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系統識別號 U0026-1107201311005900
論文名稱(中文) 第四型鉀氯離子共同輸送體在腫瘤轉移上所扮演的角色
論文名稱(英文) The potential role of potassium chloride cotransporter 4 in tumor metastasis
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 101
學期 2
出版年 102
研究生(中文) 翁子玉
研究生(英文) Tzu-Yu Weng
學號 s58951358
學位類別 博士
語文別 英文
論文頁數 78頁
口試委員 指導教授-周振陽
口試委員-劉校生
召集委員-鄭宏祺
口試委員-沈孟儒
口試委員-張榮賢
口試委員-戴明泓
中文關鍵字 第四型鉀氯離子共同輸送體  N-linked 醣基化作用  醣基複雜度  細胞膜表現  腫瘤形成  腫瘤轉移  纖維連接蛋白 
英文關鍵字 KCC4  N-linked glycosylation  glycan complexity  membrane expression  tumor formation  tumor metastasis  fibronectin 
學科別分類
中文摘要 鉀氯離子共同輸送體 (Potassium chloride cotransporter, KCC) 對細胞體積,離子通透和維持細胞內氯離子濃度的調控是必須的。有新的證據顯示,KCCs在腫瘤生物學也扮演一定的角色,尤其是第三型鉀氯離子共同輸送體(KCC3)和第四型鉀氯離子共同輸送體(KCC4)。 KCC3曾被報導對細胞生長有關,KCC4則是對癌細胞的侵犯有關。在臨床的檢體中發現,KCC4和癌症轉移有相關性,而且其運輸到lamellipodium對細胞侵犯是有關的。在此,我們利用醣基化突變的KCC4,探討KCC4在腫瘤轉移中扮演的角色,並利用高度轉移的細胞株,進一步研究KCC4在其中參與的機制。醣基化現象會調控許多重要的功能,如細胞貼附,蛋白質摺疊,運輸和細胞膜表現。然而,KCC4的醣基化現象尚未被探討。我們利用點突變的方式去探討KCC4外圍四個N-linked醣基化位置,對KCC4半衰期,細胞膜表現,離子通透力及腫瘤形成能力是否有所影響。我們發現N-linked的三點突變 (N312/331/344/Q),和四點突變 (N312/331/344/360/Q) 會破壞KCC4的N-linked醣基化作用,並使得大部分的KCC4堆積在內質網,而不是細胞膜。我們進一步證實,突變中央的兩個醣基分支會抑制KCC4運輸到細胞膜。結構分析顯示, N331 和N344 位置是 Endo H-resistant,複雜型的醣基結構; 而N312 和 N360位置,是Endo H-sensitive,富含多量甘露醣的結構。非膜表現型的醣基化突變KCC4,在低張溶液刺激之下對氯離子濃度的平衡,體積回復的能力都有所降低。在動物實驗中,也看到腫瘤形成和肺部colonization的能力下降。 醣基化作用的複雜度,可能導致不同的醣基分支對KCC4的活性有不同貢獻度。因此,我們推測醣基化作用對KCC4的膜表現,蛋白質穩定度和生物活性是很重要的。檢驗不同轉移程度的細胞株後發現,KCC4在轉移能力強的4T1表現量比其低轉移性細胞株67NR高。我們初步的結果也顯示KCC4可以去調控纖維連接蛋白 (fibronectin) 的表現和組裝,而纖維連接蛋白對貼附到標的器官的轉移是很重要的。未來會進一步探討KCC4可能導致轉移的機制,以及其可能參與在轉移的哪一步驟中。
英文摘要 Potassium chloride cotransporter (KCC) is necessary for the regulation of cell volume, the transepithelial transport of ions, and the maintenance of intracellular Cl- concentration. Emerging evidence suggests KCCs, especially the isoforms KCC3 and KCC4 which are differentially required for cancer cell proliferation and invasiveness, also play roles in the tumor biology. The correlation of KCC4 with metastasis has been demonstrated in clinical specimen and KCC4 trafficking to lamellipodium is important for cell invasion. We used glycosylation mutation to test the role of KCC4 in metastasis and investigated the potential mechanism that could be involved by silencing KCC4 expression in highly metastatic cell. Glycosylation plays a role in regulating many biological activities, including protein folding and cell surface expression of biomolecules. However, the importance of glycosylation for KCC4 function has not previously been demonstrated. Site-directed mutagenesis was performed on the four putative extracellular N-linked glycosylation sites of KCC4 to determine the role of these sites in KCC4 half-life, cell surface expression, and transporter activity, as well as in KCC4-dependent tumor formation. We showed that triple (N312/331/344/Q) and quadruple (N312/331/344/360/Q) mutations of N-linked glycosylation sites disrupt the N-linked glycosylation of KCC4, resulting in the accumulation of KCC4, predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Further investigation indicated that mutations of the central two (N331/344/Q) N-linked glycosylation sites inhibit the membrane trafficking of KCC4. Our data suggest that the glycan moieties at the N331 and N344 sites were Endo H-resistant, complex-form structures, and that the N312 and N360 sites were Endo H-sensitive, high mannose-containing structures. Under hypotonic stress conditions, the ability to adapt to changes in intracellular chloride ion concentrations and RVD (regulatory volume decrease) activities were less efficient in cells containing the deglycosylated form of KCC4 that were not expressed at the cell surface. Deglycosylated forms of KCC4 also demonstrated decreased tumor formation and lung colonization in mouse xenografts. The difference in glycan complexity may account for the differential impact of each branch on the biological effects of KCC4. We propose that glycosylation is essential for the surface expression, stabilization, and bioactivity of KCC4. By using cell pairs that have different metastatic potential, we found that KCC4 expressed higher in cells with greater metastatic potential 4T1 cell compared with the low malignant counterpart 67NR cell. Our preliminary data showed that KCC4 also regulates fibronectin expression and assembly which is an important step that mediates cancer cells to arrest in the target organ. Further effects will be focused on the potential mechanism of KCC4-mediated metastasis and what metastasis step KCC4 may contribute to.
論文目次 Abstract ..................................................I
中文摘要..................................................III
致謝.......................................................V
Table of Contents ........................................VI
Table List ...............................................IX
Figure List ...............................................X
AAbbreviations ..........................................XII
I. Introduction ..................................... 1
1. Potassium chloride cotransporters................. 1
Characterization of KCCs.................................. 1
Related functions and disorders of KCCs................... 2
The role of KCCs in tumor biology......................... 3
2. Glycosylation .................................... 4
N-linked glycosylation.................................... 5
Functions of glycosylation.................................5
Glycosylation and tumor................................... 6
3. Metastasis and fibronectin........................ 6
II. Objective and Specific aims....................... 9
III. Materials and Methods............................ 10
1. Cell culture and reagents........................ 10
2. Plasmid constructs............................... 10
3. Site-directed mutagenesis........................ 11
4. Glycosidase assay................................ 12
5. Western blotting................................. 12
6. Immunofluorescence............................... 13
7. Cell surface biotinylation....................... 13
8. Chloride measurement............................. 14
9. Regulatory volume decrease....................... 15
10. Migration and invasion........................... 15
11. Fibronectin assembly assay....................... 16
12. Purification of fusion proteins.................. 16
13. Colony formation and animal experiments.......... 16
14. Immunohistochemistry............................. 17
15. Statistical analysis............................. 17
IV. Results ......................................... 18
1. Mutations at 4 N-linked glycosylation sites change the glycoprotein structure of KCC4....................... 18
2. M III and M IV mutants are not expressed at the cell surface.................................................. 19
3. The central two N-linked glycans of KCC4 are important for KCC4 membrane expression................... 21
4. The high-mannose form of N-glycan is degraded through the proteasome pathway........................... 22
5. M IV and N331/344/Q mutants suppress KCC4-mediated RVD and tumor formation.................................. 23
6. 4T1 shKCC4 reduced lung colonization............. 25
7. KCC4 regulated fibronectin expression and assembly................................................. 26
V. Discussion....................................... 28
1. Structure analysis of N-linked glycosylation of KCC4......................................................28
2. ER and KCC4.......................................30
3. N-linked glycosylation and complex form glycans augment the membrane expression of KCC4...................31
4. KCC4 and tumor biology............................32
5. KCCs and fibronectin..............................33
6. Perspective.......................................34
VI. Conclusion....................................... 36
VII. References....................................... 37
Tables................................................... 43
Figures ................................................. 45
Appendixes .............................................. 75
參考文獻 Aslakson CJ, Miller FR (1992). Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer research 52 (6): 1399-1405.
Bellemer A, Hirata T, Romero MF, Koelle MR (2011). Two types of chloride transporters are required for GABA(A) receptor-mediated inhibition in C. elegans. The EMBO journal 30 (9): 1852-1863.
Benarroch EE (2013). Cation-chloride cotransporters in the nervous system: General features and clinical correlations. Neurology 80 (8): 756-763.
Berglund K, Schleich W, Krieger P, Loo LS, Wang D, Cant NB et al (2006). Imaging synaptic inhibition in transgenic mice expressing the chloride indicator, Clomeleon. Brain cell biology 35 (4-6): 207-228.
Bize I (2001). Theoretical validation for a model of KCC regulation in human erythrocytes. Blood cells, molecular and disease 27 (1): 121-126.
Boassa D, Ambrosi C, Qiu F, Dahl G, Gaietta G, Sosinsky G (2007). Pannexin1 channels contain a glycosylation site that targets the hexamer to the plasma membrane. The Journal of biological chemistry 282 (43): 31733-31743.
Boettger T, Rust MB, Maier H, Seidenbecher T, Schweizer M, Keating DJ et al (2003). Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold. The EMBO journal 22 (20): 5422-5434.
Capo-Aponte JE, Wang Z, Akinci MA, Wolosin JM, Pokorny KS, Iserovish P et al (2007). Potassium-chloride cotransporter mediates cell cycle progression and proliferation of human corneal epithelial cells. Cell cycle 6 (21): 2709-2718.
Casula S, Shmukler BE, Wilhelm S, Stuart-Tilley AK, Su W, Chernova MN et al (2001). A dominant negative mutant of the KCC1 K-Cl cotransporter: both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity. The Journal of biological chemistry 276 (45): 41870-41878.
Chambers AF, Groom AC, MacDonald IC (2002). Dissemination and growth of cancer cells in metastatic sites. Nature reviews cancer 2 (8): 563-572.
Chang XB, Mengos A, Hou YX, Cui L, Jensen TJ, Aleksandrov A et al (2008). Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator. Journal of cell science 121 (Pt 17): 2814-2823.
Chen YF, Chou CY, Wilkins RJ, Ellory JC, Mount DB, Shen MR (2009). Motor protein-dependent membrane trafficking of KCl cotransporter-4 is important for cancer cell invasion. Cancer research 69 (22): 8585-8593.
Cheng HC, Abdel-Ghany M, Elble RC, Pauli BU (1998). Lung endothelial dipeptidyl peptidase IV promotes adhesion and metastasis of rat breast cancer cells via tumor cell surface-associated fibronectin. The Journal of biological chemistry 273 (37): 24207-24215.
Cheng HC, Abdel-Ghany M, Pauli BU (2003). A novel consensus motif in fibronectin mediates dipeptidyl peptidase IV adhesion and metastasis. The Journal of biological chemistry 278 (27): 24600-24607.
Clark EA, Golub TR, Lander ES, Hynes RO (2000). Genomic analysis of metastasis reveals an essential role for RhoC. Nature 406 (6795): 532-535.
Contessa JN, Bhojani MS, Freeze HH, Ross BD, Rehemtulla A, Lawrence TS (2010). Molecular imaging of N-linked glycosylation suggests glycan biosynthesis is a novel target for cancer therapy. Clinical cancer research : an official journal of the American Association for Cancer Research 16 (12): 3205-3214.
Dube DH, Bertozzi CR (2005). Glycans in cancer and inflammation--potential for therapeutics and diagnostics. Nature reviews drug discovery 4 (6): 477-488.
Dunham PB, Stewart GW, Ellory JC (1980). Chloride-activated passive potassium transport in human erythrocytes. Proceedings of the National Academy of Sciences of the United States of America 77 (3): 1711-1715.
DuPre SA, Hunter KW, Jr. (2007). Murine mammary carcinoma 4T1 induces a leukemoid reaction with splenomegaly: association with tumor-derived growth factors. Experimental and molecular pathology 82 (1): 12-24.
Ferreira CS, Cheung MC, Missailidis S, Bisland S, Gariepy J (2009). Phototoxic aptamers selectively enter and kill epithelial cancer cells. Nucleic acids research 37 (3): 866-876.
Fidler IJ (2003). The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited. Nature reviews cancer 3 (6): 453-458.
Fishman DA, Liu Y, Ellerbroek SM, Stack MS (2001). Lysophosphatidic acid promotes matrix metalloproteinase (MMP) activation and MMP-dependent invasion in ovarian cancer cells. Cancer research 61 (7): 3194-3199.
Fujii T, Takahashi Y, Itomi Y, Fujita K, Morii M, Tabuchi Y et al (2008). K+-Cl- Cotransporter-3a Up-regulates Na+,K+-ATPase in Lipid Rafts of Gastric Luminal Parietal Cells. The Journal of biological chemistry 283 (11): 6869-6877.
Fujii T, Takahashi Y, Ikari A, Morii M, Tabuchi Y, Tsukada K et al (2009). Functional association between K+-Cl- cotransporter-4 and H+,K+-ATPase in the apical canalicular membrane of gastric parietal cells. The Journal of biological chemistry 284 (1): 619-629.
Fuster MM, Esko JD (2005). The sweet and sour of cancer: glycans as novel therapeutic targets. Nature reviews cancer 5 (7): 526-542.
Gillen CM, Brill S, Payne JA, Forbush B, 3rd (1996). Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human. A new member of the cation-chloride cotransporter family. The Journal of biological chemistry 271 (27): 16237-16244.
Glozman R, Okiyoneda T, Mulvihill CM, Rini JM, Barriere H, Lukacs GL (2009). N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic. The Journal of cell biology 184 (6): 847-862.
Granovsky M, Fata J, Pawling J, Muller WJ, Khokha R, Dennis JW (2000). Suppression of tumor growth and metastasis in Mgat5-deficient mice. Nature medicine 6 (3): 306-312.
Gupta GP, Massague J (2006). Cancer metastasis: building a framework. Cell 127 (4): 679-695.
Hall MK, Cartwright TA, Fleming CM, Schwalbe RA (2011). Importance of glycosylation on function of a potassium channel in neuroblastoma cells. PloS one 6 (4): e19317.
Haltiwanger RS, Lowe JB (2004). Role of glycosylation in development. Annual review of biochemistry 73: 491-537.
Hartmann AM, Wenz M, Mercado A, Storger C, Mount DB, Friauf E et al (2010). Differences in the large extracellular loop between the K(+)-Cl(-) cotransporters KCC2 and KCC4. The Journal of biological chemistry 285 (31): 23994-24002.
Hebert SC, Mount DB, Gamba G (2004). Molecular physiology of cation-coupled Cl- cotransport: the SLC12 family. Pflugers Archiv : European journal of physiology 447 (5): 580-593.
Hekmat-Scafe DS, Lundy MY, Ranga R, Tanouye MA (2006). Mutations in the K+/Cl- cotransporter gene kazachoc (kcc) increase seizure susceptibility in Drosophila. The Journal of neuroscience : the official journal of the Society for Neuroscience 26 (35): 8943-8954.
Hendriks G, Koudijs M, van Balkom BW, Oorschot V, Klumperman J, Deen PM et al (2004). Glycosylation is important for cell surface expression of the water channel aquaporin-2 but is not essential for tetramerization in the endoplasmic reticulum. The Journal of biological chemistry 279 (4): 2975-2983.
Hiki K, D'Andrea RJ, Furze J, Crawford J, Woollatt E, Sutherland GR et al (1999). Cloning, characterization, and chromosomal location of a novel human K+-Cl- cotransporter. The Journal of biological chemistry 274 (15): 10661-10667.
Howard HC, Mount DB, Rochefort D, Byun N, Dupre N, Lu J et al (2002). The K-Cl cotransporter KCC3 is mutant in a severe peripheral neuropathy associated with agenesis of the corpus callosum. Nature genetics 32 (3): 384-392.
Hsu YM, Chen YF, Chou CY, Tang MJ, Chen JH, Wilkins RJ et al (2007a). KCl cotransporter-3 down-regulates E-cadherin/beta-catenin complex to promote epithelial-mesenchymal transition. Cancer research 67 (22): 11064-11073.
Hsu YM, Chou CY, Chen HH, Lee WY, Chen YF, Lin PW et al (2007b). IGF-1 upregulates electroneutral K-Cl cotransporter KCC3 and KCC4 which are differentially required for breast cancer cell proliferation and invasiveness. Journal of cellular physiology 210 (3): 626-636.
Hubner CA, Jentsch TJ (2002). Ion channel diseases. Human Molecular Genetics 11 (20): 2435-2445.
Jitsuhara Y, Toyoda T, Itai T, Yamaguchi H (2002). Chaperone-like functions of high-mannose type and complex-type N-glycans and their molecular basis. Journal of biochemistry 132 (5): 803-811.
Kajiya H, Okamoto F, Li JP, Nakao A, Okabe K (2006). Expression of mouse osteoclast K-Cl Co-transporter-1 and its role during bone resorption. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 21 (7): 984-992.
Kakazu Y, Akaike N, Komiyama S, Nabekura J (1999). Regulation of intracellular chloride by cotransporters in developing lateral superior olive neurons. Journal of Neuroscience 19 (8): 2843-2851.
Kaspar M, Zardi L, Neri D (2006). Fibronectin as target for tumor therapy. International journal of cancer Journal international du cancer 118 (6): 1331-1339.
Khanna R, Myers MP, Laine M, Papazian DM (2001). Glycosylation increases potassium channel stability and surface expression in mammalian cells. The Journal of biological chemistry 276 (36): 34028-34034.
Lauf PK, Adragna NC (2000). K-Cl cotransport: properties and molecular mechanism. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 10 (5-6): 341-354.
Malik G, Knowles LM, Dhir R, Xu S, Yang S, Ruoslahti E et al (2010). Plasma fibronectin promotes lung metastasis by contributions to fibrin clots and tumor cell invasion. Cancer research 70 (11): 4327-4334.
Moroz A, Delella FK, Lacorte LM, Deffune E, Felisbino SL (2013). Fibronectin induces MMP2 expression in human prostate cancer cells. Biochemical and biophysical research communications 430 (4): 1319-1321.
Mount DB, Mercado A, Song L, Xu J, George AL, Jr., Delpire E et al (1999). Cloning and characterization of KCC3 and KCC4, new members of the cation-chloride cotransporter gene family. The Journal of biological chemistry 274 (23): 16355-16362.
Ohtsubo K, Marth JD (2006). Glycosylation in cellular mechanisms of health and disease. Cell 126 (5): 855-867.
Palma E, Amici M, Sobrero F, Spinelli G, Di Angelantonio S, Ragozzino D et al (2006). Anomalous levels of Cl- transporters in the hippocampal subiculum from temporal lobe epilepsy patients make GABA excitatory. Proceedings of the National Academy of Sciences of the United States of America 103 (22): 8465-8468.
Psaila B, Lyden D (2009). The metastatic niche: adapting the foreign soil. Nature reviews cancer 9 (4): 285-293.
Sakuma K, Fujimoto I, Hitoshi S, Tanaka F, Ikeda T, Tanabe K et al (2006). An N-glycan structure correlates with pulmonary metastatic ability of cancer cells. Biochemical and biophysical research communications 340 (3): 829-835.
Shen MR, Chou CY, Ellory JC (2000). Volume-sensitive KCI cotransport associated with human cervical carcinogenesis. Pflugers Archiv : European journal of physiology 440 (5): 751-760.
Shen MR, Chou CY, Hsu KF, Liu HS, Dunham PB, Holtzman EJ et al (2001). The KCl cotransporter isoform KCC3 can play an important role in cell growth regulation. Proceedings of the National Academy of Sciences of the United States of America 98 (25): 14714-14719.
Shen MR, Chou CY, Hsu KF, Hsu YM, Chiu WT, Tang MJ et al (2003). KCl cotransport is an important modulator of human cervical cancer growth and invasion. The Journal of biological chemistry 278 (41): 39941-39950.
Shen MR, Lin AC, Hsu YM, Chang TJ, Tang MJ, Alper SL et al (2004). Insulin-like growth factor 1 stimulates KCl cotransport, which is necessary for invasion and proliferation of cervical cancer and ovarian cancer cells. The Journal of biological chemistry 279 (38): 40017-40025.
Shiraishi T, Yoshida T, Nakata S, Horinaka M, Wakada M, Mizutani Y et al (2005). Tunicamycin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human prostate cancer cells. Cancer research 65 (14): 6364-6370.
Simard CF, Bergeron MJ, Frenette-Cotton R, Carpentier GA, Pelchat ME, Caron L et al (2007). Homooligomeric and heterooligomeric associations between K+-Cl- cotransporter isoforms and between K+-Cl- and Na+-K+-Cl- cotransporters. The Journal of biological chemistry 282 (25): 18083-18093.
Sun JY, Yang H, Miao S, Li JP, Wang SW, Zhu MZ et al (2009). Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo. Phytomedicine : international journal of phytotherapy and phytopharmacology 16 (11): 1070-1074.
Sun YT, Shieh CC, Delpire E, Shen MR (2012). K(+)-Cl(-) cotransport mediates the bactericidal activity of neutrophils by regulating NADPH oxidase activation. The Journal of physiology 590 (Pt 14): 3231-3243.
Vagin O, Turdikulova S, Sachs G (2004). The H,K-ATPase beta subunit as a model to study the role of N-glycosylation in membrane trafficking and apical sorting. The Journal of biological chemistry 279 (37): 39026-39034.
Vagin O, Kraut JA, Sachs G (2009). Role of N-glycosylation in trafficking of apical membrane proteins in epithelia. American journal of physiology renal physiology 296 (3): F459-469.
Wei WC, Akerman CJ, Newey SE, Pan J, Clinch NW, Jacob Y et al (2011). The potassium-chloride cotransporter 2 promotes cervical cancer cell migration and invasion by an ion transport-independent mechanism. The Journal of physiology 589 (Pt 22): 5349-5359.
Yeh HH, Lai WW, Chen HH, Liu HS, Su WC (2006). Autocrine IL-6-induced Stat3 activation contributes to the pathogenesis of lung adenocarcinoma and malignant pleural effusion. Oncogene 25 (31): 4300-4309.
Yu M, Ting DT, Stott SL, Wittner BS, Ozsolak F, Paul S et al (2012). RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis. Nature 487 (7408): 510-513.
Zaarour N, Demaretz S, Defontaine N, Mordasini D, Laghmani K (2009). A highly conserved motif at the COOH terminus dictates endoplasmic reticulum exit and cell surface expression of NKCC2. The Journal of biological chemistry 284 (32): 21752-21764.
Zhu J, Yan J, Thornhill WB (2012). N-Glycosylation promotes the cell surface expression of Kv1.3 potassium channels. The FEBS journal 279 (15): 2632-264
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