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Mechanisms of Notch4-induced inhibition of endothelial sprouting Mackenzie, Farrell Ian
Abstract
Notch proteins comprise a family of transmembrane receptors. Ligand activation of Notch releases an intracellular region of the receptor that translocates to the nucleus and regulates transcription through interaction with the DNA-binding protein CBF1. CBF1- independent Notch signalling has also been described. Previously, the human Notch4 intracellular region (N4IC) was shown to inhibit endothelial sprouting in vitro and angiogenesis in vivo. The objective of this study was to determine the N4IC domains required for inhibition of endothelial sprouting and to resolve whether this inhibition involves CBFl-dependent signalling. N4IC contains a RAM domain and six ankyrin repeats for protein binding and a Cterminal region (CT) that has poorly defined function. The necessity of each domain for Notch4 activity in endothelial cells was analysed using human dermal microvascular endothelial cells (HMEC-1) expressing N4IC or N4IC deletion constructs. As shown by immunofluorescent staining, mutants lacking the RAM domain had reduced nuclear localisation. Nuclear targeting was restored by fusion of a viral nuclear localisation signal. The angiogenic effect of the N4IC mutants was quantitated using an in vitro endothelial sprouting assay. Deletion of the ankyrin domain, but not the RAM or CT, abrogated the inhibition of FGF-2- and VEGF-induced sprouting, while the ankyrin repeats alone partially blocked sprouting. N4IC decreased VEGF Receptor 2 (VEGFR2) mRNA expression but did not alter FGF Receptor 1 (FGFR1) levels. Conversely, constructs lacking the RAM or CT increased VEGFR2 mRNA compared to controls. Therefore, Notch4 appears to block endothelial sprouting through mechanisms other than mere downregulation of VEGFR2 and FGFR1. The ankyrin repeats, but not the RAM or CT, were also required for upregulation of CBFl-dependent gene expression as shown by luciferase reporter assays and RT-PCR for endogenous CBF1 targets. The ankyrin domain alone was sufficient to upregulate some, but not all, CBFl-dependent genes. Fusion of CBF1 to a viral transactivation domain created a construct that induced expression of target genes independently of N4IC. This constitutively-active CBF1 significantly inhibited HMEC-1 sprouting, but not as strongly as N4IC. Therefore, the inhibition of endothelial sprouting by N4IC requires the ankyrin repeats and appears to involve CBFl-dependent and -independent signalling.
Item Metadata
Title |
Mechanisms of Notch4-induced inhibition of endothelial sprouting
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2003
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Description |
Notch proteins comprise a family of transmembrane receptors. Ligand activation of
Notch releases an intracellular region of the receptor that translocates to the nucleus and
regulates transcription through interaction with the DNA-binding protein CBF1. CBF1-
independent Notch signalling has also been described. Previously, the human Notch4
intracellular region (N4IC) was shown to inhibit endothelial sprouting in vitro and angiogenesis
in vivo. The objective of this study was to determine the N4IC domains required for inhibition of
endothelial sprouting and to resolve whether this inhibition involves CBFl-dependent signalling.
N4IC contains a RAM domain and six ankyrin repeats for protein binding and a Cterminal
region (CT) that has poorly defined function. The necessity of each domain for Notch4
activity in endothelial cells was analysed using human dermal microvascular endothelial cells
(HMEC-1) expressing N4IC or N4IC deletion constructs. As shown by immunofluorescent
staining, mutants lacking the RAM domain had reduced nuclear localisation. Nuclear targeting
was restored by fusion of a viral nuclear localisation signal. The angiogenic effect of the N4IC
mutants was quantitated using an in vitro endothelial sprouting assay. Deletion of the ankyrin
domain, but not the RAM or CT, abrogated the inhibition of FGF-2- and VEGF-induced
sprouting, while the ankyrin repeats alone partially blocked sprouting. N4IC decreased VEGF
Receptor 2 (VEGFR2) mRNA expression but did not alter FGF Receptor 1 (FGFR1) levels.
Conversely, constructs lacking the RAM or CT increased VEGFR2 mRNA compared to
controls. Therefore, Notch4 appears to block endothelial sprouting through mechanisms other
than mere downregulation of VEGFR2 and FGFR1. The ankyrin repeats, but not the RAM or
CT, were also required for upregulation of CBFl-dependent gene expression as shown by
luciferase reporter assays and RT-PCR for endogenous CBF1 targets. The ankyrin domain alone
was sufficient to upregulate some, but not all, CBFl-dependent genes. Fusion of CBF1 to a viral
transactivation domain created a construct that induced expression of target genes independently
of N4IC. This constitutively-active CBF1 significantly inhibited HMEC-1 sprouting, but not as
strongly as N4IC. Therefore, the inhibition of endothelial sprouting by N4IC requires the
ankyrin repeats and appears to involve CBFl-dependent and -independent signalling.
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Extent |
5784210 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-10-28
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0091088
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2003-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.