Identification of Vascular Endothelial Growth Factor (VEGF) Receptor-2 (Flk-1) Promoter/Enhancer Sequences Sufficient for Angioblast and Endothelial Cell-Specific Transcription in Transgenic Mice

Abstract

The vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) is the first endothelial receptor tyrosine kinase to be expressed in angioblast precursors, and its function is essential for the differentiation of endothelial cells and hematopoietic precursors. We have identified cis-acting regulatory elements of the murineFlk-1 gene that mediate endothelium-specific expression of a LacZ reporter gene in transgenic mice. Sequences within the 5′-flanking region of the Flk-1 gene, in combination with sequences located in the first intron, specifically targeted transgene expression to angioblasts and endothelial cells of transgenic mice. The intronic regulatory sequences functioned as an autonomous endothelium-specific enhancer. Sequences of the 5′-flanking region contributed to a strong, uniform, and reproducible transgene expression and were stimulated by the transcription factor HIF-2. The Flk-1 gene regulatory elements described in this study should allow the elucidation of the molecular mechanisms involved in endothelial cell differentiation and angiogenesis.

The transcription factor complex LMO2/TAL1 regulates branching and endothelial cell migration in sprouting angiogenesis

“…We further explored the fine regulation of VEGFR2 expression by LYL1 or TAL1 transcription factors using luciferase reporter with VEGFR2 intronic enhancer sequence having TAL1 binding sites in it (pGL3 VEGFR2 enh-pro) in dual luciferase assay. This sequence is responsible for endothelium specific expression of VEGFR2 in mouse 38 . Overexpression of LYL1 achieved by co-transfection of LYL1 expression vector in 293 T cells results in the similar luciferase activity as endogenous one.…”

“…pGL3-VEGFR2-enh-pro (VEGFR2-enh-pro) was used as a reporter, in which a firefly luciferase reporter gene was driven by a VEGRF2 promoter and endothelium specific intronic enhancer 38 . In this vector, human VEGFR2 promoter sequence about 500 bp including 5ʹ UTR amplified with primers 5ʹ-CAAAGTTGTTGCTCTGGGATG and 5ʹ-GGGAGCCGGTTCTTTCTCCCA was cloned into multiple cloning site of pGL3Basic vector (Promega) and intronic enhancer sequence about 500 bp was cloned into SalI site of the same vector.…”

ETV2/ER71 regulates the generation of FLK1+ cells from mouse embryonic stem cells through miR-126-MAPK signaling

“…To get a detailed insight into how the MAPK pathway activated by ETV2 regulates the generation of FLK1 + cells, we examined the regulatory elements such as promoter and enhancer of Flk1 gene [23] and found several potential activator protein 1 (AP1) binding sequences in Flk1 enhancer which is critical for controlling the endogenous expression of Flk1 (Fig. 4a).…”

Functional Repercussions of Hypoxia-Inducible Factor-2α in Idiopathic Pulmonary Fibrosis

“…The HIF-1α and 2α isoforms are known to be homologous and functionally equally involved in angiogenesis; however, there are some differences since HIF-1α regulates and promotes proliferation and migration in early angiogenesis, while HIF-2α participates in remodeling and microvasculature, controlling vascular morphogenesis, integrity, and assembly [69]. The expression of growth factors such as vascular endothelial growth factor (VEGF) is dependent on HIFs, where HIF-2α regulates the level of the VEGFR-2 receptor and participates in endothelial differentiation and angiogenesis [70], and HIF-1α regulates the VEGFR-1 receptor [71]. The induction of angiogenesis can be regulated at the transcriptional level by activating HERs through HIFs; however, in some cases, this induction does not depend on the activation of HERs but on hypoxic conditions or overexpression of HIF-1α through the activation of some transcriptional cofactors [68].…”