Changes in Oxidative Phosphorylation Activity in Fibroblasts at p38 MAPK Pathway Inhibition

ORIGINAL ARTICLE
Irina A Shurygina, Irina S Trukhan, Nataliya N Dremina, Michael G Shurygin

 
International Journal of Biomedicine. 2019;9(4):350-355.
DOI: 10.21103/Article9(4)_OA15
Originally published December 15, 2019

Abstract: 

Background: Mitochondrial oxidative phosphorylation (OxPhos) accounts for more than 90% of the cellular ATP production, plays the role in reactive oxygen species (ROS) generation and programmed cell death. In addition, it contributes to such cellular processes as proliferation, differentiation and cell aging. Currently, several signaling systems are known to participate in regulation of OxPhos and activity of cytochrome c-oxidase (CcO), the terminal enzyme of the mitochondrial electron transport chain. However, data on mechanisms and key units involved in the signal transduction are still being supplemented.
Methods: Peritoneal fibroblasts were isolated from the omentum of Wistar rats by fragmenting the dissected tissue and disaggregating the fragments in collagenase solution (200 U/ml). The primary culture of fibroblasts was cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1% antibiotic/antimycotic at 37°C, 80% RH and 5% СО2 in Biostation CT, Nikon. To obtain a culture, the fibroblasts were subcultured every 7 days. After the third passage the culture was treated with SB203580 at the concentration of 10 μM or with SB203580 in combination with bFGF at the dose of 133 pg/ml. Cells for immunofluorescent studies were fixed with 70% ethanol and stained with antibodies to CcO subunit I.
Results: When exposed to SB203580 or the combination of SB203580 and bFGF, marked changes were observed in the fibroblast culture: in both cases there was intensive collagen destruction; attached fibroblasts rounded and detached from the substrate. When exposed to SB203580, non-rounded cells started to vacuolate actively while vacuoles occupied the entire cytoplasm. Introduction of the p38 inhibitor into the culture of activated fibroblasts caused a more intensive cell detachment and collagen destruction. Moreover, the formation of large conglomerates containing several dozens of cells connected with collagen fibers was observed in the areas characterized by the highest cell density. Immunofluorescent staining made it possible to reveal a certain increase in the cell area occupied by CcO after fibroblasts exposure to SB203580 and a significant increase in the area of the enzyme distribution in the studied cells (more than 5-fold in comparison with the control group) when simultaneously adding  SB203580 and bFGF. In addition, one-way ANOVA test demonstrated a statistically significant increase in the CcO fluorescent staining intensity in both cases.
Conclusion: The analysis of the results indicates that SB203580, a p38 MAPK inhibitor, influences both peritoneal fibroblast morphology and the energy status of the cells under study increasing the amount of CcO, the terminal enzyme of the mitochondrial electron transport chain, in the cells, although no cell change to active proliferation or apoptosis was observed. Fibroblast culture stimulation by bFGF significantly enhances the effect of SB203580, which implies a greater OxPhos complex expression in case of impaired p38 MAPK signaling pathway activation.

Keywords: 
peritoneal fibroblast • oxidative phosphorylation • р38 МАРК • SB203580 • bFGF • OxPhos
References: 
  1. Jezek P, Hlavata L. Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism.  Int J Biochem. Cell Biol. 2005;37(12):2478–503.
  2. Hüttemann M, Lee I, Grossman LI, Doan JW, Sanderson TH. Phosphorylation of mammalian cytochrome С and cytochrome С oxidase in the regulation of cell destiny: respiration, apoptosis, and human disease. Adv Exp Med Biol. 2012;748:237–64. doi: 10.1007/978-1-4614-3573-0_10.
  3. Srinivasan S, Avadhani NG. Cytochrome c oxidase dysfunction in oxidative stress. Free Radic Biol Med. 2012;53(6):1252-63. doi: 10.1016/j.freeradbiomed.2012.07.021.
  4. Vallée A, Lecarpentier Y, Vallée JN.  Thermodynamic aspects and reprogramming cellular energy metabolism during the fibrosis process. Int J Mol Sci. 2017;18(12). pii: E2537. doi: 10.3390/ijms18122537
  5. Greco M, Villani G, Mazzucchelli F, Bresolin N, Papa S, Attardi G. Marked aging-related decline in efficiency of oxidative phosphorylation in human skin fibroblasts. FASEB J. 2003;17(12):1706-8. doi: 10.1096/fj.02-1009fje.
  6. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324(5930):1029–33. doi: 10.1126/science.1160809.
  7. Yao CH, Wang R, Wang Y, Kung CP, Weber JD, Patti GJ. Mitochondrial fusion supports increased oxidative phosphorylation during cell proliferation. Elife. 2019;8. pii: e41351. doi: 10.7554/eLife.41351.
  8. Abdel-Haleem AM, Lewis NE, Jamshidi N, Mineta K, Gao X, Gojobori T. The emerging facets of non-cancerous warburg effect. Front Endocrinol (Lausanne). 2017;8:279. doi: 10.3389/fendo.2017.00279.
  9. Ashton TM, McKenna WG, Kunz-Schughart LA, Higgins GS. Oxidative phosphorylation as an emerging target in cancer therapy. Clin Cancer Res. 2018;24(11):2482-90. doi: 10.1158/1078-0432.CCR-17-3070.
  10. Dawson TL, Gores GJ, Nieminen AL, Herman B, Lemasters JJ. Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes. Am J Physiol. 1993; 264(4 Pt 1):C961–7. doi: 10.1152/ajpcell.1993.264.4.C961.
  11. Fukuda R, Zhang H, Kim JW, Shimoda L, Dang CV, Semenza GL. HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. Cell. 2007;129(1):111-22. doi: 10.1016/j.cell.2007.01.047.
  12. Khalimonchuk O, Bird A, Winge DR. Evidence for a pro-oxidant intermediate in the assembly of cytochrome oxidase. J Biol Chem. 2007;282(24):17442-9. doi: 10.1074/jbc.M702379200.
  13. Castello PR, David PS, McClure T, Crook Z, Poyton RO. Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: implications for oxygen sensing and hypoxic signaling in eukaryotes. Cell Metab. 2006;3:277–87. doi: 10.1016/j.cmet.2006.02.011.
  14. Yang WL, Iacono L, Tang WM, Chin KV. Novel function of the regulatory subunit of protein kinase A: regulation of cytochrome c oxidase activity and cytochrome c release. Biochemistry. 1998; 37(40):14175–80. doi: 10.1021/bi981402a.
  15. Lee I, Salomon AR, Ficarro S, Mathes I, Lottspeich F, Grossman LI, Huttemann M. cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity. J Biol Chem. 2005; 280(7):6094–100. doi: 10.1074/jbc.M411335200.
  16. Boerner JL, Demory ML, Silva C, Parsons SJ. Phosphorylation of Y845 on the epidermal growth factor receptor mediates binding to the mitochondrial protein cytochrome c oxidase subunit II. Mol Cell Biol. 2004;24(16):7059-71. doi: 10.1128/MCB.24.16.7059-7071.2004.
  17. Pang L, Qiu T, Cao X, Wan M. Apoptotic role of TGF-β mediated by Smad4 mitochondria translocation and cytochrome c oxidase subunit II interaction. Exp Cell Res. 2011;317(11):1608-20. doi: 10.1016/j.yexcr.2011.02.004.
  18. Samavati L, Lee I, Mathes I, Lottspeich F, Huttemann M. Tumor necrosis factor alpha inhibits oxidative phosphorylation through tyrosine phosphorylation at subunit I of cytochrome c oxidase. J Biol Chem. 2008;283(30):21134-44. doi: 10.1074/jbc.M801954200.;
  19. Shurygin MG, Shurygina IA, Kanya OV, Dremina NN, Lushnikova EL, Nepomnyashchikh RD. Morphological evaluation of oxidative phosphorylation system in myocardial infarction under conditions of modified vascular endothelial growth factor concentration. Bull  Exp Biol Med. 2015;159 (3):402-5. doi: 10.1007/s10517-015-2974-x.
  20. Shurygin MG, Shurygina IA, Granina GB, Zelenin NV, Ayushinova NI. Using laser confocal microscopy to assess the activity of MAP kinase systems in the reparative process. Bulletin of the Russian Academy of Sciences: Physics. 2016;.80(1):14-6. doi: 10.3103/S1062873816010214.
  21. Zhang W, Liu HT. MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res. 2002;12(1):9-18. doi: 10.1038/sj.cr.7290105.
  22. Shurygina IA, Shurygin MG, Ayushinova NI, Granina GB, Zelenin NV. Mechanisms of connective tissue formation and blocks of mitogen activated protein kinase. Frontiers of Chemical Science and Engineering. 2012;6(2):232-7. doi: 10.1007/s11705-012-1286-1.
  23. Shurygina IA, Shurygin MG, Zelenin NV, Ayushinova NI Influence on mitogen-activated protein kinases as a new direction of connective tissue growth regulation. Byulleten Sibirskoy Meditsiny. 2017;16(4):86-93. doi: 10.20538/1682-0363-2017-4-86-93.
  24. Shurygina IA, Aushinova NI, Shurygin MG. Effect of p38 MAPK inhibition on apoptosis marker expression in the process of peritoneal adhesion formation. International Journal of Biomedicine. 2018;8(4):342-6. doi: 10.21103/Article8(4)_OA15.
  25. Takenaka K, Mcriguchi T, Nishida E. Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest. Science. 1998;280(5363):599-602. doi: 10.1126/science.280.5363.599.
  26. Molkentin JD, Bugg D, Ghearing N, Dorn LE, Kim P, Sargent MA et al. Fibroblast-specific genetic manipulation of p38 mitogen-activated protein kinase in vivo reveals its central regulatory role in fibrosis. Circulation. 2017;136(6):549-61. doi: 10.1161/CIRCULATIONAHA.116.026238.
  27. Ju X, Wen Y, Metzger D, Jung M. The role of p38 in mitochondrial respiration in male and female mice. Neurosci Lett. 2013;544:152-6. doi: 10.1016/j.neulet.2013.04.004.
  28. Ronda AC, Vasconsuelo A, Boland R. Extracellular-regulated kinase and p38 mitogen-activated protein kinases are involved in the antiapoptotic action of 17beta-estradiol in skeletal muscle cells. J Endocrinol. 2010;206(2):235-46. doi: 10.1677/JOE-09-0429.
  29. Lu Z, Zhou H, Zhang S, Dai W, Zhang Y, Hong L et al. Activation of reactive oxygen species-mediated mitogen-activated protein kinases pathway regulates both extrinsic and intrinsic apoptosis induced by arctigenin in Hep G2. J Pharm Pharmacol. 2020;72(1):29-43. doi: 10.1111/jphp.13180.
  30. Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer. 2000;7(3):165-97.
  31. Shurygin MG, Shurygina IA. Influence of FGF2 level on the inflammation phase dynamics at the postinfarction cardiosclerosis. Patol Fiziol Eksp Ter. 2010;(4):34-7.
  32. Shurygin MG, Kanya OV, Dremina NN, Shurygina IA. Morphometric analysis of the growth factors influence on fibroblastic phase of inflammation at experimental myocardial infraction. Acta Biomedica Scientifica. 2014.97(3):105-8.
  33. Matsumoto T, Turesson I, Book M, Gerwins P, Claesson-Welsh L. p38 MAP kinase negatively regulates endothelial cell survival, proliferation, and differentiation in FGF-2-stimulated angiogenesis. J Cell Biol. 2002;156(1):149-60. doi: 10.1083/jcb.200103096.
  34. Raucci A, Laplantine E, Mansukhani A, Basilico C. Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes. J Biol Chem. 2004;279(3):1747-56. doi: 10.1074/jbc.M310384200.
  35. Maher P. p38 mitogen-activated protein kinase activation is required for fibroblast growth factor-2-stimulated cell proliferation but not differentiation. J Biol Chem. 1999;274(25):17491-8. doi: 10.1074/jbc.274.25.17491.
  36. Williamson AJ, Dibling BC, Boyne JR, Selby P, Burchill SA. Basic fibroblast growth factor-induced cell death is effected through sustained activation of p38MAPK and up-regulation of the death receptor p75NTR. J Biol Chem. 2004;279(46):47912-28. doi: 10.1074/jbc.M409035200.
  37. Johansson N, Ala-aho R, Uitto V, Grénman R, Fusenig NE, López-Otín C, Kähäri VM. Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase. J Cell Sci. 2000;113 Pt 2:227-35.

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Received October 28, 2019.
Accepted November 27, 2019.
©2019 International Medical Research and Development Corporation.