Fibronectin Increases Matrix Metalloproteinase 9 Expression through Activation of c-Fos via Extracellular-regulated Kinase and Phosphatidylinositol 3-Kinase Pathways in Human Lung Carcinoma Cells*

Enhanced expression of matrix metalloproteinase-9 (MMP-9) is associated with human lung tumor invasion and/or metastasis. We have demonstrated that fibronectin (FN), a matrix glycoprotein, stimulates human non-small cell lung carcinoma (NSCLC) cell proliferation. The current study examines the effect of FN on MMP-9 expression in NSCLC cells. We show that FN increases MMP-9 protein, mRNA expression, and gelatinolytic activity in NSCLC cells. The integrin α5β1 mediated the effects of FN because α5 small interfering RNA blocked FN-stimulated MMP-9 protein expression, and also abrogated FN-induced phosphorylation of ERK and phosphatidylinositol 3-kinase (PI3K) signals. The inhibitor of ERK, PD98095, and of PI3K, wortmannin, but not that of protein kinase A, H89, of Rho kinase, Y-27632, of mTOR, rapamycin, or of JNK, SP600125, prevented FN-induced MMP-9 gelatinolytic activity and gene expression. FN enhanced MMP-9 gene promoter activity; however, there was no response to FN in DNA constructs with an AP-1 site mutation. FN increased AP-1 DNA binding activity, and this was abrogated by cyclic AMP response element decoy oligonucleotides, which also diminished FN-induced MMP-9 promoter activity. FN increased the expression of the AP-1 subunit c-Fos protein, but not in the presence of PD98095 and wortmannin. The AP-1 inhibitor, nordihydroguaiaretic acid, and a c-Fos small interfering RNA eliminated the effect of FN on MMP-9 expression. This study indicates that FN, by binding to the integrin α5β1 receptor, stimulates the expression of MMP-9 through increased AP-1/DNA binding and c-Fos protein expression via ERK and PI3K signaling pathways. The data unveils a novel mechanism by which FN could promote NSCLC cell invasion and metastasis.

Matrix metalloproteinases (MMPs) 2 are a family of zinc enzymes responsible for degradation of extracellular matrix components including basement membrane collagen, interstitial collagens, fibronectin (FN), and various proteoglycans during normal remodeling and repair processes (1,2). The potent proteolytic activities of MMPs are mainly regulated by the concomitant expression of specific tissue inhibitors of matrix metalloproteinases (3). Excessive or inappropriate expression of MMPs may contribute to the pathogenesis of tissue destructive processes in a wide variety of diseases including lung disorders like bronchial asthma, chronic obstructive pulmonary disease, acute lung injury, pulmonary hypertension, and interstitial lung diseases (2,4). In addition, MMPs have been implicated in the progression and metastases of different tumors including lung cancer (see later in text).
One member of the MMP family is the 92-kDa type IV collagenase MMP-9. The MMP-9 gene is encoded on chromosome 20 and its expression is under the control of a 2.2-kb upstream regulatory sequence harboring binding sites for AP-1, NF-B, Sp1, and others (5). MMP-9 expression is increased in malignant cancers when compared with benign tumors and non-invasive ones, and there is compelling in vitro and in vivo evidence for a role of MMP-9 in tumor invasion and angiogenesis (6,7). Its dramatic overexpression in cancer and various inflammatory conditions suggest that this protease is a potential target for the development of novel therapeutic interventions (8).
Because of its perceived importance, our research focuses on the factors that increase MMP-9 expression in the lung. Our search led us to FN, a matrix glycoprotein highly expressed in tobacco-related lung disease that has been shown to stimulate lung carcinoma cell growth through several mechanisms (9 -11). Cell adhesion to FN results in MMP secretion in normal and tumor cell systems (12)(13)(14). These studies suggest that tumor cell interactions with FN might lead to MMP-9 expression thereby promoting cancer cell migration, invasion, and related processes. However, the mechanisms by which FN stim-ulates MMP-9 expression in tumor cells have not been explored in detail. Here, we report that FN, by binding to its ␣5␤1 integrin receptor, stimulates MMP-9 expression through activation of extracellular-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) dual signaling pathways followed by induction of AP-1 binding activity and c-Fos expression that stimulate MMP-9 gene transcription.

MATERIALS AND METHODS
Cultures and Chemicals-The human non-small cell lung carcinoma cell lines H1838 and H2106 were obtained from American Type Culture Collection (Manassas, VA) and were grown in RPMI 1640 medium supplemented with 10% heatinactivated fetal bovine serum, HEPES buffer, 50 IU/ml penicillin/streptomycin, and 1 g of amphotericin (complete medium) (15). Afterward, cells were harvested and replated in serum-free medium on FN-coated culture plates for experiments described later. [methyl-3 H]Thymidine and poly(dI-dC) were purchased from Amersham Biosciences. [␥-32 P]ATP was purchased from PerkinElmer Life Sciences, Inc. Collagen type 1 and polyclonal antibodies against the integrin ␣5 (H-104) and MMP-9 (H129) were purchased from Santa Cruz Biotechnology, Inc. Polyclonal antibodies specific for Akt, ERK1, ERK2, and their phosphorylated forms ( p-Akt Thr 308 , p-ERK1/2 Thr 202 /Tyr 204 ) and rapamycin were purchased from Cell Signaling Inc. (Beverly, MA). The AP-1 inhibitor, nordihydroguaiaretic acid (NDGA), the PI3K inhibitor wortmannin, the ERK1/2 inhibitor PD98095, the Rho kinase inhibitor Y-27632, the protein kinase A inhibitor H89, and the c-Jun NH 2 -terminal kinase (JNK) inhibitor SP600125, were obtained from Calbiochem. Gel shift assay system and the Dual Luciferase Reporter assay kit were obtained from Promega. All reverse transcriptase (RT)-PCR kit components were obtained from PerkinElmer. FN (derived from human fibroblasts) and all other chemicals were purchased from Sigma, unless otherwise indicated.
Gelatin Zymography-Gelatin zymography was performed by using a 9% SDS-PAGE gel saturated with 1 mg/ml gelatin (Sigma, 300 bloom) as previously described (16). Samples with equal protein concentration (10 g) were loaded onto the gel and electrophoresed at a constant 150 V for 1.5 h. The gels were incubated for 1 h at room temperature in 2.5% Triton X-100, followed by an overnight incubation at 37°C in gelatinase substrate buffer (50 mM Tris, 10 mM CaCl 2 , and 0.02% NaN 2 , pH 8.0). The gels were stained with 0.5% Coomassie Blue followed by subsequent destaining with 50% methanol. The gels were dried onto cellophane and scanned under a densitometer for determination of gelatinolytic activity.
RT-Polymerase Chain Reaction-Total RNA was prepared from human lung carcinoma cells treated with FN or collagen type 1 (20 g/ml each) using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. To amplify the 590-bp MMP-9 and 200-bp GAPDH cDNA fragments, the sequences of PCR primers (Sigma Genosys, Woodlands, Texas) were as follows: for MMP-9 sense, 5Ј-CACTGTCCACCCCT-CAGAGC, and antisense, 5Ј-GCCACTTGTCGGCGATA-AGG; and for GAPDH sense, 5Ј-CCATGGAGAAGGCTG-GGG, and antisense, 5Ј-CAAAGTTGTCATGGATGACC, according to published data (15,17). The RT-PCR was carried out as previously described (15). The samples were first denatured at 95°C for 30 s, followed by 32 PCR cycles, each with temperature variations as follows: 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The last cycle was followed by an additional extension incubation of 7 min at 72°C. Analysis of amplicons was accomplished on 1% agarose gel containing 0.2 g/l ethidium bromide and visualized under a UV transilluminator.
Real-time RT-PCR-This procedure was described previously (15). Briefly, total RNA was prepared from H1838 cells with TRIzol reagent according to the manufacturer's instructions. Final results were expressed as n-fold differences in MMP-9 gene expression relative to the GAPDH gene. All PCR using the LightCycler-FastStart DNA Master SYBR Green I kit were performed in the Cepheid Smart Cycler real-time PCR cycler (Sunnyvale, CA) (15). Experiments were performed in triplicate for each data point.
Western Blot Analysis-The procedure was performed as previously described (18). Protein concentrations were determined by the Bio-Rad protein assay. Equal amounts of protein from whole cell lysates (50 g) were solubilized in 2ϫ SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 5-10% 2-mercaptoethanol, and 0.004% bromphenol blue), and separated on SDS-8% polyacrylamide gels. The separated proteins were transferred onto nitrocellulose using a Bio-Rad TransBlot semidry transfer apparatus for 1 h at 25 voltage, blocked with Blotto (1ϫ TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl)) with or without 5% bovine serum albumin, 5% nonfat dry milk, and 0.1% Tween 20 overnight at 4°C, and washed twice for 5 min with wash buffer (1ϫ TBS and 0.1% Tween 20). Blots were incubated with polyclonal antibodies raised against human MMP-9, integrin ␣5 (1:4000), Akt, ERK1, ERK2, and their phosphor forms, c-Jun, or c-Fos (1:1000) for 2 h at room temperature. After washing, the blots were washed three times for 5 min with wash buffer and incubated with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase (1:2000, Cell Signaling) for 1 h at room temperature. The blots were washed, transferred to freshly made ECL solution (Amersham Biosciences) for 1 min and exposed to x-ray film. Protein bands were quantified by densitometric scanning using a Bio-Rad GS-800 calibrated densitometer. In controls, the antibodies were omitted or replaced by rabbit IgG.
Transient Transfection Assays-Human NSCLC cells were seeded at a density of 1 ϫ 10 5 cells/well in 6-well dishes and grown to 60% confluence. For each well, the plasmid DNA (1 to 2 g) containing wild-type, or deleted or mutated MMP-9 promoter constructs, and 0.2 g of the internal control plasmid phRL-SV40 (Renilla luciferase gene) were cotransfected into the cells using FuGENE 6 lipofection reagent as described in our earlier work (15). After 24 h of incubation, cells were exposed to FN or collagen type 1-coated plates for an additional 24 h. The preparation of cell extracts and measurement of luciferase activities were carried out using the Dual Luciferase Reporter Kit according to recommendations by the manufacturer. The assays for firefly luciferase activity and Renilla luciferase activ-ity were performed sequentially using a luminometer with two injectors (Thermo Labsystems, Helsinki, Finland). Changes in firefly luciferase activity were calculated and plotted after normalization with changes in Renilla luciferase activity in the same sample.
Electrophoretic Mobility Shift Assay (EMSA)-Nuclear protein extracts were prepared for EMSA as described earlier (15). The protein content of the nuclear extract was determined using the Bradford protein assay kit (Bio-Rad). EMSA experiments were performed as described before (15). The probes of double-stranded oligonucleotides for NF-B, Sp1, and AP-1 were synthesized by Sigma Genosys, and were based on the human MMP-9 promoter sequence (20) as follows: wild-type NF-B (5Ј-TGGAATTCCCAG); mutant NF-B (5Ј-TTTAATTCCCAG); wild-type Sp1 (5Ј-CCTTCCGCCCCC); mutant Sp1 (5Ј-CCTTC-CAACCCC); wild-type AP-1 (5Ј-CCTGAGTCAGCA); and mutated Ap-1(5Ј-CCTTTGTCAGCA). The complimentary oligonucleotides were annealed and purified following the manufacturer's instructions. The NF-B, Sp1, and AP-1 oligonucleotides were end-labeled with [␥-32 P]ATP using T4 polynucleotide kinase as recommended by the manufacturer (Promega). Five g of nuclear proteins from control or treated cells were incubated with 32 P-labeled oligonucleotide probe under binding conditions (Promega) for 20 min at room temperature in a final volume of 20 l. For cold competition, a 100-fold excess of the respective unlabeled consensus oligonucleotide was added in reaction buffer containing nuclear protein for 10 min before adding probe. The same amount of mutated oligonucleotide probe was used as another control. After binding, protein-DNA complexes were electrophoresed on a native 4.5% polyacrylamide gel using 1ϫ Tris glycine buffer. Each gel was then dried and subjected to autoradiography at Ϫ80°C.
Treatment of Cells in Culture with Cyclic AMP Response Element (CRE) Oligonucleotides-CRE-decoy (CRE) and control oligonucleotides (cREC) used in the present studies are phosphorothioate oligonucleotides based on published data (21). Their sequences are as follows: CRE, 24-mer CRE-palindrome: 5Ј-TGA-CGTCATGACGTCATGACGTCA; cREC, 24-mer nonsense-sequence palindrome control oligonucleotide: 5Ј-CTAGCTAGCT-AGCTAGCTAGCTAG, which was synthesized by Sigma Genosys. Cells (0.25-1 ϫ 10 5 cells/well) were plated in 6-well plates containing the growth medium and grown to 70% confluence. CRE-decoy or control oligonucleotides were transfected into cells using FuGENE 6 lipofection reagent as described in our earlier work (15). After 24 h of incubation, cells were exposed to the culture plates coated with FN for an additional 24 h. The preparation of cell extracts and measurement of luciferase activities were carried out using the Dual Luciferase Reporter Kit according to recommendations by the manufacturer.
Integrin Subunit ␣5, c-Fos, and c-Jun Small Interfering RNA (siRNA) Treatments-The integrin subunit ␣5 siRNA (catalog number sc-29372), the AP-1 subunit c-Fos siRNA duplexes (catalog number sc-29221), c-Jun (catalog number sc-29223), and negative control siRNA (catalog number sc-37007) were purchased from Santa Cruz Biotechnology, Inc. For the transfection procedure, cells were grown to 60% confluence and ␣5, c-Fos, and c-Jun siRNAs or control siRNA were transfected using Lipofectamine TM 2000 (Invitrogen) according to the FIGURE 1. The effect of FN on MMP-9 gelatinolytic activity and gene expression in human lung carcinoma cells. A, dose-dependent effect of FN on MMP-9 expression. Culture medium and cellular proteins were isolated from H1838 cells exposed to increased concentrations of FN in FN-coated culture plates as indicated, followed by gelatin zymograph and Western blot for MMP-9 activity and protein. B, time-dependent effect of FN on MMP-9 expression. Culture medium and cellular proteins were isolated from H1838 cells exposed to FN (20 g/ml)-coated culture plates for the indicated time periods. Afterward, gelatin zymograph and Western blot analysis were performed to determine MMP-9 activity and protein, respectively. Actin served as internal control for normalization purposes. The right panel summarizes the data by showing densitometric analysis of at least three separate experiments. C, dose-dependent effect of FN on MMP-9 mRNA expression. Total RNA was isolated from H1838 cells exposed to increased concentrations of FN-coated culture plates, then subjected to RT-PCR analysis. GAPDH expression was evaluated as internal control. D, effect of FN on MMP-9 mRNA expression as determined by real-time RT-PCR. Total RNA was isolated from H1838 cells cultured on plastic or FN or collagen type 1 (20 g/ml each)coated tissue culture plates, followed by real-time PCR analysis. GAPDH served as internal control for normalization purposes. * denotes significant differences from control ( p Ͻ 0.05). Con, indicates untreated control cells.
manufacturer's instructions. Briefly, Lipofectamine 2000 reagent was incubated with serum-free medium for 10 min. Subsequently, a mixture of respective siRNA was added. After incubation for 15 min at room temperature, the mixture was diluted with culture medium and added to each well. The final concentration of siRNA in each well was 100 nM. After culturing for 30 h, cells were washed, resuspended in new culture media, and were exposed to the culture plates coated with FN for an additional 24 h. Afterward, cells were harvested by Western blot, transfection luciferase activity assays, and [H 3 ]thymidine incorporation assays.
Statistical Analysis-All experiments were repeated a minimum of three times. All data collected from electrophoresis gel mobility shift assays, luciferase activity assays, real-time RT-PCR, and Western blot were expressed as mean Ϯ S.D. The data presented in some figures are from a representative experiment, which was qualitatively similar in the replicate experiments. Statistical significance was determined with Student's t test (two-tailed) comparison between two groups of data sets. Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control condition ( p Ͻ 0.05, see figure legends).

FN Stimulates MMP-9 Gelatinolytic Activity and Gene
Expression in Human NSCLC Cells-We began by exploring the effects of FN on MMP-9 gelatinolytic activity and gene expression. As shown in Fig. 1, A and B, cultures of H1838 cells on FN-coated plates showed an increase in MMP-9 gelatinolytic activity and protein synthesis in a dose-and time-dependent manner with maximal stimulation at 20 g/ml in 24 h as determined by gelatin zymography and Western blot analysis. FN also stimulated MMP-9 mRNA expression in a dose-dependent manner as assessed by RT-PCR (Fig. 1C). The stimulatory effect of FN on MMP-9 mRNA levels was confirmed by real-time RT-PCR ( Fig. 1D). Similar results were obtained with H2106 cells (not shown). Note that collagen type 1 had no effect.
FN Stimulates MMP-9 Expression through ␣5␤1-dependent Signals That Include ERK and PI3K Activation-Because FN function is mediated largely through its integrin receptor ␣5␤1, we determined if blockade of the ␣5 subunit abrogates the effect of FN on MMP-9 expression. H1838 cells transfected with ␣5 siRNA were exposed to FN for an additional 24 h. Afterward, the ␣5 and MMP-9 protein levels were evaluated. As shown in Fig. 2A, ␣5 siRNA blocked endogenous ␣5 protein expression in H1838 cells. FN had no effect on induction of MMP-9 expression in ␣5 silenced cells, whereas control siRNA had no effect on FN-induced MMP-9 ( Fig. 2A). FN has been shown to affect several kinases including ERK and PI3K/Akt in cell systems including human lung carcinoma cells (7,15,22). Here, we show that FN-induced phosphorylation of ERK1/2 (Fig.  2B) and Akt (Fig. 2C) were abrogated in ␣5 silenced NSCLC cells. This, together with the finding above, suggested that the ␣5 integrin signal was critical in mediating the effect of FN.
We then tested whether regulation of MMP-9 protein and activity by FN was mediated by ERK and PI3K. We found MMP-9-related gelatinolytic activity present in conditioned medium, and this was increased in the setting of FN exposure (Fig. 3). Treatment with PD98095, an inhibitor of ERK, and with wortmannin, an inhibitor of PI3K, blocked the stimulatory effect of FN on MMP-9 protein (Fig. 3A) and mRNA expression (Fig. 3, B and C). In contrast, the inhibitor of Rho kinase, Y27632, and that of protein kinase A, H89, did not affect FNinduced MMP-9 protein and mRNA expression (Fig. 3, D-F). We previously identified a role for the mTOR signal cascade in mediating the effect of FN on NSCLC cell growth (9). However, we found that rapamycin, an inhibitor of mTOR, had no effect on blockade of FN-induced MMP-9 protein (Fig. 3G) and mRNA (Fig. 3, H and I) expression. We also tested whether JNK was involved in the effect of FN on MMP-9, and found that the inhibitor of JNK, SP600125, had no effect on blockade of FNinduced MMP-9 protein (Fig. 3J) and mRNA expression (Fig.  3K). Similar results were obtained with H2106 cells (not shown).
FN Increases MMP-9 Promoter Activity-We next examined whether the effects of FN on MMP-9 expression occur at the transcriptional level. The MMP-9 promoter constructs used contain multiple transcription factor binding sites including NF-B, Sp1, and AP-1 (Fig. 4, A and B). These sites have been shown to be differentially responsive to various stimuli (19,20,23). As shown in Fig. 4C, we found that H1838 cells transfected with the wild-type MMP-9 promoter luciferase reporter construct and exposed to FN showed increased activity. Collagen type 1 had no effect. The FN-induced MMP-9 promoter activity signals on FN-induced MMP-9 protein expression. Cellular protein was isolated from H1838 cells transfected with control or ␣5 siRNA (100 nM each) for 30 h before exposing the cells to FN (20 g/ml)-coated culture plates for an additional 24 h, and then subjected to Western blot analysis for ␣5, MMP-9, and actin proteins. B, effect of blocking ␣5 signals on FN-induced phosphorylation of ERK. Cellular protein was isolated from H1838 cells transfected with control or ␣5 siRNA (100 nM each) for 30 h before exposing the cells to FN (20 g/ml)-coated culture plates for an additional 2 h, followed by Western blot analysis with antibodies against ␣5, total ERK1/2, and phosphorylated ERK1/2 (p-ERK1/2). C, effect of blocking ␣5 signals on FN-induced phosphorylation of Akt. Cellular protein was isolated from H1838 cells transfected with control or ␣5 siRNA (100 nM each) for 30 h before exposing the cells to FN (20 g/ml)-coated culture plates for an additional 2 h, followed by Western blot analysis with antibodies against ␣5, total Akt, and phosphorylated Akt (p-Akt). Actin served as internal control for normalization purposes. Con, indicates untreated control cells.

Induction of MMP-9 by Fibronectin in Human Lung Carcinoma Cells
was still observed with one MMP-9 deletion construct (Ϫ90/ ϩ34 bp). However, there was a decreased response to FN with the smallest MMP-9 deletion construct (Ϫ73/ϩ34 bp) (Fig.  4C). By using site-directed mutated MMP-9 promoter constructs connected to the luciferase reporter gene in which the NF-B, Sp1, and AP-1 sites were separately mutated, we found that FN had no effect on MMP-9 promoter activity when the AP-1 site was mutated, whereas FN-induced activity was detected when the other two were tested (Fig. 4D). This suggests the involvement of AP-1 in mediating the stimulatory effect of FN on MMP-9 expression. Similar results were obtained with H2106 cells (not shown).
AP-1, but Not NF-B or Sp1 Sites in the MMP-9 Promoter, Is Involved in FN-induced MMP-9 Gene Expression-To further explore FN regulation of MMP-9 promoter activity, electrophoretic mobility shift assays were performed to identify the transcription factors regulated by FN. As shown in Fig. 5, H1838 cells exposed to FN (20 g/ml) for 24 h showed a significant increase in AP-1 DNA binding (A), whereas very little effect on Sp1 (B) and NF-B (C) nuclear protein binding activities were noted when compared with solvent controls. In contrast, collagen type 1 (20 g/ml) had no effect. The specific bands for AP-1, Sp-1, or NF-B were attenuated by a 100-fold molar excess of unlabeled consensus oligonucleotides, but were not inhibited by the mutated unlabeled oligonucleotide (mut). Oligonucleotides containing mutated AP-1 (Mut AP-1), Sp1 (Mut Sp1), NF-B (Mut NF-B) sites were end labeled with [␥-32 P]ATP and used as another control to confirm the binding specificity. The addition of anti-c-Fos antibody resulted in a supershift band, whereas an anti-c-Jun antibody had a lesser effect (Fig. 5D). The natural product, NDGA, has been shown to inhibit binding of the Jun/AP-1 protein to the AP-1 site (24). As shown in Fig. 6, A and B, NDGA (0.5 M) blocked FN-induced MMP-9 protein production (A), and MMP-9 promoter activity (B). The synthetic double-stranded phosphorothioate oligonucleotides with high affinity for a target transcription factor can be introduced into cells as decoy cis-elements to bind the factors and alter gene expression. The CRE oligonucleotides have been shown to inhibit CRE-and AP-1-directed gene transcription and promote growth inhibition in vitro and in vivo in a broad spectrum of cancer cells (25). Using CRE-decoy oligonucleotides transfected into cells, we found that the CRE palindromic oligonucleotides eliminated the FN-induced AP-1/ DNA binding (Fig. 6C) and MMP-9 promoter activity (Fig. 6D), whereas the control CRE oligonucleotides had no effects. This suggests that the CRE-decoy competed with AP-1 and interfered with FN-induced AP-1/DNA binding and MMP-9 promoter activity. Similar results were obtained with H2106 cells (not shown).
c-Fos, but Not c-Jun, Mediates the Effects of FN on MMP-9 Expression-In Fig. 5D, we showed that FN stimulates c-Fos protein. Next, we found that FN increased nuclear c-Fos protein expression in a time-and dose-dependent manner (Fig. 7,  A and B), whereas it had little effects on c-Jun. The inhibitor of ERK, PD98095, and PI3K, wortmannin, blocked FN-induced c-Fos protein expression (Fig. 7C). We also found that NDGA inhibited FN-induced c-Fos protein expression (Fig. 7D). To determine the role of c-Fos in mediating the effects of FN on MMP-9, we blocked c-Fos expression by a siRNA approach. As shown in Fig. 7E, c-Fos siRNA completely abrogated the production of c-Fos protein. Furthermore, this siRNA eliminated the stimulatory effect of FN on MMP-9 protein expression, whereas the control siRNA had no effect (Fig. 7E). It also attenuated the effects of FN on MMP-9 promoter activity (Fig. 7F). However, silencing c-Jun by siRNA had no effect on FN- C, effect of inhibitor of ERK1/2 and PI3K on FN-induced MMP-9 mRNA as determined by real-time RT-PCR. Total RNA was isolated from H1838 cells cultured for up to 2 h in the presence or absence of PD98095 (25 M) or wortmannin (100 nM) before exposure of the cells to FN-coated culture plates for an additional 24 h, followed by real-time PCR analysis. GAPDH served as internal control for normalization purposes. *, denotes significant differences from control ( p Ͻ 0.05). **, indicates significance of combination treatment as compared with FN alone ( p Ͻ 0.05). Con, indicates untreated control cells. D, effect of protein kinase A and Rho kinase inhibitors on FN-induced MMP-9 gelatinolytic activity and protein. Culture medium and cellular protein were isolated from H1838 cells incubated for up to 2 h in the presence or absence of H89 (10 M) and Y27632 (10 M) before exposure of the cells to FN-coated culture plates for an additional 24 h, then subjected to gelatin zymograph for gelatinolytic activity and Western blot analysis for MMP-9 protein. E, effect of PKA and Rho kinase inhibitors on FN-induced MMP-9 mRNA levels. Total RNA was isolated from H1838 cells cultured for up to 2 h in the presence or absence of H89 (10 M) and Y27632 (10 M) before exposing the cells to FN-coated culture plates for an additional 24 h, then subjected to RT-PCR analysis. GAPDH expression was evaluated as internal control. F, effect of PKA and Rho kinase inhibitors on FN-induced MMP-9 mRNA as determined by real-time RT-PCR. Total RNA was isolated from H1838 cells cultured for up to 2 h in the presence or absence of H89 (10 M) and Y27632 (10 M) before exposure of the cells to FN-coated culture plates for an additional 24 h, followed by real-time PCR analysis. GAPDH served as internal control for normalization purposes. G, effect of mTOR inhibitor on FN-induced MMP-9 gelatinolytic activity and protein levels. Culture medium and cellular protein were isolated from H1838 cells cultured for up to 24 h in the presence or absence of mTOR inhibitor, rapamycin (10 nM), before exposure of the cells to FN-coated culture plates for an additional 24 h, and then subjected to gelatin zymograph for gelatinolytic activity and Western blot analysis for MMP-9 protein. H, effect of mTOR inhibitor on FN-induced MMP-9 mRNA levels. Total RNA was isolated from H1838 cells cultured for up to 24 h in the presence or absence of rapamycin (10 nM) before exposure of the cells to FN-coated culture plates for an additional 24 h, and then subjected to RT-PCR analysis. GAPDH expression was evaluated as internal control. I, effect of the mTOR inhibitor on FN-induced MMP-9 mRNA as determined by real-time RT-PCR. Total RNA was isolated from H1838 cells incubated for up to 24 h in the presence or absence of rapamycin (10 nM) before exposure of the cells to FN-coated culture plates for an additional 24 h, followed by real-time PCR analysis. GAPDH served as internal control for normalization purposes. *, denotes significant differences from control ( p Ͻ 0.05). J, effect of inhibitors of JNK on FN-induced MMP-9 protein. Cellular protein was isolated from H1838 cells incubated for up to 2 h in the presence or absence of SP600125 (10 M) before exposure of the cells to FN-coated culture plates for an additional 24 h, then subjected to Western blot analysis for MMP-9 protein. Actin served as internal control for normalization purposes. K, effect of JNK inhibitor on FN-induced MMP-9 mRNA levels. Total RNA was isolated from H1838 cells cultured for up to 24 h in the presence or absence of SP600125 (10 M) before exposure of the cells to FN-coated culture plates for an additional 24 h, and then subjected to RT-PCR analysis. GAPDH expression was evaluated as internal control. Con, indicates untreated control cells.

DISCUSSION
The expression of MMP-9 is regulated by growth factors, hormones, cytokines, and cellular transformation. MMP-9 is highly regulated at three different levels: transcriptional regulation, activation of latent MMP-9, and inhibition of MMP-9 activity. Compared with that of control subjects, MMP-9 levels are significantly higher in the plasma of NSCLC patients (26). The enhanced expression of MMP-9 is associated with human lung cancer invasion and/or metastasis (26,27). MMP-9 null mice showed an 81% reduction in Lewis lung carcinoma tumor as compared with wild-type controls (28). Homogeneous MMP-9 expression in cancer cells is an independent prognostic factor in operable NSCLC, so MMP-9 may be considered as target for adjuvant anticancer therapy in operable NSCLC using selective MMP inhibitors with high specificity for MMP-9. MMP-9 inhibitors and an adenovirus-mediated transfer of antisense MMP-9 have been shown to inhibit invasion, angiogenesis, growth, and metastasis in NSCLC cells (29,30). In human lung cancer cells, high expression of MMP-9 is associated with growth, metastasis, and progression, and blockade of MMP-9 has been shown to control lymph node metastasis and prolong the life span of lung cancer patients (29 -32). At present, the mechanisms responsible for the regulation of MMP-9 in tumors remain incompletely elucidated.
In this study, we show that the matrix component FN increased the expression and gelatinolytic activity of MMP-9 in NSCLC cells. FN is a heterodimeric extracellular matrix glycoprotein implicated in a number of physiological events during embryogenesis, angiogenesis, thrombosis, inflammation, and tumor invasion (33,34). The adhesion of lung carcinoma cells to FN enhances tumorigenicity and confers resistance to apoptosis induced by standard chemotherapeutic agents (35). FN has been shown to affect MMP-9 function and gene expression in several other systems (12,13,36,37). For example, monocyte activation within the glomerulus was mediated by binding to mesangial matrix components, particularly FN, and this resulted in activation of MMP-9 (13). FN secreted from peritoneum increased MMP-9 activity and expression, and, in turn, invasiveness of ovarian cancer cells through multiple signal pathways (14). Induction of cell adhesion and MMP-9 gene expression in human HL-60 myeloid leukemia cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to FN and its receptor ␣5␤1 integrin (38). We found that 20 g/ml concentration of FN gives a maximal induction of MMP-9 protein, mRNA, and gelatinolytic activity, whereas higher doses of FN had no further effect (Fig. 1, A and B). This is well in the dose range of FN used in experiments presented in the literature. The drop in MMP-9 observed at higher doses may be due to desensitization of the cells exposed to higher doses of FN used in this study, and this needs further exploration.
FN has been shown to activate several kinase signals in different cell systems (7,15,22,39,40). We previously demonstrated that this molecule increased NSCLC cell growth by activating the ERK and PI3K signals (9,15). Both signal pathways mediating the effects of FN on MMP-9 expression have been shown to be important in several other studies suggesting that activation of these dual signaling pathways is required for the FNdependent activation of MMP-9 secretion and gene expression (14,37,41). Knockdown of Akt using siRNA also abrogated FNinduced MMP-9 expression (not shown). Head and neck squamous cell carcinoma adhesion to FN and induction of MMP-9 secretion were increased by the chemokine CXCCL12, whereas the addition of an inhibitor of ERK-1/2 signaling, PD98059, reduced the effects of CXCL12 (42). Also, treatment of cells with ERK inhibitors, U0126 and PD98059, and PI3K inhibitors, wortmannin and LY294002, dramatically suppressed the secretion of MMP-9 induced by FN. In contrast, a specific PKC inhibitor (GF109203X) did not inhibit FN-dependent MMP-9 secretion and mutated reporter constructs of the human MMP-9 promoter (B). These regions contain several transcription factor binding sites including NF-B, Sp1, and AP-1. C, effects of FN on MMP-9 promoter activity. H1838 lung cancer cells (1 ϫ 10 5 cells) were cotransfected with a full-length or several human MMP-9 promoter deletion constructs ligated to the luciferase reporter gene and an internal control phRL-TK synthetic Renilla luciferase reporter vector as described under "Materials and Methods" for 24 h, then exposed the cells to the vehicle control (Con), 20 g/ml FN, or collagen type 1-coated culture plates for an additional 24 h. D, effects of FN and collagen type 1 on mutated MMP-9 promoter activity. H1838 cells were transfected with specific MMP-9 mutated constructs in respective sites (NF-Bm, Sp1m, and AP-1m) for 24 h, and then exposed the cells to FN-or collagen type 1 (20 g/ml, each)-coated culture plates for an additional 24 h. The ratio of firefly luciferase to Renilla luciferase activity was quantified as described under "Materials and Methods." The bars represent the mean Ϯ S.D. of at least four independent experiments for each condition. * indicates significance as compared with controls. ** indicates significance of combination treatment as compared with FN alone ( p Ͻ 0.05). Con, indicates untreated control cells. (14). These observations by others are consistent with the results of this study and indicate that activation of these two signal pathways play an important role in mediating the effect of FN on MMP-9 expression. In contrast, the effects of FN on MMP-9 were not affected by blockade of Rho kinase, mTOR, and JNK signals, although these signals are involved in controlling cell migration, invasion, and gene expression, and in promoting NSCLC cell growth by FN (11,43,44,45).
The function of FN is largely mediated through its integrin receptor ␣5␤1. We have found that blockade of this receptor eliminated the stimulatory effect of FN on MMP-9 and on phosphorylation of ERK and PI3K/Akt signals suggesting ␣5␤1 is critical in mediating the effect of FN on these processes. Blockade of this integrin receptor has been shown to affect FN-mediated cell adhesion, survival, proliferation, and gene expression (9, 46 -48). Monocyte adhesion was abrogated in cells treated with specific neutralizing anti-␣5␤1 integrin monoclonal antibody (46). Antibodies against ␣5␤1 have been shown to block the effect of FN on phosphorylation of p70 S6K, a downstream signal of mTOR (9). Antisense oligonucleotides to the integrin receptor subunit ␣5 decreased FN fragment-mediated cartilage chondrolysis (49). Chondrosarcoma-derived chondrocytic cell adhesion to FN was abolished by a blocking antibody against ␣5␤1 integrin, but not by one against anti-␣v␤3 integrin, suggesting the specificity of this integrin in mediating many FN functions (50).
The MMP-9 promoter contains multiple transcription factor binding sites including NF-B, Sp1, and AP-1. These sites have been shown to be differentially responsive to various stimuli (23,51). MMP-9 has been demonstrated to be regulated at the level of gene transcription in different cell types (23,52). To investigate whether FN-mediated up-regulation of MMP-9 reflected transactivation of the gene, we performed transient transfection experiments utilizing human MMP-9 promoter constructs connected to a luciferase reporter gene. We found that FN, not collagen type 1, increased MMP-9 promoter activity. Furthermore, we provide evidence in support of AP-1 controlling MMP-9 expression. Data obtained from different experimental models in vitro and in vivo indicate that the AP-1 protein functions as an important regulator of cell proliferation, differentiation, apoptosis, and transformation (53). AP-1 activity is regulated in a given cell by a broad range of physiological and pathological stimuli including cytokines, growth factors, stress signals, and infections, as well as oncogenic stimuli (54). Mutation of the AP-1 site in the MMP-9 promoter region severely diminishes the stimulatory effect of FN on MMP-9 expression; this indicates that increased AP-1 DNA binding activity is necessary for up-regulation of MMP-9 by FN. The role of the AP-1 site in regulation of MMP-9 expression has been reported by others in other systems (19,55). Serum amyloid A-activating factor-1, a novel transcription factor, and the AP-1 family of proteins cooperatively regulate cytokinemediated induction of MMP-9 in the resident cells of the joint capsule, whereas the mutation of these two elements results in severe reduction in cytokine responsiveness of the MMP-9 promoter activity (23). The ginseng saponin metabolite suppresses phorbol ester-induced matrix MMP-9 expression through inhi- Oligonucleotides containing the AP-1 site were end labeled with [␥-32 P]ATP and incubated with nuclear extracts (5 g) from H1838 cells exposed to FN-or collagen type 1 (20 g/ml each)-coated culture plates for an additional 24 h. B, effect of FN on Sp1 binding activities. Oligonucleotides containing the Sp1 site were end labeled with [␥-32 P]ATP and incubated with nuclear extracts (5 g) from H1838 cells exposed to FN-or collagen type 1 (20 g/ml each)-coated culture plates for an additional 24 h. C, effect of FN on NF-B binding activities. Oligonucleotides containing the NF-B site were end labeled with [␥-32 P]ATP and incubated with nuclear extracts (5 g) from H1838 cells exposed to FN-or collagen type 1 (20 g/ml each)-coated culture plates for an additional 24 h. D, anti-c-Fos antibody supershift. Oligonucleotides containing AP-1 sites were end labeled with [␥-32 P]ATP and incubated with nuclear extracts (5 g), c-Fos, and c-Jun antibodies (2 g/l each) for 24 h. For competition assays, a molar excess (ϫ100) of consensus or mutated AP-1 (Cold AP-1 and mut AP-1), Sp1 (Cold Sp1 and mut Sp1), or NF-B (Cold NF-B and mut NF-B) were added to the binding reaction. Oligonucleotides containing mutated AP-1 (Mut AP-1), Sp1 (Mut Sp1), or NF-B (Mut NF-B) sites end labeled with [␥-32 P]ATP were also used to confirm binding specificity. Con, indicates untreated control cells.
bition of AP-1 and mitogen-activated protein kinase signaling pathways in human astroglioma cells (55). This study further indicates an important role of AP-1 in mediating the effect of FN on MMP-9 expression.
The CRE transcription factor complex is a pleiotropic activator that participates in the induction of a wide variety of cellular and viral genes. Because the CRE cis-element, TGACGTCA, is palindromic, a synthetic single-stranded oligonucleotide composed of the CRE sequence self-hybridizes to form a duplex/hairpin. This oligonucleotide inhibits CRE-and AP-1-directed gene transcription and promotes growth inhibition in vitro and in vivo in a broad spectrum of cancer cells, without adversely affecting normal cell growth (25,56). CRE- A, effect of AP-1 inhibitor on FN-induced MMP-9 protein expression. Cellular protein was isolated from H1838 cells treated with a chemical AP-1 inhibitor, NDGA (0.5 M), for 24 h before exposure of the cells to FN-coated culture plates for an additional 24 h. Afterward, Western blot analysis was performed to examine for MMP-9 protein. B, effect of the AP-1 inhibitor on MMP-9 promoter activity. H1838 cells were transfected with MMP-9 constructs (Ϫ670/ ϩ34), together with the AP-1 inhibitor NDGA (0.5 M) for 24 h, then exposed to FN (20 g/ml)-coated culture plates for an additional 24 h. C, effect of CRE-decoy oligonucleotide on AP-1/DNA binding. Oligonucleotides containing the AP-1 site were end labeled with [␥-32 P]ATP and incubated with nuclear extracts (5 g) from H1838 cells transfected with CRE-decoy (dCRE) or control oligonucleotides (cCRE, 150 nM each) for 24 h before exposure of the cells to culture plates coated with FN (20 g/ml each) for an additional 24 h. Binding site specificity was tested by EMSA. For competition assays, a molar excess (ϫ100) of consensus or mutated AP-1 (Cold AP-1 and mut AP-1) were added to the binding reaction. D, effect of CRE-decoy oligonucleotides on MMP-9 promoter activity. H1838 cells (1 ϫ 10 5 cells) were cotransfected with CRE-decoy (CRE) or control oligonucleotides (CREC, 150 nM each) and wildtype human MMP-9 promoter reporter constructs ligated to a luciferase reporter gene and an internal control phRL-TK synthetic Renilla luciferase reporter vector for 24 h, then treated as indicated with vehicle control (Con), or FN (20 g/ml)-coated culture plates for an additional 24 h. The ratio of firefly luciferase to Renilla luciferase activity was quantified as described under "Materials and Methods. " The bars represent the mean Ϯ S.D. of at least four independent experiments for each condition. * indicates significance as compared with controls. ** indicates significance of combination treatment as compared with FN alone ( p Ͻ 0.05). Con, indicates untreated control cells. were transfected with control c-Fos siRNA (100 nM each) and with wild-type human MMP-9 promoter constructs ligated to luciferase reporter gene and an internal control phRL-TK synthetic Renilla luciferase reporter vector as described under "Materials and Methods" for up to 30 h before exposure of the cells to the culture plates coated with FN (20 g/ml) for an additional 24 h. The ratio of firefly luciferase to Renilla luciferase activity was quantified as described under "Materials and Methods. " Data are expressed as mean Ϯ S.D. of at least three independent experiments. * indicates significant difference from control. ** indicates significance of a combination treatment as compared with FN alone ( p Ͻ 0.05). G, effect of blocking the c-Jun signal on FN-induced MMP-9 protein expression. Cellular protein was isolated from H1838 cells transfected with control or c-Jun siRNA (100 nM each) for 30 h before exposure of the cells to FN (20 g/ml)-coated culture plates for an additional 24 h, and then subjected to Western blot analysis for c-Jun and MMP-9 proteins. Actin served as internal control for normalization purposes. Con, indicates untreated control cells.
decoy oligonucleotides may provide a powerful new means of combating cancers, and other pathological conditions by regulating the expression of cAMP-responsive genes (25). Phenylephrine stimulated DNA binding activity of AP-1 and increased protein synthesis in cardiomyocytes of adult rats, and inhibition of AP-1 binding activity by CRE-decoy oligonucleotides abolished both of these growth responses (57). In another study, the CRE-decoy oligonucleotide treatment was shown to inhibit ovarian cancer cell growth and caused a marked reduction in MMP-9 activity (58). Consistent with this, we found that CRE palindromic oligonucleotides blocked the FN-induced effect on AP-1/DNA binding and MMP-9 promoter activity, whereas the control CRE oligonucleotide had no effect. Together, these observations suggest that the CRE-decoy competed with AP-1 and interfered with FN-induced AP-1/DNA binding and MMP-9 promoter activity.
We also found that the AP-1 subunit c-Fos played a critical role in mediating the effect of FN on controlling MMP-9 expression. c-Fos has oncogenic activity and is frequently overexpressed in tumor cells (53). Numerous experiments have demonstrated that AP-1 subunits, in addition to their pro-apoptotic function, are also critically involved in survival signaling. c-Fos expression negatively correlates with increased neuronal cell death in the hippocampus during kainic acid-induced seizure, indicating an anti-apoptotic role for the protein in this scenario (60). Osteoblast binding to extracellular matrix proteins such as FN through integrins induces c-Fos and c-Jun expression via protein kinase C and phosphotyrosine kinase signaling pathways (61). FN is required for vascular endothelial growth factor-induced c-Fos induction, mitogenic response, and cell migration in T47D breast cancer cells (62). We found that the inhibitors of ERK, PD98095, of PI3K, wortmannin, and of AP-1, NDGA, blocked FN-induced c-Fos protein expression. Cellular interactions with the extracellular matrix play an important role in activating ERK and c-Fos-dependent processes. There is a positive correlation between survival and adhesion to FN, as well as activation of PI3K and ERK, and induced expression of c-Fos in bone marrow-derived mast cells, and wortmannin blocked these effects (63). Increased c-Fos expression was largely affected by inhibition of ERK and/or PI3K signals in other studies as well (64,65). We also found that c-Fos siRNA alleviated the effects of FN on enhancing MMP-9 promoter activity. The c-Fos antisense oligonucleotides blocked thrombin-induced expression of MMP-9 mRNA as well as AP-1 binding activities in cultured human mesangial cells (59). Together, these studies show that c-Fos is a critical transactivator for MMP-9 gene expression. Note that the MMP-9 basal activity was still observed in c-Fos-silenced cells, suggesting that transcription factors other than AP-1 (e.g. Sp1 and NF-B) might play a role in maintaining the MMP-9 basal promoter activity (19,20). FN had little effect on c-Jun expression and silencing c-Jun by siRNA did not block the FN-induced MMP-9 protein expression. These results indicated that c-Jun played no significant role in mediating the effect of FN on MMP-9 expression.
In summary, our study indicates that FN, by binding to the integrin ␣5␤1 receptor, stimulates the expression of MMP-9 through increasing AP-1/DNA binding activity and c-Fos protein expression via the activation of PI3K and ERK signaling pathways. The CRE-decoy abrogated FN-induced AP-1/DNA binding and MMP-9 promoter activity. Blockade of c-Fos signal also ablated the stimulatory effect of FN (Fig. 8). This study reveals a novel molecular mechanism by which FN modulates NSCLC cell functions.