Activation Of Wnt5a/Ca²⁺ Signaling Pathway By Exogenous Wnt5a In K562 Cells And The Biologic Effects

ObjectiveWnt5a, a family member of the Wnt proteins, plays important roles in body development, cell adhesion, motility, proliferation, differentiation, polarity and death, even was proposed to be close relative to tumorigenesis. Nowadays, studies indicates Wnt5a, which can activate the Wnt/β-catenin signaling pathway or noncanonical Wnt5a/Ca²⁺ signaling pathway, is overexpressed in some tumors and also suppressed in other ones. So, Wnt5a maybe is both oncogene and antioncogene. We found rare studies on Wnt5a's expression and it's function in hematopoietic disease, and haven't found report about what effect exogenous Wnt5a would contribute to leukemic cells. Therefor, we detected the expression of Wnt5a andβ-catenin in hematopoietic disease samples and leukemic cell lines, and choiced K562 cell lines as research model to observe whether exogenous Wnt5a could activate noncanonical Wnt5a/Ca²⁺ signaling pathway and it's biologic effects. Through these studies, we try to uncover the function and mechanism of Wnt5a in leukemiagenesis and to seek new target for leukemia therapy.Methods1. The expression of Wnt5a in 31 hematopoietic disease samples and 3 kinds of leukemic cell lines, and that ofβ-catenin in 11 myeloid leukemic samples and 3 kinds of leukemic cell lines, were detected by RT-PCR.2. Amplification of AdWnt5a and AdGFP (both labelled with GFP tag gene) were prepared by infecting HEK293 cells respectively. The Wnt5a and GFP condition medium were separately prepared by collecting the culture supernatants medium of CHO cells infected with AdWnt5a and AdGFP. The expression of Wnt5a protein in condition medium were identified by western blot.3. To observe whether exogenous Wnt5a could activate noncanonical Wnt5a/Ca²⁺ signaling pathway, K562 cells were previously stained with fluorescence dyestuff Fluo-3/AM, then the influence on Ca²⁺ inflow in K562 cells treated with Wnt5a and GFP condition medium were checked with laser confocal scanning microscope.4. To observe the relationship between exogenous Wnt5a and Wnt/β-catenin signaling pathway, K562 cells were separately treated with Wnt5a and GFP condition medium for 1 to 5 days, then the expression ofβ-catenin and cyclin D1 in K562 cells were detected by RT-PCR, immunochemistical staining and western blot respectively.5. Separately treated with Wnt5a and GFP condition medium for 1 to 5 days after, the biologic effects of exogenous Wnt5a on K562 cells were observed as follow: counting cells number each day and figuring out proliferation curve to observe the influence of exogenous Wnt5a on cell proliferation; detecting the cell cycle distribution of cells by flow cytometry to observe the influence of exogenous Wnt5a on cell cycle; observing the influence of exogenous Wnt5a on cell apoptosis by DNA Ladder electrophoresis and with laser confocal scanning microscope after AO/EB staining; observing cellular morphologic changes with light microscope after Wright's staining and detecting the expression changes of CD13, CD41, CD68 and GlyA by immunochemistical staining to observe the influence of exogenous Wnt5a on cell differentiation.Results1. Wnt5a expression was lost in myeloid leukemic samples and K562, HL-60 cells, butβ-catenin were overexpressed in most myeloid leukemic samples, K562 cells and Jurkat cells.2. The Wnt5a and GFP condition medium were successfully prepared with AdWnt5a, AdGFP to infect CHO cells separately. Identified by western blot, Wnt5a protein was found expressed in Wnt5a condition medium but not in GFP condition medium.3. GFP condition medium couldn't stimulate K562 cells Ca²⁺ inflow but Wnt5a condition medium could, and the Ca²⁺ inflow could be blocked with Wnt5a antibody.4. Wnt5a condition medium did not down-regulate the expression ofβ-catenin mRNA, but suppressed the expression ofβ-catenin and cyclin D1 protein in K562 cells.5. The biologic effects of Wnt5a condition medium on K562 cells: cell proliferation was significantly inhibit; the cells number of G1 phase increased and that of S phase and G2 phase decreased; mature morphologic differentiational features was detected and the positive rate of CD68 significantly increased, but that of CD13, CD41 and GlyA had no obvious changes; no obvious trapeziform band was detected in DNA Ladder electrophoresis and apoptosis rate didn't obviously rise observed with laser confocal scanning microscope.Conclusions1. The loss of Wnt5a expression and overexpression ofβ-catenin may be is relative to leukemiagenesis.2. Exogenous Wnt5a can activate the noncanonical Wnt5a/Ca²⁺ signaling pathway and downregulated the expression ofβ-catenin and cyclin D1 proteins in K562 cells. It indicate that exogenous Wnt5a can inhibit Wnt/β-catenin signaling pathway in K562 cells.3. Exogenous Wnt5a can induce K562 cells arise G1 phase arrest, inhibit K562 cells proliferation, and induce K562 cells differentiate to monocyte, but can't induce K562 cells apoptosis.