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To whom correspondence should be addressed: The Division of Surgical Research, Royal Victoria Hospital, Room H6.25, 687 Pine Ave. W., Montreal, Quebec Canada H3A 1A1. Tel.: 514-842-1231 (ext. 36692); Fax: 514-843-1411;
Departments of Surgery and Université de Montréal, Montreal, Quebec H3A 1A1, CanadaMedicine, McGill University Health Center, the Royal Victoria Hospital and the Université de Montréal, Montreal, Quebec H3A 1A1, Canada
* This study was supported by Grant MT-10505 from the Canadian Institute for Health Research and Grant 11423 (a Terry Fox New Frontiers Initiative grant) from the National Cancer Institute of Canada (to P. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The matrix metalloproteinase (MMP)-2 has been recognized as a major mediator of basement membrane degradation, angiogenesis, tumor invasion, and metastasis. The factors that regulate its expression have not, however, been fully elucidated. We previously identified the type I insulin-like growth factor (IGF-I) receptor as a regulator of MMP-2 synthesis. The objective of the present study was to investigate the signal transduction pathway(s) mediating this regulation. We show here that in Lewis lung carcinoma subline H-59 cells treated with IGF-I (10 ng/ml), the PI 3-kinase (phosphatidylinositol 3′-kinase) /protein kinase B (Akt) and C-Raf/ERK pathways were activated, and MMP-2 promoter activity, mRNA, and protein synthesis were induced. MMP-2 induction was blocked by the PI 3-kinase inhibitors LY294002 and wortmannin, by overexpression of a dominant-negative Akt or wild-type PTEN (phosphatase and tensin homologue deleted on chromosome 10), and by rapamycin. In contrast, a MEK inhibitor PD98059 failed to reduce MMP-2 promoter activation and actually increased MMP-2 mRNA and protein synthesis by up to 30%. Interestingly, suppression of PI 3-kinase signaling by a dominant-negative Akt enhanced ERK activity in cells stimulated with 10 ng/ml but not with 100 ng/ml IGF-I. Furthermore, at the higher (100 ng/ml) IGF-I concentration, C-Raf and ERK, but not PI 3-kinase activation, was enhanced, and this resulted in down-regulation of MMP-2 synthesis. This effect was reversed in cells expressing a dominant-negative ERK mutant. The results suggest that IGF-I can up-regulate MMP-2 synthesis via PI 3-kinase/Akt/mTOR (the mammalian target of rapamycin) signaling while concomitantly transmitting a negative regulatory signal via the Raf/ERK pathway. The outcome of IGF-IR (the receptor for IGF-I) activation may ultimately depend on factors, such as ligand bioavailability, that can shift the balance preferentially toward one pathway or the other.
The breakdown of the extracellular matrix by proteinases is an essential step in the processes of cancer invasion and metastasis. Malignant progression is frequently associated with up-regulated production and/or activity of one or several matrix metalloproteinases (MMP),
). The 72-kDa type IV collagenase, MMP-2, is thought to play a critical role in tumor cell invasion by facilitating the degradation of basement membrane type IV collagen, by exposing cryptic, growth-affecting sites on extracellular matrix proteins, and by increasing the bioavailability of matrix-associated growth factors (
Similarly to other members of the metalloproteinase family, MMP-2 synthesis and function are regulated at multiple levels including transcriptional activation, post-transcriptional processing, and positive or negative regulation of proteolytic activity by membrane type-MMPs (MT-MMPs) and a family of endogenous inhibitors collectively known as tissue inhibitors of metalloproteinases (TIMPs) (
). MMP-2 is secreted as an inactive, 72-kDa zymogen and is activated by proteolytic cleavage extracellularly. This process involves MT1-MMP that binds MMP-2 on the cell membrane in a multimeric complex with TIMP-2 (
). Although the MMP-2 promoter lacks well characterized regulatory elements, several transcription factors including Sp1, Sp3, and AP-2 were recently implicated in its transcriptional activation in astroglioma cells (
The receptor for the type 1 insulin-like growth factor (IGF-IR) and its ligands IGF-I and IGF-II play critical roles in the regulation of cellular proliferation, apoptosis, and transformation. IGFs have increasingly been recognized as important mitogens for many tumor types and overexpression and/or constitutive activation of IGF-IR in non-malignant cells resulted in the acquisition of a transformed phenotype and tumorigenic potential (
). In vivo, IGF-I-dependent tumor cell growth can be regulated in a paracrine manner by serum or stromal cell-derived IGFs, or it may be regulated through an autocrine loop. Indeed, increased expression of IGF-IR, IGF-I, and IGF-II or a combination thereof has been documented in many animal and human malignancies (
). In several animal tumor models, inhibition of IGF-IR or IGF-I expression by various strategies including antisense oligodeoxynucleotides, antisense RNA, or IGF-I peptide analogues caused a suppression of tumor cell growth in vivo (
). Ligand binding to the IGF-IR can trigger multiple signaling pathways including the Ras/Raf/ERK pathway implicated in receptor-mediated mitogenesis and transformation and the PI 3-kinase/Akt pathway implicated in the transmission of cell survival signals (
). All cell lines were cultured in RPMI 1640 supplemented with 10% fetal calf serum. Transfected cells were maintained in medium containing 200 μg/ml G 418. All cell lines were routinely tested for infectious agents and were found to be free of infection during the course of this study. Recombinant hIGF-I was from United States Biological (Swampscott, MA), LY294002, PD98059, and rapamycin were from Calbiochem, and wortmannin was from Sigma.
Reverse Transcription PCR (RT-PCR)—Total cellular RNA was extracted using Trizol (Invitrogen). RT-PCR was performed using the ThermoScript™ RT-PCR system, Platinum TaqDNA polymerase (both from Invitrogen), and the following primers: for MMP-2, sense, 5′-GAG TTG GCA GTG CAA TAC CT-3′, and antisense, 5′-GCC GTC CTT CTC AAA GTT GT-3′ (expected product 665 bp); and for GAPDH, sense, 5′-GGT GAA GGT CGG TGT GAA CGG ATT T-3′, and antisense, 5′-AAT GCC AAA GTT GTC ATG GAT GAC C-3′ (expected product 520 bp). The cDNA was amplified using 30 cycles for MMP-2 (25 cycles for GAPDH) each of 30 s at 94 °C, 30sat58 °C, and 30 s at 72 °C, and this was followed by a 10-min incubation at 72 °C. The amplified DNA fragments were analyzed by electrophoresis on a 1.5% agarose gel.
Real-time Quantitative PCR—PCR was performed with the LightCycler FastStart DNA Master SYBR Green I® (Roche Applied Science) in a standard PCR reaction mixture containing 0.5 μm of each primer (described above), 3 mm MgCl2, and 2 μl of cDNA. Amplification and detection were performed in a LightCycler® instrument (Roche Applied Science) as follows: 20 μl of the reaction mixture were initially incubated for 10 min at 95 °C to denature the DNA and activate the Fast Start TaqDNA; amplification was then performed for 43 cycles of denaturation (95 °C, 10 s, ramp rate 20 °C/s), annealing (58 °C, 5 s, ramp rate 20 °C/s), and extension (72 °C, 26 s, ramp rate 20 °C/s). A single fluorescence reading was taken at each extension step. The crossing points, marking the cycle when the fluorescence of a given sample significantly exceeded the baseline signal, were recorded and expressed as a function of the cycle number. The crossing points were plotted against known concentrations determined on the basis of a pre-established standard curve. The identity of the amplification products was confirmed by determining the melting curve. The melting point of the MMP-2 amplification product (665 bp) was 88 ± 0.5 °C.
Plasmids and Transfection—Wild-type and dominant-negative Akt (DN-Akt) constructs were gifts from Dr. David Kaplan (University of Toronto, Toronto, Ontario, Canada). The DN-Akt cDNA encoding a catalytically inactive mutant was constructed by a lysine-methionine substitution (residue 179) within the ATP-binding domain (
). It was a gift from Dr. Nahum Sonenberg (McGill University, Montreal, Quebec, Canada). The expression vectors encoding the hemagglutinin-tagged wild-type ERK1 or a dominant-negative mutant, in which threonine 192 was replaced by alanine have been characterized elsewhere (
). Transient transfections of H-59 cells seeded in 10-cm culture dishes, at a density of 106 cells/dish, were performed using the LipofectAMINE Plus reagent (Invitrogen) and 5 μg of DNA/dish, as suggested by the manufacturer. The transfected cells were incubated first for 5 h in RPMI 1640 medium containing 10% fetal calf serum and then for 18 h in serum-depleted medium, prior to stimulation with IGF-I.
Chloramphenicol Acetyltransferase (CAT)-Reporter Assay—Tumor cells were co-transfected with a pCAT basic vector expressing the CAT reporter gene downstream of a 680-bp (nucleotides –390 to +290) fragment of the human MMP-2 promoter (
) and a pSV40-Luciferase plasmid expressing the luciferase gene under the control of an RSV promoter, using the LipofectAMINE Plus reagent (Invitrogen). The transfected cells were incubated for 5 h in complete medium and then in serum-depleted RPMI medium for 18 h. To each dish, fresh medium was then added with or without 10 ng/ml hIGF-I, and the cells were incubated for another 6–48 h. Cell lysates were prepared using the Reporter lysis buffer (Promega), and CAT activity was measured using the [3H]chloramphenicol assay (
). Transfection efficiencies were normalized relative to luciferase activity as measured using an MLX microtiter plate luminometer (LKB Instruments).
Western Blot Assay—Subconfluent monolayers (∼8 × 106 cells) were washed extensively to remove serum, and the cells were cultured for 48 h at 37 °C in serum-free medium with or without the indicated concentration of recombinant hIGF-I (United States Biological). For analysis of MMP-2, 50-fold concentrated conditioned media were used, and 50 or 100 μg of protein (as specified) were loaded per lane and separated by electrophoresis on a 10% SDS-polyacrylamide gel. The separated proteins were transferred onto nitrocellulose membranes and probed with a monoclonal antibody to MMP-2 (manufactured by Fuji Co. for Medicorp, Montreal, Quebec, Canada) or with a rabbit antiserum raised against human cathepsin L (a kind of a gift from Dr. John Mort, Shriners Hospital, McGill University, Montreal, Quebec, Canada (
)). A peroxidase-conjugated affiniPure goat anti-mouse IgG or donkey anti-rabbit IgG was used as secondary antibody. For detection of phospho-Akt, ERK, C-Raf, and tubulin, the cells were incubated with IGF-I, washed with ice-cold phosphate-buffered saline, and lysed in 1 ml of lysis buffer containing 30 mm Tris-HCl (pH 8.0), 1% Triton X-100, 150 mm NaCl, 1 mm Na3VO4, 1 mm NaF, 1 mm iodoacetamide, 0.5% deoxycholic acid sodium salt, 1 mm phenylmethylsulfonyl fluoride, and 0.01% aprotinin. Proteins (50 or 100 μg/lane) were separated on a 10% SDS-polyacrylamide gel and probed with antibodies to Akt, phospho-Akt (Thr308), ERK1/2 (p42/44 MAPK), phospho-ERK1/2, and phospho-C-Raf (Ser259) (all from Cell Signal Technology), C-Raf and phospho-C-Raf (Tyr340/341) (both from Santa Cruz Biotechnology), and tubulin and hemagglutinin (from Sigma). Horseradish peroxidase-conjugated affiniPure donkey anti-rabbit, donkey anti-goat, or goat anti-mouse IgG antisera were used as required, as secondary antibodies.
Gelatin Zymography—The gelatinolytic activity of MMP-2 was analyzed by zymography, as we described previously (
). Briefly, concentrated conditioned media (50×) were electrophoresed on a 10% SDS-polyacrylamide gel containing 1 mg/ml gelatin. The gels were stained with Coomassie Blue R250 and destained with 10% acetic acid-20% methanol until the desired color intensity was obtained. The gelatinolytic activity, seen as a clear zone on the blue background, was quantified by laser densitometry using photographic negatives of the gels.
In Vitro Kinase Assay—Stimulation with IGF-I and cell lysis was performed as described above. C-Raf activity was measured by the Raf-1 immunoprecipitation kinase cascade assay Kit (Upstate Biotechnology). Briefly, cell lysates (1 ml) were immunoprecipitated with a polyclonal anti-C-Raf antibody and 50% (v/v) protein G agarose. The precipitate was washed in ice-cold lysis buffer and incubated for 30 min at 30 °C in assay buffer containing inactive MEK-1 and -2, and 4 μl of the mixture were added to the assay buffer containing [γ-32P]ATP and 2 mg/ml myelin basic protein as the substrate, for a 10-min incubation at 30 °C. From each reaction mixture, 25 μl were spotted onto the center of a 2-cm2 P81 filter paper, and the papers were washed with 0.75% phosphoric acid and transferred into the scintillation mixture for counting. Reaction mixtures containing no enzyme were used as controls.
Statistical Analysis—The Student's t test was used for analysis of the CAT assay and real-time PCR data.
IGF-I Induces MMP-2 mRNA and Protein Synthesis—The effect of IGF-I on MMP-2 expression was measured at the mRNA and protein levels. An RT-PCR analysis showed that in response to 10 ng/ml IGF-I, MMP-2 mRNA expression in both H-59 and M-27IGF-IR, but not in control, M-27 cells began to rise at 2 h, reached maximal levels at 8 h, and persisted for up to 24 h (Fig. 1A). A quantitative real-time PCR analysis confirmed these results, showing a 4-fold increase in MMP-2 transcription at 8 h (Fig. 1B). The increase in MMP-2 mRNA expression was reflected in increased protein production and enzyme activity, as determined by Western blotting (Fig. 1C, I) and gelatin zymography (Fig. 1C, II) respectively, performed on medium conditioned by H-59 cells. Gelatin zymography revealed two bands corresponding to the native (72-kDa) and activated (64-kDa) forms of MMP-2 (Fig. 1C, II). In contrast, tumor cell treatment with 100 ng/ml IGF-I reduced MMP-2 mRNA expression by 70%, as measured by real-time PCR (Fig. 1B), and this resulted in a corresponding reduction in protein synthesis (Fig. 1C).
MMP-2 promoter activation by IGF-I was subsequently analyzed in H-59 cells that were transiently transfected with a pCAT basic vector in which a 680-bp fragment of the MMP-2 promoter (nucleotides –390 to +290) was cloned upstream of the CAT reporter gene. This promoter region was selected because it contains the Sp1- and AP-2-binding sites that were previously identified as critical for MMP-2 gene transcription (
). To control for transfection efficiency, the cells were cotransfected with a second plasmid vector expressing the luciferase gene under the control of a constitutive RSV promoter. CAT activity in the transfected cells increased 2.4-fold relative to controls when the cells were treated with 10 ng/ml IGF-I for 6 h (Fig. 2, p < 0.01). The activity remained elevated relative to controls for up to 24 h, declining to basal levels by 36 h (data not shown). A similar IGF-I-induced increase in CAT activity was seen in M-27IGF-IR cells (p < 0.01), but no response was observed in control, M-27 cells (p > 0.05, Fig. 2).
The PI 3-Kinase/Akt and MEK/MAPK Pathways Transmit Opposing Signals for MMP-2 Synthesis in Response to IGF-I— Signal transduction by the IGF-IR is known to involve both the ERK and the PI 3-kinase pathways (
). To identify the pathway(s) regulating MMP-2 induction by IGF-I, we first tested the effects of pharmacological inhibitors of these pathways on MMP-2 expression, using a combination of RT-PCR, Western blotting, and the CAT-MMP-2 promoter assay. Results in Fig. 3 show that treatment of H-59 cells with the PI 3-kinase inhibitors LY294002 (20 μm) and wortmannin (5 nm), under conditions that had no deleterious effect on cell viability (
), blocked IGF-I-induced MMP-2 mRNA synthesis (Fig. 3A) and reduced MMP-2 protein synthesis by 82 and >90%, respectively, relative to non-treated, IGF-I stimulated cells. Moreover, in H-59 cells that were transiently transfected with plasmid vectors expressing a dominant-negative (DN) Akt mutant or wild-type PTEN, IGF-I-mediated MMP-2 induction was completely blocked, whereas it increased by 3–3.6-fold in non-transfected or wild-type Akt-transfected cells (Fig. 3, C and E), confirming the involvement of the PI 3-kinase/Akt pathway in the induction. The decrease in MMP-2 induction in DN-Akt transfected cells was seen despite increased levels of phospho-ERK in these cells (Fig. 3D). In addition, treatment with PD98059 (20 μm) increased MMP-2 mRNA and protein levels, as measured by real-time PCR (Fig. 3A) and by Western blot analysis (Fig. 3B). Moreover, cell pretreatment with LY294002 and wortmannin, but not with PD98059, blocked IGF-I-induced MMP-2 promoter activation, as measured by the CAT reporter assay (Fig. 3F). Taken together, the data indicate that IGF-I can up-regulate MMP-2 synthesis via PI 3-kinase/Akt signaling while also transmitting a negative regulatory signal via C-Raf/ERK signaling.
Inhibition of IGF-I-mediated MMP-2 Protein but Not mRNA Synthesis by Rapamycin—The mammalian target of rapamycin (mTOR) is a substrate downstream of Akt that is involved in the regulation of translation initiation (
). To evaluate its involvement in IGF-I-induced MMP-2 synthesis, the cells were treated with rapamycin at non-toxic concentrations prior to stimulation with IGF-I (10 ng/ml). This treatment blocked the increase in MMP-2 protein synthesis (a decrease of 70 and 85% in cells treated with 20 and 100 ng/ml rapamycin, respectively, relative to non-treated cells, Fig. 4A) while not affecting MMP-2 mRNA synthesis (Fig. 4B).
Changes in Signaling in Response to High Dose IGF-I, Evidence for Inter-pathway Cross-talk—Because IGF-I at a concentration of 100 ng/ml decreased, rather than enhanced, MMP-2 synthesis (Fig. 1, B and C), it was of interest to compare the signals generated at this dose to those seen at the MMP-2-inducing concentration of 10 ng/ml. We observed that in cells treated with 10 ng/ml IGF-I, the PI 3-kinase/Akt and Raf/ERK pathways were both activated. Increased Akt phosphorylation was observed by 2 min following the treatment, returning to basal levels by 20 min. Phospho-ERK levels began to increase at 5 min, were maximal at 20 min, concomitantly with maximal C-Raf kinase activity, and returned to basal levels at 40–60 min (Fig. 5, A and B). This pattern was altered at 100 ng/ml IGF-I. At this concentration, there was no measurable increase in phospho-Akt levels, whereas C-Raf activation and ERK phosphorylation were both enhanced and more sustained and could be detected for up to 1 and 2 h, respectively, after IGF-I treatment (Fig. 5B, I and II).
C-Raf was recently identified as an Akt substrate in several cell lines including human breast carcinoma line MCF-7 (
), and in the present study, an increase in phospho-ERK was seen in cells transiently transfected with DN-Akt (Fig. 3D), it was of interest to investigate whether IGF-I in fact triggered cross-pathway phosphorylation in our cells. When C-Raf activation was measured in cells treated with 10 or 100 ng/ml IGF-I, we found that at the lower IGF-I concentration, C-Raf was transiently phosphorylated on Tyr residues 340 and 341, and this coincided with an initial decrease in phospho-Ser259 levels and C-Raf activation. However, an increase in phospho-Ser259 was subsequently seen following Akt activation, causing a decline in C-Raf activity to below baseline levels by 60 min and leading to MMP-2 induction. On the other hand, treatment with 100 ng/ml IGF-I resulted in a decrease in phospho-Akt levels by 20 min, and this coincided with a sustained decrease in phospho-Ser259 C-Raf levels, a more sustained C-Raf activity (Fig. 5, A and B), and a decrease in MMP-2 production. The negative regulatory role played by ERK signaling was also confirmed in H-59 cells that were transiently transfected with plasmid vectors expressing the wild type or a dominant-negative mutant of ERK-1. In response to 100 ng/ml IGF-I, overexpression of wild-type ERK caused a further reduction in MMP-2 levels, as compared with controls, whereas a dominant-negative ERK-1 reversed the IGF-I-induced suppression.
Our results show that IGF-I can regulate MMP-2 synthesis at several levels. It can activate the MMP-2 promoter and also enhance protein synthesis through activation of mTOR, both via the PI-3K/Akt pathway. At the same time, it can also transmit a down-modulating signal via the Raf/MEK/ERK pathway. The net effect of these opposing regulatory signals appears to depend, among others, on the ligand load. At a concentration of 10 ng/ml, which is optimal for mitogenesis, cellular proliferation, and motility (
), the PI-3K/Akt pathway is activated, leading to Raf kinase inactivation and a short-lived ERK response, and this appears to shift the balance in favor of MMP-2 induction. At the higher dose, however, activation of the ERK pathway appears to dominate, and this leads to a down-regulation of MMP-2 synthesis (diagrammatic representation in Fig. 6). These results are consistent with a recent report of MMP-2 down-regulation in mesangial cells treated with 100 ng/ml IGF-I (
) where PI-3 kinase signaling in MCF-7 cells was shown to predominate at 100 ng/ml IGF-I, causing an inhibition of Raf/ERK signaling at this concentration, but not at 10 ng/ml IGF-I. The mechanisms responsible for the preferential activation of one pathway or the other at different ligand concentrations are not presently clear. However, differences in cell surface receptor numbers and the expression of downstream substrates are likely to play a role (
). The IGF-I concentration that is available to tumor cells growing in vivo cannot be readily assessed because it will ultimately depend on factors such as the level of circulating and tissue IGFBP, autocrine IGF-I production, and the level of IGF-I produced by the stroma.
PI 3-kinase has been identified as the major transducer of the IGF-IR signal in other cellular systems, and its activity was shown to be critical for IGF-I-induced cell survival, mitogenesis, and transcriptional and translational regulation of protein synthesis (
). The degree to which different cells utilize these pathways to convey IGF-IR signals may be cell context-dependent and could be determined, among others, by the levels and accessibility of downstream substrates such as insulin receptor substrate-1 or Shc (
The transcription factors involved in positive and negative IGF-I-dependent MMP-2 regulation in our model remain to be identified. Recently, it has been shown that the transcription factors Sp1, Sp3, and AP-2 were required for constitutive MMP-2 gene expression (
D. Zhang, E. N. Benveniste, and P. Brodt, unpublished observation.
suggesting that this element may be exerting a negative regulatory effect on MMP-2 gene expression. Interestingly, other studies have recently shown that the activity of the Ets transcription factor PEA3 is regulated by two distinct MAPK pathways, namely the ERK and the Jun kinase (JNK) pathways (
). When taken together with the present findings, these observations suggest that PEA3 may be the element that exerts a negative regulatory effect on MMP-2 synthesis downstream of ERK, whereas Sp1 may be involved in positive regulation (
), their role in IGF-I signaling is unclear. In this respect, it should be noted that although we observed an increase in MMP-2 transcription in the presence of the MEK inhibitor PD98059 (Fig. 3A), this inhibitor did not significantly alter the level of promoter activation by IGF-I (Fig. 3F). This difference may be related to the use of a partial promoter construct (nucleotides –390 to +290) in the CAT reporter assay. This portion of the promoter, although it contains the AP-2 and Sp1 elements implicated in positive regulation of MMP-2 transcription (
) that may exert the negative regulatory effects. The possibility that the observed increase in MMP-2 mRNA levels in the presence of a MEK inhibitor is due, at least in part, to increased mRNA stability cannot at present be ruled out.
We recently reported that in the present tumor model, IGF-I can also induce MT1-MMP expression in a PI 3-kinase-/Akt signaling-dependent manner, but it does not affect TIMP-2 expression (
). The concomitant activation of MMP-2 and MT1-MMP synthesis by IGF-I, in the absence of a corresponding increase in TIMP-2 levels, could shift the balance in favor of increased production of catalytically active MMP-2 and is likely responsible for the increased invasion observed in these cells (
), the coordinated regulation of MMP-2 and MT1-MMP by IGF-I may also contribute to the central role that the IGF-IR plays in embryogenesis and development.
We are indebted to Dr. David Kaplan for the Akt constructs, Dr. Nahum Sonenberg for the PTEN plasmid and helpful discussions, Dr. Hanna Hyman Friedman for help with the real-time PCR, and Dr. Eric Chevet for helpful discussions.