Cl--Channel Is Essential for LDL-induced Cell Proliferation via the Activation of Erk1/2 and PI3K/Akt and the Upregulation of Egr-1 in Human Aortic Smooth Muscle Cells

  • Heo, Kyung-Sun (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Ryoo, Sung-Woo (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Lila (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Nam, Miyoung (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Baek, Seung-Tae (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Lee, Hyemi (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Lee, Ah-Reum (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Park, Song-Kyu (Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Park, Youngwoo (Therapeutic Antibody Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Myung, Chang-Seon (Department of Pharmacy, Chungnam National University) ;
  • Kim, Dong-Uk (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Hoe, Kwang-Lae (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology)
  • Received : 2008.04.30
  • Accepted : 2008.08.14
  • Published : 2008.11.30

Abstract

Low-density lipoprotein (LDL) induces cell proliferation in human aortic smooth muscle cells (hAoSMCs), which may be involved in atherogenesis and intimal hyperplasia. Recent studies have demonstrated that $Cl^-$ channels are related to vessel cell proliferation induced by a variety of stimuli. In this study, we investigated a potential role of $Cl^-$ channels in the signaling pathway of LDL effects on hAoSMC proliferation with a focus on the activation of Erk1/2-PI3K/Akt and the subsequent upregulation of Egr-1. $Cl^-$ channel blockers, DIDS, but neither NPPB nor Furosemide, completely abolished the LDL-induced DNA synthesis and cell proliferation. Moreover, DIDS, but not NPPB, significantly decreased LDL-stimulated $Cl^-$ concentration, as judged by flow cytometry analysis using MQAE as a $Cl^-$-detection dye. DIDS pretreatment completely abolished the activation of Erk1/2 and PI3K/Akt in a dose-dependent manner that is the hallmark of LDL activation, as judged by Western blot and proliferation assays. Moreover, pretreatment with DIDS ($Cl^-$ channel blockers) but not LY294002 (PI3K inhibitors) completely abolished the LDL-induced upregulation of Egr-1 to the same extent as PD98059 (MEK inhibitors to inhibit Erk), as judged by Western blot and luciferase reporter assays. This is the first report, to our knowledge, that DIDS-sensitive $Cl^-$-channels play a key role in the LDL-induced cell proliferation of hAoSMCs via the activation of Erk1/2 and PI3K/Akt and the upregulation of Egr-1.

Keywords

$Cl^-$ channel;DIDS;Egr-1;low-density lipoprotein;PI3K

References

  1. Guillemot, L., Levy, A., Raymondjean, M., and Rothhut, B. (2001). Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT(1A) cells. J. Biol. Chem. 276, 39394-39403 https://doi.org/10.1074/jbc.M103862200
  2. Heo, K.S., Kim, D.U., Ryoo, S., Nam, M., Baek, S.T., Kim, L., Park, S.K., Myung, C.S., and Hoe, K.L. (2007). PPAR$\gamma$ activation abolishes LDL-induced proliferation of human aortic smooth muscle cells via SOD-mediated down-regulation of superoxide. Biochem. Biophys. Res. Commun. 359, 1017-1023 https://doi.org/10.1016/j.bbrc.2007.06.006
  3. Li, R.C., Cindrova-Davies, T., Skepper, J.N., and Sellers, L.A. (2004). Prostacyclin induces apoptosis of vascular smooth muscle cells by a cAMP-mediated inhibition of extracellular signalregulated kinase activity and can counteract the mitogenic activity of endothelin-1 or basic fibroblast growth factor. Circ. Res. 94, 759-767 https://doi.org/10.1161/01.RES.0000121568.40692.97
  4. Locher, R., Brandes, R.P., Vetter, W., and Barton, M. (2002). Native LDL induces proliferation of human vascular smooth muscle cells via redox-mediated activation of ERK 1/2 mitogen-activated protein kinases. Hypertension 39, 645-650 https://doi.org/10.1161/hy0202.103473
  5. Nilius, B., and Droogmans, G. (2003). Amazing chloride channels: an overview. Acta Physiol. Scand. 177, 119-147 https://doi.org/10.1046/j.1365-201X.2003.01060.x
  6. Ross, R. (1999). Atherosclerosis--an inflammatory disease. N. Engl. J. Med. 340, 115-126 https://doi.org/10.1056/NEJM199901143400207
  7. Rouzaire-Dubois, B., Milandri, J.B., Bostel, S., and Dubois, J.M. (2000). Control of cell proliferation by cell volume alterations in rat C6 glioma cells. Pflugers Arch. 440, 881-888 https://doi.org/10.1007/s004240000371
  8. Sachinidis, A., Schulte, K.B., Ko, Y., Seul, C., Meyer zu Brickwedde, M.K., Dusing, R., and Vetter, H. (1996). Oligodeoxynucleotides directed to early growth response gene-1 mRNA inhibit DNA synthesis in the smooth muscle cell. Eur. J. Pharmacol. 309, 95-105 https://doi.org/10.1016/0014-2999(96)00323-8
  9. Santiago, F.S., Lowe, H.C., Kavurma, M.M., Chesterman, C.N., Baker, A., Atkins, D.G., and Khachigian, L.M. (1999). New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury. Nat. Med. 5, 1264-1269 https://doi.org/10.1038/15215
  10. Voets, T., Szucs, G., Droogmans, G., and Nilius, B. (1995). Blockers of volume-activated Cl- currents inhibit endothelial cell proliferation. Pflugers Arch. 431, 132-134 https://doi.org/10.1007/BF00374387
  11. Xiao, G.N., Guan, Y.Y., and He, H. (2002). Effects of Cl- channel blockers on endothelin-1-induced proliferation of rat vascular smooth muscle cells. Life Sci. 70, 2233-2241 https://doi.org/10.1016/S0024-3205(02)01508-4
  12. Rocic, P., Govindarajan, G., Sabri, A., and Lucchesi, P.A. (2001). A role for PYK2 in regulation of ERK1/2 MAP kinases and PI 3-kinase by ANG II in vascular smooth muscle. Am. J. Physiol. Cell. Physiol. 280, C90-99 https://doi.org/10.1152/ajpcell.2001.280.1.C90
  13. Kamimura, M., Bea, F., Akizawa, T., Katus, H.A., Kreuzer, J., and Viedt, C. (2004). Platelet-derived growth factor induces tissue factor expression in vascular smooth muscle cells via activation of Egr-1. Hypertension 44, 944-951 https://doi.org/10.1161/01.HYP.0000146908.75091.99
  14. Auge, N., Maupas-Schwalm, F., Elbaz, M., Thiers, J.C., Waysbort, A., Itohara, S., Krell, H.W., Salvayre, R., and Negre-Salvayre, A. (2004). Role for matrix metalloproteinase-2 in oxidized lowdensity lipoprotein-induced activation of the sphingomyelin/ ceramide pathway and smooth muscle cell proliferation. Circulation 110, 571-578 https://doi.org/10.1161/01.CIR.0000136995.83451.1D
  15. Goncharova, E.A., Ammit, A.J., Irani, C., Carroll, R.G., Eszterhas, A.J., Panettieri, R.A., and Krymskaya, V.P. (2002). PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells. Am. J. Physiol. Lung Cell Mol. Physiol. 283, L354-363 https://doi.org/10.1152/ajplung.00010.2002
  16. Koncz, C., and Daugirdas, J.T. (1994). Use of MQAE for measurement of intracellular [Cl-] in cultured aortic smooth muscle cells. Am. J. Physiol. 267, H2114-2123
  17. McCaffrey, T.A., Fu, C., Du, B., Eksinar, S., Kent, K.C., Bush, H., Jr., Kreiger, K., Rosengart, T., Cybulsky, M.I., Silverman, E.S., et al. (2000). High-level expression of Egr-1 and Egr-1-inducible genes in mouse and human atherosclerosis. J. Clin. Invest. 105, 653-662 https://doi.org/10.1172/JCI8592
  18. Watanabe, T., Pakala, R., Koba, S., Katagiri, T., and Benedict, C.R. (2001). Lysophosphatidylcholine and reactive oxygen species mediate the synergistic effect of mildly oxidized LDL with serotonin on vascular smooth muscle cell proliferation. Circulation 103, 1440-1445 https://doi.org/10.1161/01.CIR.103.10.1440
  19. Castelli, W.P. (1984). Epidemiology of coronary heart disease: the Framingham study. Am. J. Med. 76, 4-12 https://doi.org/10.1016/0002-9343(84)90952-5
  20. Calabro, P., Samudio, I., Willerson, J.T., and Yeh, E.T. (2004).Resistin promotes smooth muscle cell proliferation through activation of extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase pathways. Circulation 110, 3335-3340 https://doi.org/10.1161/01.CIR.0000147825.97879.E7
  21. Bortner, C.D., and Cidlowski, J.A. (2004). The role of apoptotic volume decrease and ionic homeostasis in the activation and repression of apoptosis. Pflugers Arch. 448, 313-318 https://doi.org/10.1007/s00424-004-1266-5
  22. Sarosi, G.A., Jr., Jaiswal, K., Herndon, E., Lopez-Guzman, C., Spechler, S.J., and Souza, R.F. (2005). Acid increases MAPKmediated proliferation in Barrett's esophageal adenocarcinoma cells via intracellular acidification through a Cl-/HCO3- exchanger. Am. J. Physiol. Gastrointest. Liver Physiol. 289, G991-997 https://doi.org/10.1152/ajpgi.00215.2005
  23. Guan, Y.Y., Wang, G.L., and Zhou, J.G. (2006). The ClC-3 Cl(-) channel in cell volume regulation, proliferation and apoptosis in vascular smooth muscle cells. Trends Pharmacol. Sci. 27, 290-296 https://doi.org/10.1016/j.tips.2006.04.008
  24. Zhou, J.G., Ren, J.L., Qiu, Q.Y., He, H., and Guan, Y.Y. (2005). Regulation of intracellular Cl- concentration through volumeregulated ClC-3 chloride channels in A10 vascular smooth muscle cells. J. Biol. Chem. 280, 7301-7308 https://doi.org/10.1074/jbc.M412813200
  25. Sachinidis, A., Ko, Y., Wieczorek, A., Weisser, B., Locher, R., Vetter, W., and Vetter, H. (1993). Lipoproteins induce expression of the early growth response gene-1 in vascular smooth muscle cells from rat. Biochem. Biophys. Res. Commun. 192, 794-799 https://doi.org/10.1006/bbrc.1993.1484
  26. Wetzker, R., and Bohmer, F.D. (2003). Transactivation joins multiple tracks to the ERK/MAPK cascade. Nat. Rev. Mol. Cell. Biol. 4, 651-657 https://doi.org/10.1038/nrm1173
  27. Gervais, M., Dugourd, C., Muller, L., Ardidie, C., Canton, B., Loviconi, L., Corvol, P., Chneiweiss, H., and Monnot, C. (2006). Akt down-regulates ERK1/2 nuclear localization and angiotensin IIinduced cell proliferation through PEA-15. Mol. Biol. Cell 17, 3940-3951 https://doi.org/10.1091/mbc.E06-06-0501
  28. Voets, T., Wei, L., De Smet, P., Van Driessche, W., Eggermont, J., Droogmans, G., and Nilius, B. (1997). Downregulation of volume- activated Cl- currents during muscle differentiation. Am. J. Physiol. 272, C667-674 https://doi.org/10.1152/ajpcell.1997.272.2.C667
  29. Sachinidis, A., Mengden, T., Locher, R., Brunner, C., and Vetter, W. (1990). Novel cellular activities for low density lipoprotein in vascular smooth muscle cells. Hypertension 15, 704-711 https://doi.org/10.1161/01.HYP.15.6.704
  30. Duan, C., Bauchat, J.R., and Hsieh, T. (2000). Phosphatidylinositol 3-kinase is required for insulin-like growth factor-I-induced vascular smooth muscle cell proliferation and migration. Circ. Res. 86, 15-23 https://doi.org/10.1161/01.RES.86.1.15
  31. Wondergem, R., Gong, W., Monen, S.H., Dooley, S.N., Gonce, J.L., Conner, T.D., Houser, M., Ecay, T.W., and Ferslew, K.E. (2001). Blocking swelling-activated chloride current inhibits mouse liver cell proliferation. J. Physiol. 532, 661-672 https://doi.org/10.1111/j.1469-7793.2001.0661e.x
  32. Heo, K.S., Kim, D.U., Kim, L., Nam, M., Baek, S.T., Park, S.K., Park Y., Myung, C.S., Hwang S.O., and Hoe, K.L. (2008). Activation of PKC${\beta}_{II}$ and PKC$\theta$ is essential for LDL-induced cell proliferation of human aortic smooth muscle cells via Gi-mediated Erk1/2 activation and Egr-1 upregulation. Biochem. Biophys. Res. Commun. 368, 126-131 https://doi.org/10.1016/j.bbrc.2008.01.050
  33. Ryoo, S.W., Kim, D.U., Won, M., Chung, K.S., Jang, Y.J., Oh, G.T., Park, S.K., Maeng, P.J., Yoo, H.S., and Hoe, K.L. (2004). Native LDL induces interleukin-8 expression via H2O2, p38 Kinase, and activator protein-1 in human aortic smooth muscle cells. Cardiovasc. Res. 62, 185-193 https://doi.org/10.1016/j.cardiores.2004.01.002