Kruppel-like factor 4 regulates developmental angiogenesis through disruption of the RBP-J–NICD–MAML complex in intron 3 of Dll4
Angiogenesis is a multistep process that requires highly regulated endothelial cell (EC) behavior. The transcription factor Krüppel-like factor 4 (KLF4) is a critical regulator of several basic EC functions; we have recently shown that KLF4 disturbs pathological (tumor) angiogenesis by mediating the expression of members of VEGF and Notch signaling pathways. Notch signaling is central to orchestration of sprouting angiogenesis but little is known about the upstream regulation of Notch itself. To determine the role of KLF4 in normal (developmental) angiogenesis, we used a mouse retinal angiogenesis model. We found that endothelial-specific overexpression of KLF4 in transgenic mice (EC-K4 Tg) leads to increased vessel density, branching and number of tip cell filopodia as assessed on postnatal day 6 (P6). The hypertrophic vasculature seen with sustained KLF4 overexpression is not stable and undergoes prominent remodeling during P7–P12 resulting in a normal appearing retinal vasculature in adult EC-K4 Tg mice. We find that KLF4 inhibits Delta-like 4 (DLL4) expression in the angiogenic front during retinal vascular development. Furthermore, in an oxygen-induced retinopathy model, overexpression of KLF4 results in decreased vaso-obliteration and neovascular tuft formation that is similar to genetic or pharmacologic DLL4 inhibition. Mechanistically, we show that KLF4 disables the activity of the essential Notch transcriptional activator RBP-J by interfering with binding of co-activators NICD and MAML at intron 3 of the Notch ligand DLL4. In summary, our experimental results demonstrate a regulatory role of KLF4 in developmental angiogenesis through regulation of DLL4 transcription.
KeywordsAngiogenesis Notch signaling Endothelium Kruppel-like factor 4 Transcriptional regulation
We are grateful for the support and encouragement provided by Dr. Mukesh K. Jain. We thank Dr. Christina Antonopoulos-Buzzy (Case Western Reserve University School of Medicine) for kindly providing technical support, discussion, and review of the manuscript. We thank Kevin P. Montgomery for assistance with figures. This work is supported by NIH R01HL113570.
EB designed, performed, and analyzed experiments, and drafted the manuscript. HZ, JP, and MAS provided input into experimental results and reviewed the manuscript. RHA provided crucial reagents and reviewed the manuscript. AH conceived and coordinated the studies, designed, and analyzed the experiments, and revised the manuscript. All authors approved the final version of the manuscript.