Feed-forward signaling by membrane-bound ligand receptor circuit: the case of NOTCH DELTA-like 4 ligand in endothelial cells.
The DELTA like-4 ligand (DLL4) belongs to the highly conserved NOTCH family and is specifically expressed in the endothelium. DLL4 regulates crucial processes in vascular growth, including endothelial cell (EC) sprouting and arterial specification. Its expression is increased by VEGF-A. In the present study, we show that VEGF-induced DLL4 expression depends on NOTCH activation. VEGF-induced DLL4 expression was prevented by the blockage of NOTCH signaling with γ-secretase or ADAM inhibitors in human cardiac microvascular ECs. Similar to VEGF-A, recombinant DLL4 itself stimulated NOTCH signaling and resulted in up-regulation of DLL4, suggesting a positive feed-forward mechanism. These effects were abrogated by NOTCH inhibitors but not by inhibition of VEGF signaling. NOTCH activation alone suffices to induce DLL4 expression as illustrated by the positive effect of NOTCH intracellular domain (NICD)-1 or -4 overexpression. To discriminate between NICD/RBP-Jκ and FOXC2-regulated DLL4 expression, DLL4 promoter activity was assessed in promoter deletion experiments. NICD induced promoter activity was dependent on RBP-Jκ site but independent of the FOXC2 binding site. Accordingly, constitutively active FOXC2 did not affect DLL4 expression. The notion that the positive feed-forward mechanism might propagate NOTCH activation to neighboring ECs was supported by our observation that DLL4-eGFP-transfected ECs induced DLL4 expression in nontransfected cells in their vicinity. In summary, our data provide evidence for a mechanism by which VEGF or ligand-induced NOTCH signaling up-regulates DLL4 through a positive feed-forward mechanism. By this mechanism, DLL4 could propagate its own expression and enable synchronization of NOTCH expression and signaling between ECs.
Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, 6229 ER Maastricht, The Netherlands.
Vincenza
V
Caolo
Nynke M S
NM
van den Akker
Sanne
S
Verbruggen
Marjo M P C
MM
Donners
Geertje
G
Swennen
Henny
H
Schulten
Johannes
J
Waltenberger
Mark J
MJ
Post
Daniel G M
DG
Molin
D048868:Adaptor Proteins, Signal Transducing; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002450:Cell Communication / Q000502:physiology*; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D017434:Protein Structure, Tertiary; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D015854:Up-Regulation / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C424126:DLL4 protein, human; D051858:Forkhead Transcription Factors; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D036341:Intercellular Signaling Peptides and Proteins; C497570:RBPJ protein, human; D051880:Receptors, Notch; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C082078:mesenchyme fork head 1 protein; D053829:Amyloid Precursor Protein Secretases