ERDAL BALCAN, Özlem ARSLAN, Ayça GÜMÜŞ, Mesut ŞAHİN

Tunikamisinin embriyonik ve yenidoğan fare dalak dokularına etkisi

Tunikamisin proteinlerdeki asparagin gruplarının N-glikozilasyonunu inhibe etme yeteneği ve endoplazmik retikulum stresini arttırması nedeniyle hücre biyolojisi çalışmalarında yoğun olarak kullanılan bir antibiyotiktir. Bu çalışmada tunikamisinin embriyonik hayatın 17. günü ile 1. ve 3. postnatal günlere ait fare dalak dokularındaki etkileri, 1) glikozaminoglikan moleküllerindeki değişiklikler, 2) apoptozis ve 3) laminin moleküllerindeki değişiklikler olmak üzere 3 yapısal ve fizyolojik parametre ile araştırılmıştır. Alcian blueperiyodik asit-Schiff metodu ile kontrol grubuna ait dalak parankiminin karboksillenmiş glikozaminoglikanları içerdiği, ancak bu moleküllerin tunikamisin uygulanan tüm gruplarda hafifçe azaldığı belirlenmiştir. Öte yandan, tunikamisin uygulanan gruplarda hücreler arası alanların genişlediği görülmüştür. Ek olarak, immunoblotting analizler ile gelişen dalak dokularında laminin moleküllerinin ekspresyonlarının tunikamisinin uygulamasıyla azaldığı belirlenmiştir. Tunikamisinin apoptotik etkilerini belirlemek için TUNEL yöntemi kullanılmış ve gelişen dalak dokularında apoptotik hücre sayısının tunikamisin uygulaması ile büyük ölçüde arttığı görülmüştür. Bu bulgular, hücre-ekstrasellüler matriks etkileşimlerini düzenleyen hücre dışı proteinlerin (örneğin lamininlerin) glikozilasyon durumunun ve ekstrasellüler matriksin glikozaminoglikan içeriğindeki değişimlerin, dalak gelişimi sürecinde hücre fizyolojisini ve morfogenezini belirleyen son derece önemli faktörler olduğunu göstermiştir.

Anahtar Kelimeler:

yenidoğan hayvanlar, tunikamisin, dalak, fareler, laminin, immunoblatlama, glikozaminoglikan, glikoprotein, programlanmış hücre ölümü, antibiyotikler

The effect of Tunicamycin on embryonic and newborn murine spleen tissues

Tunicamycin is an antibiotic that widely used in cell biology for its ability to inhibit N-linked glycosylation of asparagine residues on proteins and to induce endoplasmic reticulum stress. In the present study, the effects of tunicamycin on murine splenic tissues at 17th embryonic day and 1st and 3rd postnatal days were evaluated with three structural and physiological parameters: 1) alterations in glycosaminoglycan molecules, 2) apoptosis and 3) alterations in laminin molecules. It was shown that splenic microenvironments of control groups contain carboxylated glycosaminoglycans, but their content slightly decreased in all tunicamycin treated groups by alcian blue-periodic acid-Schiff method. On the other hand, there was an increase in the interstitial space among the cells of tunicamycin treated groups. In addition, it was shown by immunoblotting analyses that expression levels of laminin molecules were decreased by tunicamycin tratment in developing spleen tissues. In order to determine apoptotic effects of tunicamycin, TUNEL assay was performed and it was found that the number of apoptotic cells in developing spleen was strongly increased with tunicamycin treatments. These results suggest that, during the spleen development, the alterations of glycosaminoglycan contents in the extracellular matrix and the glycosylation status of extracellular glycoproteins (e.g. laminins) that mediate cell-extracellular matrix interactions are very important factors that seal the fate of cell physiology and morphogenesis.

Keywords:

newborn animals, tunicamycin, spleen, mice, laminins, immunoblotting, glycosaminoglycans, glycoproteins, apoptosis, antibiotics,

PDF

___

1.Damsky C, Sutherland A, Fisher S: Extracellular matrix 5: adhesive interactions in early mammalian embryogenesis, implantation, and placentation. FASEB J, 7 (14): 1320-1329, 1993.
2.Aumailley M, Gayraud B: Structure and biological activity of the extracellular matrix. J Mol Med, 76 (3-4): 253-265, 1998.
3.Daley WP, Peters SB, Larsen M: Extracellular matrix dynamics in development and regenerative medicine. J Cell Sci, 121, 255-264, 2008.
4.Kleinman HK, Philp D, Hoffman MP: Role of the extracellular matrix in morphogenesis. Curr Opin Biotechnol, 14 (5): 526-532, 2003.
5.Streuli C: Extracellular matrix remodelling and cellular differentiation. Curr Opin Cell Biol, 11 (5): 634-640, 1999.
6.Hall H, Schachner M: Laminins and their ligands: involvement of carbohydrates in formation of the extracellular matrix and in cell adhesion. Trends Glycosci Glycotechnol, 10 (55): 361-382, 1998.
7.Timpl R, Rohde H, Robey PG, Rennard SI, Foidart JM, Martin GR: Laminin-a glycoprotein from basement membranes. J Biol Chem, 254 (19): 9933-9937, 1979.
8.Dean JW 3rd, Chandrasekaran S, Tanzer ML: A biological role of the carbohydrate moieties of laminin. J Biol Chem, 265 (21): 12553-12562, 1990.
9.Mercurio AM: Laminin receptors: Achieving specificity through cooperation. Trends Cell Biol, 5 (11): 419-423 1995.
10.Colognato H, Yurchenco PD: Form and function: the laminin family of heterotrimers. Dev Dyn, 218 (2): 213-234, 2000.
11.Davies JA, Fisher CE, Barnett MW: Glycosaminoglycans in the study of mammalian organ development. Biochem Soc Trans, 29, 166-171, 2001.
12.Hardingham TE, Fosang AJ: Proteoglycans: Many forms and many functions. FASEB J, 6 (3): 861-870, 1992.
13.Yanagishita M: Function of proteoglycans in the extracellular matrix. Acta Pathol Jpn, 43 (6): 283-293, 1993
14.Har-el R, Tanzer ML: Extracellular matrix. 3: Evolution of the extracellular matrix in invertebrates. FASEB J, 7 (12): 1115-1123, 1993.
15.Harrisson F: The extracellular matrix and cell surface, mediators of cell interactions in chicken gastrulation. Int J Dev Biol, 33 (4): 417-438, 1989.
16.Zagris N: Extracellular matrix in development of the early embryo. Micron, 32 (4): 427-438, 2001.
17.Kolset SO, Gallagher JT: Proteoglycans in haemopoietic cells. Biochim Biophys Acta, 1032 (2-3): 191-211, 1990.
18.Kolset SO, Prydz K, Pejler G: Intracellular proteoglycans. Biochem J, 379, 217-227, 2004.
19.Iozzo RV, Murdoch AD: Proteoglycans of the extracellular environment: Clues from the gene and protein side offer novel perspectives in molecular diversity and function. FASEB J, 10 (5): 598-614, 1996.
20.Jalkanen M, Rapraeger A, Bernfield M: Mouse mammary epithelial cells produce basement membrane and cell surface heparan sulfate proteoglycans containing distinct core proteins. J Cell Biol, 106 (3): 953-962, 1988.
21.Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P: Cell junctions, cell adhesions, and the extracellular matrix. In, Gibbs S (Ed): Molecular Biology of the Cell. 5th ed. pp. 11311204, Garland Science, New York, 2008.
22.Prydz K, Dalen KT: Synthesis and sorting of proteoglycans. J Cell Sci, 113, 193-205, 2000.
23.Lehninger AL, Nelson DL, Cox MM: Carbohydrates. In, Nelson DL, Cox MM (Eds): Principles of Biochemistry. 2nd ed. pp. 298-323, Worth Publishers, New York, 1993.
24.Timar J, Jeney A, Kovalszky I I, Kopper L: Role of Proteoglycans in Tumor Progression. Pathol Oncol Res, 1 (1): 85-93, 1995.
25.Toole BP: Hyaluronan in morphogenesis. Semin Cell Dev Biol, 12 (2): 79-87, 2001.
26.King IA, Tabiowo A: Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro. Biochem J, 198 (2): 331-338, 1981.
27.Takatsuki A, Kohno K, Tamura G: Inhibition of biosynthesis of polyisoprenol sugars in chick embryo microsomes by tunicamycin. Agric Biol Chem, 39, 20892091, 1975.
28.Tkacz JS, Lampen O: Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem Biophys Res Commun, 65, 248-257, 1975.
29.Lehle L, Tanner W: The specific site of tunicamycin inhibition in the formation of dolichol-bound Nacetylglucosamine derivatives. FEBS Lett, 72 (1): 167-170, 1976.
30.Tsvetanova BC, Kiemle DJ, Price NP: Biosynthesis of tunicamycin and metabolic origin of the 11-carbon dialdose sugar, tunicamine. J Biol Chem, 277 (38): 35289-35296, 2002.
31.Scott JE, Dorling J: Differential staining of acid glycosaminoglycans (mucopolysaccharides) by alcian blue in salt solutions. Histochimie, 5 (3): 221-233, 1965.
32.Mowry RW: The special value of methods that color both acidic and vicinal hydroxyl groups in the histochemical study of mucins. With revised directions for the colloidal iron stain, the use of alcian blue 8GX, and their combination with the periodic-acid-Schiff reaction. Ann N Y Acad Sci, 106, 402423, 1963.
33.Spicer SS, Horn RG, Leppi TJ: Histochemistry of connective tissue mucopolysaccharides. In, Wagner BM, Smith DE (Eds): The Connective Tissue. pp. 251-303, Williams and Wilkins Co, Baltimore, 1967.
34.Yamabayashi S: Periodic acid-Schiff-alcian blue: A method for the differential staining of glycoproteins. Histochem J, 19 (10-11): 565-571, 1987.
35.Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227 (5259): 680-685, 1970.
36.Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA, 76 (9): 4350-4354, 1979.
37.Takagaki K, Tazawa T, Munakata H, Nakamura T, Endo M: Characterization of beta-D-xyloside-initiated glycosaminoglycan synthesized by human skin fibroblasts in the presence of tunicamycin. Glycoconj J, 15 (5): 483-489, 1998.
38.Hart GW, Lennarz WJ: Effects of tunicamycin on the biosynthesis of glycosaminoglycans by embryonic chick cornea. J Biol Chem, 253 (16): 5795-5801, 1978.
39.Straus AH, Nader HB, Dietrich CP: Absence of heparin or heparin-like compounds in mast-cell-free tissues and animals. Biochim Biophys Acta, 717 (3): 478-485, 1982.
40.Liakka A, Apaja-Sarkkinen M, Karttunen T, Autio-Harmainen H: Distribution of laminin and types IV and III collagen in fetal, infant and adult human spleens. Cell Tissue Res, 263 (2): 245-252, 1991.
41.van den Berg TK, van der Ende M, Döpp EA, Kraal G, Dijkstra CD: Localization of beta 1 integrins and their extracellular ligands in human lymphoid tissues. Am J Pathol, 143 (4): 1098-1110, 1993.
42.Liakka KA: The integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha V, beta 1 and beta 3 in fetal, infant and adult human spleen as detected by immunohistochemistry. Differentiation, 56 (3): 183-190, 1994.
43.Jaspars LH, De Melker AA, Bonnet P, Sonnenberg A, Meijer CJ: Distribution of laminin variants and their integrin receptors in human secondary lymphoid tissue. Colocalization suggests that the alpha 6 beta 4-integrin is a receptor for laminin-5 in lymphoid follicles. Cell Adhes Commun, 4 (4-5): 269-279, 1996.
44.Smyth N, Vatansever HS, Murray P, Meyer M, Frie C, Paulsson M, Edgar D: Absence of basement membranes after targeting the LAMC1 gene results in embryonic lethality due to failure of endoderm differentiation. J Cell Biol, 144 (1): 151-160, 1999.
45.Miner JH, Li C, Mudd JL, Go G, Sutherland AE: Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation. Development, 131 (10): 2247-2256, 2004.
46. Guo LT, Zhang XU, Kuang W, Xu H, Liu LA, Vilquin JT, Miyagoe-Suzuki Y, Takeda S, Ruegg MA, Wewer UM, Engvall E: Laminin alpha2 deficiency and muscular dystrophy; genotype-phenotype correlation in mutant mice. Neuromuscul Disord, 13 (3): 207-215, 2003.
47. Määttä M, Liakka A, Salo S, Tasanen K, Bruckner-Tuderman L, Autio-Harmainen H: Differential expression of basement membrane components in lymphatic tissues. J Histochem Cytochem, 52 (8): 1073-1081, 2004.
48.ten Dam GB, Hafmans T, Veerkamp JH, van Kuppevelt TH: Differential expression of heparan sulfate domains in rat spleen. J Histochem Cytochem, 51 (6): 727-739, 2003.
49.Lokmic Z, Lämmermann T, Sixt M, Cardell S, Hallmann R, Sorokin L: The extracellular matrix of the spleen as a potential organizer of immune cell compartments. Semin Immunol, 20 (1): 4-13, 2008.
50.Morohashi K, Tsuboi-Asai H, Matsushita S, Suda M, Nakashima M, Sasano H, Hataba Y, Li CL, Fukata J, Irie J, Watanabe T, Nagura H, Li E: Structural and functional abnormalities in the spleen of an mFtz-F1 gene-disrupted mouse. Blood, 93 (5): 1586-94, 1999.
51.Howe CC: Functional role of laminin carbohydrate. Mol Cell Biol, 4 (1): 1-7, 1984.
52.Tiganis T, Leaver DD, Ham K, Friedhuber A, Stewart P, Dziadek M: Functional and morphological changes induced by tunicamycin in dividing and confluent endothelial cells. Exp Cell Res, 198 (2): 191-200, 1992.
53.Reed JC: Mechanisms of apoptosis. Am J Pathol, 157 (5): 1415-1430, 2000.
54.Raff MC: Social controls of cell survival and cell death. Nature, 356 (6368): 397-400, 1992.
55.Vaux DL, Strasser A: The molecular biology of apoptosis. Proc Natl Acad Sci U S A, 93 (6): 2239-2244, 1996.
56.Renehan AG, Booth C, Potten CS: What is apoptosis, and why is it important? BMJ, 322 (7301): 1536-1538, 2001.
57.Maag RS, Hicks SW, Machamer CE: Death from within: apoptosis and the secretory pathway. Curr Opin Cell Biol, 15 (14): 456-461, 2003.
58.Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T, Korsmeyer SJ: BAX and BAK regulation of endoplasmic reticulum Ca2+: A control point for apoptosis. Sci, 300 (5616): 135-139, 2003.
59.Nakagawa T, Yuan J: Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J Cell Biol, 150 (4): 887-94, 2000.
60.Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, Yuan J: Caspase-12 mediates endoplasmic-reticulumspecific apoptosis and cytotoxicity by amyloid-beta. Nature, 403 (6765): 98-103, 2000.
61.Cheung HH, Lynn Kelly N, Liston P, Korneluk RG: Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: A role for the IAPs. Exp Cell Res, 312 (12): 2347-2357, 2006.
62. Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D: Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell, 5 (5): 897-904, 2000.
63.Walker BK, Lei H, Krag SS: A functional link between N-linked glycosylation and apoptosis in Chinese hamster ovary cells. Biochem Biophys Res Commun, 250 (2): 264-270, 1998.
64.Yoshimi M, Sekiguchi T, Hara N, Nishimoto T: Inhibition of N-linked glycosylation causes apoptosis in hamster BHK21 cells. Biochem Biophys Res Commun, 276 (3): 965-969, 2000.
65.Frisch SM, Francis H: Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol, 124 (4): 619-626, 1994.
66.Khwaja A, Rodriguez-Viciana P, Wennström S, Warne PH, Downward J: Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J, 16 (10): 2783-2793, 1997.
67.Frisch SM, Ruoslahti E: Integrins and anoikis. Curr Opin Cell Biol, 9 (5): 701-706, 1997.
68.Miner JH, Yurchenco PD: Laminin functions in tissue morphogenesis. Annu Rev Cell Dev Biol, 20, 255-284, 2004.
69.Vachon PH, Loechel F, Xu H, Wewer UM, Engvall E: Merosin and laminin in myogenesis; specific requirement for merosin in myotube stability and survival. J Cell Biol, 134 (6): 1483-1497, 1996.
70.Talhouk RS, Bissell MJ, Werb Z: Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution. J Cell Biol, 118 (5): 1271-1282, 1992.
71.Boudreau N, Sympson CJ, Werb Z, Bissell MJ: Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Sci, 267 (5199): 891-893, 1995.
72. Jones PL, Boudreau N, Myers CA, Erickson HP, Bissell MJ: Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments. J Cell Sci, 108: 519-527, 1995.
73.Lund LR, Rømer J, Thomasset N, Solberg H, Pyke C, Bissell MJ, Danø K, Werb Z: Two distinct phases of apoptosis in mammary gland involution: Proteinase-independent and dependent pathways. Development, 122 (1): 181-193, 1996.
74.Faraldo MM, Deugnier MA, Lukashev M, Thiery JP, Glukhova MA: Perturbation of beta1-integrin function alters the development of murine mammary gland. EMBO J, 17 (8): 2139-2147, 1998.
75.Hansen RK, Bissell MJ: Tissue architecture and breast cancer: The role of extracellular matrix and steroid hormones. Endocr Relat Cancer, 7 (2): 95-113, 2000.