Limits...
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.

Show MeSH

Related in: MedlinePlus

Plasminogen binding and activation on MCF-7 cells. After culture for 16 hours in 5% foetal calf serum/RPMI containing 100 nM PMA (PMA-stimulated) or vehicle alone (control), cells were detached and pre-incubated for 10 minutes at room temperature in the absence or presence of 50 nM active urokinase plasminogen activator (uPA) or PMSF-inactivated uPA (PMSF-uPA), washed, and then analysed for cell-surface plg binding or activation. (a) Cell-surface, lysine-dependent plg binding to PMA-stimulated MCF-7 cells is shown as a percentage increase compared to unstimulated MCF-7 cells in the absence of uPA. (b) Cell-surface plasmin (pln) generation. Pln activity assays were performed using Spectrozyme PL in the presence of α2-antiplasmin to inhibit any solution-phase pln generation. Activity in the presence of aprotinin (pln inhibitor) was also measured and subtracted from all values to determine pln-dependent activity. (c) Cell-surface lysine-dependent fluorescein isothiocyanate-plg binding was measured in the presence or absence of aprotinin. Percentages show the proportion of binding due to pln activity at the cell surface (that is, pln-dependent binding calculated as total binding minus binding in the presence of aprotinin; open bars) and the proportion that is independent of pln activity (that is, pln-independent binding calculated as residual binding in the presence of aprotinin; hatched bars), which together constitute total lysine-dependent plg binding. *Significant increase compared to unstimulated control cells not pre-incubated with uPA or PMSF-uPA (p < 0.05). **Significant increase compared to PMA-stimulated cells not pre-incubated with uPA or PMSF-uPA. MFI, mean fluorescence intensity; PMA, 12-O-tetradecanoylphorbol-13-acetate; PMSF, alpha-toluenesulfonyl fluoride.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1851380&req=5

Figure 5: Plasminogen binding and activation on MCF-7 cells. After culture for 16 hours in 5% foetal calf serum/RPMI containing 100 nM PMA (PMA-stimulated) or vehicle alone (control), cells were detached and pre-incubated for 10 minutes at room temperature in the absence or presence of 50 nM active urokinase plasminogen activator (uPA) or PMSF-inactivated uPA (PMSF-uPA), washed, and then analysed for cell-surface plg binding or activation. (a) Cell-surface, lysine-dependent plg binding to PMA-stimulated MCF-7 cells is shown as a percentage increase compared to unstimulated MCF-7 cells in the absence of uPA. (b) Cell-surface plasmin (pln) generation. Pln activity assays were performed using Spectrozyme PL in the presence of α2-antiplasmin to inhibit any solution-phase pln generation. Activity in the presence of aprotinin (pln inhibitor) was also measured and subtracted from all values to determine pln-dependent activity. (c) Cell-surface lysine-dependent fluorescein isothiocyanate-plg binding was measured in the presence or absence of aprotinin. Percentages show the proportion of binding due to pln activity at the cell surface (that is, pln-dependent binding calculated as total binding minus binding in the presence of aprotinin; open bars) and the proportion that is independent of pln activity (that is, pln-independent binding calculated as residual binding in the presence of aprotinin; hatched bars), which together constitute total lysine-dependent plg binding. *Significant increase compared to unstimulated control cells not pre-incubated with uPA or PMSF-uPA (p < 0.05). **Significant increase compared to PMA-stimulated cells not pre-incubated with uPA or PMSF-uPA. MFI, mean fluorescence intensity; PMA, 12-O-tetradecanoylphorbol-13-acetate; PMSF, alpha-toluenesulfonyl fluoride.

Mentions: Several studies in other cell types have shown that limited proteolysis of the cell surface by pln can reveal cryptic plg-binding sites [21,22,40]. To assess the effect of cell-surface proteolytic activity on plg binding, a number of experiments were performed on MCF-7 cells with modulated active and inactive exogenous uPA levels. No significant difference in lysine-dependent plg binding was observed in control cells pre-incubated with either uPA or PMSF-uPA (data not shown). However, lysine-dependent plg binding on MCF-7 cells after PMA stimulation increased to approximately 183% compared to control cells (Figure 5a). Subsequent addition of exogenous uPA caused a further approximately 37% increase in plg binding, which was not observed with the addition of PMSF-uPA (Figure 5a). Given that the direct, non-active-site-dependent interaction of plg with uPA was precluded through the use of urine-derived uPA, this clearly demonstrates that the additional plg binding observed after uPA treatment of PMA-stimulated cells is entirely dependent on uPA catalytic activity. This effect is functionally important because PMA stimulation alone (which increased plg binding) had no effect on plg activation, whereas uPA treatment of these cells caused a very large (15-fold) increase in cell-surface pln generation (Figure 5b). In addition, the pln generated represents activated cell-surface-bound plg as solution-phase pln was inhibited by the presence of α2-antiplasmin. Hence, without uPA catalytic activity at the cell surface, increased plg binding alone is not sufficient for increased plg activation. Nevertheless, increased lysine-dependent plg binding has been clearly linked to increased plg activation in the presence of uPA activity [27].


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)

Plasminogen binding and activation on MCF-7 cells. After culture for 16 hours in 5% foetal calf serum/RPMI containing 100 nM PMA (PMA-stimulated) or vehicle alone (control), cells were detached and pre-incubated for 10 minutes at room temperature in the absence or presence of 50 nM active urokinase plasminogen activator (uPA) or PMSF-inactivated uPA (PMSF-uPA), washed, and then analysed for cell-surface plg binding or activation. (a) Cell-surface, lysine-dependent plg binding to PMA-stimulated MCF-7 cells is shown as a percentage increase compared to unstimulated MCF-7 cells in the absence of uPA. (b) Cell-surface plasmin (pln) generation. Pln activity assays were performed using Spectrozyme PL in the presence of α2-antiplasmin to inhibit any solution-phase pln generation. Activity in the presence of aprotinin (pln inhibitor) was also measured and subtracted from all values to determine pln-dependent activity. (c) Cell-surface lysine-dependent fluorescein isothiocyanate-plg binding was measured in the presence or absence of aprotinin. Percentages show the proportion of binding due to pln activity at the cell surface (that is, pln-dependent binding calculated as total binding minus binding in the presence of aprotinin; open bars) and the proportion that is independent of pln activity (that is, pln-independent binding calculated as residual binding in the presence of aprotinin; hatched bars), which together constitute total lysine-dependent plg binding. *Significant increase compared to unstimulated control cells not pre-incubated with uPA or PMSF-uPA (p < 0.05). **Significant increase compared to PMA-stimulated cells not pre-incubated with uPA or PMSF-uPA. MFI, mean fluorescence intensity; PMA, 12-O-tetradecanoylphorbol-13-acetate; PMSF, alpha-toluenesulfonyl fluoride.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Plasminogen binding and activation on MCF-7 cells. After culture for 16 hours in 5% foetal calf serum/RPMI containing 100 nM PMA (PMA-stimulated) or vehicle alone (control), cells were detached and pre-incubated for 10 minutes at room temperature in the absence or presence of 50 nM active urokinase plasminogen activator (uPA) or PMSF-inactivated uPA (PMSF-uPA), washed, and then analysed for cell-surface plg binding or activation. (a) Cell-surface, lysine-dependent plg binding to PMA-stimulated MCF-7 cells is shown as a percentage increase compared to unstimulated MCF-7 cells in the absence of uPA. (b) Cell-surface plasmin (pln) generation. Pln activity assays were performed using Spectrozyme PL in the presence of α2-antiplasmin to inhibit any solution-phase pln generation. Activity in the presence of aprotinin (pln inhibitor) was also measured and subtracted from all values to determine pln-dependent activity. (c) Cell-surface lysine-dependent fluorescein isothiocyanate-plg binding was measured in the presence or absence of aprotinin. Percentages show the proportion of binding due to pln activity at the cell surface (that is, pln-dependent binding calculated as total binding minus binding in the presence of aprotinin; open bars) and the proportion that is independent of pln activity (that is, pln-independent binding calculated as residual binding in the presence of aprotinin; hatched bars), which together constitute total lysine-dependent plg binding. *Significant increase compared to unstimulated control cells not pre-incubated with uPA or PMSF-uPA (p < 0.05). **Significant increase compared to PMA-stimulated cells not pre-incubated with uPA or PMSF-uPA. MFI, mean fluorescence intensity; PMA, 12-O-tetradecanoylphorbol-13-acetate; PMSF, alpha-toluenesulfonyl fluoride.
Mentions: Several studies in other cell types have shown that limited proteolysis of the cell surface by pln can reveal cryptic plg-binding sites [21,22,40]. To assess the effect of cell-surface proteolytic activity on plg binding, a number of experiments were performed on MCF-7 cells with modulated active and inactive exogenous uPA levels. No significant difference in lysine-dependent plg binding was observed in control cells pre-incubated with either uPA or PMSF-uPA (data not shown). However, lysine-dependent plg binding on MCF-7 cells after PMA stimulation increased to approximately 183% compared to control cells (Figure 5a). Subsequent addition of exogenous uPA caused a further approximately 37% increase in plg binding, which was not observed with the addition of PMSF-uPA (Figure 5a). Given that the direct, non-active-site-dependent interaction of plg with uPA was precluded through the use of urine-derived uPA, this clearly demonstrates that the additional plg binding observed after uPA treatment of PMA-stimulated cells is entirely dependent on uPA catalytic activity. This effect is functionally important because PMA stimulation alone (which increased plg binding) had no effect on plg activation, whereas uPA treatment of these cells caused a very large (15-fold) increase in cell-surface pln generation (Figure 5b). In addition, the pln generated represents activated cell-surface-bound plg as solution-phase pln was inhibited by the presence of α2-antiplasmin. Hence, without uPA catalytic activity at the cell surface, increased plg binding alone is not sufficient for increased plg activation. Nevertheless, increased lysine-dependent plg binding has been clearly linked to increased plg activation in the presence of uPA activity [27].

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