| Objective:Wnt5a, one member of the Wnt family, has been found to be correlated with tumorigenesis, but the specific relations and the mechanism are still not clear, especially concerning with leukemia. Our previous studies reported that Wnt5a was doesn't expressed or down-regulated in myeloid and lymphoid leukemia, and myeloid leukemia cell lines. However, Wnt5a was found to be expressed in leukemias cases of complete remission. Wnt5a gene promoter region methylation in K562, HL-60 and U937 cell lines, also in multiple myeloma, myeloid and lymphoid leukemia cases, but was not found in normal and patients with complete remission. In addition, we have demonstrated that exogenous Wnt5a inhibits the proliferation of K562 cells and induces differentiation. We hypothesis that Wnt5a may be associated with leukemia. The K562 cells that stably overexpress Wnt5a protein were constructed successfully, and our current study shows that Wnt5a activate noncanonical or canonical Wnt signaling pathways in K562 cells through specific coupling of different receptor. Thus, we think that Wnt5a may play different role in the progression of leukemia.This study established the foundation for wnt5a as a tumor marker and a gene therapy target for leukemia.Methods:1. Wnt5a regulation Ror2, Frz4, LRP5 receptor expression.1.1 Western blotting, immunofluorescence, immunocytochemistry were used for detecting Wnt5a regulating Ror2, Frz4, LRP5 receptor expression and localization in K562 cells.1.2 K562?293?HepG2?L02 cells were analyzed for the localization of Ror2 by immunofluorescence. 1.3 Wnt5a induction Ror2 receptor internalization was observed by Immune electron microscopy in K562 cells.2. Wnt5a activates noncanonical or canonical Wnt signaling pathways through specific coupling of related coreceptors.2.1 Wnt5a mediated Ror2/Frz4 co-receptor to activate noncanonical pathway and inhibi classical Wnt signaling pathway.2.1.1 Western blotting, immunoprecipitation were used to observe that Wnt5a carrying out diverse roles via Ror2/Frz4 co-receptor.2.1.2 Western blotting, immunofluorescence were used for analyzing the expression and distribution of?-catenin in total cells, nuclei and cytoplasm of K562 cells.2.1.3 Western blotting were used for observing the expression of?-catenin phosphorylation and cyclinD1, and?-catenin transcriptional activity was used for detecting by dual-luciferase assay.2.2 Wnt5a activates classical Wnt signaling pathway through Frz4/LRP5 co-receptor.2.2.1 Ror2 receptor leads to blocking the Wnt5a/Ror2/Frz4 nonclassical pathway through specific coupling of Ror2 antibody in K562 cells.2.2.2 To make it clear whether the Wnt5a/Ror2/Frz4 signal blocked was evaluated by Western blotting, immunoprecipitation.2.2.3 We assessed whether Wnt5a carrying out its diverse roles via Frz4/LRP5 co-receptor was detected by Western blotting, immunoprecipitation.2.2.4 Western blotting was used for detecting changes of?-catenin, p-?-catenin and cyclinD1 expression.2.2.5 MTT cell proliferation assay, flow cytometry were used for observing the proliferation and growth cycle of K562 cells.Results:1.Ror2 and Frz4 were found strongly expresseing more strongly in K562-Wnt5a than in control cells, but no change of the expression of LRP5 occurred. Furthermore, Ror2 localized in foci of the nuclear invagination area in K562-Wnt5a. Frz4, LRP5 localized in the cell membrane and cytoplasm.2. Immune electron microscopy showed that immunoreactive particulate matter occurred on the plasmalemma of K562-Wnt5a cells, which was internalized into the cytoplasm, and localized in the nuclear invagination cytoplasmic area.3. Compared with the control cells, Wnt5a and Ror2 co-expressed, and enhanced binding of each other in K562-Wnt5a cells. Wnt5a also enhanced binding to Frz4. Wnt5a played its roles via Ror2/Frz4 co-receptor.4. Wnt5a has no effect on total and cytoplasmic?-catenin expression, but can decrease expression of?-catenin in the nucleus.5. Phosphotyrosine of?-catenin was detected in K562-Wnt5a cells, and TOPflash reporter assay demonstrated significant down-regulation of?-catenin transcriptional activity and leaded to downregulation cyclinD1.6. Ror2 receptor blocked the Wnt5a/Ror2/Frz4 nonclassical pathway through specific coupling of Ror2 antibody. Subsequently, Wnt5a induced increased expression of Frz4, and enhanced interaction between Frz4 and LRP5 proteins. We think that Wnt5a play a role through Frz4/LRP5 co-receptors when Ror2 expression was absenced or reduced.7. Wnt5a induced Increased expression of?-catenin and cyclin D1, and P-?-catenin was not detected in K562-Wnt5a cells. We think that Wnt5a carry out its roles by classical Wnt signaling pathway via Frz4/LRP5 co-receptor.8. MTT and flow cytometry assay results indicate that compared with control, overexpression Wnt5a promoted the proliferation of K562 cells.Conclusion:1. Overexpression Wnt5a resulted in increasing expression of Ror2 and Frz4. Wnt5a activates nonclassical signaling and inhibit the classical Wnt pathway by Frz4 synergistic through Ror2 internalization. Tyrosine phosphorylation of?-catenin was detected in Wnt5a-K562 cells. Results show that?-catenin transcriptional activity was negatively regulated by Wnt5a through decreasing the localization and expression of?-catenin in the nucleus, and leaded to decreasing cyclinD1 expression levels.2. Wnt5a activated canonical signaling pathways through Ror2/Frz4 co-receptor when Ror2 expression was absence or reduced. We think that Frz4 is required for Wnt5a coupling Frz4 to LRP5 or Ror2 co-receptors, to induce noncanonical or canonical Wnt signaling pathways. 3. Wnt5a proteins can lead to different outcomes in tumorigenesis. Wnt5a carry out diverse roles by different signaling pathways via specific to coupling different receptor in different cells. which is also the mechanism for Wnt5a in leukemia genesis. |
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