The Molecular Mechanism Of Erythroid Differentiation Affected By T-2 Toxin On K562 Cells

As one of the primary members of type-A trichothecenes, T-2 toxin is produced mainly by Fusarium genus. T-2 toxin is the greatest toxic for animals among trichothecenes, and it could contaminate cereals such as wheat, barley, oats, and maize. Toxic effect of T-2 toxin is characterized by vomiting, anemia, leukopenia, hematopoietic system atrophy and immunosuppression reaction, inhibiting protein synthesis, and even causing cancer. Importantly, T-2 toxin could also inhibit the synthesis of protein, DNA and RNA through conjugating with ribosomes as well as the damage of mitochondria.One of the main toxic target of T-2 toxin is hematopoietic system, and it can cause lesions in animal bone marrow and spleens, depletion hematopoietic stem cells (CFUs), granulo-monocytic progenitors (CFU-GM), colony-forming unit erythroid (CFU-E), erythroblastic progenitors (BFU-E) in hematopoietic system, and decrease the counts of red blood cells and platelet. However, the research of hematopoietic system damage induced by T-2 toxin remains undiscovered, and the molecular mechanism and signal transduction of T-2 toxin inhibiting the differentiation of hematopoietic stem cell is not clear.K562 cells, a human erythroleukemia cell line was used as the research modes in our previous research. It was found that T-2 toxin inhibits erythroid differentiation of K562 cells.981 differentially expressed genes and 22 differentially expressed proteins were identified by microarray and two-dimensional electrophoresis technology (2-DE). On the basis of the result of previous study in our laboratory, eight differentially expressed genes (PI3K, MS4A3, EPAS1, ZNF382, SQSTM1, INSIG1, KDM4D and GPSM2) and four differentially expressed proteins (HSP27, PRDX3, PSMC2 and ACTR1A) were found that they might be involved in erythroid differentiation on K562 cells. The mechanism was investigated by using real-time PCR, western bolt, RNA interference and flow cytometry technology. The study of signaling pathways will provide further information for the potential toxicity mechanism of T-2 toxin on hematopoiesis as well as the toxicity mechanisms of blood system induced by trichothecenes.Flow cytometry was used to detect the numbers of erythroid differentiated cells of K562 cells, which could specificly bind to CD235a-FITC antibody. By comparing the CD235a expression in the different treatment groups, it was found that erythroid differentiation of K562 cells was inhibited by T-2 toxin through the JAK2-STAT3, p38, ERK and JNK signaling pathway after 24 h, while JAK2-STAT3, p38, JNK and NF-κB signaling pathways were found to be activated after 48 h.To detect the time-effect relationship between T-2 toxin and genes (or proteins), K562 cells were incubated with 15 nM T-2 toxin for 2 h,6 h,12 h,24 h and 48 h, respectively. Cells were incubated with T-2 toxin and inhibitors of JAK2, STAT3, p38, ERK and NF-κB signaling pathways associated with erythroid differentiation on the time point which showed the most significant changes on gene expressions. The gene expression levels of HSP27, PSMC2, SQSTM1 and ZNF382 were found to be positively regulated by JAK2 signaling pathway, whereas the gene expression of ACTR1A was negatively regulated. Gene expressions of PI3K, ACTR1 A, INSIG1 and PRDX3 were inhibited by STAT3 signaling pathway. Low level of HSP27 expression and high level of INSIG1 and PRDX3 expressions were detected in K562 cells incubated by inhibitor of p38 pathway. The gene expression levels of SQSTM1 and ZNF382 were found to be positively regulated and the gene expression of ACTR1A was negatively regulated by JNK signaling pathway. SQSTM1, EPAS1 and ZNF382 were activated by T-2 toxin through ERK signaling pathway. Gene expression levels of KDM4D, SQSTM1 and ZNF382 were significantly upregulated induced by T-2 toxin through NF-κB signaling pathway, but the expression level of ACTR1A was suppressed.It was found that HSP27 was phosphorylated in a p38-dependent manner in the erythroid differentiation of K562 cells. This research firstly discovered that MS4A3 plays a positive role in the erythroid differentiation of K562 cells used RNA interference technology. It was also found that MS4A3 can regulate the expression levels of HSP27, GATA-1, ZNF382 and SQSTM1. More specifically, we demonstrate that MS4A3 plays a pivotal role in the erythroid differentiation through negative regulation of HSP27 and GATA-1, indicating T-2 toxin can inhibit erythroid differentiation of K562 cells through this process.In summary, the differentially expressed genes and proteins were screened and they were found to be involved in erythroid differentiation on the basis of the result of previous study in our laboratory. It was found that NF-κB and JAK2-STAT3 signaling pathway regulate the erythroid differentiation of K562 cells, and JAK2-STAT3, p38, ERK, JNK and NF-κB signaling pathways negatively regulates the erythroid differentiation of K562 cells induced by T-2 toxin. More importantly, it was for the first time discovered the key role of MS4A3 in erythroid differentiation. This study illustrated the molecular mechanism of erythroid differentiation inhibited by T-2 toxin on K562 cells, and provided some crucial information for the toxicity mechanism of hematopoietic system induced by trichothecene toxin.