P120-catenin Isoform 3 Regulates Subcellular Localization Of Kaiso And Promotes Invasion In Lung Cancer Cells Via A Phosphorylation-dependent Mechanism

IntroductionThe Armadillo catenine (p120 catenine, hereafter p120ctn) regulates E-cadherin stability at plasma membrane and interacts with Rho GTPase to regulate cytoskeleton organization in the cytoplasm. Recent increasing evident has revealed that p120ctn also shuttles into nucleus to regulate gene expression via interaction with transcription factors Kaiso. Kaiso putative target genes include matrilysin (MMP-7) and MTA2.Increasing evident show that phosphorylation of p120ctn as a mechanism of regulation E-cadherin stability and Rho GTPase activity, but little research has been done on the phosphorylated p120ctn in nucleus.Several investigors have indicated p120ctn is capable of entering and shuttling out of the nucleus via nuclear localization signal (NLS) and nuclear export signal (NES). However, Kaiso have NLS, its NES remains unexplored, but its nuclear or cytoplasmic distribution couples with p120ctn, moreover, the phosphorylation of serine 288 site (Ps288) on p120ctn appears likely in the nucleus, so we postulated that Kaiso shuttled out of nucleus via NES on p120ctn and Ps288 affected p120ctn interacting with Kaiso.Here, we overexpress p120ctn plasmids in lung cancer cell lines (A549 and H460), use reagents to inhibit p120ctn shuttling out of nucleus, to alter the phosphorylated state of p120ctn, then observe nuclear-cytoplasmic distribution of p120ctn and Kaiso, furthermore, make a scientific approach to the role of pl20ctn isoforms and its phosphorylated state on binding affinity with Kaiso, Kaiso putative target gene Materials and Methods1.Cell culture and cell treatmentBE1 and LH7 cell lines established from a human pulmonary giant cell carcinoma (a gift from Dr. J. Chen, Medical College of Beijing University, China). Human pulmonary carcinoma cell lines NCI-H460, NCI-661, LTEP-a-2, SPC-A-1, SK-MES-1 and NCI-H446 were obtained from the Cell Bank of Chinese Academy (Shanghai, China). Immortal bronchial epithelial HBE and adenocarcinoma A549 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). The cell lines were maintained in DMEM (GIBCO Inc., LosAngeles, CA, USA), supplemented with 10% FBS (GIBCO). For serum starvation experiments, cells were starved in DMEM contain 0.1% FBS. Leptomycin (Sigma-Aldrich Inc., St. Louis, MO, USA,50 nM), PMA (Sigma,200 nM) or BisⅠ(Sigma,1μM) was added to medium.2. Plamid constructs and transfectionFull-length murine p120ctn isoform 1/3A were generated by PCR using RacCMV-Kpnl/p120-1/3A (a gift from AB Reynolds, Vanderbilt University Medical School, Nashville, TN) as template, then were subcloned into nucleus-localization expression vector pCMV/myc/nuc (Invitrogen, Carlsbad, CA, USA) to generate the pCMV/p120ctn-lA/nuc (p120-NLS-1A) and pCMV/p120ctn-3A/nuc (p120-NLS-3A) plamids. pBluescript-Kaiso plasmid (a gift from Juliet M. Daniel, McMaster University, Hamilton, Canada). Human specific p120ctn siRNA sequence A: 5'-GGATCACAGTCACCTTCTA-3'; 5'-TAGAAGGTGACTGTGATCC-3 B: 5'-GCACTTGTATTACAGACAA-3'; 5'-TTGTCTGTAATACAAGTGC-3'C: 5'-GGATAACAAGATTGCCATA-3'; 5'-TATGGCAATCTTGTTATCC-3', were synthesized by TaKaRa (TaKaRa Biotechnology Co., Ltd., Dalian, China). The DNA plasmids were transfected with LipofectamineTM 2000 (Invitrogen) according to the manufacturers'protocols. 3. ImmunofluorescenceTwenty-four hours post-transfection, cells were fixed, permeabilized and blocked before incubated with anti-pp120ctn and anti-p120ctn-ps288 mAb (1.25 mg/ml, BD Biosciences), anti-Kaiso pAb (2 mg/ml, Santa Cruze Biotechnology, Santa Cruze, CA, USA) and TRITC-phalloidin F-actin (1:1000, Invitrogen). Then, followed by incubated with a second antibodies conjugated to TRITC/FITC-label (1:200, Zhongshan GoldennBridge Biotechnology Co., Beijing, China). Bisbenzimide (Hoechst 33342, Sigma) was used for nuclear counterstainings.4. Nuclear and cytoplasmic Extraction, immunoprecipitation and immunoblot analysisNuclear and cytoplasmic Extraction (Pierce Biotechnology, Inc, USA) were performed according to the manufacturers'protocols. For immunoprecipitation and immunoblotting were performed using established protocols with anti-p120ctn(6H11, pp120), anti-Ps288, anti-Kaiso (Upstate Biotechnology, LakePlacid, NY, USA), anti-LaminB1 (Santa Cruze), anti-Tubulin (Santa Cruze), anti-actin (Upstate) mAb and anti-Kaiso pAb, at a dilution 1:500-800. Bound antibody was detected by incubaton with HRP conjugated secondary antibody (1:5,000-1:10,000, Zhongshan GoldennBridge) and processed using ECL. When necessary, blots were stripped and reprobed with additional antibodies.5. RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR)Total RNA of the cultured cells was prepared using the TRIzol reagent (Invitrogen). The RT-PCR was done with TaKaRa RNA PCR Kit version 3.0 (TaKaRa) according to the manufacturers' protocols. The PCR products were electrophoresed in a 1.5% agarose gel containing 0.1μg/μl ethidium bromide. The grayscale intensity value for each target band was normalized toβ-actin, to provide a value for the transcriptional level of each gene. 6. Matrigel invasion assayMatrigel (BD Biosciences) and Transwell inserts of 8.0cm pore size (Corning Inc., Corning, NY, USA) were used to assess the invasive ability of cells. The upper chamber were seeded with 5×105 cells/ml and allowed to migrate at 37℃for 48 h towards a lower reservoir of DMEM with 10% FBS before fixing, staining and counting cell on the lower surface filters were counted in 10 random high power fields (magnification:400×).7. Statistical analysisAll data were expressed as mean±standard deviation (S.D.) for in vitro experiments performed at least 3 times, and the one-way analysis of variance followed by a least significant different test (LSD) for multiple comparisons was used for statistical analysis via SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). P values less than 0.05 were considered statistically significant.Results1. Western-blot showed that p120ctn protein expressed in various lung cancer cell lines, predominantly were p120ctn isoform 1 (120kD) and isoform 3 (100kD); A nuclear-cytoplasmic extraction assay and an immunoblotting analysis were performed that p120ctn isoform 1 and 3 both localized nucleus and cytoplasm. The immunoprecipitation analysis demonstrated that Kaiso interacts only with the smaller p120ctn isoform 3.2. Then, nuclear-cytoplasmic extraction and immunoblotting analysis showed that overexpressed p120-3A, resulted in a significant shift of Kaiso from nucleus (P<0.01) to cytoplasm (P<0.05). However, overexpressed p120-1 A, there was no effect on Kaiso subcellular distribution (P>0.05).3. Nuclear-cytoplasmic extraction and immunoblotting analysis showed that nuclear p120ctn was increase owing to p120-3A overexpression and Leptomycin (LMB) treatment, meanwhile, Kaiso emerged a subsellular redistribution of nuclear increase and cytoplasmic decrease compared to p120-3A overexpression. However, the subcellular distribution of Kaiso was not affected by p120ctn-1A, though LMB also partially inhibited nuclear export of p120ctn isoform 1.4. The RT-PCR showed that p120-3A overexpression up-regulated MMP-7 and MTA2 mRNA expression (P＜0.05).P120-1A overexpression also up-regulated MMP-7 and MTA2 expression level and higher than p120-3A overexpression (P＜0.05)5. Overexpressed p120-1A/3A and treated with serum starvation, PMA and BisⅠin stable transfection A549 cell lines. The nuclear-cytoplasmic extraction showed that the phosphorylation of S288 on p120ctn isoform 3 regulate the subcellular redistribution of Kaiso, futhermore, also enhanced relieving Kaiso-mediated transcriptional repression, and was stronger than p120-3A.6. The immunofluorecence and transwell invasion assay results showed that phosphorylated state of p120ctn serine/threonine (S/T) site enhanced protrusion formation and the invasive cell in transwell corning (P<0.05).Conclusions1. Nuclear-cytoplasmic shuttling of p120ctn isoform 3 interacts with nuclear-cytoplasmic shuttling Kaiso directly2. Nuclear-cytoplasmic shuttling of p120ctn isoform 3 regulates subcellular localization of Kaiso3. The Kaiso enrichment in cytoplasm by p120ctn isoform 3 is largely CRM-dependent4. P120ctn isoform 3 relieve Kaiso-mediated transcriptional repression5. The phosphorylation of serine 288 on p120ctn regulate subcellular redistribution of Kaiso and Kaiso-mediated transcriptional repression6. The phosphorylation of serine and threonine site on p120ctn promote lung cancer cells invasion...