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Galectins pp 451-463 | Cite as

Analysis of the Intracellular Role of Galectins in Cell Growth and Apoptosis

  • Daniel K. Hsu
  • Ri-Yao Yang
  • Jun Saegusa
  • Fu-Tong LiuEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1207)

Abstract

Galectins are a family of animal lectins with conserved carbohydrate-recognition domains that recognize β-galactosides. Despite structural similarities, these proteins have diverse functions in a variety of cellular processes. While a large number of extracellular functions have been demonstrated for galectins, the existence of intracellular functions has been clearly shown for a number of galectins, including regulation of cell growth and apoptosis; these latter functions may not involve glycan binding. There is considerable interest in intracellular regulation by galectins of cell growth and apoptosis, as these are fundamental cellular processes in normal homeostasis. Their dysregulation can cause pathologies such as autoimmune disorders, cancer, and neural degenerative diseases. Here we describe methods that we routinely perform in the laboratory to investigate the role of galectins in cell growth and apoptosis. These include methods for cell isolation, cell maintenance, and genetic manipulations to perturb galectin gene expression, as well as assays for cell growth and apoptosis.

Key words

Galectin Intracellular regulation Cell growth Apoptosis 

References

  1. 1.
    Zhang X, Goncalves R, Mosser DM. The isolation and characterization of murine macrophages. In: Coligan JE (ed) Current protocols in immunology, vol 14.1, 2008 edn. Wiley, New York, pp. 1–14Google Scholar
  2. 2.
    Leffler H, Barondes SH (1986) Specificity of binding of three soluble rat lung lectins to substituted and unsubstituted mammalian b-galactosides. J Biol Chem 261:10119–10126PubMedGoogle Scholar
  3. 3.
    Sparrow CP, Leffler H, Barondes SH (1987) Multiple soluble b-galactoside-binding lectins from human lung. J Biol Chem 262:7383–7390PubMedGoogle Scholar
  4. 4.
    Perillo NL, Pace KE, Seilhamer JJ, Baum LG (1995) Apoptosis of T cells mediated by galectin-1. Nature 378:736–739PubMedCrossRefGoogle Scholar
  5. 5.
    Zhou Q, Cummings RD (1993) L-14 lectin recognition of laminin and its promotion of in vitro cell adhesion. Arch Biochem Biophys 300:6–17PubMedCrossRefGoogle Scholar
  6. 6.
    Puche AC, Poirier F, Hair M, Bartlett PF, Key B (1996) Role of galectin-1 in the developing mouse olfactory system. Dev Biol 179:274–287PubMedCrossRefGoogle Scholar
  7. 7.
    Adams L, Scott GK, Weinberg CS (1996) Biphasic modulation of cell growth by recombinant human galectin-1. Biochim Biophys Acta Mol Cell Res 1312:137–144CrossRefGoogle Scholar
  8. 8.
    Yang RY, Hsu DK, Liu FT (1996) Expression of galectin-3 modulates T cell growth and apoptosis. Proc Natl Acad Sci U S A 93:6737–6742PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Moutsatsos IK, Davis JM, Wang JL (1986) Endogenous lectins from cultured cells: Subcellular localization of carbohydrate-binding protein 35 in 3T3 fibroblasts. J Cell Biol 102:477–483PubMedCrossRefGoogle Scholar
  10. 10.
    Moutsatsos IK, Wade M, Schindler M, Wang JL (1987) Endogenous lectins from cultured cells: Nuclear localization of carbohydrate-binding protein 35 in proliferating 3T3 fibroblasts. Proc Natl Acad Sci U S A 84:6452–6456PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Park JW, Voss PG, Grabski S, Wang JL, Patterson RJ (2001) Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein. Nucleic Acids Res 29(17):3595–3602PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Elad-Sfadia G, Haklai R, Ballan E, Kloog Y (2004) Galectin-3 augments K-Ras activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity. J Biol Chem 279:34922–34930PubMedCrossRefGoogle Scholar
  13. 13.
    Shimura T, Takenaka Y, Tsutsumi S, Hogan V, Kikuchi A, Raz A (2004) Galectin-3, a novel binding partner of beta-catenin. Cancer Res 64(18):6363–6367. doi: 10.1158/0008-5472.CAN-04-1816 PubMedCrossRefGoogle Scholar
  14. 14.
    Chen HY, Fermin A, Vardhana S, Weng IC, Lo KF, Chang EY, Maverakis E, Yang RY, Hsu DK, Dustin ML, Liu FT (2009) Galectin-3 negatively regulates TCR-mediated CD4+ T-cell activation at the immunological synapse. Proc Natl Acad Sci U S A 106(34):14496–14501, doi:0903497106 [pii] 10.1073/pnas.0903497106PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Liu W, Hsu DK, Chen HY, Yang RY, Carraway KL III, Isseroff RR, Liu FT (2012) Galectin-3 regulates intracellular trafficking of EGFR through Alix and promotes keratinocyte migration. J Invest Dermatol 132(12):2828–2837. doi: 10.1038/jid.2012.211 PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Cecchinelli B, Lavra L, Rinaldo C, Iacovelli S, Gurtner A, Gasbarri A, Ulivieri A, Del Prete F, Trovato M, Piaggio G, Bartolazzi A, Soddu S, Sciacchitano S (2006) Repression of the antiapoptotic molecule galectin-3 by homeodomain-interacting protein kinase 2-activated p53 is required for p53-induced apoptosis. Mol Cell Biol 26(12):4746–4757PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Fukumori T, Kanayama HO, Raz A (2007) The role of galectin-3 in cancer drug resistance. Drug Resist Updat 10(3):101–108. doi: 10.1016/j.drup.2007.04.001 PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Liu FT, Rabinovich GA (2005) Galectins as modulators of tumour progression. Nat Rev Cancer 5(1):29–41PubMedCrossRefGoogle Scholar
  19. 19.
    Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B (1997) A model for p53-induced apoptosis. Nature 389:300–305PubMedCrossRefGoogle Scholar
  20. 20.
    Kuwabara I, Kuwabara Y, Yang RY, Schuler M, Green DR, Zuraw BL, Hsu DK, Liu FT (2002) Galectin-7 (PIG1) exhibits pro-apoptotic function through JNK activation and mitochondrial cytochrome c release. J Biol Chem 277(5):3487–3497PubMedCrossRefGoogle Scholar
  21. 21.
    Sato S, Ouellet M, St-Pierre C, Tremblay MJ (2012) Glycans, galectins, and HIV-1 infection. Ann N Y Acad Sci 1253:133–148. doi: 10.1111/j.1749-6632.2012.06475.x PubMedCrossRefGoogle Scholar
  22. 22.
    Rabinovich GA, Croci DO (2012) Regulatory circuits mediated by lectin-glycan interactions in autoimmunity and cancer. Immunity 36(3):322–335. doi: 10.1016/j.immuni.2012.03.004 PubMedCrossRefGoogle Scholar
  23. 23.
    Liu FT, Yang RY, Hsu DK (2012) Galectins in acute and chronic inflammation. Ann N Y Acad Sci 1253:80–91. doi: 10.1111/j.1749-6632.2011.06386.x PubMedCrossRefGoogle Scholar
  24. 24.
    Vasta GR (2012) Galectins as pattern recognition receptors: structure, function, and evolution. Adv Exp Med Biol 946:21–36. doi: 10.1007/978-1-4614-0106-3_2 PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    de Boer RA, Yu L, van Veldhuisen DJ (2010) Galectin-3 in cardiac remodeling and heart failure. Curr Heart Fail Rep 7(1):1–8. doi: 10.1007/s11897-010-0004-x PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Kato MA, Fahey TJ III (2009) Molecular markers in thyroid cancer diagnostics. Surg Clin North Am 89(5):1139–1155. doi: 10.1016/j.suc.2009.06.012 PubMedCrossRefGoogle Scholar
  27. 27.
    Delacour D, Koch A, Jacob R (2009) The role of galectins in protein trafficking. Traffic 10(10):1405–1413, doi:TRA960 [pii] 10.1111/j.1600-0854.2009.00960.xPubMedCrossRefGoogle Scholar
  28. 28.
    Haudek KC, Spronk KJ, Voss PG, Patterson RJ, Wang JL, Arnoys EJ (2010) Dynamics of galectin-3 in the nucleus and cytoplasm. Biochim Biophys Acta 1800(2):181–189, doi:S0304-4165(09)00194-9 [pii] 10.1016/j.bbagen.2009.07.005PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Stillman BN, Hsu DK, Pang M, Brewer CF, Johnson P, Liu FT, Baum LG (2006) Galectin-3 and galectin-1 bind distinct cell surface glycoprotein receptors to induce T cell death. J Immunol 176(2):778–789PubMedCrossRefGoogle Scholar
  30. 30.
    Fukumori T, Takenaka Y, Yoshii T, Kim HR, Hogan V, Inohara H, Kagawa S, Raz A (2003) CD29 and CD7 mediate galectin-3-induced type II T-cell apoptosis. Cancer Res 63(23):8302–8311PubMedGoogle Scholar
  31. 31.
    Paclik D, Berndt U, Guzy C, Dankof A, Danese S, Holzloehner P, Rosewicz S, Wiedenmann B, Wittig BM, Dignass AU, Sturm A (2008) Galectin-2 induces apoptosis of lamina propria T lymphocytes and ameliorates acute and chronic experimental colitis in mice. J Mol Med 86(12):1395–1406. doi: 10.1007/s00109-007-0290-2 PubMedCrossRefGoogle Scholar
  32. 32.
    Paclik D, Danese S, Berndt U, Wiedenmann B, Dignass A, Sturm A (2008) Galectin-4 controls intestinal inflammation by selective regulation of peripheral and mucosal T cell apoptosis and cell cycle. PLoS One 3(7):e2629. doi: 10.1371/journal.pone.0002629 PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    Zhu C, Anderson AC, Schubart A, Xiong H, Imitola J, Khoury SJ, Zheng XX, Strom TB, Kuchroo VK (2005) The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol 6(12):1245–1252PubMedCrossRefGoogle Scholar
  34. 34.
    Hadari YR, Arbel-Goren R, Levy Y, Amsterdam A, Alon R, Zakut R, Zick Y (2000) Galectin-8 binding to integrins inhibits cell adhesion and induces apoptosis. J Cell Sci 113(Pt 13):2385–2397PubMedGoogle Scholar
  35. 35.
    Buttke TM, McCubrey JA, Owen TC (1993) Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines. J Immunol Methods 157:233–240PubMedCrossRefGoogle Scholar
  36. 36.
    Ozdemir O, Ravindranath Y, Savasan S (2003) Cell-mediated cytotoxicity evaluation using monoclonal antibody staining for target or effector cells with annexinV/propidium iodide colabeling by fluorosphere-adjusted counts on three-color flow cytometry. Cytometry 56(1):53–60. doi: 10.1002/cyto.a.10081 PubMedCrossRefGoogle Scholar
  37. 37.
    Baum C et al (1994) An optimized electroporation protocol applicable to a wide range of cell lines. Biotechniques 17(6):1058–1062PubMedGoogle Scholar
  38. 38.
    Goldberg GS, Lau AF (1993) Transfection of mammalian cells with PEG-purified plasmid DNA. Biotechniques 14(4):548–550PubMedGoogle Scholar
  39. 39.
    Wang LF, Voysey R, Yu M (1994) Simplified large-scale alkaline lysis preparation of plasmid DNA with minimal use of phenol. Biotechniques 17(1):26–28PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Daniel K. Hsu
    • 1
  • Ri-Yao Yang
    • 1
  • Jun Saegusa
    • 2
  • Fu-Tong Liu
    • 1
    • 3
    Email author
  1. 1.Department of DermatologyUniversity of California, DavisDavisCalifornia
  2. 2.Department of Rheumatology, School of MedicineKobe UniversityKobeJapan
  3. 3.Department of DermatologyUniversity of California Davis, School of MedicineSacramentoUSA

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