Please use this identifier to cite or link to this item:
標題: H9c2大鼠心臟細胞Gelsolin影響細胞形態和caveolin訊號路徑抗雙氧水誘導的氧化壓力
Gelsolin Affects Cell Morphology and Caveolin Signaling Against Hydrogen Peroxide-induced Oxidative Stress in H9c2 Rat Cardiomyoblasts
作者: 何舒婷
He, Shu-Ting
關鍵字: 大鼠心臟細胞株H9c2
H9c2 Rat Cardiac cells
gelsolin (GSN)
caveolins (cavs)
Gelsolin (GSN)
Caveolins (cavs)
Oxidative stress
Actin filaments
stress fiber
出版社: 生命科學系所
引用: Aggeli, I.K., Gaitanaki, C., and Beis, I. (2006). Involvement of JNKs and p38-MAPK/MSK1 pathways in H2O2-induced upregulation of heme oxygenase-1 mRNA in H9c2 cells. Cellular signalling 18, 1801-1812. Ahuja, P., Sdek, P., and MacLellan, W.R. (2007). Cardiac myocyte cell cycle control in development, disease, and regeneration. Physiological reviews 87, 521-544. Alnemri, E.S., Livingston, D.J., Nicholson, D.W., Salvesen, G., Thornberry, N.A., Wong, W.W., and Yuan, J. (1996). Human ICE/CED-3 protease nomenclature. Cell 87, 171. Amit, Y., and Boneh, A. (1993). Bilirubin inhibits protein kinase C activity and protein kinase C-mediated phosphorylation of endogenous substrates in human skin fibroblasts. Clinica chimica acta; international journal of clinical chemistry 223, 103-111. Azuma, T., Koths, K., Flanagan, L., and Kwiatkowski, D. (2000). Gelsolin in complex with phosphatidylinositol 4,5-bisphosphate inhibits caspase-3 and -9 to retard apoptotic progression. The Journal of biological chemistry 275, 3761-3766. Boyd, N.L., Park, H., Yi, H., Boo, Y.C., Sorescu, G.P., Sykes, M., and Jo, H. (2003). Chronic shear induces caveolae formation and alters ERK and Akt responses in endothelial cells. American journal of physiology Heart and circulatory physiology 285, H1113-1122. Burtnick, L.D., Koepf, E.K., Grimes, J., Jones, E.Y., Stuart, D.I., McLaughlin, P.J., and Robinson, R.C. (1997). The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation. Cell 90, 661-670. Burtnick, L.D., Urosev, D., Irobi, E., Narayan, K., and Robinson, R.C. (2004). Structure of the N-terminal half of gelsolin bound to actin: roles in severing, apoptosis and FAF. The EMBO journal 23, 2713-2722. Chen, Q.M., Tu, V.C., Wu, Y., and Bahl, J.J. (2000). Hydrogen peroxide dose dependent induction of cell death or hypertrophy in cardiomyocytes. Archives of biochemistry and biophysics 373, 242-248. Chiarugi, P. (2005). PTPs versus PTKs: the redox side of the coin. Free radical research 39, 353-364. Chien, A.J., Gao, T., Perez-Reyes, E., and Hosey, M.M. (1998). Membrane targeting of L-type calcium channels. Role of palmitoylation in the subcellular localization of the beta2a subunit. The Journal of biological chemistry 273, 23590-23597. Choe, H., Burtnick, L.D., Mejillano, M., Yin, H.L., Robinson, R.C., and Choe, S. (2002). The calcium activation of gelsolin: insights from the 3A structure of the G4-G6/actin complex. Journal of molecular biology 324, 691-702. Choi, J.W., Lee, K.H., Kim, S.H., Jin, T., Lee, B.S., Oh, J., Won, H.Y., Kim, S.Y., Kang, S.M., and Chung, J.H. (2011). C-reactive protein induces p53-mediated cell cycle arrest in H9c2 cardiac myocytes. Biochemical and biophysical research communications 410, 525-530. Cohen, A.W., Hnasko, R., Schubert, W., and Lisanti, M.P. (2004). Role of caveolae and caveolins in health and disease. Physiological reviews 84, 1341-1379. Cohen, A.W., Park, D.S., Woodman, S.E., Williams, T.M., Chandra, M., Shirani, J., Pereira de Souza, A., Kitsis, R.N., Russell, R.G., Weiss, L.M., et al. (2003). Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts. American journal of physiology Cell physiology 284, C457-474. Coue, M., Brenner, S.L., Spector, I., and Korn, E.D. (1987). Inhibition of actin polymerization by latrunculin A. FEBS letters 213, 316-318. Cunningham, C.C., Stossel, T.P., and Kwiatkowski, D.J. (1991). Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin. Science 251, 1233-1236. Das, M., Gherghiceanu, M., Lekli, I., Mukherjee, S., Popescu, L.M., and Das, D.K. (2008). Essential role of lipid raft in ischemic preconditioning. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 21, 325-334. Dore, S., Takahashi, M., Ferris, C.D., Zakhary, R., Hester, L.D., Guastella, D., and Snyder, S.H. (1999). Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury. Proceedings of the National Academy of Sciences of the United States of America 96, 2445-2450. Echarri, A., Muriel, O., Pavon, D.M., Azegrouz, H., Escolar, F., Terron, M.C., Sanchez-Cabo, F., Martinez, F., Montoya, M.C., Llorca, O., et al. (2012). Caveolar domain organization and trafficking is regulated by Abl kinases and mDia1. Journal of cell science 125, 3097-3113. Edelmann, H.M., Kuhne, C., Petritsch, C., and Ballou, L.M. (1996). Cell cycle regulation of p70 S6 kinase and p42/p44 mitogen-activated protein kinases in Swiss mouse 3T3 fibroblasts. The Journal of biological chemistry 271, 963-971. Fielding, C.J., Bist, A., and Fielding, P.E. (1997). Caveolin mRNA levels are up-regulated by free cholesterol and down-regulated by oxysterols in fibroblast monolayers. Proceedings of the National Academy of Sciences of the United States of America 94, 3753-3758. Force, T. (2008). The weakness of a big heart. Nature medicine 14, 244-245. Galbiati, F., Volonte, D., Meani, D., Milligan, G., Lublin, D.M., Lisanti, M.P., and Parenti, M. (1999). The dually acylated NH2-terminal domain of gi1alpha is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains. Palmitoylation of caveolin-1 is required for the recognition of dually acylated g-protein alpha subunits in vivo. The Journal of biological chemistry 274, 5843-5850. glioma C6 cellsNakamura, M., Sunagawa, M., Kosugi, T., and Sperelakis, N. (2000). Actin filament disruption inhibits L-type Ca(2+) channel current in cultured vascular smooth muscle cells. American journal of physiology Cell physiology 279, C480-487. Goshima, M., Kariya, K., Yamawaki-Kataoka, Y., Okada, T., Shibatohge, M., Shima, F., Fujimoto, E., and Kataoka, T. (1999). Characterization of a novel Ras-binding protein Ce-FLI-1 comprising leucine-rich repeats and gelsolin-like domains. Biochemical and biophysical research communications 257, 111-116. Green, D.R., and Reed, J.C. (1998). Mitochondria and apoptosis. Science 281, 1309-1312. Grinshtein, N., Bamm, V.V., Tsemakhovich, V.A., and Shaklai, N. (2003). Mechanism of low-density lipoprotein oxidation by hemoglobin-derived iron. Biochemistry 42, 6977-6985. Grynkiewicz, G., Poenie, M., and Tsien, R.Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. The Journal of biological chemistry 260, 3440-3450. Ha, H., and Pak, Y. (2005). Modulation of the caveolin-3 and Akt status in caveolae by insulin resistance in H9c2 cardiomyoblasts. Experimental & molecular medicine 37, 169-178. Horikawa, Y.T., Patel, H.H., Tsutsumi, Y.M., Jennings, M.M., Kidd, M.W., Hagiwara, Y., Ishikawa, Y., Insel, P.A., and Roth, D.M. (2008). Caveolin-3 expression and caveolae are required for isoflurane-induced cardiac protection from hypoxia and ischemia/reperfusion injury. Journal of molecular and cellular cardiology 44, 123-130. Hsu, Y.C., and Liou, Y.M. (2011). The anti-cancer effects of (-)-epigallocatechin-3-gallate on the signaling pathways associated with membrane receptors in MCF-7 cells. Journal of cellular physiology 226, 2721-2730. Hu, X., Wang, J., and Jiang, H. (2012). Heme oxygenase-1: An important therapeutic target for protecting against myocardial ischemia and reperfusion injury. International journal of cardiology. Insel, P.A., and Patel, H.H. (2007). Do studies in caveolin-knockouts teach us about physiology and pharmacology or instead, the ways mice compensate for ''lost proteins''? British journal of pharmacology 150, 251-254. Ishikawa, K., Sugawara, D., Wang, X., Suzuki, K., Itabe, H., Maruyama, Y., and Lusis, A.J. (2001). Heme oxygenase-1 inhibits atherosclerotic lesion formation in ldl-receptor knockout mice. Circulation research 88, 506-512. Johnson, B.W., and Boise, L.H. (1999). Bcl-2 and caspase inhibition cooperate to inhibit tumor necrosis factor-alpha-induced cell death in a Bcl-2 cleavage-independent fashion. The Journal of biological chemistry 274, 18552-18558. Jung, C., Martins, A.S., Niggli, E., and Shirokova, N. (2008). Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways. Cardiovascular research 77, 766-773. Jurgensmeier, J.M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D., and Reed, J.C. (1998). Bax directly induces release of cytochrome c from isolated mitochondria. Proceedings of the National Academy of Sciences of the United States of America 95, 4997-5002. Kawabe, J., Okumura, S., Lee, M.C., Sadoshima, J., and Ishikawa, Y. (2004). Translocation of caveolin regulates stretch-induced ERK activity in vascular smooth muscle cells. American journal of physiology Heart and circulatory physiology 286, H1845-1852. Kazzaz, J.A., Xu, J., Palaia, T.A., Mantell, L., Fein, A.M., and Horowitz, S. (1996). Cellular oxygen toxicity. Oxidant injury without apoptosis. The Journal of biological chemistry 271, 15182-15186. Kerr, J.F., Wyllie, A.H., and Currie, A.R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British journal of cancer 26, 239-257. Kukreja, R.C., Jesse, R.L., and Hess, M.L. (1992). Singlet oxygen: a potential culprit in myocardial injury? Molecular and cellular biochemistry 111, 17-24. Lanks, K.W., and Kasambalides, E.J. (1983). Dexamethasone induces gelsolin synthesis and altered morphology in L929 cells. The Journal of cell biology 96, 577-581. Lee, S.H., and Dominguez, R. (2010). Regulation of actin cytoskeleton dynamics in cells. Molecules and cells 29, 311-325. Lee, Y.J., and Keng, P.C. (2005). Studying the effects of actin cytoskeletal destabilization on cell cycle by cofilin overexpression. Molecular biotechnology 31, 1-10. Lefer, A.M., and Lefer, D.J. (1996). The role of nitric oxide and cell adhesion molecules on the microcirculation in ischaemia-reperfusion. Cardiovascular research 32, 743-751. Li, G., Chen, Y., Saari, J.T., and Kang, Y.J. (1997). Catalase-overexpressing transgenic mouse heart is resistant to ischemia-reperfusion injury. The American journal of physiology 273, H1090-1095. Li, G.H., Shi, Y., Chen, Y., Sun, M., Sader, S., Maekawa, Y., Arab, S., Dawood, F., Chen, M., De Couto, G., et al. (2009). Gelsolin regulates cardiac remodeling after myocardial infarction through DNase I-mediated apoptosis. Circulation research 104, 896-904. Li, Y., Huang, T.T., Carlson, E.J., Melov, S., Ursell, P.C., Olson, J.L., Noble, L.J., Yoshimura, M.P., Berger, C., Chan, P.H., et al. (1995). Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nature genetics 11, 376-381. Lu, C., Chan, S.L., Fu, W., and Mattson, M.P. (2002). The lipid peroxidation product 4-hydroxynonenal facilitates opening of voltage-dependent Ca2+ channels in neurons by increasing protein tyrosine phosphorylation. The Journal of biological chemistry 277, 24368-24375. Maciel, E.N., Vercesi, A.E., and Castilho, R.F. (2001). Oxidative stress in Ca(2+)-induced membrane permeability transition in brain mitochondria. Journal of neurochemistry 79, 1237-1245. Magenta, A., Fasanaro, P., Romani, S., Di Stefano, V., Capogrossi, M.C., and Martelli, F. (2008). Protein phosphatase 2A subunit PR70 interacts with pRb and mediates its dephosphorylation. Molecular and cellular biology 28, 873-882. Maines, M.D. (1993). Carbon Monoxide: An Emerging Regulator of cGMP in the Brain. Molecular and cellular neurosciences 4, 389-397. Marzo, I., Brenner, C., Zamzami, N., Jurgensmeier, J.M., Susin, S.A., Vieira, H.L., Prevost, M.C., Xie, Z., Matsuyama, S., Reed, J.C., et al. (1998). Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281, 2027-2031. Matveev, S., van der Westhuyzen, D.R., and Smart, E.J. (1999). Co-expression of scavenger receptor-BI and caveolin-1 is associated with enhanced selective cholesteryl ester uptake in THP-1 macrophages. Journal of lipid research 40, 1647-1654. Michel, V., and Bakovic, M. (2007). Lipid rafts in health and disease. Biology of the cell / under the auspices of the European Cell Biology Organization 99, 129-140. Munoz, C.M., van Meeteren, L.A., Post, J.A., Verkleij, A.J., Verrips, C.T., and Boonstra, J. (2002). Hydrogen peroxide inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. Free radical biology & medicine 33, 1061-1072. Muriel, O., Echarri, A., Hellriegel, C., Pavon, D.M., Beccari, L., and Del Pozo, M.A. (2011). Phosphorylated filamin A regulates actin-linked caveolae dynamics. Journal of cell science 124, 2763-2776. Nishio, R., and Matsumori, A. (2009). Gelsolin and cardiac myocyte apoptosis: a new target in the treatment of postinfarction remodeling. Circulation research 104, 829-831. Nobel, C.S., Kimland, M., Nicholson, D.W., Orrenius, S., and Slater, A.F. (1997). Disulfiram is a potent inhibitor of proteases of the caspase family. Chemical research in toxicology 10, 1319-1324. Nusco, G.A., Chun, J.T., Ercolano, E., Lim, D., Gragnaniello, G., Kyozuka, K., and Santella, L. (2006). Modulation of calcium signalling by the actin-binding protein cofilin. Biochemical and biophysical research communications 348, 109-114. Ohtsu, M., Sakai, N., Fujita, H., Kashiwagi, M., Gasa, S., Shimizu, S., Eguchi, Y., Tsujimoto, Y., Sakiyama, Y., Kobayashi, K., et al. (1997). Inhibition of apoptosis by the actin-regulatory protein gelsolin. The EMBO journal 16, 4650-4656. Patel, H.H., Tsutsumi, Y.M., Head, B.P., Niesman, I.R., Jennings, M., Horikawa, Y., Huang, D., Moreno, A.L., Patel, P.M., Insel, P.A., et al. (2007). Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin-1. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 21, 1565-1574. Pena-Silva, R.A., Miller, J.D., Chu, Y., and Heistad, D.D. (2009). Serotonin produces monoamine oxidase-dependent oxidative stress in human heart valves. American journal of physiology Heart and circulatory physiology 297, H1354-1360. Peterson, T.E., Poppa, V., Ueba, H., Wu, A., Yan, C., and Berk, B.C. (1999). Opposing effects of reactive oxygen species and cholesterol on endothelial nitric oxide synthase and endothelial cell caveolae. Circulation research 85, 29-37. Petit, P.X., Zamzami, N., Vayssiere, J.L., Mignotte, B., Kroemer, G., and Castedo, M. (1997). Implication of mitochondria in apoptosis. Molecular and cellular biochemistry 174, 185-188. Pike, L.J. (2003). Lipid rafts: bringing order to chaos. Journal of lipid research 44, 655-667. Pope, B., Maciver, S., and Weeds, A. (1995). Localization of the calcium-sensitive actin monomer binding site in gelsolin to segment 4 and identification of calcium binding sites. Biochemistry 34, 1583-1588. Pope, B., Way, M., and Weeds, A.G. (1991). Two of the three actin-binding domains of gelsolin bind to the same subdomain of actin. Implications of capping and severing mechanisms. FEBS letters 280, 70-74. Reshetnikova, G., Barkan, R., Popov, B., Nikolsky, N., and Chang, L.S. (2000). Disruption of the actin cytoskeleton leads to inhibition of mitogen-induced cyclin E expression, Cdk2 phosphorylation, and nuclear accumulation of the retinoblastoma protein-related p107 protein. Experimental cell research 259, 35-53. Rothberg, K.G., Heuser, J.E., Donzell, W.C., Ying, Y.S., Glenney, J.R., and Anderson, R.G. (1992). Caveolin, a protein component of caveolae membrane coats. Cell 68, 673-682. Sabala, P., Targos, B., Caravelli, A., Czajkowski, R., Lim, D., Gragnaniello, G., Santella, L., and Baranska, J. (2002). Role of the actin cytoskeleton in store-mediated calcium entry in glioma C6 cells. Biochemical and biophysical research communications 296, 484-491. Shieh, D.B., Li, R.Y., Liao, J.M., Chen, G.D., and Liou, Y.M. (2010). Effects of genistein on beta-catenin signaling and subcellular distribution of actin-binding proteins in human umbilical CD105-positive stromal cells. Journal of cellular physiology 223, 423-434. Shiomi, T., Tsutsui, H., Matsusaka, H., Murakami, K., Hayashidani, S., Ikeuchi, M., Wen, J., Kubota, T., Utsumi, H., and Takeshita, A. (2004). Overexpression of glutathione peroxidase prevents left ventricular remodeling and failure after myocardial infarction in mice. Circulation 109, 544-549. Singer, S.J. (1972). A fluid lipid-globular protein mosaic model of membrane structure. Annals of the New York Academy of Sciences 195, 16-23. Song, K.S., Tang, Z., Li, S., and Lisanti, M.P. (1997). Mutational analysis of the properties of caveolin-1. A novel role for the C-terminal domain in mediating homo-typic caveolin-caveolin interactions. The Journal of biological chemistry 272, 4398-4403. Suhler, E., Lin, W., Yin, H.L., and Lee, W.M. (1997). Decreased plasma gelsolin concentrations in acute liver failure, myocardial infarction, septic shock, and myonecrosis. Critical care medicine 25, 594-598. Teng, C.H., Huang, W.N., and Meng, T.C. (2007). Several dual specificity phosphatases coordinate to control the magnitude and duration of JNK activation in signaling response to oxidative stress. The Journal of biological chemistry 282, 28395-28407. Thornberry, N.A. (1998). Caspases: key mediators of apoptosis. Chemistry & biology 5, R97-103. van Eickels, M., Grohe, C., Cleutjens, J.P., Janssen, B.J., Wellens, H.J., and Doevendans, P.A. (2001). 17beta-estradiol attenuates the development of pressure-overload hypertrophy. Circulation 104, 1419-1423. Verkade, P., and Simons, K. (1997). Robert Feulgen Lecture 1997. Lipid microdomains and membrane trafficking in mammalian cells. Histochemistry and cell biology 108, 211-220. Wang, G., Hamid, T., Keith, R.J., Zhou, G., Partridge, C.R., Xiang, X., Kingery, J.R., Lewis, R.K., Li, Q., Rokosh, D.G., et al. (2010). Cardioprotective and antiapoptotic effects of heme oxygenase-1 in the failing heart. Circulation 121, 1912-1925. Waschke, J., Golenhofen, N., Kurzchalia, T.V., and Drenckhahn, D. (2006). Protein kinase C-mediated endothelial barrier regulation is caveolin-1-dependent. Histochemistry and cell biology 126, 17-26. Way, M., Pope, B., Gooch, J., Hawkins, M., and Weeds, A.G. (1990). Identification of a region in segment 1 of gelsolin critical for actin binding. The EMBO journal 9, 4103-4109. Way, M., Pope, B., and Weeds, A.G. (1992). Are the conserved sequences in segment 1 of gelsolin important for binding actin? The Journal of cell biology 116, 1135-1143. Weeds, A.G., Gooch, J., McLaughlin, P., Pope, B., Bengtsdotter, M., and Karlsson, R. (1995). Identification of the trapped calcium in the gelsolin segment 1-actin complex: implications for the role of calcium in the control of gelsolin activity. FEBS letters 360, 227-230. Wei, Y., Zhang, Z., Andersen, C.H., Schmelzer, E., Gregersen, P.L., Collinge, D.B., Smedegaard-Petersen, V., and Thordal-Christensen, H. (1998). An epidermis/papilla-specific oxalate oxidase-like protein in the defence response of barley attacked by the powdery mildew fungus. Plant molecular biology 36, 101-112. Williams, T.M., and Lisanti, M.P. (2004). The caveolin proteins. Genome biology 5, 214. Witke, W., Sharpe, A.H., Hartwig, J.H., Azuma, T., Stossel, T.P., and Kwiatkowski, D.J. (1995). Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin. Cell 81, 41-51. Wyllie, A.H. (1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555-556. Wyse, B.D., Prior, I.A., Qian, H., Morrow, I.C., Nixon, S., Muncke, C., Kurzchalia, T.V., Thomas, W.G., Parton, R.G., and Hancock, J.F. (2003). Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane. The Journal of biological chemistry 278, 23738-23746. Xi, Q., Adebiyi, A., Zhao, G., Chapman, K.E., Waters, C.M., Hassid, A., and Jaggar, J.H. (2008). IP3 constricts cerebral arteries via IP3 receptor-mediated TRPC3 channel activation and independently of sarcoplasmic reticulum Ca2+ release. Circulation research 102, 1118-1126. Yang, B., Oo, T.N., and Rizzo, V. (2006). Lipid rafts mediate H2O2 prosurvival effects in cultured endothelial cells. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20, 1501-1503. Yang, J., Moravec, C.S., Sussman, M.A., DiPaola, N.R., Fu, D., Hawthorn, L., Mitchell, C.A., Young, J.B., Francis, G.S., McCarthy, P.M., et al. (2000). Decreased SLIM1 expression and increased gelsolin expression in failing human hearts measured by high-density oligonucleotide arrays. Circulation 102, 3046-3052. Yin, H.L., and Stossel, T.P. (1979). Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein. Nature 281, 583-586. Zhang, X., Shan, P., Otterbein, L.E., Alam, J., Flavell, R.A., Davis, R.J., Choi, A.M., and Lee, P.J. (2003). Carbon monoxide inhibition of apoptosis during ischemia-reperfusion lung injury is dependent on the p38 mitogen-activated protein kinase pathway and involves caspase 3. The Journal of biological chemistry 278, 1248-1258.
摘要: Gelsolin (GSN)是一個廣泛分佈,具有鍵結肌動蛋白與調控肌動蛋白絲的組裝與拆卸。GSN也具有促凋亡和抗凋亡的功能;蛋白分子的 N端具有促凋亡的特性,但全長的GSN會與PIP2形成複合物則有抗凋亡的特性。但是到目前為止對於GSN在心臟細胞中所扮演的功能與分子調控機轉還不清楚。另外, caveolins (cavs)在心臟疾病上扮演保護的角色;能增加抗氧化能力與減少氧化傷害。本研究利用過量表現GSN (GSN op)與緘默GSN (si-GSN)的H9c2細胞株,藉由外加雙氧水使細胞面臨氧化壓力的模式下,探討GSN是否會影響cavs在H9c2細胞株內的功能。首先,反轉錄聚合酶連鎖反應確認pcDNA6-GSN有轉染送入到H9c2細胞內(GSN op);西方轉漬墨點法也顯示GSN蛋白表現有增加的情形。螢光光譜儀和倒立式顯微鏡檢測細胞內鈣離子濃度和細胞形態,結果發現過量表現GSN會增加胞內鈣離子濃度和使細胞形態變得較短小。隨後MTT assay檢測細胞增長時間,再利用免疫螢光顯微鏡術檢測細胞內肌動蛋白絲的分子組裝組裝與拆卸的變化,再以流式細胞儀檢測細胞週期。結果發現:過量表現GSN會減緩細胞生長,且細胞分布在S期和G2期的比例上升,還有增加細胞內肌動蛋白絲去聚合反應。因此,GSN在H9c2細胞內過度表現可能經由影響肌動蛋白絲動態組裝與卸載的機轉因而影響細胞週期與生長。另外,過量表現GSN也會增加cavs的表現;相反地,緘默GSN則抑制cavs的表現。進一步探討過量表現GSN增加細胞內cavs對細胞抗氧化壓力的影響;因此,檢測細胞內自由基與SOD活性變化,結果發現在雙氧水誘導氧化壓力增加的正常與GSN過量表現的H9c2細胞,過量表現GSN能減緩雙氧水減少cavs蛋白表現和SOD活性,以及細胞內自由基的產生,終而增加細胞抗凋亡的能力。綜合以上結果,過量GSN表現可能增加細胞內切割和加帽的能力,增加肌動蛋白絲的降解,使降低細胞分裂的能力,進而延長細胞生長時間。另外,也影響了細胞內cavs 傳導路徑,進而促使細胞內抗氧化能力增加,達到保護的效果。由於cavs傳導路徑在心臟保護上可能扮演很重要的角色,所以未來經由影響細胞骨架重組來調控cavs訊號路徑可作為研究心臟保護的新方向。
Gelsolin (GSN) is a Ca2+-dependent actin-regulatory protein that can sever actin filaments and cap the quickly growing ends of filaments, and thus promoting actin disassembly. Accordingly, GSN plays a role in the organization of the cytoskeleton assembly/disassembly, cell motility, cell growth, and apoptosis. The biological function of GSN and its interaction with scafold proteins-caveolin in cardiac cells is not clear thus far. Caveolin (cav) is known to play an important role in myocardial protection signaling for controling intracellular SOD activity and modulating oxidative stress in cardic cells. It is of interest to define the role of GSN and to establiah relationship between the stress fiber formation by cytoskeleton and the expression of Cavs in cardic cells. In this study, GSN over-expressed (GSN op) and GSN silenced (si-GSN) H9c2 rat cardiomyoblasts in a simulated condition with oxidative stress by hydrogen peroxide were used to explore the potential mechanism of GSN in cardiac protection aganist oxidative stress. A pcDNA6-GSN containing full-length human GSN transfected into H9c2 cells caused increases in intracellular calcium concentration and alteration of cell morphology and actin filament-associated structure. GSN over-expressing cells changed morphology to a “shorter and broader” form, while GSN silencing cells displayed a less change in cell shape as compared with wild-type cells. MTT assay and flow cytometry indicated that GSN over-expression slowed down cell proliferation and increased cell populations sorted in S and G2 phases. These results suggested that GSN over-expression may enhance the ability of capping and severing F-actin such as to increase dissembly of actin filaments, and to slow down cell division and cell growth. In addition, GSN was found to modulate the expression of Cavs and their cellular functions in protecting cardic cells from oxidative stress induced by H2O2. The experiments designed to investigate the impact of an increase intracellular Cavs on anti-oxidative effects in H9c2 cells showed that GSN over-expressing cells increased their anti-oxidative capacity in H2O2-induced oxidative stress by preventing the down-regulation of Cavs, and by attenuating the H2O2-increased intracellular ROS levels, and by elevating mRNA expression for associated anti-oxidants such as HO-1, Cu-SOD, and catalase, and by increasing anti-oxidative SOD activity. Taken together, the results found in this study suggested that GSN might induce remodeling of actin filaments and that linked to mediate the Cav signaling for anti-oxidative effects in cardiac cells.
其他識別: U0005-2108201319465700
Appears in Collections:生命科學系所



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.