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J. Biol. Chem., Vol. 277, Issue 49, 46845-46848, December 6, 2002

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ACCELERATED PUBLICATION
Maspin Inhibits Cell Migration in the Absence of Protease Inhibitory Activity^*

Rosemary Bass, Ana-María Moreno Fernández, and Vincent Ellis

From the School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

Received for publication, September 19, 2002, and in revised form, October 13, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Maspin is a member of the serpin family of protease inhibitors and is a tumor suppressor gene acting at the level of tumor invasion and metastasis. This in vivo activity correlates with the ability of maspin to inhibit cell migration in vitro. This behavior suggests that maspin inhibits matrix-degrading proteases, such as those of the plasminogen activation system, in a similar manner to the serpin PAI-1. However, there is controversy concerning the protease inhibitory activity of maspin. It is devoid of activity against a wide range of proteases, in common with other non-inhibitory serpins, but has recently been reported to inhibit plasminogen activators associated with cells and other biological surfaces (Sheng, S. J., Truong, B., Fredrickson, D., Wu, R. L., Pardee, A. B., and Sager, R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 499-504; McGowen, R., Biliran, H., Jr., Sager, R., and Sheng, S. (2000) Cancer Res. 60, 4771-4778). We have compared the effects of maspin with those of PAI-1 in a range of situations in which plasminogen activation is potentiated, reflecting the biological context of this proteolytic system: urokinase-type plasminogen activator bound to its receptor on the surface of tumor cells, tissue-type plasminogen activator specifically bound to vascular smooth muscle cells, fibrin, and the prion protein. Maspin was found to have no inhibitory effect in any of these situations, in contrast to the efficient inhibition observed with PAI-1, but nevertheless maspin inhibited the migration of both tumor and vascular smooth muscle cells. We conclude that maspin is a non-inhibitory serpin and that protease inhibition does not account for its activity as a tumor suppressor.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Proteolytic activity is a key event in cell migration and invasion, being required to dynamically modulate interactions between the cell and its surrounding extracellular matrix (1). Multiple protease systems are implicated in this process, including the serine proteases of the plasminogen activation system (2). In the pericellular environment the activity of this system is regulated by binding of the proteases or their zymogens to specific cell-surface receptors or binding sites. The plasminogen activator uPA¹ binds to uPAR, a well characterized glycosylphosphatidylinositol-anchored membrane protein (3); tPA binds to cell-surface proteins on cell types including endothelial (4) and VSMC (5, 6); and plasminogen binds to multiple cell-surface molecules (7). These interactions lead to assembly of complexes on the cell surface that greatly increase plasmin generation (5, 8, 9). The activity of tPA is also potentiated by binding to protein cofactors, including fibrin (10) and PrP (11). This powerful proteolytic system is inhibited by members of the serpin (serine protease inhibitor) family, in particular PAI-1 (SERPINE1), which can inhibit free, cofactor-bound and cell-associated plasminogen activators (6, 12). The protease inhibitory activity of PAI-1 has been shown to be of importance in vivo, inhibiting VSMC migration (13) and regulating tumor angiogenesis (14), and its expression correlates with disease progression and prognosis in human cancers (15).

Some serpins have biological activities independent of protease inhibition. For example, PAI-1 binds to vitronectin, modulating cell adhesion and migration (16). Other serpins lack intrinsic inhibitory activity. Examples of this are ovalbumin, thyroid-binding globulin (SERPINA6), angiotensinogen (SERPINA8), and pigment epithelium-derived factor (SERPINF1), which has neurotrophic and anti-angiogenic activity (17, 18). Maspin (SERPINB5) is thought to be another non-inhibitory serpin.

Maspin was first identified as a class II tumor suppressor in human breast cancer (19), and transfection of maspin into carcinoma cells reduces their metastatic potential in vivo (19, 20). It is a predominantly cytoplasmic protein, but is also secreted to the cell surface (21), where it has been shown to reduce the migration of various cell types in vitro (22, 23) and to inhibit angiogenesis in both in vitro and in vivo models (24). These activities of maspin are consistent with those of a protease inhibitor, yet extensive biochemical characterization has failed to demonstrate a protease target for maspin, and it lacks key features of inhibitory serpins (25). Therefore the mechanisms underlying its biological activities are considered to be largely unresolved (26). However, recent studies have suggested that maspin does exhibit inhibitory activity toward the plasminogen activators uPA and tPA, but only when these proteases are bound to macromolecular cofactors, that is tPA bound to fibrin (27) and uPA on the cell surface (28, 29).

Using techniques that we have previously established to investigate the activity and inhibition of cell-surface plasminogen activators, we demonstrate here that maspin has no inhibitory activity against these protease in either cellular environments or other situations in which their activities are potentiated and that reflect the biological context of this proteolytic system. Nevertheless, maspin was able to inhibit cell migration, strongly suggesting that this activity of maspin is not dependent on protease inhibition.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Recombinant maspin expressed in Saccharomyces cerevisiae (25) was obtained from Andy Robertson (Department of Biochemistry, University of Iowa). tPA (Actilyse) was obtained from Boehringer-Ingleheim (Ingleheim, Germany). Recombinant PAI-1 was obtained from Calbiochem and its concentration determined by titration against tPA (6). Lys-plasminogen (i.e. with Lys⁷⁷ as N terminus) was obtained from Enzyme Research Laboratories (Swansea, UK). The soluble fibrin fragment preparation Desafib-X was obtained from American Diagnostica (Greenwich, CT). Recombinant PrP was prepared as described previously (11). The fibrosarcoma cell line HT-1080 and DU 145 prostate carcinoma cells were from ATCC, and VSMC of aortic origin were isolated and cultures as described previously (5).

Determination of Cell-surface Plasminogen Activation-- Plasminogen activation by uPAR-bound uPA on the surface of HT-1080 and DU 145 cells was determined as described previously (8). In brief, cells grown to confluence in 24-well plates were washed in phosphate-buffered saline to remove unbound uPA and incubated at 37 °C with varying fixed concentrations of plasminogen (20-200 nM), the plasmin specific substrate H-d-Val-Leu-Lys-AMC (0.25 mM), and varying concentrations of maspin or PAI-1. Plasmin generated by endogenously bound uPA was measured continuously as change in fluorescence in a SpectraMAX Gemini microplate reader (Molecular Device, Sunnyvale, CA) at  360/440 nm. Plasmin concentration was determined as F and plasmin generation represented as F versus time. Second-order inhibition rate constants were calculated from inhibition curves according to (30), as described previously (6, 12).

Plasminogen activation by tPA bound to VSMC was determined essentially as described previously (5, 6). In brief, cells grown to confluence in 24-well plates were incubated with tPA (10 nM) for 20 min at 37 °C, washed extensively to remove unbound tPA, and plasminogen activation determined as described above. In these experiments plasmin generation was represented as F versus time².

Determination of Cofactor-stimulated Plasminogen Activation-- tPA-catalyzed plasminogen activation stimulated by fibrin was determined by incubation of tPA (1.5 nM), Lys-plasminogen (25 nM), and varying concentrations of fibrin fragments in 0.05 M Tris-HCl, 0.1 M NaCl, pH 7.4, containing H-d-Val-Leu-Lys-AMC (0.25 mM). In preliminary experiments the fibrin concentration giving maximal stimulation (~250-fold) was determined and found to be 250 µg/ml. This concentration was used for all subsequent experiments. Varying concentrations of either maspin or PAI-1 were included in these experiments and inhibition of plasminogen activation determined as described above.

Similar experiments were performed to determine the effect of PrP on tPA inhibition by maspin. Recombinant PrP in its divalent metal ion-free form (apo-PrP) was included, in place of fibrin, at an optimal concentration of 50 µg/ml leading to more than a 250-fold stimulation of plasmin generation (11).

Cell Migration Assays-- Cell migration was determined using time-lapse video microscopy. VSMC were seeded into four-well plates at a density of 7500 cells/ml/well in Medium 231 containing Smooth Muscle Cell Growth Supplement (Cascade Biologics, Portland, OR). After 24 h the medium was changed to L15 air-buffered medium (Sigma), 0.1% bovine serum albumin containing varying concentrations of maspin (0-200 nM). Cell movement was recorded by computerized time-lapse video microscopy (Nikon, Kingston upon Thames, UK) with images acquired every 5 min for 15 h. 10-20 cells were tracked per movie and cell movement quantified using Lucia 32G/Magic 4.11 software (Nikon) and expressed as micrometer/hour.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of Maspin on uPAR-bound uPA-- The best characterized pericellular proteolytic system is the uPA/uPAR system, which specifically activates cell-associated plasminogen. We have previously shown that uPA bound to cellular uPAR is efficiently inhibited by PAI-1, with kinetics similar to those in solution (12). Fig. 1A shows the inhibition of endogenous uPA on HT-1080 fibrosarcoma cells. Increasing concentrations of PAI-1 (up to 20 nM) lead to a complete inhibition of uPA activity in a time-dependent manner, consistent with the standard serpin inhibitory mechanism. The calculated second-order inhibition rate constant, 4.1 × 10⁶ M¹ s¹, compares with 7.9 × 10⁶ M¹ s¹ determined for uPA in solution.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Comparison of inhibitory effects of maspin and PAI-1. A and B, inhibition of uPAR-bound uPA on HT-1080 cells. Washed cells were incubated with Lys-plasminogen (30 nM) and Val-Leu-Lys-AMC and plasmin generation by endogenously bound uPA determined in the presence of 0 (), 0.2 (), 0.5 (), 2 (), 5 () and 20 nM () PAI-1 or 0 (), 10 (), 50, (), 100 (), and 200 nM () maspin. C and D, inhibition of tPA bound to VSMC. Cells were incubated tPA (10 nM), washed extensively, and activation of Lys-plasminogen (30 nM) determined as above in the presence of 0 (), 0.5 (), 5 (), and 50 nM () PAI-1 or 0 (), 10 (), 100 (), and 200 nM () maspin. E and F, inhibition tPA bound to fibrin. tPA (1.5 nM) was incubated with fibrin fragments (250 µg/ml), Lys-plasminogen (25 nM), and Val-Leu-Lys-AMC and plasmin generation determined in the presence of 0 (), 0.1 (circo]), 1, (), and 10 nM () PAI-1 or 0 (), 5 (), 50 (), and 500 nM () maspin. Experiments similar to those shown here were also performed using native Glu-plasminogen with comparable results.

In sharp contrast to this, maspin at concentrations up to 200 nM completely failed to inhibit uPA activity (Fig. 1B). Decreasing the concentration of plasminogen in the experiment to greater than 10-fold below K_m, to minimize possible competitive effects on the reaction with maspin, did not lead to an observable inhibitory effect. In the absence of cells, uPA bound to recombinant soluble uPAR was also not inhibited by maspin (data not shown). From these data in Fig. 1B it can be estimated (assuming a minimum detection level of 5% inhibition) that the maximum value of the rate constant for uPA inhibition by maspin is ~400 M¹ s¹, 4 orders of magnitude less than for inhibition by PAI-1.

Experiments were also performed using DU 145 prostate carcinoma cells (as used in the study of McGowen et al. (28)), and a similar lack of inhibition by maspin was observed (data not shown).

Effect of Maspin on tPA Bound to VSMC-- tPA is also known to associate with certain cell types, and we have shown that VSMC bind tPA and stimulate its activity more than 100-fold (5, 6). This involves a putative receptor-mediated mechanism analogous to the uPAR-dependent mechanism for the activation of cell-associated plasminogen. Fig. 1, C and D, show that tPA specifically bound to VSMC is efficiently inhibited by PAI-1, but again no inhibition was detectable at maspin concentrations of up to 200 nM.

Plasmin generation at the highest maspin concentration was consistently increased, but was also observed with the non-inhibitory serpin ovalbumin (data not shown), suggesting an additional stimulation possibly by cleaved serpin. A similar effect has previously been observed (27), and high concentrations of both maspin and ovalbumin led to a small stimulation of tPA activity in solution (data not shown).

Effect of Maspin on tPA Bound to Fibrin-- tPA activity is stimulated, in the absence of cells, by binding to fibrin. This involves the binding of tPA and plasminogen to fibrin as a catalytic "template" and direct effects on catalytic activity. In the presence of this very specific stimulatory mechanism, PAI-1 is still an effective inhibitor of tPA (Fig. 1E), but maspin once again failed to manifest inhibitory activity.

Effect of Maspin on tPA Bound to PrP-- We have recently shown that certain conformations of PrP can enhance tPA-catalyzed plasminogen activation by greater than 300-fold by a template mechanism involving independent and specific interactions of PrP with plasminogen and tPA (11). Maspin was also unable to inhibit tPA activity in this environment (data not shown).

Maspin Inhibits Cell Migration in the Absence of Protease Inhibitory Activity-- Previous studies have correlated the inhibitory effects of maspin on cell migration to the inhibition of plasminogen activator activity (28, 29). We have determined the effect of maspin on the migration of VSMC using time-lapse video microscopy. Fig. 2 shows that maspin inhibited VSMC migration in a biphasic manner, consistent with previous observations on other cell types (22, 28). Migration of HT-1080 cells was also inhibited in a similar manner (data not shown). Interestingly, the time course of migration in the presence of maspin was linear (Fig. 2, inset), suggesting that maspin exerts an immediate inhibitory effect. These experiments both verify the biological activity of the maspin used in these experiments and more importantly demonstrate that the inhibitory effect of maspin on cell migration, thought to be intimately involved in its tumor suppressing activity, is not a consequence of protease inhibition.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Maspin inhibits VSMC migration. The effect of varying concentrations of maspin on VSMC migration was assessed by time-lapse video microscopy. Data shown represent the means and S.E. of three independent experiments performed over 15 h. The inset shows the time course of migration in the presence of 50 nM maspin () compared with control ().

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The activity of the plasminogen activation system is regulated by two opposing mechanisms: cell-surface-binding sites and protein cofactors, which facilitate productive catalytic interactions with plasminogen and thereby potentiate plasmin generation, and serpin inhibitors, which temporally and spatially restrict the activities of the proteases. These mechanisms have been shown to have a complex interplay in vivo, for example, in the regulation of angiogenesis (31). The ability of maspin to inhibit cell migration and its tumor suppressing activity in vivo are consistent with the inhibition of pericellular proteases, particularly the plasminogen activators. However the data presented here show that in a wide variety of situations in which the functional activity of the plasminogen activation system is highly up-regulated, and in which PAI-1 is an extremely effective inhibitor, maspin has no detectable inhibitory effect. Despite this lack of protease inhibitory activity, maspin was nevertheless able to inhibit the migration of both VSMC and tumor cells in a biphasic manner consistent with previous reports (22).

Our observations are consistent with the molecular characteristics of maspin in relation to current knowledge of serpin mechanisms. This involves complex formation between protease and serpin and cleavage at the P1 residue of the reactive-site loop (RSL) followed by insertion of this loop into the major -sheet as a new central strand and translocation of the protease to the opposite pole of the serpin leading to structural alterations in the now covalently bound protease (32). This mechanism is critically dependent on a number of features of the serpin RSL, one being its length (32, 33). In the far majority of inhibitory serpins the RSL has 17 residues (determined from the Glu residue of the proximal hinge region to the reactive-site P1 residue) and 16 residues in the remainder (Fig. 3). Although a three-dimensional structure is not available for maspin, its sequence suggests that it has the shortest RSL of both the inhibitory and non-inhibitory serpins. Arg³⁴⁰ is the putative P1 residue in maspin, giving an RSL of just 13 residues. A potential alternative P1 residue for cleavage by serine proteases with trypsin-like specificity is Lys³⁴⁵, which would give an 18-residue RSL. The length of neither of these RSLs appears to be compatible with protease inhibition. For maspin to have an RSL of 16 or 17 residues, the P1 residue would be either Gln³⁴³ or His³⁴⁴, neither of which is a suitable P1 residue for serine protease inhibition. Gln is not found as a P1 residue in any serpin, and His is found only in the "fast" isoform of ₁-PI from guinea pig, a species with multiple ₁-PI isoforms and homologs (34), suggesting that this protein may not be inhibitory.

View larger version (62K): [in this window] [in a new window]   Fig. 3.   Comparison of serpin RSL sequences. The sequence of various serpins is shown from the Glu residue that marks the start of the RSL (P17 in most serpins) to the C-terminal Pro residue (those serpins that are extended beyond this are marked). Sequences are grouped according to RSL length (either 17 residues or 16 residues in a small subset of serpins) and compared with maspin and the known non-inhibitory serpins. The reactive site cleavage is shown as | but is left blank for the non-inhibitory serpins, and residues strictly conserved in the inhibitory serpins are highlighted in bold. The trivial name is shown to the right of the sequence, and the gene name is shown to the left (all sequences are human with the exceptions of ovalbumin and Serpin 2 from Myxoma virus).

Another critical feature of the RSL is its sequence, as incorporation of the RSL into the -sheet requires residues to be compatible with adopting  conformation and not to involve burial of unfavorable side chains (33). Maspin lacks the Ala-rich sequence found in the RSL of most inhibitory serpins, instead having bulky or charged residues including Ile³³⁴ and Glu³³⁵. Pro³³⁷ at the P8 position is particularly unfavorable, being a Thr residue in the majority of inhibitory serpins and a critical determinant of RSL insertion (35). P14 is also important in regulating serpin inhibitory function and is also a Thr residue in the inhibitory serpins but Gly in maspin. The introduction of a P14 Thr  Gly mutation in PAI-1 leads to a significant reduction in inhibitory activity (36). Maspin also lacks the hinge region P12 Ala residue found in all inhibitory, but never in non-inhibitory, serpins. A corollary of the serpin mechanism is that RSL cleavage by non-target proteases induces a transition from a "stressed" (S) to a "relaxed" (R) form by incorporation of the cleaved RSL into the major -sheet, equivalent to the insertion occurring during the inhibitory mechanism. However, it has previously been shown that maspin does not undergo this hallmark S  R transition on cleavage at Arg³⁴⁰, the putative P1 residue (25), consistent with the preceding structural considerations. Other non-inhibitory serpins also fail to undergo this conformational transition (37-39). These observations strongly suggest that maspin cannot be an inhibitory serpin, in agreement with our failure to detect inhibition of plasminogen activator activity under a wide range of conditions.

Our conclusions differ from those of Sheng and co-workers, who claimed that maspin has protease inhibitory activity against both tPA bound to a fibrin surface (27) and uPA associated with tumor cells (28, 29). This is not easily reconciled, but two lines of argumentation can be proposed that support our conclusions. The first is that the previously reported effects were not characteristic of the standard covalent serpin inhibitory mechanism, being more suggestive of competitive inhibition. Non-inhibitory serpins act as protease substrates and, at sufficiently high concentrations, will act as competitive inhibitors in the same way as other competing substrates. However, as the kinetic mechanism underlying the stimulation of plasminogen activation in the various situations studied here is a large reduction in the K_m for plasminogen, the reaction with substrate plasminogen is highly favored and reactions with potential competing substrates equally disfavored. We have used plasminogen concentrations both above and below K_m, but no effects indicative of maspin behaving as a competing substrate were observed. Therefore, our data suggest that surface-bound plasminogen activators are no different to the soluble proteases in their reactivity with maspin, with neither being inhibited. The second consideration, in the case of uPA, is that our observations are consistent with the known independence of the C-terminal catalytic domain from the N-terminal uPAR-binding domain (40) and our previous observations on the mechanism of enhancement of plasminogen activation by uPAR. We have shown that the catalytic activity of uPA is not affected by uPAR and that the enhanced plasminogen activation is due to the formation of catalytically favored complexes with cell-associated plasminogen (8, 9, 41). A consequence of this is that both uPAR-bound uPA and uPA in solution are inhibited by the plasminogen activator inhibitors PAI-1 and PAI-2 with similar kinetics (12). Therefore, the reaction of receptor-bound uPA with other serpins would be expected to be similarly unaffected, i.e. maspin would not be expected to inhibit either free or cell-associated uPA.

Maspin has been reported to bind specifically to the cell surface (23, 28), raising the possibility that this interaction conformationally converts maspin into an inhibitory form or facilitates a reaction between uPA and maspin by close juxtaposition of the proteins. Neither of these possibilities are compatible with the previous considerations regarding the requirements for an inhibitory RSL, although the latter could favor competitive substrate-like behavior. However, our observations provide no evidence for such an effect under conditions where the inhibitory effects of PAI-1 are readily detected. Although it cannot be completely excluded that specific conditions favor inhibitory-like activity in maspin, the observations here that maspin inhibits cell migration in the absence of detectable protease inhibitory activity demonstrates that this is not the mechanism responsible for the biological activity of maspin.

Our data are consistent with reports that maspin both directly and indirectly affects cell adhesion, a critical event in the regulation of cell motility. Maspin has recently been shown to bind directly to collagen, an interaction that may contribute to cell adhesion (42). Interestingly, maspin has also been shown to alter the expression profile of integrins in breast carcinoma cells, in particular inducing the expression of the ₅₁ fibronectin receptor (43). Although we have shown here that maspin does not inhibit the activity of uPAR-bound uPA, the reported increase in ₅₁ expression by maspin may potentially lead to an indirect effect on this proteolytic system. uPAR is known to associate with ₅₁ (44), and recent observations in this laboratory indicate that this interaction may lead to a reduction in uPA binding and a concomitant reduction in cell-surface plasminogen activation.² Therefore, despite lacking protease inhibitory activity, it is possible that maspin can indirectly influence the activity of the cell-surface plasminogen activation system and that this mechanism may contribute to its function as a tumor suppressor.

	ACKNOWLEDGEMENT

We thank Andy Robertson (University of Iowa) for the gift of recombinant maspin.

	FOOTNOTES

^* This work was supported by Grant PG/1999079 from the British Heart Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Senior Research Fellow of the British Heart Foundation. To whom correspondence should be addressed: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK. Tel.: 44-1603-592570; Fax: 44-1603-592250; E-mail: v.ellis@uea.ac.uk.

Published, JBC Papers in Press, October 15, 2002, DOI 10.1074/jbc.C200532200

² R. Bass, F. Berditchevski, and V. Ellis, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: uPA, urokinase-type plasminogen activator; uPAR, cellular receptor for uPA; tPA, tissue-type plasminogen activator; RSL, reactive-site loop; PrP, prion protein; VSMC, vascular smooth muscle cells; AMC, amido-4-methylcoumarin; ₁-PI, ₁-proteinase inhibitor; S, stressed; R, relaxed.

	REFERENCES

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