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

Ying Wang, Luke H. Hoeppner, Ramcharan Singh Angom, Enfeng Wang, Shamit Dutta, Heike R. Doeppler, Fei Wang, Tao Shen, Isobel A. Scarisbrick, Sushovan Guha, Peter Storz, Resham Bhattacharya, Debabrata Mukhopadhyay

Research output: Contribution to journalArticle

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 AP2β 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 AP2β up-regulation decreases VEGFR-2 expression and that loss of AP2β enhances VEGFR-2 expression in HUVECs. In vivo experiments confirmed increased VEGFR-2 immunostaining in the spinal cord of AP2β knockout mouse embryos. Mechanistically, we observed that PKD phosphorylates AP2β at Ser258 and Ser277 and suppresses its nuclear accumulation. Inhibition of PKD activity with a pan-PKD inhibitor increased AP2β nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2β nuclear localization through a Ser258- and Ser277-dependent mechanism. Furthermore, substitution of Ser277 in AP2β 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.

Original languageEnglish (US)
Pages (from-to)15759-15767
Number of pages9
JournalJournal of Biological Chemistry
Volume294
Issue number43
DOIs
StatePublished - Jan 1 2019

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Transcription Factor AP-2
Vascular Endothelial Growth Factor Receptor-2
Vascular Endothelial Growth Factor Receptor
Endothelial cells
Transcription
Vascular Endothelial Growth Factor A
Up-Regulation
Endothelial Cells
Human Umbilical Vein Endothelial Cells
Binding Sites
Activating Transcription Factors
Physiological Phenomena
protein kinase D
Cell proliferation
Bioinformatics
Computational Biology
Luciferases
Knockout Mice
Cell Movement
Assays

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Protein kinase D up-regulates transcription of VEGF receptor-2 in endothelial cells by suppressing nuclear localization of the transcription factor AP2β. / Wang, Ying; Hoeppner, Luke H.; Angom, Ramcharan Singh; Wang, Enfeng; Dutta, Shamit; Doeppler, Heike R.; Wang, Fei; Shen, Tao; Scarisbrick, Isobel A.; Guha, Sushovan; Storz, Peter; Bhattacharya, Resham; Mukhopadhyay, Debabrata.

In: Journal of Biological Chemistry, Vol. 294, No. 43, 01.01.2019, p. 15759-15767.

Research output: Contribution to journalArticle

Wang, Ying ; Hoeppner, Luke H. ; Angom, Ramcharan Singh ; Wang, Enfeng ; Dutta, Shamit ; Doeppler, Heike R. ; Wang, Fei ; Shen, Tao ; Scarisbrick, Isobel A. ; Guha, Sushovan ; Storz, Peter ; Bhattacharya, Resham ; Mukhopadhyay, Debabrata. / Protein kinase D up-regulates transcription of VEGF receptor-2 in endothelial cells by suppressing nuclear localization of the transcription factor AP2β. In: Journal of Biological Chemistry. 2019 ; Vol. 294, No. 43. pp. 15759-15767.
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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 AP2β 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 AP2β up-regulation decreases VEGFR-2 expression and that loss of AP2β enhances VEGFR-2 expression in HUVECs. In vivo experiments confirmed increased VEGFR-2 immunostaining in the spinal cord of AP2β knockout mouse embryos. Mechanistically, we observed that PKD phosphorylates AP2β at Ser258 and Ser277 and suppresses its nuclear accumulation. Inhibition of PKD activity with a pan-PKD inhibitor increased AP2β nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2β nuclear localization through a Ser258- and Ser277-dependent mechanism. Furthermore, substitution of Ser277 in AP2β 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.",
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T1 - Protein kinase D up-regulates transcription of VEGF receptor-2 in endothelial cells by suppressing nuclear localization of the transcription factor AP2β

AU - Wang, Ying

AU - Hoeppner, Luke H.

AU - Angom, Ramcharan Singh

AU - Wang, Enfeng

AU - Dutta, Shamit

AU - Doeppler, Heike R.

AU - Wang, Fei

AU - Shen, Tao

AU - Scarisbrick, Isobel A.

AU - Guha, Sushovan

AU - Storz, Peter

AU - Bhattacharya, Resham

AU - Mukhopadhyay, Debabrata

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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 AP2β 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 AP2β up-regulation decreases VEGFR-2 expression and that loss of AP2β enhances VEGFR-2 expression in HUVECs. In vivo experiments confirmed increased VEGFR-2 immunostaining in the spinal cord of AP2β knockout mouse embryos. Mechanistically, we observed that PKD phosphorylates AP2β at Ser258 and Ser277 and suppresses its nuclear accumulation. Inhibition of PKD activity with a pan-PKD inhibitor increased AP2β nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2β nuclear localization through a Ser258- and Ser277-dependent mechanism. Furthermore, substitution of Ser277 in AP2β 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.

AB - 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 AP2β 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 AP2β up-regulation decreases VEGFR-2 expression and that loss of AP2β enhances VEGFR-2 expression in HUVECs. In vivo experiments confirmed increased VEGFR-2 immunostaining in the spinal cord of AP2β knockout mouse embryos. Mechanistically, we observed that PKD phosphorylates AP2β at Ser258 and Ser277 and suppresses its nuclear accumulation. Inhibition of PKD activity with a pan-PKD inhibitor increased AP2β nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2β nuclear localization through a Ser258- and Ser277-dependent mechanism. Furthermore, substitution of Ser277 in AP2β 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.

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