1 Department of Cell and Molecular Physiology and 2 Center for Gastrointestinal Biology and Disease, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545
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ABSTRACT |
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Insulin-like growth factor I (IGF-I)
and transforming growth factor-1 (TGF-
1) are upregulated in
myofibroblasts at sites of fibrosis in experimental enterocolitis and
in Crohn's disease (CD). We compared the sites of expression of IGF-I
and TGF-
1 in a rat peptidoglycan-polysaccharide (PG-PS) model of
chronic granulomatous enterocolitis and fibrosis. We used the human
colonic CCD-18Co fibroblast/myofibroblast cell line to test the
hypothesis that TGF-
1 and IGF-I interact to regulate proliferation,
collagen synthesis, and activated phenotype typified by expression of
-smooth muscle actin and organization into stress fibers.
IGF-I potently stimulated while TGF-
1 inhibited basal DNA synthesis.
TGF-
1 and IGF-I each had similar but not additive effects to induce type I collagen. TGF-
1 but not IGF-I potently stimulated expression of
-smooth muscle actin and stress fiber formation. IGF-I in combination with TGF-
1 attenuated stress fiber formation without reducing
-smooth muscle actin expression. Stress fibers were not a
prerequisite for increased collagen synthesis. TGF-
1 upregulated IGF-I mRNA, which led us to examine the effects of IGF-I in cells previously activated by TGF-
1 pretreatment. IGF-I potently
stimulated proliferation of TGF-
1-activated myofibroblasts without
reversing activated fibrogenic phenotype. We conclude that TGF-
1 and
IGF-I both stimulate type I collagen synthesis but have differential effects on activated phenotype and proliferation. We propose that during intestinal inflammation, regulation of activated phenotype and
proliferation may require sequential actions of TGF-
1 and IGF-I, but
they may act in concert to increase collagen deposition.
Crohn's disease; -smooth muscle actin; fibrosis
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INTRODUCTION |
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FIBROSIS IS A SERIOUS COMPLICATION of Crohn's disease (CD) that involves excessive transmural deposition of collagen and disorganized overgrowth of the intestinal smooth muscle layers (7, 13, 14, 34).
Activated mesenchymal cells with myofibroblast phenotype, characterized
by the presence of -smooth muscle actin organized in prominent
stress fibers (19, 20, 25, 26), play a major role in wound
healing and fibrosis in a number of organs (19, 20).
Transient appearance of activated myofibroblasts is a feature of normal
wound healing (19, 20, 26), but the persistence of these
cell types is associated with excessive collagen deposition and
fibrosis. In some organs, like the liver, it is generally accepted that
activation of
-smooth muscle actin-negative hepatic stellate cells
to an
-smooth muscle actin-positive phenotype and expansion of this
modified cell population lead to chronic liver fibrosis
(5). Myofibroblasts and fibroblasts are increased in areas
of fibrosis in the involved regions of the intestine in patients with
CD (22) and in a rat model of peptidoglycan-polysaccharide (PG-PS)-induced granulomatous enterocolitis and fibrosis
(31). This provides evidence that myofibroblasts mediate
or contribute to fibrosis associated with chronic intestinal
inflammation (20, 22). In the intestine, however, the
origin of myofibroblasts present at sites of fibrosis is unknown. They
may derive from resident collagen producing fibroblasts in the
submucosa, resident subepithelial myofibroblasts, smooth muscle cells,
or interstitial cells of Cajal (19, 20, 22, 23, 26).
A considerable amount of evidence suggests that transforming growth
factor (TGF)-1 and insulin-like growth factor (IGF)-I play a role in
intestinal fibrosis (13, 20). Their expression is
increased in regions of collagen deposition in CD (1, 22). In PG-PS-induced enterocolitis (31, 36, 37), both TGF-
1 (31) and IGF-I (36, 37) have been localized
to myofibroblasts surrounding submucosal granulomas. IGF-I and TGF-
1
both induce collagen synthesis in multiple mesenchymal cell types
(20), including rat intestinal myofibroblasts (6,
31, 34). The role of TGF-
1 in activation of fibroblasts to
-smooth muscle actin-expressing myofibroblast phenotype in vivo and
in vitro is well established (3, 6, 19, 20). TGF-
1,
however, has known antiproliferative effects (19) while
fibroblasts and myofibroblasts increase in number in intestinal
fibrosis (20, 22, 31). A different mitogen, therefore, may
act independently of TGF-
1 to stimulate growth of fibrogenic
mesenchymal cells. Increased expression of IGF-I in intestinal fibrosis
and its documented proliferative effects on intestinal mesenchymal
cells (10, 27, 34) indicate that IGF-I could serve as such mitogen.
A family of IGF-binding proteins (IGFBPs) is known to modulate the
actions of IGF-I. Depending on whether they are secreted or associated
with cell surface or extracellular matrix (ECM), they can inhibit or
potentiate IGF-I action (2, 15). IGFBPs, in particular
IGFBP-3, can also have IGF-independent effects (11). Three
IGFBPs, IGFBP-3, IGFBP-4, and IGFBP-5, are present in the normal
intestine in vivo (29, 33-35). Increased expression
of IGFBP-5 occurs in CD (35), suggesting that IGFBP-5
could modulate IGF-I action on fibrosis. IGF-I has been shown to
stimulate expression of all three IGFBPs in intestinal smooth muscle
cells in culture (10, 34), and TGF-1 has been shown to
induce an inhibitory IGFBP-3 in skin fibroblasts (16). The
effects of IGF-I and TGF-
1 on IGFBPs synthesized in intestinal
myofibroblasts are not defined.
The present study examined whether IGF-I and TGF-1 show similar or
overlapping sites of expression in the rat PG-PS model of intestinal
inflammation and fibrosis. Partially overlapping sites of expression
suggested that the two peptides may interact to regulate fibrosis in
vivo. A well-characterized CCD-18Co cell line that can exhibit
fibroblast (27) or myofibroblast (30) phenotype was therefore used to directly assess interactions between TGF-
1, IGF-I, and IGFBPs in regulating proliferation, collagen synthesis, and phenotype.
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MATERIALS AND METHODS |
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Materials.
TGF-1 was purchased from Sigma Chemical (St. Louis, MO), human
recombinant IGFBP-4 and -5 from Austral Biologicals (San Ramon, CA),
and IGFBP-3 from Upstate Biotechnology (Lake Placid, NY). Rabbit
polyclonal antibodies to IGFBP-3, -4, and -5 were purchased from
Upstate Biotechnology, mouse monoclonal antibody specific for
-smooth muscle actin (Clone 1A4) from Dako (Carpinteria, CA),
affinity-purified rabbit polyclonal antibody specific for procollagen
1(I) from Rockland Immunochemicals (Gilbertsville, PA), monoclonal
antibody to
-tubulin (Clone Tub 2.1) from Sigma Chemicals, and goat
polyclonal anti-human LAP TGF-
1 antibody from R&D Systems
(Minneapolis, MN). Biotinylated horse anti-mouse antibody and Texas Red
avidin D were purchased from Vector Laboratories (Burlingame, CA),
peroxidase-conjugated donkey anti-rabbit, rabbit anti-goat, and rabbit
anti-mouse immunoglobulins from Jackson ImmunoResearch Laboratories
(West Grove, PA), enhanced chemiluminescence (ECL) reagent from New
England Nuclear (Boston, MA), and [3H]thymidine and
[125I]iodine from Amersham (Arlington Heights, IL).
Polyclonal antibody to rat IGF-I precursor Ea-domain and human
recombinant IGF-I (Genentech, San Francisco, CA) were kind gifts from
Dr. L. E. Underwood (University of North Carolina, Chapel Hill, NC).
Localization of TGF-1 and IGF-I in inflamed fibrotic intestine
in vivo.
Immunoreactivity for TGF-
1 or IGF-I precursor was localized in
adjacent sections of the cecum of rats with chronic PG-PS-induced enterocolitis (22, 37). Paraformaldehyde (4%)-fixed,
paraffin-embedded tissue was sectioned at 6 µm. After quenching of
endogenous peroxidase with methanol and hydrogen peroxide, sections
were blocked with 4% normal rabbit serum (for IGF-I) or normal goat
serum (for TGF-
1 antibody) for 30 min at room temperature (RT).
Overnight incubation with anti-IGF-I antibody, diluted 1:300, and with
anti-TGF-
1 antibody, diluted 1:100, in Triton X-100-containing
phosphate-buffered saline (PBS, pH 7.4) was carried out overnight at
4°C. Sections were washed, then incubated with biotinylated secondary
antibodies for 90 min at RT, followed by 90 min with avidin-biotin
complex (Vectastain Kit, Vector Laboratories, Burlingame, CA).
Peroxidase was visualized by diaminobenzidine treatment, and tissues
were counterstained with Mayer's hematoxylin. Normal serum (4%)
substituted for the primary antisera gave uniformly negative results.
Adjacent sections were stained with hematoxylin and eosin for
morphology and Sirius red for collagen.
Cell culture.
CCD-18Co cells (CRL 1459) at passage 6 were obtained from
American Type Culture Collection (Rockville, MD) and used between passages 8 and 15. Cells were routinely split 1:3
when they reached 70-80% confluence and then were maintained in
Dulbecco's modified Eagle's medium, supplemented with 10% fetal
bovine serum (FBS), 50 U/ml penicillin, and 50 µg/ml streptomycin.
For experiments, cells were plated at a density of 1 × 104 cells per well in 24-well tissue culture plates in
medium containing FBS. Cells were grown overnight, then serum-deprived
for 24 h, followed by treatment with serum-free medium with
addition of indicated peptides. Concentrations of peptides required to
induce maximal effects, IGF-I (10 ng/ml), TGF-1 (1 ng/ml), and
IGFBPs (5 nM), were established in preliminary experiments. In
experiments where treatments were continued for longer than 24 h,
medium was replaced every 24 h.
Assays of cell proliferation. Incorporation of [3H]thymidine into DNA was used as a measure of mitogenic effects of peptide treatments. [3H]thymidine (2 µCi/ml) was added for the last 24 h of incubation in each experiment. To evaluate [3H]thymidine incorporation into DNA, medium was aspirated, and cells were washed twice with PBS and fixed with 10% trichloroacetic acid. Total cell extracts were harvested in 0.2 N NaOH and 0.1% SDS. Radioactivity incorporated into DNA was quantified by scintillation counting. All assays were performed in triplicate or quadruplicate and replicated in at least two separate experiments.
Western immunoblot or radioligand blot.
For immunoblots of whole cell lysates, cells were solubilized in SDS
sample buffer. Equivalent amounts of protein were size fractionated on
8.5% SDS-polyacrylamide gels and transferred onto Immobilon-P
polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA)
using a Semi-Phor system (Hoefer Scientific Instruments, San Francisco,
CA). After blocking in PBS containing 3% non-fat dry milk, blots were
incubated with primary antisera against -smooth muscle actin or
procollagen
1(I) for 16 h at 4°C, washed in PBS containing
0.05% Tween, and then incubated with peroxidase conjugated secondary
antibodies for 30 min at RT. Immunoreactive proteins were identified
using the ECL detection system. Intensity of bands was quantified by
densitometry of X-ray films using NIH Image software (version 1.61).
Immunohistochemistry for -smooth muscle actin.
Cells were grown in four-well Lab-Tech II plastic chambers (Nunc,
Naperville, IL) and treated for different lengths of time (2-7
days) with various combinations of TGF-
and/or IGF-I. After treatment, cells were fixed in methanol (
20°C) for 10 min, blocked with 5% normal horse serum in PBS for 1 h at RT, and then
incubated with the mouse monoclonal
-smooth muscle actin antibody
(diluted 1:100 in PBS) for 2 h at RT. After washing in PBS, slides
were incubated with biotinylated horse anti-mouse immunoglobulin,
followed by Texas red-labeled avidin D (1:50 in PBS) for 30 min at RT. Fluorescent cells were visualized using a Zeiss Axiphot microscope with
FITC (excitation = 488 nm, emission = 530/30BP) and Texas red
(excitation = 568 nm, emission = 590LP) filters.
RNA extraction and analyses. Total RNA was extracted from cells grown in 100-mm tissue culture dishes (Corning, Corning, NY) using the TRIzol Reagent (GIBCO BRL, Grand Island, NY), according to manufacturer's instructions. RT-PCR for mRNAs of human IGF-I, IGF-II, and constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed on reverse-transcribed RNA using procedures described by Simmons et al. (27). Oligomers used for PCR were human IGF-I sense (5'-CCTCGCCTCTCTTCTACCTGGC-3') and antisense (5'-CATGTCACTCTTCACTCCTCAGG-3'), human IGF-II sense (5'-CCTGGAGACGTACTGTGCTACC-3') and antisense (5'-GCTCACTTCCGATTGCTGG-3'), and human GAPDH sense (5'-CTACTGGCGCTGCCAAGGCTGT-3') and antisense (5'-GCCATGAGGTCCACCACCCTGTTG-3'). PCR conditions were 40 cycles with denaturation at 95°C, annealing at 55°C, and amplification at 72°C, each for 30 s.
Statistical methods.
Values for [3H]thymidine incorporation and abundance
of procollagen 1(I) or
-smooth muscle actin were expressed as a
ratio of levels in the serum-free control within the same experiment, and means ± SE were calculated across replicate
experiments. Comparisons between treatments were analyzed by
one-way ANOVA and post hoc pairwise comparisons using Tukey's test. A
P value of <0.05 was considered statistically significant.
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RESULTS |
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TGF-1 and IGF-I show similar but not overlapping sites of
expression during PG-PS-induced enterocolitis and fibrosis.
Figure 1 shows immunohistochemical
localization of TGF-
1 and IGF-I precursor in areas of submucosal
fibrosis in rats with chronic PG-PS-induced enterocolitis. Both
peptides localize to cells surrounding granulomas, previously
identified as myofibroblasts (31, 36, 37), but their sites
of expression do not fully overlap. IGF-I precursor-positive cells are
more numerous. In the granuloma, IGF-I precursor immunoreactivity
localizes to mesenchymal cells that lie primarily at the periphery of
the granuloma while TGF-
1 localizes more centrally, in closer
proximity to immune cells. These findings suggest that some mesenchymal
cells are exposed to TGF-
1 alone, others to TGF-
1 and IGF-I, and
others primarily to IGF-I. This prompted us to examine the effects of TGF-
1 and IGF-I alone or in combination.
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Effects of TGF-1 and IGF-I on DNA synthesis.
IGF-I potently stimulated DNA synthesis in CCD-18Co cells, whereas
TGF-
1 inhibited basal and IGF-I-stimulated DNA synthesis (Fig.
2).
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Effects of TGF-1 and IGF-I on type I collagen and
-smooth
muscle actin expression.
Figure 3 shows the time course of
TGF-
1 action on type I collagen and
-smooth muscle actin. A
prominent band of 175,000 kDa, corresponding in size to procollagen
1(I) (35), was detected in serum-deprived cells, which
increased in abundance within 8 h of treatment with TGF-
1
followed by further increases to maximum abundance between 24 and
48 h of continuous exposure to TGF-
1. Low-level expression of
-smooth muscle actin was observed in serum-deprived cells and
increased progressively over the course of 48 h of treatment with
TGF-
1 (Fig. 3).
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TGF-1 induces IGF-I in CCD-18Co cells.
We have previously reported that serum-deprived CCD-18Co cells express
endogenous IGF-I and IGF-II that stimulate proliferation in an
autocrine fashion (27). We used RT-PCR to test whether expression of IGFs is altered by TGF-
1. Indeed, treatment with TGF-
1 stimulated expression of endogenous IGF-I but not IGF-II (Fig.
6). This effect has been observed in
multiple intestinal myofibroblast cell lines (unpublished data).
TGF-
1 induction of IGF-I may account for the lack of additive effect
of TGF-
1 and IGF-I on collagen accumulation and indicates that
TGF-
1 and IGF-I could act sequentially in regulating activated
myofibroblast phenotype or proliferation. We therefore examined the
effects of exogenous IGF-I on cells that had been pretreated with
TGF-
1 followed by removal of conditioned medium before addition of
IGF-I.
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IGF-I stimulates proliferation of cells activated with TGF-1
without reversing activated phenotype.
CCD-18Co cells were pretreated with TGF-
1 for 24 h or 7 days,
times when we had established that few (<5% at 1 day) or many (96%
at 7 days) of the TGF-
1-treated CCD-18Co cells show activated phenotype. TGF-
1 was then removed and IGF-I added. Cells, pretreated with TGF-
1, showed robust increases in DNA synthesis when
subsequently exposed to IGF-I, even when TGF-
1 pretreatment was as
long as 7 days (Fig. 7A).
Next, we asked if cells responding to IGF-I maintain the activated
phenotype induced by TGF-
1. Cells pretreated with TGF-
1 for 7 days and then exposed to serum-free medium or IGF-I for 3 days were
immunostained for
-smooth muscle actin (Fig. 7B). A large
percentage of the 96% of cells that exhibit stress fibers after 7 days
of TGF-
1 treatment still maintained stress fibers after exposure for
3 days to serum-free medium (79 ± 3%) or IGF-I (82 ± 9.8%). Activated phenotype is therefore not reversed spontaneously or
by IGF-I over this time frame.
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IGF-I and TGF-1 regulate IGFBPs expressed in CCD-18Co cells.
Radioligand and immunoblot data show that CCD-18Co cells express
IGFBP-3, IGFBP-4, and IGFBP-5 (Fig. 9),
the same IGFBPs that have been demonstrated in intestinal mesenchymal
cells in vivo (13). IGF-I increased secretion of IGFBP-5
into cell-conditioned medium (Fig. 9, A and B),
without affecting the association of any of the IGFBP with cells or ECM
(Fig. 9C). TGF-
1 induced IGFBP-3 secretion into medium as
well as its association with ECM. TGF-
1 and IGF-I, in combination,
resulted in increases in both IGFBP-3 and IGFBP-5 in medium and similar
association of IGFBP-3 with ECM as observed with TGF-
1.
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IGFBP-3 and -4 inhibit IGF-I-dependent proliferation of CCD-18Co
cells without affecting phenotype.
Figure 10 summarizes the effects of
IGFBPs on basal and IGF-I-stimulated DNA synthesis. Addition of IGFBP-3
to serum-deprived CCD-18Co modestly inhibited and IGFBP-5 modestly
enhanced basal DNA synthesis. When coincubated with IGF-I, IGFBP-3 and
IGFBP-4 significantly inhibited IGF-I-stimulated DNA synthesis while
IGFBP-5 had no effect. IGFBPs did not affect the inhibitory effect of TGF-1 on basal DNA synthesis and did not affect basal or IGF-I- or
TGF-
1-stimulated expression of
-smooth muscle actin and type I
collagen (data not shown).
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DISCUSSION |
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TGF-1 is a well-established mediator of wound healing and
fibrosis in a number of organs, including skin, lungs, and the liver
(3, 5, 12, 19). In the intestine, however, increased expression of TGF-
1 accompanies ulcerative colitis (UC), which generally is not associated with fibrosis, and CD, where fibrosis is a
common complication (1). Our recent findings of IGF-I upregulation in the intestine of patients with CD, and not in UC
(22), suggest a specific role of IGF-I in the fibrosis
associated with CD. We have also shown that an expanded population of
myofibroblasts and fibroblasts heavily populates areas of fibrosis in
CD (22). We therefore used a human CCD-18Co cell line that
can exhibit fibroblast (27) or myofibroblast
(30) phenotype to directly test the potential interactions
between TGF-
1 and IGF-I in regulating proliferation, phenotype, and
collagen synthesis.
Our findings indicate that, like in other systems, TGF-1 potently
activates CCD-18Co cells to a myofibroblast phenotype and induces
expression of type I collagen, a major component of ECM in fibrotic
intestine in CD (17, 22, 28) and in animal models of
chronic intestinal inflammation (31). TGF-
1, however,
inhibits proliferation of CCD-18Co cells. These antiproliferative
effects of TGF-
1 in CCD-18Co cells are shared by other mesenchymal
cells (8, 19). Thus, while TGF-
1 may have a prominent
role in the induction of ECM synthesis during intestinal inflammation, it is not likely to be a mediator of expansion of fibrogenic cells. In
the liver, where TGF-
1 does stimulate growth of activated hepatic
stellate cells (5), it also induces other peptide growth factors that further increase proliferation (5). Our novel observations that TGF-
1 induces expression of IGF-I in CCD-18Co cells and that IGF-I stimulates growth of CCD-18Co cells activated to a
myofibroblast phenotype by TGF-
1 suggest that this model may be
operative in the intestine. This is further supported by our prior
(31, 36, 37) and current observations that TGF-
1 and
IGF-I are both expressed in myofibroblasts surrounding submucosal granulomas in the PG-PS model of chronic enterocolitis. Close, but not
overlapping, localization is consistent with a model whereby TGF-
1
could induce IGF-I in neighboring mesenchymal cells, which, in turn,
could stimulate proliferation of mesenchymal cells in an autocrine or
paracrine fashion.
Our study supports a role of IGF-I, along with TGF-1, as a mediator
of increased ECM synthesis during intestinal inflammation. Our study,
however, provides no evidence for additive or synergistic effects of
TGF-
1 and IGF-I on collagen synthesis in intestinal myofibroblasts
as has been observed for type II collagen synthesis in cultured
chondrocytes (32). This may reflect the fact that TGF-
1-induced IGF-I may, in part, mediate the actions of TGF-
1 on
type I collagen synthesis.
The expression of IGFBP-3, IGFBP-4, and IGFBP-5 by CCD-18Co cells is
consistent with observations that these are the primary IGFBPs
expressed in the intestine in vivo (18, 29, 33, 35). As
IGFBP-3 inhibits DNA synthesis and is induced by TGF-1, it may
contribute to TGF-
1-mediated inhibition of basal or IGF-I-dependent proliferation. It is noteworthy that the effect of TGF-
1 to induce both IGF-I and IGFBP-3 indicates that, if such an effect occurs in
vivo, the spatial and temporal distribution of IGF-I and IGFBP-3 may
have major effects on the degree to which fibrogenic populations of
myofibroblasts are expanded. Other than modest effects to enhance basal
DNA synthesis, our observations provide no evidence that IGFBP-5
potentiates IGF-I-mediated proliferation or collagen synthesis in
intestinal myofibroblasts, which contrasts with its potentiating effects on IGF-I action in vascular smooth muscle (2).
The functional role of -smooth muscle actin in myofibroblasts during
wound healing and fibrosis is not well defined. Transient appearance of
-smooth muscle actin-positive myofibroblasts plays a role in wound
contraction in the skin (3) and is accompanied by brief
collagen gene induction (3, 4). In contrast, continuous activation of myofibroblasts results in fibrosis and excessive tissue
scarring (3, 5). Our findings demonstrate that TGF-
1 stimulates increases in collagen synthesis, coincident with increases in
-smooth muscle actin expression, but at times that precede the
organization of
-smooth muscle actin into stress fibers that typify
activated phenotype. Furthermore, IGF-I strongly induces collagen but
has very weak effects on
-smooth muscle actin expression or stress
fiber formation. Thus, in intestinal myofibroblasts/fibroblasts, the
appearance of
-smooth muscle actin stress fibers is not a prerequisite for increased collagen synthesis. Consistent with these
findings, a recent report demonstrated that vanadate, an inhibitor of
tyrosine phosphatases, reduced the appearance of
-smooth muscle
actin stress fibers in skin myofibroblasts without impeding wound
healing or ECM deposition (4). Inhibition of organization
of
-smooth muscle actin into stress fibers has also been shown to
enhance cell motility (24). This may suggest that factors
that limit organization of
-smooth muscle actin into stress fibers,
like IGF-I, may promote myofibroblast migration and could contribute to
the disorganized mesenchymal overgrowth observed in fibrosis associated
with chronic intestinal inflammation. This is speculative at present
but worthy of further investigation.
In the intestine, the sequence of cellular events that underlie
fibrosis is not well defined due, in part, to the complexity of
mesenchymal cell subtypes. Subepithelial myofibroblasts and enteric
smooth muscle cells (19) both are -smooth muscle actin positive, making it difficult to trace activated myofibroblasts during
intestinal inflammation (23). Nonetheless, our findings in
a simple model, CCD-18Co cells, indicate that the relative abundance
and spatial and temporal relationship of TGF-
1 and IGF-I could
profoundly influence the phenotype and number of fibrogenic cells.
These findings, in conjunction with localization of TGF-
1 and IGF-I
to distinct mesenchymal cell populations in submucosal granulomas at
regions of intestinal fibrosis in vivo, support a model put forward in
Fig. 11. Initially, TGF-
1 expressed
in immune cells recruits fibroblasts, converts them to activated phenotype, and induces IGF-I in these or neighboring
fibroblasts/myofibroblasts. The combined actions of TGF-
1 and IGF-I
would stimulate collagen synthesis and healing, but proliferation would
be limited by TGF-
1 and IGFBP-3 induced by TGF-
1. The persistent
expression of IGF-I in cells exposed to TGF-
1, but at sites distant
from TGF-
1 expression, would permit expansion of
collagen-producing myofibroblasts and fibrosis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Balfour Sartor for provision of fixed tissue from PG-PS-treated rats and K. McNaughton for assistance with histology.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-40247 and DK-47769 and Minority Opportunities in Research Division of the National Institutes of General Medical Sciences Grant GM-000678. The study was facilitated by the molecular histopathology core of the Center for Gastrointestinal Biology and Disease (NIH P30-DK-34987) and the tissue culture and DNA synthesis cores of the Lineberger Cancer Center (NIH CA-16086).
Address for reprint requests and other correspondence: J. G. Simmons, Dept. of Cell and Molecular Physiology, The Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: jgs{at}med.unc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 24, 2002;10.1152/ajpgi.00057.2002
Received 11 February 2002; accepted in final form 15 April 2002.
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