Smad 4 Restoration Is Involved In Regulatory Process Of Wnt/β-catenin Signal Pathway In Human Colon Carcinoma Cells

Background and objectivesColorectal cancer(CRC) is one of the major malignancies in the world,the incidence rate of CRC is the third most common type of cancer.With the many changes having taken place in people’s diet and lifestyle,CRC has become the fourth most frequent cause of death due to cancer.in china,and the number of new cases arising each year is still increasing.At present,it is considered that the development of cancer has the relation to the failure to anti-oncogene、activation of oncogene because of deletion and mutation. Smad4,originally characterized as a central intracellular signal transduction component of the transforming growth factor-β(TGF-β) family in epithelial cells,is regarded as a tumor suppressor gene predominantly involved in gastrointestinal carcinogenesis.Loss of Smad4 function either due to loss of its expression or genetic mutation is considered to be a genetically late step and occurs in a significant proportion of colon and pancreatic cancer.The Wnt/β-catenin signaling is implicated in colon carcinogenesis and suppression of this signal pathway is associated with a reduction of cellular proliferation and the induction of cellular differentiation.β-catenin performs dual functions,which include a crucial role in cell-cell adhesion and the Wnt/β-catenin pathway.Activation of Wnt signaling inhibits GSK dependent phosphorylation ofβ-catenin and results in an increase inβ-catenin protein levels.The accumulation ofβ-catenin promotes its nuclear translocation where it regulates Wnt target genes transcription in collaboration with factors from the Tcf/Lef family.Both TGF-βand Wnt signaling pathways are thought to play critical roles in regulating the differentiation processes of colonic epithelial cells.Increasing evidence of cross talk between Smad signaling and the Wnt pathway has been reported during the past years.Nevertheless,the relationship between the migration-suppressive responses of Smad4 and the activity of Wnt signaling pathway is not thoroughly understood.Recent evidences have suggested additional mechanisms underlying the effects of Smad4,for example,re-expression of Smad4 in the Smad4-deficient SW480 colon carcinoma cells resulted in loss of tumorigenicity of these cells in nude mice that was accompanied by the restoration of a more epithelioid morphology and induced transcription of E-cadherin and P-cadherin,in addition,it was recently shown that a novel mechanism underlying Smad4 invasion-suppressive function through regulation of a potential metastatic modulator,Claudin-1,in a TGF-βindependent manner.Cell-cell adhesion in epithelial cell sheets is maintained mainly through adherens junctions and tight junctions.E-cadherin is a key player in cell—cell adhesion connecting adjacent cells via the cadherin—catenin adhesion complex. Claudin-1,the most apical cell-cell contacts and the most important tight junction for barrier function in colon cancer,was recently identified as a target of Wnt/β-catenin signaling.Based on these studies,we hypothesized that there was a relationship between Smad4,a tumor suppressor protein,and Wnt/β-catenin signaling in colorectal carcinoma.The objective of this study was to investigate the impact of enhanced Smad4 expression on Wnt/β-catenin signaling activity in colon cancer cells.In this study,we reported that Smad4 directly suppressed the Wnt/β-catenin signal pathway in Smad4-responsive human colon carcinoma cells by down-regulating the cytoplasmic and nucleus expression ofβ-catenin and increasing localization ofβ-catenin to the plasma membrane.We further showed that Smad4 induced the expression of the intercellular adhesion molecule E-cadherin and reduced transcriptional activity ofβ-catenin/Tcf target genes,such as Claudin-1 and MMP-7.The data suggested that restoration of Smad4 in Smad4-deficient cells may provide a potential therapeutic strategy for intervention of colon cancer progress.Materials and methods1.Construction of eukaryotic expression plasmidA cDNA of human Smad4 was amplified by RT-PCR and constructed into the eukaryotic expression vector pcDNA3.1(+),the recombinant plasmid pcDNA3.1(+)-Stand4 was identified by enzyme digestion、PCR amplification and DNA sequencing.2.Overexpression of Stand4 by stable transfectionCells were transfected using Lipofectamine2000 according to the manufacturer’s instructions and selected in complete medium containing 1mg/ml G418 sulfate at 48h after transfection,G418-resistant cell clones were isolated for 2 weeks.Two clones that expressed similar levels of Smad4 in protein concentrations were selected for subsequent experiments.SW480 cells that were stably transfected with pcDNA3.1(+) were used as a control.The expression changes of Smad4 were detected using RT-PCR.A fluorescence microscope was used to detected the distribution of Smad4.3.Transient transfection and luciferase assaysCells were cotransfected with a firefly luciferase reporter construct Topflash or Fopflash,and a reference construct that contains Renilla reniformis luciferase, pRL-SV40,using Lipofectamine 2000.Forty-eight hours later,luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit in a GloMax Luminometry System.Tcf-mediated gene transcription was determined by the ratio of pTopflash to pFopflash luciferase activity.Firefly luciferase activity was normalized to Renilla reniformis luciferase activity and plotted as mean±SD from three independent experiments.4.Semiquantitative RT-PCRReverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit.PCR analysis was performed using gene-specific primers,such asβ-catenin、Claudin-1、MMP-7,etc.5.Immunoblot analysisEqual amounts of protein samples were electrophoretically separated by SDS-PAGE in separation gels and transferred to nitrocellulose sheets.They were incubated overnight at 4℃with the primary antibodies diluted in the same buffer (Smad4 1:400;E-cadherin 1:600;β-catenin 1:1000;Claudin-1 1:1000;β-actin 1:3000 dilutions).Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies(1:5000 dilutions) for enhanced chemiluminescence detection using an ECL detection kit.6.ImmunofluorescenceImmunofluorescence were performed with the following primary antibodies: anti-β-catenin、-claudin-1 and -E-cadherin.FITC anti- mouse and anti-rabbit、Rhodamine Red-X anti-rabbit were used as secondary antibodies at 1:200 dilutions, respectively.Stained glass coverslips were viewed and photographed using an OLYMPUS fluorescence microscope.7.Statistical analysisEach experiment was repeated at least three times.Differences in mean values were evaluated using One-way ANOVA tests,Bonferrori analysis were performed in groups comparison.All statistical analyses were performed using SPSS 13.0 software. P＜0.05 was considered to be statistically significant.Results1.The DNA oligonucleotides encoding Smad4 mRNA were synthesized,and cloned into the eukaryotic expression plasmid,which were identified by restriction enzyme digestion analysis and DNA sequencing.Recombinant Smad4 eukaryotic expression vector pcDNA3.1(+)-Smad4 was constructed successfully.2.SW480 cells which were transfected with pcDNA3.1(+)-Smad4 were screened by G418,two clones of SW480 with stable expression of Smad4 were established using Smad4 eukaryotic expression plasmid.The results of RT-PCR demonstrated that the mRNA level of Smad4 was increased notably in the Smad4-SW480 cells, and the result of immunofluorescence showed that Smad4 was predominantly localized in cytoplasm and nucleus.3.We analyzed Wnt/β-catenin signaling activity in SW480、pc-SW480 and Smad4-SW480 cells by transiently transfecting Tcf and mutant Tcf reporter plasmids(Topflash and Fopflash,respectively).Relative luciferase activities were calculated as described in Methods.The data show that the relative transcriptional activity of theβ-catenin/Lef complex was decreased(3- to 4-fold) in Smad4-SW480 cells compared with SW480 or pc-SW480 cells.This analysis confirmed that Wnt signaling activity was significantly suppressed by Smad4 expression.4.There was lessβ-catenin mRNA expression in Smad4-transfected cells than Smad4-negative colon carcinoma cells(P＜0.05).Relatively lessβ-catenin was detected in the cytoplasmic and nuclear fractions of Smad4-transfected cells compared with SW480 and pc-SW480 groups(P＜0.05).As the function ofβ-catenin in signal transduction depends on its cytoplasmic/nuclear localization, we further analyzed the distribution of theβ-catenin protein through indirect immunofluorescence.SW480 cells showed strong staining forβ-catenin in the cytoplasm and nucleus.Smad4 re-expression cells,in contrast,showed staining in the plasma membrane.The data demonstrated that up-regulation of Smad4 caused the relocalization ofβ-catenin from cytoplasm and nucleus to the plasma membrane in SW480 cells.5.Low levels of E-cadherin protein were observed in SW480 cells and its control cell lines(pc-SW480) with relatively more E-cadherin detected in cells that had increased Smad4(P＜0.05).Inverse data were found in cellular Claudin-1 concentration,we observed reduced expression of claudin-1 in clones transfected with Smad4 when compared with the Smad4-negative cells(P＜0.05).We performed immunofluorescence analysis to further investigate whether up-regulation of Smad4 expression changed the localization of E-cadherin and Claudin-1.The localization of E-cadherin and Claudin-1 was not altered in Smad4-SW480 cells.The results of these two studies indicated a decrease in the expression of Claudin-1 but a robust increase in E-cadherin expression in Smad4-SW480 clones compared with SW480 cells.6.There was less MMP-7 and Claudin-1 mRNA in Smad4-SW480 clones than SW480 or pc-SW480 cells(P＜0.05).Transfection of Smad4 resulted in reduced MMP-7 and Claudin-1 mRNA concentration as compared to cells transfected with the control or nonsense.Conclusions1.Recombinant Smad4 eukaryotic expression vector pcDNA3.1(+)-Smad4 was constructed successfully.2.Stable colon cancer cells that have increased Smad4 expression were created using a eukaryotic vector containing Smad4-pcDNA3.1(+). 3.Re-expression of Smad4 in SW480 cells leads to a reduction in Wnt/β-catenin signaling activity.4.Smad4 restoration decreasesβ-catenin expression(cytoplasm and nuclus) and alters its localization from cytoplasm to the plasma membrane in SW480 cells.5.Up-regulation of Smad4 also increased the expression of E-cadherin and decreased the expression of Claudin-1.6.Smad4 restoration in SW480 cells decreases mRNA expression of Wnt/β-catenin signaling target genes,such as Claudin-1 and MMP-7.7.The data suggested that restoration of Smad4 in Smad4-deficient cells may provide a potential therapeutic strategy for intervention of colon cancer progress.