Limits...
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.

Show MeSH

Related in: MedlinePlus

uPA promotes cellular migration by a uPAR-dependent mechanism which is inhibited by PD098059 in parent and uPAR-overexpressing MCF-7 cells. Each of the designated cell types was treated with 10 nM DIP-uPA (+) or with vehicle (−) and allowed to migrate for 6 h on serum-coated Transwell membranes. Some cells were treated with uPA-specific antibody, uPAR-specific antibody, nonimmune IgG (each at 25 μg/ml), or with PD098059 (50 μM). Cellular migration is expressed as a percentage of that observed with control cells, which were not treated with DIP-uPA, antibodies, or PD098059 .
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199739&req=5

Figure 1: uPA promotes cellular migration by a uPAR-dependent mechanism which is inhibited by PD098059 in parent and uPAR-overexpressing MCF-7 cells. Each of the designated cell types was treated with 10 nM DIP-uPA (+) or with vehicle (−) and allowed to migrate for 6 h on serum-coated Transwell membranes. Some cells were treated with uPA-specific antibody, uPAR-specific antibody, nonimmune IgG (each at 25 μg/ml), or with PD098059 (50 μM). Cellular migration is expressed as a percentage of that observed with control cells, which were not treated with DIP-uPA, antibodies, or PD098059 .

Mentions: We demonstrated previously that uPA activates ERK1/2, rapidly but transiently, in MCF-7 cells and stimulates MCF-7 cell migration (Nguyen et al. 1998). The selective MEK inhibitor, PD098059, blocked the ability of uPA to stimulate MCF-7 cell migration without affecting the basal level of cellular migration, suggesting that ERK activation is essential in the pathway by which uPA increases MCF-7 cell motility. Fig. 1 A confirms and extends our original observations by demonstrating that DIP-uPA (10 nM) increases MCF-7 cell migration, in serum-coated Transwells, and that this activity is blocked by antibodies (25 μg/ml) which bind to uPA or uPAR and prevent uPAR ligation. The uPA- and uPAR-specific antibodies did not affect MCF-7 cell migration in the absence of exogenously added uPA. Furthermore, nonimmune IgG did not affect MCF-7 cell migration, in the presence or absence of uPA. Thus, the motility-stimulating activity of uPA, in MCF-7 cells, requires uPA-binding to uPAR.


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)

uPA promotes cellular migration by a uPAR-dependent mechanism which is inhibited by PD098059 in parent and uPAR-overexpressing MCF-7 cells. Each of the designated cell types was treated with 10 nM DIP-uPA (+) or with vehicle (−) and allowed to migrate for 6 h on serum-coated Transwell membranes. Some cells were treated with uPA-specific antibody, uPAR-specific antibody, nonimmune IgG (each at 25 μg/ml), or with PD098059 (50 μM). Cellular migration is expressed as a percentage of that observed with control cells, which were not treated with DIP-uPA, antibodies, or PD098059 .
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199739&req=5

Figure 1: uPA promotes cellular migration by a uPAR-dependent mechanism which is inhibited by PD098059 in parent and uPAR-overexpressing MCF-7 cells. Each of the designated cell types was treated with 10 nM DIP-uPA (+) or with vehicle (−) and allowed to migrate for 6 h on serum-coated Transwell membranes. Some cells were treated with uPA-specific antibody, uPAR-specific antibody, nonimmune IgG (each at 25 μg/ml), or with PD098059 (50 μM). Cellular migration is expressed as a percentage of that observed with control cells, which were not treated with DIP-uPA, antibodies, or PD098059 .
Mentions: We demonstrated previously that uPA activates ERK1/2, rapidly but transiently, in MCF-7 cells and stimulates MCF-7 cell migration (Nguyen et al. 1998). The selective MEK inhibitor, PD098059, blocked the ability of uPA to stimulate MCF-7 cell migration without affecting the basal level of cellular migration, suggesting that ERK activation is essential in the pathway by which uPA increases MCF-7 cell motility. Fig. 1 A confirms and extends our original observations by demonstrating that DIP-uPA (10 nM) increases MCF-7 cell migration, in serum-coated Transwells, and that this activity is blocked by antibodies (25 μg/ml) which bind to uPA or uPAR and prevent uPAR ligation. The uPA- and uPAR-specific antibodies did not affect MCF-7 cell migration in the absence of exogenously added uPA. Furthermore, nonimmune IgG did not affect MCF-7 cell migration, in the presence or absence of uPA. Thus, the motility-stimulating activity of uPA, in MCF-7 cells, requires uPA-binding to uPAR.

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