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Published on: May 19, 2016
Maspin retards cell detachment via a novel interaction with the urokinase-type plasminogen activator/urokinase-type
Shuping Yin1, Jaron Lockett, Yonghong Meng
1Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Abstract:
It is well documented that tumor suppressive maspin inhibits tumor cell invasion and extracellular matrix remodeling. Maspin is a cytosolic, cell surface-associated, and secreted protein in the serine protease inhibitor superfamily. Although several molecules have been identified as candidate intracellular maspin targets, the extracellular maspin target(s) remains elusive. Although maspin does not directly inhibit urokinase-type plasminogen activator (uPA) activity, we have shown evidence that maspin may block the pericellular proteolysis mediated by cell surface-associated uPA. In the current study, maspin significantly inhibited the Ca2+ reduction-induced detachment of DU145 cells. This maspin effect was associated with increased and sustained levels of mature focal adhesion contacts (FAC). We noted that maspin (a) colocalized with uPA and uPA receptor (uPAR), (b) enhanced the interaction between uPAR and low-density lipoprotein receptor related protein, and (c) induced rapid internalization of uPA and uPAR. The maspin effects on surface-associated uPA and uPAR required the interaction between uPA and uPAR. Further biochemical and biophysical analyses revealed that maspin specifically bound to pro-uPA with a deduced K(d) of 270 nmol/L and inhibited the plasmin-mediated pro-uPA cleavage. Interestingly, substitution of maspin p1' site Arg340 in the reactive site loop (RSL) with alanine not only abolished the binding to pro-uPA but also diminished the maspin effects on pro-uPA cleavage and cell detachment. These data show an important role of maspin RSL in regulating the uPA/uPAR-dependent cell detachment. Together, our data led to a new hypothesis that maspin may stabilize mature FACs by quenching localized uPA/uPAR complex before uPA activation.
Insights
Maspin, a tumor suppressor, stabilizes cell adhesion by interacting with urokinase-type plasminogen activator (uPA) and its receptor (uPAR). This interaction prevents cell detachment and extracellular matrix remodeling, highlighting maspin
Area of Science:
- Oncology
- Cell Biology
- Biochemistry
Background:
- Maspin is a known tumor suppressor protein involved in inhibiting cancer cell invasion and extracellular matrix remodeling.
- While intracellular targets of maspin are identified, its extracellular targets and mechanisms remain largely unknown.
- Maspin's potential role in modulating cell surface-associated urokinase-type plasminogen activator (uPA) activity has been suggested but not fully elucidated.
Purpose of the Study:
- To investigate the extracellular targets and mechanisms of maspin in regulating cell adhesion and invasion.
- To determine how maspin affects the urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) system and focal adhesion contacts (FACs).
- To elucidate the role of maspin's reactive site loop (RSL) in its interaction with pro-uPA and its functional consequences.
Main Methods:
- Cell detachment assays using DU145 cells treated with maspin and calcium reduction.
- Immunofluorescence microscopy to assess maspin colocalization with uPA/uPAR and focal adhesion contacts (FACs).
- Biochemical and biophysical analyses, including surface plasmon resonance, to determine maspin's binding affinity to pro-uPA and its effect on pro-uPA cleavage.
Main Results:
- Maspin significantly inhibited calcium-induced cell detachment, correlating with increased mature focal adhesion contacts (FACs).
- Maspin colocalized with uPA and uPAR, enhanced uPAR interaction with LRP, and induced rapid internalization of uPA/uPAR complexes.
- Maspin specifically bound to pro-uPA (K(d) = 270 nmol/L) and inhibited its plasmin-mediated cleavage, with the maspin RSL being critical for these interactions.
Conclusions:
- Maspin stabilizes mature focal adhesion contacts (FACs) by interacting with and potentially sequestering the uPA/uPAR complex before uPA activation.
- The reactive site loop (RSL) of maspin is crucial for its binding to pro-uPA and its inhibitory effects on cell detachment.
- These findings reveal a novel mechanism by which maspin regulates pericellular proteolysis and cell adhesion, impacting tumor progression.
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