After 15 min, total cell lysates (TCL) were subjected to immunoblotting with the indicated antibodies to determine the phosphorylation of p38 (top). of metastatic lung nodules (bottom). The columns symbolize the imply ( s.d.) quantity of lung metastatic nodules (= 3). * 0.05, ** 0.01. Shed syndecan-2 extracellular domain name contributes MK-8998 to syndecan-2-associated malignancy activity regulation To directly assess the role of syndecan-2 extracellular domain name shedding, we investigated whether shed syndecan-2 itself promotes colon cancer cell activities. The level of shed syndecan-2 was increased in HT29 cells transfected with a vector expressing Flag-tagged WT-SDC2, compared with vector-transfected cells, but not in cells expressing Flag-tagged NC-SDC2 (Physique ?(Physique2A,2A, top). Cell migration was markedly increased in HT29 and HCT116 cells treated with WT-SDC2-expressing HT29 cell conditioned media (Physique ?(Physique2A,2A, bottom). Consistently, treatment with WT-SDC2-expressing HT29 cell conditioned media enhanced the migration of HCT116 cells, but depletion of shed syndecan-2 in the conditioned media abolished increased cell migration of HCT116 cells (Physique ?(Figure2B).2B). In addition, purified shed syndecan-2 from HT29 MK-8998 cell conditioned media (Physique ?(Physique2C,2C, left) directly enhanced the migration (Physique ?(Physique2C,2C, right) and the real-time cell migration rates of both HT29 and HCT116 cells (Physique ?(Figure2D).2D). Treatment with purified His-tagged syndecan-2 extracellular domain name showed significant effects, on migration and anchorage-independent growth of colon cancer cells, without affecting cell proliferation MK-8998 (Supplementary Physique S3). Consistently, transfection with an Fc receptor-shed syndecan-2 chimera (sS2E-Fc) enhanced cell migration of HCT116 cells, sS2E-Fc proteins were detected in the conditioned media, and sS2E-Fc treatment enhanced cell migration and colony forming activities of HCT116 cells (Supplementary Physique S4). These data suggest that shed syndecan-2 extracellular domain name contributes to syndecan-2-associated malignancy activity regulation. Open in a separate window Physique 2 Shedding of syndecan-2 plays a critical role in colon cancer cell migration(A) HT29 cells were transfected with indicated cDNAs, and syndecan-2 mRNA expression was evaluated by RT-PCR. Conditioned media were subjected to slot blotting with the anti-Flag antibody (top). HT29 and HCT116 cells were treated with HT29 conditioned media (final 10% v/v) from VEC, WT-or NC-syndecan-2 mutant transfected cells and allowed to migrate on Transwell apparatus (bottom). = 5; * 0.05, ** 0.01. (B) Conditioned media were immunodepleted with control IgG- or anti-syndecan-2 antibody-conjugated protein G beads. The supernatants were subjected to slot blotting with anti-syndecan-2 antibody (top). Mixture of HCT116 cells with each supernatant (final 10% v/v) were added to the upper chambers of CIM-plates and migration curves were monitored using the xCELLigence system. The rates of cell migration over 24 hr were analyzed using the RTCA software to each RTCA CIM-Plate wells (bottom). (C) Shed syndecan-2 in HT29 cell conditioned media was isolated by DEAE-Sepharose column chromatography. Final elution fractions were digested by heparinase and analyzed by immunoblotting Tgfbr2 using anti-syndecan-2 antibody (left). Cells were treated with 0.2 g/ml of purified shed syndecan-2, and MK-8998 Transwell migration assay was performed (right). = 5; *= 0.05, ** 0.01. (D) Cells were MK-8998 treated with 0.2 g/ml of purified shed syndecan-2, and a real-time migration assay was analyzed by xCELLigence system. = 5; *= 0.05, ** 0.01. Shed syndecan-2 synthetic peptide is sufficient for potentiating main tumor growth and metastasis We next constructed a series of recombinant deletion mutants of shed syndecan-2, a C-terminal deletion mutant, N2E-Fc, and an N-terminal deletion mutant, C2E-Fc of shed syndecan-2, expressed each in HEK293T cells, and collected the conditioned media. Treatment of HCT116 cells with the conditioned media containing C2E-Fc caused a remarkable increase in migration and anchorage-independent growth of HCT116 cells (Supplementary Physique S5ACS5C). When we further constructed a C2E-Fc deletion mutant, N-terminus residues 89C104 (L89TSAAPEVETMTLKTQ104, C2EQ104-Fc), the conditioned media from your C2EQ104-Fc-expressing cells enhanced migration of HCT116 cells (Supplementary Physique S5D), suggesting that this tumorigenic activity of shed syndecan-2 resides in the C-terminus of the extracellular domain name. Expectedly, treatment of HCT116 cells with synthetic peptides corresponding to human sequence (hS2LQ) caused a remarkable increase in cell migration compared with the control peptide (hS2EA), without affecting cell proliferation (Physique ?(Figure3A).3A). In addition, hS2LQ-treated.