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Abstract
Human thrombopoietin (TPO) is the primary regulator of megakaryopoiesis. It is known that TPO levels in blood are regulated by a mechanism involving its uptake by platelet TPO receptors (c-MPL) and its subsequent intraplatelet destruction. However, this mechanism does not fully explain TPO regulation and there is evidence of an additional regulatory mechanism such as a negative feedback transcriptional mechanism at the level of the bone marrow stroma. This thesis focuses on further investigation of this transcriptional regulation. The aims of the investigation are to: i) characterize the potential repressor(s) of thrombopoietin (TPO) gene transcription; and ii) define the platelet factor 4 (PF4)-mediated pathway that causes suppression of TPO gene expression.
A potential TPO gene repressor, ZEB1, was firstly studied. Promoter analysis showed that there was a small region that greatly reduced TPO promoter activity and this region carries a cluster of four conserved ZEB1 binding motifs. However further analysis revealed that repression of TPO promoter activity did not depend on this ZEB1 cluster, suggesting that ZEB1 is unlikely to be the repressor. This promoter region also contains binding motifs for MZF1. Electrophoretic mobility shift assay (EMSA) showed the binding of MZF1 to the putative binding motif in this region. siRNA knockdown of MZF1 expression reversed the reduced TPO promoter activity. A significant increase of TPO expression was also observed in MZF1 siRNA transfected OP9 cells (surrogate for bone marrow stromal cells). The signaling pathway that mediates PF4-induced repression of TPO expression was then investigated. I found that PF4 is the key factor in the serum that suppresses TPO expression. This suppression is dose-dependent and further experiments revealed that this suppression was through the interaction with LRP1 expressed on OP9 cells. PF4/LRP1 interaction triggered PI3K/Akt signaling pathway with phosphorylation of Akt. I suggest that PI3K/Akt activation possibly then results in MZF1 activation which then likely migrates into the nucleus and mediates repression of TPO transcription.
In conclusion, the current study identifies a novel intronic transcription repressor (MZF1) of human TPO expression. TPO repression is mediated by PF4 which binds to LRP1, and activates PI3K/Akt signal transduction pathway.