1 Pulmonary and Critical Care Medicine, Department of Medicine, Mount Sinai
School of Medicine, New York, NY 10029, USA
2 Departments of Medicine and Cell Biology, NYU School of Medicine, New York, NY
10016, USA
3 Pulmonary and Critical Care Medicine, Harborview Medical Center, University of
Washington, Seattle, WA 98104, USA
* Author for correspondence (e-mail: lschnapp{at}u.washington.edu)
Accepted 3 September 2002
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Summary |
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Key words: Integrin, LAP-TGF-ß, 8ß1, Cell signaling
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Introduction |
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Recent work showed that LAP-TGFß1 binds to the integrins
vß1 (Munger et al.,
1998
) and
vß6
(Munger et al., 1999
). Binding
of
vß6 to LAP-TGFß1 activates TGFß1, independent of
protease activity. Mice lacking the ß6 integrin subunit are protected
from the development of pulmonary fibrosis due to the inability to activate
TGFß1 (Munger et al.,
1999
).
We previously characterized the human integrin subunit, 8, which
pairs exclusively with ß1 to form the heterodimer
8ß1
(Schnapp et al., 1995a
). The
integrin
8ß1 interacts with the RGD sequences in several matrix
proteins including fibronectin, vitronectin, tenascin, osteopontin
(Denda et al., 1998
;
Muller et al., 1995
;
Schnapp et al., 1995b
) and
nephronectin (Brandenberger et al.,
2001
).
8ß1 is expressed in alveolar interstitial cells
and is upregulated during pulmonary and hepatic fibrosis
(Levine et al., 2000
). We now
report that LAP-TGFß1 is a ligand for
8ß1 and that binding of
LAP-TGFß1 to
8ß1 increases spreading and proliferation of
cells and increases phosphorylation of the proteins FAK and ERK.
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Materials and Methods |
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Mink lung epithelial cells (Mv1Lu) stably transfected with a portion of the
plasminogen activator inhibitor 1 (PAI-1) promoter upstream of a luciferase
reporter gene were used as previously described
(Abe et al., 1994). Recombinant
LAP-TGFß1 and RGE-LAP-TGFß1 were produced in a baculovirus system as
described (Munger et al.,
1998
). Production of TGFß3 cDNA expression construct was
previously reported (Annes et al.,
2002
). TGFß3 was cloned into pCDNA-Fc vector and used for
protein purification as previously described
(Annes et al., 2002
).
Fibronectin (FN) was purchased from Boerhinger Mannheim and poly-L-lysine
was purchased from Sigma. Rabbit polyclonal antibody to FAK and HRP-conjugated
anti-phosphotyrosine antibody (clone 4G10) were obtained from Upstate
Biotechnology. Antibody to phosphorylated ERK was obtained from Santa Cruz.
Polyclonal antibody to recombinant human LAP-TGFß1 (AF-246-NA) was
obtained from R&D Systems. The integrin-activating antibody 8A2 and the
ß1 integrin blocking antibody 5D1 were a generous gift from John Harlan,
University of Washington (Seattle, WA). The v integrin blocking
antibody L230 was prepared from hybridoma cells obtained from American Type
Culture Collection (ATCC). Working dilutions for antibodies were determined
for each application to optimize the results.
Adhesion assays
The assays were performed as previously described
(Schnapp et al., 1995b).
Briefly, untreated polystyrene 96-well flat bottom microtiter plates
(Evergreen) were coated with increasing concentrations (0.3, 1, 3, 10, 20
µg/ml) of protein (LAP-TGFß1, LAP-TGFß3, FN) or 0.01%
poly-L-lysine. As a negative control, wells were coated with 1% BSA. Wells
coated at 37°C for 1 hour were washed with phosphate buffered saline (PBS:
137 mM NaCl, 2.7 mM KCl and 10 mM Na2HPO4, pH 7.4) and
non-specific protein binding sites were saturated with 1% BSA for 30 minutes
at 37°C. Cells were detached with 2 mM EDTA, washed with PBS and
resuspended in serum-free DMEM (pH 7.4) with or without 5 mM Mn2+.
In some experiments, cells were preincubated with integrin blocking antibodies
L230 or 5D1, or integrin-activating antibody 8A2 for 15 minutes on ice, prior
to addition to wells. 50,000 cells were added to each well, centrifuged at 10
g for 3 minutes to ensure uniform settling of cells and
incubated for 1 hour at 37°C. Non-adherent cells were then removed by
centrifugation (top-side down) at 10 g for 5 minutes. The
attached cells were fixed and stained with 1% formaldehyde/0.5% crystal
violet/20% methanol for 30 minutes at RT. After washing with PBS, adherence
was determined by absorption at 595 nm in a Microplate Reader (Bio-Rad,
Richmond, CA). The data were reported as the mean absorbance of triplicate
wells±s.e., minus the mean absorbance of BSA-coated wells.
LAP TGFß1 ELISA
Lungs were extracted from C57BL/6 mice (n=3) after perfusion with
PBS/ heparin through the RV outflow tract until the lungs blanched, to remove
blood. Lungs were weighed, placed in 2 ml PBS and homogenized 30 seconds with
tissue homogenizer. Samples were filtered through a 0.45 micron filter to
remove debris. Ninety-six well plates (Nunc-immunoplate, maxisorp surface)
were coated overnight at 4°C with serial dilutions of lung homogenate in
duplicate. Wells were coated with BSA alone as negative controls. To generate
a standard curve, wells were coated with serial dilutions of recombinant
LAP-TGFß1 protein. Non-specific binding sites were then saturated with 3%
BSA /PBS for 1 hour at 37°C. Wells were washed with PBS-0.05% Tween and
then incubated with 50 µl of anti-LAP-TGFß1 IgG antibody (0.5
µg/ml) (R&D Systems) at RT for 2 hours. Unbound protein was removed by
washing with PBS/0.05% Tween. Biotinylated rabbit anti-goat IgG (0.15
µg/ml) was added to wells for 1 hour at RT, followed by addition of
streptavidin AH-Biotin complex solution (Zymed SABC kit). Color development
was performed using TMB Microwell Peroxidase Substrate system (KPL) and read
at 450 nm after addition of stop solution (1 M phosphoric acid). The detection
limit was approximately 60 pg/well. The concentration of LAP-TGFß1 in the
samples was determined by interpolation from the standard curve.
TGFß bioassays
TGF-ß1 bioassay was performed as previously described
(Munger et al., 1999).
Briefly, 100 µl of Mv1Lu reporter cells were plated at a density of
105 cells/ml and allowed to adhere for 1 hour at 37°C in DMEM
containing 10% FCS. Equal number of test cells were added to wells and
cultured for 16 hours. In some experiments, test cells were incubated with the
ß1 integrin-activating antibody 8A2 for 15 minutes prior to addition to
Mv1Lu reporter cells. Lysates were assayed for luciferase activity using
Luciferase Assay System (Promega). As a positive control, Mv1Lu reporter cells
were cultured with recombinant TGFß1 (gift of Dan Rifkin). To determine
whether
8ß1 expression affected the activation of TGFß1 by
vß6, we incubated Mv1Lu reporter cells with ß6-transfected
SW480 (0.5x105 cells) and either
8-transfected SW480
or mock-transfected SW480 cells, and assayed for luciferase activity as
described above.
To measure TGF-ß3 activation, we transfected mock or 8
transfectants with TGFß3 cDNA expression vector or control vector using
Lipofectamine Plus (Life Technology) (Annes
et al., 2002
). After 16 hours, cells were added to reporter cells
for 24 hours and luciferase activity was measured as above. When high amounts
of TGFß3 cDNA were used for transfection, autoactivation of TGF-ß3
occurred. Therefore, we titered the amount of cDNA and found that transfection
with 100 ng of TGFß3 cDNA eliminated autoactivation and resulted in
detectable amounts of TGFß3 in supernatants.
Immunoprecipitation and western blot analysis
For FAK and ERK phosphorylation, cells were plated on ligands for 30
minutes, and then lysed in buffer containing 50 mM Tri-HCl (pH 7.4), 150 mM
NaCl, 0.25% sodium deoxychloate, 1% IGEPAL CA-630 (non-ionic, non-denaturing
detergent, Sigma), 1 mM EGTA, 1 mM PMSF, 1 mM NaVO3, 1 mM NaF, 1
mg/ml each of aprotonin, leupeptin, pepstatin. Samples were incubated with
antibodies for 1-2 hours at 4°C. Immune complexes were captured with
Protein A sepharose (Pharmacia). Beads were washed 5 times, boiled for 5
minutes in Laemli sample buffer and then proteins were separated by SDS-PAGE.
Gels were transferred to Immobilon and non-specific binding sites were
saturated with 3% BSA for 1 hour. Blots were incubated with primary antibody
for 1 hour, followed by peroxidase conjugated secondary antibody for 1 hour
and then developed with ECL (Amersham).
Proliferation assays
5x103 AtT20 or AtT20 8-transfected cells were
plated in serum-free media in 96 well plates coated with 5 µg/ml
LAP-TGFß1, FN, or 0.01% poly-L-lysine. Proliferation was assayed at
indicated times using the Roche Cell Proliferation Kit (MTT) per
manufacturer's instructions. Four independent clones of AtT20
8 were
tested. All experiments were performed in triplicate and presented as the
mean±s.e.
Immunohistochemistry
Lungs were obtained from 8-week-old C57BL/6 mice as previously described
(Madtes et al., 2001).
Briefly, the lungs was inflated with 4% neutral buffered paraformaldehyde
instilled at 30 cm H2O pressure through the trachea for 120
minutes. The trachea was then tied and the lung immersed in the RNAse-free, 4%
buffered paraformaldehyde for 24 hours before embedding in paraffin. 5-µm
sections of lung fixed with 4% (wt/vol) paraformaldehyde were deparaffinized
and rehydrated. Endogenous peroxidase and biotin activity was saturated by
incubation of the sections in Peroxoblock (Zymed), followed by Avidin-Biotin
Blocking Reagent (Zymed). The sections were incubated overnight at 4°C
with affinity purified goat anti-human LAP-TGFß1 IgG antibody (2.5
µg/ml) (R&D Systems). Primary antibody was detected with biotinylated
rabbit anti-goat IgG antibody (Zymed Laboratories) (0.15 µg/ml). Bound
antibody was visualized with ABC peroxidase (Vector Laboratories). The
sections were counterstained with hematoxylin. As a negative control, adjacent
serial sections were stained in the absence of primary antibody.
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Results |
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In contrast to LAP-TGFß1, fibronectin (a known RGD-containing ligand
for vß6 and
8ß1) supported adhesion of unactivated
SW480ß6 and SW480
8 cells equally well
(Fig. 1C). Cell surface
expression of
8 and ß6 was similar in SW480 cells (data not
shown). To confirm that the RGD site in LAP-TGFß1 was involved in binding
to
8ß1, we examined adhesion of
8-transfected cells to a
recombinant LAP-TGFß1 in which the RGD site was mutated to RGE. Mutation
of RGD to RGE eliminated
8ß1-mediated adhesion to LAP-TGFß1
in all cell lines tested (Fig.
1D).
LAP-TGFß1 and LAP-TGFß3 support 8ß1 adhesion
with similar efficacy
Because LAP-TGFß3 contains an RGD sequence in a similar location as
LAP-TGFß1, we asked whether LAP-TGFß3 was also a ligand for
8ß1. We examined the adhesion of AtT20
8 cells and AtT20
mock cells to recombinant LAP-TGFß3. We found that AtT20
8 cells
adhered to LAP-TGFß3 significantly better than mock transfected cells
(Fig. 1E). The adhesion of
8-transfected cells to LAP-TGFß3 was similar to adhesion to
LAP-TGFß1 (Fig. 1E).
Adhesion to LAP-TGFß1 results in cell signaling
Integrin mediated signaling results in alterations in the cytoskeleton,
leading to cell shape changes. When AtT20 cells were plated on LAP-TGFß1,
cells became flat and spread on the substrate, and developed long extensions
(Fig. 2i). Focal adhesion
kinase (FAK) is present at focal contacts and becomes phosphorylated after
integrin-mediated cell adhesion and plays a role as an adapter protein for
integrin-mediated cell signaling. We hypothesized that if LAP-TGFß1 is a
ligand for 8ß1, adhesion of
8ß1-expressing cells to
LAP-TGFß1 would lead to FAK phosphorylation. We plated mock-transfected
or
8-transfected AtT20 cells on plates coated with poly-L-lysine (which
allows non-integrin mediated adhesion), fibronectin (a known ligand for
8ß1) and LAP-TGFß1 and RGE-LAP-TGFß1 for 30 minutes in
serum-free media. Cells were lysed in the presence of phosphatase and protease
inhibitors, and immunoprecipitated with anti-FAK antibody, followed by
blotting with anti-phosphotyrosine antibody or FAK antibody. We found that
interaction of LAP-TGFß1 with
8ß1 leads to tyrosine
phosphorylation of FAK comparable to phosphorylation seen after fibronectin
adhesion (Fig. 2ii).
Furthermore, mutation of the LAP-TGFß1 RGD site to RGE eliminated FAK
phosphorylation. Mock transfected cells did not show FAK phosphorylation when
grown on LAP-TGFß1 or fibronectin.
|
LAP-TGFß1-8ß1 mediates cell proliferation
We then asked whether cell behaviors such as proliferation were affected by
adhesion to LAP-TGFß1. We found that 8-transfected cells
proliferated significantly better when grown on LAP-TGFß1 compared to
mock transfected cells grown on LAP-TGFß1 in serum-free media
(Fig. 3A). The degree of
proliferation was similar to that of cells grown on fibronectin. To insure
that the enhanced proliferation was not due to clonal variation, we tested
four independent clones of AtT20
8 transfectants. All showed a
significant increase in proliferation compared to mock transfected or wild
type cells (Fig. 3A and data
not shown) The average fold increase in proliferation was 1.9 compared to mock
transfected cells. We examined whether ERK was phosphorylated in response to
8ß1-LAP-TGFß1 binding. We found an increase in phospho-ERK
levels in
8-transfected cells adherent to LAP-TGFß1, compared to
8-transfected cells adherent to poly-L-lysine, or mock-transfected
cells (Fig. 3B).
|
8ß1 binding to LAP-TGFß1 does not affect activation
of TGFß1
We asked whether binding of 8ß1 to LAP-TGFß1 activated
TGFß1, as described for
vß6
(Munger et al., 1999
). As an
indicator of TGFß1 activation, we used Mv1Lu reporter cells transfected
with a portion of the plasminogen activator inhibitor-1 (PAI-1) promoter
upstream of a luciferase reporter gene. The PAI-1 promoter contains a
well-characterized TGFß-responsive element. Therefore, if active
TGFß1 is present, an increase in luciferase activity will be detected.
Mv1Lu reporter cells were co-cultured with mock-transfected SW480 cells or
8-transfected SW480 cells. As a positive control, Mv1Lu reporter cells
were cultured with TGFß1, or with SW480ß6 cells. Luciferase activity
did not increase when
8-transfected cells were cultured with MLEC,
suggesting that adhesion of LAP-TGFß1 to
8ß1 was not
sufficient to activate TGFß1 (Fig.
4A). Addition of 8A2 or Mn2+, which enhanced adhesion
of
8 cells to LAP-TGFß1, did not affect TGF-ß activation
(Fig. 4A, data not shown).
|
We then asked whether adhesion of 8ß1 to LAP-TGFß1
affected the activation of TGFß1 mediated by
vß6, by
competing for LAP-TGFß1. We set up a triple co-culture system using Mv1Lu
reporter cells cultured with SW480ß6 and either
8-transfected
SW480 cells or mock-transfected SW480 cells. No difference in
vß6-mediated activation of TGF-ß1 was found between
8-transfected SW480 cells and mock-transfected SW480 cells
(Fig. 4B). Since we found
adhesion of
8-transfected cells to LAP-TGFß3, we asked whether
that interaction led to activation of LAP-TGFß3. Recent reports showed
that
vß6, which activated TGFß1, also binds and activates
TGFß3 (Annes et al.,
2002
). However, we found no difference in activation of
LAP-TGFß3 when we compared CHO and CHO-
8 cells transfected with
LAP-TGFß3 (Fig. 4C).
Similar negative results were observed with 293 and 293
8 cells (data
not shown). These results suggest that
8ß1 does not activate
LAP-TGFß3.
Immunolocalization and concentration of LAP-TGFß1 in lung
We previously showed that 8 is localized to lung interstitial cells
and is upregulated during pulmonary fibrosis. Using an antibody specific for
LAP-TGFß1, we examined the immunolocalization of LAP-TGFß1 in normal
lung tissue (Fig. 5).
Immunoreactivity for LAP was detected along the interstitial cells, in a
patchy pattern similar to alpha 8 immunolocalization, as well as in
macrophages. To estimate the relative concentration of LAP-TGFß1 in mouse
lung, we developed an ELISA for LAP-TGFß1 for use on whole mouse lung
homogenates. The measurements ranged from 0.5 to 8 µg/mg lung tissue with
an average of 3.38 µg of LAP-TGFß1 per mg lung tissue.
|
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Discussion |
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Ligands for integrins include ECM proteins such as fibronectin, collagens,
and laminin, and cell surface counter receptors such as immunoglobulin
superfamily members. However, the ligand repertoire of integrins may be
considerably greater, considering the number of proteins that contain
potential integrin binding motifs. For example, several viruses contain
conserved RGD sequences in their envelope, which interact with integrins and
contribute to viral adhesion and entry
(Chiu et al., 1999;
Jackson et al., 2000
;
Neff et al., 1998
).
Disintegrins also contain RGD sites that are used to interact with integrins
(Gould et al., 1990
;
McLane et al., 1998
;
Niewiarowski et al.,
1994
).
We show that 8ß1 recognizes LAP-TGFß1 via the RGD
sequence.
8, along with
5,
v and
IIb, form a
subfamily of integrin subunits that are related based on sequence homology,
binding to RGD sequences and absence of I domain. Three other family members,
vß1,
vß6 and
vß8, also bind
LAP-TGFß1. However, only
vß6 or
vß8 binding to
LAP-TGFß1 activates TGFß1 (Mu et
al., 2002
; Munger et al.,
1999
). Thus, the adhesion of
8ß1 more closely
resembles the adhesion of
vß1 to LAP-TGFß1. Although
8ß1 did not result in activation of TGFß1 by the assay
performed, it may facilitate activation by another mechanism. Binding of
LAP-TGFß1 to
8ß1 may localize LAP-TGFß1 to the cell
surface and lead to activation by other pathways, such as proteolytic cleavage
or by thrombospondin. The binding avidity of the integrin to LAP-TGFß1
may determine whether TGFß1 activation occurs. Both
8ß1 and
vß1 only bind to LAP-TGFß1 at higher concentrations than
vß6. However, we are able to increase adhesion of
8ß1
to LAP-TGFß1 to levels comparable to
vß6 by Mn2+
or integrin ß1-activating antibody and despite the increased adhesion,
activation of TGFß1 did not occur. Another possibility is that a second
binding determinant is required. For example, interaction of the disintegrin
echistatin with ß1 and ß3 integrins involves a secondary binding
determinant on the C-terminus in addition to the RGD site
(Wierzbicka-Patynowski et al.,
1999
). Recently, the sequence DLXXL was reported as a ligand for
vß6 (Kraft et al.,
1999
). A similar sequence is found adjacent to the RGD site in
LAP-TGFß1 (RGDLXXI), LAP-TGF-ß3 (RGDLXXL) and adjacent to the RGD
site in Foot and Mouth Disease Virus, another recently described ligand for
vß6 (Jackson et al.,
2000
). Therefore, activation of TGFß1 by integrin binding may
be determined by sequence adjacent to RGD sequence. Because LAP-TGFß3
also contains an RGD sequence, we examined adhesion of
8ß1 to
LAP-TGFß3. We found
8-transfected cells adhered similarly to
LAP-TGFß3 as to LAP-TGFß1. However, similar to
8ß1-LAP-TGFß1 interaction,
8ß1-LAP-TGFß3
interaction was not sufficient to activate LAP-TGFß3.
Integrins such as 8ß1 and
vß1 that bind but do not
activate LAP-TGFß1 may negatively regulate TGFß1 activity by
sequestering latent TGFß1 and preventing access to an activating integrin
such as
vß6. Although we did not see this affect in vitro, excess
TGFß1 is present in serum and may overcome sequestration by
8ß1. Another explanation for the lack of affect of
8ß1
on
vß6-mediated activation was that
8ß1 and
vß6 were expressed on different cells. However, this mimics the in
vivo situation, where
vß6 is expressed on epithelial cells and
8ß1 is expressed on interstitial cells
(Breuss et al., 1995
;
Levine et al., 2000
).
Is the concentration of LAP-TGFß1 required to see an effect
physiologically relevant? We have several lines of evidence to suggest it
might be. Measurements of TGFß1 concentrations in bronchoalveolar lavage
fluid (BALF) from 67 patients with persistent acute respiratory distress
syndrome (ARDS) showed values as high as 973 pg/ml, with an average
concentration of 124 pg/ml +/-182 pg/ml (Personal communication, Richard B.
Goodman, University of Washington). Significant immunoreactivity was only
detected after acid activation of BALF, indicating that the measured
TGFß1 was present in the latent form. Because bronchoalveolar lavage in
humans has been reported to dilute lung fluid by 100- fold
(Miller et al., 1992), we
estimate the lung fluid concentrations of LAP TGFß1 in these patients to
be as high as 0.1 µg/ml, with an average concentration of 12 ng/ml. This
value is likely to underestimate the LAP-TGFß1 concentration within the
microenvironment of the lung parenchyma, as the distribution of LAP-TGFß1
is heterogeneous (Fig. 5).
Next, we measured LAP-TGFß1 in mouse lung homogenates and detected a
range of LAP-TGFß1 from 0.5 to 8 µg/mg lung tissue, with an average of
3.38 µg±4. Finally, in vitro interactions of the integrin
vß6 with LAP-TGFß1 occur with coating concentrations in the
microgram range and this interaction has important physiological consequences
in the regulation of lung inflammation
(Munger et al., 1999
). Thus,
we conclude that coating concentrations of LAP-TGFß1 used in this report
are in the range of concentrations potentially encountered in vivo.
LAP-TGFß1 distribution in the lung interstitium was similar to
8 distribution suggesting that
8ß1-LAP-TGFß1
interactions can occur in vivo. The ability of
8ß1-LAP-TGFß1
interactions to induce FAK and ERK phosphorylation and promote cell
proliferation strongly argues that LAP may be a relevant biological ligand in
vivo and that interactions of cells with ECM-bound LAP may result in
alterations in cell behavior. LAP-TGFß1 ligation to
8ß1
resulted in cell spreading, adhesion, proliferation, and phosphorylation of
FAK and ERK. LAP-TGFß3 is likely to have similar effects. Thus,
independent of its role in regulating the amount of active TGFß1,
LAP-TGFß1 may have a novel role in regulation of cell behavior via
interaction with integrins such as
8ß1.
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Acknowledgments |
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References |
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