Activation of Epithelial CD98 Glycoprotein Perpetuates Colonic Inflammation

Laboratory Investigation (2005) 85, 932–941 & 2005 USCAP, Inc All rights reserved 0023-6837/05 $30.00 www.laboratoryinvestigation.org Activation of epithelial CD98 glycoprotein perpetuates colonic inflammation Torsten Kucharzik1, Andreas Lugering1, Yutao Yan2, Adel Driss2, Laetitia Charrier2, Shanthi Sitaraman2 and Didier Merlin2 1Department of Medicine B, Mu¨nster University of Mu¨nster, Germany and 2Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GA, USA Anomalies in the regulation and function of integrins have been implicated in the etiology of various pathologic conditions, including inflammatory disorders such as irritable bowel disease. Several classes of cell surface glycoproteins such as CD98 have been shown to play roles in integrins-mediated events. Here, we investigated the role of CD98 in intestinal inflammation using both in vivo and in vitro approaches. We found that in Caco2- BBE monolayers and colonic tissues, expression of CD98 was upregulated by the proinflammatory cytokine, interferon gamma (INF c). Furthermore, CD98 was highly upregulated in colonic tissues from mice with active colitis induced by dextran sodium sulfate (DSS), but not in DSS-treated INF c À/À mice. Administration of an anti-CD98 antibody worsened DSS-induced colitis in mice but had no effect on untreated control mice. Finally, we used Caco2-BBE cell monolayers to model intestinal epithelial wound healing, and found that activation of epithelial CD98 in DSS-treated monolayers inhibited monolayer reconstitution, but had no affect on untreated control monolayers. Our data collectively indicate that (i) CD98 upregulation is mediated by INF c during intestinal inflammation and (ii) activation of epithelial CD98 protein aggravates intestinal inflammation by reducing intestinal epithelial reconstitution. Overall, our data suggest that epithelial CD98 plays an important role in the perpetuation of intestinal inflammation. Laboratory Investigation (2005) 85, 932–941. doi:10.1038/labinvest.3700289; Published online 9 May 2005 Keywords: CD98; DSS model; Caco2-BBE monolayers The regulation and establishment of the major barriers.8–10 We previously demonstrated that b1 epithelial permeability barrier is the end result of a integrins, which are also basolaterally polarized in cascade of events triggered by cell–cell and cell– intestinal epithelial cells, associate with CD98/LAT-2, extracellular matrix (ECM) interactions. Anomalies and that CD98 influences b1 integrins distribution in the regulation and function of integrins have been as well as cell shape and cytoskeletal organization, implicated in the etiology of various pathologic which depend on the function of b1 integrins.9 conditions, including inflammatory disorders such Interestingly, CD98 interacts specifically with b1 as inflammatory bowel disease (IBD).1–3 integrins, but not with the muscle-specific splice Several classes of cell surface glycoproteins have variant b1D, or the leukocyte-specific b7 integrins.11 been shown to play a role in integrins-mediated The basolateral location of CD98 suggests that this events, including CD98, CD36, CD63, CD9. Of these, protein could be involved in some form(s) of cell CD98 is a cell surface heterodimer formed by signaling where binding of a ligand to the extra- covalent linkage of a B80 kDa heavy chain (hc) cellular loop of CD98 results in cellular changes and B40 kDa light chain (lc).4,5 CD98 was initially through regulation of an amino-acid transporter characterized as a T-cell activation marker, and was (LAT-2 in the intestine) and the function of b1 later shown to function as an amino-acid transpor- integrins.9,10 We previously demonstrated that ter.6,7 Recent work demonstrated that a CD98/LAT-2 ICAM-1 co-precipitates with the CD98/LAT2 hetero- heterodimer is found in tissues containing epithelial dimer in intestinal epithelial cells, suggesting that ICAM-1 functions not only as an individual receptor but also as a component of supramolecular com- Correspondence: Dr D Merlin, PhD, Department of Medicine, plexes at the plasma membrane in epithelial cells.12 Division of Digestive Diseases, Emory University, 615 Michael In the context of intestinal inflammation, CD98 Street, Atlanta, GA 30322, USA. E-mail: [email protected] protein expression in human colonic epithelium Received 1 February 2005; revised 30 March 2005; accepted 31 was shown to be upregulated by proinflammatory March 2005; published online 9 May 2005 cytokines such as Interferon g (INF g),13 and Role of CD98 in intestinal inflamation T Kucharzik et al 933 increased expression of lymphocyte activation anti- inhibitor cocktail (Boehringer Mannheim), EDTA, gens for CD98 was found on the cell surface of SDS, sodium orthovanadate, and sodium fluoride. intestinal B cells, T cells, CD4 þ T cells, and CD8 þ T Proteins were separated by SDS-polyacrylamide cells isolated from patients with Crohn’s disease and gel electrophoresis on 4–20% gradient gels (BioRad, ulcerative colitis.14 These and other studies have Hercules, CA, USA), and then transferred to nitro- revealed that markedly increased intestinal lympho- cellulose membranes. The blots were blocked for 1 h cyte activation is a major immunological alteration with 5% nonfat milk in blocking buffer, and then in IBD.15 In addition, 5-aminosalicylic acid (which incubated for 1 h with 1/100 dilutions of goat is used for the treatment of intestinal inflammation polyclonal anti-human CD98 (Santa Cruz Biotech- in IBD) dose-dependently inhibited the expression nology, Santa Cruz, CA, USA), goat polyclonal anti- of the CD98 cell-surface activation antigen on mouse CD98 (Santa Cruz) or goat polyclonal mitogen-activated peripheral blood lymphocytes, anti-ERK (Santa Cruz). After the blots were washed further suggesting that CD98 plays an important three times for 15 min in blocking buffer, they were inflammatory role.14 However, although these further incubated for 1 h with the corresponding previous studies have clearly shown that CD98 secondary horseradish peroxidase (HPR) conjugated likely plays an important role in intestinal inflam- anti-goat antibody (diluted 1:2000). The membranes mation, no previous work has studied the role of were then washed three times (20 min each) epithelial CD98 in intestinal inflammation. Here, we in blocking buffer and then probed using a used a combination of in vivo and in vitro chemiluminescence system (ECL; Amersham, approaches to investigate the role of epithelial Piscataway, NJ, USA). CD98 in intestinal inflammation. Membrane Extractions Materials and methods Caco2-BBE pellets were resuspended and homoge- nized in HEPES (5 mM) containing protease inhibi- Cell Culture and Treatments tors. The samples were then incubated for 30 min at 1 1 16–19 4 C and centrifuged at 13 000 g for 30 min at 4 C. Caco2-BBE cells (between passages 30 and 50) The resulting pellet was suspended in PBS by were grown in high glucose Dulbecco’s Vogt Mod- repeated passage through an 18-gauge needle. The ified Eagle’s Media (DMEM; Invitrogen, Carlsbad, protein solution was boiled for 5 min at 1001Cin CA, USA) supplemented with 14 mmol/l NaHCO3, Laemmli buffer supplemented with 0.5% b-mercap- and 10% newborn calf serum. Cells were incubated toethanol and then used for protein separation on 1 at 37 Cin5%CO2 and 90% humidity, and the SDS-polyacrylamide gel electrophoresis. medium was changed daily. Monolayers were sub- cultured every 7 days with 0.1% trypsin and 0.9 mmol/l ethylenediaminetetraacetic acid (EDTA) Confocal Immunofluorescence in Ca2 þ /Mg2 þ -free phosphate-buffered saline (PBS). Experiments were performed with confluent mono- Caco2-BBE cells grown on filters were washed twice layers plated on permeable supports (area 1 cm2; in Hank’s balanced salt solution (HBSS), pH 7.4, and pore size 0.4 mm; Transwell-Clear polyester mem- then fixed with 3.7% paraformaldehyde in HBSS þ branes; Costar (Life Sciences, Acton, MA, USA) and with calcium, pH 7.4 (HBSS ). The cells were then examined 15 days postplating. IFN g (100 U/ml in permeabilized with 0.5% Triton for 30 min at 251C, serum-free medium) was added to the basolateral rinsed and incubated with rhodamine-phalloidin aspect of the monolayers and incubated for 48 h. (Molecular Probes, Eugene, OR, USA) diluted 1:60 Recombinant IFN g (produced in Escherichia coli for 40 min. The samples were then blocked for 1 h in and having a specific activity of 2 Â 10 (REF) U/mg) a blocking solution containing 0.2% gelatin and þ was obtained from Genentech (San Francisco, CA, 0.08% saponin in HBSS and incubated for 1 h with USA). 2 mg/ml goat anti-human CD98 antibody (Santa Cruz, CA, USA). The monolayers were stained with the appropriate fluorescein isothiocyanate antibody diluted 1:1000 (Molecular Probes, OR, USA). Micro- Western Blot Analysis scopy was performed with a Zeiss epifluorescence Colonic mucosal biopsies were collected from microscope equipped with a BioRad MRC600 patients who underwent colonoscopy for polyp or confocal unit, computer and laser scanning micro- cancer surveillance. All subjects were given in- scope (LSM) image analysis software (Zeiss, Jena, formed consent, as approved by Human Investiga- Germany). tions Committee of Emory University Atlanta, GA. Human colonic mucosa biopsies and mouse colonic Animals mucosa (the muscle layer was carefully removed using pointed forceps) were lysed with PBS contain- All studies were carried out in female C57BL/6 mice ing 1% Triton X-100 and 1% NP-40 (v/v), protease (8 weeks, 18–22 g) or female C57BL/6 IFN gÀ/À mice Laboratory Investigation (2005) 85, 932–941 Role of CD98 in intestinal inflamation T Kucharzik et al 934 (8 weeks, 18–22 g) obtained from Jackson Labora- Wound-Healing Assays tories (Bar Harbor, ME, USA). Mice were group- housed under a controlled temperature (251C) and Wound-healing assays were performed with the photoperiod (12:12-h light–dark cycle). The mice Electric Cell-Substrate Impedance Sensing (ECIS were allowed unrestricted access to standard mouse Model 1600R (Applied BioPhysics, Inc., Troy, NY, chow and tap water.

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