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Biochemistry and Biophysics |
Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Maspin has a sequence homology with members in the serpin family including PAI-1 and PAI-2 (1) . In 1998, Sheng et al. (7) reported that purified recombinant maspin specifically inhibits tPA that is associated with fibrinogen or poly-lysine. However, purified maspin does not act as a classical serpin in cell-free solutions, i.e., it does not inhibit a series of serine proteases including tPA and uPA (7 , 8) . On the basis of the specific interaction between maspin and fibrinogen-associated tPA, it was speculated that maspin may target plasminogen activators that are bound to a biological surface such as the plasma membrane. Recently, McGowen et al. (9) described first evidence that the tumor cell surface-associated uPA is inhibited by recombinant maspin. The inhibitory effect of maspin on DU145 cell-mediated uPA activity was similar to that of uPA-neutralizing antibody, and was reversed by the polyclonal antibody made against the RSL sequence of maspin. Furthermore, the proteolytic inhibitory effect of recombinant maspin was quantitatively consistent with the inhibitory effect of maspin in cell migration.
It is intriguing to speculate that endogenous maspin may exert its tumor suppressive activity by targeting the cell surface-associated plasminogen activator(s). It is worth noting that, to date, the role of tPA in cell surface-mediated plasminogen activation remains unclear. However, uPA and its cell surface receptor uPAR have been shown to promote tumor metastasis by mediating pericellular plasminogen activation (10 , 11) . We report here that expression of endogenous maspin in prostate carcinoma cells DU145 resulted in a significant inhibition of uPA-mediated pericellular plasminogen activation. A maspin/uPA complex was detected both on the cell surface and in the conditioned medium of maspin transfectant clones. Consistently, the two active forms of uPA, HMW and LMW were significantly reduced in the conditioned medium. Interestingly, although the endogenous expression of maspin did not alter the transcription of the endogenous uPA and uPAR, it lead to a significantly reduced level of cell-associated uPAR protein. The maspin-induced reduction of secreted uPA and cell surface uPAR appears to result from the LRP-mediated internalization. Taken together, our data demonstrates a pleiotropic inhibitory effect of maspin on pericellular uPA system, thus supporting the hypothesis that maspin exerts its tumor suppressive function by inhibiting pericellular proteolysis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chain and ß-chain) were obtained from American Diagnostica (Greenwich, CT). The purified glutathione S-transferase-receptor associated protein fusion protein was kindly provided by Dr. Steven Gonias (University of Virginia Health Sciences Center, Charlottesville, VA). Reagents for protein concentration analysis and protein gel electrophoresis were obtained from Bio-Rad (Hercules, CA). Sulforhodamine B and other chemicals and reagents of the highest purity, unless otherwise specified, were purchased from Sigma (St. Louis, MO).
Cell Lines and Cell Culture.
Multiple stable maspin transfectant clones were generated by an established method (1)
using human prostate carcinoma cells DU145 (American Type Culture Collection) as the parental cell line. The transcription of maspin in these transfectant cells is under the control of the cytomegalovirus promoter. In parallel, mock transfectants were generated using the empty plasmid. Transfected cells were first selected based on their resistance to geneticin (G418) at 600 µg/ml and subsequently maintained at 300 µg/ml G418. The transfected cells were additionally screened for the expression of maspin using Western blot analysis and RT-PCR.
Both DU145 cells and the DU145-derived transfectant clones were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 5% fetal bovine serum. An additional 300 mg/ml of G418 was added to the culture medium for the stable transfectant clones. The immortalized normal human prostate epithelial cells MLC8891 (9) were cultured in serum-free KGM medium (Life Technologies, Inc.). All of the cell cultures were kept in a humidifier incubator at 37°C with 6.5% CO2.
To investigate whether the LRP regulates the secretion of uPA and the internalization and catabolism of uPAR in maspin expressing transfectants, equal number of maspin-expressing transfectant clones and a mock control clone were incubated for 24 h in KGM-SF in the presence or absence of 200 nM of glutathione S-transferase-receptor associated protein, a specific LRP inhibitor (12 , 13) . The resulting conditioned medium was collected and concentrated, whereas the cells were lysed in a low salt, protease inhibitor-rich buffer [4 mM NaHCO3, 100 mM NaF, 20 mM KH2PO4, 2 mM sodium orthovanadate, 5 mM EDTA, 5 mM diisopropylfluorophosphate, 2 mM phenylmethylsulfonyl fluoride, 2 ug/ml aprotinin, 2 ug/ml leupeptin (pH 7.2); Ref. 14 ] containing 1% Triton X-100. The resulting protein samples were used for Western analyses.
Protein Fractionation and Western Blotting.
Western analyses were performed as described (1)
using the cell surface eluates, total cell lysate protein, and SF-CM. The protein fractionation is briefly as follows. When the cells in culture reached approximately 70–80% confluence, the maintenance culture medium was replaced with KGM-SF. After 24 h of continued incubation, the conditioned medium was collected and subsequently concentrated using the Centricon-10 filter units (Amicon, Bedford, MA). In the meantime, the cells were detached with trypsin/EDTA followed by neutralization with trypsin inhibitor. The cell lysate was prepared by DONCE homogenization in a low salt, protease inhibitor-rich lysis buffer [4 mM NaHCO3, 100 mM NaF, 20 mM KH2PO4, 2 mM sodium orthovanadate, 5 mM EDTA, 5 mM diisopropylfluorophosphate, 2 mM phenylmethylsulfonyl fluoride, 2 ug/ml aprotinin, 2 ug/ml leupeptin (pH 7.2); Ref. 14
] containing 1% Triton X-100, followed by centrifugation. To elute the cell surface-bound uPA, confluent cultures were treated with 50 mM glycine-HCl (pH 3.0) containing 0.1 M NaCl for 3 min at 23°C (15)
. The acid eluate was neutralized with 0.5 M Tris-HCl (pH 7.8) and was subsequently concentrated using the Centricon-10 filter units. Equal amounts of the protein samples were denatured in the presence of DTT at 85°C for 5 min, resolved by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The blots were subsequently blotted for detecting maspin, uPA, and uPAR using the corresponding specific primary antibodies as indicated. The bound primary polyclonal antibody was then probed with HRP-conjugated antirabbit IgG (Amersham, Arlington Heights, IL), used at 2000-fold dilution. The bound primary monoclonal antibody was probed with HRP-conjugated antimouse IgG (Amersham) used at 1500-fold dilution. Finally, the immunoreactive bands were detected using the ECL detection system (Pierce) according to the manufacturer’s instruction.
RT-PCR Analysis.
The total RNA extracted from each cell line using the RNeasy Mini kit (Qiagen) was reversed transcribed with the AMV reverse transcriptase (Promega) in the presence of oligo(dT)15 primer as described by the manufacturer’s instruction. The resulting cDNA preparation was subjected to PCR amplification using template-specific upstream and downstream primers. The following oligonucleotide primers synthesized and purified by Life Technologies, Inc., were: maspin, 5'-GGGGAATTCCATGGATGCCCTGCAACT-3' and 5'-CCGGTCTAGACATGGGCTATGCCACTT-3; uPA, 5'-GGCAGCAATGAACTTCATCAAGTTCC-3' and 5'TATTTCACAGTGCTGCCCTCCG-3'; tPA, 5'-CCAGCAACATCAGTCATGGC-3' and 5'-GCACTTCCCAGCAAATCCTTC-3'; uPAR, 5'-ACAGGAGCTGCCCTCGCGAC-3' and 5-GAGGGGGATTTCAGGTTTAGG-3'; and GAPDH, 5'-ACGGATTTGGTCGTATTGGG-3' and 5'-TGATTTTGGAGGGATCTCGC-3'. Each RT-PCR cycle included a denaturation step at 94°C for 30 s; a primer-annealing step at 55°C for 45 s (maspin), at 56°C for 30 s (uPA), at 60°C for 1 min (uPAR and tPA), or at 62°C for 1 min (GAPDH); and an extension step at 72°C for 45 s. The numbers of thermal cycles were: 20 cycles for maspin; 25 cycles for uPA, uPAR, and tPA, and 30 cycles for GAPDH. Reactions were performed using a Genius programmable thermal controller model (Techne Incorporated, Duxford, Cambridge, United Kingdom). The PCR products were analyzed by electrophoresis on a 1% agarose gel containing EtBr, and photographed under UV light.
Northern Blot Analysis.
The cDNA probes for uPA, uPAR, and GADPH, generated by RT-PCR using specific upstream and downstream primers as described above, were labeled with HRP using the North2South Direct HRP Labeling kit (Pierce). Total cellular RNA was isolated from 70–80% confluent cell cultures using the RNeasy Mini kit (Qiagen). Samples of RNA (20 µg/each) were electrophoresed on a 1% formaldehyde agarose gel and blotted onto nylon membranes (Amersham Life Science). The RNA blots were hybridized at 55°C for 4 h with HRP-labeled cDNA probes for uPA, uPAR, and GADPH, respectively. After hybridization, the membrane blots were washed three times in 2 x SSC/0.1% SDS at 55°C for 15 min, and three times in 2 x SSC at room temperature for 15 min. The blots were then incubated with North2South chemiluminescent working solution for 5 min at room temperature and subsequently exposed to X-ray film.
Gelatinolytic Zymogram.
Equal numbers of cells from each cell (or clonal) line were plated and cultured in the maintenance medium for 48 h. Then the culture medium were replaced with KGM-SF. After 24 h of continued incubation, the conditioned medium were collected and subsequently concentrated using the Centricon-10 filter units. The resulting concentrated conditioned medium was analyzed by plasminogen-dependent and metalloprotease gelatinolytic zymograms, as described previously (16
, 17)
. Similarly, acidic eluates from different clonal lines were concentrated and analyzed by plasminogen-dependent zymography.
In Vitro Motility and Invasion Assays Using MICS.
Briefly, the serum-starved cells (24 h) from different clonal lines were seeded onto the 8-µm polycarbonate insert (precoated with 50 µg/ml Matrigel for motility and 4 mg/ml for invasion). To verify the specificity of the maspin effect in inhibiting tumor cell invasion, cells in suspension were preincubated with the neutralizing polyclonal antibody directed against maspin RSL sequence (Abs4A) at 0, 10, and 20 µg/ml, respectively. The MICS chamber was incubated for 6 h at 37°C with 6.5% CO2 to allow the cells to migrate. On the other hand, the MICS chamber was incubated for 48 h at 37°C with 6.5% CO2 to assess the invasive potential of the different clonal lines. Cells attached to the bottom side of the polycarbonate membrane insert were fixed, stained, and counted under the microscope as described previously (5)
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Others.
Experiments that were performed as described by McGowen et al. (9)
include coupled colorimetric assay for plasminogen activation in conditioned culture medium and coupled colorimetric assay for cell surface-associated plasminogen activation. Cell staining with sulforhodamine B was performed as described by Garbin et al. (18)
. Protein concentrations were determined using the Bio-Rad Protein concentration dye as instructed by the manufacturer.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As shown in Fig. 1A
, maspin was detected in both the total cell lysates and the conditioned medium of multiple maspin transfectant clones. The expression of maspin appeared to be less than that in normal immortalized prostate epithelial cells (MLC8891) and varied slightly among different maspin transfectant clones. The mock transfectant clone and the parental cell line DU145 expressed no detectable amount of maspin protein in either fraction. The endogenously expressed maspin had a molecular mass of 42 kDa, identical to that found in normal prostate epithelial cells MLC8891. Subsequent quantitative RT-PCR (Fig. 1B)
showed a similar maspin expression pattern as shown by Western blot. The stable DU145 cell-derived maspin transfectants as well as the mock transfectant cells had similar growth rate and similar morphological features as the parental cell line (data not shown).
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, as compared with parental cells DU145, all three of the maspin transfectant clones tested were inhibited by 
60% in the motility assay. In contrast, the mock transfectant clone exhibited a similar migratory potential as the parental cells. Similarly, a significant reduction of about 40–50% in the invasive potential was noted in maspin-expressing transfectants compared with that of the neo control (Fig. 1D)
. As shown in Fig. 1E
, the inhibitory effect of endogenous maspin on tumor cell invasion appeared to be specific. The maspin-neutralizing antibody Abs4A had no significant effect on the invasive potential of the mock transfectant clone. In parallel, however, Abs4A reversed the subdued invasive potential of a maspin transfectant clone in a dose-dependent manner. These data are in line with the notion that the anti-invasive effect of maspin is localized on the cell surface and depends on maspin RSL sequence.
Endogenous Expression of Maspin Inhibits the Cell Surface-bound uPA.
To examine whether the biological activity of the endogenously expressed maspin in inhibiting cell motility and invasion correlates with a specific inhibition of uPA on the cell surface, we measured the cell surface-associated plasminogen activation using the coupled colorimetric plasminogen activation assay. As shown in Fig. 2A
, the cell surface-mediated plasminogen activation activity in three maspin transfectant clones was significantly inhibited compared with that of either parental or a mock transfectant clone. The cell surface-mediated plasminogen activation in each case was effectively blocked by a uPA-neutralizing antibody but not by a tPA neutralizing antibody. These data are consistent with the earlier evidence that DU145 cell-mediated plasminogen activation depends primarily on uPA (9)
. A plasminogen-dependent zymogram was subsequently performed using the cell surface eluates to examine the maspin effect on uPA presentation on the cell surface. Compared with the uPA standard, the HMW uPA (activity) in the eluate derived from maspin transfectants was significantly diminished compared with that of the parental and neo control (Fig. 2B)
. Thus, the inhibition of the cell surface-mediated plasminogen activation correlates with a reduced level of cell-associated uPA in maspin-expressing transfectants.
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, a uPA ß-chain specific antibody detected a 54 kDa band, possibly a mixture of pro-uPA and the active HMW-uPA. This 54 kDa was greatly reduced in maspin transfectants as compared with both the parental and the neo control. The uPA antibody also detected an upper band of 100 kDa, which coincides with the combined molecular weight of maspin (42 kDa) and uPA (54 kDa). Importantly, this upper band was only detected in the eluate of maspin transfectant clones. In parallel, the monoclonal antibody to the chain of uPA similarly detected an upper band in the eluate derived from maspin transfectants but not in that of the parental or neo control (data not shown). Because others have shown that uPA forms an SDS-resistant 1:1 complex with inhibitory serpins PAI-1 and PAI-2 (20, 21, 22)
, it is likely that the uPA containing upper band detected in maspin transfectants represents the stable complex of maspin and uPA.
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may be a mixture of pro-uPA and HMW-uPA, the decreased level of 54 kDa uPA in maspin transfectants as detected under the nonreducing condition may result from the loss of active HMW-uPA because of the formation of a stable maspin/uPA complex. However, the possibility exists that endogenous maspin expression also led to a reduced level of pro-uPA. To address this possibility, Western blotting under a reducing condition was performed using the uPA ß-chain-specific antibody. It is known that the two subunits (24 kDa chain and 33 kDa ß-chain) in the active HMW-uPA are linked by disulfide bonds and can be separated on reduction (19)
. As shown in Fig. 3B
, the 33 kDa uPA ß-chain was detected in the active HMW-uPA standard, indicating that the two-chain active HMW-uPA had been successfully dissociated into two subunits under our reducing condition. The level of uPA ß-chain was significantly decreased in the eluate of maspin transfectant clones compared with that of the parental and neo control, indicating that less active HMW-uPA was associated with maspin transfectants. In addition, the level of pro-uPA (54 kDa) decreased significantly in the cell surface-associated protein fraction of maspin transfectant clones as compared with that of parental cells or mock control. These results indicate that maspin expression leads to the reduction of both pro-uPA and active HMW-uPA that are associated with the cell surface.
Decreased Level of Cell-associated uPAR in Maspin Transfectant Clones.
It has been well documented that the proteolytic activation of uPA (from pro- to active HMW-uPA) is mediated by its cell membrane-anchored uPAR (23
, 24)
. To test the possible involvement of uPAR in the inhibitory effect of maspin on the cell surface-bound uPA, cell lysates were analyzed by Western blotting of uPAR. As shown in Fig. 4A
, the polyclonal anti-uPAR (#399R; American Diagnostica, Greenwich, CT) detected a specific uPAR band of 54 kDa in both the mock transfectant clone and the parental cell line. However, little or no signal was detected in the lysates of three maspin transfectants clones. To additionally examine whether the reduction of uPAR protein in maspin transfectants was a result of the decreased transcription of uPAR, quantitative RT-PCR of uPAR was performed using an established method (25)
. As shown in Fig. 4B
, the steady-state levels of uPAR mRNA in DU145/M clones are comparable with that of the parental (DU145) and mock control (DU145/Neo1). Quantitative RT-PCR (Fig. 4B)
and Northern blot analyses (Fig. 4C)
were also performed to evaluate the steady-state level of mRNAs of uPA, tPA, and uPAR. All of the clonal lines tested expressed the mRNAs of tPA, uPA, and uPAR. Furthermore, the steady-state levels of the mRNAs of uPA and uPAR in maspin transfectant clones were similar to those found in the parental cells or transfection control cells, respectively. Thus, it appears that maspin affects uPA and uPAR at the protein level but not at the transcription step.
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54 kDa and 36 kDa, respectively (Fig. 6A)
, in the SF-CM of DU145 and neo control cells. The 54 kDa band represents a mixture of both the secreted pro-uPA and HMW-uPA, whereas the 36 kDa band represents the LMW-uPA in which ß-chain is linked to a small fragment of chain through disulfide bond (26
, 27)
. Both the 54 kDa band and the 36 kDa band were significantly reduced in the SF-CM of the maspin transfectant clones as compared with that in the SF-CM of DU145 cells and the mock transfectant control cells. In fact, the LMW-uPA species was barely detectable in the SF-CM of the maspin transfectant clones. In addition, an upper band was detected in the SF-CM of all three of the maspin transfectant clones but not of DU145 cells or the mock transfectant clone. Similarly, under nonreducing condition, a monoclonal antibody to maspin detected this upper band in the conditioned medium derived from maspin transfectants but not from the mock control cell line (Fig. 6C)
. The molecular weight of this upper band was estimated as 100 kDa, approximately the combined molecular weight of maspin and uPA.
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54 kDa and 33 kDa, respectively (Fig. 6B)
. The 54 kDa band represents the single chain pro-uPA, whereas the polypeptide 33 kDa corresponds to the ß-chain of uPA derived from both the active HMW-uPA and the active LMW-uPA. Both the pro-uPA and the dissociated uPA ß-chain were significantly reduced in the SF-CM of maspin transfectant clones as compared with that of DU145 cells or mock control cells. It is worth noting that the upper band of 100 kDa detected in the SF-CM of maspin transfectant clones under nonreducing condition was not detected under the reducing condition. Consistent results were obtained from Western analyses using uPA -chain-specific antibody (data not shown).
To examine whether the 36 kDa LMW-uPA detected by Western blotting has the expected enzyme activity, plasminogen-dependent gelatinolytic zymogram was performed. As shown in Fig. 6D
, in addition to a clear zone corresponding to the 54 kDa HMW-uPA, the clear zone of the 36 kDa LMW-uPA was detected in the SF-CM of the mock transfectant control cells. Both HMW-uPA and LMW-uPA decreased dramatically in the SF-CM of maspin transfectant clones. In parallel, gelatinolytic zymogram of metalloproteinase showed a similar level of pro-matrix metalloprotease-2 and pro-matrix metalloprotease-9, respectively, in all of the SF-CM samples tested (data not shown), additionally confirming an equal sample loading.
Previous studies have shown that the LRP promotes the endocytosis of uPA/uPAR complex on the interaction of uPA with its inhibitor such as PAI-1 (12
, 13)
. As shown in Fig. 7
, treatment with receptor-associated protein (RAP), a specific inhibitor of LRP, significantly increased the steady-state level of secreted uPA in the conditioned medium as well as cell surface uPAR protein in maspin transfectants. In contrast, no significant increase in secreted uPA and cell surface uPAR expression by the LRP inhibitor RAP was observed in the mock transfectant control cell line. This data suggests that the reduction of secreted uPA and cell surface-associated uPAR protein in maspin transfectants is in part attributable to the increased LRP-mediated clearance.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The current paradigm of the inhibitory interaction between a serpin and its target protease states that the catalytic unit of the protease binds to the serpin RSL, forming a stable serpin:protease complex. Our previous data showed that the tumor suppressive and biochemical activity of maspin is dependent on its intact RSL, supporting the notion that maspin acts as an inhibitory serpin (5
, 7)
. In the current study, a maspin/uPA complex was detected on the cell surface and in the conditioned medium of maspin transfectants by immunoblotting only under nonreducing conditions but not under a reducing condition. Our data indicate that maspin can complex only with the intact heterodimeric HMW-uPA. Thus, the inhibitory effect of maspin on cell-associated uPA may involve not only the interaction between the RSL of maspin with the catalytic site containing ß-chain of uPA but may also require an association between another segregated functional domain of maspin with the -chain subunit of uPA. Consistently, Sheng et al. (7)
have shown that recombinant maspin inhibited the sctPA associated with poly-lysine or fibrinogen through its RSL but stimulated the free sctPA via its NH2-terminal 38 kDa fragment. This biphasic effect of recombinant maspin on sctPA derives from its two segregated domains interacting with the catalytic and the regulatory domains of sctPA, respectively.
Two other potent biological inhibitors of uPA, PAI-1 and PAI-2, have been shown to form a stable complex with uPA (20, 21, 22) . The detection of an uPA/maspin complex on the cell surface in this study indicates that endogenous maspin indeed acted as an inhibitory serpin similar to PAI-1 and PAI-2 (28 , 29) . However, unlike PAI-1 and PAI-2 that also inhibit soluble uPA, maspin does not inhibit free uPA in solution (7 , 8) or in conditioned cell culture medium (9) . The current study, along with previous in vitro evidence (9) , supports a hypothesis that direct interaction between endogenous maspin and uPA on the cell surface causes pleiotropic changes in the molecular profile of the pericellular uPA system and leads to a dramatic decrease of uPA-medicated plasminogen activation both on the cell surface and in the conditioned medium.
A body of literature demonstrates that the molecular interactions among factors in the pericellular uPA system are dynamically regulated (30 , 31) . The proteolytic activation of pro-uPA to two-chain active uPA on the cell surface is mediated by uPAR (32 , 33) . Maspin forms a stable complex with uPA on the cell surface and inhibits the uPA-mediated cell surface plasminogen activation. In addition, we noticed that maspin expression led to a significant and simultaneous reduction of cell surface-bound uPA and cell-associated uPAR. These data suggest an involvement of uPAR in the interaction between maspin with uPA. Our results showed that the expression of neither uPA nor uPAR at the mRNA level was altered by the constitutive expression of maspin. In addition, the presence of the LRP inhibitor RAP resulted in a significant increase of both secreted uPA and cell surface uPAR protein in maspin transfectants but not in the mock control clone. Thus, it is likely that the reduced level of secreted uPA and cell surface uPAR in maspin transfectant cells may in part be attributable to the increased LRP-dependent internalization of a maspin/uPA/uPAR complex. Our data are additionally in line with the earlier evidence with recombinant PAI-1 that the binding of PAI-1 to the uPAR-bound uPA triggers LRP-dependent endocytosis and enhances the catabolism of uPA and uPAR (12 , 13 , 34, 35, 36, 37, 38) .
The previous study by McGowen et al. (9)
showed that purified maspin does not form a complex with secreted uPA in the conditioned culture medium. Thus, it is likely that the maspin/uPA complex detected in the SF-CM of maspin transfectants resulted from the shedding from the cell surface. In the case of maspin transfectants, as the stable maspin/uPA complex is continuously shed from the cell surface and accumulates in the conditioned medium (Fig. 6)
, a smaller amount of active HMW-uPA would be secreted to the conditioned medium (Fig. 6)
. Because active HMW-uPA is derived from the secreted pro-uPA, as more maspin/uPA complex is formed, more pro-uPA would be depleted from the conditioned medium (as compared with the parental and mock transfectant control, Fig. 6
). Moreover, it has been shown that the active HMW-uPA undergoes additional cleavage, producing an active LMW-uPA (26
, 27)
. Our Western blotting and zymographic analysis revealed that endogenous maspin expression correlates with a dramatically reduced level of the LMW-uPA in the conditioned medium. It is possible that the complex formation between maspin and uPA on the cell surface may, by reducing the availability of the active HMW-uPA, also limit the formation and shedding of the LMW-uPA.
Whereas our new evidence addresses the biochemical characteristics of maspin as a novel inhibitory serpin against pericellular uPA system, the changes of molecular profile in this system provoked by endogenous maspin expression may lead to additional indirect changes in other cellular pathways. For example, because uPAR has been shown to directly interact with and regulate the function of integrins, the transmembrane receptors of extracellular matrix proteins (39 , 40) , the reduction of cell-associated uPAR in the presence of maspin may help explain an earlier observation that maspin treatment changed the integrin profile on breast carcinoma cell surface and altered cell adhesion to specific extracellular substrata in favor of a normal epithelial phenotype (6) .
Expression of uPA by malignant cells correlates with an aggressive phenotype including increased tumor cell invasion and metastasis, presumably through the activation of plasminogen and the resulting pericellular matrix degradation (10 , 11) . Consistent with our earlier evidence with purified recombinant maspin, data from the current study demonstrated an important role of epithelial cell surface in mediating the inhibitory interaction between endogenous maspin and pericellular uPA. The proteolytic inhibitory effect of endogenous maspin coincided with a significant inhibition of cell motility and invasion potential in vitro. Furthermore, the anti-invasive effect of maspin appeared to be localized on the cell surface and dependent on its RSL sequence. Whereas extensive studies are under way to investigate whether uPA produced by other types of carcinoma cells is inhibited by maspin, it is intriguing to hypothesize that novel maspin-based therapeutic strategies may prove useful to specifically target human malignancies that are associated with markedly elevated uPA. To this end, it is important to point out, however, that different plasminogen activator inhibitors may play distinct roles in tumor progression. For example, PAI-1 along with uPA and uPAR, is causatively involved in the progression of breast cancer. In contrast, maspin, which is down-regulated in several types of carcinomas, has a tumor suppressive activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by a Ruth Sager Memorial Fund (to S. S.), a Prostate Cancer Pilot Grant CA69845 from National Cancer Institute/Wayne State University (to S. S.), NIH Grant CA84176 (to S. S.), and a Virtual Discovery Grant from the Karmanos Cancer Institute (to S. S.). 
2 To whom requests for reprints should be addressed, at Wayne State University, Department of Pathology, Gordon H. Scott Hall, Basic Medical Sciences, 540 E. Canfield Avenue, Detroit, MI 48201. Phone: (313) 993-8197; Fax: (313) 993-4112; E-mail: ssheng{at}med.wayne.edu
3 The abbreviations used are: serpin, serine protease inhibitor; uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor; PAI, plasminogen activator inhibitor; RSL, reactive site loop; HMW, high molecular weight; LMW, low molecular weight; LRP, low-density lipoprotein receptor-related protein; KGM-SF, serum-free KGM; HRP, horseradish peroxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EtBr, ethidium bromide; MICS, membrane invasion culture system; SF-CM, serum-free conditioned medium; sctPA, single-chain tissue-type plasminogen activator.
Received 5/30/01. Accepted 10/17/01.
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 R.-A. Toillon, C. Lagadec, A. Page, V. Chopin, P.-E. Sautiere, J.-M. Ricort, J. Lemoine, M. Zhang, H. Hondermarck, and X. Le Bourhis Proteomics Demonstration That Normal Breast Epithelial Cells Can Induce Apoptosis of Breast Cancer Cells through Insulin-like Growth Factor-binding Protein-3 and Maspin Mol. Cell. Proteomics, July 1, 2007; 6(7): 1239 - 1247. [Abstract] [Full Text] [PDF]
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 E. Tsoli, P. K Tsantoulis, A. Papalambros, B. Perunovic, D. England, D. A Rawlands, G. M Reynolds, D. Vlachodimitropoulos, S. L Morgan, C. A Spiliopoulou, et al. Simultaneous evaluation of maspin and CXCR4 in patients with breast cancer J. Clin. Pathol., March 1, 2007; 60(3): 261 - 266. [Abstract] [Full Text] [PDF]
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X. Li, S. Yin, Y. Meng, W. Sakr, and S. Sheng Endogenous inhibition of histone deacetylase 1 by tumor-suppressive maspin. Cancer Res., September 15, 2006; 66(18): 9323 - 9329. [Abstract] [Full Text] [PDF]
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 N. Cella, A. Contreras, K. Latha, J. M. Rosen, and M. Zhang Maspin is physically associated with {beta}1 integrin regulating cell adhesion in mammary epithelial cells FASEB J, July 1, 2006; 20(9): 1510 - 1512. [Abstract] [Full Text] [PDF]
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S. Yin, J. Lockett, Y. Meng, H. Biliran Jr., G. E. Blouse, X. Li, N. Reddy, Z. Zhao, X. Lin, J. Anagli, et al. Maspin Retards Cell Detachment via a Novel Interaction with the Urokinase-Type Plasminogen Activator/Urokinase-Type Plasminogen Activator Receptor System. Cancer Res., April 15, 2006; 66(8): 4173 - 4181. [Abstract] [Full Text] [PDF]
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T. D. Shellenberger, A. Mazumdar, Y. Henderson, K. Briggs, M. Wang, C. Chattopadhyay, A. Jayakumar, M. Frederick, and G. L. Clayman Headpin: A Serpin with Endogenous and Exogenous Suppression of Angiogenesis Cancer Res., December 15, 2005; 65(24): 11501 - 11509. [Abstract] [Full Text] [PDF]
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S. Yin, X. Li, Y. Meng, R. L. Finley Jr., W. Sakr, H. Yang, N. Reddy, and S. Sheng Tumor-suppressive Maspin Regulates Cell Response to Oxidative Stress by Direct Interaction with Glutathione S-Transferase J. Biol. Chem., October 14, 2005; 280(41): 34985 - 34996. [Abstract] [Full Text] [PDF]
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 J Schedel, O Distler, M Woenckhaus, R E Gay, B Simmen, B A Michel, U Muller-Ladner, and S Gay Discrepancy between mRNA and protein expression of tumour suppressor maspin in synovial tissue may contribute to synovial hyperplasia in rheumatoid arthritis Ann Rheum Dis, October 1, 2004; 63(10): 1205 - 1211. [Abstract] [Full Text] [PDF]
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 K. Tanaga, H. Bujo, Y. Zhu, T. Kanaki, S. Hirayama, K. Takahashi, M. Inoue, K. Mikami, W. J. Schneider, and Y. Saito LRP1B Attenuates the Migration of Smooth Muscle Cells by Reducing Membrane Localization of Urokinase and PDGF Receptors Arterioscler Thromb Vasc Biol, August 1, 2004; 24(8): 1422 - 1428. [Abstract] [Full Text] [PDF]
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J. Liu, S. Yin, N. Reddy, C. Spencer, and S. Sheng Bax Mediates the Apoptosis-Sensitizing Effect of Maspin Cancer Res., March 1, 2004; 64(5): 1703 - 1711. [Abstract] [Full Text] [PDF]
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 Y. Akiyama, C. Maesawa, S. Ogasawara, M. Terashima, and T. Masuda Cell-Type-Specific Repression of the Maspin Gene Is Disrupted Frequently by Demethylation at the Promoter Region in Gastric Intestinal Metaplasia and Cancer Cells Am. J. Pathol., November 1, 2003; 163(5): 1911 - 1919. [Abstract] [Full Text] [PDF]
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C. Ngamkitidechakul, D. J. Warejcka, J. M. Burke, W. J. O'Brien, and S. S. Twining Sufficiency of the Reactive Site Loop of Maspin for Induction of Cell-Matrix Adhesion and Inhibition of Cell Invasion: CONVERSION OF OVALBUMIN TO A MASPIN-LIKE MOLECULE J. Biol. Chem., August 22, 2003; 278(34): 31796 - 31806. [Abstract] [Full Text] [PDF]
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 M. L. Cher, H. R. Biliran Jr., S. Bhagat, Y. Meng, M. Che, J. Lockett, J. Abrams, R. Fridman, M. Zachareas, and S. Sheng Maspin expression inhibits osteolysis, tumor growth, and angiogenesis in a model of prostate cancer bone metastasis PNAS, June 24, 2003; 100(13): 7847 - 7852. [Abstract] [Full Text] [PDF]
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R. Bass, A.-M. M. Fernandez, and V. Ellis Maspin Inhibits Cell Migration in the Absence of Protease Inhibitory Activity J. Biol. Chem., November 27, 2002; 277(49): 46845 - 46848. [Abstract] [Full Text] [PDF]
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), 10 µg/ml (
), and 20 µg/ml (
) of Abs4A. D and E, cell invasion is expressed as the total number of cells that invaded Matrigel in a modified Boyden chamber assay. C–E, data represent an average of three repeats; bars, ± SD.
A550 nm). Data represent an average of three experiments; bars, ± SD. B, plasminogen-dependent gelatinolytic zymogram using the acidic cell eluates derived from different clonal lines. The purified HMW-uPA was used as a standard. Equal amount of concentrated eluate (5 µg) was loaded per lane.
), DU145/Neo1 cells (
), DU145/M3 cells (
), DU145/M7 (
). The data represent an average of triplicate repeats. SEs are not shown for clarity.
