Fucosyltransferase IV Enhances Expression of MMP-12 Stimulated by EGF via the ERK 1 / 2 , p 38 and NF-kB Pathways in A 431 Cells

Endometrial carcinoma is the most common malignancy in the female genital tract. The invasion of endometrial adenocarcinoma into endometrial tissue and metastasis to the nearby pelvic or distal tissues and organs are crucial factors affecting the prognosis and mortality of women. The synthesis and secretion of several proteases are up-regulated in endometrial carcinoma, including matrix metalloproteinase (MMP) (Shaco-Levy et al., 2008; Oh et al., 2009). Matrix metalloproteinases (MMPs), a family of more than 20 extracellular, zinc-dependant proteases that degrade the multiple structural components of the extracellular matrix (ECM), have been shown to engage in tumor invasion. MMPs play essential roles in many aspects of biology, including cell proliferation, differentiation, apoptosis, and migration. These processing enzymes have clear links to cancer and tumor progression, in which proteolysis of the ECM is required to accommodate increased growth, migration, and invasion of tumor cells (Korpos et al., 2009; Thrailkill et al., 2009; Tonti et al., 2009). In a previous study we showed that increased expression of MMP-12 was associated with the extent


Introduction
Endometrial carcinoma is the most common malignancy in the female genital tract.The invasion of endometrial adenocarcinoma into endometrial tissue and metastasis to the nearby pelvic or distal tissues and organs are crucial factors affecting the prognosis and mortality of women.The synthesis and secretion of several proteases are up-regulated in endometrial carcinoma, including matrix metalloproteinase (MMP) (Shaco-Levy et al., 2008;Oh et al., 2009).
Matrix metalloproteinases (MMPs), a family of more than 20 extracellular, zinc-dependant proteases that degrade the multiple structural components of the extracellular matrix (ECM), have been shown to engage in tumor invasion.MMPs play essential roles in many aspects of biology, including cell proliferation, differentiation, apoptosis, and migration.These processing enzymes have clear links to cancer and tumor progression, in which proteolysis of the ECM is required to accommodate increased growth, migration, and invasion of tumor cells (Korpos et al., 2009;Thrailkill et al., 2009;Tonti et al., 2009).
In a previous study we showed that increased expression of MMP-12 was associated with the extent 1 Department of Biochemistry and Molecular Biology, Liaoning Provincial Core Lab of Glycobiology and Glycoengineering, 2 First Affiliated Hospital of Dalian Medical University, Dalian, China *For correspondence: yxmsnow@126.com,yanqiu2083@126.com
IkB is phosphorylated and degraded following stimulation by growth factors, cytokines, hormones, or other agents, resulting in dissociation from NF-kB and nuclear translocation of NF-kB.Constitutive activation of NF-kB via persistent nuclear NF-kB localization, and thus NF-kB-dependent transcription, has been detected in many cancers, including endometrial cancer (Oh et al., 2009).In addition, NF-kB also regulates the expression and activation of MMPs, which play a significant role in ECM degradation and in facilitating cell motility, tumor growth, and metastasis (Karin et al., 2002).Thus, we predict that compounds that block NF-kB activity will be useful for inhibiting MMP-dependent tumor growth and invasion.
LeY is a difucosylated oligosaccharide, which is overexpressed in the majority of carcinomas (Cao et al., 2001;Escrevente et al., 2006)4and has the following chemical structure: [Fucα14 2Galβ14 4(Fucα14 3) GlcNAcβ14 R].Arai (Arai &Nishida, 2003), andSkovlund (1997) showed that LeY is highly expressed in endometrial cancer and related to tumor grade.Theα1,3 fucosylation of LeY is catalyzed by fucosyltransferase IV (FUT4).FUT4 is a critical enzyme that controls LeY oligosaccharide synthesis (Taniguchi et al., 2000;Wang et al., 2001).In a previous study, we reported that MMP-12 is an important oncogene in high-stage and high-grade endometrial adenocarcinoma; however, the relationship between FUT4 and MMP-12 is not clear.
We investigated the possible signaling pathways involved in EGF-induced, NF-kB-mediated MMP-12 expression in A431 cells and tested the effect of FUT4 during the process.Our results suggest significantly increased EGF-induced MMP-12 expression via ERK1/2 and p38 MAPK-mediated phosphorylation and degradation of IkBα and NF-kB activation in A431 cells.

Preparation of Nuclear and Cytosolic Fractions
The cells (6×10 5 ) were cultured on a 100 mm culture dish in 10% FBS DMEM/F12 for 24 hr.These cells were incubated in serum-free DMEM/F12 for another 24 hr t and treated with EGF at designated times.Cells were washed twice with cold PBS and then scraped to an eppendorf in 1 ml of PBS, centrifuged at 4 ℃, 12,000 rpm for 5 min.Discarding the supernatant, the pellet was homogenized in 300 μl of hypotonic lysis buffer (HEPES (pH 7.6) 10 mM, EDTA 0.1 mM, dithiothreitol (DTT) 1 mM, PMSF 0.5 mM).After repeated homogenization, the homogenate was centrifuged at 4 ℃, 12,000 rpm for 10 min.The supernatant was the cytosolic fraction and was kept at -70 ℃ overnight.The pellet was washed twice in 20 μl of hypotonic lysis buffer to remove residual cytosolic proteins.After washing, the pellet was dissolved in 30 μl of hypertonic lysis buffer (HEPES (pH 7.6) 20 mM, EDTA 1 mM, DTT 1 mM PMSF 0.5 mM, 25% glycerol, 0.4 M NaCl), pipetted to homogeneity, fiercely vortexed, and then stored at -70 ℃ overnight.Next day, the solution was centrifuged to collect the supernatant, which was the nuclear fraction.The cytosolic and nuclear fractions were quantified, and equal amounts of protein were subjected to Western blot.

Western Blot Analysis
The nuclear and cytoplasmic extracts (30 μg) were resolved by SDS-PAGE and then electrotransferred to the nitrocellulose membrane.Western blot analysis was done as described elsewhere (Yang et al., 2007b).

Statistics
Results are expressed as the mean±the standard error of the mean (SEM) of at least three independent experiments.Statistical significance of difference between test groups was assessed by one-way ANOVA followed by Scheffe's test (post hoc).Statistical significance was defined at P< 0.05.

EGF increased A431 cell migration
To assess the effect of EGF on cell morphology, A431 cells were grown on 6-well plates and serum- deprived for 24 hr, followed by stimulation with different concentrations of EGF (0, 50, 100, or 200 ng/ml) for 24 hr.Microscopy revealed that addition of EGF resulted in an alteration of cell morphology.As shown in Figure 1A, EGF induced an elongated and fibroblastoid phenotype.The increase in concentration of EGF led to a more rapid and pronounced alteration in cell morphology (Figure 1Ab, c, d), suggesting that EGF stimulation was associated with a change of cell motility.
To confirm the transformation from the epithelial-tofibroblastoid phenotype, we examined the expression of MMP-12.MMP-12 is a member of the family of MMPs, which are zinc-dependent endopeptidases.To delineate if MMP-12 was implicated in the increase of cell migration, A431 cells were stimulated with EGF (100 ng/ml) for 24 hr and the expression of MMP-12 was analyzed.The effect of EGF on MMP-12 expression was determined by RT-PCR and Western blot analysis.The data demonstrated that EGF stimulation can increase the expression of MMP-12, and there were no differences at different concentrations (Figure 1B, 1C).

EGF induced nuclear translocation of NF-kB subunits p65 and p50, resulting in phosphorlation of IkBα
Transcription factor NF-kB has been reported to control MMP-12 gene expression (Churg et al., 2001).Thus, we examined the localization of NF-kB in nuclear and cytyosolic fractions from A431 cells to determine whether or not NF-kB was involved in regulating MMP-12 gene expression.A431 cells were treated with 100 ng/ml of EGF for 24 hr.Next, nuclear and cytoplasmic fractions were prepared from the cells.As shown in Figure 2, we showed that EGF induced NF-kB nuclear translocation from the cytosol in a time-dependent manner.In the nontreated control cells, p65 and p50 were primarily localized to the cytoplasm.Following EGF treatment, p65 and p50 translocated to the nucleus in a time-dependent manner.Ninety and 120 min after EGF treatment p65 and p50 were predominantly localized to the nucleus, respectively (Figure 2A).Next, it was determined whether or not EGFinduced NF-kB activation was due to phosphorylation and subsequent degradation of IkBα.The results showed that EGF treatment increased the amount of phospho-IkBα after 30 min and approached to the maximum at 60 min, then decreased (Figure 2B).Taken together, these results showed that EGF treatment resulted in nuclear translocation of NF-kB in A431 cells.

Effect of EGF on MMP-12 activity through the MAPK/ NF-kB signaling pathway
Several effector pathways for the EGF receptor have been reported.Among them, the mitogen-activated protein kinase (MAPK) cascade is considered to be most crucial (Seger & Krebs, 1995).To determine if the effector pathways were involved in the stimulation of MMP-12 activity by EGF, we first examined the kinetic profile of ERK activation upon stimulation by EGF using phospho-specific ERK1/2 and p38 antibodies.The results of Western blot analysis demonstrated that EGF (100 μg/ ml) induced a sharp and transient activation of the ERK and p38 signals, which was rapidly detected 15 or 30 min following the addition of EGF, then gradually declined (Figure 3A).
To further confirm that MAPK was an intermediate in the pathway that links EGF exposure to NF-kB activation, endogenous ERK1/2 and p38 expression were silenced in A431 cells an ERK inhibitor (PD98059) and/or a p38 inhibitor (SB203580).A431 cells were pretreated with an ERK inhibitor (10-6 μM PD98059) and/or a p38 inhibitor (10-6 μM SB203580) followed by EGF for 1 day.Inhibition of ERK1/2 and p38 by inhibitors resulted in reducing EGF-mediated NF-kB nuclear translocation compared with cells stimulated by EGF only (Figure 3B).NF-kB was inhibited by the same inhibitors.The results of Western blot analysis for MMP-12 revealed that PD98059 and SB203580 blocked the stimulatory effect of EGF on MMP-12 expression (Figure 3B).Taken together, these results indicated that MAPK is a critical mediator of EGF-induced, NF-kB-dependent MMP-12 expression in A431 cells.

Effect of FUT4 on MMP-12 stimulated by EGF via the MAPK signaling pathway
In previous studies we have shown that FUT4 promotes cell proliferation through the MAPK signaling pathway (Yang et al., 2010).FUT4 overexpression increased the activation of ERK1/2 and p38.To test if FUT4 could increase EGF-induced p65 and p50 translocation, A431 cells, FUT4 overexpression cells, and FUT4-RNAi cells were treated with or without EGF for 24 hr.Nuclear and cytoplasmic fractionation were prepared from the cells.The results showed that FUT4 promoted EGF-induced nuclear translocation of the NF-kB subunits, p65 and p50, in A431 cells (Figure 4).Next, we determined whether or not FUT4 increased EGF-induced MMP-12 expression.The results showed that FUT4 overexpression could increase MMP-12 expression induced by EGF (Figure 4).Taken together, the data showed that FUT4 effectively increased EGF-induced, NF-kB-dependent MMP-12 expression.

Discussion
The delicate balance between MMPs and inhibitors is maintained in normal physiologic conditions.Excessive production of MMPs could increase the invasion and mobility of tumor cells.A recent study has shown that MMP-12 is overexpresssed in various human cancers, e.g., epithelial ovarian carcinoma (Li et al., 2009), prostate cancer (Nabha et al., 2008), endometrial adenocarcinoma (Yang et al., 2007a), glioma cell (Sarkar et al., 2006), non-small cell lung cancer (Hofmann et al., 2005), oral verrucous and squamous cell cancer (Impola et al., 2004) and squamous cell carcinoma (Kerkela et al., 2002).In addition, FUT4 is mainly expressed in leukocytes and some epithelial cells (Allahverdian et al., 2006).Increases in the expression of FUT4 level are seen in different cancers, e.g., gastric carcinoma (Petretti et al., 1999), colorectal cancer (Kudo et al., 1998), pancreatic cancer (Taniguchi et al., 2000) and lung adnocarcinoma (Martin-Satue et al., 1998).FUT4 has an effect on cell invasion and mobility (Yang et al., 2007b).However, little is known about FUT4-mediated regulation of MMP-12 expression in epithelial-derived A431 cells.In this study we characterized the mechanism of EGF-induced, NF-kBmediated MMP-12 secretion in A431 cells.Our results showed that EGF induced phosphorylation of IkBα, which ultimately led to phosphorylation and degradation of IkBα and NF-kB nuclear translocation and transcriptional activation.Moreover, inhibition of MAPK decreased EGF-induced NF-kB activation and MMP-12 secretion.In addition, FUT4 activated NF-kB and stimulated MMP-12 secretion through the MAPK signaling pathway.
Up-regulation of MMP-12 expression by FUT4 in A431 cells may lead to cell mobility and invasion, which depend on both cellular properties and ECM composition.The expression and secretion of ECM-modifying MMPs is an important determinant of tumor invasion.MMP expression is regulated at the transcriptional level and the MMP promoters contain NF-kB (Chen et al., 2009;Oh et al., 2009), which have been shown to regulate MMP-12 promoter activity (Castrillo et al., 2003;Sampath et al., 2006).
The NF-kB transcription factor family regulates number of genes involved in diverse cellular processes, including inflammation, immune response, cell proliferation, and apoptosis.NF-kB activity is regulated by the endogenous inhibitor, IkBα, and interaction with IkBα blocks the nuclear localization of NF-kB, keeping NF-kB it sequestered in the cytoplasm.In the current study we showed that EGF induced phosphorylation of IkB, which led to degradation of IkBα and to NF-kB nuclear translocation and transcriptional activation in A431 cells (Kim et al., 2008).
Cell motility and invasiveness are still dependent on high doses of EGF (Rijken et al. 1991;Peppelenbosch et al., 1993).The early response of A431 cells to EGF induces rapid alterations in the organization of the actin microfilament system that result in extensive ruffling, lamellipodia formation, and cortical actin polymerization.EGF potentiates the signal-transduction pathway for cell proliferation, which differs from the pathway for migration.Mitogen-activated protein kinases (MAPKs) are rapidly activated in cells stimulated by a variety of mitogens or motogens, including EGF, which delivers essential signals via Ras to a protein kinase network involving the Raf-1 kinase, MEK, and MAPK (Klemke et al., 1997;Kawahara et al., 2002).A431 cells, human Biophys Res Commun, 324, 534-46. Li Y, Jia JH, Kang S, et al (2009).The functional polymorphisms on promoter region of matrix metalloproteinase-12, -13 genes may alter the of epithelial ovarian carcinoma in Chinese.Int J Gynecol Cancer, 19, 129-33. Lyu J, Joo CK (2005)

Figure 1 .
Figure 1.Effect of EGF on Invasion of A431 Cells.(A) A431 cells were treated with various concentrations of EGF (0, 50, 100, or 200 ng/ml) for 24 hr.In vitro invasion assay was performed according to the manufacturer's instructions and photographed under a microscope at 100×.(B) (C) The effect of various concentrations of EGF on the expression of MMP-12.A431 cells were treated with various concentrations of EGF (0, 50, 100, or 200 ng/ml) for 24 hr.Then, the proteins were collected and analyzed by Western blot (B) with antibody against MMP-12 and RT-PCR analysis (C)