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Transforming growth factor induces fibroblast fibrillin-1 matrix formation.

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Vol. 46, No. 11, November 2002, pp 3000–3009
DOI 10.1002/art.10621
© 2002, American College of Rheumatology
Transforming Growth Factor ␤ Induces
Fibroblast Fibrillin-1 Matrix Formation
Eugene Y. Kissin, Raphael Lemaire, Joseph H. Korn, and Robert Lafyatis
normal or scleroderma fibroblasts. However, TGF␤ did
not alter the expression of either soluble fibrillin protein
or fibrillin mRNA.
Conclusion. Our data show that TGF␤ induces
fibrillin protein incorporation into the extracellular
matrix without affecting fibrillin gene expression or
protein synthesis, suggesting that fibrillin matrix assembly is regulated extracellularly. TGF␤ might increase fibrillin matrix by activating myofibroblasts.
Such TGF␤-mediated effects could account for the
increased fibrillin matrix observed in SSc skin.
Objective. Fibrillin, an extracellular matrix protein implicated in dermal fibrosis, is increased in the
reticular dermis of systemic sclerosis (SSc) skin. We
undertook this study to investigate the hypothesis that
transforming growth factor ␤ (TGF␤) or other cytokines regulate fibrillin matrix formation by normal and
SSc fibroblasts. We further investigated the mechanism
of TGF␤-induced fibrillin fibrillogenesis and its relationship to myofibroblasts.
Methods. Fibrillin and fibronectin matrix deposition and ␣-smooth muscle actin expression by fibroblast
cultures from normal and SSc skin treated with TGF␤
or other cytokines were analyzed by immunofluorescence. Supernatant and extracellular matrix from normal and SSc fibroblasts treated with or without TGF␤
were evaluated by Western blot and Northern blot for
fibrillin protein and messenger RNA (mRNA) expression, respectively.
Results. Immunofluorescence demonstrated increased fibrillin matrix formation by normal and scleroderma fibroblasts after TGF␤ treatment. Other cytokines, including tumor necrosis factor ␣, interleukin-1␤
(IL-1 ␤ ), IL-4, granulocyte–macrophage colonystimulating factor, and platelet-derived growth factor,
did not affect fibrillin fibrillogenesis. Fibrillin matrix
formed in proximity to myofibroblasts and independently of up-regulation of fibronectin matrix or cell
number. Western blot analysis of extracellular matrix
confirmed increased fibrillin after TGF␤ stimulation of
Transforming growth factor ␤ (TGF␤) is an
important mediator of fibrosis. TGF␤ stimulates the
synthesis and assembly of matrix proteins such as fibronectin and collagen (1,2). TGF␤ also promotes the
formation of myofibroblasts, activated fibroblasts responsible for the generation of contractile force (3).
Many studies have suggested a role for TGF␤ in
the pathogenesis of scleroderma, or systemic sclerosis
(SSc), a systemic disease that leads to fibrosis of the skin
and internal organs (4). The effects of TGF␤ in vitro and
in vivo mimic many of the observed changes in SSc skin
pathology. These include increased collagen and fibronectin matrix formation (1,4) and increased connective tissue growth factor expression (5). Elevated levels
of TGF␤ in several organs in SSc patients have been
reported. TGF␤2 has been shown in some studies, but
not in all, to be overexpressed in SSc skin (6–9). Some
reports, but not all, also indicate increased levels of
TGF␤ (particularly TGF␤1) in bronchoalveolar lavage
fluids from SSc patients with pulmonary fibrosis (10,11).
Other studies have indicated that fibroblasts from SSc
patients are more sensitive to TGF␤ than are normal
fibroblasts (12,13). A potential mechanism for increased
sensitivity was suggested through the observation that
TGF␤ receptor type I (TGF␤RI) and TGF␤RII are
overexpressed in scleroderma fibroblasts compared with
control fibroblasts (14). Genetic studies have also sup-
Supported by USPHS grants AR-32343 and AR-07598, by the
Scleroderma Research Fund, and by an Arthritis Foundation Basic
Science Research Grant.
Eugene Y. Kissin, MD, Raphael Lemaire, PhD, Joseph H.
Korn, MD, Robert Lafyatis, MD: Boston University School of Medicine, Boston, Massachusetts.
Address correspondence and reprint requests to Robert
Lafyatis, MD, Boston University Medical Center, Arthritis Center,
K-5, 80 East Concord Street, Boston, MA 02118. E-mail: rlafyatis@
Submitted for publication February 15, 2002; accepted in
revised form August 5, 2002.
ported a role for TGF␤ in SSc. Certain alleles for
TGF␤2 and TGF␤3 are encountered more frequently in
patients with SSc (15).
Fibrillin is a connective tissue protein produced
by fibroblasts and smooth muscle cells that helps form
extracellular microfibrils and has also been implicated in
the pathogenesis of fibrosis (16). Fibrillin-containing
microfibrils interact both with basement membranes and
with nearby elastic fibers and may help stabilize the
extracellular matrix by forming a scaffold (17).
Several studies have implicated fibrillin in the
pathogenesis of scleroderma (18). In biopsy samples
from affected skin, fibrillin lacking associated elastin is
markedly increased in the lower reticular dermis in a
randomly arranged pattern (19). Further supporting the
role of fibrillin in scleroderma is the observation that the
murine tight skin 1 (Tsk1) phenotype is due to an
in-frame duplication of exons 17–40 of the fibrillin-1
gene (20). Dermal pathology in these mice has similarities to human SSc (21). Studies in humans have also
supported a genetic link between fibrillin mutations and
SSc. A study of Choctaw American Indians, a population
with a high prevalence of SSc, has shown an association
of a 2-cM haplotype that contains 2 markers for the
fibrillin-1 gene with SSc (22). Further studies have
indicated that a single-nucleotide polymorphism in the
5⬘-untranslated region of fibrillin is strongly associated
with SSc (23). Together, these observations suggest that
abnormal fibrillin deposition might contribute to fibrosis
in SSc.
We have investigated the potential role on fibrillin regulation of a variety of cytokines implicated in
fibrosis or SSc. We show here that TGF␤, but not other
cytokines tested, stimulates the incorporation of fibrillin
into a fibrillar matrix by both normal and SSc fibroblasts.
Strikingly, we show that TGF␤ stimulates fibrillin fibrillogenesis without affecting messenger RNA (mRNA)
expression or protein secretion. Thus, TGF␤ stimulates
fibrillin fibrillogenesis at the step of fibrillin incorporation into the extracellular matrix.
Cell culture. Fibroblasts were grown from skin biopsy
samples obtained from the forearms of normal volunteers or
patients with SSc. The epidermis of the biopsy specimen was
removed and the dermis was sectioned into submillimeter
pieces. These dermal fragments were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% heatinactivated fetal calf serum and penicillin G (100 units/ml)/
streptomycin (100 ␮g/ml) at 37°C in an incubator in 8% CO2.
Cells reached confluence in 3–4 weeks and were used at
passages 3–7.
Immunofluorescence. Cultured human dermal fibroblasts (0.2 ml of 3 ⫻ 105/ml ⫽ 6 ⫻ 104/well) were plated onto
8-well chamber slides in 10% fetal bovine serum (FBS) containing DMEM. Chamber slides were examined 24 hours after
plating to ensure confluent cell cultures prior to replacement
of the media with serum-free DMEM. Human TGF␤1 (R&D
Systems, Minneapolis, MN), platelet-derived growth factor
type BB (PDGF-BB; Gibco BRL, Rockville, MD),
interleukin-1␤ (IL-1␤; Endogen, Cambridge, MA), IL-4 (R&D
Systems), tumor necrosis factor ␣ (TNF␣; Gibco BRL), or
granulocyte–macrophage colony-stimulating factor (GM-CSF;
McKesson BioServices, Rockville, MD) was added to some
chamber wells at the indicated concentrations.
After 4 days, supernatants were aspirated and the cells
were fixed with 100% methanol for 5 minutes. Cells were
washed with Tris buffered saline (TBS; 50 mM Tris [pH 8.0],
150 mM NaCl) and then incubated with rabbit antifibrillin
antibody (pAb9543; kindly provided by Dr. Lynn Sakai [17,24])
at 1:250 dilution in TBS for 2 hours at 37°C. This antibody was
raised against the amino-terminal half of human fibrillin-1 and
cross-reacts with mouse fibrillin-1, but not with fibrillin-2
(17,24). Some cells were also incubated with mouse monoclonal anti–␣-smooth muscle actin (anti–␣-SMA) antibodies
(clone 1A4; Sigma, St. Louis, MO) at a 1:250 dilution in TBS
during the same time period. Cells were washed 3 times and
incubated with rhodamine-conjugated donkey anti-rabbit antibody as well as with either fluorescein isothiocyanate (FITC)–
conjugated goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA) or FITC-conjugated mouse
antifibronectin antibody (Jackson ImmunoResearch) for 1
hour. Cells were washed again and nuclei were stained for 1
minute using Hoechst reagent (100 ng/ml in phosphate buffered saline [PBS]).
Chambers were removed from the slides, and cells
were visualized using a fluorescence microscope (Olympus,
Lake Success, NY) and photographed using a DC 120 Zoom
digital camera (Eastman Kodak, Rochester, NY). Immunofluorescence results were quantified using image analysis software (version 1.62; NIH Image, National Institutes of Health,
Bethesda, MD; online at
Images were analyzed by setting the density slice option so that
positively stained fibers were measured.
Western blot. Dermal fibroblasts were passaged in
10% serum containing DMEM for 24 hours, and the media
were replaced with serum-free DMEM. Cells were then
treated with 5 ng/ml of TGF␤ or were left untreated. After
48 hours, supernatants, cell lysates, and extracellular matrix
were prepared for sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE) analysis similar to that described
by Kitahama et al (25).
Supernatants (1 ml) were mixed with 273 ␮l of 10%
trichloroacetic acid for 30 minutes on ice and centrifuged at
16,000g for 20 minutes at 4°C, and the resulting pellet was
resuspended in 100 ␮l of 2⫻ SDS-PAGE sample buffer
(0.125M Tris [pH 6.8], 20% [weight/volume] glycerol, 4.6%
SDS). Cells and extracellular matrix were scraped in 400 ␮l of
1% Nonidet P40 in 50 mM Tris, pH 8.0, supplemented with 1
mM phenylmethylsulfonyl fluoride. Fifty microliters of sample
was saved for bicinchoninic acid assay for total protein con-
Figure 1. Induction of fibrillin fibrillogenesis in dermal fibroblasts by transforming growth factor ␤ (TGF␤). A, Left panels show nuclei stained with
Hoechst reagent (blue); right panels show overlays with nuclei stained with Hoechst reagent (blue) and fibrillin fibers stained with rhodamine (red).
Fibrillin can be seen most prominently in areas of greatest cell density. Cultures were left untreated (control) or treated with 5 ng/ml TGF␤, as
indicated. B, Rhodamine immunofluorescence in A is shown graphically after image analysis. C, In contrast to cells treated with 5 ng/ml TGF␤,
treatment with tumor necrosis factor ␣ (TNF␣; 10 ng/ml), interleukin-1␤ (IL-1; 1 ng/ml), IL-4 (5 ng/ml), or granulocyte–macrophage
colony-stimulating factor (GM-CSF; 5 ng/ml) did not increase matrix fibrillin (red fibers) over that seen in control cells. (Original magnification ⫻ 20
in A and C.)
centration according to the supplied protocol (Micro BCA
Protein Assay; Pierce, Rockford, IL), while the remainder was
vortexed and centrifuged at 16,000g for 10 minutes at 4°C. The
supernatant, cell lysate fraction, was mixed with 400 ␮l of 2⫻
SDS-PAGE sample buffer. The pellet, matrix fraction, was
resuspended in 100 ␮l of 1.2⫻ SDS-PAGE sample buffer
containing 10% ␤-mercaptoethanol.
Samples were heated to 95°C for 3 minutes. Fifteen
micrograms of total protein from normal fibroblast culture
supernatants and matrix fractions and 40 ␮g of total protein
from scleroderma fibroblast culture supernatants and matrix
fractions were analyzed on 5% Tris/glycine SDS–
polyacrylamide gels. Proteins were transferred to nitrocellulose in 10 mM borax buffer (50 mM Tris, 380 mM glycine, 0.1%
SDS, 20% methanol, and 10 mM borax). Membranes were
blocked with 5% nonfat milk in PBS and incubated with rabbit
antifibrillin primary antibody (pAb9543, described above) at a
1:1,000 dilution in PBS for matrix protein and a 1:4,000
dilution in PBS for supernatant protein for 2 hours. After
washing in 0.05% PBS–Tween (PBST), blots were incubated
with peroxidase-conjugated anti-goat antibody (Jackson
ImmunoResearch) for 1 hour at 1:1,000 dilution in PBS for
matrix protein and 1:4,000 dilution in PBS for supernatant
protein and again washed in 0.05% PBST. Blots were developed by enhanced chemiluminescence detection (SuperSignal,
West PICO chemiluminescent substrate; Pierce).
Northern blot. Cultured human dermal fibroblasts
were passaged in DMEM containing 10% FBS for 24 hours
and the media replaced with serum-free DMEM supplemented with 0.1 mg/ml bovine serum albumin. For Northern
blot analyses, cells were harvested for RNA using the RNeasy
mini protocol for isolation of total RNA according to the
supplied protocol (Qiagen, Valencia, CA). RNAs were analyzed by formaldehyde/1% agarose gel electrophoresis and
Figure 2. Induction of fibrillin fibrillogenesis in systemic sclerosis (SSc) fibroblasts by transforming growth factor ␤ (TGF␤). Shown is a representative SSc fibroblast culture stained for fibrillin
with rhodamine (red) and counterstained with Hoechst reagent (blue). SSc fibroblasts show little
baseline deposition of fibrillin matrix (control), but considerable fibrillin matrix formed after
treatment with TGF␤. (Original magnification ⫻ 40.)
transferred to Nytran as described previously (26). Fibrillin
mRNA was detected by hybridization to a 32P-labeled complementary DNA fragment of a 593-nucleotide human fibrillin-1
complementary DNA. By BLAST comparison, this region
shows no homology with fibrillin-2. This fragment was obtained by reverse transcription of poly(dT)-primed human
dermal fibroblast RNA followed by polymerase chain reaction
using primers (hFBN-1 5⬘, bp 1970–1991, 5⬘-TGCGGAGCACATGCTATGGTGG-3⬘ and hFBN-1 3⬘, bp 2562–2539,
1970–2562 of the fibrillin-1 mRNA. Signals were detected
using a Cyclone phosphorimager (Hewlett-Packard, McMinnville, OR) and quantified using the OptiQuant software
Fibrillin fibrillogenesis stimulated by TGF␤, but
not by other cytokines. To investigate the effect of
cytokines implicated in fibrosis or SSc, cultured dermal
fibroblasts from normal volunteers were stimulated with
TGF␤, IL-4, TNF␣, IL-1␤, GM-CSF, or PDGF-BB.
Only stimulation with TGF␤ resulted in increased fibrillin fiber formation (Figures 1A and B). After 4 days,
TGF␤-stimulated fibroblasts appeared more dense and
aggregated to discrete areas on the slide surface. These
cultures revealed heavy fibrillin deposition over cell
clusters, with more sparse deposition in other areas.
While this pattern was observed in cultures both with
and without TGF␤ treatment, cultures treated with
TGF␤ showed increased cell number, increased cell
aggregation, and increased fibrillin deposition (Figures
1A and B). Low-level fibrillin staining in control cultures
may have been partly due to lower plating density of
cells used in the current study compared with that used
in other studies (27). Dermal fibroblasts from skin
affected by scleroderma also showed increased fibrillin
matrix with TGF␤ (Figure 2).
Next, to determine whether TGF␤ increases
fibrillin production simply by increasing the cell number,
we counterstained cells for nuclei. We were thus able to
compare cell clusters with roughly equivalent cell densities. We found that TGF␤ increased fibrillin deposition
even when cell density was controlled for (Figure 3A,
panels a and b). To further control for the effects of cell
number, normal fibroblasts were stimulated with PDGF,
another known fibroblast mitogen. While PDGF also
increased the cell number, its effect on fibrillin matrix
deposition was minimal compared with that of TGF␤
Figure 3. No dependence on increased cell number of transforming
growth factor ␤ (TGF␤) induction of fibrillin incorporation into
matrix. A, Identical regions of normal dermal fibroblasts costained for
fibrillin (with rhodamine; red) (a–c) and fibronectin (with fluorescein
isothiocyanate; green) (d–f). Fibroblast cultures were treated with 5
ng/ml TGF␤ (b and e) or 5 ng/ml platelet-derived growth factor
(PDGF) (c and f). Hoechst staining (blue) shows nuclei in all panels.
To control for the effect of TGF␤ on cell proliferation, areas of the
culture slide containing approximately equal numbers of cells with and
without TGF␤ treatment are shown (92 control cells in a and d; 75
TGF␤-treated cells in b and e). PDGF induces fibronectin fibrillogenesis (f), but not fibrillin fibrillogenesis (c). Arrowheads in a and d
indicate colocalization of some fibrillin and fibronectin matrix strands
(original magnification ⫻ 20). B, Fibrillin and fibronectin immunofluorescence in A shown graphically after image analysis.
(Figure 3A, panels b and c). Therefore, the effect of
TGF␤ on fibrillin matrix formation is not mediated only
through changes in cell number.
Since other groups of investigators have shown
that fibronectin colocalizes with fibrillin and have suggested that fibronectin may act as a template for fibrillin
fibrillogenesis (28), we tested whether TGF␤ increases
fibrillin matrix as a byproduct of increasing fibronectin
formation. We first costained for fibrillin and fibronectin
matrix in cultured dermal fibroblasts and found partial
colocalization of the two proteins (Figure 3A, panels a
and d). Investigators in our group have previously shown
that PDGF and TGF␤ stimulated fibronectin fibrillogenesis by synovial fibroblasts (29). We therefore compared the effects of TGF␤ with those of PDGF on
fibrillin and fibronectin formation. PDGF induced substantially greater fibronectin fibrillogenesis with relatively little effect on fibrillin fibrillogenesis, while TGF␤
up-regulated both processes (Figures 3A, panels b, c, e,
and f, and 3B).
Association of fibrillin fibrillogenesis with myofibroblasts. Fibronectin and other matrix proteins are
produced at increased levels by myofibroblasts. Since
TGF␤ is known to induce myofibroblast formation, we
costained cell cultures for ␣-SMA, a marker of myofibroblasts, in order to determine whether fibrillin matrix
is associated with myofibroblast formation. Untreated
cultures showed only occasional ␣-SMA–positive cells.
Cultures treated with TGF␤ showed a dramatic increase
in ␣-SMA–positive cells (Figure 4), as previously reported (30). TGF␤-induced fibrillin fibers were generally associated with ␣-SMA–positive cells (Figure 4,
white arrows). Myofibroblasts were associated with cell
aggregates, and such foci also showed increased fibrillin.
Although immunofluorescent staining for fibrillin generally localized to culture regions containing myofibroblasts, the overlap was not complete (Figure 4, blue
arrow). Some ␣-SMA–positive cells showed no fibrillin,
and some fibrillin fibers could be seen in association with
␣-SMA–negative cells.
Induction of fibrillin fibrillogenesis by TGF␤
without change in fibrillin mRNA or protein expression.
Many effects of TGF␤ are mediated through changes in
gene expression. To further elucidate the mechanism by
which TGF␤ exerts its effect on fibrillin matrix formation, we analyzed the effect of TGF␤ on fibrillin mRNA
expression. Surprisingly, TGF␤ had no effect on fibrillin
mRNA expression by either normal or SSc dermal
fibroblasts (Figure 5).
We next determined whether TGF␤ affects fibrillin fibrillogenesis through increased fibrillin protein
synthesis or secretion. We analyzed supernatant and
matrix fractions from dermal fibroblasts with and
without TGF␤ treatment. Matrix was solubilized using
SDS-PAGE sample buffer containing 10%
Figure 4. Fibrillin matrix production by myofibroblasts. Dermal fibroblasts that were untreated (top panels) or were treated with
5 ng/ml of transforming growth factor ␤ (TGF␤) (bottom panels) were costained for fibrillin fibers with rhodamine (red) (left panels)
and for myofibroblasts with fluorescein isothiocyanate (green) (right panels). Cell cultures were costained with ␣-smooth muscle actin
(␣-SMA), a marker of myofibroblasts, to determine whether fibrillin matrix is associated with myofibroblast formation. White arrows
indicate colocalization of myofibroblasts with fibrillin matrix (␣-SMA–positive cells). Blue arrow (upper right panel) indicates
myofibroblasts without surrounding fibrillin fibers. Stimulation with TGF␤ produced increased numbers of myofibroblasts and
surrounding fibrillin fiber formation (bottom panels). (Original magnification ⫻ 20 .)
␤-mercaptoethanol. Reduction of disulfide bonds by
␤-mercaptoethanol extracts a proportion of fibrillin
bound to matrix, although perhaps not all (31). In
normal fibroblast cell lines, TFG␤ did not significantly
affect the amount of fibrillin in the soluble/supernatant
fraction of cell cultures (Figure 6A). Despite this, TGF␤
increased the amount of fibrillin in the matrix fraction of
cultured fibroblasts, as expected from our immunofluorescence results (compare Figure 6 with Figures 1–3).
Antibody specificity for fibrillin was confirmed in control experiments using fibroblast supernatants from control (pa/pa) and Tsk1 (tsk/pa) mice. Control mouse
fibroblast supernatants showed a single high molecular
weight band of the same size as that seen in the human
immunoblots (Figure 6B). An additional higher molecular weight band was seen in the supernatant from Tsk1
fibroblasts corresponding to mutated fibrillin, containing
a large in-frame insertion (Figure 6B).
Similar results were obtained from SSc fibroblast
cell lines (Figure 6C). As in normal fibroblasts, matrix
fibrillin was increased in all cell lines. Although TGF␤
led to a modest increase in soluble fibrillin in one SSc
cell line, the change in matrix-associated protein was
much more dramatic. In all cell lines tested, the amount
of soluble fibrillin greatly exceeded that found in the
matrix. This finding suggests that the availability of
soluble fibrillin is not the limiting step in fibrillin fibrillogenesis. There was no consistent difference in basal or
TGF␤-induced soluble or matrix-associated fibrillin between normal and scleroderma cell lines (data not
shown). Differences in matrix formation at baseline and
after TGF␤ treatment correlated with the passage number. Both normal and SSc fibroblast cultures of low
passage formed greater amounts of fibrillin matrix than
did those of higher passage (data not shown).
The generation of extracellular matrix begins
with synthesis of matrix proteins and is followed by
Figure 5. Fibrillin expression in fibroblasts treated with TGF␤. Normal and SSc dermal fibroblast cultures were left untreated or were
treated with 5 ng/ml of TGF␤ for 48 hours, and then mRNA was
purified, blotted, and hybridized with a fibrillin-1 cDNA probe. Signal
was detected using a phosphorimager. The blot was then rehybridized
with an 18S rRNA probe, and the signal was detected again on a
phosphorimager. Signal intensities were analyzed using OptiQuant
software, fibrillin expression was normalized to the 18S rRNA signal,
and fibrillin expression in TGF␤-treated cells was normalized to
fibrillin-1 expression in untreated cells. See Figure 2 for definitions.
formation of an insoluble matrix. Incorporation of collagen and fibronectin into the extracellular matrix provides examples of matrix fibrillogenesis. Type I collagen
assembles passively into matrix by self-polymerization
(32), although certain proteins, including other collagen
types, fibromodulin, lumican, decorin, and tenascin, can
modify this process (33–35). In contrast, fibronectin
matrix assembly is an active process and requires several
conditions: an intact intracellular cytoskeleton, an activated fibronectin-binding integrin receptor, and tension
across the developing fibrillar structure (36,37). Investigators in our group showed previously that TGF␤ and
PDGF stimulate fibronectin matrix assembly, possibly
through changes in integrin receptor activity (29).
Our results demonstrate that TFG␤ also stimulates fibrillin fibrillogenesis. The action of TGF␤ on
fibrillin was not mediated through increased cell number
alone, since stimulation of fibroblasts with PDGF increased cell number and density, but had no effect on
fibrillin fibril formation. Further, TGF␤ did not change
fibrillin mRNA expression or secretion, despite dramatically increasing fibrillin fibrillogenesis. Thus, our data
indicate that TGF␤ induces fibrillar fibrillin by affecting
the incorporation of soluble fibrillin into cross-linked
fibrillin matrix. Our understanding of this process is
incomplete, but the process starts with release of soluble
fibrillin, which is then incorporated into matrix fibrils.
Like fibronectin, fibrillin incorporation into matrix fibrils involves several steps, including covalent
cross-linking through intermolecular cysteine bonds
(31). Heparin inhibits fibrillin fibrillogenesis, suggesting
that as-yet-undefined active mechanisms might regulate
fibrillin matrix assembly (38). Our data, which show that
the formation of fibrillin matrix is independent of soluble fibrillin concentration in the media, suggest that, like
fibronectin matrix formation, fibrillin matrix formation
is an active process. Our data showing that TGF␤
stimulates this process further support active, perhaps
cell-directed, fibrillin matrix assembly. The mechanisms
that underlie this process and the effect of TGF␤ remain
unclear. Some investigators have suggested that fibrillin
matrix depends on fibronectin matrix assembly (28).
While a minimal amount of fibronectin may be necessary for fibrillin scaffold formation, up-regulation of
fibronectin with PDGF did not produce similar increased fibrillin matrix. It is noteworthy, however, that
fibrillin, like fibronectin, contains an RGD integrin
binding site (39), with highest affinity for ␣v␤3 receptors, suggesting that like fibronectin, fibrillin binding to
integrins might stimulate fibrillin matrix formation.
Fibrillin is found in increased amounts in the
lower reticular dermis of scleroderma skin. Previous
investigation has shown that TGF␤ regulates collagen
and fibronectin matrix formation and suggests that it
may induce fibrosis in SSc skin. The present study did
not show a difference in basal or TGF␤-induced fibrillin
matrix formation between normal and SSc fibroblasts,
consistent with the observations by Wallis et al (40).
Wallis et al also showed that fibrillin matrix produced by
SSc fibroblasts is less stable than that produced by
normal fibroblasts (40). Although these intriguing observations are similar to an observed decrease in stability
of fibrillin produced by Tsk1 mouse fibroblasts (41), they
do not readily explain the presence of increased fibrillin
in the dermis of SSc skin. Our results suggest that the
excess fibrillin found in SSc skin may be the result of
increased TGF␤ secreted by endogenous or infiltrating
cells in SSc skin, or of increased sensitivity to TGF␤.
Potentially, TGF␤ may trigger both fibroblast production of excess collagen and excess fibrillin fibrillogenesis.
Our observation that TGF␤, but not other cytokines that
induce collagen and/or are implicated in SSc (including
PDGF, TNF␣, IL-1␤, IL-4, or GM-CSF), causes increased fibrillin fibrillogenesis supports the notion that
TGF␤ plays a particularly prominent role in this disease.
Fibrillin incorporation into the matrix was asso-
Figure 6. Increased extracellular matrix accumulation, but not secreted fibrillin, induced in dermal
fibroblasts by TGF␤. A, Normal or C, SSc fibroblasts were left untreated or treated with 5 ng/ml
TGF␤ for 24 hours, and proteins were precipitated from supernatants (soluble) or extracted from
extracellular matrix (matrix). Total protein was assayed and equal amounts were loaded in each
lane. Nonreduced (soluble) and reduced (matrix) proteins were analyzed by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis and blotted to nitrocellulose, and fibrillin was detected
as described in Materials and Methods. B, Proteins from wild-type (WT) control (pa/pa) mouse or
tight skin 1 (tsk/pa) mouse fibroblast supernatants were precipitated and analyzed as in A and C.
See Figure 2 for other definitions.
ciated with myofibroblasts, and TGF␤ is known to
induce myofibroblast formation. The ability of TGF␤ to
up-regulate ␣-SMA expression depends on the cellular
growth phase. In proliferating fibroblast cultures at low
density, TGF␤ stimulates less production of ␣-SMA
than it does under nonproliferating, high-density conditions (42). This may explain the association of fibrillin
matrix with regions of increased cell density, if myofibroblasts are responsible for integrating soluble fibrillin
into matrix. Myofibroblasts share many properties with
smooth muscle cells, and others have shown that fibrillin
matrix is assembled faster by smooth muscle cells than
by fibroblasts (43). Furthermore, ultrastructural analyses
revealed fibrillin fibers assembled by smooth muscle
cells to be of greater length than those assembled by skin
fibroblasts. Myofibroblasts are also increased in SSc and
could therefore account for increased fibrillin matrix in
SSc skin. Alternatively, fibrillin matrix might protect
myofibroblasts from apoptosis. Jelaska and Korn have
shown previously that prolonged treatment with TGF␤
induces myofibroblasts and protects dermal fibroblasts
from apoptosis (30). Perhaps fibrillin, alone or through
interactions with other matrix proteins, protects myofibroblasts from apoptosis (44–46).
Observations in mice with mutations in fibrillin
suggest that fibrillin may play a more primary role in SSc
by regulating connective tissue homeostasis and fibrosis.
Most significantly, Tsk1 mice show dermal fibrosis histologically similar to that seen in SSc skin (20). It is not
known how mutant fibrillin leads to fibrosis, but this
observation suggests that fibrillin may normally regulate
connective tissue structure. Pereira et al have suggested
a similar role for fibrillin based on the vascular pathology of mice harboring a targeted homozygous mutation
in fibrillin that leads to an internally deleted protein
(47). Homozygous mutant mice develop Marfan’s
syndrome–like features and die perinatally due to vascular complications. Surprisingly, despite markedly disorganized structure of the vascular wall, these animals
have relatively preserved elastic fibers. These and other
results have challenged the previously held belief that
fibrillin functions primarily in elastic fibers, and suggest
that fibrillin helps to organize connective tissue structure
(48). Therefore, not only does increased fibrillin deposition suggest increased TGF␤ activity, it also suggests
that TGF␤ may mediate increased fibrosis in SSc skin.
In summary, we show that TGF␤ induces markedly increased fibrillin matrix by stimulating soluble
fibrillin incorporation into the extracellular matrix.
These observations indicate a possible role for TGF␤ in
both human and murine fibrotic diseases through increased accumulation of matrix fibrillin.
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