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Departments of Urology (X.L., J.B.T., J.H., B.L.) and Pathology (J.P.), Kansas Cancer Institute, University of Kansas Medical Center, Kansas City, Kansas 66160; and Department of Chemistry and Biochemistry (Q.-X.A.S.), Florida State University, Tallahassee, Florida 32306
Address all correspondence and requests for reprints to: Benyi Li, M.D./Ph.D., Kansas University Medical Center Urology, 3901 Rainbow Boulevard, Lied 1042, Kansas City, Kansas 66160. E-mail: bli{at}kumc.edu.
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Introduction |
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The matrix metalloproteinase (MMP) family is comprised of secreted and membrane-associated zinc-dependent endopeptidases that can selectively degrade extracellular matrix (ECM) protein and nonmatrix proteins. Currently, up to 25 members of the MMP family have been reported, and the broad range of their substrates conveys a pivotal role for the MMP involvement in normal physiological processes and pathological states including tumor metastasis and angiogenesis (reviewed in Ref. 4). MMP-2, also called gelatinase A, is produced as a latent form (pro-MMP-2), and the activation process is mediated at least partially by MT1-MMP on the cell surface (5). It has been shown that MMP-2 is secreted by the human prostate gland, both in vivo and in vitro, and higher expression levels of MMP-2 are associated with increasing Gleason score, tumor metastasis, and aggressive behavior of prostate cancer (Refs. 6 and 7 and reviewed in Ref. 8).
To understand the role of the androgen receptors (ARs) in prostate cancer development and progression, it is important first to determine the AR signaling cascades and the genes that are regulated by AR. In view of the evidence for the association of MMP-2 expression and prostate cancer behavior, we evaluated the expression of pro-MMP-2 after androgen treatment in human prostate cancer LNCaP and LAPC-4 cells, which express a functional AR.
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Materials and Methods |
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Western blot analysis
For immunoblot analysis, cells were washed in PBS and lysed in a radioimmunoprecipitation assay buffer supplied with protease inhibitors (CytoSignal, Irvine, CA). Equal amounts of protein were separated on an 8% sodium dodecyl sulfate-polyacrylamide gel and blotted onto a polyvinyl difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA). Membranes were blocked in a Tris-buffered saline solution with 5% nonfat dry milk and incubated with antibodies overnight at 4 C. Immunoreactive signals were detected by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) followed by chemiluminescent detection (SuperSignal West Dura substrate kit, Pierce Chemical Co., Rockford, IL).
Assay of gelatin-degrading MMPs by zymography
Unconcentrated conditioned media from the cell cultures were analyzed for MMP gelatinolytic activities by gelatin zymography as described previously (11). Briefly, conditioned media (mixed with 5x sample buffer) were fractionated by SDS-PAGE on a 10% gel containing 1.0 mg/ml gelatin (Sigma) under nonreducing conditions. After two washes in Tris buffer (50 mM Tris, 200 mM NaCl, 10 mM CaCl2, 1 mM ZnCl2, 1% Triton X-100, pH 7.5), the gel was incubated in the same buffer in the absence of Triton X-100 for 18 h at 37 C. After being stained with Coomassie Brilliant Blue R-250, the gel was destained with 10% (vol/vol) acetic acid, and the nonstaining bands resulting from digestion of the substrate by gelatinase enzymes were then visualized.
Cell migration assay
After serum starvation, the cells were trypsinized and resuspended in RPMI 1640 with 5% cFBS. A Transwell insert with 8-µM pore (Nunc, Naperville, IL) was coated with collagen (50 µg/ml in PBS) for 2 h at 37 C. A total of 1.0 x 105 cells were then seeded in the upper chambers of the Transwells. R1881 was added in both the upper and lower chambers containing RPMI 1640 supplied with 5% cFBS. Where indicated, cells were preincubated with inhibitors (5.0 µM OA-Hy or 10 µM LY294002) for 30 min at room temperature before seeding in the Transwell. Cells were incubated for 48 h, and then the chamber was disassembled. Cells on the upper surface were removed, and cells invaded to the lower surface of the filters were fixed, stained, and counted as described (12).
Luciferase and SEAP reporter assay
A luciferase reporter plasmid controlled by the 1716-bp length of the human MMP-2 promoter (MMP2-LUC) was obtained from Dr. Yi Sun (13, 14). The human MMP-9 gene promoter-luciferase vector (MMP9-LUC) was obtained from Dr. Yasuyuki Sasaguri (15). The reporter vector pCMV-SEAP, expressing secreted alkaline phosphatase (SEAP) under the control of the cytomegalovirus (CMV) promoter, was a kind gift from Dr. David Spencer (16) and was used as an internal reference control. The cells were plated in 6-well tissue culture plates and transfected the following day with 2.0 µg MMP-2 reporter construct and 0.5 µg pCMV-SEAP construct by using the Cytofectene reagent (Bio-Rad Laboratories, Inc.) according to manufacturer’s protocol. After 24 h, the cells were serum starved for another 24 h and then treated with R1881 (1.0 nM) or PMA (50 µM) in 2% cFBS. After 24 h, culture supernatants were harvested and assayed for SEAP activity as described previously (17). Cells were lysed with a lysis buffer supplied by a luciferase assay system (catalog no. 4030, Promega Corp., Madison, WI). Protein concentration in the cell lysates was measured by a protein assay kit (Bio-Rad Laboratories, Inc.). An equal amount of protein from each cell lysate was assayed in triplicate for luciferase enzyme activity by using the luciferase assay system (Promega Corp.) and Lumat LB9501 reader (Berthold, Oak Ridge, TN). The luciferase activity of each sample was normalized against the corresponding SEAP activity before the fold induction value relative to control cells was calculated.
Statistical analysis
All experiments were repeated two or three times. Zymographic data and Western blot results are presented from a representative experiment. The mean and SD from two experiments for cell migration and luciferase assay are shown. The number of migrating cells in the absence of either R1881 or inhibitors is assigned a relative value of 100%. The significant differences between groups were analyzed using the SPSS computer software (SPSS, Inc., Chicago, IL).
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Results |
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Under serum-free condition and the solvent control, there was no detectable MMP-2 gelatinolytic activity in the conditioned media from the cell culture (Fig. 1
, A and E, lanes 1 and 2). On R1881 addition, MMP gelatinolytic activity corresponding to pro-MMP-2 was gradually increased in a dose-dependent manner (lanes 3–6). Furthermore, an additional active form of MMP-2 appeared after higher doses of R1881 in LNCaP cells (1.0–10 nM in lanes 5 and 6) but not in LAPC-4 cells, which may reflect a cell-based specificity. To confirm the MMP-2 induction by R1881 stimulation, MMP-2 protein levels in the whole-cell lysates were determined by Western blot analysis. As shown in Fig. 1B, on R1881 stimulation, the cellular level of pro-MMP-2 protein was increased in a dose-dependent manner. The protein levels of pro-MMP-2 matched well with the gelatinolytic activities secreted into the conditioned media. However, MMP-9 gelatinolytic activity was not detectable under our experimental conditions (Fig. 1, A and E), which is consistent with our previous report (20). The protein level of cellular pro-MMP-9 (Fig. 1C) was unchanged by R1881 treatment. These data indicate that androgen induces pro-MMP-2 but not pro-MMP-9 expression in LNCaP and LAPC-4 cells.
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, as is the case for PSA, pretreatment with bicalutamide completely abolished androgen-induced pro-MMP-2 expression in LNCaP cells (lane 3 vs. lane 2). Paralleling the protein expression, androgen-induced MMP-2 gelatinolytic activity was also suppressed in the presence of bicalutamide (Fig. 2A, zymography panel). However, bicalutamide itself when added alone had no effect on MMP-2 expression (data not shown).
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, lanes 4 and 5 vs. lane 2). However, addition of actinomycin D or cycloheximide did not affect AR protein levels (Fig. 2A, AR panel, lanes 4 and 5 vs. lane 2), indicating that suppression of R1881-induced pro-MMP-2 expression by the two inhibitors is not due to reduced expression of AR protein. In contrast, AR protein was slightly increased in the presence of R1881 (Fig. 2A, AR panel, lane 1 vs. lanes 2–5), which is consistent with a previous report (24). These results indicate that androgen induction of MMP-2 is due to AR-mediated gene expression (in other words, AR transactivation).
To further confirm the involvement of AR transactivation in androgen induction of pro-MMP-2, we used a luciferase reporter construct under the control of human MMP-2 promoter (1659 bp of the 5' region on MMP-2 gene) to define the stimulating effect of androgen on MMP-2 promoter activity (13). After transfection with the reporter constructs, LNCaP cells were serum starved for 24 h and then stimulated with R1881 (1.0 nM). Luciferase activity in the cell extracts was measured 24 h later. As shown in Fig. 2B, R1881 treatment induced about 3.5-fold increase of the luciferase activity, which was abolished by bicalutamide addition (10 µM), consistent with the results as seen in zymography and Western blot (Fig. 2A). As expected, MMP-9 promoter activity was not affected by R1881 treatment but was strongly induced by a well-known MMP-9 stimulator PMA (25). In addition, PMA suppressed the basal activity of the MMP-2 promoter by almost 50% (Fig. 2B), which is in accordance with a previous report (26). When LAPC-4 cells were used for those luciferase assays, a very similar result was also observed (Fig. 2C). These results clearly suggest that androgen-stimulated pro-MMP-2 expression is mediated through AR transactivation.
MMP-2 is produced in a latent form, which is activated by the membrane type MMP, MT1-MMP, in participation with tissue inhibitor of MMP-2 (5). Because we observed an active form of MMP-2 in gelatinolytic zymography assays when LNCaP cells were treated with higher doses of R1881 (Fig. 1A), we checked the MT1-MMP expression to rule out the possibility that MT1-MMP is activated after R1881 treatment. As expected, MT1-MMP was expressed at a relatively low level in LNCaP cells (Fig. 3), which is consistent with a previous report (27). After R1881 treatment, however, there was no significant alteration to the protein levels of either the pro-form (65 kDa) or the active form (63 kDa) of MT1-MMP (28). These results indicate that MT1-MMP is constitutively expressed in LNCaP cells, and its activity or expression level is not regulated by AR signaling. Appearance of the MMP-2 active form after R1881 treatment at higher doses might be due to increased pro-MMP-2 expression, which in turn leads to subsequent accumulation of the active form in the media.
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, among the inhibitors used in this study, only the PI3K inhibitor LY294002 completely abolished the androgen induction of MMP-2 in LNCaP cells. To examine whether the inhibitory effect of LY294002 on androgen-induced MMP-2 gelatinolytic activity is in parallel with the protein expression but not caused by inhibition of MMP-2 secretion, protein levels of pro-MMP-2 from equal amounts of whole-cell lysates were determined by Western blot. As shown in Fig. 4B, two commonly used PI3K inhibitors (LY294002 and wortmannin) totally abolished pro-MMP-2 and PSA expression. MEK1 inhibitor PD98059 had no effect on pro-MMP-2 expression, which was consistent with the zymography data. In addition, the inhibitors alone did not cause any change, as shown in the right panel of Fig. 4B. These results suggest that PI3K is involved in AR-mediated pro-MMP-2 expression, which is consistent with previous reports that PI3K activity is required for the AR transactivation (29, 30, 31).
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) and gelatin zymography (data not shown). As expected, FGF-2 stimulation increased pro-MMP-2 expression dramatically. In contrast to androgen stimulation, pro-MMP-2 induction after FGF-2 stimulation was not suppressed by either the androgen antagonist bicalutamide or PI3K inhibitor LY294002 but by MEK1 inhibitor PD98059 (Fig. 4C). A similar result was also observed when LAPC-4 cells were used (data not shown). These results indicate that the regulatory mechanism for MMP-2 gene expression is stimulus specific in cells.
Inhibition of MMP or PI3K suppresses LNCaP cell migration in response to R1881
Recently, MMPs have been shown to be correlated with tumor dissemination because of their proteolytic activity on ECM, and tumor cell migration is one of the most important events contributing to tumor dissemination (reviewed in Ref. 35). Because we observed androgen induction of pro-MMP-2 expression in LNCaP cells, we next asked whether MMP-2 inhibition could suppress cell migration in response to R1881 stimulation. Using a collagen-coated Transwell chamber assay, we assessed the ability of LNCaP cells to undergo unstimulated, steroid-depleted serum-stimulated, or R1881-stimulated migration. Without serum stimulation, almost no sign of migration was observed for LNCaP cells (data not shown), which is consistent with a previous report (36). However, addition of charcoal-stripped serum (steroids were depleted) stimulated cell migration. When R1881 (1.0 nM) was added in the culture, cell migration was significantly enhanced by almost 2-fold, compared with that incubated with charcoal-stripped serum alone (Fig. 5). To define the responsibility for MMP-2 in R1881-enhanced cell migration, we evaluated the effect of PI3K inhibitor LY294002 and a potent MMP-2 specific inhibitor OA-Hy (37) on LNCaP cell migration. Indeed, R1881-enhanced LNCaP cell migration was almost completely abolished by pretreatment with either OA-Hy (5.0 µM) or LY294002 (10 µM). In addition, we noticed that LY294002 alone or LY294002 plus R1881 resulted in a slight decrease (not statistical significant) of cell migration, compared with charcoal-stripped serum control, which is consistent with a previous report that PI3K activity is fundamental for serum-stimulated cell migration (38).
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Discussion |
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Historically, MMP-2 has been considered a constitutive gene because of a lack of well-characterized regulatory elements in the MMP-2 promoter region (reviewed in Ref. 39). However, recent studies of the MMP-2 promoter sequence analyses have revealed a number of potential cis-acting regulatory elements including p53, activator protein-1, Ets-1, CCAAT/enhancer-binding protein, cAMP response element-binding protein, polymavirus enhancer activator 3, surfactant protein 1, and activator protein-2 that could be involved in regulation of MMP-2 expression (13, 14, 40). Several factors, such as TGFß1 (7), UVB/IL-8 (41), concanavalin A (42), short-term exposure to 
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-interferons (43), and transfection of c-Ha-ras (44) or Akt1 (45), have been previously reported to induce MMP-2 expression. In contrast, others including retinoic acid (46), PMA (26), long-term exposure to - and -interferons (43), and the calcium influx inhibitor carboxy amidotriazole (47) were shown to suppress MMP-2 expression. We demonstrated for the first time in this study that MMP-2 expression was elevated when prostate cancer LNCaP and LAPC-4 cells were stimulated with androgen, adding androgen as a new member to the list of MMP-2 modulators. Our results are consistent with previous reports that long-term treatment with androgen increased prostate MMP-2 content in rats (48). However, another sex hormone, estrogen, was reported to inhibit MMP-2 expression in human fibroblast-derived cells (49), further suggesting that MMP-2 regulation is cell and stimulus specific.
Androgens such as R1881 produce most cellular responses through their cognate nuclear receptor, AR. On binding to the hormone, the AR forms homodimer. The dimerized protein then interacts with specific DNA sequences directly through an androgen-responsive element (ARE; reviewed in Ref. 50) or indirectly through other transcription factors that bind DNA in the regulatory region of the target gene promoters, such as Ets (51). The result is an alteration in protein synthesis and the generation of a cellular response. In this study, we demonstrated that R1881-stimulated pro-MMP-2 expression was abolished by the androgen antagonist bicalutamide that can block AR-mediated gene transcription (23) and by RNA transcription-specific inhibitor actinomycin D and protein translation inhibitor cycloheximide. Furthermore, in a luciferase reporter assay, human MMP-2 promoter activity was induced by R1881 stimulation that was also abolished by bicalutamide addition. These data all indicate that the expression of MMP-2 gene is regulated by androgen via an AR transactivation mechanism, although the possibility of an indirect effect of AR on pro-MMP-2 expression could not be fully ruled out. Recently interaction of AR with Ets protein has been reported to negatively modulate MMP-1, 3, and 7 (51), and an Ets-1-binding site was found in the MMP-2 promoter region (14). However, it might be unlikely that androgen-stimulated pro-MMP-2 expression is also via an interaction between AR and Ets-1 because androgen positively induces pro-MMP-2 expression.
The location, sequence, and number of AREs associated with a given androgen target gene varies, although androgen-responsive regions typically contain multiple nonconsensus AREs (5'-TGTTCT-3'; Ref. 52). By analyzing the published sequence of the MMP-2 gene promoter (13), we noticed that there are two potential ARE-like motifs located in the promoter of the MMP-2 gene (-1539-TGTTcCT-1503, and -609-TGTaTCT-603) with one nucleotide mismatch (italicized). Further characterization of the MMP-2 promoter for ARE elements is being carried out currently by our group.
It has been shown that overexpression of the Akt1 gene induces MMP-2 activity in mouse mammary epithelial cells (45), but reintroduction of the PTEN gene reduces MMP-2 gene expression in human glioma cells (53). In human prostate cancer LNCaP cells, PI3K-Akt activity is elevated because of an inactive mutant of the PTEN gene (54). Although the molecular basis in AR signaling is not fully understood, an involvement of PI3K-Akt and PTEN pathways was recently reported (29, 30, 31). Consistent with those reports, we also observed that AR-mediated pro-MMP-2 expression is PI3K dependent, further demonstrating that PI3K activity is required for AR transactivation. However, the detailed mechanism for PI3K-Akt’s involvement needs further investigation.
Extensive work on the mechanisms of tumor invasion and metastasis has determined the MMP’s function as key factors in the process of tumor dissemination. In this regard, the increased expression of various MMPs is strongly associated with tumor invasiveness. Specifically, MMP-2 expression is elevated in metastatic prostate cancers (reviewed in Ref. 8). Previous studies have pointed out that tumor cells achieve increased motility through mechanisms that circumvent the requirement for exogenous motogenic factors (e.g. androgen or other serum-derived factors). Among the signal cascades initiated by the factors, the PI3K-dependent pathway is essential in cell migration (38). The LNCaP cell line is the one among those cell lines that require serum stimulation to migrate (36). The failure of LNCaP cells to migrate was not due to their inability to adhere to or spread on ECM but to a requirement of extracellular factors to activate intracellular signals necessary for motility (36). In this study, we also observed androgen-enhanced LNCaP cell migration. Androgen-enhanced cell migration was totally blocked by the MMP-2 specific inhibitor OA-Hy, and the PI3K inhibitor LY294002. These results suggest that androgen-induced MMP-2 played a role in androgen-enhanced cell migration.
In conclusion, we have presented for the first time that pro-MMP-2 expression is induced by androgen-stimulated AR transactivation in human prostate cancer LNCaP and LAPC-4 cells. In addition, AR-mediated pro-MMP-2 expression, like PSA, is dependent on an active PI3K pathway, which is consistent with previous reports (29, 30, 31). Moreover, androgen-enhanced LNCaP cell migration is suppressed by the MMP-2 inhibitor OA-Hy or PI3K inhibitor LY294002. These findings suggest that AR-mediated pro-MMP-2 expression may participate in the process of prostate development or prostate cancer invasion/metastasis.
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Acknowledgments |
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Footnotes |
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Abbreviations: AR, Androgen receptor; ARE, androgen-responsive element; cFBS, charcoal-stripped FBS; CMV, cytomegalovirus; ECM, extracellular matrix; FBS, fetal bovine serum; FGF, fibroblast growth factor; MEK, MAPK and ERK kinase; MMP, matrix metalloproteinase; MMP9-LUC, human MMP-9 gene promoter-luciferase vector; PI3K, phosphatidylinositol 3'-kinase; PMA, phorbol 12-myristate 13-acetate; pro-MMP-2, latent form of MMP-2; PSA, prostate-specific antigen; SEAP, secreted alkaline phosphatase.
Received December 16, 2002.
Accepted for publication January 22, 2003.
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