Expression of lysosome-associated membrane protein 1 (Lamp-1) and galectins in human keratinocytes is regulated by differentiation

Original Paper


Lysosomes and their components are suspected to be involved in epidermal differentiation. In this study, lysosomal enzyme activities, expression of the lysosome-associated membrane protein 1 (Lamp-1) and expression of the epidermal galectins-1, -3 and -7 were investigated in human keratinocytes cultured at different cell densities (subconfluence, confluence and postconfluence) in order to induce differentiation. Detected by Western blot and immunofluorescence, Lamp-1 expression is transiently upregulated at culture confluence, but reduced at postconfluence. Northern blot analyses performed on subconfluent, confluent and post-confluent cultures of keratinocytes show that Lamp-1 mRNA expression is also upregulated at culture confluence, but downregulated at postconfluence. Measurements of lysosomal enzyme activities indicate a transient upregulation at culture confluence, whereas cathepsins B, C and L are particularly downregulated at postconfluence. Cell density and differentiation of epidermal cells also differentially regulates galectin expression in autocrine cultures. As the expression of galectin-1 mRNA is high in subconfluent cells, it is assumed to be associated with their proliferative state. On the other hand, as the mRNA levels for galectins-3 and -7 are notably upregulated at culture confluence (galectin-7) or at postconfluence (galectin-3), their expression is thought to be related to the differentiated state of keratinocytes. However, we collected evidence by confocal microscopy that galectin-3 and Lamp-1 do not colocalize in vitro in keratinocytes. Altogether, our results suggest that the upregulated Lamp-1 expression at confluence could be involved in keratinocyte differentiation, but apparently not through interaction with galectin-3.


Keratinocytes Lysosomes Lamp-1 Galectins Culture confluence 



The authors are grateful to Prof. M. Jadot for his helpful ideas and critical remarks. The technical assistance provided by R. Deom, F. Dubois and F. Herphelin is gratefully acknowledged. We thank Dr B. Bienfait (Namur-Bouge) for providing skin specimens. Special thanks are addressed to Drs D.R. Roop (Houston), R.L. Eckert (Cleveland) and F. Van Den Brûle (Liège) for their gift of keratins, involucrin and galectins cDNA probes, respectively.


  1. 1.
    Akimoto Y, Hirabayashi J, Kasai K, Hirano H (1995) Expression of the endogenous 14-kDa beta-galactoside-binding lectin galectin in normal human skin. Cell Tissue Res 280:1–10PubMedCrossRefGoogle Scholar
  2. 2.
    Barret AJ, Kirschke H (1981) Cathepsin B, cathepsin H and cathepsin L. Methods Enzymol 80:535–561PubMedCrossRefGoogle Scholar
  3. 3.
    Brown R, Wu WW, Bernstein IA (1987) Changes in lectin binding by differentiating cutaneous keratinocytes from the newborn rat. J Invest Dermatol 88:719–726CrossRefPubMedGoogle Scholar
  4. 4.
    Brysk MM, Snider JM (1982) The effect of the state of differentiation on labeling of epidermal cell surface glycoproteins. J Invest Dermatol 78:366–370CrossRefPubMedGoogle Scholar
  5. 5.
    Cella N, Cornejo-Uribe RR, Montes GS, Hynes NE, Chammas R (1996) The lysosomal-associated membrane protein Lamp-1 is a novel differentiation marker for HC11 mouse mammary epithelial cells. Differentiation 61:113–120CrossRefPubMedGoogle Scholar
  6. 6.
    Dagher SF, Wang JL, Patterson RJ (1995) Identification of galectin-3 as a factor in pre-mRNA splicing. Proc Natl Acad Sci USA 92:1213–1217PubMedCrossRefGoogle Scholar
  7. 7.
    de Duve C, Pressman BC, Gianetto R, Wattiaux R, Appelmans F (1955) Tissue fractionation studies. 6. Intracellular distribution pattern of enzymes in rat liver tissue. Biochem J 60:604–617PubMedGoogle Scholar
  8. 8.
    Delacour D, Cramm-Behrens CI, Drobecq H, Le Bivic A, Naim HY, Jacob R (2006) Requirement for galectin-3 in apical protein sorting. Curr Biol 16:408–414CrossRefPubMedGoogle Scholar
  9. 9.
    de Stefanis D, Demoz M, Dragonetti A, Houri JJ, Ogier-Denis E, Codogno P, Baccino FM, Isidoro C (1997) Differentiation-induced changes in the content, secretion, and subcellular distribution of lysosomal cathepsins in the human colon cancer HT-29 cell line. Cell Tissue Res 289:109–117CrossRefPubMedGoogle Scholar
  10. 10.
    Dumic J, Lauc G, Hadzija M, Flogel M (2000) Transfer to in vitro conditions influences expression and intracellular distribution of galectin-3 in murine peritoneal macrophages. Z Naturforsch 55:261–266Google Scholar
  11. 11.
    Eckert RL, Green H (1986) Structure and evolution of the human involucrin gene. Cell 46:583–589PubMedCrossRefGoogle Scholar
  12. 12.
    Eckert RL, Crish JF, Banks EB, Welter JF (1997) The epidermis: genes on—genes off. J Invest Dermatol 109:501–509CrossRefPubMedGoogle Scholar
  13. 13.
    Esposito C, Caputo I (2005) Mammalian transglutaminases. Identification of substrates as a key to physiological function and physiopathological relevance. FEBS J 272:615–631CrossRefPubMedGoogle Scholar
  14. 14.
    Freinkel RK, Traczyk TN (1985) Lipid composition and acid hydrolase content of lamellar granules of fetal rat epidermis. J Invest Dermatol 85:295–298CrossRefPubMedGoogle Scholar
  15. 15.
    Fuchs E (1993) Epidermal differentiation and keratin gene expression. J Cell Sci Suppl 17:197–208PubMedGoogle Scholar
  16. 16.
    Glombitza GJ, Becker E, Kaiser HW, Sandhoff K (1997) Biosynthesis, processing, and intracellular transport of GM2 activator protein in human epidermal keratinocytes. J Biol Chem 272:5199–5207PubMedCrossRefGoogle Scholar
  17. 17.
    Goletz S, Hanisch F-G, Karsten U (1997) Novel αGalNAc containing glycans on cytokeratins are recognized in vitro by galectins with type II carbohydrate recognition domains. J Cell Sci 110:1585–1596PubMedGoogle Scholar
  18. 18.
    Grayson S, Johnson-Winegar AG, Wintroub BU, Isseroff RR, Epstein EH Jr, Elias PM (1985) Lamellar body-enriched fractions from neonatal mice: preparative techniques and partial characterization. J Invest Dermatol 85:289–294CrossRefPubMedGoogle Scholar
  19. 19.
    Hertle MD, Kubler MD, Leigh IM, Watt FM (1992) Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis. J Clin Invest 89:1892–1901PubMedCrossRefGoogle Scholar
  20. 20.
    Hotchin NA, Gandarillas A, Watt FM (1995) Regulation of cell surface β1 integrin levels during keratinocyte terminal differentiation. J Cell Biol 128:1209–1219CrossRefPubMedGoogle Scholar
  21. 21.
    Horikoshi T, Arany I, Rajaraman S, Chen SH, Brysk H, Lei G, Tyring SK, Brysk MM (1998) Isoforms of cathepsin D and human epidermal differentiation. Biochimie 80:605–612CrossRefPubMedGoogle Scholar
  22. 22.
    Jadot M, Wattiaux-De Coninck S, Wattiaux R (1985) Effect on lysosomes of invertase endocytosed by rat liver. Eur J Biochem 151:485–488CrossRefPubMedGoogle Scholar
  23. 23.
    Jans R, Sartor M, Jadot M, Poumay Y (2004) Calcium entry into keratinocytes induces exocytosis of lysosomes. Arch Dermatol Res 296:30–41CrossRefPubMedGoogle Scholar
  24. 24.
    Kanan K, Stewart RM, Bounds W, Carlsson SR, Fukuda M, Betzing KW, Holcombe RF (1996) Lysosome-associated membrane proteins h-Lamp-1 (CD107a) and h-Lamp-2 (CD107b) are activation-dependent cell surface glycoproteins in human peripheral blood mononuclear cells, which mediate cell adhesion to vascular endothelium. Cell Immunol 171:10–19CrossRefPubMedGoogle Scholar
  25. 25.
    Kolly C, Suter MM, Müller EJ (2005) Proliferation, cell cycle exit, and onset of terminal differentiation in cultured keratinocytes: pre-programmed pathways in control of c-Myc and Notch1 prevail over extracellular calcium signals. J Invest Dermatol 124:1014–1025CrossRefPubMedGoogle Scholar
  26. 26.
    Krzeslak A, Lipinska A (2004) Galectin-3 as a multifunctional protein. Cell Mol Biol Lett 9:305–328PubMedGoogle Scholar
  27. 27.
    Laborda J (1991) 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO. Nucleic Acids Res 19:3998PubMedCrossRefGoogle Scholar
  28. 28.
    Madison KC, Sando GN, Howard EJ, True CA, Gilbert D, Swartzendruber DC, Wertz PW (1998) Lamellar granule biogenesis: a role for ceramide glucosyltransferase, lysosomal enzyme transport, and the Golgi. J Investig Dermatol Symp Proc 3:80–86PubMedGoogle Scholar
  29. 29.
    Magnaldo T, Bernerd F, Darmon M (1995) Galectin-7, a human 14-kD S-lectin, specifically expressed in keratinocytes and sensitive to retinoic acid. Dev Biol 168:259–271CrossRefPubMedGoogle Scholar
  30. 30.
    Nangia-Makker P, Conklin J, Hogan V, Raz A (2002) Carbohydrate-binding proteins in cancer, and their ligands as therapeutic agents. Trends Mol Med 8:187–192CrossRefPubMedGoogle Scholar
  31. 31.
    Ochieng J, Leite-Browning ML, Warfield P (1998) Regulation of cellular adhesion to extracellular matrix proteins by galectin-3. Biochem Biophys Res Commun 246:788–791CrossRefPubMedGoogle Scholar
  32. 32.
    Orlow SJ (1995) Melanosomes are specialized members of the lysosomal lineage of organelles. J Invest Dermatol 105:3–7CrossRefPubMedGoogle Scholar
  33. 33.
    Perillo NL, Marcus ME, Baum LG (1998) Galectins: versatile modulators of cell adhesion, cell proliferation, and cell death. J Mol Med 76:402–412CrossRefPubMedGoogle Scholar
  34. 34.
    Peters TJ, Müller M, de Duve C (1972) Lysosomes of the arterial wall. J Exp Med 136:1117–1139CrossRefPubMedGoogle Scholar
  35. 35.
    Poumay Y, Pittelkow MR (1995) Cell density and culture factors regulate keratinocyte commitment to differentition and expression of suprabasal K1/K10 keratins. J Invest Dermatol 104:271–276CrossRefPubMedGoogle Scholar
  36. 36.
    Poumay Y, Herphelin F, Smits P, De Potter I, Pittelkow MR (1999) High-cell-density phorbol ester and retinoic acid upregulate involucrin and downregulate suprabasal keratin 10 in autocrine cultures of human epidermal keratinocytes. Mol Cell Biol Res Commun 2:138–144CrossRefPubMedGoogle Scholar
  37. 37.
    Rabinovich GA, Baum LG, Tinari N, Paganelli R, Natoli C, Liu FT, Iacobelli S (2002) Galectins and their ligands: amplifiers, silencers or tuners of the inflammatory response? Trends Immunol 23:313–320CrossRefPubMedGoogle Scholar
  38. 38.
    Roop DR, Krieg TM, Mehrel T, Cheng CK, Yuspa SH (1988) Transcriptional control of high molecular weight keratin gene expression in multistage mouse skin carcinogenesis. Cancer Res 48:3245–3252PubMedGoogle Scholar
  39. 39.
    Saitoh O, Wang WC, Lottan R, Fukuda M (1992) Differential glycosylation and cell surface expression of lysosomal membrane glycoproteins in sublines of a human colon cancer exhibiting distinct metastatic potentials. J Biol Chem 267:5700–5711PubMedGoogle Scholar
  40. 40.
    Sando GN, Zhu H, Weis JM, Richman JT, Wertz PW, Madison KC (2003) Caveolin expression and localization in human keratinocytes suggest a role in lamellar granule biogenesis. J Invest Dermatol 120:531–541CrossRefPubMedGoogle Scholar
  41. 41.
    Sarafian V, Jadot M, Foidart JM, Letesson JJ, van den Brule F, Castronovo V, Wattiaux R, Wattiaux-De Coninck S (1998) Expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in human tumor cells. Int J Cancer 75:105–111CrossRefPubMedGoogle Scholar
  42. 42.
    Sawada R, Jardine KA, Fukuda M (1993) The genes of major lysosomal membrane glycoproteins, lamp-1 and lamp-2. Flanking sequence of lamp-2 gene and comparison of exon organization in two genes. J Biol Chem 268:9014–9022PubMedGoogle Scholar
  43. 43.
    Smits P, Poumay Y, Karperien M, Tylzanowski P, Wauters J, Huylebroeck D, Ponec M, Merregaert J (2000) Differentiation-dependent alternative splicing and expression of the extracellular matrix protein 1 gene in human keratinocytes. J Invest Dermatol 114:718–724CrossRefPubMedGoogle Scholar
  44. 44.
    Szebenyi G, Rotwein P (1991) Differential regulation of mannose 6-phosphate receptors and their ligands during the myogenic development of C2 cells. J Biol Chem 266:5534–5539PubMedGoogle Scholar
  45. 45.
    Tanabe H, Kumagai N, Tsukahara T, Ishiura S, Kominami E, Nishina H, Sugita H (1991) Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation. Biochim Biophys Acta 1094:281–287CrossRefPubMedGoogle Scholar
  46. 46.
    Tobin DJ, Foitzik K, Reinheckel T, Mecklenburg L, Botchkarev VA, Peters C, Paus R (2002) The lysosomal protease cathepsin L is an important regulator of keratinocyte and melanocyte differentiation during hair follicle morphogenesis and cycling. Am J Pathol 160:1807–1821PubMedGoogle Scholar
  47. 47.
    Vaes G (1966) Subcellular localization of glycosidases in lysosomes. Methods Enzymol 8:509–514Google Scholar
  48. 48.
    van den Brûle FA, Buicu C, Baldet M, Sobel ME, Cooper DN, Marschal P, Castronovo V (1995) Galectin-1 modulates human melanoma cell adhesion to laminin. Biochem Biophys Res Commun 209:760–767CrossRefPubMedGoogle Scholar
  49. 49.
    van den Brûle FA, Fernandez PL, Buicu C, Liu FT, Jackers P, Lambotte R, Castronovo V (1997) Differential expression of galectin-1 and galectin-3 during first trimester human embryogenesis. Dev Dyn 209:399–405CrossRefPubMedGoogle Scholar
  50. 50.
    van den Brûle F, Waltregny DW, Liu FT, Castronovo V (2000) Alteration of the cytoplasmic/nuclear expression pattern of galectin-3 correlates with prostate carcinoma progression. Int J Cancer 89:361–367CrossRefPubMedGoogle Scholar
  51. 51.
    van der Zee J, Dubbelman TMAR, Raap TK, Van Steveninck J (1987) Toxic effects of ozone on murine L929 fibroblasts. Biochem J 242:707–712PubMedGoogle Scholar
  52. 52.
    Watt FM (1989) Terminal differentiation of epidermal keratinocytes. Curr Opin Cell Biol 1:1107–1115PubMedCrossRefGoogle Scholar
  53. 53.
    Welss T, Sun J, Irving JA, Blum R, Smith AI, Whisstock JC, Pike RN, von Mikecz A, Ruzicka T, Bird PI, Abts HF (2003) Hurpin is a selective inhibitor of lysosomal cathepsin L and protects keratinocytes from ultraviolet-induced apoptosis. Biochemistry 42:7381–7389CrossRefPubMedGoogle Scholar
  54. 54.
    Wille JJ Jr, Pittelkow MR, Shipley GD, Scott RE (1984) Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics, and cell cycle studies. J Cell Physiol 121:31–44CrossRefPubMedGoogle Scholar
  55. 55.
    Wollenberg A, de la Salle H, Hanau D, Liu FT, Bieber T (1993) Human keratinocytes release the endogenous beta-galactoside-binding soluble lectin immunoglobulin E (IgE-binding protein) which binds to Langerhans cells where it modulates their binding capacity for IgE glycoforms. J Exp Med 178:777–785CrossRefPubMedGoogle Scholar
  56. 56.
    Youakim A, Romero PA, Yee K, Carlsson SR, Fukuda M, Herscovics A (1989) Decrease in polylactosaminoglycans associated with lysosomal membrane glycoproteins during differentiation of CaCo-2 human colonic adenocarcinoma cells. Cancer Res 49:6889–6895PubMedGoogle Scholar
  57. 57.
    Zhang Y, Wick DA, Haas AL, Seetharam B, Dahms NM (1995) Regulation of lysosomal and ubiquitin degradative pathways in differentiating human intestinal Caco-2 cells. Biochim Biophys Acta 1267:15–24CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  1. 1.Department of Histologie-EmbryologieFacultés Universitaires Notre-Dame de la PaixNamurBelgium
  2. 2.Department of BiologyMedical University-PlovdivPlovdivBulgaria

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