Overexpresssion of Tyrosine Kinase Discoidin Domain Receptor I (DDR1 ) in Transitional Cell Carcinoma

Overexpresssion of Tyrosine Kinase Discoidin domain receptor I (DDR1 ) in Transitional cell Carcinoma Szu-Ting Chen,* and Shie-Liang Hsieh* Institute and Department of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan; To whom proofs are to be sent: Shie-Liang Hsieh, Department and Institute of Microbiology and Immunology, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan. E-mail address: [email protected] Telephone number: 886-2-28267161 Fax number: 886-2-28277933 INTRODUCTION DDR1, discoidin domain receptor 1, belongs to the novel subfamily of tyrosine kinase receptor, which forms homodimer upon ligand engagement. DDR1 is distinguished from other receptor tyrosine kinase by the discoidin domain in their extracellular domain, which is a homology region originally identified in Dictyostelium discoideum (slime mold) protein, discoidin I, and involves in cells aggregation. Discoidin-1 has binding specificity toward galactose and N-acetyl galactosamine and is essential for slime mold cells adhesion, migration and aggregation during its development, suggesting that DDR1 shared similar biologically function in mammalian [1-3]. DDR1 has been found mainly distributed and in human tissue epithelia, such as kidney, breast, lung [3], bronchial [4] and keratinocytes [5]. Furthermore, DDR1 has been also reported its expression in immune system like the monocyte-derived dendritic cells, annotated as CD167 [6] and macrophage[7]. However, recently, the overexpression of DDR1 has been detected in several human cancers, such as primary breast cancer [1, 8, 9] ovarian[10, 11], brain[12], esophageal cancer [13], and TCC (our data unpublished) in which raising the possibility that DDR1 may play a important role in tumorigenesis [14]. Ligand binding to RTKs is though to induce receptors dimerization, and dimerized receptors subsequently autophosphorylated specific tyrosine residues in cytoplasmic domain each other. Collagen type I to IV can bind to discoidin domain of DDR1 and activate this receptor; however, unlike other RTKs, the activation process was so delayed that required ligand activation for 18 hrs to reach maximal tyrosine phosphorylation [3, 15]. Collagen acts as a ligand for DDR1, implying that DDR1 may involved in arterial wound repair and primary vascular smooth muscle cells migration. The downstream genes MMP2 and MMP9, which related to the extracellular matrix degradation and injury repair, were further connected to DDR1 signaling pathway and regulated by activated DDR1 [16, 17]. Recently, there has been hypothesized that DDR1 involved in the regulation of leukocytes in response to inflammation microenvironment. By degrading membrane basement, leukocytes extravasated the vascular endothelia wall toward inflammatory site; and DDR1 expression was induced by proinflammatory cytokine simultaneously. Due to basement destruction and extracellular matrix exposure, the interaction between DDR1 and collagen, which mediated the migration of tissue infiltrating leukocytes[7, 18]. In addition; DDR1 also plays an essential role in coordinating other function of leucocytes, such like facilitating dendritic cells maturation, promoting macrophages differentiation [7, 19], and up-regulation of IL-1 and IL-8 production by human macrophages [20]. As mentioned previously, overexpression of DDR1 has been found in various types of cancer cells. Receptor overexpression mimicked the ligand engagement and resulted in the dimerization and autophosphorylation of these activated receptors. Therefore, basing on the constitutively activated form results from DDR1 overexpression, we are interested to figure out the possibility of which results in tumorogenesis. According to the sequence similarity, there has been predicted that, tyrosine residues at 792, 796 and 797 may participate in autophosphorylation [21]. Owing to the phosphorylated tyrosine located in DDR1 kinas domain (a.a. 610-915), implying 1) these tyrosine residues may involve in receptor activation; and 2) the expression of downstream gene may be regulated through these specific tyrosine residues in response to DDR1 activation. Since the role of tyrosine residue in mediating DDR1 activation has not been illustrated yet, we characterized these specific tyrosine residues by using side-directed mutagenesis, and assayed the behavior of downstream gene in response to DDR1 activation. In the present study, constitutively activated DDR1 promoted IL-8 secretion and cells migration by modifying the tyrosine residues at site of 792, 796, and 797 of DDR1. These three tyrosine residues exhibited different level of contribution to mediating DDR1 activation, particularly the Y796. This is a pilot experiment, in which supported the information of which tyrosine should be chosen to lead the DDR1 constitutive activation. It will be a useful model in studying the mechanism of bladder cancer formation through the DDR1 molecule. EXPERIMENTAL PROCEDURES Reagents, Cells and antibodies —DDR1 cDNA were cloned and PCR amplified by using Pfu Turbo polymerase (Stratagene). The TransFastTM reagent was purchased from Promega Biotechnology, Inc. (U.S.A.) in the utilization of all cell type transient transfection. The human embryonic kidney 293 cells (with or without large T antigen) and mouse fibroblast NIH3T3 were obtained from American Tissue Culture Collection and cultivated under the recommended conditions. Bovine type I collagen and all other reagents were purchased from Sigma. Antibody to DDR1 (amino acids 894-913) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), monoclonal anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology, Inc. and monoclonal anti-MMP2 antibody which recognized pro- and active MMP2 simultaneously was obtained form CHEMICON International Inc. Plasmid construction and site-directed mutagenesis --DDR1 full-length cDNA was cloned from U373MG cells with oligonucleotide primers harboring EcoRI and BamHI restriction enzyme sequence. In this study, we used standard overlapping PCR mutagenesis methods to construct the dominant active DDR1 expression vector. Briefly, DDR1-Y792E mutated fragments were PCR amplified with two primer pairs: (i) 5’-GTGAGG TCGACAGTCCCTCA-3’ sense primer harboring SalI site and the Y792E antisense primer, containing mutated bases shown in bold: 5’-CACACGGTAATAGTCCCCAGCCTCGAGG TTCCGGCTCATGCCAAAG-3’, (ii) the Y792E sense, 5’-CTTTGGCATGAGC CGAACCTCGAGGCTGGGGACTATTACCGTGTG-3’and 5’-TACGTGAGTT GTGCCACACT-3’ primer containing an engineered BamHI site. PCR amplification condition was considered as usual, and these two fragments were overlapped in 15 cycles consisting of 1min at 95℃, 1 min at 55℃ and 1 min at 72℃. Overlapping PCR products were subjected to another amplification with the restriction enzyme harboring sense and antisense primers as previously described. The construction of DDR1 double-‘Y792/796E’ and triple- ‘Y792/796/797E’mutants were generated by replacing the DDR1wild type and DDR1-Y792/796E mutant with the corresponding mutagenized fragments, respectively. Point mutation was introduced with the following oligonucleotides: 5’-CGGAACCTCGAGGCTGGGGAGTACCGTGTGCAGGGCCGGCAG-3’, 5’-CTGCCCGGCCCTGCACACGGTACTCGTCCCCAGCCTCGAGGTTCC- 3’were for Y792/796E and 5’-CCTCGAGGCTGGGGACGAGGACGTG TGCAGGGCCGGGCAGTG-3’, 5’-CACTGCCCGGCCCTGCA1CACGTTCC CGTCCCCACCTCGAGG-3’ were for Y792/796/797E. All cDNA were subcloned to pFlagCMV2 mammalian expression vector. Transfection and western blotting---Semi-confluent 293T cells were independently transfected with wild type and tyrosine mutants of DDR1 full-length expression plasmids by using TransFastTM reagent. Sixteen hours later, cells were transferred to serum-free media for another 20 hrs. Cells were stimulated with 10μg/ml type I collagen for 90min and lysed with 1% Triton X-100, 0.1% SDS, 150mM NaCl, 5mM EDTA, 50mM Tris-HCl (pH7.5), 10mM NaF, 1mM sodium orthovanadate, 3μM H2O2 lysis buffer containing proteinase inhibitor cocktail tablet. Equal amounts of total protein were subjected to immunoprecipitation with anti-DDR1 antibody (C-20) for 4hrs at 4℃ on a rotating wheel. The immunocomplex was washed three times and separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membrane and immuoblotted with anti-phosphotyrosine antibody diluted 1:1000 (4G10). Western blots were developed using HRP-conjugated second antibody and enhanced chemiluminescence (Amersham). For reprobing, the membrane was stripped with strong re-probe kit (Chemicon) and immunoblotted with DDR1 c-20 antibody diluted 1:400. DDR1 transient expression in 293 cell and IL8 secretion assay---Semi-confluent 293 cells (3×105) were seeded in 6-well plate and independently transfected with wild type and tyrosine mutants of DDR1 expression plasmids. Sixteen hours later, the wild type transfectants were treated with or without 50μg/ml type I bovine collagen. Harvesting the supernatants at 24, 36 and 48 hr after transfection procedure. IL8 concentration was determined by using human IL8 ELISA set (BD OptEIATM,BD Bioscience). Cell migration assay---Mouse fibroblasts NIH3T3 were independently transfected with wild type and tyrosine mutants of DDR1 expression plasmids. Cells (5×104) were suspended in DMEM plus 1% fetal bovine serum and placed onto the upper inserts of transwell (8-μm pores, o.33cm2, Costar), previously coated with 0.01% gelatin (Sigma). DMEM containing 10% fetal bovine serum in the lower compartment was used as chemoattractant. In some experiments, cells were transferred directly on the uncoated inserts

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