Biochemistry and Molecular Biology CXCR4 and CXCR7 Mediate TFF3-Induced Cell Migration Independently From the ERK1/2 Signaling Pathway

Julia Dieckow,1,2 Wolfgang Brandt,3 Kirsten Hattermann,4 Stefan Schob,2,5 Ute Schulze,6 Rolf Mentlein,4 Philipp Ackermann,7 Saadettin Sel,8 and Friedrich P. Paulsen2

1Department of Ophthalmology, University of Leipzig, Leipzig, Germany 2Department of Anatomy II, Friedrich Alexander University Erlangen-Nurnberg,¨ Erlangen, Germany 3Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle, Germany 4Institute of Anatomy, Christian Albrecht University of Kiel, Kiel, Germany 5Department of Neuroradiology, University of Leipzig, Leipzig, Germany 6Department of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg, Halle, Germany 7Department of Ophthalmology, Heinrich Heine University Dusseldorf,¨ Dusseldorf,¨ Germany 8Department of Ophthalmology, Ruprecht Karl University Heidelberg, Heidelberg, Germany

Correspondence: Friedrich P. Paul- PURPOSE. Trefoil factor family (TFF) peptides, and in particular TFF3, are characteristic sen, Department of Anatomy II, secretory products of mucous epithelia that promote antiapoptosis, epithelial migration, Friedrich Alexander University Er- restitution, and wound healing. For a long time, a receptor for TFF3 had not yet been langen-Nurnberg,¨ Faculty of Medi- identified. However, the chemokine receptor CXCR4 has been described as a low affinity cine, Universit¨atsstraße 19, D-91054 receptor for TFF2. Additionally, CXCR7, which is able to heterodimerize with CXCR4, has also Erlangen, Germany; [email protected]. been discussed as a potential TFF2 receptor. Since there are distinct structural similarities Julia Dieckow, Department of Oph- between the three known TFF peptides, this study evaluated whether CXCR4 and CXCR7 thalmology, University of Leipzig, may also act as putative TFF3 receptors. Liebigstr. 10-14, 04103 Leipzig, Ger- METHODS. We evaluated the expression of both CXCR4 and CXCR7 in samples of human many; ocular surface tissues and cell lines, using RT-PCR, immunohistochemistry, and Western blot [email protected]. de. analysis. Furthermore, we studied possible binding interactions between TFF3 and the receptor in an x-ray structure-based modeling system. Functional studies of TFF3– Submitted: September 6, 2015 CXCR4/CXCR7 interaction were accomplished by cell culture–based migration assays, flow Accepted: November 30, 2015 cytometry, and evaluation of activation of the mitogen-activated (MAP) kinase Citation: Dieckow J, Brandt W, Hat- signaling cascade. termann K, et al. CXCR4 and CXCR7 mediate TFF3-induced cell migration RESULTS. We detected both receptors at mRNA and protein level in all analyzed ocular surface independently from the ERK1/2 sig- tissues, and in lesser amount in ocular surface cell lines. X-ray structure-based modeling naling pathway. Invest Ophthalmol revealed CXCR4 and CXCR7 dimers as possible binding partners to TFF3. Cell culture–based Vis Sci. 2016;57:56–65. DOI:10.1167/ assays revealed enhanced cell migration under TFF3 stimulation in a conjunctival epithelial iovs.15-18129 cell line, which was completely suppressed by blocking CXCR4 and/or CXCR7. Flow cytometry showed increased proliferation rates after TFF3 treatment, while blocking both receptors had no effect on this increase. Trefoil factor family 3 also activated the MAP kinase signaling cascade independently from receptor activity.

CONCLUSIONS. Dimers CXCR4 and CXCR7 are involved in TFF3-dependent activation of cell migration, but not cell proliferation. The ERK1/2 pathway is activated in the process, but not influenced by CXCR4 or CXCR7. These results implicate a dependence of TFF3 activity as to cell migration on the chemokine receptors CXCR4 and CXCR7 at the ocular surface. Keywords: TFF3, trefoil factor, chemokine receptors

refoil factor family peptide 3 (TFF3, intestinal trefoil factor) Trefoil factor family peptides are associated with mucus. T is one of the three known trefoil factor family peptides first Human TFF3 has been found in nearly all mucosae and several detected in the mammalian intestine. At mucosal surfaces, TFF glands (e.g., the gastrointestinal, respiratory, and urogenital peptides have been shown to carry out different functions (for tracts; the mammary and salivary glands), but also in non- review, see Ref. 1) like proliferation and migration enhance- epithelial structures such as articular cartilage.2,4,5 The natural ment, antiapoptosis, and wound healing. However, catabolic concentration of TFF3 in human body fluids varies between 12 functions such as activation of matrix metalloproteinases and nmol/L (~94 ng/mL, gastric juice) and 870 nmol/L (~6.8 lg/mL proapoptotic effects have also been described outside of cervical mucus).6 mucosae.2 The three known TFF peptides (TFF1, -2, and -3) With regard to the eye, which our group uses as a model are very consistent in structure, all of them contain a particular system, TFF3 has been described in goblet cells of the trefoil domain consisting of 40 to 41 amino acids with three conjunctiva, the tear film, and the epithelium of the nasolac- preserved cysteine residues.3 rimal ducts.7,8 It is not detectable in healthy cornea, but is

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induced under pathologic conditions like keratoconus and inhibitory effect of CXCR4 blockade via the competitive herpetic keratitis,9 as well as after experimental epithelial antagonist AMD3100 on gastric wound healing induced by damage in the remaining epithelial cells at the defect rim.10 In TFF3 in TFF2 null mice. human epithelial cell lines, TFF3 is additionally induced under The present study uses ocular surface tissues as a model exposure to UV-B light, after stimulation with proinflammatory system in which the presence and effects of TFF3 have been cytokines (tumor necrosis factor [TNF]-b, interleukin [IL]-1b, extensively studied, while until now the expression of CXCR4 interferon c) and under hyperosmolar conditions.11 and CXCR7 has only been investigated marginally. Here, we The functions of TFF3 in the body are various. Its dimeric investigate whether there is a dependence of TFF3 activity on more than its monomeric isoform is thought to interact directly the chemokine receptors CXCR4 and CXCR7. with mucins, increasing viscosity and elasticity of mucin- containing fluids like the tear film.8,12 It has been hypothesized MATERIALS AND METHODS that this may be their leading mechanism of action. The motogenic effects of TFF3 play an important role during Modeling of TFF3 and CXCR4/CXCR7 Interaction epithelial restitution and ensure rapid sealing of the epithelial layer after injuries.10 The x-ray structure of the homodimer CXCR4 (pdb-entry: For a long time receptors for TFFs have been unknown. In 3ODU)31 crystallized as lysozyme chimera was used to study 2009 the chemokine receptor type 4 (CXCR4, also stromal interactions with TFF3. Since the lysozyme part is located in cell–derived factor 1 [SDF-1] receptor) was described as a low the intracellular domain, it was assumed that it does not affinity receptor for TFF2.13 Chemokine receptor type 4 is an influence the docking behavior of TFF3, which is expected to almost ubiquitously present membrane receptor that, in be located on the extracellular side. The x-ray structure of TFF3 combination with its main substrate SDF-1 (CXCL12), is has also been determined previously (pdb-entry: 1PE3).32 involved in a variety of chemotactic processes, thus regulating Hydrogen atoms were added to both proteins by applying the precisely the fate of migrating cells.14 Its role in stem cell 3D-protonate option of a molecular operating environment trafficking, mobilization, and targeted migration to their (MOE, 2012.10; Chemical Computing Group, Inc., Montreal, reservoirs during development and adulthood is strongly QC, Canada). Since there is no x-ray structure for CXCR7, a illustrated by its various effects on organogenesis, regenera- homology model for the dimeric structure was built using the tion, damage repair, tumorigenesis and metastasis.15 In the eye, x-ray structure of CXCR4 (3ODU) as a template. This structure presence of CXCR4 has previously been reported in limbal and was modeled by using MOE with the new force field corneal epithelial and stromal cells, where it contributes to the (AMBER12 EHT, new amber version implemented in MOE; attraction of stromal niche cells to epithelial stem cells to Chemical Computing Group, Inc.). The quality of the model prevent their differentiation.16 It is also present in giant was checked with PROCHECK33 and fulfilled all stereochem- papillae in atopic keratoconjunctivitis.17 ical criteria for a reasonable model. Subsequently, rigid body Chemokine receptor type 4 consists of seven transmem- protein–protein docking studies were performed with brane helices and signals mainly through activation of G PLANTS34 bychoosinganatominthecenterofthe proteins. While SDF-1 was long considered the only CXCR4 extracellularly located loops as origin, with a radius of 40A,˚ ligand, there has been recent evidence that it has in fact to define the putative binding site of TFF3. Fifty different poses multiple substrates (e.g., the macrophage migration inhibitory were generated for each docking run. Finally, the best poses factor [MIF]).18 Beta defensin 3 is also a competitor with SDF-1 were energy minimized with the force field (Chemical for CXCR4, and acts as a natural antagonist at the receptor.19 Computing Group, Inc.) to allow induced fit and final Chemokine receptor type 7 (CXCR7) is of the same formation of hydrogen bonds and salt bridges which could subfamily and also contains seven transmembrane helices. It not be reached perfectly during the rigid body docking. is present in T lymphocytes and regulates SDF-1–mediated chemotaxis.20 High levels of CXCR7 were found in the heart, Ocular Sample Preparation brain, spleen, kidney, lung, bladder, skeletal muscles, Langer- 21–23 Tissues of the ocular surface and the lacrimal apparatus as well hans islets, cartilage, synovia, testes, ovary, and placenta. as lung tissue for positive controls were obtained from Just like CXCR4, it is overexpressed in a variety of tumor cells 24 cadavers donated to the Department of Anatomy and Cell and facilitates tumorigenesis in multiple different tissues. Biology, Martin Luther University Halle-Wittenberg, Germany. Chemokine receptor type 7 also has been shown to be 16 The donors (age 67–82 years) were free of recent trauma, eye expressed by corneal and limbal epithelial and stromal cells. and nasal infections, and diseases involving or affecting It has been described to bind SDF-1 and ITAC (CXCL11), the lacrimal function. All tissues were dissected from the cadavers latter with a 10-fold lower affinity than SDF-1. In contrast to within a time frame of 4 to 24 hours post mortem. After CXCR4, CXCR7 has been shown to be unable to recruit G- 24 dissection, the tissues were prepared for paraffin embedding proteins and mobilize calcium influx after ligand binding. For (right eye) by 4% paraformaldehyde fixation or were used for a considerable amount of time, its only function had been molecular biological investigation (left eye) and were immedi- assumed to be modulating the CXCR4 effects, probably by ately frozen at 808C. scavenging or sequestering SDF-1, thus creating SDF-1 gradi- 25 ents leading to altered CXCR4 signaling. This view had to be Cell Lines revised, as its activation by SDF-1 alone was described as inducing intracellular signaling events, with CXCR7 acting as a Simian vacuolating virus 40–transformed human corneal ‘‘biased receptor,’’ (e.g., through b-arrestins), thus activating epithelial (HCE) cells (provided by Kaoru Araki-Sasaki, Tane MAP kinases.26,27 Receptors CXCR4 and CXCR7 may dimerize, Memorial Eye Hospital, Osaka, Japan),35 as well as a homologously as well as heterologously, creating a variety of spontaneously immortalized epithelial cell line from normal binding possibilities.27,28 Chemokine receptor type 7 has been human conjunctiva (IOBA-NHC, here referred to as HCjE cells; suggested as a possible binding partner for TFF2 as well29 and provided by Yolanda Diebold, University Institute of Applied may therefore also qualify as a putative TFF3 receptor. Ophthalmobiology, University of Valladolid, Valladolid, Spain)36 In addition to the knowledge that CXCR4 acts as a low- were cultured as monolayers and used for stimulation affinity receptor for TFF2, there is evidence that it also engages experiments. We cultivated HCE cells in Dulbecco’s modified with the sister peptide TFF3. Xue et al.30 demonstrated the Eagle’s medium (DMEM)/HAM’S F12 1:1 medium, supplement-

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ed with 10% fetal calf serum (FCS; growing medium). The After a 2-hour incubation period at room temperature with the growing medium for HCjE cells also contained 1 lg/mL bovine peroxidase-conjugated goat anti-rabbit IgG secondary antibody insulin and 5 lg/mL hydrocortisone. The human breast cancer (1:10,000; DAKO, Glostrup, Denmark), positive binding was cell line MCF7 that served as a positive control was obtained detected using the ECL chemiluminescence substrate (ECL and cultivated as described.37 Western blotting detection kit, Amersham RPN 2106; Amer- sham Life Technologies). RNA Extraction, cDNA Synthesis, and Polymerase Chain Reaction Immunohistochemistry For reverse transcriptase polymerase chain reaction (RT-PCR), Immunohistochemistry on paraffin-embedded sections (7 lm) the following samples of ocular surface tissue and lacrimal of human lacrimal gland, cornea, conjunctiva, and lung tissue apparatus were investigated: cornea, conjunctiva, and lacrimal (positive control; n ¼ 5) was performed with a monoclonal gland (n ¼ 3 for each tissue). Tissue samples were first crushed mouse antibody to human CXCR4 (1:10 in TBS, MAB173; R&D in an agate mortar under liquid nitrogen and subsequently Systems, Inc., Minneapolis, MN, USA) and a monoclonal mouse homogenized (Polytron, Norcross, GA, USA). Total RNA was antibody to human CXCR7 (1:20 in TBS, MAB4227, R&D extracted following the manufacturer’s protocol (RNeasy Mini Systems, Inc.). These were applied with a standard peroxidase- Kit; Qiagen, Hilden, Germany). Reverse transcription of the labeled streptavidin-biotin technique, using conventional RNA samples to first-strand cDNA was performed according to methods with trypsinization. Nuclei were counterstained with the manufacturer’s protocol (RevertAid H Minus M-MuL V hematoxylin and slides finally mounted in aqueous medium Reverse Transcriptase Kit; Fermentas, St. Leon-Rot, Germany). (Aquatex; Roche, Mannheim, Germany). Two negative control For each reaction, 2 lg total RNA and 10 pmol Oligo (dT) sections, one incubated with the secondary antibody only, the primer (Fermentas) were used. Each PCR reaction was other with the primary antibody only, were used for each prepared with 2 lL cDNA, 9.8 lLH2O, 2 lL 50 mM MgCl2,2 tissue. Furthermore, control sections were incubated with lL 10 mM dNTPs, 2 lL 10x PCR buffer, 0.2 lL(5U)Taq- nonimmune IgG to determine possible nonspecific binding of Polymerase (Invitrogen, Darmstadt, Germany) and 2 lL10 mouse or rabbit IgG. All slides were examined and photomi- pmol primer mix. The following primers were used: crographs were taken (Keyence Biozero BZ-8100; Keyence Corporation, Osaka, Japan). CXCR4: forward 50-GGTGGTCTATGTTGGCGTCT-30 and reverse 50-TGGAGTGTGACAGCTTGGAG-30 (prod- Quantitative RT-PCR of Cell Lines uct: 227 base pairs [bp]); and CXCR7: forward 50-TCGTCTGCATCCTGGTGTGG-30 and We isolated RNA using TRIzol reagent (Life Technologies, reverse 50-CTGTGCTTCTCCTGGTCACTGG-30 Karlsruhe, Germany), DNA was digested by DNase, and cDNA (product: 259 bp). was synthesized. For quantitative RT-PCR TaqMan master mix and primer probes were used (Applied Biosystems, Inc.): For PCR control samples, 2 lL DNase-free water was used hGAPDH (Hs99999905_m1), hCXCR4 (Hs00607978_s1) and instead of cDNA. Lung tissue served as a positive control. The hCXCR7 (Hs00664172_s1). Analyses (n ¼ 3) were performed polymerase chain reaction included an initial cycle at 958Cfor with an ABI 7500 Fast detection system. Values of DCT ¼ 5 minutes followed by 35 cycles of 958C for 15 seconds, 648C CTGene of interest CTGAPDH (glyceraldehyde-3-phosphate for 30 seconds, 728C for 25 seconds, and a final elongation at dehydrogenase, housekeeping ). A value of DCT equal to 728C for 5 minutes, followed by a temperature hold at 48C. We 3.33 corresponds to one magnitude lower loaded 10 lL PCR product on a 2% agarose gel containing compared with GAPDH. ethidium bromide, and the amplified products were visualized under UV light after electrophoresis. values were Immunocytochemistry of Cell Lines compared with GenBank data. We confirmed PCR products by DNA sequencing using a sequencing kit (BigDye Terminator Cells were seeded on poly-D-lysine coated cover slips, grown Cycle Sequencing Kit; Applied Biosystems, Inc., Foster City, for 24 hours and differentiated for 24 hours (n ¼ 2). Medium CA, USA). was changed to DMEM þ 1% bovine serum albumin (BSA), and cells kept for 1 hour at 378C, then gradually cooled down to Western Blot Analysis 48C. Antibodies against CXCR4 (rabbit anti-CXCR4, 1:150; Imgenex, San Diego, CA, USA) and CXCR7 (mouse anti-CXCR7, For Western blot analysis, tissue samples of cornea, conjunctiva 1.100; R&D Systems, Wiesbaden, Germany) were applied in and lacrimal gland (n ¼ 4) were crushed in an agate mortar DMEM þ 4% horse serum at 48C for 1 hour. Cells were carefully under liquid nitrogen, then homogenized in 300 lL 1% Triton washed with ice-cold PBS þ 1% BSA and incubated with the buffer (Triton X-100; Carl Roth, Karlsruhe, Germany) with a secondary antibodies (donkey anti-mouse AlexaFluor 488 and protease and phosphatase inhibitor cocktail (Fermentas). The donkey anti-rabbit AlexaFluor 555, 1:800; Life Technologies) samples were centrifuged at 17.99g for 30 minutes, and the for 1 hour at 48C. Cells were washed again and fixed with supernatant was stored at 808C. The total protein concentra- Zamboni fixative (freshly prepared, 4% paraformaldehyde, tion was measured based on the Bradford dye-binding 17.5% picrinic acid in phosphate buffer) at room temperature procedure (Bio-Rad, Hercules, CA, USA). Total protein (40 for 30 minutes, washed, and nuclei were counterstained with lg) was analyzed by Western blot according to the following DAPI (Sigma-Aldrich, Munich, Germany). Cover slips were protocol: Samples were diluted in reducing Laemmli buffer, washed, desalted in distilled water, and embedded with denatured for 5 minutes at 958C, separated by SDS-PAGE on immumount (ThermoShandon, Frankfurt, Germany). 10% acrylamide separating gels, and transferred to Hybond-ECL nitrocellulose membranes (Amersham Life Technologies, Ar- Migration Assay (Scratch Assay) lington Heights, IL, USA). Protein blots were then incubated with anti-CXCR4 or anti-CXCR7 antibodies (CXCR4: rabbit We cultivated HCjE cells in DMEM/HAM’S F12 1:1 medium, polyclonal to CXCR4, ab2074; Abcam, Inc., Cambridge, UK; supplemented with 10% FCS, 1 lg/mL bovine insulin, and 5 lg/ CXCR7: rabbit polyclonal to GPCR RDC1, ab72100, Abcam, mL hydrocortisone. After reaching confluence, the medium was Inc.) diluted 1:500 in PBS containing 5% milk overnight at 48C. switched to equally supplemented, but FCS-free medium

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FIGURE 1. (A) Docking arrangement of TFF3 (above, space-fill representation) to the x-ray structure of CXCR4 (below, secondary structure visualization of the seven trans-membrane helices in red). (B) Docking arrangement of TFF3 (above, space-fill representation) to the model of CXCR7 (below, secondary structure visualization).

(starvation medium) for 3 hours. Where applicable, the receptor Tyr204, 1:500; Cell Signaling, Danvers, MA, USA) and antagonists AMD3100 (CXCR4, 100 ng/mL; Sigma-Aldrich Corp., horseradish peroxidase (HRP)–labeled secondary antibody St. Louis, MO, USA) or CCX733 (CXCR7, 10 nM; provided by (anti-rabbit, 1:30,000; Santa Cruz Biotechnologies, Dallas, TX, ChemoCentryx, Inc., Mountain View, CA, USA) were added to USA). Signals were detected with Amersham ECL Advance and the media for the last 30 minutes of the starvation time. Using Amersham Hyperfilm ECL (GE Healthcare Life Sciences, pipette tips, the cell layer was scratched several times, creating Uppsala, Sweden). To ensure equal loading, membranes were ‘‘wounds’’ of similar width. The cells were washed twice with stripped (Reblot Plus Strong Stripping solution; Millipore, PBS to remove debris, and starvation medium (with or without Darmstadt, Germany) and reprobed with anti-ERK2 (1:500; antagonists) was applied. Images of wounded areas were taken Santa Cruz Biotechnologies) and HRP-labeled secondary (Keyence Biozero BZ-8100) and areas were marked for later antibody (1:30,000 anti-mouse; Santa Cruz Biotechnologies). observation. Cells were subsequently stimulated with recombi- nant human (rh) TFF3 (10, 30, 100, 300, 1000 lg/mL). Controls ESULTS contained no rhTFF3, but adequate volumes of rhTFF3/ R AMD3100/CCX773 solvents (PBS or DMSO). Bovine serum Dimers of CXCR4 or CXCR7 Show High Affinity for albumin (300 lg/mL) served as a protein control. In order to ensure only migration, but not proliferation, mitomycin C was TFF3 in X-Ray Structure-Based Modeling added to the cells at a concentration of 10 lg/mL. The Todefinepossibleinteractionsbetweenthechemokine previously imaged areas were again photographed after 24 receptors and TFF3 we used published x-ray structure data hours of stimulation. The wounded area was determined at 0 and performed 3D modeling structural analysis. The most hours as well as 24 hours using graphics editing software probable protein–protein docking arrangement between TFF3 (Adobe Photoshop; Adobe Systems, Mountain View, CA, USA). and CXCR4 is displayed in Figure 1A. This complex is strongly Stimulated samples were compared with control values. stabilized by the formation of several strong salt bridges Statistical significance was determined by 1-way ANOVA and between amino acid side chains of TFF3 and CXCR4 Tukey’s multiple comparisons test using commercial scientific (R34:D193 chain B, D1:K271 chain A, E30:K271 chain B), statistics software (InStat; GraphPad Software, Inc., San Diego, hydrogen bonds (N33:E277 chain B, Y23:E268 chain B), and CA, USA); n ¼ 6 for each condition. some hydrophobic interactions (e.g., F59:F189 chain B). These interactions of TFF3 mainly occur with the extracellular loops Proliferation Assay (Flow Cytometry) of CXCR4 but also partly in the pore formed by the trans- We cultivated and treated HCjE cells as mentioned above membrane helices. The docking behavior of TFF3 with the (migration assay). After the respective treatment, cells were model of CXCR7 (Fig. 1B) is also characterized by the detached using trizol, and stained with propidium iodide (PI) formation of several strong salt bridges and hydrogen bonds using PI/RNase staining buffer (BD Biosciences, San Jose, CA, (R41:E202A, S40:E202B, N33:N191B, F99C terminus, K40B USA) following the manufacturer’s protocol. Resulting PI and N191B.E56: R288B. R18:D39B) and occurs also with the staining was subsequently read using a flow cytometer (BD extracellular loops at the surface. These software modeling FACSCalibur; BD Biosciences); n ¼ 4 for each condition. results indicate that CXCR4 and CXCR7 may have the possibility to interact with the TFF3 molecule. Western Blot on ERK1/2 Phosphorylation CXCR4 and CXCR7 Are Present at the Ocular We differentiated HCjE cells (n ¼ 3) for 24 hours, washed Surface serum-free (2 3 1hourwithDMEMþ 1% BSA), and preincubated for 1 hour with inhibitors: 1 lM AMD3100 or The presence of mRNA for chemokine receptors CXCR4 and 0.1 lM CCX733 or 0.1% DMSO (solvent control). Cells were CXCR7 was demonstrated by RT-PCR in human lacrimal gland, stimulated with 10 lg/mL rhTFF3 (as this concentration cornea, and conjunctiva (Fig. 2A, n ¼ 3 for each tissue). proved most efficient in the migration assay) for 15 minutes Additionally, Western blot analysis revealed protein biosynthe- while inhibitor concentrations were maintained. Samples were sis in the named tissues (n ¼ 4). Immunohistochemistry (n ¼ 5 lysed and subjected to SDS-PAGE with subsequent immuno- for each tissue) showed the following distribution of both blotting with antiphosphorylated ERK1/2 (pERK, Tyr202/ proteins: in the cornea, the reactivity of the CXCR4 antibody is

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FIGURE 2. (A) Detection of CXCR4 and CXCR7 in tissues of the ocular surface. Analysis of RT-PCR (n ¼ 3) shows receptor transcription in all tested tissues. Protein synthesis is shown by Western blot (n ¼ 4); one lacrimal gland, two cornea, three conjunctiva. (B) Immunohistochemistry of CXCR4 and CXCR7 in human lacrimal gland, cornea, and conjunctiva (n ¼ 5). Lung tissue served as a positive control. The CXCR4 antibody shows reactivity throughout the corneal epithelium—both membrane-bound (arrowheads) and cytoplasmic (arrows)—while binding in conjunctival tissue occurs predominantly in the apical cell layer. The lacrimal gland synthesizes CXCR4 weakly, mostly in acinar and myoepithelial cells (asterisks). The CXCR7 receptor is produced by corneal cells as well, showing mostly intracellular localization. In the conjunctiva, the receptor is mainly expressed intracellularly by apical cell layers. In the lacrimal gland it is produced by myoepithelial cells, ductal cells with an apical localization and a cytoplasmic localization inside the acini. Ac, acinus; Alv, alveolus; B, Bowman’s layer; D, duct; Ep, epithelium; FC, fat cell; St, stroma.

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(data not shown). Only migratory effects, but not proliferation, seem to be mediated by CXCR4 and/or CXCR7. Cell proliferation assays testing for cycling cells using PI staining and subsequent flow cytometry showed increased proliferation rates (higher number of cells undergoing S- and G2/M-phase) in HCjE cells after rhTFF3 stimulation (Fig. 5A, n ¼ 4). The addition of the specific receptor antagonists AMD3100 (for CXCR4) and/or CCX733 (for CXCR7) did not significantly affect the increase in cycling cells, neither alone nor added in combination (Fig. 5B).

rhTFF3 Stimulation Results in Activation of the MAP Kinase Signaling Cascade Independently FIGURE 3. (A) Quantitative RT-PCR of CXCR4 and CXCR7 in human From CXCR4 or CXCR7 conjunctival epithelial cell line (HCjE) and MCF7 cell line (positive control; n ¼ 3). Expression is detectable in both cell lines; in HCjE, To evaluate whether rhTFF3 had any effect on the MAP kinase however, in a lower relative concentration at the detection limit. (B) pathway, we investigated the phosphorylation status of ERK1/ Only few receptor antibody signals are detectable by immunocyto- 2 proteins by Western blot analysis in HCjE cells (n ¼ 3). This chemistry in a cultured HCjE cell compared to a cultured MCF7 cell revealed an upregulation of pERK after stimulation of HCjE that was used as positive control (n ¼ 2). cells with rhTFF3 (Fig. 6). This increase in pERK by rhTFF3 was also seen in the presence of the specific CXCR4 and visible as membrane-bound as well as partly cytoplasmic CXCR7 antagonists AMD3100 and CCX733. There was also an throughout the epithelium, while the CXCR7 antibody shows increase in signal intensity after AMD3100 treatment alone. primarily a weak cytoplasmic binding. The conjunctiva shows a stronger apical localization of both receptors, again DISCUSSION membrane-bound as well as intracellular. For the lacrimal gland we detected a weak cytoplasmic signal of both CXCR4 Chemokine receptors CXCR4 and CXCR7 are present in a and CXCR7 inside the acini as well as strong reactivity with variety of tissues throughout the human body. At the ocular myoepithelial cells, while the signal of ductal cells had a more surface, their presence had only been shown in corneal and apical localization (Fig. 2B). limbal epithelial and corneal stromal cells16 and, under pathologic conditions, during atopic keratoconjunctivitis.17 CXCR4 and CXCR7 Are Not Expressed in the HCE In the present study, we demonstrated the expression and Cell Line, but in the HCjE Cell Line synthesis of CXCR4 and CXCR7 at the healthy ocular surface. Under physiological conditions, both receptors were detected Human immortalized corneal epithelial cells did not express in human cornea, conjunctiva and lacrimal gland, and in lesser CXCR4 and CXCR7, as evaluated by quantitative RT-PCR and amounts in the conjunctival epithelial cell line used. The immunocytochemistry (data not shown). Human immortalized presence of TFF3 in these tissues has already been shown.7,8 conjunctival epithelial cells expressed both receptors. Howev- Their similar expression patterns in these tissues allow for er, the expression level of CXCR4 as well as CXCR7 in HCjE initial speculation about putative interactions. cell line was not pronounced compared with the expression in Chemokine receptor type 4 is present on most hematopoi- MCF7 cell line (a human breast cancer cell line), which was etic cell types, where it plays an important role in recruitment used as control cell line. In HCjE, quantitative RT-PCR yielded of immune cells during inflammation. It is known that CXCR4, but not SDF-1, is upregulated by proinflammatory cytokines, signals at the detection limit (Fig. 3A, n ¼ 3) and immunocy- 38 tochemistry revealed only few positive signals at the surface of like TNF-a or IL-1b. A comparable situation was found for gastric ulcer healing, where SDF-1 levels decrease in the cultivated cells (Fig. 3B, n ¼ 2). The control cell line MCF7 39 showed strong signals for CXCR4 and CXCR7 in both analyses. process, while CXCR4 levels increase. At the ocular surface, TFF3 is induced during corneal infection9 and after corneal 10 rhTFF3 Promotes Cell Migration and Proliferation damage. Corneal epithelial cells upregulate TFF3 when stimulated with TNF-a and IL-1b.40 The fact that CXCR4, but of HCjE Cells in a Concentration-Dependent not its main ligand SDF-1, is upregulated during inflammation Manner leads to the hypothesis that another ligand may be activating the receptor under these conditions. The described upregu- For the following experiments, only the receptor expressing lation of TFF3 leaves room for further theorizing about a cell line HCjE was further investigated. The cell culture–based possible CXCR4-TFF3 interplay. migration assays revealed increased migration of HCjE cells in As stated earlier, CXCR4 and CXCR7 have been discussed as the presence of rhTFF3 (Figs. 4A, 4B; n ¼ 6). The effect seen low affinity receptors for TFF2.13,29 The well-studied structural with lower concentrations, however, decreased with increas- similarities between the different TFF peptides allow for ing concentrations of rhTFF3. When adding the specific speculation about a possible interaction with TFF3 as well. chemokine receptor antagonists, the observed rhTFF3 effect As for CXCR4, this has been shown recently by Xue et al.30:In was completely neutralized (Fig. 4C; n ¼ 6). Significant wild-type mice, AMD3100 significantly slowed restitution of differences were seen between rhTFF3 stimulation and the gastric mucosa after experimental injury. In TFF2 knockout addition of AMD3100 and CCX733 in every rhTFF3 concen- animals, topic addition of rat TFF3 peptide partly compensated tration tested. There are no measurable differences in for the loss of TFF2, an effect that was also strongly inhibited migration rates between the two antagonists: The remaining by adding AMD3100. This suggests that both trefoils (i.e., TFF3 wound areas in both cases resemble those of controls. as well), engage the CXCR4 receptor during wound healing.30 Simultaneous blockade of both receptors has been tested for To elucidate these theoretic interactions, we used an x-ray a rhTFF3 concentration of 300 lg/mL and yielded a migration based computer modeling system to determine possible impairing effect comparable to blocking either one receptor binding conformations of both receptors with the TFF3

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FIGURE 4. (A) Scratch assay on HCjE cells (representative pictures, n ¼ 6). Confluent cell layers were scratched with a pipette tip, wounded area was photographed and measured (0 hours). After 24 hours of incubation, remaining wound area was again photographed and measured (24 hours). Values of cells incubated with different concentrations of rhTFF3, with or without addition of AMD3100 or CCX733, were compared with control values. (B) Restored wound area after scratch and 24-hour incubation with rhTFF3, compared with control values. The wound healing rates are significantly higher under stimulation with 10, 30, 100, and 300 lg/mL and 1 mg/mL rhTFF3 compared with no TFF3 control as well as BSA protein control. Lower concentrations seem to have better effects on cell migration, while effect regresses with increasing concentrations. *P < 0.05; **P < 0.01; ***P < 0.001 compared with control; 8P < 0.05; 88P < 0.01; 888P < 0.001 compared with BSA protein control. (C) Restored wound area after scratch and 24-hour incubation with rTFF3 and the CXCR4 antagonist AMD3100 or the CXCR7 antagonist CCX733, compared with control values. Enhancement of migration, seen with rTFF3, is significantly inhibited by addition of either antagonist. No significant differences in migration rates between the two antagonists were seen. *P < 0.05; **P < 0.01; ***P < 0.001.

peptide. The modeling identified the CXCR4 homodimer and stronger in an experimental system that mimics the in vivo the CXCR7 homodimer as probable binding partners for TFF3 situation more precisely. Nevertheless, our results are of great in each case, showing that the main TFF3-receptor interaction interest as they indicate an interplay of TFF3 with CXCR4 and was taking place between the substrate TFF3 and basically only CXCR7 that can be more pronounced in other tissues and cell one of the involved monomers (referred to as chain B in each lines which need to be determined in the future. receptor, see above). Therefore, an interaction of TFF3 with In our model system, we found opposite results: investigat- the heterodimer also seems possible, as TFF3 may interact ing proliferation rates by analyzing cell cycle parameters with independently with each monomer involved. flow cytometry, we found elevated levels of cycling cells after The most often described effect of TFF3 is enhancement of rhTFF3 stimulation with or without addition of AMD3100 or wound healing. This complex process can be understood as a CCX733, suggesting independence from the chemokine synergy of, among others, cell proliferation and migration in receptors. On the contrary, our cell culture–based migration order to cover and repair an injured tissue. Here, we assay yielded most different results: stimulation of wounded investigated both subprocesses independently to evaluate a ocular surface cells with rhTFF3 resulted in accelerated cell putative chemokine receptor function. In general, it is to be migration. In accordance with the findings of Xue et al.,30 the noted that the expression levels of CXCR4 and CXCR7 in the blockage of CXCR4 completely abrogated the rhTFF3 effect used HCjE cells were low, so that interpretation of the in vitro when compared with control values. In addition, we show results with this cell line needs to be done with caution. here that antagonizing CXCR7 with CCX733 had a comparable However, according to the immunohistochemistry results effect: Wound healing rates that had increased under rhTFF3 obtained in conjunctiva, the in vivo receptor density is stimulation were significantly impaired when CXCR7 was markedly higher than in the HCjE cell line. Therefore, the blocked. The migration assay revealed that the effect of rhTFF3 obtained reactions to stimulation with rhTFF3 may prove even was fully diminished not only by blocking both receptors at

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FIGURE 5. Flow cytometry on PI stained HCjE cells (n ¼ 4). (A) Shows cell cycle distribution in G0/G1-, S- and G2/M-phase. Representative graphs are shown for control cells as well as after stimulation with rTFF3 or rTFF3 þ AMD3100. FL2-A: emitted fluorescent light of the DNA dye (FL2) measured as pulse-area (FL2-A). (B) Number of cells in S- or G2/M-phase compared with control after the addition of rhTFF3 and/or the cytokine receptor antagonists. Addition of rhTFF3 alone leads to a significant increase of cells undergoing S- and G2/M-phase. Addition of AMD3100 and/or CCX733 does not significantly influence this activation of the cell cycle. **P < 0.01; 888P < 0.001 compared with respective control.

once, but by blocking either one of the receptors, despite the membrane of human embryonic kidney cells in the absence of fact that the other receptor in each case was still fully a ligand, with additional evidence of further intracellular functional. heterodimer pools. Levoye et al.28 confirmed the constitutive There have been previous investigations in which a formation of heterodimers, and indicated the same efficiency blockade of either receptor, CXCR4 or CXCR7, resulted in for homo- and heterodimerization, suggesting that the levels of impaired effects of a substrate, or even in complete abrogation CXCR4 and CXCR7 biosynthesis determine the incidences of 41 of the effect, respectively. Yan et al. showed that blocking homo- and heterodimers. Heterodimerization on the cell CXCR7 with CCX733 impaired the SDF-1 induced adhesion of surface and combined internalization of both receptors has endothelial progenitor cells to active human umbilical vein also been shown by fluorescence light and electron microsco- endothelial cells (HUVECs). Antagonist AMD3100 completely py on MCF-7 breast cancer cells.37 Besides detecting CXCR4- abolished the adhesion, while the inhibitory effect of blocking CXCR7 heterodimers using immunoprecipitation, Decaillot´ et both receptors resembled that of AMD3100 alone.41 Moreover, al.27 described the ability of CXCR7 to alter CXCR4/SDF-1– Watanabe et al.42 demonstrated results even more congruent mediated inhibition of adenylyl cyclase and subsequent cAMP with those gathered in the current study: SDF-1 was confirmed production, and instead switch to constitutively recruiting - to induce tube-forming activity in HUVECs, which was b completely suppressed by blocking either CXCR4 or CXCR7, arrestin complexes. In fact, b-arrestin recruiting was notably and to a comparable extent. Blocking both receptors resulted stronger in CXCR4 and CXCR7 coexpressing cells than in cells in similarly low levels of tube formation compared to blocking expressing CXCR7 alone. So a strong b-arrestin response is only one receptor alone.42 In the latter study, the authors dependent on the presence, function, and heterodimerization suggest that signaling from both receptors was necessary for of both CXCR receptors. the SDF-1 effect to take place. In summary, we found that cell proliferation of rhTFF3 Another possible explanation involves the previously stimulated HCjE cells occurred independently from the studied ability of CXCR4 and CXCR7 to form functional functionality of CXCR4 and CXCR7, while the observed heterodimers. Using FRET analysis, Sierro et al.43 demonstrated migratory effects were dependent on both chemokine the presence of preformed CXCR4-CXCR7 dimers on the cell receptors, and possibly on their homo- or heterodimerization.

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of a complex substrate-receptor interplay with multiple cellular signaling targets involved, with CXCR4 and CXCR7 just being two of them. In the given model system, they promote cell migration in a MAP kinase–independent manner. Further experiments are needed to determine alternative receptors that activate the MAP kinase pathway, as well as the subsequent signaling chain after CXCR4/CXCR7 activation by TFF3.

Acknowledgments The authors thank Susann M¨oschter, Stephanie Beilecke, Martin Schicht, and Katja Dieckow, Tamara Grummet, and Andrea Sehr for their helpful assistance and advice. Supported by the German Research Foundation (DFG, Program Grant PA738/9-2 and PA738/11-1, and ME758/10-1, RM, and SE1995/1-1, SS); a special fund for scientific work at Friedrich Alexander University of Erlangen-Nurnberg¨ (FP); an MD fellowship of the Boehringer Ingelheim Fonds, Foundation for Basic Research in Medicine, Heidesheim, Germany (JD); and Sicca Forschungsf¨or- derung of the Association of German Ophthalmologists (JD). The

FIGURE 6. Western blot of ERK1/2 phosphorylation (upper panel) and present work was performed in fulfillment of the requirements for respective densitometry (lower panel; n ¼ 3). Stimulation of HCjE cells obtaining the degree ‘‘Dr med.’’ with rhTFF3 results in significantly higher levels of pERK, an effect that Disclosure: J. Dieckow, None; W. Brandt, None; K. Hatter- is not abrogated by addition of the specific receptor antagonists mann, None; S. Schob, None; U. Schulze, None; R. Mentlein, AMD3100 or CCX733. *P < 0.05 compared with control. DMSO, None; P. Ackermann, None; S. Sel, None; F.P. Paulsen, None solvent control; NS, not significant. References A closer look into the signaling chains of CXCR4, CXCR7 and TFF3 further elucidates the possibility of interaction. The 1. Kjellev S. The trefoil factor family - small peptides with multiple functionalities. 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