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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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Sustained ERK phosphorylation requires the continuous presence of uPA–PAI-1 complex. MCF-7 cells were serum starved for 12 h and then pulse-exposed to 5 nM uPA–PAI-1 complex for 1 min at 37°C. Cultures were then processed for ERK analysis (1 min) or washed and incubated in fresh medium without uPA–PAI-1 complex for the indicated times. Control cells were not treated with uPA–PAI-1 complex. Phosphorylated and total ERK1 and ERK2 were detected by immunoblot analysis.
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Figure 6: Sustained ERK phosphorylation requires the continuous presence of uPA–PAI-1 complex. MCF-7 cells were serum starved for 12 h and then pulse-exposed to 5 nM uPA–PAI-1 complex for 1 min at 37°C. Cultures were then processed for ERK analysis (1 min) or washed and incubated in fresh medium without uPA–PAI-1 complex for the indicated times. Control cells were not treated with uPA–PAI-1 complex. Phosphorylated and total ERK1 and ERK2 were detected by immunoblot analysis.

Mentions: A second model to explain the sustained activation of ERK in uPA–PAI-1 complex-treated MCF-7 cells involves the ability of the VLDLr to mediate uPAR endocytosis and recycling when uPA–PAI-1 complex binds to uPAR (Conese et al. 1995; Nykjær et al. 1997; Webb et al. 1999). Because uPA-initiated ERK activation in MCF-7 cells is transient, due to processes such as Shc–Grb2–Sos dissociation, uPAR endocytosis and recycling may provide a constant pool of unliganded uPAR to initiate new signaling events. If this model is correct, then a continuous source of free uPA–PAI complex should be necessary to sustain ERK phosphorylation. To test this hypothesis, MCF-7 cells were pulse-exposed to 5 nM uPA–PAI-1 complex for 1 min, washed, and then incubated in fresh medium without uPA–PAI-1 complex. As shown in Fig. 6, ERK was phosphorylated in the early time points. However, by 15 min, the level of phosphorylated ERK returned to baseline. Thus, a continuous source of free uPA–PAI-1 complex is necessary to sustain ERK phosphorylation in MCF-7 cells.


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)

Sustained ERK phosphorylation requires the continuous presence of uPA–PAI-1 complex. MCF-7 cells were serum starved for 12 h and then pulse-exposed to 5 nM uPA–PAI-1 complex for 1 min at 37°C. Cultures were then processed for ERK analysis (1 min) or washed and incubated in fresh medium without uPA–PAI-1 complex for the indicated times. Control cells were not treated with uPA–PAI-1 complex. Phosphorylated and total ERK1 and ERK2 were detected by immunoblot analysis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195772&req=5

Figure 6: Sustained ERK phosphorylation requires the continuous presence of uPA–PAI-1 complex. MCF-7 cells were serum starved for 12 h and then pulse-exposed to 5 nM uPA–PAI-1 complex for 1 min at 37°C. Cultures were then processed for ERK analysis (1 min) or washed and incubated in fresh medium without uPA–PAI-1 complex for the indicated times. Control cells were not treated with uPA–PAI-1 complex. Phosphorylated and total ERK1 and ERK2 were detected by immunoblot analysis.
Mentions: A second model to explain the sustained activation of ERK in uPA–PAI-1 complex-treated MCF-7 cells involves the ability of the VLDLr to mediate uPAR endocytosis and recycling when uPA–PAI-1 complex binds to uPAR (Conese et al. 1995; Nykjær et al. 1997; Webb et al. 1999). Because uPA-initiated ERK activation in MCF-7 cells is transient, due to processes such as Shc–Grb2–Sos dissociation, uPAR endocytosis and recycling may provide a constant pool of unliganded uPAR to initiate new signaling events. If this model is correct, then a continuous source of free uPA–PAI complex should be necessary to sustain ERK phosphorylation. To test this hypothesis, MCF-7 cells were pulse-exposed to 5 nM uPA–PAI-1 complex for 1 min, washed, and then incubated in fresh medium without uPA–PAI-1 complex. As shown in Fig. 6, ERK was phosphorylated in the early time points. However, by 15 min, the level of phosphorylated ERK returned to baseline. Thus, a continuous source of free uPA–PAI-1 complex is necessary to sustain ERK phosphorylation in MCF-7 cells.

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