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Possible role of HIWI2 in modulating tight junction proteins in retinal pigment epithelial cells through Akt signaling pathway

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Abstract

PIWI subfamily of proteins is shown to be primarily expressed in germline cells. They maintain the genomic integrity by silencing the transposable elements. Although the role of PIWI proteins in germ cells has been documented, their presence and function in somatic cells remains unclear. Intriguingly, we detected all four members of PIWI-like proteins in human ocular tissues and somatic cell lines. When HIWI2 was knocked down in retinal pigment epithelial cells, the typical honeycomb morphology was affected. Further analysis showed that the expression of tight junction (TJ) proteins, CLDN1, and TJP1 were altered in HIWI2 knockdown. Moreover, confocal imaging revealed disrupted TJP1 assembly at the TJ. Previous studies report the role of GSK3β in regulating TJ proteins. Accordingly, phospho-kinase proteome profiler array indicated increased phosphorylation of Akt and GSK3α/β in HIWI2 knockdown, suggesting that HIWI2 might affect TJ proteins through Akt-GSK3α/β signaling axis. Moreover, treating the HIWI2 knockdown cells with wortmannin increased the levels of TJP1 and CLDN1. Taken together, our study demonstrates the presence of PIWI-like proteins in somatic cells and the possible role of HIWI2 in preserving the functional integrity of epithelial cells probably by modulating the phosphorylation status of Akt.

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Acknowledgements

We acknowledge the financial assistance provided by Department of Science and Technology under the project “SR/FT/LS-145/2010”, Indian Council of Medical Research “BMS/FW/BIOCHEM/2015-23270/OCT-2015/24/TN/PVT” and Max Planck-India mobility grant ‘IGSTC/MPG/FS(SC)2012’. CS thanks University Grants Commission, New Delhi for the award of Assistant Professorship under its Faculty Recharge Program (UGC-FRP). We acknowledge the facilities, the scientific technical assistance of Advanced Microscopy facility at NCTB, Department of Biotechnology, IIT Madras, Chennai, India. We thank Dr. R.Baskaran, Department of Biochemistry and Molecular Biology, Pondicherry University, for critical reading of the manuscript.

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Authors and Affiliations

  1. RS Mehta Jain Department of Biochemistry and Cell Biology, Vision Research Foundation, Chennai, India

    Suganya Sivagurunathan, Karthikka Palanisamy & Subbulakshmi Chidambaram

  2. School of Chemical and Biotechnology, SASTRA University, Thanjavur, India

    Suganya Sivagurunathan & Karthikka Palanisamy

  3. SN ONGC Department of Genetics and Molecular Biology, Vision Research Foundation, Chennai, India

    Jayamuruga Pandian Arunachalam

  4. Department of Biochemistry and Molecular Biology, Pondicherry University, Pondicherry, India

    Subbulakshmi Chidambaram

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  1. Suganya Sivagurunathan
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Correspondence to Subbulakshmi Chidambaram.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. If applicable (where such a committee exists): “All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.”

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11010_2016_2906_MOESM1_ESM.tif

Supplementary material 1: S1 figure. Specificity of the primers. a) Agarose gel images showing the specificity of the primers. b) Melting curve of the real time PCR primers showing the amplification was only due to PIWIL transcripts (TIFF 3196 kb)

11010_2016_2906_MOESM2_ESM.tif

Supplementary material 2: S2 figure. Validation of HIWI and HILI antibodies. a) Expression of HIWI in rat testis and rat liver as a positive and negative control respectively. The band at 99 kDa in rat testis has reduced intensity when the HIWI antibody was incubated with the respective blocking peptide. b) Expression of HILI in rat testis and rat liver as a positive and negative control respectively. The band at 110 kDa in rat testis has reduced intensity when the HILI antibody was incubated with the respective blocking peptide. (Positive control (PC) – rat testis; Negative control (NC)—Rat liver; BP—blocking peptide). (TIFF 1947 kb)

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Sivagurunathan, S., Palanisamy, K., Arunachalam, J.P. et al. Possible role of HIWI2 in modulating tight junction proteins in retinal pigment epithelial cells through Akt signaling pathway. Mol Cell Biochem 427, 145–156 (2017). https://doi.org/10.1007/s11010-016-2906-8

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