or LPS (100 ng/ml) were added
and the cells cultured for 72 h at 37 °C. Supernatants were
collected and stored at
80 °C until assayed for IL-10 using a
specific ELISA (Endogen, Inc.).
 |
RESULTS AND DISCUSSION |
We first characterized the activities of recombinant fibronectin
type III repeat domains on cells that express TLR4, human THP-1
monocyte/macrophage cells, using production of MMP-9 as an index of the
cellular response. Gelatin zymography performed on conditioned
medium demonstrated that recombinant EDA (1 µM) strongly induced pro-MMP-9 expression (Fig.
1A). Recombinant EDB at
similar concentrations caused only a modest increase in pro-MMP-9 release, whereas recombinant III11 domain, a 7-kDa
recombinant protein representing the C terminus of repeat
III1 (III1C), poly-L-histidine, and
human fibroblast cellular fibronectin had no major effect on pro-MMP-9
release (Fig. 1A). MMP-2 was detected in the medium in some experiments, but recombinant EDA did not have a significant effect on its expression (Fig. 1A). Note that the stronger
MMP-2 activities and weak MMP-9 band appearing in the conditioned
medium of cells cultured with cellular fibronectin represent
contaminants in the cellular fibronectin preparation as they were
present on zymographic analysis of medium containing cellular
fibronectin that never was exposed to THP-1 cells (data not shown). At
a concentration of 1 µM, EDA stimulated a progressive
accumulation of pro-MMP-9 over a 72 h incubation period with a
marked increase in pro-MMP-9 detectable within 24 h (Fig.
2). A shorter time course revealed induction of pro-MMP-9 within 6 h of EDA treatment (data not
shown). Concentrations of recombinant EDA as low as 300 nM
stimulated pro-MMP-9 production, and the response of the THP-1 cells
was dose-dependent to concentrations of EDA up to 10 µM (Fig. 2). Quantitation of MMP-9 released by THP-1
cells by ELISA confirmed the stimulatory effect of EDA and the failure
of other recombinant fibronectin domains to markedly induce MMP-9
expression (Fig. 1B). The recombinant fibronectin domains
contained <1 ng of LPS/nmol of protein, and there was no detectable
hsp 60 in the preparations. The LPS antagonist, E5531, substantially
reduced production of MMP-9 by LPS-stimulated THP-1 cells but had no
appreciable impact on secretion of MMP-9 by EDA-treated cells (Fig.
3). None of the treatments promoted
significant differentiation/attachment of the cells, which mostly
remained in suspension; no more than 5% of cells in any of the
treatments became adherent and spread.

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Fig. 1.
EDA stimulates MMP-9 production by THP-1
cells. THP-1 cells (5 × 105 cells/well) were
seeded into each well of a 24-well plate in serum-free culture medium.
Cells were either untreated (control) or treated with 1 µM of different recombinant fibronectin type III repeats,
poly-L-histidine, or cellular fibronectin (CFN)
for 48 h. A, gelatinase activities released from THP-1
cells analyzed using gelatin zymography. B, MMP-9 in
conditioned medium measured by ELISA and expressed as the
mean ± S.D. from triplicate cultures.
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Fig. 2.
Time course (A) and
dose-response (B) of MMP-9 expression by THP-1 cells
treated with EDA. A, THP-1 cells were incubated with
poly-L-histidine (1 µM) or EDA (1 µM) for the indicated times. Zymography was performed on
conditioned medium. B, a dose-response study was
conducted with the indicated concentrations of
poly-L-histidine and EDA. Cells were incubated for 48 h and zymography was performed.
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Fig. 3.
The LPS antagonist, E5531, inhibits
LPS-stimulated MMP-9 production but does not block the action of
EDA. THP-1 cells (5 × 105 cells/well) were
cultured for 24 h in the presence of LPS (200 ng/ml), 256 nM E5531, EDA (300 nM), or a combination of LPS + E5531 or EDA + E5531 as indicated. Zymography was carried out on
conditioned medium to demonstrate gelatinase activity.
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NF-
B participates in the induction of MMP-9 gene
transcription (24), and NF-
B is activated in response to LPS
stimulation of TLR4. We found that components of NF-
B were increased
in the nuclei of THP-1 cells as early as 15 min after addition of EDA, reaching peak levels at 1 h that were maintained up to 3 h
(Fig. 4).

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Fig. 4.
Nuclear accumulation of
NF- B in THP-1 cells treated with EDA.
THP-1 cells (10 × 106 cells/well) were incubated with
1 µM EDA for 0, 15, 30, 60, or 180 min. Nuclear extracts
were obtained and analyzed by Western blotting for NF- B p50
(A) or p65 (B).
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To determine whether recombinant EDA can activate TLR4, we transfected
HEK 293 cells, which do not normally express TLR4, with expression
plasmids for TLR4 and its accessory proteins and the reporter
construct, pELAM-1-luc, which contains a fragment of the
NF-
B-responsive E-selectin promoter coupled to luciferase (14) and then challenged the transfected cells with recombinant fibronectin domains or LPS. LPS in the presence of sCD14, which binds
LPS and presumably presents it to TLR4, produced the expected strong
reporter response in cells transfected with TLR4 and MD-2 (14, 15) but
not in cells that did not receive the TLR4 expression plasmid (Fig.
5). Recombinant EDA, but not
III11 or EDB, produced a response of the reporter construct
in TLR4/MD-2 transfectants that was ~40-45% of the LPS response.
Like LPS, cotransfection with TLR4 and MD-2 was required for a vigorous
response of the reporter construct to EDA (Fig.
6).

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Fig. 5.
EDA stimulates NF- B
activation via TLR4. HEK 293 cells were plated at a density of
2 × 105 cells/well in 48-well plates and transfected
with TLR4 cDNA (striped bars) or empty vector
(filled bars) and MD-2 cDNA plus pELAM-1-luc.
The following day, the medium was replaced with DMEM plus 0.5%
FBS, and the cells were either left untreated (basal) or incubated with
1 µM of the indicated recombinant fibronectin domain or
100 ng/ml LPS plus 10 nM sCD14 for 18 h. Cell lysates
were assayed for luciferase activity. Values are means ± S.D.
from quadruplicate cultures in two different experiments (A
and B).
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Fig. 6.
MD-2 is required for EDA activation of
TLR4. HEK 293 cells were plated at a density of 2 × 105 cells/well in 48-well plates and transfected with empty
vector (Vector), TLR4, MD-2 cDNA, or TLR4 and MD-2 cDNA plus
pELAM-1-luc. The following day, the medium was replaced with
DMEM plus 0.5% FBS, and the cells were either left untreated or
incubated with recombinant III11 repeat, 1 µM
EDA, or 100 ng/ml LPS plus 10 nM sCD14 for 18 h. Cell
lysates were assayed for luciferase activity. Values are means ± S.D. from quadruplicate cultures.
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To further verify that recombinant EDA activates TLR4, we isolated
splenocytes from C3H/HeJ mice, which have an inactivating mutation in
the Tlr4 gene, and C3H/HeN control mice and challenged them
with LPS, recombinant EDA, EDB, and III11 and measured
IL-10 production. LPS (100 ng/ml) and EDA (1 µM)
stimulated IL-10 production by C3H/HeN splenocytes, whereas recombinant
EDB and III11 had minimal stimulatory effects (Table
I). The relative activity of EDA (1 µM) compared with 100 ng/ml LPS in stimulating IL-10 release by C3H/HeN splenocytes was somewhat lower than the relative response that we observed in activation of TLR4 in the HEK 293 cells.
This may be because of differences in ligand specificity between the
human and murine TLR4s (e.g. taxol activates murine Tlr4 but
not human TLR4) or to the presence of a higher concentration of serum
(10% FBS) in the splenocyte assay compared with 0.5% FBS in the HEK
293 studies (and serum-free conditions with THP-1 cells). We have found
that serum blunts the response to recombinant EDA but not to LPS. The
mechanism underlying the serum effect on EDA action is not known but
could reflect the presence of an EDA-binding protein or antagonist.
Importantly, the actions of LPS and recombinant EDA on IL-10 production
were significantly blunted in C3H/HeJ splenocytes, consistent with an
action of LPS and EDA via Tlr4.
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Table I
Relative stimulation of IL-10 secretion by mouse splenocytes
Spleen cells were stimulated as described under "Experimental
Procedures." The relative stimulation of IL-10 is calculated by
dividing the pg/ml of IL-10 released in an experimental well by the
amount released in medium alone. Medium control values ranged from 67 to 500 pg/ml. ND, not determined.
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Using stable TLR4/MD-2 HEK 293 transfectants, we found as expected that
LPS in the absence of sCD14 had a minimal effect on TLR4-mediated
reporter gene activation (Fig. 7), but
there was a dramatic increase in the response to LPS when sCD14 was
added. In the absence of sCD14, polymyxin B and the LPS antagonist,
E5564, had no major impact on EDA activation of TLR4. However, the same concentrations of polymyxin B and E5564 completely blocked the stimulatory effects of LPS in the presence of sCD14. sCD14 (10 ng/ml)
also increased the response to recombinant EDA (Fig. 7). This sCD14
augmentation of EDA activation of TLR4 was inhibited by polymyxin B and
E5564, raising the possibility that the increased activity of EDA in
the presence of sCD14 could be due, in part, to LPS contamination. Heat
treatment ablated the TLR4-stimulating activity of recombinant EDA but
had no effect on LPS. Collectively, these experiments demonstrate that
EDA activation of TLR4 cannot be accounted for by LPS contamination in
the recombinant protein preparations.

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Fig. 7.
Effects of heat treatment, polymyxin B, and
the LPS antagonist, E5564, on EDA activation of TLR4.
HEK-TLR4/MD2/ELAM-1-luc cells were seeded in 96-well plates and
maintained as described under "Experimental Procedures." Cells were
incubated with EDA (1 µM) or LPS (10 ng/ml) in the
absence or presence of 10 nM sCD14 with phosphate-buffered
saline (PBS), 10 µg/ml polymyxin B (Polymix),
or 1 µM E5564 as indicated for 18 h. Heat indicates
that EDA and LPS were heated (95 °C for 60 min) before addition to
the cells. Following the incubation period, cell lysates were
immediately analyzed for luciferase activity. Values are means ± S.D. from quadruplicate cultures from a representative
experiment.
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Other investigators have examined the effects of fibronectin as well as
fibronectin fragments not containing EDA on various cells and concluded
that the responses are a consequence of fibronectin binding to integrin
fibronectin receptors (25-27). Fibronectin fragment effects have also
been linked to cellular differentiation, adhesion, and changes in the
organization of actin filaments (25-27). Our observations suggest an
alternative signaling pathway for fibronectin molecules containing the
EDA domain.
The known TLR4 activators require accessory proteins to turn on TLR4
signaling. Like LPS, EDA activation of TLR4 requires MD-2, suggesting
shared features of the mechanism of TLR4 activation. LPS requires CD14
for efficient activation of TLR4. Although we found that sCD14 enhanced
the EDA activation of TLR4, we cannot rule out the possibility that
sCD14 exposed the activity of minor LPS contaminants. It is of interest
that Asea et al. (28) recently found that hsp 70 acts
as a cytokine employing a CD14-dependent pathway,
suggesting that CD14, a known lipid-binding protein, can also augment
the action of proteins. We have been unable to demonstrate binding of
EDA to sCD14.2 Therefore, if
CD14 does increase the action of EDA, it must do so through a mechanism
that does not involve high affinity interactions between these
molecules. Importantly, polymyxin B and LPS antagonists did not have a
major impact on EDA-stimulated THP-1 cell MMP-9 production and EDA
activation of TLR4. The activity of EDA was destroyed by heat
whereas LPS activity was unaffected. Thus, the effects of the EDA
domain can be clearly distinguished from those of LPS.
Saito et al. (4) recently reported that recombinant EDA
domains, but not intact cellular fibronectin, induce proinflammatory cytokines and MMP expression by rabbit synovial cells. Likewise, we
found that intact cellular fibronectin did not have a major impact on
MMP-9 expression in THP-1 cells. Saito et al. (4) also isolated a fibronectin fragment from human placenta containing the
EDA domain in its C terminus. This fibronectin fragment displayed activities similar to that of the recombinant EDA domains. Their observations lend credence to the notion that endogenously generated EDA-containing fibronectin fragments serve as signaling molecules and
indicate that domains in the fibronectin protein distal to the EDA
domain may suppress the ability of fibronectin to activate signaling.
Further investigation is needed to elucidate 1) the mechanism by which
EDA stimulates TLR4 activity and 2) the structure of endogenous
EDA-containing fibronectin fragments.
 |
FOOTNOTES |
*
This work was supported by National Institutes of
Health Grant HD-34612 and by a grant from the Bill and Melinda
Gates Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: 1354 BRB II/III,
University of Pennsylvania Medical Center, 421 Curie Blvd.,
Philadelphia, PA 19104. Tel.: 215-898-0147; Fax: 215-573-5408; E-mail:
jfs3@mail.med.upenn.edu.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M100099200
2
Y. Okamura and J. F. Strauss,
unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
EDA, extra domain A;
EDB, extra domain B;
LPS, lipopolysaccharide;
TLR4, Toll-like receptor
4;
IL-1, interleukin 1;
NF-
B, nuclear factor
B;
III1C, recombinant fibronectin type III repeat 1 C-terminus;
III 11, fibronectin type III repeat 11;
FBS, fetal bovine serum;
MMP, matrix metalloproteinase;
ELISA, enzyme-linked
immunosorbent assay;
DMEM, Dulbecco's modified Eagle's medium;
sCD14, soluble CD14.
 |
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