Expression of Tissue Factor Is up-Regulated in U937/PR9 Cells by the PML/RARα Fusion Protein through Its Interaction with the Regulatory Region of Tissue Factor Promoter.

Life-threatening bleeding remains a challenging complication of acute promyelocytic leukemia (APL), which is characterized by the t(15;17) translocation producing a fusion gene encoding the PML/RARα fusion protein, and is mainly ascribable to the increased levels of tissue factor (TF) in APL cells. Previous studies have shown that the TF expression levels are elevated in NB4 (APL) cells or in U937 (monoblastic) cells ectopically expressing PML/RARα indicating that the TF expression in these cells is regulated in response to PML/RARα. The molecular mechanisms by which PML/RARα regulates the TF expression are, however, still poorly understood. To examine whether the PML/RARα fusion protein regulates the TF expression through an interaction with TF promoter, a series of cell lines stably expressing luciferase reporter gene under control of different 5’ flanking regions of TF promoter were established with U937/PR9 cells, a U937 cell line expresses Zn-inducible PML/RARα. In luciferase assays a Zn-induced transcriptional activity was present in cells with truncated TF promoters (-2106~+128), (-1386~+128), (-684~+128), (-384~+128) and (-247~+128) but absent in those with a shorter version of TF promoter (-201~+128). These results suggested that the sequences within -247 and -201 of TF promoter were necessary for the regulation of TF gene expression regulated by PML/RARα in U937/PR9 cells. In ChIP experiments a markedly increased amount of PCR products in response to the expression of PML/RARα were amplified by the primers flanking the regulatory region (-247~-201) from the chromatins immunoprecipitated with an anti-RARα antibody, suggesting that the PML/RARα fusion protein interacts with TF promoter concomitant with the elevated luciferase activity. The EMSA data using probes containing distal or proximal AP-1 consensus sequences located in the regulatory region (-247~-201) showed that PML/RARα did not directly bind to this region but enhanced the binding ability of c-Jun and Jun D to the proximal AP-1 site. Collectively, our data showed that the PML/RARα fusion protein up-regulates the expression of TF through the interaction with the regulatory region of TF promoter. The more accurate localization of the regulatory region in TF promoter and the related molecular mechanisms regarding the regulatory effects of the PML/RARα fusion protein on TF expression are under investigation.