fibroblasts expression in human colonic 2 Cytokine ...

Cytokine-mediated PGE2expression in human colonic fibroblasts

Edward C. Kim, Yingting Zhu, Valerie Andersen, Daniela Sciaky, H. James Cao, Heather Meekins, Terry J. Smith and Peter Lance Am J Physiol Cell Physiol 275:C988-C994, 1998. ; You might find this additional info useful... This article cites 33 articles, 17 of which you can access for free at: http://ajpcell.physiology.org/content/275/4/C988.full#ref-list-1 This article has been cited by 6 other HighWire-hosted articles: http://ajpcell.physiology.org/content/275/4/C988#cited-by Updated information and services including high resolution figures, can be found at: http://ajpcell.physiology.org/content/275/4/C988.full

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Cytokine-mediated PGE2 expression in human colonic fibroblasts EDWARD C. KIM,1 YINGTING ZHU,1 VALERIE ANDERSEN,1 DANIELA SCIAKY,2 H. JAMES CAO,2HEATHER MEEKINS,2 TERRY J. SMITH,2 AND PETER LANCE1 1Division of Gastroenterology, Department of Medicine, Veterans Affairs Medical Center and Buffalo General Hospital, State University of New York, Buffalo 14215; and 2Division of Molecular and Cellular Medicine, Department of Medicine, Albany Medical College and Samuel S. Stratton Veterans Affairs Medical Center, Albany, New York 12208

colorectal adenocarcinoma; primary fibroblast cultures; colon cancer cell lines; prostaglandin H synthases

ASPIRIN AND OTHER nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the cyclooxygenase enzymes, PGH synthase-1 and -2 (PGHS-1 and -2; EC 1.14.99) (33). PGE2 and other prostanoids are generated through activity of these bifunctional enzymes, which are usually rate limiting (14). Clinical (9) and experimental (27) evidence of inhibition of colonic neoplastic progression by NSAIDs is consistent with the epidemiological evidence that aspirin can reduce colorectal cancer mortality (10). Although PGHS-1 is constitutively expressed in a wide range of tissues and there is little modulation in the levels of expression, PGHS-2 is highly inducible by cytokines and other participants in inflammatory and neoplastic processes (16). PGHS-2 mRNA and protein expression are increased in colorectal adenocarcinomas compared with levels in adjacent normal tissue (17). A causal role for PGHS-2 in murine colorectal carcinogenesis was demonstrated experimentally in vivo, and the biological effects of PGHS-2 upregulation were mediated predominantly through increased PGE2 production (24). However, the cells from which prostanoids

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emanate in normal or neoplastic colorectal tissue have not been defined. DuBois et al. (7) predicted that transformed epithelial cells will prove to be a major site of colorectal PGHS-2 expression and PG synthesis. In support of this hypothesis, immunohistological localization of PGHS-2 in colorectal adenocarcinoma tissue sections was most intense in the carcinoma cells themselves (28), and PGE2 synthesis is detectable in some, but not all, colon cancer cell lines (15). Suggestive of an alternative source, PGHS-2 was expressed essentially in interstitial cells rather than in the intestinal epithelium of mice from a knockout strain that develops numerous intestinal adenomas due to a truncation mutation of the Apc gene (25). Uncertainty about the source of prostanoids in benign or malignant colorectal tissue prompted us to consider the synthetic capacity of resident cell types other than epithelial cells. New concepts concerning the functions of fibroblasts have been defined recently (30). Fibroblasts express many receptors for cytokines, growth factors, and hormones (8). Those from a single tissue do not constitute a homogeneous phenotypic or functional population, and fibroblasts from different tissues have characteristic phenotypes. For example, cultured orbital fibroblasts were shown to differ metabolically from dermal fibroblasts with regard to extracellular matrix production and morphological changes elicited by PGE2 (31). Fibroblasts, regardless of their tissue of origin, express PGHS-1 at relatively high levels, accounting for basal PGE2 in these cells (30, 34). In contrast, the tissue of origin is a crucial determinant of fibroblast PGHS-2 expression induced by cytokines and other agents. Pulmonary, synovial, and orbital fibroblasts express highly inducible PGHS-2, but induction in dermal fibroblasts is much less robust. Colonic fibroblasts have not previously been examined for this property. Intestinal fibroblasts were shown to synthesize and release somatomedin (insulin-like growth factor) that functioned by paracrine pathways to influence the growth and differentiation of a number of adjacent cell types (12). In a further example of the importance of mesenchymal-epithelial interactions, fibroblasts induced organization and differentiation of colonic epithelial (T84) cells in vitro (11). However, little is known of the colonic fibroblast and its potential contribution to diseases such as colorectal cancer and inflammatory bowel disease (IBD).

0363-6143/98 $5.00 Copyright r 1998 the American Physiological Society


Kim, Edward C., Yingting Zhu, Valerie Andersen, Daniela Sciaky, H. James Cao, Heather Meekins, Terry J. Smith, and Peter Lance. Cytokine-mediated PGE2 expression in human colonic fibroblasts. Am. J. Physiol. 275 (Cell Physiol. 44): C988–C994, 1998.—We investigated prostanoid biogenesis in human colonic fibroblasts (CCD-18Co and 5 primary fibroblast cultures) and epithelial cell lines (NCM460, T84, HT-29, and LS 174T) and the effect of PGE2 on fibroblast morphology. Cytokine-stimulated PGE2 production was measured. PGH synthase-1 and -2 (PGHS-1 and -2) protein and mRNA expression were evaluated. Basal PGE2 levels were low in all cell types (0.15–6.47 ng/mg protein). Treatment for 24 h with interleukin-1b (IL-1b; 10 ng/ml) or tumor necrosis factor-a (50 ng/ml), respectively, elicited maximal 25- and 6-fold inductions of PGE2 synthesis in CCD-18Co cultures and similar results in primary fibroblast cultures; maximal inductions with IL-1b in colonic epithelial cell lines were from zero to fivefold. Treatment of CCD-18Co fibroblasts with IL-1b caused maximal 21- and 53-fold increases, respectively, in PGHS-2 protein and mRNA levels without altering PGHS-1 expression. PGE2 (0.1 µmol/l) elicited a dramatic shape change in selected fibroblasts. Colonic fibroblasts are potentially important as cytokine targets and a source of and target for colonic prostanoids in vivo.

PG EXPRESSION IN COLONIC FIBROBLASTS

In the present study, we investigated PGHS-2 mRNA and protein induction by cytokines in cultured human colonic fibroblasts. We report that interleukin-1b (IL1b) in particular can induce dramatic PGHS-2 expression, with a consequent 25-fold increase in PGE2 production. The response of PGHS-2 and PGE2 synthesis to IL-1b in colonic fibroblasts was considerably more robust than that observed in neoplastic and nonneoplastic colonic epithelial cells. These findings indicate the potentially prominent role of fibroblasts in prostanoid synthesis in the colon and define another cellular target for the beneficial actions of NSAIDs in colorectal neoplasia. MATERIALS AND METHODS

were performed on fibroblast strains from the third to the sixth passage in culture. Immunohistochemical stains performed on representative cells from each strain supported the fibroblast cell type. A vimentin stain was positive, whereas pancytokeratin, factor VIII, antichymotrypsin, a-antitrypsin, and S-100 stains were negative for each strain. PGE2 assay. Fibroblasts were seeded at a density of 5 3 105 cells/cm2 and grown to confluence in 24-well plastic culture plates in medium supplemented with 10% FBS. It is well recognized that serum is a potent inducer of PGHS-2 and thus PGE2 synthesis (18). Therefore, confluent cultured fibroblasts were shifted from medium supplemented with 10% FBS to medium containing 1% FBS for the 48 h before harvesting that included the period of treatment with test compounds. To determine the effects of cytokines, cultures were incubated without addition of a cytokine or in medium supplemented with IL-1b (10 ng/ml) or TNF-a (50 ng/ml). The following compounds, added at the same time as IL-1b, were used in some experiments at the final concentrations indicated: dexamethasone (10 nmol/l), indomethacin (10 µmol/l) and SC58125 (5 µmol/l). Before harvesting of material for measurement of PGE2 levels, medium was replaced with PBS supplemented with 1% gelatin for the final 30 min of incubation; test compounds were present during this final incubation period. PGE2 levels in harvested PBS-gelatin were determined using RIA kits according to the manufacturer’s instructions. Protein content in the harvest was determined by the method of Bradford (3). Western analysis of PGHS-2 protein expression. Relative levels of the cyclooxygenase proteins were determined as described (34). Briefly, confluent cultured fibroblasts were shifted from medium supplemented with 10% FBS to medium containing 1% FBS for a total of 48 h during treatment with test compounds and then harvested. Lysates were subjected to gel electrophoresis, and the separated proteins were transferred to a membrane. Membranes were incubated with primary monoclonal antibodies specific for PGHS-2, generously provided by Dr. J. Maclouf (IFR CirculationLariboisiere, INSERM 348, Paris, France) or purchased from Cayman (Ann Arbor, MI). Bound antibodies were detected by chemiluminescence using ECL reagents purchased from Amersham. The resulting bands were analyzed densitometrically. Isolation of fibroblast RNA and Northern analysis. Isolation of total cellular RNA, gel electrophoresis, Northern blotting, hybridization with cDNA probes, and quantitation of mRNA levels were performed as described (29). Fibroblasts were cultivated to confluence in 100-mm-diameter plastic culture plates, treated with test compounds, and harvested for isolation of total RNA. The human PGHS-1 and -2 cDNAs utilized for hybridizations were generously supplied by Drs. D. Young (University of Rochester, Rochester, NY) and T. Hla (American Red Cross, Washington, DC). The densities of the DNA-RNA hybrids were determined by scanning the resulting radiographs, and results were normalized for intensity of staining with ethidium bromide. Effects of PGE2 on fibroblast morphology. Fibroblast cultures were grown to confluence in 24-well plates in medium supplemented with 10% FBS. Confluent cultures were shifted to medium containing 1% FBS 24 h before addition of PGE2 at a final concentration of 0.1 µmol/l. Phase-contrast analysis of cellular morphology was performed using a Zeiss Axiovert microscope (Carl Zeiss, Thornwood, NY) equipped with a 35-mm camera. Photographs were taken on Kodak Technical Pan film (Eastman Kodak, Rochester, NY) at a final magnification of 3200.


Reagents. IL-1b, Klenow enzyme, random primer, and oligonucleotides were purchased from Boehringer Mannheim (Indianapolis, IN). Tumor necrosis factor-a (TNF-a), dexamethasone, and indomethacin were from Sigma (St. Louis, MO). SC-58125 was kindly provided by Dr. P. Isakson of Searle (Skokie, IL). Cesium chloride and guanidinium isothiocyanate were purchased from Bethesda Research Laboratories (Bethesda, MD). DMEM, fetal bovine serum (FBS), L-glutamine, sodium pyruvate, penicillin, D-glucose, HEPES, streptomycin, amphotericin, and nonessential amino acids were purchased from GIBCO Laboratories (Grand Island, NY). Kits for measurement of PGE2 levels by RIA were purchased from Amersham (Arlington Heights, IL). Cell culture. CCD-18Co, a human colonic fibroblast strain derived from normal tissue, and HT-29, LS 174T, and T84 colon cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). NCM460, an immortalized line established from normal human colonic epithelium, was a generous gift of Dr. M. P. Moyer (University of Texas, San Antonio, TX) (23). Other fibroblast cultures were initiated from colonoscopic biopsy or orbital tissue explants, obtained with approval of the Institutional Review Boards of the Buffalo Veterans Administration Medical Center and the Albany Medical College. Colonic fibroblast cultures were established from five patients. Biopsies in four patients were taken from endoscopically normal mucosa and in one (patient 2) from a tubular adenoma. Endoscopic features were patient 1, normal colonoscopy; patient 2, tubular adenoma (from which fibroblasts were cultured); patient 3, segmental focal colitis (fibroblasts cultured from normal mucosa at a distance from colitis); patient 4, tubular adenoma (fibroblasts cultured from normal mucosa remote from the adenoma); and patient 5, hyperplastic polyp (fibroblasts cultured from normal mucosa remote from the polyp). Two biopsies taken at each colonic site were immediately placed in DMEM fortified with 5% FBS, L-glutamine (2 mmol/l), sodium pyruvate (1 mmol/l), penicillin (200 units), and streptomycin (200 µg/ml). Biopsy tissue was dispersed by mechanical teasing with a glass pipette, centrifuged at 2,000 rpm for 2 min, and washed four times in medium. The tissue explant was then blotted on a plastic culture dish and covered with medium. All cell cultures were maintained at 37°C in a humidified 5% CO2 incubator. The medium, supplemented now with 100 µg/ml streptomycin, 100 units penicillin, and 20% FBS, was replaced the next day and subsequently changed twice weekly. Once cells that were morphologically characteristic of fibroblasts appeared and began to propagate, the remainder of the tissue sample was discarded. Studies

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RESULTS

Fig. 1. Effects of interleukin-1b (IL-1b) on PGE2 synthesis in CCD18Co fibroblasts. Cells were incubated for times indicated in presence of IL-1b (10 ng/ml) alone or IL-1b and one other compound. Other compounds were dexamethasone (DEX; 100 nmol/l), indomethacin (INDO; 10 µmol/l), and SC-58125 (5 µmol/l). PGE2 levels in harvested medium were determined by RIA. Levels plotted for IL-1b and IL-1b with each additional compound are means 6 SE of triplicate cultures for each point from a representative time course experiment.

Fig. 2. Effect of IL-1b on PGE2 synthesis in human primary fibroblast cultures from colonoscopic biopsies obtained from 5 patients (1–5). Biopsies of 4 of 5 patients were from endoscopically normal mucosa. Biopsies in patient 2 were from a colonic adenoma. Clinical and endoscopic details of patients 1–5 are given in MATERIALS AND METHODS. Fibroblasts were cultured in presence of IL-1b (10 ng/ml) for times indicated. PGE2 levels plotted at each time point are means 6 SE of triplicate cultures from a representative experiment.

creases in fibroblast PGE2 synthesis in response to IL-1b were discernible at 4 h and were maximal from 8 to 24 h after its addition to cultures. Levels of induction with IL-1b at 24 h ranged from 4- to 10-fold over basal. Characteristics of IL-1b-induced PGE2 synthesis in the fibroblast strain propagated from a colonic adenoma did not appear to be different from the four strains established from normal colorectal epithelium. Average maximal PGE2 induction at 24 h with TNF-a (threefold) was less than with IL-1b (data not shown). The maximal level and time course of IL-1b-induced PGE2 synthesis in the CCD-18Co strain were within the ranges delineated for the five primary fibroblast cultures. Comparison of effects of IL-1b on PGE2 synthesis in epithelial cell lines and fibroblasts. PGE2 synthesis in colonic fibroblasts (CCD-18Co) in response to IL-1b was compared with the levels in colonic nonneoplastic (NCM460) and malignant (HT-29, LS 174T, and T84) epithelial cell lines and an orbital fibroblast strain (Fig. 3). Basal levels of PGE2 in the epithelial cell lines were 0.15–1.3 ng/mg protein. The basal level in the orbital fibroblast strain was 0.64 6 0.05 ng/mg protein. Maximal induction of PGE2 synthesis by IL-1b in orbital fibroblasts was 12-fold, consistent with our earlier report on orbital fibroblasts (34). In contrast, IL-1b increased PGE2 synthesis only twofold over a 24-h period in the nonneoplastic (NCM460) cell line. In one of the colon cancer cell lines, T84, there was no significant induction of PGE2 synthesis by IL-1b. In HT-29 and LS 174T, respectively, IL-1b caused five- and threefold increases in PGE2 production at 8 h, returning to basal levels by 24 h. To compare PGE2 synthesis in colonic fibroblasts and epithelial cell lines, maximal levels of ,20–40 ng/ml among 5 fibroblast strains (Fig. 2) were 7- to 13-fold higher than the maximal level of ,3 ng/ml in HT-29 cells after IL-1b stimulation (Fig. 3).


Treatment of human colonic fibroblast strains with IL-1b or TNF-a results in substantial increases in PGE2 synthesis. IL-1 plays a widespread and potent role in mediating immune and inflammatory responses (21) and is produced by a wide variety of cells, including fibroblasts (5). The stimulatory effects of proinflammatory cytokines, such as IL-1b and TNF-a, on PGE2 synthesis in various cell types, including fibroblasts, are well documented (4, 13, 34). We examined PGE2 synthesis in the CCD-18Co human colonic fibroblast strain (Fig. 1). The mean basal PGE2 level from seven separate experiments was 1.40 6 0.27 ng/mg protein. In an additional experiment to characterize PGE2 synthesis in unstimulated cells, levels at 8 and 24 h, respectively, were 2.4 and 2.3 ng/mg protein. The PGE2 level increased 25-fold in response to IL-1b at the physiologically relevant concentration of 10 ng/ml (5). Increased PGE2 synthesis was evident at 2 h and peaked at 24 h. TNF-a (50 ng/ml) elicited an almost sixfold induction in PGE2 levels (data not shown). NSAIDs, such as indomethacin, inhibit PGE2 synthesis by inhibition of PGHS-1 and -2 activity. SC-58125 is a highly selective PGHS-2 inhibitor. Glucocorticoids, such as dexamethasone, inhibit PGE2 synthesis by multiple mechanisms; prominent among these is an attenuation of PGHS-2 gene expression (1). Effects of indomethacin, SC-58125, and dexamethasone on IL-1b-induced PGE2 synthesis in colonic fibroblasts are shown in Fig. 1.All the compounds tested caused nearly complete attenuation of the PGE2 response to IL-1b when added simultaneously with the cytokine. This blockade persisted for the duration of the experimental period (24 h). Five primary fibroblast cultures were established from human colonoscopic biopsies. Mean basal PGE2 production in these five strains was 5.4 ng/mg protein (Fig. 2) with a range of 2.6–9.2 ng/mg protein. In-


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IL-1b increases PGHS-2 protein levels in normal colonic fibroblasts. Western analysis was performed to determine whether exposure of colonic fibroblasts to IL-1b led to increased expression of PGHS-2 protein (Fig. 4). PGHS-2 protein was almost undetectable in untreated cells. A maximal 21-fold increase in PGHS-2 enzyme level was seen after exposure to IL-1b (10 ng/ml) for 16 h. PGHS-1 protein levels, in contrast, were unaffected by cytokine treatment (data not shown). Treatment with IL-1b or TNF-a increases PGHS-2 mRNA levels in normal colonic fibroblasts. To characterize further the cyclooxygenase pathway and its regulation in colonic fibroblasts, Northern blots were performed to analyze basal expression and upregulation of PGHS-1 and PGHS-2 mRNA in response to IL-1b (Fig. 5) in CCD-18Co cells. With IL-1b (10 ng/ml), PGHS-2 mRNA expression, barely detectable in cellular mRNA from untreated CCD-18Co fibroblasts, increased 10fold at 2 h (data not shown) and reached a level at least 50-fold greater than baseline after exposure to IL-1b

Fig. 4. Effect of IL-1b on PGH synthase (PGHS)-2 protein levels in CCD-18Co colonic fibroblasts. Lysates from cells incubated for times indicated with IL-1b (10 ng/ml) were electrophoresed and transferred to membranes, which were then incubated with monoclonal antibodies for PGHS-2. Bound antibodies were detected by chemiluminescence.

Fig. 5. Effects of IL-1b on PGHS-2 (A) and PGHS-1 (B) mRNA levels in CCD-18Co colonic fibroblasts. Cells were incubated for 24 h without or with IL-1b (10 ng/ml), alone or in combination with dexamethasone (100 nmol/l) or indomethacin (10 µmol/l). Total RNA was isolated, electrophoresed, transferred, and hybridized with PGHS-1 or -2 probes. Densitometric intensities of PGHS-1 or -2 mRNA bands were normalized for staining with ethidium bromide (Et Br in A; data not shown in B).

for 8 h. The level of induction remained 48-fold at 24 h. In contrast, PGHS-1 mRNA levels were unaffected by IL-1b treatment. TNF-a (50 ng/ml) produced a 15-fold induction of the PGHS-2 mRNA level at 18 h (data not shown). Glucocorticoids, such as dexamethasone, exhibit transcriptional and posttranscriptional downregulation of PGHS-2 and attenuate phospholipase A2 expression (1, 26), whereas NSAIDs inhibit cyclooxygenase enzyme activity without affecting transcriptional activity (22). As expected, dexamethasone (10 nmol/l) caused neartotal blockade of the upregulatory effect of IL-1b on PGHS-2 mRNA expression in CCD-18Co fibroblasts, whereas indomethacin (10 µmol/l) had no effect (Fig. 5). As in control cultures, cellular mRNA from CCD-18Co fibroblasts treated with dexamethasone or indomethacin alone remained barely detectable (data not shown). PGE2 elicits a shape change in colonic fibroblasts. We reported previously that human orbital fibroblasts undergo a morphological change when incubated with PGE2 (32). Figure 6 shows typical microscopic fields of untreated CCD-18Co colonic fibroblasts and fibroblasts that were incubated with PGE2 (0.1 µmol/l) for 4 h. As with orbital fibroblasts, when the culture medium of colonic fibroblasts was supplemented with PGE2, some cells underwent a profound change in appearance; the central nucleus-containing region was condensed, and prominent spidery cytoplasmic processes developed. Other cells were unaffected. Increasing the concentration to 10 µmol/l did not appear to further increase the shape-changing properties of PGE2.


Fig. 3. Comparative effects of IL-1b (10 ng/ml) on PGE2 synthesis by orbital fibroblasts (Orbital) and colonic epithelial cell lines (NCM460, HT-29, LS 174T, and T84). Results plotted are means 6 SE of duplicate or triplicate cultures for each point from a representative experiment. Note change in scale for PGE2 level compared with Figs. 1 and 2.

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DISCUSSION

In conjunction with experimental evidence of a causal relationship between increased PGHS-2 expression and colorectal carcinogenesis, immunohistological localization of the enzyme was reported in the interstitial but not the epithelial cells of murine colonic adenomas (24). We have demonstrated that, fully consistent with the murine immunohistological data, human colonic fibroblasts not only are highly sensitive and more responsive to proinflammatory cytokine-mediated induction of PGHS-2 expression than are epithelial cell lines under identical experimental conditions in vitro but are capable of much higher levels of PGE2 production. Our findings indicate that colonic fibroblasts share with pulmonary, synovial, and orbital fibroblasts the property of expressing highly inducible PGHS-2. Further, we report that colonic fibroblasts, like orbital fibroblasts, are able to undergo dramatic morphological changes in vitro in response to concentrations of PGE2 that have physiological relevance. Whether these responses to PGE2 are relevant to the normal function of colonic tissue or to any disease state remains to be determined. Clearly, establishment of cultures in vitro could have selected and accentuated cellular behavior patterns that may not accurately reflect the patterns of fibroblast gene expression in vivo. Nonetheless, five of six fibroblast strains utilized in this study (the American


Fig. 6. Phase-contrast microscopic examination of colonic (CCD18Co) fibroblasts untreated (A) or treated (B) with PGE2 (0.1 µmol/l) for 4 h. Confluent cultures in 24-well plates were shifted to medium containing 1% fetal bovine serum 24 h before addition of PGE2 and examination of untreated and treated preparations. Arrows denote cells undergoing shape change. Final magnification, 3200.

Type Culture Collection strain and four of five primary fibroblast cultures established in our laboratory) were initiated from histologically normal epithelium. Colorectal adenocarcinomas are clonal in origin and develop through accumulated malfunction of multiple genes. The capacity for PGHS-2 expression exhibited by the normal colonic fibroblast strains on which we report suggests that clones of neoplastic epithelial cells in the earliest stages of histological development (aberrant crypt foci and early adenomas) could be exposed to prostanoid products from adjacent cells, especially those found in the interstitium, with consequent potential for enhancement of neoplastic progression. Consistent with the potentially important influence of fibroblasts and other interstitial components in colorectal carcinogenesis, intestinal microadenomas in mice carrying a truncated Apc gene originated from the antiluminal aspect of crypt epithelium as outpockets growing into the intravillous space that is populated by fibroblasts and other interstitial cells (25). PGHS-2 expression, as reflected by PGE2 synthesis, was responsive to cytokines in all the colonic fibroblast strains examined, but the degree of inducibility varied considerably. Although increased PG levels have been widely reported in colon cancer specimens, the stage of neoplastic transformation at which increased PGE2 is first apparent is debated. Oshima et al. (24) did not detect PGHS-2 protein in polyps ,2 mm, but Boolbol et al. (2) reported increased levels of the enzyme in histologically normal colonic epithelium from Min mice compared with normal littermates lacking the Apc mutation. To reiterate, five of the six fibroblast strains that were examined in this study were from normal mucosa. It will be necessary to study fibroblasts from multiple additional subjects without colonic disease and from patients with adenomas, adenocarcinomas, and other colonic diseases to define the range of colonic fibroblast responsiveness to cytokine stimulation and the temporal relationship of increased PG synthesis to the stage of colon carcinogenesis. It may be that the distinct phenotypic attributes of an individual’s colonic fibroblasts, such as relative sensitivity to cytokine stimulation, can have a protective role or contribute to the pathogenesis of large bowel disease. Knowledge of the behavior of colonic fibroblasts in patients with IBD is limited. Although intestinal mucosal PG levels are elevated in IBD (20), indomethacin and other cyclooxygenase inhibitors are clinically ineffective in these diseases. In the absence of evidence that prostaglandins are important mediators of inflammation in IBD, interest has focused on arachidonic acid metabolites of the 5-lipoxygenase pathway. Leukotriene B4, in particular, has been incriminated as a potential inflammatory mediator in the intestine, but elucidation of the pathophysiological mechanisms of IBD is far from complete. In light of their profound responsiveness to proinflammatory cytokines, further investigations of colonic fibroblasts from patients with IBD may provide useful new insights.


We thank H. Hameer for immunohistochemical characterization of primary fibroblast cultures. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43649, Veterans Affairs Central Office Merit Review, and a grant from the Margaret Duffy and Robert Cameron Troup Memorial Fund for Cancer Research of the Buffalo General Hospital (to P. Lance) and by National Eye Institute Grants RO1-EY-08976 and RO1-EY-11708 and Veterans Affairs Central Office Merit Review (to T. J. Smith). A portion of this work was presented at the annual meeting of the American Gastroenterological Association in May 1997 and published in abstract form (17a). Address for reprint requests: P. Lance, Division of Gastroenterology, Buffalo General Hospital, 100 High St., Buffalo, NY 14203. Received 5 December 1997; accepted in final form 10 June 1998. REFERENCES 1. Angel, J., F. Berenbaum, C. Le Denmat, T. Nevalainen, J. Masliah, and C. Fournier. Interleukin-1-induced prostaglandin E2 biosynthesis in human synovial cells involves the activation of cytosolic phospholipase A2 and cyclooxygenase-2. Eur. J. Biochem. 222: 125–131, 1994. 2. Boolbol, S. K., A. J. Dannenberg, A. Chadburn, C. Martucci, X. Guo, J. T. Ramonetti, M. Abreu-Goris, H. L. Newmark, M. L. Lipkin, J. J. DeCosse, and M. M. Bertagnolli. Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis. Cancer Res. 56: 2556–2560, 1996. 3. Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254, 1976. 4. Casey, M. L., K. Korte, and P. C. MacDonald. Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase. J. Biol. Chem. 263: 7846–7854, 1988. 5. Crofford, L. J., R. L. Wilder, A. P. Ristima¨ki, H. Sano, E. F. Remmers, H. R. Epps, and T. Hla. Cyclooxygenase-1 and -2 expression in rheumatoid synovial tissues. J. Clin. Invest. 93: 1095–1101, 1994. 6. Diaz, A., A. M. Reginato, and S. A. Jimenez. Alternative splicing of human prostaglandin G/H synthase mRNA and evidence of differential regulation of the resulting transcripts by transforming growth factor b1, interleukin 1b, and tumor necrosis factor a. J. Biol. Chem. 267: 10816–10822, 1993.

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The finding that fibroblast PGHS-1 mRNA and protein levels were unaffected by any of the cytokine treatments is consistent with the view that this cyclooxygenase is constitutively expressed in a wide variety of tissues and not necessarily involved in the inflammatory response (14). However, evidence from experiments utilizing knockout mice with disruptions of PGHS-1 or -2 gene expression raises the possibility that PGHS-2 expression and inducibility alone may not entirely account for prostanoid-dependent inflammatory responses (19); PGHS-1 expression may also be involved. This possibility is supported by evidence for differential regulation of PGHS-1 expression by cytokines, including IL-1b and TNF-a, through alternative mRNA splicing (6). It is increasingly evident that fibroblasts and their products are crucial determinants of epithelial cell behavior. We suggest that fibroblasts could be important cytokine targets in the colon. Furthermore, they may both represent a potentially important site of colonic prostanoid biogenesis in vivo and be targets for PGE2.

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