Mesalamine blocks tumor necrosis factor growth inhibition and nuclear factor κB activation in mouse colonocytes

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Mesalamine Blocks Tumor Necrosis Factor Growth Inhibition and Nuclear Factor ␬B Activation in Mouse Colonocytes GREG C. KAISER, FANG YAN, and D. BRENT POLK Division of Gastroenterology and Nutrition, Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee

Background & Aims: Derivatives of 5-aminosalicylic acid (mesalamine) represent a mainstay in inflammatory bowel disease therapy, yet the precise mechanism of their therapeutic action is unknown. Because tumor necrosis factor (TNF)-␣ is important in the pathogenesis of inflammatory bowel disease, we investigated the effect of mesalamine on TNF-␣–regulated signal transduction and proliferation in intestinal epithelial cells. Methods: Young adult mouse colon cells were studied with TNF-␣, epidermal growth factor, or ceramide in the presence or absence of mesalamine. Proliferation was studied by hemocytometry. Mitogenactivated protein (MAP) kinase activation and I␬B␣ expression were determined by Western blot analysis. Nuclear transcription factor ␬B (NF-␬B) nuclear translocation was determined by confocal laser immunofluorescent microscopy. Results: The antiproliferative effects of TNF-␣ were blocked by mesalamine. TNF-␣ and ceramide activation of MAP kinase were inhibited by mesalamine, whereas epidermal growth factor activation of MAP kinase was unaffected. TNF-␣–stimulated NF-␬B activation and nuclear translocation and the degradation of I␬-B␣ were blocked by mesalamine. Conclusions: Mesalamine inhibits TNF-␣–mediated effects on intestinal epithelial cell proliferation and activation of MAP kinase and NF-␬B. Therefore, it may function as a therapeutic agent based on its ability to disrupt critical signal transduction events in the intestinal cell necessary for perpetuation of the chronic inflammatory state.

umor necrosis factor (TNF)-␣, a 17-kilodalton polypeptide hormone, is a regulatory cytokine in the pathogenesis of inflammatory bowel disease (IBD).1 TNF-␣ exerts its effects through two glycoprotein receptors on the cell membrane that lack any known catalytic domain: a 55-kilodalton receptor (TNF-␣R1) and a 75-kilodalton receptor (TNF-␣R2).2,3 To date, TNF-␣ is the only cytokine that, when specifically inhibited, leads to remission in Crohn’s disease.4 Investigations have primarily focused on the role of TNF-␣ in orchestrating an exaggerated inflammatory response in individuals predisposed to the development

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of IBD.5–8 Our laboratory has recently shown that TNF-␣ directly regulates intestinal epithelial cell proliferation in a concentration-dependent manner, with low-dose TNF-␣ (0.006–0.06 nmol/L) having a proliferative effect through TNF-␣R2 activation and high-dose TNF-␣ (6–60 nmol/L) exerting an antiproliferative effect through TNF-␣R1 activation. In addition, high-dose TNF-␣ inhibits growth factor–stimulated mitogenesis through TNF-␣R1.9 Although the intracellular signaling of TNF-␣ is not completely understood, evidence shows TNF-␣ activates at least two signaling pathways, including several members of the evolutionarily conserved mitogen-activated protein (MAP) kinase family10 and the nuclear transcription factor ␬B (NF-␬B). These pathways may be critical in regulation of cellular proliferation and inflammation, respectively.11–13 TNF-␣ has also been shown to stimulate translocation of NF-␬B from the cytosol into the nucleus to enhance expression of proinflammatory cytokines and adhesion molecules.13,14 NF-␬B is held in an inactive state in the cytosol bound to its inhibitory protein (I␬B) until activation of signaling pathways that result in I␬B phosphorylation. Once phosphorylated, I␬B is ubiquitinated and targeted for degradation via the proteosome, releasing NF-␬B for nuclear translocation.15–17 Derivatives of mesalamine represent a mainstay of IBD therapy, although the precise mechanism of action of therapeutic action remains unknown. TNF-␣ levels decreased in patients treated with mesalamine compounds,18 and intraepithelial colonic lymphocytes seemed to decrease cytokine transcription of TNF-␣ and interferon gamma (IFN-␥) in the presence of mesalamine.19 In addition, sulfasalazine, but not mesalamine, inhibited the binding of TNF-␣ to its receptor.20 Recently, sodium salicylate was shown to inhibit the activation of ERK1/ Abbreviations used in this paper: BSA, bovine serum albumin; EGF, epidermal growth factor; FBS, fetal bovine serum; HRP, horseradish peroxidase; IFN, interferon; MAP, mitogen-activated protein; NF-␬B, nuclear transcription factor ␬B; SDS, sodium dodecyl sulfate; TNF, tumor necrosis factor; YAMC, young adult mouse colon. r 1999 by the American Gastroenterological Association 0016-5085/99/$10.00

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ERK2 by TNF-␣ in fibroblasts21 and to inhibit the nuclear translocation of NF-␬B in murine T and B cells.22 We studied the effect of mesalamine on TNF-␣– initiated signaling events in young adult mouse colon (YAMC) cells. The findings indicate mesalamine inhibits TNF-␣ regulation of intestinal epithelial cell proliferation, MAP kinase activation, and NF-␬B nuclear activation and translocation, effects that may be important in the intestinal epithelial cell role in IBD.

Materials and Methods Materials Recombinant murine TNF-␣ was purchased from Pepro Tech, Inc. (Rocky Hill, NJ). Murine epidermal growth factor (EGF) was the generous gift of Stanley Cohen (Vanderbilt University). C8-Ceramide was supplied by Biomol Research Laboratories, Inc. (Plymouth Meeting, PA); rabbit antiactive MAP kinase (ERK1/ERK2) polyclonal antibody from Promega (Madison, WI); and murine anti–pan ERK1/ ERK2 monoclonal antibody from Transduction Laboratories (Lexington, KY). Recombinant protein A–horseradish peroxidase (HRP)-conjugated antibody was supplied by Zymed Laboratories, Inc. (San Francisco, CA). Rabbit phosphospecific JNK/SAPK polyclonal antibody, rabbit pan-JNK/SAPK polyclonal antibody, and anti-rabbit immunoglobulin (Ig) G–HRP conjugated polyclonal antibody were purchased from New England Biolaboratories (Beverly, MA). Rabbit anti-human NF-␬B p65 polyclonal antibody and fetal bovine serum (FBS) were obtained from Upstate Biotechnology (Lake Placid, NY); rabbit anti-human I␬B␣ polyclonal antibody from Santa Cruz Biotechnology (Santa Cruz, CA); and Cy3-conjugated donkey anti-rabbit polyclonal antibody and normal donkey serum from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Cell culture dishes, rat tail collagen type I, and ITS culture media supplement were obtained from Collaborative Biomedical Products (Bedford, MA). Thermanox tissue culture slides were supplied by Nalge Nunc International (Rochester, NY). Recombinant murine IFN-␥ was obtained from Life Technologies, Inc. (Gaithersburg, MD). Other media additives were purchased from Mediatech, Inc. (Herndon, VA). RPMI 1640 media, mesalamine, and all other materials were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell Culture All reported experiments were performed using YAMC cells (passage 12–35), a conditionally immortalized murine colon cell line isolated from the H-2kb-tsA58 mouse expressing a heat-labile simian virus 40 large T antigen with an IFN-␥–inducible promoter.9,23,24 Cells were grown on either culture dishes or Thermanox culture chamber slides coated with rat tail collagen type I (5 µg/cm2) using RPMI 1640 media (pH 7.4) supplemented with 5% FBS, 3 g/L NaHCO3, 6.25 mg/L insulin, 6.25 mg/L transferrin, 6.25 µg/L selenous acid, 1.25 g/L bovine serum albumin (BSA), 5.35 mg/L linoleic acid, 100,000 IU/L penicillin, 100 mg/L streptomycin, and 5

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U/mL of murine IFN-␥. The cells were cultured under permissive (transforming) conditions at 33°C in a humidified atmosphere with 5% CO2 as described previously.9 Before use in all experiments, cell monolayers were cultured in serum-free (0.5% FBS) and IFN-␥–free media under nonpermissive (nontransforming) conditions of 37°C for 24 hours. Confirmatory studies in IEC-6 cells were also performed using cell culture methods previously described by this laboratory.25

Experimental Cell Culture Protocols YAMC cells were cultured with murine TNF-␣, EGF, or additives for various periods of time at 37°C. Except where indicated, TNF-␣ was added to the media 30 minutes after addition of mesalamine and EGF was added 30 minutes after TNF-␣. Media containing mesalamine were prepared by adding mesalamine to a final concentration of 200 mmol/L, then heated to 37°C for 30 minutes on a stirring apparatus with pH adjusted to 6.65 with NaOH. Subsequent dilutions were prepared using RPMI 1640. Medium containing mesalamine was protected from light using aluminum foil, and cell culture was carried out in a dark culture chamber. For 4-aminosalicylic acid, solubility in media was at 100 mmol/L at 37°C, and this was used as the initial concentration for preparation of subsequent dilutions.

Cell Counting and Proliferation Analysis The number of cells was determined by counting trypsinized cell suspensions on a hemocytometer as reported previously.9 Briefly, cell monolayers were washed twice with phosphate-buffered saline (PBS) and exposed to trypsin 1⫻ in EDTA at 37°C for 2 minutes. The cells were then suspended in a known volume of media and counted in quadruplicate. The change in cell number between untreated samples from the start to the end of an experiment was used as a control to define baseline proliferation (standardized as 100% proliferation). The change in cell number in treated cell samples over the course of an experiment was then reported as a percentage relative to the control.

Preparation of Cellular Lysates Cell monolayers were washed twice with ice-cold PBS and then lysed in cell lysis buffer containing 1% Triton X-100, 20 mmol/L HEPES (pH 7.4), 0.5 mmol/L Na3VO4, 50 mmol/L ␤-glycerolphosphate, 10 mmol/L Na4P2O7, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 17.4 µg/mL phenylmethylsulfonyl fluoride. The Triton-soluble fraction was collected after a 20-minute incubation on ice with occasional gentle agitation. Protein content was determined by Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA) and subsequent spectrophotometry using a Spectronic 601 by Milton Roy (Rochester, NY). An equal volume of 2⫻ Laemmli buffer26 was added to each sample, and equivalent concentrations of protein were loaded into each well of a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins were subsequently separated by electrophoresis. Cytosol fractions for I␬B Western blot analysis were prepared using a 20-minute incubation on ice with hyposmotic

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buffer containing 10 mmol/L HEPES, 10 mmol/L KCl, 0.1 mmol/L EDTA, and 0.1 mmol/L ethylene bis(oxyethylenenitrilo)-tetraacetic acid followed subsequently by differential centrifugation as reported previously.27

Western Blot Analysis After SDS-PAGE gel electrophoresis, protein transfer to an enhanced chemiluminescence nitrocellulose membrane from Amersham Life Science (Arlington Heights, IL) was accomplished by semidry technique. The membrane was blocked overnight in a solution of 2% nonfat, dried milk in PBS with 0.05% Tween. Before immunoblotting, the membrane was washed twice in PBS. Western blotting was conducted using either an antiactive MAP kinase (ERK1/ ERK2) antibody (1:10,000), anti–pan ERK1/ERK2 antibody (1:5000), antiphosphospecific JNK/SAPK antibody (1:1000), anti–pan JNK/SAPK antibody (1:1000), or anti-I␬B␣ antibody (1:1000) for 2 hours at room temperature in PBS with 0.05% Tween. The membrane was then washed three times in 1% nonfat, dried milk in PBS with 0.05% Tween for 15, 5, and 5 minutes, respectively. Secondary antibody incubations were then carried out using either an anti-rabbit IgG-HRP– conjugated antibody (1:2000) or a recombinant protein A-HRP– conjugated antibody (1:10,000) for 2 hours at room temperature in PBS with 0.05% Tween. After three washings in 1% nonfat, dried milk in PBS with 0.05% Tween for 15, 5, and 5 minutes, respectively, proteins were visualized by enhanced chemiluminescent detection from Dupont NEN (Wilmington, DE).

Immunofluorescence YAMC cells were cultured on collagen-coated Thermanox plastic tissue culture slides, and experiments were conducted as described above. At the conclusion of all experiments, the cells were washed twice with ice-cold PBS and fixed by immersing the slides in ⫺10°C methanol for 5 minutes. Once the slides had air-dried, the cells were incubated in 5% normal donkey serum in PBS with 0.2% BSA for 20 minutes to block nonspecific antibody binding. The cells were then incubated with rabbit anti-human NF-␬B p65 polyclonal antibody (1:50) in PBS with 0.2% BSA for 60 minutes at room temperature. The slides were then washed three times in PBS with 0.2% BSA for 5 minutes at room temperature. Cells were then incubated with Cy3-conjugated donkey anti-rabbit polyclonal antibody (1:300) in PBS with 0.2% BSA for 45 minutes at room temperature. The slides were then washed three times in PBS with 0.2% BSA for 5 minutes at room temperature, dried, and mounted with glass coverslips using Vectashield mounting medium from Vector Laboratories (Burlingame, CA). Immunofluorescence was observed by using an LSM410 Confocal Laser Scanning Microscope from Carl Zeiss, Inc. (Oberkochen, Germany).

Statistical Analysis The statistical significance of the differences between cell proliferation at various conditions was determined using

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the Student t test analysis from StatWorks 1.2 for Macintosh (Cricket Software, Inc., Philadelphia, PA). The level of statistical significance was set at P ⬍ 0.05. Each individual experiment was repeated on at least three separate occasions with similar results obtained. Proliferation data are presented as the mean and standard deviation of triplicate samples from a representative experiment.

Results Antiproliferative Effect of High-Dose TNF-␣ Is Inhibited by Mesalamine To determine the effect of mesalamine on the antiproliferative response of TNF-␣ in intestinal epithelial cells, YAMC cells were cultured either with or without mesalamine (20 mmol/L) in serum-starved (0.5% FBS) RPMI 1640 media in the presence or absence of EGF and murine TNF-␣ for 24 hours at 37°C. Cells were trypsinized and counted by hemocytometry as described previously.9 As shown in Figure 1, mesalamine blocks both the direct antiproliferative effect of high-dose TNF-␣ (100 ng/mL) and the inhibition of EGF-mediated mitogenesis by TNF-␣. However, the proliferative response to either EGF or low-dose TNF-␣ (1 ng/mL) is unaffected by the presence of mesalamine compared with the respective controls. Activation of ERK1/ERK2 by TNF-␣ Is Blocked by Mesalamine in Intestinal Epithelial Cells To study the effect of mesalamine on EGF and TNF-␣ activation of ERK1/ERK2 in intestinal epithelial cells, YAMC cells were cultured with mesalamine in

Figure 1. The antiproliferative effect of high-dose TNF-␣ is inhibited by mesalamine. YAMC monolayers were serum-starved at nonpermissive conditions for 24 hours before use. Cells were incubated for 24 hours at 37°C with EGF and/or TNF-␣ in the presence or absence of mesalamine (5-ASA) pretreatment. Cell numbers were determined as described in Materials and Methods. *P ⬍ 0.005 compared with control; ⌽P ⬍ 0.001 compared with 100 ng/mL TNF-␣; ␻P ⬍ 0.001 compared with 10 ng/mL EGF; ⌺P ⬍ 0.001 compared with 1 ng/mL TNF-␣ and 10 ng/mL EGF; and ␰P ⬍ 0.001 compared with 100 ng/mL TNF-␣ and 10 ng/mL EGF.

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Figure 2. TNF-␣–stimulated ERK1/ERK2 activation is blocked by mesalamine. YAMC cells were incubated with EGF for 2 minutes or TNF-␣ for 15 minutes in the presence or absence of mesalamine (5-ASA) at 37°C. Triton-soluble lysates were obtained, proteins separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane, and Western blot analysis was conducted with an antiactive ERK1/ERK2 antibody (top panel ) and an anti–pan ERK1/ERK2 antibody (lower panel ) as described in Materials and Methods. Proteins were visualized by enhanced chemiluminescence.

RPMI 1640 media with 0.5% FBS 30 minutes before either the addition of murine TNF-␣ for 15 minutes or murine EGF for 2 minutes at 37°C. Triton-soluble cellular lysates were prepared as described in Materials and Methods for SDS-PAGE and Western blot analysis (shown in Figure 2), with an antibody to the dually phosphorylated ERK1/ERK2 in the upper panel or anti–pan ERK1/ERK2 antibody in the lower panel. As shown in Figure 2, both EGF and TNF-␣ stimulate the activation of ERK1/ERK2. Interestingly, mesalamine completely inhibits TNF-␣ activation of ERK1/ERK2 but has no effect on the stimulation of ERK1/ERK2 by EGF. Inhibition of TNF-␣–Stimulated ERK1/ERK2 and JNK/SAPK by Mesalamine Is Both Concentration and Time Dependent To evaluate the kinetics and dose requirements of mesalamine inhibition of TNF-␣–stimulated ERK1/ ERK2 and JNK/SAPK activation, YAMC cells were cultured with varying concentrations of mesalamine at various time points. As shown in the upper panels of Figure 3A and B, mesalamine concentrations of 20–200 mmol/L inhibit the dual phosphorylation of ERK1/ ERK2 and JNK/SAPK by TNF-␣, respectively. The

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Figure 4. ERK1/ERK2 activation by ceramide is blocked by mesalamine. YAMC cells were treated with TNF-␣ or ceramide in dimethyl sulfoxide for 15 minutes at 37°C in the presence or absence of mesalamine (5-ASA). Antiactive ERK1/ERK2 was detected as described in Figure 2.

lower panels of Figure 3A and B show that preincubation of cells with mesalamine for 15 minutes or greater is required to inhibit TNF-␣ activation of these MAP kinases. The inhibition of TNF-␣–stimulated ERK1/ ERK2 and JNK/SAPK activation by mesalamine is sustained for at least 120 minutes. Activation of ERK1/ERK2 by Ceramide Is Inhibited by Mesalamine Because it has been suggested that the activation of MAP kinase by TNF-␣ is mediated by ceramide production, we determined the effect of mesalamine on ceramide activation of ERK1/ERK2 in intestinal epithelial cells.28,29 YAMC cells were cultured with mesalamine in RPMI 1640 media with 0.5% FBS for 30 minutes before addition of either 0.3 µmol/L C8 ceramide or 100 ng/mL murine TNF-␣ for 15 minutes at 37°C. As shown in Figure 4, ceramide activates ERK1/ERK2 similar to TNF-␣. The solvent for ceramide, dimethyl sulfoxide, has no effect on ERK1/ERK2, but mesalamine blocks the activation of ERK1/ERK2 by both ceramide and TNF-␣. TNF-␣–Stimulated Nuclear Translocation of NF-␬B Is Inhibited by Mesalamine To determine the effect of mesalamine on TNF-␣– stimulated nuclear translocation of NF-␬B in intestinal epithelial cells, YAMC cells were cultured in the presence or absence of 20 mmol/L mesalamine in serum-starved (0.5% FBS) RPMI 1640 media for 30 minutes before the addition of 100 ng/mL murine TNF-␣ for 30 minutes at

Figure 3. Inhibition of TNF-␣–stimulated ERK1/ERK2 and JNK/SAPK activation by mesalamine is both concentration and time dependent. In the top panels, YAMC cells were incubated with various concentrations of mesalamine (5-ASA), then cultured with TNF-␣ for 15 minutes at 37°C. (A ) Antiactive ERK1/ERK2 was detected as described in Figure 2. (B ) Antiactive JNK/SAPK was identified by Western blot analysis of Triton-soluble lysates as described in Materials and Methods.

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37°C. Cells were fixed and incubated with anti–NF-␬B p65 and detected by incubation with an anti-rabbit IgG antibody conjugated to Cy3 for detection by confocal laser immunofluorescent microscopy. As shown in Figure 5A, NF-␬B is predominantly cytoplasmic before the addition of TNF-␣. After treatment of cells with TNF-␣ for 30 minutes, there is almost complete nuclear translocation of NF-␬B (Figure 5B), which is blocked by pretreatment with mesalamine (Figure 5D). I␬B Degradation Is Inhibited by Mesalamine The principal regulatory mechanism for NF-␬B activation seems to be I␬B degradation; therefore, we studied the effects of mesalamine on I␬-B␣ degradation. YAMC cells were cultured in the presence or absence of mesalamine in serum-starved (0.5% FBS) RPMI 1640 media for 30 minutes before addition of 100 ng/mL murine TNF-␣ for 0, 5, or 15 minutes at 37°C. Triton-soluble cellular lysates were prepared for Western blot analysis with an anti–I␬-B␣ antibody. As shown in Figure 6A, cellular lysates from cells treated with TNF-␣ show a progressive loss of I␬-B␣ within 5 and 15 minutes, respectively. In cells pretreated with mesalamine, however, I␬-B␣ was still clearly detectable at both 5 and 15 minutes. The ability of mesalamine to inhibit TNF-␣– stimulated I␬B degradation was detectable at 20 mmol/L and greater concentrations, as shown in Figure 6B.

Figure 6. I␬-B␣ degradation is inhibited by mesalamine. YAMC cells were treated with 100 ng/mL TNF-␣ for various times at 37°C as indicated in the presence or absence of (A ) mesalamine (5-ASA) or (B ) treated with TNF-␣ for 15 minutes in the presence of mesalamine at various concentrations. Cytosolic fractions were prepared; proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane; and Western blot analysis was conducted with an anti–I␬-B␣ antibody as described in Materials and Methods. Proteins were visualized by enhanced chemiluminescence.

TNF-␣–Stimulated I␬B Degradation and MAP Kinase Activation Are not Affected by pH or Osmolarity Because metabolic pathways can be influenced by both pH and osmotic shifts, these effects were studied on TNF-␣–stimulated I␬B degradation and MAP kinase activation. TNF-␣–stimulated I␬B degradation and MAP kinase activation were determined in the presence of 20 mmol/L butyric acid (pH 6.65), 200 mmol/L mannitol, or various concentrations of 4-aminosalicylic acid. The combined effects of pH and osmolarity were studied in the presence of both butyric acid and mannitol containing media corrected to a pH of 6.65 (same pH corrected as 200 mmol/L mesalamine). As shown in Figure 7, although mesalamine blocked the activation of MAP kinase by TNF-␣, neither pH nor osmolarity shifts independently or in combination inhibited either degradation of I␬B or the dual phosphorylation of ERK1/ ERK2 by TNF-␣. Interestingly, 4-aminosalicylic acid showed a concentration-dependent inhibition of both effects, similar to mesalamine.

Discussion Figure 5. TNF-␣–stimulated nuclear translocation of NF-␬B is inhibited by mesalamine. YAMC cells were grown on collagen-coated Thermanox culture slides at 37°C and received (A ) no treatment, (B ) 100 ng/mL TNF-␣ for 30 minutes, (C ) 20 mmol/L mesalamine for 30 minutes, or (D ) the combination of TNF-␣ and mesalamine. Cells were then fixed, probed with an anti–NF-␬B p65 antibody, and incubated with a secondary antibody linked to Cy3 as described in Materials and Methods. Images were obtained using confocal laser immunofluorescent microscopy.

Since their introduction in the 1940s, mesalaminecontaining agents have proven effective in both the maintenance and treatment of IBD.30 Mesalamine inhibits cyclooxygenase and lipoxygenase pathways to reduce the production of prostaglandins and leukotrienes, respectively; however, the exact mechanism of action in IBD has eluded investigators.31 In this study, we show that mesalamine reverses the antiproliferative effects of TNF-␣

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Figure 7. TNF-␣–stimulated degradation of I␬B and activation of MAP kinase are not affected by pH nor osmolarity. YAMC cells were treated with TNF-␣ for 15 minutes alone or in combination with a 30-minute pretreatment with mesalamine (5-ASA), equivalent osmotic concentrations of mannitol, equivalent pH-corrected media with butyric acid, or the combined osmotic and pH effects or various concentrations of 4-aminosalicylic acid at 37°C. Cell lysates were prepared for Western blot analysis with anti-I␬B or antiactive ERK1/ERK2 or anti-ERK1/ ERK2, as indicated.

and profoundly inhibits TNF-␣ signaling events including the activation of MAP kinase and NF-␬B in intestinal cells. Thus, mesalamine may directly disrupt the effect of cytokines by reducing intestinal cell transcription of inflammatory mediators. Intestinal epithelial cells produce a wide array of cytokines, which act to regulate the immunologic response observed in colitis.32 Both MAP kinase and NF-␬B have been implicated as targets of intestinal epithelial cytokines in the inflammatory response to colonic infection.33 Thus, the intestinal epithelium plays an important role in the regulation and activation of the immune system. Additionally, there is evidence for regulation of intestinal epithelial cell growth and development by intraepithelial lymphocytes and cytokines.34–36 The effects of mesalamine on TNF-␣ antiproliferative signaling are concentration dependent with inhibition between 2 and 20 mmol/L (data not shown). Similar concentrations have been reported in the stools of patients receiving therapeutic doses of mesalamine.31,37 Reversal of TNF-␣ inhibition on growth factor–stimulated proliferation may be biologically relevant because EGF and other growth factors seem important for both cytoprotection and repair of intestinal mucosa.38–40 It seems unlikely that the mesalamine effects are mediated by osmolarity or pH changes because, as shown in Figure 7, TNF-␣ was able to stimulate both MAP kinase activity and I␬B degradation in media containing equimolar mannitol and pH-adjusted butyric acid. The parent compound, sulfasalazine, from which me-

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salamine can be derived by bacterial cleavage of the azo bond to produce sulfapyridine and mesalamine, has also been shown to inhibit TNF-␣ signaling.20,41 This may be through sulfasalazine inhibition of TNF-␣ receptor binding, shown at 1 mmol/L sulfasalazine, but not at up to 100 mmol/L mesalamine.20 Our findings suggest that mesalamine does not functionally inhibit TNF-␣ receptor ligand binding. As shown in Figure 3, mesalamine inhibition of MAP kinase is time-dependent with TNF-␣ activation still detected in the presence of 200 mmol/L mesalamine for up to 15 minutes after pretreatment with mesalamine. However, after 30 minutes of exposure to mesalamine, TNF-␣–stimulated activation of MAP kinase is completely inhibited, suggesting a requirement for either cellular uptake or metabolism. It is also worth noting that 4-aminosalicylic acid, which has similar therapeutic efficacy to mesalamine,42 also inhibits TNF␣–stimulated I␬B degradation and MAP kinase activation (Figure 7). Similar results were seen for mesalamine on TNF-␣ antiproliferative and signal transduction pathways in IEC-6 cells (data not shown). Ceramide, generated by sphingomyelinase activity, has been proposed as a second messenger for TNF-␣ receptor signaling pathways.43 In our studies, ceramide activation of MAP kinase was also inhibited by mesalamine. Interestingly, the ability of EGF to activate ERK1/ERK2 was unaffected by mesalamine, clearly indicating the effect of mesalamine to be upstream of MAP kinase in TNF-␣ or ceramide signal transduction, perhaps inhibiting the ceramide-activated protein kinase signaling pathways as well.44 The role of ceramide in TNF-␣ signaling is not clear, but it seems to be necessary for NF-␬B activation in some cell types but not others.45–47 The direct role of ceramide-activated protein kinase in these pathways remains to be determined.44 Nuclear translocation of NF-␬B in response to TNF-␣, or other cytokines, is a principal regulatory step in the transcription of various inflammatory mediators.13,48 A recent report demonstrated increased NF-␬B activation in epithelial cells of inflamed mucosa in patients with Crohn’s disease and ulcerative colitis, but not in adjacent uninvolved mucosa.49 As shown in Figure 5, TNF-␣– stimulated NF-␬B nuclear translocation is inhibited by mesalamine in intestinal cells. Similar inhibition of TNF-␣–stimulated nuclear translocation of NF-␬B was also shown by gel shift assay analysis of nuclear extracts with a 32P-labeled NF-␬B probe (data not shown). The effect of mesalamine in this pathway seems to be inhibition of I␬B degradation. Glucocorticoids, which are potent anti-inflammatory agents in the treatment of IBD, have been shown to both increase the synthesis and stabilize the expression of I␬-B␣.50–52 The mechanism

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whereby mesalamine inhibits degradation of I␬B in our studies is unclear but is the focus of ongoing investigation. Our findings show direct effects of mesalamine on the intestinal epithelial cell including the inhibition of TNF-␣ signal transduction pathways regulating proliferation and transcription. This leads us to propose that mesalamine may be a therapeutic agent based on its ability to disrupt signal transduction events in the intestinal cell necessary for perpetuation of the chronic inflammatory state.

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Received July 8, 1998. Accepted November 24, 1998. Address requests for reprints to: D. Brent Polk, M.D., Division of Gastroenterology and Nutrition, Department of Pediatrics, 21st and Garland Avenue, S-4322 MCN, Nashville, Tennessee 37232-2576. e-mail: [email protected]; fax: (615) 343-8915. Supported by the National Institutes of Health grants T32 DK07673 and DK02212, and a Crohn’s and Colitis Foundation of America research grant. Immunofluorescent images were collected in part through the use of the Vanderbilt University Medical Center Imaging Core Research Laboratory, supported by National Institutes of Health grants CA68485 and DK20593. The authors thank Robert Whitehead and the Ludwig Institute (Melbourne, Australia) for generously providing YAMC cells, Lei Sun for his technical skills in performing gel shift assays, and Peter Dempsey and Jonathan Sheehan for advice in immunofluorescent and confocal laser microscopy.