1 Department of Physiology and 2 Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545
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ABSTRACT |
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Paracrine and autocrine actions of the insulin-like growth factors (IGFs) are inferred by local expression within the bowel. CCD-18Co cells, IEC-6 cells, and immunoneutralization were used to analyze whether IGFs have direct autocrine or paracrine effects on proliferation of cultured intestinal fibroblasts and epithelial cells. Growth factor expression was analyzed by ribonuclease protection assay and RT-PCR. Extracellular matrix (ECM) was analyzed for effects on cell proliferation. CCD-18Co cells express IGF-II mRNAs and low levels of IGF-I mRNA. Conditioned medium from CCD-18Co cells (CCD-CM) stimulated proliferation of IEC-6 and CCD-18Co cells. Neutralization of IGF immunoreactivity in CCD-CM reduced but did not abolish this effect. RT-PCR and immunoneutralization demonstrated that other growth factors contribute to mitogenic activity of CCD-CM. Preincubation of CCD-CM with ECM prepared from IEC-6 or CCD-18Co cells reduced its mitogenic activity. ECM from CCD-18Co cells enhanced growth factor-dependent proliferation of IEC-6 cells. IEC-6 cell ECM inhibited IGF-I action on CCD-18Co cells. We conclude that IGF-II is a potent autocrine mitogen for intestinal fibroblasts. IGF-II interacts with other fibroblast-derived growth factors and ECM to stimulate proliferation of intestinal epithelial cells in a paracrine manner.
intestinal epithelial cells; intestinal fibroblasts; extracellular matrix; conditioned medium
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INTRODUCTION |
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IN THE INTESTINAL MUCOSA, connective tissue, comprising
fibroblasts or myofibroblasts, lies beneath the epithelial cells, and
the two cell types are separated by a basement membrane composed of
extracellular matrix (ECM) (10, 25, 26). Normal morphogenesis and
differentiation of the intestinal epithelium during development require
interactions between epithelial cells and these neighboring mesenchymal
cells (10, 25). It is generally accepted that the intestinal
mesenchymal cells provide trophic factors that regulate crypt cell
proliferation and continuous renewal of the intestinal epithelium. The
precise mesenchymal cell-derived growth factors that regulate these
processes are not well defined. Little is known about the growth
factors that regulate proliferation of intestinal fibroblasts or
myofibroblasts themselves. The insulin-like growth factors (IGFs) are
good candidates as mesenchymal cell-derived growth factors that may
modulate proliferation of intestinal crypt epithelial cells in a
paracrine manner and/or have autocrine effects on intestinal
mesenchymal cells. Accumulating evidence indicates that the IGFs are
trophic factors for the small intestine (11, 13). IGF-I and IGF-II are
widely expressed in many tissues, including the small and large
intestine (5, 12, 33). Available evidence indicates that the primary
sites of expression of the IGFs within the small and large bowel are
mesenchymal cells within the lamina propria of normal bowel (5, 33) or
mesenchymal cells within lamina propria, submucosa, or smooth muscle
layer of bowel during experimentally induced inflammation and damage (15, 35, 37). These sites of IGF expression support a hypothesis that
IGF-I derived from intestinal mesenchymal cells may exert paracrine
actions to regulate growth and function of neighboring epithelial
cells. An alternative, and not mutually exclusive, possibility is that
IGFs regulate proliferation and function of intestinal mesenchymal
cells themselves. These hypotheses are supported by observations that
levels of locally expressed IGF-I mRNA within the intestine correlate
with changes in growth induced by fasting and refeeding (33), bowel
resection, damage, or disease (14, 15, 23, 36, 37). More direct
evidence for autocrine actions of IGF-I on intestinal mesenchymal cells
derives from recent observations in transgenic mice that have germline
transmission of a chimeric transgene composed of the -smooth muscle
actin promoter and a rat IGF-I cDNA (32). In these mice, the transgene is expressed at high levels in intestinal smooth muscle cell layers and
this leads to increased mass of enteric smooth muscle and increased
bowel length (32). The present study used model in vitro cell culture
systems to test whether IGF-I or IGF-II expressed in intestinal
fibroblasts have direct proliferative actions on intestinal epithelial
cells or on fibroblasts themselves.
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MATERIALS AND METHODS |
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Cell lines. IEC-6 cells, an epithelial cell line derived from neonatal rat intestine (20), and CCD-18Co cells, a fibroblast cell line derived from human colon (30), were used as model systems. IEC-6 cells at passage 9 were kindly provided by Dr. Andrea Quaroni (Cornell University Medical Center, Ithaca, NY). IEC-6 cells (CRL 1592) at passage 13 were obtained from the American Type Culture Collection (ATCC, Rockville, MD). CCD-18Co cells (CRL 1459, normal human colon fibroblasts) at passage 6 were obtained from ATCC. Both cell types were propagated in 75- or 150-cm2 flasks at 37°C in 5% CO2-95% humidity. IEC-6 cells were propagated in growth medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin), and ITS [insulin (5 µg/ml), transferrin (5 µg/ml), selenium (5 ng/ml), Boehringer Mannheim, Indianapolis, IN]. CCD-18Co cells were grown in the same medium without ITS. All experiments were performed on IEC-6 cells between passage 14 and 20 and on CCD-18Co cells between passage 8 and 13. For experiments, both cell types were switched to serum-free DMEM supplemented with 0.1% BSA and 5 µg/ml transferrin. For experiments with conditioned medium (CM), the CM was collected from subconfluent cells after 24 h of incubation in serum-free medium and used immediately or stored at 4°C until use. CM was used routinely within 1 wk of collection.
Primary cultures of human intestinal fibroblasts. A subset of experiments was performed on early passage human intestinal fibroblast cultures. These cells were derived from primary cultures of grossly normal tissue at the margins of diseased small intestine obtained from a patient with Crohn's disease after necessary surgical resection. Cells were prepared according to methods described by Stallmach et al. (29). Briefly, the fibroblast-containing lamina propria was dissected away from other layers of the resected bowel after extensive washing in PBS (pH 7.4) plus 0.5% sodium hypochlorite at 4°C. Minced fragments of the lamina propria were incubated in DMEM containing collagenase, neuraminidase, and hyaluronidase for 60 min at 37°C. After enzyme treatment tissue fragments were cultured on plastic dishes with DMEM plus 10% FBS and antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin, 2.5 µg/ml amphotericin B). Monolayers surrounding explants were subcultured after 10-14 days. Subconfluent cells (HIF-4) were studied at passages 4-6. Cells were deprived of serum for 24 h, and CM was collected as previously described for CCD-18Co cells. Serum-deprived HIF-4 cells were also collected for preparation of total RNA (see RNA analysis).
Growth factors, IGFBP-3, and antibodies.
Human recombinant insulin-like growth factor I (IGF-I) was obtained
from Genentech (San Francisco, CA) and human recombinant insulin-like
growth factor II (IGF-II) was obtained from GIBCO BRL (Kalamazoo, MI).
Basic fibroblast growth factor (bFGF, from bovine pituitary gland),
human recombinant keratinocyte growth factor [KGF or fibroblast growth
factor-7 (FGF-7)], human recombinant epidermal growth factor (EGF),
and human recombinant hepatocyte growth factor (HGF) were obtained from
Sigma Chemical (St. Louis, MO). Human recombinant IGF binding protein 3 (IGFBP-3) was obtained from Upstate Biotechnology (Lake Placid, NY). A
mouse monoclonal antibody (Sm 1.2) that immunoneutralizes human IGF-I
and IGF-II (31) was kindly provided by Dr. J. Van Wyk (University of
North Carolina at Chapel Hill, NC). Immunoneutralizing mouse monoclonal antibody to human bFGF was purchased from Calbiochem (San Diego, CA),
and immunoneutralizing monoclonals for human KGF-FGF-7 and human HGF
were purchased from R&D Systems (Minneapolis, MN). Monoclonal antibodies to human vimentin and human -smooth muscle actin were obtained from Dako (Carpenteria, CA). An anti-actin polyclonal antibody
to the conserved COOH terminus of cytoskeletal-contractile actin was
purchased from Sigma and used as a negative control in
immunoneutralization assays.
Immunocytochemisty.
Phenotype of CCD-18Co cells was examined by immunocytochemistry for
vimentin and -smooth muscle actin. Vimentin is a cytoskeletal marker
expressed in fibroblasts (4, 17).
-Smooth muscle actin is expressed
in cells with myofibroblast or smooth muscle phenotype (4, 27, 30).
Cells were grown on plastic slides and serum deprived for 24 h and then
washed in PBS and fixed in 100% methanol (
20°C). Slides
were blocked with PBS containing 1% horse serum for 30 min at room
temperature and then incubated for 1 h at room temperature with 1:100
dilutions of antibodies to vimentin or
-smooth muscle actin. Slides
were then incubated with a Texas red-labeled goat anti-rabbit secondary
antibody (1:200, Vector Laboratories, Burlingame, CA) and visualized by
fluorescence microscopy (Nikon Diaphot inverted fluorescence microscope).
Assays of cell proliferation. CCD-18Co cells or IEC-6 cells were seeded into 24-well plates at densities of 1 × 104 per well and grown for 16-24 h in medium with FBS. Cells were switched to serum-free medium for 24 h, and then medium was aspirated and replaced with fresh serum-free medium with or without growth factors or CM. For assays of DNA synthesis, medium was supplemented with 2 µCi/ml [3H]thymidine for the entire duration (16-24 h) of treatment with serum-free medium with or without growth factor or CM. Thymidine incorporation into DNA was measured by washing cells twice in PBS, fixing with 10% TCA, and harvesting of precipitated DNA in 0.2 N NaOH and 0.1% SDS. Radioactivity was quantified by scintillation counting. Assays were perfomed in triplicate or quadruplicate and were replicated in at least two separate experiments. To quantify cell number, serum-deprived cells were incubated with or without growth factors or CM for 24 or 48 h and then washed once with versene (PBS plus 50 mM EDTA) and trypsinized (0.25% trypsin-50 mM EDTA). Cell numbers were assessed using a hemocytometer.
RNA analyses.
We previously have analyzed IEC-6 cells for expression of IGF-I and
IGF-II and found no detectable expression of IGF-I or IGF-II mRNAs
using highly sensitive RNase protection assays on IEC-6 cell
polyadenylated RNA (24). To evaluate expression of the IGFs in CCD-18Co
cells and HIF-4 cells, the cells were grown to subconfluence (~70%)
in 150-cm2 flasks in DMEM plus
10% FBS, serum-deprived for 24 h, and then washed in PBS and harvested
into guanidine thiocyanate (12). Total RNA was pelleted by
centrifugation over 5.7 M CsCl as previously described (12). Expression
of IGF-I and IGF-II was measured by RNase protection assays (33, 37)
using 32P-labeled human IGF-I and
IGF-II antisense RNA probes (6, 16). RT-PCR reaction was performed to
assess expression of bFGF, HGF, and KGF mRNAs in CCD-18Co and HIF-4
cells. Sense and antisense oligomers used for RT-PCR of these mRNAs and
control glyceraldehyde-3-phosphate dehydrogenase mRNA are shown in
Table 1. Total RNA was
reverse-transcribed into cDNA using oligo(dT) and avian myeloblastosis
virus reverse transcriptase (Promega, Madison, WI) and then amplified
by PCR using the GeneAmp PCR System 2400 (Perkin-Elmer, Norwalk,
CT). Conditions for PCR reactions were denaturation for 2 min at 94°C, followed by 40 cycles of denaturation at 94°C (30 s), annealing at 55°C (30 s), and amplification at 72°C (30 s).
PCR products were visualized on 2% agarose gels containing ethidium
bromide.
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Immunoneutralization assays. To assess whether IGFs or other growth factors contribute to mitogenic activity in CM from CCD-18Co cells (CCD-CM), [3H]thymidine incorporation was measured in IEC-6 cells or CCD-18Co cells that were treated with CCD-CM or CCD-CM plus 6 µg/ml of anti-IGF monoclonal Sm 1.2 or CCD-CM plus neutralizing doses of antibodies to bFGF (0.6 µg/ml), HGF (3 µg/ml), or KGF (3 µg/ml) (Sigma), as recommended by the manufacturer. CCD-CM plus an anti-actin polyclonal antibody (3 µg/ml) was used as a negative control. As an additional test for IGF bioactivity in CCD-CM, IGFBP-3, an IGF binding protein that binds IGF-I and IGF-II with high affinity (7), was added to CCD-CM before assays of effects on thymidine incorporation in IEC-6 cells. Similar neutralization assays with Sm 1.2 and IGFBP-3 were performed on CM from HIF-4 cells to assess if IGFs are mitogenic factors in CM from early passage cultures of human intestinal fibroblasts as well as the CCD-18Co cell line.
Isolation of ECM. IEC-6 and CCD-18Co cells were seeded in 12-well plates and grown to confluence and then serum deprived for 24 h. Cells were lysed, and ECM was prepared according to Kramer et al. (8). Briefly, cell monolayers were washed once with PBS plus 1% BSA, followed by two 5-min washes with hypotonic buffer [10 mM Tris · HCl, 0.1% BSA, 0.1 mM CaCl2, pH 7.5, plus protease inhibitors, 1 mM phenylmethylsulfonyl fluoride and 1 mM N-ethylmaleimide]. Cell membranes were dissolved in hypotonic solution plus 0.5% nonidet P-40 (NP-40, Sigma). This was followed by two 2-min extractions in hypotonic buffer with 0.2% sodium deoxycholate. As described by Kramer et al. (8) and confirmed in this study by light microscopy, ECM at this point was free of membranes, nuclei, and cellular debris. For experiments, cells were plated on freshly prepared ECM immediately after preparation.
Statistics. All data are expressed as means ± SE; statistical comparisons were made by Student's t-test or one-factor ANOVA for comparisons among multiple groups. Statistical significance is set at P < 0.05.
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RESULTS |
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Characterization of CCD-18Co cells for phenotype and IGF expression.
Subconfluent, serum-deprived CCD-18Co cells uniformly stain positive
for vimentin, but only a few scattered cells (less than 5%) show
fibrillar immunostaining for -smooth muscle actin (Fig. 1). Primarily negative immunostaining for
-smooth muscle actin indicates fibroblast phenotype. The presence of
a small subset of
-smooth muscle actin-positive cells is a common
feature of fibroblast cell lines, even clonally derived cell lines,
indicating plasticity of phenotype and ability to spontaneously
transform into myofibroblasts (3, 4, 22).
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Mitogenic effects of IGF-I, IGF-II, and other peptide growth factors
in CCD-18Co and IEC-6 cells.
Effects of different doses of IGF-I and IGF-II on DNA synthesis were
compared in CCD-18Co and IEC-6 cells. Both IGFs exert mitogenic effects
on CCD-18Co and IEC-6 cells. IGF-I was more potent than IGF-II in
CCD-18Co cells, but maximum responses to both growth factors were
observed at doses between 5 and 10 ng/ml (Fig.
2A).
IGF-I and IGF-II were equipotent in IEC-6 cells with maximum effects
also observed at doses between 5 and 10 ng/ml (Fig.
2B). IGF-I, rather than IGF-II, was
used in subsequent assays of cell proliferation because it is more
readily available. In addition, all known mitogenic effects of IGF-I
and IGF-II are mediated by the type 1 receptor, which interacts with
both IGFs with similar affinity (11). IGF-II, and not IGF-I, can
interact with a type 2 IGF receptor, which is identical to the
cation-independent mannose-6 phosphate receptor (11). At present, the
biological role of the type 2 IGF receptor is not known. By using IGF-I
rather than IGF-II in subsequent assays of cell proliferation, we
excluded any possible type 2 IGF receptor-mediated effects. Effects of maximal doses of IGF-I and IGF-II (10 ng/ml) on DNA synthesis in
CCD-18Co and IEC-6 cells are shown in Fig.
3. Effects of the IGFs also were compared
with several other growth factors. All growth factors were used at
doses shown to elicit maximum responses in preliminary experiments.
Growth factors were tested alone and in combinations. In CCD-18Co
cells, IGF-I and IGF-II had more potent effects on DNA synthesis than
bFGF or EGF (Fig. 3A). HGF and KGF
did not significantly stimulate DNA synthesis above background. Responses to IGF-I in combination with EGF or bFGF did not differ significantly from responses to IGF-I alone. Responses to EGF and bFGF
were less than additive when the two growth factors were added in
combination.
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CCD-CM has mitogenic effects on IEC-6 and CCD-18Co cells.
We examined the mitogenic effects of CCD-CM on IEC-6 and CCD-18Co cells
to assess potential paracrine or autocrine effects on cell
proliferation. Inclusion of 25% CCD-CM in serum-free medium stimulated
DNA synthesis in both IEC-6 and CCD-18Co cells. The effect observed in
IEC-6 cells was much greater (23.4 ± 3.6-fold is maximum response)
than in CCD-18Co cells, which showed maximum responses of only 2.8 ± 0.1-fold. CM from IEC-6 cells had no effect on DNA synthesis in
either IEC-6 or CCD-18Co cells (data not shown). As shown in Fig.
4A, the
effect of CCD-CM on DNA synthesis in IEC-6 cells was dose dependent.
CCD-CM also stimulated proliferation and/or survival of IEC-6 cells.
IEC-6 cells plated in serum-free medium survive for 2 days but then
begin to detach and die by day 3 of
culture (Fig. 4B). Addition of
CCD-CM promoted an increase in cell number by day
2 of incubation and then maintained survival of cells
after 3 days in culture in serum-free medium, a time when IEC-6 cells
grown in serum-free medium alone began to die. The mitogenic activity
of CCD-CM was still evident, although diminished, after storage for up
to 4 wk at 4°C, indicating that the mitogenic activity is
reasonably stable.
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IGFs are one component of complex mitogenic activity secreted by
intestinal fibroblasts.
To assess if IGFs are expressed in CCD-18Co cells or primary cultures
of human intestinal fibroblasts, RNase protection analyses were
performed on total RNA. Both CCD-18Co and HIF-4 cells express significant levels of IGF-II mRNA and low levels of IGF-I mRNA (Fig.
5).
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Effects of ECM on proliferation of IEC-6 and CCD-18Co cells.
Mesenchymal cell ECM is known to have profound effects on survival and
differentiation of fetal intestinal endoderm (26). We therefore
compared growth factor-dependent proliferation of IEC-6 cells cultured
on ECM prepared from CCD-18Co cells (CCD-ECM) and cells cultured on
plastic. Culture of IEC-6 cells on CCD-ECM enhanced basal DNA synthesis
and DNA synthesis stimulated by IGF-I, EGF, or IGF-I and EGF in
combination (Fig. 8). One
potential concern was whether this effect is merely due to nonspecific
effects of ECM to bind or sequester
[3H]thymidine and
artifactually enhance basal or growth factor-stimulated DNA synthesis.
We believe that this is unlikely because cells cultured on CCD-ECM show
a much greater absolute increase in
[3H]thymidine
incorporation in the presence of growth factors than in serum-free
medium alone (Fig. 8A), the latter
presumably reflecting the possible nonspecific effects of CCD-ECM on
thymidine uptake and/or stimulatory effects of CCD-ECM on basal DNA
synthesis. Furthermore, CCD-ECM-enhanced growth factor mediated
increases in cell number, further suggesting a specific effect on cell
proliferation. When IEC-6 cells were grown on plastic, IGF-I and EGF
alone and even IGF-I and EGF in combination failed to induce
significant increases in cell number (Fig.
8B). In contrast, when cells were grown on CCD-ECM, there was a small but significant increase in cell
number with IGF-I or EGF alone relative to serum-free control and IGF-I
plus EGF induced a 2.5 ± 0.1-fold increase in cell number (Fig.
8B). Interestingly, when CCD-18Co
cells were plated on ECM prepared from IEC-6 cells (IEC-ECM), this
reduced basal and IGF-I-induced DNA synthesis relative to cells grown
on plastic (Fig. 8C).
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Effect of preabsorbing CCD-CM on ECM.
Stimulatory effects of CCD-ECM on IEC-6 cell proliferation may reflect
sequestration of growth factors onto the ECM. We therefore tested the
effects of preabsorption of CCD-CM onto ECM prepared from CCD-18Co or
IEC-6 cells. CCD-CM was incubated with ECM for 16 h at 4°C and then
was collected and applied to serum-deprived IEC-6 cells followed by
assay of [3H]thymidine
incorporation. Preabsorption of CCD-CM onto ECM prepared from IEC-6
cells reduced the subsequent effect of CCD-CM on DNA synthesis in IEC-6
cells by 88.7 ± 5.5% (Fig. 9).
Preabsorption of CCD-CM onto ECM prepared from CCD-18Co cells reduced
the subsequent effect on DNA synthesis in IEC-6 cells by 55.9 ± 6.7% (Fig. 9). This suggests that ECM from both cell types can bind or
sequester the mitogenic activity present in CCD-CM.
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DISCUSSION |
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We have used the CCD-18Co intestinal fibroblast cell line and the IEC-6
intestinal epithelial cell line as simple model systems to study the
direct and autocrine actions of the IGFs on proliferation of intestinal
fibroblasts and to gain insight into the role of IGFs in mediating
paracrine interactions between intestinal fibroblasts and epithelial
cells. IEC-6 cells are one of few nontransformed epithelial cell lines
derived from the small intestine that are used widely to study
proliferation of intestinal epithelial cells (18). CCD-18Co cells are
fibroblasts derived from human colon that can exhibit fibroblast or
myofibroblast phenotype (19, 30). Myofibroblasts and fibroblasts both
stain positively for vimentin but only myofibroblasts show intense
fibrillar immunostaining for -smooth muscle actin (4, 19). Under the
conditions used here to analyze fibroblast-derived mitogenic factors,
the CCD-18Co cells exhibit primarily fibroblast phenotype as evidenced
by intense staining for vimentin and the presence of only scattered
cells that stain positive for fibrillar
-smooth muscle actin. We
note that Valentich et al. (30) detected
-smooth muscle actin in serum-deprived CCD-18Co cells by Western blot assays. Our results are
not inconsistent because Western blot analysis would detect
-smooth
muscle actin even in the small population of positively stained cells.
In vivo data indicate that the predominant sites of expression of IGF-I or IGF-II in intestine are mesenchymal cells and that epithelial cells in normal small or large bowel are not major sites of IGF expression (5, 33, 37). Our findings that IGF-II is expressed at high levels in CCD-18Co fibroblasts and prior findings that IGF-II mRNA is undetectable in IEC-6 cells (24) indicate that these two cell lines retain phenotypic characteristics of the normal bowel in vivo with respect to IGF expression and therefore represent suitable model systems for the present studies. Early passage fibroblasts cultured from human small intestine (HIF-4 cells), like CCD-18Co cells, express IGF-II, indicating that the CCD-18Co cell line shares features in common with fibroblasts isolated from human small intestine. IGF-I is expressed at only very low levels in CCD-18Co cells and HIF-4 cells. This is typical of cultured cells because few cells maintained in culture express IGF-I (11). Available evidence indicates that IGF-I and IGF-II share identical mitogenic effects mediated by the type 1 IGF receptor (11).
Comparisons between growth factor responses of CCD-18Co intestinal fibroblasts and IEC-6 intestinal epithelial cells provide evidence for a cell-specific pattern of responsiveness to IGFs and other peptide growth factors in the intestine. IGF-I and IGF-II were the most potent mitogens for CCD-18Co cells when compared with a number of other growth factors tested. In contrast to IEC-6 cells, CCD-18Co cells show no synergistic mitogenic response when the IGFs are added in combination with EGF or bFGF and are poorly responsive to the fibroblast-derived growth factors HGF and KGF. Together with observations that CCD-18Co cells express IGF-II and that a neutralizing IGF antibody inhibits basal DNA synthesis in CCD-18Co cells, these observations suggest that IGF-II can serve as a major autocrine growth factor for intestinal fibroblasts.
IEC-6 cells differ from CCD-18Co cells in that IGF-I and IGF-II added alone have only modest stimulatory effects on DNA synthesis and are weaker mitogens in these cells than EGF or bFGF. Immunoneutralization of IGF-II has no significant effect on basal DNA synthesis in IEC-6 cells, indicating that IGF-II does not serve as a major autocrine growth factor for IEC-6 cells. This observation is consistent with prior observations that IGFs are not expressed at significant levels in IEC-6 cells (24). IGF-I does, however, elicit synergistic mitogenic effects in IEC-6 cells when present in combination with EGF or bFGF and has additive mitogenic effects with HGF and KGF. We have reported previously that the synergy between EGF and IGF-I reflects in part effects of EGF to maintain expression of the type 1 IGF receptor and to downregulate an inhibitory IGF binding protein, as well as distinct effects of EGF and IGF-I on pathways leading to transcriptional activation of the transcription factor AP-1 (24). It remains to be established whether synergy between bFGF and IGF-I reflects similar mechanisms of interaction. Support for this possibility stems from the current observations that EGF and bFGF in combination produce less than additive effects on DNA synthesis in IEC-6 cells, indicating that they share a common mechanism of action.
The interactions among exogenous IGF-I, bFGF, HGF, and KGF in mitogenic
effects on IEC-6 cells and prior observations in vivo that the mRNAs
encoding these growth factors are expressed predominantly in
mesenchymal cells in intestine or other systems (1, 11, 13, 28)
indicates that intestinal fibroblasts may secrete a complex combination
of mitogens that exert potent paracrine activity on neighboring
epithelial cells. Localization of IGF-I (5, 13), KGF (1), and HGF (28)
mRNAs to intestinal mesenchyme in vivo only infers and does not
directly prove a paracrine action of these growth factors. Our current
findings provide direct evidence that the CCD-18Co intestinal
fibroblast cell line and early passage intestinal fibroblast cultures
from human small intestine (HIF-4 cells) synthesize multiple growth
factors that exert paracrine mitogenic effects on intestinal epithelial
cells. One major goal of our study was to test the hypothesis that IGF
synthesized by intestinal fibroblasts serves as a paracrine mediator of
proliferation of intestinal epithelial cells. A number of observations
support this hypothesis. Two separate inhibitors of IGF bioactivity, a neutralizing monoclonal antibody (Sm 1.2) and IGFBP-3, reduce the
mitogenic effects of CCD-CM on IEC-6 cells. Preliminary data in
SMP8-IGF-I transgenic mice that express IGF-I under the control of an
-smooth muscle actin promoter also indicate that paracrine effects
of mesenchymal cell-derived IGF-I are operative in vivo. Recent
preliminary data demonstrate that the SMP8-IGF-I transgene is expressed
in mucosal subepithelial cells, as well as in smooth muscle cells, and
that the SMP8-IGF-I transgenics exhibit overgrowth of intestinal mucosa
as well as smooth muscle (P. K. Lund, K. L. Williams, and J. A. Fagin,
unpublished data). The current RT-PCR and
immunoneutralization data indicate that in addition to the IGFs, bFGF,
HGF, and KGF all are produced by CCD-18Co cells and contribute to the
mitogenic effects of CCD-CM on IEC-6 cells. HGF mRNA has been localized
to embryonic intestinal mesenchymal cells in vivo, in proximity to
epithelial cells that express the HGF receptor c-met (28). The mRNA
encoding keratinocyte growth factor (KGF/FGF-7), a member of the FGF
family of growth factors, has been localized to lamina propria
fibroblasts in adult human bowel, especially inflamed bowel (1). To our
knowledge, bFGF mRNA has not been previously localized in intestine or
identified as a secretory product of intestinal fibroblasts. Our
current findings demonstrate also that early passage fibroblast
cultures isolated from adult human small intestine also express IGF,
HGF, KGF, and bFGF mRNAs, indicating that this property of the CCD-18Co cell line is shared by normal intestinal fibroblasts.
We recognize that RT-PCR analysis is not quantitative and that detection of mRNA by RT-PCR does not directly prove that the encoded protein is synthesized or secreted. However, immunological approaches to assay growth factors at the protein level are problematic because of rapid secretion, high affinity for ECM, and the existence of soluble binding proteins (1, 11-13, 28). The majority of information about growth factor expression derives from mRNA analyses (1, 11-13, 28). In the present study therefore as well as RNase protection assays and RT-PCR to demonstrate mRNAs, immunoneutralization was used to functionally inhibit the action of secreted protein. The mRNA and immunoneutralization data provide two independent pieces of evidence that bFGF, HGF, and KGF are synthesized and secreted by cultured intestional fibroblasts even though secreted protein was not definitively identified.
Major problems in analyses of the biology of intestinal epithelial cells are the limited life span and limited proliferative capabilities of nontransformed intestinal epithelial cells isolated from normal bowel. This has led to the reliance of most investigators on transformed colon cancer cell lines or the limited numbers of available nontransformed epithelial cell lines derived from small intestine (18, 21). CM from mesenchymal cell lines such as CCD-18Co cells may prove useful in overcoming some of the prior difficulties associated with the limited life span of normal intestinal epithelial cells because it represents a source of multiple growth factors with potent mitogenic activity.
A series of elegant studies has defined the important role of ECM components derived from intestinal mesenchyme in morphogenesis and differentiation of intestinal epithelium (36). Little is known about the role of these components in regulating cell proliferation. In addition, a growing body of evidence indicates that growth factors themselves interact with ECM components (7). Findings that preabsorption of CCD-CM onto ECM prepared from IEC-6 cells or ECM prepared from CCD-18Co cells reduced its subsequent mitogenic effects on IEC-6 cells demonstrate that mitogenic factors present in CCD-CM can bind to ECM of both IEC-6 cells and CCD-18Co cells. The inhibitory effect was greater when CCD-CM was preabsorbed onto IEC-6 cell-ECM compared with preabsorption onto ECM from CCD-18Co cells. This may indicate some preferential affinity of mesenchymal cell-derived mitogenic factor(s) for ECM from intestinal epithelial cells. It is also possible that CCD-18Co cell ECM already contains these factors and as a result has a reduced capacity to bind additional activity in CCD-CM. Observations that culture of IEC-6 cells on ECM prepared from serum-deprived CCD-18Co cells enhances DNA synthesis in response to IGF-I, EGF, or IGF-I plus EGF further suggest that ECM of CCD-18Co cells may contain or bind mitogens. In addition, IGF-I and EGF in combination were able to support proliferation of IEC-6 cells when cultured on ECM from CCD-18Co cells but not when the cells were cultured on plastic. This effect seems to be distinct from other matrices such as matrigel isolated from a mouse sarcoma which inhibits growth factor responsiveness of IEC-18 cells (34) or, in some instances, has been reported to stimulate differentiation of IEC-6 cells (2). In our studies, culture of IEC-6 cells on ECM from CCD-18Co cells did not alter cell morphology (data not shown). IEC-6 cells have limited capacity for differentiation, and in the present study we have used them primarily as a model to study growth factor-dependent proliferation. We therefore cannot draw conclusions as to whether ECM from CCD-18Co cells can modulate differentiated function of intestinal epithelial cells. It will be of interest, however, to establish if ECM from CCD-18Co cells alters proliferation or differentiated phenotype of other intestinal epithelial cells such as human H-4 cells that have been shown to differentiate on matrigel (21).
A recent report indicates that clonally derived mesenchymal cells from
rat fetal intestine exhibit two distinct phenotypes that differentially
modulate proliferation and differentiation of fetal endoderm (4). One
intestinal fibroblast cell line (F1:G9) exhibits fibrillar -smooth
muscle actin immunostaining in response to transforming growth
factor-
(TGF-
) and has low capacity to support growth of fetal
endoderm but effectively stimulates morphogenesis and differentiation
(4). Another fibroblast cell line (A1:F1) does not exhibit fibrillar
immunostaining with
-smooth muscle actin even in the presence of
TGF-
and supports growth but not morphogenesis or differentiation of
fetal endoderm (4). CCD-18Co cells share phenotypic characteristics
common to both of these rat fibroblast cell lines, dependent on the
culture conditions. Serum-deprived CCD-18Co cells are
-smooth
actin-negative like A1:F1 cells. It will be of interest to establish if
the growth-promoting properties of A1:F1 cells reflect a common profile
of secreted growth factors as serum-deprived CCD-18Co cells. TGF-
stimulates transformation of CCD-18Co cells into myofibroblast,
-smooth muscle actin-expressing phenotype (19), a characteristic
shared by F1:G9 intestinal fibroblasts that stimulate differentiation of fetal intestinal endoderm (4). Future studies will establish if
TGF-
-treated CCD-18Co cells share the differentiative actions of
F1:G9 cells and if TGF-
treatment alters the profile of growth factors expressed by CCD-18Co cells. The current data and these future
studies should yield important insights into the contribution of the
IGFs and other growth factors to the functional effects of different
mesenchymal cell phenotypes on proliferation and differentiation of
intestinal epithelium.
Paracrine signals may presumably pass in either direction between intestinal mesenchymal cells and epithelial cells. The juxtaposition of epithelial cell ECM and mesenchymal cells should permit effects of epithelial cell ECM on proliferation or function of intestinal mesenchymal cells. When CCD-18Co cells were cultured on ECM from IEC-6 cells, this reduced basal and growth factor-stimulated DNA synthesis. These observations indicate that ECM from intestinal epithelial cells may inhibit growth of intestinal mesenchyme. We note that this warrants further study because it may be relevant to understanding the mechanisms of mesenchymal responses or fibrosis in response to epithelial damage as occurs, for example, in inflammatory bowel disease.
In conclusion, we have demonstrated that the IGFs are potent autocrine mitogens produced by intestinal fibroblasts. We present direct evidence that IGFs derived from intestinal fibroblasts interact with other fibroblast-derived growth factors to stimulate proliferation of intestinal epithelial cells in a paracrine manner. We also present evidence that ECM from the CCD-18Co intestinal fibroblast cell line enhances survival and growth factor-dependent proliferation of intestinal epithelial cells and that binding or sequestration of fibroblast-derived growth factors may contribute to this effect. Finally, ECM from IEC-6 cells inhibits basal or IGF-stimulated DNA synthesis in CCD-18Co cells, supporting the concept that there may be bidirectional communication between intestinal epithelial cells and underlying mesenchyme and that ECM from intestinal epithelial cells may modulate proliferation of intestinal mesenchymal cells.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the contributions of Eileen Hoyt, Dr. Judson J. Van Wyk, Deborah Carver, and Chris DaCosta to the completion of this work. We also acknowledge input and use of facilities available in the molecular biology and tissue culture cores of The Center for Gastrointestinal Biology and Disease and the DNA synthesis core of the Lineberger Cancer Center.
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FOOTNOTES |
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-40247 and DK-47769 to P. K. Lund and DK-02402 to J. B. Pucilowska.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. K. Lund, Dept. of Physiology, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: empk{at}med.unc.edu).
Received 13 October 1998; accepted in final form 23 November 1998.
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REFERENCES |
---|
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---|
1.
Bajaj-Elliott, M.,
E. Breese,
R. Poulsom,
P. D. Fairclough,
and
T. T. MacDonald.
Keratinocyte growth factor in inflammatory bowel disease. Increased mRNA transcripts in ulcerative colitis compared with Crohn's disease in biopsies and isolated mucosal myofibroblasts.
Am. J. Pathol.
151:
1469-1476,
1997[Abstract].
2.
Carrol, K. M.,
T. T. Wong,
D. L. Drabik,
and
E. B. Chang.
Differentiation of rat small intestine epithelial cells by extracellular matrix.
Am. J. Physiol.
254 (Gastrointest. Liver Physiol. 17):
G355-G360,
1988
3.
Desmouliere, A.,
L. Rubbia-Brandt,
A. Abdiu,
T. Walz,
and
A. Macieira-Coelho.
Alpha-smooth muscle actin is expressed in a subpopulation of cultured and cloned fibroblasts and is modulated by gamma-interferon.
Exp. Cell Res.
201:
64-73,
1992[Medline].
4.
Fritsch, C.,
P. Simon-Assmann,
M. Kedinger,
and
G. S. Evans.
Cytokines modulate fibroblast phenotype and epithelial-stroma interactions in rat intestine.
Gastroenterology
112:
826-838,
1997[Medline].
5.
Han, V. K.,
A. J. D'Ercole,
and
P. K. Lund.
Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus.
Science
236:
193-197,
1987[Medline].
6.
Jansen, M.,
F. M. van Schaik,
and
A. T. Ricker.
Sequence of cDNA encoding human insulin-like growth factor I precursor.
Nature
306:
609-611,
1983[Medline].
7.
Jones, J. I.,
and
D. R. Clemmons.
Insulin-like growth factors and their binding proteins: biological actions.
Endocr. Rev.
16:
3-34,
1995[Medline].
8.
Kramer, R. H.,
G. M. Fuh,
K. G. Bensch,
and
M. A. Karasek.
Synthesis of extracellular matrix glycoproteins by cultured microvascular endothelial cells isolated from the dermis of neonatal and adult skin.
J. Cell. Physiol.
123:
1-9,
1985[Medline].
9.
Li, L.,
P. K. Lund,
J. B. Pucilowska,
and
E. M. Zimmermann.
IGF-I and IGF binding protein 5 in Crohn's disease (Abstract).
Gastroenterology
114:
A4184,
1998.
10.
Louvard, D.,
M. Kedinger,
and
H. P. Hauri.
The differentiating intestinal epithelial cell: establishment and maintenance of functions through interactions between cellular structures.
Annu. Rev. Cell Biol.
8:
157-195,
1992.
11.
Lund, P. K.
Insulin-like growth factors.
In: Gut Peptides: Biochemistry and Physiology, edited by G. Dockray,
and J. H. Walsh. New York: Raven, 1994, p. 587-613.
12.
Lund, P. K.,
B. M. Moats-Staats,
M. A. Hynes,
J. G. Simmons,
M. Jansen,
A. J. D'Ercole,
and
J. J. van Wyk.
Somatomedin-C/insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues.
J. Biol. Chem.
261:
14539-14544,
1986
13.
Lund, P. K.,
and
E. M. Zimmermann.
Insulin-like growth factors and inflammatory bowel disease.
Baillieres Clin. Gastroenterol.
10:
83-96,
1996[Medline].
14.
Mantell, M. P.,
T. R. Ziegler,
W. T. Adamson,
J. A. Roth,
W. Zhang,
W. Frankel,
J. C. Chow,
R. J. Smith,
and
J. L. Rombeau.
Resection-induced colonic adaptation is augmented by IGF-I and associated with upregulation of colonic IGF-I mRNA.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G974-G980,
1995
15.
Mohapatra, N. K., M. H. Ulshen, C. R. Fuller, E. M. Zimmermann, K. Haldeman, and P. K. Lund. Cell specific expression and regulation of
insulin-like growth factor I (IGF-I) and IGF binding proteins (IGFBPs)
during adaptive growth of small intestine. Endocri.
Soc. Abst, 1995, OR37-5, p. 96.
16.
Ohneda, K.,
M. H. Ulshen,
C. R. Fuller,
A. J. D'Ercole,
and
P. K. Lund.
Enhanced growth of small bowel in transgenic mice expressing human insulin-like growth factor I.
Gastroenterology
112:
444-454,
1997[Medline].
17.
Osborn, M.,
E. Debus,
and
K. Weber.
Monoclonal antibodies specific for vimentin.
Eur. J. Cell Biol.
34:
137-143,
1984[Medline].
18.
Podolsky, D. K.
Regulation of intestinal epithelial proliferation: a few answers, many questions.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G179-G186,
1993
19.
Pucilowska, J.,
J. G. Simmons,
R. B. Sartor,
and
P. K. Lund.
IGF-I and TGF have different effects on phenotype and proliferation of intestinal fibroblasts: a potential role of IGF binding proteins (IGFBPs) (Abstract).
Gastroenterology
112:
A719,
1997.
20.
Quaroni, A.,
J. Wands,
R. L. Trelstad,
and
K. J. Isselbacher.
Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria.
J. Cell Biol.
80:
248-265,
1979[Abstract].
21.
Sanderson, I. R.,
R. M. Ezzell,
M. Kedinger,
M. Erlanger,
Z. X. Xu,
E. Pringault,
D. Louvard,
and
W. A. Walker.
Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.
Proc. Natl. Acad. Sci. USA
93:
7717-7722,
1996
22.
Sappino, A. P.,
W. Schurch,
and
G. Gabbiani.
Differentiation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic modulations.
Lab. Invest.
63:
144-161,
1990[Medline].
23.
Savendahl, L.,
L. E. Underwood,
K. M. Haldeman,
M. H. Ulshen,
and
P. K. Lund.
Fasting prevents experimental murine colitis produced by dextran sulfate sodium and decreases interleukin-1 beta and insulin-like growth factor I messenger ribonucleic acid.
Endocrinology
138:
734-740,
1997
24.
Simmons, J. G.,
E. C. Hoyt,
J. K. Westwick,
D. A. Brenner,
J. B. Pucilowska,
and
P. K. Lund.
Insulin-like growth factor-I and epidermal growth factor interact to regulate growth and gene expression in IEC-6 intestinal epithelial cells.
Mol. Endocrinol.
9:
1157-1165,
1995[Abstract].
25.
Simon-Assmann, P.,
and
M. Kedinger.
Heterotypic cellular cooperation in gut morphogenesis and differentiation.
Semin. Cell Biol.
4:
221-230,
1993[Medline].
26.
Simon-Assmann, P.,
M. Kedinger,
A. De Arcangelis,
V. Rousseau,
and
P. Simo.
Extracellular matrix components in intestinal development.
Experientia
51:
883-900,
1995[Medline].
27.
Skalli, O.,
P. Ropraz,
A. Trzeciak,
G. Benzonana,
D. Gillessen,
and
G. Gabbiani.
A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.
J. Cell Biol.
103:
2787-2796,
1986[Abstract].
28.
Sonnenberg, E.,
D. Meyer,
K. M. Weidner,
and
C. Birchmeier.
Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development.
J. Cell Biol.
123:
223-235,
1993[Abstract].
29.
Stallmach, A.,
D. Schuppan,
H. H. Riese,
H. Matthes,
and
E. O. Riecken.
Increased collagen type III synthesis by fibroblasts isolated from strictures of patients with Crohn's disease.
Gastroenterology
102:
1920-1929,
1992[Medline].
30.
Valentich, J. D.,
V. Popov,
J. I. Saada,
and
D. W. Powell.
Phenotypic characterization of an intestinal subepithelial myofibroblast cell line.
Am. J. Physiol.
272 (Cell Physiol. 41):
C1513-C1524,
1997
31.
van Wyk, J. J.,
and
E. T. Bruton.
Molecular basis for species and ligand specificity of a monoclonal antibody raised against human IGF-I.
Endocrinology
138:
4521-4523,
1997
32.
Wang, J.,
W. Niu,
Y. Nikiforov,
S. Naito,
S. Chernausek,
D. Witte,
D. LeRoith,
A. Strauch,
and
J. A. Fagin.
Targeted overexpression of IGF-I evokes distinct patterns of organ remodeling in smooth muscle cell tissue beds of transgenic mice.
J. Clin. Invest.
100:
1425-1439,
1997
33.
Winesett, D. E.,
M. H. Ulshen,
E. C. Hoyt,
N. K. Mohapatra,
C. R. Fuller,
and
P. K. Lund.
Regulation and localization of the insulin-like growth factor system in small bowel during altered nutrient status.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G631-G640,
1995
34.
Wolpert, S.,
M. L. Wong,
and
B. L. Bass.
Matrix alters the proliferative response of enterocytes to growth factors.
Am. J. Surg.
171:
109-112,
1996[Medline].
35.
Zhee, J. M.,
N. Mohapatra,
P. K. Lund,
V. E. Eysselein,
and
J. A. McRoberts.
Differential expression and localization of IGF-I and IGF binding proteins in inflamed rat colon.
J. Recept. Signal Transduct. Res.
18:
265-280,
1998[Medline].
36.
Ziegler, T. R.,
M. P. Mantell,
J. C. Chow,
J. L. Rombeau,
and
R. J. Smith.
Intestinal adaptation after extensive small bowel resection: differential changes in growth and insulin-like growth factor system messenger ribonucleic acids in jejunum and ileum.
Endocrinology
139:
3119-3126,
1998[Medline].
37.
Zimmermann, E. M.,
R. B. Sartor,
R. D. McCall,
M. Pardo,
D. Bender,
and
P. K. Lund.
Insulinlike growth factor I and interleukin 1 beta messenger RNA in a rat model of granulomatous enterocolitis and hepatitis.
Gastroenterology
105:
399-409,
1993[Medline].