Maspin Retards Cell Detachment via a Novel Interaction with the Urokinase-Type Plasminogen Activator/Urokinase-Type Plasminogen Activator Receptor System

Shuping Yin, Jaron Lockett, Yonghong Meng, Hector Biliran, Grant E. Blouse, Xiaohua Li, Neelima Reddy, Zimin Zhao, Xinli Lin, John Anagli, Michael L. Cher, Shijie Sheng
2006 Cancer Research  
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
more » ... aspin may block the pericellular proteolysis mediated by cell surface-associated uPA. In the current study, maspin significantly inhibited the Ca 2+ 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 plasminmediated pro-uPA cleavage. Interestingly, substitution of maspin p 1 V site Arg 340 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. (Cancer Res 2006; 66(8): 4173-81) (S. Yin). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Figure 6. A critical role of maspin RSL in the interaction with pro-uPA, the inhibition of pro-uPA cleavage, and the inhibition of cell detachment. A, ELISA detection of protein-protein interaction. a, ELISA detection of rMaspin and Mas R340A bound to immobilized pro-uPA. The bindings of rMaspin and Mas R340A were normalized against that of BSA and are presented as fold differences. b, ELISA detection of rMaspin and Mas R340A bound to immobilized plasmin. The bindings of rMaspin and Mas R340A were normalized against that of BSA and are presented as fold differences. c, Coomassie blue-stained SDS-PAGE loaded with 50 Ag lysate of insect cells infected with the mock baculovirus (lane 1), 50 Ag lysate of insect cells infected with Mas R340A -encoding baculovirus (lane 2 ), 5 Ag purified Mas R340A (lane 3), and 5 Ag purified rMaspin (lane 4). Molecular weight standards (left ) . B, equilibrium binding of pro-uPA to maspin ÀFL . Changes of fluorescence of maspin ÀFL are plotted against the concentration of pro-uPA. Inset, dose-dependent replacement of pro-uPA-bound maspin ÀFL (formed by incubating 10 nmol/L maspin ÀFL and 50 nmol/L pro-uPA) by unlabeled rMaspin. C, effects of rMaspin and Mas R340A on in vitro plasmin-mediated pro-uPA cleavage. Proteins resulting from the indicated reaction conditions were resolved by nonreducing SDS-PAGE for Western blot analyses. The same membrane was probed for both uPA (top ) and maspin (bottom ). D, effects of Mas R340A and rPAI-1 on DU145 detachment. Adherent DU145 cells were analyzed by the sulforhodamine B assay after the detachment treatment in the presence of Mas R340A ( ) and rPAI-1 (n) at the indicated concentrations. The cell adherence data were normalized against the control data obtained with BSA at the corresponding concentrations. The result of a parallel detachment experiment in the presence of 20 Ag/mL rMaspin (5) is included as a reference. Columns/points, averages of four independent titrations; bars, SE. SEs are not shown in the inset of (C ) for clarity.
doi:10.1158/0008-5472.can-05-3514 pmid:16618739 fatcat:cs5zpia2kza3tce2bofhwwqlyu