Skip to main content
Springer Nature Link
Log in

Signal transducer and activator of transcription-3 mediated neuroprotective effect of interleukin-6 on cobalt chloride mimetic hypoxic cell death in R28 cells

  • Short Communication
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Background

Hypoxic injury to retinal ganglionic cells and adjoining glia is implicated in glaucomatous optic neuropathy. The present study evaluates the effect of IL-6 on R28 retinal precursor cell line exposed to hypoxic injury.

Methods and results

Apoptotic cell death induced by hypoxia mimetic CoCl2 in R28 cells with or without IL-6 treatment was measured using cell viability assays and apoptotic markers. Oxidative stress was also measured. Hypoxia induced by mimetic CoCl2 led to a time and concentration dependent apoptosis of cells mediated by disruption of mitochondrial membrane potential and activation of caspase 3. Cells pre-treated with IL-6 demonstrated significantly higher viability and mitochondrial integrity under hypoxic conditions. A critical role of STAT3 was observed in mediating the cytoprotective effects of IL-6. Treatment of cells with IL-6 led to STAT3-mediated expression of the Bcl-2 family proteins and MnSOD.

Conclusions

The data from the present study indicate cytoprotective role of IL-6 and suggest a previously unreported mechanism of neuroprotection via STAT3 mediated signaling.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+
from $39.99 /Month
  • Starting from 10 chapters or articles per month
  • Access and download chapters and articles from more than 300k books and 2,500 journals
  • Cancel anytime
View plans

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Explore related subjects

Discover the latest articles, books and news in related subjects, suggested using machine learning.

Data availability

The data generated in the study is included in the manuscript.

References

  1. Almasieh M, Wilson AM, Morquette B et al (2012) The molecular basis of retinal ganglion cell death in glaucoma. Prog Retin Eye Res 31:152–181. https://doi.org/10.1016/j.preteyeres.2011.11.002

    Article  CAS  PubMed  Google Scholar 

  2. Diekmann H, Fischer D (2013) Glaucoma and optic nerve repair. Cell Tissue Res 353:327–337. https://doi.org/10.1007/s00441-013-1596-8

    Article  PubMed  Google Scholar 

  3. Willis EF, MacDonald KPA, Nguyen QH et al (2020) Repopulating microglia promote brain repair in an IL-6-dependent manner. Cell. https://doi.org/10.1016/j.cell.2020.02.013

    Article  PubMed  Google Scholar 

  4. Sappington RM, Chan M, Calkins DJ (2006) Interleukin-6 protects retinal ganglion cells from pressure-induced death. Invest Ophthalmol Vis Sci 47:2932–2942. https://doi.org/10.1167/iovs.05-1407

    Article  PubMed  Google Scholar 

  5. Mendonça Torres PM, de Araujo EG (2001) Interleukin-6 increases the survival of retinal ganglion cells in vitro. J Neuroimmunol 117:43–50. https://doi.org/10.1016/S0165-5728(01)00303-4

    Article  PubMed  Google Scholar 

  6. Müller A, Hauk TG, Fischer D (2007) Astrocyte-derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation. Brain 130:3308–3320. https://doi.org/10.1093/brain/awm257

    Article  PubMed  Google Scholar 

  7. Leibinger M, Müller A, Gobrecht P et al (2013) Interleukin-6 contributes to CNS axon regeneration upon inflammatory stimulation. Cell Death Dis 4:e609. https://doi.org/10.1038/cddis.2013.126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Leibinger M, Andreadaki A, Diekmann H, Fischer D (2013) Neuronal STAT3 activation is essential for CNTF- and inflammatory stimulation-induced CNS axon regeneration. Cell Death Dis 4:e805. https://doi.org/10.1038/cddis.2013.310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pernet V, Joly S, Jordi N et al (2013) Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve. Cell Death Dis 4:e734. https://doi.org/10.1038/cddis.2013.266

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pandey RK, Bhatt KH, Dahiya Y, Sodhi A (2011) Mycobacterium indicus pranii supernatant induces apoptotic cell death in mouse peritoneal macrophages in vitro. PLoS ONE 6:17093. https://doi.org/10.1371/journal.pone.0017093

    Article  CAS  Google Scholar 

  11. Seigel GM, Mutchler AL, Imperato EL (1996) Expression of glial markers in a retinal precursor cell line. Mol Vis 2:2

    CAS  PubMed  Google Scholar 

  12. Seigel GM (2014) Review: R28 retinal precursor cells: the first 20 years. Mol Vis 20:301–306

    PubMed  PubMed Central  Google Scholar 

  13. Seigel GM, Salvi RJ (2013) A microarray dataset of genes expressed by the R28 retinal precursor cell line. Dataset Pap Neurosci. https://doi.org/10.7167/2013/261063

    Article  Google Scholar 

  14. Seigel GM, Sun W, Wang J et al (2004) Neuronal gene expression and function in the growth-stimulated R28 retinal precursor cell line. Curr Eye Res 28(4):257–269. https://doi.org/10.1076/ceyr.28.4.257.27831

    Article  CAS  PubMed  Google Scholar 

  15. Wu D, Yotnda P (2011) Induction and testing of hypoxia in cell culture. J Vis Exp 12(54):2899. https://doi.org/10.3791/2899

    Article  Google Scholar 

  16. Kamiya T, Hara H, Inagaki N, Adachi T (2010) The effect of hypoxia mimetic cobalt chloride on the expression of EC-SOD in 3T3-L1 adipocytes. Redox Rep 15(3):131–137. https://doi.org/10.1179/174329210X12650506623483

    Article  CAS  PubMed  Google Scholar 

  17. Fuhrmann DC, Brüne B (2017) Mitochondrial composition and function under the control of hypoxia. Redox Biol. https://doi.org/10.1016/j.redox.2017.02.012

    Article  PubMed  PubMed Central  Google Scholar 

  18. Kang S, Narazaki M, Metwally H, Kishimoto T (2020) Historical overview of the interleukin-6 family cytokine. J Exp Med. https://doi.org/10.1084/jem.20190347

    Article  PubMed  PubMed Central  Google Scholar 

  19. Tanaka T, Narazaki M, Kishimoto T (2014) Il-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol. https://doi.org/10.1101/cshperspect.a016295

    Article  PubMed  PubMed Central  Google Scholar 

  20. Jourdan M, de Vos J, Mechti N, Klein B (2000) Regulation of Bcl-2-family proteins in myeloma cells by three myeloma survival factors: Interleukin-6, interferon-alpha and insulin-like growth factor 1. Cell Death Differ 7(12):1244–1252. https://doi.org/10.1038/sj.cdd.4400758

    Article  CAS  PubMed  Google Scholar 

  21. Slomp A, Peperzak V (2018) Role and regulation of pro-survival BCL-2 proteins in multiple myeloma. Front Oncol 8:533. https://doi.org/10.3389/fonc.2018.00533

    Article  PubMed  PubMed Central  Google Scholar 

  22. Holley AK, Bakthavatchalu V, Velez-Roman JM, StClair DK (2011) Manganese superoxide dismutase: guardian of the powerhouse. Int J Mol Sci 12(10):7114–7162. https://doi.org/10.3390/ijms12107114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Candas D, Li JJ (2014) MnSOD in oxidative stress response-potential regulation via mitochondrial protein influx. Antioxid Redox Signal 20(10):1599–617. https://doi.org/10.1089/ars.2013.5305

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ono M, Kohda H, Kawaguchi T et al (1992) Induction of Mn-superoxide dismutase by tumor necrosis factor, Interleukin-1 and Interleukin-6 in human hepatoma cells. Biochem Biophys Res Commun 182(3):1100–1107. https://doi.org/10.1016/0006-291X(92)91845-H

    Article  CAS  PubMed  Google Scholar 

  25. Joo EJ, Gab SK, Narasimhan P et al (2009) Regulation of Mn-superoxide dismutase activity and neuroprotection by STAT3 in mice after cerebral ischemia. J Neurosci 29(21):7003–7014. https://doi.org/10.1523/JNEUROSCI.1110-09.2009

    Article  CAS  Google Scholar 

  26. van Bergen NJ, Wood JPM, Chidlow G et al (2009) Recharacterization of the RGC-5 retinal ganglion cell line. Invest Ophthalmol Vis Sci 50(9):4267–4272. https://doi.org/10.1167/iovs.09-3484

    Article  PubMed  Google Scholar 

Download references

Funding

The study was supported by Grant No. SB/YS/LS-140/2014 by the Department of Science and Technology, Science and Engineering Research Board (DST-SERB), Start-up BSR grant and Special Assistance Program (SAP) by University Grants Commission (UGC) and Department of Science and Technology, Promotion of University Research and Scientific Excellence (DST-PURSE) (NO.SR/PURSE Phase 2/18(G)). Author NT received senior research fellowship from Indian Council of Medical Research (ICMR).

Author information

Authors and Affiliations

  1. Department of Human Genetics, Guru Nanak Dev University, Amritsar, Punjab, 143005, India

    Nanamika Thakur & Sanjana Mehrotra

  2. Thermo Fisher Scientific, Bangalore, Karnataka, 560066, India

    Rajeev Kumar Pandey

Authors
  1. Nanamika Thakur
    View author publications

    Search author on:PubMed Google Scholar

  2. Rajeev Kumar Pandey
    View author publications

    Search author on:PubMed Google Scholar

  3. Sanjana Mehrotra
    View author publications

    Search author on:PubMed Google Scholar

Contributions

RKP and SM conceived and planned the study. All authors performed the experiments and analyzed the data. All authors wrote the manuscript and approved the final version. SM acquired financial funding for the study.

Corresponding author

Correspondence to Sanjana Mehrotra.

Ethics declarations

Conflict of interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

11033_2021_6586_MOESM1_ESM.tif

Supplementary file1 Supplementary Fig.S1. IL-6 mediated protection against CoCl2 induced cell death in R28 cells. (a) Cells were treated with mentioned doses of CoCl2 for 24 hours or (b) 0.5 mM dose of CoCl2 for 2, 4, 6, 12 and 24 hours and MTT assay was performed to calculate % viability based on the readings at 540 nm. The plots represent data from 3 independent experiments and the error bars indicate SEM values. (c) Cells were treated without or in presence of different concentrations of IL-6 with CoCl2 and MTT assay was performed after 24 hours. (d) Cells left untreated or pretreated with 10 ng/mL of IL-6 for 1 hour were treated with CoCl2 for 12 or 24 hours. MTT assay was performed and % viability was plotted. (e) Similarly, at the completion of treatment, cells were stained with Annexin V and observed under fluorescence microscope and counted for Annexin V positive cells. Percentage of Annexin V-positive cells has been plotted on the Y-Axis. (f) Cells, untreated or pretreated with of IL-6 for 1 hour were incubated with CoCl2 for 24 hours, fixed with 4% PFA, stained with DAPI and observed under fluorescence microscope. Cells with fragmented nuclei were counted and plotted on Y-axis as percentage cells with nuclear fragmentation. A minimum of 20 fields or 250 cells were observed. The statistical significance of difference between the test groups in the all figures was analyzed by student’s t-test (two tailed). p-value of less than 0.05 is considered significant. The error bars of the values represent SEM from the replicates (TIF 528 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Thakur, N., Pandey, R.K. & Mehrotra, S. Signal transducer and activator of transcription-3 mediated neuroprotective effect of interleukin-6 on cobalt chloride mimetic hypoxic cell death in R28 cells. Mol Biol Rep 48, 6197–6203 (2021). https://doi.org/10.1007/s11033-021-06586-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-021-06586-5

Keywords