AJP - GI Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 273: G875-G882, 1997;
0193-1857/97 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zimmermann, E. M.
Right arrow Articles by Bitar, K. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zimmermann, E. M.
Right arrow Articles by Bitar, K. N.
Vol. 273, Issue 4, G875-G882, October 1997

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

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 alpha 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

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

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-beta -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 alpha 1(I) (25) and human alpha -smooth muscle actin (alpha -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.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

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).


View larger version (43K):
[in this window]
[in a new window]
  		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.

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).


View larger version (124K):
[in this window]
[in a new window]
 

View larger version (128K):
[in this window]
[in a new window]
 

View larger version (127K):
[in this window]
[in a new window]
 

View larger version (135K):
[in this window]
[in a new window]
 

View larger version (74K):
[in this window]
[in a new window]
 

View larger version (140K):
[in this window]
[in a new window]
 

View larger version (124K):
[in this window]
[in a new window]
 

View larger version (111K):
[in this window]
[in a new window]
  		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.

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 alpha -sm actin. There was no evidence of decreasing expression of alpha -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 alpha 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).


View larger version (35K):
[in this window]
[in a new window]
  		Fig. 3.   IGF-I increases IGFBP-5 and procollagen alpha 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 alpha 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.


View larger version (38K):
[in this window]
[in a new window]
 

View larger version (37K):
[in this window]
[in a new window]
  		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).


View larger version (31K):
[in this window]
[in a new window]
  		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.


View larger version (33K):
[in this window]
[in a new window]
  		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.

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.


View larger version (19K):
[in this window]
[in a new window]
 

View larger version (41K):
[in this window]
[in a new window]
  		Fig. 7.   Effect of 5,6-dichloro-1-beta -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.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Camacho-Hubner, C., W. H. Busby, Jr., R. H. McCusker, G. Wright, and D. R. Clemmons. Identification of the forms of insulin-like growth factor binding proteins produced by human fibroblasts and the mechanisms that regulate their secretion. J. Biol. Chem. 267: 11949-11950, 1992[Abstract/Free Full Text].

2.   Cheung, P. T., E. P. Smith, S. Shimasaki, N. Ling, and S. D. Chernausek. Characterization of an insulin-like growth factor binding protein (IGFBP-4) produced by the B104 rat neuronal cell line: chemical and biological properties and differential synthesis by sublines. Endocrinology 129: 1006-1015, 1991[Abstract].

3.   Chirgwin, J., A. Przybyla, R. Macdonald, and W. Rutter. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5249-5299, 1979.

4.   Clemmons, D. R. IGF binding proteins and their functions. Mol. Reprod. Dev. 35: 368-374, 1993[Medline].

5.   Cohen, J. A., E. M. Zimmermann, R. B. Sartor, and P. K. Lund. IGF-I and IGF-II are overexpressed in inflamed and strictured intestine in Crohn's disease (Abstract). Gastroenterology 104: A683, 1993.

6.   Conover, C. A., L. K. Bale, J. T. Clarkson, and O. Torring. Regulation of insulin-like growth factor binding protein-5 messenger ribonucleic acid expression and protein availability in rat osteoblast-like cells. Endocrinology 132: 2525-2530, 1993[Abstract].

7.   Conover, C. A., and M. C. Kiefer. Regulation and biological effect of endogenous insulin-like growth factor binding protein-5 in human osteoblastic cells. J. Clin. Endocrinol. Metab. 76: 1153-1159, 1993[Abstract].

8.   Dong, Y., and E. Canalis. Insulin-like growth factor (IGF) I and retinoic acid induce the synthesis of IGF-binding protein 5 in rat osteoblastic cells. Endocrinology 136: 2000-2006, 1995[Abstract].

9.   Duan, C., S. B. Hawes, T. Prevette, and D. R. Clemmons. Insulin-like growth factor-I (IGF-I) regulates IGF-binding protein-5 synthesis through transcriptional activation of the gene in aortic smooth muscle cells. J. Biol. Chem. 271: 4280-4288, 1996[Abstract/Free Full Text].

10.   Dvorak, A. M., J. E. Osage, R. A. Monahan, and D. R. Dickersin. Crohn's disease: transmission electron microscopic studies. Hum. Pathol. 11: 620-634, 1980[Medline].

11.   Elgin, R. G., W. H. Busby, Jr., and D. R. Clemmons. An insulin-like growth factor (IGF) binding protein enhances the biologic response to IGF-I. Proc. Natl. Acad. Sci. USA 84: 3254-3258, 1987[Abstract/Free Full Text].

12.   Feldman, E. V., and A. E. Randolph. Regulation of insulin-like growth factor binding protein synthesis and secretion in human retinal pigment epithelial cells. J. Cell. Physiol. 158: 198-204, 1994[Medline].

13.   Gillery, P., A. Leperre, F. X. Maquart, and J. P. Borel. Insulin-like growth factor-I (IGF-I) stimulates protein synthesis and collagen gene expression in monolayer and lattice cultures of fibroblasts. J. Cell. Physiol. 152: 389-396, 1992[Medline].

14.   Goldstein, R. H., C. F. Poliks, P. F. Pilch, B. D. Smith, and A. Fine. Stimulation of collagen formation by insulin and insulin-like growth factor I in cultures of human lung fibroblasts. Endocrinology 124: 964-970, 1989[Abstract].

15.   Graham, M. F., R. F. Diegelmann, C. O. Elson, K. N. Bitar, and H. P. Ehrlich. Isolation and culture of human intestinal smooth muscle cells. Proc. Soc. Exp. Biol. Med. 176: 503-506, 1984[Abstract].

16.   Graham, M. F., R. F. Diegelmann, C. O. Elson, W. J. Lindblad, N. Gotschalk, S. Gay, and R. Gay. Collagen content and types in the intestinal strictures of Crohn's disease. Gastroenterology 94: 257-265, 1988[Medline].

17.   Graham, M. F., D. E. M. Drucker, R. F. Diegelmann, and C. O. Elson. Collagen synthesis by human intestinal smooth muscle cells in culture. Gastroenterology 92: 400-405, 1987[Medline].

18.   Hock, J. M., M. Centrella, and E. Canalis. Insulin-like growth factor I has independent effects on bone matrix formation and cell replication. Endocrinology 122: 254-260, 1988[Abstract].

19.   Jones, J. I., A. Gockerman, W. H. Busby, Jr., C. Camacho-Hubner, and D. R. Clemmons. Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I. J. Cell Biol. 121: 679-687, 1993[Abstract/Free Full Text].

20.   Rechler, M. Insulin-like growth factor binding proteins. Vitam. Horm. 47: 1-114, 1993[Medline].

21.   Ross, J. mRNA stability on mammalian cells. Microbiol. Rev. 59: 423-450, 1995[Abstract/Free Full Text].

22.   Sartor, R. B., W. J. Cromartie, D. W. Powell, and J. H. Schwab. Granulomatous enterocolitis induced in rats by purified bacterial cell wall fragments. Gastroenterology 89: 587-596, 1985[Medline].

23.   Zeeh, J. M., P. Hoffman, M. Sottili, H. Ennes, V. E. Eysselein, and J. A. McRoberts. Up-regulation of insulinlike growth factor-I binding proteins in experimental colitis in rats. Gastroenterology 108: 644-652, 1995[Medline].

24.   Zeeh, J. M., F. Procaccino, P. Hoffman, T. T. Zittel, V. E. Eysselein, and J. A. McRoberts. Colonic smooth muscle of rats is a major target of insulin-like growth factor-I in vivo and in vitro (Abstract). Gastroenterology 108: A1019, 1995.

25.   Zhang, K., M. Gharaee-Kermani, B. McGarry, and S. H. Phan. In situ hybridization analysis of rat lung alpha 1(I) and alpha 2(I) collagen gene expression in pulmonary fibrosis induced by endotracheal bleomycin injection. Lab. Invest. 70: 192-202, 1994[Medline].

26.   Zhang, K., M. D. Rekhter, D. Gordon, and S. H. Phan. Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. Am. J. Pathol. 145: 1-12, 1994[Medline].

27.   Zimmermann, E. M., R. D. Sartor, R. D. McCall, M. Pardo, D. Bender, and P. K. Lund. Insulinlike growth factor I and interleukin 1beta messenger RNA in a rat model of granulomatous enterocolitis and hepatitis. Gastroenterology 105: 399-409, 1993[Medline].

AJP Gastroint Liver Physiol 273(4):G875-G882
0193-1857/97 $5.00 Copyright © 1997 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
J. G. Simmons, Y. Ling, H. Wilkins, C. R. Fuller, A. J. D'Ercole, J. Fagin, and P. K. Lund
Cell-specific effects of insulin receptor substrate-1 deficiency on normal and IGF-I-mediated colon growth
Am J Physiol Gastrointest Liver Physiol, November 1, 2007; 293(5): G995 - G1003.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
S. G. Murali, X. Liu, D. W. Nelson, A. K. Hull, M. Grahn, M. K. Clayton, J. E. Pintar, and D. M. Ney
Intestinotrophic effects of exogenous IGF-I are not diminished in IGF binding protein-5 knockout mice
Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2007; 292(6): R2144 - R2150.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. Fruchtman, J. G. Simmons, C. Z. Michaylira, M. E. Miller, C. J. Greenhalgh, D. M. Ney, and P. K. Lund
Suppressor of cytokine signaling-2 modulates the fibrogenic actions of GH and IGF-I in intestinal mesenchymal cells
Am J Physiol Gastrointest Liver Physiol, August 1, 2005; 289(2): G342 - G350.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
X. Xin, Y. T. Hou, L. Li, P. Schmiedlin-Ren, G. M. Christman, H.-L. Cheng, K. N. Bitar, and E. M. Zimmermann
IGF-I increases IGFBP-5 and collagen {alpha}1(I) mRNAs by the MAPK pathway in rat intestinal smooth muscle cells
Am J Physiol Gastrointest Liver Physiol, May 1, 2004; 286(5): G777 - G783.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
K. L. Williams, C. R. Fuller, J. Fagin, and P. K. Lund
Mesenchymal IGF-I overexpression: paracrine effects in the intestine, distinct from endocrine actions
Am J Physiol Gastrointest Liver Physiol, October 1, 2002; 283(4): G875 - G885.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
J. G. Simmons, J. B. Pucilowska, T. O. Keku, and P. K. Lund
IGF-I and TGF-beta 1 have distinct effects on phenotype and proliferation of intestinal fibroblasts
Am J Physiol Gastrointest Liver Physiol, September 1, 2002; 283(3): G809 - G818.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
C. A. Shoubridge, C.-B. Steeb, and L. C. Read
IGFBP mRNA expression in small intestine of rat during postnatal development
Am J Physiol Gastrointest Liver Physiol, December 1, 2001; 281(6): G1378 - G1384.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
E. M. Zimmermann, L. Li, Y. T. Hou, N. K. Mohapatra, and J. B. Pucilowska
Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease
Am J Physiol Gastrointest Liver Physiol, May 1, 2001; 280(5): G1022 - G1029.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
J. B. Pucilowska, K. K. McNaughton, N. K. Mohapatra, E. C. Hoyt, E. M. Zimmermann, R. B. Sartor, and P. K. Lund
IGF-I and procollagen alpha 1(I) are coexpressed in a subset of mesenchymal cells in active Crohn's disease
Am J Physiol Gastrointest Liver Physiol, December 1, 2000; 279(6): G1307 - G1322.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
J. F. Kuemmerle and B. Teng
Regulation of IGFBP-4 levels in human intestinal muscle by an IGF-I-activated, confluence-dependent protease
Am J Physiol Gastrointest Liver Physiol, November 1, 2000; 279(5): G975 - G982.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
J. B. Pucilowska, K. L. Williams, and P. K. Lund
Fibrogenesis IV. Fibrosis and inflammatory bowel disease: cellular mediators and animal models
Am J Physiol Gastrointest Liver Physiol, October 1, 2000; 279(4): G653 - G659.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
J. F. Kuemmerle
Endogenous IGF-I regulates IGF binding protein production in human intestinal smooth muscle cells
Am J Physiol Gastrointest Liver Physiol, May 1, 2000; 278(5): G710 - G717.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
D. W. Powell, R. C. Mifflin, J. D. Valentich, S. E. Crowe, J. I. Saada, and A. B. West
Myofibroblasts. I. Paracrine cells important in health and disease
Am J Physiol Cell Physiol, July 1, 1999; 277(1): C1 - C19.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
T. L. Bushman and J. F. Kuemmerle
IGFBP-3 and IGFBP-5 production by human intestinal muscle: reciprocal regulation by endogenous TGF-beta 1
Am J Physiol Gastrointest Liver Physiol, December 1, 1998; 275(6): G1282 - G1290.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
J. F. Kuemmerle and T. L. Bushman
IGF-I stimulates intestinal muscle cell growth by activating distinct PI 3-kinase and MAP kinase pathways
Am J Physiol Gastrointest Liver Physiol, July 1, 1998; 275(1): G151 - G158.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow