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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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uPA–PAI-1 complex promotes MCF-7 cell growth. (A) MCF-7 cells were cultured in serum-supplemented medium for 48 h. The cells were then washed and cultured in serum-free medium supplemented with DIP–uPA, uPA–PAI-1 complex, PAI-1, or vehicle (“C”). After 30 h, the cells were pulse-exposed to [3H]thymidine for 1 h at 37°C. The cells were then washed and incubated with 10% trichloroacetic acid. Cell-associated radioactivity was recovered in 1 M NaOH. (B) MCF-7 cells were cultured in serum-supplemented medium for 48 h, washed, and treated with uPA, uPA–PAI-1 complex, PAI-1, uPA–PAI-1R76E complex, or vehicle (“C”) in serum free–defined medium. After culturing for an additional 36 h, MTT assays were performed. The graph shows the increase in cell number relative to the increase observed in control cultures. (C) MCF-7 cells were pulse-exposed to uPA–PAI-1 complex for 4 h, in the presence or absence of RAP. After culturing for an additional 32 h, cell growth was determined by MTT assay.
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Figure 8: uPA–PAI-1 complex promotes MCF-7 cell growth. (A) MCF-7 cells were cultured in serum-supplemented medium for 48 h. The cells were then washed and cultured in serum-free medium supplemented with DIP–uPA, uPA–PAI-1 complex, PAI-1, or vehicle (“C”). After 30 h, the cells were pulse-exposed to [3H]thymidine for 1 h at 37°C. The cells were then washed and incubated with 10% trichloroacetic acid. Cell-associated radioactivity was recovered in 1 M NaOH. (B) MCF-7 cells were cultured in serum-supplemented medium for 48 h, washed, and treated with uPA, uPA–PAI-1 complex, PAI-1, uPA–PAI-1R76E complex, or vehicle (“C”) in serum free–defined medium. After culturing for an additional 36 h, MTT assays were performed. The graph shows the increase in cell number relative to the increase observed in control cultures. (C) MCF-7 cells were pulse-exposed to uPA–PAI-1 complex for 4 h, in the presence or absence of RAP. After culturing for an additional 32 h, cell growth was determined by MTT assay.

Mentions: The length of time that ERK phosphorylation is sustained may determine whether active ERK translocates to the nucleus and promotes cell growth (Lenormand et al. 1993). We demonstrated previously that uPA does not stimulate MCF-7 cell proliferation (Nguyen et al. 1998). To determine whether uPA–PAI-1 complex functions as a mitogen, [3H]thymidine incorporation experiments were conducted. Fig. 8 A shows that [3H]thymidine incorporation was increased 3.5 ± 0.2-fold (n = 4) in MCF-7 cells that were exposed to 5 nM uPA–PAI-1 complex for 30 h. Under equivalent conditions, DIP–uPA and free PAI-1 had no effect on [3H]thymidine incorporation.


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)

uPA–PAI-1 complex promotes MCF-7 cell growth. (A) MCF-7 cells were cultured in serum-supplemented medium for 48 h. The cells were then washed and cultured in serum-free medium supplemented with DIP–uPA, uPA–PAI-1 complex, PAI-1, or vehicle (“C”). After 30 h, the cells were pulse-exposed to [3H]thymidine for 1 h at 37°C. The cells were then washed and incubated with 10% trichloroacetic acid. Cell-associated radioactivity was recovered in 1 M NaOH. (B) MCF-7 cells were cultured in serum-supplemented medium for 48 h, washed, and treated with uPA, uPA–PAI-1 complex, PAI-1, uPA–PAI-1R76E complex, or vehicle (“C”) in serum free–defined medium. After culturing for an additional 36 h, MTT assays were performed. The graph shows the increase in cell number relative to the increase observed in control cultures. (C) MCF-7 cells were pulse-exposed to uPA–PAI-1 complex for 4 h, in the presence or absence of RAP. After culturing for an additional 32 h, cell growth was determined by MTT assay.
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Related In: Results  -  Collection

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Figure 8: uPA–PAI-1 complex promotes MCF-7 cell growth. (A) MCF-7 cells were cultured in serum-supplemented medium for 48 h. The cells were then washed and cultured in serum-free medium supplemented with DIP–uPA, uPA–PAI-1 complex, PAI-1, or vehicle (“C”). After 30 h, the cells were pulse-exposed to [3H]thymidine for 1 h at 37°C. The cells were then washed and incubated with 10% trichloroacetic acid. Cell-associated radioactivity was recovered in 1 M NaOH. (B) MCF-7 cells were cultured in serum-supplemented medium for 48 h, washed, and treated with uPA, uPA–PAI-1 complex, PAI-1, uPA–PAI-1R76E complex, or vehicle (“C”) in serum free–defined medium. After culturing for an additional 36 h, MTT assays were performed. The graph shows the increase in cell number relative to the increase observed in control cultures. (C) MCF-7 cells were pulse-exposed to uPA–PAI-1 complex for 4 h, in the presence or absence of RAP. After culturing for an additional 32 h, cell growth was determined by MTT assay.
Mentions: The length of time that ERK phosphorylation is sustained may determine whether active ERK translocates to the nucleus and promotes cell growth (Lenormand et al. 1993). We demonstrated previously that uPA does not stimulate MCF-7 cell proliferation (Nguyen et al. 1998). To determine whether uPA–PAI-1 complex functions as a mitogen, [3H]thymidine incorporation experiments were conducted. Fig. 8 A shows that [3H]thymidine incorporation was increased 3.5 ± 0.2-fold (n = 4) in MCF-7 cells that were exposed to 5 nM uPA–PAI-1 complex for 30 h. Under equivalent conditions, DIP–uPA and free PAI-1 had no effect on [3H]thymidine incorporation.

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