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Protein kinase D up-regulates transcription of VEGF receptor-2 in endothelial cells by suppressing nuclear localization of the transcription factor AP2?.

ABSTRACT: Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGF receptor-2 (VEGFR-2) to control vasculogenesis and angiogenesis, key physiological processes in cardiovascular disease and cancer. In human umbilical vein endothelial cells (HUVECs), knockdown of protein kinase D-1 (PKD1) or PKD2 down-regulates VEGFR-2 expression and inhibits VEGF-induced cell proliferation and migration. However, how PKD regulates VEGF signaling is unclear. Previous bioinformatics analyses have identified binding sites for the transcription factor activating enhancer-binding protein 2 (AP2) in the VEGFR-2 promoter. Using ChIP analyses, here we found that PKD knockdown in HUVECs increases binding of AP2? to the VEGFR-2 promoter. Luciferase reporter assays with serial deletions of AP2-binding sites within the VEGFR-2 promoter revealed that its transcriptional activity negatively correlates with the number of these sites. Next we demonstrated that AP2? up-regulation decreases VEGFR-2 expression and that loss of AP2? enhances VEGFR-2 expression in HUVECs. In vivo experiments confirmed increased VEGFR-2 immunostaining in the spinal cord of AP2? knockout mouse embryos. Mechanistically, we observed that PKD phosphorylates AP2? at Ser258 and Ser277 and suppresses its nuclear accumulation. Inhibition of PKD activity with a pan-PKD inhibitor increased AP2? nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2? nuclear localization through a Ser258- and Ser277-dependent mechanism. Furthermore, substitution of Ser277 in AP2? increased its binding to the VEGFR-2 promoter. Our findings uncover evidence of a molecular pathway that regulates VEGFR-2 expression, insights that may shed light on the etiology of diseases associated with aberrant VEGF/VEGFR signaling.

SUBMITTER: Wang Y

PROVIDER: S-EPMC6816101 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Protein kinase D up-regulates transcription of <i>VEGF receptor-2</i> in endothelial cells by suppressing nuclear localization of the transcription factor AP2β.

Wang Ying Y Hoeppner Luke H LH Angom Ramcharan Singh RS Wang Enfeng E Dutta Shamit S Doeppler Heike R HR Wang Fei F Shen Tao T Scarisbrick Isobel A IA Guha Sushovan S Storz Peter P Bhattacharya Resham R Mukhopadhyay Debabrata D

The Journal of biological chemistry 20190906 43

Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGF receptor-2 (VEGFR-2) to control vasculogenesis and angiogenesis, key physiological processes in cardiovascular disease and cancer. In human umbilical vein endothelial cells (HUVECs), knockdown of protein kinase D-1 (PKD1) or PKD2 down-regulates VEGFR-2 expression and inhibits VEGF-induced cell proliferation and migration. However, how PKD regulates VEGF signaling is unclear. Previous bioinformatics an ...[more]

PMID: 31492751

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Project description:Vascular permeability triggered by inflammation or ischemia promotes edema, exacerbates disease progression, and impairs tissue recovery. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability. VEGF plays an integral role in regulating vascular barrier function physiologically and in pathologies, such as cancer, ischemic stroke, cardiovascular disease, retinal conditions, and COVID-19-associated pulmonary edema and sepsis, which often leads to acute lung injury, including acute respiratory distress syndrome. However, after initially stimulating permeability, VEGF subsequently mediates angiogenesis to repair damaged tissue. Consequently, understanding temporal molecular regulation of VEG-Finduced vascular permeability will facilitate developing therapeutics that achieve the delicate balance of inhibiting vascular permeability while preserving tissue repair. Here, we demonstrate that VEGF signals through signal transducer and activator of transcription 3 (STAT3) to promote vascular permeability. Specifically, we show that genetic STAT3 ablation reduces vascular permeability in STAT3-deficient endothelium of mice and VEGF-inducible zebrafish crossed with CRISPR/Cas9 generated genomic STAT3 knockout zebrafish. Importantly, STAT3 deficiency does not impair vascular development and function in vivo. We identify intercellular adhesion molecule 1 (ICAM-1) as a STAT3-dependent transcriptional regulator and show VEGF-dependent STAT3 activation is regulated by JAK2. Pyrimethamine, an FDA-approved antimicrobial agent that inhibits STAT3-dependent transcription, substantially reduces VEGF-induced vascular permeability in zebrafish, mouse, and human endothelium. Indeed, pharmacologically targeting STAT3 increases vascular barrier integrity using two additional compounds, atovaquone and C188-9. Collectively, our findings suggest that the VEGF, VEGFR-2, JAK2, and STAT3 signaling cascade regulates vascular barrier integrity, and inhibition of STAT3-dependent activity reduces VEGF-induced vascular permeability in vertebrate models.

Dataset Information

Protein kinase D up-regulates transcription of VEGF receptor-2 in endothelial cells by suppressing nuclear localization of the transcription factor AP2?.

Publications

Protein kinase D up-regulates transcription of <i>VEGF receptor-2</i> in endothelial cells by suppressing nuclear localization of the transcription factor AP2β.

Similar Datasets

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