Protein kinase C regulates FLT1 abundance and stimulates its cleavage in vascular endothelial cells with the release of a soluble PlGF/VEGF antagonist

Raikwar, Nandita S.; Liu, Kang Z.; Thomas, Christie P.

doi:10.1016/j.yexcr.2013.07.005

Cited by 28 publications

(34 citation statements)

References 29 publications

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“…The extracellular domain can bind VEGF and PlGF and is very similar to the transcriptionally derived sFlt1 [15]. We also demonstrated that PMA stimulated an increase in the abundance of Flt1 in HUVEC which was completely inhibited by GF109203X.…”

Section: Discussionmentioning

confidence: 56%

N-Terminal Cleavage and Release of the Ectodomain of Flt1 Is Mediated via ADAM10 and ADAM 17 and Regulated by VEGFR2 and the Flt1 Intracellular Domain

2014

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Flt is one of the cell surface VEGF receptors which can be cleaved to release an N-terminal extracellular fragment which, like alternately transcribed soluble Flt1 (sFlt1), can antagonize the effects of VEGF. In HUVEC and in HEK293 cells where Flt1 was expressed, metalloprotease inhibitors reduced Flt1 N-terminal cleavage. Overexpression of ADAM10 and ADAM17 increased cleavage while knockdown of ADAM10 and ADAM17 reduced N-terminal cleavage suggesting that these metalloproteases were responsible for Flt1 cleavage. Protein kinase C (PKC) activation increased the abundance and the cleavage of Flt1 but this did not require any residues within the intracellular portion of Flt1. ALLN, a proteasomal inhibitor, increased the abundance of Flt1 which was additive to the effect of PKC. Removal of the entire cytosolic region of Flt1 appeared to stimulate cleavage of Flt1 and Flt1 was no longer sensitive to ALLN suggesting that the cytosolic region contained a degradation domain. Knock down of c-CBL, a ring finger ubiquitin ligase, in HEK293 cells increased the expression of Flt1 although it did not appear to require a previously published tyrosine residue (1333Y) in the C-terminus of Flt1. Increasing VEGFR2 expression increased VEGF-stimulated sFlt1 expression and progressively reduced the cleavage of Flt1 with Flt1 staying bound to VEGFR2 as a heterodimer. Our results imply that secreted sFlt1 and cleaved Flt1 will tend to have local effects as a VEGF antagonist when released from cells expressing VEGFR2 and more distant effects when released from cells lacking VEGFR2.

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Section: Discussionmentioning

confidence: 56%

N-Terminal Cleavage and Release of the Ectodomain of Flt1 Is Mediated via ADAM10 and ADAM 17 and Regulated by VEGFR2 and the Flt1 Intracellular Domain

2014

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“…We have reported that s FLT1 was expressed, at least at the mRNA level, in CTBs, at about 1000 fold more than FLT1 (4). We have also previously reported that, in addition to its transcriptional origin, sFLT1 can be derived by post-translational cleavage of FLT1(6). We were not able to detect FLT1 by immunoblotting in CTBs, presumably because of its low abundance (Figure 1A).…”

Section: Resultsmentioning

confidence: 90%

“…C-terminal GFP-tagged open reading frame (ORF) clone of full length human VEGFR2 was purchased from OriGene (Rockville, MD). Recombinant adenovirus expressing FLT, sFLT1-i13, sFLT1-e15a and control virus were generated by the vector Core Facility at the University of Iowa and has been previously described (4, 6). …”

Section: Methodsmentioning

confidence: 99%

“…Further processing of conditioned media and preparation of whole cell lysates from monolayers for western blotting were performed as previously described. (6)…”

Section: Methodsmentioning

confidence: 99%

“…These forms of sFLT1 are naturally occurring VEGF/PlGF antagonists. We, and others, have recently identified a novel sFLT1 which is post-translationally cleaved from FLT1 (6, 7). This cleaved form of sFLT1 is also released into the extracellular milieu and can function as a VEGF antagonist.…”

Section: Introductionmentioning

confidence: 95%

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Aspirin inhibits expression of sFLT1 from human cytotrophoblasts induced by hypoxia, via cyclo-oxygenase 1

Li¹,

Raikwar

Santillan³

et al. 2015

Placenta

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Introduction Elevated circulating soluble FLT1 (sFLT1) levels in preeclampsia may play a role in its development. Aspirin is recommended for prevention of preeclampsia. We hypothesized that aspirin may inhibit the production of sFlt1. Methods Placentas from women with and without preeclampsia were collected. Primary cytotrophoblasts (CTBs) were cultured from normal placentas and treated with aspirin, sc-560, a COX1 inhibitor or celecoxib, a COX-2 inhibitor. The expression of sFLT1, FLT1, COX1, COX2 was studied. The effect of aspirin on sFlt1 expression was also studied in HEK293 cells and in HTR-8/SVNeo cells. Results The expression of sFLT1 was increased in preeclamptic placentas compared to control placentas and the expression and release of sFLT1 increased in CTBs exposed to 2% O2 compared to controls. Aspirin at 3 and 12 mM concentration reduced the expression and release of sFLT1 in CTBs. Aspirin also inhibited sFlt1 expression from HTR-8/SVNeo and HEK293 cells. Sc-560, but not celecoxib, reduced sFLT1 expression and release from CTBs. Aspirin and sc-560 also reduced hypoxia-induced FLT1 mRNA expression and inhibited COX1 mRNA in CTBs. Discussion This study confirms that sFLT1 expression is increased in preeclamptic placentas and in CTBs exposed to hypoxia. Aspirin inhibits the production sFLT1 in CTBs and in HTR-8/SVNeo. Sc-560 recapitulated the effects of aspirin on sFLT1 expression and release in CTBs suggesting that the aspirin effect may be mediated via inhibition of COX1. The study increases our understanding of the mechanisms regulating sFlt1 expression and provides a plausible explanation for the effect of aspirin to prevent preeclampsia.

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Irreversible modifications of receptor tyrosine kinases

2018

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Each group of the 56 receptor tyrosine kinases (RTK) binds with one or more soluble growth factors and coordinates a vast array of cellular functions. These outcomes are tightly regulated by inducible post-translational events, such as tyrosine phosphorylation, ubiquitination, ectodomain shedding, and regulated intramembrane proteolysis. Because of the delicate balance required for appropriate RTK function, cells may become pathogenic upon dysregulation of RTKs themselves or their post-translational covalent modifications. For example, reduced ectodomain shedding and decreased ubiquitination of the cytoplasmic region, both of which enhance growth factor signals, characterize malignant cells. Whereas receptor phosphorylation and ubiquitination are reversible, proteolytic cleavage events are irreversible, and either modification might alter the subcellular localization of RTKs. Herein, we focus on ectodomain shedding by metalloproteinases (including ADAM family proteases), cleavage within the membrane or cytoplasmic regions of RTKs (by gamma-secretases and caspases, respectively), and complete receptor proteolysis in lysosomes and proteasomes. Roles of irreversible modifications in RTK signaling, pathogenesis, and pharmacology are highlighted.

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Protein kinase C regulates FLT1 abundance and stimulates its cleavage in vascular endothelial cells with the release of a soluble PlGF/VEGF antagonist

Cited by 28 publications

References 29 publications

N-Terminal Cleavage and Release of the Ectodomain of Flt1 Is Mediated via ADAM10 and ADAM 17 and Regulated by VEGFR2 and the Flt1 Intracellular Domain

N-Terminal Cleavage and Release of the Ectodomain of Flt1 Is Mediated via ADAM10 and ADAM 17 and Regulated by VEGFR2 and the Flt1 Intracellular Domain

Aspirin inhibits expression of sFLT1 from human cytotrophoblasts induced by hypoxia, via cyclo-oxygenase 1

Irreversible modifications of receptor tyrosine kinases

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