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Plasminogen binding and activation at the breast cancer cell surface: the integral role of urokinase activity.

Stillfried GE, Saunders DN, Ranson M - Breast Cancer Res. (2007)

Bottom Line: Using techniques that preserve cell integrity, we characterise the role of uPA as both a plasminogen receptor and activator and quantify the relative contribution of pre-formed and cryptic plasminogen receptors to plasminogen binding.Cell-surface plasminogen binding was significantly enhanced in the presence of elevated levels of uPA in an activity-dependent manner and was greatly attenuated in the presence of the plasmin inhibitor aprotinin.Nevertheless, a relatively modest increase in plasminogen-binding capacity coupled with an increase in uPA led to a dramatic increase in the proteolytic capacity of these cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, University of Wollongong, Northfields Avenue, Wollongong, NSW, 2522, Australia.

ABSTRACT

Introduction: The regulation of extracellular proteolytic activity via the plasminogen activation system is complex, involving numerous activators, inhibitors, and receptors. Previous studies on monocytic and colon cell lines suggest that plasmin pre-treatment can increase plasminogen binding, allowing the active enzyme to generate binding sites for its precursor. Other studies have shown the importance of pre-formed receptors such as annexin II heterotetramer. However, few studies have used techniques that exclusively characterise cell-surface events and these mechanisms have not been investigated at the breast cancer cell surface.

Methods: We have studied plasminogen binding to MCF-7 in which urokinase plasminogen activator receptor (uPAR) levels were upregulated by PMA (12-O-tetradecanoylphorbol-13-acetate) stimulation, allowing flexible and transient modulation of cell-surface uPA. Similar experiments were also performed using MDA-MB-231 cells, which overexpress uPAR/uPA endogenously. Using techniques that preserve cell integrity, we characterise the role of uPA as both a plasminogen receptor and activator and quantify the relative contribution of pre-formed and cryptic plasminogen receptors to plasminogen binding.

Results: Cell-surface plasminogen binding was significantly enhanced in the presence of elevated levels of uPA in an activity-dependent manner and was greatly attenuated in the presence of the plasmin inhibitor aprotinin. Pre-formed receptors were also found to contribute to increased plasminogen binding after PMA stimulation and to co-localise with uPA/uPAR and plasminogen. Nevertheless, a relatively modest increase in plasminogen-binding capacity coupled with an increase in uPA led to a dramatic increase in the proteolytic capacity of these cells.

Conclusion: We show that the majority of lysine-dependent plasminogen binding to breast cancer cells is ultimately regulated by plasmin activity and is dependent on the presence of significant levels of active uPA. The existence of a proteolytic positive feedback loop in plasminogen activation has profound implications for the ability of breast cancer cells expressing high amounts of uPA to accumulate a large proteolytic capacity at the cell surface, thereby conferring invasive potential.

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PMA stimulation prior to urokinase plasminogen activator (uPA) incubation mediates increased plasminogen (plg) binding at the MCF-7 cell surface. Confocal microscopy images of control and PMA-stimulated (100 nM, 16 hours) MCF-7 cells both incubated with 50 nM uPA for 10 minutes at room temperature, washed, and probed for annexin II (AnnII), uPA, and Glu-plg binding. Areas of co-localisation of annexin II, uPA, and Glu-plg are shown in white in the merged image. Arrows indicate areas of concentrated expression. Inset shows non-lysine-dependent plg binding (that is, plg binding in the presence of 5 mM tranexamic acid). Glu, glutamic acid; PAS, plasminogen activation system; PMA, 12-O-tetradecanoylphorbol 13-acetate.
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Figure 4: PMA stimulation prior to urokinase plasminogen activator (uPA) incubation mediates increased plasminogen (plg) binding at the MCF-7 cell surface. Confocal microscopy images of control and PMA-stimulated (100 nM, 16 hours) MCF-7 cells both incubated with 50 nM uPA for 10 minutes at room temperature, washed, and probed for annexin II (AnnII), uPA, and Glu-plg binding. Areas of co-localisation of annexin II, uPA, and Glu-plg are shown in white in the merged image. Arrows indicate areas of concentrated expression. Inset shows non-lysine-dependent plg binding (that is, plg binding in the presence of 5 mM tranexamic acid). Glu, glutamic acid; PAS, plasminogen activation system; PMA, 12-O-tetradecanoylphorbol 13-acetate.

Mentions: To investigate the spatial relationship between cell-surface-bound plg and putative plg receptors, we performed co-localisation experiments using immunofluorescence staining and confocal microscopy. Results for both control and PMA-stimulated MCF-7 cells pre-incubated with exogenous uPA are shown (Figure 4). In the absence of exogenous uPA, regardless of PMA stimulation, very little uPA staining was observed (data not shown).


Plasminogen binding and activation at the breast cancer cell surface: the integral role of urokinase activity.

Stillfried GE, Saunders DN, Ranson M - Breast Cancer Res. (2007)

PMA stimulation prior to urokinase plasminogen activator (uPA) incubation mediates increased plasminogen (plg) binding at the MCF-7 cell surface. Confocal microscopy images of control and PMA-stimulated (100 nM, 16 hours) MCF-7 cells both incubated with 50 nM uPA for 10 minutes at room temperature, washed, and probed for annexin II (AnnII), uPA, and Glu-plg binding. Areas of co-localisation of annexin II, uPA, and Glu-plg are shown in white in the merged image. Arrows indicate areas of concentrated expression. Inset shows non-lysine-dependent plg binding (that is, plg binding in the presence of 5 mM tranexamic acid). Glu, glutamic acid; PAS, plasminogen activation system; PMA, 12-O-tetradecanoylphorbol 13-acetate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851380&req=5

Figure 4: PMA stimulation prior to urokinase plasminogen activator (uPA) incubation mediates increased plasminogen (plg) binding at the MCF-7 cell surface. Confocal microscopy images of control and PMA-stimulated (100 nM, 16 hours) MCF-7 cells both incubated with 50 nM uPA for 10 minutes at room temperature, washed, and probed for annexin II (AnnII), uPA, and Glu-plg binding. Areas of co-localisation of annexin II, uPA, and Glu-plg are shown in white in the merged image. Arrows indicate areas of concentrated expression. Inset shows non-lysine-dependent plg binding (that is, plg binding in the presence of 5 mM tranexamic acid). Glu, glutamic acid; PAS, plasminogen activation system; PMA, 12-O-tetradecanoylphorbol 13-acetate.
Mentions: To investigate the spatial relationship between cell-surface-bound plg and putative plg receptors, we performed co-localisation experiments using immunofluorescence staining and confocal microscopy. Results for both control and PMA-stimulated MCF-7 cells pre-incubated with exogenous uPA are shown (Figure 4). In the absence of exogenous uPA, regardless of PMA stimulation, very little uPA staining was observed (data not shown).

Bottom Line: Using techniques that preserve cell integrity, we characterise the role of uPA as both a plasminogen receptor and activator and quantify the relative contribution of pre-formed and cryptic plasminogen receptors to plasminogen binding.Cell-surface plasminogen binding was significantly enhanced in the presence of elevated levels of uPA in an activity-dependent manner and was greatly attenuated in the presence of the plasmin inhibitor aprotinin.Nevertheless, a relatively modest increase in plasminogen-binding capacity coupled with an increase in uPA led to a dramatic increase in the proteolytic capacity of these cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, University of Wollongong, Northfields Avenue, Wollongong, NSW, 2522, Australia.

ABSTRACT

Introduction: The regulation of extracellular proteolytic activity via the plasminogen activation system is complex, involving numerous activators, inhibitors, and receptors. Previous studies on monocytic and colon cell lines suggest that plasmin pre-treatment can increase plasminogen binding, allowing the active enzyme to generate binding sites for its precursor. Other studies have shown the importance of pre-formed receptors such as annexin II heterotetramer. However, few studies have used techniques that exclusively characterise cell-surface events and these mechanisms have not been investigated at the breast cancer cell surface.

Methods: We have studied plasminogen binding to MCF-7 in which urokinase plasminogen activator receptor (uPAR) levels were upregulated by PMA (12-O-tetradecanoylphorbol-13-acetate) stimulation, allowing flexible and transient modulation of cell-surface uPA. Similar experiments were also performed using MDA-MB-231 cells, which overexpress uPAR/uPA endogenously. Using techniques that preserve cell integrity, we characterise the role of uPA as both a plasminogen receptor and activator and quantify the relative contribution of pre-formed and cryptic plasminogen receptors to plasminogen binding.

Results: Cell-surface plasminogen binding was significantly enhanced in the presence of elevated levels of uPA in an activity-dependent manner and was greatly attenuated in the presence of the plasmin inhibitor aprotinin. Pre-formed receptors were also found to contribute to increased plasminogen binding after PMA stimulation and to co-localise with uPA/uPAR and plasminogen. Nevertheless, a relatively modest increase in plasminogen-binding capacity coupled with an increase in uPA led to a dramatic increase in the proteolytic capacity of these cells.

Conclusion: We show that the majority of lysine-dependent plasminogen binding to breast cancer cells is ultimately regulated by plasmin activity and is dependent on the presence of significant levels of active uPA. The existence of a proteolytic positive feedback loop in plasminogen activation has profound implications for the ability of breast cancer cells expressing high amounts of uPA to accumulate a large proteolytic capacity at the cell surface, thereby conferring invasive potential.

Show MeSH
Related in: MedlinePlus