FEBS Letters 580 (2006) 1431–1438 In vitro and in vivo studies on CCR10 regulation by Annexin A1 Flavia Cristina Rodrigues-Lisonia,b, Devinder K. Mehemeta, Paulo Peitl Jrc, Christopher D. Johna, Eloiza Tajarad, Julia C. Buckinghama, Egle Solitoa,* a Department of Cellular and Molecular Neuroscience, Division of Neuroscience and Mental Health, Faculty of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK b UNESP-Sao Paulo State University, IBILCE, São José do Rio Preto, SP, Brazil c Faculdade de Medicina, USP/Ribeirão Preto, Brazil d Faculdade de Medicina, de São José do Rio Preto, SP, Brazil Received 13 December 2005; revised 20 January 2006; accepted 23 January 2006 Available online 31 January 2006 Edited by Beat Imhof Abstract The mode of action of annexin A1 (ANXA1) is poorly understood. By using rapid subtraction hybridization we studied the effects of human recombinant ANXA1 and the N-terminal ANXA1 peptide on gene expression in a human larynx cell line. Three genes showed strong downregulation after treatment with ANXA1. In contrast, expression of CCR10, a seven transmem- brane G-protein coupled receptor for chemokine CCL27 involved in mucosal immunity, was increased. Moreover the reduction in CCR10 expression induced by ANXA1 gene deletion was res- cued by intravenous treatment with low doses of ANXA1. These findings provide new evidence that ANXA1 modulates gene expression. � 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Gene expression; Host defence; Annexin A1-null mice; Epithelial cells; Annexin A1 1. Introduction Annexin A1 (ANXA1) is a 37 kDa member of a family of Ca2+-regulated lipid-binding proteins [1]. Functional studies have identified roles for ANXA1 and its N-terminal domain [2,3] in the regulation of inflammation [4], the cell cycle [5] and as a mediator of glucocorticoid action on the release of cor- ticotrophin (ACTH) from the anterior pituitary gland [6,7]. Many of the regulatory actions ofANXA1 in these systems have been shown to emerge promptly, i.e., within minutes to hours of contact with the protein [6]. Whilst some have been ascribed to the ability of ANXA1 to act intracellularly to disrupt signalling cascades [8], others appear to reflect interactions with cell sur- face receptors [9–11]. Recent studies suggest that members of the formyl peptide receptor (FPR) family of G-protein coupled receptors are particularly important in this regard [12,13]. Three members of this receptor family have been identified in human, termed FPR, FPRL1 and FPRL2. Receptor-specific ligands are not yet available, but FPR is highly sensitive to the bacterial peptide formyl-Met-Leu-Phe (fMLP), while FPRL1 shows some selectivity for lipoxin A4, a lipid metabolite, in addition to responding to high concentrations of fMLP [14]. *Corresponding author. Fax: +44 208 3838032. E-mail address: e.solito@imperial.ac.uk (E. Solito). 0014-5793/$32.00 � 2006 Federation of European Biochemical Societies. Pu doi:10.1016/j.febslet.2006.01.072 Despite the evidence that ANXA1 might influence cellular responsiveness, the possibility that ANXA1 regulates gene expression has received scant attention. Interestingly however, the recently generated ANXA1 knockout mouse shows signif- icant alterations in the expression of a number of gene prod- ucts associated with inflammation [15,16], a finding that concurs with earlier reports that ANXA1 reduces the expres- sion of inducible nitric oxide synthase [17]. We have used rapid subtraction hybridisation (RaSH) [18] to investigate the effects of ANXA1Ac2–26 on gene expression in a human cell line of epithelial origin. In addition, we have compared the effects of hrANXA1 and ANXA1Ac2–26 with those of fMLP (a bacterial peptide) and the stable analogue of lipoxin A4 [15-epi-16-(para-fluoro)-phenoxy-LXA4-methyl ester, ATLa-ME], an arachidonic acid metabolite, in order to explore the role of the FPR family in effecting the response to ANXA1. The data obtained from the cell line in vitro have been evaluated in vivo by studies on Anxa1 null mice. Our findings strongly support a role for ANXA1 in the regulation of gene expression, particularly those genes concerned with the inflammatory response. 2. Materials and methods 2.1. Drugs Human recombinant annexin A1, [19] was produced, purified and tested by SDS–PAGE for the presence of cleaved forms which were not detected, by Scientific Proteins, Witterswil, Switzerland. Further- more, the recombinant protein did not show any phosphorylation at the N-terminal as reported previously [19]. HumanANXA1Ac2–26 (acet- ylated peptide at the N-terminal) was synthesized in the Advanced Bio- technology Centre, Imperial College London, UK. The peptide fMLP was purchased from Sigma (Sigma–Aldrich Corp., Poole, Dorset, UK) and 15-epi-16-(para-fluoro)-phenoxy-ATLA-methyl ester (ATLa, the methyl ester of lipoxin A4) was a generous gift from Professor C. Serhan (Harvard Medical School, Boston, USA). The doses of acety- lated peptide (ANXA1Ac2–26; 2 lM) were chosen on the basis of previ- ous studies, performed both in vitro and in vivo, in which we showed that comparable biological activity were obtained when the ratio be- tween fMLP or ATLa and ANXA1Ac2–26 was 1:20 [13,20] furthermore such molar ratio was of 1:15 between the entire recombinant ANXA1 and the peptide Ac2–26 as described previously [21]. 2.2. Cell culture The Hep-2 cell lines were derived from a human larynx epidermoid carcinoma cells (ATCC Rockville, MD, USA) and used for RaSH analysis. The PDFS cell line derived from a clinically non-functioning pituitary macroadenoma was used to confirm and compare the analy- sis performed on Hep-2 cells (see supplement Table 2) [22]. blished by Elsevier B.V. All rights reserved. mailto:e.solito@imperial.ac.uk 1432 F.C. Rodrigues-Lisoni et al. / FEBS Letters 580 (2006) 1431–1438 2.3. Animals Male Anxa1-null mice aged approximately 4 months old [15] and weighing 30 ± 4 g were used together with age- and sex-matched wild type controls. All animals were fed on a standard chow pellet diet. All procedures were carried out under licence in accordance with the UK Animals (Scientific Procedures) Act, 1986. For tissue collection the animals were killed by cervical dislocation and their lower back re- gion was shaved. Longitudinal fragments of the dorsal skin (after shav- ing), trachea and ileum were obtained from each mouse (6 animals per tissue) and frozen immediately in liquid nitrogen for subsequent extraction of RNA and protein. Anxa1 null mice were injected intravenously via the lateral tail vein with hrANXA1 (10 ng/mouse), ANXA1Ac2–26 (14 ng/mouse) or a cor- responding volume of the saline vehicle (100 ll). Mice (three animals per treatment) were killed with CO2, 24 h after drug administration, and tissues collected exactly as described above. 2.4. Rapid subtraction hybridization (RaSH) and RT-PCR analysis Hep-2 cells were plated at a concentration of 106 cells/in six well plates (Corning Incorporated, NY, USA) and 24 h later in culture, stimulated with ANXA1Ac2–26 (2 lM; 72 h). Total RNA isolated by RNeasy (Qiagen, West Sussex, England) from control and treated cells were used for double-stranded cDNA synthesis using a standard pro- tocol [23]. The DNA was then digested and mixed with the adaptors XPDN-14/XPDN-12 (Sigma Genosys; final concentration 20 lM) as described by Jiang et al. [18]. 100 ng tester cDNA (e.g., untreated cells) was mixed with 5 lg driver cDNA (e.g., ANXA1Ac2–26 treated cells, see also Table 1 ) in hybridization solution [18,24]. After extraction and precipitation the hybridization mixtures (1 lg) were ligated to an XhoI digested pCDNA3 plasmid and transformed in competent bacteria. Bacterial colonies were selected randomly and amplified by PCR. The sequences of these clones were determined using the automated cy- cle sequencer in the Advanced Biotechnology Centre, Imperial College London. Sequences were compared with the NCBI nucleotide bank (GenBank) allowing identification of any genes up or down regulated. Experiments were then set up (n = 3) in the same conditions described above and RT-PCR with specific oligonucleotides (supplementary data Table 1) for the genes identified by RaSH was performed. 2.5. Semi quantitative RNA analysis We first determined the linear range of amplification (25, 20, 35, 40 cycles) of cDNA using each of the primer sets. Amplification was with- in the linear range for all genes studies when 35 PCR cycles were used and this level of amplification was therefore used in all further studies. For each sample 5 ll of the PCR amplification products was analysed on 2% agarose gels and stained with ethidium bromide. The intensities of the bands were compared to GAPDH in each sample and evaluated using the Image Master Software (SYDR-1990, SYNGENE, USA). 2.6. Western blot analysis Expression of CCR10 (GPR2) protein (15 lg) was examined in the cell lines and murine tissue extracts by SDS–PAGE, then transferred electrophoretically to nitrocellulose paper [25]. The blots were incu- bated overnight at 4 �C in incubation buffer in presence of an anti- CCR10 antiserum (diluted 1:1000, goat anti-mouse polyclonal, Abcam; or anti human CCR10 diluted 1:1000, Imgenex San Diego, CA, USA). Immunoreactive protein bands were detected by chemilu- minescence (GE Healthcare, UK) after incubation with secondary Table 1 Genes isolated by RaSH Symbol Locus Upregulated 100 ng of tester (ANXA1Ac2–26 treated cells) versus 5 lg of driver (untreated cells) CCR10 (NM_016602) 2826 Downregulated 100 ng of tester (ANXA1Ac2–26 treated cells) versus 5 lg of driver (untreated cells) TIFA (NM_052864) 92610 FARSLA (NM_004461) 2193 ITGB1BP1 (NM_004763) 9270 antibody (rabbit anti-goat peroxidase; 1: 100000, Biomeda; or goat anti-rabbit peroxidase conjugated antiserum, 1:25000, Sigma–Aldrich Corp.). The blots were then scanned and analyzed (HP Scanjet 5200 with Adobe Photodeluxe Business Edition, version 1.1; Cupertino, CA). The intensities of the bands were compared to b-actin (mouse monoclonal 1:5000, Abcam). 2.7. FACS analysis For detection of FPR and FPRL1 on the cell surface of Hep-2 and PDFS cells, aliquots of suspensions of the cells (105 cells in 100 ll) were added to a 96-flat well plate in triplicate and incubated at 4 �C for 1 h in the presence or absence (controls) of either: (a) a monoclonal anti- human FPR antibody (1:100, BD Pharmingen) or mouse IgG1 clone NOPC-21 (isotype-matched control antibody 1:100, BD Pharmingen, UK) or (b) a rabbit anti-human FPRL1 polyclonal antiserum raised against the peptide TVWLKEMLFYGKYKIIDILVNP from the 3rd extracellular domain of the receptor (1:100, Neosystem Strasburg, France). Non-specific binding was minimized by the addition of hu- man IgG (1.6 mg/ml, Roche Diagnostics, Lewes, UK). After incuba- tion with the primary antiserum, the cells were incubated on ice for 30 min with FITC-conjugated rabbit anti-mouse or sheep anti-rabbit IgG (1:300, Serotec, Kidlington, Oxford, UK). The cell surface fluores- cence was analyzed immediately by flow cytometry using a Becton Dickinson FACScan II analyzer. At least 10000 cells per sample were counted. The data were analyzed as units of fluorescence measured in the FL1 channel (mean fluorescence intensity). A similar method was used to detect and quantify expression of the cell surface receptor CCR10 (GPR2) using a goat anti-rabbit polyclonal antibody to CCR10 (GPR2, 1:100, Imgenex San Diego, CA, USA) as a probe and FITC-conjugated sheep anti-rabbit IgG (1:300, Serotec) for detection. 2.8. Statistical analysis Statistical significance was assessed by ANOVA followed by Bonfer- roni’s t test, and P < 0.05 was taken as statistically significant. 3. Results and discussion 3.1. Annexin A1 modulates gene expression in cell line Subtractive hybridisation performed on the human cell line Hep-2, identified three genes, which were downregulated by hrANXA1 (100 nM) and ANXA1Ac2–26 (2 lM) treatment. The first was ITGB1BP1 (previously named ICAP-1A, Table 1 and Fig. 1) a protein specifically associated with the b1 cyto- plasmic domain of integrins [26] containing putative phosphor- ylation sites. The phosphorylated form probably impairs cell adhesion and spreading [27], but over-expression of ITGB1BP1 stimulates cell migration [28]. Thus, the ability of ANXA1 to impair monocytes on the endothelium in vitro [21] may be med- iated, at least in part, through ITGB1BP1 downregulation. Since ANXA1 and ANXA1-derived peptides are potential ligands for members of the formyl peptide receptor (FPR) family, we explored the potential role of these receptors in ID Function A G protein coupled receptor for chemokine CCL27 mostly involved in T cell mediated skin inflammation A TRAF2 binding protein involved in the TNF-mediated signalling A tRNA synthetase family expressed in a cell cycle stage and differentiation-dependent manner Accessory protein for the integrin beta 1 Fig. 1. Comparison of the effect of ANXA1 and FPRs ligands: fMLP and ATLa on ITGB1BP1, TIFA and FARSLA in Hep-2 genes isolated by RaSH. Cells were plated and treated as reported for RaSH analysis. After 72 h of stimulation with (A) ANXA1 (100 nM) or ANXA1Ac2–26 (2 lM) or (B) fMLP (100 nM) and ATLa (100 nM) total RNA was extracted and RT-PCR performed using specific oligos for the above genes. Semi- quantitative normalization was performed using GAPDH expression. The data are representative of six independent experiments. F.C. Rodrigues-Lisoni et al. / FEBS Letters 580 (2006) 1431–1438 1433 mediating the genomic effects of ANXA1 reported above. First, we used RT-PCR and FACS analysis to determine whether Hep-2 cells express FPR and FPRL1. Our data dem- onstrated mRNA and protein for both receptors in Hep-2 cells (Fig. 2A and B). Subsequently, we used ligands of FPR and FPRL1, fMLP and the lipoxin A4 analogue (ATLa), to ex- plore whether activation of these receptors modulated the expression of ITGB1BP1 (Fig. 1B and Table 3B supplement Fig. 2. Hep-2 cells express both FPR and FPRL1 receptors. (A) RT-PCR an the entire coding sequences for FPR and FPRL1, respectively. Lanes 2 and 3 FACS analysis of FPR and FPRL1 expression on the surface of the cells. T data). Neither fLMP nor ATLa, ligands for FPR and FPRL1, modified the expression of ITGB1BP1 (Fig. 1B). Although we cannot exclude the possibility that higher concentrations of these compounds may have been effective they would be ex- pected to act at the concentrations tested [13] and indeed have been shown to modify the expression of other genes in this study. We therefore suggest that the regulatory actions of ANXA1 and ANXA1Ac2–26 on the expression of ITGB1BP1 alysis lanes 1 and 4 represent the control amplified plasmids containing amplified product in Hep-2 cells for FPR and FPRL1 respectively. (B) he data are representative of three independent analyses. 1434 F.C. Rodrigues-Lisoni et al. / FEBS Letters 580 (2006) 1431–1438 were mediated via a mechanism independent of FPR or FPRL1. A second gene downregulated by both hrANXA1 and AN- XA1Ac2–26 was a phenylalanine-tRNA synthetase (FARSLA). Unlike ITGB1BP1 this gene was also downregulated by fMLP and ATLa, suggesting a role for the FPR family in mediating the response (Fig. 1A and B). In vivo FARSLA is expressed in a tumour-selective, cell cycle stage and differentiation-depen- dent manner and its expression is augmented in tumourigenic versus non-tumourigenic variant of the same cell line [29]. Interactions between ANXA1 and aminoacyl-tRNa synthase have been reported in patients with dermatomyositis [30] and between ANXA1 and tryptophanyl-tRNA synthetase [31]. To- gether, these results raise the possibility that ANXA1 plays a role in cell transformation as well as in protein translation and may explain why it is highly expressed in some cancers and not in others [32]. Interestingly, it has been proposed that ANXA1 and tRNA synthase share some physiological role in process related to myositis and immune response [31]. Fig. 3. CCR10 expression in Hep 2 cells. RT-PCR analysis of CCR10 expre for 72 h. The data are representative of six independent experiments. Fig. 4. Western blot and FACS analysis of CCR10 expression on Hep-2 ce protein cell extract from treated or untreated cells were analysed by Wester number of cells) showing the data from one of three independent experimen A third gene downregulated by ANXAAc2–26 and fMLP, but not by ANXA1 or ATLa (Fig. 1A and B) was a TRAF-inter- acting protein with a forkhead-associated domain termed TIFA or T2BP. Although merely speculative, as we do not know how the acetylation may affect the structure and func- tion of the peptide, the downregulation of TIFA suggests that ANXA1 may modulate inflammatory processes via TNFa and TIFA, supporting our previous findings on the role of ANXA1 in systemic endotoxemia [33]. The data on the full protein (hrANXA1) or ATLa (ligand of FPRL1 receptor), which did not affect the expression of this gene, suggest that the full- length protein (hrANXA1) and the N-terminal peptide (AN- XA1Ac2–26) act via different mechanisms and thus support a preferential binding of ANXA1 to FPRL1 receptor [13]. We have recently published data on the differential effects of the full-length protein and its peptide in the firm adhesion of hu- man PMNs on the endothelium under flow [20] and the results demonstrated here reinforce the dichotomy in the biological activity of the acetylated peptide versus the entire molecule. ssion after stimulation with ANXA1, ANXA1Ac2–26, fMLP and ATLa lls treated with recombinant hANXA1 and ANXA1Ac2–26. (A) Total n blot. (B) FACS analysis: histogram (intensity of fluorescence versus ts which all showed similar results. F.C. Rodrigues-Lisoni et al. / FEBS Letters 580 (2006) 1431–1438 1435 In contrast to it effects on ITGB1BP1, FARSLA and TIFA, ANXA1 (which is strongly expressed in epithelial cells and skin [34]), had a positive effect on the expression of the Fig. 5. CCR10 expression in Anxa 1 null mice. Left: RT-PCR analysis and r Anxa1 null and wild type control mice. Histograms represent a semi-quant relative WT. CCR10 gene (Table 1), a skin homing receptor for chemokine CCL27 [35]. This effect was mimicked by ANXA1Ac2–26, fMLP and ATLa (Fig. 3 A and B), supporting a role for the FPR ight: Western blot analysis of CCR10 in trachea, skin and ileum from itative analysis performed on 6 mice per genotype, * P < 0.005 versus 1436 F.C. Rodrigues-Lisoni et al. / FEBS Letters 580 (2006) 1431–1438 family in mediating the positive effect of ANXA1. Support for a role of ANXA1 in CCR10 gene regulation was provided by the Western blot and FACS analysis of the protein expression and membrane localisation (Fig. 4A and B) indicating the po- tential for a ligand binding activity. 3.2. Analysis of CCR10 expression in vivo Our studies on Anxa1-null mice and wild type controls re- veal a marked reduction in CCR10 expression in the Anxa1- null mice versus control (Fig. 5) and show that this is reversed by treatment with hrANXA1 or ANXA1Ac2–26 (Fig. 6). How- Fig. 6. Exogenous recombinant ANXA1 augments the expression of CCR1 analysis Right panel: Western blot of CCR10 in trachea and ileum of wild typ or ANXA1Ac2–26 (10 ng/mouse, and 14 ng/ mouse, respectively, iv, 24 h befor experiments performed on three animals per group. ever Anxa1 null mice did not show any phenotype that could readily be linked to downregulation of CCR10 and further studies are necessary to explore the significance of these find- ings. However, our own data (John et al. manuscript in prep- aration) and a recent report showing the LPS susceptibility of the Anxa1 null mice [36] open new avenues on the possible sig- nificance of changes in CCR10 expression. As ANXA1 is commonly viewed as an anti-inflammatory protein and CCL27 is pro-inflammatory, it might have been predicted that ANXA1 would downregulate CCR10 expres- sion. However recent studies have reported that CCR10 0 in trachea and ileum from Anxa 1 null mice. Left panel: RT-PCR e (WT) and Anxa1 null mice treated with human recombinant ANXA1 e animals were killed). The data are representative of two independent F.C. Rodrigues-Lisoni et al. / FEBS Letters 580 (2006) 1431–1438 1437 induction on pathogen-specific IgA-secreting B cells may be an important factor for successful protection following local mucosal infection [37]. 4. Conclusions Taken together, the data provide new evidence that ANXA1 modulates gene expression. The mechanisms responsible re- quire further investigation. While our data support a role for the FPR family in mediating some of the effects, the failure of fLMP and ATLa to suppress ITGB1BP1 suggest other mechanisms are involved. Furthermore, the dissociation between the activities of hrANXA1 and ANXA1Ac2–26 support the premise that the actions of ANXA1, but not ANXA1Ac2–26, are akin to those of the FPRL1 ligand LXA4. Acknowledgements: We are grateful to the Wellcome Trust (Grant No. 069234/B/02/2) CAPES, CNPq and Fapesp for their financial support. We thank: Prof Charlie Serhan (Center for Experimental Therapeutics & Reperfusion Injury, Department of Anesthesiology, Perioperative & Pain Medicine. Brighan and Women’s Hospital, Harvard Medical School, Boston USA) for providing the 15-epi-16-(para-fluoro)- phenoxy-ATLA-methyl ester. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2006.01.072. 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(2005) Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation. Am. J. Pathol. 166, 1607–1617. [37] Kunkel, E.J., Kim, C.H., Lazarus, N.H., Vierra, M.A., Soler, D., Bowman, E.P. and Butcher, E.C. (2003) CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells. J. Clin. Invest. 111, 1001–1010. In vitro and in blank vivo studies on CCR10 regulation by Annexin A1 Introduction Materials and methods Drugs Cell culture Animals Rapid subtraction hybridization (RaSH) and RT-PCR analysis Semi quantitative RNA analysis Western blot analysis FACS analysis Statistical analysis Results and discussion Annexin A1 modulates gene expression in cell line Analysis of CCR10 expression in blank vivo Conclusions Acknowledgements Supplementary data References