Suppression of Kinesin Expression Disrupts Adenomatous Polyposis Coli (APC) Localization and Affects β-Catenin Turnover in Young Adult Mouse Colon (YAMC) Epithelial Cells

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Suppression of Kinesin Expression Disrupts Adenomatous Polyposis Coli (APC) Localization and Affects β-Catenin Turnover in Young Adult Mouse Colon (YAMC) Epithelial Cells
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  Suppression of Kinesin Expression Disrupts Adenomatous PolyposisColi (APC) Localization and Affects   -Catenin Turnoverin Young Adult Mouse Colon (YAMC) Epithelial Cells Hongyi Cui, Mei Dong, Devaki N. Sadhu, and Daniel W. Rosenberg 1 Center for Molecular Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030-3101  Mutational inactivationoftheadenomatouspolypo-sis coli (APC) protein initiates most hereditary andsporadic colon cancers.Thetumor-suppressiveeffectof APC is mediated by promoting degradation of theoncogenic transcriptional activator   -catenin, andlossof APC function often resultsin nuclear accumu-lation of    -catenin in cancer cells. APC is a nuclear-cytoplasmicshuttlingproteinandmovesalongmicro-tubules in the cytoplasm. However, the molecularmotor proteinsresponsiblefor APC translocationandtheimplicationsofAPC traffickingon   -catenin turn-over areunknown.Hereweshow that APC protein isassociated with microtubules and is colocalized withkinesinheavychain(KHC)and  -catenintoclustersof puncta at the tip regions of cellular extensions in aconditionallyimmortalizedmousecolonepithelialcellline,youngadult mousecolon (YAMC, APC   /  ).Inhi-bition of KHC expression usingan antisenseoligonu-cleotidedisruptsperipheral translocationofAPC andinducesnucleocytoplasmicaccumulationof   -catenin.Thesedata indicatethat KHC-mediated APC translo-cation istightly coordinated with   -catenin turnoverinthecell.  ©2002Elsevier Science (USA) Key Wor ds:   APC;   -catenin; microtubules; kinesinheavy chain;YAMC cell. INTRODUCTION The majority of sporadic and hereditary colorectaltumors exhibit loss of adenomatous polyposis coli(APC) 2 function [1, 2]. Colorectal cancer cells with mu-tant APC often contain high levels of free   -cateninwhich can be downregulated by expression of wild-typeAPC protein in the cell [3]. Stabilized   -catenin medi-ates malignant transformation by entering the nucleusand stimulating the transcription of a panel of Wnt-targeted oncogenes, including  MYC, cyclin D1  , and PPAR    [4–8]. The major tumor suppressor effect ofAPC protein lies in its ability to destabilize   -cateninin the cell [9, 10].The large APC protein comprises multiple functionaldomains, and two motifs have been shown to interactwith   -catenin [11, 12]. APC protein also contains twofunctional nuclear export signals (NES) and can shut-tle   -catenin out of the nucleus for degradation [13–15]. In the cytoplasm, Axin, APC,   -catenin, and gly-cogen synthase kinase 3  (GSK3  ) together with manyother proteins assemble into a multiprotein structuretermed the   -catenin destruction complex [12, 16, 17],which triggers the proteasomal degradation of   -cate-nin. Recently, a part of the   -catenin destruction com-plex has been successfully isolated by subcellular frac-tionation [18]. It is likely that   -catenin is sorted anddegraded in the cell periphery, since the major scaffoldprotein of the destruction complex, Axin, has beenshown to localize to the apical submembrane compart-ment in  Xenopus   epithelial cells and  Xenopus   embry-onic cells [19, 20]. Another component of the   -catenindestruction complex, dishevelled, has also been shownto be transported along microtubules and to localize tothe dorsal side of the  Xenopus   embryos [21].APC protein is localized to apical membrane regionsof both colonic and noncolonic epithelial cells [18, 22].Recent studies have shown that APC protein is associ-ated with microtubules and is transported to the grow-ing plus ends [23–25]. However, the motor proteinsresponsible for APC translocation and the associationof APC trafficking with its known functional role in  -catenin degradation are not known.In this study, we analyzed the subcellular localiza-tion of APC protein with respect to microtubules, KHC,and   -catenin, and the effects of suppressing KHCexpression on APC and   -catenin expression in youngadult mouse colon (YAMC) cells. YAMC cells are de-rived from normal colonic epithelial cells that are con-ditionally immortalized by virtue of expression of atemperature-sensitive simian virus 40 (SV40) large Tantigen [26]. The cells proliferate continuously at the 1 To whom correspondence and reprint requests should be ad-dressed. Fax: (860)-679-7639. E-mail: rosenberg@nso2.uchc.edu. 2 Abbreviations used: APC, adenomatous polyposis coli; KHC, ki-nesin heavy chain; YAMC, young adult mouse colon; GSK3  , glyco-gen synthase kinase 3  ; NES, nuclear export signal.12 0014-4827/02 $35.00© 2002 Elsevier Science (USA)All rights reserved. Experimental Cell Research  280, 12–23 (2002)doi:10.1006/excr.2002.5506  permissive temperature (33 ° C), but stop proliferatingand detach at a nonpermissive temperature (39 ° C).They retain some of the properties of the tissue ofsrcin as evidenced by their ability to synthesize ker-atin, brush border peptidases, and a disaccharidaseenzyme [26]. Even under proliferative conditions, thecells are maintained in a nontransformed state and arenontumsrcenic in nude mice [27]. They exhibit thewild-type APC genotype ( APC   /  ) and express normalAPC protein as shown by cytogenetic and Western blotanalysis [27]. Thus it is likely that YAMC cells possessmetabolic and physiological properties that are morerepresentative of colon epithelial cells  in vivo   thanheterologous cell systems that have been used in pre-vious studies of APC. YAMC cells plated at a lowerdensity proliferate rapidly under permissive condi-tions, with the majority of cells establishing long mi-grating  fi lopodia and thin lamella extensions [26, 27].This polarized morphology provides an excellent exper-imental system for using advanced imaging techniquesto analyze the subcellular localization of proteins. Wehave established that the endogenous APC protein inYAMC cells is associated with microtubules, KHC, and  -catenin, and is transported to the tip regions of cel-lular extensions. Suppression of KHC expression usingan antisense oligonucleotide abolishes peripheraltranslocation of APC and induces cellular accumula-tion of   -catenin. Our  fi ndings indicate that KHC-me-diated peripheral translocation of the APC gene prod-uct is tightly coordinated with   -catenin turnover inthe cell. MATERIALS AND METHODS (1) Cell culture, antibodies, and staining.  YAMC cells were grownto 40 – 50%con fl uence in RPMI 1640 glutaMAX media (GIBCO BRL,Gaithersburg, MD) supplemented with 10%fetal bovine serum, 100units/ml penicillin/streptomycin (GIBCO BRL), 10 units/ml recom-binant murine interferon     (Life Technologies, Inc., Rockville, MD),and ITS-BSA-linoleic acid (BD Biosciences, Franklin Lakes, NJ ) at33 ° C in the presence of 5% CO 2 . Cells were transferred to 39 ° Cwithout interferon 48 h prior to experimentation.Primary antibodies used for immuno fl uorescence microscopy wereas follows: APC, anti-human APC C-terminus (diluted 1:50) (C-20,rabbit polyclonal IgG; Santa Cruz Biotechnology, Inc., Santa Cruz,CA); APC, anti-human/mouse APC N-terminus (diluted 1:50) (N-15,rabbit polyclonal IgG; Santa Cruz Biotechnology);   -tubulin, anti-bovine-  -tubulin (diluted 1:50) (clone 236-10501, mouse monoclonalIgG  1 ; Molecular Probes, Eugene, OR); KHC, anti-bovine brain KHCN-terminus (diluted 1:50) (H2, mouse monoclonal IgG  2b ; ChemiconInternational, Inc., Temecula, CA);   -catenin, anti-human or mouse  -catenin (diluted 1:50) (C2206, rabbit polyclonal IgG; Sigma, St.Louis, MO). Secondary antibodies were as follows: Alexa Fluor 488goat anti-rabbit IgG (H    L) conjugate (diluted 1:100) (A-11008,Molecular Probes); Alexa Fluor 568 goat anti-mouse IgG (H    L)conjugate (diluted 1:100) (A-11031, Molecular Probes).For immunostaining, cells were  fi xed for 20 min in 3.7%formal-dehyde and permeabilized with 0.1% nonylphenol ethoxylate-40(NP40) for 3 min. Permeabilized cells were incubated with primaryantibodies for 30 min at room temperature, followed by correspond-ing secondary antibodies for 30 min at room temperature in the dark.As controls for APC or   -catenin staining, normal rabbit IgG wassubstituted for the primary antibodies. (2) Fluorescence confocal microscopy and image analysis.  Fluo-rescence was visualized by confocal microscopy using a Zeiss LSM410 with a 63    1.4 N.A. planapochromat objective. Dual channelimages were collected simultaneously using dual excitation at 488nm (for Alexa Fluor 488) and 568 nm (for Alexa Fluo 568) withappropriate emission  fi lters. Dual channel images were then merged,maintaining a full 8-bit dynamic range in each image, and displayedwith red/green pseudocolor. Fluorescence quantitation was per-formed using NIH Image 1.62 software (Wayne Rasband Analytics,Research Services Branch, NIH, Bethesda, MD). (3) Antisense treatment.  The antisense oligonucleotide used wasas follows: 5  -ACTCCGCCGGGTCCGCCATCTTTTG-3  , which is theinverse complement of nucleotides   6 to   16 in the mouse KHC(locus MUSKHCSA, Accession No. L27153) [31, 32]. The sense oli-gonucleotide consisted of the inverse complement of the antisenseoligonucleotide. Oligonucleotides were purchased from GIBCO/BRL.Treatment of YAMC cells with the oligonucleotides was as follows.The cells were allowed to attach and establish polarized morphologyat 33 ° C, and then were transferred to 39 ° C without interferon for48 h prior to adding the oligonucleotide. The cells were  fi rst treatedwith 50   M oligonucleotide for 12 h, followed by addition of 25   Moligonucleotide for an additional 12 h. For control experiments, equalamounts of a random 25-nt oligonucleotide was added and the cellswere incubated for the same period of time as the experimentalgroups. (4) Western blot analysis.  Cultured YAMC cells were incubated inRIPA buffer containing protease inhibitors on ice for 30 min and thenpassed through a 21-gauge needle  fi ve times. Cell lysates were cen-trifuged at 10,000 g   for 15 min, and the total protein content in thesupernatants was quanti fi ed using the Bio-Rad DC protein assay kit.For APC, 40   g of the isolated protein was separated on a 5%polyacrylamide gel containing 25% glycerol [51]. For KHC and  -catenin, total protein was separated on a 10%SDS – PAG. Electro-transferring onto nitrocellulose membrane was performed followingthe standard protocol. The membrane was probed with anti-APCantibodies (C-20/N-15, 1:300, Santa Cruz), anti-KHC antibody (1:1,000, Chemicon), anti-  -catenin antibody (1:1,000, Sigma), or anti-  -actin antibody (1:5,000, Sigma) as a loading control for 1 h andthen incubated with the relevant horseradish peroxidase-conjugatedsecondary antibody for 30 min. The membranes were visualizedusing the ECL Western blot analysis system (Santa Cruz Biotech-nology). Densitometry analysis was performed using NIH ImageSoftware 1.62. RESULTS Localization of APC Protein with Respect to Microtubules  APC protein localizes to apical membrane regions ofcolonic epithelial cells [18] and the migrating edges ofMDCK cells [22]. APC has also been shown to associatewith microtubules in  Xenopus   kidney epithelial cellsand colorectal tumor cells [23 – 25]. To determine thesubcellular localization of APC protein with respect tomicrotubules in YAMC cells, cells were double-stainedwith antibodies against APC protein and   -tubulin andvisualized by dual-channel confocal microscopy. Thestaining patterns for APC using two different antibod-ies (C-20, N-15) were indistinguishable (data notshown); thus all subsequent analyses for APC were13 KHC IN APC LOCALIZATION AND   -CATENIN TURNOVER  performed using antibody C-20. Furthermore, the mor-phology of YAMC cells was not altered by either  fi xa-tion or the staining procedures used (data not shown).Laser-scanning confocal microscopy revealed thatAPC protein was distributed throughout the cyto-plasm, but highly accumulated in the perikaryon aswell as in clusters of puncta near the margin of pro-truding membrane structures (Fig. 1A). These data arebroadly consistent with the observations of previousstudies [18, 22], although the perikaryal accumulationof APC protein has not been reported previously. Thisis presumably due to a constitutively higher APC ex-pression level that occurs in YAMC cells comparedwith other cell types. FIG.1.  Localization of APC protein with respect to microtubules in YAMC cells. YAMC cells were double stained with antibodies againstAPC and   -tubulin and visualized by dual-channel confocal microscopy. The subcellular distribution of APC protein (A, stained with C-20)with respect to microtubules (B) in a representative group of cells. (C) merged image of (A) and (B). Arrows show APC protein clusters ofpuncta with respect to microtubule bundles in thin lamellae regions. (D – F) Distribution of APC (D) and microtubules (E) in an enlargedmembrane lamella. (F) is the merged image of (D) and (E). Scale bars: (C) and (F) 5 and 2   m, respectively. 14  CUI ET AL.  The overall subcellular distribution of APC proteinwas very similar to that of the microtubules within thecell (Figure 1B). In thin lamella regions, APC proteinappeared to concentrate at microtubule bundles nearthe margins of the cell (Figs. 1A – 1C, arrows). Sincemicrotubules are generally oriented with their growing FIG.2.  Localization of APC protein with respect to KHC in YAMC cells. YAMC cells were double stained with antibodies against APCprotein and KHC and visualized by dual-channel confocal microscopy. (A – C) Subcellular distribution of APC (A, stained with C-20) proteinwith respect to KHC (B) in a representative group of cells. (C) Merged image of (A) and (B). (D – F) Higher magni fi cation view of APC proteinclusters of puncta (D) with respect to KHC (E) in a thin lamellae region. (F) Merged image of (D) and (E). Arrows indicate colocalization ofAPC with KHC in clusters of puncta. Scale bars: (C) and (F), 10 and 2   m, respectively. 15 KHC IN APC LOCALIZATION AND   -CATENIN TURNOVER  plus ends facing the cell periphery [28], this indicatesthat intrinsic APC protein is localized to the microtu-bule plus ends at the cell periphery. In certain cellularcompartments, APC protein appears to decorate micro-tubules along their entire length (Figs. 1D – 1F), indi-cating that endogenous APC protein is associated withmicrotubules in YAMC cells as demonstrated in othercell types [23 – 25]. Intriguingly, treatment with no-codazole (10   g/ml for up to 4 h) did not abolish APClocalization in the cell periphery prior to signi fi cant FIG.3.  Effect of suppression of KHC expression on the overall cell morphology and the distribution of microtubules in YAMC cells. YAMCcells were treated with either sense or antisense KHC oligonucleotides; the morphology of the cell and the distribution of microtubules wereanalyzed by dual-channel differential interference contrast (DIC) and  fl uorescent confocal microscopy. (A) A DIC image of control cells(treated with equal amounts of random 25-nt oligonucleotide). (B) Microtubule distribution in cells shown in (A). (C) DIC image of cellstreated with sense KHC oligonucleotide. (D)Microtubule distribution in cells shown in (C). (E)DIC image of cells treated with antisense KHColigonucleotide. (F) Microtubule staining in cells shown in (E). Scale bar: (F), 5   m. 16  CUI ET AL.
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