Abstract
The Notch1 pathway plays important roles in modulating erythroid and megakaryocyte differentiation. To screen the Notch1-related genes that regulate differentiation fate of K562 and HEL cells, the expression of transient receptor potential ankyrin 1 (TRPA1) was induced by Notch1 receptor intracellular domain (N1IC), the activated form of Notch1 receptor. N1IC and v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1) bound to TRPA1 promoter region to regulate transcription in K562 cells. Transactivation of TRPA1 promoter by N1IC depended on the methylation status of TRPA1 promoter. N1IC and Ets-1 suppressed the DNA methyltransferase 3B (DNMT3B) level in K562 cells. Inhibition of TRPA1 expression after Notch1 knockdown could be attenuated by nanaomycin A, an inhibitor of DNMT3B, in K562 and HEL cells. Functionally, hemin-induced erythroid differentiation could be suppressed by TRPA1, and the reduction of erythroid differentiation of both cells by N1IC and Ets-1 occurred via TRPA1. However, PMA-induced megakaryocyte differentiation could be enhanced by TRPA1, and the surface markers of megakaryocytes could be elevated by nanaomycin A. Megakaryocyte differentiation could be reduced by Notch1 or Ets-1 knockdown and relieved by TRPA1 overexpression. The results suggest that Notch1 and TRPA1 might be critical modulators that control the fate of erythroid and megakaryocyte differentiation.
Highlights
The Notch pathway regulates several biological functions, including proliferation, differentiation, apoptosis, and tumorigenesis[1,2]; exerts complex and multi-faceted functions; and plays either oncogenic or tumor-suppressive roles in tumorigenesis[3,4]
TRPA1, one of the differentially expressed genes, showed elevated transcript (Fig. 1A, left) and protein (Fig. 1A, right) levels in K562/HA-Notch1 receptor intracellular domain (N1IC) cells compared with K562/pcDNA3 control cells
The results showed that N1IC overexpression decreased the mRNA levels of DNMT3B but not DNMT1 or DNMT3A in N1IC-expressing K562/HA-N1IC cells compared with K562/pcDNA3 control cells
Summary
The Notch pathway regulates several biological functions, including proliferation, differentiation, apoptosis, and tumorigenesis[1,2]; exerts complex and multi-faceted functions; and plays either oncogenic or tumor-suppressive roles in tumorigenesis[3,4]. Mounting lines of evidence have suggested that activation of the Notch[1] pathway modulates erythroid[7,8,9] and megakaryocyte[8] differentiation. The intracellular domain subsequently translocates into the nucleus to modulate target gene expression via mechanisms both dependent and independent of C promoter binding factor-1 (CBF1)/ recombination signal binding protein-Jκ (RBP-Jκ)[1,3,4]. Previous reports have demonstrated that TRPA1 expression was restricted to sensory neurons[10]. Deletion of glycoprotein 130 (the subunit of interleukin-6 receptor) down-regulates TRPA1 expression in small sensory neurons[24]. Tumor necrosis factor-α and interleukin-1α induce TRPA1 levels in human fibroblast-like synoviocytes[25]. The involvement of Notch[1] pathway-mediated TRPA1 expression in erythroid and megakaryocyte differentiation was investigated in this work
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