Abstract
Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-XL overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxindeficient (Metmut) and Bcl-XL-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaΔTM/C) or Bcl-XL in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-XL overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-XL appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-XL may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-XL overespression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.
Article PDF
Similar content being viewed by others
References
Adams, J.M., and Cory, S. (1998). The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322–1326.
Antos, N., Budzinska, M., and Kmita, H. (2001). An interplay between the TOM complex and porin isoforms in the yeast Saccharomy cescerevisiae mitochondria. FEBS Lett 500, 12–16.
Armstrong, L.C., Komiya, T., Bergman, B.E., Mihara, K., and Bornstein, P. (1997). Metaxin is a component of a preprotein import complexin the outer membrane of the mammalian mitochondrion. J Biol Chem 272, 6510–6518.
Bernal, S.D., Lampidis, T.J., Summerhayes, I.C., and Chen, L.B. (1982). Rhodamine-123 selectively reduces clonal growth of carcinoma cells in vitro. Science 218, 1117–1119.
Beutler, B., and Cerami, A. (1986). Cachectin and tumour necrosis factor as two sides of the same biological coin. Nature 320, 584–588.
Beutler, B., and Cerami, A. (1988). Tumor necrosis, cachexia, shock, and inflammation: A common mediator. Ann Rev Biochem 57, 505–518.
Beyaert, R. and Fiers, W. (1994). Molecular mechanisms of tumor necrosis factor-induced cytotoxicity. What we do understand and what we do not. FEBS 340, 9–16.
Boldin, M.P., Goncharov, T.M., Goltsev, Y.V., and Wallach, D. (1996). Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1 and TNF receptor-induced cell death. Cell 85, 803–815.
Bornstein, P., McKinney, C.E., LaMarca, M.E., Winfield, S., Shingu, T., Devarayalu, S., Vos, H.L., and Ginns, E.I. (1995). Metaxin, a gene contiguous to both thrombospondin 3 and glucocerebrosidase, is required for embryonic development in the mouse: implications for Gaucher disease. Proc Natl Acad Sci U S A 92, 4547–4551.
Brekke, O.L., Shalaby, M.R., Sundan, A., Espevik, T., and Bjerve, K. S. (1992). Butylated hydroxyanisole specifically inhibits tumornecrosis factor-induced cytotoxicity and growth enhancement. Cytokine 4, 269–280.
Carswell, E.A., Old, L.J., Kassel, R.L., Green, S., Fiore, N., and Williamson, B. (1975). An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A 72, 3666–3670.
Cho, Y.S., Challa, S., Moquin, D., Genga, R., Ray, T.D., Guildford, M., and Chan, F.K. (2009). Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virusinduced inflammation. Cell 137, 1112–1123.
Fiers, W., Beyaert, R., Declercq, W., and Vandenabeele, P. (1999). More than one way to die: apoptosis, necrosis and reactive oxygen damage. Oncogene 18, 7719–7730.
Galluzzi, L., Kepp, O., and Kroemer, G. (2009). RIP kinases initiate programmed necrosis. J Mol Cell Biol 1, 8–10.
Gardner, T. S. and Collins, J. J. (2000). Neutralizing noise in gene networks. Nature 405, 520–521.
Golstein, P., Ojcius, D.M., and Young, J.D. (1991). Cell death mechanisms and the immune system. Immunol Rev 121, 29–65.
Goossens, V., De Vos, K., Vercammen, D., Steemans, M., Vancompernolle, K., Fiers, W., Vandenabeele, P., and Grooten, J. (1999). Redox regulation of TNF signaling. Biofactors 10, 145–156.
Goossens, V., Grooten, J., De Vos, K., and Fiers, W. (1995). Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc Natl Acad Sci U S A 92, 8115–8119.
Gross, A., Pilcher, K., Blachly-Dyson, E., Basso, E., Jockel, J., Bassik, M.C., Korsmeyer, S.J., and Forte, M. (2000). Biochemical and genetic analysis of the mitochondrial response of yeast to BAX and BCL-X(L). Mol Cell Biol 20, 3125–3136.
Harris, M.H., Vander Heiden, M.G., Kron, S.J., and Thompson, C.B. (2000). Role of oxidative phosphorylation in Bax toxicity. Mol Cell Biol 20, 3590–3596.
He, S., Wang, L., Miao, L., Wang, T., Du, F., Zhao, L., and Wang, X. (2009). Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 137, 1100–1111.
Holler, N., Zaru, R., Micheau, O., Thome, M., Attinger, A., Valitutti, S., Bodmer, J.L., Schneider, P., Seed, B., and Tschopp, J. (2000). Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1, 489–495.
Janicke, R.U., Sprengart, M.L., Wati, M.R., and Porter, A.G. (1998). Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 273, 9357–9360.
Kmita, H. and Budzinska, M. (2000). Involvement of the TOM complex in external NADH transport into yeast mitochondria depleted of mitochondrial porin1. Biochim Biophys Acta 1509, 86–94.
Kroemer, G., Dallaporta, B., and Resche-Rigon, M. (1998). Themitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60, 619–642.
Kroemer, G. and Reed, J.C. (2000). Mitochondrial control of cell death. Nat Med 6, 513–519.
Kunz, J. and Hall, M.N. (1993). Cyclosporin A, FK506 and rapamycin: more than just immunosuppression. Trends Biochem Sci 18, 334–338.
Li, H. and Yuan, J. (1999). Deciphering the pathways of life and death. Curr Opin Cell Biol 11, 261–266.
Lin, Y., Choksi, S., Shen, H.M., Yang, Q.F., Hur, G.M., Kim, Y.S., Tran, J.H., Nedospasov, S.A., and Liu, Z.G. (2004). Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation. J Biol Chem 279, 10822–10828.
Liu, M.Y. and Colombini, M. (1992). Regulation of mitochondrial respiration by controlling the permeability of the outer membrane through the mitochondrial channel, VDAC. Biochim Biophys Acta 1098, 255–260.
Lotem, J., Kama, R., and Sachs, L. (1999). Suppression or induction of apoptosis by opposing pathways downstream from calciumactivated calcineurin. Proc Natl Acad Sci U S A 96, 12016–12020.
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., and Kroemer, G. (1998). Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281, 2027–2031.
Minn, A.J., Velez, P., Schendel, S.L., Liang, H., Muchmore, S.W., Fesik, S.W., Fill, M., and Thompson, C.B. (1997). Bcl-x(L) forms an ion channel in synthetic lipid membranes. Nature 385, 353–357.
Muchmore, S.W., Sattler, M., Liang, H., Meadows, R.P., Harlan, J.E., Yoon, H.S., Nettesheim, D., Chang, B.S., Thompson, C.B., Wong, S.L., et al. (1996). X-ray and NMR structureof human Bcl-xL, an inhibitor of programmed cell death. Nature 381, 335–341.
Nadakavukaren, K.K., Nadakavukaren, J.J., and Chen, L.B. (1985). Increased rhodamine 123 uptake by carcinoma cells. Cancer Res 45, 6093–6039.
Neale, M.L., Fiera, R.A., and Matthews, N. (1988). Involvement of phospholipase A2 activation in tumour cell killing by tumour necrosis factor. Immunology 64, 81–85.
Old, L.J. (1985). Tumor necrosis factor (TNF). Science 230, 630–632.
Ono, K., Wang, X., and Han, J. (2001). Resistance to tumornecrosis factor-induced cell death mediated by PMCA4 deficiency. Mol Cell Biol 21, 8276–88.
Pastorino, J.G., Simbula, G., Yamamoto, K., Glascott, P.A.J., Rothman, R.J., and Farber, J.L. (1996). The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition. J Biol Chem 271, 29792–29798.
Pfanner, N. and Geissler, A. (2001). Versatility of the mitochondrial protein import machinery. Nat Rev Mol Cell Biol 2, 339–349.
Ravagnan, L., Marzo, I., Costantini, P., Susin, S.A., Zamzami, N., Petit, P.X., Hirsch, F., Goulbern, M., Poupon, M.F., Miccoli, L., et al. (1999). Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore. Oncogene 18, 2537–2546.
Rothe, J., Gehr, G., Loetscher, H., and Lesslauer, W. (1992). Tumornecrosis factor receptors-structure and function. Immunol Res 11, 81–90.
Schnaitman, C. and Greenawalt, J.W. (1968). Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J Cell Biol 38, 158–175.
Schulze-Osthoff, K., Bakker, A.C., Vanhaesebroeck, B., Beyaert, R., Jacob, W.A., and Fiers, W. (1992). Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J Biol Chem 267, 5317–5323.
Shimizu, S., Narita, M., and Tsujimoto, Y. (1999). Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 399, 483–487.
Strasser, A., O’Connor, L., and Dixit, V.M. (2000). Apoptosis signaling. Annu Rev Biochem 69, 217–245.
Tartaglia, L.A., Ayres, T.M., Wong, G.H., and Goeddel, D.V. (1993). A novel domain within the 55 kD TNF receptor signals cell death. Cell 74, 845–853.
Tartaglia, L.A. and Goeddel, D.V. (1992). Two TNF receptors. Immunol Today 13, 151–153.
Tewari, M. and Dixit, V.M. (1995). Fas- and tumor necrosis factor-induced apoptosis ininhibited by the poxvirus crmA gene product. J Biol Chem 270, 3255–3260.
Tracey, K.J. and Cerami, A. (1993). Tumor necrosis factor, other cytokines and disease. Annu Rev Cell Biol 9, 317–343.
Vander Heiden, M.G., Chandel, N.S., Li, X.X., Schumacker, P.T., Colombini, M., and Thompson, C.B. (2000). Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival. Proc Natl Acad Sci US A 97, 4666–4671.
Vander Heiden, M.G. and Thompson, C.B. (1999). Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis? Nat Cell Biol 1, E209–216.
Vercammen, D., Beyaert, R., Denecker, G., Goossens, V., Van Loo, G., Declercq, W., Grooten, J., Fiers, W., and Vandenabeele, P. (1998). Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med 187, 1477–1485.
Wang, X., Ono, K., Kim, S.O., Kravchenko, V., Lin, S.C., and Han, J. (2001). Metaxin is required for tumor necrosis factor-induced cell death. EMBO Rep 2, 628–633.
Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M., Cai, J., Peng, T.I., Jones, D.P., and Wang, X. (1997). Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129–1132.
Zamzami, N. and Kroemer, G.(2001). The mitochondrion in apoptosis: how Pandora’s box opens. Nat Rev Mol Cell Biol 2, 67–71.
Zhang, D.W., Shao, J., Lin, J., Zhang, N., Lu, B.J., Lin, S.C., Dong, M. Q., and Han, J. (2009). RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325, 332–336.
Author information
Authors and Affiliations
Corresponding author
Additional information
An erratum to this article can be found at http://dx.doi.org/10.1007/s13238-010-0145-4
Rights and permissions
About this article
Cite this article
Ono, K., Wang, X., Kim, S.O. et al. Metaxin deficiency alters mitochondrialmembrane permeability and leads to resistance to TNF-induced cell killing. Protein Cell 1, 161–173 (2010). https://doi.org/10.1007/s13238-010-0017-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13238-010-0017-y