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Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX–MAPK pathway

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Abstract

Impaired wound healing is a major diabetes-related complication. Keratinocytes play an important role in wound healing. Multiple factors have been proposed that can induce dysfunction in keratinocytes. The focus of present research is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing human immortalized keratinocyte (HaCaT) cell apoptosis and the cellular mechanism underlying the proapoptotic effect of AOPPs. HaCaT cells were treated with increasing concentrations of AOPP–human serum albumin or for increasing time durations. The cell viability was measured using the thiazolyl blue tetrazolium bromide method, and flow cytometry was used to assess the rate of cell apoptosis. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed through a confocal laser scanning microscope system, and the level of ROS generation was determined using a microplate reader. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)4, extracellular signal–regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and apoptosis-related downstream protein interactions were investigated using the Western blot analysis. We found that AOPPs triggered HaCaT cell apoptosis and MMP loss. After AOPP treatment, intracellular ROS generation increased in a time- and dose-dependent manner. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and poly(ADP-ribose) polymerase (PARP)-1 were activated, whereas anti-apoptotic Bcl-2 protein was downregulated. AOPPs also increased NOX4, ERK1/2, and p38 MAPK expression. Taken together, these findings suggest that extracellular AOPP accumulation triggered NOX-dependent ROS production, which activated ERK1/2 and p38 MAPK, and induced HaCaT cell apoptosis by activating caspase 3 and PARP-1.

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References

  1. Velnar T, Bailey T, Smrkolj V (2009) The wound healing process: an overview of the cellular and molecular mechanisms. J Int Med Res 37:1528–1542

    Article  CAS  PubMed  Google Scholar 

  2. Lundvig DM, Pennings SW, Brouwer KM et al (2015) Cytoprotective responses in HaCaT keratinocytes exposed to high doses of curcumin. Exp Cell Res 336:298–307

    Article  CAS  PubMed  Google Scholar 

  3. Stojadinovic O, Pastar I, Vukelic S et al (2008) Deregulation of keratinocyte differentiation and activation: a hallmark of venous ulcers. J Cell Mol Med 12:2675–2690

    Article  PubMed  PubMed Central  Google Scholar 

  4. Lamers ML, Almeida ME, Vicente-Manzanares M, Horwitz AF, Santos MF (2011) High glucose-mediated oxidative stress impairs cell migration. PLoS ONE 6:e22865

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Benavente CA, Jacobson EL (2008) Niacin restriction upregulates NADPH oxidase and reactive oxygen species (ROS) in human keratinocytes. Free Radic Biol Med 44:527–537

    Article  CAS  PubMed  Google Scholar 

  6. Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87:245–313

    Article  CAS  PubMed  Google Scholar 

  7. Chamulitrat W, Stremmel W, Kawahara T et al (2004) A constitutive NADPH oxidase-like system containing gp91phox homologs in human keratinocytes. J Invest Dermatol 122:1000–1009

    Article  CAS  PubMed  Google Scholar 

  8. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J (2007) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39:44–84

    Article  CAS  PubMed  Google Scholar 

  9. Nam HJ, Park YY, Yoon G, Cho H, Lee JH (2010) Co-treatment with hepatocyte growth factor and TGF-beta1 enhances migration of HaCaT cells through NADPH oxidase-dependent ROS generation. Exp Mol Med 42:270–279

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ott M, Gogvadze V, Orrenius S, Zhivotovsky B (2007) Mitochondria, oxidative stress and cell death. Apoptosis 12:913–922

    Article  CAS  PubMed  Google Scholar 

  11. Chiu TM, Huang CC, Lin TJ, Fang JY, Wu NL, Hung CF (2009) In vitro and in vivo anti-photoaging effects of an isoflavone extract from soybean cake. J Ethnopharmacol 126:108–113

    Article  CAS  PubMed  Google Scholar 

  12. Rodriguez PG, Felix FN, Woodley DT, Shim EK (2008) The role of oxygen in wound healing: a review of the literature. Dermatol Surg 34:1159–1169

    CAS  PubMed  Google Scholar 

  13. Nowotny K, Jung T, Hohn A, Weber D, Grune T (2015) Advanced glycation end products and oxidative stress in type 2 diabetes mellitus. Biomolecules 5:194–222

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Taylor EL, Armstrong KR, Perrett D, Hattersley AT, Winyard PG (2015) Optimisation of an advanced oxidation protein products assay: its application to studies of oxidative stress in diabetes mellitus. Oxid Med Cell Longev 2015:496271

    Article  PubMed  PubMed Central  Google Scholar 

  15. Witko-Sarsat V, Friedlander M, Nguyen KT et al (1998) Advanced oxidation protein products as novel mediators of inflammation and monocyte activation in chronic renal failure. J Immunol 161:2524–2532

    CAS  PubMed  Google Scholar 

  16. Porto ML, Lirio LM, Dias AT et al (2015) Increased oxidative stress and apoptosis in peripheral blood mononuclear cells of fructose-fed rats. Toxicol in Vitro 29:1977–1981

    Article  CAS  PubMed  Google Scholar 

  17. Xie F, Sun S, Xu A et al (2014) Advanced oxidation protein products induce intestine epithelial cell death through a redox-dependent, c-jun N-terminal kinase and poly (ADP-ribose) polymerase-1-mediated pathway. Cell Death Dis 5:e1006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Valente AJ, Yoshida T, Clark RA, Delafontaine P, Siebenlist U, Chandrasekar B (2013) Advanced oxidation protein products induce cardiomyocyte death via Nox2/Rac1/superoxide-dependent TRAF3IP2/JNK signaling. Free Radic Biol Med 60:125–135

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tabak O, Gelisgen R, Erman H et al (2011) Oxidative lipid, protein, and DNA damage as oxidative stress markers in vascular complications of diabetes mellitus. Clin Invest Med 34:E163–E171

    CAS  PubMed  Google Scholar 

  20. Chawla D, Bansal S, Banerjee BD, Madhu SV, Kalra OP, Tripathi AK (2014) Role of advanced glycation end product (AGE)-induced receptor (RAGE) expression in diabetic vascular complications. Microvasc Res 95:1–6

    Article  CAS  PubMed  Google Scholar 

  21. Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106:761–771

    Article  CAS  PubMed  Google Scholar 

  22. Koybasi S, Senkal CE, Sundararaj K et al (2004) Defects in cell growth regulation by C18:0-ceramide and longevity assurance gene 1 in human head and neck squamous cell carcinomas. J Biol Chem 279:44311–44319

    Article  CAS  PubMed  Google Scholar 

  23. Lee YH, Su SB, Huang CC et al (2014) N-acetylcysteine attenuates hexavalent chromium-induced hypersensitivity through inhibition of cell death, ROS-related signaling and cytokine expression. PLoS ONE 9:e108317

    Article  PubMed  PubMed Central  Google Scholar 

  24. Firth JD, Putnins EE (2004) Keratinocyte growth factor 1 inhibits wound edge epithelial cell apoptosis in vitro. J Invest Dermatol 122:222–231

    Article  CAS  PubMed  Google Scholar 

  25. Amar SK, Goyal S, Mujtaba SF et al (2015) Role of type I & type II reactions in DNA damage and activation of caspase 3 via mitochondrial pathway induced by photosensitized benzophenone. Toxicol Lett 235:84–95

    Article  CAS  PubMed  Google Scholar 

  26. Fischer TW, Zmijewski MA, Wortsman J, Slominski A (2008) Melatonin maintains mitochondrial membrane potential and attenuates activation of initiator (casp-9) and effector caspases (casp-3/casp-7) and PARP in UVR-exposed HaCaT keratinocytes. J Pineal Res 44:397–407

    Article  CAS  PubMed  Google Scholar 

  27. Hseu YC, Lo HW, Korivi M, Tsai YC, Tang MJ, Yang HL (2015) Dermato-protective properties of ergothioneine through induction of Nrf2/ARE-mediated antioxidant genes in UVA-irradiated Human keratinocytes. Free Radic Biol Med 86:102–117

    Article  CAS  PubMed  Google Scholar 

  28. Yang CT, Zhao Y, Xian M et al (2014) A novel controllable hydrogen sulfide-releasing molecule protects human skin keratinocytes against methylglyoxal-induced injury and dysfunction. Cell Physiol Biochem 34:1304–1317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chipuk JE, Bouchier-Hayes L, Green DR (2006) Mitochondrial outer membrane permeabilization during apoptosis: the innocent bystander scenario. Cell Death Differ 13:1396–1402

    Article  CAS  PubMed  Google Scholar 

  30. Ago T, Nunoi H, Ito T, Sumimoto H (1999) Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47(phox). Triple replacement of serines 303, 304, and 328 with aspartates disrupts the SH3 domain-mediated intramolecular interaction in p47(phox), thereby activating the oxidase. J Biol Chem 274:33644–33653

    Article  CAS  PubMed  Google Scholar 

  31. Cross AR, Erickson RW, Curnutte JT (1999) The mechanism of activation of NADPH oxidase in the cell-free system: the activation process is primarily catalytic and not through the formation of a stoichiometric complex. Biochem J 341(Pt 2):251–255

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Cheng G, Cao Z, Xu X, van Meir EG, Lambeth JD (2001) Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene 269:131–140

    Article  CAS  PubMed  Google Scholar 

  33. Pearson G, Robinson F, Beers GT et al (2001) Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22:153–183

    CAS  PubMed  Google Scholar 

  34. Kyriakis JM, Avruch J (2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81:807–869

    CAS  PubMed  Google Scholar 

  35. Chouinard N, Valerie K, Rouabhia M, Huot J (2002) UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53. BiochemJ 365:133–145

    Article  CAS  Google Scholar 

  36. Lee ER, Kang YJ, Kim JH, Lee HT, Cho SG (2005) Modulation of apoptosis in HaCaT keratinocytes via differential regulation of ERK signaling pathway by flavonoids. J Biol Chem 280:31498–31507

    Article  CAS  PubMed  Google Scholar 

  37. Lu Z, Xu S (2006) ERK1/2 MAP kinases in cell survival and apoptosis. IUBMB Life 58:621–631

    Article  CAS  PubMed  Google Scholar 

  38. Gotway MB, Freemer MM, King TJ (2007) Challenges in pulmonary fibrosis. 1: use of high resolution CT scanning of the lung for the evaluation of patients with idiopathic interstitial pneumonias. Thorax 62:546–553

    Article  PubMed  PubMed Central  Google Scholar 

  39. Descamps-Latscha B, Witko-Sarsat V (2001) Importance of oxidatively modified proteins in chronic renal failure. Kidney Int Suppl 78:S108–S113

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by Science and Technology Planning Project of Guangdong Province, China (2013B040401013).

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    Authors and Affiliations

    1. Post-graduate School, Southern Medical University, Guangzhou, Guangdong, China

      Baihui Sun & Bulin Wang

    2. Plastic & Laser Center, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong, China

      Baihui Sun, Wenlin Yu, Yanhong Wu & Qin Li

    3. Spine Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China

      Ruoting Ding

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    1. Baihui Sun
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    Correspondence to Qin Li.

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    Baihui Sun and Ruoting Ding have contributed equally to this work.

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    Sun, B., Ding, R., Yu, W. et al. Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX–MAPK pathway. Apoptosis 21, 825–835 (2016). https://doi.org/10.1007/s10495-016-1245-2

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