Generation and characterization of urokinase receptor-deficient mice.

Dewerchin, Mieke; Nuffelen, An Van; Wallays, Goedele; Bouché, Ann; Moons, Lieve; Carmeliet, Peter; Mulligan, Richard C.; Collen, Désiré

doi:10.1172/jci118489

Cited by 225 publications

(148 citation statements)

References 33 publications

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“…Low-density lipoprotein-receptor-deficient (LDLR -/ -) ( Jackson Laboratories) and urokinase receptor-deficient (uPAR -/ -) mice [14] were crossbred into the C57Bl/6J-background (F10). Double heterozygous offspring were bred to obtain uPAR -/ -/LDLR -/ -mice and uPAR + / + /LDLR -/ -control mice.…”

Section: Animal Experimentsmentioning

confidence: 99%

Urokinase Receptor Mediates Osteogenic Differentiation of Mesenchymal Stem Cells and Vascular Calcification via the Complement C5a Receptor

Anaraki

Patecki²,

Larmann³

et al. 2014

Stem Cells and Development

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Vascular calcification is a severe consequence of several pathological processes with a lack of effective therapy. Recent studies suggest that circulating and resident mesenchymal stem cells (MSC) contribute to the osteogenic program of vascular calcification. Molecular mechanisms underlying MSC osteogenic potential and differentiation remain, however, sparsely explored. We investigated a role for the complement receptor C5aR in these processes. We found that expression of C5aR was upregulated upon differentiation of human MSC to osteoblasts. C5aR inhibition by silencing and specific antagonist impaired osteogenic differentiation. We demonstrate that C5aR expression upon MSC differentiation was regulated by the multifunctional urokinase receptor (uPAR). uPAR targeting by siRNA resulted in complete abrogation of C5aR expression and consequently in the inhibition of MSC-osteoblast differentiation. We elucidated the NFkB pathway as the mechanism utilized by the uPAR-C5aR axis. MSC treatment with the NFkB inhibitor completely blocked the differentiation process. Nuclear translocation of the p65 RelA component of the NFkB complex was induced under osteogenic conditions and impaired by the inhibition of uPAR or C5aR. Dual-luciferase reporter assays demonstrated enhanced NFkB signaling upon MSC differentiation, whereas uPAR and C5aR downregulation lead to inhibition of the NFkB activity. We show involvement of the Erk1/2 kinase in this cascade. In vivo studies in a uPAR/LDLR double knockout mouse model of diet-induced atherosclerosis revealed impaired C5aR expression and calcification in aortic sinus plaques in uPAR -/ -/LDLR -/ -versus uPAR + / + /LDLR -/ -control animals. These results suggest that uPAR-C5aR axis via the underlying NFkB transcriptional program controls osteogenic differentiation with functional impact on vascular calcification in vivo.

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Section: Animal Experimentsmentioning

confidence: 99%

Urokinase Receptor Mediates Osteogenic Differentiation of Mesenchymal Stem Cells and Vascular Calcification via the Complement C5a Receptor

Anaraki

Patecki²,

Larmann³

et al. 2014

Stem Cells and Development

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show abstract

“…Homozygous uPAR þ / þ and uPAR À/À mice (75% C57B/6 Â 25% 129/Sv background) were provided by Dr Peter Carmeliet (Leuven, Belgium) (Dewerchin et al, 1996). E13.5 MEFs were isolated from uPAR þ / þ , uPAR þ /À and uPAR À/À littermate embryos as described (Todaro and Green, 1963).…”

Section: Mefs Derivation and Culture Conditionsmentioning

confidence: 99%

A direct link between expression of urokinase plasminogen activator receptor, growth rate and oncogenic transformation in mouse embryonic fibroblasts

et al. 2006

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In addition to its role in invasion and metastasis of several tumors, the multifunctional urokinase receptor uPAR (urokinase plasminogen activator receptor) is directly involved in the growth of several cancer cells in vitro and in vivo. We have compared growth rate and oncogenic transformation in wild-type (wt) or uPAR À/À mouse embryonic fibroblasts (MEFs). Surprisingly, uPAR À/À MEFs grew faster than wt MEFs. This agreed with elevated levels of cell cycle mediators like extracellular signal-regulated protein kinase, p38, AP1 and Cyclin D1. Infection with a uPAR retrovirus reverted the effect, decreasing the growth rate.When MEFs were transformed with H-Ras V12 and E1A oncogenes, the efficiency of transformation in uPAR À/À MEFs was higher than in wt. UPAR À/À MEFs grew faster at low serum, produced more colonies in agar and produced tumors in vivo in nude mice with a lower latency period. The properties of the heterozygous uPAR þ /À MEFs were always intermediate. We conclude therefore that in MEFs uPAR concentration controls cell proliferation and the transforming activity of some oncogenes.

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“…Hence abolishment of single components of the plasminogen activation system, i.e. uPA (13), tPA (13), and uPAR (14,15), by gene targeting does not affect the viability of the homozygous knock-out animals as they by and large exhibit normal phenotypes with respect to growth, reproduction, and survival. In contrast, mice with combined deficiency in uPA and tPA are characterized by retarded growth, reduced fertility, and shortened life span (13).…”

mentioning

confidence: 99%

Antibody-mediated Targeting of the Urokinase-type Plasminogen Activator Proteolytic Function Neutralizes Fibrinolysis in Vivo

Lund

Jögi

Rønø

et al. 2008

Journal of Biological Chemistry

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Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using genetargeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPAdependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo.

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Generation and characterization of urokinase receptor-deficient mice.

Cited by 225 publications

References 33 publications

Urokinase Receptor Mediates Osteogenic Differentiation of Mesenchymal Stem Cells and Vascular Calcification via the Complement C5a Receptor

Urokinase Receptor Mediates Osteogenic Differentiation of Mesenchymal Stem Cells and Vascular Calcification via the Complement C5a Receptor

A direct link between expression of urokinase plasminogen activator receptor, growth rate and oncogenic transformation in mouse embryonic fibroblasts

Antibody-mediated Targeting of the Urokinase-type Plasminogen Activator Proteolytic Function Neutralizes Fibrinolysis in Vivo

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