Involvement of the [uPAR:uPA:PAI-1:LRP] Complex in Human Myogenic Cell Motility

doi:10.1006/excr.2000.4934

Experimental Cell Research

Volume 258, Issue 2, 1 August 2000, Pages 237-244

https://doi.org/10.1006/excr.2000.4934 Get rights and content

Abstract

The urokinase-type plasminogen activator system is a proteolytic system involved in tissue remodeling and cell migration. At the cell surface, receptor (uPAR)-bound urokinase (uPA) binds its inhibitor PAI-1, localized in the matrix, and the complex is internalized by endocytic receptors, such as the low-density lipoprotein receptor-related protein (LRP). We previously proposed a nonproteolytic role for the uPA system in human myogenic cell differentiation in vitro, i.e., cell fusion, and showed that myogenic cells can use PAI-1 as an adhesion matrix molecule. The aim of this study was to define the role of the uPA system in myogenic cell migration that is necessary for fusion. Using a two-dimensional motility assay and microcinematography, we showed that any interference with the [uPAR:uPA:PAI-1] complex formation, and interference with LRP binding to this complex, markedly decreased myogenic cell motility. This phenomenon was reversible and independent of plasmin activity. Inhibition of cell motility was associated with suppression of both filopodia and membrane ruffling activity. [uPAR:uPA:PAI-1:LRP] complex formation involves high-affinity molecular interactions and results in quick internalization of the complex. It is likely that this complex supports the membrane ruffling activity involved in the guidance of the migrating cell toward appropriate sites for attachment.

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Our finding that LRP1 silencing resulted in a significant decrease in Ang II-induced RAoSMCs migration is consistent with a previous report that LRP1 is essential to human vascular smooth muscle cell migration [10]. Moreover, functional blocking of LRP1 by RAP also led to decreased cell migration, which is also consistent with reports that RAP treatment decreased migration of breast cancer cells, myogenic cells [8,21], as well as smooth muscle cells [9,22], indicating the ligand binding activity of LRP1 is associated with cellular migratory mechanism. In contrast, there are reports that knockdown or deletion of LRP1 increased cell migration, likely by decreasing the catabolic rate of the uPA:PAI-1:uPAR complex [23,24].
Angiotensin II (Ang II), one of the main vasoactive hormones of the renin-angiotensin system, contributes to the development and progression of atherosclerosis by inducing vascular smooth muscle cells (VSMCs) migration. Although previous studies have shown that Ang II upregulates low density lipoprotein receptor-related protein 1 (LRP1) expression in VSMCs and increases VSMCs migration, the role of LRP1 in Ang II-induced VSMCs migration remains unclear. Here, we reveal a novel mechanism by which LRP1 induces the expression of matrix metalloproteinase 2 (MMP2) and thereby promotes the migration of rat aortic SMCs (RAoSMCs). Knockdown of LRP1 expression greatly decreased RAoSMCs migration, which was rescued by forced expression of a functional LRP1 minireceptor, suggesting that LRP1 is a key regulator of Ang II-induced RAoSMCs migration. Inhibition of ligand binding to LRP1 by the specific antagonist receptor-associated protein (RAP) also led to reduced RAoSMCs migration. Because MMPs play critical roles in RAoSMCs migration, we examined the expression of several MMPs and found that the expression of functional MMP2 was selectively increased by Ang II treatment and decreased in LRP1-knockdown RAoSMCs. More interestingly, reduced MMP2 expression in LRP1-knockdown cells was completely rescued by exogenous expression of mLRP4, suggesting that MMP2 is a downstream regulator of LRP1 in Ang II-induced RAoSMCs migration. Together, our data strongly suggest that LRP1 promotes the migration of RAoSMCs by regulating the expression and function of MMP2.
The plasminogen activator inhibitor "paradox" in cancer
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Proteolysis in general and specifically the plasminogen activating system regulated by urokinase (uPA) its specific receptor, the GPI membrane anchored urokinase receptor (uPAR) and the specific plasminogen activator inhibitor 1 (PAI-1) plays a major role in tumorigenesis, tumor progression, tumor invasion and metastasis formation. This is exemplified by a body of published work showing a positive correlation between the expression of uPA or uPAR in several tumors and their malignancy. It is generally assumed that such a “pro-malignant” effect of the uPA–uPAR system is mediated by increased local proteolysis thus favoring tumor invasion, by a pro-angiogenic effect of this system and also by uPA–uPAR signaling towards the tumor thereby shifting the tumor phenotype to a more “malignant” one. However, when tumor patients are analyzed for long term survival, those with high levels of the inhibitor of the system, PAI-1 have a much worse prognosis than those with lower PAI-1 levels. This indicates that increased overall proteolysis alone cannot be made responsible for the adverse effects of the plasminogen activating system in tumors. Moreover, it becomes increasingly evident that components of the fibrinolytic system secreted by the tumor cells themselves are not solely responsible for a correlation between the plasminogen activating system and tumor malignancy; components of the plasminogen activating system secreted by stroma cells or cells of the immune system such as macrophages contribute also to the impact of fibrinolysis on malignancy.
This review summarizes the evidence for the role of plasminogen activator inhibitor-1 in mediating the malignant phenotype and possible mechanism thereby trying to explain the “PAI-1 paradox in cancer” on a molecular level.
A new pro-migratory activity on human myogenic precursor cells for a synthetic peptide within the E domain of the mechano growth factor
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Duchenne muscular dystrophy (DMD) is an inherited disease that leads to progressive muscle wasting. Myogenic precursor cell transplantation is an approach that can introduce the normal dystrophin gene in the muscle fibers of the patients. Unfortunately, these myogenic precursor cells do not migrate well in the muscle and thus many injections have to be done to enable a good graft success. Recent reports have shown that there is extensive splicing of the IGF-1 gene in muscles. The MGF isoform contains a C-terminal 24 amino acids peptide in the E domain (MGF-Ct24E) that has intrinsic properties. It can promote the proliferation while delaying the differentiation of C₂C₁₂ cells.
Here, we demonstrated that this synthetic peptide is a motogenic factor for human precursor myogenic cells in vitro and in vivo. Indeed, MGF-Ct24E peptide can modulate members of the fibrinolytic and metalloproteinase systems, which are implicated in the migration of myogenic cells. MGF-Ct24E peptide enhances the expression of u-PA, u-PAR and MMP-7 while reducing PAI-1 activity. Moreover, it has no effect on the gelatinases MMP-2 and -9. Those combined effects can favour cell migration. Finally, we present some results suggesting that the MGF-Ct24E peptide induces these cell responses through a mechanism that does not involve the IGF-1 receptor.
Thus, this MGF-Ct24E peptide has a new pro-migratory activity on human myogenic precursor cells that may be helpful in the treatment of DMD. Those results reinforce the possibility that the IGF-1Ec isoform may produce an E domain peptide that can act as a cytokine.
The PAI-1 swing: Microenvironment and cancer cell migration
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Le processus cancéreux est une dynamique complexe, dont l'intégration de tous les paramètres nécessite de mieux comprendre les événements observés aux différentes échelles et d'en modéliser les interactions. La modification d'assemblages moléculaires « matricellulaires » peut déclencher le processus métastatique. In vitro, un microenvironnement cellulaire, très enrichi en PAI-1 matriciel, entraîne une modification du comportement des cellules qui adoptent le mode de migration amiboïde et présentent toutes les caractéristiques morphologiques de l'état post-transition mésenchymateuse-amiboïde et ses conséquences. PAI-1 apparaît donc comme une protéine matricellulaire capable de déclencher le processus à l'échelle moléculaire, cellulaire, et tissulaire, susceptible d'engager un pronostic concernant l'organisme entier. Pour citer cet article : M. Malo et al., C. R. Biologies 329 (2006).
Cancer is a complex and dynamic process caused by a cellular dysfunction leading to a whole organ or even organism vital perturbation. To better understand this process, we need to study each one of the levels involved, which allows the scale change, and to integrate this knowledge. A matricellular protein, PAI-1, is able to induce in vitro cell behaviour modifications, morphological changes, and to promote cell migration. PAI-1 influences the mesenchymo-amaeboid transition. This matricellular protein should be considered as a potential ‘launcher’ of the metastatic process acting at the molecular, cellular, tissular levels and, as a consequence, at the organism's level. To cite this article: M. Malo et al., C. R. Biologies 329 (2006).
Dual role for plasminogen activator inhibitor type 1 as soluble and as matricellular regulator of epithelial alveolar cell wound healing
2006, American Journal of Pathology
Citation Excerpt :
PAI-1 is found either soluble or matrix-bound,26,49–51 where it plays diverse roles in adhesion depending on the cell type or the experimental conditions. Matrix-bound PAI-1 can either promote cell adhesion21,25,27 and subsequent migration24,28,40,52 or deadhesion.20,22,23,53 The inhibition of healing in the presence of blocking antibodies against PAI-1, dependent on the concentration (up to 40% with 50 μg/ml), together with the dramatic increase in matrix bound PAI-1 (Figure 4D) suggests that a nonproteolytic action of the uPA system is involved in the KGF-induced wound healing.
Epithelium repair, crucial for restoration of alveolo-capillary barrier integrity, is orchestrated by various cytokines and growth factors. Among them keratinocyte growth factor plays a pivotal role in both cell proliferation and migration. The urokinase plasminogen activator (uPA) system also influences cell migration through proteolysis during epithelial repair. In addition, the complex formed by uPAR-uPA and matrix-bound plasminogen activator inhibitor type-1 (PAI-1) exerts nonproteolytic roles in various cell types. Here we present new evidence about the dual role of PAI-1 under keratinocyte growth factor stimulation using an in vitro repair model of rat alveolar epithelial cells. Besides proteolytic involvement of the uPA system, the availability of matrix-bound-PAI-1 is also required for an efficient healing. An unexpected decrease of healing was shown when PAI-1 activity was blocked. However, the proteolytic action of uPA and plasmin were still required. Moreover, immediately after wounding, PAI-1 was dramatically increased in the newly deposited matrix at the leading edge of wounds. We thus propose a dual role for PAI-1 in epithelial cell wound healing, both as a soluble inhibitor of proteolysis and also as a matrix-bound regulator of cell migration. Matrix-bound PAI-1 could thus be considered as a new member of the matricellular protein family.
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Different molecules are available to recruit new neighboring myogenic cells to the site of regeneration. Formerly called B cell stimulatory factor-1, IL-4 can now be included in the list of motogenic factors. The present report demonstrates that human IL-4 is not required for fusion between mononucleated myoblasts but is required for myotube maturation. In identifying IL-4 as a pro-migratory agent for myogenic cells, these results provide a mechanism which partly explains IL-4 demonstrated activity during differentiation.
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Therefore, IL-4 co-injection with transplanted myoblasts might be an approach to enhance the migration of transplanted cells for the treatment of a damaged myocardium or of a Duchenne Muscular Dystrophy patient.

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Regular ArticleInvolvement of the [uPAR:uPA:PAI-1:LRP] Complex in Human Myogenic Cell Motility

Abstract

Cell. Differ. Dev.

Immunol. Today

Curr. Opin. Cell Biol.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Exp. Cell Res.

Biol. Cell.

Thromb Res.

Dev. Biol.

Cell

J. Biol. Chem.

J. Biol. Chem.

Exp. Cell Res.

Curr. Opin. Cell Biol.

J. Biol. Chem.

Cell

Extracellular matrix remodeling by metalloproteinases and plasminogen activators

Kidney Int.

The plasminogen activator/plasmin system

J. Clin. Invest.

The cell biology of the plasminogen system

FASEB J.

Characterization of the binding of different conformational forms of plasminogen activator inhibitor-1 to vitronectin

J. Biol. Chem.

α2-Macroglobulin receptor/LDL receptor-related protein (Lrp)-dependent internalization of the urokinase receptor

J. Cell Biol.

Regular Article
Involvement of the [uPAR:uPA:PAI-1:LRP] Complex in Human Myogenic Cell Motility

α₂-Macroglobulin receptor/LDL receptor-related protein (Lrp)-dependent internalization of the urokinase receptor