Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Mar 1;378(Pt 2):649–656. doi: 10.1042/BJ20031398

Overexpression of phospholipase D prevents actinomycin D-induced apoptosis through potentiation of phosphoinositide 3-kinase signalling pathways in Chinese-hamster ovary cells.

Momoko Yamada 1, Yoshiko Banno 1, Yoh Takuwa 1, Masahiro Koda 1, Akira Hara 1, Yoshinori Nozawa 1
PMCID: PMC1223985  PMID: 14640974

Abstract

To examine the roles of PLD (phospholipase D) in the regulation of the apoptotic process, PLD1 and PLD2 were stably overexpressed in S1P3-CHO cells [CHO (Chinese-hamster ovary) cells expressing the S1P (sphingosine 1-phosphate) receptor S1P3]. Treatment of S1P3-CHO cells with ActD (actinomycin D) induced apoptosis, as shown by the occurrence of nuclear fragmentation and the caspase-dependent proteolytic cleavage of PARP [poly(ADP-ribose) polymerase] and protein kinase Cd. Overexpression of either PLD1 or PLD2 protected S1P3-CHO cells from ActD-induced apoptosis, as demonstrated by an increased number of viable cells and inhibition of PARP and protein kinase Cd cleavage. However, in the early phase of apoptosis, ActD induced an increase in PLD activity and activation of key factors in the cell-survival signalling pathways, such as PI3K (phosphoinositide 3-kinase), Akt, p70S6K (p70 S6 kinase) and ERK (extracellular-signal-regulated kinase). Furthermore, the ActD-induced activation of these survival signalling enzymes was potentiated by overexpression of either PLD1 or PLD2. The PI3K inhibitor LY294002 inhibited the ActD-induced activation of Akt and p70S6K, and completely abolished the effects of PLD1 or PLD2, whereas inhibition of ERK activity by the MEK inhibitor U0126 had a milder effect. The ActD-induced activation of p70S6K and ERKs was blocked by 1-butanol, but not by t-butanol; similar to S1P, exogenous PLD suppressed the ActD-induced events in the apoptosis signalling pathways. These results show that, in S1P3-CHO cells, increased expression of PLDs prevents ActD-induced apoptosis by enhanced activation of the PI3K signalling pathways.

Full Text

The Full Text of this article is available as a PDF (334.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. An S., Zheng Y., Bleu T. Sphingosine 1-phosphate-induced cell proliferation, survival, and related signaling events mediated by G protein-coupled receptors Edg3 and Edg5. J Biol Chem. 2000 Jan 7;275(1):288–296. doi: 10.1074/jbc.275.1.288. [DOI] [PubMed] [Google Scholar]
  2. Anantharam Vellareddy, Kitazawa Masashi, Wagner Jarrad, Kaul Siddharth, Kanthasamy Anumantha G. Caspase-3-dependent proteolytic cleavage of protein kinase Cdelta is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl. J Neurosci. 2002 Mar 1;22(5):1738–1751. doi: 10.1523/JNEUROSCI.22-05-01738.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andresen Bradley T., Rizzo Mark A., Shome Kuntala, Romero Guillermo. The role of phosphatidic acid in the regulation of the Ras/MEK/Erk signaling cascade. FEBS Lett. 2002 Oct 30;531(1):65–68. doi: 10.1016/s0014-5793(02)03483-x. [DOI] [PubMed] [Google Scholar]
  4. Azuma T., Koths K., Flanagan L., Kwiatkowski D. Gelsolin in complex with phosphatidylinositol 4,5-bisphosphate inhibits caspase-3 and -9 to retard apoptotic progression. J Biol Chem. 2000 Feb 11;275(6):3761–3766. doi: 10.1074/jbc.275.6.3761. [DOI] [PubMed] [Google Scholar]
  5. Banno Y., Fujita H., Ono Y., Nakashima S., Ito Y., Kuzumaki N., Nozawa Y. Differential phospholipase D activation by bradykinin and sphingosine 1-phosphate in NIH 3T3 fibroblasts overexpressing gelsolin. J Biol Chem. 1999 Sep 24;274(39):27385–27391. doi: 10.1074/jbc.274.39.27385. [DOI] [PubMed] [Google Scholar]
  6. Banno Y., Takuwa Y., Akao Y., Okamoto H., Osawa Y., Naganawa T., Nakashima S., Suh P. G., Nozawa Y. Involvement of phospholipase D in sphingosine 1-phosphate-induced activation of phosphatidylinositol 3-kinase and Akt in Chinese hamster ovary cells overexpressing EDG3. J Biol Chem. 2001 Jul 23;276(38):35622–35628. doi: 10.1074/jbc.M105673200. [DOI] [PubMed] [Google Scholar]
  7. Banno Yoshiko. Regulation and possible role of mammalian phospholipase D in cellular functions. J Biochem. 2002 Mar;131(3):301–306. doi: 10.1093/oxfordjournals.jbchem.a003103. [DOI] [PubMed] [Google Scholar]
  8. Banno Yoshiko, Takuwa Yoh, Yamada Momoko, Takuwa Noriko, Ohguchi Kenji, Hara Akira, Nozawa Yoshinori. Involvement of phospholipase D in insulin-like growth factor-I-induced activation of extracellular signal-regulated kinase, but not phosphoinositide 3-kinase or Akt, in Chinese hamster ovary cells. Biochem J. 2003 Jan 15;369(Pt 2):363–368. doi: 10.1042/BJ20021368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brodie C., Blumberg P. M. Regulation of cell apoptosis by protein kinase c delta. Apoptosis. 2003 Jan;8(1):19–27. doi: 10.1023/a:1021640817208. [DOI] [PubMed] [Google Scholar]
  10. Cockcroft S. Signalling roles of mammalian phospholipase D1 and D2. Cell Mol Life Sci. 2001 Oct;58(11):1674–1687. doi: 10.1007/PL00000805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Datta S. R., Dudek H., Tao X., Masters S., Fu H., Gotoh Y., Greenberg M. E. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 1997 Oct 17;91(2):231–241. doi: 10.1016/s0092-8674(00)80405-5. [DOI] [PubMed] [Google Scholar]
  12. Diehl J. A., Cheng M., Roussel M. F., Sherr C. J. Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev. 1998 Nov 15;12(22):3499–3511. doi: 10.1101/gad.12.22.3499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Exton J. H. Regulation of phospholipase D. Biochim Biophys Acta. 1999 Jul 30;1439(2):121–133. doi: 10.1016/s1388-1981(99)00089-x. [DOI] [PubMed] [Google Scholar]
  14. Fang Y., Vilella-Bach M., Bachmann R., Flanigan A., Chen J. Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science. 2001 Nov 30;294(5548):1942–1945. doi: 10.1126/science.1066015. [DOI] [PubMed] [Google Scholar]
  15. Foster David A., Xu Lizhong. Phospholipase D in cell proliferation and cancer. Mol Cancer Res. 2003 Sep;1(11):789–800. [PubMed] [Google Scholar]
  16. Frohman M. A., Morris A. J. Phospholipase D structure and regulation. Chem Phys Lipids. 1999 Apr;98(1-2):127–140. doi: 10.1016/s0009-3084(99)00025-0. [DOI] [PubMed] [Google Scholar]
  17. Goetzl E. J., Kong Y., Mei B. Lysophosphatidic acid and sphingosine 1-phosphate protection of T cells from apoptosis in association with suppression of Bax. J Immunol. 1999 Feb 15;162(4):2049–2056. [PubMed] [Google Scholar]
  18. Honda A., Nogami M., Yokozeki T., Yamazaki M., Nakamura H., Watanabe H., Kawamoto K., Nakayama K., Morris A. J., Frohman M. A. Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation. Cell. 1999 Nov 24;99(5):521–532. doi: 10.1016/s0092-8674(00)81540-8. [DOI] [PubMed] [Google Scholar]
  19. Hong J. H., Oh S. O., Lee M., Kim Y. R., Kim D. U., Hur G. M., Lee J. H., Lim K., Hwang B. D., Park S. K. Enhancement of lysophosphatidic acid-induced ERK phosphorylation by phospholipase D1 via the formation of phosphatidic acid. Biochem Biophys Res Commun. 2001 Mar;281(5):1337–1342. doi: 10.1006/bbrc.2001.4517. [DOI] [PubMed] [Google Scholar]
  20. Ito Y., Nakashima S., Nozawa Y. Hydrogen peroxide-induced phospholipase D activation in rat pheochromocytoma PC12 cells: possible involvement of Ca2+-dependent protein tyrosine kinase. J Neurochem. 1997 Aug;69(2):729–736. doi: 10.1046/j.1471-4159.1997.69020729.x. [DOI] [PubMed] [Google Scholar]
  21. Iwasaki-Bessho Y., Banno Y., Yoshimura S., Ito Y., Kitajima Y., Nozawa Y. Decreased phospholipase D (PLD) activity in ceramide-induced apoptosis of human keratinocyte cell line HaCaT. J Invest Dermatol. 1998 Apr;110(4):376–382. doi: 10.1038/jid.1998.2. [DOI] [PubMed] [Google Scholar]
  22. Joseph Troy, Bryant Annika, Frankel Paul, Wooden Roger, Kerkhoff Eugen, Rapp Ulf R., Foster David A. Phospholipase D overcomes cell cycle arrest induced by high-intensity Raf signaling. Oncogene. 2002 May 16;21(22):3651–3658. doi: 10.1038/sj.onc.1205380. [DOI] [PubMed] [Google Scholar]
  23. Kang J. H., Shin I., Han J. S. Changes of phospholipase D activity in TNF-alpha and anti-Fas/Apo1 monoclonal antibody induced apoptosis in HL-60 and A20 cells. Exp Mol Med. 1998 Mar 31;30(1):21–27. doi: 10.1038/emm.1998.3. [DOI] [PubMed] [Google Scholar]
  24. Kasai T., Ohguchi K., Nakashima S., Ito Y., Naganawa T., Kondo N., Nozawa Y. Increased activity of oleate-dependent type phospholipase D during actinomycin D-induced apoptosis in Jurkat T cells. J Immunol. 1998 Dec 15;161(12):6469–6474. [PubMed] [Google Scholar]
  25. Kennedy S. G., Wagner A. J., Conzen S. D., Jordán J., Bellacosa A., Tsichlis P. N., Hay N. The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev. 1997 Mar 15;11(6):701–713. doi: 10.1101/gad.11.6.701. [DOI] [PubMed] [Google Scholar]
  26. Kim S., Jung Y., Kim D., Koh H., Chung J. Extracellular zinc activates p70 S6 kinase through the phosphatidylinositol 3-kinase signaling pathway. J Biol Chem. 2000 Aug 25;275(34):25979–25984. doi: 10.1074/jbc.M001975200. [DOI] [PubMed] [Google Scholar]
  27. Kleeff J., Kornmann M., Sawhney H., Korc M. Actinomycin D induces apoptosis and inhibits growth of pancreatic cancer cells. Int J Cancer. 2000 May 1;86(3):399–407. doi: 10.1002/(sici)1097-0215(20000501)86:3<399::aid-ijc15>3.0.co;2-g. [DOI] [PubMed] [Google Scholar]
  28. Kwun Hyun Jin, Lee Jae Hwa, Min Do Sik, Jang Kyung Lib. Transcriptional repression of cyclin-dependent kinase inhibitor p21 gene by phospholipase D1 and D2. FEBS Lett. 2003 Jun 5;544(1-3):38–44. doi: 10.1016/s0014-5793(03)00446-0. [DOI] [PubMed] [Google Scholar]
  29. Le Stunff Hervé, Peterson Courtney, Liu Hong, Milstien Sheldon, Spiegel Sarah. Sphingosine-1-phosphate and lipid phosphohydrolases. Biochim Biophys Acta. 2002 May 23;1582(1-3):8–17. doi: 10.1016/s1388-1981(02)00132-4. [DOI] [PubMed] [Google Scholar]
  30. Lee S. D., Lee B. D., Han J. M., Kim J. H., Kim Y., Suh P. G., Ryu S. H. Phospholipase D2 activity suppresses hydrogen peroxide-induced apoptosis in PC12 cells. J Neurochem. 2000 Sep;75(3):1053–1059. doi: 10.1046/j.1471-4159.2000.0751053.x. [DOI] [PubMed] [Google Scholar]
  31. Liscovitch M., Czarny M., Fiucci G., Tang X. Phospholipase D: molecular and cell biology of a novel gene family. Biochem J. 2000 Feb 1;345(Pt 3):401–415. [PMC free article] [PubMed] [Google Scholar]
  32. Loreni F., Thomas G., Amaldi F. Transcription inhibitors stimulate translation of 5' TOP mRNAs through activation of S6 kinase and the mTOR/FRAP signalling pathway. Eur J Biochem. 2000 Nov;267(22):6594–6601. doi: 10.1046/j.1432-1327.2000.01753.x. [DOI] [PubMed] [Google Scholar]
  33. Martelli A. M., Bortul R., Bareggi R., Tabellini G., Grill V., Baldini G., Narducci P. The pro-apoptotic drug camptothecin stimulates phospholipase D activity and diacylglycerol production in the nucleus of HL-60 human promyelocytic leukemia cells. Cancer Res. 1999 Aug 15;59(16):3961–3967. [PubMed] [Google Scholar]
  34. Mayo L. D., Donner D. B. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc Natl Acad Sci U S A. 2001 Aug 14;98(20):11598–11603. doi: 10.1073/pnas.181181198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mejillano M., Yamamoto M., Rozelle A. L., Sun H. Q., Wang X., Yin H. L. Regulation of apoptosis by phosphatidylinositol 4,5-bisphosphate inhibition of caspases, and caspase inactivation of phosphatidylinositol phosphate 5-kinases. J Biol Chem. 2000 Oct 20;276(3):1865–1872. doi: 10.1074/jbc.M007271200. [DOI] [PubMed] [Google Scholar]
  36. Min D. S., Kwon T. K., Park W. S., Chang J. S., Park S. K., Ahn B. H., Ryoo Z. Y., Lee Y. H., Lee Y. S., Rhie D. J. Neoplastic transformation and tumorigenesis associated with overexpression of phospholipase D isozymes in cultured murine fibroblasts. Carcinogenesis. 2001 Oct;22(10):1641–1647. doi: 10.1093/carcin/22.10.1641. [DOI] [PubMed] [Google Scholar]
  37. Nozawa Yoshinori. Roles of phospholipase D in apoptosis and pro-survival. Biochim Biophys Acta. 2002 Dec 30;1585(2-3):77–86. doi: 10.1016/s1388-1981(02)00327-x. [DOI] [PubMed] [Google Scholar]
  38. Okamoto H., Takuwa N., Yatomi Y., Gonda K., Shigematsu H., Takuwa Y. EDG3 is a functional receptor specific for sphingosine 1-phosphate and sphingosylphosphorylcholine with signaling characteristics distinct from EDG1 and AGR16. Biochem Biophys Res Commun. 1999 Jun 24;260(1):203–208. doi: 10.1006/bbrc.1999.0886. [DOI] [PubMed] [Google Scholar]
  39. Pyne S., Pyne N. J. Sphingosine 1-phosphate signalling in mammalian cells. Biochem J. 2000 Jul 15;349(Pt 2):385–402. doi: 10.1042/0264-6021:3490385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rizzo M. A., Shome K., Vasudevan C., Stolz D. B., Sung T. C., Frohman M. A., Watkins S. C., Romero G. Phospholipase D and its product, phosphatidic acid, mediate agonist-dependent raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway. J Biol Chem. 1999 Jan 8;274(2):1131–1139. doi: 10.1074/jbc.274.2.1131. [DOI] [PubMed] [Google Scholar]
  41. Takuwa Y., Okamoto H., Takuwa N., Gonda K., Sugimoto N., Sakurada S. Subtype-specific, differential activities of the EDG family receptors for sphingosine-1-phosphate, a novel lysophospholipid mediator. Mol Cell Endocrinol. 2001 May 25;177(1-2):3–11. doi: 10.1016/s0303-7207(01)00441-5. [DOI] [PubMed] [Google Scholar]
  42. Uchida N., Okamura S., Kuwano H. Phospholipase D activity in human gastric carcinoma. Anticancer Res. 1999 Jan-Feb;19(1B):671–675. [PubMed] [Google Scholar]
  43. Xu Y., Bialik S., Jones B. E., Iimuro Y., Kitsis R. N., Srinivasan A., Brenner D. A., Czaja M. J. NF-kappaB inactivation converts a hepatocyte cell line TNF-alpha response from proliferation to apoptosis. Am J Physiol. 1998 Oct;275(4 Pt 1):C1058–C1066. doi: 10.1152/ajpcell.1998.275.4.C1058. [DOI] [PubMed] [Google Scholar]
  44. Yamakawa H., Banno Y., Nakashima S., Sawada M., Yamada J., Yoshimura S., Nishimura Y., Nozawa Y., Sakai N. Increased phospholipase D2 activity during hypoxia-induced death of PC12 cells: its possible anti-apoptotic role. Neuroreport. 2000 Nov 9;11(16):3647–3650. doi: 10.1097/00001756-200011090-00049. [DOI] [PubMed] [Google Scholar]
  45. Yoshida M., Okamura S., Kodaki T., Mori M., Yamashita S. Enhanced levels of oleate-dependent and Arf-dependent phospholipase D isoforms in experimental colon cancer. Oncol Res. 1998;10(8):399–406. [PubMed] [Google Scholar]
  46. Zhao Y., Ehara H., Akao Y., Shamoto M., Nakagawa Y., Banno Y., Deguchi T., Ohishi N., Yagi K., Nozawa Y. Increased activity and intranuclear expression of phospholipase D2 in human renal cancer. Biochem Biophys Res Commun. 2000 Nov 11;278(1):140–143. doi: 10.1006/bbrc.2000.3719. [DOI] [PubMed] [Google Scholar]
  47. Zhong Minghao, Shen Yingjie, Zheng Yang, Joseph Troy, Jackson Desmond, Foster David A. Phospholipase D prevents apoptosis in v-Src-transformed rat fibroblasts and MDA-MB-231 breast cancer cells. Biochem Biophys Res Commun. 2003 Mar 14;302(3):615–619. doi: 10.1016/s0006-291x(03)00229-8. [DOI] [PubMed] [Google Scholar]
  48. Zhou H., Li X. M., Meinkoth J., Pittman R. N. Akt regulates cell survival and apoptosis at a postmitochondrial level. J Cell Biol. 2000 Oct 30;151(3):483–494. doi: 10.1083/jcb.151.3.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. van Dijk M. C., Postma F., Hilkmann H., Jalink K., van Blitterswijk W. J., Moolenaar W. H. Exogenous phospholipase D generates lysophosphatidic acid and activates Ras, Rho and Ca2+ signaling pathways. Curr Biol. 1998 Mar 26;8(7):386–392. doi: 10.1016/s0960-9822(98)70157-5. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES