July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Core Clock Protein Bmal 1 Controls Reactive Oxygen Species Homeostasis And Oxidative Responses By Transregulating Prdx6 Expression
Author Affiliations & Notes
  • Dhirendra P Singh
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska, United States
  • Eri Kubo
    Ophthalmology, kanazawa Medical University, Kanazawa, Ishikawa, Japan
  • Bhavana Chhunchha
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska, United States
  • Footnotes
    Commercial Relationships   Dhirendra Singh, None; Eri Kubo, None; Bhavana Chhunchha, None
  • Footnotes
    Support  EY024589
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 1127. doi:
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      Dhirendra P Singh, Eri Kubo, Bhavana Chhunchha; Core Clock Protein Bmal 1 Controls Reactive Oxygen Species Homeostasis And Oxidative Responses By Transregulating Prdx6 Expression. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1127.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Protective protein Peroxiredoxin 6 (Prdx6) rescues lens epithelial cells (LECs) from various stressors and delays lens opacity by regulating reactive oxygen species (ROS) homeostasis. Herein, we revealed, for the first time, that the core molecular clock protein, Bmal1, controls Prdx6 mRNA expression via direct E-box binding to its promoter.

Methods : The regulatory effects of Bmal1 on Prdx6 expression were assessed in vivo in eye lenses and LECs isolated from C57BL/6 mice (maintained at 12:12 light/dark) at different Zeitgeber times (ZTs), in human (h) LECs overexpressing pGFP/Bmal1 and in Bmal1 knockdown LECs (using Bmal1-shRNA) by qPCR and Western blot. The effect of Bmal1 expression on cell viability and ROS during oxidative stress induced by H2O2 (0-200μM) was examined by MTS and H2DCF-DA assays. In silico analysis (MatInspector, Genomatix) was used to spot out Bmal1/E-box binding site in hPrdx6 promoter. The promoter (-918/+30nt) and its mutant at E-Box site (-341CA(T)CGT(A)G-336) cloned into pGL2-basic plasmid were used for transactivation assay. ChIP and Gel shift assays were performed to assess Bmal1/Ebox binding. Two-tailed Student’s t-test and one-way ANOVA were used for statistical analysis.

Results : qRT-PCR revealed that the clock oscillated in LECs in vivo, and Bmal1 and Prdx6 expression levels were downregulated during ZTs 8-14 and increased during ZT20-ZT2 (ZT0 is when the lights were turned on), which was anti-phasic with ROS levels. Expression assays showed Bmal 1-dependent expression of Prdx6 mRNA and protein. DNA binding and transactivation assays disclosed that Bmal1 bound to E-box sequences and activated Prdx6 gene transcription, while mutant promoter (mutant E-box, CTCGTG) resulted in significant reduction of transcriptional activity in LECs (p<0.001), suggesting that Bmal1 directly bound and transregulated Prdx6 promoter. Intriguingly, Bmal1 knockdown LECs showed repression of Prdx6 expression and were more vulnerable to oxidative stress-induced death.

Conclusions : Our findings indicate that Bmal1 has a clear role in regulating Prdx6 in LECs and affecting oxidative responses, and provide a first insight into a plausible role for the molecular clock controls of Prdx6 and oxidative stress. Results suggest that Bmal1 is required for LEC homeostasis by mitigating oxidative stress through Prdx6.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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