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Formyl Peptide Receptor-1 Activation Enhances Intestinal Epithelial Cell Restitution through Phosphatidylinositol 3-Kinase-Dependent Activation of Rac1 and This information is current as Cdc42 of September 24, 2021. Brian A. Babbin, Algirdas J. Jesaitis, Andrei I. Ivanov, Daina Kelly, Mike Laukoetter, Porfirio Nava, Charles A. Parkos and Asma Nusrat

J Immunol 2007; 179:8112-8121; ; Downloaded from doi: 10.4049/jimmunol.179.12.8112 http://www.jimmunol.org/content/179/12/8112 http://www.jimmunol.org/ References This article cites 81 articles, 39 of which you can access for free at: http://www.jimmunol.org/content/179/12/8112.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Formyl Peptide Receptor-1 Activation Enhances Intestinal Epithelial Cell Restitution through Phosphatidylinositol 3-Kinase-Dependent Activation of Rac1 and Cdc421

Brian A. Babbin,2* Algirdas J. Jesaitis,† Andrei I. Ivanov,* Daina Kelly,* Mike Laukoetter,* Porﬁrio Nava,* Charles A. Parkos,* and Asma Nusrat2*

Inflammatory disorders of the gastrointestinal tract result in the breakdown of the intestinal epithelial barrier in the form of erosion and ulceration. To reestablish the epithelial barrier, the epithelium must efficiently migrate to reseal wounds. Numerous signaling cascades are involved in the induction and regulation of this complex process. N-formyl peptide receptors comprise a group of Gi-coupled receptors that regulate innate immune responses. Previously, we identified the expression of functional

N-formyl peptide receptors in model SK-CO15 intestinal epithelial cells and observed a role for activation of these receptors in Downloaded from regulating cellular invasive behavior. In these studies, we performed formyl peptide receptor-1 (FPR) localization and evaluated its role in regulating intestinal epithelial cell wound closure. Immunolocalization studies using a recently developed specific monoclonal anti-FPR Ab demonstrated its localization along the lateral membrane of crypt epithelial cells in normal human colonic epithelium. In vitro studies using the classical FPR agonist fMLF showed that FPR activation significantly enhances model intestinal epithelial cell restitution and that FPR localized along actin filaments in lamellipodial and filopodial extrusions. The increase in cell migration was associated with activation of PI3K, Rac1, and Cdc42. Pharmacologic inhibition of PI3K activity http://www.jimmunol.org/ abrogated the fMLF-induced increase in wound closure and activation of both Rac1 and Cdc42. Inhibition of Rac1 and Cdc42 using pharmacologic inhibitors and dominant negative mutants also inhibited the fMLF-induced increase in cell migration. Taken together, theses results support a novel role for FPR stimulation in enhancing intestinal epithelial cell restitution through PI3K- dependent activation of Rac1 and Cdc42. The Journal of Immunology, 2007, 179: 8112–8121.

variety of inflammatory gastrointestinal disorders, in- kinases and G protein-coupled receptors (GPCRs)3 have both been cluding infectious colitis and inflammatory bowel dis- shown to regulate epithelial migratory responses including those of ease, result in breakdown of the intestinal epithelial bar- intestinal epithelial cells (3–6). For example, stimulation of the A by guest on September 24, 2021 rier with resultant erosion and ulceration. To reestablish the tyrosine kinase receptor c-Met with hepatocyte growth factor has epithelial barrier and gastrointestinal function, the epithelium must been shown to stimulate or enhance intestinal epithelial cell resti- efficiently reseal such discontinuities. A critical component by tution through activation of RhoA (6). Lysophosphatidic acid stim- which mucosal wound healing and regeneration occur is through ulation of its GPCR has been also been shown to regulate epithelial intestinal epithelial cell migration referred to as “restitution.” Res- cell migration (7, 8). titution and has been observed both in vitro and in vivo. Restitution PI3K is a critical signaling mediator that is activated by several is a complex process that is regulated by numerous proteins and is extracellular ligands through both receptor tyrosine kinases and dependent on the ability of cells to remodel the actin cytoskeleton GPCRs (9–11). In different cell types, including epithelial cells, and dynamically adhere to the underlying matrix (1, 2). A variety PI3K regulates cell migration through a variety of downstream of physical and chemical signals sensed by cell surface receptors signaling molecules including Rho GTPases (12–14). Rho GTPase can initiate intracellular signaling pathways that coordinate the cel- family members (Rac1, Cdc42, and RhoA) are central regulators lular changes observed during cell migration. Receptor tyrosine of F-actin reorganization and thus play a crucial role in the migration of various cell types (15–18). These monomeric signal transduction proteins have inducible activity that is dependent on reversible interactions with triphosphorylated guanine nucleotides *Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory (GTPs). Guanine nucleotide exchange factors (GEFs) regulate the Medicine, Emory University, Atlanta, GA 30322; and †Department of Microbiology, Montana State University, Bozeman, MT 59715 activity of Rho GTPases by catalyzing the exchange of GDP for Received for publication June 21, 2007. Accepted for publication October 11, 2007. GTP. The lipid product of PI3K, phosphatidylinositol 3,4,5- The costs of publication of this article were defrayed in part by the payment of page trisphosphate (PI(3,4,5)P3 or PIP3), has been shown to regulate charges. This article must therefore be hereby marked advertisement in accordance GEF function and thus provides a mechanistic link between PI3K with 18 U.S.C. Section 1734 solely to indicate this fact. activity and Rho GTPase activation (12, 19). 1 This work was supported by National Institutes of Health Career Development Award K08 DK074706-01 (to B.A.B.), a career development award from the Crohn’s and Colitis Foundation of America (to A.I.I.), Digestive Disease Minicenter Grant DK 064399, Public Health Service RO1 Awards AI22735 and AI26711 (to A.J.J.), 3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; Boc, N- DK61379, HL72124, and DK72564 (to C.A.P.), and DK55679 and DK59888 tert-butoxycarbonyl-Met-Leu-Phe; EGFP, enhanced GFP; FPR, formyl peptide (to A.N.). receptor-1; FPRL1, formyl peptide receptor-like-1; GEF, guanine nucleotide ex- 2 Address correspondence and reprint requests to Dr. Brian A. Babbin or Dr. Asma change factor; IEC-6, intestinal epithelial cell line; MLB, magnesium lysis buffer; Nusrat, Department of Pathology and Laboratory Medicine, Emory University, NFPR2, mouse anti-FPR mAb. Whitehead Biomedical Building, 615 Michael Street, Atlanta, GA 30322. E-mail addresses: [email protected] and [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 8113

N-formyl peptide receptors comprise a group of GPCRs that regulate innate inflammatory responses. Three human N-formyl peptide receptors have been identified and include formyl peptide receptor-1 (FPR), formyl peptide receptor-like-1 (FPRL1), and formyl peptide receptor-like-2 (20). FPR is the prototypical high- affinity formyl peptide receptor that regulates leukocyte activation and chemotactic responses. Upon ligand binding, FPR undergoes a conformation change enabling interactions with Gi family proteins. Downstream of these heterotrimeric G proteins, a complex cascade of signaling is initiated that involves phospholipases, protein kinase C (PKC) isoforms, and MAPKs, as well as PI3K and the Rho GTPases discussed above (21, 22). In leukocytes, PI3K and Rho GTPase activities are critical in the regulation of their activation and emigration into tissue compartments (23–29). Agonist-induced signaling also results in phosphorylation of C-terminal residues via GPCR kinases and other protein kinases (PKA and PKC) leading to receptor desensitization and internalization (30–32). Al- though FPR has been classically thought of as a leukocyte chemotactic receptor, it has been shown to be expressed in a variety Downloaded from of cell types other than leukocytes (33–36). However, the biological significance of FPR expression in such cells is not fully understood. In a previous study, we identified the expression of functional FPR in a model intestinal epithelial cell line, SK-CO15 (37). In this current study, we sought to explore the role of FPR in regulating epithelial wound healing responses following injury. FIGURE 1. FPR localizes to the lateral membrane of human colonic http://www.jimmunol.org/ Our studies demonstrate, for the first time, distribution of an active crypt epithelial cells. Frozen sections of normal human colonic mucosa FPR pool in native human intestinal crypt epithelium. In vitro stud- were labeled for nonphosphorylated FPR using the NFPR2 Ab (green) and ies support an important role of FPR activation in enhancing in- nuclei were highlighted with To-Pro-3 iodide (blue). As shown at lower testinal epithelial cell migration and wound closure that requires magnification (A, ϫ100), NFPR2 labels the epithelial cells along the axis PI3K-dependent activation of both Rac1 and Cdc42. of the crypts (bracket). Surface enterocytes did not show a significant amount of labeling (B, ϫ400). Upon higher magnification (C, ϫ400), FPR is found along the lateral membrane of the crypt epithelial cells (arrows; L, Materials and Methods lumen). Cell culture by guest on September 24, 2021 The human intestinal epithelial cell line SK-CO15 was grown in high glu- cose (4.5 g/L) DMEM supplemented with 10% FBS, 100 U/ml penicillin, ␮ ␮g/ml), pertussis toxin (1 ␮g/ml), wortmannin (100 nM), LY294002 (25 100 g/ml streptomycin, 15 mM HEPES (pH 7.4), 2 mM L-glutamine, and ␮ ␮ ␮ 1% nonessential amino acids as previously described (38, 39). The non- M), NSC23766 (100 M), or secramine A (20 M). For assays using tumorigenic rat intestinal epithelial cell line IEC-6 was grown in the same SK-CO15 and IEC-6 cells transfected with adenoviral constructs, mono- medium as for SK-CO15 cells (40). Cells were passaged and seeded on layers were incubated in low calcium medium (calcium-free Eagle’s MEM Ͻ ␮ 2ϩ - collagen-coated permeable supports or tissue culture-treated plates (Costar; with 10% dialyzed FBS containing 10 mol/L extracellular Ca ) over night. Cells were then placed back in complete medium containing viral Corning). particles diluted to ϳ5 ϫ 105 PFU/ml and incubated for 2 days before use. Abs and other reagents Sites at which wounds were to be measured were marked on the under surface of the wells to ensure that measurements were taken at the same Mouse anti-FPR mAb, NFPR2, was generated as previously described place. Wounds were imaged at 0, 6, and 24 h on a Zeiss Axiovert micro- (41). This Ab recognizes the C-terminal tail sequence 337-NSTLPSAEUA- scope with an attached charge-coupled device camera. Wound widths were 346, which is sensitive to phosphorylation and thus recognizes a nonphos- measured from the images using Scion Image software. Ten measurements phorylated receptor (41). Rabbit polyclonal anti-Rac1 and Cdc42 were ob- along the wound length were averaged to determine wound widths and the tained from Santa Cruz Biotechnology. Rabbit polyclonal anti-hPepT1 Abs distance (␮m) the wound edges migrated into the wound space, which were obtained from Rockland Immunochemicals. Anti-Akt and anti-phospho- controls for any minor variation in the widths of initial wounds. Akt/protein kinase B Abs were obtained from Cell Signaling. Pertussis toxin, wortmannin, and LY294002 were obtained through Calbiochem. Monolayer wounding, immunoblot analysis, and densitometry The FPR antagonist N-tert-butoxycarbonyl-Met-Leu-Phe (Boc) was ob- 2 - tained through MP Biomedicals (Aurora, OH). Adenoviral vectors express- SK-CO15 cell monolayers were grown on collagen-coated, 5-cm perme able supports and wounded using a specialized wounding comb that es- ing dominant negative N-terminal myc-tagged Rac1 and Cdc42 (N17-Rac1 sentially converts the entire monolayer into spreading and migrating cells and N17-Cdc42) plus enhanced GFP (EGFP) as well as control viruses (6). Medium was changed after wounding. Cells were harvested and expressing EGFP only were a generous gift of Dr. J. Bamburg (Colorado dounced in lysis buffer (20 mM Tris HCL, 150 mM NaCl, 2 mM EDTA, State University, Boulder, CO). The Rac1-activation inhibitor, NSC23766, 1% Triton X-100, 0.1% SDS, 0.2 mM EGTA, and 1% sodium deoxy- was a generous gift of Dr. Y. Zheng (Children’s Hospital Research Foun- cholate (pH 7.4)) containing protease and phosphatase inhibitor mixtures dation, Cincinnati, OH) (42), and the Cdc42 inhibitor, secramine A, was a (Sigma-Aldrich). Insoluble material was removed by centrifugation generous gift from Drs. T. Kirchhausen, M. D. Shair, H. E. Pelish, and (16,000 ϫ g for 10 min at 4o G. B. Hammond (43, 44). The specific Rho inhibitor, DC3B, was a gen- C). Lysates were normalized for protein concentration using a bicinchoninic acid assay (Pierce) and subjected to erous gift of Dr. P. Boquet (45). Western Blot analysis. PI3K activity was assessed through analysis of Akt Restitution assay phosphorylation as previously described (46, 47). For these studies, lysates were generated in PhosphoSafe buffer (Chemicon) with an added protease SK-CO15 and IEC-6 cells were grown in collagen-I coated, 24-well culture inhibitor mixture (Sigma-Aldrich) and subjected to Western blot analysis. plates. When cells reached 100% confluency, a single linear wound was Membranes were blocked with 3% BSA in TBS with Tween 20 and in- created through the monolayer with a sterile pipette tip. Monolayers were cubated with Akt and phospho-Akt Abs at 4°C overnight. Densitometric washed to remove cellular debris and then treated with vehicle only (0.1% analysis was performed using the UN-SCAN-IT automated digitizing sys- DMSO) or fMLF (500 nM) with or without the FPR antagonist Boc (100 tem (Silk Scientific). 8114 FPR REGULATES INTESTINAL EPITHELIAL CELL RESTITUTION

FIGURE 2. FPR activation enhances SK-CO15 cell wound closure. A, Confluent SK-CO15 cell monolayers were wounded and incubated with fMLF (500 nM) or vehicle only (0.1% DMSO). Wound widths were determined at 6 and 24 h. As shown in the differential interference contrast microscopy images, the presence of fMLF significantly enhanced wound closure at 6 and 24 h. B, fMLF induced a 1.8- and a 1.3-fold increase in Ͻ ء wound closure at 6 and 24 h, respectively ( , p 0.05; Downloaded from n ϭ 9). C and D, The increase in SK-CO15 wound closure was inhibited in the presence of 100 ␮g/ml FPR antagonist Boc (C) as well as 1 ␮g/ml pertussis toxin (D) (time, 6 h; n ϭ 6). Boc or pertussis toxin alone did not significantly affect SK-CO15 cell restitution. E, Similar results were obtained using IEC-6 cells; however, pertussis toxin alone inhibited IEC-6 wound clo- http://www.jimmunol.org/ .(p Ͻ 0.05; n ϭ 9 ,ء) sure by guest on September 24, 2021

Rho GTPase activation assays paired Student t tests were used to compare compiled results from different trials. To determine Rac1/Cdc42 or RhoA, RhoB, or RhoC activity, commercially available activation assays were used (Upstate). Briefly, monolayers were washed in cooled TBS before lysis in supplied magnesium lysis buffer Results (MLB) containing protease inhibitors. Lysates were incubated at 4°C with FPR localization in intestinal epithelial cells and normal human gentle agitation before centrifugation (14,000 ϫ g for 5 min). Supernatants colonic mucosa were normalized for protein concentrations using a bicinchoninic acid assay (Pierce) and incubated with either recombinant PAK1-GST (for Rac1/ One limitation in studying FPR has been the lack of specific mAbs. Cdc42) or Rhotekin-GST (for RhoA, RhoB, or RhoC) coupled to agarose Recently, a monoclonal Ab (NFPR2) has been generated against beads (45 min, at 4°C with rotation). Beads were washed with MLB and purified hexahistidine-tagged recombinant FPR (41). NFPR2 binds resuspended in SDS sample buffer for Western blot analysis using anti- Rac1, anti-Cdc42, or anti-RhoA, -RhoB, or -RhoC Abs. the C-terminal domain of FPR and does not cross-react with FPRL1. Binding of NFPR2 to its epitope is phosphorylation sen- Immunofluoresence and image analysis sitive and thus it recognizes a nonphosphorylated receptor. Be- Immunofluorescence studies were performed on cells grown on 0.33-cm2 cause agonist-induced phosphorylation of the cytoplasmic C-ter- polycarbonate collagen-coated permeable supports and frozen sections of minal tail results in desensitization and inhibition of FPR normal human colonic mucosa. Cells and tissues were fixed/permeabilized signaling, NFPR2 thus recognizes an active receptor pool. Using in 100% ethanol or methanol, respectively, for 20 min at Ϫ20°C. All subse- this Ab, we sought to characterize active FPR localization in nor- quent steps were performed at room temperature. Cells/tissues were washed with HBSSϩ and blocked in HBSSϩ with 3% BSA for 1 h. Primary Ab mal human colonic mucosa. In frozen sections of normal human reactions were performed in HBSSϩ with 3% BSA for1h(4␮g/ml NFPR2). colonic mucosa fixed with 100% methanol, NFPR2 labeling was Secondary Abs and Alexa 488-conjugated phalloidin were diluted 1/1000 in identified in the crypt epithelium only where it was distributed in 3% BSA and incubated with monolayers for 45 min. To-Pro-3 iodide was the lateral membrane (Fig. 1, A–C, arrows). No staining was ob- ϩ diluted 1/1000 in HBSS and added to the tissue sections for 5 min after served in tissues incubated with secondary Ab alone as expected secondary reactions. Monolayers/tissues were then washed and mounted. Con- focal microscopy was performed using the Zeiss LSM 510 microscope. (Fig. 1D). The lack of NFPR2 labeling in surface enterocytes could be attributed to loss of FPR expression or that FPR is phosphor- Statistics ylated and thus inactive in these cells. Thus, differential labeling of Experiments were performed independently at least three times. Results are crypt vs surface epithelial cells with NFPR2 might be attributed to expressed as the mean Ϯ SEM. One-way ANOVA with post hoc testing or differences in exposure to luminal formyl peptides. The Journal of Immunology 8115

FIGURE 3. FPR colocalizes with F-actin in lamellipodial and ﬁlopodial extrusions of migrating SK-CO15 cells. Wounded SK-CO15 cell monolayers were ﬁxed with 100% ethanol and labeled with NFPR2 (red) and

FITC-phalloidin (green). In migrating cells FPR localized to lamellipodial Downloaded from extrusions at the leading wound edge, some of which localized along actin filaments (a–c, arrows; bar ϭ 20 ␮m). FPR also localized along actin filaments in filopodial extrusions (d–f; bar ϭ 20 ␮m).

FPR activation enhances intestinal epithelial cell wound closure http://www.jimmunol.org/ Because N-formyl peptide receptors are known to regulate innate immune responses, we sought to define the role of N-formyl pep- FIGURE 4. FPR stimulation increases PI3K activation in migrating SK- tide receptor family members in regulating intestinal epithelial cell CO15 cells. Wounded SK-CO15 cell monolayers were incubated with responses to injury. In this study we focused on formyl peptide fMLF (500 nM), fMLF (500 nM) plus Boc (100 ␮g/ml), or vehicle only receptor 1, FPR. FPR was specifically activated using fMLF at (0.1% DMSO) for 6 h. PI3K activity was assessed as a function of Akt nanomolar concentration (500 nM) and thus does not activate the phosphorylation A, Confluent or nonwounded monolayers were designated as baseline. Western blot analysis revealed a significant increase in Akt low-affinity fMLF receptor, FPRL1 (48, 49). To examine the role phosphorylation in wounded control monolayers compared with baseline. of FPR in regulating intestinal epithelial cell restitution, a single Phospho-Akt levels were further increased in wounded SK-CO15 cells in by guest on September 24, 2021 linear wound was created through the confluent SK-CO15 cell the presence of fMLF, the effect of which was inhibited in the presence of monolayer using a sterile pipette tip. Monolayers were treated with Boc. No significant changes in total Akt were observed. Immunoblotting for either fMLF (500 nM) or vehicle only (DMSO, 0.1% final). actin was used as a loading control. B, Based on densitometric analysis of Wounds were imaged at 0, 6, and 24 h and the distance the wound Western blots from these experiments (n ϭ 3), wounded control monolayers edges migrated into the wound space was determined. As shown in exhibited, on average, a 2.3-fold increase in Akt phosphorylation compared Fig. 2, A and B, fMLF significantly increased the rate of wound with confluent monolayers. However, wounded monolayers treated with fMLF closure. At 6 h postwounding, the fMLF-treated group exhibited a exhibited an average 3.7-fold increase in phospho-Akt compared with base- 1.9-fold increase in wound closure compared with control (350 vs line. The fMLF plus Boc-treated group exhibited an increase in phospho-Akt .(p Ͻ 0.05 ,ء) similar to that of control wounded monolayers 185 ␮m). At 24 h, wounds in the fMLF-treated group were gen- erally closed and, on average, migrated a distance of 216 ␮m greater (1.4 times) than the wounded control monolayers (Fig. 2, A Given that FPR activation enhanced epithelial cell migration and B). We did observe that SK-CO15 cells express the peptide and wound closure, we examined the distribution of this receptor transporter, hPepT1, based on Western blot analysis using rabbit in migrating cells. Interestingly, NFPR2 labeling was identified in polyclonal Abs (data not shown). This transporter is known to lamellipodial extrusions, some of which appeared to be distributed mediate the uptake of fMLF (50, 51) which could play a role in the along actin filaments in these cellular protrusions. (Fig. 3, a–c, response of SK-CO15 cells to fMLF. However, the fMLF-induced arrows). FPR also localized along actin filaments in filopodial ex- increase in wound closure was abrogated in the presence of the trusions (Fig. 3, d–f, arrows). We did not observe any morphologic FPR receptor antagonist Boc (100 ␮g/ml) as well as pertussis toxin changes in migrating cells treated with fMLF compared with con- (1 ␮g/ml), demonstrating that this effect is indeed mediated trols (data not shown). through FPR activation (Fig. 2, C and D). Boc or pertussis toxin alone did not significantly affect SK-CO15 cell wound closure. FPR stimulation increases PI3K activation in migrating Thus, FPR activation significantly enhances intestinal epithelial SK-CO15 cells cell restitution. The lack of inhibition of SK-CO15 cell restitution Because FPR stimulation increased SK-CO15 cell wound closure, by Boc or pertussis toxin indicates that endogenous FPR ligands do we analyzed the signaling mechanisms that mediate this effect. It not contribute to the induction/stimulation of wound closure. Sim- is well known that activation of FPR induces PI3K activity im- ilar results were obtained using IEC-6 cells, which were also found portant for downstream signaling events during neutrophil chemo- to express hPepT1 (Fig. 2E and data not shown). Interestingly taxis and activation (26, 27, 29). Furthermore, PI3K activity has however, pertussis toxin alone, but not Boc, inhibited IEC-6 cell been shown to regulate epithelial cell migration, including the mi- restitution by 54%, suggesting that autocrine/paracrine stimulation gration of intestinal epithelial cells (5, 9, 52). We therefore exam- of other Gi-coupled receptors plays a role in the wound healing ined whether FPR activation stimulates PI3K activity in our sys- response of these cells. tem and determined whether PI3K is required for the increase in 8116 FPR REGULATES INTESTINAL EPITHELIAL CELL RESTITUTION

FIGURE 5. PI3K activity is required for the enhancement of intestinal epithelial cell restitution induced by FPR activation. A, SK-CO15 cell monolayers were wounded and incubated with fMLF (500 nM), fMLF (500 nM) plus wortmannin (100 nM), wortmannin (100 nM) alone, or vehicle only (0.1% DMSO). Wound widths were measured at 0 and 6 h. As shown in the representative differential interference contrast images, the presence of fMLF signiﬁcantly enhanced SK- CO15 cell wound closure, which was inhibited by wortmannin. Wortmannin alone did not signiﬁcantly affect wound closure. B, On average, fMLF induced a 1.75- fold increase in SK-CO15 cell wound closure (382 vs ␮ ϭ

235 m), which was blocked by wortmannin (n 6). C, Downloaded from The fMLF-induced increase in wound closure was also inhibited by LY294002, and the LY294002-treated group averaged 30% less wound closure compared with -p Ͻ 0.05). D, The fMLF ,ء) the vehicle only treated group induced increase in IEC-6 cell restitution was also inhibited by wortmannin (n ϭ 6). Wortmannin alone inhibited

/p Ͻ 0.05). http://www.jimmunol.org ,ء) IEC-6 wound closure by 45%

restitution following FPR stimulation. SK-CO15 cell monolayers (100 nM) (53, 54). A single linear wound was created through by guest on September 24, 2021 were wounded and incubated with vehicle only (0.1% DMSO), confluent SK-CO15 cell monolayers that were incubated with ve- fMLF (500 nM), or fMLF (500 nM) plus Boc (100 ␮g/ml) for 6 h. hicle only (0.1% DMSO) or fMLF (500 nM) with or without wort- Lysates were harvested in PhosphoSafe buffer (Chemicon), nor- mannin (100 nM) for 6 h. As shown in Fig. 5, A and B, fMLF malized for protein concentration, and subject to Western blot increased epithelial would closure by 1.75-fold compared with analysis for phospho-Akt and total Akt. As shown in Fig. 4A, controls (migration of 382 vs 235 ␮m). The presence of wortman- increased phosphorylated Akt was observed in wounded control nin abrogated the fMLF-induced increase in wound closure, and monolayers compared with baseline (nonwounded monolayers). wortmannin alone did not have a significant effect on wound clo- However, significantly increased levels of phospho-Akt were ob- sure over the observed time of 6 h. These findings support our served in epithelial cells migrating in the presence of fMLF when conclusion that activation of PI3K is required for the enhanced compared with control migrating cells. This result was inhibited in wound closure induced by FPR activation. These studies were also the presence of Boc, supporting the role of FPR activation in in- performed using another PI3K inhibitor, LY294002 (20 ␮M) (55, ducing this effect. Based on densitometric analysis, wounded con- 56), which showed similar results (Fig. 5, B and C). LY294002 trol monolayers showed a 2.3-fold increase in the phosphorylation of alone did induce ϳ30% reduction in average wound closure Akt compared with baseline, whereas the fMLF-treated group showed (Fig. 5C). In IEC-6 cells, wortmannin also abrogated the fMLF- a 3.7-fold increase (Fig. 4B). The fMLF plus Boc-treated group ex- induced increase in wound closure and inhibited restitution by hibited a 2.4-fold increase in phospho-Akt similar to that of the 45% (Fig. 5D). wounded control group. There was a 1.6-fold increase in phosphorylation of Akt in wounded fMLF-treated cells compared with control The induction of SK-CO15 cell migration is associated with the migrating cells. No significant changes in total Akt were observed in activation of Rho GTPases that is enhanced by FPR stimulation any of the above groups. Thus, the induction of SK-CO15 cell mi- Rho GTPases are known to play a central role in regulating the gration is associated with increased PI3K activity that is further in- cellular functions underlying cell migration (15, 16, 57). FPR stim- creased by FPR stimulation. We did not detect a significant increase ulation has been shown to involve PI3K-dependent activation of in phospho-Akt levels in confluent monolayers incubated with fMLF Rho GTPase family members during neutrophil chemotaxis (24, (data not shown). 29). Similarly, PI3K-dependent activation of Rac1 has been shown to play an important role in regulating the migration of epithelial PI3K activity is required for the enhancement of restitution cells (5, 12). We therefore sought to determine whether such because of FPR stimulation downstream PI3K signaling is occurring in fMLF-treated SK- To determine whether PI3K activity is required for the FPR-in- CO15 cells and whether this is required for the increase in resti- duced increase in wound closure, restitution assays were per- tution induced by FPR stimulation. Wounded monolayers were formed in the presence of the selective PI3K inhibitor wortmannin incubated with vehicle only (0.1% DMSO) or fMLF (500 nM) for The Journal of Immunology 8117

FIGURE 6. The induction of SK-CO15 cell migration is associated with the activation of Rho GTPases that is enhanced by FPR stimulation. SK-CO15 cell monolayers were wounded and incubated for 6 h before analysis of Rho GTPase activation. A, Western blot analysis demonstrated increased levels of active Rac1 and Cdc42 compared with nonwounded monolayers. fMLF stimulation (500 nM) further increased the levels of active Rac1 and Cdc42. B, Densitometric analysis of Western blots from these experiments (n ϭ 3) demonstrated that control migrating SK-CO15 cells exhibited

a 1.8 and 1.3 increase in the levels of active Rac1 and Downloaded from Cdc42, respectively, compared with nonwounded monolayers. fMLF-treated wounded monolayers exhibited a 4.4 and 3.4-fold increase in the respective levels of Rac1 and Cdc42 compared with nonwounded monolayers. C and D, The fMLF-induced enhancement of Rac1 and Cdc42 activity was inhibited by Boc (100 ␮g/ ml) (n ϭ 3). http://www.jimmunol.org/ by guest on September 24, 2021

6 h. Lysates were then generated in MLB buffer, normalized for mannin (100 nM) for 6 h before analysis of Rac1/Cdc42 activity. protein concentration, and incubated with PAK-agarose or Rho- As shown in Fig. 7, A and B, fMLF induced significantly higher tekin-agarose beads to determine activity of Rac1/Cdc42 and Rac1 and Cdc42 activity compared with control migrating cells RhoA, RhoB, and RhoC, respectively. As shown in Fig. 6, A and (ϳ3-fold increase). The presence of wortmannin in the fMLF- B, the induction of cell migration in control monolayers induced a treated group abrogated the increase in Rac1 and Cdc42 activation, 1.8- and 1.5-fold increase in the amount of active Rac1 and Cdc42, and wortmannin alone did not significantly effect their activation respectively, based on densitometric analysis. However, wounded compared with control. Similar results were obtained using monolayers incubated with fMLF demonstrated a 4.4- and 3.4-fold LY294002 (20 ␮M) (Fig. 7, C and D). Interestingly however, in increase in the respective activities of Rac1 and Cdc42 compared these experiments treatment with both fMLF and LY294002 lead with nonwounded monolayers, significantly higher activities than to diminished activity of Rac1 and Cdc42. This could be due to an that of the control wounded group (Fig. 6, A and B). The ability of imbalance between activating and counter-regulatory signaling be- fMLF to increase the activity of Rac1 and Cdc42 was inhibited by cause of simultaneous stimulation of FPR and blockade of PI3K. Boc, again supporting the hypothesis that this effect is mediated Also, this could be related to the inhibition of other kinases by through FPR activation (Fig. 6, C and D). Boc alone did not have LY294002, as this compound is not entirely specific to PI3K (58). a significant effect on Rho GTPase activation. We did not observe Given that FPR stimulation resulted in the increased cell migra- a statistically significant difference in RhoA, RhoB, and RhoC ac- tion associated with activation of Rac1 and Cdc42, we next deter- tivation under the above conditions (data not shown). Thus, the mined whether these GTPases are required for the increase in SK- induction of SK-CO15 cell migration is associated with increased CO15 cell wound closure induced by fMLF. For these studies, we Rac1 and Cdc42 activity that is further enhanced by FPR stimu- performed restitution assays using the selective Rho GTPase acti- lation. Enhanced Rac1 and Cdc42 activity due to FPR stimulation vator inhibitors NSC23766 (100 ␮M) and secramine A (20 ␮M), was also identified in migrating IEC-6 cells (data not shown). which respectively inhibit the activation of Rac1 and Cdc42 (42– 44). A single linear wound was created in confluent SK-CO15 cell FPR stimulation induces Rac1 and Cdc42 GTPase activity in a monolayers that were incubated with fMLF with and without PI3K-dependent manner that is required for the fMLF-induced NSC23766 or secramine A. Wound widths were measured at 0- increase in SK-CO15 cell restitution and 6-h time points and the degree of wound closure was deter- We next determined whether PI3K activation is required for the mined. As shown in Fig. 8, A and B, inhibition of Rac1 alone increased activation of Rac1 and Cdc42 due to FPR stimulation. decreased wound closure by ϳ30%, consistent with its known role As above, monolayers were wounded and incubated with vehicle in regulating epithelial cell migration (15, 18). Additionally, only (0.1% DMSO) or fMLF (500 nM) with and without wort- NSC23766 inhibited the fMLF-induced increase in wound closure. 8118 FPR REGULATES INTESTINAL EPITHELIAL CELL RESTITUTION

FIGURE 7. The increased Rho GTPase activity induced by FPR stimulation is PI3K-dependent. SK- CO15 cell monolayers were wounded and incubated with vehicle only (0.1% DMSO) or fMLF (500 nM) with or without wortmannin (100 nM) for 6 h before analysis of Rac1 and Cdc42 activity. A, Western blot analysis demonstrated signiﬁcant increases the amount of active Rac1 and Cdc42 compared with control, which was blocked in the presence of wortmannin. B, Densi- tometric analysis of Western blots from these experiments (n ϭ 3), revealed that fMLF-treated wounded Downloaded from monolayers exhibited ϳ3-fold increase in the activity of both Rac1 and Cdc42 compared with control, which was abrogated by wortmannin. Wortmannin alone did signiﬁcantly affect Rac1/Cdc42 activity. C and D, Sim- ilar results were seen using LY294002 (20 ␮M) (n ϭ 3). However, the combined treatment of fMLF plus LY294002 resulted in decreased Rac1 and Cdc42 http://www.jimmunol.org/ activities. by guest on September 24, 2021

Inhibition of Cdc42 using secramine A did not significantly affect though some increase in restitution was observed in cells express- wound closure. However, secramine A abrogated the increase in ing either dominant negative Rac1 or dominant negative Cdc42 wound closure induced by fMLF stimulation (Fig. 8, C and D). treated with fMLF, this could be due to activation of the endoge- These experiments were also performed under serum-free condi- nous pool of Rac1 and Cdc42. tions with similar results (data not shown). Thus, independent inhibition of Rac1 and Cdc42 activities abrogates the enhancement Discussion of SK-CO15 cell wound closure due to FPR activation. Inhibition The intestinal epithelial barrier regulates the uptake of nutrients of RhoA, RhoB, and RhoC activity using DC3B (45) did not sig- and ions while restricting underlying tissues from luminal Ags. nificantly affect the restitution of control monolayers nor did it Inflammatory disorders of the gastrointestinal tract result in dys- abrogate the fMLF-induced increase in wound closure (data not function of the intestinal epithelium and breakdown of its barrier shown). properties. To confirm the requirement of Rac1 and Cdc42 for the FPR- Formylated peptides generated from luminal bacteria and mito- stimulated increase in wound closure, we used adenoviral vectors chondrial proteins released from injured cells are potent agonists encoding N-terminal myc-tagged dominant negative Rac1 (N17- of FPR and thus lead to recruitment and activation of leukocytes at Rac1) and Cdc42 (N17-Cdc42) plus EGFP and EGFP alone. As sites of mucosal injury (59–61). Beyond the regulation of leuko- shown in Fig. 8E, expression of EGFP alone did not affect the cyte function, a more global role for FPR in regulating innate ability of fMLF to increase SK-CO15 cell restitution. Expression inflammatory responses is becoming evident as the expression of of dominant negative Rac1 inhibited wound closure by ϳ30% FPR has been identified in a variety of cell types other than leu- compared with control infected cells, consistent with the pharma- kocytes. RT-PCR based studies, analysis of the Human Expressed cologic studies described above. Cells expressing DN-Rac1 treated Sequence Tags database (National Center for Biotechnology In- with fMLF demonstrated a restitution response similar to that of formation, Bethesda, MD), and immunostaining studies using control infected cells. Expression of DN-Cdc42 alone did not sig- rabbit polyclonal antiserum have identified expression of FPR nificantly impact wound closure but did attenuate the fMLF-in- in fibroblasts, lung alveolar epithelia, smooth muscle cells, duced increase in restitution. Similar results were obtained using hepatocytes, Kupffer cells, vascular endothelia, and glial tissues IEC-6 cells (Fig. 8F). Together, these findings support the hypoth- (33–36). Although the biologic significance of this is not fully esis that both Rac1 and Cdc42 are required for the enhancement of understood, stimulation of FPR with bacterial and host-derived intestinal epithelial cell restitution due to FPR stimulation. Al- ligands in some of these cell types has been shown to induce the The Journal of Immunology 8119

C terminus of FPR, NFPR2, which does not exhibit cross-reactiv- ity with FPRL1 (41). The binding of NFPR2 to its epitope is sensitive to phosphorylation and thus binds a nonphosphorylated receptor. Because agonist-induced phosphorylation of cytoplasmic C-terminal serine and threonine residues results in desensitization and inhibition of FPR signaling, NFPR2 can be considered to react with an active receptor pool (41). Interestingly, we identified NFPR2 labeling in the lateral membrane of crypt epithelial cells of normal human colonic mucosa, whereas surface enterocytes did not bind NFPR2. This finding could be explained by either the loss of FPR expression in surface enterocytes or a difference in the exposure of surface enterocytes to luminal formyl peptides leading to FPR phosphorylation and desensitization. Such differential expression of active FPR could possibly play a role in epithelial migration and/or maturation along the crypt-surface axis under normal conditions. However, FPR knockout mice do not exhibit morphological changes in gastrointestinal tissues or functional impairment of gastrointestinal function under normal conditions (62). This could be reflective of alternative FPR-like proteins that could Downloaded from perform analogous functions. Increased exposure of crypt epithelial cells to luminal contents may be reflective of an early event in mucosal injury/impairment, and the stimulation of FPR in crypt epithelial cells could play an important role in mucosal responses to such injury and the subsequent inflammatory response. We did observe that incubation of http://www.jimmunol.org/ wounded SK-CO15 monolayers with the nonpathogenic bacteria Lactobacillus rhamnosus (ϳ9 ϫ 109 CFU/ml) resulted in enhanced restitution compared with nontreated monolayers (data not shown). Furthermore, we observed that FPR localizes to lamellipodial and filopodial extrusions of migrating SK-CO15 cells and that FPR stimulation significantly enhanced their wound closure rates. This also suggests that this population of receptors is part of

an active pool maintained even during fMLF exposure at normally by guest on September 24, 2021 saturating concentrations. Of note, studies examining restitution of single cell defects introduced into the mouse colonic surface epithelium did not identify enhancement of wound closure following exposure to nonpathogenic Escherichia coli supernatants (63). However, this study examined surface enterocytes that are not na- ive to luminal contents and may therefore lack functional FPR or do not express FPR, similar to what we found in human colonic mucosa. Furthermore, the mechanisms by which single cell defects reseal has been reported to differ from that of larger contiguous multicellular defects (64). Additionally, we observed that FPR localized along actin filaments in lamellipodial/filopodial extrusions FIGURE 8. Rac1 and Cdc42 activities are independently required for of migrating SK-CO15 cells. There is evidence to suggest that FPR the enhancement of SK-CO15 and IEC-6 cell restitution induced by FPR forms molecular complexes with the actin cytoskeleton that regu- activation. A and B, SK-CO15 cell monolayers were wounded and incubated with vehicle only (0.1% DMSO) or fMLF (500 nM) with or without lates its ligand affinity, plasma membrane distribution, and possi- NSC23766 (100 ␮M) or secramine A (20 ␮M). Inhibition of Rac1 with bly actin polymerization (65–70). Thus, this pool of receptor may NSC23766 resulted in a 30% reduction in wound closure and inhibited the be bound to the actin cytoskeleton, and characterization of such fMLF-induced increase in restitution (n ϭ 6). C and D, Although inhibition interaction in this system is an interesting focus of future study. of Cdc42 alone with secramine A did not affect SK-CO15 cell wound Given that FPR significantly enhanced intestinal epithelial cell closure, it did abrogate the fMLF-induced increase in wound closure (n ϭ restitution, we sought to define the key signaling mechanisms by 6). E and F, Similar results were obtained using dominant negative (DN) which this occurs. PI3K-dependent signaling has been shown to ϭ constructs of Rac1 and Cdc42 in both SKCO-15 and IEC6 cells (n 6). play crucial roles in regulating the diverse biologic effects of various receptor-ligand interactions, including cell migration (9–11). A variety of signaling mediators are elaborated in the microenvi- production of acute phase reactants, stimulate motility, and regu- ronment following mucosal injury and inflammation including late actin polymerization (34–36). growth factors (epidermal growth factor, fibroblast growth factor, We previously identified expression of functional FPR in the vascular endothelial growth factor, and hepatocyte growth factor), model intestinal epithelial cell line SK-CO15 via RT-PCR, West- cytokines (IL-1, IL-2, and TGF-␤), and other compounds such as ern blot analysis, and intracellular calcium release studies (37). As lysophosphatidic acid, which has growth factor-like properties (8, part of this study, we analyzed the distribution of FPR in normal 71, 72). EGF and EGF receptor ligands, as well as lysophospha- human colonic mucosa using a recently generated mAb against the tidic acid via its GPCRs, are some examples of autocrine/paracrine 8120 FPR REGULATES INTESTINAL EPITHELIAL CELL RESTITUTION signaling factors known to promote intestinal epithelial cell resti- In summary, our studies have, for the first time, identified an tution via stimulation of PI3K-mediated signaling (73, 74). Simi- active FPR pool in the lateral membrane of crypt epithelial cells larly, it is well established that FPR stimulation leads to activation that is not present in surface enterocytes. Using an in vitro model of PI3K, which plays an important role in regulating the polarized of intestinal epithelial restitution, we demonstrate that FPR stim- F-actin restructuring required for leukocyte transmigration (14, 26, ulation significantly enhanced intestinal epithelial cell migration 27, 29). We therefore examined whether FPR stimulation in mi- and wound closure that required PI3K-dependent activation of grating SK-CO15 cells induces PI3K activity. Although the induc- both Rac1 and Cdc42. tion of SK-CO15 cell migration was associated with PI3K activation, this was significantly enhanced in the presence of fMLF. Disclosures Furthermore, pharmacologic inhibition of PI3K abrogated the The authors have no financial conflict of interest. fMLF-induced enhancement of restitution in both SK-CO15 and IEC-6 cells, demonstrating the critical role of PI3K in mediating References the effect of FPR stimulation on intestinal epithelial cell wound 1. Lauffenburger, D. A., and A. F. Horwitz. 1996. Cell migration: a physically closure. integrated molecular process. Cell 84: 359–369. 2. Fenteany, G., P. A. Janmey, and T. P. Stossel. 2000. Signaling pathways and cell The conversion of phosphatidylinositol 3,4-diphosphate to mechanics involved in wound closure by epithelial cell sheets. Curr. 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