Mechanisim Of Cross-Talk Between Wnt/β-catenin Signaling Pathway And Core Fucosylation In HEK293t Cell

Fucosyltransferase 8(FUT8), which is also called core α1,6-fucosyltransferase,transfers fucose from GDP-L-fucose to the innermost asparagine-linked Glc NAc of N-glycan. Unlike other members in FUT family, FUT8 draws an extraordinary importance due to no functional redundancy. FUT8 is wildly involved in many physiological and pathological progresses, including cell signaling pathway, cell proliferation, invasion and migration, cell-cell adhesion, and immune-reaction. There has been reported that the expression and activity of FUT8 are abnormal in many diseases, including hepatocellular and colon carcinoma, lung cancer, prostrate cancer,gastric cancer, ovarian cancer, and also renal fibrosis. Besides, FUT8 also plays a key role in neurite formation and aging progress.Wnt signaling pathway controls many biological progresses including cell proliferation, differentiation and adult stem cell self-maintenance. In colorectal cancer,hepatocellular carcinoma and hair follicle tumor, mutations in Wnt signaling pathway are often observed. By now the canonical Wnt/β-catenin cascade is best studied among the three cascades that have been known.Even though FUT8 and Wnt/β-catenin signaling pathway are both up-regulated in many diseases such as liver cancer and colon cancer, the molecular mechanism of possible synergism between these two is still unclear. We use HEK293 T cell line, whichhas a normal level FUT8 expression and an intact Wnt pathway, to study the cross talk of these two elements by overexpression certain protein, to provide a possible explanation of this phenomenon.Methods:1) Western-blot and HPLC are used to exam the level of FUT8 protein expression and catalytic activity after Wnt/β-catenin signaling pathway is activated by transfectingβ-catenin and Wnt3 a overexpression plasmid in HEK293 T cell.2) Western-blot, luciferase assay, cell immunofluorescence assay and clone formation assay are used to exam the status of Wnt/β-catenin signaling pathway and cell proliferation ability after FUT8 overexpression.3) Protein synthesis inhibitor Cycloheximide, and proteasome inhibitor MG132 are used to exam the stability and ubiquitylation of β-catenin after FUT8 overexpression.Immunoprecipitation, Western-blot and Lectin-blot are used to exam the effect of core fucosylation on β-catenin.Result:1) In HEK293 T cell line, when Wnt/β-catenin signaling pathway is activated,FUT8 is up-regulated both in protein expression and catalytic activity.2) Western-blot results show that critical proteins in Wnt/β-catenin signaling pathway like wnt3 a, Dvl2, β-catenin, active β-catenin and also its target proteins are all up-regulated significantly after overexpression FUT8 in HEK293 T cell line. Besides,luciferase assay, cell immunofluorescence assay and clone formation assay show that overexpression FUT8 can active Wnt/β-catenin signaling pathway and stimulate cell proliferation.3) Western-blot results show that FUT8 also promotes the stability of β-catenin,inhibits the ubiqutylation in whole cell level. Immunoprecipitation and Lectin-blot indicate that β-catenin or some other proteins in Wnt/β-catenin signaling pathway is fucosylated.Conclusion:FUT8 is a target candidate of Wnt/β-catenin signaling pathway in HEK293 T cellline, whose both protein expression and catalytic activity are up-reguated when the pathway is activated. On the other hand, FUT8 can activate Wnt/β-catenin signaling pathway by stabilize β-catenin, inhibit ubiquitylation and finally stimulate cell proliferation. The possible molecular mechanism is that β-catenin or some other protein in Wnt/β-catenin signaling pathway is fucosylated, thus its function is changed and Wnt/β-catenin signaling pathway is activated.