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1 From the University of Texas Southwestern Medical Center at Dallas; and the 2 University of Cincinnati, Ohio.
Abstract
PURPOSE. Recent studies indicate that transforming growth factor
(TGF)ß is a potent inducer of corneal myofibroblast
differentiation and expression of smooth muscle–specific, 
-actin
(-SMA). Although TGFß is known to enhance synthesis of
extracellular matrix proteins and receptors, little is known about how
it modulates the expression of smooth muscle proteins in nonmuscle
cells. The purpose of this study was to identify the role of
Arg-Gly-Asp (RGD)-dependent tyrosine phosphorylation in regulating
-SMA gene expression and ultimately myofibroblast development.
METHODS. Because cell culture in serum-containing media mimics myofibroblast
transformation, all experiments were performed on freshly isolated
rabbit keratocytes plated in defined, serum-free media. Cells were
exposed to TGFß (1 ng/ml), Gly-Arg-Gly-Asp-D-Ser-Pro
(GRGDdSP, 50 µM), Gly-Arg-AL-Asp-Ser-Pro (GRADSP; 100 µM), or
herbimycin A (0.1–10 nM) at 24 hours (sparse) or 7 days (confluent).
Cells were evaluated by immunocytochemistry and proteins and RNA
collected for western and northern blot analyses using antibodies
specific for -SMA, fibronectin, focal adhesion proteins, and
phosphotyrosine (clones 4G10 and PY20); and probes directed against
rabbit -SMA. All experiments were repeated at least three times.
RESULTS. Keratocytes exposed to TGFß showed expression of -SMA
that coincided with the intracellular reorganization of the actin
cytoskeleton and the extracellular assembly of fibronectin fibrils.
Addition of RGD containing but not control peptides blocked the
organization of intracellular actin, extracellular fibronectin, and
-SMA protein and mRNA. Immunoprecipitation of cell proteins with
4G10 or PY20 identified the TGFß-associated tyrosine
phosphorylation of paxillin, pp125fak, p130, PLC
, and
tensin, which was blocked by addition of GRGDdSP. Addition of
herbimycin A to keratocytes exposed to TGFß showed a
dose-dependent loss of -SMA protein and mRNA which correlated with
loss of tyrosine phosphorylation, absence of actin reorganization, and
fibronectin assembly.
CONCLUSIONS. The data suggest that TGFß-mediated -SMA gene
expression leading to myofibroblast transformation may involve an
RGD-dependent phosphotyrosine signal transduction
pathway.
During the process of wound healing, invading corneal fibroblasts
exhibit unique ultrastructural and physiological characteristics
similar to smooth muscle cells, including prominent intracellular
microfilament bundles and in vitro contractile responses to smooth
muscle agonists.1
2
These characteristics have been used
to define these cells phenotypically as myofibroblasts. It has been
further recognized that corneal myofibroblast differentiation involves
the expression of the -isoform of actin, specific for vascular
smooth muscle cells.3
Many aspects of myofibroblast function have yet to be understood fully;
however, of particular interest is the regulation and function of
smooth muscle–specific actins in nonmuscle fibroblastic cells. There
are two classes of differentially expressed actin isoforms present in
mammalian cells, comprising the nonmuscle actins ß and (class 1)
and the three isoforms of actins present in cardiac, skeletal, and
vascular muscle (class 2).4
Isoactins show considerable
sequence homology, differing predominantly at the
NH2-terminal sequence for which -SMA contains
an Ac-EEED sequence important in -SMA polymerization.5
In general, -actins appear to localize preferentially to
microfilament bundles—that is, stress fibers, rather than the
actin-rich cortex or lamellipodial ruffles that contain nonmuscle
ß and actin.6
-SMA can be selectively removed
from stress fibers by microinjection of either monoclonal antibodies
specific for the Ac-EEED epitope or by the injection of small peptides
containing the Ac-EEED sequence.5
Displacement of -SMA
from stress fibers leads to physiologically enhanced cell motility and
loss of focal contacts, suggesting that -SMA plays a role in
regulating cell adhesivity.7
Expression of -SMA is both developmentally and environmentally
regulated. It appears in high concentration early in development of
cardiac and skeletal muscle, disappears later,8
9
and
is expressed at the leading edge of migrating neural crest cells
invading the primary corneal stroma.10
During wound
healing, expression is exclusively localized to cells within the
corneal wound and excluded from undamaged regions, even in migrating
cells containing stress fibers.3
In studies in vitro,
-SMA expression in nonmuscle cells has been shown to be cell-density
dependent,11
12
influenced by cell substrate
composition,13
and sensitive to regulation by various
cytokines.14
15
16
Recently, interest has focused on transforming growth factor
(TGF)ß as a potent inducer of -SMA
expression and myofibroblast differentiation of dermal,16
breast,17
palatal,18
lung,11
and
corneal stromal fibroblasts19
and of other nonfibroblastic
cells, including lens epithelial and liver Ito cells.20
21
TGFß plays an important role in the wound
healing response and has been shown to: enhance the synthesis and
deposition of extracellular matrix proteins, including type I collagen
and fibronectin22
23
24
; decrease the degradation of
extracellular matrix by decreasing the synthesis of matrix
metalloproteinases (e.g., collagenase and stromelysin) and enhance the
synthesis of protease inhibitors25
26
; and promote
cell–matrix interaction by upregulating the synthesis of membrane
surface receptors including 5ß1 integrin.27
28
Moreover, inactivation of TGFß by the
application of neutralizing antibodies to dermal or corneal wounds in
vivo blocks the development of fibrosis or scarring, inhibits the
deposition of collagen and fibronectin, and overall, decreases the
number of wound-healing fibroblasts or
myofibroblasts.29
30
31
32
More recent studies indicate that the ED-A domain of fibronectin is
crucial for induction of -SMA expression by
TGFß, suggesting an outside–in signaling
mechanism involving extracellular fibronectin.33
In the
present study we used a serum-free culture system to evaluate the
effect of fibronectin assembly and Arg-Gly-Asp (RGD)-dependent
signaling on the TGFß-mediated expression of
-SMA and myofibroblast differentiation of rabbit corneal
keratocytes. Overall, the data indicate that the upregulation of
-SMA expression by TGFß involves
RGD-dependent, tyrosine phosphorylation consistent with an outside–in
signal transduction pathway and environmental control of myofibroblast
differentiation.
Materials and Methods
Cell Culture
Whole rabbit eyes were obtained from Pel Freez (Rogers, AR). The
surface epithelium was initially scraped from the cornea with a no. 10
Bard Parker blade (Lance, Sheffield, UK), the corneas excised, and the
endothelium removed with a sterile cotton-tipped applicator soaked in
ethanol. The corneas were then digested in sterile 2.0 mg/ml
collagenase (Gibco, Gaithersburg, MD) and 0.5 mg/ml hyaluronidase
(Worthington, Freehold, NJ) in minimum essential medium (MEM; Gibco)
overnight at 37oC. Corneal keratocytes were then
plated in MEM in serum-free media supplemented with RPMI vitamin mix
and glutathione, nonessential amino acids, pyruvic acid, 1% glutamine
and penicillin-streptomycin (Gibco) at 5.0 x
104 cells/cm2. Keratocytes
were plated onto 100- mm dishes (Falcon Primaria; Becton Dickinson,
Lincoln Park, NJ) for biochemical analyses or 12-mm diameter glass
coverslips coated with collagen (Vitrogen; Collagen, Freemont, CA) for
immunocytochemical localization. Primary serum-free cultures were used
exclusively in all experiments.
To evaluate the temporal effects of TGFß on myofibroblast differentiation, primary keratocytes were treated with 1 ng/ml TGFß1 (Gibco) after overnight plating or after 7 days in culture to allow cells to establish cell–cell contacts before exposure to TGFß. Cells were then evaluated between 8 and 72 hours after exposure. To evaluate the effects of RGD-dependent cell–matrix interactions, cells were plated overnight and then treated with TGFß1 (1 ng/ml) in combination with the following peptides from Gibco: Gly-Arg-Gly-Asp-Asn-Pro (GRGDNP, 10–500 µM), Gly-Arg-Gly-Asp-D-Ser-Pro (GRGDdSP, 50 µM), Gly-Arg-AL-Asp-Ser-Pro (GRADSP, 50–100 µM), and Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-AL (GpenGRGDSPCA, 1–1000 µM). Cells were then cultured for 72 hours and evaluated. To study the effects of herbimycin A (Gibco), a tyrosine phosphokinase inhibitor, cells were plated overnight and then exposed to TGFß.(1 ng/ml) in combination with herbimycin A at various concentrations from 0.1 nM to 600 nM. All conditions were evaluated in triplicate, and experiments were repeated at least two times.
Immunocytochemistry
Keratocytes grown on glass coverslips were rinsed once in
phosphate-buffered saline (PBS; pH 7.4) fixed in 1% paraformaldehyde
in PBS, rinsed in PBS, and extracted in cold acetone
(-20oC). Cells were rehydrated in PBS and
nonantigenic sites blocked by incubating with 1% ovalbumin (Sigma, St.
Louis, MO) or 10 µg/ml goat serum (Cappel, Durham, NC). Cells were
then reacted with primary antibodies including, mouse monoclonal
anti--SMA (1:100), clone 1A4 (Sigma); mouse monoclonal anti-human
vinculin (1:100), clone V284 (Serotec, Washington, DC); and fluorescein
isothiocyanate or rhodamine-conjugated goat anti-human fibronectin
(1:20; Binding Site, San Diego, CA) for 60 minutes. Cells were then
washed in PBS and reacted with appropriate affinity-purified
fluorescein-conjugated goat anti-mouse IgG (1:20; Cappel). Cells were
also doubled labeled with rhodamine-conjugated phalloidin (Molecular
Probes, Eugene, OR) to identify f-actin filaments. Cells were then
washed in PBS, mounted onto glass slides using a 1:1 solution of
glycerol-PBS containing 1 mg/ml phenylenediamine (Sigma), and observed
with an epifluorescence microscope (Diaplan; Leica, Deerfield, IL).
Western Blot Analysis
Proteins for western blot analysis were solubilized in buffer
containing 25 mM Tris-HCl (pH 7.4) containing 1 mM EDTA, 1 mM EGTA, 10
mM dithiothreitol, 1% sodium dodecyl sulfate (SDS), 5 µg/ml
antipain, 5 µg/ml pepstatin A, and 1 mM phenyl methyl sulfonyl
fluoride (PMSF). Cells were kept on ice and scraped using a rubber
policeman. After protein determination, samples were boiled for 5
minutes and run on a 10% acrylamide gel to identify -SMA and on a
7.5% acrylamide gel to identify tyrosine-phosphorylated proteins.
Proteins were then transferred to nitrocellulose paper, blocked with
5% dry milk in Tris-saline, and incubated for 2 to 3 hours with
monoclonal antibody to -SMA (Sigma) or antibodies to phosphotyrosine
(clone 4G10; UBI, Lake Placid, NY). The nitrocellulose paper was then
washed in Tris-saline and incubated in horseradish
peroxidase–conjugated goat anti-mouse IgG (1:1000) for 1 hour
(Cappel). Proteins were then visualized by enhanced chemiluminescence
reagent (ECL; Amersham, Arlington Heights, IL).
Immunoprecipitation of Tyrosine-Phosphorylated Proteins
Immunoprecipitation of tyrosine-phosphorylated proteins was
performed on cell lysates diluted to 1 µg/µl, total cell protein,
with immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM
Tris [pH 7.4], 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM
PMSF, and 0.5% NP-40). Tyrosine-phosphorylated proteins were
precipitated with 1 to 5 µg 4G10 or PY20 (ICN Biomedicals, Costa
Mesa, CA) monoclonal antibody. Immunoprecipitated proteins were run on
7.5% polyacrylamide one-dimensional sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred
to nitrocellulose paper, and probed with anti-chicken tensin (p210;
UBI), anti-p130 (Transduction Laboratories, Lexington, KY), anti-FAK
(Transduction Laboratories), anti-paxillin (ICN Biomedicals), and
anti-PLC (Transduction Laboratories).
Generation of a cDNA Probe for Rabbit -SMA mRNA
Rabbit corneal keratocyte RNA was used as a template for RT-PCR
reactions to produce a cDNA product directed toward -SMA mRNA.
Primers used to generate RT-PCR products were designed against regions
of the rabbit -SMA mRNA sequence extending upstream from base pair
607 to 630 (5'-GTGACTACTGCTGAACGTGAGATT-3') and downstream from base
pair 1157 to 1180 (5'-TAGCCCACAACTGTGAATGTGTTG-3').34
The
PCR reaction product was then cloned into a vector (pCR II-TOPO;
Invitrogen, Carlsbad, CA), sequenced to confirm the identity, and
32P labeled using random-primer labeling
(Stratagene, La Jolla, CA).
Northern Blot Hybridization
RNA was extracted from cultured keratocytes by using the RNazol
method (TRI reagent; Molecular Research, Cincinnati, OH). RNA was then
electrophoresed (10 µg/lane) through an agarose–formaldehyde gel,
transferred overnight onto a nylon membrane (GeneScreen; New England
Nuclear, Boston, MA) in 20x SSC, rinsed in 2x SSC, UV cross-linked,
and dried in an 80oC oven for 2 hours. Membranes
were prehybridized in 50% formamide, 5x SSC, 0.1% SDS, and 5x
Denhardt’s reagent at 42oC for 8 hours and
hybridized to 32P-labeled probes overnight at
42oC. Blots were washed three times in 0.1x SSC
and 0.1% SDS at 42oC and autoradiographed.
Results
Temporal Response of Corneal Keratocytes to TGFß
Primary corneal keratocytes cultured under defined, serum-free
conditions maintained a dendritic morphology (Fig. 1
A) characteristic of in situ keratocytes.35
The
cytoskeletal organization of actin within these cells showed a
predominantly a cortical localization (Fig. 1B)
with few, if any,
stress fibers and no focal adhesions (Fig. 1C)
. Interestingly,
keratocytes grown under these defined serum-free conditions failed to
express -SMA (Fig. 1D)
, whereas approximately 10% of keratocytes
grown in the presence of serum show -SMA expression,19
a prevalence similar to that observed in cultured dermal
fibroblasts.14
When exposed to 1 ng/ml TGFß,
serum-free cultured keratocytes appeared to retract their dendritic
processes and extend fewer, but broader, cellular processes, assuming a
more fibroblastic cell shape (Fig. 1E)
that was apparent as early as 24
hours after exposure to TGFß in confluent cultures.
Concomitant with the marked change in cell shape there was a striking
reorganization of the f-actin into prominent stress fibers (Fig. 1F)
that terminated at focal adhesion complexes, visualized by
anti-vinculin antibody staining (Fig. 1G
, arrows). Reorganized actin
filaments also showed staining with antibodies to -SMA (Fig. 1H)
,
suggesting the development of a myofibroblast phenotype.
|
-SMA, which was not immunocytochemically detectable
until 72 hours after treatment (Fig. 2
A, 2B, 2C). Interestingly, it is during this early phase, before the
appearance of -SMA, that fibronectin, synthesized by keratocytes in
response to TGFß,19
is deposited and
organized into fibronectin fibrils within the extracellular matrix
(Fig. 2E
2F)
. As shown previously,19
keratocytes cultured
under defined serum-free conditions synthesize and deposit little if
any fibronectin which after 4 hours of exposure to TGFß
is limited to sparse, punctate deposits and rare, poorly formed
fibronectin fibrils (Fig. 2D
, arrows). By 24 hours there was increased
fibronectin deposited within the extracellular matrix that was
organized into fibrils running parallel to cellular processes (Fig. 2E
,
arrows). With continued culture there was increasingly more fibrillar
fibronectin deposited around the cells (Fig. 2F
, arrows).
|
-SMA
Expression-SMA staining suggests that fibronectin may play
a role in modulating the expression of -SMA. This possibility is
also supported by the in vivo finding that -SMA expression in wound
healing keratocytes is exclusively localized to the
wound,3
suggesting that environmental, extracellular
matrix factors unique to the wound, and not solely soluble cytokines,
play an important role in regulating -SMA expression. Cell binding
to fibronectin is mediated by cell surface integrin receptor
recognition of the RGD sequence of fibronectin in the cell-binding
domain.36
Addition of soluble RGD-containing peptides is
known to block this cell–fibronectin interaction and thereby prevent
cell attachment and fibronectin fibril assembly.36
37
When
peptides containing the RGD sequences were added with
TGFß to primary serum-free cultured
keratocytes, there was complete inhibition of
TGFß-mediated change in cell shape, actin
reorganization, and fibronectin fibril assembly (Fig. 3
, Table 1
). TGFß treated cells cultured in the
presence of either GRGDNP, which prevents cell attachment to
fibronectin and vitronectin, or GRGDdSP, which prevents cell attachment
to fibronectin, maintained a dendritic, keratocyte morphology (Fig. 3A)
and neither -SMA expression (Fig. 3B)
or stress fibers and
fibronectin fibrils developed (data not shown). TGFß
upregulation of -SMA was not effected by the addition of either
control, non-RGD containing peptides, GRADSP (Fig. 3C)
, or the
RGD-containing peptide, GPenGRGDSPCA, that blocks cell binding to
vitronectin but not fibronectin (Fig. 3D)
. Overall, the effect of RGD
peptides was dose dependent with maximal inhibition of -SMA
expression observed at concentrations that were an order of magnitude
below the required dose for inhibition of cell attachment (Table 1)
.36
|
|
-SMA. In control, untreated keratocyte
cultures there was no detectable -SMA protein (Fig. 4
A, lane 1), whereas cultures treated with 1 ng/ml TGFß for
3 days showed staining of a prominent protein band that migrated at
approximately 45 kDa (Fig. 4A
, lane 2), consistent with smooth muscle
actin. Cultures treated with GRGDNP (50 µM) in combination with
TGFß (1 ng/ml) showed no detectable levels of -SMA
protein (Fig. 4A , lane 3), similar to that observed in the untreated
control cultures. Cultures treated with the control peptide, GRADSP at
100 µM (Fig. 4A
, lane 4), showed a level of expression of -SMA
similar to that observed in cultures treated with TGFß
alone. Northern blot analysis of RNA isolated from parallel cultures
showed low-level binding of the -SMA cDNA probe to a 1.7-kb RNA
species of a size consistent with that expected for rabbit -SMA
(Fig. 4B , lane 1). When keratocytes were treated with
TGFß alone (1 ng/ml) for 3 days, there was a marked
increase in the level of binding of the -SMA cDNA (Fig. 4B
, lane 2),
comparable to the level of binding detected in RNA from serum-cultured
keratocytes (Fig. 4B
, lane 4). When keratocytes were cotreated with
GRGDdSP (50 µM) and TGFß (1 ng/ml), binding was
substantially reduced to levels observed in control, untreated cells.
These findings suggest that the upregulation of -SMA mRNA induced by
TGFß is blocked when the binding of fibronectin to
surface membrane receptors is inhibited and that the decrease in
-SMA protein observed after GRGDdSP treatment may be caused by a
block in the upregulation of -SMA message by TGFß.
However, because the isoforms of actin show considerable sequence
homology, further work is necessary to demonstrate gene regulation at
the transcriptional level.
|
(lane 3), p130 (lane 4), pp125fak (lane 5), and
paxillin (lane 6). The two bands identified for p130 are consistent
with the presence of both phosphorylated (130-kDa) and unphosphorylated
(125-kDa) forms,38
whereas the lower molecular weight
bands stained by anti-tensin antibodies most likely represent
degradation products. Blots stained with antibodies to irrelevant
proteins (actin or -SMA, lane 7) showed no staining, indicating that
the immunoprecipitates contained predominantly tyrosine-phosphorylated
protein complexes. When keratocytes were treated with
TGFß and GRGDdSP (50 µM), the RGD-containing
peptides blocked the tyrosine phosphorylation (Fig. 5A
, lane 3).
Control peptides, GRADSP (Fig. 5A
, lane 4), had no effect on
TGFß-related tyrosine phosphorylation of
proteins.
|
and ß subunits that mediate cell adhesion to extracellular
matrix.40
Signal transduction mediated by integrin
receptors involves initial clustering of receptors and recruitment of
tensin and pp125fak followed by tyrosine
phosphorylation and focal accumulation of actin, actin-binding
proteins, and at least 19 signal transduction molecules in the
formation of focal adhesions.41
42
Inhibition of tyrosine
phosphorylation by herbimycin A, an inhibitor of the Src family of
tyrosine kinases, not only blocks aggregation of signaling molecules
but also inhibits the formation of focal adhesions and stress
fibers.43 We therefore evaluated the role of focal adhesion assembly in mediating myofibroblast differentiation by exposing keratocytes to herbimycin A in combination with TGFß. Treatment of serum-free cultured keratocytes with herbimycin A alone at a range of concentrations appeared to have no effect on keratocyte morphology or actin organization. However, when herbimycin A at 600 nM, a dose previously shown to block tyrosine phosphorylation of paxillin and pp125fak in fibroblasts,43 was added simultaneously with TGFß (1 ng/ml) to 7-day-old cultures, herbimycin A completely inhibited the formation of stress fibers and focal adhesions (data not shown). When herbimycin was removed from the culture media, continued treatment with TGFß led to stress fiber and focal adhesion formation, indicating that the effect of herbimycin A was reversible and not toxic. The lowest dose of herbimycin A that inhibited stress fiber formation (Fig. 6f -Actin) and focal adhesion assembly (Fig. 6 , Vinculin) after TGFß treatment (1 ng/ml) was 10 nM. At lower doses there was partial (1.0 nM) to no (0.1 nM) inhibition of the stress fiber and focal adhesion formation induced by TGFß, indicating a dose-response effect.
|
-SMA expression in a
dose-dependent fashion (Fig. 6
, -SM). Western blot analysis of
proteins (Fig. 7
A) obtained from TGFß alone (lane 1) and
TGFß in combination with herbimycin A at 0.1 nM
(lane 2), 1.0 nM (lane 3), and 10 nM (lane 4) confirmed that the loss
of -SMA protein expression was dose dependent and correlated with
the observed block of actin reorganization and focal adhesion assembly.
Furthermore, northern blot analysis of RNA from similarly treated
cultures (Fig. 7B)
also showed a dose-dependent loss in binding of the
-SMA cDNA, consistent with the inhibition of -SMA mRNA expression
by herbimycin A in TGFß-treated cultures.
Overall, the data support the observations made when using RGD peptides
and indicate that actin reorganization and focal adhesion assembly play
an important role in downstream modulation of -SMA expression.
|
This study suggests that the induction of corneal myofibroblast
differentiation and expression of -SMA is environmentally modulated;
explaining the unique localization of this cell type to regions
associated with corneal matrix organization and wound contraction.
Specifically, TGFß appears to enhance the
biosynthetic activity of cells, leading to synthesis of extracellular
matrix proteins including fibronectin.19
Later,
RGD-dependent interactions between the extracellular matrix and the
appropriate cell surface receptors lead to actin reorganization, focal
adhesion formation, and extracellular matrix assembly, which appear to
precede or occur coincident with expression of -SMA. Blockage of
either matrix assembly using RGD peptides or focal adhesion and stress
fiber formation using herbimycin A inhibits -SMA protein expression
and reduces mRNA levels that bind cDNA probes for -SMA. Overall,
these findings support the view that TGFß
regulates myofibroblast differentiation by modifying important
cell–matrix signaling pathways controlling extracellular matrix
assembly, cell shape, and actin organization that lead to expression of
-SMA consistent with the downstream regulation of gene
transcription.
The finding that extracellular matrix interactions are important in
regulating the expression of -SMA and myofibroblast differentiation
in corneal keratocytes is consistent with previous work by Newcomb and
Herman,13
showing that matrix synthesized by the vascular
endothelium and defined extracellular matrix constituents modulated
-SMA protein and mRNA expression in cultured pericytes. They are
also consistent with the more recent observations of Serini et
al.33
who showed that the ED-A domain of fibronectin was
crucial for the TGFß-mediated induction of
-SMA expression in dermal fibroblasts. Cell density has also been
shown to modulate -SMA expression, with both high cell
density11
and very low cell density appearing to increase
expression.12
However, using serum-free culture conditions
in the present experiments, neither density nor original substrate
conditions influenced expression of -SMA. Maintaining cells in
culture for 7 days to establish cell–cell contacts, plating on
fibronectin and collagen, or wounding cultures failed to induce -SMA
expression in serum-free cultured primary keratocytes (unpublished
results). Only treatment of serum-free cells with
TGFß, regardless of cell density, age, or substrate,
induced a change in expression.
The absence of response of serum-free cultured keratocytes to
environmental conditions known to modulate -SMA expression is most
likely related, in part, to the types of extracellular matrix receptors
expressed by these cells. Although both in situ corneal keratocytes and
serum-free cultured keratocytes show no detectable expression of the
fibronectin receptor (i.e., 5ß1 or vß3
integrin,3
35
44
) there is a marked upregulation of both
integrins on exposure to serum or TGFß in the absence of
serum.19
44
Both 5ß1 and vß3 localize to focal
adhesions and are involved in the assembly of fibronectin
fibrils.45
46
Interestingly, in wound tissue
myofibroblasts, 5ß1 is also an important constituent of the
fibronexus junctions that link intracellular actin to extracellular
matrix,47
48
forming a putative contractile apparatus that
exerts mechanical force and plays a role in wound matrix organization
and wound contraction.49
Induction of 5ß1–vß3
integrin expression by TGFß may therefore play a
physiologically critical role in the downstream signaling of -SMA
expression and myofibroblast differentiation by establishing a
mechanochemical signaling pathway.50
Such a pathway may
involve tyrosine phosphorylation of pp125fak and p130 and
downstream activation of the ras/MAP kinase signal transduction
pathways.41
Clearly, additional work is necessary to
understand more fully the role of integrins in regulating -SMA
expression.
Although TGFß’s effects on integrin and
extracellular matrix expression may be important, other factor(s)
undoubtedly play a role in regulating -SMA expression and
myofibroblast differentiation. As shown in various fibroblastic cells,
serum starvation results in loss of focal adhesion and stress fibers
that rapidly reappear on addition of serum or other factors, including
LPA and bombesin, a response shown to be dependent on Rho
activation.51
More important, the activation of Rho has
been shown to be necessary in focal adhesion assembly, downstream gene
transcription in the presence of integrin-extracellular matrix
interaction,52
and cell contractility.53
Therefore, expression of extracellular matrix and appropriate matrix
receptors by TGFß may not be sufficient by themselves to
initiate actin–focal adhesion assembly and downstream signaling.
Overall, the activation and signaling by Rho is complex and involves,
in part, the activation of G-protein–coupled receptors, leading to
activation of phospholipase C, which in turn activates a tyrosine
kinase that signals downstream conversion of Rho-guanosine diphosphate
to Rho-guanosine triphosphate, a process also controlled by guanine
exchange factors.54
Activated Rho may then induce the
tyrosine phosphorylation of pp125fak, p130, and
paxillin55
and phosphorylation of myosin light-chain
kinase,53
thus potentially regulating both focal adhesion
formation and contractility, respectively.
Finally, studies of myofibroblast differentiation using low-density
cell culture suggest that cell–cell contacts may play a role in
-SMA expression.12
56
Fibroblasts expressing -SMA
also appear to show upregulation of cadherins, suggesting important
changes in cell–cell adhesion complexes in myofibroblastic cells.
Interestingly, myofibroblasts in corneal wound tissue show both the
upregulation of connexin 43, shown by immunostaining49
and
the presence of functional gap junctions, shown by dye
injection.57
Myofibroblasts within wounds appear to
maintain a remarkable degree of interconnected structure, with actin
filaments appearing at times to extend between cells, supporting the
contention that cadherin junctions may play an important role in
myofibroblast function in wound contraction. Whether communication
mediated through gap junctions or other intercellular adhesions plays a
role in regulating myofibroblast differentiation has yet to be
determined.
Footnotes
Reprint requests: James V. Jester, University of Texas, Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235.
Supported by Grants EY07348 and EY10556 and Senior Scientist Awards (JVJ, HDC) from the National Institutes of Health; a Manpower Award (WMP) and by an unrestricted grant from Research to Prevent Blindness.
Submitted for publication November 2, 1998; revised February 23, 1999; accepted April 1, 1999.
Proprietary interest category: N.
References
5ß1 integrin fibronectin receptor, but not the a5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly J Biol Chem 268,21883-218885ß1) antibodies J Cell Biol 111,699-708This article has been cited by other articles:
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L. Shelton and J. A. Summers Rada Inhibition of Human Scleral Fibroblast Cell Attachment to Collagen Type I by TGFBIp Invest. Ophthalmol. Vis. Sci., August 1, 2009; 50(8): 3542 - 3552. [Abstract] [Full Text] [PDF]
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J. Buhren, L. Nagy, J. N. Swanton, S. Kenner, S. MacRae, R. P. Phipps, and K. R. Huxlin Optical Effects of Anti-TGF{beta} Treatment after Photorefractive Keratectomy in a Cat Model Invest. Ophthalmol. Vis. Sci., February 1, 2009; 50(2): 634 - 643. [Abstract] [Full Text] [PDF]
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L. J. Dawes, J. A. Eldred, I. K. Anderson, M. Sleeman, J. R. Reddan, G. Duncan, and I. M. Wormstone TGF{beta}-Induced Contraction Is Not Promoted by Fibronectin-Fibronectin Receptor Interaction, or {alpha}SMA Expression Invest. Ophthalmol. Vis. Sci., February 1, 2008; 49(2): 650 - 661. [Abstract] [Full Text] [PDF]
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 A. M. Bernstein, S. S. Twining, D. J. Warejcka, E. Tall, and S. K. Masur Urokinase Receptor Cleavage: A Crucial Step in Fibroblast-to-Myofibroblast Differentiation Mol. Biol. Cell, July 1, 2007; 18(7): 2716 - 2727. [Abstract] [Full Text] [PDF]
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C. Meltendorf, G. J. Burbach, J. Buhren, R. Bug, C. Ohrloff, and T. Deller Corneal Femtosecond Laser Keratotomy Results in Isolated Stromal Injury and Favorable Wound-Healing Response Invest. Ophthalmol. Vis. Sci., May 1, 2007; 48(5): 2068 - 2075. [Abstract] [Full Text] [PDF]
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E. Guerriero, J. Chen, Y. Sado, R. R. Mohan, S. E. Wilson, J. L. Funderburgh, and N. SundarRaj Loss of Alpha3(IV) Collagen Expression Associated with Corneal Keratocyte Activation Invest. Ophthalmol. Vis. Sci., February 1, 2007; 48(2): 627 - 635. [Abstract] [Full Text] [PDF]
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 G Perrella, P Brusini, R Spelat, P Hossain, A Hopkinson, and H S Dua Expression of haematopoietic stem cell markers, CD133 and CD34 on human corneal keratocytes Br J Ophthalmol, January 1, 2007; 91(1): 94 - 99. [Abstract] [Full Text] [PDF]
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 R. S. Greenberg, A. M. Bernstein, M. Benezra, I. H. Gelman, L. Taliana, and S. K. Masur FAK-dependent regulation of myofibroblast differentiation FASEB J, May 1, 2006; 20(7): 1006 - 1008. [Abstract] [Full Text] [PDF]
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K. Izumi, D. Kurosaka, T. Iwata, Y. Oguchi, Y. Tanaka, Y. Mashima, and K. Tsubota Involvement of Insulin-like Growth Factor-I and Insulin-like Growth Factor Binding Protein-3 in Corneal Fibroblasts during Corneal Wound Healing Invest. Ophthalmol. Vis. Sci., February 1, 2006; 47(2): 591 - 598. [Abstract] [Full Text] [PDF]
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J. Y. Kim, M. J. Kim, T.-i. Kim, H.-j. Choi, J. H. Pak, and H. Tchah A Femtosecond Laser Creates a Stronger Flap than a Mechanical Microkeratome Invest. Ophthalmol. Vis. Sci., February 1, 2006; 47(2): 599 - 604. [Abstract] [Full Text] [PDF]
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S. Yoshida, S. Shimmura, J. Shimazaki, N. Shinozaki, and K. Tsubota Serum-Free Spheroid Culture of Mouse Corneal Keratocytes Invest. Ophthalmol. Vis. Sci., May 1, 2005; 46(5): 1653 - 1658. [Abstract] [Full Text] [PDF]
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P. Ottino, J. He, T. W. Axelrad, and H. E. P. Bazan PAF-Induced Furin and MT1-MMP Expression Is Independent of MMP-2 Activation in Corneal Myofibroblasts Invest. Ophthalmol. Vis. Sci., February 1, 2005; 46(2): 487 - 496. [Abstract] [Full Text] [PDF]
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L. Taliana, M. Benezra, R. S. Greenberg, S. K. Masur, and A. M. Bernstein ZO-1: Lamellipodial Localization in a Corneal Fibroblast Wound Model Invest. Ophthalmol. Vis. Sci., January 1, 2005; 46(1): 96 - 103. [Abstract] [Full Text] [PDF]
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S. E. Wilson, R. R. Mohan, M. Netto, V. Perez, D. Possin, J. Huang, R. Kwon, A. Alekseev, and J. P. Rodriguez-Perez RANK, RANKL, OPG, and M-CSF Expression in Stromal Cells during Corneal Wound Healing Invest. Ophthalmol. Vis. Sci., July 1, 2004; 45(7): 2201 - 2211. [Abstract] [Full Text] [PDF]
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 R. B. Hough and J. Piatigorsky Preferential Transcription of Rabbit Aldh1a1 in the Cornea: Implication of Hypoxia-Related Pathways Mol. Cell. Biol., February 1, 2004; 24(3): 1324 - 1340. [Abstract] [Full Text] [PDF]
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D. G. Ryan, L. Taliana, L. Sun, Z.-G. Wei, S. K. Masur, and R. M. Lavker Involvement of S100A4 in Stromal Fibroblasts of the Regenerating Cornea Invest. Ophthalmol. Vis. Sci., October 1, 2003; 44(10): 4255 - 4262. [Abstract] [Full Text] [PDF]
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 S. C. Hubchak, C. E. Runyan, J. I. Kreisberg, and H. W. Schnaper Cytoskeletal Rearrangement and Signal Transduction in TGF-{beta}1-Stimulated Mesangial Cell Collagen Accumulation J. Am. Soc. Nephrol., August 1, 2003; 14(8): 1969 - 1980. [Abstract] [Full Text] [PDF]
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J. V. Jester, J. Huang, S. Fisher, J. Spiekerman, J. H. Chang, W. E. Wright, and J. W. Shay Myofibroblast Differentiation of Normal Human Keratocytes and hTERT, Extended-Life Human Corneal Fibroblasts Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 1850 - 1858. [Abstract] [Full Text] [PDF]
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S. Saika, T. Miyamoto, S.-i. Tanaka, T. Tanaka, I. Ishida, Y. Ohnishi, A. Ooshima, T. Ishiwata, G. Asano, T.-i. Chikama, et al. Response of Lens Epithelial Cells to Injury: Role of Lumican in Epithelial-Mesenchymal Transition Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 2094 - 2102. [Abstract] [Full Text] [PDF]
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B. L. Berryhill, R. Kader, B. Kane, D. E. Birk, J. Feng, and J. R. Hassell Partial Restoration of the Keratocyte Phenotype to Bovine Keratocytes Made Fibroblastic by Serum Invest. Ophthalmol. Vis. Sci., November 1, 2002; 43(11): 3416 - 3421. [Abstract] [Full Text] [PDF]
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 J.-T. Kim and C.-K. Joo Involvement of Cell-Cell Interactions in the Rapid Stimulation of Cas Tyrosine Phosphorylation and Src Kinase Activity by Transforming Growth Factor-beta 1 J. Biol. Chem., August 23, 2002; 277(35): 31938 - 31948. [Abstract] [Full Text] [PDF]
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 L. Ronnov-Jessen, R. Villadsen, J. C. Edwards, and O. W. Petersen Differential Expression of a Chloride Intracellular Channel Gene, CLIC4, in Transforming Growth Factor-{beta}1-Mediated Conversion of Fibroblasts to Myofibroblasts Am. J. Pathol., August 1, 2002; 161(2): 471 - 480. [Abstract] [Full Text] [PDF]
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L. You and F. E. Kruse Differential Effect of Activin A and BMP-7 on Myofibroblast Differentiation and the Role of the Smad Signaling Pathway Invest. Ophthalmol. Vis. Sci., January 1, 2002; 43(1): 72 - 81. [Abstract] [Full Text] [PDF]
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J. L. Funderburgh, M. L. Funderburgh, M. M. Mann, L. Corpuz, and M. R. Roth Proteoglycan Expression during Transforming Growth Factor beta -induced Keratocyte-Myofibroblast Transdifferentiation J. Biol. Chem., November 16, 2001; 276(47): 44173 - 44178. [Abstract] [Full Text] [PDF]
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O. Maltseva, P. Folger, D. Zekaria, S. Petridou, and S. K. Masur Fibroblast Growth Factor Reversal of the Corneal Myofibroblast Phenotype Invest. Ophthalmol. Vis. Sci., October 1, 2001; 42(11): 2490 - 2495. [Abstract] [Full Text] [PDF]
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P. A. Folger, D. Zekaria, G. Grotendorst, and S. K. Masur Transforming Growth Factor-{beta}-Stimulated Connective Tissue Growth Factor Expression during Corneal Myofibroblast Differentiation Invest. Ophthalmol. Vis. Sci., October 1, 2001; 42(11): 2534 - 2541. [Abstract] [Full Text] [PDF]
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 S. E. Wilson, R. R. Mohan, J.-W. Hong, J.-S. Lee, R. Choi, and R. R. Mohan The Wound Healing Response After Laser In Situ Keratomileusis and Photorefractive Keratectomy: Elusive Control of Biological Variability and Effect on Custom Laser Vision Correction Arch Ophthalmol, June 1, 2001; 119(6): 889 - 896. [Abstract] [Full Text] [PDF]
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T. Nagano, J.-L. Hao, M. Nakamura, N. Kumagai, M. Abe, T. Nakazawa, and T. Nishida Stimulatory Effect of Pseudomonal Elastase on Collagen Degradation by Cultured Keratocytes Invest. Ophthalmol. Vis. Sci., May 1, 2001; 42(6): 1247 - 1253. [Abstract] [Full Text]
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