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Plasminogen activator inhibitor 1 functions as a urokinase response modifier at the level of cell signaling and thereby promotes MCF-7 cell growth.

Webb DJ, Thomas KS, Gonias SL - J. Cell Biol. (2001)

Bottom Line: This effect required the cooperative function of uPAR and the very low density lipoprotein receptor (VLDLr).When MCF-7 cells were treated with uPA-PAI-1 complex in the presence of the VLDLr antagonist, receptor-associated protein, or with uPA-PAI-1(R76E) complex, which binds to the VLDLr with greatly decreased affinity, transient ERK phosphorylation (<5 min) was observed, mimicking the uPA response.Our results demonstrate that PAI-1 may regulate uPA-initiated cell signaling by a mechanism that requires VLDLr recruitment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, and Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Plasminogen activator inhibitor 1 (PAI-1) is a major inhibitor of urokinase-type plasminogen activator (uPA). In this study, we explored the role of PAI-1 in cell signaling. In MCF-7 cells, PAI-1 did not directly activate the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2, but instead altered the response to uPA so that ERK phosphorylation was sustained. This effect required the cooperative function of uPAR and the very low density lipoprotein receptor (VLDLr). When MCF-7 cells were treated with uPA-PAI-1 complex in the presence of the VLDLr antagonist, receptor-associated protein, or with uPA-PAI-1(R76E) complex, which binds to the VLDLr with greatly decreased affinity, transient ERK phosphorylation (<5 min) was observed, mimicking the uPA response. ERK phosphorylation was not induced by tissue-type plasminogen activator-PAI-1 complex or by uPA-PAI-1 complex in the presence of antibodies that block uPA binding to uPAR. uPA-PAI-1 complex induced tyrosine phosphorylation of focal adhesion kinase and Shc and sustained association of Sos with Shc, whereas uPA caused transient association of Sos with Shc. By sustaining ERK phosphorylation, PAI-1 converted uPA into an MCF-7 cell mitogen. This activity was blocked by receptor-associated protein and not observed with uPA-PAI-1(R76E) complex, demonstrating the importance of the VLDLr. uPA promoted the growth of other cells in which ERK phosphorylation was sustained, including beta3 integrin overexpressing MCF-7 cells and HT 1080 cells. The MEK inhibitor, PD098059, blocked the growth-promoting activity of uPA and uPA-PAI-1 complex in these cells. Our results demonstrate that PAI-1 may regulate uPA-initiated cell signaling by a mechanism that requires VLDLr recruitment. The kinetics of ERK phosphorylation in response to uPAR ligation determine the function of uPA and uPA-PAI-1 complex as growth promoters.

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Activation of ERK1 and ERK2 in uPA–PAI-1 complex-treated MCF-7 cells. MCF-7 cells were serum starved for 12 h and then treated with 5 nM DIP–uPA (A), 5 nM uPA–PAI-1 complex (B), or vehicle (Control; A and B) for the indicated times. (C) Cultures were incubated with uPA- or uPAR-specific antibodies (+) or vehicle (−) for 15 min at 37°C and then exposed to uPA–PAI-1 complex (5 nM) or vehicle for 5 min. Phosphorylated ERK1 and ERK2 were detected by immunoblot analysis. The nitrocellulose membranes were then stripped and reprobed with a separate antibody that detects total ERK.
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Figure 1: Activation of ERK1 and ERK2 in uPA–PAI-1 complex-treated MCF-7 cells. MCF-7 cells were serum starved for 12 h and then treated with 5 nM DIP–uPA (A), 5 nM uPA–PAI-1 complex (B), or vehicle (Control; A and B) for the indicated times. (C) Cultures were incubated with uPA- or uPAR-specific antibodies (+) or vehicle (−) for 15 min at 37°C and then exposed to uPA–PAI-1 complex (5 nM) or vehicle for 5 min. Phosphorylated ERK1 and ERK2 were detected by immunoblot analysis. The nitrocellulose membranes were then stripped and reprobed with a separate antibody that detects total ERK.

Mentions: We demonstrated previously that ERK1 and ERK2 are rapidly phosphorylated and activated in uPA-treated MCF-7 cells (Nguyen et al. 1998, Nguyen et al. 1999). The response requires uPA binding to uPAR and is highly transient since levels of phosphorylated ERK return to baseline in <5 min (Nguyen et al. 1998). In the present study, we confirmed our previous results, demonstrating rapid but transient ERK phosphorylation in MCF-7 cells treated with 5 nM DIP–uPA (Fig. 1 A). ERK phosphorylation was not observed in MCF-7 cells that were treated with free PAI-1 (5 nM) in its active conformation (results not shown).


Plasminogen activator inhibitor 1 functions as a urokinase response modifier at the level of cell signaling and thereby promotes MCF-7 cell growth.

Webb DJ, Thomas KS, Gonias SL - J. Cell Biol. (2001)

Activation of ERK1 and ERK2 in uPA–PAI-1 complex-treated MCF-7 cells. MCF-7 cells were serum starved for 12 h and then treated with 5 nM DIP–uPA (A), 5 nM uPA–PAI-1 complex (B), or vehicle (Control; A and B) for the indicated times. (C) Cultures were incubated with uPA- or uPAR-specific antibodies (+) or vehicle (−) for 15 min at 37°C and then exposed to uPA–PAI-1 complex (5 nM) or vehicle for 5 min. Phosphorylated ERK1 and ERK2 were detected by immunoblot analysis. The nitrocellulose membranes were then stripped and reprobed with a separate antibody that detects total ERK.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195772&req=5

Figure 1: Activation of ERK1 and ERK2 in uPA–PAI-1 complex-treated MCF-7 cells. MCF-7 cells were serum starved for 12 h and then treated with 5 nM DIP–uPA (A), 5 nM uPA–PAI-1 complex (B), or vehicle (Control; A and B) for the indicated times. (C) Cultures were incubated with uPA- or uPAR-specific antibodies (+) or vehicle (−) for 15 min at 37°C and then exposed to uPA–PAI-1 complex (5 nM) or vehicle for 5 min. Phosphorylated ERK1 and ERK2 were detected by immunoblot analysis. The nitrocellulose membranes were then stripped and reprobed with a separate antibody that detects total ERK.
Mentions: We demonstrated previously that ERK1 and ERK2 are rapidly phosphorylated and activated in uPA-treated MCF-7 cells (Nguyen et al. 1998, Nguyen et al. 1999). The response requires uPA binding to uPAR and is highly transient since levels of phosphorylated ERK return to baseline in <5 min (Nguyen et al. 1998). In the present study, we confirmed our previous results, demonstrating rapid but transient ERK phosphorylation in MCF-7 cells treated with 5 nM DIP–uPA (Fig. 1 A). ERK phosphorylation was not observed in MCF-7 cells that were treated with free PAI-1 (5 nM) in its active conformation (results not shown).

Bottom Line: This effect required the cooperative function of uPAR and the very low density lipoprotein receptor (VLDLr).When MCF-7 cells were treated with uPA-PAI-1 complex in the presence of the VLDLr antagonist, receptor-associated protein, or with uPA-PAI-1(R76E) complex, which binds to the VLDLr with greatly decreased affinity, transient ERK phosphorylation (<5 min) was observed, mimicking the uPA response.Our results demonstrate that PAI-1 may regulate uPA-initiated cell signaling by a mechanism that requires VLDLr recruitment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, and Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Plasminogen activator inhibitor 1 (PAI-1) is a major inhibitor of urokinase-type plasminogen activator (uPA). In this study, we explored the role of PAI-1 in cell signaling. In MCF-7 cells, PAI-1 did not directly activate the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2, but instead altered the response to uPA so that ERK phosphorylation was sustained. This effect required the cooperative function of uPAR and the very low density lipoprotein receptor (VLDLr). When MCF-7 cells were treated with uPA-PAI-1 complex in the presence of the VLDLr antagonist, receptor-associated protein, or with uPA-PAI-1(R76E) complex, which binds to the VLDLr with greatly decreased affinity, transient ERK phosphorylation (<5 min) was observed, mimicking the uPA response. ERK phosphorylation was not induced by tissue-type plasminogen activator-PAI-1 complex or by uPA-PAI-1 complex in the presence of antibodies that block uPA binding to uPAR. uPA-PAI-1 complex induced tyrosine phosphorylation of focal adhesion kinase and Shc and sustained association of Sos with Shc, whereas uPA caused transient association of Sos with Shc. By sustaining ERK phosphorylation, PAI-1 converted uPA into an MCF-7 cell mitogen. This activity was blocked by receptor-associated protein and not observed with uPA-PAI-1(R76E) complex, demonstrating the importance of the VLDLr. uPA promoted the growth of other cells in which ERK phosphorylation was sustained, including beta3 integrin overexpressing MCF-7 cells and HT 1080 cells. The MEK inhibitor, PD098059, blocked the growth-promoting activity of uPA and uPA-PAI-1 complex in these cells. Our results demonstrate that PAI-1 may regulate uPA-initiated cell signaling by a mechanism that requires VLDLr recruitment. The kinetics of ERK phosphorylation in response to uPAR ligation determine the function of uPA and uPA-PAI-1 complex as growth promoters.

Show MeSH
Related in: MedlinePlus