Molecular Biology Reports

, Volume 45, Issue 4, pp 533–540 | Cite as

Level of hydrogen peroxide affects expression and sub-cellular localization of Pax6

  • Sachin Shukla
  • Rajnikant Mishra
Original Article


The Pax6 is a multifunctional pairedbox and homeobox containing transcription factor which is involved in several functions of brain, eyes, and pancreas. It regulates expression of genes involved in cell proliferation, differentiation, inflammation, oxidative stress management, and neuropathy. Dynamic changes in the sub-cellular localization of Pax6 are proposed to regulate its activity, however, the underlying mechanism remains poorly understood. The oxidative stress mediated changes were studied in sub-cellular localization of Pax6 in cultured cells derived from the eye (cornea) and pancreas. The impact of induced oxidative stress was investigated on reactive oxygen species scavenger molecules, Superoxide dismutase1 (SOD1) and Catalase, and a critical cell signalling molecule Transforming growth factor-beta (TGF-β1). The cells were treated with three different concentrations of H2O2, viz., 0.3, 1.5, and 3.0 mM. The cell viability was analysed through Trypan blue dye exclusion assay. The localization of Pax6 was observed by immunofluorescence labeling, and alterations in levels of Pax6, SOD1, Catalase, and TGF-β1 were investigated by semi-quantitative RT-PCR. Nucleo-cytoplasmic shuttling of Pax6 was observed in cells of corneal epithelial (SIRC) and pancreatic origins (MIA-PaCa2). The percentage distribution of Pax6 in nuclear and cytoplasmic compartments of SIRC and MIA-PaCa2 cells was analyzed through ImageJ software. Level of hydrogen peroxide affects expression and sub-cellular localization of Pax6. Expression of Pax6 and TGF-β1 are directly associated with changes in sub-cellular localization of Pax6 and modulation in expression of Catalase. This may be the result of a cellular protective mechanism against peroxide-dependent cellular stress.


Pax6 Oxidative stress Sub-cellular localization Nucleo-cytoplasmic shuttling 



Authors are highly thankful to Prof. M. K. Thakur, Biochemistry& Molecular Biology Unit, Department of Zoology, Banaras Hindu University, Varanasi, India, for providing the fluorescence microscope facility.


This work was funded by the grants from the Council of Scientific & Industrial Research (CSIR) (37(1521)/12/EMR-II) India. Sachin Shukla acknowledges Senior Research Fellowship from the Council of Scientific & Industrial Research (9/13(442)/2012-EMR-I), New Delhi, India.

Compliance with ethical standards

Conflict of interest

Authors declare that no conflict of interest exist.


  1. 1.
    Grindley JC, Davidson DR, Hill RE (1995) The role of Pax-6 in eye and nasal development. Development 121:1433–1442PubMedGoogle Scholar
  2. 2.
    Kioussi C, O’Connell S, St-Onge L, Treier M, Gleiberman AS, Gruss P, Rosenfeld MG (1999) Pax6 is essential for establishing ventral-dorsal cell boundaries in pituitary gland development. Proc Natl Acad Sci USA 96:14378–14382CrossRefPubMedGoogle Scholar
  3. 3.
    Walther C, Gruss P (1991) Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113:1435–1449PubMedGoogle Scholar
  4. 4.
    Thomas MG, Welch C, Stone L, Allan P, Barker RA, White RB (2016) PAX6 expression may be protective against dopaminergic cell loss in Parkinson’s disease. CNS Neurol Disord Drug Targets 15:73–79CrossRefPubMedGoogle Scholar
  5. 5.
    Chang JY, Hu Y, Siegel E, Stanley L, Zhou YH (2007) PAX6 increases glioma cell susceptibility to detachment and oxidative stress. J Neurooncol 84:9–19CrossRefPubMedGoogle Scholar
  6. 6.
    Zhang SJ, Li YF, Tan RR, Tsoi B, Huang WS, Huang YH, Tang XL, Hu D, Yao N, Yang X, Kurihara H, Wang Q, He RR (2016) A new gestational diabetes mellitus model: hyperglycemia-induced eye malformation via inhibition of Pax6 in the chick embryo. Dis Model Mech 9:177–186CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lou MF (2003) Redox regulation in the lens. Prog Retin Eye Res 22:657–682CrossRefPubMedGoogle Scholar
  8. 8.
    Truscott RJ (2005) Age-related nuclear cataract-oxidation is the key. Exp Eye Res 80:709–725CrossRefPubMedGoogle Scholar
  9. 9.
    Yao K, Tan J, Gu WZ, Ye PP, Wang KJ (2007) Reactive oxygen species mediates the apoptosis induced by transforming growth factor beta(2) in human lens epithelial cells. Biochem Biophys Res Commun 354:278–283CrossRefPubMedGoogle Scholar
  10. 10.
    Shubham K, Mishra R (2012) Pax6 interacts with SPARC and TGF-beta in murine eyes. Mol Vis 18:951–956PubMedPubMedCentralGoogle Scholar
  11. 11.
    Mishra S, Maurya SK, Srivastava K, Shukla S, Mishra R (2015) Pax6 influences expression patterns of genes involved in neuro- degeneration. Ann Neurosci 22:226–231CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wang J, Wang Y, Lu L (2012) De-SUMOylation of CTCF in hypoxic stress-induced human corneal epithelial cells. J Biol Chem. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ou J, Lowes C, Collinson JM (2010) Cytoskeletal and cell adhesion defects in wounded and Pax6+/- corneal epithelia. Invest Ophthalmol Vis Sci 51:1415–1423CrossRefPubMedGoogle Scholar
  14. 14.
    Chamberlain CG, Mansfield KJ, Cerra A (2009) Glutathione and catalase suppress TGFbeta-induced cataract-related changes in cultured rat lenses and lens epithelial explants. Mol Vis 15:895–905PubMedPubMedCentralGoogle Scholar
  15. 15.
    Ou J, Walczysko P, Kucerova R, Rajnicek AM, McCaig CD, Zhao M, Collinson JM (2008) Chronic wound state exacerbated by oxidative stress in Pax6+/- aniridia-related keratopathy. J Pathol 215:421–430CrossRefPubMedGoogle Scholar
  16. 16.
    Peng Y, Yang PH, Guo Y, Ng SS, Liu J, Fung PC, Tay D, Ge J, He ML, Kung HF, Lin MC (2004) Catalase and peroxiredoxin 5 protect Xenopus embryos against alcohol-induced ocular anomalies. Invest Ophthalmol Vis Sci 45:23–29CrossRefPubMedGoogle Scholar
  17. 17.
    Jia ZM, Xu W, Yu L, Zhang JJ, Lu L, Lei LS, Wu SG (2005) Effects of cyclosporin A on gene expression profiles of NIT-1 pancreatic beta cell line. Di Yi Jun Yi Da Xue Xue Bao 25:853–857PubMedGoogle Scholar
  18. 18.
    Dreja T, Jovanovic Z, Rasche A, Kluge R, Herwig R, Tung YC, Joost HG, Yeo GS, Al-Hasani H (2010) Diet-induced gene expression of isolated pancreatic islets from a polygenic mouse model of the metabolic syndrome. Diabetologia 53:309–320CrossRefPubMedGoogle Scholar
  19. 19.
    Okladnova O, Syagailo YV, Mossner R, Riederer P, Lesch KP (1998) Regulation of PAX-6 gene transcription: alternate promoter usage in human brain. Brain Res Mol Brain Res 60:177–192CrossRefPubMedGoogle Scholar
  20. 20.
    Duan D, Fu Y, Paxinos G, Watson C (2012) Spatiotemporal expression patterns of Pax6 in the brain of embryonic, newborn, and adult mice. Brain Struct Funct. CrossRefPubMedGoogle Scholar
  21. 21.
    Yu AL, Fuchshofer R, Birke M, Kampik A, Bloemendal H, Welge-Lussen U (2008) Oxidative stress and TGF-beta2 increase heat shock protein 27 expression in human optic nerve head astrocytes. Invest Ophthalmol Vis Sci 49:5403–5411CrossRefPubMedGoogle Scholar
  22. 22.
    Hebert-Schuster M, Cottart CH, Laguillier-Morizot C, Raynaud-Simon A, Golmard JL, Cynober L, Beaudeux JL, Fabre EE, Nivet-Antoine V (2011) Catalase rs769214 SNP in elderly malnutrition and during renutrition: is glucagon to blame? Free Radic Biol Med 51:1583–1588CrossRefPubMedGoogle Scholar
  23. 23.
    Parisi I, Collinson JM (2012) Regulation of Merkel cell development by Pax6. Int J Dev Biol 56:341–350CrossRefPubMedGoogle Scholar
  24. 24.
    Pinson J, Simpson TI, Mason JO, Price DJ (2006) Positive autoregulation of the transcription factor Pax6 in response to increased levels of either of its major isoforms, Pax6 or Pax6(5a), in cultured cells. BMC Dev Biol 6:25CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Shukla S, Mishra R (2011) Functional analysis of missense mutations G36A and G51A in PAX6, and PAX6(5a) causing ocular anomalies. Exp Eye Res 93:40–49CrossRefPubMedGoogle Scholar
  26. 26.
    Xu ZP, Saunders GF (1997) Transcriptional regulation of the human PAX6 gene promoter. J Biol Chem 272:3430–3436CrossRefPubMedGoogle Scholar
  27. 27.
    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 39:44–84CrossRefPubMedGoogle Scholar
  28. 28.
    Dillon N (2008) The impact of gene location in the nucleus on transcriptional regulation. Dev Cell 15:182–186CrossRefPubMedGoogle Scholar
  29. 29.
    Prochiantz A (2000) Messenger proteins: homeoproteins, TAT and others. Curr Opin Cell Biol 12:400–406CrossRefPubMedGoogle Scholar
  30. 30.
    Prochiantz A, Joliot A (2003) Can transcription factors function as cell-cell signalling molecules? Nat Rev Mol Cell Biol 4:814–819CrossRefPubMedGoogle Scholar
  31. 31.
    Dreos R, Ambrosini G, Groux R, Cavin Périer R, Bucher P (2017) The eukaryotic promoter database in its 30th year: focus on non-vertebrate organisms. Nucleic Acids Res 45(D1):D51–D55CrossRefPubMedGoogle Scholar
  32. 32.
    Maurya SK, Mishra R (2017) Pax6 binds to promoter sequence elements associated with immunological surveillance and energy homeostasis in brain of aging mice. Ann Neurosci 24(1):20–25CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Biochemistry and Molecular Biology Laboratory, Department of Zoology, Institute of ScienceBanaras Hindu UniversityVaranasiIndia
  2. 2.Schepens Eye Research Institute, Massachusetts Eye and Ear InfirmaryHarvard Medical SchoolBostonUSA

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