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College of Pharmacy, Duksung Women’s University, Seoul 132-714, Korea [M-S. K., E-J. L., A. M.], and Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201 [H-R. C. K.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To unveil ras downstream signaling pathways critical for tumor progression, we investigated previously the effects of H-, and N-ras on cancerous phenotypic changes using MCF10A human breast epithelial cell lines in which H- and N-ras are constitutively activated by mutation of gly to asp in the amino acid codon 12 in H- and N-ras, respectively (7) . We showed that H-ras, but not N-ras, induced an invasive phenotype in MCF10A human breast epithelial cells, whereas both induced transformed phenotype as determined by anchorage-independent growth and foci formation (7) .
Downstream effector molecules of ras include members of the MAPK family, composed of JNK, ERK, and stress-activated protein kinase-2 (p38). MAPKs are among the central elements that transduce extracellular signaling into cellular responses (8 , 9) and are believed to play pivotal roles in proliferation, apoptosis, differentiation, cytoskeleton remodeling, and cell cycle regulation (10, 11, 12, 13) . Recent evidences suggest a role for the MAPK members in invasive phenotype and/or cell motility in various cell systems. p38 has been demonstrated to be an important mediator of the cell motility responses in endothelial cells (14 , 15) and smooth muscle cells (16) . The ERK pathway was shown to be involved in the invasive or migrative behavior of human glioblastoma cells (17) , vascular smooth muscle cells (18) , and FG carcinoma cells (19) , whereas Matsumoto et al. (15) reported that ERK, unlike p38, is not a mediator of porcine aortic endothelial cell motility responses elicited by platelet-derived growth factor.
Members of the MMP family play a critical role in tumor invasion and metastasis formation, especially, MMP-2 (Mr 72,000 type IV collagenase, gelatinase A) and MMP-9 (Mr 92,000 type IV collagenase, gelatinase B; Refs. 20 , 21 ). MMP-9 has been suggested to be critical for the induction of an invasive phenotype in rat embryonic fibroblast cells (22 , 23) . In contrast, our previous study demonstrated that H-ras-induced invasive phenotype of MCF10A cells is associated more closely with the expression of MMP-2 than MMP-9 (7) . Whereas the significance of MAPK family members in the regulation of MMP-9 expression has been well documented (24, 25, 26, 27) , the regulatory mechanisms responsible for the MMP-2 expression are poorly understood at present.
In the present study, we investigated the role of the MAPK family members in H-ras-induced invasive phenotype and motility of MCF10A cells. Here, we report that H-ras-mediated cell migration and invasive phenotype involves activation of both p38 and ERKs, whereas N-ras activated ERKs are insufficient to induce cell migration and invasive phenotype. We also present evidence suggesting that the activation of p38 and, to a lesser degree, ERKs may contribute to a more invasive phenotype possibly via up-regulation of MMP-2 expression in human breast epithelial cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transfection.
Transfection was performed using Lipofectamine reagent (Life Technologies, Inc., Rockville, MD) following the manufacturer’s instruction. The DN construct of p38 (28)
was kindly provided by Dr. Young-Joon Surh (College of Pharmacy, Seoul National University, Seoul, Korea). DN MEK-1 (29)
and DN JNK-1 (30)
were kindly provided by Dr. Yong J. Lee (Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA).
Immunoblot Analysis.
Equal amounts of protein extracts in SDS-lysis buffer were subjected to 12% SDS-PAGE analysis and electrophoretically transferred to nitrocellulose membrane. Anti-JNK-1, anti-phosphorylated JNK, anti-ERK-1,2, antiphosphorylated ERK-1,2, and antiphosphorylated p38 antibodies were purchased from New England Biolabs (Beverly, MA). Anti-p38 antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY). Enhanced chemiluminescence (Amersham-Pharmacia, Buckinghamshire, United Kingdom) system was used for detection. Relative band intensities were determined by quantitation of each band with an Image Analyzer (Vilber Lourmet, Marne-la-Vallee Cedex 1, France).
Gelatin Zymography.
Cells were cultured in serum-free DMEM/F12 medium for 48 h. Conditioned medium was collected and centrifuged at 3000 rpm for 10 min to remove cell debris. The protein concentration was measured using BCA protein assay reagents (Pierce, Rockford, IL). Equal amounts of protein of conditioned media were mixed with 2x Laemmli nonreducing sample buffer, incubated for 15 min at room temperature, and then electrophoresed on 10% SDS-PAGE gels containing 1 mg/ml gelatin. After electrophoresis, the gels were washed with 2.5% Triton X-100 three times for 30 min, rinsed for 15 min with 50 mM Tris-HCl buffer (pH 7.6) containing 5 mM CaCl2, 0,02% Brij-35, and 0.2% sodium azide, and incubated overnight at 37°C. The gels were stained with 0.5% Coomassie Brilliant Blue R-250 solution containing 10% acetic acid and 20% methanol for 30 min and destained with 10% acetic acid solution. Areas of gelatinase activity were detected as clear bands against the blue-stained gelatin background.
MTT Assay.
Cells (1 x 104) cultured in a 96-well plate were treated with SB203580 or PD98059. MTT solution purchased from Promega Corporation (Madison, WI; 5 mg/ml) was added to the medium, and the cells were additionally incubated for 4 h. After 100 µl of supernatant was replaced with 100 µl of DMSO, absorbance of each well was measured at 540 nm with a micro-ELISA reader (Molecular Devices, Sunnyvale, CA). Percentage of cell survival was determined as the relative absorbance of treated versus untreated cells.
In Vitro Invasion Assay.
In vitro invasion assay was performed using 24-well Transwell unit with polycarbonate filters (Corning Costar, Cambridge, MA) as described previously (7)
. The lower side of the filter was coated with type I collagen, and the upper side was coated with Matrigel (Collaborative Research, Lexington, KY). Lower compartment was filled with serum-free medium containing 0.1% BSA. Cells were placed in the upper part of the Transwell plate, incubated for 17 h, fixed with methanol, and stained with hematoxylin for 10 min followed briefly by eosin. The invasive phenotypes were determined by counting the cells that migrated to the lower side of the filter with microscopy at x400. Thirteen fields were counted for each filter, and each sample was assayed in triplicate.
In Vitro Migration Assay Using Transwell.
In vitro migration assay was performed using a 24-well Transwell unit with polycarbonate filters. Experimental procedures are the same as the in vitro invasion assay described above except that the filter was not coated with Matrigel for the migration assay.
Wound Migration Assay.
Cells were pretreated with mitomycin C (25 µg/ml) for 30 min before injury line was made. The injury line was made with a tip with 2 mm in width on the cells plated in culture dishes at 90% confluency. After being rinsed with PBS, cells were allowed to migrate in complete medium, and photographs were taken (x40) at indicated time points.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Transwell chamber (Fig. 1B)
, motility of MCF10A cells was greatly induced by H-ras, but not by N-ras. It should be mentioned that both H-ras and N-ras increased cell proliferation rates and anchorage-independent cell growth at comparable levels (7)
,6
suggesting that H-ras-mediated migrative property of MCF10A cells is unlikely to result from increased cell proliferation rates. To ensure this, the wound migration assay was performed using cells pretreated with mitomycin C, a cell cycle blocker at the S phase (Ref. 31
; Fig. 1A
). These results demonstrate that H-ras, but not N-ras, enhances cell motility in MCF10A cells.
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is the activation of MAPK family members in the parental, H-ras, and N-ras MCF10A cells. Whereas JNK-1,2 were activated neither by H-ras nor by N-ras, ERK-1,2 were effectively activated by both H-ras and N-ras. Interestingly, prominent activation of p38 was shown only in H-ras-activated cells but not in N-ras MCF10A cells, suggesting the possible involvement of p38 in H-ras-induced invasiveness and/or motility.
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, SB203580 specifically inhibited the activation of p38 with little effects on the phosphorylated forms of JNK-1 or ERK-1,2. To evaluate whether p38 is critical for H-ras-induced invasion, we examined the effect of SB203580 on the invasive phenotype of H-ras MCF10A cells by performing in vitro invasion assay. Treatment of 50 µM SB203580 significantly reduced the number of invaded cells by 
43% (Fig. 3B)
. Next we examined the role of p38 in the migratory property of H-ras MCF10A cells. Width of injury line was measured and plotted as percentage of control for quantification of the inhibitory effect of SB203580 on cell migration. H-ras MCF10A cell migration was significantly inhibited (0% versus 80%) in the presence of 50 µM SB203580 after 11 h as shown by wound migration assay (Fig. 3C)
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, indicating that the inhibition of invasion and motility was not because of a cytotoxic effect of SB203580. These findings indicate a critical role for p38 in H-ras-induced invasive phenotype and migration in human breast epithelial cells.
To additionally investigate the role for p38 in cellular invasion and migration, we transfected H-ras MCF10A cells with DN p38 in which the dual phosphorylated motif, Thr-Gly-Tyr, was mutated to Ala-Gly-Phe (28)
. Specific inactivation of p38 in DN p38-transfected cells was confirmed by immunoblot analysis (Fig. 4A)
. Phosphorylation of p38 was inhibited prominently in DN p38 transfectants, whereas phosphorylated levels of JNK-1 or ERK-1,2 were not altered by DN p38 transfection. To additionally test a notion that the p38 pathway plays a critical role for H-ras-mediated cell migration, we performed wound migration assay using DN p38 transfectants. Cell migration was clearly reduced in DN p38 transfectants as shown in Fig. 4B
. The migration end point was 24 h instead of 11 h (as in Fig. 1A
and Fig. 3C
), because the transfected cells migrated slower than nontransfectants. Invasive phenotype was also inhibited by transfection with DN p38 construct (data not shown). As shown in Fig. 4C
, DN p38 transfection did not lead to a significant reduction in viability, showing that DN p38-mediated inhibition of H-ras MCF10A cell invasion/motility was not because of a cytotoxic effect. The results demonstrate that activation of p38 pathway by H-ras is critical for ras-induced breast epithelial cell migration and invasion.
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. Treatment of PD98059 resulted in inhibition of both invasiveness (Fig. 5B)
and cell migration (Fig. 5C)
without affecting cell survival (Fig. 5D)
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, ERK-1,2 were specifically inactivated in DN MEK-1 transfectants, whereas the levels of phosphorylated JNK-1 and p38 were not affected. Selective inhibition of the ERK pathway by DN MEK-1 transfection significantly inhibited invasive phenotype (Fig. 6B)
and motility (Fig. 6C)
, demonstrating the significance of the ERK pathway in H-ras-induced invasion and migration in MCF10A cells. Given that ERK-1,2 were activated not only in H-ras MCF10A cells but also in noninvasive/nonmigrative N-ras MCF10A cells (Fig. 2)
, these results suggest that the activation of ERK-1,2 is not sufficient but required for H-ras-induced invasion and motility.
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. Interfering with JNK-1 activation by DN JNK-1 transfection did not affect H-ras MCF10A cell invasiveness (data not shown) and migration (Fig. 7B)
, indicating that the H-ras-induced invasion and motility are not dependent on JNK-1 pathway.
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80% (Fig. 8A)
. Interestingly, the MMP-9 expression levels were slightly increased after the treatment with low concentrations of SB203580 (1–10 µM). When ERK activation was inhibited by 25 µM PD98059, the MMP-9 and MMP-2 expression levels were decreased by 80% and 40%, respectively (Fig. 8B)
. Of note, whereas PD98059 is a specific inhibitor of ERKs at lower concentrations (up to 25 µM), it also inhibited other signaling molecules such as JNK and p38 at 50 µM concentration (data not shown). These results suggest that activation of p38 and, to a lesser degree ERKs, is required for enhanced MMP-2 expression. However, ERK activation is more critical than p38 activation for MMP-9 induction. Taken together, we propose that H-ras activation of both p38 and ERK induces MMP-2 and MMP-9 expression, resulting in phenotypic conversion of noninvasive MCF10A cells to an invasive phenotype (Fig. 8C)
. In contrast, N-ras-mediated ERK activation alone leading to MMP-9 induction is not sufficient for invasive phenotype of MCF10A cells (7)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study demonstrated that constitutive activation of the H-ras pathway, but not the N-ras pathway, was sufficient to convert the nonmigrative, noninvasive phenotype of MCF10A breast epithelial cells to a motile and invasive phenotype. Although its molecular action is unclear at present, only H-ras, but not N-ras, effectively activated the p38 pathway in MCF10A cells, whereas both activated the ERK pathway. N-ras activation of the ERKs was insufficient to induce a migrative and invasive phenotype (7) . However, ERKs activation was required for H-ras-mediated cell migration and invasive phenotype in MCF10A cells. Unlike p38 and ERKs, involvement of JNK in cell invasiveness or migration has not been reported extensively. JNK-dependent signaling molecules were shown recently to regulate invasive behavior and MMP-9 production of human glioblastoma cells (17) . However, JNK-1 played no significant role in ras-mediated migrative and invasive behavior of H-ras MCF10A cells. Consistently, inhibition of JNK pathway in ras-transformed NIH3T3 cells had no effect on the ability to invade Matrigel (34) .
Previous studies have shown that p38 lies downstream of the Ras-related GTP-binding proteins Rac and Cdc42, and is directly activated by MKK3, MKK6, and MKK4 (35, 36, 37, 38)
. The relationship between the two MAPK pathways, p38 and ERK, has been elucidated recently in Ras-induced cellular responses. The existence of a Ras/p38 pathway independent of the classical Ras/ERK pathway has been suggested in platelet-derived growth factor-induced cell motility (15)
. However, Chen et al. (39)
have reported that Ras-activation of MEK/ERK is a prerequisite for p38 pathway, suggesting ERK as an upstream regulator of p38. In MCF10A cells, however, the DN MEK-1 mutant prevented ERK activation without any effect on p38 activation (Fig. 6A)
, ruling out the possibility that ERK is an upstream regulator of the p38 pathway. It appears that the p38-mediated signaling pathway is independent of the ERK-mediated pathway, and both pathways cooperate in H-ras-mediated migrative and invasive responses in MCF10A cells.
An essential part of invasion and metastasis includes degradation of the basement membrane and the stromal ECM by members of the MMP family (32) . Our previous study suggested the involvement of MMP-2 in H-ras-induced invasive phenotype of MCF10A cells (7) . The present study suggests that activation of p38 may contribute to a more invasive phenotype via the secretion of MMP-2 in H-ras MCF10A cells. Consistently, a recent report (40) shows that the inhibition of p38 by SB203580 results in MMP-2 down-regulation in malignant melanoma cells. In contrast, ERK and p38 signaling pathways had little effect on MMP-2 expression (41) or were shown to inhibit MMP-2 expression (42) in different cell systems. Deletion and site-directed mutagenesis analyses of the MMP-2 promoter demonstrated that the transcription factors SP1, Sp3, and AP-2, rather than AP-1, were functionally important for MMP-2 gene expression in astroglioma cells (43) . It would be of interest to determine the p38-responsive promoter sequences of the MMP-2 gene. Additional studies to delineate MMP-2 regulation by the p38 pathway remain to be performed.
We showed previously that N-ras induced MMP-9 expression more effectively than H-ras (7)
. Interestingly, MMP-9 expression is enhanced on down-regulation of the p38 pathway by low concentrations of SB203580 in H-ras-activated MCF10A cells (Fig. 8A)
. This is consistent with the observation that N-ras activation of ERK without p38 activation induces MMP-9 expression more effectively than H-ras activation of both ERKs and p38. N-ras-mediated ERK activation and MMP-9 induction were insufficient for induction of invasive phenotype (7)
. Consistently, when MMP-2 expression was down-regulated by inhibition of the p38 pathway, MMP-9 expression alone was insufficient for induction of cell motility and invasive phenotype of H-ras MCF10A cells (Figs. 3
and 8
). However, inhibition of H-ras MCF10A cell invasion was more drastic when both MMP-2 and MMP-9 were down-regulated on inhibition of the ERK pathway by 25 µM PD98059 (60% loss, Fig. 5B
; Fig. 8B
) compared with the inhibition of MCF10A cell invasiveness when MMP-2 expression alone was inhibited using antisense MMP-2 oligodeoxyribonucleotide (30% loss; Ref. 7
). Thus, for completion of H-ras-induced invasion of human breast epithelial cells, not only MMP-2 but also other gene products such as MMP-9 may be required, although MMP-9 alone is not sufficient.
Many studies have identified the PI3K as a key regulator for cell growth and survival. Involvement of PI3K activity in other aspects of tumor progression including invasion and metastasis has been suggested recently (44) . We are currently investigating the functional role of PI3K pathway in H-ras-induced invasive phenotype and motility of MCF10A cells.
In this report, we show that p38 and ERK signaling can regulate H-ras MCF10A cell migration and invasion, suggesting overlapping signaling pathways for regulation of cell motility and ECM degrading activities. Our studies establish an experimental system in which ras-mediated overlapping and unique signaling pathways for cell motility and ECM-degrading activities can be additionally analyzed at the molecular levels.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a Korea Health 21 R&D, Ministry of Health and Welfare Grant (02-PJ1-PG10-20801-0001; to A. M.) and by a National Cancer Institute NIH Grant (CA-64139; to H-R. C. K.). 
2 These authors equally contributed to this study.
3 Present address: Institute of Science and Technology, CJ Corp., Inchonsi, Kyonggi-Do 467-810, Korea.
4 To whom requests for reprints should be addressed, at College of Pharmacy, Duksung Women’s University, 419 Ssangmun-Dong, Tobong-Ku, Seoul 132-714, Korea. Phone: 82-2-901-8394; Fax: 82-2-901-8386; E-mail: armoon{at}duksung.ac.kr
5 The abbreviations used are: ECM, extracellular matrix; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal protein kinase; ERK, extracellular signal-regulating kinase; MMP, matrix metalloproteinase; DN, dominant-negative; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MEK, mitogen-activated protein/extracellular signal-regulating kinase kinase; PI3K, phosphatidylinositol 3'-kinase.
Received 3/19/03. Revised 5/22/03. Accepted 6/ 3/03.
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REFERENCES |
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