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• W2022389224 abstract "Loss of Wnt-5a protein expression is associated with shorter recurrence-free survival in breast carcinoma patients and increased motility in mammary cell lines. Based on sequence analysis of Wnt-5a, we identified 14 peptide fragments and investigated their ability to mimic the effects of Wnt-5a on mammary cell adhesion and migration. Two of these peptides significantly increased adhesion and impaired migration in the non-tumorigenic HB2 breast epithelial cell line and in the MDA-MB-468 breast cancer cell line, both of which show little endogenous expression of the Wnt-5a protein. We removed two amino acids at a time from the N terminus of the shorter of these two peptides to identify the shortest peptide that still inhibited migration. The influence on tumor cell adhesion was gradually lost and was no longer detectable when only six amino acids remained. However, formylation of the N-terminal methionine of this hexapeptide restored its effect on adhesion and reduced tumor cell motility via a Frizzled-5 receptor-dependent mechanism, even at a low pH such as encountered in breast tumor tissue. This formylated hexapeptide ligand induced a rapid cytosolic calcium signal, whereas it did not affect the cellular levels of unphosphorylated β-catenin or active JNK. The novel formyl-Met-Asp-Gly-Cys-Glu-Leu peptide ligand is not only a valuable experimental tool but has also a potential role in antimetastatic treatment of the 50% of human breast cancer patients that have reduced endogenous Wnt-5a protein expression. Loss of Wnt-5a protein expression is associated with shorter recurrence-free survival in breast carcinoma patients and increased motility in mammary cell lines. Based on sequence analysis of Wnt-5a, we identified 14 peptide fragments and investigated their ability to mimic the effects of Wnt-5a on mammary cell adhesion and migration. Two of these peptides significantly increased adhesion and impaired migration in the non-tumorigenic HB2 breast epithelial cell line and in the MDA-MB-468 breast cancer cell line, both of which show little endogenous expression of the Wnt-5a protein. We removed two amino acids at a time from the N terminus of the shorter of these two peptides to identify the shortest peptide that still inhibited migration. The influence on tumor cell adhesion was gradually lost and was no longer detectable when only six amino acids remained. However, formylation of the N-terminal methionine of this hexapeptide restored its effect on adhesion and reduced tumor cell motility via a Frizzled-5 receptor-dependent mechanism, even at a low pH such as encountered in breast tumor tissue. This formylated hexapeptide ligand induced a rapid cytosolic calcium signal, whereas it did not affect the cellular levels of unphosphorylated β-catenin or active JNK. The novel formyl-Met-Asp-Gly-Cys-Glu-Leu peptide ligand is not only a valuable experimental tool but has also a potential role in antimetastatic treatment of the 50% of human breast cancer patients that have reduced endogenous Wnt-5a protein expression. The Wnts are a family of secreted glycoproteins that have molecular masses of 39–46 kDa and participate in development and tumorigenesis via autocrine or paracrine routes (for reviews see Refs. 1Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1727) Google Scholar, 2Veeman M.T. Axelrod J.D. Moon R.T. Dev. Cell. 2003; 5: 367-377Abstract Full Text Full Text PDF PubMed Scopus (1144) Google Scholar, 3Nelson W.J. Nusse R. Science. 2004; 303: 1483-1487Crossref PubMed Scopus (2208) Google Scholar). Secreted Wnt proteins bind to and activate G-protein-coupled receptors of the Frizzled (Frz) 2The abbreviations used are: Frz, Frizzled; CM, Wnt-5a-conditioned medium; DDR1, discoidin domain receptor 1; F-actin, filamentous actin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; Ab, antibody; JNK, c-Jun N-terminal kinase. 2The abbreviations used are: Frz, Frizzled; CM, Wnt-5a-conditioned medium; DDR1, discoidin domain receptor 1; F-actin, filamentous actin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; Ab, antibody; JNK, c-Jun N-terminal kinase. family (4Yang-Snyder J. Miller J.R. Brown J.D. Lai C.J. Moon R.T. Curr. Biol. 1996; 6: 1302-1306Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 5Wang Y. Macke J.P. Abella B.S. Andreasson K. Worley P. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. J. Biol. Chem. 1996; 271: 4468-4476Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar), and it is presumed that the low density lipoprotein (LDL) receptor-related proteins LRP5 and LRP6 act as coreceptors in that context (6Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. Nature. 2000; 407: 530-535Crossref PubMed Scopus (1078) Google Scholar, 7Mao J. Wang J. Liu B. Pan W. Farr 3rd, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). Based on differences in the ability to transform mouse mammary epithelial cells (8Shimizu H. Julius M.A. Giarre M. Zheng Z. Brown A.M. Kitajewski J. Cell Growth Differ. 1997; 8: 1349-1358PubMed Google Scholar), the Wnt proteins can in the present context be divided into the following three distinct classes: Wnt-1, Wnt-3a, and Wnt-7a have the greatest transforming capacity; Wnt-2, Wnt-5b, and Wnt-7b have an intermediate transforming capacity; and Wnt-4, Wnt-5a, and Wnt-6 are non-transforming.We have previously reported that low level expression of Wnt-5a protein in primary invasive breast carcinomas is associated with higher histological grade (poor differentiation) and shortened recurrence-free survival because of more rapid development of distant metastases (9Jönsson M. Dejmek J. Bendahl P.O. Andersson T. Cancer Res. 2002; 62: 409-416PubMed Google Scholar). This association cannot be caused by an effect of the protein on proliferation, because no correlation has been found between loss of Wnt-5a protein expression and presence of the proliferative marker Ki67 (10Dejmek J. Leandersson K. Manjer J. Bjartell A. Emdin S.O. Vogel W.F. Landberg G. Andersson T. Clin. Cancer Res. 2005; 11: 520-528PubMed Google Scholar). Currently, the only available explanation for the ability of Wnt-5a to reduce the metastatic capacity of invasive breast cancer is that this protein enhances adhesion and thus reduces the migration of these tumor cells. However, it is possible that as yet undefined mechanisms also contribute to the mentioned clinical effect of Wnt-5a on tumor progression. Nevertheless, it has been shown that Wnt-5a-dependent activation of the adhesion receptor discoidin domain receptor 1 (DDR1) could be responsible for the decrease in the motility and the invasive potential of breast epithelial cells (11Jönsson M. Andersson T. J. Cell Sci. 2001; 114: 2043-2053PubMed Google Scholar). Breast epithelial cells are normally firmly adherent, but reduced expression of Wnt-5a leads to decreased adhesion and thereby increases motility. In accordance with that, we have previously observed that introduction of a Wnt-5a expression vector into a MCF-7 breast cancer cell line, which exhibits little endogenous Wnt-5a expression, was required for collagen-induced activation of the adhesion receptor DDR1 (11Jönsson M. Andersson T. J. Cell Sci. 2001; 114: 2043-2053PubMed Google Scholar). In good agreement, non-tumor mammary epithelial HB2 cells transfected to express increased and decreased Wnt-5a protein levels, respectively, exhibited in the former case increased and the latter decreased adhesion to collagen I (11Jönsson M. Andersson T. J. Cell Sci. 2001; 114: 2043-2053PubMed Google Scholar). It has not yet been established which of the different Frz receptors mediates the effects of Wnt-5a in human mammary cells. However, experiments using a Frz-5-specific blocking Ab have shown that this particular receptor is responsible for the effects induced by Wnt-5a in both fibroblast-like synoviocytes and in malignant melanoma cells (12Sen M. Chamorro M. Reifert J. Corr M. Carson D.A. Arthritis Rheum. 2001; 44: 772-781Crossref PubMed Scopus (158) Google Scholar, 13Weeraratna A.T. Jiang Y. Hostetter G. Rosenblatt K. Duray P. Bittner M. Trent J.M. Cancer Cell. 2002; 1: 279-288Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar).To develop a treatment that is based on the functional role of Wnt-5a and will target breast cancer metastasis, it is important to understand the cellular and molecular events that underlie the impact of this protein on such epithelial tumors. One of the critical and initial events in metastasis is when the transformed cells detach from each other and also from the ECM components of the basement membrane (14DiMilla P.A. Barbee K. Lauffenburger D.A. Biophys. J. 1991; 60: 15-37Abstract Full Text PDF PubMed Scopus (470) Google Scholar). In the presence of Wnt-5a, the adhesion receptor DDR1 in breast epithelial cells can be activated by collagen, hence this receptor is a likely candidate for mediating the Wnt-5a-induced increase in anchoring of these cells to collagen (11Jönsson M. Andersson T. J. Cell Sci. 2001; 114: 2043-2053PubMed Google Scholar). It is plausible that the elevated metastatic activity seen in breast carcinomas that lack Wnt-5a is caused by reduced adhesion and a subsequent increase in the migratory capacity of these tumor cells. Therefore, one approach to reduce the metastatic activity in Wnt-5a-deficient breast carcinomas might be to find a way to overcome the translational defect responsible for the loss of Wnt-5a protein expression (10Dejmek J. Leandersson K. Manjer J. Bjartell A. Emdin S.O. Vogel W.F. Landberg G. Andersson T. Clin. Cancer Res. 2005; 11: 520-528PubMed Google Scholar) and another to reconstitute the Wnt-5a-induced signaling and activation of the cells. Experiments have shown that adding recombinant Wnt-5a to differentiated thyroid carcinomas induces tumor suppressor activity (15Kremenevskaja N. von Wasielewski R. Rao A.S. Schofl C. Andersson T. Brabant G. Oncogene. 2005; 24: 2144-2154Crossref PubMed Scopus (235) Google Scholar). However, the fact that Wnt-5a is a relatively large protein (43 kDa) makes it attractive to search for a small molecule that can mimic the signaling and functional effects of Wnt-5a.Based on sequence analysis of Wnt-5a, we identified and investigated 14 peptide fragments regarding their ability to mimic the effects of the native protein on mammary cell adhesion and locomotion. Our goal was to find a substance that can serve as a possible model in the development of novel antimetastatic treatment strategies for breast carcinomas that exhibit reduced endogenous expression of Wnt-5a.EXPERIMENTAL PROCEDURESCell Culture—We used the HB2 mammary epithelial cell line, which is a subclone of the MTSV-1.7 line developed in the laboratory of Dr. J. Taylor-Papadimitriou (16Bartek J. Bartkova J. Kyprianou N. Lalani E.N. Staskova Z. Shearer M. Chang S. Taylor-Papadimitriou J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3520-3524Crossref PubMed Scopus (142) Google Scholar). HB2 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 5 units/ml penicillin, 0.5 units/ml streptomycin, 2 mm glutamine, 10 μg/ml bovine insulin, and 5 μg/ml hydrocortisone. The human mammary carcinoma cell lines MCF-7 and MDA-MB-468 were grown in DMEM supplemented with 10% fetal bovine serum, 5 units/ml penicillin, 0.5 units/ml streptomycin, and 2 mm glutamine. The pH experiments were done in MEM supplemented with 10 mm Tris, 0.5% BSA, 5 units/ml penicillin, 0.5 units/ml streptomycin, and 2 mm glutamine. The pH 7.4 medium, but not the pH 6.7 medium, was further supplemented with 4 mm sodium bicarbonate. All cells were incubated at 37 °C in 95% air and 5% CO2 except for the fura-2-loaded cells from which we recorded the fluorescence response to Wnt-3a and a formylated hexapeptide (Fig. 9C).Chemicals and Reagents—The following Abs and chemicals were used (sources within parentheses): anti-actin monoclonal Ab C4 (MP Biomedicals, Irvine, CA); anti-β-catenin dephospho amino acids 35–50 monoclonal Ab 7A7 (Alexis Biochemicals, Lausen, Switzerland); anti-β-catenin monoclonal Ab and collagen type I from rat tail (BD Biosciences, San José, CA); anti-β1-integrin monoclonal Ab P5D2 (Chemicon, Temecula, CA); anti-phospho SAPK/JNK Thr183/Tyr185 monoclonal Ab and anti-SAPK/JNK Ab (Cell Signaling Technology, Beverly, MA); anti-phosphotyrosine monoclonal Ab 4G10 (Upstate Biotech. Inc. Lake Placid, NY); recombinant mouse Wnt-3a and recombinant mouse Wnt-5a (R&D Systems, Abingdon, UK); SDS-PAGE reagents (Bio-Rad); fura-2/AM (Molecular Probes, Eugene, OR).Sequence Analysis—Amino acid sequences of Wnt-5a from different species were investigated interactively and using multiple sequence alignment, secondary/solvent accessible surface predictions according to the PHD method (17Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar) to identify potentially solvent-exposed loop segments.Peptide Synthesis—Different peptide fragments derived from the Wnt-5a molecule that were used in the experiments are outlined in Table 1. Most of them were synthesized by Eurogentec (Seraing, Belgium), although the shorter peptides and the formylated peptide derived from the 12-amino acid long pept175 were from Pepscan systems (Lelystad, Netherlands). The synthesized peptides were quality controlled by RP-HPLC and mass spectrometry. To reach different purity levels, peptides were purified by preparative RP-HPLC. The crude (60–70% pure) peptides were synthesized at two different occasions whereas the pure (>95% pure) peptides were synthesized three times. Regardless of the batch used, we obtained similar results with peptides derived from different syntheses. All other laboratory reagents were of analytical grade and obtained from Sigma Aldrich.TABLE 1The peptide sequences and their corresponding positions in the human Wnt-5a proteinPeptide (pept)Amino acid sequencePosition in the Wnt-5a sequence (amino acids)No. of amino acids167SRAARPKDLPRDWLW150–16415168DARERERIHAKGSYE183–19715169ADFRKVGDALKEKYD242–25615170aThe three cysteines in the native Wnt-5a sequence were replaced with alanines (A) in the peptideVKAKKATEIVDQFVA350–36415171SQLAGLSQGQKKL55–6713172GDNIDYGYRFAKE168–18013173RGYDQFKTVQTER327–33913174bExtended sequence based on pept175LGTQGRLCNKTSEGMDGCEL303–32220175NKTSEGMDGCEL311–32212176YQDHMQYIGE71–8010177QYQFRHRRWN90–9910178RVMQIGSRET111–12010179HNNEAGRR206–2138180NSRGKLVQ264–2718a The three cysteines in the native Wnt-5a sequence were replaced with alanines (A) in the peptideb Extended sequence based on pept175 Open table in a new tab Receptor-blocking Antibodies—The peptide CPILKESHPLYNKVRTGQVPN, corresponding to amino acids 198–217 in the ectodomain of the human Frz-5 receptor was used as previously described to generate a Frz-5 receptor-blocking Ab (12Sen M. Chamorro M. Reifert J. Corr M. Carson D.A. Arthritis Rheum. 2001; 44: 772-781Crossref PubMed Scopus (158) Google Scholar), and the peptide CPRVLKVPSYLSYKFLGERD, corresponding to amino acids 202–220 in the ectodomain of the human Frz-2 receptor, was used to produce a control anti-Frz-2 Ab. (In the peptide designations, letters in italics indicate the Frz-5 and Frz-2 sequences, whereas the initial non-italic cysteine (C) in each peptide was added for subsequent purifications of the Abs.) The productions of rabbit anti-Frz-5 and anti-Frz-2 antisera were managed by AgriSera (Vännäs, Sweden; permit A69–04 from Umeå Animal Ethics Committee). The antisera were affinity-purified in our laboratory by coupling the respective peptide antigen to Sulfolink-Sepharose over which the relevant antiserum was eluted.Immunoprecipitation and Western Blotting—Western blot analysis was performed to evaluate expression of the proteins Wnt-5a, Frz-2, Frz-5, dephospho β-catenin, total β-catenin, actin, phospho-JNK, and total JNK in HB2 and/or MDA-MB-468 cells. The batch of MDA-MB-468 cells we used evidently expressed only a low level of Wnt-5a. The Wnt-5a Ab was produced in our laboratory as previously described (9Jönsson M. Dejmek J. Bendahl P.O. Andersson T. Cancer Res. 2002; 62: 409-416PubMed Google Scholar, 11Jönsson M. Andersson T. J. Cell Sci. 2001; 114: 2043-2053PubMed Google Scholar). The cells were lysed and boiled in 2× concentrated Laemmli buffer. The protein content in each sample was determined and adjusted to ensure loading of equal amounts of protein in each lane. Thereafter, 50 mm dithiothreitol were added, and the lysates were boiled for 10 min and then separated by SDS gel electrophoresis. The proteins were subsequently transferred to polyvinylidene difluoride membranes, which were blocked in 3% BSA or 5% nonfat milk for 45 min, incubated for 1 h with the primary Ab in 1% BSA or 1% nonfat milk (diluted 1:1,000 for phospho-JNK, total JNK, dephospho β-catenin, total β-catenin, and actin; 1:2,000 for Wnt-5a; 1:5,000 for phosphotyrosine, Frz-2 and Frz-5), and then for 1 h with a horseradish peroxidase-conjugated secondary Ab. Ab-antigen complexes were detected using an enhanced chemilu-minescence kit from Amersham Biosciences. Before reprobing, the membranes were stripped with a Reblot Strong solution from Chemicon.The phosphorylation status of DDR1 was analyzed as previously described (18Dejmek J. Dib K. Jönsson M. Andersson T. Int. J. Cancer. 2003; 103: 344-351Crossref PubMed Scopus (69) Google Scholar), with the following modifications. The cells were subjected to serum depletion for 16 h and then detached with Versene and allowed to adhere to collagen I for 60 (MDA-MB-468 cells) or 90 (HB2 cells) min at 37 °C. The experiments were terminated by disrupting both adherent and non-adherent cells with a lysis buffer (50 mm Tris, pH 7.4, 1% Triton X-100, 5 mm EDTA, 5 mm EGTA, 50 mm NaCl, 42 μg/ml aprotinin, 2.5 mm benzamidine, 1 mm pefabloc, 10 μg/ml leupeptin, and 2mm Na3VO4). In immunoblotting, an anti-DDR1 rabbit polyclonal Ab, sc-532 (Santa Cruz Biotechnology) was used at a dilution of 1:10,000. The Ab recognizes DDR1b/c, which is essential because collagen only induces phosphorylation of the b and/or c isoforms of DDR1 in mammary epithelial cells (11Jönsson M. Andersson T. J. Cell Sci. 2001; 114: 2043-2053PubMed Google Scholar).Adhesion Assay—The adhesion assay was carried out as previously described (18Dejmek J. Dib K. Jönsson M. Andersson T. Int. J. Cancer. 2003; 103: 344-351Crossref PubMed Scopus (69) Google Scholar). Briefly, cells were serum-depleted for 16 h, detached with Versene, and allowed to adhere individually to collagen I for 60 (MDA-MB-468 cells) or 90 (HB2 cells) min at 37 °C. Non-adherent cells were removed by washing with phosphate-buffered saline. The adherent cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) in a dye reduction assay (Promega), and absorbance was measured at 490 nm using a Fluostar microplate reader from BMG LabTechnologies (GmbH, Offenberg, Germany). A single value was calculated for each experiment, based on the mean of adhesion from six separate wells after subtracting the adhesion to plastic alone. The Wnt-5a-conditioned media (CM) used as a positive control in the adhesion assays was obtained from confluent stably transfected Wnt-5a-overexpressing HB2 cells (for experiments with Wnt-5a antisense HB2 cells) or Wnt-5a-overexpressing MCF-7 tumor cells (for use with MDA-MB-468 tumor cells). The CM was always used within 3 days of collection. The specificity of the CMs was checked by incubating the medium with either the anti-Wnt-5a Ab or a control Ab for 45 min at 37 °C. Subsequently, 10 μl of protein A-Sepharose was added, and 30 min later the protein A-bound antibodies were removed by centrifugation leaving the Wnt-5a-depleted supernatants to be used in experiments.Cell Migration Assay—Cell migration was analyzed in a modified Boyden chamber (Transwell®; Costar, Cambridge, MA) in which the two chambers were separated by a polycarbonate membrane (10-μm thick and with a pore diameter of 8.0 μm). In some experiments (as indicated in figure legends) the membrane was precoated with collagen by adding a collagen I solution (10 μg/ml in 0.25% acetic acid) to the upper chamber for 1 h at 37°C and thereafter washing the membrane three times with phosphate-buffered saline. At the onset of each experiment, the cells were detached by treatment with Versene for 10–20 min at 37 °C, washed, and resuspended as single cells in serum-free DMEM medium supplemented with 0.5% BSA. A 0.2-ml aliquot of such a cell suspension (containing 25,000 MDA-MB-468 or 50,000 HB2 cells) was added to the upper transwell chamber together with the agonist of interest. The lower chamber was filled with 0.6 ml of serum-free, BSA-containing DMEM medium supplemented with the chemoattractant IGF-I (1 ng/ml). As a control, IGF-I was excluded from some of the wells. The cells were allowed to migrate in the Boyden chamber for 18 h at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Discarding the medium, using a cotton-tipped applicator to remove non-migratory cells and cut the membrane out of the chamber, terminated an experiment. The cells that had migrated through the membrane and were located on its lower surface were stained for 20 min with 0.5% crystal violet diluted in 20% methanol, 80% water. The membrane was subsequently washed, and the stained cells were counted (number per membrane) in an inverted microscope. The results for each experiment are presented as the median value for three separate wells.Fluorescence Microscopy—MDA-MB-468 cells were detached from the tissue culture dishes by incubating with Versene for 10 min at 37 °C, washed, resuspended as single cells in serum-free DMEM medium supplemented with 0.5% BSA and 100 μm of the agonist of interest, and plated on a cover glass precoated with 10 μg/ml collagen-I in a cell culture dish. These experiments were terminated after 18 h by fixing the cells with 4% paraformaldehyde for 10 min. The cells were then permeabilized by incubation for 5 min with 0.5% Trition X-100 and blocked by a 45-min incubation in a 3% BSA solution supplemented with 0.3% Triton X-100. The cells were stained in the dark by incubating for 40 min with AlexaFluor® 488 phalloidin (diluted 1:500; Molecular Probes) in the presence of 1% BSA, and then washed extensively and mounted with Dako Cytomation fluorescent mounting medium. The samples were examined and photographed in a Nikon Eclipse 800 microscope using a ×60 oil immersion objective.Determination of Cytosolic Free Calcium Levels—MDA-MB-468 cells grown on cover glasses were incubated with 4 μm fura-2/AM in culture medium for 30 min at 37 °C (19Nielsen C.K. Massoumi R. Sonnerlind M. Sjölander A. Exp. Cell Res. 2005; 302: 31-39Crossref PubMed Scopus (21) Google Scholar). After fura-2 loading of the cells, the cover glasses were washed and mounted in a specially designed chamber to which we added a calcium-containing medium (136 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 1.1 mm CaCl2, 1.2 KH2PO4, 5 mm NaHCO3, 5.5 mm glucose, and 20 mm Hepes, pH 7.4). The chamber was then placed in a system consisting of a Nikon Diaphot microscope connected to a Photon Technology International (PTI) imaging system. The cells were first allowed to rest for 10 min before any stimulation was performed. Fura-2 fluorescence was then recorded from the cells before and after stimulation with either Wnt-3a (100 ng/ml) or the formylated hexapeptide (100 μm) using an excitation wavelength rapidly alternating between 340 and 380 nm, whereas the emission was recorded at 510 nm. The fluorescence intensity ratios (340/380 nm) were subsequently calculated and analyzed using the PTI Image Master Software.RESULTSPeptide Sequences Based on PHD Prediction—We first predicted the most likely exposed amino acid residues by the PHD method (17Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar). The results are shown in Fig. 1 together with the synthesized peptides and corresponding identification number (see also Table 1). Several segments are likely to be solvent-exposed because of the presence of several charged/polar/short residues and most were investigated.FIGURE 1A schematic outline and alignment of the amino acid residues of Wnt-5a from human, mouse, rat, urodele amphibian (plewa), Xenopus laevis (xenla), and Ambystoma mexicanum (ambme). The figure illustrates the mature Wnt-5a protein sequences from the different species. The sequence homologies between human and the other species are shown at the top of the figure. Residues predicted to be solvent-exposed by the PHD method are indicated by gray boxes under the given sequences. Based on these predictions and the presence of cysteine residues (bolded) we decided to investigate possible functional effects of the indicated peptide fragments (black lines above the sequences). The identification numbers used in the present article are given above each indicated segment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Peptide-induced Effects on Adhesion and Migration of Wnt-5a Antisense HB2 Cells—Table 1 lists the initial 14 peptide fragments, designated pept167 through pept180, which were investigated regarding their ability to mimic the effects of the Wnt-5a protein on mammary cell adhesion and motility. All the peptides were derived from the amino acid sequence of Wnt-5a. In a previous study (18Dejmek J. Dib K. Jönsson M. Andersson T. Int. J. Cancer. 2003; 103: 344-351Crossref PubMed Scopus (69) Google Scholar), we had shown that Wnt-5a is required for collagen-induced activation of the adhesion receptor DDR1 in mammary cells and for maximal adhesion of these cells to collagen. Therefore, in the current experiments, we analyzed DDR1 phosphorylation and adhesion as a means of screening for the ability of the peptides to mimic the effects mediated by Wnt-5a in Wnt-5a antisense HB2 cells. We chose these cells because they contained a low level of Wnt-5a, which made it easier to discern Wnt-5a mimicking effects of the peptides. The representative blots and the accumulated results of densitometric analysis shown in Fig. 2 demonstrate that pept168, pept169, pept170, and pept175 (100 μm) caused significant phosphorylation of DDR1. According to HPLC analysis (data not shown), the purity of these four peptides was ∼65% in the initial screens (Figs. 2 and 3). Additionally, we conducted experiments to determine the percentage of cells adhering to collagen I in the presence of pept168, pept169, pept170, or pept175. We also included pept171 as a negative control and incubated the cells in Wnt-5a-CM as a positive control. The results show that adhesion was not affected by pept168, but it was significantly influenced by pept171, which we had presumed to be a negative control peptide (Fig. 3A). Inasmuch as there was no DDR1 phosphorylation in the presence of pept171 (Fig. 2A), we deduced that the mechanism underlying the impact of this peptide on cell adhesion does not require the activity of DDR1. Earlier studies had shown that β1-integrins are not involved in activation of DDR1 (20Vogel W. Brakebusch C. Fassler R. Alves F. Ruggiero F. Pawson T. J. Biol. Chem. 2000; 275: 5779-5784Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and that Wnt-5a-dependent adhesion of mammary cells is insensitive to preincubation with a blocking anti-β1-integrin Ab (18Dejmek J. Dib K. Jönsson M. Andersson T. Int. J. Cancer. 2003; 103: 344-351Crossref PubMed Scopus (69) Google Scholar). Therefore, we pretreated suspended HB2 cells with the previously used monoclonal anti-β1-integrin Ab for 45 min at 37 °C to block the integrins. The results strongly suggested that the increased adhesion caused by the control pept171 was caused by β1-integrin activation (Fig. 3B), which is in contrast to the effects of pept175 (Fig. 3B). In this context, it should be pointed out that, at 65% purity, none of the four peptides in question (pept168, pept169, pept170, and pept175) increased HB2 cell adhesion to the same extent as the CM did (Fig. 3A). To ascertain whether the effects of CM that are illustrated in Fig. 3 were actually mediated by Wnt-5a, we pretreated CM with an anti-Wnt-5a Ab and protein A to deplete the content of Wnt-5a, and then we tested the ability of the medium to induce cell adhesion. The results of these control experiments revealed that the Wnt-5a-depleted CM had no effect on adhesion (data not shown).FIGURE 2Effects of crude Wnt-5a-derived peptides on collagen-induced DDR1 phosphorylation in Wnt-5a antisense HB2 cells. Single cells were allowed to adhere to collagen-I-coated plates in the presence of the indicated crude peptides (100 μm). Results are illustrated for experiments performed in the presence of pept167–173 (A) and pept175–180 (B). After 90 min, the cells were lysed, and DDR1 was immunoprecipitated. The immunoprecipitates were analyzed by Western blotting with an antiphosphotyrosine Ab, after which the membranes were stripped and reprobed with an anti-DDR1 Ab to ascertain equal loading. The diagrams depict densitometric ratios of DDR1 tyrosine phosphorylation normalized against the total amount of DDR1, expressed as percent of the control value obtained in the absence of any peptide. The blots shown are representative of three separate" @default.
• W2022389224 created "2016-06-24" @default.
• W2022389224 creator A5021772465 @default.
• W2022389224 creator A5025935464 @default.
• W2022389224 creator A5033949988 @default.
• W2022389224 creator A5047777714 @default.
• W2022389224 creator A5051589659 @default.
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• W2022389224 date "2006-02-01" @default.
• W2022389224 modified "2023-10-16" @default.
• W2022389224 title "A Formylated Hexapeptide Ligand Mimics the Ability of Wnt-5a to Impair Migration of Human Breast Epithelial Cells" @default.
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