During myeloid cell maturation expression
During myeloid cell maturation, expression of 5-LO mRNA and protein is strongly induced (60–130-fold) by calcitriol (1,25(OH)2D3) and transforming growth factor-β (TGFβ) [6,7]. The upregulation of 5-LO mRNA transcription is mainly due to transcript elongation and mRNA maturation [8,9]. Interestingly, subsequent studies on the mechanism of 5-LO mRNA induction by TGFβ demonstrated the involvement of the 5-LO coding sequence in TGFβ/SMAD signalling and identified regulatory elements in the distal part of 5-LO gene which seems to be relevant for the regulation of 5-LO expression by TGFβ and SMAD3/4 . So far, no induction of 5-LO promoter activity by TGFβ could be detected [8,10]. In Heparin to this, the histone deacetylase inhibitor trichostatin A increases 5-LO promoter activity by recruitment of the transcription factors SP1/3 to the proximal GC-boxes of the 5-LO promoter [11,12]. Thereby, an increased acetylation of histones H3 and H4 at the 5-LO core promoter and an increase in trimethylation of histone 3 lysine 4 (H3K4me3) was found which strongly correlated with 5-LO mRNA induction. The mixed lineage leukemia protein (MLL/KTM2A) which catalyzes the trimethylation of histone 3 at lysine 4 (H3K4me3) is also recruited to the 5-LO promoter. Arbitrary activation of the MLL protein complex by class I HDAC inhibitors induces 5-LO promoter activity [13,14]. As 5-LO gene overexpression has been linked with distinct forms of leukemias, the influence of leukemic MLL fusion proteins on 5-LO promoter activation was recently investigated as well [14,15]. For the MLL gene, a large number of chromosomal rearrangements are described in patients with acute lymphoblastic leukemia. In particular, the chromosomal translocation t(4;11)(q21;q23) with the AF4 gene (AFF1) is the most frequently diagnosed reciprocal chromosomal translocation of the human MLL gene . MLL-AF4 acts as a constitutively active form of wildtype MLL and was shown to strongly stimulate 5-LO promoter activity (47-fold) .
Material and methods
Discussion Previous studies demonstrated the functional importance of calcitriol and TGFβ for the transcriptional regulation of the endogenous 5-LO gene in differentiating MM6 cells [6,9]. However, no induction of 5-LO gene transcription by either TGFβ or calcitriol was ever detected with reporter gene constructs containing only the pN10 promoter region [8,10,17]. Consistent with previous publications the effect of TGFβ and calcitriol was associated with post-transcriptional events, such as enhanced transcript elongation and mRNA maturation [8,9]. When the role of TGFβ and its downstream effectors SMAD3 and SMAD4 on 5-LO regulation were systematically investigated by qPCR analysis, a rapid induction of 5-LO mRNA expression was observed in MM6 cells after treatment with TGFβ, demonstrating that 5-LO is a primary TGFβ target. Several SBEs in the 5-LO coding sequence were identified in silico which are relevant for the regulation of 5-LO gene expression by TGFβ and SMAD3/4 . Here, we studied the role of SMAD3/4 for the regulation of the 5-LO promoter. In EMSA studies, we demonstrate that SMAD3/4 binds to two identified SBEs within the 5-LO promoter. This was validated by performing a ChIP experiment with MM6 cells, demonstrating the in vivo binding of SMAD4 to the endogenous 5-LO promoter. Studies on the regulation of 5-LO promoter activity by TGFβ and SMAD proteins were hampered in the past because the pGL3 plasmid and the luciferase reporter gene contain multiple SMAD binding sites leading to a very high background SMAD activity which prevents the detection of promoter specific SMAD effects and which might explain previous failures to detect TGFβ effects on 5-LO promoter activity. Here, we recloned the pN10 5-LO promoter fragment into a pGL4 vector and used Luc2 to detect reporter activity. Both have obviously no SMAD binding sites. This allowed the sensitive detection of SMAD-dependent 5-LO promoter activation with rather low background activity. Using this approach, we identified two novel SMAD binding sites, SBE-1 and SBE-2, in the proximal part of the 5-LO promoter.