IGF-I induces collagen and IGFBP-5 mRNA in rat intestinal
smooth muscle
E. M.
Zimmermann1,
L.
Li1,
Y. T.
Hou1,
M.
Cannon1,
G. M.
Christman2, and
K. N.
Bitar3
Departments of 1 Internal
Medicine, 2 Obstetrics and
Gynecology, and 3 Pediatrics,
University of Michigan Medical School, Ann Arbor, Michigan 48109-0586
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ABSTRACT |
Insulin-like
growth factor (IGF) binding protein 5 (IGFBP-5) mRNA was studied in
intestines of rats with peptidoglycan-polysaccharide enterocolitis by
Northern analysis and in situ hybridization. IGFBP-5
mRNA was increased 2.4 ± 0.5-fold in inflamed rat colon compared
with controls and was highly expressed in smooth muscle. Cultured rat
intestinal smooth muscle cells were used to study the regulation of
IGFBP-5 and type I collagen synthesis. IGF-I (100 ng/ml) increased
IGFBP-5 mRNA (1.9 ± 0.1-fold) and collagen type
1(I) mRNA (1.6 ± 0.2-fold)
in cultured smooth muscle cells. IGF-I induced a dose- and
time-dependent increase in IGFBP-5 in conditioned medium by Western
ligand blot and by immunoblot. IGF-I did not affect the IGFBP-5
mRNA decay rate after transcriptional blockade. Cycloheximide abolished
IGFBP-5 mRNA. In conclusion, IGFBP-5 mRNA is expressed by
intestinal smooth muscle and is increased during chronic
inflammation. IGF-I increases IGFBP-5 and collagen mRNAs in
intestinal smooth muscle cells.
inflammatory bowel disease; Crohn's disease; intestinal fibrosis; growth factors; insulin-like growth factor I; insulin-like growth
factor binding protein 5
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INTRODUCTION |
PATIENTS WITH Crohn's disease develop intestinal
fibrosis that often leads to stricture formation and intestinal
obstruction. The mechanism of fibrosis that results from chronic
intestinal inflammation is poorly understood. Morphological studies
suggest that smooth muscle cell proliferation contributes to intestinal wall thickening and that smooth muscle cells are important sites of
intestinal collagen synthesis in inflamed and fibrotic bowel (10, 16).
Insulin-like growth factor (IGF) I is a potent fibrogenic peptide that
may be important in the pathogenesis of intestinal fibrosis in
inflammatory bowel disease (IBD). IGF-I is mitogenic for fibroblasts
and smooth muscle cells and induces collagen synthesis by these cells
in vitro (11, 13, 14, 18). IGF-I mRNA is increased in inflamed and
fibrotic intestines of patients with Crohn's disease and in animals
with experimental enterocolitis (5, 23, 27). Infusion of exogenous
recombinant human IGF-I in vivo increases the thickness of the
muscularis externa in normal rats, suggesting that intestinal smooth
muscle may be an important site for IGF-I actions in the intestine
(24).
Actions of IGF-I are modulated by a unique class of binding proteins
[IGF binding protein (IGFBP)-1 to IGFBP-6]. Circulating binding proteins act as carriers that prolong the plasma half-life of
IGF-I and limit the insulin-like endocrine actions of IGF-I (4, 20).
The IGFBPs are expressed in a wide range of peripheral tissues in which
the physiological roles are less certain. One of the binding proteins,
IGFBP-5, may be particularly relevant to the development of intestinal
fibrosis. IGFBP-5 has been shown to be unique among the IGFBPs in its
ability to enhance the actions of IGF-I on fibroblasts and smooth
muscle cells (11, 19). This effect appears to be related to the ability
of IGFBP-5 to associate with the extracellular matrix (ECM). It is
postulated that ECM-associated IGFBP-5 may enhance IGF-I actions by
increasing local concentrations of IGF-I, by modulating the
interactions of IGF-I with its receptor, or by protecting IGF-I from
proteolytic degradation (4, 20).
Evidence suggests that IGF-I is induced in chronic inflammation and
acts in an autocrine or paracrine manner on intestinal smooth muscle to
increase collagen synthesis and to promote fibrogenesis. Based on in
vitro data from other systems, IGFBP-5 may play an important role in
the enhancement of the fibrogenic actions of IGF-I. To gain insight
into the role of IGF-I in the inflamed intestine and the potential role
of IGFBP-5 in the pathogenesis of intestinal fibrosis, we
studied IGFBP-5 expression in intestinal tissue from rats with
peptidoglycan-polysaccharide (PG-PS)-induced enterocolitis. Here we
demonstrate increased IGFBP-5 mRNA in inflamed intestines from rats
with PG-PS enterocolitis compared with control rats. In this model,
IGFBP-5 mRNA is expressed in smooth muscle cells of the muscularis
externa and in other cells in the inflamed intestinal wall. We used
cultured rat intestinal smooth muscle cells (RISM) to study the effects
of IGF-I on collagen expression and regulation of IGFBP-5 mRNA.
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METHODS |
Animals.
PG-PS enterocolitis was induced by the method of Sartor et al. (22) and
Zimmermann et al. (27). Briefly, Lewis strain rats (135-150 g body
wt, female, specific pathogen free; Charles River Laboratories,
Wilmington, MA) were anesthetized (50 mg/kg ketamine and 5 mg/kg body
wt xylazine by im injection) and underwent laparotomy using an aseptic
technique. Rats received a total dose of either 37.5 µg/g body wt
PG-PS (10S preparation; Lee Laboratories, Grayson, GA) or a control
solution of human serum albumin (HSA; Baxter Health Care, Glendale, CA)
by subserosal injection in seven standardized sites in the distal ileum
and cecum (27). After laparotomy, the rats were given free access to
food and water and were cared for according to standards established by
the University Committee for the Use and Care of Animals at the
University of Michigan. Rear ankle joint diameter was measured three
times per week. Rear ankle arthritis has been shown to correlate with
the development of chronic intestinal inflammation (22, 27) and developed in 90% of PG-PS injected rats ~15 days after laparotomy. Rats were killed by inhalation of 100%
CO2 28 days after laparotomy. The
cecum was removed, rinsed in ice-cold phosphate-buffered saline (PBS),
and frozen in liquid N2 for
Northern analysis or placed in plastic capsules of optimum cutting
temperature compound (Miles, Elkhart, IN), and frozen in isopentane at
50°C for in situ hybridization. Tissues were stored at
80°C until the time of study.
Smooth muscle cell culture.
RISM were prepared by a modification of an established method (15).
Briefly, two Lewis strain rats were killed by inhalation of 100%
CO2, and the colons were dissected
from the peritoneal reflection to the cecum. The colon was slit
longitudinally with tonotomy scissors and rinsed with ice-cold PBS with
3% penicillin-streptomycin (P/S; GIBCO-BRL, Gaithersburg, MD) and then
in cold 70% ethanol, followed by PBS with 3% P/S. Mucosa was gently
scraped from the deep intestinal layers with a scalpel blade and
discarded. Fat and connective tissue were removed from the serosal
surface. Tissue was minced by hand into
~5-mm2 pieces and was placed in
a 25-cm2 culture flask (Corning,
Corning, NY) containing 10-12 ml of sterile Hanks' balanced salt
solution (GIBCO-BRL) with 1 mg/ml collagenase (type 2; Worthington
Biochemical, Freehold, NJ) and 3% P/S. Tissue was incubated in a 5%
CO2 incubator for 2 h at 37°C.
Tissue fragments were rinsed in Dulbecco's modified Eagle's medium
(DMEM) with 15% fetal bovine serum (FBS; GIBCO-BRL) and 3% P/S and
were triturated using a sterile plastic 10-ml pipette. Isolated cells
were collected by filtration. Approximately 5 ml of the cell suspension
were placed in a 25-cm2 culture
flask and incubated at 37°C in 5%
CO2. Culture medium was changed
every 3 days, and cells were passed when they were 80% confluent.
Experiments were performed on cell
passages
6-12.
For culture experiments, cells were passed into 100-mm diameter dishes
and were grown in DMEM with 15% FBS. When they were 80% confluent,
cells were washed three times with serum-free DMEM to remove serum
IGFBPs and then were incubated in serum-free DMEM for 2 h. Medium was
removed, fresh serum-free DMEM was added, and the cells were incubated
for 24 h. Cells were exposed in triplicate to 10-200 ng/ml IGF-I
(UBI, Lake Placid, NY) for 4 to 24 h. Conditioned medium was collected
for Western ligand blot and immunoblot analysis, and RNA was extracted
from cells for Northern analysis. In some experiments, 5 µg/ml
actinomycin D, 5-10 µg/ml cycloheximide, or 75 µM
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole
(DRB) were added at the same time or 24 h after addition of IGF-I.
Cells were collected for RNA analysis from 0 to 24 h after addition of
actinomycin D, cycloheximide, or DRB.
In situ hybridization.
The plasmid containing the rat IGFBP-5 cDNA (kindly provided by Drs. N. Ling and S. Shimisaki, Whittier Institute, San Diego, CA) was
linearized using appropriate restriction enzymes. Sense and antisense
35S-labeled probes were generated
using the T3 and T7 DNA-dependent RNA polymerases (GIBCO-BRL),
respectively, in a standard in vitro transcription protocol (27).
In situ hybridization was performed on 10-µm-thick sections of rat
cecum using methods previously described (27). Briefly, sections were
fixed in 4% neutral buffered formaldehyde for 30 min, washed in 0.1 M
PBS, and treated with 1 µg/µl proteinase K for 10 min. Slides were
exposed to triethanolamine and acetic anhydride (Sigma Chemical, St.
Louis, MO) for 10 min, washed, and dehydrated through graded alcohols.
Sections were exposed to standard hybridization buffer (27) containing
75% formamide (GIBCO-BRL) and 1-2 × 106 counts/min (cpm)
radiolabeled probe for 18 h at 55°C. After hybridization, coverslips were removed, and the slides were treated with 200 µg/ml
ribonuclease (RNase) A (Sigma Chemical) for 30 min, then washed in
increasingly stringent standard sodium citrate (SSC) buffers with the most stringent being 0.5× SSC for 1 h at
55°C. Slides were dehydrated, exposed to X-ray film for 24 h,
dipped in radiographic emulsion, and maintained at 4°C for 2 wk.
Slides were developed and then viewed and photographed under light- and dark-field illumination using a Zeiss axiophot microscope (Carl Zeiss,
Thornwood, NJ). Negative controls performed included slides exposed to
RNase A for 60 min before hybridization with the antisense probe and
slides hybridized with a sense probe.
RNA analysis.
RNA was extracted from ~0.5 g of whole rat cecum using the method of
Chirgwin et al. (3) with minor modifications (27). The RNA from cecal
tissue was enriched for poly(A)+ using oligo(dT)
cellulose chromatography (27). For RNA extraction from RISM, cells were
grown to 80% confluence in a 100-mm dish. Cells were washed and lysed
with 500 µl of guanidine isothiocyanate. A rubber spatula was used to
collect the lysed cells. RNA was extracted with phenol and
chloroform-isoamylalcohol (49:1) and was precipitated with isopropanol.
The pellet was dissolved in guanidine isothiocyanate and reprecipitated
in ethanol. The pellet was washed with 70% ethanol, air-dried, and
dissolved in diethyl pyrocarbonate-treated
H2O. The optical density at 260 nm
was used to determine the RNA concentration of each sample.
The IGFBP-5 cDNA was isolated from the IGFBP-5/pBluescript SK(+)
plasmid (Stratagene, La Jolla, CA) using appropriate restriction enzymes. The cDNA was purified from 1% agarose gel by electroelution (IBI, New Haven, CT) and was radiolabeled with
32P (Amersham, Arlington Heights,
IL) using a random priming kit (Boehringer Mannheim, Indianapolis, IN).
Antisense oligonucleotide probes for rat procollagen
1(I) (25) and human
-smooth
muscle actin (
-sm actin; see Ref. 26) were synthesized by the
University of Michigan Biomedical Research DNA Synthesis Core Facility
using an automated synthesizer (Applied Biosystems, Foster City, CA). Oligonucleotides were purified by high-performance liquid
chromatography and were 32P
5'-end labeled by the kinase reaction (Boehringer Mannheim).
For Northern analysis, RNA was electrophoresed on 1% agarose gel
(GIBCO-BRL) with 6% formaldehyde (Sigma Chemical). The gel was soaked
in H2O to decrease the
formaldehyde concentration, stained with ethidium bromide for 30 min,
and then destained for 3 h. The presence of sharp bands corresponding
to the 18S and 28S ribosomal RNAs were confirmed by ultraviolet
illumination. RNA was transferred overnight to Nytran (Schleicher & Schuell, Keene, NH) using capillary action, and the blots were baked at 80°C. For slot blots, RNA was loaded onto the slot-blot apparatus (Schleicher & Schuell) and transferred to the membrane. Blots were
prehybridized for 3 h and then hybridized overnight in buffer containing 50% formamide, 5× SSC, 150 µg/ml salmon sperm DNA, [32P]cDNA probe, and a
buffer containing 250 mM tris(hydroxmethyl)aminomethane (pH 7.5), 0.5%
sodium pyrophosphate, 5% sodium dodecyl sulfate, 1%
polvinylpyrrolidone, 1% Ficoll, 25 mM EDTA, and 1% bovine serum albumin. Membranes were washed in increasingly stringent SSC washes, with the most stringent being 0.5× SSC and 0.1% SDS at 55°C
for 30 min. Membranes were exposed to radiographic film overnight at
80°C with intensifying screens. The autoradiogram was
digitized by flatbed scanning and was imported for densitometric
analysis into NIH Image (National Institutes of Health, Bethesda, MD). The relative densitometric value for each band was adjusted for minor
variations in loading by using the corresponding signal for blots
probed with a
[32P]cDNA probe for
glyceraldehyde-3-phosphate dehydrogenase (American Type Culture
Collection, Rockville, MD).
Western ligand blot and immunoblot.
Western ligand blot for detection of IGFBP-5 was performed as
previously described (12). Conditioned medium was centrifuged to remove
cellular debris, then concentrated using a Centriprep 10 concentrator
(Amicon, Beverly, MA) in the presence of 1 mg/ml aprotinin (Boehringer
Mannheim) and 20 mg/ml phenylmethylsulfonyl fluoride (Boehringer
Mannheim). The protein concentration was determined using the Bio-Rad
detergent compatible protein assay (Bio-Rad, Hercules,
CA). Samples were diluted to 26.5 µg of total protein in 50 µl of
concentrated medium. For Western ligand blot, samples were
electrophoresed on a 10% polyacrylamide gel containing 0.1% SDS.
Proteins were transferred to nitrocellulose (0.2 µm; Schleicher & Schuell) and were then incubated overnight with
105 cpm/ml
125I-labeled IGF-I (Amersham).
Blots were washed, dried, and exposed to radiographic film at
80°C for 2-4 days. Protein molecular weight standards
were included on each gel (Amersham).
Samples of conditioned medium for immunoblot were collected and
concentrated as for Western ligand blot. Samples were electrophoresed on a 12% polyacrylamide gel with 0.1% SDS. Proteins were transferred to nitrocellulose (0.2 µm; Schleicher & Schuell). Blots were
incubated for 3 h with primary antibody: rabbit anti-human IGFBP-5
(1:400 dilution; UBI). Blots were washed, then exposed to the secondary antibody: goat anti-rabbit immunoglobulin G (1:1000 dilution; Santa
Cruz Biotechnology, Santa Cruz, CA) for 1 h. Blots were washed, and
specific antibody was detected by enhanced chemiluminescence detection
system (Amersham) and exposed to X-ray film for 1-10 min.
Statistical analysis.
Comparisons were made between groups of unpaired samples by the
Mann-Whitney U-test and between groups
of paired samples by Wilcoxon's signed rank test. Linear regression
analysis was used to describe dose- and time-dependent relationships.
Data were considered significant if P < 0.05.
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RESULTS |
IGFBP-5 mRNA in normal and inflamed rat intestine.
The abundance of IGFBP-5 mRNA was studied in intestinal tissue from
PG-PS-injected and control rats. Northern analysis of RNA extracted
from the ceca of rats showed a single 6.0-kb IGFBP-5 mRNA transcript
consistent with prior reports of IGFBP-5 mRNA size (2). There was a 2.4 ± 0.5-fold increase in IGFBP-5 mRNA in RNA extracted from
PG-PS-injected rats compared with control rats (Fig.
1; n = 6 pairs of rats; P = 0.02).

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Fig. 1.
Insulin-like growth factor (IGF) binding protein 5 (IGFBP-5) mRNA is increased in ceca of rats with
peptidoglycan-polysaccharide (PG-PS) enterocolitis. Each lane
represents 20 µg of poly(A)+ RNA from the cecum of a
different PG-PS-injected or human serum albumin (HSA)-injected
(control) rat. Top: Northern analysis
shows 6.0-kb IGFBP-5 transcript in all samples with a 2.4 ± 0.5-fold increase in IGFBP-5 mRNA in PG-PS-injected rats compared with
HSA-injected controls. Bottom:
ethidium bromide-stained agarose gel of RNA used for Northern analysis.
Autoradiograms were exposed overnight at 80°C with
intensifying screens.
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In situ hybridization was used to determine the cellular sites of
IGFBP-5 synthesis in inflamed and control bowel. In situ hybridization
using the antisense probe for IGFBP-5 demonstrated IGFBP-5 mRNA in
smooth muscle cells of the muscularis externa of PG-PS- and
HSA-injected rat colons (Fig. 2). IGFBP-5
mRNA was expressed in inflamed submucosa, including in macrophage-like cells within PG-PS-induced granulomas (Fig. 2; see Ref. 27). There was
also expression of IGFBP-5 mRNA in individual cells within the expanded
submucosa and serosa (Fig. 2). Many of these cells had large pale
nuclei similar to the cells at the center of granulomas and likely
represent macrophages. Inflamed submucosa and serosa contain a variety
of tightly packed cell types, making it impossible to determine with
certainty which cell types expressed IGFBP-5 mRNA by this technique.
IGFBP-5 mRNA was expressed in inflamed foci in the lamina propria of
the PG-PS injected rat colonic mucosa but was undetectable in the
lamina propria of control rat intestines (data not shown). There was no
detectable expression of IGFBP-5 mRNA in the epithelial layer of the
inflamed or normal gut. Sections hybridized with the sense probe or
pretreated with RNase A before hybridization with the antisense probe
showed only low-level background labeling over the tissue (Fig.
2E).

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Fig. 2.
Localization of IGFBP-5 mRNA in inflamed and normal
bowel by in situ hybridization. Luminal surface of each section is at
top of the photomicrograph.
A: low-powered light-field
photomicrograph of a section of cecum from a PG-PS-injected rat 28 days
after laparotomy hybridized with the antisense probe for IGFBP-5. There
is expansion of the submucosa (Sm) and serosa (S) with chronic
inflammatory cells and collagen. A granuloma (arrowheads) is seen in
the submucosa. M, mucosa, ME, muscularis externa.
B: corresponding dark-field
photomicrograph demonstrating expression of IGFBP-5 mRNA in the ME and
in the inflamed submucosa and serosa.
C: higher-powered photomicrograph of
section shown in A and
B. Serosal inflammatory cells that
express IGFBP-5 mRNA are shown by arrowheads.
D: dark-field photomicrograph of
section shown in C showing intense
hybridization of the antisense probe in the smooth muscle of the ME
with less intense hybridization in cells within the submucosa and
serosa. E: section adjacent to section
shown in
A-D
hybridized with the IGFBP-5 sense probe showing background labeling.
F: high-powered photomicrograph
showing hybridization with the antisense probe in the circular
(MEC) and longitudinal
(MEL) layers of the ME.
Hybridization of individual cells in the submucosa is also demonstrated
(arrowheads). G and
H: light and dark-field
photomicrographs, respectively, of sections of HSA-injected (control)
rats with histologically normal bowel hybridized with the IGFBP-5
antisense probe. Hybridization is seen in the ME and the submucosa.
Sections were exposed to radiographic emulsion for 14 days. Scale bars = 100 µm.
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Northern analysis and in situ hybridization demonstrated increased
IGFBP-5 mRNA in inflamed regions of the intestinal mucosa, submucosa,
and serosa with the highest expression in smooth muscle cells of the
muscularis externa.
IGFBP-5 and procollagen synthesis in cultured intestinal smooth
muscle cells.
Given the expression of IGFBP-5 mRNA in intestinal smooth muscle and
the data from Crohn's disease suggesting that smooth muscle cells are
important sites of intestinal collagen synthesis (16), we cultured
smooth muscle cells from the rat muscularis externa for use as an in
vitro system to study the expression of IGFBP-5 and collagen. RISM
maintained a smooth muscle phenotype in culture as determined by
continued expression of
-sm actin. There was no evidence of
decreasing expression of
-sm actin mRNA at least through
passage
15 (data not shown;
n = 3, r2 = 0.1, P = 0.32). Confluent cells exhibited
typical growth characteristics, including the development of ridges and
valleys (15).
RISM expressed IGFBP-5 mRNA consistent with in situ hybridization data
that demonstrate IGFBP-5 mRNA in normal smooth muscle of the muscularis
externa (Fig. 2, D and
E). There was a 1.9 ± 0.1-fold
increase in IGFBP-5 mRNA after 24 h of exposure to 100 ng/ml IGF-I
(n = 3 experiments in triplicate,
P < 0.01; Fig.
3A). IGF-I also induced procollagen
1(I) mRNA in RISM 1.6 ± 0.2-fold (n = 2 experiments in
triplicate, P = 0.04; Fig.
3B). There was a dose-dependent
increase in IGFBP-5 mRNA with increasing doses of IGF-I
(r2 = 0.55, P = 0.006; Fig.
4). Consistent with mRNA data from in situ
hybridization and Northern analysis, IGFBP-5 was barely detectable in
medium from control cells, as determined by Western ligand blot and
immunoblot (Figs. 5 and
6). There was a dose-dependent increase in
IGFBP-5 with increasing doses of IGF-I (Fig. 5). IGFBP-5 accumulated
in conditioned medium of RISM exposed to IGF-I for increasing lengths
of time from 4 to 24 h (Fig. 6). There was a 1.6 ± 0.2-fold
increase in IGFBP-5 mRNA 8 h after IGF-I exposure compared with control
cells at the same time point (n = 3 experiments in triplicate; P = 0.04). IGFBP-5 mRNA remained increased at least 24 h after IGF-1
exposure (Fig. 3A).

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Fig. 3.
IGF-I increases IGFBP-5 and procollagen
1(I) mRNAs in rat intestinal
smooth muscle cells (RISM). Cells were exposed to 100 ng/ml IGF-I for
24 h. RNA was isolated from RISM, and Northern analysis was performed
using 10 µg of RNA in each lane. A:
there was a 1.9 ± 0.1-fold increase in IGFBP-5 mRNA (6.0 kb) in
cells exposed to IGF-I compared with cells exposed to serum-free medium
alone (control; P < 0.01;
n = 3 experiments in triplicate).
B: procollagen
1(I) mRNA was increased in
cells exposed to IGF-I (1.6 + 0.2-fold;
n = 2 experiments in
triplicate, P = 0.04). mRNA abundance
was determined by densitometry and was normalized with the signal for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
bottom). Autoradiograms were exposed
overnight at 80°C with intensifying screens.
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Fig. 4.
Dose-dependent increase in IGFBP-5 mRNA in RISM
exposed to IGF-I. Cells were exposed to 10-200 ng/ml IGF-I for 24 h.
RNA was isolated from RISM, and Northern analysis was performed using
10 µg of RNA in each lane. A:
representative autoradiogram of blots hybridized with IGFBP-5 probe
(top) and probe for GAPDH
(bottom). Autoradiograms were
exposed overnight at 80°C with intensifying screens.
B: mRNA abundance was determined by
densitometry and was normalized with the signal for GAPDH. Data are
means ± SE of 2-3 experiments in triplicate. mRNA abundance
after 100 and 200 ng/ml IGF-I exposure was significantly different from
control (untreated cells; P = 0.03 and
P = 0.01, respectively).
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Fig. 5.
Dose-dependent increase in IGFBP-5 in RISM exposed to IGF-I. Cells were
exposed to 10-200 ng/ml IGF-I for 24 h. Conditioned medium was
collected and Western ligand blot (A;
exposed to film for 2 days) and immunoblot
(B; exposed to film for 20 min) were
performed. Each lane represents concentrated conditioned medium from a
different cell culture dish (26.5 µg protein/lane). IGFBP-5 (32 kDa)
in conditioned medium of control cells was barely detectable. IGFBP-5
in conditioned medium increased with increasing concentration of IGF-I.
A faint 24-kDa band was detectable in cells exposed to 200 ng/ml IGF-I,
consistent with the size for IGFBP-4 (18). A representative of 2 experiments is shown.
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Fig. 6.
IGFBP-5 in RISM conditioned medium increased with
increasing duration of IGF-I exposure. RISM were exposed to 100 ng/ml
IGF-I for 4-24 h. Control cells were cultured for 24 h in
serum-free DMEM. Conditioned medium was collected, and Western ligand
blot and immunoblot were performed. Each lane in Western ligand blot
and immunoblot represents concentrated conditioned medium from a
different cell culture dish (26.5 µg protein/lane). IGFBP-5 was
barely detectable in control cells and increased with increasing length
of exposure to IGF-I as shown by Western ligand blot
(A; exposed to film for 3 days) and
immunoblot (B; exposed to film for 5 min). Shown are representative blots of 2 experiments.
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Western ligand blot demonstrated a 32-kDa protein consistent with the
size for IGFBP-5 (1) and a second faint 24-kDa band that is consistent
with the published size for IGFBP-4 (Figs. 5A and
6A; see Ref. 2). The 32-kDa band was
confirmed by immunoblot to be IGFBP-5; the 24-kDa band was not seen on
immunoblot and was not characterized further.
Effect of transcriptional and translational blockade on IGFBP-5 mRNA
abundance.
The transcriptional blocker actinomycin D was used to determine the
effect of blocking new gene transcription on the abundance of IGFBP-5
mRNA. When mRNA abundance was studied 24 h after addition of IGF-I and
actinomycin D, actinomycin D had little effect on IGFBP-5 mRNA
abundance. When studied at earlier time points (2 and 4 h), however,
actinomycin D caused an increase in IGFBP-5 mRNA (data not shown). This
paradoxical increase in mRNA with the transcriptional blocker has been
previously described (21) and made actinomycin D noninformative in our
system. DRB, another blocker of transcription, caused a decrease in
IGFBP-5 mRNA when mRNA abundance was studied 0, 4, 8, and 24 h after
addition of DRB (Fig.
7A). The
slopes of the curves for cells with and without exposure to IGF-I were
no different, suggesting that IGF-I did not increase IGFBP-5 mRNA
abundance by increasing mRNA stability and thereby supporting the
hypothesis that IGF-I increases transcription of IGFBP-5 mRNA.
Cycloheximide, an inhibitor of protein synthesis, abolished IGFBP-5
mRNA (Fig. 7B), suggesting that new
protein synthesis is important for integrity of IGFBP-5 mRNA.

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Fig. 7.
Effect of
5,6-dichloro-1- -D-ribofuranosylbenzimidazole (DRB)
and cycloheximide (C) on mRNA abundance. A: RISM were
incubated alone or with 100 ng/ml IGF-I. After 24 h, 75 µM DRB were
added. RNA was extracted from cells collected at different times (0, 4, 8, and 24 h) after DRB addition to RISM. Ten micrograms of RNA were
loaded in each slot-blot well. IGFBP-5 mRNA was quantitated by
densitometry and was normalized to signal for GAPDH. Each point
represents means of 3 experiments in duplicate. B: 100 ng/ml
IGF-I was added to RISM alone or with cycloheximide (5 or 10 µg/ml).
After 24 h, cells were collected, and RNA was extracted. IGFBP-5 mRNA
abundance was determined by Northern analysis using 10 µg of RNA in
each lane. Cycloheximide completely abolished IGFBP-5 mRNA.
Hybridization of blots with GAPDH probe confirms integrity of RNA in
all lanes.
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DISCUSSION |
Morphological studies of strictured intestine from patients with IBD
suggest that smooth muscle cells are major sites of collagen synthesis
(10, 16). Recent studies demonstrate that IGF-I stimulates
proliferation of smooth muscle cells in vivo, suggesting that smooth
muscle cells are important targets for IGF-I actions (24). We have
previously shown that IGF-I mRNA is increased in inflamed and fibrotic
intestine from patients with IBD and rats with PG-PS enterocolitis (5,
27). Here, we demonstrate that IGF-I increases expression of type I
collagen mRNA in cultured intestinal smooth muscle cells. These data
are consistent with a mechanism whereby IGF-I is increased during the
development of chronic inflammation and acts on smooth muscle cells to
increase collagen synthesis and promote fibrogenesis.
IGF binding proteins are emerging as key factors in the determination
of the sites of IGF-I actions and the magnitude or extent of the
biological effects of IGF-I (4, 20). Classically, the IGFBPs inhibit
the actions of IGF-I in vitro probably by competing for IGF-I with the
target cell IGF type I receptor. Like other IGFBPs, IGFBP-5 has been
shown to inhibit the mitogenic actions of IGF-I in vitro; however,
IGFBP-5 is unique in that, under certain experimental conditions, it
enhances the mitogenic actions of IGF-I. The potentiating effects of
IGFBP-5 are seen when IGFBP-5 is associated with ECM underlying the
target cell monolayer (19). ECM-associated IGFBP-5 may enhance IGF-I
actions by accumulating IGF-I near its receptor and/or
protecting it from proteolysis (19). In addition, ECM-associated
IGFBP-5 has a lower affinity for IGF-I than soluble IGFBP-5 and may act
as a less effective competitor for IGF-I, thereby
facilitating the interaction of IGF-I with its receptor (19). Here we
demonstrate expression of IGFBP-5 mRNA in rat intestinal smooth muscle
and increased IGFBP-5 mRNA in chronically inflamed intestine.
Cultured intestinal smooth muscle cells were shown to express
IGFBP-5 and type I collagen mRNAs, and these mRNAs increased after
exposure of the cells to IGF-I. IGF-I increased IGFBP-5 mRNA
and secretion of IGFBP-5 into conditioned medium. Our hypothesis is
that IGF-I is increased in inflamed intestine and, in turn, induces
synthesis of IGFBP-5 and collagen by smooth muscle cells. Secreted
IGFBP-5 associates with the ECM adjacent to smooth muscle
cells and, as demonstrated in vitro, enhances the action of IGF-I to
increase collagen synthesis, thereby promoting fibrogenesis. In vivo
modulation of IGF-I or IGFBP-5 synthesis in the inflamed
intestinal environment will be required to definitively determine the
roles of IGF-I and IGFBP-5 in intestinal fibrogenesis.
The effect of IGF-I on IGFBP-5 mRNA and protein levels in conditioned
medium appears to depend on the cell system studied. In human U-2
osteosarcoma cells (7) and human fibroblasts (1), IGF-I increased
IGFBP-5 in conditioned medium but had little effect on IGFBP-5 mRNA. In
other cell systems, including vascular smooth muscle, IGF-I increased
IGFBP-5 mRNA and protein accumulation, as was observed in our study (6,
8, 9). Depending on the cell system, the effect of IGF-I on IGFBP-5 has
been determined to be the result of increased transcription (6, 8, 9) or posttranslational mechanisms (1, 7). Our results differ slightly
from prior studies in that the IGFBP-5 protein accumulation in response
to IGF-I was much more pronounced than the mRNA induction. This may be
related to lower activity of IGFBP-5 proteolysis in our system than in
others. In vascular smooth muscle, a 22-kDa IGFBP-5 immunoreactive
proteolytic fragment, but not intact IGFBP-5, was detectable in control
cells (cells not exposed to IGF-I or heparin), and proteolytic
fragments were as abundant as intact IGFBP-5 in conditioned medium from
cells exposed to IGF-I (9). In RISM, IGFBP-5 fragments are not
detectable, even with long exposures of blots, in medium from control
cells or cells exposed to IGF-I.
Factors that increase mRNA abundance and ultimately IGFBP-5
concentrations may be key to the determination of the actions of IGF-I
in vivo. In RISM, IGF-I does not increase the half-life of IGFBP-5 mRNA
as determined by comparison of mRNA abundance in IGF-I-treated and
control cells after addition of the transcriptional blocker DRB. The
finding that IGF-I did not affect mRNA stability indirectly supports
the hypothesis that IGF-I increases transcription of IGFBP-5.
Direct methods of analysis would include nuclear run-on assay. However,
this proved technically difficult in our system because IGF-I induced
only a twofold increase in IGFBP-5 mRNA. Data from nuclear run-on
assays in vascular smooth muscle (9) suggest that IGF-I mediates the
increase in IGFBP-5 through increased IGFBP-5 gene transcription. The
finding that cycloheximide completely abolished IGFBP-5 mRNA in RISM is
interesting. It suggests an important role for synthesis of one or more
intermediumte proteins in maintaining IGFBP-5 integrity. In vascular
smooth muscle, cycloheximide abolished only the IGFBP-5 mRNA induced by
IGF-I (9), whereas, in RISM, all IGFBP-5 mRNA was abolished by
cycloheximide. This difference may be related to differences between
regulation of IGFBP-5 mRNA in vascular and intestinal smooth muscle and
may indicate important regulatory proteins affecting intestinal IGFBP-5 mRNA.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Eva Feldman for critical review of this manuscript,
Dr. Thomas Chen for assistance with the Western blot procedures, and
Dr. P. K. Lund for advice and encouragement.
 |
FOOTNOTES |
This research was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants K08 DK-02131 and R29 DK-49628 (E. M. Zimmermann) and a pilot grant from the Michigan Gastrointestinal
Peptide Core Center (to E. M. Zimmermann).
Address for reprint requests: E. M. Zimmermann, 4410 Kresge III, Univ.
of Michigan, Ann Arbor, MI 48109-0586.
Received 13 November 1996; accepted in final form 24 June
1997.
 |
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