Abstract
Notch is a critical mediator of endothelial-to-mesenchymal transition (EndMT) during cardiac cushion development. Slug, a transcriptional repressor that is a Notch target, is an important Notch effector of EndMT in the cardiac cushion. Here, we report that the runt-related transcription factor RUNX3 is a novel direct Notch target in the endothelium. Ectopic expression of RUNX3 in endothelium induces Slug expression and EndMT independent of Notch activation. Interestingly, RUNX3 physically interacts with CSL, the Notch-interacting partner in the nucleus, and induces Slug in a CSL-dependent, but Notch-independent manner. Although RUNX3 may not be required for the initial induction of Slug and EndMT by Notch, because RUNX3 has a much longer half-life than Slug, it sustains the expression of Slug thereby maintaining the mesenchymal phenotype. CSL binds to the Runx3 promoter in the atrioventricular canal in vivo, and inhibition of Notch reduces RUNX3 expression in the cardiac cushion of embryonic hearts. Taken together, our results suggest that induction of RUNX3 may be a mechanism to maintain Notch-transformed mesenchymal cells during heart development.
Highlights
Endothelial-to-mesenchymal transition (EndMT)5 is a critical process during heart development [1] and plays a role in some pathologic conditions such as cancer and cardiac fibrosis [2, 3]
Our studies indicate that RUNX3 is a direct target gene of Notch in endothelial cells and may play a role in sustaining the mesenchymal phenotype in cardiac cushion cells during heart development
To investigate whether RUNX proteins are downstream targets of Notch in endothelial cells, we activated Notch signaling in human microvascular endothelial cells (HMEC) by overexpression of constitutively-active Notch (NICD) or by coculture of parental HMEC with Notch ligand Dll4- or Jagged1-expressing HMEC at a 1:1 ratio (Dll4 or Jag1 coculture) [15, 22]
Summary
Antibodies and Reagents—Rabbit anti-RUNX1 antibody was purchased from Active Motif. Mouse anti-RUNX3 antibody (R3–5G4) was obtained from Abcam. Chromatin Immunoprecipitation Assay—ChIP to examine the binding of CSL to the RUNX3 promoter in HMEC was conducted using an anti-FLAG antibody as described previously [24]. Enrichment of the RUNX3 promoter DNA was detected by qPCR using primers flanking the CSL bind sites in RUNX3 or SMA promoter, normalized again the input DNA, and expressed as the relative signal with vector/antibody samples designated as 1. ChIP assay to examine the CSL binding to the Runx promoter of mouse embryonic hearts was conducted using a protocol described by Wederell et al [25]. After the cross-linking and sonication, equal amount of chromatin (15 g) was used for ChIP assay using 1 g CSL antibody (ProteinTech Group, Inc.) or 1 g normal rabbit IgG (Vector Laboratories, Burlingame, CA) as negative control. Cell numbers by counting the DAPI-stained nuclei in the same area
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