Transforming Growth Factor β1 Signaling via Interaction with Cell Surface Hyal-2 and Recruitment of WWOX/WOX1
2009; Elsevier BV; Volume: 284; Issue: 23 Linguagem: Inglês
10.1074/jbc.m806688200
ISSN1083-351X
AutoresLi‐Jin Hsu, Lori Schultz, Qunying Hong, Kris Van Moer, John K. 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,
Tópico(s)Developmental Biology and Gene Regulation
ResumoTransforming 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 determined by immunoelectron microscopy. This binding resulted in recruitment of proapoptotic WOX1 (also named WWOX or FOR) and formation of Hyal-2·WOX1 complexes for relocation to the nuclei. TGF-β1 strengthened the binding of the catalytic domain of Hyal-2 with the N-terminal Tyr-33-phosphorylated WW domain of WOX1, as determined by time lapse fluorescence resonance energy transfer analysis in live cells, co-immunoprecipitation, and yeast two-hybrid domain/domain mapping. In promoter activation assay, ectopic WOX1 or Hyal-2 alone increased the promoter activity driven by Smad. In combination, WOX1 and Hyal-2 dramatically enhanced the promoter activation (8–9-fold increases), which subsequently led to cell death (>95% of promoter-activated cells). TGF-β1 supports L929 fibroblast growth. In contrast, transiently overexpressed WOX1 and Hyal-2 sensitized L929 to TGF-β1-induced apoptosis. Together, TGF-β1 invokes a novel signaling by engaging cell surface Hyal-2 and recruiting WOX1 for regulating the activation of Smad-driven promoter, thereby controlling cell growth and death. 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 determined by immunoelectron microscopy. This binding resulted in recruitment of proapoptotic WOX1 (also named WWOX or FOR) and formation of Hyal-2·WOX1 complexes for relocation to the nuclei. TGF-β1 strengthened the binding of the catalytic domain of Hyal-2 with the N-terminal Tyr-33-phosphorylated WW domain of WOX1, as determined by time lapse fluorescence resonance energy transfer analysis in live cells, co-immunoprecipitation, and yeast two-hybrid domain/domain mapping. In promoter activation assay, ectopic WOX1 or Hyal-2 alone increased the promoter activity driven by Smad. In combination, WOX1 and Hyal-2 dramatically enhanced the promoter activation (8–9-fold increases), which subsequently led to cell death (>95% of promoter-activated cells). TGF-β1 supports L929 fibroblast growth. In contrast, transiently overexpressed WOX1 and Hyal-2 sensitized L929 to TGF-β1-induced apoptosis. Together, TGF-β1 invokes a novel signaling by engaging cell surface Hyal-2 and recruiting WOX1 for regulating the activation of Smad-driven promoter, thereby controlling cell growth and death. Transforming growth factor β (TGF-β) 4The abbreviations used are:TGF-βtransforming growth factor βTβRIItype II TGF-β receptorEGFPenhanced green fluorescence proteinEYFPenhanced yellow fluorescence proteinECFPenhanced cyan fluorescence proteinERKextracellular signal-regulated kinaseDNdominant-negativeMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseFRETfluorescence resonance energy transfer. 4The abbreviations used are:TGF-βtransforming growth factor βTβRIItype II TGF-β receptorEGFPenhanced green fluorescence proteinEYFPenhanced yellow fluorescence proteinECFPenhanced cyan fluorescence proteinERKextracellular signal-regulated kinaseDNdominant-negativeMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseFRETfluorescence resonance energy transfer. plays a dual role in cell growth and 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. 2005; 17: 49-54Crossref PubMed Scopus (159) Google Scholar). TGF-β inhibits mammary epithelial cell growth. In contrast, invasive cancer cells frequently overproduce TGF-β to promote growth and metastasis (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. 2005; 17: 49-54Crossref PubMed Scopus (159) Google Scholar). The underlying mechanism is largely unknown. TGF-β induces the development of metastatic phenotypes, i.e. stimulation of epithelial-mesenchymal transitions in cancerous mammary epithelial cells (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. 2005; 17: 49-54Crossref PubMed Scopus (159) Google Scholar). These cells are normally devoid of functional type II TGF-β receptor (TβRII), suggesting that TGF-β binds to an alternative receptor for signaling. transforming growth factor β type II TGF-β receptor enhanced green fluorescence protein enhanced yellow fluorescence protein enhanced cyan fluorescence protein extracellular signal-regulated kinase dominant-negative mitogen-activated protein kinase/extracellular signal-regulated kinase kinase fluorescence resonance energy transfer. transforming growth factor β type II TGF-β receptor enhanced green fluorescence protein enhanced yellow fluorescence protein enhanced cyan fluorescence protein extracellular signal-regulated kinase dominant-negative mitogen-activated protein kinase/extracellular signal-regulated kinase kinase fluorescence resonance energy transfer. Hyaluronan is the major components of pericellular coat and plays a key role in affecting cell morphology, communication, and behavior (3.Udabage L. Brownlee G.R. Nilsson S.K. Brown T.J. Exp. Cell Res. 2005; 310: 205-217Crossref PubMed Scopus (179) Google Scholar, 4.Cattaruzza S. Perris R. Matrix Biol. 2005; 24: 400-417Crossref PubMed Scopus (90) Google Scholar, 5.Stern R. Jedrzejas M.J. Chem. Rev. 2006; 106: 818-839Crossref PubMed Scopus (569) Google Scholar). Up-regulation of hyaluronan and hyaluronidases Hyal-1, Hyal-2, and PH-20 is associated with cancer metastasis (3.Udabage L. Brownlee G.R. Nilsson S.K. Brown T.J. Exp. Cell Res. 2005; 310: 205-217Crossref PubMed Scopus (179) Google Scholar, 4.Cattaruzza S. Perris R. Matrix Biol. 2005; 24: 400-417Crossref PubMed Scopus (90) Google Scholar, 5.Stern R. Jedrzejas M.J. Chem. Rev. 2006; 106: 818-839Crossref PubMed Scopus (569) Google Scholar). Hyaluronidases counteract the activity of TGF-β1 (6.Chang N.S. Am. J. Physiol. 1997; 273: C1987-C1994Crossref PubMed Google Scholar, 7.Chang N.S. Int. J. Mol. Med. 1998; 2: 653-659PubMed Google Scholar, 8.Chang N.S. BMC Cell Biol. 2002; 3: 8Crossref PubMed Scopus (37) Google Scholar). TGF-β1 suppresses the proliferation of normal epithelial cells, whereas PH-20 blocks the TGF-β1 effect (6.Chang N.S. Am. J. Physiol. 1997; 273: C1987-C1994Crossref PubMed Google Scholar). Hyal-1 and Hyal-2 enhance the cytotoxic function of TNF and block TGF-β1-mediated protection of murine L929 fibroblasts from TNF cytotoxicity (6.Chang N.S. Am. J. Physiol. 1997; 273: C1987-C1994Crossref PubMed Google Scholar, 7.Chang N.S. Int. J. Mol. Med. 1998; 2: 653-659PubMed Google Scholar, 8.Chang N.S. BMC Cell Biol. 2002; 3: 8Crossref PubMed Scopus (37) Google Scholar). Hyaluronidases PH-20, Hyal-1, and Hyal-2 induce the expression of tumor suppressor WW domain-containing oxidoreductase, known as WWOX, FOR or WOX1 (8.Chang N.S. BMC Cell Biol. 2002; 3: 8Crossref PubMed Scopus (37) Google Scholar, 9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 10.Chang N.S. Hsu L.J. Lin Y.S. Lai F.J. Sheu H.M. Trends Mol. Med. 2007; 13: 12-22Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 11.Lokeshwar V.B. Cerwinka W.H. Isoyama T. Lokeshwar B.L. Cancer Res. 2005; 65: 7782-7789Crossref PubMed Scopus (102) Google Scholar). Human WWOX gene is located on a chromosomal fragile site 16q23 and encodes WWOX/FOR/WOX1 and isoforms (9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 10.Chang N.S. Hsu L.J. Lin Y.S. Lai F.J. Sheu H.M. Trends Mol. Med. 2007; 13: 12-22Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12.Bednarek A.K. Keck-Waggoner C.L. Daniel R.L. Laflin K.J. Bergsagel P.L. Kiguchi K. Brenner A.J. Aldaz C.M. Cancer Res. 2001; 61: 8068-8073PubMed Google Scholar, 13.Ried K. Finnis M. Hobson L. Mangelsdorf M. Dayan S. Nancarrow J.K. Woollatt E. Kremmidiotis G. Gardner A. Venter D. Baker E. Richards R.I. Hum. Mol. Genet. 2000; 9: 1651-1663Crossref PubMed Scopus (244) Google Scholar, 14.Smith D.I. McAvoy S. Zhu Y. Perez D.S. Semin. Cancer Biol. 2007; 17: 31-41Crossref PubMed Scopus (86) Google Scholar, 15.Sze C.I. Su M. Pugazhenthi S. Jambal P. Hsu L.J. Heath J. Schultz L. Chang N.S. J. Biol. Chem. 2004; 279: 30498-30506Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16.Mahajan N.P. Whang Y.E. Mohler J.L. Earp H.S. Cancer Res. 2005; 65: 10514-10523Crossref PubMed Scopus (159) Google Scholar). The full-length 46-kDa WOX1 possesses two N-terminal WW domains (containing conserved tryptophan residues), a nuclear localization sequence between the WW domains, and a C-terminal short chain alcohol dehydrogenase/reductase domain. Numerous exogenous stimuli, including sex steroid hormones, TNF, anisomycin, UV light, and apoptosis inducers, induce WOX1 activation via phosphorylation at Tyr-33 and nuclear translocation both in vivo and in vitro (9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18.Chang N.S. Doherty J. Ensign A. Schultz L. Hsu L.J. Hong Q. J. Biol. Chem. 2005; 280: 43100-43108Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 19.Chang N.S. Schultz L. Hsu L.J. Lewis J. Su M. Sze C.I. Oncogene. 2005; 24: 714-723Crossref PubMed Scopus (73) Google Scholar, 20.Lai F.J. Cheng C.L. Chen S.T. Wu C.H. Hsu L.J. Lee J.Y. Chao S.C. Sheen M.C. Shen C.L. Chang N.S. Sheu H.M. Clin. Cancer Res. 2005; 11: 5769-5777Crossref PubMed Scopus (53) Google Scholar). Human and mouse WWOX/WOX1 appears to play a dual role in regulating cell survival and death (for review, see Ref. 10.Chang N.S. Hsu L.J. Lin Y.S. Lai F.J. Sheu H.M. Trends Mol. Med. 2007; 13: 12-22Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Ectopic WOX1 exerts apoptosis in vitro (9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18.Chang N.S. Doherty J. Ensign A. Schultz L. Hsu L.J. Hong Q. J. Biol. Chem. 2005; 280: 43100-43108Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 19.Chang N.S. Schultz L. Hsu L.J. Lewis J. Su M. Sze C.I. Oncogene. 2005; 24: 714-723Crossref PubMed Scopus (73) Google Scholar, 20.Lai F.J. Cheng C.L. Chen S.T. Wu C.H. Hsu L.J. Lee J.Y. Chao S.C. Sheen M.C. Shen C.L. Chang N.S. Sheu H.M. Clin. Cancer Res. 2005; 11: 5769-5777Crossref PubMed Scopus (53) Google Scholar, 21.Qin H.R. Iliopoulos D. Semba S. Fabbri M. Druck T. Volinia S. Croce C.M. Morrison C.D. Klein R.D. Huebner K. Cancer Res. 2006; 66: 6477-6481Crossref PubMed Scopus (91) Google Scholar, 22.Fabbri M. Iliopoulos D. Trapasso F. Aqeilan R.I. Cimmino A. Zanesi N. Yendamuri S. Han S.Y. Amadori D. Huebner K. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15611-15616Crossref PubMed Scopus (114) Google Scholar) and inhibits tumor growth in vivo (12.Bednarek A.K. Keck-Waggoner C.L. Daniel R.L. Laflin K.J. Bergsagel P.L. Kiguchi K. Brenner A.J. Aldaz C.M. Cancer Res. 2001; 61: 8068-8073PubMed Google Scholar, 22.Fabbri M. Iliopoulos D. Trapasso F. Aqeilan R.I. Cimmino A. Zanesi N. Yendamuri S. Han S.Y. Amadori D. Huebner K. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15611-15616Crossref PubMed Scopus (114) Google Scholar). Targeted deletion of murine Wwox gene to exons 2–4 induces spontaneous tumor formation in the lung and bone marrow (23.Aqeilan R.I. Trapasso F. Hussain S. Costinean S. Marshall D. Pekarsky Y. Hagan J.P. Zanesi N. Kaou M. Stein G.S. Lian J.B. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3949-3954Crossref PubMed Scopus (176) Google Scholar). The whole body Wwox gene-ablated mice can only survive for approximately 1 month (23.Aqeilan R.I. Trapasso F. Hussain S. Costinean S. Marshall D. Pekarsky Y. Hagan J.P. Zanesi N. Kaou M. Stein G.S. Lian J.B. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3949-3954Crossref PubMed Scopus (176) Google Scholar). Later on, murine Wwox gene was shown to be essential for postnatal survival and normal bone metabolism in mice (24.Aqeilan R.I. Hassan M.Q. de Bruin A. Hagan J.P. Volinia S. Palumbo T. Hussain S. Lee S.H. Gaur T. Stein G.S. Lian J.B. Croce C.M. J. Biol. Chem. 2008; 283: 21629-21639Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and development of the reproductive system (25.Ludes-Meyers J.H. Kil H. Nuñez M.I. Conti C.J. Parker-Thornburg J. Bedford M.T. Aldaz C.M. Genes Chromosomes Cancer. 2007; 46: 1129-1136Crossref PubMed Scopus (58) Google Scholar, 26.Aqeilan R.I. Hagan J.P. de Bruin A. Rawahneh M. Salah Z. Gaudio E. Siddiqui H. Volinia S. Alder H. Lian J.B. Stein G.S. Croce C.M. Endocrinology. 2009; 150: 1530-1535Crossref PubMed Scopus (65) Google Scholar). Indeed, several prior reports have clearly indicated that endogenous human and mouse WWOX/WOX1 is up-regulated at both gene and protein levels during embryonic development (27.Chen S.T. Chuang J.I. Wang J.P. Tsai M.S. Li H. Chang N.S. Neuroscience. 2004; 124: 831-839Crossref PubMed Scopus (52) Google Scholar) and in the early stages of hyperplasia and cancerous progression of human breast and prostate (17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 28.Watanabe A. Hippo Y. Taniguchi H. Iwanari H. Yashiro M. Hirakawa K. Kodama T. Aburatani H. Cancer Res. 2003; 63: 8629-8633PubMed Google Scholar, 29.Guler G. Uner A. Guler N. Han S.Y. Iliopoulos D. Hauck W.W. McCue P. Huebner K. Cancer. 2004; 100: 1605-1614Crossref PubMed Scopus (117) Google Scholar, 30.Płuciennik E. Kusińska R. Potemski P. Kubiak R. Kordek R. Bednarek A.K. Eur. J. Surg. Oncol. 2006; 32: 153-157Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 31.Driouch K. Prydz H. Monese R. Johansen H. Lidereau R. Frengen E. Oncogene. 2002; 21: 1832-1840Crossref PubMed Scopus (92) Google Scholar). Also, WWOX/WOX1 is up-regulated during normal skin keratinocyte differentiation as well as in the early stages of UVB-induced formation of squamous cell carcinoma in humans and mice (20.Lai F.J. Cheng C.L. Chen S.T. Wu C.H. Hsu L.J. Lee J.Y. Chao S.C. Sheen M.C. Shen C.L. Chang N.S. Sheu H.M. Clin. Cancer Res. 2005; 11: 5769-5777Crossref PubMed Scopus (53) Google Scholar). Again, these observations support the dual functional roles of WWOX/WOX1 in supporting cell survival, differentiation, and organogenesis and yet controlling cancer growth. WOX1 binds numerous proteins in the stress signaling and apoptotic responses and factors in gene transcription (for reviews, see Refs. 10.Chang N.S. Hsu L.J. Lin Y.S. Lai F.J. Sheu H.M. Trends Mol. Med. 2007; 13: 12-22Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar and 32.Aqeilan R.I. Croce C.M. J. Cell Physiol. 2007; 212: 307-310Crossref PubMed Scopus (101) Google Scholar). In this study we investigated whether TGF-β1 signals via a pathway independently of TβRII and examined whether this signaling activates WOX1 for cell growth regulation. Here, we demonstrated a novel signaling involving the binding of TGF-β1 with membrane Hyal-2 and then recruiting WOX1. The resulting Hyal-2·WOX1 complexes relocate to the nuclei for controlling the activation of Smad-driven promoter, thereby regulating cell growth and death. Murine L929 fibroblasts and human TβRII-deficient colorectal HCT116 cells from American Type Culture Collections have been maintained in our laboratory (8.Chang N.S. BMC Cell Biol. 2002; 3: 8Crossref PubMed Scopus (37) Google Scholar, 9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) and used in this study. Platelet-derived TGF-β1 was from R & D Systems, and recombinant TGF-β1 was from PeproTek. 4′,6-Diamidino-2-phenylindole was from Calbiochem. We have generated polyclonal antibodies against 1) an N-terminal segment of WOX1 at the first WW domain (pan-specific for human, rat, and mouse) (17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), 2) a WOX1 peptide with Tyr-33 phosphorylation at the first WW domain (pan-specific for human, rat, and mouse) (17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), and 3) the unique C terminus of human WWOX/WOX1 (15.Sze C.I. Su M. Pugazhenthi S. Jambal P. Hsu L.J. Heath J. Schultz L. Chang N.S. J. Biol. Chem. 2004; 279: 30498-30506Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In addition, a synthetic peptide of murine Hyal-2, NH2-CPDVEVARNDQLAWL-COOH (amino acids 227–241) was made (Genemed Synthesis). We generated polyclonal antibodies against this peptide in rabbits using an EZ antibody production and purification kit (Pierce), as described (9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 15.Sze C.I. Su M. Pugazhenthi S. Jambal P. Hsu L.J. Heath J. Schultz L. Chang N.S. J. Biol. Chem. 2004; 279: 30498-30506Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The selected amino acid sequence of murine Hyal-2 is identical to that in human, pig, and rat. Also, this region is predicted to be a helical, surface-exposed segment, according to homology searching in the GenBank™ data base for the three-dimensional structure of the lyco_hydro_56 domain (or catalytic domain) in hyaluronidase. Additional specific antibodies used in Western blotting were against the following proteins: Smad4 (mouse and rabbit IgG from Zymed Laboratories Inc., Cell Signaling Technology, Upstate Biotechnology, and Santa Cruz Laboratories), Tyr-204-phosphorylated extracellular signal-regulated kinase (p-ERK, Santa Cruz Laboratories), CD44 (Chemicon); α-tubulin (Accurate Chemicals). WWOX IgG antibodies were gifts from Santa Cruz Laboratories and Abcam. The following expression constructs were made; 1) murine EGFP-WOX1, full-length coding region of WOX1 cDNA tagged with enhanced green fluorescence protein (EGFP) in p-EGFP-C1 vector (Clontech) (9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar); 2) DN-WOX1, murine dominant-negative WOX1 tagged with EGFP (17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar); 3) EYFP-Hyal-2(GLYH), the catalytic domain of murine Hyal-2, tagged with enhanced yellow fluorescence protein (EYFP) in a p-EYFP-C1 vector (Clontech); 4) EGFP-Smad4, murine Smad4 tagged with EGFP (GenBankTM accession AY493561). In addition, we constructed an EGFP-tagged Hyal-2(EGF) construct in pEGFP-C1 vector (Clontech). This construct expresses the EGF domain (epidermal growth factor-like domain) of Hyal-2 (225 bp for the cDNA insert). We selected an antisense construct which allows expression of EGFP and a short stretch of antisense mRNA for Hyal-2, designated EGFP-Hyal-2(as). Primers for designing this construct were 1) forward 5′-TCGAATTCTATGTATTGCAGTTGGACCAG, and 2) reverse 5′-CAGAATTCGATTATTGGCACTGCTCGCCACC. This antisense construct suppressed Hyal-2 protein expression in human, mouse, and rat. L929 or HCT116 cells were electroporated with the above constructs (200 volt, 50 ms; Square Wave BTX ECM830, Genetronics) (9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18.Chang N.S. Doherty J. Ensign A. Schultz L. Hsu L.J. Hong Q. J. Biol. Chem. 2005; 280: 43100-43108Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), grown overnight, treated with TGF-β1, and then subjected to extraction and separation of cytosolic and nuclear fractions using the NE-PER Nuclear and Cytoplasmic Extraction reagent (Pierce). Albumin (0.5%) was included during electroporation to enhance electroporation efficiency (300–500% increase) and to limit electric shock-mediated cell death (18.Chang N.S. Doherty J. Ensign A. Schultz L. Hsu L.J. Hong Q. J. Biol. Chem. 2005; 280: 43100-43108Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Where indicated, these cell preparations were subjected to co-immunoprecipitation and Western blotting (9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18.Chang N.S. Doherty J. Ensign A. Schultz L. Hsu L.J. Hong Q. J. Biol. Chem. 2005; 280: 43100-43108Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In addition, cells were transfected with cDNA constructs using liposome-based FuGENE 6 (Roche Applied Science) or Genefector (VennNova). Both reagents were equally effective in gene transfection. Epifluorescence microscopy was carried out in some experiments using a Nikon Eclipse TE-2000U inverted microscope. Confocal microscopy was also performed to determine protein localization (Olympus FV1000). HCT116 cells cultured on Petri dishes were washed 3 times with phosphate-buffered saline (PBS) and incubated with 0.25 μg/ml purified platelet TGF-β1 (in PBS containing 10 μg/ml bovine serum albumin; R&D Systems) on ice for 90 min. Disuccinimidyl suberate (Pierce), a chemical cross-linker, was prepared fresh and added to the cells (0.4 mm final concentration). The cells were kept on ice for 2 h, then rinsed with ice-cold phosphate-buffered saline and fixed with 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.2) on ice for 1 h. The samples were dehydrated in a graded series of ethanol and embedded in LR White resin (London Resin) in a 50 °C oven (33.Chuang J.I. Chen S.T. Chang Y.H. Jen L.S. J. Chem. Neuroanat. 2001; 21: 215-223Crossref PubMed Scopus (13) Google Scholar). Ultrathin sections (70–80 nm; ultramicrotome, from Reichert-Jung) were prepared and incubated with selected combinations of two of the following antibodies: rabbit polyclonal anti-WOX1, anti-Hyal-2, goat polyclonal anti-human WWOX (Santa Cruz Biotechnologies), or mouse monoclonal anti-TGF-β (R&D systems). These sections were then stained with anti-rabbit IgG (conjugated with 20-nm gold particle) and anti-goat or anti-mouse IgG (conjugated with 10-nm gold particle) antibodies (BB International Ltd). Specimens were counterstained with saturated aqueous uranyl acetate and lead citrate at room temperature and examined under a transmission electron microscopy (JEOL JEM-1200EX, Japan) at 100 kV. Ras rescue-based CytoTrap yeast two-hybrid analysis (Stratagene) was performed to map the domain/domain interaction between Hyal-2 and WOX1, as described (9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18.Chang N.S. Doherty J. Ensign A. Schultz L. Hsu L.J. Hong Q. J. Biol. Chem. 2005; 280: 43100-43108Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Briefly, bait protein was tagged with Sos, and target protein was tagged with a membrane-anchoring signal sequence (Stratagene). Binding of the cytosolic Sos-tagged bait protein to the cell membrane-anchored target activates the Sos/Ras/Raf/MEK/ERK signal pathway, thereby allowing the growth of mutant yeast cdc25H at 37 °C under a synthetic defined galactose media (−Ura,−Leu) in agarose plates. That is, the occurrence of positive protein/protein binding and subsequent activation of the Sos/Ras/Raf/MEK/ERK signaling promotes yeast cells to grow at 37 °C, whereas at room temperature (22 °C) all yeast cells grow in the selective medium (9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18.Chang N.S. Doherty J. Ensign A. Schultz L. Hsu L.J. Hong Q. J. Biol. Chem. 2005; 280: 43100-43108Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Yeast cells were transfected with a bait construct (e.g. the full-length WOX1, the N-terminal first WW domain, or a Y33R mutant of the WW domain (in a pSos vector; Stratagene) and the Hyal-2 construct for membrane-anchoring (in a pMyr vector; Stratagene). Colony growth and selection were then performed (9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18.Chang N.S. Doherty J. Ensign A. Schultz L. Hsu L.J. Hong Q. J. Biol. Chem. 2005; 280: 43100-43108Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). For positive controls, WOX1/p53 physical association and MafB self-interaction were carried out. Empty pSos/pMyr vectors were regarded as negative controls, which yielded no yeast colonies (9.Chang N.S. Pratt N. Heath J. Schultz L. Sleve D. Carey G.B. Zevotek N. J. Biol. Chem. 2001; 276: 3361-3370Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 17.Chang N.S. Doherty J. Ensign A. J. Biol. Chem. 2003; 278: 9195-9202Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18.Chang N.S. Doherty J. Ensign A. Schultz L. Hsu L.J. Hong Q. J. Biol. Chem. 2005; 280: 43100-43108Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The full-length murine WOX1 and the catalytic domain of murine Hyal-2 were constructed in-frame with ECFP and EYFP expression vectors (Clontech), respectively. L929 and HCT116 cells were transfected with both constructs by liposome-based Genefector (VennNova) and cultured for 24–48 h. FRET analysis was performed using an inverted fluorescence microscope (Nikon Eclipse TE-2000U). Cells were stimulated with an excitation wavelength 440 nm. FRET signals were detected at an emission wavelength 535 nm (see Fig. 6A for the schematic). ECFP and EYFP were used as donor and acceptor fluorescent molecules, respectively. The FRET images were corrected for background fluorescence from an area free of cells and spectral bleed-through. The spectrally corrected FRET concentration (FRETc) was calculated by Youvan's equation (using a software program Image-Pro 6.1, Media Cybernetics): FRETc = [fret − bk(fret)] − cf(don) × [don − bk(don)] − cf(acc) × [acc − bk(acc)], where fret = fret image, bk = background, cf = correction factor, don = donor image, and acc = acceptor image. The equation normalizes the FRET signals to the expression levels of the fluorescent proteins. An assay kit for the promoter function driven by Smad was from SABiosciences. HCT116 and L929 cells were transfected with a promoter construct using green fluorescent protein as a reporter by electroporation (200 volt and 50 ms; using a BTX ECM830 electroporator from Genetronics) or using liposome-based FuGENE 6 (Roche Applied Science) or Genefector (VennNova). Also, expression constructs for WOX1, Hyal-2(GLYH), DN-WOX1, and/or Smad4 were included in the mixtures for electroporation. The cells were cultured for 24 h. Promoter activation was examined under fluorescence microscopy. Both positive and negative controls from the assay kit were also tested in each experiment. We examined whether TGF-β1 stimulates relocation of WOX1 to the nuclei in murine L929 fibroblasts. These cells are responsive to TGF-β1-induced proliferation (6.Chang N.S. Am. J. Physiol. 1997; 273: C1987-C1994Crossref PubMed Google Scholar, 7.Chang N.S. Int. J. Mol. Med. 1998; 2: 653-659PubMed Google Scholar, 8.Chang N.S. BMC Cell Biol. 2002; 3: 8Crossref PubMed Scopus (37) Google Scholar). Also, TGF-β1 protects L929 cells from the cytotoxic effect of tumor necrosis factor (6.Chang N.S. Am. J. Physiol. 1997; 273: C1987-C1994Crossref PubMed Google Scholar, 7.Chang N.S. Int. J. Mol. Med. 1998; 2: 653-659PubMed Google Scholar, 8.Chang N.S. BMC Cell Biol. 2002; 3: 8Crossref PubMed Scopus (37) Google Scholar). L929 fibroblasts were cultured overnight i
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