PML/RARα plays a role for basal activity and retinoid-induced repression of the tissue factor promoter in acute promyelocytic leukemia cells

Abstract

Constitutive expression of tissue factor (TF) by acute promyelocytic leukemia (APL) cells may contribute to thrombotic complications. In this study we examined the transcriptional mechanisms of all-trans retinoic acid (ATRA)-induced down-regulation of TF in the APL cell line NB4, by analysis of stable clones expressing the luciferase gene under the control of 5' flanking regions of the TF gene. We show that the TF promoter is constitutively active in NB4 cells, and that ATRA induces rapid suppression of the promoter. Basal activity and ATRA-induced suppression of TF promoter is determined by the proximal -383 to +121 bp of the promoter. Electrophoretic mobility shift assays demonstrate the binding of Fos/Jun complexes to two TF promoter AP-1 sites in this region. Both complexes were suppressed by ATRA treatment. The ectopic expression of the APL-specific PML/RARalpha oncoprotein in U-937 cells results in induction of TF mRNA and promoter activity. Interestingly, this PML/RARalpha-mediated increase in TF promoter activity is sensitive to ATRA treatment. These data indicate that TF expression in APL cells is exacerbated by the presence of the PML/RARalpha fusion protein, and implicates the loss of Fos/Jun binding to the TF promoter in ATRA-induced suppression of TF.

PML/RARα fusion protein transactivates the tissue factor promoter through a GAGC-containing element without direct DNA association

“…In luciferase assays PML/RARα induced a moderate but reproducible TF promoter transactivation in U937-PR9 cells indicating that, in line with the previous report (22), the extent of transactivation does not necessarily reflect the functional importance of the regulation. Moreover, the magnitude of the promoter transactivation in this work corroborates the results in a published study, in which U937-PR9 cells stably transfected with native promoter constructs that usually lead to moderate levels of transactivation (23) also were used (17). The conventional ChIP protocols cannot localize the target sequences precisely and are not applicable to the recombinant mutational analysis.…”

“…To establish the stable cell lines, transfected U937-PR9 cells were selected with hygromycin B (Calbiochem). Cells were subcloned and selected by increased luciferase activity in response to lipopolysaccharide (LPS) (Sigma) (17). In this study, data reported for each stable cell line represent at least three clones.…”

“…Previous work (17) has established that the TF promoter is constitutively active in the promyelocytic NB4 cells and undergoes a down-regulation with ATRA treatment dependent on its proximal −383 to +121 bp. Thus it is possible that PML/RARα might regulate the expression of TF through an interaction with the TF promoter in a region proximal to −383.…”

“…The monoblastic cell line U937 constitutively expresses TF, and this expression is markedly increased when the cells are transfected with PML/ RARα fusion gene (17), suggesting that the fusion protein is involved in the regulation of excessive TF expression in U937 cells. U937-PR9 is a subclone of U937 cells stably transfected with the PML/RARα cDNA under the control of a zinc-inducible promoter and thus expresses PML/RARα only in the presence of zinc Author contributions: S.C., Z.C., and X.X.…”