Reciprocal Targeting of Hath1 and β-Catenin by Wnt Glycogen Synthase Kinase 3β in Human Colon Cancer
2006; Elsevier BV; Volume: 132; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2006.10.031
ISSN1528-0012
AutoresKiichiro Tsuchiya, Tetsuya Nakamura, Ryuichi Okamoto, Takanori Kanai∥, Mamoru Watanabe,
Tópico(s)Kruppel-like factors research
ResumoBackground & Aims: The transcription factor Hath1 plays a crucial role in the differentiation program of the human gut epithelium. The present study was conducted to investigate the molecular mechanism of Hath1 expression and its close association with β-catenin/glycogen synthase kinase 3β (GSK3β) under the Wnt pathway in human colonocytes. Methods: Tissue distribution of Hath1 messenger RNA in human tissues was examined by Northern blot. Stability of Hath1 protein was analyzed by expression of FLAG-tagged Hath1 in human cell lines. Targeting of Hath1 protein by GSK3β was determined by specific inhibition of GSK-3β function. Expression of Hath1 protein in colorectal cancers was examined by immunohistochemistry. Results: Hath1 messenger RNA expression was confined to the lower gastrointestinal tract in human adult tissues. In colon cancer cells, although Hath1 messenger RNA was also detected, Hath1 protein was positively degradated by proteasome-mediated proteolysis. Surprisingly, the GSK3β-dependent protein degradation was switched between Hath1 and β-catenin by Wnt signaling, leading to the dramatic alteration of cell status between proliferation and differentiation, respectively. Hath1 protein was detected exclusively in normal colon tissues but not in cancer tissues, where nuclear-localized β-catenin was present. Conclusions: The present study suggests a novel function of the canonical Wnt signaling in human colon cancer cells, regulating cell proliferation and differentiation by GSK3β-mediated, reciprocal degradation of β-catenin or Hath1, respectively, which further emphasizes the importance of aberrant Wnt signaling in colonocyte transformation. Background & Aims: The transcription factor Hath1 plays a crucial role in the differentiation program of the human gut epithelium. The present study was conducted to investigate the molecular mechanism of Hath1 expression and its close association with β-catenin/glycogen synthase kinase 3β (GSK3β) under the Wnt pathway in human colonocytes. Methods: Tissue distribution of Hath1 messenger RNA in human tissues was examined by Northern blot. Stability of Hath1 protein was analyzed by expression of FLAG-tagged Hath1 in human cell lines. Targeting of Hath1 protein by GSK3β was determined by specific inhibition of GSK-3β function. Expression of Hath1 protein in colorectal cancers was examined by immunohistochemistry. Results: Hath1 messenger RNA expression was confined to the lower gastrointestinal tract in human adult tissues. In colon cancer cells, although Hath1 messenger RNA was also detected, Hath1 protein was positively degradated by proteasome-mediated proteolysis. Surprisingly, the GSK3β-dependent protein degradation was switched between Hath1 and β-catenin by Wnt signaling, leading to the dramatic alteration of cell status between proliferation and differentiation, respectively. Hath1 protein was detected exclusively in normal colon tissues but not in cancer tissues, where nuclear-localized β-catenin was present. Conclusions: The present study suggests a novel function of the canonical Wnt signaling in human colon cancer cells, regulating cell proliferation and differentiation by GSK3β-mediated, reciprocal degradation of β-catenin or Hath1, respectively, which further emphasizes the importance of aberrant Wnt signaling in colonocyte transformation. The gut epithelium undergoes continual renewal throughout adult life, maintaining the proper architecture and function of the intestinal crypts. This process involves highly coordinated regulation of the induction of cellular differentiation and the cessation of proliferation, and vice versa.1Booth C. Brady G. Potten C.S. Crowd control in the crypt.Nat Med. 2002; 8: 1360-1361Google Scholar, 2El-Assal O.N. Besner G.E. HB-EGF enhances restitution after intestinal ischemia/reperfusion via PI3K/Akt and MEK/ERK1/2 activation.Gastroenterology. 2005; 129: 609-625Google Scholar, 3Haramis A.P. Begthel H. van den Born M. van Es J. Jonkheer S. Offerhaus G.J. Clevers H. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine.Science. 2004; 303: 1684-1686Google Scholar The intestinal epithelium consists of cells of 4 lineages: goblet cells, enteroendocrine cells, Paneth cells, and enterocytes.4Cheng H. Leblond C.P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types.Am J Anat. 1974; 141: 537-561Google Scholar Cellular differentiation into the former 3 lineages is believed to be regulated by a basic helix-loop-helix transcription factor called "Math1" in mice and "Hath1" in humans (officially termed as "ATOH1"). Math1 and Hath1 are known to play crucial roles in differentiation of various cells in other tissues, such as dorsal interneurons in the spinal cord,5Gowan K. Helms A.W. Hunsaker T.L. Collisson T. Ebert P.J. Odom R. Johnson J.E. Crossinhibitory activities of Ngn1 and Math1 allow specification of distinct dorsal interneurons.Neuron. 2001; 31: 219-232Google Scholar granule cells in the cerebellum,6Ben-Arie N. Bellen H.J. Armstrong D.L. McCall A.E. Gordadze P.R. Guo Q. Matzuk M.M. Zoghbi H.Y. Math1 is essential for genesis of cerebellar granule neurons.Nature. 1997; 390: 169-172Google Scholar Merkel cells in the skin,7Leonard J.H. Cook A.L. Van Gele M. Boyle G.M. Inglis K.J. Speleman F. Sturm R.A. Proneural and proneuroendocrine transcription factor expression in cutaneous mechanoreceptor (Merkel) cells and Merkel cell carcinoma.Int J Cancer. 2002; 101: 103-110Google Scholar and inner hair cells in the auditory systems.8Bermingham N.A. Hassan B.A. Price S.D. Vollrath M.A. Ben-Arie N. Eatock R.A. Bellen H.J. Lysakowski A. Zoghbi H.Y. Math1: an essential gene for the generation of inner ear hair cells.Science. 1999; 284: 1837-1841Google Scholar In mice intestine, the Math1 gene promotes the differentiation of epithelial cells to secretory lineage cells without affecting absorptive cell differentiation and is expressed in Ki-67–positive proliferating cells of the crypt, indicating a role of Math1 at an early stage of lineage commitment.9Yang Q. Bermingham N.A. Finegold M.J. Zoghbi H.Y. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine.Science. 2001; 294: 2155-2158Google Scholar Expression of Math1 seems to be regulated at its transcriptional level, because forced expression of Notch intracellular domain in murine intestinal epithelial cells causes a decrease of Math1 messenger RNA (mRNA) expression and subsequent depletion of goblet cells in vivo.10Fre S. Huyghe M. Mourikis P. Robine S. Louvard D. Artavanis-Tsakonas S. Notch signals control the fate of immature progenitor cells in the intestine.Nature. 2005; 435: 964-968Google Scholar Conversely, depletion of Hes1, another basic helix-loop-helix transcription factor known as a downstream target of Notch intracellular domain, up-regulates Math1 mRNA expression in murine intestine.11Jensen J. Pedersen E.E. Galante P. Hald J. Heller R.S. Ishibashi M. Kageyama R. Guillemot F. Serup P. Madsen O.D. Control of endodermal endocrine development by Hes-1.Nat Genet. 2000; 24: 36-44Google Scholar Thus, it is likely that Math1 gene expression is regulated at the mRNA level by Notch signaling, leading to subsequent control of intestinal epithelial cell lineage decision of the crypt cells. It was recently reported that Hath1, a human homologue of Math1, up-regulates gastric mucin gene expression in gastric cells12Sekine A. Akiyama Y. Yanagihara K. Yuasa Y. Hath1 up-regulates gastric mucin gene expression in gastric cells.Biochem Biophys Res Commun. 2006; 344: 1166-1171Google Scholar; however, the regulation of Hath1 expression is less understood in human intestine. The canonical Wnt signaling is another signaling pathway known to regulate cell differentiation and proliferation of the intestinal crypt cells.13Batlle E. Henderson J.T. Beghtel H. van den Born M.M. Sancho E. Huls G. Meeldijk J. Robertson J. van de Wetering M. Pawson T. Clevers H. Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB.Cell. 2002; 111: 251-263Google Scholar It is believed that Wnt proteins induce inactivation of glycogen synthase kinase 3β (GSK3β), a component of the so-called destruction complex that also contains adenomatous polyposis coli (APC) and Axin, and the resultant dephosphorylation and stabilization of its substrate β-catenin leads to the transcription of genes targeted by the nuclear β-catenin/T-cell factor (TCF) complex.14Huelsken J. Behrens J. The Wnt signalling pathway.J Cell Sci. 2002; 115: 3977-3978Google Scholar, 15Polakis P. Wnt signaling and cancer.Genes Dev. 2000; 14: 1837-1851Crossref Google Scholar However, in intestinal cells, it has not been shown whether activation of Wnt signaling simply inactivates general kinase activity of GSK3β or could possibly change the substrate specificity instead of kinase activity, thereby stabilizing the β-catenin protein. Constitutive activation of Wnt signaling is assumed to be essential for both continuous proliferation and maintenance of the undifferentiated state in intestinal stem cells.16Marshman E. Booth C. Potten C.S. The intestinal epithelial stem cell.Bioessays. 2002; 24: 91-98Google Scholar, 17Pinto D. Gregorieff A. Begthel H. Clevers H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.Genes Dev. 2003; 17: 1709-1713Google Scholar Of note, the biological impact of the Wnt pathway lies in its close association with the carcinogenesis of colorectal cancer. Mutations that perturb the assembly or function of the destruction complex, such as truncation of APC, are present in approximately more than 90% of colorectal tumors. These mutations lead to constitutive activation of Wnt signaling, and the downstream genes that are transcriptionally up-regulated by the β-catenin/TCF complex are implicated in the growth-promoting properties of the tumor cells.15Polakis P. Wnt signaling and cancer.Genes Dev. 2000; 14: 1837-1851Crossref Google Scholar, 18Polakis P. The oncogenic activation of beta-catenin.Curr Opin Genet Dev. 1999; 9: 15-21Google Scholar However, it has not been well understood how constitutive Wnt signaling could maintain colorectal cancer cells at an undifferentiated state. A previous study reported that inhibition of Wnt signaling in a human colon cancer–derived cell line, HT-29, up-regulated both Hath1 and MUC2 gene mRNA expression.19Leow C.C. Romero M.S. Ross S. Polakis P. Gao W.Q. Hath1, down-regulated in colon adenocarcinomas, inhibits proliferation and tumorigenesis of colon cancer cells.Cancer Res. 2004; 64: 6050-6057Google Scholar This suggested that Hath1 expression may be suppressed at the mRNA level by the aberrant Wnt signaling, thereby maintaining the undifferentiated state of colorectal cancer cells. However, in the same study, it was also suggested that some colorectal cancers did express Hath1 mRNA at an amount comparable to the neighboring normal colon tissue but maintained an undifferentiated state at the same time. These data prompted us to prove that Hath1 gene function is regulated by the aberrant Wnt signaling, not only by the mRNA level but also by an unknown posttranscriptional or posttranslational mechanism in human colon cancer cells. Here, we present the evidence that Hath1 protein expression is regulated by Wnt signaling via GSK3β-mediated protein degradation. Our results suggest that the reciprocal regulation of Hath1 and β-catenin protein stability is mediated by GSK3β, which functions as a molecular switch regulating the proliferation and differentiation of colon cancer cells in vitro and in vivo. These results present a novel function of the Wnt-GSK3β pathway and further emphasize the importance of aberrant Wnt signaling in colonocyte transformation. Human colon cancer–derived SW480, DLD-1, and HT-29 cells and human embryonic kidney-derived 293T cells were grown in Dulbecco's modified Eagle medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. In all experiments, 1 × 106 cells were seeded onto 6-cm culture dishes 36 hours before the experiment. All transfection experiments of DNA constructs and small interfering RNA (siRNA) oligonucleotides were performed by using TransIT transfection reagent (Mirus, Madison, WI) according to the manufacturer's instructions. pcDNA3-Myc-ubiquitin20Shimura H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase.Nat Genet. 2000; 25: 302-305Google Scholar was a kind gift from Dr K. Tanaka (Tokyo Metropolitan Institute, Tokyo, Japan). pMX-IRES-GFP21Onishi M. Kinoshita S. Morikawa Y. Shibuya A. Phillips J. Lanier L.L. Gorman D.M. Nolan G.P. Miyajima A. Kitamura T. Applications of retrovirus-mediated expression cloning.Exp Hematol. 1996; 24: 324-329Google Scholar was a kind gift from Dr T. Kitamura (University of Tokyo, Tokyo, Japan). Series of expression vectors encoding mutants for APC genes (pCS2-APC2, -APC3, and -APC25)22Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein.Proc Natl Acad Sci U S A. 1995; 92: 3046-3050Google Scholar and a pRL5-Wnt123Nishita M. Hashimoto M.K. Ogata S. Laurent M.N. Ueno N. Shibuya H. Cho K.W. Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann's organizer.Nature. 2000; 403: 781-785Google Scholar were kind gifts from Dr H Shibuya (Tokyo Medical and Dental University, Tokyo, Japan). Expression plasmids encoding N-terminally Flag-tagged WT-Hath1 (pCMV-Flag-WT-Hath1) or enhanced green fluorescent protein (EGFP) (pCMV-Flag-EGFP) were generated by inserting the polymerase chain reaction (PCR)-amplified Hath1 gene or EGFP gene, respectively, into the EcoRI/BamHI site of a pCMV-Flag vector (Stratagene, La Jolla, CA) in frame. Plasmids for various mutants that lack either the N- or C-terminal region of Hath1 (N1-5, C1, and C2 mutants; Figure 3A) and the mutant 54/58SA-Hath1, in which both 54S and 58S are substituted to alanines, were constructed by PCR-mediated mutagenesis by using pCMV-Flag-WT-Hath1 as a starting material. pMX-Flag-WT-Hath1-IRES-GFP was generated by inserting a fragment encoding the N-terminally Flag-tagged Hath1 gene, which was amplified by PCR using pCMV-Flag-WT-Hath1 as a template, into the pMX-IRES-GFP vector. A reporter plasmid E-box Luc was generated by inserting a 77–base pair oligonucleotide containing 7 repeats of the E-box (kE sites) (AGGCAGGTGGC) into an SmaI site of the pTA-Luc vector (Clontech, Mountain View, CA). Reporter plasmids TOPflash and FOPflash were obtained from Upstate Biotechnology (Charlottesville, VA). All plasmids constructed were verified by sequencing. Cells were transfected with 1 μg of pCMV-Flag vector (control), pCMV-Flag-EGFP, pCMV-Flag-Hath1, or various mutants of pCMV-Flag-Hath1. In cotransfection experiments, 1 μg of either pcDNA3-Myc-ubiquitin or one of the expression plasmids for mutant APC (pCS2-APC2, -APC25, or -APC3) or pRL5-Wnt1 was transfected along with 1 μg of pCMV-Flag vector (control) or pCMV-Flag-Hath1. In each cotransfection experiment, the total amount of DNA was equalized by adding the appropriate amount of empty expression vector. After 12 hours of transfection, cells were cultured for 12 hours under the usual conditions or in the presence of 10 μmol/L lactacystin (Calbiochem, San Diego, CA), 10 μmol/L MG132 (Calbiochem), 5 μmol/L calpain inhibitor (Calbiochem), 100 μmol/L chloroquine (Sigma-Aldrich, St Louis, MO), 100 μmol/L RO-31-8220 (Calbiochem), 100 μmol/L staurosporine (Calbiochem), 1 μmol/L UO126 (Calbiochem), 30 mmol/L LiCl (Sigma-Aldrich), 30 mmol/L kenpaullone (Calbiochem), or 5 μmol/L BIO (Calbiochem). For siRNA experiments, SW480 cells were transfected with 100 nmol/L siRNA oligonucleotide along with 1 μg of pCMV-Flag-WT-Hath1 for 12 hours, cultured for an additional 12 hours under the usual conditions, and then treated with MG132 or left untreated for 12 hours. A siRNA oligonucleotide specific for human GSK3β was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and a negative control (nonsense siRNA) oligonucleotide was synthesized as described elsewhere.24Oshima S. Nakamura T. Namiki S. Okada E. Tsuchiya K. Okamoto R. Yamazaki M. Yokota T. Aida M. Yamaguchi Y. Kanai T. Handa H. Watanabe M. Interferon regulatory factor 1 (IRF-1) and IRF-2 distinctively up-regulate gene expression and production of interleukin-7 in human intestinal epithelial cells.Mol Cell Biol. 2004; 24: 6298-6310Google Scholar After transfection, cells were harvested, washed with phosphate-buffered saline (PBS) once, and incubated on ice for 15 minutes in 1% sodium dodecyl sulfate (SDS)-containing radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl [pH 8.0], 1% Triton X-100, 1% SDS, 0.1% sodium deoxycholate, 1 mmol/L EDTA, 0.5 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 140 mmol/L NaCl). After brief sonication of the lysates to shear genomic DNA, the samples were centrifuged for 20 minutes and the supernatant was used as whole cell extract. The protein concentration in each sample was determined by using protein assay reagent (Pierce, Rockford, IL). For immunoblotting, 50 μg or 100 μg of whole cell extract was separated in 12% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, blocked, and probed according to standard procedures.24Oshima S. Nakamura T. Namiki S. Okada E. Tsuchiya K. Okamoto R. Yamazaki M. Yokota T. Aida M. Yamaguchi Y. Kanai T. Handa H. Watanabe M. Interferon regulatory factor 1 (IRF-1) and IRF-2 distinctively up-regulate gene expression and production of interleukin-7 in human intestinal epithelial cells.Mol Cell Biol. 2004; 24: 6298-6310Google Scholar The following antibodies and dilutions were used: mouse anti-Flag M2 (Sigma Chemical Co, St Louis, MO), 1:5000; mouse anti–dephospho-β-catenin (Alexis, San Diego, CA), 1:500; mouse anti-GSK3 (Calbiochem), 1:1000; rabbit anti-USF2 (loading control for the amount of nuclear proteins; Santa Cruz Biotechnology), 1:1000; and mouse anti–β-actin (loading control for the whole cell extracts; Sigma Chemical Co), 1:5000. Horseradish peroxidase–conjugated secondary antibodies were used for mouse (Amersham Biosciences UK, Buckinghamshire, England) and rabbit immunoglobulin G (Cell Signaling Technology, Danvers, MA). Blots were visualized with the ECL Plus System (Amersham Biosciences UK) by using a Lumi-Imager F1 system (Roche Diagnostics, Rotkreutz, Switzerland). For immunoprecipitation assays, 300 μg of whole cell extract in 1% SDS-containing RIPA buffer was diluted with 9 vol of non–SDS-containing RIPA buffer to give an SDS concentration of 0.1%, and then the total volume was adjusted to 1 mL by adding an appropriate amount of 0.1% SDS-containing RIPA buffer. The lysates were precleared by incubation with 40 μL of protein G/Sepharose (50% slurry in 0.1% SDS-RIPA buffer) for 1 hour, and then the supernatants were incubated with 1 μg of anti-Flag M2 antibody (Sigma Chemical Co) overnight. A 40-μL aliquot of 50% protein G/Sepharose slurry was added to each sample and incubated for 2 hours at 4°C. Precipitates were washed 3 times in 0.1% SDS-containing RIPA buffer, resolved by SDS/polyacrylamide gel electrophoresis, and analyzed by immunoblotting using a mouse anti-myc antibody (Invitrogen, Carlsbad, CA) at 1:1000 dilution. Protein visualization was performed as described previously. Total RNA was isolated by using TRIzol reagent (Invitrogen). Aliquots of 5 μg of total RNA were used for complementary DNA synthesis in 21 μL of reaction volume by using oligo dT primers. One microliter of complementary DNA was amplified with 0.25 U of LA Taq polymerase (Takara Bio, Otsu, Japan) in a 25-μL reaction. Sense and antisense primers and the cycle numbers for the amplification of each gene were as follows: sense Flag, 5′-CACCATGGATTACAAGGATGACGACGAT-3′ and antisense Hath1, 5′-TTGCCCGCGCCCCCTTCATAG-3′ for the fragment covering the region for Flag-Hath1 (20 cycles); sense Flag, 5′-CACCATGGATTACAAGGATGACGACGAT-3′ (common for the S primer for the Flag-Hath1 fragment) and antisense Flag-EGFP, 5′-AGGATGTTGCCGTCCTCC-3′ for Flag-EGFP (20 cycles); sense GSK3β, 5′-ATCTTAATCTGGTGCTGGACTATGT-3′ and antisense GSK3β, 5′-TTGAGTGGTGAAGTTGAAGAGTGCA-3′ for GSK3β (25 cycles); sense MUC2, 5′-CTGCACCAAGACCGTCCTCATG-3′ and antisense MUC2, 5′-GCAAGGACTGAACAAAGACTCAGAC-3′ for MUC2 (25 cycles); sense c-Myc, 5′-CTTCTGCTGGAGGCCACAGCAAACCTCCTC-3′ and antisense-c-Myc, 5′-CCAACTCCGGGATCTGGTCACGCAGGG -3′ for c-Myc (25 cycles); sense Hath1, 5′-AAGACGTTGCAGAAGAGACCCG-3′ and antisense Hath1, 5′- TTGCCCGCGCCCCCTTCATAG-3′ (common for the antisense primer for Flag-Hath1 fragment) for endogenous Hath1 (25 cycles); and sense glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and antisense G3PDH, 5′-CATGTGGGCCATGAGGTCCACCAC-3′ for glyceraldehyde-3-phosphate dehydrogenase (17 cycles). The amplification for each gene was logarithmic under these conditions. PCR products were separated on 1.5% agarose gels, stained with ethidium bromide, and visualized with a Lumi-Imager F1 (Roche Diagnostics). Expression levels of Hath1 mRNA in human tissues were analyzed by using 2 human multiple tissue blots (BioChain Institute, Hayward, CA). The complementary DNA probe corresponding to nucleotides +1/+749 for the Hath1 gene was generated by reverse-transcription (RT)-PCR from an RNA sample obtained from human colonic tissues. The probe for G3PDH was also generated by RT-PCR. The probes were labeled with [α-32P]deoxycytidine triphosphate by random priming using RediPrime II (Amersham Biosciences UK) according to the manufacturer's instructions. Hybridization was performed in Ultra Hyb solution (Ambion, Austin, TX) at 42°C overnight for Hath1 and at 55°C for 2 hours for β-actin. Visualization of the hybridized signals was conducted with the BAS-2000 image analyzing system (Fuji Film, Tokyo, Japan). SW480 cells were transiently transfected with 10 ng of renilla luciferase reporter plasmid pRL-TK-Luc (Promega) along with 100 ng of either TOPflash, FOPflash, or an E-box-Luc reporter plasmid. One microgram of the expression plasmid pCMV-Flag-WT-Hath1 or its empty control, and the same amount of pCS2-APC2 or its empty control, were also cotransfected, keeping the total amount of plasmid per transfection constant. Transfections of 293T cells were performed identically, except for substituting the pCS2-APC2 with the pRL5-Wnt1 and the pCS2 vector with the pRL5 vector, respectively. After 12 hours of transfection, cells were cultured for 24 hours and lysed by 3 cycles of freezing and thawing. Firefly luciferase activity was normalized with renilla luciferase activity in each sample by using the Dual Luciferase Kit (Promega). The E-box–dependent luciferase activities were shown as arbitrary units normalized by renilla luciferase activity, and the β-catenin/TCF–dependent luciferase activities were shown as a ratio of TOPflash and FOPflash. SW480 cells were cotransfected on a sterile glass coverslip with 1 μg of a bicistronic expression vector pMX-Flag-Hath1-IRES-GFP or its empty control together with 1 μg of a pCS2-APC2 or pCS2 vector as indicated. Twelve hours after transfection, cells were fixed with 4.0% paraformaldehyde, rinsed twice with PBS, and permeabilized with 0.2% Triton X-100 in PBS, followed by incubation for 1 hour in 3% bovine serum albumin–containing PBS to block nonspecific antibody binding. The samples were incubated for 3 hours at 37°C with either mouse anti-Flag antibody (1 μg/mL) or mouse anti-MUC2 antibody (1 μg/mL, Ccp58; Santa Cruz Biotechnology), washed twice with PBS, and then incubated for 1 hour at 37°C with Alexa 594–conjugated anti-mouse fluorescent secondary antibodies (Molecular Probes, Eugene, OR). The cells were also counterstained with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) to visualize nuclei. The samples were washed 3 times with PBS and analyzed with an epifluorescence microscope (BX-50; Olympus, Tokyo, Japan) equipped with a PDMC device camera (Polaroid, Waltham, MA) for coexistence of the fluorescent signals of the secondary antibodies (Flag-Hath1 or MUC2 protein) and green fluorescent protein, the latter of which is translated from the IRES element fused downstream of the coding region for Flag-Hath1 protein. Images were processed in Adobe Photoshop software (Adobe Systems Inc, San Jose, CA). Normal and cancerous colonic mucosae were obtained from 4 patients with colorectal cancer who underwent colectomy. Written informed consent was obtained from all patients, and these experiments were approved by the Tokyo Medical and Dental University Hospital Ethics Committee on Human Subjects. Immunohistochemistry for β-catenin was performed as described elsewhere,25Matsumoto T. Okamoto R. Yajima T. Mori T. Okamoto S. Ikeda Y. Mukai M. Yamazaki M. Oshima S. Tsuchiya K. Nakamura T. Kanai T. Okano H. Inazawa J. Hibi T. Watanabe M. Increase of bone marrow-derived secretory lineage epithelial cells during regeneration in the human intestine.Gastroenterology. 2005; 128: 1851-1867Abstract Full Text Full Text PDF Scopus (73) Google Scholar using anti–β-catenin antibody (BD Transduction, San Diego, CA) and the standard ABC method (Vectastain; Vector Laboratories). Staining was developed by addition of diaminobenzidine (Vector Laboratories). Hath-1 antibody was generated by immunizing rabbits with Hath-1 peptide (247–265). Samples were fixed with 4% paraformaldehyde and subjected for staining using a TSA Signal Amplifying Kit (Molecular Probes) following the manufacturer's instructions. Staining was developed by addition of Alexa 488–conjugated tyramide. Sections were also counterstained with 4′,6-diamidino-2-phenylindole (Vector Laboratories) to visualize nuclei. Stained samples were analyzed with an epifluorescence microscope (BX-50; Olympus) equipped with a PDMC device camera (Polaroid). The mRNA expression of Math1 and Hath1 is reported to be confined to the gastrointestinal tract in adult mice26Akazawa C. Ishibashi M. Shimizu C. Nakanishi S. Kageyama R. A mammalian helix-loop-helix factor structurally related to the product of Drosophila proneural gene atonal is a positive transcriptional regulator expressed in the developing nervous system.J Biol Chem. 1995; 270: 8730-8738Abstract Full Text Full Text PDF Scopus (334) Google Scholar and humans,19Leow C.C. Romero M.S. Ross S. Polakis P. Gao W.Q. Hath1, down-regulated in colon adenocarcinomas, inhibits proliferation and tumorigenesis of colon cancer cells.Cancer Res. 2004; 64: 6050-6057Google Scholar respectively. However, precise analysis of the expression of Hath1 mRNA within the gastrointestinal tract has never been reported. Thus, we compared mRNA expression of Hath1 in each section of the adult human gastrointestinal tract by Northern blotting. Results revealed that Hath1 mRNA is indeed exclusively expressed in the gastrointestinal tract, from the jejunum to the rectum (Figure 1). Moreover, the amount of Hath1 mRNA expression was significantly increased in the colon, compared with the jejunum or the ileum, where the population of secretory lineage epithelial cells is relatively increased. These results suggested that Hath1 expression is strictly regulated by mRNA expression, at least in the normal adult human body, and may have critical roles especially in the differentiation of colonocytes into secretory lineage cells. To further analyze the functional role of Hath1 in colonocyte differentiation, we first asked whether an overexpression of Hath1 could change any phenotype of human colon-derived epithelial cells. Because the results of the former section suggested that expression of Hath1 mRNA may directly lead to Hath1 protein expression and function to regulate differentiation in colonocytes, we introduced an expression plasmid vector encoding Flag-tagged Hath1 (Flag-WT-Hath1) into various human colon cancer–derived epithelial cell lines. Surprisingly, significant expression of FLAG-Hath1 protein could not be observed in all 3 colon cancer cell lines examined, which were SW480, DLD1, and HT-29. This was not due to low efficiency of transfection or poor sensitivity of the immunoblot, because expression of Flag-EGFP protein could be easily detected by introducing the same amount of the expression plasmid having the same plasmid backbone (Figure 2A). Furthermore, semiquantitative RT-PCR showed an equal amount of Flag-Hath1 or Flag-EGFP mRNA expression in every colon cancer cell line, confirming the efficient transfection of the FLAG-Hath1 gene. However, Flag-Hath1 and Flag-EGFP showed equal expression of both mRNA and protein in 293T cells. These results suggested that there might be a posttranscriptional regulation of Hath1 expression involving protein degradation, specifically in colon cancer cells (Figure 2A). To confirm the involvement of proteolysis in the significantly decreased expression of Hath1 protein in colon cancer cell lines, we used various pharmacologic inhibitors of the cellular proteolytic system. When SW480 cells were treated with these inhibitors after transfection of Flag-Hath1 expression vectors, inhibitors such as calpain inhibitor or chloroquine had no effect on protein expression of Flag-Hath1 (Figure 2B). In sharp contrast, treatment with proteasome inhibitors such as MG132 or lactacystin significantly increased the protein expression of Flag-Hath1, suggesting that Hath1 protein is degradated by the proteasome-mediated proteolysis in colon cancer cells (Figure 2B). Because proteasome-mediated p
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