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Apelin Receptor Signaling Protects GT1-7 GnRH Neurons Against Oxidative Stress In Vitro

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Abstract

Hypothalamic–pituitary–adrenal (HPA) axis regulates stress response in the body and abnormal increase in oxidative stress contributes to the various disease pathogenesis. Although hypothalamic distribution of Apelin receptor (APLNR) has been studied, the potential regulatory role in hormone releasing function of hypothalamus in response to stress is not well elucidated yet. To determine whether APLNR is involved in the protection of the hypothalamus against oxidative stress, gonadotropin-releasing hormone (GnRH) cells were used as an in vitro model system. GT1-7 mouse hypothalamic neuronal cell line was subjected to H2O2 and hypoxia induced oxidative stress under various circumstances including APLNR overexpression, knockdown and knockout. Overexpression and activation of APLNR in GnRH producing neurons caused an increase in cell proliferation under oxidative stress. In addition, blockage of APLNR function by siRNA reduced GnRH release. Activation of APLNR initiated AKT kinase pathway as a proliferative response against hypoxic culture conditions and blocked apoptosis. Although expression and activation of APLNR have not been related to GnRH neuron differentiation during development, positive contribution of activated APLNR signaling to GnRH release in mouse embryonic stem cell derived GnRH neurons was observed in the present study. Sustained overexpression and complete deletion of APLNR in mouse embryonic stem cell derived GnRH neurons reduced GnRH release in vitro. The present findings suggest that expression and activation of APLNR in GnRH releasing GT1-7 neurons might induce a protective mechanism against oxidative stress induced cell death and APLNR signaling may play a role in GnRH neurons.

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Acknowledgements

This study was supported by Yeditepe University. We would like to thank to Professor Pamela Mellon for donation of GT1-7 cell line to Bayram Yilmaz lab. We are grateful to Dr. Michael Kyba for sharing A2Lox.cre mouse embryonic stem (ES) cells. We would like to thank to Dr. Terry P. Yamaguchi for processing A2Lox.cre cell line propagation and transfer, respectively.

Funding

This study was supported by Yeditepe University.

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  1. Hatice Burcu Şişli and Taha Bartu Hayal have contributed equally to this manuscript.

Authors and Affiliations

  1. Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey

    Hatice Burcu Şişli, Taha Bartu Hayal, Selinay Şenkal, Binnur Kıratlı, Derya Sağraç, Selin Seçkin, Murat Özpolat, Fikrettin Şahin & Ayşegül Doğan

  2. Department of Physiology, Faculty of Medicine, Yeditepe University, Istanbul, Turkey

    Bayram Yılmaz

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  1. Hatice Burcu Şişli
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Contributions

HBŞ and TBH conducted most of the cell culture experiments and oxidative stress modeling. SŞ performed siRNA experiments. BK completed all light and confocal microscopy experiments. DS and SS conducted knockout and overexpression experiments, respectively. MÖ did all flow cytometry experiments and data analysis. BY provided GT1-7 cells and conducted GT1-7 hypoxia experiments. FŞ and AD completed mouse embryonic stem cell differentiation and characterization experiments. The first draft of the manuscript was written by HBŞ and AD and all authors commented on previous versions of the manuscript. BY and FŞ edited the manuscript before submission. All authors read and approved the final manuscript. AD is corresponding author and designed all experiments and conducted in vitro assays together with team.

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Correspondence to Ayşegül Doğan.

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10571_2020_968_MOESM1_ESM.tif

Supplementary Fig. 1. Total and phosphorylated Akt protein levels of GT1-7 cells under normoxia and oxidative stress conditions in vitro. The western blot band intensities of (A) Aplnr activation, (B) Aplnr knockdown, (C) Aplnr knockout, (D) Aplnr overexpression. Notes: Apelin-13: 1µM, ML233: 0.1µM, Protein amounts was normalized to β-actin, *P<0.05, **P<0.005, ***P<0.001, Akt: Protein kinase B, p-Akt: Phosphorylated Protein kinase B. (TIF 366 kb)

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Şişli, H.B., Hayal, T.B., Şenkal, S. et al. Apelin Receptor Signaling Protects GT1-7 GnRH Neurons Against Oxidative Stress In Vitro. Cell Mol Neurobiol 42, 753–775 (2022). https://doi.org/10.1007/s10571-020-00968-2

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