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Cell Stress and Chaperones

, Volume 23, Issue 6, pp 1283–1294 | Cite as

Palmitic acid induces human osteoblast-like Saos-2 cell apoptosis via endoplasmic reticulum stress and autophagy

  • Lei YangEmail author
  • Gaopeng Guan
  • Lanjie Lei
  • Qizhuang Lv
  • Shengyuan Liu
  • Xiuwen Zhan
  • Zhenzhen Jiang
  • Xiang GuEmail author
Original Paper

Abstract

Palmitic acid (PA) is the most common saturated long-chain fatty acid in food that causes cell apoptosis. However, little is known about the molecular mechanisms of PA toxicity. In this study, we explore the effects of PA on proliferation and apoptosis in human osteoblast-like Saos-2 cells and uncover the signaling pathways involved in the process. Our study showed that endoplasmic reticulum (ER) stress and autophagy are involved in PA-induced Saos-2 cell apoptosis. We found that PA inhibited the viability of Saos-2 cells in a dose- and time-dependent manner. At the same time, PA induced the expression of ER stress marker genes (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)), altered autophagy-related gene expression (microtubule-associated protein 1 light chain 3 (LC3), ATG5, p62, and Beclin), promoted apoptosis-related gene expression (Caspase 3 and BAX), and affected autophagic flux. Inhibiting ER stress with 4-PBA diminished the PA-induced cell apoptosis, activated autophagy, and increased the expression of Caspase 3 and BAX. Inhibiting autophagy with 3-MA attenuated the PA and ER stress-induced cell apoptosis and the apoptosis-related gene expression (Caspase 3 and BAX), but seemed to have no obvious effects on ER stress, although the CHOP expression was downregulated. Taken together, our results suggest that PA-induced Saos-2 cell apoptosis is activated via ER stress and autophagy, and the activation of autophagy depends on the ER stress during this process.

Keywords

Palmitic acid Endoplasmic reticulum stress Autophagy Saos-2 cells 

Notes

Funding information

This research study was funded by the National Natural Science Foundation of China (No. 81660152) and Doctoral Research Start-Up Foundation of Jiujiang University (No. 8879522).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12192_2018_936_MOESM1_ESM.docx (16 kb)
Supplementary Table 1 (DOCX 16 kb)
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Fig. S1

The morphology of Saos-2 cells after treatment with different doses of PA (0–800 μM) for 24 h. (PNG 18533 kb)

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High resolution image (TIF 26708 kb)
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Fig. S2

Effects of PA on ER Stress, autophagy, and apoptosis related protein expression. (A) The expression of GRP78 and CHOP in Saos-2 cells after treatment with different doses of PA (0–800 μM) for 24 h; (B) The expression of GRP78 and CHOP after treatment with 200 μM PA for different times (0–48 h); (C) The expression of Beclin1, LC3, ATG5 and p62 in Saos-2 cells after treatment with different doses of PA (0–800 μM) for 24 h; (D) The expression of Beclin1, LC3, ATG5 and p62 after treatment with 200 μM PA for different times (0–48 h); (E) The expression of BAX in Saos-2 cells after treatment with different dose of PA (0–800 μM) for 24 h; (F) The expression of BAX after treatment with 200 μM PA for different times (0–48 h). (PNG 395 kb)

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High resolution image (TIF 810 kb)
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Fig. S3

The related protein expression of ER Stress, autophagy, and apoptosis in different treatment groups. (A) Effect of 4-PBA in PA-treated Saos-2 cells; (B) Effect of 3-MA in PA-treated Saos-2 cells; (C) Effect of 3-MA in TG-treated Saos-2 cells. (PNG 1311 kb)

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Fig. S4

Amplified Fig. 5D. (PNG 2133 kb)

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High resolution image (TIF 2977 kb)
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Fig. S5

Amplified Fig. 6D. (PNG 1843 kb)

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Fig. S6

Amplified Fig. 7D. (PNG 4088 kb)

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High resolution image (TIF 7559 kb)

References

  1. Ben SI, Prola A, Boussabbeh M, Guilbert A, Bacha H, Abid-Essefi S, Lemaire C (2015) Crocin and quercetin protect HCT116 and HEK293 cells from Zearalenone-induced apoptosis by reducing endoplasmic reticulum stress. Cell Stress Chaperones 20:927–938CrossRefGoogle Scholar
  2. Cawley K, Deegan S, Samali A, Gupta S (2011) Assays for detecting the unfolded protein response. Methods Enzymol 490:31–51CrossRefGoogle Scholar
  3. Chamolstad H, Yu JE, Lee SH, Kim JG, Sung KS, Hwang J, Yoo YD, Lee YJ, Kim ST, Lee DH (2016) Modulation of SQSTM1/p62 activity by N-terminal arginylation of the endoplasmic reticulum chaperone HSPA5/GRP78/BiP. Autophagy 12:426–428CrossRefGoogle Scholar
  4. Chen YJ, Liu WH, Kao PH, Wang JJ, Chang LS (2010) Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells. Toxicon 55:1306–1316CrossRefGoogle Scholar
  5. Chen F, Li Q, Zhang Z, Lin P, Lei L, Wang A, Jin Y (2015) Endoplasmic reticulum stress cooperates in zearalenone-induced cell death of RAW 264.7 macrophages. Int J Mol Sci 16:19780–19795CrossRefGoogle Scholar
  6. Chen F, Lin P, Wang N, Yang D, Wen X, Zhou D, Wang A, Jin Y (2016) Herp depletion inhibits zearalenone-induced cell death in RAW 264.7 macrophages. Toxicol in Vitro 32:115–122CrossRefGoogle Scholar
  7. Cunha DAD, Hekerman P, Ladriere L, Bazarracastro A, Ortis F, Wakeham MC, Moore F, Rasschaert J, Cardozo AK, Bellomo EA (2008) Initiation and execution of lipotoxic ER stress in pancreatic beta-cells. J Cell Sci 121:2308–2318CrossRefGoogle Scholar
  8. Dikic I, Elazar Z (2018) Mechanism and medical implications of mammalian autophagy. Nat Rev Mol Cell Biol 19:349–364CrossRefGoogle Scholar
  9. Feng D, Wang B, Wang L, Abraham N, Tao K, Huang L, Shi W, Dong Y, Qu Y (2017) Pre-ischemia melatonin treatment alleviated acute neuronal injury after ischemic stroke by inhibiting ER stress-dependent autophagy via PERK and IRE1 signalings. J Pineal Res 62:e12395CrossRefGoogle Scholar
  10. Gunn P (2007) ER stress triggers apoptosis by activating BH3-only protein Bim. Cell 129:1337–1349CrossRefGoogle Scholar
  11. Gwak H, Kim S, Dhanasekaran DN, Song YS (2016) Resveratrol triggers ER stress-mediated apoptosis by disrupting N-linked glycosylation of proteins in ovarian cancer cells. Cancer Lett 371:347–353CrossRefGoogle Scholar
  12. Han J, Wang Y (2017) mTORC1 signaling in hepatic lipid metabolism. Protein Cell 1–7Google Scholar
  13. He Y, Zhou L, Fan Z, Liu S, Fang W (2018) Palmitic acid, but not high-glucose, induced myocardial apoptosis is alleviated by N-acetylcysteine due to attenuated mitochondrial-derived ROS accumulation-induced endoplasmic reticulum stress. Cell Death Dis 9:568CrossRefGoogle Scholar
  14. Hino S, Kondo S, Yoshinaga K, Saito A, Murakami T, Kanemoto S, Sekiya H, Chihara K, Aikawa Y, Hara H (2010) Regulation of ER molecular chaperone prevents bone loss in a murine model for osteoporosis. J Bone Miner Metab 28:131–138CrossRefGoogle Scholar
  15. Hsiao YH, Lin CI, Liao H, Chen YH, Lin SH (2014) Palmitic acid-induced neuron cell cycle G2/M arrest and endoplasmic reticular stress through protein palmitoylation in SH-SY5Y human neuroblastoma cells. Int J Mol Sci 15:20876–20899CrossRefGoogle Scholar
  16. Hsu HC, Li SJ, Chen CY, Chen MF (2017) Eicosapentaenoic acid protects cardiomyoblasts from lipotoxicity in an autophagy-dependent manner. Cell Biol Toxicol 1–13Google Scholar
  17. Huang Z, Wu S, Kong F, Cai X, Ye B, Shan P, Huang W (2017) MicroRNA-21 protects against cardiac hypoxia/reoxygenation injury by inhibiting excessive autophagy in H9c2 cells via the Akt/mTOR pathway. J Cell Mol Med 21:467–474CrossRefGoogle Scholar
  18. Jaishy B, Abel ED (2016) Lipids, lysosomes and autophagy. J Lipid Res 57:1619–1635CrossRefGoogle Scholar
  19. Jiang H, Liang C, Liu X, Jiang Q, He Z, Wu J, Pan X, Ren Y, Fan M, Li M (2010) Palmitic acid promotes endothelial progenitor cell apoptosis via p38 and JNK mitogen-activated protein kinase pathways. Atherosclerosis 210:71–77CrossRefGoogle Scholar
  20. Jiang X, Chen X, Wan J, Gui H, Ruan X, Du X (2017) Autophagy protects against palmitic acid-induced apoptosis in podocytes in vitro. Sci Rep 7:42764CrossRefGoogle Scholar
  21. Katsoulieris E, Mabley JG, Samai M, Green IC, Chatterjee PK (2009) Alpha-linolenic acid protects renal cells against palmitic acid lipotoxicity via inhibition of endoplasmic reticulum stress. Eur J Pharmacol 623:107–112CrossRefGoogle Scholar
  22. Lakhani SA, Masud A, Kuida K, Jr PG, Booth CJ, Mehal WZ, Inayat I, Flavell RA (2006) Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311:847–851CrossRefGoogle Scholar
  23. Las G, Shirihai OS (2010) The role of autophagy in β-cell lipotoxicity and type 2 diabetes. Diabetes Obes Metab 12:15–19CrossRefGoogle Scholar
  24. Leroy C, Tricot S, Lacour B, Grynberg A (2008) Protective effect of eicosapentaenoic acid on palmitate-induced apoptosis in neonatal cardiomyocytes. BBA-Biomembranes 1781:685–693PubMedGoogle Scholar
  25. Levine B, Yuan J (2005) Autophagy in cell death: an innocent convict? J Clin Invest 115:2679–2688CrossRefGoogle Scholar
  26. Li S, Li J, Shen C, Zhang X, Sun S, Cho M, Sun C, Song Z (2014) Tert-butylhydroquinone (tBHQ) protects hepatocytes against lipotoxicity via inducing autophagy independently of Nrf2 activation. BBA-Biomembranes 1841:22–33PubMedGoogle Scholar
  27. Li J, Yang S, Li X, Liu D, Wang Z, Guo J, Tan N, Gao Z, Zhao X, Zhang J (2017) Role of endoplasmic reticulum stress in disuse osteoporosis. Bone 97:2–14CrossRefGoogle Scholar
  28. Liang Q, Kobayashi S (2016) Mitochondrial quality control in the diabetic heart. J Mol Cell Cardiol 95:57–69CrossRefGoogle Scholar
  29. Lin P, Yang Y, Li X, Chen F, Cui C, Hu L, Li Q, Liu W, Jin Y (2012) Endoplasmic reticulum stress is involved in granulosa cell apoptosis during follicular atresia in goat ovaries. Mol Reprod Dev 79:423–432CrossRefGoogle Scholar
  30. Lin P, Chen F, Sun J, Zhou J, Wang X, Wang N, Li X, Zhang Z, Wang A, Jin Y (2015) Mycotoxin zearalenone induces apoptosis in mouse Leydig cells via an endoplasmic reticulum stress-dependent signalling pathway. Reprod Toxicol 52:71–77CrossRefGoogle Scholar
  31. Lisse TS, Thiele F, Fuchs H, Hans W, Przemeck GKH, Abe K, Rathkolb B, Quintanillamartinez L, Hoelzlwimmer G, Helfrich M (2008) ER stress-mediated apoptosis in a new mouse model of osteogenesis imperfecta. PLoS Genet 4:e7CrossRefGoogle Scholar
  32. Liu Y, Levine B (2015) Autosis and autophagic cell death: the dark side of autophagy. Cell Death Differ 22:367–376CrossRefGoogle Scholar
  33. Lu J, Wang Q, Huang L, Dong H, Lin L, Lin N, Zheng F, Tan J (2012) Palmitate causes endoplasmic reticulum stress and apoptosis in human mesenchymal stem cells: prevention by AMPK activator. Endocrinology 153:5275–5284CrossRefGoogle Scholar
  34. Ma Y, Shimizu Y, Mann MJ, Jin Y, Hendershot LM (2010) Plasma cell differentiation initiates a limited ER stress response by specifically suppressing the PERK-dependent branch of the unfolded protein response. Cell Stress Chaperones 15:281–293CrossRefGoogle Scholar
  35. Ma XH, Piao SF, Dey S, Mcafee Q, Karakousis G, Villanueva J, Hart LS, Levi S, Hu J, Zhang G (2014) Targeting ER stress-induced autophagy overcomes BRAF inhibitor resistance in melanoma. J Clin Invest 124:1406–1417CrossRefGoogle Scholar
  36. Maeyashiki C, Oshima S, Otsubo K, Kobayashi M, Nibe Y, Matsuzawa Y, Onizawa M, Nemoto Y, Nagaishi T, Okamoto R (2017) HADHA, the alpha subunit of the mitochondrial trifunctional protein, is involved in long-chain fatty acid-induced autophagy in intestinal epithelial cells. Biochem Bioph Res Commun 484:636–641CrossRefGoogle Scholar
  37. Martinez SC, Tanabe K, Cras-Méneur C, Abumrad NA, Bernal-Mizrachi E, Permutt MA (2008) Inhibition of Foxo1 protects pancreatic islet beta-cells against fatty acid and endoplasmic reticulum stress-induced apoptosis. Diabetes 57:846–859CrossRefGoogle Scholar
  38. Matsui Y, Takagi H, Qu X, Abdellatif M, Sakoda H, Asano T, Levine B, Sadoshima J (2007) Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ Res 100:914–922CrossRefGoogle Scholar
  39. Mei S, Ni H, Manley S, Bockus A, Kassel KM, Luyendyk JP, Copple BL, Ding W (2011) Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes. J Pharmacol Exp Ther 339:487–498CrossRefGoogle Scholar
  40. Momoi T (2006) ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation. Cell Death Differ 14:230–239PubMedGoogle Scholar
  41. Mu YM, Yanase T, Nishi Y, Tanaka A, Saito M, Jin CH, Mukasa C, Okabe T, Nomura M, Goto K (2001) Saturated FFAs, palmitic acid and stearic acid, induce apoptosis in human granulosa cells. Endocrinology 142:3590–3597CrossRefGoogle Scholar
  42. Porter AG, Jänicke RU (1999) Emerging roles of caspase-3 in apoptosis. Cell Death Differ 6:99–104CrossRefGoogle Scholar
  43. Qaisiya M, Brischetto C, Jašprová J, Vitek L, Tiribelli C, Bellarosa C (2016) Bilirubin-induced ER stress contributes to the inflammatory response and apoptosis in neuronal cells. Arch Toxicol 91:1–12Google Scholar
  44. Szegezdi E, Logue SE, Gorman AM, Samali A (2006) Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep 7:880–885CrossRefGoogle Scholar
  45. Szpigel A, Hainault I, Carlier A, Venteclef N, Batto AF, Hajduch E, Bernard C, Ktorza A, Gautier JF, Ferré P (2018) Lipid environment induces ER stress, TXNIP expression and inflammation in immune cells of individuals with type 2 diabetes. Diabetologia 61:399–412CrossRefGoogle Scholar
  46. Tan SH, Shui G, Zhou J, Li JJ, Bay BH, Wenk MR, Shen HM (2012) Induction of autophagy by palmitic acid via protein kinase C-mediated signaling pathway independent of mTOR (mammalian target of rapamycin). J Biol Chem 287:14364–14376CrossRefGoogle Scholar
  47. Tu QQ, Zheng RY, Li J, Hu L, Chang YX, Li L, Li MH, Wang RY, Huang DD, Wu MC (2014) Palmitic acid induces autophagy in hepatocytes via JNK2 activation. Acta Pharmacol Sin 35:504–512CrossRefGoogle Scholar
  48. Walter P, Ron D (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science 334:1081–1086CrossRefGoogle Scholar
  49. Wang X, Lin P, Yin Y, Zhou J, Lei L, Zhou X, Jin Y, Wang A (2015) Brucella suis vaccine strain S2-infected immortalized caprine endometrial epithelial cell lines induce non-apoptotic ER-stress. Cell Stress Chaperones 20:399–409CrossRefGoogle Scholar
  50. Wang X, Lin P, Li Y, Xiang C, Yin Y, Chen Z, Du Y, Zhou D, Jin Y, Wang A (2016) Brucella suis vaccine strain 2 induces endoplasmic reticulum stress that affects intracellular replication in goat trophoblast cells in vitro. Front Cell Infect Microbiol 6:19PubMedPubMedCentralGoogle Scholar
  51. Wen H, Wu Z, Hu H, Wu Y, Yang G, Lu J, Yang G, Guo G, Dong Q (2017) The anti-tumor effect of pachymic acid on osteosarcoma cells by inducing PTEN and Caspase 3/7-dependent apoptosis. J Nat Med 72:1–7Google Scholar
  52. Xia X, Kar R, Gluhakheinrich J, Yao W, Lane NE, Bonewald LF, Biswas SK, Lo W, Jiang JX (2010) Glucocorticoid-induced autophagy in osteocytes. J Bone Miner Res 25:2479–2488CrossRefGoogle Scholar
  53. Yamada H, Nakajima T, Domon H, Honda T, Yamazaki K (2015) Endoplasmic reticulum stress response and bone loss in experimental periodontitis in mice. J Periodontal Res 50:500–508CrossRefGoogle Scholar
  54. Yang Y, Cheung HH, Tu J, Miu KK, Chan W (2016a) New insights into the unfolded protein response in stem cells. Oncotarget 7:54010–54027PubMedPubMedCentralGoogle Scholar
  55. Yang Y, Pei X, Jin Y, Wang Y, Zhang C (2016b) The roles of endoplasmic reticulum stress response in female mammalian reproduction. Cell Tissue Res 363:589–597CrossRefGoogle Scholar
  56. Yong Z, Xue R, Zhang Z, Xia Y, Shi H (2012) Palmitic and linoleic acids induce ER stress and apoptosis in hepatoma cells. Lipids Health Dis 11:1CrossRefGoogle Scholar
  57. Zeng M, Sang W, Chen S, Chen R, Zhang H, Xue F, Li Z, Liu Y, Gong Y, Zhang H (2017) 4-PBA inhibits LPS-induced inflammation through regulating ER stress and autophagy in acute lung injury models. Toxicol Lett 271:26–37CrossRefGoogle Scholar
  58. Zhang Y, Yang X, Shi H, Dong L, Bai J (2011) Effect of α-linolenic acid on endoplasmic reticulum stress-mediated apoptosis of palmitic acid lipotoxicity in primary rat hepatocytes. Lipids Health Dis 10:122CrossRefGoogle Scholar
  59. Zhang W, Meng H, Yang R, Yang M, Sun G, Liu J, Shi P, Liu F, Yang B (2016) Melatonin suppresses autophagy in type 2 diabetic osteoporosis. Oncotarget 7:52179–52194PubMedPubMedCentralGoogle Scholar
  60. Zhao L, Jiang H, Papasian CJ, Maulik D, Drees BM, Hamilton JJ, Deng H (2007a) Correlation of obesity and osteoporosis: effect of fat mass on the determination of osteoporosis. J Bone Miner Res 23:17–29CrossRefGoogle Scholar
  61. Zhao L, Liu Y, Liu P, Hamilton JJ, Recker RR, Deng H (2007b) Relationship of obesity with osteoporosis. J Clin Endocrinol Metab 92:1640–1646CrossRefGoogle Scholar
  62. Zheng YZ, Cao ZG, Hu X, Shao ZM (2014) The endoplasmic reticulum stress markers GRP78 and CHOP predict disease-free survival and responsiveness to chemotherapy in breast cancer. Breast Cancer Res Treat 145:349–358CrossRefGoogle Scholar
  63. Zhu Q, Yang J, Zhu R, Jiang X, Li W, He S, Jin J (2016) Dihydroceramide-desaturase-1-mediated caspase 9 activation through ceramide plays a pivotal role in palmitic acid-induced HepG2 cell apoptosis. Apoptosis 21:1033–1044CrossRefGoogle Scholar

Copyright information

© Cell Stress Society International 2018

Authors and Affiliations

  1. 1.College of Basic MedicalJiujiang UniversityJiujiangChina
  2. 2.Key Laboratory of System Bio-medicine of Jiangxi ProvinceJiujiang UniversityJiujiangChina
  3. 3.Affiliated Hospital of Jiujiang UniversityJiujiang UniversityJiujiangChina
  4. 4.Medicine Graduate SchoolNanchang UniversityNanchangChina
  5. 5.College of Biology & PharmacyYulin Normal UniversityYulinChina

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