Effects And Mechanisms Of β-catenin On Monocyte-macrophage Differentiation

V Leukemia, a kind of malignant tumor occurring in hematopoietic system with abnormal proliferation, blocked differentiation and apoptosis in HSCs and blood cells is one of the most common malignant cancera and is severely harm to human health and also is the primary causes of child and adolescent cancer morbidity and mortality which has attracted worldwide attention. Although a growing number of researchers working on the molecular mechanisms of leukemia, the pathogenic mechanism is still not entirely clear.Studys has shown that activation of the canonical Wnt/b-catenin signaling pathway induced leukemia and is related to poor prognosis. The signaling is composed of Wnt, FZD family of membrane receptors, cytosolic Dsh, β-catenin, APC, Axin, GSK3β and mediate the adhesion of cells to each other.This pathway significantly plays an important role in embryonic development and regulates self-renewal, proliferation and differentiation of adult mammalian tissue stem and progenitor cells. Tight regulation of canonical Wnt/b-catenin signaling is critical for normal hematopoiesis and its deregulation contributes to leukemia development. β-catenin, a central molecule in the canonical Wnt/β-catenin signaling pathway, acts as a nuclear transcriptional coactivator with lymphoid enhancer factor/T cell factor(LEF/TCF) family to stimulate transcription of various target genes. In addition, β-catenin overexpression has been observed in myeloid cell lines, as well as primary AML blasts and expression of β-catenin is associated with poor survival. But the role of β-catenin overexpression in leukemogenesis is unclear.APC is a negative regulator of Wnt/b-catenin signaling pathway and Suppression of APC can activate Wnt/b-catenin signaling pathway. Studys shown that marked decrease of Mac-1+Gr-1+ myeloid cells and rapid depletion of granulocytemacrophage progenitors(GMPs) but not megakaryocyte-erythrocyte progenitors(MEPs) were observed in both Apc-null and transgenic mice with expression of stabilized β-catenin. However, whether decrease of myeloid cells is due to a blockage of differentiation of granulocyte-macrophage progenitors or the depletion of granulocyte-macrophage progenitors in vivo or both has not been defined.So in this study, we mainly study on the effect of activation of the Wnt/ β-catenin signaling pathway on monocyte-macrophage differentiation of PUER cells, mouse HSCs and human monocytic cell line, and elucidate the potential molecular mechanisms underlying the effect of β-catenin overexpression on monocytemacrophage differentiation. Our studies provide new insights into the pathogenic role of β-catenin in the development of myeloid leukemia. It mainly including three parts in this study.Part Ⅰ Effects of β-catenin overexpression on monocyte-macrophage differentiation of PUER cells and mouse HSCsThe PUER cells represent an experimentally manipulable cellular model system for analyzing molecular mechanisms that underlie macrophage differentiation in vitro. The PUER cells were originally derived from mouse PU.1-/- myeloid progenitors with expression of a conditionally active PU.1 protein fused to the ligand binding domain of the estrogen receptor(PUER). Addition of 4-hydroxy-tamoxifen(4-OHT) leads to activation of PU.1 and differentiation of PUER cells. In this study, we first expressed stabilized b-catenin mutant or APC sh RNA in PUER cells, The expression of b-catenin and APC was confirmed by Western Blot. After induction of differentiation by 4-OHT, cellular morphology and differentiation rate of PUER cells were analyzed by Flow cytometric analysis and May-Grünwald giemsa staining. The results suggest that stabilized b-catenin or knockdown of the Apc gene blocks PU.1-induced macrophage differentiation of PUER cells. To determine whether stabilized b-catenin affects the macrophage differentiation of primary bone marrow progenitor cells, we infected Lin-primary bone marrow cells, isolated from C57Bl6 mice, with retrovirus expressing control vector or b-catenin. After treatment of GM-CSF, known to promote macrophage differentiation for 24 hours the bone marrow cells expressing control vector differentiate into macrophages. The result show that stabilized b-catenin blocks GM-CSF-driven macrophage differentiation of mouse primary bone marrow cells.Part Ⅱ Efffects of β-catenin overexpression on monocyte-macrophage differentiation of human U937 cellsIn this part, we determined whether β-catenin overexpression inhibits differentiation of human U973 cells exposed to PMA. U937 cells were infected with control or β-catenin vectors and cultured for 48 h in the presence of vehicle or 50 ng·m L-1 PMA. The expression of β-catenin was confirmed by Western blot. After induction of differentiation by PMA, cellular morphology and differentiation rate of U937 cells were analyzed by Flow cytometric analysis, RT-PCR and May-Grünwald giemsa staining. As determined by flow cytometric analysis, the percentage of CD14+ and CD11b+ cells in vector-expressed U937 cells increased significantly after PMA treatment, indicating PMA induced monocyte/macrophage differentiation of U937 cells. Notably, the percentage of CD14+ and CD11b+cells in β-catenin-expressed U937 cells was significantly lower than it was in vector-expressed U937. The morphological features associated with mature monocytes were observed in U937 cells expressing control vector but not β-cat-S33 Y after PMA treatment for 48 hours. Furthermore, stabilized β-catenin inhibits m RNA expression of CD14, Mac-1 and CSF1 R genes induced by PMA in U937 cells, as determined by q RT-PCR. We next determined the consequence of APC knockdown on monocyte/macrophage differentiation of U937 cells. knockdown APC by APC-sh RNA 1 or 2 led to stabilization of the endogenous β-catenin in U937 cells. U937 cells expressing APC sh RNA 1 or 2 had a low percentage of CD11b+ or CD14+ cells compared with U937 cells expressing control vector. Together, these results suggest that either APC knockdown or expression of stabilized β-catenin inhibits PMA-induced monocyte/macrophage differentiation of U937 cells.Part Ⅲ Mechanism of β-catenin on monocyte-macrophage differentiationTo understand the mechanism by which b-catenin inhibits PU.1-mediated monocyte differentiation, we performed microarray analysis of gene expression profiling of PUER cells with expression of stabilized β-catenin or control vector before and after 24 h induction of PU.1 expression. Using Cluster 3.0 analysis and R program, we found that 505 genes were markedly up-regulated while 390 genes were significantly down-regulated by PU.1 gene expression. Induction of PU.1 expression in PUER cells with expression of stabilized β-catenin for 24 hours resulted in 505 genes up-regulated by PU.1 with 219 genes significantly inhibited by stabilized β-catenin. However, of 390 genes down-regulated by PU.1, 72 genes were up-regulated by stabilized β-catenin, indicating that β-catenin significantly impairs PU.1-targeted gene transcription. Egr2 is a transcriptional master regulator of monocyte/macrophage differentiation. To determine whether stabilized β-catenin inhibited Egr2 expression in PUER and U937 cells during monocyte/macrophage differentiation, we expressed β-catenin in these two cells and EGR2 expression is determined by q RT-PCR before and after induction of monocyte/macrophage differentiation. We found that β-catenin markedly suppressed EGR2 expression during U937 cell or PUER cell differentiation. To determine whether regulation of Egr2 plays a critical role in mediating the inhibitory function of β-catenin in monocyte differentiation, we co-expressed Egr2 and β-catenin in PUER and U937 cells. The results showed forced-expression of Egr2 reversed β-catenin-induced inhibition of monocyte/macrophage differentiation of both PUER and U937 cells. There are several predicted TCF4 binding sites identified on the promoter region of Erg2. So next, we performed Luciferase reporter assay and chromatin immunoprecipitation(Ch IP) assay followed by PCR. The results showed that Luciferase reporter showed that Egr2 promoter activity was repressed significantly with an increased amount of plasmids expressing stabilized β-catenin and Ch IP assay followed by PCR results revealed that both TCF4 and β-catenin binds directly to the predicted TCF4 binding sites in Egr2 promoter.Stabilized β-catenin inhibits Egr2 expression in both PUER and U937 cells while forced expression of Egr2 released the blockage of monocyte-macrophage differentiation induced by stabilized β-catenin, suggesting an important role of Egr2 in mediating the negative effect of β-catenin overexpression in monocyte-macrophage differentiation. This study provides a new molecular mechanism on the development of leukemia,which can be used to study on a new target for the diagnosis and treatment of leukemia, as well as for the study of the pathogenesis of leukemia caused by xenobiotics.