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Department of Medicine, Arthritis Center, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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v integrin receptors
mediated this down-regulation upon adhesion to fibronectin. Loss of
cell adhesion and TNF- stimulation synergistically increased
collagenase expression. Increased collagenase expression upon
nonadherence was mimicked by treatment with cytochalasin B, suggesting
that the loss of cytoskeletal structure associated with a change in
cell shape mediates increased collagenase in nonadherent cells. Thus,
although increased fibronectin in the lining layer in RA might be
expected to inhibit collagenase expression, the change in cell shape
associated with this multilayer structure might actually lead to
increased collagenase expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synovial tissue expansion creating the pannus in rheumatoid tissue results in part from the proliferation of FLSs (7). Transformed-like cellular growth has been proposed as a model for erosion of bone by the expanding pannus (2, 8, 9). In vitro studies of synovial fibroblast transformation have relied on assay of anchorage-independent cell growth. These studies showed that synovial fibroblasts require serum factors, notably platelet-derived growth factor (PDGF), to grow without anchorage. PDGF similarly stimulated cells derived from normal or arthritic joints to grow under anchorage-independent conditions (8). On the basis of these studies a concept of cytokine-dependent cellular transformation was proposed (8).
Cellular transformation as assayed by anchorage-independent cell growth measures cell proliferation not requiring adhesion to plastic. Cell adhesion to plastic relies on the binding of the serum proteins fibronectin and vitronectin to plastic, followed by the binding of cells to these proteins through surface integrin receptors (10). Signaling through integrin receptors is thus an important stimulus of cell proliferation in normal cells that is bypassed or constitutively activated in neoplastic cells (11, 12). Why FLSs can grow under anchorage-independent conditions in response to PDGF is not known, but might reflect local fibronectin matrix formation within the agarose gel (13, 14).
Integrin receptors also regulate metalloproteinase expression. In
rabbit synovial fibroblasts engagement of the
5ß1 integrin increases and that of the
4ß1 integrin inhibits collagenase (15),
and in dermal fibroblasts engagement of the
2ß1 receptor increases collagenase (16).
Thus, integrin interactions with ECM might be expected to regulate
metalloproteinase of FLSs in RA synovial tissues.
Several histological studies have examined ECM and integrin expression
in rheumatoid synovium. Several groups have observed increased levels
of fibronectin in rheumatoid synovial tissues (17, 18, 19, 20, 21). This is
particularly pronounced in the synovial lining layer and the luminal
surface of endothelial cells, but also occurs in the sublining layer.
Vitronectin is increased in the synovial lining layer in both OA and RA
tissues compared with that in normal tissues (22). Expression of
several integrin receptor subunits is also increased in situ;
rheumatoid synovial tissues express high levels of the
3, 4, 5, 6,
and ß1 integrin subunits, but low levels of the
v integrin subunit (23, 24, 25).
We hypothesized that cell interactions with ECM control FLS phenotype
in RA and developed a system to address the impact of cell adhesion on
FLS growth and metalloproteinase expression. We found that FLS cell
adhesion to any of several ECM components is essential for cell
division. Fibronectin, vitronectin, collagen, and laminin were each
capable of providing a signal complementary to a signal provided by
PDGF to induce FLS proliferation. Adhesion to fibronectin, collagen, or
an Arg-Gly-Asp (RGD) peptide down-regulated collagenase expression.
Cell adhesion also affected the sensitivity of synovial fibroblasts to
TNF- and TGF-ß. TNF- and nonadherence or cytoskeletal
disruption synergistically increased collagenase expression. Our data
indicate that FLS/ECM interactions likely regulate FLS collagenase
production and proliferation in RA synovial tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synovial fibroblasts were obtained from patients with
rheumatoid arthritis (RA) at the time of elective surgery for knee
replacement in accordance with an approved institutional review
board protocol. Tissue (0.2–0.5 g) was minced finely, digested
with collagenase (4 mg/ml; Worthington Biochemical, Freehold, NJ) for
4–6 h, centrifuged, and resuspended in DMEM supplemented with 10% FBS
and penicillin (100 U/ml)/streptomycin (100 µg/ml). Cells were
passaged upon confluence and were used between passages 4–8. Porcine
TGF-ß1 was provided by Dr. Michael Sporn, and human recombinant
PDGF-BB was obtained from Life Technologies (Gaithersburg, MD). mAb to
human integrin v (Chemicon, Temecola,
CA; MAB1980), human integrin 5 (Becton Dickinson,
Mountain View, CA; mAb16), human integrin ß1 (Coulter,
Hialeah, FL; 4B4), and control Ab (Organon Teknika, Durham, NC;
MOPC 141) were used at a concentration of 10 µg/ml for blocking
experiments.
Preparation of nonadherent surfaces and ligand-coated beads
To eliminate attachment of cells to culture dishes, wells were coated with poly(2-hydroxyethyl methacrylate) (HEMA). One-half milliliter of a solution of 0.12 g/ml HEMA in 95% ethanol was added to each well of a six-well cluster dish, or 0.05 ml was added to each well of a 96-well dish, and the ethanol was allowed to dry over several days. ECM ligands and peptides were covalently linked to activated agarose beads (AminoLink Coupling Gel, Pierce, Rockland, IL) according to the manufacturer’s instructions. Briefly, an equal volume of washed beads was added to 1.0 mg/ml of ECM protein or peptide in 0.1 M NaPO4 (pH 7.0). A 0.02 vol of 1.0 M NaCNBH3 was added, and the suspension was incubated at room temperature on a rotator for 2 h. The resulting gel was washed, incubated with 1.0 M Tris, pH 7.4, to block unreacted sites, and washed again in complete medium three times before use. ECM proteins used as ligands were type I collagen (from calf skin, Sigma, St. Louis, MO), fibronectin (from human plasma, Life Technologies), or vitronectin (from human plasma, provided by C. Peterson). Peptide containing the RGD tripeptide (Gly-Arg-Gly-Asp-Ser-Pro-Lys) and control peptide (Gly-Arg-Ala-Asp-Ser-Pro-Lys) were purchased from Sigma.
Assays of cell proliferation
Cells (
2 x 104) were added to 96-well
plates coated with HEMA or left uncoated in DMEM supplemented with 10%
FBS. In other experiments about 106 cells were added to
HEMA-coated wells of six-well plates, and ligand-coated beads (20 µl
packed volume) were added to each well. The cells were allowed to
adhere overnight to the ligand-coated bead or plastic surfaces. The
next day the cells were washed three times with serum-free DMEM. For
the cells adherent to plastic, medium was removed and replaced in the
well (three times). For cells in HEMA-coated wells, the complex of
beads and cells were transferred to a 15-ml conical tube and
centrifuged at 1000 rpm for 5 min, the supernatant was aspirated, and
serum-free DMEM was added (wash repeated three times). Cells were then
left in serum-free medium for 3 days before adding PDGF or TGF-ß, and
[3H]thymidine. After the addition of cytokines, the cells
were left for another 3 days, and then [3H]thymidine
incorporation was assayed by collection and lysis of cells on a cell
harvester, and counting on a scintillation counter.
To assay nonadherent cell viability, FLSs cultured on HEMA-coated plates were treated with trypsin to separate the cells, the trypsin was neutralized by addition of medium supplemented with serum, trypan blue was added to 0.02%, and 100 cells were counted for viability by dye exclusion.
Preparation of RNA, Northern blotting, and phosphorimaging
Total RNAs were prepared from cells by direct lysis in 400 µl of RT-lysis buffer and purification according to the procedure described for isolation of total RNA (RNeasy Total RNA kit, Qiagen, Chatsworth, CA). RNA was eluted from the columns in a volume of 40 µl and was stored at -70°C. RNAs were analyzed on agarose/formaldehyde gels and blotted to nitrocellulose as previously described (26). Blotted RNAs were serially hybridized to cDNA probes of collagenase (metalloproteinase-1) and collagen and then to a probe specific for 18S ribosomal RNA (rRNA) using the method of Church et al. (27). An 18S human rRNA probe was generated by RT followed by PCR using the 5' primer (5'-ACGTCTGCCCTATCAACTTTCGA-3'; bp 450–472) and the 3' primer (5'-CCTCACTAAACCATCCAATCGG-3'; bp 1817–1838), yielding an amplified product of 1388 bp. For each hybridization signals were quantified by phosphorimaging and normalized to rRNA expression.
Statistical analyses
Data from proliferation studies were analyzed by one- or two-way analysis of variance and Tukey’s method for multiple comparison post-tests (using Prism software, GraphPad, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To understand more completely the potential contribution of cell
adhesion to phenotypic abnormalities expressed by RA FLSs,
proliferation and collagenase expression of FLSs were assayed under
adherent and nonadherent conditions. Primary cell cultures were
passaged to plastic wells or wells coated with HEMA, a polymer that
inhibits cell attachment to plastic (28). Nonadherent cells formed cell
clumps (Fig. 1
, C and
D). In the absence of adhesion, cell proliferation declined
and became unresponsive to PDGF, a potent mitogen for FLSs (Fig. 1A). As previously described, PDGF enhanced FLS
proliferation of cells adherent to plastic (29). In contrast, FLSs
cultured under nonadherent conditions highly increased collagenase mRNA
expression (Fig. 1B). We have seen similar increases in
collagenase in FLSs derived from three other patients with RA and from
five patients with OA and in dermal fibroblast cell lines. Although
loss of adhesion induces cell death or ankoisis in some cell types,
synovial fibroblasts remained fully viable during these experiments by
trypan blue exclusion. This is consistent with the observation that
nontransformed fibroblasts are resistant to apoptosis induced by
nonadherence (30).
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We next investigated whether the ECM proteins shown to be
responsible for cell binding to plastic, fibronectin and vitronectin,
could reproduce proliferation dependent on adhesion to plastic. The
addition of these proteins in a soluble form did not induce
proliferation when added alone or together with PDGF (data not shown).
Since proliferation might require cytoskeletal formation dependent on
cell spreading, FLSs were then cultured in HEMA-coated wells with
fibronectin- or vitronectin-coated beads. Cells attached to beads
coated with either fibronectin (Fig. 2B) or vitronectin (not
shown), but not to control beads (Fig. 2A). Cell
proliferation was measured after the addition of PDGF, TGF-ß, or both
growth factors. Experiments were conducted using concentrations of PDGF
and TGF-ß previously found to maximally affect FLS growth and
collagenase expression (8, 31). In early experiments the addition of
ligand-coated beads without any exogenous growth factor(s) increased
proliferation (for example, see Fig. 3B). However, upon
extended serum starvation the addition of either ligand-coated beads or
PDGF alone did not stimulate cell proliferation (Fig. 3A; p > 0.05
for the difference between untreated (without cytokine) cells with
control beads vs untreated cells on each of the ECM-coated beads; also
p > 0.05 for the difference between untreated vs
PDGF-treated cells with control beads). Cell proliferation increased
after addition of both ligand-coated beads and PDGF (Fig. 3A; p < 0.05 for the difference between
untreated vs PDGF-treated cells on fibronectin- and vitronectin-coated
beads). TGF-ß synergistically stimulated the proliferation of
PDGF-stimulated cells (Fig. 3A; p < 0.001
for the difference between PDGF vs PDGF + TGF-ß for fibronectin- and
vitronectin-coated beads; p > 0.05 for control beads).
TGF-ß had little effect alone (Fig. 3A; p
> 0.05 for the difference between TGF-ß vs untreated cells on all
ECM-coated and control beads).
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1, 5, and
vß3 integrins, but not 2,
3, or 6 receptors (32). Related to this
integrin-specific activation, proliferation increased upon cell binding
to fibronectin or vitronectin, but not laminin. Therefore, to define
the variety of ECM proteins able to synergistically activate FLS
proliferation with PDGF, beads coated with collagen or laminin were
also tested. Cells bound readily to beads coated with collagen (Fig. 2
C) or laminin (not shown) and responded to PDGF and TGF-ß
similarly to cells attached to fibronectin or vitronectin (Fig. 3A; p < 0.01 and p < 0.05
for the difference between control vs PDGF-treated cells on,
respectively, collagen- and laminin-coated beads; p
< 0.001 for the difference between control vs PDGF- and
TGF-ß-treated cells on collagen- or laminin-coated beads).
To confirm that FLSs maintained viability under these conditions
of prolonged serum starvation, untreated or TGF-ß- or PDGF-treated
FLSs cultured under nonadherent, serum-free conditions were analyzed
after 1, 2, 3, 4, 5, 7, and 10 days for cell death by trypsin
separation of the cell aggregates and trypan blue exclusion. No cell
death was detected at any time point (<1%). After 10 days under these
conditions the cells were placed in medium supplemented with serum on
plastic tissue culture dishes. The cells attached to the surface
overnight (at 10% confluence) and grew to confluence.
Results for cells growing on fibronectin-coated beads were similar to
those for cells grown on fibronectin-coated wells (Fig. 3C).
However, the ECM ligand(s) providing the signal to cells grown on
fibronectin-coated wells is not as well defined, since cells can
bind to tissue culture plastic in the absence of the fibronectin
coating.
Studies of FLSs from two other patients with RA and two patients with
OA showed similar matrix and cytokine requirements. Thus, OA FLSs also
required both adhesion to an ECM protein and PDGF to stimulate
proliferation, indicating that OA and RA FLSs have the same
requirements for proliferation (see Table I).
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To better understand the role of serum in this system, cell
proliferation was studied under serum-free or serum containing
conditions. In control nonadherent cells serum had little effect. The
presence of serum resulted in increased proliferation upon the addition
of collagen beads, but blunted the effect of TGF-ß and PDGF (Fig. 3B). This is probably due to the presence of TGF-ß and
PDGF in serum.
Although shown to markedly stimulate collagenase expression (see
below), TNF- had no significant effect on proliferation of FLS bound
to ECM-coated beads under serum-free conditions (Fig. 3B and
data not shown). TNF- slightly inhibited the proliferation of FLSs
bound to ECM beads in the presence of serum; however, this did not
reach statistical significance (Fig. 3B and data not shown).
This is similar to the effect of IL-1 in the presence of serum. In the
case of IL-1, inhibition of FLS proliferation is mediated by
PGE2, which is induced by both IL-1 and TNF- and
inhibits PDGF-stimulated proliferation of FLSs (33).
Binding of FLSs to fibronectin or collagen inhibits collagenase expression
To further define the importance of integrin engagement,
collagenase expression by FLSs was measured under nonadherent
conditions or after adhesion to ligand-coated beads. Adhesion to
plastic, fibronectin, or collagen decreased collagenase expression
compared with that of nonadherent cells (Fig. 4). TGF-ß inhibits basal and stimulated
FLS collagenase expression (31). The addition of TGF-ß decreased
collagenase expression by both adherent and nonadherent cells, but did
not completely inhibit the high levels of collagenase induced in
nonadherent cells (Fig. 4). Cell integrin receptors bind to several
different peptide motifs on ECM proteins, including the
arginine-glycine-aspartatic acid (RGD) tripeptide found in fibronectin
and vitronectin (34). Collagen molecules, although not containing this
tripeptide, bind through a conformational analogue to this motif (35, 36). An RGD-containing peptide known to bind highly to fibronectin and
vitronectin receptors was covalently bound to beads, and similar to the
full-length proteins stimulated cell binding (not shown) and inhibited
collagenase mRNA expression (Fig. 4). Control peptide beads
slightly inhibited collagenase expression compared with control beads
(Fig. 4). This correlated with slight binding of the cells to these
beads compared with the complete absence of binding to control beads.
Apparently integrin receptors are able to bind weakly to the mutated
peptide. We have seen similar inhibition of collagenase expression by
all cell lines tested from patients with RA and OA (Table I). Although
there is some variability, collagen beads and fibronectin beads
generally inhibited collagenase expression to a similar degree (Table I).
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The preincubation of Abs against various integrin receptors
permitted assignment of the integrins responsible for the observed
effects. FLSs preincubated with blocking Abs to the v,
and 5 or ß1 integrin subunits no longer
attached to fibronectin-coated beads, but preincubation with
v, 5, or ß1 Abs alone had
no observable effect on cell binding to the beads (Figs. 5 and 6A). These results
indicate that FLSs bind to fibronectin by
5ß1 and vß1,
vß3, and/or
vß5 integrin receptors. Other experiments
showed that binding of FLSs to vitronectin was blocked by Ab to
v (but did not require Ab to the 5
subunit; data not shown), indicating that FLSs bind to vitronectin by
vß1, vß3,
and/or vß5 integrin receptors (Fig. 6A). The receptors used by
FLSs are thus similar to those reported for adhesion of dermal
fibroblasts to fibronectin and vitronectin (37). These results were
consistent in all OA (five of five) and most RA (four of five) FLS
lines tested. Ab to only the v integrin subunit was
sufficient to block binding to fibronectin (and vitronectin) by one RA
cell line, highlighting the importance of v receptors in
binding to both fibronectin and vitronectin. Antibody to the
ß1 subunit (but not by any combination of the
receptor blocking Abs) blocked binding to collagen (Fig. 6A), indicating that FLSs bind to collagen through undefined
ß1 receptors (probably 1ß1
and/or 2ß1) (38).
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v or ß1 integrin subunits
increased collagenase expression by FLSs bound to fibronectin, but
blocking Ab to 5 alone had no effect (Fig. 6B). The combination of blocking Abs to both
v and 5 increased collagenase expression
to a level slightly higher than that seen in fibronectin-adherent FLSs
treated with Ab to v alone (Fig. 6B). Control
mAb MOPC 141 had no effect on collagenase expression (data not shown).
TNF- and loss of integrin engagement synergistically enhance
collagenase expression
Blocking of TNF- leads to dramatic clinical improvement
of patients with RA, implicating a central role for this cytokine in RA
pathogenesis (39, 40). It has been long recognized that TNF-
potently induces collagenase expression by FLSs (41). To determine the
potential contribution of TNF- and ECM adhesion to collagenase
expression in vivo, nonadherent FLSs or FLSs adherent to fibronectin or
collagen beads were treated with TNF- or TGF-ß, and collagenase
expression was analyzed. As described above, adhesion to collagen or
fibronectin beads inhibited collagenase expression relative to
nonadherent cells (Figs. 4 and 7).
TGF-ß further inhibited collagenase levels in both adherent and
nonadherent cells. TNF- stimulated increased collagenase expression
by both adherent and nonadherent FLSs (Figs. 4 and 7). Similar effects
were seen in two other cell lines. In other experiments FLSs were
treated with PDGF or TGF-ß plus PDGF under adherent and nonadherent
conditions. PDGF had no significant effect on collagenase expression
under these conditions (data not shown).
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synergistically
increase collagenase expression
Integrin engagement stimulates the formation of the actin-based
cytoskeleton that terminates in focal adhesions composed of several
intracellular cytoskeletal proteins, including -actinin, talin,
vinculin, paxillin, and tensin. These proteins bind to the
ß1 integrin tail at the interior of the cell membrane.
The observation that adhesion to any of several different integrins
leads to decreased collagenase expression suggested that formation of
the cytoskeleton might mediate this signal. The addition of
cytochalasin B, an inhibitor of cytoskeletal formation, up-regulated
collagenase expression by FLSs. This up-regulation was similar in
magnitude to that seen upon culture of the cells under nonadherent
conditions (Fig. 8). The addition of
TNF- to cytochalasin B-treated FLSs resulted in a synergistic
increase in collagenase expression similar to that seen upon treatment
of nonadherent cells with TNF- (Fig. 8). Similar effects were seen
on two other cell lines.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We show that FLS proliferation requires a signal provided by ECM in addition to a signal provided by a mitogenic cytokine, the most potent being PDGF. These data reinforce earlier observations that FLSs from RA and OA patients have the same requirements for anchorage-dependent and anchorage-independent growth (8, 29). Mutations in genes regulating cellular growth and transformation should change the dependence of FLS proliferation on adhesion and/or cytokines. By these measures RA FLSs need the same stimuli as OA FLSs for proliferation. Our data do not support the idea of a mutational event or a transforming virus contributing to the pathogenesis of synovial hyperplasia in RA (2).
Any of several different ECM proteins, including fibronectin,
collagen, vitronectin, or laminin can provide the ECM signal required
for FLS proliferation. The integrin receptors used by different ECM
proteins are different, but overlapping, and depend on the cell type
analyzed (38). Through blocking experiments we found that synovial
fibroblasts bind to fibronectin through
5ß1 and v integrins, to
vitronectin through v integrins, and to collagen though
ß1 integrins. Our results do not support an integrin
receptor specificity of a signal for FLS cell cycle entry as has been
described for HUVEC (32). These cells do not proliferate or activate a
Fos promoter-luciferase construct upon cultivation on laminin 1, in
contrast to cultivation on fibronectin or vitronectin. The difference
between our results and these findings was not due to differences in
the type or the preparation of laminin because we used the same source
of laminin 1 as these investigators (32). Possibly FLSs bind to laminin
through receptors different from those found on HUVEC. Alternatively, a
broader array of integrin receptors on FLSs are able to activate the
required costimulus for proliferation.
Previous analyses have shown that fibroblast adhesion permits progression through the G1/S phase of the cell cycle (11). In this regard FLS cell growth is similar to NIH-3T3 or BALB/c 3T3 fibroblasts, which require an ECM stimulus for proliferation. In contrast, treatment with TGF-ß can bypass this need for an ECM stimulus in NRK cells, permitting the cells to grow without anchorage (42). TGF-ß stimulation of anchorage-independent growth by NRK cells has been attributed to its stimulation of ECM formation (13). In contrast, TGF-ß inhibits anchorage-independent growth of FLSs (8), even though it stimulates FLS collagen formation and fibrillar fibronectin formation (14). PDGF can also promote fibrillar fibronectin matrix formation by FLSs, and thus local formation of ECM may contribute to PDGF-stimulated anchorage-independent growth of FLSs (14). Apparently other intracellular signals elicited by TGF-ß selectively inhibit FLS growth under anchorage-independent conditions.
The ECM highly regulates collagenase expression. In rheumatoid
synovial tissues collagenase expression is particularly pronounced in
the synovial lining layer. In RA, the cells in this region form a
multilayer structure of macrophages and FLSs with little intervening
extracellular space. Despite this structure, this region stains highly
with Abs to fibronectin. We suggest that the high levels of collagenase
in this region may be related to this structure. We show that ECM
engagement without attendant cytoskeletal formation leads to high level
collagenase expression associated with a change in cell shape. Probably
cytokines, including TNF- secreted by macrophages in this region,
may act synergistically with cytoskeletal changes to increase FLS
metalloproteinase expression (43, 44).
Werb et al. have shown that collagenase expression by rabbit synovial
fibroblasts is cooperatively regulated by binding of the
4 and 5 integrins to fibronectin (15).
Our results show human RA FLSs do not adhere to fibronectin by
4ß1, but use v and
5ß1 integrin receptors. In contrast to
rabbit synovial fibroblasts in which 5 engagement
increases collagenase expression, 5 engagement modestly
inhibits RA FLS collagenase expression. Engagement of v
integrin receptors is more important in the down-regulation of
collagenase expression upon FLS binding to fibronectin. This result is
consistent with similar collagenase down-regulation upon FLS binding to
vitronectin; binding to vitronectin only required v
receptors.
Recently, Wang et al. have reported data suggesting that
v, 5, and 4 receptors are
important in IL-1ß-stimulated invasion of cartilage (45). However,
4 blocking Abs had no effect on unstimulated FLSs.
Possibly, IL-1 stimulated FLS to express increased 4
integrin receptors, and thus this receptor may contribute to regulation
of proliferation and/or collagenase expression in IL-1-stimulated FLSs.
Further studies will be needed to clarify this possibility.
Integrins use at least two intracellular signaling pathways and a
plethora of signaling molecules to deliver signals intracellularly.
Early studies identified FAK as a potential mediator of integrin
signaling. Inhibition of FAK is associated with disruption or
reorganization of the actin cytoskeleton (46, 47), and a constitutively
activated form of FAK confers resistance to anoikis on epithelial cell
lines (48). Intracellular pathways not involving FAK can be activated
upon integrin stimulation. Integrin engagement activates
mitogen-activated protein (MAP) kinases, Erk1 and Erk2, through a
Ras-dependent pathway (49). Although previously thought to be
downstream from FAK activation, Ras-dependent integrin signals
represent a separate signaling pathway (32, 49). The adaptor protein,
Shc, may mediate this pathway by recruiting Grb2 to ß1
integrins (32). Surprisingly, triggering this pathway does not require
the ß1 integrin cytoplasmic tail that binds to
cytoskeletal proteins to form the focal adhesion complex. However,
formation of the cytoskeleton plays a key role in signal transduction,
since phosphorylation of both MAP kinases (50) and FAK (51) upon
integrin engagement depends on cytoskeletal formation. Clustering of
Src and MAP kinases and Ras upon integrin aggregation also depends on
the formation of a cytoskeleton (52, 53). Apparently cytoskeletal
formation is requisite for propagation of Ras-dependent signals,
although it is not required for early aspects of signal transmission.
Thus, our data do not distinguish between Ras- and FAK-dependent
pathways in mediating integrin signaling intracellularly on FLS
collagenase expression. Our data do suggest that one of these
intracellular signaling pathways interacts with TNF- signals to
synergistically regulate collagenase gene expression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert Lafyatis, The Arthritis Center, Boston University School of Medicine, Building K-5, 80 East Concord Ave., Boston, MA 02118. E-mail address:
3 Abbreviations used in this paper: FLS, fibroblast-like synoviocytes; PDGF, platelet-derived growth factor; ECM, extracellular matrix; RA, rheumatoid arthritis; OA, osteoarthritis; RGD, Arg-Gly-Asp; HEMA, poly(2-hydroxyethyl methacrylate); rRNA, ribosomal ribonucleic acid; MAP, mitogen-activated protein.
Received for publication April 20, 1998. Accepted for publication October 14, 1998.
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5ß1 and 4ß1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. J. Cell Biol. 129:867.2ß1 is a positive regulator of collagenase (MMP-1) and collagen 1(I) gene expression. J. Biol. Chem. 22:13548.
v subfamily of integrins and their putative ligands in the synovial cell layer. J. Rheum. 22:16.[Medline]
1, 3 and 5 chains of ß1 integrins. Virchows Arch. 425:171.[Medline]
2ß1 integrin in collagen. J. Biol. Chem. 266:7363.v and ß1 integrins in the adhesion of human dermal fibroblasts to provisional matrix proteins fibronectin, vitronectin, and fibrinogen. J. Invest. Dermatol. 106:102.[Medline]
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(cA2) versus placebo in rheumatoid arthritis. Lancet 344:1105.[Medline]
gene expression in human rheumatoid synoviocytes and normal monocytes by in situ hybridization. J. Immunol. 1454:154.
in synovial tissues and at the cartilage-pannus junction in patients with rheumatoid arthritis. Arthritis Rheum. 34:1125.[Medline]
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O. Sanchez-Pernaute, M. J. Lopez-Armada, E. Calvo, I. Diez-Ortego, R. Largo, J. Egido, and G. Herrero-Beaumont Fibrin generated in the synovial fluid activates intimal cells from their apical surface: a sequential morphological study in antigen-induced arthritis Rheumatology, January 1, 2003; 42(1): 19 - 25. [Abstract] [Full Text] [PDF]
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T C A Tolboom, E Pieterman, W H van der Laan, R E M Toes, A L Huidekoper, R G H H Nelissen, F C Breedveld, and T W J Huizinga Invasive properties of fibroblast-like synoviocytes: correlation with growth characteristics and expression of MMP-1, MMP-3, and MMP-10 Ann Rheum Dis, November 1, 2002; 61(11): 975 - 980. [Abstract] [Full Text] [PDF]
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 M. Walther, H. Harms, V. Krenn, S. Radke, S. Kirschner, and F. Gohlke Synovial Tissue of the Hip at Power Doppler US: Correlation between Vascularity and Power Doppler US Signal Radiology, October 1, 2002; 225(1): 225 - 231. [Abstract] [Full Text] [PDF]
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 Y. Matsumoto, K. Tanaka, G. Hirata, M. Hanada, S. Matsuda, T. Shuto, and Y. Iwamoto Possible Involvement of the Vascular Endothelial Growth Factor-Flt-1-Focal Adhesion Kinase Pathway in Chemotaxis and the Cell Proliferation of Osteoclast Precursor Cells in Arthritic Joints J. Immunol., June 1, 2002; 168(11): 5824 - 5831. [Abstract] [Full Text] [PDF]
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D Baeten, P Demetter, C Cuvelier, F Van den Bosch, E Kruithof, N Van Damme, G Verbruggen, H Mielants, E M Veys, and F De Keyser Comparative study of the synovial histology in rheumatoid arthritis, spondyloarthropathy, and osteoarthritis: influence of disease duration and activity Ann Rheum Dis, December 1, 2000; 59(12): 945 - 953. [Abstract] [Full Text]
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 G. G. Vaday, R. Hershkoviz, M. A. Rahat, N. Lahat, L. Cahalon, and O. Lider Fibronectin-bound TNF-{alpha} stimulates monocyte matrix metalloproteinase-9 expression and regulates chemotaxis J. Leukoc. Biol., November 1, 2000; 68(5): 737 - 747. [Abstract] [Full Text]
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 L Yan, M. Moses, S Huang, and D. Ingber Adhesion-dependent control of matrix metalloproteinase-2 activation in human capillary endothelial cells J. Cell Sci., January 11, 2000; 113(22): 3979 - 3987. [Abstract] [PDF]
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Y. T Konttinen, T. F. Li, J. W. Xu, M. Tagaki, L. Pirilä, T. Silvennoinen, S. Santavirta, and I. Virtanen Expression of laminins and their integrin receptors in different conditions of synovial membrane and synovial membrane-like interface tissue Ann Rheum Dis, November 1, 1999; 58(11): 683 - 690. [Abstract] [Full Text]
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 M. Sen, K. Lauterbach, H. El-Gabalawy, G. S. Firestein, M. Corr, and D. A. Carson Expression and function of wingless and frizzled homologs in rheumatoid arthritis PNAS, March 14, 2000; 97(6): 2791 - 2796. [Abstract] [Full Text] [PDF]
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