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Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.

Nguyen DH, Catling AD, Webb DJ, Sankovic M, Walker LA, Somlyo AV, Weber MJ, Gonias SL - J. Cell Biol. (1999)

Bottom Line: When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration.Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration.Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a beta1-integrin (probably alphaVbeta1) and alphaVbeta5. When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

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αVβ5 and β1-containing integrins mediate MCF-7 cell adhesion to vitronectin. MCF-7 cells were allowed to adhere to vitronectin-coated surfaces for 1 h in the presence (+) or absence (−) of 10 nM DIP-uPA. Some MCF-7 cells were treated with blocking antibodies (each at 32 μg/ml) against αVβ3 (LM609), αVβ5 (P1F6), and/or β1 subunit (6S6). Adherent cells were stained with Calcein AM and measured by fluorescence emission using a Cytofluor 2350. Cellular adhesion was quantitated as a percentage of that observed with control cells that were not exposed to DIP-uPA or antibodies.
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Figure 9: αVβ5 and β1-containing integrins mediate MCF-7 cell adhesion to vitronectin. MCF-7 cells were allowed to adhere to vitronectin-coated surfaces for 1 h in the presence (+) or absence (−) of 10 nM DIP-uPA. Some MCF-7 cells were treated with blocking antibodies (each at 32 μg/ml) against αVβ3 (LM609), αVβ5 (P1F6), and/or β1 subunit (6S6). Adherent cells were stained with Calcein AM and measured by fluorescence emission using a Cytofluor 2350. Cellular adhesion was quantitated as a percentage of that observed with control cells that were not exposed to DIP-uPA or antibodies.

Mentions: Meyer et al. 1998 demonstrated that MCF-7 cells express αVβ5 and αVβ1, but not αVβ3. Our flow cytometry experiments, with antibodies directed against αVβ5 and αVβ3, confirmed their results (data not shown). We also demonstrated substantial levels of cell-surface β1 subunit; however, our antibody was not specific for αVβ1. To compare the function of various integrins in MCF-7 cell adhesion to vitronectin, in the presence and absence of DIP-uPA (10 nM), integrin-neutralizing antibodies were used. In the absence of uPA, antibody P1F6, which blocks αVβ5, substantially inhibited MCF-7 cell adhesion whereas LM609, which blocks αVβ3, was ineffective as anticipated (Fig. 9). Interestingly, when the β1-integrin–blocking antibody, 6S6, was added in combination with P1F6, the extent of inhibition was significantly increased (P < 0.05). These results suggest that a β1 subunit–containing integrin (probably αVβ1) plays a significant supporting role in MCF-7 cell adhesion to vitronectin. DIP-uPA neither promoted nor inhibited MCF-7 cell adhesion in the presence or absence of any of the antibodies, suggesting that uPA does not affect the function of the major vitronectin-binding integrins as mediators of MCF-7 cell adhesion.


Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.

Nguyen DH, Catling AD, Webb DJ, Sankovic M, Walker LA, Somlyo AV, Weber MJ, Gonias SL - J. Cell Biol. (1999)

αVβ5 and β1-containing integrins mediate MCF-7 cell adhesion to vitronectin. MCF-7 cells were allowed to adhere to vitronectin-coated surfaces for 1 h in the presence (+) or absence (−) of 10 nM DIP-uPA. Some MCF-7 cells were treated with blocking antibodies (each at 32 μg/ml) against αVβ3 (LM609), αVβ5 (P1F6), and/or β1 subunit (6S6). Adherent cells were stained with Calcein AM and measured by fluorescence emission using a Cytofluor 2350. Cellular adhesion was quantitated as a percentage of that observed with control cells that were not exposed to DIP-uPA or antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199739&req=5

Figure 9: αVβ5 and β1-containing integrins mediate MCF-7 cell adhesion to vitronectin. MCF-7 cells were allowed to adhere to vitronectin-coated surfaces for 1 h in the presence (+) or absence (−) of 10 nM DIP-uPA. Some MCF-7 cells were treated with blocking antibodies (each at 32 μg/ml) against αVβ3 (LM609), αVβ5 (P1F6), and/or β1 subunit (6S6). Adherent cells were stained with Calcein AM and measured by fluorescence emission using a Cytofluor 2350. Cellular adhesion was quantitated as a percentage of that observed with control cells that were not exposed to DIP-uPA or antibodies.
Mentions: Meyer et al. 1998 demonstrated that MCF-7 cells express αVβ5 and αVβ1, but not αVβ3. Our flow cytometry experiments, with antibodies directed against αVβ5 and αVβ3, confirmed their results (data not shown). We also demonstrated substantial levels of cell-surface β1 subunit; however, our antibody was not specific for αVβ1. To compare the function of various integrins in MCF-7 cell adhesion to vitronectin, in the presence and absence of DIP-uPA (10 nM), integrin-neutralizing antibodies were used. In the absence of uPA, antibody P1F6, which blocks αVβ5, substantially inhibited MCF-7 cell adhesion whereas LM609, which blocks αVβ3, was ineffective as anticipated (Fig. 9). Interestingly, when the β1-integrin–blocking antibody, 6S6, was added in combination with P1F6, the extent of inhibition was significantly increased (P < 0.05). These results suggest that a β1 subunit–containing integrin (probably αVβ1) plays a significant supporting role in MCF-7 cell adhesion to vitronectin. DIP-uPA neither promoted nor inhibited MCF-7 cell adhesion in the presence or absence of any of the antibodies, suggesting that uPA does not affect the function of the major vitronectin-binding integrins as mediators of MCF-7 cell adhesion.

Bottom Line: When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration.Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration.Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a beta1-integrin (probably alphaVbeta1) and alphaVbeta5. When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

Show MeSH
Related in: MedlinePlus