- Open Access
Taurine protection of PC12 cells against endoplasmic reticulum stress induced by oxidative stress
© rentice and Wu; licensee BioMed Central Ltd. 2010
- Published: 24 August 2010
Taurine is a free amino acid present in high concentrations in a variety of organs of mammalians. As an antioxidant, taurine has been found to protect cells against oxidative stress, but the underlying mechanism is still unclear.
In this report, we present evidence to support the conclusion that taurine exerts a protective function against endoplasmic reticulum (ER) stress induced by H2O2 in PC 12 cells. Oxidative stress was introduced by exposure of PC 12 cells to 250 uM H2O2 for 4 hours.
It was found that the cell viability of PC 12 cells decreased with an increase of H2O2 concentration ranging from approximately 76% cell viability at 100 uM H2O2 down to 18% at 500 uM H2O2. At 250 uM H2O2, cell viability was restored to 80% by taurine at 25 mM. Furthermore, H2O2 treatment also caused a marked reduction in the expression of Bcl-2 while no significant change of Bax was observed. Treatment with taurine restored the reduced expression of Bcl-2 close to the control level without any obvious effect on Bax. Furthermore, taurine was also found to suppress up-regulation of GRP78, GADD153/CHOP and Bim induced by H2O2, suggesting that taurine may also exert a protective function against oxidative stress by reducing the ER stress.
In summary, taurine was shown to protect PC12 cells against oxidative stress induced by H2O2. ER stress was induced by oxidative stress and can be suppressed by taurine.
- PC12 Cell
- Endoplasmic Reticulum Stress
- Unfolded Protein Response
- Endoplasmic Reticulum Stress Response
Taurine, a sulfur-containing amino acid, is a free amino acid present in high concentrations in a variety of organs of most mammals, including brain, heart, kidneys . Taurine mediates many physiological functions, such as neuro-modulation, regulation of calcium-dependent processes, osmoregulation, thermoregulation, membrane stabilization and detoxication, neurotransmission and neuroprotection [2–6]. Taurine is known as an antioxidant to counteract oxidative stress, which is involved in many diseases, such as chronic lung disease, diabetes, Alzheimer's disease, Parkinson's disease and heart failure [7, 8]. A recent paper revealed that taurine plays an important role in reducing ER stress in C2C12 and 3T3L1 cells .
The ER is a key cell organelle that is responsible for synthesis and folding of proteins destined for secretion, cell membrane, Golgi apparatus, lysosomes and elsewhere, intracellular calcium homeostasis, and cell death signaling activation . Physiological or pathological processes that disturb protein folding in the ER lumen are referred to as ER stress, and a set of signaling pathways responding to ER stress is termed the Unfolded Protein Response (UPR) . ER stress has been recently implicated in inflammation, ischemia, heart disease, liver disease, kidney disease and neurodegenerative diseases, which include Parkinson’s, Alzheimer’s disease and polyglutamine disease [12–14]. The predominant signaling pathways associated with ER stress are initiated by the ER membrane-associated proteins, protein kinase R [PKR]-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6), which in turn activate distinct signaling cascades mediating the ER stress response [15–17]. Among these three major UPR signal transduction pathways, the IRE-1 and ATF-6 pathways increase the expression of the ER-resident chaperone, glucose-regulated protein 78 (GRP78) [18, 19], and all of these three pathways up-regulate the transcription factor C/EBP homologous protein (CHOP), also known as growth arrest and DNA damage-inducible gene 153 (GADD153) . CHOP/GADD153 regulates expression of several Bcl-2 family members. For example, CHOP decreases expression of antiapoptotic Bcl-2 , but increases expression of the proapoptotic Bim , thus contributing to cell death. The PERK pathway can also activate caspase-12, which plays an essential role in programmed cell death progression during the proapoptotic phase of the ER stress response . Recently, it has been suggested that oxidative stress and ER stress are closely linked events, although the molecular pathways that couple these processes are poorly understood . Moreover, GRP78 was shown to protect neurons against excitotoxicity and to suppress oxidative stress . In the present study, we demonstrated that taurine exerts a protective function against ER stress induced by oxidative stress in PC 12 cells.
F-12K media, trypsin-EDTA solution, horse serum and rat phenocromocytoma PC12 cell line were purchased from ATCC (Manassas, VA, USA). Fetal bovine serum, poly-D-lysine, taurine, Penicillin-Streptomycin and other chemicals were purchased from Sigma (St. Louis, MO, USA). Mouse anti-actin, rabbit anti-Bax, rabbit anti–Bcl-2, rabbit anti-GRP78, rabbit anti-CHOP/GADD153 antibodies, and secondary mouse and rabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-Bim antibody was purchased from assay designs (Ann Arbor, Michigan, USA). Adenosine 5’-triphosphate (ATP) Bioluminescent Assay Kit and 3, (4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide (MTT) assay kit were purchased from Promega (Madison, WI, USA) and ATCC (Manassas, VA, USA) respectively. RIPA buffer was purchased from thermo scientific (Rockford, IL, USA).
PC12 cells were maintained at 37oC/5% CO2 in F12-K medium supplemented with 2.5% (v/v) fetal bovine serum (FBS), 15% (v/v) heat-inactivated horse serum (HS) and 1% (v/v) penicillin-streptomycin solution. All experiments were performed on undifferentiated cells plated in 96-well plates at a density of approximately 5×104 cells/ml for the ATP assay, 1×105 cells/ml for the MTT assay and in 60mm petri dishes at 5×105 cells/well for western blot for 24 hours before starting the experiments. The 96-well plates or petri dishes were precoated with poly-D lysine before plating.
Measurement of cell viability
PC12 cells in 96-well plates were treated with or without 25 mM taurine for 1 hour, and then cells were exposed to 100-500 uM H2O2 for 4 hours to induce cell death. ATP solution (Promega) was added to each well and cells were incubated for 10 minutes, then the amount of ATP was quantified through a luciferase reaction. The luminescence intensity was determined using a luminometer (SpectraMax, Molecular Devices) after transferring the lysate to a standard opaque walled multi-well plate. The ATP content was determined by running an internal standard and expressed as a percentage of untreated cells (control).
PC12 cells in 96 well plates were treated with 25 mM Taurine for 1 hour and then cells were exposed to 250 uM H2O2 for 4 hours to induce cell death. Subsequently, 10 ul MTT reagent (ATCC) was added to each well and cells were incubated for 4 hours until a purple precipitate was visible. Then 100 ul detergent reagent was added and the solution was left at room temperature in the dark for 2 hours. The absorbance was detected with a microtiter plate reader at 570 nm.
Western blot analysis
PC12 cells were lysed in RIPA buffer (25 mM Tris_HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing 1% (v/v) mammalian protease inhibitor cocktail from Sigma and separated on SDS-PAGE, following by transferring to a nitrocellulose membrane. The membrane was then blocked in blocking buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, 5% milk) for 1.5 hours at room temperature. After blocking, corresponding primary antibody was incubated for one hour, followed by a one hour incubation with the corresponding HRP-conjugated secondary antibody at room temperature. Extensive washes with a blocking buffer were performed between each step. The protein immuno-complex was visualized by ECL detection reagents.
All data presented in the figures were expressed as the mean±SEM. The Student’s t-test or one-way ANOVA was used to compare means between groups. Differences of P<0.05 were considered statistically significant.
Dose dependent toxicity of H2O2 in PC12 cells
Taurine protected PC12 cells against H2O2 induced oxidative stress
Extracellular taurine elicited protection of PC12 cells against H2O2 induced oxidative stress
Taurine restored the expression of Bcl-2 and had no significant effect on the expression of Bax
Taurine reversed the H2O2-induced up-regulation of GRP78, CHOP/GADD 153 and Bim in PC12 cells
This paper presents evidence indicating that 1) extracellular taurine exerted a protective function against oxidative stress induced by H2O2 in PC12 cells, 2) Bcl-2 expression in PC12 cells was restored but not Bax expression after treatment with taurine, 3) H2O2 induced ER stress by up-regulation of GRP78, Bim and CHOP/GADD153 and 4) Taurine protected PC12 cells from ER stress induced by H2O2 through downregulation of GRP78, Bim and CHOP/GADD153.
As a naturally occurring antioxidant, taurine was investigated to treat oxidative stress trigged by different inducers, such as age-related retinal degeneration, a high cholesterol diet, lead poisoning and nitric oxide [32–35]. Although a number of studies proved that taurine has a protective function against oxidative stress, the mechanism underlying its protection is still not fully understood. Li et al. (2009)  demonstrated that taurine treatment alleviated the oxidative injury of the kidney, improved SOD and GSH-Px activities and prevented mitochondrial membrane injury. They found that taurine protected the kidney from oxidative injury through a mitochondrial-linked pathway . Here, we demonstrate that the function of taurine as a protectant against oxidative stress induced by H2O2 is via the alleviation of ER stress.
In summary, the results of our present study shed a light on the sequential relationship between oxidative stress and ER stress and the mechanism underlying protection by taurine against oxidative stress. Further study is warranted to examine in detail which specific pathway in ER can be activated or inhibited by taurine. In addition, it remains to be known exactly how oxidative stress triggers ER stress. Further scrutiny is necessary to elucidate the precise mechanism behind the oxidative stress-elicited ER stress. Our model suggests a key role for extracellular taurine in preventing ER stress induced by H2O2.
This work was supported in part by the James and Esther King Biomedical Research Program, Florida Department of Health.
This article has been published as part as part of Journal of Biomedical Science Volume 17 Supplement 1, 2010: Proceedings of the 17th International Meeting of Taurine. The full contents of the supplement are available online at http://www.jbiomedsci.com/supplements/17/S1.
- Huxtable RJ: Physiological actions of taurine. Physiol Rev. 1992, 72: 101-163.PubMedGoogle Scholar
- Schaffer SW, Azuma J: Myocardial physiological effects of taurine and their significance. Adv Exp Med Biol. 1992, 315: 105-120.View ArticlePubMedGoogle Scholar
- Schaffer S, Solodushko V, Azuma J: Taurine-deficient cardiomyopathy: role of phospholipids, calcium and osmotic stress. Adv Exp Med Biol. 2000, 483: 57-69. full_text.View ArticlePubMedGoogle Scholar
- Wu H, Jin Y, Wei J, Jin H, Sha D, Wu J-Y: Mode of action of taurine as a neuroprotector. Brain Res. 2005, 1038: 123-131. 10.1016/j.brainres.2005.01.058.View ArticlePubMedGoogle Scholar
- Leon R, Wu H, Jin Y, Wei J, Buddhala C, Prentice H, Wu J-Y: Protective function of taurine in glutamate-induced apoptosis in cultured neurons. J Neurosci Res. 2009, 87: 1185-1194. 10.1002/jnr.21926.View ArticlePubMedGoogle Scholar
- Chepkova AN, Doreulee N, Yanovsky Y, Mukhopadhyay D, Haas HL, Sergeeva OA: Long-lasting enhancement of corticostriatal neurotransmission by taurine. Eur J Neurosci. 2002, 16: 1523-1530. 10.1046/j.1460-9568.2002.02223.x.View ArticlePubMedGoogle Scholar
- Tsutsui H, Ide T, Shiomi T, Kang D, Hayashidani Shunji, Suematsu N, Wen J, Utsumi H, Hamasaki N, Takeshita : A 8-Oxo-dGTPase, which prevents oxidative stress-induced DNA damage, increases in the mitochondria from failing hearts. Circulation. 2001, 104: 2883-2885. 10.1161/hc4901.101347.View ArticlePubMedGoogle Scholar
- Spector A: Oxidative stress and disease. J Ocul Pharmacol Ther. 2000, 16: 193-201. 10.1089/jop.2000.16.193.View ArticlePubMedGoogle Scholar
- Song H, Kim H, Park T, Lee DH: Characterization of myogenic differentiation under endoplasmic reticulum stress and taurine treatment. Adv Exp Med Biol. 2009, 643: 253-61. full_text.View ArticlePubMedGoogle Scholar
- Baumann O, Walz BB: Endoplasmic reticulum of animal cells and its organization into structural and functional domains. Int Rev Cytol. 2001, 205: 149-214. full_text.View ArticlePubMedGoogle Scholar
- Lai E, Teodoro T, Volchuk A: Endoplasmic reticulum stress: Signaling the unfolded protein response. Physiology (Bethesda). 2007, 22: 193-201.View ArticleGoogle Scholar
- Hoozemans JJ, Veerhuis R, Van Haastert ES, Rozemuller JM, Baas F, Eikelenboom P, Scheper W: The unfolded protein response is activated in Alzheimer’s disease. Acta Neuropathol. 2005, 110: 165-172. 10.1007/s00401-005-1038-0.View ArticlePubMedGoogle Scholar
- Imai Y, Soda M, Inoue H, Hattori N, Mizuno Y, Takahashi R: An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin. Cell. 2001, 105: 891-902. 10.1016/S0092-8674(01)00407-X.View ArticlePubMedGoogle Scholar
- Yoshida H: ER stress and diseases. FEBS J. 2007, 274: 630-658. 10.1111/j.1742-4658.2007.05639.x.View ArticlePubMedGoogle Scholar
- Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D: PERK is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell. 2000, 5: 897-904. 10.1016/S1097-2765(00)80330-5.View ArticlePubMedGoogle Scholar
- Wang XZ, Harding HP, Zhang Y, Jolicoeur EM, Kuroda M, Ron D: Cloning of mammalian Ire1 reveals diversity in the ER stress responses. EMBO J. 1998, 17: 5708-5717. 10.1093/emboj/17.19.5708.PubMed CentralView ArticlePubMedGoogle Scholar
- Yoshida H, Haze K, Yanagi H, Yura T, Mori K: Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins Involvement of basic leucine zipper transcription factors. J Biol Chem. 1998, 273: 33741-33749. 10.1074/jbc.273.50.33741.View ArticlePubMedGoogle Scholar
- Kaufman RJ: Orchestrating the unfolded protein response in health and disease. J. Clin. Invest. 2002, 110: 1389-1398.PubMed CentralView ArticlePubMedGoogle Scholar
- Ji C, Kaplowitz N: Hyperhomocysteinemia, endoplasmic reticulum stress, and alcoholic liver injury. World J Gastroenterol. 2004, 10: 1699-1708.PubMed CentralView ArticlePubMedGoogle Scholar
- Oyadomari S, Mori M: Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004, 11: 381-389. 10.1038/sj.cdd.4401373.View ArticlePubMedGoogle Scholar
- McCullough KD, Martindale JL, Klotz LO, Aw TY, Holbrook NJ: Gadd153 sensitizes cells to endoplasmic reticulum stress by downregulating Bcl2 and perturbing the cellular redox state. Mol Cell Biol. 2001, 21: 1249-1259. 10.1128/MCB.21.4.1249-1259.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Puthalakath H, O’Reilly LA, Gunn P, Lee L, Kelly PN, Huntington ND, Hughes PD, Michalak EM, McKimm-Breschkin J, Motoyama N, Gotoh T, Akira S, Bouillet P, Strasser A: ER stress triggers apoptosis by activating BH3-only protein Bim. Cell. 2007, 129: 1337-1349. 10.1016/j.cell.2007.04.027.View ArticlePubMedGoogle Scholar
- Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, Yuan J: Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature. 2000, 403: 98-103. 10.1038/47513.View ArticlePubMedGoogle Scholar
- Zhang K: Integration of ER stress, oxidative stress and the inflammatory response in health and disease. Int J Clin Exp Med. 2010, 3: 33-40.PubMed CentralPubMedGoogle Scholar
- Yu Z, Luo H, Fu W, Mattson PM: The endoplasmic reticulum Stress-Responsive Protein GRP78 Protects Neurons Against Excitotoxicity and apoptosis: Suppression of oxidative stress and stabilization of calcium homeostasis. Exp Neurol. 1999, 155: 302-314. 10.1006/exnr.1998.7002.View ArticlePubMedGoogle Scholar
- Barakat L, Wang D, Bordey A: Carrier-mediated uptake and release of taurine from Bergmann glia in rat cerebellar slices. J Physiol. 2002, 541: 753-767. 10.1113/jphysiol.2001.015834.PubMed CentralView ArticlePubMedGoogle Scholar
- Anderson C, Howard A, Walters J, Ganapathy V, Thwaites D: Taurine uptake across the human intestinal brush-border membrane is via two transporters: H+-coupled PAT1(SLC36A1) and Na+ - and Cl- - dependent TauT(SlC6A6). J Physiol. 2009, 587: 731-744. 10.1113/jphysiol.2008.164228.PubMed CentralView ArticlePubMedGoogle Scholar
- Chesney RW, Budreau AM: Inhibitors of anion exchanger activity reduce sodium chloride-dependent taurine transport by brush border vesicles. Adv Exp Med Biol. 1994, 359: 111-120.View ArticlePubMedGoogle Scholar
- Oltvai ZN, Milliman CL, Korsmeyer SJ: Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993, 74: 609-619. 10.1016/0092-8674(93)90509-O.View ArticlePubMedGoogle Scholar
- Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ: Bad, a heterodimeric partner for Bcl-xL and Bcl-2, displaces Bax and promotes cell death. Cell. 1995, 80: 285-291. 10.1016/0092-8674(95)90411-5.View ArticlePubMedGoogle Scholar
- Wang XZ, Lawson B, Brewer JW, Zinszner H, Sanjay A: Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153). Mol Cell Biol. 1996, 16: 4273-4280.PubMed CentralView ArticlePubMedGoogle Scholar
- Militante J, Lombardini J: Age-related retinal degeneration in animal models of aging: Possible involvement of taurine deficiency and oxidative stress. Neurochem Res. 2004, 29: 151-160. 10.1023/B:NERE.0000010444.97959.1b.View ArticlePubMedGoogle Scholar
- Balkan J, Kanbagli O, Hatipoglu A, Kucuk M, Cevikbas U, Aykac-Toker G, Uysal M: Improving Effect of dietary taurine supplementation on the oxidative stress and lipid levels in the plasma, liver and aorta of rabbits fed on a high-cholesterol diet. Biosci Biotechnol Biochem. 2002, 66: 1755-1758. 10.1271/bbb.66.1755.View ArticlePubMedGoogle Scholar
- Gurer H, Ozgunes H, Saygin E, Ercal N: Antioxidant effect of taurine against lead-induced oxidative stress. Arch Environ Contam Toxicol. 2001, 41: 397-402. 10.1007/s002440010265.View ArticlePubMedGoogle Scholar
- Merezak S, Hardikar AA, Yajnik CS, Remacle C, Reusens B: Intrauterine low protein diet increases fetal β-cell sensitivity to NO and IL-1β: the protective role of taurine. J Endocrinol. 2001, 171: 299-308. 10.1677/joe.0.1710299.View ArticlePubMedGoogle Scholar
- Li CY, Deng YL, Sun BH: Taurine protected kidney from oxidative injury through mitochondrial-linked pathway in a rat model of nephrolithiasis. Urol Res. 2009, 37: 211-220. 10.1007/s00240-009-0197-1.View ArticlePubMedGoogle Scholar
- Kohen R, Nyska A: Oxidation of Biological Systems: Oxidative Stress Phenomena, Antioxidants, Redox Reactions, and Methods for Their Quantitation. Toxicol Pathol. 2002, 30: 620-650. 10.1080/01926230290166724.View ArticlePubMedGoogle Scholar
- Hayashi T, Saito A, Okuno S, Ferrand-Drake M, Dodd LR, Chan HP: Damage to the endoplasmic reticulum and activation of apoptotic machinery by oxidative stress in ischemic neurons. J Cerebr Blood Flow Metab. 2005, 25: 41-53. 10.1038/sj.jcbfm.9600005.View ArticleGoogle Scholar
- Zhang K: Integration of ER stress, oxidative stress and the inflammatory response in health and disease. Int J Clin Exp Med. 2010, 3: 33-40.PubMed CentralPubMedGoogle Scholar
- Ilieva VE, Ayala V, Mariona J, Dalfo E, Cacabelos D, Povedano M, Maria JB, Isidre F, Pamplona R, Portero-Otin M: Oxidative and endoplasmic reticulum stress interplay in sporadic amyotrophic lateral sclerosis. . Brain. 2007, 130: 3111-3123. 10.1093/brain/awm190.View ArticlePubMedGoogle Scholar
- He S, Yaung J, Kim YH, Barron E, Ryan JS, Hinton RD: Endoplasmic reticulum stress induced by oxidative stress in retinal pigment epithelial cells. Graefes Arch Clin Exp Ophthalmol. 2008, 246: 677-683. 10.1007/s00417-008-0770-2.View ArticlePubMedGoogle Scholar
- Hung CC, Ichimura T, Stevens LJ, Bonventre VJ: Protection of retinal epithelial cells against oxidative injury by endoplasmic reticulum stress preconditioning is mediated by ERK1/2 activation. J Biol Chem. 2003, 278: 29317-29326. 10.1074/jbc.M302368200.View ArticlePubMedGoogle Scholar
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