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Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells
R W Stephens1, H Tapiovaara, T Reisberg
1Department of Virology, University of Helsinki, Finland.
Abstract:
Human RC2A myelomonocytic leukemia cells are able to activate the prourokinase (pro-u-PA) they secrete so that active u-PA is present both in serum-free conditioned medium from these cells, as well as on the cell surface. When the cells are grown in serum-containing medium, no u-PA activity can be found in the medium but active u-PA is found bound to the cell surface where it can generate bound plasmin. This distribution of u-PA activity was shown to be, first, the net result of slow inactivation of free active u-PA by serum inhibitor(s) and simultaneous rapid uptake of u-PA onto the cell surface. Binding to cells was at least six times faster than inactivation by 10% serum. The principal serum inhibitor of u-PA was identified as alpha 2-macroglobulin (alpha 2M), and prior inactivation of u-PA by purified human alpha 2M was also shown to prevent uptake of u-PA activity onto cells. Second, although endogenous u-PA could form covalent complexes with purified alpha 2M in the culture medium of RC2A cells, covalent alpha 2M complexes were not formed by u-PA on the cell surface; the u-PA taken up in this compartment was protected against alpha 2M inhibition. u-PA anchored to plastic surfaces via monoclonal antibodies to the amino-terminal region of u-PA was also protected against alpha 2M, suggesting that the protection of cell surface u-PA results from a steric effect. These results provide evidence as to how the active u-PA produced by leukemia cells can contribute to proteolytic activity on their cell surface in the presence of serum inhibitors.
Insights
Human leukemia cells activate secreted prourokinase (pro-u-PA) into active urokinase plasminogen activator (u-PA). Cell surface binding protects u-PA from serum inhibitors like alpha 2-macroglobulin, enabling localized proteolytic activity.
Area of Science:
- Biochemistry
- Cell Biology
- Hematology
Background:
- Human RC2A myelomonocytic leukemia cells secrete prourokinase (pro-u-PA).
- Pro-u-PA can be activated to active urokinase plasminogen activator (u-PA).
- u-PA plays a role in extracellular matrix degradation and cell migration.
Purpose of the Study:
- To investigate the distribution and regulation of u-PA activity in RC2A leukemia cells.
- To determine the role of serum inhibitors and cell surface interactions in u-PA regulation.
- To elucidate the mechanism protecting cell-surface u-PA from inhibition.
Main Methods:
- Culturing RC2A cells in serum-free and serum-containing media.
- Measuring u-PA activity in conditioned medium and on the cell surface.
- Identifying serum inhibitors of u-PA using purified alpha 2-macroglobulin (alpha 2M).
- Investigating u-PA binding kinetics and complex formation with alpha 2M.
- Anchoring u-PA to plastic surfaces to mimic cell surface interactions.
Main Results:
- Active u-PA was found in serum-free medium and on the cell surface, but only on the cell surface in serum-containing medium.
- Serum rapidly inactivates free u-PA, while cell surface binding is a faster process.
- Alpha 2-macroglobulin (alpha 2M) was identified as the primary serum inhibitor of u-PA.
- Cell surface-bound u-PA is protected from alpha 2M inhibition, unlike u-PA in solution.
- Protection of cell surface u-PA from alpha 2M is likely due to steric hindrance.
Conclusions:
- Leukemia cell surface binding is a critical mechanism for maintaining active u-PA in the presence of serum inhibitors.
- This localized u-PA activity on the cell surface may contribute to leukemia cell invasion and progression.
- The findings highlight a novel regulatory mechanism for u-PA activity at the cell-host matrix interface.