Transforming Growth Factor β1 Signaling via Interaction with Cell Surface Hyal-2 and Recruitment of WWOX/WOX1

• DOI: 10.1074/jbc.m806688200
• Publication Date: 2009-04-15T00:13:09Z
• Authors: Li-Jin Hsu; Lori Schultz; Qunying Hong; Kris Van Moer; John Heath; Meng-Yen Li; Feng-Jie Lai; Sing-Ru Lin; Ming-Hui Lee; Cheng-Peng Lo; Yee-Shin Lin; Shur-Tzu Chen; Nan-Shan Chang
• Keywords: WWOX; Factor (programming language)

Transforming growth factor β (TGF-β) initiates multiple signal pathways and activates many downstream kinases. Here, we determined that TGF-β1 bound cell surface hyaluronidase Hyal-2 on microvilli in type II TGF-β receptor-deficient HCT116 cells, as by immunoelectron microscopy. This binding resulted recruitment of proapoptotic WOX1 (also named WWOX or FOR) formation Hyal-2·WOX1 complexes for relocation to the nuclei. strengthened catalytic domain with N-terminal Tyr-33-phosphorylated WW WOX1, time lapse fluorescence resonance energy transfer analysis live co-immunoprecipitation, yeast two-hybrid domain/domain mapping. In promoter activation assay, ectopic alone increased activity driven Smad. combination, dramatically enhanced (8–9-fold increases), which subsequently led death (>95% promoter-activated cells). supports L929 fibroblast growth. contrast, transiently overexpressed sensitized TGF-β1-induced apoptosis. Together, invokes a novel signaling engaging recruiting regulating Smad-driven promoter, thereby controlling death. 4The abbreviations used are:TGF-βtransforming βTβRIItype receptorEGFPenhanced green proteinEYFPenhanced yellow proteinECFPenhanced cyan proteinERKextracellular signal-regulated kinaseDNdominant-negativeMEKmitogen-activated protein kinase/extracellular kinase kinaseFRETfluorescence transfer. plays dual role tumorigenesis (1.Muraoka-Cook R.S. Dumont N. Arteaga C.L. Clin. Cancer Res. 2005; 11: 937S-943SPubMed Google Scholar, 2.Bachman K.E. Park B.H. Curr. Opin. Oncol. 17: 49-54Crossref PubMed Scopus (159) Scholar). inhibits mammary epithelial invasive cancer cells frequently overproduce promote metastasis The underlying mechanism is largely unknown. induces development metastatic phenotypes, i.e. stimulation epithelial-mesenchymal transitions cancerous These are normally devoid functional receptor (TβRII), suggesting binds an alternative signaling. transforming extracellular dominant-negative mitogen-activated Hyaluronan major components pericellular coat key affecting morphology, communication, behavior (3.Udabage L. Brownlee G.R. Nilsson S.K. Brown T.J. Exp. Cell 310: 205-217Crossref (179) 4.Cattaruzza S. Perris R. Matrix Biol. 24: 400-417Crossref (90) 5.Stern Jedrzejas M.J. Chem. Rev. 2006; 106: 818-839Crossref (569) Up-regulation hyaluronan hyaluronidases Hyal-1, Hyal-2, PH-20 associated Hyaluronidases counteract (6.Chang N.S. Am. J. Physiol. 1997; 273: C1987-C1994Crossref 7.Chang Int. Mol. Med. 1998; 2: 653-659PubMed 8.Chang BMC 2002; 3: 8Crossref (37) suppresses proliferation normal whereas blocks effect Hyal-1 enhance cytotoxic function TNF block TGF-β1-mediated protection murine fibroblasts from cytotoxicity PH-20, induce expression tumor suppressor domain-containing oxidoreductase, known WWOX, FOR (8.Chang 9.Chang Pratt Heath Schultz Sleve D. Carey G.B. Zevotek 2001; 276: 3361-3370Abstract Full Text PDF (188) 10.Chang Hsu L.J. Lin Y.S. Lai F.J. Sheu H.M. Trends 2007; 13: 12-22Abstract (104) 11.Lokeshwar V.B. Cerwinka W.H. Isoyama T. Lokeshwar B.L. 65: 7782-7789Crossref (102) Human gene located chromosomal fragile site 16q23 encodes WWOX/FOR/WOX1 isoforms (9.Chang 12.Bednarek A.K. Keck-Waggoner Daniel R.L. Laflin K.J. Bergsagel P.L. Kiguchi K. Brenner A.J. Aldaz C.M. 61: 8068-8073PubMed 13.Ried Finnis M. Hobson Mangelsdorf Dayan Nancarrow J.K. Woollatt E. Kremmidiotis G. Gardner A. Venter Baker Richards R.I. Hum. Genet. 2000; 9: 1651-1663Crossref (244) 14.Smith D.I. McAvoy Zhu Y. Perez D.S. Semin. 31-41Crossref (86) 15.Sze C.I. Su Pugazhenthi Jambal P. Chang 2004; 279: 30498-30506Abstract (93) 16.Mahajan N.P. Whang Y.E. Mohler J.L. Earp H.S. 10514-10523Crossref full-length 46-kDa possesses two domains (containing conserved tryptophan residues), nuclear localization sequence between domains, C-terminal short chain alcohol dehydrogenase/reductase domain. Numerous exogenous stimuli, including sex steroid hormones, TNF, anisomycin, UV light, apoptosis inducers, via phosphorylation at Tyr-33 translocation both vivo vitro 17.Chang Doherty Ensign 2003; 278: 9195-9202Abstract (108) 18.Chang Hong Q. 280: 43100-43108Abstract (99) 19.Chang Lewis Sze Oncogene. 714-723Crossref (73) 20.Lai Cheng Chen S.T. Wu C.H. Lee J.Y. Chao S.C. Sheen M.C. Shen 5769-5777Crossref (53) mouse WWOX/WOX1 appears play survival (for review, see Ref. Ectopic exerts 21.Qin H.R. Iliopoulos Semba Fabbri Druck Volinia Croce Morrison C.D. Klein R.D. Huebner 66: 6477-6481Crossref (91) 22.Fabbri Trapasso F. Aqeilan Cimmino Zanesi Yendamuri Han S.Y. Amadori Proc. Natl. Acad. Sci. U. 102: 15611-15616Crossref (114) Scholar) (12.Bednarek Targeted deletion Wwox exons 2–4 spontaneous lung bone marrow (23.Aqeilan Hussain Costinean Marshall Pekarsky Hagan J.P. Kaou Stein G.S. Lian J.B. 104: 3949-3954Crossref (176) whole body gene-ablated mice can only survive approximately 1 month Later on, was shown be essential postnatal metabolism (24.Aqeilan Hassan M.Q. de Bruin Palumbo S.H. Gaur 2008; 283: 21629-21639Abstract reproductive system (25.Ludes-Meyers J.H. Kil H. Nuñez M.I. Conti C.J. Parker-Thornburg Bedford M.T. Genes Chromosomes Cancer. 46: 1129-1136Crossref (58) 26.Aqeilan Rawahneh Salah Z. Gaudio Siddiqui Alder Endocrinology. 2009; 150: 1530-1535Crossref (65) Indeed, several prior reports have clearly indicated endogenous human up-regulated levels during embryonic (27.Chen Chuang J.I. Wang Tsai M.S. Li Neuroscience. 124: 831-839Crossref (52) early stages hyperplasia progression breast prostate (17.Chang 28.Watanabe Hippo Taniguchi Iwanari Yashiro Hirakawa Kodama Aburatani 63: 8629-8633PubMed 29.Guler Uner Guler Hauck W.W. McCue 100: 1605-1614Crossref (117) 30.Płuciennik Kusińska Potemski Kubiak Kordek Bednarek Eur. Surg. 32: 153-157Abstract (78) 31.Driouch Prydz Monese Johansen Lidereau Frengen 21: 1832-1840Crossref (92) Also, skin keratinocyte differentiation well UVB-induced squamous carcinoma humans (20.Lai Again, these observations support roles supporting survival, differentiation, organogenesis yet numerous proteins stress apoptotic responses factors transcription reviews, Refs. Scholar 32.Aqeilan 212: 307-310Crossref (101) this study investigated whether signals pathway independently TβRII examined regulation. demonstrated involving membrane then WOX1. resulting relocate nuclei Murine TβRII-deficient colorectal American Type Culture Collections been maintained our laboratory study. Platelet-derived R & D Systems, recombinant PeproTek. 4′,6-Diamidino-2-phenylindole Calbiochem. We generated polyclonal antibodies against 1) segment first (pan-specific human, rat, mouse) Scholar), 2) peptide 3) unique C terminus (15.Sze addition, synthetic NH2-CPDVEVARNDQLAWL-COOH (amino acids 227–241) made (Genemed Synthesis). rabbits using EZ antibody production purification kit (Pierce), described selected amino acid identical pig, rat. region predicted helical, surface-exposed segment, according homology searching GenBank™ data base three-dimensional structure lyco_hydro_56 (or domain) hyaluronidase. Additional specific Western blotting were following proteins: Smad4 (mouse rabbit IgG Zymed Laboratories Inc., Signaling Technology, Upstate Biotechnology, Santa Cruz Laboratories), Tyr-204-phosphorylated (p-ERK, CD44 (Chemicon); α-tubulin (Accurate Chemicals). gifts Abcam. constructs made; EGFP-WOX1, coding cDNA tagged (EGFP) p-EGFP-C1 vector (Clontech) Scholar); DN-WOX1, EGFP EYFP-Hyal-2(GLYH), (EYFP) p-EYFP-C1 (Clontech); 4) EGFP-Smad4, (GenBankTM accession AY493561). constructed EGFP-tagged Hyal-2(EGF) construct pEGFP-C1 (Clontech). expresses EGF (epidermal factor-like (225 bp insert). antisense allows stretch mRNA designated EGFP-Hyal-2(as). Primers designing forward 5′-TCGAATTCTATGTATTGCAGTTGGACCAG, reverse 5′-CAGAATTCGATTATTGGCACTGCTCGCCACC. suppressed mouse, electroporated above (200 volt, 50 ms; Square Wave BTX ECM830, Genetronics) grown overnight, treated TGF-β1, subjected extraction separation cytosolic fractions NE-PER Nuclear Cytoplasmic Extraction reagent (Pierce). Albumin (0.5%) included electroporation efficiency (300–500% increase) limit electric shock-mediated (18.Chang Where indicated, preparations co-immunoprecipitation transfected liposome-based FuGENE 6 (Roche Applied Science) Genefector (VennNova). Both reagents equally effective transfection. Epifluorescence microscopy carried out some experiments Nikon Eclipse TE-2000U inverted microscope. Confocal also performed determine (Olympus FV1000). cultured Petri dishes washed 3 times phosphate-buffered saline (PBS) incubated 0.25 μg/ml purified platelet (in PBS containing 10 bovine serum albumin; R&D Systems) ice 90 min. Disuccinimidyl suberate chemical cross-linker, prepared fresh added (0.4 mm final concentration). kept 2 h, rinsed ice-cold fixed 4% paraformaldehyde 0.1 m phosphate buffer (pH 7.2) h. samples dehydrated graded series ethanol embedded LR White resin (London Resin) °C oven (33.Chuang Y.H. Jen L.S. Neuroanat. 215-223Crossref (13) Ultrathin sections (70–80 nm; ultramicrotome, Reichert-Jung) combinations antibodies: anti-WOX1, anti-Hyal-2, goat anti-human (Santa Biotechnologies), monoclonal anti-TGF-β (R&D systems). stained anti-rabbit (conjugated 20-nm gold particle) anti-goat anti-mouse 10-nm (BB International Ltd). Specimens counterstained saturated aqueous uranyl acetate lead citrate room temperature under transmission electron (JEOL JEM-1200EX, Japan) 100 kV. Ras rescue-based CytoTrap (Stratagene) map interaction Briefly, bait Sos, target membrane-anchoring (Stratagene). Binding Sos-tagged membrane-anchored Sos/Ras/Raf/MEK/ERK pathway, allowing mutant cdc25H 37 defined galactose media (−Ura,−Leu) agarose plates. That is, occurrence positive protein/protein subsequent promotes grow °C, (22 °C) all selective medium Yeast (e.g. domain, Y33R pSos vector; Stratagene) pMyr Stratagene). Colony selection For controls, WOX1/p53 physical association MafB self-interaction out. Empty pSos/pMyr vectors regarded negative yielded no colonies in-frame ECFP EYFP (Clontech), respectively. (VennNova) 24–48 FRET microscope (Nikon TE-2000U). Cells stimulated excitation wavelength 440 nm. detected emission 535 nm (see Fig. 6A schematic). donor acceptor fluorescent molecules, images corrected background area free spectral bleed-through. spectrally concentration (FRETc) calculated Youvan's equation (using software program Image-Pro 6.1, Media Cybernetics): FRETc = [fret − bk(fret)] cf(don) × [don bk(don)] cf(acc) [acc bk(acc)], where fret image, bk background, cf correction factor, don acc image. normalizes proteins. An assay Smad SABiosciences. reporter volt ECM830 electroporator Hyal-2(GLYH), and/or mixtures electroporation. 24 Promoter controls tested each experiment. stimulates fibroblasts. responsive protects necrosis overnight i