| BackgroundAs a tumor suppressor signal, the TGF-β/SMAD signaling pathway was involvedin abnormal expression in a variety of solid tumors, which also found abnormalexpression in leukemia cells. In the process of abnormal hematopoiesis, theconstituent parts of the TGF-β/SMAD signaling pathway mutate or deactivate and theexpression disorder of downstream target genes which resulted to the TGF-β/SMADsignaling pathways blocked, the proliferation of leukemia cells was abnormal anddifferentiation was blocked.There is significant evidence that proves theTGF-β/SMAD signaling pathway plays a crucial role in leukemia cell differentiationinduced by TGF-β, vitamin D and retinoic acid(ATRA) inducing leukemia cellsdifferentiation.In addition, there are some upstream transcription factors could regulate theTGF-β/SMAD signaling pathway transduction, such as early growth response factor1(Egr-1) and the Wilms’ tumour1(WT1). In addition, according the literature, Egr-1and WT1coding protein structure containing zinc finger domain structure, similar tothat of the two has extensive homology. It occurs frequently that these factors have a corresponding binding site on TGF-β1. They can bind to the DNA binding area so asto regulate the transcription of the target genes.P21is one of the cell cycle dependentkinase inhibitors which located in the downstream of P53and it mainly acts as atumor suppressor. Research shows that: P21plays an important role in tumordifferentiation. In leukemia cells, P21is in lower expression level. This may related tothe abnormal regulation level in the transcription and translation. According to areport in the literature, TGF-β can be mediated the expression of P21. VEGF（vascularendothelial growth factor）plays an important role in promoting tumor angiogenesisand destroys the steady state of the hematopoietic microenvironment, then led to theoccurrence of leukemia and other malignancies. In vitro experiments, VEGF promotesleukemia cells proliferation and inhibits apoptosis. As the targets of the downstreamof TGF-β/SMAD signaling pathway, the expressions of P21and VEGF areinfluenced by the signaling pathway.According to the above background information, the TGF-β/SMAD signalingpathway was considered to be the breakthrough point of research. The purpose ofthese studies was to observe the regulation function of Egr-1and WT1to TGF-β1andthe effect they had on the TGF-β/SMAD signaling pathway in the process ofdifferentiation of leukemia cells into macrophage lineage.ObjectiveTGF-β/SMAD signaling pathway was considered as the breakthrough point ofresearch. To observe the levels of TGF-β1and its’ downstream genes, such as Earlygrowth response gene1(Egr-1) and the Wilms’ tumor1(WT1) in macrophagedifferentiation which was induced by phorbol ester (PMA). As well as the regulationfunction of Egr-1and WT1to TGF-β1and the transduction of TGF-β/SMAD signaling pathways.Then further discussion the influence of them to TGF-β/SMADsignaling pathway.MethodsWe established macrophage differentiation models of K562, HL-60and THP-1cells treated with PMA respectively. The cell proliferation of K562cells which treadby different concentration at different times was detected by CCK-8assay and theexpression of CD11b and CD14in K562, HL-60and THP-1cells induced by PMAwere detected by flow cytometry assay. Cell morphology changes were obtained byWright-Giemsa staining. In addition, Egr-1, WT1, TGF-β1and the factors, such asSMAD3, SMAD4and the downstream factors P21and VEGF were detected byRT-PCR. Egr-1, WT1and TGF-β1were also detected by Western blot analysis. Andthe chromatin immunoprecipitation (ChIP) assay was used to reveal the roles of Egr-1and WT1binding their common target gene TGF-β1.ResultsOur previous study revealed that PMA can inhibit the proliferation of leukemiacell lines and promote cell differentiation into macrophages in vitro.The medianeffective concentration (IC50) of PMA treated for72hours was about100nmol/L. Itwas shown that the three leukemia cell lines appear to similar changes which wereenlarged in size and cytoplasm, nuclear/cytoplasmic ratios reduced, cell surface drapeincreased. It was also noted that cytoplasm staining decreased and the number ofmature cells was increased. The expression of CD11b and CD14in K562, HL-60andTHP-1cells were increased, either the mRNA and protein expression of TGF-β1,SMAD3, SMAD4were gradually increased in response to treatment with PMA ascompared with that of control while the level of WT1was decreased. The expressionof Egr-1was quickly increased, then appeared a downward trend and maintained a period of time. PMA treatment suppressed the binding of WT1on TGF-β1promoterwhile promoting the binding of Egr-1to TGF-β1promoter which was indicated byChIP assay.ConclusionsIn conclusion, PMA plays an important role in inhibition of proliferation andinduction of committed macrophage differentiation of leukemia cells. Egr-1cancompetitively inhibit WT1bind on TGF-β1promoter so as to promote TGF-β/SMADsignaling pathway transduction during the process of the differentiation. |
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