1
Department of Biochemistry, The Holland Laboratory, American Red Cross,
Rockville, MD 20855, USA
2
Department of Biochemistry and Molecular Biology, The George Washington
University, Washington, DC 20037, USA
*
Author for correspondence (e-mail:
belkina{at}usa.redcross.org
)
Accepted May 3, 2001
![]() |
SUMMARY |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Key words: Transgutaminase, Integrin, Fibronectin assembly
![]() |
INTRODUCTION |
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Interaction of the III9III10 modules of Fn with
integrins is crucial for Fn polymerization (Pierschbaher and Ruoslahti, 1984;
Akiyama et al., 1989; Giancotti and Ruoslahti,
1990). Peptides that contain
the Arg-Gly-Asp (RGD) sequence and antibodies against the major Fn-binding
integrin
5ß1 strongly inhibit the assembly of Fn into matrix
(Fogerty et al., 1990
).
Although the
5ß1 integrin has a predominant role in Fn assembly,
other Fn-binding integrins such as
Vß3 can mediate fibril
formation (Yang and Hynes,
1996
; Wu,
1997
). Integrin activation and
integrin-cytoskeletal association induce Fn assembly (Wu et al.,
1995
). In addition to the
RGD-containing module III10 and module III9, which
includes a synergy site (Obara et al.,
1988
; Sechler et al.,
1997
), several other regions
of Fn are important for fibrillogenesis. These include a 29 kDa N-terminal
I1-5 domain of Fn, which binds directly to cell surfaces and
inhibits the assembly (McKeown-Longo and Mosher,
1985
; Quade and McDonald,
1988
; Christopher et al.,
1997
). A recent work suggested
that the surface matrix assembly sites interacting with the 29 kDa N-terminal
Fn domain represent integrins as the
5ß1 integrin reportedly binds
to this part of Fn (Hocking et al.,
1998
). The 29 kDa N-terminal
Fn fragment decreases Fn binding to cell layers and therefore is likely to be
involved in initiation of assembly (Sottile et al.,
1991
; Sottile and Mosher
1997
). In addition, Fn
constructs lacking the N-terminal domain are not incorporated into fibrillar
matrix, indicating a role for this domain in Fn-Fn interactions (Schwarzbauer,
1991
).
The III1 module is another region of Fn. It is involved in
homophylic Fn interactions and has been shown to modulate fibril formation
(Chernousov et al., 1991; Morla
and Ruoslahti, 1992
). The
native isolated III1 module interacts with the denatured
III10 module (Hocking et al.,
1996
) as well as with
III7 and III15 modules (Ingham et al.,
1997
). In turn, a cryptic site
within the III1 module reportedly binds the N-terminal
I1-5 domain of Fn (Hocking et al.,
1994
), yet the interactions of
the III1 module with other Fn domains and its role in the assembly
of Fn fibrils remain controversial. The I9III1 modules
contain a cryptic site involved in Fn self-assembly and exposed in fibrillar
Fn by cell-generated tension via activation of Rho (Zhong et al.,
1998
). Finally, Fn
fibrillogenesis involves the III12-14 modules of the C-terminal
heparin II domain of Fn, although its interactions with other Fn fragments
remain unknown (Bultmann et al.,
1998
).
Enzymes of the transglutaminase family are implicated in the formation and
modification of Fn matrices (Mosher et al.,
1992). Tissue transglutaminase
(tTG) is a member of a family of Ca2+-dependent crosslinking
enzymes (Folk, 1980
). It is
expressed in a variety of cell types and covalently crosslinks several ECM
proteins (Aeschlimann and Paulsson,
1991
; Martinez et al.,
1994
; Kleman et al.,
1995
; Kaartinen et al.,
1997
). tTG is localized
primarily in the cytoplasm, yet it is also present on the cell surface
(Upchurch et al., 1991
;
Verderio et al., 1998
; Gaudry
et al., 1999
). Because tTG
lacks a leader sequence, the mechanisms of its externalization remain unclear.
tTG binds in vitro with high affinity to the 42 kDa gelatin-binding region of
Fn that consists of I6II1,2I7-9 modules
(Turner and Lorand, 1989
;
Radek et al., 1993
). Several
studies documented the ability of surface tTG to crosslink Fn into high
molecular weight polymers (Martinez et al.,
1994
; Jones et al.,
1997
; Verderio et al.,
1998
). These works established
that tTG enzymatic activity on the surface is involved in the covalent
modification of Fn, but did not analyze whether tTG modulates Fn
polymerization, influencing a preceding stage of matrix assembly. Some data
also implicated surface tTG in cell adhesion. Overexpression of tTG increased
cell spreading (Gentile et al.,
1992
), whereas downregulation
of tTG or addition of tTG-inactivating monoclonal antibody (mAb) inhibited
adhesion and spreading on Fn (Jones et al.,
1997
; Verderio et al.,
1998
). Recently, we found that
tTG interacts with ß1 and ß3 integrins during biosynthesis and that
integrin-tTG complexes accumulate on the cell surface and in focal adhesions
(Akimov et al., 2000
). tTG
independently mediates the association of integrins with Fn due to its
high-affinity interaction with both proteins, thereby functioning as an
adhesion coreceptor for Fn.
Here we report that cell-surface tTG enhances Fn matrix formation mediated
by 5ß1 integrin. This stimulation of Fn assembly does not require
its enzymatic activity and is thus distinct from the reported tTG-mediated
covalent crosslinking of Fn (Jones et al.,
1997
; Verderio et al.,
1998
). We also show that tTG
exerts its effects on Fn fibrillogenesis via interaction with the
gelatin-binding region of the molecule, for the first time implicating this
domain of Fn in matrix assembly. Finally, we provide evidence that
upregulation of surface tTG and its association with ß1 integrins in
fibroblasts promotes Fn assembly in response to transforming growth factor
ß (TGFß). This suggests a role for surface tTG in the enhancement of
Fn matrix deposition during normal wound healing and fibroproliferative
diseases.
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MATERIALS AND METHODS |
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Purified 42 kDa Fn fragment was biotinylated using EZ-LinkTM Sulfo-NHS-Biotin (Pierce). Fn and the 42 kDa fragment were labeled with [125I]Na (ICN, Irvine, CA) using IODO-BEADS® (Pierce).
Cell culture and transfection
Swiss 3T3 and WI-38 fibroblasts were obtained from ATCC and cultured by
standard methods. Swiss 3T3 cells were transfected with tTG or tTG(C277S)
cDNAs in pcDNA3.1-zeo plazmid (Invitrogen, Carlsbad, CA) using
SuperfectTM (Quaigen, Valensia, CA), and stable populations of tTG
transfectants (vector, tTG[1], tTG[2], tTGC277S[1] and
tTGC277S[2]) were selected in 100 µg/ml zeocinTM
(Invitrogen). In some experiments, WI-38 fibroblasts were treated with 2 ng/ml
recombinant human TGFß1 (R&D Systems, Minneapolis, MN).
Analysis of tTG association with integrins by
co-immunoprecipitation
Immunoprecipitation of tTG, 5ß1 and
Vß3 integrins
from RIPA lysates of Swiss 3T3 transfectants or untreated and
TGFß-treated WI-38 fibroblasts was performed as described (Akimov et al.,
2000
) with mAbs BMA5 (for mouse
5ß1), JBS5 (for human
5ß1) and LM609 (for human
Vß3), using 0.5 mg of total protein for each sample. The resulting
samples were run on 10% SDS gels, proteins were transferred to nitrocellulose
and blotted for tTG, ß1 and ß3 integrins.
Flow cytometry
Swiss 3T3 or WI-38 fibroblasts were detached with EDTA. Live
nonpermeabilized cells were stained at 4°C with 10 µg/ml
affinity-purified anti-tTG antibody or mAbs against 5ß1,
Vß3 integrin or individual integrin subunits. After incubation
with secondary fluorescein-labeled IgG, cells were analyzed in FACScanTM
flowcytometer (Becton Dickinson, San Jose, CA).
Binding of Fn and 42 kDa Fn fragment to cells in suspension and
adherent cells
Binding of [125I]Fn and [125I]42 kDa Fn fragment to
intact cells in suspension was performed as described (Wu et al.,
1995).
Cycloheximide-pretreated cells were resuspended in modified Tyrode's buffer
(10 mM HEPES, 150 mM NaCl, 2.5 mM KCl, 2 mM NaHCO3, 2 mM
MgCl2, 2 mM CaCl2, 1 mg/ml BSA, 1 mg/ml dextrose, pH
7.4). Cells (2x106 in 200 µl) were incubated with 1 nM-0.5
µM [125I]Fn (sp. act. 2.5x106 cpm/µg) or
with 1 nM-1 µM [125I]42 kDa Fn fragment (sp. act.
1.6x106 cpm/µg) for 1 hour at 37°C on rotator. 5 µM
unlabeled Fn or 42 kDa fragment were used to determine nonspecific background
binding, which was subtracted from the obtained values. Before incubation with
[125I]Fn or [125I]42 kDa, some samples were pretreated
for 30 minutes with 10 µg/ml anti-tTG antibody, blocking
anti-
5ß1 integrin mAbs BMA5 (for mouse cells) or JBS5 (for human
cells) or control nonimmune IgG, which were kept in the samples during the
incubation. Cells were then layered on 0.5 ml of 20% sucrose in modified
Tyrode's buffer and centrifuged for 5 minutes at 10,000 rpm. Cell-associated
125I-radioactivity in the pellets was quantitated in a gamma
counter. All measurements were performed in triplicate.
Alternatively, binding of [125I]42 kDa Fn fragment to adherent
Swiss 3T3 transfectants plated for 2 or 72 hours in DMEM with Fn-depleted 10%
bovine calf serum (BCS) was studied as described (Sottile and Mosher,
1997). After 1 hour incubation
at 37°C, adherent cells were washed with PBS, solubilized in 1% SDS and
radioactivity was counted in a gamma counter. Nonspecific binding in the
presence of excess unlabeled 42 kDa fragment was subtracted from the obtained
values.
Measurements of Fn incorporation into deoxycholate-insoluble
matrix
105 EDTA-detached cells were plated on 6-well plates (Costar),
either untreated or pretreated for 30 minutes with 2 µM 42 kDa Fn fragment
or 20 µg/ml affinity-purified anti-tTG antibody in serum-free DMEM. In some
experiments cells were plated in the presence of 10 µg/ml blocking mAbs
BMA5 (to mouse 5ß1 integrin), JBS5 (to human
5ß1), or
2
M 110 kDa cell-binding fragment of fibronectin. The antibodies or Fn
fragments were kept in the medium during the assay. WI-38 fibroblasts were
plated for Fn assembly experiments on dishes with or without 2 ng/ml
TGFß. Three hours after plating, attached and spread cells were
supplemented with 10% Fn-depleted fetal bovine serum (FBS) or Fn-depleted BCS
and incubated with 50 nM [125I]Fn (sp. act.
0.5x106 cpm/µg) for 6-36 hours either with or without the
above-indicated proteins. Following the incubations, the cells were extracted
on ice with (1) 3% Triton X-100 in PBS with 2 mM EDTA, 0.5 mM PMSF, 0.5 mM
benzamidine, 10 µg/ml leupeptin, 10 µg/ml aprotinin; (2) 100 µg/ml
DNAse I in 50 mM TrisCl, pH 7.4, 1 M NaCl, 10 mM MnCl2; and (3) 2%
Nadeoxycholate in TrisCl, pH 8.8, 10 mM EDTA, 0.5 mM PMSF. [125I]Fn
incorporated into deoxycholate-insoluble matrix was lysed and boiled in 2% SDS
with 1% ß-mercaptoethanol and analyzed by SDS-PAGE on 5% gels without
stacking parts. To generate Fn polymers in vitro, 10 µg [125I]Fn
was treated with 0.1 unit of tTG for 30 minutes at 37°C in 50 mM TrisCl,
pH 8.0 with 5 mM CaCl2 and 100 mM NaCl. [125I]labeled
monomeric and polymeric tTG-crosslinked Fn were analyzed together with
deoxycholate-insoluble matrix Fn. [125I]Fn bands were visualized by
autoradiography, cut out and counted in a gamma counter. The total amounts of
deoxycholate-insoluble Fn (both monomer and tTG-crosslinked polymer) were
determined in three independent experiments performed with each cell type and
treatment.
Analysis of Fn matrix by immunofluorescence
2x105 Swiss 3T3 transfectants or 0.5x105
WI-38 fibroblasts were plated on glass coverslips and grown for 6-36 hours in
the media containing 10% Fn-depleted FBS or BCS. Three hours after plating,
cells were supplemented with 50 nM exogenous Fn. In some samples, 2 µM 42
kDa Fn fragment or 20 µg/ml affinity-purified anti-tTG antibody were
premixed with Swiss 3T3 or WI-38 fibroblasts in serum-free DMEM 30 minutes
before plating and then kept in the medium for the following 24-36 hours.
After the incubation, the wells were washed with PBS, fixed with 3%
paraformaldehyde and stained with 10 µg/ml rabbit anti-Fn antibody,
followed by rhodamine-conjugated secondary IgG. Cells were photographed using
Nikon Eclipse E800 microscope and Spot RT digital camera.
Visualization of the cell-surface binding sites for the 42 kDa Fn
fragment
Swiss 3T3 transfectants (vector or tTG[2], 0.5x105 cells
in 0.5 ml), were incubated in Tyrode's buffer with 25 µg/ml biotinylated 42
kDa Fn fragment for 1 hour at 37°C on rotator. Cells were extensively
washed to remove unbound 42 kDa fragment and then plated in DMEM with 10%
Fn-depleted BCS for 0-36 hours on uncoated or Fn-coated glass coverslips. To
visualize cell-bound biotinylated 42 kDa Fn fragment, fixed nonpermeabilized
cells were double-stained with rhodamine-avidin and mAb HMß1-1 (for mouse
ß1 integrins) or mAbs TG100/CUB7402 (for surface tTG), followed by
fluoresceinlabeled secondary IgG. Alternatively, cells were costained with
fluorescein-avidin and anti-Fn antibody, followed by rhodamine-labeled
secondary IgG.
To study surface distribution of tTG and Fn, cells spread for 3 hours on glass coverslips were incubated for 2-36 hours with 20 µg/ml human plasma Fn. Fixed nonpermeabilized cells were double-stained with mAbs TG100/CUB7402 and rabbit anti-Fn antibody, followed by fluorescein-labeled anti-mouse IgG and Alexa-Fluor 350-labeled anti-rabbit IgG. To analyze the relationship between surface-bound 42 kDa Fn fragment, tTG and Fn matrices, cells were costained as described above, whereas the cell-bound biotinylated 42 kDa fragment was visualized with avidin-rhodamine.
Quantitation of cell area
The outlines and cell areas of randomly chosen nonadjacent 100-120 vector
and tTG[2] Swiss 3T3 transfectants plated for 3 hours on Fn, laminin and
vitronectin were analyzed using Image-Pro Plus microscopy software (Media
Cybernetics, Baltimore, MD). The software was calibrated with an Applied Micro
Stage micrometer (Applied Image, Inc.). The accuracy of the area measurements
was confirmed with a measurement slide containing etched squares of known
dimensions.
Analysis of biosynthesis of ß1 and ß3 integrins, tTG and
Fn
WI-38 fibroblasts grown with or without TGFß were labeled for 16 hours
with 50 µCi/ml [35S]Translabel (ICN Biologicals, Irvine, CA) in
methionine, cysteine-free DMEM (ICN). Cells were washed in PBS, lyzed in RIPA
buffer and 0.2 mg (1.8x108 cpm) of cell lysates were
subjected to immunoprecipitation with mAb TS2/16 against ß1 integrin, mAb
25E11 against ß3 integrin, affinity-purified anti-tTG or anti-Fn antibody
or anti-actin mAb 1501, followed by incubation with Protein G-Sepharose (Gibco
BRL). Alternatively, following 24 hour labeling with 20 µCi/ml
[35S]Translabel, 35S-labeled Fn was immunoprecipitated
from 100 µl (0.2x108 cpm) of growth medium. The
immunoprecipitates were washed and boiled in SDS-PAGE sample buffer. The
resulting samples were run on 10% gels and 35S-labeled bands were
visualized by gel treatment with Autofluor (Amersham-Pharmacia Biotech,
Piscataway, NJ) and fluorography. 35S-labeled transfectants were
analyzed for the rates of Fn biosynthesis by immunoprecipitation of
[35S]Fn from 0.2 mg (1.3x108 cpm) of cell
lysates.
![]() |
RESULTS |
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|
|
The enhancement of matrix assembly by tTG depends on its interaction
with the 42 kDa gelatin-binding domain of Fn
We tested binding of [125I]42 kDa fragment of Fn to vector,
[tTG]2 and tTGC277S[2] transfectants in suspension
(Fig. 2A). The 42 kDa Fn
fragment bound to the transfectants expressing tTG or tTG(C277S), but not to
the cells lacking tTG. This binding was specific, concentration-dependent and
saturable with calculated Kd=7.08±1.11 nM. The
amounts of cell-bound 42 kDa fragment was proportional to the surface levels
of tTG (Table 1 and
Fig. 2B) and the estimated
number of 5ß1 integrin/tTG complexes
(2.7±0.2x105 and 1.3±0.1x105
per cell for tTG[2] and tTG[1] transfectants, respectively). The observed
binding of the 42 kDa fragment to the tTG[2] transfectants was inhibited by
preincubation of cells with the anti-tTG antibody, but not by blocking mAb
BMA5 against
5ß1 integrin or control nonimmune IgG
(Fig. 2B). Similar results were
obtained with transfectants expressing catalytically inactive tTG (data not
shown). To test whether surface expression of tTG alters binding of Fn to
cells, we determined the binding of [125I]Fn to vector and tTG[2]
transfectants (Fig. 2C). tTG
significantly enhanced the binding of [125I]Fn to the cell
surfaces, with about a 2-fold increase in the number of binding sites and a
decrease in calculated Kd from 34.0±3.2 nM for
vector-transfected cells to 20.2±1.8 nM for tTG[2] transfectants
(Fig. 2C). Preincubation of the
tTG[2] transfectants with the anti-tTG antibody or excess unlabeled 42 kDa
fragment decreased the binding of [125I]Fn to the levels observed
for the vector-expressing cells (Fig.
2C). By contrast, neither the 42 kDa fragment nor the antibody
against tTG altered the binding of [125I]Fn to the cells lacking
tTG, therefore proving the specific role of surface tTG in the enhanced Fn
binding (Fig. 2C).
|
We also studied binding of the 42 kDa fragment of Fn to adherent
transfectants plated for 2 hours and to confluent monolayers of transfectants
grown for 72 hours (Fig. 2D).
tTG[2] and tTGC277S[2] transfectants plated on culture dishes for 2
hours bound the 42 kDa fragment, although the binding was decreased by
10-15% compared with that for the same cell populations in suspension
(Fig. 2A,D). By contrast,
regardless of tTG expression, no specific binding of this Fn fragment was
observed to the monolayers of transfectants cultured for 72 hours. The lack of
binding of the 42 kDa fragment to the monolayers can be explained by blocking
the Fn-binding site on surface tTG with the excess of secreted Fn and
inability of this fragment to interact with Fn (Sottile and Mosher,
1997
).
The 42 kDa Fn fragment and the anti-tTG antibody were used in Fn assembly
assays with the transfectants (Fig.
3). Neither reagent had a significant effect on the amounts of
[125I]Fn incorporated into the deoxycholate-insoluble fraction in
vector-transfected cells (Fig.
3A,B). By contrast, both treatments markedly decreased Fn assembly
by tTG[2] transfectants to the levels characteristic for the
vector-transfected cells (Fig.
3A,B). In control experiments, the use of blocking
anti-5ß1 integrin mAb BMA5 or excess 110 kDa integrin-binding Fn
fragment strongly suppressed matrix formation regardless of tTG expression
(Fig. 3A,B). Notably,
treatments with anti-tTG or blocking anti-
5ß1 integrin antibodies,
as well as with the 42 kDa or the 110 kDa fragments, decreased both the
amounts of deoxycholate-insoluble Fn monomers and the crosslinked Fn multimers
assembled by the tTG[2] transfectants. However, these treatments did not alter
the ratio between the two forms of deoxycholate-insoluble Fn (
6:1 to
10:1, monomer to multimers). This suggests that the Fn crosslinking
activity of tTG is separate and independent from its ability to bind Fn. When
the formation of Fn matrices was tested by immunostaining, the 42 kDa fragment
and the anti-tTG antibody inhibited fibril formation by the transfectants
expressing tTG or tTG(C277S), but not by the cells lacking tTG
(Fig. 3C).
|
Several studies documented that the 70 kDa N-terminal fragment of Fn
I1-6II1,2I7-9 binds to cells (McKeown-Longo
and Mosher, 1985; Christopher
et al., 1997
). This binding was
primarily attributed to its 29 kDa N-terminal region consisting of modules
I1-5, but not to its other region, the 42 kDa fragment containing
modules I6II1,2I7-9 (McKeown-Longo and
Mosher, 1985
; Quade and
McDonald, 1988
; Hocking et
al., 1998
). To re-examine the
role of N-terminal (I1-5) and gelatin-binding
(I6II1,2I7-9) domains of Fn in matrix
assembly depending on the presence of surface tTG and to reconcile these
previous data with our findings, we tested the effects of the 29 and 42 kDa
fragments on Fn matrix formation (Fig.
4). In vector transfectants, the 29 kDa fragment inhibited the
incorporation of [125I]Fn into deoxycholate-insoluble pool
(IC50
130 nM), decreasing it in a concentration-dependent manner
to
35-40% of the control. Yet, the 42 kDa fragment displayed no
inhibitory effect nor did it potentiate the effect of the 29 kDa fragment in
these cells lacking tTG (Fig.
4A). By contrast, the 29 kDa fragment had only a weak effect on
the higher level of matrix-incorporated Fn in cells expressing surface tTG,
decreasing it by less than 20% (Fig.
4B). However, the 42 kDa Fn fragment inhibited by
70-75% the
incorporation of Fn into the deoxycholate-insoluble pool by the tTG[2]
transfectants (IC50
12 nM). This potent inhibition of matrix
assembly by the 42 kDa fragment in these transfectants was only slightly
increased by combining it with the 29 kDa fragment
(Fig. 4B). Therefore, the 29
kDa and 42 kDa fragments have dissimilar effects on Fn assembly with the 42
kDa fragment disrupting association of Fn with surface tTG.
|
Binding sites for the 42 kDa gelatin-binding Fn fragment colocalize
with surface tTG and Fn fibrils during matrix assembly
We visualized binding sites for the 42 kDa Fn fragment on the surface of
the transfectants (Fig. 5). No
specific binding of this Fn fragment was observed to cells lacking tTG
(Fig. 5A,C). When tTG[2]
transfectants preincubated with the 42 kDa Fn fragment were plated on
Fn-coated coverslips, cell-bound 42 kDa fragment codistributed with ß1
integrins at focal adhesions on the ventral cell surfaces
(Fig. 5A). This is in agreement
with the localization of surface tTG at focal adhesions in cells plated on Fn
(Akimov et al., 2000). When Fn
was added for 2 hours to the tTG[2] transfectants spread on glass coverslips,
surface tTG was colocalized with nascent Fn fibrils on the dorsal surfaces
(Fig. 5B, left panels, arrows).
In the tTG[2] transfectants plated on coverslips for 12 hours, the 42 kDa
fragment of Fn codistributed with both ß1 integrins and clusters of
surface tTG at the dorsal surfaces of cell monolayers
(Fig. 5B, middle and right
panels). An extensive colocalization of surface tTG with Fn matrices was
detected after 36 hour incubation of tTG[2] transfectants with exogenous Fn
(Fig. 5D). The cell-bound 42
kDa fragment still could be detected at this timepoint of assembly and
remained colocalized with surface tTG. At the same time, clusters of newly
synthesized surface tTG not associated with bound 42 kDa fragment coincided
with most prominent clusters of Fn fibrils assembled on the dorsal surfaces of
cell monolayers (Fig. 5D,
arrows). Therefore, surface tTG accumulates at the sites where Fn fibrils
start to form and remains codistributed with Fn matrices during the assembly
process.
|
Stimulation of Fn assembly by tTG is separate from its effects on
cell spreading
Integrin-bound surface tTG enhances cell spreading on Fn via interaction
with its gelatin-binding domain (Akimov et al.,
2000). To distinguish
stimulation of Fn assembly by tTG from the effects of tTG on cell spreading,
we plated vector and tTG[2] transfectants on vitronectin- and laminin-coated
surfaces for 3 hours (Fig. 6). As neither tTG nor
5ß1 interact with vitronectin or laminin, they
remained dispersed throughout the cell surface, whereas integrins other than
5ß1 were involved in adhesion, spreading and focal contact
formation. A 6 hour incubation of these cells with Fn induced co-clustering of
5ß1 integrin and tTG with growing Fn fibrils on the dorsal cell
surfaces (Fig. 5B and data not
shown) and led to enhanced deposition of Fn matrices by tTG[2] transfectants
(Fig. 6A,B). However, no
difference in the extent of cell spreading was found for these transfectants
adherent on vitronectin or laminin (Fig.
6C). Thus, the effects on Fn assembly are separate from the
ability of surface tTG to promote cell spreading.
|
TGFß induces tTG association with ß1 integrins and
increases surface expression of tTG
To search for a physiologically relevant model of Fn assembly involving
tTG, we employed WI-38 human lung fibroblasts that express cell-surface tTG
(Upchurch et al., 1991) and
adhere on the 42 kDa Fn fragment (Akimov et al.,
2000
). Using metabolic labeling
and immunoprecipitation, we tested whether treatment of WI-38 fibroblasts with
TGFß altered expression of integrins, tTG and Fn
(Fig. 7A). Synthesis of
1,
5,
V, ß1 or ß3 integrin subunits was
slightly enhanced following this treatment, whereas the amount of synthesized
actin was unaffected by TGFß (Fig.
7A). TGFß moderately (
1.3-2.2-fold) elevated the amounts
of tTG synthesized by WI-38 fibroblasts
(Fig. 7A) and the levels of Fn
biosynthesis and secretion (Fig.
7A,B). By contrast, the amounts of tTG associated with
5ß1 integrin increased markedly
(Fig. 7C). This effect was
specific for ß1 integrins, as a slight decrease in tTG compex formation
with
Vß3 integrin was observed
(Fig. 7C). In parallel, flow
cytometry confirmed a drastic
3-fold increase in the surface levels of
tTG following a 48 hour treatment of cells with TGFß, whereas the levels
of
5, ß1 subunits or
5ß1 and
Vß3 integrins
remained similar or raised insignificantly
(Table 2). Notably, the amounts
of surface tTG increased
1.5-2-fold 2 hours after stimulation with
TGFß and this rapid increase could not be blocked by cycloheximide (data
not shown). These data indicate that TGFß specifically stimulates the tTG
interaction with ß1 integrins and tTG expression on the cell surface.
|
|
Cell-surface tTG is involved in the TGFß-mediated increase of Fn
assembly
We studied the effects of TGFß on the binding of [125I]42
kDa Fn fragment and [125I]Fn to WI-38 fibroblasts in suspension
(Fig. 8A,B). Treatment with
TGFß markedly enhanced the binding of the 42 kDa fragment to the cell
surfaces, whereas the antibody against tTG abolished the binding of the 42 kDa
fragment to the cells regardless of the TGFß treatment
(Fig. 8A). Accordingly, the
treatment with TGFß increased, although to a lesser extent, the binding
of Fn to fibroblasts in suspension and this increase was again blocked by the
anti-tTG antibody (Fig.
8B).
|
Analysis of [125I]Fn incorporation into the
deoxycholate-insoluble pool of WI-38 fibroblasts showed a sharp increase in Fn
assembly in response to TGFß (Fig.
9A,B). Coincubation of cells with unlabeled 42 kDa fragment or
anti-tTG antibody suppressed the Fn assembly by 3-3.5-fold in the case of
untreated cells. When the same treatments were applied to the cells grown with
TGFß, they caused an even more potent
8-fold inhibition of
[125I]Fn incorporation into the deoxycholate-insoluble fraction,
abolishing the entire stimulatory effect of the cytokine
(Fig. 9A,B). Yet, regardless of
the levels of surface tTG, the assembly of Fn was entirely dependent on
integrin-Fn interaction, as blocking anti-
5ß1 integrin mAb and the
110 kDa integrin-binding Fn fragment caused a strongest inhibition of matrix
formation (Fig. 9A,B).
Immunostaining for Fn revealed a strong increase in fibril formation by
TGFß-treated WI-38 fibroblasts (Fig.
9C). Again, the 42 kDa fragment or the anti-tTG antibody inhibited
Fn fibrillogenesis by the untreated cells and even more so by the cells grown
with TGFß (Fig. 9C).
Therefore, surface tTG is involved in the TGFß-mediated increase of Fn
assembly by WI-38 fibroblasts.
|
![]() |
DISCUSSION |
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We employed a noncatalytic mutant of tTG, tTG(C277S), which binds integrins
and promotes cell adhesion and spreading on Fn (Akimov et al.,
2000). The catalytically
inactive tTG stimulates Fn incorporation into deoxycholate-insoluble matrix as
efficiently as the wild-type protein, indicating that the effects of surface
tTG on Fn polymerization do not require its crosslinking activity, although
such activity leads to a production of deoxycholate-insoluble nonreducible Fn
multimers. The use of the tTG(C277S) mutant allowed us to distinguish the tTG
effects on Fn polymerization from the tTG-dependent covalent crosslinking of
Fn and led us to conclude that integrin-bound surface tTG promotes these
processes independently.
The interaction of surface tTG with the 42 kDa gelatin-binding domain of Fn
mediates cell adhesion and limited spreading on this part of Fn (Akimov et
al., 2000). In this study, we
found that the 42 kDa Fn fragment binds to cells and this binding strictly
depends on the presence of surface tTG. Two lines of evidence suggest a role
for interaction between cell-surface tTG and the 42 kDa fragment in
cell-mediated assembly of Fn fibrils. First, increased binding of this Fn
fragment correlates with the stimulation of binding of whole Fn to cells
expressing surface tTG. Second, competition experiments with excess 42 kDa
fragment showed its potent inhibition of Fn matrix formation in the
tTG-expressing cells, whereas the lower level of assembly observed in cells
lacking tTG was unaffected. Notably, to efficiently decrease Fn assembly by
the tTG transfectants and WI-38 fibroblasts, the excess 42 kDa fragment or
anti-tTG antibody had to be added to the cells prior to plating and kept in
the medium during the assay. Thus, most of the inhibitory effect was due to
competition with binding of exogenous Fn to the surface tTG. By contrast, very
little or no binding of the 42 kDa fragment to monolayers of confluent
transfectants expressing tTG was observed due to blocking surface tTG by Fn
present in the medium. In addition, exogenous tTG did not promote Fn
polymerization (data not shown); this was probably due to its inability to
bind to the cell surface. Together, our data indicate that tTG stimulates
initiation of assembly rather than elongation of Fn fibrils.
Surprisingly, we found that 29 kDa N-terminal Fn fragment I1-5
had little effect on matrix assembly in cells expressing surface tTG, whereas
in agreement with previous reports it strongly inhibited the lower level of
assembly in cells lacking tTG (McKeown-Longo and Mosher,
1985; Sottile et al.,
1991
; Sottile and Mosher,
1997
). This suggests that
there are at least two modes of incorporation of Fn into the
deoxycholate-insoluble pool, utilizing two distinct sites within the 70 kDa
N-terminal fragment of Fn. One of them, located within the 29 kDa Fn fragment,
is critical for matrix formation by a wide variety of cell types and may
involve a direct interaction of this part of Fn with matrix assembly receptor
(Mosher et al., 1992
), which
probably represents
5ß1 integrin (Hocking et al.,
1998
). The other, within the
42 kDa gelatin-binding fragment, binds to cell surfaces and plays a role in Fn
polymerization only in cells expressing tTG on their surface. Dual interaction
of incoming Fn protomer with integrins via the III9III10
modules and the N-terminal 29 kDa domain might be required for `activation' of
Fn protomers for polymerization, likely via exposure of cryptic self-assembly
site(s) such as the one located within the III1 module (Ingham et
al., 1997
; Zhong et al.,
1998
). An inefficient
inhibition of Fn assembly by the 29 kDa Fn fragment in cells expressing tTG
suggests that high-affinity binding of tTG to the gelatin-binding domain of Fn
can substitute for interaction between integrin (or other hypothetical Fn
assembly receptor) and the 29 kDa N-terminal domain of Fn in the `activation'
of cell-bound Fn protomers. Thus, previous findings on the inability of the 42
kDa fragment to interfere with matrix assembly might be explained by the low
levels or complete absence of tTG on the surface of the cells employed
(Schwarzbauer, 1991
; Sottile
and Mosher, 1997
). The
observed stimulation of Fn assembly by surface tTG is due primarily to
increased binding of Fn to cell surfaces and therefore tTG likely promotes the
initial stages of Fn fibrillogenesis. This is in agreement with our
localization data showing tTG codistributed with emerging Fn fibrils on the
cell surface at early timepoints of matrix assembly. Notably, for surface tTG,
its stimulatory effect on Fn assembly exceeded that on binding of Fn to the
cell surfaces. This suggests that, besides initiation of assembly, tTG
promotes elongation of Fn fibrils. Therefore, the tTG-mediated pathway of Fn
matrix formation may also include an exposure of cryptic self-assembly sites
in Fn protomers (Zhong et al.,
1998
), driven by enhanced
formation of focal adhesions and stress fibers (Akimov et al.,
2000
). Although association
with tTG may also affect the activation state of integrins important for Fn
assembly (Wu et al., 1995
), we
could not detect tTG-dependent changes in integrin affinity for the
RGD-containing Fn fragment (data not shown). Together, our results indicate a
previously unrecognized mode of Fn assembly, which involves a high-affinity
interaction of surface tTG with the gelatin-binding domain of Fn. Surface tTG
is unable to efficiently polymerize Fn by itself, but its coreceptor function
in matrix assembly depends on integrin-Fn interaction and, possibly, on the
formation of ternary integrin-tTG-Fn complexes (Akimov et al.,
2000
).
On the basis of these observations, we proposed that modulation of surface
tTG by growth factors and cytokines might contribute to alterations in the
assembly of Fn matrix. Several studies reported that the multifunctional
growth factor TGFß is a potent stimulator of Fn synthesis and assembly
(Allen-Hoffmann et al., 1988;
Roberts et al., 1988
). It also
enhances tTG expression in some cells (George et al.,
1990
), likely due to the
presence of a TGFß response element in the tTG gene (Ritter and Davies,
1998
). We found that treatment
of fibroblasts with TGFß markedly increases the association of tTG with
ß1 integrins and surface expression of tTG. Our data suggest that
TGFß promotes tTG externalization and/or retention on the cell surface,
causing a rapid and sustainable increase in its surface expression and the
number of Fn-binding sites. Thus, an earlier reported effect of TGFß on
Fn binding and assembly (Allen-Hoffmann et al.,
1988
) might involve the
elevation of surface tTG. A strong inhibition of Fn assembly in
TGFß-treated cells by the 42 kDa Fn fragment or anti-tTG antibody
suggests that the enhanced expression of integrin-bound surface tTG strongly
contributes to increased matrix formation mediated by TGFß. tTG is
involved in activation of latent TGFß (Kojima et al.,
1993
), and
Vß6 and
Vß1 integrins bind and activate latent TGFß (Munger et al.,
1998
; Munger et al.,
1999
). This suggests a
positive feedback loop between tTG and TGFß production by cells and,
together with the results presented here, implies a cooperation between
integrin-tTG complexes and latent TGFß on the cell surface. This
functional collaboration among tTG, integrins and TGFß can play an
essential role in dermal wound healing in vivo, a process in which tTG has
been shown to participate (Haroon et al.,
1999
), as well as in fibrotic
disorders accompanied by an increased deposition of ECM. The data presented in
this study provide a novel explanation for the well-documented effects of
TGFß on Fn assembly and suggest a previously uncharacterized pathway of
Fn polymerization that involves interaction of surface tTG with the
gelatin-binding region of Fn.
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ACKNOWLEDGMENTS |
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