1 Department of Medicine-Vascular Medicine Unit, University of Rochester School
of Medicine and Dentistry, Rochester, NY 14642, USA
2 Department of Pathology and Laboratory Medicine, University of Rochester
School of Medicine and Dentistry, Rochester, NY 14642, USA
3 Department of Pharmacology and Physiology, University of Rochester School of
Medicine and Dentistry, Rochester, NY 14642, USA
4 Center for Cardiovascular Research, University of Rochester School of Medicine
and Dentistry, Rochester, NY 14642, USA
5 Department of Microbiology and Immunology, University of Rochester School of
Medicine and Dentistry, Rochester, NY 14642, USA
Author for correspondence (e-mail: pj_simpsonhaidaris{at}urmc.rochester.edu)
Accepted 24 October 2001
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Summary |
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Key words: Fibrinogen, Fibronectin, Heparan sulfate proteoglycans, Extracellular matrix, Lysophasphatidic acid, RhoA GTPase, Wound repair
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Introduction |
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The extracellular matrix (ECM) is composed of two main classes of
macromolecules: proteoglycans and adhesive glycoproteins
(Teti, 1992). Some common
adhesive proteins found in the ECM or basement membrane include FN, collagen
and laminin. Although these proteins differ in primary structure, they each
display functional motifs that contribute to their adhesive properties for
cells and other proteins, as well as to the ability to organize into fibrillar
structures (Engel, 1991
). The
ECM provides structure and elasticity for tissues, compartmentalizes different
cell types and serves as a reservoir for growth factors by sequestering and
protecting them in the microenvironment (Mosher, 1992). It is clear that the
functions of the ECM are not exclusively structural. The ECM is a dynamic
environment that elicits distinct cellular phenotypes. ECM constituents
interact with specific adhesion receptors on cell surfaces and regulate
multiple cell functions, including adhesion, migration, proliferation and
differentiation.
FBG is typically considered a soluble plasma protein produced by
hepatocytes. However, extrahepatic synthesis of intact FBG occurs in
epithelial cell lines from the intestine (Molmenti, 1993), the cervix
(Lee et al., 1996), the lung
(Simpson-Haidaris, 1997
) as
well as in lung alveolar epithelial cells (Simpson-Haidaris, 1998). We found
that FBG expression is upregulated 5-10 fold in a lung epithelial cell line
(A549) following induction with the proinflammatory mediators of FBG
gene expression during the acute phase response
(Simpson-Haidaris, 1997
). In
addition, A549 cells synthesize and secrete FBG basolaterally
(Guadiz et al., 1997a
), which
becomes incorporated into detergent-insoluble matrix fibrils, independently of
either thrombin or plasmin enzymatic action
(Guadiz et al., 1997b
). FBG
assembled into the ECM is conformationally altered to expose a cryptic epitope
on the Bß chain (Guadiz et al.,
1997b
). This epitope falls within residues ß15-42 that
constitutes the neo-N-terminus of the fibrin ß chain, as well as the
heparin binding domain (HBD), which is exposed after thrombin cleavage
(Odrljin, 1996a
;
Odrljin et al., 1996b
). FBG
synthesized by lung epithelial cells or plasma FBG exogenously added to
fibroblast monolayers is assembled into matrix fibrils that colocalize with
FN, heparan sulfate proteoglycans (HSPG), collagen IV, and laminin
(Guadiz et al., 1997b
). Other
matrix glycoproteins such as fibulin-1 and tenascin-C colocalize with FN in
the ECM (Chung and Erickson,
1997
; Chung et al.,
1995
; Godyna et al.,
1995
). Because a FN matrix is required for assembly of fibulin-1
and tenascin-C into the ECM (Chung and
Erickson, 1997
; Godyna et al.,
1995
), we hypothesized that the deposition of FBG into the ECM is
also dependent on the presence of a FN matrix. In this study, we determine
whether FN plays a role in mediating the assembly of FBG into matrix
fibrils.
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Materials and Methods |
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Protein purification
Human plasma FN was purified as previously described
(McKeown-Longo and Etzler,
1987). Human FBG was purchased from CalBiochem (San Diego, CA) and
further purified by lysine-Sepharose affinity chromatography to remove
contaminating plasminogen, followed by gelatin-Sepharose affinity
chromatography in tandem with an anti-human FN-Sepharose affinity column to
remove contaminating FN. Removal of detectable FN was confirmed by western
blot and enzyme linked immunosorbent assay. FBG was labeled with the
fluorophore Oregon-GreenTM (Molecular Probes, Eugene, OR) as described
(Odrljin et al., 2001
); the
resulting conjugate was designated FBG-Oregon Green.
Immunofluorescent detection
HFF and FN-null cells were seeded on round glass coverslips, grown to
confluence and treated further as described in the figure legends. Rabbit
anti-human FN antibody (Sigma, St. Louis, MO), which was affinity purified
over FBG-Sepharose to remove contaminating antibodies to FBG, was used at 5-10
µg/ml. Rabbit anti-human FBG antibody (Dako Corp., Carpenteria, CA) was
purified to remove contaminating antibodies to serum proteins and FN as
previously described (Simpson-Haidaris,
1997) and used at 40 µg/ml. Monoclonal antibody (MoAb) against
human FN (Sigma) was used at 70 µg/ml. MoAb against heparan sulfate (mouse
IgM) (Seikagaku America, Falmouth, MA) was used at 50 µg/ml. Secondary
antibodies were fluorescein- or rhodamine-conjugated goat anti-rabbit antibody
(Cappel, Durham, NC) and rhodamine-conjugated goat anti-mouse polyvalent Ig
antiserum (Chemicon, Temecula, CA). FBG-Oregon Green was detected by direct
epifluorescence. Microscopy was carried out with a Nikon Eclipse E800
phase-contrast microscope equipped with single and dual band filters for
epifluorescence. A cooled color digital camera, the Spot II from Diagnostic
Instruments (Sterling Heights, MI) and a Hewlett Packard Pentium III computer
with color monitor were used to capture images. Analysis was carried out using
IP Lab image analysis software (Scanalytics Inc, Fairfax, VA).
Lysophosphatidic acid, phospholipase B and C3 transferase
treatments
Lysophosphatidic acid (LPA), phospholipase B (PLB) and Clostridium
botulinum C3 transferase were purchased from Sigma. HFF were grown to
confluence on gelatin coated glass coverslips, and 24 hours prior to the
addition of 200 or 500 nM LPA (Checovich
and Mosher, 1993; Zhang et
al., 1997
) the cells were deprived of serum. After 24 hours of
serum deprivation, medium containing LPA, but no FBS, or a range of 0.5% to
10% FBS was supplemented with 40 µg/ml FBG-Oregon Green then added to the
cells and incubated for an additional 18 hours. Confluent HFF were
serum-starved for 24 hours and then treated for an additional 24 hours with 1%
serum-containing medium supplemented with 40 µg/ml FBG-Oregon Green in the
absence or presence 0.1 U/ml PLB, which specifically hydrolyzes LPA
(Checovich and Mosher, 1993
;
Zhang, 1997). To inhibit Rhomediated signaling, C3 transferase (2 µg/ml)
was incubated with LipofectAMINE (Life Technologies) (10 µg/ml) for 30
minutes at room temperature before application in serum-free medium to 24-hour
serum starved HFF and incubated for one hour at 37°C
(Wenk et al., 2000
;
Zhang et al., 1997
;
Zhong et al., 1998
). After
this, the HFF were incubated for 4 hours with 30 µg/ml FBG-Oregon Green in
medium containing 1% serum to induce Rho activation.
MoAb 9D2 modulation of FBG and FN matrix assembly
Murine anti-human FN MoAb 9D2, which recognizes an epitope on FN's type
III-1 module, was employed to inhibit FN-FN self association, a critical step
in the assembly of a FN matrix (Chernousov
et al., 1991). To determine whether inhibition of FN assembly in
HFF-modulated deposition of FBG into the ECM, confluent HFF were treated with
9D2 (30 µg/ml) for 18 hours. This was followed by incubation for an
additional 24 hours with 30 µg/ml FBG-Oregon Green in the continued
presence of 9D2. To determine whether the inhibitory effect of 9D2 was
reversible, after 18 hours, another set of coverslips were washed three times
with medium to remove unbound 9D2 and incubated with FBG-Oregon Green for an
additional 24 hours. FN-null cells were grown to confluence on vitronectin
coated glass coverslips and incubated with FBG (20 µg/ml) and FN (25.8
µg/ml) in the presence of 35 µg/ml of either 9D2 or nonimmune mouse
IgG1 (Sigma) for 24 hours.
Cell binding assays
Iodination of FN and FBG was performed using Iodo-Gen® Reagent
according to the manufacturer's protocol (Pierce, Rockford, IL). Specific
activities of 125I-FN and 125I-FBG were 0.19 mCi/mg
(4.2x108 cpm/mg) and 2.2 mCi/mg (48.9x108
cpm/mg), respectively. Binding studies were performed as previously described
(Chernousov et al., 1991) on
confluent monolayers of FN-null cells grown in 48-well plates. To determine
the total amount of labeled ligand associated with the cells, the washed cell
monolayers were solubilized in 1N NaOH. Nonspecific binding of
125I-FN was measured in the presence of 500 µg/ml unlabeled FN.
Nonspecific binding was subtracted from total binding to obtain specific
binding. Scatchard analysis was performed using the software package
Equilibrate version 1.2.16 available as freeware from
http://equilibrate.homestead.com/files.
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Results |
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|
FBG assembly into the ECM requires LPA
LPA, at least in part, regulates assembly of FN into ECM fibrils
(Checovich and Mosher, 1993;
Zhang et al., 1997
).
Therefore, we sought to determine whether LPA signaling was required to
support FBG deposition into the ECM. Confluent monolayers of HFF were
serum-starved for 24 hours then medium containing either 10% FBS
(Fig. 2A,B), no FBS
(Fig. 2C,D) or 500 nM LPA
(Fig. 2E,F), each supplemented
with 40 µg/ml FBG-Oregon-GreenTM, was added to cells and incubated for
an additional 18 hours. The results show that in serum starved cells, very
little deposition of FBG into ECM occurred
(Fig. 2C) compared to the 10%
serum condition (Fig. 2A). The
FBG that was deposited in the matrix of starved cells appeared in short
fibrils or in patches on cell surfaces
(Fig. 2C). In the presence of
500 nM LPA in place of FBS, fibrillar FBG assembly was partially restored
(Fig. 2E).
|
Because HSPG play a role in the assembly of FN in the ECM (Bultmann, 1998;
Chung and Erickson, 1997;
Hocking et al., 1999
;
Klass et al., 2000
;
Sottile et al., 2000
), we
analyzed the colocalization of FBG with HSPG in the ECM in the presence or
absence of serum. The results show that FBG colocalized with HSPG matrix
fibrils in the presence of 10% FBS (Fig. 2A
compared with 2B). Although serum starvation significantly reduced
assembly of FBG into matrix fibrils, significant levels of HSPG
(Fig. 2C compared with 2D) that
were pre-established into matrix fibrils over seven days of cell growth
remained after serum deprivation. LPA treatment of serum starved HFF resulted
in restoration of FBG assembly in thick matrix fibrils that colocalize with
HSPG fibrils (Fig. 2E compared with
2F). Moreover, the data indicate that the presence of HSPG in the
pre-established ECM was not sufficient to support assembly of FBG into matrix
fibrils in the absence of serum stimulation.
PLB specifically hydrolyzes LPA, the component in serum that induces
incorporation of FN into the ECM (Checovich
and Mosher, 1993; Zhang et
al., 1997
). To confirm that LPA is one of the major serum
components responsible for signaling to HFF to assemble FBG in their matrix,
confluent HFF were serum starved then treated with 1% FBS containing medium
with or without 0.1 U/ml PLB during the subsequent 24 hour incubation with
FBG-Oregon Green. FBG-Oregon Green in the presence of 1% serum assembled into
fibrils in the ECM (Fig. 3B);
however, in the presence of PBL-treated 1% serum containing medium, little or
no incorporation of FBG-Oregon Green into matrix fibrils was observed
(Fig. 3A). These data indicate
that LPA is a major serum component responsible for outside-in signaling
during FBG assembly into a fibrillar ECM.
|
Inhibition of Rho activation by C3 transferase reduced FBG assembly
into matrix fibrils
LPA has dramatic effects on actin polymerization, stress fiber formation
and focal adhesion assembly (Hall et al.,
1993; Nobes and Hall,
1995
). Activation of the small GTPases, Rho and Rac, by LPA plays
a role in mediating these cellular events. C3 transferase, a bacterial toxin
from Clostridium botulinum, is used to inhibit Rho-mediated signal
transduction; C3 transferase treatment reduces FN binding to cell surfaces and
subsequent assembly into a fibrillar matrix by inhibiting Rho-mediated cell
contractility (Zhang et al.,
1997
; Zhong et al.,
1998
). Therefore, 24-hour serum starved cells were treated with C3
transferase for 1 hour to inhibit Rho activation to determine whether such
treatment prevents or reduces the subsequent assembly of FBG into mature
matrix fibrils in the presence of serum containing medium. The results show
that treatment of HFF with C3 transferase
(Fig. 3C) for 1 hour partially
inhibited both the amount of FBG deposited and the extent of FBG fibril
elongation achieved in the presence of 1% serum containing medium
(Fig. 3D). In the presence of
C3 transferase (Fig. 3C), the
FBG appeared in patches, which suggests cell surface binding, and in short
stitch-like fibers instead of the thicker and longer fibrils associated with
mature matrix. A quantitative analysis of the relative fluorescence of matrix
fibrils from three experiments revealed that 1 hour of C3 transferase
treatment reduced the amount of FBG deposited from 1% serum-containing medium
into the matrix by an average of 38.4±14%. Taken together, these data
suggest that LPA is the major serum component that induces Rho-dependent
signaling to permit assembly of FBG into the ECM.
Active FN matrix assembly is required for FBG incorporation into
matrix
To determine whether active FN assembly plays a role in FBG assembly into
the ECM, we examined whether inhibition of FN matrix assembly would alter FBG
incorporation into the ECM. Deposition of FN into the ECM of HFF was inhibited
by the anti-human FN MoAb 9D2, which inhibits assembly of FN into the matrix
but not the initial binding of FN to the cell surface
(Chernousov et al., 1991).
Following pretreatment with 30 µg/ml 9D2 for 18 hours, confluent HFF were
incubated with 30 µg/ml FBG-Oregon Green in the continued presence of 9D2
for 24 hours. At each time point analyzed in the presence of 9D2, matrix FN
was predominantly organized into short, linear arrays
(Fig. 4, panels B,D,F) compared
with FN fibrils found over the same time course in the absence of 9D2
(Fig. 1B,D,F). These results
suggested that extensive fibrillar formation was reduced by MoAb 9D2; the FN
fibrils remaining are probably those pre-established in the matrix of HFF
prior to 9D2 treatment. In the presence of 9D2, FBG incorporation into the ECM
was negligible at all time points (Fig.
4A,C,E) compared to the amount of FBG assembled into matrix
fibrils in the absence of 9D2 (Fig.
1A,C,E). The results suggest that 9D2 inhibition of ongoing FN
matrix assembly prohibited the deposition of FBG into the matrix of HFF. To
test whether removal of 9D2 would allow for recovery of FN and FBG deposition
into the matrix, HFF, which were treated as described above, were washed to
remove 9D2 from the medium and further incubated in the presence of FBG-Oregon
Green for 24 hours. The restoration of both FN and FBG assembly into matrix
after removing 9D2 from the medium was observed as changing from short
stitch-like fibers at 1 hour (Fig.
4G,H) to thicker, longer and more branched fibrils by 24 hours
(Fig. 4K,L). However, the
restoration of FBG fibril formation was not as robust 24 hours after washing
out 9D2 as at 24 hours in the absence of 9D2
(Fig. 4K compared with
Fig. 1E). This is probably due
to the residual 9D2 bound to FN at the surface of the cells. Nonetheless, the
data suggest that cell-associated contractility mediated by ongoing FN
polymerization (Hocking, 2000) is only transiently inhibited by the presence
of 9D2.
|
To further test whether there is an absolute dependence on active FN matrix
assembly for FBG incorporation into the ECM, the following experiments were
performed using mouse embryonic FN-null cells. These cells synthesize and
deposit HSPG into the ECM, which is required for assembly of a FN matrix
(Sottile et al., 2000) but do
not synthesize or secrete endogenous FN. Nonetheless, they assemble
exogenously added human FN into mature matrix fibrils
(Sottile et al., 1998
). In the
presence of added FN (Fig. 5A),
FBG was incorporated into matrix fibrils
(Fig. 5B). However, in the
absence of FN (Fig. 5D), FBG
was not incorporated into the ECM of the FN-null cells
(Fig. 5E). Phase contrast
images (Fig. 5C,F) demonstrate
that the FN-null cells remained as a confluent monolayer during the assembly
of FN and FBG into the ECM. These data provide evidence that assembly of a FN
matrix plays a critical role in the incorporation of FBG into matrix
fibrils.
|
Kinetics of FN binding to FN-null cells
To determine whether human FN bound to mouse embryonic FN-null cells with
the same affinity as human FN for human fibroblasts, we characterized the
binding of 125I-FN to the null cells. FN-null cells were incubated
with increasing amounts of 125I-FN with or without an excess of
unlabeled FN for 1 hour at 37°C as described previously
(Chernousov et al., 1991). The
results indicate that specific binding of FN to the cell surface of FN-null
cells was both dose dependent and saturable, implying a receptor-mediated
binding event (Fig. 6).
Scatchard analysis of the data indicates that human FN protomers bind to the
mouse embryonic FN-null cells with an average Kd of 98 nM; human FN
was shown previously to bind to human fibroblasts with an average
Kd of 62 nM (McKeown-Longo and
Mosher, 1989
).
|
9D2 inhibition of FBG binding to FN-null cells
Previous studies show that MoAb 9D2 does not interfere with the initial
binding of FN to the cell surface matrix assembly sites, instead 9D2 inhibits
FN elaboration into a fibrillar matrix by blocking FN-FN homotypic binding
interactions (Chernousov et al.,
1991). To analyze whether FBG assembly into the ECM was dependent
on the polymerization of FN, we utilized MoAb 9D2 to halt FN-FN homotypic self
association. FN-null cells were treated with increasing concentrations of 9D2
in the presence of constant amounts of 125I-FBG and nonlabeled FN.
Exogenous FN was added to the cells to allow FN binding to the cell surface
matrix assembly sites. Control experiments demonstrated that, in the presence
of nonlabeled FBG, increasing concentrations of 9D2 did not significantly
alter the binding of iodinated FN to the FN-null cells (not shown). In
contrast, MoAb 9D2 inhibited, either directly or indirectly, the association
of 125I-FBG to FN-null cells by 60%
(Fig. 7). Nonimmune mouse
IgG1 had no significant effect on the binding of
125I-FBG to the FN-null cells
(Fig. 7). These data indicate
that FBG assembly into the ECM was dependent on FN-FN self-association for FN
fibril elaboration, a step that is also dependent on Rho-mediated cell
contractility (Zhang et al.,
1997
; Zhong et al.,
1998
).
|
To visualize the inhibitory effects of MoAb 9D2 on FN and FBG fibril elongation, FN-null cells were treated with and without 9D2 or nonimmune IgG1 in the presence of exogenous FN and FBG for 24 hours. MoAb 9D2 effectively inhibited matrix fibril formation of FBG (Fig. 8D) and FN (Fig. 8E), as compared with cells that received no treatment (Fig. 8A,B) and those treated with nonimmune IgG1 (Fig. 8G,H). In the presence of 9D2, both FBG and FN bound to the cell surfaces and between cells (Fig. 8D-F). The effects of 9D2 on fibril formation are shown most dramatically by the dual fluorescence images (Fig. 8C,F,I). In the control condition and with cells treated with nonimmune IgG1, significant colocalization of FBG and FN fibrils is demonstrated by the yellow-orange fluorescence (Fig. 8C,I, respectively). However, 9D2 treatment reduced FN and FBG `cofibril' formation as denoted by the dramatic reduction of yellow-orange fluorescence; only a few short stitch-like fibrils of both FN and FBG are observed in this representative field (Fig. 8F). Furthermore, residual FBG staining is revealed in green fluorescence, whereas little to no red fluorescence, which is indicative of only FN staining, was observed. These data suggest that FBG binds to cell surface and cell-cell contact sites other than cell surface FN matrix assembly sites (Fig. 8F). Together, these data support the hypothesis that assembly of FBG into complex matrix fibrils is dependent on the active assembly of a fibrillar FN matrix and suggest further that FBG binds to cell surface sites distinct from those bound by FN.
|
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Discussion |
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In this study, we present evidence that FBG assembly into the ECM of
adherent cells requires the active assembly of a FN matrix. We pursued this
line of investigation because our earlier data indicated that the mechanisms
of FBG assembly into the ECM showed striking similarities to that of FN.
Extraction of confluent monolayers of cells with 1% deoxycholate will remove
cells and soluble matrix proteins although it leaves behind the complex
detergent-insoluble matrix containing FN and HSPG
(McKeown-Longo and Mosher,
1983). We determined that this cell-free residual matrix produced
by HFF was not sufficient to support the deposition of FBG-Oregon Green into
matrix fibrils (unpublished data). Newly synthesized and secreted FBG binds to
the alveolar epithelial cell surface in a saturable manner, suggesting a
receptor-mediated binding event. This newly synthesized FBG remains cell
and/or matrix-associated in a trypsin-sensitive fraction, which, over time,
becomes incorporated into a deoxycholate-insoluble matrix fraction
(Guadiz et al., 1997b
) similar
to FN (McKeown-Longo and Mosher,
1983
). We have shown that, like FN
(Chernousov et al., 1985
;
McDonald et al., 1987
), FBG
assembly into the ECM requires metabolically active cells but not new
synthesis of a cell surface receptor or matrix constituent
(Pereira and Simpson-Haidaris,
2001
). Other similarities between FN and FBG incorporation into
matrix were found. FBG secretion from alveolar epithelial cells
(Guadiz et al., 1997a
) and FN
secretion from endothelial cells
(Kowalczyk et al., 1990
) is
polarized to the basolateral face of the cells, directing these glycoproteins
to the ECM. In addition, both cellular and plasma FN are known to incorporate
into ECM of heterologous cell types
(Peters et al., 1990
).
Similarly, lung-cell-derived FBG and purified plasma FBG incorporate into the
ECM of fibroblasts and lung epithelial cells. The deposition of FBG into the
ECM results in exposure of the ß15-21 fibrin-specific epitope
independently of thrombin or plasmin cleavage
(Guadiz et al., 1997b
). FN
also undergoes conformational changes during assembly into the ECM exposing
cryptic sites that are important for fibril elongation
(Erickson, 1994
;
Hocking et al., 1996
;
Hocking et al., 1994
;
Morla et al., 1994
;
Sechler et al., 1996
).
Finally, FBG and FN are extensively colocalized in the ECM of HFF and lung
epithelial cells (Guadiz et al.,
1997b
), suggesting a heterotypic association between FN and FBG in
matrix fibrils. Collectively, these data led us to hypothesize that FN plays
an essential role in FBG incorporation into the ECM.
Cultured cells rapidly assemble focal adhesions in response to serum
components, such as LPA, which activate the Rho-family of small GTPases
(Hall et al., 1993;
Nobes and Hall, 1995
) through
a G-protein-coupled cell surface receptor
(Ridley and Hall, 1992
). In
this report, we show that FBG assembly into the ECM of HFF is modulated by
Rho-dependent signaling,, a requirement for assembly of FN into the ECM as
well (Zhong, 1998). Furthermore, the inhibition of Rho-mediated signaling with
C3 transferase from C. botulinum inhibited the assembly of FBG into
the ECM of fibroblasts, but left the pre-established matrix components largely
intact. During wound healing, matrix deposition and remodeling create tensile
forces that modulate integrin-mediated cell function (Bultmann, 1998;
Hocking et al., 2000
). Indeed,
FN polymerization stimulates cell spreading and triggers a significant
increase in cytoskeletal contractility in a Rho-dependent manner
(Hocking et al., 2000
). Taken
together, these data indicate that the cell-dependent processes necessary for
promoting assembly of FBG into the ECM probably involve the concerted action
of FN polymerization and regulation of the actin cytoskeleton through
Rho-dependent pathways.
The FN-specific MoAb 9D2 inhibits FN polymer elongation into matrix fibrils
without inhibiting the initial binding of FN to the cell surface
(Chernousov et al., 1991). To
determine whether MoAb 9D2 inhibition of FN assembly would also inhibit FBG
assembly into the ECM, we treated HFF with 9D2. Over time, 9D2 inhibited the
formation of long and thick fibrils of FN, which was accompanied by a striking
change in the pattern and decrease in the amount of FBG assembled into the
ECM. Inhibition of both FN and FBG assembly into the matrix of HFF was
reversible, as removal of 9D2 allowed recovery of the cells' ability to
assemble a mature matrix composed of extensive fibrils of both FN and FBG.
Because HFF produce and secrete endogenous FN, experiments were conducted
using FN-null cells to clarify the role of FN in mediating the incorporation
of FBG into the ECM. In the complete absence of FN, exogenously added FBG was
unable to assemble into the ECM of FN-null cells. However, when FBG was added
concomitantly with FN, extensive FBG matrix fibrils, colocalizing with those
of FN, were present in the ECM of FN-null cells. Similar to HFF, MoAb 9D2
significantly inhibited assembly of FN and FBG into matrix fibrils in the ECM
of FN-null cells. Human FN bound to the mouse embryonic FN-null cells with
comparable affinity to that of human FN binding to HFF
(McKeown-Longo and Mosher,
1983
). Furthermore, FN binding to the FN-null cell monolayers was
concentration-dependent and saturable, indicating the involvement of specific
cell surface receptors. We conclude from these data that the incorporation of
FBG into the ECM is dependent on the active assembly of a FN matrix.
Although understanding the functional significance of FBG as a matrix
protein remains a focus of our continued investigations, we do not know the
mechanism of FBG interactions with FN that are essential for assembly of FBG
into complex matrix fibrils. There are two fibrin-binding sites on each FN
subunit that may play a role in the assembly of the conformationally altered
FBG in matrix (McKeown-Longo and Mosher,
1989). Previously, we found that the MoAb T2G1, which has always
been considered a fibrin-specific antibody
(Kudryk et al., 1984
), reacts
with intact FBG assembled into the ECM of HFF
(Guadiz et al., 1997b
). FBG
recovered from HFF ECM showed no evidence of thrombin or plasmin enzymatic
action, confirming that FBG, not fibrin, is assembled into the matrix.
Therefore, upon incorporation into the matrix, FBG undergoes conformational
changes exposing a cryptic epitope on the Bß chain normally only exposed
after FBG is proteolytically converted to fibrin. Importantly,
thrombin-generated exposure of the ß15-42 region promotes cell spreading
and enhances cell proliferation on a fibrin matrix
(Francis et al., 1993
). In
addition, the ß15-42 HBD of fibrin monomers binds to the surface of
endothelial cells in a heparin-dependent manner
(Odrljin et al., 1996a
). We
hypothesize that exposure of the HBD in matrix-associated FBG may be important
for modulating cellular responses to matrix FBG.
The data obtained from both HFF and FN-null cells suggest that FBG assembly
into the ECM is dependent on active FN polymerization and not on the presence
of a pre-established matrix or the initial binding of FN to the cell surface.
The results support the hypothesis that FBG assembly into matrix fibrils
requires FN-FBG heterotypic-association. FBG deposition into the ECM occurs in
the absence of covalent crosslinking to itself or other matrix constituents.
Furthermore, non-reducing SDS-PAGE analysis of 125I-FBG recovered
from ECM of HFF and A549 cells indicates that this FBG is not multimerized by
new disulfide bond formation to itself or any other matrix molecules. Soluble
FBG does not self polymerize; however, thrombin cleavage of FBG results in
conformational changes in the fibrin monomer that lead to lateral self
association and fibrin polymers that are stabilized by factor
XIIIa-mediated covalent crosslinking. Thrombin cleavage of FBG also
enhances exposure of the HBD, which comprises residues ß15-42
(Guadiz, 1997b). Because
exposure of ß15-42 by thrombin cleavage is accompanied by fibrin monomer
self association, we cannot rule out the possibility that exposure of
ß15-42 during deposition of FBG into the ECM promotes FBG-FBG homotypic
interactions.
The role of fibrin in both hemostasis and homeostasis is well documented.
Following vascular injury, FBG plays a role in controlling blood loss by
promoting platelet aggregation as well as forming an insoluble fibrin clot.
Although fibrin is the predominant protein, other adhesive glycoproteins such
as FN are constituents of the provisional clot matrix
(Clark et al., 1982). The
crosslinking of FN to the fibrin clot enhances the stability of the clot, and
both fibrin and FN act in concert to promote cell migration into the clot and
modulate gene expression of cells within the clot
(Knox et al., 1986
). Altering
the composition of a fibrin clot with FN promotes matrix composition-specific
modulation of cellular responses (Corbett
et al., 1996
). We hypothesize that FBG deposition rapidly changes
the topology of the ECM to provide a surface/conduit for cell migration during
tissue repair. Furthermore, the exposure of new epitopes on matrix FBG
probably signals to cells to alter their morphology in response to changes in
the microenvironment while potentially masking epitopes on matrix FN or other
matrix components to modulate signal-transduction pathways and ultimately
target gene expression.
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Acknowledgments |
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