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Bile Salt Stimulates Intestinal Epithelial Cell Migration through TGFβ after Wounding
Journal of Surgical Research 97, 49 –53 (2001) doi:10.1006/jsre.2001.6110, available online at http://www.idealibrary.com on
Bile Salt Stimulates Intestinal Epithelial Cell Migration through TGF after Wounding Eric D. Strauch, 1 Jian–Ying Wang, and Barbara L. Bass Department of Surgery, University of Maryland and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201 Presented at the Annual Meeting of the Association for Academic Surgery, Tampa, Florida, November 2– 4, 2000; published online March 28, 2001
function and in preservation of an intact mucosa. © 2001 Academic Press Key Words: restitution; bile salt; transforming growth factor-; intestinal growth.
Background. In addition to aiding in the digestion of fats, luminal bile salts have been shown to modulate gastrointestinal epithelial growth, differentiation, and other functions. We hypothesized that bile acids could modulate the intestinal mucosal repair process of restitution. We investigated the effect of the bile salt taurodeoxycholic acid on epithelial migration and identified a role for TGF, a widely expressed cytokine in the intestinal villus, in this repair process. Methods. Using a well-established model of epithelial restitution, IEC-6 cells were plated on 60-mm Matrigel-coated plastic dishes and grown to confluence. The epithelium was wounded by scraping with a 6-mm-wide blade to create a smooth denuded edge and cell migration was measured 8 h later. Cells were grown in control DMEM with 5% FBS with or without 0.01–2 mM taurodeoxycholic acid (TDCA). In parallel experiments, cells were harvested for Northern analysis of TGF and GAPDH expression; [ 3H]thymidine uptake was used to measure proliferation. Anti-TGF antibody was added to cells grown in the presence of 0.05 mM TDCA and migration was measured at 8 h. Results. TDCA at physiologic luminal concentrations augments IEC-6 cell migration, with a maximal effect at 0.05 mM. TDCA inhibited proliferation at these concentrations. TGF expression increased in response to bile acid, while wounding had less of an effect on TGF expression. Blockade of TGF function with TGF antibody eliminated the effect of bile on cell migration. Conclusions. Bile acid at physiologic concentrations augments small intestinal epithelial cell migration. The process is dependent on TGF and is independent of cell division. The data further support a role for bile acids and TGF in differentiated intestinal cell
INTRODUCTION
Small intestinal digestive and secretory functions require an intact mucosa. Minor disruption of mucosal integrity is common and may result from superficial erosions from luminal contents, medications, or infections. More severe injury may occur as a result of ischemia, mechanical obstruction, or necrotizing infections. While severe injuries must heal by cell proliferation, the more common superficial injuries heal by a process known as restitution. Restitution is that process by which normal adjacent intestinal epithelial cells migrate over a denuded area to reseal and repair the epithelium. Migration of the cells occurs as a sheet to reform cell contacts and reestablish barrier function. While all the elements in epithelial migration are as yet unknown, the cytoskeletal proteins, adhesion receptors, and extracellular matrix glycoproteins are involved in this process. The stimuli and mediators of migration have been the subject of considerable investigation in recent years. Intestinal epithelial restitution occurs in a matter of hours following injury and does not require cell proliferation [1–3]. In contrast, repair of deep or extensive mucosal injuries requires cell proliferation and several days to complete repairs. Transforming growth factor  (TGF) is a multifunctional cytokine widely distributed in tissues. TGF is produced by many cell types including the gastrointestinal epithelium [4, 5]. TGF has been shown to inhibit cell proliferation, to stimulate epithelial migration, and to increase the production of extracellular matrix, all processes which promote restitution [4 –9].
1
To whom correspondence should be addressed at Department of Surgery, University of Maryland School of Medicine, 22 South Greene Street, Baltimore, MD 21201. Fax: (410) 328-0652.
49
0022-4804/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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JOURNAL OF SURGICAL RESEARCH: VOL. 97, NO. 1, MAY 1, 2001
Bile salts are normally found within the gastrointestinal succus entericus where their primary function is to aid in the digestion of lipids and lipid-soluble vitamins [10]. In the lumen, most bile salts are present in micellar form, but some exist as free bile salts. Recent studies have shown that bile salts have various biologic effects independent of their role in digestion. The bile salt deoxycholic acid has been shown to induce apoptosis in a colon cancer cell line [11]. Taurodeoxycholic acid (TDCA) increases esophageal mucosal growth in a rabbit explant model [12]. Bile salts may also modulate gene expression; for example, deoxycholate acid has been shown to modulate p53 gene expression in colonic adenoma cell lines [13]. These suggest potent cellular effects of bile salts operative at the gene expression and growth regulatory level. There are no data regarding the effects of bile salts on the process of intestinal mucosal restitution. The current study was designed to test the hypothesis that bile salt at physiologic luminal conditions would augment the process of intestinal mucosal restitution. We chose to study the bile salt taurodeoxycholate because it is a common soluble bile salt found in the succus entericus. We have also previously demonstrated growth stimulatory effects for this bile salt at concentrations that are found in the intestinal lumen [12]. To test our hypothesis we examined the effect of TDCA on intestinal epithelial cell migration in a well-established in vitro model of restitution using cultured IEC-6 rat intestinal crypt cells. When a stimulatory effect of bile salts was noted on migration we then sought to determine if this enhanced migration involved TGF and determined if this migration was in fact dependent on TGF expression. MATERIAL AND METHODS Materials. Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed fetal bovine serum were from GIBCO (Grand Island, NY). Biochemicals were purchased from Sigma (St. Louis, MO). Tritiated thymidine was purchased from NEN Life Sciences, Inc. (Boston MA). AntiTGF was purchased from R&D Systems (Minneapolis, MN). Cell culture and experimental protocol. The IEC-6 cell line was purchased from ATCC at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. [14]. IEC-6 cells originated from intestinal crypt cells as judged by morphological and immunological criteria. They are nontumorigenic and retain the undifferentiated character of epithelial stem cells. Stock cells were maintained in T-150 flasks in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated FBS with 1% antibiotic. Flasks were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2. Stock cells were subcultured once a week at 1:2; medium was change three times weekly. The cells were restarted from original frozen stock. Passages 16 –19 were used in the experiments. There were no significant changes of biologic function and characterizations from passages 15–20. The general protocol of the wounding model used was described previously [15]. Briefly, IEC-6 cells were plated at 6.25 ⫻ 10 4 cells/ cm 2 in DMEM plus 5% FBS on 60-mm plates thinly coated with
Matrigel and grown until confluent. The epithelium is scraped using a sterile 6-mm-wide blade to create a smooth denuded wound, and cell migration is allowed to occur over the denuded area for 8 h. Cell migration is measured by counting the cells in the denuded area in a randomized-blinded fashion. Results are reported as the number of cells per 0.25 mm of scratch. Effect of TDCA on intestinal cell migration. IEC-6 cells were grown as above. Following the standard wounding protocol, culture media were changed immediately after wounding to either control media or media containing TDCA at a concentration ranging from 0.01 to 2 mM. Cell migration was measured in a standard fashion by counting the cells in a denuded area in a randomized blinded manner. Cell proliferation determination by tritiated thymidine uptake. IEC-6 cells were plated at 6.25 ⫻ 10 4 cells/cm 2 in DMEM plus 5% FBS for 24 h. Tritiated thymidine (1 ci/ml) was added to the media for 24 h in the presence of the IEC-6 cells in DMEM with 5% FBS and TDCA in concentrations ranging from 0.01 to 1 mM. Cell proliferation was determined by measuring the number of counts per minute in the IEC-6 cells per microgram of protein. To determine if the growth arrest noted in cells exposed to TDCA was a result of toxicity, resumption of cell proliferation was determined by exposing the IEC-6 cells to the same concentrations of TDCA for 24 h and then washing the TDCA from the IEC-6 cells. Tritiated thymidine incorporation of the cells in the new media was then determined. TGF gene expression. Cell were grown as for the wounding protocol in control media and a media with 0.05 mM TDCA. After wounding cells were harvested at 8 h for RNA extraction as described below. Immunoneutralization with anti-TGF. To determine if migration was dependent on TGF, migrating cells were grown with and without anti-TGF antibody. IEC-6 cells were grown in DMEM with 5% FBS with and without 0.05 mM of TDCA at the time of wounding. Cell migration was determined 8 h after wounding as described above. RNA isolation and Northern blot analysis. Total RNA was extracted with guanidinium isothiocyanate solution and purified by CsCl density gradient ultracentrifugation as described by Chirgwin et al. [16]. Briefly the monolayer of cells was washed in DPBS and lysed in 4 M guanidinium isothiocyanate. The lysates were brought to 2.4 M CsCl concentration and centrifuged through a 5.7 M CsCl cushion at 150,000g at 20°C for 24 h. After centrifugation, the supernatant was aspirated and the tube cut 0.5 cm from the bottom with a flamed scalpel. The resulting RNA pellet was dissolved in Tris 䡠 HCL (pH 7.5) containing 1 mM EDTA, 5% sodium laurylsarcosine, and 5% phenol (added just before use). The addition of 0.1 vol of 3 M sodium acetate and 2.5 vol of ethanol precipitated the purified RNA from the aqueous phase in sequence. Final RNA was dissolved in water and estimated from its ultraviolet absorbance at 260 nm by using a conversion factor of 40 units. In most cases, 30 g of total cellular RNA was denatured and fractionated electrophoretically by using 1.2% agarose gel containing 3% formaldehyde and transferred by blotting to nitrocellulose filters. Blots were prehybridized for 24 h at 42°C with 5X Denhardt’s solution and 5X standard saline sperm DNA. CDNA probes for TGF and GAPDH were labeled with [␣- 32P]dCTP by using a standard nick-translation procedure. Hybridization was carried out overnight at 42°C in the same solution containing 10% dextran sulfate and 32P-labeled DNA probes. Blots were washed with two changes of 1X saline sodium citrate-0.1% SDS at room temperature. After the final wash, the filters were autoradiographed with intensifying screens at ⫺70°C. The signal was quantified by densitometry analysis of the autoradiograms. Statistics. Values are means ⫾ SE from six dishes. Autoradiographed results were repeated. The significance of the difference between means was determined by ANOVA.
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The Effect of TDCA on TGF Gene Expression To determine if TDCA were altering migration via an effect on TGF, we examined TGF gene expression in migrating cells in the presence or absence of TDCA. In control cells, there was a slight increase in TGF mRNA level 8 h after wounding (Fig. 3). When 0.05 mM TDCA was given immediately after wounding, the levels for TGF mRNA increased significantly compared with migrating cells treated without TDCA. To determine if increased migration was dependent on TGF, migration was measured in cells with and without anti-TGF antibody. TDCA-stimulated migration was significantly decreased in the presence of antiTGF. The rate of cell migration was similar to that of the control group after treatment with TDCA plus antiTGF antibody (Fig. 4). These results suggest that TDCA stimulates intestinal epithelial cell migration after wounding, at least partially, through the TGF pathway. DISCUSSION FIG. 1. Effect of TDCA on cell migration in cultured IEC-6 cells. (A) Photographs of migrating cells in the absence (a) or presence of 0.05 mM TDCA (b). Cells were grown in DMEM containing 5% serum for 72 h and cell migration was assayed 8 h after removal of part of cell layer. (B) Rates of cell migration after exposure to different concentrations of TDCA. Values are mean ⫾ SE from 6 dishes. *P ⬍ 0.05 compared with control group.
RESULTS
The Effect of TDCA on Migration in IEC-6 Cells TDCA at concentrations ranging from 0.01 to 0.05 mM increased cell migration. The maximal response was achieved at 0.05 mM (Fig. 1). TDCA at 0.5 mM did not affect IEC-6 cell migration after wounding, while higher concentrations of TDCA (2 mM and higher) were toxic to the IEC-6 cells and resulted in cell death.
Early mucosal restitution is an important primary repair modality in the gastrointestinal tract. The process of mucosal restitution is the rapid resealing of superficial wounds and occurs as a consequence of epithelial cell migration into the injury, a process not requiring cell proliferation. The role of the gastrointestinal luminal contents in this process has not been determined. Bile salts are physiologically active components within the gastrointestinal lumen. Reflux of bile salts into the esophagus causes mucosal injury and is implicated in the development of reflux esophagitis [17]. On the other hand, bile acid at low concentration results in hyperproliferation of the colonic epithelium and is involved in the regulation of colonic mucosal growth [11, 18 –21]. Thus bile salts have cellular effects on the gastrointestinal epithelium. We chose to study
The Effect of TDCA on Proliferation in IEC-6 Cells In order to determine possible involvement of cell proliferation in this repair process in vitro, DNA synthesis was measured after wounding. Figure 2 clearly shows that the stimulation of cell migration by TDCA did not result from the change in cell proliferation. Exposure to TDCA at concentrations ranging from 0.01 to 1 mM for 8 h after wounding dose-dependently inhibited DNA synthesis as measured by [ 3H]thymidine incorporation techniques, indicating that TDCA inhibited IEC-6 cell growth during restitution. The inhibitory effect of TDCA on DNA synthesis is not a simple toxic effect up to 1 mM because cell growth resumed when the TDCA was washed out (data not shown). The rate of IEC-6 cell growth returned to normal within 16 h after removal of the TDCA.
FIG. 2. Effect of various concentrations of TDCA on DNA synthesis in IEC-6 cells. Cells were grown for 24 h and exposed to different concentration of TDCA. DNA synthesis was assayed 24 h after the treatment by measurement of [ 3H]thymidine incorporation. Values are mean ⫾ SE from 6 dishes. *P ⬍ 0.05 compared with control group.
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FIG. 3. TGF mRNA levels in migrating cells in the presence or absence of TDCA. (A) Representative autoradiograms of Northern blot analysis. Cells were grown for 72 h and 0.05 mM TDCA was added immediately after removal of part of the cell layer. Total RNA was extracted 8 h after wounding and TGF mRNA levels were measured by Northern blot analysis by using TGF1 cDNA probe. (B) Quantitative analysis of the ratio of TGF mRNA to GAPDH derived from densitometric analysis of autoradiograms described in A.
TDCA, a common conjugated bile salt, found to have other cellular effects at physiologic concentrations in the gastrointestinal tract for our studies.
FIG. 4.
These data reveal that the bile salt TDCA at physiologic concentrations augments the migration of intestinal epithelial cells in an in vitro model that mimics the early cell division-independent stages of epithelial restitution. When TDCA was given immediately after wounding, the bile salt dose-dependently increased intestinal epithelial cell migration in a statistically significant manner. At higher concentrations of TDCA (up to 1 mM) epithelial cell migration returned to baseline levels. Very high concentration (2 mM) were toxic to the cells. The concentration of free luminal bile salts varies from 0.01 to 0.5 mM. At higher concentration bile salts are found as micelles. Thus, free TDCA at concentrations that the intestinal epithelial cells may be exposed to in vivo augments intestinal epithelial cell migration in vitro. This stimulatory effect is independent from cell proliferation because the treatment with similar doses of TDCA inhibited DNA synthesis in a dose-dependent reversible manner in IEC-6 cells. Migration of intestinal epithelial cells is an important component of the restitution process, the mechanism of intestinal repair after superficial injury [1–3]. Although the exact mechanism of restitution has not been elucidated, the cytokine TGF has been shown to play an important role in the stimulation of cell migration after wounding. TGF is a multifunctional cytokine that is produced in the gastrointestinal mucosal epithelial cells and plays an active role in the regulation of wound repair. TGF gene expression increases after wounding and is necessary for intestinal epithelial cell migration [16]. Exogenous TGF has been shown to inhibit cell proliferation [6], stimulate cell migration [7, 9], and increase the production of extracellular matrix [5, 8]. The stimulation of cell migration
Effect of immunoneutralizing anti-TGFb antibody added to cultures on cell migration in IEC-6 cells.
STRAUCH, WANG, AND BASS: TDCA AND EPITHELIAL RESTITUTION 7.
Basson, J. A., Modlin, I. M., Flynn, S. D., Jena, B. P., and Madri, J. A. Independent modulation of migration and proliferation by growth factors, matrix proteins and pharmacological agents in an in vitro model of mucosal healing. Surgery 112: 299, 1992.
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Barnard, J. A., Lyons, R. M., and Moses, H. L. The cell biology of tranforming growth factor . Biochim. Biophys. Acta 1032: 79, 1990.
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Ciacci, C., Lind, S. E., and Podolsky, D. K. Transforming growth factor  regulation of migration in wounded rat intestinal epithelial monolayers. Gastroenterology 105: 93, 1993.
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Hoffman, A. F. Intestinal absorption of bile acids and biliary constituents. Physiol. Gastrointestinal Tract 2: 1845, 1994.
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Martinez, J. D., Stratagoules, E. D., LaRue, J. M., Powell, A. A., Gause, P. R., Craven, M. T., Payne, C. M., Powell, M. B., Gerner, E. W., and Earnest, D. L. Different bile acids exhibit distinct biological effects: The tumor promoter deoxycholic acid induces apoptosis and he chemopreventive agent ursodeosycholic acid inhibits cell proliferation. Nutr. Cancer 31: 111, 1998.
12.
D’Addio, V., Sayles, J. M., Alvarez, C., Lally, K., and Bass, B. L. Bile: A trophic factor for rabbit esophageal epithelium in vitro. Surg. Forum 647: 125, 1996.
13.
Palmer, D. G., Paraskeva, C., and Williams, A. C. Modulation of p53 expression in cultured colonic adenoma cell lines by the naturally occurring lumenal factors butyrate and deoxycholate. Int. J. Cancer 73: 702, 1997.
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Quaroni, A., Wands, J., Trelstad R. L., and Isselbacher, K. J. Epithelial cell cultures from rat small intestine. J. Cell Biol. 80: 248, 1979.
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McCormack, S. A., Wang, J.-Y., and Johnson L. R. Polyamine deficiency causes reorgainzation of F-actin and tropomyosin in IEC-6 cells. Am J. Physiol. 267: C715, 1994.
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Feil, W., Wenzl, E., Vattay, P., Starlinger, M., Sogukoglu, T., and Schiessel, R. Repair of rabbit duodenal mucosa after acid injury in vivo and in vitro. Gastroenterology 92: 1973, 1987.
Chirgwin, J. M., Przybyla, A. E., Macdonald, R. J., and Rutter, W. J. Isolation of biologically active ribonucleic acid sources enriched in ribonucleases. Biochemistry 18: 5294, 1979.
17.
Rutten, M. J., and Ito, S. Morphology and electrophysiology of guinea pig gastric mucosal repair in vitro. Am. J. Physiol. 244: G171, 1983.
Vaezi, M. F., Singh, S, and Richter, J. E. Role of acid and duodenogastric reflux in esophageal mucosal injury: A review of animal and human studies. Gastroenterology 108: 1897, 1995.
18.
Huang, X. P, Fan, X. T., Desjeux, J. F., and Castagna, M. Bile acids, non-phorbol-ester-type tumor promoters, stimulate the phosphorylation of protein kinase C substrates in human platelets and colon cell line Ht29. Int. J. Cancer 52: 444, 1992.
by TDCA correlates with a significant increase in expression of the TGF gene. Furthermore, the increase in cell migration seen in the presence of TDCA is TGF dependent as inhibition of the activity of TGF by anti-TGF antibody significantly prevented the stimulatory effect of TDCA on cell migration after wounding. These findings suggest that the bile acid TDCA induces intestinal epithelial migration, at least partially, through the TGF-dependent pathway. Thus, the bile salt TDCA contributes to the maintenance of intestinal mucosal integrity by augmenting intestinal epithelial restitution by activating a signaling pathway, yet to be defined, involved in the restitution process through TGF. In summary, these results indicate that exposure to TDCA, at low concentrations that the intestinal epithelium may encounter during normal function, significantly stimulates small intestinal epithelial cell migration in an in vitro system. TDCA also increases expression of the TGF gene in migrating cells and the increase in epithelial cell migration by TDCA is TGF dependent. The exact mechanism by which TDCA stimulates TGF gene expression has not been elucidated but further studies to determine signaling pathways leading to upregulation of TGF gene expression are warranted.
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Silen, W., and Ito, S. Mechanism for rapid-epithelialization of the gastric mucosal surface. Annu. Rev. Physiol. 47: 217, 1985.
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Armendariz-Borunda, J., Katai, H., Jones, C. M., Seuer, J. M., Kang, A. H., and Raghow, R. Transforming growth factor- gene is transiently enhanced at a critical stage during liver regeneration after CCl 4 treatment. Lab. Invest. 69: 283, 1993.
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Fitzer, C. J., O’Brian, C. A., Guillem, J. G., and Weinstein, I. B. The regulation of protein kinase C by chenodeoxycholate, deoxycholate and several structurally related bile acids. Carcinogenesis 8: 217, 1987.
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Ignotz, R. A., and Massague, J. Transforming growth factor  stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J. Biol. Chem. 261: 4337, 1986.
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Craven, P. A., Pfanstiel, J., and DeRubertis, F. R. Role of activation of protein kinase C in the stimulation of colonic epithelial proliferation and reactive oxygen formation by bile acids. J. Clin. Invest. 79: 532, 1987.
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Peitenpol, J. A., Holt, J. T., Stein, R. W., and Moses, H. L. Transforming growth factor 1 suppression of c-myc gene transcription: Role in inhibition of keratinocyte proliferation. Proc. Natl. Acad. Sci. USA 87: 3758, 1990.
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Pongracz, J., Clark, P., Neoptolemos, J. P., and Lord, J. M. Expression of protein kinase C isoenzymes in colorectal cancer tissue and their differential activation by different bile acids. Int. J. Cancer 61: 35, 1995.
Bile Salt Stimulates Intestinal Epithelial Cell Migration through TGF after Wounding Eric D. Strauch, 1 Jian–Ying Wang, and Barbara L. Bass Department of Surgery, University of Maryland and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201 Presented at the Annual Meeting of the Association for Academic Surgery, Tampa, Florida, November 2– 4, 2000; published online March 28, 2001
function and in preservation of an intact mucosa. © 2001 Academic Press Key Words: restitution; bile salt; transforming growth factor-; intestinal growth.
Background. In addition to aiding in the digestion of fats, luminal bile salts have been shown to modulate gastrointestinal epithelial growth, differentiation, and other functions. We hypothesized that bile acids could modulate the intestinal mucosal repair process of restitution. We investigated the effect of the bile salt taurodeoxycholic acid on epithelial migration and identified a role for TGF, a widely expressed cytokine in the intestinal villus, in this repair process. Methods. Using a well-established model of epithelial restitution, IEC-6 cells were plated on 60-mm Matrigel-coated plastic dishes and grown to confluence. The epithelium was wounded by scraping with a 6-mm-wide blade to create a smooth denuded edge and cell migration was measured 8 h later. Cells were grown in control DMEM with 5% FBS with or without 0.01–2 mM taurodeoxycholic acid (TDCA). In parallel experiments, cells were harvested for Northern analysis of TGF and GAPDH expression; [ 3H]thymidine uptake was used to measure proliferation. Anti-TGF antibody was added to cells grown in the presence of 0.05 mM TDCA and migration was measured at 8 h. Results. TDCA at physiologic luminal concentrations augments IEC-6 cell migration, with a maximal effect at 0.05 mM. TDCA inhibited proliferation at these concentrations. TGF expression increased in response to bile acid, while wounding had less of an effect on TGF expression. Blockade of TGF function with TGF antibody eliminated the effect of bile on cell migration. Conclusions. Bile acid at physiologic concentrations augments small intestinal epithelial cell migration. The process is dependent on TGF and is independent of cell division. The data further support a role for bile acids and TGF in differentiated intestinal cell
INTRODUCTION
Small intestinal digestive and secretory functions require an intact mucosa. Minor disruption of mucosal integrity is common and may result from superficial erosions from luminal contents, medications, or infections. More severe injury may occur as a result of ischemia, mechanical obstruction, or necrotizing infections. While severe injuries must heal by cell proliferation, the more common superficial injuries heal by a process known as restitution. Restitution is that process by which normal adjacent intestinal epithelial cells migrate over a denuded area to reseal and repair the epithelium. Migration of the cells occurs as a sheet to reform cell contacts and reestablish barrier function. While all the elements in epithelial migration are as yet unknown, the cytoskeletal proteins, adhesion receptors, and extracellular matrix glycoproteins are involved in this process. The stimuli and mediators of migration have been the subject of considerable investigation in recent years. Intestinal epithelial restitution occurs in a matter of hours following injury and does not require cell proliferation [1–3]. In contrast, repair of deep or extensive mucosal injuries requires cell proliferation and several days to complete repairs. Transforming growth factor  (TGF) is a multifunctional cytokine widely distributed in tissues. TGF is produced by many cell types including the gastrointestinal epithelium [4, 5]. TGF has been shown to inhibit cell proliferation, to stimulate epithelial migration, and to increase the production of extracellular matrix, all processes which promote restitution [4 –9].
1
To whom correspondence should be addressed at Department of Surgery, University of Maryland School of Medicine, 22 South Greene Street, Baltimore, MD 21201. Fax: (410) 328-0652.
49
0022-4804/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
50
JOURNAL OF SURGICAL RESEARCH: VOL. 97, NO. 1, MAY 1, 2001
Bile salts are normally found within the gastrointestinal succus entericus where their primary function is to aid in the digestion of lipids and lipid-soluble vitamins [10]. In the lumen, most bile salts are present in micellar form, but some exist as free bile salts. Recent studies have shown that bile salts have various biologic effects independent of their role in digestion. The bile salt deoxycholic acid has been shown to induce apoptosis in a colon cancer cell line [11]. Taurodeoxycholic acid (TDCA) increases esophageal mucosal growth in a rabbit explant model [12]. Bile salts may also modulate gene expression; for example, deoxycholate acid has been shown to modulate p53 gene expression in colonic adenoma cell lines [13]. These suggest potent cellular effects of bile salts operative at the gene expression and growth regulatory level. There are no data regarding the effects of bile salts on the process of intestinal mucosal restitution. The current study was designed to test the hypothesis that bile salt at physiologic luminal conditions would augment the process of intestinal mucosal restitution. We chose to study the bile salt taurodeoxycholate because it is a common soluble bile salt found in the succus entericus. We have also previously demonstrated growth stimulatory effects for this bile salt at concentrations that are found in the intestinal lumen [12]. To test our hypothesis we examined the effect of TDCA on intestinal epithelial cell migration in a well-established in vitro model of restitution using cultured IEC-6 rat intestinal crypt cells. When a stimulatory effect of bile salts was noted on migration we then sought to determine if this enhanced migration involved TGF and determined if this migration was in fact dependent on TGF expression. MATERIAL AND METHODS Materials. Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed fetal bovine serum were from GIBCO (Grand Island, NY). Biochemicals were purchased from Sigma (St. Louis, MO). Tritiated thymidine was purchased from NEN Life Sciences, Inc. (Boston MA). AntiTGF was purchased from R&D Systems (Minneapolis, MN). Cell culture and experimental protocol. The IEC-6 cell line was purchased from ATCC at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. [14]. IEC-6 cells originated from intestinal crypt cells as judged by morphological and immunological criteria. They are nontumorigenic and retain the undifferentiated character of epithelial stem cells. Stock cells were maintained in T-150 flasks in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated FBS with 1% antibiotic. Flasks were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2. Stock cells were subcultured once a week at 1:2; medium was change three times weekly. The cells were restarted from original frozen stock. Passages 16 –19 were used in the experiments. There were no significant changes of biologic function and characterizations from passages 15–20. The general protocol of the wounding model used was described previously [15]. Briefly, IEC-6 cells were plated at 6.25 ⫻ 10 4 cells/ cm 2 in DMEM plus 5% FBS on 60-mm plates thinly coated with
Matrigel and grown until confluent. The epithelium is scraped using a sterile 6-mm-wide blade to create a smooth denuded wound, and cell migration is allowed to occur over the denuded area for 8 h. Cell migration is measured by counting the cells in the denuded area in a randomized-blinded fashion. Results are reported as the number of cells per 0.25 mm of scratch. Effect of TDCA on intestinal cell migration. IEC-6 cells were grown as above. Following the standard wounding protocol, culture media were changed immediately after wounding to either control media or media containing TDCA at a concentration ranging from 0.01 to 2 mM. Cell migration was measured in a standard fashion by counting the cells in a denuded area in a randomized blinded manner. Cell proliferation determination by tritiated thymidine uptake. IEC-6 cells were plated at 6.25 ⫻ 10 4 cells/cm 2 in DMEM plus 5% FBS for 24 h. Tritiated thymidine (1 ci/ml) was added to the media for 24 h in the presence of the IEC-6 cells in DMEM with 5% FBS and TDCA in concentrations ranging from 0.01 to 1 mM. Cell proliferation was determined by measuring the number of counts per minute in the IEC-6 cells per microgram of protein. To determine if the growth arrest noted in cells exposed to TDCA was a result of toxicity, resumption of cell proliferation was determined by exposing the IEC-6 cells to the same concentrations of TDCA for 24 h and then washing the TDCA from the IEC-6 cells. Tritiated thymidine incorporation of the cells in the new media was then determined. TGF gene expression. Cell were grown as for the wounding protocol in control media and a media with 0.05 mM TDCA. After wounding cells were harvested at 8 h for RNA extraction as described below. Immunoneutralization with anti-TGF. To determine if migration was dependent on TGF, migrating cells were grown with and without anti-TGF antibody. IEC-6 cells were grown in DMEM with 5% FBS with and without 0.05 mM of TDCA at the time of wounding. Cell migration was determined 8 h after wounding as described above. RNA isolation and Northern blot analysis. Total RNA was extracted with guanidinium isothiocyanate solution and purified by CsCl density gradient ultracentrifugation as described by Chirgwin et al. [16]. Briefly the monolayer of cells was washed in DPBS and lysed in 4 M guanidinium isothiocyanate. The lysates were brought to 2.4 M CsCl concentration and centrifuged through a 5.7 M CsCl cushion at 150,000g at 20°C for 24 h. After centrifugation, the supernatant was aspirated and the tube cut 0.5 cm from the bottom with a flamed scalpel. The resulting RNA pellet was dissolved in Tris 䡠 HCL (pH 7.5) containing 1 mM EDTA, 5% sodium laurylsarcosine, and 5% phenol (added just before use). The addition of 0.1 vol of 3 M sodium acetate and 2.5 vol of ethanol precipitated the purified RNA from the aqueous phase in sequence. Final RNA was dissolved in water and estimated from its ultraviolet absorbance at 260 nm by using a conversion factor of 40 units. In most cases, 30 g of total cellular RNA was denatured and fractionated electrophoretically by using 1.2% agarose gel containing 3% formaldehyde and transferred by blotting to nitrocellulose filters. Blots were prehybridized for 24 h at 42°C with 5X Denhardt’s solution and 5X standard saline sperm DNA. CDNA probes for TGF and GAPDH were labeled with [␣- 32P]dCTP by using a standard nick-translation procedure. Hybridization was carried out overnight at 42°C in the same solution containing 10% dextran sulfate and 32P-labeled DNA probes. Blots were washed with two changes of 1X saline sodium citrate-0.1% SDS at room temperature. After the final wash, the filters were autoradiographed with intensifying screens at ⫺70°C. The signal was quantified by densitometry analysis of the autoradiograms. Statistics. Values are means ⫾ SE from six dishes. Autoradiographed results were repeated. The significance of the difference between means was determined by ANOVA.
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The Effect of TDCA on TGF Gene Expression To determine if TDCA were altering migration via an effect on TGF, we examined TGF gene expression in migrating cells in the presence or absence of TDCA. In control cells, there was a slight increase in TGF mRNA level 8 h after wounding (Fig. 3). When 0.05 mM TDCA was given immediately after wounding, the levels for TGF mRNA increased significantly compared with migrating cells treated without TDCA. To determine if increased migration was dependent on TGF, migration was measured in cells with and without anti-TGF antibody. TDCA-stimulated migration was significantly decreased in the presence of antiTGF. The rate of cell migration was similar to that of the control group after treatment with TDCA plus antiTGF antibody (Fig. 4). These results suggest that TDCA stimulates intestinal epithelial cell migration after wounding, at least partially, through the TGF pathway. DISCUSSION FIG. 1. Effect of TDCA on cell migration in cultured IEC-6 cells. (A) Photographs of migrating cells in the absence (a) or presence of 0.05 mM TDCA (b). Cells were grown in DMEM containing 5% serum for 72 h and cell migration was assayed 8 h after removal of part of cell layer. (B) Rates of cell migration after exposure to different concentrations of TDCA. Values are mean ⫾ SE from 6 dishes. *P ⬍ 0.05 compared with control group.
RESULTS
The Effect of TDCA on Migration in IEC-6 Cells TDCA at concentrations ranging from 0.01 to 0.05 mM increased cell migration. The maximal response was achieved at 0.05 mM (Fig. 1). TDCA at 0.5 mM did not affect IEC-6 cell migration after wounding, while higher concentrations of TDCA (2 mM and higher) were toxic to the IEC-6 cells and resulted in cell death.
Early mucosal restitution is an important primary repair modality in the gastrointestinal tract. The process of mucosal restitution is the rapid resealing of superficial wounds and occurs as a consequence of epithelial cell migration into the injury, a process not requiring cell proliferation. The role of the gastrointestinal luminal contents in this process has not been determined. Bile salts are physiologically active components within the gastrointestinal lumen. Reflux of bile salts into the esophagus causes mucosal injury and is implicated in the development of reflux esophagitis [17]. On the other hand, bile acid at low concentration results in hyperproliferation of the colonic epithelium and is involved in the regulation of colonic mucosal growth [11, 18 –21]. Thus bile salts have cellular effects on the gastrointestinal epithelium. We chose to study
The Effect of TDCA on Proliferation in IEC-6 Cells In order to determine possible involvement of cell proliferation in this repair process in vitro, DNA synthesis was measured after wounding. Figure 2 clearly shows that the stimulation of cell migration by TDCA did not result from the change in cell proliferation. Exposure to TDCA at concentrations ranging from 0.01 to 1 mM for 8 h after wounding dose-dependently inhibited DNA synthesis as measured by [ 3H]thymidine incorporation techniques, indicating that TDCA inhibited IEC-6 cell growth during restitution. The inhibitory effect of TDCA on DNA synthesis is not a simple toxic effect up to 1 mM because cell growth resumed when the TDCA was washed out (data not shown). The rate of IEC-6 cell growth returned to normal within 16 h after removal of the TDCA.
FIG. 2. Effect of various concentrations of TDCA on DNA synthesis in IEC-6 cells. Cells were grown for 24 h and exposed to different concentration of TDCA. DNA synthesis was assayed 24 h after the treatment by measurement of [ 3H]thymidine incorporation. Values are mean ⫾ SE from 6 dishes. *P ⬍ 0.05 compared with control group.
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JOURNAL OF SURGICAL RESEARCH: VOL. 97, NO. 1, MAY 1, 2001
FIG. 3. TGF mRNA levels in migrating cells in the presence or absence of TDCA. (A) Representative autoradiograms of Northern blot analysis. Cells were grown for 72 h and 0.05 mM TDCA was added immediately after removal of part of the cell layer. Total RNA was extracted 8 h after wounding and TGF mRNA levels were measured by Northern blot analysis by using TGF1 cDNA probe. (B) Quantitative analysis of the ratio of TGF mRNA to GAPDH derived from densitometric analysis of autoradiograms described in A.
TDCA, a common conjugated bile salt, found to have other cellular effects at physiologic concentrations in the gastrointestinal tract for our studies.
FIG. 4.
These data reveal that the bile salt TDCA at physiologic concentrations augments the migration of intestinal epithelial cells in an in vitro model that mimics the early cell division-independent stages of epithelial restitution. When TDCA was given immediately after wounding, the bile salt dose-dependently increased intestinal epithelial cell migration in a statistically significant manner. At higher concentrations of TDCA (up to 1 mM) epithelial cell migration returned to baseline levels. Very high concentration (2 mM) were toxic to the cells. The concentration of free luminal bile salts varies from 0.01 to 0.5 mM. At higher concentration bile salts are found as micelles. Thus, free TDCA at concentrations that the intestinal epithelial cells may be exposed to in vivo augments intestinal epithelial cell migration in vitro. This stimulatory effect is independent from cell proliferation because the treatment with similar doses of TDCA inhibited DNA synthesis in a dose-dependent reversible manner in IEC-6 cells. Migration of intestinal epithelial cells is an important component of the restitution process, the mechanism of intestinal repair after superficial injury [1–3]. Although the exact mechanism of restitution has not been elucidated, the cytokine TGF has been shown to play an important role in the stimulation of cell migration after wounding. TGF is a multifunctional cytokine that is produced in the gastrointestinal mucosal epithelial cells and plays an active role in the regulation of wound repair. TGF gene expression increases after wounding and is necessary for intestinal epithelial cell migration [16]. Exogenous TGF has been shown to inhibit cell proliferation [6], stimulate cell migration [7, 9], and increase the production of extracellular matrix [5, 8]. The stimulation of cell migration
Effect of immunoneutralizing anti-TGFb antibody added to cultures on cell migration in IEC-6 cells.
STRAUCH, WANG, AND BASS: TDCA AND EPITHELIAL RESTITUTION 7.
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Martinez, J. D., Stratagoules, E. D., LaRue, J. M., Powell, A. A., Gause, P. R., Craven, M. T., Payne, C. M., Powell, M. B., Gerner, E. W., and Earnest, D. L. Different bile acids exhibit distinct biological effects: The tumor promoter deoxycholic acid induces apoptosis and he chemopreventive agent ursodeosycholic acid inhibits cell proliferation. Nutr. Cancer 31: 111, 1998.
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by TDCA correlates with a significant increase in expression of the TGF gene. Furthermore, the increase in cell migration seen in the presence of TDCA is TGF dependent as inhibition of the activity of TGF by anti-TGF antibody significantly prevented the stimulatory effect of TDCA on cell migration after wounding. These findings suggest that the bile acid TDCA induces intestinal epithelial migration, at least partially, through the TGF-dependent pathway. Thus, the bile salt TDCA contributes to the maintenance of intestinal mucosal integrity by augmenting intestinal epithelial restitution by activating a signaling pathway, yet to be defined, involved in the restitution process through TGF. In summary, these results indicate that exposure to TDCA, at low concentrations that the intestinal epithelium may encounter during normal function, significantly stimulates small intestinal epithelial cell migration in an in vitro system. TDCA also increases expression of the TGF gene in migrating cells and the increase in epithelial cell migration by TDCA is TGF dependent. The exact mechanism by which TDCA stimulates TGF gene expression has not been elucidated but further studies to determine signaling pathways leading to upregulation of TGF gene expression are warranted.
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