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Acylated and unacylated ghrelin protect MC3T3-E1 cells against tert-butyl hydroperoxide-induced oxidative injury: pharmacological characterization of ghrelin receptor and possible epigenetic involvement

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Abstract

Increasing evidence suggests a role for oxidative stress in age-related decrease in osteoblast number and function leading to the development of osteoporosis. This study was undertaken to investigate whether ghrelin, previously reported to stimulate osteoblast proliferation, counteracts tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in MC3T3-E1 osteoblastic cells as well as to characterize the ghrelin receptor (GHS-R) involved in such activity. Pretreatment with ghrelin (10−7–10−11 M) significantly increased viability and reduced apoptosis of MC3T3-E1 cells cultured with t-BHP (250 μM) for three hours at the low concentration of 10−9 M as shown by MTT assay and Hoechst-33258 staining. Furthermore, ghrelin prevented t-BHP-induced osteoblastic dysfunction and changes in the cytoskeleton organization evidenced by the staining of the actin fibers with Phalloidin-FITC by reducing reactive oxygen species generation. The GHS-R type 1a agonist, EP1572 (10−7–10−11 M), had no effect against t-BHP-induced cytotoxicity and pretreatment with the selective GHS-R1a antagonist, d-Lys3-GHRP-6 (10−7 M), failed to remove ghrelin (10−9 M)-protective effects against oxidative injury, indicating that GHS-R1a is not involved in such ghrelin activity. Accordingly, unacylated ghrelin (DAG), not binding GHS-R1a, displays the same protective actions of ghrelin against t-BHP-induced cytotoxicity. Preliminary observations indicate that ghrelin increased the trimethylation of lys4 on histones H3, a known epigenetic mark activator, which may regulate the expression of some genes limiting oxidative damage. In conclusion, our data demonstrate that ghrelin and DAG promote survival of MC3T3-E1 cell exposed to t-BHP-induced oxidative damage. Such effect is independent of GHS-R1a and is likely mediated by a common ghrelin/DAG binding site.

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Acknowledgments

This work was supported by funds from FIRST 2009 to VS and from Project ASI DC-DTE-2011-033 for biomedical and biotechnological studies to FC.

Conflict of interest

The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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Authors and Affiliations

  1. Department of Medical Biotechnology and Translational Medicine, Medical Pharmacology Unit, Universita`degli Studi di Milano, Via Vanvitelli, 32, 20129, Milan, Italy

    Elisa Dieci, Lavinia Casati, Francesca Pagani & Valeria Sibilia

  2. Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy

    Fabio Celotti

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  1. Elisa Dieci
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  2. Lavinia Casati
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  3. Francesca Pagani
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  4. Fabio Celotti
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  5. Valeria Sibilia
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Correspondence to Valeria Sibilia.

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Dieci, E., Casati, L., Pagani, F. et al. Acylated and unacylated ghrelin protect MC3T3-E1 cells against tert-butyl hydroperoxide-induced oxidative injury: pharmacological characterization of ghrelin receptor and possible epigenetic involvement. Amino Acids 46, 1715–1725 (2014). https://doi.org/10.1007/s00726-014-1734-y

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