Coronary vessel development requires activation of the TrkB neurotrophin receptor by the Wilms' tumor transcription factor Wt1

Abstract

The formation of intramyocardial blood vessels is critical for normal heart development and tissue repair after infarction. We report here expression of the Wilms' tumor gene-1, Wt1, in coronary vessels, which could contribute to the defective cardiac vascularization in Wt1 −/− mice. Furthermore, the high-affinity neurotrophin receptor TrkB, which is expressed in the epicardium and subepicardial blood vessels, was nearly absent from Wt1-deficient hearts. Activation of Wt1 in an inducible cell line significantly enhanced TrkB expression. The promoter of NTRK2, the gene encoding TrkB, was stimulated ∼10-fold by transient cotransfection of a Wt1 expression construct. The critical DNA-binding site for activation of the NTRK2 promoter by Wt1 was delineated by DNase I footprint analysis and electrophoretic mobility shift assay. Transgenic experiments revealed that the identified Wt1 consensus motif in the NTRK2 promoter was necessary to direct expression of a reporter gene to the epicardium and the developing vasculature of embryonic mouse hearts. Finally, mice with a disrupted Ntrk2 gene lacked a significant proportion of their intramyocardial blood vessels. These findings demonstrate that transcriptional activation of the TrkB neurotrophin receptor gene by the Wilms' tumor suppressor Wt1 is a crucial mechanism for normal vascularization of the developing heart.

The Wilms' tumour suppressor WT1 is involved in endothelial cell proliferation and migration: expression in tumour vessels in vivo

“…This extends a recent study, which described WT1 expression in WT1 activates ETS-1 in endothelial cells N Wagner et al angiogenic tumours of the skin (Timar et al, 2005). The partial co-localization of WT1 with proliferation markers in tumour vessels and the diminished proliferation, migration and tube formation of endothelial cells in vitro upon silencing of WT1 is in agreement with our recent observations that WT1 is required for coronary vessel formation during embryonic development (Wagner et al, 2005) and it is expressed in microvessels after myocardial infarction, but not in vessels of adult control hearts (Wagner et al, 2002). These data suggest that WT1 is involved actively in proliferation of endothelial cells and vessel formation rather than being only a novel endothelial cell marker.…”

“…The Wilms' tumour suppressor WT1 is expressed in vessels of the most common tumours Prompted by a recent report on WT1 expression in angiogenic tumours of the skin (Timar et al, 2005) and our observation on the role of WT1 for cardiac vessel development (Wagner et al, 2005), we reasoned that WT1 might play a general role in tumour vessel formation. We show here that vascular WT1 expression was detectable in 95% of 113 tumour samples investigated (Table 1, Figure 1).…”

“…As reported earlier, WT1 showed some additional expression in vascular smooth muscle cells (Wagner et al, 2002). To investigate whether WT1 might be able to directly transcriptionally activate ETS-1, we cloned the published mouse ETS-1 promoter (Jorcyk et al, 1997) into a luciferase reporter vector and performed transient co-transfection experiments with expression plasmids encoding the WT1(ÀKTS) or WT1( þ KTS) splice variants as described by Wagner et al (2005). HEK293 cells were chosen, because low levels of WT1 are transcribed in these cells, suggesting that the transacting factors required for normal WT1 expression are operating in this cell line.…”

“…Alternatively, 48 h after transfection, cells were fixed and immunohistochemical detection of PCNA was performed as described (Wagner et al, 2005) with counterstaining of nuclei using DAPI (Vector Laboratories). PCNA-positive cells in five random optical fields from three independent experiments each were counted at  400 magnification.…”

“…Apoptotic cells were detected by TUNEL staining 48 h after WT1 silencing using the in situ Cell Death Detection Kit (Roche Diagnostics) as described by Wagner et al (2005). Alternatively, HUVECs were incubated with fluorescein isothiocyanate (FITC)-conjugated annexin V (Roche Molecular Biochemicals) 48 h after transfection according to the manufacturer's instructions and counterstained with propidiumiodide to distinguish necrotic from apoptotic cell death.…”